Frequency of Nucleic Acid Amplification Test Positivity Among Men Who Have Sex With Men Returning for a Test-of-Cure Visit 7 to 30 Days After Treatment of Laboratory-Confirmed Neisseria gonorrhoeae Infection at 2 Public Sexual Health Clinics, New York City, 2013 to 2016.

PubMed

Okah, Ebiere; Westheimer, Emily F; Jamison, Kelly; Schillinger, Julia A

2018-03-01

The Centers for Disease Control and Prevention 2015 Sexually Transmitted Disease Treatment Guidelines recommend that clinicians consider cephalosporin treatment failure in patients who deny interval sexual exposure and are nucleic acid amplification test (NAAT) positive for Neisseria gonorrhoeae (NG) at least 7 days after adequate treatment. We evaluate the real-world implications of the interval the Centers for Disease Control and Prevention recommends for a NAAT test-of-cure (TOC), by ascertaining the frequency of NG NAAT positivity at different anatomic sites among men who have sex with men (MSM) at TOC 7 to 30 days after treatment. We analyzed data from the medical records of MSM with laboratory-confirmed NG who were presumptively treated for NG during the period from June 2013 to April 2016 and returned for a TOC visit within 30 days. Data examined included symptoms, site of NG specimen collection, treatment regimen, follow-up testing, and intervening sexual activity. There were 1027 NG-positive specimens obtained from 763 MSM patients at 889 presumptive treatment visits. Of these, 44% (337/763) MSM returned for 1 or more TOC visits, and 413 specimens were collected a median of 10 days after presumptive treatment. Three percent (14/413) of specimens collected were NG NAAT positive at TOC a median of 13 days after treatment: 5% (12/256) of urethral specimens, 1% (1/147) of anorectal specimens (P = 0.037, urethral vs. anorectal), and 10% (1/10) of oropharyngeal specimens (P = 0.40, urethral vs. oropharyngeal). A small percent of patients were NG NAAT positive at TOC. Compared with anorectal specimens, urethral specimens were more frequently still positive at TOC. A large proportion of MSM will return for a TOC visit as part of standard clinical care.

Rapid change in the ciprofloxacin resistance pattern among Neisseria gonorrhoeae strains in Nuuk, Greenland: time to reconsider preventive and treatment strategies.

PubMed

Rolskov, Anne Skjerbæk; Bjorn-Mortensen, Karen; Mulvad, Gert; Poulsen, Peter; Jensen, Jørgen Skov; Pedersen, Michael Lynge

2015-01-01

Sexually transmitted infections (STIs), including infections with Neisseria gonorrhoeae (GC), are highly incident in Greenland. Since January 2011, GC testing has been performed on urine with nucleic acid amplification tests (NAATs) by strand displacement amplification (Becton Dickinson ProbeTec). Monitoring of GC antibiotic susceptibility by culture was introduced in Nuuk in 2012. Until 2014, no cases of ciprofloxacin-resistant GC strains were reported. In this paper, we report the finding of ciprofloxacin-resistant GC and describe the most recent incidence of GC infections in Greenland. The number of urine NAATs and culture-positive swabs from January to October 2014 were obtained from the Central Laboratory at Queens Ingrid's Hospital in Nuuk and stratified on gender, place and period of testing. Incidence rates were estimated as number of urine NAAT * (12/10) per 100,000 inhabitants. Men in Nuuk with a positive NAAT for GC were encouraged to provide a urethral swab for culture and susceptibility testing. From January to October 2014, a total of 5,436 urine GC NAATs were performed on patients from Nuuk and 9,031 from the rest of Greenland. Of these, 422 (8%) and 820 (9%) were positive, respectively. From January to August, 6 (15%) cultures from Nuuk were ciprofloxacin resistant while in September and October, 26 (59%) were ciprofloxacin resistant (p<0.01). In total, 35 (40%) of 88 culture-positive isolates showed ciprofloxacin resistance. GC incidence in Nuuk was 3,017 per 100,000 inhabitants per year, compared to 2,491 per 100,000 inhabitants per year in the rest of Greenland. Within a short period, a rapid and dramatic change in ciprofloxacin susceptibility among GC strains isolated in Nuuk was documented and recommendation for first line treatments has changed. Continued monitoring and rethinking of primary and secondary preventive initiatives is highly recommended in this high GC incidence setting.

Evaluation of Xpert C. difficile, BD MAX Cdiff, IMDx C. difficile for Abbott m2000, and Illumigene C. difficile Assays for Direct Detection of Toxigenic Clostridium difficile in Stool Specimens.

PubMed

Shin, Bo-Moon; Yoo, Sun Mee; Shin, Won Chang

2016-03-01

We evaluated the performance of four commercial nucleic acid amplification tests (NAATs: Xpert C. difficile, BD MAX Cdiff, IMDx C. difficile for Abbott m2000, and Illumigene C. difficile) for direct and rapid detection of Clostridium difficile toxin genes. We compared four NAATs on the same set of 339 stool specimens (303 prospective and 36 retrospective specimens) with toxigenic culture (TC). Concordance rate among four NAATs was 90.3% (306/339). Based on TC results, the sensitivity and specificity were 90.0% and 92.9% for Xpert; 86.3% and 89.3% for Max; 84.3% and 94.4% for IMDx; and 82.4% and 93.7% for Illumigene, respectively. For 306 concordant cases, there were 11 TC-negative/NAATs co-positive cases and 6 TC-positive/NAATs co-negative cases. Among 33 discordant cases, 18 were only single positive in each NAAT (Xpert, 1; Max, 12; IMDx, 1; Illumigene, 4). Positivity rates of the four NAATs were associated with those of semi-quantitative cultures, which were maximized in grade 3 (>100 colony-forming unit [CFU]) compared with grade 1 (<10 CFU). Commercial NAATs may be rapid and reliable methods for direct detection of tcdA and/or tcdB in stool specimens compared with TC. Some differences in the sensitivity of the NAATs may partly depend on the number of toxigenic C. difficile in stool specimens.

Detection of antibiotic resistance is essential for gonorrhoea point-of-care testing: a mathematical modelling study.

PubMed

Fingerhuth, Stephanie M; Low, Nicola; Bonhoeffer, Sebastian; Althaus, Christian L

2017-07-26

Antibiotic resistance is threatening to make gonorrhoea untreatable. Point-of-care (POC) tests that detect resistance promise individually tailored treatment, but might lead to more treatment and higher levels of resistance. We investigate the impact of POC tests on antibiotic-resistant gonorrhoea. We used data about the prevalence and incidence of gonorrhoea in men who have sex with men (MSM) and heterosexual men and women (HMW) to calibrate a mathematical gonorrhoea transmission model. With this model, we simulated four clinical pathways for the diagnosis and treatment of gonorrhoea: POC test with (POC+R) and without (POC-R) resistance detection, culture and nucleic acid amplification tests (NAATs). We calculated the proportion of resistant infections and cases averted after 5 years, and compared how fast resistant infections spread in the populations. The proportion of resistant infections after 30 years is lowest for POC+R (median MSM: 0.18%, HMW: 0.12%), and increases for culture (MSM: 1.19%, HMW: 0.13%), NAAT (MSM: 100%, HMW: 99.27%), and POC-R (MSM: 100%, HMW: 99.73%). Per 100 000 persons, NAAT leads to 36 366 (median MSM) and 1228 (median HMW) observed cases after 5 years. Compared with NAAT, POC+R averts more cases after 5 years (median MSM: 3353, HMW: 118). POC tests that detect resistance with intermediate sensitivity slow down resistance spread more than NAAT. POC tests with very high sensitivity for the detection of resistance are needed to slow down resistance spread more than by using culture. POC with high sensitivity to detect antibiotic resistance can keep gonorrhoea treatable longer than culture or NAAT. POC tests without reliable resistance detection should not be introduced because they can accelerate the spread of antibiotic-resistant gonorrhoea.

A recommendation for timing of repeat Chlamydia trachomatis test following infection and treatment in pregnant and nonpregnant women

PubMed Central

Lazenby, Gweneth B; Korte, Jeffrey E; Tillman, Sarah; Brown, Florence K; Soper, David E

2018-01-01

The objective of this study was to describe the time required to obtain a negative chlamydia test in pregnant and nonpregnant women following treatment to inform test-of-cure collection and recommend an abstinence period to avoid reinfection. Seventy-two women with Chlamydia trachomatis infection, 36 pregnant and 36 nonpregnant, were enrolled in a prospective cohort study. Women were excluded less than 18 years of age, if they had been treated for chlamydia, reported an allergy to macrolide antibiotics, or if they had Myasthenia Gravis. Women were treated for chlamydia with single-dose therapy and submitted weekly vaginal chlamydia nucleic acid amplification tests (NAATs). Once NAATwere negative, the participants completed the study. Forty-seven women completed the study per protocol. The primary outcome was to determine the time to a negative chlamydia NAAT following treatment, with secondary outcomes of determining the appropriate time to collect a test-of-cure following chlamydia treatment and to recommend an appropriate abstinence period following treatment to avoid reinfection. Results showed that the time to a negative chlamydia NAAT was significantly different between groups (log-rank p = 0.0013). The median number of days to obtain a negative chlamydia NAAT was 8 days (IQR 7–14) in pregnant and 7 days (IQR 6–10) in nonpregnant women (WRST p = 0.04). All participants had a negative chlamydia NAAT by day 29 post-treatment. Following single-dose treatment for chlamydia, both pregnant and nonpregnant women should test negative with NAAT by 30 days post-treatment. Clinicians should collect a test-of-cure in pregnant women no earlier than 1 month. To avoid reinfection, women should avoid condomless intercourse for at least 1 month. PMID:27864473

High Incidence of Chikungunya Virus and Frequency of Viremic Blood Donations during Epidemic, Puerto Rico, USA, 2014

PubMed Central

Brès, Vanessa; Lu, Kai; Liss, Nathan M.; Brambilla, Donald J.; Ryff, Kyle R.; Bruhn, Roberta; Velez, Edwin; Ocampo, Derrek; Linnen, Jeffrey M.; Latoni, Gerardo; Petersen, Lyle R.; Williamson, Phillip C.; Busch, Michael P.

2016-01-01

Chikungunya virus (CHIKV) caused large epidemics throughout the Caribbean in 2014. We conducted nucleic acid amplification testing (NAAT) for CHIKV RNA (n = 29,695) and serologic testing for IgG against CHIKV (n = 1,232) in archived blood donor samples collected during and after an epidemic in Puerto Rico in 2014. NAAT yields peaked in October with 2.1% of donations positive for CHIKV RNA. A total of 14% of NAAT-reactive donations posed a high risk for virus transmission by transfusion because of high virus RNA copy numbers (104–109 RNA copies/mL) and a lack of specific IgM and IgG responses. Testing of minipools of 16 donations would not have detected 62.5% of RNA-positive donations detectable by individual donor testing, including individual donations without IgM and IgG. Serosurveys before and after the epidemic demonstrated that nearly 25% of blood donors in Puerto Rico acquired CHIKV infections and seroconverted during the epidemic. PMID:27070192

High Incidence of Chikungunya Virus and Frequency of Viremic Blood Donations during Epidemic, Puerto Rico, USA, 2014.

PubMed

Simmons, Graham; Brès, Vanessa; Lu, Kai; Liss, Nathan M; Brambilla, Donald J; Ryff, Kyle R; Bruhn, Roberta; Velez, Edwin; Ocampo, Derrek; Linnen, Jeffrey M; Latoni, Gerardo; Petersen, Lyle R; Williamson, Phillip C; Busch, Michael P

2016-07-01

Chikungunya virus (CHIKV) caused large epidemics throughout the Caribbean in 2014. We conducted nucleic acid amplification testing (NAAT) for CHIKV RNA (n = 29,695) and serologic testing for IgG against CHIKV (n = 1,232) in archived blood donor samples collected during and after an epidemic in Puerto Rico in 2014. NAAT yields peaked in October with 2.1% of donations positive for CHIKV RNA. A total of 14% of NAAT-reactive donations posed a high risk for virus transmission by transfusion because of high virus RNA copy numbers (10 (4) -10 (9) RNA copies/mL) and a lack of specific IgM and IgG responses. Testing of minipools of 16 donations would not have detected 62.5% of RNA-positive donations detectable by individual donor testing, including individual donations without IgM and IgG. Serosurveys before and after the epidemic demonstrated that nearly 25% of blood donors in Puerto Rico acquired CHIKV infections and seroconverted during the epidemic.

Point-Counterpoint: What Is the Optimal Approach for Detection of Clostridium difficile Infection?

PubMed Central

Wilcox, Mark H.

2017-01-01

INTRODUCTION In 2010, we published an initial Point-Counterpoint on the laboratory diagnosis of Clostridium difficile infection (CDI). At that time, nucleic acid amplification tests (NAATs) were just becoming commercially available, and the idea of algorithmic approaches to CDI was being explored. Now, there are numerous NAATs in the marketplace, and based on recent proficiency test surveys, they have become the predominant method used for CDI diagnosis in the United States. At the same time, there is a body of literature that suggests that NAATs lack clinical specificity and thus inflate CDI rates. Hospital administrators are taking note of institutional CDI rates because they are publicly reported. They have become an important metric impacting hospital safety ratings and value-based purchasing; hospitals may have millions of dollars of reimbursement at risk. In this Point-Counterpoint using a frequently asked question approach, Ferric Fang of the University of Washington, who has been a consistent advocate for a NAAT-only approach for CDI diagnosis, will discuss the value of a NAAT-only approach, while Christopher Polage of the University of California Davis and Mark Wilcox of Leeds University, Leeds, United Kingdom, each of whom has recently written important articles on the value of toxin detection in the diagnosis, will discuss the impact of toxin detection in CDI diagnosis. PMID:28077697

The performance of non-NAAT point-of-care (POC) tests and rapid NAAT tests for chlamydia and gonorrhoea infections. An assessment of currently available assays.

PubMed

Brook, Gary

2015-12-01

To identify point-of-care (POC) and rapid nucleic acid amplification techniques (NAATs) for the diagnosis of chlamydia and gonorrhoea and assess their utility. Literature search for available POC and rapid NAATs. The performance from the best-performing assays were applied hypothetically to patients in the clinic in which 100 consecutive patients with chlamydia and 100 with gonorrhoea were diagnosed in 1737 and 4575 patients respectively, with 44/100 and 54/100 treated at first attendance, respectively. 11 POC and 1 rapid NAAT were identified. Published performances for the best POC for chlamydia (CRT) were: sensitivity 41%-87%, specificity 89%-99.6%. Our data suggest that if this assay was used instead of our current NAAT, for every 100 patients diagnosed currently, 23-46 extra patients would be treated at first attendance; 10-35 would go undiagnosed with 7-191 false-positives. Best chlamydia rapid NAAT (GeneXpert): sensitivity 97.5%-98.7%, specificity 99.4%-99.9%. Anticipated performance for every 100 patients diagnosed currently: 0 extra patients treated at first attendance, 1-3 undiagnosed, 0-2 false-positives. Best POC for gonorrhoea (GC Check): sensitivity 54%-70%, specificity 97%-98%. Anticipated performance for every 100 patients diagnosed currently: 14-18 extra patients treated at first attendance, 28-32 undiagnosed, 92-137 false-positives. Best rapid NAAT for gonorrhoea (GeneXpert): sensitivity 96%-100%, specificity 99.9%-100%. Anticipated performance for every 100 patients diagnosed currently: 0 extra patients treated at first attendance, 0-4 undiagnosed, 0-5 false-positives. Rapid NAAT would reduce time to treatment by 4 days for initially untreated patients. POC assays would need to be used in conjunction with a NAAT, increasing early treatment rates expense and false-positive results. The rapid NAAT could be used alone, with a reduction in average time-to-treat and a small reduction in sensitivity and specificity. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Improved sexual history taking in the 2012 BASHH asymptomatic screening re-audit.

PubMed

Menon-Johansson, A S; McClean, H; Carne, C A; Estreich, S; Knapper, C; Sethi, G; Smith, A; Sullivan, A K

2014-04-01

Effective asymptomatic screening for sexually transmitted infections is an important public health service because a significant proportion of sexually transmitted infections do not present with symptoms. In 2009, the National Audit Group of the British Association of Sexual Health and HIV (BASHH) audited the management of asymptomatic patients and recommended increased documentation about oral and anal sex, regional strategies for nucleic acid amplification test (NAAT) use for gonorrhoea, improved screening for hepatitis B in men who have sex with men and an increase in screening for HIV. The 2012 audit used web-based forms to collect submissions from 180 consultant-led centres (65% response rate) that included episodes of care from 6669 asymptomatic patients. An improvement was demonstrated for all the areas measured during the 2009 audit. A doubling of gonorrhoea testing using NAATs was seen and yet 10% of asymptomatic patients continued to have microscopy despite these tests not being recommended by BASHH guidelines. This audit recommends universal adoption of gonorrhoea NAATs across the United Kingdom.

Performance of different mono- and multiplex nucleic acid amplification tests on a multipathogen external quality assessment panel.

PubMed

Loens, K; van Loon, A M; Coenjaerts, F; van Aarle, Y; Goossens, H; Wallace, P; Claas, E J C; Ieven, M

2012-03-01

An external quality assessment (EQA) panel consisting of a total of 48 samples in bronchoalveolar lavage (BAL) fluid or transport medium was prepared in collaboration with Quality Control for Molecular Diagnostics (QCMD) (www.qcmd.org). The panel was used to assess the proficiency of the three laboratories that would be responsible for examining the 6,000 samples to be collected in the GRACE Network of Excellence (www.grace-lrti.org). The main objective was to decide on the best-performing testing approach for the detection of influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus (RSV), human metapneumovirus, coronavirus, rhinovirus, adenovirus, Chlamydophila pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila by nucleic acid amplification techniques (NAATs). Two approaches were chosen: (i) laboratories testing samples using their in-house procedures for extraction and amplification and (ii) laboratories using their in-house amplification procedures on centrally extracted samples. Furthermore, three commercially available multiplex NAAT tests-the ResPlex (Qiagen GmbH, Hilden, Germany), RespiFinder plus (PathoFinder, Maastricht, The Netherlands), and RespiFinder Smart 21 (PathoFinder) tests-were evaluated by examination of the same EQA panel by the manufacturer. No large differences among the 3 laboratories were noticed when the performances of the assays developed in-house in combination with the in-house extraction procedures were compared. Also, the extraction procedure (central versus local) had little effect on performance. However, large differences in amplification efficacy were found between the commercially available tests; acceptable results were obtained by using the PathoFinder assays.

Mapping patient pathways and estimating resource use for point of care versus standard testing and treatment of chlamydia and gonorrhoea in genitourinary medicine clinics in the UK.

PubMed

Adams, Elisabeth J; Ehrlich, Alice; Turner, Katherine M E; Shah, Kunj; Macleod, John; Goldenberg, Simon; Meray, Robin K; Pearce, Vikki; Horner, Patrick

2014-07-23

We aimed to explore patient pathways using a chlamydia/gonorrhoea point-of-care (POC) nucleic acid amplification test (NAAT), and estimate and compare the costs of the proposed POC pathways with the current pathways using standard laboratory-based NAAT testing. Workshops were conducted with healthcare professionals at four sexual health clinics representing diverse models of care in the UK. They mapped out current pathways that used chlamydia/gonorrhoea tests, and constructed new pathways using a POC NAAT. Healthcare professionals' time was assessed in each pathway. The proposed POC pathways were then priced using a model built in Microsoft Excel, and compared to previously published costs for pathways using standard NAAT-based testing in an off-site laboratory. Pathways using a POC NAAT for asymptomatic and symptomatic patients and chlamydia/gonorrhoea-only tests were shorter and less expensive than most of the current pathways. Notably, we estimate that POC testing as part of a sexual health screen for symptomatic patients, or as stand-alone chlamydia/gonorrhoea testing, could reduce costs per patient by as much as £16 or £6, respectively. In both cases, healthcare professionals' time would be reduced by approximately 10 min per patient. POC testing for chlamydia/gonorrhoea in a clinical setting may reduce costs and clinician time, and may lead to more appropriate and quicker care for patients. Further study is warranted on how to best implement POC testing in clinics, and on the broader clinical and cost implications of this technology. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Targeting screening and social marketing to increase detection of acute HIV infection in men who have sex with men in Vancouver, British Columbia.

PubMed

Gilbert, Mark; Cook, Darrel; Steinberg, Malcolm; Kwag, Michael; Robert, Wayne; Doupe, Glenn; Krajden, Mel; Rekart, Michael

2013-10-23

The contribution of acute HIV infection (AHI) to transmission is widely recognized, and increasing AHI diagnosis capacity can enhance HIV prevention through subsequent behavior change or intervention. We examined the impact of targeted pooled nucleic acid amplification testing (NAAT) and social marketing to increase AHI diagnosis among men who have sex with men (MSM) in Vancouver. Observational study. We implemented pooled NAAT following negative third-generation enzyme immunoassay (EIA) testing for males above 18 years in six clinics accessed by MSM, accompanied by two social marketing campaigns developed by a community gay men's health organization. We compared test volume and diagnosis rates for pre-implementation (April 2006-March 2009) and post-implementation (April 2009-March 2012) periods. After implementation, we used linear regression to examine quarterly trends and calculated diagnostic yield. After implementation, the AHI diagnosis rate significantly increased from 1.03 to 1.84 per 1000 tests, as did quarterly HIV test volumes and acute to non-acute diagnosis ratio. Of the 217 new HIV diagnoses after implementation, 54 (24.9%) were AHIs (25 detected by pooled NAAT only) for an increased diagnostic yield of 11.5%. The average number of prior negative HIV tests (past 2 years) increased significantly for newly diagnosed MSM at the six study clinics compared to other newly diagnosed MSM in British Columbia, per quarter. Targeted implementation of pooled NAAT at clinics accessed by MSM is effective in increasing AHI diagnoses compared to third-generation EIA testing. Social marketing campaigns accompanying pooled NAAT implementation may contribute to increasing AHI diagnoses and frequency of HIV testing.

Impact of deploying multiple point-of-care tests with a 'sample first' approach on a sexual health clinical care pathway. A service evaluation.

PubMed

Harding-Esch, Emma M; Nori, Achyuta V; Hegazi, Aseel; Pond, Marcus J; Okolo, Olanike; Nardone, Anthony; Lowndes, Catherine M; Hay, Phillip; Sadiq, S Tariq

2017-09-01

To assess clinical service value of STI point-of-care test (POCT) use in a 'sample first' clinical pathway (patients providing samples on arrival at clinic, before clinician consultation). Specific outcomes were: patient acceptability; whether a rapid nucleic acid amplification test (NAAT) for Chlamydia trachomatis/Neisseria gonorrhoeae (CT/NG) could be used as a POCT in practice; feasibility of non-NAAT POCT implementation for Trichomonas vaginalis (TV) and bacterial vaginosis (BV); impact on patient diagnosis and treatment. Service evaluation in a south London sexual health clinic. Symptomatic female and male patients and sexual contacts of CT/NG-positive individuals provided samples for diagnostic testing on clinic arrival, prior to clinical consultation. Tests included routine culture and microscopy; CT/NG (GeneXpert) NAAT; non-NAAT POCTs for TV and BV. All 70 (35 males, 35 females) patients approached participated. The 'sample first' pathway was acceptable, with >90% reporting they were happy to give samples on arrival and receive results in the same visit. Non-NAAT POCT results were available for all patients prior to leaving clinic; rapid CT/NG results were available for only 21.4% (15/70; 5 males, 10 females) of patients prior to leaving clinic. Known negative CT/NG results led to two females avoiding presumptive treatment, and one male receiving treatment directed at possible Mycoplasma genitalium infection causing non-gonococcal urethritis. Non-NAAT POCTs detected more positives than routine microscopy (TV 3 vs 2; BV 24 vs 7), resulting in more patients receiving treatment. A 'sample first' clinical pathway to enable multiple POCT use was acceptable to patients and feasible in a busy sexual health clinic, but rapid CT/NG processing time was too long to enable POCT use. There is need for further development to improve test processing times to enable POC use of rapid NAATs. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Can a toxin gene NAAT be used to predict toxin EIA and the severity of Clostridium difficile infection?

PubMed

Garvey, Mark I; Bradley, Craig W; Wilkinson, Martyn A C; Holden, Elisabeth

2017-01-01

Diagnosis of C. difficile infection (CDI) is controversial because of the many laboratory methods available and their lack of ability to distinguish between carriage, mild or severe disease. Here we describe whether a low C. difficile toxin B nucleic acid amplification test (NAAT) cycle threshold (CT) can predict toxin EIA, CDI severity and mortality. A three-stage algorithm was employed for CDI testing, comprising a screening test for glutamate dehydrogenase (GDH), followed by a NAAT, then a toxin enzyme immunoassay (EIA). All diarrhoeal samples positive for GDH and NAAT between 2012 and 2016 were analysed. The performance of the NAAT CT value as a classifier of toxin EIA outcome was analysed using a ROC curve; patient mortality was compared to CTs and toxin EIA via linear regression models. A CT value ≤26 was associated with ≥72% toxin EIA positivity; applying a logistic regression model we demonstrated an association between low CT values and toxin EIA positivity. A CT value of ≤26 was significantly associated ( p  = 0.0262) with increased one month mortality, severe cases of CDI or failure of first line treatment. The ROC curve probabilities demonstrated a CT cut off value of 26.6. Here we demonstrate that a CT ≤26 indicates more severe CDI and is associated with higher mortality. Samples with a low CT value are often toxin EIA positive, questioning the need for this additional EIA test. A CT ≤26 could be used to assess the potential for severity of CDI and guide patient treatment.

Molecular testing for viral and bacterial enteric pathogens: gold standard for viruses, but don't let culture go just yet?

PubMed

Bloomfield, Maxim G; Balm, Michelle N D; Blackmore, Timothy K

2015-04-01

Contemporary diagnostic microbiology is increasingly adopting molecular methods as front line tests for a variety of samples. This trend holds true for detection of enteric pathogens (EP), where nucleic acid amplification tests (NAAT) for viruses are well established as the gold standard, and an increasing number of commercial multi-target assays are now available for bacteria and parasites. NAAT have significant sensitivity and turnaround time advantages over traditional methods, potentially returning same-day results. Multiplex panels offer an attractive 'one-stop shop' that may provide workflow and cost advantages to laboratories processing large sample volumes. However, there are a number of issues which need consideration. Reflex culture is required for antibiotic susceptibility testing and strain typing when needed for food safety and other epidemiological investigations. Surveillance systems will need to allow for differences in disease incidence due to the enhanced sensitivity of NAAT. Laboratories should be mindful of local epidemiology when selecting which pathogens to include in multiplex panels, and be thoughtful regarding which pathogens will not be detected. Multiplex panels may not be appropriate in certain situations, such as hospital-onset diarrhoea, where Clostridium difficile testing might be all that is required, and laboratories may wish to retain the flexibility to run single tests in such situations. The clinical impact of rapid results is also likely to be relatively minor, as infective diarrhoea is a self-limiting illness in the majority of cases. Laboratories will require strategies to assist users in the interpretation of the results produced by NAAT, particularly where pathogens are detected at low levels with uncertain clinical significance. These caveats aside, faecal NAAT are increasingly being used and introduce a new era of diagnosis of gastrointestinal infection.

Evaluation of a rapid, real-time intrapartum group B Streptococcus assay

PubMed Central

YOUNG, Brett C.; DODGE, Laura E.; GUPTA, Munish; RHEE, Julie S.; HACKER, Michele R.

2011-01-01

OBJECTIVE To evaluate an intrapartum nucleic acid amplification test (NAAT) for Group B streptococcus (GBS). STUDY DESIGN Prospective cohort study of 559 women comparing intrapartum GBS culture with antepartum culture and intrapartum NAAT. RESULTS GBS prevalence was 19.5% by antepartum culture and 23.8% by intrapartum culture. Compared with intrapartum culture, antepartum culture had 69.2% sensitivity (60.6–76.9) and 96.0% specificity (93.7–97.7). The NAAT demonstrated sensitivity of 90.8% (84.6–95.2), specificity of 97.6% (95.6–98.8), and predictive values exceeding 92%. The incidence of discordant cultures was 10.4%. Of the women with negative antepartum and positive intrapartum cultures, only 1 (2.4%) received intrapartum antibiotics. Compared with white women, black (P=0.02) and Hispanic (P=0.02) women were more likely to have discordant cultures. CONCLUSION This intrapartum NAAT has excellent characteristics. It may be superior to antepartum culture for detecting intrapartum GBS—allowing more accurate management of laboring mothers and reducing neonatal GBS sepsis. PMID:21864820

Strategies for Optimizing the Diagnostic Predictive Value of Clostridium difficile Molecular Diagnostics.

PubMed

Kociolek, Larry K

2017-05-01

Because nucleic acid amplification tests (NAATs) do not distinguish Clostridium difficile infection (CDI) and asymptomatic C. difficile carriage, the diagnostic predictive value of NAATs is limited when used in patients with a low probability of CDI. In this issue of the Journal of Clinical Microbiology , Truong et al. (J. Clin. Microbiol., 55:1276-1284, 2017, https://doi.org/10.1128/JCM.02319-16) report significant reductions in hospital-onset CDI and oral vancomycin utilization at their institution following implementation of a novel intervention that leveraged their clinical bioinformatics resources to prevent C. difficile testing of stools from patients without clinically significant diarrhea and in patients with recent laxative use. Copyright © 2017 American Society for Microbiology.

Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time

PubMed Central

Gandelman, Olga A.; Church, Vicki L.; Moore, Cathy A.; Kiddle, Guy; Carne, Christopher A.; Parmar, Surendra; Jalal, Hamid; Tisi, Laurence C.; Murray, James A. H.

2010-01-01

Background The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. Principal Findings Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. Conclusions The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings. PMID:21152399

Staff, associate specialist and specialty doctors' national audit on the management of gonorrhoea in the United Kingdom, 2015.

PubMed

Mullan, Helen; Richards, Jane; Lee, John

2017-12-01

The British Association for Sexual Health and HIV (BASHH) revised United Kingdom national guideline for the management of gonorrhoea in adults, 2011, identified five auditable outcome measures, namely, that all patients should receive first-line treatment, be screened or treated for chlamydial infection, have a test of cure (TOC), be offered written information and have partner notification carried out. The UK National Guideline for Gonorrhoea Testing, Clinical Effectiveness Group, BASHH, 2012, recommended in addition that all reactive nucleic acid amplification tests (NAATs) from pharynx and rectum should be confirmed by supplementary testing, using a second NAAT which detects a different nucleic acid target, all those with a positive NAAT for gonorrhoea should have culture and antimicrobial susceptibility testing and that TOC should be done by two weeks. Staff, associate specialist and specialty doctors performed a national audit against these standards. Data from 3233 cases were submitted; 8% of cases of gonorrhoea diagnosed in England, Scotland and Wales over this period. We found that 83% patients received first-line treatment with a reason for not doing so provided for 11%. TOC was documented for 62% and written information was offered to 41%. Results about supplementary testing were inconsistent. The results for the other outcomes were satisfactory.

Detecting asymptomatic Trichomonas vaginalis in females using the BD ProbeTec™ Trichomonas vaginalis Qx nucleic acid amplification test.

PubMed

Lord, Emily; Newnham, Tana; Dorrell, Lucy; Jesuthasan, Gerald; Clarke, Lorraine; Jeffery, Katie; Sherrard, Jackie

2017-03-01

Trichomonas vaginalis (TV) rates in women are increasing and many are asymptomatic. Nucleic acid amplification tests (NAATs) are becoming the 'gold standard' for diagnosis. We aimed to establish our asymptomatic TV rates by testing all women attending Oxfordshire's Sexual Health service, regardless of symptoms, using the BD ProbeTec™ TV Q x NAATs (BDQ x ). During BDQ x 's verification process, the sensitivity and specificity were calculated using results of 220 endocervical samples from symptomatic women, compared with culture. BDQ x was subsequently implemented and prospectively evaluated over 6 months in female attendees. Wet mount microscopy was also performed in symptomatics. Demographic and clinical characteristics of those diagnosed were analysed. From 220 samples tested by BDQ x and culture: 5 were positive on both and one solely using BDQ x , giving a sensitivity and specificity of 100% and 99.53%, respectively. In the prospective cohort, of 5775 BDQ x tests, 33 (0.57%) were positive. 11/33 (33%) patients were asymptomatic. All patients diagnosed had risk factors: age >25 years (85%), residence in a deprived area (79%) and black ethnicity (21%). Despite BDQ x being highly sensitive and specific, with our low TV prevalence universal screening may not be justified. Targeted screening using local demographic data merits further investigation.

Simple Approaches to Minimally-Instrumented, Microfluidic-Based Point-of-Care Nucleic Acid Amplification Tests

PubMed Central

Mauk, Michael G.; Song, Jinzhao; Liu, Changchun; Bau, Haim H.

2018-01-01

Designs and applications of microfluidics-based devices for molecular diagnostics (Nucleic Acid Amplification Tests, NAATs) in infectious disease testing are reviewed, with emphasis on minimally instrumented, point-of-care (POC) tests for resource-limited settings. Microfluidic cartridges (‘chips’) that combine solid-phase nucleic acid extraction; isothermal enzymatic nucleic acid amplification; pre-stored, paraffin-encapsulated lyophilized reagents; and real-time or endpoint optical detection are described. These chips can be used with a companion module for separating plasma from blood through a combined sedimentation-filtration effect. Three reporter types: Fluorescence, colorimetric dyes, and bioluminescence; and a new paradigm for end-point detection based on a diffusion-reaction column are compared. Multiplexing (parallel amplification and detection of multiple targets) is demonstrated. Low-cost detection and added functionality (data analysis, control, communication) can be realized using a cellphone platform with the chip. Some related and similar-purposed approaches by others are surveyed. PMID:29495424

Diagnostic testing for Giardia infections.

PubMed

Heyworth, Martin F

2014-03-01

The traditional method for diagnosing Giardia infections involves microscopic examination of faecal specimens for Giardia cysts. This method is subjective and relies on observer experience. From the 1980s onwards, objective techniques have been developed for diagnosing Giardia infections, and are superseding diagnostic techniques reliant on microscopy. Detection of Giardia antigen(s) by immunoassay is the basis of commercially available diagnostic kits. Various nucleic acid amplification techniques (NAATs) can demonstrate DNA of Giardia intestinalis, and have the potential to become standard approaches for diagnosing Giardia infections. Of such techniques, methods involving either fluorescent microspheres (Luminex) or isothermal amplification of DNA (loop-mediated isothermal amplification; LAMP) are especially promising.

A Review of Methods for Detection of Hepatozoon Infection in Carnivores and Arthropod Vectors.

PubMed

Modrý, David; Beck, Relja; Hrazdilová, Kristýna; Baneth, Gad

2017-01-01

Vector-borne protists of the genus Hepatozoon belong to the apicomplexan suborder Adeleorina. The taxonomy of Hepatozoon is unsettled and different phylogenetic clades probably represent evolutionary units deserving the status of separate genera. Throughout our review, we focus on the monophyletic assemblage of Hepatozoon spp. from carnivores, classified as Hepatozoon sensu stricto that includes important pathogens of domestic and free-ranging canine and feline hosts. We provide an overview of diagnostic methods and approaches from classical detection in biological materials, through serological tests to nucleic acid amplification tests (NAATs). Critical review of used primers for the 18S rDNA is provided, together with information on individual primer pairs. Extension of used NAATs target to cover also mitochondrial genes is suggested as a key step in understanding the diversity and molecular epidemiology of Hepatozoon infections in mammals.

Comparison of Abbott RealTime genotype II, GeneMatrix restriction fragment mass polymorphism and Sysmex HISCL HCV Gr assays for hepatitis C virus genotyping.

PubMed

Han, Mi-Soon; Park, Yongjung; Kim, Hyon-Suk

2017-07-26

Hepatitis C virus (HCV) genotype is a predictive marker for treatment response. We sequentially evaluated the performances of two nucleic acid amplification tests (NAATs) and one serology assay for HCV genotype: Abbott RealTime genotype II (RealTime II), GeneMatrix restriction fragment mass polymorphism (RFMP), and Sysmex HISCL HCV Gr (HISCL Gr). We examined 281 clinical samples with three assays. The accuracy was assessed using the HCV Genotype Performance Panel PHW204 (SeraCare Life Sciences) for two NAATs. Discrepant cases were re-genotyped by the Versant HCV v.2.0 (line probe 2.0) assay. With the RealTime II assay, clinic samples were analyzed as follows: genotypes 1b (43.1%), 2 (40.2%), 1 subtypes other than 1a and 1b (12.5%), 3 (1.8%), 4 (1.4%), 1a (0.7%), 6 (0.4%), and mixed (1.1%). The RealTime II and RFMP assays showed a type concordance rate of 97.5% (274/281) (κ=0.80) and no significant discordance (p=0.25). Both assays accurately genotyped all samples in the Performance Panel by the subtype level. The HISCL Gr assay showed concordance rates of about 91% (κ<0.40) and statistically significant discordances with two NAATs (p<0.05). In confirmation tests, the results of RFMP assay were the most consistent with those of Versant 2.0 assay. The three HCV assays provided genotyping and serotyping results with good concordance rates. The two NAATs (RealTime II and RFMP) showed comparable performance and good agreement. However, the results of the HISCL Gr assay showed statistically significant differences with those of the NAATs.

Chlamydial and gonococcal infection in men without polymorphonuclear leukocytes on gram stain: implications for diagnostic approach and management.

PubMed

Geisler, William M; Yu, Shuying; Hook, Edward W

2005-10-01

Gram stain is used to detect urethral inflammation, suggestive of infection, in men and guide therapeutic decisions. In the absence of signs, symptoms, or polymorphonuclear leukocytes (PMNs) on urethral Gram stain, treatment and sometimes testing is deferred. Determine the proportion of men with chlamydia or gonorrhea diagnosed by nucleic acid amplification testing (NAAT) or culture who lack Gram stain evidence of inflammation and compare their clinical characteristics to men with inflammation. Records from 2629 men presenting for routine sexually transmitted disease care with urethral PMN count and NAAT data were retrospectively analyzed. A subpopulation tested by NAAT and culture was analyzed. Men receiving antibiotics within the prior month or those reporting a sexual partner with trichomoniasis were excluded. Among 2266 eligible men, 353 (16%) had chlamydia and 462 (20%) had gonorrhea. Among chlamydia-infected men, PMNs per oil-immersion field (oif) on Gram stain were > or =5 in 291 (82%), 1 to 4 in 20 (6%), and none in 42 (12%). In men with gonorrhea, PMNs/oif were > or =5 in 433 (94%), 1 to 4 in 6 (1%), and none in 23 (5%). Urethral symptoms, discharge, and/or > or =5 PMNs/oif were absent in 47 (13%) and 22 (5%) of chlamydial and gonococcal infections, respectively (including no PMNs/oif and 1-4 PMNs/oif). None of these 47 chlamydial-infected men and only 4 of 22 men with gonorrhea received therapy at the time of initial examination. Twelve percent of chlamydial and 5% of gonococcal infections had no Gram stain evidence of urethral inflammation. Absence of symptoms and discharge is not uncommon in chlamydial infection detected by NAAT, and without testing, many infections will go untreated, furthering the possibility of complications or partner transmission.

Calibration of BK Virus Nucleic Acid Amplification Testing to the 1st WHO International Standard for BK Virus

PubMed Central

Tan, Susanna K.; Milligan, Stephen; Sahoo, Malaya K.; Taylor, Nathaniel

2017-01-01

ABSTRACT Significant interassay variability in the quantification of BK virus (BKV) DNA precludes establishing broadly applicable thresholds for the management of BKV infection in transplantation. The 1st WHO International Standard for BKV (primary standard) was introduced in 2016 as a common calibrator for improving the harmonization of BKV nucleic acid amplification testing (NAAT) and enabling comparisons of biological measurements worldwide. Here, we evaluated the Altona RealStar BKV assay (Altona) and calibrated the results to the international unit (IU) using the Exact Diagnostics BKV verification panel, a secondary standard traceable to the primary standard. The primary and secondary standards on Altona had nearly identical linear regression equations (primary standard, Y = 1.05X − 0.28, R2 = 0.99; secondary standard, Y = 1.04X − 0.26, R2 = 0.99) and conversion factors (primary standard, 1.11 IU/copy; secondary standard, 1.09 IU/copy). A comparison of Altona with a laboratory-developed BKV NAAT assay in IU/ml versus copies/ml using Passing-Bablok regression revealed similar regression lines, no proportional bias, and improvement in the systematic bias (95% confidence interval of intercepts: copies/ml, −0.52 to −1.01; IU/ml, 0.07 to −0.36). Additionally, Bland-Altman analyses revealed a clinically significant reduction of bias when results were reported in IU/ml (IU/ml, −0.10 log10; copies/ml, −0.70 log10). These results indicate that the use of a common calibrator improved the agreement between the two assays. As clinical laboratories worldwide use calibrators traceable to the primary standard to harmonize BKV NAAT results, we anticipate improved interassay comparisons with a potential for establishing broadly applicable quantitative BKV DNA load cutoffs for clinical practice. PMID:28053213

Nucleic acid amplification tests (NAATs) for gonorrhoea diagnosis in women: experience of a tertiary care hospital in north India.

PubMed

Sood, Seema; Verma, Rachna; Mir, Shazia Shaheen; Agarwal, Madhav; Singh, Neeta; Kar, Hemanta Kumar; Sharma, Vinod Kumar

2014-11-01

Gonorrhoea is among the most frequent of the estimated bacterial sexually transmitted infections (STIs) and has significant health implications in women. The use of nucleic acid amplification tests (NAATs) has been shown to provide enhanced diagnosis of gonorrhoea in female patients. However, it is recommended that an on-going assessment of the test assays should be performed to check for any probable sequence variation occurring in the targeted region. In this study, an in-house PCR targeting opa-gene of Neisseria gonorrhoeae was used in conjunction with 16S ribosomal PCR to determine the presence of gonorrhoea in female patients attending the tertiary care hospitals. Endocervical samples collected from 250 female patients with complaints of vaginal or cervical discharge or pain in lower abdomen were tested using opa and 16S ribosomal assay. The samples were also processed by conventional methods. Of the 250 female patients included in the study, only one was positive by conventional methods (microscopy and culture) whereas 17 patients were found to be positive based on PCR results. The clinical sensitivity of conventional methods for the detection of N. gonorrhoeae in female patients was low. The gonococcal detection rates increased when molecular method was used giving 16 additional positives. Studies should be done to find out other gene targets that may be used in the screening assays to detect the presence of gonorrhoea.

The value of confirmatory testing in early infant HIV diagnosis programmes in South Africa: A cost-effectiveness analysis.

PubMed

Dunning, Lorna; Francke, Jordan A; Mallampati, Divya; MacLean, Rachel L; Penazzato, Martina; Hou, Taige; Myer, Landon; Abrams, Elaine J; Walensky, Rochelle P; Leroy, Valériane; Freedberg, Kenneth A; Ciaranello, Andrea

2017-11-01

The specificity of nucleic acid amplification tests (NAATs) used for early infant diagnosis (EID) of HIV infection is <100%, leading some HIV-uninfected infants to be incorrectly identified as HIV-infected. The World Health Organization recommends that infants undergo a second NAAT to confirm any positive test result, but implementation is limited. Our objective was to determine the impact and cost-effectiveness of confirmatory HIV testing for EID programmes in South Africa. Using the Cost-effectiveness of Preventing AIDS Complications (CEPAC)-Pediatric model, we simulated EID testing at age 6 weeks for HIV-exposed infants without and with confirmatory testing. We assumed a NAAT cost of US$25, NAAT specificity of 99.6%, NAAT sensitivity of 100% for infants infected in pregnancy or at least 4 weeks prior to testing, and a mother-to-child transmission (MTCT) rate at 12 months of 4.9%; we simulated guideline-concordant rates of testing uptake, result return, and antiretroviral therapy (ART) initiation (100%). After diagnosis, infants were linked to and retained in care for 10 years (false-positive) or lifelong (true-positive). All parameters were varied widely in sensitivity analyses. Outcomes included number of infants with false-positive diagnoses linked to ART per 1,000 ART initiations, life expectancy (LE, in years) and per-person lifetime HIV-related healthcare costs. Both without and with confirmatory testing, LE was 26.2 years for HIV-infected infants and 61.4 years for all HIV-exposed infants; clinical outcomes for truly infected infants did not differ by strategy. Without confirmatory testing, 128/1,000 ART initiations were false-positive diagnoses; with confirmatory testing, 1/1,000 ART initiations were false-positive diagnoses. Because confirmatory testing averted costly HIV care and ART in truly HIV-uninfected infants, it was cost-saving: total cost US$1,790/infant tested, compared to US$1,830/infant tested without confirmatory testing. Confirmatory testing remained cost-saving unless NAAT cost exceeded US$400 or the HIV-uninfected status of infants incorrectly identified as infected was ascertained and ART stopped within 3 months of starting. Limitations include uncertainty in the data used in the model, which we examined with sensitivity and uncertainty analyses. We also excluded clinical harms to HIV-uninfected infants incorrectly treated with ART after false-positive diagnosis (e.g., medication toxicities); including these outcomes would further increase the value of confirmatory testing. Without confirmatory testing, in settings with MTCT rates similar to that of South Africa, more than 10% of infants who initiate ART may reflect false-positive diagnoses. Confirmatory testing prevents inappropriate HIV diagnosis, is cost-saving, and should be adopted in all EID programmes.

Evaluation of the Efficiency of the Sample Inactivation Reagent in the Abbott RealTime MTB Assay for Inactivation of Mycobacterium tuberculosis

PubMed Central

Wallis, Carole; Pahalawatta, Vihanga; Frank, Andrea; Ramdin, Neeshan; Viana, Raquel; Abravaya, Klara; Leckie, Gregor; Tang, Ning

2015-01-01

The Abbott RealTime MTB assay is a nucleic acid amplification test (NAAT) for the detection of Mycobacterium tuberculosis complex DNA. The sample inactivation procedure used in the assay, consisting of one part sample treated with 3 parts inactivation reagent for 60 min, effectively reduced viscosity and inactivated M. tuberculosis in clinical specimens. PMID:26085611

Detection of Neisseria gonorrhoeae in the pharynx and saliva: implications for gonorrhoea transmission.

PubMed

Chow, Eric P F; Lee, David; Tabrizi, Sepehr N; Phillips, Samuel; Snow, Anthony; Cook, Stuart; Howden, Benjamin P; Petalotis, Irene; Bradshaw, Catriona S; Chen, Marcus Y; Fairley, Christopher K

2016-08-01

This study aimed to determine the proportion of untreated pharyngeal swabs or saliva samples positive by culture or nucleic acid amplification tests (NAATs) for Neisseria gonorrhoeae up to 14 days after an initial culture-positive pharyngeal swab. Men who have sex with men who tested positive for pharyngeal gonorrhoea at Melbourne Sexual Health Centre (MSHC) and returned to MSHC for treatment within 14 days between 13 October 2014 and 25 March 2015 were included in this study. Pharyngeal swabs and saliva samples were collected for culture and NAAT. Of 33 initially culture-positive pharyngeal swabs, 32 saliva samples and 31 pharyngeal swabs were positive by NAAT and 14 pharyngeal and 6 saliva samples were positive by culture within 14 days. There was a significant decline in the proportion of repeated pharyngeal culture samples positive by culture over time (p<0.001). The rapid decline suggests pharyngeal gonorrhoea is short-lived, and the finding of gonorrhoea commonly in the saliva implicates this body fluid in its transmission without direct throat inoculation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Evaluation of gonorrhea test of cure at 1 week in a Los Angeles community-based clinic serving men who have sex with men.

PubMed

Beymer, Matthew R; Llata, Eloisa; Stirland, Ali M; Weinstock, Hillard S; Wigen, Christine L; Guerry, Sarah L; Mejia, Everardo; Bolan, Robert K

2014-10-01

Because of the decreasing susceptibility of Neisseria gonorrhoeae to cephalosporin therapy, the Centers for Disease Control and Prevention recommends test of cure (TOC) 1 week after gonorrhea (GC) treatment if therapies other than ceftriaxone are used. In addition, the Centers for Disease Control and Prevention asks clinicians, particularly those caring for men who have sex with men (MSM) on the west coast, to consider retesting all MSM at 1 week. However, it is unclear if this is acceptable to providers and patients or if nucleic acid amplification tests (NAATs) are useful for TOC at 7 days. Between January and July 2012, MSM with GC were advised to return 1 week after treatment for TOC using NAAT. A multivariate logistic regression model was used to determine demographic and behavioral differences between MSM who returned for follow-up and MSM who did not. Of 737 men with GC, 194 (26.3%) returned between 3 and 21 days of treatment. Individuals who returned were more likely to have no GC history (P = 0.0001) and to report no initial symptoms (P = 0.02) when compared with individuals who did not return for TOC. Of those who returned, 0% of urethral samples, 7.4% of rectal samples, and 5.3% of pharyngeal samples were NAAT positive at TOC. Although TOC may be an important strategy in reducing complications and the spread of GC, low return rates may make implementation challenging. If implemented, extra efforts should be considered to enhance return rates among individuals with a history of GC. If TOCs are recommended at 1 week and NAATs are used, the interpretation of positive results, particularly those from extragenital sites, may be difficult.

Assigning Cytomegalovirus (CMV) Status in Children Awaiting Organ Transplant: Viral Shedding, CMV-Specific T-cells and CD27-CD28-CD4+ T-cells.

PubMed

Burton, Catherine E; Sester, Martina; Robinson, Joan L; Eurich, Dean T; Preiksaitis, Jutta K; Urschel, Simon

2018-05-24

Passive antibodies, maternal or transfusion-acquired, make serologic determination of pre-transplant cytomegalovirus (CMV) status unreliable. We evaluated 3 assays un-affected by passive antibodies, in assignment of CMV infection status in children awaiting solid organ transplant and in controls: i) CMV Nucleic Acid Amplification Testing (NAAT), quantification of ii) CMV-specific CD4+T-cells, and iii) CD27-CD28-CD4+T-cells. Our results highlight that CMV NAAT, from urine and oropharynx, is useful in confirming positive CMV status. Detection of CMV-specific CD4+T-cells was sensitive and specific in children >18 months but was less sensitive in children <12 months. CD27-CD28- CD4+T-cells are not likely useful in CMV risk-stratification in children.

Genotype-Specific Concordance of Chlamydia trachomatis Genital Infection Within Heterosexual Partnerships.

PubMed

Schillinger, Julia A; Katz, Barry P; Markowitz, Lauri E; Braslins, Phillip G; Shrier, Lydia A; Madico, Guillermo; Van Der Pol, Barbara; Orr, Donald P; Rice, Peter A; Batteiger, Byron E

2016-12-01

Sexual transmission rates of Chlamydia trachomatis (Ct) cannot be measured directly; however, the study of concordance of Ct infection in sexual partnerships (dyads) can help to illuminate factors influencing Ct transmission. Heterosexual men and women with Ct infection and their sex partners were enrolled and partner-specific coital and behavioral data collected for the prior 30 days. Microbiological data included Ct culture, and nucleic acid amplification testing (NAAT), quantitative Ct polymerase chain reaction, and ompA genotyping. We measured Ct concordance in dyads and factors (correlates) associated with concordance. One hundred twenty-one women and 125 men formed 128 dyads. Overall, 72.9% of male partners of NAAT-positive women and 68.6% of female partners of NAAT-positive men were Ct-infected. Concordance was more common in dyads with culture-positive members (78.6% of male partners, 77% of female partners). Partners of women and men who were NAAT-positive only had lower concordance (33.3%, 46.4%, respectively). Women in concordant dyads had significantly higher median endocervical quantitative Ct polymerase chain reaction values (3,032) compared with CT-infected women in discordant dyads (1013 inclusion forming units DNA equivalents per mL; P < 0.01). Among 54 Ct-concordant dyads with ompA genotype data for both members, 96.2% had identical genotypes. Higher organism load appears associated with concordance among women. Same-genotype chlamydial concordance was high in sexual partnerships. No behavioral factors were sufficiently discriminating to guide partner services activities. Findings may help model coitus-specific transmission probabilities.

Laboratory Capacity for Antimicrobial Susceptibility Surveillance of Neisseria gonorrhoeae-District of Columbia, 2007-2012.

PubMed

Garrett, Tiana A; Davies-Cole, John; Furness, Bruce

2015-08-01

In the District of Columbia (DC), Neisseria gonorrhoeae (gonorrhea) infections accounted for more than 25% of 9321 incident sexually transmitted infections reported in 2011; untreated infections can lead to reproductive complications and a higher risk for HIV transmission. In DC, limited capacity to measure the prevalence of antibiotic-resistant N. gonorrhoeae is available; culture-based antibiotic susceptibility testing (AST) is needed to monitor antimicrobial resistance. We examined the capacity of laboratories that report to the DC Department of Health to perform AST for ongoing surveillance of antibiotic-resistant N. gonorrhoeae and to identify suspected treatment failures. We created a survey about diagnostic methods for gonorrhea testing and identified 33 laboratories that reported gonorrhea results to Department of Health in 2007 to 2012. Laboratories were assessed for use of bacterial culture or nucleic acid amplification testing (NAAT) for gonorrhea testing, prevalence of AST on gonorrhea-positive cultures, and types of antibiotics tested during AST. We estimated the prevalence of laboratory practices on the basis of self-report by staff. Nineteen (58%) laboratories completed the survey, representing 92% of the gonorrhea reporting. Seventeen (89%) of 19 laboratories conducted testing by culture; only 6 (35%) performed AST; 79% performed NAAT. Barriers to AST included longer completion times and limited number of provider requests for AST. Commercial laboratories (32%) were more likely to conduct both culture and NAAT, compared with health care facilities (11%). We report a low prevalence of laboratories performing AST because of multiple barriers. State-specific strategies addressing these barriers are needed to improve detection of antibiotic-resistant gonorrhea stains circulating among the population.

Chlamydia trachomatis infection in African American women who exclusively have sex with women.

PubMed

Muzny, Christina A; Kapil, Richa; Austin, Erika L; Brown, LaDraka; Hook, Edward W; Geisler, William M

2016-10-01

Little is known about whether Chlamydia trachomatis can be sexually transmitted between women or how often it occurs in women who have sex with women (WSW). We investigated Chlamydia trachomatis prevalence and serum Chlamydia trachomatis-specific antibody responses among African American WSW who reported a lifetime history of sex only with women (exclusive WSW) (n = 21) vs. an age-matched group of women reporting sex with women and men (WSWM) (n = 42). Participants completed a survey, underwent a pelvic examination in which a cervical swab was collected for Chlamydia trachomatis nucleic acid amplification testing (NAAT), and had serum tested for anti-Chlamydia trachomatis IgG1 and IgG3 antibodies using a Chlamydia trachomatis elementary body-based ELISA. No exclusive WSW had a positive Chlamydia trachomatis NAAT vs. 5 (11.9%) WSWM having a positive Chlamydia trachomatis NAAT (p = 0.16). Compared with WSWM, WSW were significantly less likely to be Chlamydia trachomatis seropositive (7 [33.3%] vs. 29 [69%], p = 0.007). Among Chlamydia trachomatis seropositive women, all were seropositive by IgG1, and the magnitude of Chlamydia trachomatis-specific IgG1 responses did not differ in Chlamydia trachomatis-seropositive WSW vs. WSWM. In conclusion, Chlamydia trachomatis seropositivity was relatively common in exclusive African American WSW, though significantly less common than in African American WSWM. © The Author(s) 2016.

Evaluation of the Efficiency of the Sample Inactivation Reagent in the Abbott RealTime MTB Assay for Inactivation of Mycobacterium tuberculosis.

PubMed

Qi, Chao; Wallis, Carole; Pahalawatta, Vihanga; Frank, Andrea; Ramdin, Neeshan; Viana, Raquel; Abravaya, Klara; Leckie, Gregor; Tang, Ning

2015-09-01

The Abbott RealTime MTB assay is a nucleic acid amplification test (NAAT) for the detection of Mycobacterium tuberculosis complex DNA. The sample inactivation procedure used in the assay, consisting of one part sample treated with 3 parts inactivation reagent for 60 min, effectively reduced viscosity and inactivated M. tuberculosis in clinical specimens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Diagnostic accuracy of nucleic acid amplification based assays for tuberculous meningitis: A meta-analysis.

PubMed

Gupta, Renu; Talwar, Puneet; Talwar, Pumanshi; Khurana, Sarbjeet; Kushwaha, Suman; Jalan, Nupur; Thakur, Rajeev

2018-05-25

Numerous in-house and commercial nucleic acid amplification tests (NAAT) have been evaluated using variable reference standards for diagnosis of TBM but their diagnostic potential is still not very clear. We conducted a meta-analysis to assess the diagnostic accuracy of different NAAT based assays for diagnosing TBM against 43 data sets of confirmed TBM (n = 1066) and 61 data sets of suspected TBM (n = 3721) as two reference standards. The summary estimate of the sensitivity and the specificity were obtained using the bivariate model. QUADAS-2 tool was used to perform the Quality assessment for bias and applicability. Publication bias was assessed with Deeks' funnel plot. Studies with confirmed TBM had better summary estimates as compared to studies with clinically suspected TBM irrespective of NAAT and index tests used. Among in-house assays, MPB as the gene target had best summary estimates in both confirmed [sensitivity:90%(83-95), specificity:97-%(87-99), DOR:247 (50-1221), AUC:99%(97-100), PLR:38.8-(6.6-133), NLR:0.11(0.05-0.18), I 2   = 15%] and clinically suspected [sensitivity:69%(47-85), specificity:96%(90-98), DOR:62(16.8-232), AUC:94%(92-97), PLR:16.9(6.5-36.8), NLR:0.33(0.16-0.56), I 2 :15.3%] groups. GeneXpert revealed good diagnostic accuracy only in confirmed TBM group [sensitivity = 57%(38-74), specificity = 98%(89-100), DOR = 62(7-589), AUC = 87%(79-96), PLR = 33.2(3.8-128), NLR = 0.45(0.26-0.68), I 2   = 0%]. This meta-analysis identified potential role of MPB gene among in-house assays and GeneXpert as commercial assay for diagnosing TBM. Copyright © 2018. Published by Elsevier Ltd.

A 30-Min Nucleic Acid Amplification Point-of-Care Test for Genital Chlamydia trachomatis Infection in Women: A Prospective, Multi-center Study of Diagnostic Accuracy.

PubMed

Harding-Esch, E M; Cousins, E C; Chow, S-L C; Phillips, L T; Hall, C L; Cooper, N; Fuller, S S; Nori, A V; Patel, R; Thomas-William, S; Whitlock, G; Edwards, S J E; Green, M; Clarkson, J; Arlett, B; Dunbar, J K; Lowndes, C M; Sadiq, S T

2018-02-01

Rapid Point-Of-Care Tests for Chlamydia trachomatis (CT) may reduce onward transmission and reproductive sexual health (RSH) sequelae by reducing turnaround times between diagnosis and treatment. The io® single module system (Atlas Genetics Ltd.) runs clinical samples through a nucleic acid amplification test (NAAT)-based CT cartridge, delivering results in 30min. Prospective diagnostic accuracy study of the io® CT-assay in four UK Genito-Urinary Medicine (GUM)/RSH clinics on additional-to-routine self-collected vulvovaginal swabs. Samples were tested "fresh" within 10days of collection, or "frozen" at -80°C for later testing. Participant characteristics were collected to assess risk factors associated with CT infection. CT prevalence was 7.2% (51/709) overall. Sensitivity, specificity, positive and negative predictive values of the io® CT assay were, respectively, 96.1% (95% Confidence Interval (CI): 86.5-99.5), 97.7% (95%CI: 96.3-98.7), 76.6% (95%CI: 64.3-86.2) and 99.7% (95%CI: 98.9-100). The only risk factor associated with CT infection was being a sexual contact of an individual with CT. The io® CT-assay is a 30-min, fully automated, high-performing NAAT currently CE-marked for CT diagnosis in women, making it a highly promising diagnostic to enable specific treatment, initiation of partner notification and appropriately intensive health promotion at the point of care. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

In Silico PCR Tools for a Fast Primer, Probe, and Advanced Searching.

PubMed

Kalendar, Ruslan; Muterko, Alexandr; Shamekova, Malika; Zhambakin, Kabyl

2017-01-01

The polymerase chain reaction (PCR) is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. The principle of this technique has been further used and applied in plenty of other simple or complex nucleic acid amplification technologies (NAAT). In parallel to laboratory "wet bench" experiments for nucleic acid amplification technologies, in silico or virtual (bioinformatics) approaches have been developed, among which in silico PCR analysis. In silico NAAT analysis is a useful and efficient complementary method to ensure the specificity of primers or probes for an extensive range of PCR applications from homology gene discovery, molecular diagnosis, DNA fingerprinting, and repeat searching. Predicting sensitivity and specificity of primers and probes requires a search to determine whether they match a database with an optimal number of mismatches, similarity, and stability. In the development of in silico bioinformatics tools for nucleic acid amplification technologies, the prospects for the development of new NAAT or similar approaches should be taken into account, including forward-looking and comprehensive analysis that is not limited to only one PCR technique variant. The software FastPCR and the online Java web tool are integrated tools for in silico PCR of linear and circular DNA, multiple primer or probe searches in large or small databases and for advanced search. These tools are suitable for processing of batch files that are essential for automation when working with large amounts of data. The FastPCR software is available for download at http://primerdigital.com/fastpcr.html and the online Java version at http://primerdigital.com/tools/pcr.html .

Intestinal Carriage of Carbapenemase-Producing Organisms: Current Status of Surveillance Methods

PubMed Central

Jacobs, Michael R.; Wilson, Brigid; Kaye, Keith; Donskey, Curtis J.; Perez, Federico

2015-01-01

SUMMARY Carbapenemases have become a significant mechanism for broad-spectrum β-lactam resistance in Enterobacteriaceae and other Gram-negative bacteria such as Pseudomonas and Acinetobacter spp. Intestinal carriage of carbapenemase-producing organisms (CPOs) is an important source of transmission. Isolation of carriers is one strategy that can be used to limit the spread of these bacteria. In this review, we critically examine the clinical performance, advantages, and disadvantages of methods available for the detection of intestinal carriage of CPOs. Culture-based methods (Centers for Disease Control and Prevention [CDC] protocols, chromogenic media, specialized agars, and double-disk synergy tests) for detecting carriage of CPOs are convenient due to their ready availability and low cost, but their limited sensitivity and long turnaround time may not always be optimal for infection control practices. Contemporary nucleic acid amplification techniques (NAATs) such as real-time PCR, hybridization assays, loop-mediated isothermal amplification (LAMP), or a combined culture and NAAT approach may provide fast results and/or added sensitivity and specificity compared with culture-based methods. Infection control practitioners and clinical microbiologists should be aware of the strengths and limitations of available methods to determine the most suitable approach for their medical facility to fit their infection control needs. PMID:26511484

Anal lymphogranuloma venereum infection screening with IgA anti-Chlamydia trachomatis-specific major outer membrane protein serology.

PubMed

de Vries, Henry J C; Smelov, Vitaly; Ouburg, Sander; Pleijster, Jolein; Geskus, Ronald B; Speksnijder, Arjen G C L; Fennema, Johannes S A; Morré, Servaas A

2010-12-01

Anal lymphogranuloma venereum (LGV) infections, caused by Chlamydia trachomatis biovar L (Ct+/LGV+), are endemic among men who have sex with men (MSM). Anal non-LGV biovar Ct infections (Ct+/LGV-) can be eradicated with 1 week doxycycline, whereas Ct+/LGV+ infections require 3-week doxycycline. To differentiate Ct+/LGV+ from Ct+/LGV- infections, biovar-specific Nucleic Acid Amplification Test (NAAT) are standard, but also expensive and laborious. A chlamydia-specific serological assay could serve as an alternative test. MSM were screened for anal Ct+/LGV+ and Ct+/LGV- infections with a commercial nonspecific NAAT and an in house biovar L-specific NAAT. Serum samples were evaluated with chlamydia-specific anti-Major Outer Membrane Protein (MOMP) and antilipopolysaccharide assays of IgA and IgG classes. Asymptomatic patients were identified as: (1) no anal complaints or (2) no microscopic inflammation (i.e., <10 leucocytes per high power field in anal smears). The best differentiating assay was subsequently evaluated in 100 Ct+/LGV+ and 100 Ct+/LGV- MSM using different cut-off points. The anti-MOMP IgA assay was the most accurate to differentiate Ct+/LGV+ (n = 42) from Ct+/LGV- (n = 19) with 85.7% sensitivity (95% confidence interval [CI], 72.2-93.3) and 84.2% specificity (95% CI, 62.4-94.5), even among asymptomatic patients. In a population comprising 98 Ct+/LGV+ and 105 Ct+/LGV- patients, the anti-MOMP IgA assay scored most accurate when the cut-off point was set to 2.0 with 75.5% (95% CI, 65.8-83.6) sensitivity and 74.3% (95% CI, 64.8-82.3) specificity. The IgA anti-MOMP assay can identify a considerable proportion of the (asymptomatic) anal LGV infections correctly. Yet, biovar L-specific NAAT are still the preferred diagnostic tests in clinical settings.

Market assessment of tuberculosis diagnostics in China in 2012.

PubMed

Zhao, Y-L; Pang, Y; Xia, H; Du, X; Chin, D; Huan, S-T; Dong, H-Y; Zhang, Z-Y; Ginnard, J; Perkins, M D; Boehme, C C; Jefferson, C; Pantoja, A; Qin, Z Z; Chedore, P; Denkinger, C M; Pai, M; Kik, S V

2016-03-01

To assess the 2012 served available market for tuberculosis (TB) diagnostics in China in the sector served by the China Centre for Disease Control and Prevention (CDC) and the hospital sector in China, including both designated TB hospitals and general hospitals. Test volumes and unit costs were assessed for tuberculin skin tests, interferon-gamma release assays (IGRAs), smear microscopy, serology, cultures, speciation tests, nucleic-acid amplification tests (NAATs), drug susceptibility tests and adenosine-deaminase tests (ADA). Data were obtained from electronic databases (CDC sector) and through surveys (hospital sector), and were estimated for the two sectors and for the country as a whole. Test costs were estimated by staff at China CDC, and using published literature. In 2012, the China CDC and hospital sectors performed a total of 44 million TB diagnostic tests at an overall value of US$294 million. Tests used by the CDC sector were smear microscopy, solid and liquid culture and DST, while the hospital sector also used IGRAs, NAATs, ADA and serology. The hospital sector accounted for 76% of the overall test volume and 94% of the market value. China has a very large TB diagnostic market that encompasses a wide range of diagnostic tests, with the majority being performed in Chinese hospitals.

Trichomonas vaginalis: Clinical relevance, pathogenicity and diagnosis.

PubMed

Edwards, Thomas; Burke, Patricia; Smalley, Helen; Hobbs, Glyn

2016-05-01

Trichomonas vaginalis is the etiological agent of trichomoniasis, the most prevalent non-viral sexually transmitted disease worldwide. Trichomoniasis is a widespread, global health concern and occurring at an increasing rate. Infections of the female genital tract can cause a range of symptoms, including vaginitis and cervicitis, while infections in males are generally asymptomatic. The relatively mild symptoms, and lack of evidence for any serious sequelae, have historically led to this disease being under diagnosed, and under researched. However, growing evidence that T. vaginalis infection is associated with other disease states with high morbidity in both men and women has increased the efforts to diagnose and treat patients harboring this parasite. The pathology of trichomoniasis results from damage to the host epithelia, caused by a variety of processes during infection and recent work has highlighted the complex interactions between the parasite and host, commensal microbiome and accompanying symbionts. The commercial release of a number of nucleic acid amplification tests (NAATs) has added to the available diagnostic options. Immunoassay based Point of Care testing is currently available, and a recent initial evaluation of a NAAT Point of Care system has given promising results, which would enable testing and treatment in a single visit.

Evaluation of the CDC proposed laboratory HIV testing algorithm among men who have sex with men (MSM) from five US metropolitan statistical areas using specimens collected in 2011.

PubMed

Masciotra, Silvina; Smith, Amanda J; Youngpairoj, Ae S; Sprinkle, Patrick; Miles, Isa; Sionean, Catlainn; Paz-Bailey, Gabriela; Johnson, Jeffrey A; Owen, S Michele

2013-12-01

Until recently most testing algorithms in the United States (US) utilized Western blot (WB) as the supplemental test. CDC has proposed an algorithm for HIV diagnosis which includes an initial screen with a Combo Antigen/Antibody 4th generation-immunoassay (IA), followed by an HIV-1/2 discriminatory IA of initially reactive-IA specimens. Discordant results in the proposed algorithm are resolved by nucleic acid-amplification testing (NAAT). Evaluate the results obtained with the CDC proposed laboratory-based algorithm using specimens from men who have sex with men (MSM) obtained in five metropolitan statistical areas (MSAs). Specimens from 992 MSM from five MSAs participating in the CDC's National HIV Behavioral Surveillance System in 2011 were tested at local facilities and CDC. The five MSAs utilized algorithms of various screening assays and specimen types, and WB as the supplemental test. At the CDC, serum/plasma specimens were screened with 4th generation-IA and the Multispot HIV-1/HIV-2 discriminatory assay was used as the supplemental test. NAAT was used to resolve discordant results and to further identify acute HIV infections from all screened-non-reactive missed by the proposed algorithm. Performance of the proposed algorithm was compared to site-specific WB-based algorithms. The proposed algorithm detected 254 infections. The WB-based algorithms detected 19 fewer infections; 4 by oral fluid (OF) rapid testing and 15 by WB supplemental testing (12 OF and 3 blood). One acute infection was identified by NAAT from all screened-non-reactive specimens. The proposed algorithm identified more infections than the WB-based algorithms in a high-risk MSM population. OF testing was associated with most of the discordant results between algorithms. HIV testing with the proposed algorithm can increase diagnosis of infected individuals, including early infections. Published by Elsevier B.V.

Non-Instrumented Incubation of a Recombinase Polymerase Amplification Assay for the Rapid and Sensitive Detection of Proviral HIV-1 DNA

PubMed Central

Lillis, Lorraine; Lehman, Dara; Singhal, Mitra C.; Cantera, Jason; Singleton, Jered; Labarre, Paul; Toyama, Anthony; Piepenburg, Olaf; Parker, Mathew; Wood, Robert; Overbaugh, Julie; Boyle, David S.

2014-01-01

Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25–43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS. PMID:25264766

Non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral HIV-1 DNA.

PubMed

Lillis, Lorraine; Lehman, Dara; Singhal, Mitra C; Cantera, Jason; Singleton, Jered; Labarre, Paul; Toyama, Anthony; Piepenburg, Olaf; Parker, Mathew; Wood, Robert; Overbaugh, Julie; Boyle, David S

2014-01-01

Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25-43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS.

Correlation of Leukorrhea and Trichomonas vaginalis Infection

PubMed Central

Soper, David E.; Nolte, Frederick S.

2013-01-01

Trichomonas vaginalis is a common sexually transmitted infection (STI) causing vaginitis. Microscopy has poor sensitivity but is used for diagnosis of trichomoniasis in resource-poor settings. We aimed to provide a more reliable diagnosis of trichomoniasis by investigating an association with leukorrhea. Women presenting for evaluation of vaginal discharge, STI exposure, or preventative gynecologic examination were evaluated for Trichomonas infection. Vaginal pH was determined and microscopy was performed by the provider, who recorded the number of polymorphonuclear leukocytes (PMNLs) per epithelial cell and the presence of clue cells, yeast, and/or motile trichomonads. Leukorrhea was defined as greater than one PMNL per epithelial cell. Culture and a nucleic acid amplification test (NAAT) were used to detect T. vaginalis. Patients were evaluated for Chlamydia trachomatis and Neisseria gonorrhoeae using NAATs and bacterial vaginosis using Gram stains. Two hundred ninety-four women were enrolled, and 16% were found to have Trichomonas (46/294). Trichomonas infection was more common in parous non-Hispanic, black women, who reported low rates of contraceptive use (33% versus 17%; P = 0.02) and a STI history (85% versus 55%; P = 0.002). These women were more likely to report vaginal discharge (76% versus 59%; P = 0.02) and have an elevated vaginal pH (87% versus 48%; P < 0.001) and gonorrhea infection (15% versus 4%; P = 0.002). Leukorrhea was associated with a 4-fold-increased risk of Trichomonas infection. Leukorrhea on microscopy was associated with Trichomonas vaginitis. Patients with leukorrhea should be evaluated with more-sensitive tests for T. vaginalis, preferably NAATs, if microscopy is negative. PMID:23678058

Lymphogranuloma venereum detected from the pharynx in four London men who have sex with men.

PubMed

Dosekun, O; Edmonds, S; Stockwell, S; French, P; White, J A

2013-06-01

Lymphogranuloma venereum (LGV) is an established cause of proctitis in men who have sex with men (MSM). Currently in the UK, testing for pharyngeal Chlamydia trachomatis (CT) is not routine, and LGV typing is usually only performed in patients with anorectal symptoms. We report four cases where LGV-associated CT DNA was detected from the pharynx in MSM, demonstrating that nucleic acid amplification testing (NAAT) can be used for detecting and typing pharyngeal CT infection. These cases also highlight other possible routes of infection for LGV, and add to the broad spectrum of clinical presentations associated with this infection.

Sero-epidemiological assessment of Chlamydia trachomatis infection and sub-fertility in Samoan women.

PubMed

Menon, S; Stansfield, S H; Walsh, M; Hope, E; Isaia, L; Righarts, A A; Niupulusu, T; Temese, S V A; Iosefa-Siitia, L; Auvaa, L; Tapelu, S A; Motu, M F; Suaalii-Sauni, T; Timms, P; Hill, P C; Huston, W M

2016-04-21

In our recent village-based cross-sectional study, the prevalence of nucleic acid amplification technique (NAAT) diagnosed Chlamydia trachomatis (CT) in sexually active Samoan women was very high (36 %), and test positivity was associated with sub-fertility. We conducted a serological and epidemiological analysis in these participants to identify if serological data can provide further insight into the potential contribution of CT to sub-fertility in this population. Serological prediction of CT associated sub-fertility was conducted using a series of commercial tests. The correlation between fertility or sub-fertility, behavioral factors, and serologically predicted CT associated sub-fertility was determined. A positive antibody reaction against the Chlamydia Major Outer Membrane Protein (MOMP) was significantly associated with sub-fertility, with 50 % of infertile women being positive. Serum IgG and IgA antibodies against MOMP correlated with current infection measured by urine NAAT, suggesting longer term infections are common in this population. Chlamydia pneumoniae antibodies were frequently detected in this population (84 %), and unexpectedly, were significantly associated with sub-fertility. The high prevalence of chlamydial infection and of positive chlamydial sub-fertility results suggests that CT is an important and frequent contributory factor to sub-fertility in this population.

Premarket evaluations of the IMDx C. difficile for Abbott m2000 Assay and the BD Max Cdiff Assay.

PubMed

Stellrecht, K A; Espino, A A; Maceira, V P; Nattanmai, S M; Butt, S A; Wroblewski, D; Hannett, G E; Musser, K A

2014-05-01

Clostridium difficile-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing C. difficile has been difficult, as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAATs) are quickly becoming the standard of care. We compared the performance of two automated investigational/research use only (IUO/RUO) NAATs for the detection of C. difficile toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD Max Cdiff Assay (Max). A prospective analysis of 111 stool specimens received in the laboratory for C. difficile testing by the laboratory's test of record (TOR), the BD GeneOhm Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for C. difficile were included in the study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods, 9 were positive by TOR and Max, and 3 were positive by TOR only. One negative specimen was initially inhibitory by Max. The remaining 95 specimens were negative by all methods. Toxigenic C. difficile culture was performed on the 12 discrepant samples. True C. difficile-positive status was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for Max and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance, and each has individual benefits, which will ensure that they will both have a niche in clinical laboratories.

Seven days of doxycycline is an effective treatment for asymptomatic rectal Chlamydia trachomatis infection.

PubMed

Elgalib, A; Alexander, S; Tong, C Y W; White, J A

2011-08-01

There are no evidence-based guidelines for the specific management of rectal Chlamydia trachomatis (CT) infection. All men who have sex with men (MSM) diagnosed with asymptomatic rectal CT by nucleic acid amplification test (NAAT) at a large London genitourinary (GU) medicine clinic between September 2006 and September 2009 were offered oral doxycycline 100 mg twice daily for seven days and invited for a test of cure (TOC) by CT NAAT four weeks after treatment. A total of 487 asymptomatic rectal CT infections were diagnosed and analysis was restricted to 165 TOCs from men whose only treatment had been doxycycline for seven days. The median time post-treatment for TOC was 45 days (interquartile range [IQR], 34-88). Only two patients tested CT-positive at follow-up. One had taken doxycycline only for three days; the other attended for TOC 240 days after the completion of doxycycline treatment and at this time presented with new symptoms in the context of ongoing high sexual risk. Our findings show that doxycycline 100 mg twice daily for seven days is highly effective treatment for asymptomatic rectal CT infection, achieving clearance of CT in 98.8% (163/165; 95% CI 95.4-99.9%) of cases. We advocate doxycycline for seven days as first-line therapy for asymptomatic rectal CT.

Evaluation of the hyplex® TBC PCR test for detection of Mycobacterium tuberculosis complex in clinical samples

PubMed Central

2010-01-01

Background Tuberculosis (TB) is one of the major public health concerns worldwide. The detection of the pathogen Mycobacterium tuberculosis complex (MTBC) as early as possible has a great impact on the effective control of the spread of the disease. In our study, we evaluated the hyplex® TBC PCR test (BAG Health Care GmbH), a novel assay using a nucleic acid amplification technique (NAAT) with reverse hybridisation and ELISA read out for the rapid detection of M. tuberculosis directly in clinical samples. Results A total of 581 respiratory and non-respiratory specimens from our pneumological hospital and the National TB Institute of Uzbekistan were used for the evaluation of the PCR assay. Of these, 292 were classified as TB samples and 289 as non-TB samples based on the results of the TB cultures as reference method. The PCR results were initially used to optimise the cut-off value of the hyplex® TBC test system by means of a ROC analysis. The overall sensitivity of the assay was determined to be 83.1%. In smear-positive TB samples, the sensitivity of the hyplex® TBC PCR test was estimated to 93.4% versus 45.1% in smear-negative samples. The specificity of the test was 99.25%. Of the two specimens (0.75%) with false-positive PCR results, one yielded a culture positive for non-tuberculous mycobacteria. Based on the assumption of a prevalence of 8% TB positives among the samples in our diagnostic TB laboratory, the positive and negative predictive values were estimated to 90.4% and 98.5%, respectively. Conclusions The hyplex® TBC PCR test is an accurate NAAT assay for a rapid and reliable detection of M. tuberculosis in various respiratory and non-respiratory specimens. Compared to many other conventional NAAT assays, the hyplex® TBC PCR test is in a low price segment which makes it an attractive option for developing and emerging countries with high TB burdens. PMID:20356361

Systematic review of the clinical effectiveness and cost-effectiveness of rapid point-of-care tests for the detection of genital chlamydia infection in women and men.

PubMed

Hislop, J; Quayyum, Z; Flett, G; Boachie, C; Fraser, C; Mowatt, G

2010-06-01

To assess whether or not the Chlamydia Rapid Test (CRT) could improve detection of genital chlamydia, and whether it is more effective than current practice using nucleic acid amplification tests (NAATs), in terms of the number of cases of chlamydia that are detected and treated and the proportion of partners identified and treated. Eleven electronic bibliographic databases (including MEDLINE and EMBASE) were searched until November 2008, as well as relevant websites. Studies of sexually active adolescent and adult women and men suspected of having or being tested for genital chlamydia infection were considered. The tests considered were the CRT and other comparator point-of-care tests identified, using a NAAT as a reference standard. Summary sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratios for each model were reported as a median and a 95% confidence interval (CI). Effectiveness was measured in terms of the absolute numbers of true-positives, false-positives, false-negatives (and other positive cases missed) and true-negatives detected. Costs were considered from the health service's perspective. Incremental cost-effectiveness ratios were used to examine the relative cost-effectiveness, and values of the major parameters of the models were varied in a sensitivity analysis. Thirteen studies enrolling 8817 participants were included in the analysis. In the pooled estimates for the CRT, sensitivity (95% CI) was 80% (73% to 85%) for vaginal swab specimens and 77% (59% to 89%) for first void urine (FVU) specimens. Specificity was 99% (99% to 100%) for vaginal swab specimens and 99% (98% to 99%) for FVU specimens. In the pooled estimates for a comparator point-of-care test (Clearview Chlamydia), sensitivity (95% CI) was 52% (39% to 65%) for vaginal, cervical and urethral swab specimens combined, and 64% (47% to 77%) for cervical specimens alone. Specificity was 97% (94% to 100%) for vaginal, cervical and urethral swab specimens combined, and 97% (88% to 99%) for cervical specimens alone. The results of the economic evaluation showed that for a hypothetical cohort of 1000 people, using the current practice of polymerase chain reaction testing would result in 12.63 people who were offered testing being correctly treated and having their sexual partners contacted, at a cost of 7070 pounds (for the whole cohort). For the CRT, the number being correctly treated would be 10.98, at a cost of 7180 pounds. For the Clearview Chlamydia test, the number correctly treated would be 7.14, at a cost of 7170 pounds. Both point-of-care tests were therefore more costly and less effective than current practice. The limited evidence available suggests that NAATs are still the most accurate and cost-effective method for diagnosing chlamydia infection. There may be circumstances in which point-of-care tests could be provided in addition to existing NAAT services, but there is currently little evidence on point-of-care methods in such settings. Robust evidence of the diagnostic accuracy of point-of-care tests for different types of samples is also still required, as are studies evaluating clinical effectiveness outcomes for these tests in comparison with NAATs.

Commercial products to preserve specimens for tuberculosis diagnosis: a systematic review.

PubMed

Reeve, B W P; McFall, S M; Song, R; Warren, R; Steingart, K R; Theron, G

2018-07-01

Eliminating tuberculosis in high-burden settings requires improved diagnostic capacity. Important tests such as Xpert® MTB/RIF and culture are often performed at centralised laboratories that are geographically distant from the point of specimen collection. Preserving specimen integrity during transportation, which could affect test performance, is challenging. To conduct a systematic review of commercial products for specimen preservation for a World Health Organization technical consultation. Databases were searched up to January 2018. Methodological quality was assessed using Quality Assessment of Technical Studies, a new technical study quality-appraisal tool, and Quality Assessment of Diagnostic Accuracy Studies-2. Studies were analysed descriptively in terms of the different products, study designs and diagnostic strategies used. Four products were identified from 16 studies: PrimeStore-Molecular-Transport-Medium (PS-MTM), FTA card, GENO•CARD (all for nucleic acid amplification tests [NAATs]) and OMNIgene•SPUTUM (OMS; culture, NAATs). PS-MTM, but not FTA card or GENO•CARD, rendered Mycobacterium tuberculosis non-culturable. OMS reduced Löwenstein-Jensen but not MGIT™ 960™ contamination, led to delayed MGIT time-to-positivity, resulted in Xpert performance similar to cold chain-transported untreated specimens, and obviated the need for N-acetyl-L-cysteine-sodium hydroxide decontamination. Data from paucibacillary specimens were limited. Evidence that a cold chain improves culture was mixed and absent for Xpert. The effect of the product alone could be discerned in only four studies. Limited evidence suggests that transport products result in test performance comparable to that seen in cold chain-transported specimens.

Impact of Molecular Diagnostics for Tuberculosis on Patient-Important Outcomes: A Systematic Review of Study Methodologies

PubMed Central

Schumacher, Samuel G.; Sohn, Hojoon; Qin, Zhi Zhen; Gore, Genevieve; Davis, J. Lucian; Denkinger, Claudia M.; Pai, Madhukar

2016-01-01

Background Several reviews on the accuracy of Tuberculosis (TB) Nucleic Acid Amplification Tests (NAATs) have been performed but the evidence on their impact on patient-important outcomes has not been systematically reviewed. Given the recent increase in research evaluating such outcomes and the growing list of TB NAATs that will reach the market over the coming years, there is a need to bring together the existing evidence on impact, rather than accuracy. We aimed to assess the approaches that have been employed to measure the impact of TB NAATs on patient-important outcomes in adults with possible pulmonary TB and/or drug-resistant TB. Methods We first develop a conceptual framework to clarify through which mechanisms the improved technical performance of a novel TB test may lead to improved patient outcomes and outline which designs may be used to measure them. We then systematically review the literature on studies attempting to assess the impact of molecular TB diagnostics on such outcomes and provide a narrative synthesis of designs used, outcomes assessed and risk of bias across different study designs. Results We found 25 eligible studies that assessed a wide range of outcomes and utilized a variety of experimental and observational study designs. Many potentially strong design options have never been used. We found that much of the available evidence on patient-important outcomes comes from a small number of settings with particular epidemiological and operational context and that confounding, time trends and incomplete outcome data receive insufficient attention. Conclusions A broader range of designs should be considered when designing studies to assess the impact of TB diagnostics on patient outcomes and more attention needs to be paid to the analysis as concerns about confounding and selection bias become relevant in addition to those on measurement that are of greatest concern in accuracy studies. PMID:26954678

Fully 3D printed integrated reactor array for point-of-care molecular diagnostics.

PubMed

Kadimisetty, Karteek; Song, Jinzhao; Doto, Aoife M; Hwang, Young; Peng, Jing; Mauk, Michael G; Bushman, Frederic D; Gross, Robert; Jarvis, Joseph N; Liu, Changchun

2018-06-30

Molecular diagnostics that involve nucleic acid amplification tests (NAATs) are crucial for prevention and treatment of infectious diseases. In this study, we developed a simple, inexpensive, disposable, fully 3D printed microfluidic reactor array that is capable of carrying out extraction, concentration and isothermal amplification of nucleic acids in variety of body fluids. The method allows rapid molecular diagnostic tests for infectious diseases at point of care. A simple leak-proof polymerization strategy was developed to integrate flow-through nucleic acid isolation membranes into microfluidic devices, yielding a multifunctional diagnostic platform. Static coating technology was adopted to improve the biocompatibility of our 3D printed device. We demonstrated the suitability of our device for both end-point colorimetric qualitative detection and real-time fluorescence quantitative detection. We applied our diagnostic device to detection of Plasmodium falciparum in plasma samples and Neisseria meningitides in cerebrospinal fluid (CSF) samples by loop-mediated, isothermal amplification (LAMP) within 50 min. The detection limits were 100 fg for P. falciparum and 50 colony-forming unit (CFU) for N. meningitidis per reaction, which are comparable to that of benchtop instruments. This rapid and inexpensive 3D printed device has great potential for point-of-care molecular diagnosis of infectious disease in resource-limited settings. Copyright © 2018 Elsevier B.V. All rights reserved.

Clinical Diagnostic Utility of IP-10 and LAM Antigen Levels for the Diagnosis of Tuberculous Pleural Effusions in a High Burden Setting

PubMed Central

Dheda, Keertan; Van-Zyl Smit, Richard N.; Sechi, Leonardo A.; Badri, Motasim; Meldau, Richard; Symons, Gregory; Khalfey, Hoosein; Carr, Igshaan; Maredza, Alice; Dawson, Rodney; Wainright, Helen; Whitelaw, Andrew; Bateman, Eric D.; Zumla, Alimuddin

2009-01-01

Background Current tools for the diagnosis of tuberculosis pleural effusions are sub-optimal. Data about the value of new diagnostic technologies are limited, particularly, in high burden settings. Preliminary case control studies have identified IFN-γ-inducible-10kDa protein (IP-10) as a promising diagnostic marker; however, its diagnostic utility in a day-to-day clinical setting is unclear. Detection of LAM antigen has not previously been evaluated in pleural fluid. Methods We investigated the comparative diagnostic utility of established (adenosine deaminase [ADA]), more recent (standardized nucleic-acid-amplification-test [NAAT]) and newer technologies (a standardized LAM mycobacterial antigen-detection assay and IP-10 levels) for the evaluation of pleural effusions in 78 consecutively recruited South African tuberculosis suspects. All consenting participants underwent pleural biopsy unless contra-indicated or refused. The reference standard comprised culture positivity for M. tuberculosis or histology suggestive of tuberculosis. Principal Findings Of 74 evaluable subjects 48, 7 and 19 had definite, probable and non-TB, respectively. IP-10 levels were significantly higher in TB vs non-TB participants (p<0.0001). The respective outcomes [sensitivity, specificity, PPV, NPV %] for the different diagnostic modalities were: ADA at the 30 IU/L cut-point [96; 69; 90; 85], NAAT [6; 93; 67; 28], IP-10 at the 28,170 pg/ml ROC-derived cut-point [80; 82; 91; 64], and IP-10 at the 4035 pg/ml cut-point [100; 53; 83; 100]. Thus IP-10, using the ROC-derived cut-point, missed ∼20% of TB cases and mis-diagnosed ∼20% of non-TB cases. By contrast, when a lower cut-point was used a negative test excluded TB. The NAAT had a poor sensitivity but high specificity. LAM antigen-detection was not diagnostically useful. Conclusion Although IP-10, like ADA, has sub-optimal specificity, it may be a clinically useful rule-out test for tuberculous pleural effusions. Larger multi-centric studies are now required to confirm our findings. PMID:19277111

Chlamydia positivity in New Orleans public high schools, 1996-2005: implications for clinical and public health practices.

PubMed

Nsuami, M Jacques; Nsa, Musheni; Brennan, Christine; Cammarata, Catherine L; Martin, David H; Taylor, Stephanie N

2013-01-01

To describe the trends in chlamydia positivity among New Orleans high school students tested in a schoolwide screening between 1996 and 2005, and to determine factors associated with chlamydia positivity among students during the 10-year period. Between school years 1995-1996 and 2004-2005, students in New Orleans public high schools were tested for chlamydia using nucleic acid amplification tests (NAAT) in urine specimens (LCx assay until 1999-2000; BD assay from 2000-2001 to 2004-2005). For each year, we calculated chlamydia positivity by dividing the number of students testing positive by the total number of students tested. Data were analyzed separately by gender. Logistic regressions were performed to determine independent predictors of chlamydia positivity during the 10-year period. Between 1996 and 2005, the average chlamydia positivity was 7.0% (95% confidence interval 6.6-7.4) in boys and 13.1% (95% confidence interval 12.6-13.7) in girls (P < .001). Chlamydia detection increased with the switch from LCx to BD assay. In multivariate analyses, chlamydia positivity among boys and girls was significantly associated with age, black race, and gonorrhea coinfection. Additionally, positivity was significantly different by school year among boys (P = .03) and by NAAT used among girls (P = .008). The trends in chlamydia positivity observed between 1996 and 2005 more likely reflected a high and stable prevalence of chlamydia in the New Orleans school-age adolescent population. Any benefit of screening on individuals tested was likely to be mitigated by participants' uninterrupted social interactions with the dynamic forces that sustain the sexual transmission of chlamydia in the population. Copyright © 2013 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.

The cobas® 6800/8800 System: a new era of automation in molecular diagnostics.

PubMed

Cobb, Bryan; Simon, Christian O; Stramer, Susan L; Body, Barbara; Mitchell, P Shawn; Reisch, Natasa; Stevens, Wendy; Carmona, Sergio; Katz, Louis; Will, Stephen; Liesenfeld, Oliver

2017-02-01

Molecular diagnostics is a key component of laboratory medicine. Here, the authors review key triggers of ever-increasing automation in nucleic acid amplification testing (NAAT) with a focus on specific automated Polymerase Chain Reaction (PCR) testing and platforms such as the recently launched cobas® 6800 and cobas® 8800 Systems. The benefits of such automation for different stakeholders including patients, clinicians, laboratory personnel, hospital administrators, payers, and manufacturers are described. Areas Covered: The authors describe how molecular diagnostics has achieved total laboratory automation over time, rivaling clinical chemistry to significantly improve testing efficiency. Finally, the authors discuss how advances in automation decrease the development time for new tests enabling clinicians to more readily provide test results. Expert Commentary: The advancements described enable complete diagnostic solutions whereby specific test results can be combined with relevant patient data sets to allow healthcare providers to deliver comprehensive clinical recommendations in multiple fields ranging from infectious disease to outbreak management and blood safety solutions.

Diagnostic accuracy of nucleic acid amplification tests (NAATs) in urine for genitourinary tuberculosis: a systematic review and meta-analysis.

PubMed

Altez-Fernandez, Carlos; Ortiz, Victor; Mirzazadeh, Majid; Zegarra, Luis; Seas, Carlos; Ugarte-Gil, Cesar

2017-06-05

Genitourinary tuberculosis is the third most common form of extrapulmonary tuberculosis. Diagnosis is difficult because of unspecific clinical manifestations and low accuracy of conventional tests. Unfortunately, the delayed diagnosis impacts the urinary tract severely. Nucleic acid amplification tests yield fast results, and among these, new technologies can also detect drug resistance. There is lack of consensus regarding the use of these tests in genitourinary tuberculosis; we therefore aimed to assess the accuracy of nucleic acid amplification tests in the diagnosis of genitourinary tuberculosis and to evaluate the heterogeneity between studies. We did a systematic review and meta-analysis of research articles comparing the accuracy of a reference standard and a nucleic acid amplification test for diagnosis of urinary tract tuberculosis. We searched Medline, EMBASE, Web of Science, LILACS, Cochrane Library, and Scopus for articles published between Jan 1, 1990, and Apr 14, 2016. Two investigators identified eligible articles and extracted data for individual study sites. We analyzed data in groups with the same index test. Then, we generated pooled summary estimates (95% CIs) for sensitivity and specificity by use of random-effects meta-analysis when studies were not heterogeneous. We identified eleven relevant studies from ten articles, giving information on PCR, LCR and Xpert MTB/RIF tests. All PCR studies were "in-house" tests, with different gene targets and had several quality concerns therefore we did not proceed with a pooled analysis. Only one study used LCR. Xpert studies were of good quality and not heterogeneous, pooled sensitivity was 0·87 (0·66-0·96) and specificity was 0·91 (0·84-0·95). PCR studies were highly heterogeneous. Among Xpert MTB/RIF studies, specificity was favorable with an acceptable confidence interval, however new studies can update meta-analysis and get more precise estimates. Further high-quality studies are urgently needed to improve diagnosis of genitourinary tuberculosis. PROSPERO CRD42016039020.

Rapid molecular assays for detection of tuberculosis.

PubMed

Eddabra, Rkia; Ait Benhassou, Hassan

2018-01-01

Tuberculosis (TB) is an infectious disease that remains an important public health problem at the global level. It is one of the main causes of morbidity and mortality, due to the emergence of antibiotic resistant Mycobacterium strains and HIV co-infection. Over the past decade, important progress has been made for better control of the disease. While microscopy and culture continue to be indispensible for laboratory diagnosis of tuberculosis, the range of several molecular diagnostic tests, including the nucleic acid amplification test (NAAT) and whole-genome sequencing (WGS), have expanded tremendously. They are becoming more accessible not only for detection and identification of Mycobacterium tuberculosis complex in clinical specimens, but now extend to diagnosing multi-drug resistant strains. Molecular diagnostic tests provide timely results useful for high-quality patient care, low contamination risk, and ease of performance and speed. This review focuses on the current diagnostic tests in use, including emerging technologies used for detection of tuberculosis in clinical specimens. The sensitivity and specificity of these tests have also been taken into consideration.

Molecular Diagnosis of Tuberculosis.

PubMed

Nurwidya, Fariz; Handayani, Diah; Burhan, Erlina; Yunus, Faisal

2018-01-01

Tuberculosis (TB) is one of the leading causes of adult death in the Asia-Pacific Region, including Indonesia. As an infectious disease caused by Mycobacterium tuberculosis (MTB), TB remains a major public health issue especially in developing nations due to the lack of adequate diagnostic testing facilities. Diagnosis of TB has entered an era of molecular detection that provides faster and more cost-effective methods to diagnose and confirm drug resistance in TB cases, meanwhile, diagnosis by conventional culture systems requires several weeks. New advances in the molecular detection of TB, including the faster and simpler nucleic acid amplification test (NAAT) and whole-genome sequencing (WGS), have resulted in a shorter time for diagnosis and, therefore, faster TB treatments. In this review, we explored the current findings on molecular diagnosis of TB and drug-resistant TB to see how this advancement could be integrated into public health systems in order to control TB.

Clinical Evaluation of the Cepheid Xpert TV Assay for Detection of Trichomonas vaginalis with Prospectively Collected Specimens from Men and Women

PubMed Central

Gaydos, C. A.; Davis, T.; Marrazzo, J.; Furgerson, D.; Taylor, S. N.; Smith, B.; Bachmann, L. H.; Ackerman, R.; Spurrell, T.; Ferris, D.; Burnham, C.-A. D.; Reno, H.; Lebed, J.; Eisenberg, D.; Kerndt, P.; Philip, S.; Jordan, J.; Quigley, N.

2017-01-01

ABSTRACT Trichomoniasis is the most prevalent curable sexually transmitted disease (STD). It has been associated with preterm birth and the acquisition and transmission of HIV. Recently, nucleic acid amplification tests (NAAT) have been FDA cleared in the United States for detection of Trichomonas vaginalis in specimens from both women and men. This study reports the results of a multicenter study recently conducted using the Xpert TV (T. vaginalis) assay to test specimens from both men and women. On-demand results were available in as little as 40 min for positive specimens. A total of 1,867 women and 4,791 men were eligible for inclusion in the analysis. In women, the performance of the Xpert TV assay was compared to the patient infected status (PIS) derived from the results of InPouch TV broth culture and Aptima NAAT for T. vaginalis. The diagnostic sensitivities and specificities of the Xpert TV assay for the combined female specimens (urine samples, self-collected vaginal swabs, and endocervical swabs) ranged from 99.5 to 100% and 99.4 to 99.9%, respectively. For male urine samples, the diagnostic sensitivity and specificity were 97.2% and 99.9%, respectively, compared to PIS results derived from the results of broth culture for T. vaginalis and bidirectional gene sequencing of amplicons. Excellent performance characteristics were seen using both female and male specimens. The ease of using the Xpert TV assay should result in opportunities for enhanced screening for T. vaginalis in both men and women and, hopefully, improved control of this infection. PMID:29167292

Evaluation of a Rapid and Completely Automated Real-Time Reverse Transcriptase PCR Assay for Diagnosis of Enteroviral Meningitis▿

PubMed Central

Nolte, Frederick S.; Rogers, Beverly B.; Tang, Yi-Wei; Oberste, M. Steven; Robinson, Christine C.; Kehl, K. Sue; Rand, Kenneth A.; Rotbart, Harley A.; Romero, Jose R.; Nyquist, Ann-Christine; Persing, David H.

2011-01-01

Nucleic acid amplification tests (NAATs) for enterovirus RNA in cerebrospinal fluid (CSF) have emerged as the new gold standard for diagnosis of enteroviral meningitis, and their use can improve the management and decrease the costs for caring for children with enteroviral meningitis. The Xpert EV assay (Cepheid, Sunnyvale, CA) is a rapid, fully automated real-time PCR test for the detection of enterovirus RNA that was approved by the U.S. Food and Drug Administration for in vitro diagnostic use in March 2007. In this multicenter trial we established the clinical performance characteristics of the Xpert EV assay in patients presenting with meningitis symptoms relative to clinical truth. Clinical truth for enteroviral meningitis was defined as clinical evidence of meningitis, the absence of another detectable pathogen in CSF, and detection of enterovirus in CSF either by two reference NAATs or by viral culture. A total of 199 prospectively and 235 retrospectively collected specimens were eligible for inclusion in this study. The overall prevalence of enteroviral meningitis was 26.04%. The Xpert EV assay had a sensitivity of 94.69% (90% confidence interval [CI] = 89.79 to 97.66%), specificity of 100% (90% CI = 99.07 to 100%), positive predictive value of 100%, negative predictive value of 98.17, and an accuracy of 98.62% relative to clinical truth. The Xpert EV assay demonstrated a high degree of accuracy for diagnosis of enteroviral meningitis. The simplicity and on-demand capability of the Xpert EV assay should prove to be a valuable adjunct to the evaluation of suspected meningitis cases. PMID:21159942

Evaluation of a rapid and completely automated real-time reverse transcriptase PCR assay for diagnosis of enteroviral meningitis.

PubMed

Nolte, Frederick S; Rogers, Beverly B; Tang, Yi-Wei; Oberste, M Steven; Robinson, Christine C; Kehl, K Sue; Rand, Kenneth A; Rotbart, Harley A; Romero, Jose R; Nyquist, Ann-Christine; Persing, David H

2011-02-01

Nucleic acid amplification tests (NAATs) for enterovirus RNA in cerebrospinal fluid (CSF) have emerged as the new gold standard for diagnosis of enteroviral meningitis, and their use can improve the management and decrease the costs for caring for children with enteroviral meningitis. The Xpert EV assay (Cepheid, Sunnyvale, CA) is a rapid, fully automated real-time PCR test for the detection of enterovirus RNA that was approved by the U.S. Food and Drug Administration for in vitro diagnostic use in March 2007. In this multicenter trial we established the clinical performance characteristics of the Xpert EV assay in patients presenting with meningitis symptoms relative to clinical truth. Clinical truth for enteroviral meningitis was defined as clinical evidence of meningitis, the absence of another detectable pathogen in CSF, and detection of enterovirus in CSF either by two reference NAATs or by viral culture. A total of 199 prospectively and 235 retrospectively collected specimens were eligible for inclusion in this study. The overall prevalence of enteroviral meningitis was 26.04%. The Xpert EV assay had a sensitivity of 94.69% (90% confidence interval [CI] = 89.79 to 97.66%), specificity of 100% (90% CI = 99.07 to 100%), positive predictive value of 100%, negative predictive value of 98.17, and an accuracy of 98.62% relative to clinical truth. The Xpert EV assay demonstrated a high degree of accuracy for diagnosis of enteroviral meningitis. The simplicity and on-demand capability of the Xpert EV assay should prove to be a valuable adjunct to the evaluation of suspected meningitis cases.

Characterisation of the nicotianamine aminotransferase and deoxymugineic acid synthase genes essential to Strategy II iron uptake in bread wheat (Triticum aestivum L.)

PubMed Central

Johnson, Alexander A. T.

2017-01-01

Iron (Fe) uptake in graminaceous plant species occurs via the release and uptake of Fe-chelating compounds known as mugineic acid family phytosiderophores (MAs). In the MAs biosynthetic pathway, nicotianamine aminotransferase (NAAT) and deoxymugineic acid synthase (DMAS) enzymes catalyse the formation of 2’-deoxymugineic acid (DMA) from nicotianamine (NA). Here we describe the identification and characterisation of six TaNAAT and three TaDMAS1 genes in bread wheat (Triticum aestivum L.). The coding sequences of all six TaNAAT homeologs consist of seven exons with ≥88.0% nucleotide sequence identity and most sequence variation present in the first exon. The coding sequences of the three TaDMAS1 homeologs consist of three exons with ≥97.8% nucleotide sequence identity. Phylogenetic analysis revealed that the TaNAAT and TaDMAS1 proteins are most closely related to the HvNAAT and HvDMAS1 proteins of barley and that there are two distinct groups of TaNAAT proteins—TaNAAT1 and TaNAAT2 –that correspond to the HvNAATA and HvNAATB proteins, respectively. Quantitative reverse transcription-PCR analysis revealed that the TaNAAT2 genes are expressed at highest levels in anther tissues whilst the TaNAAT1 and TaDMAS1 genes are expressed at highest levels in root tissues of bread wheat. Furthermore, the TaNAAT1, TaNAAT2 and TaDMAS1 genes were differentially regulated by plant Fe status and their expression was significantly upregulated in root tissues from day five onwards during a seven-day Fe deficiency treatment. The identification and characterization of the TaNAAT1, TaNAAT2 and TaDMAS1 genes provides a valuable genetic resource for improving bread wheat growth on Fe deficient soils and enhancing grain Fe nutrition. PMID:28475636

The levonorgestrel-releasing intrauterine system is associated with delayed endocervical clearance of Chlamydia trachomatis without alterations in vaginal microbiota.

PubMed

Liechty, Emma R; Bergin, Ingrid L; Bassis, Christine M; Chai, Daniel; LeBar, William; Young, Vincent B; Bell, Jason D

2015-11-01

Progestin-based contraception may impact women's susceptibility to sexually transmitted infection. We evaluated the effect of the levonorgestrel intrauterine system (LNG-IUS) on cervical persistence of Chlamydia trachomatis (CT) in a baboon model. Female olive baboons (Papio anubis) with or without an LNG-IUS received CT or sham inoculations. CT was detected in cervical epithelium with weekly nucleic acid amplification testing (NAAT) and culture. Presence of the LNG-IUS was associated with prolonged persistence of CT. Median time to post-inoculation clearance of CT as detected by NAAT was 10 weeks (range 7-12) for animals with an LNG-IUS and 3 weeks (range 0-12) for non-LNG-IUS animals (P = 0.06). Similarly, median time to post-inoculation clearance of CT by culture was 9 weeks (range 3-12) for LNG-IUS animals and 1.5 weeks (range 0-10) for non-LNG-IUS animals (P = 0.04). We characterized the community structure of the vaginal microbiota with the presence of the LNG-IUS to determine if alterations in CT colonization dynamics were associated with changes in vaginal commensal bacteria. Vaginal swabs were collected weekly for microbiome analysis. Endocervical CT infection was not correlated with alterations in the vaginal microbiota. Together, these results suggest that LNG-IUS may facilitate CT endocervical persistence through a mechanism distinct from vaginal microbial alterations. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The levonorgestrel-releasing intrauterine system is associated with delayed endocervical clearance of Chlamydia trachomatis without alterations in vaginal microbiota

PubMed Central

Liechty, Emma R.; Bergin, Ingrid L.; Bassis, Christine M.; Chai, Daniel; LeBar, William; Young, Vincent B.; Bell, Jason D.

2015-01-01

Progestin-based contraception may impact women's susceptibility to sexually transmitted infection. We evaluated the effect of the levonorgestrel intrauterine system (LNG-IUS) on cervical persistence of Chlamydia trachomatis (CT) in a baboon model. Female olive baboons (Papio anubis) with or without an LNG-IUS received CT or sham inoculations. CT was detected in cervical epithelium with weekly nucleic acid amplification testing (NAAT) and culture. Presence of the LNG-IUS was associated with prolonged persistence of CT. Median time to post-inoculation clearance of CT as detected by NAAT was 10 weeks (range 7–12) for animals with an LNG-IUS and 3 weeks (range 0–12) for non-LNG-IUS animals (P = 0.06). Similarly, median time to post-inoculation clearance of CT by culture was 9 weeks (range 3–12) for LNG-IUS animals and 1.5 weeks (range 0–10) for non-LNG-IUS animals (P = 0.04). We characterized the community structure of the vaginal microbiota with the presence of the LNG-IUS to determine if alterations in CT colonization dynamics were associated with changes in vaginal commensal bacteria. Vaginal swabs were collected weekly for microbiome analysis. Endocervical CT infection was not correlated with alterations in the vaginal microbiota. Together, these results suggest that LNG-IUS may facilitate CT endocervical persistence through a mechanism distinct from vaginal microbial alterations. PMID:26371177

Occupational HIV Transmission Among Male Adult Film Performers - Multiple States, 2014.

PubMed

Wilken, Jason A; Ried, Christopher; Rickett, Pristeen; Arno, Janet N; Mendez, Yesenia; Harrison, Robert J; Wohlfeiler, Dan; Bauer, Heidi M; Joyce, M Patricia; Switzer, William M; Heneine, Walid; Shankar, Anupama; Mark, Karen E

2016-02-12

In 2014, the California Department of Public Health was notified by a local health department of a diagnosis of acute human immunodeficiency virus (HIV) infection* and rectal gonorrhea in a male adult film industry performer, aged 25 years (patient A). Patient A had a 6-day history of rash, fever, and sore throat suggestive of acute retroviral syndrome at the time of examination. He was informed of his positive HIV and gonorrhea test results 6 days after his examination. Patient A had a negative HIV-1 RNA qualitative nucleic acid amplification test (NAAT)(†) 10 days before symptom onset. This investigation found that during the 22 days between the negative NAAT and being informed of his positive HIV test results, two different production companies directed patient A to have condomless sex with a total of 12 male performers. Patient A also provided contact information for five male non-work-related sexual partners during the month before and after his symptom onset. Patient A had additional partners during this time period for which no locating information was provided. Neither patient A nor any of his interviewed sexual partners reported taking HIV preexposure prophylaxis (PrEP). Contact tracing and phylogenetic analysis of HIV sequences amplified from pretreatment plasma revealed that a non-work-related partner likely infected patient A, and that patient A likely subsequently infected both a coworker during the second film production and a non-work-related partner during the interval between his negative test and receipt of his positive HIV results. Adult film performers and production companies, medical providers, and all persons at risk for HIV should be aware that testing alone is not sufficient to prevent HIV transmission. Condom use provides additional protection from HIV and sexually transmitted infections (STIs). Performers and all persons at risk for HIV infection in their professional and personal lives should discuss the use of PrEP with their medical providers.

Will targeting oropharyngeal gonorrhoea delay the further emergence of drug-resistant Neisseria gonorrhoeae strains?

PubMed

Lewis, D A

2015-06-01

Gonorrhoea is an important sexually transmitted infection associated with serious complications and enhanced HIV transmission. Oropharyngeal infections are often asymptomatic and will only be detected by screening. Gonococcal culture has low sensitivity (<50%) for detecting oropharyngeal gonorrhoea, and, although not yet approved commercially, nucleic acid amplification tests (NAAT) are the assay of choice. Screening for oropharyngeal gonorrhoea should be performed in high-risk populations, such as men-who-have-sex-with-men(MSM). NAATs have a poor positive predictive value when used in low-prevalence populations. Gonococci have repeatedly thwarted gonorrhoea control efforts since the first antimicrobial agents were introduced. The oropharyngeal niche provides an enabling environment for horizontal transfer of genetic material from commensal Neisseria and other bacterial species to Neisseria gonorrhoeae. This has been the mechanism responsible for the generation of mosaic penA genes, which are responsible for most of the observed cases of resistance to extended-spectrum cephalosporins (ESC). As antimicrobial-resistant gonorrhoea is now an urgent public health threat, requiring improved antibiotic stewardship, laboratory-guided recycling of older antibiotics may help reduce ESC use. Future trials of antimicrobial agents for gonorrhoea should be powered to test their efficacy at the oropharynx as this is the anatomical site where treatment failure is most likely to occur. It remains to be determined whether a combination of frequent screening of high-risk individuals and/or laboratory-directed fluoroquinolone therapy of oropharyngeal gonorrhoea will delay the further emergence of drug-resistant N. gonorrhoeae strains. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Clinical Evaluation of the Cepheid Xpert TV Assay for Detection of Trichomonas vaginalis with Prospectively Collected Specimens from Men and Women.

PubMed

Schwebke, Jane R; Gaydos, C A; Davis, T; Marrazzo, J; Furgerson, D; Taylor, S N; Smith, B; Bachmann, L H; Ackerman, R; Spurrell, T; Ferris, D; Burnham, C A; Reno, H; Lebed, J; Eisenberg, D; Kerndt, P; Philip, S; Jordan, J; Quigley, N

2018-02-01

Trichomoniasis is the most prevalent curable sexually transmitted disease (STD). It has been associated with preterm birth and the acquisition and transmission of HIV. Recently, nucleic acid amplification tests (NAAT) have been FDA cleared in the United States for detection of Trichomonas vaginalis in specimens from both women and men. This study reports the results of a multicenter study recently conducted using the Xpert TV ( T. vaginalis ) assay to test specimens from both men and women. On-demand results were available in as little as 40 min for positive specimens. A total of 1,867 women and 4,791 men were eligible for inclusion in the analysis. In women, the performance of the Xpert TV assay was compared to the patient infected status (PIS) derived from the results of InPouch TV broth culture and Aptima NAAT for T. vaginalis The diagnostic sensitivities and specificities of the Xpert TV assay for the combined female specimens (urine samples, self-collected vaginal swabs, and endocervical swabs) ranged from 99.5 to 100% and 99.4 to 99.9%, respectively. For male urine samples, the diagnostic sensitivity and specificity were 97.2% and 99.9%, respectively, compared to PIS results derived from the results of broth culture for T. vaginalis and bidirectional gene sequencing of amplicons. Excellent performance characteristics were seen using both female and male specimens. The ease of using the Xpert TV assay should result in opportunities for enhanced screening for T. vaginalis in both men and women and, hopefully, improved control of this infection. Copyright © 2018 Schwebke et al.

Persistence after treatment of pharyngeal gonococcal infections in patients of the STI clinic, Amsterdam, the Netherlands, 2012–2015: a retrospective cohort study

PubMed Central

De Vries, Henry John Christiaan; van Dam, Alje P; van Rooijen, Martijn Sebastiaan; Soebono, Hardyanto; Schim van der Loeff, Maarten Franciscus

2017-01-01

Introduction Infection of Neisseria gonorrhoeae in the pharynx (pharyngeal Ng) is associated with gonococcal transmission and development of antimicrobial resistance. We assessed proportion of and determinants for persistence after treatment of pharyngeal Ng. Methods At the STI clinic of Amsterdam, the Netherlands, females-at-risk and men who have sex with men are routinely screened for pharyngeal Ng using an RNA-based nucleic acid amplification test (NAAT; Aptima Combo 2). Patients with pharyngeal Ng were invited for a test-of-cure (TOC) 7 days after treatment with a 500 mg ceftriaxone intramuscularly. We retrospectively examined medical records of patients with pharyngeal Ng (January 2012–August 2015) who returned for a TOC 7–28 days after treatment. Persistence was defined as a positive NAAT at TOC. Results Out of 2204 pharyngeal Ng cases recorded in the study period, 781 cases (median time between first treatment and TOC of 8 (IQR 7–12) days) were included in the analysis. Persistence after treatment was found in 36 (4.6%) and was less likely among patients who received ceftriaxone in combination with other antibiotics (vs monotherapy) (adjusted OR (aOR) 0.36, 95% CI 0.12 to 1.04) and with longer time from treatment to TOC (aOR 0.74, 95% CI 0.60 to 0.90, per extra day). In those with a TOC 15–28 days after treatment, Ng persisted in only 1.0% (1/105 cases). Conclusion A small proportion of pharyngeal Ng persists despite appropriate treatment. Combining ceftriaxone with other antibiotics appears to lead to faster clearance. A TOC for pharyngeal Ng 7 days after treatment may be too soon. PMID:28822976

Determinants of Clostridium difficile Infection Incidence Across Diverse United States Geographic Locations

PubMed Central

Lessa, Fernanda C.; Mu, Yi; Winston, Lisa G.; Dumyati, Ghinwa K.; Farley, Monica M.; Beldavs, Zintars G.; Kast, Kelly; Holzbauer, Stacy M.; Meek, James I.; Cohen, Jessica; McDonald, L. Clifford; Fridkin, Scott K.

2014-01-01

Background  Clostridium difficile infection (CDI) is no longer restricted to hospital settings, and population-based incidence measures are needed. Understanding the determinants of CDI incidence will allow for more meaningful comparisons of rates and accurate national estimates. Methods  Data from active population- and laboratory-based CDI surveillance in 7 US states were used to identify CDI cases (ie, residents with positive C difficile stool specimen without a positive test in the prior 8 weeks). Cases were classified as community-associated (CA) if stool was collected as outpatients or ≤3 days of admission and no overnight healthcare facility stay in the past 12 weeks; otherwise, cases were classified as healthcare-associated (HA). Two regression models, one for CA-CDI and another for HA-CDI, were built to evaluate predictors of high CDI incidence. Site-specific incidence was adjusted based on the regression models. Results  Of 10 062 cases identified, 32% were CA. Crude incidence varied by geographic area; CA-CDI ranged from 28.2 to 79.1/100 000 and HA-CDI ranged from 45.7 to 155.9/100 000. Independent predictors of higher CA-CDI incidence were older age, white race, female gender, and nucleic acid amplification test (NAAT) use. For HA-CDI, older age and a greater number of inpatient-days were predictors. After adjusting for relevant predictors, the range of incidence narrowed greatly; CA-CDI rates ranged from 30.7 to 41.3/100 000 and HA-CDI rates ranged from 58.5 to 94.8/100 000. Conclusions  Differences in CDI incidence across geographic areas can be partially explained by differences in NAAT use, age, race, sex, and inpatient-days. Variation in antimicrobial use may contribute to the remaining differences in incidence. PMID:25734120

Determinants of Clostridium difficile Infection Incidence Across Diverse United States Geographic Locations.

PubMed

Lessa, Fernanda C; Mu, Yi; Winston, Lisa G; Dumyati, Ghinwa K; Farley, Monica M; Beldavs, Zintars G; Kast, Kelly; Holzbauer, Stacy M; Meek, James I; Cohen, Jessica; McDonald, L Clifford; Fridkin, Scott K

2014-09-01

Clostridium difficile infection (CDI) is no longer restricted to hospital settings, and population-based incidence measures are needed. Understanding the determinants of CDI incidence will allow for more meaningful comparisons of rates and accurate national estimates. Data from active population- and laboratory-based CDI surveillance in 7 US states were used to identify CDI cases (ie, residents with positive C difficile stool specimen without a positive test in the prior 8 weeks). Cases were classified as community-associated (CA) if stool was collected as outpatients or ≤3 days of admission and no overnight healthcare facility stay in the past 12 weeks; otherwise, cases were classified as healthcare-associated (HA). Two regression models, one for CA-CDI and another for HA-CDI, were built to evaluate predictors of high CDI incidence. Site-specific incidence was adjusted based on the regression models. Of 10 062 cases identified, 32% were CA. Crude incidence varied by geographic area; CA-CDI ranged from 28.2 to 79.1/100 000 and HA-CDI ranged from 45.7 to 155.9/100 000. Independent predictors of higher CA-CDI incidence were older age, white race, female gender, and nucleic acid amplification test (NAAT) use. For HA-CDI, older age and a greater number of inpatient-days were predictors. After adjusting for relevant predictors, the range of incidence narrowed greatly; CA-CDI rates ranged from 30.7 to 41.3/100 000 and HA-CDI rates ranged from 58.5 to 94.8/100 000. Differences in CDI incidence across geographic areas can be partially explained by differences in NAAT use, age, race, sex, and inpatient-days. Variation in antimicrobial use may contribute to the remaining differences in incidence.

[Clinical application of testing methods on acid-fast bacteria].

PubMed

Ichiyama, Satoshi; Suzuki, Katsuhiro

2005-02-01

Clinical bacteriology pertaining to acid-fast bacteria has made marked advances over the past decade, initiated by the development of a DNA probe kit for identification of acid-fast bacteria. Wide-spread use of nucleic acid amplification for rapid detection of tubercle bacillus contributed more greatly than any other factor to such advances in this field. At present, 90% of all kits used for nucleic acid amplification in the world are consumed in Japan. Unfortunately, not a few clinicians in Japan have a false idea that the smear method and nucleic acid amplification are necessary but culture is not. In any event nucleic acid amplification has exerted significant impacts on the routine works at bacteriology laboratories. Among others, collecting bacteria by pretreatment with NALC-NaOH has simplified the introduction of the collective mode smear method and liquid media. Furthermore, as clinicians have become increasingly more experienced with various methods of molecular biology, it now seems possible to apply these techniques for detection of genes encoding drug resistance and for utilization of molecular epidemiology in routine laboratory works. Meanwhile, attempts to diagnose acid-fast bacteriosis by checking blood for antibody have also been made, primarily in Japan. At present, two kits for detecting antibodies to glycolipids (LAM, TDM, etc.) are covered by national health insurance in Japan. We have an impression that in Japan clinicians do not have adequate knowledge and skill to make full use of these new testing methods clinically. We, as the chairmen of this symposium, hope that this symposium will help clinicians increase their skill related to new testing methods, eventually leading to stimulation of advances in clinical practices related to acid-fast bacteria in Japan. 1. Smear microscopy by concentration method and broth culture system: Kazunari TSUYUGUCHI (Clinical Research Center, National Hospital Organization Kinki-chuo Chest Medical Center) Smear microscopy and culture still remain the cornerstone to diagnose tuberculosis. However, the classical methods in Japan using direct microscopy and Ogawa solid media were not sufficient for clinical use. In recent years substantial advance has been made in these fields. Concentration of clinical samples by centrifugation improves the sensitivity of smear microscopy with excellent reproducibility. The Mycobacteria Growth Indicator Tube (MGIT) system using liquid media yields high sensitivity and rapidity. Using these methods, more and more tuberculosis cases would be correctly diagnosed and treated adequately based on drug susceptibility testing. 2. New technologies for anti-tuberculosis drug susceptibility testing: Satoshi MITARAI (Bacteriology Division, Reference Centre for Mycobacterium, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association) Several new technologies have been developed to obtain anti-tuberculosis drug susceptibility testing (AST) results rapidly, utilising liquid culture and molecular technologies. Mycobacterium Growth Indicator Tube (MGIT), as a popular liquid culturing and AST system, was evaluated for its accuracy and usefulness. As for isoniazid, MGIT showed 12.6% of discordant result comparing with standard method. These MGIT resistant and Ogawa susceptible strains had relatively high MICs ranging 0.13 to 2.0 microg/ml. The molecular detection of resistant gene mutation is also a useful method to estimate drug resistance rapidly. The rpoB mutation detection is reliable with high sensitivity and specificity. 3. Nucleic acid amplification and novel diagnostic methods: Shunji TAKAKURA (Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine) Sensitivities of nucleic acid amplification tests (NAATs) for the diagnosis of tuberculosis meet clinical requirement that patients with high-risk of transmission should be identified within a day. Comparison of the performance of various NAATs is difficult because of the difference in sample processing and in samples tested among methods and reports. Considering the limitations of NAATs (low sensitivity compared with culture, inability to differentiate dead bacilli from the living), further advances would be expected when novel technologies could confer additional information, such as drug susceptibility, quantity, viability, and genotype. 4. Serodiagnosis of Mycobacterium avium complex lung disease: Seigo KITADA (Department of Internal Medicine, National Hospital Organization Toneyama National Hospital) Mycobacterium avium complex (MAC) organisms are ubiquitous in environment and a contamination in respiratory tracts is sometimes observed, and that complex the diagnosis. We developed a serodiagnostic method for MAC disease using an enzyme immunoassay with the MAC-specific glycopeptidolipid (GPL) core as antigen. A significant increase in GPL core antibodies was detected in sera of patients with MAC pulmonary diseases compared to patients who were colonized with MAC, patients with M. kansasii disease and tuberculosis and healthy subjects. The serodiagnosis is useful for diagnosis of MAC lung disease. 5. Molecular epidemiologic tools for tuberculosis: IS6110 RFLP, Spoligotyping, and VNTR: Tomoshige MATSUMOTO, Hiromi ANO, Tetsuya TAKASHIMA, Izuo TSUYUGUCHI (Osaka Prefectural Medical Center for Respiratory and Allergic Diseases) We have performed molecular typing on about 1,300 culture positive clinical isolates that made up the majority of tuberculosis strains in part of southeast Osaka since 2001 until now. By spoligotyping, about 75% of entire strains belonged to the Beijing strain. Particular spoligotyping descriptions, which were not described in SpolDBIII, were found in the strains with lower than 6 copies of IS6110 RFLP. We described them as Osaka type. We could also show that direct typing from Tb PCR positive sputum of patients with tuberculosis was possible by VNTR and that VNTR with 16 loci was useful in tuberculosis typing in Osaka.

[Laboratory diagnosis of lymphocytic meningitis].

PubMed

Marí, José María Navarro; Ruiz, Mercedes Pérez; Anza, Diego Vicente

2010-01-01

Lymphocytic meningitis, mainly those with an acute and benign course, are caused by viruses. In our area, the most commonly involved agents are enteroviruses, herpes simplex, varicella zoster and Toscana viruses. Nucleic acids amplification techniques (NAAT) are the methods of choice to diagnose viral meningitis from cerebrospinal fluid (CSF) samples. They are more rapid and sensitive, and indeed, they are not influenced by the viability of the virus in the clinical specimen as traditional methods are. The development of commercial equipments, the degree of automation, and the use of real-time polymerase chain reaction (PCR) systems are the most important premises to choose the molecular method in each laboratory. Recently, commercial kits of real-time PCR are available for the detection of enteroviruses and herpesviruses, which are the most frequently viruses involved in meningitis. Although NAAT from the clinical sample have replaced cell culture for diagnostic purposes, the combination of both methods remain useful. When the detection of the causal agent from the CSF sample is not possible, other specimens (pharyngeal exudates, stools) or serological methods can be used. Serology is the reference method for meningitis caused by West Nile virus and lymphocytic choriomeningitis virus, which are less frequently detected in our area. 2010 Elsevier España S.L. All rights reserved.

Evaluation of the Architect HIV Ag/Ab Combo Assay in a low-prevalence setting: The role of samples with a low S/CO ratio.

PubMed

Alonso, Roberto; Pérez-García, Felipe; Gijón, Paloma; Collazos, Ana; Bouza, Emilio

2018-06-01

The Architect HIV Ag/Ab Combo Assay, a fourth-generation ELISA, has proven to be highly reliable for the diagnosis of HIV infection. However, its high sensitivity may lead to false-positive results. To evaluate the diagnostic performance of Architect in a low-prevalence population and to assess the role of the sample-to-cutoff ratio (S/CO) in reducing the frequency of false-positive results. We conducted a retrospective study of samples analyzed by Architect between January 2015 and June 2017. Positive samples were confirmed by immunoblot (RIBA) or nucleic acid amplification tests (NAATs). Different S/CO thresholds (1, 2.5, 10, 25, and 100) were analyzed to determine sensitivity, specificity, and negative and positive predictive values (NPV, PPV). ROC analysis was used to determine the optimal S/CO. A total of 69,471 samples were analyzed. 709 (1.02%) were positive by Architect. Of these, 63 (8.89%) were false-positive results. Most of them (93.65%) were in samples with S/CO < 100. However, most confirmations by NAATs (12 out of 19 cases) were also recorded for these samples. The optimal S/CO was 2.5, which provided the highest area under the ROC curve (0.9998) and no false-negative results. With this S/CO, sensitivity and specificity were 100.0%, and PPV and NPV were 95.8% and 100.0%, respectively. In addition, the frequency of false-positive results decreased significantly to 4.15%. Although Architect generates a relatively high number of false-positive results, raising the S/CO limit too much to increase specificity can lead to false-negative results, especially in newly infected individuals. Copyright © 2018 Elsevier B.V. All rights reserved.

Trachoma Rapid Assessments in Unity and Northern Bahr-el-Ghazal States, Southern Sudan

PubMed Central

Robinson, Emily; Kur, Lucia W.; Ndyaba, Aggrey; Lado, Mounir; Shafi, Juma; Kabare, Emmanuel; McClelland, R. Scott; Kolaczinski, Jan H.

2010-01-01

Background Trachoma is thought to be endemic over large parts of Southern Sudan, but empirical evidence is limited. While some areas east of the Nile have been identified as highly endemic, few trachoma surveys have been conducted in the remainder of the country. This study aimed to determine whether trachoma constitutes a problem to public health in Northern Bahr-el-Ghazal and Unity State, both located west of the Nile. Methods and Principal Findings Trachoma rapid assessments (TRA) were conducted between July and September 2009. Seven villages in Northern Bahr-el-Ghazal State and 13 villages in Unity State were surveyed; an average of 50 children (age 1–9 years) and 44 women (age 15 years and above) were examined per village. Samples for analysis using the APTIMA Combo-2 nucleic acid amplification test (NAAT) were collected from participants with active trachoma in eight villages in Unity State. In Northern Bahr-el-Ghazal State, only three children with active trachoma (trachomatous inflammation follicular (TF) and/or trachomatous inflammation intense (TI)) and two women with trichiasis (TT) were found, in two of the seven villages surveyed. In Unity State, trachoma was endemic in all thirteen villages surveyed; the proportion of children with active trachoma ranged from 33% to 75% between villages, while TF in children ranged from 16% to 44%. Between 4% to 51% of examined women showed signs of TT. Samples from active trachoma cases tested using the NAAT were positive for Chlamydia trachomatis infection for 46.6% of children and 19.0% of women. Conclusions Trachoma presents a major problem to public health Unity State, while the disease is of low priority in Northern-Bahr-el-Ghazal State. Implementation of a population-based prevalence survey is now required in Unity State to generate baseline prevalence data so that trachoma interventions can be initiated and monitored over time. PMID:20957205

Persistence after treatment of pharyngeal gonococcal infections in patients of the STI clinic, Amsterdam, the Netherlands, 2012-2015: a retrospective cohort study.

PubMed

Hananta, I Putu Yuda; De Vries, Henry John Christiaan; van Dam, Alje P; van Rooijen, Martijn Sebastiaan; Soebono, Hardyanto; Schim van der Loeff, Maarten Franciscus

2017-11-01

Infection of Neisseria gonorrhoeae in the pharynx (pharyngeal Ng) is associated with gonococcal transmission and development of antimicrobial resistance. We assessed proportion of and determinants for persistence after treatment of pharyngeal Ng. At the STI clinic of Amsterdam, the Netherlands, females-at-risk and men who have sex with men are routinely screened for pharyngeal Ng using an RNA-based nucleic acid amplification test (NAAT; Aptima Combo 2). Patients with pharyngeal Ng were invited for a test-of-cure (TOC) 7 days after treatment with a 500 mg ceftriaxone intramuscularly. We retrospectively examined medical records of patients with pharyngeal Ng (January 2012-August 2015) who returned for a TOC 7-28 days after treatment. Persistence was defined as a positive NAAT at TOC. Out of 2204 pharyngeal Ng cases recorded in the study period, 781 cases (median time between first treatment and TOC of 8 (IQR 7-12) days) were included in the analysis. Persistence after treatment was found in 36 (4.6%) and was less likely among patients who received ceftriaxone in combination with other antibiotics (vs monotherapy) (adjusted OR (aOR) 0.36, 95% CI 0.12 to 1.04) and with longer time from treatment to TOC (aOR 0.74, 95% CI 0.60 to 0.90, per extra day). In those with a TOC 15-28 days after treatment, Ng persisted in only 1.0% (1/105 cases). A small proportion of pharyngeal Ng persists despite appropriate treatment. Combining ceftriaxone with other antibiotics appears to lead to faster clearance. A TOC for pharyngeal Ng 7 days after treatment may be too soon. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Trachoma rapid assessments in Unity and Northern Bahr-el-Ghazal States, Southern Sudan.

PubMed

Robinson, Emily; Kur, Lucia W; Ndyaba, Aggrey; Lado, Mounir; Shafi, Juma; Kabare, Emmanuel; McClelland, R Scott; Kolaczinski, Jan H

2010-10-01

Trachoma is thought to be endemic over large parts of Southern Sudan, but empirical evidence is limited. While some areas east of the Nile have been identified as highly endemic, few trachoma surveys have been conducted in the remainder of the country. This study aimed to determine whether trachoma constitutes a problem to public health in Northern Bahr-el-Ghazal and Unity State, both located west of the Nile. Trachoma rapid assessments (TRA) were conducted between July and September 2009. Seven villages in Northern Bahr-el-Ghazal State and 13 villages in Unity State were surveyed; an average of 50 children (age 1-9 years) and 44 women (age 15 years and above) were examined per village. Samples for analysis using the APTIMA Combo-2 nucleic acid amplification test (NAAT) were collected from participants with active trachoma in eight villages in Unity State. In Northern Bahr-el-Ghazal State, only three children with active trachoma (trachomatous inflammation follicular (TF) and/or trachomatous inflammation intense (TI)) and two women with trichiasis (TT) were found, in two of the seven villages surveyed. In Unity State, trachoma was endemic in all thirteen villages surveyed; the proportion of children with active trachoma ranged from 33% to 75% between villages, while TF in children ranged from 16% to 44%. Between 4% to 51% of examined women showed signs of TT. Samples from active trachoma cases tested using the NAAT were positive for Chlamydia trachomatis infection for 46.6% of children and 19.0% of women. Trachoma presents a major problem to public health Unity State, while the disease is of low priority in Northern-Bahr-el-Ghazal State. Implementation of a population-based prevalence survey is now required in Unity State to generate baseline prevalence data so that trachoma interventions can be initiated and monitored over time.

Detection of gonococcal infection : pros and cons of a rapid test.

PubMed

Vickerman, Peter; Peeling, Rosanna W; Watts, Charlotte; Mabey, David

2005-01-01

WHO estimates that 62 million cases of gonorrhea occur annually worldwide. Untreated infection can cause serious long-term complications, especially in women. In addition, Neisseria gonorrheae infection can facilitate HIV transmission, and babies born to infected mothers are at risk of ocular infection, which can lead to blindness. Where diagnostic facilities are lacking, gonorrhea can be treated syndromically. However, this inevitably leads to over-treatment, especially in women in whom the syndrome of vaginal discharge may be due not to N. gonorrheae infection but to several other more prevalent conditions. Over-treatment is a major concern because of widespread N. gonorrheae antibiotic resistance. Moreover, a high proportion of gonorrhea cases are asymptomatic and so do not present for syndromic management. Such cases will only be detected by screening tests. The gold standard test for the detection of N. gonorrheae is culture, which has high sensitivity and specificity. However, it requires well trained staff and its performance is affected by specimen transport conditions. Other options include microscopy and tests that detect gonococcal antigen or nucleic acid. Nucleic acid amplification tests (NAATs) have higher sensitivity and can be used on non-invasive samples (urine). However, they can cross-react with other Neisseria species and are expensive, requiring highly trained staff and sophisticated equipment. In settings where patients are asked to return for laboratory results, some infected patients never receive treatment as they fail to return for their test results. This reduction in treatment, and the possible onward transmission of N. gonorrheae during any delay in treatment, means that a rapid test of lower sensitivity may be more effective if it results in patients being treated at the initial visit. Indeed, even with the low sensitivity of currently available rapid tests (50-70%), modeling shows that they can outperform gold standard tests in populations with high sexual activity and/or low return rates. Unfortunately, however, most of the rapid tests currently available are immunoassays that are quite expensive and involve many steps, which limit their current usefulness. In summary, the pros and cons of using a rapid test are dependent on the setting. Culture or NAATs remain the best choice in an ideal setting. However, in settings where laboratory facilities are not available, or in high-risk populations where return rates are low, rapid tests may be the most effective way of diagnosing gonorrhea. Their optimal use in these settings requires the development of simpler and cheaper rapid tests.

Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes.

PubMed

Gadkar, Vijay J; Goldfarb, David M; Gantt, Soren; Tilley, Peter A G

2018-04-03

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification (iNAAT) technique known for its simplicity, sensitivity and speed. Its low-cost feature has resulted in its wide scale application, especially in low resource settings. The major disadvantage of LAMP is its heavy reliance on indirect detection methods like turbidity and non-specific dyes, which often leads to the detection of false positive results. In the present work, we have developed a direct detection approach, whereby a labelled loop probe quenched in its unbound state, fluoresces only when bound to its target (amplicon). Henceforth, referred to as Fluorescence of Loop Primer Upon Self Dequenching-LAMP (FLOS-LAMP), it allows for the sequence-specific detection of LAMP amplicons. The FLOS-LAMP concept was validated for rapid detection of the human pathogen, Varicella-zoster virus, from clinical samples. The FLOS-LAMP had a limit of detection of 500 copies of the target with a clinical sensitivity and specificity of 96.8% and 100%, respectively. The high level of specificity is a major advance and solves one of the main shortcomings of the LAMP technology, i.e. false positives. Self-quenching/de-quenching probes were further used with other LAMP primer sets and different fluorophores, thereby demonstrating its versatility and adaptability.

Design of the FemCure study: prospective multicentre study on the transmission of genital and extra-genital Chlamydia trachomatis infections in women receiving routine care.

PubMed

Dukers-Muijrers, Nicole H T M; Wolffs, Petra F G; Eppings, Lisanne; Götz, Hannelore M; Bruisten, Sylvia M; Schim van der Loeff, Maarten F; Janssen, Kevin; Lucchesi, Mayk; Heijman, Titia; van Benthem, Birgit H; van Bergen, Jan E; Morre, Servaas A; Herbergs, Jos; Kok, Gerjo; Steenbakkers, Mieke; Hogewoning, Arjan A; de Vries, Henry J; Hoebe, Christian J P A

2016-08-08

In women, anorectal infections with Chlamydia trachomatis (CT) are about as common as genital CT, yet the anorectal site remains largely untested in routine care. Anorectal CT frequently co-occurs with genital CT and may thus often be treated co-incidentally. Nevertheless, post-treatment detection of CT at both anatomic sites has been demonstrated. It is unknown whether anorectal CT may play a role in post-treatment transmission. This study, called FemCure, in women who receive routine treatment (either azithromycin or doxycycline) aims to understand the post-treatment transmission of anorectal CT infections, i.e., from their male sexual partner(s) and from and to the genital region of the same woman. The secondary objective is to evaluate other reasons for CT detection by nucleic acid amplification techniques (NAAT) such as treatment failure, in order to inform guidelines to optimize CT control. A multicentre prospective cohort study (FemCure) is set up in which genital and/or anorectal CT positive women (n = 400) will be recruited at three large Dutch STI clinics located in South Limburg, Amsterdam and Rotterdam. The women self-collect anorectal and vaginal swabs before treatment, and at the end of weeks 1, 2, 4, 6, 8, 10, and 12. Samples are tested for presence of CT-DNA (by NAAT), load (by quantitative polymerase chain reaction -PCR), viability (by culture and viability PCR) and CT type (by multilocus sequence typing). Sexual exposure is assessed by online self-administered questionnaires and by testing samples for Y chromosomal DNA. Using logistic regression models, the impact of two key factors (i.e., sexual exposure and alternate anatomic site of infection) on detection of anorectal and genital CT will be assessed. The FemCure study will provide insight in the role of anorectal chlamydia infection in maintaining the CT burden in the context of treatment, and it will provide practical recommendations to reduce avoidable transmission. Implications will improve care strategies that take account of anorectal CT. ClinicalTrials.gov Identifier: NCT02694497 .

Nanoporous array anodic titanium-supported co-polymeric ionic liquids as high performance solid-phase microextraction sorbents for hydrogen bonding compounds.

PubMed

Jia, Jing; Liang, Xiaojing; Wang, Licheng; Guo, Yong; Liu, Xia; Jiang, Shengxiang

2013-12-13

A nanoporous array anodic titanium-supported co-polymeric ionic liquids (NAAT/PILs) solid-phase microextraction (SPME) fiber was prepared in situ on the titanium wire. NAAT was selected as the substrate, in view of its high surface-to-volume ratio, easy preparation, mechanical stability, and rich titanol groups on its surface which can anchor silica coupling agent containing vinyl and then introduce ionic liquid copolymers as sorbents. In this work, 1-vinyl-3-nonanol imidazolium bromide ([C9OHVIm]Br) and 1,4-di(3-vinylimidazolium) butane dibromide ([(VIM)2C4]2[Br]) were synthesized and used as monomer and crosslinker, respectively. Extraction properties of the NAAT/PILs fiber for polar alcohols and volatile fatty acids (VFAs) in aqueous matrix were examined using gaseous sampling-SPME (GS-SPME) and headspace SPME (HS-SPME) mode, respectively. Combining the superior properties of NAAT substrate and the strong hydrogen bond interaction of PILs to polar compounds, the NAAT/PILs SPME fiber showed much higher adsorption affinity to aliphatic alcohols than bare NAAT and pure PILs fibers. The detection limits (LOD) of established GS-SPME-GC-FID method are in the range of 0.35-17.30ngL(-1) with a linear range from 0.01 to 500ngmL(-1). Also, it showed high extraction performance toward volatile fatty acids (VFAs) compounds from aqueous matrix. Under the optimized SPME conditions, wide linear ranges were obtained with correlation coefficients (R(2)) greater than 0.99 and limits of detection were in the range of 0.85-8.74ngL(-1). Moreover, real-world samples were analyzed and good results were obtained. Copyright © 2013. Published by Elsevier B.V.

The effect of epileptic seizures on proton MRS visible neurochemical concentrations.

PubMed

Simister, Robert J; McLean, Mary A; Salmenpera, Tuuli M; Barker, Gareth J; Duncan, John S

2008-09-01

To investigate post-ictal changes in cerebral metabolites. We performed a longitudinal quantitative proton magnetic resonance spectroscopy (MRS) study in 10 patients with epilepsy and 10 control subjects. The patients were studied on two occasions: immediately following a seizure, and on a second occasion at least 7h after the most recent seizure. Each study measured N-acetyl aspartate plus N-acetyl aspartyl glutamate (NAAt), Creatine plus phosphocreatine (Cr), Choline containing compounds (Cho) and glutamate plus glutamine (GLX) concentrations using a short-echo time sequence (TE=30ms), and NAAt, Cr and lactate using a second sequence with longer echo time (TE=144ms). The control group was studied on two occasions using the same sequences. No inter-scan differences were observed for the control group. NAAt and NAAt/Cr levels were lower in the patient group at both measured TEs but did not change significantly between studies. The ratio of Cr at TE 144ms to TE 30ms (Cr(144)/Cr(30)) and GLX/Cr were higher and Cho lower in the post-ictal scan compared to the inter-ictal study. Change in Cr(144)/Cr(30) and NAAt(144)/Cr(144) correlated with the post-ictal interval. Lactate measurement at longer TE was not informative. Proton MRS is sensitive to metabolite changes following epileptic seizures within the immediate post-ictal period. The ratio Cr(144)/Cr(30) is the most sensitive measure of metabolic disturbance and is highest in the post-ictal period but appears to normalise within 2h of the most recent seizure.

Comparison of 4th-Generation HIV Antigen/Antibody Combination Assay With 3rd-Generation HIV Antibody Assays for the Occurrence of False-Positive and False-Negative Results.

PubMed

Muthukumar, Alagarraju; Alatoom, Adnan; Burns, Susan; Ashmore, Jerry; Kim, Anne; Emerson, Brian; Bannister, Edward; Ansari, M Qasim

2015-01-01

To assess the false-positive and false-negative rates of a 4th-generation human immunodeficiency virus (HIV) assay, the Abbott ARCHITECT, vs 2 HIV 3rd-generation assays, the Siemens Centaur and the Ortho-Clinical Diagnostics Vitros. We examined 123 patient specimens. In the first phase of the study, we compared 99 specimens that had a positive screening result via the 3rd-generation Vitros assay (10 positive, 82 negative, and 7 indeterminate via confirmatory immunofluorescent assay [IFA]/Western blot [WB] testing). In the second phase, we assessed 24 HIV-1 RNA-positive (positive result via the nuclear acid amplification test [NAAT] and negative/indeterminate results via the WB test) specimens harboring acute HIV infection. The 4th-generation ARCHITECT assay yielded fewer false-positive results (n = 2) than the 3rd-generation Centaur (n = 9; P = .02) and Vitros (n = 82; P <.001) assays. One confirmed positive case had a false-negative result via the Centaur assay. When specimens from the 24 patients with acute HIV-1 infection were tested, the ARCHITECT assay yielded fewer false-negative results (n = 5) than the Centaur (n = 10) (P = .13) and the other 3rd-generation tests (n = 16) (P = .002). This study indicates that the 4th-generation ARCHITECT HIV assay yields fewer false-positive and false-negative results than the 3rd-generation HIV assays we tested. Copyright© by the American Society for Clinical Pathology (ASCP).

Population-based study of chlamydial and gonococcal infections among women in Shenzhen, China: Implications for programme planning.

PubMed

Luo, Zhen-Zhou; Li, Wu; Wu, Qiu-Hong; Zhang, Li; Tian, Li-Shan; Liu, Lan-Lan; Ding, Yi; Yuan, Jun; Chen, Zhong-Wei; Lan, Li-Na; Wu, Xiao-Bing; Cai, Yu-Mao; Hong, Fu-Chang; Feng, Tie-Jian; Zhang, Min; Chen, Xiang-Sheng

2018-01-01

This study was aimed to estimate the prevalences of chlamydia (CT) and gonococcal (NG) infections and explore risk factors associated with the CT infection among women in Shenzhen, China. We collected socio-demographic and clinical data from women (aged 20-60) and determined positivity of CT or NG by nucleic acid amplification test (NAAT) with self-collected urine specimens. We estimated prevalence of CT and NG and determined risk factors associated with CT infection. Among 9,207 participants, 4.12% (95% confidence interval [CI], 3.71%-4.53%) tested positive for CT and 0.17% (95% CIs, 0.09%-0.25%) for NG. Factors significantly associated with CT infection included being an ethnic minority (ethnicity other than Han China) (Adjusted odds ratio [AOR], 1.9; 95% CI, 1.2-3.0), using methods other than condom for contraception (AOR, 1.5; 95% CI, 1.2-1.8), having a history of adverse pregnancy outcomes (AOR, 1.4; 95% CI, 1.1-1.8), and experiencing reproductive tract symptoms in the past three months (AOR, 1.3; 95% CI, 1.0-1.7). we found that CT infection is prevalent among women in Shenzhen, China and associated with both demographic and behavioral factors. A comprehensive CT screening, surveillance and treatment programme targeting this population is warranted.

Exploring the Benefits of Molecular Testing for Gonorrhoea Antibiotic Resistance Surveillance in Remote Settings.

PubMed

Hui, Ben B; Ryder, Nathan; Su, Jiunn-Yih; Ward, James; Chen, Marcus Y; Donovan, Basil; Fairley, Christopher K; Guy, Rebecca J; Lahra, Monica M; Law, Mathew G; Whiley, David M; Regan, David G

2015-01-01

Surveillance for gonorrhoea antimicrobial resistance (AMR) is compromised by a move away from culture-based testing in favour of more convenient nucleic acid amplification test (NAAT) tests. We assessed the potential benefit of a molecular resistance test in terms of the timeliness of detection of gonorrhoea AMR. An individual-based mathematical model was developed to describe the transmission of gonorrhoea in a remote Indigenous population in Australia. We estimated the impact of the molecular test on the time delay between first importation and the first confirmation that the prevalence of gonorrhoea AMR (resistance proportion) has breached the WHO-recommended 5% threshold (when a change in antibiotic should occur). In the remote setting evaluated in this study, the model predicts that when culture is the only available means of testing for AMR, the breach will only be detected when the actual prevalence of AMR in the population has already reached 8 - 18%, with an associated delay of ~43 - 69 months between first importation and detection. With the addition of a molecular resistance test, the number of samples for which AMR can be determined increases facilitating earlier detection at a lower resistance proportion. For the best case scenario, where AMR can be determined for all diagnostic samples, the alert would be triggered at least 8 months earlier than using culture alone and the resistance proportion will have only slightly exceeded the 5% notification threshold. Molecular tests have the potential to provide more timely warning of the emergence of gonorrhoea AMR. This in turn will facilitate earlier treatment switching and more targeted treatment, which has the potential to reduce the population impact of gonorrhoea AMR.

Exploring the Benefits of Molecular Testing for Gonorrhoea Antibiotic Resistance Surveillance in Remote Settings

PubMed Central

Hui, Ben B.; Ryder, Nathan; Su, Jiunn-Yih; Ward, James; Chen, Marcus Y.; Donovan, Basil; Fairley, Christopher K.; Guy, Rebecca J.; Lahra, Monica M.; Law, Mathew G.; Whiley, David M.; Regan, David G.

2015-01-01

Background Surveillance for gonorrhoea antimicrobial resistance (AMR) is compromised by a move away from culture-based testing in favour of more convenient nucleic acid amplification test (NAAT) tests. We assessed the potential benefit of a molecular resistance test in terms of the timeliness of detection of gonorrhoea AMR. Methods and Findings An individual-based mathematical model was developed to describe the transmission of gonorrhoea in a remote Indigenous population in Australia. We estimated the impact of the molecular test on the time delay between first importation and the first confirmation that the prevalence of gonorrhoea AMR (resistance proportion) has breached the WHO-recommended 5% threshold (when a change in antibiotic should occur). In the remote setting evaluated in this study, the model predicts that when culture is the only available means of testing for AMR, the breach will only be detected when the actual prevalence of AMR in the population has already reached 8 – 18%, with an associated delay of ~43 – 69 months between first importation and detection. With the addition of a molecular resistance test, the number of samples for which AMR can be determined increases facilitating earlier detection at a lower resistance proportion. For the best case scenario, where AMR can be determined for all diagnostic samples, the alert would be triggered at least 8 months earlier than using culture alone and the resistance proportion will have only slightly exceeded the 5% notification threshold. Conclusions Molecular tests have the potential to provide more timely warning of the emergence of gonorrhoea AMR. This in turn will facilitate earlier treatment switching and more targeted treatment, which has the potential to reduce the population impact of gonorrhoea AMR. PMID:26181042

Urethral inflammatory response to ureaplasma is significantly lower than to Mycoplasma genitalium and Chlamydia trachomatis.

PubMed

Moi, Harald; Reinton, Nils; Randjelovic, Ivana; Reponen, Elina J; Syvertsen, Line; Moghaddam, Amir

2017-07-01

A non-syndromic approach to treatment of people with non-gonococcal urethritis (NGU) requires identification of pathogens and understanding of the role of those pathogens in causing disease. The most commonly detected and isolated micro-organisms in the male urethral tract are bacteria belonging to the family of Mycoplasmataceae, in particular Ureaplasma urealyticum and Ureaplasma parvum. To better understand the role of these Ureaplasma species in NGU, we have performed a prospective analysis of male patients voluntarily attending a drop in STI clinic in Oslo. Of 362 male patients who were tested for NGU using microscopy of urethral smears, we found the following sexually transmissible micro-organisms: 16% Chlamydia trachomatis, 5% Mycoplasma genitalium, 14% U. urealyticum, 14% U. parvum and 5% Mycoplasma hominis. We found a high concordance in detecting in turn U. urealyticum and U. parvum using 16s rRNA gene and ureD gene as targets for nucleic acid amplification testing (NAAT). Whilst there was a strong association between microscopic signs of NGU and C. trachomatis infection, association of M. genitalium and U. urealyticum infections in turn were found only in patients with severe NGU (>30 polymorphonuclear leucocytes, PMNL/high powered fields, HPF). U. parvum was found to colonise a high percentage of patients with no or mild signs of NGU (0-9 PMNL/HPF). We conclude that urethral inflammatory response to ureaplasmas is less severe than to C. trachomatis and M. genitalium in most patients and that testing and treatment of ureaplasma-positive patients should only be considered when other STIs have been ruled out.

Meatal Swabs Contain Less Cellular Material and Are Associated with a Decrease in Gram Stain Smear Quality Compared to Urethral Swabs in Men.

PubMed

Jordan, Stephen J; Schwebke, Jane R; Aaron, Kristal J; Van Der Pol, Barbara; Hook, Edward W

2017-07-01

Urethral swabs are the samples of choice for point-of-care Gram stain testing to diagnose Neisseria gonorrhoeae infection and nongonococcal urethritis (NGU) in men. As an alternative to urethral swabs, meatal swabs have been recommended for the collection of urethral discharge to diagnose N. gonorrhoeae and Chlamydia trachomatis infection in certain populations by nucleic acid amplification testing (NAAT), as they involve a less invasive collection method. However, as meatal swabs could be sampling a reduced surface area and result in fewer collected epithelial cells compared to urethral swabs, the adequacy of meatal swab specimens to collect sufficient cellular material for Gram stain testing remains unknown. We enrolled 66 men who underwent either urethral or meatal swabbing and compared the cellular content and Gram stain failure rate. We measured the difference in swab cellular content using the Cepheid Xpert CT/NG sample adequacy control crossing threshold (SAC CT ) and determined the failure rate of Gram stain smears (GSS) due to insufficient cellular material. In the absence of discharge, meatal smears were associated with a significant reduction in cellular content ( P = 0.0118), which corresponded with a GSS failure rate significantly higher than that for urethral swabs (45% versus 3%, respectively; P < 0.0001). When discharge was present, there was no difference among results from urethral and meatal swabs. Therefore, if GSS testing is being considered for point-of-care diagnosis of N. gonorrhoeae infection or NGU in men, meatal swabs should be avoided in the absence of a visible discharge. Copyright © 2017 American Society for Microbiology.

The performance and clinical utility of cervical microscopy for the diagnosis of gonorrhoea in women in the era of the NAAT.

PubMed

Thorley, Nicola; Radcliffe, Keith

2015-08-01

We evaluated the performance and clinical utility of cervical microscopy for diagnosing genital gonorrhoea (GC) in women, using the APTIMA Combo 2 dual GC/Chlamydia trachomatis assay as the gold standard. Test performance was assessed overall and for patient subgroups (symptomatic, GC contacts, pelvic inflammatory disease [PID] diagnosis). It was found that 93 women had positive GC genital NAAT results; 15 had positive cervical microscopy (8 were GC contacts). In all subgroups except GC contacts, the PPV of cervical microscopy was below the nationally accepted target of 90%. Compared to APTIMA Combo 2, cervical microscopy has poor sensitivity for diagnosing GC in women (16.1% overall, 17.3% symptomatic, 25% GC contacts, 10.5% PID). GC contacts accounted for over 50% of the positive microscopy results and would have received epidemiological treatment for GC, regardless of the microscopy result. Discontinuing the routine use of cervical microscopy would improve patient flow through the sexual health clinic and resources could be utilised more effectively. © The Author(s) 2014.

Detection of new HIV infections in a multicentre HIV antiretroviral pre-exposure prophylaxis trial.

PubMed

Fransen, Katrien; de Baetselier, Irith; Rammutla, Elizabeth; Ahmed, Khatija; Owino, Frederick; Agingu, Walter; Venter, Gustav; Deese, Jen; Van Damme, Lut; Crucitti, Tania

2017-08-01

Monthly specimens collected from FEM-PrEP-a Phase III trial [1] were investigated for the detection of acute HIV (AHI) infection. To evaluate the efficiency of the study-specific HIV algorithm in detecting AHI, and the performance of each of the serological and molecular tests used in diagnosing new infections, and their contribution to narrowing the window period. A total of 83 pre-seroconversion specimens from 61 seroconverters from the FEM-PrEP trial were further analyzed in a sub-study. During the trial, HIV seroconversion was diagnosed on site using a testing algorithm with simple/rapid tests (SRTs) and confirmed with a gold standard testing algorithm (see short communication: Fig. 1). The infection date was determined more accurately by the use of standard ELISAs and Nucleic Acid Amplification Tests (NAAT) in a look-back procedure. For this sub-study, the international central laboratory repeated the study algorithm using SRTs. A total of 83 pre-seroconversions specimens from 61 seroconverters were analyzed in a look-back procedure. RNA was detected in 35/61 seroconverters at the visit before the seroconversion visit as determined at the study sites. Four seroconversion dates were inaccurate at one study site as the international central laboratory detected the HIV infection one visit earlier using the same test algorithm. Using the gold standard, an additional seroconversion was detected at an earlier visit. The combined antigen/antibody and the single antigen test had a higher sensitivity compared to the SRTs in detecting acute infections. In the FEM-PrEP trial, the international central laboratory detected a small number of seroconversions one month earlier than the study sites using the same study algorithm. Standard tests are still the most sensitive tests in detecting pre-seroconversion or acute HIV infection, but they are costly, time consuming and not recommended for use on-site in a clinical trial. Copyright © 2017 Elsevier B.V. All rights reserved.

Diagnosis and monitoring of HCV infection using the cobas® HCV test for use on the cobas® 6800/8800 systems.

PubMed

Yao, Joseph D; Young, Stephen; Heilek, Gabrielle M; Marino, Enrique; Paxinos, Ellen E; Marins, Ed G; Valsamakis, Alexandra

2018-05-01

Accurate, sensitive, and specific tests for detection and monitoring of hepatitis C virus (HCV) RNA concentrations are essential for diagnosis and management of HCV infections. We evaluated the next-generation reverse-transcription real-time PCR test, cobas ® HCV test for use with the cobas ® 6800/8800 systems ("cobas HCV") by determining its analytical performance characteristics and clinical utility for the diagnosis and therapeutic monitoring of chronic HCV infections. The limit of detection (LOD), linearity, precision, specificity, matrix equivalence of plasma and serum, and quantitative agreement with the COBAS ® AmpliPrep/COBAS ® TaqMan ® HCV Test version 2.0 ("CAP/CTM HCV v2") were evaluated. Clinical utility for the diagnosis of chronic HCV infection was demonstrated by testing plasma from HCV seropositive individuals and comparing results to a nucleic acid amplification test (NAAT) approved for use in the diagnosis of chronic hepatitis C. Clinical specificity was investigated by testing plasma from HCV antibody negative subjects with non-HCV related liver diseases. Utility for monitoring treatment response was defined by testing plasma collected during treatment of HCV genotypes (GT) 1, 2, and 3 and determining positive predictive value (PPV), negative predictive value (NPV) and the odds ratio (OR) for predicting cure (sustained virologic response 12 weeks after treatment cessation, "SVR12"). The cobas HCV test demonstrated an LOD of at least 15 IU/mL and measurable range from 15 to at least 1.0E + 08 IU/mL (1.2-8.0 log 10 IU/mL) for GT 1-6, with high accuracy (≤0.16 log 10 difference) and precision (standard deviation 0.04-0.14 log 10 ) throughout the linear range. Paired plasma and serum samples showed highly correlated performance (R 2  = 0.97). Quantification was 100% specific for HCV in analytical studies. Correlation with CAP/CTM HCV v2 was high in patient samples (mean titer difference: 0.05 log 10 with a 95% CI: 0.03-0.06 log 10 ). For the diagnosis of chronic HCV, positive and negative percent agreement between cobas HCV and the comparator NAAT were 98.8-100% on the cobas 6800 and 8800 systems. Clinical specificity of cobas HCV using samples from HCV antibody negative subjects with non-HCV related liver diseases was 99.6% and 100% on cobas 6800 and 8800 systems. In therapeutic monitoring and SVR12 prediction during experimental treatment for chronic HCV GT 1 infections, undetectable HCV RNA by cobas HCV at different on-treatment weeks had a PPV 76.8%-79.4%, NPV 29.9%-100%, and OR 1.64-47.52. During therapy of HCV GT 2 and GT 3, treatment week 4 and 12 results were: PPV, 84.7% and 75.3%; NPV, 47.8% and 50.0%; OR, 5.09 and 3.05. The cobas HCV test is highly sensitive, specific, and accurate HCV RNA test for GT 1-6. It demonstrates excellent correlation with the FDA-approved CAP/CTM HCV v2 test. It is useful clinically for detection of active HCV infection in individuals that have had a positive anti-HCV antibody test result and in monitoring treatment response. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Azithromycin treatment failure for Chlamydia trachomatis among heterosexual men with nongonococcal urethritis

PubMed Central

Kissinger, Patricia; White, Scott; Manhart, Lisa E.; Schwebke, Jane; Taylor, Stephanie N; Mena, Leandro; Khosropour, Christine M; Wilcox, Larissa; Schmidt, Norine; Martin, David H

2016-01-01

Background Three recent prospective studies have suggested that the 1 g dose of azithromycin for Chlamydia trachomatis (Ct) was less effective than expected, reporting a wide range of treatment failure rates (5.8%–22.6%). Reasons for the disparate results could be attributed to geographic or methodological differences. The purpose of this study was to re-examine the studies and attempt to harmonize methodologies to reduce misclassification as a result of false positives from early test-of-cure (TOC) or reinfection as a result of sexual exposure rather than treatment failure. Methods Men who had sex with women, who received 1 g azithromycin under directly observed therapy (DOT) for presumptive treatment of nongonococcal urethritis (NGU) with confirmed Ct were included. Baseline screening was performed on urethral swabs or urine and TOC screening was performed on urine using nucleic acid amplification tests (NAAT). Post-treatment vaginal sexual exposure was elicited at TOC. Data from the three studies was obtained and re-analyzed. Rates of Ct re-test positive were examined for all cases and a sensitivity analysis was conducted to either reclassify potential false positives/reinfections as negative or remove them from the analysis. Results The crude treatment failure rate was 12.8% (31/242). The rate when potential false positives/reinfections were reclassified as negative was 6.2% (15/242) or when these were excluded from analysis was 10.9% (15/138). Conclusion In these samples of men who have sex with women with Ct-related NGU, azithromycin treatment failure was between 6.2% and 12.8%. This range of failure is lower than previously published but higher than the desired World Health Organization’s target chlamydia treatment failure rate of < 5%. PMID:27631353

The Evolution of Advanced Molecular Diagnostics for the Detection and Characterization of Mycoplasma pneumoniae.

PubMed

Diaz, Maureen H; Winchell, Jonas M

2016-01-01

Over the past decade there have been significant advancements in the methods used for detecting and characterizing Mycoplasma pneumoniae, a common cause of respiratory illness and community-acquired pneumonia worldwide. The repertoire of available molecular diagnostics has greatly expanded from nucleic acid amplification techniques (NAATs) that encompass a variety of chemistries used for detection, to more sophisticated characterizing methods such as multi-locus variable-number tandem-repeat analysis (MLVA), Multi-locus sequence typing (MLST), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), single nucleotide polymorphism typing, and numerous macrolide susceptibility profiling methods, among others. These many molecular-based approaches have been developed and employed to continually increase the level of discrimination and characterization in order to better understand the epidemiology and biology of M. pneumoniae. This review will summarize recent molecular techniques and procedures and lend perspective to how each has enhanced the current understanding of this organism and will emphasize how Next Generation Sequencing may serve as a resource for researchers to gain a more comprehensive understanding of the genomic complexities of this insidious pathogen.

Early HIV Infections Among Men Who Have Sex with Men in Five Cities in the United States

PubMed Central

Smith, A.; Masciotra, S.; Zhang, W.; Bingham, T.; Flynn, C.; German, D.; Al-Tayyib, A.; Magnus, M.; LaLota, M.; Rose, C. E.; Owen, S. M.

2016-01-01

We tested blood samples from men who have sex with men (MSM) to detect early HIV infection. Early HIV included both acute (infected past 30 days) and recent (estimated recency past 240 days). Acute infections were defined as screen immunoassay (IA) negative/NAAT-positive or IA-positive/Multispot-negative/NAAT-positive. Recent infections were defined as avidity index cutoff <30 % on an avidity-based IA and, (1) not reporting antiretroviral therapy use or, (2) HIV RNA >150 copies/mL. Of 937 samples, 26 % (244) were HIV-infected and of these 5 % (12) were early. Of early infections, 2 were acute and 10 recent; most (8/12) were among black MSM. Early infection was associated with last partner of black race [adjusted relative risk (ARR) = 4.6, confidence intervals (CI) 1.2–17.3], receptive anal sex at last sex (ARR = 4.3, CI 1.2–15.0), and daily Internet use to meet partners/ friends (ARR = 3.3, CI 1.1–9.7). Expanding prevention and treatment for black MSM will be necessary for reducing incidence in the United States. PMID:25680518

Utility of adenosine deaminase (ADA), PCR & thoracoscopy in differentiating tuberculous & non-tuberculous pleural effusion complicating chronic kidney disease.

PubMed

Kumar, Sravan; Agarwal, Ritesh; Bal, Amanjit; Sharma, Kusum; Singh, Navneet; Aggarwal, Ashutosh N; Verma, Indu; Rana, Satyawati V; Jha, Vivekanand

2015-03-01

Pleural effusion is a common occurrence in patients with late-stage chronic kidney disease (CKD). In developing countries, many effusions remain undiagnosed after pleural fluid analysis (PFA) and patients are empirically treated with antitubercular therapy. The aim of this study was to evaluate the role of adenosine deaminase (ADA), nucleic acid amplification tests (NAAT) and medical thoracoscopy in distinguishing tubercular and non-tubercular aetiologies in exudative pleural effusions complicating CKD. Consecutive stage 4 and 5 CKD patients with pleural effusions underwent PFA including ADA and PCR [65 kDa gene; multiplex (IS6110, protein antigen b, MPB64)]. Patients with exudative pleural effusion undiagnosed after PFA underwent medical thoracoscopy. All 107 patients underwent thoracocentesis with 45 and 62 patients diagnosed as transudative and exudative pleural effusions, respectively. Twenty six of the 62 patients underwent medical thoracoscopy. Tuberculous pleurisy was diagnosed in six while uraemic pleuritis was diagnosed in 20 subjects. The sensitivity and specificity of pleural fluid ADA, 65 kDa gene PCR, and multiplex PCR were 66.7 and 90 per cent, 100 and 50 per cent, and 100 and 100 per cent, respectively. Thoracoscopy was associated with five complications in three patients. Uraemia remains the most common cause of pleural effusion in CKD even in high TB prevalence country. Multiplex PCR and thoracoscopy are useful investigations in the diagnostic work-up of pleural effusions complicating CKD while the sensitivity and/or specificity of ADA and 65 kDa gene PCR is poor.

Lymphogranuloma venereum among patients presenting at the HIV/STI clinic in Antwerp, Belgium : a case series.

PubMed

Apers, Ludwig; Florence, Eric; Crucitti, Tania; Anwar, Nabila

2017-01-01

Objective of this study was to describe the patient characteristics and clinical presentation of laboratory confirmed lymphogranuloma venereum (LGV) cases, diagnosed at the Institute of Tropical Medicine (ITM), Antwerp, Belgium. Demographic and biomedical characteristics of all patients with chlamydia-positive sample results were retrieved for the years 2013 and 2014. Samples were obtained from both symptomatic and asymptomatic patients who consulted at the HIV/STI clinic. Fifty four patients with laboratory confirmed LGV were detected among 3885 nucleic acid amplification tests (NAATs) performed for the detection of chlamydia during the two years under review. Fifty three were men and equally fifty three had sex with men only (MSM). HIV (87%) and HCV (31.5%) were common concomitant infections, whilst anal gonorrhoea and syphilis were detected at the moment of the LGV diagnosis among 19 (35.2%) and 6 (11.0%) cases respectively. All cases were symptomatic, except one. The most frequent symptoms that were recorded could be categorised as proctitis (in 40 patients (74%)). Lymphadenopathy, anal and genital ulcers were signs that were present in 7 (13.0%), 4 (7.4%) and 2 patients (3.7%) respectively. LGV remains an important sexually transmitted disease among MSM. In this retrospective study, the far majority of LGV was detected amongst symptomatic persons. HCV, HIV, anal gonorrhoea and syphilis were associated co-infections. Proctitis in a high risk patient should alert the clinician for the possibility of an STI. © Acta Gastro-Enterologica Belgica.

A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

PubMed

Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

2016-12-15

The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management. Copyright © 2016 Elsevier B.V. All rights reserved.

"Pee-in-a-Pot": acceptability and uptake of on-site chlamydia screening in a student population in the Republic of Ireland

PubMed Central

2010-01-01

Background The aim of the study was to explore the acceptability and uptake of on-campus screening using a youth friendly approach in two Third Level higher education institutions (HEIs). This study is part of wider research exploring the optimal setting for chlamydia screening in Ireland. Methods Male and female students were given the opportunity to take a free anonymous test for chlamydia during a one week programme of "pee-in-a-pot" days at two HEI campuses in the West of Ireland. The study was set up after extensive consultation with the two HEIs and advertised on the two campuses using a variety of media in the two weeks preceding the screening days. Screening involved the provision and distribution of testing packs at communal areas and in toilet facilities. In Ireland, chlamydia notifications are highest amongst 20-29 year olds and hence the screening criterion was aimed at 18-29 year olds. Urine samples were tested using a nucleic acid amplification test (NAAT). Following the screening days, qualitative in-depth interviews were conducted with participants about their experiences of the event. Results Out of 1,249 test kits distributed in two HEIs, 592 specimens were collected giving a return rate of 47.5%. Tests excluded (54) were due to labelling errors or ineligibility of participants' age. Two thirds of those tested were females and the mean age was 21 years. Overall,3.9% (21/538) of participants tested positive, 5% (17/336) among females and 2% (4/191) among males. Participant interviews identified factors which enhanced student participation such as anonymity, convenience, accessibility of testing, and the informal and non-medical approach to testing. Conclusions Screening for chlamydia using on-campus "pee-in-a-pot" days is an acceptable strategy in this population. This model can detect and treat asymptomatic cases of chlamydia and avoid many of the barriers associated with testing for sexually transmitted infections (STIs) in clinical settings. PMID:21070646

Lymphogranuloma venereum in an Italian MSM: concurrent pharyngeal and rectal infection.

PubMed

Foschi, Claudio; Filippini, Andrea; D'Antuono, Antonietta; Compri, Monica; Macca, Francesca; Banzola, Nicoletta; Marangoni, Antonella

2014-07-01

An Italian HIV-positive man having sex with men (MSM) attended the STIs Outpatients Clinic of Sant'Orsola Hospital in Bologna complaining of anal pain and constipation. According to patient's sexual history and repertoires, NAAT testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) was performed. Pharyngeal and anal swabs resulted positive for CT DNA detection and the following molecular genotyping identified a L2 serovar, coming to the final diagnosis of pharyngeal and rectal lymphogranuloma venereum (LGV) infection. After an antibiotic therapy with doxycycline 100 mg twice a day for 3 weeks, the patient completely recovered and the test of cure was negative for LGV infection.

Acute Hepatitis E Virus infection with coincident reactivation of Epstein-Barr virus infection in an immunosuppressed patient with rheumatoid arthritis: a case report.

PubMed

Schultze, Detlev; Mani, Bernhard; Dollenmaier, Günter; Sahli, Roland; Zbinden, Andrea; Krayenbühl, Pierre Alexandre

2015-10-29

Hepatitis E virus (HEV) is the most recently discovered of the hepatotropic viruses, and is considered an emerging pathogen in developed countries with the possibility of fulminant hepatitis in immunocompromised patients. Especially in the latter elevated transaminases should be taken as a clue to consider HEV infection, as it can be treated by discontinuation of immunosuppression and/or ribavirin therapy. To our best knowledge, this is a unique case of autochthonous HEV infection with coincident reactivation of Epstein-Barr virus (EBV) infection in an immunosuppressed patient with rheumatoid arthritis (RA). A 68-year-old Swiss woman with RA developed hepatitis initially diagnosed as methotrexate-induced liver injury, but later diagnosed as autochthonous HEV infection accompanied by reactivation of her latent EBV infection. She showed confounding serological results pointing to three hepatotropic viruses (HEV, Hepatitis B virus (HBV) and EBV) that could be resolved by detection of HEV and EBV viraemia. The patient recovered by temporary discontinuation of immunosuppressive therapy. In immunosuppressed patients with RA and signs of liver injury, HEV infection should be considered, as infection can be treated by discontinuation of immunosuppression. Although anti-HEV-IgM antibody assays can be used as first line virological tools, nucleic acid amplification tests (NAAT) for detection of HEV RNA are recommended--as in our case--if confounding serological results from other hepatotropic viruses are obtained. After discontinuation of immunosuppressive therapy, our patient recovered from both HEV infection and reactivation of latent EBV infection without sequelae.

[Non-viral sexually transmitted infections - Epidemiology, clinical manifestations, diagnostics and therapy : Part 2: Chlamydia and mycoplasma].

PubMed

Nenoff, P; Manos, A; Ehrhard, I; Krüger, C; Paasch, U; Helmbold, P; Handrick, W

2017-01-01

Chlamydia trachomatis is the most common pathogen of sexually transmitted bacterial infections worldwide. Every year in Germany approximately 300,000 new infections are to be expected. Chlamydia infections occur nearly exclusively in the postpubertal period. The peak age group is 15-25 years. The infection usually runs an asymptomatic course and the diagnosis is made by nucleic acid amplification techniques (NAAT) often after chlamydial screening or if complications occur. For treatment of chlamydial infections oral doxycycline 100 mg twice daily over 7 days is initially used or alternatively oral azithromycin 1.5 g as a single dose is recommended. The sexual partner should also be investigated and treated. Genital Mycoplasma infections are caused by Ureaplasma urealyticum (pathogen of urethritis and vaginitis), Ureaplasma parvum (mostly saprophytic and rarely a cause of urethritis) and Mycoplasma hominis (facultative pathogenic). Mycoplasma genitalium represents a relatively new sexually transmitted Mycoplasma species. Doxycycline is effective in Ureaplasma infections or alternatively clarithromycin and azithromycin. Doxycycline can be ineffective in Mycoplasma hominis infections and an alternative is clindamycin. Non-gonococcal and non-chlamydial urethritis due to Mycoplasma genitalium can now be diagnosed by molecular biological techniques using PCR and should be treated by azithromycin.

Chlamydia and lymphogranuloma venereum in Canada: 2003-2012 Summary Report

PubMed Central

Totten, S; MacLean, R; Payne, E; Severini, A

2015-01-01

Background Chlamydia continues to be the most commonly reported sexually transmitted infection in Canada. Lymphogranuloma venereum (LGV), caused by certain serovars of Chlamydia trachomatis, is becoming established in some populations in a number of Western countries. Objective To identify trends in reported cases of chlamydia and LGV in Canada from January 1, 2003 to December 31, 2012. Methods Notifiable disease data on chlamydia were submitted to the Public Health Agency of Canada by provincial and territorial epidemiological units and summarized at the national level by age and sex. Confirmatory testing for suspected LGV cases and serovar subtyping were performed by the National Microbiology Laboratory (NML). Where possible, provincial/territorial health authorities use a standardized national case report form to collect enhanced epidemiological data on each case and to submit the data to the Agency. Results Rates of reported cases of chlamydia increased by 57.6%, from 189.6 to 298.7 per 100,000 between 2003 and 2012. The rate of reported cases of chlamydia among females (383.5 per 100,000) was almost twice as high as that among males (212.0 per 100,000), although the highest relative rate increase occurred among males. In both males and females, the rates of chlamydia were highest in those aged 20 to 24 years. From 2004 to 2012, 170 cases of LGV were reported to the Agency by provincial health authorities (including 104 confirmed and 66 probable cases). In 2012, case reports were received on 12 confirmed and probable cases, compared to 38 laboratory-positive cases confirmed by the NML. Conclusion In Canada, as in many countries, chlamydia rates have markedly increased over the last 10 years, in part due to improved diagnosis through nucleic acid amplification (NAAT) testing. Consistent with trends in Europe and other countries, LGV is emerging in Canada among men who have sex with men (MSM). PMID:29769927

Humidification of the Arctic: Effects of more open ocean water on land temperatures and tundra productivity along continental and maritime bioclimate transects

NASA Astrophysics Data System (ADS)

Bhatt, U. S.; Walker, D. A.; Raynolds, M. K.; Epstein, H. E.

2017-12-01

Amplified Arctic warming linked to declining sea-ice extent led to generally enhanced productivity of the tundra biome during the period 1982-2008. After about 2002, coinciding with a recent precipitous decline in sea ice, large areas of the Arctic began showing reversals of previous positive productivity trends. To better understand these recent vegetation productivity declines and whether they are associated with differences in a general humidification of portions of the Arctic, we focus analysis on two transects with ground information: the more continental North America Arctic Transect (NAAT) and the more maritime Eurasia Arctic Transect (EAT). We compare ground information with satellite-derived trends in open water, summer terrestrial temperatures, and vegetation greenness and changes in continentality of the two transects, as indicated by the differences in the annual maximum and minimum mean monthly temperatures. Areas adjacent to perennial sea ice along in the northern parts of the NAAT exhibit climates with positive trends in summer warmth, but negative greening trends, possibly due to soil drying. Southern parts of the NAAT in the vicinity of more open water show positive greenness trends. Along the EAT, cooling midsummer conditions and reduced greenness appear to be caused by cloudier conditions, and possibly later snow melt during the period of maximum potential photosynthesis. Ground-based environmental and vegetation data indicate that biomass, particularly moss biomass is much greater along the more maritime EAT, indicating a buffering effect of the vegetation that will act to damp productivity as humidification of the Arctic proceeds. This multi-scale analysis is one step in the direction of understanding the drivers of tundra vegetation productivity in the Arctic.

Optimizing the diagnostic testing of Clostridium difficile infection.

PubMed

Bouza, Emilio; Alcalá, Luis; Reigadas, Elena

2016-09-01

Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhea and is associated with a considerable health and cost burden. However, there is still not a clear consensus on the best laboratory diagnosis approach and a wide variation of testing methods and strategies can be encountered. We aim to review the most practical aspects of CDI diagnosis providing our own view on how to optimize CDI diagnosis. Expert commentary: Laboratory diagnosis in search of C. difficile toxins should be applied to all fecal diarrheic samples reaching the microbiology laboratory in patients > 2 years old, with or without classic risk factors for CDI. Detection of toxins either directly in the fecal sample or in the bacteria isolated in culture confirm CDI in the proper clinical setting. Nuclear Acid Assay techniques (NAAT) allow to speed up the process with epidemiological and therapeutic consequences.

Nucleic Acid Amplification Testing in Suspected Child Sexual Abuse

ERIC Educational Resources Information Center

Esernio-Jenssen, Debra; Barnes, Marilyn

2011-01-01

The American Academy of Pediatrics recommends that site-specific cultures be obtained, when indicated, for sexually victimized children. Nucleic acid amplification testing is a highly sensitive and specific methodology for identifying sexually transmitted infections. Nucleic acid amplification tests are also less invasive than culture, and this…

Age Differences in STDs, Sexual Behaviors, and Correlates of Risky Sex Among Sexually Experienced Adolescent African-American Females

PubMed Central

Brown, Jennifer L.; DiClemente, Ralph J.; Davis, Teaniese L.; Kottke, Melissa J.; Rose, Eve S.

2012-01-01

Objective To explore age differences in factors associated with positive sexually transmitted diseases (STD) status among a sample of African-American adolescent females. Methods Data were collected via ACASI from 701 African-American adolescent females (14–20 years) seeking services at reproductive health clinics. Adolescents provided self-collected vaginal swabs assayed using NAAT to assess the prevalence of three STDs. Results Younger adolescents (14–17 years) had significantly higher rates of STDs than older adolescents (18–20 years), but older adolescents had significantly higher levels of STD-associated risk behavior. In controlled analysis, having a casual sex partner was the only variable significantly associated with a positive STD test for younger adolescents, and prior history of STD and higher impulsivity were significantly associated with testing STD positive among older adolescents. Conclusions These findings suggest that developmentally tailored STD/HIV prevention interventions are needed for younger and older subgroups of adolescent females to help reduce their risk of infection. PMID:21933811

HIV single staging algorithm: integration and maximization of resources by reducing time between HIV diagnosis and treatment.

PubMed

Bennett, Berry; Hardy, Bonnie; Fordan, Sally; Haddock-Morilla, Lizzette; Rowlinson, Marie-Claire; Crowe, Susanne

2013-12-01

Early HIV diagnosis, linkage and engagement into care are vital to improved personal health outcomes. The initiation of antiretroviral therapy, with retention in care and drug adherence leads to viral load suppression, a significant decrease in HIV transmission rates and ultimately a reduction in HIV incidence rates. In the U.S only 51% of those diagnosed with HIV infection are retained in care and 28% have a suppressed viral load. Reducing the time and number of visits from HIV diagnosis to entry into care, has the potential to engage and treat an increased number of infected individuals. (1) Evaluate the feasibility of conducting HIV-1 supplemental testing concurrently with baseline clinical management testing; (2) to evaluate whether all tests could be completed and reported prior to the traditional posttest counseling appointment; (3) to monitor the return activity for posttest and medical provider appointments. Baseline CD4 and HIV-1 viral load tests were performed concurrently with an HIV-1/2 antibody immunoassay (IA) and HIV-1 Western blot (WB) on 105 individuals with preliminary positive rapid test results. Participating study-sites were located in high-risk, high-morbidity locations: a county jail, a county mobile unit and a county hospital emergency department. Based on the individual's self-reporting statement of "No" to a previous HIV diagnosis and the POC preliminary positive rapid test result, blood specimens were processed via the Single Staging Algorithm. Study site data and medical record review established time intervals between the rapid test and subsequent visits. Of the 105 individuals with HIV-1 preliminary positive rapid test results, 102 were confirmed positive with HIV-1 WB (plus 3rd generation IA repeatedly reactive) and one was confirmed by an HIV-1 WB indeterminate (gp160), HIV-1 Nucleic Acid Amplification Test (NAAT) reactive (an algorithm-defined early infection). The concordance between POC preliminary positive rapid tests and the confirmatory test of the single staging algorithm was 98%. Ninety-six (91%) HIV-1 baseline viral load test results and 82 (78%) CD4/CD8 absolute counts were performed and made available to the provider prior to posttest counseling. The average number of visits for posttest counseling at 14 days was 44.7% (range 37.9-56.5%) with an additional 31.1% (range 22.7-37.9%) returning within 30 days. The average number of clients that returned for the medical provider appointment was 55.4%. A high percentage of HIV-1 clinical management baseline results (78-91%) and 100% confirmatory diagnostic results were completed and reported prior to the traditional posttest counseling appointment. Additional data and analysis is needed to determine the impact of the Single Staging Algorithm on medical provider appointments if the posttest appointment is more than 30 days after the preliminary HIV diagnosis. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparison of patients diagnosed with gonorrhoea through community screening with those self-presenting to the genitourinary medicine clinic.

PubMed

Cook, Penny A; Evans-Jones, John; Mallinson, Harry; Wood, Martyn; Alloba, Fath; Jones, Kathy; Strodtbeck, Sara; Hanna-Bashara, Layla

2014-03-14

To compare the clinical, socioeconomic and demographic characteristics of individuals diagnosed with Neisseria gonorrhoeae (NG) in the community using a concomitant nucleic acid amplification test (NAAT, AptimaCombo2) as part of the (community-based) UK Chlamydia Screening Programme (CSP), with those diagnosed in hospital-based genitourinary medicine (GUM) services. A retrospective case note review of all 643 patients treated for NG at a GUM in north west England (January 2007-April 2009). All 643 treated for NG (including CSP cases, since all cases were referred to GUM for treatment). Limited data were available for 13 CSP cases who failed to attend GUM. Whether the case was detected in the community or GUM services. Predictors were demographics (age, gender, postcode for deprivation analysis), sexual history (eg, number of partners) and clinical factors (eg, culture positivity). 131 cases were diagnosed by CSP (13 of whom did not attend GUM). A further four cases were contacts of these. The GUM caseload was thus inflated by 23% (from 521 to 643). Community cases were overwhelmingly female (85% vs 27% in GUM, p<0.001) and younger (87% females were <25 years vs 70% GUM females, p=0.001). Logistic regression analysis restricted to the target age of the CSP (<25 years) revealed that CSP cases, compared with GUM cases, were more likely to reside in deprived areas (adjusted OR=5.6, 95% CI 1.4 to 21.8 and 5.3, CI 1.7 to 16.6 for the most and second most deprived group respectively, compared with the averagely deprived group, p=0.037) and be asymptomatic (adjusted OR=1.9, CI 1.1 to 3.4, p=0.02). Community screening for NG led to a 79% increase in the number of infections detected in women aged <25 years. Screening is targeted at young people, and tends to disproportionately attract young women, a group under-represented at GUM. Screening also contributed further to case detection in deprived areas.

High pre-diagnosis attrition among patients with presumptive MDR-TB: an operational research from Bhopal district, India.

PubMed

Shewade, Hemant Deepak; Kokane, Arun M; Singh, Akash Ranjan; Verma, Manoj; Parmar, Malik; Chauhan, Ashish; Chahar, Sanjay Singh; Tiwari, Manoj; Khan, Sheeba Naz; Gupta, Vivek; Tripathy, Jaya Prasad; Nagar, Mukesh; Singh, Sanjai Kumar; Mehra, Pradeep Kumar; Kumar, Ajay Mv

2017-04-04

Pre-diagnosis attrition needs to be addressed urgently if we are to make progress in improving MDR-TB case detection and achieve universal access to MDR-TB care. We report the pre-diagnosis attrition, along with factors associated, and turnaround times related to the diagnostic pathway among patient with presumptive MDR-TB in Bhopal district, central India (2014). Study was conducted under the Revised National Tuberculosis Control Programme setting. It was a retrospective cohort study involving record review of all registered TB cases in Bhopal district that met the presumptive MDR-TB criteria (eligible for DST) in 2014. In quarter 1, Line Probe Assay (LPA) was used if sample was smear/culture positive. Quarter 2 onwards, LPA and Cartridge-based Nucleic Acid Amplification Test (CbNAAT) was used for smear positive and smear negative samples respectively. Pre-diagnosis attrition was defined as failure to undergo DST among patients with presumptive MDR-TB (as defined by the programme). Of 770 patients eligible for DST, 311 underwent DST and 20 patients were diagnosed as having MDR-TB. Pre-diagnosis attrition was 60% (459/770). Among those with pre-diagnosis attrition, 91% (417/459) were not identified as 'presumptive MDR-TB' by the programme. TAT [median (IQR)] to undergo DST after eligibility was 4 (0, 10) days. Attrition was more than 40% across all subgroups. Age more than 64 years; those from a medical college; those eligible in quarter 1; patients with presumptive criteria 'previously treated - recurrent TB', 'treatment after loss-to-follow-up' and 'previously treated-others'; and patients with extra-pulmonary TB were independent risk factors for not undergoing DST. High pre-diagnosis attrition was contributed by failure to identify and refer patients. Attrition reduced modestly with time and one factor that might have contributed to this was introduction of CbNAAT in quarter 2 of 2014. General health system strengthening which includes improvement in identification/referral and patient tracking with focus on those with higher risk for not undergoing DST is urgently required.

The effect of main urine inhibitors on the activity of different DNA polymerases in loop-mediated isothermal amplification.

PubMed

Jevtuševskaja, Jekaterina; Krõlov, Katrin; Tulp, Indrek; Langel, Ülo

2017-04-01

The use of rapid amplification methods to detect pathogens in biological samples is mainly limited by the amount of pathogens present in the sample and the presence of inhibiting substances. Inhibitors can affect the amplification efficiency by either binding to the polymerase, interacting with the DNA, or interacting with the polymerase during primer extension. Amplification is performed using DNA polymerase enzymes and even small changes in their activity can influence the sensitivity and robustness of molecular assays Methods: The main purpose of this research was to examine which compounds present in urine inhibit polymerases with strand displacement activity. To quantify the inhibition, we employed quantitative loop-mediated isothermal amplification Results: The authors found that the presence of BSA, Mg 2+, and urea at physiologically relevant concentrations, as well as acidic or alkaline conditions did not affect the activity of any of the tested polymerases. However, addition of salt significantly affected the activity of the tested polymerases. These findings may aid in the development of more sensitive, robust, cost effective isothermal amplification based molecular assays suitable for both point-of-care testing and on-site screening of pathogens directly from unprocessed urine which avoid the need for long and tedious DNA purification steps prior to amplification.

[Principle of LAMP method--a simple and rapid gene amplification method].

PubMed

Ushikubo, Hiroshi

2004-06-01

So far nucleic acid test (NAT) has been employed in various fields, including infectious disease diagnoses. However, due to its complicated procedures and relatively high cost, it has not been widely utilized in many actual diagnostic applications. We have therefore developed a simple and rapid gene amplification technology, Loop-mediated Isothermal Amplification (LAMP) method, which has shown prominent results of surpassing the performance of the conventional gene amplification methods. LAMP method acquires three main features: (1) all reaction can be carried out under isothermal conditions; (2) the amplification efficiency is extremely high and tremendous amount of amplification products can be obtained; and (3) the reaction is highly specific. Furthermore, developed from the standard LAMP method, a rapid LAMP method, by adding in the loop primers, can reduce the amplification time from the previous 1 hour to less than 30 minutes. Enormous amount of white precipitate of magnesium pyrophosphate is produced as a by-product of the amplification, therefore, direct visual detection is possible without using any reaction indicators and detection equipments. We believe LAMP technology, with the integration of these features, can rightly apply to clinical genetic testing, food and environmental analysis, as well as NAT in different fields.

Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach.

PubMed

Zahradnik, Celine; Kolm, Claudia; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

2014-11-01

In 2003 the European Commission introduced a 0.9% threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5%. A false-negative rate of only 5% for 1% GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP.

Horizontal localization and speech intelligibility with bilateral and unilateral hearing aid amplification.

PubMed

Köbler, S; Rosenhall, U

2002-10-01

Speech intelligibility and horizontal localization of 19 subjects with mild-to-moderate hearing loss were studied in order to evaluate the advantages and disadvantages of bilateral and unilateral hearing aid (HA) fittings. Eight loudspeakers were arranged in a circular array covering the horizontal plane around the subjects. Speech signals of a sentence test were delivered by one, randomly chosen, loudspeaker. At the same time, the other seven loudspeakers emitted noise with the same long-term average spectrum as the speech signals. The subjects were asked to repeat the speech signal and to point out the corresponding loudspeaker. Speech intelligibility was significantly improved by HAs, bilateral amplification being superior to unilateral. Horizontal localization could not be improved by HA amplification. However, bilateral HAs preserved the subjects' horizontal localization, whereas unilateral amplification decreased their horizontal localization abilities. Front-back confusions were common in the horizontal localization test. The results indicate that bilateral HA amplification has advantages compared with unilateral amplification.

Use of APTIMA Combo 2: The Experience of a Child Advocacy Center

ERIC Educational Resources Information Center

Leder, M. Ranee; Leber, Amy L.; Marcon, Mario J.; Scribano, Philip V.

2013-01-01

The Centers for Disease Control and Prevention recommends nucleic acid amplification testing for chlamydia and gonorrhea in sexually abused girls. No studies describe performance of APTIMA Combo 2 Assay with second target confirmation on the same testing platform. This nucleic acid amplification testing is evaluated within a large child advocacy…

Quality control for quantitative PCR based on amplification compatibility test.

PubMed

Tichopad, Ales; Bar, Tzachi; Pecen, Ladislav; Kitchen, Robert R; Kubista, Mikael; Pfaffl, Michael W

2010-04-01

Quantitative qPCR is a routinely used method for the accurate quantification of nucleic acids. Yet it may generate erroneous results if the amplification process is obscured by inhibition or generation of aberrant side-products such as primer dimers. Several methods have been established to control for pre-processing performance that rely on the introduction of a co-amplified reference sequence, however there is currently no method to allow for reliable control of the amplification process without directly modifying the sample mix. Herein we present a statistical approach based on multivariate analysis of the amplification response data generated in real-time. The amplification trajectory in its most resolved and dynamic phase is fitted with a suitable model. Two parameters of this model, related to amplification efficiency, are then used for calculation of the Z-score statistics. Each studied sample is compared to a predefined reference set of reactions, typically calibration reactions. A probabilistic decision for each individual Z-score is then used to identify the majority of inhibited reactions in our experiments. We compare this approach to univariate methods using only the sample specific amplification efficiency as reporter of the compatibility. We demonstrate improved identification performance using the multivariate approach compared to the univariate approach. Finally we stress that the performance of the amplification compatibility test as a quality control procedure depends on the quality of the reference set. Copyright 2010 Elsevier Inc. All rights reserved.

A misleading false-negative result using Neisseria gonorrhoeae opa MGB multiplex PCR assay in patient's rectal sample due to partial mutations of the opa gene.

PubMed

Vahidnia, Ali; van Empel, Pieter Jan; Costa, Sandra; Oud, Rob T N; van der Straaten, Tahar; Bliekendaal, Harry; Spaargaren, Joke

2015-07-01

A 53-year-old homosexual man presented at his general practitioner (GP) practice with a suspicion of sexually transmitted infection. Initial NAAT screening was performed for Chlamydia trachomatis and Neisseria gonorrhoeae. The patient was positive for Neisseria gonorrhoeae both for his urine and rectal sample. The subsequent confirmation test for Neisseria gonorrhoeae by a second laboratory was only confirmed for the urine sample and the rectal sample was negative. We report a case of a potential false-negative diagnosis of Neisseria gonorrhoeae due to mutations of DNA sequence in the probe region of opa-MGB assay of the rectal sample. The patient did not suffer any discomfort as diagnosis of Neisseria gonorrhoeae in his urine sample had already led to treatment by prescribing the patient with Ceftriaxone 500 mg IV dissolved in 1 ml lidocaine 2% and 4 mL saline. The patient also received a prescription for Azithromycin (2x500 mg).

Differential Item Functioning Amplification and Cancellation in a Reading Test

ERIC Educational Resources Information Center

Bao, Han; Dayton, C. Mitchell; Hendrickson, Amy B.

2009-01-01

When testlet effects and item idiosyncratic features are both considered to be the reasons of DIF in educational tests using testlets (Wainer & Kiely, 1987) or item bundles (Rosenbaum, 1988), it is interesting to investigate the phenomena of DIF amplification and cancellation due to the interactive effects of these two factors. This research…

Microfluidic "Pouch" Chips for Immunoassays and Nucleic Acid Amplification Tests.

PubMed

Mauk, Michael G; Liu, Changchun; Qiu, Xianbo; Chen, Dafeng; Song, Jinzhao; Bau, Haim H

2017-01-01

Microfluidic cassettes ("chips") for processing and analysis of clinical specimens and other sample types facilitate point-of-care (POC) immunoassays and nucleic acid based amplification tests. These single-use test chips can be self-contained and made amenable to autonomous operation-reducing or eliminating supporting instrumentation-by incorporating laminated, pliable "pouch" and membrane structures for fluid storage, pumping, mixing, and flow control. Materials and methods for integrating flexible pouch compartments and diaphragm valves into hard plastic (e.g., acrylic and polycarbonate) microfluidic "chips" for reagent storage, fluid actuation, and flow control are described. We review several versions of these pouch chips for immunoassay and nucleic acid amplification tests, and describe related fabrication techniques. These protocols thus offer a "toolbox" of methods for storage, pumping, and flow control functions in microfluidic devices.

Parvovirus B19 is a bystander in adult myocarditis.

PubMed

Koepsell, Scott A; Anderson, Daniel R; Radio, Stanley J

2012-01-01

The genomic DNA of parvovirus B19, a small single-stranded DNA virus of the genus Erythrovirus, has been shown to persist in solid tissues of constitutionally healthy, immunocompetent individuals. Despite these data, many case reports and series have linked the presence of parvovirus B19 genomic DNA, detected through nucleic acid amplification testing, with myocarditis and cardiomyopathy. Herein, we use multiple tools to better assess the relationship between parvovirus B19 and myocarditis and cardiomyopathy. Nucleic acid amplification testing, immunohistochemistry, in situ hybridization, and electron microscopy were used to assess the location and activity of parvovirus B19 in cases of myocarditis and in cases with no significant cardiac disease. Nucleic acid amplification testing for parvovirus B19 genomic DNA was positive in 73% of patients with myocarditis/cardiomyopathy and in 26% of patients with no significant disease. In situ hybridization and immunohistochemistry showed that, in cases with amplifiable parvovirus B19 DNA, parvovirus B19 genomic DNA and viral protein production were present in rare mononuclear cells. In a majority of cases of myocarditis and a significant number of otherwise normal hearts, nucleic acid amplification testing detected persistent parvovirus B19 genomic DNA that did not play a significant pathogenic role. The source of parvovirus B19 DNA appeared to be interstitial mononuclear inflammatory cells and not myocardial or endothelial cells. Therefore, nucleic acid amplification testing alone is not diagnostically helpful for determining the etiology of adult myocarditis. Copyright © 2012 Elsevier Inc. All rights reserved.

Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization.

PubMed

Pauletti, G; Godolphin, W; Press, M F; Slamon, D J

1996-07-04

Amplification and overexpression of the HER-2/neu gene occurs in 25-30% of human breast cancers. This genetic alteration is associated with a poor clinical prognosis in women with either node negative or node positive breast cancers. The initial studies testing this association were somewhat controversial and this controversy was due in large part to significant heterogeneity in both the methods and/or reagents used in testing archival material for the presence of the alteration. These methods included a number of solid matrix blotting techniques for DNA, RNA and protein as well as immunohistochemistry. Fluorescence in situ hybridization (FISH) represents the newest methodologic approach for testing for this genetic alteration. In this study, FISH is compared to Southern, Northern and Western blot analyses as well as immunohistochemistry in a large cohort of archival human breast cancer specimens. FISH was found to be superior to all other methodologies tested in assessing formalin fixed, paraffin embedded material for HER-2/neu amplification. The results from this study also confirm that overexpression of HER-2/neu rarely occurs in the absence of gene amplification in breast cancer (approximately 3% of cases). This method of analysis is rapid, reproducible and extremely reliable in detecting presence of HER-2/neu gene amplification and should have clinical utility.

Amplification in the rehabilitation of unilateral deafness: speech in noise and directional hearing effects with bone-anchored hearing and contralateral routing of signal amplification.

PubMed

Lin, Li-Mei; Bowditch, Stephen; Anderson, Michael J; May, Bradford; Cox, Kenneth M; Niparko, John K

2006-02-01

Vibromechanical stimulation with a semi-implantable bone conductor (Entific BAHA device) overcomes some of the head-shadow effects in unilateral deafness. What specific rehabilitative benefits are observed when the functional ear exhibits normal hearing versus moderate sensorineural hearing loss (SNHL)? The authors conducted a prospective trial of subjects with unilateral deafness in a tertiary care center. This study comprised adults with unilateral deafness (pure-tone average [PTA] > 90 dB; Sp.D. < 20%) and either normal monaural hearing (n = 18) or moderate SNHL (PTA = 25-50 dB: Sp.D. > 75%) in the contralateral functional ear (n = 5). Subjects were fit with contralateral routing of signal (CROS) devices for 1 month and tested before (mastoid) implantation, fitting, and testing with a bone-anchored hearing aid (BAHA). Outcome measures were: 1) subjective benefit; 2) source localization tests (Source Azimuth Identification in Noise Test [SAINT]); 3) speech discrimination in quiet and in noise assessed with Hearing In Noise Test (HINT) protocols. There was consistent satisfaction with BAHA amplification and poor acceptance of CROS amplification. General directional hearing decreased with CROS use and was unchanged by BAHA and directional microphone aids. Relative to baseline and CROS, BAHA produced significantly better speech recognition in noise. Twenty-two of 23 subjects followed up in this study continue to use their BAHA device over an average follow-up period of 30.24 months (range, 51-12 months). BAHA amplification on the side of a deaf ear yields greater benefit in subjects with monaural hearing than does CROS amplification. Advantages likely related to averting the interference of speech signals delivered to the better ear, as occurs with conventional CROS amplification, while alleviating the negative head-shadow effects of unilateral deafness. The advantages of head-shadow reduction in enhancing speech recognition with noise in the hearing ear outweigh disadvantages inherent in head-shadow reduction that can occur by introducing noise from the deaf side. The level of hearing impairment correlates with incremental benefit provided by the BAHA. Patients with a moderate SNHL in the functioning ear perceived greater increments in benefit, especially in background noise, and demonstrated greater improvements in speech understanding with BAHA amplification.

Outbreak or illusion: consequences of 'improved' diagnostics for gonorrhoea.

PubMed

Bennett, Amy; Jeffery, Katie; O'Neill, Eunan; Sherrard, Jackie

2017-06-01

The sexual health service in Oxford introduced gonorrhoea nucleic amplification acid testing using the BD Viper XTR™ System. For practical reasons, a confirmatory nucleic amplification acid testing using a different platform was not used initially. Following the introduction of nucleic amplification acid testing, the rates of gonorrhoea increased threefold. Concerns were raised that this increase represented an outbreak. A retrospective review of cases over six months suggested that there may have been a number of false-positive results. A prospective study was then undertaken over six months, where all gonorrhoea positive samples were sent for confirmatory testing. This evaluation of all gonorrhoea cases in an English county found that the overall presumptive false-positive rates for gonorrhoea nucleic amplification acid testing using BD Viper XTR™ in our population are significant at 27% of female samples, 13.2% of heterosexual male samples, 3.5% of anogenital multiple site men who have sex with men samples and 62.8% of pharyngeal only men who have sex with men samples. The data demonstrate the need for confirmatory testing using a second nucleic acid target, as per BASHH/Public Health England guidelines, especially in low-prevalence settings and extragenital sites, due to cross-reactivity with commensal Neisseria species and low positive predictive values.

PCR amplification on microarrays of gel immobilized oligonucleotides

DOEpatents

Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

2003-11-04

The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

Modular development of a prototype point of care molecular diagnostic platform for sexually transmitted infections.

PubMed

Branavan, Manoharanehru; Mackay, Ruth E; Craw, Pascal; Naveenathayalan, Angel; Ahern, Jeremy C; Sivanesan, Tulasi; Hudson, Chris; Stead, Thomas; Kremer, Jessica; Garg, Neha; Baker, Mark; Sadiq, Syed T; Balachandran, Wamadeva

2016-08-01

This paper presents the design of a modular point of care test platform that integrates a proprietary sample collection device directly with a microfluidic cartridge. Cell lysis, within the cartridge, is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample using a serpentine channel. Results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/µL and 100ng/µL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification and recombinase polymerase amplification. A low cost benchtop real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Results show that the platform is capable of detecting 1.32×10(6) of sample DNA through thermophillic helicase dependant amplification and 1×10(5) copy numbers Chlamydia trachomatis genomic DNA within 10min through recombinase polymerase nucleic acid amplification tests. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Development and Comparison of a Rapid Isothermal Nucleic Acid Amplification Test for Typing of Herpes Simplex Virus Types 1 and 2 on a Portable Fluorescence Detector

PubMed Central

Tong, Yanhong; McCarthy, Kaitlin; Kong, Huimin; Lemieux, Bertrand

2013-01-01

We have developed a rapid and simple molecular test, the IsoGlow HSV Typing assay, for the detection and typing of herpes simplex virus (type 1 and 2) from genital or oral lesions. Clinical samples suspended in viral transport mediums are simply diluted and then added to a helicase-dependent amplification master mix. The amplification and detection were performed on a portable fluorescence detector called the FireFly instrument. Detection of amplification products is based on end-point analysis using cycling probe technology. An internal control nucleic acid was included in the amplification master mix to monitor the presence of amplification inhibitors in the samples. Because the device has only two fluorescence detection channels, two strategies were developed and compared to detect the internal control template: internal control detected by melting curve analysis using a dual-labeled probe, versus internal control detection using end-point fluorescence release by a CPT probe at a lower temperature. Both have a total turnaround time of about 1 hour. Clinical performance relative to herpes viral culture was evaluated using 176 clinical specimens. Both formats of the IsoGlow HSV typing assay had sensitivities comparable to that of the Food and Drug Administration–cleared IsoAmp HSV (BioHelix Corp., Beverly MA) test and specificity for the two types of HSV comparable to that of ELVIS HSV (Diagnostic Hybrids, Athens, OH). PMID:22951487

A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection.

PubMed

Tang, Ruihua; Yang, Hui; Gong, Yan; You, MinLi; Liu, Zhi; Choi, Jane Ru; Wen, Ting; Qu, Zhiguo; Mei, Qibing; Xu, Feng

2017-03-29

Nucleic acid testing (NAT) has been widely used for disease diagnosis, food safety control and environmental monitoring. At present, NAT mainly involves nucleic acid extraction, amplification and detection steps that heavily rely on large equipment and skilled workers, making the test expensive, time-consuming, and thus less suitable for point-of-care (POC) applications. With advances in paper-based microfluidic technologies, various integrated paper-based devices have recently been developed for NAT, which however require off-chip reagent storage, complex operation steps and equipment-dependent nucleic acid amplification, restricting their use for POC testing. To overcome these challenges, we demonstrate a fully disposable and integrated paper-based sample-in-answer-out device for NAT by integrating nucleic acid extraction, helicase-dependent isothermal amplification and lateral flow assay detection into one paper device. This simple device allows on-chip dried reagent storage and equipment-free nucleic acid amplification with simple operation steps, which could be performed by untrained users in remote settings. The proposed device consists of a sponge-based reservoir and a paper-based valve for nucleic acid extraction, an integrated battery, a PTC ultrathin heater, temperature control switch and on-chip dried enzyme mix storage for isothermal amplification, and a lateral flow test strip for naked-eye detection. It can sensitively detect Salmonella typhimurium, as a model target, with a detection limit of as low as 10 2 CFU ml -1 in wastewater and egg, and 10 3 CFU ml -1 in milk and juice in about an hour. This fully disposable and integrated paper-based device has great potential for future POC applications in resource-limited settings.

Detection of mycobacterium tuberculosis in paraffin-embedded pleural biopsy specimens by commercial ribosomal RNA and DNA amplification kits.

PubMed

Ruiz-Manzano, J; Manterola, J M; Gamboa, F; Calatrava, A; Monsó, E; Martínez, C; Ausina, V

2000-09-01

To evaluate the utility of two gene amplification systems in historical paraffin-embedded pleural biopsy (PEB) tissues from patients with pleural tuberculosis, and to compare the results to those obtained with conventional histologic and microbiological methods. A retrospective study. Seventy-four formalin-fixed PEB tissues collected and stored over 12 years (1984 through 1995) were retrieved. Gene amplifications were performed in 57 tissues from patients with diagnoses of pleural tuberculosis and in 17 from patients with carcinoma as controls, using the first version of the Amplified Mycobacterium tuberculosis Direct Test (AMTDT; Gen-Probe; San Diego, CA) and the LCx Mycobacterium tuberculosis Assay (LCxMTB; Abbott Laboratories; Abbott Park, IL). The sensitivities of the AMTDT and LCxMTB were 52.6% and 63.2%, respectively (p = not statistically significant). The specificity of both tests was 100%. Twenty tissue samples (35.1%) were positive by both systems, and 10 tissues (17.5%) were positive only by the AMTDT, while 16 tissues (28.1%) were positive only by the LCxMTB. Both tests gave negative results for 11 specimens (19.3%). When both tests were used, a positive diagnosis was achieved in 80.7% of the samples. Diagnosis of 73.7% of patient conditions had previously been made by smear examination of pleural biopsy and sputum, pleural liquid, or biopsy culture. The overall diagnostic yield with both culture and amplification techniques was 96.5% (55 of 57 patients) for pleural tuberculosis, with amplification techniques adding 22.8% of the diagnoses. Amplification techniques are useful in archival PEB tissues, providing additional diagnoses beyond culturing, although the sensitivity should be improved, possibly by standardizing protocols.

Screening for Trichomonas vaginalis in a Large High-Risk Population: Prevalence Among Men and Women Determined by Nucleic Acid Amplification Testing.

PubMed

Schwebke, Jane; Merriweather, Anthony; Massingale, Sharon; Scisney, Mary; Hill, Craig; Getman, Damon

2018-05-01

Men and women attending family planning and sexually transmitted disease clinics for sexually transmitted infection screening in 2012 to 2013 were tested for Trichomonas vaginalis (TV) using a sensitive nucleic acid amplification test. T. vaginalis prevalence in urogenital samples was 11.3% in 77,740 women and 6.1% in 12,604 men, and increased with age in both sexes.

Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based analytical systems for rapid pathogen detection.

PubMed

Barreda-García, Susana; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Lobo-Castañón, María Jesús

2018-01-01

Highly sensitive testing of nucleic acids is essential to improve the detection of pathogens, which pose a major threat for public health worldwide. Currently available molecular assays, mainly based on PCR, have a limited utility in point-of-need control or resource-limited settings. Consequently, there is a strong interest in developing cost-effective, robust, and portable platforms for early detection of these harmful microorganisms. Since its description in 2004, isothermal helicase-dependent amplification (HDA) has been successfully applied in the development of novel molecular-based technologies for rapid, sensitive, and selective detection of viruses and bacteria. In this review, we highlight relevant analytical systems using this simple nucleic acid amplification methodology that takes place at a constant temperature and that is readily compatible with microfluidic technologies. Different strategies for monitoring HDA amplification products are described. In addition, we present technological advances for integrating sample preparation, HDA amplification, and detection. Future perspectives and challenges toward point-of-need use not only for clinical diagnosis but also in food safety testing and environmental monitoring are also discussed. Graphical Abstract Expanding the analytical toolbox for the detection of DNA sequences specific of pathogens with isothermal helicase dependent amplification (HDA).

Increased Sensitivity of HIV-1 p24 ELISA Using a Photochemical Signal Amplification System.

PubMed

Bystryak, Simon; Santockyte, Rasa

2015-10-01

In this study we describe a photochemical signal amplification method (PSAM) for increasing of the sensitivity of enzyme-linked immunosorbent assay (ELISA) for determination of HIV-1 p24 antigen. The photochemical signal amplification method is based on an autocatalytic photochemical reaction of a horseradish peroxidase (HRP) substrate, orthophenylenediamine (OPD). To compare the performance of PSAM-boosted ELISA with a conventional colorimetric ELISA for determination of HIV-1 p24 antigen we employed a PerkinElmer HIV-1 p24 ELISA kit, using conventional ELISA alongside ELISA + PSAM. In the present study, we show that PSAM technology allows one to increase the analytical sensitivity and dynamic range of a commercial HIV-1 p24 ELISA kit, with and without immune-complex disruption, by a factor of approximately 40-fold. ELISA + PSAM is compatible with commercially available microtiter plate readers, requires only an inexpensive illumination device, and the PSAM amplification step takes no longer than 15 min. This method can be used for both commercially available and in-house ELISA tests, and has the advantage of being considerably simpler and less costly than alternative signal amplification methods. This method can be used for both commercially available and in-house ELISA tests, and has the advantage of being considerably simpler and less costly than alternative signal amplification methods.

Prospective Clinical Evaluation of the Serologic Tuberculous Glycolipid Test in Combination with the Nucleic Acid Amplification Test

PubMed Central

Maekura, Ryoji; Kohno, Hiroaki; Hirotani, Atsushi; Okuda, Yoshinari; Ito, Masami; Ogura, Takeshi; Yano, Ikuya

2003-01-01

We have conducted a prospective controlled multicenter study to evaluate differences in the levels of clinical utility of the tuberculous glycolipid (TBGL) serodiagnostic test and the nucleic acid amplification test in patients with smear-negative active pulmonary tuberculosis (TB). The TBGL test and the PCR test were individually not so useful for the rapid diagnosis of smear-negative active pulmonary TB. However, clinical utility was considerably improved by using the TBGL test and the PCR test in combination, especially in patients with smear-negative and culture-negative active pulmonary TB and in patients with minimally advanced lesions. PMID:12624077

Development and comparison of a rapid isothermal nucleic acid amplification test for typing of herpes simplex virus types 1 and 2 on a portable fluorescence detector.

PubMed

Tong, Yanhong; McCarthy, Kaitlin; Kong, Huimin; Lemieux, Bertrand

2012-11-01

We have developed a rapid and simple molecular test, the IsoGlow HSV Typing assay, for the detection and typing of herpes simplex virus (type 1 and 2) from genital or oral lesions. Clinical samples suspended in viral transport mediums are simply diluted and then added to a helicase-dependent amplification master mix. The amplification and detection were performed on a portable fluorescence detector called the FireFly instrument. Detection of amplification products is based on end-point analysis using cycling probe technology. An internal control nucleic acid was included in the amplification master mix to monitor the presence of amplification inhibitors in the samples. Because the device has only two fluorescence detection channels, two strategies were developed and compared to detect the internal control template: internal control detected by melting curve analysis using a dual-labeled probe, versus internal control detection using end-point fluorescence release by a CPT probe at a lower temperature. Both have a total turnaround time of about 1 hour. Clinical performance relative to herpes viral culture was evaluated using 176 clinical specimens. Both formats of the IsoGlow HSV typing assay had sensitivities comparable to that of the Food and Drug Administration-cleared IsoAmp HSV (BioHelix Corp., Beverly MA) test and specificity for the two types of HSV comparable to that of ELVIS HSV (Diagnostic Hybrids, Athens, OH). Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

NASBA: A detection and amplification system uniquely suited for RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sooknanan, R.; Malek, L.T.

1995-06-01

The invention of PCR (polymerase chain reaction) has revolutionized our ability to amplify and manipulate a nucleic acid sequence in vitro. The commercial rewards of this revolution have driven the development of other nuclei acid amplification and detection methodologies. This has created an alphabet soup of technologies that use different amplification methods, including NASBA (nucleic acid sequence-based amplification), LCR (ligase chain reaction), SDA (strand displacement amplification), QBR (Q-beta replicase), CPR (cycling probe reaction), and bDNA (branched DNA). Despite the differences in their processes, these amplification systems can be separated into two broad categories based on how they achieve their goal:more » sequence-based amplification systems, such as PCR, NASBA, and SDA, amplify a target nucleic acid sequence. Signal-based amplification systems, such as LCR, QBR, CPR and bDNA, amplify or alter a signal from a detection reaction that is target-dependent. While the various methods have relative strengths and weaknesses, only NASBA offers the unique ability to homogeneously amplify an RNA analyte in the presence of homologous genomic DNA under isothermal conditions. Since the detection of RNA sequences almost invariably measures biological activity, it is an excellent prognostic indicator of activities as diverse as virus production, gene expression, and cell viability. The isothermal nature of the reaction makes NASBA especially suitable for large-scale manual screening. These features extend NASBA`s application range from research to commercial diagnostic applications. Field test kits are presently under development for human diagnostics as well as the burgeoning fields of food and environmental diagnostic testing. These developments suggest future integration of NASBA into robotic workstations for high-throughput screening as well. 17 refs., 1 tab.« less

Site Amplification in the Central U.S.: Towards and understanding of factors influencing the site effect

NASA Astrophysics Data System (ADS)

Yassminh, R.; Sandvol, E. A.

2017-12-01

We have mapped site amplification using a Reverse Two Station (RTS) approach across much of the Central United States. We have found several unexpected results including a lack of amplification in Paleozoic basins such as the Illinois and Michigan basins. In general, we found that the amplification of high frequency regional waves is related to the topography. We also suggest that the HVSR spectra are primarily a function of the shallow velocity structure. The Central United States Seismic Observatory (CUSSO) is a vertical seismic array located adjacent to the central segment of the NMSZ. CUSSO data gives us the opportunity to understand the amplification of the ground motion at different depths within the uppermost crust. Simulating ground motions throughout the CUSSO borehole and examining the factors affecting the ground amplification, such as the velocity and thicknesses of the model layers and the source sizes, is an effective way to understand the role different factors playing in modifying the ground motion for both the local and regional seismic phases. We have used the spectral-element method (SEMs) with a 1D crustal velocity structure derived from logging data taken from CUSSO borehole. This model is comprised of near surface sediment layers and a Paleozoic basement. Utilizing the software package SPECFEM2D with virtual seismometers located on the surface and in the bottom of the different sediment layers, we have computed the true synthetic site amplification for frequencies between 0.01-3 Hz. For the local model, we have tested the sensitivity of the ground motion amplification to the source magnitude. For frequencies>0.6, the ground motions have been amplified with decreasing the magnitudes while for HZ<0.6 the the horizontal amplification increases with increasing the magnitudes. The effect of the velocity of the upper 30m was tested and the result shows that decreasing the Vs30m resulted in amplifying of the ground motion and decreasing of the resonant frequency. The velocity of the lower part of the sediment basin modifies the ground motion amplification and the result shows that increasing the velocity resulted in shifting the horizontal amplification peak frequency toward a lower frequency and increasing the amplification for frequencies >2Hz.

Diagnostic Accuracy of Molecular Amplification Tests for Human African Trypanosomiasis—Systematic Review

PubMed Central

Boer, Kimberly R.; Dyserinck, Heleen C.; Büscher, Philippe; Schallig, Henk D. H. F.; Leeflang, Mariska M. G.

2012-01-01

Background A range of molecular amplification techniques have been developed for the diagnosis of Human African Trypanosomiasis (HAT); however, careful evaluation of these tests must precede implementation to ensure their high clinical accuracy. Here, we investigated the diagnostic accuracy of molecular amplification tests for HAT, the quality of articles and reasons for variation in accuracy. Methodology Data from studies assessing diagnostic molecular amplification tests were extracted and pooled to calculate accuracy. Articles were included if they reported sensitivity and specificity or data whereby values could be calculated. Study quality was assessed using QUADAS and selected studies were analysed using the bivariate random effects model. Results 16 articles evaluating molecular amplification tests fulfilled the inclusion criteria: PCR (n = 12), NASBA (n = 2), LAMP (n = 1) and a study comparing PCR and NASBA (n = 1). Fourteen articles, including 19 different studies were included in the meta-analysis. Summary sensitivity for PCR on blood was 99.0% (95% CI 92.8 to 99.9) and the specificity was 97.7% (95% CI 93.0 to 99.3). Differences in study design and readout method did not significantly change estimates although use of satellite DNA as a target significantly lowers specificity. Sensitivity and specificity of PCR on CSF for staging varied from 87.6% to 100%, and 55.6% to 82.9% respectively. Conclusion Here, PCR seems to have sufficient accuracy to replace microscopy where facilities allow, although this conclusion is based on multiple reference standards and a patient population that was not always representative. Future studies should, therefore, include patients for which PCR may become the test of choice and consider well designed diagnostic accuracy studies to provide extra evidence on the value of PCR in practice. Another use of PCR for control of disease could be to screen samples collected from rural areas and test in reference laboratories, to spot epidemics quickly and direct resources appropriately. PMID:22253934

Diagnostic accuracy of molecular amplification tests for human African trypanosomiasis--systematic review.

PubMed

Mugasa, Claire M; Adams, Emily R; Boer, Kimberly R; Dyserinck, Heleen C; Büscher, Philippe; Schallig, Henk D H F; Leeflang, Mariska M G

2012-01-01

A range of molecular amplification techniques have been developed for the diagnosis of Human African Trypanosomiasis (HAT); however, careful evaluation of these tests must precede implementation to ensure their high clinical accuracy. Here, we investigated the diagnostic accuracy of molecular amplification tests for HAT, the quality of articles and reasons for variation in accuracy. Data from studies assessing diagnostic molecular amplification tests were extracted and pooled to calculate accuracy. Articles were included if they reported sensitivity and specificity or data whereby values could be calculated. Study quality was assessed using QUADAS and selected studies were analysed using the bivariate random effects model. 16 articles evaluating molecular amplification tests fulfilled the inclusion criteria: PCR (n = 12), NASBA (n = 2), LAMP (n = 1) and a study comparing PCR and NASBA (n = 1). Fourteen articles, including 19 different studies were included in the meta-analysis. Summary sensitivity for PCR on blood was 99.0% (95% CI 92.8 to 99.9) and the specificity was 97.7% (95% CI 93.0 to 99.3). Differences in study design and readout method did not significantly change estimates although use of satellite DNA as a target significantly lowers specificity. Sensitivity and specificity of PCR on CSF for staging varied from 87.6% to 100%, and 55.6% to 82.9% respectively. Here, PCR seems to have sufficient accuracy to replace microscopy where facilities allow, although this conclusion is based on multiple reference standards and a patient population that was not always representative. Future studies should, therefore, include patients for which PCR may become the test of choice and consider well designed diagnostic accuracy studies to provide extra evidence on the value of PCR in practice. Another use of PCR for control of disease could be to screen samples collected from rural areas and test in reference laboratories, to spot epidemics quickly and direct resources appropriately.

Detection of HIV-1 p24 Gag in plasma by a nanoparticle-based bio-barcode-amplification method.

PubMed

Kim, Eun-Young; Stanton, Jennifer; Korber, Bette T M; Krebs, Kendall; Bogdan, Derek; Kunstman, Kevin; Wu, Samuel; Phair, John P; Mirkin, Chad A; Wolinsky, Steven M

2008-06-01

Detection of HIV-1 in patients is limited by the sensitivity and selectivity of available tests. The nanotechnology-based bio-barcode-amplification method offers an innovative approach to detect specific HIV-1 antigens from diverse HIV-1 subtypes. We evaluated the efficacy of this protein-detection method in detecting HIV-1 in men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). The method relies on magnetic microparticles with antibodies that specifically bind the HIV-1 p24 Gag protein and nanoparticles that are encoded with DNA and antibodies that can sandwich the target protein captured by the microparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated to remove the conjugated barcode DNA. The DNA barcodes (hundreds per target) were identified by a nanoparticle-based detection method that does not rely on PCR. Of 112 plasma samples from HIV-1-infected subjects, 111 were positive for HIV-1 p24 Gag protein (range: 0.11-71.5 ng/ml of plasma) by the bio-barcode-amplification method. HIV-1 p24 Gag protein was detected in only 23 out of 112 men by the conventional ELISA. A total of 34 uninfected subjects were negative by both tests. Thus, the specificity of the bio-barcode-amplification method was 100% and the sensitivity 99%. The bio-barcode-amplification method detected HIV-1 p24 Gag protein in plasma from all study subjects with less than 200 CD4(+) T cells/microl of plasma (100%) and 19 out of 20 (95%) HIV-1-infected men who had less than 50 copies/ml of plasma of HIV-1 RNA. In a separate group of 60 diverse international isolates, representative of clades A, B, C and D and circulating recombinant forms CRF01_AE and CRF02_AG, the bio-barcode-amplification method identified the presence of virus correctly. The bio-barcode-amplification method was superior to the conventional ELISA assay for the detection of HIV-1 p24 Gag protein in plasma with a breadth of coverage for diverse HIV-1 subtypes. Because the bio-barcode-amplification method does not require enzymatic amplification, this method could be translated into a robust point-of-care test.

Genomic Analysis Reveals a Common Breakpoint in Amplifications of the Plasmodium vivax Multidrug Resistance 1 Locus in Thailand

PubMed Central

Auburn, Sarah; Serre, David; Pearson, Richard D.; Amato, Roberto; Sriprawat, Kanlaya; To, Sheren; Handayuni, Irene; Suwanarusk, Rossarin; Russell, Bruce; Drury, Eleanor; Stalker, Jim; Miotto, Olivo; Kwiatkowski, Dominic P.; Nosten, Francois; Price, Ric N.

2016-01-01

In regions of coendemicity for Plasmodium falciparum and Plasmodium vivax where mefloquine is used to treat P. falciparum infection, drug pressure mediated by increased copy numbers of the multidrug resistance 1 gene (pvmdr1) may select for mefloquine-resistant P. vivax. Surveillance is not undertaken routinely owing in part to methodological challenges in detection of gene amplification. Using genomic data on 88 P. vivax samples from western Thailand, we identified pvmdr1 amplification in 17 isolates, all exhibiting tandem copies of a 37.6–kilobase pair region with identical breakpoints. A novel breakpoint-specific polymerase chain reaction assay was designed to detect the amplification. The assay demonstrated high sensitivity, identifying amplifications in 13 additional, polyclonal infections. Application to 132 further samples identified the common breakpoint in all years tested (2003–2015), with a decline in prevalence after 2012 corresponding to local discontinuation of mefloquine regimens. Assessment of the structure of pvmdr1 amplification in other geographic regions will yield information about the population-specificity of the breakpoints and underlying amplification mechanisms. PMID:27456706

Electricity-free amplification and detection for molecular point-of-care diagnosis of HIV-1.

PubMed

Singleton, Jered; Osborn, Jennifer L; Lillis, Lorraine; Hawkins, Kenneth; Guelig, Dylan; Price, Will; Johns, Rachel; Ebels, Kelly; Boyle, David; Weigl, Bernhard; LaBarre, Paul

2014-01-01

In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens.

Electricity-Free Amplification and Detection for Molecular Point-of-Care Diagnosis of HIV-1

PubMed Central

Singleton, Jered; Osborn, Jennifer L.; Lillis, Lorraine; Hawkins, Kenneth; Guelig, Dylan; Price, Will; Johns, Rachel; Ebels, Kelly; Boyle, David; Weigl, Bernhard; LaBarre, Paul

2014-01-01

In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens. PMID:25426953

Hearing aid fitting for visual and hearing impaired patients with Usher syndrome type IIa.

PubMed

Hartel, B P; Agterberg, M J H; Snik, A F; Kunst, H P M; van Opstal, A J; Bosman, A J; Pennings, R J E

2017-08-01

Usher syndrome is the leading cause of hereditary deaf-blindness. Most patients with Usher syndrome type IIa start using hearing aids from a young age. A serious complaint refers to interference between sound localisation abilities and adaptive sound processing (compression), as present in today's hearing aids. The aim of this study was to investigate the effect of advanced signal processing on binaural hearing, including sound localisation. In this prospective study, patients were fitted with hearing aids with a nonlinear (compression) and linear amplification programs. Data logging was used to objectively evaluate the use of either program. Performance was evaluated with a speech-in-noise test, a sound localisation test and two questionnaires focussing on self-reported benefit. Data logging confirmed that the reported use of hearing aids was high. The linear program was used significantly more often (average use: 77%) than the nonlinear program (average use: 17%). The results for speech intelligibility in noise and sound localisation did not show a significant difference between type of amplification. However, the self-reported outcomes showed higher scores on 'ease of communication' and overall benefit, and significant lower scores on disability for the new hearing aids when compared to their previous hearing aids with compression amplification. Patients with Usher syndrome type IIa prefer a linear amplification over nonlinear amplification when fitted with novel hearing aids. Apart from a significantly higher logged use, no difference in speech in noise and sound localisation was observed between linear and nonlinear amplification with the currently used tests. Further research is needed to evaluate the reasons behind the preference for the linear settings. © 2016 The Authors. Clinical Otolaryngology Published by John Wiley & Sons Ltd.

MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES

EPA Science Inventory

We have measured the levels of amplification of oncogenes and tumor marker genes or other genes of interest in nine human lung tumor cell lines in comparison to normal human bronchial epithelial cells or normal blood lymphocytes to test the hypothesis that aberrant amplification ...

EGFR amplification and expression in oral squamous cell carcinoma in young adults.

PubMed

Costa, V; Kowalski, L P; Coutinho-Camillo, C M; Begnami, M D; Calsavara, V F; Neves, J I; Kaminagakura, E

2018-07-01

The aim of this study was to investigate epidermal growth factor receptor (EGFR) gene alterations in two groups of patients with oral squamous cell carcinoma (OSCC) (a test group of subjects aged ≤40 years and a control group of subjects aged ≥50 years) and to associate the results with EGFR immunostaining, clinicopathological features, and the prognosis. Sixty cases of OSCC were selected (test group, n=21; control group, n=39). The tissue microarray technique was applied to ensure the uniformity of results. Gene amplification was analyzed by fluorescence in situ hybridization (FISH), and immunohistochemical staining for EGFR was analyzed using an automated imaging system. EGFR amplification was higher in the test group than in the control group (P=0.018) and was associated with advanced clinical stage (P=0.013), regardless of age. Patients with EGFR overexpression had worse survival rates, as did patients who had T3-T4 tumours and positive margins. EGFR overexpression has a negative impact on disease progression. Despite the higher amplification of EGFR in young adults, it does not significantly impact the survival rates of affected patients. Copyright © 2018 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Accelerated isothermal nucleic acid amplification in betaine-free reaction.

PubMed

Ma, Cuiping; Wang, Yifan; Zhang, Pansong; Shi, Chao

2017-08-01

Betaine was used as a common additive to isothermal nucleic acid amplification reactions because of lowering the melting temperature (Tm) of DNA. Herein, we reported a novel finding that betaine was inhibiting the reaction efficiency of isothermal amplification reactions. In this work, we have verified this finding by classical loop-mediated isothermal amplification that the addition of 0.8 M betaine inhibited the efficiency of reaction dropping to approximately 1%. Additionally, we clarified the mechanism of betaine hindering isothermal amplification reactions with a molecular barrier to lower associate rate constant K1 for intermolecular hybridization. This finding would be very significant for studies on the interaction between small molecule substance and DNA, and the development of point-of-care testing because of simplifying reaction system and increasing reaction efficiency. Copyright © 2017 Elsevier Inc. All rights reserved.

Construction and Evaluation of Internal Control DNA for PCR Amplification of Chlamydia trachomatis DNA from Urine Samples

PubMed Central

Betsou, Fotini; Beaumont, Katy; Sueur, Jean Marie; Orfila, Jeanne

2003-01-01

An internal control DNA (ICD) with the same primer binding sequences as the target Chlamydia trachomatis DNA was constructed and evaluated in a PCR assay with immunoenzymatic detection. One hundred urine specimens were tested, and 23 were found to contain inhibitors of the PCR, if not subjected to DNA extraction prior to amplification. Coamplification and detection of the ICD appeared to be a useful method for estimating the effects of inhibitors on C. trachomatis DNA amplification. PMID:12624066

[Research status and prospects of DNA test on difficult specimens].

PubMed

Dang, Hua-Wei; Mao, Jiong; Wang, Hui; Huang, Jiang-Ping; Bai, Xiao-Gang

2012-02-01

This paper reviews the advances of DNA detection on three types of difficult biological specimens including degraded samples, trace evidences and mixed samples. The source of different samples, processing methods and announcements were analyzed. New methods such as mitochondrial test system, changing the original experimental conditions, low-volume PCR amplification and new technologies such as whole genome amplification techniques, laser capture micro-dissection, and mini-STR technology in recent years are introduced.

Application of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cow Components Adulterated in Buffalo Milk/Meat.

PubMed

Deb, Rajib; Sengar, Gyanendra Singh; Singh, Umesh; Kumar, Sushil; Alyethodi, R R; Alex, Rani; Raja, T V; Das, A K; Prakash, B

2016-12-01

Loop-mediated isothermal amplification (LAMP) is a diagnostic method for amplification of DNA with rapid and minimal equipment requirement. In the present study, we applied the LAMP assay for rapid detection of cow components adulteration in buffalo milk/meat samples. The test can be completed within around 1 h 40 min starting from DNA extraction and can be performed in water bath without requirement of thermocycler. The cow DNA in buffalo samples were identified in the developed LAMP assay by either visualizing with SYBR Green I/HNB dyes or observing the typical ladder pattern on gel electrophoresis. The test can detect up to 5 % level of cow milk/meat mixed in buffalo counterparts. Due to the simplicity and specificity, the developed LAMP test can be easily adapted in any laboratory for rapid detection of cow species identification in livestock by products.

MYC and MYCN amplification can be reliably assessed by aCGH in medulloblastoma.

PubMed

Bourdeaut, Franck; Grison, Camille; Maurage, Claude-Alain; Laquerriere, Annie; Vasiljevic, Alexandre; Delisle, Marie-Bernadette; Michalak, Sophie; Figarella-Branger, Dominique; Doz, François; Richer, Wilfrid; Pierron, Gaelle; Miquel, Catherine; Delattre, Olivier; Couturier, Jérôme

2013-04-01

As prognostic factors, MYC and MYCN amplifications are routinely assessed in medulloblastomas. Fluorescence in situ hybridization (FISH) is currently considered as the technique of reference. Recently, array comparative genomic hybridization (aCGH) has been developed as an alternative technique to evaluate genomic abnormalities in other tumor types; however, this technique has not been widely adopted as a replacement for FISH in medulloblastoma. In this study, 34 tumors were screened by both FISH and aCGH. In all cases showing amplification by FISH, aCGH also unambiguously revealed the abnormality. The aCGH technique was also performed on tumors showing no amplification by FISH, and the absence of amplification was confirmed in all cases. Interestingly, one tumor showed a subclonal MYC amplification by FISH. This subclonal amplification was observed in approximately 20% of tumor cells and was clearly evident on aCGH. In conclusion, our analysis confirms that aCGH is as safe as FISH for the detection of MYC/MYCN gene amplification. Given its cost efficiency in comparison to two FISH tests and the global genomic information additionally provided by an aCGH experiment, this reproducible technique can be safely retained as an alternative to FISH for routine investigation of medulloblastoma. Copyright © 2013 Elsevier Inc. All rights reserved.

Correlation of MET gene amplification and TP53 mutation with PD-L1 expression in non-small cell lung cancer

PubMed Central

Albitar, Maher; Sudarsanam, Sucha; Ma, Wanlong; Jiang, Shiping; Chen, Wayne; Funari, Vincent; Blocker, Forrest; Agersborg, Sally

2018-01-01

Background The role of MET amplification in lung cancer, particularly in relation to checkpoint inhibition and EGFR WT, has not been fully explored. In this study, we correlated PD-L1 expression with MET amplification and EGFR, KRAS, or TP53 mutation in primary lung cancer. Methods In this retrospective study, tissue collected from 471 various tumors, including 397 lung cancers, was tested for MET amplification by FISH with a MET/centromere probe. PD-L1 expression was evaluated using clone SP142 and standard immunohistochemistry, and TP53, KRAS, and EGFR mutations were tested using next generation sequencing. Results Our results revealed that PD-L1 expression in non-small cell lung cancer is inversely correlated with EGFR mutation (P=0.0003), and positively correlated with TP53 mutation (P=0.0001) and MET amplification (P=0.004). Patients with TP53 mutations had significantly higher MET amplification (P=0.007), and were more likely (P=0.0002) to be EGFR wild type. There was no correlation between KRAS mutation and overall PD-L1 expression, but significant positive correlation between PD-L1 expression and KRAS with TP53 co-mutation (P=0.0002). A cut-off for the ratio of MET: centromere signal was determined as 1.5%, and 4% of lung cancer patients were identified as MET amplified. Conclusions This data suggests that in lung cancer both MET and TP53 play direct roles in regulating PD-L1 opposing EGFR. Moreover, KRAS and TP53 co-mutation may cooperate to drive PD-L1 expression in lung cancer. Adding MET or TP53 inhibitors to checkpoint inhibitors may be an attractive combination therapy in patients with lung cancer and MET amplification. PMID:29568386

The Las Vegas Valley Seismic Response Project: Ground Motions in Las Vegas Valley from Nuclear Explosions at the Nevada Test Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodgers, A; Tkalcic, H; McCallen, D

2005-03-18

Between 2001-2004 the Las Vegas Seismic Response Project has sought to understand the response of Las Vegas Valley (LVV) to seismic excitation. In this study, the author report the findings of this project with an emphasis on ground motions in LVV from nuclear explosions at the Nevada Test Site (NTS). These ground motions are used to understand building structural response and damage as well as human perception. Historical nuclear explosion observations are augmented with earthquake recordings from a temporary deployment of seismometers to improve spatial coverage of LVV. The nuclear explosions were conducted between 1968 and 1989 and were recordedmore » at various sites within Las Vegas. The data from past nuclear tests were used to constrain ground motions in LVV and to gain a predictive capability of ground motions for possible future nuclear tests at NTS. Analysis of ground motion data includes peak ground motions (accelerations and velocities) and amplification of basin sites relative to hard rock sites (site response). Site response was measured with the Standard Spectral Ratios (SSR) technique relative to hard rock reference sites on the periphery of LVV. The site response curves indicate a strong basin amplification of up to a factor of ten at frequencies between 0.5-2 Hz. Amplifications are strongest in the central and northern portions of LVV, where the basin is deeper than 1 km based on the reported basin depths of Langenheim et al (2001a). They found a strong correlation between amplification and basin depth and shallow shear wave velocities. Amplification below 1 Hz is strongly controlled by slowness-averaged shear velocities to depths of 30 and 100 meters. Depth averaged shear velocities to 10 meters has modest control of amplifications between 1-3 Hz. Modeling reveals that low velocity material in the shallow layers (< 200 m) effectively controls amplification. They developed a method to scale nuclear explosion ground motion time series to sites around LVV that have no historical record of explosions. The method is also used to scale nuclear explosion ground motions to different yields. They also present a range of studies to understand basin structure and response performed on data from the temporary deployment.« less

Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection

PubMed Central

Roskos, Kristina; Hickerson, Anna I.; Lu, Hsiang-Wei; Ferguson, Tanya M.; Shinde, Deepali N.; Klaue, Yvonne; Niemz, Angelika

2013-01-01

Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden. PMID:23922706

Frequent amplification of receptor tyrosine kinase genes in welldifferentiated/ dedifferentiated liposarcoma.

PubMed

Asano, Naofumi; Yoshida, Akihiko; Mitani, Sachiyo; Kobayashi, Eisuke; Shiotani, Bunsyo; Komiyama, Motokiyo; Fujimoto, Hiroyuki; Chuman, Hirokazu; Morioka, Hideo; Matsumoto, Morio; Nakamura, Masaya; Kubo, Takashi; Kato, Mamoru; Kohno, Takashi; Kawai, Akira; Kondo, Tadashi; Ichikawa, Hitoshi

2017-02-21

Well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) are closely related tumors commonly characterized by MDM2/CDK4 gene amplification, and lack clinically effective treatment options when inoperable. To identify novel therapeutic targets, we performed targeted genomic sequencing analysis of 19 WDLPS and 37 DDLPS tumor samples using a panel of 104 cancer-related genes (NCC oncopanel v3) developed specifically for genomic testing to select suitable molecular targeted therapies. The results of this analysis indicated that these sarcomas had very few gene mutations and a high frequency of amplifications of not only MDM2 and CDK4 but also other genes. Potential driver mutations were found in only six (11%) samples; however, gene amplification events (other than MDM2 and CDK4 amplification) were identified in 30 (54%) samples. Receptor tyrosine kinase (RTK) genes in particular were amplified in 18 (32%) samples. In addition, growth of a WDLPS cell line with IGF1R amplification was suppressed by simultaneous inhibition of CDK4 and IGF1R, using palbociclib and NVP-AEW541, respectively. Combination therapy with CDK4 and RTK inhibitors may be an effective therapeutic option for WDLPS/DDLPS patients with RTK gene amplification.

Genomic Analysis Reveals a Common Breakpoint in Amplifications of the Plasmodium vivax Multidrug Resistance 1 Locus in Thailand.

PubMed

Auburn, Sarah; Serre, David; Pearson, Richard D; Amato, Roberto; Sriprawat, Kanlaya; To, Sheren; Handayuni, Irene; Suwanarusk, Rossarin; Russell, Bruce; Drury, Eleanor; Stalker, Jim; Miotto, Olivo; Kwiatkowski, Dominic P; Nosten, Francois; Price, Ric N

2016-10-15

In regions of coendemicity for Plasmodium falciparum and Plasmodium vivax where mefloquine is used to treat P. falciparum infection, drug pressure mediated by increased copy numbers of the multidrug resistance 1 gene (pvmdr1) may select for mefloquine-resistant P. vivax Surveillance is not undertaken routinely owing in part to methodological challenges in detection of gene amplification. Using genomic data on 88 P. vivax samples from western Thailand, we identified pvmdr1 amplification in 17 isolates, all exhibiting tandem copies of a 37.6-kilobase pair region with identical breakpoints. A novel breakpoint-specific polymerase chain reaction assay was designed to detect the amplification. The assay demonstrated high sensitivity, identifying amplifications in 13 additional, polyclonal infections. Application to 132 further samples identified the common breakpoint in all years tested (2003-2015), with a decline in prevalence after 2012 corresponding to local discontinuation of mefloquine regimens. Assessment of the structure of pvmdr1 amplification in other geographic regions will yield information about the population-specificity of the breakpoints and underlying amplification mechanisms. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

In-field Raman amplification on coherent optical fiber links for frequency metrology.

PubMed

Clivati, C; Bolognini, G; Calonico, D; Faralli, S; Mura, A; Levi, F

2015-04-20

Distributed Raman amplification (DRA) is widely exploited for the transmission of broadband, modulated signals used in data links, but not yet in coherent optical links for frequency metrology, where the requirements are rather different. After preliminary tests on fiber spools, in this paper we deeper investigate Raman amplification on deployed in-field optical metrological links. We actually test a Doppler-stabilized optical link both on a 94 km-long metro-network implementation with multiplexed ITU data channels and on a 180 km-long dedicated fiber haul connecting two cities, where DRA is employed in combination with Erbium-doped fiber amplification (EDFA). The performance of DRA is detailed in both experiments, indicating that it does not introduce noticeable penalties for the metrological signal or for the ITU data channels. We hence show that Raman amplification of metrological signals can be compatible with a wavelength division multiplexing architecture and that it can be used as an alternative or in combination with dedicated bidirectional EDFAs. No deterioration is noticed in the coherence properties of the delivered signal, which attains frequency instability at the 10(-19) level in both cases. This study can be of interest also in view of the undergoing deployment of continental fiber networks for frequency metrology.

New Fpg probe chemistry for direct detection of recombinase polymerase amplification on lateral flow strips.

PubMed

Powell, Michael L; Bowler, Frank R; Martinez, Aurore J; Greenwood, Catherine J; Armes, Niall; Piepenburg, Olaf

2018-02-15

Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings. Copyright © 2017. Published by Elsevier Inc.

Effects of Instantaneous Multiband Dynamic Compression on Speech Intelligibility

NASA Astrophysics Data System (ADS)

Herzke, Tobias; Hohmann, Volker

2005-12-01

The recruitment phenomenon, that is, the reduced dynamic range between threshold and uncomfortable level, is attributed to the loss of instantaneous dynamic compression on the basilar membrane. Despite this, hearing aids commonly use slow-acting dynamic compression for its compensation, because this was found to be the most successful strategy in terms of speech quality and intelligibility rehabilitation. Former attempts to use fast-acting compression gave ambiguous results, raising the question as to whether auditory-based recruitment compensation by instantaneous compression is in principle applicable in hearing aids. This study thus investigates instantaneous multiband dynamic compression based on an auditory filterbank. Instantaneous envelope compression is performed in each frequency band of a gammatone filterbank, which provides a combination of time and frequency resolution comparable to the normal healthy cochlea. The gain characteristics used for dynamic compression are deduced from categorical loudness scaling. In speech intelligibility tests, the instantaneous dynamic compression scheme was compared against a linear amplification scheme, which used the same filterbank for frequency analysis, but employed constant gain factors that restored the sound level for medium perceived loudness in each frequency band. In subjective comparisons, five of nine subjects preferred the linear amplification scheme and would not accept the instantaneous dynamic compression in hearing aids. Four of nine subjects did not perceive any quality differences. A sentence intelligibility test in noise (Oldenburg sentence test) showed little to no negative effects of the instantaneous dynamic compression, compared to linear amplification. A word intelligibility test in quiet (one-syllable rhyme test) showed that the subjects benefit from the larger amplification at low levels provided by instantaneous dynamic compression. Further analysis showed that the increase in intelligibility resulting from a gain provided by instantaneous compression is as high as from a gain provided by linear amplification. No negative effects of the distortions introduced by the instantaneous compression scheme in terms of speech recognition are observed.

Functional characterization of the 19q12 amplicon in grade III breast cancers

PubMed Central

2012-01-01

Introduction The 19q12 locus is amplified in a subgroup of oestrogen receptor (ER)-negative grade III breast cancers. This amplicon comprises nine genes, including cyclin E1 (CCNE1), which has been proposed as its 'driver'. The aim of this study was to identify the genes within the 19q12 amplicon whose expression is required for the survival of cancer cells harbouring their amplification. Methods We investigated the presence of 19q12 amplification in a series of 313 frozen primary breast cancers and 56 breast cancer cell lines using microarray comparative genomic hybridisation (aCGH). The nine genes mapping to the smallest region of amplification on 19q12 were silenced using RNA interference in phenotypically matched breast cancer cell lines with (MDA-MB-157 and HCC1569) and without (Hs578T, MCF7, MDA-MB-231, ZR75.1, JIMT1 and BT474) amplification of this locus. Genes whose silencing was selectively lethal in amplified cells were taken forward for further validation. The effects of cyclin-dependent kinase 2 (CDK2) silencing and chemical inhibition were tested in cancer cells with and without CCNE1 amplification. Results 19q12 amplification was identified in 7.8% of ER-negative grade III breast cancer. Of the nine genes mapping to this amplicon, UQCRFS1, POP4, PLEKHF1, C19ORF12, CCNE1 and C19ORF2 were significantly over-expressed when amplified in primary breast cancers and/or breast cancer cell lines. Silencing of POP4, PLEKHF1, CCNE1 and TSZH3 selectively reduced cell viability in cancer cells harbouring their amplification. Cancer cells with CCNE1 amplification were shown to be dependent on CDK2 expression and kinase activity for their survival. Conclusions The 19q12 amplicon may harbour more than a single 'driver', given that expression of POP4, PLEKHF1, CCNE1 and TSZH3 is required for the survival of cancer cells displaying their amplification. The observation that cancer cells harbouring CCNE1 gene amplification are sensitive to CDK2 inhibitors provides a rationale for the testing of these chemical inhibitors in a subgroup of patients with ER-negative grade III breast cancers. PMID:22433433

E2F activators signal and maintain centrosome amplification in breast cancer cells.

PubMed

Lee, Mi-Young; Moreno, Carlos S; Saavedra, Harold I

2014-07-01

Centrosomes ensure accurate chromosome segregation by directing spindle bipolarity. Loss of centrosome regulation results in centrosome amplification, multipolar mitosis and aneuploidy. Since centrosome amplification is common in premalignant lesions and breast tumors, it is proposed to play a central role in breast tumorigenesis, a hypothesis that remains to be tested. The coordination between the cell and centrosome cycles is of paramount importance to maintain normal centrosome numbers, and the E2Fs may be responsible for regulating these cycles. However, the role of E2F activators in centrosome amplification is unclear. Because E2Fs are deregulated in Her2(+) cells displaying centrosome amplification, we addressed whether they signal this abnormal process. Knockdown of E2F1 or E2F3 in Her2(+) cells decreased centrosome amplification without significantly affecting cell cycle progression, whereas the overexpression of E2F1, E2F2, or E2F3 increased centrosome amplification in MCF10A mammary epithelial cells. Our results revealed that E2Fs affect the expression of proteins, including Nek2 and Plk4, known to influence the cell/centrosome cycles and mitosis. Downregulation of E2F3 resulted in cell death and delays/blocks in cytokinesis, which was reversed by Nek2 overexpression. Nek2 overexpression enhanced centrosome amplification in Her2(+) breast cancer cells silenced for E2F3, revealing a role for the E2F activators in maintaining centrosome amplification in part through Nek2.

Development and Initial Results of a Low Cost, Disposable, Point-of-Care Testing Device for Pathogen Detection

PubMed Central

Dugan, Lawrence C.; Baker, Brian R.; Hall, Sara B.; Ebert, Katja; Mioulet, Valerie; Madi, Mikidache; King, Donald P.

2011-01-01

Development of small footprint, disposable, fast, and inexpensive devices for pathogen detection in the field and clinic would benefit human and veterinary medicine by allowing evidence-based responses to future out breaks. We designed and tested an integrated nucleic acid extraction and amplification device employing a loop-mediated isothermal amplification (LAMP) or reverse transcriptase-LAMP assay. Our system provides a screening tool with polymerase-chain-reaction-level sensitivity and specificity for outbreak detection, response, and recovery. Time to result is ~90 min. The device utilizes a swab that collects sample and then transfers it to a disc of cellulose-based nucleic acid binding paper. The disc is positioned within a disposable containment tube with a manual loading port. In order to test for the presence of target pathogens, LAMP reagents are loaded through the tube’s port into contact with the sample containing cellulose disc. The reagents then are isothermally heated to 63°C for ~1 h to achieve sequence-specific target nucleic acid amplification. Due to the presence of a colorimetric dye, amplification induces visible color change in the reagents from purple to blue. As initial demonstrations, we detected methicillin resistant Staphylococcus aureus genomic DNA, as well as recombinant and live foot-and-mouth disease virus. PMID:21342806

Standardisation of polymerase chain reaction for the detection of Salmonella typhi in typhoid fever.

PubMed Central

Chaudhry, R; Laxmi, B V; Nisar, N; Ray, K; Kumar, D

1997-01-01

To improve the diagnosis of Salmonella typhi infection, a polymerase chain reaction (PCR) assay was developed for the amplification of the dH flagellin gene of S typhi. Primers were designed from dH flagellin gene sequence which will give an amplification product of 486 base pairs. In tests to study the specificity of the assay, no amplification was seen in non-salmonella strains or salmonella strains with flagellar gene other than "d". Sensitivity tests determined that 28 pg of S typhi target DNA or 3 x 10(2) target bacteria could be detected by the PCR assay. Subsequently, the PCR technique was used for detection of S typhi in blood or clot cultures from 84 patients clinically suspected of having typhoid fever, and from 20 healthy control subjects. Twenty five of 84 samples from clinically suspected cases were positive by PCR; four of which were culture negative. No amplification was seen in samples from patients who were culture positive for organisms other than S typhi or from controls. The time taken for each sample for PCR analysis was less than 48 hours compared with three to five days for blood or clot culture. PCR appeared to be a promising diagnostic test for typhoid fever. Images PMID:9215131

Development of one-step Loop-Mediated Isothermal Amplification (LAMP) for the detection of norovirus in oysters

USDA-ARS?s Scientific Manuscript database

The aim of this study was to develop a simple and rapid technique for detecting human norovirus (NoV). The loop-mediated isothermal amplification (LAMP) technique was evaluated and found to be sensitive, highly specific, and useful for routine oyster testing. Reverse transcription-LAMP (RT-LAMP) pri...

Adolescents Exiting Homelessness over Two Years: The Risk Amplification and Abatement Model

ERIC Educational Resources Information Center

Milburn, Norweeta G.; Rice, Eric; Rotheram-Borus, Mary Jane; Mallett, Shelley; Rosenthal, Doreen; Batterham, Phillip; May, Susanne J.; Witkin, Andrea; Duan, Naihua

2009-01-01

The Risk Amplification and Abatement Model (RAAM) demonstrates that negative contact with socializing agents amplify risk, while positive contact abates risk for homeless adolescents. To test this model, the likelihood of exiting homelessness and returning to familial housing at 2 years and stably exiting over time are examined with longitudinal…

A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

PubMed Central

Zhang, Yunqing; Zhang, Xinju; Xu, Xiao; Kang, Zhihua; Li, Shibao; Zhang, Chen; Su, Bing

2014-01-01

A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process. PMID:24729973

Prevention effect of rare ginsenosides against stress-hormone induced MTOC amplification

PubMed Central

Lee, Jee-Hyun; Cheong, Kyu Jin; Jung, Youn-Sang; Woo, Tae-Gyun; Yoon, Min-Ho; Oh, Ah-Young; Kang, So-Mi; Lee, Chunghui; Sun, Hokeun; Hwang, Jihwan; Song, Gyu-Yong; Park, Bum-Joon

2016-01-01

Stress has been suggested as one of important cause of human cancer without molecular biological evidence. Thus, we test the effect of stress-related hormones on cell viability and mitotic fidelity. Similarly to estrogen, stress hormone cortisol and its relative cortisone increase microtubule organizing center (MTOC) number through elevated expression of γ-tubulin and provide the Taxol resistance to human cancer cell lines. However, these effects are achieved by glucocorticoid hormone receptor (GR) but not by estrogen receptor (ER). Since ginsenosides possess steroid-like structure, we hypothesized that it would block the stress or estrogen-induced MTOC amplification and Taxol resistance. Among tested chemicals, rare ginsenoside, CSH1 (Rg6) shows obvious effect on inhibition of MTOC amplification, γ-tubulin induction and Taxol resistance. Comparing to Fulvestant (FST), ER-α specific inhibitor, this chemical can block the cortisol/cortisone-induced MTOC deregulation as well as ER-α signaling. Our results suggest that stress hormone induced tumorigenesis would be achieved by MTOC amplification, and CSH1 would be useful for prevention of stress-hormone or steroid hormone-induced chromosomal instability. PMID:27147573

An exo probe-based recombinase polymerase amplification assay for the rapid detection of porcine parvovirus.

PubMed

Wang, Jian-Chang; Liu, Li-Bing; Han, Qing-An; Wang, Jin-Feng; Yuan, Wan-Zhe

2017-10-01

Recombinase polymerase amplification (RPA), an isothermal amplification technology, has been developed as an alternative to PCR in pathogen detection. A real-time RPA assay (rt-RPA) was developed to detect the porcine parvovirus (PPV) using primers and exo probe specific for the VP2 gene. The amplification was performed at 39°C for 20min. There was no cross-reaction with other pathogens tested. Using the recombinant plasmid pPPV-VP2 as template, the analytical sensitivity was 103 copies. The assay performance was evaluated by testing 115 field samples by rt-RPA and a real-time PCR assay. The diagnostic agreement between assays was 100%, and PPV DNA was detected in 94 samples. The R 2 value of rt-RPA and real-time PCR was 0.909 by linear regression analysis. The developed rt-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of PPV in diagnostic laboratories and at point-of-care, especially in remote and rural areas. Copyright © 2017 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strizhkov, B. N.; Drobyshev, A. L.; Mikhailovich, V. M.

PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency,more » the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.« less

Pre-amplification in the context of high-throughput qPCR gene expression experiment.

PubMed

Korenková, Vlasta; Scott, Justin; Novosadová, Vendula; Jindřichová, Marie; Langerová, Lucie; Švec, David; Šídová, Monika; Sjöback, Robert

2015-03-11

With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm). For evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted. We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step.

Development of a Rapid Point-of-Use DNA Test for the Screening of Genuity® Roundup Ready 2 Yield® Soybean in Seed Samples.

PubMed

Chandu, Dilip; Paul, Sudakshina; Parker, Mathew; Dudin, Yelena; King-Sitzes, Jennifer; Perez, Tim; Mittanck, Don W; Shah, Manali; Glenn, Kevin C; Piepenburg, Olaf

2016-01-01

Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings.

Novel Degenerate PCR Method for Whole-Genome Amplification Applied to Peru Margin (ODP Leg 201) Subsurface Samples

PubMed Central

Martino, Amanda J.; Rhodes, Matthew E.; Biddle, Jennifer F.; Brandt, Leah D.; Tomsho, Lynn P.; House, Christopher H.

2011-01-01

A degenerate polymerase chain reaction (PCR)-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. While optimized here for use with Roche 454 technology, the general framework presented may be applicable to other next generation sequencing systems as well (e.g., Illumina, Ion Torrent). The method, which we have called random amplification metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3′ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10×. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa identified illustrates well the generally accepted view that community analysis is sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low-biomass samples. PMID:22319519

Loop-Mediated Isothermal Amplification for Detection of Endogenous Sad1 Gene in Cotton: An Internal Control for Rapid Onsite GMO Testing.

PubMed

Singh, Monika; Bhoge, Rajesh K; Randhawa, Gurinderjit

2018-04-20

Background : Confirming the integrity of seed samples in powdered form is important priorto conducting a genetically modified organism (GMO) test. Rapid onsite methods may provide a technological solution to check for genetically modified (GM) events at ports of entry. In India, Bt cotton is the commercialized GM crop with four approved GM events; however, 59 GM events have been approved globally. GMO screening is required to test for authorized GM events. The identity and amplifiability of test samples could be ensured first by employing endogenous genes as an internal control. Objective : A rapid onsite detection method was developed for an endogenous reference gene, stearoyl acyl carrier protein desaturase ( Sad1 ) of cotton, employing visual and real-time loop-mediated isothermal amplification (LAMP). Methods : The assays were performed at a constant temperature of 63°C for 30 min for visual LAMP and 62ºC for 40 min for real-time LAMP. Positive amplification was visualized as a change in color from orange to green on addition of SYBR ® Green or detected as real-time amplification curves. Results : Specificity of LAMP assays was confirmed using a set of 10 samples. LOD for visual LAMP was up to 0.1%, detecting 40 target copies, and for real-time LAMP up to 0.05%, detecting 20 target copies. Conclusions : The developed methods could be utilized to confirm the integrity of seed powder prior to conducting a GMO test for specific GM events of cotton. Highlights : LAMP assays for the endogenous Sad1 gene of cotton have been developed to be used as an internal control for onsite GMO testing in cotton.

Lack of correlation between reaction speed and analytical sensitivity in isothermal amplification reveals the value of digital methods for optimization: validation using digital real-time RT-LAMP

PubMed Central

Khorosheva, Eugenia M.; Karymov, Mikhail A.; Selck, David A.; Ismagilov, Rustem F.

2016-01-01

In this paper, we asked if it is possible to identify the best primers and reaction conditions based on improvements in reaction speed when optimizing isothermal reactions. We used digital single-molecule, real-time analyses of both speed and efficiency of isothermal amplification reactions, which revealed that improvements in the speed of isothermal amplification reactions did not always correlate with improvements in digital efficiency (the fraction of molecules that amplify) or with analytical sensitivity. However, we observed that the speeds of amplification for single-molecule (in a digital device) and multi-molecule (e.g. in a PCR well plate) formats always correlated for the same conditions. Also, digital efficiency correlated with the analytical sensitivity of the same reaction performed in a multi-molecule format. Our finding was supported experimentally with examples of primer design, the use or exclusion of loop primers in different combinations, and the use of different enzyme mixtures in one-step reverse-transcription loop-mediated amplification (RT-LAMP). Our results show that measuring the digital efficiency of amplification of single-template molecules allows quick, reliable comparisons of the analytical sensitivity of reactions under any two tested conditions, independent of the speeds of the isothermal amplification reactions. PMID:26358811

Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

PubMed

Boyle, David S; McNerney, Ruth; Teng Low, Hwee; Leader, Brandon Troy; Pérez-Osorio, Ailyn C; Meyer, Jessica C; O'Sullivan, Denise M; Brooks, David G; Piepenburg, Olaf; Forrest, Matthew S

2014-01-01

Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets.

PubMed

Seok, Youngung; Joung, Hyou-Arm; Byun, Ju-Young; Jeon, Hyo-Sung; Shin, Su Jeong; Kim, Sanghyo; Shin, Young-Beom; Han, Hyung Soo; Kim, Min-Gon

2017-01-01

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 10 2 -10 5 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets

PubMed Central

Seok, Youngung; Joung, Hyou-Arm; Byun, Ju-Young; Jeon, Hyo-Sung; Shin, Su Jeong; Kim, Sanghyo; Shin, Young-Beom; Han, Hyung Soo; Kim, Min-Gon

2017-01-01

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing. PMID:28740546

Identification of Clinical Coryneform Bacterial Isolates: Comparison of Biochemical Methods and Sequence Analysis of 16S rRNA and rpoB Genes▿

PubMed Central

Adderson, Elisabeth E.; Boudreaux, Jan W.; Cummings, Jessica R.; Pounds, Stanley; Wilson, Deborah A.; Procop, Gary W.; Hayden, Randall T.

2008-01-01

We compared the relative levels of effectiveness of three commercial identification kits and three nucleic acid amplification tests for the identification of coryneform bacteria by testing 50 diverse isolates, including 12 well-characterized control strains and 38 organisms obtained from pediatric oncology patients at our institution. Between 33.3 and 75.0% of control strains were correctly identified to the species level by phenotypic systems or nucleic acid amplification assays. The most sensitive tests were the API Coryne system and amplification and sequencing of the 16S rRNA gene using primers optimized for coryneform bacteria, which correctly identified 9 of 12 control isolates to the species level, and all strains with a high-confidence call were correctly identified. Organisms not correctly identified were species not included in the test kit databases or not producing a pattern of reactions included in kit databases or which could not be differentiated among several genospecies based on reaction patterns. Nucleic acid amplification assays had limited abilities to identify some bacteria to the species level, and comparison of sequence homologies was complicated by the inclusion of allele sequences obtained from uncultivated and uncharacterized strains in databases. The utility of rpoB genotyping was limited by the small number of representative gene sequences that are currently available for comparison. The correlation between identifications produced by different classification systems was poor, particularly for clinical isolates. PMID:18160450

E2F Activators Signal and Maintain Centrosome Amplification in Breast Cancer Cells

PubMed Central

Lee, Mi-Young; Moreno, Carlos S.

2014-01-01

Centrosomes ensure accurate chromosome segregation by directing spindle bipolarity. Loss of centrosome regulation results in centrosome amplification, multipolar mitosis and aneuploidy. Since centrosome amplification is common in premalignant lesions and breast tumors, it is proposed to play a central role in breast tumorigenesis, a hypothesis that remains to be tested. The coordination between the cell and centrosome cycles is of paramount importance to maintain normal centrosome numbers, and the E2Fs may be responsible for regulating these cycles. However, the role of E2F activators in centrosome amplification is unclear. Because E2Fs are deregulated in Her2+ cells displaying centrosome amplification, we addressed whether they signal this abnormal process. Knockdown of E2F1 or E2F3 in Her2+ cells decreased centrosome amplification without significantly affecting cell cycle progression, whereas the overexpression of E2F1, E2F2, or E2F3 increased centrosome amplification in MCF10A mammary epithelial cells. Our results revealed that E2Fs affect the expression of proteins, including Nek2 and Plk4, known to influence the cell/centrosome cycles and mitosis. Downregulation of E2F3 resulted in cell death and delays/blocks in cytokinesis, which was reversed by Nek2 overexpression. Nek2 overexpression enhanced centrosome amplification in Her2+ breast cancer cells silenced for E2F3, revealing a role for the E2F activators in maintaining centrosome amplification in part through Nek2. PMID:24797070

Gonorrhea Test

MedlinePlus

... for Targeted Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker ... NAT Neisseria gonorrhoeae Nucleic Acid Amplification Test, Culture, Gram Stain, DNA Probe Formal Name Neisseria gonorrhoeae This article ...

The implementation of nucleic acid amplification technology testing for living tissue donors.

PubMed

Westby, J; Lomas, R J; Kearney, J N

2010-05-01

There is a significant requirement within the United Kingdom for tissue grafts from living donors. To ensure safety, blood samples from these donors are tested for pathogens at donation, and at 180 days post donation. Nucleic acid amplification technology (NAT) permits more sensitive detection of pathogens in blood samples than serum antigen testing. NAT testing can be applied to samples from living tissue donors to eliminate the need to re-test these donors 180 days post-donation before grafts can be implanted. This has major financial and operational advantages for a tissue bank, and this manuscript describes how NAT testing was assessed and implemented by NHSBT Tissue Services. When compared to traditional serum antigen testing, NAT testing was more cost effective, more convenient for donors and resulted in a greater proportion of donated grafts being made available for transplant.

Polymer-based microfluidic chips for isothermal amplification of nucleic acids

NASA Astrophysics Data System (ADS)

Posmitnaya, Y. S.; Rudnitskaya, G. E.; Tupik, A. N.; Lukashenko, T. A.; Bukatin, A. C.; Evstrapov, A. A.

2017-11-01

Creation of low-cost compact devices based on microfluidic platforms for biological and medical research depends on the degree of development and enhancement of prototyping technologies. Two designs of polymer and hybrid microfluidic devices fabricated by soft lithography and intended for isothermal amplification and polymerase chain reaction are presented in this paper. The digital helicase-dependent isothermal amplification was tested in the device containing a droplet generator. Polymerase chain reaction was carried out in the hybrid microfluidic device having ten reaction chambers. A synthesized cDNA fragment of GAPDH housekeeping gene was used as a target.

Loop-Mediated Isothermal Amplification Test for Trypanosoma gambiense Group 1 with Stem Primers: A Molecular Xenomonitoring Test for Sleeping Sickness.

PubMed

Njiru, Zablon K; Mbae, Cecilia K; Mburugu, Gitonga N

2017-01-01

The World Health Organization has targeted Human African Trypanosomiasis (HAT) for elimination by 2020 with zero incidence by 2030. To achieve and sustain this goal, accurate and easy-to-deploy diagnostic tests for Gambian trypanosomiasis which accounts for over 98% of reported cases will play a crucial role. Most needed will be tools for surveillance of pathogen in vectors (xenomonitoring) since population screening tests are readily available. The development of new tests is expensive and takes a long time while incremental improvement of existing technologies that have potential for xenomonitoring may offer a shorter pathway to tools for HAT surveillance. We have investigated the effect of including a second set of reaction accelerating primers (stem primers) to the standard T. brucei gambiense LAMP test format. The new test format was analyzed with and without outer primers. Amplification was carried out using Rotorgene 6000 and the portable ESE Quant amplification unit capable of real-time data output. The stem LAMP formats indicated shorter time to results (~8 min), were 10-100-fold more sensitive, and indicated higher diagnostic sensitivity and accuracy compared to the standard LAMP test. It was possible to confirm the predicted product using ESE melt curves demonstrating the potential of combining LAMP and real-time technologies as possible tool for HAT molecular xenomonitoring.

Human epidermal growth factor receptor 2 testing in invasive breast cancer: should histological grade, type and oestrogen receptor status influence the decision to repeat testing?

PubMed

Rakha, Emad A; Pigera, Marian; Shin, Sandra J; D'Alfonso, Timothy; Ellis, Ian O; Lee, Andrew H S

2016-07-01

The recent American Society of Clinical Oncology/College of American Pathologists guidelines for human epidermal growth factor receptor 2 (HER2) testing in breast cancer recommend repeat testing based on tumour grade, tumour type, and hormone receptor status. The aim of this study was to test the value of these criteria. HER2 status was concordant in the core biopsies and excision specimens in 392 of 400 invasive carcinomas. The major reasons for discordance were amplification around the cut-off for positivity and tumour heterogeneity. Of 116 grade 3 carcinomas that were HER2-negative in the core biopsy, four were HER2-positive in the excision specimen. Three of these four either showed borderline negative amplification in the core biopsy or were heterogeneous. None of the 55 grade 1 carcinomas were HER2-positive. Review of repeat testing of HER2 in routine practice suggested that it may also be of value for multifocal tumours and if recommended by the person assessing the in-situ hybridization. Mandatory repeat HER2 testing of grade 3 HER2-negative carcinomas is not appropriate. This is particularly true if repeat testing is performed after borderline negative amplification in the core biopsy or in HER2-negative heterogeneous carcinomas. © 2015 John Wiley & Sons Ltd.

AccuCopy quantification combined with pre-amplification of long-distance PCR for fast analysis of intron 22 inversion in haemophilia A.

PubMed

Ding, Qianlan; Wu, Xi; Lu, Yeling; Chen, Changming; Shen, Rui; Zhang, Xi; Jiang, Zhengwen; Wang, Xuefeng

2016-07-01

To develop a digitalized intron 22 inversion (Inv22) detection in patients with severe haemophilia A. The design included two tests: A genotyping test included two multiplex pre-amplification of LD-PCR (PLP) with two combinations of five primers to amplify wild-type and chimeric int22h alleles; a carrier mosaicism test was similar to the genotyping test except only amplification of chimeric int22h alleles by removing one primer from each of two combinations. AccuCopy detection was used to quantify PLP products. PLP product patterns in the genotyping test allowed identifying all known Inv22. Quantitative patterns accurately represented the product patterns. The results of 164 samples detected by the genotyping test were consistent with those obtained by LD-PCR detection. Limit of detection (LOD) of the carrier mosaicism test was at least 2% of heterozygous cells with Inv22. Performing the test in two obligate mothers with negative Inv22 from two sporadic pedigrees mosaic rates of blood and hair root of the mother from pedigree 1 were 8.3% and >20%, respectively and negative results were obtained in pedigree 2. AccuCopy quantification combined with PLP (AQ-PLP) method was confirmed to be rapid and reliable for genotyping Inv22 and highly sensitive to carrier mosaicism detection. Copyright © 2016 Elsevier B.V. All rights reserved.

Word Recognition and Learning: Effects of Hearing Loss and Amplification Feature

PubMed Central

Stewart, Elizabeth C.; Willman, Amanda P.; Odgear, Ian S.

2017-01-01

Two amplification features were examined using auditory tasks that varied in stimulus familiarity. It was expected that the benefits of certain amplification features would increase as the familiarity with the stimuli decreased. A total of 20 children and 15 adults with normal hearing as well as 21 children and 17 adults with mild to severe hearing loss participated. Three models of ear-level devices were selected based on the quality of the high-frequency amplification or the digital noise reduction (DNR) they provided. The devices were fitted to each participant and used during testing only. Participants completed three tasks: (a) word recognition, (b) repetition and lexical decision of real and nonsense words, and (c) novel word learning. Performance improved significantly with amplification for both the children and the adults with hearing loss. Performance improved further with wideband amplification for the children more than for the adults. In steady-state noise and multitalker babble, performance decreased for both groups with little to no benefit from amplification or from the use of DNR. When compared with the listeners with normal hearing, significantly poorer performance was observed for both the children and adults with hearing loss on all tasks with few exceptions. Finally, analysis of across-task performance confirmed the hypothesis that benefit increased as the familiarity of the stimuli decreased for wideband amplification but not for DNR. However, users who prefer DNR for listening comfort are not likely to jeopardize their ability to detect and learn new information when using this feature. PMID:29169314

Prognostic relevance of Fédération Nationale des Centres de Lutte Contre le Cancer grade and MDM2 amplification levels in dedifferentiated liposarcoma: a study of 50 cases.

PubMed

Jour, George; Gullet, Ashley; Liu, Mingdong; Hoch, Benjamin L

2015-01-01

Dedifferentiated liposarcoma represents a form of liposarcoma composed of a non-lipogenic sarcoma associated with well-differentiated liposarcoma. The prognostic significance of histological grading of the dedifferentiated component remains to be elucidated due to vague grading criteria employed in previous studies. Molecular markers of tumor behavior, including amplification levels of murine double minute-2 (MDM2) and cyclin-dependent kinase-4 (CDK4) genes, have been explored in a limited number of cases. Here we investigate whether 'Fédération Nationale des Centres de Lutte Contre le Cancer' (FNCLCC) grade and MDM2 gene amplification levels have prognostic value in dedifferentiated liposarcoma in terms of local recurrence and disease-specific survival. Fifty cases were retrieved, reviewed and FNCLCC grade was scored for the dedifferentiated component. Testing for MDM2 gene amplification was performed by fluorescence in situ hybridization. Amplification was categorized as high level (≥20 copies) and as low level (<20 copies). Follow-up data was obtained through chart review. Log-rank test and Cox proportional hazard models were used to determine the effect of grade and level of MDM2 amplification on outcomes. Our series includes 50 patients (male n=28, female n=22) with an average age of 63 years (range, 28-88) and a median follow-up of 28 months (range, 2-120). Tumors were graded as grade 1 (6%), grade 2 (58%), and grade 3 (36%). When adjusted for age, sex, site, tumor size, and margin status, grade 3 patients had a higher recurrence rate than grades 1 and 2 (HR=2.07, 95% CI: 1.24, 7.62; P=0.015). Patients with high-level MDM2 amplification had higher recurrence rate on univariate analysis (P=0.028), but not on multivariate analysis (HR=1.69, 95% CI: 0.73, 3.94; P=0.221). FNCLCC grade 3 dedifferentiation confers a worse prognosis in dedifferentiated liposarcoma in terms of local recurrence. MDM2 amplification level remains a useful diagnostic tool in dedifferentiated liposarcoma, but has no prognostic value in terms of local recurrence.

Comparison of measurement methods for capacitive tactile sensors and their implementation

NASA Astrophysics Data System (ADS)

Tarapata, Grzegorz; Sienkiewicz, Rafał

2015-09-01

This paper presents a review of ideas and implementations of measurement methods utilized for capacity measurements in tactile sensors. The paper describes technical method, charge amplification method, generation and as well integration method. Three selected methods were implemented in dedicated measurement system and utilised for capacitance measurements of ourselves made tactile sensors. The tactile sensors tested in this work were fully fabricated with the inkjet printing technology. The tests result were presented and summarised. The charge amplification method (CDC) was selected as the best method for the measurement of the tactile sensors.

Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

PubMed Central

Ullrich, Thomas; Ermantraut, Eugen; Schulz, Torsten; Steinmetzer, Katrin

2012-01-01

Background State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests. PMID:22539973

Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA

NASA Technical Reports Server (NTRS)

Polacek, Denise C.; Passerini, Anthony G.; Shi, Congzhu; Francesco, Nadeene M.; Manduchi, Elisabetta; Grant, Gregory R.; Powell, Steven; Bischof, Helen; Winkler, Hans; Stoeckert, Christian J Jr;

Direct solar-pumped iodine laser amplifier

NASA Technical Reports Server (NTRS)

Han, Kwang S.; Hwang, In Heon

1990-01-01

The optimum conditions of a solar pumped iodine laser are found in this research for the case of a continuous wave operation and a pulsed operation. The optimum product of the pressure(p) inside the laser tube and the tube diameter(d) was pd=40 approx. 50 torr-cm on the contrary to the case of a high intensity flashlamp pumped iodine laser where the optimum value of the product is known to be pd=150 torr-cm. The pressure-diameter product is less than 1/3 of that of the high power iodine laser. During the research period, various laser materials were also studied for solar pumping. Among the laser materials, Nd:YAG is found to have the lowest laser threshold pumping intensity of about 200 solar constant. The Rhodamine 6G was also tested as the solar pumped laser material. The threshold pumping power was measured to be about 20,000 solar constant. The amplification experiment for a continuously pumped iodine laser amplifier was performed using Vortek solar simulator and the amplification factors were measured for single pass amplification and triple pass amplification of the 15 cm long amplifier tube. The amplification of 5 was obtained for the triple pass amplification.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

PubMed

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges.

PubMed

Qing, Taiping; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Wen, Li; Shangguan, Jingfang; Mao, Zhengui; Lei, Yanli

2016-04-01

Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

PubMed Central

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

2008-01-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains.

PubMed

Di Bernardo, Giovanni; Del Gaudio, Stefania; Cammarota, Marcella; Galderisi, Umberto; Cascino, Antonino; Cipollaro, Marilena

2002-02-15

Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (epsilon globin, p53 and gamma interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to alpha(lambda)mu(omicron)sigma(tau) 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed.

Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains

PubMed Central

Di Bernardo, Giovanni; Del Gaudio, Stefania; Cammarota, Marcella; Galderisi, Umberto; Cascino, Antonino; Cipollaro, Marilena

2002-01-01

Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (ɛ globin, p53 and γ interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to αλµοστ 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed. PMID:11842122

Femtosecond optical parametric amplification in BBO and KTA driven by a Ti:sapphire laser for LIDT testing and diagnostic development

NASA Astrophysics Data System (ADS)

Meadows, Alexander R.; Cupal, Josef; Hříbek, Petr; Durák, Michal; Kramer, Daniel; Rus, Bedřich

2017-05-01

We present the design of a collinear femtosecond optical parametric amplification (OPA) system producing a tunable output at wavelengths between 1030 nm and 1080 nm from a Ti:Sapphire pump laser at a wavelength of 795 nm. Generation of a supercontinuum seed pulse is followed by one stage of amplification in Beta Barium Borate (BBO) and two stages of amplification in Potassium Titanyle Arsenate (KTA), resulting in a 225 μJ output pulse with a duration of 90 fs. The output of the system has been measured by self-referenced spectral interferometry to yield the complete spectrum and spectral phase of the pulse. When compared to KTP, the greater transparency of KTA in the spectral range from 3 - 4 μm allows for reduced idler absorption and enhanced gain from the OPA process when it is pumped by the fundamental frequency of a Ti:sapphire laser. In turn, the use of the Ti:sapphire fundamental at 795 nm as a pump improves the efficiency with which light can be converted to wavelengths between 1030 nm and 1080 nm and subsequently used to test components for Nd-based laser systems. This OPA system is operated at 1 kHz for diagnostic development and laser-induced damage threshold testing of optical components for the ELI-Beamlines project.

Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones

PubMed Central

2016-01-01

Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests. PMID:26900709

System for portable nucleic acid testing in low resource settings

NASA Astrophysics Data System (ADS)

Lu, Hsiang-Wei; Roskos, Kristina; Hickerson, Anna I.; Carey, Thomas; Niemz, Angelika

2013-03-01

Our overall goal is to enable timely diagnosis of infectious diseases through nucleic acid testing at the point-of-care and in low resource settings, via a compact system that integrates nucleic acid sample preparation, isothermal DNA amplification, and nucleic acid lateral flow (NALF) detection. We herein present an interim milestone, the design of the amplification and detection subsystem, and the characterization of thermal and fluidic control and assay execution within this system. Using an earlier prototype of the amplification and detection unit, comprised of a disposable cartridge containing flexible pouches, passive valves, and electrolysis-driven pumps, in conjunction with a small heater, we have demonstrated successful execution of an established and clinically validated isothermal loop-mediated amplification (LAMP) reaction targeting Mycobacterium tuberculosis (M.tb) DNA, coupled to NALF detection. The refined design presented herein incorporates miniaturized and integrated electrolytic pumps, novel passive valves, overall design changes to facilitate integration with an upstream sample preparation unit, and a refined instrument design that automates pumping, heating, and timing. Nucleic acid amplification occurs in a two-layer pouch that facilitates fluid handling and appropriate thermal control. The disposable cartridge is manufactured using low-cost and scalable techniques and forms a closed system to prevent workplace contamination by amplicons. In a parallel effort, we are developing a sample preparation unit based on similar design principles, which performs mechanical lysis of mycobacteria and DNA extraction from liquefied and disinfected sputum. Our next step is to combine sample preparation, amplification, and detection in a final integrated cartridge and device, to enable fully automated sample-in to answer-out diagnosis of active tuberculosis in primary care facilities of low-resource and high-burden countries.

Pretreatment Dynamic Susceptibility Contrast MRI Perfusion in Glioblastoma: Prediction of EGFR Gene Amplification.

PubMed

Gupta, A; Young, R J; Shah, A D; Schweitzer, A D; Graber, J J; Shi, W; Zhang, Z; Huse, J; Omuro, A M P

2015-06-01

Molecular and genetic testing is becoming increasingly relevant in GBM. We sought to determine whether dynamic susceptibility contrast (DSC) magnetic resonance imaging (MRI) perfusion imaging could predict EGFR-defined subtypes of GBM. We retrospectively identified 106 consecutive glioblastoma (GBM) patients with known EGFR gene amplification, and a subset of 65 patients who also had known EGFRvIII gene mutation status. All patients underwent T2* DSC MRI perfusion. DSC perfusion maps and T2* signal intensity time curves were evaluated, and the following measures of tumor perfusion were recorded: (1) maximum relative cerebral blood volume (rCBV), (2) relative peak height (rPH), and (3) percent signal recovery (PSR). The imaging metrics were correlated to EGFR gene amplification and EGFRvIII mutation status using univariate analyses. EGFR amplification was present in 44 (41.5 %) subjects and absent in 62 (58.5 %). Among the 65 subjects who had undergone EGFRvIII mutation transcript analysis, 18 subjects (27.7 %) tested positive for the EGFRvIII mutation, whereas 47 (72.3 %) did not. Higher median rCBV (3.31 versus 2.62, p = 0.01) and lower PSR (0.70 versus 0.78, p = 0.03) were associated with high levels of EGFR amplification. Higher median rPH (3.68 versus 2.76, p = 0.03) was associated with EGFRvIII mutation. DSC MRI perfusion may have a role in identifying patients with EGFR gene amplification and EGFRvIII gene mutation status, potential targets for individualized treatment protocols. Our results raise the need for further investigation for imaging biomarkers of genetically unique GBM subtypes.

Hearing Loss and Cognitive-Communication Test Performance of Long-Term Care Residents With Dementia: Effects of Amplification

ERIC Educational Resources Information Center

Hopper, Tammy; Slaughter, Susan E.; Hodgetts, Bill; Ostevik, Amberley; Ickert, Carla

2016-01-01

Purpose: The study aims were (a) to explore the relationship between hearing loss and cognitive-communication performance of individuals with dementia, and (b) to determine if hearing loss is accurately identified by long-term care (LTC) staff. The research questions were (a) What is the effect of amplification on cognitive-communication test…

Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

PubMed

Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

2015-02-01

In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on paraffin-embedded material to test for other diagnostically, prognostically, or therapeutically relevant genomic mutations in lipomatous tumors.

Lack of correlation between reaction speed and analytical sensitivity in isothermal amplification reveals the value of digital methods for optimization: validation using digital real-time RT-LAMP.

PubMed

Khorosheva, Eugenia M; Karymov, Mikhail A; Selck, David A; Ismagilov, Rustem F

2016-01-29

In this paper, we asked if it is possible to identify the best primers and reaction conditions based on improvements in reaction speed when optimizing isothermal reactions. We used digital single-molecule, real-time analyses of both speed and efficiency of isothermal amplification reactions, which revealed that improvements in the speed of isothermal amplification reactions did not always correlate with improvements in digital efficiency (the fraction of molecules that amplify) or with analytical sensitivity. However, we observed that the speeds of amplification for single-molecule (in a digital device) and multi-molecule (e.g. in a PCR well plate) formats always correlated for the same conditions. Also, digital efficiency correlated with the analytical sensitivity of the same reaction performed in a multi-molecule format. Our finding was supported experimentally with examples of primer design, the use or exclusion of loop primers in different combinations, and the use of different enzyme mixtures in one-step reverse-transcription loop-mediated amplification (RT-LAMP). Our results show that measuring the digital efficiency of amplification of single-template molecules allows quick, reliable comparisons of the analytical sensitivity of reactions under any two tested conditions, independent of the speeds of the isothermal amplification reactions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Error amplification to promote motor learning and motivation in therapy robotics.

PubMed

Shirzad, Navid; Van der Loos, H F Machiel

2012-01-01

To study the effects of different feedback error amplification methods on a subject's upper-limb motor learning and affect during a point-to-point reaching exercise, we developed a real-time controller for a robotic manipulandum. The reaching environment was visually distorted by implementing a thirty degrees rotation between the coordinate systems of the robot's end-effector and the visual display. Feedback error amplification was provided to subjects as they trained to learn reaching within the visually rotated environment. Error amplification was provided either visually or through both haptic and visual means, each method with two different amplification gains. Subjects' performance (i.e., trajectory error) and self-reports to a questionnaire were used to study the speed and amount of adaptation promoted by each error amplification method and subjects' emotional changes. We found that providing haptic and visual feedback promotes faster adaptation to the distortion and increases subjects' satisfaction with the task, leading to a higher level of attentiveness during the exercise. This finding can be used to design a novel exercise regimen, where alternating between error amplification methods is used to both increase a subject's motor learning and maintain a minimum level of motivational engagement in the exercise. In future experiments, we will test whether such exercise methods will lead to a faster learning time and greater motivation to pursue a therapy exercise regimen.

Evaluation of the efficacy of constitutional array-based comparative genomic hybridization in the diagnosis of aneuploidy using genomic and amplified DNA.

PubMed

Tan, Niap H; Palmer, Rodger; Wang, Rubin

2010-02-01

Array-based comparative genomic hybridization (array CGH) is a new molecular technique that has the potential to revolutionize cytogenetics. However, use of high resolution array CGH in the clinical setting is plagued by the problem of widespread copy number variations (CNV) in the human genome. Constitutional microarray, containing only clones that interrogate regions of known constitutional syndromes, may circumvent the dilemma of detecting CNV of unknown clinical significance. The present study investigated the efficacy of constitutional microarray in the diagnosis of trisomy. Test samples included genomic DNA from trisomic cell lines, amplification products of 50 ng of genomic DNA and whole genome amplification products of single cells. DNA amplification was achieved by means of multiple displacement amplification (MDA) over 16 h. The trisomic and sex chromosomes copy number imbalances in the genomic DNA were correctly identified by the constitutional microarrays. However, there was a failure to detect the trisomy in the amplification products of 50 ng of genomic DNA and whole genome amplification products of single cells. Using carefully selected clones, Spectral Genomics constitutional microarray was able to detect the chromosomal copy number imbalances in genomic DNA without the confounding effects of CNV. The diagnostic failure in amplified DNA samples could be attributed to the amplification process. The MDA duration of 16 h generated excessive amount of biases and shortening the duration might minimize the problem.

Rapid diagnosis of equine influenza by highly sensitive silver amplification immunochromatography system.

PubMed

Yamanaka, Takashi; Nemoto, Manabu; Bannai, Hiroshi; Tsujimura, Koji; Kondo, Takashi; Matsumura, Tomio; Fu, Tao Qi Huang; Fernandez, Charlene Judith; Gildea, Sarah; Cullinane, Ann

2017-06-16

Equine influenza (EI) is a respiratory disease caused by equine influenza A virus (EIV, H3N8) infection. Rapid diagnosis is essential to limit the disease spread. We previously reported that some rapid antigen detection (RAD) tests are fit for diagnosing EI although their sensitivity is not optimal. Here, we evaluated the performance of the newly developed RAD test using silver amplification immunochromatography (Quick Chaser Auto Flu A, B: QCA) to diagnose EI. The detection limits of QCA for EIVs were five-fold lower than the conventional RAD tests. The duration of virus antigen detection in the infected horses was longer than the conventional RAD tests. We conclude that QCA could be a valuable diagnostic method for EI.

Development of a Rapid Point-of-Use DNA Test for the Screening of Genuity® Roundup Ready 2 Yield® Soybean in Seed Samples

PubMed Central

Chandu, Dilip; Paul, Sudakshina; Parker, Mathew; Dudin, Yelena; King-Sitzes, Jennifer; Perez, Tim; Mittanck, Don W.; Shah, Manali; Glenn, Kevin C.; Piepenburg, Olaf

2016-01-01

Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings. PMID:27314015

Laboratory Diagnosis of Zika Virus Infection.

PubMed

Landry, Marie Louise; St George, Kirsten

2017-01-01

-The rapid and accurate diagnosis of Zika virus infection is an international priority. -To review current recommendations, methods, limitations, and priorities for Zika virus testing. -Sources include published literature, public health recommendations, laboratory procedures, and testing experience. -Until recently, the laboratory diagnosis of Zika infection was confined to public health or research laboratories that prepared their own reagents, and test capacity has been limited. Furthermore, Zika cross-reacts serologically with other flaviviruses, such as dengue, West Nile, and yellow fever. Current or past infection, or even vaccination with another flavivirus, will often cause false-positive or uninterpretable Zika serology results. Detection of viral RNA during acute infection using nucleic acid amplification tests provides more specific results, and a number of commercial nucleic acid amplification tests have received emergency use authorization. In addition to serum, testing of whole blood and urine is recommended because of the higher vial loads and longer duration of shedding. However, nucleic acid amplification testing has limited utility because many patients are asymptomatic or present for testing after the brief period of Zika shedding has passed. Thus, the greatest need and most difficult challenge is development of accurate antibody tests for the diagnosis of recent Zika infection. Research is urgently needed to identify Zika virus epitopes that do not cross-react with other flavivirus antigens. New information is emerging at a rapid pace and, with ongoing public-private and international collaborations and government support, it is hoped that rapid progress will be made in developing robust and widely applicable diagnostic tools.

Assessment of HER2 status in breast cancer biopsies is not affected by accelerated tissue processing.

PubMed

Bulte, Joris P; Halilovic, Altuna; Kalkman, Shona; van Cleef, Patricia H J; van Diest, Paul J; Strobbe, Luc J A; de Wilt, Johannes H W; Bult, Peter

2018-03-01

To establish whether core needle biopsy (CNB) specimens processed with an accelerated processing method with short fixation time can be used to determine accurately the human epidermal growth factor receptor 2 (HER2) status of breast cancer. A consecutive case-series from two high-volume breast clinics was created. We compared routine HER2 immunohistochemistry (IHC) assessment between accelerated processing CNB specimens and routinely processed postoperative excision specimens. Additional amplification-based testing was performed in cases with equivocal results. The formalin fixation time was less than 2 h and between 6 and 72 h, respectively. Fluorescence in-situ hybridisation and multiplex ligation-dependent probe amplification were used for amplification testing. One hundred and forty-four cases were included, 15 of which were HER2-positive on the routinely processed excision specimens. On the CNB specimens, 44 were equivocal on IHC and required an amplification-based test. Correlation between the CNB specimens and the corresponding excision specimens was high for final HER2 status, with an accuracy of 97% and a kappa of 0.85. HER2 status can be determined reliably on CNB specimens with accelerated processing time using standard clinical testing methods. Using this accelerated technology the minimum 6 h of formalin fixation, which current guidelines consider necessary, can be decreased safely. This allows for a complete and expedited histology-based diagnosis of breast lesions in the setting of a one-stop-shop, same-day breast clinic. © 2018 The Authors. Histopathology Published by John Wiley & Sons Ltd.

Development of a highly sensitive immunochromatographic detection kit for H5 influenza virus hemagglutinin using silver amplification.

PubMed

Wada, Atsuhiko; Sakoda, Yoshihiro; Oyamada, Takayoshi; Kida, Hiroshi

2011-12-01

H5N1, a highly pathogenic avian influenza virus (HPAIV), has become a serious epizootic threat to the poultry population in Asia. In addition, significant numbers of human cases of HPAIV infection have been reported to date. To prevent the spread of HPAIV among humans and to allow for timely medical intervention, a rapid and high sensitive method is needed to detect and subtype the causative HPAIVs. In the present study, a silver amplification technique used in photographic development was combined with immunochromatography technologies and a highly sensitive and rapid diagnostic test to detect the hemagglutinin of H5 influenza viruses was developed. The sensitivity of the test kit was increased 500 times by silver amplification. The sensitivity of the method was more than 10 times higher than those of conventional rapid influenza diagnostic tests, which detect viral nucleoproteins. The diagnostic system developed in the present study can therefore provide rapid and highly sensitive results and will be useful for diagnosis of H5 HPAIV infection in humans and animals. Copyright © 2011 Elsevier B.V. All rights reserved.

chipPCR: an R package to pre-process raw data of amplification curves.

PubMed

Rödiger, Stefan; Burdukiewicz, Michał; Schierack, Peter

2015-09-01

Both the quantitative real-time polymerase chain reaction (qPCR) and quantitative isothermal amplification (qIA) are standard methods for nucleic acid quantification. Numerous real-time read-out technologies have been developed. Despite the continuous interest in amplification-based techniques, there are only few tools for pre-processing of amplification data. However, a transparent tool for precise control of raw data is indispensable in several scenarios, for example, during the development of new instruments. chipPCR is an R: package for the pre-processing and quality analysis of raw data of amplification curves. The package takes advantage of R: 's S4 object model and offers an extensible environment. chipPCR contains tools for raw data exploration: normalization, baselining, imputation of missing values, a powerful wrapper for amplification curve smoothing and a function to detect the start and end of an amplification curve. The capabilities of the software are enhanced by the implementation of algorithms unavailable in R: , such as a 5-point stencil for derivative interpolation. Simulation tools, statistical tests, plots for data quality management, amplification efficiency/quantification cycle calculation, and datasets from qPCR and qIA experiments are part of the package. Core functionalities are integrated in GUIs (web-based and standalone shiny applications), thus streamlining analysis and report generation. http://cran.r-project.org/web/packages/chipPCR. Source code: https://github.com/michbur/chipPCR. stefan.roediger@b-tu.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Recombinase Polymerase Amplification (RPA) of CaMV-35S Promoter and nos Terminator for Rapid Detection of Genetically Modified Crops

PubMed Central

Xu, Chao; Li, Liang; Jin, Wujun; Wan, Yusong

2014-01-01

Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37–42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S) promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos) terminator, which are widely incorporated in genetically modified (GM) crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15–25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean). With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops. PMID:25310647

Chronic centrosome amplification without tumorigenesis

PubMed Central

Vitre, Benjamin; Holland, Andrew J.; Kulukian, Anita; Shoshani, Ofer; Hirai, Maretoshi; Wang, Yin; Maldonado, Marcus; Cho, Thomas; Boubaker, Jihane; Swing, Deborah A.; Tessarollo, Lino; Evans, Sylvia M.; Fuchs, Elaine; Cleveland, Don W.

2015-01-01

Centrosomes are microtubule-organizing centers that facilitate bipolar mitotic spindle assembly and chromosome segregation. Recognizing that centrosome amplification is a common feature of aneuploid cancer cells, we tested whether supernumerary centrosomes are sufficient to drive tumor development. To do this, we constructed and analyzed mice in which centrosome amplification can be induced by a Cre-recombinase–mediated increase in expression of Polo-like kinase 4 (Plk4). Elevated Plk4 in mouse fibroblasts produced supernumerary centrosomes and enhanced the expected mitotic errors, but proliferation continued only after inactivation of the p53 tumor suppressor. Increasing Plk4 levels in mice with functional p53 produced centrosome amplification in liver and skin, but this did not promote spontaneous tumor development in these tissues or enhance the growth of chemically induced skin tumors. In the absence of p53, Plk4 overexpression generated widespread centrosome amplification, but did not drive additional tumors or affect development of the fatal thymic lymphomas that arise in animals lacking p53. We conclude that, independent of p53 status, supernumerary centrosomes are not sufficient to drive tumor formation. PMID:26578792

Recombinase polymerase amplification (RPA) of CaMV-35S promoter and nos terminator for rapid detection of genetically modified crops.

PubMed

Xu, Chao; Li, Liang; Jin, Wujun; Wan, Yusong

2014-10-10

Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37-42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S) promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos) terminator, which are widely incorporated in genetically modified (GM) crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15-25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean). With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops.

Levers and linkages: mechanical trade-offs in a power-amplified system.

PubMed

Anderson, Philip S L; Claverie, Thomas; Patek, S N

2014-07-01

Mechanical redundancy within a biomechanical system (e.g., many-to-one mapping) allows morphologically divergent organisms to maintain equivalent mechanical outputs. However, most organisms depend on the integration of more than one biomechanical system. Here, we test whether coupled mechanical systems follow a pattern of amplification (mechanical changes are congruent and evolve toward the same functional extreme) or independence (mechanisms evolve independently). We examined the correlated evolution and evolutionary pathways of the coupled four-bar linkage and lever systems in mantis shrimp (Stomatopoda) ultrafast raptorial appendages. We examined models of character evolution in the framework of two divergent groups of stomatopods-"smashers" (hammer-shaped appendages) and "spearers" (bladed appendages). Smashers tended to evolve toward force amplification, whereas spearers evolved toward displacement amplification. These findings show that coupled biomechanical systems can evolve synergistically, thereby resulting in functional amplification rather than mechanical redundancy. © 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.

Application of a molecular beacon based real-time isothermal amplification (MBRTIA) technology for simultaneous detection of Bacillus cereus and Staphylococcus aureus.

PubMed

Mandappa, I M; Joglekar, Prasanna; Manonmani, H K

2015-07-01

A multiplex real-time isothermal amplification assay was developed using molecular beacons for the detection of Bacillus cereus and Staphylococcus aureus by targeting four important virulence genes. A correlation between targeting highly accessible DNA sequences and isothermal amplification based molecular beacon efficiency and sensitivity was demonstrated using phi(Φ)29 DNA polymerase at a constant isothermal temperature of 30 °C. It was very selective and consistently detected down to 10(1) copies of DNA. The specificity and sensitivity of this assay, when tested with pure culture were high, surpassing those of currently used PCR assays for the detection of these organisms. The molecular beacon based real-time isothermal amplification (MBRTIA) assay could be carried out entirely in 96 well plates or well strips, enabling a rapid and high-throughput detection of food borne pathogens.

A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification

PubMed Central

Craw, Pascal; Mackay, Ruth E.; Naveenathayalan, Angel; Hudson, Chris; Branavan, Manoharanehru; Sadiq, S. Tariq; Balachandran, Wamadeva

2015-01-01

Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings. PMID:26389913

Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences.

PubMed

Lee, David; La Mura, Maurizio; Allnutt, Theo R; Powell, Wayne

2009-02-02

The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

Yoctomole electrochemical genosensing of Ebola virus cDNA by rolling circle and circle to circle amplification.

PubMed

Carinelli, S; Kühnemund, M; Nilsson, M; Pividori, M I

2017-07-15

This work addresses the design of an Ebola diagnostic test involving a simple, rapid, specific and highly sensitive procedure based on isothermal amplification on magnetic particles with electrochemical readout. Ebola padlock probes were designed to detect a specific L-gene sequence present in the five most common Ebola species. Ebola cDNA was amplified by rolling circle amplification (RCA) on magnetic particles. Further re-amplification was performed by circle-to-circle amplification (C2CA) and the products were detected in a double-tagging approach using a biotinylated capture probe for immobilization on magnetic particles and a readout probe for electrochemical detection by square-wave voltammetry on commercial screen-printed electrodes. The electrochemical genosensor was able to detect as low as 200 ymol, corresponding to 120 cDNA molecules of L-gene Ebola virus with a limit of detection of 33 cDNA molecules. The isothermal double-amplification procedure by C2CA combined with the electrochemical readout and the magnetic actuation enables the high sensitivity, resulting in a rapid, inexpensive, robust and user-friendly sensing strategy that offers a promising approach for the primary care in low resource settings, especially in less developed countries. Copyright © 2016 Elsevier B.V. All rights reserved.

Natural laminar flow flight experiments on a swept wing business jet-boundary layer stability analyses

NASA Technical Reports Server (NTRS)

Rozendaal, R. A.

1986-01-01

The linear boundary layer stability analyses and their correlation with data of 18 cases from a natural laminar flow (NLF) flight test program using a Cessna Citation 3 business jet are described. The transition point varied from 5% to 35% chord for these conditions, and both upper and lower wing surfaces were included. Altitude varied from 10,000 to 43,000 ft and Mach number from 0.3 to 0.8. Four cases were at nonzero sideslip. Although there was much scatter in the results, the analyses of boundary layer stability at the 18 conditions led to the conclusion that crossflow instability was the primary cause of transition. However, the sideslip cases did show some interaction of crossflow and Tollmien-Schlichting disturbances. The lower surface showed much lower Tollmien-Schlichting amplification at transition than the upper surface, but similar crossflow amplifications. No relationship between Mach number and disturbance amplification at transition could be found. The quality of these results is open to question from questionable wing surface quality, inadequate density of transition sensors on the wing upper surface, and an unresolved pressure shift in the wing pressure data. The results of this study show the need for careful preparation for transition experiments. Preparation should include flow analyses of the test surface, boundary layer disturbance amplification analyses, and assurance of adequate surface quality in the test area. The placement of necessary instruments and usefulness of the resulting data could largely be determined during the pretest phase.

European Society of Clinical Microbiology and Infectious Diseases: update of the diagnostic guidance document for Clostridium difficile infection.

PubMed

Crobach, M J T; Planche, T; Eckert, C; Barbut, F; Terveer, E M; Dekkers, O M; Wilcox, M H; Kuijper, E J

2016-08-01

In 2009 the first European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guideline for diagnosing Clostridium difficile infection (CDI) was launched. Since then newer tests for diagnosing CDI have become available, especially nucleic acid amplification tests. The main objectives of this update of the guidance document are to summarize the currently available evidence concerning laboratory diagnosis of CDI and to formulate and revise recommendations to optimize CDI testing. This update is essential to improve the diagnosis of CDI and to improve uniformity in CDI diagnosis for surveillance purposes among Europe. An electronic search for literature concerning the laboratory diagnosis of CDI was performed. Studies evaluating a commercial laboratory test compared to a reference test were also included in a meta-analysis. The commercial tests that were evaluated included enzyme immunoassays (EIAs) detecting glutamate dehydrogenase, EIAs detecting toxins A and B and nucleic acid amplification tests. Recommendations were formulated by an executive committee, and the strength of recommendations and quality of evidence were graded using the Grades of Recommendation Assessment, Development and Evaluation (GRADE) system. No single commercial test can be used as a stand-alone test for diagnosing CDI as a result of inadequate positive predictive values at low CDI prevalence. Therefore, the use of a two-step algorithm is recommended. Samples without free toxin detected by toxins A and B EIA but with positive glutamate dehydrogenase EIA, nucleic acid amplification test or toxigenic culture results need clinical evaluation to discern CDI from asymptomatic carriage. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Development and evaluation of an in-house single step loop-mediated isothermal amplification (SS-LAMP) assay for the detection of Mycobacterium tuberculosis complex in sputum samples from Moroccan patients.

PubMed

Bentaleb, El Mehdi; Abid, Mohammed; El Messaoudi, My Driss; Lakssir, Brahim; Ressami, El Mostafa; Amzazi, Saaïd; Sefrioui, Hassan; Ait Benhassou, Hassan

2016-09-27

Tuberculosis (TB) is a major global health problem and remains the leading cause of morbidity and mortality in developing countries. Routinely used TB diagnostic methods, in most endemic areas, are time-consuming, often less-sensitive, expensive and inaccessible to most patients. Therefore, there is an urgent need for the development of early, easy to use and effective diagnosis tools of TB, which can be effectively integrated into resource limited settings, to anticipate the early treatment and limit further spread of the disease. Over the last decade, Loop-mediated isothermal amplification (LAMP) assays have become a powerful tool for rapid diagnosis of infectious diseases because of the simplicity of device requirements. Indeed, LAMP is a simple, quick and cost effective Isothermal Nucleic Acid Amplification diagnostic test (INAAT) that has the potential to be used in TB endemic settings of resource-poor countries. In the present study, we have developed a simple and rapid TB molecular diagnostic test using a Single-Step Loop-mediated isothermal DNA amplification (SS-LAMP) method for the detection of Mycobacterium tuberculosis complex (MTBC) strains, with a simplified sample preparation procedure, eliminating DNA extraction prior to LAMP amplification, DNA initial denaturation and enzymatic inactivation steps during the amplification process. To perform our in-house SS-LAMP assay, a set of six specific primers was specifically designed to recognize eight distinct regions on the MTBC species-specific repetitive insertion sequence 6110 (IS6110). The amplification of the targeted DNA was carried out under isothermal conditions at 65 °C within 1 h. Our protocol was firstly optimized using 60 of confirmed MTBC isolates and a recombinant pGEMeasy-IS6110 vector for sensitivity testing. Thereafter, the assay was evaluated on liquefied sputum specimens collected from 157 Moroccan patients suspected of having TB. Our SS-LAMP developed assay was able to detect MTBC DNA directly from liquefied sputum samples without any prior DNA extraction, denaturation nor the final enzymatic inactivation step. When compared to routinely used Löwenstein Jensen (LJ) Culture method, our SS-LAMP assay is rapid and showed specificity and sensitivity of 99.14 % and 82.93 % respectively which are within the international standards. In addition, the limit of detection of our assay was found to be as little as 10 copies of bacterial DNA. To our knowledge, this is the first study using a single step LAMP (SS-LAMP) procedure as a rapid, easy to perform and cost effective testing for TB early detection. This innovative assay could be suitable for low-income countries with restricted health equipment facilities.

Deployment of a Reverse Transcription Loop-Mediated Isothermal Amplification Test for Ebola Virus Surveillance in Remote Areas in Guinea.

PubMed

Kurosaki, Yohei; Magassouba, N'Faly; Bah, Hadja Aïssatou; Soropogui, Barré; Doré, Amadou; Kourouma, Fodé; Cherif, Mahamoud Sama; Keita, Sakoba; Yasuda, Jiro

2016-10-15

To strengthen the laboratory diagnostic capacity for Ebola virus disease (EVD) in the remote areas of Guinea, we deployed a mobile field laboratory and implemented reverse transcription loop-mediated isothermal amplification (RT-LAMP) for postmortem testing. We tested 896 oral swab specimens and 21 serum samples, using both RT-LAMP and reverse transcription-polymerase chain reaction (RT-PCR). Neither test yielded a positive result, and the results from RT-LAMP and RT-PCR were consistent. More than 95% of the samples were tested within 2 days of sample collection. These results highlight the usefulness of the RT-LAMP assay as an EVD diagnostic testing method in the field or remote areas. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Strong Effects of Vs30 Heterogeneity on Physics-Based Scenario Ground-Shaking Computations

NASA Astrophysics Data System (ADS)

Louie, J. N.; Pullammanappallil, S. K.

2014-12-01

Hazard mapping and building codes worldwide use the vertically time-averaged shear-wave velocity between the surface and 30 meters depth, Vs30, as one predictor of earthquake ground shaking. Intensive field campaigns a decade ago in Reno, Los Angeles, and Las Vegas measured urban Vs30 transects with 0.3-km spacing. The Clark County, Nevada, Parcel Map includes urban Las Vegas and comprises over 10,000 site measurements over 1500 km2, completed in 2010. All of these data demonstrate fractal spatial statistics, with a fractal dimension of 1.5-1.8 at scale lengths from 0.5 km to 50 km. Vs measurements in boreholes up to 400 m deep show very similar statistics at 1 m to 200 m lengths. When included in physics-based earthquake-scenario ground-shaking computations, the highly heterogeneous Vs30 maps exhibit unexpectedly strong influence. In sensitivity tests (image below), low-frequency computations at 0.1 Hz display amplifications (as well as de-amplifications) of 20% due solely to Vs30. In 0.5-1.0 Hz computations, the amplifications are a factor of two or more. At 0.5 Hz and higher frequencies the amplifications can be larger than what the 1-d Building Code equations would predict from the Vs30 variations. Vs30 heterogeneities at one location have strong influence on amplifications at other locations, stretching out in the predominant direction of wave propagation for that scenario. The sensitivity tests show that shaking and amplifications are highly scenario-dependent. Animations of computed ground motions and how they evolve with time suggest that the fractal Vs30 variance acts to trap wave energy and increases the duration of shaking. Validations of the computations against recorded ground motions, possible in Las Vegas Valley due to the measurements of the Clark County Parcel Map, show that ground motion levels and amplifications match, while recorded shaking has longer duration than computed shaking. Several mechanisms may explain the amplification and increased duration of shaking in the presence of heterogeneous spatial distributions of Vs: conservation of wave energy across velocity changes; geometric focusing of waves by low-velocity lenses; vertical resonance and trapping; horizontal resonance and trapping; and multiple conversion of P- to S-wave energy.

Investigation of Extended Bandwidth Hearing Aid Amplification on Speech Intelligibility and Sound Quality in Adults with Mild-to-Moderate Hearing Loss.

PubMed

Seeto, Angeline; Searchfield, Grant D

2018-03-01

Advances in digital signal processing have made it possible to provide a wide-band frequency response with smooth, precise spectral shaping. Several manufacturers have introduced hearing aids that are claimed to provide gain for frequencies up to 10-12 kHz. However, there is currently limited evidence and very few independent studies evaluating the performance of the extended bandwidth hearing aids that have recently become available. This study investigated an extended bandwidth hearing aid using measures of speech intelligibility and sound quality to find out whether there was a significant benefit of extended bandwidth amplification over standard amplification. Repeated measures study designed to examine the efficacy of extended bandwidth amplification compared to standard bandwidth amplification. Sixteen adult participants with mild-to-moderate sensorineural hearing loss. Participants were bilaterally fit with a pair of Widex Mind 440 behind-the-ear hearing aids programmed with a standard bandwidth fitting and an extended bandwidth fitting; the latter provided gain up to 10 kHz. For each fitting, and an unaided condition, participants completed two speech measures of aided benefit, the Quick Speech-in-Noise test (QuickSIN™) and the Phonak Phoneme Perception Test (PPT; high-frequency perception in quiet), and a measure of sound quality rating. There were no significant differences found between unaided and aided conditions for QuickSIN™ scores. For the PPT, there were statistically significantly lower (improved) detection thresholds at high frequencies (6 and 9 kHz) with the extended bandwidth fitting. Although not statistically significant, participants were able to distinguish between 6 and 9 kHz 50% better with extended bandwidth. No significant difference was found in ability to recognize phonemes in quiet between the unaided and aided conditions when phonemes only contained frequency content <6 kHz. However significant benefit was found with the extended bandwidth fitting for recognition of 9-kHz phonemes. No significant difference in sound quality preference was found between the standard bandwidth and extended bandwidth fittings. This study demonstrated that a pair of currently available extended bandwidth hearing aids was technically capable of delivering high-frequency amplification that was both audible and useable to listeners with mild-to-moderate hearing loss. This amplification was of acceptable sound quality. Further research, particularly field trials, is required to ascertain the real-world benefit of high-frequency amplification. American Academy of Audiology

Assessment of self taken swabs versus clinician taken swab cultures for diagnosing gonorrhoea in women: single centre, diagnostic accuracy study.

PubMed

Stewart, Catherine M W; Schoeman, Sarah A; Booth, Russell A; Smith, Susan D; Wilcox, Mark H; Wilson, Janet D

2012-12-12

To compare gonorrhoea detection by self taken vulvovaginal swabs (tested with nucleic acid amplification tests) with the culture of urethral and endocervical samples taken by clinicians. Prospective study of diagnostic accuracy. 1 sexual health clinic in an urban setting (Leeds Centre for Sexual Health, United Kingdom), between March 2009 and January 2010. Women aged 16 years or older, attending the clinic for sexually transmitted infection (STI) testing and consenting to perform a vulvovaginal swab themselves before routine examination. During examination, clinicians took urethral and endocervical samples for culture and an endocervical swab for nucleic acid amplification testing. Urethra and endocervix samples were analysed by gonococcal culture. Vulvovaginal swabs and endocervical swabs were analysed by the Aptima Combo 2 (AC2) assay; positive results from this assay were confirmed with a second nucleic acid amplification test. Positive confirmation of gonorrhoea. Of 3859 women with complete data and test results, 96 (2.5%) were infected with gonorrhoea (overall test sensitivities: culture 81%, endocervical swabs with AC2 96%, vulvovaginal swabs with AC2 99%). The AC2 assays were more sensitive than culture (P<0.001), but the endocervical and vulvovaginal assays did not differ significantly (P=0.375). Specificity of all Aptima Combo 2 tests was 100%. Of 1625 women who had symptoms suggestive of a bacterial STI, 56 (3.4%) had gonorrhoea (culture 84%, endocervical AC2 100%, vulvovaginal AC2 100%). The AC2 assays were more sensitive than culture (P=0.004), and the endocervical and vulvovaginal assays were equivalent to each other. Of 2234 women who did not have symptoms suggesting a bacterial STI, 40 (1.8%) had gonorrhoea (culture 78%, endocervical AC2 90%, vulvovaginal AC2 98%). The vulvovaginal swab was more sensitive than culture (P=0.008), but there was no difference between the endocervical and vulvovaginal AC2 assays (P=0.375) or between the endocervical AC2 assay and culture (P=0.125). The endocervical swab assay performed less well in women without symptoms of a bacterial STI than in those with symptoms (90% v 100%, P=0.028), whereas the vulvovaginal swab assay performed similarly (98% v 100%, P=0.42). Self taken vulvovaginal swabs analysed by nucleic acid amplification tests are significantly more sensitive at detecting gonorrhoea than culture of clinician taken urethral and endocervical samples, and are equivalent to endocervical swabs analysed by nucleic acid amplification tests. Self taken vulvovaginal swabs are the sample of choice in women without symptoms and have the advantage of being non-invasive. In women who need a clinical examination, either a clinician taken or self taken vulvovaginal swab is recommended.

Point of care nucleic acid detection of viable pathogenic bacteria with isothermal RNA amplification based paper biosensor

NASA Astrophysics Data System (ADS)

Liu, Hongxing; Xing, Da; Zhou, Xiaoming

2014-09-01

Food-borne pathogens such as Listeria monocytogenes have been recognized as a major cause of human infections worldwide, leading to substantial health problems. Food-borne pathogen identification needs to be simpler, cheaper and more reliable than the current traditional methods. Here, we have constructed a low-cost paper biosensor for the detection of viable pathogenic bacteria with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper biosensor to perform a visual test, in which endpoint detection was performed using sandwich hybridization assays. When the RNA products migrated along the paper biosensor by capillary action, the gold nanoparticles accumulated at the designated Test line and Control line. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from Listeria monocytogenes could be detected. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. The developed method is suitable for point-of-care applications to detect food-borne pathogens, as it can effectively overcome the false-positive results caused by amplifying nonviable Listeria monocytogenes.

A Transport Equation Approach to Modeling the Influence of Surface Roughness on Boundary Layer Transition

NASA Astrophysics Data System (ADS)

Langel, Christopher Michael

A computational investigation has been performed to better understand the impact of surface roughness on the flow over a contaminated surface. This thesis highlights the implementation and development of the roughness amplification model in the flow solver OVERFLOW-2. The model, originally proposed by Dassler, Kozulovic, and Fiala, introduces an additional scalar field roughness amplification quantity. This value is explicitly set at rough wall boundaries using surface roughness parameters and local flow quantities. This additional transport equation allows non-local effects of surface roughness to be accounted for downstream of rough sections. This roughness amplification variable is coupled with the Langtry-Menter model and used to modify the criteria for transition. Results from flat plate test cases show good agreement with experimental transition behavior on the flow over varying sand grain roughness heights. Additional validation studies were performed on a NACA 0012 airfoil with leading edge roughness. The computationally predicted boundary layer development demonstrates good agreement with experimental results. New tests using varying roughness configurations are being carried out at the Texas A&M Oran W. Nicks Low Speed Wind Tunnel to provide further calibration of the roughness amplification method. An overview and preliminary results are provided of this concurrent experimental investigation.

Amplification of intrinsic fluctuations by the Lorenz equations

NASA Astrophysics Data System (ADS)

Fox, Ronald F.; Elston, T. C.

1993-07-01

Macroscopic systems (e.g., hydrodynamics, chemical reactions, electrical circuits, etc.) manifest intrinsic fluctuations of molecular and thermal origin. When the macroscopic dynamics is deterministically chaotic, the intrinsic fluctuations may become amplified by several orders of magnitude. Numerical studies of this phenomenon are presented in detail for the Lorenz model. Amplification to macroscopic scales is exhibited, and quantitative methods (binning and a difference-norm) are presented for measuring macroscopically subliminal amplification effects. In order to test the quality of the numerical results, noise induced chaos is studied around a deterministically nonchaotic state, where the scaling law relating the Lyapunov exponent to noise strength obtained for maps is confirmed for the Lorenz model, a system of ordinary differential equations.

Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

PubMed

Giuffrida, Maria Chiara; Zanoli, Laura Maria; D'Agata, Roberta; Finotti, Alessia; Gambari, Roberto; Spoto, Giuseppe

2015-02-01

Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.

Paternity testing in case of brother-sister incest.

PubMed

Macan, Marijana; Uvodić, Petra; Botica, Vladimir

2003-06-01

We performed a paternity test in a case of incest between brother and sister. DNA from blood samples of the alleged parents and their two children was obtained with Chelex DNA extraction method and quantified with Applied Biosystems QuantiBlot quantitation kit. Polymerase chain reaction (PCR) amplification of DNA samples was performed with AmpFlSTR SGM Plus PCR amplification kit and GenePrint PowerPlex PCR amplification kit. The amplified products were separated and detected by using the Perkin Elmer's ABI PRISM trade mark 310 Genetic Analyser. DNA and data analysis of 17 loci and Amelogenin confirmed the suspicion of brother-sister incest. Since both children had inherited all of the obligate alleles from the alleged father, we could confirm with certainty of 99.999999% that the oldest brother in the family was the biological father of both children. Calculated data showed that even in a case of brother-sister incest, paternity could be proved by the analysis of Amelogenin and 17 DNA loci.

Determining the sensitivity of the amplitude source location (ASL) method through active seismic sources: An example from Te Maari Volcano, New Zealand

NASA Astrophysics Data System (ADS)

Walsh, Braden; Jolly, Arthur; Procter, Jonathan

2017-04-01

Using active seismic sources on Tongariro Volcano, New Zealand, the amplitude source location (ASL) method is calibrated and optimized through a series of sensitivity tests. By applying a geologic medium velocity of 1500 m/s and an attenuation value of Q=60 for surface waves along with amplification factors computed from regional earthquakes, the ASL produced location discrepancies larger than 1.0 km horizontally and up to 0.5 km in depth. Through the use of sensitivity tests on input parameters, we show that velocity and attenuation models have moderate to strong influences on the location results, but can be easily constrained. Changes in locations are accommodated through either lateral or depth movements. Station corrections (amplification factors) and station geometry strongly affect the ASL locations laterally, horizontally and in depth. Calibrating the amplification factors through the exploitation of the active seismic source events reduced location errors for the sources by up to 50%.

Quantum Hash function and its application to privacy amplification in quantum key distribution, pseudo-random number generation and image encryption

NASA Astrophysics Data System (ADS)

Yang, Yu-Guang; Xu, Peng; Yang, Rui; Zhou, Yi-Hua; Shi, Wei-Min

2016-01-01

Quantum information and quantum computation have achieved a huge success during the last years. In this paper, we investigate the capability of quantum Hash function, which can be constructed by subtly modifying quantum walks, a famous quantum computation model. It is found that quantum Hash function can act as a hash function for the privacy amplification process of quantum key distribution systems with higher security. As a byproduct, quantum Hash function can also be used for pseudo-random number generation due to its inherent chaotic dynamics. Further we discuss the application of quantum Hash function to image encryption and propose a novel image encryption algorithm. Numerical simulations and performance comparisons show that quantum Hash function is eligible for privacy amplification in quantum key distribution, pseudo-random number generation and image encryption in terms of various hash tests and randomness tests. It extends the scope of application of quantum computation and quantum information.

Quantum Hash function and its application to privacy amplification in quantum key distribution, pseudo-random number generation and image encryption

PubMed Central

Yang, Yu-Guang; Xu, Peng; Yang, Rui; Zhou, Yi-Hua; Shi, Wei-Min

2016-01-01

Quantum information and quantum computation have achieved a huge success during the last years. In this paper, we investigate the capability of quantum Hash function, which can be constructed by subtly modifying quantum walks, a famous quantum computation model. It is found that quantum Hash function can act as a hash function for the privacy amplification process of quantum key distribution systems with higher security. As a byproduct, quantum Hash function can also be used for pseudo-random number generation due to its inherent chaotic dynamics. Further we discuss the application of quantum Hash function to image encryption and propose a novel image encryption algorithm. Numerical simulations and performance comparisons show that quantum Hash function is eligible for privacy amplification in quantum key distribution, pseudo-random number generation and image encryption in terms of various hash tests and randomness tests. It extends the scope of application of quantum computation and quantum information. PMID:26823196

Quantum Hash function and its application to privacy amplification in quantum key distribution, pseudo-random number generation and image encryption.

PubMed

Yang, Yu-Guang; Xu, Peng; Yang, Rui; Zhou, Yi-Hua; Shi, Wei-Min

2016-01-29

Quantum information and quantum computation have achieved a huge success during the last years. In this paper, we investigate the capability of quantum Hash function, which can be constructed by subtly modifying quantum walks, a famous quantum computation model. It is found that quantum Hash function can act as a hash function for the privacy amplification process of quantum key distribution systems with higher security. As a byproduct, quantum Hash function can also be used for pseudo-random number generation due to its inherent chaotic dynamics. Further we discuss the application of quantum Hash function to image encryption and propose a novel image encryption algorithm. Numerical simulations and performance comparisons show that quantum Hash function is eligible for privacy amplification in quantum key distribution, pseudo-random number generation and image encryption in terms of various hash tests and randomness tests. It extends the scope of application of quantum computation and quantum information.

Moving toward rapid and low-cost point-of-care molecular diagnostics with a repurposed 3D printer and RPA.

PubMed

Chan, Kamfai; Wong, Pui-Yan; Parikh, Chaitanya; Wong, Season

2018-03-15

Traditionally, the majority of nucleic acid amplification-based molecular diagnostic tests are done in centralized settings. In recent years, point-of-care tests have been developed for use in low-resource settings away from central laboratories. While most experts agree that point-of-care molecular tests are greatly needed, their availability as cost-effective and easy-to-operate tests remains an unmet goal. In this article, we discuss our efforts to develop a recombinase polymerase amplification reaction-based test that will meet these criteria. First, we describe our efforts in repurposing a low-cost 3D printer as a platform that can carry out medium-throughput, rapid, and high-performing nucleic acid extraction. Next, we address how these purified templates can be rapidly amplified and analyzed using the 3D printer's heated bed or the deconstructed, low-cost thermal cycler we have developed. In both approaches, real-time isothermal amplification and detection of template DNA or RNA can be accomplished using a low-cost portable detector or smartphone camera. Last, we demonstrate the capability of our technologies using foodborne pathogens and the Zika virus. Our low-cost approach does not employ complicated and high-cost components, making it suitable for resource-limited settings. When integrated and commercialized, it will offer simple sample-to-answer molecular diagnostics. Copyright © 2018 Elsevier Inc. All rights reserved.

Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification.

PubMed

Poon, Leo L M; Wong, Bonnie W Y; Ma, Edmund H T; Chan, Kwok H; Chow, Larry M C; Abeyewickreme, Wimal; Tangpukdee, Noppadon; Yuen, Kwok Y; Guan, Yi; Looareesuwan, Sornchai; Peiris, J S Malik

2006-02-01

Malaria is one of the most important parasitic infections in humans. A sensitive diagnostic test for malaria that could be applied at the community level could be useful in programs to control the disease. The aim of the present work was to develop a simple, inexpensive molecular test for Plasmodium falciparum. Blood was collected from controls (n = 100) and from patients diagnosed with falciparum malaria infection (n = 102), who were recruited to the study. Heat-treated blood samples were tested by a loop-mediated isothermal amplification (LAMP) assay for P. falciparum. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. To evaluate the assay, DNA from these samples was purified and tested by PCR. Results from the LAMP and PCR assays were compared. The LAMP assay detected P. falciparum directly from heat-treated blood. The quantitative data from the assay correlated to the parasite counts obtained by blood-film microscopic analyses. When we used the PCR assay as the comparison method, the sensitivity and specificity of the LAMP assay were 95% and 99%, respectively. Unlike PCR, the LAMP assay does not require purified DNA for efficient DNA amplification, thereby reducing the cost and turnaround time for P. falciparum diagnosis. The assay requires only basic instruments, and assay positivity can be verified by visual inspection.

HER2 Status in Advanced or Metastatic Gastric, Esophageal, or Gastroesophageal Adenocarcinoma for Entry to the TRIO-013/LOGiC Trial of Lapatinib.

PubMed

Press, Michael F; Ellis, Catherine E; Gagnon, Robert C; Grob, Tobias J; Buyse, Marc; Villalobos, Ivonne; Liang, Zhiyong; Wu, Shafei; Bang, Yung-Jue; Qin, Shu-Kui; Chung, Hyun Cheol; Xu, Jianming; Park, Joon Oh; Jeziorski, Krzysztof; Afenjar, Karen; Ma, Yanling; Estrada, Monica C; Robinson, Douglas M; Scherer, Stefan J; Sauter, Guido; Hecht, J Randolph; Slamon, Dennis J

2017-01-01

HER2/ERBB2 status is used to select patients for HER2-targeted therapy. HER2/ERBB2 amplification/overexpression of upper gastrointestinal (UGI) adenocarcinomas was determined locally or in two central laboratories to select patients for the TRIO-013/LOGiC trial of chemotherapy with or without lapatinib. Patients selected locally had central laboratory confirmation of HER2 amplification for inclusion in the primary efficacy population. HER2 was assessed with PathVysion or IQ PharmDx FISH and HercepTest immunohistochemistry assays. Associations with outcomes were retrospectively evaluated. Overall, HER2 status was determined in UGI cancers from 4,674 patients in a central laboratory for eligibility (1,995 cases) and for confirmation of local HER2 results (333 cases). Of 1,995 adenocarcinomas screened centrally, 322 (16.1%) had HER2-amplified disease with 29 (1.5%) showing HER2 genomic heterogeneity. Men and older patients had higher rates of amplification. Of 545 patients accrued to the trial (gastric, 87.3%; GEJ, 8.3% and esophageal cancer, 4.4%), 487 patients (89%) were centrally confirmed as having HER2-amplified disease. Concordance between central and local HER2 testing was 83%. Concordance between PathVysion and IQ PharmDx FISH assays was 99% and FISH in the two central laboratories was 95%. Lapatinib-treated Asian participants and those less than 60 years had significant improvement in progression-free survival (PFS), particularly among those whose cancers had 5.01-10.0 and >10.0-fold amplification of HER2 In conclusion, HER2 is commonly amplified in UGI adenocarcinomas with amplification highly correlated to overexpression, and HER2 amplification levels correlated with PFS. While HER2 genomic heterogeneity occurs, its prevalence is low. Mol Cancer Ther; 16(1); 228-38. ©2016 AACR. ©2016 American Association for Cancer Research.

Geometric Effects on the Amplification of First Mode Instability Waves

NASA Technical Reports Server (NTRS)

Kirk, Lindsay C.; Candler, Graham V.

2013-01-01

The effects of geometric changes on the amplification of first mode instability waves in an external supersonic boundary layer were investigated using numerical techniques. Boundary layer stability was analyzed at Mach 6 conditions similar to freestream conditions obtained in quiet ground test facilities so that results obtained in this study may be applied to future test article design to measure first mode instability waves. The DAKOTA optimization software package was used to optimize an axisymmetric geometry to maximize the amplification of the waves at first mode frequencies as computed by the 2D STABL hypersonic boundary layer stability analysis tool. First, geometric parameters such as nose radius, cone half angle, vehicle length, and surface curvature were examined separately to determine the individual effects on the first mode amplification. Finally, all geometric parameters were allowed to vary to produce a shape optimized to maximize the amplification of first mode instability waves while minimizing the amplification of second mode instability waves. Since first mode waves are known to be most unstable in the form of oblique wave, the geometries were optimized using a broad range of wave frequencies as well as a wide range of oblique wave angles to determine the geometry that most amplifies the first mode waves. Since first mode waves are seen most often in flows with low Mach numbers at the edge of the boundary layer, the edge Mach number for each geometry was recorded to determine any relationship between edge Mach number and the stability of first mode waves. Results indicate that an axisymmetric cone with a sharp nose and a slight flare at the aft end under the Mach 6 freestream conditions used here will lower the Mach number at the edge of the boundary layer to less than 4, and the corresponding stability analysis showed maximum first mode N factors of 3.

Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

PubMed Central

2011-01-01

Background Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products. Methods Reagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested. Results Amplification and fluorescence detection by real-time PCR were optimal with 40× SYBR® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA. Conclusions The methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings. PMID:21851640

A Paper and Plastic Device for Performing Recombinase Polymerase Amplification of HIV DNA

PubMed Central

Rohrman, Brittany A.; Richards-Kortum, Rebecca R.

2013-01-01

Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HIV DNA test is currently available. We have developed a device constructed of layers of paper, glass fiber, and plastic that is capable of performing isothermal, enzymatic amplification of HIV DNA. The device is inexpensive, small, light-weight, and easy to assemble. The device stores lyophilized enzymes, facilitates mixing of reaction components, and supports recombinase polymerase amplification in five steps of operation. Using commercially available lateral flow strips as a detection method, we demonstrate the ability of our device to amplify 10 copies of HIV DNA to detectable levels in 15 minutes. Our results suggest that our device, which is designed to be used after DNA extraction from dried-blood spots, may serve in conjunction with lateral flow strips as part of a point-of-care HIV DNA test to be used in low resource settings. PMID:22733333

A paper and plastic device for performing recombinase polymerase amplification of HIV DNA.

PubMed

Rohrman, Brittany A; Richards-Kortum, Rebecca R

2012-09-07

Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HIV DNA test is currently available. We have developed a device constructed of layers of paper, glass fiber, and plastic that is capable of performing isothermal, enzymatic amplification of HIV DNA. The device is inexpensive, small, light-weight, and easy to assemble. The device stores lyophilized enzymes, facilitates mixing of reaction components, and supports recombinase polymerase amplification in five steps of operation. Using commercially available lateral flow strips as a detection method, we demonstrate the ability of our device to amplify 10 copies of HIV DNA to detectable levels in 15 min. Our results suggest that our device, which is designed to be used after DNA extraction from dried-blood spots, may serve in conjunction with lateral flow strips as part of a point-of-care HIV DNA test to be used in low resource settings.

Origins of DNA Replication and Amplification in the Breast Cancer Genome

DTIC Science & Technology

2012-09-01

W81XWH-10-1-0463 TITLE: Origins of DNA Replication and Amplification in the...2. REPORT TYPE Final 3. DATES COVERED 1 Sep 2010 – 31 Aug 2012 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Origins of DNA Replication and...described in the DOD funded parent grant, to test our hypothesis we need to map origins of DNA replication in the genome and ask which of these

Amplification of the Gp41 gene for detection of mutations conferring resistance to HIV-1 fusion inhibitors on genotypic assay

NASA Astrophysics Data System (ADS)

Tanumihardja, J.; Bela, B.

2017-08-01

Fusion inhibitors have potential for future use in HIV control programs in Indonesia, so the capacity to test resistance to such drugs needs to be developed. Resistance-detection with a genotypic assay began with amplification of the target gene, gp41. Based on the sequence of the two most common HIV subtypes in Indonesia, AE and B, a primer pair was designed. Plasma samples containing both subtypes were extracted to obtain HIV RNA. Using PCR, the primer pair was used to produce the amplification product, the identity of which was checked based on length under electrophoresis. Eleven plasma samples were included in this study. One-step PCR using the primer pair was able to amplify gp41 from 54.5% of the samples, and an unspecific amplification product was seen in 1.1% of the samples. Amplification failed in 36.4% of the samples, which may be due to an inappropriate primer sequence. It was also found that the optimal annealing temperature for producing the single expected band was 57.2 °C. With one-step PCR, the designed primer pair amplified the HIV-1 gp41 gene from subtypes AE and B. However, further research should be done to determine the conditions that will increase the sensitivity and specificity of the amplification process.

The use of short and long PCR products for improved detection of prunus necrotic ringspot virus in woody plants.

PubMed

Rosner, A; Maslenin, L; Spiegel, S

1997-09-01

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of prunus necrotic ringspot virus (PNRSV) in dormant peach and almond trees by the application of two different pairs of primers yielding a short and a long product, respectively. The relative amount of the short (200 base pair, bp) product was higher than the longer (785 bp) product. PNRSV was detected better in plant tissues with a low virus concentration (e.g. dormant trees) by amplification of the short PCR product, whereas the long product was product was produced at higher virus titers. Simultaneous amplification of both short and long products was demonstrated using a three-primer mixture in a single reaction tube. In this assay, amplification of either PCR product indicated the presence of PNRSV-specific sequences in the plant tissue examined, thus covering a wide range of virus concentrations in a single test. Dilution of the RNA extracted from infected plant material resulted in a steep decline in the amplification of both short and long PCR products. In contrast, serial dilutions of the intermediate cDNA template differentially affected the amplification patterns: the relative amount of the short product increased whereas that of the long product decreased. These results may explain the preferential amplification of the short PCR product observed in samples containing low virus concentrations.

Isothermal amplification of environmental DNA (eDNA) for direct field-based monitoring and laboratory confirmation of Dreissena sp.

PubMed

Williams, Maggie R; Stedtfeld, Robert D; Engle, Cathrine; Salach, Paul; Fakher, Umama; Stedtfeld, Tiffany; Dreelin, Erin; Stevenson, R Jan; Latimore, Jo; Hashsham, Syed A

2017-01-01

Loop-mediated isothermal amplification (LAMP) of aquatic invasive species environmental DNA (AIS eDNA) was used for rapid, sensitive, and specific detection of Dreissena sp. relevant to the Great Lakes (USA) basin. The method was validated for two uses including i) direct amplification of eDNA using a hand filtration system and ii) confirmation of the results after DNA extraction using a conventional thermal cycler run at isothermal temperatures. Direct amplification eliminated the need for DNA extraction and purification and allowed detection of target invasive species in grab or concentrated surface water samples, containing both free DNA as well as larger cells and particulates, such as veligers, eggs, or seeds. The direct amplification method validation was conducted using Dreissena polymorpha and Dreissena bugensis and uses up to 1 L grab water samples for high target abundance (e.g., greater than 10 veligers (larval mussels) per L for Dreissena sp.) or 20 L samples concentrated through 35 μm nylon screens for low target abundance, at less than 10 veligers per liter water. Surface water concentrate samples were collected over a period of three years, mostly from inland lakes in Michigan with the help of a network of volunteers. Field samples collected from 318 surface water locations included i) filtered concentrate for direct amplification validation and ii) 1 L grab water sample for eDNA extraction and confirmation. Though the extraction-based protocol was more sensitive (resulting in more positive detections than direct amplification), direct amplification could be used for rapid screening, allowing for quicker action times. For samples collected between May and August, results of eDNA direct amplification were consistent with known presence/absence of selected invasive species. A cross-platform smartphone application was also developed to disseminate the analyzed results to volunteers. Field tests of the direct amplification protocol using a portable device (Gene-Z) showed the method could be used in the field to obtain results within one hr (from sample to result). Overall, the direct amplification has the potential to simplify the eDNA-based monitoring of multiple aquatic invasive species. Additional studies are warranted to establish quantitative correlation between eDNA copy number, veliger, biomass or organismal abundance in the field.

Isothermal amplification of environmental DNA (eDNA) for direct field-based monitoring and laboratory confirmation of Dreissena sp.

PubMed Central

Stedtfeld, Robert D.; Engle, Cathrine; Salach, Paul; Fakher, Umama; Stedtfeld, Tiffany; Dreelin, Erin; Stevenson, R. Jan; Latimore, Jo; Hashsham, Syed A.

2017-01-01

Loop-mediated isothermal amplification (LAMP) of aquatic invasive species environmental DNA (AIS eDNA) was used for rapid, sensitive, and specific detection of Dreissena sp. relevant to the Great Lakes (USA) basin. The method was validated for two uses including i) direct amplification of eDNA using a hand filtration system and ii) confirmation of the results after DNA extraction using a conventional thermal cycler run at isothermal temperatures. Direct amplification eliminated the need for DNA extraction and purification and allowed detection of target invasive species in grab or concentrated surface water samples, containing both free DNA as well as larger cells and particulates, such as veligers, eggs, or seeds. The direct amplification method validation was conducted using Dreissena polymorpha and Dreissena bugensis and uses up to 1 L grab water samples for high target abundance (e.g., greater than 10 veligers (larval mussels) per L for Dreissena sp.) or 20 L samples concentrated through 35 μm nylon screens for low target abundance, at less than 10 veligers per liter water. Surface water concentrate samples were collected over a period of three years, mostly from inland lakes in Michigan with the help of a network of volunteers. Field samples collected from 318 surface water locations included i) filtered concentrate for direct amplification validation and ii) 1 L grab water sample for eDNA extraction and confirmation. Though the extraction-based protocol was more sensitive (resulting in more positive detections than direct amplification), direct amplification could be used for rapid screening, allowing for quicker action times. For samples collected between May and August, results of eDNA direct amplification were consistent with known presence/absence of selected invasive species. A cross-platform smartphone application was also developed to disseminate the analyzed results to volunteers. Field tests of the direct amplification protocol using a portable device (Gene-Z) showed the method could be used in the field to obtain results within one hr (from sample to result). Overall, the direct amplification has the potential to simplify the eDNA-based monitoring of multiple aquatic invasive species. Additional studies are warranted to establish quantitative correlation between eDNA copy number, veliger, biomass or organismal abundance in the field. PMID:29036210

Limited efficiency of universal mini-barcode primers for DNA amplification from desert reptiles, birds and mammals.

PubMed

Arif, I A; Khan, H A; Al Sadoon, M; Shobrak, M

2011-10-31

In recent years, DNA barcoding has emerged as a powerful tool for species identification. We report an extended validation of a universal DNA mini-barcode for amplification of 130-bp COI segments from 23 specimens collected from a desert environment, including 11 reptiles, five mammals and seven birds. Besides the standard double-annealing protocol, we also tested a more stringent single-annealing protocol. The PCR success rate for the amplification of the mini-barcode region was: mammals (4/5), reptiles (5/11) and birds (4/7). These findings demonstrate the limited utility of universal primers for mini-barcoding, at least for these vertebrate taxa that we collected from the Saudi Arabian desert.

Protein detection using biobarcodes.

PubMed

Müller, Uwe R

2006-10-01

Over the past 50 years the development of assays for the detection of protein analytes has been driven by continuing demands for higher levels of sensitivity and multiplexing. The result has been a progression of sandwich-type immunoassays, starting with simple radioisotopic, colorimetric, or fluorescent labeling systems to include various enzymatic or nanostructure-based signal amplification schemes, with a concomitant sensitivity increase of over 1 million fold. Multiplexing of samples and tests has been enabled by microplate and microarray platforms, respectively, or lately by various molecular barcoding systems. Two different platforms have emerged as the current front-runners by combining a nucleic acid amplification step with the standard two-sided immunoassay. In both, the captured protein analyte is replaced by a multiplicity of oligonucleotides that serve as surrogate targets. One of these platforms employs DNA or RNA polymerases for the amplification step, while detection is by fluorescence. The other is based on gold nanoparticles for both amplification as well as detection. The latter technology, now termed Biobarcode, is completely enzyme-free and offers potentially much higher multiplexing power.

Signal amplification of FISH for automated detection using image cytometry.

PubMed

Truong, K; Boenders, J; Maciorowski, Z; Vielh, P; Dutrillaux, B; Malfoy, B; Bourgeois, C A

1997-05-01

The purpose of this study was to improve the detection of FISH signals, in order that spot counting by a fully automated image cytometer be comparable to that obtained visually under the microscope. Two systems of spot scoring, visual and automated counting, were investigated in parallel on stimulated human lymphocytes with FISH using a biotinylated centromeric probe for chromosome 3. Signal characteristics were first analyzed on images recorded with a coupled charge device (CCD) camera. Number of spots per nucleus were scored visually on these recorded images versus automatically with a DISCOVERY image analyzer. Several fluochromes, amplification and pretreatments were tested. Our results for both visual and automated scoring show that the tyramide amplification system (TSA) gives the best amplification of signal if pepsin treatment is applied prior to FISH. Accuracy of the automated scoring, however, remained low (58% of nuclei containing two spots) compared to the visual scoring because of the high intranuclear variation between FISH spots.

Detection of Entamoeba histolytica by Recombinase Polymerase Amplification

PubMed Central

Nair, Gayatri; Rebolledo, Mauricio; White, A. Clinton; Crannell, Zachary; Richards-Kortum, R. Rebecca; Pinilla, A. Elizabeth; Ramírez, Juan David; López, M. Consuelo; Castellanos-Gonzalez, Alejandro

2015-01-01

Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions. PMID:26123960

Cloning and sequencing of the histidine decarboxylase genes of gram-negative, histamine-producing bacteria and their application in detection and identification of these organisms in fish.

PubMed

Takahashi, Hajime; Kimura, Bon; Yoshikawa, Miwako; Fujii, Tateo

2003-05-01

The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products. To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed. A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (hdc) was developed. A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested. Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography. Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of Citrobacter braakii. In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product. Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product. A phylogenetic tree of the isolates constructed using newly determined sequences of partial hdc was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences. Histamine accumulation occurred when PCR amplification of hdc was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification. The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.

Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics

PubMed Central

Radonić, Aleksandar; Kocak Tufan, Zeliha; Domingo, Cristina

2017-01-01

Background We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics. Methodology An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. Principal findings The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1–10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours. Conclusions Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring. PMID:29155823

Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics.

PubMed

Brinkmann, Annika; Ergünay, Koray; Radonić, Aleksandar; Kocak Tufan, Zeliha; Domingo, Cristina; Nitsche, Andreas

2017-11-01

We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics. An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1-10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours. Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.

Determining the profiles and parameters for gene amplification testing of growth factor receptors in lung cancer.

PubMed

Pros, Eva; Lantuejoul, Sylvie; Sanchez-Verde, Lydia; Castillo, Sandra D; Bonastre, Ester; Suarez-Gauthier, Ana; Conde, Esther; Cigudosa, Juan C; Lopez-Rios, Fernando; Torres-Lanzas, Juan; Castellví, Josep; Ramon y Cajal, Santiago; Brambilla, Elisabeth; Sanchez-Cespedes, Montse

2013-08-15

Growth factor receptors (GFRs) are amenable to therapeutic intervention in cancer and it is important to select patients appropriately. One of the mechanisms for activation of GFRs is gene amplification (GA) but discrepancies arising from the difficulties associated with data interpretation and the lack of agreed parameters confound the comparison of results from different laboratories. Here, we attempt to establish appropriate conditions for standardization of the determination of GA in a panel of GFRs. A NSCLC tissue microarray panel containing 302 samples was screened for alterations at ALK, FGFR1, FGFR2, FGFR3, ERBB2, IGF1R, KIT, MET and PDGFRA by FISH, immunostaining and/or real-time quantitative RT-PCR. Strong amplification was found for FGFR1, ERBB2, KIT/PDFGRA and MET, with frequencies ranging from 1 to 6%. Thresholds for overexpression and GA were established. Strong immunostaining was found in most tumors with ERBB2, MET and KIT amplification, although some tumors underwent strong immunostaining in the absence of GA. KIT and PDFGRA were always coamplified, but only one tumor showed PDGFRA overexpression, indicating that KIT is the main target. Amplification of FGFR1 predominated in squamous cell carcinomas, although the association with overexpression was inconclusive. Interestingly, alterations at ALK, MET, EGFR, ERBB2 and KRAS correlated with augmented levels of phospho-S6 protein, suggesting activation of the mTOR pathway, which may prove useful to pre-select tumors for testing. Overall, here, we provide with parameters for the determination of GA at ERBB2, MET, KIT and PDGFRA which could be implemented in the clinic to stratify lung cancer patients for specific treatments. Copyright © 2013 UICC.

"Paper Machine" for Molecular Diagnostics.

PubMed

Connelly, John T; Rolland, Jason P; Whitesides, George M

2015-08-04

Clinical tests based on primer-initiated amplification of specific nucleic acid sequences achieve high levels of sensitivity and specificity. Despite these desirable characteristics, these tests have not reached their full potential because their complexity and expense limit their usefulness to centralized laboratories. This paper describes a device that integrates sample preparation and loop-mediated isothermal amplification (LAMP) with end point detection using a hand-held UV source and camera phone. The prototype device integrates paper microfluidics (to enable fluid handling) and a multilayer structure, or a "paper machine", that allows a central patterned paper strip to slide in and out of fluidic path and thus allows introduction of sample, wash buffers, amplification master mix, and detection reagents with minimal pipetting, in a hand-held, disposable device intended for point-of-care use in resource-limited environments. This device creates a dynamic seal that prevents evaporation during incubation at 65 °C for 1 h. This interval is sufficient to allow a LAMP reaction for the Escherichia coli malB gene to proceed with an analytical sensitivity of 1 double-stranded DNA target copy. Starting with human plasma spiked with whole, live E. coli cells, this paper demonstrates full integration of sample preparation with LAMP amplification and end point detection with a limit of detection of 5 cells. Further, it shows that the method used to prepare sample enables concentration of DNA from sample volumes commonly available from fingerstick blood draw.

Protocol for the use of a rapid real-time PCR method for the detection of HIV-1 proviral DNA using double-stranded primer.

PubMed

Pau, Chou-Pong; Wells, Susan K; Granade, Timothy C

2012-01-01

This chapter describes a real-time PCR method for the detection of HIV-1 proviral DNA in whole blood samples using a novel double-stranded primer system. The assay utilizes a simple commercially available DNA extraction method and a rapid and easy-to-perform real-time PCR protocol to consistently detect a minimum of four copies of HIV-1 group M proviral DNA in as little as 90 min after sample (whole blood) collection. Co-amplification of the human RNase P gene serves as an internal control to monitor the efficiency of both the DNA extraction and amplification. Once the assay is validated properly, it may be suitable as an alternative confirmation test for HIV-1 infections in a variety of HIV testing venues including the mother-to-child transmission testing sites, clinics, and diagnostic testing centers.

Rapid Detection of Prunus Necrotic Ringspot Virus by Reverse Transcription-cross-priming Amplification Coupled with Nucleic Acid Test Strip Cassette.

PubMed

Huo, Ya-Yun; Li, Gui-Fen; Qiu, Yan-Hong; Li, Wei-Min; Zhang, Yong-Jiang

2017-11-23

Prunus necrotic ringspot virus (PNRSV) is one of the most devastating viruses to Prunus spp. In this study, we developed a diagnostic system RT-CPA-NATSC, wherein reverse transcription-cross-priming amplification (RT-CPA) is coupled with nucleic acid test strip cassette (NATSC), a vertical flow (VF) visualization, for PNRSV detection. The RT-CPA-NATSC assay targets the encoding gene of the PNRSV coat protein with a limit of detection of 72 copies per reaction and no cross-reaction with the known Prunus pathogenic viruses and viroids, demonstrating high sensitivity and specificity. The reaction is performed on 60 °C and can be completed less than 90 min with the prepared template RNA. Field sample test confirmed the reliability of RT-CPA-NATSC, indicating the potential application of this simple and rapid detection method in routine test of PNRSV.

Comparison of DNA aneuploidy, chromosome 1 abnormalities, MYCN amplification and CD44 expression as prognostic factors in neuroblastoma.

PubMed

Christiansen, H; Sahin, K; Berthold, F; Hero, B; Terpe, H J; Lampert, F

1995-01-01

A comparison of the prognostic impact of five molecular variables in a large series was made, including tests of their nonrandom association and multivariate analysis. Molecular data were available for 377 patients and MYCN amplification, cytogenetic chromosome 1p deletion, loss of chromosome 1p heterozygosity, DNA ploidy and CD44 expression were investigated. Their interdependence and influence on event-free survival was tested uni- and multivariately using Pearson's chi 2-test, Kaplan-Meier estimates, log rank tests and the Cox's regression model. MYCN amplification was present in 18% (58/322) of cases and predicted poorer prognosis in localised (P < 0.001), metastatic (P = 0.002) and even 4S (P = 0.040) disease. CD44 expression was found in 86% (127/148) of cases, and was a marker for favourable outcome in patients with neuroblastoma stages 1-3 (P = 0.003) and 4 (P = 0.017). Chromosome 1p deletion was cytogenetically detected in 51% (28/55), and indicated reduced event-free survival in localised neuroblastoma (P = 0.020). DNA ploidy and loss of heterozygosity on chromosome 1p were of less prognostic value. Most factors of prognostic significance were associated with each other. By multivariate analysis, MYCN was selected as the only relevant factor. Risk estimation of high discriminating power is, therefore, possible for patients with localised and metastatic neuroblastoma using stage and MYCN.

Relative Accuracy of Nucleic Acid Amplification Tests and Culture in Detecting Chlamydia in Asymptomatic Men

PubMed Central

Cheng, Hong; Macaluso, Maurizio; Vermund, Sten H.; Hook, Edward W.

2001-01-01

Published estimates of the sensitivity and specificity of PCR and ligase chain reaction (LCR) for detecting Chlamydia trachomatis are potentially biased because of study design limitations (confirmation of test results was limited to subjects who were PCR or LCR positive but culture negative). Relative measures of test accuracy are less prone to bias in incomplete study designs. We estimated the relative sensitivity (RSN) and relative false-positive rate (RFP) for PCR and LCR versus cell culture among 1,138 asymptomatic men and evaluated the potential bias of RSN and RFP estimates. PCR and LCR testing in urine were compared to culture of urethral specimens. Discordant results (PCR or LCR positive, but culture negative) were confirmed by using a sequence including the other DNA amplification test, direct fluorescent antibody testing, and a DNA amplification test to detect chlamydial major outer membrane protein. The RSN estimates for PCR and LCR were 1.45 (95% confidence interval [CI] = 1.3 to 1.7) and 1.49 (95% CI = 1.3 to 1.7), respectively, indicating that both methods are more sensitive than culture. Very few false-positive results were found, indicating that the specificity levels of PCR, LCR, and culture are high. The potential bias in RSN and RFP estimates were <5 and <20%, respectively. The estimation of bias is based on the most likely and probably conservative parameter settings. If the sensitivity of culture is between 60 and 65%, then the true sensitivity of PCR and LCR is between 90 and 97%. Our findings indicate that PCR and LCR are significantly more sensitive than culture, while the three tests have similar specificities. PMID:11682509

Absence of estrogen receptor alpha (ESR1) gene amplification in a series of breast cancers in Taiwan.

PubMed

Chen, Jim-Ray; Hsieh, Tsan-Yu; Chen, Huang-Yang; Yeh, Kun-Yan; Chen, Kuo-Su; ChangChien, Yi-Che; Pintye, Mariann; Chang, Liang-Che; Hwang, Cheng-Cheng; Chien, Hui-Ping; Hsu, Yuan-Chun

2014-06-01

Immunohistochemical expression of ERα, encoded by the ESR1 (estrogen receptor 1) gene located at 6q25.1, is the most important determinant of responsiveness to endocrine therapy in breast cancer. The prevalence and significance of ESR1 amplification in breast cancer remain controversial. We set out to assess ESR1 status and its relevance in breast cancer in Taiwan. We tested tissue samples from 311 invasive carcinomas in a tissue microarray for ESR1 status by fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH). In order to examine its association with ERα and ESR1 status, HER2 status was determined by FISH. Of the carcinomas, 58.8 % (183/311) was ERα positive. None of the carcinomas showed amplification of ESR1 by either method, whereas 24.1 % (75/311) of the carcinomas harbored HER2 amplification. Of the carcinomas, 9.6 % (26/301) showed ESR1 gain (1.3 ≤ ratio ESR1/chromosome 6 < 2) by FISH and 10 % (24/299) by CISH. FISH and CISH results showed a good correlation (κ-coefficient = 0.786). ESR1 gain by FISH and CISH was significantly associated with high-grade (P = 0.0294 and 0.0417, respectively) but not with ERα expression, HER2 status, or overall survival. ERα positivity was significantly associated with better overall survival (P = 0.039). HER2 amplification was significantly related with poor overall survival (P = 0.002). Our data confirm that in breast cancer, HER2 amplification is a frequent genetic aberration and a negative prognostic factor, and show that ESR1 amplification is not a key genetic abnormality in the tumorigenesis of breast cancer in Taiwan.

Differential impact of ionic and coordinate covalent chromium (Cr)-DNA binding on DNA replication.

PubMed

Fornsaglio, Jamie L; O'Brien, Travis J; Patierno, Steven R

2005-11-01

The reactive species produced by the reduction of Cr(VI), particularly Cr(III), can form both ionic and coordinate covalent complexes with DNA. These Cr(III)-DNA interactions consist of Cr-DNA monoadducts, Cr-DNA ternary adducts, and Cr-DNA interstrand cross-links (Cr-ICLs), the latter of which are DNA polymerase arresting lesions (PALs). We sought to determine the impact of Cr-DNA interactions on the formation of replication blocking lesions in S. cerevisiae using a PCR-based method. We found that target sequence (TS) amplification using DNA isolated from Cr(VI)-treated yeast actually increased as a function of Cr(VI) concentration. Moreover, the enhanced TS amplification was reproduced in vitro using Cr(III)-treated DNA. In contrast, PCR amplification of TS from DNA isolated from yeast exposed to equitoxic doses of the inorganic DNA cross-linking agent cisplatin (CDDP), was decreased in a concentration-dependent manner. This paradox suggested that a specific Cr-DNA interaction, such as an ionic Cr-DNA complex, was responsible for the enhanced TS amplification, thereby masking the replication-blocking effect of certain ternary Cr-DNA adducts (i.e. interstrand cross-links). To test this possibility, we removed ionically associated Cr from the DNA using salt extraction prior to PCR analysis. This procedure obviated the increased amplification and revealed a dose-dependent decrease in TS amplification and an increase in Cr-PALs. These data from DNA analyzed ex vivo after treatment of intact cells indicate that ionic interactions of Cr with DNA result in increased DNA amplification whereas coordinate-covalent Cr-DNA complexes lead to formation of Cr-PALs. Thus, these results suggest that treatment of living cells with Cr(VI) leads to two modes of Cr-binding, which may have conflicting effects on DNA replication.

Amplification of the NSD3-BRD4-CHD8 pathway in pelvic high-grade serous carcinomas of tubo-ovarian and endometrial origin.

PubMed

Jones, Derek H; Lin, Douglas I

2017-08-01

Identification of novel therapeutics in pelvic high-grade serous carcinoma (HGSC) has been hampered by a paucity of actionable point mutations in target genes. The aim of the present study was to investigate the extent of amplification of the therapeutically targetable NSD3-CHD8-BRD4 pathway in pelvic HGSC, and to determine whether amplification is associated with worse prognosis. The Cancer Genome Atlas (TCGA) ovarian and endometrial cancer cohorts were retrospectively analyzed via online data-mining tools to test the association of NSD3 , CHD8 and BRD4 genomic alterations with survival of pelvic HGSC patients. It was demonstrated that amplification of the NSD3-CHD8-BRD4 pathway in the ovarian HGSC cohort (observed in 18% of the cases, 88/489) was significantly associated with worse overall and progression-free survival compared with non-amplified cases. In addition, amplification of NSD3 , CHD8 and BRD4 also occurred in 9% (21/232) of overall endometrial cancer TCGA cases, which was associated with worse overall survival. In the endometrial cancer TCGA cohort, NSD3 , CHD8 and BRD4 amplification occurred specifically in the serous carcinoma (25%, 13/53) and 'serous-like' copy number high endometrial carcinoma (33%, 20/60) subgroups, compared with the polymerase e (0%, 0/17), microsatellite instability high (0%, 0/65) or low copy number (1%, 1/90) subgroups. These findings support the hypothesis that amplification of the NSD3-BRD4-CDH8 axis is frequent in pelvic HGSC of both ovarian and endometrial origin, and that this pathway is potentially targetable in a subset of HGSC patients.

A three-phase amplification of the cosmic magnetic field in galaxies

NASA Astrophysics Data System (ADS)

Martin-Alvarez, Sergio; Devriendt, Julien; Slyz, Adrianne; Teyssier, Romain

2018-06-01

Arguably the main challenge of galactic magnetism studies is to explain how the interstellar medium of galaxies reaches energetic equipartition despite the extremely weak cosmic primordial magnetic fields that are originally predicted to thread the inter-galactic medium. Previous numerical studies of isolated galaxies suggest that a fast dynamo amplification might suffice to bridge the gap spanning many orders of magnitude in strength between the weak early Universe magnetic fields and the ones observed in high redshift galaxies. To better understand their evolution in the cosmological context of hierarchical galaxy growth, we probe the amplification process undergone by the cosmic magnetic field within a spiral galaxy to unprecedented accuracy by means of a suite of constrained transport magnetohydrodynamical adaptive mesh refinement cosmological zoom simulations with different stellar feedback prescriptions. A galactic turbulent dynamo is found to be naturally excited in this cosmological environment, being responsible for most of the amplification of the magnetic energy. Indeed, we find that the magnetic energy spectra of simulated galaxies display telltale inverse cascades. Overall, the amplification process can be divided in three main phases, which are related to different physical mechanisms driving galaxy evolution: an initial collapse phase, an accretion-driven phase, and a feedback-driven phase. While different feedback models affect the magnetic field amplification differently, all tested models prove to be subdominant at early epochs, before the feedback-driven phase is reached. Thus the three-phase evolution paradigm is found to be quite robust vis-a-vis feedback prescriptions.

Instrument for Real-Time Digital Nucleic Acid Amplification on Custom Microfluidic Devices

PubMed Central

Selck, David A.

2016-01-01

Nucleic acid amplification tests that are coupled with a digital readout enable the absolute quantification of single molecules, even at ultralow concentrations. Digital methods are robust, versatile and compatible with many amplification chemistries including isothermal amplification, making them particularly invaluable to assays that require sensitive detection, such as the quantification of viral load in occult infections or detection of sparse amounts of DNA from forensic samples. A number of microfluidic platforms are being developed for carrying out digital amplification. However, the mechanistic investigation and optimization of digital assays has been limited by the lack of real-time kinetic information about which factors affect the digital efficiency and analytical sensitivity of a reaction. Commercially available instruments that are capable of tracking digital reactions in real-time are restricted to only a small number of device types and sample-preparation strategies. Thus, most researchers who wish to develop, study, or optimize digital assays rely on the rate of the amplification reaction when performed in a bulk experiment, which is now recognized as an unreliable predictor of digital efficiency. To expand our ability to study how digital reactions proceed in real-time and enable us to optimize both the digital efficiency and analytical sensitivity of digital assays, we built a custom large-format digital real-time amplification instrument that can accommodate a wide variety of devices, amplification chemistries and sample-handling conditions. Herein, we validate this instrument, we provide detailed schematics that will enable others to build their own custom instruments, and we include a complete custom software suite to collect and analyze the data retrieved from the instrument. We believe assay optimizations enabled by this instrument will improve the current limits of nucleic acid detection and quantification, improving our fundamental understanding of single-molecule reactions and providing advancements in practical applications such as medical diagnostics, forensics and environmental sampling. PMID:27760148

An Innovative Field-Applicable Molecular Test to Diagnose Cutaneous Leishmania Viannia spp. Infections

PubMed Central

Saldarriaga, Omar A.; Castellanos-Gonzalez, Alejandro; Porrozzi, Renato; Baldeviano, Gerald C.; Lescano, Andrés G.; de Los Santos, Maxy B.; Fernandez, Olga L.; Saravia, Nancy G.; Costa, Erika; Melby, Peter C.; Travi, Bruno L.

2016-01-01

Cutaneous and mucosal leishmaniasis is widely distributed in Central and South America. Leishmania of the Viannia subgenus are the most frequent species infecting humans. L. (V.) braziliensis, L. (V.) panamensis are also responsible for metastatic mucosal leishmaniasis. Conventional or real time PCR is a more sensitive diagnostic test than microscopy, but the cost and requirement for infrastructure and trained personnel makes it impractical in most endemic regions. Primary health systems need a sensitive and specific point of care (POC) diagnostic tool. We developed a novel POC molecular diagnostic test for cutaneous leishmaniasis caused by Leishmania (Viannia) spp. Parasite DNA was amplified using isothermal Recombinase Polymerase Amplification (RPA) with primers and probes that targeted the kinetoplast DNA. The amplification product was detected by naked eye with a lateral flow (LF) immunochromatographic strip. The RPA-LF had an analytical sensitivity equivalent to 0.1 parasites per reaction. The test amplified the principal L. Viannia species from multiple countries: L. (V.) braziliensis (n = 33), L. (V.) guyanensis (n = 17), L. (V.) panamensis (n = 9). The less common L. (V.) lainsoni, L. (V.) shawi, and L. (V.) naiffi were also amplified. No amplification was observed in parasites of the L. (Leishmania) subgenus. In a small number of clinical samples (n = 13) we found 100% agreement between PCR and RPA-LF. The high analytical sensitivity and clinical validation indicate the test could improve the efficiency of diagnosis, especially in chronic lesions with submicroscopic parasite burdens. Field implementation of the RPA-LF test could contribute to management and control of cutaneous and mucosal leishmaniasis. PMID:27115155

Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

PubMed Central

Gulliksen, Anja; Keegan, Helen; Martin, Cara; O'Leary, John; Solli, Lars A.; Falang, Inger Marie; Grønn, Petter; Karlgård, Aina; Mielnik, Michal M.; Johansen, Ib-Rune; Tofteberg, Terje R.; Baier, Tobias; Gransee, Rainer; Drese, Klaus; Hansen-Hagge, Thomas; Riegger, Lutz; Koltay, Peter; Zengerle, Roland; Karlsen, Frank; Ausen, Dag; Furuberg, Liv

2012-01-01

The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection. PMID:22235204

Enhanced solid-phase recombinase polymerase amplification and electrochemical detection.

PubMed

Del Río, Jonathan Sabaté; Lobato, Ivan Magriñà; Mayboroda, Olena; Katakis, Ioanis; O'Sullivan, Ciara K

2017-05-01

Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10 -15 M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.

Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea

PubMed Central

Kurosaki, Yohei; Magassouba, N’Faly; Oloniniyi, Olamide K.; Cherif, Mahamoud S.; Sakabe, Saori; Takada, Ayato; Hirayama, Kenji; Yasuda, Jiro

2016-01-01

Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure. PMID:26900929

Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea.

PubMed

Kurosaki, Yohei; Magassouba, N'Faly; Oloniniyi, Olamide K; Cherif, Mahamoud S; Sakabe, Saori; Takada, Ayato; Hirayama, Kenji; Yasuda, Jiro

2016-02-01

Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure.

The influence of mass configurations on velocity amplified vibrational energy harvesters

NASA Astrophysics Data System (ADS)

O'Donoghue, D.; Frizzell, R.; Kelly, G.; Nolan, K.; Punch, J.

2016-05-01

Vibrational energy harvesters scavenge ambient vibrational energy, offering an alternative to batteries for the autonomous operation of low power electronics. Velocity amplified electromagnetic generators (VAEGs) utilize the velocity amplification effect to increase power output and operational bandwidth, compared to linear resonators. A detailed experimental analysis of the influence of mass ratio and number of degrees-of-freedom (dofs) on the dynamic behaviour and power output of a macro-scale VAEG is presented. Various mass configurations are tested under drop-test and sinusoidal forced excitation, and the system performances are compared. For the drop-test, increasing mass ratio and number of dofs increases velocity amplification. Under forced excitation, the impacts between the masses are more complex, inducing greater energy losses. This results in the 2-dof systems achieving the highest velocities and, hence, highest output voltages. With fixed transducer size, higher mass ratios achieve higher voltage output due to the superior velocity amplification. Changing the magnet size to a fixed percentage of the final mass showed the increase in velocity of the systems with higher mass ratios is not significant enough to overcome the reduction in transducer size. Consequently, the 3:1 mass ratio systems achieved the highest output voltage. These findings are significant for the design of future reduced-scale VAEGs.

Adult hearing-aid users with cochlear dead regions restricted to high frequencies: Implications for amplification.

PubMed

Pepler, Anna; Lewis, Kathryn; Munro, Kevin J

2016-01-01

Cochlear dead regions (DR) are common in adult hearing-aid users, but are usually restricted to high frequencies. The aim was to determine the benefit of high-frequency amplification for ears with and without high-frequency DRs. Participants were fitted with the study hearing aid and tested under four conditions: unfiltered (NAL-NL2 prescription), and low-pass filtered at 1.5, 2, and 3 kHz. VCV stimuli were presented at 65 dB (A) in quiet and in 20-talker babble at a signal-to-babble ratio of 0 dB. Experienced adult hearing-aid users: one group of 18 with a DR edge frequency above 1.5 kHz, and a group of 18 matched controls. Overall performance was best in the unfiltered condition. There was no significant difference in mean performance between the two groups when tested in quiet. However, the DR group obtained less benefit from high-frequency amplification when tested in babble: the mean difference between the unfiltered and 3-kHz filtered condition was 6% and 13% for the DR group and controls, respectively. In adults with a moderate hearing loss and a restricted DR, speech recognition was always best in the unfiltered condition, although mean performance in babble was lower for the DR group.

Detection of Entamoeba histolytica by Recombinase Polymerase Amplification.

PubMed

Nair, Gayatri; Rebolledo, Mauricio; White, A Clinton; Crannell, Zachary; Richards-Kortum, R Rebecca; Pinilla, A Elizabeth; Ramírez, Juan David; López, M Consuelo; Castellanos-Gonzalez, Alejandro

2015-09-01

Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions. © The American Society of Tropical Medicine and Hygiene.

'The waiting game': are current chlamydia and gonorrhoea near-patient/point-of-care tests acceptable to service users and will they impact on treatment?

PubMed

Atkinson, Lindsay M; Vijeratnam, Dayan; Mani, Reena; Patel, Raj

2016-07-01

The objective of this study was to assess the length of time service users were prepared to wait for chlamydia and gonorrhoea (CT/GC) near-patient/point-of-care test (NP-POCT) results and to determine the possible effect on management. Individuals attending two UK clinics from November 2013 to February 2014 were surveyed asking the maximum length of time they would wait for CT/GC NP-POCT results after consultation. Linked CT/GC prevalence and treatment rates were analysed. A total of 1817 participants were surveyed, and 1356 provided CT/GC NAAT samples, in which it was found that 115 (8.5%) could wait over 90 minutes in clinic for their result. 115 received treatment at consultation, of which 50 were CT/GC negative and 12 were treated for urethritis or cervicitis; 38 attended as CT/GC contacts. Six of this population would have waited over 90 minutes were NP-POCTs available. A total of 129 tested CT/GC positive, of whom 65 were treated at their consultation, 61 at a later date, and three were untreated. Twelve of these 129 patients would also have waited over 90 minutes for a NP-POCT result. We conclude that 90-minute NP-POCTs are not acceptable to most clinic attendees and would not have impacted on treatment rates or inappropriate prescribing, and 20-minute NP-POCTs show a marginal benefit in treating CT/GC. While NP-POCTs for CT/GC are promising, they must meet client expectations and enhance disease management in order to be accepted by patients and clinicians. © The Author(s) 2015.

Measures of Attitudes Toward and Communication about Condom Use: Their Relationships With Sexual Risk Behavior Among Young Black MSM

PubMed Central

Crosby, Richard A.; Graham, Cynthia A.; Yarber, William L.; Sanders, Stephanie A.; Milhausen, Robin R.; Mena, Leandro

2015-01-01

Objective To construct and test measures of psychosocial mediators that could be used in intervention studies seeking to promote safer sex behavior among young Black men who have sex with men (YBMSM). Methods YBMSM (N=400), ages 18–29 years, were recruited from an STI clinic, in the Southern U.S. All men had engaged in penile-anal sex with a male as a “top” in the past 6 months. Men completed an audio-computer assisted self-interview and provided specimens used for NAAT testing to detect Chlamydia and gonorrhea. Four measures were constructed and tested for criterion validity (Safer Sex Communication, Condom Turn-Offs, Condom Pleasure Scale, and a single item assessing frequency of condom use discussions before sexual arousal). Results With the exception of Safer Sex Communication, all of the measures showed criterion validity for both unprotected anal insertive, and unprotected anal receptive sex. With the exception of the Condom Turn-Offs, the three other measures were supported by criterion validity for oral sex. Both the Condom Turn-Offs and Condom Pleasure Scale were significantly related to whether or not men reported multiple partners as a “top” but only the Condom Pleasure Scale was associated with reports of multiple partners as a “bottom.” Only the Condom Turn-Offs Scale was positively associated with having been diagnosed with either Chlamydia or gonorrhea. Conclusion Findings provide three brief scales and a single item that can be used in intervention studies targeting YBMSM. Perceptions about condoms a turn off and about condoms enhancing pleasure showed strong association with sexual risk behaviors. PMID:26766525

Centrifugal LabTube platform for fully automated DNA purification and LAMP amplification based on an integrated, low-cost heating system.

PubMed

Hoehl, Melanie M; Weißert, Michael; Dannenberg, Arne; Nesch, Thomas; Paust, Nils; von Stetten, Felix; Zengerle, Roland; Slocum, Alexander H; Steigert, Juergen

2014-06-01

This paper introduces a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA purification platform (LabTube). We demonstrate LabTube-based fully automated DNA purification of as low as 100 cell-equivalents of verotoxin-producing Escherichia coli (VTEC) in water, milk and apple juice in a laboratory centrifuge, followed by integrated and automated LAMP amplification with a reduction of hands-on time from 45 to 1 min. The heating system consists of two parallel SMD thick film resistors and a NTC as heating and temperature sensing elements. They are driven by a 3 V battery and controlled by a microcontroller. The LAMP reagents are stored in the elution chamber and the amplification starts immediately after the eluate is purged into the chamber. The LabTube, including a microcontroller-based heating system, demonstrates contamination-free and automated sample-to-answer nucleic acid testing within a laboratory centrifuge. The heating system can be easily parallelized within one LabTube and it is deployable for a variety of heating and electrical applications.

Development of a recombinase polymerase amplification assay for Vibrio parahaemolyticus detection with an internal amplification control.

PubMed

Yang, Huan-Lan; Wei, Shuang; Gooneratne, Ravi; Mutukumira, Anthony N; Ma, Xue-Jun; Tang, Shu-Ze; Wu, Xi-Yang

2018-04-01

A novel RPA-IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 10 3 CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA-IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.

The thermal history of human fossils and the likelihood of successful DNA amplification.

PubMed

Smith, Colin I; Chamberlain, Andrew T; Riley, Michael S; Stringer, Chris; Collins, Matthew J

2003-09-01

Recent success in the amplification of ancient DNA (aDNA) from fossil humans has led to calls for further tests to be carried out on similar material. However, there has been little systematic research on the survival of DNA in the fossil record, even though the environment of the fossil is known to be of paramount importance for the survival of biomolecules over archaeological and geological timescales. A better understanding of aDNA survival would enable research to focus on material with greater chances of successful amplification, thus preventing the unnecessary loss of material and valuable researcher time. We argue that the thermal history of a fossil is a key parameter for the survival of biomolecules. The thermal history of a number of northwest European Neanderthal cave sites is reconstructed here and they are ranked in terms of the relative likelihood of aDNA survival at the sites, under the assumption that DNA depurination is the principal mechanism of degradation. The claims of aDNA amplification from material found at Lake Mungo, Australia, are also considered in the light of the thermal history of this site.

Portable nucleic acid thermocyclers.

PubMed

Almassian, David R; Cockrell, Lisa M; Nelson, William M

2013-11-21

A nucleic acid thermal cycler is considered to be portable if it is under ten pounds, easily carried by one individual, and battery powered. Nucleic acid amplification includes both polymerase chain reaction (e.g. PCR, RT-PCR) and isothermal amplification (e.g. RPA, HDA, LAMP, NASBA, RCA, ICAN, SMART, SDA). There are valuable applications for portable nucleic acid thermocyclers in fields that include clinical diagnostics, biothreat detection, and veterinary testing. A system that is portable allows for the distributed detection of targets at the point of care and a reduction of the time from sample to answer. The designer of a portable nucleic acid thermocycler must carefully consider both thermal control and the detection of amplification. In addition to thermal control and detection, the designer may consider the integration of a sample preparation subsystem with the nucleic acid thermocycler. There are a variety of technologies that can achieve accurate thermal control and the detection of nucleic acid amplification. Important evaluation criteria for each technology include maturity, power requirements, cost, sensitivity, speed, and manufacturability. Ultimately the needs of a particular market will lead to user requirements that drive the decision between available technologies.

Polydimethylsiloxane microfluidic chemiluminescence immunodevice with the signal amplification strategy for sensitive detection of human immunoglobin G.

PubMed

Li, Huifang; Zhao, Mei; Liu, Wei; Chu, Weiru; Guo, Yumei

2016-01-15

A polydimethylsiloxane (PDMS) microfluidic chemiluminescence (CL) immunodevice for sensitive detection of human immunoglobin G (IgG) with the signal amplification strategy was developed in this work. The immunodevice was prepared by covalently immobilizing capture antibodies (Abs) on the silanized microchannel of microfluidic chip. Gold nanoparticles (AuNPs) functionalized with a high molar ratio of horseradish peroxidase (HRP) were used as an Ab label for signal amplification. Using a sandwich immunoassay, the multi-HRP conjugated AuNPs can catalyze the luminol-H2O2 CL system to achieve the high sensitivity. In addition, the double spiral flow-channel was adopted here, which can still contribute to the high sensitivity. Based on signal amplification strategy, the performance of human IgG tests revealed a lower detection limit (DL) of 0.03ng/mL and showed an increase of 7.4-fold in detection sensitivity compared to a commercial Ab-HRP conjugation. This microfluidic immunodevice can provide an alternative approach for sensitive detection of human IgG in the field of clinic diagnostic and therapeutic. Copyright © 2015 Elsevier B.V. All rights reserved.

Towards in vivo amplification: Overcoming hurdles in the use of hematopoietic stem cells in transplantation and gene therapy

PubMed Central

Nagree, Murtaza S; López-Vásquez, Lucía; Medin, Jeffrey A

2015-01-01

With the advent of safer and more efficient gene transfer methods, gene therapy has become a viable solution for many inherited and acquired disorders. Hematopoietic stem cells (HSCs) are a prime cell compartment for gene therapy aimed at correcting blood-based disorders, as well as those amenable to metabolic outcomes that can effect cross-correction. While some resounding clinical successes have recently been demonstrated, ample room remains to increase the therapeutic output from HSC-directed gene therapy. In vivo amplification of therapeutic cells is one avenue to achieve enhanced gene product delivery. To date, attempts have been made to provide HSCs with resistance to cytotoxic drugs, to include drug-inducible growth modules specific to HSCs, and to increase the engraftment potential of transduced HSCs. This review aims to summarize amplification strategies that have been developed and tested and to discuss their advantages along with barriers faced towards their clinical adaptation. In addition, next-generation strategies to circumvent current limitations of specific amplification schemas are discussed. PMID:26730268

Transferability of microsatellite primers developed for stingless bees to four other species of the genus Melipona.

PubMed

Viana, M V C; Miranda, E A; de Francisco, A K; Carvalho, C A L; Waldschmidt, A M

2011-11-22

Microsatellite markers are a useful tool for ecological monitoring of natural and managed populations. A technical limitation is the necessity for investment in the development of primers. Heterologous primers can provide an alternative to searching for new loci. In bees, these markers have been used in populational and intracolonial genetic analyses. The genus Melipona has the largest number of species among bee genera, about 70, occurring throughout the Neotropical region. However, only five species of the genus Melipona have specific microsatellite markers. Given the great diversity of this genus, this number is not representative. We analyzed the transferability of 49 microsatellite loci to four other species of the genus Melipona (M. scutellaris, M. mondury, M. mandacaia, and M. quadrifasciata). Four individuals of each species, from different localities, were used in amplification tests. Primer pairs described for five Melipona species and for Trigona carbonaria were tested. Among the 49 loci, 22 gave amplification products for all four species, while three gave nonspecific bands and five showed no amplification products. The remaining loci varied in the pattern of amplification, according to the species examined. The number of alleles ranged from 1 to 6. The results demonstrate the possibility of using these heterologous markers in other Melipona species, increasing the number of loci that can be analyzed and contributing to further genetic analyses of intra- and intercolonial structure, which is required for conservation measure planning, genetic improvement and resolution of taxonomic problems.

The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections.

PubMed

Gray, J; Coupland, L J

2014-01-01

On 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG® Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene - in-vitro diagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

Rapid word-learning in normal-hearing and hearing-impaired children: effects of age, receptive vocabulary, and high-frequency amplification.

PubMed

Pittman, A L; Lewis, D E; Hoover, B M; Stelmachowicz, P G

2005-12-01

This study examined rapid word-learning in 5- to 14-year-old children with normal and impaired hearing. The effects of age and receptive vocabulary were examined as well as those of high-frequency amplification. Novel words were low-pass filtered at 4 kHz (typical of current amplification devices) and at 9 kHz. It was hypothesized that (1) the children with normal hearing would learn more words than the children with hearing loss, (2) word-learning would increase with age and receptive vocabulary for both groups, and (3) both groups would benefit from a broader frequency bandwidth. Sixty children with normal hearing and 37 children with moderate sensorineural hearing losses participated in this study. Each child viewed a 4-minute animated slideshow containing 8 nonsense words created using the 24 English consonant phonemes (3 consonants per word). Each word was repeated 3 times. Half of the 8 words were low-pass filtered at 4 kHz and half were filtered at 9 kHz. After viewing the story twice, each child was asked to identify the words from among pictures in the slide show. Before testing, a measure of current receptive vocabulary was obtained using the Peabody Picture Vocabulary Test (PPVT-III). The PPVT-III scores of the hearing-impaired children were consistently poorer than those of the normal-hearing children across the age range tested. A similar pattern of results was observed for word-learning in that the performance of the hearing-impaired children was significantly poorer than that of the normal-hearing children. Further analysis of the PPVT and word-learning scores suggested that although word-learning was reduced in the hearing-impaired children, their performance was consistent with their receptive vocabularies. Additionally, no correlation was found between overall performance and the age of identification, age of amplification, or years of amplification in the children with hearing loss. Results also revealed a small increase in performance for both groups in the extended bandwidth condition but the difference was not significant at the traditional p = 0.05 level. The ability to learn words rapidly appears to be poorer in children with hearing loss over a wide range of ages. These results coincide with the consistently poorer receptive vocabularies for these children. Neither the word-learning or receptive-vocabulary measures were related to the amplification histories of these children. Finally, providing an extended high-frequency bandwidth did not significantly improve rapid word-learning for either group with these stimuli.

Specific detection of Xanthomonas axonopodis pv. dieffenbachiae in anthurium (Anthurium andreanum) tissues by nested PCR.

PubMed

Robène-Soustrade, Isabelle; Laurent, Philippe; Gagnevin, Lionel; Jouen, Emmanuel; Pruvost, Olivier

2006-02-01

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (10(3) CFU ml(-1)) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.

Novel microsatellite loci for Agave parryi and cross-amplification in Agave palmeri (Agavaceae).

PubMed

Lindsay, Denise L; Edwards, Christine E; Jung, Michael G; Bailey, Pamela; Lance, Richard F

2012-07-01

To examine the foraging behavior of nectarivorous bats in southeastern Arizona, we developed microsatellite primers in Agave parryi. These markers were also tested for cross-amplification and applicability to assess patterns of genetic diversity and structure in A. palmeri. Utilizing DNA sequence data from 454 shotgun sequencing, we identified seven novel polymorphic microsatellite loci in A. parryi and screened them for cross-amplification in A. palmeri. These markers were characterized in two populations of 30 individuals each for each species. In A. parryi, all primers were polymorphic and amplified between three and 12 alleles per population. In A. palmeri, all primers amplified, six were polymorphic, and allelic diversity ranged from one to 16 alleles per population. Our results demonstrate the applicability of these microsatellite primers for population genetics studies in both A. parryi and A. palmeri.

Polymerization-based signal amplification under ambient conditions with thirty-five second reaction times.

PubMed

Kaastrup, Kaja; Sikes, Hadley D

2012-10-21

Although polymerization-based amplification (PBA) has demonstrated promise as an inexpensive technique for use in molecular diagnostics, oxygen inhibition of radical photopolymerization has hindered its implementation in point-of-care devices. The addition of 0.3-0.7 μM eosin to an aqueous acrylate monomer solution containing a tertiary amine allows an interfacial polymerization reaction to proceed in air only near regions of a test surface where additional eosin initiators coupled to proteins have been localized as a function of molecular recognition events. The dose of light required for the reaction is inversely related to eosin concentration. This system achieves sensitivities comparable to those reported for inert gas-purged systems and requires significantly shorter reaction times. We provide several comparisons of this system with other implementations of polymerization-based amplification.

Self-Reported Use of Mouthwash and Pharyngeal Gonorrhoea Detection by Nucleic Acid Amplification Test.

PubMed

Chow, Eric P F; Walker, Sandra; Read, Tim R H; Chen, Marcus Y; Bradshaw, Catriona S; Fairley, Christopher K

2017-10-01

Use of alcohol-containing mouthwash has been found to have an inhibitory effect against pharyngeal gonorrhoea. The aim of this study was to investigate the association between self-reported mouthwash use and pharyngeal gonorrhoea detection among men who have sex with men (MSM). A cross-sectional survey was conducted between March 23, 2015, and June 30, 2015 among MSM attending the Melbourne Sexual Health Centre in Australia. Men who have sex with men were invited to complete a short questionnaire on mouthwash use and they were also tested for pharyngeal gonorrhoea by nucleic acid amplification test. Multivariate logistic regression was performed to examine the association between mouthwash use and pharyngeal gonorrhoea detection. Of the 823 MSM, pharyngeal gonorrhoea detection decreased significantly with increasing age group (≤24 years, 14.5%; 25-34 years, 10.7%; ≥35 years, 6.0%; ptrend = 0.003). The proportion reporting daily use of mouthwash increased significantly with increasing age group (from 10.1% to 14.5% to 19.8%; ptrend = 0.005). However, there was no significant association between pharyngeal gonorrhoea detection and daily use of mouthwash after adjusting for age, number of male sexual partners, human immunodeficiency virus status, and type of mouthwash use. Although the proportion of daily use of mouthwash increased with age, and pharyngeal gonorrhoea detection decreased with age, the association between self-reported mouthwash use and pharyngeal gonorrhoea detection by nucleic acid amplification test was not statistically significant.

One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

PubMed

Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

2013-01-02

Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs.

Amplification of transcutaneous and percutaneous bone-conduction devices with a test-band in an induced model of conductive hearing loss.

PubMed

Park, Marn Joon; Lee, Jae Ryung; Yang, Chan Joo; Yoo, Myung Hoon; Jin, In Suk; Choi, Chi Ho; Park, Hong Ju

2016-11-01

Transcutaneous devices have a disadvantage, the dampening effect by soft tissue between the bone and devices. We investigated hearing outcomes with percutaneous and transcutaneous devices using test-bands in an induced unilateral conductive hearing loss. Comparison of hearing outcomes of two devices in the same individuals. The right ear was plugged in 30 subjects and a test-band with devices (Cochlear™ Baha® BP110 Power and Sophono® Alpha-2 MPO™) was applied on the right mastoid tip with the left ear masked. Sound-field thresholds, speech recognition thresholds (SRTs), and word recognition scores (WRSs) were compared. Aided thresholds of Sophono were significantly better than those of Baha at most frequencies. Sophono WRSs (86 ± 12%) at 40 dB SPL and SRTs (14 ± 5 dB HL) were significantly better than those (73 ± 24% and 23 ± 8 dB HL) of Baha. However, Sophono WRSs (98 ± 3%) at 60 dB SPL did not differ from Baha WRSs (95 ± 12%). Amplifications of the current transcutaneous device were not inferior to those of percutaneous devices with a test-band in subjects with normal bone-conduction thresholds. Since the percutaneous devices can increase the gain when fixed to the skull by eliminating the dampening effect, both devices are expected to provide sufficient hearing amplification.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stobinska, M.; Institute for Theoretical Physics II, Erlangen-Nuernberg University, Erlangen; Sekatski, P.

Quantum correlations may violate the Bell inequalities. Most experimental schemes confirming this prediction have been realized in all-optical Bell tests suffering from the detection loophole. Experiments which simultaneously close this loophole and the locality loophole are highly desirable and remain challenging. An approach to loophole-free Bell tests is based on amplification of the entangled photons (i.e., on macroscopic entanglement), for which an optical signal should be easy to detect. However, the macroscopic states are partially indistinguishable by classical detectors. An interesting idea to overcome these limitations is to replace the postselection by an appropriate preselection immediately after the amplification. Thismore » is in the spirit of state preprocessing revealing hidden nonlocality. Here, we examine one of the possible preselections, but the presented tools can be used for analysis of other schemes. Filtering methods making the macroscopic entanglement useful for Bell tests and quantum protocols are the subject of an intensive study in the field nowadays.« less

An expanded nuclear phylogenomic PCR toolkit for Sapindales1

PubMed Central

Collins, Elizabeth S.; Gostel, Morgan R.; Weeks, Andrea

2016-01-01

Premise of the study: We tested PCR amplification of 91 low-copy nuclear gene loci in taxa from Sapindales using primers developed for Bursera simaruba (Burseraceae). Methods and Results: Cross-amplification of these markers among 10 taxa tested was related to their phylogenetic distance from B. simaruba. On average, each Sapindalean taxon yielded product for 53 gene regions (range: 16–90). Arabidopsis thaliana (Brassicales), by contrast, yielded product for two. Single representatives of Anacardiaceae and Rutacaeae yielded 34 and 26 products, respectively. Twenty-six primer pairs worked for all Burseraceae species tested if highly divergent Aucoumea klaineana is excluded, and eight of these amplified product in every Sapindalean taxon. Conclusions: Our study demonstrates that customized primers for Bursera can amplify product in a range of Sapindalean taxa. This collection of primer pairs, therefore, is a valuable addition to the toolkit for nuclear phylogenomic analyses of Sapindales and warrants further investigation. PMID:28101434

The principle and application of new PCR Technologies

NASA Astrophysics Data System (ADS)

Yu, Miao; Cao, Yue; Ji, Yubin

2017-12-01

Polymerase chain reaction (PCR) is essentially a selective DNA amplification technique commonlyapplied for genetic testing and molecular diagnosis because of its high specificity and sensitivity.PCR technologies as the key of molecular biology, has realized that the qualitative detection of absolute quantitative has been changed. It has produced a variety of new PCR technologies, such as extreme PCR, photonic PCR, o-amplification at lower denaturation temperature PCR, nanoparticle PCR and so on. In this paper, the principle and application of PCR technologies are reviewed, and its development is prospected too.

Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of Rickettsia spp.

PubMed

Hanaoka, Nozomu; Matsutani, Minenosuke; Satoh, Masaaki; Ogawa, Motohiko; Shirai, Mutsunori; Ando, Shuji

2017-01-24

We developed a novel loop-mediated isothermal amplification (LAMP) method to detect Rickettsia spp., including Rickettsia prowazekii and R. typhi. Species-specific LAMP primers were developed for orthologous genes conserved among Rickettsia spp. The selected modified primers could detect all the Rickettsia spp. tested. The LAMP method was successfully used to detect 100 DNA copies of Rickettsia spp. within approximately 60 min at 63℃. Therefore, this method may be an excellent tool for the early diagnosis of rickettsiosis in a laboratory or in the field.

Loop-mediated isothermal amplification test for detection of Neisseria gonorrhoeae in urine samples and tolerance of the assay to the presence of urea.

PubMed

Edwards, Thomas; Burke, Patricia A; Smalley, Helen B; Gillies, Liz; Hobbs, Glyn

2014-06-01

A loop-mediated isothermal amplification (LAMP) assay for open reading frame 1 (ORF1) of the glutamine synthetase gene of Neisseria gonorrhoeae was able to tolerate urea concentrations of ≤ 1.8 M, compared with a PCR assay that was functional at concentrations of <100 mM. The LAMP assay was as sensitive as the PCR assay while being faster and simpler to perform. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Enzyme-catalysed deposition of ultrathin silver shells on gold nanorods: a universal and highly efficient signal amplification strategy for translating immunoassay into a litmus-type test.

PubMed

Yang, Xinjian; Gao, Zhiqiang

2015-04-25

On the basis of enzyme-catalysed reduction of silver ions and consequent deposition of ultrathin silver shells on gold nanorods, a highly efficient signal amplification method for immunoassay is developed. For a model analyte prostate-specific antigen, a 10(4)-fold improvement over conventional enzyme-linked immunosorbent assay is accomplished by leveraging on the cumulative nature of the enzymatic reaction and the sensitive response of plasnomic gold nanorods to the deposition the silver shells.

An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases

PubMed Central

Liu, Changchun; Geva, Eran; Mauk, Michael; Qiu, Xianbo; Abrams, William R.; Malamud, Daniel; Curtis, Kelly; Owen, S. Michele; Bau, Haim H.

2015-01-01

A simple, point of care, inexpensive, disposable cassette for the detection of nucleic acids extracted from pathogens was designed, constructed, and tested. The cassette utilizes a single reaction chamber for isothermal amplification of nucleic acids. The chamber is equipped with an integrated, flow-through, Flinders Technology Associates (Whatman FTA®) membrane for the isolation, concentration, and purification of DNA and/or RNA. The nucleic acids captured by the membrane are used directly as templates for amplification without elution, thus simplifying the cassette’s flow control. The FTA membrane also serves another critical role—enabling the removal of inhibitors that dramatically reduce detection sensitivity. Thermal control is provided with a thin film heater external to the cassette. The amplification process was monitored in real time with a portable, compact fluorescent reader. The utility of the integrated, single-chamber cassette was demonstrated by detecting the presence of HIV-1 in oral fluids. The HIV RNA was reverse transcribed and subjected to loop-mediated, isothermal amplification (LAMP). A detection limit of less than 10 HIV particles was demonstrated. The cassette is particularly suitable for resource poor regions, where funds and trained personnel are in short supply. The cassette can be readily modified to detect nucleic acids associated with other pathogens borne in saliva, urine, and other body fluids as well as in water and food. PMID:21455542

An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases.

PubMed

Liu, Changchun; Geva, Eran; Mauk, Michael; Qiu, Xianbo; Abrams, William R; Malamud, Daniel; Curtis, Kelly; Owen, S Michele; Bau, Haim H

2011-05-21

A simple, point of care, inexpensive, disposable cassette for the detection of nucleic acids extracted from pathogens was designed, constructed, and tested. The cassette utilizes a single reaction chamber for isothermal amplification of nucleic acids. The chamber is equipped with an integrated, flow-through, Flinders Technology Associates (Whatman FTA®) membrane for the isolation, concentration, and purification of DNA and/or RNA. The nucleic acids captured by the membrane are used directly as templates for amplification without elution, thus simplifying the cassette's flow control. The FTA membrane also serves another critical role-enabling the removal of inhibitors that dramatically reduce detection sensitivity. Thermal control is provided with a thin film heater external to the cassette. The amplification process was monitored in real time with a portable, compact fluorescent reader. The utility of the integrated, single-chamber cassette was demonstrated by detecting the presence of HIV-1 in oral fluids. The HIV RNA was reverse transcribed and subjected to loop-mediated, isothermal amplification (LAMP). A detection limit of less than 10 HIV particles was demonstrated. The cassette is particularly suitable for resource poor regions, where funds and trained personnel are in short supply. The cassette can be readily modified to detect nucleic acids associated with other pathogens borne in saliva, urine, and other body fluids as well as in water and food.

Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

PubMed

Marine, Rachel; McCarren, Coleen; Vorrasane, Vansay; Nasko, Dan; Crowgey, Erin; Polson, Shawn W; Wommack, K Eric

2014-01-30

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested. Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes. MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination

PubMed Central

Karthik, K.; Rathore, Rajesh; Thomas, Prasad; Arun, T.R.; Viswas, K.N.; Dhama, Kuldeep; Agarwal, R.K.

2014-01-01

Loop mediated isothermal amplification (LAMP) assay, a promising diagnostic test, has been developed for detection of different pathogens of human as well as animals. Various positive points support its use as a field level test but the major problem is product cross contamination leading to false positive results. Different methods were adopted by various researchers to control this false positive amplification due to cross contamination but all have their own advantages and disadvantages. A new closed tube LAMP assay based on agar dye capsule was developed in the present study and this technique has some advantages over the other closed tube technique.•Agar at the concentration of 1.5% was used to sandwich SYBR green dye I with the aid of intradermal syringe. This agar dye capsule was placed over the LAMP reaction mixture before it was amplified.•To eliminate the hazardous nature of Ultra Violet (UV) light during result visualization of LAMP products, the present study demonstrates the use of Light Emitting Diode (LED) lights for result visualization.•LAMP was carried out for Brucella species detection using this modified techniques yielding good results without any cross contamination and LED showed similar fluorescence compared to UV. PMID:26150945

New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination.

PubMed

Karthik, K; Rathore, Rajesh; Thomas, Prasad; Arun, T R; Viswas, K N; Dhama, Kuldeep; Agarwal, R K

2014-01-01

Loop mediated isothermal amplification (LAMP) assay, a promising diagnostic test, has been developed for detection of different pathogens of human as well as animals. Various positive points support its use as a field level test but the major problem is product cross contamination leading to false positive results. Different methods were adopted by various researchers to control this false positive amplification due to cross contamination but all have their own advantages and disadvantages. A new closed tube LAMP assay based on agar dye capsule was developed in the present study and this technique has some advantages over the other closed tube technique.•Agar at the concentration of 1.5% was used to sandwich SYBR green dye I with the aid of intradermal syringe. This agar dye capsule was placed over the LAMP reaction mixture before it was amplified.•To eliminate the hazardous nature of Ultra Violet (UV) light during result visualization of LAMP products, the present study demonstrates the use of Light Emitting Diode (LED) lights for result visualization.•LAMP was carried out for Brucella species detection using this modified techniques yielding good results without any cross contamination and LED showed similar fluorescence compared to UV.

Comparison of point-of-care-compatible lysis methods for bacteria and viruses.

PubMed

Heiniger, Erin K; Buser, Joshua R; Mireles, Lillian; Zhang, Xiaohong; Ladd, Paula D; Lutz, Barry R; Yager, Paul

2016-09-01

Nucleic acid sample preparation has been an especially challenging barrier to point-of-care nucleic acid amplification tests in low-resource settings. Here we provide a head-to-head comparison of methods for lysis of, and nucleic acid release from, several pathogenic bacteria and viruses-methods that are adaptable to point-of-care usage in low-resource settings. Digestion with achromopeptidase, a mixture of proteases and peptidoglycan-specific hydrolases, followed by thermal deactivation in a boiling water bath, effectively released amplifiable nucleic acid from Staphylococcus aureus, Bordetella pertussis, respiratory syncytial virus, and influenza virus. Achromopeptidase was functional after dehydration and reconstitution, even after eleven months of dry storage without refrigeration. Mechanical lysis methods proved to be effective against a hard-to-lyse Mycobacterium species, and a miniature bead-mill, the AudioLyse, is shown to be capable of releasing amplifiable DNA and RNA from this species. We conclude that point-of-care-compatible sample preparation methods for nucleic acid tests need not introduce amplification inhibitors, and can provide amplification-ready lysates from a wide range of bacterial and viral pathogens. Copyright © 2016. Published by Elsevier B.V.

Analysis on Two Typical Landslide Hazard Phenomena in The Wenchuan Earthquake by Field Investigations and Shaking Table Tests.

PubMed

Yang, Changwei; Zhang, Jianjing; Liu, Feicheng; Bi, Junwei; Jun, Zhang

2015-08-06

Based on our field investigations of landslide hazards in the Wenchuan earthquake, some findings can be reported: (1) the multi-aspect terrain facing empty isolated mountains and thin ridges reacted intensely to the earthquake and was seriously damaged; (2) the slope angles of most landslides was larger than 45°. Considering the above disaster phenomena, the reasons are analyzed based on shaking table tests of one-sided, two-sided and four-sided slopes. The analysis results show that: (1) the amplifications of the peak accelerations of four-sided slopes is stronger than that of the two-sided slopes, while that of the one-sided slope is the weakest, which can indirectly explain the phenomena that the damage is most serious; (2) the amplifications of the peak accelerations gradually increase as the slope angles increase, and there are two inflection points which are the point where the slope angle is 45° and where the slope angle is 50°, respectively, which can explain the seismic phenomenon whereby landslide hazards mainly occur on the slopes whose slope angle is bigger than 45°. The amplification along the slope strike direction is basically consistent, and the step is smooth.

Loop-mediated amplification of the Clavibacter michiganensis subsp. michiganensis micA gene is highly specific.

PubMed

Yasuhara-Bell, Jarred; Kubota, Ryo; Jenkins, Daniel M; Alvarez, Anne M

2013-12-01

Loop-mediated amplification (LAMP) was used to specifically identify Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial canker of tomato. LAMP primers were developed to detect micA, a chromosomally stable gene that encodes a type II lantibiotic, michiganin A, which inhibits growth of other C. michiganensis subspecies. In all, 409 bacterial strains (351 C. michiganensis subsp. michiganensis and 58 non-C. michiganensis subsp. michiganensis) from a worldwide collection were tested with LAMP to determine its specificity. LAMP results were compared with genetic profiles established using polymerase chain reaction (PCR) amplification of seven genes (dnaA, ppaJ, pat-1, chpC, tomA, ppaA, and ppaC). C. michiganensis subsp. michiganensis strains produced eight distinct profiles. The LAMP reaction identified all C. michiganensis subsp. michiganensis strains and discriminated them from other C. michiganensis subspecies and non-Clavibacter bacteria. LAMP has advantages over immunodiagnostic and other molecular detection methods because of its specificity and isothermal nature, which allows for easy field application. The LAMP reaction is also not affected by as many inhibitors as PCR. This diagnostic tool has potential to provide an easy, one-step test for rapid identification of C. michiganensis subsp. michiganensis.

Isolation, amplification and characterization of foodborne pathogen disease bacteria gene for rapid kit test development

NASA Astrophysics Data System (ADS)

Nurjayadi, M.; Santoso, I.; Kartika, I. R.; Kurniadewi, F.; Saamia, V.; Sofihan, W.; Nurkhasanah, D.

2017-07-01

There is a lot of public concern over food safety. Food-safety cases recently, including many food poisoning cases in both the developed and developing countries, considered to be the national security threats which involved police investigation. Quick and accurate detection methods are needed to handle the food poisoning cases with a big number of sufferers at the same time. Therefore, the research is aimed to develop a specific, sensitive, and rapid result molecular detection tool for foodborne pathogen bacteria. We, thus, propose genomic level approach with Polymerase Chain Reaction. The research has successfully produced a specific primer to perform amplification to fim-C S. typhi, E. coli, and pef Salmonella typhimurium genes. The electrophoresis result shows that amplification products are 95 base pairs, 121 base pairs, and 139 base pairs; and all three genes are in accordance with the size of the in silico to third genes bacteria. In conclusion, the research has been successfully designed a specific detection tool to three foodborne pathogen bacteria genes. Further stages test and the uses of Real-time PCR in the detection are still in the trial process for better detection method.

Single-Use, Electricity-Free Amplification Device for Detection of HIV-1

PubMed Central

Curtis, Kelly A.; Rudolph, Donna L.; Morrison, Daphne; Guelig, Dylan; Diesburg, Steven; McAdams, David; Burton, Robert A.; LaBarre, Paul; Owen, Michele

2016-01-01

Early and accurate diagnosis of HIV is key for the reduction of transmission and initiation of patient care. The availability of a rapid nucleic acid test (NAT) for use at the point-of-care (POC) will fill a gap in HIV diagnostics, improving the diagnosis of acute infection and HIV in infants born to infected mothers. In this study, we evaluated the performance of non-instrumented nucleic acid amplification, single-use disposable (NINA-SUD) devices for the detection of HIV-1 in whole blood using reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) with lyophilized reagents. The NINA-SUD heating device harnesses the heat from an exothermic chemical reaction initiated by the addition of saline to magnesium iron powder. Reproducibility was demonstrated between NINA-SUD units and comparable, if not superior, performance for detecting clinical specimens was observed as compared to the thermal cycler. The stability of the lyophilized HIV-1 RT-LAMP reagents was also demonstrated following storage at −20, 4, 25, and 30°C for up to one month. The single-use, disposable NAT minimizes hands-on time and has the potential to facilitate HIV-1 testing in resource-limited settings or at the POC. PMID:27616198

Molecular diagnosis of protozoan parasites by Recombinase Polymerase Amplification.

PubMed

Castellanos-Gonzalez, A; White, A C; Melby, P; Travi, B

2018-06-01

Infections caused by protozoan parasites affect millions of people around the world. Traditionally, diagnosis was made by microscopy, which is insensitive and in some cases not specific. Molecular methods are highly sensitive and specific, but equipment costs and personnel training limit its availability only to specialized centers, usually far from populations with the highest risk of infection. Inexpensive methods that can be applied at the point of care (POC), especially in places with limited health infrastructure, would be a major advantage. Isothermal amplification of nucleic acids does not require thermocyclers and is relatively inexpensive and easy to implement. Among isothermal methods, recombinase polymerase amplification (RPA) is sensitive and potentially applicable at POC. We and others have developed RPA diagnostic tests to detect protozoan parasites of medical importance. Overall, our results have shown high specificity with limits of detection similar to PCR. Currently, the optimization of RPA for use at the POC is under development, and in the near future the tests should become available to detect protozoan infections in the field. In this review we discuss the current status, challenges, and future of RPA in the field of molecular diagnosis of protozoan parasites. Copyright © 2018 Elsevier B.V. All rights reserved.

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

PubMed

Strauss, Christian; Endimiani, Andrea; Perreten, Vincent

2015-01-01

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance. Copyright © 2014 Elsevier B.V. All rights reserved.

Establishment and application of cross-priming isothermal amplification coupled with lateral flow dipstick (CPA-LFD) for rapid and specific detection of red-spotted grouper nervous necrosis virus.

PubMed

Su, Zi Dan; Shi, Cheng Yin; Huang, Jie; Shen, Gui Ming; Li, Jin; Wang, Sheng Qiang; Fan, Chao

2015-09-26

Red-spotted grouper nervous necrosis virus (RGNNV) is an important pathogen that causes diseases in many species of fish in marine aquaculture. The larvae and juveniles are more easily infected by RGNNV and the cumulative mortality is as high as 100 % after being infected with RGNNV. This virus imposes a serious threat to aquaculture of grouper fry. This study aimed to establish a simple, accurate and highly sensitive method for rapid detection of RGNNV on the spot. In this study, the primers specifically targeting RGNNV were designed and cross-priming isothermal amplification (CPA) system was established. The product amplified by CPA was detected through visualization with lateral flow dipstick (LFD). Three important parameters, including the amplification temperature, the concentration of dNTPs and the concentration of Mg(2+) for the CPA system, were optimized. The sensitivity and specificity of this method for RGNNV were tested and compared with those of the conventional RT-PCR and real-time quantitative RT-PCR (qRT-PCR). The optimized conditions for the CPA amplification system were determined as follows: the optimal amplification temperature, the optimized concentration of dNTPs and the concentration for Mg(2+) were 69 °C, 1.2 mmol/L and 5 mmol/L, respectively. The lowest limit of detection (LLOD) of this method for RGNNV was 10(1) copies/μL of RNA sample, which was 10 times lower than that of conventional RT-PCR and comparable to that of RT-qPCR. This method was specific for RGNNV in combination with SJNNV and had no cross-reactions with 8 types of virus and bacterial strains tested. This method was successfully applied to detect RGNNV in fish samples. This study established a CPA-LFD method for detection of RGNNV. This method is simple and rapid with high sensitivity and good specificity and can be widely applied for rapid detection of this virus on the spot.

Coastal Amplification Laws for the French Tsunami Warning Center: Numerical Modeling and Fast Estimate of Tsunami Wave Heights Along the French Riviera

NASA Astrophysics Data System (ADS)

Gailler, A.; Hébert, H.; Schindelé, F.; Reymond, D.

2017-11-01

Tsunami modeling tools in the French tsunami Warning Center operational context provide rapidly derived warning levels with a dimensionless variable at basin scale. A new forecast method based on coastal amplification laws has been tested to estimate the tsunami onshore height, with a focus on the French Riviera test-site (Nice area). This fast prediction tool provides a coastal tsunami height distribution, calculated from the numerical simulation of the deep ocean tsunami amplitude and using a transfer function derived from the Green's law. Due to a lack of tsunami observations in the western Mediterranean basin, coastal amplification parameters are here defined regarding high resolution nested grids simulations. The preliminary results for the Nice test site on the basis of nine historical and synthetic sources show a good agreement with the time-consuming high resolution modeling: the linear approximation is obtained within 1 min in general and provides estimates within a factor of two in amplitude, although the resonance effects in harbors and bays are not reproduced. In Nice harbor especially, variation in tsunami amplitude is something that cannot be really assessed because of the magnitude range and maximum energy azimuth of possible events to account for. However, this method is well suited for a fast first estimate of the coastal tsunami threat forecast.

Genomic screening for blood-borne viruses in transfusion settings.

PubMed

Allain, J P

2000-02-01

The residual risk of post-transfusion human immunodeficiency virus (HIV) infection is low but slightly higher for hepatitis B virus (HBV) and hepatitis C virus (HCV), the main reason being viraemia during the window period preceding antibody or antigen detection by enzyme immunoassays. Immunosilent-infected individuals and carriers of distant viral variants also play an unquantifiable role. Multiple techniques, e.g. reverse transcription-polymerase chain reaction (RT-PCR), PCR, ligase-chain reaction, nucleic acid sequence-based amplification (NASBA) and transcription-mediated amplification (TMA) have been developed to amplify and detect viral genomes as single or multiplex assays. Equipment providing various degrees of automation has been adapted to these techniques. Applying nucleic acid amplification techniques (NAT) to blood screening, two main approaches have been advocated: plasma pool and single-donation testing. Pool testing presents the advantage of lower cost and readily available equipment although it is prone to false negative and positive reactions. The time required to identify infected donations is incompatible with blood component release, and may lead to product waste. Single-unit testing, although appealing, is not yet fully automated and potentially very costly unless a systematic multiplex approach is taken. Although technically feasible, NAT applied to the blood supply needs to be clinically evaluated and its cost efficiency assessed in the general public health context. However, pool NAT is currently implemented in continental Europe and the USA.

Coastal amplification laws for the French tsunami Warning Center: numerical modeling and fast estimate of tsunami wave heights along the French Riviera

NASA Astrophysics Data System (ADS)

Gailler, A.; Schindelé, F.; Hebert, H.; Reymond, D.

2017-12-01

Tsunami modeling tools in the French tsunami Warning Center operational context provide for now warning levels with a no dimension scale, and at basin scale. A new forecast method based on coastal amplification laws has been tested to estimate the tsunami onshore height, with a focus on the French Riviera test-site (Nice area). This fast prediction tool provides a coastal tsunami height distribution, calculated from the numerical simulation of the deep ocean tsunami amplitude and using a transfer function derived from the Green's law. Due to a lack of tsunami observation in the western Mediterranean basin, coastal amplification parameters are here defined regarding high resolution nested grids simulations. The first encouraging results for the Nice test site on the basis of 9 historical and fake sources show a good agreement with the time-consuming high resolution modeling: the linear approximation provides within in general 1 minute estimates less a factor of 2 in amplitude, although the resonance effects in harbors and bays are not reproduced. In Nice harbor especially, variation in tsunami amplitude is something that cannot be really appreciated because of the magnitude range and maximum energy azimuth of possible events to account for. However, this method suits well for a fast first estimate of the coastal tsunami threat forecast.

Coastal Amplification Laws for the French Tsunami Warning Center: Numerical Modeling and Fast Estimate of Tsunami Wave Heights Along the French Riviera

NASA Astrophysics Data System (ADS)

Gailler, A.; Hébert, H.; Schindelé, F.; Reymond, D.

2018-04-01

Tsunami modeling tools in the French tsunami Warning Center operational context provide rapidly derived warning levels with a dimensionless variable at basin scale. A new forecast method based on coastal amplification laws has been tested to estimate the tsunami onshore height, with a focus on the French Riviera test-site (Nice area). This fast prediction tool provides a coastal tsunami height distribution, calculated from the numerical simulation of the deep ocean tsunami amplitude and using a transfer function derived from the Green's law. Due to a lack of tsunami observations in the western Mediterranean basin, coastal amplification parameters are here defined regarding high resolution nested grids simulations. The preliminary results for the Nice test site on the basis of nine historical and synthetic sources show a good agreement with the time-consuming high resolution modeling: the linear approximation is obtained within 1 min in general and provides estimates within a factor of two in amplitude, although the resonance effects in harbors and bays are not reproduced. In Nice harbor especially, variation in tsunami amplitude is something that cannot be really assessed because of the magnitude range and maximum energy azimuth of possible events to account for. However, this method is well suited for a fast first estimate of the coastal tsunami threat forecast.

EGFR gene amplification is relatively common and associates with outcome in intestinal adenocarcinoma of the stomach, gastro-oesophageal junction and distal oesophagus.

PubMed

Birkman, Eva-Maria; Ålgars, Annika; Lintunen, Minnamaija; Ristamäki, Raija; Sundström, Jari; Carpén, Olli

2016-07-07

Approximately 50 % of gastric adenocarcinomas belong to a molecular subgroup characterised by chromosomal instability and a strong association with the intestinal histological subtype. This subgroup typically contains alterations in the receptor tyrosine kinase-RAS pathway, for example EGFR or HER2 gene amplifications leading to protein overexpression. In clinical practice, HER2 overexpressing metastatic gastric cancer is known to respond to treatment with anti-HER2 antibodies. By contrast, anti-EGFR antibodies have not been able to provide survival benefit in clinical trials, which, however, have not included patient selection based on the histological subtype or EGFR gene copy number analysis of the tumours. To examine the role of EGFR as a potential biomarker, we studied the prevalence, clinicopathological associations as well as prognostic role of EGFR and HER2 expression and gene amplification in intestinal adenocarcinomas of the stomach, gastro-oesophageal junction and distal oesophagus. Tissue samples from 220 patients were analysed with EGFR and HER2 immunohistochemistry. Those samples with moderate/strong staining intensity were further analysed with silver in situ hybridization to quantify gene copy numbers. The results were associated with clinical patient characteristics and survival. Moderate/strong EGFR protein expression was found in 72/220 (32.7 %) and EGFR gene amplification in 31/220 (14.1 %) of the tumours, while moderate/strong HER2 protein expression was detected in 31/220 (14.1 %) and HER2 gene amplification in 29/220 (13.2 %) of the tumours. EGFR and HER2 genes were co-amplified in eight tumours (3.6 %). EGFR gene amplification was more common in tumours of distal oesophagus/gastro-oesophageal junction/cardia than in those of gastric corpus (p = 0.013). It was associated with shortened time to cancer recurrence (p = 0.026) and cancer specific survival (p = 0.033). EGFR gene amplification is relatively common in intestinal adenocarcinomas and associates with decreased survival. It is rarely concurrent with HER2 gene amplification, suggesting that anti-EGFR therapies might be applicable to some patients not eligible for anti-HER2 treatment. Analogous to HER2 testing, determination of EGFR gene amplification status in concert with immunohistochemistry could improve the specificity of patient selection when investigating the possible benefits of anti-EGFR therapies in the treatment of gastric adenocarcinomas.

High amplification of FGFR1 gene is a delayed poor prognostic factor in early stage ESCC patients

PubMed Central

Song, Qi; Liu, Yalan; Jiang, Dongxian; Wang, Haixing; Huang, Jie; Xu, Yifan; Sujie, Akesu; Zeng, Haiying; Xu, Chen; Hou, Yingyong

2017-01-01

Amplification of the fibroblast growth factor receptor 1 (FGFR1) is believed to predict response to FGFR inhibitors. The aim of this study was to investigate the frequency and the prognostic impact of FGFR1 amplification in patients with resected esophageal squamous cell carcinoma (ESCC) by using fluorescent in situ hybridization. Microarrayed paraffin embedded blocks were constructed, and the cohort of tissues came from 506 patients with ESCC. FGFR1 high amplification (FGFR1high) was defined by an FGFR1/centromere 8 ratio of ≥ 2.0, or average number of FGFR1 signals/tumor cell nucleus ≥ 6.0, or percentage of tumor cells containing ≥ 15 FGFR1 signals, or large cluster in ≥ 10% of cancer cells. FGFR1 low amplification was defined by ≥ 5 FGFR1 signals in ≥ 50% of cancer cells. Kaplan-Meier curves with log-rank tests and Cox proportional hazards model were used to analyze patients’ survival. Among 506 patients, high amplification, low amplification, and disomy were detected in 8.7%, 3.6% and 87.7%, respectively. In general, the FGFR1high group trended towards worse disease-free survival (DFS) and overall survival (OS) compared to the FGFR1 low amplification/disomy (FGFR1low/disomy) group (DFS, P=0.108; OS, P=0.112), but this trend was amplified for patients with DFS ≥ 30 months (DFS, P=0.009; OS, P=0.007). Furthermore, when patients were stratified into stage I-II and stage III-IV, the FGFR1high group directly presented with adverse DFS and OS than the FGFR1low/disomy group in stage I-II patients (DFS, P=0.019; OS, P=0.034), especially with DFS ≥ 30 months (DFS, P=0.002; OS, P=0.001). However, for patients in stage III-IV, FGFR1high had no effect on prognosis regardless of DFS time. FGFR1high occurs in a minority of ESCC, and it predicts delayed poor prognosis in stage I and II ESCC patients. PMID:29088806

HER2 Amplification and HER2 Mutation Are Distinct Molecular Targets in Lung Cancers.

PubMed

Li, Bob T; Ross, Dara S; Aisner, Dara L; Chaft, Jamie E; Hsu, Meier; Kako, Severine L; Kris, Mark G; Varella-Garcia, Marileila; Arcila, Maria E

2016-03-01

Human epidermal growth factor receptor 2 gene (HER2 [also known as ERBB2]) alterations have been identified as oncogenic drivers and potential therapeutic targets in lung cancers. The molecular associations of HER2 gene amplification, mutation, and HER2 protein overexpression in lung cancers have not been distinctly defined. To explore these associations, Memorial Sloan Kettering Cancer Center and the University of Colorado combined their data on HER2 alterations in lung cancers. Tumor specimens from 175 patients with lung adenocarcinomas and no prior targeted therapy were evaluated for the presence of HER2 amplification and mutation and HER2 protein overexpression. Amplification was assessed by fluorescence in situ hybridization (FISH) and defined as an HER2-to-chromosome enumeration probe 17 ratio of at least 2.0. Mutation was assessed by fragment analysis, mass spectrometry genotyping, and Sanger sequencing. Overexpression was assessed by immunohistochemical (IHC) staining. The frequencies of HER2 amplification and mutation and HER2 overexpression were calculated and their overlap examined. HER2 amplification was detected by FISH in 5 of 175 cases (3%). HER2 mutation was detected in 4 of 148 specimens (3%), including three identical 12-base pair insertions (p.A775_G776insYVMA) and a 9-base pair insertion, all in exon 20. None of the HER2-mutant cases was amplified. HER2 overexpression (2+ or 3+) on IHC staining was not detected in the 25 specimens available for testing, and negative IHC staining correlated with the negative results according to FISH. HER2 mutations are not associated with HER2 amplification, thus suggesting a distinct entity and therapeutic target. HER2-positive lung cancer may not be an adequate term, and patient cohorts for the study of HER2-targeted agents should be defined by the specific HER2 alteration present. Copyright © 2015 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Multicenter Clinical Evaluation of the Novel Alere i Strep A Isothermal Nucleic Acid Amplification Test.

PubMed

Cohen, Daniel M; Russo, Michael E; Jaggi, Preeti; Kline, Jennifer; Gluckman, William; Parekh, Amisha

2015-07-01

Rapid detection of group A beta-hemolytic streptococcus (GAS) is used routinely to help diagnose and treat pharyngitis. However, available rapid antigen detection tests for GAS have relatively low sensitivity, and backup testing is recommended in children. Newer assays are more sensitive yet require excessive time for practical point-of-care use as well as laboratory personnel. The Alere i strep A test is an isothermal nucleic acid amplification test designed to offer highly sensitive results at the point of care within 8 min when performed by nonlaboratory personnel. The performance of the Alere i strep A test was evaluated in a multicenter prospective trial in a Clinical Laboratory Improvement Amendments (CLIA)-waived setting in comparison to bacterial culture in 481 children and adults. Compared to culture, the Aleri i strep A test had 96.0% sensitivity and 94.6% specificity. Discrepant results were adjudicated by PCR and found the Alere i strep A test to have 98.7% sensitivity and 98.5% specificity. Overall, the Alere i strep A test could provide a one-step, rapid, point-of-care testing method for GAS pharyngitis and obviate backup testing on negative results. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Diagnosis and follow-up of Whipple's disease by amplification of the 16S rRNA gene of Tropheryma whippelii.

PubMed

Pron, B; Poyart, C; Abachin, E; Fest, T; Belanger, C; Bonnet, C; Capelle, P; Bretagne, J F; Fabianek, A; Girard, L; Hagège, H; Berche, P

1999-01-01

Amplification of the 16S rRNA gene of Tropheryma whippelii was performed in eight patients with Whipple's disease and 34 control patients to confirm a diagnosis of Whipple's disease and to monitor the course of disease. Polymerase chain reaction (PCR) tests were positive before treatment in 13 of 15 tissue samples from Whipple's disease patients (gut 8/8; lymph nodes 2/2; bone marrow 1/2; peripheral blood 2/3), in contrast to none of 54 tissue samples from controls. PCR tests converted to negative within 4-6 months in six of the Whipple's disease patients undergoing therapy. These results show that PCR is a reliable and useful tool for diagnosis of Whipple's disease and for monitoring bacterial elimination during antibiotic therapy.

Amplification, Decoherence, and the Acquisition of Information by Spin Environments

PubMed Central

Zwolak, Michael; Riedel, C. Jess; Zurek, Wojciech H.

2016-01-01

Quantum Darwinism recognizes the role of the environment as a communication channel: Decoherence can selectively amplify information about the pointer states of a system of interest (preventing access to complementary information about their superpositions) and can make records of this information accessible to many observers. This redundancy explains the emergence of objective, classical reality in our quantum Universe. Here, we demonstrate that the amplification of information in realistic spin environments can be quantified by the quantum Chernoff information, which characterizes the distinguishability of partial records in individual environment subsystems. We show that, except for a set of initial states of measure zero, the environment always acquires redundant information. Moreover, the Chernoff information captures the rich behavior of amplification in both finite and infinite spin environments, from quadratic growth of the redundancy to oscillatory behavior. These results will considerably simplify experimental testing of quantum Darwinism, e.g., using nitrogen vacancies in diamond. PMID:27193389

Amplification, Decoherence, and the Acquisition of Information by Spin Environments

NASA Astrophysics Data System (ADS)

Zwolak, Michael; Riedel, C. Jess; Zurek, Wojciech H.

2016-05-01

Quantum Darwinism recognizes the role of the environment as a communication channel: Decoherence can selectively amplify information about the pointer states of a system of interest (preventing access to complementary information about their superpositions) and can make records of this information accessible to many observers. This redundancy explains the emergence of objective, classical reality in our quantum Universe. Here, we demonstrate that the amplification of information in realistic spin environments can be quantified by the quantum Chernoff information, which characterizes the distinguishability of partial records in individual environment subsystems. We show that, except for a set of initial states of measure zero, the environment always acquires redundant information. Moreover, the Chernoff information captures the rich behavior of amplification in both finite and infinite spin environments, from quadratic growth of the redundancy to oscillatory behavior. These results will considerably simplify experimental testing of quantum Darwinism, e.g., using nitrogen vacancies in diamond.

Integrating DNA strand displacement circuitry to the nonlinear hybridization chain reaction.

PubMed

Zhang, Zhuo; Fan, Tsz Wing; Hsing, I-Ming

2017-02-23

Programmable and modular attributes of DNA molecules allow one to develop versatile sensing platforms that can be operated isothermally and enzyme-free. In this work, we present an approach to integrate upstream DNA strand displacement circuits that can be turned on by a sequence-specific microRNA analyte with a downstream nonlinear hybridization chain reaction for a cascading hyperbranched nucleic acid assembly. This system provides a two-step amplification strategy for highly sensitive detection of the miRNA analyte, conducive for multiplexed detection. Multiple miRNA analytes were tested with our integrated circuitry using the same downstream signal amplification setting, showing the decoupling of nonlinear self-assembly with the analyte sequence. Compared with the reported methods, our signal amplification approach provides an additional control module for higher-order DNA self-assembly and could be developed into a promising platform for the detection of critical nucleic-acid based biomarkers.

Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin.

PubMed

Mecklenburg, M; Lindbladh, C; Li, H; Mosbach, K; Danielsson, B

1993-08-01

A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.

Recombinase polymerase amplification: Emergence as a critical molecular technology for rapid, low-resource diagnostics.

PubMed

James, Ameh; Macdonald, Joanne

2015-01-01

Isothermal molecular diagnostics are bridging the technology gap between traditional diagnostics and polymerase chain reaction-based methods. These new techniques enable timely and accurate testing, especially in settings where there is a lack of infrastructure to support polymerase chain reaction facilities. Despite this, there is a significant lack of uptake of these technologies in developing countries where they are highly needed. Among these novel isothermal technologies, recombinase polymerase amplification (RPA) holds particular potential for use in developing countries. This rapid nucleic acid amplification approach is fast, highly sensitive and specific, and amenable to countries with a high burden of infectious diseases. Implementation of RPA technology in developing countries is critically required to assess limitations and potentials of the diagnosis of infectious disease, and may help identify impediments that prevent adoption of new molecular technologies in low resource- and low skill settings. This review focuses on approaching diagnosis of infectious disease with RPA.

The depression distress amplification model in adolescents: A longitudinal examination of anxiety sensitivity cognitive concerns, depression and suicidal ideation

PubMed Central

Capron, Daniel W.; Allan, Nicholas P.; Ialongo, Nicholas S.; Leen-Feldner, Ellen; Schmidt, Norman B.

2015-01-01

Adolescents with comorbid anxiety and depression are at significantly increased risk of suicide. The recently proposed depression distress amplification model appears to have promise for explaining the relations between anxiety, depression, and suicidality, but it has not been tested in adolescents. Participants were 524 adolescents followed over two years. Baseline data for the current report were collected by trained interviewers while the adolescents were in eighth grade. Data were obtained in the same manner when the adolescents were in tenth grade. Baseline anxiety sensitivity cognitive concerns significantly predicted suicidal ideation two years later, above and beyond baseline suicidal ideation and depression. Further, consistent with the depression distress amplification model, anxiety sensitivity cognitive concerns interacted with depressive symptoms to predict suicidal ideation. This report extends the empirical and theoretical support for a relationship between anxiety sensitivity cognitive concerns and suicidality. PMID:25754194

Broad-Spectrum Molecular Detection of Fungal Nucleic Acids by PCR-Based Amplification Techniques.

PubMed

Czurda, Stefan; Lion, Thomas

2017-01-01

Over the past decade, the incidence of life-threatening invasive fungal infections has dramatically increased. Infections caused by hitherto rare and emerging fungal pathogens are associated with significant morbidity and mortality among immunocompromised patients. These observations render the coverage of a broad range of clinically relevant fungal pathogens highly important. The so-called panfungal or, perhaps more correctly, broad-range nucleic acid amplification techniques do not only facilitate sensitive detection of all clinically relevant fungal species but are also rapid and can be applied to analyses of any patient specimens. They have therefore become valuable diagnostic tools for sensitive screening of patients at risk of invasive fungal infections. This chapter summarizes the currently available molecular technologies employed in testing of a wide range of fungal pathogens, and provides a detailed workflow for patient screening by broad-spectrum nucleic acid amplification techniques.

Role of monkeys in the sylvatic cycle of chikungunya virus in Senegal.

PubMed

Althouse, Benjamin M; Guerbois, Mathilde; Cummings, Derek A T; Diop, Ousmane M; Faye, Ousmane; Faye, Abdourahmane; Diallo, Diawo; Sadio, Bakary Djilocalisse; Sow, Abdourahmane; Faye, Oumar; Sall, Amadou A; Diallo, Mawlouth; Benefit, Brenda; Simons, Evan; Watts, Douglas M; Weaver, Scott C; Hanley, Kathryn A

2018-03-13

Arboviruses spillover into humans either as a one-step jump from a reservoir host species into humans or as a two-step jump from the reservoir to an amplification host species and thence to humans. Little is known about arbovirus transmission dynamics in reservoir and amplification hosts. Here we elucidate the role of monkeys in the sylvatic, enzootic cycle of chikungunya virus (CHIKV) in the region around Kédougou, Senegal. Over 3 years, 737 monkeys were captured, aged using anthropometry and dentition, and tested for exposure to CHIKV by detection of neutralizing antibodies. Infant monkeys were positive for CHIKV even when the virus was not detected in a concurrent survey of mosquitoes and when population immunity was too high for monkeys alone to support continuous transmission. We conclude that monkeys in this region serve as amplification hosts of CHIKV. Additional efforts are needed to identify other hosts capable of supporting continuous circulation.

Biochip for Real-Time Monitoring of Hepatitis B Virus (HBV) by Combined Loop-Mediated Isothermal Amplification and Solution-Phase Electrochemical Detection

NASA Astrophysics Data System (ADS)

Tien, Bui Quang; Ngoc, Nguyen Thy; Loc, Nguyen Thai; Thu, Vu Thi; Lam, Tran Dai

2017-06-01

Accurate in situ diagnostic tests play a key role in patient management and control of most infectious diseases. To achieve this, use of handheld biochips that implement sample handling, sample analysis, and result readout together is an ideal approach. We present herein a fluid-handling biochip for real-time electrochemical monitoring of nucleic acid amplification based on loop-mediated isothermal amplification and real-time electrochemical detection on a microfluidic platform. Intercalation between amplifying DNA and free redox probe in solution phase was used to monitor the number of DNA copies. The whole diagnostic process is completed within 70 min. Our platform offers a fast and easy tool for quantification of viral pathogens in shorter time and with limited risk of all potential forms of cross-contamination. Such diagnostic tools have potential to make a huge difference to the lives of millions of people worldwide.

Quantification of HIV-1 DNA using real-time recombinase polymerase amplification.

PubMed

Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

2014-06-17

Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic acids in point-of-care applications, RPA has not yet been implemented to quantify sample concentration using a standard curve. Here, we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes real-time fluorescence data to quantify HIV-1 DNA. We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within 1 order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that quantitative RPA (qRPA) may serve as a powerful tool for quantifying nucleic acids and may be adapted for use in single-sample point-of-care diagnostic systems.

Update on Clostridium difficile infections.

PubMed

Le Monnier, A; Zahar, J-R; Barbut, F

2014-08-01

Clostridium difficile infections (CDI) occur primarily in hospitalized patients with risk factors such as concomitant or recent use of antibiotics. CDI related additional costs are important for the global population and health-care facilities. CDI epidemiology has changed since 2003: they became more frequent boosted by large outbreaks, more severe, more resistant to antibiotic treatment, and spread to new groups of population without any risk factor. This is partly due to the emergence and worldwide dissemination of new and more virulent C. difficile strains such as the epidemic clone 027/NAP1/BI. The host immune response plays a central role in the pathogenesis of CDI and could also be involved in the occurrence of recurrent or severe forms. New guidelines including new molecular tests (NAAT) have recently clarified and simplified the diagnostic strategies for the microbiological diagnosis of CDI. The CDI incidence was proven to be related to the level of clinical suspicion and the frequency of microbiological screening for C. difficile. The current recommendations for the treatment of CDI mention oral metronidazole as the first line treatment for mild to moderate diarrhea. Oral vancomycin use should be restricted to severe cases. In the absence of consensus, the treatment of multiple recurrences remains a major concern. New and more targeted antibiotics and innovative therapeutic strategies (fecal transplantation, monoclonal antibodies, and vaccination) have emerged as new therapies for CDI. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Randomness Amplification under Minimal Fundamental Assumptions on the Devices

NASA Astrophysics Data System (ADS)

Ramanathan, Ravishankar; Brandão, Fernando G. S. L.; Horodecki, Karol; Horodecki, Michał; Horodecki, Paweł; Wojewódka, Hanna

2016-12-01

Recently, the physically realistic protocol amplifying the randomness of Santha-Vazirani sources producing cryptographically secure random bits was proposed; however, for reasons of practical relevance, the crucial question remained open regarding whether this can be accomplished under the minimal conditions necessary for the task. Namely, is it possible to achieve randomness amplification using only two no-signaling components and in a situation where the violation of a Bell inequality only guarantees that some outcomes of the device for specific inputs exhibit randomness? Here, we solve this question and present a device-independent protocol for randomness amplification of Santha-Vazirani sources using a device consisting of two nonsignaling components. We show that the protocol can amplify any such source that is not fully deterministic into a fully random source while tolerating a constant noise rate and prove the composable security of the protocol against general no-signaling adversaries. Our main innovation is the proof that even the partial randomness certified by the two-party Bell test [a single input-output pair (u* , x* ) for which the conditional probability P (x*|u*) is bounded away from 1 for all no-signaling strategies that optimally violate the Bell inequality] can be used for amplification. We introduce the methodology of a partial tomographic procedure on the empirical statistics obtained in the Bell test that ensures that the outputs constitute a linear min-entropy source of randomness. As a technical novelty that may be of independent interest, we prove that the Santha-Vazirani source satisfies an exponential concentration property given by a recently discovered generalized Chernoff bound.

An integrated paper-based sample-to-answer biosensor for nucleic acid testing at the point of care.

PubMed

Choi, Jane Ru; Hu, Jie; Tang, Ruihua; Gong, Yan; Feng, Shangsheng; Ren, Hui; Wen, Ting; Li, XiuJun; Wan Abas, Wan Abu Bakar; Pingguan-Murphy, Belinda; Xu, Feng

2016-02-07

With advances in point-of-care testing (POCT), lateral flow assays (LFAs) have been explored for nucleic acid detection. However, biological samples generally contain complex compositions and low amounts of target nucleic acids, and currently require laborious off-chip nucleic acid extraction and amplification processes (e.g., tube-based extraction and polymerase chain reaction (PCR)) prior to detection. To the best of our knowledge, even though the integration of DNA extraction and amplification into a paper-based biosensor has been reported, a combination of LFA with the aforementioned steps for simple colorimetric readout has not yet been demonstrated. Here, we demonstrate for the first time an integrated paper-based biosensor incorporating nucleic acid extraction, amplification and visual detection or quantification using a smartphone. A handheld battery-powered heating device was specially developed for nucleic acid amplification in POC settings, which is coupled with this simple assay for rapid target detection. The biosensor can successfully detect Escherichia coli (as a model analyte) in spiked drinking water, milk, blood, and spinach with a detection limit of as low as 10-1000 CFU mL(-1), and Streptococcus pneumonia in clinical blood samples, highlighting its potential use in medical diagnostics, food safety analysis and environmental monitoring. As compared to the lengthy conventional assay, which requires more than 5 hours for the entire sample-to-answer process, it takes about 1 hour for our integrated biosensor. The integrated biosensor holds great potential for detection of various target analytes for wide applications in the near future.

Evaluating whole transcriptome amplification for gene profiling experiments using RNA-Seq.

PubMed

Faherty, Sheena L; Campbell, C Ryan; Larsen, Peter A; Yoder, Anne D

2015-07-30

RNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN's Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats (Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates. We found that 93% of expressed genes were identical between all unamplified versus matched amplified comparisons, also finding that gene density is similar across all comparisons. Our sequencing experiment and downstream bioinformatic analyses using the Tuxedo analysis pipeline resulted in the assembly of 25,543 high-quality transcripts. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively. Although significant differentially expressed genes (P < 0.05) were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison. Transcriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove challenging.

High amplification levels of MDM2 and CDK4 correlate with poor outcome in patients with dedifferentiated liposarcoma: A cytogenomic microarray analysis of 47 cases.

PubMed

Ricciotti, Robert W; Baraff, Aaron J; Jour, George; Kyriss, McKenna; Wu, Yu; Liu, Yuhua; Li, Shao-Chun; Hoch, Benjamin; Liu, Yajuan J

2017-12-01

Dedifferentiated liposarcoma (DDLS) is characterized at the molecular level by amplification of genes within 12q13-15 including MDM2 and CDK4. However, other than FNCLCC grade, prognostic markers are limited. We aim to identify molecular prognostic markers for DDLS to help risk stratify patients. To this end, we studied 49 cases of DDLS in our institutional archives and performed cytogenomic microarray analysis on 47 cases. Gene copy numbers for 12 loci were evaluated and correlated with outcome data retrieved from our institutional electronic medical records. Using cut point analysis and comparison of Kaplan-Meier survival curves by log rank tests, high amplification levels of MDM2 (>38 copies) and CDK4 (>30 copies) correlated with decreased disease free survival (DFS) (P = .0168 and 0.0169 respectively) and disease specific survival (DSS) (P = .0082 and 0.0140 respectively). Additionally, MDM2 and CDK4 showed evidence of a synergistic effect so that each additional copy of one enhances the effect on prognosis of each additional copy of the other for decreased DFS (P = .0227, 0.1% hazard). High amplification of JUN (>16 copies) also correlated with decreased DFS (P = .0217), but not DSS. The presence of copy number alteration at 3q29 correlated with decreased DSS (P = .0192). The presence of >10 mitoses per 10 high power fields and FNCLCC grade 3 also correlated with decreased DFS (P = .0310 and 0.0254 respectively). MDM2 and CDK4 gene amplification levels, along with JUN amplification and copy alterations at 3q29, can be utilized for predicting outcome in patients with DDLS. Published by Elsevier Inc.

Identification of root rot fungi in nursery seedlings by nested multiplex PCR.

PubMed Central

Hamelin, R C; Bérubé, P; Gignac, M; Bourassa, M

1996-01-01

The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated. PMID:8899993

Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Simultaneous Detection of mecA, nuc, and femB by Loop-Mediated Isothermal Amplification (LAMP).

PubMed

Chen, Changguo; Zhao, Qiangyuan; Guo, Jianwei; Li, Yanjun; Chen, Qiuyuan

2017-08-01

The aim of this study was to develop a rapid detection assay to identify methicillin-resistant Staphylococcus aureus by simultaneous testing for the mecA, nuc, and femB genes using the loop-mediated isothermal amplification (LAMP) method. LAMP primers were designed using online bio-software ( http://primerexplorer.jp/e/ ), and amplification reactions were performed in an isothermal temperature bath. The products were then examined using 2% agarose gel electrophoresis. MecA, nuc, and femB were confirmed by triplex TaqMan real-time PCR. For better naked-eye inspection of the reaction result, hydroxy naphthol blue (HNB) was added to the amplification system. Within 60 min, LAMP successfully amplified the genes of interest under isothermal conditions at 63 °C. The results of 2% gel electrophoresis indicated that when the Mg 2+ concentration in the reaction system was 6 μmol, the amplification of the mecA gene was relatively good, while the amplification of the nuc and femB genes was better at an Mg 2+ concentration of 8 μmol. Obvious color differences were observed by adding 1 μL (3.75 mM) of HNB into 25 μL reaction system. The LAMP assay was applied to 128 isolates cases of methicillin-resistant Staphylococcus aureus, which were separated from the daily specimens and identified by Vitek microbial identification instruments. The results were identical for both LAMP and PCR. LAMP offers an alternative detection assay for mecA, nuc, and femB and is faster than other methods.

Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms.

PubMed

Moura-Melo, Suely; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Dos Santos Junior, J Ribeiro; da Silva Fonseca, Rosana A; Lobo-Castañón, Maria Jesús

2015-08-18

Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas.

2D dynamic studies combined with the surface curvature analysis to predict Arias Intensity amplification

NASA Astrophysics Data System (ADS)

Torgoev, Almaz; Havenith, Hans-Balder

2016-07-01

A 2D elasto-dynamic modelling of the pure topographic seismic response is performed for six models with a total length of around 23.0 km. These models are reconstructed from the real topographic settings of the landslide-prone slopes situated in the Mailuu-Suu River Valley, Southern Kyrgyzstan. The main studied parameter is the Arias Intensity (Ia, m/sec), which is applied in the GIS-based Newmark method to regionally map the seismically-induced landslide susceptibility. This method maps the Ia values via empirical attenuation laws and our studies investigate a potential to include topographic input into them. Numerical studies analyse several signals with varying shape and changing central frequency values. All tests demonstrate that the spectral amplification patterns directly affect the amplification of the Ia values. These results let to link the 2D distribution of the topographically amplified Ia values with the parameter called as smoothed curvature. The amplification values for the low-frequency signals are better correlated with the curvature smoothed over larger spatial extent, while those values for the high-frequency signals are more linked to the curvature with smaller smoothing extent. The best predictions are provided by the curvature smoothed over the extent calculated according to Geli's law. The sample equations predicting the Ia amplification based on the smoothed curvature are presented for the sinusoid-shape input signals. These laws cannot be directly implemented in the regional Newmark method, as 3D amplification of the Ia values addresses more problem complexities which are not studied here. Nevertheless, our 2D results prepare the theoretical framework which can potentially be applied to the 3D domain and, therefore, represent a robust basis for these future research targets.

Amplification of the 20q Chromosomal Arm Occurs Early in Tumorigenic Transformation and May Initiate Cancer

PubMed Central

Buganim, Yosef; Solomon, Hilla; Goldfinger, Naomi; Hovland, Randi; Ke, Xi-Song; Oyan, Anne M.; Kalland, Karl-H.; Rotter, Varda; Domany, Eytan

2011-01-01

Duplication of chromosomal arm 20q occurs in prostate, cervical, colon, gastric, bladder, melanoma, pancreas and breast cancer, suggesting that 20q amplification may play a causal role in tumorigenesis. According to an alternative view, chromosomal imbalance is mainly a common side effect of cancer progression. To test whether a specific genomic aberration might serve as a cancer initiating event, we established an in vitro system that models the evolutionary process of early stages of prostate tumor formation; normal prostate cells were immortalized by the over-expression of human telomerase catalytic subunit hTERT, and cultured for 650 days till several transformation hallmarks were observed. Gene expression patterns were measured and chromosomal aberrations were monitored by spectral karyotype analysis at different times. Several chromosomal aberrations, in particular duplication of chromosomal arm 20q, occurred early in the process and were fixed in the cell populations, while other aberrations became extinct shortly after their appearance. A wide range of bioinformatic tools, applied to our data and to data from several cancer databases, revealed that spontaneous 20q amplification can promote cancer initiation. Our computational model suggests that 20q amplification induced deregulation of several specific cancer-related pathways including the MAPK pathway, the p53 pathway and Polycomb group factors. In addition, activation of Myc, AML, B-Catenin and the ETS family transcription factors was identified as an important step in cancer development driven by 20q amplification. Finally we identified 13 "cancer initiating genes", located on 20q13, which were significantly over-expressed in many tumors, with expression levels correlated with tumor grade and outcome suggesting that these genes induce the malignant process upon 20q amplification. PMID:21297939

Problem-Solving Test: Real-Time Polymerase Chain Reaction

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2009-01-01

Terms to be familiar with before you start to solve the test: polymerase chain reaction, DNA amplification, electrophoresis, breast cancer, "HER2" gene, genomic DNA, "in vitro" DNA synthesis, template, primer, Taq polymerase, 5[prime][right arrow]3[prime] elongation activity, 5[prime][right arrow]3[prime] exonuclease activity, deoxyribonucleoside…

Use of microsatellite markers in an American beech (Fagus grandifolia) population and paternity testing

Treesearch

Jennifer Koch; Dave Carey; M.E. Mason

2010-01-01

Cross-species amplification of six microsatellite markers from European beech (Fagus sylvatica Linn) and nine markers from Japanese beech (Fagus crenata Blume) was tested in American beech (Fagus grandifolia Ehrh.). Three microsatellites from each species were successfully adapted for use in American beech...

New nucleic acid testing devices to diagnose infectious diseases in resource-limited settings.

PubMed

Maffert, P; Reverchon, S; Nasser, W; Rozand, C; Abaibou, H

2017-10-01

Point-of-care diagnosis based on nucleic acid testing aims to incorporate all the analytical steps, from sample preparation to nucleic acid amplification and detection, in a single device. This device needs to provide a low-cost, robust, sensitive, specific, and easily readable analysis. Microfluidics has great potential for handling small volumes of fluids on a single platform. Microfluidic technology has recently been applied to paper, which is already used in low-cost lateral flow tests. Nucleic acid extraction from a biological specimen usually requires cell filtration and lysis on specific membranes, while affinity matrices, such as chitosan or polydiacetylene, are well suited to concentrating nucleic acids for subsequent amplification. Access to electricity is often difficult in resource-limited areas, so the amplification step needs to be equipment-free. Consequently, the reaction has to be isothermal to alleviate the need for a thermocycler. LAMP, NASBA, HDA, and RPA are examples of the technologies available. Nucleic acid detection techniques are currently based on fluorescence, colorimetry, or chemiluminescence. For point-of-care diagnostics, the results should be readable with the naked eye. Nowadays, interpretation and communication of results to health professionals could rely on a smartphone, used as a telemedicine device. The major challenge of creating an "all-in-one" diagnostic test involves the design of an optimal solution and a sequence for each analytical step, as well as combining the execution of all these steps on a single device. This review provides an overview of available materials and technologies which seem to be adapted to point-of-care nucleic acid-based diagnosis, in low-resource areas.

Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP(®) using reverse transcription-recombinase polymerase amplification.

PubMed

Zhang, Shulu; Ravelonandro, Michel; Russell, Paul; McOwen, Nathan; Briard, Pascal; Bohannon, Seven; Vrient, Albert

2014-10-01

Plum pox virus (PPV) causes the most destructive viral disease known as plum pox or Sharka disease in stone fruit trees. As an important regulated pathogen, detection of PPV is thus of critical importance to quarantine and eradication of the spreading disease. In this study, the innovative development of two AmplifyRP(®) tests is reported for a rapid isothermal detection of PPV using reverse transcription-recombinase polymerase amplification. In an AmplifyRP(®) test, all specific recombination and amplification reactions occur at a constant temperature without thermal cycling and the test results are either recorded in real-time with a portable fluorescence reader or displayed using a lateral flow strip contained inside an amplicon detection chamber. The major improvement of this assay is that the entire test from sample preparation to result can be completed in as little as 20min and can be performed easily both in laboratories and in the field. The results from this study demonstrated the ability of the AmplifyRP(®) technique to detect all nine PPV strains (An, C, CR, D, EA, M, Rec, T, or W). Among the economic benefits to pathogen surveys is the higher sensitivity of the AmplifyRP(®) to detect PPV when compared to the conventional ELISA and ImmunoStrip(®) assays. This is the first report describing the use of such an innovative technique to detect rapidly plant viruses affecting perennial crops. Copyright © 2014 Elsevier B.V. All rights reserved.

Hindering the illegal trade in dog and cat furs through a DNA-based protocol for species identification.

PubMed

Garofalo, Luisa; Mariacher, Alessia; Fanelli, Rita; Fico, Rosario; Lorenzini, Rita

2018-01-01

In Western countries dogs and cats are the most popular pets, and people are increasingly opposed to their rearing for the fur industry. In 2007, a Regulation of the European Union (EU) banned the use and trade of dog and cat furs, but an official analytical protocol to identify them as source species was not provided, and violations of law are still frequent in all Member States. In this paper we report on the development and validation of a simple and affordable DNA method for species detection in furs to use as an effective tool to combat illegal trade in fur products. A set of mitochondrial primers was designed for amplification of partial cytochrome b, control region and ND1 gene in highly degraded samples, like furs and pelts. Our amplification workflow involved the use of a non-specific primer pair to perform a first test to identify the species through sequencing, then the application of species-specific primer pairs to use in singleplex end-point PCRs as confirmation tests. The advantage of this two-step procedure is twofold: on the one hand it minimises the possibility of negative test results from degraded samples, since failure of amplification with a first set of primers can be offset by successful amplification of the second, and on the other it adds confidence and reliability to final authentication of species. All designed primers were validated on a reference collection of tissue samples, obtaining solid results in terms of specificity, sensitivity, repeatability and reproducibility. Application of the protocol on real caseworks from seized furs yielded successful results also from old and dyed furs, suggesting that age and chemical staining do not necessarily affect positive amplifications. Major pros of this approach are: (1) sensitive and informative primer sets for detection of species; (2) short PCR amplicons for the analysis of poor quality DNA; (3) binding primers that avoid contamination from human DNA; (4) user-friendly protocol for any laboratory equipped for analysis of low-copy-number DNA. Our molecular procedure proved to be a good starting point for enforcing the EU Regulation against dog and cat fur trade in forensic contexts where source attribution is essential to the assignment of responsibilities.

A paper and plastic device for the combined isothermal amplification and lateral flow detection of Plasmodium DNA.

PubMed

Cordray, Michael S; Richards-Kortum, Rebecca R

2015-11-26

Isothermal amplification techniques are emerging as a promising method for malaria diagnosis since they are capable of detecting extremely low concentrations of parasite target while mitigating the need for infrastructure and training required by other nucleic acid based tests. Recombinase polymerase amplification (RPA) is promising for further development since it operates in a short time frame (<30 min) and produces a product that can be visually detected on a lateral flow dipstick. A self-sealing paper and plastic system that performs both the amplification and detection of a malaria DNA sequence is presented. Primers were designed using the NCBI nBLAST tools and screened using gel electrophoresis. Paper and plastic devices were prototyped using commercial design software and parts were cut using a laser cutter and assembled by hand. Synthetic copies of the Plasmodium 18S gene were spiked into solution and used as targets for the RPA reaction. To test the performance of the device the same samples spiked with synthetic target were run in parallel both in the paper and plastic devices and using conventional bench top methods. Novel RPA primers were developed that bind to sequences present in the four species of Plasmodium which infect humans. The paper and plastic devices were found to be capable of detecting as few as 5 copies/µL of synthetic Plasmodium DNA (50 copies total), comparable to the same reaction run on the bench top. The devices produce visual results in an hour, cost approximately $1, and are self-contained once the device is sealed. The device was capable of carrying out the RPA reaction and detecting meaningful amounts of synthetic Plasmodium DNA in a self-sealing and self-contained device. This device may be a step towards making nucleic acid tests more accessible for malaria detection.

Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue.

PubMed

Miles, Timothy D; Martin, Frank N; Coffey, Michael D

2015-02-01

Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing, thereby reducing the time necessary for accurate diagnostics and making management decisions.

Point of care diagnosis of multiple schistosome parasites: Species-specific DNA detection in urine by loop-mediated isothermal amplification (LAMP).

PubMed

Lodh, Nilanjan; Mikita, Kei; Bosompem, Kwabena M; Anyan, William K; Quartey, Joseph K; Otchere, Joseph; Shiff, Clive J

2017-09-01

Schistosomes are easily transmitted and high chance of repeat infection, so if control strategies based on targeted mass drug administration (MDA) are to succeed it is essential to have a test that is sensitive, accurate and simple to use. It is known and regularly demonstrated that praziquantel does not always eliminate an infection so in spite of the successes of control programs a residual of the reservoir survives to re-infect snails. The issue of diagnostic sensitivity becomes more critical in the assessment of program effectiveness. While serology, such as antigen capture tests might improve sensitivity, it has been shown that the presence of species-specific DNA fragments will indicate, most effectively, the presence of active parasites. Polymerase chain reaction (PCR) can amplify and detect DNA from urine residue captured on Whatman No. 3 filter paper that is dried after filtration. Previously we have detected S. mansoni and S. haematobium parasite-specific small repeat DNA fragment from filtered urine on filter paper by PCR. In the current study, we assessed the efficacy of detection of 86 urine samples for either or both schistosome parasites by PCR and loop-mediated isothermal amplification (LAMP) that were collected from a low to moderate transmission area in Ghana. Two different DNA extraction methods, standard extraction kit and field usable LAMP-PURE kit were also evaluated by PCR and LAMP amplification. With S. haematobium LAMP amplification for both extractions showed similar sensitivity and specificity when compared with PCR amplification (100%) verified by gel electrophoresis. For S. mansoni sensitivity was highest for LAMP amplification (100%) for standard extraction than PCR and LAMP with LAMP-PURE (99% and 94%). The LAMP-PURE extraction produced false negatives, which require further investigation for this field usable extraction kit. Overall high positive and negative predictive values (90% - 100%) for both species demonstrated a highly robust approach. The LAMP approach is close to point of care use and equally sensitive and specific to detection of species-specific DNA by PCR. LAMP can be an effective means to detect low intensity infection due to its simplicity and minimal DNA extraction requirement. This will enhance the effectiveness of surveillance and MDA control programs of schistosomiasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Exponential isothermal amplification of nucleic acids and amplified assays for proteins, cells, and enzyme activities.

PubMed

Reid, Michael S; Le, X Chris; Zhang, Hongquan

2018-04-27

Isothermal exponential amplification techniques, such as strand-displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA), have great potential for on-site, point-of-care, and in-situ assay applications. These amplification techniques eliminate the need for temperature cycling required for polymerase chain reaction (PCR) while achieving comparable amplification yield. We highlight here recent advances in exponential amplification reaction (EXPAR) for the detection of nucleic acids, proteins, enzyme activities, cells, and metal ions. We discuss design strategies, enzyme reactions, detection techniques, and key features. Incorporation of fluorescence, colorimetric, chemiluminescence, Raman, and electrochemical approaches enables highly sensitive detection of a variety of targets. Remaining issues, such as undesirable background amplification resulting from non-specific template interactions, must be addressed to further improve isothermal and exponential amplification techniques. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Assessing the performance of a Loop Mediated Isothermal Amplification (LAMP) assay for the detection and subtyping of high-risk suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification.

PubMed

Rohatensky, Mitchell G; Livingstone, Devon M; Mintchev, Paul; Barnes, Heather K; Nakoneshny, Steven C; Demetrick, Douglas J; Dort, Joseph C; van Marle, Guido

2018-02-08

Oropharyngeal Squamous Cell Carcinoma (OPSCC) is increasing in incidence despite a decline in traditional risk factors. Human Papilloma Virus (HPV), specifically subtypes 16, 18, 31 and 35, has been implicated as the high-risk etiologic agent. HPV positive cancers have a significantly better prognosis than HPV negative cancers of comparable stage, and may benefit from different treatment regimens. Currently, HPV related carcinogenesis is established indirectly through Immunohistochemistry (IHC) staining for p16, a tumour suppressor gene, or polymerase chain reaction (PCR) that directly tests for HPV DNA in biopsied tissue. Loop mediated isothermal amplification (LAMP) is more accurate than IHC, more rapid than PCR and is significantly less costly. In previous work we showed that a subtype specific HPV LAMP assay performed similar to PCR on purified DNA. In this study we examined the performance of this LAMP assay without DNA purification. We used LAMP assays using established primers for HPV 16 and 18, and new primers for HPV 31 and 35. LAMP reaction conditions were tested on serial dilutions of plasmid HPV DNA to confirm minimum viral copy number detection thresholds. LAMP was then performed directly on different human cell line samples without DNA purification. Our LAMP assays could detect 10 5 , 10 3 , 10 4 , and 10 5 copies of plasmid DNA for HPV 16, 18, 31, and 35, respectively. All primer sets were subtype specific, with no cross-amplification. Our LAMP assays also reliably amplified subtype specific HPV DNA from samples without requiring DNA isolation and purification. The high risk OPSCC HPV subtype specific LAMP primer sets demonstrated, excellent clinically relevant, minimum copy number detection thresholds with an easy readout system. Amplification directly from samples without purification illustrated the robust nature of the assay, and the primers used. This lends further support HPV type specific LAMP assays, and these specific primer sets and assays can be further developed to test for HPV in OPSCC in resource and lab limited settings, or even bedside testing.

RPA using a multiplexed cartridge for low cost point of care diagnostics in the field.

PubMed

Ereku, Luck Tosan; Mackay, Ruth E; Craw, Pascal; Naveenathayalan, Angel; Stead, Thomas; Branavan, Manorharanehru; Balachandran, Wamadeva

2018-04-15

A point of care device utilising Lab-on-a-Chip technologies that is applicable for biological pathogens was designed, fabricated and tested showing sample in to answer out capabilities. The purpose of the design was to develop a cartridge with the capability to perform nucleic acid extraction and purification from a sample using a chitosan membrane at an acidic pH. Waste was stored within the cartridge with the use of sodium polyacrylate to solidify or gelate the sample in a single chamber. Nucleic acid elution was conducted using the RPA amplification reagents (alkaline pH). Passive valves were used to regulate the fluid flow and a multiplexer was designed to distribute the fluid into six microchambers for amplification reactions. Cartridges were produced using soft lithography of silicone from 3D printed moulds, bonded to glass substrates. The isothermal technique, RPA is employed for amplification. This paper shows the results from two separate experiments: the first using the RPA control nucleic acid, the second showing successful amplification from Chlamydia Trachomatis. Endpoint analysis conducted for the RPA analysis was gel electrophoresis that showed 143 base pair DNA was amplified successfully for positive samples whilst negative samples did not show amplification. End point analysis for Chlamydia Trachomatis samples was fluorescence detection that showed successful detection of 1 copy/μL and 10 copies/μL spiked in a MES buffer. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

Influence of sequence and size of DNA on packaging efficiency of parvovirus MVM-based vectors.

PubMed

Brandenburger, A; Coessens, E; El Bakkouri, K; Velu, T

1999-05-01

We have derived a vector from the autonomous parvovirus MVM(p), which expresses human IL-2 specifically in transformed cells (Russell et al., J. Virol 1992;66:2821-2828). Testing the therapeutic potential of these vectors in vivo requires high-titer stocks. Stocks with a titer of 10(9) can be obtained after concentration and purification (Avalosse et al., J. Virol. Methods 1996;62:179-183), but this method requires large culture volumes and cannot easily be scaled up. We wanted to increase the production of recombinant virus at the initial transfection step. Poor vector titers could be due to inadequate genome amplification or to inefficient packaging. Here we show that intracellular amplification of MVM vector genomes is not the limiting factor for vector production. Several vector genomes of different size and/or structure were amplified to an equal extent. Their amplification was also equivalent to that of a cotransfected wild-type genome. We did not observe any interference between vector and wild-type genomes at the level of DNA amplification. Despite equivalent genome amplification, vector titers varied greatly between the different genomes, presumably owing to differences in packaging efficiency. Genomes with a size close to 100% that of wild type were packaged most efficiently with loss of efficiency at lower and higher sizes. However, certain genomes of identical size showed different packaging efficiencies, illustrating the importance of the DNA sequence, and probably its structure.

Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

PubMed

Ball, Cameron S; Light, Yooli K; Koh, Chung-Yan; Wheeler, Sarah S; Coffey, Lark L; Meagher, Robert J

2016-04-05

Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.

Surmounting a PCR challenge using a Contradictory matrix from the Theory of Inventive Problem Solving (TRIZ).

PubMed

Drábek, Jiří

2016-01-01

In this paper I tested whether Contradictory Matrix with 40 Inventive Principles, the simplest instrument from the Theory of Inventive Problem Solving (TRIZ), is a useful approach to a real-life PCR scenario. The PCR challenge consisted of standardization of fluorescence melting curve measurements in Competitive Amplification of Differentially Melting Amplicons (CADMA) PCR for multiple targets. Here I describe my way of using the TRIZ Matrix to generate seven alternative solutions from which I can choose the successful solution, consisting of repeated cycles of amplification and melting in a single PCR run.

Signs or Symptoms of Acute HIV Infection in a Cohort Undergoing Community-Based Screening.

PubMed

Hoenigl, Martin; Green, Nella; Camacho, Martha; Gianella, Sara; Mehta, Sanjay R; Smith, Davey M; Little, Susan J

2016-03-01

We analyzed signs and symptoms in 90 patients diagnosed with acute HIV infection in a community-based program that offered universal HIV-1 nucleic acid amplification testing. Forty-seven (52%) patients reported ongoing signs or symptoms at the time of testing. Another 25 (28%) reported signs or symptoms that had occurred during the 14 days before testing.

Development of the polymerase chain reaction for diagnosis of chancroid.

PubMed Central

Chui, L; Albritton, W; Paster, B; Maclean, I; Marusyk, R

1993-01-01

The published nucleotide sequences of the 16S rRNA gene of Haemophilus ducreyi were used to develop primer sets and probes for the diagnosis of chancroid by polymerase chain reaction (PCR) DNA amplification. One set of broad specificity primers yielded a 303-bp PCR product from all bacteria tested. Two 16-base probes internal to this sequence were species specific for H. ducreyi when tested with 12 species of the families Pasteurellaceae and Enterobacteriaceae. The two probes in combination with the broad specificity primers were 100% sensitive with 51 strains of H. ducreyi isolated from six continents over a 15-year period. The direct detection of H. ducreyi from 100 clinical specimens by PCR showed a sensitivity of 83 to 98% and a specificity of 51 to 67%, depending on the number of amplification cycles. Images PMID:8458959

Analysis of multiple cell upset sensitivity in bulk CMOS SRAM after neutron irradiation

NASA Astrophysics Data System (ADS)

Pan, Xiaoyu; Guo, Hongxia; Luo, Yinhong; Zhang, Fengqi; Ding, Lili

2018-03-01

In our previous studies, we have proved that neutron irradiation can decrease the single event latch-up (SEL) sensitivity of CMOS SRAM. And one of the key contributions to the multiple cell upset (MCU) is the parasitic bipolar amplification, it bring us to study the impact of neutron irradiation on the SRAM’s MCU sensitivity. After the neutron experiment, we test the devices’ function and electrical parameters. Then, we use the heavy ion fluence to examine the changes on the devices’ MCU sensitivity pre- and post-neutron-irradiation. Unfortunately, neutron irradiation makes the MCU phenomenon worse. Finally, we use the electric static discharge (ESD) testing technology to deduce the experimental results and find that the changes on the WPM region take the lead rather than the changes on the parasitic bipolar amplification for the 90 nm process.

Point-of-care testing system enabling 30 min detection of influenza genes.

PubMed

Abe, Tomoteru; Segawa, Yuji; Watanabe, Hidetoshi; Yotoriyama, Tasuku; Kai, Shinichi; Yasuda, Akio; Shimizu, Norio; Tojo, Naoko

2011-03-21

We developed a portable and easy-to-use nucleic acid amplification test (NAT) system for use in point-of-care testing (POCT). The system shows sensitivity that is sufficiently higher than that of the currently available rapid diagnostic kit and is comparable to that of real-time reverse transcription polymerase chain reaction (RT-PCR) for influenza testing. This journal is © The Royal Society of Chemistry 2011

A novel monolithic piezoelectric actuated flexure-mechanism based wire clamp for microelectronic device packaging.

PubMed

Liang, Cunman; Wang, Fujun; Tian, Yanling; Zhao, Xingyu; Zhang, Hongjie; Cui, Liangyu; Zhang, Dawei; Ferreira, Placid

2015-04-01

A novel monolithic piezoelectric actuated wire clamp is presented in this paper to achieve fast, accurate, and robust microelectronic device packaging. The wire clamp has compact, flexure-based mechanical structure and light weight. To obtain large and robust jaw displacements and ensure parallel jaw grasping, a two-stage amplification composed of a homothetic bridge type mechanism and a parallelogram leverage mechanism was designed. Pseudo-rigid-body model and Lagrange approaches were employed to conduct the kinematic, static, and dynamic modeling of the wire clamp and optimization design was carried out. The displacement amplification ratio, maximum allowable stress, and natural frequency were calculated. Finite element analysis (FEA) was conducted to evaluate the characteristics of the wire clamp and wire electro discharge machining technique was utilized to fabricate the monolithic structure. Experimental tests were carried out to investigate the performance and the experimental results match well with the theoretical calculation and FEA. The amplification ratio of the clamp is 20.96 and the working mode frequency is 895 Hz. Step response test shows that the wire clamp has fast response and high accuracy and the motion resolution is 0.2 μm. High speed precision grasping operations of gold and copper wires were realized using the wire clamper.

Analysis on Two Typical Landslide Hazard Phenomena in The Wenchuan Earthquake by Field Investigations and Shaking Table Tests

PubMed Central

Yang, Changwei; Zhang, Jianjing; Liu, Feicheng; Bi, Junwei; Jun, Zhang

2015-01-01

Based on our field investigations of landslide hazards in the Wenchuan earthquake, some findings can be reported: (1) the multi-aspect terrain facing empty isolated mountains and thin ridges reacted intensely to the earthquake and was seriously damaged; (2) the slope angles of most landslides was larger than 45°. Considering the above disaster phenomena, the reasons are analyzed based on shaking table tests of one-sided, two-sided and four-sided slopes. The analysis results show that: (1) the amplifications of the peak accelerations of four-sided slopes is stronger than that of the two-sided slopes, while that of the one-sided slope is the weakest, which can indirectly explain the phenomena that the damage is most serious; (2) the amplifications of the peak accelerations gradually increase as the slope angles increase, and there are two inflection points which are the point where the slope angle is 45° and where the slope angle is 50°, respectively, which can explain the seismic phenomenon whereby landslide hazards mainly occur on the slopes whose slope angle is bigger than 45°. The amplification along the slope strike direction is basically consistent, and the step is smooth. PMID:26258785

Sensitive and rapid detection of Chlamydia trachomatis by recombinase polymerase amplification directly from urine samples.

PubMed

Krõlov, Katrin; Frolova, Jekaterina; Tudoran, Oana; Suhorutsenko, Julia; Lehto, Taavi; Sibul, Hiljar; Mäger, Imre; Laanpere, Made; Tulp, Indrek; Langel, Ülo

2014-01-01

Chlamydia trachomatis is the most common sexually transmitted human pathogen. Infection results in minimal to no symptoms in approximately two-thirds of women and therefore often goes undiagnosed. C. trachomatis infections are a major public health concern because of the potential severe long-term consequences, including an increased risk of ectopic pregnancy, chronic pelvic pain, and infertility. To date, several point-of-care tests have been developed for C. trachomatis diagnostics. Although many of them are fast and specific, they lack the required sensitivity for large-scale application. We describe a rapid and sensitive form of detection directly from urine samples. The assay uses recombinase polymerase amplification and has a minimum detection limit of 5 to 12 pathogens per test. Furthermore, it enables detection within 20 minutes directly from urine samples without DNA purification before the amplification reaction. Initial analysis of the assay from clinical patient samples had a specificity of 100% (95% CI, 92%-100%) and a sensitivity of 83% (95% CI, 51%-97%). The whole procedure is fairly simple and does not require specific machinery, making it potentially applicable in point-of-care settings. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification.

PubMed

Silva, Gonçalo; Bömer, Moritz; Nkere, Chukwuemeka; Kumar, P Lava; Seal, Susan E

2015-09-15

Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam production globally. Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential component of disease control. Current serological and PCR-based diagnostic methods for YMV are time consuming involving a succession of target detection steps. In this study, a novel assay for specific YMV detection is described that is based on isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown to be reproducible and able to detect as little as 14 pg/μl of purified RNA obtained from an YMV-infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages over the RT-PCR; positive samples can be detected in less than 30 min, and amplification only requires a single incubation temperature (optimum 37°C). These features make the RT-exoRPA assay a promising candidate for adapting into a field test format to be used by yam breeding programmes or certification laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

The nature of combustion noise: Stochastic or chaotic?

NASA Astrophysics Data System (ADS)

Gupta, Vikrant; Lee, Min Chul; Li, Larry K. B.

2016-11-01

Combustion noise, which refers to irregular low-amplitude pressure oscillations, is conventionally thought to be stochastic. It has therefore been modeled using a stochastic term in the analysis of thermoacoustic systems. Recently, however, there has been a renewed interest in the validity of that stochastic assumption, with tests based on nonlinear dynamical theory giving seemingly contradictory results: some show combustion noise to be stochastic while others show it to be chaotic. In this study, we show that this contradiction arises because those tests cannot distinguish between noise amplification and chaos. We further show that although there are many similarities between noise amplification and chaos, there are also some subtle differences. It is these subtle differences, not the results of those tests, that should be the focus of analyses aimed at determining the true nature of combustion noise. Recognizing this is an important step towards improved understanding and modeling of combustion noise for the study of thermoacoustic instabilities. This work was supported by the Research Grants Council of Hong Kong (Project No. 16235716 and 26202815).

Amplification of ST50 gene using dry-reagent-based polymerase chain reaction for the detection of Salmonella typhi.

PubMed

Aziah, Ismail; Ravichandran, Manickam; Ismail, Asma

2007-12-01

Conventional polymerase chain reaction (PCR) testing requires many pipetting steps and has to be transported and stored in cold chain. To overcome these limitations, we designed a ready-to-use PCR test for Salmonella typhi using PCR reagents, primers against the ST50 gene of S. typhi, a built-in internal amplification control (IAC), and gel loading dye mixed and freeze-dried in a single tube. The 2-step dry-reagent-based assay was used to amplify a 1238-bp target gene and an 810-bp IAC gene from 73 BACTEC blood culture broths (33 true positives for S. typhi and 40 true negatives for non-S. typhi). The sensitivity, specificity, positive predictive value, and negative predictive value of the PCR assay were 87.9%, 100%, 100%, and 90.9%, respectively. We suggest that this rapid 2-step PCR test could be used for the rapid diagnosis of typhoid fever.

Early Detection of Dengue Virus by Use of Reverse Transcription-Recombinase Polymerase Amplification

PubMed Central

Teoh, Boon-Teong; Sam, Sing-Sin; Tan, Kim-Kee; Danlami, Mohammed Bashar; Shu, Meng-Hooi; Johari, Jefree; Hooi, Poh-Sim; Brooks, David; Piepenburg, Olaf; Nentwich, Oliver; Wilder-Smith, Annelies; Franco, Leticia; Tenorio, Antonio

2015-01-01

A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue. PMID:25568438

Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thissen, James B.; McLoughlin, Kevin; Gardner, Shea

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA wasmore » able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish.

PubMed

Saleh, Mona; Soliman, Hatem; El-Matbouli, Mansour

2008-08-12

Enteric Redmouth (ERM) disease also known as Yersiniosis is a contagious disease affecting salmonids, mainly rainbow trout. The causative agent is the gram-negative bacterium Yersinia ruckeri. The disease can be diagnosed by isolation and identification of the causative agent, or detection of the Pathogen using fluorescent antibody tests, ELISA and PCR assays. These diagnostic methods are laborious, time consuming and need well trained personnel. A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for detection of Y. ruckeri the etiological agent of enteric red mouth (ERM) disease in salmonids. The assay was optimised to amplify the yruI/yruR gene, which encodes Y. ruckeri quorum sensing system, in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63 degrees C for one hour. Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons. Digestion with HphI restriction enzyme demonstrated that the amplified product was unique. The specificity of the assay was verified by the absence of amplification products when tested against related bacteria. The assay had 10-fold higher sensitivity compared with conventional PCR and successfully detected Y. ruckeri not only in pure bacterial culture but also in tissue homogenates of infected fish. The ERM-LAMP assay represents a practical alternative to the microbiological approach for rapid, sensitive and specific detection of Y. ruckeri in fish farms. The assay is carried out in one hour and needs only a heating block or water bath as laboratory furniture. The advantages of the ERM-LAMP assay make it a promising tool for molecular detection of enteric red mouth disease in fish farms.

A novel, integrated forensic microdevice on a rotation-driven platform: Buccal swab to STR product in less than 2 h.

PubMed

Cox, Jordan O; DeCarmen, Teresa Sikes; Ouyang, Yiwen; Strachan, Briony; Sloane, Hillary; Connon, Cathey; Gibson, Kemper; Jackson, Kimberly; Landers, James P; Cruz, Tracey Dawson

2016-12-01

This work describes the development of a novel microdevice for forensic DNA processing of reference swabs. This microdevice incorporates an enzyme-based assay for DNA preparation, which allows for faster processing times and reduced sample handling. Infrared-mediated PCR (IR-PCR) is used for STR amplification using a custom reaction mixture, allowing for amplification of STR loci in 45 min while circumventing the limitations of traditional block thermocyclers. Uniquely positioned valves coupled with a simple rotational platform are used to exert fluidic control, eliminating the need for bulky external equipment. All microdevices were fabricated using laser ablation and thermal bonding of PMMA layers. Using this microdevice, the enzyme-mediated DNA liberation module produced DNA yields similar to or higher than those produced using the traditional (tube-based) protocol. Initial microdevice IR-PCR experiments to test the amplification module and reaction (using Phusion Flash/SpeedSTAR) generated near-full profiles that suffered from interlocus peak imbalance and poor adenylation (significant -A). However, subsequent attempts using KAPA 2G and Pfu Ultra polymerases generated full STR profiles with improved interlocus balance and the expected adenylated product. A fully integrated run designed to test microfluidic control successfully generated CE-ready STR amplicons in less than 2 h (<1 h of hands-on time). Using this approach, high-quality STR profiles were developed that were consistent with those produced using conventional DNA purification and STR amplification methods. This method is a smaller, more elegant solution than current microdevice methods and offers a cheaper, hands-free, closed-system alternative to traditional forensic methods. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

DOE PAGES

Thissen, James B.; McLoughlin, Kevin; Gardner, Shea; ...

2014-06-01

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA wasmore » able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

Simultaneous identification and DNA barcoding of six Eimeria species infecting turkeys using PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus.

PubMed

Hafeez, Mian A; Shivaramaiah, Srichaitanya; Dorsey, Kristi Moore; Ogedengbe, Mosun E; El-Sherry, Shiem; Whale, Julia; Cobean, Julie; Barta, John R

2015-05-01

Species-specific PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus were generated that allow for the specific identification of the most common Eimeria species infecting turkeys (i.e., Eimeria adenoeides, Eimeria meleagrimitis, Eimeria gallopavonis, Eimeria meleagridis, Eimeria dispersa, and Eimeria innocua). PCR reaction chemistries were optimized with respect to divalent cation (MgCl2) and dNTP concentrations, as well as PCR cycling conditions (particularly anneal temperature for primers). Genomic DNA samples from single oocyst-derived lines of six Eimeria species were tested to establish specificity and sensitivity of these newly designed primer pairs. A mixed 60-ng total DNA sample containing 10 ng of each of the six Eimeria species was used as DNA template to demonstrate specific amplification of the correct product using each of the species-specific primer pairs. Ten nanograms of each of the five non-target Eimeria species was pooled to provide a non-target, control DNA sample suitable to test the specificity of each primer pair. The amplifications of the COI region with species-specific primer pairs from pooled samples yielded products of expected sizes (209 to 1,012 bp) and no amplification of non-target Eimeria sp. DNA was detected using the non-target, control DNA samples. These primer pairs specific for Eimeria spp. of turkeys did not amplify any of the seven Eimeria species infecting chickens. The newly developed PCR primers can be used as a diagnostic tool capable of specifically identifying six turkey Eimeria species; additionally, sequencing of the PCR amplification products yields sequence-based genotyping data suitable for identification and molecular phylogenetics.

Advantages of binaural amplification to acceptable noise level of directional hearing aid users.

PubMed

Kim, Ja-Hee; Lee, Jae Hee; Lee, Ho-Ki

2014-06-01

The goal of the present study was to examine whether Acceptable Noise Levels (ANLs) would be lower (greater acceptance of noise) in binaural listening than in monaural listening condition and also whether meaningfulness of background speech noise would affect ANLs for directional microphone hearing aid users. In addition, any relationships between the individual binaural benefits on ANLs and the individuals' demographic information were investigated. Fourteen hearing aid users (mean age, 64 years) participated for experimental testing. For the ANL calculation, listeners' most comfortable listening levels and background noise level were measured. Using Korean ANL material, ANLs of all participants were evaluated under monaural and binaural amplification with a counterbalanced order. The ANLs were also compared across five types of competing speech noises, consisting of 1- through 8-talker background speech maskers. Seven young normal-hearing listeners (mean age, 27 years) participated for the same measurements as a pilot testing. The results demonstrated that directional hearing aid users accepted more noise (lower ANLs) with binaural amplification than with monaural amplification, regardless of the type of competing speech. When the background speech noise became more meaningful, hearing-impaired listeners accepted less amount of noise (higher ANLs), revealing that ANL is dependent on the intelligibility of the competing speech. The individuals' binaural advantages in ANLs were significantly greater for the listeners with longer experience of hearing aids, yet not related to their age or hearing thresholds. Binaural directional microphone processing allowed hearing aid users to accept a greater amount of background noise, which may in turn improve listeners' hearing aid success. Informational masking substantially influenced background noise acceptance. Given a significant association between ANLs and duration of hearing aid usage, ANL measurement can be useful for clinical counseling of binaural hearing aid candidates or unsuccessful users.

A Simple, Inexpensive Device for Nucleic Acid Amplification without Electricity—Toward Instrument-Free Molecular Diagnostics in Low-Resource Settings

PubMed Central

LaBarre, Paul; Hawkins, Kenneth R.; Gerlach, Jay; Wilmoth, Jared; Beddoe, Andrew; Singleton, Jered; Boyle, David; Weigl, Bernhard

2011-01-01

Background Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation). Methodology/Principal Findings In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented. Conclusions/Significance We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA) heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes. PMID:21573065

Detection and identification of occult HBV in blood donors in Taiwan using a commercial, multiplex, multi-dye nucleic acid amplification technology screening test.

PubMed

Lin, K T; Chang, C L; Tsai, M H; Lin, K S; Saldanha, J; Hung, C M

2014-02-01

The ability of a new generation commercial, multiplex, multi-dye test from Roche, the cobas TaqScreen MPX test, version 2.0, to detect and identify occult HBV infections was evaluated using routine donor samples from Kaohsiung Blood Bank, Taiwan. A total of 5973 samples were tested by nucleic acid amplification technology (NAT); 5898 in pools of six, 66 in pools of less than six and nine samples individually. NAT-reactive samples were retested with alternative NAT tests, and follow-up samples from the donors were tested individually by NAT and for all the HBV serological markers. Eight NAT-only-reactive donors were identified, and follow-up samples were obtained from six of the donors. The results indicated that all eight donors had an occult HBV infection with viral loads <12 IU/ml. The cobas(®) TaqScreen MPX test, version 2.0, has an advantage over the current Roche blood screening test, the cobas TaqScreen MPX test, for screening donations in countries with a high prevalence of occult HBV infections since the uncertainty associated with identifying samples with very low viremia is removed by the ability of the test to identify the viral target in samples that are reactive with the cobas TaqScreen MPX test, version 2.0. © 2013 International Society of Blood Transfusion.

Prevalence and predictors of Lymphogranuloma venereum in a high risk population attending a STD outpatients clinic in Italy.

PubMed

Foschi, Claudio; Marangoni, Antonella; D'Antuono, Antonietta; Nardini, Paola; Compri, Monica; Bellavista, Sara; Filippini, Andrea; Bacchi Reggiani, Maria Letizia; Cevenini, Roberto

2014-04-09

We evaluated LGV prevalence and predictors in a high risk population attending a STI Outpatients Clinic in the North of Italy. A total of 108 patients (99 MSM and 9 women), with a history of unsafe anal sexual intercourses, were enrolled. Anorectal swabs and urine samples were tested for Chlamydia trachomatis (CT) DNA detection by Versant CT/GC DNA 1.0 Assay (Siemens Healthcare Diagnostics Terrytown, USA). RFLP analysis was used for CT molecular typing. L2 CT genotype was identified in 13/108 (12%) rectal swabs. All LGV cases were from MSM, declaring high-risk sexual behaviour and complaining anorectal symptoms. Patients first attending the STI Outpatient Clinic received a significant earlier LGV diagnosis than those first seeking care from general practitioners or gastroenterologists (P = 0.0046). LGV prevalence and characteristics found in our population are in agreement with international reports. Statistical analysis showed that LGV positive patients were older (P = 0.0008) and presented more STIs (P = 0.0023) than LGV negative ones, in particular due to syphilis (P < 0.001), HIV (P < 0.001) and HBV (P = 0.001).Multivariate logistic regression analysis revealed that HIV and syphilis infections are strong risk factors for LGV presence (respectively, P = 0.001 and P = 0.010). Even if our results do not provide sufficient evidence to recommend routine screening of anorectal swabs in high-risk population, they strongly suggest to perform CT NAAT tests and genotyping on rectal specimens in presence of ulcerative proctitis in HIV and/or syphilis-positive MSM. In this context, CT DNA detection by Versant CT/GC DNA 1.0 Assay, followed by RFLP analysis for molecular typing demonstrated to be an excellent diagnostic algorithm for LGV identification.

Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay.

PubMed

Goud, Kalal Iravathy; Dayakar, Seetha; Vijayalaxmi, Kolanupaka; Babu, Saidam Jangu; Reddy, P Vijay Anand

2012-03-01

Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER 2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis. A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy. Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4. Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated.

Direct amplification of casework bloodstains using the Promega PowerPlex(®) 21 PCR amplification system.

PubMed

Gray, Kerryn; Crowle, Damian; Scott, Pam

2014-09-01

A significant number of evidence items submitted to Forensic Science Service Tasmania (FSST) are blood swabs or bloodstained items. Samples from these items routinely undergo phenol:chloroform:isoamyl alcohol organic extraction and quantitative Polymerase Chain Reaction (qPCR) testing prior to PowerPlex(®) 21 amplification. This multi-step process has significant cost and timeframe implications in a fiscal climate of tightening government budgets, pressure towards improved operating efficiencies, and an increasing emphasis on rapid techniques better supporting intelligence-led policing. Direct amplification of blood and buccal cells on cloth and Whatman FTA™ card with PowerPlex(®) 21 has already been successfully implemented for reference samples, eliminating the requirement for sample pre-treatment. Scope for expanding this method to include less pristine casework blood swabs and samples from bloodstained items was explored in an endeavour to eliminate lengthy DNA extraction, purification and qPCR steps for a wider subset of samples. Blood was deposited onto a range of substrates including those historically found to inhibit STR amplification. Samples were collected with micro-punch, micro-swab, or both. The potential for further fiscal savings via reduced volume amplifications was assessed by amplifying all samples at full and reduced volume (25 and 13μL). Overall success rate data showed 80% of samples yielded a complete profile at reduced volume, compared to 78% at full volume. Particularly high success rates were observed for the blood on fabric/textile category with 100% of micro-punch samples yielding complete profiles at reduced volume and 85% at full volume. Following the success of this trial, direct amplification of suitable casework blood samples has been implemented at reduced volume. Significant benefits have been experienced, most noticeably where results from crucial items have been provided to police investigators prior to interview of suspects, and a coronial identification has been successfully completed in a short timeframe to avoid delay in the release of human remains to family members. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A multivariate test of disease risk reveals conditions leading to disease amplification.

PubMed

Halliday, Fletcher W; Heckman, Robert W; Wilfahrt, Peter A; Mitchell, Charles E

2017-10-25

Theory predicts that increasing biodiversity will dilute the risk of infectious diseases under certain conditions and will amplify disease risk under others. Yet, few empirical studies demonstrate amplification. This contrast may occur because few studies have considered the multivariate nature of disease risk, which includes richness and abundance of parasites with different transmission modes. By combining a multivariate statistical model developed for biodiversity-ecosystem-multifunctionality with an extensive field manipulation of host (plant) richness, composition and resource supply to hosts, we reveal that (i) host richness alone could not explain most changes in disease risk, and (ii) shifting host composition allowed disease amplification, depending on parasite transmission mode. Specifically, as predicted from theory, the effect of host diversity on parasite abundance differed for microbes (more density-dependent transmission) and insects (more frequency-dependent transmission). Host diversity did not influence microbial parasite abundance, but nearly doubled insect parasite abundance, and this amplification effect was attributable to variation in host composition. Parasite richness was reduced by resource addition, but only in species-rich host communities. Overall, this study demonstrates that multiple drivers, related to both host community and parasite characteristics, can influence disease risk. Furthermore, it provides a framework for evaluating multivariate disease risk in other systems. © 2017 The Author(s).

swga: a primer design toolkit for selective whole genome amplification.

PubMed

Clarke, Erik L; Sundararaman, Sesh A; Seifert, Stephanie N; Bushman, Frederic D; Hahn, Beatrice H; Brisson, Dustin

2017-07-15

Population genomic analyses are often hindered by difficulties in obtaining sufficient numbers of genomes for analysis by DNA sequencing. Selective whole-genome amplification (SWGA) provides an efficient approach to amplify microbial genomes from complex backgrounds for sequence acquisition. However, the process of designing sets of primers for this method has many degrees of freedom and would benefit from an automated process to evaluate the vast number of potential primer sets. Here, we present swga , a program that identifies primer sets for SWGA and evaluates them for efficiency and selectivity. We used swga to design and test primer sets for the selective amplification of Wolbachia pipientis genomic DNA from infected Drosophila melanogaster and Mycobacterium tuberculosis from human blood. We identify primer sets that successfully amplify each against their backgrounds and describe a general method for using swga for arbitrary targets. In addition, we describe characteristics of primer sets that correlate with successful amplification, and present guidelines for implementation of SWGA to detect new targets. Source code and documentation are freely available on https://www.github.com/eclarke/swga . The program is implemented in Python and C and licensed under the GNU Public License. ecl@mail.med.upenn.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

The MicroActive project: automatic detection of disease-related molecular cell activity

NASA Astrophysics Data System (ADS)

Furuberg, Liv; Mielnik, Michal; Johansen, Ib-Rune; Voitel, Jörg; Gulliksen, Anja; Solli, Lars; Karlsen, Frank; Bayer, Tobias; Schönfeld, Friedhelm; Drese, Klaus; Keegan, Helen; Martin, Cara; O'Leary, John; Riegger, Lutz; Koltay, Peter

2007-05-01

The aim of the MicroActive project is to develop an instrument for molecular diagnostics. The instrument will first be tested for patient screening for a group of viruses causing cervical cancer. Two disposable polymer chips with reagents stored on-chip will be inserted into the instrument for each patient sample. The first chip performs sample preparation of the epithelial cervical cells while mRNA amplification and fluorescent detection takes place in the second chip. More than 10 different virus markers will be analysed in one chip. We report results on sub-functions of the amplification chip. The sample is split into smaller droplets, and the droplets move in parallel channels containing different dried reagents for the different analyses. We report experimental results on parallel droplet movement control using one external pump only, combined with hydrophobic valves. Valve burst pressures are controlled by geometry. We show droplet control using valves with burst pressures between 800 and 4500 Pa. We also monitored the re-hydration times for two necessary dried reagents. After sample insertion, uniform concentration of the reagents in the droplet was reached after respectively 60 s and 10 min. These times are acceptable for successful amplification. Finally we have shown positive amplification of HPV type 16 using dried enzymes stored in micro chambers.

Trifunctional molecular beacon-mediated quadratic amplification for highly sensitive and rapid detection of mercury(II) ion with tunable dynamic range.

PubMed

Zhao, Yue; Liu, Huaqing; Chen, Feng; Bai, Min; Zhao, Junwu; Zhao, Yongxi

2016-12-15

Analyses of target with low abundance or concentration varying over many orders of magnitude are severe challenges faced by numerous assay methods due to their modest sensitivity and limited dynamic range. Here, we introduce a homogeneous and rapid quadratic polynomial amplification strategy through rational design of a trifunctional molecular beacon, which serves as not only a reporter molecule but also a bridge to couple two stage amplification modules without adding any reaction components or process other than basic linear amplification. As a test bed for our studies, we took mercury(II) ion as an example and obtained a high sensitivity with detection limit down to 200 pM within 30min. In order to create a tunable dynamic range, homotropic allostery is employed to modulate the target specific binding. When the number of metal binding site varies from 1 to 3, signal response is programmed accordingly with useful dynamic range spanning 50, 25 and 10 folds, respectively. Furthermore, the applicability of the proposed method in river water and biological samples are successfully verified with good recovery and reproducibility, indicating considerable potential for its practicality in complex real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification.

PubMed

del Río, Jonathan Sabaté; Yehia Adly, Nouran; Acero-Sánchez, Josep Lluis; Henry, Olivier Y F; O'Sullivan, Ciara K

2014-04-15

Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 μL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 μL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers. © 2013 Elsevier B.V. All rights reserved.

A lab-on-a-chip system with integrated sample preparation and loop-mediated isothermal amplification for rapid and quantitative detection of Salmonella spp. in food samples.

PubMed

Sun, Yi; Quyen, Than Linh; Hung, Tran Quang; Chin, Wai Hoe; Wolff, Anders; Bang, Dang Duong

2015-04-21

Foodborne disease is a major public health threat worldwide. Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture or molecular-based methods are time consuming and usually take a few hours to days to complete. In response to the demand for rapid on line or on site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic bead-based sample preparation and loop-mediated isothermal amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time, will greatly enhance the practical applicability of the LOC system for rapid on-site screening of Salmonella for applications in food safety control, environmental surveillance, and clinical diagnostics.

GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

PubMed

Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

2015-06-01

Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

Forensic DNA testing.

PubMed

Butler, John M

2011-12-01

Forensic DNA testing has a number of applications, including parentage testing, identifying human remains from natural or man-made disasters or terrorist attacks, and solving crimes. This article provides background information followed by an overview of the process of forensic DNA testing, including sample collection, DNA extraction, PCR amplification, short tandem repeat (STR) allele separation and sizing, typing and profile interpretation, statistical analysis, and quality assurance. The article concludes with discussions of possible problems with the data and other forensic DNA testing techniques.

Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings.

PubMed

Jordan, Jeanne A; Ibe, Christine O; Moore, Miranda S; Host, Christel; Simon, Gary L

2012-05-01

In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner. Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS. 60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells. The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470 copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS. Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay. Copyright © 2012 Elsevier B.V. All rights reserved.

Sin Nombre Virus and Rodent Species Diversity: A Test of the Dilution and Amplification Hypotheses

PubMed Central

Clay, Christine A.; Lehmer, Erin M.; Jeor, Stephen St.; Dearing, M. Denise

2009-01-01

Background Species diversity is proposed to greatly impact the prevalence of pathogens. Two predominant hypotheses, the “Dilution Effect” and the “Amplification Effect”, predict divergent outcomes with respect to the impact of species diversity. The Dilution Effect predicts that pathogen prevalence will be negatively correlated with increased species diversity, while the Amplification Effect predicts that pathogen prevalence will be positively correlated with diversity. For many host-pathogen systems, the relationship between diversity and pathogen prevalence has not be empirically examined. Methodology/Principal Findings We tested the Dilution and Amplification Effect hypotheses by examining the prevalence of Sin Nombre virus (SNV) with respect to diversity of the nocturnal rodent community. SNV is directly transmitted primarily between deer mice (Peromyscus maniculatus). Using mark-recapture sampling in the Spring and Fall of 2003–2005, we measured SNV prevalence in deer mice at 16 landscape level sites (3.1 hectares each) that varied in rodent species diversity. We explored several mechanisms by which species diversity may affect SNV prevalence, including reduced host density, reduced host persistence, the presence of secondary reservoirs and community composition. We found a negative relationship between species diversity and SNV prevalence in deer mice, thereby supporting the Dilution Effect hypothesis. Deer mouse density and persistence were lower at sites with greater species diversity; however, only deer mouse persistence was positively correlated with SNV prevalence. Pinyon mice (P. truei) may serve as dilution agents, having a negative effect on prevalence, while kangaroo rats (Dipodomys ordii), may have a positive effect on the prevalence of SNV, perhaps through effects on deer mouse behavior. Conclusions/Significance While previous studies on host-pathogen systems have found patterns of diversity consistent with either the Dilution or Amplification Effects, the mechanisms by which species diversity influences prevalence have not been investigated. Our study indicates that changes in host persistence, coupled with interspecific interactions, are important mechanisms through which diversity may influence patterns of pathogens. Our results reveal the complexity of rodent community interactions with respect to SNV dynamics. PMID:19649283

Developmental validation of a Cannabis sativa STR multiplex system for forensic analysis.

PubMed

Howard, Christopher; Gilmore, Simon; Robertson, James; Peakall, Rod

2008-09-01

A developmental validation study based on recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM) was conducted on a multiplex system of 10 Cannabis sativa short tandem repeat loci. Amplification of the loci in four multiplex reactions was tested across DNA from dried root, stem, and leaf sources, and DNA from fresh, frozen, and dried leaf tissue with a template DNA range of 10.0-0.01 ng. The loci were amplified and scored consistently for all DNA sources when DNA template was in the range of 10.0-1.0 ng. Some allelic dropout and PCR failure occurred in reactions with lower template DNA amounts. Overall, amplification was best using 10.0 ng of template DNA from dried leaf tissue indicating that this is the optimal source material. Cross species amplification was observed in Humulus lupulus for three loci but there was no allelic overlap. This is the first study following SWGDAM validation guidelines to validate short tandem repeat markers for forensic use in plants.

Designing a Micromixer for Rolling Circle Amplification in Cancer Biomarker Detection

NASA Astrophysics Data System (ADS)

Altural, Hayriye

2015-03-01

Rolling circle amplification (RCA) is an alternative method to the Polymerase Chain Reaction based amplification for point-of-care (POC) diagnosis. In future personalized cancer diagnostic for POC applications, smaller, faster and cheaper methods are needed instead of costly and time-consuming laboratory tests. Microfluidic chips can perform the detection of cancer biomarkers within less analysis time, and provide for improvement in the sensitivity and specificity required for biochemical analysis as well. Rapid mixing is essential in the chips used in cancer diagnostic. The goal of this study is to design a micromixer for rapid RCA-based analysis and develop the assay time in cancer biomarker detection. By combining assays with micromixers, multi-step bioreactions in microfluidic chips may be achieved with minimal external control. Here, simulation results related to the micromixer are obtained by COMSOL software. The Scientific and Technological Research Council of Turkey (TUBITAK) is acknowledged for granting of H. Altural postdoctoral study in the framework of TUBITAK-BIDEB 2219-International Postdoctoral Research Scholarship Program.

EnVision+, a new dextran polymer-based signal enhancement technique for in situ hybridization (ISH).

PubMed

Wiedorn, K H; Goldmann, T; Henne, C; Kühl, H; Vollmer, E

2001-09-01

Seventy paraffin-embedded cervical biopsy specimens and condylomata were tested for the presence of human papillomavirus (HPV) by conventional in situ hybridization (ISH) and ISH with subsequent signal amplification. Signal amplification was performed either by a commercial biotinyl-tyramide-based detection system [GenPoint (GP)] or by the novel two-layer dextran polymer visualization system EnVision+ (EV), in which both EV-horseradish peroxidase (EV-HRP) and EV-alkaline phosphatase (EV-AP) were applied. We could demonstrate for the first time, that EV in combination with preceding ISH results in a considerable increase in signal intensity and sensitivity without loss of specificity compared to conventional ISH. Compared to GP, EV revealed a somewhat lower sensitivity, as measured by determination of the integrated optical density (IOD) of the positively stained cells. However, EV is easier to perform, requires a shorter assay time, and does not raise the background problems that may be encountered with biotinyl-tyramide-based amplification systems. (J Histochem Cytochem 49:1067-1071, 2001)

Comparison of Two Methods of RNA Extraction from Formalin-Fixed Paraffin-Embedded Tissue Specimens

PubMed Central

Gouveia, Gisele Rodrigues; Ferreira, Suzete Cleusa; Ferreira, Jerenice Esdras; Siqueira, Sheila Aparecida Coelho; Pereira, Juliana

2014-01-01

The present study aimed to compare two different methods of extracting RNA from formalin-fixed paraffin-embedded (FFPE) specimens of diffuse large B-cell lymphoma (DLBCL). We further aimed to identify possible influences of variables—such as tissue size, duration of paraffin block storage, fixative type, primers used for cDNA synthesis, and endogenous genes tested—on the success of amplification from the samples. Both tested protocols used the same commercial kit for RNA extraction (the RecoverAll Total Nucleic Acid Isolation Optimized for FFPE Samples from Ambion). However, the second protocol included an additional step of washing with saline buffer just after sample rehydration. Following each protocol, we compared the RNA amount and purity and the amplification success as evaluated by standard PCR and real-time PCR. The results revealed that the extra washing step added to the RNA extraction process resulted in significantly improved RNA quantity and quality and improved success of amplification from paraffin-embedded specimens. PMID:25105117

Isolation and characterization of ten novel microsatellite loci in the red-winged tinamou (Rhynchotus rufescens, Tinamiformes, Aves) and cross-amplification in other tinamous.

PubMed

Santos, Dimas O; Moreira, Lucas R; Tonhati, Humberto; Caparroz, Renato

2012-04-01

We describe the isolation and characterization of ten microsatellite loci from the red-winged tinamou (Rhynchotus rufescens) and also evaluated the cross-amplification of these loci and other ten loci previously developed for the great tinamou (Tinamus major) in other tinamous. Genetic variability was assessed using 24 individuals. Six loci were polymorphic with moderate to high number of alleles per locus (2-12 alleles) and showed expected heterozygosity (HE) ranging from 0.267 to 0.860. All loci conformed to the Hardy-Weinberg expectation and linkage disequilibrium was not significant for any pair of loci. This battery of polymorphic loci showed high paternity exclusion probability (0.986) and low genetic identity probability (4.95 × 10(-5)), proving to be helpful for parentage tests and population analyses in the red-winged tinamou. The cross-amplification was moderate where of the 160 locus/taxon combinations, 46 (28.75%) successfully amplified.

Development of a recombinase polymerase amplification assay for the detection of pathogenic Leptospira.

PubMed

Ahmed, Ahmed; van der Linden, Hans; Hartskeerl, Rudy A

2014-05-08

Detection of leptospires based on DNA amplification techniques is essential for the early diagnosis of leptospirosis when anti-Leptospira antibodies are below the detection limit of most serological tests. In middle and low income countries where leptospirosis is endemic, routine implementation of real-time PCR is financially and technically challenging due to the requirement of expensive thermocycler equipment. In this study we report the development and evaluation of a novel isothermal recombinase polymerase amplification assay (RPA) for detection of pathogenic Leptospira based on TwistAmp chemistry. RPA enabled the detection of less than two genome copies per reaction. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity and specificity of 94.7% and 97.7%, respectively) compared to culturing as the reference standard. RPA presents a powerful tool for the early diagnosis of leptospirosis in humans and in animals. Furthermore, it enables the detection of the causative agent in reservoirs and environment, and as such is a valuable adjunct to current tools for surveillance and early outbreak warning.

Development of a Recombinase Polymerase Amplification Assay for the Detection of Pathogenic Leptospira

PubMed Central

Ahmed, Ahmed; van der Linden, Hans; Hartskeerl, Rudy A.

2014-01-01

Detection of leptospires based on DNA amplification techniques is essential for the early diagnosis of leptospirosis when anti-Leptospira antibodies are below the detection limit of most serological tests. In middle and low income countries where leptospirosis is endemic, routine implementation of real-time PCR is financially and technically challenging due to the requirement of expensive thermocycler equipment. In this study we report the development and evaluation of a novel isothermal recombinase polymerase amplification assay (RPA) for detection of pathogenic Leptospira based on TwistAmp chemistry. RPA enabled the detection of less than two genome copies per reaction. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity and specificity of 94.7% and 97.7%, respectively) compared to culturing as the reference standard. RPA presents a powerful tool for the early diagnosis of leptospirosis in humans and in animals. Furthermore, it enables the detection of the causative agent in reservoirs and environment, and as such is a valuable adjunct to current tools for surveillance and early outbreak warning. PMID:24814943

Quenching of unincorporated amplification signal reporters in reverse-transcription loop-mediated isothermal amplification enabling bright, single-step, closed-tube, and multiplexed detection of RNA viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ball, Cameron S.; Light, Yooli K.; Koh, Chung -Yan

Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of themore » reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read “quasar”), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). As a result, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.« less

Sex and PRNP genotype determination in preimplantation caprine embryos.

PubMed

Guignot, F; Perreau, C; Cavarroc, C; Touzé, J-L; Pougnard, J-L; Dupont, F; Beckers, J-F; Rémy, B; Babilliot, J-M; Bed'Hom, B; Lamorinière, J M; Mermillod, P; Baril, G

2011-08-01

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo. © 2010 Blackwell Verlag GmbH.

Profiling In Situ Microbial Community Structure with an Amplification Microarray

PubMed Central

Knickerbocker, Christopher; Bryant, Lexi; Golova, Julia; Wiles, Cory; Williams, Kenneth H.; Peacock, Aaron D.; Long, Philip E.

2013-01-01

The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO3−) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures. The succession of the microbial community was evident with and consistent between the two microarray platforms. Amplification microarray analysis of acetate-treated groundwater showed elevated levels of iron-reducing bacteria (Flexibacter, Geobacter, Rhodoferax, and Shewanella) relative to the average background profile, as expected. Identical molecular signatures were evident in the transect treated with acetate plus NaHCO3, but at much lower signal intensities and with a much more rapid decline (to nondetection). Azoarcus, Thaurea, and Methylobacterium were responsive in the acetate-only transect but not in the presence of bicarbonate. Observed differences in microbial community composition or response to bicarbonate amendment likely had an effect on measured rates of U reduction, with higher rates probable in the part of the field experiment that was amended with bicarbonate. The simplification in microarray-based work flow is a significant technological advance toward entirely closed-amplicon microarray-based tests and is generally extensible to any number of environmental monitoring applications. PMID:23160129

Quenching of unincorporated amplification signal reporters in reverse-transcription loop-mediated isothermal amplification enabling bright, single-step, closed-tube, and multiplexed detection of RNA viruses

DOE PAGES

Ball, Cameron S.; Light, Yooli K.; Koh, Chung -Yan; ...

2016-03-16

Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of themore » reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read “quasar”), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). As a result, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.« less

Continuous in vitro evolution of catalytic function.

PubMed

Wright, M C; Joyce, G F

1997-04-25

A population of RNA molecules that catalyze the template-directed ligation of RNA substrates was made to evolve in a continuous manner in the test tube. A simple serial transfer procedure was used to achieve approximately 300 successive rounds of catalysis and selective amplification in 52 hours. During this time, the population size was maintained against an overall dilution of 3 x 10(298). Both the catalytic rate and amplification rate of the RNAs improved substantially as a consequence of mutations that accumulated during the evolution process. Continuous in vitro evolution makes it possible to maintain laboratory "cultures" of catalytic molecules that can be perpetuated indefinitely.

Continuous in vitro evolution of catalytic function

NASA Technical Reports Server (NTRS)

Wright, M. C.; Joyce, G. F.

1997-01-01

A population of RNA molecules that catalyze the template-directed ligation of RNA substrates was made to evolve in a continuous manner in the test tube. A simple serial transfer procedure was used to achieve approximately 300 successive rounds of catalysis and selective amplification in 52 hours. During this time, the population size was maintained against an overall dilution of 3 x 10(298). Both the catalytic rate and amplification rate of the RNAs improved substantially as a consequence of mutations that accumulated during the evolution process. Continuous in vitro evolution makes it possible to maintain laboratory "cultures" of catalytic molecules that can be perpetuated indefinitely.

Validation of a fully automated HER2 staining kit in breast cancer.

PubMed

Moelans, Cathy B; Kibbelaar, Robby E; van den Heuvel, Marius C; Castigliego, Domenico; de Weger, Roel A; van Diest, Paul J

2010-01-01

Testing for HER2 amplification and/or overexpression is currently routine practice to guide Herceptin therapy in invasive breast cancer. At present, HER2 status is most commonly assessed by immunohistochemistry (IHC). Standardization of HER2 IHC assays is of utmost clinical and economical importance. At present, HER2 IHC is most commonly performed with the HercepTest which contains a polyclonal antibody and applies a manual staining procedure. Analytical variability in HER2 IHC testing could be diminished by a fully automatic staining system with a monoclonal antibody. 219 invasive breast cancers were fully automatically stained with the monoclonal antibody-based Oracle HER2 Bond IHC kit and manually with the HercepTest. All cases were tested for amplification with chromogenic in situ hybridization (CISH). HercepTest yielded an overall sharper membrane staining, with less cytoplasmic and stromal background than Oracle in 17% of cases. Overall concordance between both IHC techniques was 89% (195/219) with a kappa value of 0.776 (95% CI 0.698-0.854), indicating a substantial agreement. Most (22/24) discrepancies between HercepTest and Oracle showed a weaker staining for Oracle. Thirteen of the 24 discrepant cases were high-level HER2 amplified by CISH, and in 12 of these HercepTest IHC better reflected gene amplification status. All the 13 HER2 amplified discrepant cases were at least 2+ by HercepTest, while 10/13 of these were at least 2+ for Oracle. Considering CISH as gold standard, sensitivity of HercepTest and Oracle was 91% and 83%, and specificity was 94% and 98%, respectively. Positive and negative predictive values for HercepTest and Oracle were 90% and 95% for HercepTest and 96% and 91% for Oracle, respectively. Fully-automated HER2 staining with the monoclonal antibody in the Oracle kit shows a high level of agreement with manual staining by the polyclonal antibody in the HercepTest. Although Oracle shows in general some more cytoplasmic staining and may be slightly less sensitive in picking up HER2 amplified cases, it shows a higher specificity and may be considered as an alternative method to evaluate the HER2 expression in breast cancer with potentially less analytical variability.

A silent allele in the locus D5S818 contained within the PowerPlex®21 PCR Amplification Kit.

PubMed

Chen, Ling; Tai, Yunchun; Qiu, Pingming; Du, Weian; Liu, Chao

2015-11-01

Three paternity tests cases were found with a single locus mismatch at the locus D5S818 with PowerPlex®21 PCR Amplification Kit (Promega). Forward and reverse primers were redesigned to type the samples again and to evaluate if there were alleles dropped out. The results showed the existence of a silent allele 12 in all the three families, due to a point mutation that changed cytosine to adenine at 90 nucleotides upstream from the 5' end of the AGAT repeat sequences in all the six individuals. A single locus mismatch due to a silent allele may occur in any locus using any kit. Therefore, we recommend using multiple kits to confirm the results in paternity testing cases with mismatches, especially when there is a single locus mismatch with homozygote involved. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khalili, Farid; Danilishin, Stefan; Mueller-Ebhardt, Helge

We consider enhancing the sensitivity of future gravitational-wave detectors by using double optical spring. When the power, detuning and bandwidth of the two carriers are chosen appropriately, the effect of the double optical spring can be described as a 'negative inertia', which cancels the positive inertia of the test masses and thus increases their response to gravitational waves. This allows us to surpass the free-mass standard quantum limit (SQL) over a broad frequency band, through signal amplification, rather than noise cancellation, which has been the case for all broadband SQL-beating schemes so far considered for gravitational-wave detectors. The merit ofmore » such signal amplification schemes lies in the fact that they are less susceptible to optical losses than noise-cancellation schemes. We show that it is feasible to demonstrate such an effect with the Gingin High Optical Power Test Facility, and it can eventually be implemented in future advanced GW detectors.« less

Miniaturized isothermal nucleic acid amplification, a review.

PubMed

Asiello, Peter J; Baeumner, Antje J

2011-04-21

Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.

Sentence recognition in noise and perceived benefit of noise reduction on the receiver and transmitter sides of a BICROS hearing aid.

PubMed

Oeding, Kristi; Valente, Michael

2013-01-01

In the past, bilateral contralateral routing of signals (BICROS) amplification incorporated omnidirectional microphones on the transmitter and receiver sides and some models utilized noise reduction (NR) on the receiver side. Little research has examined the performance of BICROS amplification in background noise. However, previous studies examining contralateral routing of signals (CROS) amplification have reported that the presence of background noise on the transmitter side negatively affected speech recognition. Recently, NR was introduced as a feature on the receiver and transmitter sides of BICROS amplification, which has the potential to decrease the impact of noise on the wanted speech signal by decreasing unwanted noise directed to the transmitter side. The primary goal of this study was to examine differences in the reception threshold for sentences (RTS in dB) using the Hearing in Noise Test (HINT) in a diffuse listening environment between unaided and three aided BICROS conditions (no NR, mild NR, and maximum NR) in the Tandem 16 BICROS. A secondary goal was to examine real-world subjective impressions of the Tandem 16 BICROS compared to unaided. A randomized block repeated measures single blind design was used to assess differences between no NR, mild NR, and maximum NR listening conditions. Twenty-one adult participants with asymmetric sensorineural hearing loss (ASNHL) and experience with BICROS amplification were recruited from Washington University in St. Louis School of Medicine. Participants were fit with the National Acoustic Laboratories' Nonlinear version 1 prescriptive target (NAL-NL1) with the Tandem 16 BICROS at the initial visit and then verified using real-ear insertion gain (REIG) measures. Participants acclimatized to the Tandem 16 BICROS for 4 wk before returning for final testing. Participants were tested utilizing HINT sentences examining differences in RTS between unaided and three aided listening conditions. Subjective benefit was determined via the Abbreviated Profile of Hearing Aid Benefit (APHAB) questionnaire between the Tandem 16 BICROS and unaided. A repeated measures analysis of variance (ANOVA) was utilized to analyze the results of the HINT and APHAB. Results revealed no significant differences in the RTS between unaided, no NR, mild NR, and maximum NR. Subjective impressions using the APHAB revealed statistically and clinically significant benefit with the Tandem 16 BICROS compared to unaided for the Ease of Communication (EC), Background Noise (BN), and Reverberation (RV) subscales. The RTS was not significantly different between unaided, no NR, mild NR, and maximum NR. None of the three aided listening conditions were significantly different from unaided performance as has been reported for previous studies examining CROS hearing aids. Further, based on comments from participants and previous research studies with conventional hearing aids, manufacturers of BICROS amplification should consider incorporating directional microphones and independent volume controls on the receiver and transmitter sides to potentially provide further improvement in signal-to-noise ratio (SNR) for patients with ASNHL. American Academy of Audiology.

The use of acid phosphatase test papers for DNA profiling.

PubMed

Reshef, A; Barash, M; Gallili, N; Michael, A; Brauner, P

2005-01-01

The acid phosphatase (AP) test is a routine assay used to screen casework items for the possible presence of semen. This colour test is carried out on filter paper which is retained after testing. Two-year-old AP test papers were found to contain sufficient DNA for short tandem repeat (STR) profiling. Prior to polymerase chain reaction (PCR) amplification, the DNA was preferentially separated into sperm depleted and sperm enriched cell fractions. The implication of these findings for past and present cases is discussed.

[Companion diagnostics in the era of personalized medicine--chairmen's introductory remarks].

PubMed

Fukutsuka, Katsuhiro; Takubo, Takayuki

2014-04-01

Personalized medicine is a medical model that proposes the customization of treatment for individual patients. In this model, diagnostic tests are essential for selecting safer and more efficacious treatments. The term "companion diagnostics" has been used to describe these tests, whereby molecular assays that measure the levels of proteins or specific gene mutations are used to provide a specific therapy for an individual by stratifying the disease status, selecting the proper medication, and tailoring dosages. Examples of companion diagnostics in the field of cancer medicine for molecular targeted therapy include tests for the ALK-fusion gene in non-small cell lung cancer and expression of CCR4 in adult T-cell leukemia. For breast cancer, the expression of HER2 protein is evaluated by immunohistochemistry (IHC), and gene amplification of HER2 is tested by fluorescence in situ hybridization (FISH); both tests consist of pre-analysis, analysis, and post-analysis processes that require quality control to ensure the reliability of the results. This symposium includes: 1) future aspects of companion diagnostics addressing many of the problems that must be overcome, 2) companion diagnostics using FISH focusing on HER2 amplification and ALK alteration, 3) newly developed diagnostic tests using tumor specimens and cell-free DNA in serum, and 4) CCR4 expression detected by IHC and flow cytometry.

[Automated RNA amplification for the rapid identification of Mycobacterium tuberculosis complex in respiratory specimens].

PubMed

Drouillon, V; Houriez, F; Buze, M; Lagrange, P; Herrmann, J-L

2006-01-01

Rapid and sensitive detection of Mycobacterium tuberculosis complex (MTB) directly on clinical respiratory specimens is essential for a correct management of patients suspected of tuberculosis. For this purpose PCR-based kits are available to detect MTB in respiratory specimen but most of them need at least 4 hours to be completed. New methods, based on TRC method (TRC: Transcription Reverse transcription Concerted--TRCRapid M. Tuberculosis--Tosoh Bioscience, Tokyo, Japon) and dedicated monitor have been developed. A new kit (TRC Rapid M. tuberculosis and Real-time monitor TRCRapid-160, Tosoh Corporation, Japan) enabling one step amplification and real-time detection of MTB 16S rRNA by a combination of intercalative dye oxazole yellow-linked DNA probe and isothermal RNA amplification directly on respiratory specimens has been tested in our laboratory. 319 respiratory specimens were tested in this preliminary study and results were compared to smear and culture. Fourteen had a positive culture for MTB. Among theses samples, smear was positive in 11 cases (78.6%) and TRC process was positive in 8 cases (57.1%). Overall sensitivity of TRC compared to smear positive samples is 73%. Theses first results demonstrated that a rapid identification of MTB was possible (less than 2 processing hours for 14 specimens and about 1 hour for 1 specimen) in most cases of smear positive samples using ready to use reagents for real time detection of MTB rRNA in clinical samples. New pretreatment and extraction reagents kits to increase the stability of the sputum RNA and the extraction efficiency are now tested in our laboratory.

A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA.

PubMed

Ramos, Andrea Estefanía; Muñoz, Marina; Cortés-Vecino, Jesús Alfredo; Barato, Paola; Patarroyo, Manuel Alfonso

2017-11-29

Neospora caninum is a cyst-forming, coccidian parasite which is known to cause neurological disorders in dogs and abortion and neonatal mortality in cows and other livestock. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay based on the Neospora caninum Nc-5 gene and compares its efficacy for detecting DNA to that of a semi-nested PCR test. Six primers were designed based on the Nc-5 repeat region of N. caninum. Specific LAMP primers led to successful amplification of N. caninum DNA at 63 °C in 30 min. The LAMP assay was highly specific (i.e. it did not reveal cross-reactivity with other parasite species) and had a low N. caninum plasmid DNA limit of detection (1 fg), which is ten times higher than that for the semi-nested PCR. LAMP applicability was evaluated using a set of naturally-infected samples (59 from canine faeces and five from bovine abortions). Thirty-nine percent (25/64) of the naturally-infected samples were positive for N. caninum DNA by LAMP and 36% (23/64) by semi-nested PCR. However, the LAMP assay is much faster to perform than semi-nested PCR and provides results in 30 min. The optimized reaction conditions described in this study resulted in a sensitive, specific and rapid technique for detecting N. caninum DNA. Considering the advantages of LAMP for detecting N. caninum DNA, further assays aimed at testing its usefulness on a wider range of field samples are recommended.

HER2 and TOP2A amplification in a hospital-based cohort of breast cancer patients: associations with patient and tumor characteristics.

PubMed

Fasching, Peter A; Weihbrecht, Sebastian; Haeberle, Lothar; Gasparyan, Armen; Villalobos, Ivonne E; Ma, Yanling; Ekici, Arif B; Wachter, David L; Hartmann, Arndt; Beckmann, Matthias W; Slamon, Dennis J; Press, Michael F

2014-05-01

Gene amplification is an important factor for altered gene expression in breast cancers. TOP2A-amplification often occurs concomitantly with HER2 amplification, and it has been suggested to be predictive for the response to anthracycline chemotherapy. This study assessed the correlation between HER2 status and TOP2A co-amplification, the possible association of TOP2A single-nucleotide polymorphisms with the frequency of this co-amplification as well as confirmation of association with outcome. HER2 and TOP2A amplification were analyzed in a tissue microarray from a clinical cohort study. Additionally, a common genetic variant (rs13695) in the TOP2A gene was genotyped in germline DNA. HER2 gene amplification was compared with HER2-IHC findings assessed during clinical routine work, and the association between all the biomarkers analyzed and the clinical outcome was determined. As an exploratory aim, rs13695 genotypes were compared with TOP2A amplification status. HER2 amplification was seen in 101 of 628 (16.1 %) and TOP2A amplification in 32 (5.1 %) cancers. No TOP2A amplification occurred without HER2 co-amplification. HER2 amplification was found in 8, 13.6, and 55.1 % of patients with HER2-IHC 0/1+, 2+, and 3+ tumors, respectively. HER2-IHC was not associated with an effect on the prognosis, but HER2-FISH was. There was an association between the rs13695 genotype and TOP2A amplification status (P = 0.03). Although there was a significant correlation between HER2 status determined by IHC and HER2 by FISH, only HER2 gene amplification status by FISH was correlated with outcome indicating greater utility for FISH in routine clinical settings.

Diagnosis and treatment of histoplasmosis in solid organ transplant patients.

PubMed

Gajurel, Kiran; Dhakal, Reshika; Deresinski, Stan

2018-05-05

Unlike immunocompetent hosts, solid organ transplant (SOT) recipients with posttransplant histoplasmosis (PTH) often present with disseminated disease and have an attributable mortality of approximately 10%. In this review, we discuss currently available diagnostic tests and treatment strategies in PTH. None of the available tests have a 100% diagnostic accuracy. Histoplasma antigen assays are the most sensitive commercially available tests. However, crossreactivity of histoplasma antigen with aspergillus galactomannan and false positive histoplasma antigen tests because of rabbit antithymocyte globulin may cause difficulty in interpreting positive test results in transplant recipients. Molecular assays such as amplification and sequencing of 'panfungal' portions of the 28S ribosomal RNA from clinical specimens appear to be promising.Lipid formulations of amphotericin B and itraconazole are the drugs of choice in the treatment of PTH. Other extended spectrum azoles also appear to be effective, but, like itraconazole, problems with drug interactions and prolongation of the QTc interval (except for isavuconazole, which shortens the QTc interval) remain. Mycophenolate therapy is associated with severe disease and should be stopped during active disease and, if feasible, calcineurin inhibitors and steroids should be reduced. A combination of various tests (culture, antigen tests, nucleic amplification tests, etc.) should be used to optimize diagnostic yield. The role of unbiased next generation sequencing for early diagnosis and newer azoles in the treatment needs to be further explored.

Diagnostic performance of a novel loop-mediated isothermal amplification (LAMP) assay targeting the apicoplast genome for malaria diagnosis in a field setting in sub-Saharan Africa.

PubMed

Oriero, Eniyou C; Okebe, Joseph; Jacobs, Jan; Van Geertruyden, Jean-Pierre; Nwakanma, Davis; D'Alessandro, Umberto

2015-10-09

New diagnostic tools to detect reliably and rapidly asymptomatic and low-density malaria infections are needed as their treatment could interrupt transmission. Isothermal amplification techniques are being explored for field diagnosis of malaria. In this study, a novel molecular tool (loop-mediated isothermal amplification-LAMP) targeting the apicoplast genome of Plasmodium falciparum was evaluated for the detection of asymptomatic malaria-infected individuals in a rural setting in The Gambia. A blood was collected from 341 subjects (median age 9 years, range 1-68 years) screened for malaria. On site, a rapid diagnostic test (RDT, SD Bioline Malaria Antigen P.f) was performed, thick blood films (TBF) slides for microscopy were prepared and dry blood spots (DBS) were collected on Whatman(®) 903 Specimen collection paper. The TBF and DBS were transported to the field laboratory where microscopy and LAMP testing were performed. The latter was done on DNA extracted from the DBS using a crude (methanol/heating) extraction method. A laboratory-based PCR amplification was done on all the samples using DNA extracted with the Qiagen kit and its results were taken as reference for all the other tests. Plasmodium falciparum malaria prevalence was 37 % (127/341) as detected by LAMP, 30 % (104/341) by microscopy and 37 % (126/341) by RDT. Compared to the reference PCR method, sensitivity was 92 % for LAMP, 78 % for microscopy, and 76 % for RDT; specificity was 97 % for LAMP, 99 % for microscopy, and 88 % for RDT. Area under the receiver operating characteristic (ROC) curve in comparison with the reference standard was 0.94 for LAMP, 0.88 for microscopy and 0.81 for RDT. Turn-around time for the entire LAMP assay was approximately 3 h and 30 min for an average of 27 ± 9.5 samples collected per day, compared to a minimum of 10 samples an hour per operator by RDT and over 8 h by microscopy. The LAMP assay could produce reliable results the same day of the screening. It could detect a higher proportion of low density malaria infections than the other methods tested and may be used for large campaigns of systematic screening and treatment.

Accurate clinical genetic testing for autoinflammatory diseases using the next-generation sequencing platform MiSeq.

PubMed

Nakayama, Manabu; Oda, Hirotsugu; Nakagawa, Kenji; Yasumi, Takahiro; Kawai, Tomoki; Izawa, Kazushi; Nishikomori, Ryuta; Heike, Toshio; Ohara, Osamu

2017-03-01

Autoinflammatory diseases occupy one of a group of primary immunodeficiency diseases that are generally thought to be caused by mutation of genes responsible for innate immunity, rather than by acquired immunity. Mutations related to autoinflammatory diseases occur in 12 genes. For example, low-level somatic mosaic NLRP3 mutations underlie chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID). In current clinical practice, clinical genetic testing plays an important role in providing patients with quick, definite diagnoses. To increase the availability of such testing, low-cost high-throughput gene-analysis systems are required, ones that not only have the sensitivity to detect even low-level somatic mosaic mutations, but also can operate simply in a clinical setting. To this end, we developed a simple method that employs two-step tailed PCR and an NGS system, MiSeq platform, to detect mutations in all coding exons of the 12 genes responsible for autoinflammatory diseases. Using this amplicon sequencing system, we amplified a total of 234 amplicons derived from the 12 genes with multiplex PCR. This was done simultaneously and in one test tube. Each sample was distinguished by an index sequence of second PCR primers following PCR amplification. With our procedure and tips for reducing PCR amplification bias, we were able to analyze 12 genes from 25 clinical samples in one MiSeq run. Moreover, with the certified primers designed by our short program-which detects and avoids common SNPs in gene-specific PCR primers-we used this system for routine genetic testing. Our optimized procedure uses a simple protocol, which can easily be followed by virtually any office medical staff. Because of the small PCR amplification bias, we can analyze simultaneously several clinical DNA samples with low cost and can obtain sufficient read numbers to detect a low level of somatic mosaic mutations.

Comparative application of IS711-based polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for canine brucellosis diagnosis.

PubMed

Batinga, Maria Cryskely Agra; de Lima, Julia Teresa Ribeiro; Gregori, Fabio; Diniz, Jaqueline Assumpção; Muner, Kerstin; Oliveira, Trícia M F S; Ferreira, Helena Lage; Soares, Rodrigo Martins; Keid, Lara Borges

2018-06-01

Canine brucellosis is caused by Brucella canis, a gram negative and facultative intracellular bacterium that is commonly associated with reproductive failures in dogs. The accurate diagnosis of the infection relies on the use of serological tests associated with blood culturing to guarantee sensitivity. The polymerase chain reaction (PCR) can replace the culturing procedure for the direct diagnosis of the infection because of its speed, high specificity and sensitivity values; however, it depends on some laboratory infrastructure to be conducted. The loop-mediated isothermal amplification (LAMP) may be an alternative method for DNA amplification in a shorter period, using simpler equipment, and with a lower cost. This study evaluated the potential of molecular tools based on PCR and LAMP using primers targeting the insertion sequence IS711 for Brucella detection in three groups of dogs (infected, non-infected and suspected of brucellosis), which were determined according to the results of blood culturing and clinical examination. The performance of the three diagnostic tests was also determined using McNemar test and Kappa coefficient. The proportion of positive samples detected by blood culturing, PCR and LAMP was respectively 31.57% (18/57), 33.34% (19/57), and 14.03% (8/57). The agreement between blood culturing and PCR was almost perfect, while the agreement of PCR and blood culturing compared to LAMP was fair. The diagnostic sensitivity of PCR and LAMP was respectively 100% (18/18) and 44.44% (8/18), while the diagnostic specificity of both tests was 100% (21/21). LAMP performance was not satisfactory for canine brucellosis diagnosis because of the low diagnostic sensitivity of the test. The IS711 based PCR, otherwise, showed high values of sensitivity and specificity, which makes it a good alternative for use for the rapid diagnosis of canine brucellosis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Evaluation of HER-2/neu gene amplification and overexpression: comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens.

PubMed

Press, Michael F; Slamon, Dennis J; Flom, Kerry J; Park, Jinha; Zhou, Jian-Yuan; Bernstein, Leslie

2002-07-15

To compare and evaluate HER-2/neu clinical assay methods. One hundred seventeen breast cancer specimens with known HER-2/neu amplification and overexpression status were assayed with four different immunohistochemical assays and two different fluorescence in situ hybridization (FISH) assays. The accuracy of the FISH assays for HER-2/neu gene amplification was high, 97.4% for the Vysis PathVision assay (Vysis, Inc, Downers Grove, IL) and 95.7% for the the Ventana INFORM assay (Ventana, Medical Systems, Inc, Tucson, AZ). The immunohistochemical assay with the highest accuracy for HER-2/neu overexpression was obtained with R60 polyclonal antibody (96.6%), followed by immunohistochemical assays performed with 10H8 monoclonal antibody (95.7%), the Ventana CB11 monoclonal antibody (89.7%), and the DAKO HercepTest (88.9%; Dako, Corp, Carpinteria, CA). Only the sensitivities, and therefore, overall accuracy, of the DAKO Herceptest and Ventana CB11 immunohistochemical assays were significantly different from the more sensitive FISH assay. Based on these findings, the FISH assays were highly accurate, with immunohistochemical assays performed with R60 and 10H8 nearly as accurate. The DAKO HercepTest and the Ventana CB11 immunohistochemical assay were statistically significantly different from the Vysis FISH assay in evaluating these previously molecularly characterized breast cancer specimens.

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens

PubMed Central

Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert

2018-01-01

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology. PMID:29425124

Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

PubMed

Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

2016-10-01

Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens.

PubMed

Higgins, Owen; Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert; Smith, Terry J

2018-02-09

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae , Neisseria meningitidis and Haemophilus influenzae . Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae , N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

Pulsatile plasma filtration and cell-free DNA amplification using a water-head-driven point-of-care testing chip.

PubMed

Lee, Yonghun; Kim, Dong-Min; Li, Zhenglin; Kim, Dong-Eun; Kim, Sung-Jin

2018-03-13

We demonstrate a microfiltration chip that separates blood plasma by using water-head-driven pulsatile pressures rather than any external equipment and use it for on-chip amplification of nucleic acids. The chip generates pulsatile pressures to significantly reduce filter clogging without hemolysis, and consists of an oscillator, a plasma-extraction pump, and filter units. The oscillator autonomously converts constant water-head pressure to pulsatile pressure, and the pump uses the pulsatile pressure to extract plasma through the filter. Because the pulsatile pressure can periodically clear blood cells from the filter surface, filter clogging can be effectively reduced. In this way, we achieve plasma extraction with 100% purity and 90% plasma recovery at 15% hematocrit. During a 10 min period, the volume of plasma extracted was 43 μL out of a 243 μL extraction volume at 15% hematocrit. We also studied the influence of the pore size and diameter of the filter, blood loading volume, oscillation period, and hematocrit level on the filtration performance. To demonstrate the utility of our chip for point-of-care testing (POCT) applications, we successfully implemented on-chip amplification of a nucleic acid (miDNA21) in plasma filtered from blood. We expect our chip to be useful not only for POCT applications but also for other bench-top analysis tools using blood plasma.

Gortler vortices and transition in wall boundary layers of two Mach 5 nozzles

NASA Technical Reports Server (NTRS)

Beckwith, I. E.; Holley, B. B.

1981-01-01

The onset of transition in the wall boundary layers of two axisymmetric Mach 5 wind-tunnel nozzles has been measured under conditions of extremely low incident disturbance levels. The range of test unit Reynolds numbers, based on conditions at the nozzle exit, was from 6 x 10 to the 6th power m to 2.5 x 10 to the 7th power m. When the nozzle walls were maintained in a polished and clean condition, transition moved gradually upstream as the test Reynolds number was increased. When transition occurred in the supersonic concave wall region, the values of the local Gortler parameter at transition varied from about 5 to 6, whereas the momentum thickness Reynolds number varied from about 750 to 1050. Oil flow patterns obtained near the exit of the nozzles indicated that Gortler vortices were always present when the wall boundary layers were laminar. Calculations for the growth of Gortler vortices based on new results from linear theory for supersonic flat-plate profiles gave amplification ratios to transition from e to the 4th power to e to the 15th power. Possible reasons for this wide range in amplification ratios are discussed, but no definite conclusions are yet possible regarding the values of n in a simple e to the nth power type theory for the assumed linear amplification of Gortler vortices to transition in supersonic nozzles.

Dynamics and control of DNA sequence amplification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marimuthu, Karthikeyan; Chakrabarti, Raj, E-mail: raj@pmc-group.com, E-mail: rajc@andrew.cmu.edu; Division of Fundamental Research, PMC Advanced Technology, Mount Laurel, New Jersey 08054

2014-10-28

DNA amplification is the process of replication of a specified DNA sequence in vitro through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reactionmore » are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal temperature profile. Strategies for the optimal synthesis of the DNA amplification control trajectory are proposed. Analogous methods can be used to formulate control problems for more advanced amplification objectives corresponding to the design of new types of DNA amplification reactions.« less

Methylation-sensitive amplification polymorphism analysis of fat and muscle tissues in pigs.

PubMed

Ma, J D; Li, M Z; Zhou, S L; Zhou, C W; Li, X W

2012-09-26

DNA methylation may be involved in regulating the expression of protein-coding genes, resulting in different fat and muscle phenotypes. Using a methylation-sensitive amplified polymorphism approach, we obtained 7423 bands by selective amplification of genomic DNA from six different fat depots and two heterogeneous muscle types from Duroc/Landrace/Yorkshire cross-bred pigs. The degrees of DNA methylation, determined by the percentages of hemi- and fully methylated sites relative to the total number of CCGG sites, were similar in male and female pigs for each specific tissue [χ(2) test; P (two-tailed) > 0.05]. Gender bias was therefore ignored. There were significant differences in the degree of DNA methylation among the eight tissue types [χ(2) test; P(total) (two-tailed) = 0.009]. However, similar degrees of methylation were observed among the six fat depots [χ(2) test; P(fat) (two-tailed) = 0.24 > 0.05]and between the two muscle types [χ(2) test; P(muscle) (two-tailed) = 0.76 > 0.05]. We conclude that the degree of DNA methylation differs between porcine fat and muscle tissue, but that the methylation status of a particular tissue type is similar, despite being deposited at different body sites.

Determining putative vectors of the Bogia Coconut Syndrome phytoplasma using loop-mediated isothermal amplification of single-insect feeding media

PubMed Central

Lu, Hengyu; Wilson, Bree A. L.; Ash, Gavin J.; Woruba, Sharon B.; Fletcher, Murray J.; You, Minsheng; Yang, Guang; Gurr, Geoff M.

2016-01-01

Phytoplasmas are insect vectored mollicutes responsible for disease in many economically important crops. Determining which insect species are vectors of a given phytoplasma is important for managing disease but is methodologically challenging because disease-free plants need to be exposed to large numbers of insects, often over many months. A relatively new method to detect likely transmission involves molecular testing for phytoplasma DNA in sucrose solution that insects have fed upon. In this study we combined this feeding medium method with a loop-mediated isothermal amplification (LAMP) assay to study 627 insect specimens of 11 Hemiptera taxa sampled from sites in Papua New Guinea affected by Bogia coconut syndrome (BCS). The LAMP assay detected phytoplasma DNA from the feeding solution and head tissue of insects from six taxa belonging to four families: Derbidae, Lophopidae, Flatidae and Ricaniidae. Two other taxa yielded positives only from the heads and the remainder tested negative. These results demonstrate the utility of combining single-insect feeding medium tests with LAMP assays to identify putative vectors that can be the subject of transmission tests and to better understand phytoplasma pathosystems. PMID:27786249

Comparison of Three Different Sensitive Assays for Hepatitis B Virus DNA in Monitoring of Responses to Antiviral Therapy

PubMed Central

Chan, Henry L. Y.; Leung, Nancy W. Y.; Lau, Tracy C. M.; Wong, May L.; Sung, Joseph J. Y.

2000-01-01

The aim of our study was to compare the performances of two new hepatitis B virus (HBV) DNA assays, a cross-linking assay (NAXCOR) and a hybrid-capture amplification assay (Digene), versus the widely used branched-DNA (bDNA) assay (Chiron) in the monitoring of HBV DNA levels during antiviral treatment. Serial serum samples from 12 chronically HBV infected patients undergoing a phase II trial of an antiviral drug, 2′,3′-dideoxy-5-fluoro-3′-thiacytidine (FTC), were studied. A total of 96 serum samples were tested for HBV DNA using the cross-linking, hybrid-capture amplification, and bDNA assays. In the comparison of the cross-linking and bDNA assays, concordant results were found in 77 (80.3%) samples, no significant difference was found between the median log10 HBV DNA levels (6.66 versus 7.17 meq/ml), and the results of the two assays were closely correlated (r = 0.95). In the comparison of the hybrid-capture amplification and bDNA assays, concordant results were found in 79 (82.3%) samples, no significant difference was found between the median log10 HBV DNA levels (6.98 versus 6.99 meq/ml), and the results of the two assays were closely correlated (r = 0.99). Six (6.3%) samples by the cross-linking assay and 10 (10.4%) samples by the bDNA assay required retesting because of unacceptably high within-run coefficients of variance. No sample required retesting in the hybrid-capture amplification assay according to the internal validation. In conclusion, the cross-linking and hybrid-capture amplification assays were as sensitive as the bDNA assay for HBV DNA detection and can be recommended for monitoring of HBV DNA levels during antiviral treatment. PMID:10970358

Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria.

PubMed

Lucchi, Naomi W; Demas, Allison; Narayanan, Jothikumar; Sumari, Deborah; Kabanywanyi, Abdunoor; Kachur, S Patrick; Barnwell, John W; Udhayakumar, Venkatachalam

2010-10-29

Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.

Stronger transferability but lower variability in transcriptomic- than in anonymous microsatellites: evidence from Hylid frogs.

PubMed

Dufresnes, Christophe; Brelsford, Alan; Béziers, Paul; Perrin, Nicolas

2014-07-01

A simple way to quickly optimize microsatellites in nonmodel organisms is to reuse loci available in closely related taxa; however, this approach can be limited by the stochastic and low cross-amplification success experienced in some groups (e.g. amphibians). An efficient alternative is to develop loci from transcriptome sequences. Transcriptomic microsatellites have been found to vary in their levels of cross-species amplification and variability, but this has to date never been tested in amphibians. Here, we compare the patterns of cross-amplification and levels of polymorphism of 18 published anonymous microsatellites isolated from genomic DNA vs. 17 loci derived from a transcriptome, across nine species of tree frogs (Hyla arborea and Hyla cinerea group). We established a clear negative relationship between divergence time and amplification success, which was much steeper for anonymous than transcriptomic markers, with half-lives (time at which 50% of the markers still amplify) of 1.1 and 37 My, respectively. Transcriptomic markers are significantly less polymorphic than anonymous loci, but remain variable across diverged taxa. We conclude that the exploitation of amphibian transcriptomes for developing microsatellites seems an optimal approach for multispecies surveys (e.g. analyses of hybrid zones, comparative linkage mapping), whereas anonymous microsatellites may be more informative for fine-scale analyses of intraspecific variation. Moreover, our results confirm the pattern that microsatellite cross-amplification is greatly variable among amphibians and should be assessed independently within target lineages. Finally, we provide a bank of microsatellites for Palaearctic tree frogs (so far only available for H. arborea), which will be useful for conservation and evolutionary studies in this radiation. © 2013 John Wiley & Sons Ltd.

Evaluation of four novel isothermal amplification assays towards simple and rapid genotyping of chloroquine resistant Plasmodium falciparum.

PubMed

Chahar, Madhvi; Anvikar, Anup; Dixit, Rajnikant; Valecha, Neena

2018-07-01

Loop mediated isothermal amplification (LAMP) assay is sensitive, prompt, high throughput and field deployable technique for nucleic acid amplification under isothermal conditions. In this study, we have developed and optimized four different visualization methods of loop-mediated isothermal amplification (LAMP) assay to detect Pfcrt K76T mutants of P. falciparum and compared their important features for one-pot in-field applications. Even though all the four tested LAMP methods could successfully detect K76T mutants of P. falciparum, however considering the time, safety, sensitivity, cost and simplicity, the malachite green and HNB based methods were found more efficient. Among four different visual dyes uses to detect LAMP products accurately, hydroxynaphthol blue and malachite green could produce long stable color change and brightness in a close tube-based approach to prevent cross-contamination risk. Our results indicated that the LAMP offers an interesting novel and convenient best method for the rapid, sensitive, cost-effective, and fairly user friendly tool for detection of K76T mutants of P. falciparum and therefore presents an alternative to PCR-based assays. Based on our comparative analysis, better field based LAMP visualization method can be chosen easily for the monitoring of other important drug targets (Kelch13 propeller region). Copyright © 2018 Elsevier Inc. All rights reserved.

Development of a quantitative fluorescence single primer isothermal amplification-based method for the detection of Salmonella.

PubMed

Wang, Jianchang; Li, Rui; Hu, Lianxia; Sun, Xiaoxia; Wang, Jinfeng; Li, Jing

2016-02-16

Food-borne disease caused by Salmonella has long been, and continues to be, an important global public health problem, necessitating rapid and accurate detection of Salmonella in food. Real time PCR is the most recently developed approach for Salmonella detection. Single primer isothermal amplification (SPIA), a novel gene amplification technique, has emerged as an attractive microbiological testing method. SPIA is performed under a constant temperature, eliminating the need for an expensive thermo-cycler. In addition, SPIA reactions can be accomplished in 30 min, faster than real time PCR that usually takes over 2h. We developed a quantitative fluorescence SPIA-based method for the detection of Salmonella. Using Salmonella Typhimurium genomic DNA as template and a primer targeting Salmonella invA gene, we showed the detection limit of SPIA was 2.0 × 10(1)fg DNA. Its successful amplification of different serotypic Salmonella genomic DNA but not non-Salmonella bacterial DNA demonstrated the specificity of SPIA. Furthermore, this method was validated with artificially contaminated beef. In conclusion, we showed high sensitivity and specificity of SPIA in the detection of Salmonella, comparable to real time PCR. In addition, SPIA is faster and more cost-effective (non-use of expensive cyclers), making it a potential alternative for field detection of Salmonella in resource-limited settings that are commonly encountered in developing countries. Copyright © 2015 Elsevier B.V. All rights reserved.

Fitting new technologies into the safety paradigm: use of microarrays in transfusion.

PubMed

Fournier-Wirth, C; Coste, J

2007-01-01

Until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. The recent emergence of Nucleic acid Amplification Technologies (NAT) has revolutionised viral diagnosis, not only by increasing the level of sensitivity but also by facilitating the detection of several viruses in parallel by multiplexing specific primers. In more complex biological situations, when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. High throughput systems, such as DNA Arrays, permit a conceptually new approach. These miniaturised micro systems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, a reduction in the number of confirmation tests and a simplification of data interpretation. However, the systems currently available require additional instrumentation and reagents for sample preparation and target amplification prior to detection on the DNA array. A major challenge in the area of DNA detection is the development of methods that do not rely on target amplification systems. Likewise, the advances of protein microarrays have lagged because of poor stability of proteins, complex coupling chemistry and weak detection signals. Emerging technologies like Biosensors and nano-particle based DNA or Protein Bio-Barcode Amplification Assays are promising diagnostic tools for a wide range of clinical applications, including blood donation screening.

Rapid detection of Porcine circovirus 2 by recombinase polymerase amplification.

PubMed

Wang, Jianchang; Wang, Jinfeng; Liu, Libing; Li, Ruiwen; Yuan, Wanzhe

2016-09-01

Porcine circovirus-associated disease, caused primarily by Porcine circovirus 2 (PCV-2), has become endemic in many pig-producing countries and has resulted in significant economic losses to the swine industry worldwide. Tests for PCV-2 infection include PCR, nested PCR, competitive PCR, and real-time PCR (rtPCR). Recombinase polymerase amplification (RPA) has emerged as an isothermal gene amplification technology for the molecular detection of infectious disease agents. RPA is performed at a constant temperature and therefore can be carried out in a water bath. In addition, RPA is completed in ~30 min, much faster than PCR, which usually takes >60 min. We developed a RPA-based method for the detection of PCV-2. The detection limit of RPA was 10(2) copies of PCV-2 genomic DNA. RPA showed the same sensitivity as rtPCR but was 10 times more sensitive than conventional PCR. Successful amplification of PCV-2 DNA, but not other viral templates, demonstrated high specificity of the RPA assay. This method was also validated using clinical samples. The results showed that the RPA assay had a diagnostic agreement rate of 93.7% with conventional PCR and 100% with rtPCR. These findings suggest that the RPA assay is a simple, rapid, and cost-effective method for PCV-2 detection, which could be potentially applied in clinical diagnosis and field surveillance of PCV-2 infection. © 2016 The Author(s).

Duplex recombinase polymerase amplification assays incorporating competitive internal controls for bacterial meningitis detection.

PubMed

Higgins, Owen; Clancy, Eoin; Forrest, Matthew S; Piepenburg, Olaf; Cormican, Martin; Boo, Teck Wee; O'Sullivan, Nicola; McGuinness, Claire; Cafferty, Deirdre; Cunney, Robert; Smith, Terry J

2018-04-01

Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technology that provides rapid and robust infectious disease pathogen detection, ideal for point-of-care (POC) diagnostics in disease-prevalent low-resource countries. We have developed and evaluated three duplex RPA assays incorporating competitive internal controls for the detection of leading bacterial meningitis pathogens. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae singleplex RPA assays were initially developed and evaluated, demonstrating 100% specificity with limits of detection of 4.1, 8.5 and 3.9 genome copies per reaction, respectively. Each assay was further developed into internally controlled duplex RPA assays via the incorporation of internal amplification control templates. Clinical performance of each internally controlled duplex RPA assay was evaluated by testing 64 archived PCR-positive clinical samples. Compared to real-time PCR, all duplex RPA assays demonstrated 100% diagnostic specificity, with diagnostic sensitivities of 100%, 86.3% and 100% for the S. pneumoniae, N. meningitidis and H. influenzae assays, respectively. This study details the first report of internally controlled duplex RPA assays for the detection of bacterial meningitis pathogens: S. pneumoniae, N. meningitidis and H. influenzae. We have successfully demonstrated the clinical diagnostic utility of each duplex RPA assay, introducing effective diagnostic technology for POC bacterial meningitis identification in disease-prevalent developing countries. Copyright © 2018 Elsevier Inc. All rights reserved.

Evaluation of Loop-Mediated Isothermal Amplification Suitable for Molecular Monitoring of Schistosome-Infected Snails in Field Laboratories

PubMed Central

Hamburger, Joseph; Abbasi, Ibrahim; Kariuki, Curtis; Wanjala, Atsabina; Mzungu, Elton; Mungai, Peter; Muchiri, Eric; King, Charles H.

2013-01-01

We previously described loop-mediated isothermal amplification (LAMP) for detection of Schistosoma haematobium and S. mansoni DNA in infected snails. In the present study, we adapted the LAMP assay for application in field laboratories in schistosomiasis-endemic areas. Isolation of DNA was simplified by blotting snail tissue (extracted in NaOH/sodium dodecyl sulfate) onto treated membranes, which enabled preservation at ambient temperatures. A ready-mix of LAMP reagents, suitable for shipment at ambient temperature and storage in minimal refrigeration, was used. Local survey teams without experience in molecular biology acquired operational expertise with this test within a few hours. Fifty-four field-caught snails were tested locally by LAMP and 59 were tested at similar conditions in Jerusalem. The LAMP results were consistent with those of a polymerase chain reaction; only four samples showed false-negative results. Results indicate that LAMP assays are suitable for detection of S. haematobium and S. mansoni in low-technology parasitology laboratories in which schistosomiasis elimination activities are undertaken. PMID:23208875

Chlamydia trachomatis infection positivity rates determined by nucleic acid amplification test in patients of hospitals in the northeastern region of Ukraine.

PubMed

Belozorov, Alexei; Fedets, Olga; Chastii, Tatjana; Milutina, Elena; Sokol, Oksana; Grigorova, Ritsa; Unuchko, Sergey

2017-12-01

There are no accurate data regarding the prevalence of Chlamydia trachomatis infection in Ukraine. This study aims to estimate the prevalence in the northeastern region of the country through reviewing nucleic acid amplification test results in patients of medical institutions in the Kharkov region during 2014-2016. Samples from 6920 patients (5028 women and 1892 men) aged 12-76 years were tested. The overall positivity rate was 4.5% (95% CI 4.0-5.0): 3.9% (95% CI 3.4-4.5) in women and 6.1% (95% CI 5.1-7.3) in men. The highest prevalence was found in the 16-20 (8.5%, CI 6.3-11.4) and 21-25 (8.0%, CI 6.7-9.4) year age groups. The prevalence in men was higher than in women in all investigated groups. The results show the need for more attention to the prevention, diagnosis, and treatment of chlamydial infection in these age groups of women and men in this region.

Evaluation of the Illumigene Malaria LAMP: A Robust Molecular Diagnostic Tool for Malaria Parasites

PubMed Central

Lucchi, Naomi W.; Gaye, Marie; Diallo, Mammadou Alpha; Goldman, Ira F.; Ljolje, Dragan; Deme, Awa Bineta; Badiane, Aida; Ndiaye, Yaye Die; Barnwell, John W.; Udhayakumar, Venkatachalam; Ndiaye, Daouda

2016-01-01

Isothermal nucleic acid amplification assays such as the loop mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to amplify the DNA. To further facilitate the use of LAMP assays in remote settings, simpler sample preparation methods and lyophilized reagents are required. The performance of a commercial malaria LAMP assay (Illumigene Malaria LAMP) was evaluated using two sample preparation workflows (simple filtration prep (SFP)) and gravity-driven filtration prep (GFP)) and pre-dispensed lyophilized reagents. Laboratory and clinical samples were tested in a field laboratory in Senegal and the results independently confirmed in a reference laboratory in the U.S.A. The Illumigene Malaria LAMP assay was easily implemented in the clinical laboratory and gave similar results to a real-time PCR reference test with limits of detection of ≤2.0 parasites/μl depending on the sample preparation method used. This assay reliably detected Plasmodium sp. parasites in a simple low-tech format, providing a much needed alternative to the more complex molecular tests for malaria diagnosis. PMID:27827432

[Multiplex Ligation - dependent Probe Amplification (MLPA) as a screening test in children with developmental defects and intellectual disability of unknown etiology].

PubMed

Laczmańska, Izabela; Jakubiak, Aleksandra; Slęzak, Ryszard; Pesz, Karolina; Stembalska, Agnieszka; Laczmański, Lukasz; Sąsiadek, Maria M; Smigiel, Robert

2011-01-01

Developmental delay and intellectual disability are significant medical and social problems which concern 1-3% of population. The etiology remains unknown in over half of the cases. To evaluate the efficiency of MLPA (Multiplex Ligation-dependent Probe Amplification) as a screening test in diagnosis of patients with developmental delay and/or intellectual disability. 313 MLPA tests were performed in 256 patients with developmental delay and/ or intellectual disability with unknown etiology. MLPA test was made after exclusion of genetic disorders possible to diagnose by dysmorphological examination or using specifi c genetic tests. Positive results were confirmed by FISH analysis with appropriate probes. Chromosomal microaberrations were identifi ed in 15 patients (4,8%): deletions of 1p36 in 4 cases, in one case deletion of 22q11.21, 22q13.33, SNRPN1, 4ptel, 6qtel, 7q11.23, 16ptel, 18qtel as well as one ca se of deletion 3ptel/duplication 15qtel; deletion 18qtel/duplication Xqtel, and also duplication 7q11.23. Detail clinical analysis was performed in patients with diagnosed microaberrations in MLPA test. The molecular MLPA test, screening for chromosomal microaberration syndromes, should be performed in each patient with developmental delay and/or intellectual disability of unknown etiology and normal cytogenetic analysis, even if congenital defects and positive familial history do not exist.

Droplet microfluidics for amplification-free genetic detection of single cells.

PubMed

Rane, Tushar D; Zec, Helena C; Puleo, Chris; Lee, Abraham P; Wang, Tza-Huei

2012-09-21

In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are tested using a synthetic target mimicking the actual expression level of 16S rRNA in single cells. The capability of the device to perform "sample-to-answer" pathogen detection of single cells is demonstrated using E. coli as a model pathogen.

Improved multiple displacement amplification (iMDA) and ultraclean reagents.

PubMed

Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W

2014-06-06

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome. The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay

PubMed Central

Goud, Kalal Iravathy; Dayakar, Seetha; Vijayalaxmi, Kolanupaka; Babu, Saidam Jangu; Vijay, Anand Reddy P.

2012-01-01

Background & objectives: Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis. Methods: A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy. Results: Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4. Interpretation & conclusions: Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated. PMID:22561616

Evaluation of Site Amplification Functions For Generalized Soil Types Using Earthquake Records and Spectral Models

NASA Astrophysics Data System (ADS)

Sokolov, V.; Loh, C. H.; Wen, K. L.

When evaluating the local site influence on seismic ground motion, in certain cases (e.g. building codes provisions) it is sufficient to describe the variety of soil condi- tions by a few number of generalized site classes. The site classification system that is widely used at present is based on on the properties of top 30 m of soil column, dis- regarding the characteristics of the deeper geology. Six site categories are defined on the basis of averaged shear waves velocity, namely: A - hard rock; B - rock; C - very dense or stiff soil; D - stiff soil; E - soft soil; F - soils requiring special studies. The generalized site amplification curves were developed for several site classes in west- ern US (Boore and Joyner, 1997) and Greece (Klimis et al., 1999) using available geotechnical data from near-surface boreholes. We propose to evaluate the amplifica- tion functions as the ratios between the spectra of real earthquakes recordings and the spectra modeled for "very hard rock" (VHR) conditions. The VHR spectra (regional source scaling and attenuation models) are constructed on the basis of ground motion records. The approach allows, on the one hand, to analyze all obtained records. On the other hand, it is possible to test applicability of the used spectral model. Moreover, the uncertainty of site response may be evaluated and described in terms of random variable characteristics to be considered in seismic hazard analysis. The results of the approach application are demonstrated for the case of Taiwan region. The char- acteristics of site amplification functions (mean values and standard deviation) were determined and analyzed in frequency range of 0.2-13 Hz for site classes B and C us- ing recordings of the 1999 Chi-Chi, Taiwan, earthquake (M=7.6), strong aftershocks (M=6.8), and several earthquakes (M < 6.5) occurred in the region in 1995-1998. When comparing the empirical amplification function resulting from the Taiwan data with that proposed for western US, it has been shown that, for both class B and class C, the US amplification functions exhibit lower values than Taiwan class B function for frequencies 1-8 Hz. The Hellenic class C amplification shows, in general, the sim- ilar shape and amplitude as that evaluated for Taiwan region. Thus, the generalized site amplification curves should be also considered as region-dependent functions.

Evidence of high-elevation amplification versus Arctic amplification

NASA Astrophysics Data System (ADS)

Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

2016-01-01

Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961-2010 period, we find that the warming for the world’s high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction.

Evidence of high-elevation amplification versus Arctic amplification

PubMed Central

Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

2016-01-01

Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961–2010 period, we find that the warming for the world’s high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction. PMID:26753547

Isolation and Characterization of Microsatellite Loci for Stys's Bush-Cricket, Isophya stysi, and Cross-Species Amplification in Closely Related Species from the Phaneropteridae Family

PubMed Central

Iorgu, Elena I.; Popa, Oana P.; Krapal, Ana-Maria; Popa, Luis O.

2013-01-01

Ten microsatellite loci were isolated and characterized for Stys's bush-cricket, Isophya stysi Cejchan (Orthoptera: Tettigoniidae), an endemic Orthoptera species to the Carpathian Basin, using an enriched genomic library procedure. The polymorphism of these loci were tested in two populations of I. stysi, and the number of alleles per locus varied from 4 to 16. The expected and observed heterozygosities ranged from 0.612 to 0.925 and from 0.625 to 1.000, respectively. The interspecific applicability of these microsatellites was evaluated by amplification in 20 related species: Isophya camptoxypha, Isophya sicula, Isophya ciucasi, Isophya pienensis, Isophya harzi, Isophya kraussii, Isophya zubovskii, Isophya rectipennis, Isophya modesta, Isophya longicaudata, Isophya dobrogensis, Isophya hospodar, Isophya speciosa, Isophya modestior, Poecilimon fussii, Poecilimon affinis, Polysarcus denticauda, Barbitistes constrictus, Leptophyes discoidalis, Phaneroptera falcata. All primer pairs for the 10 loci yielded successful amplifications in at least one other taxon from the Isophya genus. This set of microsatellite loci would be useful for genetic studies in I. stysi and other species of the genus Isophya. PMID:23906094

A powerless on-the-spot detection protocol for transgenic crops within 30 min, from leaf sampling up to results.

PubMed

Wang, Liu; Wang, Rui; Yu, Yonghua; Zhang, Fang; Wang, Xiaofu; Ying, Yibin; Wu, Jian; Xu, Junfeng

2016-01-01

The requirement of power-dependent instruments or excessive operation time usually restricts current nucleic acid amplification methods from being used for detection of transgenic crops in the field. In this paper, an easy and rapid detection method which requires no electricity supply has been developed. The time-consuming process of nucleic acid purification is omitted in this method. DNA solution obtained from leaves with 0.5 M sodium hydroxide (NaOH) can be used for loop-mediated isothermal amplification (LAMP) only after simple dilution. Traditional instruments like a polymerase chain reaction (PCR) amplifier and water bath used for DNA amplification are abandoned. Three kinds of dewar flasks were tested and it turned out that the common dewar flask was the best. Combined with visual detection of LAMP amplicons by phosphate (Pi)-induced coloration reaction, the whole process of detection of transgenic crops via genetically pure material (leaf material of one plant) could be accomplished within 30 min. The feasibility of this method was also verified by analysis of practical samples.

Rapid detection of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) by loop-mediated isothermal amplification (LAMP).

PubMed

Bhat, A I; Siljo, A; Deeshma, K P

2013-10-01

The loop-mediated isothermal amplification (LAMP) assay for Piper yellow mottle virus and the reverse transcription (RT) LAMP assay for Cucumber mosaic virus each consisted of a set of five primers designed against the conserved sequences in the viral genome. Both RNA and DNA isolated from black pepper were used as a template for the assay. The results were assessed visually by checking turbidity, green fluorescence and pellet formation in the reaction tube and also by gel electrophoresis. The assay successfully detected both viruses in infected plants whereas no cross-reactions were recorded with healthy plants. Optimum conditions for successful amplification were determined in terms of the concentrations of magnesium sulphate and betaine, temperature, and duration. The detection limit for both LAMP and RT-LAMP was up to 100 times that for conventional PCR and up to one-hundredth of that for real-time PCR. The optimal conditions arrived at were validated by testing field samples of infected vines of three species from different regions. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral fluid by quantitative reverse transcriptase real-time PCR.

PubMed

Decorte, Inge; Van der Stede, Yves; Nauwynck, Hans; De Regge, Nick; Cay, Ann Brigitte

2013-08-01

This study evaluated the effect of extraction-amplification methods, storage temperature and saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus (PRRSV) RNA by quantitative reverse transcriptase real-time PCR (qRT-PCR) in porcine oral fluid. The diagnostic performance of different extraction-amplification methods was examined using a dilution series of oral fluid spiked with PRRSV. To determine RNA stability, porcine oral fluid, with or without commercially available saliva stabilisers, was spiked with PRRSV, stored at 4°C or room temperature and tested for the presence of PRRSV RNA by qRT-PCR. PRRSV RNA could be detected in oral fluid using all extraction-amplification combinations, but the limit of detection varied amongst different combinations. Storage temperature and saliva stabilisers had an effect on the stability of PRRSV RNA, which could only be detected for 7 days when PRRSV spiked oral fluid was kept at 4°C or stabilised at room temperature with a commercial mRNA stabiliser. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular identification of Neofabraea species associated with bull's-eye rot on apple using rolling-circle amplification of partial EF-1α sequence.

PubMed

Lin, Huijiao; Jiang, Xiang; Yi, Jianping; Wang, Xinguo; Zuo, Ranling; Jiang, Zide; Wang, Weifang; Zhou, Erxun

2018-01-01

A rolling-circle amplification (RCA) method with padlock probes targeted on EF-1α regions was developed for rapid detection of apple bull's-eye rot pathogens, including Neofabraea malicorticis, N. perennans, N. kienholzii, and N. vagabunda (synonym: N. alba). Four padlock probes (PLP-Nm, PLP-Np, PLP-Nk, and PLP-Nv) were designed and tested against 28 samples, including 22 BER pathogen cultures, 4 closely related species, and 2 unrelated species that may cause serious apple decays. The assay successfully identified all the bull's-eye rot pathogenic fungi at the level of species, while no cross-reaction was observed in all target species and no false-positive reaction was observed with all strains used for reference. This study showed that the use of padlock probes and the combination of probe signal amplification by RCA provided an effective and sensitive method for the rapid identification of Neofabraea spp. The method could therefore be a useful tool for monitoring bull's-eye rot pathogens in port quarantine and orchard epidemiological studies.

YAP1 and VGLL3, encoding two cofactors of TEAD transcription factors, are amplified and overexpressed in a subset of soft tissue sarcomas.

PubMed

Hélias-Rodzewicz, Zofia; Pérot, Gaëlle; Chibon, Frédéric; Ferreira, Céline; Lagarde, Pauline; Terrier, Philippe; Coindre, Jean-Michel; Aurias, Alain

2010-12-01

In a series of 404 adult soft tissue sarcomas, analyzed by array-CGH, we have observed in approximately 10% of them a genomic amplification of either chromosome bands 11q22 or 3p12. These two amplicons likely target the YAP1 and VGLL3 genes, respectively. Both genes encode proteins that are cofactors of the TEAD family of transcription factors. Very good correlations between amplification and expression levels were observed. Welch test analyses of transcriptome data demonstrate that tumors with amplicons share a large set of upregulated and downregulated genes. Inhibition of YAP1 and VGLL3 in cell lines with these amplifications/overexpressions leads to similar phenotypes: decrease of proliferation rate, and to a lesser extent decrease of migration properties. These data, and the fact that these amplicons are observed either in de-differentiated liposarcomas or in undifferentiated pleomorphic sarcomas, suggest that these genetics events could be involved in oncogenesis and progression of soft tissue sarcomas. © 2010 Wiley-Liss, Inc.

Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip

PubMed Central

Yeh, Erh-Chia; Fu, Chi-Cheng; Hu, Lucy; Thakur, Rohan; Feng, Jeffrey; Lee, Luke P.

2017-01-01

Portable, low-cost, and quantitative nucleic acid detection is desirable for point-of-care diagnostics; however, current polymerase chain reaction testing often requires time-consuming multiple steps and costly equipment. We report an integrated microfluidic diagnostic device capable of on-site quantitative nucleic acid detection directly from the blood without separate sample preparation steps. First, we prepatterned the amplification initiator [magnesium acetate (MgOAc)] on the chip to enable digital nucleic acid amplification. Second, a simplified sample preparation step is demonstrated, where the plasma is separated autonomously into 224 microwells (100 nl per well) without any hemolysis. Furthermore, self-powered microfluidic pumping without any external pumps, controllers, or power sources is accomplished by an integrated vacuum battery on the chip. This simple chip allows rapid quantitative digital nucleic acid detection directly from human blood samples (10 to 105 copies of methicillin-resistant Staphylococcus aureus DNA per microliter, ~30 min, via isothermal recombinase polymerase amplification). These autonomous, portable, lab-on-chip technologies provide promising foundations for future low-cost molecular diagnostic assays. PMID:28345028

Cross-subtype Detection of HIV-1 Using Reverse Transcription and Recombinase Polymerase Amplification

PubMed Central

Lillis, Lorraine; Lehman, Dara A.; Siverson, Joshua B.; Weis, Julie; Cantera, Jason; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie; Boyle, David S.

2016-01-01

A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at the point of care could facilitate early treatment, improving outcomes. However, many infant HIV diagnostics can only be performed in laboratory settings. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that can rapidly amplify proviral DNA from multiple subtypes of HIV-1 in under twenty minutes without complex equipment. In this study we added reverse transcription (RT) to RPA to allow detection of both HIV-1 RNA and DNA. We show that this RT-RPA HIV-1 assay has a limit of detection of 10 to 30 copies of an exact sequence matched DNA or RNA, respectively. In addition, at 100 copies of RNA or DNA, the assay detected 171 of 175 (97.7 %) sequence variants that represent all the major subtypes and recombinant forms of HIV-1 Groups M and O. This data suggests that the application of RT-RPA for the combined detection of HIV-1 viral RNA and proviral DNA may prove a highly sensitive tool for rapid and accurate diagnosis of infant HIV. PMID:26821087

Topoisomerase expression and amplification in solid tumours: Analysis of 24,262 patients

PubMed Central

Heestand, Gregory M.; Schwaederle, Maria; Gatalica, Zoran; Arguello, David; Kurzrock, Razelle

2017-01-01

Background Topoisomerase I (TOPO1) and topoisomerase IIα (TOP2A) are specific targets of multiple chemotherapy drugs. Increased expression of TOPO1 protein and amplification of the TOP2A gene have been associated with treatment response in colorectal and breast cancers, respectively. TOPO1 and TOP2A may be potential therapeutic targets in other malignancies as well. Summary of methods We analysed TOPO1 protein expression and TOP2A gene amplification in patients (n = 24,262 specimens) with diverse cancers. Since HER2 and TOP2A co-amplification have been investigated for predictive value regarding anthracycline benefit, we analysed specimens for HER2 amplification as well. Results Overexpressed TOPO1 protein was present in 51% of the tumours. Four percent of the tumours had TOP2A amplification, with gallbladder tumours and gastroesophageal/oesophageal tumours having rates over 10%. Overall, 4903 specimens were assessed for both TOP2A and HER2 amplification; 129 (2.6%) had co-amplification. High rates (>40%) of HER2 amplification were seen in patients with TOP2A amplification in breast, ovarian, gastroesophageal/oesophageal and pancreatic cancer. Conclusion Our data indicate that increased TOPO1 expression and TOP2A amplification, as well as HER2 co-alterations, are present in multiple malignancies. The implications of these observations regarding sensitivity to chemotherapy not traditionally administered to these tumour types merits investigation. PMID:28728050

Joint Service Aircrew Mask (JSAM) - Tactical Aircraft (TA) A/P22P-14A Respirator Assembly (V)3: Noise Attenuation and Speech Intelligibility Performance with Double Hearing Protection, HGU-68/P Flight Helmet

DTIC Science & Technology

2017-03-31

dB Sound Pressure Level (SPL) background pink noise. The speech intelligibility tests shall result in a Modified Rhyme Test (MRT) score as listed...below. Speech intelligibility testing shall be measured per ANSI S3.2 for each background pink noise level using a minimum of ten talkers and of ten...listeners. The test shall be conducted wearing the JSAM-TA using appropriate communication amplification. Test must include the configurations

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

PubMed

Zhang, Xin; Zhang, He; Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

2013-01-01

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods.

PubMed

Shanks, Orin C; Kelty, Catherine A; Oshiro, Robin; Haugland, Richard A; Madi, Tania; Brooks, Lauren; Field, Katharine G; Sivaganesan, Mano

2016-05-01

There is growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (no-template and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria proposed in this study should help scientists, managers, reviewers, and the public evaluate the technical quality of future findings against an established benchmark. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Recombinase Polymerase Amplification Combined with Lateral Flow Strip for Listeria monocytogenes Detection in Food.

PubMed

Du, Xin-Jun; Zang, Yu-Xuan; Liu, Hai-Bin; Li, Ping; Wang, Shuo

2018-04-01

Listeria monocytogenes is an important food-borne pathogenic bacterium that causes human disease, resulting in economic losses worldwide. The current detection methods for L. monocytogenes are not well suited for direct field testing because they involve complicated, time-consuming operations. A simple, efficient method is vital for L. monocytogenes detection. In this study, we combined isothermal recombinase polymerase amplification (RPA) with a lateral flow (LF) strip to rapidly and reliably detect L. monocytogenes. In the presence of biotin- and digoxin-modified primers, RPA produced numerous digoxin- and biotin-attached duplex DNA products. These products were detected on an LF strip via dual immunoreactions (digoxin on the duplex DNA reacted with the anti-digoxin antibody on the gold nanoparticle (Au-NP) and the biotin on the duplex DNA captured by the streptavidin on the LF test zone). The accumulation of Au-NPs produced characteristic bands, enabling the visual detection of L. monocytogenes without instrumentation. This assay could be used to detect L. monocytogenes within 15 min, including DNA amplification with RPA for 10 min at 39 °C and visualization of the amplicons by LF strips for 5 min. Experiments confirmed a detection limit as low as 300 fg of DNA and 1.5 × 10 1 CFU in pure cultures. Furthermore, RPA-LF exhibited no cross-reactions with pathogens. Evaluation of the method with food samples indicated that the detection limit was substantially improved to 1.5 × 10° CFU for the original bacterial content in 25 g/mL samples after enrichment for 6 hr. RPA-LF can be used as a sensitive and rapid detection technique for L. monocytogenes. Recombinase polymerase amplification (RPA) can amplify target DNA at 37 to 42 °C without a thermal cycler. Lateral flow (LF) strips are portable, cheap and easy to operate. RPA combined with LF strips to detect Listeria monocytogenes can be widely used in remote areas. © 2018 Institute of Food Technologists®.

Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media.

PubMed

Shamonki, Mousa I; Jin, Helen; Haimowitz, Zachary; Liu, Lian

2016-11-01

To assess whether preimplantation genetic screening (PGS) is possible by testing for free embryonic DNA in spent IVF media from embryos undergoing trophectoderm biopsy. Prospective cohort analysis. Academic fertility center. Seven patients undergoing IVF and 57 embryos undergoing trophectoderm biopsy for PGS. On day 3 of development, each embryo was placed in a separate media droplet. All biopsied embryos received a PGS result by array comparative genomic hybridization. Preimplantation genetic screening was performed on amplified DNA extracted from media and results were compared with PGS results for the corresponding biopsy. [1] Presence of DNA in spent IVF culture media. [2] Correlation between genetic screening result from spent media and corresponding biopsy. Fifty-five samples had detectable DNA ranging from 2-642 ng/μL after a 2-hour amplification. Six samples with the highest DNA levels underwent PGS, rendering one result with a derivative log ratio SD (DLRSD) of <0.85 (a quality control metric of oligonucleotide array comparative genomic hybridization). The fluid sample and trophectoderm results were identical demonstrating (45XY, -13). Three samples were reamplified 1 hour later and tested showing improving DLRSD. One of the three samples with a DLRSD of 0.85 demonstrated (46XY), consistent with the biopsy. Overnight DNA amplification showed DNA in all samples. We demonstrate two novel findings: the presence of free embryonic DNA in spent media and a result that is consistent with trophectoderm biopsy. Improvements in DNA collection, amplification, and testing may allow for PGS without biopsy in the future. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites

PubMed Central

Lucchi, Naomi W.; Ljolje, Dragan; Silva-Flannery, Luciana; Udhayakumar, Venkatachalam

2016-01-01

Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1–8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed. PMID:26967908

Cognitive Processing Speed, Working Memory, and the Intelligibility of Hearing Aid-Processed Speech in Persons with Hearing Impairment

PubMed Central

Yumba, Wycliffe Kabaywe

2017-01-01

Previous studies have demonstrated that successful listening with advanced signal processing in digital hearing aids is associated with individual cognitive capacity, particularly working memory capacity (WMC). This study aimed to examine the relationship between cognitive abilities (cognitive processing speed and WMC) and individual listeners’ responses to digital signal processing settings in adverse listening conditions. A total of 194 native Swedish speakers (83 women and 111 men), aged 33–80 years (mean = 60.75 years, SD = 8.89), with bilateral, symmetrical mild to moderate sensorineural hearing loss who had completed a lexical decision speed test (measuring cognitive processing speed) and semantic word-pair span test (SWPST, capturing WMC) participated in this study. The Hagerman test (capturing speech recognition in noise) was conducted using an experimental hearing aid with three digital signal processing settings: (1) linear amplification without noise reduction (NoP), (2) linear amplification with noise reduction (NR), and (3) non-linear amplification without NR (“fast-acting compression”). The results showed that cognitive processing speed was a better predictor of speech intelligibility in noise, regardless of the types of signal processing algorithms used. That is, there was a stronger association between cognitive processing speed and NR outcomes and fast-acting compression outcomes (in steady state noise). We observed a weaker relationship between working memory and NR, but WMC did not relate to fast-acting compression. WMC was a relatively weaker predictor of speech intelligibility in noise. These findings might have been different if the participants had been provided with training and or allowed to acclimatize to binary masking noise reduction or fast-acting compression. PMID:28861009

Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies

PubMed Central

Op De Beeck, Michiel; Lievens, Bart; Busschaert, Pieter; Declerck, Stéphan; Vangronsveld, Jaco; Colpaert, Jan V.

2014-01-01

Current metabarcoding studies aiming to characterize microbial communities generally rely on the amplification and sequencing of relatively short DNA regions. For fungi, the internal transcribed spacer (ITS) region in the ribosomal RNA (rRNA) operon has been accepted as the formal fungal barcode. Despite an increasing number of fungal metabarcoding studies, the amplification efficiency of primers is generally not tested prior to their application in metabarcoding studies. Some of the challenges that metabarcoding primers should overcome efficiently are the amplification of target DNA strands in samples rich in non-target DNA and environmental pollutants, such as humic acids, that may have been co-extracted with DNA. In the current study, three selected primer pairs were tested for their suitability as fungal metabarcoding primers. The selected primer pairs include two primer pairs that have been frequently used in fungal metabarcoding studies (ITS1F/ITS2 and ITS3/ITS4) and a primer pair (ITS86F/ITS4) that has been shown to efficiently amplify the ITS2 region of a broad range of fungal taxa in environmental soil samples. The selected primer pairs were evaluated in a 454 amplicon pyrosequencing experiment, real-time PCR (qPCR) experiments and in silico analyses. Results indicate that experimental evaluation of primers provides valuable information that could aid in the selection of suitable primers for fungal metabarcoding studies. Furthermore, we show that the ITS86F/ITS4 primer pair outperforms other primer pairs tested in terms of in silico primer efficiency, PCR efficiency, coverage, number of reads and number of species-level operational taxonomic units (OTUs) obtained. These traits push the ITS86F/ITS4 primer pair forward as highly suitable for studying fungal diversity and community structures using DNA metabarcoding. PMID:24933453

Systematic review: Comparison of Xpert MTB/RIF, LAMP and SAT methods for the diagnosis of pulmonary tuberculosis.

PubMed

Yan, Liping; Xiao, Heping; Zhang, Qing

2016-01-01

Technological advances in nucleic acid amplification have led to breakthroughs in the early detection of PTB compared to traditional sputum smear tests. The sensitivity and specificity of loop-mediated isothermal amplification (LAMP), simultaneous amplification testing (SAT), and Xpert MTB/RIF for the diagnosis of pulmonary tuberculosis were evaluated. A critical review of previous studies of LAMP, SAT, and Xpert MTB/RIF for the diagnosis of pulmonary tuberculosis that used laboratory culturing as the reference method was carried out together with a meta-analysis. In 25 previous studies, the pooled sensitivity and specificity of the diagnosis of tuberculosis were 93% and 94% for LAMP, 96% and 88% for SAT, and 89% and 98% for Xpert MTB/RIF. The I(2) values for the pooled data were >80%, indicating significant heterogeneity. In the smear-positive subgroup analysis of LAMP, the sensitivity increased from 93% to 98% (I(2) = 2.6%), and specificity was 68% (I(2) = 38.4%). In the HIV-infected subgroup analysis of Xpert MTB/RIF, the pooled sensitivity and specificity were 79% (I(2) = 72.9%) and 99% (I(2) = 64.4%). In the HIV-negative subgroup analysis for Xpert MTB/RIF, the pooled sensitivity and specificity were 72% (I(2) = 49.6%) and 99% (I(2) = 64.5%). LAMP, SAT and Xpert MTB/RIF had comparably high levels of sensitivity and specificity for the diagnosis of tuberculosis. The diagnostic sensitivity and specificity of three methods were similar, with LAMP being highly sensitive for the diagnosis of smear-positive PTB. The cost effectiveness of LAMP and SAT make them particularly suitable tests for diagnosing PTB in developing countries. Copyright © 2015 Elsevier Ltd. All rights reserved.

A new TaqMan method for the reliable diagnosis of Ehrlichia spp. in canine whole blood.

PubMed

Thomson, Kirsty; Yaaran, Tal; Belshaw, Alex; Curson, Lucia; Tisi, Laurence; Maurice, Sarah; Kiddle, Guy

2018-06-18

Ehrlichiosis is an important emerging infectious disease of the canid family and humans worldwide. To date, no extensive evaluation or validation of a molecular diagnostic test for ehrlichiosis has been published. Here, we present data for a newly designed TaqMan assay and compare its performance to a commercial technology (PCRun®). Both of these real-time methods of analysis were evaluated using a comprehensive number of prospective and retrospective samples collected from dogs exhibiting symptoms of ehrlichiosis. Whole blood samples collected from dogs, retrospectively in the United Kingdom and prospectively in Israel, were analysed for the presence of Ehrlichia canis and Ehrlichia minasensis DNA using the TaqMan PCR, developed specifically for this study. The results were compared to those of a real time commercial isothermal amplification method (PCRun® system developed by Biogal Galed Labs ACS, Galed, Israel). The sensitivity and specificity (CI: 95%) of the TaqMan PCR and PCRun® were both determined to be 100% and absolute, for all of the samples tested. Interestingly, both tests were demonstrated to be highly comparable, irrespective of differences in amplification chemistry or sequences targeted. Host differences, incidence of disease and geographical location of the isolates had little impact on the positivity recorded by each of the diagnostic methods. It was evident that both amplification methods were equally suited for diagnosing canine ehrlichiosis and while the PCRun® clearly amplified all clinically relevant Ehrlichia species known to infect dogs and humans, the TaqMan method was more specific for E. canis and E. minasensis. This work demonstrates that despite good analytical sensitivities and specificities for Ehrlichia spp. neither method could fully account for the clinical diagnosis of thrombocytopenia.

Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification.

PubMed

Wang, Jianchang; Wang, Jinfeng; Li, Ruiwen; Liu, Libing; Yuan, Wanzhe

2017-08-15

Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens. A real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR. The RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min-12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R 2 value of the positive results was 0.947. The results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings.

Potential for palivizumab interference with commercially available antibody-antigen based respiratory syncytial virus diagnostic assays.

PubMed

Deming, Damon J; Patel, Nita; McCarthy, Michael P; Mishra, Lalji; Shapiro, Alan M; Suzich, JoAnn A

2013-10-01

Palivizumab is a monoclonal antibody indicated for the prevention of serious lower respiratory tract disease caused by respiratory syncytial virus infection in infants. The potential for palivizumab to interfere with commercially available respiratory syncytial virus diagnostic tests was demonstrated. Negative test results in palivizumab-treated subjects should be interpreted with caution and confirmed by a nucleic acid amplification-based assay.

A Novel Molecular Test to Diagnose Canine Visceral Leishmaniasis at the Point of Care

PubMed Central

Castellanos-Gonzalez, Alejandro; Saldarriaga, Omar A.; Tartaglino, Lilian; Gacek, Rosana; Temple, Elissa; Sparks, Hayley; Melby, Peter C.; Travi, Bruno L.

2015-01-01

Dogs are the principal reservoir hosts of zoonotic visceral leishmaniasis (VL) but current serological methods are not sensitive enough to detect all subclinically infected animals, which is crucial to VL control programs. Polymerase chain reaction (PCR) methods have greater sensitivity but require expensive equipment and trained personnel, impairing its implementation in endemic areas. We developed a diagnostic test that uses isothermal recombinase polymerase amplification (RPA) to detect Leishmania infantum. This method was coupled with lateral flow (LF) reading with the naked eye to be adapted as a point-of-care test. The L. infantum RPA-LF had an analytical sensitivity similar to real time-PCR, detecting DNA of 0.1 parasites spiked in dog blood, which was equivalent to 40 parasites/mL. There was no cross amplification with dog or human DNA or with Leishmania braziliensis, Leishmania amazonensis, or Trypanosoma cruzi. The test also amplified Leishmania donovani strains (N = 7). In a group of clinically normal dogs (N = 30), RPA-LF detected more subclinical infections than rK39 strip test, a standard serological method (50% versus 13.3% positivity, respectively; P = 0.005). Also, RPA-LF detected L. infantum in noninvasive mucosal samples of dogs with a sensitivity comparable to blood samples. This novel molecular test may have a positive impact in leishmaniasis control programs. PMID:26240156

Immunohistochemistry and alternative FISH testing in breast cancer with HER2 equivocal amplification.

PubMed

Agersborg, Sally; Mixon, Christopher; Nguyen, Thanh; Aithal, Sramila; Sudarsanam, Sucha; Blocker, Forrest; Weiss, Lawrence; Gasparini, Robert; Jiang, Shiping; Chen, Wayne; Hess, Gregory; Albitar, Maher

2018-03-22

While HER2 testing is well established in directing appropriate treatment for breast cancer, a small percentage of cases show equivocal results by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Alternative probes may be used in equivocal cases. We present a single community-based institution's experience in further evaluating these cases. Between 2014 and 2016, 4255 samples were submitted for HER2 amplification testing by alternative probes, TP53, RAI1, and RARA. Of the patients tested by FISH, 505/3908 (12.9%) also had IHC data. Most (73.9%) FISH equivocal cases remained equivocal after IHC testing. However, 50.5% of equivocal cases were classified as HER2 amplified by alternative probes. Most cases were positive by more than one probe: 78% of positive cases by RAI1 and 73.9% by TP53. There was a significant difference between IHC and FISH alternative testing (p < 0.0001) among the equivocal cases by conventional FISH testing, 44% of IHC negative cases became positive while 36% of the positive IHC cases became negative by alternative FISH testing. Available data showed that 41% of patients were treated with palbociclib and were positive by alternative FISH. The prevalence of double HER2 equivocal cases and the discrepancy between IHC and alternative FISH testing suggest that FISH alternative testing using both RAI1 and TP53 probes is necessary for conclusive classification. Because almost half of FISH equivocal cases converted to HER2 amplified upon alternative testing, clinical studies to determine the benefit of anti-HER2 therapy in these patients are urgently needed.

The G1 phase Cdks regulate the centrosome cycle and mediate oncogene-dependent centrosome amplification

PubMed Central

2011-01-01

Because centrosome amplification generates aneuploidy and since centrosome amplification is ubiquitous in human tumors, a strong case is made for centrosome amplification being a major force in tumor biogenesis. Various evidence showing that oncogenes and altered tumor suppressors lead to centrosome amplification and aneuploidy suggests that oncogenes and altered tumor suppressors are a major source of genomic instability in tumors, and that they generate those abnormal processes to initiate and sustain tumorigenesis. We discuss how altered tumor suppressors and oncogenes utilize the cell cycle regulatory machinery to signal centrosome amplification and aneuploidy. PMID:21272329

Identification and Treatment of Very Young Children with Hearing Loss.

ERIC Educational Resources Information Center

Madell, Jane R.

1988-01-01

Hearing loss in infants and young children can be identified through behavioral observation audiometry, visual reinforcement audiometry, or auditory brainstem response testing. Habilitation may involve amplification with hearing aids, other assistive listening devices, or cochlear implants. Expectations for children with different degrees of…

Partial gravity simulation using a pneumatic actuator with closed loop mechanical amplification

NASA Technical Reports Server (NTRS)

Ray, David M.

1994-01-01

To support future manned missions to the surface of the Moon and Mars or missions requiring manipulation of payloads and locomotion in space, a training device is required to simulate the conditions of both partial and microgravity as compared to the gravity on Earth. The focus of this paper is to present the development, construction, and testing of a partial gravity simulator which uses a pneumatic actuator with closed loop mechanical amplification. Results of the testing show that this type of simulator maintains a constant partial gravity simulation with a variation of the simulated body force between 2.2 percent and 10 percent, depending on the type of locomotion inputs. The data collected using the simulator show that mean stride frequencies at running speeds at lunar and Martian gravity levels are 12 percent less than those at Earth gravity. The data also show that foot/ground reaction forces at lunar and Martian gravity are, respectively, 62 percent and 51 percent less than those on Earth.

Microwave Driven Actuators Power Allocation and Distribution

NASA Technical Reports Server (NTRS)

Forbes, Timothy; Song, Kyo D.

2000-01-01

Design, fabrication and test of a power allocation and distribution (PAD) network for microwave driven actuators is presented in this paper. Development of a circuit that would collect power from a rectenna array amplify and distribute the power to actuators was designed and fabricated for space application in an actuator array driven by a microwave. A P-SPICE model was constructed initially for data reduction purposes, and was followed by a working real-world model. A voltage up - converter (VUC) is used to amplify the voltage from the individual rectenna. The testing yielded a 26:1 voltage amplification ratio with input voltage at 9 volts and a measured output voltage 230VDC. Future work includes the miniaturization of the circuitry, the use of microwave remote control, and voltage amplification technology for each voltage source. The objective of this work is to develop a model system that will collect DC voltage from an array of rectenna and propagate the voltage to an array of actuators.

Correction of a Technical Error in the Golf Swing: Error Amplification Versus Direct Instruction.

PubMed

Milanese, Chiara; Corte, Stefano; Salvetti, Luca; Cavedon, Valentina; Agostini, Tiziano

2016-01-01

Performance errors drive motor learning for many tasks. The authors' aim was to determine which of two strategies, method of amplification of error (MAE) or direct instruction (DI), would be more beneficial for error correction during a full golfing swing with a driver. Thirty-four golfers were randomly assigned to one of three training conditions (MAE, DI, and control). Participants were tested in a practice session in which each golfer performed 7 pretraining trials, 6 training-intervention trials, and 7 posttraining trials; and a retention test after 1 week. An optoeletronic motion capture system was used to measure the kinematic parameters of each golfer's performance. Results showed that MAE is an effective strategy for correcting the technical errors leading to a rapid improvement in performance. These findings could have practical implications for sport psychology and physical education because, while practice is obviously necessary for improving learning, the efficacy of the learning process is essential in enhancing learners' motivation and sport enjoyment.

Ureaplasma parvum causing life-threatening disease in a susceptible patient.

PubMed

Korytny, Alexander; Nasser, Roni; Geffen, Yuval; Friedman, Tom; Paul, Mical; Ghanem-Zoubi, Nesrin

2017-08-16

A 56-year-old man with lymphoma developed orchitis followed by septic arthritis of his right glenohumeral joint. Synovial fluid cultures were negative but PCR amplification test was positive for Ureaplasmaparvum. The patient was treated with doxycycline. Two and a half years later, the patient presented with shortness of breath and grade III/IV diastolic murmur on auscultation. Echocardiography revealed severely dilated left heart chambers, severe aortic regurgitation and several mobile masses on the aortic valve cusps suspected to be vegetations. He underwent valve replacement; valve tissue culture was negative but the 16S rRNA gene amplification test was positive for U. parvum He was treated again with doxycycline. In an outpatient follow-up 1 year and 3 months later, the patient was doing well. Repeated echocardiography showed normal aortic prosthesis function. © BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Comparison of an assay using signal amplification of the heat-dissociated p24 antigen with the Roche Monitor human immunodeficiency virus RNA assay.

PubMed

Pascual, Alvaro; Cachafeiro, Ada; Funk, Michele L; Fiscus, Susan A

2002-07-01

We compared an assay using signal amplification of a heat-dissociated p24 antigen (HDAg) with the Roche Monitor human immunodeficiency virus (HIV) RNA assay. The two assays gave comparable results when 130 specimens from 130 patients were tested (r = 0.60, P < 0.0001). The HDAg assay was almost as sensitive (85%) as the Roche HIV RNA kit (95%), just as specific (25 negative results from 25 HIV seronegative volunteers [100%]), less variable (mean log standard deviation of 0.07 compared to 0.11 when eight specimens were tested three or four times), and less expensive (reagent and labor costs, $8 versus $75). The assay appeared to be useful for monitoring established patients (n = 17) and identifying seroconverters (n = 4). HIV subtypes A to F were all recognized. This assay should be useful for monitoring patients in resource-poor countries and for monitoring vaccine recipients.

Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification.

PubMed

Boyle, David S; Lehman, Dara A; Lillis, Lorraine; Peterson, Dylan; Singhal, Mitra; Armes, Niall; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie

2013-04-02

Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes.

Increased robustness of single-molecule counting with microfluidics, digital isothermal amplification, and a mobile phone versus real-time kinetic measurements.

PubMed

Selck, David A; Karymov, Mikhail A; Sun, Bing; Ismagilov, Rustem F

2013-11-19

Quantitative bioanalytical measurements are commonly performed in a kinetic format and are known to not be robust to perturbation that affects the kinetics itself or the measurement of kinetics. We hypothesized that the same measurements performed in a "digital" (single-molecule) format would show increased robustness to such perturbations. Here, we investigated the robustness of an amplification reaction (reverse-transcription loop-mediated amplification, RT-LAMP) in the context of fluctuations in temperature and time when this reaction is used for quantitative measurements of HIV-1 RNA molecules under limited-resource settings (LRS). The digital format that counts molecules using dRT-LAMP chemistry detected a 2-fold change in concentration of HIV-1 RNA despite a 6 °C temperature variation (p-value = 6.7 × 10(-7)), whereas the traditional kinetic (real-time) format did not (p-value = 0.25). Digital analysis was also robust to a 20 min change in reaction time, to poor imaging conditions obtained with a consumer cell-phone camera, and to automated cloud-based processing of these images (R(2) = 0.9997 vs true counts over a 100-fold dynamic range). Fluorescent output of multiplexed PCR amplification could also be imaged with the cell phone camera using flash as the excitation source. Many nonlinear amplification schemes based on organic, inorganic, and biochemical reactions have been developed, but their robustness is not well understood. This work implies that these chemistries may be significantly more robust in the digital, rather than kinetic, format. It also calls for theoretical studies to predict robustness of these chemistries and, more generally, to design robust reaction architectures. The SlipChip that we used here and other digital microfluidic technologies already exist to enable testing of these predictions. Such work may lead to identification or creation of robust amplification chemistries that enable rapid and precise quantitative molecular measurements under LRS. Furthermore, it may provide more general principles describing robustness of chemical and biological networks in digital formats.

Recombinase Polymerase Amplification Assay-A Simple, Fast and Cost-Effective Alternative to Real Time PCR for Specific Detection of Feline Herpesvirus-1.

PubMed

Wang, Jianchang; Liu, Libing; Wang, Jinfeng; Sun, Xiaoxia; Yuan, Wanzhe

2017-01-01

Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection.

Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification

PubMed Central

Brewer, Bonita J.; Payen, Celia; Di Rienzi, Sara C.; Higgins, Megan M.; Ong, Giang; Dunham, Maitreya J.; Raghuraman, M. K.

2015-01-01

DNA replication errors are a major driver of evolution—from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model—Origin-Dependent Inverted-Repeat Amplification (ODIRA)—proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error—the ligation of leading and lagging nascent strands to create “closed” forks—can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent—a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution. PMID:26700858

Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification.

PubMed

Brewer, Bonita J; Payen, Celia; Di Rienzi, Sara C; Higgins, Megan M; Ong, Giang; Dunham, Maitreya J; Raghuraman, M K

2015-12-01

DNA replication errors are a major driver of evolution--from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model--Origin-Dependent Inverted-Repeat Amplification (ODIRA)-proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error-the ligation of leading and lagging nascent strands to create "closed" forks-can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent--a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution.

Recombinase Polymerase Amplification Assay—A Simple, Fast and Cost-Effective Alternative to Real Time PCR for Specific Detection of Feline Herpesvirus-1

PubMed Central

Wang, Jianchang; Liu, Libing; Wang, Jinfeng; Sun, Xiaoxia; Yuan, Wanzhe

2017-01-01

Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection. PMID:28045956

Detection of proteins and bacteria using an array of feedback capacitance sensors.

PubMed

Mehta, Manav; Hanumanthaiah, Chandra Sekar; Betala, Pravin Ajitkumar; Zhang, Hong; Roh, SaeWeon; Buttner, William; Penrose, William R; Stetter, Joseph R; Pérez-Luna, Victor H

2007-12-15

An integrated array of micron-dimension capacitors, originally developed for biometric applications (fingerprint identification), was engineered for detection of biological agents such as proteins and bacteria. This device consists of an array of 93,184 (256 x 364) individual capacitor-based sensing elements located underneath a thin (0.8 microm) layer of glass. This glass layer can be functionalized with organosilane-based monolayers to provide groups amenable for the immobilization of bioreceptors such as antibodies, enzymes, peptides, aptamers, and nucleotides. Upon functionalization with antibodies and in conjunction with signal amplification schemes that result in perturbation of the dielectric constant around the captured antigens, this system can be used as a detector of biological agents. Two signal amplification schemes were tested in this work: one consisted of 4 microm diameter latex immunobeads and a second one was based on colloidal gold catalyzed reduction of silver. These signal amplification approaches were demonstrated and show that this system is capable of specific detection of bacteria (Escherichia coli) and proteins (ovalbumin). The present work shows proof-of-principle demonstration that a simple fingerprint detector based on feedback capacitance measurements can be implemented as a biosensor. The approach presented could be easily expanded to simultaneously test for a large number of analytes and multiple samples given that this device has a large number of detectors. The device and required instrumentation is highly portable and does not require expensive and bulky instrumentation because it relies purely on electronic detection.

A norovirus detection architecture based on isothermal amplification and expanded genetic systems.

PubMed

Yaren, Ozlem; Bradley, Kevin M; Moussatche, Patricia; Hoshika, Shuichi; Yang, Zunyi; Zhu, Shu; Karst, Stephanie M; Benner, Steven A

2016-11-01

Noroviruses are the major cause of global viral gastroenteritis with short incubation times and small inoculums required for infection. This creates a need for a rapid molecular test for norovirus for early diagnosis, in the hope of preventing the spread of the disease. Non-chemists generally use off-the shelf reagents and natural DNA to create such tests, suffering from background noise that comes from adventitious DNA and RNA (collectively xNA) that is abundant in real biological samples, especially feces, a common location for norovirus. Here, we create an assay that combines artificially expanded genetic information systems (AEGIS, which adds nucleotides to the four in standard xNA, pairing orthogonally to A:T and G:C) with loop-mediated isothermal amplification (LAMP) to amplify norovirus RNA at constant temperatures, without the power or instrument requirements of PCR cycling. This assay was then validated using feces contaminated with murine norovirus (MNV). Treating stool samples with ammonia extracts the MNV RNA, which is then amplified in an AEGIS-RT-LAMP where AEGIS segments are incorporated both into an internal LAMP primer and into a molecular beacon stem, the second lowering background signaling noise. This is coupled with RNase H nicking during sample amplification, allowing detection of as few as 10 copies of noroviral RNA in a stool sample, generating a fluorescent signal visible to human eye, all in a closed reaction vessel. Copyright © 2016 Elsevier B.V. All rights reserved.

Clinical Characteristics and Outcome of Patients with Neuroblastoma Presenting Genomic Amplification of Loci Other than MYCN

PubMed Central

Guimier, Anne; Ferrand, Sandrine; Pierron, Gaëlle; Couturier, Jérôme; Janoueix-Lerosey, Isabelle; Combaret, Valérie; Mosseri, Véronique; Thebaud, Estelle; Gambart, Marion; Plantaz, Dominique; Marabelle, Aurélien; Coze, Carole; Rialland, Xavier; Fasola, Sylvie; Lapouble, Eve; Fréneaux, Paul; Peuchmaur, Michel; Michon, Jean; Delattre, Olivier; Schleiermacher, Gudrun

2014-01-01

Background Somatically acquired genomic alterations with MYCN amplification (MNA) are key features of neuroblastoma (NB), the most common extra-cranial malignant tumour of childhood. Little is known about the frequency, clinical characteristics and outcome of NBs harbouring genomic amplification(s) distinct from MYCN. Methods Genomic profiles of 1100 NBs from French centres studied by array-CGH were re-examined specifically to identify regional amplifications. Patients were included if amplifications distinct from the MYCN locus were seen. A subset of NBs treated at Institut Curie and harbouring MNA as determined by array-CGH without other amplification was also studied. Clinical and histology data were retrospectively collected. Results In total, 56 patients were included and categorised into 3 groups. Group 1 (n = 8) presented regional amplification(s) without MNA. Locus 12q13-14 was a recurrent amplified region (4/8 cases). This group was heterogeneous in terms of INSS stages, primary localisations and histology, with atypical clinical features. Group 2 (n = 26) had MNA as well as other regional amplifications. These patients shared clinical features of those of a group of NBs MYCN amplified (Group 3, n = 22). Overall survival for group 1 was better than that of groups 2 and 3 (5 year OS: 87.5%±11% vs 34.9%±7%, log-rank p<0.05). Conclusion NBs harbouring regional amplification(s) without MNA are rare and seem to show atypical features in clinical presentation and genomic profile. Further high resolution genetic explorations are justified in this heterogeneous group, especially when considering these alterations as predictive markers for targeted therapy. PMID:25013904

[Applying competitive polymerase chain reaction to the detection of hepatitis B virus DNA].

PubMed

Wang, Ling; Yang, Peng; Li, Shuang-qing; Xu, Shu-hui; Cao, Gui-qun; Zhang, Fa-qiang; Zhang, Mei-xia; Chen, Qing-ying; Xia, Qing-jie; Liu, Kai; Tang, Fang; Zhang, Yuan-zheng

2004-11-01

To reduce the rate of accidental false negative result in the HBV DNA PCR test on clinical serum samples. A competitive polymerase chain reaction (C-PCR) was used to decrease the false negative ratio. In the C-PCR, a constructed inner control DNA was added for co-amplification with the HBV target DNA. In a 20 microl C-PCR system, about 60 to 200 copies of inner control DNA could give apparent co-amplification signal band after electrophoresis on a 2% agarose gel. Five of 120 samples of clinical serum (4.2%) could not be amplified. C-PCR has the advantage of yielding information on false negative in the HBV DNA PCR assay of clinical serum samples.

Dielectrophoretic manipulation of particles for use in microfluidic devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belgrader, P; Bettencourt, K; Hamilton, J

1999-06-23

Amplification and hybridization of DNA are commonly used techniques to verify the presence of a specific DNA sequence in a test sample. Automatic sample handling to concentrate and purify sample prior to amplification is desirable both from the cost standpoint and from the standpoint of reducing the possibility of sample contamination. This paper explores the use of the dielectrophoretic force to manipulate DNA, Bacillus globigii spores, and Erwinia herbicola bacteria to provide concentration and purification as part of the sample handling functions in biological monitoring equipment. It was found that for what would be considered a typical microfabricated structure withmore » electrode gaps at 30 {micro}m operating at 5V, that concentration of the particles is very effective.« less