Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films.

PubMed

Proudfoot, Owen; Drew, Nathan; Scholzen, Anja; Xiang, Sue; Plebanski, Magdalena

2008-04-21

Daily assessment of the percentage of erythrocytes that are infected ('percent-parasitaemia') across a time-course is a necessary step in many experimental studies of malaria, but represents a time-consuming and unpopular task among researchers. The most common method is extensive microscopic examination of Giemsa-stained thin blood-films. This study explored a method for the assessment of percent-parasitaemia that does not require extended periods of microscopy and results in a descriptive and permanent record of parasitaemia data that is highly amenable to subsequent 'data-mining'. Digital photography was utilized in conjunction with a basic purpose-written computer programme to test the viability of the concept. Partial automation of the determination of percent parasitaemia was then explored, resulting in the successful customization of commercially available broad-spectrum image analysis software towards this aim. Lastly, automated discrimination between infected and uninfected RBCs based on analysis of digital parameters of individual cell images was explored in an effort to completely automate the calculation of an accurate percent-parasitaemia.

Improvement of malaria diagnostic system based on acridine orange staining.

PubMed

Kimura, Masatsugu; Teramoto, Isao; Chan, Chim W; Idris, Zulkarnain Md; Kongere, James; Kagaya, Wataru; Kawamoto, Fumihiko; Asada, Ryoko; Isozumi, Rie; Kaneko, Akira

2018-02-07

Rapid diagnosis of malaria using acridine orange (AO) staining and a light microscope with a halogen lamp and interference filter was deployed in some malaria-endemic countries. However, it has not been widely adopted because: (1) the lamp was weak as an excitation light and the set-up did not work well under unstable power supply; and, (2) the staining of samples was frequently inconsistent. The halogen lamp was replaced by a low-cost, blue light-emitting diode (LED) lamp. Using a reformulated AO solution, the staining protocol was revised to make use of a concentration gradient instead of uniform staining. To evaluate this new AO diagnostic system, a pilot field study was conducted in the Lake Victoria basin in Kenya. Without staining failure, malaria infection status of about 100 samples was determined on-site per one microscopist per day, using the improved AO diagnostic system. The improved AO diagnosis had both higher overall sensitivity (46.1 vs 38.9%: p = 0.08) and specificity (99.0 vs 96.3%) than the Giemsa method (N = 1018), using PCR diagnosis as the standard. Consistent AO staining of thin blood films and rapid evaluation of malaria parasitaemia with the revised protocol produced superior results relative to the Giemsa method. This AO diagnostic system can be set up easily at low cost using an ordinary light microscope. It may supplement rapid diagnostic tests currently used in clinical settings in malaria-endemic countries, and may be considered as an inexpensive tool for case surveillance in malaria-eliminating countries.

Cytological diagnosis of basal cell carcinoma and actinic keratosis, using Papanicolaou and May-Grünwald-Giemsa stained cutaneous tissue smear.

PubMed

Christensen, E; Bofin, A; Gudmundsdóttir, I; Skogvoll, E

2008-10-01

Cytology may become the diagnostic method of choice with the advent of new non-invasive treatments for non-melanoma skin cancer, as the sampling technique for cytology entails little tissue disfiguration. The aim of this study was to compare and evaluate the diagnostic performance of scrape cytology using two different cytological staining techniques, and to evaluate additional touch imprint cytology, with that of histopathology of basal cell carcinoma (BCC) and actinic keratosis (AK). We investigated 50 BCC and 28 AK histologically verified lesions, from 41 and 25 patients, respectively. Two separate skin scrape samples and one touch imprint sample were taken from each lesion. The smears were stained with Papanicolaou (Pap) or May-Grünwald-Giemsa (MGG) stains. All cytological specimens were examined in random order by pathologists without knowledge of the histology. Cytodiagnostic results were compared with the histopathological report. Scrape cytodiagnosis agreed with histopathology in 48 (Pap) and 47 (MGG) of the 50 BCC cases, and in 26 of 28 (Pap) and 21 of 26 (MGG) AK cases, yielding sensitivities of 96%, 94%, 93% and 81%, respectively. No significant difference in sensitivity between the two staining methods was found but a trend towards higher Pap sensitivity for AK was noted (P = 0.10). Touch imprint cytology confirmed histopathology in 38 of the 77 cases of BCC and AK. Cytological diagnosis with either Pap or MGG stain for BCC and AK is reliable, and differentiates well between BCC and AK. Imprint cytology proved to be non-diagnostic in half of the examined cases.

Morphological characteristics of developmental stages of Acanthamoeba and Naegleria species before and after staining by various techniques.

PubMed

Ithoi, Init; Ahmad, Arine-Fadzlun; Mak, J W; Nissapatorn, Veeranoot; Lau, Yee-Ling; Mahmud, Rohela

2011-11-01

Seven stains were studied to determine the best color and contrast for staining the developmental stages of free living pathogenic Acanthamoeba and Naegleria species. The acid-fast bacilli stain (AFB) produced a blue color without contrast; trichrome-eosin and modified Field's showed various color contrasts; Giemsa, iron-hematoxylin, modified AFB and Gram produced only one color which distinguished the nucleus, nucleolus, cytoplasm, food- and water-vacuoles. The motile organs (acanthopodia, pseudopodia, lobopodia and flagella) were also clearly differentiated but produced a similar color as the cytoplasm. These motile organelles were first induced by incubating at 37 degrees C for at least 15 minutes and then fixing with methanol in order to preserve the protruding morphology prior to staining. The trichrome-eosin and iron-hematoxylin stains showed good color contrast for detecting all three stages, the trophozoite, cyst and flagellate; Giemsa and Gram stained the trophozoite and flagellate stages; the modified Field's and modified AFB stains stained only the trophozoite stage. Depending on the purpose, all these stains (except the AFB stain) can be used to identify the developmental stages of Acanthamoeba and Naegleria for clinical, epidemiological or public health use.

Reliability of a rapid hematology stain for sputum cytology*

PubMed Central

Gonçalves, Jéssica; Pizzichini, Emilio; Pizzichini, Marcia Margaret Menezes; Steidle, Leila John Marques; Rocha, Cristiane Cinara; Ferreira, Samira Cardoso; Zimmermann, Célia Tânia

2014-01-01

Objective: To determine the reliability of a rapid hematology stain for the cytological analysis of induced sputum samples. Methods: This was a cross-sectional study comparing the standard technique (May-Grünwald-Giemsa stain) with a rapid hematology stain (Diff-Quik). Of the 50 subjects included in the study, 21 had asthma, 19 had COPD, and 10 were healthy (controls). From the induced sputum samples collected, we prepared four slides: two were stained with May-Grünwald-Giemsa, and two were stained with Diff-Quik. The slides were read independently by two trained researchers blinded to the identification of the slides. The reliability for cell counting using the two techniques was evaluated by determining the intraclass correlation coefficients (ICCs) for intraobserver and interobserver agreement. Agreement in the identification of neutrophilic and eosinophilic sputum between the observers and between the stains was evaluated with kappa statistics. Results: In our comparison of the two staining techniques, the ICCs indicated almost perfect interobserver agreement for neutrophil, eosinophil, and macrophage counts (ICC: 0.98-1.00), as well as substantial agreement for lymphocyte counts (ICC: 0.76-0.83). Intraobserver agreement was almost perfect for neutrophil, eosinophil, and macrophage counts (ICC: 0.96-0.99), whereas it was moderate to substantial for lymphocyte counts (ICC = 0.65 and 0.75 for the two observers, respectively). Interobserver agreement for the identification of eosinophilic and neutrophilic sputum using the two techniques ranged from substantial to almost perfect (kappa range: 0.91-1.00). Conclusions: The use of Diff-Quik can be considered a reliable alternative for the processing of sputum samples. PMID:25029648

Evaluation of Papanicolaou stain for studying micronuclei in buccal cells under field conditions.

PubMed

Ayyad, Sohair B A; Israel, Ebenezer; El-Setouhy, Maged; Nasr, Ghada Radwan; Mohamed, Mostafa K; Loffredo, Christopher A

2006-01-01

To compare Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stain as 2 techniques for staining for buccal mucosal cells to detect micronuclei (MN) infield studies. Eighty cytologic smears (2 per individual) were taken from the buccal mucosa of 40 cigarette smokers recruited at a rural village in Egypt. Forty smears were stained with Pap stain and 40 with MGG stain. All were assessed for cellularity and scored for MN. Pap stain was faster and easier to process and transport in the field study than was MGG stain. Regarding MGG smears, bacteria and cell debris masked the MN as compared to Pap smears, in which the fixative destroyed the bacteria and made the cell boundaries clearly demarcated. Using Pap stain, MN were seen easily in transparent cytoplasm. Pap stain is the preferred method infield studies for scoring and detecting MN in cells of buccal mucosa.

Comparison between conventional and molecular methods for diagnosis of bovine babesiosis (Babesia bovis infection) in tick infested cattle in upper Egypt.

PubMed

Al-Hosary, Amira A T

2017-03-01

Ticks and tick-borne diseases are the main problems affecting the livestock production in Egypt. Bovine babesiosis has adverse effects on the animal health and production. A comparison of Giemsa stained blood smears, polymerase chain reaction (PCR) and nested PCR (nPCR) assays for detection of Babesia bovis infection in Egyptian Baladi cattle ( Bos taurus ) in reference to reverse line blot was carried out. The sensitivity of PCR and nested PCR (nPCR) assays were 65 and 100 % respectively. Giemsa stained blood smears showed the lowest sensitivity (30 %). According to these results using of PCR and nPCR target for B. bovis , [BBOV-IV005650 (BV5650)] gene are suitable for diagnosis of B. bovis infection. The 18Ss rRNA partial sequence confirmed that all the positive samples were Babesia bovis and all of them were deposited in the GenBank databases (Accession No: KM455548, KM455549 and KM455550).

Molecular identification of leishmania species using samples obtained from negative stained smears.

PubMed

Mohaghegh, Ma; Fata, A; Salehi, Gh; Berenji, F; Bazzaz, M Mousavi; Rafatpanah, H; Parian, M; Movahedi, A

2013-04-01

Cutaneous Leishmaniasis (CL) is a parasitic skin disease. Diagnosis primarily is based on clinical signs and microscopic observation of parasite on direct stained smears or tissue sections. Sensitivity of direct smear is not as high as molecular methods. The aim of this study was to identify and characterize Leishmania species among the negative direct smears obtained from skin ulcers suspected to CL by PCR method. Among 81 patients with suspicious skin lesions to CL referred to the Parasitology lab, negative Giemsa stained smears were collected. DNA extraction performed by scraping stained smears, then PCR was performed. Among the DNA extracted from smears, L. tropica was isolated from 9 (11.1%) of the smears and L.major was not isolated from any samples. Direct microscopy on stained smears for diagnosis of leishmaniasis is not enough accurate. PCR is recommended for clinically suspected lesions with negative result of direct smear.

Comparative evaluation of fluorescent in situ hybridization and Giemsa microscopy with quantitative real-time PCR technique in detecting malaria parasites in a holoendemic region of Kenya.

PubMed

Osoga, Joseph; Waitumbi, John; Guyah, Bernard; Sande, James; Arima, Cornel; Ayaya, Michael; Moseti, Caroline; Morang'a, Collins; Wahome, Martin; Achilla, Rachel; Awinda, George; Nyakoe, Nancy; Wanja, Elizabeth

2017-07-24

Early and accurate diagnosis of malaria is important in treatment as well as in the clinical evaluation of drugs and vaccines. Evaluation of Giemsa-stained smears remains the gold standard for malaria diagnosis, although diagnostic errors and potential bias estimates of protective efficacy have been reported in practice. Plasmodium genus fluorescent in situ hybridization (P-Genus FISH) is a microscopy-based method that uses fluorescent labelled oligonucleotide probes targeted to pathogen specific ribosomal RNA fragments to detect malaria parasites in whole blood. This study sought to evaluate the diagnostic performance of P-Genus FISH alongside Giemsa microscopy compared to quantitative reverse transcription polymerase chain reaction (qRT-PCR) in a clinical setting. Five hundred study participants were recruited prospectively and screened for Plasmodium parasites by P-Genus FISH assay, and Giemsa microscopy. The microscopic methods were performed by two trained personnel and were blinded, and if the results were discordant a third reading was performed as a tie breaker. The diagnostic performance of both methods was evaluated against qRT-PCR as a more sensitive method. The number of Plasmodium positive cases was 26.8% by P-Genus FISH, 33.2% by Giemsa microscopy, and 51.2% by qRT-PCR. The three methods had 46.8% concordant results with 61 positive cases and 173 negative cases. Compared to qRT-PCR the sensitivity and specificity of P-Genus FISH assay was 29.3 and 75.8%, respectively, while microscopy had 58.2 and 93.0% respectively. Microscopy had a higher positive and negative predictive values (89.8 and 68.0% respectively) compared to P-Genus FISH (56.0 and 50.5%). In overall, microscopy had a good measure of agreement (76%, k = 0.51) compared to P-Genus FISH (52%, k = 0.05). The diagnostic performance of P-Genus FISH was shown to be inferior to Giemsa microscopy in the clinical samples. This hinders the possible application of the method in the field despite the many advantages of the method especially diagnosis of low parasite density infections. The P-Genus assay has great potential but application of the method in clinical setting would rely on extensive training of microscopist and continuous proficiency testing.

[Pneumocystis pneumonia biological diagnosis at Fann Teaching Hospital in Dakar, Senegal].

PubMed

Dieng, Y; Dieng, T; Sow, D; Wlouhou, S; Sylla, K; Tine, R; Ndiaye, M; Ndiaye, J L; Faye, B; Faye, O; Gaye, O

2016-03-01

Data relative to Pneumocystis pneumonia in sub-Saharan Africa are not well known. Weakness of the technical material and use of little sensitive biological tools of diagnosis are among the evoked reasons. The objective of this study is to update the data of the disease at the Fann Teaching Hospital in Dakar and to estimate biological methods used in diagnosis. A descriptive longitudinal study was carried out from January 5th, 2009 to October 31st, 2011 in the parasitology and mycology laboratory of the Fann Teaching Hospital in Dakar. The bronchoalveolar lavages received in the laboratory were examined microscopically for Pneumocystis jirovecii by indirect fluorescent assay or after Giemsa or toluidine blue O staining. One hundred and eighty-three bronchoalveolar lavages withdrawn from 183 patients were received in the laboratory. Sixteen were positive for P. jirovecii at 9% frequency. Four among these patients were HIV positive. Indirect fluorescent assay allowed finding of P. jirovecii among 16 patients while Giemsa staining discovered P. jirovecii only in a single patient. No case was diagnosed by toluidine blue O staining. Pneumocystis pneumonia in Parasitology and Mycology Laboratory of Fann Teaching Hospital at Dakar was mainly diagnosed among HIV patients. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

A "two-objective, one-area" procedure in absorption microphotometry and its application using an inverted microscope.

PubMed

Chaubal, K A

1988-08-01

A 'two-objective, one-area' method and related equations are suggested to measure absorbance of microscopic stained objects. In such work, the measuring field invariably includes an image of the object and some clear area surrounding the image. The total intensity in the two areas is measured photometrically, using two different objectives, and substituted in the equation for absorbance. The equation is independent of the term representing intensity from the clear area and hence the error in the measurement of absorbance is reduced. The limitations of the 'two-objective, one-area' method are discussed and its pragmatic operation described with an experimental setup involving an inverted microscope. The method permits measurement of intensity in a part of a stained cell while the rest of the cell remains in the field of view. The method is applied to measure absorbance in Giemsa stained ascites cells and Feulgen stained liver and Human Amnion cells.

Opportunistic parasites among immunosuppressed children in Minia District, Egypt.

PubMed

Abdel-Hafeez, Ekhlas H; Ahmad, Azza K; Ali, Basma A; Moslam, Fadia A

2012-03-01

A total of 450 stool samples were collected from inpatient and outpatient clinics of Pediatric Department, Minia University Hospital, Minia District, Egypt. Two groups of patients were studied, including 200 immunosuppressed and 250 immunocompetent children. Stool samples were subjected to wet saline and iodine mounts. A concentration technique (formol-ether sedimentation method) was carried out for stool samples diagnosed negative by wet saline and iodine mounts. Samples were stained by 2 different methods; acid fast stain (modified Ziehl-Neelsen stain) and Giemsa stain. Total 188 cases (94%) were diagnosed positive for parasitic infections among immunosuppressed children, whereas 150 cases (60%) were positive in immunocompetent children (P<0.0001). The most common protozoan infection in immunosuppressed group was Cryptosporidium parvum (60.2%), followed by Blastocystis hominis (12.1%), Isospora belli (9.7%), and Cyclospora caytenensis (7.8%). On the other hand, Entamoeba histolytica (24.6%) and Giardia lamblia (17.6%) were more common than other protozoans in immunocompetent children.

Staining human lymphocytes and onion root cell nuclei with madder root.

PubMed

Cücer, N; Guler, N; Demirtas, H; Imamoğlu, N

2005-01-01

We performed staining experiments on cells using natural dyes and different mordants using techniques that are used for wool and silk dyeing. The natural dye sources were madder root, daisy, corn cockle and yellow weed. Ferrous sulfate, copper sulfate, potassium tartrate, urea, potassium aluminum sulfate and potassium dichromate were used as mordants. Distilled water, distilled water plus ethanol, heptane, and distilled water plus methanol were used as solvents. All dye-mordant-solvent combinations were studied at pH 2.4, 3.2 and 4.2. The generic staining procedure was to boil 5-10 onion roots or stimulated human lymphocyte (SHL) preparations in a dye bath on a hot plate. Cells were examined at every half hour. For multicolor staining, madder-dyed lymphocytes were decolorized, then stained with Giemsa. The AgNOR technique was performed following the decolorization of Giemsa stained lymphocytes. Good results were obtained for both onion root cells and lymphocytes that were boiled for 3 h in a dye bath that included 4 g madder root, 4 g ferrous sulfate as mordant in 50 ml of 1:1 (v/v) methanol:distilled water. The pH was adjusted to 4.2 with 6 ml acetic acid. We conclude that madder root has potential as an alternative dye for staining biological materials.

External quality assessment of malaria microscopy in the Democratic Republic of the Congo

PubMed Central

2011-01-01

Background External quality assessments (EQA) are an alternative to cross-checking of blood slides in the quality control of malaria microscopy. This study reports the findings of an EQA of malaria microscopy in the Democratic Republic of the Congo (DRC). Methods After validation, an EQA slide panel and a questionnaire were delivered to diagnostic laboratories in four provinces of DRC. The panel included three samples for diagnosis (sample 1: Plasmodium falciparum, 177,000/μl, sample 2: P. falciparum, 2,500/μl, sample 3: no parasites seen), one didactic sample (Howell-Jolly bodies) and one sample for assessing the quality of staining. Participating laboratories were addressed and selected through the network of the National Tuberculosis Control Programme. Participants were asked to return the responses together with a stained thin and thick blood film for evaluation of Giemsa stain quality. Results Among 174 participants (response rate 95.1%), 26.2% scored samples 1, 2 and 3 correctly and 34.3%, 21.5% and 5.8% of participants reported major errors in one, two or three samples respectively. Major errors included reporting "no malaria" or "non-falciparum malaria" for Plasmodium falciparum-positive samples 1 and 2 (16.1% and 34.9% of participants respectively) and "P. falciparum" for Plasmodium negative sample 3 (24.0%). Howell-Jolly bodies (didactic sample) were not recognized by any of the participants but reported as "P. falciparum" by 16.7% of participants. With parasite density expressed according to the "plus system", 16.1% and 21.5% of participants scored one "+" different from the reference score for samples 1 and 2 respectively and 9.7% and 2.9% participants scored more than two "+" different. When expressed as counts of asexual parasites/μl, more than two-thirds of results were outside the mean ± 2SD reference values. The quality of the Giemsa stain was poor, with less than 20% slides complying with all criteria assessed. Only one quarter of participants purchase Giemsa stain from suppliers of documented reliability and half of participants use a buffered staining solution. One third of participants had participated in a formal training about malaria diagnosis, half of them earlier than 2007. Conclusion The present EQA revealed a poor quality of malaria microscopy in DRC. PMID:22008378

Bacillus Anthracis Spores of the bclA Mutant Exhibit Increased Adherence to Epithelial Cells, Fibroblasts, and Endothelial Cells but not to Macrophages

DTIC Science & Technology

2007-09-01

immunofluorescence (IFM) and light microscopy. Samples were fixed in forma- lin, stained with immunofluorescent dyes (as described below) or spore stain ( malachite ...BEC. Adherence was assessed by microscopic observation of the infected cells stained with malachite green and counterstaining of the BEC. For enzymatic...this significant difference, BEC infected with spores were stained with malachite green and counter- stained with Wright-Giemsa (Fig. 1B and C). This

Guidelines for May-Grünwald-Giemsa staining in haematology and non-gynaecological cytopathology: recommendations of the French Society of Clinical Cytology (SFCC) and of the French Association for Quality Assurance in Anatomic and Cytologic Pathology (AFAQAP).

PubMed

Piaton, E; Fabre, M; Goubin-Versini, I; Bretz-Grenier, M-F; Courtade-Saïdi, M; Vincent, S; Belleannée, G; Thivolet, F; Boutonnat, J; Debaque, H; Fleury-Feith, J; Vielh, P; Egelé, C; Bellocq, J-P; Michiels, J-F; Cochand-Priollet, B

2016-10-01

Since the guidelines of the International Committee for Standardisation in Haematology (ICSH) in 1984 and those of the European Committee for External Quality Assessment Programmes in Laboratory Medicine (EQALM) in 2004, no leading organisation has published technical recommendations for the preparation of air-dried cytological specimens using May-Grünwald-Giemsa (MGG) staining. Literature data were retrieved using reference books, baseline-published studies, articles extracted from PubMed/Medline and Google Scholar, and online-available industry datasheets. The present review addresses all pre-analytical issues concerning the use of Romanowsky's stains (including MGG) in haematology and non-gynaecological cytopathology. It aims at serving as actualised, best practice recommendations for the proper handling of air-dried cytological specimens. It, therefore, appears complementary to the staining criteria of the non-gynaecological diagnostic cytology handbook edited by the United Kingdom National External Quality Assessment Service (UK-NEQAS) in February 2015. © 2016 John Wiley & Sons Ltd.

[Human chromosome banding with raw extract of fruits or leaves of papaya].

PubMed

Solís, M V

2001-01-01

One week old human chromosome preparations were treated with filtrate from one liquefied leaf (53 g) of papaya (Carica papaya) in 100 ml of distilled water, and stained with 1.5% Giemsa (pH 6.8). Good chromosome banding was obtained after 2 min of treatment. Solutions that have been frozen even for years are effective and the method is cheaper and easier than others.

Lymphatic Filariasis

MedlinePlus

... stained with Giemsa. Center: Photograph of a female Aedes aegypti mosquito as she was in the process of obtaining a “blood meal.” Laboratory strains of Aedes aegypti can be infected with Brugia. Credit: DPDx , ...

Opportunistic Parasites among Immunosuppressed Children in Minia District, Egypt

PubMed Central

Ahmad, Azza K.; Ali, Basma A.; Moslam, Fadia A.

2012-01-01

A total of 450 stool samples were collected from inpatient and outpatient clinics of Pediatric Department, Minia University Hospital, Minia District, Egypt. Two groups of patients were studied, including 200 immunosuppressed and 250 immunocompetent children. Stool samples were subjected to wet saline and iodine mounts. A concentration technique (formol-ether sedimentation method) was carried out for stool samples diagnosed negative by wet saline and iodine mounts. Samples were stained by 2 different methods; acid fast stain (modified Ziehl-Neelsen stain) and Giemsa stain. Total 188 cases (94%) were diagnosed positive for parasitic infections among immunosuppressed children, whereas 150 cases (60%) were positive in immunocompetent children (P<0.0001). The most common protozoan infection in immunosuppressed group was Cryptosporidium parvum (60.2%), followed by Blastocystis hominis (12.1%), Isospora belli (9.7%), and Cyclospora caytenensis (7.8%). On the other hand, Entamoeba histolytica (24.6%) and Giardia lamblia (17.6%) were more common than other protozoans in immunocompetent children. PMID:22451735

Trypanosoma evansi isolated from capybara (Hidrochaeris hidrochaeris).

PubMed

Muñoz, K; Chávez, A

2001-10-01

A study was conducted to determine the morphological and biometric characteristics of Trypanosoma isolated from 50 capybaras animals, raised in captivity in the Peruvian Amazon. Trypanosoma was found in 14 blood samples using the microhaematocrit, wide drop, and Giemsa-stain methods and T. evansi was identified through morphological details in all 14 positive samples (the subterminal kinetoplast, the developed undulating membrane, and a long free flagellum were used for the identification of the agent).

The occurrence of hepatozoon in the gray squirrel (Sciurus carolinensis)

USGS Publications Warehouse

Herman, C.M.; Price, D.L.

1955-01-01

Hepatozoon sciuri (Coles, 1914) is reported from gray squirrels (Sciurus carolinensis) in Washington, D.C. and Maryland. Blood smears stained with Giemsa's stain revealed a parasitemia in 16 to 71% of the squirrels examined. A technique for laking the red cells and concentrating the white cells in blood samples demonstrated this protozoon to be present in every squirrel so tested.

Toluidine Blue with a Synergistic Effect in Morphological Assessment of Oral Cytosmears.

PubMed

Ratna Kumari, T V N; Ahmed Mujib, B R

2018-01-01

One percent toluidine blue being the most effective adjunct is often used to detect dysplasia. Not much attention has been given to the effect of toluidine blue in enhancement of cytological smears. The present study assessed the smears before and after application of toluidine blue in smokers against non-smokers using three different stains [Papanicolaou (PAP), Hematoxylin and Eosin (H and E), and Giemsa]. Twenty-five individuals from each group participated in the study. The oral cytosmears were obtained before and after application of toluidine blue and assessed for clumping of squamous cells, cellular and nuclear pleomorphism, micronuclei, binucleation, bacterial colony units, and keratin flakes. In smokers, the maximum enhancement in cytological smears post-toluidine blue application was shown by Giemsa stain than PAP and H and E stains. Among the individual parameters, nuclear pleomorphism exhibited greatest significant difference between smokers and non-smokers. Toluidine blue enhanced the staining characteristics both in terms of sensitivity and specificity and thereby was found to be synergistic in assessment of cytosmears. The cellular alterations noticed in the smears of smokers with clinically normal buccal mucosa can be used as a means of education tool in counselling for smoking cessation.

Toluidine Blue with a Synergistic Effect in Morphological Assessment of Oral Cytosmears

PubMed Central

Ratna Kumari, T V. N.; Ahmed Mujib, B. R.

2018-01-01

Objectives: One percent toluidine blue being the most effective adjunct is often used to detect dysplasia. Not much attention has been given to the effect of toluidine blue in enhancement of cytological smears. The present study assessed the smears before and after application of toluidine blue in smokers against non-smokers using three different stains [Papanicolaou (PAP), Hematoxylin and Eosin (H and E), and Giemsa]. Study Design: Twenty-five individuals from each group participated in the study. The oral cytosmears were obtained before and after application of toluidine blue and assessed for clumping of squamous cells, cellular and nuclear pleomorphism, micronuclei, binucleation, bacterial colony units, and keratin flakes. Results: In smokers, the maximum enhancement in cytological smears post-toluidine blue application was shown by Giemsa stain than PAP and H and E stains. Among the individual parameters, nuclear pleomorphism exhibited greatest significant difference between smokers and non-smokers. Conclusion: Toluidine blue enhanced the staining characteristics both in terms of sensitivity and specificity and thereby was found to be synergistic in assessment of cytosmears. The cellular alterations noticed in the smears of smokers with clinically normal buccal mucosa can be used as a means of education tool in counselling for smoking cessation. PMID:29403163

Biological dosimetry of ionizing radiation: Evaluation of the dose with cytogenetic methodologies by the construction of calibration curves

NASA Astrophysics Data System (ADS)

Zafiropoulos, Demetre; Facco, E.; Sarchiapone, Lucia

2016-09-01

In case of a radiation accident, it is well known that in the absence of physical dosimetry biological dosimetry based on cytogenetic methods is a unique tool to estimate individual absorbed dose. Moreover, even when physical dosimetry indicates an overexposure, scoring chromosome aberrations (dicentrics and rings) in human peripheral blood lymphocytes (PBLs) at metaphase is presently the most widely used method to confirm dose assessment. The analysis of dicentrics and rings in PBLs after Giemsa staining of metaphase cells is considered the most valid assay for radiation injury. This work shows that applying the fluorescence in situ hybridization (FISH) technique, using telomeric/centromeric peptide nucleic acid (PNA) probes in metaphase chromosomes for radiation dosimetry, could become a fast scoring, reliable and precise method for biological dosimetry after accidental radiation exposures. In both in vitro methods described above, lymphocyte stimulation is needed, and this limits the application in radiation emergency medicine where speed is considered to be a high priority. Using premature chromosome condensation (PCC), irradiated human PBLs (non-stimulated) were fused with mitotic CHO cells, and the yield of excess PCC fragments in Giemsa stained cells was scored. To score dicentrics and rings under PCC conditions, the necessary centromere and telomere detection of the chromosomes was obtained using FISH and specific PNA probes. Of course, a prerequisite for dose assessment in all cases is a dose-effect calibration curve. This work illustrates the various methods used; dose response calibration curves, with 95% confidence limits used to estimate dose uncertainties, have been constructed for conventional metaphase analysis and FISH. We also compare the dose-response curve constructed after scoring of dicentrics and rings using PCC combined with FISH and PNA probes. Also reported are dose response curves showing scored dicentrics and rings per cell, combining PCC of lymphocytes and CHO cells with FISH using PNA probes after 10 h and 24 h after irradiation, and, finally, calibration data of excess PCC fragments (Giemsa) to be used if human blood is available immediately after irradiation or within 24 h.

Single step modified ink staining for Tzanck test: quick detection of herpetic giant cells in Tzanck smear.

PubMed

Mizutani, Hitoshi; Akeda, Tomoko; Yamanaka, Kei-Ichi; Isoda, Kenichi; Gabazza, Esteban C

2012-02-01

Tzanck test has been recently re-evaluated as a method for the diagnosis of herpes virus infection. Giemsa staining for the Tzanck test is time-consuming and laborious. There is a need to develop simple and quick staining methods for bedside diagnosis of this disease. We report a single step and quick method for staining herpes giant cells in Tzanck smears using routinely available inks and physiological saline. A keratinocyte cell line (HaCaT) was cultured on a slide glass and stained with various commercially available blue, blue-black and black inks serially diluted with physiological saline. Clinical smear samples from herpes lesions were also stained with these solutions without specific pretreatment. The nuclei of HaCaT were clearly stained showing high contrast with the cytoplasm using 5% Parker-Quink blue-black ink saline solution. Concentration of ink solution higher or lower than 5% resulted in less contrast. Blue or black inks or other manufacturers' inks can also be used, but staining of the cultured keratinocytes was less clear. Smear of clinical samples from herpes lesions were also stained with 5% ink solution. The nuclei of the multinucleated giant cells were clearly stained, and the sample could be immediately used for microscopic examination. One step staining of Tzanck smear using this diluted ink solution is an inexpensive and a convenient bedside diagnostic tool for the dermatologist. © 2011 Japanese Dermatological Association.

Identification criteria of the rare multi-flagellate Lophomonas blattarum: comparison of different staining techniques.

PubMed

Alam-Eldin, Yosra Hussein; Abdulaziz, Amany Mamdouh

2015-09-01

Bronchopulmonary lophomoniasis (BPL) is an emerging disease of potential importance. BPL is presented by non-specific clinical picture and is usually accompanied by immunosuppression. Culture of Lophomonas blattarum is difficult and its molecular diagnosis has not yet been developed. Therefore, microscopic examination of respiratory samples, e.g., bronchoalveolar lavage (BAL) or sputum, is the mainstay of BPL diagnosis. Creola bodies and ciliocytophthoria are two forms of bronchial cells which occur in chest diseases with non-specific clinical picture like that of BPL. Both forms could be misrecognized as multi-flagellates because of their motile cilia in the wet mounts and due to shape variability of L. blattarum in stained smears. The aim of the study is to compare different staining techniques for visualizing L. blattarum to improve the recognition and diagnosis of BPL, to distinguish respiratory epithelial cells from L. blattarum and to decide which stain is recommended in suspected cases of BPL. BAL samples from patients which contain L. blattarum, creola bodies, and ciliocytophthoria were collected then wet mounts were examined. The BAL samples were also stained by Papanicolaou (PAP), Giemsa, hematoxylin and eosin (H & E), trichrome, Gram, and Diff-Quik (DQ) stains. The different staining techniques were compared regarding the stain quality. In wet mounts, the ciliary movement was coordinate and synchronous while the flagellar movement was wavy and leaded to active swimming of L. blattarum. In stained slides, bronchial cells were characterized by the presence of basal nucleus and the terminal bar from which the cilia arise. Trichrome was the best stain in demonstration of cellular details of L. blattarum. H & E, PAP, and Giemsa stains showed good quality of stains. Gram and DQ stains showed only pale hues of L. blattarum. We recommended adding Wheatley's trichrome staining to the differential diagnosis workup of cases of non-specific chest infections, especially when BPL is suspected, to avoid overdiagnosis or underdiagnosis of it.

Fluorescence microscope (Cyscope) for malaria diagnosis in pregnant women in Medani Hospital, Sudan.

PubMed

Hassan, Saad El-Din H; Haggaz, Abd Elrahium D; Mohammed-Elhassan, Ehab B; Malik, Elfatih M; Adam, Ishag

2011-09-24

Accuracy of diagnosis is the core for malaria control. Although microscopy is the gold standard in malaria diagnosis, its reliability is largely dependent on user skill. We compared performance of Cyscope fluorescence microscope with the Giemsa stained light microscopy for the diagnosis of malaria among pregnant women at Medani Hospital in Central Sudan. The area is characterized by unstable malaria transmission. Socio-demographic characteristics and obstetrics history were gathered using pre-tested questionnaires. Blood samples were collected from febrile pregnant women who were referred as malaria case following initial diagnosis by general microscopist. During the study period 128 febrile pregnant women presented at the hospital. Among them, Plasmodium falciparum malaria was detected in 82 (64.1%) and 80 (62.5%) by the Giemsa-stained light microscopy and the Cyscope fluorescence microscope, respectively. The sensitivity of the Cyscope fluorescence microscope was 97.6% (95% CI: 92.2%-99.6%). Out of 46 which were negative by Giemsa-stained light microscopy, 5 were positive by the Cyscope fluorescence microscope. This is translated in specificity of 89.1% (95% CI: 77.5%-95.9%). The positive and negative predictive value of Cyscope fluorescence microscope was 94.1% (95% CI: 87.4% -97.8%) and 95.3% (95% CI: 85.4% - 99.2%), respectively. This study has shown that Cyscope fluorescence microscope is a reliable diagnostic, sensitive and specific in diagnosing P. falciparum malaria among pregnant women in this setting. Further studies are needed to determine effectiveness in diagnosing other Plasmodium species and to compare it with other diagnostic tools e.g. rapid diagnostic tests and PCR.

The evolution of chromosomal instability in Chinese hamster cells: a changing picture?

NASA Technical Reports Server (NTRS)

Ponnaiya, B.; Limoli, C. L.; Corcoran, J.; Kaplan, M. I.; Hartmann, A.; Morgan, W. F.

1998-01-01

PURPOSE: To investigate the kinetics of chromosomal instability induced in clones of Chinese hamster cells following X-irradiation. MATERIALS AND METHODS: X-irradiated clones of GM10115, human-hamster hybrid cells containing a single human chromosome 4 (HC4), have been previously established. These clones were defined as unstable if they contained > or = three subpopulations of cells with unique rearrangements of HC4 as detected by FISH. Stable and unstable clones were analysed by FISH and Giemsa staining at various times post-irradiation. RESULTS: While most of the stable clones continued to show chromosomal stability of HC4 over time, one became marginally unstable at approximately 45 population doublings post-irradiation. Clones exhibiting chromosomal instability had one of several fates. Many of the unstable clones were showed similar levels of instability over time. However, one unstable clone became stable with time in culture, while another became even more unstable over time. Cytogenetic analyses of all clones after Giemsa staining indicated that in some clones the hamster chromosomes were rearranged independent of HC4, demonstrating increased frequencies of chromatid breaks and dicentric chromosomes. The majority of the unstable clones also had higher yields of chromatid gaps. CONCLUSIONS: These data demonstrate the dynamic nature of chromosomal instability as measured by two different cytogenetic assays.

Histological Stains: A Literature Review and Case Study

PubMed Central

Alturkistani, Hani A; Tashkandi, Faris M; Mohammedsaleh, Zuhair M

2016-01-01

The history of histology indicates that there have been significant changes in the techniques used for histological staining through chemical, molecular biology assays and immunological techniques, collectively referred to as histochemistry. Early histologists used the readily available chemicals to prepare tissues for microscopic studies; these laboratory chemicals were potassium dichromate, alcohol and the mercuric chloride to harden cellular tissues. Staining techniques used were carmine, silver nitrate, Giemsa, Trichrome Stains, Gram Stain and Hematoxylin among others. The purpose of this research was to assess past and current literature reviews, as well as case studies, with the aim of informing ways in which histological stains have been improved in the modern age. Results from the literature review has indicated that there has been an improvement in histopathology and histotechnology in stains used. There has been a rising need for efficient, accurate and less complex staining procedures. Many stain procedures are still in use today, and many others have been replaced with new immunostaining, molecular, non-culture and other advanced staining techniques. Some staining methods have been abandoned because the chemicals required have been medically proven to be toxic. The case studies indicated that in modern histology a combination of different stain techniques are used to enhance the effectiveness of the staining process. Currently, improved histological stains, have been modified and combined with other stains to improve their effectiveness. PMID:26493433

Histological Stains: A Literature Review and Case Study.

PubMed

Alturkistani, Hani A; Tashkandi, Faris M; Mohammedsaleh, Zuhair M

2015-06-25

The history of histology indicates that there have been significant changes in the techniques used for histological staining through chemical, molecular biology assays and immunological techniques, collectively referred to as histochemistry. Early histologists used the readily available chemicals to prepare tissues for microscopic studies; these laboratory chemicals were potassium dichromate, alcohol and the mercuric chloride to harden cellular tissues. Staining techniques used were carmine, silver nitrate, Giemsa, Trichrome Stains, Gram Stain and Hematoxylin among others. The purpose of this research was to assess past and current literature reviews, as well as case studies, with the aim of informing ways in which histological stains have been improved in the modern age. Results from the literature review has indicated that there has been an improvement in histopathology and histotechnology in stains used. There has been a rising need for efficient, accurate and less complex staining procedures. Many stain procedures are still in use today, and many others have been replaced with new immunostaining, molecular, non-culture and other advanced staining techniques. Some staining methods have been abandoned because the chemicals required have been medically proven to be toxic. The case studies indicated that in modern histology a combination of different stain techniques are used to enhance the effectiveness of the staining process. Currently, improved histological stains, have been modified and combined with other stains to improve their effectiveness.

[Standardization of Blastocystis hominis diagnosis using different staining techniques].

PubMed

Eymael, Dayane; Schuh, Graziela Maria; Tavares, Rejane Giacomelli

2010-01-01

The present study was carried out from March to May 2008, with the aim of evaluating the effectiveness of different techniques for diagnosing Blastocystis hominis in a sample of the population attended at the Biomedicine Laboratory of Feevale University, Novo Hamburgo, Rio Grande do Sul. On hundred feces samples from children and adults were evaluated. After collection, the samples were subjected to the techniques of spontaneous sedimentation (HPJ), sedimentation in formalin-ether (Ritchie) and staining by means of Gram and May-Grünwald-Giemsa (MGG). The presence of Blastocystis hominis was observed in 40 samples, when staining techniques were used (MGG and Gram), while sedimentation techniques were less efficient (32 positive samples using the Ritchie technique and 20 positive samples using the HPJ technique). Our results demonstrate that HPJ was less efficient than the other methods, thus indicating the need to include laboratory techniques that enable parasite identification on a routine basis.

Wright-Giemsa staining to observe phagocytes in Locusta migratoria infected with Metarhizium acridum.

PubMed

Yu, Ying; Cao, Yueqing; Xia, Yuxian; Liu, Feihong

2016-09-01

Hemocytes are the first line of defense in the invertebrate immune system. Understanding their roles in cellular immunity is important for developing more efficient mycoinsecticides. However, the exact classification of hemocytes has been inconsistent and the various types of phagocytes in Locusta migratoria are poorly defined. Herein, the Wright-Giemsa staining method and microscopy were employed to characterize the hemocytes of L. migratoria following infection by Metarhizium acridum. Hemocytes were classified into four types, including granulocytes, plasmatocytes, prohemocytes, and oenocytoids, based on size, morphology, and dye-staining properties. Each type of hemocyte was classified into several subtypes according to different ultrastructural features. At least four subtypes of granulocytes or plasmatocytes, including small-nucleus plasmatocytes, basophil vacuolated plasmatocytes, homogeneous plasmatocytes, and eosinophilic granulocytes, carried out phagocytosis. The percentage of total phagocytes increased two days after infection by M. acridum, then gradually declined during the next two days, and then increased sharply again at the fifth day. Our data suggested that plasmatocytes and granulocytes may be the major phagocytes that protect against invasion by a fungal pathogen in L. migratoria. Total hemocytes in locusts significantly increased in the initial days after infection and decreased in the late period of infection compared to controls. In the hemocoel, hyphal bodies were recognized, enwrapped, and digested by the phagocytes. Then, the broken hyphal pieces were packaged as vesicles to be secreted from the cell. Moreover, locusts might have a sensitive and efficient cellular immune system that can regulate phagocyte differentiation and proliferation before fungi colonize the host hemolymph. Copyright © 2016 Elsevier Inc. All rights reserved.

Inactivation of Orientia tsutsugamushi in red blood cells, plasma, and platelets with riboflavin and light, as demonstrated in an animal model.

PubMed

Rentas, Francisco; Harman, Ronald; Gomez, Charlotte; Salata, Jeanne; Childs, Joseph; Silva, Tonya; Lippert, Lloyd; Montgomery, Joshua; Richards, Allen; Chan, Chye; Jiang, Ju; Reddy, Heather; Li, John; Goodrich, Raymond

2007-02-01

Treatment of blood products with riboflavin and light has been used to reduce the number of certain pathogens. Orientia (formerly Rickettsia) tsutsugamushi, the scrub typhus agent, is an obligate intracellular bacterium that grows free in the cytoplasm of infected cells. This study evaluated the capability of riboflavin and light to inactivate O. tsutsugamushi in red blood cells (RBCs), platelets (PLTs), and plasma, as measured by mouse infectivity. A total of 108 mice, equally divided into groups receiving RBCs, plasma, and PLTs, received untreated products infected with 10(0) to 10(5) organisms. Eighteen mice received products infected with 10(5) organisms and were subsequently treated with riboflavin and light. Mice were monitored daily for up to 17 days for signs and symptoms of infection (e.g., lethargy, labored breathing, rough coat) and killed upon appearance of symptoms or on Day 17 after infection. Real-time polymerase chain reaction (PCR) on blood and Giemsa stains from peritoneal exudates were performed. A total of 102 of 108 mice receiving the untreated products developed signs and symptoms of infection and had positive PCR and Giemsa stain results. None of the 18 animals receiving riboflavin and light-treated blood products exhibited signs or symptoms of infection, nor was infection observed by PCR testing or Giemsa staining. Riboflavin and light are effective in reducing O. tsutsugamushi. Mice injected with blood products inoculated with 10(5) organisms and treated with riboflavin and light did not experience any signs or symptoms of infection, 17 days after inoculation. A 5-log reduction of this organism in blood was achieved as assayed in an animal model.

Spirochaetemia in a HIV positive patient.

PubMed

Okwori, E E

2006-01-01

Borreliosis, caused by Borrelia recurrentis and several other Borrelia species is not a commonly reported case in our environment, but the search for the cause of recurrent pyrexia in this patient made it possible to discover the spirochete as the cause of the disease. A 38 year old married HIV positive woman presented with recurrent fever in a private clinic. Six thin smears were made out of the patient serum and dried in the air. Three slides each were stained with 0.12% Leishman and 20% Giemsa stains and examined under the light microscope. Three of the Giemsa slides were positive for spirochetes (4-5 spirals), which were constituents with Borrelia species. The patient responded very well to tetracycline and serum became negative for the organism after ten days of treatment. Borrelia was discovered to be the cause of the recurrent pyrexia in this patient who responded very well to tetracycline. Borrelia should be looked for in cases of pyrexia of unknown origin

New Advanced Technology for Muscular Dystrophy

DTIC Science & Technology

2008-03-01

muscles per population) than for M-MDSCs ( gray bars; RI = 109 ± 18; mean ± SEM; n = 10 M-MDSC populations; two to six muscles per population; P = 0.035...experiments) of the latter in 2-week cultures of whole muscle cells ( gray bars) and sorted myogenic cells (black bars). On average, 15–35% of cells...subsequently GTG stained (brief trypsin treatment followed by Giemsa stain). At least ten metaphases were counted for each cell fraction and the

New Advanced Technology for Muscular Dystrophy

DTIC Science & Technology

2009-11-01

per population) than for M-MDSCs ( gray bars; RI = 109 ± 18; mean ± SEM; n = 10 M-MDSC populations; two to six muscles per population; P = 0.035; t...latter in 2-week cultures of whole muscle cells ( gray bars) and sorted myogenic cells (black bars). On average, 15–35% of cells coexpressing... GTG stained (brief trypsin treatment followed by Giemsa stain). At least ten metaphases were counted for each cell fraction and the chromosomes from

[Analysis of on-call consultations with clinical pathologists--identification of customer's satisfaction].

PubMed

Yanai, M

2000-09-01

One aspect whereby effectiveness of clinical pathologists can be measured is customer service and satisfaction. Clinical pathologist should identify their customers, their processes and procedures to meet these needs to the customer's satisfaction. To identify customer's satisfaction, the records of on-call consultations with clinical pathologists were analyzed. Between January 1996 and December 1998, 1327 consultations were recorded, 40% of which were consultations from physicians, 50% from medical technologists. Physicians requested interpretation of laboratory data obtained, and clinical knowledge mainly concerning the microbiology and hematology during office hours. On holidays, physicians needed help performing emergency tests such as Gram stain and Wright-Giemsa stain. During office hours, medical technologists requested clinical information concerning patients in whom unreasonable data would be reported and the contact to the clinical side. Furthermore, technologists inquired about the methodology of laboratory tests during day duty on holidays. These results indicated that the clinical pathologist in our hospital could satisfy the customer(physicians and medical technologists), by providing 1) a wide range of clinical knowledge concerning not only the laboratory medicine but clinical medicine including therapeutics, 2) capability of performing emergency tests such as Gram stain and Wright-Giemsa stain, and 3) capability of interpreting the results obtained. Although these would not be adopted in every hospital, every clinical pathologist should examine his role in the hospital.

[Utility of chromosome banding with ALU I enzyme for identifying methylated areas in breast cancer].

PubMed

Rojas-Atencio, Alicia; Yamarte, Leonard; Urdaneta, Karelis; Soto-Alvarez, Marisol; Alvarez Nava, Francisco; Cañizalez, Jenny; Quintero, Maribel; Atencio, Raquel; González, Richard

2012-12-01

Cancer is a group of disorders characterized by uncontrolled cell growth which is produced by two successive events: increased cell proliferation (tumor or neoplasia) and the invasive capacity of these cells (metastasis). DNA methylation is an epigenetic process which has been involved as an important pathogenic factor of cancer. DNA methylation participates in the regulation of gene expression, directly, by preventing the union of transcription factors, and indirectly, by promoting the "closed" structure of the chromatine. The objectives of this study were to identify hypermethyled chromosomal regions through the use of restriction Alu I endonuclease, and to relate cytogenetically these regions with tumor suppressive gene loci. Sixty peripheral blood samples of females with breast cancer were analyzed. Cell cultures were performed and cytogenetic spreads, previously digested with Alu I enzyme, were stained with Giemsa. Chromosomal centromeric and not centromeric regions were stained in 37% of cases. About 96% of stained hypermethyled chromosomal regions (1q, 2q, 6q) were linked with methylated genes associated with breast cancer. In addition, centromeric regions in chromosomes 3, 4, 8, 13, 14, 15 and 17, usually unstained, were found positive to digestion with Alu I enzime and Giemsa staining. We suggest the importance of this technique for the global visualization of the genome which can find methylated genes related to breast cancer, and thus lead to a specific therapy, and therefore a better therapeutic response.

Adenoid hypertrophy and chronic rhinosinusitis: Helicobacter pylori on antral lavages, adenoid tissue and salival inmunoglobuline A on paediatric patients.

PubMed

Cedeño, Eleazar E Graterón; Ortiz-Princz, Diana; Figueredo, Sinay A Ceballos; Porro, María Eugenia Cavazza

2016-01-01

To determine Helicobacter pylori presence on antral lavages, adenoids and salival inmunoglobuline A on paediatric patients with chronic rhinosinusitis without nasal polyps (CRSsNP) and adenoid hypertrophy. Adenoid tissue, liquid obtained from antral lavages and saliva from 28 children diagnosed with CRSsNP, from the paediatric otorhinolaryngology practice of "Dr. Domingo Luciani" Hospital was taken and processed by means of polymerase chain reaction (PCR) using cagA, vacA and babA primers, also anatomopathological examination using Giemsa stain of the adenoids, determination of salivary specific secretory inmunoglobuline A (sIgA), socio-economic condition using the Graffar scale and associated gastrointestinal symptoms were assessed. No evidence of Helicobacter pylori neither in antral lavages liquid nor adenoid tissue was found using PCR and Giemsa stain. sIgA was present in 28.6% of the subjects. The most frequently found symptoms were, diarrhea in 17.9%, distension and abdominal pain in 10.7%, 64.3% of the patients were in working (28.6%) and low middle (35.7%) classes. Helicobacter pylori is not present neither in maxillary sinuses nor adenoid tissue of the evaluated patients, sIgA it is a non-invasive method for assessment of immunologic challenge with the bacteria, not the presence of acute or chronic infection. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A rapid method for counting nucleated erythrocytes on stained blood smears by digital image analysis

USGS Publications Warehouse

Gering, E.; Atkinson, C.T.

2004-01-01

Measures of parasitemia by intraerythrocytic hematozoan parasites are normally expressed as the number of infected erythrocytes per n erythrocytes and are notoriously tedious and time consuming to measure. We describe a protocol for generating rapid counts of nucleated erythrocytes from digital micrographs of thin blood smears that can be used to estimate intensity of hematozoan infections in nonmammalian vertebrate hosts. This method takes advantage of the bold contrast and relatively uniform size and morphology of erythrocyte nuclei on Giemsa-stained blood smears and uses ImageJ, a java-based image analysis program developed at the U.S. National Institutes of Health and available on the internet, to recognize and count these nuclei. This technique makes feasible rapid and accurate counts of total erythrocytes in large numbers of microscope fields, which can be used in the calculation of peripheral parasitemias in low-intensity infections.

Karyotype and identification of sex in two endangered crane species

USGS Publications Warehouse

Goodpasture, C.; Seluja, G.; Gee, G.; Wood, Don A.

1992-01-01

A laboratory procedure for sex identification of monomorphic birds was developed using modern cytological methods of detecting chromosome abnormalities in human amniotic fluid samples. A pin feather is taken from a pre-fledging bird for tissue culture and karyotype analysis. Through this method, the sex was identified and the karyotype described of the whooping crane (Grus americana) and the Mississippi sandhill crane (G. canadensis pulla). Giemsa-stained karyotypes of these species showed an identical chromosome constitution with 2n = 78 + 2. However, differences in the amount of centromeric heterochromatin were observed in the Mississippi sandhill crane when compared to the whooping crane C-banded karyotype.

Uniform staining of Cyclospora oocysts in fecal smears by a modified safranin technique with microwave heating.

PubMed Central

Visvesvara, G S; Moura, H; Kovacs-Nace, E; Wallace, S; Eberhard, M L

1997-01-01

Cyclospora, a coccidian protist, is increasingly being identified as an important, newly emerging parasite that causes diarrhea, flatulence, fatigue, and abdominal pain leading to weight loss in immunocompetent persons with or without a recent travel history as well as in patients with AIDS. Modified Kinyoun's acid-fast stain is the most commonly used stain to identify the oocyst of this parasite in fecal smears. Oocysts of Cyclospora stain variably by the modified acid-fast procedure, resulting in the possible misidentification of this parasite. We examined fecal smears stained by six different procedures that included Giemsa, trichrome, chromotrope, Gram-chromotrope, acid-fast, and safranin stains. We report on safranin-based stain that uniformly stains oocysts of Cyclospora a brilliant reddish orange, provided that the fecal smears are heated in a microwave oven prior to staining. This staining procedure, besides being superior to acid-fast staining, is fast, reliable, and easy to perform in most clinical laboratories. PMID:9041421

Hepatozoon langii n. sp. and Hepatozoon vacuolatus n. sp. (Apicomplexa: Adele-orina: Hepatozoidae) from the crag lizard (Sauria: Cordylidae) Pseudocordylus langi from the North Eastern Drakensberg escarpment, Eastern Free State, South Africa.

PubMed

Van As, Johann; Davies, Angela J; Smit, Nico J

2013-01-22

Two new haemogregarine species, Hepatozoon langii n. sp. and Hepatozoon vacuolatus n. sp., are described from the pe-ripheral blood of the high altitude crag lizard, Pseudocordylus langi, collected between October 2006 and April 2009 from the North Eastern Drakensberg, Eastern Free State. Hepatozoon langii n. sp. has maturing and mature gamonts that appear encapsulated and have narrow, curved tails. Their cytoplasm stains pinkish-purple with Giemsa, while their nuclei are pur-ple stained with stranded chromatin. Mature gamonts measure 19.1 ± 1.0 (15.4-28.1) μm long by 6.2 ± 1.1 (3.5-7.9) μm wide. Hepatozoon vacuolatus n. sp. gamonts are mostly broader at one pole than the other, have bluish-pink cytoplasm characterised by distinctive rounded and oval vacuoles, and demonstrate pink granules with Giemsa staining. Nuclei stain purple and are mainly coarsely granular. Mature gamonts measure 16.5 ± 1.0 (14.7 - 17.6) μm long by 5.9 ± 1.2 (4.0 - 7.7) μm wide. Both species parasitize erythroblasts, as well as erythrocytes and can dehaemoglobinize the cytoplasm of their host cells. Hepatozoon langii n. sp occurred in the absence of H. vacuolatus n. sp., but the latter haemogregarine always formed mixed infections with the former; no stages intermediate between the two haemogregarine types were observed.

A Study for the Feature Selection to Identify GIEMSA-Stained Human Chromosomes Based on Artificial Neural Network

DTIC Science & Technology

2001-10-25

neural network (ANN) has been adopted for the human chromosome classification. It is important to select optimum features for training neural network...Many studies for computer-based chromosome analysis have shown that it is possible to classify chromosomes into 24 subgroups. In addition, artificial

First Molecular Identification and Genetic Characterization of Theileria lestoquardi in Sheep of the Maghreb Region.

PubMed

Rjeibi, M R; Darghouth, M A; Rekik, M; Amor, B; Sassi, L; Gharbi, M

2016-06-01

Theileria lestoquardi is the most prominent Theileria species in small ruminants that causes malignant theileriosis of sheep in Africa and Asia. In the present survey, blood samples and ticks were collected in Kebili (southern Tunisia) from 166 Queue Fine de l'Ouest sheep. Giemsa-stained blood smears, immunofluorescent antibody test (IFAT) and PCR were performed. The DNA was extracted from blood and analysed by PCR targeting 18S rRNA gene of Theileria spp. and then sequenced. A total number of 140 ticks were collected from a total number of 166 sheep during the four seasons. The ticks belonged to two genera and 4 species; the most frequent tick was Hyalomma excavatum 84.3% (118/140) and then Rhipicephalus spp. 15.7% (22/140). Only two animals had positive Giemsa-stained blood smears, and they were also positive by IFAT. The amplicons had 99.3 and 99.6% homology with the BLAST published T. lestoquardi amplicons. To our knowledge, this is the first report of T. lestoquardi in small ruminants within the Maghreb region. © 2014 Blackwell Verlag GmbH.

Identification of the facultative heterochromatic X chromosome in females of 25 rodent species.

PubMed

Kanda, N; Yosida, T H

1979-01-01

Treatment of the chromosomes of 25 rodent species with a 50 degrees C hypotonic solution and Giemsa staining permitted identification of the heterochromatic X chromosome in 24 species. With this technique, the facultative of the heterochromatic X chromosome or the facultative portion of large, composite-type X chromosoms is stained darker than the other chromosomes, allowing it to be distinguished from the homologous euchromatic X chromosome in female metaphase cells. Intense staining of the single X chromosome was not observed in male metaphase cells. It is suggested that this differential staining of one of the two X chromosomes might be due to qualitative differences in chromosomal proteins rather than to differences in the degree of chromosomal condensation or in DNA base sequence.

Comparison of the novel Partec rapid malaria test to the conventional Giemsa stain and the gold standard real-time PCR.

PubMed

Nkrumah, Bernard; Agyekum, Alex; Acquah, Samuel E K; May, Jürgen; Tannich, Egbert; Brattig, Norbert; Nguah, Samuel Blay; von Thien, Heidrun; Adu-Sarkodie, Yaw; Huenger, Frank

2010-08-01

Malaria remains the single most frequent cause of death in Africa, killing one child every 30 s, but treatment decisions are often made only on clinical diagnosis, as laboratory techniques to confirm the clinical suspicion are labor intensive and costly. In this study, we evaluated the recently developed Partec rapid malaria test (PM) for the detection of Plasmodium spp. in human blood from patients in an area where malaria is endemic and compared the results with those of thick blood film Giemsa stain (GS) in terms of its performance and operational characteristics, using real-time (RT) PCR as the gold standard. The sensitivities of the PM and the GS were 62.2% (95% CI, 56.3 to 67.8) and 61.8% (95% CI, 55.9 to 67.4), respectively, while the specificities were 96.0% (95% CI, 92.3 to 98.3) and 98% (95% CI, 95.0 to 99.5), respectively. There was an excellent agreement between the results for the PM and those of the GS (k [level of agreement] = 0.96; P < 0.001). The results for the PM were obtained more quickly and at less cost than those for the GS. The performance characteristics of the PM were almost equal to those of the GS, but the operational characteristics were better, and the PM can therefore be considered as an alternative method for GS.

Cut-off optimization for 13C-urea breath test in a community-based trial by mathematic, histology and serology approach.

PubMed

Li, Zhe-Xuan; Huang, Lei-Lei; Liu, Cong; Formichella, Luca; Zhang, Yang; Wang, Yu-Mei; Zhang, Lian; Ma, Jun-Ling; Liu, Wei-Dong; Ulm, Kurt; Wang, Jian-Xi; Zhang, Lei; Bajbouj, Monther; Li, Ming; Vieth, Michael; Quante, Michael; Zhou, Tong; Wang, Le-Hua; Suchanek, Stepan; Soutschek, Erwin; Schmid, Roland; Classen, Meinhard; You, Wei-Cheng; Gerhard, Markus; Pan, Kai-Feng

2017-05-18

The performance of diagnostic tests in intervention trials of Helicobacter pylori (H.pylori) eradication is crucial, since even minor inaccuracies can have major impact. To determine the cut-off point for 13 C-urea breath test ( 13 C-UBT) and to assess if it can be further optimized by serologic testing, mathematic modeling, histopathology and serologic validation were applied. A finite mixture model (FMM) was developed in 21,857 subjects, and an independent validation by modified Giemsa staining was conducted in 300 selected subjects. H.pylori status was determined using recomLine H.pylori assay in 2,113 subjects with a borderline 13 C-UBT results. The delta over baseline-value (DOB) of 3.8 was an optimal cut-off point by a FMM in modelling dataset, which was further validated as the most appropriate cut-off point by Giemsa staining (sensitivity = 94.53%, specificity = 92.93%). In the borderline population, 1,468 subjects were determined as H.pylori positive by recomLine (69.5%). A significant correlation between the number of positive H.pylori serum responses and DOB value was found (r s  = 0.217, P < 0.001). A mathematical approach such as FMM might be an alternative measure in optimizing the cut-off point for 13 C-UBT in community-based studies, and a second method to determine H.pylori status for subjects with borderline value of 13 C-UBT was necessary and recommended.

Malaria over-diagnosis in Cameroon: diagnostic accuracy of Fluorescence and Staining Technologies (FAST) Malaria Stain and LED microscopy versus Giemsa and bright field microscopy validated by polymerase chain reaction.

PubMed

Parsel, Sean M; Gustafson, Steven A; Friedlander, Edward; Shnyra, Alexander A; Adegbulu, Aderosoye J; Liu, Ying; Parrish, Nicole M; Jamal, Syed A; Lofthus, Eve; Ayuk, Leo; Awasom, Charles; Henry, Carolyn J; McArthur, Carole P

2017-04-04

Malaria is a major world health issue and its continued burden is due, in part, to difficulties in the diagnosis of the illness. The World Health Organization recommends confirmatory testing using microscopy-based techniques or rapid diagnostic tests (RDT) for all cases of suspected malaria. In regions where Plasmodium species are indigenous, there are multiple etiologies of fever leading to misdiagnoses, especially in populations where HIV is prevalent and children. To determine the frequency of malaria infection in febrile patients over an 8-month period at the Regional Hospital in Bamenda, Cameroon, we evaluated the clinical efficacy of the Flourescence and Staining Technology (FAST) Malaria stain and ParaLens Advance TM microscopy system (FM) and compared it with conventional bright field microscopy and Giemsa stain (GS). Peripheral blood samples from 522 patients with a clinical diagnosis of "suspected malaria" were evaluated using GS and FM methods. A nested PCR assay was the gold standard to compare the two methods. PCR positivity, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined. Four hundred ninety nine samples were included in the final analysis. Of these, 30 were positive via PCR (6.01%) with a mean PPV of 19.62% and 27.99% for GS and FM, respectively. The mean NPV was 95.01% and 95.28% for GS and FM, respectively. Sensitivity was 26.67% in both groups and specificity was 92.78% and 96.21% for GS and FM, respectively. An increased level of diagnostic discrepancy was observed between technicians based upon skill level using GS, which was not seen with FM. The frequency of malarial infections confirmed via PCR among patients presenting with fever and other symptoms of malaria was dramatically lower than that anticipated based upon physicians' clinical suspicions. A correlation between technician skill and accuracy of malaria diagnosis using GS was observed that was less pronounced using FM. Additionally, FM increased the specificity and improved the PPV, suggesting this relatively low cost approach could be useful in resource-limited environments. Anecdotally, physicians were reluctant to not treat all patients symptomatically before results were known and in spite of a negative microscopic diagnosis, highlighting the need for further physician education to avoid this practice of overtreatment. A larger study in an area with a known high prevalence is being planned to compare the two microscopy methods against available RDTs.

Fluorescent microscopy and Ziehl-Neelsen staining of bronchoalveolar lavage, bronchial washings, bronchoscopic brushing and post bronchoscopic sputum along with cytological examination in cases of suspected tuberculosis.

PubMed

Bodal, Vijay Kumar; Bal, Manjit S; Bhagat, Sunita; Kishan, Jai; Brar, Rupinder K

2015-01-01

Ever since the discovery of Mycobacterium tuberculosis in 1882, many diagnostic methods have been developed. However "The gold standard" for the diagnosis of tuberculosis (TB) is still the demonstration of acid fast Bacilli (AFB) by microscopic examination of smear or bacteriological confirmation by culture method. In suspected 75 patients with active pulmonary TB, the materials obtained bronchoscopically, were bronchoalveolar lavage (BAL), bronchial brushings, bronchial washings and post bronchoscopic sputum. Four smears were made from each of the specimen. Fluorescent Staining, Ziehl-Neelsen (ZN), Pap and May Grunwald-Giemsa (MGG) stains were carried out for cytological examination. Fluorescent stain yielded maximum AFB positivity in all the methods, that is 36 (48%) in post fibre-optic bronchoscopy (FOB) sputum and 19 (25.33%) by fluorescence microscopy in both bronchial brushings and bronchial washings. Maximum yield of AFB with ZN staining 12 (16%) was equal to the post FOB sputum and bronchial brushings samples. It was followed by 6 cases (8%) in BAL and 4 (5.3%) in bronchial washings. The cytological examination was suggestive of TB in only 8 (10.66%) cases in bronchial washings and 6 (8%) cases in post FOB collection. It was equal in BAL and Bronchial brushings each that is 5 (6.67%). Bronchoscopy is a useful diagnostic tool and fluorescent microscopy is more sensitive than ZN and cytology. On X-ray examination, other diseases like malignancy or fungus can also mimick TB. So apart from ZN staining or fluorescence microscopy, Pap and MGG stain will be worthwhile to identify other microorganisms.

Estimating the parasitaemia of Plasmodium falciparum: experience from a national EQA scheme

PubMed Central

2013-01-01

Background To examine performance of the identification and estimation of percentage parasitaemia of Plasmodium falciparum in stained blood films distributed in the UK National External Quality Assessment Scheme (UKNEQAS) Blood Parasitology Scheme. Methods Analysis of performance for the diagnosis and estimation of the percentage parasitaemia of P. falciparum in Giemsa-stained thin blood films was made over a 15-year period to look for trends in performance. Results An average of 25% of participants failed to estimate the percentage parasitaemia, 17% overestimated and 8% underestimated, whilst 5% misidentified the malaria species present. Conclusions Although the results achieved by participants for other blood parasites have shown an overall improvement, the level of performance for estimation of the parasitaemia of P. falciparum remains unchanged over 15 years. Possible reasons include incorrect calculation, not examining the correct part of the film and not examining an adequate number of microscope fields. PMID:24261625

Comparison of diagnostic methods to detect Histoplasma capsulatum in serum and blood samples from AIDS patients

PubMed Central

da Silva, Marcos Vinicius; Criado, Paulo Ricardo; Luiz, Olinda do Carmo; Vicentini, Adriana Pardini

2018-01-01

Background Although early and rapid detection of histoplasmosis is essential to prevent morbidity and mortality, few diagnostic tools are available in resource-limited areas, especially where it is endemic and HIV/AIDS is also epidemic. Thus, we compared conventional and molecular methods to detect Histoplasma capsulatum in sera and blood from HIV/AIDS patients. Methodology We collected a total of 40 samples from control volunteers and patients suspected of histoplasmosis, some of whom were also infected with other pathogens. Samples were then analyzed by mycological, serological, and molecular methods, and stratified as histoplasmostic with (group I) or without AIDS (group II), uninfected (group III), and infected with HIV and other pathogens only (group IV). All patients were receiving treatment for histoplasmosis and other infections at the time of sample collection. Results Comparison of conventional methods with nested PCR using primers against H. capsulatum 18S rRNA (HC18S), 5.8S rRNA ITS (HC5.8S-ITS), and a 100 kDa protein (HC100) revealed that sensitivity against sera was highest for PCR with HC5.8S-ITS, followed by immunoblotting, double immunodiffusion, PCR with HC18S, and PCR with HC100. Specificity was equally high for double immunodiffusion, immunoblotting and PCR with HC100, followed for PCR with HC18S and HC5.8-ITS. Against blood, sensitivity was highest for PCR with HC5.8S-ITS, followed by PCR with HC18S, Giemsa staining, and PCR with HC100. Specificity was highest for Giemsa staining and PCR with HC100, followed by PCR with HC18S and HC5.8S-ITS. PCR was less efficient in patients with immunodeficiency due to HIV/AIDS and/or related diseases. Conclusion Molecular techniques may detect histoplasmosis even in cases with negative serology and mycology, potentially enabling early diagnosis. PMID:29342162

Optimization of Trichomonas vaginalis Diagnosis during Pregnancy at a University Hospital, Argentina.

PubMed

Testardini, Pamela; Vaulet, María Lucía Gallo; Entrocassi, Andrea Carolina; Menghi, Claudia; Eliseht, Martha Cora; Gatta, Claudia; Losada, Mirta; Touzón, María Sol; Corominas, Ana; Vay, Carlos; Tatti, Silvio; Famiglietti, Angela; Fermepin, Marcelo Rodriguez; Perazzi, Beatriz

2016-04-01

The aim of this study was to evaluate different methods for Trichomonas vaginalis diagnosis during pregnancy in order to prevent maternal and perinatal complications. A total of 386 vaginal exudates from pregnant women were analyzed. T. vaginalis was investigated by 3 types of microscopic examinations direct wet mount with physiologic saline solution, prolonged May-Grunwald Giemsa (MGG) staining, and wet mount with sodium-acetate-formalin (SAF)/methylene blue method. PCR for 18S rRNA gene as well as culture in liquid medium were performed. The sensitivity and specificity of the microscopic examinations were evaluated considering the culture media positivity or the PCR techniques as gold standard. The frequency of T. vaginalis infection was 6.2% by culture and/or PCR, 5.2% by PCR, 4.7% by culture, 3.1% by SAF/methylene blue method and 2.8% by direct wet smear and prolonged MGG staining. The sensitivities were 83.3%, 75.0%, 50.0%, and 45.8% for PCR, culture, SAF/methylene blue method, and direct wet smear-prolonged MGG staining, respectively. The specificity was 100% for all the assessed methods. Microscopic examinations showed low sensitivity, mainly in asymptomatic pregnant patients. It is necessary to improve the detection of T. vaginalis using combined methods providing higher sensitivity, such as culture and PCR, mainly in asymptomatic pregnant patients, in order to prevent maternal and perinatal complications.

Water extract of Semecarpus parvifolia Thw. leaves inhibits cell proliferation and induces apoptosis on HEp-2 cells.

PubMed

Soysa, Preethi; Jayarthne, Panchima; Ranathunga, Imali

2018-03-05

Semecarpus parvifolia Thw is used as an ingredient of poly herbal decoctions to treat cancer in traditional medicine. The present study aims to investigate the antiproliferative activity on HEp 2 cells by the water extract of S. parvifolia leaves and to evaluate potential mechanisms. The plant extract was exposed to S. parvifolia for 24 hours and antiproliferative activity was quantified by Sulforhodamine B (SRB), 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Lactate dehydrogenase (LDH) assays. Morphological changes were observed after staining cells with ethidium bromide/acridine orange (EB/AO) and Giemsa dye. Comet assay was performed to evaluate the DNA damage. The toxicity of the plant extract was determined by brine shrimp lethality assay. S. parvifolia leaves reduced the cell proliferation in a dose and time dependent manner. A two fold increase in NO level was observed at higher concentrations. Morphological changes characteristic to apoptosis were observed in light microscopy, Giemsa and EB/AO stained cells. Fragmented DNA further confirmed its capacity to induce apoptosis. No lethality was observed with brine shrimps. The results suggest that Semecarpus parvifolia Thw induces apoptosis in HEp-2 cells through a NO dependent pathway.

Additional corpus biopsy enhances the detection of Helicobacter pylori infection in a background of gastritis with atrophy

PubMed Central

2012-01-01

Background The best sites for biopsy-based tests to evaluate H. pylori infection in gastritis with atrophy are not well known. This study aimed to evaluate the site and sensitivity of biopsy-based tests in terms of degree of gastritis with atrophy. Methods One hundred and sixty-four (164) uninvestigated dyspepsia patients were enrolled. Biopsy-based tests (i.e., culture, histology Giemsa stain and rapid urease test) and non-invasive tests (anti-H. pylori IgG) were performed. The gold standard of H. pylori infection was defined according to previous criteria. The sensitivity, specificity, positive predictive rate and negative predictive rate of biopsy-based tests at the gastric antrum and body were calculated in terms of degree of gastritis with atrophy. Results The prevalence rate of H. pylori infection in the 164 patients was 63.4%. Gastritis with atrophy was significantly higher at the antrum than at the body (76% vs. 31%; p<0.001). The sensitivity of biopsy-based test decreased when the degree of gastritis with atrophy increased regardless of biopsy site (for normal, mild, moderate, and severe gastritis with atrophy, the sensitivity of histology Giemsa stain was 100%, 100%, 88%, and 66%, respectively, and 100%, 97%, 91%, and 66%, respectively, for rapid urease test). In moderate to severe antrum or body gastritis with atrophy, additional corpus biopsy resulted in increased sensitivity to 16.67% compare to single antrum biopsy. Conclusions In moderate to severe gastritis with atrophy, biopsy-based test should include the corpus for avoiding false negative results. PMID:23272897

Diagnosis of clinical bovine mastitis by fine needle aspiration followed by staining and scanning electron microscopy in a Prototheca zopfii outbreak.

PubMed

da Costa, Elizabeth Oliveira; Ribeiro, Márcio Garcia; Ribeiro, Andréa Rentz; Rocha, Noeme Sousa; de Nardi Júnior, Geraldo

2004-07-01

Biopsy by fine needle aspiration together with microbiological examination and scanning electron microscopy were evaluated in diagnosis of clinical bovine mastitis in a Prototheca zopfii outbreak. Fine needle aspiration was performed in 21 mammary quarters from ten Holstein cows presenting clinical mastitis caused by P. zopfii. The algae were previously identified in the microbiological examination of milk collected from these cows. Material aspirated from these 21 mammary glands was submitted to cytological staining (Gram, Giemsa and/or Shor staining). Fine needle aspiration enabled cytological identification of the algae in these 21 mammary glands, from which P. zopfii was isolated in the milk. Simultaneously, five mammary fragments collected by fine needle aspiration from these 21 mammary glands presenting clinical mastitis were also submitted to microbiological examination. P. zopfii was also isolated from these five fragments. Scanning electron microscopy technique also identified three of these five P zopfii strains isolated from mammary fragments collected by cytological aspiration. These results suggest that fine needle aspiration may be an alternative method for the diagnosis of clinical mastitis.

Investigation of Helicobacter pylori in tonsillary tissue with Pronto Dry test and pathologic examination.

PubMed

Aslan, Sundus; Yilmaz, Ismail; Bal, Nebil; Sener, Mesut; Butros, Reha; Demirhan, Beyhan; Ozluoglu, Levent N

2007-09-01

The objectives of this clinical study were to identify, by means of the Pronto Dry test and pathologic examination, Helicobacter pylori (HP) in tonsillary tissue and to establish the role of HP in tonsillary microbiology by identifying that bacterium in the tonsillary mucosa or within the tonsil core. The subjects consisted of 52 patients (25 men and 27 women; age range, 3-65 years; mean age, 15.1+/-14.5 years) who were scheduled to undergo tonsillectomy for the treatment of chronic tonsillitis and who had not been treated with an antibiotic or a bismuth-containing compound for 6 months before the initiation of the study. In each patient, two specimens (one 4 mm x 4 mm tissue sample from the nonmucosal tonsil core and one 4 mm x 4 mm sample of mucosal tissue) were excised from both tonsils immediately after tonsillectomy. The specimens were placed in the Pronto Dry test kit, and the test results were obtained 1 h later. The remaining tonsillary tissues were submitted for pathologic analysis via hematoxylin-eosin stain, Giemsa stain, Warthin-Starry silver stain, and staining for inducible nitric oxide synthase (iNOS). The results of the Pronto Dry test were positive for HP in 42% (n=22) of the excised mucosal tissue and in 47% (n=24) of the excised core tissue. In 27% (n=14) of the patients, both the core and the mucosal tissues tested positive for HP. There was no significant difference between the positive Pronto Dry test ratios of the biopsies obtained from the mucosa and those obtained from the core (P=0.693). iNOS staining showed that macrophage iNOS activity was significantly higher (P=0.025) in biopsied mucosal tissues with a positive Pronto Dry test result than in those with a negative result. Light microscopy revealed no HP in samples stained with hematoxylin-eosin stain, Giemsa stain, or Warthin-Starry silver stain. Positive Pronto Dry test results and the results of iNOS staining showed that HP contributes to chronic tonsillitis, especially at the mucosal layer. Although HP does not colonize, it contributes to the chronic tonsillary inflammatory process as a triggering agent by affecting macrophages in the tonsil and thus increasing iNOS expression.

Spectral imaging perspective on cytomics.

PubMed

Levenson, Richard M

2006-07-01

Cytomics involves the analysis of cellular morphology and molecular phenotypes, with reference to tissue architecture and to additional metadata. To this end, a variety of imaging and nonimaging technologies need to be integrated. Spectral imaging is proposed as a tool that can simplify and enrich the extraction of morphological and molecular information. Simple-to-use instrumentation is available that mounts on standard microscopes and can generate spectral image datasets with excellent spatial and spectral resolution; these can be exploited by sophisticated analysis tools. This report focuses on brightfield microscopy-based approaches. Cytological and histological samples were stained using nonspecific standard stains (Giemsa; hematoxylin and eosin (H&E)) or immunohistochemical (IHC) techniques employing three chromogens plus a hematoxylin counterstain. The samples were imaged using the Nuance system, a commercially available, liquid-crystal tunable-filter-based multispectral imaging platform. The resulting data sets were analyzed using spectral unmixing algorithms and/or learn-by-example classification tools. Spectral unmixing of Giemsa-stained guinea-pig blood films readily classified the major blood elements. Machine-learning classifiers were also successful at the same task, as well in distinguishing normal from malignant regions in a colon-cancer example, and in delineating regions of inflammation in an H&E-stained kidney sample. In an example of a multiplexed ICH sample, brown, red, and blue chromogens were isolated into separate images without crosstalk or interference from the (also blue) hematoxylin counterstain. Cytomics requires both accurate architectural segmentation as well as multiplexed molecular imaging to associate molecular phenotypes with relevant cellular and tissue compartments. Multispectral imaging can assist in both these tasks, and conveys new utility to brightfield-based microscopy approaches. Copyright 2006 International Society for Analytical Cytology.

Detection of small number of Giardia in biological materials prepared from stray dogs.

PubMed

Esmailikia, Leila; Ebrahimzade, Elahe; Shayan, Parviz; Amininia, Narges

2017-12-20

Giardia lamblia is an intestinal protozoa with intermittent and low shedding especially in dogs, and the detection of Giardia is accompanied with problems such as sampling and diagnostic method. The objective of this study was to detection of Giardia in biological materials with low number of parasite using parasitological and molecular methods, and also to determine whether the examined stray dogs harbor known zoonotic genotype of Giardia. For this aim 85 fecal and duodenal samples were studied from which 1 was positive by Trichrome staining of stool, 4 were positive by staining of duodenal samples. The nested PCR analysis with primers derived from 18 SrRNA showed that the specific PCR product could be amplified in 4 stool and 4 duodenal samples. All positive samples in staining analysis were also positive in nested PCR. No amplification could be observed by nested PCR with primers derived from β giardin gene due to the single copy of gene. Interestingly, the extracted DNA from old fixed stained Giardia positive smears could be also amplified with primers derived from 18SrRNA gene. The sequence analysis of nested PCR products showed that they belong to the genotype D. In conclusion, it is to denote that the Trichrome or Giemsa methods were not suitable for the detection of small number of this parasite in stool and the nested PCR with primers derived from 18S rRNA gene can replace the traditional methods successfully. For detection of Giardia in stool, primers derived from β giardin will not be recommended.

Morphologic aspects of Tetratrichomonas didelphidis isolated from opossums Didelphis marsupialis and Lutreolina crassicaudata.

PubMed

Tasca, T; De Carli, G A; Glock, L; Jeckel-Neto, E A

2001-02-01

Tetratrichomonas didelphidis (Hegner & Ratcliffe, 1927) Andersen & Reilly, 1965 is a flagellate protozoan found in the intestine, cecum, and colon of Didelphis marsupialis. The parasitic protozoa used in this study was found and isolated in the intestine of opossums in Pavlova starch-containing medium in Florianópolis, State of Santa Catarina, Brazil, from D. marsupialis and Lutreolina crassicaudata. The strains were cultivated in Diamond medium without maltose and with starch solution, pH 7.5 at 28 degrees C. The specimens were stained by the Giemsa method and Heidenhain's iron hematoxylin. The light microscopy study of the trophozoites revealed the same morphologic characteristics as specimens previously described.

No significant level of inheritable interchromosomal aberrations in the progeny of bystander primary human fibroblasts after alpha particle irradiation

NASA Astrophysics Data System (ADS)

Hu, Burong; Zhu, Jiayun; Zhou, Hongning; Hei, Tom K.

2013-02-01

A major concern for bystander effects is the probability that normal healthy cells adjacent to the irradiated cells become genomically unstable and undergo further carcinogenesis after therapeutic irradiation or space mission where astronauts are exposed to low dose of heavy ions. Genomic instability is a hallmark of cancer cells. In the present study, two irradiation protocols were performed in order to ensure pure populations of bystander cells and the genomic instability in their progeny were investigated. After irradiation, chromosomal aberrations of cells were analyzed at designated time points using G2 phase premature chromosome condensation (G2-PCC) coupled with Giemsa staining and with multiplex fluorescent in situ hybridization (mFISH). Our Giemsa staining assay demonstrated that elevated yields of chromatid breaks were induced in the progeny of pure bystander primary fibroblasts up to 20 days after irradiation. mFISH assay showed no significant level of inheritable interchromosomal aberrations were induced in the progeny of the bystander cell groups, while the fractions of gross aberrations (chromatid breaks or chromosomal breaks) significantly increased in some bystander cell groups. These results suggest that genomic instability occurred in the progeny of the irradiation associated bystander normal fibroblasts exclude the inheritable interchromosomal aberration.

Isolation and morphology of Stem Cells from Deciduous Tooth (SHED) and Human Dental Pulp Stem Cells (hDPSC)

NASA Astrophysics Data System (ADS)

Ariffin, Shahrul Hisham Zainal; Manogaran, Thanaletchumi; Abidin, Intan Zarina Zainol; Senafi, Sahidan; Wahab, Rohaya Megat Abdul

2016-11-01

Dental pulp is a tissue obtained from pulp chamber of deciduous and permanent tooth which contain stem cells. Stem cell isolation procedure is performed to obtain cells from tissue using enzymatic digestion. The aim of this study is to isolate and observe the morphology of stem cells during passage 0 and passage 3. Dental pulp from deciduous and permanent tooth was enzymatically digested using collagenase Type I and cells obtained were cultured in DMEM-KO that contains 10% fetal bovine serum, 1% antibiotic-antimycotic solution and 0.001× GlutaMax®. During culture, cell morphology was observed under the microscope on day 3, 16 and 33 and captured using cellB software. Giemsa staining was conducted on cells at passage 3. Cells attached at the bottom of the flask on day 3 and started forming small colonies. Cells became confluent after approximately 4 weeks. Both Stem Cells from Deciduous Tooth (SHED) and Human Dental Pulp Stem Cells (hDPSC) exhibited fibroblast-like morphology during passage 0 and passage 3. Meanwhile, Giemsa staining at passage 3 revealed single intact nucleus surrounded by fibroblastic cytoplasm structure. It can be concluded that SHED and hDPSC showed consistent fibroblast-like morphology throughout culture period.

No significant level of inheritable interchromosomal aberrations in the progeny of bystander primary human fibroblast after alpha particle irradiation.

PubMed

Hu, Burong; Zhu, Jiayun; Zhou, Hongning; Hei, Tom K

2013-02-01

A major concern for bystander effects is the probability that normal healthy cells adjacent to the irradiated cells become genomically unstable and undergo further carcinogenesis after therapeutic irradiation or space mission where astronauts are exposed to low dose of heavy ions. Genomic instability is a hallmark of cancer cells. In the present study, two irradiation protocols were performed in order to ensure pure populations of bystander cells and the genomic instability in their progeny were investigated. After irradiation, chromosomal aberrations of cells were analyzed at designated time points using G 2 phase premature chromosome condensation (G 2 -PCC) coupled with Giemsa staining and with multiplex fluorescent in situ hybridization (mFISH). Our Giemsa staining assay demonstrated that elevated yields of chromatid breaks were induced in the progeny of pure bystander primary fibroblasts up to 20 days after irradiation. MFISH assay showed no significant level of inheritable interchromosomal aberrations were induced in the progeny of the bystander cell groups, while the fractions of gross aberrations (chromatid breaks or chromosomal breaks) significantly increased in some bystander cell groups. These results suggest that genomic instability occurred in the progeny of the irradiation associated bystander normal fibroblasts exclude the inheritable interchromosomal aberration.

Clinicoepidemiologic pattern of cutaneous leishmaniasis and molecular characterization of its causative agent in Hajjah governorate, northwest of Yemen.

PubMed

Mogalli, Nabil M; El Hossary, Shabaan S; Khatri, Mishri Lal; Mukred, Abdualdaim M; Kassem, Hala A; El Sawaf, Bahira M; Ramadan, Nadia F

2016-11-01

The clinicoepidemiologic profile of 143 cases (93 males and 50 females) with cutaneous leishmaniasis from 18 villages of Hajjah governorate, Yemen was studied. Dry-type lesions were seen in 98.6% and wet-type lesions in 1.4% of patients. Lesions were localized in all cases with different morphological patterns. Microscopic examination of Giemsa-stained slit smears revealed amastigotes in 74.1% of patients with dry-type lesions and 0% in patients with wet-type lesions. The burden of the parasites in the lesions was high indicating active transmission of the disease. Most cases were from villages with moderate altitude range (8001-1600m). All age groups were affected, but most cases were seen in ages from 5 to 15 years. Leishmania species identification was done for all cases by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The biopsic material was scraped from both Giemsa-stained and methanol-fixed smears. The molecular characterization of Leishmania species revealed Leishmania tropica as the causative agent of cutaneous leishmaniasis in Hajjah, Yemen. The risk factors associated with the transmission of the disease and recommendations for improving case detection were discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

External quality assessment of Giemsa-stained blood film microscopy for the diagnosis of malaria and sleeping sickness in the Democratic Republic of the Congo

PubMed Central

Mukadi, Pierre; Gillet, Philippe; Lukuka, Albert; Atua, Benjamin; Sheshe, Nicole; Kanza, Albert; Mayunda, Jean Bosco; Mongita, Briston; Senga, Raphaël; Ngoyi, John; Muyembe, Jean-Jacques; Jacobs, Jan

2013-01-01

Abstract Objective To report the findings of a second external quality assessment of Giemsa-stained blood film microscopy in the Democratic Republic of the Congo, performed one year after the first. Methods A panel of four slides was delivered to diagnostic laboratories in all provinces of the country. The slides contained: (i) Plasmodium falciparum gametocytes; (ii) P. falciparum trophozoites (reference density: 113 530 per µl); (iii) Trypanosoma brucei subspecies; and (iv) no parasites. Findings Of 356 laboratories contacted, 277 (77.8%) responded. Overall, 35.0% of the laboratories reported all four slides correctly but 14.1% reported correct results for 1 or 0 slides. Major errors included not diagnosing trypanosomiasis (50.4%), not recognizing P. falciparum gametocytes (17.5%) and diagnosing malaria from the slide with no parasites (19.0%). The frequency of serious errors in assessing parasite density and in reporting false-positive results was lower than in the previous external quality assessment: 17.2% and 52.3%, respectively, (P < 0.001) for parasite density and 19.0% and 33.3%, respectively, (P < 0.001) for false-positive results. Laboratories that participated in the previous quality assessment performed better than first-time participants and laboratories in provinces with a high number of sleeping sickness cases recognized trypanosomes more frequently (57.0% versus 31.2%, P < 0.001). Malaria rapid diagnostic tests were used by 44.3% of laboratories, almost double the proportion observed in the previous quality assessment. Conclusion The overall quality of blood film microscopy was poor but was improved by participation in external quality assessments. The failure to recognize trypanosomes in a country where sleeping sickness is endemic is a concern. PMID:24052681

The Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections

PubMed Central

Peterson, Tracy S.; Spitsbergen, Jan M.; Feist, Stephen W.; Kent, Michael L.

2014-01-01

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation or small spores in nuclei (i.e., Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores are recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite’s acid fast, Giemsa, and H&E stains on eight aquatic microsporidian organisms that were readily available in our two laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum and an unidentified microsporidian from E. sinensis, UK. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the two microsporidia from invertebrates, Steinhausia mytilovum and the unidentified microsporidian from E. sinensis. PMID:21848126

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections.

PubMed

Peterson, Tracy S; Spitsbergen, Jan M; Feist, Stephen W; Kent, Michael L

2011-06-16

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite's acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.

An in vivo cytogenetic analysis of human oral squamous cell carcinoma

PubMed Central

Mohanta, Abhimanyu; Mohanty, Prafulla K.; Parida, Gadadhar

2015-01-01

Background: Oral cancer ranks in the top three of all cancers in India, which accounts for over 30% of all cancers reported in the country. The micronucleus test (MNT) is one of the most widely applied short term tests used in genetic toxicology to evaluate the mutagenicity and carcinogenicity. Aims: The present study aims at an in vivo cytogenetic analysis of human oral squamous cell carcinoma and to assess the applicability of MNT in diagnosing early detection of oral carcinoma. Materials and Methods: Exfoliated scrape smears were collected from the clinically diagnosed 136 patients suffering from oral precancerous and cancerous lesions. The wet fixed smears were stained by adopting Papanicolaou's staining protocol and counter-stained with Giemsa's solution. Results: The frequency of micronucleated cells has been observed to be in increasing order with the increase of the age-groups and from control to precancerous to cancerous cases significantly in both sexes. Conclusion: Micronucleus formation in the oral mucosa could be a biomarker of genetic damage and also a potential onco-indicator in the long run of oral carcinogenesis. Therefore, MNT can be applied for the early detection of oral carcinoma in the human being. PMID:26942142

Symptomatic candidiasis: Using self sampled vaginal smears to establish the presence of Candida, lactobacilli, and Gardnerella vaginalis.

PubMed

Engberts, M K; Boon, M E; van Haaften, M; Heintz, A P M

2007-10-01

In a prospective cohort study, 10 symptomatic women with recurrent vulvovaginal candidiasis were taught how to prepare vaginal smears of their own vaginal fluids on days 7, 14, 21, and 28. The 40 smears were stained with the PAS-method and examined by three different cytopathologists for presence of Candida. Thereafter, the smears were restained with Giemsa-stain to determine presence of lactobacilli, Gardnerella vaginalis ("clue cells") and neutrophils. All three cytopathologists unequivocally established Candida blastospores and (pseudo)hyphae in 27 out of the 40 PAS-stained vaginal smears, whereas in the remaining 13 smears Candida was not found. All 10 patients had Candida in their smears during the second half of their menstrual cycle.Self sampled smears prove to be reliable for establishing the presence of Candida in symptomatic patients with candidiasis. Candida is associated with a lactobacillus-predominated vaginal flora, but with the absence of Gardnerella vaginalis. Further studies may be directed towards the interaction between the various members of the vaginal flora. This study should open molecular methodology for determining the possible interactions of lactobacilli and Candida. (c) 2007 Wiley-Liss, Inc.

Detection of the HTLV-I gene on cytologic smear slides.

PubMed

Kashima, Kenji; Nagahama, Junji; Sato, Keiji; Tanamachi, Hiroyuki; Gamachi, Ayako; Daa, Tsutomu; Nakayama, Iwao; Yokoyama, Shigeo

2002-01-01

To apply the polymerase chain reaction (PCR) for detection of the HTLV-I gene from cytologic smear slides. Samples were from seven cases of serum anti-ATL antibody (ATLA)-positive T-cell lymphoma and three from ATLA-negative T-cell lymphoma. Six of the seven ATLA-positive cases were confirmed to be ATLL by Southern blotting. From the seventh case a fresh sample for blotting could not obtained. DNA was extracted from the cytologic smear slides of all 10 cases; they had been stained with Papanicolaou or May-Giemsa stain, digested with proteinase K and precipitated with phenol and ethanol. The target sequence in the pX region of the HTLV-I gene was amplified by PCR. All seven ATLA-positive cases, including one that had not yet been confirmed by Southern blotting, showed a single band, as predicted, while the three ATLA-negative cases showed no band. If cytologic smear slides are available but a fresh sample is not, the PCR method should provide evidence that the virus is present since in our study sufficient DNA templates were successfully extracted from the stained cytologic smear slides for detection of the virus.

Standardization of blood smears prepared in transparent acetate: an alternative method for the microscopic diagnosis of malaria.

PubMed

Mello, Marcia B C; Luz, Francisco C; Leal-Santos, Fabio A; Alves, Eduardo R; Gasquez, Thamires M; Fontes, Cor J F

2014-06-17

Due to students' initial inexperience, slides are frequently broken and blood smears are damaged in microscopy training, leading to the need for their constant replacement. To minimize this problem a method of preparing blood smears on transparent acetate sheets was developed with the goal of implementing appropriate and more readily available teaching resources for the microscopic diagnosis of malaria. Acetate sheets derived from polyester were used to standardize the preparation and staining of thin and thick blood smears on transparent acetate sheets. Thick and thin blood smears were also prepared using the conventional method on glass slides. The staining was conducted using Giemsa staining for the thick and thin smears. Microscopic examination (1,000x) of the thin and thick blood smears prepared on transparent acetate produced high-quality images for both the parasites and the blood cells. The smears showed up on a clear background and with minimal dye precipitation. It was possible to clearly identify the main morphological characteristics of Plasmodium, neutrophils and platelets. After 12 months of storage, there was no change in image quality or evidence of fungal colonization. Preparation of thin and thick blood smears in transparent acetate for the microscopic diagnosis of malaria does not compromise the morphological and staining characteristics of the parasites or blood cells. It is reasonable to predict the applicability of transparent acetate in relevant situations such as the training of qualified professionals for the microscopic diagnosis of malaria and the preparation of positive specimens for competency assessment (quality control) of professionals and services involved in the diagnosis of malaria.

Counting malaria parasites with a two-stage EM based algorithm using crowsourced data.

PubMed

Cabrera-Bean, Margarita; Pages-Zamora, Alba; Diaz-Vilor, Carles; Postigo-Camps, Maria; Cuadrado-Sanchez, Daniel; Luengo-Oroz, Miguel Angel

2017-07-01

Malaria eradication of the worldwide is currently one of the main WHO's global goals. In this work, we focus on the use of human-machine interaction strategies for low-cost fast reliable malaria diagnostic based on a crowdsourced approach. The addressed technical problem consists in detecting spots in images even under very harsh conditions when positive objects are very similar to some artifacts. The clicks or tags delivered by several annotators labeling an image are modeled as a robust finite mixture, and techniques based on the Expectation-Maximization (EM) algorithm are proposed for accurately counting malaria parasites on thick blood smears obtained by microscopic Giemsa-stained techniques. This approach outperforms other traditional methods as it is shown through experimentation with real data.

Morphological and molecular characterization of a marine fish trypanosome from South Africa, including its development in a leech vector

PubMed Central

2014-01-01

Background Trypanosomes are ubiquitous blood parasites of marine and freshwater fishes, typically transmitted by aquatic leeches. Phylogenetic studies have been dominated by examples derived from freshwater fishes, with few marine representatives. Furthermore, life cycle studies on marine fish trypanosomes have focused on those of the northern hemisphere. In this investigation, we have examined the life cycle and molecular taxonomy of a marine fish trypanosome from South Africa. Methods To locate trypanosome stages, leeches were removed from fishes captured on the west and south coasts of South Africa, and fish blood films and leech squashes were Giemsa-stained and screened; leeches were also examined histologically. To determine whether trypanosome stages in fishes and leeches were of the same genotype, DNA was extracted from Giemsa-stained fish blood films and leech squashes, and from fish whole blood. Fragments of the 18S rRNA gene were amplified by PCR using trypanosome-specific primers and sequenced. Resulting sequence data were compared with each other and with published trypanosome 18S rDNA sequences, and used for phylogenetic analysis. Results Trypanosomes were detected in blood films from fishes of the families Clinidae, Blenniidae and Gobiidae. The flagellates ranged in size and staining properties within the films and across fish hosts. In squashes and histological sections of adult and juvenile leeches, identified as Zeylanicobdella arugamensis, trypanosome developmental stages were predominantly slender epimastigotes. Sequence data showed that trypanosomes derived from fishes were identical, irrespective of whether they were small or large forms; sequences derived largely from leech epimastigotes were also identical to those obtained from fish trypanosomes. Fish and leech trypanosome sequences fell into a marine fish aquatic clade, and aligned most closely with two trypanosome sequences from marine fishes off Norway. Conclusions Combined morphological and molecular methods indicate that the trypanosomes examined here represent a single pleomorphic species, rather than the three species described originally. This species is identified as Trypanosoma nudigobii Fantham, 1919 with the leech Z. arugamensis as its vector, and T. capigobii Fantham, 1919 and T. blenniclini Fantham, 1930 are regarded as junior synonyms of the species. Phylogenetic analysis establishes its affinity with marine fish trypanosomes off Norway. PMID:24460725

Morphological and molecular characterization of a marine fish trypanosome from South Africa, including its development in a leech vector.

PubMed

Hayes, Polly M; Lawton, Scott P; Smit, Nico J; Gibson, Wendy C; Davies, Angela J

2014-01-24

Trypanosomes are ubiquitous blood parasites of marine and freshwater fishes, typically transmitted by aquatic leeches. Phylogenetic studies have been dominated by examples derived from freshwater fishes, with few marine representatives. Furthermore, life cycle studies on marine fish trypanosomes have focused on those of the northern hemisphere. In this investigation, we have examined the life cycle and molecular taxonomy of a marine fish trypanosome from South Africa. To locate trypanosome stages, leeches were removed from fishes captured on the west and south coasts of South Africa, and fish blood films and leech squashes were Giemsa-stained and screened; leeches were also examined histologically. To determine whether trypanosome stages in fishes and leeches were of the same genotype, DNA was extracted from Giemsa-stained fish blood films and leech squashes, and from fish whole blood. Fragments of the 18S rRNA gene were amplified by PCR using trypanosome-specific primers and sequenced. Resulting sequence data were compared with each other and with published trypanosome 18S rDNA sequences, and used for phylogenetic analysis. Trypanosomes were detected in blood films from fishes of the families Clinidae, Blenniidae and Gobiidae. The flagellates ranged in size and staining properties within the films and across fish hosts. In squashes and histological sections of adult and juvenile leeches, identified as Zeylanicobdella arugamensis, trypanosome developmental stages were predominantly slender epimastigotes. Sequence data showed that trypanosomes derived from fishes were identical, irrespective of whether they were small or large forms; sequences derived largely from leech epimastigotes were also identical to those obtained from fish trypanosomes. Fish and leech trypanosome sequences fell into a marine fish aquatic clade, and aligned most closely with two trypanosome sequences from marine fishes off Norway. Combined morphological and molecular methods indicate that the trypanosomes examined here represent a single pleomorphic species, rather than the three species described originally. This species is identified as Trypanosoma nudigobii Fantham, 1919 with the leech Z. arugamensis as its vector, and T. capigobii Fantham, 1919 and T. blenniclini Fantham, 1930 are regarded as junior synonyms of the species. Phylogenetic analysis establishes its affinity with marine fish trypanosomes off Norway.

Operational research to inform a sub-national surveillance intervention for malaria elimination in Solomon Islands

PubMed Central

2012-01-01

Background Successful reduction of malaria transmission to very low levels has made Isabel Province, Solomon Islands, a target for early elimination by 2014. High malaria transmission in neighbouring provinces and the potential for local asymptomatic infections to cause malaria resurgence highlights the need for sub-national tailoring of surveillance interventions. This study contributes to a situational analysis of malaria in Isabel Province to inform an appropriate surveillance intervention. Methods A mixed method study was carried out in Isabel Province in late 2009 and early 2010. The quantitative component was a population-based prevalence survey of 8,554 people from 129 villages, which were selected using a spatially stratified sampling approach to achieve uniform geographical coverage of populated areas. Diagnosis was initially based on Giemsa-stained blood slides followed by molecular analysis using polymerase chain reaction (PCR). Local perceptions and practices related to management of fever and treatment-seeking that would impact a surveillance intervention were also explored using qualitative research methods. Results Approximately 33% (8,554/26,221) of the population of Isabel Province participated in the survey. Only one subject was found to be infected with Plasmodium falciparum (Pf) (96 parasites/μL) using Giemsa-stained blood films, giving a prevalence of 0.01%. PCR analysis detected a further 13 cases, giving an estimated malaria prevalence of 0.51%. There was a wide geographical distribution of infected subjects. None reported having travelled outside Isabel Province in the previous three months suggesting low-level indigenous malaria transmission. The qualitative findings provide warning signs that the current community vigilance approach to surveillance will not be sufficient to achieve elimination. In addition, fever severity is being used by individuals as an indicator for malaria and a trigger for timely treatment-seeking and case reporting. In light of the finding of a low prevalence of parasitaemia, the current surveillance system may not be able to detect and prevent malaria resurgence. Conclusion An adaption to the malERA surveillance framework is proposed and recommendations made for a tailored provincial-level surveillance intervention, which will be essential to achieve elimination, and to maintain this status while the rest of the country catches up. PMID:22462770

Web-based scoring of the dicentric assay, a collaborative biodosimetric scoring strategy for population triage in large scale radiation accidents.

PubMed

Romm, H; Ainsbury, E; Bajinskis, A; Barnard, S; Barquinero, J F; Barrios, L; Beinke, C; Puig-Casanovas, R; Deperas-Kaminska, M; Gregoire, E; Oestreicher, U; Lindholm, C; Moquet, J; Rothkamm, K; Sommer, S; Thierens, H; Vral, A; Vandersickel, V; Wojcik, A

2014-05-01

In the case of a large scale radiation accident high throughput methods of biological dosimetry for population triage are needed to identify individuals requiring clinical treatment. The dicentric assay performed in web-based scoring mode may be a very suitable technique. Within the MULTIBIODOSE EU FP7 project a network is being established of 8 laboratories with expertise in dose estimations based on the dicentric assay. Here, the manual dicentric assay was tested in a web-based scoring mode. More than 23,000 high resolution images of metaphase spreads (only first mitosis) were captured by four laboratories and established as image galleries on the internet (cloud). The galleries included images of a complete dose effect curve (0-5.0 Gy) and three types of irradiation scenarios simulating acute whole body, partial body and protracted exposure. The blood samples had been irradiated in vitro with gamma rays at the University of Ghent, Belgium. Two laboratories provided image galleries from Fluorescence plus Giemsa stained slides (3 h colcemid) and the image galleries from the other two laboratories contained images from Giemsa stained preparations (24 h colcemid). Each of the 8 participating laboratories analysed 3 dose points of the dose effect curve (scoring 100 cells for each point) and 3 unknown dose points (50 cells) for each of the 3 simulated irradiation scenarios. At first all analyses were performed in a QuickScan Mode without scoring individual chromosomes, followed by conventional scoring (only complete cells, 46 centromeres). The calibration curves obtained using these two scoring methods were very similar, with no significant difference in the linear-quadratic curve coefficients. Analysis of variance showed a significant effect of dose on the yield of dicentrics, but no significant effect of the laboratories, different methods of slide preparation or different incubation times used for colcemid. The results obtained to date within the MULTIBIODOSE project by a network of 8 collaborating laboratories throughout Europe are very promising. The dicentric assay in the web based scoring mode as a high throughput scoring strategy is a useful application for biodosimetry in the case of a large scale radiation accident.

Storage and qualification of viable intact human amniotic graft and technology transfer to a tissue bank.

PubMed

Laurent, Romain; Nallet, Aurélie; Obert, Laurent; Nicod, Laurence; Gindraux, Florelle

2014-06-01

Human amniotic membrane (hAM) is known to have good potential to help the regeneration of tissue. It has been used for over 100 years in many medical disciplines because of its properties, namely a scaffold containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. In order to use this "boosted membrane" as an advanced therapeutic medicinal product for bone repair, we aimed to observe the influence of tissue culture and/or cryopreservation on cell viability and tissue structure, and secondly, to adapt to a tissue bank, identify easy processes to store hAM containing viable cells and to verify the quality of the graft before its release for use. To this end, we tested different published culture or cryopreservation storage conditions and cell viability assays. Tissue structure was evaluated by Giemsa staining and was compared to histological analysis. Preliminary results show no dramatic decrease in cell viability in cultured hAM as compared to cryopreserved hAM, but tissue structure alterations were observed with both storage conditions. Histological and immunohistochemical data highlight that tissue damage was associated with significantly modified protein expression, which could lead to a possible loss of differentiation potential. Finally, we report that trypan blue and Giemsa staining could constitute controls that are "materially and easily transferable" to a tissue bank.

Epidemiological analysis of Leishmania tropica strains and giemsa-stained smears from Syrian and Turkish leishmaniasis patients using multilocus microsatellite typing (MLMT)

PubMed Central

Nasereddin, Abed; Onay, Hüseyin; Karaca, Emin; Özkeklikçi, Ahmet; Jaffe, Charles L.; Kuhls, Katrin; Özbilgin, Ahmet; Ertabaklar, Hatice; Demir, Samiye; Özbel, Yusuf; Töz, Seray

2017-01-01

Turkey is located in an important geographical location, in terms of the epidemiology of vector-borne diseases, linking Asia and Europe. Cutaneous leishmaniasis (CL) is one of the endemic diseases in a Turkey and according to the Ministry Health of Turkey, 45% of CL patients originate from Şanlıurfa province located in southeastern Turkey. Herein, the epidemiological status of CL, caused by L. tropica, in Turkey was examined using multilocus microsatellite typing (MLMT) of strains obtained from Turkish and Syrian patients. A total of 38 cryopreserved strains and 20 Giemsa-stained smears were included in the present study. MLMT was performed using 12 highly specific microsatellite markers. Delta K (ΔK) calculation and Bayesian statistics were used to determine the population structure. Three main populations (POP A, B and C) were identified and further examination revealed the presence of three subpopulations for POP B and C. Combined analysis was performed using the data of previously typed L. tropica strains and Mediterranean and Şanlıurfa populations were identified. This finding suggests that the epidemiological status of L. tropica is more complicated than expected when compared to previous studies. A new population, comprised of Syrian L. tropica samples, was reported for the first time in Turkey, and the data presented here will provide new epidemiological information for further studies. PMID:28403153

Surveillance of vector-borne pathogens under imperfect detection: lessons from Chagas disease risk (mis)measurement.

PubMed

Minuzzi-Souza, Thaís Tâmara Castro; Nitz, Nadjar; Cuba, César Augusto Cuba; Hagström, Luciana; Hecht, Mariana Machado; Santana, Camila; Ribeiro, Marcelle; Vital, Tamires Emanuele; Santalucia, Marcelo; Knox, Monique; Obara, Marcos Takashi; Abad-Franch, Fernando; Gurgel-Gonçalves, Rodrigo

2018-01-09

Vector-borne pathogens threaten human health worldwide. Despite their critical role in disease prevention, routine surveillance systems often rely on low-complexity pathogen detection tests of uncertain accuracy. In Chagas disease surveillance, optical microscopy (OM) is routinely used for detecting Trypanosoma cruzi in its vectors. Here, we use replicate T. cruzi detection data and hierarchical site-occupancy models to assess the reliability of OM-based T. cruzi surveillance while explicitly accounting for false-negative and false-positive results. We investigated 841 triatomines with OM slides (1194 fresh, 1192 Giemsa-stained) plus conventional (cPCR, 841 assays) and quantitative PCR (qPCR, 1682 assays). Detections were considered unambiguous only when parasitologists unmistakably identified T. cruzi in Giemsa-stained slides. qPCR was >99% sensitive and specific, whereas cPCR was ~100% specific but only ~55% sensitive. In routine surveillance, examination of a single OM slide per vector missed ~50-75% of infections and wrongly scored as infected ~7% of the bugs. qPCR-based and model-based infection frequency estimates were nearly three times higher, on average, than OM-based indices. We conclude that the risk of vector-borne Chagas disease may be substantially higher than routine surveillance data suggest. The hierarchical modelling approach we illustrate can help enhance vector-borne disease surveillance systems when pathogen detection is imperfect.

Laboratory diagnostics of malaria

NASA Astrophysics Data System (ADS)

Siahaan, L.

2018-03-01

Even now, malaria treatment should only be administered after laboratory confirmation. There are several principal methods for diagnosing malaria. All these methods have their disadvantages.Presumptive treatment of malaria is widely practiced where laboratory tests are not readily available. Microscopy of Giemsa-stained thick and thin blood films remains the gold standard for the diagnosis of malaria infection. The technique of slide preparation, staining and reading are well known and standardized, and so is the estimate of the parasite density and parasite stages. Microscopy is not always available or feasible at primary health services in limited resource settings due to cost, lack of skilled manpower, accessories and reagents required. Rapid diagnostic tests (RDTs) are potential tools for parasite-based diagnosis since the tests are accurate in detecting malaria infections and are easy to use. The test is based on the capture of parasite antigen that released from parasitized red blood cells using monoclonal antibodies prepared against malaria antigen target. Polymerase Chain Reaction (PCR), depend on DNA amplification approaches and have higher sensitivity than microscopy. PCR it is not widely used due to the lack of a standardized methodology, high costs, and the need for highly-trained staff.

Characterization of intracellular inclusions in the urothelium of mice exposed to inorganic arsenic.

PubMed

Dodmane, Puttappa R; Arnold, Lora L; Muirhead, David E; Suzuki, Shugo; Yokohira, Masanao; Pennington, Karen L; Dave, Bhavana J; Lu, Xiufen; Le, X Chris; Cohen, Samuel M

2014-01-01

Inorganic arsenic (iAs) is a known human carcinogen at high exposures, increasing the incidences of urinary bladder, skin, and lung cancers. In most mammalian species, ingested iAs is excreted mainly through urine primarily as dimethylarsinic acid (DMA(V)). In wild-type (WT) mice, iAs, DMA(V), and dimethylarsinous acid (DMA(III)) exposures induce formation of intramitochondrial urothelial inclusions. Arsenite (iAs(III)) also induced intranuclear inclusions in arsenic (+3 oxidation state) methyltransferase knockout (As3mt KO) mice. The arsenic-induced formation of inclusions in the mouse urothelium was dose and time dependent. The inclusions do not occur in iAs-treated rats and do not appear to be related to arsenic-induced urothelial cytotoxicity. Similar inclusions in exfoliated urothelial cells from humans exposed to iAs have been incorrectly identified as micronuclei. We have characterized the urothelial inclusions using transmission electron microscopy (TEM), DNA-specific 4',6-diamidino-2-phenylindole (DAPI), and non-DNA-specific Giemsa staining and determined the arsenical content. The mouse inclusions stained with Giemsa but not with the DAPI stain. Analysis of urothelial mitochondrial- and nuclear-enriched fractions isolated from WT (C57BL/6) and As3mt KO mice exposed to arsenate (iAs(V)) for 4 weeks showed higher levels of iAs(V) in the treated groups. iAs(III) was the major arsenical present in the enriched nuclear fraction from iAs(V)-treated As3mt KO mice. In conclusion, the urothelial cell inclusions induced by arsenicals appear to serve as a detoxifying sequestration mechanism similar to other metals, and they do not represent micronuclei.

Role of FNA and Special Stains in Rapid Cytopathological Diagnosis of Pulmonary Nocardiosis.

PubMed

Sood, Ridhi; Tyagi, Ruchita; Selhi, Pavneet Kaur; Kaur, Gursheen; Kaur, Harpreet; Singh, Akashdeep

2018-01-01

Nocardia, a gram-positive aerobic bacillus of the Actinomycetales family, is a significant opportunistic pathogen in immunocompromised individuals. Clinical and radiological features of pulmonary nocardiosis are nonspecific and can be misdiagnosed as tuberculosis, pneumocystis, staphylococcal or fungal infections, or as malignancy. Aspiration cytology with special stains is a quick and effective approach for accurate diagnosis. We present 7 cases of pulmonary nocardiosis, admitted to the pathology department in a tertiary-care hospital in Punjab. Clinical findings, immune status, laboratory tests, chest radiographs, and computed tomography scans were reviewed. Cytologically, special stains like 1% Ziehl-Neelsen (ZN), 20% ZN, periodic acid-Schiff (PAS), Grocott methenamine silver (GMS), and reticulin stains were studied along with May-Grünwald Giemsa, Papanicolaou, and hematoxylin and eosin. All the patients were immunocompromised. The radiological changes were nonspecific. Cytomorphology showed acute and chronic inflammatory infiltrates with necrosis. None of the cases showed well-defined granulomas. GMS, modified 1% ZN and, Gordon and Sweet reticulin stains highlighted the delicate filamentous bacteria in all cases. PAS and 20% ZN stain for tuberculous bacilli were uniformly negative. FNAC can provide a quick and accurate diagnosis of nocardiosis and thereby facilitate timely medical management. © 2018 S. Karger AG, Basel.

Comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma.

PubMed

Ankle, Madhuri R; Kale, Alka D; Charantimath, Seema

2007-01-01

Mitosis of cells gives rise to tissue integrity. Defects during mitosis bring about abnormalities. Excessive proliferation of cells due to increased mitosis is one such outcome, which is the hallmark in precancer and cancer. The localization of proliferating cells or their precursors may not be obvious and easy. Establishing an easy way to distinguish these mitotic cells will help in grading and understanding their biological potential. Although immunohistochemistry is an advanced method in use, the cost and time factor makes it less feasible for many laboratories. Selective histochemical stains like toluidine blue, giemsa and crystal violet have been used in tissues including the developing brain, neural tissue and skin. 1) To compare the staining of mitotic cells in haematoxylin and eosin with that in crystal violet. 2) To compare the number of mitotic figures present in normal oral mucosa, epithelial dysplasia and oral squamous cell carcinoma in crystal violet-stained sections with that in H and E-stained sections. Ten tissues of normal oral mucosa and 15 tissues each of oral epithelial dysplasia seen in tobacco-associated leukoplakia and squamous cell carcinoma were studied to evaluate the selectivity of 1% crystal violet for mitotic figures. The staining was compared with standard H and E staining. Statistical analysis was done using Mann-Whitney U test. A statistically significant increase in the mean mitotic count was observed in crystal violet-stained sections of epithelial dysplasia as compared to the H and E-stained sections (p=0.0327). A similar increase in the mitotic counts was noted in crystal violet-stained sections of oral squamous cell carcinoma as compared to the H and E-stained sections.(p=0.0443). No significant difference was found in the mitotic counts determined in dysplasia or carcinoma by either the crystal violet (p=0.4429) or the H and E-staining techniques (p=0.2717). One per cent crystal violet provides a definite advantage over the H and E-stained sections in selectively staining the mitotic figures.

Relationship of gastric Helicobacter pylori infection to Barrett’s esophagus and gastro-esophageal reflux disease in Chinese

PubMed Central

Zhang, Jun; Chen, Xiao-Li; Wang, Kang-Min; Guo, Xiao-Dan; Zuo, Ai-Li; Gong, Jun

2004-01-01

AIM: To evaluate the relationship of Helicobacter pylori infection to reflux esophagitis (RE), Barrett’s esophagus (BE) and gastric intestinal metaplasia (IM). METHODS: RE, BE and gastric IM were determined by upper endoscopy. Patients were divided into 2 groups; those with squamocolumnar junction (SCJ) beyond gastroesophageal junction (GEJ) ≥ 3 cm (group A), and those with SCJ beyond GEJ < 3 cm (group B). Biopsy specimens were obtained endoscopically from just below the SCJ, gastric antrum along the greater and lesser curvature. Pathological changes and H pylori infection were determined by HE staining, Alcian blue staining and Giemsa staining. RESULTS: The prevalence of H pylori infection was 46.93%. There was no difference in the prevalence between males and females. The prevalence of H pylori infection decreased stepwise significantly from RE grade I to III. There was no difference in the prevalence between the two groups, and between long-segment and short-segment BE. In distal stomach, prevalence of H pylori infection was significantly higher in patients with IM than those without IM. CONCLUSION: There is a protective role of H pylori infection to GERD. There may be no relationship between H pylori infection of stomach and BE. H pylori infection is associated with the development of IM in the distal stomach. PMID:14991936

[Morphofunctional characteristics of immunocompetent cells in dysplasia of the breast].

PubMed

Dikshteĭn, E A; Burlak, Iu P; Shevchenko, N I

1985-01-01

Immunocompetent cells were studied in the stroma and epithelium of 34 cases of mammary gland dysplasia. The following stainings were used for light microscopy: hematoxylin and eosin, methods of Brachet, van Gieson, Romanovsky-Giemsa, hallocyanine alums, Gomori, PAS-reaction as well as the determination of acid and alkaline phosphatases, glucose-6-phosphate and succinate dehydrogenase were used. 14 cases were studied ultrastructurally. Two types of small B lymphocytes and one type of large lymphocytes, stromal macrophages are described. Their morphofunctional characteristics is given and their properties in proliferating and non-proliferating fibroadenomatosis are shown. Interaction of intraepithelial large granular lymphocytes (normal killers) with immature epithelial cells resulting in the death of these epitheliocytes is described. The results obtained are regarded as a morphological manifestation of the immune surveillance.

Epidemiological characteristics of Malassezia folliculitis and use of the May-Grünwald-Giemsa stain to diagnose the infection.

PubMed

Durdu, Murat; Güran, Mümtaz; Ilkit, Macit

2013-08-01

Various bacterial, fungal, parasitic, and viral pathogens can cause folliculitis, which is often mistakenly treated with antibiotics for months or even years. A laboratory diagnosis is required before therapy can be planned. Here, we describe the prevalence and risk factors, as well as the clinical, cytological, and mycological characteristics, of patients with Malassezia folliculitis (MF) in Adana, Turkey. We also report the treatment responses of the MF patients and describe the Malassezia spp. using culture-based molecular methods. Cytological examinations were performed in 264 folliculitis patients, 49 of whom (18.5%) were diagnosed with MF. The positivity of the May-Grünwald-Giemsa (MGG) smear was higher (100%) than that of the potassium hydroxide test (81.6%). Using Wood's light, yellow-green fluorescence was observed in 66.7% of the MF patients. Identification using the rDNA internal transcribed spacer region revealed that Malassezia globosa was the most common species, followed by Malassezia sympodialis, Malassezia restricta, and Malassezia furfur. The MF patients were treated with itraconazole capsules (200 mg/d) for 2 weeks. Complete recovery was observed in 79.6% of the patients. These novel findings help improve our current understanding of the epidemiological characteristics of MF and establish MGG as a practical tool for the diagnosis of MF. Copyright © 2013 Elsevier Inc. All rights reserved.

Pneumocystis jiroveci in HIV/AIDS patients: detection by FTA filter paper together with PCR in noninvasive induced sputum specimens.

PubMed

Jaijakul, Siraya; Saksirisampant, Wilai; Prownebon, Juraratt; Yenthakam, Sutin; Mungthin, Mathirut; Leelayoova, Saovanee; Nuchprayoon, Surang

2005-09-01

To detect P. jiroveci (previously named P. carinii) by PCR using FTA filter paper to extract the DNA, from noninvasive induced sputum samples of HIV/AIDS patients. Fifty two HIV/AIDS patients suspected of Pneumocystis jiroveci pneumonia (PJP) in King Chulalongkorn Memorial Hospital were recruited. Both cytological method and PCR with FTA filter paper technique were performed to detect P jiroveci from each specimen. The detectability rate of P. jiroveci infection was 21%. The PCR with FTA filter paper method was 4 folds much more sensitive than Giemsa staining technique. P. jiroveci was detected in 18% of the HIV/AIDS patients in spite of receiving standard PJP prophylaxis. Detection of P. jiroveci by using FTA filter paper together with PCR in induced sputum samples could detect more cases of P. jiroveci infection than by using cytological method. DNA extraction using the FTA filter paper was more rapid and convenient than other extraction methods. The causes of failure of PJP prophylaxis should be evaluated.

Dermatophilus congolensis infection in sheep and goats in Delta region of Tamil Nadu

PubMed Central

Chitra, M. Ananda; Jayalakshmi, K.; Ponnusamy, P.; Manickam, R.; Ronald, B. S. M.

2017-01-01

Aim: The study was conducted to isolate and identify Dermatophilus congolensis (DC) using conventional and molecular diagnostic techniques in scab materials collected from skin infections of sheep and goats in the Delta region of Tamil Nadu. Materials and Methods: A total of 20 scab samples collected from 18 goats and 2 sheep from Nagapattinam, Thanjavur, and Tiruvarur districts of Tamil Nadu. Smears were made from softened scab materials and stained by either Gram’s or Giemsa staining. Isolation was attempted on blood agar plates, and colonies were stained by Gram’s staining for morphological identification. Identification was also done by biochemical tests and confirmed by 16S rRNA polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the amplified product. Results: The peculiar laddering arrangement of coccoid forms in stained smears prepared from scab materials revealed the presence of DC. Isolated colonies from scab materials of sheep and goats on bovine blood agar plate were small, hemolytic, rough, adherent, and bright orange-yellow in color, but some colonies were white to cream color. Gram-staining of cultured organisms revealed Gram-positive branching filaments with various disintegration stages of organisms. 16S rRNA PCR yielded 500 bp amplicon specific for DC. Sequence analysis of a sheep DC isolate showed 99-100% sequence homology with other DC isolates available in NCBI database, and phylogenetic tree showed a close cluster with DC isolates of Congo, Nigeria, and Angola of Africa. Genes for virulence factors such as serine protease and alkaline ceramidase could not be detected by PCR in any of the DC strains isolated of this study. Conclusion: The presence of dermatophilosis in Tamil Nadu was established from this study. PMID:29263591

Operational research to inform a sub-national surveillance intervention for malaria elimination in Solomon Islands.

PubMed

Atkinson, Jo-An; Johnson, Marie-Louise; Wijesinghe, Rushika; Bobogare, Albino; Losi, L; O'Sullivan, Matthew; Yamaguchi, Yuka; Kenilorea, Geoffrey; Vallely, Andrew; Cheng, Qin; Ebringer, Andrew; Bain, Lisa; Gray, Karen; Harris, Ivor; Whittaker, Maxine; Reid, Heidi; Clements, Archie; Shanks, Dennis

2012-03-30

Successful reduction of malaria transmission to very low levels has made Isabel Province, Solomon Islands, a target for early elimination by 2014. High malaria transmission in neighbouring provinces and the potential for local asymptomatic infections to cause malaria resurgence highlights the need for sub-national tailoring of surveillance interventions. This study contributes to a situational analysis of malaria in Isabel Province to inform an appropriate surveillance intervention. A mixed method study was carried out in Isabel Province in late 2009 and early 2010. The quantitative component was a population-based prevalence survey of 8,554 people from 129 villages, which were selected using a spatially stratified sampling approach to achieve uniform geographical coverage of populated areas. Diagnosis was initially based on Giemsa-stained blood slides followed by molecular analysis using polymerase chain reaction (PCR). Local perceptions and practices related to management of fever and treatment-seeking that would impact a surveillance intervention were also explored using qualitative research methods. Approximately 33% (8,554/26,221) of the population of Isabel Province participated in the survey. Only one subject was found to be infected with Plasmodium falciparum (Pf) (96 parasites/μL) using Giemsa-stained blood films, giving a prevalence of 0.01%. PCR analysis detected a further 13 cases, giving an estimated malaria prevalence of 0.51%. There was a wide geographical distribution of infected subjects. None reported having travelled outside Isabel Province in the previous three months suggesting low-level indigenous malaria transmission. The qualitative findings provide warning signs that the current community vigilance approach to surveillance will not be sufficient to achieve elimination. In addition, fever severity is being used by individuals as an indicator for malaria and a trigger for timely treatment-seeking and case reporting. In light of the finding of a low prevalence of parasitaemia, the current surveillance system may not be able to detect and prevent malaria resurgence. An adaption to the malERA surveillance framework is proposed and recommendations made for a tailored provincial-level surveillance intervention, which will be essential to achieve elimination, and to maintain this status while the rest of the country catches up.

Micronucleus Assay: An Early Diagnostic Tool to Assess Genotoxic Changes in Patients with Tobacco Use, Oral Leukoplakia and Oral Submucous Fibrosis

PubMed Central

Ahuja, Puneet; Mehendiratta, Monica; Sharma, Mohit; Dutta, Jahnobi

2017-01-01

Introduction Micronuclei (MNi) are acentric chromatid or chromosome fragments produced via genetic damage through genotoxic agents contained in tobacco and betel nut. Evidently, the various Oral Potentially Malignant Disorders (OPMDs) like oral lichen Planus, oral leukoplakia and Oral Submucous Fibrosis (OSMF) demonstrate MNi, as a substantiation of genetic damage. As these changes can be easily appreciated in oral exfoliated cells, an exfoliated cell based MNi assay might be utilized as handy and non invasive biomonitoring tool for gauging the genetic damage and hence the propensity for malignant transformation in OPMDs. To this end, MNi are definitely easier to evaluate when compared to chromosome aberrations. Aim To compare the MNi frequency in normal mucosa, in individuals using various tobacco forms without oral leukoplakia, individuals using various tobacco forms with oral leukoplakia, and areca nut chewers with OSMF, using three different stains. Materials and Methods Oral exfoliated cells from 50 cases of normal mucosa (Group I), 50 cases of tobacco chewing people without Oral Leukoplakia (Group II), 50 cases of people with Oral Leukoplakia (Group III) and 50 cases of areca nut chewers with OSMF (Group IV) were taken. MNi frequencies were compared in these groups using three different stains i.e., Papanicolaou (PAP) stain, May Grunwald Giemsa (MGG) stain and Feulgen stain. The data between cases (Group II, III and IV) and control groups (Group I) was analyzed by Kruskal-Wallis Test. The comparison between two independent groups was done by Mann-Whitney U test and interstain comparison between cases and control was done by Wilcoxon Signed Rank Test and the individual p-value was obtained. Results A significant increase in the count was observed during transition of normal mucosa to OPMDs. The best stain for detecting MNi was PAP stain followed by MGG stain and Feulgen stain. Conclusion The higher mean MNi count for PAP stain and MGG stain could be attributed to nonspecific staining. Further study using a larger sample size on quantitative assessment of MNi count in various OPMDs is warranted. PMID:29207828

[Microbiological diagnosis of imported malaria].

PubMed

Torrús, Diego; Carranza, Cristina; Manuel Ramos, José; Carlos Rodríguez, Juan; Rubio, José Miguel; Subirats, Mercedes; Ta-Tang, Thuy-Huong

2015-07-01

Current diagnosis of malaria is based on the combined and sequential use of rapid antigen detection tests (RDT) of Plasmodium and subsequent visualization of the parasite stained with Giemsa solution in a thin and thick blood smears. If an expert microscopist is not available, should always be a sensitive RDT to rule out infection by Plasmodium falciparum, output the result immediately and prepare thick smears (air dried) and thin extensions (fixed with methanol) for subsequent staining and review by an expert microscopist. The RDT should be used as an initial screening test, but should not replace microscopy techniques, which should be done in parallel. The diagnosis of malaria should be performed immediately after clinical suspicion. The delay in laboratory diagnosis (greater than 3 hours) should not prevent the initiation of empirical antimalarial treatment if the probability of malaria is high. If the first microscopic examination and RDT are negative, they must be repeated daily in patients with high suspicion. If suspicion remains after three negative results must be sought the opinion of an tropical diseases expert. Genomic amplification methods (PCR) are useful as confirmation of microscopic diagnosis, to characterize mixed infections undetectable by other methods, and to diagnose asymptomatic infections with submicroscopic parasitaemia. Copyright © 2015 Elsevier España, S.L.U. All rights reserved.

Malaria problem in Afghanistan: malaria scanning results of the Turkish medical aid group after the war.

PubMed

Oner, Yaşar Ali; Okutan, Salih Erkan; Artinyan, Elizabeth; Kocazeybek, Bekir

2005-04-01

Malaria is a parasitic infection caused by Plasmodium species and it is especially seen in tropical and subtropical areas. We aimed to evaluate the effects of the infection in Afghanistan, which is an endemic place for malaria and had severe socio-economical lost after the war. We also compared these data with the ones that were recorded before the war. Blood samples were taken from 376 malaria suspected patients who come to the health center, established by the medical group of Istanbul Medical Faculty in 2002, Afghanistan. Blood samples were screened using the OPTIMAL Rapid Malaria Test and Giemsa staining method. In 95 (25.3%) patients diagnosis was malaria. In 65 patients (17.3%) the agent of the infection was P. falciparum and in 30 patients (8%) agents were other Plasmodium species.

A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

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

Moen, Scott T.; Hatcher, Christopher L.; Singh, Anup K.

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. Furthermore, this platform has great potential in both medical diagnostics and researchmore » applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.« less

A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

DOE PAGES

Moen, Scott T.; Hatcher, Christopher L.; Singh, Anup K.

2016-04-07

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. Furthermore, this platform has great potential in both medical diagnostics and researchmore » applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.« less

Comparative effectiveness of light-microscopic techniques and PCR in detecting Thelohania solenopsae (Microsporidia) infections in red imported fire ants (Solenopsis invicta).

PubMed

Milks, Maynard L; Sokolova, Yuliya Y; Isakova, Irina A; Fuxa, James R; Mitchell, Forrest; Snowden, Karen F; Vinson, S Bradleigh

2004-01-01

The main goal of this study was to compare the effectiveness of three staining techniques (calcofluor white M2R, Giemsa and modified trichrome), and the polymerase chain reaction (PCR) in detecting the microsporidium Thelohania solenopsae in red imported fire ants (Solenopsis invicta). The effect of the number of ants in a sample on the sensitivity of the staining techniques and the PCR, and the effect of three DNA extraction protocols on the sensitivity of PCR were also examined. In the first protocol, the ants were macerated and the crude homogenate was used immediately in the PCR. In the second protocol, the homogenate was placed on a special membrane (FTA card) that traps DNA, which is subsequently used in the PCR. In the third protocol, the DNA was purified from the homogenate by traditional phenol-chloroform extraction. Except for PCR using FTA cards, the sensitivity (number of samples positive for T. solenopsae) of all detection techniques increased with the number of ants in the sample. Overall, Giemsa was the least sensitive of all detection techniques. Calcofluor was more sensitive than modified trichrome with ants from one site and was equally as sensitive as PCR with crude DNA or a FTA card with ants from both sites. Trichrome staining was equally as sensitive as PCR with a FTA card at both sites, but it was less sensitive than PCR with crude DNA at one site. PCR on FTA cards was less sensitive than PCR with crude DNA for ants from one site but not the other. There was no difference whether crude or phenol-chloroform purified DNA was used as template. In summary, the results of this study show that PCR based on a crude DNA solution is equal to or more sensitive in detecting T. solenopsae than the other detection techniques investigated, and that it can be used as a reliable diagnostic tool for screening field samples of S. invicta for T. solenopsae. Nevertheless, ant smear stained with calcofluor or modified trichrome should be used to buttress findings from PCR.

Chlamydia trachomatis in the conjunctiva of children living in three rural areas in Mexico.

PubMed

Goldschmidt, Pablo; Vanzzini Zago, Virginia; Diaz Vargas, Lidia; Espinoza Garcia, Laura; Morales Montoya, Carlos; Peralta, Beatríz; Mercado, Mario

2007-07-01

Chlamydia trachomatis infections, in the context of extreme poverty, may trigger trachoma. Because the levels of C. trachomatis eye infections in Mexico are unknown, this study sought to determine if C. trachomatis was present in the conjunctiva of children living in three poor, rural areas of the country. Clinical diagnosis of conjunctival follicles in children was conducted during the 2004 visual acuity assessment campaigns in rural areas of the states of Chiapas, Oaxaca, and Zacatecas. C. trachomatis detection was carried out by sampling the children with follicles and examining the specimens after Giemsa or microimmunofluorescence (MIF) staining. A total of 941 children from 6 to 12 years of age were examined in 2004. Of the 484 in Chiapas, 30% were found to have follicles; of the 181 in Zacatecas, 22%; and of the 276 in Oaxaca, 42%. C. trachomatis was detected at levels ranging between 2% and 5%; positive by Giemsa in 4.5% of the children with follicles, and by MIF in 15.5%. Considering that the chlamydiae sampling procedures and detection methods used in this study were not the most sensitive, the results underestimate the chlamydial eye infections and represent a conservative assessment of a potential risk for preventable visual impairment. Because C. trachomatis was detected here at levels similar to those reported for low-endemic trachoma areas, health authorities should be prepared to implement appropriate measures should it be confirmed that the visual health of Mexico's children is at risk.

The new situation of cutaneous leishmaniasis after Syrian civil war in Gaziantep city, Southeastern region of Turkey.

PubMed

Özkeklikçi, Ahmet; Karakuş, Mehmet; Özbel, Yusuf; Töz, Seray

2017-02-01

Cutaneous leishmaniasis (CL) is an important public health problem with around 2.000 autochthonous reported cases each year in Turkey. Due to the civil war in Syria, Turkey received around three million refugees and they are mainly located at either camps or homes in south/southeastern part of Turkey. In the present study, we aimed to collect samples from CL suspected patients admitting to State Hospital in Gaziantep City and perform parasitological and DNA-based techniques for diagnosis as well as species identification of the parasite for better understanding the prevalence of each species among Turkish and Syrian patients in the region. The collection of samples was carried out between January 2009 and July 2015. The lesion aspiration samples were taken and stained with Giemsa stain followed by microscopical examination for parasitological diagnosis. After the DNA extraction from Giemsa stained slides, real time and semi-nested PCRs both targeting ITS1 region were performed for molecular diagnosis and species identification. A total of 567 people were admitted to the hospital with the suspicion of CL and 263 (46.4%) of them were found to be positive by parasitological examination. One hundred seventy-four (66.15%), 88 (33.46%) and 1 (0.38%) of them were Turkish, Syrians and Afghan, respectively. Slide samples obtained from 34 CL suspected patients were analyzed by PCR and 20 of them were found positive. Eighteen (13 Turkish and 13 Syrians) of the positive samples were identified as L. tropica, while two (1 Turkish and 1 Syrian) of them were L. infantum. In conclusion, the effects of Syrian civil war on the epidemiology of CL in Gaziantep city is demonstrated in the present study. The use of molecular tool in the diagnosis of leishmaniasis is effective, sensitive and time saving which will enable the species typing. Species typing of the causative agent in endemic areas will bring valuable data to epidemiological knowledge. Copyright © 2016 Elsevier B.V. All rights reserved.

Quality assessment of malaria laboratory diagnosis in South Africa.

PubMed

Dini, Leigh; Frean, John

2003-01-01

To assess the quality of malaria diagnosis in 115 South African laboratories participating in the National Health Laboratory Service Parasitology External Quality Assessment Programme we reviewed the results from 7 surveys from January 2000 to August 2002. The mean percentage incorrect result rate was 13.8% (95% CI 11.3-16.9%), which is alarmingly high, with about 1 in 7 blood films being incorrectly interpreted. Most participants with incorrect blood film interpretations had acceptable Giemsa staining quality, indicating that there is less of a problem with staining technique than with blood film interpretation. Laboratories in provinces in which malaria is endemic did not necessarily perform better than those in non-endemic areas. The results clearly suggest that malaria laboratory diagnosis throughout South Africa needs strengthening by improving laboratory standardization and auditing, training, quality assurance and referral resources.

Nucleolar organizer regions in Sittasomus griseicapillus and Lepidocolaptes angustirostris (Aves, Dendrocolaptidae): Evidence of a chromosome inversion.

PubMed

de Oliveira Barbosa, Marcelo; da Silva, Rubens Rodrigues; de Sena Correia, Vanessa Carolina; Dos Santos, Luana Pereira; Garnero, Analía Del Valle; Gunski, Ricardo José

2013-03-01

Cytogenetic studies in birds are still scarce compared to other vertebrates. Woodcreepers (Dendrocolaptidae) are part of a highly specialized group within the Suboscines of the New World. They are forest birds exclusive to the Neotropical region and similar to woodpeckers, at a comparable evolutionary stage. This paper describes for the first time the karyotypes of the Olivaceous and the Narrow-billed Woodcreeper using conventional staining with Giemsa and silver nitrate staining of the nucleolar organizer regions (Ag-NORs). Metaphases were obtained by fibular bone marrow culture. The chromosome number of the Olivaceous Woodcreeper was 2n = 82 and of the Narrow-billed Woodcreeper, 2n = 82. Ag-NORs in the largest macrochromosome pair and evidence of a chromosome inversion are described herein for the first time for this group.

The genetic diversity of metronidazole susceptibility in Trichomonas vaginalis clinical isolates in an Egyptian population.

PubMed

Abdel-Magied, Aida A; El-Kholya, El-Said I; Abou El-Khair, Salwa M; Abdelmegeed, Eman S; Hamoudaa, Marwa M; Mohamed, Sara A; El-Tantawy, Nora Labeeb

2017-11-01

Trichomoniasis is the most common curable sexually transmitted disease worldwide. Resistance to metronidazole in treating trichomoniasis is a problematic health issue. We aimed to determine the minimum lethal concentration (MLC) of metronidazole for Trichomonas vaginalis isolates detected in Mansoura, Egypt and studied the genotypic profile of these isolates. Vaginal swab specimens were obtained from 320 symptomatic and 100 asymptomatic females, for whom clinical examination, vaginal discharge wet mount, Giemsa stain, and culture in modified Diamond's media were performed. Metronidazole susceptibility testing by an aerobic tube assay was performed. Both sensitive and resistant isolates were examined by PCR amplification followed by restriction fragment length polymorphism (RFLP). Trichomonas vaginalis was identified in 49/420 (11.7%) using either culture or PCR, while wet mount and Giemsa stain detected the parasite in 8.1 and 7.6% of participants, respectively. After 48 h incubation, most isolates were sensitive to metronidazole with a minimal lethal concentration (MLC) of 1 μg/ml. Mild resistance was observed in two isolates with MLCs of 64 μg\\ml and mild to moderate resistance was observed in an additional two isolates with MLCs of 128 μg/ml. The four isolates that demonstrated low to moderate metronidazole resistance displayed a unique genotype band pattern by RFLP compared to the other 45 samples that were metronidazole sensitive. Our results highlight the presence of in vitro metronidazole tolerance in a few T. vaginalis isolates in Mansoura, Egypt that may lead to the development of drug resistance as well as the possibility of an identifying RFLP pattern in the isolates.

Targeting a Newly Established Spontaneous Feline Fibrosarcoma Cell Line by Gene Transfer

PubMed Central

Nande, Rounak; De Carlo, Flavia; Carper, Miranda; Claudio, Charlene D.; Denvir, Jim; Valluri, Jagan; Duncan, Gary C.; Claudio, Pier Paolo

2012-01-01

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas. PMID:22666387

Targeting a newly established spontaneous feline fibrosarcoma cell line by gene transfer.

PubMed

Nande, Rounak; Di Benedetto, Altomare; Aimola, Pierpaolo; De Carlo, Flavia; Carper, Miranda; Claudio, Charlene D; Denvir, Jim; Valluri, Jagan; Duncan, Gary C; Claudio, Pier Paolo

2012-01-01

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas.

Evaluation of the utility value of three diagnostic methods in the detection of malaria parasites in endemic area.

PubMed

Ugah, Uchenna Iyioku; Alo, Moses Nnaemeka; Owolabi, Jacob Oluwabusuyi; Okata-Nwali, Oluchi DivineGift; Ekejindu, Ifeoma Mercy; Ibeh, Nancy; Elom, Michael Okpara

2017-05-06

Malaria is a debilitating disease with high morbidity and mortality in Africa, commonly caused by different species of the genus Plasmodium in humans. Misdiagnosis is a major challenge in endemic areas because of other disease complications and technical expertise of the medical laboratory staff. Microscopic method using Giemsa-stained blood film has been the mainstay of diagnosis of malaria. However, since 1993 when rapid diagnostic test (RDT) kits were introduced, they have proved to be effective in the diagnosis of malaria. This study was aimed at comparing the accuracy of microscopy and RDTs in the diagnosis of malaria using nested PCR as the reference standard. Four hundred and twenty (420) venous blood specimens were collected from patients attending different General Hospitals in Ebonyi State with clinical symptoms of malaria. The samples were tested with Giemsa-stained microscopy and three RDTs. Fifty specimens were randomly selected for molecular analysis. Using different diagnostic methods, the prevalence of malaria among the subjects studied was 25.95% as detected by microscopy, prevalence found among the RDTs was 22.90, 15.20 and 54.80% for Carestart, SD Bioline PF and SD Bioline PF/PV, respectively. Molecular assay yielded a prevalence of 32%. The major specie identified was Plasmodium falciparum; there was co-infection of P. falciparum with Plasmodium malariae and Plasmodium ovale. The sensitivity and specificity of microscopy was 50.00 and 70.59% while that of the RDTs were (25.00 and 85.29%), (25.00 and 94.12%) and (68.75 and 52.94%) for Carestart, SD Bioline PF and SD Bioline PF/PV, respectively. Cohen's kappa coefficient was used to measure the level of agreement of the methods with nested PCR. Microscopy showed a moderate measure of agreement (k = 0.491), Carestart showed a good measure of agreement (k = 0.611), SD Bioline PF showed a fair measure of agreement (k = 0.226) while SD Bioline PF/PV showed a poor measure of agreement (k = 0.172). This study recommends that the policy of malaria diagnosis be changed such that the routine diagnosis of malaria is done by a combination of both microscopy and a RDT kit of high sensitivity and specificity so as to complement the errors associated with either of the methods. The finding of P. ovale in the study area necessitates an expanded molecular epidemiology of malaria within the study area.

A flow cytometric method for assessing viability of intraerythrocytic hemoparasites.

PubMed

Wyatt, C R; Goff, W; Davis, W C

1991-06-24

We have developed a rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites. The assay involves the selective uptake and metabolic conversion of hydroethidine to ethidium by live parasites present in intact erythrocytes. The red fluorescence imparted by ethidium intercalated into the DNA of the parasite permits the use of flow cytometry to distinguish infected erythrocytes with viable parasites from uninfected erythrocytes and erythrocytes containing dead parasites. Comparison of the fluorochromasia technique of enumerating the number and viability of hemoparasites in cultured erythrocytes with enumeration in Giemsa-stained films and uptake of [3H]hypoxanthine demonstrated the fluorochromasia technique yields comparable results. Studies with the hemoparasite, Babesia bovis, have shown the fluorochromasia technique can also be used to monitor the effect of parasiticidal drugs on parasites in vitro. The cumulative studies with the fluorochromasia assay suggest the assay will also prove useful in investigations focused on analysis of the immune response to hemoparasites and growth in vitro.

Endoparasites of Wild Rodents in Southeastern Iran

PubMed Central

Nateghpour, Mehdi; Motevalli-Haghi, Afsaneh; Akbarzadeh, Kamran; Akhavan, Amir Ahmad; Mohebali, Mehdi; Mobedi, Iraj; Farivar, Leila

2015-01-01

Background: This study was aimed to collect wild rodents for endoparasites determination in some parts of Sistan and Baluchistan Province, southeastern Iran nearby Pakistan and Afghanistan countries. Methods: A total of 100 wild rodents were captured alive with cage traps. Various samples were collected from blood and feces, also impression smear prepared from different organs. The samples were prepared by formalin-ether or stained with Giemsa, after that were examined under microscope. Results: All the caught rodents (47 Tatera indica, 44 Meriones hurriana, 5 Gerbilus nanus and 4 Meriones libycus) were studied for endoparasites emphasizing to their zoonotic aspects. Endoparasites including Spirurida, Hymenolepis diminuta, Hymenolepis nana feraterna, Trichuris trichiura, Skerjabino taenia, Trichostrongylus spp, Entamoeba muris, Chilomastix mesnili and Leishmania spp were parasitologically identified. Conclusion: Among 9 genera or species of the identified parasites at least 5 of them have zoonotic and public health importance. PMID:26114139

Improvement of a PCR test to diagnose infection by Mansonella ozzardi.

PubMed

Vera, Luana Janaína Souza; Basano, Sergio de Almeida; Camargo, Juliana de Souza Almeida Aranha; França, Andonai Krauze de; Ferreira, Ricardo de Godoi Mattos; Casseb, Almeida Andrade; Medeiros, Jansen Fernandes; Fontes, Gilberto; Camargo, Luís Marcelo Aranha

2011-01-01

Mansonelliasis is caused by Mansonella ozzardi. It is widespread in the Amazon region, with a high prevalence. The common exam of thick blood smears stained with Giemsa shows low efficacy levels and has been an obstacle to diagnosing individuals with low blood parasitemia. In order to increase diagnosis efficacy, the PCR technique was improved. PCR demonstrated the best performance, with sensitivity and negative predictive values (NPV) of 100%, followed by blood filtration through membrane filters, which showed a sensitivity of 88.9% and a NPV of 84.6%, when compared to thick blood smears.

"Filarial dance sign" real-time ultrasound diagnosis of filarial oophoritis.

PubMed

Panditi, Surekha; Shelke, Ashwini G; Thummalakunta, Laxmi Narasimha Praveen

2016-10-01

Filariasis is a parasitic disease caused by Filarial nematodes (Wuchereria bancrofti, Brugia malayi, and Brugia timori) that commonly causes lymphatic obstruction resulting in edema and increase in the size of the affected organ. Filariasis is diagnosed by identifying microfilariae on Giemsa stain. The immunochromatographic card test is diagnostic. Ultrasound is the imaging modality of choice for detecting adult filarial worms/microfilaria in the lymphatic system, which are responsible for the classic "filarial dance sign" caused by twirling movements of the microfilariae. © 2016 Wiley Periodicals, Inc. J Clin Ultrasound 44:500-501, 2016. © 2016 Wiley Periodicals, Inc.

Detection of species and molecular typing of Leishmania in suspected patients by targeting cytochrome b gene in Zahedan, southeast of Iran.

PubMed

Mirahmadi, Hadi; Rezaee, Nasrin; Mehravaran, Ahmad; Heydarian, Peyman; Raeghi, Saber

2018-05-01

Cutaneous leishmaniasis (CL) is one of the most important health problems that are capable of involving both tropical and subtropical areas, especially in Iran. This cross-sectional study aimed to differentiate the species that are able to cause CL in Zahedan city by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. It was conducted on 145 suspected CL patients in Zahedan city between 2014 and 2016. The smears were initially prepared, air-dried, fixed with absolute methanol, and stained with 10% Giemsa. Then, we examined the stained samples by a light microscope under 1000× magnifications. PCR assay targeted cytochrome b (cyt b ) gene using LCBF1 and LCBR2 primers and the products digested by Ssp1 enzymes. From 145 suspected CL patients, 76 (52.4%) were positive in microscopic examination. In addition, we detected gene of interest (cyt b ) in 98 (67.5%). The results of PCR-RFLP indicated that 53/98 (54%) cases were Leishmania major and 45/98 (46%) were Leishmania tropica , and the main species in these areas was L. major . We concluded that the microscopic examination is not sensitive enough and is not able to distinguish between different Leishmania species. Instead, molecular methods like PCR-RFLP can be appropriately used with promising results.

The comparison of detection methods of asymptomatic malaria in hypoendemic areas

NASA Astrophysics Data System (ADS)

Siahaan, L.; Panggabean, M.; Panggabean, Y. C.

2018-03-01

Malaria is still a problem that disrupts public health in North Sumatera. Late diagnosis will increase the chances of increased morbidity and mortality due to malaria. The early detection of asymptomatic malaria is one of the best efforts to reduce the transmission of the disease. Early detection is certainly must be done on suspect patients who have no malaria complaints. Passive Case Detection (PCD) methods seem hard to find asymptomatic malaria. This study was conducted to compare ACD (Active Case Detection) and PCD methods in asymptomatic malaria detection in the hypoendemic areas of malaria. ACD method is done by going to the sample based on secondary data. Meanwhile, PCD is done on samples that come to health services. Samples were taken randomly and diagnosis was confirmed by microscopic examination with 3% Giemsa staining, as gold standard of malaria diagnostics. There was a significant difference between ACD and PCD detection methods (p = 0.034), where ACD method was seen superior in detecting malaria patients in all categories, such as: clinical malaria (65.2%), asymptomatic malaria (65.1%) and submicroscopic malaria (58.5%). ACD detection methods are superior in detecting malaria sufferers, especially asymptomatic malaria sufferers.

Experimental reproduction of Potomac horse fever in horses with a newly isolated Ehrlichia organism.

PubMed

Dutta, S K; Myrup, A C; Rice, R M; Robl, M G; Hammond, R C

1985-08-01

Potomac horse fever, a recently recognized disease of equines, characterized by high fever, leukopenia, and a profuse diarrhea, was studied for its etiology. An Ehrlichia organism was isolated in equine macrophage-fibroblast cell cultures and mouse macrophage cell cultures from the mononuclear cells of blood of infected horses. The agent was continuously propagated in mouse macrophage cell cultures. The organism multiplied in the cytoplasm of mouse macrophage cells and was identified by Giemsa staining, acridine orange staining, and by indirect immunofluorescence with convalescent sera from infected horses. The disease was experimentally reproduced in horses inoculated with Ehrlichia-infected cell culture material. The Ehrlichia organism was reisolated from the blood of these infected horses during the course of the disease. Antibody against the organism was detected in the sera of experimentally infected horses. This study confirmed that the new Ehrlichia organism is the etiological agent of Potomac horse fever.

Cytomorphology of Circulating Colorectal Tumor Cells:A Small Case Series

PubMed Central

Marrinucci, Dena; Bethel, Kelly; Lazar, Daniel; Fisher, Jennifer; Huynh, Edward; Clark, Peter; Bruce, Richard; Nieva, Jorge; Kuhn, Peter

2010-01-01

Several methodologies exist to enumerate circulating tumor cells (CTCs) from the blood of cancer patients; however, most methodologies lack high-resolution imaging, and thus, little is known about the cytomorphologic features of these cells. In this study of metastatic colorectal cancer patients, we used immunofluorescent staining with fiber-optic array scanning technology to identify CTCs, with subsequent Wright-Giemsa and Papanicolau staining. The CTCs were compared to the corresponding primary and metastatic tumors. The colorectal CTCs showed marked intrapatient pleomorphism. In comparison to the corresponding tissue biopsies, cells from all sites showed similar pleomorphism, demonstrating that colorectal CTCs retain the pleomorphism present in regions of solid growth. They also often retain particular cytomorphologic features present in the patient's primary and/or metastatic tumor tissue. This study provides an initial analysis of the cytomorphologic features of circulating colon cancer cells, providing a foundation for further investigation into the significance and metastatic potential of CTCs. PMID:20111743

Longitudinal differentiation in Melipona mandacaia (Hymenoptera, Meliponini) chromosomes.

PubMed

Rocha, M P; Cruz, M P; Fernandes, A; Waldschmidt, A M; Silva-Júnior, J C; Pompolo, S G

2003-01-01

Melipona mandacaia is a stingless bee endemic to northeast Brasil. We describe the M. mandacaia karyotype using C-banding technique. fluorochrome staining and treatment with restriction enzymes and discuss the position of this species in the context of the phylogeny of the genus. Melipona mandacaia has 2n = 18 (14 SM + 2 M + 2 A). Heterochromatin was detected in the pericentromeric region of pairs 1, 2 and 8 and in the form of small blocks in the remaining pairs. Staining with base-specific fluorochromes showed that this heterochromatin was rich AT (QM and DAPI), except in the region corresponding to the NOR which was rich GC (CMA3) and was cleaved by the HaeIII enzyme. Melipona mandacaia is a member of Group I Melipona. Treatment with DraI/Giemsa discloses a larger number of bands than treatment with DraI/QM. Pre-cleavage with DraI gave rise to a larger number of bands following QM staining; a circumstance evidently due to a removal of the DNA-protein complex that prevented the association of the fluorochrome with AT-rich DNA. The results highlight the complex nature of heterochromatin.

Micronucleus Assay in Exfoliated Buccal Epithelial Cells Using Liquid Based Cytology Preparations in Building Construction Workers.

PubMed

Arul, P; Smitha, Shetty; Masilamani, Suresh; Akshatha, C

2018-01-01

Cytogenetic damage in exfoliated buccal epithelial cells due to environmental and occupational exposure is often monitored by micronucleus (MN) assay using liquid based cytology (LBC) preparations. This study was performed to evaluate MN in exfoliated buccal epithelial cells of building construction workers using LBC preparations. LBC preparations of exfoliated buccal epithelial cells from 100 subjects [50 building construction workers (cases) and 50 administrative staffs (controls)] was evaluated by May-Grunwald Giemsa, Hematoxylin and Eosin and Papanicolaou stains. Student's t test was used for statistical analysis and a P value of <0.05 was considered as statistically significant. The mean frequencies of MN for cases were significantly higher than controls regardless of staining methods used. There were statistically significant differences between smokers and non-smokers of the controls as well as duration of working exposure (<5 and >5 years) and smokers and non-smokers of cases (P=0.001). However, there were meaningful differences regarding mean frequencies of MN between smokers, non-smokers, those with alcohol consumption or not in cases and controls using various stains (P=0.001). There was an increased risk of cytogenetic damage in building construction workers. However, evaluation of MN of exfoliated buccal epithelial cells in building construction workers serve as a minimally invasive biomarker for cytogenetic damage. LBC preparations can be applied for MN assay as it improves the quality of smears and cell morphology, decreases the confounding factors and reduces false positive results.

A case of loiasis in Rome.

PubMed

Morrone, A; Franco, G; Toma, L; Tchangmena, O B; Marangi, M

2002-05-01

Owing to the increase of an immigrant population and of Italian citizens travelling for tourism or on business, it is nowadays possible to observe clinical pictures characteristic of tropical regions, often with indistinct symptoms. One of these is Loa loa infestation, or loiasis, a form of filariasis caused by Loa loa and transmitted by the Chrysops fly. We present the case of a male immigrant from Cameroon. Characteristic symptoms were intense xerosis, mostly of the third inferior part of the legs, intensely pruritic, with numerous lesions from scratching. No benefit was obtained by emollient topics, anti-acarus and systemic antihistamines. Serum samples and Giemsa, haematoxylin, haematoxylin + Giemsa concentration-on-membrane stains, have evidenced the presence of Loa loa microfilariae. A diagnosis for L. loa (loiasis) infestation was made. At the beginning of the migration phenomenon, particularly from Africa, Italian physicians, especially dermatologists, were eagerly looking for 'tropical' diseases; this approach can be defined as 'Salgari's syndrome' from the name of the Italian novelist who, though never travelling out of Italy, had perfectly described environments and habits typical of far away countries. Now, conversely, we have to avoid the opposite approach of considering real tropical diseases as related to social or psychological difficult conditions.

Molecular Characterization of Leishmania Parasites in Giemsa-Stained Slides from Cases of Human Cutaneous and Visceral Leishmaniasis, Eastern Algeria.

PubMed

Beldi, Nadia; Mansouri, Roukaya; Bettaieb, Jihene; Yaacoub, Alia; Souguir Omrani, Hejer; Saadi Ben Aoun, Yusr; Saadni, Farida; Guizani, Ikram; Guerbouj, Souheila

2017-06-01

In Algeria, visceral leishmaniasis (VL) is due to Leishmania (L.) infantum, while three cutaneous forms (CL) are caused by Leishmania major, Leishmania tropica and Leishmania infantum. In this study, the use of Giemsa-stained slides was evaluated with two PCR techniques, in Eastern Algeria. A total of 136 samples corresponding to 100 CL smears (skin scrapings) and 36 VL slides (bone marrow aspirates) collected from 2008 to 2014 were tested. Upon DNA extraction, two PCRs were used to amplify the ribosomal Internal Transcribed Spacer 1 (ITS1) and mini-exon genes. Amplified products were digested (PCR-RFLP) and profiles analyzed for Leishmania species identification. A statistical analysis was also performed. ITS1-PCR was found significantly more sensitive than mini-exon-PCR (77.95% positives vs. 67.65%; p = 0.001). Comparison of PCR positivity showed statistically significant differences between old and recently prepared slides suggesting a better use of recent slides in PCR analyses. For species identification, PCR-restriction fragment length polymorphism (RFLP) results of ITS1 and mini-exon were concordant. L. infantum was identified from VL cases and L. infantum, L. major, and L. tropica from CL ones. According to geographical origin, L. infantum was found in North-Eastern provinces, while L. major was distributed from the North to the Center-East of Algeria. Interestingly, two L. tropica samples were identified in Annaba, located far North-East Algeria. Distribution of leishmaniasis in Eastern parts of Algeria, besides finding of L. tropica in the far North, is in this study described for the first time using molecular tools, thus confirming the usefulness of slides for PCR identification of Leishmania parasites in retrospective epidemiological investigations.

Molecular cytogenetic of the Amoy croaker, Argyrosomus amoyensis (Teleostei, Sciaenidae)

NASA Astrophysics Data System (ADS)

Liao, Mengxiang; Zheng, Jiao; Wang, Zhiyong; Wang, Yilei; Zhang, Jing; Cai, Mingyi

2017-08-01

The family Sciaenidae is remarkable for its species richness and economic importance. However, the cytogenetic data available in this fish group are still limited, especially those obtained using fluorescence in situ hybridization (FISH). In the present study, the chromosome characteristics of a sciaenid species, Argyrosomus amoyensis, were examined with several cytogenetic methods, including dual-FISH with 18S and 5S rDNA probes, and a self-genomic in situ hybridization procedure (Self-GISH). The karyotype of A. amoyensis comprised 2n=48 acrocentric chromosomes. A single pair of nucleolar organizer regions (NORs) was located at the proximal position of chromosome 1, which was positive for silver nitrate impregnation (AgNO3) staining and denaturation-propidium iodide (DPI) staining but negative for Giemsa staining and 4',6-diamidino-2-phenylindole (DAPI) staining, and was confirmed by FISH with 18S rDNA probes. The 5S rDNA sites were located at the centromeric region of chromosome 3. Telomeric FISH signals were detected at all chromosome ends with different intensities, but internal telomeric sequences (ITSs) were not found. Self-GISH resulted in strong signals distributed at the centromeric regions of all chromosomes. C-banding revealed not only centromeric heterochromatin, but also heterochromatin that located on NORs, in interstitial and distal telomeric regions of specific chromosomes. These results suggest that the karyotype of Amoy croaker was relatively conserved and primitive. By comparison with the reported cytogenetic data of other sciaenids, it can be deduced that although the karyotypic macrostructure and chromosomal localization of 18S rDNA are conserved, the distribution of 5S rDNA varies dynamically among sciaenid species. Thus, the 5S rDNA sites may have different evolutionary dynamics in relation to other chromosomal regions, and have the potential to be effective cytotaxonomic markers in Sciaenidae.

Chronic Gastritis and its Association with H. Pylori Infection.

PubMed

Fatema, J; Khan, A H; Uddin, M J; Rahman, M H; Saha, M; Safwath, S A; Alam, M J; Mamun, M A

2015-10-01

This cross sectional study was designed to see association of chronic gastritis including its type with H. pylori infection. Consecutive patients undergoing endoscopic examination having histopathological evidence of chronic gastritis were enrolled in the study and was done in Sylhet MAG Osmani Medical College from July 2011 to June 2012. Biopsies were taken from antrum, body and fundus in all patients. Histopathological examinations were done using H-E stain and for detection of H. pylori, rapid urease test, anti-H.pylori antibody test and histopathological test with modified Giemsa stain were done. Patients having results positive in at least two methods were considered infected by H. pylori. Total 80 dyspeptic patients having chronic gastritis were evaluated. Out of them 67(83.8%) had H. pylori infection and 13(16.2%) were H. pylori negative. Among all patients 57(71.2%) had pangastritis and 23(28.8%) had antral gastritis with female and male predominance respectively. H. pylori infection was present in 49(86.0%) cases of pangastritis and 18(78.3%) cases of antral gastritis. H. pylori infection was a little higher among males (34, 50.7%) females (33, 49.3%). H. pylori infection is the predominant cause of chronic gastritis and pangastritis is the major type.

Immature granulocyte detection by the SE-9000 haematology analyser during pregnancy.

PubMed

Fernández-Suárez, A; Pascual, V T; Gimenez, M T F; Hernández, J F S

2003-12-01

The objective of this study was to determine the nature of the alarm for immature granulocytes appearing in haemograms from pregnant women, as detected by the immature cell information channel (IMI) of the SE-9000 automated haematology analyser. Of all tests run on pregnant women in a 4-month period (n = 698), the first 100 haemograms with immature granulocyte alarms (14.33%) were collected. Each of these samples was then stained with Wright-Giemsa stain. The following variables were also analysed: age of the mother, trimester and days of gestation, type of delivery, weight and sex of the baby, and Apgar score. Most pregnant women were in the third trimester of gestation (82%) when an alarm was noted on the IMI channel. Of the patients, 62% had normal deliveries. The most frequent complication was obstructed delivery (23%). Mean percentages by microscopic counts of band cells, metamyelocytes, and myelocytes were 2.99, 0.45, and 0.19%, respectively. There was a statistically significant correlation for all cell types between the SE-9000 and the manual count method. No association was observed between the presence of immature granulocytes and the clinical variables analysed. The SE-9000 analyser shows high sensitivity in the IMI channel for detection of immature forms.

Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens.

PubMed

Garcia, L S; Bruckner, D A; Brewer, T C; Shimizu, R Y

1983-07-01

Due to increasing numbers of patients with documented infections with Cryptosporidium and other coccidia, it is important for the physician and clinical laboratory to be aware of the appropriate diagnostic techniques necessary for organism recovery and identification. Although Cryptosporidium is found in the gastrointestinal tract, tissue biopsies may be insufficient for organism recovery; the examination of stool specimens is a noninvasive procedure and will provide better overall opportunities for organism recovery. Human clinical specimens were examined from 45 patients with confirmed cryptosporidiosis or suspected of having the infection. Tissue biopsy sections, fecal wet preparations, and permanent stained smears were examined. Stool specimens were submitted in 10% Formalin, 2.5% potassium dichromate, and polyvinyl alcohol and were examined for oocysts by using 15 different methods: phase-contrast and light microscopy; Sheather's sugar flotation; Formalin concentration techniques; 10% potassium hydroxide; Giemsa; trichrome; periodic acid-Schiff; modified periodic acid-Schiff; silver methenamine; acridine orange; auramine-rhodamine; Kinyoun acid-fast; Ziehl-Neelsen carbolfuchsin; and a modified acid-fast procedure. Each technique or combination of techniques was assessed by organism quantitation, organism morphology, and ease of visual recognition. Based on these comparative studies, the modified Ziehl-Neelsen carbolfuchsin stain on 10% Formalin-preserved stool is recommended for the recovery and identification of Cryptosporidium.

Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens.

PubMed Central

Garcia, L S; Bruckner, D A; Brewer, T C; Shimizu, R Y

1983-01-01

Due to increasing numbers of patients with documented infections with Cryptosporidium and other coccidia, it is important for the physician and clinical laboratory to be aware of the appropriate diagnostic techniques necessary for organism recovery and identification. Although Cryptosporidium is found in the gastrointestinal tract, tissue biopsies may be insufficient for organism recovery; the examination of stool specimens is a noninvasive procedure and will provide better overall opportunities for organism recovery. Human clinical specimens were examined from 45 patients with confirmed cryptosporidiosis or suspected of having the infection. Tissue biopsy sections, fecal wet preparations, and permanent stained smears were examined. Stool specimens were submitted in 10% Formalin, 2.5% potassium dichromate, and polyvinyl alcohol and were examined for oocysts by using 15 different methods: phase-contrast and light microscopy; Sheather's sugar flotation; Formalin concentration techniques; 10% potassium hydroxide; Giemsa; trichrome; periodic acid-Schiff; modified periodic acid-Schiff; silver methenamine; acridine orange; auramine-rhodamine; Kinyoun acid-fast; Ziehl-Neelsen carbolfuchsin; and a modified acid-fast procedure. Each technique or combination of techniques was assessed by organism quantitation, organism morphology, and ease of visual recognition. Based on these comparative studies, the modified Ziehl-Neelsen carbolfuchsin stain on 10% Formalin-preserved stool is recommended for the recovery and identification of Cryptosporidium. Images PMID:6193138

Use of a thin-layer technique in thyroid fine needle aspiration.

PubMed

Malle, Despoina; Valeri, Rosalia-Maria; Pazaitou-Panajiotou, Kalliopi; Kiziridou, Anastasia; Vainas, Iraklis; Destouni, Charicleia

2006-01-01

To investigate the efficacy of the ThinPrep Processor (Cytyc Corporation, Boxborough, Massachusetts, U.S.A) in fine needle aspiration (FNA) of thyroid gland lesions. This study included 459 thyroid FNA specimens obtained from patients who came to our endocrinology department with various thyroid disorders over 3 years. The cytologic material was prepared using both the conventional and ThinPrep method in the first 2 years (285 cases), while in the last one only the ThinPrep method was used (1 74 cases). The smears were stained using a modified Papanicolaou procedure and May-Grünwald-Giemsa stain. Immunocytochemistry was performed on thin-layer slides using specific monoclonal antibodies when needed. Thin-layer and direct smear diagnoses were compared with the final cytologic or histologic diagnoses, when available. Our cases included 279 adenomatoid nodules, 15 cases of Hashimoto thyroiditis, 45 follicular neoplasms, 14 Hürthle cell tumors, 58 papillary carcinomas and 1 5 anaplastic carcinomas. Thin-layer preparations showed a trend toward a lower proportion of inadequate specimens and a lower false negative rate. Cytomorphologic features showed some differences between the 2 methods. Colloid was less frequently observed on ThinPrep slides, while nuclear detail and micronucleoli were more easily detected with this technique. Moreover, ThinPrep appeared to be the appropriate method for the use of ancillary techniques in suspicious cases. Thin-layer cytology improves the diagnostic accuracy of thyroid FNA and offers the possibility of performing new techniques, such as immunocytochemistry, on the same sample in order to detect malignancy as well as the type and origin of thyroid gland neoplasms.

Theileria parva: effects of irradiation on a culture of parasitized bovine lymphoid cells. [Gamma radiation

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

Irvin, A.D.; Brown, C.G.D.; Stagg, D.A.

1975-01-01

Aliquots of a culture of Theileria parva-infected bovine lymphoid cells were irradiated at 0, 300, 600, 900, and 1200 rads. The short-term effects of irradiation were evaluated on examination of Giemsa-stained smears and on autoradiography of cells labeled with (/sup 3/H)thymidine. Irradiation inhibited cell division but parasite division did not appear to be inhibited and macroschizont nuclear particles increased in number, frequently to several hundred per schizont. There was no evidence of an increased percentage switch from macro- to microschizont. Apparently viable cells were still present in all cultures 4 days after irradiation.

A novel trypanoplasm-like flagellate Jarrellia atramenti n. g., n. sp. (Kinetoplastida: Bodonidae) and ciliates from the blowhole of a stranded pygmy sperm whale Kogia breviceps (Physeteridae): morphology, life cycle and potential pathogenicity.

PubMed

Poynton, S L; Whitaker, B R; Heinrich, A B

2001-04-10

The successful 6 mo rehabilitation of a stranded juvenile pygmy sperm whale Kogia breviceps afforded the opportunity to study the poorly known protozoan fauna of the upper respiratory tract of cetaceans. Mucus samples were collected by holding either a petri dish or glass slides over the blowhole for 3 to 5 exhalations; preparations were examined as wet mounts, and then stained with Wrights-Giemsa or Gram stain. Blood smears were stained with Wrights-Giemsa. Unidentified spindle-shaped and unidentified broad ciliates, reported from the blowhole of the pygmy sperm whale for the first time, were seen only initially, while yeast-like organisms and bacteria were seen intermittently. Epithelial cells and white blood cells were often present in the blowhole mucus, but red blood cells were never seen. A novel trypanoplasm-like bodonid kinetoplastid biflagellate (Order Kinetoplastida) was commonly encountered in the blowhole mucus, but never in the blood. Both mature flagellates and those undergoing longitudinal binary fission were present. The elongate flagellate had a long whiplash anterior flagellum; the recurrent flagellum was attached along at least two-thirds of the body length, forming a prominent undulating membrane, and the trailing portion was short. The kinetoplast was irregularly fragmented. The flagellates were either free-swimming, or attached to host material via the free portion of the posterior flagellum. The prominent undulating membrane was characteristic of Trypanoplasma, while the fragmented kinetoplast was characteristic of some species of Cryptobia. For the novel bodonid kinetoplastid, with its unique combination of morphological features (prominent undulating membrane and fragmented kinetoplast), we propose the creation of a new genus Jarrellia. We believe this to be the first published description of a flagellate from a marine mammal, and among the first reports of a trypanoplasm-like flagellate from a warm-blooded host. We expect that a diversity of flagellates and ciliates are commonly present in the blowhole of cetaceans. Future studies on the identity of the protozoans and the health of their cetacean hosts, which are readily studied in captivity, are necessary to establish their status as commensals or parasites.

A technique to improve diagnostic information from fine-needle aspirations: immunohistochemistry on cytoscrape.

PubMed

Skov, Birgit Guldhammer; Kiss, Katalin; Ramsted, Julie; Linnemann, Dorte

2009-04-25

Cytologic examination of fine-needle aspiration (FNA) material is being used increasingly for the diagnosis of pulmonary lesions. Accurate distinction between nonsmall cell lung cancer (NSCLC), including subgroups, and small cell lung cancer and between primary lung cancer and metastases has therapeutic impact. However, the distinction between these groups may be difficult on smears. In this report, the authors describe a simple method, called cytoscrape (CS), which can be used on virtually any smear to produce material useful for ancillary methods, including immunohistochemistry. Aspirates from 47 patients who had possible malignant infiltrates identified on computed tomography scans of the chest were included. Smears were stained by May-Grunwald-Giemsa and Diff-Quick for diagnostic purposes. CS material was obtained by gently scraping cells off the slides. Clots were made, and the sections were stained for thyroid transcription factor-1 (TTF-1) and mucin. The utility of the CS technique was evaluated by assessing the sensitivity and specificity of the method and by quantifying the extra diagnostic information obtained by the method relative to smears alone. Malignant tumor cells in the CS material were identified in 43 aspirates (91%). Both the sensitivity and the specificity for TTF-1 were 100%. The sensitivity for mucin was 60%, and the specificity for mucin was 100%. The diagnoses made on smears were improved by CS in 31 patients (72%), in that more precise separation of subgroups of NSCLC was possible or information on primary tumors was obtained. The CS technique improved the diagnostic information from FNA in a clinically relevant way. The method is simple, quick, and inexpensive. (c) 2009 American Cancer Society.

A "down under" lesion on the muzzle of a dog.

PubMed

Twomey, Leanne N; Wuerz, Julia A; Alleman, A Rick

2005-06-01

A 10-year-old, castrated, male Labrador Retriever was presented to a local veterinary practice for investigation of a firm, deeply pigmented, alopecic, subcutaneous mass (8 mm in diameter) on the left side of the muzzle. A fine-needle aspirate of the mass was submitted for cytologic evaluation to the University of Florida. Microscopically, the preparation contained a predominant population of histiocytes that contained variable numbers of intracytoplasmic, negative-staining, filamentous structures consistent with Mycobacterium sp. A presumptive diagnosis of canine leproid granuloma syndrome was based on the cytologic findings and location of the lesion. Acid-fast staining revealed bright pink, acid-fast organisms within the histiocytic cells, supporting the diagnosis. The bacteria were not detected in histopathologic sections or by a polymerase chain reaction (PCR) test 1 week later, however, possibly because of spontaneous remission. Canine leproid granuloma syndrome is a common disease in Australia, but is uncommon in dogs in North America. It is caused by a novel, unnamed Mycobacterium species and usually affects the skin and subcutaneous tissues of the head and ears. A diagnosis usually can be made in Wright's-Giemsa and acid-fast-stained cytologic specimens; however, definitive diagnosis requires PCR testing at a specialized laboratory.

Mixed Ehrlichia canis, Hepatozoon canis, and presumptive Anaplasma phagocytophilum infection in a dog.

PubMed

Mylonakis, Mathio E; Koutinas, Alex F; Baneth, Gad; Polizopoulou, Zoe; Fytianou, Anna

2004-01-01

A 5-month-old, female, mongrel dog was admitted to the Clinic of Companion Animal Medicine, Aristotle University of Thessaloniki, Greece, with depression, anorexia, fever, peripheral lymphadenopathy, splenomegaly, oculonasal discharge, nonregenerative anemia, and mild thrombocytopenia. Cytology of Giemsa-stained buffy coat, bone marrow, and lymph node aspiration smears revealed numerous morulae in mononuclear leukocytes and in neutrophils, and Hepatozoon canis gamonts in neutrophils. The dog was seropositive to Ehrlichia canis (immunofluorescence assay [IFA]) and Hepatozoon canis (ELISA) but not to Anaplasma phagocytophilum (IFA). A nested polymerase chain reaction performed on bone marrow aspirates was positive for E canis. This method was not applied for the detection of A phagocytophilum. Treatment with doxycycline and imidocarb dipropionate resulted in both clinical and parasitologic cure. This is the first reported case of a mixed infection with E canis, H canis, and presumptive A phagocytophilum. The findings emphasize the value of cytology in offering a quick and inexpensive diagnosis in mixed tick-borne infections of dogs.

[Construction and characterization of enterohemorrhagic Escherichia coli O157:H7 ppk- deleted strain].

PubMed

Han, Peng; Sun, Qi; Zhao, Suhui; Zhang, Qiwei; Wan, Chengsong

2014-06-01

To construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics. The gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157: H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD600 value and Giemsa staining. We established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.

American ginseng tea protects cellular DNA within 2 h from consumption: results of a pilot study in healthy human volunteers.

PubMed

Szeto, Yim Tong; Sin, Yuk Shan Pauline; Pak, Sok Cheon; Kalle, Wouter

2015-01-01

The acute genoprotective effect of Panax quinquefolius (American ginseng) has been investigated. The experiment was carried out to explore the DNA protective effect after a single dose of American ginseng tea bag infusion. Fourteen subjects (6 males and 8 females) were recruited in this study. Seven of them (3 males and 4 females) were asked to drink a cup of freshly prepared American ginseng infusions. Water was taken by the remaining subjects as the control group. Blood samples of both groups were taken before and 2 h post-ingestion. The blood samples were challenged with ultraviolet B irradiation followed by using comet assay. Completed slides were stained with Giemsa stain and DNA damage was assessed. Results showed a significant decrease in comet score after American ginseng supplementation and no change in the control group. The current study demonstrated a cup of American ginseng infusion could protect cellular DNA from oxidative stress at least within 2 h.

CYTOPATHIC EFFECT OF THE ATYPICAL PNEUMONIA ORGANISM IN CULTURES OF HUMAN TISSUE

PubMed Central

Eaton, Monroe D.; Farnham, Ann E.; Levinthal, Jeana D.; Scala, Anthony R.

1962-01-01

Eaton, Monroe D. (Harvard Medical School, Boston, Mass.), Ann E. Farnham, Jeana D. Levinthal, and Anthony R. Scala. Cytopathic effect of the atypical pneumonia organism in cultures of human tissue. J. Bacteriol. 84:1330–1337. 1962.—Three strains of the atypical pneumonia agent were adapted to grow in continuous cell cultures of human amnion or human embryonic lung, with production of initial increased acidity followed by destruction of the cells. Evidence is presented that cytopathic effects of the organism were associated with intracellular growth and formation of microcolonies. Clumps of organisms stained specifically with fluorescein-labeled antibody, and showed distinctive tinctorial reactions with the May Grünwald-Giemsa stain. The cytopathic effect was prevented by fresh serum from a rabbit immunized with an egg-passage strain of the atypical pneumonia agent. Heating the immune serum to 56 C for 30 min abolished the neutralizing effect. The significance of heat-labile serum constituents in killing or inhibition of mycoplasma is discussed. Images PMID:16561984

Clinical, Cytological, Histological and Immunohistochemical Features of Cutaneous Mast Cell Tumours in Ferrets (Mustela putorius furo).

PubMed

Vilalta, L; Meléndez-Lazo, A; Doria, G; Ramis, A; Solano-Gallego, L; Pastor, J; Martorell, J

2016-11-01

Cutaneous mast cell tumours (cMCTs) are one of the most common cutaneous tumours in ferrets (Mustela putorius furo). However, limited information is available regarding cytological and histological features of these tumours and studies evaluating KIT expression are lacking in this species. The aims of this prospective study were to describe the most common clinical, cytological and histological features of cMCTs in ferrets and to compare the usefulness of different staining techniques in the diagnosis of these tumours in ferrets as well as evaluating KIT expression in neoplastic mast cells (MCs) by immunohistochemistry. Macroscopically, the tumours were small, round to plaque-like and frequently associated with surface crusting. The most common locations were the extremities and the trunk. MC granules were stained in all cases using toluidine blue (TB) and Wright-Giemsa stains in cytological specimens, but none stained with modified Wright's stain. Haematoxylin and eosin and TB on histological sections failed to stain MC granules in all the cases. Cytological and histological examination revealed low to moderate anisocytosis and anisokaryosis. An infiltrative rather than a delineated or encapsulated growth pattern was noted histologically in all cases. Eosinophilic infiltration was not uncommon and 'collagenolysis' was detected on cytological and histological examination. KIT expression was detected in all cases evaluated. In approximately one third of the cases the MCs exhibited KIT labelling pattern I and in the remaining ferrets, KIT pattern III. No correlation was found between KIT expression pattern and biological behaviour. Copyright © 2016 Elsevier Ltd. All rights reserved.

Gastric carcinoma metastatic to the bone marrow: immunoperoxidase identification of KMO-1 antigen in MGG-destained aspirate.

PubMed

Kobayashi, T K; Yakushiji, M

1991-01-01

A case is presented that illustrates the application of the immunoperoxidase technique to the May-Grünwald-Giemsa (MGG)-destained bone marrow aspirate. The cytologic findings in a MGG-stained smear of the bone marrow suggested a metastatic epithelial tumor. Subsequently, a positive reaction to KMO-1, a monoclonal antibody raised against a colon carcinoma cell line, was demonstrated in tumor cells in the MGG-destained smear sample as well as in the paraffin-embedded section of the primary gastric cancer. The demonstration of the cancer-associated antigen in the MGG-destained material may be useful in establishing the diagnosis of metastatic tumor in the bone marrow.

Application of liquid-based cytology preparation in micronucleus assay of exfoliated buccal epithelial cells in road construction workers.

PubMed

Arul, P

2017-01-01

Asphalts are bitumens that consist of complex of hydrocarbon mixtures and it is used mainly in road construction and maintenance. This study was undertaken to evaluate the micronucleus (MN) assay of exfoliated buccal epithelial cells in road construction workers using liquid-based cytology (LBC) preparation. Three different stains (May-Grunwald Giemsa, hematoxylin and eosin, and Papanicolaou) were used to evaluate the frequency of MN in exfoliated buccal epithelial of 100 participants (fifty road construction workers and fifty administrative staff) using LBC preparation. Statistical analysis was performed with Student's t-test, and P< 0.05 was considered statistically significant. The mean frequency of MN for cases was significantly higher than that of controls (P = 0.001) regardless of staining method used and also cases with exposure period of more than 5 years had statistically significant difference (P < 0.05) than cases with Conclusion: The present study concluded that workers exposed to asphalts during road construction exhibit a higher frequency of MN in exfoliated buccal epithelial cells and they are under the significant risk of cytogenetic damage. LBC preparation has potential application for the evaluation of frequency of MN. This technique may be advocated in those who are occupationally exposed to potentially carcinogenic agents in view of improvement in the smear quality and visualization of cell morphology.

Biomimetic mineral-organic composite scaffolds with controlled internal architecture.

PubMed

Manjubala, I; Woesz, Alexander; Pilz, Christine; Rumpler, Monika; Fratzl-Zelman, Nadja; Roschger, Paul; Stampfl, Juergen; Fratzl, Peter

2005-12-01

Bone and cartilage generation by three-dimensional scaffolds is one of the promising techniques in tissue engineering. One approach is to generate histologically and functionally normal tissue by delivering healthy cells in biocompatible scaffolds. These scaffolds provide the necessary support for cells to proliferate and maintain their differentiated function, and their architecture defines the ultimate shape. Rapid prototyping (RP) is a technology by which a complex 3-dimensional (3D) structure can be produced indirectly from computer aided design (CAD). The present study aims at developing a 3D organic-inorganic composite scaffold with defined internal architecture by a RP method utilizing a 3D printer to produce wax molds. The composite scaffolds consisting of chitosan and hydroxyapatite were prepared using soluble wax molds. The behaviour and response of MC3T3-E1 pre-osteoblast cells on the scaffolds was studied. During a culture period of two and three weeks, cell proliferation and in-growth were observed by phase contrast light microscopy, histological staining and electron microscopy. The Giemsa and Gömöri staining of the cells cultured on scaffolds showed that the cells proliferated not only on the surface, but also filled the micro pores of the scaffolds and produced extracellular matrix within the pores. The electron micrographs showed that the cells covering the surface of the struts were flattened and grew from the periphery into the middle region of the pores.

[Clinical features of four atypical pediatric cases of endemic typhus with pneumonia].

PubMed

Liu, Jin-rong; Xu, Bao-ping; Li, Shao-gang; Liu, Jun; Tian, Bao-lin; Zhao, Shun-ying

2013-10-01

To analyze clinical manifestations, treatment and prognosis of 4 cases with endemic typhus. The clinical data of four endemic typhus patients in prognosis were retrospectively analyzed. These four atypical cases of endemic typhus with pneumonia were treated in our department from October 2011 to March 2012. They were all male, with an age range of 15 months to 7 years. The four patients had long history, mild respiratory symptom and no improvement was found after treatment with cephalosporins. There were no evidences of bacterial, viral, or fungal infections and we thought they might have infection with other pathogen. Three were from rural areas. Routine blood tests, Weil-Felix reaction, blood smear (Giemsa staining) , and indirect immunofluorescence assay were performed. Blood smear and IFA tests showed evidences for endemic typhus. The clinical presentations were atypical, the patients had no headache, but all had fever, rash, and pneumonia of varying severity. None of the patients had a severe cough, but bronchial casts were observed in one case. Recurrent fever was reported in three cases. Physical examinations showed no eschars, but one patient had a subconjunctival hemorrhage, and one had skin scratches, cervical lymphadenopathy, pleural effusion, pericardial effusion, and cardiac dilatation. Two patients had remarkably increased peripheral blood leukocyte counts; both these patients also had high alanine aminotransferase (ALT) levels and one had a high C-reactive protein (CRP) level. Weil-Felix testing was negative or the OX19 titer was low. The peripheral blood smear (Giemsa stain) showed intracellular pathogens in all four cases. After combined therapy with doxycycline and macrolide antibiotics, all four patients recovered well. The endemic typhus children often come from rural areas. The clinical presentations were atypical, they usually have no headache, but have fever (often Periodic fever) , rash, and pneumonia of varying severity in these four cases. Combined therapy with doxycycline and macrolide antibiotics was effective in all four patients.

Dermatophilus congolensis infection in sheep and goats in Delta region of Tamil Nadu.

PubMed

Chitra, M Ananda; Jayalakshmi, K; Ponnusamy, P; Manickam, R; Ronald, B S M

2017-11-01

The study was conducted to isolate and identify Dermatophilus congolensis (DC) using conventional and molecular diagnostic techniques in scab materials collected from skin infections of sheep and goats in the Delta region of Tamil Nadu. A total of 20 scab samples collected from 18 goats and 2 sheep from Nagapattinam, Thanjavur, and Tiruvarur districts of Tamil Nadu. Smears were made from softened scab materials and stained by either Gram's or Giemsa staining. Isolation was attempted on blood agar plates, and colonies were stained by Gram's staining for morphological identification. Identification was also done by biochemical tests and confirmed by 16S rRNA polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the amplified product. The peculiar laddering arrangement of coccoid forms in stained smears prepared from scab materials revealed the presence of DC. Isolated colonies from scab materials of sheep and goats on bovine blood agar plate were small, hemolytic, rough, adherent, and bright orange-yellow in color, but some colonies were white to cream color. Gram-staining of cultured organisms revealed Gram-positive branching filaments with various disintegration stages of organisms. 16S rRNA PCR yielded 500 bp amplicon specific for DC. Sequence analysis of a sheep DC isolate showed 99-100% sequence homology with other DC isolates available in NCBI database, and phylogenetic tree showed a close cluster with DC isolates of Congo, Nigeria, and Angola of Africa. Genes for virulence factors such as serine protease and alkaline ceramidase could not be detected by PCR in any of the DC strains isolated of this study. The presence of dermatophilosis in Tamil Nadu was established from this study.

Treatment of low-grade gastric MALT lymphoma using Helicobacter pylori eradication.

PubMed

Grgov, Saša; Katić, Vuka; Krstić, Miljan; Nagorni, Aleksandar; Radovanović-Dinić, Biljana; Tasić, Tomislav

2015-05-01

Lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) of the stomach usually occurs as a consequence of Helicobacter pylori (H. pylori) infection. The aim of this study was to investigate the long-term effect of treatment of low-grade gastric MALT lymphoma with the H. pylori eradication method. In the period 2002-2012 in 20 patients with dyspepsia, mean age 55.1 years, the endoscopic and histologic diagnosis of gastric MALT lymphoma in the early stages were made. Histological preparations of endoscopic biopsy specimens were stained with hematoxyllineosin (HE), histochemical and immunohistochemical methods. Endoscopic findings of gastritis were documented in 25% of the patients, and 75% of the patients had hypertrophic folds, severe mucosal hyperemia, fragility, nodularity, exulcerations and rigidity. Histopathologically, pathognomonic diagnostic criterion were infiltration and destruction of glandular epithelium with neoplastic lymphoid cells, the so-called lymphoepithelial lesions. In all 20 patients H. pylori was verified by rapid urease test and Giemsa stain. After the triple eradication therapy complete remission of MALT lymphoma was achieved in 85% of the patients, with no recurrence of lymphoma and H. pyloi infection in the average follow-up period of 48 months. In 3 (15%) of the patients, there was no remission of MALT lymphoma 12 months after the eradication therapy. Of these 3 patients 2 had progression of MALT lymphoma to diffuse large-cell lymphoma. Durable complete remission of low-grade gastric MALT lymphoma is achieved in a high percentage after eradication of H. pylori infection, thus preventing the formation of diffuse large-cell lymphoma and gastric adenocarcinoma.

Reliable enumeration of malaria parasites in thick blood films using digital image analysis.

PubMed

Frean, John A

2009-09-23

Quantitation of malaria parasite density is an important component of laboratory diagnosis of malaria. Microscopy of Giemsa-stained thick blood films is the conventional method for parasite enumeration. Accurate and reproducible parasite counts are difficult to achieve, because of inherent technical limitations and human inconsistency. Inaccurate parasite density estimation may have adverse clinical and therapeutic implications for patients, and for endpoints of clinical trials of anti-malarial vaccines or drugs. Digital image analysis provides an opportunity to improve performance of parasite density quantitation. Accurate manual parasite counts were done on 497 images of a range of thick blood films with varying densities of malaria parasites, to establish a uniformly reliable standard against which to assess the digital technique. By utilizing descriptive statistical parameters of parasite size frequency distributions, particle counting algorithms of the digital image analysis programme were semi-automatically adapted to variations in parasite size, shape and staining characteristics, to produce optimum signal/noise ratios. A reliable counting process was developed that requires no operator decisions that might bias the outcome. Digital counts were highly correlated with manual counts for medium to high parasite densities, and slightly less well correlated with conventional counts. At low densities (fewer than 6 parasites per analysed image) signal/noise ratios were compromised and correlation between digital and manual counts was poor. Conventional counts were consistently lower than both digital and manual counts. Using open-access software and avoiding custom programming or any special operator intervention, accurate digital counts were obtained, particularly at high parasite densities that are difficult to count conventionally. The technique is potentially useful for laboratories that routinely perform malaria parasite enumeration. The requirements of a digital microscope camera, personal computer and good quality staining of slides are potentially reasonably easy to meet.

Plasmodium carmelinoi n. sp. (Haemosporida: Plasmodiidae) of the lizard Ameiva ameiva (Squamata: Teiidae) in Amazonian Brazil.

PubMed

Lainson, R; Franco, C M; Da Matta, R

2010-06-01

Plasmodium carmelinoi n. sp. is described in the teiid lizard Ameiva ameiva from north Brazil. Following entry of the merozoites into the erythrocyte, the young, uninucleated trophozoites are at first tear-shaped and already possess a large vacuole: with growth, they may assume an irregular shape, but eventually become spherical or broadly ovoid. The vacuole reduces the cytoplasm of the parasite to a narrow peripheral band in which nuclear division produces a schizont with 8-12 nuclei. At first the dark, brownish-black pigment granules are restricted to this rim of cytoplasm but latterly become conspicuously concentrated within the vacuole. The mature schizonts are spherical to ovoid and predominantly polar in their position in the erythrocyte. They average 5.4 x 4,9 microm (4.4 x 4.4 - 6.6 x 5,9 microm), shape index 1.1, n = 50: 8-12 merozoites are produced and measure approximately 2.0 x 1,0 microm. Mature gametocytes are also polar in position, and spherical to subspherical. The macrogametocytes measure 5.7 x 5,2 microm (4.4 x 4.0- 5.9 x 5,1 microm), shape index 1.1, n = 50 and, following staining by Giemsa's method, possess a compact, pink-staining nucleus and a clear blue, faintly stained cytoplasm. Microgametocytes are slightly larger, 6.0 x 5,0 microm (5.2 x 4.4 - 6.2 x 5,2 microm), shape index 1.2, n = 45. They stain an over-all pink colour due to the dispersed nuclear chromatin. The vacuoles in both the macro- and microgametocytes are considerably smaller than those of the schizonts and of ovoid or spindle shape: they contain most of the pigment granules. The sex ratio, as seen in an inicial intense infection, was 1 male to 2.2 females. Prevalence of infection was low (5%) but, due to the very low parasitaemia which may result in a failure to detect parasites, it is probably higher than this.

Design of an optically stable pH sensor based on immobilization of Giemsa on triacetylcellulose membrane.

PubMed

Khodadoust, Saeid; Kouri, Narges Cham; Talebiyanpoor, Mohammad Sharif; Deris, Jamile; Pebdani, Arezou Amiri

2015-12-01

In this work a simple, inexpensive, and sensitive optical sensor based on triacetylcellulose membrane as solid support was developed by using immobilization of Giemsa indicator for pH measurement. In this method, the influence variables on the membrane performance including pH concentration of indicator, response time, ionic strength, and reversibility were investigated. At optimum values of all variables the response of optical pH sensor is linear in the pH range of 3.0-12.0. This optical sensor was produced through simultaneous binding of the Giemsa on the activated triacetylcellulose membrane which responded to the pH changes in a broader linear range within less than 2.0 min and suitable reproducibility (RSD<5%). Stability results showed that this sensor was stable after 6 months of storage in the water/ethanol (50:50, v/v) solution without any measurable divergence in response properties (less than 5% RSD). Copyright © 2015 Elsevier B.V. All rights reserved.

Male infertility associated with de novo pericentric inversion of chromosome 1.

PubMed

Balasar, Özgür; Zamani, Ayşe Gül; Balasar, Mehmet; Acar, Hasan

2017-12-01

Inversion occurs after two breaks in a chromosome have happened and the segment rotates 180° before reinserting. Inversion carriers have produced abnormal gametes if there is an odd number crossing- over between the inverted and the normal homologous chromosomes causing a duplication or deletion. Reproductive risks such as infertility, abortion, stillbirth and birth of malformed child would be expected in that case. A 54-year- old male patient was consulted to our clinic for primary infertility. The routine chromosome study were applied using peripheral blood lymphocyte cultures and analyzed by giemsa-trypsin-giemsa (GTG) banding, and centromer banding (C-banding) stains. Y chromosome microdeletions in the azoospermia factor (AZF) regions were analyzed with polymerase chain reaction. Additional test such as fluorescence in situ hybridization (FISH) was used to detect the sex-determining region of the Y chromosome (SRY). Semen analysis showed azoospermia. A large pericentric inversion of chromosome 1 46,XY, inv(1) (p22q32) was found in routine chromosome analysis. No microdeletions were seen in AZF regions. In our patient the presence of SRY region was observed by using FISH technique with SRY-specific probe. Men who have pericentric inversion of chromosome 1, appear to be at risk for infertility brought about by spermatogenic breakdown. The etiopathogenic relationship between azoospermia and pericentric inversion of chromosome 1 is discussed.

Simple method for culture of peripheral blood lymphocytes of Testudinidae.

PubMed

Silva, T L; Silva, M I A; Venancio, L P R; Zago, C E S; Moscheta, V A G; Lima, A V B; Vizotto, L D; Santos, J R; Bonini-Domingos, C R; Azeredo-Oliveira, M T V

2011-12-06

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

Dientamoeba fragilis in swine population: a preliminary investigation.

PubMed

Crotti, D; Sensi, M; Crotti, S; Grelloni, V; Manuali, E

2007-04-30

Dientamoeba fragilis, a protozoan with worldwide distribution is considered to be responsible for enteric disease in humans. A wide spectrum of clinical symptoms including; diarrhoea (acute or prolonged), flatulence, abdominal pains and other unspecific bowel symptoms have been ascribed to this parasite. Asymptomatic infection has also been reported. Dientamoeba fragilis is as its name indicates an extremely delicate protozoon and only the trophozoite has ever been demonstrated in stool samples. The definitive diagnosis of this infection is based on demonstration in permanently stained stool samples. In Italy examination of ova and parasite (O&P) samples are not currently performed. This protozoan is extremely difficult to cultivate but molecular techniques such as the Polymerase Chain Reaction offer promise as a means of diagnosing infection. The epidemiology of Dientamoebiasis is not clear. This paper will present preliminary results from a study looking for the parasite's presence in swine faeces. The possible role of pigs as a reservoir of infection was studied; 121 faecal samples from breeding and fattening pigs were examined using a Giemsa permanent stain. Dientamoeba fragilis was found in 53 (43.8%) of the stool samples examined.

[Multiplication of Brucella abortus and production of nitric oxide in two macrophage cell lines of different origin].

PubMed

Serafino, J; Conde, S; Zabal, O; Samartino, L

2007-01-01

Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with the rough strain RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably affects the intracellular growth of B. abortus.

Toxoplasma gondii bradyzoites and tachyzoites isolation from vitreous of atypical necrotizing retinitis.

PubMed

Kharel Sitaula, Ranju; Joshi, Sagun Narayan; Sah, Ranjit; Khadka, Sushila; Khatri Kc, Anadi; Pokharel, Bharat Mani

2018-06-15

Detection of Toxoplasma gondii cysts in vitreous of immunocompetent patient with necrotizing retinitis is extremely rare. We herein report the isolation of Toxoplasma bradyzoites and tachyzoites from the vitreous of healthy person. A 19-year-old immunocompetent female presented with sudden loss of vision in left eye since 1 week. The BCVA was 6/6 and HM in right and left eye. The left eye finding was suggestive of diffuse necrotizing retinitis with retinal detachment. The IgM and IgG for TORCH infection were negative and HIV, HCV and HBsAg tests were also non reactive. The patient underwent diagnostic and therapeutic vitrectomy with silicon oil installation. The vitreous toxoplasma IgG titre was found to be significantly raised to 1:16. Bradyzoites of toxoplasma were identified in H&E staining and tachyzoites of Toxoplasma were identified in Giemsa staining of vitreous sample. She received oral clindamycin and oral corticosteroid but the vision could not be restored in left eye. Hence, atypical toxoplasmosis with necrotizing retinitis is a fulminant condition with the diagnostic and therapeutic challenge.

Recent Progress in the Development of Diagnostic Tests for Malaria.

PubMed

Krampa, Francis D; Aniweh, Yaw; Awandare, Gordon A; Kanyong, Prosper

2017-09-19

The impact of malaria on global health has continually prompted the need to develop effective diagnostic strategies. In malaria endemic regions, routine diagnosis is hampered by technical and infrastructural challenges to laboratories. These laboratories lack standard facilities, expertise or diagnostic supplies; thus, therapy is administered based on clinical or self-diagnosis. There is the need for accurate diagnosis of malaria due to the continuous increase in the cost of medication, and the emergence and spread of drug resistant strains. However, the widely utilized Giemsa-stained microscopy and immunochromatographic tests for malaria are liable to several drawbacks, including inadequate sensitivity and false-positive outcomes. Alternative methods that offer improvements in performance are either expensive, have longer turnaround time or require a level of expertise that makes them unsuitable for point-of-care (POC) applications. These gaps necessitate exploration of more efficient detection techniques with the potential of POC applications, especially in resource-limited settings. This minireview discusses some of the recent trends and new approaches that are seeking to improve the clinical diagnosis of malaria.

Should fine needle aspiration biopsy be the first pathological investigation in the diagnosis of a bone lesion? An algorithmic approach with review of literature

PubMed Central

Mehrotra, Ravi; Singh, Mamta; Singh, Premala A; Mannan, Rahul; Ojha, Vinod K; Singh, Pradumyn

2007-01-01

Background Fine needle aspiration biopsy (FNAB) is gaining increasing popularity in the diagnosis of musculoskeletal lesions; and in many patients, a definitive diagnosis can be rendered from aspiration smears alone. Its applicability in bone pathology, however, has been controversial due to a high percentage of inadequate smears, difficulty in evaluation of tissue architecture and nonspecific results in the diagnosis of primary bone lesions. In this study, the value of aspiration as the first pathological investigation in the diagnosis of a bone lesion was evaluated. Methods 91 cases of clinically suspected cases of bone lesions were aspirated over a period of two years. Direct or cytospin smears were fixed in 95% alcohol and stained by Hematoxylin and Eosin or air-dried and later fixed in methanol for May Grŭnwald Giemsa staining. Results Of the 91 patients who were subjected to FNAB, 81 were considered satisfactory and 10.9 % (10) were inadequate\\inconclusive for diagnosis. Cyto-histological concordance was obtained in 78.5 % (51/65) patients. Positive and negative predictive values were 87.5% and 97.2 % respectively. Sensitivity as a preliminary diagnostic technique was 93.3%, whereas specificity was 94.5 %. Overall, diagnostic accuracy was 94.2 %. Metastatic lesions were detected with 100% accuracy. Two cases were reported as false positive and one case as false negative. Conclusion Cytology provides valuable information to the clinician to make an informed decision regarding appropriate therapy. We conclude that time-consuming and costly investigations may be reduced by choosing FNAB as the initial pathological diagnostic method for skeletal lesions of unknown origin. The choice of radiological examinations, laboratory tests and surgical biopsies can be determined after the FNAB diagnosis. PMID:17439659

[Importance of studying the balance of vaginal content (BAVACO) in the preventive control of sex workers].

PubMed

Bologno, Romina; Díaz, Yanina M; Giraudo, María C; Fernández, Rosa; Menéndez, Viviana; Brizuela, Juan C; Gallardo, Adriana A; Alvarez, Laura A; Estevao Belchior, Silvia G

2011-01-01

The aim of this work was to study the vaginal microenvironment in sex workers from Comodoro Rivadavia, Chubut. For that purpose, BAVACO procedures were applied. A total of 229 female sex workers attended public health centers. Vaginal secretions were analyzed by Gram and Giemsa stains. The following results were obtained: normal microbiota 35.37 %, intermediate microbiota 15.72 %, bacterial vaginosis 23.14 %, microbial non-specific vaginitis, Donders'"aerobic vaginitis" 10.48 %, yeast vulvovaginitis 8.30 %, and trichomoniasis 6.99 %. The intermediate microbiota was characterized by a decrease in the number of lactobacilli and the presence of diphtheroid bacilli cell types. The population studied shared increased values of vaginal dysfunctions. These results are considered risk factors for obstetric and gynecologic diseases.

Ancient pathogens in museal dry bone specimens: analysis of paleocytology and aDNA.

PubMed

Gaul, Johanna Sophia; Winter, Eduard; Grossschmidt, Karl

2015-04-01

Bone samples investigated in this study derive from the pathologic-anatomical collection of the Natural History Museum of Vienna. In order to explore the survival of treponemes and treponemal ancient DNA in museal dry bone specimens, we analyzed three individuals known to have been infected with Treponema pallidum pallidum. No reproducible evidence of surviving pathogen's ancient DNA (aDNA) was obtained, despite the highly sensitive extraction and amplification techniques (TPP15 and arp). Additionally, decalcification fluid of bone sections was smear stained with May-Gruenwald-Giemsa. The slides were examined using direct light microscope and dark field illumination. Remnants of spirochetal structures were detectable in every smear. Our results demonstrate that aDNA is unlikely to survive, but spirochetal remains are stainable and thus detectable.

An automatic device for detection and classification of malaria parasite species in thick blood film.

PubMed

Kaewkamnerd, Saowaluck; Uthaipibull, Chairat; Intarapanich, Apichart; Pannarut, Montri; Chaotheing, Sastra; Tongsima, Sissades

2012-01-01

Current malaria diagnosis relies primarily on microscopic examination of Giemsa-stained thick and thin blood films. This method requires vigorously trained technicians to efficiently detect and classify the malaria parasite species such as Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) for an appropriate drug administration. However, accurate classification of parasite species is difficult to achieve because of inherent technical limitations and human inconsistency. To improve performance of malaria parasite classification, many researchers have proposed automated malaria detection devices using digital image analysis. These image processing tools, however, focus on detection of parasites on thin blood films, which may not detect the existence of parasites due to the parasite scarcity on the thin blood film. The problem is aggravated with low parasitemia condition. Automated detection and classification of parasites on thick blood films, which contain more numbers of parasite per detection area, would address the previous limitation. The prototype of an automatic malaria parasite identification system is equipped with mountable motorized units for controlling the movements of objective lens and microscope stage. This unit was tested for its precision to move objective lens (vertical movement, z-axis) and microscope stage (in x- and y-horizontal movements). The average precision of x-, y- and z-axes movements were 71.481 ± 7.266 μm, 40.009 ± 0.000 μm, and 7.540 ± 0.889 nm, respectively. Classification of parasites on 60 Giemsa-stained thick blood films (40 blood films containing infected red blood cells and 20 control blood films of normal red blood cells) was tested using the image analysis module. By comparing our results with the ones verified by trained malaria microscopists, the prototype detected parasite-positive and parasite-negative blood films at the rate of 95% and 68.5% accuracy, respectively. For classification performance, the thick blood films with Pv parasite was correctly classified with the success rate of 75% while the accuracy of Pf classification was 90%. This work presents an automatic device for both detection and classification of malaria parasite species on thick blood film. The system is based on digital image analysis and featured with motorized stage units, designed to easily be mounted on most conventional light microscopes used in the endemic areas. The constructed motorized module could control the movements of objective lens and microscope stage at high precision for effective acquisition of quality images for analysis. The analysis program could accurately classify parasite species, into Pf or Pv, based on distribution of chromatin size.

Fine needle aspiration cytology in lymphadenopathy of HIV-positive cases.

PubMed

Saikia, U N; Dey, P; Jindal, B; Saikia, B

2001-01-01

To evaluate the role of fine needle aspiration biopsy (FNAB) material in 25 HIV-positive cases with lymphadenopathy. We selected 25 cases for the present study who were enzyme-linked immunosorbent assay positive for HIV (HIV-1). FNAB was performed as a routine, outdoor procedure with informed consent of the patient. For each case, along with routine May-Grünwald-Giemsa and hematoxylin and eosin staining, Ziehl-Neelsen staining for acid-fast bacilli and periodic acid-Schiff staining for fungi were performed wherever necessary. A total of 28 sites were aspirated from 25 HIV patients. All these patients were heterosexual, and none had a history of drug abuse. FNAB was performed under ultrasound guidance in all four cases of a retroperitoneal group of lymph nodes. The most common FNAB diagnosis was reactive lymphoid hyperplasia (10), followed by tuberculosis (8). There were three cases diagnosed as fungal infection (two, Cryptococcus; one, histoplasmosis). FNAB of a case of lymph node was suggestive of tuberculosis. There was one case each diagnosed as non-Hodgkin's lymphoma and squamous cell carcinoma (metastatic). One case of a small axillary lymph node did not yield representative material. FNAB is a relatively inexpensive initial investigative technique in the diagnosis and management of HIV-positive patients. It can obviate the need for surgical excision and enable immediate treatment of specific infections.

Cytological diagnostic of lymphadenitis tuberculosis by eosinophilic material

NASA Astrophysics Data System (ADS)

Delyuzar; Amir, Z.; Kusumawati, L.

2018-03-01

AFB sputum and chest X-ray are used to identify patients with pulmonary TB. For extrapulmonary TB, fine needle aspiration cytology is needed, even though occasionally found not atypical feature in the form of eosinophilic material with dark brown particles, suspected as TB. This research was to show that eosinophilic material with dark brown particles is accurate as new criteria for the cytological diagnosis of TB. By performing fine needle aspiration biopsy stained with Giemsa, if an eosinophilic material with dark brown particles was encountered, we continued with Ziehl-Neelsen AFB stain and confirmed with PCR. To assess accuracy, we used a diagnostic test to evaluate sensitivity and specificity of eosinophilic material with dark brown particles by using AFB and PCR as the gold standard. The sensitivity and specificity of cytological diagnosis in tuberculosis of eosinophilic material with dark brown particles were 93.65% and 70.99%, respectively if confirmed with AFB. On the other hand, if confirmed with PCR using Mycobacterium tuberculosis DNA, the sensitivity and specificity were 98.95% and 96.79%, respectively. In conclusion, eosinophilic masses with dark brown particles is accurate as new criteria of TB diagnostic cytology with high sensitivity and specificity confirmed with AFB and PCR test.

Comparative cytogenetics of two endangered leuciscine fish, Squalius aradensis and S. torgalensis (Teleostei, Cyprinidae), from the Iberian Peninsula

PubMed Central

Nabais, Catarina; Rampin, Massimiliano; Collares-Pereira, Maria João

2013-01-01

Abstract In this study, the description of the karyotypes of the endangered chubs Squalius aradensis (Coelho, Bogutskaya, Rodrigues and Collares-Pereira, 1998) and Squalius torgalensis (Coelho, Bogutskaya, Rodrigues and Collares-Pereira, 1998) is presented by means of conventional (Giemsa-staining, Chromomycin A3 (CMA3)-fluorescence, Silver-impregnation (Ag-NORs)) and molecular (fluorescence in situ hybridization (FISH) with 18S rDNA probe) protocols. These endemic sister-species have an allopatric but adjacent distribution in the most southwestern part of the Iberian Peninsula. Diploid chromosome number was invariably 2n = 50 and karyotypes of both species were grossly similar, composed of metacentric and submetacentric elements with a reduced number of acrocentric pairs. Sequential staining using FISH with an 18S rDNA probe, CMA3 and Ag-NORs treatments revealed consistent positive signals located at the end of the short arms of a submetacentric chromosome pair, likely homologous in both species. While providing useful cytogenetic comparative data against other members of the genus Squalius Bonaparte, 1837, the work aimed to draw attention towards the conservation of two narrow-range and highly confined fish species. PMID:24260688

Occupational contact dermatitis to a limonene-based solvent in a histopathology technician.

PubMed

Foti, Caterina; Zambonin, Carlo G; Conserva, Anna; Casulli, Claudia; D'Accolti, Lucia; Angelini, Gianni

2007-02-01

Recently, D-limonene-based solvents are used as a safe alternative to xylene for histological and cytological application to dissolve paraffin. We report the case of a histopathology technician with a recalcitrant hand contact dermatitis strictly related to the use of a limonene-based solvent agent. Patch tests with SIDAPA (Italian Society of Allergological, Professional and Environmental Dermatology) standard series, limonene-based solvent used by the patient and D- and L-limonene (both oxidized and nonoxidized form) and with Giemsa and methylene blue stains were performed. Patch testing gave positive results to oxidized D- and L-limonene. The patient retired from work and promptly improved and healed the hand eczema. Subsequently, the potential occurrence of limonene oxidation products in the incriminated preparation was investigated using gas chromatography-mass spectrometry. While patch test showed positive reaction to oxidized limonene, chemical analysis failed to detect oxidized limonene in the preparations used by the patient. Considering the strict relation between the use of the preparations and the appearance of symptoms, we can assume that oxidized limonene may be produced during the handling of limonene-based products, especially in the presence of oxidants stains, frequently used in histological laboratories.

Leishmania major: Genetic Profiles of the Parasites Isolated from Chabahar, Southeastern Iran by PPIP-PCR

PubMed Central

SHARIFI-RAD, Mehdi; DABIRZADEH, Mansour; SHARIFI, Iraj; BABAEI, Zahra

2016-01-01

Background: Leishmaniasis is important vector-borne parasitic disease worldwide, caused by the genus Leishmania. The objective of the current study was to identify genetic polymorphism in L. major, one of the species causing cutaneous leishmaniasis (CL), isolated from southeastern Iran, using Permissively Primed Intergenic Polymorphic-Polymerase Chain Reaction (PPIP-PCR) method. Methods: Overall, 340 patients with suspected CL were examined. They referred to the Central Laboratory in Chabahar, Iran during Apr 2013 to Feb 2014. Microscopic examination of Giemsa-stained slides from lesions as well as aspirates cultured in Novy- Mac Neal-Nicolle (NNN) Media was employed in order to diagnose CL in these patients. Our analyses detected 86 suspected subjects as having CL from which 35 isolates were cultured successfully. PPIP-PCR method was performed on extracted genomic DNA from selected isolates in order to determine the genetic polymorphism among L. major isolates. Results: The electrophoresis patterns demonstrated two genetic profiles including A or A1 patterns between all samples tested. Frequency of A and A1 sub-types were 33 (94.3%) and two (5.7%), respectively. Conclusion: Both host and parasite factors may contribute to the clinical profile of human leishmaniasis in the endemic foci of the disease. Here we showed that genetic variations pertaining to the Leishmania parasites might determine, in part, the clinical outcomes of human leishmaniasis. PMID:28127333

First Molecular Characterization of Leishmania Species Causing Visceral Leishmaniasis among Children in Yemen

PubMed Central

Mahdy, Mohammed A. K.; Al-Mekhlafi, Abdulsalam M.; Abdul-Ghani, Rashad; Saif-Ali, Reyadh; Al-Mekhlafi, Hesham M.; Al-Eryani, Samira M.; Lim, Yvonne A. L.; Mahmud, Rohela

2016-01-01

Visceral leishmaniasis (VL) is a debilitating, often fatal disease caused by Leishmania donovani complex; however, it is a neglected tropical disease. L. donovani complex comprises two closely related species, L. donovani that is mostly anthroponotic and L. infantum that is zoonotic. Differentiation between these two species is critical due to the differences in their epidemiology and pathology. However, they cannot be differentiated morphologically, and their speciation using isoenzyme-based methods poses a difficult task and may be unreliable. Molecular characterization is now the most reliable method to differentiate between them and to determine their phylogenetic relationships. The present study aims to characterize Leishmania species isolated from bone marrows of Yemeni pediatric patients using sequence analysis of the ribosomal internal transcribed spacer-1 (ITS1) gene. Out of 41 isolates from Giemsa-stained bone marrow smears, 25 isolates were successfully amplified by nested polymerase chain reaction and sequenced in both directions. Phylogenetic analysis using neighbor joining method placed all study isolates in one cluster with L. donovani complex (99% bootstrap). The analysis of ITS1 for microsatellite repeat numbers identified L. infantum in 11 isolates and L. donovani in 14 isolates. These data suggest the possibility of both anthroponotic and zoonotic transmission of VL-causing Leishmania species in Yemen. Exploring the possible animal reservoir hosts is therefore needed for effective control to be achieved. PMID:26966902

Evaluation of PCR for cutaneous leishmaniasis diagnosis and species identification using filter paper samples in Panama, Central America.

PubMed

Miranda, A; Saldaña, A; González, K; Paz, H; Santamaría, G; Samudio, F; Calzada, J E

2012-09-01

Cutaneous leishmaniasis (CL) is a major vectorborne disease in Panama. In this study, the diagnostic performance and usefulness of two DNA extraction procedures from skin scraping samples collected on FTA filter paper for subsequent PCR diagnosis of CL was evaluated. A positive CL laboratory diagnosis was based on a positive parasitological test (Giemsa-stained smears or in vitro culture) and/or positive PCR test performed from skin scrapings collected in TE buffer (PCR-TE). Of 100 patients with skin lesions suggestive of CL, 82 (82%) were confirmed as CL positive. The sensitivity was calculated for each of the PCR approaches from samples collected on filter paper. The highest sensitivity was achieved by PCR-FTA processed by Chelex 100 (PCR-Chelex) (0.94). PCR-FTA extracted using the FTA purification reagent presented a lower sensitivity (0.60). Good concordance between routine PCR-TE and PCR-Chelex was observed (percent agreement=0.88, κ index=0.65). In conclusion, use of FTA filter paper for skin scraping collection combined with PCR is a reliable and convenient method for CL diagnosis in Panama, with comparable performance to the routine PCR method and with improved sensitivity compared with those of conventional parasitological methods. Copyright © 2012 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Comparative Study of Genotoxicity in Different Tobacco Related Habits using Micronucleus Assay in Exfoliated Buccal Epithelial Cells

PubMed Central

Guruprasad, Yadavalli; Jose, Maji; Saxena, Kartikay; K, Deepa; Prabhu, Vishnudas

2014-01-01

Background: Oral cancer is one of the most debilitating diseases afflicting mankind. Consumption of tobacco in various forms constitutes one of the most important etiological factors in initiation of oral cancer. When the focus of today’s research is to determine early genotoxic changes in human cells, micronucleus (MN) assay provides a simple, yet reliable indicator of genotoxic damage. Aims and Objectives: To identify and quantify micronuclei in the exfoliated cells of oral mucosa in individuals with different tobacco related habits and control group, to compare the genotoxicity of different tobacco related habits between each group and also with that of control group. Patients and Methods: In the present study buccal smears of 135 individuals with different tobacco related habits & buccal smears of 45 age and sex matched controls were obtained, stained using Giemsa stain and then observed under 100X magnification in order to identify and quantify micronuclei in the exfoliated cells of oral mucosa. Results: The mean Micronucleus (MN) count in individuals having smoking habit were 3.11 while the count was 0.50, 2.13, and 1.67 in normal control, smoking with beetle quid and smokeless tobacco habit respectively. MN count in smokers group was 2.6 times more compared to normal controls. MN count was more even in other groups when compared to normal control but to a lesser extent. Conclusion: From our study we concluded that tobacco in any form is genotoxic especially smokers are of higher risk and micronucleus assay can be used as a simple yet reliable marker for genotoxic evaluation. PMID:24995238

Performance of a malaria microscopy image analysis slide reading device

PubMed Central

2012-01-01

Background Viewing Plasmodium in Romanovsky-stained blood has long been considered the gold standard for diagnosis and a cornerstone in management of the disease. This method however, requires a subjective evaluation by trained, experienced diagnosticians and establishing proficiency of diagnosis is fraught with many challenges. Reported here is an evaluation of a diagnostic system (a “device” consisting of a microscope, a scanner, and a computer algorithm) that evaluates scanned images of standard Giemsa-stained slides and reports species and parasitaemia. Methods The device was challenged with two independent tests: a 55 slide, expert slide reading test the composition of which has been published by the World Health Organization (“WHO55” test), and a second test in which slides were made from a sample of consenting subjects participating in a malaria incidence survey conducted in Equatorial Guinea (EGMIS test). These subjects’ blood was tested by malaria RDT as well as having the blood smear diagnosis unequivocally determined by a worldwide panel of a minimum of six reference microscopists. Only slides with unequivocal microscopic diagnoses were used for the device challenge, n = 119. Results On the WHO55 test, the device scored a “Level 4” using the WHO published grading scheme. Broken down by more traditional analysis parameters this result was translated to 89% and 70% sensitivity and specificity, respectively. Species were correctly identified in 61% of the slides and the quantification of parasites fell within acceptable range of the validated parasitaemia in 10% of the cases. On the EGMIS test it scored 100% and 94% sensitivity/specificity, with 64% of the species correct and 45% of the parasitaemia within an acceptable range. A pooled analysis of the 174 slides used for both tests resulted in an overall 92% sensitivity and 90% specificity with 61% species and 19% quantifications correct. Conclusions In its current manifestation, the device performs at a level comparable to that of many human slide readers. Because its use requires minimal additional equipment and it uses standard stained slides as starting material, its widespread adoption may eliminate the current uncertainty about the quality of microscopic diagnoses worldwide. PMID:22559294

A method for reducing the sloughing of thick blood films for malaria diagnosis.

PubMed

Norgan, Andrew P; Arguello, Heather E; Sloan, Lynne M; Fernholz, Emily C; Pritt, Bobbi S

2013-07-08

The gold standard for malaria diagnosis is the examination of thick and thin blood films. Thick films contain 10 to 20 times more blood than thin films, correspondingly providing increased sensitivity for malaria screening. A potential complication of thick film preparations is sloughing of the blood droplet from the slide during staining or rinsing, resulting in the loss of sample. In this work, two methods for improving thick film slide adherence ('scratch' (SCM) and 'acetone dip' (ADM) methods) were compared to the 'standard method' (SM) of thick film preparation. Standardized blood droplets from 26 previously examined EDTA whole blood specimens (22 positive and four negative) were concurrently spread on glass slides using the SM, ADM, and SCM. For the SM and ADM prepared slides, the droplet was gently spread to an approximate 22 millimeters in diameter spot on the slide using the edge of a second glass slide. For the SCM, the droplet was spread by carefully grinding (or scratching) it into the slide with the point of a second glass slide. Slides were dried for one hour in a laminar flow hood. For the ADM, slides were dipped once in an acetone filled Coplin jar and allowed to air dry. All slides were then Giemsa-stained and examined in a blinded manner. Adherence was assessed by blinded reviewers. No significant or severe defects were observed for slides prepared with the SCM. In contrast, 8 slides prepared by the ADM and 3 prepared using the SM displayed significant or severe defects. Thick films prepared by the three methods were microscopically indistinguishable and concordant results (positive or negative) were obtained for the three methods. Estimated parasitaemia of the blood samples ranged from 25 to 429,169 parasites/μL of blood. The SCM is an inexpensive, rapid, and simple method that improves the adherence of thick blood films to standard glass slides without altering general slide preparation, microscopic appearance or interpretability. Using the SCM, thick films can be reliably examined less than two hours after sample receipt. This represents a significant diagnostic improvement over protocols requiring extended drying periods.

Comparison of ELISA antigen preparations alone or in combination for serodiagnosing Helicobacter pylori infections.

PubMed Central

Hirschl, A M; Rathbone, B J; Wyatt, J I; Berger, J; Rotter, M L

1990-01-01

The immunoglobulin G antibody response to Helicobacter pylori was assessed in 78 patients with non-ulcer dyspepsia using five different antigen preparations. All patients were endoscoped and biopsied. The H pylori state was determined histologically on at least two endoscopic biopsy specimens using a modified Giemsa stain. The ultracentrifuged cell sonicate, acid glycine extract, and 120 kilodalton protein antigens were specific in diagnosing infection (95-98%), but had only moderate sensitivity (70-84%). By mixing either of the two complex antigens with the 120 kilodalton protein, the sensitivity of the test was increased to 97% without affecting the high specificity. The combination of ultracentrifuged sonicate or acid glycine extract with the 120 kilodalton protein therefore seems to be superior to the individual antigen preparations and is particularly suitable for the serodiagnosis of H pylori infection. PMID:2380396

Stable and unstable chromosomal aberrations among Finnish nuclear power plant workers.

PubMed

Lindholm, C

2001-01-01

Twenty nuclear power plant workers with relatively high recorded cumulative doses were studied using FISH chromosome painting and dicentric analysis after solid Giemsa staining. The results indicated that chronic exposure to ionising radiation can be detected on the group level using translocation analysis after chromosome painting, although the mean cumulative dose was approximately 100 mSv. A significant association between translocation frequency and cumulative dose was observed. Variability in the translocation yields among workers with similar recorded doses was large, resulting in a poor correlation between translocation frequencies and documented doses on the individual level. The yields of dicentric and acentric chromosomes were not correlated with the cumulative dose, indicating the inability of unstable aberrations to monitor long-term exposures. It was also shown that the unstable aberrations were not correlated with the most recent annual dose.

FNAC Versus Core Needle Biopsy: A Comparative Study in Evaluation of Palpable Breast Lump

PubMed Central

Saha, Abhijit; Mukhopadhyay, Madhumita; Sarkar, Koushik; Saha, Ashis Kumar; Sarkar, Diptendra KR

2016-01-01

Introduction Breast carcinoma is the most common malignant tumour and the leading cause of carcinoma death in women in world. The main purpose of FNAC or CNB of breast lumps is to confirm cancer preoperatively and to avoid unnecessary surgery in specific benign conditions. Aims and Objective The objective of the study was to compare between Fine Needle Aspiration Cytology (FNAC) and Core Needle Biopsy (CNB) in the diagnosis of breast carcinoma with final histological diagnosis from excision specimen as it is gold standard. Materials and Methods A prospective study was done on 50 cases. Patients undergoing all three procedures (Fine Needle Aspiration Cytology and Core Needle Biopsy done at Department of Pathology; subsequent excision surgeries done at Department of General Surgery) were selected. May Grunwald Giemsa (MGG) and Papaniculou (PAP) staining were performed on cytology smears. Haematoxylin and Eosin (H&E) staining was done on both the CNB and tissue specimens obtained from subsequent excision surgeries to see the histological features. Results FNAC showed sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were 69%, 100%, 100%, 38.1%, and 74% respectively in diagnosing carcinoma. CNB had sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of 88.3%, 100%, 100%, 53.3% and 86%. Both FNAC and CNB showed statistically significant correlation with confirmatory HPE of excision specimen (p-value <0.05) in the diagnosis of breast carcinoma. Conclusion Fine needle aspiration cytology (FNAC) is a rapid, less complicated, economical, reliable and relevant method for the preoperative pathological diagnosis of breast carcinoma in a developing nation like ours. If the initial FNAC is inadequate, core needle biopsy (CNB) can be a useful second line method of pathological diagnosis in order to minimize the chance of missed diagnosis of breast cancer. PMID:27042469

Retrospective study of clinical and hematological aspects associated with dogs naturally infected by Hepatozoon canis in Ludhiana, Punjab, India

PubMed Central

Chhabra, Sushma; Uppal, Sanjeev Kumar; Singla, Lachhman Das

2013-01-01

Objective To evaluate clinical and hematological aspects of dogs naturally infected with Hepatozoon canis (H. canis) presented at the Small Animal Clinics of Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana. Methods Blood films of 34 naturally infected dogs were examined for haematological alterations and parasitaemia. Signalment and clinical signs were recorded from the animals. Clinical histories were filled out during the consultation. Results Of the 34 positive dogs by Giemsa stained peripheral blood films, 88.23% presented parasitaemia by H. canis only, while 11.77% had the combination of H. canis, Babesia sp. and Ehrlichia sp. Young male dogs less than one-year-old, of non-descript breed, were the most commonly affected. And 26.47% were presented with anorexia/inappetence as the only clinical symptom. Other clinical symptoms were mild to moderate fever, pale mucosae and lethargy; a few were also showing the signs of vomiting and diarrhoea. Haematological alterations showed mainly normochromic-normocytic anaemia, leukocytosis and neutrophilia. Conclusions The findings of this study substantiate that H. canis caused clinical and haematological alterations of the varied intensity in dogs, even with low parasitaemia, should be taken into consideration. PMID:23730562

Plasmodium ovale infection in Malaysia: first imported case

PubMed Central

2010-01-01

Background Plasmodium ovale infection is rarely reported in Malaysia. This is the first imported case of P. ovale infection in Malaysia which was initially misdiagnosed as Plasmodium vivax. Methods Peripheral blood sample was first examined by Giemsa-stained microscopy examination and further confirmed using a patented in-house multiplex PCR followed by sequencing. Results and Discussion Initial results from peripheral blood smear examination diagnosed P. vivax infection. However further analysis using a patented in-house multiplex PCR followed by sequencing confirmed the presence of P. ovale. Given that Anopheles maculatus and Anopheles dirus, vectors of P. ovale are found in Malaysia, this finding has significant implication on Malaysia's public health sector. Conclusions The current finding should serve as an alert to epidemiologists, clinicians and laboratory technicians in the possibility of finding P. ovale in Malaysia. P. ovale should be considered in the differential diagnosis of imported malaria cases in Malaysia due to the exponential increase in the number of visitors from P. ovale endemic regions and the long latent period of P. ovale. It is also timely that conventional diagnosis of malaria via microscopy should be coupled with more advanced molecular tools for effective diagnosis. PMID:20929588

Absence of Asymptomatic Malaria Infection in a Cross-sectional Study in Iranshahr District, Iran under Elimination Programmes

PubMed Central

PIRAHMADI, Sakineh; ZAKERI, Sedigheh; RAEISI, Ahmad

2017-01-01

Background: Asymptomatic malaria infection provides a reservoir of parasites, causing the persistence of malaria transmission. It accounts an important challenge for successful management of the control, elimination, and eradication programmes in any malaria-endemic region. This investigation was designed to assess the presence and the prevalence of asymptomatic carriers in Iranshahr district of Sistan and Baluchistan Province (2013–2014), with a considerable population movement, during the malaria elimination phase in Iran. Methods: Finger-prick blood samples were collected from symptomless (n=250) and febrile (n=50) individuals residing in Iranshahr district, easthern Iran (Hoodian, Mand, Chah-e Giji, Jolgehashem, Esfand, Dalgan and Chahshour) during Jan 2013 to Dec 2014, and Plasmodium infections were detected using light microscopic and highly sensitive nested-PCR techniques. Results: Thick and thin Giemsa-stained blood smears were negative for Plasmodium parasites. In addition, based on nested-PCR analysis, no P. vivax, P. falciparum, and P. malariae parasites were detected among the studied individuals. Conclusion: Investigation the absence of asymptomatic carriers in Iranshahr district was illustrated and achieving malaria elimination in this area is feasible in a near future. PMID:28761465

A study of peripheral blood in hedgehogs in Turkey.

PubMed

Ozparlak, Haluk; Celik, Ilhami; Sur, Emrah; Ozaydin, Tuğba; Arslan, Atilla

2011-09-01

The aim of this study was to determine diameters of blood cells, differential counts of peripheral blood leukocytes, alpha-naphthyl acetate esterase (ANAE), acid phosphatase (ACP-ase) activity of some leukocyte types, and enzymatic positivity percentages of peripheral blood lymphocytes in two hedgehogs species, Hemiechinus auritus, the long-eared hedgehog, and Erinaceus concolor, the southern white-breasted hedgehog. Air-dried peripheral blood smears were stained with May-Grünwald-Giemsa stain. ANAE and ACP-ase were stained in glutaraldehyde-acetone-fixed smears. ANAE-positive lymphocytes displayed a dot-like positivity pattern characterized with 1-5 reddish brown cytoplasmic granules, whereas ACP-ase positive lymphocytes displayed a dot-like positivity pattern characterized with 1-3 pinkish cytoplasmic granules. Monocytes gave a diffuse and strong reaction while neutrophils displayed a weak positive reaction for ANAE and ACP-ase. No difference was observed in mean diameters of peripheral blood cells of these species. It was found that lymphocytes made up the majority (64.3% and 65.5%) of leukocytes, followed by neutrophils (23.9% and 23.3%), eosinophils (9.0% and 7.6%), monocytes (1.8% and 2.3%), and basophils (1.0% and 1.3%) in H. auritus and E. concolor, respectively. Mean ANAE positivity oflymphocytes was 36.6% and 51.3% and ACP-ase positivity was 32.1% and 37.5% for H. auritus and E. concolor, respectively. The ANAE positivity of lymphocytes in E. concolor was significantly (P < 0.05) higher than that of H. auritus.

Recommendations, evaluation and validation of a semi-automated, fluorescent-based scoring protocol for micronucleus testing in human cells.

PubMed

Seager, Anna L; Shah, Ume-Kulsoom; Brüsehafer, Katja; Wills, John; Manshian, Bella; Chapman, Katherine E; Thomas, Adam D; Scott, Andrew D; Doherty, Ann T; Doak, Shareen H; Johnson, George E; Jenkins, Gareth J S

2014-05-01

Micronucleus (MN) induction is an established cytogenetic end point for evaluating structural and numerical chromosomal alterations in genotoxicity testing. A semi-automated scoring protocol for the assessment of MN preparations from human cell lines and a 3D skin cell model has been developed and validated. Following exposure to a range of test agents, slides were stained with 4'-6-diamidino-2-phenylindole (DAPI) and scanned by use of the MicroNuc module of metafer 4, after the development of a modified classifier for selecting MN in binucleate cells. A common difficulty observed with automated systems is an artefactual output of high false positives, in the case of the metafer system this is mainly due to the loss of cytoplasmic boundaries during slide preparation. Slide quality is paramount to obtain accurate results. We show here that to avoid elevated artefactual-positive MN outputs, diffuse cell density and low-intensity nuclear staining are critical. Comparisons between visual (Giemsa stained) and automated (DAPI stained) MN frequencies and dose-response curves were highly correlated (R (2) = 0.70 for hydrogen peroxide, R (2) = 0.98 for menadione, R (2) = 0.99 for mitomycin C, R (2) = 0.89 for potassium bromate and R (2) = 0.68 for quantum dots), indicating the system is adequate to produce biologically relevant and reliable results. Metafer offers many advantages over conventional scoring including increased output and statistical power, and reduced scoring subjectivity, labour and costs. Further, the metafer system is easily adaptable for use with a range of different cells, both suspension and adherent human cell lines. Awareness of the points raised here reduces the automatic positive errors flagged and drastically reduces slide scoring time, making metafer an ideal candidate for genotoxic biomonitoring and population studies and regulatory genotoxic testing.

Blood donors screening for malaria in non-endemic area in the Kingdom of Saudi Arabia: Is it necessary to introduce immunological testing?

PubMed

Elyamany, Ghaleb; Al Gharawi, Ali; Alrasheed, Mohammed; Alsuhaibani, Omar

2016-02-01

In Saudi Arabia, where malaria is not endemic, the incidence is very low. However, malaria transmission cases have been reported, mainly in Asir and Jazan provinces along the Southwestern border with Yemen. Imported cases also have been reported. The aims of this study were to determine the prevalence of malaria in blood donors in a tertiary care hospital in the central area of Saudi Arabia and to assess the effectiveness of malaria screening methods used by transfusion services in Prince Sultan Military Medical City. This study was conducted on 180,000 people who donated blood during 2006-2015. All blood smears from blood donors were screened for malaria infection using Giemsa staining, low power and high power microscopic examinations, and using oil immersion lens. The data were analyzed and reported in descriptive statistics and prevalence. From the total of 180,000 blood donors who were screened for malaria, 156,000 (87%) and 23.400 (13%) were Saudi Arabia citizens and non-Saudi residents, respectively. The mean age of the blood donors was 32 (ranging from 18 to 65), 97% and 3% were male and female, respectively. Using our current method for malaria screening, the prevalence of malaria in the study population was zero. The current methods of malaria screening in blood donors is not suitable for screening low-level parasiotemia. Adding the immunoassay and molecular screening methods is suggested.

Severe human Babesia divergens infection in Norway.

PubMed

Mørch, K; Holmaas, G; Frolander, P S; Kristoffersen, E K

2015-04-01

Human babesiosis is a rare but potentially life-threatening parasitic disease transmitted by ixodid ticks, and has not previously been reported in Norway. We report a case of severe babesiosis that occurred in Norway in 2007. The patient had previously undergone a splenectomy. He was frequently exposed to tick bites in an area endemic for bovine babesiosis in the west of Norway. The patient presented with severe haemolysis and multiorgan failure. Giemsa-stained blood smears revealed 30% parasitaemia with Babesia spp. He was treated with quinine in combination with clindamycin, apheresis, and supportive treatment with ventilatory support and haemofiltration, and made a complete recovery. This is the first case reported in Norway; however Babesia divergens seroprevalence in cattle in Norway is high, as is the risk of Ixodes ricinus tick bite in the general population. Babesiosis should be considered in the differential diagnosis of unexplained febrile haemolytic disease. Copyright © 2015. Published by Elsevier Ltd.

Ovarian dysgenesis in an alpaca with a minute chromosome 36.

PubMed

Fellows, Elizabeth; Kutzler, Michelle; Avila, Felipe; Das, Pranab J; Raudsepp, Terje

2014-01-01

A 4-year-old female alpaca (Lama pacos [LPA]) was presented to the Oregon State Veterinary Teaching Hospital for failure to display receptive behavior to males. Although no abnormalities were found on physical examination, transrectal ultrasonographic examination of the reproductive tract revealed uterine hypoplasia and ovarian dysgenesis. Cytogenetic analysis demonstrated a normal female 74,XX karyotype with 1 exceptionally small (minute) homologue of autosome LPA36. Chromosome analysis by Giemsa staining and DAPI- and C-banding revealed that the minute LPA36 was submetacentric, AT-rich, and largely heterochromatic. Because of the small size and lack of molecular markers, it was not possible to identify the origin of the minute. There is a need to improve molecular cytogenetic tools to further study the phenomenon of this minute chromosome and its relation to female reproduction in alpacas and llamas. © The American Genetic Association. 2012. All rights reserved. For permissions, please email: journals.permissions@oup.com.

[Application of Nested PCR in the Diagnosis of Imported Plasmodium Ovale Infection].

PubMed

Huang, Bing-cheng; Xu, Chao; Li, Jin; Xiao, Ting; Yin, Kun; Liu, Gong-zhen; Wang, Wei-yan; Zhao, Gui-hua; Wei, Yan-bin; Wang, Yong-bin; Zhao, Chang-lei; Wei, Qing-kuan

2015-02-01

To identity Plasmodium ovale infection by 18S rRNA gene nested PCR. Whole blood and filter paper blood samples of malaria patients in Shandong Province were collected during 2012-2013. The parasites were observed under a microscope with Giemsa staining. The genome DNA of blood samples were extracted as PCR templates. Genus- and species-specific primers were designed according to the Plasmodium 18S rRNA gene sequences. Plasmodium ovale-positive specimens were identified by nested PCR as well as verified by sequencing. There were 7 imported cases of P. ovale infection in the province during 2012-2013. Nested PCR results showed that the P. ovale specific band (800 bp) was amplified in all the 7 specimens. Blast results indicated that the PCR products were consistent with the Plasmodium ovale reference sequence in GenBank. Seven imported cases of ovale malaria in Shandong Province in 2012-2013 are confirmed by nested PCR.

Morphological and morphometrical characterization of gametocytes of Hepatozoon procyonis Richards, 1961 (Protista, Apicomplexa) from a Brazilian wild procionid Nasua nasua and Procyon cancrivorus (Carnivora, Procyonidae).

PubMed

Soares Ferreira Rodrigues, André Flávio; Daemon, Erik; Massard, Carlos Luiz

2007-01-01

The species Hepatozoon procyonis Richards, 1961 was described in Procyon lotor in the USA and then in other reports in the USA, while in Panama H. procyonis has been described in Procyon cancrivorus. The objective of this paper is to report the occurrence of this species in the Brazilian procionids P. cancrivorus and Nasua nausa and to describe the morphology and morphometrics of the gametocytes. The analysis was based on blood smears, stained with Giemsa, which were examined under a photonic microscope. The morphometry was done with an ocular micrometer. It was based on the morphological characteristics and morphometric data on the gametocyte. It can be concluded that the species of the genus Hepatozoon that occurs in Brazilian procionids is the same as that occurring in procionids in Central and North America.

[A case of donovanosis acquired in France].

PubMed

Okhremchuk, I; Marmottant, E; Abed, S; Nguyen, A-T; Fournier, B; Boye, T; Morand, J-J

2016-11-01

Donovanosis (granuloma inguinale) is a bacterial infection caused by Klebsiella granulomatis that occurs mainly in the genital area and is primarily sexually transmitted; it is seen predominantly in the tropics. Herein, we report a case of the disease contracted in metropolitan France. A 47-year-old man presented with painless ulceration of the glans, present for one month, with progressive extension; there was no history of any recent trip abroad. Skin biopsy with Whartin-Starry and Giemsa staining revealed Donovan bodies in the cytoplasm of macrophages. Based on these findings, further questioning of the patient revealed unprotected sexual contact two months earlier in France. Treatment was initiated with azithromycin 1g on the first day followed by 500mg per day for three weeks. The clinical outcome was spectacular, with almost complete regression of the ulcer at 7 days. This case demonstrates that donovanosis can occur in metropolitan France. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Morphological and populational characteristics of hemocytes of Ornithodoros moubata nymphs during the ecdysial phase.

PubMed

Kadota, K; Walter, S; Claveria, F G; Igarashi, I; Taylor, D; Fujisaki, K

2003-11-01

The ultrastructure and characteristics of hemocytes of argasid tick species, Ornithodoros moubata, during the ecdysdial phase are herein presented. Hemocyte classes/populations characterized based on their affinity with Giemsa stain and ultrastructural differences comprised the prohemocytes, nongranular cells (Nc), eosinophilic granular cells (Ec), basophilic granular cells (Bc), and unidentified cells. Significant changes/shift in the ratio of hemocyte classes/population was apparent in ticks before and after the ecdysial phase. The granule-scant basophilic granular cells (sBc) constituted the most abundant hemocyte population in the ecdysial phase. Nymphs in ecdysis showed increases in Nc and sBc and decrease in Ec, a phenomenon that was reversed in unengorged nymphs and adults ticks. The significant increase in total Bc population in ecdysis relative to nonengorged ticks clearly point to blastogenesis of Bc taking place during the ecdysial phase and Bc's important role in the process of tissue remodeling.

Chromosome characterization and variability in some Iridaceae from Northeastern Brazil

PubMed Central

Alves, Lânia Isis F.; Lima, Saulo Antônio A.; Felix, Leonardo P.

2011-01-01

The chromosomes of 15 species of Iridaceae of the genera Alophia, Cipura, Eleutherine, Neomarica and Trimezia (subfamily Iridoideae) were examined after conventional Giemsa staining. The karyotypes of Alophia drummondii (2n = 14+1B, 28, 42 and 56), Cipura paludosa (2n = 14), C. xanthomelas (2n = 28) and Eleutherine bulbosa (2n = 12) were asymmetric; Neomarica candida, N. caerulea, N. humilis, N. glauca, N. gracilis, N. northiana and Neomarica sp. (2n = 18); N. cf. paradoxa (2n = 28), Trimezia fosteriana (2n = 52), T. martinicensis (2n = 54) and T. connata (2n = 82) were all generally symmetric. New diploid numbers of 2n = 56 for Alophia drummondii, 2n = 18 for N. candida, N. humilis, N. glauca, and N. gracilis, 2n = 28 for N. cf. paradoxa, and 2n = 82 for T. connata are reported. The karyotypic evolution of the studied species is discussed. PMID:21734827

Endogenous Life Cycle of Eimeria melanomytis (Apicomplexa: Eimeriidae) from the Dusky Rice Rat, Melanomys caliginosus (Rodentia: Cricetidae: Sigmodontinae) in Costa Rica.

PubMed

Chinchilla, Misael; Valerio, Idalia; Sánchez, Ronald; Duszynski, Donald W

2017-02-01

Endogenous stages of the life cycle of Eimeria melanomytis, infecting the peripheral epithelial cells of villi of the small intestine of experimentally infected young dusky rice rats, Melanomys caliginosus , were studied. Giemsa-stained mucosal scrapings and histological sections were examined for all the stages. Eimeria melanomytis has 3 generations of meronts (M), different in size, shape, and number of merozoites (m); and in size, shape, and location of the nuclei within the cytoplasm of the meronts. The 3 meront types, M 1 -M 3 , respectively, had 11-14 (m 1 ), 7-10 (m 2 ), and 20-30 (m 3 ) merozoites. Macrogametocytes and microgametocytes, as well as macrogametes and microgametes, complete the sexual cycle forming the unsporulated oocysts. This parasite's endogenous development produced severe intestinal lesions in experimentally infected dusky rice rats.

Role of intraoperative imprint cytology in diagnosis of suspected ovarian neoplasms.

PubMed

Dey, Soumit; Misra, Vatsala; Singh, P A; Mishra, Sanjay; Sharma, Nishant

2010-01-01

The present study was conducted to assess whether cytology can help in rapid diagnosis of ovarian neoplasms and thus facilitate individualised treatment. A prospective investigation was performed on 30 cases of suspected ovarian neoplasms. Imprint smears were made intraperatively from fresh samples from various representative areas, and stained with Leishman Giemsa for air-dried smears, and with hematoxylin and eosin and Papanicolaou for alcohol-fixed smears. A rapid opinion regarding the benign or malignant nature of the lesion and the type of tumour was given. The overall sensitivity was 96.2%, specificity 75%, positive predictive value 96.3%, and diagnostic accuracy of 83.3%. Characteristic cytological patterns were noted in various epithelial and germ cell tumours. Imprint cytology can be used as an adjunct to histopathology for rapid and early diagnosis in the operation theatre, thus helping better management of patients.

Microscopic and Molecular Detection of Theileria (Babesia) Equi Infection in Equids of Kurdistan Province, Iran

PubMed Central

HABIBI, Gholamreza; ESMAEILNIA, Kasra; HABLOLVARID, Mohammad Hasan; AFSHARI, Asghar; ZAMEN, Mohsen; BOZORGI, Soghra

2016-01-01

Background: Equine piroplasmosis (EP) is the cause of persistent tick-borne infection with no symptoms, but the most important problem of EP is due to the persistent carrier state. Carrier animals to Babesia (Theileria) equi (Laveran 1901) and B. caballi (Nuttall, 1910) infestation could be identified by extremely sensitive PCR-based method. The purpose of this study was to identify the causative agents of equine piroplasmosis based on molecular and microscopic assays in equids from Kurdistan Province, Iran. Methods: Thirty one horse and mule blood samples were used with history of living in Kurdistan Province of Iran. The blood specimens were utilized for T. equi and B. caballi DNA identification by PCR and Giemsa stained smears for microscopic observation. Results: The results clearly showed the presence of B. (Theileria) equi DNA in 30 of 31 blood samples (96.77%), but the microscopic examination revealed the 3 of 31 positive Babesia like organisms in the red blood cells (9.67%). Conclusion: The obtained results demonstrated the presence of hidden B. (Theileria) equi infection in horses with previous habitance in Kurdistan Province of Iran. The carrier animals became a main source of infection and can transmit the disease. Therefore, hidden infection might be considered as a health threatening and limiting factor in animals used in therapeutic antisera research and production centers. PMID:27095973

Splanchnic Th(2) and Th(1) cytokine redistribution in microsurgical cholestatic rats.

PubMed

García-Dominguez, José; Aller, María-Angeles; García, Cruz; de Vicente, Felipe; Corcuera, Maria-Teresa; Gómez-Aguado, Fernando; Alonso, María José; Vara, Elena; Arias, Jaime

2010-08-01

Long-term extrahepatic cholestasis in the rat induces ductular proliferation and fibrosis in the liver, portal hypertension, splenomegaly, portosystemic collateral circulation, and ascites. These splanchnic alterations could have an inflammatory pathophysiology. We measured serum levels of hepatobiliary injury markers and the acute phase proteins, alpha-1-major acid protein (alpha(1)-MAP) and alpha-1-acid glycoprotein (alpha(1)-GPA) in rats 6 wk after microsurgical extrahepatic cholestasis. We also assayed Th(1) (TNF-alpha and IL-1beta) and Th(2) (IL-4 and IL-10) cytokine levels in the liver, ileum, spleen, and mesenteric lymph complex by enzyme-linked immunosorbent assay (ELISA) techniques. Liver fibrosis was measured by Sirius red stain and by using an image system computer-assisted method and mast cell liver infiltration by Giemsa stain. The cholestatic rats showed an increase (P<0.001) in serum levels of bile acids, total and direct bilirubin, AST, ALT, AST/ALT index, gamma-GT, alkaline phosphatase, alpha(1)- MAP, alpha(1)-GPA, and LDH (P<0.05) in relation to sham-operated rats. TNF-alpha, IL-1beta, IL-4, and IL-10 increased in the ileum (P<0.01) and mesenteric lymph complex (P<0.001), and decreased in the liver (P<0.001). A marked bile proliferation associated with fibrosis (P<0.001) and mast cell infiltration was also shown in the liver of cholestatic rats. The splanchnic redistribution of cytokines, with an increase of Th(1) and Th(2) production in the small bowel and in the mesenteric lymph complex, supports the key role of inflammatory mechanisms in rats with secondary biliary fibrosis. Copyright 2010 Elsevier Inc. All rights reserved.

Imprint cytology on microcalcifications excised by vacuum-assisted breast biopsy: a rapid preliminary diagnosis.

PubMed

Fotou, Maria; Oikonomou, Vassiliki; Zagouri, Flora; Sergentanis, Theodoros N; Nonni, Afroditi; Athanassiadou, Pauline; Drouveli, Theodora; Atsouris, Efstratios; Kotzia, Evagelia; Zografos, George C

2007-04-03

To evaluate imprint cytology in the context of specimens with microcalcifications derived from Vacuum-Assisted Breast Biopsy (VABB). A total of 93 women with microcalcifications BI-RADS 3 and 4 underwent VABB and imprint samples were examined. VABB was performed on Fischer's table using 11-gauge Mammotome vacuum probes. A mammogram of the cores after the procedure confirmed the excision of microcalcifications. For the application of imprint cytology, the cores with microcalcifications confirmed by mammogram were gently rolled against glass microscope slides and thus imprint smears were made. For rapid preliminary diagnosis Diff-Quick stain, modified Papanicolaou stain and May Grunwald Giemsa were used. Afterwards, the core was dipped into a CytoRich Red Collection fluid for a few seconds in order to obtain samples with the use of the specimen wash. After the completion of cytological procedures, the core was prepared for routine histological study. The pathologist was blind to the preliminary cytological results. The cytological and pathological diagnoses were comparatively evaluated. According to the pathological examination, 73 lesions were benign, 15 lesions were carcinomas (12 ductal carcinomas in situ, 3 invasive ductal carcinomas), and 5 lesions were precursor: 3 cases of atypical ductal hyperplasia (ADH) and 2 cases of lobular neoplasia (LN). The observed sensitivity and specificity of the cytological imprints for cancer were 100% (one-sided, 97.5% CI: 78.2%-100%). Only one case of ADH could be detected by imprint cytology. Neither of the two LN cases was detected by the imprints. The imprints were uninformative in 11 out of 93 cases (11.8%). There was no uninformative case among women with malignancy. Imprint cytology provides a rapid, accurate preliminary diagnosis in a few minutes. This method might contribute to the diagnosis of early breast cancer and possibly attenuates patients' anxiety.

Seroprevalence of Pediatric Malaria in Quetta, Balochistan, Pakistan

PubMed Central

Hussain, K; Shafee, M; Khan, N; Jan, S; Tareen, AM; Khan, MA

2013-01-01

Background Malaria is one of the most devastating protozoal diseases in under developing countries like Pakistan where health facilities are scarce. It is the second most frequently reported disease with 4.5 million suspected cases in Pakistan. The current study was designed to determine the incidence of pediatric malaria in Quetta, Balochistan. Methods The study was conducted at Children Hospital Quetta (CHQ) during July 2011march 2012. Blood samples were collected from 3418 clinically suspected and were evaluated using thin and thick blood films stained with Giemsa stain. Results Out of 3418 total of 230 (6.72%) children were found positive for any of the malarial parasitic infestation. Plasmodium vivax was observed to be more common 54.34% (n= 125/230) than P. falciparum 44.78% (n = 103/230). Male children were 65.21% (150/230) i.e. two times more commonly affected than female 34.78% (80/230) children. The prevalence among age groups was 7.41% (n = 89/1200) in preschool-aged children aged 1-5 years, 7.11% (n = 75/1054) in school-aged children aged 6—10 years while 6.78% (n = 46/678) in 11-15 years-old children, and 6.66% (n = 20/300) in >15 year-olds children. Peak prevalence was noted in summer and mild in winter. Mixed infection of (0.86%: 2/230) P. vivax and P. falciparum was also observed in two cases although no case of P. malariae or P. ovale infection was seen during entire study. Conclusion The results reflect the higher prevalence of malaria in Quetta, Pakistan that poses a significant health threat and requires urgent attention of high-ups to launch programme to control the disease in the area. PMID:23914251

Pathological Study of Blood Parasites in Rice Field Frogs, Hoplobatrachus rugulosus (Wiegmann, 1834)

PubMed Central

Sailasuta, Achariya; Satetasit, Jetjun; Chutmongkonkul, Malinee

2011-01-01

One hundred and forty adult rice field frogs, Hoplobatrachus rugulosus (Wiegmann, 1834), were collected in Srakaew province, Thailand. For blood parasite examination, thin blood smears were made and routinely stained with Giemsa. The results showed that 70% of the frogs (98/140) were infected with 5 species of blood parasites, including a Trypanosoma rotatorium-like organism, Trypanosoma chattoni, Hepatozoon sp. a, Hepatozoon sp. b, and Lankesterella minima. Pathological examination of the liver, lung, spleen, and kidney of the frogs that were apparently infected with one of these blood parasites were collected and processed by routine histology and subsequently stained with haematoxylin and eosin. Histopathological findings associated with the Trypanosoma rotatorium-like organism and Trypanosoma chattoni-infected frogs showed no pathological lesions. Hepatozoon sp. a and Hepatozoon sp. b-infected frogs developed inflammatory lesions predominantly in the liver, demonstrating granuloma-like lesions with Hepatozoon sp. meronts at the centre. Tissue sections of Lankesterella minima-infected frogs also showed lesions. Liver and spleen showed inflammatory lesions with an accumulation of melanomacrophage centres (MMCs) surrounding the meronts and merozoites. It is suggested that Hepatozoon sp. a, Hepatozoon sp. b, and Lankesterella minima-infections are capable of producing inflammatory lesions in the visceral organs of rice field frogs, and the severity of lesions is tentatively related to levels of parasitemia. PMID:21918731

Chromatin differentiation between Theobroma cacao L. and T. grandiflorum Schum

PubMed Central

2010-01-01

A comparative analysis of mitotic chromosomes of Theobroma cacao (cacao) and T. grandiflorum (cupuaçu) was performed aiming to identify cytological differences between the two most important species of this genus. Both species have symmetric karyotypes, with 2n = 20 metacentric chromosomes ranging in size from 2.00 to 1.19 μm (cacao) and from 2.21 to 1.15 μm (cupuaçu). The interphase nuclei of both species were of the arreticulate type, displaying up to 20 chromocentres, which were more regularly shaped in cacao than in cupuaçu. Prophase chromosomes of both species were more condensed in the proximal region, sometimes including the whole short arm. Both species exhibited only one pair of terminal heterochromatic bands, positively stained with chromomycin A 3 , which co-localized with the single 45S rDNA site. Each karyotype displayed a single 5S rDNA site in the proximal region of another chromosome pair. Heterochromatic bands were also observed on the centromeric/pericentromeric regions of all 20 chromosomes of cacao after C-banding followed by Giemsa or DAPI staining, whereas in cupuaçu they were never detected. These data suggest that the chromosomes of both species have been largely conserved and their pericentromeric chromatin is the only citologically differentiated region. PMID:21637611

First report of birds infection by intestinal parasites in Khorramabad, west Iran.

PubMed

Badparva, Ebrahim; Ezatpour, Behrouz; Azami, Mehdi; Badparva, Masoud

2015-12-01

Parasitic infections in birds are omnipresent, even when they occur in low amounts, may result in subclinical diseases. There aren't any studies, based on Iranian data, investigating the prevalence of intestinal parasitic infections in some birds' species. We conducted a cross-sectional study between December 2011 and December 2012. The fecal samples were taken from 451 birds including hen, turkey, sparrow, pigeon and decorative birds. The samples screened for intestinal parasitic infections using direct smear, formalin-ether concentration technique, modified Ziehl-Neelsen staining, Culture in RPMI 1640 medium, sporulation with potassium dichromate and Trichrome and Giemsa staining. Out of 451 birds' species, 157 (34.8 %), were infected with one or more type of intestinal parasites. We identified two nematode, two cestoda species and five protozoan parasites species. No trematodes were found in the samples studied. The parasites identified among birds involved Raillietina spp. (4.2 %) and Eimeria spp. (7.1 %) were the most common helminthes and protozoa respectively. From total of birds study, 12 (2.7 %) and 6 (1.3 %) have two and three mixed infections respectively. Intestinal parasitic infections are common in birds in west Iran. The future studies are needed in order to determine to which extent the infections influence mortality and performance of the birds.

Human fetal bone cells in delivery systems for bone engineering.

PubMed

Tenorio, Diene M H; Scaletta, Corinne; Jaccoud, Sandra; Hirt-Burri, Nathalie; Pioletti, Dominique P; Jaques, Bertrand; Applegate, Lee Ann

2011-11-01

The aim of this study was to culture human fetal bone cells (dedicated cell banks of fetal bone derived from 14 week gestation femurs) within both hyaluronic acid gel and collagen foam, to compare the biocompatibility of both matrices as potential delivery systems for bone engineering and particularly for oral application. Fetal bone cell banks were prepared from one organ donation and cells were cultured for up to 4 weeks within hyaluronic acid (Mesolis®) and collagen foams (TissueFleece®). Cell survival and differentiation were assessed by cell proliferation assays and histology of frozen sections stained with Giemsa, von Kossa and ALP at 1, 2 and 4 weeks of culture. Within both materials, fetal bone cells could proliferate in three-dimensional structure at ∼70% capacity compared to monolayer culture. In addition, these cells were positive for ALP and von Kossa staining, indicating cellular differentiation and matrix production. Collagen foam provides a better structure for fetal bone cell delivery if cavity filling is necessary and hydrogels would permit an injectable technique for difficult to treat areas. In all, there was high biocompatibility, cellular differentiation and matrix deposition seen in both matrices by fetal bone cells, allowing for easy cell delivery for bone stimulation in vivo. Copyright © 2011 John Wiley & Sons, Ltd.

Infection and propagation of lymphocystis virus isolated from the cultured flounder Paralichthys olivaceus in grass carp cell lines.

PubMed

Zhang, Qi-Ya; Ruan, Hong-Mei; Li, Zhen-Qiu; Yuan, Xiu-Ping; Gui, Jian-Fang

2003-12-03

The causative agent of lymphocystis disease that frequently occurs in cultured flounder Paralichthys olivaceus in China is lymphocystis virus (LV). In this study, 13 fish cell lines were tested for their susceptibility to LV. Of these, 2 cell lines derived from the freshwater grass carp Ctenopharyngodon idellus proved susceptible to the LV, and 1 cell line, GCO (grass carp ovary), was therefore used to replicate and propagate the virus. An obvious cytopathic effect (CPE) was first observed in cell monolayers at 1 d post-inoculation, and at 3 d this had extended to about 75% of the cell monolayer. However, no further CPE extension was observed after 4 d. Cytopathic characteristics induced by the LV were detected by Giemsa staining and fluorescence microscopic observation with Hoechst 33258 staining. The propagated virus particles were also observed by electron microscopy. Ultrastructure analysis revealed several distinct cellular changes, such as chromatin compaction and margination, vesicle formation, cell-surface convolution, nuclear fragmentation and the occurrence of characteristic 'blebs' and cell fusion. This study provides a detailed report of LV infection and propagation in a freshwater fish cell line, and presents direct electron microscopy evidence for propagation of the virus in infected cells. A possible process by which the CPEs are controlled is suggested.

Molecular diagnosis of canine visceral leishmaniasis: a comparative study of three methods using skin and spleen from dogs with natural Leishmania infantum infection.

PubMed

Reis, Levi Eduardo Soares; Coura-Vital, Wendel; Roatt, Bruno Mendes; Bouillet, Leoneide Érica Maduro; Ker, Henrique Gama; Fortes de Brito, Rory Cristiane; Resende, Daniela de Melo; Carneiro, Mariângela; Giunchetti, Rodolfo Cordeiro; Marques, Marcos José; Carneiro, Cláudia Martins; Reis, Alexandre Barbosa

2013-11-08

Polymerase chain reaction (PCR) and its variations represent highly sensitive and specific methods for Leishmania DNA detection and subsequent canine visceral leishmaniasis (CVL) diagnosis. The aim of this work was to compare three different molecular diagnosis techniques (conventional PCR [cPCR], seminested PCR [snPCR], and quantitative PCR [qPCR]) in samples of skin and spleen from 60 seropositive dogs by immunofluorescence antibody test and enzyme-linked immunosorbent assay. Parasitological analysis was conducted by culture of bone marrow aspirate and optical microscopic assessment of ear skin and spleen samples stained with Giemsa, the standard tests for CVL diagnosis. The primers L150/L152 and LINR4/LIN17/LIN19 were used to amplify the conserved region of the Leishmania kDNA minicircle in the cPCR, and snPCR and qPCR were performed using the DNA polymerase gene (DNA pol α) primers from Leishmania infantum. The parasitological analysis revealed parasites in 61.7% of the samples. Sensitivities were 89.2%, 86.5%, and 97.3% in the skin and 81.1%, 94.6%, and 100.0% in spleen samples used for cPCR, snPCR, and qPCR, respectively. We demonstrated that the qPCR method was the best technique to detect L. infantum in both skin and spleen samples. However, we recommend the use of skin due to the high sensitivity and sampling being less invasive. Copyright © 2013 Elsevier B.V. All rights reserved.

Biodosimetry estimate for high-LET irradiation.

PubMed

Wang, Z Z; Li, W J; Zhi, D J; Jing, X G; Wei, W; Gao, Q X; Liu, B

2007-08-01

The purpose of this paper is to prepare for an easy and reliable biodosimeter protocol for radiation accidents involving high-linear energy transfer (LET) exposure. Human peripheral blood lymphocytes were irradiated using carbon ions (LET: 34.6 keV microm(-1)), and the chromosome aberrations induced were analyzed using both a conventional colcemid block method and a calyculin A induced premature chromosome condensation (PCC) method. At a lower dose range (0-4 Gy), the measured dicentric (dics) and centric ring chromosomes (cRings) provided reasonable dose information. At higher doses (8 Gy), however, the frequency of dics and cRings was not suitable for dose estimation. Instead, we found that the number of Giemsa-stained drug-induced G2 prematurely condensed chromosomes (G2-PCC) can be used for dose estimation, since the total chromosome number (including fragments) was linearly correlated with radiation dose (r = 0.99). The ratio of the longest and the shortest chromosome length of the drug-induced G2-PCCs increased with radiation dose in a linear-quadratic manner (r = 0.96), which indicates that this ratio can also be used to estimate radiation doses. Obviously, it is easier to establish the dose response curve using the PCC technique than using the conventional metaphase chromosome method. It is assumed that combining the ratio of the longest and the shortest chromosome length with analysis of the total chromosome number might be a valuable tool for rapid and precise dose estimation for victims of radiation accidents.

Use of calcofluor-blue brightener for the diagnosis of Pneumocystis jirovecii pneumonia in bronchial-alveolar lavage fluids: A single-center prospective study.

PubMed

Desoubeaux, Guillaume; Franck-Martel, Claire; Caille, Agnès; Drillaud, Nicolas; Lestrade Carluer de Kyvon, Marie-Alix; Bailly, Éric; Chandenier, Jacques

2017-04-01

The biological diagnosis of Pneumocystis jirovecii pneumonia (PjP) is based on the investigation of respiratory fluids by conventional staining methods and/or molecular biology. Diagnostic performance of an in-house technique based on calcofluor-blue brightener for the direct detection of P. jirovecii cysts was prospectively assessed in bronchial-alveolar lavage fluids (BALF) from patients with a suspected PjP infection over a three-year period in a single center: the diagnostic yield was compared to that of a commercial kit based on monoclonal immunofluorescence assay (IFA) on replicate smears. May-Grünwald Giemsa (MGG) staining and quantitative Polymerase Chain Reaction (qPCR) were also performed. The gold standard for each patient was the definitive diagnosis of PjP infection by an independent committee based on clinical, radiological, and biological data. Overall, 481 BALF were assessed: 42 were found to be positive for the detection of P. jirovecii by at least one laboratory technique, but only 35 were actually judged to be in agreement with the definitive diagnosis of PjP infection. The sensitivity of the calcofluor-blue brightener technique was 74.3% vs. 60.0%, 34.6%, and 82.9% for IFA, MGG, and qPCR, respectively; and its specificity was 99.6% vs. 99.3%, 100.0%, and 99.4% for IFA, MGG, and qPCR. No technique was shown to be statistically superior to calcofluor-blue brightener. Further validation of the test through multicenter studies is now required, but in light of its low cost and easy preparation, the use of calcofluor-blue brightener in BALF appears to be a valuable alternative method for the routine first-line diagnosis of PjP infection. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A simple reliable procedure for obtaining metaphases from human leukemic bone-marrow aspirates suitable for Giemsa banding.

PubMed

Srivastava, A K; Smith, R D

1980-02-01

Short incubation of heparinized human leukemic bone-marrow cells in phosphate buffered saline containing colcemid and overnight chilling of fixed cells yields metaphases with elongated and well-spread chromosomes. This technique enables us to do trypsin-Giemsa banding of chromosomes obtained from leukemic marrow cells otherwise difficult to band.

Nonaspiration fine needle cytology and its histologic correlation in canine skin and soft tissue tumors.

PubMed

Chalita, M C; Matera, J M; Alves, M T; Longatto Filho, A

2001-12-01

To analyze the findings of nonaspiration fine needle (NAFN) cytology as compared with the histopathologic findings in evaluating canine skin and soft tissue tumors. NAFN (21-27 gauge) cytology was performed on 213 cases. Smears were air dried and stained by the Rosenfeld method (May-Grünwald-Giemsa modification). Histopathologic evaluation was available for comparison in 40% of cases. NAFN cytology and histopathology results were compared in 85 dogs. The size of the 117 lesions varied from 0.5 to 2 cm (n=39), 2.1 to 5 cm (n=43), and > or = 5.1 (n=35). There were 22 nonneoplastic lesions, mostly inflammatory processes and cysts. Neoplastic lesions were classified as epithelial (36%), mesenchymal (30%), round cell tumor (n=13) and melanocytic (2%). Among 40 malignant lesions, mast cell tumor (n=14) and hemangiopericytoma (n=9) were the most frequent. Lipoma (n=14) and trichoblastoma (n=10) were the most common benign neoplastic lesions. Cytology showed sensitivity of 89%, specificity of 100%, positive and negative predictive value of 100% and 96%, respectively, and efficacy of 97%. NAFN cytology is extremely useful and accurate. It is safe and avoids the use of anesthesia. Further, it is easy to perform and noninvasive and usually provides a high-quality sample.

Microscopic and Molecular Detection of Theileria (Babesia) Equi Infection in Equids of Kurdistan Province, Iran.

PubMed

Habibi, Gholamreza; Esmaeilnia, Kasra; Hablolvarid, Mohammad Hasan; Afshari, Asghar; Zamen, Mohsen; Bozorgi, Soghra

2016-01-01

Equine piroplasmosis (EP) is the cause of persistent tick-borne infection with no symptoms, but the most important problem of EP is due to the persistent carrier state. Carrier animals to Babesia (Theileria) equi (Laveran 1901) and B. caballi (Nuttall, 1910) infestation could be identified by extremely sensitive PCR-based method. The purpose of this study was to identify the causative agents of equine piroplasmosis based on molecular and microscopic assays in equids from Kurdistan Province, Iran. Thirty one horse and mule blood samples were used with history of living in Kurdistan Province of Iran. The blood specimens were utilized for T. equi and B. caballi DNA identification by PCR and Giemsa stained smears for microscopic observation. The results clearly showed the presence of B. (Theileria) equi DNA in 30 of 31 blood samples (96.77%), but the microscopic examination revealed the 3 of 31 positive Babesia like organisms in the red blood cells (9.67%). The obtained results demonstrated the presence of hidden B. (Theileria) equi infection in horses with previous habitance in Kurdistan Province of Iran. The carrier animals became a main source of infection and can transmit the disease. Therefore, hidden infection might be considered as a health threatening and limiting factor in animals used in therapeutic antisera research and production centers.

Pneumocystis pneumonia (PCP) and Pneumocystis jirovecii carriage in renal transplantation patients: a single-centre experience.

PubMed

Maruschke, Matthias; Riebold, Diana; Holtfreter, Martha Charlotte; Sombetzki, Martina; Mitzner, Steffen; Loebermann, Micha; Reisinger, Emil Christian; Hakenberg, Oliver W

2014-12-01

The Pneumocystis pneumonia is an increasing problem in transplanted patients: up to 25% suffer from Pneumocystis pneumonia, occurring during the first 6 months after transplantation. From 2001 to 2009, we investigated 21 patients with pneumonia after renal transplantation for the presence of Pneumocystis jirovecii. The laboratory diagnosis was established by Grocott and Giemsa staining methods and Pneumocystis-specific mitochondrial transcribed large subunit nested polymerase chain reaction (PCR). The PCR was also used for the differentiation of Pneumocystis pneumonia from Pneumocystis carriage. Of 21 patients, 7 had a Pneumocystis pneumonia, 6 were Pneumocystis carriers and 8 patients were negative. Four out of seven Pneumocystis pneumonia patients and two out of six patients with Pneumocystis carriage had a delayed graft function. An acute cytomegalovirus infection after transplantation was not detectable in the patients with Pneumocystis pneumonia, but in three patients with Pneumocystis carriage. Pneumocystis pneumonia was present in 33.3% of transplanted patients with suspected pneumonia. An association between acute rejection or co-infections and Pneumocystis pneumonia or carriage in patients after renal transplantation cannot be excluded. In three out of seven Pneumocystis pneumonia patients, an overlapping of hospitalisation times and an onset of Pneumocystis pneumonia 6 months after transplantation was found. Thus, person-to-person transmission seems probable in these cases.

Performance evaluation of rapid diagnostic test for malaria in high malarious districts of Amhara region, Ethiopia.

PubMed

Beyene, Belay Bezabih; Yalew, Woyneshet Gelaye; Demilew, Ermias; Abie, Getent; Tewabe, Tsehaye; Abera, Bayeh

2016-03-01

Malaria is one of the leading public health challenges in Ethiopia. To address this, the Federal Ministry of Ethiopia launched a laboratory diagnosis programme for promoting use of either rapid diagnostic tests (RDTs) or Giemsa microscopy to all suspected malaria cases. This study was conducted to assess the performance of RDT and influencing factors for Giemsa microscopic diagnosis in Amhara region. A cross-sectional study was conducted in 10 high burden malaria districts of Amhara region from 15 May to 15 June 2014. Data were collected using structured questionnaire. Blood samples were collected from 1000 malaria suspected cases in 10 health centers. RDT (SD BIOLINE) and Giemsa microscopy were performed as per standard procedures. Kappa value, logistic regression and chi-square test were used for statistical analysis. The overall positivity rate (PR) of malaria parasites by RDT and Giemsa microscopy was 17.1 and 16.5% respectively. Compared to Giemsa microscopy as "gold standard", RDT showed 83.9% sensitivity and 96% specificity. The level of agreement between first reader and second reader for blood film microscopy was moderate (Kappa value = 0.74). Logistic regression showed that male, under five year of age and having fever more than 24 h prior to malaria diagnosis had statistically significant association with malaria positivity rate for malaria parasites. The overall specificity and negative predictive values of RDT for malaria diagnosis were excellent. However, the sensitivity and positive predictive values of RDT were low. Therefore, in-service training, quality monitoring of RDTs, and adequate laboratory supplies for diagnostic services of malaria would be crucial for effective intervention measures.

Molecular Epidemiological Survey of Cutaneous Leishmaniasis in Two Highly Endemic Metropolises of Iran, Application of FTA Cards for DNA Extraction From Giemsa-Stained Slides

PubMed Central

Izadi, Shahrokh; Mirhendi, Hossein; Jalalizand, Niloufar; Khodadadi, Hossein; Mohebali, Mehdi; Nekoeian, Shahram; Jamshidi, Ali; Ghatee, Mohammad Amin

2016-01-01

Background: PCR has been used for confirmation of leishmaniasis in epidemiological studies, but complexity of DNA extraction and PCR approach has confined its routine use in developing countries. Objectives: In this study, recent epidemiological situation of cutaneous leishmaniasis (CL) in two hyper-endemic metropolises of Shiraz and Isfahan in Iran was studied using DNA extraction by commercial FTA cards and kinetoplastid DNA (kDNA)-PCR amplification for detection/identification of Leishmania directly from stained skin scraping imprints. Patients and Methods: Fifty four and 30 samples were collected from clinically diagnosed CL patients referred to clinical laboratories of leishmaniasis control centers in Isfahan and Shiraz cities, respectively. The samples were examined by direct microscopy and then scrapings of the stained smears were applied to FTA cards and used directly as DNA source in a nested-PCR to amplify kDNA to detect and identify Leishmania species. Results: Fifty four of 84 (64.2%) slides obtained from patients had positive results microscopically, while 79/84 (94%) of slides had positive results by FTA card-nested-PCR. PCR and microscopy showed a sensitivity of 96.4% and 64.2% and specificity of 100% and 100%, respectively. Interestingly, Leishmania major as causative agent of zoonotic CL was identified in 100% and 90.7% of CL cases from Isfahan and Shiraz cities, respectively, but L. tropica was detected from only 9.3% of cases from Shiraz city. All cases from central regions of Shiraz were L. tropica and no CL case was found in Isfahan central areas. Conclusions: Filter paper-based DNA extraction can facilitate routine use of PCR for diagnosis of CL in research and diagnostic laboratories in Iran and countries with similar conditions. Epidemiologic changes including dominancy of L. major in suburbs of Shiraz and Isfahan metropolises where anthroponotic CL caused by L. tropica had been established, showed necessity of precise studies on CL epidemiology in old urban and newly added districts in the suburbs. PMID:27127596

Performance of a malaria microscopy image analysis slide reading device.

PubMed

Prescott, William R; Jordan, Robert G; Grobusch, Martin P; Chinchilli, Vernon M; Kleinschmidt, Immo; Borovsky, Joseph; Plaskow, Mark; Torrez, Miguel; Mico, Maximo; Schwabe, Christopher

2012-05-06

Viewing Plasmodium in Romanovsky-stained blood has long been considered the gold standard for diagnosis and a cornerstone in management of the disease. This method however, requires a subjective evaluation by trained, experienced diagnosticians and establishing proficiency of diagnosis is fraught with many challenges. Reported here is an evaluation of a diagnostic system (a "device" consisting of a microscope, a scanner, and a computer algorithm) that evaluates scanned images of standard Giemsa-stained slides and reports species and parasitaemia. The device was challenged with two independent tests: a 55 slide, expert slide reading test the composition of which has been published by the World Health Organization ("WHO55" test), and a second test in which slides were made from a sample of consenting subjects participating in a malaria incidence survey conducted in Equatorial Guinea (EGMIS test). These subjects' blood was tested by malaria RDT as well as having the blood smear diagnosis unequivocally determined by a worldwide panel of a minimum of six reference microscopists. Only slides with unequivocal microscopic diagnoses were used for the device challenge, n = 119. On the WHO55 test, the device scored a "Level 4" using the WHO published grading scheme. Broken down by more traditional analysis parameters this result was translated to 89% and 70% sensitivity and specificity, respectively. Species were correctly identified in 61% of the slides and the quantification of parasites fell within acceptable range of the validated parasitaemia in 10% of the cases. On the EGMIS test it scored 100% and 94% sensitivity/specificity, with 64% of the species correct and 45% of the parasitaemia within an acceptable range. A pooled analysis of the 174 slides used for both tests resulted in an overall 92% sensitivity and 90% specificity with 61% species and 19% quantifications correct. In its current manifestation, the device performs at a level comparable to that of many human slide readers. Because its use requires minimal additional equipment and it uses standard stained slides as starting material, its widespread adoption may eliminate the current uncertainty about the quality of microscopic diagnoses worldwide.

[Investigation of the prevalence of Trichomonas vaginalis among female Syrian refugees with the complaints of vaginitis aged between 15-49 years].

PubMed

Yentür Doni, Nebiye; Aksoy, Mustafa; Şimşek, Zeynep; Gürses, Gülcan; Hilali, Neşe Gül; Yıldız Zeyrek, Fadile; Özek, Behire; Yıldırımkaya, Gökhan

2016-10-01

Since the Syrian civil war began in 2011, most of the Syrian refugees have immigrated to Turkey due to its open gate policy and the width of the border. By the end of 2015, it was estimated that there were 2.5 million Syrian refugees in Turkey. Many of the Syrian refugees live in Sanliurfa due to its location on the border with Syria. Trichomonas vaginalis, apart from viral agents is the most common parasite among sexually transmitted infection agents. The aim of this study was to determine the prevalence of T.vaginalis among female married Syrian refugees living outside of the camps in Sanliurfa city center, aged between 15-49 years with complaints of vaginitis. This multi-purpose survey was carried out between February and March of 2015, in collaboration with the United Nations Population Fund and Harran University. This study was approved under the heading of "General Health Status of Female Syrian Refugees" by the Ethics Committee of Harran University Faculty of Medicine. A total of 460 Syrian refugees house were selected using the probability cluster sampling method, with a 95% confidence level and a 5% confidence interval with a design effect. Two women refused to participate in the study, and the response rate was 99.6%. Two Syrian nurses, one laboratory technician, and one interpreter who knew Kurdish and Arabic were hired for the field survey. A structured questionnaire written in Turkish was translated to Arabic and used to collect the sociodemographic data during face to face interviews. According to the questionnaire data, the women with the complaints of vaginal discharge, unusual vaginal bleeding and/or dyspareunia were invited to the Gynecology Department of Harran University Research and Training Hospital for a medical examination. During gynecological examination, swab samples obtained from posterior fornix were evaluated by direct microscopy and Giemsa staining methods for the presence of T.vaginalis trophozoites. Of 458 women who have participated the questionnaire survey, 232 (50.6%) have declared that they had vaginitis complaints. Accordingly, 157 symptomatic and non-pregnant women were invited to the hospital, however only 89 (56.7%) accepted the invitation. T.vaginalis infection was detected in 19 (21.3%) by direct microscopy, and in 32 (36%) by Giemsa staining of the samples taken during the examination of those 89 women (mean age: 31.6 ± 8.7 years). In the gynecological examination, 56.2% (50/89) of the women were clinically diagnosed as vaginitis. A statistically significant association was detected between T.vaginalis positivity and the cases with or without the clinical vaginitis diagnosis (p< 0.001). Our data indicated that the prevalence of T.vaginalis (36%) detected in the female Syrian refugees is higher than the prevalence (3-13%) of our general population, but it is close to the prevalence (40%) in groups with risky behaviors (sex workers). In conclusion, health screening studies and health educations about safe sex life for Syrian refugees would be useful in the prevention of sexually transmitted diseases.

[Effect of Corynebacterium non diphtheriae on functional activity and apoptosis of macrophages].

PubMed

Kharseeva, G G; Voronina, N A; Tiukavkina, S Iu

2014-01-01

Determine the ability of Corynebacterium non diphtheriae to induce phagocytosis and apoptosis of macrophages and evaluate regulatory effect of nuetrophilokines (NPK) induced by Corynebacterium non diphtheriae on these processes. The ability of Corynebacterium non diphtheriae, isolated from upper respiratory tract, skin and urogenital tract (UGT) were studied for the ability to induce phagocytosis and apoptosis of mice macrophages (MP; in vitro during staining by May-Grunwald with additional staining by Romanowsky-Giemsa) before and after the addition of NPK induced by Corynebacterium non diphtheriae. Phagocytic index (PI) was the same for all the Corynebacterium non diphtheriae species, phagocytic number (PN) and index of phagocytosis completion (IPC)--were minimal relative to corynebacteria isolated from UGT. All the studied corynebacteria species induced MP apoptosis; the most pronounced apoptogenic effect was detected in Corynebacterium pseudotuberculosis isolated from UGT. NPK increased PN against corynebacteria isolated from the studied biotopes, IPC--only during studies of corynebacteria isolated from skin. The effect of NPK resulted in a reduction of apoptogenic effect for almost all the Corynebacterium non diphtheriae, regardless of the isolation location. A pronounced apoptogenic effect and insufficiency of phagocytosis processes induced by corynebacteria are the means of realization of Corynebacterium non diphtheriae pathogenic effect. NPK use is possible for immune correction of immune deficiency conditions developing against the background of diseases determined by Corynebacterium non diphtheriae.

DNA damage, lysosomal degradation and Bcl-xL deamidation in doxycycline- and minocycline-induced cell death in the K562 leukemic cell line.

PubMed

Fares, Mona; Abedi-Valugerdi, Manuchehr; Hassan, Moustapha; Potácová, Zuzana

2015-07-31

We investigated mechanisms of cytotoxicity induced by doxycycline (doxy) and minocycline (mino) in the chronic myeloid leukemia K562 cell line. Doxy and mino induced cell death in exposure-dependent manner. While annexin V/propidium iodide staining was consistent with apoptosis, the morphological changes in Giemsa staining were more equivocal. A pancaspase inhibitor Z-VAD-FMK partially reverted cell death morphology, but concurrently completely prevented PARP cleavage. Mitochondrial involvement was detected as dissipation of mitochondrial membrane potential and cytochrome C release. DNA double strand breaks detected with γH2AX antibody and caspase-2 activation were found early after the treatment start, but caspase-3 activation was a late event. Decrement of Bcl-xL protein levels and electrophoretic shift of Bcl-xL molecule were induced by both drugs. Phosphorylation of Bcl-xL at serine 62 was ruled out. Similarly, Bcr/Abl tyrosine kinase levels were decreased. Lysosomal inhibitor chloroquine restored Bcl-xL and Bcr/Abl protein levels and inhibited caspase-3 activation. Thus, the cytotoxicity of doxy and mino in K562 cells is mediated by DNA damage, Bcl-xL deamidation and lysosomal degradation with activation of mitochondrial pathway of apoptosis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Effect of The Receptor Activator of Nuclear Factor кB and RANK Ligand on In Vitro Differentiation of Cord Blood CD133(+) Hematopoietic Stem Cells to Osteoclasts.

PubMed

Kalantari, Nasim; Abroun, Saeid; Soleimani, Masoud; Kaviani, Saeid; Azad, Mehdi; Eskandari, Fatemeh; Habibi, Hossein

2016-01-01

Receptor activator of nuclear factor-kappa B ligand (RANKL) appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells (HSCs). This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor (CTR) on cord blood HSC surface. In this experimental study, CD133(+) hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase (TRAP) staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction (RT-PCR) assay for specific genes. Hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells. Presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast.

Prevalence of mabDAS-1 positivity in biopsy specimens from the esophagogastric junction.

PubMed

Rogge-Wolf, Claudia; Seldenrijk, Cornelis A; Das, Kiron M; Timmer, Robin; Breumelhof, Ronald; Smout, André J P M; Amenta, Peter S; Griffel, Louis H

2002-12-01

Intestinal metaplasia (IM) is a precursor for malignancies at the esophagogastric junction. A monoclonal antibody, mAbDAS-1, can probably identify cellular characteristics of IM before the appearance of goblet cells. The aim of this study was to examine the prevalence of mAbDAS-1 positivity in biopsies from the squamocolumnar junction (SCJ) and to correlate this positivity with the presence of IM and clinical findings. In 559 patients, reflux symptoms were scored, and the presence of reflux esophagitis and hiatus hernia was evaluated during endoscopy. Two biopsy specimens were obtained from the SCJ. In a subset of patients (n = 99), biopsies from the endoscopically defined cardiac region (2 cm distal to proximal margin of gastric folds) were available. Biopsy specimens were stained with hematoxylin and eosin, Alcian Blue, modified Giemsa, and mAbDAS-1. mAbDAS-1 positivity was observed in the SCJ biopsies of 201 of 486 (41.4%) patients without IM and in 64 of 73 (87.7%) patients with IM. Patients without IM but with antibody positivity showed similar histological characteristics as patients with IM at the SCJ. Biopsies of 123 of 559 patients (22%) revealed a columnar-cuboidal epithelium, which was found to be mAbDAS-1 positive in 64.2% (77 of 123). Tissue specimens from the cardiac region without IM stained positive in 14.2% (13 of 91), 12 of those also stained at the SCJ. In patients without IM, a high prevalence of mAbDAS-1 positivity was observed. Biopsies of these patients showed similar histological characteristics as patients with IM. Although not all patients exhibiting this reactivity may develop IM, mAbDAS-1 reactivity may help in the understanding of the histogenesis of IM at the SCJ.

Identification by real-time PCR with SYBR Green of Leishmania spp. and Serratia marcescens in canine 'sterile' cutaneous nodular lesions.

PubMed

Cornegliani, Luisa; Corona, Antonio; Vercelli, Antonella; Roccabianca, Paola

2015-06-01

Noninfectious, non-neoplastic, nodular to diffuse, so-called 'sterile' granulomatous/pyogranulomatous skin lesions (SGPSLs) are infrequently identified in dogs and may represent a diagnostic challenge. Their correct identification is based on history, histopathology and absence of intralesional foreign bodies and micro-organisms. The aim of this study was to investigate the presence of Leishmania spp., Mycobacterium spp., Serratia marcescens and Nocardia spp. by real-time PCR in canine nodular skin lesions histologically diagnosed as putatively sterile. Formalin-fixed skin biopsies were collected from 40 dogs. All samples were associated with an SGPSL diagnosis characterized by multifocal, nodular to diffuse, periadnexal and perifollicular pyogranulomas/granulomas. Neither micro-organisms nor foreign bodies were detected with haematoxylin and eosin staining, under polarized light. Further analyses included periodic acid Schiff, Ziehl-Neelsen, Fite Faraco, Giemsa and Gram histochemical stains; anti-Bacillus Calmette-Guérin (BCG) and Leishmania spp. immunohistochemistry; and real-time PCR analysis for Leishmania spp., Mycobacterium spp., S. marcescens and Nocardia spp. Special stains and BCG/immunohistochemistry were negative in all samples. Real-time PCR was positive for Leishmania spp. in four of 40 biopsies and for S. marcescens in two of 40 samples. Real-time PCR for Mycobacterium spp. and Nocardia spp. was negative. No correlation between real-time PCR positivity and a specific histological pattern was identified. Leishmania spp. have been previously identified as possible agents of certain SGPSLs, while the involvement of S. marcescens has not been investigated previously. According to our findings, Serratia spp. should be included in the list of agents possibly associated with a subgroup of granulomatous/pyogranulomatous skin lesions in dogs. © 2015 ESVD and ACVD.

Human-induced pluripotent stem cell-derived macrophages and their immunological function in response to tuberculosis infection.

PubMed

Hong, Danping; Ding, Jiongyan; Li, Ouyang; He, Quan; Ke, Minxia; Zhu, Mengyi; Liu, Lili; Ou, Wen-Bin; He, Yulong; Wu, Yuehong

2018-02-26

Induced pluripotent stem cells (iPS) represent an innovative source for the standardized in vitro generation of macrophages (Mφ). Mφ show great promise in disease pathogenesis, particularly tuberculosis. However, there is no information about human iPS-derived (hiPS) macrophages (hiPS-Mφ) in response to tuberculosis infection. In the present study, macrophages derived from hiPS were established via embryoid body (EB) formation by using feeder-free culture conditions, and the human monocyte cell line THP-1 (THP-1-Mφ) was used as control. iPS-Mφ were characterized by using morphology, Giemsa staining, nonspecific esterase staining (α-NAE), phagocytosis, and surface phenotype. Additionally, after treatment with Bacillus Calmette-Guérin (BCG) for 24 h, cell apoptosis was detected by using an Annexin V-FITC Apoptosis Detection assay. The production of nitric oxide (NO), expression of tumor necrosis factor alpha (TNF-α), activity of apoptosis-related protein cysteine-3 (Caspase-3) and expression of B-cell lymphoma-2 (Bcl-2) were analyzed. With respect to morphology, surface phenotype, and function, the iPS-Mφ closely resembled their counterparts generated in vitro from a human monocyte cell line. iPS-Mφ exhibited the typically morphological characteristics of macrophages, such as round, oval, fusiform and irregular characteristics. The cells were Giemsa-stained-positive, α-NAE-positive, and possessed phagocytic ability. iPS-Mφ express high levels of CD14, CD11b, CD40, CD68, and major histocompatibility complex II (MHC-II). Moreover, with regard to the apoptotic rate, the production of NO, expression of TNF-α, and activity of Caspase-3 and Bcl-2, iPS-Mφ closely resemble that of their counterparts generated in vitro from human monocyte cell line in response to BCG infection. The rate of apoptosis of BCG-treated iPS-Mφ was 37.77 ± 7.94% compared to that of the untreated group at 4.97 ± 1.60% (P < 0.01) by using Annexin V-FITC Apoptosis Detection. Additionally, the rate of apoptosis of BCG-treated THP-1-Mφ was 37.1 ± 2.84% compared to that of the untreated group at 6.19 ± 1.68% (P < 0.001). The expression of TNF-α and the production of NO were significantly increased (P < 0.001), and the activity of Caspase-3 was increased. However, the expression of Bcl-2 was inhibited (P < 0.001). Our results demonstrate that Mφ derived from hiPS perform the immunological function in response to Bacillus Calmette-Guérin infection by undergoing apoptosis, increasing the production of NO and expression of TNF-α. Thus, our study may help to overcome the limitations of research into certain rare diseases due to the lack of adequate supply of disease-specific primary cells.

[Entomological study of Trypanosoma cruzi vectors in the rural communities of Sucre state, Venezuela].

PubMed

García-Jordán, Noris; Berrizbeitia, Mariolga; Concepción, Juan Luis; Aldana, Elis; Cáceres, Ana; Quiñones, Wilfredo

2015-01-01

The ecological niche of Reduvidae vectors has been modified due to environmental changes and human encroachment into the rural areas. This study evaluates the current entomological indices of triatomines responsible for Trypanosoma cruzi infection in Sucre State, Venezuela. A cross-sectional and prospective study was conducted in 95 towns and 577 dwellings in the 15 municipalities of the state of Sucre, Venezuela, from August to November, 2008. Triatomine bugs were identified on the basis of morphological characteristics, and their feces examined for T. cruzi infection through direct microscopy. Positive slides were stained with Giemsa and parasites were identified by morphologic characterization. The entomological indices expressing the highest values were dispersion (16.67%) and household colonization (33.33%). The triatomine species captured were: Rhodnius prolixus , Rhodnius main intradomiciliary vector. Despite the low index of vector infection (1.72%), the existence of species with domiciliary and peridomiciliary reproductive success ensures the persistence of the epidemiological chain both for the disease and the parasite.

[Study of mastocytes in 1298 bone biopsies. Relationship between mastocytes and osteoporosis].

PubMed

Grardel, B; Flautre, B; Sutter, B; Duriez, J; Hardouin, P

1991-11-30

The relationship between the bone damage in systemic mastocytosis and reactional mastocytosis is still poorly understood. The purpose of this study was to determine the incidence of excessive mastocytes in a series of bone biopsies and their significance in cases of osteoporosis. The mastocytes were routinely counted in 1,298 successive biopsies stained with May Grumwald Giemsa: 131 biopsies had more than 5 mastocytes/mm2, i.e., 10% of all samples for all diagnoses combined. In 11 patients (13 bone biopsies) with a large excess of mastocytes (more than 15/mm2) and osteoporosis, the biopsies were examined again to look for mastocytic nodules suggesting bone mastocytosis: mastocytic nodules of this type were found in only 4 cases. The mastocyte is an active cell which may play a role in bone metabolism through the intermediary of its mediators. In osteoporosis, the incidence and significance of excessive mastocytes is not yet understood; this excess of mastocytes appears to correspond to reactive mastocytosis rather than systemic mastocytosis.

Meningoencephalitis due to the amoeboflagellate Naegleria fowleri in ruminants in Algeria.

PubMed

Benterki, Mohamed Seghir; Ayachi, Ammar; Bennoune, Omar; Régoudis, Estelle; Pélandakis, Michel

2016-01-01

Primary amoebic meningoencephalitis (PAM) is a fatal infection in most cases, caused by the amoeba flagellate Naegleria fowleri. This report describes the first cases of PAM in Algeria, in a cow and a ewe from Batna, north-eastern Algeria. The death of both ruminants occurred a week after the first clinical manifestations. The cerebrospinal fluid, after staining with May-Grünwald-Giemsa, showed the presence of amoebae cells. Histological sections revealed numerous amoebae in all parts of the brain. The presence of N. fowleri was confirmed using a species-specific real-time PCR in histological tissue sections. The two PAM cases were reported during the hot season, and the source of infection is very likely the water where the cattle came to drink. Particular attention should be focused on this type of infection in aquatic environments when the temperature is high and preventive measures must be taken to avoid the proliferation of N. fowleri. © M. Benterki et al., published by EDP Sciences, 2016.

Hemocytes of the cochineal insect: ultrastructure.

PubMed

Caselín-Castro, Sandra; Llanderal-Cázares, Celina; Méndez-Gallegos, Santiago de Jesús; Ramírez-Cruz, Arturo; Hernández-Hernández, Fidel de la Cruz

2010-03-01

Using transmission electron microscopy, light microscopy (Giemsa May-Grumwald), and the Periodic Acid-Schif (PAS) and Sudan Black B staining techniques, hemocytes in the hemolymph of adult female Dactylopius coccus were characterized. The following, in order of abundance, were found: granulocytes, plasmatocytes, prohemocytes, and oenocytoids. Granulocytes varied in size with granulations in the cytoplasm, a large quantity of mitochondria, rugose endoplasmatic reticulum, ribosomes and vesicles, central or exocentric, spherical and occasionally lobulate nucleus. Plasmatocytes were polymorphic with irregularities in the plasma membrane; cytoplasm contained mitochondria, rugose endoplasmatic reticulum and vesicles, and exocentric, spherical, or irregular nucleus. In both types of hemocytes, scant polysaccharides and lipids were found. Prohemocytes were small and spherical with homogeneous cytoplasm and large exocentric nuclei. Oenocytoids were oval or irregular with dense homogeneous cytoplasm and elongated exocentric nuclei. The percentages of granulocytes on different days (d 1 and 10) during the life of the adult female were significantly different, as were those of plasmatocytes on d 30 and 50 and prohemocytes on d 1 and 50. (c) 2010 Wiley Periodicals, Inc.

White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis.

PubMed

Wang, Y T; Liu, W; Seah, J N; Lam, C S; Xiang, J H; Korzh, V; Kwang, J

2002-12-10

White spot syndrome virus (WSSV) was specifically detected by PCR in Penaeus merguiensis hemocytes, hemolymph and plasma. This suggested a close association between the shrimp hemolymph and the virus. Three types of hemocyte from shrimp were isolated using flow cytometry. Dynamic changes of the hemocyte subpopulations in P. merguiensis at different times after infection were observed, indicating that the WSSV infection selectively affected specific subpopulations. Immunofluorescence assay (IFA) and a Wright-Giemsa double staining study of hemocyte types further confirmed the cellular localization of the virus in the infected hemocytes. Electron microscopy revealed virus particles in both vacuoles and the nucleus of the semigranular cells (SGC), as well as in the vacuoles of the granular cells (GC). However, no virus could be detected in the hyaline cells (HC). Our results suggest that the virus infects 2 types of shrimp hemocytes--GCs and SGCs. The SGC type contains higher virus loads and exhibits faster infection rates, and is apparently more susceptible to WSSV infection.

Meiotic Chromosome Analysis of the Giant Water Bug, Lethocerus indicus

PubMed Central

Wisoram, Wijit; Saengthong, Pradit; Ngernsiri, Lertluk

2013-01-01

The giant water bug, Lethocerus indicus (Lepeletier and Serville) (Heteroptera: Belostomatidae), a native species of Southeast Asia, is one of the largest insects belonging to suborder Heteroptera. In this study, the meiotic chromosome of L. indicus was studied in insect samples collected from Thailand, Myanmar, Loas, and Cambodia. Testicular cells stained with lacto-acetic orcein, Giemsa, DAPI, and silver nitrate were analyzed. The results revealed that the chromosome complement of L. indicus was 2n = 22A + neo-XY + 2m, which differed from that of previous reports. Each individual male contained testicular cells with three univalent patterns. The frequency of cells containing neo-XY chromosome univalent (∼5%) was a bit higher than that of cells with autosomal univalents (∼3%). Some cells (∼0.5%) had both sex chromosome univalents and a pair of autosomal univalents. None of the m-chromosome univalents were observed during prophase I. In addition, this report presents clear evidence about the existence of m-chromosomes in Belostomatidae. PMID:23895100

Meningoencephalitis due to the amoeboflagellate Naegleria fowleri in ruminants in Algeria

PubMed Central

Benterki, Mohamed Seghir; Ayachi, Ammar; Bennoune, Omar; Régoudis, Estelle; Pélandakis, Michel

2016-01-01

Primary amoebic meningoencephalitis (PAM) is a fatal infection in most cases, caused by the amoeba flagellate Naegleria fowleri. This report describes the first cases of PAM in Algeria, in a cow and a ewe from Batna, north-eastern Algeria. The death of both ruminants occurred a week after the first clinical manifestations. The cerebrospinal fluid, after staining with May-Grünwald-Giemsa, showed the presence of amoebae cells. Histological sections revealed numerous amoebae in all parts of the brain. The presence of N. fowleri was confirmed using a species-specific real-time PCR in histological tissue sections. The two PAM cases were reported during the hot season, and the source of infection is very likely the water where the cattle came to drink. Particular attention should be focused on this type of infection in aquatic environments when the temperature is high and preventive measures must be taken to avoid the proliferation of N. fowleri. PMID:26979770

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

Sousa, Debora Batista Pinheiro, E-mail: deborabpsousa@gmail.com; Neta, Raimunda Nonata Fortes Carvalho

This study used micronucleus assays and erythrocyte indices in the freshwater fish tambaqui, Colossoma macropomum, to assess environmental impacts in the Environmental Protection Area at Maracanã, São Luis, Brazil. Fish were sampled from two locations within the protected area, Serena Lagoon and Ambude River, on four occasions. Biometric data (length and weight) and an aliquot of blood were collected from each fish for analysis. Erythrocyte indices including: mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were calculated, and blood samples were examined for micronuclei and nuclear morphological changes. Micronuclei were found in fish from both locations, althoughmore » the frequency was higher in fish from Ambude River. Nuclear morphological changes were identified only in fish collected from Ambude River. Several nuclear morphological changes were found in erythrocytes stained with Giemsa, including: micronuclei and binucleate nuclei. On average, erythrocyte indices were lower in fish collected from Ambude River than in those from Serena Lagoon. Our results indicate that micronuclei and erythrocyte indices can be used in C. macropomum as indicators of environmental health.« less

Leukocyte profiles for western fence lizards, Sceloporus occidentalis, naturally infected by the malaria parasite Plasmodium mexicanum.

PubMed

Motz, Victoria L; Lewis, William D; Vardo-Zalik, Anne M

2014-10-01

Plasmodium mexicanum is a malaria parasite that naturally infects the western fence lizard, Sceloporus occidentalis , in northern California. We set out to determine whether lizards naturally infected with this malaria parasite have different leukocyte profiles, indicating an immune response to infection. We used 29 naturally infected western fence lizards paired with uninfected lizards based on sex, snout-to-vent length, tail status, and the presence-absence of ectoparasites such as ticks and mites, as well as the presence-absence of another hemoparasite, Schellackia occidentalis. Complete white blood cell (WBC) counts were conducted on blood smears stained with Giemsa, and the proportion of granulocytes per microliter of blood was estimated using the Avian Leukopet method. The abundance of each WBC class (lymphocytes, monocytes, heterophils, eosinophils, and basophils) in infected and uninfected lizards was compared to determine whether leukocyte densities varied with infection status. We found that the numbers of WBCs and lymphocytes per microliter of blood significantly differed (P < 0.05) between the 2 groups for females but not for males, whereas parasitemia was significantly correlated with lymphocyte counts for males, but not for females. This study supports the theory that infection with P. mexicanum stimulates the lizard's immune response to increase the levels of circulating WBCs, but what effect this has on the biology of the parasite remains unclear.

ZnO Nanoparticles Treatment Induces Apoptosis by Increasing Intracellular ROS Levels in LTEP-a-2 Cells.

PubMed

Wang, Caixia; Hu, Xiaoke; Gao, Yan; Ji, Yinglu

2015-01-01

Owing to the wide use of novel nanoparticles (NPs) such as zinc oxide (ZnO) in all aspects of life, toxicological research on ZnO NPs is receiving increasing attention in these days. In this study, the toxicity of ZnO NPs in a human pulmonary adenocarcinoma cell line LTEP-a-2 was tested in vitro. Log-phase cells were exposed to different levels of ZnO NPs for hours, followed by colorimetric cell viability assay using tetrazolium salt and cell survival rate assay using trypan blue dye. Cell morphological changes were observed by Giemsa staining and light microscopy. Apoptosis was detected by using fluorescence microscopy and caspase-3 activity assay. Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method. Results showed that ZnO NPs (≥ 0.01 μg/mL) significantly inhibited proliferation (P < 0.05) and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure. The intracellular ROS level rose up to 30-40% corresponding to significant depletion (approximately 70-80%) in GSH content in LTEP-a-2 cells (P < 0.05), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production. This study elucidates the toxicological mechanism of ZnO NPs in human pulmonary adenocarcinoma cells and provides reference data for application of nanomaterials in the environment.

In vitro cultivation and cryopreservation of Babesia bigemina sporokinetes in hemocytes of Rhipicephalus microplus.

PubMed

de Rezende, Jania; Rangel, Charles P; McIntosh, Douglas; Silveira, Júlia A G; Cunha, Nathalie C; Ramos, Carlos A N; Fonseca, Adivaldo H

2015-09-15

Cultures of tick hemocytes represent alternative cell lines for the isolation and cultivation of a variety of hemoparasites. The present study reports the development and evaluation of methods for the in vitro culture and maintenance of sporokinetes of Babesia bigemina in association with hemocytes of the tick Rhipicephalus microplus. Hemolymph, from engorged females infected with B. bigemina sporokinetes, was incubated at 28 °C in L15 culture medium supplemented with 40% fetal bovine serum. Adherence of hemocytes to flask surfaces and the development of B. bigemina sporokinetes commenced on the first day of cultivation. The protozoa demonstrated clear motility and the capacity to adhere to hemocyte membranes for up to 25 days, at which time the hemocytes began to show signs of degeneration. Examination of Giemsa stained hemocyte cultures, revealed the presence of pyriformis forms, as well as mature and immature sporokinetes with dark red nuclei, centralized or near the apical extremities. Sporokinetes harvested from culture supernatants were cryopreserved in liquid nitrogen. Inoculation of parasite-free hemocyte cultures with defrosted sporokinetes, demonstrated the viability and interaction of the protozoa with the hemocytes over 21 days. Cultured hemocytes of R. microplus hold potential for development as a tool in the study of host parasite interactions and as a substrate for the in vitro maintenance of B. bigemina sporokinetes. Copyright © 2015 Elsevier B.V. All rights reserved.

Biodosimetry results from space flight Mir-18.

PubMed

Yang, T C; George, K; Johnson, A S; Durante, M; Fedorenko, B S

1997-11-01

Astronauts are classified as radiation workers due to the presence of ionizing radiation in space. For the assessment of health risks, physical dosimetry has been indispensable. However, the change of the location of dosimeters on the crew members, the variation in dose rate with location inside the spacecraft and the unknown biological effects of microgravity can introduce significant uncertainties in estimating exposure. To circumvent such uncertainty, a study on the cytogenetic effects of space radiation in human lymphocytes was proposed and conducted for Mir-18, a 115-day mission. This study used fluorescence in situ hybridization (FISH) with whole-chromosome painting probes to score chromosomal exchanges and the Giemsa staining method to determine the frequency of dicentrics. The growth kinetics of cells and sister chromatid exchanges (SCEs) were examined to ensure that chromosomal aberrations were scored in the first mitosis and were induced primarily by space radiation. Our results showed that the frequency of chromosomal aberrations increased significantly in postflight samples compared to samples drawn prior to flight, and that the frequency of SCEs was similar for both pre- and postflight samples. Based on a dose-response curve for preflight samples exposed to gamma rays, the absorbed dose received by crew members during the mission was estimated to be about 14.75 cSv. Because the absorbed dose measured by physical dosimeters is 5.2 cGy for the entire mission, the RBE is about 2.8.

Biodosimetry results from space flight Mir-18

NASA Technical Reports Server (NTRS)

Yang, T. C.; George, K.; Johnson, A. S.; Durante, M.; Fedorenko, B. S.

1997-01-01

Astronauts are classified as radiation workers due to the presence of ionizing radiation in space. For the assessment of health risks, physical dosimetry has been indispensable. However, the change of the location of dosimeters on the crew members, the variation in dose rate with location inside the spacecraft and the unknown biological effects of microgravity can introduce significant uncertainties in estimating exposure. To circumvent such uncertainty, a study on the cytogenetic effects of space radiation in human lymphocytes was proposed and conducted for Mir-18, a 115-day mission. This study used fluorescence in situ hybridization (FISH) with whole-chromosome painting probes to score chromosomal exchanges and the Giemsa staining method to determine the frequency of dicentrics. The growth kinetics of cells and sister chromatid exchanges (SCEs) were examined to ensure that chromosomal aberrations were scored in the first mitosis and were induced primarily by space radiation. Our results showed that the frequency of chromosomal aberrations increased significantly in postflight samples compared to samples drawn prior to flight, and that the frequency of SCEs was similar for both pre- and postflight samples. Based on a dose-response curve for preflight samples exposed to gamma rays, the absorbed dose received by crew members during the mission was estimated to be about 14.75 cSv. Because the absorbed dose measured by physical dosimeters is 5.2 cGy for the entire mission, the RBE is about 2.8.

Clinical and Laboratory Features of Six Cases of Candida and Dermatophyte Folliculitis and a Review of Published Studies.

PubMed

Durdu, Murat; Güran, Mümtaz; Kandemir, Hazal; Ilkit, Macit; Seyedmousavi, Seyedmojtaba

2016-02-01

Although some studies have investigated the epidemiological characteristics of Malassezia folliculitis (MF), little is known about the clinical features and laboratory characteristics of folliculitis caused by other fungi. In this prospective study, 158 patients with folliculitis were identified, and cytological and mycological examinations were performed. The positive fungal cultures were confirmed using conventional methods, ITS sequencing and HWP1 analysis. Additionally, an in vitro antifungal susceptibility test was performed. Of 158 patients with folliculitis, 65 (41.1 %) were found to have fungal folliculitis. The most common (90.8 %) fungal folliculitis was MF. Non-MF fungal folliculitis was detected in 6 (9.2 %) patients. Four patients were diagnosed with dermatophytic folliculitis (Trichophyton rubrum in three patients and Arthroderma vanbreuseghemii in one patient), and two patients were diagnosed with Candida albicans folliculitis. Although only 5 of the 6 samples were found to be positive via a potassium hydroxide test, all May-Grünwald-Giemsa-stained samples were positive. Both of the C. albicans isolates demonstrated a susceptibility profile to itraconazole, and all four dermatophytes were susceptible to terbinafine. All six patients completely recovered with systemic and topical treatment. This study revealed that dermatophytes and C. albicans are the primary causative agents of non-Malassezia fungal folliculitis. We compared our findings with published reports on fungal folliculitis.

Genotoxic assessment of chlorhexidine mouthwash on exfoliated buccal epithelial cells in chronic gingivitis patients

PubMed Central

Khan, Saif; Khan, Asad Ullah; Hasan, Sadaf

2016-01-01

Background: Chlorhexidine (CHX) is the gold standard of all chemical plaque control agents and the most commonly prescribed mouthwash. However, several studies have shown cytotoxic and genotoxic effects of CHX on various eukaryotic cells. In this study, we have used micronuclei as a biomarker of DNA damage in buccal epithelial cells of chronic gingivitis patients who were given adjunct 0.2% CHX for plaque control. Materials and Methods: Chronic gingivitis patients who were exclusively on mechanical plaque control methods were taken as control (Group A) (n = 101), and chronic gingivitis patients who along with mechanical plaque control measures were taking 0.2% chlorhexidine mouthwash as adjunct were taken as cases (Group B) (n = 255). The Group B was further divided into 5 subgroups (B1, B2, B3, B4, B5) (n = 51) on increasing duration of usage of CHX from ≤1 week to 24 weeks. Buccal epithelial cells were gently scrapped from the buccal mucosa using soft toothbrush. The epithelial cells were collected in buffer solution and centrifuged at 8000 rpm for 5 min. The buccal epithelial cells were air dried, fixed, and stained with 5% Giemsa stain on preheated glass microscopic slides and observed under microscope to screen 2000 nucleated cells per individual for number of micronucleated cells and micronuclei as genotoxic measure. Results: The mean number of micronucleated cells was found to be 0.41 ± 0.71 for Group A as compared values ranging from 1.65 ± 2.09 (Group B1) to 11.7 ± 1.87 (Group B5) in different subgroups of Group B, and similarly, the mean number of micronuclei was found to be 0.48 ± 0.80 for Group A as compared to values ranging from 2.57 ± 1.64 (Group B1) to 14.5 ± 2.49 (Group B5) in different subgroups of Group B using analysis of variance (P < 0.001). Conclusion: We conclude that CHX mouthwash is genotoxic to buccal epithelial cells and there is incremental trend in genotoxicity as the duration of usage is increased. PMID:29238137

[The diagnosis of malaria by the thick film and the QBC: a comparative study of both technics].

PubMed

Cabezos, J; Bada, J L

1993-06-12

The diagnosis of paludism is important because of the severity of the clinical picture caused by Plasmodium falciparum, the increasing number of travellers to endemic zones and the emigration from these zones. A comparative study of the QBC techniques (staining with acridin orange and observation with ultraviolet light) and the thick film with Giemsa staining was carried out. The QBC and thick film were performed parallelly for 17 months in a total of 623 samples pertaining to subjects from endemic zones of paludism (emigrants, immigrants and travellers). Of the 623 samples studied 49 were positive for paludism by both techniques. Ten were positive with only the thick film and six were positive only with QBC. The sensitivity of QBC versus thick film was 83% and specificity 98.9%. The time used to determine diagnosis with the QBC technique ranged from 6 to 12 minutes from withdrawal of the sample, while with the thick film the time spent was more than 2 hours. The cases positive by thick film and negative with QBC corresponded to patients with very low parasitation. The intensity of parasitation was difficult to determine quantitatively by QBC. Although the QBC technique has the advantage of speed it is inexact with respect to the quantification of parasitemia. Moreover, it is less sensitive than the thick film in patient with very low parasitations and cannot thus substitute the thick film.

Anticancer activity of 7-epiclusianone, a benzophenone from Garcinia brasiliensis, in glioblastoma.

PubMed

Sales, Leilane; Pezuk, Julia Alejandra; Borges, Kleiton Silva; Brassesco, María Sol; Scrideli, Carlos Alberto; Tone, Luiz Gonzaga; dos Santos, Marcelo Henrique; Ionta, Marisa; de Oliveira, Jaqueline Carvalho

2015-10-30

Glioblastoma is the most common tumor of the central nervous system and one of the hardest tumors to treat. Consequently, the search for novel therapeutic options is imperative. 7-epiclusianone, a tetraprenylated benzophenone isolated from the epicarp of the native plant Garcinia brasiliensis, exhibits a range of biological activities but its prospect anticancer activity is underexplored. Thus, the aim of the present study was to evaluate the influence of 7-epiclusianone on proliferation, clonogenic capacity, cell cycle progression and induction of apoptosis in two glioblastoma cell lines (U251MG and U138MG). Cell viability was measured by the MTS assay; for the clonogenic assay, colonies were stained with Giemsa and counted by direct visual inspection; For cell cycle analysis, cells were stained with propidium iodide and analyzed by cytometry; Cyclin A expression was determined by immunoblotting; Apoptotic cell death was determined by annexin V fluorescein isothiocyanate labeling and Caspase-3 activity in living cells. Viability of both cell lines was drastically inhibited; moreover, the colony formation capacity was significantly reduced, demonstrating long-term effects even after removal of the drug. 7-epiclusianone treatment at low concentrations also altered cell cycle progression, decreased the S and G2/M populations and at higher concentrations increased the number of cells at sub-G1, in concordance with the increase of apoptotic cells. The present study demonstrates for the first time the anticancer potential of 7-epiclusianone against glioblastoma cells, thus meriting its further investigation as a potential therapeutic agent.

Morphological changes in vascular and circulating blood cells following exposure to detergent sclerosants.

PubMed

Cooley-Andrade, O; Connor, D E; Ma, D D F; Weisel, J W; Parsi, K

2016-04-01

To investigate morphological changes in vascular and circulating blood cells following exposure to detergent sclerosants sodium tetradecyl sulfate and polidocanol. Samples of whole blood, isolated leukocytes, platelets, endothelial cells, and fibroblasts were incubated with varying concentrations of sclerosants. Whole blood smears were stained with Giemsa and examined by light and bright field microscopy. Phalloidin and Hoechst stains were used to analyze cytoplasmic and nuclear morphology by fluorescence microscopy. Endothelial cell and fibroblasts were analyzed by live cell imaging. Higher concentrations of sclerosants induced cell lysis. Morphological changes in intact cells were observed at sublytic concentrations of detergents. Low concentration sodium tetradecyl sulfate induced erythrocyte acanthocytosis and macrocytosis, while polidocanol induced Rouleaux formation and increased the population of target cells and stomatocytes. Leukocytes showed swelling, blebbing, vacuolation, and nuclear degradation following exposure to sodium tetradecyl sulfate, while polidocanol induced pseudopodia formation, chromatin condensation, and fragmentation. Platelets exhibited pseudopodia with sodium tetradecyl sulfate and a "fried egg" appearance with polidocanol. Exposure to sodium tetradecyl sulfate resulted in size shrinkage in both endothelial cell and fibroblasts, while endothelial cell developed distinct spindle morphology. Polidocanol induced cytoplasmic microfilament bundles in both endothelial cell and fibroblasts. Patchy chromatin condensation was observed following exposure of fibroblasts to either agent. Detergent sclerosants are biologically active at sublytic concentrations. The observed morphological changes are consistent with cell activation, apoptosis, and oncosis. The cellular response is concentration dependent, cell-specific, and sclerosant specific. © The Author(s) 2015.

First confirmed report of outbreak of theileriosis/anaplasmosis in a cattle farm in Henan, China.

PubMed

Cui, Yanyan; Wang, Xiaoxing; Zhang, Yan; Yan, Yaqun; Dong, Haiju; Jian, Fuchun; Shi, Ke; Zhang, Longxian; Wang, Rongjun; Ning, Changshen

2018-01-01

Tick-borne diseases (TBDs) impose a significant constraint to livestock production world widely. In this paper, we presented a case of TBD in a cattle farm in Henan, China. 35 blood samples (7 samples sent by veterinarian, 28 samples gathered by our colleagues) were collected from ill, surviving and asymptomatic cattle and microscopic observation and PCR assays were conducted to characterize the pathogens. Genus Ixodes feeding on these cattle were collected and identified. Theileria annulata-like and Anaplasma marginale-like pathogens were observed in the blood smears stained with Giemsa staining under microscope. Furthermore, 5 out of 7 cattle blood samples were found to be positive for T. annulata by PCR. In the 28 blood specimens, three were positive for T. annulata, while A. marginale DNA was detected in nine blood DNA samples. Besides, 56 ticks feeding on cattle were collected from this farm and were all identified as Rhipisephalus microplus, meanwhile, 10 of them were found to be positive for A. marginale. In addition, phylogenetic analysis of the msp4 gene sequences of A. marginale obtained in this study showed that the isolate from cattle (KX840009) fell in the same clade with that of R. microplus (KX904527), sharing 100% similarity. To the best of our knowledge, this is the first confirmed report of outbreak of theileriosis/anaplasmosis in cattle farms in Henan, China. Copyright © 2017. Published by Elsevier B.V.

Assessing the reliability of microscopy and rapid diagnostic tests in malaria diagnosis in areas with varying parasite density among older children and adult patients in Nigeria.

PubMed

Ayogu, E E; Ukwe, C V; Nna, E O

2016-01-01

Current malaria control strategies are based on early diagnosis and appropriate treatment of malaria cases. The study aimed at comparing the performance of blood film microscopy and rapid diagnostic test (RDT) in Plasmodium falciparum detection in patients ≥6 years of age. A total of 154 consecutive pyretic patients aged 6-62 years were enrolled, sampled, and tested for malaria using RDT (first response) and microscopy by Giemsa staining. Genomic DNA was extracted after saponin hemolysis and nested polymerase chain reaction (PCR) was used to detect Plasmodium falciparum. The endpoints were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Of the 154 patients, 80 (51.9%) had fever of ≥37.5°C. 106 (68.8%) were positive by First response® , 132 (85.7%) by microscopy, and 121 (78.6%) by PCR. The sensitivity, specificity, PPV, and NPV of first response compared to microscopic method were 82.2%, 100.0%, 100.0%, and 34.3%, respectively, while it was 75.4%, 75.0%, 95.3%, and 31.2%, respectively, when compared to PCR. The sensitivity, specificity, PPV, and NPV of the microscopic method compared to PCR were 92.3%, 50.0%, 90.91%, and 54.5%, respectively. There was a significant difference in the performance of RDT and film microscopy methods (P ≤ 0.05). Microscopy performed better and is more reliable than first response (RDT) in areas with low parasite density among patients ≥6 years of age. Rapid diagnostic tests could be useful in aareas with high parasite density as an alternative to smear microscopy.

Automated and unsupervised detection of malarial parasites in microscopic images.

PubMed

Purwar, Yashasvi; Shah, Sirish L; Clarke, Gwen; Almugairi, Areej; Muehlenbachs, Atis

2011-12-13

Malaria is a serious infectious disease. According to the World Health Organization, it is responsible for nearly one million deaths each year. There are various techniques to diagnose malaria of which manual microscopy is considered to be the gold standard. However due to the number of steps required in manual assessment, this diagnostic method is time consuming (leading to late diagnosis) and prone to human error (leading to erroneous diagnosis), even in experienced hands. The focus of this study is to develop a robust, unsupervised and sensitive malaria screening technique with low material cost and one that has an advantage over other techniques in that it minimizes human reliance and is, therefore, more consistent in applying diagnostic criteria. A method based on digital image processing of Giemsa-stained thin smear image is developed to facilitate the diagnostic process. The diagnosis procedure is divided into two parts; enumeration and identification. The image-based method presented here is designed to automate the process of enumeration and identification; with the main advantage being its ability to carry out the diagnosis in an unsupervised manner and yet have high sensitivity and thus reducing cases of false negatives. The image based method is tested over more than 500 images from two independent laboratories. The aim is to distinguish between positive and negative cases of malaria using thin smear blood slide images. Due to the unsupervised nature of method it requires minimal human intervention thus speeding up the whole process of diagnosis. Overall sensitivity to capture cases of malaria is 100% and specificity ranges from 50-88% for all species of malaria parasites. Image based screening method will speed up the whole process of diagnosis and is more advantageous over laboratory procedures that are prone to errors and where pathological expertise is minimal. Further this method provides a consistent and robust way of generating the parasite clearance curves.

A CASE OF THEILERIOSIS IN THE TIBETIAN YAK (P. GRUNNEUS),

DTIC Science & Technology

Smears taken from the dead and recovered yaks were coloured by Romanovsky-Giemsa and Leishman method but the Theileria of Tibetian yaks proved to be...different from Theileria annulata; in smears rodlike, anaplasmoid, commashaped, and punctiform; the round or ringshaped forms that predominate in... Theileria annalata were very rarely found. Pearshaped forms were not revealed in smears. The parasites are mononuclear. The nucleus is located in one

Image analysis approach for development of a decision support system for detection of malaria parasites in thin blood smear images.

PubMed

Prasad, Keerthana; Winter, Jan; Bhat, Udayakrishna M; Acharya, Raviraja V; Prabhu, Gopalakrishna K

2012-08-01

This paper describes development of a decision support system for diagnosis of malaria using color image analysis. A hematologist has to study around 100 to 300 microscopic views of Giemsa-stained thin blood smear images to detect malaria parasites, evaluate the extent of infection and to identify the species of the parasite. The proposed algorithm picks up the suspicious regions and detects the parasites in images of all the views. The subimages representing all these parasites are put together to form a composite image which can be sent over a communication channel to obtain the opinion of a remote expert for accurate diagnosis and treatment. We demonstrate the use of the proposed technique for use as a decision support system by developing an android application which facilitates the communication with a remote expert for the final confirmation on the decision for treatment of malaria. Our algorithm detects around 96% of the parasites with a false positive rate of 20%. The Spearman correlation r was 0.88 with a confidence interval of 0.838 to 0.923, p<0.0001.

[Efficacy of a rapid test to diagnose Plasmodium vivax in symptomatic patients of Chiapas, Mexico].

PubMed

González-Cerón, Lilia; Rodríguez, Mario H; Betanzos, Angel F; Abadía, Acatl

2005-01-01

To evaluate, under laboratory conditions, the sensitivity and specificity of a rapid diagnostic test (OptiMAL), based on immunoreactive strips, to detect Plasmodium vivax infection in febrile patients in Southern Chiapas, Mexico. The presence of parasites in blood samples of 893 patients was investigated by Giemsa-stained thick blood smear microscopic examination (gold standard). A blood drop from the same sample was smeared on immunoreactive strips to investigate the presence of the parasite pLDH. Discordant results were resolved by PCR amplification of the parasite's 18S SSU rRNA, to discard infection. OptiMAL had an overall sensitivity of 93.3% and its specificity was 99.5%. Its positive and negative predictive values were 96.5% and 98.9%, respectively. Signal intensity in OptiMAL strips correlated well with the parasitemia density in the blood samples (r = 0.601, p = 0.0001). This rapid test had acceptable sensitivity and specificity to detect P. vivax under laboratory conditions and could be useful for malaria diagnosis in field operations in Mexico.

BLOOD VESSELS IN GANGLIA IN HUMAN ESOPHAGUS MIGHT EXPLAIN THE HIGHER FREQUENCY OF MEGAESOPHAGUS COMPARED WITH MEGACOLON

PubMed Central

Adad, Sheila Jorge; Etchebehere, Renata Margarida; Jammal, Alessandro Adad

2014-01-01

This study aimed to determine the existence of blood vessels within ganglia of the myenteric plexus of the human esophagus and colon. At necropsy, 15 stillborns, newborns and children up to two years of age, with no gastrointestinal disorders, were examined. Rings of the esophagus and colon were analyzed and then fixed in formalin and processed for paraffin. Histological sections were stained by hematoxylin-eosin, Giemsa and immunohistochemistry for the characterization of endothelial cells, using antibodies for anti-factor VIII and CD31. Blood vessels were identified within the ganglia of the myenteric plexus of the esophagus, and no blood vessels were found in any ganglia of the colon. It was concluded that the ganglia of the myenteric plexus of the esophagus are vascularized, while the ganglia of the colon are avascular. Vascularization within the esophageal ganglia could facilitate the entrance of infectious agents, as well as the development of inflammatory responses (ganglionitis) and denervation, as found in Chagas disease and idiopathic achalasia. This could explain the higher frequency of megaesophagus compared with megacolon. PMID:25351549

Cytological and cytogenetical studies on brain tumors. V. Preferential loss of sex chromosomes in human meningiomas.

PubMed

Zankl, H; Seidel, H; Zang, K D

1975-01-01

Twelve out of 88 cytogenetically examined meningiomas of female patients showed, in addition to the typical loss of a chromosome 22, a loss of 1 or more chromosomes of group C. Among them 8 tumors had less than 8% cells with Barr-body-like particles, whereas in one tumor 12% and in 3 others over 20% Barr bodies were found, which, based on control studies, were classified as sex-chromatin negative, partly positive, and positive, respectively. In one case the loss of an X chromosome was verified by Giemsa banding. In 6 out of 24 meningiomas of male origin, the chromosomal morphology and association pattern strongly indicated that besides the loss of a chromosome 22, the Y chromosome was also missing. Moreover, the loss of the male sex chromosome could be ascertained in 4 tumors by the conspicuous absence of Y fluorescence in interphase nuclei and in metaphase plates after fluorescence staining. The findings are discussed in connection with the gonosomal loss in other human tumors and in old age.

[Study on sperm damage caused by trichloroethylene in male rats].

PubMed

Wu, De-sheng; Yang, Lin-qing; Huang, Sui; Liu, Jian-jun; Xu, Xin-yun; Huang, Hai-yan; Gong, Chun-mei; Hu, Gong-hua; Liu, Qing-cheng; Yang, Xi-fei; Hong, Wen-xu; Zhou, Li; Huang, Xin-feng; Yuan, Jian-hui; Zhuang, Zhi-xiong

2013-11-01

To study in vitro sperm damage caused by trichloroethylene in male rats. Sperms of Sprague-Dawley (SD) rats were collected 4 hours after being contaminated by trichloroethylene of 0, 2, 4, 6, 8, and 10 mmol/L in vitro. Giemsa staining was performed to observe the morphological changes of sperms, and flow cytometer was used to detect the changes in mitochondrial membrane potential. The sperm motilities in 6, 8, and 10 mmol/L trichloroethylene groups decreased significantly compared with that in control group (P <0.01); the sperm aberration rates in 8 and 10 mmol/L trichloroethylene groups were significantly higher than that in control group (P<0.01). With the increase in exposure dose, the proportion of sperms with reduced mitochondrial membrane potential increased, and there were significant differences in sperm apoptosis rate between the 4, 6, 8, and 10 mmol/L trichloroethylene groups and control group (P<0.01). In vitro exposure to trichloroethylene can reduce sperm motility and increase the aberration rate and apoptosis rate of sperms in male SD rats.

Description and phylogeny of a new microsporidium from the elm leaf beetle, Xanthogaleruca luteola Muller, 1766 (Coleoptera: Chrysomelidae).

PubMed

Bekircan, Çağrı; Bülbül, Ufuk; Güler, Halil I; Becnel, James J

2017-02-01

This study describes a new genus and species of microsporidia which is a pathogen of the elm leaf beetle, Xanthogaleruca luteola Muller, 1776 (Coleoptera: Chrysomelidae). The beetles were collected from Istanbul in Turkey. All developmental stages are uninucleate and in direct contact with the host cell cytoplasm. Giemsa-stained mature spores are oval in shape and measured 3.40 ± 0.37 μm in length and 1.63 ± 0.20 μm in width. These uninucleate spores have an isofilar polar filament with 11 turns. The spore wall was trilaminar (75 to 115 nm) with a rugose, electron-dense exospore (34 to 45 nm) and a thickened, electron-lucent endospore (65 to 80 nm) overlaying the plasmalemma. Morphological, ultrastructural, and molecular features indicate that the described microsporidium is dissimilar to all known microsporidian taxa and confirm that it has different taxonomic characters than other microsporidia infecting X. luteola and is named here as Rugispora istanbulensis n. gen., n. sp.

A model of chromosome aberration induction: applications to space research.

PubMed

Ballarini, Francesca; Ottolenghi, Andrea

2005-10-01

A mechanistic model and Monte Carlo code simulating chromosome aberration induction in human lymphocytes is presented. The model is based on the assumption that aberrations arise from clustered DNA lesions and that only the free ends of clustered lesions created in neighboring chromosome territories or in the same territory can join and produce exchanges. The lesions are distributed in the cell nucleus according to the radiation track structure. Interphase chromosome territories are modeled as compact intranuclear regions with volumes proportional to the chromosome DNA contents. Both Giemsa staining and FISH painting can be simulated, and background aberrations can be taken into account. The good agreement with in vitro data provides validation of the model in terms of both the assumptions adopted and the simulation techniques. As an application in the field of space research, the model predictions were compared with aberration yields measured among crew members of long-term missions on board Mir and ISS, assuming an average radiation quality factor of 2.4. The agreement obtained also validated the model for in vivo exposure scenarios and suggested possible applications to the prediction of other relevant aberrations, typically translocations.

Morphometric study of the fibrosis and mast cell count in the circular colon musculature of chronic Chagas patients with and without megacolon.

PubMed

Pinheiro, Simone Wanderley; Rua, Adilha Misson de Oliveira; Etchebehere, Renata Margarida; Cançado, Cristiane Gobbo; Chica, Javier Em lio Lazo; Lopes, Edison Reis; Adad, Sheila Jorge

2003-01-01

A morphometric study of the circular colon musculature was performed, in which the mast cell count was determined and the connective fibrous tissue in this layer was measured. The objective was to gain better understanding of Chagas megacolon morphology and contribute towards the knowledge of fibrosis pathogenesis in Chagas megas. An evaluation was made of 15 distal sigmoid rings from Chagas patients with megacolon (MCC), 15 without megacolon (CSMC) and 15 non-Chagas patients (NC). The rings were fixed in formol, embedded in paraffin, and 7mm thick sections were cut and stained using Azan-Heidenhain and Giemsa. The mast cell count and fibrosis were greater in the MCC group than in the CSMC and NC groups (p< 0,05; Kruskal-Wallis test) and there was no significant difference between the latter two. The fibrosis and increased mast cell count in the colon musculature of the MCC group possibly indicates that there is a relationship between mastocytosis and fibrosis, as has already been demonstrated in other pathologies.

Naegleria fowleri That Induces Primary Amoebic Meningoencephalitis: Rapid Diagnosis and Rare Case of Survival in a 12-Year-Old Caucasian Girl.

PubMed

Dunn, Andrew L; Reed, Tameika; Stewart, Charlotte; Levy, Rebecca A

2016-05-01

Primary amoebic meningoencephalitis (PAM) is a rare and almost always fatal disease that is caused by Naegleria fowleri, a freshwater thermophilic amoeba. Our case involves an adolescent female who presented with fever of unknown origin. A lumbar puncture was performed, and the Wright-Giemsa and Gram stained cerebrospinal fluid (CSF) cytospin slides showed numerous organisms. Experienced medical technologists in the microbiology and hematology laboratories identified the organisms as morphologically consistent with Naegleria species. The laboratory made a rapid diagnosis and alerted emergency department care providers within 75 minutes. The patient was treated for PAM with amphotericin, rifampin, azithromycin, fluconazole and aggressive supportive therapy including dexamethasone. The Centers for Disease Control and Prevention (CDC) was contacted, and miltefosine, an investigational medication, was started. Additional treatment included an intraventricular shunt and controlled hypothermia in order to mitigate potential cerebral edema. Our patient is a rare success story, as she was diagnosed swiftly, successfully treated, and survived PAM. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

[Canine visceral leishmaniasis in dog from Caldas Novas, Goiás].

PubMed

Azevedo, Elisa M R; Linhares, Guido F C; Duarte, Sabrina C; Jayme, Valéria D S; Oliveira, Helton F; Oliveira, Vilma F

2008-09-01

The objective of the present work was to describe a visceral case of leishmaniasis in a dog from Caldas Novas, GO, region until then considered as a disease free area. The animal, attended in the Veterinarian Hospital of the Federal University of Goiás, presented loss of weight, alopecic area of irregular format at the nasal back, onicogrifosis, atrophy of the heads muscles and failure of the right popliteus lymphonode. For the laboratorial diagnosis, samples were collected by biopsy from the complete skin of the auricular region, by punsion from the popliteus lymphonode for preparation of Giemsa stained blades and 5 mL of the blood were also collected for serum diagnosis. The direct microscopy revealed, from the evaluation of the imprint obtained from the skin fragment and from the squash of the inhaled lymphonode, great amount of amastigoste forms of Leishmania in the cytoplasm of macrophages. Based on the clinical presentation, on the parasitologic and on the serology examination of the samples, we concluded the exams with the diagnosis of calazar disease.

Endogenous Life Cycle of Eimeria marmosopos (Apicomplexa: Eimeriidae) from the Opossum, Didelphis marsupialis (Didelphimorphia: Didelphidae) in Costa Rica.

PubMed

Chinchilla, Misael; Valerio, Idalia; Duszynski, Donald

2015-08-01

The endogenous life cycle of Eimeria marmosopos was studied in experimentally infected young opossums, Didelphis marsupialis . All the endogenous stages were located in the epithelial cells of villi in the small intestine. Giemsa-stained mucosal scrapings and histological sections were studied for the diagnosis of all the life cycle stages. Eimeria marmosopos has 3 generations of meronts (M) that differ by size, shape, and number of merozoites (m), which also differ in their size, shape, and location of their nuclei within the cytoplasm of the meronts. The 3 meront types, M(1)-M(3), respectively, had 8-15 (m(1)), 4-9 (m(2)), and 22-30 (m(3)) merozoites. Macrogametocytes and microgametocytes, as well as macrogametes and microgametes, completed the sexual cycle, finishing with the formation of unsporulated oocysts. This parasite's endogenous development produced severe intestinal lesions in experimentally infected opossums. There are 56 Eimeria species known from all marsupials worldwide, but this is the first complete life cycle in which both the asexual and sexual stages have been documented.

Absence of Asymptomatic Malaria Infection in a Cross-sectional Study in Iranshahr District, Iran under Elimination Programmes.

PubMed

Pirahmadi, Sakineh; Zakeri, Sedigheh; Raeisi, Ahmad

2017-01-01

Asymptomatic malaria infection provides a reservoir of parasites, causing the persistence of malaria transmission. It accounts an important challenge for successful management of the control, elimination, and eradication programmes in any malaria-endemic region. This investigation was designed to assess the presence and the prevalence of asymptomatic carriers in Iranshahr district of Sistan and Baluchistan Province (2013-2014), with a considerable population movement, during the malaria elimination phase in Iran. Finger-prick blood samples were collected from symptomless (n=250) and febrile (n=50) individuals residing in Iranshahr district, easthern Iran (Hoodian, Mand, Chah-e Giji, Jolgehashem, Esfand, Dalgan and Chahshour) during Jan 2013 to Dec 2014, and Plasmodium infections were detected using light microscopic and highly sensitive nested-PCR techniques. Thick and thin Giemsa-stained blood smears were negative for Plasmodium parasites. In addition, based on nested-PCR analysis, no P. vivax , P. falciparum, and P. malariae parasites were detected among the studied individuals. Investigation the absence of asymptomatic carriers in Iranshahr district was illustrated and achieving malaria elimination in this area is feasible in a near future.

Plasmodium ovale infection in Malaysia: first imported case.

PubMed

Lim, Yvonne A L; Mahmud, Rohela; Chew, Ching Hoong; T, Thiruventhiran; Chua, Kek Heng

2010-10-08

Plasmodium ovale infection is rarely reported in Malaysia. This is the first imported case of P. ovale infection in Malaysia which was initially misdiagnosed as Plasmodium vivax. Peripheral blood sample was first examined by Giemsa-stained microscopy examination and further confirmed using a patented in-house multiplex PCR followed by sequencing. Initial results from peripheral blood smear examination diagnosed P. vivax infection. However further analysis using a patented in-house multiplex PCR followed by sequencing confirmed the presence of P. ovale. Given that Anopheles maculatus and Anopheles dirus, vectors of P. ovale are found in Malaysia, this finding has significant implication on Malaysia's public health sector. The current finding should serve as an alert to epidemiologists, clinicians and laboratory technicians in the possibility of finding P. ovale in Malaysia. P. ovale should be considered in the differential diagnosis of imported malaria cases in Malaysia due to the exponential increase in the number of visitors from P. ovale endemic regions and the long latent period of P. ovale. It is also timely that conventional diagnosis of malaria via microscopy should be coupled with more advanced molecular tools for effective diagnosis.

Inter-rater reliability of malaria parasite counts and comparison of methods

PubMed Central

2009-01-01

Background The introduction of artemesinin-based treatment for falciparum malaria has led to a shift away from symptom-based diagnosis. Diagnosis may be achieved by using rapid non-microscopic diagnostic tests (RDTs), of which there are many available. Light microscopy, however, has a central role in parasite identification and quantification and remains the main method of parasite-based diagnosis in clinic and hospital settings and is necessary for monitoring the accuracy of RDTs. The World Health Organization has prepared a proficiency testing panel containing a range of malaria-positive blood samples of known parasitaemia, to be used for the assessment of commercially available malaria RDTs. Different blood film and counting methods may be used for this purpose, which raises questions regarding accuracy and reproducibility. A comparison was made of the established methods for parasitaemia estimation to determine which would give the least inter-rater and inter-method variation Methods Experienced malaria microscopists counted asexual parasitaemia on different slides using three methods; the thin film method using the total erythrocyte count, the thick film method using the total white cell count and the Earle and Perez method. All the slides were stained using Giemsa pH 7.2. Analysis of variance (ANOVA) models were used to find the inter-rater reliability for the different methods. The paired t-test was used to assess any systematic bias between the two methods, and a regression analysis was used to see if there was a changing bias with parasite count level. Results The thin blood film gave parasite counts around 30% higher than those obtained by the thick film and Earle and Perez methods, but exhibited a loss of sensitivity with low parasitaemia. The thick film and Earle and Perez methods showed little or no bias in counts between the two methods, however, estimated inter-rater reliability was slightly better for the thick film method. Conclusion The thin film method gave results closer to the true parasite count but is not feasible at a parasitaemia below 500 parasites per microlitre. The thick film method was both reproducible and practical for this project. The determination of malarial parasitaemia must be applied by skilled operators using standardized techniques. PMID:19939271

Molecular Epidemiological Survey of Cutaneous Leishmaniasis in Two Highly Endemic Metropolises of Iran, Application of FTA Cards for DNA Extraction From Giemsa-Stained Slides.

PubMed

Izadi, Shahrokh; Mirhendi, Hossein; Jalalizand, Niloufar; Khodadadi, Hossein; Mohebali, Mehdi; Nekoeian, Shahram; Jamshidi, Ali; Ghatee, Mohammad Amin

2016-02-01

PCR has been used for confirmation of leishmaniasis in epidemiological studies, but complexity of DNA extraction and PCR approach has confined its routine use in developing countries. In this study, recent epidemiological situation of cutaneous leishmaniasis (CL) in two hyper-endemic metropolises of Shiraz and Isfahan in Iran was studied using DNA extraction by commercial FTA cards and kinetoplastid DNA (kDNA)-PCR amplification for detection/identification of Leishmania directly from stained skin scraping imprints. Fifty four and 30 samples were collected from clinically diagnosed CL patients referred to clinical laboratories of leishmaniasis control centers in Isfahan and Shiraz cities, respectively. The samples were examined by direct microscopy and then scrapings of the stained smears were applied to FTA cards and used directly as DNA source in a nested-PCR to amplify kDNA to detect and identify Leishmania species. Fifty four of 84 (64.2%) slides obtained from patients had positive results microscopically, while 79/84 (94%) of slides had positive results by FTA card-nested-PCR. PCR and microscopy showed a sensitivity of 96.4% and 64.2% and specificity of 100% and 100%, respectively. Interestingly, Leishmania major as causative agent of zoonotic CL was identified in 100% and 90.7% of CL cases from Isfahan and Shiraz cities, respectively, but L. tropica was detected from only 9.3% of cases from Shiraz city. All cases from central regions of Shiraz were L. tropica and no CL case was found in Isfahan central areas. Filter paper-based DNA extraction can facilitate routine use of PCR for diagnosis of CL in research and diagnostic laboratories in Iran and countries with similar conditions. Epidemiologic changes including dominancy of L. major in suburbs of Shiraz and Isfahan metropolises where anthroponotic CL caused by L. tropica had been established, showed necessity of precise studies on CL epidemiology in old urban and newly added districts in the suburbs.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System.

PubMed

Miousse, Isabelle R; Koturbash, Igor; Chalbot, Marie-Cécile; Hauer-Jensen, Martin; Kavouras, Ilias; Pathak, Rupak

2016-12-21

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Role of morphometry in the cytological differentiation of benign and malignant thyroid lesions

PubMed Central

Khatri, Pallavi; Choudhury, Monisha; Jain, Manjula; Thomas, Shaji

2017-01-01

Context: Thyroid nodules represent a common problem, with an estimated prevalence of 4–7%. Although fine needle aspiration cytology (FNAC) has been accepted as a first line diagnostic test, the rate of false negative reports of malignancy is still high. Nuclear morphometry is the measurement of nuclear parameters by image analysis. Image analysis can merge the advantages of morphologic interpretation with those of quantitative data. Aims: To evaluate the nuclear morphometric parameters in fine needle aspirates of thyroid lesions and to study its role in differentiating benign from malignant thyroid lesions. Material and Methods: The study included 19 benign and 16 malignant thyroid lesions. Image analysis was performed on Giemsa-stained FNAC slides by Nikon NIS-Elements Advanced Research software (Version 4.00). Nuclear morphometric parameters analyzed included nuclear size, shape, texture, and density parameters. Statistical Analysis: Normally distributed continuous variables were compared using the unpaired t-test for two groups and analysis of variance was used for three or more groups. Tukey or Tamhane's T2 multiple comparison test was used to assess the differences between the individual groups. Categorical variables were analyzed using the chi square test. Results and Conclusion: Five out of the six nuclear size parameters as well as all the texture and density parameters studied were significant in distinguishing between benign and malignant thyroid lesions (P < 0.05). Cut-off values were derived to differentiate between benign and malignant cases. PMID:28182069

Atmospheric plasma surface modifications of electrospun PCL/chitosan/PCL hybrid scaffolds by nozzle type plasma jets for usage of cell cultivation

NASA Astrophysics Data System (ADS)

Surucu, Seda; Masur, Kai; Turkoglu Sasmazel, Hilal; Von Woedtke, Thomas; Weltmann, Klaus Dieter

2016-11-01

This paper reports Ar gas, Ar + O2, Ar + O2 + N2 gas mixtures and dry air plasma modifications by atmospheric pressure argon driven kINPen and air driven Diener (PlasmaBeam) plasma jets to alter surface properties of three dimensional (3D), electrospun PCL/Chitosan/PCL layer by layer hybrid scaffolds to improve human fibroblast (MRC5) cell attachment and growth. The characterizations of the samples were done by contact angle (CA) measurements, scanning electron microscopy (SEM), X-Ray Photoelectron spectroscopy (XPS) analysis. The results showed that the plasma modification carried out under dry air and Ar + O2 + N2 gas mixtures were altered effectively the nanotopography and the functionality of the material surfaces. It was found that the samples treated with Ar + O2 + N2 gas mixtures for 1 min and dry air for 9 min have better hydrophilicity 78.9° ± 1.0 and 75.6° ± 0.1, respectively compared to the untreated samples (126.5°). Biocompatibility performance of the scaffolds was determined with alamarBlue (aB) assay and MTT assay methods, Giemsa staining, fluorescence microscope, confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) analyses. The results showed that plasma treated samples increased the hydrophilicity and oxygen functionality and topography of the surfaces significantly, thus affecting the cell viability and proliferation on/within scaffolds.

Serological responses and immunity to superinfection with avian malaria in experimentally-infected Hawaii Amakihi

USGS Publications Warehouse

Atkinson, Carter T.; Dusek, Robert J.; Lease, Julie K.

2001-01-01

Six of seven Hawaii Amakihi (Hemignathus virens) with chronic malarial infections had no increases in peripheral parasitemia, declines in food consumption, or loss of body weight when rechallenged with the homologous isolate of Plasmodium relictum 61 to 62 days after initial infection. Five uninfected control amakihi exposed at the same time to infective mosquito bites developed acute infections with high parasitemias. Reductions in food consumption and loss of body weight occurred in all control birds and three of these individuals eventually died. When surviving birds were rechallenged >2 yr later with either the same parasite isolate or an isolate of P. relictum collected on the island of Kauai, all individuals were immune to superinfection. Chronically infected birds developed antibodies to a common suite of malarial antigens ranging in size from 22 to 170 kDa that were detectable as early as 8 days post infection on immunoblots of SDS-polyacrylamide gels. Antibodies to this suite of malarial antigens persisted as long as 1,248 days after initial infection and were consistently detectable at times when parasites were not easily found by microscopy on Giemsa-stained blood smears. The immunoblotting method that is described here appears to be an effective technique for identifying birds with chronic, low-intensity malarial infections when circulating parasites are not easily detectable by microscopy. Hawaiian honeycreepers that are capable of recovering from acute infections develop concomitant immunity to superinfection, making them functionally immune in areas where malaria transmission has become endemic.

Amplification of Mitochondrial DNA for detection of Plasmodiumvivax in Balochistan.

PubMed

Shahwani, Muhammad Naeem; Nisar, Samia; Aleem, Abdul; Panezai, Marina; Afridi, Sarwat; Malik, Shaukat Iqbal

2017-05-01

To access a new step using PCR to amplify the targeted mtDNA sequence for detecting specifically Plasmodium vivax and its co-infections, false positive and false negative results with Plasmodium falciparum. In this study we have standardized a new technical approach in which the target mitochondrial DNA sequence (mtDNA) was amplified by using a PCR technique as a tool to detect Plasmodium spp. Species specific primers were designed to hybridize with cytochrome c oxidase gene of P. vivax (cox I) and P. falciparum (cox III). Two hundred blood samples were collected on the basis of clinical symptoms which were initially examined through microscopic analysis after preparing Giemsa stained thick and thin blood smears. Afterwards genomic DNA was extracted from all samples and was then subjected to PCR amplification by using species specific primers and amplified segments were sequenced for confirmation of results. One-hundred and thirty-two blood samples were detected as positive for malaria by PCR, out of which 64 were found to be positive by PCR and 53 by both microscopy and PCR for P.vivax infection. Nine samples were found to be false negative, one P.vivax mono infection was declared as co infection by PCR and 3 samples identified as having P.falciparum gametes were confirmed as P.vivax by PCR amplification. Sensitivity and specificity were found to be 85% and 92% respectively. Results obtained through PCR method were comparatively better and reliable than microscopy.

Investigative modalities in infectious keratitis.

PubMed

Gupta, Noopur; Tandon, Radhika

2008-01-01

Standard recommended guidelines for diagnosis of infectious keratitis do exist. Based on an extensive Medline literature search, the various investigative modalities available for aiding the diagnosis of microbial keratitis have been reviewed and described briefly. Preferred practice patterns have been outlined and the importance of routine pre-treatment cultures in the primary management of infectious keratitis has been highlighted. Corneal scraping, tear samples and corneal biopsy are few of the specimens needed to carry out the investigative procedures for diagnosis and for initiating therapy in cases of microbial keratitis. In bacterial, fungal and amoebic keratitis, microscopic examination of smears is essential for rapid diagnosis. Potassium hydroxide (KOH) wet mount, Gram's stain and Giemsa stain are widely used and are important for clinicians to start empirical therapy before microbial culture results are available. The usefulness of performing corneal cultures in all cases of suspected infectious keratitis has been well established. In cases of suspected viral keratitis, therapy can be initiated on clinical judgment alone. If a viral culture is needed, scrapings should directly be inoculated into the viral transport media. In vivo confocal microscopy is a useful adjunct to slit lamp bio-microscopy for supplementing diagnosis in most cases and establishing early diagnosis in many cases of non-responding fungal and amoebic keratitis. This is a non-invasive, high resolution technique which allows rapid detection of Acanthamoeba cysts and trophozoites and fungal hyphae in the cornea long before laboratory cultures give conclusive results. Other new modalities for detection of microbial keratitis include molecular diagnostic techniques like polymerase chain reaction, and genetic finger printing by pulsed field gel electrophoresis.

Isolation and characterization of a rhabdovirus from starry flounder (Platichthys stellatus) collected from the northern portion of Puget Sound, Washington, USA

USGS Publications Warehouse

Mork, Christina; Hershberger, Paul K.; Kocan, Richard; Batts, William N.; Winton, James R.

2004-01-01

The initial characterization of a rhabdovirus isolated from a single, asymptomatic starry flounder (Platichthys stellatus) collected during a viral survey of marine fishes from the northern portion of Puget Sound, Washington, USA, is reported. Virions were bullet-shaped and approximately 100 nm long and 50 nm wide, contained a lipid envelope, remained stable for at least 14 days at temperatures ranging from -80 to 5 degrees C and grew optimally at 15 degrees C in cultures of epithelioma papulosum cyprini (EPC) cells. The cytopathic effect on EPC cell monolayers was characterized by raised foci containing rounded masses of cells. Pyknotic and dark-staining nuclei that also showed signs of karyorrhexis were observed following haematoxylin and eosin, May-Grunwald Giemsa and acridine orange staining. PAGE of the structural proteins and PCR assays using primers specific for other known fish rhabdoviruses, including Infectious hematopoietic necrosis virus, Viral hemorrhagic septicemia virus, Spring viremia of carp virus, and Hirame rhabdovirus, indicated that the new virus, tentatively termed starry flounder rhabdovirus (SFRV), was previously undescribed in marine fishes from this region. In addition, sequence analysis of 2678 nt of the amino portion of the viral polymerase gene indicated that SFRV was genetically distinct from other members of the family Rhabdoviridae for which sequence data are available. Detection of this virus during a limited viral survey of wild fishes emphasizes the void of knowledge regarding the diversity of viruses that naturally infect marine fish species in the North Pacific Ocean.

Automated discrimination of dicentric and monocentric chromosomes by machine learning-based image processing.

PubMed

Li, Yanxin; Knoll, Joan H; Wilkins, Ruth C; Flegal, Farrah N; Rogan, Peter K

2016-05-01

Dose from radiation exposure can be estimated from dicentric chromosome (DC) frequencies in metaphase cells of peripheral blood lymphocytes. We automated DC detection by extracting features in Giemsa-stained metaphase chromosome images and classifying objects by machine learning (ML). DC detection involves (i) intensity thresholded segmentation of metaphase objects, (ii) chromosome separation by watershed transformation and elimination of inseparable chromosome clusters, fragments and staining debris using a morphological decision tree filter, (iii) determination of chromosome width and centreline, (iv) derivation of centromere candidates, and (v) distinction of DCs from monocentric chromosomes (MC) by ML. Centromere candidates are inferred from 14 image features input to a Support Vector Machine (SVM). Sixteen features derived from these candidates are then supplied to a Boosting classifier and a second SVM which determines whether a chromosome is either a DC or MC. The SVM was trained with 292 DCs and 3135 MCs, and then tested with cells exposed to either low (1 Gy) or high (2-4 Gy) radiation dose. Results were then compared with those of 3 experts. True positive rates (TPR) and positive predictive values (PPV) were determined for the tuning parameter, σ. At larger σ, PPV decreases and TPR increases. At high dose, for σ = 1.3, TPR = 0.52 and PPV = 0.83, while at σ = 1.6, the TPR = 0.65 and PPV = 0.72. At low dose and σ = 1.3, TPR = 0.67 and PPV = 0.26. The algorithm differentiates DCs from MCs, overlapped chromosomes and other objects with acceptable accuracy over a wide range of radiation exposures. © 2016 Wiley Periodicals, Inc.

Inclusion bodies in loggerhead erythrocytes are associated with unstable hemoglobin and resemble human Heinz bodies.

PubMed

Basile, Filomena; Di Santi, Annalisa; Caldora, Mercedes; Ferretti, Luigi; Bentivegna, Flegra; Pica, Alessandra

2011-08-01

The aim of this study was to clarify the role of the erythrocyte inclusions found during the hematological screening of loggerhead population of the Mediterranean Sea. We studied the erythrocyte inclusions in blood specimens collected from six juvenile and nine adult specimens of the loggerhead turtle, Caretta caretta, from the Adriatic and Tyrrhenian Seas. Our study indicates that the percentage of mature erythrocytes containing inclusions ranged from 3 to 82%. Each erythrocyte contained only one round inclusion body. Inclusion bodies stained with May Grünwald-Giemsa show that their cytochemical and ultrastructure characteristics are identical to those of human Heinz bodies. Because Heinz bodies originate from the precipitation of unstable hemoglobin (Hb) and cause globular osmotic resistance to increase, we analyzed loggerhead Hb using electrophoresis and high-performance liquid chromatography to detect and quantitate Hb fractions. We also tested the resistance of Hb to alkaline pH, heat, isopropanol denaturation, and globular osmosis. Our hemogram results excluded the occurrence of any infection, which could be associated with an inclusion body, in all the specimens. Negative Feulgen staining indicated that the inclusion bodies are not derived from DNA fragmentation. We hypothesize that amino acid substitutions could explain why loggerhead Hb precipitates under normal physiologic conditions, forming Heinz bodies. The identification of inclusion bodies in loggerhead erythrocytes allow us to better understand the haematological characteristics and the physiology of these ancient reptiles, thus aiding efforts to conserve such an endangered species. Copyright © 2011 Wiley-Liss, Inc., A Wiley Company.

Utility of rapid on-site cytologic evaluation during endobronchial ultrasound with a guide sheath for peripheral pulmonary lesions.

PubMed

Izumo, Takehiro; Matsumoto, Yuji; Sasada, Shinji; Chavez, Christine; Nakai, Toshiyuki; Tsuchida, Takaaki

2017-03-01

The utility of rapid on-site evaluation during endobronchial ultrasound with a guide sheath for peripheral pulmonary lesions is unclear. The aim of this study was to evaluate the role of rapid on-site evaluation during endobronchial ultrasound with a guide sheath for peripheral pulmonary lesions. Consecutive patients who underwent endobronchial ultrasound with a guide sheath for the diagnosis of peripheral pulmonary lesions at our hospital between September 2012 and July 2014 were included in this retrospective study. Cytology slides were air-dried, and modified Giemsa (Diff-Quik) staining was used for rapid on-site evaluation. Additional smears were prepared for Papanicolaou staining and tissue samples were placed in formalin for histologic evaluation. The results of rapid on-site evaluation were compared with the final diagnoses of endobronchial ultrasound with a guide sheath. A total of 718 cases were included in the study population. The sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of rapid on-site evaluation during endobronchial ultrasound with a guide sheath for peripheral pulmonary lesions was 88.6%, 65.9%, 81.2%, 77.7% and 80.1%, respectively. There were no procedure-related deaths. Rapid on-site evaluation during endobronchial ultrasound with a guide sheath had high sensitivity for peripheral pulmonary lesions. When carrying out rapid on-site evaluation of transbronchial biopsy samples from peripheral pulmonary lesions, careful interpretation and clinical correlation are necessary. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Sympatry influence in the interaction of Trypanosoma cruzi with triatomine.

PubMed

Dworak, Elaine Schultz; Araújo, Silvana Marques de; Gomes, Mônica Lúcia; Massago, Miyoko; Ferreira, Érika Cristina; Toledo, Max Jean de Ornelas

2017-01-01

Trypanosoma cruzi, the etiologic agent of Chagas disease, is widely distributed in nature, circulating between triatomine bugs and sylvatic mammals, and has large genetic diversity. Both the vector species and the genetic lineages of T. cruzi present a varied geographical distribution. This study aimed to verify the influence of sympatry in the interaction of T. cruzi with triatomines. Methods: The behavior of the strains PR2256 (T. cruzi II) and AM14 (T. cruzi IV) was studied in Triatoma sordida (TS) and Rhodnius robustus (RR). Eleven fifth-stage nymphs were fed by artificial xenodiagnosis with 5.6 × 103 blood trypomastigotes/0.1mL of each T. cruzi strain. Every 20 days, their excreta were examined for up to 100 days, and every 30 days, the intestinal content was examined for up to 120 days, by parasitological (fresh examination and differential count with Giemsa-stained smears) and molecular (PCR) methods. Rates of infectivity, metacyclogenesis and mortality, and mean number of parasites per insect and of excreted parasites were determined. Sympatric groups RR+AM14 and TS+PR2256 showed higher values of the four parameters, except for mortality rate, which was higher (27.3%) in the TS+AM14 group. General infectivity was 72.7%, which was mainly proven by PCR, showing the following decreasing order: RR+AM14 (100%), TS+PR2256 (81.8%), RR+PR2256 (72.7%) and TS+AM14 (36.4%). Our working hypothesis was confirmed once higher infectivity and vector capacity (flagellate production and elimination of infective metacyclic forms) were recorded in the groups that contained sympatric T. cruzi lineages and triatomine species.

Morphological analysis of circulating tumour cells in patients undergoing surgery for non-small cell lung carcinoma using the isolation by size of epithelial tumour cell (ISET) method.

PubMed

Hofman, V; Long, E; Ilie, M; Bonnetaud, C; Vignaud, J M; Fléjou, J F; Lantuejoul, S; Piaton, E; Mourad, N; Butori, C; Selva, E; Marquette, C H; Poudenx, M; Sibon, S; Kelhef, S; Vénissac, N; Jais, J P; Mouroux, J; Molina, T J; Vielh, P; Hofman, P

2012-02-01

Recurrence rates after surgery for non-small cell lung cancer (NSCLC) range from 25 to 50% and 5-year survival is only 60-70%. Because no biomarkers are predictive of recurrence or the onset of metastasis, pathological TNM (pTNM) staging is currently the best prognostic factor. Consequently, the preoperative detection of circulating tumour cells (CTCs) might be useful in tailoring therapy. The aim of this study was to characterize morphologically any circulating non-haematological cells (CNHCs) in patients undergoing surgery for NSCLC using the isolation by size of epithelial tumour cell (ISET) method. Of 299 blood samples tested, 250 were from patients with resectable NSCLC and 59 from healthy controls. The presence of CNHCs was assessed blindly and independently by 10 cytopathologists on May-Grünwald-Giemsa stained filters and the cells classified into three groups: (i) malignant cells, (ii) uncertain malignant cells, and (iii) benign cells. We assessed interobserver agreement using Kappa (κ) analysis as the measure of agreement. A total of 123 out of 250 (49%) patients showed CNHCs corresponding to malignant, uncertain malignant and benign cells, in 102/250 (41%), 15/250 (6%) and 6/250 (2%) cases, respectively. No CNHCs were detected in the blood of healthy subjects. Interobserver diagnostic variability was absent for CNHCs, low for malignant cells and limited for uncertain malignant and benign cells. Identification of CTCs in resectable NSCLC patients, using ISET technology and according to cytopathological criteria of malignancy, appears to be a new and promising field of cytopathology with potential relevance to lung oncology. © 2011 Blackwell Publishing Ltd.

Atypical Strain of Toxoplasma gondii Causing Fatal Reactivation after Hematopoietic Stem Cell Transplantion in a Patient with an Underlying Immunological Deficiency

PubMed Central

Štajner, Tijana; Vasiljević, Zorica; Vujić, Dragana; Marković, Marija; Ristić, Goran; Mićić, Dragan; Pašić, Srdjan; Ivović, Vladimir; Ajzenberg, Daniel

2013-01-01

In immunocompromized patients, including hematopoietic stem cell transplant (HSCT) recipients, life-threatening toxoplasmosis may result from reactivation of previous infection. We report a case of severe disseminated toxoplasmosis that developed early after allogeneic HSCT for T-cell lymphoblastic leukemia/lymphoma in a 15-year-old Toxoplasma gondii-seropositive boy with Nijmegen breakage syndrome, a rare genetic DNA repair disorder associated with immunodeficiency. The donor was the patient's HLA-identical brother. Prophylaxis with cotrimoxazole was discontinued a day before the HSCT procedure. Signs of lung infection appeared as early as day 14 post-HSCT. The presence of tachyzoite-like structures on Giemsa-stained bronchoalveolar lavage (BAL) fluid smears suggested toxoplasmosis. Real-time PCR targeted at the T. gondii AF146527 gene revealed extremely high parasite burdens in both blood and BAL fluid. Although immediate introduction of specific treatment resulted in a marked reduction of the parasite load and transient clinical improvement, the patient deteriorated and died of multiple organ failure on day 39 post-HSCT. Direct genotyping of T. gondii DNA from blood and BAL fluid with the PCR-restriction fragment length polymorphism method revealed type II alleles with SAG1, SAG2, and GRA6 markers but alleles of both type I and type II with GRA7. Additional analysis with 15 microsatellite markers showed that the T. gondii DNA was atypical and genetically divergent from that of the clonal type I, II, and III strains. This is the first report of increased clinical severity of toxoplasmosis associated with an atypical strain in the setting of immunosuppression, which emphasizes the need to diagnose and monitor toxoplasmosis by quantitative molecular methods in cases of reactivation risk. PMID:23761151

Evaluation of a Microculture Method for Isolation of Leishmania Parasites from Cutaneous Lesions of Patients in Peru▿

PubMed Central

Boggild, Andrea K.; Miranda-Verastegui, Cesar; Espinosa, Diego; Arevalo, Jorge; Adaui, Vanessa; Tulliano, Gianfranco; Llanos-Cuentas, Alejandro; Low, Donald E.

2007-01-01

Traditional culture of Leishmania spp. is labor intensive and has poor sensitivity. We evaluated a microculture method for the diagnosis of cutaneous leishmaniasis in consecutive patients presenting to the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Peru, for evaluation of skin lesions. Lesion aspirates were cultured in duplicate and parallel in traditional culture tubes containing modified Novy-MacNeal-Nicolle (NNN) medium or Roswell Park Memorial Institute medium 1640 with 10% fetal bovine serum (10% RPMI) and in 70-μl capillary tubes containing a mixture of lesion aspirate and 10% RPMI. For sensitivity analysis, the consensus standard was considered to be a positive result in any two of the following four tests: Giemsa-stained lesion smear, culture, kinetoplast DNA PCR, or leishmanin skin test. The outcome measures were sensitivity and time to culture positivity. Forty-five patients with 62 skin lesions were enrolled in the study, of which 53 lesions fulfilled the consensus criteria for a final diagnosis of cutaneous leishmaniasis. Of these 53 lesions, 39 were culture positive: 38 in capillary tubes, 29 in traditional culture tubes with modified NNN medium, and 19 in traditional culture tubes with 10% RPMI medium. The sensitivity of microculture was 71.7%, versus 54.7% for traditional culture with NNN (P, 0.038) and 35.8% with 10% RPMI (P, <0.001). The mean times to culture positivity were 4.2 days by microculture, 5.2 days in NNN, and 6 days in 10% RPMI (P, 0.009). We have demonstrated that microculture is a more sensitive and time-efficient means of isolating Leishmania parasites from cutaneous lesions than traditional culture. PMID:17881557

Epidemiological and clinical correlates of malaria-helminth co-infections in southern Ethiopia

PubMed Central

2013-01-01

Background In many areas of the world, including Ethiopia, malaria and helminths are co-endemic, therefore, co-infections are common. However, little is known how concurrent infections affect the epidemiology and/or pathogenesis of each other. Therefore, this study was conducted to assess the effects of intestinal helminth infections on the epidemiology and clinical patterns of malaria in southern Ethiopia where both infections are prevalent. Methods A cross-sectional study was conducted in 2006 at Wondo Genet Health Center and Bussa Clinic, southern Ethiopia. Consecutive blood film positive malaria patients (N=230) and malaria negative asymptomatic individuals (N=233) were recruited. Malaria parasite detection and quantification was diagnosed using Giemsa-stained thick and thin blood films, respectively. Helminths were detected using direct microscopy and formol-ether concentration techniques. Coarse quantification of helminths ova was made using Kato Katz method. Results The over all magnitude of intestinal parasitic infection was high irrespective of malaria infection (67% among malaria positive patients versus 53.1% among malaria non-infected asymptomatic individuals). Trichuris trichiura infection was associated with increased malaria prevalence while increased worm burden of helminths as expressed by egg intensity was associated with increased malaria parasitaemia which could be a potential factor for development of severe malarial infection with the course of the disease. Majority (77%) of the subjects had multiple helminths infection. T. trichiura, Ascaris lumbricoides, Schistosoma mansoni, and hookworm infestation accounted for 64.5, 57.7 %, 28.4%, and 12.2% of the infections, respectively. Conclusions Populations in malaria-endemic areas of southern Ethiopia are multi-parasitized with up to four helminths. Mass deworming may be a simple practical approach in endemic areas in reducing the risk of severe malarial attack particularly for those at high risk of both infections. PMID:23822192

SPIROCHAETA MORSUS MURIS, N.SP., THE CAUSE OF RAT-BITE FEVER : SECOND PAPER.

PubMed

Futaki, K; Takaki, I; Taniguchi, T; Osumi, S

1917-01-01

1. Since our first report on the discovery of the cause of rat-bite fever, we have been able to prove the existence of the same spirochete in five out of six more cases which have come under our observation. 2. The clinical symptoms of rat-bite fever are inflammation of the bitten parts, paroxysms of fever of the relapsing type, swelling of the lymph glands, and eruption of the skin, all occurring after an incubation period usually of from 10 to 22 days, or longer. 3. Our spirochete is present in the swollen local lesion of the skin and the enlarged lymph glands. But as the spirochetes are so few in number it is exceedingly difficult to discover them directly in material taken from patients. It is therefore better to inoculate the material into a mouse. In some cases the organism is found in the blood of the inoculated animal after a lapse of 5 to 14 days, or at the latest 4 weeks. 4. Generally speaking, the spirochetes present thick and short forms of about 2 to 5 micro and have flagella at both ends. Including the flagella, they measure 6 to 10 micro in length. Some forms in the cultures reach 12 to 19 micro excluding the flagella. The curves are regular, and the majority have one curve in 1 micro. Smaller ones are found in the blood and larger ones in the tissues. 5. The spirochetes stain easily. With Giemsa's stain they take a deep violet-red; they also stain with ordinary aniline dyes. The flagella, too, take Giemsa's stain. 6. The movements of our spirochetes are very rapid, resembling those of a vibrio, and distinguish them from all other kinds of spirochetes. When, however, the movements become a little sluggish, they begin to present movements characteristic of ordinary spirochetes. 7. For experimental purposes, mice, house rats, white rats, and monkeys are the most suitable animals. Monkeys have intermittent fever after infection, and spirochetes can be found in their blood, but they are not so numerous as in the blood of mice. Mice are the most suitable animals for these experiments, and they appear, as a rule, to escape fatal consequences. 8. The spirochete is markedly affected by salvarsan. 9. The organism is not present in the blood of all rats, and there is no relation between the species of the rat and the ratio of infection. We have never found the spirochete in healthy guinea pigs or mice. By permitting a rat infected with the spirochete to bite a guinea pig, the latter develops the disease. 10. We have succeeded in cultivating the spirochete in Shimamine's medium. 11. Among the spirochetes described in the literature or discovered in the blood of rats and mice, there may be some resembling our spirochete, but none of the descriptions agree with it fully. Hence we have named our organism Spirochaeta morsus muris and regard it as belonging to the Spironemacea (Gross) of the nature of treponema. 12. The spirochete can be detected in the bodies of patients. In seven cases out of eight, it disappears on recovery, only to reappear during the relapse. 13. The spirochete can be detected in about 3 per cent of house rats. These facts enable us to identify the cause of the disease. 14. There may be other causes than the spirochete for diseases following the bite of a rat. The cause, however, of rat-bite fever in the form most common in Japan is, we believe, the spirochete which we have described.

Photoinactivation effect of eosin methylene blue and chlorophyllin sodium-copper against Staphylococcus aureus and Escherichia coli.

PubMed

Caires, Cynthia S A; Leal, Cassia R B; Ramos, Carlos A N; Bogo, Danielle; Lima, Alessandra R; Arruda, Eduardo J; Oliveira, Samuel L; Caires, Anderson R L; Nascimento, Valter A

2017-07-01

The use of eosin methylene blue according to Giemsa as photosensitizer is presented for the first time in this paper. The present study evaluated the potential application of chlorophyllin sodium copper salt (CuChlNa) and eosin methylene blue according to Giemsa (EMB) as antimicrobial photosensitizers (aPS) for photodynamic inactivation (PDI) of Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria. The experiments were performed using S. aureus stain ATCC 25923 and E. coli ATCC 25922 in which five aPS concentrations (0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 μM for S. aureus and 0.0, 5.0, 10.0, 20.0, 40.0, and 50.0 μM for E. coli) were prepared and added in 2 mL of a saline solution containing the bacterial inoculum. After aPS incubation, the samples were divided into two groups, one kept in the dark and another submitted to the illumination. Then, the bacterial inactivation was determined 18 h after the incubation at 37 °C by counting the colony-forming units (CFU). The results revealed that both EMB and CuChlNa can be used as aPS for the photoinactivation of S. aureus, while only EMB was able to photoinactivate E. coli. Nevertheless, a more complex experimental setup was needed for photoinactivation of E. coli. The data showed that EMB and CuChlNa presented similar photoinactivation effects on S. aureus, in which bacterial growth was completely inhibited at photosensitizer (PS) concentrations over 5 μM, when samples were previously incubated for 30 min and irradiated by a light dose of 30 J cm -2 as a result of an illumination of 1 h at 8.3 mW cm -2 by using a red light at 625 nm with a 1 cm beam diameter and output power of 6.5 mW. In the case of E. coli, bacterial growth was completely inhibited only when combining a PS incubation period of 120 min with concentrations over 20 μM.

Morphological and molecular identification of Sarcocystis arctica sarcocysts in three red foxes (Vulpes vulpes) from the Czech Republic.

PubMed

Pavlásek, Ivan; Máca, Ondřej

2017-10-01

Muscular sarcocystosis by Sarcocystis arctica was found for the first time in the Czech Republic, in different muscles of red fox (Vulpes vulpes). Cysts were slim, elongated, thread-like, whitish, 1-7mm long, and 206-270μm wide; bradyzoites were 7.9×2.7μm in unstained wet mounts and 9.2×2.9μm in cyst Giemsa-stained smears. The cyst wall was thin, with short villi-like protrusions, and no host response was observed in the histological sections. Examination of the distribution and intensity of sarcocysts in 17 different muscle groups revealed that the highest intensity was in the cranial tibial muscle (>15 cysts in compressoria), followed by the diaphragm, forearm, and other groups (with intensities of 3-15 cysts in compressoria). Sarcocysts were detected in 3 out of 86 foxes. Genetic characterization at 18S rRNA, 28S rRNA, ITS1 and cox1, consistently showed that the species was identical with S. arctica. Interestingly, this protozoan was also detected as a co-infection in 3 foxes with the nematode Trichinella spp. for the first time. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular and parasitological survey of Hepatozoon canis (Apicomplexa: Hepatozoidae) in dogs from rural area of Sao Paulo state, Brazil.

PubMed

Rubini, Adriano Stefani; dos Santos Paduan, Karina; Von Ah Lopes, Viviane; O'Dwyer, Lucia Helena

2008-04-01

Hepatozoon canis is a protozoan that infects dogs and is transmitted by the ingestion of the brown dog tick, Rhipicephalus sanguineus. Two distinct species of Hepatozoon genus can infect dogs, H. canis and H. americanum. Routine tests to detect the disease are based on direct examination of gametocytes on Giemsa-stained blood smears. The objectives of this study were the investigation of infection prevalence in rural area dogs, the comparison of diagnostics by blood smear examination and polymerase chain reaction (PCR), and the association of infection with tick infestation. Blood smears, collected by puncture of the cephalic vein and ear margin capillary bed from 150 dogs, were examined. This technique detected 17 positive animals (11.3%), with 14 (9.3%) in peripheral blood and seven (4.7%) in cephalic vein blood. PCR tests detected 80 (53.3%) positive animals. R. sanguineus and Amblyomma spp. were found in 36 of the dogs (24%), in equal proportions. The identified species for Amblyomma genus were A. cajennense and A. ovale. Data analysis showed that PCR was much more sensitive when compared to blood smear examination. Hepatozoon species was previously identified as closely related to H. canis.

Retrospective study of clinical and hematological aspects associated with dogs naturally infected by Hepatozoon canis in Ludhiana, Punjab, India.

PubMed

Chhabra, Sushma; Uppal, Sanjeev Kumar; Singla, Lachhman Das

2013-06-01

To evaluate clinical and hematological aspects of dogs naturally infected with Hepatozoon canis (H. canis) presented at the Small Animal Clinics of Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana. Blood films of 34 naturally infected dogs were examined for haematological alterations and parasitaemia. Signalment and clinical signs were recorded from the animals. Clinical histories were filled out during the consultation. Of the 34 positive dogs by Giemsa stained peripheral blood films, 88.23% presented parasitaemia by H. canis only, while 11.77% had the combination of H. canis, Babesia sp. and Ehrlichia sp. Young male dogs less than one-year-old, of non-descript breed, were the most commonly affected. And 26.47% were presented with anorexia/inappetence as the only clinical symptom. Other clinical symptoms were mild to moderate fever, pale mucosae and lethargy; a few were also showing the signs of vomiting and diarrhoea. Haematological alterations showed mainly normochromic-normocytic anaemia, leukocytosis and neutrophilia. The findings of this study substantiate that H. canis caused clinical and haematological alterations of the varied intensity in dogs, even with low parasitaemia, should be taken into consideration.

First case of Plasmodium knowlesi infection in a Japanese traveller returning from Malaysia.

PubMed

Tanizaki, Ryutaro; Ujiie, Mugen; Kato, Yasuyuki; Iwagami, Moritoshi; Hashimoto, Aki; Kutsuna, Satoshi; Takeshita, Nozomi; Hayakawa, Kyoko; Kanagawa, Shuzo; Kano, Shigeyuki; Ohmagari, Norio

2013-04-15

This is the first case of Plasmodium knowlesi infection in a Japanese traveller returning from Malaysia. In September 2012, a previously healthy 35-year-old Japanese man presented to National Center for Global Health and Medicine in Tokyo with a two-day history of daily fever, mild headaches and mild arthralgia. Malaria parasites were found in the Giemsa-stained thin blood smear, which showed band forms similar to Plasmodium malariae. Although a nested PCR showed the amplification of the primer of Plasmodium vivax and Plasmodium knowlesi, he was finally diagnosed with P. knowlesi mono-infection by DNA sequencing. He was treated with mefloquine, and recovered without any complications. DNA sequencing of the PCR products is indispensable to confirm P. knowlesi infection, however there is limited access to DNA sequencing procedures in endemic areas. The extent of P. knowlesi transmission in Asia has not been clearly defined. There is limited availability of diagnostic tests and routine surveillance system for reporting an accurate diagnosis in the Asian endemic regions. Thus, reporting accurately diagnosed cases of P. knowlesi infection in travellers would be important for assessing the true nature of this emerging human infection.

Mobile-Based Analysis of Malaria-Infected Thin Blood Smears: Automated Species and Life Cycle Stage Determination.

PubMed

Rosado, Luís; da Costa, José M Correia; Elias, Dirk; Cardoso, Jaime S

2017-09-21

Microscopy examination has been the pillar of malaria diagnosis, being the recommended procedure when its quality can be maintained. However, the need for trained personnel and adequate equipment limits its availability and accessibility in malaria-endemic areas. Rapid, accurate, accessible diagnostic tools are increasingly required, as malaria control programs extend parasite-based diagnosis and the prevalence decreases. This paper presents an image processing and analysis methodology using supervised classification to assess the presence of malaria parasites and determine the species and life cycle stage in Giemsa-stained thin blood smears. The main differentiation factor is the usage of microscopic images exclusively acquired with low cost and accessible tools such as smartphones, a dataset of 566 images manually annotated by an experienced parasilogist being used. Eight different species-stage combinations were considered in this work, with an automatic detection performance ranging from 73.9% to 96.2% in terms of sensitivity and from 92.6% to 99.3% in terms of specificity. These promising results attest to the potential of using this approach as a valid alternative to conventional microscopy examination, with comparable detection performances and acceptable computational times.

Gastrointestinal and blood parasite determination in the guanaco (Lama guanicoe) under semi-captivity conditions.

PubMed

Correa, Loreto; Zapata, Beatriz; Soto-Gamboa, Mauricio

2012-01-01

The breeding of wild animals for commercial purposes is becoming more frequent nowadays. This situation has led to an increase in contact rates between wild and domestic animals, with subsequent reciprocal transmission of parasites. In this study, we characterized the gastrointestinal and blood parasites of a group of 15 semi-captive guanacos (Lama guanicoe). We characterized gastrointestinal parasites by analyzing fecal samples through the sedimentation-flotation technique and hemoparasites by using blood smears stained with Giemsa. We found several gastrointestinal parasites including Nematoda and protozoans. The most frequently found parasites were Nematodirus sp. and Eimeria sp. In contrast with previous studies, neither Cestoda nor Fasciola were found. The only hemoparasite detected was Mycoplasma haemolamae, a parasite already described in llamas and alpacas. We conclude that the most frequent gastrointestinal parasites of semi-captive guanacos were nematodes and protozoans. Also, the hemoparasite M. haemolamae seems to be prevalent among captive populations of South American camelids. Finally, captive guanacos share several parasites with the traditional livestock. Therefore, keeping captive or semi-captive guanacos without an adequate sanitary protocol might have adverse consequences to adjacent traditional cattle farming and/or for wild animals.

Prevalence of Malaria in Pregnant Women in Lagos, South-West Nigeria

PubMed Central

Agomo, Chimere O.; Anorlu, Rose I.; Agomo, Philip U.

2009-01-01

Prevalence rates reported for malaria in pregnancy in Nigeria vary considerably. The accuracy of results of malaria diagnosis is dependent on training, experience, and motivation of the microscopist as well as the laboratory facility available. Results of training programmes on malaria microscopy have shown low levels of sensitivity and specificity of those involved in malaria diagnosis routinely and for research. This study was done to ascertain the true prevalence of malaria in pregnancy in Lagos, South-West Nigeria. A total of 1,084 pregnant women were recruited into this study. Blood smears stained with Giemsa were used for malaria diagnosis by light microscopy. Malaria infection during pregnancy presents mostly as asymptomatic infection. The prevalence of malaria in this population was 7.7% (95% confidence interval; 6.2-9.4%). Factors identified to increase the risk of malaria infection include young maternal age (< 20 years), and gravidity (primigravida). In conclusion, this study exposes the over-diagnosis of malaria in pregnancy and the need for training and retraining of laboratory staffs as well as establishing the malaria diagnosis quality assurance programme to ensure the accuracy of malaria microscopy results at all levels. PMID:19488427

Rapid diagnosis of liver cancer by ultrasound-guided fine-needle aspiration biopsy.

PubMed

Huber, K; Heuhold, N

1987-01-01

Because of the relatively favorable prognosis to the patient with early detected hepatocarcinoma followed by surgical treatment if resection is possible, it is important to differentiate quickly between primary and secondary liver cancer. Ultrasound-guided percutaneous fine needle aspiration biopsy (US-FNAB) was used as a first diagnostic measure in patients with sonographic evidence of liver tumors. Biopsies were done under sonographic control and antiseptic conditions from the center and the border zone of solid tumors of the liver, and the aspirated cell material was air dried on glass slides and Giemsa stained. The cytologic diagnosis was proved by clinical course and in most cases by surgical or autoptic histology. Cytologic evaluation lead in 15 cases to the diagnosis of definitive or suspicious malignant liver disease; the sensitivity was 93% and the specificity was 87%. One case classified as suspicious for malignancy by cytologic examination could be identified as cirrhotic nodule by further investigations. In none of the patients did we find complications from the biopsy procedure. From these data it is concluded that US-FNAB can serve as a rapid, inexpensive, safe, and highly accurate first diagnostic step in patients with solid lesions of the liver.

Intraspecific and interspecific polyploidy of Brazilian species of the genus Inga (Leguminosae: Mimosoideae).

PubMed

Figueiredo, M F; Bruno, R L A; Barros e Silva, A E; Nascimento, S; Oliveira, I G; Felix, L P

2014-04-29

We investigated the karyotypes of 13 species of six sections of the genus Inga (Leguminosae-Mimosoideae) from Brazil. We used conventional Giemsa staining to identify numerical chromosomal variations and looked for karyotypic evolutionary patterns. The karyotypes generally had small chromosomes, varying from metacentric to submetacentric, with a basic number x=13. Nine of the species showed 2n=2x=26 (I. thibaudiana, I. cayennensis, I. ingoides, I. edulis, I. vera, I. subnuda, I. striata, I. bollandii, and Inga sp), while 2n=4x=52 was seen in a population of Inga cylindrical and of I. capitata, and in five populations of I. laurina. Additionally, 2n=8x=104 was observed in a population of I. cayennensis. Eight of these counts were new, while the counts of 2n=52 for I. laurina and 2n=26 for I. marginata, I. vera, I. subnuda, and I. edulis confirmed previous studies. We did not find cytological stability among the sections studied, with occurrence of significant intra- and inter-specific numerical variations. We conclude that polyploidy has played a significant role in karyotypic evolution in this group and that it occurred independently in several sections of the genus.

Haemoproteus syrnii in Strix aluco from France: morphology, stages of sporogony in a hippoboscid fly, molecular characterization and discussion on the identification of Haemoproteus species

PubMed Central

Karadjian, Grégory; Puech, Marie-Pierre; Duval, Linda; Chavatte, Jean-Marc; Snounou, Georges; Landau, Irène

2013-01-01

In France, Haemoproteus syrnii is frequently found in the Tawny Owl, Strix aluco. Additional and complementary features of this species, and in particular the characteristics of volutin, are presented. The authors consider the volutin granules as constant in a given species, and discuss their taxonomic value. These cytoplasmic inclusions appear early during the first stages of development of the gametocytes as an initial granule which multiplies as the parasite develops. They were reported in some species of Haemoproteus but are seldom considered as a specific character and described with precision. Sporogony from ookinete to apparently mature sporozoites appears to take place in a pupiparous hippoboscid (Ornithomyia sp.). One specimen was crushed between two slides and stained with Giemsa. Gametocytes of H. syrnii, many ookinetes, an immature oocyst and mature sporozoites were observed spread all over the smear. This would allow classifying this species in the Haemoproteus subgenus. We provide associated molecular data derived from the cyt b and cox 1 gene from this parasite. We discuss the problems of multiple infections and the difficulties in identifying Haemoproteus species and in deriving conclusions from sequences deposited in databases. PMID:24029169

Enhancement of caffeic acid phenethyl ester on all-trans retinoic acid-induced differentiation in human leukemia HL-60 cells

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

Kuo, H.-C.; Kuo, W.-H.; Lee, Y.-J.

2006-10-01

All-trans retinoic acid (ATRA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL); however, the response is sometimes very slow. Furthermore, relapse and resistance to treatment often occur despite continued treatment with ATRA. Thereafter, combination treatment strategies have been suggested to circumvent these problems. The present study demonstrates that caffeic acid phenethyl ester (CAPE), a major component of honeybee propolis, enhanced ATRA-induced granulocytic differentiation in HL-60, a human promyelocytic cell line. The differentiation was assessed by Wright-Giemsa stain, nitroblue tetrazolium reduction, and membrane differentiation marker CD11b. In addition, CAPE enhanced ATRA-induced cell cycle arrest atmore » the G1 phase by decreasing the association of cdk2-cyclin E complex. Finally, it was demonstrated that CAPE promoted the ATRA-mediated nuclear transcription activation of RAR{alpha} assessed by EMSA assay and enhanced the expression of target genes including RAR{alpha}, C/EBP{epsilon}, and p21 protein resulting in the differentiation development of leukemia. It is suggested that CAPE possesses the potential to enhance the efficiency of ATRA in the differentiation therapy of APL.« less

Diagnostic accuracy of lymphoma established by fine-needle aspiration cytological biopsy

NASA Astrophysics Data System (ADS)

Delyuzar; Amir, Z.; Suryadi, D.

2018-03-01

Based on Globocan data in 2012, it is estimated that about 14,495 Indonesians suffer from lymphoma, both Hodgkin’s lymphoma, and non-Hodgkin’s lymphoma. Some areas of specialization still doubt the accuracy of cytology diagnosis of fine needle aspiration biopsy.This study is a diagnostic test with a cross sectional analytic design to see how the cytology diagnostic accuracy of fine needle aspiration aspirate in lymphoma. It was in Department of Anatomical Pathology Faculty of Medicine USU, Haji Adam Malik Hospital, Dr.Pirngadi hospital, or private clinic in Medan. Peripheral cytology technique biopsy of fine needle aspiration on lymph node subsequently stained with Giemsa, when the cytology of lymphoma is obtained and confirmed by histopathologic examination. Cytology and histopathologic examination will be tested by Diagnostic Test and assessed for its sensitivity and specificity. The diagnostic of lymphoma cytology provides 93.33% sensitivity and 92.31% specificity when confirmed by histopathological examination. Positive predictive value and negative predictive value of 96.55% and 85.71% respectively. In conclusion, the cytology of fine needle aspiration biopsy is accurate enough to be used as a diagnostic tool, so it is advisable to establish a lymphoma diagnosis to perform a needle aspiration biopsy examination.

Mobile-Based Analysis of Malaria-Infected Thin Blood Smears: Automated Species and Life Cycle Stage Determination

PubMed Central

da Costa, José M. Correia; Elias, Dirk

2017-01-01

Microscopy examination has been the pillar of malaria diagnosis, being the recommended procedure when its quality can be maintained. However, the need for trained personnel and adequate equipment limits its availability and accessibility in malaria-endemic areas. Rapid, accurate, accessible diagnostic tools are increasingly required, as malaria control programs extend parasite-based diagnosis and the prevalence decreases. This paper presents an image processing and analysis methodology using supervised classification to assess the presence of malaria parasites and determine the species and life cycle stage in Giemsa-stained thin blood smears. The main differentiation factor is the usage of microscopic images exclusively acquired with low cost and accessible tools such as smartphones, a dataset of 566 images manually annotated by an experienced parasilogist being used. Eight different species-stage combinations were considered in this work, with an automatic detection performance ranging from 73.9% to 96.2% in terms of sensitivity and from 92.6% to 99.3% in terms of specificity. These promising results attest to the potential of using this approach as a valid alternative to conventional microscopy examination, with comparable detection performances and acceptable computational times. PMID:28934170

Cutaneous leishmaniasis in three Dutch military cohorts following jungle training in Belize.

PubMed

van Thiel, P P A M; Zeegelaar, J E; van Gool, T; Faber, W R; Kager, P A

2011-05-01

Skin lesions occur frequently in travelers to tropical countries. Military personnel acquire skin lesions regularly during jungle training as did Dutch troops who trained in the jungle of Belize in 1998, 2004 and 2009, in an area endemic for cutaneous leishmaniasis. Demographic and clinical data were collected retrospectively. Diagnostic investigations for cutaneous leishmaniasis included Giemsa stain, culture, PCR and NASBA and histopathology of biopsies. Treatment of leishmaniasis was with sodium stibogluconate, given intravenously or intralesionally, the latter with cryotherapy. In 1998 and 2004 cutaneous leishmaniasis due to Leishmania braziliensis and Leishmania mexicana infection was diagnosed in 25 persons out of 99 (attack rate 25.2%) and 14 persons out of 80 (attack rate 17.5%) respectively. In 2009 cutaneous leishmaniasis was not acquired. Skin problems were common during and after jungle training. Cutaneous leishmaniasis was important in the first two cohorts but not observed in the third cohort. Factors that could have played a role in the absence of cutaneous leishmaniasis in the third cohort include variability in transmission and availability of better preventive measures and adherence to these. Sodium stibogluconate treatment, intralesional or intravenous, was effective. Copyright © 2011 Elsevier Ltd. All rights reserved.

RDT accuracy based on age group in hypoendemic malaria

NASA Astrophysics Data System (ADS)

Siahaan, L.; Panggabean, M.; Panggabean, Y. C.

2018-03-01

Malaria is still one of the problem of community health in Sumatera. This study was carried out to compare RDT accuracy in some groups of age in hypoendemic malaria. The microscopy test was investigated by 3% Giemsa Staining and examined by a trained laboratory technician. RDT was carried out by using Monotes Test Drive. The accuracy of RDT diagnostic was commonly significant in all groups of age, exceptin thegroup of age > 65 years old (p=0.393). The highest sensitivity of RDT was commonly inagroup of age ≤ 5 years old and decreased in the older group of age. Otherwise, the lowest specificity was found in agroup of age ≤ 5 years old and the highest in agroup of age 6-15 years old.The highest PPV and NPV was found inagroup of age 16-65 years old and ≤ 5 years old, respectively. The highest of parasite density was found in a group of age ≤ 5 years old (644.4±494.5parasite/μl) and the lowest in agroup of age > 65 years (400±490.71parasite/μl). The accurate diagnosis of RDT reduces by increasing of age.

Nanosilica induced dose-dependent cytotoxicity and cell type-dependent multinucleation in HepG2 and L-02 cells

NASA Astrophysics Data System (ADS)

Yu, Yongbo; Duan, Junchao; Li, Yang; Yu, Yang; Hu, Hejing; Wu, Jing; Zhang, Yannan; Li, Yanbo; CaixiaGuo; Zhou, Xianqing; Sun, Zhiwei

2016-11-01

The prevalent exposure to nanosilica gained concerns about health effects of these particles on human beings. Although nanosilica-induced multinucleation has been confirmed previously, the underlying mechanism was still not clear; this study was to investigate the origination of multinucleated cells caused by nanosilica (62 nm) in both HepG2 and L-02 cells. Cell viability and cellular uptake was determined by MTT assay and transmission electron microscope (TEM), respectively. Giemsa staining was applied to detect multinucleation. To clarify the origination of multinucleated cells, fluorescent probes, PKH26 and PKH67, time-lapse observation were further conducted by confocal microscopy. Results indicated that nanosilica particles were internalized into cells and induced cytotoxicity in a dose-dependent manner. Quantification analysis showed that nanosilica significantly increased the rates of binucleated and multinucleated cells, which suggested mitotic catastrophe induction. Moreover, dynamic visualization verified that multinucleation resulted from cell fusion in HepG2 cells not in L-02 cells after nanosilica exposure, suggesting cell type-dependent multinucleation formation. Both multinucleation and cell fusion were involved in genetic instability, which emphasized the significance to explore the multinucleation induced by nanosilica via environmental, occupational and consumer product exposure.

Lymphoid follicles in children with Helicobacter pylori-negative gastritis

PubMed Central

Broide, Efrat; Richter, Vered; Mendlovic, Sonia; Shalem, Tzippora; Eindor-Abarbanel, Adi; Moss, Steven F; Shirin, Haim

2017-01-01

Purpose The prevalence of Helicobacter pylori gastritis has been declining, whereas H. pylori-negative gastritis has become more common. We evaluated chronic gastritis in children with regard to H. pylori status and celiac disease (CD). Patients and methods Demographic, clinical, endoscopic, and histologic features of children who underwent elective esophagogastroduodenoscopy were reviewed retrospectively. Gastric biopsies from the antrum and corpus of the stomach were graded using the Updated Sydney System. H. pylori presence was defined by hematoxylin and eosin, Giemsa, or immunohistochemical staining and urease testing. Results A total of 184 children (61.9% female) met the study criteria with a mean age of 10 years. A total of 122 (66.3%) patients had chronic gastritis; 74 (60.7%) were H. pylori-negative. Children with H. pylori-negative gastritis were younger (p=0.003), were less likely to present with abdominal pain (p=0.02), and were mostly of non-Arabic origin (p=0.011). Nodular gastritis was found to be less prevalent in H. pylori-negative gastritis (6.8%) compared with H. pylori-positive gastritis (35.4%, p<0.001). The grade of mononuclear infiltrates and neutrophil density was more severe in the H. pylori-positive group (p<0.001). Pan-gastritis and lymphoid follicles were associated most commonly with H. pylori. Although less typical, lymphoid follicles were demonstrated in 51.3% of H. pylori-negative patients. The presence or absence of CD was not associated with histologic findings in H. pylori-negative gastritis. Conclusion Our findings suggest that lymphoid follicles are a feature of H. pylori-negative gastritis in children independent of their CD status. PMID:28860835

Quality of Pinzgau bull spermatozoa following different periods of cryostorage.

PubMed

Chrenek, P; Spaleková, E; Olexikova, L; Makarevich, A; Kubovicova, E

2017-04-01

The aim of this work was to examine the influence of cryostorage duration of Pinzgau bull's insemination doses (IDs) on some sperm traits. The IDs were frozen by a slow freezing method and stored in liquid nitrogen for different periods: less than 8 years (group 1), 8-13 years (group 2) and 14-18 years (group 3). Motility (CASA), pathological sperm rate (Giemsa staining), apoptotic (Yo-Pro-1-positive) and necrotic (propidium iodide-positive) cell occurrence and fertilizing ability (penetration/fertilization test) of spermatozoa were evaluated post-thaw. The average post-thaw sperm motility in all examined groups was over 40%. No significant influence of storage length either on the sperm total motility or progressive movement was revealed. In each tested group the average rate of malformed spermatozoa did not exceed 20%. No effect of cryostorage length on the occurrence of apoptotic or necrotic sperm was noted. Similarly, penetrating/fertilizing ability of sperm did not differ among the groups, excepting differences in the rate of pronuclei (PN) formation. In group 1, 72.9% of eggs showed two visible PN following 20 h incubation with sperm, whilst in groups 2 and 3 only 67 and 54.5% of zygotes, respectively, had both PN at this time. These results revealed no influence of storage time on the bull spermatozoa in all parameters excepting the rate of PN formation. As high inter-male variability was observed in the susceptibility of bull sperm to cryostorage, individual differences should be taken into account when semen from individual bulls is to be stored for a long time.

Epidemiological Study on Cutaneous Leishmaniasis in an Endemic Area, of Qom Province, Central Iran

PubMed Central

Saghafipour, Abedin; Vatandoost, Hassan; Zahraei-Ramazani, Ali Reza; Yaghoobi-Ershadi, Mohammad Reza; Jooshin, Moharram Karami; Rassi, Yavar; Shirzadi, Mohammad Reza; Akhavan, Amir Ahmad; Hanafi-Bojd, Ahmad Ali

2017-01-01

Background: Cutaneous leishmaniasis (CL) is one of the most important health problems in many areas of Iran. There are two forms of the disease in Iran, anthroponotic and zoonotic CL. This study conducted to assess the epidemiological situation of CL in an endemic area of Qom Province, central Iran from Apr to Nov 2015. Methods: The sticky paper traps and aspirating tubes were used for collecting adult sand flies. Sherman traps and small insect nets were used to capture rodents and small mammals. Giemsa staining was used for preparing the expanded smear and followed by PCR for identifying the causative agent in human, vectors, and reservoirs. In this study, relative frequency of CL was also calculated. Results: Fourteen species of Phlebotomine sand flies were collected. Phlebotomus papatasi (61.74%) was the predominant species through the period of activity. Overall, 62 Meriones libycus, 8 Nesokia indica, 4 Mus musculus, 16 Allactaga elater and 2 Hemiechinus auritis were caught. PCR technique showed 6 out of 150 P. papatasi (2%), two out of 62 M. libycus (3.23%) and all of suspected human’s skin tissue samples (100%) were infected with Leishmania major. The relative frequency of CL was 0.30%. Conclusion: This is the first detection of L. major within P. papatasi, M. libycus and human in Kahak District in Qom Province of Iran. Zoonotic cycle of CL exists in this area, L. major is the causative agent, P. papatasi is the main vector and M. libycus is the main reservoir of the disease. PMID:29322057

A cell line resource derived from honey bee (Apis mellifera) embryonic tissues.

PubMed

Goblirsch, Michael J; Spivak, Marla S; Kurtti, Timothy J

2013-01-01

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz's L15 medium and incubated at 32(°)C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10-14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

Novel flowcytometry-based approach of malignant cell detection in body fluids using an automated hematology analyzer

PubMed Central

Tabe, Yoko; Takemura, Hiroyuki; Kimura, Konobu; Takahashi, Toshihiro; Yang, Haeun; Tsuchiya, Koji; Konishi, Aya; Uchihashi, Kinya; Horii, Takashi; Ohsaka, Akimichi

2018-01-01

Morphological microscopic examinations of nucleated cells in body fluid (BF) samples are performed to screen malignancy. However, the morphological differentiation is time-consuming and labor-intensive. This study aimed to develop a new flowcytometry-based gating analysis mode “XN-BF gating algorithm” to detect malignant cells using an automated hematology analyzer, Sysmex XN-1000. XN-BF mode was equipped with WDF white blood cell (WBC) differential channel. We added two algorithms to the WDF channel: Rule 1 detects larger and clumped cell signals compared to the leukocytes, targeting the clustered malignant cells; Rule 2 detects middle sized mononuclear cells containing less granules than neutrophils with similar fluorescence signal to monocytes, targeting hematological malignant cells and solid tumor cells. BF samples that meet, at least, one rule were detected as malignant. To evaluate this novel gating algorithm, 92 various BF samples were collected. Manual microscopic differentiation with the May-Grunwald Giemsa stain and WBC count with hemocytometer were also performed. The performance of these three methods were evaluated by comparing with the cytological diagnosis. The XN-BF gating algorithm achieved sensitivity of 63.0% and specificity of 87.8% with 68.0% for positive predictive value and 85.1% for negative predictive value in detecting malignant-cell positive samples. Manual microscopic WBC differentiation and WBC count demonstrated 70.4% and 66.7% of sensitivities, and 96.9% and 92.3% of specificities, respectively. The XN-BF gating algorithm can be a feasible tool in hematology laboratories for prompt screening of malignant cells in various BF samples. PMID:29425230

Novel flowcytometry-based approach of malignant cell detection in body fluids using an automated hematology analyzer.

PubMed

Ai, Tomohiko; Tabe, Yoko; Takemura, Hiroyuki; Kimura, Konobu; Takahashi, Toshihiro; Yang, Haeun; Tsuchiya, Koji; Konishi, Aya; Uchihashi, Kinya; Horii, Takashi; Ohsaka, Akimichi

2018-01-01

Morphological microscopic examinations of nucleated cells in body fluid (BF) samples are performed to screen malignancy. However, the morphological differentiation is time-consuming and labor-intensive. This study aimed to develop a new flowcytometry-based gating analysis mode "XN-BF gating algorithm" to detect malignant cells using an automated hematology analyzer, Sysmex XN-1000. XN-BF mode was equipped with WDF white blood cell (WBC) differential channel. We added two algorithms to the WDF channel: Rule 1 detects larger and clumped cell signals compared to the leukocytes, targeting the clustered malignant cells; Rule 2 detects middle sized mononuclear cells containing less granules than neutrophils with similar fluorescence signal to monocytes, targeting hematological malignant cells and solid tumor cells. BF samples that meet, at least, one rule were detected as malignant. To evaluate this novel gating algorithm, 92 various BF samples were collected. Manual microscopic differentiation with the May-Grunwald Giemsa stain and WBC count with hemocytometer were also performed. The performance of these three methods were evaluated by comparing with the cytological diagnosis. The XN-BF gating algorithm achieved sensitivity of 63.0% and specificity of 87.8% with 68.0% for positive predictive value and 85.1% for negative predictive value in detecting malignant-cell positive samples. Manual microscopic WBC differentiation and WBC count demonstrated 70.4% and 66.7% of sensitivities, and 96.9% and 92.3% of specificities, respectively. The XN-BF gating algorithm can be a feasible tool in hematology laboratories for prompt screening of malignant cells in various BF samples.

Low incidence of Pneumocystis jirovecii pneumonia in an unprophylaxed liver transplant cohort.

PubMed

Sarwar, S; Carey, B; Hegarty, J E; McCormick, P A

2013-10-01

Liver transplant recipients are managed with a range of immunosuppressive regimens that place them at heightened risk of life-threatening opportunistic infections such as Pneumocystis jirovecii pneumonia (PJP). No routine PJP prophylaxis is used at out institution. We reviewed the incidence of PJP in this cohort of unprophylaxed liver transplant recipients. We examined all liver transplants performed between January 2000 and January 2012 in Ireland's National Liver Transplant Centre, St. Vincent's University Hospital, Dublin. Cases were identified through a computerized database and through the histopathology and microbiology registration system. The diagnosis of PJP was confirmed by identification of Pneumocystis cysts in bronchoalveolar lavage (BAL) fluid or on autopsy specimens using Grocott-Gomori methenamine-silver nitrate or modified Wright-Giemsa staining methods. During the study period, 687 liver transplants were performed. We found 7 cases of PJP with an incidence rate of 0.84 per 1000 person transplant years. Five cases occurred within 12 months of transplant with 2 cases occurring at 56 and 60 months, respectively. Two cases were diagnosed at postmortem; 1 previously had negative cytology from BAL, while the other could not be bronchoscoped because of rapid deterioration in the clinical condition. Three of the 5 treated patients died. The incidence of PJP in this cohort was very low, but the case fatality rate was high. Two cases occurred well after the usual recommended period of prophylaxis. In institutions with a very low risk of infection, targeted rather than universal prophylaxis may be reasonable. © 2013 John Wiley & Sons A/S.

Genetic alterations in benign lesions: Chronic gastritis and gastric ulcer

PubMed Central

César, Ana Cristina Gobbo; Calmon, Marília de Freitas; Cury, Patrícia Maluf; Caetano, Alaor; Borim, Aldenis Albaneze; Silva, Ana Elizabete

2006-01-01

AIM: To investigate the occurrence of chromosome 3, 7, 8, 9, and 17 aneuploidies, TP53 gene deletion and p53 protein expression in chronic gastritis, atrophic gastritis and gastric ulcer, and their association with H pylori infection. METHODS: Gastric biopsies from normal mucosa (NM, n = 10), chronic gastritis (CG, n = 38), atrophic gastritis (CAG, n=13) and gastric ulcer (GU, n = 21) were studied using fluorescence in situ hybridization (FISH) and immunohistochemical assay. A modified Giemsa staining technique and PCR were used to detect H pylori. An association of the gastric pathologies and aneuploidies with H pylori infection was assessed. RESULTS: Aneuploidies were increasingly found from CG (21%) to CAG (31%) and to GU (62%), involving mainly monosomy and trisomy 7, trisomies 7 and 8, and trisomies 7, 8 and 17, respectively. A significant association was found between H pylori infection and aneuploidies in CAG (P = 0.0143) and GU (P = 0.0498). No TP53 deletion was found in these gastric lesions, but p53-positive immunoreactivity was detected in 45% (5/11) and 12% (2/17) of CG and GU cases, respectively. However, there was no significant association between p53 expression and H pylori infection. CONCLUSION: The occurrence of aneuploidies in benign lesions evidences chromosomal instability in early stages of gastric carcinogenesis associated with H pylori infection, which may confer proliferative advantage. The increase of p53 protein expression in CG and GU may be due to overproduction of the wild-type protein related to an inflammatory response in mucosa. PMID:16489680

Benefits and limitations of fine needle aspiration cytology in the diagnosis and classification of leprosy in primary and secondary healthcare settings.

PubMed

Ray, R; Mondal, R K; Pathak, S

2015-08-01

The goal of the World Health Organization (WHO) is to eliminate leprosy as a public health problem. This will only be possible when all patients are detected and cured using multidrug therapy, which requires accurate diagnosis prior to treatment. The objective of this study was to evaluate the possibility of the diagnosis of leprosy lesions by fine needle aspiration cytology according to a modification of the Ridley-Jopling scale, as it can be used in primary and secondary healthcare centres, especially in low-resource settings in which leprosy is prevalent. A prospective study comprising 54 cases with cardinal features of leprosy was performed. Among the 54 cases, 27 patients consented to a histopathological biopsy procedure. The slides were stained with Giemsa, modified Ziehl-Neelsen, Papanicolaou and haematoxylin and eosin methods. Among the 54 cases, 34 were reported as tuberculoid leprosy, five as mid-borderline (BB), three as borderline lepromatous (BL) and eight as lepromatous leprosy (LL); four were unsatisfactory. Histopathological study was performed in 27 cases, which showed cyto-histological correlation in 21 cases (78%). Agreement between histological and cytological diagnosis was achieved in 12 of the 15 tuberculoid cases, one of the three BB cases, one of the two BL cases and all seven LL cases. With the implementation of the WHO classification based on patch counting, there is the possibility of the over-treatment of paucibacillary cases and under-treatment of multibacillary cases. Cytology in terms of cellular type morphology and bacteriological study can complement the WHO classification. © 2014 John Wiley & Sons Ltd.

Detection and Species Determination of Malaria Parasites by PCR: Comparison with Microscopy and with ParaSight-F and ICT Malaria Pf Tests in a Clinical Environment

PubMed Central

Tham, Jill M.; Lee, Szu Hee; Tan, Theresa M. C.; Ting, Robert C. Y.; Kara, Ursula A. K.

1999-01-01

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients. Plasmodium infections were identified with a genus-specific primer set, and species differentiation between Plasmodium falciparum and Plasmodium vivax was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed P. falciparum-P. vivax infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays for P. falciparum, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive for P. vivax at the time of discharge were later readmitted to the hospital with a recurrence of P. vivax infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria. PMID:10203469

"Holostei versus Halecostomi" Problem: Insight from Cytogenetics of Ancient Nonteleost Actinopterygian Fish, Bowfin Amia calva.

PubMed

Majtánová, Zuzana; Symonová, Radka; Arias-Rodriguez, Lenin; Sallan, Lauren; Ráb, Petr

2017-11-01

Bowfin belongs to an ancient lineage of nonteleost ray-finned fishes (actinopterygians) and is the only extant survivor of a once diverged group, the Halecomorphi or Amiiformes. Owing to the scarcity of extant nonteleost ray-finned lineages, also referred as "living fossils," their phylogenetic interrelationships have been the target of multiple hypotheses concerning their sister group relationships. Molecular and morphological data sets have produced controversial results; bowfin is considered as either the sister group to genome-duplicated teleosts (together forming the group of Halecostomi) or to gars (Lepisosteiformes; together forming the group of Holostei). However, any detailed cytogenetic analysis of bowfin chromosomes has never been performed to address this issue. Here we examined bowfin chromosomes by conventional (Giemsa-staining, C-banding, base-specific fluorescence and silver staining) and molecular (FISH with rDNA probes) cytogenetic protocols. We identified diploid chromosome number 2n = 46 with a middle-sized submetacentric chromosome pair as the major ribosomal DNA-bearing (45S rDNA), GC-positive and silver-positive element. The minor rDNA (5S rDNA) sites were localized in the pericentromeric region of one middle-sized acrocentric chromosome pair. Comparison with available cytogenetic data of other nonteleost actinopterygians (bichirs, sturgeons, gars) and teleost species including representative of basally branching lineages showed bowfin chromosomal characteristics more similar to the teleost type than to any other nonteleosts. Particularly striking differences were identified between bowfin and gars, the latter of which were found to mimic mammalian AT/GC genomic organisation. Such conclusion however contradicts the most recent phylogenomic results and raises the question what states are ancestral and what are derived. © 2017 Wiley Periodicals, Inc.

Agaritine from Agaricus blazei Murrill induces apoptosis in the leukemic cell line U937.

PubMed

Akiyama, Hidehiko; Endo, Masahiro; Matsui, Taei; Katsuda, Itsurou; Emi, Nobuhiko; Kawamoto, Yasuko; Koike, Takaaki; Beppu, Hidehiko

2011-05-01

Agaricus blazei Murrill (ABM) has been shown to exhibit immunostimulatory and anti-cancer activities; however, its mechanism of action is poorly understood. We recently found that the diffusible fraction of hot-water extract of ABM exhibits anti-tumor activity toward leukemic cells, and identified it as agaritine, a hydrazine-containing compound. In the present study, we examined the morphological and cytochemical effects of agaritine on U937 cells to elucidate the tumoricidal mechanism of agaritine. Surface expression of phosphatidylserine (evaluated by annexin V binding), Fas antigen, DNA cleavage using TUNEL staining, changes in caspase activities and cytochrome c release, before and after treatment with agaritine, were examined using U937 cells. Nuclear damage, DNA fragmentation, was observed by Wright-Giemsa, TUNEL staining and agarose gel electrophoresis when U937 cells were incubated with 10μg/mL of agaritine for 48h. Flow cytometric analysis indicated that agaritine augments the proportion of annexin V-positive U937 cells without significant change in Fas antigen expression. Activities of caspase-3, -8 and -9 were gradually increased after the addition of agaritine. In the presence of caspase-3 or granzyme B inhibitor, except for the caspase-8 inhibitor, annexin V expression was significantly decreased, suggesting that mainly caspase-3 and -9 participate in the apoptotic pathway. Furthermore, cytochrome c release was detected by western blotting analysis after agaritine treatment. These results strongly suggest that the ABM constituent agaritine moderately induces apoptosis in U937 leukemic cells via caspase activation through cytochrome c release from mitochondria. This is the first report suggesting that the anti-tumor effect of agaritine is mediated through apoptosis. The present results might provide helpful suggestions for the design of anti-tumor drugs toward leukemia patients. Copyright © 2011 Elsevier B.V. All rights reserved.

Secondary pulmonary syphilis: Case report and review of literature.

PubMed

Kassem Youssef, H; Blind, A; Chouta Ngaha, F; Drenou, B; Nojavan, H; Michel, C

2018-04-01

Syphilis is a sexually transmitted disease that can affect numerous organs in its secondary or tertiary stages. We describe a case of secondary syphilis with pulmonary involvement and we present a literature review. A 69-year-old male patient was admitted to hospital for dyspnoea and extended papular exanthema with palmoplantar involvement. The serological test for syphilis was positive. Ocular examination showed bilateral papillitis and retinal haemorrhage. Chest radiography revealed an interstitial alveolar infiltrate predominantly in the upper lobes, mild pleural effusion and hilar adenopathy. These infiltrates were slightly hypermetabolic on PET scan suggesting inflammatory or infectious origin. Treatment with intravenous penicillin G was effective on cutaneous, ocular and pulmonary manifestations. Lung involvement in secondary syphilis is poorly known and rarely described. We found 27 cases of pulmonary syphilis reported in English and the main European languages since 1967. Mean age at diagnosis was 46 years with clear male predominance (89%). HIV co-infection was declared in 5 cases. Treponema pallidum was found in 6 patients using PCR on bronchoalveolar lavage (BAL) (3 patients) or on a lung biopsy (1 patient), immunohistochemistry (IHC) on BAL (1 patient) and Giemsa staining on a pleural fluid sample (1 patient). Chest X-rays may show unilateral or bilateral infiltrates or nodules with or without pleural effusion or hilar adenopathy. Sub-pleural involvement is frequent and penicillin is the treatment of choice. Pulmonary syphilitic involvement should be suspected where pulmonary symptoms or radiological changes occur in secondary syphilis. IHC, special staining or PCR on BAL, pleural fluid or lung tissue are useful for the identification of spirochetes. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Histopathological observation of immunized rhesus macaques with plague vaccines after subcutaneous infection of Yersinia pestis.

PubMed

Tian, Guang; Qiu, Yefeng; Qi, Zhizhen; Wu, Xiaohong; Zhang, Qingwen; Bi, Yujing; Yang, Yonghai; Li, Yuchuan; Yang, Xiaoyan; Xin, Youquan; Li, Cunxiang; Cui, Baizhong; Wang, Zuyun; Wang, Hu; Yang, Ruifu; Wang, Xiaoyi

2011-04-29

In our previous study, complete protection was observed in Chinese-origin rhesus macaques immunized with SV1 (20 µg F1 and 10 µg rV270) and SV2 (200 µg F1 and 100 µg rV270) subunit vaccines and with EV76 live attenuated vaccine against subcutaneous challenge with 6×10(6) CFU of Y. pestis. In the present study, we investigated whether the vaccines can effectively protect immunized animals from any pathologic changes using histological and immunohistochemical techniques. In addition, the glomerular basement membranes (GBMs) of the immunized animals and control animals were checked by electron microscopy. The results show no signs of histopathological lesions in the lungs, livers, kidneys, lymph nodes, spleens and hearts of the immunized animals at Day 14 after the challenge, whereas pathological alterations were seen in the corresponding tissues of the control animals. Giemsa staining, ultrastructural examination, and immunohistochemical staining revealed bacteria in some of the organs of the control animals, whereas no bacterium was observed among the immunized animals. Ultrastructural observation revealed that no glomerular immune deposits on the GBM. These observations suggest that the vaccines can effectively protect animals from any pathologic changes and eliminate Y. pestis from the immunized animals. The control animals died from multi-organ lesions specifically caused by the Y. pestis infection. We also found that subcutaneous infection of animals with Y. pestis results in bubonic plague, followed by pneumonic and septicemic plagues. The histopathologic features of plague in rhesus macaques closely resemble those of rodent and human plagues. Thus, Chinese-origin rhesus macaques serve as useful models in studying Y. pestis pathogenesis, host response and the efficacy of new medical countermeasures against plague.

Helicobacter pylori and primary gastric lymphoma. A histopathologic and immunohistochemical analysis of 237 patients.

PubMed

Nakamura, S; Yao, T; Aoyagi, K; Iida, M; Fujishima, M; Tsuneyoshi, M

1997-01-01

Few previous articles have analyzed the relation between infection with Helicobacter pylori (H. pylori) and primary gastric lymphoma in a large number of patients. Resected and biopsied specimens from 237 patients with primary gastric lymphoma were investigated for H. pylori using hematoxylin and eosin stain, modified Giemsa stain, and immunohistochemistry. These specimens were compared with specimens from 29 patients with chronic active gastritis, 33 with peptic ulcers, and 41 with gastric carcinoma. H. pylori was detected in 145 of 237 patients (61%) with gastric lymphoma. The frequency of H. pylori positivity was higher in patients with lymphoma restricted to the mucosa and submucosa (76%) than in those with lymphoma invading beyond the submucosa (48%) (P < 0.001), and was also higher in patients with low grade mucosa-associated lymphoid tissue lymphoma (72%) than in those with high grade tumors (55%) (P < 0.05). The frequency of H. pylori positivity in patients with lymphoma was lower than in those with chronic active gastritis (100%) (P < 0.001) or peptic ulcer (91%) (P < 0.05). In patients with lymphoma restricted to the mucosa and the superficial portion of the submucosa, the frequency of H. pylori positivity (90%) was as high as that observed in patients with chronic active gastritis and peptic ulcer. The H. pylori grading score for patients with lymphoma (0.9 +/- 1.0) was lower than for those with chronic active gastritis (1.9 +/- 0.8) (P < 0.001), peptic ulcers (2.2 +/- 1.0) (P < 0.001), or gastric carcinoma (1.2 +/- 1.1) (P < 0.05). These results suggest that H. pylori is more likely to be associated with early states of primary gastric lymphoma than with advanced states. Thus, H. pylori may disappear during the progression of primary gastric lymphoma.

[Postgraduates' training as laboratory physicians/clinical pathologists in Japan--board certification of JSLM as a mandatory requirement for chairpersons of laboratory medicine].

PubMed

Kumasaka, Kazunari

2002-04-01

The educational committee of the Japanese Society of Laboratory Medicine(JSLM) proposed a revised laboratory medicine residency curriculum in 1999 and again in 2001. The committee believes that present undergraduate clinical training is insufficient and that Japanese medical graduates need clinical training for two years after graduation. This two years training should be a precondition for further postgraduate training in laboratory medicine and should include fundamental clinical skills(communication skills, physical examination and common laboratory procedures such as Gram's stain, Wright-Giemsa stain and urinalysis). After the two years training, the minimal training period of laboratory medicine should be three years, and should include: 1) Principles, instrumentation and techniques of each discipline including clinical chemistry, clinical hematology, clinical microbiology, clinical immunology, blood banking and other specific areas. 2) The use of laboratory information in a medical setting. 3) Interaction of the laboratory physician with laboratory staff, physicians and patients. With good on-the-job training and 24 hours on-call duties, laboratory physicians are expected to perform their tasks, including laboratory management, effectively. They should have appropriate educational background and should be well motivated. The background and duties of the laboratory physicians often reflect the institutional needs and personal philosophy of the chairperson of their department. At the moment, few senior physicians in Japan have qualifications in laboratory medicine and are unable, therefore, to provide the necessary guidance to help the laboratory physicians in their work. I therefore believe that the board certification of JSLM should be regarded as mandatory for chairpersons of laboratory medicine. Our on-call service system can enhance the training in laboratory medicine, and improve not only laboratory quality assurance but patients' care as well.

Mitochondrial DNA from the eradicated European Plasmodium vivax and P. falciparum from 70-year-old slides from the Ebro Delta in Spain

PubMed Central

Gelabert, Pere; Sandoval-Velasco, Marcela; Olalde, Iñigo; Fregel, Rosa; Rieux, Adrien; Escosa, Raül; Aranda, Carles; Paaijmans, Krijn; Mueller, Ivo; Gilbert, M. Thomas P.; Lalueza-Fox, Carles

2016-01-01

Phylogenetic analysis of Plasmodium parasites has indicated that their modern-day distribution is a result of a series of human-mediated dispersals involving transport between Africa, Europe, America, and Asia. A major outstanding question is the phylogenetic affinity of the malaria causing parasites Plasmodium vivax and falciparum in historic southern Europe—where it was endemic until the mid-20th century, after which it was eradicated across the region. Resolving the identity of these parasites will be critical for answering several hypotheses on the malaria dispersal. Recently, a set of slides with blood stains of malaria-affected people from the Ebro Delta (Spain), dated between 1942 and 1944, have been found in a local medical collection. We extracted DNA from three slides, two of them stained with Giemsa (on which Plasmodium parasites could still be seen under the microscope) and another one consisting of dried blood spots. We generated the data using Illumina sequencing after using several strategies aimed at increasing the Plasmodium DNA yield: depletion of the human genomic (g)DNA content through hybridization with human gDNA baits, and capture-enrichment using gDNA derived from P. falciparum. Plasmodium mitochondrial genome sequences were subsequently reconstructed from the resulting data. Phylogenetic analysis of the eradicated European P. vivax mtDNA genome indicates that the European isolate is closely related to the most common present-day American haplotype and likely entered the American continent post-Columbian contact. Furthermore, the European P. falciparum mtDNA indicates a link with current Indian strains that is in agreement with historical accounts. PMID:27671660

Plasmodium knowlesi Skeleton-Binding Protein 1 Localizes to the 'Sinton and Mulligan' Stipplings in the Cytoplasm of Monkey and Human Erythrocytes.

PubMed

Lucky, Amuza Byaruhanga; Sakaguchi, Miako; Katakai, Yuko; Kawai, Satoru; Yahata, Kazuhide; Templeton, Thomas J; Kaneko, Osamu

2016-01-01

The malaria parasite, Plasmodium, exports protein products to the infected erythrocyte to introduce modifications necessary for the establishment of nutrient acquisition and surface display of host interaction ligands. Erythrocyte remodeling impacts parasite virulence and disease pathology and is well documented for the human malaria parasite Plasmodium falciparum, but has been less described for other Plasmodium species. For P. falciparum, the exported protein skeleton-binding protein 1 (PfSBP1) is involved in the trafficking of erythrocyte surface ligands and localized to membranous structures within the infected erythrocyte, termed Maurer's clefts. In this study, we analyzed SBP1 orthologs across the Plasmodium genus by BLAST analysis and conserved gene synteny, which were also recently described by de Niz et al. (2016). To evaluate the localization of an SBP1 ortholog, we utilized the zoonotic malaria parasite, Plasmodium knowlesi. Immunofluorescence assay of transgenic P. knowlesi parasites expressing epitope-tagged recombinant PkSBP1 revealed a punctate staining pattern reminiscent of Maurer's clefts, following infection of either monkey or human erythrocytes. The recombinant PkSBP1-positive puncta co-localized with Giemsa-stained structures, known as 'Sinton and Mulligan' stipplings. Immunoelectron microscopy also showed that recombinant PkSBP1 localizes within or on the membranous structures akin to the Maurer's clefts. The recombinant PkSBP1 expressed in P. falciparum-infected erythrocytes co-localized with PfSBP1 at the Maurer's clefts, indicating an analogous trafficking pattern. A member of the P. knowlesi 2TM protein family was also expressed and localized to membranous structures in infected monkey erythrocytes. These results suggest that the trafficking machinery and induced erythrocyte cellular structures of P. knowlesi are similar following infection of both monkey and human erythrocytes, and are conserved with P. falciparum.

Seroprevalence of pediatric malaria in quetta, balochistan, pakistan.

PubMed

Hussain, K; Shafee, M; Khan, N; Jan, S; Tareen, Am; Khan, Ma

2013-04-01

Malaria is one of the most devastating protozoal diseases in under developing countries like Pakistan where health facilities are scarce. It is the second most frequently reported disease with 4.5 million suspected cases in Pakistan. The current study was designed to determine the incidence of pediatric malaria in Quetta, Balochistan. The study was conducted at Children Hospital Quetta (CHQ) during July 2011march 2012. Blood samples were collected from 3418 clinically suspected and were evaluated using thin and thick blood films stained with Giemsa stain. Out of 3418 total of 230 (6.72%) children were found positive for any of the malarial parasitic infestation. Plasmodium vivax was observed to be more common 54.34% (n= 125/230) than P. falciparum 44.78% (n = 103/230). Male children were 65.21% (150/230) i.e. two times more commonly affected than female 34.78% (80/230) children. The prevalence among age groups was 7.41% (n = 89/1200) in preschool-aged children aged 1-5 years, 7.11% (n = 75/1054) in school-aged children aged 6-10 years while 6.78% (n = 46/678) in 11-15 years-old children, and 6.66% (n = 20/300) in >15 year-olds children. Peak prevalence was noted in summer and mild in winter. Mixed infection of (0.86%: 2/230) P. vivax and P. falciparum was also observed in two cases although no case of P. malariae or P. ovale infection was seen during entire study. The results reflect the higher prevalence of malaria in Quetta, Pakistan that poses a significant health threat and requires urgent attention of high-ups to launch programme to control the disease in the area.

Mitochondrial DNA from the eradicated European Plasmodium vivax and P. falciparum from 70-year-old slides from the Ebro Delta in Spain.

PubMed

Gelabert, Pere; Sandoval-Velasco, Marcela; Olalde, Iñigo; Fregel, Rosa; Rieux, Adrien; Escosa, Raül; Aranda, Carles; Paaijmans, Krijn; Mueller, Ivo; Gilbert, M Thomas P; Lalueza-Fox, Carles

2016-10-11

Phylogenetic analysis of Plasmodium parasites has indicated that their modern-day distribution is a result of a series of human-mediated dispersals involving transport between Africa, Europe, America, and Asia. A major outstanding question is the phylogenetic affinity of the malaria causing parasites Plasmodium vivax and falciparum in historic southern Europe-where it was endemic until the mid-20th century, after which it was eradicated across the region. Resolving the identity of these parasites will be critical for answering several hypotheses on the malaria dispersal. Recently, a set of slides with blood stains of malaria-affected people from the Ebro Delta (Spain), dated between 1942 and 1944, have been found in a local medical collection. We extracted DNA from three slides, two of them stained with Giemsa (on which Plasmodium parasites could still be seen under the microscope) and another one consisting of dried blood spots. We generated the data using Illumina sequencing after using several strategies aimed at increasing the Plasmodium DNA yield: depletion of the human genomic (g)DNA content through hybridization with human gDNA baits, and capture-enrichment using gDNA derived from P. falciparum Plasmodium mitochondrial genome sequences were subsequently reconstructed from the resulting data. Phylogenetic analysis of the eradicated European P. vivax mtDNA genome indicates that the European isolate is closely related to the most common present-day American haplotype and likely entered the American continent post-Columbian contact. Furthermore, the European P. falciparum mtDNA indicates a link with current Indian strains that is in agreement with historical accounts.

A malaria diagnostic tool based on computer vision screening and visualization of Plasmodium falciparum candidate areas in digitized blood smears.

PubMed

Linder, Nina; Turkki, Riku; Walliander, Margarita; Mårtensson, Andreas; Diwan, Vinod; Rahtu, Esa; Pietikäinen, Matti; Lundin, Mikael; Lundin, Johan

2014-01-01

Microscopy is the gold standard for diagnosis of malaria, however, manual evaluation of blood films is highly dependent on skilled personnel in a time-consuming, error-prone and repetitive process. In this study we propose a method using computer vision detection and visualization of only the diagnostically most relevant sample regions in digitized blood smears. Giemsa-stained thin blood films with P. falciparum ring-stage trophozoites (n = 27) and uninfected controls (n = 20) were digitally scanned with an oil immersion objective (0.1 µm/pixel) to capture approximately 50,000 erythrocytes per sample. Parasite candidate regions were identified based on color and object size, followed by extraction of image features (local binary patterns, local contrast and Scale-invariant feature transform descriptors) used as input to a support vector machine classifier. The classifier was trained on digital slides from ten patients and validated on six samples. The diagnostic accuracy was tested on 31 samples (19 infected and 12 controls). From each digitized area of a blood smear, a panel with the 128 most probable parasite candidate regions was generated. Two expert microscopists were asked to visually inspect the panel on a tablet computer and to judge whether the patient was infected with P. falciparum. The method achieved a diagnostic sensitivity and specificity of 95% and 100% as well as 90% and 100% for the two readers respectively using the diagnostic tool. Parasitemia was separately calculated by the automated system and the correlation coefficient between manual and automated parasitemia counts was 0.97. We developed a decision support system for detecting malaria parasites using a computer vision algorithm combined with visualization of sample areas with the highest probability of malaria infection. The system provides a novel method for blood smear screening with a significantly reduced need for visual examination and has a potential to increase the throughput in malaria diagnostics.

Methods for chromosome-specific staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

1995-01-01

Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

Methods and compositions for chromosome-specific staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

2003-07-22

Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

Safer staining method for acid fast bacilli.

PubMed Central

Ellis, R C; Zabrowarny, L A

1993-01-01

To develop a method for staining acid fast bacilli which excluded highly toxic phenol from the staining solution. A lipophilic agent, a liquid organic detergent, LOC High Studs, distributed by Amway, was substituted. The acid fast bacilli stained red; nuclei, cytoplasm, and cytoplasmic elements stained blue on a clear background. These results compare very favourably with acid fast bacilli stained by the traditional method. Detergents are efficient lipophilic agents and safer to handle than phenol. The method described here stains acid fast bacilli as efficiently as traditional carbol fuchsin methods. LOC High Suds is considerably cheaper than phenol. Images PMID:7687254

Safer staining method for acid fast bacilli.

PubMed

Ellis, R C; Zabrowarny, L A

1993-06-01

To develop a method for staining acid fast bacilli which excluded highly toxic phenol from the staining solution. A lipophilic agent, a liquid organic detergent, LOC High Studs, distributed by Amway, was substituted. The acid fast bacilli stained red; nuclei, cytoplasm, and cytoplasmic elements stained blue on a clear background. These results compare very favourably with acid fast bacilli stained by the traditional method. Detergents are efficient lipophilic agents and safer to handle than phenol. The method described here stains acid fast bacilli as efficiently as traditional carbol fuchsin methods. LOC High Suds is considerably cheaper than phenol.

Short Nissl staining for incubated cryostat sections of the brain.

PubMed

Lindroos, O F

1991-01-01

Nissl stain often binds poorly to cryostat sections which have been incubated in solutions of radiolabeled ligands. Such incubation is used in receptor autoradiography of the brain when using the in vitro method. We have developed a rapid (16 min) modification of Nissl staining for sections that bind stain poorly, e.g., incubated sections. The method stains well sections which cannot be stained with other rapid Nissl staining methods.

Discriminative staining methods for the nervous system: luxol fast blue--periodic acid-Schiff--hematoxylin triple stain and subsidiary staining methods.

PubMed

Goto, N

1987-09-01

This paper describes a new series of staining methods which can discriminatively demonstrate every structure of the nervous system, including axons and capillaries, in animal and human materials. Methods described in this paper consist of one primary stain, luxol fast blue-periodic acid Schiff-hematoxylin (LPH) and six different subsidiary staining methods. The LPH triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc.), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, and others. Subsidiary staining methods are also described briefly. Applications are discussed in the context of staining technology and neuromorphological research.

Methods for validating the presence of and characterizing proteins deposited onto an array

DOEpatents

Schabacker, Daniel S.

2010-09-21

A method of determining if proteins have been transferred from liquid-phase protein fractions to an array comprising staining the array with a total protein stain and imaging the array, optionally comparing the staining with a standard curve generated by staining known amounts of a known protein on the same or a similar array; a method of characterizing proteins transferred from liquid-phase protein fractions to an array including staining the array with a post-translational modification-specific (PTM-specific) stain and imaging the array and, optionally, after staining the array with a PTM-specific stain and imaging the array, washing the array, re-staining the array with a total protein stain, imaging the array, and comparing the imaging with the PTM-specific stain with the imaging with the total protein stain; stained arrays; and images of stained arrays.

Methods for chromosome-specific staining

DOEpatents

Gray, J.W.; Pinkel, D.

1995-09-05

Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogeneous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include ways for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes. 3 figs.

Fluorometric method of quantitative cell mutagenesis

DOEpatents

Dolbeare, Frank A.

1982-01-01

A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

Fluorometric method of quantitative cell mutagenesis

DOEpatents

Dolbeare, F.A.

1980-12-12

A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

The use of a differential fluorescent staining method to detect bacteriuria.

PubMed

Ciancaglini, Ettore; Fazii, Paolo; Sforza, Giuseppe Riario

2004-01-01

This report describes a differential staining method which distinguishes gram-positive from gram-negative bacteria in fluorescence. Gram-positive bacteria appear yellow and gram-negative bacteria appear green. The method is based on two fluorochromes, one acting in the wavelength of red, i.e. the acridine orange, and another acting in the wavelength of green, i.e. the fluorescein, which together form a red/ green system. In this report we compared the accuracy of the differential fluorescent staining method and the Gram stain in screening for bacteriuria, as detected by conventional cultures. A total of 1487 urine samples were tested. 289 cultures were positive. 237 specimens grew a single organism at 10(5) and 10(4) CFU/ml. 224 smears were detected by the differential fluorescent staining method and 162 were detected by Gram stain. 1198 samples failed to grow organisms at 10(5) and 10(4) CFU/ml. 107 smears were falsely positive by the fluorescent staining procedure and 289 were falsely positive by the Gram stain. On the basis of the culture results, the sensitivity of the differential fluorescent staining method was 94.5% and that of the Gram stain 68.3%. The specificity of the fluorescent staining procedure was 91.6% and that of the Gram stain 75.8%. The positive predictive value and the negative predictive value of the fluorescent staining method were 67.6% and 98.8%, respectively. Those of the Gram stain were 35.9% and 92.3%, respectively. A wide range of microbiological and chemical techniques are available to identify bacteria in urine. This fluorescent staining method represents a simple, rapid, reliable method with low-running costs. The main advantage of this technique is that it enables the microbiologist to exclude the presence of bacteria in the urine within a short time after specimen receipt and to eliminate a large number of specimens for culture with significant cost saving. Another advantage of the method is that it allows to distinguish gram-positive from gram-negative bacteria in positive slides on the same day the sample is obtained. The stained smears were easily interpreted, even when the bacterial counts in the specimen were low.

Differential fluorescent staining method for detection of bacteria in blood cultures, cerebrospinal fluid and other clinical specimens.

PubMed

Fazii, P; Ciancaglini, E; Riario Sforza, G

2002-05-01

The aim of this study was to evaluate a differential staining method to distinguish gram-positive from gram-negative bacteria in fluorescence. The method is based on two fluorochromes, one acting in the wavelength of red, i.e. the acridine orange, and another acting in the wavelength of green, i.e. the fluorescein. With this method, gram-positive bacteria appear yellow and gram-negative bacteria appear green. In view of the importance of a rapid aetiological diagnosis in cases of septicaemia, the differential staining method in fluorescence was compared with Gram stain for the detection of bacteria in blood. Of 5,820 blood cultures entered into the study and identified by the Bactec 9120 fluorescent series instrument (Becton Dickinson Europe, France), 774 were positive. Of the 774 positive cultures, 689 yielded only a single organism. The differential staining method in fluorescence detected 626 of the 689 cultures, while Gram stain detected 468. On the basis of these results, the sensitivity of the differential staining method in fluorescence was 90.9%, while that of Gram stain was 67.9%. The difference between the two methods was statistically significant ( P<0.001). The differential fluorescent staining method was more sensitive than Gram stain in the detection of bacteria in blood cultures during the incubation period. This technique provides a rapid, simple and highly sensitive staining method that can be used in conjunction with subculture methods. Whereas subculture requires an incubation period of 18-24 h, the fluorescent staining technique can detect bacteria on the same day that smears are prepared and examined. The differential fluorescent staining method was also evaluated for its ability to detect microorganisms in cerebrospinal fluid and other clinical specimens. The microorganisms were easily detected, even when bacterial counts in the specimens were low.

From Blood Islands to Blood Vessels: Morphologic Observations and Expression of Key Molecules during Hyaloid Vascular System Development

PubMed Central

McLeod, D. Scott; Hasegawa, Takuya; Baba, Takayuki; Grebe, Rhonda; Galtier d'Auriac, Ines; Merges, Carol; Edwards, Malia; Lutty, Gerard A.

2012-01-01

Purpose. The mode of development of the human hyaloid vascular system (HVS) remains unclear. Early studies suggested that these blood vessels formed by vasculogenesis, while the current concept seems to favor angiogenesis as the mode of development. We examined embryonic and fetal human HVS using a variety of techniques to gain new insights into formation of this vasculature. Methods. Embryonic and fetal human eyes from 5.5 to 12 weeks gestation (WG) were prepared for immunohistochemical analysis or for light and electron microscopy. Immunolabeling of sections with a panel of antibodies directed at growth factors, transcription factors, and hematopoietic stem cell markers was employed. Results. Light microscopic examination revealed free blood islands (BI) in the embryonic vitreous cavity (5.5–7 WG). Giemsa stain revealed that BI were aggregates of mesenchymal cells and primitive nucleated erythroblasts. Free cells were also observed. Immunolabeling demonstrated that BI were composed of mesenchymal cells that expressed hemangioblast markers (CD31, CD34, C-kit, CXCR4, Runx1, and VEGFR2), erythroblasts that expressed embryonic hemoglobin (Hb-ε), and cells that expressed both. Few cells were proliferating as determined by lack of Ki67 antigen. As development progressed (12 WG), blood vessels became more mature structurally with pericyte investment and basement membrane formation. Concomitantly, Hb-ε and CXCR4 expression was down-regulated and von Willebrand factor expression was increased with the formation of Weibel-Palade bodies. Conclusions. Our results support the view that the human HVS, like the choriocapillaris, develops by hemo-vasculogenesis, the process by which vasculogenesis, erythropoiesis, and hematopoiesis occur simultaneously from common precursors, hemangioblasts. PMID:23092923

Growth factor and proteinase profile of Vivostat® platelet-rich fibrin linked to tissue repair.

PubMed

Agren, M S; Rasmussen, K; Pakkenberg, B; Jørgensen, B

2014-07-01

Autologous platelet-rich fibrin (PRF(®)) is prepared by the automatic Vivostat(®) system. Conflicting results with Vivostat PRF in acute wound healing prompted us to examine its cellular and biomolecular composition. Specifically, platelets, selected growth factors and matrix metalloproteinase (MMP)-9 were quantified using novel analytical methods. Ten healthy non-thrombocytopenic volunteers donated blood for generation of intermediate fibrin-I and final PRF. Anticoagulated whole blood and serum procured in parallel served as baseline controls. Leucocyte, erythrocyte and platelet counts in whole blood and fibrin-I were determined by automated haematology analyser. Platelet concentration in PRF was quantified manually by stereologic analysis of Giemsa-stained tissue sections, and the total content of five growth factors and MMP-9 by enzyme-linked immunosorbent assays. The number of leucocytes and erythrocytes was reduced (P < 0·001), whereas platelets increased (P < 0·001) in fibrin-I versus whole blood. PRF contained 982 ± 206 × 10(9) platelets/l representing 3·9-fold (P < 0·001) enrichment relative to whole blood. Growth factor abundance in Vivostat PRF and serum was in descending order: transforming growth factor-β1 [5·1-fold higher in PRF than serum, P < 0·001] > platelet-derived growth factor (PDGF)-AB [2·5-fold, P < 0·01] > PDGF-BB [1·6-fold, P < 0·05] > vascular endothelial growth factor > basic fibroblast growth factor [75-fold, P < 0·001]. MMP-9 was reduced 139-fold (P < 0·001) compared with serum, reflecting leucocyte depletion in PRF. The gained knowledge on platelet enrichment and biomolecular constituents may guide clinicians in their optimal use of Vivostat PRF for tissue regenerative applications. © 2013 International Society of Blood Transfusion.

First Report on Isolation and Characterization of Leishmania major from Meriones hurrianae (Rodentia: Gerbillidae) of A Rural Cutaneous leishmaniasis Focus in South-Eastern Iran

PubMed Central

Kassiri, Hamid; Naddaf, Saied Reza; Javadian, Ezat–Aldin; Mohebali, Mehdi

2013-01-01

Background Zoonotic Cutaneous Leishmaniasis (ZCL) is an endemic health problem in many rural areas of Iran, with doubled number of incidences over the last decade. Different species of rodents serve as natural reservoir host for ZCL. The disease is considered as a major health problem in rural areas of Mirjaveh, Chabahar, and Konarak Counties of Sistan va Baluchistan Province. Objectives This study describes the identity of Leishmania species, isolated from Meriones hurrianae from Chabahar County using RAPD-PCR methodology. Materials and Methods Rodents were entrapped by live traps baited with roasted walnut, tomato, and cucumber during spring and summer. All rodents were identified based on external features including fur color, ears characteristics, tail length, hind feet, body measurements, and internal features of teeth and cranium. Giemsa-stained impressions from rodents’ ears were examined for amastigotes microscopically. The samples from infected rodents were cultured in NNN+LIT medium and then the harvested parasites at the stationary phase were subjected to DNA extraction followed by amplification with RAPD-PCR. Results All the 28 entrapped animals were identified as M. hurrianae. Five animals showed to harbor Leishmania parasite by microscopy. Leishmania DNA isolated from five M. hurrianae produced distinctive bands of L. major with four primers. However, the products that were amplified with primers AB1-07, 327, and 329 were stable and reproducible. This is the first report on the isolation and identification of L. major from M. hurrianae from Iran. Conclusions Regarding infection rate of 17.8%, M. hurrianae seems to play the major role in the maintenance and transmission of disease to humans in this area. PMID:24616787

Helicobacter pylori eradication in patients with chronic immune thrombocytopenic purpura

PubMed Central

Noonavath, Ravinder Naik; Lakshmi, Chandrasekharan Padma; Dutta, Tarun Kumar; Kate, Vikram

2014-01-01

AIM: To assess the effect of Helicobacter pylori (H. pylori) eradication on platelet counts in patients with chronic immune thrombocytopenic purpura (cITP). METHODS: A total of 36 cITP patients were included in the study. The diagnosis of H. pylori was done by rapid urease test and Giemsa staining of the gastric biopsy specimen. All H. pylori positive patients received standard triple therapy for 14 d and were subjected for repeat endoscopy at 6 wk. Patients who continued to be positive for H. pylori on second endoscopy received second line salvage therapy. All the patients were assessed for platelet response at 6 wk, 3rd and 6th months. RESULTS: Of the 36 patients, 17 were positive for H. pylori infection and eradication was achieved in 16 patients. The mean baseline platelet count in the eradicated patients was 88615.38 ± 30117.93/mm3 and platelet count after eradication at 6 wk, 3 mo and 6 mo was 143230.77 ± 52437.51/mm3 (P = 0.003), 152562.50 ± 52892.3/mm3 (P = 0.0001), 150187.50 ± 41796.68/mm3 (P = 0.0001) respectively and in the negative patients, the mean baseline count was 71000.00 ± 33216.46/mm3 and at 6 wk, 3rd and 6th month follow up was 137631.58 ± 74364.13/mm3 (P = 0.001), 125578.95 ± 71472.1/mm3 (P = 0.005), 77210.53 ± 56892.28/mm3 (P = 0.684) respectively. CONCLUSION: Eradication of H. pylori leads to increase in platelet counts in patients with cITP and can be recommended as a complementary treatment with conventional therapy. PMID:24944483

Invasive Salmonella Infections in Areas of High and Low Malaria Transmission Intensity in Tanzania

PubMed Central

Biggs, Holly M.; Lester, Rebecca; Nadjm, Behzad; Mtove, George; Todd, Jim E.; Kinabo, Grace D.; Philemon, Rune; Amos, Ben; Morrissey, Anne B.; Reyburn, Hugh; Crump, John A.

2014-01-01

Background. The epidemiology of Salmonella Typhi and invasive nontyphoidal Salmonella (NTS) differs, and prevalence of these pathogens among children in sub-Saharan Africa may vary in relation to malaria transmission intensity. Methods. We compared the prevalence of bacteremia among febrile pediatric inpatients aged 2 months to 13 years recruited at sites of high and low malaria endemicity in Tanzania. Enrollment at Teule Hospital, the high malaria transmission site, was from June 2006 through May 2007, and at Kilimanjaro Christian Medical Centre (KCMC), the low malaria transmission site, from September 2007 through August 2008. Automated blood culture, malaria microscopy with Giemsa-stained blood films, and human immunodeficiency virus testing were performed. Results. At Teule, 3639 children were enrolled compared to 467 at KCMC. Smear-positive malaria was detected in 2195 of 3639 (60.3%) children at Teule and 11 of 460 (2.4%) at KCMC (P < .001). Bacteremia was present in 336 of 3639 (9.2%) children at Teule and 20 of 463 (4.3%) at KCMC (P < .001). NTS was isolated in 162 of 3639 (4.5%) children at Teule and 1 of 463 (0.2%) at KCMC (P < .001). Salmonella Typhi was isolated from 11 (0.3%) children at Teule and 6 (1.3%) at KCMC (P = .008). With NTS excluded, the prevalence of bacteremia at Teule was 5.0% and at KCMC 4.1% (P = .391). Conclusions. Where malaria transmission was intense, invasive NTS was common and Salmonella Typhi was uncommon, whereas the inverse was observed at a low malaria transmission site. The relationship between these pathogens, the environment, and the host is a compelling area for further research. PMID:24336909

BMP-7 Preserves Surface Integrity of Degradable-ceramic Cranioplasty in a Göttingen Minipig Model

PubMed Central

Schulz, Peter; Klünter, Tim; Deisinger, Ulrike; Diez, Claudius; Waiss, Waltraud; Kirschneck, Christian; Reichert, Torsten E.; Detsch, Rainer

2017-01-01

Background: The aim of the study was to evaluate the integrity of a craniotomy grafted site in a minipig model using different highly porous calcium phosphate ceramic scaffolds either loaded or nonloaded with bone morphogenetic protein-7 (BMP-7). Methods: Four craniotomies with a diameter of 15 mm (critical-size defect) were grafted with different highly porous (92–94 vol%) calcium phosphate ceramics [hydroxyapatite (HA), tricalcium phosphate (TCP), and biphasic calcium phosphate (BCP; a mixture of HA and TCP)] in 10 Göttingen minipigs: (a) group I (n = 5): HA versus BCP; (b) group II (n = 5): TCP versus BCP. One scaffold of each composition was supplied with 250 μg of BMP-7. In vivo computed tomography scan and fluorochrome bone labeling were performed. Specimens were evaluated 14 weeks after surgery by environmental scanning electron microscopy, fluorescence microscopy, and Giemsa staining histology. Results: BMP-7 significantly enhanced bone formation in TCP (P = 0.047). Slightly enhanced bone formation was observed in BCP (P = 0.059) but not in HA implants. BMP-7 enhanced ceramic degradation in TCP (P = 0.05) and BCP (P = 0.05) implants but not in HA implants. Surface integrity of grafted site was observed in all BMP-7-loaded implants after successful creeping substitution by the newly formed bone. In 9 of 10 HA implants without BMP-7, partial collapse of the implant site was observed. All TCP implants without BMP-7 collapsed. Fluorescent labeling showed bone formation at week 1 in BMP-7-stimulated implants. Conclusions: BMP-7 supports bone formation, ceramic degradation, implant integration, and surface integrity of the grafted site. PMID:28458969

Role of intercellular communications in breast cancer multicellular tumor spheroids after chemotherapy.

PubMed

Oktem, G; Bilir, A; Ayla, S; Yavasoglu, A; Goksel, G; Saydam, G; Uysal, A

2006-01-01

Tumor heterogeneity is an important feature that is especially involved in tumor aggressiveness. Multicellular tumor spheroids (MTS) may provide some benefits in different steps for investigation of the aggregation, organization, differentiation, and network formation of tumor cells in 3D space. This model offers a unique opportunity for improvements in the capability of a current strategy to detect the effect of an appropriate anticancer agent. The aim of this study was to investigate the cellular interactions and morphological changes following chemotherapy in a 3D breast cancer spheroid model. Distribution of the gap junction protein "connexin-43" and the tight junction protein "occludin" was investigated by immunohistochemistry. Cellular interactions were examined by using transmission and scanning electron microscopies as well as light microscopy with Giemsa staining after treating cells with doxorubicin, docetaxel, and doxorubicin/docetaxel combination. Statistical analyses showed significant changes and various alterations that were observed in all groups; however, the most prominent effect was detected in the doxorubicin/docetaxel combination group. Distinct composition as a vessel-like structure and a pseudoglandular pattern of control spheroids were detected in drug-administered groups. Immunohistochemical results were consistent with the ultrastructural changes. In conclusion, doxorubicin/docetaxel combination may be more effective than the single drug usage as shown in a 3D model. The MTS model has been found to be an appropriate and reliable method for the detection of the changes in the expression of cellular junction proteins as well as other cellular proteins occurring after chemotherapy. The MTS model can be used to validate the effects of various combinations or new chemotherapeutic agents as well as documentation of possible mechanisms of new drugs.

Prevalence of Haemoproteus spp. in Tumbler Pigeons (Columba livia domestica) in Kirikkale Province, Turkey.

PubMed

Sürsal, Neslihan; Atan, Perçem; Gökpınar, Sami; Duru, Özkan; Çakmak, Ayşe; Yıldız, Kader

2017-06-01

Haemoproteus spp. are common blood parasites of pigeons. They have been reported in pigeons in many regions worldwide, including Turkey. Pigeon breeding is a popular hobby in Kirikkale province, and there is no information about the prevalence of Haemoproteus spp. The present study aimed to determine the prevalence of Haemoproteus spp. in tumbler pigeons in Kirikkale province (Kırıkkale and Yahsihan district). Blood samples were taken from the wing vein of pigeons (n: 173) through microcapillary (with/heparin) tubes between February and March 2016. Blood smears were stained with 5% Giemsa solution. Ectoparasites of the pigeons were collected in separate sealed boxes. Epidemiological data of the sampled pigeons (age and sex) were obtained from the breeders. In total, 23 (%13.2) of 173 pigeons were infected with Haemoproteus spp. Parasite was detected in 73.9% of pigeons over 1 year old and 26.1% of pigeon under 1 year age. Haemoproteus spp. was observed in 56.2% of females (13/23) and 43.4% of males (10/23), Sex-related differences were not observed (p = 0.821). Ectoparasites of the pigeons were identified as Columbicola spp. To the best of our knowledge, this is the first study in Kirikkale province that reported the prevalence of Haemoproteus spp. in pigeons.

["Mixed" and "miscellaneous" vulvovaginitis: diagnostics and therapy of vaginal administration of nystatin and nifuratel].

PubMed

Cepický, P; Malina, J; Líbalová, Z; Kuzelová, M

2005-05-01

The evaluation of combined and miscellaneous vulvovaginal infections incidence and their treatment with combined vaginal products containing nifuratel and nystatin. Prospective study. Gynecologic outpatient department LEVRET, Prague; Laboratories of Microbiology AescuLab, Prague. 70 consecutive patients were examined with complaint of vaginal fluor and/or pruritus. We established macroscopic features of fluor, pH, amine test and mounts stained with Giemsa and Gram. We qualified the cases with more diagnostic criteria (mycosis, lactobacillosis, anaerobic vaginosis, aerobic vaginitis) as combined infection, those with no diagnostic criteria as miscellaneous. We treated all patients with vaginal tablets nystatin + nifuratel (Macmiror complex). We prescribed clotrimazol cream, if pruritus was present. We evaluated withdrawals of symptoms and relapses during 3 months after treatment. Combined infection was found in 21 patients from 70 (30%). The most frequent combination was that of mycosis and aerobic vaginitis (13/70, 18.6%) or mycosis and anaerobic vaginosis (4/70, 5.7%); 11 patients fulfilled criteria of no diagnosis. We concluded them as "miscelaneous". The treatment was successful in all cases, 10 women relapsed in 3 months. Combined vaginal infection findings are present very often (30%), likewise miscellaneous ones (15%) occur. The treatment of these women in successful with vaginal tablets with nystatin + nifuratel.

Intracranial hemangiopericytoma: Case study with cytogenetics and genome wide SNP-A analysis.

PubMed

Holland, Heidrun; Livrea, Michela; Ahnert, Peter; Koschny, Ronald; Kirsten, Holger; Meixensberger, Jürgen; Bauer, Manfred; Schober, Ralf; Fritzsch, Dominik; Krupp, Wolfgang

2011-05-15

The tumor entity of hemangiopericytoma is not universally recognized as a nosological entity by pathologists, and there is a trend toward reassigning it to other categories gradually. However, hemangiopericytomas occurring in the nervous system are included in the new WHO classification of brain tumors, and are distinguished from both meningioma and fibrous tumors. Since there are few genetic studies, we performed a comprehensive cytogenetic analysis of an infratentorial hemangiopericytoma in a 55-year-old female. It was originally classified as a grade II tumor but recurred as a grade III tumor with a proliferation index of 20%. Using trypsin-Giemsa staining (GTG-banding) and multicolor fluorescence in situ hybridization (M-FISH), we could confirm the loss of chromosomal material 10q, which has been previously described in hemangiopericytoma, and we identified de novo chromosomal aberrations on chromosome 8. Applying genome-wide high-density single nucleotide polymorphism array (SNP-A) analysis, we detected segments with loss or gain, as well as clonal deletions or regions suggestive of segmental uniparental disomy. These findings, together with the results of conventional histological and immunohistochemical characterization, provide additional evidence for the nosological separation of hemangiopericytoma in the central nervous system as a biologically different entity. Copyright © 2011 Elsevier GmbH. All rights reserved.

Sister-chromatid exchanges in lymphocytes in women with cancer of the breast.

PubMed

Husum, B; Wulf, H C; Niebuhr, E

1981-10-01

Examination of sister-chromatid exchanges (SCE) in lymphocytes may be useful for the evaluation of exposure to mutagens/carcinogens. Information of a possible association between SCE and cancer is scarce. We therefore examined SCE in peripheral lymphocytes in 131 women, aged 17-90 years (median 51.8 years), coming to operation because of a tumor of the breast. Venous blood samples were cultivated during PHA stimulation in the presence of BrdU. After treatment with colcemid (R), fixation, treatment with bisbenzimide and staining with Giemsa, 30 metaphases were scored in each specimen. 52 patients with peroperatively demonstrated carcinoma of the breast had 9.39 +/- 0.17 SCE/cell and the remaining 79 women with non-malignant fibroadenomatosis had 9.88 +/- 0.18 SCE/cell. By multiple regression analysis it appeared that the character of the tumor, the patient's age, hormone treatment and preoperative examination by mammography all were without significant influence on the SCE rate. A statistically significant correlation was found between SCE and cigarette smoking. THe 45 cigarette-smoking patients had 10.49 +/- 0.23 SCE/cell compared with 9.26 +/- 0.13 SCE/cell in the 86 non-smokers. It was concluded that spontaneous SCE in lymphocytes is not an indicator of carcinoma of the breast.

Prevalence and spatial distribution of intraerythrocytic parasite(s) in Puget Sound rockfish (Sebastes emphaeus) from the San Juan Archipelago, Washington (USA)

USGS Publications Warehouse

Van Der Straaten, N.; Jacobson, A.; Halos, D.; Hershberger, P.; Kocan, A.A.; Kocan, R.

2005-01-01

Two morphologically distinct forms of an intraerythrocytic parasite(s) were detected by microscopic observation of Giemsa-stained blood films in 45.7% of 119 rockfish (Sebastes emphaeus) from the San Juan Archipelago (Washington State, U.S.A.). Infection prevalence for both forms was 53% in males, 44% in females, and 33% in fish of undetermined gender. A binucleate "ring-stage" was present at all 4 geographic sites, with a mean prevalence of 45.7%, while mean prevalence of a larger gamont-like form from the same sites was 5.1%. The relationship of the 2 forms to each other could not be determined. Neither schizogony nor binary fission was evident in any of the infected erythrocytes and the parasites contained no obvious pigment. The possibility of the 2 morphologic forms being 2 distinct species is supported by the observation that no difference in parasitemia was seen in the binucleate form among sites (1.6-1.9%), while parasitemia of the gamont-like form varied significantly among sites, ranging from a high of 4% to a low of 0.1%. Taxonomic status of either form could not be determined at this time based on limited existing morphologic data. ?? American Society of Parasitologists 2005.

A PCR-based survey on Phytomonas (Euglenozoa: Trypanosomatidae) in phytophagous hemipterans of the Amazon region.

PubMed

Godoi, Mara M I; Serrano, Myrna G; Teixeira, Marta M G; Camargo, Erney P

2002-01-01

We have surveyed 244 hemipterans from Western Brazilian Amazĵnia for the presence of trypanosomatids and identification of members of the genus Phytomonas. Examination by phase microscopy of squashes of insect salivary glands (SG) and digestive tubes (DT) revealed that 44% (108/244) of insects from seven families harbored trypanosomatids. Infections were 5 times more frequent in Coreidae than in all other families together. Smears of SG and DT of the dissected insects were fixed on glass slides with methanol and stained with Giemsa for morphological analysis. DNA was recovered from these preparations and submitted to a PCR assay that permitted amplification of all trypanosomatid genera using primers of conserved sequences flanking a segment of the spliced leader (SL) gene. Upon PCR amplification of the recovered DNA, amplicons were hybridized with an oligonucletide probe (SL3') complementary to a SL intron sequence specific for flagellates of the genus Phytomonas. Among the trypanosomatid-positive insects, 38.8% harbored Phytomonas spp., corresponding to an overall Phytomonas prevalence of 17.1% among phytophagous bugs, their putative vectors. Since many Phytomonas are pathogenic in plants, this high prevalence in their vectors emphasizes the permanent risk of exposure to disease by native and cultured plants of the Amazon region.

Molecular Identification of Hemoprotozoan Parasites in Camels (Camelus dromedarius) of Iran.

PubMed

Sazmand, Alireza; Eigner, Barbara; Mirzaei, Mohammad; Hekmatimoghaddam, Seyed Hossein; Harl, Josef; Duscher, Georg Gerhard; Fuehrer, Hans-Peter; Joachim, Anja

2016-01-01

Although camels represent a valuable source of food, wool and hide in many countries, in-depth information about their vector-borne pathogens is scarce compared to other animals. The aim of the current study was to characterize vector-borne protozoa in the blood of dromedaries from Iran by molecular tools. From June to July 2014, 200 peripheral blood samples were collected from asymptomatic one-humped camels in two provinces of Kerman and Sistan- va-Baloochestan in central and southeastern Iran. Microscopic examination was performed on Giemsa-stained blood smears, and drops of blood were spotted on Whatman FTA ® cards for further analyses. Genomic DNA was extracted from the cards, and PCR was carried out for the detection of piroplasms and trypanosomes, followed by sequence analysis of positive samples. One sample was positive Trypanosoma spp. trypomastigotes in light microscopy. PCR results revealed one positive sample each with Theileria annulata and Trypanosoma evansi . Camels were identified as hosts for bovine Mediterranean theileriosis in the investigated area. The presence of Tr. evansi , the causative agent of surra disease, was also confirmed in camels of Iran. Further studies are recommended in order to investigate their impact on the health and productivity of camels and other livestock in this region.

[Evaluation of ectoparasites and hemoparasites in dogs kept in apartments and houses with yards in the city of Juiz de Fora, Minas Gerais, Brazil].

PubMed

Soares, Aline O; Souza, Aline D; Feliciano, Eveline A; Rodrigues, André F S F; D'Agosto, Marta; Daemon, Erik

2006-01-01

Fleas and ticks transmit various pathogens while feeding on the blood of dogs. This study sought to verify the occurrence of ectoparasitism and hemoparasitism in dogs from two urban areas in the city of Juiz de Fora, Minas Gerais, Brazil. Between February and August 2003, 101 dogs were studied: 50 came from apartments in the downtown region and 51 from houses with grassy yards. The ectoparasites were collected and conserved in etanol 70%. The occurrence of hemoparasites was verified by examining blood smears from sample taken from the dogs'ears. The blood smears were stained with Giemsa and 100 fields per slide were examined, studying the erythrocytes to determine parasitism. From among the dogs living in apartments, we found (with respective prevalence and mean intensity): Ctenocephalides felis (12%), (3.3+/-2.0); Rhipicephalus sanguineus (2%); and ixodid nymphs (2%). In this environment in the dogs were not found hemoparasites. From the houses with grassy yards, we observed the following prevalence levels and mean intensities: C. felis (14%), (2.28+/-1.9); R. sanguineus (35%), (7.8+/-9.8); ixodid nymph (18%), (1.4+/-0.7); and ixodid larvae (4%), (12+/-14.4). The hemoparasites found were: Ehrlichia canis (16%) and Babesia canis (2%).

Defense cells profile of cervical mucous during follicular and luteal phases of estrus cycle in river buffalo

PubMed Central

Ayen, Esmail; Hasanzadeh, Shapour; Tabatabaei, Saleh

2012-01-01

The aim of this study was to evaluate the defense cells changes of cervical mucous during follicular and luteal phases of estrus cycle in river buffalo. Reproductive organs of the adult and apparently healthy female buffaloes were collected from the slaughterhouse. By visual investigation of both the ovaries for presence of corpus luteum and growing follicles, the luteal and follicular phase of each buffalo was specified. Cervical discharge samples were collected by sterile swabs and then spread over the glass slides, dried and fixed with methanol. The specimens were undergone Giemsa staining. The percentage of lymphocytes, neutrophils, monocytes (macrophages), eosinophils and basophils in each case (for both the follicular and luteal phases) were obtained at 20 microscopic fields. The percentage of lymphocytes, neutrophils and basophils in luteal phase were higher than the follicular phase. The percentage of eosinophils in follicular phase was higher than the luteal phase. The percentage of monocytes (macrophages) in luteal and follicular phases was nearly equal. The statistical analysis showed that the differences of all cells between follicular and luteal phase were not significant (P > 0.05). The most defense cells in discharges of external os of cervix (both follicular and luteal phases) were neutrophils and lymphocytes. PMID:25653745

Determining Proportion of Exfoliative Vaginal Cell during Various Stages of Estrus Cycle Using Vaginal Cytology Techniques in Aceh Cattle

PubMed Central

Siregar, Tongku N.; Melia, Juli; Rohaya; Thasmi, Cut Nila; Masyitha, Dian; Wahyuni, Sri; Rosa, Juliana; Nurhafni; Panjaitan, Budianto; Herrialfian

2016-01-01

The aim of this study was to investigate the period of estrus cycle in aceh cattle, Indonesia, based on vaginal cytology techniques. Four healthy females of aceh cattle with average weight of 250–300 kg, age of 5–7 years, and body condition score of 3-4 were used. All cattle were subjected to ultrasonography analysis for the occurrence of corpus luteum before being synchronized using intramuscular injections of PGF2 alpha 25 mg. A vaginal swab was collected from aceh cattle, stained with Giemsa 10%, and observed microscopically. Period of estrus cycle was predicted from day 1 to day 24 after estrus synchronization was confirmed using ultrasonography analysis at the same day. The result showed that parabasal, intermediary, and superficial epithelium were found in the vaginal swabs collected from proestrus, metestrus, and diestrus aceh cattle. Proportions of these cells in the particular period of estrus cycle were 36.22, 32.62, and 31.16 (proestrus); 21.33, 32.58, and 46.09 (estrus); 40.75, 37.58, and 21.67 (metestrus); and 41.07, 37.38, and 21.67 (diestrus), respectively. In conclusion, dominant proportion of superficial cell that occurred in estrus period might be used as the base for determining optimal time for insemination. PMID:26977335

Prevalence and Clinical Manifestations of Malaria in Aligarh, India

PubMed Central

Asma, Umm-e; Taufiq, Farha

2014-01-01

Malaria is one of the most widespread infectious diseases of tropical countries with an estimated 207 million cases globally. In India, there are endemic pockets of this disease, including Aligarh. Hundreds of Plasmodium falciparum and P. vivax cases with severe pathological conditions are recorded every year in this district. The aim of this study is to find out changes in liver enzymes and kidney markers. Specific diagnosis for P. falciparum and P. vivax was made by microscopic examination of Giemsa stained slides. Clinical symptoms were observed in both of these infections. Liver enzymes, such as AST, ALT, and ALP, and kidney function markers, such as creatinine and urea, were estimated by standard biochemical techniques. In Aligarh district, P. vivax, P. falciparum, and mixed infections were 64%, 34%, and 2%, respectively. In case of P. falciparum infection, the incidences of anemia, splenomegaly, renal failure, jaundice, and neurological sequelae were higher compared to those in P. vivax infection. Recrudescence and relapse rates were 18% and 20% in P. falciparum and P. vivax infections, respectively. Liver dysfunctions and renal failures were more common in P. falciparum patients, particularly in elderly patients. Artesunate derivatives must, therefore, be introduced for the treatment of P. falciparum as they resist to chloroquine as well as sulfadoxine-pyrimethamine combinations. PMID:25548413

Prevalence and clinical manifestations of malaria in Aligarh, India.

PubMed

Asma, Umm-e; Taufiq, Farha; Khan, Wajihullah

2014-12-01

Malaria is one of the most widespread infectious diseases of tropical countries with an estimated 207 million cases globally. In India, there are endemic pockets of this disease, including Aligarh. Hundreds of Plasmodium falciparum and P. vivax cases with severe pathological conditions are recorded every year in this district. The aim of this study is to find out changes in liver enzymes and kidney markers. Specific diagnosis for P. falciparum and P. vivax was made by microscopic examination of Giemsa stained slides. Clinical symptoms were observed in both of these infections. Liver enzymes, such as AST, ALT, and ALP, and kidney function markers, such as creatinine and urea, were estimated by standard biochemical techniques. In Aligarh district, P. vivax, P. falciparum, and mixed infections were 64%, 34%, and 2%, respectively. In case of P. falciparum infection, the incidences of anemia, splenomegaly, renal failure, jaundice, and neurological sequelae were higher compared to those in P. vivax infection. Recrudescence and relapse rates were 18% and 20% in P. falciparum and P. vivax infections, respectively. Liver dysfunctions and renal failures were more common in P. falciparum patients, particularly in elderly patients. Artesunate derivatives must, therefore, be introduced for the treatment of P. falciparum as they resist to chloroquine as well as sulfadoxine-pyrimethamine combinations.

Antitumor activity of Brazilian red propolis fractions against Hep-2 cancer cell line.

PubMed

Frozza, Caroline Olivieri da Silva; Santos, Denis Amilton; Rufatto, Luciane Corbellini; Minetto, Luciane; Scariot, Fernando Joel; Echeverrigaray, Sergio; Pich, Claus Tröger; Moura, Sidnei; Padilha, Francine Ferreira; Borsuk, Sibele; Savegnago, Lucielli; Collares, Tiago; Seixas, Fabiana Kömmling; Dellagostin, Odir; Roesch-Ely, Mariana; Henriques, João Antonio Pêgas

2017-07-01

Continuous increases in the rates of tumor diseases have highlighted the need for identification of novel and inexpensive antitumor agents from natural sources. In this study, we investigated the effects of enriched fraction from hydroalcoholic Brazilian red propolis extract against Hep-2 cancer cell line. Initially 201 fractions were arranged in 12 groups according to their chromatographic characteristics (A-L). After an in vitro cell viability screening, J and L were further selected as promising enriched fractions for this study. The chemical characterization was performed and Biochanin A, Formononetin, and Liquiritigenin compounds were quantified. Through MTT viability assay and morphological changes observed by Giemsa and DAPI staining, the results showed that red propolis inhibited cancer cells growth. Flow cytometry results indicated effects that were partly mediated through programmed cell death as confirmed by externalization of phosphatidylserine, DNA cleaved assay, increase at SUB G1-G0 phase in cell cycle analysis and loss of mitochondrial membrane potential. In conclusion, our results demonstrated that red propolis enriched fractions promoted apoptotic effects in human cancer cells through the mechanisms involving mitochondrial perturbation. Therefore, red propolis fractions contain candidate agents for adjuvant cancer treatment, which further studies should elucidate the comprehensive mechanistic pathways. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Tularemia in differential diagnosis of cervical lymphadenopathy: cytologic features of tularemia lymphadenitis.

PubMed

Markoc, Fatma; Koseoglu, Resid Dogan; Koc, Sema; Gurbuzler, Levent

2014-01-01

Tularemia can cause cervical lymphadenopathy. Fine-needle aspiration (FNA) cytology is the first step in the workup for cervical lymphadenopathy; however, little has been published regarding the cytomorphological features of tularemia lymphadenitis. The aim of this study was to evaluate the FNA cytology of tularemia lymphadenitis. Review of medical records identified 36 patients with serologically proven tularemia, and who had undergone lymph node FNA. In each case, the original May-Grünwald-Giemsa-stained FNA smears from enlarged cervical lymph node were reevaluated. Suppuration and cytolysis were frequent cytological findings. Twenty-three (63.8%) of the 36 cases were assessed as suppurative inflammation. In 10 of these cases (27.8% of the total), cytolysis was prominent. In 7 cases (19.4%) the smears featured microgranulomas as well as suppuration, and 2 of these (5.6%) also featured giant cells. In 1 case (2.8%), there was caseous necrosis. In 2 cases (5.6%), the cytopathological findings were consistent with reactive lymphoid hyperplasia. Three aspirates (8.3%) were inadequate for evaluation. Cytopathological findings on FNA of tularemia lymphadenitis are nonspecific; however, in regions where tularemia is endemic, this disease should be considered in the differential diagnosis for suppurative lymphadenitis. © 2013 S. Karger AG, Basel.

The prevalence of protozoa in the gut of German cockroaches (Blattella germanica) with special reference to Lophomonas blattarum.

PubMed

Martínez-Girón, Rafael; Martínez-Torre, Cristina; van Woerden, Hugo Cornelis

2017-11-01

The German cockroach (Blattella germanica) is a common domestic pest, which produces allergens that have been associated with broncho-pulmonary disease. Various protozoan species have been identified in the intestine of this cockroach and it has been hypothesised that these protozoa, or their proteases, may contribute to the burden of cockroach-associated allergens and adjuvants present in domestic dust. The aim of this study was therefore to determine the prevalence of protozoan species in the intestine of Blattella germanica. German cockroaches were anesthetised and dissected and gut contents are used to produce wet slides for microscopy. Both, Giemsa and Papanicolaou stains were used to confirm correct identification of Lophomonas blattarum. Representatives of four genera of protozoa were identified in 110 cockroaches: Nyctoterus sp. was observed in 91.8% of cases, Gregarina sp. in 64.5%, Amoeba sp. in 25.4% and Lophomonas blattarum in 13.6%. Nyctoterus and Gregarina were statistically significantly more likely to be found in diseased cockroaches compared to Amoeba or Lophomonas. The prevalence of Lophomonas blattarum was similar to that in published studies of a different species of cockroach, Periplaneta americana. Further work is needed to assess the interplay between protozoa, cockroaches and broncho-pulmonary diseases.

LC-MS analysis of Hep-2 and Hek-293 cell lines treated with Brazilian red propolis reveals differences in protein expression.

PubMed

da Silva Frozza, Caroline O; da Silva Brum, Emyle; Alving, Anjali; Moura, Sidnei; Henriques, João A P; Roesch-Ely, Mariana

2016-08-01

Red propolis, an exclusive variety of propolis found in the northeast of Brazil has shown to present antitumour activity, among several other biological properties. This article aimed to help to evaluate the underlying molecular mechanisms of the potential anticancer effects of red propolis on tumour, Hep-2, and non-tumour cells, Hek-293. Differentially expressed proteins in human cell lines were identified through label-free quantitative MS-based proteomic platform, and cells were stained with Giemsa to show morphological changes. A total of 1336 and 773 proteins were identified for Hep-2 and Hek-293, respectively. Among the proteins here identified, 16 were regulated in the Hep-2 cell line and 04 proteins in the Hek-293 line. Over a total of 2000 proteins were identified under MS analysis, and approximately 1% presented differential expression patterns. The GO annotation using Protein Analysis THrough Evolutionary Relationships classification system revealed predominant molecular function of catalytic activity, and among the biological processes, the most prominent was associated to cell metabolism. The proteomic profile here presented should help to elucidate further molecular mechanisms involved in inhibition of cancer cell proliferation by red propolis, which remain unclear to date. © 2016 Royal Pharmaceutical Society.

Effect of cell passage on the susceptibility of BALB/3T3 clone A31-1-1 cells to 3-methylcholanthrene-induced morphological transformation

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

Sheu, C.W.; Moreland, F.M.; Dunkel, V.C.

1987-01-01

The response of BALB/3T3 clone A31-1-1 cells to chemically induced morphological transformation was evaluated using 3-methylcholanthrene (MCA). Stock cultures were initiated from cryopreserved cells, grown in T25 flasks containing 5 ml of medium, and replated at subconfluency. Serially transferred cells were then subjected to transformation assay. After 24-hr seeding, cells were incubated 48 hr with MCA in a 5% CO/sub 2/ incubator. They were then rinsed and incubated for an additional 4 weeks with twice weekly medium change. Type III foci were scored after fixation and staining with Giemsa. With serial passage from the frozen state, cells of passages 3-14more » had a low level of spontaneous transformation; zero to 6 type III foci per 20 dishes were counted. In the MCA-treated cultures the number of transformed foci, however, increased with passage. Such passage-related sensitivity to MCA was demonstrated for cells cultured in two batches of sera: one from MA Bioproducts (Lot no. 2E052) and the other from Armour Pharmaceuticals (Lot no. Y65801). The passage-related increase in number of transformed foci was not related to doubling time, cloning efficiency, or MCA-induced growth inhibition.« less

Probing Cytological and Reproductive Phenomena by Means of Bryophytes.

ERIC Educational Resources Information Center

Newton, M. E.

1985-01-01

Describes procedures (recommended for both secondary and college levels) to study mitosis, Giemsa C-banding, reproductive phenomena (including alternation of generations), and phototropism in mosses and liverworts. (JN)

Fungal infections of the eye--laboratory diagnosis and treatment.

PubMed

Nayak, N

2008-03-01

Infections of the eye give rise to severe ocular morbidity and blindness include keratitis, orbital cellulites, endophthalmitis and dacryocystitis. Corneal blindness, in developing countries is predominantly associated with infections. In India, nearly 30-35% of all culture positive infectious keratitis are caused fungi. Laboratory diagnosis mainly depends upon proper collection and transport of clinical specimens. In fungal keratitis, corneal scraping is the ideal sample, but occasionally corneal biopsy or anterior chamber aspirate may also be needed. Corneal scraping is usually by Kimura spatula, under a slit lamp examination, after anaesthetizing the cornea with topical anaesthetic like 0.4% proparcaine. Corneal biopsy is done by a minor trephining and AC aspirate using a sterile tuberculin syringe. In case of endophthalmitis, 150-200 ìl of aqueous humour is collected. Vitreous fluid (500-1000 ìl), however, is collected by pars plana vitrectomy onto sterile tuberculin syringe, the needle is then fixed to a sterile rubber bung after expelling air from the syringe. The collected sample is immediately transported to the laboratory. Swabs from the regurgitating lacrimnal sacs and wound aspirate/swabs are the ideal specimens for dacryocystitis and orbital cellulites, respectively. These samples are cultured onto SDA slants following standard procedures. The main draw back of culture is its long incubation time (5 to 14 days), though it is indispensable from the view point of the specificity. Direct examination (KOH wet mount, Gram's, Giemsa or calcofluor fluorescent staining methods) of the specimen, however, is quick and immensely helpful for ophthalmologist. The newer rapid methods, such as molecular techniques are also available and the management of patients can be according to the results obtained. With the advent of novel antifungal agents such as newer azoles and cell wall acting antifungals like echinocandins, the clinician has the wider option of selecting the therapeutic modality. In the event of the increasing reports of in vitro drug resistance to much frequently used azoles, polyenes and 5-fluorocytosines, clinical applicability of the newer antifungal agents seems to be quite promising.

Schistosoma mansoni Infections, Undernutrition and Anaemia among Primary Schoolchildren in Two Onshore Villages in Rorya District, North-Western Tanzania.

PubMed

Munisi, David Zadock; Buza, Joram; Mpolya, Emmanuel A; Kinung'hi, Safari M

2016-01-01

Undernutrition and anaemia remains to be a major public health problem in many developing countries, where they mostly affect children. Intestinal parasitic infections are known to affect both growth and haemoglobin levels. Much has been reported on the impact of geohelminths on anaemia and undernutrition, leaving that of Schistosoma mansoni not well studied. Therefore this study intended to determine the association between S.mansoni infections, anaemia and undernutrition among schoolchildren in Rorya district, Northwestern Tanzania. A cross-sectional study was carried among schoolchildren in two onshore villages namely Busanga and Kibuyi in Rorya district. Single stool specimens were collected from 513 randomly selected schoolchildren and processed for microscopic examination using the Kato-Katz method. Nutritional status was determined by anthropometry. Blood samples were also collected and examined for malaria parasites and haemoglobin levels using the Giemsa stain and HaemoCue methods, respectively. A pretested questionnaire was used to collect socio-demographic data and associated factors. The prevalence of S. mansoni infection and malaria was 84.02% and 9.16%, respectively. Other parasites found were Ascaris lumbricoides (1.36%) and Hookworm (1.36%). The prevalence of stunting and wasting was 38.21% and 14.42%, respectively. The prevalence of anaemia was 29.43%, whereby 0.58% had severe anaemia. S. mansoni infection was not found to be associated with undernutrition or anaemia (p>0.05). The risk of stunting and wasting increased with increasing age (p<0.001). Anaemia was associated with age, sex and village of residence (p<0.05). S.mansoni, undernutrition and anaemia are highly prevalent in the study area. The observed rates of undernutrition and anaemia were seen not to be associated with S.mansoni infection suggesting possibly being a result of poor dietary nutrients. This study suggests that policy makers should consider Rorya district for inclusion into national schistosomiasis control and school feeding programmes.

Parasites and vector-borne pathogens in client-owned dogs in Albania. Blood pathogens and seroprevalences of parasitic and other infectious agents.

PubMed

Hamel, Dietmar; Shukullari, Enstela; Rapti, Dhimitër; Silaghi, Cornelia; Pfister, Kurt; Rehbein, Steffen

2016-02-01

Knowledge on the epidemiology of parasitic and vector-borne infections is still very limited for Albania, a country located in the Balkan Peninsula in southeast Europe. Recent publications indicated prevalence rates of up to 52% for vector-borne infections in less-cared dogs in Albania. To provide data on the epidemiological situation in dogs under veterinary care, a total of 602 client-owned dogs presented to four small animal clinics between March 2010 and April 2011 in Tirana, Albania, were screened by examination of Giemsa-stained blood smears, PCR, and serological methods for the presence of arthropod-borne infections, as well as Neospora caninum and Toxoplasma gondii. Eight different pathogens, namely Babesia vogeli, Hepatozoon canis, Leishmania infantum, Dirofilaria immitis, Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, and Mycoplasma haemocanis, were detected by direct methods with prevalence rates ranging from 1 to 9%. Seroprevalence for Babesia spp., L. infantum, Anaplasma spp., and E. canis were 6.6, 5.1, 24.1, and 20.8%, respectively. Dogs >1 year of age were positive for vector-borne infections significantly more often than younger dogs (p = 0.003). More than half (51.7%) of the dogs were seroreactive to T. gondii and 18.3% to N. caninum. This is the first report on the detection of A. phagocytophilum, A. platys, E. canis, and M. haemocanis by PCR as well as the serological confirmation of exposure of dogs to N. caninum and T. gondii in Albania. The spectrum of pathogens and the seroprevalences for N. caninum and T. gondii in client-owned dogs from Tirana, Albania, are comparable to that reported in other countries in the Mediterranean Basin. The prevalence rates of vector-borne pathogens are at the lower range of that reported in studies from this geographical region. This is probably due to increased awareness of the owners of pet dogs, including better husbandry conditions and ectoparasiticidal treatment, thus limiting exposure of dogs to vectors.

Centrifuge-operated specimen staining method and apparatus

NASA Technical Reports Server (NTRS)

Feeback, Daniel L. (Inventor); Clarke, Mark S. F. (Inventor)

1999-01-01

A method of staining preselected, mounted specimens of either biological or nonbiological material enclosed within a staining chamber where the liquid staining reagents are applied and removed from the staining chamber using hypergravity as the propelling force. In the preferred embodiment, a spacecraft-operated centrifuge and method of diagnosing biological specimens while in orbit, characterized by hermetically sealing a shell assembly. The assembly contains slide stain apparatus with computer control therefor, the operative effect of which is to overcome microgravity, for example on board an International Space Station.

Mass spectrometry-compatible silver staining of histones resolved on acetic acid-urea-Triton PAGE.

PubMed

Pramod, Khare Satyajeet; Bharat, Khade; Sanjay, Gupta

2009-05-01

Acetic acid-Urea-Triton (AUT) PAGE is commonly used method to separate histone variants and their post-translationally modified forms. Coomassie staining is the preferred method for protein visualization; however, its sensitivity is less than that of silver staining. Though silver staining of histones in AUT-PAGE has been reported, the method is time-consuming, dependent on prior staining by Amido black and has not been reported suitable for mass spectrometry. Here, we propose 'SDS-Silver' method for rapid, sensitive and mass spectrometry-compatible staining of histones resolved on AUT-PAGE.

Helicobacter pylori and precancerous conditions of the stomach: the frequency of infection in a cross-sectional study of 79 consecutive patients with chronic antral gastritis in Yaoundé, Cameroon

PubMed Central

Ankouane, Firmin; Noah, Dominique Noah; Enyime, Félicien Ntoné; Ndjollé, Carole Menzy; Djapa, Roger Nsenga; Nonga, Bernadette Ngo; Njoya, Oudou; Ndam, Elie Claude Ndjitoyap

2015-01-01

Introduction The study aimed at determining the different types of precancerous conditions of the stomach and searches the frequency of Helicobacter pylori in these lesions in patients with chronic antral gastritis in Yaounde, Cameroon. Methods Five gastric biopsies were performed during upper gastrointestinal endoscopy for pathology and fixed in formol 10% before being coated in paraffin. Both the modified Giemsa and Periodic acid of Shift – Alkaline blue stains were used for the histological diagnosis of Helicobacter pylori infection. Hematoxylyn and eosin stain was used to determine the activity of gastritis, atrophic gastritis and intestinal metaplasia in accordance to the Sydney's classification of gastritis. Data were analysed using both the Epi info 6.04 and Excel 2007 softwares. Means and their standard deviations, medians and their interquartiles (IQR) were calculated. Proportions were established for qualitative variables and chi square analysis done in this study with a p value set at 0.05. Results Seventy-nine patients with chronic antral gastritis were enrolled, of which 43 (54.4%) were male, median age: 43 years (range from 21 to 70 years). The rate of atrophic gastritis was 74.7% (59/79). The activity of atrophic gastritis was mild in 47.5% (28/59) of cases, moderate in 47.5% (28/59) and severe in 5% (5/59). Intestinal metaplasia and follicular gastritis were present in 6.3% (5/79), and 10.1% (8/79), respectively. Concerning Helicobacter pylori infection, 71.2% (42/59) of patients with atrophic gastritis tested positive against 28.8% (17/59) who tested negative (p = 0.00003). Helicobacter pylori infection was related to the severity of gastric atrophy (p = 0.0001). Among patients with intestinal metaplasia and follicular gastritis, the proportion of those who tested positive for Helicobacter pylori infection was 80% (4/5), and 75% (6/8), respectively. There were no significant differences in the occurrence of atrophic gastritis according to age groups (p = 0.908). Conclusion This study concludes that atrophic gastritis, which is most often caused by Helicobacter pylori, is the most frequent precancerous condition of stomach in Cameroon. Routine gastric sampling for pathologic analysis is mandatory for effective diagnosis and surveillance of Helicobacter pylori infection and precancerous conditions of the stomach. PMID:26090010

A robust, efficient and flexible method for staining myelinated axons in blocks of brain tissue.

PubMed

Wahlsten, Douglas; Colbourne, Frederick; Pleus, Richard

2003-03-15

Previous studies have demonstrated the utility of the gold chloride method for en bloc staining of a bisected brain in mice and rats. The present study explores several variations in the method, assesses its reliability, and extends the limits of its application. We conclude that the method is very efficient, highly robust, sufficiently accurate for most purposes, and adaptable to many morphometric measures. We obtained acceptable staining of commissures in every brain, despite a wide variety of fixation methods. One-half could be stained 24 h after the brain was extracted and the other half could be stained months later. When staining failed because of an exhausted solution, the brain could be stained successfully in fresh solution. Relatively small changes were found in the sizes of commissures several weeks after initial fixation or staining. A half brain stained to reveal the mid-sagittal section could then be sectioned coronally and stained again in either gold chloride for myelin or cresyl violet for Nissl substance. Uncertainty, arising from pixelation of digitized images was far less than errors arising from human judgments about the histological limits of major commissures. Useful data for morphometric analysis were obtained by scanning the surface of a gold chloride stained block of brain with an inexpensive flatbed scanner.

Comparison of different diagnostic techniques for the detection of cryptosporidiosis in bovines

PubMed Central

Rekha, K. M. H.; Puttalakshmamma, G. C.; D’Souza, Placid E.

2016-01-01

Aim: Aim of the present study was to compare different methods, viz., Sheather's sugar flotation (SSF), Ziehl-Neelsen (ZN), Kinyoun's acid-fast method (KAF), safranin-methylene blue staining (SMB), and negative staining techniques such as nigrosin staining, light green staining, and malachite green staining for the detection of Cryptosporidium spp. oocysts in bovines. Materials and Methods: A total of 455 fecal samples from bovines were collected from private, government farms and from the clinical cases presented to Department of Medicine, Veterinary College, Bengaluru. They were subjected for SSF, ZN, KAF, SMB and negative staining methods. Results: Out of 455 animal fecal samples screened 5.71% were found positive for Cryptosporidium spp. oocysts. The species were identified as Cryptosporidium parvum in calves and Cryptosporidium andersoni in adults based on the morphological characterization and micrometry of the oocysts. Conclusions: Of all the techniques, fecal flotation with sheather's was found to be more specific and sensitive method for the detection of Cryptosporidium spp. oocysts. Among the conventional staining methods, the SMB gives better differentiation between oocysts and yeast. Among the three negative staining methods, malachite green was found sensitive over the other methods. PMID:27051211

[Comparison of the quick Gram stain method to the B&M modified and favor methods].

PubMed

Osawa, Kayo; Kataoka, Nobumasa; Maruo, Toshio

2011-01-01

The Gram stain is an established method for bacterial identification, but the time needed to carry out this stain is 2-3 min. We attempted to shorten this time and stained a total of 70 clinical specimens isolated from using the Bartholomew & Mittwer (B&M) modified or Favor methods with a 3 s duration for washing and staining steps. Results were plotted and analyzed using a Hue Saturation Intensity (HSI) model. The range based on a plot of the two methods with the HSI model was presented as a reference interval. Our results indicated that 100% (35/35) of strains were Gram positive and 97.1% (34/35) were Gram negative for the quick B&M modified method. In the quick Favor method, 80.0% (28/35) were Gram positive and 68.6% (24/35) of strains were Gram negative. We propose that the quick B&M modified method is equivalent to the standard Gram staining method and is superior to the quick Favor method.

Gram stain method shows better sensitivity than clinical criteria for detection of bacterial vaginosis in surveillance of pregnant, low-income women in a clinical setting.

PubMed Central

Tam, M T; Yungbluth, M; Myles, T

1998-01-01

OBJECTIVE: The purpose of the study is to determine whether the Gram stain method is superior to the clinical criteria for the diagnosis of bacterial vaginosis in low-income pregnant women seen in a resident clinic setting. The clinical criteria is the current diagnostic method employed to diagnose bacterial vaginosis. STUDY DESIGN: In this study, 51 pregnant women with vaginal discharge were prospectively evaluated. All were screened using the clinical criteria, Gram stain method, and culture of the discharge. The modified scoring system instituted by Nugent et al. (J Clin Microbiol 29:297-301, 1991) was employed in reading the Gram stain smears. The clinical criteria were then compared with the Gram stain method. Isolation of moderate to many Gardnerella vaginalis growth by culture was used as the confirmatory finding. RESULTS: Sensitivity of the Gram stain method (91%) was significantly higher than that of the clinical criteria (46%), (sign test P = 0.0023, < 0.01). The Gram stain method also has both a low false-negative (4%) and high negative predictive value (96%), making it an ideal diagnostic test. CONCLUSION: The Gram stain method is a rapid and cost-effective test that is also highly reproducible and readily available in many laboratories. These features make the Gram stain method a more desirable screening procedure for bacterial vaginosis in a clinic population. PMID:9894174

Effect of controlled and uncontrolled rate of cooling, prior to controlled rate of freezing, on motion characteristics and acrosomal integrity of cryopreserved ram spermatozoa.

PubMed

Joshi, Anil; Kumar, Davendra; Naqvi, S M K; Maurya, V P

2008-12-01

A programmable cell freezer provides ideal cryobiological conditions for controlled-rate cooling and freezing of ram spermatozoa. The purpose of this study was to investigate the effects of controlled (Group 1) and uncontrolled (Group 2) cooling conditions prior to programmable freezing of ram semen on post-thaw sperm motion characteristics and acrosomal integrity of ram spermatozoa. Semen samples of good initial motility obtained from adult Malpura rams were pooled, diluted to 1 × 10(9) spermatozoa per milliliter with Egg yolk-TEST-glycerol extender, and packaged in 0.25 mL straws. Straws representing Group 1 were cooled in a programmable cell freezer from 25°C to 5°C at the rate of -0.15°C per minute followed by a holding time of 2 h for equilibration, while straws of Group 2 were allowed to cool slowly up to 5°C and equilibrate for 2 h in the cold cabinet. After equilibration, straws of Group 2 were also loaded in the cell freezer for freezing straws of both the treatment groups simultaneously from 5°C to -125°C at the rate of -25°C per minute. Thawing of straws was done at 50°C for 10 s and the quality of frozen-thawed spermatozoa was objectively assessed by using sperm motility analyzer. Thawed samples were also evaluated for acrosomal integrity after staining the dried semen smears with Giemsa stain. The average post-thaw motility of straws was significantly higher (P < 0.05) in samples frozen after controlled cooling, compared with samples frozen after uncontrolled rate of cooling. The percent of spermatozoa with normal acrosome was also significantly (P < 0.05) higher in Group 1, compared to Group 2. The results indicate that controlled-rate cooling has a significant effect on post-thaw motility and acrosomal integrity of frozen-thawed ram spermatozoa, compared to uncontrolled-rate cooling prior to programmable freezing.

[Regulative effects of hydrogen-rich medium on monocytic adhesion and vascular endothelial permeability].

PubMed

Wang, Wei-na; Xie, Ke-liang; Chen, Hong-guang; Han, Huan-zhi; Wang, Guo-lin; Yu, Yong-hao

2013-11-19

To explore the regulative effects of hydrogen-rich medium on lipopolysaccharide (LPS)-induced monocytes adhesion to human umbilical vein endothelial cells (HUVEC) and vascular endothelial permeability in vitro. Endothelial cells were seeded in 6-well plates and randomly divided into 4 groups (n = 42 each):control (A), hydrogen-rich medium (B), LPS (C) and LPS+hydrogen-rich medium (D). Cells were cultured in plain culture medium in groups A and C or in hydrogen-saturated culture medium in groups B and D.LPS 1 µg/ml was added into groups C and D.When forming a monolayer, monocytes were added into each group after 6, 12 and 24 h respectively. After a 90-minute co-culturing, adhesion status was detected by Wright-Giemsa stain.Supernatants were collected to detect the concentrations of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin by enzyme-linked immunosorbent assay (ELISA). The expression of VE-cadherin was measured by Western blot. Cells were stained with immunofluorescence to show the distribution of VE-cadherin after a 24-hour incubation. Compared with group A, the adhesion of monocytes to endothelial cells increased (P < 0.05) in group C, the levels of E-selectin and VCAM-1 became elevated (P < 0.05) while the expression of VE-cadherin decreased significantly (P < 0.05). Compared with group C, adhesion decreased in group D (P < 0.05), the levels of E-selectin and VCAM-1 decreased (P < 0.05) while there was an increased expression of VE-cadherin (P < 0.05). Three timepoints showed the same tendency. The results of 24 h fluorescence indicated that, compared with group A, VE-cadherin was incomplete in cell-cell connections in group C.However it was complete and well-distributed in group D versus group C. Hydrogen-rich medium may reduce the LPS-induced release of adhesion molecules, lessen monocytic adhesion to HUVEC and regulate the expression of VE-cadherin to protect vascular permeability.

Comparison of spontaneous and idoxuridine-induced micronuclei by chromosome painting.

PubMed

Fauth, E; Zankl, H

1999-04-06

Fluorescence in situ hybridisation (FISH) technique with chromosome specific library (CSL) DNA probes for all human chromosomes were used to study about 9000 micronuclei (MN) in normal and idoxuridine (IUdR)-treated lymphocyte cultures of female and male donors. In addition, MN rates and structural chromosome aberrations were scored in Giemsa-stained chromosome spreads of these cultures. IUdR treatment (40 microg/ml) induced on the average a 12-fold increase of the MN rate. Metaphase analysis revealed no distinct increase of chromosome breaks but a preferential decondensation at chromosome 9q12 (28-79%) and to a lower extend at 1q12 (8-21%). Application of FISH technique with CSL probes to one male and one female untreated proband showed that all human chromosomes except chromosome 12 (and to a striking high frequency chromosomes 9, X and Y) occurred in spontaneous MN. In cultures containing IUdR, the chromosomal spectrum found in MN was reduced to 10 chromosomes in the male and 13 in the female proband. Eight chromosomes (2, 6, 12, 13, 14, 15, 17 and 18) did not occur in MN of both probands. On the contrary chromosomes 1 and especially 9 were found much more frequently in the MN of IUdR-treated cultures than in MN of control cultures. DAPI-staining revealed heterochromatin signals in most of the IUdR-induced MN. In an additional study, spontaneous and IUdR-induced MN were investigated in lymphocytes of another female donor using CSL probes only for chromosomes 1, 6, 9, 15, 16 and X. The results confirmed the previous finding that chromosomes 1 and 9 occur very often in MN after IUdR-treatment. The results indicate that decondensation of heterochromatic regions on chromosomes 1 and 9 caused by IUdR treatment strongly correlates with MN formation by these chromosomes. Copyright 1999 Elsevier Science B.V.

The value of Tzanck smear test in diagnosis of erosive, vesicular, bullous, and pustular skin lesions.

PubMed

Durdu, Murat; Baba, Mete; Seçkin, Deniz

2008-12-01

Tzanck smear is generally used for the diagnosis of the pemphigus group of autoimmune bullous diseases and mucocutaneous herpesvirus infections. There are only a few studies in the literature investigating its diagnostic value. We aimed to investigate Tzanck smear findings and to determine the diagnostic value of this test in moist (erosive, vesicular, bullous, and pustular) skin lesions. We also aimed to develop an algorithmic approach for the diagnosis of these types of skin lesions according to the Tzanck smear findings. Samples were stained with May-Grünwald-Giemsa and evaluated by the same dermatologist. In some patients, methylene blue and Gram staining or direct immunofluorescence examinations were additionally performed. In all of the study cases, after the evaluation of clinical and laboratory findings (including, when appropriate, potassium hydroxide examination; viral serology; bacterial and fungal cultures; histopathology; direct and indirect immunofluorescence; patch testing), the definite diagnosis was established. We also determined the sensitivity and the specificity of certain Tzanck smear findings. Tzanck smear was performed in a total of 400 patients with moist skin lesions. The sensitivities of multinucleated giant cells and acantholytic cells in herpetic infections, dyskeratotic acantholytic cells and cocci in bullous impetigo, pseudohyphae in candidiasis, acantholytic cells in pemphigus and more than 10 tadpole cells (magnification x100) in spongiotic dermatitis were 84.7%, 92%, 100%, 100%, and 81.5%, respectively. Because Tzanck smears were evaluated by the same dermatologist, no comment could be made regarding the interobserver reliability of this test and how the level of experience with this technique might affect the results. Also, the sensitivity and the specificity of Tzanck smear test findings for certain diseases could not be calculated because of an insufficient number of patients. The Tzanck smear test is an inexpensive, useful, and an easy diagnostic tool for certain skin diseases.

Plasmodium knowlesi Skeleton-Binding Protein 1 Localizes to the ‘Sinton and Mulligan’ Stipplings in the Cytoplasm of Monkey and Human Erythrocytes

PubMed Central

Lucky, Amuza Byaruhanga; Sakaguchi, Miako; Katakai, Yuko; Kawai, Satoru; Yahata, Kazuhide; Templeton, Thomas J.

2016-01-01

The malaria parasite, Plasmodium, exports protein products to the infected erythrocyte to introduce modifications necessary for the establishment of nutrient acquisition and surface display of host interaction ligands. Erythrocyte remodeling impacts parasite virulence and disease pathology and is well documented for the human malaria parasite Plasmodium falciparum, but has been less described for other Plasmodium species. For P. falciparum, the exported protein skeleton-binding protein 1 (PfSBP1) is involved in the trafficking of erythrocyte surface ligands and localized to membranous structures within the infected erythrocyte, termed Maurer's clefts. In this study, we analyzed SBP1 orthologs across the Plasmodium genus by BLAST analysis and conserved gene synteny, which were also recently described by de Niz et al. (2016). To evaluate the localization of an SBP1 ortholog, we utilized the zoonotic malaria parasite, Plasmodium knowlesi. Immunofluorescence assay of transgenic P. knowlesi parasites expressing epitope-tagged recombinant PkSBP1 revealed a punctate staining pattern reminiscent of Maurer's clefts, following infection of either monkey or human erythrocytes. The recombinant PkSBP1-positive puncta co-localized with Giemsa-stained structures, known as ‘Sinton and Mulligan’ stipplings. Immunoelectron microscopy also showed that recombinant PkSBP1 localizes within or on the membranous structures akin to the Maurer's clefts. The recombinant PkSBP1 expressed in P. falciparum-infected erythrocytes co-localized with PfSBP1 at the Maurer's clefts, indicating an analogous trafficking pattern. A member of the P. knowlesi 2TM protein family was also expressed and localized to membranous structures in infected monkey erythrocytes. These results suggest that the trafficking machinery and induced erythrocyte cellular structures of P. knowlesi are similar following infection of both monkey and human erythrocytes, and are conserved with P. falciparum. PMID:27732628

Quantification of histochemical stains using whole slide imaging: development of a method and demonstration of its usefulness in laboratory quality control.

PubMed

Gray, Allan; Wright, Alex; Jackson, Pete; Hale, Mike; Treanor, Darren

2015-03-01

Histochemical staining of tissue is a fundamental technique in tissue diagnosis and research, but it suffers from significant variability. Efforts to address this include laboratory quality controls and quality assurance schemes, but these rely on subjective interpretation of stain quality, are laborious and have low reproducibility. We aimed (1) to develop a method for histochemical stain quantification using whole slide imaging and image analysis and (2) to demonstrate its usefulness in measuring staining variation. A method to quantify the individual stain components of histochemical stains on virtual slides was developed. It was evaluated for repeatability and reproducibility, then applied to control sections of an appendix to quantify H&E staining (H/E intensities and H:E ratio) between automated staining machines and to measure differences between six regional diagnostic laboratories. The method was validated with <0.5% variation in H:E ratio measurement when using the same scanner for a batch of slides (ie, it was repeatable) but was not highly reproducible between scanners or over time, where variation of 7% was found. Application of the method showed H:E ratios between three staining machines varied from 0.69 to 0.93, H:E ratio variation over time was observed. Interlaboratory comparison demonstrated differences in H:E ratio between regional laboratories from 0.57 to 0.89. A simple method using whole slide imaging can be used to quantify and compare histochemical staining. This method could be deployed in routine quality assurance and quality control. Work is needed on whole slide imaging devices to improve reproducibility. 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.

Color normalization of histology slides using graph regularized sparse NMF

NASA Astrophysics Data System (ADS)

Sha, Lingdao; Schonfeld, Dan; Sethi, Amit

2017-03-01

Computer based automatic medical image processing and quantification are becoming popular in digital pathology. However, preparation of histology slides can vary widely due to differences in staining equipment, procedures and reagents, which can reduce the accuracy of algorithms that analyze their color and texture information. To re- duce the unwanted color variations, various supervised and unsupervised color normalization methods have been proposed. Compared with supervised color normalization methods, unsupervised color normalization methods have advantages of time and cost efficient and universal applicability. Most of the unsupervised color normaliza- tion methods for histology are based on stain separation. Based on the fact that stain concentration cannot be negative and different parts of the tissue absorb different stains, nonnegative matrix factorization (NMF), and particular its sparse version (SNMF), are good candidates for stain separation. However, most of the existing unsupervised color normalization method like PCA, ICA, NMF and SNMF fail to consider important information about sparse manifolds that its pixels occupy, which could potentially result in loss of texture information during color normalization. Manifold learning methods like Graph Laplacian have proven to be very effective in interpreting high-dimensional data. In this paper, we propose a novel unsupervised stain separation method called graph regularized sparse nonnegative matrix factorization (GSNMF). By considering the sparse prior of stain concentration together with manifold information from high-dimensional image data, our method shows better performance in stain color deconvolution than existing unsupervised color deconvolution methods, especially in keeping connected texture information. To utilized the texture information, we construct a nearest neighbor graph between pixels within a spatial area of an image based on their distances using heat kernal in lαβ space. The representation of a pixel in the stain density space is constrained to follow the feature distance of the pixel to pixels in the neighborhood graph. Utilizing color matrix transfer method with the stain concentrations found using our GSNMF method, the color normalization performance was also better than existing methods.

First case of a naturally acquired human infection with Plasmodium cynomolgi

PubMed Central

2014-01-01

Since 1960, a total of seven species of monkey malaria have been reported as transmissible to man by mosquito bite: Plasmodium cynomolgi, Plasmodium brasilianum, Plasmodium eylesi, Plasmodium knowlesi, Plasmodium inui, Plasmodium schwetzi and Plasmodium simium. With the exception of P. knowlesi, none of the other species has been found to infect humans in nature. In this report, it is described the first known case of a naturally acquired P. cynomolgi malaria in humans. The patient was a 39-year-old woman from a malaria-free area with no previous history of malaria or travel to endemic areas. Initially, malaria was diagnosed and identified as Plasmodium malariae/P. knowlesi by microscopy in the Terengganu State Health Department. Thick and thin blood films stained with 10% Giemsa were performed for microscopy examination. Molecular species identification was performed at the Institute for Medical Research (IMR, Malaysia) and in the Malaria & Emerging Parasitic Diseases Laboratory (MAPELAB, Spain) using different nested PCR methods. Microscopic re-examination in the IMR showed characteristics of Plasmodium vivax and was confirmed by a nested PCR assay developed by Snounou et al. Instead, a different PCR assay plus sequencing performed at the MAPELAB confirmed that the patient was infected with P. cynomolgi and not with P. vivax. This is the first report of human P. cynomolgi infection acquired in a natural way, but there might be more undiagnosed or misdiagnosed cases, since P. cynomolgi is morphologically indistinguishable from P. vivax, and one of the most used PCR methods for malaria infection detection may identify a P. cynomolgi infection as P. vivax. Simian Plasmodium species may routinely infect humans in Southeast Asia. New diagnostic methods are necessary to distinguish between the human and monkey malaria species. Further epidemiological studies, incriminating also the mosquito vector(s), must be performed to know the relevance of cynomolgi malaria and its implication on human public health and in the control of human malaria. The zoonotic malaria cannot be ignored in view of increasing interactions between man and wild animals in the process of urbanization. PMID:24564912

First case of a naturally acquired human infection with Plasmodium cynomolgi.

PubMed

Ta, Thuy H; Hisam, Shamilah; Lanza, Marta; Jiram, Adela I; Ismail, NorParina; Rubio, José M

2014-02-24

Since 1960, a total of seven species of monkey malaria have been reported as transmissible to man by mosquito bite: Plasmodium cynomolgi, Plasmodium brasilianum, Plasmodium eylesi, Plasmodium knowlesi, Plasmodium inui, Plasmodium schwetzi and Plasmodium simium. With the exception of P. knowlesi, none of the other species has been found to infect humans in nature. In this report, it is described the first known case of a naturally acquired P. cynomolgi malaria in humans.The patient was a 39-year-old woman from a malaria-free area with no previous history of malaria or travel to endemic areas. Initially, malaria was diagnosed and identified as Plasmodium malariae/P. knowlesi by microscopy in the Terengganu State Health Department. Thick and thin blood films stained with 10% Giemsa were performed for microscopy examination. Molecular species identification was performed at the Institute for Medical Research (IMR, Malaysia) and in the Malaria & Emerging Parasitic Diseases Laboratory (MAPELAB, Spain) using different nested PCR methods.Microscopic re-examination in the IMR showed characteristics of Plasmodium vivax and was confirmed by a nested PCR assay developed by Snounou et al. Instead, a different PCR assay plus sequencing performed at the MAPELAB confirmed that the patient was infected with P. cynomolgi and not with P. vivax.This is the first report of human P. cynomolgi infection acquired in a natural way, but there might be more undiagnosed or misdiagnosed cases, since P. cynomolgi is morphologically indistinguishable from P. vivax, and one of the most used PCR methods for malaria infection detection may identify a P. cynomolgi infection as P. vivax.Simian Plasmodium species may routinely infect humans in Southeast Asia. New diagnostic methods are necessary to distinguish between the human and monkey malaria species. Further epidemiological studies, incriminating also the mosquito vector(s), must be performed to know the relevance of cynomolgi malaria and its implication on human public health and in the control of human malaria.The zoonotic malaria cannot be ignored in view of increasing interactions between man and wild animals in the process of urbanization.

Comparison of culture-based, vital stain and PMA-qPCR methods for the quantitative detection of viable hookworm ova.

PubMed

Gyawali, P; Sidhu, J P S; Ahmed, W; Jagals, P; Toze, S

2017-06-01

Accurate quantitative measurement of viable hookworm ova from environmental samples is the key to controlling hookworm re-infections in the endemic regions. In this study, the accuracy of three quantitative detection methods [culture-based, vital stain and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR)] was evaluated by enumerating 1,000 ± 50 Ancylostoma caninum ova in the laboratory. The culture-based method was able to quantify an average of 397 ± 59 viable hookworm ova. Similarly, vital stain and PMA-qPCR methods quantified 644 ± 87 and 587 ± 91 viable ova, respectively. The numbers of viable ova estimated by the culture-based method were significantly (P < 0.05) lower than vital stain and PMA-qPCR methods. Therefore, both PMA-qPCR and vital stain methods appear to be suitable for the quantitative detection of viable hookworm ova. However, PMA-qPCR would be preferable over the vital stain method in scenarios where ova speciation is needed.

[Investigation of Pneumocystis jirovecii pneumonia and colonization in iatrogenically immunosuppressed and immunocompetent patients].

PubMed

Özkoç, Soykan; Bayram Delibaş, Songül

2015-04-01

Pneumocystis pneumonia (PCP) is a potentially life-threatening infection for the immunocompromized patients. However, Pneumocystis jirovecii colonization can also be detected in healthy individuals and in patients with various underlying lung diseases. The aim of this study was to evaluate the immunocompetent and iatrogenically immunosuppressed patients in terms of PCP and P.jirovecii colonization. A total of 92 patients (66 male, 26 female; age range: 18-93 years, median: 58.5) who underwent bronchoscopy due to various pulmonary symptoms between January 2011-April 2014, were included in the study. Of these patients, 65 were under immunosuppressive therapy (38 were treated with anti-cancer drugs, 15 with anti-rejection/immunomodulatory drugs and 12 with corticosteroids), while 27 were immunocompetent. Bronchoalveolar lavage (BAL) fluids were evaluated for the presence of P.jirovecii mitochondrial gene coding ribosomal large subunit (mtLSUrRNA) with nested PCR (nPCR) method. All of the samples were also examined by Giemsa and Gomori's methenamine silver (GMG) staining methods. P.jirovecii DNA was detected in 31 (33.7%) out of 92 BAL samples by nPCR. Although six immunosuppressed patients were positive in the first round of amplification, 26 of 65 (40%) immunosuppressed and five of 27 (18.5%) immunocompetent patients were positive with nPCR. P.jirovecii cysts and trophozoites were detected in only five (16.1%) of the 31 nPCR positive samples. The probability of being immunosuppressive among nPCR positive cases was statistically higher than nPCR negative cases (χ²= 3.940; p= 0.047). This difference was more significant in organ transplant recipients and patients under anti-rejection/immunomodulatory treatment (χ²= 6.715, p= 0.01; χ²= 5.550, p= 0.018, respectively). When clinical, laboratory and radiological findings of nPCR positive patients were considered, five patients (2 kidney transplant, 1 bone marrow transplant, 1 interstitial lung disease and 1 lung cancer case) in immunosuppressed group were interpreted as "definite PCP" and eight patients (2 kidney transplant, 1 leukemia, 1 connective tissue disease, 1 Wegener's granulomatosus, 2 rheumatoid arthritis and 1 lung cancer case) were interpreted as "probable PCP". Other 18 (19.6%) nPCR positive patients, of them 13 were immunosuppressive and five were immunocompetent, were considered as "P.jirovecii colonization". The colonization rate was determined as 50% (13/26) in immunosuppressive patients, and was mostly detected in patients with hematological malignancies (4/13), followed by patients with solid tumors (3/13) and organ transplantations (3/13). On the other hand, all of the nPCR positive immunocompetent patients (5/5) were evaluated as colonization. In this study significant data was obtained about P.jirovecii epidemiology in our country. Our results also showed that iatrogenically immunosuppressed patients are under risk of PCP and nPCR method is more sensitive than conventional PCR and classical staining methods in the diagnosis of these patients.

Fluorogenic Ag+–Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining

PubMed Central

Xie, Sheng; Wong, Alex Y. H.; Kwok, Ryan T. K.; Li, Ying; Su, Huifang; Lam, Jacky W. Y.

2018-01-01

Abstract Silver staining, which exploits the special bioaffinity and the chromogenic reduction of silver ions, is an indispensable visualization method in biology. It is a most popular method for in‐gel protein detection. However, it is limited by run‐to‐run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) analysis; limitations that are largely attributed to the tricky chromogenic visualization. Herein, we reported a novel water‐soluble fluorogenic Ag+ probe, the sensing mechanism of which is based on an aggregation‐induced emission (AIE) process driven by tetrazolate‐Ag+ interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains. PMID:29575702

Usefulness of Leifson Staining Method in Diagnosis of Helicobacter pylori Infection

PubMed Central

Piccolomini, Raffaele; Di Bonaventura, Giovanni; Neri, Matteo; Di Girolamo, Arturo; Catamo, Giovanni; Pizzigallo, Eligio

1999-01-01

The Leifson staining method was used to diagnose Helicobacter pylori infection and was compared to histology, culture, and the rapid urease test (RUT). Histology gave the best sensitivity (98%), compared to Leifson staining (97%), culture (92%), and RUT (85%) (P < 0.005). Leifson staining is a sensitive, rapid, economical method for diagnosis of H. pylori infection in dyspeptic patients. PMID:9854090

A multiple translocation event in a patient with hexadactyly, facial dysmorphism, mental retardation and behaviour disorder characterised comprehensively by molecular cytogenetics. Case report and review of the literature.

PubMed

Seidel, Jörg; Heller, Anita; Senger, Gabriele; Starke, Heike; Chudoba, Ilse; Kelbova, Christina; Tönnies, Holger; Neitzel, Heidemarie; Haase, Claudia; Beensen, Volkmar; Zintl, Felix; Claussen, Uwe; Liehr, Thomas

2003-09-01

We report a 13-year-old female patient with multiple congenital abnormalities (microcephaly, facial dysmorphism, anteverted dysplastic ears and postaxial hexadactyly), mental retardation, and adipose-gigantism. Ultrasonography revealed no signs of a heart defect or renal abnormalities. She showed no speech development and suffered from a behavioural disorder. CNS abnormalities were excluded by cerebral MRI. Initial cytogenetic studies by Giemsa banding revealed an aberrant karyotype involving three chromosomes, t(2;4;11). By high resolution banding and multicolour fluoresence in-situ hybridisation (M-FISH, MCB), chromosome 1 was also found to be involved in the complex chromosomal aberrations, confirming the karyotype 46,XX,t(2;11;4).ish t(1;4;2;11)(q43;q21.1;p12-p13.1;p14.1). To the best of our knowledge no patient has been previously described with such a complex translocation involving 4 chromosomes. This case demonstrates that conventional chromosome banding techniques such as Giemsa banding are not always sufficient to characterise complex chromosomal abnormalities. Only by the additional utilisation of molecular cytogenetic techniques could the complexity of the present chromosomal rearrangements and the origin of the involved chromosomal material be detected. Further molecular genetic studies will be performed to clarify the chromosomal breakpoints potentially responsible for the observed clinical symptoms. This report demonstrates that multicolour-fluorescence in-situ hybridisation studies should be performed in patients with congenital abnormalities and suspected aberrant karyotypes in addition to conventional Giemsa banding.

COMPARISON OF PERMANENT STAINING METHODS FOR THE LABORATORY DIAGNOSIS OF TRICHOMONIASIS

PubMed Central

MENEZES, Camila Braz; MELLO, Mariana dos Santos; TASCA, Tiana

2016-01-01

Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in the world. The diagnosis is based on wet mount preparation and direct microscopy on fixed and stained clinical specimens. The aim of this study was to compare the performance of different fixing and staining techniques used in the detection of T. vaginalis in urine. The smears were fixed and submitted to different methods of permanent staining and then, the morphological aspects of the parasites were analyzed and compared. The Papanicolaou staining with ethanol as the fixative solution showed to be the best method of permanent staining. Our data suggest that staining techniques in association with wet mount examination of fresh specimens contribute to increase the sensitivity in the diagnosis of trichomoniasis. PMID:26910452

Differences in staining intensities affect reported occurrences and concentrations of Giardia spp. in surface drinking water sources.

PubMed

Alderisio, K A; Villegas, L F; Ware, M W; McDonald, L A; Xiao, L; Villegas, E N

2017-12-01

USEPA Method 1623, or its equivalent, is currently used to monitor for protozoan contamination of surface drinking water sources worldwide. At least three approved staining kits used for detecting Cryptosporidium and Giardia are commercially available. This study focuses on understanding the differences among staining kits used for Method 1623. Merifluor and EasyStain labelling kits were used to monitor Cryptosporidium oocyst and Giardia cyst densities in New York City's raw surface water sources. In the year following a change to the approved staining kits for use with Method 1623, an anomaly was noted in the occurrence of Giardia cysts in New York City's raw surface water. Specifically, Merifluor-stained samples had higher Giardia cyst densities as compared with those stained with EasyStain. Side by side comparison revealed significantly lower fluorescence intensities of Giardia muris as compared with Giardia duodenalis cysts when labelled with EasyStain. This study showed very poor fluorescence intensity signals by EasyStain on G. muris cysts resulting in lower cyst counts, while Merifluor, with its broader Giardia cyst staining specificity, resulted in higher cyst counts, when using Methods 1623. These results suggest that detected Giardia cyst concentrations are dependent on the staining kits used, which can result in a more or less conservative estimation of occurrences and densities of zoonotic Giardia cysts by detecting a broader range of Giardia species/Assemblages. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Quantitative cytochemistry of nuclear and cytoplasmic proteins using the Naphthol Yellow S and dinitrofluorobenzene staining methods.

PubMed

Tas, J; James, J

1981-09-01

The 'total protein staining' of biological specimens with the electrostatically binding Naphthol Yellow S or the covalently binding dinitrofluorobenzene must be interpreted as methods which yield data on the specific amino acid pool of the proteins concerned. Both dyes bind to certain free amino-acid side-chains, giving different dye--protein ratios for various proteins. In the presence of DNA, dinitrofluorobenzene stains all proteins present in cell nuclei, whereas Naphthol Yellow S only stains the majority of the non-histone proteins. When protein staining methods are combined with the Feulgen--Pararosanile (SO2) procedure for DNA, decreased Feulgen--DNA contents were measured in dinitrofluorobenzene-stained isolated nuclei and lymphocytes.

Comparative evaluation of two rapid field tests for malaria diagnosis: Partec Rapid Malaria Test® and Binax Now® Malaria Rapid Diagnostic Test

PubMed Central

2011-01-01

Background About 90% of all malaria deaths in sub-Saharan Africa occur in children under five years. Fast and reliable diagnosis of malaria requires confirmation of the presence of malaria parasites in the blood of patients with fever or history suggestive of malaria; hence a prompt and accurate diagnosis of malaria is the key to effective disease management. Confirmation of malaria infection requires the availability of a rapid, sensitive, and specific testing at an affordable cost. We compared two recent methods (the novel Partec Rapid Malaria Test® (PT) and the Binax Now® Malaria Rapid Diagnostic Test (BN RDT) with the conventional Giemsa stain microscopy (GM) for the diagnosis of malaria among children in a clinical laboratory of a hospital in a rural endemic area of Ghana. Methods Blood samples were collected from 263 children admitted with fever or a history of fever to the pediatric clinic of the Agogo Presbyterian Hospital. The three different test methods PT, BN RDT and GM were performed independently by well trained and competent laboratory staff to assess the presence of malaria parasites. Results were analyzed and compared using GM as the reference standard. Results In 107 (40.7%) of 263 study participants, Plasmodium sp. was detected by GM. PT and BN RDT showed positive results in 111 (42.2%) and 114 (43.4%), respectively. Compared to GM reference standard, the sensitivities of the PT and BN RDT were 100% (95% CI: 96.6-100) and 97.2% (95% CI: 92.0-99.4), respectively, specificities were 97.4% (95% CI: 93.6-99.3) and 93.6% (95% CI: 88.5-96.9), respectively. There was a strong agreement (kappa) between the applied test methods (GM vs PT: 0.97; p < 0.001 and GM vs BN RDT: 0.90; p < 0.001). The average turnaround time per tests was 17 minutes. Conclusion In this study two rapid malaria tests, PT and BN RDT, demonstrated a good quality of their performance compared to conventional GM. Both methods require little training, have short turnaround times, are applicable as well as affordable and can therefore be considered as alternative diagnostic tools in malaria endemic areas. The species of Plasmodium cannot be identified. PMID:21605401

A procedure for Alcian blue staining of mucins on polyvinylidene difluoride membranes.

PubMed

Dong, Weijie; Matsuno, Yu-ki; Kameyama, Akihiko

2012-10-16

The isolation and characterization of mucins are critically important for obtaining insight into the molecular pathology of various diseases, including cancers and cystic fibrosis. Recently, we developed a novel membrane electrophoretic method, supported molecular matrix electrophoresis (SMME), which separates mucins on a polyvinylidene difluoride (PVDF) membrane impregnated with a hydrophilic polymer. Alcian blue staining is widely used to visualize mucopolysaccharides and acidic mucins on both blotted membranes and SMME membranes; however, this method cannot be used to stain mucins with a low acidic glycan content. Meanwhile, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, but is incompatible with glycan analysis, which is indispensable for mucin characterizations. Here we describe a novel staining method, designated succinylation-Alcian blue staining, for visualizing mucins on a PVDF membrane. This method can visualize mucins regardless of the acidic residue content and shows a sensitivity 2-fold higher than that of Pro-Q Emerald 488, a fluorescent periodate Schiff-base stain. Furthermore, we demonstrate the compatibility of this novel staining procedure with glycan analysis using porcine gastric mucin as a model mucin.

Hepatoprotective Potential of Some Local Medicinal Plants against 2-Acetylaminoflourene-Induced Damage in Rat.

PubMed

Adetutu, Adewale; Olorunnisola, Olubukola S

2013-01-01

The in vivo micronucleus assay was used to examine the anticlastogenic effects of crude extracts of Bridelia ferruginea, Vernonia amygdalina, Tridax procumbens, Ocimum gratissimum, and Lawsonia inermis in Wistar albino rats. Extracts of doses of 100 mg/kg body weight were given to rats in five groups for seven consecutive days followed by a single dose of 2-AAF (0.5 mmol/kg body weight). The rats were sacrificed after 24 hours and their bone marrow smears were prepared on glass slides stained with Giemsa. The micronucleated polychromatic erythrocyte cells (mPCEs) were thereafter recorded. The hepatoprotective effects of the plant extracts against 2-AAF-induced liver toxicity in rats were evaluated by monitoring the levels of alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), and histopathological analysis. The results of the 2-AAF-induced liver toxicity experiments showed that rats treated with the plant extracts (100 mg/kg) showed a significant decrease in mPCEs as compared with the positive control. The rats treated with the plant extracts did not show any significant change in the concentration of ALP and GGT in comparison with the negative control group whereas the 2-AAF group showed a significant increase (P < 0.05) in these parameters. Some of the leaf extracts also showed protective effects against histopathological alterations. This study suggests that the leaf extracts have hepatoprotective potential, thereby justifying their ethnopharmacological uses.

Helicobacter pylori: a risk and severity factor of non-steroidal anti-inflammatory drug induced gastropathy.

PubMed Central

Heresbach, D; Raoul, J L; Bretagne, J F; Minet, J; Donnio, P Y; Ramée, M P; Siproudhis, L; Gosselin, M

1992-01-01

This prospective study aimed to determine the prevalence of Helicobacter pylori infection in relation to the occurrence and severity of NSAIDs induced gastropathy. A total of 111 patients were studied-66 were taking NSAIDs and 45 were control patients. All patients underwent endoscopy during which antral biopsy specimens were taken to determine H pylori status (Gram and Giemsa staining, urease test, and cultures). The NSAID group comprised: group I, patients without mucosal damage (n = 28); group II, patients with gastropathy (n = 26); and group III, patients with bleeding associated with NSAID induced gastropathy (n = 12). Control patients had neither dyspeptic symptoms nor endoscopic lesions. There were no differences in age, sex ratio, or presence of H pylori (26% v 24%) between the NSAID and the control groups. Among patients taking NSAIDs, H pylori infection was more frequently (p < 0.02) diagnosed in those who presented with gastropathy (groups II and III: 37%) than in those without lesions (group I: 11%). The frequency of H pylori infection increased significantly with the severity of gastropathy (group I = 11%; group II = 31%; group III = 50%; p < 0.03). H pylori infection was associated with chronic active gastritis (group I = 21%; group II = 35%; group III = 67%; p < 0.05). These data suggest that H pylori may be a risk factor of NSAID induced gastropathy. PMID:1487160

Rapid immunochromatographic diagnosis and Rolling Back Malaria--experiences from an African control program.

PubMed

Durrheim, D N; Govere, J; la Grange, J J; Mabuza, A

2001-01-01

Malaria is a re-emerging disease in much of Africa. In response, the World Health Organization launched the Roll Back Malaria (RBM) initiative. One of six key principles adopted is the early detection of malaria cases. However, the importance of definitive diagnosis and potential value of field deployment of rapid malaria tests in RBM has been largely ignored. The Lowveld Region of Mpumalanga Province, South Africa, is home to a predominantly non-immune population, of approximately 850000 inhabitants, who are at risk of seasonal Plasmodium falciparum malaria. Malaria treatment in this area is usually only initiated on detection of malaria parasites in the peripheral bloodstream, as many other rickettsial and viral febrile illness mimic malaria. The malaria control programme traditionally relied on light microscopy of Giemsa-stained thick blood films for malaria diagnosis. This review summarizes operational research findings that led to the introduction of rapid malaria card tests for primary diagnosis of malaria throughout the Mpumalanga malaria area. Subsequent operational research and extensive experience over a four-year period since introducing the ICT Malaria Pf test appears to confirm the local appropriateness of this diagnostic modality. A laboratory is not required and clinic staff are empowered to make a prompt definitive diagnosis, limiting delays in initiating correct therapy. The simple, accurate and rapid non-microscopic means now available for diagnosing malaria could play an important role in Rolling Back Malaria in selected areas.

Can hemozoin alone cause host anaemia?

PubMed

Sun, Jun; Wang, Su-Wen; Jin, Chang-Long; Zeng, Xiao-Li; Piao, Xing-Yu; Bai, Ling; Tang, Dan-Li; Ji, Chang-Le

2016-12-01

Both schistosomes and malaria parasites produce hemozoin and cause host anaemia. However, the relationship between anaemia and hemozoin is unclear. Although some studies have proposed that hemozoin is related to anaemia in malaria patients, whether hemozoin alone can cause anaemia in patients infected by malaria parasites or schistosomes is uncertain. To investigate the effect of hemozoin on hosts, β-haematin was injected intravenously to normal mice. Then, liver and spleen tissues were observed. Mouse blood was examined. Red blood cells (RBCs), white blood cells (WBCs) and haemoglobin were analysed. Macrophage changes in the spleens and marrow cells were compared using immunofluorescence and H&E or Giemsa stain, respectively. We found that after 15 injections of β-haematin, a large amount of β-haematin was observed to deposit in the livers and spleens. Splenomegaly and bone marrow mild hyperplasia were detected. The average number of RBCs, average number of WBCs and average concentration of haemoglobin decreased significantly from 9.36 × 10 12 cells/L to 8.7 × 10 12 cells/L, 3.8 × 10 9 cells/L to 1.7 × 10 9 cells/L and 142.8 g/L to 131.8 g/L, respectively. In specific, the number of macrophages in the spleens greatly increased after β-haematin infection. The results showed that injections of β-haematin alone can cause anaemia possibly through hypersplenism.

Preliminary characterization of a virus causing infectious anemia among stocks of salmonid fish in the western United States

USGS Publications Warehouse

Arakawa, C.K.; Hursh, D.A.; Lannan, C.N.; Rohovec, J.S.; Winton, J.R.; Ahne, Winfried; Kurstak, E.

1989-01-01

Since 1982, anemias occurring in stocks of yearling coho (Oncorhynchus kisutch) and chinook salmon (Oncorhynchus tshawytscha) have been associated with serious losses at hatcheries in the Pacific Northwest, USA. The anemia is often accompanied by infections with external fungus (e.g. Saprolegnia) or the bacterial pathogens Cytophaga psychrophila or Renibacterium salmoninarum (Holt and Rohovec 1984, Leek 1987). The losses associated with the anemia are thought to be caused by these secondary infections.Blood smears that were made from anemic fish and stained with Giemsa or pinacyanol chloride showed erythrocytic inclusions ranging in size from 1 to 8 um and varying in number per cell. Thin sections of infected red blood cells (RBC) examined by electron microscopy revealed virus particles approximately 70 nm in diameter. The virions were scattered in the cytoplasm of the RBC or contained within membrane bound organelles. These virus particles were morphologically distinct from the iridovirus, erythrocytic necrosis virus (ENV), which is also associated with anemia (Holt and Rohovec 1984, Leek 1987). Evidence suggests that the etiological agent of this new anemic disease, termed erythrocytic inclusion body syndrome (EIBS) by Leek (1987), is a previously undescribed virus infecting salmon. The purpose of this study was to experimentally transmit the disease to healthy fish, to determine the blood parameters associated with infection, and to investigate the nature of the virus associated with EIBS.

Trypanosoma cf. varani in an imported ball python (Python reginus) from Ghana.

PubMed

Sato, Hiroshi; Takano, Ai; Kawabata, Hiroki; Une, Yumi; Watanabe, Haruo; Mukhtar, Maowia M

2009-08-01

Peripheral blood from a ball python (Python reginus) imported from Ghana was cultured in Barbour-Stoenner-Kelly (BSK) medium for Borrelia spp. isolation, resulting in the prominent appearance of free, and clusters of, trypanosomes in a variety of morphological forms. The molecular phylogenetic characterization of these cultured trypanosomes, using the small subunit rDNA, indicated that this python was infected with a species closely related to Trypanosoma varani Wenyon, 1908, originally described in the Nile monitor lizard (Varanus niloticus) from Sudan. Furthermore, nucleotide sequences of glycosomal glyceraldehyde-3-phosphate dehydrogenase gene of both isolates showed few differences. Giemsa-stained blood smears, prepared from the infected python 8 mo after the initial observation of trypanosomes in hemoculture, contained trypomastigotes with a broad body and a short, free flagellum; these most closely resembled the original description of T. varani, or T. voltariae Macfie, 1919 recorded in a black-necked spitting cobra (Naja nigricollis) from Ghana. It is highly possible that lizards and snakes could naturally share an identical trypanosome species. Alternatively, lizards and snakes in the same region might have closely related, but distinct, Trypanosoma species as a result of sympatric speciation. From multiple viewpoints, including molecular phylogenetic analyses, reappraisal of trypanosome species from a wide range of reptiles in Africa is needed to clarify the relationship of recorded species, or to unmask unrecorded species.

Selective induction of apoptosis in human gastric cancer cells by Lactobacillus kefiri (PFT), a novel kefir product

PubMed Central

GHONEUM, MAMDOOH; FELO, NOURAN

2015-01-01

The present study was undertaken to evaluate the effect of Lactobacillus kefiri (PFT), a novel kefir product, on apoptosis of gastric cancer cells (AGS), breast cancer cells (4T1), and human peripheral blood mononuclear cells (PBMCs). Cells were cultured with PFT and apoptosis was determined by flow cytometry using 7-AAD dye and cytospin preparation. Mitochondrial dysfunction and expression of Bcl2 were monitored by flow cytometry. Results showed that PFT induced apoptosis in AGS gastric cancer cells in a dose-dependent manner. Apoptosis was detected at a concentration of 0.3 mg/ml (20.8%), increased to 25.8% at 0.6 mg/ml, 37% at 1.2 mg/ml, 53.1% at 2.5 mg/ml, and peaked at 66.3% at 5.0 mg/ml. Apoptosis is associated with the decreased polarization of mitochondrial membrane potential (MMP) and decreased Bcl2 expression. PFT-treated AGS cells manifested membrane blebbing, nuclear condensation, and fragmentation as identified in cytospin cytocentrifuge Giemsa stained preparations. On the other hand, flow cytometry analysis showed that PFT did not induce apoptosis in 4T1 breast cancer cells nor in PBMCs. These results suggest that PFT is safe for white blood cells and selectively induces apoptotic effects in gastric cancer cells. Hence, it may have potential as a therapeutic agent for the treatment of gastric cancers. PMID:26251956

Peroxisome proliferator-activated receptor-γ is expressed in eosinophils in nasal polyps.

PubMed

Asaka, Chikara; Honda, Kohei; Ito, Eiko; Fukui, Naoko; Chihara, Junichi; Ishikawa, Kazuo

2011-01-01

Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptors, which regulate fatty acid metabolites. One of the natural ligands for PPARγ is 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a major metabolite of prostaglandin D(2) (PGD(2)). Recently, PPARγ has been shown to play an important role in anti-inflammatory reactions, including within-airway allergic diseases, in a mouse model. Our aim was to clarify the expression and localization of PPARγ and PGD(2) synthase, which produces ligands of PPARγ, in nasal polyps by immunohistochemical analysis. Nasal polyps of chronic rhinosinusitis patients (6 asthmatic patients and 6 nonasthmatic patients) were obtained during surgery. May-Grünwald-Giemsa staining was performed to evaluate the eosinophil infiltration of the polyps. To identify the cells expressing PPARγ protein and PGD(2) synthase, double immunostaining was performed using anti-PPARγ antibody, monoclonal antileukocyte antibodies, and PGD(2) synthase antibody. The number of eosinophils and the number of PPARγ-positive cells in the nasal polyps of the asthmatic patients were significantly higher than those in the nonasthmatic patients. PPARγ was expressed on eosinophils and T cells of the infiltrating cells in the nasal polyps. PGD(2) synthase was also expressed on PPARγ-positive cells. PPARγ is involved in nasal polyposis pathogenesis, acting on eosinophils and T cells. Copyright © 2011 S. Karger AG, Basel.

Karyotype morphology suggests that the Nyctibius griseus (Gmelin, 1789) carries an ancestral ZW-chromosome pair to the order Caprimulgiformes (Aves)

PubMed Central

Nieto, Leonardo Martin; Kretschmer, Rafael; Ledesma, Mario Angel; Garnero, Analía Del Valle; Gunski, Ricardo José

2012-01-01

Abstract Studies of karyotypes have been revealing important information on the taxonomic relationships and evolutionary patterns in various groups of birds. However, the order Caprimulgiformes is one of the least known in terms of its cytotaxonomy. So far, there are no cytogenetic data in the literature on birds belonging to 3 of 5 families of this order -Nyctibiidae, Steatornithidae and Aegothelidae. For this reason, the aim of our study was to describe the karyotype of Nyctibius griseus (Gmelin, 1789) (Aves, Nyctibiidae, Caprimulgiformes) and contribute with new data that could help to clarify the evolutionary relationships in this group. Bone marrow was cultured directly to obtain material for the chromosome study. C-banding was used to visualize the constitutive heterochromatin and Ag-NOR-banding to reveal nucleolus organizer regions. The diploid number observed was 2n=86±. Using sequential Giemsa/C-banding staining, we determined that the W chromosome was entirely C-band positive with the two most prominent markers in the interstitial and distal regions of the long arm. The nucleolus organizer regions showed a typical location in a pair of microchromosomes that exhibited Ag-NOR.The results obtained for Nyctibius griseus suggest that, of all the species studied in the references cited, it has the most ancestral sex chromosome composition of the order Caprimulgiformes. PMID:24260678

In vitro evaluation of photodynamic therapy using curcumin on Leishmania major and Leishmania braziliensis.

PubMed

Pinto, Juliana Guerra; Fontana, Letícia Correa; de Oliveira, Marco Antonio; Kurachi, Cristina; Raniero, Leandro José; Ferreira-Strixino, Juliana

2016-07-01

Cutaneous leishmaniasis is an infectious disease caused by the Leishmania protozoan. The conventional treatment is long-lasting and aggressive, in addition to causing harmful effect. Photodynamic therapy has emerged as a promising alternative treatment, which allows local administration with fewer side effects. This study investigated the photodynamic activity of curcumin on Leishmania major and Leishmania braziliensis promastigote. Both species were submitted to incubation with curcumin in serial dilutions from 500 μg/ml up to 7.8 μg/ml. Control groups were kept in the dark while PDT groups received a fluency of 10 J/cm(2) at 450 nm. Mitochondrial activity was assessed by MTT assay 18 h after light treatment, and viability was measured by Trypan blue dye exclusion test. Morphological alterations were observed by Giemsa staining. Confocal microscopy showed the uptake of curcumin by both tested Leishmania species. Mitochondrial activity was inconclusive to determine viability; however, Trypan blue test was able to show that curcumin photodynamic treatment had a significant effect on viability of parasites. The morphology of promastigotes was highly affected by the photodynamic therapy. These results indicated that curcumin may be a promising alternative photosensitizer, because it presents no toxicity in the dark; however, further tests in co-culture with macrophages and other species of Leishmania should be conducted to determine better conditions before in vivo tests are performed.

Sequence analysis of DBL2β domain of vargene of Indonesian Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Sulistyaningsih, E.; Romadhon, B. D.; Palupi, I.; Hidayah, F.; Dewi, R.; Prasetyo, A.

2018-03-01

Malaria is a major health problem in tropical countries including Indonesia. The most deadly agent is Plasmodium falciparum. In P. falciparum infection, PfEMP1 is supposed to play an important role in the pathogenesis of malaria. PfEMP1 is encoded by var gene family, it is a polymorphic protein where the extra-cellular portion contains of three distinct binding domains: Duffy binding-like (DBL), Cysteine-rich interdomain regions (CIDR) and C2. PfEMP1 varies in domain composition and binding specificity. The study explored the characteristic of Indonesian DBL2β-var genes and investigated its role to the malaria outcome. Twenty blood samples from clinically mild to severe malaria patients in Jember, East Java were collected for DNA extraction. Diagnosis was confirmed by Giemsa-stained thick blood smear. PCR was conducted using specific primer targeting on the full-length of DBL2ß and resulted approximately single band of 1,7 kb in a sample. This band was observed only from severe malaria sample. Sequence analysis directly from PCR product showed 74-99% similarities with previous sequences in Gene Bank. In conclusion, the DBL2β domain of vargene of Indonesian isolates was 1603 nucleotides in length and there was a possible association of the existence of DBL2β domain with the severity of malaria outcome.

Cultivation of Anaplasma ovis in the HL-60 human promyelocytic leukemia cell line.

PubMed

Wei, Ran; Liu, Hong-Bo; Jongejan, Frans; Jiang, Bao-Gui; Chang, Qiao-Cheng; Fu, Xue; Jiang, Jia-Fu; Jia, Na; Cao, Wu-Chun

2017-09-20

The tick-borne bacterium Anaplasma ovis is a widely distributed pathogen affecting sheep, goats and wild ruminants. Here, the HL-60 human promyelocytic leukemia cell line was used to isolate A. ovis from PCR-positive sheep and goats in Heilongjiang Province, China. Two weeks after inoculation, morulae were observed in cytoplasmic vacuoles in four different HL-60 cultures. Confocal microscopy using a Cy3-labeled A. ovis-specific probe confirmed that the HL-60 cells were infected with A. ovis. Cells from the 6th HL-60 subculture displayed positive fluorescence when incubated with A. ovis antiserum in the indirect fluorescent antibody assay. PCR amplification and sequencing of 16S rRNA, groEL, gltA, msp2 and msp4 Anaplasma genes revealed that the four A. ovis culture isolates were identical. Phylogenetic analysis showed that the sequences clustered with other A. ovis strains but could clearly be distinguished from other Anaplasma species. When the 18th subculture of infected HL-60 cells was examined by electron microscopy, lysosomes were often observed near the vacuoles. After the 24th subculture, Giemsa staining and PCR indicated that the HL-60 cells were negative for A. ovis. Although A. ovis can infect HL-60 cells for only four months, the ability of the organism to infect and multiply in HL-60 cells provides a tool to study intra-erythrocytic Anaplasma and host cell interactions.

[LE cells in synovial fluid: prevalence and diagnostic usefulness in rheumatic diseases].

PubMed

Puszczewicz, Mariusz; Białkowska-Puszczewicz, Grazyna

2010-01-01

This study was undertaken to determine the prevalence of LE cells in synovial fluid and their importance for the diagnosis of rheumatic disease. Synovial fluid was obtained from 631 patients: 31 with systemic lupus erythematosus (SLE), 337 with rheumatoid arthritis (RA), 4 with Still's disease, 9 with systemic scleroderma (SS), 27 with the overlap syndrome (RA/SLE), 132 with ankylosing spondylitis (AS), 57 with Reiter's syndrome, and 34 with psoriatic arthritis (PA). The fluid was centrifuged, precipitate smears were done and were May-Grünwald-Giemsa stained for cytologic assessment. The supernatant was collected for antinuclear antibody (ANA) testing. Physicochemical and serologic properties of the synovial fluid were routinely determined. All synovial fluids demonstrated signs of inflammation. The presence of LE cells was ascertained in five patients with SLE and nine patients with the overlap syndrome. In these cases, LE cells were accompanied by ANA. In addition, hematoxylin bodies were revealed in SLE patients. LE cells were observed in 2.6% of patients with RA but were not accompanied by ANA. Patients with SS, Still's disease, AS, Reiter's syndrome, and PA tested negative for LE cells. It appears from these results that LE cells are rarely present in the synovial fluid of patients with rheumatic diseases. In contrast, they occur in more than 40% of patients with the overlap syndrome and may thus be regarded as important for the diagnosis of this condition.

Establishment of Mouse Model of MYH9 Disorders: Heterozygous R702C Mutation Provokes Macrothrombocytopenia with Leukocyte Inclusion Bodies, Renal Glomerulosclerosis and Hearing Disability

PubMed Central

Suzuki, Nobuaki; Kunishima, Shinji; Ikejiri, Makoto; Maruyama, Shoichi; Sone, Michihiko; Takagi, Akira; Ikawa, Masahito; Okabe, Masaru; Kojima, Tetsuhito; Saito, Hidehiko; Naoe, Tomoki; Matsushita, Tadashi

2013-01-01

Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. MYH9 disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation. We generated knock-in mice expressing the Myh9 R702C mutation. R702C knock-in hetero mice (R702C+/− mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/− mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May–Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm. In other organs, R702C+/− mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response. These findings indicate that Myh9 R702C knock-in mice mirror features of human MYH9 disorders arising from the R702C mutation. PMID:23976996

Establishment of mouse model of MYH9 disorders: heterozygous R702C mutation provokes macrothrombocytopenia with leukocyte inclusion bodies, renal glomerulosclerosis and hearing disability.

PubMed

Suzuki, Nobuaki; Kunishima, Shinji; Ikejiri, Makoto; Maruyama, Shoichi; Sone, Michihiko; Takagi, Akira; Ikawa, Masahito; Okabe, Masaru; Kojima, Tetsuhito; Saito, Hidehiko; Naoe, Tomoki; Matsushita, Tadashi

2013-01-01

Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. MYH9 disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation. We generated knock-in mice expressing the Myh9 R702C mutation. R702C knock-in hetero mice (R702C+/- mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/- mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May-Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm. In other organs, R702C+/- mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response. These findings indicate that Myh9 R702C knock-in mice mirror features of human MYH9 disorders arising from the R702C mutation.

Molecular identification and phylogenetic analysis of Dipetalonema evansi (LEWIS, 1882) in camels (Camelus dromedarius) of Iran.

PubMed

Sazmand, Alireza; Eigner, Barbara; Mirzaei, Mohammad; Hekmatimoghaddam, Seyedhossein; Harl, Josef; Duscher, Georg Gerhard; Fuehrer, Hans-Peter; Joachim, Anja

2016-04-01

Despite the economic importance of camels, the parasites that affect them have not received adequate attention so far and molecular studies are scarce compared to other livestock. In this study, we characterized peripheral blood microfilariae in 200 healthy one-humped camels (Camelus dromedarius) from south-east Iran by microscopy and molecular tools to receive a more detailed insight into prevalence and species that affect them. Moreover, adult specimens of the filarial nematode Dipetalonema evansi were collected from the carcass of an infected animal. Microscopic examination was performed on Giemsa-stained blood smears, and blood was also spotted on Whatman FTA(®) cards for DNA analysis. Genomic DNA was extracted, and PCR was carried out for the detection of filaroid helminths, followed by sequence analysis of positive samples. Four samples were positive for microfilariae by microscopy, while 16 animals (8 %) were positive by PCR. Sequence analysis revealed D. evansi in all cases. Phylogenetic analysis of a cytochrome C oxidase subunit I (COI) sequence of filaroid nematodes showed that most species in a single genus cluster in the same clade; however, D. evansi and D. gracile are not monophyletic and branch rather at the base of the tree. Further studies on the life cycle of D. evansi, specifically the identification of intermediate host(s), have become feasible with the provision of the first specific COI sequences in this study.

Distinct classical and molecular cytogenetics of Astyanax marionae and A. fasciatus (Characiformes: Characidae): a comparative study of the organization of heterochromatin and repetitive genes.

PubMed

Piscor, Diovani; Centofante, Liano; Parise-Maltempi, Patricia Pasquali

2017-09-01

Genus Astyanax is well distributed in Neotropical freshwater environments and its taxonomic position is uncertain, as is the case with other Characidae genera allocated in the group incertae sedis. This study aimed to analyse the karyotype of different populations of Astyanax fasciatus (Corumbataí River basin) using Giemsa staining, C-band technique, and fluorescence in situ hybridization for the H3 histone and 5S rRNA genes, in addition we describe for the first time the chromosomal organization of H3 histone and 5S rRNAgenes in A. marionae (ParaguayRiver basin). Chromosomes of three A. fasciatus populations were analysed (two with 2n = 50 and one with 2n = 48) and the heterochromatin was organized in two forms (blocks with blurred boundaries and distinct blocks). H3 histone and 5S rRNA genes were observed in all the three populations of A. fasciatus on two chromosome pairs (one metacentric chromosome showing H3 histone and 5S rRNA gene clusters). In A. marionae (2n = 48), H3 histone and 5S rRNA genes were observed in one acrocentric chromosome pair (different pairs). Further, differences between karyotypes and heterochromatin, as well as the chromosomal organization of H3 histone and 5S rRNA genes in Astyanax species, focussing on chromosome evolution in the group are discussed.

The first report of Anaplasma phagocytophilum and a novel Theileria spp. co-infection in a South African giraffe.

PubMed

Zhang, Yan; Li, Tongyi; Cui, Yanyan; Wang, Jinhong; Lv, Yali; Wang, Rongjun; Jian, Fuchun; Zhang, Longxian; Wang, Jiantang; Yang, Guangcheng; Ning, Changshen

2016-08-01

Organisms of the genera Anaplasma and Theileria are important intracellular bacteria and parasites that cause various tick-borne diseases, threatening the health of numerous animals as well as human beings. In the present study, a 12-month-old male wild South African giraffe (Giraffa camelopardalis giraffa) originating from South Africa, and living in Zhengzhou Zoo (located in the urban district of Zhengzhou in the provincial capital of Henan), suddenly developed an unknown fatal disease and died 1day after the onset of the clinical signs. By microscopic examination of Giemsa-stained blood smears combined with nested PCR and DNA sequence analysis, Anaplasma phagocytophilum, Anaplasma bovis and a novel Theileria spp. were found in the blood of this giraffe. The six other Cervidae animals in the zoo and three ruminants living in the same colony house with them were found to be negative for both Anaplasma and Theileria in their blood specimens. We report on the first case of an A. phagocytophilum infection and the occurrence of a novel Theileria spp. in the blood of a giraffe. This is the first reported case of a multi-infection of A. bovis, A. phagocytophilum and Theileria spp. in a giraffe, as revealed by microscopic examination of blood smears and the results of nested PCR and DNA sequencing. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

First microscopical and molecular-based characterization of Leishmania major within naturally infected Phlebotomus salehi (Diptera; Psychodidae) in Fars province, southern Iran

PubMed Central

DAVAMI, M H; MOTAZEDIAN, M H; KALANTARI, M; ASGARI, Q; BADZOHRE, A; MOHAMMADPOUR, I

2011-01-01

Zoonotoc cutaneous leishmaniasis is endemic in several parts of Iran. Jahrom district is one of the most important endemic foci of leishmaniasis located in Fars province, southern Iran. To identify the vectors of leishmaniasis in this area, a total of 349 sandflies were collected during May to August 2009. They were caught from outdoors in five regions of Jahrom district including villages of Mousavieh, Ghotb-Abad, Heydar-Abad, Fath-Abad and Jahrom County. Eleven species of Phlebotomine (three Phlebotomus spp. and eight Sergentomyia spp.) were detected. To determine the sandflies naturally infected by Leishmania spp., 122 female sandflies were dissected and evaluated microscopically using Giemsa-stained slides. Natural infection of 2 out of 38 (5.26%) P. papatasi and 1 out of 8 (12.5%) P. salehi to Leishmania major was confirmed in the region. Sequencing and nested polymerase chain reaction-based detection of Leishmania were carried out to confirm the microscopic findings. Five (13.16%) P. papatasi and two (25%) P. salehi were positive in nested polymerase chain reaction assay. All positive samples were shown 72–76% similarity with L. major Friedlin. On the basis of our knowledge, this is the first molecular detection of L. major within naturally infected P. salehi in this region in southern Iran. PMID:22185942

[Prevalence of Bancroftian filariasis in seven villages of the Bonassama Health District in the Wouri Estuary, littoral province of Cameroon].

PubMed

Moyou-Somo, R; Ouambe, M Antoine; Fon, E; Bema, J

2003-01-01

Lymphatic filariasis is one of the 6 diseases targeted for global eradication by the World Health Organization. In 2000 Cameroon was selected for inclusion in the eradication program. As a prerequisite for the program, epidemiological mapping was undertaken to determine the prevalence of Wuchereria bancrofti in 7 villages located in the mangrove area of the Wouri estuary littoral province of Cameroon. Informed consent was obtained from each participant or from parents of minors. Night blood specimens were collected for thick and thin blood films, stained using the Giemsa solution, and microscopically examined to identify microfilariae of W. bancrofti. The study population included 924 subjects (336 males and 558 females). Their age ranged from 1 to 79 years (mean, 26.9 years). W. bancrofti was identified in 4 of the 7 villages with prevalence rates ranging from 0.7% to 3.25% (mean, 0.97%). All positive subjects were over 30 years old. Despite night blood specimen collection, other blood microfilariae were detected including Loa loa in 54 cases (prevalence, 5.84%) and Dipetalonema perstans in 10 (prevalence, 1.1%). Only a small number of subjects presented clinical manifestations of lymphatic filariasis (e.g., pruritus). Elephantiasis of the lower limb was identified in only one subject but there was hearsay evidence of other cases involving subjects embarrassed to come forward.

First description of the karyotype and localization of major and minor ribosomal genes in Rhoadsia altipinna Fowler, 1911 (Characiformes, Characidae) from Ecuador

PubMed Central

Sánchez-Romero, Omar; Abad, César Quezada; Cordero, Patricio Quizhpe; de Sene, Viviani França; Nirchio, Mauro; Oliveira, Claudio

2015-01-01

Abstract Karyotypic features of Rhoadsia altipinna Fowler, 1911 from Ecuador were investigated by examining metaphase chromosomes through Giemsa staining, C-banding, Ag-NOR, and two-color-fluorescence in situ hybridization (FISH) for mapping of 18S and 5S ribosomal genes. The species exhibit a karyotype with 2n = 50, composed of 10 metacentric, 26 submetacentric and 14 subtelocentric elements, with a fundamental number FN=86 and is characterized by the presence of a larger metacentric pair (number 1), which is about 2/3 longer than the average length of the rest of the metacentric series. Sex chromosomes were not observed. Heterochromatin is identifiable on 44 chromosomes, distributed in paracentromeric position near the centromere. The first metacentric pair presents two well-defined heterochromatic blocks in paracentromeric position, near the centromere. Impregnation with silver nitrate showed a single pair of Ag-positive NORs localized at terminal regions of the short arms of the subtelocentric chromosome pair number 12. FISH assay confirmed these localization of NORs and revealed that minor rDNA clusters occur interstitially on the larger metacentric pair number 1. Comparison of results here reported with those available on other Characidae permit to hypothesize that the presence of a very large metacentric pair might represent a unique and derived condition that characterize one of four major lineages molecularly identified in this family. PMID:26140168

Genetic variation of pfhrp2 in Plasmodium falciparum isolates from Yemen and the performance of HRP2-based malaria rapid diagnostic test.

PubMed

Atroosh, Wahib M; Al-Mekhlafi, Hesham M; Al-Jasari, Adel; Sady, Hany; Al-Delaimy, Ahmed K; Nasr, Nabil A; Dawaki, Salwa; Abdulsalam, Awatif M; Ithoi, Init; Lau, Yee Ling; Fong, Mun Yik; Surin, Johari

2015-07-22

The genetic variation in the Plasmodium falciparum histidine-rich protein 2 (pfhrp2) gene that may compromise the use of pfhrp2-based rapid diagnostic tests (RDTs) for the diagnosis of malaria was assessed in P. falciparum isolates from Yemen. This study was conducted in Hodeidah and Al-Mahwit governorates, Yemen. A total of 622 individuals with fever were examined for malaria by CareStart malaria HRP2-RDT and Giemsa-stained thin and thick blood films. The Pfhrp2 gene was amplified and sequenced from 180 isolates, and subjected to amino acid repeat types analysis. A total of 188 (30.2%) participants were found positive for P. falciparum by the RDT. Overall, 12 different amino acid repeat types were identified in Yemeni isolates. Six repeat types were detected in all the isolates (100%) namely types 1, 2, 6, 7, 10 and 12 while types 9 and 11 were not detected in any of the isolates. Moreover, the sensitivity and specificity of the used PfHRP2-based RDTs were high (90.5% and 96.1%, respectively). The present study provides data on the genetic variation within the pfhrp2 gene, and its potential impact on the PfHRP2-based RDTs commonly used in Yemen. CareStart Malaria HRP2-based RDT showed high sensitivity and specificity in endemic areas of Yemen.

Malaria rapid diagnostic test in children: The Zamfara, Nigeria experience.

PubMed

Abdulkadir, Isa; Rufai, Hafsah Ahmad; Ochapa, Sunday Onazi; Malam, Mado Sani; Garba, Bilkisu Ilah; Oloko, Adebayo Ganiyu Yusuf; George, Idemudia Itoya

2015-01-01

Malaria remains a major cause of under-five morbidity and mortality in Nigeria, and prompt diagnosis occupies a strategic position in its management. Malaria rapid diagnostic test (RDT), a nontechnical, easy to perform test promises to meet this need. It is important to locally document the usefulness of the use of RDT in making prompt malaria diagnosis in children. To determine the prevalence of malaria and evaluate the diagnostic performance of malaria RDT kit in febrile under-five children presenting to a Tertiary Health Facility in Gusau, North-Western Nigeria. A cross-sectional study of children aged 6-59 months, evaluated for malaria in a tertiary health facility from August 2012 to January 2013. Information was obtained from care providers of all subjects with fever and a presumptive diagnosis of malaria. All subjects were investigated using Giemsa stain microscopy and Carestart™ malaria RDT. The prevalence of malaria in 250 febrile under-five children was 54%. Three-quarter (79%) of the children received inappropriate nonrecommended antimalaria prior to their presentation, including 20% who received chloroquine. The overall sensitivity of RDT was 40.3%. The specificity, positive and negative predictive values were 89.6%, 81.8%, and 56.5%, respectively. Use of RDT should be encouraged for screening and diagnosis using a protocol such that febrile children with positive RDT results are confirmed as having malaria while those with negative results are further evaluated using microscopy.

Comparative Analysis Reveals Potential Utility of Digital Microscopy in the Evaluation of Peripheral Blood Smears With Some Barriers to Implementation.

PubMed

Gomez-Gelvez, Juan C; Kryvenko, Oleksandr N; Chabot-Richards, Devon S; Foucar, Kathryn; Inamdar, Kedar V; Karner, Kristin H

2015-07-01

Evaluation of the peripheral blood smear (PBS) is an essential diagnostic test in current medical practice. We aimed to evaluate the use of digital microscopy for the examination of PBS as an option to provide expert interpretation to remote sites and in "on-call" situations. We collected 100 Wright-Giemsa-stained PBS slides representing normal and abnormal findings seen at a community-based hospital. Four hematopathologists independently evaluated the cases using conventional light and digital microscopy. When comparing digital vs light microscopy, most of the cellular features evaluated showed at least a moderate degree of agreement in at least three of the reviewers. Discrepancies in final diagnosis were identified in a minority of the cases, most of which were attributed to the poorer resolution of digital microscopy at high magnification (×400). These results support the limited use of digital microscopy for evaluation and triage of peripheral blood smears as a practical option to obtain expert opinion in locations where experienced staff is not available on site. Our results indicate that while digital microscopy is well suited for basic triage of these blood smears, limitations in quality of imaging at higher magnification as well as large file size may limit its utility in certain settings and situations. Copyright© by the American Society for Clinical Pathology.

Morphologic examination of CD3-CD4(bright) cells in rat liver.

PubMed

Yamamoto, Satoshi; Sato, Yosinobu; Abo, Toru; Hatakeyama, Katsuyosi

2002-01-01

Recently, we found CD3-CD4(bright) cells with comparative specificity for normal rat liver. In the current study, we investigated the type and form of both CD3-CD4(bright) cells and CD3-CD4(dull) cells in the rat liver. The surface phenotype of hepatic mononuclear cells in Lewis rats was identified by using monoclonal antibodies including anti-CD4, anti-CD3, and antimacrophage in conjunction with two- or three-color immunofluorescence analysis. CD3-CD4(bright) cells and CD3-CD4(dull) cells were examined morphologically using May-Giemsa staining and scanning electron microscopy. The distribution of CD3-CD4(bright) cells and CD3-CD4(dull) cells 48 hours after intravenous administration of liposome-encapsulated dichloromethylene diphosphate was also investigated. In comparison to CD3-CD4(dull) cells, CD3-CD4(bright) cells were slightly larger macrophages with abundant cytoplasmic granules, being present with comparative specificity for normal rat liver and showing negligible effects by intravenous liposome-encapsulated dichloromethylene diphosphate administration. These data suggest that in normal young rat liver these CD3-CD4(dull) and CD3-CD4(bright) cells may be dendritic cells and Kupffer cells that shift from the liver to the spleen or vice versa. These cells may also be able to locally proliferate in liver or spleen due to changes in the developing liver.

Prognostic impact of circulating plasma cells in patients with multiple myeloma: implications for plasma cell leukemia definition

PubMed Central

Granell, Miquel; Calvo, Xavier; Garcia-Guiñón, Antoni; Escoda, Lourdes; Abella, Eugènia; Martínez, Clara Mª; Teixidó, Montserrat; Gimenez, Mª Teresa; Senín, Alicia; Sanz, Patricia; Campoy, Desirée; Vicent, Ana; Arenillas, Leonor; Rosiñol, Laura; Sierra, Jorge; Bladé, Joan; de Larrea, Carlos Fernández

2017-01-01

The presence of circulating plasma cells in patients with multiple myeloma is considered a marker for highly proliferative disease. In the study herein, the impact of circulating plasma cells assessed by cytology on survival of patients with multiple myeloma was analyzed. Wright-Giemsa stained peripheral blood smears of 482 patients with newly diagnosed myeloma or plasma cell leukemia were reviewed and patients were classified into 4 categories according to the percentage of circulating plasma cells: 0%, 1–4%, 5–20%, and plasma cell leukemia with the following frequencies: 382 (79.2%), 83 (17.2%), 12 (2.5%) and 5 (1.0%), respectively. Median overall survival according to the circulating plasma cells group was 47, 50, 6 and 14 months, respectively. At multivariate analysis, the presence of 5 to 20% circulating plasma cells was associated with a worse overall survival (relative risk 4.9, 95% CI 2.6–9.3) independently of age, creatinine, the Durie-Salmon system stage and the International Staging System (ISS) stage. Patients with ≥5% circulating plasma cells had lower platelet counts (median 86×109/L vs. 214×109/L, P<0.0001) and higher bone marrow plasma cells (median 53% vs. 36%, P=0.004). The presence of ≥5% circulating plasma cells in patients with multiple myeloma has a similar adverse prognostic impact as plasma cell leukemia. PMID:28255016

Parasitological and molecular diagnostic of a clinical Babesia caballi outbreak in Southern Romania.

PubMed

Ionita, Mariana; Nicorescu, Isabela Madalina; Pfister, Kurt; Mitrea, Ioan Liviu

2018-05-15

Equine piroplasmosis (EP) is a tick-borne disease of equids caused by Babesia caballi and/or Theileria equi, which is endemic in many tropical and temperate areas of the world. However, clinical outbreaks of EP in Romania during the last decades have not been reported Therefore, the aim of this paper is (i) to describe a clinical B. caballi outbreak in horses on several farms in Southern Romania using a diagnostic and therapeutic approach and (ii) the molecular diagnostic of EP in an endemic area of Romania. In the first case, a 10-month-old stallion male was presented with lethargy, anorexia, fever (40.9 °C), pale mucosal/mucous/membranes and a marked anemia. In the subsequent weeks, three horses from other farms located in the same area, displayed similar clinical signs. B. caballi was diagnosed in all the horses based on Giemsa-stained blood smears and the diagnosis was further confirmed by polymerase chain reaction (PCR), using a single-round and multiplex PCR and sequencing. All four horses were treated with imidocarb dipropionate, at a dose rate of 2.2 mg/kg body weight (two injections at 48 h apart), and all horses clinically recovered within 24-48 h, post-treatment. This report presents the first molecularly characterized B. caballi outbreak in Romania in clinically affected horses, confirmed by DNA sequencing.

Selective induction of apoptosis in human gastric cancer cells by Lactobacillus kefiri (PFT), a novel kefir product.

PubMed

Ghoneum, Mamdooh; Felo, Nouran

2015-10-01

The present study was undertaken to evaluate the effect of Lactobacillus kefiri (PFT), a novel kefir product, on apoptosis of gastric cancer cells (AGS), breast cancer cells (4T1), and human peripheral blood mononuclear cells (PBMCs). Cells were cultured with PFT and apoptosis was determined by flow cytometry using 7-AAD dye and cytospin preparation. Mitochondrial dysfunction and expression of Bcl2 were monitored by flow cytometry. Results showed that PFT induced apoptosis in AGS gastric cancer cells in a dose-dependent manner. Apoptosis was detected at a concentration of 0.3 mg/ml (20.8%), increased to 25.8% at 0.6 mg/ml, 37% at 1.2 mg/ml, 53.1% at 2.5 mg/ml, and peaked at 66.3% at 5.0 mg/ml. Apoptosis is associated with the decreased polarization of mitochondrial membrane potential (MMP) and decreased Bcl2 expression. PFT-treated AGS cells manifested membrane blebbing, nuclear condensation, and fragmentation as identified in cytospin cytocentrifuge Giemsa stained preparations. On the other hand, flow cytometry analysis showed that PFT did not induce apoptosis in 4T1 breast cancer cells nor in PBMCs. These results suggest that PFT is safe for white blood cells and selectively induces apoptotic effects in gastric cancer cells. Hence, it may have potential as a therapeutic agent for the treatment of gastric cancers.

Preparation of colloidal gold for staining proteins electrotransferred onto nitrocellulose membranes.

PubMed

Yamaguchi, K; Asakawa, H

1988-07-01

This paper describes a simple method of preparing colloidal gold for staining protein blots. Colloidal gold was prepared from 0.005 or 0.01% HAuCl4 by the addition of formalin as a reductant and potassium hydroxide. Staining of small cell carcinoma tissue extract blotted onto nitrocellulose membranes with this colloidal gold solution resulted in the appearance of a large number of clear wine-red bands. The sensitivity of gold staining was 60 times higher than that of Coomassie brilliant blue staining and almost comparable to that of silver staining of proteins in polyacrylamide gel. The sensitivity of this method was also satisfactory in comparison with that of enzyme immunoblotting. The colloidal gold prepared by this method is usable for routine work.

Cutaneous Leishmaniasis

DTIC Science & Technology

2011-06-01

macrophage. Note also a few extracellular organisms (arrows). Giemsa x750 Figure 4.3 Female sandfly ( Lutzomyia longipalpis) taking blood meal from...Old World; Lutzomyia sp (Fig 4.3) and Psychodopygus sp in the New World) and transmitted to a host during a blood meal (Fig 4.4). Promastigotes

Exploring methods for prevention of oxidative stain in soft maple

Treesearch

Michael C. Wiemann; Richard D. Bergman; Mark Knaebe; Scott A. Bowe

2009-01-01

Interior gray enzymatic oxidative stain for white woods such as maple has plagued the wood industry for many years because methods that have been found to reduce stain are hard to scale up to industrial levels. We examined possible alternative treatments to eliminate stain in soft maple (Acer rubrum L.), and found that exposure to sulfur dioxide gas eliminates interior...

Improved Nissl method to stain formaldehyde or glutaraldehyde-fixed material.

PubMed

Böck, P

1979-05-15

Nissl staining of paraffin sections from formaldehyde- or glutaraldehyde-fixed specimens is significantly intensified when sections are kept in a 50% (w/v) aqueous solution of potassium metabisulfite before being stained by a conventional Nissl method.

Evaluation of detection methods for Campylobacter infections among under-fives in Mwanza City, Tanzania

PubMed Central

Mushi, Martha Fidelis; Paterno, Laurent; Tappe, Dennis; Deogratius, Anna Pendo; Seni, Jeremiah; Moremi, Nyambura; Mirambo, Mariam Mwijuma; Mshana, Stephen Eliatosha

2014-01-01

Introduction Campylobacter species are recognized as a major cause of acute gastroenteritis in humans throughout the world. The diagnosis is mainly based on stool culture. This study was done to evaluate the effectiveness of staining methods (Gram stain using 0.3% carbol fuchsin as counter stain and 1% carbol fuchsin direct stain) versus culture as the gold standard. Methods A total of 300 children attending Bugando Medical Centre (BMC) and the Sekou Toure regional hospital with acute watery diarrhea were enrolled. Two sets of slides were prepared stained with 1% carbol fuchsin for 30 seconds first set, and the second set stained with Gram's stain using 0.3% carbol fuchsin as counter stain for five minutes. Concurrently, stool samples were inoculated on Preston Agar selective. Results Of 300 stool specimens, 14(4.7%) showed positive culture after 48 hours of incubation and 28 (9.3%) shows typical morphology of Campylobacter species by both Gram stain and direct stain. The sensitivity of the Gram stain using 0.3% carbol fuchsin as counter stain and 1% carbol fuchsin simple stain versus culture as gold standard was 64.3%, with a specificity of 93.4%. The positive predictive value and negative predictive value were 32.1% and 98.2% respectively. Conclusion The detection of Campylobacter by 1% carbol fuchsin is simple, inexpensive, and fast, with both a high sensitivity and specificity. Laboratories in settings with high prevalence of campylobacteriosis and/or limited resources can employ 1% carbol fuchsin direct stain in detecting campylobacter infections. PMID:25995788

Pericardial fluid Gram stain

MedlinePlus

... stain To use the sharing features on this page, please enable JavaScript. Pericardial fluid Gram stain is a method of staining a sample of fluid taken from the pericardium. This is the sac surrounding ...

Counterion dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic gel digestion of stained protein for mass spectrometry.

PubMed

Cong, Wei-Tao; Wang, Xu; Hwang, Sun-Young; Jin, Li-Tai; Choi, Jung-Kap

2012-01-01

A fast and matrix-assisted laser desorption/ionization mass spectrometry compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon and ethyl violet, to form an ion-pair complex. The protocol, including fixing, staining, and quick washing steps, can be completed in 1-1.5 h, depending upon gel thickness. It has the sensitivity comparable to the colloidal Coomassie Brilliant Blue G stain using phosphoric acid as a component of staining solution (4-8 ng). The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from mass spectrometry. Considering the speed, sensitivity, and compatibility with mass spectrometry, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.

Comparative evaluation of two rapid field tests for malaria diagnosis: Partec Rapid Malaria Test® and Binax Now® Malaria Rapid Diagnostic Test.

PubMed

Nkrumah, Bernard; Acquah, Samuel Ek; Ibrahim, Lukeman; May, Juergen; Brattig, Norbert; Tannich, Egbert; Nguah, Samuel Blay; Adu-Sarkodie, Yaw; Huenger, Frank

2011-05-23

About 90% of all malaria deaths in sub-Saharan Africa occur in children under five years. Fast and reliable diagnosis of malaria requires confirmation of the presence of malaria parasites in the blood of patients with fever or history suggestive of malaria; hence a prompt and accurate diagnosis of malaria is the key to effective disease management. Confirmation of malaria infection requires the availability of a rapid, sensitive, and specific testing at an affordable cost. We compared two recent methods (the novel Partec Rapid Malaria Test® (PT) and the Binax Now® Malaria Rapid Diagnostic Test (BN RDT) with the conventional Giemsa stain microscopy (GM) for the diagnosis of malaria among children in a clinical laboratory of a hospital in a rural endemic area of Ghana. Blood samples were collected from 263 children admitted with fever or a history of fever to the pediatric clinic of the Agogo Presbyterian Hospital. The three different test methods PT, BN RDT and GM were performed independently by well trained and competent laboratory staff to assess the presence of malaria parasites. Results were analyzed and compared using GM as the reference standard. In 107 (40.7%) of 263 study participants, Plasmodium sp. was detected by GM. PT and BN RDT showed positive results in 111 (42.2%) and 114 (43.4%), respectively. Compared to GM reference standard, the sensitivities of the PT and BN RDT were 100% (95% CI: 96.6-100) and 97.2% (95% CI: 92.0-99.4), respectively, specificities were 97.4% (95% CI: 93.6-99.3) and 93.6% (95% CI: 88.5-96.9), respectively. There was a strong agreement (kappa) between the applied test methods (GM vs PT: 0.97; p < 0.001 and GM vs BN RDT: 0.90; p < 0.001). The average turnaround time per tests was 17 minutes. In this study two rapid malaria tests, PT and BN RDT, demonstrated a good quality of their performance compared to conventional GM. Both methods require little training, have short turnaround times, are applicable as well as affordable and can therefore be considered as alternative diagnostic tools in malaria endemic areas. The species of Plasmodium cannot be identified.

Selection and application of exterior stains for wood

Treesearch

R. Sam Williams; William C. Feist

1999-01-01

Exterior stains for wood protect the wood surface from sunlight and moisture. Because stains are formulated to penetrate the wood surface, they are not prone to crack or peel as can film-forming finishes, such as paints. This publication describes the properties of stains and wood, methods for applying stains, and the expected service life of stains.

Fresh and aged human lymphocyte metaphase slides are equally usable for GTG banding.

PubMed

Sajjad, Naheed; Haque, Sayedul; SBurney, Syed Intesar; Shahid, Syed Muhammad; Zehra, Sitwat; Azhar, Abid

2014-09-01

The identification of chromosomes for routine cytogenetic analysis is based on quality of metaphases and good banding pattern. Fresh slides of human lymphocytes have been shown to produce good bands for the identification of chromosomes morphology. G-bands by Trypsin using Giemsa (GTG) banding of aged slides is generally considered hard to get desired band pattern of chromosomes persistently. The current study is focused on GTG banding of aged slides. A total of 340 subjects including 290 primary infertile and 50 fertile were selected. The blood samples were drawn aseptically for cytogenetic analysis. Lymphocytes were cultured and GTG banding was done on 1440 glass slides. Giemsa trypsin banding of aged slides were done by adjusting average trypsin time for each month according to the slide age and metaphase concentration. Correlation analyses showed a significant and positive correlation between slide ageing and trypsin pre-treatment time. The results of this study suggest that, the fresh and aged human lymphocyte metaphases are equally usable for GTG banding.

Automated single-slide staining system

NASA Technical Reports Server (NTRS)

Mills, S. M.; Wilkins, J. R.

1974-01-01

Apparatus developed to Gram-stain single slides automatically is flexible enough to accommodate other types of staining procedures. Method frees operator and eliminates necessity for subjective evaluations as to length of staining or decolorizing time.

Two novel deletions (array CGH findings) in pigment dispersion syndrome.

PubMed

Mikelsaar, Ruth; Molder, Harras; Bartsch, Oliver; Punab, Margus

2007-12-01

We report the first male with pigment dispersion syndrome and a balanced translocation t(10;15)(p11.1;q11.1). Cytogenetic analyses using Giemsa banding and FISH methods, and array CGH were performed. Array CGH analyses did not show altered DNA sequences in the breakpoints of the translocation, but revealed two novel deletions in 2q22.1 and 18q22.1. We suppose that the coexistence of t(10;15) and pigment dispersion syndrome in our patient is a coincidence. The deletion in 2q22.1, where the gene LRP1B has been located, may play a major role in the dysembryogenesis of the eye and cause the disorder.

The Bacterial Endospore Stain on Schaeffer Fulton using Variation of Methylene Blue Solution

NASA Astrophysics Data System (ADS)

Oktari, A.; Supriatin, Y.; Kamal, M.; Syafrullah, H.

2017-02-01

Endospores staining is the type of staining to recognize the presence spore in bacterial vegetative cells. The bacterial endospores need a staining which can penetrate wall thickness of spore bacteria. A method of endospores staining is Schaeffer Fulton method that used Malachite Green. It is an alkaline substance staining that can staining the spore bacteria. In this research, it have found the alternative staining that can replace Malachite Green solution in spore bacterial stain. The alternative staining used is Methylene Blue solution (0,5 %, 0,7%, and 1% concentration) with pH variation (10, 11, and 12), and varyous heating time (3, 4, and 5 minutes). The all treatments staining have been effect on bacterial spores staining results. The warming time greatly affect the dye to penetrate the walls of bacterial spores, this can be seen in the results with various concentration at pH 10, indicates that the not long warm-up time 3 and 4 minutes, bacterial spores are not stained, while in the longer heating time is 5 minutes bacterial spores stained. This is caused because the longer heating time can make the pores of spore wall is open so that can facilitate the dye to get into the bacterial spores.

Efficacy of evaluation of rooster sperm morphology using different staining methods.

PubMed

Lukaszewicz, E; Jerysz, A; Partyka, A; Siudzińska, A

2008-12-01

This work focused on inexpensive methods of evaluation fowl sperm morphology, based on eosin-nigrosin smears, which can determine disorders in spermatogenesis and can be recommended for evaluating the fertilising potency and selecting males in flocks reproduced by artificial insemination. Four fowl breeds (Black Minorca, Italian Partridge, Forwerk and Greenleg Partridge) were used to determine the efficacy of sperm morphology evaluation using four eosin-nigrosin staining methods (according to Blom, Bakst and Cecil, Morisson, Jaśkowski) and three examiners of different experience (high, medium, novice). There were significant (P< or = 0.01) differences in sperm morphology between Blom's staining method and those of Bakst and Cecil, Morisson or Jaśkowski, irrespective of fowl breed and examiners experience. Blom stain caused sperm head swelling and showed a drastic reduction in the proportion of live spermatozoa with normal morphology. The staining method had a greater influence on sperm morphology evaluation than the experience of the examiners.

A new technique for Gram staining paraffin-embedded tissue.

PubMed Central

Engbaek, K; Johansen, K S; Jensen, M E

1979-01-01

Five techniques for Gram staining bacteria in paraffin sections were compared on serial sections of pulmonary tissues from eight bacteriological necropsies. Brown and Hopp's method was the most satisfactory for distinguishing Gram-positive and Gram-negative bacteria. However, this method cannot be recommended as the preparations were frequently overstained, and the Gram-negative bacteria were stained indistinctly. A modification of Brown and Hopps' method was developed which stains larger numbers of Gram-negative bacteria and differentiates well between different cell types and connective tissue, and there is no risk of overstaining. PMID:86548

Whole blood staining in suspension for nonspecific esterase and alkaline phosphatase analyzed with a Technicon H-1.

PubMed

Ross, D W; Bishop, C; Henderson, A; Kaplow, L

1990-01-01

We adapted previously published methods for nonspecific esterase and alkaline phosphatase staining of white blood cells in suspension for use on a Technicon H-1 hematology analyzer. The objective was to develop a semiautomated method using whole blood that could be employed on a large scale for hematology laboratory applications, including toxicology studies, measurement of neutrophil left shift, and cytochemical classification of myeloid leukemias. The nonspecific esterase method uses the pararosaniline stain, generating the unstable substrate from two stable precursors. Whole blood is added to the substrate plus dye mix. Next, acid lysis and fixation steps destroy red cells and stabilize the monocyte staining. The alkaline phosphatase stain employs a stable naphthyl phosphate substrate and fast blue B coupling dye. The red cells are lysed with a pH 10.3 propanediol buffer, and the white blood cells are then stabilized with formalin fixation. For both methods the staining is performed off-line, and the sample is then diluted with propanediol to match the refractive index of the sheath on the H-1 analyzer, before aspiration into the direct cytometry port. A cytogram of scattered versus absorbed light is obtained. The number of cells staining and the intensity of the stain can be quantified from the cytogram.

Evaluation of staining methods for cytologic diagnosis of oral lesions.

PubMed

Almeida, Janete Dias; Lima, Celina Faig; Brandão, Adriana Aigotti Haberbeck; Cabral, Luiz Antonio Guimarães

2008-01-01

To compare the efficacy ofPapanicolaou, hematoxylin-eosin (H-E), Leishman and periodic acid-Schiff (PAS) staining for cytologic diagnosis of oral lesions. Patients from the Discipline of Stomatology, São José dos Campos Dental School, from the wards of Hosapital Heliópolis and from the dentistry outpatient clinic of the University Hospital, University of São Paulo Medical School, with the following diseases, were selected: erythematous candidiasis (n=9), pseudomembranous candidiasis (n=10), squamous cell carcinoma (n=19), herpes simplex (n=8), paracoccidioidomycosis (n=8) and pemphigus vulgaris (n=1). The different staining methods were compared regarding the quality of definition of cytoplasmic and nuclear morphologic characteristics and the identification of bacteria, fungi, inflammatory cells and secretions. Papanicolaou and H-E staining were considered better methods. In cases of fungal infections, PAS staining is useful and should be applied as a complementary method. Within the limitations of this study, it can be concluded that the cytologic diagnosis of oral lesions along with different staining methods is a useful tool for oral diagnosis.

Guinea-pig interpubic joint (symphysis pubica) relaxation at parturition: Underlying cellular processes that resemble an inflammatory response

PubMed Central

Rodríguez, Horacio A; Ortega, Hugo H; Ramos, Jorge G; Muñoz-de-Toro, Mónica; Luque, Enrique H

2003-01-01

Background At term, cervical ripening in coordination with uterine contractions becomes a prerequisite for a normal vaginal delivery. Currently, cervical ripening is considered to occur independently from uterine contractions. Many evidences suggest that cervical ripening resembles an inflammatory process. Comparatively little attention has been paid to the increased flexibility of the pelvic symphysis that occurs in many species to enable safe delivery. The aim of this study was to investigate whether the guinea-pig interpubic joint relaxation process observed during late pregnancy and parturition resembles an inflammatory process. Methods Samples of pubic symphysis were taken from pregnant guinea-pigs sacrificed along gestation, parturition and postpartum. Serial sections of paraffin-embedded tissues were used to measure the interpubic distance on digitalized images, stained with Giemsa to quantify leukocyte infiltration and to describe the vascular area changes, or studied by the picrosirius-polarization method to evaluate collagen remodeling. P4 and E2 serum levels were measured by a sequential immunometric assay. Results Data showed that the pubic relaxation is associated with an increase in collagen remodeling. In addition, a positive correlation between E2 serum levels and the increase in the interpubic distance was found. On the other hand, a leukocyte infiltration in the interpubic tissue around parturition was described, with the presence of almost all inflammatory cells types. At the same time, histological images show an increase in vascular area (angiogenesis). Eosinophils reached their highest level immediately before parturition; whereas for the neutrophilic and mononuclear infiltration higher values were recorded one day after parturition. Correlation analysis showed that eosinophils and mononuclear cells were positively correlated with E2 levels, but only eosinophilic infiltration was associated with collagen remodeling. Additionally, we observed typical histological images of dissolution of the connective tissue matrix around eosinophils. Conclusion The present study shows that a timely regulated influx of infiltrating leukocytes is associated with an extensive collagen remodeling process that allows the pubic separation for a normal delivery in guinea-pig. Thus, the findings in this study support the hypothesis that the guinea-pig pubic symphyseal relaxation at parturition resembles an inflammatory process. PMID:14633278

Staining Methods for Normal and Regenerative Myelin in the Nervous System.

PubMed

Carriel, Víctor; Campos, Antonio; Alaminos, Miguel; Raimondo, Stefania; Geuna, Stefano

2017-01-01

Histochemical techniques enable the specific identification of myelin by light microscopy. Here we describe three histochemical methods for the staining of myelin suitable for formalin-fixed and paraffin-embedded materials. The first method is conventional luxol fast blue (LFB) method which stains myelin in blue and Nissl bodies and mast cells in purple. The second method is a LBF-based method called MCOLL, which specifically stains the myelin as well the collagen fibers and cells, giving an integrated overview of the histology and myelin content of the tissue. Finally, we describe the osmium tetroxide method, which consist in the osmication of previously fixed tissues. Osmication is performed prior the embedding of tissues in paraffin giving a permanent positive reaction for myelin as well as other lipids present in the tissue.

Methods of biological dosimetry employing chromosome-specific staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

2000-01-01

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Further, methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. Still further, the invention provides for automated means to detect and analyse chromosomal abnormalities.

Methods And Compositions For Chromosome-Specific Staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

2003-08-19

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Further, methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. Still further, the invention provides for automated means to detect and analyse chromosomal abnormalities.

Unsupervised color normalisation for H and E stained histopathology image analysis

NASA Astrophysics Data System (ADS)

Celis, Raúl; Romero, Eduardo

2015-12-01

In histology, each dye component attempts to specifically characterise different microscopic structures. In the case of the Hematoxylin-Eosin (H&E) stain, universally used for routine examination, quantitative analysis may often require the inspection of different morphological signatures related mainly to nuclei patterns, but also to stroma distribution. Nevertheless, computer systems for automatic diagnosis are often fraught by color variations ranging from the capturing device to the laboratory specific staining protocol and stains. This paper presents a novel colour normalisation method for H&E stained histopathology images. This method is based upon the opponent process theory and blindly estimates the best color basis for the Hematoxylin and Eosin stains without relying on prior knowledge. Stain Normalisation and Color Separation are transversal to any Framework of Histopathology Image Analysis.

Gram stain of tissue biopsy

MedlinePlus

... biopsy To use the sharing features on this page, please enable JavaScript. Gram stain of tissue biopsy test involves using crystal violet stain to test a sample of tissue taken from a biopsy . The Gram stain method can ...

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy

PubMed Central

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin

2016-01-01

Objectives We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. Methods We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. Results An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. Conclusions The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis. PMID:27525165

Digital simulation of staining in histopathology multispectral images: enhancement and linear transformation of spectral transmittance.

PubMed

Bautista, Pinky A; Yagi, Yukako

2012-05-01

Hematoxylin and eosin (H&E) stain is currently the most popular for routine histopathology staining. Special and/or immuno-histochemical (IHC) staining is often requested to further corroborate the initial diagnosis on H&E stained tissue sections. Digital simulation of staining (or digital staining) can be a very valuable tool to produce the desired stained images from the H&E stained tissue sections instantaneously. We present an approach to digital staining of histopathology multispectral images by combining the effects of spectral enhancement and spectral transformation. Spectral enhancement is accomplished by shifting the N-band original spectrum of the multispectral pixel with the weighted difference between the pixel's original and estimated spectrum; the spectrum is estimated using M < N principal component (PC) vectors. The pixel's enhanced spectrum is transformed to the spectral configuration associated to its reaction to a specific stain by utilizing an N × N transformation matrix, which is derived through application of least mean squares method to the enhanced and target spectral transmittance samples of the different tissue components found in the image. Results of our experiments on the digital conversion of an H&E stained multispectral image to its Masson's trichrome stained equivalent show the viability of the method.

[Louse-borne-relapsing-fever in refugees from the Horn of Africa; a case series of 25 patients].

PubMed

Seilmaier, M; Guggemos, W; Wieser, A; Fingerle, V; Balzer, L; Fenzl, T; Hoch, M; von Both, U; Schmidt, H U; Wendtner, C M; Strobel, E

2016-07-01

Background | Relapsing fever is divided into tick borne relapsing fever (TBRF) and louse borne relapsing fever (LBRF). This report describes 25 refugees from East Africa who were diagnosed to suffer from LBRF within a period of 6 month only at a single hospital in Munich / Germany. Material & Methods | The aim was to point out common clinical features as well as laboratory findings and clinical symptoms before and after initiation of treatment in 25 patients with louse borne relapsing fever (LBRF) who were diagnosed and treated at Klinikum München Schwabing from August 2015 to January 2016. To the best of our knowledge this is the largest case series of LBRF in the western world for decades. Main focus of the investigation was put on clinical aspects. Results | All 25 patients suffered from acute onset of high fever with chills, headache and severe prostration. Laboratory analysis showed high CRP and a marked thrombocytopenia. A Giemsa blood stain was procured immediately in order to look for malaria. In the blood smear spirochetes with typical shape and aspect of borrelia species could be detected.The further PCR analysis confirmed infection with Borrelia recurrentis. Treatment with Doxycycline was started forthwith. The condition improved already on the second day after treatment was started and all were restored to health in less than a week. Apart from a mild to moderate Jarisch-Herxheimer-reaction we didn`t see any side effects of the therapy. Conclusion | LBRF has to be taken into account in feverish patients who come as refugees from East-Africa. It seems that our patients belong to a cluster which probably has its origin in Libya and more patients are to be expected in the near future. As LBRF might cause outbreaks in refugee camps it is pivotal to be aware of this emerging infectious disease in refugees from East-Africa. © Georg Thieme Verlag KG Stuttgart · New York.

Trypanosoma janseni n. sp. (Trypanosomatida: Trypanosomatidae) isolated from Didelphis aurita (Mammalia: Didelphidae) in the Atlantic Rainforest of Rio de Janeiro, Brazil: integrative taxonomy and phylogeography within the Trypanosoma cruzi clade.

PubMed

Lopes, Camila Madeira Tavares; Menna-Barreto, Rubem Figueiredo Sadok; Pavan, Márcio Galvão; Pereira, Mirian Cláudia De Souza; Roque, André Luiz R

2018-01-01

Didelphis spp. are a South American marsupial species that are among the most ancient hosts for the Trypanosoma spp. We characterise a new species (Trypanosoma janseni n. sp.) isolated from the spleen and liver tissues of Didelphis aurita in the Atlantic Rainforest of Rio de Janeiro, Brazil. The parasites were isolated and a growth curve was performed in NNN and Schneider's media containing 10% foetal bovine serum. Parasite morphology was evaluated via light microscopy on Giemsa-stained culture smears, as well as scanning and transmission electron microscopy. Molecular taxonomy was based on a partial region (737-bp) of the small subunit (18S) ribosomal RNA gene and 708 bp of the nuclear marker, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. Maximum likelihood and Bayesian inference methods were used to perform a species coalescent analysis and to generate individual and concatenated gene trees. Divergence times among species that belong to the T. cruzi clade were also inferred. In vitro growth curves demonstrated a very short log phase, achieving a maximum growth rate at day 3 followed by a sharp decline. Only epimastigote forms were observed under light and scanning microscopy. Transmission electron microscopy analysis showed structures typical to Trypanosoma spp., except one structure that presented as single-membraned, usually grouped in stacks of three or four. Phylogeography analyses confirmed the distinct species status of T. janseni n. sp. within the T. cruzi clade. Trypanosoma janseni n. sp. clusters with T. wauwau in a well-supported clade, which is exclusive and monophyletic. The separation of the South American T. wauwau + T. janseni coincides with the separation of the Southern Super Continent. This clade is a sister group of the trypanosomes found in Australian marsupials and its discovery sheds light on the initial diversification process based on what we currently know about the T. cruzi clade.

Burden of malaria in mobile populations in the Greater Accra region, Ghana: a cross- sectional study.

PubMed

Diallo, Nouhoum; Akweongo, Patricia; Maya, Ernest; Aikins, Moses; Sarfo, Bismark

2017-03-09

The burden of malaria in mobile populations remains poorly documented in sub-Saharan Africa. This study determined the prevalence of malaria among hawkers and long-distance truck drivers in the Greater Accra region of Ghana. A cross-sectional design using consecutive sampling method between June and July 2016 in Accra and Tema in Ghana was used in this study. The study population was hawkers who roam and sleep in the Market Streets, and long-distance truck drivers. Participants completed closed ended interview questionnaires on socio-demographic characteristics, primary residence and knowledge about malaria. Rapid diagnostic test and thick blood smears of each participant were stained with Giemsa and read using microscopy. Geographical position system (GPS) was used to collect the station locations of these mobile populations. The overall prevalence of malaria was 15.1% and Plasmodium falciparum was responsible for all malaria infection. The malaria prevalence was 18.9 and 10.9% respectively among hawkers and truck drivers (p < 0.05). The hawkers, the single and the no formal educated participants were more likely to get malaria than the long-distance truck drivers (OR = 1.91, 95% CI 1.07-3.42), the married (OR = 1.94 95% CI 1.11-3.40) and the educated participants (OR = 2.56 95% CI 1.10-5.93), respectively. After controlling for other variables, marital status (OR = 2.60 95% CI 1.43- 4.73) and educational level (OR = 2.70 95% CI 1.08-6.77) were statistically significantly associated with malaria. This study demonstrated that the prevalence of malaria is high among hawkers and long distance truck drivers. Sociodemographic characteristics, such as marital status, occupation and educational level are significantly associated with malaria. The station locations as determined by GPS technology will make these mobile populations easier to reach for any malaria intervention.

Symptomatic malaria diagnosis overestimate malaria prevalence, but underestimate anaemia burdens in children: results of a follow up study in Kenya.

PubMed

Choge, Joseph K; Magak, Ng'wena G; Akhwale, Willis; Koech, Julius; Ngeiywa, Moses M; Oyoo-Okoth, Elijah; Esamai, Fabian; Osano, Odipo; Khayeka-Wandabwa, Christopher; Kweka, Eliningaya J

2014-04-09

The commonly accepted gold standard diagnostic method for detecting malaria is a microscopic reading of Giemsa-stained blood films. However, symptomatic diagnosis remains the basis of therapeutic care for the majority of febrile patients in malaria endemic areas. This study aims to compare the discrepancy in malaria and anaemia burdens between symptomatic diagnosed patients with those diagnosed through the laboratory. Data were collected from Western Kenya during a follow-up study of 887 children with suspected cases of malaria visiting the health facilities. In the laboratory, blood samples were analysed for malaria parasite and haemoglobin levels. Differences in malaria prevalence between symptomatic diagnosis and laboratory diagnosis were analysed by Chi-square test. Bayesian probabilities were used for the approximation of the malaria and anaemia burdens. Regression analysis was applied to: (1) determine the relationships between haemoglobin levels, and malaria parasite density and (2) relate the prevalence of anaemia and the prevalence of malaria. The prevalence of malaria and anaemia ranged from 10% to 34%, being highest during the rainy seasons. The predominant malaria parasite was P. falciparum (92.3%), which occurred in higher density in children aged 2‒5 years. Fever, high temperature, sweating, shivering, vomiting and severe headache symptoms were associated with malaria during presumptive diagnosis. After conducting laboratory diagnosis, lower malaria prevalence was reported among the presumptively diagnosed patients. Surprisingly, there were no attempts to detect anaemia in the same cohort. There was a significant negative correlation between Hb levels and parasite density. We also found a positive correlation between the prevalence of anaemia and the prevalence of malaria after laboratory diagnosis indicating possible co-occurrence of malaria and anaemia. Symptomatic diagnosis of malaria overestimates malaria prevalence, but underestimates the anaemia burden in children. Good clinical practice dictates that a laboratory should confirm the presence of parasites for all suspected cases of malaria.

Influence of Helicobacter pylori Colonization on Histological Grading of Chronic Gastritis in Korean Patients with Peptic Ulcer

PubMed Central

Park, Joongwon; Kim, Mi Kyung; Park, Sill Moo

1995-01-01

Objectives: We conducted an analysis of correlation between histological grading of chronic gastritis and the presence of H. pylori infection to investigate if H. pylori influences histological severity of chronic gastritis in Korean patients with peptic ulcers. Methods: Gastroscopic antral biopsy specimens and peripheral venous blood were taken from 80 patients with gastric or duodenal ulcers. H. pylori was identified microscopically in sections with Giemsa staining and quantitative grading of cultured H. pylori was reported on a scale 0 to 3. The histopathological features of biopsy specimens were reported according to the Sydney classification of chronic gastritis. Serum gastritis and pepsinogen concentrations were measured by radioimmunoassay. Results: H. pylori was identified in 62.5% (20 of 32 GU, 30 of 48 DU) of the study group. Gastric clonization rate of H. pylori did not increased with age. Forty of 50 biopsy specimens with H. pylori and also 23 of 30 biopsy specimens without H. pylori showed active chronic gastritis. There was no significant correlation overall between the presence of H. pylori and histological grading of chronic gastritis, including activity, and also no association was found between the quantitative grading of H. pylori and the histological grading of chronic gastritis. With and without H. pylori, a mean of serum gastritis concentration (79.4±43.0 pg/ml and 80.2±31.9 pg/ml) showed no significant difference, but a mean of serum pepsinogen concentration (87.7±41.6 ng/ml and 119±34.4 ng/ml) showed significant difference between the populations with and without H. pylori (p=0.001) Conclusions: The influence of H. pylori on histological grading of chronic gastritis in Korean is less than that in prior studies of Western countries, and further investigation of pathogenesis of H. pylori in chronic gastritis and peptic ulceration is necessary. PMID:7495770

Prevalence and Predictors of Asymptomatic Malaria Parasitemia among Pregnant Women in the Rural Surroundings of Arbaminch Town, South Ethiopia

PubMed Central

Nega, Desalegn; Dana, Daniel; Tefera, Tamirat; Eshetu, Teferi

2015-01-01

Background In Sub-Saharan African countries, including Ethiopia, malaria in pregnancy is a major public health threat which results in significant morbidities and mortalities among pregnant women and their fetuses. In malaria endemic areas, Plasmodium infections tend to remain asymptomatic yet causing significant problems like maternal anemia, low birth weight, premature births, and still birth. This study was conducted to determine the prevalence and predictors of asymptomatic Plasmodium infection among pregnant women in the rural surroundings of Arba Minch Town, Southern Ethiopia. Methods A community based cross-sectional study comprising multistage sampling was conducted between April and June, 2013. Socio-demographic data were collected by using a semi-structured questionnaire. Plasmodium infection was diagnosed by using Giemsa-stained blood smear microscopy and a rapid diagnostic test (SD BIOLINE Malaria Ag Pf/Pv POCT, standard diagnostics, inc., Korea). Results Of the total 341 pregnant women participated in this study, 9.1% (31/341) and 9.7% (33/341) were confirmed to be infected with Plasmodium species by microscopy and rapid diagnostic tests (RDTs), respectively. The geometric mean of parasite density was 2392 parasites per microliter (μl); 2275/ μl for P. falciparum and 2032/ μl for P. vivax. Parasitemia was more likely to occur in primigravidae (Adjusted odds ratio (AOR): 9.4, 95% confidence interval (CI): 4.3–60.5), secundigravidae (AOR: 6.3, 95% CI: 2.9–27.3), using insecticide treated bed net (ITN) sometimes (AOR: 3.2, 95% CI: 1.8- 57.9), not using ITN at all (AOR: 4.6, 95% CI: 1.4–14.4) compared to multigravidae and using ITN always, respectively. Conclusion Asymptomatic malaria in this study is low compared to other studies’ findings. Nevertheless, given the high risk of malaria during pregnancy, pregnant women essentially be screened for asymptomatic Plasmodium infection and be treated promptly via the antenatal care (ANC) services. PMID:25849587

Simultaneous detection of Plasmodium vivax and Plasmodium falciparum gametocytes in clinical isolates by multiplex-nested RT-PCR

PubMed Central

2012-01-01

Background Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax. Methods A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR. Results The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates. Conclusions The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa. PMID:22682065

Malaria and helminth co-infection and nutritional status of febrile patients in Southern Ethiopia.

PubMed

Degarege, Abraham; Animut, Abebe; Legesse, Mengistu; Medhin, Girmay; Erko, Berhanu

2014-02-01

Because the mechanisms by which Plasmodium and helminth parasites affect nutritional status are different, these parasites likely have additive effects when they co-exist in a host. This study aimed to compare the prevalence of undernutrition in patients infected with either Plasmodium or helminths and those co-infected with the two types of parasites. Acute febrile patients suspected of having malaria who attended the outpatient clinic at Dore Bafeno Health Center between December 2010 and February 2011 were examined for Plasmodium parasites using Giemsa-stained thick and thin blood smears and for helminths using the thick Kato-Katz method. Nutritional status was determined using anthropometric indices generated from height and weight measurements. Of the 702 patients examined, 34.5% were infected with helminths alone, 12.3% were infected with Plasmodium alone, and 19.4% co-infected with Plasmodium and intestinal helminths. Out of the patients examined, 44.9% were undernourished. The prevalence of undernutrition was not significantly different between those patients not infected with Plasmodium or helminth species and those infected with Plasmodium or helminth species. The differences in the odds of undernutrition were also not significant between patients who were co-infected with different Plasmodium and helminth species and those with single infections with Plasmodium or helminth species in our multivariable logistic regression model adjusted for the confounding effects of age and sex. The prevalence of undernutrition was comparable in patients infected with Plasmodium or helminths alone and those co-infected with Plasmodium and helminths in Dore Bafeno Health Center, Southern Ethiopia. However, further studies are needed in areas of intense transmission where both parasites are endemic to elucidate whether the impact of Plasmodium and helminth co-infection on undernutrition is additive or multiplicative. Copyright © 2013 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Facile preparation of biphasic-induced magnetic icariin-loaded composite microcapsules by automated in situ click technology.

PubMed

Pan, Panpan; Chen, Jingdi; Fan, Tiantang; Hu, Yimin; Wu, Tao; Zhang, Qiqing

2016-04-01

This research aims to prepare the biphasic-induced magnetic composite microcapsules (BIMCM) as a promising environmental stimuli-responsive delivery vehicle to dispose the problem of drug burst effect. The paper presented a novel automated in situ click technology of magnetic chitosan/nano hydroxyapatite (CS/nHA) microcapsules. Fe3O4 magnetic nanoparticles (MNP) and nHA were simultaneously in situ crystallized by one-step process. Icariin (ICA), a plant-derived flavonol glycoside, was combined to study drug release properties of BIMCM. BIMCM were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and Thermal gravimetric analysis/Differential Scanning Calorimetry(TGA/DSC) in order to reveal their component and surface morphology as well as the role of the in situ generated Fe3O4 MNP and nHA. The magnetic test showed the BIMCM were super-paramagnetic. Both in situ generated Fe3O4 MNP and nHA serve as stable inorganic crosslinkers in BIMCM to form many intermolecular crosslinkages for the movability of the CS chains. This makes ICA loaded microcapsules take on a sustained release behavior and results in the self-adjusting of surface morphology, decreasing of swelling and degradation rates. In addition, in vitro tests were systematically carried out to examine the biocompatibility of the microcapsules by MTT test, Wright-Giemsa dying assay and AO/EB fluorescent staining method. These results demonstrated that successful introduction of the in situ click Fe3O4 MNP provided an alternative strategy because of magnetic sensitivity and sustained release. As such, the novel ICA loaded biphasic-induced magnetic CS/nHA/MNP microcapsules are expected to find potential applications in drug delivery system for bone repair. Copyright © 2015 Elsevier B.V. All rights reserved.

Cytokine-associated neutrophil extracellular traps and antinuclear antibodies in Plasmodium falciparum infected children under six years of age

PubMed Central

Baker, Virginia S; Imade, Godwin E; Molta, Norman B; Tawde, Pallavi; Pam, Sunday D; Obadofin, Michael O; Sagay, Soloman A; Egah, Daniel Z; Iya, Daniel; Afolabi, Bangmboye B; Baker, Murray; Ford, Karen; Ford, Robert; Roux, Kenneth H; Keller, Thomas CS

2008-01-01

Background In Plasmodium falciparum-infected children, the relationships between blood cell histopathology, blood plasma components, development of immunocompetence and disease severity remain poorly understood. Blood from Nigerian children with uncomplicated malaria was analysed to gain insight into these relationships. This investigation presents evidence for circulating neutrophil extracellular traps (NETs) and antinuclear IgG antibodies (ANA). The presence of NETs and ANA to double-stranded DNA along with the cytokine profiles found suggests autoimmune mechanisms that could produce pathogenesis in children, but immunoprotection in adults. Methods Peripheral blood smear slides and blood samples obtained from 21 Nigerian children under six years of age, presenting with uncomplicated malaria before and seven days after initiation of sulphadoxine-pyrimethamine (SP) treatment were analysed. The slides were stained with Giemsa and with DAPI. Levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF, CRP, and IL-6, select anti-inflammatory cytokines TGF-β and IL-10, and ANA were determined by immunoassay. Results The children exhibited circulating NETs with adherent parasites and erythrocytes, elevated ANA levels, a Th2 dominated cytokine profile, and left-shifted leukocyte differential counts. Nonspecific ANA levels were significant in 86% of the children pretreatment and in 100% of the children seven days after SP treatment, but in only 33% of age-matched control samples collected during the season of low parasite transmission. Levels of ANA specific for dsDNA were significant in 81% of the children both pre-treatment and post treatment. Conclusion The results of this investigation suggest that NET formation and ANA to dsDNA may induce pathology in falciparum-infected children, but activate a protective mechanism against falciparum malaria in adults. The significance of in vivo circulating chromatin in NETs and dsDNA ANA as a causative factor in the hyporesponsiveness of CpG oligonucleotide-based malaria vaccines is discussed. PMID:18312656

Mucocutaneous Manifestations in Patients with Chronic Kidney Disease: A Cross-sectional Study

PubMed Central

Rashpa, Rattan S.; Mahajan, Vikram K.; Kumar, Pankaj; Mehta, Karaninder S.; Chauhan, Pushpinder S.; Rawat, Ritu; Sharma, Vikas

2018-01-01

Background: Chronic kidney disease (CKD)-associated mucocutaneous manifestations significantly impair the quality of life but often remain understudied. They may also vary across regions, socioeconomic and nutritional status, and racial differences. Objectives: To study the patterns of mucocutaneous disorders and their prevalence in CKD patients irrespective of clinical stage or dialysis status. Materials and Methods: 122 (M:F = 77:45) patients aged 21‒85 (Mean ± SD = 57.5 ± 14.0) years having CKD for 3 month to 5 years were studied for mucocutaneous manifestations. Fifty (41%) patients were on hemodialysis for 1‒42 months. Detailed medical history, clinical and mucocutaneous examination, and lab investigations were performed. KOH mounts, skin biopsy, Gram's and Giemsa staining, bacterial or fungal cultures were performed as required. Results: Xerosis in 93 (76.2%), skin pallor in 61 (50%), pruritus in 57 (46.7%), pigmentation in 47 (38.5%), and purpura in 18 (14.8%) patients were the major dermatoses. Bullous lesions and perforating folliculitis occurred in 3 (2.5%) patients each. Major nail abnormalities were pallor (in 35.2%), absent lunula (in 23.8%), nail discoloration (in 18%), and “half-and-half nails” in 16.4% patients, respectively. Hair abnormalities included sparse scalp and body hairs (in 35.2% and 13.1%, respectively) and lusterless hair in 12.3% patients. Coated tongue (in 14.8%), xerostomia (in 12.3%), and macroglossia with teeth indention (in 7.4%) patients were the mucosal manifestations. Conclusions: Xerosis, pruritus, skin pallor/pigmentary changes, nail pallor, absent lunula, nail discoloration, sparse hairs, coated tongue, xerostomia, macroglossia, and infections were the most common mucocutaneous manifestations in the studied patients irrespective of hemodialysis status. Cold and dry climates might be additional aggravators for xerosis/pruritus. Lifelong follow-up may be needed to reduce the morbidity associated with CKD/hemodialysis specific dermatoses appearing over a period. PMID:29441293

The Effect of Intestinal Parasitic Infection on the Clinical Outcome of Malaria in Coinfected Children in Cameroon

PubMed Central

Kwenti, Tebit E.; Nkume, Franklin A.; Tanjeko, Ajime T.; Kwenti, Tayong D. B.

2016-01-01

Background The interaction between intestinal parasites and malaria is still not clear. Data in published literature are conflicting. We studied the effect of intestinal parasitic infection (IPI) on the clinical outcome of malaria in coinfected children. Methods In a cross sectional study performed between October 2014 and September 2015, children infected with malaria, as demonstrated by the presence of asexual parasites in Giemsa stained blood films, were enrolled. Stool samples were obtained from participants and subjected to the formol-ether concentration technique for the detection of intestinal parasites. The Complete blood count was performed using an automated haematology analyser (Mindray, BC-2800). The risk ratio, Pearson’s chi-square and the student T test were all performed as part of the statistical analyses. Statistical significance was set at p < 0.05. Results In all, 405 children successfully took part in the study. The children were between 1 week and 120 months of age (mean ± SD = 41.5 ± 33.5). Coinfection with intestinal parasites was observed in 11.6%. The rate of severe malaria (SM) attack in this study was 10.9%. SM was not observed to be associated with age (p = 0.377) or gender (p = 0.387), meanwhile coinfection with intestinal parasites was associated with age (p = 0.003). Among SM cases, IPI prevalence was higher in children with mild (WHO group 3) severe malaria (p = 0.027). Overall, IPI was not observed to be associated with SM (p = 0.656) or malaria parasite density (p = 0.185) or haemoglobin concentration (p = 0.205). The main clinical features of SM observed were hyperpyrexia (68.2%), severe malarial anaemia (61.4%), and multiple convulsion (52.3%). Conclusion IPI was not observed to be associated with the severity of malaria, the malaria parasite density, and the haemoglobin concentration in coinfected children in Cameroon. The clinical outcome of malaria in children coinfected with intestinal parasites may depend on the geographical setting after all. PMID:27128975

Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact.

PubMed

Gallo, Alessandra; Boni, Raffaele; Tosti, Elisabetta

2018-01-01

The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

Application of immunohistochemical staining to detect antigen destruction as a measure of tissue damage.

PubMed

Onul, Abdullah; Colvard, Michael D; Paradise, William A; Elseth, Kim M; Vesper, Benjamin J; Gouvas, Eftychia; Deliu, Zane; Garcia, Kelly D; Pestle, William J; Radosevich, James A

2012-09-01

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, "antigen destruction immunohistochemistry" (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method.

Application of Immunohistochemical Staining to Detect Antigen Destruction as a Measure of Tissue Damage

PubMed Central

Onul, Abdullah; Colvard, Michael D.; Paradise, William A.; Elseth, Kim M.; Vesper, Benjamin J.; Gouvas, Eftychia; Deliu, Zane; Garcia, Kelly D.; Pestle, William J.

2012-01-01

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, “antigen destruction immunohistochemistry” (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method. PMID:22723525

Comparison of methylene blue/gentian violet stain to Gram's stain for the rapid diagnosis of gonococcal urethritis in men.

PubMed

Taylor, Stephanie N; DiCarlo, Richard P; Martin, David H

2011-11-01

We compared a simple, one-step staining procedure using a mixture of methylene blue and gentian violet to Gram stain for the detection of gonococcal urethritis. The sensitivity and specificity of both Gram stain and methylene blue/gentian violet stain were 97.3% and 99.6%, respectively. There was a 100% correlation between the 2 methods.

Demonstration of lipofuscin and Nissl bodies in crystal violet stained sections using a fluorescence technique or pyronin Y stain.

PubMed

Terr, L I

1986-09-01

This paper presents two simple, reliable methods for identification of lipofuscin and Nissl bodies in the same section. One method shows that lipofuscin stained with crystal violet retains its ability to fluoresce and can be observed under the fluorescence microscope after the stain has faded. Fading is accompanied by a gradual increase in the intensity of the fluorescence and is complete in about 5 min. Exciting illumination from this part of the spectrum also substantially fades staining of other autofluorescing tissue elements, such as lipids. Nonfluorescing structures, such as Nissl bodies, remain stained. By changing from transillumination with tungsten light to epifluorescent illumination and vice versa, both types of structures--Nissl bodies and lipofuscin--can be identified in the same section. The second technique uses pyronin Y for staining Nissl bodies in preparations previously stained with crystal violet. Nissl bodies are stained pink but lipofuscin remains violet. Lipofuscin in these sections also remains autofluorescent after the crystal violet stain has faded under violet or near-UV light.

Rapid contrast evaluation method based on affinity beads and backscattered electron imaging for the screening of electron stains.

PubMed

Kaku, Hiroki; Inoue, Kanako; Muranaka, Yoshinori; Park, Pyoyun; Ikeda, Kenichi

2015-10-01

Uranyl salts are toxic and radioactive; therefore, several studies have been conducted to screen for substitutes of electron stains. In this regard, the contrast evaluation process is time consuming and the results obtained are inconsistent. In this study, we developed a novel contrast evaluation method using affinity beads and a backscattered electron image (BSEI), obtained using scanning electron microscopy. The contrast ratios of BSEI in each electron stain treatment were correlated with those of transmission electron microscopic images. The affinity beads bound to cell components independently. Protein and DNA samples were enhanced by image contrast treated with electron stains; however, this was not observed for sugars. Protein-conjugated beads showed an additive effect of image contrast when double-stained with lead. However, additive effect of double staining was not observed in DNA-conjugated beads. The varying chemical properties of oligopeptides showed differences in image contrast when treated with each electron stain. This BSEI-based evaluation method not only enables screening for alternate electron stains, but also helps analyze the underlying mechanisms of electron staining of cellular structures. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Circular Mixture Modeling of Color Distribution for Blind Stain Separation in Pathology Images.

PubMed

Li, Xingyu; Plataniotis, Konstantinos N

2017-01-01

In digital pathology, to address color variation and histological component colocalization in pathology images, stain decomposition is usually performed preceding spectral normalization and tissue component segmentation. This paper examines the problem of stain decomposition, which is a naturally nonnegative matrix factorization (NMF) problem in algebra, and introduces a systematical and analytical solution consisting of a circular color analysis module and an NMF-based computation module. Unlike the paradigm of existing stain decomposition algorithms where stain proportions are computed from estimated stain spectra using a matrix inverse operation directly, the introduced solution estimates stain spectra and stain depths via probabilistic reasoning individually. Since the proposed method pays extra attentions to achromatic pixels in color analysis and stain co-occurrence in pixel clustering, it achieves consistent and reliable stain decomposition with minimum decomposition residue. Particularly, aware of the periodic and angular nature of hue, we propose the use of a circular von Mises mixture model to analyze the hue distribution, and provide a complete color-based pixel soft-clustering solution to address color mixing introduced by stain overlap. This innovation combined with saturation-weighted computation makes our study effective for weak stains and broad-spectrum stains. Extensive experimentation on multiple public pathology datasets suggests that our approach outperforms state-of-the-art blind stain separation methods in terms of decomposition effectiveness.

[Histochemical stains for minerals by hematoxylin-lake method].

PubMed

Miyagawa, Makoto

2013-04-01

The present study was undertaken to establish the experimental animal model by histological staining methods for minerals. After intraperitoneal injections of minerals, precipitates deposited on the surface of the liver. Liver tissues were fixed in paraformaldehyde, embedded in paraffin and cut into thin sections which were used as minerals containing standard section. Several reagents for histological stains and spectrophotometry for minerals were applied in both test-tube experiments and stainings of tissue sections to test for minerals. Hematoxylin-lake was found of capable of staining minerals in tissue. A simple technique used was described for light microscopic detection of minerals.

A Novel Contrast Stain for the Rapid Diagnosis of Pityriasis Versicolor: A Comparison of Chicago Sky Blue 6B Stain, Potassium Hydroxide Mount and Culture

PubMed Central

Lodha, Nikita; Poojary, Shital Amin

2015-01-01

Background: The mycological study of pityriasis versicolor is usually done by potassium hydroxide (KOH) mount and culture. However, KOH mount lacks a color contrast and requires a trained eye to interpret, while culture is difficult to perform, time consuming and has low sensitivity. Chicago Sky Blue 6B (CSB) is a new contrast stain that highlights the fungal hyphae and spores, blue against a purplish background. Aims and Objectives: This study was done to compare the utility of a novel contrast stain (CSB stain) with KOH mount and culture. Materials and Methods: Skin scrapings from the lesions of 100 clinically diagnosed cases of P. versicolor were subjected to (1) KOH mount and CSB stain for direct microscopic examination and (2) culture using Sabouraud's dextrose agar. The statistical analysis of CSB stain and culture was done using KOH mount as the reference method, as it is the most commonly performed and practical diagnostic test available for P. versicolor. An interrater reliability analysis using the Cohen's Kappa statistic was performed to determine consistency (agreement) among the different modalities. Observations and Results: Direct microscopy with CSB stain, KOH mount and mycological culture showed positive results in 98 (98%), 92 (92%) and 56 (56%) patients, respectively. Using KOH mount as the reference method, CSB stain had a sensitivity of 100% which was significantly higher than culture (60.9%). Statistically significant fair agreement was found between CSB stain and KOH mount (94% with κ=0.38, P < 0.001). Negligible agreement was found between CSB stain and culture (66%, κ=0.199, P = 0.001) as well as between KOH mount and culture (64%, κ=0.051, P = 0.107). Conclusion: CSB staining of skin scrapings is the most sensitive method for the diagnosis of pityriasis versicolor. Due to the distinct contrast provided by CSB, it is easy to perform, rapid and qualitatively superior to KOH mount. PMID:26288400

More experiences with the Tzanck smear test: cytologic findings in cutaneous granulomatous disorders.

PubMed

Durdu, Murat; Baba, Mete; Seçkin, Deniz

2009-09-01

Granulomatous dermatitis is a distinctive histopathologic cutaneous reaction pattern against various infectious and noninfectious agents. Cytologically, granulomatous dermatitis shows granulomas and multinucleated giant cells. Various etiologic agents of granulomatous diseases can also be identified. We aimed to investigate Tzanck smear findings in granulomatous skin diseases. Patients who had granulomas and/or multinucleated giant cells of Langhans, foreign body- and/or Touton type in Tzanck smear tests were included in the study. In these patients, Tzanck preparations were then further evaluated for additional cytologic findings. Samples stained with May-Grünwald-Giemsa stain were evaluated by the same dermatologist throughout the study. In some patients, methylene blue, Gram and/or Erlich-Ziehl-Nielsen stains were also performed. In all of the study cases, the final diagnosis was established after the evaluation of clinical and laboratory findings (including, when appropriate, potassium hydroxide examination; bacterial, leishmanial, and fungal cultures; histopathology; tuberculosis and leishmania polymerase chain reaction). We also calculated the sensitivity and specificity of the Leishman-Donovan body for cutaneous leishmaniasis. Over a 2-year period, 94 of 950 patients (9.9%) in whom Tzanck smear tests were performed had cytologic findings consistent with a granulomatous reaction. In 74 (78.7%) and 20 (21.3%) patients, the granulomatous reaction was due to infectious and noninfectious causes, respectively. Infectious causes included cutaneous leishmaniasis in 65 patients (87.8%), candidal granuloma in two patients, botyromycosis in two patients, and aspergillosis, blastomycosis, mucormycosis, leprosy, and cutaneous tuberculosis in one patient each. In 58 of 74 patients (78.4%) with infectious granulomatous dermatitis, the causes of the granulomas were identified. Noninfectious granulomatous reactions were due to granuloma annulare in 7 patients, sarcoidosis in 5 patients, a foreign body in 4 patients, necrobiosis lipoidica in 2 patients, and juvenile xanthogranuloma in 2 patients. In 17 of 20 patients (85%) with noninfectious granulomatous reactions, the cytologic findings were characteristic of the final diagnoses. The sensitivity and specificity of Leishman-Donovan bodies for cutaneous leishmaniasis were 76.9% and 100%, respectively. All of the samples were evaluated by the same dermatologist throughout the study; therefore no comment could be made regarding the reliability of the Tzanck smear test. In addition, the sensitivity and specificity of Tzanck smear test findings for diseases other than cutaneous leishmaniasis could not be calculated because of an insufficient number of patients. The Tzanck smear test may be a useful diagnostic tool for certain granulomatous skin diseases.

Compositions for chromosome-specific staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

1998-01-01

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Further, methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. Still further, the invention provides for automated means to detect and analyse chromosomal abnormalities.

Better detection of Demodex mites by Löffler’s alkaline methylene blue staining in patients with blepharitis

PubMed Central

Kiuchi, Katsuji

2018-01-01

Purpose To determine whether the Löffler’s alkaline methylene blue staining method is better than no staining in detecting Demodex mites in the eyelashes of patients with blepharitis. Materials and methods Eyelashes were collected from 22 patients with blepharitis. The mean age of the patients was 82.5±6.2 years (± SD) with a range from 71 to 93 years. Eyelashes were epilated by forceps and placed individually on microscope slides. The number of Demodex mites was determined by conventional optical microscopy before and immediately after the addition of the methylene blue staining solution. Results The mean Demodex count before the addition of the methylene blue solution was 2.9±2.9, and it was 4.4±3.9 after the addition of the methylene blue solution (P<0.01, Wilcoxon test). Conclusion The methylene blue staining method is a simple and useful method in detecting the presence and quantifying the number of Demodex mites. We recommend the methylene blue staining method not only for the diagnosis of the presence of Demodex mites but also to evaluate the therapeutic effects of medications to eliminate the mite infestation. PMID:29713140

Compositions for chromosome-specific staining

DOEpatents

Gray, J.W.; Pinkel, D.

1998-05-26

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. The methods produce staining patterns that can be tailored for specific cytogenetic analyses. The probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. The invention provides for automated means to detect and analyze chromosomal abnormalities. 17 figs.

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy.

PubMed

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin; Sohn, Dae Kyung

2016-07-01

We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis.

Reassignment of the land tortoise haemogregarine Haemogregarina fitzsimonsi Dias 1953 (Adeleorina: Haemogregarinidae) to the genus Hepatozoon Miller 1908 (Adeleorina: Hepatozoidae) based on parasite morphology, life cycle and phylogenetic analysis of 18S rDNA sequence fragments.

PubMed

Cook, Courtney A; Lawton, Scott P; Davies, Angela J; Smit, Nico J

2014-06-13

SUMMARY Research was undertaken to clarify the true taxonomic position of the terrestrial tortoise apicomplexan, Haemogregarina fitzsimonsi (Dias, 1953). Thin blood films were screened from 275 wild and captive South African tortoises of 6 genera and 10 species between 2009-2011. Apicomplexan parasites within films were identified, with a focus on H. fitzsimonsi. Ticks from wild tortoises, especially Amblyomma sylvaticum and Amblyomma marmoreum were also screened, and sporogonic stages were identified on dissection of adult ticks of both species taken from H. fitzsimonsi infected and apparently non-infected tortoises. Parasite DNA was extracted from fixed, Giemsa-stained tortoise blood films and from both fresh and fixed ticks, and PCR was undertaken with two primer sets, HEMO1/HEMO2, and HepF300/HepR900, to amplify parasite 18S rDNA. Results indicated that apicomplexan DNA extracted from tortoise blood films and both species of tick had been amplified by one or both primer sets. Haemogregarina  fitzsimonsi 18S rDNA sequences from tortoise blood aligned with those of species of Hepatozoon, rather than those of species of Haemogregarina or Hemolivia. It is recommended therefore that this haemogregarine be re-assigned to the genus Hepatozoon, making Hepatozoon fitzsimonsi (Dias, 1953) the only Hepatozoon known currently from any terrestrial chelonian. Ticks are its likely vectors.

Biological dosimetry in a group of radiologists by the analysis of dicentrics and translocations.

PubMed

Montoro, A; Rodríguez, P; Almonacid, M; Villaescusa, J I; Verdú, G; Caballín, M R; Barrios, L; Barquinero, J F

2005-11-01

The results of a cytogenetic study carried out in a group of nine radiologists are presented. Chromosome aberrations were detected by fluorescence plus Giemsa staining and fluorescence in situ hybridization. Dose estimates were obtained by extrapolating the yield of dicentrics and translocations to their respective dose-effect curves. In seven individuals, the 95% confidence limits of the doses estimated by dicentrics did not include 0 Gy. The 99 dicentrics observed in 17,626 cells gave a collective estimated dose of 115 mGy (95% confidence limits 73-171). For translocations, five individuals had estimated doses that were clearly higher than the total accumulated recorded dose. The 82 total apparently simple translocations observed in 9722 cells gave a collective estimated dose of 275 mGy (132-496). The mean genomic frequencies (x100 +/- SE) of complete and total apparently simple translocations observed in the group of radiologists (1.91 +/- 0.30 and 2.67 +/- 0.34, respectively) were significantly higher than those observed in a matched control group (0.53 +/- 0.10 and 0.87 +/- 0.13, P < 0.01 in both cases) and in another occupationally exposed matched group (0.79 +/- 0.12 and 1.14 +/-0.14, P < 0.03 and P < 0.01, respectively). The discrepancies observed between the physically recorded doses and the biologically estimated doses indicate that the radiologists did not always wear their dosimeters or that the dosimeters were not always in the radiation field.

Hepatoprotective Potential of Some Local Medicinal Plants against 2-Acetylaminoflourene-Induced Damage in Rat

PubMed Central

Adetutu, Adewale; Olorunnisola, Olubukola S.

2013-01-01

The in vivo micronucleus assay was used to examine the anticlastogenic effects of crude extracts of Bridelia ferruginea, Vernonia amygdalina, Tridax procumbens, Ocimum gratissimum, and Lawsonia inermis in Wistar albino rats. Extracts of doses of 100 mg/kg body weight were given to rats in five groups for seven consecutive days followed by a single dose of 2-AAF (0.5 mmol/kg body weight). The rats were sacrificed after 24 hours and their bone marrow smears were prepared on glass slides stained with Giemsa. The micronucleated polychromatic erythrocyte cells (mPCEs) were thereafter recorded. The hepatoprotective effects of the plant extracts against 2-AAF-induced liver toxicity in rats were evaluated by monitoring the levels of alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), and histopathological analysis. The results of the 2-AAF-induced liver toxicity experiments showed that rats treated with the plant extracts (100 mg/kg) showed a significant decrease in mPCEs as compared with the positive control. The rats treated with the plant extracts did not show any significant change in the concentration of ALP and GGT in comparison with the negative control group whereas the 2-AAF group showed a significant increase (P < 0.05) in these parameters. Some of the leaf extracts also showed protective effects against histopathological alterations. This study suggests that the leaf extracts have hepatoprotective potential, thereby justifying their ethnopharmacological uses. PMID:24163694

Rejoining of isochromatid breaks induced by heavy ions in G2-phase normal human fibroblasts

NASA Technical Reports Server (NTRS)

Kawata, T.; Durante, M.; Furusawa, Y.; George, K.; Ito, H.; Wu, H.; Cucinotta, F. A.

2001-01-01

We reported previously that exposure of normal human fibroblasts in G2 phase of the cell cycle to high-LET radiation produces a much higher frequency of isochromatid breaks than exposure to gamma rays. We concluded that an increase in the production of isochromatid breaks is a signature of initial high-LET radiation-induced G2-phase damage. In this paper, we report the repair kinetics of isochromatid breaks induced by high-LET radiation in normal G2-phase human fibroblasts. Exponentially growing human fibroblasts (AG1522) were irradiated with gamma rays or energetic carbon (290 MeV/nucleon), silicon (490 MeV/nucleon), or iron (200 MeV/nucleon) ions. Prematurely condensed chromosomes were induced by calyculin A after different postirradiation incubation times ranging from 0 to 600 min. Chromosomes were stained with Giemsa, and aberrations were scored in cells at G2 phase. G2-phase fragments, the result of the induction of isochromatid breaks, decreased quickly with incubation time. The curve for the kinetics of the rejoining of chromatid-type breaks showed a slight upward curvature with time after exposure to 440 keV/microm iron particles, probably due to isochromatid-isochromatid break rejoining. The formation of chromatid exchanges after exposure to high-LET radiation therefore appears to be underestimated, because isochromatid-isochromatid exchanges cannot be detected. Increased induction of isochromatid breaks and rejoining of isochromatid breaks affect the overall kinetics of chromatid-type break rejoining after exposure to high-LET radiation.

Silica nanoparticles induce multinucleation through activation of PI3K/Akt/GSK-3β pathway and downregulation of chromosomal passenger proteins in L-02 cells

NASA Astrophysics Data System (ADS)

Geng, Weijia; Li, Yang; Yu, Yongbo; Yu, Yang; Duan, Junchao; Jiang, Lizhen; Li, Qiuling; Sun, Zhiwei

2016-04-01

Silica nanoparticles (SNPs) are applicable in various fields due to their unique physicochemical characteristics. However, concerns over their potential adverse effects have been raised. In our previous studies, we reported that SNPs could induce abnormal high incidence of multinucleation. The aim of this study is to further investigate the mechanisms of multinucleation induced by SNPs (68 nm) in human normal liver L-02 cells (L-02 cells). In order to determine the cytotoxicity of SNPs, MTT assay was performed, and the cell viability was decreased in a dose-dependent manner. The intracellular reactive oxygen species (ROS) detected by flow cytometry and multinucleation observed by Giemsa stain showed that ROS generation and rate of multinucleated cells increased after SNPs exposure. N-acetyl-cysteine (NAC), a glutathione precursor against SNP-induced toxicity, was used as a ROS inhibitor to elucidate the relationship between ROS and multinucleation. The presence of NAC resulted in inhibition of both ROS generation and rate of multinucleation. Moreover, Western blot analysis showed that the protein levels of Cdc20, Aurora B, and Survivin were down-regulated, and the PI3K/Akt/GSK-3β pathway was activated by SNPs. In conclusion, our findings strongly suggested that multinucleation induced by SNPs was related to PI3K/Akt/GSK-3β signal pathway activation and downregulation of G2/M phase-related protein and chromosomal passenger proteins.

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae).

PubMed

Bello, Felio J; Mejía, Astrid J; Corena, María del Pilar; Ayala, Martha; Sarmiento, Ladys; Zuñiga, Claudio; Palau, María T

2005-10-01

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37 masculineC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

Antagonism between granulocytic maturation and deacetylase inhibitor-induced apoptosis in acute promyelocytic leukaemia cells.

PubMed

Hennig, D; Müller, S; Wichmann, C; Drube, S; Pietschmann, K; Pelzl, L; Grez, M; Bug, G; Heinzel, T; Krämer, O H

2015-01-20

Transcriptional repression is a key mechanism driving leukaemogenesis. In acute promyelocytic leukaemia (APL), the fusion protein promyelocytic leukaemia-retinoic acid receptor-α fusion (PML-RARα) recruits transcriptional repressors to myeloid differentiation genes. All-trans-retinoic acid (ATRA) induces the proteasomal degradation of PML-RARα and granulocytic differentiation. Histone deacetylases (HDACs) fall into four classes (I-IV) and contribute to the transcription block caused by PML-RARα. Immunoblot, flow cytometry, and May-Grünwald-Giemsa staining were used to analyze differentiation and induction of apoptosis. A PML-RARα- and ATRA-dependent differentiation programme induces granulocytic maturation associated with an accumulation of the myeloid transcription factor CCAAT/enhancer binding protein (C/EBP)ɛ and of the surface protein CD11b. While this process protects APL cells from inhibitors of class I HDAC activity, inhibition of all Zinc-dependent HDACs (classes I, II, and IV) with the pan-HDACi (histone deacetylase inhibitor(s)) LBH589 induces apoptosis of immature and differentiated APL cells. LBH589 can eliminate C/EBPɛ and the mitochondrial apoptosis regulator B-cell lymphoma (BCL)-xL in immature and differentiated NB4 cells. Thus, BCL-xL and C/EBPɛ are newly identified molecular markers for the efficacy of HDACi against APL cells. Our results could explain the therapeutic limitations occurring with ATRA and class I HDACi combinations. Pro-apoptotic effects caused by pan-HDAC inhibition are not blunted by ATRA-induced differentiation and may provide a clinically interesting alternative.

In vivo stimulation of granulopoiesis by recombinant human granulocyte colony-stimulating factor

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

Cohen, A.M.; Zsebo, K.M.; Inoue, H.

1987-04-01

Osmotic pumps containing Escherichia coli-derived recombinant human granulocyte colony-stimulating factor (rhG-CSF) were attached to indwelling jugular vein catheters and implanted subcutaneously into Golden Syrian hamsters. Within 3 days, peripheral granulocyte counts had increased > 10-fold with a concomitant 4-fold increase in total leukocytes. Microscopic examination of Wright-Giemsa-stained blood smears from rhG-CSF hamsters showed that only the neutrophil subpopulation of granulocytes had increased. After subcutaneous injection at /sup 35/S-labeled rhG-CSF doses of up to 10 ..mu..g x kg/sup -1/ x day/sup -1/ only granulocyte counts were affected. However, at higher dose levels, a transient thrombocytopenia was noted. Erythrocyte and lymphocyte/monocyte countsmore » remained unaffected by rhG-CSF over the entire dose range studied. Total leukocyte counts increased 3-fold within 12 hr after a single s.c. injection of rhG-CSF. This early effect was associated with an increase in the total number of colony-forming cells and the percent of active cycling cells in the marrow. A sustained elevation of peripheral leukocyte and marrow progenitor counts was observed following seven daily s.c. injections of rhG-CSF. The ability of rhG-CSF to increase the production and release of granulocytes from the marrow may underlie the beneficial effect it produced on the restoration of peripheral leukocyte counts in hamsters made leukopenic by treatment with 5-fluorouracil.« less

Changes associated with percutaneous ethanol injection in the treatment of thyroid nodules.

PubMed

Schrut, Gianna Carla Alberti; Miasaki, Fabíola Yukiko; Paz-Filho, Gilberto; Cavalcanti, Teresa Cristina Santos; Graf, Hans; de Carvalho, Gisah Amaral

2011-06-01

Percutaneous ethanol injection (PEI) is an alternative therapy for thyroid nodules (TN). However, some concern is raised on its carcinogenic effects. To evaluate the cytological and clinical changes caused by PEI in patients with benign TN. Thirty-nine patients with TN (23.1% hyperfunctioning) were submitted to a median of three PEI sessions. After a median of 17 months, patients were reassessed. A new ultrasound-guided fine needle biopsy (US-FNB) was performed, and the smears were analyzed after May-Grünwald-Giemsa staining. The diagnostic findings and the cellular characteristics were compared before and after treatment. There was an increase in the proportion of nondiagnostic/unsatisfactory results (from 2.5% to 18.9%). No malignant cases were observed. The proportion of moderate/intense macrophage infiltration decreased from 60% to 15%. Before treatment, 23.1% patients had hyperthyroidism, which was completely or partially resolved in 66.7%. By ultrasound, the percentage of homogeneous nodules decreased from 64.0% to 38.4% (p=0.0235), and the mean nodule volume decreased from 13.4 ± 12.2 to 5.3 ± 5.1 cm(3). We demonstrate that PEI increases the proportion of nondiagnostic/unsatisfactory results from US-FNB. Therefore, cytological findings after PEI must be evaluated with caution. Our results also suggest that PEI is an efficacious and safe therapeutic option, with no carcinogenic effects observed on cytological evaluations. Safety and efficacy must be evaluated in larger studies with longer follow-up periods.

Micronuclei and erythrocytic abnormalities frequencies of freshwater fishes: Establishing a baseline for health status

NASA Astrophysics Data System (ADS)

Sousa, Debora Batista Pinheiro; Torres, Audalio Rebelo; Oliveira, Suelen Rosana Sampaio; Castro, Jonatas da Silva; Neta, Raimunda Nonata Fortes Carvalho

2017-11-01

Majority papers shows that micronucleus test and erythrocyte abnormalities are excellent tools such as tools for monitor fish health and the level of impact in aquatic ecosystems. Nevertheless, still do not know the baseline for those changes in freshwater fishes communities in the Brazilian Northeastern river. In this study, we show the level of basis of two species of freshwater fishes (Colossoma macropomum -tambaqui and Oreochromis niloticus - tilápia) with the aim of establish levels of background these species. The animals were collected from Ambude river in the protected area and blood collected from all fish for analysis. Erythrocyte indices—mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)—were calculated. Blood samples from all fish were examined for micronuclear changes after Giemsa staining. Micronuclei were found in fish from from Ambude River. The baseline values determined for tambaqui was (micronuclei= 0.0071±0.0026; MCV=0.0073±0.0037; MCHV=0.0071±0.0024) and tilapia (micronuclei= 0.0061±0.0026; MCV=0.0037±0.0017; MCHV=0.056±0.0036). We belive that, we propose using the genotoxic approach for estimating fish health status as the technique allows examination in locus of live fish without the need for animal euthanasia. Besides, baseline level can be to establish levels of background and patterns to pathological and physiological research of these species in future biomonitoring programs.

Pathogenic waterborne free-living amoebae: An update from selected Southeast Asian countries

PubMed Central

Abdul Majid, Mohamad Azlan; Mahboob, Tooba; Mong, Brandon G. J.; Jaturas, Narong; Richard, Reena Leeba; Tian-Chye, Tan; Phimphila, Anusorn; Mahaphonh, Panomphanh; Aye, Kyaw Nyein; Aung, Wai Lynn; Chuah, Joon; Ziegler, Alan D.; Yasiri, Atipat; Sawangjaroen, Nongyao; Lim, Yvonne A. L.; Nissapatorn, Veeranoot

2017-01-01

Data on the distribution of free-living amoebae is still lacking especially in Southeast Asian region. The aquatic environment revealed a high occurrence of free-living amoebae (FLA) due to its suitable condition and availability of food source, which subsequently causes infection to humans. A total of 94 water samples consisted of both treated and untreated from Laos (31), Myanmar (42), and Singapore (21) were investigated for the presence of pathogenic FLA. Each water sample was filtered and cultured onto non-nutrient agar seeded with live suspension of Escherichia coli and incubated at room temperature. Morphological identification was conducted for both trophozoites and cysts via microscopic stains (Giemsa and immunofluorescence). The presence of Naegleria-like structures was the most frequently encountered in both treated and untreated water samples, followed by Acanthamoeba-like and Vermamoeba-like features. To identify the pathogenic isolates, species-specific primer sets were applied for molecular identification of Acanthamoeba, Naegleria, and Vermamoeba. The pathogenic species of Acanthamoeba lenticulata and A. triangularis were detected from untreated water samples, while Vermamoeba vermiformis was found in both treated and untreated water samples. Our results suggested that poor water quality as well as inadequate maintenance and treatment might be the cause of this alarming problem since chlorine disinfection is ineffective in eradicating these amoebas in treated water samples. Regular monitoring and examination of water qualities are necessary in order to control the growth, hence, further preventing the widespread of FLA infections among the public. PMID:28212409

Pathogenic waterborne free-living amoebae: An update from selected Southeast Asian countries.

PubMed

Abdul Majid, Mohamad Azlan; Mahboob, Tooba; Mong, Brandon G J; Jaturas, Narong; Richard, Reena Leeba; Tian-Chye, Tan; Phimphila, Anusorn; Mahaphonh, Panomphanh; Aye, Kyaw Nyein; Aung, Wai Lynn; Chuah, Joon; Ziegler, Alan D; Yasiri, Atipat; Sawangjaroen, Nongyao; Lim, Yvonne A L; Nissapatorn, Veeranoot

2017-01-01

Data on the distribution of free-living amoebae is still lacking especially in Southeast Asian region. The aquatic environment revealed a high occurrence of free-living amoebae (FLA) due to its suitable condition and availability of food source, which subsequently causes infection to humans. A total of 94 water samples consisted of both treated and untreated from Laos (31), Myanmar (42), and Singapore (21) were investigated for the presence of pathogenic FLA. Each water sample was filtered and cultured onto non-nutrient agar seeded with live suspension of Escherichia coli and incubated at room temperature. Morphological identification was conducted for both trophozoites and cysts via microscopic stains (Giemsa and immunofluorescence). The presence of Naegleria-like structures was the most frequently encountered in both treated and untreated water samples, followed by Acanthamoeba-like and Vermamoeba-like features. To identify the pathogenic isolates, species-specific primer sets were applied for molecular identification of Acanthamoeba, Naegleria, and Vermamoeba. The pathogenic species of Acanthamoeba lenticulata and A. triangularis were detected from untreated water samples, while Vermamoeba vermiformis was found in both treated and untreated water samples. Our results suggested that poor water quality as well as inadequate maintenance and treatment might be the cause of this alarming problem since chlorine disinfection is ineffective in eradicating these amoebas in treated water samples. Regular monitoring and examination of water qualities are necessary in order to control the growth, hence, further preventing the widespread of FLA infections among the public.

Chlorin E6 phototoxicity in L. major and L. braziliensis promastigotes-In vitro study.

PubMed

Pinto, Juliana Guerra; Pereira, André Henrique Correia; de Oliveira, Marco Antonio; Kurachi, Cristina; Raniero, Leandro José; Ferreira-Strixino, Juliana

2016-09-01

Cutaneous leishmaniasis is a zoonosis caused by protozoa of the genus Leishmania. Conventional treatments are long and aggressive, and they trigger a diversity of side effects. Photodynamic Therapy was originally proposed as a treatment for cancer, and it now appears to be a promising therapy for local treatment with fewer side effects of infectious diseases. This study aimed to evaluate Chlorin e6 internalization by Leishmania major and Leishmania braziliensis promastigotes and its viability and effects on mitochondrial activity. Control groups were kept in the dark, while PDT groups received fluence of 10J/cm(2) (660nm). Chlorin internalization was evaluated using confocal microscopy after one hour of incubation for both species. The mitochondrial activity was evaluated by MTT assay, and viability was measured by the Trypan blue exclusion test. Giemsa staining was used to observe morphological changes. PS was internalized in both species and mitochondrial activity changed in all groups. However, the obtained MTT and Trypan results indicated that despite the change in mitochondrial activity in the dark groups, their viability was not affected, whereas the PDT treated groups had significantly reduced viability. Morphology was drastically altered in PDT treated groups, while groups kept in the dark exhibited the standard morphology. This study demonstrates that Chlorin has great potential for being used in PDT as a treatment for cutaneous leishmaniasis, although more studies are needed to determine in vivo application protocols. Copyright © 2016 Elsevier B.V. All rights reserved.

All lesions great and small, part 1: diagnostic cytology in veterinary medicine.

PubMed

Sharkey, Leslie C; Seelig, Davis M; Overmann, Jed

2014-06-01

Cytopathology is a minimally invasive, rapid, and cost-effective diagnostic modality with broad utilization in veterinary medicine. Primary care clinicians often screen common cutaneous and subcutaneous aspirates, with other samples most frequently evaluated by board certified veterinary clinical pathologists in reference laboratories. Wright-Giemsa stains are frequently utilized with the application of ancillary diagnostics such as cytochemistry, immunocytochemistry, flow cytometry, and molecular diagnostic techniques complicated by the need to develop and validate species specific reagents and protocols. The interpretation of veterinary cytology samples must be undertaken with extensive knowledge of the breadth of animal species, which includes familiarity with the frequency and biological behavior of inflammatory, infectious, and neoplastic lesions that are influenced by species, breed, and husbandry conditions. This review is the first of two parts that focus on the most common domestic companion animal species (dog, cat, and horse), taking an organ system approach to survey important lesions that may be unique to veterinary species or have interesting correlates in human medicine. The first of the two-part series covers skin and subcutaneous tissue, the musculoskeletal system, and lymphoid organs. The cytologic features and biological behavior of similar lesions are compared, and selected molecular mechanisms of disease and ancillary diagnostics are reviewed when characterized. Supporting figures illustrate a subset of lesions. While not a comprehensive catalog of veterinary cytology, the goal is to give cytopathologists working in human medicine a general impression of correlates in veterinary practice. Copyright © 2014 Wiley Periodicals, Inc.

Epidemiological Study on Cutaneous Leishmaniasis in an Endemic Area, of Qom Province, Central Iran.

PubMed

Saghafipour, Abedin; Vatandoost, Hassan; Zahraei-Ramazani, Ali Reza; Yaghoobi-Ershadi, Mohammad Reza; Jooshin, Moharram Karami; Rassi, Yavar; Shirzadi, Mohammad Reza; Akhavan, Amir Ahmad; Hanafi-Bojd, Ahmad Ali

2017-09-01

Cutaneous leishmaniasis (CL) is one of the most important health problems in many areas of Iran. There are two forms of the disease in Iran, anthroponotic and zoonotic CL. This study conducted to assess the epidemiological situation of CL in an endemic area of Qom Province, central Iran from Apr to Nov 2015. The sticky paper traps and aspirating tubes were used for collecting adult sand flies. Sherman traps and small insect nets were used to capture rodents and small mammals. Giemsa staining was used for preparing the expanded smear and followed by PCR for identifying the causative agent in human, vectors, and reservoirs. In this study, relative frequency of CL was also calculated. Fourteen species of Phlebotomine sand flies were collected. Phlebotomus papatasi (61.74%) was the predominant species through the period of activity. Overall, 62 Meriones libycus , 8 Nesokia indica , 4 Mus musculus , 16 Allactaga elater and 2 Hemiechinus auritis were caught. PCR technique showed 6 out of 150 P. papatasi (2%), two out of 62 M. libycus (3.23%) and all of suspected human's skin tissue samples (100%) were infected with Leishmania major . The relative frequency of CL was 0.30%. This is the first detection of L. major within P. papatasi , M. libycus and human in Kahak District in Qom Province of Iran. Zoonotic cycle of CL exists in this area, L. major is the causative agent, P. papatasi is the main vector and M. libycus is the main reservoir of the disease.

Enhanced yield of chromosome aberrations after CT examinations in paediatric patients.

PubMed

Stephan, G; Schneider, K; Panzer, W; Walsh, L; Oestreicher, U

2007-05-01

To determine whether computed tomography (CT) could enhance the chromosome aberration yields in paediatric patients. Blood samples were taken before and after CT scans from 10 children for whom the medical justifications for CT examinations were accidental injuries and not diseases as investigated in earlier studies. Chromosome analysis was carried out in lymphocytes by fluorescence plus Giemsa (FPG) staining exclusively in metaphases of the first cell cycle in vitro. The mean blood dose of the 10 children was about 12.9 mGy which was determined by a newly developed dose estimation. Based on more than 20,000 analyzed cells it was found that after CT examination the frequencies of dicentrics (dic) and excess acentric fragments (ace) in lymphocytes were significantly increased. By subdividing the children into two age groups, those with an age from 0.4 years to 9 years and from 10 - 15 years, it became obvious that the observed increase in chromosome aberrations was mainly contributed by the younger age group. In this group the frequency of dicentrics was significantly increased whereas in the older group the observed increase was not significant. Our results demonstrate that CT examinations enhance the dicentrics yields in peripheral lymphocytes of children aged up to 15 years. Since in particular significantly increased dicentric yields could be observed in children with an age from 0.4 - 9 years, it can be assumed that children younger than 10 years may be more radiation sensitive than older subjects.

Performance and usefulness of the Hexagon rapid diagnostic test in children with asymptomatic malaria living in the Mount Cameroon region.

PubMed

Wanji, Samuel; Kimbi, Helen K; Eyong, Joan E; Tendongfor, Nicholas; Ndamukong, Judith L

2008-05-22

Rapid and correct diagnosis of malaria is considered an important strategy in the control of the disease. However, it remains to be determined how well these tests can perform in those who harbour the parasite, but are asymptomatic, so that rapid diagnostic tests (RDTs) could be used in rapid mass surveillance in malaria control programmes. Microscopic and immunochromatographic diagnosis of malaria were performed on blood samples from the hyperendemic Mount Cameroon region. Thin and thick blood films were stained with Giemsa and examined under light microscopy for malaria parasites. The RDT was performed on the blood samples for the detection of Plasmodium species. In addition, the performance characteristics of the test were determined using microscopy as gold standard. Results revealed 40.32% to be positive for microscopy and 34.41% to be positive for the RDT. Parasites were detected in a greater proportion of samples as the parasite density increase. Plasmodium falciparum was the predominant Plasmodium species detected in the study population either by microscopy or by the RDT. Overall, the test recorded a sensitivity and specificity of 85.33% and 95.05% respectively, and an accuracy of 91.40%. The sensitivity and specificity of the RDT increased as parasite densities increased. The Hexagon Malaria Combi test showed a high sensitivity and specificity in diagnosing malaria in asymptomatic subjects and so could be suitable for use in mass surveillance programmes for the management and control of malaria.

Comprehensive high-resolution genomic profiling and cytogenetics of human chondrocyte cultures by GTG-banding, locus-specific FISH, SKY and SNP array.

PubMed

Wallenborn, M; Petters, O; Rudolf, D; Hantmann, H; Richter, M; Ahnert, P; Rohani, L; Smink, J J; Bulwin, G C; Krupp, W; Schulz, R M; Holland, H

2018-04-23

In the development of cell-based medicinal products, it is crucial to guarantee that the application of such an advanced therapy medicinal product (ATMP) is safe for the patients. The consensus of the European regulatory authorities is: "In conclusion, on the basis of the state of art, conventional karyotyping can be considered a valuable and useful technique to analyse chromosomal stability during preclinical studies". 408 chondrocyte samples (84 monolayers and 324 spheroids) from six patients were analysed using trypsin-Giemsa staining, spectral karyotyping and fluorescence in situ hybridisation, to evaluate the genetic stability of chondrocyte samples from non-clinical studies. Single nucleotide polymorphism (SNP) array analysis was performed on chondrocyte spheroids from five of the six donors. Applying this combination of techniques, the genetic analyses performed revealed no significant genetic instability until passage 3 in monolayer cells and interphase cells from spheroid cultures at different time points. Clonal occurrence of polyploid metaphases and endoreduplications were identified associated with prolonged cultivation time. Also, gonosomal losses were observed in chondrocyte spheroids, with increasing passage and duration of the differentiation phase. Interestingly, in one of the donors, chromosomal aberrations that are also described in extraskeletal myxoid chondrosarcoma were identified. The SNP array analysis exhibited chromosomal aberrations in two donors and copy neutral losses of heterozygosity regions in four donors. This study showed the necessity of combined genetic analyses at defined cultivation time points in quality studies within the field of cell therapy.

Prognostic impact of circulating plasma cells in patients with multiple myeloma: implications for plasma cell leukemia definition.

PubMed

Granell, Miquel; Calvo, Xavier; Garcia-Guiñón, Antoni; Escoda, Lourdes; Abella, Eugènia; Martínez, Clara Mª; Teixidó, Montserrat; Gimenez, Mª Teresa; Senín, Alicia; Sanz, Patricia; Campoy, Desirée; Vicent, Ana; Arenillas, Leonor; Rosiñol, Laura; Sierra, Jorge; Bladé, Joan; de Larrea, Carlos Fernández

2017-06-01

The presence of circulating plasma cells in patients with multiple myeloma is considered a marker for highly proliferative disease. In the study herein, the impact of circulating plasma cells assessed by cytology on survival of patients with multiple myeloma was analyzed. Wright-Giemsa stained peripheral blood smears of 482 patients with newly diagnosed myeloma or plasma cell leukemia were reviewed and patients were classified into 4 categories according to the percentage of circulating plasma cells: 0%, 1-4%, 5-20%, and plasma cell leukemia with the following frequencies: 382 (79.2%), 83 (17.2%), 12 (2.5%) and 5 (1.0%), respectively. Median overall survival according to the circulating plasma cells group was 47, 50, 6 and 14 months, respectively. At multivariate analysis, the presence of 5 to 20% circulating plasma cells was associated with a worse overall survival (relative risk 4.9, 95% CI 2.6-9.3) independently of age, creatinine, the Durie-Salmon system stage and the International Staging System (ISS) stage. Patients with ≥5% circulating plasma cells had lower platelet counts (median 86×10 9 /L vs 214×10 9 /L, P <0.0001) and higher bone marrow plasma cells (median 53% vs 36%, P =0.004). The presence of ≥5% circulating plasma cells in patients with multiple myeloma has a similar adverse prognostic impact as plasma cell leukemia. Copyright© Ferrata Storti Foundation.

Chromosome mapping of repetitive DNAs in sergeant major fishes (Abudefdufinae, Pomacentridae): a general view on the chromosomal conservatism of the genus.

PubMed

Getlekha, Nuntaporn; Cioffi, Marcelo de Bello; Yano, Cassia Fernanda; Maneechot, Nuntiya; Bertollo, Luiz Antonio Carlos; Supiwong, Weerayuth; Tanomtong, Alongklod; Molina, Wagner Franco

2016-10-01

Species of the Abudefduf genus (sergeant-majors) are widely distributed in the Indian, Pacific and Atlantic oceans, with large schools inhabiting rocky coastal regions and coral reefs. This genus consists of twenty recognized species are of generalist habit, showing typical characteristics of colonizers. Some populations maintain gene flow between large oceanic areas, a condition that may influence their cytogenetic features. A number of species have been shown to be invaders and able to hybridize with local species. However, cytogenetic data in this genus are restricted to few species. In this way, the present study includes the chromosomal investigation, using conventional (Giemsa staining, Ag-NOR and C-banding) and molecular (in situ mapping of six different repetitive DNA classes) approaches in four Abudefduf species from different oceanic regions (A. bengalensis and A. sexfasciatus from the Indo-Pacific, A. vaigiensis from the Indian and A. saxatilis from the Atlantic oceans, respectively), to investigate the evolutionary events associated with the chromosomal diversification in this group. All species share a similar karyotype (2n = 48; NF = 52), except A. sexfasciatus (2n = 48; NF = 50), which possesses a characteristic pericentric inversion in the NOR-bearing chromosomal pair. Mapping of repetitive sequences suggests a chromosomal conservatism in this genus. The high karyotypic similarity between allopatric species of Abudefduf may be related to the success of natural viable hybrids among species with recent secondary contact.

Determination of ABO blood grouping from human oral squamous epithelium by the highly sensitive immunohistochemical staining method EnVision+.

PubMed

Noda, Hiroshi; Yokota, Makoto; Tatsumi, Shinji; Sugiyama, Shizuyuki

2002-03-01

Using the highly sensitive immunohistochemical staining method EnVision+, which employs a dextran polymer reagent for the secondary antibody, the detection of the ABH antigens was attempted in the oral squamous epithelium. This new technique uses monoclonal antibody as a primary antibody and it takes about three hours for staining. The time is much shorter than conventional absorption-elution testing or absorption-inhibition testing for the determination of ABO blood grouping. Secretor saliva samples were stained at strong intensity by the antibody, which corresponded to its blood group and anti-H. On the one hand, nonsecretor saliva samples were stained at strong intensity only by the antibody that corresponded to its blood group, and at weak intensity only by anti-H. Since human oral squamous epithelium antigens were stained specifically by this method, we can examine the ABO blood group of saliva samples and perform cytodiagnosis at the same time. Our research suggested that the EnVision+ Method is a useful technique for ABO blood grouping of saliva in forensic cases.

Prevalence of amebiasis in inflammatory bowel disease in Turkey.

PubMed

Ustun, Sebnem; Dagci, Hande; Aksoy, Umit; Guruz, Yuksel; Ersoz, Galip

2003-08-01

To explore the prevalence of amebiasis in inflammatory bowel disease (IBD) in Turkey. In this study, amoeba prevalence in 160 cases of IBD, 130 of ulcerative colitis and 30 of Crohn's disease were investigated in fresh faeces by means of wet mount+Lugol's iodine staining, modified formol ethyl acetate and trichrome staining methods and to compare the diagnostic accuracy of wet mount+Lugol's iodine staining, modified formol ethyl acetate and trichrome staining methods in the diagnosis of Entamoeba histolytica (E. histolytica)/ Entamoeba dispar (E. dispar). E. histolytica/E. dispar cysts and trophozoites were found in 14 (8.75 %) of a total of 160 cases, 13 (10.0 %) of the 130 patients with ulcerative colitis and 1 (3.3 %) of the 30 patients with Crohn's disease. As for the 105 patients in the control group who had not any gastrointestinal complaints, 2 (1.90 %) patients were found to have E. histolytica /E. dispar cysts in their faeces. Parasite prevalence in the patient group was determined to be significantly higher than that in the control group (Fischer's Exact Test, P<0.05). When the three methods of determining parasites were compared with one another, the most effective one was found to be trichrome staining method (Kruskal-Wallis Test, P<0.01). Consequently, amoeba infections in IBD cases have a greater prevalence compared to the normal population. The trichrome staining method is more effective for the detection of E. histolytica /E. dispar than the wet mount+Lugol's iodine staining, modified formol ethyl acetate methods.

Propidium iodide (PI) stains Nissl bodies and may serve as a quick marker for total neuronal cell count.

PubMed

Niu, Junfei; Li, Chunman; Wu, Haihui; Feng, Xianling; Su, Qingning; Li, Shihe; Zhang, Lihong; Yew, David Tai Wai; Cho, Eric Yu Pang; Sha, Ou

2015-03-01

Propidium iodide (PI) reacts with both DNA and RNA and is a commonly used fluorescent reagent for nucleic acid staining. The aim of the study was to compare the cellular staining patterns of PI with that of Nissl staining in rat nervous tissues and to report a modified staining method that selectively labels Nissl bodies in neurons. Cryosections and paraffin sections of different tissues of normal Sprague-Dawley rats, including trigeminal ganglia, dorsal root ganglia, spinal cord, liver, and small intestine, were stained by either PI or the hematoxylin and eosin method. Some sections were treated with RNase or DNase before the above staining, and some were double stained with PI and a Nissl stain. The sections were observed by light, fluorescence or confocal microscopy. Results showed strong PI signals detected as patterns of granules in the neuronal cytoplasm of all nervous tissues, whereas the staining of neuronal nuclei was weaker. In contrast, nuclei of neuroglial cells were strongly stained by PI, while the cytoplasm was not obviously stained. Pretreatment of the neural tissue with RNase abolished the PI signals. Furthermore, the PI positive granules in neuronal cytoplasm co-localized with Nissl bodies stained by the fluorescent Nissl stain. When the tissue was pretreated with DNase, PI only stained the cytoplasmic granules of neurons, but not that of glial cells. Our results show that PI stains Nissl bodies and may serve as an economical and convenient neuron marker for neuronal cell counting when specific neural markers such as antibodies are not readily available. Copyright © 2015. Published by Elsevier GmbH.

[Comparison of four different staining methods for ear cytology of dogs with otitis externa].

PubMed

Bouassiba, C; Osthold, W; Mueller, R S

2013-01-01

Cytological examination is crucial for the diagnosis and classification of canine otitis externa. Staining should reveal micro-organisms as perpetuating factors of otitis externa. The aim of the study was to compare four different staining methods (Diff-Quik®, Diff-Quik® after dipping in acetone, Gram Quick stain® and a commercial rapid stain for otitis externa) for ear cytology of dogs with otitis externa and to investigate the agreement of cytology and culture. In a study evaluating dogs with otitis externa, five ear swabs (one for culture and four for cytology) were taken from the horizontal part of the external auditory canal of 224 affected ears and compared semi-quantitatively. Diff-Quik® with and without prior dipping in acetone as well as the Gram Quick stain® displayed a high degree of agreement in the detection of micro-organisms (cocci p = 0.2366; rods p = 0.4832; yeasts p = 0.1574), while the commercial otitis rapid stain revealed significantly less micro-organisms (p < 0.001 for all comparisons). The results of the first three stains corresponded to the culture results by >  70%; the agreement was lower with the commercial otitis rapid stain. The quickest and easiest method was staining with Diff-Quik®. Diff-Quik® with or without prior dipping in acetone and the Gram Quick stain® had a high agreement in the detection of microorganisms and can thus be considered nearly equivalent for the diagnosis of otitis externa infectiosa. The commercial otitis rapid stain is less reliable. Based on this study Diff-Quik® can be recommended for the routine cytology of ear swabs. Additionally, a culture may be indicated and must be interpreted in the context of the cytology.

A comparative study to evaluate liquid dish washing soap as an alternative to xylene and alcohol in deparaffinization and hematoxylin and eosin staining

PubMed Central

Pandey, Pinki; Dixit, Alok; Tanwar, Aparna; Sharma, Anuradha; Mittal, Sanjeev

2014-01-01

Introduction: Our study presents a new deparaffinizing and hematoxylin and eosin (H and E) staining method that involves the use of easily available, nontoxic and eco-friendly liquid diluted dish washing soap (DWS) by completely eliminating expensive and hazardous xylene and alcohol from deparaffinizing and rehydration prior to staining, staining and from dehydration prior to mounting. The aim was to evaluate and compare the quality of liquid DWS treated xylene and alcohol free (XAF) sections with that of the conventional H and E sections. Materials and Methods: A total of 100 paraffin embedded tissue blocks from different tissues were included. From each tissue block, one section was stained with conventional H and E (normal sections) and the other with XAF H and E (soapy sections) staining method. Slides were scored using five parameters: Nuclear, cytoplasmic, clarity, uniformity, and crispness of staining. Z-test was used for statistical analysis. Results: Soapy sections scored better for cytoplasmic (90%) and crisp staining (95%) with a statistically significant difference. Whereas for uniformity of staining, normal sections (88%) scored over soapy sections (72%) (Z = 2.82, P < 0.05). For nuclear (90%) and clarity of staining (90%) total scored favored soapy sections, but the difference was not statistically significant. About 84% normal sections stained adequately for diagnosis when compared with 86% in soapy sections (Z = 0.396, P > 0.05). Conclusion: Liquid DWS is a safe and efficient alternative to xylene and alcohol in deparaffinization and routine H and E staining procedure. We are documenting this project that can be used as a model for other histology laboratories. PMID:25328332

Blood culture gram stain, acridine orange stain and direct sensitivity-based antimicrobial therapy of bloodstream infection in patients with trauma.

PubMed

Behera, B; Mathur, P; Gupta, B

2010-01-01

The purpose of this study was to ascertain if the simple practice of Gram stain, acridine orange stain and direct sensitivity determination of positive blood culture bottles could be used to guide early and appropriate treatment in trauma patients with clinical suspicion of sepsis. The study also aimed to evaluate the error in interpreting antimicrobial sensitivity by direct method when compared to standard method and find out if specific antibiotic-organism combination had more discrepancies. Findings from consecutive episodes of blood stream infection at an Apex Trauma centre over a 12-month period are summarized. A total of 509 consecutive positive blood cultures were subjected to Gram staining. AO staining was done in BacT/ALERT-positive Gram-stain negative blood cultures. Direct sensitivity was performed from 369 blood culture broths, showing single type of growth in Gram and acridine orange staining. Results of direct sensitivity were compared to conventional sensitivity for errors. No 'very major' discrepancy was found in this study. About 5.2 and 1.8% minor error rates were noted in gram-positive and gram-negative bacteria, respectively, while comparing the two methods. Most of the discrepancies in gram-negative bacteria were noted in beta lactam - beta lactamase inhibitor combinations. Direct sensitivity testing was not reliable for reporting of methicillin and vancomycin resistance in Staphylococci. Gram stain result together with direct sensitivity testing is required for optimizing initial antimicrobial therapy in trauma patients with clinical suspicion of sepsis. Gram staining and AO staining proved particularly helpful in the early detection of candidaemia.

Prevalence of amebiasis in inflammatory bowel disease in Turkey

PubMed Central

Ustun, Sebnem; Dagci, Hande; Aksoy, Umit; Guruz, Yuksel; Ersoz, Galip

2003-01-01

AIM: To explore the prevalence of amebiasis in inflammatory bowel disease (IBD) in Turkey. METHODS: In this study, amoeba prevalence in 160 cases of IBD, 130 of ulcerative colitis and 30 of Crohn’s disease were investigated in fresh faeces by means of wet mount+Lugol’s iodine staining, modified formol ethyl acetate and trichrome staining methods and to compare the diagnostic accuracy of wet mount+Lugol’s iodine staining, modified formol ethyl acetate and trichrome staining methods in the diagnosis of Entamoeba histolytica (E. histolytica)/ Entamoeba dispar (E. dispar). RESULTS: E. histolytica/E. dispar cysts and trophozoites were found in 14 (8.75%) of a total of 160 cases, 13 (10.0%) of the 130 patients with ulcerative colitis and 1 (3.3%) of the 30 patients with Crohn’s disease. As for the 105 patients in the control group who had not any gastrointestinal complaints, 2 (1.90%) patients were found to have E. histolytica /E. dispar cysts in their faeces. Parasite prevalence in the patient group was determined to be significantly higher than that in the control group (Fischer’s Exact Test, P < 0.05). When the three methods of determining parasites were compared with one another, the most effective one was found to be trichrome staining method (Kruskal-Wallis Test, P < 0.01). CONCLUSION: Consequently, amoeba infections in IBD cases have a greater prevalence compared to the normal population. The trichrome staining method is more effective for the detection of E. histolytica /E. dispar than the wet mount+Lugol’s iodine staining, modified formol ethyl acetate methods. PMID:12918132

Use of two conventional staining methods to assess the acrosomal status of stallion spermatozoa.

PubMed

Runcan, E E; Pozor, M A; Zambrano, G L; Benson, S; Macpherson, M L

2014-07-01

The acrosome is a highly specialised region of the spermatozoon that is essential for fertilisation. Defects or dysfunction of this structure have been associated with fertility problems in man and various domestic species including stallions. Current methods of evaluating the acrosome of stallion spermatozoa are time consuming and require specialised equipment, which is cost prohibitive to the average practitioner. To evaluate 2 conventional stains (Dip Quick and Spermac) and determine their usefulness in assessing acrosome integrity in stallions as compared with specific acrosomal labelling with a fluorescein-conjugated lectin - a method that has been validated for acrosome status evaluation in stallions. In vivo experimental design. Semen from 6 mature Miniature horse stallions of known fertility was collected on 5 separate occasions. To increase the number of reacted acrosomes, portions of each ejaculate were incubated with the calcium ionophore, A23187. Ejaculates were divided and semen samples were processed according to recommendations for fluorescein-conjugated peanut lectin, Pisum sativum agglutin, Dip Quick, and Spermac staining methods. Slides were evaluated independently by 2 separate investigators. Spermatozoa were classified as having intact, reacting, reacted or defective acrosomes. All parameters obtained by both investigators, using all 3 staining methods were highly correlated (P<0.001). There was no statistical difference (P>0.05) between investigators or staining method for the percentages of intact or reacted acrosomes. However, there was a significant difference between investigators and staining methods for determining reacting acrosome percentages (P<0.05). Dip Quick and Spermac stains are useful for determining intact vs. reacted acrosomes for stallion spermatozoa. © 2013 EVJ Ltd.

An improved silver staining procedure for schizodeme analysis in polyacrylamide gradient gels.

PubMed

Gonçalves, A M; Nehme, N S; Morel, C M

1990-01-01

A simple protocol is described for the silver staining of polyacrylamide gradient gels used for the separation of restriction fragments of kinetoplast DNA [schizodeme analysis of trypanosomatids (Morel et al., 1980)]. The method overcomes the problems of non-uniform staining and strong background color which are frequently encountered when conventional protocols for silver staining of linear gels are applied to gradient gels. The method described has proven to be of general applicability for DNA, RNA and protein separations in gradient gels.

Staining of retinal neurons in the isolated eyecup by extracellular horseradish peroxidase injection.

PubMed

Amthor, F R; Jackson, C A

1986-01-01

A reliable method has been developed for the staining of mammalian retinal neurons in the isolated eyecup. Small injections of horseradish peroxidase are made at a number of places on the retinal surface while the hemisected eyecup preparation floats in Eagle's medium. After 30 min, the retinas are removed, fixed, and reacted with diaminobenzidine. Golgi-like staining of fine dendrites, spines and axons of ganglion cells is achieved. The method also stains amacrine, horizontal and bipolar cells.

Microscopic quantification of bacterial invasion by a novel antibody-independent staining method.

PubMed

Agerer, Franziska; Waeckerle, Stephanie; Hauck, Christof R

2004-10-01

Microscopic discrimination between extracellular and invasive, intracellular bacteria is a valuable technique in microbiology and immunology. We describe a novel fluorescence staining protocol, called FITC-biotin-avidin (FBA) staining, which allows the differentiation between extracellular and intracellular bacteria and is independent of specific antibodies directed against the microorganisms. FBA staining of eukaryotic cells infected with Gram-negative bacteria of the genus Neisseria or the Gram-positive pathogen Staphylococcus aureus are employed to validate the novel technique. The quantitative evaluation of intracellular pathogens by the FBA staining protocol yields identical results compared to parallel samples stained with conventional, antibody-dependent methods. FBA staining eliminates the need for cell permeabilization resulting in robust and rapid detection of invasive microbes. Taken together, FBA staining provides a reliable and convenient alternative for the differential detection of intracellular and extracellular bacteria and should be a valuable technical tool for the quantitative analysis of the invasive properties of pathogenic bacteria and other microorganisms.

Evaluation of detection methods for Campylobacter infections among under-fives in Mwanza City, Tanzania.

PubMed

Mushi, Martha Fidelis; Paterno, Laurent; Tappe, Dennis; Deogratius, Anna Pendo; Seni, Jeremiah; Moremi, Nyambura; Mirambo, Mariam Mwijuma; Mshana, Stephen Eliatosha

2014-01-01

Campylobacter species are recognized as a major cause of acute gastroenteritis in humans throughout the world. The diagnosis is mainly based on stool culture. This study was done to evaluate the effectiveness of staining methods (Gram stain using 0.3% carbol fuchsin as counter stain and 1% carbol fuchsin direct stain) versus culture as the gold standard. A total of 300 children attending Bugando Medical Centre (BMC) and the Sekou Toure regional hospital with acute watery diarrhea were enrolled. Two sets of slides were prepared stained with 1% carbol fuchsin for 30 seconds first set, and the second set stained with Gram's stain using 0.3% carbol fuchsin as counter stain for five minutes. Concurrently, stool samples were inoculated on Preston Agar selective. Of 300 stool specimens, 14(4.7%) showed positive culture after 48 hours of incubation and 28 (9.3%) shows typical morphology of Campylobacter species by both Gram stain and direct stain. The sensitivity of the Gram stain using 0.3% carbol fuchsin as counter stain and 1% carbol fuchsin simple stain versus culture as gold standard was 64.3%, with a specificity of 93.4%. The positive predictive value and negative predictive value were 32.1% and 98.2% respectively. The detection of Campylobacter by 1% carbol fuchsin is simple, inexpensive, and fast, with both a high sensitivity and specificity. Laboratories in settings with high prevalence of campylobacteriosis and/or limited resources can employ 1% carbol fuchsin direct stain in detecting campylobacter infections.

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy†

PubMed Central

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-soo; Torelli, Marco D.; Hamers, Robert J.; Murhpy, Catherine J.; Orr, Galya

2015-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate eficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells. PMID:24816810

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

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

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localizationmore » patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.« less

Novel method for immunofluorescence staining of mammalian eggs using non-contact alternating-current electric-field mixing of microdroplets

PubMed Central

Hiromitsu, Shirasawa; Jin, Kumagai; Emiko, Sato; Katsuya, Kabashima; Yukiyo, Kumazawa; Wataru, Sato; Hiroshi, Miura; Ryuta, Nakamura; Hiroshi, Nanjo; Yoshihiro, Minamiya; Yoichi, Akagami; Yukihiro, Terada

2015-01-01

Recently, a new technique was developed for non-catalytically mixing microdroplets. In this method, an alternating-current (AC) electric field is used to promote the antigen–antibody reaction within the microdroplet. Previously, this technique has only been applied to histological examinations of flat structures, such as surgical specimens. In this study, we applied this technique for the first time to immunofluorescence staining of three-dimensional structures, specifically, mammalian eggs. We diluted an antibody against microtubules from 1:1,000 to 1:16,000, and compared the chromatic degree and extent of fading across dilutions. In addition, we varied the frequency of AC electric-field mixing from 5 Hz to 46 Hz and evaluated the effect on microtubule staining. Microtubules were more strongly stained after AC electric-field mixing for only 5 minutes, even when the concentration of primary antibody was 10 times lower than in conventional methods. AC electric-field mixing also alleviated microtubule fading. At all frequencies tested, AC electric-field mixing resulted in stronger microtubule staining than in controls. There was no clear difference in a microtubule staining between frequencies. These results suggest that the novel method could reduce antibody consumption and shorten immunofluorescence staining time. PMID:26477850

Feasibility of a tetracycline-binding method for detecting synovial fluid basic calcium phosphate crystals.

PubMed

Rosenthal, Ann K; Fahey, Mark; Gohr, Claudia; Burner, Todd; Konon, Irina; Daft, Laureen; Mattson, Eric; Hirschmugl, Carol; Ryan, Lawrence M; Simkin, Peter

2008-10-01

Basic calcium phosphate (BCP) crystals are common components of osteoarthritis (OA) synovial fluid. Progress in understanding the role of these bioactive particles in clinical OA has been hampered by difficulties in their identification. Tetracyclines stain calcium phosphate mineral in bone. The aim of this study was to investigate whether tetracycline staining might be an additional or alternative method for identifying BCP crystals in synovial fluid. A drop of oxytetracycline was mixed with a drop of fluid containing synthetic or native BCP, calcium pyrophosphate dihydrate (CPPD), or monosodium urate (MSU) crystals and placed on a microscope slide. Stained and unstained crystals were examined by light microscopy, with and without a portable broad-spectrum ultraviolet (UV) pen light. A small set of characterized synovial fluid samples were compared by staining with alizarin red S and oxytetracycline. Synthetic BCP crystals in synovial fluid were quantified fluorimetrically using oxytetracycline. After oxytetracycline staining, synthetic and native BCP crystals appeared as fluorescent amorphous aggregates under UV light. Oxytetracycline did not stain CPPD or MSU crystals or other particulates. Oxytetracycline staining had fewer false-positive test results than did alizarin red S staining and could provide estimates of the quantities of synthetic BCP crystals in synovial fluid. With further validation, oxytetracycline staining may prove to be a useful adjunct or alternative to currently available methods for identifying BCP crystals in synovial fluid.

Seasonal changes in gastric mucosal factors associated with peptic ulcer bleeding.

PubMed

Yuan, Xiao-Gang; Xie, Chuan; Chen, Jiang; Xie, Yong; Zhang, Kun-He; Lu, Nong-Hua

2015-01-01

A close association has been established between climate and peptic ulcer bleeding (PUB). The incidence of PUB in cold climates is significantly higher than that in hot climates. In this study, gastric mucosal damage and its barrier function (through associated barrier factors) in extreme climate conditions were examined to investigate the pathogenesis of PUB in extreme cold climates. Gastric juice and biopsy specimens were collected from 176 patients with peptic ulcer. Conventional hematoxylin and eosin staining was used to exclude malignant ulcers. Helicobacter pylori infections were detected by modified Giemsa staining. pH values of the gastric juice samples were obtained on-site by precise pH dipstick readings. The protein expression levels of heat shock protein (HSP) 70, occludin, nitric oxide synthase (NOS), epidermal growth factor (EGF) and EGF receptor (EGFR) in the gastric mucosa were detected by immunohistochemistry. No significant differences were identified between the high and low bleeding risk groups in the rates of H. pylori infection and the pH values of the gastric juices in the extreme hot or cold climates. Furthermore, no statistically significant differences were identified in the protein expression levels of occludin, NOS, EGF and EGFR between the high and low bleeding risk groups. In the extreme cold climate, the expression of HSP70 and the mucus thickness of the gastric antrum in the high bleeding risk group were significantly lower than those in the low bleeding risk group. The protein expression levels of occludin, HSP70, NOS and EGFR in the extreme cold climate were significantly lower than those in the extreme hot climate, whereas the gastric acid secretion was significantly higher in the extreme cold climate than that in the extreme hot climate. In conclusion, low expression of HSP70 in the gastric mucosa and reduced gastric mucus thickness may play key roles in the mechanism of PUB in extreme cold climates. The significant decrease in barrier factors and increase in damage in extreme cold climates may be associated with the seasonal pattern of peptic ulcers.

Seasonal changes in gastric mucosal factors associated with peptic ulcer bleeding

PubMed Central

YUAN, XIAO-GANG; XIE, CHUAN; CHEN, JIANG; XIE, YONG; ZHANG, KUN-HE; LU, NONG-HUA

2015-01-01

A close association has been established between climate and peptic ulcer bleeding (PUB). The incidence of PUB in cold climates is significantly higher than that in hot climates. In this study, gastric mucosal damage and its barrier function (through associated barrier factors) in extreme climate conditions were examined to investigate the pathogenesis of PUB in extreme cold climates. Gastric juice and biopsy specimens were collected from 176 patients with peptic ulcer. Conventional hematoxylin and eosin staining was used to exclude malignant ulcers. Helicobacter pylori infections were detected by modified Giemsa staining. pH values of the gastric juice samples were obtained on-site by precise pH dipstick readings. The protein expression levels of heat shock protein (HSP) 70, occludin, nitric oxide synthase (NOS), epidermal growth factor (EGF) and EGF receptor (EGFR) in the gastric mucosa were detected by immunohistochemistry. No significant differences were identified between the high and low bleeding risk groups in the rates of H. pylori infection and the pH values of the gastric juices in the extreme hot or cold climates. Furthermore, no statistically significant differences were identified in the protein expression levels of occludin, NOS, EGF and EGFR between the high and low bleeding risk groups. In the extreme cold climate, the expression of HSP70 and the mucus thickness of the gastric antrum in the high bleeding risk group were significantly lower than those in the low bleeding risk group. The protein expression levels of occludin, HSP70, NOS and EGFR in the extreme cold climate were significantly lower than those in the extreme hot climate, whereas the gastric acid secretion was significantly higher in the extreme cold climate than that in the extreme hot climate. In conclusion, low expression of HSP70 in the gastric mucosa and reduced gastric mucus thickness may play key roles in the mechanism of PUB in extreme cold climates. The significant decrease in barrier factors and increase in damage in extreme cold climates may be associated with the seasonal pattern of peptic ulcers. PMID:25452787

An informatics model for guiding assembly of telemicrobiology workstations for malaria collaborative diagnostics using commodity products and open-source software.

PubMed

Suhanic, West; Crandall, Ian; Pennefather, Peter

2009-07-17

Deficits in clinical microbiology infrastructure exacerbate global infectious disease burdens. This paper examines how commodity computation, communication, and measurement products combined with open-source analysis and communication applications can be incorporated into laboratory medicine microbiology protocols. Those commodity components are all now sourceable globally. An informatics model is presented for guiding the use of low-cost commodity components and free software in the assembly of clinically useful and usable telemicrobiology workstations. The model incorporates two general principles: 1) collaborative diagnostics, where free and open communication and networking applications are used to link distributed collaborators for reciprocal assistance in organizing and interpreting digital diagnostic data; and 2) commodity engineering, which leverages globally available consumer electronics and open-source informatics applications, to build generic open systems that measure needed information in ways substantially equivalent to more complex proprietary systems. Routine microscopic examination of Giemsa and fluorescently stained blood smears for diagnosing malaria is used as an example to validate the model. The model is used as a constraint-based guide for the design, assembly, and testing of a functioning, open, and commoditized telemicroscopy system that supports distributed acquisition, exploration, analysis, interpretation, and reporting of digital microscopy images of stained malarial blood smears while also supporting remote diagnostic tracking, quality assessment and diagnostic process development. The open telemicroscopy workstation design and use-process described here can address clinical microbiology infrastructure deficits in an economically sound and sustainable manner. It can boost capacity to deal with comprehensive measurement of disease and care outcomes in individuals and groups in a distributed and collaborative fashion. The workstation enables local control over the creation and use of diagnostic data, while allowing for remote collaborative support of diagnostic data interpretation and tracking. It can enable global pooling of malaria disease information and the development of open, participatory, and adaptable laboratory medicine practices. The informatic model highlights how the larger issue of access to generic commoditized measurement, information processing, and communication technology in both high- and low-income countries can enable diagnostic services that are much less expensive, but substantially equivalent to those currently in use in high-income countries.

Molecular phylogeny and karyotype differentiation in Paratelmatobius and Scythrophrys (Anura, Leptodactylidae).

PubMed

Lourenço, L B; Bacci-Júnior, M; Martins, V G; Recco-Pimentel, S M; Haddad, C F B

2008-03-01

Paratelmatobius and Scythrophrys are leptodactylid frogs endemic to the Brazilian Atlantic forest and their close phylogenetic relationship was recently inferred in an analysis that included Paratelmatobius sp. and S. sawayae. To investigate the interspecific relationships among Paratelmatobius and Scythrophrys species, we analyzed a mitochondrial region (approximately 2.4 kb) that included the ribosomal genes 12S and 16S and the tRNAval in representatives of all known localities of these genera and in 54 other species. Maximum parsimony inferences were done using PAUP* and support for the clades was evaluated by bootstrapping. A cytogenetic analysis using Giemsa staining, C-banding and silver staining was also done for those populations of Paratelmatobius not included in previous cytogenetic studies of this genus in order to assess their karyotype differentiation. Our results suggested Paratelmatobius and Scythrophrys formed a clade strongly supported by bootstrapping, which corroborated their very close phylogenetic relationship. Among the Paratelmatobius species, two clades were identified and corroborated the groups P. mantiqueira and P. cardosoi previously proposed based on morphological characters. The karyotypes of Paratelmatobius sp. 2 and Paratelmatobius sp. 3 described here had diploid chromosome number 2n = 24 and showed many similarities with karyotypes of other Paratelmatobius representatives. The cytogenetic data and the phylogenetic analysis allowed the proposal/corroboration of several hypotheses for the karyotype differentiation within Paratelmatobius and Scythrophrys. Namely the telocentric pair No. 4 represented a synapomorphy of P. cardosoi and Paratelmatobius sp. 2, while chromosome pair No. 5 with interstitial C-bands could be interpreted as a synapomorphy of the P. cardosoi group. The NOR-bearing chromosome No. 10 in the karyotype of P. poecilogaster was considered homeologous to chromosome No. 10 in the karyotype of Scythrophrys sp., chromosome No. 9 in the karyotype of Paratelmatobius sp. 1, chromosome No. 8 in the karyotypes of Paratelmatobius sp. 2 and of Paratelmatobius sp. 3, and chromosome No. 7 in the karyotype of P. cardosoi. A hypothesis for the evolutionary divergence of these NOR-bearing chromosomes, which probably involved events like gain in heteochromatin, was proposed.

Thymic stromal lymphopoietin expression is increased in nasal epithelial cells of patients with mugwort pollen sensitive-seasonal allergic rhinitis.

PubMed

Zhu, Dong-dong; Zhu, Xue-wei; Jiang, Xiao-dan; Dong, Zhen

2009-10-05

Excessive expression of thymic stromal lymphopoietin (TSLP) has been demonstrated in asthmatic airway epithelia and in nasal epithelia from animal models of allergic rhinitis (AR), but the evidence of expression of TSLP in nasal epithelial cells (NECs) of patients with AR is lacking. We aimed to investigate the expression of TSLP in NECs of patients with mugwort sensitive-seasonal AR and determine whether it is associated with severity of symptoms and the number of infiltrated eosinophils in nasal mucosa. NECs specimens were obtained by scraping with plastic curettes from the nasal inferior turbinates of patients with mugwort pollen sensitive-seasonal AR (n = 22) and nonallergic controls (n = 11) during last peak mugwort pollen season. The severity of nasal symptom was assessed using a Visual Analog Scale (VAS). In addition, serum mugwort pollen IgE levels were tested from each patient. In situ hybridization (ISH) was performed to test the messenger RNA (mRNA) of TSLP in the NECs. Furthermore, immunohistochemical staining (IHC) was scored to evaluate the expression of TSLP and eosinophil cell count was made by May-Grünwald/Giemsa staining. The correlation between expression of TSLP and all other parameters was analyzed in this study. The mRNA level of TSLP was significantly increased in NECs of patients with AR compared with the nonallergic control group (P < 0.05). In addition, IHC results showed that expression of TSLP in NECs from patients with AR was up-regulated which was correlated with VAS score (r = 0.598; P < 0.05) and nasal eosinophils count (r = 0.702; P < 0.05), but it was unrelated with mugwort pollen specific IgE level. These preliminary findings indicate a potential relationship between TSLP expression, severity of symptoms and nasal eosinophils count in pathogenesis of AR, but TSLP expression did not correlate with mugwort pollen specific IgE level. The elevated expression of TSLP might play a critical role in local atopical responses of AR. In the future, the TSLP has the potential to be one of the most important molecular markers for AR diagnoses and assessment.