Science.gov

Sample records for detecting filarial infective

  1. Lymphangiosarcoma after filarial infection

    SciTech Connect

    Sordillo, E.M.; Sordillo, P.P.; Hajdu, S.I.; Good, R.A.

    1981-03-01

    A case of lymphangiosarcoma of a lower extremity is described in a patient with chronic lymphedema of that leg from a filarial infection in childhood. Histologically, the neoplasm resembled lymphangiosarcomas that arise in arms that become lymphedematous after mastectomies, but was different in that it also contained areas of calcification consistent with prior filarial infection. Calcifications were also present in muscle uninvolved by the lymphangiosarcoma of this case. The prolonged survival of this patient is unlike that of most patients with lymphangiosarcoma, which is generally shorter. Although lymphedema after filariasis is common, this is the first case of a lymphangiosarcoma arising in chronic lymphedema of filarial origin.

  2. Detection of circulating parasite-derived microRNAs in filarial infections.

    PubMed

    Tritten, Lucienne; Burkman, Erica; Moorhead, Andrew; Satti, Mohammed; Geary, James; Mackenzie, Charles; Geary, Timothy

    2014-07-01

    Filarial nematodes cause chronic and profoundly debilitating diseases in both humans and animals. Applications of novel technology are providing unprecedented opportunities to improve diagnosis and our understanding of the molecular basis for host-parasite interactions. As a first step, we investigated the presence of circulating miRNAs released by filarial nematodes into the host bloodstream. miRNA deep-sequencing combined with bioinformatics revealed over 200 mature miRNA sequences of potential nematode origin in Dirofilaria immitis-infected dog plasma in two independent analyses, and 21 in Onchocerca volvulus-infected human serum. Total RNA obtained from D. immitis-infected dog plasma was subjected to stem-loop RT-qPCR assays targeting two detected miRNA candidates, miR-71 and miR-34. Additionally, Brugia pahangi-infected dog samples were included in the analysis, as these miRNAs were previously detected in extracts prepared from this species. The presence of miR-71 and miR-34 discriminated infected samples (both species) from uninfected samples, in which no specific miRNA amplification occurred. However, absolute miRNA copy numbers were not significantly correlated with microfilaraemia for either parasite. This may be due to the imprecision of mf counts to estimate infection intensity or to miRNA contributions from the unknown number of adult worms present. Nonetheless, parasite-derived circulating miRNAs are found in plasma or serum even for those species that do not live in the bloodstream. PMID:25033073

  3. Detection of circulating parasite-derived microRNAs in filarial infections.

    PubMed

    Tritten, Lucienne; Burkman, Erica; Moorhead, Andrew; Satti, Mohammed; Geary, James; Mackenzie, Charles; Geary, Timothy

    2014-07-01

    Filarial nematodes cause chronic and profoundly debilitating diseases in both humans and animals. Applications of novel technology are providing unprecedented opportunities to improve diagnosis and our understanding of the molecular basis for host-parasite interactions. As a first step, we investigated the presence of circulating miRNAs released by filarial nematodes into the host bloodstream. miRNA deep-sequencing combined with bioinformatics revealed over 200 mature miRNA sequences of potential nematode origin in Dirofilaria immitis-infected dog plasma in two independent analyses, and 21 in Onchocerca volvulus-infected human serum. Total RNA obtained from D. immitis-infected dog plasma was subjected to stem-loop RT-qPCR assays targeting two detected miRNA candidates, miR-71 and miR-34. Additionally, Brugia pahangi-infected dog samples were included in the analysis, as these miRNAs were previously detected in extracts prepared from this species. The presence of miR-71 and miR-34 discriminated infected samples (both species) from uninfected samples, in which no specific miRNA amplification occurred. However, absolute miRNA copy numbers were not significantly correlated with microfilaraemia for either parasite. This may be due to the imprecision of mf counts to estimate infection intensity or to miRNA contributions from the unknown number of adult worms present. Nonetheless, parasite-derived circulating miRNAs are found in plasma or serum even for those species that do not live in the bloodstream.

  4. Neurological manifestations of filarial infections.

    PubMed

    Bhalla, Devender; Dumas, Michel; Preux, Pierre-Marie

    2013-01-01

    Filarial infections cause a huge public health burden wherever they are endemic. These filaria may locate anywhere in the human body. Their manifestations and pathogenic mechanisms, except the most common ones, are rarely investigated systematically. Their neurological manifestations, however, are being increasingly recognized particularly with onchocerciasis or Loa loa infections, Wuchereria bancrofti, or Mansonella perstans. The risk of developing these manifestations may also increase in cases that harbor multiple filariasis or coinfections, for instance as with Plasmodium. The microfilaria of Onchocerca and Loa loa are seen in cerebrospinal fluid. The pathogenesis of neurological manifestations of these infections is complex; however, pathogenic reactions may be caused by mechanical disruption, e.g., degeneration often followed by granulomas, causing fibrosis or mass effects on other tissues, vascular lesions, e.g., vascular block of cerebral vessels, or disordered inflammatory responses resulting in meningitis, encephalitis or localized inflammatory responses. The chances of having neurological manifestations may also depend upon the frequency and"heaviness"of infection over a lifetime. Hence, this type of infection should no longer be considered a disease of the commonly affected areas but one that may produce systemic effects or other manifestations, and these should be considered in populations where they are endemic. PMID:23829914

  5. Circulating filarial antigen detection in brugian filariasis.

    PubMed

    Tripathi, Praveen Kumar; Mahajan, Ramesh Chander; Malla, Nancy; Mewara, Abhishek; Bhattacharya, Shailja Misra; Shenoy, Ranganatha Krishna; Sehgal, Rakesh

    2016-03-01

    Human lymphatic filariasis (LF) is a major cause of disability globally. The success of global elimination programmes for LF depends upon effectiveness of tools for diagnosis and treatment. In this study on stage-specific antigen detection in brugian filariasis, L3, adult worm (AW) and microfilarial antigenaemia were detected in around 90-95% of microfilariae carriers (MF group), 50-70% of adenolymphangitis (ADL) patients, 10-25% of chronic pathology (CP) patients and 10-15% of endemic normal (EN) controls. The sensitivity of the circulating filarial antigen (CFA) detection in serum samples from MF group was up to 95%. In sera from ADL patients, unexpectedly, less antigen reactivity was observed. In CP group all the CFA positive individuals were from CP grade I and II only and none from grade III or IV, suggesting that with chronicity the AWs lose fecundity and start to disintegrate and die. Amongst EN subject, 10-15% had CFA indicating that few of them harbour filarial AWs, thus they might not be truly immune as has been conventionally believed. The specificity for antigen detection was 100% when tested with sera from various other protozoan and non-filarial helminthic infections.

  6. Filarial infection influences mosquito behaviour and fecundity

    PubMed Central

    Gleave, Katherine; Cook, Darren; Taylor, Mark J.; Reimer, Lisa J.

    2016-01-01

    Understanding vector-parasite interactions is increasingly important as we move towards the endpoint goals set by the Global Programme for the Elimination of Lymphatic Filariasis (GPELF), as interaction dynamics may change with reduced transmission pressure. Elimination models used to predict programmatic endpoints include parameters for vector-specific transmission dynamics, despite the fact that our knowledge of the host-seeking behaviour of filariasis infected mosquitoes is lacking. We observed a dynamic, stage-specific and density dependent change in Aedes aegypti behaviour towards host cues when exposed to Brugia malayi filarial parasites. Infected mosquitoes exhibited reduced activation and flight towards a host during the period of larval development (L1/L2), transitioning to a 5 fold increase in activation and flight towards a host when infective stage larvae (L3) were present (p < 0.001). In uninfected control mosquitoes, we observed a reduction in convergence towards a host during the same period. Furthermore, this behaviour was density dependent with non-activated mosquitoes harbouring a greater burden of L1 and L2 larvae while activated mosquitoes harboured a greater number of L3 (p < 0.001). Reductions in fecundity were also density-dependent, and extended to mosquitoes that were exposed to microfilariae but did not support larval development. PMID:27796352

  7. Mosquito Infection Responses to Developing Filarial Worms

    PubMed Central

    Erickson, Sara M.; Xi, Zhiyong; Mayhew, George F.; Ramirez, Jose L.; Aliota, Matthew T.; Christensen, Bruce M.; Dimopoulos, George

    2009-01-01

    Human lymphatic filariasis is a mosquito-vectored disease caused by the nematode parasites Wuchereria bancrofti, Brugia malayi and Brugia timori. These are relatively large roundworms that can cause considerable damage in compatible mosquito vectors. In order to assess how mosquitoes respond to infection in compatible mosquito-filarial worm associations, microarray analysis was used to evaluate transcriptome changes in Aedes aegypti at various times during B. malayi development. Changes in transcript abundance in response to the different stages of B. malayi infection were diverse. At the early stages of midgut and thoracic muscle cell penetration, a greater number of genes were repressed compared to those that were induced (20 vs. 8). The non-feeding, intracellular first-stage larvae elicited few differences, with 4 transcripts showing an increased and 9 a decreased abundance relative to controls. Several cecropin transcripts increased in abundance after parasites molted to second-stage larvae. However, the greatest number of transcripts changed in abundance after larvae molted to third-stage larvae and migrated to the head and proboscis (120 induced, 38 repressed), including a large number of putative, immunity-related genes (∼13% of genes with predicted functions). To test whether the innate immune system of mosquitoes was capable of modulating permissiveness to the parasite, we activated the Toll and Imd pathway controlled rel family transcription factors Rel1 and Rel2 (by RNA interference knockdown of the pathway's negative regulators Cactus and Caspar) during the early stages of infection with B. malayi. The activation of either of these immune signaling pathways, or knockdown of the Toll pathway, did not affect B. malayi in Ae. aegypti. The possibility of LF parasites evading mosquito immune responses during successful development is discussed. PMID:19823571

  8. Mosquito infection responses to developing filarial worms.

    PubMed

    Erickson, Sara M; Xi, Zhiyong; Mayhew, George F; Ramirez, Jose L; Aliota, Matthew T; Christensen, Bruce M; Dimopoulos, George

    2009-01-01

    Human lymphatic filariasis is a mosquito-vectored disease caused by the nematode parasites Wuchereria bancrofti, Brugia malayi and Brugia timori. These are relatively large roundworms that can cause considerable damage in compatible mosquito vectors. In order to assess how mosquitoes respond to infection in compatible mosquito-filarial worm associations, microarray analysis was used to evaluate transcriptome changes in Aedes aegypti at various times during B. malayi development. Changes in transcript abundance in response to the different stages of B. malayi infection were diverse. At the early stages of midgut and thoracic muscle cell penetration, a greater number of genes were repressed compared to those that were induced (20 vs. 8). The non-feeding, intracellular first-stage larvae elicited few differences, with 4 transcripts showing an increased and 9 a decreased abundance relative to controls. Several cecropin transcripts increased in abundance after parasites molted to second-stage larvae. However, the greatest number of transcripts changed in abundance after larvae molted to third-stage larvae and migrated to the head and proboscis (120 induced, 38 repressed), including a large number of putative, immunity-related genes (approximately 13% of genes with predicted functions). To test whether the innate immune system of mosquitoes was capable of modulating permissiveness to the parasite, we activated the Toll and Imd pathway controlled rel family transcription factors Rel1 and Rel2 (by RNA interference knockdown of the pathway's negative regulators Cactus and Caspar) during the early stages of infection with B. malayi. The activation of either of these immune signaling pathways, or knockdown of the Toll pathway, did not affect B. malayi in Ae. aegypti. The possibility of LF parasites evading mosquito immune responses during successful development is discussed. PMID:19823571

  9. Toward molecular parasitologic diagnosis: enhanced diagnostic sensitivity for filarial infections in mobile populations.

    PubMed

    Fink, Doran L; Fahle, Gary A; Fischer, Steven; Fedorko, Daniel F; Nutman, Thomas B

    2011-01-01

    The diagnosis of filarial infections among individuals residing in areas where the disease is not endemic requires both strong clinical suspicion and expert training in infrequently practiced parasitological methods. Recently developed filarial molecular diagnostic assays are highly sensitive and specific but have limited availability and have not been closely evaluated for clinical use outside populations residing in areas of endemicity. In this study, we assessed the performance of a panel of real-time PCR assays for the four most common human filarial pathogens among blood and tissue samples collected from a cohort of patients undergoing evaluation for suspected filarial infections. Compared to blood filtration, real-time PCR was equally sensitive for the detection of microfilaremia due to Wuchereria bancrofti (2 of 46 samples positive by both blood filtration and PCR with no discordant results) and Loa loa (24 of 208 samples positive by both blood filtration and PCR, 4 samples positive by PCR only, and 3 samples positive by blood filtration only). Real-time PCR of skin snip samples was significantly more sensitive than microscopic examination for the detection of Onchocerca volvulus microfiladermia (2 of 218 samples positive by both microscopy and PCR and 12 samples positive by PCR only). The molecular assays required smaller amounts of blood and tissue than conventional methods and could be performed by laboratory personnel without specialized parasitology training. Taken together, these data demonstrate the utility of the molecular diagnosis of filarial infections in mobile populations. PMID:20980560

  10. Evaluation of immuno diagnostic assay for the exposure of stage specific filarial infection.

    PubMed

    Ravishankaran, Rajendran; Shridharan, Radhika Nagamangalam; Vishal, Lawrence Ansel; Meenakshisundaram, Sankaranarayanan; Karande, Anjali Anoop; Kaliraj, Perumal

    2016-03-01

    Lymphatic filariasis is a debilitating diseases caused by filarial parasitic nematodes. The infection may be acquired in childhood but the symptoms become apparent only in later life. To evaluate the success of any intervention, sensitive diagnostics were used to identify infection among endemic normals that are likely to develop microfilaremia in due course of time. Capture assay was standardized using the recombinant protein Brugia malayi Abundant Larval Transcript-2 (ALT-2) specific monoclonal and poly-clonal antibodies and evaluated with serum samples of clinical groups from high and low filarial infection area individuals (HIA/LIA), Endemic Normal (EN, n = 478), microfilaeremics (MF, n = 77), chronic pathology (CP, n = 57) and non endemic normal (NEN, n = 20). In order to assess stage-specific infection, ALT-2 capture assay was compared with the early reported Venom allergen homologue (VAH) and microfilariae specific SXP-1 capture assays. Of the 632 serum samples tested, ALT-2 and VAH capture assays detected circulating filarial antigen (CFA) in 57% and 52% of HIA-EN individuals, respectively. As expected, the VAH and SXP-1 capture assays were positive for 100 % of MF individuals. The described capture assays can be useful for the detection of early and stage-specific filarial infections in endemic regions of developing countries. PMID:27078646

  11. Maternal filarial infection - a persistent risk factor for microfilaremia in offspring?

    PubMed

    Hightower, A W; Lammie, P J; Eberhard, M L

    1993-11-01

    The observation that children born to mothers that are infected with Wuchereria bancrofti ore more susceptible to filarial infection than those born to uninfected mothers, raises many questions, particularly regarding immune mechanisms. In this article, Allen Hightower, Patrick Lommie and Mark Eberhard discuss these issues and their implications for the epidemiology of filarial infection.

  12. On the escape of infective filarial larvae from the mosquitoes.

    PubMed

    Zielke, E

    1977-12-01

    Experimentally infected females of Culex pipiens fatigans carrying infective larvae of Wuchereria bancrofti were fed, on the 16th day p.i., on four different solutions, which were offered "cold" (24 degrees C) or "warm" (34 degrees C) in Petri dishes as open fluids. Thus the sucking mosquitoes did not have to bend their labia. Only the "warm" human serum stimulated any considerable number of infective larvae (24.8%) to leave the mouthparts of the mosquitoes. 1289 infective C. fatigens females lost only an estimated 6.4% of their infective larvae of W. bancrofti, when they were maintained on sugar-water until their natural death. Most of the more heavily infected mosquitoes died relatively soon after the filarial larvae had reached maturity (15-20 days p.i.). The main stimulus provoking the filarial larvae to migrate into the labium is believed to be the movement of the muscles of the pharyngeal pump. Mature larvae protrude their anterior ends from the tip of the labellum. There they seem able to distinguish between suitable and unsuitable external conditions and accordingly they will either leave the proboscis completely or retract into the labium. PMID:601855

  13. Diversity, Host Specialization, and Geographic Structure of Filarial Nematodes Infecting Malagasy Bats.

    PubMed

    Ramasindrazana, Beza; Dellagi, Koussay; Lagadec, Erwan; Randrianarivelojosia, Milijaona; Goodman, Steven M; Tortosa, Pablo

    2016-01-01

    We investigated filarial infection in Malagasy bats to gain insights into the diversity of these parasites and explore the factors shaping their distribution. Samples were obtained from 947 individual bats collected from 52 sites on Madagascar and representing 31 of the 44 species currently recognized on the island. Samples were screened for the presence of micro- and macro-parasites through both molecular and morphological approaches. Phylogenetic analyses showed that filarial diversity in Malagasy bats formed three main groups, the most common represented by Litomosa spp. infecting Miniopterus spp. (Miniopteridae); a second group infecting Pipistrellus cf. hesperidus (Vespertilionidae) embedded within the Litomosoides cluster, which is recognized herein for the first time from Madagascar; and a third group composed of lineages with no clear genetic relationship to both previously described filarial nematodes and found in M. griveaudi, Myotis goudoti, Neoromicia matroka (Vespertilionidae), Otomops madagascariensis (Molossidae), and Paratriaenops furculus (Hipposideridae). We further analyzed the infection rates and distribution pattern of Litomosa spp., which was the most diverse and prevalent filarial taxon in our sample. Filarial infection was disproportionally more common in males than females in Miniopterus spp., which might be explained by some aspect of roosting behavior of these cave-dwelling bats. We also found marked geographic structure in the three Litomosa clades, mainly linked to bioclimatic conditions rather than host-parasite associations. While this study demonstrates distinct patterns of filarial nematode infection in Malagasy bats and highlights potential drivers of associated geographic distributions, future work should focus on their alpha taxonomy and characterize arthropod vectors.

  14. Prevalence and pathogenesis of some filarial nematodes infecting donkeys in Egypt

    PubMed Central

    Radwan, A. M.; Ahmed, N. E.; Elakabawy, L. M.; Ramadan, M. Y.; Elmadawy, R. S.

    2016-01-01

    Aim: The primary objective of the present study is to determine the commonness of filarial parasites in donkeys in Egypt, identification of the filarial species tainting them and the delivered pathogenic impact connected with the infestation. Materials and Methods: A total of 188 donkeys were examined for filarial infection. The blood samples and scraping of the cutaneous bleeding lesions were collected, stained, and inspected for microfilariae all through the period from March 2011 to October 2013. The adult worms were perceived in tissue samples acquired from skin scraping, testes, eyes, tendons, peritoneal and pleural cavities, and the ligamentum nuchae. Results: On the basis of morphological identification, 163 of 188 donkeys (86.70%) were infected with Onchocerca cervicalis (82.98%), Setaria equina (31.11%), Parafilaria multipapillosa (5.32%), and Onchocerca reticulata (4.26%). There was no significant effect of the sex on the incidence of all the encounteredfilarial worms except for S. equina, where the infection rate prevailed in males versus females (40.82% vs. 35.90%). In addition, age group of 5-15 years old exhibited a fundamentally higher predominance (p< 0.05) of the recognized filarial worms versus those of < 5 years old and >15 years old. Conclusion: The preliminary results add to our comprehension of filarial species infecting donkeys in Egypt, their impact on animal execution and production. Accentuation must be taken for avoidance, control of filarial disease, and improvement of the management system of donkeys.

  15. Prevalence and pathogenesis of some filarial nematodes infecting donkeys in Egypt

    PubMed Central

    Radwan, A. M.; Ahmed, N. E.; Elakabawy, L. M.; Ramadan, M. Y.; Elmadawy, R. S.

    2016-01-01

    Aim: The primary objective of the present study is to determine the commonness of filarial parasites in donkeys in Egypt, identification of the filarial species tainting them and the delivered pathogenic impact connected with the infestation. Materials and Methods: A total of 188 donkeys were examined for filarial infection. The blood samples and scraping of the cutaneous bleeding lesions were collected, stained, and inspected for microfilariae all through the period from March 2011 to October 2013. The adult worms were perceived in tissue samples acquired from skin scraping, testes, eyes, tendons, peritoneal and pleural cavities, and the ligamentum nuchae. Results: On the basis of morphological identification, 163 of 188 donkeys (86.70%) were infected with Onchocerca cervicalis (82.98%), Setaria equina (31.11%), Parafilaria multipapillosa (5.32%), and Onchocerca reticulata (4.26%). There was no significant effect of the sex on the incidence of all the encounteredfilarial worms except for S. equina, where the infection rate prevailed in males versus females (40.82% vs. 35.90%). In addition, age group of 5-15 years old exhibited a fundamentally higher predominance (p< 0.05) of the recognized filarial worms versus those of < 5 years old and >15 years old. Conclusion: The preliminary results add to our comprehension of filarial species infecting donkeys in Egypt, their impact on animal execution and production. Accentuation must be taken for avoidance, control of filarial disease, and improvement of the management system of donkeys. PMID:27651679

  16. Sensitivity and specificity of ultrasound detection and risk factors for filarial-associated hydroceles.

    PubMed

    Tobian, Aaron A R; Tarongka, Nandao; Baisor, Moses; Bockarie, Moses; Kazura, James W; King, Christopher L

    2003-06-01

    To better understand risk factors for hydrocele as a consequence of Wuchereria bancrofti infection, 342 men more than 15 years of age in an endemic area in Papua New Guinea were evaluated. Thirty-four subjects (9.9%) had hydrocele by physical examination. Ultrasound examination detected hydroceles in 57 men (16.7%). Compared with ultrasonography, the sensitivity of physical examination was 44.3%, the specificity was 98.2%, and the positive predictive value was 73.5%. Hydrocele was independently associated with age (odds ratio [OR] = 3.3, P < 0.01) and intensity of infection as determined by filarial antigenemia (OR = 2.3, P = 0.07). Dilation of spermatic cord lymphatics detectable by ultrasound did not correlate with hydrocele, but was associated with the presence of infection. These observations suggest that filarial pathology of the male genitalia is under-reported when evaluated by physical examination alone and that duration and intensity of infection are risk factors for hydrocele.

  17. Diversity, Host Specialization, and Geographic Structure of Filarial Nematodes Infecting Malagasy Bats

    PubMed Central

    Ramasindrazana, Beza; Dellagi, Koussay; Lagadec, Erwan; Randrianarivelojosia, Milijaona; Goodman, Steven M.; Tortosa, Pablo

    2016-01-01

    We investigated filarial infection in Malagasy bats to gain insights into the diversity of these parasites and explore the factors shaping their distribution. Samples were obtained from 947 individual bats collected from 52 sites on Madagascar and representing 31 of the 44 species currently recognized on the island. Samples were screened for the presence of micro- and macro-parasites through both molecular and morphological approaches. Phylogenetic analyses showed that filarial diversity in Malagasy bats formed three main groups, the most common represented by Litomosa spp. infecting Miniopterus spp. (Miniopteridae); a second group infecting Pipistrellus cf. hesperidus (Vespertilionidae) embedded within the Litomosoides cluster, which is recognized herein for the first time from Madagascar; and a third group composed of lineages with no clear genetic relationship to both previously described filarial nematodes and found in M. griveaudi, Myotis goudoti, Neoromicia matroka (Vespertilionidae), Otomops madagascariensis (Molossidae), and Paratriaenops furculus (Hipposideridae). We further analyzed the infection rates and distribution pattern of Litomosa spp., which was the most diverse and prevalent filarial taxon in our sample. Filarial infection was disproportionally more common in males than females in Miniopterus spp., which might be explained by some aspect of roosting behavior of these cave-dwelling bats. We also found marked geographic structure in the three Litomosa clades, mainly linked to bioclimatic conditions rather than host-parasite associations. While this study demonstrates distinct patterns of filarial nematode infection in Malagasy bats and highlights potential drivers of associated geographic distributions, future work should focus on their alpha taxonomy and characterize arthropod vectors. PMID:26751792

  18. Highly heterogeneous, activated and short-lived regulatory T cells during chronic filarial infection

    PubMed Central

    Metenou, Simon; Coulibaly, Yaya I.; Sturdevant, Daniel; Dolo, Housseini; Diallo, Abdallah A.; Soumaoro, Lamine; Coulibaly, Michel E.; Kanakabandi, Kishore; Porcella, Stephen F.; Klion, Amy D.; Nutman, Thomas B.

    2016-01-01

    The mechanisms underlying the increase in the numbers of regulatory T (Treg) cells in chronic infection settings remain unclear. Here we have delineated the phenotype and transcriptional profiles of Treg cells from 18 filarial-infected (Fil+) and 19 filarial-uninfected (Fil-) subjects. We found that the frequencies of Foxp3+ Treg cells expressing CTLA-4, GITR, LAG-3 and IL-10 were significantly higher in Fil+ subjects compared with that in Fil- subjects. Foxp3-expressing Treg-cell populations in Fil+ subjects were also more heterogeneous and had higher expression of IL-10, CCL-4, IL-29, CTLA-4 and TGF-β than Fil- subjects, each of these cytokines having been implicated in immune suppression. Moreover, Foxp3-expressing Treg cells from Fil+ subjects had markedly upregulated expression of activation-induced apoptotic genes with concomitant downregulation of those involved in cell survival. To determine whether the expression of apoptotic genes was due to Treg-cell activation, we found that the expression of CTLA-4, CDk8, RAD50, TNFRSF1A, FOXO3 and RHOA were significantly upregulated in stimulated cells compared with unstimulated cells. Taken together, our results suggest that in patent filarial infection, the expanded Treg-cell populations are heterogeneous, short-lived, activated and express higher levels of molecules known to modulate immune responsiveness, suggesting that filarial infection is associated with high Treg-cell turnover. PMID:24737144

  19. The Potentials and Pitfalls of Microarrays in Neglected Tropical Diseases: A Focus on Human Filarial Infections.

    PubMed

    Kwarteng, Alexander; Ahuno, Samuel Terkper

    2016-01-01

    Data obtained from expression microarrays enables deeper understanding of the molecular signatures of infectious diseases. It provides rapid and accurate information on how infections affect the clustering of gene expression profiles, pathways and networks that are transcriptionally active during various infection states compared to conventional diagnostic methods, which primarily focus on single genes or proteins. Thus, microarray technologies offer advantages in understanding host-parasite interactions associated with filarial infections. More importantly, the use of these technologies can aid diagnostics and helps translate current genomic research into effective treatment and interventions for filarial infections. Studying immune responses via microarray following infection can yield insight into genetic pathways and networks that can have a profound influence on the development of anti-parasitic vaccines. PMID:27600086

  20. The Potentials and Pitfalls of Microarrays in Neglected Tropical Diseases: A Focus on Human Filarial Infections

    PubMed Central

    Kwarteng, Alexander; Ahuno, Samuel Terkper

    2016-01-01

    Data obtained from expression microarrays enables deeper understanding of the molecular signatures of infectious diseases. It provides rapid and accurate information on how infections affect the clustering of gene expression profiles, pathways and networks that are transcriptionally active during various infection states compared to conventional diagnostic methods, which primarily focus on single genes or proteins. Thus, microarray technologies offer advantages in understanding host-parasite interactions associated with filarial infections. More importantly, the use of these technologies can aid diagnostics and helps translate current genomic research into effective treatment and interventions for filarial infections. Studying immune responses via microarray following infection can yield insight into genetic pathways and networks that can have a profound influence on the development of anti-parasitic vaccines. PMID:27600086

  1. A Structural Biology Approach to Understand Human Lymphatic Filarial Infection

    PubMed Central

    Nagampalli, Raghavendra Sashi Krishna; Gunasekaran, Krishnasamy; Narayanan, Rangarajan Badri; Peters, Angela; Bhaskaran, Rajagopalan

    2014-01-01

    The presence of aspartic protease inhibitor in filarial parasite Brugia malayi (Bm-Aspin) makes it interesting to study because of the fact that the filarial parasite never encounters the host digestive system. Here, the aspartic protease inhibition kinetics of Bm-Aspin and its NMR structural characteristics have been investigated. The overall aim of this study is to explain the inhibition and binding properties of Bm-Aspin from its structural point of view. UV-spectroscopy and multi-dimensional NMR are the experiments that have been performed to understand the kinetic and structural properties of Bm-Aspin respectively. The human aspartic proteases that are considered for this study are pepsin, renin, cathepsin-E and cathepsin-D. The results of this analysis performed with the specific substrate [Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu (4-pyridylmethyl) ester] against aspartic proteases suggest that Bm-Aspin inhibits the activities of all four human aspartic proteases. The kinetics studies indicate that Bm-Aspin follows a competitive mode of inhibition for pepsin and cathepsin-E, non-competitive for renin and mixed mode for cathepsin-D. The triple resonance NMR experiments on Bm-Aspin suggested the feasibility of carrying out NMR studies to obtain its solution structure. The NMR titration studies on the interactions of Bm-Aspin with the proteases indicate that it undergoes fast-exchange phenomena among themselves. In addition to this, the chemical shift perturbations for some of the residues of Bm-Aspin observed from 15N-HSQC spectra upon the addition of saturated amounts of aspartic proteases suggest the binding between Bm-Aspin and human aspartic proteases. They also provide information on the variations in the intensities and mode of binding between the proteases duly corroborating with the results from the protease inhibition assay method. PMID:24516678

  2. Hypothalamic-pituitary gonadal axis and immune response imbalance during chronic filarial infections.

    PubMed

    Mavoungou, Donatien; Poaty-Mavoungou, Virginie; Ongali, Brice; Akoume, Marie Yvonne; Maka, Gontran; Mavoungou, Elie

    2005-11-01

    Bi-directional relationships operate between the hypothalamic-pituitary-gonadal axis and the immune system. Cytokines, peptide hormones and their shared receptors/ligands are used as a common biological language for communication within and between the immune and neuroendocrine systems. Such communication suggests an immunoregulatory role for the brain and a sensory function for the immune system. We used a radioimmunoassay to measure the concentrations of steroid hormones (cortisol, testosterone, estradiol and progesterone) and pituitary hormones [follicle stimulating hormone (FSH), luteinizing hormone (LH) human chorionic gonadotropin (HCG) and prolactin] in peripheral blood plasma from 78 young Gabonese women with chronic filarial infections. We used an enzyme-linked immunosorbent assay to determine the concentrations of four proinflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-1 (IL-1) and IL-6] in the same plasma samples. Progesterone was unchanged and all other steroid hormone plasma concentrations were lower in microfilaremic women than in amicrofilaremic women. The concentration of LH was higher in amicrofilaremic women, whereas the prolactin concentration was higher in microfilaremics. The plasma concentrations of TNF-alpha, IFN-gamma, IL-1 and IL-6 were higher in microfilaremic women. A strong negative correlation was found between the steroid and pituitary hormones and the pro-inflammatory cytokines. Conversely, a strong positive correlation was found between prolactin and the same cytokines. These data provide first evidence of immune system and hormonal system disturbance during chronic filarial infections and suggest that the observed imbalance should be taken into account in the diagnosis and treatment of filarial infections.

  3. Filarial infections in domestic dogs in Lusaka, Zambia.

    PubMed

    Siwila, Joyce; Mwase, Enala T; Nejsum, Peter; Simonsen, Paul E

    2015-06-15

    Filariae are common parasites of dogs in many parts of the world, but little is known about the status of these infections in sub-Saharan Africa. A study was carried out to determine the occurrence and species of filariae among 272 dogs in Lusaka, Zambia. Giemsa stained blood smear and Knott's concentration methods revealed microfilariae in 16 (5.9%) of the dogs. PCR confirmed that most of these dogs had Acanthocheilonema reconditum infection. Ten (4.0%) of the examined dogs were positive for Dirofilaria immitis circulating antigen (by DiroCHEK(®) test), but D. immitis microfilariae were not identified in any of the dogs and the status of this infection remains unclear. Further studies are needed to explore the occurrence of filariae in Zambian dogs and the zoonotic potential for humans.

  4. Allergic Sensitization Underlies Hyperreactive Antigen-Specific CD4+ T Cell Responses in Coincident Filarial Infection.

    PubMed

    Gazzinelli-Guimarães, Pedro H; Bonne-Année, Sandra; Fujiwara, Ricardo T; Santiago, Helton C; Nutman, Thomas B

    2016-10-01

    Among the various hypotheses put forward to explain the modulatory influence of helminth infection on allergic effector responses in humans, the IL-10-induced suppression of Th2-associated responses has been the leading candidate. To explore this helminth/allergy interaction more fully, parasite- and allergen-specific CD4(+) T cell responses in 12 subjects with filarial infections, and coincident allergic sensitization (filarial [Fil](+)allergy [A](+)) were compared with the responses to three appropriate control groups (Fil(-)A(-) [n = 13], Fil(-)A(+) [n = 12], Fil(+)A(-) [n = 11]). The most important findings revealed that Fil(+)A(+) had marked (p < 0.0001 for all cytokines) increases in parasite Ag-driven Th2 (IL-4, IL-5, IL-13), Th9 (IL-9), and the regulatory (IL-10) cytokines when compared with Fil(+)A(-) Moreover, using multiparameter flow cytometry, filarial parasite Ag induced a marked increase in not only the frequency of CD4(+) T cells producing IL-4, IL-5, IL-2, and TNF-α in Fil(+)A(+) when compared with Fil(+)A(-) patients, but also in the frequencies of polyfunctional Th2-like (CD4(+)IL-4(+)IL-5(+) and CD4(+)IL-2(+)IL-4(+)IL-5(+)TNF-α(+)) cells. The Th2-associated responses seen in the Fil(+)A(+) group were correlated with serum IgE levels (p < 0.01, r = 0.5165 for IL-4; p < 0.001, r = 0.5544 for IL-5; and p < 0.001, r = 0.4901 for IL-13) and levels of circulating eosinophils (p < 0.0116, r = 0.5656) and their degranulation/activation products (major basic protein [p < 0.001, r = 0.7353] and eosinophil-derived neurotoxin [p < 0.01, r = 0.7059]). CD4(+) responses to allergen were not different (to a large extent) among the groups. Taken together, our data suggest that allergic sensitization coincident with filarial infection drives parasite Ag-specific T cell hyperresponsiveness, which is characterized largely by an augmented Th2-dominated immune response. PMID:27566825

  5. Identification of Ecdysone Hormone Receptor Agonists as a Therapeutic Approach for Treating Filarial Infections

    PubMed Central

    Mhashilkar, Amruta S.; Vankayala, Sai L.; Liu, Canhui; Kearns, Fiona; Mehrotra, Priyanka; Tzertzinis, George; Palli, Subba R.; Woodcock, H. Lee; Unnasch, Thomas R.

    2016-01-01

    Background A homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets. Methodology/ Principal Findings Administration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptor agonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites. Conclusions The studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites. PMID:27300294

  6. New Insights into the Evolution of Wolbachia Infections in Filarial Nematodes Inferred from a Large Range of Screened Species

    PubMed Central

    Barbuto, Michela; Martin, Coralie; Lo, Nathan; Uni, Shigehiko; Landmann, Frederic; Baccei, Sara G.; Guerrero, Ricardo; de Souza Lima, Sueli; Bandi, Claudio; Wanji, Samuel; Diagne, Moustapha; Casiraghi, Maurizio

    2011-01-01

    Background Wolbachia are intriguing symbiotic endobacteria with a peculiar host range that includes arthropods and a single nematode family, the Onchocercidae encompassing agents of filariases. This raises the question of the origin of infection in filariae. Wolbachia infect the female germline and the hypodermis. Some evidences lead to the theory that Wolbachia act as mutualist and coevolved with filariae from one infection event: their removal sterilizes female filariae; all the specimens of a positive species are infected; Wolbachia are vertically inherited; a few species lost the symbiont. However, most data on Wolbachia and filaria relationships derive from studies on few species of Onchocercinae and Dirofilariinae, from mammals. Methodology/Principal Findings We investigated the Wolbachia distribution testing 35 filarial species, including 28 species and 7 genera and/or subgenera newly screened, using PCR, immunohistochemical staining, whole mount fluorescent analysis, and cocladogenesis analysis. (i) Among the newly screened Onchocercinae from mammals eight species harbour Wolbachia but for some of them, bacteria are absent in the hypodermis, or in variable density. (ii) Wolbachia are not detected in the pathological model Monanema martini and in 8, upon 9, species of Cercopithifilaria. (iii) Supergroup F Wolbachia is identified in two newly screened Mansonella species and in Cercopithifilaria japonica. (iv) Type F Wolbachia infect the intestinal cells and somatic female genital tract. (v) Among Oswaldofilariinae, Waltonellinae and Splendidofilariinae, from saurian, anuran and bird respectively, Wolbachia are not detected. Conclusions/Significance The absence of Wolbachia in 63% of onchocercids, notably in the ancestral Oswaldofilariinae estimated 140 mya old, the diverse tissues or specimens distribution, and a recent lateral transfer in supergroup F Wolbachia, modify the current view on the role and evolution of the endosymbiont and their hosts. Further

  7. The effect of HIV on filarial-specific antibody response before and after treatment with diethylcarbamazine in Wuchereria bancrofti infected individuals.

    PubMed

    Petersen, Heidi H; Nielsen, Nina O; Monrad, Jesper; Magesa, Stephen M; Simonsen, Paul E

    2009-06-01

    The effect of HIV on filarial-specific antibody response before and after treatment with diethylcarbamazine (DEC) was analysed by comparing two groups of Wuchereria bancrofti-infected adult individuals (positive for circulating filarial antigen) who were positive (n=15) or negative (n=21) for HIV co-infection. Prior to DEC treatment there was no significant difference in filarial-specific IgG1, IgG2, IgG4 and IgE antibody response between the HIV negative and the HIV positive group, while a five times (statistically significant) higher filarial-specific IgG3 response was observed in the HIV positive than in the HIV negative group. At 12 weeks after treatment with DEC, a significant decrease in filarial-specific IgG4 was observed in the HIV positive but not in the HIV negative group, indicating that DEC treatment had a stronger antifilarial effect in individuals co-infected with HIV. DEC treatment had no significant effect on the other classes of filarial specific antibodies, neither in the HIV negative or the HIV positive group. PMID:19567236

  8. At homeostasis filarial infections have expanded adaptive T regulatory but not classical Th2 cells.

    PubMed

    Metenou, Simon; Dembele, Benoit; Konate, Siaka; Dolo, Housseini; Coulibaly, Siaka Y; Coulibaly, Yaya I; Diallo, Abdallah A; Soumaoro, Lamine; Coulibaly, Michel E; Sanogo, Dramane; Doumbia, Salif S; Traoré, Sekou F; Mahanty, Siddhartha; Klion, Amy; Nutman, Thomas B

    2010-05-01

    Despite the well-documented immune suppression associated with human helminth infections, studies characterizing the immune response at the single-cell level are scanty. We used multiparameter flow cytometry to characterize the type of effector (Th1, Th2, and Th17) and regulatory (natural T regulatory cells [nTregs] and adaptive Treg cells [aTreg/type 1 regulatory cells (Tr1s)]) CD4(+) and CD8(+) T cells in filaria-infected (Fil(+)) and -uninfected (Fil(-)) individuals at homeostasis (in the absence of stimulation). Frequencies of CD4(+) lymphocytes spontaneously producing IL-4, IL-10, and IL-17A were significantly higher in Fil(+), as were those of IL-10(+)/IL-4(+) double-producing CD4(+) cells. Interestingly, frequencies of Th17 and aTreg/Tr1s but not classical Th1 or Th2 cells were significantly increased in Fil(+) compared to Fil(-) individuals. Although the frequency of nTreg was increased in Fil(+), IL-10 was overwhelmingly produced by CD4(+)CD25(-) cells. Moreover, the concentration of IL-10 produced spontaneously in vitro strongly correlated with the integrated geometric mean fluorescence intensity of IL-10-producing aTreg/Tr1s in Fil(+). Together, these data show that at steady state, IL-10-producing aTreg/Tr1 as well as nTreg and effector Th17 CD4(+) cells are expanded in vivo in human filarial infections. Moreover, we have established baseline ex vivo frequencies of effector and Tregs at homeostasis at a population level. PMID:20357251

  9. Filarial infection modulates the immune response to Mycobacterium tuberculosis through expansion of CD4+ IL-4 memory T cells

    PubMed Central

    Chatterjee, Soumya; Clark, Carolyn E.; Lugli, Enrico; Roederer, Mario; Nutman, Thomas B.

    2015-01-01

    Exaggerated CD4+T helper 2-specific cytokine producing memory T cell responses developing concomitantly with a T helper1 response might have a detrimental role in immunity to infection caused by Mycobacterium tuberculosis (Mtb). To assess the dynamics of antigen (Ag)-specific memory T cell compartments in the context of filarial infection we used multiparameter flow cytometry on PBMCs from 25 microfilaremic filarial -infected (Inf) and 14 filarial-uninfected (Uninf) subjects following stimulation with filarial (BmA) or with the Mycobacterium tuberculosis (Mtb)-specific Ag CFP10. Our data demonstrated that the Inf group not only had a marked increase in BmA-specific CD4+IL-4+ cells (Median net frequency compared to baseline (Fo)=0.09% vs. 0.01%, p=0.038) but also to CFP10 (Fo =0.16% vs. 0.007%, p=0.04) and Staphylococcal Enterotoxin B (SEB) (Fo =0.49% vs. 0.26%, p=0.04). The Inf subjects showed a BmA-specific expansion of CD4+CD45RO+IL-4+ producing central memory (TCM, CD45RO+CCR7+CD27+) (Fo =1.1% vs. 0.5%, p=0.04) as well as effector memory (TEM CD45RO+CCR7-CD27-) (Fo =1.5% vs. 0.2%, p=0.03) with a similar but non-significant response to CFP10. In addition, there was expansion of CD4+ IL-4+ CD45RA+ CCR7+CD27+ (naïve-like) in Inf individuals compared to Uninf subjects. Among Inf subjects with definitive latent tuberculosis , there were no differences in frequencies of IL-4 producing cells within any of the memory compartments compared to the Uninf group. Our data suggest that filarial infection induces antigen-specific, exaggerated IL-4 responses in distinct T cell memory compartments to Mtb-specific antigens, which are attenuated in subjects who are able to mount a delayed type hypersensitivity reaction to Mtb. PMID:25667413

  10. Filarial infection modulates the immune response to Mycobacterium tuberculosis through expansion of CD4+ IL-4 memory T cells.

    PubMed

    Chatterjee, Soumya; Clark, Carolyn E; Lugli, Enrico; Roederer, Mario; Nutman, Thomas B

    2015-03-15

    Exaggerated CD4(+) T helper 2-specific cytokine producing memory T cell responses developing concomitantly with a T helper 1 response might have a detrimental role in immunity to infection caused by Mycobacterium tuberculosis. To assess the dynamics of Ag-specific memory T cell compartments in the context of filarial infection, we used multiparameter flow cytometry on PBMCs from 25 microfilaremic filarial-infected (Inf) and 14 filarial-uninfected (Uninf) subjects following stimulation with filarial Ag (BmA) or with the M. tuberculosis-specific Ag culture filtrate protein-10 (CFP-10). Our data demonstrated that the Inf group had a marked increase in BmA-specific CD4(+)IL-4(+) cells (median net frequency compared with baseline [Fo] = 0.09% versus 0.01%; p = 0.038) but also to CFP-10 (Fo = 0.16% versus 0.007%; p = 0.04) and staphylococcal enterotoxin B (Fo = 0.49% versus 0.26%; p = 0.04). The Inf subjects showed a BmA-specific expansion of CD4(+)CD45RO(+)IL-4(+) producing central memory (TCM, CD45RO(+)CCR7(+)CD27(+); Fo = 1.1% versus 0.5%; p = 0.04) as well as effector memory (TEM, CD45RO(+)CCR7(-)CD27(-); Fo = 1.5% versus 0.2%; p = 0.03) with a similar but nonsignificant response to CFP-10. In addition, there was expansion of CD4(+)IL-4(+)CD45RA(+)CCR7(+)CD27(+) (naive-like) in Inf individuals compared with Uninf subjects. Among Inf subjects with definitive latent tuberculosis, there were no differences in frequencies of IL-4-producing cells within any of the memory compartments compared with the Uninf group. Our data suggest that filarial infection induces Ag-specific, exaggerated IL-4 responses in distinct T cell memory compartments to M. tuberculosis-specific Ags, which are attenuated in subjects who are able to mount a delayed type hypersensitivity reaction to M. tuberculosis. PMID:25667413

  11. Nested PCR to detect and distinguish the sympatric filarial species Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans in the Amazon Region.

    PubMed

    Tang, Thuy-Huong Ta; López-Vélez, Rogelio; Lanza, Marta; Shelley, Anthony John; Rubio, Jose Miguel; Luz, Sérgio Luiz Bessa

    2010-09-01

    We present filaria-nested polymerase chain reaction (PCR), which is based on amplification of first internal transcribed spacer rDNA to distinguish three parasitic filarial species (Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans) that can be found in the Amazon Region. Nested PCR-based identifications yielded the same results as those utilizing morphological characters. Nested PCR is highly sensitive and specific and it detects low-level infections in both humans and vectors. No cross-amplifications were observed with various other blood parasites and no false-positive results were obtained with the nested PCR. The method works efficiently with whole-blood, blood-spot and skin biopsy samples. Our method may thus be suitable for assessing the efficacy of filaria control programmes in Amazonia by recording parasite infections in both the human host and the vector. By specifically differentiating the major sympatric species of filaria, this technique could also enhance epidemiological research in the region. PMID:20945000

  12. Antigenic role of the endosymbionts of filarial nematodes: IgG response against the Wolbachia surface protein in cats infected with Dirofilaria immitis.

    PubMed Central

    Bazzocchi, C; Ceciliani, F; McCall, J W; Ricci, I; Genchi, C; Bandi, C

    2000-01-01

    Filarial nematodes harbour intracellular endosymbiotic bacteria, which have been assigned to the genus Wolbachia. These bacteria appear to play an important role in the pathogenesis of filarial diseases through their lipopolysaccharides. In view of the presence of Wolbachia endosymbionts in the body of filarial nematodes, one might also expect that proteins from these bacteria play an antigenic role in humans and animals affected by filariases. To test this hypothesis, we produced in recombinant form the surface protein WSP and a portion of the cell-cycle protein FTSZ from the Wolbachia of Dirofilaria immitis. Western immunoblot assays were then performed using cat sera to test the immunogenicity of these proteins. Sera were collected from owners' cats, which were either sero-negative or sero-positive for D. immitis and from cats before and after experimental infection with D. immitis. FTSZ was recognized in Western blots by sera from both positive and negative cats and from both uninfected and experimentally infected cats. WSP was recognized only by sera from positive cats and from cats experimentally infected with D. immitis; this protein was not recognized by sera from negative cats and from cats before experimental infection with D. immitis. The results of Western blot assays on WSP thus support the hypothesis that infection with filarial nematodes induces the production of antibodies against Wolbachia proteins. PMID:11197127

  13. Expanded numbers of circulating myeloid dendritic cells in patent human filarial infection reflect lower CCR1 expression.

    PubMed

    Semnani, Roshanak Tolouei; Mahapatra, Lily; Dembele, Benoit; Konate, Siaka; Metenou, Simon; Dolo, Housseini; Coulibaly, Michel E; Soumaoro, Lamine; Coulibaly, Siaka Y; Sanogo, Dramane; Seriba Doumbia, Salif; Diallo, Abdallah A; Traoré, Sekou F; Klion, Amy; Nutman, Thomas B; Mahanty, Siddhartha

    2010-11-15

    APC dysfunction has been postulated to mediate some of the parasite-specific T cell unresponsiveness seen in patent filarial infection. We have shown that live microfilariae of Brugia malayi induce caspase-dependent apoptosis in human monocyte-derived dendritic cells (DCs) in vitro. This study addresses whether apoptosis observed in vitro extends to patent filarial infections in humans and is reflected in the number of circulating myeloid DCs (mDCs; CD11c(-)CD123(lo)) in peripheral blood of infected microfilaremic individuals. Utilizing flow cytometry to identify DC subpopulations (mDCs and plasmacytoid DCs [pDCs]) based on expression of CD11c and CD123, we found a significant increase in numbers of circulating mDCs (CD11c(+)CD123(lo)) in filaria-infected individuals compared with uninfected controls from the same filaria-endemic region of Mali. Total numbers of pDCs, monocytes, and lymphocytes did not differ between the two groups. To investigate potential causes of differences in mDC numbers between the two groups, we assessed chemokine receptor expression on mDCs. Our data indicate that filaria-infected individuals had a lower percentage of circulating CCR1(+) mDCs and a higher percentage of circulating CCR5(+) mDCs and pDCs. Finally, live microfilariae of B. malayi were able to downregulate cell-surface expression of CCR1 on monocyte-derived DCs and diminish their calcium flux in response to stimulation by a CCR1 ligand. These findings suggest that microfilaria are capable of altering mDC migration through downregulation of expression of some chemokine receptors and their signaling functions. These observations have major implications for regulation of immune responses to these long-lived parasites. PMID:20956349

  14. Nature limits filarial transmission

    PubMed Central

    Chandra, Goutam

    2008-01-01

    Lymphatic filariasis, caused by Wuchereria bancrofti, Brugia malayi and B. timori is a public health problem of considerable magnitude of the tropics and subtropics. Presently 1.3 billion people are at risk of lymphatic filariasis (LF) infection and about 120 million people are affected in 83 countries. In this context it is worth mentioning that 'nature' itself limits filarial transmission to a great extent in a number of ways such as by reducing vector populations, parasitic load and many other bearings. Possibilities to utilize these bearings of natural control of filariasis should be searched and if manipulations on nature, like indiscriminate urbanization and deforestation, creating sites favourable for the breeding of filarial vectors and unsanitary conditions, water pollution with organic matters etc., are reduced below the threshold level, we will be highly benefited. Understandings of the factors related to natural phenomena of control of filariasis narrated in this article may help to adopt effective control strategies. PMID:18500974

  15. Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae

    PubMed Central

    Pionnier, Nicolas; Brotin, Emilie; Karadjian, Gregory; Hemon, Patrice; Gaudin-Nomé, Françoise; Vallarino-Lhermitte, Nathaly; Nieguitsila, Adélaïde; Fercoq, Frédéric; Aknin, Marie-Laure; Marin-Esteban, Viviana; Chollet-Martin, Sylvie; Schlecht-Louf, Géraldine

    2016-01-01

    Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4+/1013). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4+/1013 mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4+/1013 mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis. PMID:27111140

  16. Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae.

    PubMed

    Pionnier, Nicolas; Brotin, Emilie; Karadjian, Gregory; Hemon, Patrice; Gaudin-Nomé, Françoise; Vallarino-Lhermitte, Nathaly; Nieguitsila, Adélaïde; Fercoq, Frédéric; Aknin, Marie-Laure; Marin-Esteban, Viviana; Chollet-Martin, Sylvie; Schlecht-Louf, Géraldine; Bachelerie, Françoise; Martin, Coralie

    2016-04-01

    Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4(+/1013)). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4(+/1013) mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4(+/1013) mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis. PMID:27111140

  17. Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae.

    PubMed

    Pionnier, Nicolas; Brotin, Emilie; Karadjian, Gregory; Hemon, Patrice; Gaudin-Nomé, Françoise; Vallarino-Lhermitte, Nathaly; Nieguitsila, Adélaïde; Fercoq, Frédéric; Aknin, Marie-Laure; Marin-Esteban, Viviana; Chollet-Martin, Sylvie; Schlecht-Louf, Géraldine; Bachelerie, Françoise; Martin, Coralie

    2016-04-01

    Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4(+/1013)). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4(+/1013) mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4(+/1013) mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis.

  18. Black flies (Diptera: Simuliidae) attracted to humans and water buffalos and natural infections with filarial larvae, probably Onchocerca sp., in northern Thailand.

    PubMed

    Takaoka, H; Choochote, W; Aoki, C; Fukuda, M; Bain, O

    2003-03-01

    Several Simulium species were investigated as to their biting habits and natural infections with filarial larvae at Ban Pan Fan, Chiang Mai Province, in northern Thailand. Female adults flies landing on or flighting around a human and a water buffalo were collected during the daytime from 06.00 to 19.00 hours on 22 June 2001. As a result, 217 S. nodosum, 86 S. asakoae and two S. nigrogilvum were obtained from a human attractant, and 416 S. nodosum, 25 S. nakhonense, 16 S. asakoae, four S. fenestratum and two S. nigrogilvum, from a water buffalo. The blood-feeding was confirmed only for S. nodosum and S. nigrogilvum on humans, and for S. nodosum and S. nakhonense on water buffalos. Dissections of these simuliids showed that S. nodosum was naturally infected with developing filarial larvae. Two types of microfilariae were distinguished but only one type of infective larvae. These larvae resembled Onchocerca suzukii, a parasite from a wild Japanese bovid, suggesting that an unknown Onchocerca species from ruminants was transmitted in Thailand. Infection rates with all stages of larvae and third-stage larvae were 2.3% (14/608) and 1.0% (6/608), respectively. This is the first report of natural infections of black flies with Onchocerca larvae in Southeast Asia, and the involved black fly species is shown to be not only anthropophilic but also zoophilic in this region.

  19. Patent filarial infection modulates malaria-specific type 1 cytokine responses in an IL-10-dependent manner in a filaria/malaria-coinfected population.

    PubMed

    Metenou, Simon; Dembélé, Benoit; Konate, Siaka; Dolo, Housseini; Coulibaly, Siaka Y; Coulibaly, Yaya I; Diallo, Abdallah A; Soumaoro, Lamine; Coulibaly, Michel E; Sanogo, Dramane; Doumbia, Salif S; Wagner, Marissa; Traoré, Sekou F; Klion, Amy; Mahanty, Siddhartha; Nutman, Thomas B

    2009-07-15

    The effect of filarial infections on malaria-specific immune responses was investigated in Malian villages coendemic for filariasis (Fil) and malaria. Cytokines were measured from plasma and Ag-stimulated whole blood from individuals with Wuchereria bancrofti and/or Mansonella perstans infections (Fil(+); n = 19) and those without evidence of filarial infection (Fil(-); n = 19). Plasma levels of IL-10 (geometric mean [GM], 22.8 vs 10.4) were higher in Fil(+) compared with Fil(-), whereas levels of IFN-inducible protein (IP)-10 were lower in Fil(+) (GM, 66.3 vs 110.0). Fil(+) had higher levels of spontaneously secreted IL-10 (GM, 59.3 vs 6.8 pg/ml) and lower levels of IL-2 (1.0 vs 1.2 pg/ml) than did Fil(-). Although there were no differences in levels of Staphylococcus aureus enterotoxin B-induced cytokines between the two groups, Fil(+) mounted lower IL-12p70 (GM, 1.11 vs 3.83 pg/ml; p = 0.007), IFN-gamma (GM, 5.44 vs 23.41 pg/ml; p = 0.009), and IP-10 (GM, 29.43 vs 281.7 pg/ml; p = 0.007) responses following malaria Ag (MalAg) stimulation compared with Fil(-). In contrast, Fil(+) individuals had a higher MalAg-specific IL-10 response (GM, 7318 pg/ml vs 3029 pg/ml; p = 0.006) compared with those without filarial infection. Neutralizing Ab to IL-10 (but not to TGFbeta) reversed the down-regulated MalAg-specific IFN-gamma and IP-10 (p < 0.001) responses in Fil(+). Together, these data demonstrate that filarial infections modulate the Plasmodium falciparum-specific IL-12p70/IFN-gamma secretion pathways known to play a key role in resistance to malaria and that they do so in an IL-10-dependent manner. PMID:19561105

  20. Finding Wolbachia in Filarial larvae and Culicidae Mosquitoes in Upper Egypt Governorate.

    PubMed

    Dyab, Ahmed K; Galal, Lamia A; Mahmoud, Abeer E; Mokhtar, Yasser

    2016-06-01

    Wolbachia is an obligatory intracellular endosymbiotic bacterium, present in over 20% of all insects altering insect reproductive capabilities and in a wide range of filarial worms which is essential for worm survival and reproduction. In Egypt, no available data were found about Wolbachia searching for it in either mosquitoes or filarial worms. Thus, we aimed to identify the possible concurrent presence of Wolbachia within different mosquitoes and filarial parasites, in Assiut Governorate, Egypt using multiplex PCR. Initially, 6 pools were detected positive for Wolbachia by single PCR. The simultaneous detection of Wolbachia and filarial parasites (Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens) by multiplex PCR was spotted in 5 out of 6 pools, with an overall estimated rate of infection (ERI) of 0.24%. Unexpectedly, the highest ERI (0.53%) was for Anopheles pharoensis with related Wolbachia and W. bancrofti, followed by Aedes (0.42%) and Culex (0.26%). We also observed that Wolbachia altered Culex spp. as a primary vector for W. bancrofti to be replaced by Anopheles sp. Wolbachia within filaria-infected mosquitoes in our locality gives a hope to use bacteria as a new control trend simultaneously targeting the vector and filarial parasites. PMID:27417080

  1. Finding Wolbachia in Filarial larvae and Culicidae Mosquitoes in Upper Egypt Governorate

    PubMed Central

    Dyab, Ahmed K.; Galal, Lamia A.; Mahmoud, Abeer E.; Mokhtar, Yasser

    2016-01-01

    Wolbachia is an obligatory intracellular endosymbiotic bacterium, present in over 20% of all insects altering insect reproductive capabilities and in a wide range of filarial worms which is essential for worm survival and reproduction. In Egypt, no available data were found about Wolbachia searching for it in either mosquitoes or filarial worms. Thus, we aimed to identify the possible concurrent presence of Wolbachia within different mosquitoes and filarial parasites, in Assiut Governorate, Egypt using multiplex PCR. Initially, 6 pools were detected positive for Wolbachia by single PCR. The simultaneous detection of Wolbachia and filarial parasites (Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens) by multiplex PCR was spotted in 5 out of 6 pools, with an overall estimated rate of infection (ERI) of 0.24%. Unexpectedly, the highest ERI (0.53%) was for Anopheles pharoensis with related Wolbachia and W. bancrofti, followed by Aedes (0.42%) and Culex (0.26%). We also observed that Wolbachia altered Culex spp. as a primary vector for W. bancrofti to be replaced by Anopheles sp. Wolbachia within filaria-infected mosquitoes in our locality gives a hope to use bacteria as a new control trend simultaneously targeting the vector and filarial parasites. PMID:27417080

  2. Finding Wolbachia in Filarial larvae and Culicidae Mosquitoes in Upper Egypt Governorate.

    PubMed

    Dyab, Ahmed K; Galal, Lamia A; Mahmoud, Abeer E; Mokhtar, Yasser

    2016-06-01

    Wolbachia is an obligatory intracellular endosymbiotic bacterium, present in over 20% of all insects altering insect reproductive capabilities and in a wide range of filarial worms which is essential for worm survival and reproduction. In Egypt, no available data were found about Wolbachia searching for it in either mosquitoes or filarial worms. Thus, we aimed to identify the possible concurrent presence of Wolbachia within different mosquitoes and filarial parasites, in Assiut Governorate, Egypt using multiplex PCR. Initially, 6 pools were detected positive for Wolbachia by single PCR. The simultaneous detection of Wolbachia and filarial parasites (Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens) by multiplex PCR was spotted in 5 out of 6 pools, with an overall estimated rate of infection (ERI) of 0.24%. Unexpectedly, the highest ERI (0.53%) was for Anopheles pharoensis with related Wolbachia and W. bancrofti, followed by Aedes (0.42%) and Culex (0.26%). We also observed that Wolbachia altered Culex spp. as a primary vector for W. bancrofti to be replaced by Anopheles sp. Wolbachia within filaria-infected mosquitoes in our locality gives a hope to use bacteria as a new control trend simultaneously targeting the vector and filarial parasites.

  3. Canine filarial infections in a human Brugia malayi endemic area of India.

    PubMed

    Ravindran, Reghu; Varghese, Sincy; Nair, Suresh N; Balan, Vimalkumar M; Lakshmanan, Bindu; Ashruf, Riyas M; Kumar, Swaroop S; Gopalan, Ajith Kumar K; Nair, Archana S; Malayil, Aparna; Chandrasekhar, Leena; Juliet, Sanis; Kopparambil, Devada; Ramachandran, Rajendran; Kunjupillai, Regu; Kakada, Showkath Ali M

    2014-01-01

    A very high prevalence of microfilaremia of 42.68 per cent out of 164 canine blood samples examined was observed in Cherthala (of Alappuzha district of Kerala state), a known human Brugia malayi endemic area of south India. The species of canine microfilariae were identified as Dirofilaria repens, Brugia malayi, and Acanthocheilonema reconditum. D. repens was the most commonly detected species followed by B. pahangi. D. immitis was not detected in any of the samples examined. Based on molecular techniques, microfilariae with histochemical staining pattern of "local staining at anal pore and diffuse staining at central body" was identified as D. repens in addition to those showing acid phosphatase activity only at the anal pore. Even though B. malayi like acid phosphatase activity was observed in few dogs examined, they were identified as genetically closer to B. pahangi. Hence, the possibility of dogs acting as reservoirs of human B. malayi in this area was ruled out. PMID:24971339

  4. Immunopathogenesis of lymphatic filarial disease.

    PubMed

    Babu, Subash; Nutman, Thomas B

    2012-11-01

    Although two thirds of the 120 million people infected with lymph-dwelling filarial parasites have subclinical infections, ~40 million have lymphedema and/or other pathologic manifestations including hydroceles (and other forms of urogenital disease), episodic adenolymphangitis, tropical pulmonary eosinophilia, lymphedema, and (in its most severe form) elephantiasis. Adult filarial worms reside in the lymphatics and lymph nodes and induce changes that result in dilatation of lymphatics and thickening of the lymphatic vessel walls. Progressive lymphatic damage and pathology results from the summation of the effect of tissue alterations induced by both living and nonliving adult parasites, the host inflammatory response to the parasites and their secreted antigens, the host inflammatory response to the endosymbiont Wolbachia, and those seen as a consequence of secondary bacterial or fungal infections. Inflammatory damage induced by filarial parasites appears to be multifactorial, with endogenous parasite products, Wolbachia, and host immunity all playing important roles. This review will initially examine the prototypical immune responses engendered by the parasite and delineate the regulatory mechanisms elicited to prevent immune-mediated pathology. This will be followed by a discussion of the proposed mechanisms underlying pathogenesis, with the central theme being that pathogenesis is a two-step process-the first initiated by the parasite and host innate immune system and the second propagated mainly by the host's adaptive immune system and by other factors (including secondary infections). PMID:23053393

  5. Hyperimmunoglobulinemia E in the absence of atopy and filarial infection: the Huaorani of Ecuador.

    PubMed

    Kron, M A; Ammunariz, M; Pandey, J; Guzman, J R

    2000-01-01

    Hyperimmunoglobulinemia E (HIGE) is associated with various conditions such as atopy, dermatitis, hypersensitivity reactions, and certain parasitic infections. In the course of vaccination initiatives in the province of Napo, eastern Ecuador, blood samples were collected from one of the two remaining rural subgroups of Huaorani Indians who in 1979 were reported to have the world's highest concentrations of IgE. One subgroup of Huaorani, the Dicaron, lives in a protected Amazonian region which has reportedly suffered from extensive pollution after petroleum industry exploration. Plasma was collected from 31 members of the Dicaron (age range 15-75 years), eight non-Dicaron Huaorani, and 16 Quichua Indians from the same province, and tested for IgE, IgG, IgM, IgA, and immunoglobulin allotypes. Subjects were examined for evidence of filariasis, a group of parasitic diseases associated with HIGE. Mean IgE concentration in the Dicaron was measured by CAP ELISA at 11,850 IU/mL (range 5000-33,000) while IgA and IgM concentrations were within normal limits compared to North American controls. IgG levels were slightly elevated and there was no evidence of filariasis. Compared to the Quichua and non-Dicaron Huaorani, two other Amerindian tribes in the Ecuadorian Amazon, the highest concentrations of IgE were recorded from the Dicaron who live within the allegedly polluted section of the Amazon. We conclude that an unexplained HIGE syndrome exists among only one subgroup of Huaorani, the Dicaron. Other eastern Ecuadorian Amerindians, such as the Quichua and resettled Huaorani, have IgE concentrations expected in a population with intestinal helminthiasis. Environmental factors cannot be excluded as the cause of HIGE in the Dicaron. PMID:11191097

  6. Filarial hydropneumothorax: a strange journey.

    PubMed

    Chakraborty, Arkaprabha; Mukherjee, Anindya; Talukdar, Payel; Talukdar, Arunansu

    2015-01-01

    Filarial infection can have varied manifestations, but hydropneumothorax at presentation has not yet been reported. A 28-year-old man presented to our hospital with heaviness of the left chest for the past 10 days, which was preceded by a sudden, short stabbing pain in the left chest after straining. Chest X-ray revealed left-sided hydropneumothorax. A peripheral blood picture revealed significant eosinophilia. A pleural fluid report also showed eosinophilia and a few motile microfilaria of Wuchereria bancrofti. Microfilaria was also documented in peripheral blood. There was no evidence of other organ system involvement. The patient was diagnosed with 'Filarial Hydropneumothorax'. After treatment with a temporary chest drain and oral diethylcarbamazine citrate, there was dramatic relief of symptoms and radiological improvement. The patient has been symptom free with no features of recurrence through 8 months of follow-up. PMID:26604240

  7. "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. PMID:27130361

  8. Filarial and Wolbachia genomics.

    PubMed

    Scott, A L; Ghedin, E; Nutman, T B; McReynolds, L A; Poole, C B; Slatko, B E; Foster, J M

    2012-01-01

    Filarial nematode parasites, the causative agents for a spectrum of acute and chronic diseases including lymphatic filariasis and river blindness, threaten the well-being and livelihood of hundreds of millions of people in the developing regions of the world. The 2007 publication on a draft assembly of the 95-Mb genome of the human filarial parasite Brugia malayi- representing the first helminth parasite genome to be sequenced - has been followed in rapid succession by projects that have resulted in the genome sequencing of six additional filarial species, seven nonfilarial nematode parasites of animals and nearly 30 plant parasitic and free-living species. Parallel to the genomic sequencing, transcriptomic and proteomic projects have facilitated genome annotation, expanded our understanding of stage-associated gene expression and provided a first look at the role of epigenetic regulation of filarial genomes through microRNAs. The expansion in filarial genomics will also provide a significant enrichment in our knowledge of the diversity and variability in the genomes of the endosymbiotic bacterium Wolbachia leading to a better understanding of the genetic principles that govern filarial-Wolbachia mutualism. The goal here is to provide an overview of the trends and advances in filarial and Wolbachia genomics. PMID:22098559

  9. Patency of Litomosoides sigmodontis infection depends on Toll-like receptor 4 whereas Toll-like receptor 2 signalling influences filarial-specific CD4(+) T-cell responses.

    PubMed

    Rodrigo, Maria B; Schulz, Sandy; Krupp, Vanessa; Ritter, Manuel; Wiszniewsky, Katharina; Arndts, Kathrin; Tamadaho, Ruth S E; Endl, Elmar; Hoerauf, Achim; Layland, Laura E

    2016-04-01

    BALB/c mice develop a patent state [release of microfilariae (Mf), the transmission life-stage, into the periphery] when exposed to the rodent filariae Litomosoides sigmodontis. Interestingly, only a portion of the infected mice become patent, which reflects the situation in human individuals infected with Wuchereria bancrofti. Since those individuals had differing filarial-specific profiles, this study compared differences in immune responses between Mf(+) and Mf(-) infected BALB/c mice. We demonstrate that cultures of total spleen or mediastinal lymph node cells from Mf(+) mice produce significantly more interleukin-5 (IL-5) to filarial antigens but equal levels of IL-10 when compared with Mf(-) mice. However, isolated CD4(+) T cells from Mf(+) mice produced significantly higher amounts of all measured cytokines, including IL-10, when compared with CD4(+) T-cell responses from Mf(-) mice. Since adaptive immune responses are influenced by triggering the innate immune system we further studied the immune profiles and parasitology in infected Toll-like receptor-2-deficient (TLR2(-/-)) and TLR4(-/-) BALB/c mice. Ninety-three per cent of L. sigmodontis-exposed TLR4(-/-) BALB/c mice became patent (Mf(+)) although worm numbers remained comparable to those in Mf(+) wild-type controls. Lack of TLR2 had no influence on patency outcome or worm burden but infected Mf(+) mice had significantly lower numbers of Foxp3(+) regulatory T cells and dampened peripheral immune responses. Interestingly, in vitro culturing of CD4(+) T cells from infected wild-type mice with granulocyte-macrophage colony-stimulating factor-derived TLR2(-/-) dendritic cells resulted in an overall diminished cytokine profile to filarial antigens. Hence, triggering TLR4 or TLR2 during chronic filarial infection has a significant impact on patency and efficient CD4(+) T-cell responses, respectively. PMID:26714796

  10. Patency of Litomosoides sigmodontis infection depends on Toll-like receptor 4 whereas Toll-like receptor 2 signalling influences filarial-specific CD4(+) T-cell responses.

    PubMed

    Rodrigo, Maria B; Schulz, Sandy; Krupp, Vanessa; Ritter, Manuel; Wiszniewsky, Katharina; Arndts, Kathrin; Tamadaho, Ruth S E; Endl, Elmar; Hoerauf, Achim; Layland, Laura E

    2016-04-01

    BALB/c mice develop a patent state [release of microfilariae (Mf), the transmission life-stage, into the periphery] when exposed to the rodent filariae Litomosoides sigmodontis. Interestingly, only a portion of the infected mice become patent, which reflects the situation in human individuals infected with Wuchereria bancrofti. Since those individuals had differing filarial-specific profiles, this study compared differences in immune responses between Mf(+) and Mf(-) infected BALB/c mice. We demonstrate that cultures of total spleen or mediastinal lymph node cells from Mf(+) mice produce significantly more interleukin-5 (IL-5) to filarial antigens but equal levels of IL-10 when compared with Mf(-) mice. However, isolated CD4(+) T cells from Mf(+) mice produced significantly higher amounts of all measured cytokines, including IL-10, when compared with CD4(+) T-cell responses from Mf(-) mice. Since adaptive immune responses are influenced by triggering the innate immune system we further studied the immune profiles and parasitology in infected Toll-like receptor-2-deficient (TLR2(-/-)) and TLR4(-/-) BALB/c mice. Ninety-three per cent of L. sigmodontis-exposed TLR4(-/-) BALB/c mice became patent (Mf(+)) although worm numbers remained comparable to those in Mf(+) wild-type controls. Lack of TLR2 had no influence on patency outcome or worm burden but infected Mf(+) mice had significantly lower numbers of Foxp3(+) regulatory T cells and dampened peripheral immune responses. Interestingly, in vitro culturing of CD4(+) T cells from infected wild-type mice with granulocyte-macrophage colony-stimulating factor-derived TLR2(-/-) dendritic cells resulted in an overall diminished cytokine profile to filarial antigens. Hence, triggering TLR4 or TLR2 during chronic filarial infection has a significant impact on patency and efficient CD4(+) T-cell responses, respectively.

  11. Immunization with Wuchereria bancrofti Glutathione-S-transferase Elicits a Mixed Th1/Th2 Type of Protective Immune Response Against Filarial Infection in Mastomys.

    PubMed

    Andure, Dhananjay; Pote, Kiran; Khatri, Vishal; Amdare, Nitin; Padalkar, Ramchandra; Reddy, Maryada Venkata Rami

    2016-10-01

    Lymphatic filariasis is a mosquito borne parasitic infection and can severely affect the normal working ability of an individual. Currently there is no vaccine available to prevent this infection and the development of a potential vaccine could effectively support the on-going mass drug administration program by World Health Organization (WHO). Filarial parasites have complex mechanisms to modulate the host immune responses against them. The glutathione-S-transferases (GST) are the important enzymes effectively involved to counteract the oxidative free radicals produced by the host. In the present study, we have shown that the mastomys which are fully permissible rodents for Brugia malayi when immunized with Wuchereria bancrofti recombinant GST (rWbGST) could induce 65.5 % in situ cytotoxicity against B. malayi infective (L3) larvae. There was a balanced Th1/Th2 immune response in the vaccinated animals, characterized by higher levels of WbGST-specific IgG1 and IgG2a antibodies and pronounced IFN-γ, IL-10 and IL-4 cytokines production by the spleen cells. PMID:27605739

  12. Xenomonitoring of different filarial nematodes using single and multiplex PCR in mosquitoes from Assiut Governorate, Egypt.

    PubMed

    Dyab, Ahmed Kamal; Galal, Lamia Ahmed; Mahmoud, Abeer El-Sayed; Mokhtar, Yasser

    2015-02-01

    Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs. PMID:25748712

  13. Wolbachia filarial interactions.

    PubMed

    Taylor, Mark J; Voronin, Denis; Johnston, Kelly L; Ford, Louise

    2013-04-01

    Wolbachia pipientis is a widespread intracellular bacterial symbiont of arthropods and is common in insects. One of their more exotic and unexpected hosts is the filarial nematodes, notable for the parasites responsible for onchocerciasis (river blindness), lymphatic filariasis (elephantiasis) and dirofilariasis (heartworm). Wolbachia are only present in a subgroup of the filarial nematodes and do not extend to other groups of nematodes either parasitic or free-living. In the medically and veterinary important species that host Wolbachia, the symbiont has become an essential partner to key biological processes in the life of the nematode to the point where antibiotic elimination of the bacteria leads to a potent and effective anti-filarial drug treatment. We review the cellular and molecular basis of Wolbachia filarial interactions and highlight the key processes provided by the endosymbiont upon which the nematodes have become entirely dependent. This dependency is primarily restricted to periods of the lifecycle with heavy metabolic demands including growth and development of larval stages and embryogenesis in the adult female. Also, the longevity of filarial parasites is compromised following depletion of the symbiont, which for the first time has delivered a safe and effective treatment to kill adult parasites with antibiotics. PMID:23210448

  14. Immunopathogenesis of lymphatic filarial disease1

    PubMed Central

    Babu, Subash; Nutman, Thomas B.

    2012-01-01

    Although two-thirds of the 120 million people infected with lymph-dwelling filarial parasites have subclinical infections, ~ 40 million have lymphedema and/or other pathologic manifestations including hydroceles (and other forms of urogenital disease), episodic adenolymphangitis, tropical pulmonary eosinophilia, lymphedema, and (in its most severe form) elephantiasis. Adult filarial worms reside in the lymphatics and lymph nodes and induce changes that result in dilatation of lymphatics and thickening of the lymphatic vessel walls. Progressive lymphatic damage and pathology results from the summation of the effect of tissue alterations induced by both living and nonliving adult parasites, the host inflammatory response to the parasites and their secreted antigens, the host inflammatory response to the endosymbiont Wolbachia, and those seen as a consequence of secondary bacterial or fungal infections. Inflammatory damage induced by filarial parasites appears to be multifactorial, with endogenous parasite products, Wolbachia, and host immunity all playing important roles. This review will initially examine the prototypical immune responses engendered by the parasite and delineate the regulatory mechanisms elicited to prevent immune-mediated pathology. This will be followed by a discussion of the proposed mechanisms underlying pathogenesis, with the central theme being that pathogenesis is a two-step process - the first initiated by the parasite and host innate immune system and the second propagated mainly by the host’s adaptive immune system and by other factors (including secondary infections). PMID:23053393

  15. Molecular epidemiology, phylogeny and evolution of the filarial nematode Wuchereria bancrofti.

    PubMed

    Small, Scott T; Tisch, Daniel J; Zimmerman, Peter A

    2014-12-01

    Wuchereria bancrofti (Wb) is the most widely distributed of the three nematodes known to cause lymphatic filariasis (LF), the other two being Brugia malayi and Brugia timori. Current tools available to monitor LF are limited to diagnostic tests targeting DNA repeats, filarial antigens, and anti-filarial antibodies. While these tools are useful for detection and surveillance, elimination programs have yet to take full advantage of molecular typing for inferring infection history, strain fingerprinting, and evolution. To date, molecular typing approaches have included whole mitochondrial genomes, genotyping, targeted sequencing, and random amplified polymorphic DNA (RAPDs). These studies have revealed much about Wb biology. For example, in one study in Papua New Guinea researchers identified 5 major strains that were widespread and many minor strains some of which exhibit geographic stratification. Genome data, while rare, has been utilized to reconstruct evolutionary relationships among taxa of the Onchocercidae (the clade of filarial nematodes) and identify gene synteny. Their phylogeny reveals that speciation from the common ancestor of both B. malayi and Wb occurred around 5-6 millions years ago with shared ancestry to other filarial nematodes as recent as 15 million years ago. These discoveries hold promise for gene discovery and identifying drug targets in species that are more amenable to in vivo experiments. Continued technological developments in whole genome sequencing and data analysis will likely replace many other forms of molecular typing, multiplying the amount of data available on population structure, genetic diversity, and phylogenetics. Once widely available, the addition of population genetic data from genomic studies should hasten the elimination of LF parasites like Wb. Infectious disease control programs have benefited greatly from population genetics data and recently from population genomics data. However, while there is currently a surplus

  16. Molecular Detection of Schistosome Infections with a Disposable Microfluidic Cassette

    PubMed Central

    Song, Jinzhao; Liu, Changchun; Bais, Swarna; Mauk, Michael G.; Bau, Haim H.; Greenberg, Robert M.

    2015-01-01

    Parasitic helminths such as schistosomes, as well as filarial and soil-transmitted nematodes, are estimated to infect at least a billion people worldwide, with devastating impacts on human health and economic development. Diagnosis and monitoring of infection dynamics and efficacy of treatment depend almost entirely on methods that are inaccurate, labor-intensive, and unreliable. These shortcomings are amplified and take on added significance in mass drug administration programs, where measures of effectiveness depend on accurate monitoring of treatment success (or failure), changes in disease transmission rates, and emergence of possible drug resistance. Here, we adapt isothermal molecular assays such as loop-mediated isothermal amplification (LAMP) to a simple, hand-held, custom-made field-ready microfluidic device that allows sensitive and specific detection of schistosome cell-free nucleic acids in serum and plasma (separated with a point-of-care plasma separator) from Schistosoma mansoni-infected mice. Cell-free S. mansoni DNA was detected with our device without prior extraction from blood. Our chip exhibits high sensitivity (~2x10−17 g/μL), with a positive signal for S. mansoni DNA detectable as early as one week post infection, several weeks before parasite egg production commences. These results indicate that incorporation of isothermal amplification strategies with our chips could represent a strategy for rapid, simple, low-cost diagnosis of both pre-patent and chronic schistosome infections as well as potential monitoring of treatment efficacy. PMID:26720725

  17. A Novel Xenomonitoring Technique Using Mosquito Excreta/Feces for the Detection of Filarial Parasites and Malaria

    PubMed Central

    Pilotte, Nils; Zaky, Weam I.; Abrams, Brian P.; Chadee, Dave D.; Williams, Steven A.

    2016-01-01

    Background Given the continued successes of the world’s lymphatic filariasis (LF) elimination programs and the growing successes of many malaria elimination efforts, the necessity of low cost tools and methodologies applicable to long-term disease surveillance is greater than ever before. As many countries reach the end of their LF mass drug administration programs and a growing number of countries realize unprecedented successes in their malaria intervention efforts, the need for practical molecular xenomonitoring (MX), capable of providing surveillance for disease recrudescence in settings of decreased parasite prevalence is increasingly clear. Current protocols, however, require testing of mosquitoes in pools of 25 or fewer, making high-throughput examination a challenge. The new method we present here screens the excreta/feces from hundreds of mosquitoes per pool and provides proof-of-concept for a practical alternative to traditional methodologies resulting in significant cost and labor savings. Methodology/Principal Findings Excreta/feces of laboratory reared Aedes aegypti or Anopheles stephensi mosquitoes provided with a Brugia malayi microfilaria-positive or Plasmodium vivax-positive blood meal respectively were tested for the presence of parasite DNA using real-time PCR. A titration of samples containing various volumes of B. malayi-negative mosquito feces mixed with positive excreta/feces was also tested to determine sensitivity of detection. Real-time PCR amplification of B. malayi and P. vivax DNA from the excreta/feces of infected mosquitoes was demonstrated, and B. malayi DNA in excreta/feces from one to two mf-positive blood meal-receiving mosquitoes was detected when pooled with volumes of feces from as many as 500 uninfected mosquitoes. Conclusions/Significance While the operationalizing of excreta/feces testing may require the development of new strategies for sample collection, the high-throughput nature of this new methodology has the

  18. Molecular identification of Wolbachia from the filarial nematode Mansonella perstans.

    PubMed

    Keiser, Paul B; Coulibaly, Yaya; Kubofcik, Joseph; Diallo, Abdallah A; Klion, Amy D; Traoré, Sekou F; Nutman, Thomas B

    2008-08-01

    Wolbachiae are bacterial endosymbionts of insects and many filarial nematodes whose products trigger inflammation in filarial infections. The dependence of the parasites on their endosymbionts has also led to the use of antibiotics directed against the Wolbachiae, therapy that has been demonstrated to have a profound salutary effect on filarial infections. The identification of Wolbachiae in Mansonella species has been conclusively shown for Mansonella ozzardi (Mo), but not for Mansonella perstans (Mp). Using primers known to amplify the 16S ribosomal DNA of other filarial Wolbachiae, an identical 1393bp band was found in all samples tested. Sequence analysis of these samples demonstrated a single consensus sequence for Mp Wolbachia 16S rDNA that was most similar to Wolbachia sequences from other filarial nematodes. When aligned with the only other Mansonella Wolbachia sequence (Mo) there were only 8 nucleotide differences in the 1369bp overlapping sequence. Phylogenetic dendrograms, examining the relationship of the Mp Wolbachia to other Wolbachia 16S rDNA, showed that the Wolbachia tracked almost identically to the 5S rRNA of their parasite host. Wolbachia surface protein (WSP) was also demonstrated in protein extracted from Mp-containing whole blood. In advance of a treatment trial of Mp, a method for the quantitation of Mp Wolbachia was developed and used to demonstrate not only a relationship between microfilarial numbers and Wolbachia copy numbers, but also to demonstrate the effect of antibiotic on ridding Mp of Wolbachia. PMID:18538871

  19. How do the macrocyclic lactones kill filarial nematode larvae?

    PubMed

    Wolstenholme, Adrian J; Maclean, Mary J; Coates, Ruby; McCoy, Ciaran J; Reaves, Barbara J

    2016-09-01

    The macrocyclic lactones (MLs) are one of the few classes of drug used in the control of the human filarial infections, onchocerciasis and lymphatic filariasis, and the only one used to prevent heartworm disease in dogs and cats. Despite their importance in preventing filarial diseases, the way in which the MLs work against these parasites is unclear. In vitro measurements of nematode motility have revealed a large discrepancy between the maximum plasma concentrations achieved after drug administration and the amounts required to paralyze worms. Recent evidence has shed new light on the likely functions of the ML target, glutamate-gated chloride channels, in filarial nematodes and supports the hypothesis that the rapid clearance of microfilariae that follows treatment involves the host immune system. PMID:27279086

  20. Interleukin 1 (IL-1)- and IL-23-Mediated Expansion of Filarial Antigen-Specific Th17 and Th22 Cells in Filarial Lymphedema

    PubMed Central

    Anuradha, R.; George, P. Jovvian; Chandrasekaran, V.; Kumaran, P. Paul; Nutman, Thomas B.

    2014-01-01

    Lymphatic filarial disease is known to be associated with elevated Th1 responses and normal or diminished Th2 responses to parasite-specific antigens. The roles of Th17 cells and the recently described Th22 cells have not been examined in detail in either filarial infection itself or in filarial disease (e.g., lymphedema and elephantiasis). To explore the roles of Th17 and Th22 cells and their subsets, we examined the frequencies of these cells in individuals with filarial lymphedema (chronic pathology [CP]), in clinically asymptomatic infected (INF) individuals, and in uninfected (UN) individuals ex vivo and in response to parasite and nonparasite antigens. Those with disease (CP) had significantly expanded frequencies of Th17 and Th22 cells, compared with either INF or UN individuals, at baseline (ex vivo) and in response to parasite antigens. This antigen-driven expansion of Th17 and Th22 cells was dependent on interleukin 1 (IL-1), IL-23, and, to lesser extent, transforming growth factor β (TGF-β), as blockade of any of these cytokines resulted in significantly diminished frequencies of Th17 and Th22 cells. Our findings, therefore, suggest that filarial parasite-driven expansion of Th17 and Th22 cells is associated with the pathogenesis of filarial infections and disease. PMID:24807054

  1. Disorganized muscle protein-1 (DIM-1) of filarial parasite Brugia malayi: cDNA cloning, expression, purification, structural modeling and its potential as vaccine candidate for human filarial infection.

    PubMed

    Kushwaha, Vikas; Kumar, Vikash; Verma, Shiv K; Sharma, Rolee; Siddiqi, M I; Murthy, P K

    2014-03-26

    We have recently identified disorganized muscle protein-1 (DIM-1) in one of the proinflammatory fractions of the human filaria Brugia malayi adult worm. The present study was undertaken to characterize B. malayi DIM-1 (DIM-1bm) and explore its vaccine potential. In this study we cloned and expressed the DIM-1bm gene, investigated its sequence homology with other nematodes, constructed in silico structural model, purified the recombinant DIM-1bm (rDIM-1bm) protein, and studied the effect of immunization with rDIM-1bm on the establishment of B. malayi infection in Mastomys coucha. DIM-1bm showed similarity with DIM-1 of Caenorhabditis elegans, Ascaris suum and Loa loa. Structural modeling revealed three immunoglobulin domains in DIM-1bm indicating that it is a member of immunoglobulin superfamily (IgSF) and 'blastn' results showed that DIM-1bm coding sequence (CDS) have almost no homology with human and mouse nucleotide sequences. Immunization with rDIM-1bm partially protected M. coucha against establishment of infection as inferred by a low recovery of microfilariae (37-64%) and parasite burden (∼50%). The enhanced activity of macrophages, and IFN-γ and NO responses, and elevated levels of specific IgG, IgG1, IgG2a and IgG2b correlated with parasitological findings. This is the first report on cloning, expression, structural modeling and purification of rDIM-1bm and its ability to partially prevent establishment of B. malayi infection. DIM-1bm's almost complete lack of homology with the human counterpart makes it an attractive protein for exploring its vaccine potential. PMID:24513011

  2. Polyanhydride Nanoparticle Delivery Platform Dramatically Enhances Killing of Filarial Worms

    PubMed Central

    Binnebose, Andrea M.; Haughney, Shannon L.; Martin, Richard; Imerman, Paula M.; Narasimhan, Balaji; Bellaire, Bryan H.

    2015-01-01

    Filarial diseases represent a significant social and economic burden to over 120 million people worldwide and are caused by endoparasites that require the presence of symbiotic bacteria of the genus Wolbachia for fertility and viability of the host parasite. Targeting Wolbachia for elimination is a therapeutic approach that shows promise in the treatment of onchocerciasis and lymphatic filariasis. Here we demonstrate the use of a biodegradable polyanhydride nanoparticle-based platform for the co-delivery of the antibiotic doxycycline with the antiparasitic drug, ivermectin, to reduce microfilarial burden and rapidly kill adult worms. When doxycycline and ivermectin were co-delivered within polyanhydride nanoparticles, effective killing of adult female Brugia malayi filarial worms was achieved with approximately 4,000-fold reduction in the amount of drug used. Additionally the time to death of the macrofilaria was also significantly reduced (five-fold) when the anti-filarial drug cocktail was delivered within polyanhydride nanoparticles. We hypothesize that the mechanism behind this dramatically enhanced killing of the macrofilaria is the ability of the polyanhydride nanoparticles to behave as a Trojan horse and penetrate the cuticle, bypassing excretory pumps of B. malayi, and effectively deliver drug directly to both the worm and Wolbachia at high enough microenvironmental concentrations to cause death. These provocative findings may have significant consequences for the reduction in the amount of drug and the length of treatment required for filarial infections in terms of patient compliance and reduced cost of treatment. PMID:26496201

  3. The "filarial dance" is not characteristic of filariasis: observations of "dancing megasperm" on high-resolution sonography in patients from nonendemic areas mimicking the filarial dance and a proposed mechanism for this phenomenon.

    PubMed

    Adejolu, Margaret; Sidhu, Paul S

    2011-08-01

    The objective of this series was to show that the sonographic appearance described as the "filarial dance" is not characteristic of filariasis but occurs in nonendemic areas as a manifestation of epididymal obstruction. An experienced observer documented cases after initial observation of the filarial dance in routine clinical practice using high-frequency linear array transducers. The filarial dance was described as excessive to-and-fro movement of echogenic particles within a prominent epididymis and graded 1 to 4 according to the extent and distribution of the abnormality. The country of birth, exposure to filarial infection or travel to a filarial-endemic area, previous scrotal surgery including vasectomy, any previous or current scrotal inflammatory disease, and any congenital testicular abnormalities were recorded. Over a 10-year period, sonographic appearances consistent with the filarial dance were observed in 18 patients (bilateral in 6). The mean patient age was 47.7 (range, 28-91) years. The abnormality was graded in the 24 affected testes as follows: grade 1, n = 3; grade 2, n = 8; grade 3, n = 8; and grade 4, n = 5. No patient had a history of filariasis or travel to an endemic area. Six of 18 patients (33.3%) had bilateral vasectomies; 5 (27.8%) had a history of epididymo-orchitis in the ipsilateral testis; 3 (16.7%) had previous scrotal surgery; and 4 (22.2%) had no relevant urologic history. We have described a sonographic appearance identical to the filarial dance in men with no history of filarial infection. Most had previous scrotal surgery or infection, suggesting that the filarial dance may not always be due to movement of filarial worms. The unifying condition in patients with filariasis and our patients is lymphatic obstruction, likely the underlying cause of the appearance in both groups.

  4. The interaction between filarial parasites and human monocyte/macrophage populations.

    PubMed

    Semnani, Roshanak Tolouei

    2013-01-01

    Lymphatic filariasis is a mafor tropical disease affecting approximately 120 million people worldwide. Patent infection, by and large, is clinically asymptomatic but is associated with the inability of T cells to proliferate or produce IFN-γ in response to parasite antigen. Monocyte dysfunction is one hypothesis felt to explain the lack of an antigen-specific T cell response. In fact, monocytes from filaria-infected individuals have been shown to be studded with internalized filarial antigens. Understanding how the phenotype and the function of these monocytes are altered through the internalization of these parasite antigens is one of the areas our laboratory has focused on. In fact, the existence and/or function of alternatively activated macrophages in murine models of filarial infections have been extensively studied. Whether this population of macrophages can be induced in human filarial infections is the main focus of this review. PMID:23456837

  5. Spectral and landscape characterization of filarious and non-filarious villages in Egypt.

    PubMed

    Sowilem, Mohamed M; Bahgat, Iman M; el-Kady, Gamal A; el-Sawaf, Bahira M

    2006-08-01

    Filarial disease is endemic in Egypt in some villages of Nile Delta governorates where it is transmitted by Culex pipiens female. GIS functions are used to identify environmental indicators of high-risk village as indicated by mosquito density, human infection rate, vector species composition, mean life expectancy "e(o)" & environmental variables (geology, hydrology, soil types, etc) as well as meteorological factors (temperature, RH and rainfall) in relation to filaria transmission risk. Remote-sensing technology was used to distinguish between the two studied villages as high and non-infected, as defined by microfilarial prevalence. The results indicate that filaria transmission risk is higher at an area characterized by highly productive aquifers, silt clay soil, receiving little amount of rain with low relative humidity (RH). The results indicate that the most important landscape elements associated with prevalence are water and different vegetation. This work showed that the integration between GIS and remote sensing technologies to analyze and identify the environmental factors, associated with the disease, did not only allow mapping icurrent spatial patterns, but also predicting its distribution under expected future developmental and environmental changes. PMID:16927856

  6. Granuloma formation around filarial larvae triggered by host responses to an excretory/secretory antigen.

    PubMed

    Dash, Yashodhara; Ramesh, Manish; Kalyanasundaram, Ramaswamy; Munirathinam, Gnanasekar; Shultz, Leonard D; Rajan, T V

    2011-02-01

    In previous studies using a murine model of filarial infection, granuloma formation was found to be a most important host-protective mechanism. We have also shown that in vitro cytoadherence is a surrogate for the formation of antifilarial granulomas in vivo and that it requires "alternatively activated" host cells and a source of antifilarial antibody. We show here that antibodies against L3 excretory/secretory (E/S) products can facilitate in vitro cytoadherence. We generated a set of hybridomas reactive with filarial E/S products and screened them for their ability to mediate in vitro cytoadherence. One clone (no. 1E9) was positive in this assay. We then screened a novel expression library of filarial antigens displayed on the surface of T7 bacteriophage for reactivity with 1E9. Phage expressing two filarial antigens (TCTP and BmALT-2) reacted with 1E9. Immunization of mice showed that the cohort immunized with BmALT-2 cleared a challenge infection with infective Brugia pahangi L3 in an accelerated manner, whereas cohorts immunized with TCTP cleared larvae with the same kinetics as in unimmunized mice. These data confirm that BmALT-2 is the antigenic target of granuloma-mediated killing of B. pahangi L3. Our findings also confirm previous studies that BmALT-2 is a potential vaccine candidate for filarial infection. Our data reinforce the work of others and also provide a possible mechanism by which immune responses to BmALT-2 may provide host protection.

  7. Cloning of a cuticular antigen that contains multiple tandem repeats from the filarial parasite Dirofilaria immitis.

    PubMed Central

    Poole, C B; Grandea, A G; Maina, C V; Jenkins, R E; Selkirk, M E; McReynolds, L A

    1992-01-01

    An unusual antigen composed of tandemly repeated protein units was cloned from the filarial parasite Dirofilaria immitis. The antigen was initially identified by screening a lambda gt11 cDNA library with serum from dogs immunized with irradiated D. immitis third-stage larvae. DNA sequence analysis of the cDNA clone, Di5, revealed a continuous open reading frame composed of two 399-base-pair repeats arranged in tandem. Southern blot analysis of genomic D. immitis DNA showed that the gene coding for Di5 is composed of a tandem array of 25-50 copies of this same 399-base-pair repeat. Antiserum raised against recombinant Di5 protein detected a protein "ladder," from about 14 to greater than 200 kDa with steps approximately 15 kDa apart, on immunoblots of D. immitis extract. Metabolic labeling of adult parasites with [35S]methionine showed that Di5 is synthesized as a large precursor that is subsequently cleaved to produce the ladder-like array. These results suggest that the characteristic ladder is created by proteolytic cleavage of the precursor at the same site in each monomer. The Di5 antigen was localized to the cuticle and hypodermis of adult D. immitis by immunoelectron microscopy. Both male and female parasites were found to release Di5 when cultured in vitro. DNA hybridization analysis demonstrated that Di5 is a member of a gene family present in many filarial parasites that infect both animal and human populations. Images PMID:1631084

  8. Early detection of CLas infections in citrus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    “Early” detection of CLas infection is essential to minimize the risk of Huanglongbing (HLB) epidemics in areas where the pathogen has been recently introduced. Any delay in confirmation of CLas infection results in delays of regulatory and management actions, and increased spread of the pathogen ev...

  9. Loop-mediated isothermal amplification for rapid and semiquantitative detection of Loa loa infection.

    PubMed

    Drame, Papa M; Fink, Doran L; Kamgno, Joseph; Herrick, Jesica A; Nutman, Thomas B

    2014-06-01

    Rapid and accurate tests are currently needed to identify individuals with high levels of Loa loa microfilaria (mf), so that these individuals may be excluded from mass ivermectin administration campaigns against onchocerciasis and lymphatic filariasis being conducted in areas where Onchocerca volvulus, Wuchereria bancrofti, and L. loa are coendemic. To address this need, colorimetric loop-mediated isothermal amplification (LAMP) assays targeting the L. loa-specific gene sequences LLMF72 and LLMF342 were developed for the detection and quantification of L. loa microfilaremia. Both LAMP assays were highly specific (100%) for L. loa infection compared to the absence of infection or infection with related filarial pathogens. The LLMF72-based LAMP assay showed greater analytic sensitivity (limit of detection, 0.1 pg/ml of genomic DNA [gDNA] and/or 5 mf/ml) than the LLMF342-based LAMP assay (10 pg/ml of gDNA and/or 50 mf/ml), and its analytic sensitivity was similar to that of LLMF72-based quantitative PCR (qPCR). A high level of correlation was observed between microfilaria counts as determined by LLMF72-based qPCR and time to positivity by the LAMP assay, and performance measures of sensitivity, specificity, and positive and negative predictive values were similar for both assays when applied to field-collected clinical samples. By simply varying the run time, the LAMP assay was able to accurately distinguish individuals at risk for serious adverse events (SAEs) after exposure to ivermectin, using thresholds of >5,000 mf/ml and >30,000 mf/ml as indicators of increasing levels of risk. In summary, LLMF72 LAMP represents a new molecular diagnostic tool that is readily applicable as a point-of-care method for L. loa microfilarial detection and quantification in resource-limited countries where L. loa infection is endemic. PMID:24696020

  10. Loop-Mediated Isothermal Amplification for Rapid and Semiquantitative Detection of Loa loa Infection

    PubMed Central

    Drame, Papa M.; Fink, Doran L.; Kamgno, Joseph; Herrick, Jesica A.

    2014-01-01

    Rapid and accurate tests are currently needed to identify individuals with high levels of Loa loa microfilaria (mf), so that these individuals may be excluded from mass ivermectin administration campaigns against onchocerciasis and lymphatic filariasis being conducted in areas where Onchocerca volvulus, Wuchereria bancrofti, and L. loa are coendemic. To address this need, colorimetric loop-mediated isothermal amplification (LAMP) assays targeting the L. loa-specific gene sequences LLMF72 and LLMF342 were developed for the detection and quantification of L. loa microfilaremia. Both LAMP assays were highly specific (100%) for L. loa infection compared to the absence of infection or infection with related filarial pathogens. The LLMF72-based LAMP assay showed greater analytic sensitivity (limit of detection, 0.1 pg/ml of genomic DNA [gDNA] and/or 5 mf/ml) than the LLMF342-based LAMP assay (10 pg/ml of gDNA and/or 50 mf/ml), and its analytic sensitivity was similar to that of LLMF72-based quantitative PCR (qPCR). A high level of correlation was observed between microfilaria counts as determined by LLMF72-based qPCR and time to positivity by the LAMP assay, and performance measures of sensitivity, specificity, and positive and negative predictive values were similar for both assays when applied to field-collected clinical samples. By simply varying the run time, the LAMP assay was able to accurately distinguish individuals at risk for serious adverse events (SAEs) after exposure to ivermectin, using thresholds of >5,000 mf/ml and >30,000 mf/ml as indicators of increasing levels of risk. In summary, LLMF72 LAMP represents a new molecular diagnostic tool that is readily applicable as a point-of-care method for L. loa microfilarial detection and quantification in resource-limited countries where L. loa infection is endemic. PMID:24696020

  11. Filarial excretory-secretory products induce human monocytes to produce lymphangiogenic mediators.

    PubMed

    Weinkopff, Tiffany; Mackenzie, Charles; Eversole, Rob; Lammie, Patrick J

    2014-07-01

    The nematodes Wuchereria bancrofti and Brugia spp. infect over 120 million people worldwide, causing lymphedema, elephantiasis and hydrocele, collectively known as lymphatic filariasis. Most infected individuals appear to be asymptomatic, but many exhibit sub-clinical manifestations including the lymphangiectasia that likely contributes to the development of lymphedema and elephantiasis. As adult worm excretory-secretory products (ES) do not directly activate lymphatic endothelial cells (LEC), we investigated the role of monocyte/macrophage-derived soluble factors in the development of filarial lymphatic pathology. We analyzed the production of IL-8, IL-6 and VEGF-A by peripheral blood mononuclear cells (PBMC) from naïve donors following stimulation with filarial ES products. ES-stimulated PBMCs produced significantly more IL-8, IL-6 and VEGF-A compared to cells cultured in medium alone; CD14(+) monocytes appear to be the primary producers of IL-8 and VEGF-A, but not IL-6. Furthermore, IL-8, IL-6 and VEGF-A induced in vitro tubule formation in LEC Matrigel cultures. Matrigel plugs supplemented with IL-8, IL-6, VEGF-A, or with supernatants from ES-stimulated PBMCs and implanted in vivo stimulated lymphangiogenesis. Collectively, these data support the hypothesis that monocytes/macrophages exposed to filarial ES products may modulate lymphatic function through the secretion of soluble factors that stimulate the vessel growth associated with the pathogenesis of filarial disease.

  12. Human Infections and Detection of Plasmodium knowlesi

    PubMed Central

    Daneshvar, Cyrus

    2013-01-01

    SUMMARY Plasmodium knowlesi is a malaria parasite that is found in nature in long-tailed and pig-tailed macaques. Naturally acquired human infections were thought to be extremely rare until a large focus of human infections was reported in 2004 in Sarawak, Malaysian Borneo. Human infections have since been described throughout Southeast Asia, and P. knowlesi is now recognized as the fifth species of Plasmodium causing malaria in humans. The molecular, entomological, and epidemiological data indicate that human infections with P. knowlesi are not newly emergent and that knowlesi malaria is primarily a zoonosis. Human infections were undiagnosed until molecular detection methods that could distinguish P. knowlesi from the morphologically similar human malaria parasite P. malariae became available. P. knowlesi infections cause a spectrum of disease and are potentially fatal, but if detected early enough, infections in humans are readily treatable. In this review on knowlesi malaria, we describe the early studies on P. knowlesi and focus on the epidemiology, diagnosis, clinical aspects, and treatment of knowlesi malaria. We also discuss the gaps in our knowledge and the challenges that lie ahead in studying the epidemiology and pathogenesis of knowlesi malaria and in the prevention and control of this zoonotic infection. PMID:23554413

  13. Diagnostic Procedures to Detect Chlamydia trachomatis Infections.

    PubMed

    Meyer, Thomas

    2016-08-05

    The intracellular life style of chlamydia and the ability to cause persistent infections with low-grade replication requires tests with high analytical sensitivity to directly detect C. trachomatis (CT) in medical samples. Nucleic acid amplification tests (NAATs) are the most sensitive assays with a specificity similar to cell culture and are considered the method of choice for CT detection. In addition, NAATs can be performed on various clinical specimens that do not depend on specific transport and storage conditions, since NAATs do not require infectious bacteria. In the case of lower genital tract infections, first void urine and vaginal swabs are the recommended specimens for testing males and females, respectively. Infections of anorectal, oropharyngeal and ocular epithelia should also be tested by NAAT analysis of corresponding mucosal swabs. In particular, anorectal infections of men who have sex with men (MSM) should include evaluation of lymphogranuloma venereum (LGV) by identification of genotypes L1, L2 or L3. Detection of CT antigens by enzyme immunoassay (EIAs) or rapid diagnostic tests (RDTs) are unsuitable due to insufficient sensitivity and specificity. Recent PCR-based RDTs, however, are non-inferior to standard NAATs, and might be used at the point-of-care. Serology finds application in the diagnostic work-up of suspected chronic CT infection but is inappropriate to diagnose acute infections.

  14. Diagnostic Procedures to Detect Chlamydia trachomatis Infections

    PubMed Central

    Meyer, Thomas

    2016-01-01

    The intracellular life style of chlamydia and the ability to cause persistent infections with low-grade replication requires tests with high analytical sensitivity to directly detect C. trachomatis (CT) in medical samples. Nucleic acid amplification tests (NAATs) are the most sensitive assays with a specificity similar to cell culture and are considered the method of choice for CT detection. In addition, NAATs can be performed on various clinical specimens that do not depend on specific transport and storage conditions, since NAATs do not require infectious bacteria. In the case of lower genital tract infections, first void urine and vaginal swabs are the recommended specimens for testing males and females, respectively. Infections of anorectal, oropharyngeal and ocular epithelia should also be tested by NAAT analysis of corresponding mucosal swabs. In particular, anorectal infections of men who have sex with men (MSM) should include evaluation of lymphogranuloma venereum (LGV) by identification of genotypes L1, L2 or L3. Detection of CT antigens by enzyme immunoassay (EIAs) or rapid diagnostic tests (RDTs) are unsuitable due to insufficient sensitivity and specificity. Recent PCR-based RDTs, however, are non-inferior to standard NAATs, and might be used at the point-of-care. Serology finds application in the diagnostic work-up of suspected chronic CT infection but is inappropriate to diagnose acute infections. PMID:27681919

  15. Diagnostic Procedures to Detect Chlamydia trachomatis Infections.

    PubMed

    Meyer, Thomas

    2016-01-01

    The intracellular life style of chlamydia and the ability to cause persistent infections with low-grade replication requires tests with high analytical sensitivity to directly detect C. trachomatis (CT) in medical samples. Nucleic acid amplification tests (NAATs) are the most sensitive assays with a specificity similar to cell culture and are considered the method of choice for CT detection. In addition, NAATs can be performed on various clinical specimens that do not depend on specific transport and storage conditions, since NAATs do not require infectious bacteria. In the case of lower genital tract infections, first void urine and vaginal swabs are the recommended specimens for testing males and females, respectively. Infections of anorectal, oropharyngeal and ocular epithelia should also be tested by NAAT analysis of corresponding mucosal swabs. In particular, anorectal infections of men who have sex with men (MSM) should include evaluation of lymphogranuloma venereum (LGV) by identification of genotypes L1, L2 or L3. Detection of CT antigens by enzyme immunoassay (EIAs) or rapid diagnostic tests (RDTs) are unsuitable due to insufficient sensitivity and specificity. Recent PCR-based RDTs, however, are non-inferior to standard NAATs, and might be used at the point-of-care. Serology finds application in the diagnostic work-up of suspected chronic CT infection but is inappropriate to diagnose acute infections. PMID:27681919

  16. Diagnostic Procedures to Detect Chlamydia trachomatis Infections

    PubMed Central

    Meyer, Thomas

    2016-01-01

    The intracellular life style of chlamydia and the ability to cause persistent infections with low-grade replication requires tests with high analytical sensitivity to directly detect C. trachomatis (CT) in medical samples. Nucleic acid amplification tests (NAATs) are the most sensitive assays with a specificity similar to cell culture and are considered the method of choice for CT detection. In addition, NAATs can be performed on various clinical specimens that do not depend on specific transport and storage conditions, since NAATs do not require infectious bacteria. In the case of lower genital tract infections, first void urine and vaginal swabs are the recommended specimens for testing males and females, respectively. Infections of anorectal, oropharyngeal and ocular epithelia should also be tested by NAAT analysis of corresponding mucosal swabs. In particular, anorectal infections of men who have sex with men (MSM) should include evaluation of lymphogranuloma venereum (LGV) by identification of genotypes L1, L2 or L3. Detection of CT antigens by enzyme immunoassay (EIAs) or rapid diagnostic tests (RDTs) are unsuitable due to insufficient sensitivity and specificity. Recent PCR-based RDTs, however, are non-inferior to standard NAATs, and might be used at the point-of-care. Serology finds application in the diagnostic work-up of suspected chronic CT infection but is inappropriate to diagnose acute infections.

  17. Expanding the MDx toolbox for filarial diagnosis and surveillance.

    PubMed

    Alhassan, Andy; Li, Zhiru; Poole, Catherine B; Carlow, Clotilde K S

    2015-08-01

    Filarial parasites are tissue-dwelling nematodes responsible for some of the most important neglected tropical diseases. All are transmitted by blood-sucking arthropod. Onchocerciasis and lymphatic filariasis in particular are the cause of much disfigurement and morbidity. Accurate parasite detection is essential for the success of filariasis control programs. The current toolbox for diagnosis and surveillance is limited because many of the available tools suffer from lack of sensitivity and specificity, and/or are cost-prohibitive. We review the methods currently in use and discuss the prospects for developing new molecular diagnostic (MDx) tools based on nucleic acid detection. We briefly describe recent developments in isothermal nucleic acid amplification and detection, and focus on emerging technologies that are field-deployable or suitable for low-resource settings.

  18. Tropical vaginal hydroceles: are they all filarial in origin?

    PubMed

    Sivam, N S; Jayanthi, S; Ananthakrishnan, N; Elango, A; Yuvaraj, J; Hoti, S L; Pani, S P

    1995-12-01

    Hydrocele of the tunica vaginalis testis has been conventionally used as an absolute indicator of filarial disease in most clinical surveys. The prevalence of filarial etiology in 100 consecutive hydroceles was studied using clinical, parasitological, histopathological and immunological parameters. Filarial etiology could be proved in 57% of hydrocele cases using major criteria: presence of microfilaria in hydrocele fluid, presence of chyle in hydrocele fluid, demonstration of adult worm in tunica, ratio of fluid antibody titer to serum antibody titer more than 2 and presence of filarial antigen in hydrocele fluid. The results of other tests in these 57 cases were used to define the minor criteria. In the other 43 cases, based on the minor criteria, 12 hydroceles could be classified as likely to be due to filariasis and the rest were probably non-filarial. Thus only 69% of hydroceles were definitely or probably filarial.

  19. Mansonella ozzardi: a neglected New World filarial nematode.

    PubMed

    Lima, Nathália F; Veggiani Aybar, Cecilia A; Dantur Juri, María J; Ferreira, Marcelo U

    2016-05-01

    Mansonella ozzardi (Nematoda: Onchocercidae) is an understudied filarial nematode, originally described by Patrick Manson in 1897, that can be transmitted by two families of dipteran vectors, biting midges (most of them members of the genus Culicoides) and black flies (genus Simulium). With a patchy geographic distribution from southern Mexico to northwestern Argentina, human infection with M. ozzardi is highly prevalent in some of the Caribbean islands, along riverine communities in the Amazon Basin, and on both sides of the border between Bolivia and Argentina. There is no clinical entity unequivocally associated with M. ozzardi infection, although fever, arthralgia, headache, cold lower extremities, and itchy cutaneous rashes are occasionally mentioned in case report series. More recently, ocular manifestations (especially keratitis) have been associated with mansonelliasis, opening an important area of investigation. Here, we briefly review the biology, epidemiology, pathogenesis, and clinical aspects of M. ozzardi infection and point to some existing knowledge gaps, aiming to stimulate a research agenda to help filling them. PMID:27376501

  20. Detection and Characterization of Infections and Infection Susceptibility

    ClinicalTrials.gov

    2016-10-26

    Immune Disorders; Chronic Granulomatous Disease; Genetic Immunological Deficiencies; Hyperimmunoglobulin-E Recurrent Infection Syndrome; Recurrent Infections; Unknown Immune Deficiency; GATA2 Deficiency (MonoMAC); Nontuberculous Mycobacterial Infections; Hyper IgE (Job s) Syndrome; Leukocyte Adhesion Deficiency; Susceptibility to Disseminated Infections; Primary Immune Deficiency Disease (PIDD)

  1. Detection and characterization of Wolbachia infections in laboratory and natural populations of different species of tsetse flies (genus Glossina)

    PubMed Central

    2012-01-01

    Background Wolbachia is a genus of endosymbiotic α-Proteobacteria infecting a wide range of arthropods and filarial nematodes. Wolbachia is able to induce reproductive abnormalities such as cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization and male killing, thus affecting biology, ecology and evolution of its hosts. The bacterial group has prompted research regarding its potential for the control of agricultural and medical disease vectors, including Glossina spp., which transmits African trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals. Results In the present study, we employed a Wolbachia specific 16S rRNA PCR assay to investigate the presence of Wolbachia in six different laboratory stocks as well as in natural populations of nine different Glossina species originating from 10 African countries. Wolbachia was prevalent in Glossina morsitans morsitans, G. morsitans centralis and G. austeni populations. It was also detected in G. brevipalpis, and, for the first time, in G. pallidipes and G. palpalis gambiensis. On the other hand, Wolbachia was not found in G. p. palpalis, G. fuscipes fuscipes and G. tachinoides. Wolbachia infections of different laboratory and natural populations of Glossina species were characterized using 16S rRNA, the wsp (Wolbachia Surface Protein) gene and MLST (Multi Locus Sequence Typing) gene markers. This analysis led to the detection of horizontal gene transfer events, in which Wobachia genes were inserted into the tsetse flies fly nuclear genome. Conclusions Wolbachia infections were detected in both laboratory and natural populations of several different Glossina species. The characterization of these Wolbachia strains promises to lead to a deeper insight in tsetse flies-Wolbachia interactions, which is essential for the development and use of Wolbachia-based biological control methods. PMID:22376025

  2. Moxidectin causes adult worm mortality of human lymphatic filarial parasite Brugia malayi in rodent models.

    PubMed

    Verma, Meenakshi; Pathak, Manisha; Shahab, Mohd; Singh, Kavita; Mitra, Kalyan; Misra-Bhattacharya, Shailja

    2014-12-01

    Moxidectin is a macrocyclic lactone belonging to milbemycin family closely related to ivermectin and is currently progressing towards Phase III clinical trial against human infection with the filaria Onchocerca volvulus (Leuckart, 1894). There is a single report on the microfilaricidal and embryostatic activity of moxidectin in case of the human lymphatic filarial parasite Brugia malayi (Brug, 1927) in Mastomys coucha (Smith) but without any adulticidal action. In the present study, the in vitro and in vivo antifilarial efficacy of moxidectin was evaluated on, B. malayi. In vitro moxidectin showed 100% reduction in adult female worm motility at 0.6 μM concentration within 7 days with 68% inhibition in the reduction of MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye) (which is used to detect viability of worms). A 50% inhibitory concentration (IC50) of moxidectin for adult female parasite was 0.242 μM, for male worm 0.186 μM and for microfilaria IC50 was 0.813 μM. In adult B. malayi-transplanted primary screening model (Meriones unguiculatus Milne-Edwards), moxidectin at a single optimal dose of 20 mg/kg by oral and subcutaneous route was found effective on both adult parasites and microfilariae. In secondary screening (M coucha, subcutaneously inoculated with infective larvae), moxidectin at the same dose by subcutaneous route brought about death of 49% of adult worms besides causing sterilisation in 54% of the recovered live female worms. The treated animals exhibited a continuous and sustained reduction in peripheral blood microfilaraemia throughout the observation period of 90 days. The mechanism of action of moxidectin is suggested to be similar to avermectins. The in silico studies were also designed to explore the interaction of moxidectin with glutamate-gated chloride channels of B. malayi. The docking results revealed a close interaction of moxidectin with various GluCl ligand sites of B. malayi. PMID:25651699

  3. Point-of-care quantification of blood-borne filarial parasites with a mobile phone microscope.

    PubMed

    D'Ambrosio, Michael V; Bakalar, Matthew; Bennuru, Sasisekhar; Reber, Clay; Skandarajah, Arunan; Nilsson, Lina; Switz, Neil; Kamgno, Joseph; Pion, Sébastien; Boussinesq, Michel; Nutman, Thomas B; Fletcher, Daniel A

    2015-05-01

    Parasitic helminths cause debilitating diseases that affect millions of people in primarily low-resource settings. Efforts to eliminate onchocerciasis and lymphatic filariasis in Central Africa through mass drug administration have been suspended because of ivermectin-associated serious adverse events, including death, in patients infected with the filarial parasite Loa loa. To safely administer ivermectin for onchocerciasis or lymphatic filariasis in regions co-endemic with L. loa, a strategy termed "test and (not) treat" has been proposed whereby those with high levels of L. loa microfilariae (>30,000/ml) that put them at risk for life-threatening serious adverse events are identified and excluded from mass drug administration. To enable this, we developed a mobile phone-based video microscope that automatically quantifies L. loa microfilariae in whole blood loaded directly into a small glass capillary from a fingerprick without the need for conventional sample preparation or staining. This point-of-care device automatically captures and analyzes videos of microfilarial motion in whole blood using motorized sample scanning and onboard motion detection, minimizing input from health care workers and providing a quantification of microfilariae per milliliter of whole blood in under 2 min. To validate performance and usability of the mobile phone microscope, we tested 33 potentially Loa-infected patients in Cameroon and confirmed that automated counts correlated with manual thick smear counts (94% specificity; 100% sensitivity). Use of this technology to exclude patients from ivermectin-based treatment at the point of care in Loa-endemic regions would allow resumption/expansion of mass drug administration programs for onchocerciasis and lymphatic filariasis in Central Africa.

  4. Point-of-care quantification of blood-borne filarial parasites with a mobile phone microscope.

    PubMed

    D'Ambrosio, Michael V; Bakalar, Matthew; Bennuru, Sasisekhar; Reber, Clay; Skandarajah, Arunan; Nilsson, Lina; Switz, Neil; Kamgno, Joseph; Pion, Sébastien; Boussinesq, Michel; Nutman, Thomas B; Fletcher, Daniel A

    2015-05-01

    Parasitic helminths cause debilitating diseases that affect millions of people in primarily low-resource settings. Efforts to eliminate onchocerciasis and lymphatic filariasis in Central Africa through mass drug administration have been suspended because of ivermectin-associated serious adverse events, including death, in patients infected with the filarial parasite Loa loa. To safely administer ivermectin for onchocerciasis or lymphatic filariasis in regions co-endemic with L. loa, a strategy termed "test and (not) treat" has been proposed whereby those with high levels of L. loa microfilariae (>30,000/ml) that put them at risk for life-threatening serious adverse events are identified and excluded from mass drug administration. To enable this, we developed a mobile phone-based video microscope that automatically quantifies L. loa microfilariae in whole blood loaded directly into a small glass capillary from a fingerprick without the need for conventional sample preparation or staining. This point-of-care device automatically captures and analyzes videos of microfilarial motion in whole blood using motorized sample scanning and onboard motion detection, minimizing input from health care workers and providing a quantification of microfilariae per milliliter of whole blood in under 2 min. To validate performance and usability of the mobile phone microscope, we tested 33 potentially Loa-infected patients in Cameroon and confirmed that automated counts correlated with manual thick smear counts (94% specificity; 100% sensitivity). Use of this technology to exclude patients from ivermectin-based treatment at the point of care in Loa-endemic regions would allow resumption/expansion of mass drug administration programs for onchocerciasis and lymphatic filariasis in Central Africa. PMID:25947164

  5. Supergroup C Wolbachia, mutualist symbionts of filarial nematodes, have a distinct genome structure

    PubMed Central

    Comandatore, Francesco; Cordaux, Richard; Bandi, Claudio; Blaxter, Mark; Darby, Alistair; Makepeace, Benjamin L.; Montagna, Matteo; Sassera, Davide

    2015-01-01

    Wolbachia pipientis is possibly the most widespread endosymbiont of arthropods and nematodes. While all Wolbachia strains have historically been defined as a single species, 16 monophyletic clusters of diversity (called supergroups) have been described. Different supergroups have distinct host ranges and symbiotic relationships, ranging from mutualism to reproductive manipulation. In filarial nematodes, which include parasites responsible for major diseases of humans (such as Onchocerca volvulus, agent of river blindness) and companion animals (Dirofilaria immitis, the dog heartworm), Wolbachia has an obligate mutualist role and is the target of new treatment regimens. Here, we compare the genomes of eight Wolbachia strains, spanning the diversity of the major supergroups (A–F), analysing synteny, transposable element content, GC skew and gene loss or gain. We detected genomic features that differ between Wolbachia supergroups, most notably in the C and D clades from filarial nematodes. In particular, strains from supergroup C (symbionts of O. volvulus and D. immitis) present a pattern of GC skew, conserved synteny and lack of transposable elements, unique in the Wolbachia genus. These features could be the consequence of a distinct symbiotic relationship between C Wolbachia strains and their hosts, highlighting underappreciated differences between the mutualistic supergroups found within filarial nematodes. PMID:26631376

  6. Supergroup C Wolbachia, mutualist symbionts of filarial nematodes, have a distinct genome structure.

    PubMed

    Comandatore, Francesco; Cordaux, Richard; Bandi, Claudio; Blaxter, Mark; Darby, Alistair; Makepeace, Benjamin L; Montagna, Matteo; Sassera, Davide

    2015-12-01

    Wolbachia pipientis is possibly the most widespread endosymbiont of arthropods and nematodes. While all Wolbachia strains have historically been defined as a single species, 16 monophyletic clusters of diversity (called supergroups) have been described. Different supergroups have distinct host ranges and symbiotic relationships, ranging from mutualism to reproductive manipulation. In filarial nematodes, which include parasites responsible for major diseases of humans (such as Onchocerca volvulus, agent of river blindness) and companion animals (Dirofilaria immitis, the dog heartworm), Wolbachia has an obligate mutualist role and is the target of new treatment regimens. Here, we compare the genomes of eight Wolbachia strains, spanning the diversity of the major supergroups (A-F), analysing synteny, transposable element content, GC skew and gene loss or gain. We detected genomic features that differ between Wolbachia supergroups, most notably in the C and D clades from filarial nematodes. In particular, strains from supergroup C (symbionts of O. volvulus and D. immitis) present a pattern of GC skew, conserved synteny and lack of transposable elements, unique in the Wolbachia genus. These features could be the consequence of a distinct symbiotic relationship between C Wolbachia strains and their hosts, highlighting underappreciated differences between the mutualistic supergroups found within filarial nematodes. PMID:26631376

  7. A survey of canine filarial diseases of veterinary and public health significance in India

    PubMed Central

    2010-01-01

    Background Dirofilaria spp., Acanthocheilonema spp. and Brugia spp. have all been reported in Indian dogs. In previous studies, diagnosis was made by morphological identification only. This is the first geographically stratified cross-sectional study in India to determine the prevalence and geographical distribution of canine filarial species of veterinary and public health importance, using a combination of conventional and molecular diagnostic techniques. Results A total of 139 from 525 dogs (26.5%; 95% CI 22.7, 30.3) were positive for microfilariae. The most common species of canine filaria identified in this study was A. reconditum (9.3%) followed by D. repens (6.7%) and D. immitis (1.5%). Three out of 525 dogs were found to have mixed infections on PCR. The morphological and molecular evidence on the sequence of the 18S gene and phylogenetic analysis of the ITS-2 region provided strong evidence that the canine microfilariae discovered in the Himalayan city of Ladakh belong to a novel species of Acanthocheilonema. Two dogs in Ladakh were also found to have mixed infections of the novel species described above and a unique microfilaria which morphologically resembled Microfilaria auquieri Foley, 1921. Conclusions At least six species of filarial nematode are now known to infect dogs in India, two of which were reported for the first time in this study. The study also confirms and extends the geographical distribution of canine heartworm (D. immitis) which overlaps with D. repens, emphasising the importance for veterinary clinicians and diagnostic laboratories to utilise immunodiagnostic tests that will not cross-react between those two filarial species. From a public health viewpoint, the distribution and prevalences of these nematodes warrant an appropriate prophylaxis to be administered to dogs. PMID:20377864

  8. Cytokine production in BALB/c mice immunized with radiation attenuated third stage larvae of the filarial nematode, Brugia pahangi

    SciTech Connect

    Bancroft, A.J.; Devaney, E. ); Grencis, R.K.; Else, K.J. )

    1993-02-15

    BALB/c mice immunized with radiation-attenuated third stage larvae of the filarial nematode Brugia pahangi are strongly immune to challenge infection. Investigation of the profile of cytokines secreted by spleen cells from immune mice stimulated in vitro with either parasite Ag or with Con A revealed high levels of IL-5 and IL-9 and moderate levels of IL-4. In contrast, secretion of IFN-[gamma] by spleen cells from immune animals was negligible. Spleen cells from control mice secreted low levels of all cytokines assayed. Levels of parasite-specific IgE were significantly elevated in immune animals and a peripheral blood eosinophilia was observed, which exhibited a biphasic distribution. Our results are consistent with the preferential expansion of Th2 cells in immune animals and provide the basis for dissecting the means by which radiation-attenuated larvae of filarial nematodes stimulate immunity. 5l refs., 3 figs., 3 tabs.

  9. Biosynthesis of isoprenoid compounds in cattle filarial parasite Setaria digitata.

    PubMed

    Kumari, G A; Santhamma, K R; Raj, R K

    1994-11-30

    The biological significance of isoprenoid compounds such as ubiquinones, prenols and sterols have been well established. The presence and biological function of the two quinones Q6 and Q8 in the cattle filarial parasite Setaria digitata have already been reported. Inhibition of the function of quinone was already shown to be an effective means of controlling the filarial parasite. Detailed investigations of the non-saponifiable lipids from S. digitata using column, thin layer, reverse phase and high performance liquid chromatography showed the presence and formation of isoprenoid compounds such as prenols and sterols, in addition to the two quinones. Blocking of the biosynthesis of these useful compounds may prove to be an additional means of control of filarial parasites.

  10. Evaluation of immune response elicited by inulin as an adjuvant with filarial antigens in mice model.

    PubMed

    Mahalakshmi, N; Aparnaa, R; Kaliraj, P

    2014-10-01

    Filariasis caused by infectious parasitic nematodes has been identified as the second leading source of permanent and long-term disability in Sub-Saharan Africa, Asia and Latin America. Several vaccine candidates were identified from infective third-stage larvae (L3) which involves in the critical transition from arthropod to human. Hitherto studies of these antigens in combination with alum adjuvant have shown to elicit its characteristic Th2 responses. Inulin is a safe, non-toxic adjuvant that principally stimulates the innate immune response through the alternative complement pathway. In the present study, the immune response elicited by inulin and alum as adjuvants were compared with filarial antigens from different aetiological agents: secreted larval acidic protein 1 (SLAP1) from Onchocerca volvulus and venom allergen homologue (VAH) from Brugia malayi as single or as cocktail vaccines in mice model. The study revealed that inulin can induce better humoral response against these antigens than alum adjuvant. Antibody isotyping disclosed inulin's ability to elevate the levels of IgG2a and IgG3 antibodies which mediates in complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC), respectively, in mice. Splenocyte analysis showed that T cells prestimulated with inulin have higher stimulation index (P < 0.05) than alum except for BmVAH antigen. In vitro ADCC assay showed that inulin formulation had induced higher cytotoxicity with filarial antigens (as single P < 0.01 and as cocktail P < 0.05, respectively) than alum. The results had confirmed the capability of inulin to deplete the levels of Treg and brought a balance in Th1/Th2 arms against filarial antigens in mice. PMID:25041426

  11. Cystatins from filarial parasites: evolution, adaptation and function in the host-parasite relationship.

    PubMed

    Gregory, William F; Maizels, Rick M

    2008-01-01

    Cystatins, together with stefins and kininogens, are members of the cystatin superfamily of cysteine protease inhibitors (CPI) present across the animal and plant kingdoms. Their role in parasitic organisms may encompass both essential developmental processes and specific interactions with the parasite's vector and/or final host. We summarise information gathered on three cystatins from the human filarial nematode Brugia malayi (Bm-CPI-1, -2 and -3), and contrast them those expressed by other parasites and by the free-living nematode Caenorhabditis elegans. Bm-CPI-2 differs from C. elegans cystatin, having acquired the additional function of inhibiting asparaginyl endopeptidase (AEP), in a manner similar to some human cystatins. Thus, we propose that Bm-CPI-2 and orthologues from related filarial parasites represent a new subset of nematode cystatins. Bm-CPI-1 and CPI-3 share only 25% amino acid identity with Bm-CPI-2, and lack an evolutionarily conserved glycine residue in the N-terminal region. These sequences group distantly from the other nematode cystatins, and represent a second novel subset of filarial cystatin-like genes. Expression analyses also show important differences between the CPI-2 and CPI-1/-3 groups. Bm-cpi-2 is expressed at all time points of the parasite life cycle, while Bm-cpi-1 and -3 expression is confined to the late stages of development in the mosquito vector, terminating within 48h of infection of the mammalian host. Hence, we hypothesise that CPI-2 has evolved to block mammalian proteases (including the antigen-processing enzyme AEP) while CPI-1 and -3 function in the milieu of the mosquito vector necessary for transmission of the parasite. PMID:18249028

  12. Evaluation of immune response elicited by inulin as an adjuvant with filarial antigens in mice model.

    PubMed

    Mahalakshmi, N; Aparnaa, R; Kaliraj, P

    2014-10-01

    Filariasis caused by infectious parasitic nematodes has been identified as the second leading source of permanent and long-term disability in Sub-Saharan Africa, Asia and Latin America. Several vaccine candidates were identified from infective third-stage larvae (L3) which involves in the critical transition from arthropod to human. Hitherto studies of these antigens in combination with alum adjuvant have shown to elicit its characteristic Th2 responses. Inulin is a safe, non-toxic adjuvant that principally stimulates the innate immune response through the alternative complement pathway. In the present study, the immune response elicited by inulin and alum as adjuvants were compared with filarial antigens from different aetiological agents: secreted larval acidic protein 1 (SLAP1) from Onchocerca volvulus and venom allergen homologue (VAH) from Brugia malayi as single or as cocktail vaccines in mice model. The study revealed that inulin can induce better humoral response against these antigens than alum adjuvant. Antibody isotyping disclosed inulin's ability to elevate the levels of IgG2a and IgG3 antibodies which mediates in complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC), respectively, in mice. Splenocyte analysis showed that T cells prestimulated with inulin have higher stimulation index (P < 0.05) than alum except for BmVAH antigen. In vitro ADCC assay showed that inulin formulation had induced higher cytotoxicity with filarial antigens (as single P < 0.01 and as cocktail P < 0.05, respectively) than alum. The results had confirmed the capability of inulin to deplete the levels of Treg and brought a balance in Th1/Th2 arms against filarial antigens in mice.

  13. Dirofilaria immitis. 5. Immunopathology of filarial nephropathy in dogs.

    PubMed Central

    Abramowsky, C. R.; Powers, K. G.; Aikawa, M.; Swinehart, G.

    1981-01-01

    Fourteen beagles infected with larvae (microfilariae) of Dirofilaria immitis, were randomly selected from another study in which the toxic effects of subfilaricidal doses of diethylcarbamazine were being evaluated. This group of 14 dogs, together with 4 uninfected control animals, were variably sacrificed between 14 and 25 months after larval inoculations, and the ensuing renal lesions were studied by light and ultrastructural microscopy and by immunofluorescence and antibody elution techniques. On the basis of these studies, two groups of animals were distinguished. The first group was characterized by a striking pattern of linear fluorescence and fine ultrastructural dense deposits along the glomerular basement membrane, poor antibody response, and an inability to clear microfilariae from the tissues and circulation. The second group, with a nonlinear pattern of fluorescence, was characterized by a strong immune response, efficient elimination of microfilariae, and immunofluorescence and ultrastructural evidence of predominantly mesangiopathic immune complex renal disease. In both groups, elution studies demonstrated tissue deposits of antiworm antibodies, suggesting a filaria-antibody immune-complex nephropathy. No evidence was found for the presence of anti-basement-membrane antibodies. On the basis of a previous experimental model, it is postulated that in the first group of animals with linear fluorescence, the observed lesions may represent a natural form of an immunopathogenic mechanism of glomerular damage in which filarial antigen becomes uniformly localized in the glomerulus and elicits an autologous antibody response. The possible role of the drug diethylcarbamazine in inducing this mechanism of immune injury is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 PMID:7020425

  14. Detection and prevention of mycoplasma hominis infection

    DOEpatents

    DelVecchio, Vito G.; Gallia, Gary L.; McCleskey, Ferne K.

    1997-01-21

    The present invention is directed to a rapid and sensitive method for detecting Mycoplasma hominis using M. hominis-specific probes, oligonucleotides or antibodies. In particular a target sequence can be amplified by in vitro nucleic acid amplification techniques, detected by nucleic acid hybridization using the subject probes and oligonucleotides or detected by immunoassay using M. hominis-specific antibodies. M. hominis-specific nucleic acids which do not recognize or hybridize to genomic nucleic acid of other Mycoplasma species are also provided.

  15. Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

    PubMed

    Kwon, Ji Yeon; Hong, Jin Sung; Kim, Min Jea; Choi, Sun Hee; Min, Byeong Eun; Song, Eun Gyeong; Kim, Hyun Hee; Ryu, Ki Hyun

    2014-09-01

    Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed.

  16. Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

    PubMed

    Kwon, Ji Yeon; Hong, Jin Sung; Kim, Min Jea; Choi, Sun Hee; Min, Byeong Eun; Song, Eun Gyeong; Kim, Hyun Hee; Ryu, Ki Hyun

    2014-09-01

    Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed. PMID:24937806

  17. On early detection of strong infections in complex networks

    NASA Astrophysics Data System (ADS)

    Yu, Yi; Xiao, Gaoxi

    2014-02-01

    Various complex systems are exposed to different kinds of infections ranging from computer viruses to rumors. An intuitive solution for limiting the damages caused by such infections is to detect the infection spreading as early as possible and then take necessary actions. In this paper, we study on how much we may expect to achieve in infection control by deploying a number of monitors in complex networks for detecting the outbreak of a strong infection at its early stage. Specifically, we consider the problem of finding the optimal locations for a given number of monitors in order to minimize the worst-case infection size. The NP-hardness of the problem is proved and a heuristic algorithm is proposed. Extensive simulations on both synthetic and real-life networks show that the worst-case infection size may be put under control by deploying a moderate number of monitors in a large complex network. Effects of a few different factors, including transmissibility of the infection, network topology and probability of detection failure, are also evaluated.

  18. Differential induction of Th2- and Th1-associated responses by filarial antigens and endosymbiotic Wolbachia in a murine model of river blindness.

    PubMed

    Gentil, K; Hoerauf, A; Pearlman, E

    2012-06-01

    Immune responses to filarial parasites like the river blindness inducing Onchocerca volvulus are obscured by combined reactions to the filarial nematodes themselves and their endosymbiont bacteria Wolbachia. Overall, infection with filarial nematodes induces a strong Th2 response characterized by IL-5 production and to a lesser degree a Th1 response and IFNγ production. Neutrophil and eosinophil infiltration into the corneal stroma are hallmark features of Onchocerca volvulus stimulation in a mouse model of river blindness. To determine the splenic and corneal response to filarial antigens in the absence of Wolbachia, C57BL/6 mice were immunized subcutaneously with either endosymbiotic Wolbachia alone, a soluble extract from the filaria Acanthocheilonema viteae that does not contain Wolbachia, or both, and injected into the corneal stroma. Neutrophil and eosinophil infiltration into the cornea was assessed by immunohistochemistry. In addition, Th1- and Th2-associated responses to filaria or Wolbachia were investigated by determining IL-5 and IFN-γ production by splenocytes. We found that A. viteae in the absence of Wolbachia induced IL-5 production and eosinophil infiltration, but not IFN-γ. Conversely, Wolbachia induced IFN-γ production and no migration of eosinophils. There was no difference in neutrophil infiltration. Together, these findings demonstrate a distinct Th-associated phenotype induced by filaria and Wolbachia. PMID:24672682

  19. Differential induction of Th2- and Th1-associated responses by filarial antigens and endosymbiotic Wolbachia in a murine model of river blindness.

    PubMed

    Gentil, K; Hoerauf, A; Pearlman, E

    2012-06-01

    Immune responses to filarial parasites like the river blindness inducing Onchocerca volvulus are obscured by combined reactions to the filarial nematodes themselves and their endosymbiont bacteria Wolbachia. Overall, infection with filarial nematodes induces a strong Th2 response characterized by IL-5 production and to a lesser degree a Th1 response and IFNγ production. Neutrophil and eosinophil infiltration into the corneal stroma are hallmark features of Onchocerca volvulus stimulation in a mouse model of river blindness. To determine the splenic and corneal response to filarial antigens in the absence of Wolbachia, C57BL/6 mice were immunized subcutaneously with either endosymbiotic Wolbachia alone, a soluble extract from the filaria Acanthocheilonema viteae that does not contain Wolbachia, or both, and injected into the corneal stroma. Neutrophil and eosinophil infiltration into the cornea was assessed by immunohistochemistry. In addition, Th1- and Th2-associated responses to filaria or Wolbachia were investigated by determining IL-5 and IFN-γ production by splenocytes. We found that A. viteae in the absence of Wolbachia induced IL-5 production and eosinophil infiltration, but not IFN-γ. Conversely, Wolbachia induced IFN-γ production and no migration of eosinophils. There was no difference in neutrophil infiltration. Together, these findings demonstrate a distinct Th-associated phenotype induced by filaria and Wolbachia.

  20. Breakdown of coevolution between symbiotic bacteria Wolbachia and their filarial hosts

    PubMed Central

    Lefoulon, Emilie; Makepeace, Benjamin L.; d’Haese, Cyrille; Uni, Shigehiko; Gavotte, Laurent

    2016-01-01

    Wolbachia is an alpha-proteobacterial symbiont widely distributed in arthropods. Since the identification of Wolbachia in certain animal-parasitic nematodes (the Onchocercidae or filariae), the relationship between arthropod and nematode Wolbachia has attracted great interest. The obligate symbiosis in filariae, which renders infected species susceptible to antibiotic chemotherapy, was held to be distinct from the Wolbachia-arthropod relationship, typified by reproductive parasitism. While co-evolutionary signatures in Wolbachia-arthropod symbioses are generally weak, reflecting horizontal transmission events, strict co-evolution between filariae and Wolbachia has been reported previously. However, the absence of close outgroups for phylogenetic studies prevented the determination of which host group originally acquired Wolbachia. Here, we present the largest co-phylogenetic analysis of Wolbachia in filariae performed to date including: (i) a screening and an updated phylogeny of Wolbachia; (ii) a co-phylogenetic analysis; and (iii) a hypothesis on the acquisition of Wolbachia infection. First, our results show a general overestimation of Wolbachia occurrence and support the hypothesis of an ancestral absence of infection in the nematode phylum. The accuracy of supergroup J is also underlined. Second, although a global pattern of coevolution remains, the signal is derived predominantly from filarial clades associated with Wolbachia in supergroups C and J. In other filarial clades, harbouring Wolbachia supergroups D and F, horizontal acquisitions and secondary losses are common. Finally, our results suggest that supergroup C is the basal Wolbachia clade within the Ecdysozoa. This hypothesis on the origin of Wolbachia would change drastically our understanding of Wolbachia evolution. PMID:27069790

  1. Breakdown of coevolution between symbiotic bacteria Wolbachia and their filarial hosts.

    PubMed

    Lefoulon, Emilie; Bain, Odile; Makepeace, Benjamin L; d'Haese, Cyrille; Uni, Shigehiko; Martin, Coralie; Gavotte, Laurent

    2016-01-01

    Wolbachia is an alpha-proteobacterial symbiont widely distributed in arthropods. Since the identification of Wolbachia in certain animal-parasitic nematodes (the Onchocercidae or filariae), the relationship between arthropod and nematode Wolbachia has attracted great interest. The obligate symbiosis in filariae, which renders infected species susceptible to antibiotic chemotherapy, was held to be distinct from the Wolbachia-arthropod relationship, typified by reproductive parasitism. While co-evolutionary signatures in Wolbachia-arthropod symbioses are generally weak, reflecting horizontal transmission events, strict co-evolution between filariae and Wolbachia has been reported previously. However, the absence of close outgroups for phylogenetic studies prevented the determination of which host group originally acquired Wolbachia. Here, we present the largest co-phylogenetic analysis of Wolbachia in filariae performed to date including: (i) a screening and an updated phylogeny of Wolbachia; (ii) a co-phylogenetic analysis; and (iii) a hypothesis on the acquisition of Wolbachia infection. First, our results show a general overestimation of Wolbachia occurrence and support the hypothesis of an ancestral absence of infection in the nematode phylum. The accuracy of supergroup J is also underlined. Second, although a global pattern of coevolution remains, the signal is derived predominantly from filarial clades associated with Wolbachia in supergroups C and J. In other filarial clades, harbouring Wolbachia supergroups D and F, horizontal acquisitions and secondary losses are common. Finally, our results suggest that supergroup C is the basal Wolbachia clade within the Ecdysozoa. This hypothesis on the origin of Wolbachia would change drastically our understanding of Wolbachia evolution. PMID:27069790

  2. Detection of transient and persistent feline leukaemia virus infections.

    PubMed

    Jarrett, O; Golder, M C; Stewart, M F

    1982-03-01

    A study was made of cats persistently or transiently viraemic with feline leukaemia virus (FeLV) following experimental oronasal infection. Cats of two ages were exposed to the virus. One group was infected when eight weeks old in the expectation that most of the cats would become persistently viraemic, and the second group when 16 weeks old, so that some would show signs of a transient infection and then recover. The periods following infection when virus was detectable in the blood and in the oropharynx were determined for each group. Three methods for detecting viraemia were compared: virus isolation, immunofluorescence on blood smears and an enzyme-linked immunosorbent assay (ELISA). There was good overall agreement among the three tests in detecting virus-positive cats. Virus was found sooner after infection by virus isolation than by the other methods, and virus appeared in the blood slightly sooner in cats which developed persistent viraemia than in transiently viraemic cats. Infectious FeLV was isolated from the oropharynx of all of the persistently viraemic cats, in most cases simultaneously with virus in the plasma. Virus was also isolated from the mouth of most transiently viraemic cats. Under field conditions such transient excretion of virus lasting only a few days would rarely be detected in a single sampling. This might explain how FeLV is maintained in free range urban cats in the absence of a large number of cats with persistent active FeLV infection. For routine diagnosis, immunofluorescence would appear to offer the best chance of differentiating transient and persistent infections by FeLV.

  3. Human filarial Wolbachia lipopeptide directly activates human neutrophils in vitro.

    PubMed

    Tamarozzi, F; Wright, H L; Johnston, K L; Edwards, S W; Turner, J D; Taylor, M J

    2014-10-01

    The host inflammatory response to the Onchocerca volvulus endosymbiont, Wolbachia, is a major contributing factor in the development of chronic pathology in humans (onchocerciasis/river blindness). Recently, the toll-like pattern recognition receptor motif of the major inflammatory ligands of filarial Wolbachia, membrane-associated diacylated lipoproteins, was functionally defined in murine models of pathology, including mediation of neutrophil recruitment to the cornea. However, the extent to which human neutrophils can be activated in response to this Wolbachia pattern recognition motif is not known. Therefore, the responses of purified peripheral blood human neutrophils to a synthetic N-terminal diacylated lipopeptide (WoLP) of filarial Wolbachia peptidoglycan-associated lipoprotein (PAL) were characterized. WoLP exposure led to a dose-dependent activation of healthy, human neutrophils that included gross morphological alterations and modulation of surface expressed integrins involved in tethering, rolling and extravasation. WoLP exposure induced chemotaxis but not chemokinesis of neutrophils, and secretion of the major neutrophil chemokine, interleukin 8. WoLP also induced and primed the respiratory burst, and enhanced neutrophil survival by delay of apoptosis. These results indicate that the major inflammatory motif of filarial Wolbachia lipoproteins directly activates human neutrophils in vitro and promotes a molecular pathway by which human neutrophils are recruited to sites of Onchocerca parasitism. PMID:24909063

  4. Human Cytomegalovirus: detection of congenital and perinatal infection in Argentina

    PubMed Central

    Distéfano, Angélica Lidia; Alonso, Alicia; Martin, Fabián; Pardon, Fabián

    2004-01-01

    Background Human cytomegalovirus (CMV) is one of the most commonly found agents of congenital infections. Primary maternal infection is associated with risk of symptomatic congenital diseases, and high morbidity is frequently associated with very low birth weight. Neonates with asymptomatic infection develop various sequelae during infancy. This is the first Argentine study performed in neonates with congenital and postnatal HCMV infection. The purpose of this study was to evaluate the performance of the polymerase chain reaction (PCR) technique with different pairs of primers, to detect cytomegalovirus isolated in tissue cultures and directly in urine and dried blood spot (DBS) specimens. Results were compared with IgM detection. Methods The study was performed between 1999 and 2001 on routine samples in the Laboratory. A total of 61 urine and 56 serum samples were selected from 61 newborns/infants, 33 patients whose samples were analyzed during the first two to three weeks of life were considered congenital infections; the remaining 28 patients whose samples were taken later than the third week were grouped as perinatal infections, although only in 4 the perinatal transmission of infection was determined unequivocally Cytomegalovirus diagnosis was made by isolating the virus from urine samples in human foreskin fibroblast cells. Three different primer pairs directed to IE, LA and gB genes were used for the HCMV PCR assay in viral isolates. Subsequently, PCR and nested PCR (nPCR) assays with gB primers were performed directly in urine and in 11 samples of dried blood spot (DBS) on Guthrie Card, these results were then compared with serology. Results The main clinical manifestations of the 33 patients with congenital infection were purpura, jaundice, hepatomegaly and anaemia. Three patients presented low birth weight as single symptom, 10, intracranial calcifications, and 2, kidney failure. In the 28 patients grouped as with perinatal infection, anaemia

  5. Filarioids infecting dogs in northeastern Brazil.

    PubMed

    Ramos, Rafael Antonio Nascimento; de Oliveira do Rêgo, Ana Gabriela; de Farias Firmino, Everton Diogo; do Nascimento Ramos, Carlos Alberto; de Carvalho, Gílcia Aparecida; Dantas-Torres, Filipe; Otranto, Domenico; Alves, Leucio Câmara

    2016-08-15

    Species of filarial nematodes belonging to the genera Dirofilaria and Acanthocheilonema are recognised as common parasites of dogs throughout the world. Recently, other filarioids featured by the presence of dermal microfilariae (e.g., Onchocerca lupi and Cercopithifilaria spp.) have been recognised in Europe. In Brazil, reports of filarioids in dogs are limited to Dirofilaria immitis, Acanthocheilonema reconditum and Cercopithifilaria bainae. To investigate the distribution of filarial infections in dogs living in an endemic region from northeastern Brazil, blood and skin samples (n=104) were microscopically (modified Knott's test and skin snip sediment examination) and molecularly evaluated. Twenty-two dogs (21.15%) were positive at microscopic and/or molecular examination for at least one filarioid species, with 21 (20.19%) animals positive for blood microfilariae at molecular and/or at microscopic examination. Microfilariae of D. immitis were detected in 12 (11.54%) animals, with co-infection of D. immitis and A. reconditum observed in four (3.85%) individuals. One animal was positive for C. bainae at both microscopic and molecular examination. Analysis of sequence obtained in the present study showed significant alignment identity with that of C. bainae from Europe. Considering that in the area of study arthropod vectors (mosquitoes, fleas and ticks) are prevalent throughout the year, preventive measures should be disposed in order to avoid the animal infestation and pathogen infection. PMID:27514878

  6. Filarioids infecting dogs in northeastern Brazil.

    PubMed

    Ramos, Rafael Antonio Nascimento; de Oliveira do Rêgo, Ana Gabriela; de Farias Firmino, Everton Diogo; do Nascimento Ramos, Carlos Alberto; de Carvalho, Gílcia Aparecida; Dantas-Torres, Filipe; Otranto, Domenico; Alves, Leucio Câmara

    2016-08-15

    Species of filarial nematodes belonging to the genera Dirofilaria and Acanthocheilonema are recognised as common parasites of dogs throughout the world. Recently, other filarioids featured by the presence of dermal microfilariae (e.g., Onchocerca lupi and Cercopithifilaria spp.) have been recognised in Europe. In Brazil, reports of filarioids in dogs are limited to Dirofilaria immitis, Acanthocheilonema reconditum and Cercopithifilaria bainae. To investigate the distribution of filarial infections in dogs living in an endemic region from northeastern Brazil, blood and skin samples (n=104) were microscopically (modified Knott's test and skin snip sediment examination) and molecularly evaluated. Twenty-two dogs (21.15%) were positive at microscopic and/or molecular examination for at least one filarioid species, with 21 (20.19%) animals positive for blood microfilariae at molecular and/or at microscopic examination. Microfilariae of D. immitis were detected in 12 (11.54%) animals, with co-infection of D. immitis and A. reconditum observed in four (3.85%) individuals. One animal was positive for C. bainae at both microscopic and molecular examination. Analysis of sequence obtained in the present study showed significant alignment identity with that of C. bainae from Europe. Considering that in the area of study arthropod vectors (mosquitoes, fleas and ticks) are prevalent throughout the year, preventive measures should be disposed in order to avoid the animal infestation and pathogen infection.

  7. Detection of viral infections using colloidal quantum dots

    NASA Astrophysics Data System (ADS)

    Bentzen, Elizabeth L.; House, Frances S.; Utley, Thomas J.; Crowe, James E., Jr.; Wright, David W.

    2006-02-01

    Fluorescence is a tool widely employed in biological assays. Fluorescent semiconducting nanocrystals, quantum dots (QDs), are beginning to find their way into the tool box of many biologist, chemist and biochemist. These quantum dots are an attractive alternative to the traditional organic dyes due to their broad excitation spectra, narrow emission spectra and photostability. Quantum dots were used to detect and monitor the progession of viral glycoproteins, F (fusion) and G (attachment), from Respiratory Syncytial Virus (RSV) in HEp-2 cells. Additionally, oligo-Qdot RNA probes have been developed for identification and detection of mRNA of the N(nucleocapsid) protein for RSV. The use of quantum dot-FISH probes provides another confirmatory route to diagnostics as well as a new class of probes for monitoring the flux and fate of viral RNA RSV is the most common cause of lower respiratory tract infection in children worldwide and the most common cause of hospitalization of infants in the US. Antiviral therapy is available for treatment of RSV but is only effective if given within the first 48 hours of infection. Existing test methods require a virus level of at least 1000-fold of the amount needed for infection of most children and require several days to weeks to obtain results. The use of quantum dots may provide an early, rapid method for detection and provide insight into the trafficking of viral proteins during the course of infection.

  8. Contemporary Diagnostic Strategies for the Detection of Helicobacter pylori Infection

    PubMed Central

    Elfant, Adam B.; Howden, Colin W.; Stollman, Neil

    2012-01-01

    Helicobacter pylori infection is highly prevalent, affecting approximately half of the world’s population. While the majority of infected individuals are asymptomatic, H. pylori infection is associated with certain diseases, including peptic ulcers (either duodenal or gastric), gastritis, and 2 malignancies—gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. Many of the epidemiologic associations between these diseases and H. pylori infection have been further validated by treatment studies, which show that effective eradication therapy correlates with a decreased risk of disease. A variety of testing strategies are used to detect H. pylori infection. Serologic techniques are widely available and inexpensive, but they are no longer preferred as they have low sensitivities and specificities, and they may show a positive result for a long period following effective therapy. The remaining testing methods are divided into 2 categories: invasive tests (which require endoscopy) and noninvasive tests. Noninvasive test methods such as the urea breath test and stool antigen test have gained popularity due to their high sensitivities and specificities. Further, both of these methods may be used to confirm the absence of infection following eradication therapy. Due to the increasing incidence of treatment failure (caused in part by antibiotic resistance), post-treatment testing is recommended to confirm H. pylori eradication. PMID:24847180

  9. Diversity and Expression of MicroRNAs in the Filarial Parasite, Brugia malayi

    PubMed Central

    Poole, Catherine B.; Gu, Weifeng; Kumar, Sanjay; Jin, Jingmin; Davis, Paul J.; Bauche, David; McReynolds, Larry A.

    2014-01-01

    Human filarial parasites infect an estimated 120 million people in 80 countries worldwide causing blindness and the gross disfigurement of limbs and genitals. An understanding of RNA-mediated regulatory pathways in these parasites may open new avenues for treatment. Toward this goal, small RNAs from Brugia malayi adult females, males and microfilariae were cloned for deep-sequencing. From ∼30 million sequencing reads, 145 miRNAs were identified in the B. malayi genome. Some microRNAs were validated using the p19 RNA binding protein and qPCR. B. malayi miRNAs segregate into 99 families each defined by a unique seed sequence. Sixty-one of the miRNA families are highly conserved with homologues in arthropods, vertebrates and helminths. Of those miRNAs not highly conserved, homologues of 20 B. malayi miRNA families were found in vertebrates. Nine B. malayi miRNA families appear to be filarial-specific as orthologues were not found in other organisms. The miR-2 family is the largest in B. malayi with 11 members. Analysis of the sequences shows that six members result from a recent expansion of the family. Library comparisons found that 1/3 of the B. malayi miRNAs are differentially expressed. For example, miR-71 is 5–7X more highly expressed in microfilariae than adults. Studies suggest that in C.elegans, miR-71 may enhance longevity by targeting the DAF-2 pathway. Characterization of B. malayi miRNAs and their targets will enhance our understanding of their regulatory pathways in filariads and aid in the search for novel therapeutics. PMID:24824352

  10. Diversity and expression of microRNAs in the filarial parasite, Brugia malayi.

    PubMed

    Poole, Catherine B; Gu, Weifeng; Kumar, Sanjay; Jin, Jingmin; Davis, Paul J; Bauche, David; McReynolds, Larry A

    2014-01-01

    Human filarial parasites infect an estimated 120 million people in 80 countries worldwide causing blindness and the gross disfigurement of limbs and genitals. An understanding of RNA-mediated regulatory pathways in these parasites may open new avenues for treatment. Toward this goal, small RNAs from Brugia malayi adult females, males and microfilariae were cloned for deep-sequencing. From ∼ 30 million sequencing reads, 145 miRNAs were identified in the B. malayi genome. Some microRNAs were validated using the p19 RNA binding protein and qPCR. B. malayi miRNAs segregate into 99 families each defined by a unique seed sequence. Sixty-one of the miRNA families are highly conserved with homologues in arthropods, vertebrates and helminths. Of those miRNAs not highly conserved, homologues of 20 B. malayi miRNA families were found in vertebrates. Nine B. malayi miRNA families appear to be filarial-specific as orthologues were not found in other organisms. The miR-2 family is the largest in B. malayi with 11 members. Analysis of the sequences shows that six members result from a recent expansion of the family. Library comparisons found that 1/3 of the B. malayi miRNAs are differentially expressed. For example, miR-71 is 5-7X more highly expressed in microfilariae than adults. Studies suggest that in C.elegans, miR-71 may enhance longevity by targeting the DAF-2 pathway. Characterization of B. malayi miRNAs and their targets will enhance our understanding of their regulatory pathways in filariads and aid in the search for novel therapeutics.

  11. Adult filarial worm from the breast aspirate of a young man.

    PubMed

    Fernandes, Hilda; Thomas, Beena Mary; Putran, Indira

    2016-06-01

    Microfilariae and adult filarial worm have been incidentally detected in fine needle aspirates of various lesions in clinically unsuspected cases. Here we report a male patient who presented with a tender breast nodule and single enlarged lymph node. Fine Needle Aspiration (FNA) yielded 1 ml of yellow coloured fluid and single thread like worm measuring 6 × 0.2 cm. A diagnosis of breast abscess with a worm morphologically consistent with filariasis was offered. A follow up visit after 2 months showed regression of the breast lesion and the lymph node. Filariasis of the breast is an uncommon condition and can cause a diagnostic dilemma at times. FNA cytology appears to be a more convenient and effective diagnostic tool in patients with mass lesions. Demonstration and identification of the parasite in smears helps in avoiding surgical excision and early institution of prompt therapy especially in young patients. PMID:27413335

  12. Comparative Analysis of the Secretome from a Model Filarial Nematode (Litomosoides sigmodontis) Reveals Maximal Diversity in Gravid Female Parasites*

    PubMed Central

    Armstrong, Stuart D.; Babayan, Simon A.; Lhermitte-Vallarino, Nathaly; Gray, Nick; Xia, Dong; Martin, Coralie; Kumar, Sujai; Taylor, David W.; Blaxter, Mark L.; Wastling, Jonathan M.; Makepeace, Benjamin L.

    2014-01-01

    Filarial nematodes (superfamily Filarioidea) are responsible for an annual global health burden of ∼6.3 million disability-adjusted life-years, which represents the greatest single component of morbidity attributable to helminths affecting humans. No vaccine exists for the major filarial diseases, lymphatic filariasis and onchocerciasis; in part because research on protective immunity against filariae has been constrained by the inability of the human-parasitic species to complete their lifecycles in laboratory mice. However, the rodent filaria Litomosoides sigmodontis has become a popular experimental model, as BALB/c mice are fully permissive for its development and reproduction. Here, we provide a comprehensive analysis of excretory-secretory products from L. sigmodontis across five lifecycle stages and identifications of host proteins associated with first-stage larvae (microfilariae) in the blood. Applying intensity-based quantification, we determined the abundance of 302 unique excretory-secretory proteins, of which 64.6% were present in quantifiable amounts only from gravid adult female nematodes. This lifecycle stage, together with immature microfilariae, released four proteins that have not previously been evaluated as vaccine candidates: a predicted 28.5 kDa filaria-specific protein, a zonadhesin and SCO-spondin-like protein, a vitellogenin, and a protein containing six metridin-like ShK toxin domains. Female nematodes also released two proteins derived from the obligate Wolbachia symbiont. Notably, excretory-secretory products from all parasite stages contained several uncharacterized members of the transthyretin-like protein family. Furthermore, biotin labeling revealed that redox proteins and enzymes involved in purinergic signaling were enriched on the adult nematode cuticle. Comparison of the L. sigmodontis adult secretome with that of the human-infective filarial nematode Brugia malayi (reported previously in three independent published studies

  13. Detection of Acute HIV-1 Infection by RT-LAMP.

    PubMed

    Rudolph, Donna L; Sullivan, Vickie; Owen, S Michele; Curtis, Kelly A

    2015-01-01

    A rapid, cost-effective diagnostic test for the detection of acute HIV-1 infection is highly desired. Isothermal amplification techniques, such as reverse-transcription loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for the development of a rapid nucleic acid amplification test (NAAT) because they are quick, easy to perform and do not require complex, dedicated equipment and laboratory space. In this study, we assessed the ability of the HIV-1 RT-LAMP assay to detect acute HIV infection as compared to a representative rapid antibody test and several FDA-approved laboratory-based assays. The HIV-1 RT-LAMP assay detected seroconverting individuals one to three weeks earlier than a rapid HIV antibody test and up to two weeks earlier than a lab-based antigen/antibody (Ag/Ab) combo enzyme immunoassay (EIA). RT-LAMP was not as sensitive as a lab-based qualitative RNA assay, which could be attributed to the significantly smaller nucleic acid input volume. To our knowledge, this is the first demonstration of detecting acute HIV infection using the RT-LAMP assay. The availability of a rapid NAAT, such as the HIV-1 RT-LAMP assay, at the point of care (POC) or in laboratories that do not have access to large platform NAAT could increase the percentage of individuals who receive an acute HIV infection status or confirmation of their HIV status, while immediately linking them to counseling and medical care. In addition, early knowledge of HIV status could lead to reduced high-risk behavior at a time when individuals are at a higher risk for transmitting the virus. PMID:25993381

  14. Detection of Polyclonality among Clinical Isolates from Prosthetic Joint Infections

    PubMed Central

    De-la-Fuente, Marta; Martinez-Perez, Marta; Gonzalez-Pallares, Iris

    2015-01-01

    Prosthetic joint infection (PJI) is an increasingly important health concern in the Western world due to the rising number of joint arthroplasties. Although most infections are considered to be monomicrobial, the introduction of sonication procedures has led to an increase in the detection of polymicrobial infections. To date, no published studies have investigated the presence of different clones of the same species in the infected patient. The objective of this study was to analyze whether the phenomenon of polyclonality, or the appearance of different clones in the same sample, occurs in PJI. Bacteria isolated by sonication of the retrieved implant from patients with theoretically monomicrobial PJI were included in the study. Two techniques (random amplified polymorphic DNA [RAPD] and matrix-assisted laser desorption ionization–time of flight [MALDI-TOF] mass spectrometry) were used to determine the presence of several clones in the same sample. Results were analyzed to determine bacterial species and infection type (acute versus chronic). RAPD showed a predominance of polyclonal cases (16 of 19). However, when performing the analysis with MALDI-TOF, all cases were shown to be polyclonal. We were unable to establish any relationship between the two methodologies. Polyclonality is a common phenomenon in acute and chronic PJI. Further studies are needed to establish the potential implications of this phenomenon on patient outcomes. PMID:26378278

  15. Mitochondrial genes for heme-dependent respiratory chain complexes are up-regulated after depletion of Wolbachia from filarial nematodes.

    PubMed

    Strübing, Uta; Lucius, Richard; Hoerauf, Achim; Pfarr, Kenneth M

    2010-08-15

    The filarial nematodes Brugia malayi, Wuchereria bancrofti and Onchocerca volvulus cause elephantiasis or dermatitis and blindness resulting in severe morbidity. Annually, 1.3 billion people are at risk of infection. Targeting the essential Wolbachia endobacteria of filarial nematodes with doxycycline has proven to be an effective therapy resulting in a block in embryogenesis, worm development and macrofilaricidal effects. However, doxycycline is contraindicated for a large portion of the at risk population. To identify new targets for anti-wolbachial therapy, understanding the molecular basis of the Wolbachia-filaria symbiosis is required. Using the B. malayi microarray we identified differentially expressed genes in the rodent filaria Litomosoides sigmodontis after depletion of Wolbachia which might have a role in symbiosis. The microarray data were filtered for regulated genes with a false discovery rate <5% and a > or = 2-fold-change. Most of the genes were differentially expressed at day 36 of tetracycline treatment, when 99.8% of Wolbachia were depleted. Several classes of genes were affected, including genes for translation, transcription, folding/sorting of proteins, motility, structure and metabolic and signalling pathways. Quantitative PCR validated 60% of the genes found to be regulated in the microarray. A nuclear encoded heme-binding protein of the globin family was up-regulated upon loss of Wolbachia. Interestingly, mitochondrial encoded subunits of respiratory chain complexes containing heme and riboflavin were also up-regulated. No change in the expression of these genes was seen in tetracycline treated Wolbachia-free Acanthocheilonema viteae. As Wolbachia synthesise heme and filaria do not, we hypothesise that without the endosymbionts no functional heme-containing enzymes can be formed, leading to loss of energy metabolism which then results in up-regulation of the mitochondrial encoded subunits in an attempt to correct the deviation from

  16. Intravascular filarial parasites elaborate cyclooxygenase-derived eicosanoids

    PubMed Central

    1990-01-01

    The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Since prostanoids formed from arachidonic acid can modulate interactions among platelets, leukocytes, and endothelial cells, we examined whether intravascular nematode parasites can elaborate prostanoids. Microfilariae of Brugia malayi utilize exogenous and endogenous arachidonic acid to generate and release two predominant prostanoids, prostacyclin and prostaglandin E2. Filarial metabolism of host fatty acids to form these vasodilatory, antiaggregatory, and immunomodulatory eicosanoids provides a means by which these helminthic parasites may influence host immune and other cellular responses. PMID:2117642

  17. Detection of hepatitis B virus infection: A systematic review

    PubMed Central

    Ghosh, Mallika; Nandi, Srijita; Dutta, Shrinwanti; Saha, Malay Kumar

    2015-01-01

    AIM: To review published methods for detection of hepatitis B virus (HBV) infection. METHODS: A thorough search on Medline database was conducted to find original articles describing different methods or techniques of detection of HBV, which are published in English in last 10 years. Articles outlining methods of detection of mutants or drug resistance were excluded. Full texts and abstracts (if full text not available) were reviewed thoroughly. Manual search of references of retrieved articles were also done. We extracted data on different samples and techniques of detection of HBV, their sensitivity (Sn), specificity (Sp) and applicability. RESULTS: A total of 72 studies were reviewed. HBV was detected from dried blood/plasma spots, hepatocytes, ovarian tissue, cerumen, saliva, parotid tissue, renal tissue, oocytes and embryos, cholangiocarcinoma tissue, etc. Sensitivity of dried blood spot for detecting HBV was > 90% in all the studies. In case of seronegative patients, HBV DNA or serological markers have been detected from hepatocytes or renal tissue in many instances. Enzyme linked immunosorbent assay and Chemiluminescent immunoassay (CLIA) are most commonly used serological tests for detection. CLIA systems are also used for quantitation. Molecular techniques are used qualitatively as well as for quantitative detection. Among the molecular techniques version 2.0 of the CobasAmpliprep/CobasTaqMan assay and Abbott’s real time polymerase chain reaction kit were found to be most sensitive with a lower detection limit of only 6.25 IU/mL and 1.48 IU/mL respectively. CONCLUSION: Serological and molecular assays are predominant and reliable methods for HBV detection. Automated systems are highly sensitive and quantify HBV DNA and serological markers for monitoring. PMID:26483870

  18. Detection of HCV Persistent Infections in the Dental Pulp: A Novel Approach for the Detection of Past and Ancient Infections

    PubMed Central

    Siravenha, Layla Gomes; Siravenha, Leonardo Quintão; Madeira, Lucimar Di Paula; Oliveira-Filho, Aldemir B.; Machado, Luiz Fernando Almeida; Martins Feitosa, Rosimar Neris; Vallinoto, Antonio Carlos Rosário; Ishak, Marluísa de Oliveira Guimarães; Ishak, Ricardo

    2016-01-01

    The dental pulp is a sterile highly vascularized tissue and has been commonly used as a biological material to detect the genome of infectious agents that reach the dental tissue. Indeed, the pulp is also used to reveal past and ancient infections in the field of paleomicrobiology. The present study aimed to detect the presence of Hepatitis C virus (HCV) in a small community (approximately 400 inhabitants) in the Amazon region of Brazil (Nossa Senhora do Perpetuo Socorro, Vizeu, Para, Brazil) and standardize a technique for the detection of the virus in the dental pulp. Serum samples were collected from 48 patients whose teeth were clinically recommended for surgical extraction. The group comprised an equal number of males and females, mostly agriculture workers and housewives, respectively. The majority (64.6%) received less than one minimum wage and were ill educated (less than four years of school years). An enzyme immune assay was used to detect antibodies to HCV and the 9 (18.8%) positive samples were submitted to nucleic acid extraction in the blood (using the EXTRAzol) and the pulp (QIAamp DNA Micro Kit e kit RNeasy Plus Micro). The pulp was removed using a modified protocol without the use of liquid nitrogen. Nucleic acid was found in 8 of the dental pulp, but in 7 of the blood samples. Sequencing of one of the samples showed the presence of genotype 1. Conclusions: A novel simplified methodology for the extraction and amplification of HCV nucleic acid was successful to detect the presence of persistent infections of the virus within the dental pulp tissue. The protocol may be helpful to detect past and ancient infections and to better understand the natural history of HCV. PMID:27783693

  19. Associations Between Helminth Infections, Plasmodium falciparum Parasite Carriage and Antibody Responses to Sexual and Asexual Stage Malarial Antigens.

    PubMed

    Ateba-Ngoa, Ulysse; Jones, Sophie; Zinsou, Jeannot Fréjus; Honkpehedji, Josiane; Adegnika, Ayola Akim; Agobe, Jean-Claude Dejon; Massinga-Loembe, Marguerite; Mordmüller, Benjamin; Bousema, Teun; Yazdanbakhsh, Maria

    2016-08-01

    Infections with helminths and Plasmodium spp. overlap in their geographical distribution. It has been postulated that helminth infections may influence malarial transmission by altering Plasmodium falciparum gametocytogenesis. This cross-sectional study assessed the effect of helminth infections on P. falciparum gametocyte carriage and on humoral immune responses to sexual stage antigens in Gabon. Schistosoma haematobium and filarial infections as well as P. falciparum asexual forms and gametocyte carriage were determined. The antibody responses measured were to sexual (Pfs230, Pfs48/45) and asexual P. falciparum antigens (AMA1, MSP1, and GLURP). A total of 287 subjects were included. The prevalence of microscopically detectable P. falciparum asexual parasites was higher in S. haematobium-infected subjects in comparison to their uninfected counterparts (47% versus 26%, P = 0.003), but this was not different when filarial infections were considered. Plasmodium falciparum gametocyte carriage was similar between Schistosoma- or filaria-infected and uninfected subjects. We observed a significant decrease of Pfs48/45 immunoglobulin G titer in S. haematobium-infected subjects (P = 0.037), whereas no difference was seen for Pfs230 antibody titer, nor for antibodies to AMA1, MSP1, or GLURP. Our findings suggest an effect of S. haematobium on antibody responses to some P. falciparum gametocyte antigens that may have consequences for transmission-blocking immunity. PMID:27273645

  20. Detection of urinary tract infections by rapid methods.

    PubMed Central

    Pezzlo, M

    1988-01-01

    A review of rapid urine screens for detection of bacteriuria and pyuria demonstrates a number of available alternatives to the culture method. Selection of one or more of these systems for routine use is dependent upon the laboratory and the patient population being tested. The laboratory approach to the diagnosis of urinary tract infection should consider the clinical diagnosis of the patient whenever possible. Keeping in mind that quantitative urine cultures alone cannot be used to detect infection in some patient populations unless lower colony counts are considered, a rapid screen may be a more practical approach. It has become accepted that 10(5) CFU/ml can no longer be used as the standard for all patient groups, that pyuria often is important in making the diagnosis of a urinary tract infection, and that most of the rapid screens are more sensitive than the culture method at 10(5) CFU/ml. Presently, no one approach can be recommended for all laboratories and all patient groups. However, each diagnostic laboratory should select one approach which is best for its situation. It is not practical, efficient, or cost effective to define a protocol for each possible clinical condition; however, all should be considered when developing a protocol. This protocol should be compatible with the patient population and communicated to the physicians. Use of a rapid screen should be beneficial to the patient, the physician, and the laboratory. PMID:3058296

  1. High Prevalence of Persistent Parasitic Infections in Foreign-Born, HIV-Infected Persons in the United States

    PubMed Central

    Hochberg, Natasha S.; Moro, Ruth N.; Sheth, Anandi N.; Montgomery, Susan P.; Steurer, Frank; McAuliffe, Isabel T.; Wang, Yun F.; Armstrong, Wendy; Rivera, Hilda N.; Lennox, Jeffrey L.; Franco-Paredes, Carlos

    2011-01-01

    Background Foreign-born, HIV-infected persons are at risk for sub-clinical parasitic infections acquired in their countries of origin. The long-term consequences of co-infections can be severe, yet few data exist on parasitic infection prevalence in this population. Methodology/Principal Findings This cross-sectional study evaluated 128 foreign-born persons at one HIV clinic. We performed stool studies and serologic testing for strongyloidiasis, schistosomiasis, filarial infection, and Chagas disease based on the patient's country of birth. Eosinophilia and symptoms were examined as predictors of helminthic infection. Of the 128 participants, 86 (67%) were male, and the median age was 40 years; 70 were Mexican/Latin American, 40 African, and 18 from other countries or regions. Strongyloides stercoralis antibodies were detected in 33/128 (26%) individuals. Of the 52 persons from schistosomiasis-endemic countries, 15 (29%) had antibodies to schistosome antigens; 7 (47%) had antibodies to S. haematobium, 5 (33%) to S. mansoni, and 3 (20%) to both species. Stool ova and parasite studies detected helminths in 5/85 (6%) persons. None of the patients tested had evidence of Chagas disease (n = 77) or filarial infection (n = 52). Eosinophilia >400 cells/mm3 was associated with a positive schistosome antibody test (OR 4.5, 95% CI 1.1–19.0). The only symptom significantly associated with strongyloidiasis was weight loss (OR 3.1, 95% CI 1.4–7.2). Conclusions/Significance Given the high prevalence of certain helminths and the potential lack of suggestive symptoms and signs, selected screening for strongyloidiasis and schistosomiasis or use of empiric antiparasitic therapy may be appropriate among foreign-born, HIV-infected patients. Identifying and treating helminth infections could prevent long-term complications. PMID:21532747

  2. Trehalose metabolism genes in Caenorhabditis elegans and filarial nematodes.

    PubMed

    Pellerone, F I; Archer, S K; Behm, C A; Grant, W N; Lacey, M J; Somerville, A C

    2003-09-30

    The sugar trehalose is claimed to be important in the physiology of nematodes where it may function in sugar transport, energy storage and protection against environmental stresses. In this study we investigated the role of trehalose metabolism in nematodes, using Caenorhabditis elegans as a model, and also identified complementary DNA clones putatively encoding genes involved in trehalose pathways in filarial nematodes. In C. elegans two putative trehalose-6-phosphate synthase (tps) genes encode the enzymes that catalyse trehalose synthesis and five putative trehalase (tre) genes encode enzymes catalysing hydrolysis of the sugar. We showed by RT-PCR or Northern analysis that each of these genes is expressed as mRNA at all stages of the C. elegans life cycle. Database searches and sequencing of expressed sequence tag clones revealed that at least one tps gene and two tre genes are expressed in the filarial nematode Brugia malayi, while one tps gene and at least one tre gene were identified for Onchocerca volvulus. We used the feeding method of RNA interference in C. elegans to knock down temporarily the expression of each of the tps and tre genes. Semiquantitative RT-PCR analysis confirmed that expression of each gene was silenced by RNA interference. We did not observe an obvious phenotype for any of the genes silenced individually but gas-chromatographic analysis showed >90% decline in trehalose levels when both tps genes were targeted simultaneously. This decline in trehalose content did not affect viability or development of the nematodes.

  3. Heme acquisition in the parasitic filarial nematode Brugia malayi

    PubMed Central

    Luck, Ashley N.; Yuan, Xiaojing; Voronin, Denis; Slatko, Barton E.; Hamza, Iqbal; Foster, Jeremy M.

    2016-01-01

    Nematodes lack a heme biosynthetic pathway and must acquire heme from exogenous sources. Given the indispensable role of heme, this auxotrophy may be exploited to develop drugs that interfere with heme uptake in parasites. Although multiple heme-responsive genes (HRGs) have been characterized within the free-living nematode Caenorhabditis elegans, we have undertaken the first study of heme transport in Brugia malayi, a causative agent of lymphatic filariasis. Through functional assays in yeast, as well as heme analog, RNAi, and transcriptomic experiments, we have shown that the heme transporter B. malayi HRG-1 (BmHRG-1) is indeed functional in B. malayi. In addition, BmHRG-1 localizes both to the endocytic compartments and cell membrane when expressed in yeast cells. Transcriptomic sequencing revealed that BmHRG-1, BmHRG-2, and BmMRP-5 (all orthologs of HRGs in C. elegans) are down-regulated in heme-treated B. malayi, as compared to non–heme-treated control worms. Likely because of short gene lengths, multiple exons, other HRGs in B. malayi (BmHRG-3–6) remain unidentified. Although the precise mechanisms of heme homeostasis in a nematode with the ability to acquire heme remains unknown, this study clearly demonstrates that the filarial nematode B. malayi is capable of transporting exogenous heme.—Luck, A. N., Yuan, X., Voronin, D., Slatko, B. E., Hamza, I., Foster, J. M. Heme acquisition in the parasitic filarial nematode Brugia malayi. PMID:27363426

  4. A comparison of two antigen-detection ELISA for detecting infection of Dirofilaria immitis in dogs.

    PubMed

    Euclid, J M; Copeman, D B

    1997-09-01

    A survey on 87 dogs necropsied in the Townsville region revealed 34 (39%) were infected with Dirofilaria immitis. Infected dogs had an average of 6.1 adult worms in the heart and associated blood vessels. The VetRED assay detected 23 of the 34 infected dogs (sensitivity 65%) and the Og4C3 ELISA detected 27 (sensitivity 80%). Sensitivity of the VetRED and Og4C3 ELISA increased to 88 and 94% respectively in dogs with three or more worms. Both tests detected correctly all uninfected dogs. Despite the higher accuracy of the Og4C3 ELISA, compared to the VetRED assay, it is unlikely to be used widely as a field test for heartworm unless it can be modified from its present plate ELISA format which takes 4 hours, into one which is more rapid and convenient. However, as a reference ELISA, it may well be worthwhile in situations which require considerable accuracy for detecting D. immitis infection.

  5. A review of methods for detect human Papillomavirus infection

    PubMed Central

    2012-01-01

    Human Papillomavirus (HPV) is the most common sexually transmitted virus. Worldwide, the most common high-risk (HR)-HPV are -16/18, and approximately 70% of cervical cancers (CC) are due to infection by these genotypes. Persistent infection by HR-HPV is a necessary but not sufficient cause of this cancer, which develops over a long period through precursor lesions, which can be detected by cytological screening. Although this screening has decreased the incidence of CC, HPV-related cervical disease, including premalignant and malignant lesions, continues to be a major burden on health-care systems. Although not completely elucidated, the HPV-driven molecular mechanisms underlying the development of cervical lesions have provided a number of potential biomarkers for both diagnostic and prognostic use in the clinical management of women with HPV-related cervical disease, and these biomarkers can also be used to increase the positive predictive value of current screening methods. In addition, they can provide insights into the biology of HPV-induced cancer and thus lead to the development of nonsurgical therapies. Considering the importance of detecting HPV and related biomarkers, a variety of methods are being developed for these purposes. This review summarizes current knowledge of detection methods for HPV, and related biomarkers that can be used to discriminate lesions with a high risk of progression to CC. PMID:23131123

  6. A review of methods for detect human Papillomavirus infection.

    PubMed

    Abreu, André L P; Souza, Raquel P; Gimenes, Fabrícia; Consolaro, Marcia E L

    2012-11-06

    Human Papillomavirus (HPV) is the most common sexually transmitted virus. Worldwide, the most common high-risk (HR)-HPV are -16/18, and approximately 70% of cervical cancers (CC) are due to infection by these genotypes. Persistent infection by HR-HPV is a necessary but not sufficient cause of this cancer, which develops over a long period through precursor lesions, which can be detected by cytological screening. Although this screening has decreased the incidence of CC, HPV-related cervical disease, including premalignant and malignant lesions, continues to be a major burden on health-care systems. Although not completely elucidated, the HPV-driven molecular mechanisms underlying the development of cervical lesions have provided a number of potential biomarkers for both diagnostic and prognostic use in the clinical management of women with HPV-related cervical disease, and these biomarkers can also be used to increase the positive predictive value of current screening methods. In addition, they can provide insights into the biology of HPV-induced cancer and thus lead to the development of nonsurgical therapies. Considering the importance of detecting HPV and related biomarkers, a variety of methods are being developed for these purposes. This review summarizes current knowledge of detection methods for HPV, and related biomarkers that can be used to discriminate lesions with a high risk of progression to CC.

  7. The therapeutic potential of the filarial nematode-derived immunodulator, ES-62 in inflammatory disease.

    PubMed

    Harnett, M M; Melendez, A J; Harnett, W

    2010-03-01

    The dramatic recent rise in the incidence of allergic or autoimmune inflammatory diseases in the West has been proposed to reflect the lack of appropriate priming of the immune response by infectious agents such as parasitic worms during childhood. Consistent with this, there is increasing evidence supporting an inverse relationship between worm infection and T helper type 1/17 (Th1/17)-based inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes and multiple sclerosis. Perhaps more surprisingly, given that such worms often induce strong Th2-type immune responses, there also appears to be an inverse correlation between parasite load and atopy. These findings therefore suggest that the co-evolution of helminths with hosts, which has resulted in the ability of worms to modulate inflammatory responses to promote parasite survival, has also produced the benefit of protecting the host from pathological lesions arising from aggressive proinflammatory responses to infection or, indeed, aberrant inflammatory responses underlying autoimmune and allergic disorders. By focusing upon the properties of the filarial nematode-derived immunomodulatory molecule, ES-62, in this review we shall discuss the potential of exploiting the immunomodulatory products of parasitic worms to identify and develop novel therapeutics for inflammation. PMID:19968663

  8. Discovery of filarial nematode DNA in Amblyomma americanum in Northern Virginia.

    PubMed

    Henning, Tyler C; Orr, John M; Smith, Joshua D; Arias, Jorge R; Rasgon, Jason L; Norris, Douglas E

    2016-03-01

    Ticks collected in 2011 were screened for the presence of filarial nematode genetic material, and positive samples were sequenced for analysis. Monanema-like filarial nematode DNA was recently discovered in Amblyomma americanum in northern Virginia, marking the first time genetic material from this parasite has been discovered in ticks in the state. Phylogenetic analysis revealed that this material was directly related to a previously discovered filarial nematode in A. americanum populations in Maryland as well as recently identified parasites in Ixodes scapularis from southern Connecticut. Further study is warranted to visually confirm the presence of these nematodes, characterize their distribution, and determine if these ticks are intermediate hosts.

  9. Deletion of Parasite Immune Modulatory Sequences Combined with Immune Activating Signals Enhances Vaccine Mediated Protection against Filarial Nematodes

    PubMed Central

    Babayan, Simon A.; Luo, HongLin; Gray, Nick; Taylor, David W.; Allen, Judith E.

    2012-01-01

    Background Filarial nematodes are tissue-dwelling parasites that can be killed by Th2-driven immune effectors, but that have evolved to withstand immune attack and establish chronic infections by suppressing host immunity. As a consequence, the efficacy of a vaccine against filariasis may depend on its capacity to counter parasite-driven immunomodulation. Methodology and Principal Findings We immunised mice with DNA plasmids expressing functionally-inactivated forms of two immunomodulatory molecules expressed by the filarial parasite Litomosoides sigmodontis: the abundant larval transcript-1 (LsALT) and cysteine protease inhibitor-2 (LsCPI). The mutant proteins enhanced antibody and cytokine responses to live parasite challenge, and led to more leukocyte recruitment to the site of infection than their native forms. The immune response was further enhanced when the antigens were targeted to dendritic cells using a single chain Fv-αDEC205 antibody and co-administered with plasmids that enhance T helper 2 immunity (IL-4) and antigen-presenting cell recruitment (Flt3L, MIP-1α). Mice immunised simultaneously against the mutated forms of LsALT and LsCPI eliminated adult parasites faster and consistently reduced peripheral microfilaraemia. A multifactorial analysis of the immune response revealed that protection was strongly correlated with the production of parasite-specific IgG1 and with the numbers of leukocytes present at the site of infection. Conclusions We have developed a successful strategy for DNA vaccination against a nematode infection that specifically targets parasite-driven immunosuppression while simultaneously enhancing Th2 immune responses and parasite antigen presentation by dendritic cells. PMID:23301106

  10. Comparative analysis of different methods to detect Clostridium difficile infection.

    PubMed

    Calderaro, Adriana; Buttrini, Mirko; Martinelli, Monica; Gorrini, Chiara; Montecchini, Sara; Medici, Maria Cristina; Arcangeletti, Maria Cristina; De Conto, Flora; Covan, Silvia; Chezzi, Carlo

    2013-01-01

    The increased incidence and severity of Clostridium difficile infection, particularly in North America and Europe, have brought renewed focus on the most appropriate method to detect C. difficile and/or its toxins in stools. This prospective study evaluated the usefulness of the Illumigene TM C. difficile assay in diagnostic practice for the detection of toxigenic C. difficile DNA in clinical samples. A total of 88 out of 306 stool samples analysed were positive both by Illumigene and the combination of toxigenic C. difficile culture (TC) and immunochromatographic assay (IC) with a concordance of 100%. Of the 218 samples negative by the combination of TC and IC, 204 were negative also by Illumigene with a concordance of 93.57%. In our experience, compared to conventional assays Illumigene assay proved to be easy to perform, accurate and prompt giving results within 1 hour at a cost of 28 euro per sample. PMID:23435816

  11. Comparison of three molecular detection methods for detection of Trichinella in infected pigs.

    PubMed

    Lin, Zhibing; Cao, Jie; Zhang, Houshuang; Zhou, Yongzhi; Deng, Mingjun; Li, Guoqing; Zhou, Jinlin

    2013-05-01

    Different molecular detection methods require diverse molecular platforms, but there is no uniform standard for people to reference in the detection of Trichinella. In this study, real-time PCR, loop-mediated isothermal amplification (LAMP), and conventional PCR were developed for the detection of Trichinella by targeting mitochondrial large subunit ribosomal DNA (mt-lsrDNA). We compared the performance of the three newly developed assays. The results revealed that the detection limits of the real-time PCR, LAMP, and conventional PCR assays were 10 and 100 fg/μL and 1 pg/μL of Trichinella spiralis genomic DNA, respectively. The assays were used in the detection of Trichinella in the field. A total of 192 samples were obtained from pigs: 75 samples from free range farming and 117 from intensive feeding factory. The infection rate was 8/192 (4.2 %), 7/192 (3.6 %), and 1/192 (1.0 %) through the real-time PCR, LAMP, and conventional PCR assays, respectively. These data indicate that Taqman real-time PCR was a rapid, specific, and sensitive tool as a preferred option for investigation of valuable samples, but that LAMP assay was closed tube, highly sensitive, cost-effective, rapid, easy-to-perform, and was the optimal choice for detection of Trichinella in the field. The results of a model of experimental infection in mice indicated that spleen can be used as sampling site for the detection of early T. spiralis infection. However, the diaphragm and myocardium were the most suitable sampling sites for the detection of T. spiralis. PMID:23334692

  12. Comparison of three molecular detection methods for detection of Trichinella in infected pigs.

    PubMed

    Lin, Zhibing; Cao, Jie; Zhang, Houshuang; Zhou, Yongzhi; Deng, Mingjun; Li, Guoqing; Zhou, Jinlin

    2013-05-01

    Different molecular detection methods require diverse molecular platforms, but there is no uniform standard for people to reference in the detection of Trichinella. In this study, real-time PCR, loop-mediated isothermal amplification (LAMP), and conventional PCR were developed for the detection of Trichinella by targeting mitochondrial large subunit ribosomal DNA (mt-lsrDNA). We compared the performance of the three newly developed assays. The results revealed that the detection limits of the real-time PCR, LAMP, and conventional PCR assays were 10 and 100 fg/μL and 1 pg/μL of Trichinella spiralis genomic DNA, respectively. The assays were used in the detection of Trichinella in the field. A total of 192 samples were obtained from pigs: 75 samples from free range farming and 117 from intensive feeding factory. The infection rate was 8/192 (4.2 %), 7/192 (3.6 %), and 1/192 (1.0 %) through the real-time PCR, LAMP, and conventional PCR assays, respectively. These data indicate that Taqman real-time PCR was a rapid, specific, and sensitive tool as a preferred option for investigation of valuable samples, but that LAMP assay was closed tube, highly sensitive, cost-effective, rapid, easy-to-perform, and was the optimal choice for detection of Trichinella in the field. The results of a model of experimental infection in mice indicated that spleen can be used as sampling site for the detection of early T. spiralis infection. However, the diaphragm and myocardium were the most suitable sampling sites for the detection of T. spiralis.

  13. Performance of commercially available serological diagnostic tests to detect Leishmania infantum infection on experimentally infected dogs.

    PubMed

    Rodríguez-Cortés, Alhelí; Ojeda, Ana; Todolí, Felicitat; Alberola, Jordi

    2013-01-31

    Leishmania infantum (syn. Leishmania chagasi) is the etiological agent of a widespread serious zoonotic disease that affects both humans and dogs. Prevalence and incidence of the canine infection are important parameters to determine the risk and the ways to control this reemergent zoonosis. Unfortunately, there is not a gold standard test for Leishmania infection. Our aim was to assess the operative validity of commercial tests used to detect antibodies to Leishmania in serum samples from experimental infections. Three ELISA tests (LEISCAN(®) Leishmania ELISA Test, INGEZIM(®) LEISHMANIA, and INGEZIM(®) LEISHMANIA VET), three immunochromatographic tests (INGEZIM(®) LEISHMACROM, SNAP(®) Leishmania, and WITNESS(®) Leishmania), and one IFAT were evaluated. LEISCAN(®) Leishmania ELISA test achieved the highest sensitivity and accuracy (both 0.98). Specificity was 1 for all tests except for IFAT. All tests but IFAT obtained a positive predictive value of 1, while the maximum negative predictive value was achieved by LEISCAN(®) Leishmania ELISA Test (0.93). The best positive likelihood ratio was obtained by INGEZIM(®) LEISHMANIA VET (30.26), while the best negative likelihood ratio was obtained by LEISCAN(®) Leishmania ELISA Test (0.02). The highest diagnostic odds ratio was achieved by LEISCAN(®) Leishmania ELISA Test (729.00). The largest area under the ROC curve was obtained by LEISCAN(®) Leishmania ELISA Test (0.981). Quantitative ELISA based tests performmed better than qualitative tests ("Rapid Tests"), and the test best suited to detect Leishmania in infected dogs and to provide clinically useful information was LEISCAN(®) Leishmania ELISA Test. This and other results point also to the need of revising the status of IFAT as a gold standard for the diagnosis of leishmaniasis.

  14. Human Plasmodium knowlesi Infection Detected by Rapid Diagnostic Tests for Malaria

    PubMed Central

    van Hellemond, Jaap J.; Rutten, Marijke; Koelewijn, Rob; Zeeman, Anne-Marie; Verweij, Jaco J.; Wismans, Pieter J.; Kocken, Clemens H.

    2009-01-01

    We describe a PCR-confirmed case of Plasmodium knowlesi infection with a high parasitemia level and clinical signs of severe malaria in a migrant worker from Malaysian Borneo in the Netherlands. Investigations showed that commercially available rapid antigen tests for detection of human Plasmodium infections can detect P. knowlesi infections in humans. PMID:19788819

  15. Genomics of Loa loa, a Wolbachia-free filarial parasite of humans.

    PubMed

    Desjardins, Christopher A; Cerqueira, Gustavo C; Goldberg, Jonathan M; Dunning Hotopp, Julie C; Haas, Brian J; Zucker, Jeremy; Ribeiro, José M C; Saif, Sakina; Levin, Joshua Z; Fan, Lin; Zeng, Qiandong; Russ, Carsten; Wortman, Jennifer R; Fink, Doran L; Birren, Bruce W; Nutman, Thomas B

    2013-05-01

    Loa loa, the African eyeworm, is a major filarial pathogen of humans. Unlike most filariae, L. loa does not contain the obligate intracellular Wolbachia endosymbiont. We describe the 91.4-Mb genome of L. loa and that of the related filarial parasite Wuchereria bancrofti and predict 14,907 L. loa genes on the basis of microfilarial RNA sequencing. By comparing these genomes to that of another filarial parasite, Brugia malayi, and to those of several other nematodes, we demonstrate synteny among filariae but not with nonparasitic nematodes. The L. loa genome encodes many immunologically relevant genes, as well as protein kinases targeted by drugs currently approved for use in humans. Despite lacking Wolbachia, L. loa shows no new metabolic synthesis or transport capabilities compared to other filariae. These results suggest that the role of Wolbachia in filarial biology is more subtle than previously thought and reveal marked differences between parasitic and nonparasitic nematodes. PMID:23525074

  16. Release of Small RNA-containing Exosome-like Vesicles from the Human Filarial Parasite Brugia malayi.

    PubMed

    Zamanian, Mostafa; Fraser, Lisa M; Agbedanu, Prince N; Harischandra, Hiruni; Moorhead, Andrew R; Day, Tim A; Bartholomay, Lyric C; Kimber, Michael J

    2015-01-01

    Lymphatic filariasis (LF) is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes. The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced. Manipulation of this interface to the detriment of the parasite is a promising potential avenue to develop disease therapies but is prevented by our very limited understanding of the host-parasite relationship. Exosomes are bioactive small vesicles (30-120 nm) secreted by a wide range of cell types and involved in a wide range of physiological processes. Here, we report the identification and partial characterization of exosome-like vesicles (ELVs) released from the infective L3 stage of the human filarial parasite Brugia malayi. Exosome-like vesicles were isolated from parasites in culture media and electron microscopy and nanoparticle tracking analysis were used to confirm that vesicles produced by juvenile B. malayi are exosome-like based on size and morphology. We show that loss of parasite viability correlates with a time-dependent decay in vesicle size specificity and rate of release. The protein cargo of these vesicles is shown to include common exosomal protein markers and putative effector proteins. These Brugia-derived vesicles contain small RNA species that include microRNAs with host homology, suggesting a potential role in host manipulation. Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1. To our knowledge, this is the first report of exosome-like vesicle release by a human parasitic nematode and our data suggest a novel mechanism by which human parasitic nematodes may actively direct the host responses to infection. Further interrogation of the makeup and function of these bioactive vesicles could seed

  17. Release of Small RNA-containing Exosome-like Vesicles from the Human Filarial Parasite Brugia malayi

    PubMed Central

    Agbedanu, Prince N; Harischandra, Hiruni; Moorhead, Andrew R; Day, Tim A; Bartholomay, Lyric C; Kimber, Michael J

    2015-01-01

    Lymphatic filariasis (LF) is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes. The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced. Manipulation of this interface to the detriment of the parasite is a promising potential avenue to develop disease therapies but is prevented by our very limited understanding of the host-parasite relationship. Exosomes are bioactive small vesicles (30–120 nm) secreted by a wide range of cell types and involved in a wide range of physiological processes. Here, we report the identification and partial characterization of exosome-like vesicles (ELVs) released from the infective L3 stage of the human filarial parasite Brugia malayi. Exosome-like vesicles were isolated from parasites in culture media and electron microscopy and nanoparticle tracking analysis were used to confirm that vesicles produced by juvenile B. malayi are exosome-like based on size and morphology. We show that loss of parasite viability correlates with a time-dependent decay in vesicle size specificity and rate of release. The protein cargo of these vesicles is shown to include common exosomal protein markers and putative effector proteins. These Brugia-derived vesicles contain small RNA species that include microRNAs with host homology, suggesting a potential role in host manipulation. Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1. To our knowledge, this is the first report of exosome-like vesicle release by a human parasitic nematode and our data suggest a novel mechanism by which human parasitic nematodes may actively direct the host responses to infection. Further interrogation of the makeup and function of these bioactive vesicles could seed

  18. Draft genome of the filarial nematode parasite Brugia malayi.

    PubMed

    Ghedin, Elodie; Wang, Shiliang; Spiro, David; Caler, Elisabet; Zhao, Qi; Crabtree, Jonathan; Allen, Jonathan E; Delcher, Arthur L; Guiliano, David B; Miranda-Saavedra, Diego; Angiuoli, Samuel V; Creasy, Todd; Amedeo, Paolo; Haas, Brian; El-Sayed, Najib M; Wortman, Jennifer R; Feldblyum, Tamara; Tallon, Luke; Schatz, Michael; Shumway, Martin; Koo, Hean; Salzberg, Steven L; Schobel, Seth; Pertea, Mihaela; Pop, Mihai; White, Owen; Barton, Geoffrey J; Carlow, Clotilde K S; Crawford, Michael J; Daub, Jennifer; Dimmic, Matthew W; Estes, Chris F; Foster, Jeremy M; Ganatra, Mehul; Gregory, William F; Johnson, Nicholas M; Jin, Jinming; Komuniecki, Richard; Korf, Ian; Kumar, Sanjay; Laney, Sandra; Li, Ben-Wen; Li, Wen; Lindblom, Tim H; Lustigman, Sara; Ma, Dong; Maina, Claude V; Martin, David M A; McCarter, James P; McReynolds, Larry; Mitreva, Makedonka; Nutman, Thomas B; Parkinson, John; Peregrín-Alvarez, José M; Poole, Catherine; Ren, Qinghu; Saunders, Lori; Sluder, Ann E; Smith, Katherine; Stanke, Mario; Unnasch, Thomas R; Ware, Jenna; Wei, Aguan D; Weil, Gary; Williams, Deryck J; Zhang, Yinhua; Williams, Steven A; Fraser-Liggett, Claire; Slatko, Barton; Blaxter, Mark L; Scott, Alan L

    2007-09-21

    Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the approximately 90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict approximately 11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during approximately 350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.

  19. Detection and diagnosis of rice-infecting viruses

    PubMed Central

    Uehara-Ichiki, Tamaki; Shiba, Takuya; Matsukura, Keiichiro; Ueno, Takanori; Hirae, Masahiro; Sasaya, Takahide

    2013-01-01

    Rice-infecting viruses have caused serious damage to rice production in Asian, American, and African countries, where about 30 rice viruses and diseases have been reported. To control these diseases, developing accurate, quick methods to detect and diagnose the viruses in the host plants and any insect vectors of the viruses is very important. Based on an antigen–antibody reaction, serological methods such as latex agglutination reaction and enzyme-linked immunosorbent assay have advanced to detect viral particles or major proteins derived from viruses. They aid in forecasting disease and surveying disease spread and are widely used for virus detection at plant protection stations and research laboratories. From the early 2000s, based on sequence information for the target virus, several other methods such as reverse transcription-polymerase chain reaction (RT-PCR) and reverse transcription-loop-mediated isothermal amplification have been developed that are sensitive, rapid, and able to differentiate closely related viruses. Recent techniques such as real-time RT-PCR can be used to quantify the pathogen in target samples and monitor population dynamics of a virus, and metagenomic analyses using next-generation sequencing and microarrays show potential for use in the diagnosis of rice diseases. PMID:24130554

  20. Infective endocarditis detection through SPECT/CT images digital processing

    NASA Astrophysics Data System (ADS)

    Moreno, Albino; Valdés, Raquel; Jiménez, Luis; Vallejo, Enrique; Hernández, Salvador; Soto, Gabriel

    2014-03-01

    Infective endocarditis (IE) is a difficult-to-diagnose pathology, since its manifestation in patients is highly variable. In this work, it was proposed a semiautomatic algorithm based on SPECT images digital processing for the detection of IE using a CT images volume as a spatial reference. The heart/lung rate was calculated using the SPECT images information. There were no statistically significant differences between the heart/lung rates values of a group of patients diagnosed with IE (2.62+/-0.47) and a group of healthy or control subjects (2.84+/-0.68). However, it is necessary to increase the study sample of both the individuals diagnosed with IE and the control group subjects, as well as to improve the images quality.

  1. Immunoradiometric assay for quantitation of Dirofilaria immitis antigen in dogs with heartworm infections

    SciTech Connect

    Hamilton, R.G.; Scott, A.L.

    1984-10-01

    An immunoradiometric assay (IRMA) was developed, optimized, and validated for detection of parasite-specific antigen in sera from hosts with filarial infections, using Dirofilaria immitis in dogs as a model. The precision, reproducibility, and parallelism of the IRMA were examined, using precision profile analysis. The IRMA had acceptable precision and reproducibility (less than 15% intra-assay coefficient of variation (CV)) over a working range of 10 to 2000 ng of D immitis-antigen (AG)/ml. The IRMA parallelism (agreement between dilutions) was acceptable (less than 10% interdilutional CV) with laboratory-spiked D immitis AG sera containing no D immitis-antibody (AB). However, it was not acceptable (greater than 20% interdilutional CV) for analysis of sera from naturally infected dogs containing D immitis AB, probably due to dissociation of immune-complexed AG with increasing serum dilution. Nonparallelism limited the accuracy of binding data interpolation from the standard curve. Specificity of the IRMA was enhanced by preabsorption of the radiolabeled detection antibody with Toxocara canis AG before use. Varying amounts of D immitis AG (22 to 1000 ng/ml) were detected in 42% (20/48) of microfilaremic dogs. The presence of AG-specific AB at concentrations as low as 1 microgram/ml reduced the ability of the IRMA to detect D immitis AG. Factors that influence the accuracy and sensitivity of immunoassays for circulating filarial antigens are discussed.

  2. Determinants of Newly Detected Human Papillomavirus Infection in HIV-Infected and HIV-Uninfected Injection Drug Using Women

    PubMed Central

    Phelan, Darcy F.; Gange, Stephen J.; Ahdieh-Grant, Linda; Mehta, Shruti H.; Kirk, Gregory D.; Shah, Keerti; Gravitt, Patti

    2009-01-01

    Background We sought to identify factors associated with newly detected human papillomavirus (HPV) infection in a high-risk cohort of injection drug using women in Baltimore, MD. Methods We studied 146 HIV-infected and 73 HIV-uninfected female participants in a 5-year prospective HIV natural history study. We examined the association of sexual and nonsexual risk factors and newly detected type-specific HPV infection as determined by consensus PCR between consecutive visits. Results Newly detected HPV was more common among HIV-infected versus HIV-uninfected women (30% and 6%, respectively; P <0.01). Among the entire cohort, recent crack use (OR, 1.7; 95% CI, 1.1−2.6) and HIV infection/CD4 cell count were independent predictors for new HPV detection (HIV-uninfected as reference, OR, 4.6; 95% CI, 2.3−8.9, OR, 5.4; 95% CI, 2.8−10.3, and OR, 10.9; 95% CI, 5.5−21.7 for HIV-infected CD4 >500, 200−500, and <200, respectively). Among HIV-uninfected women, recent marijuana use was an independent predictor of newly detected HPV infection (OR, 3.5; 95% CI, 1.3−9.5). Conclusions Newly detected HPV clearly increased with greater immunosuppression in HIV-infected injection drug users. Larger studies of HIV-uninfected and infected high-risk individuals are needed to clarify the independent associations of crack and marijuana use with new (or reactivated) HPV infection. PMID:19174735

  3. Long-Term Persistence of Prevalently Detected Human Papillomavirus Infections in the Absence of Detectable Cervical Precancer and Cancer

    PubMed Central

    Rodríguez, Ana Cecilia; Burk, Robert D.; Herrero, Rolando; Wacholder, Sholom; Hildesheim, Allan; Morales, Jorge; Rydzak, Greg; Schiffman, Mark

    2011-01-01

    Background. Detailed descriptions of long-term persistence of human papillomavirus (HPV) in the absence of cervical precancer are lacking. Methods. In a large, population-based natural study conducted in Guanacaste, Costa Rica, we studied a subset of 810 initially HPV-positive women with ≥3 years of active follow-up with ≥3 screening visits who had no future evidence of cervical precancer. Cervical specimens were tested for >40 HPV genotypes using a MY09/11 L1-targeted polymerase chain reaction method. Results. Seventy-two prevalently-detected HPV infections (5%) in 58 women (7%) persisted until the end of the follow-up period (median duration of follow-up, 7 years) without evidence of cervical precancer. At enrollment, women with long-term persistence were more likely to have multiple prevalently-detected HPV infections (P <.001) than were women who cleared their baseline HPV infections during follow-up. In a logistic regression model, women with long-term persistence were more likely than women who cleared infections to have another newly-detected HPV infection detectable at ≥3 visits (odds ratio, 2.6; 95% confidence interval, 1.2–5.6). Conclusions. Women with long-term persistence of HPV infection appear to be generally more susceptible to other HPV infections, especially longer-lasting infections, than are women who cleared their HPV infections. PMID:21343148

  4. How to diagnose hantavirus infections and detect them in rodents and insectivores.

    PubMed

    Vaheri, Antti; Vapalahti, Olli; Plyusnin, Alexander

    2008-01-01

    Hantaviruses are carried by rodents and insectivores in which they cause persistent and generally asymptomatic infections. Several hantaviruses can infect humans and many of them cause either haemorrhagic fever with renal syndrome (HFRS) in Eurasia or hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In humans hantavirus infections are diagnosed using IgM-capture tests but also by RT-PCR detection of viral RNA. For detection of hantavirus infections in rodents and insectivores, serology followed by immunoblotting of, for example, lung tissue, and RT-PCR detection of viral RNA may be used, and if of interest followed by sequencing and virus isolation. For sero/genotyping of hantavirus infections in humans and carrier animals neutralisation tests/RNA sequencing are required. Hantaviruses are prime examples of emerging and re-emerging infections and it seems likely that many new hantaviruses will be detected in the near future.

  5. An unidentified filarial species and its impact on fitness in wild populations of the black-footed ferret (Mustela nigripes).

    PubMed

    Wisely, Samantha M; Howard, JoGayle; Williams, Steven A; Bain, Odile; Santymire, Rachel M; Bardsley, Katherine D; Williams, Elizabeth S

    2008-01-01

    Disease can threaten the restoration of endangered species directly by substantially decreasing host survival or indirectly via incremental decreases in survival and reproduction. During a biomedical survey of reintroduced populations of the highly endangered black-footed ferret from 2002 to 2005, microfilariae discovered in the blood were putatively identified as Dirofilaria immitis, and widespread screening was initiated using a commercially available antigen-based ELISA test. A subset of animals (n = 16) was screened for D. immitis using a highly sensitive PCR-based assay. Microfilariae were also molecularly and morphologically characterized. Of 198 animals at six reintroduction sites, 12% had positive results using the ELISA test. No antigen-positive animals which were screened via PCR (n = 11) had positive PCR results, and all antigen-positive animals (n = 24) were asymptomatic. No significant differences were found in body mass of antigen-positive (male: 1223 +/- 82 g [mean +/- SD], female: 726 +/- 75 g) vs. antigen-negative (male: 1,198 +/- 119 g, female: 710 +/- 53 g) individuals (P = 0.4). Antigen prevalence was lower in juveniles (3%) than adults (12%; P = 0.03), and higher in in situ, captive-reared individuals (33%) than wild-born individuals (10%; P = 0.005). Morphologic analysis of microfilariae revealed they were neither D. immitis nor any other previously characterized North American species. PCR amplification of the 5S spacer region of rDNA revealed that the filarial sequence shared only 76% identity with D. immitis. This previously unidentified filarial sequence was present in all antigen positive animals (11 of 11 tested). It appears that black-footed ferrets were infected with a previously undescribed species of filaria whose antigen cross-reacted with the ELISA assay, although further analysis is needed to make a conclusive statement. Nonetheless, this previously undescribed filaria does not appear to threaten recovery for this highly

  6. Does 24bp Duplication of Human CHIT1 Gene (Chitotriosidase1) Predispose to Filarial Chyluria? A Case-Control Study

    PubMed Central

    Pant, Shriya; Agarwal, Jyotsna; Gangwar, Pravin K; Waseem, Mohammad; Gupta, Prashant; Sankhwar, Satya N; Purkait, Bimalesh

    2016-01-01

    Introduction Chyluria which is endemic in many parts of the world is mainly caused by Wuchereria bancrofti. CHIT1 (chitotriosidase) is produced by macrophages and plays an important role in the defense against chitin containing pathogen such as filarial parasite. Variation in the coding region with 24 bp duplication allele results in reduced CHIT1 activity that enhance the survival of parasite which may play a role in the occurrence of disease. Aim To examine the role of 24bp duplication of CHIT1 gene in patients of filarial chyluria (FC). Materials and Methods A case-control study was carried out where 155 confirmed FC patients and equal number of age-, sex- and residence-matched controls without any symptoms or signs of lymphatic filariasis, confirmed by negative immunochromatographic card test (ICT) and IgG/IgM combo rapid antibody test, from a hospital-based population were enrolled. Filarial aetiology was confirmed on the basis of DEC-provocative test (Giemsa staining), ICT and IgG/IgM- antifiarial antibody test. The patients positive by either of these tests were enrolled as FC cases. 24bp duplication in CHIT1 gene in FC was detected by the product size 99bp of amplified gene using polymerase chain reaction. Results The mean ages of patients and controls were 38.25±12.09 and 35.45±12.53 years, respectively while male: female ratio was 2.4:1. The mean duration of illness in chyluria patients was 62.81±60.83 months and mean number of episodes was 2.54±1.11. Homozygous wild type, heterozygous and homozygous mutant frequencies were 10.3%, 81.3% and 8.4% in FC patients and 18.7%, 75.5%, and 5.8% in controls, respectively. The 24bp duplication in CHIT1 gene showed a significant association in Heterozygous (HT) genotype with Odd Ratio (OR) of 1.95, 95% Confidence Interval (CI) (1.01-3.77); p=0.04. However, the homozygous mutant genotype (TT) was found to be non-significant with OR of 2.61, 95% CI (0.91-7.45); p=0.07. The combination of both HT+TT was also found

  7. C-cinnamoyl glycosides as a new class of anti-filarial agents.

    PubMed

    Roy, Priya; Dhara, Debashis; Parida, Pravat Kumar; Kar, Rajiv Kumar; Bhunia, Anirban; Jana, Kuladip; Sinha Babu, Santi P; Misra, Anup Kumar

    2016-05-23

    A series of C-cinnamoyl glycosides has been synthesized in good yield by the BF3·OEt2 catalyzed aldol condensation of C-glycosylated acetone derivative with a variety of aromatic aldehydes. The synthesized compounds were evaluated for their potential as anti-filarial agents against bovine filarial parasite Setaria cervi and human filariid Wuchereria bancrofti using a number of biological assays such as relative movability (RM) assessment and MTT reduction assay. Among twenty seven test compounds six compounds were found active in terms of MIC, IC50 and LC50 values. Further biological studies were carried out using three lead compounds because of their significantly low MIC values and IC50 values compared to the standard anti-filarial drug Ivermectin. In addition, structure activity relationship study of the test compounds has been carried out using 3D-QSAR analysis. PMID:27015610

  8. Detecting Rickettsia parkeri infection from eschar swab specimens.

    PubMed

    Myers, Todd; Lalani, Tahaniyat; Dent, Mike; Jiang, Ju; Daly, Patrick L; Maguire, Jason D; Richards, Allen L

    2013-05-01

    The typical clinical presentation of several spotted fever group Rickettsia infections includes eschars. Clinical diagnosis of the condition is usually made by analysis of blood samples. We describe a more sensitive, noninvasive means of obtaining a sample for diagnosis by using an eschar swab specimen from patients infected with Rickettsia parkeri.

  9. Detection of Bacteroides infection by counter immunoelectrophoresis test.

    PubMed

    Lalitha, M K; Mathai, E; Elias, L; Anandi, V; Kalpana, C R

    1991-05-01

    The counterimmunoelectrophoresis (CIE) test using sonicated antigens of Bacteroides fragilis NCTC 2553 and a B. asaccharolyticus strain, standardised in the laboratory yielded a negative result in the 50 normal sera tested, while it was positive in 24 of 34 (71%) patients with infection due to black pigmented bacteroides and in 10 of 15 (67%) with B. fragilis infection. The microagglutination test (MAT) done in parallel showed a positivity of only 44 and 40 per cent respectively. The CIE test done with B. asaccharolyticus antigen was negative in 87 per cent of patients with infection due to B. fragilis whereas MAT showed cross reactivity to a greater extent. PMID:1937595

  10. Detection of Echinococcus granulosus coproantigens in Australian canids with natural or experimental infection.

    PubMed

    Jenkins, D J; Fraser, A; Bradshaw, H; Craig, P S

    2000-02-01

    Coproparasitological and purging methods for diagnosing canids infected with the intestinal helminth Echinococcus granulosus, an important zoonotic parasite, are unreliable. Detection of coproantigens in feces of infected dogs by enzyme-linked immunosorbent assay (ELISA) is suitable for detecting patent and prepatent infections with a high degree of sensitivity and specificity. In the present study, natural and experimental infections in domestic and wild Australian canids were investigated using a coproantigen capture ELISA. Experimental infection of dogs with E. granulosus was detected at between 14 and 22 days postinfection (PI), and optical density (OD) values remained high until termination of experiments 35 days PI. After chemotherapy, coproantigen levels in infected dogs dropped rapidly, becoming negative 2-4 days after treatment. In experimentally infected red foxes (Vulpes vulpes), the coproantigen excretion profile was different, with ELISA OD levels peaking 15-17 days PI, then falling to low or undetectable levels by 30 days PI. Coproantigens were detected in the feces of naturally infected Australian wild dogs (dingoes, dingo/domestic dog hybrids) with infection levels ranging between 2 worms and 42,600. Preliminary data on the stability of coproantigen in dog feces exposed to environmental conditions indicated that there was no change in antigenicity over 6 days. The results suggest the coproantigen ELISA could be successfully used to monitor E. granulosus prevalence rates in Australian domestic dogs, foxes, and wild dogs. PMID:10701577

  11. The Performance of a Rapid Diagnostic Test in Detecting Malaria Infection in Pregnant Women and the Impact of Missed Infections

    PubMed Central

    Williams, John E.; Cairns, Matthew; Njie, Fanta; Laryea Quaye, Stephen; Awine, Timothy; Oduro, Abraham; Tagbor, Harry; Bojang, Kalifa; Magnussen, Pascal; ter Kuile, Feiko O.; Woukeu, Arouna; Milligan, Paul; Chandramohan, Daniel; Greenwood, Brian

    2016-01-01

    Background. Intermittent screening and treatment in pregnancy (ISTp) is a potential strategy for the control of malaria during pregnancy. However, the frequency and consequences of malaria infections missed by a rapid diagnostic test (RDT) for malaria are a concern. Methods. Primigravidae and secundigravidae who participated in the ISTp arm of a noninferiority trial in 4 West African countries were screened with an HRP2/pLDH RDT on enrollment and, in Ghana, at subsequent antenatal clinic (ANC) visits. Blood samples were examined subsequently by microscopy and by a polymerase chain reaction (PCR) assay. Results. The sensitivity of the RDT to detect peripheral blood infections confirmed by microscopy and/or PCR at enrollment ranged from 91% (95% confidence interval [CI], 88%, 94%) in Burkina Faso to 59% (95% CI, 48%, 70% in The Gambia. In Ghana, RDT sensitivity was 89% (95% CI, 85%, 92%), 83% (95% CI, 76%, 90%) and 77% (95% CI, 67%, 86%) at enrollment, second and third ANC visits respectively but only 49% (95% CI, 31%, 66%) at delivery. Screening at enrollment detected 56% of all infections detected throughout pregnancy. Seventy-five RDT negative PCR or microscopy positive infections were detected in 540 women; these were not associated with maternal anemia, placental malaria, or low birth weight. Conclusions. The sensitivity of an RDT to detect malaria in primigravidae and secundigravidae was high at enrollment in 3 of 4 countries and, in Ghana, at subsequent ANC visits. In Ghana, RDT negative malaria infections were not associated with adverse birth outcomes but missed infections were uncommon. PMID:26721833

  12. Evaluation of a PCR-ELISA to detect Wuchereria bancrofti in Culex pipiens from an Egyptian village with a low prevalence of filariasis.

    PubMed

    Kamal, I H; Fischer, P; Adly, M; El Sayed, A S; Morsy, Z S; Ramzy, R M

    2001-12-01

    The programmes for the elimination of bancroftian filariasis that have been implemented in the Nile delta of Egypt are expected to lead to substantial reductions in filarial loads in the treated populations. Better methods than those currently available are needed for monitoring the efficacy of these and similar efforts at intervention. A PCR-ELISA was therefore evaluated as an epidemiological tool for the detection of the Wuchereria-bancrofti-specific SspI repeat in pools of Culex pipiens collected in a village with a low prevalence of filarial infection in its human residents (2.1%). Indoor-resting mosquitoes were collected by aspiration from 114 randomly selected houses (during one to nine visits/house) and separated into 673 pools, each of which held the mosquitoes collected during one night from one house. Although 18 (2.7%) of the pools showed PCR inhibition and had to be excluded, filarial DNA was detected, using the PCR-ELISA, in 91 (13.9%) of the 655 remaining mosquito pools. The minimum prevalence of W. bancrofti infection in the mosquitoes caught (assuming one infected mosquito/positive pool) was 2.8%. The mean (S.D.) number of mosquitoes/pool did not vary significantly between positive [5.5 (3.4)] and negative [4.9 (3.5)] pools. The assay detected parasite DNA in mosquitoes from 19.3% of 114 houses when only the first visit was considered and from 73.9% of the 88 houses visited more than once. The PCR-ELISA yielded results comparable with those of the regular PCR-SspI assay. The latter assay is recommended for the routine examination, in laboratories in endemic areas, of mosquito pools from randomly selected houses, as the ELISA component of the PCR-ELISA is exceedingly time-consuming, expensive and requires special equipment. PMID:11784438

  13. Widespread infection and diverse infection patterns of Wolbachia in Chinese aphids.

    PubMed

    Wang, Zhe; Su, Xiao-Min; Wen, Juan; Jiang, Li-Yun; Qiao, Ge-Xia

    2014-06-01

    Wolbachia are intracellular symbionts that infect a wide range of arthropods and filarial nematodes. Aphids are engaged in diverse and complex relationships with their endosymbionts. Four supergroups (A, B, M and N) of Wolbachia were previously detected in aphids and supergroups M and N were only found in aphids. In this study, we detected and described Wolbachia infections in natural populations of aphids in China. Three supergroups (A, B and M) were found in the examined aphid species. Supergroup M was preponderant, whereas supergroups A and B were only detected in certain species. Supergroup N was not found in this study. There were four infection patterns of Wolbachia in aphids, namely, infection with supergroup M alone, co-infection with supergroups A and M, co-infection with supergroups B and M, and co-infection with supergroups A, B and M. The pattern of infection only with supergroup M was universal and was found in all evaluated subfamilies. Only two subfamilies, Aphidinae and Lachninae, manifested to present all four infection patterns. Three patterns were observed in Calaphidinae (M, A&M, B&M) and Eriosomatinae (M, B&M, A&B&M). Two patterns were observed in the Anoeciinae (M, A&M) and Greenideinae (M, B&M), and only one pattern (M) was observed in the remaining families and/or subfamilies of Aphidoidea. These results indicated that Wolbachia infections in Chinese aphids are widespread. Phylogenetic analyses suggest that Wolbachia supergroup M spread rapidly and recently among all host species of aphids in China. Reasons for this spread and its mechanisms are discussed along with the possible effects of Wolbachia on their aphid hosts.

  14. Detection and characterization of Wolbachia infection in silkworm

    PubMed Central

    Zha, Xingfu; Zhang, Wenji; Zhou, Chunyan; Zhang, Liying; Xiang, Zhonghuai; Xia, Qingyou

    2014-01-01

    Wolbachia naturally infects a wide variety of arthropods, where it plays important roles in host reproduction. It was previously reported that Wolbachia did not infect silkworm. By means of PCR and sequencing we found in this study that Wolbachia is indeed present in silkworm. Phylogenetic analysis indicates that Wolbachia infection in silkworm may have occurred via transfer from parasitic wasps. Furthermore, Southern blotting results suggest a lateral transfer of the wsp gene into the genomes of some wild silkworms. By antibiotic treatments, we found that tetracycline and ciprofloxacin can eliminate Wolbachia in the silkworm and Wolbachia is important to ovary development of silkworm. These results provide clues towards a more comprehensive understanding of the interaction between Wolbachia and silkworm and possibly other lepidopteran insects. PMID:25249781

  15. Detection of Zika Virus Infection in Thailand, 2012–2014

    PubMed Central

    Buathong, Rome; Hermann, Laura; Thaisomboonsuk, Butsaya; Rutvisuttinunt, Wiriya; Klungthong, Chonticha; Chinnawirotpisan, Piyawan; Manasatienkij, Wudtichai; Nisalak, Ananda; Fernandez, Stefan; Yoon, In-Kyu; Akrasewi, Passakorn; Plipat, Tanarak

    2015-01-01

    Zika virus (ZIKV) is an emerging mosquito-borne pathogen with reported cases in Africa, Asia, and large outbreaks in the Pacific. No autochthonous ZIKV infections have been confirmed in Thailand. However, there have been several cases reported in travelers returning from Thailand. Here we report seven cases of acute ZIKV infection in Thai residents across the country confirmed by molecular or serological testing including sequence data. These endemic cases, combined with previous reports in travelers, provide evidence that ZIKV is widespread throughout Thailand. PMID:26101272

  16. Detection of Zika Virus Infection in Thailand, 2012-2014.

    PubMed

    Buathong, Rome; Hermann, Laura; Thaisomboonsuk, Butsaya; Rutvisuttinunt, Wiriya; Klungthong, Chonticha; Chinnawirotpisan, Piyawan; Manasatienkij, Wudtichai; Nisalak, Ananda; Fernandez, Stefan; Yoon, In-Kyu; Akrasewi, Passakorn; Plipat, Tanarak

    2015-08-01

    Zika virus (ZIKV) is an emerging mosquito-borne pathogen with reported cases in Africa, Asia, and large outbreaks in the Pacific. No autochthonous ZIKV infections have been confirmed in Thailand. However, there have been several cases reported in travelers returning from Thailand. Here we report seven cases of acute ZIKV infection in Thai residents across the country confirmed by molecular or serological testing including sequence data. These endemic cases, combined with previous reports in travelers, provide evidence that ZIKV is widespread throughout Thailand.

  17. Detection of Vaccinia Virus in Milk: Evidence of a Systemic and Persistent Infection in Experimentally Infected Cows.

    PubMed

    de Oliveira, Tércia Moreira Ludolfo; Guedes, Maria Isabel Maldonado Coelho; Rehfeld, Izabelle Silva; Matos, Ana Carolina Diniz; Rivetti, Anselmo Vasconcelos; Alves, Pedro Augusto; Galinari, Grazielle Cossenzo Florentino; Cerqueira, Mônica Maria Oliveira Pinho; Abrahão, Jônatas Santos; Lobato, Zélia Inês Portela

    2015-11-01

    Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects lactating cows and milkers. VACV DNA and infectious particles have been detected in milk of naturally infected cows. However, the period and pattern of VACV shedding in milk is unknown, as is whether the presence of VACV in milk is due to a localized or a systemic infection. To address those questions, eight lactating cows were inoculated with VACV in previously scarified teats. The experiment was divided in two phases. In Phase 1, milk samples were collected daily for 33 days, and in Phase 2, four animals from the first phase were immunosuppressed. In both phases, milk was collected with a sterile catheter on even days and by hand milking on odd days. All animals showed typical BV lesions in the inoculated teats. All milk samples were subjected to nested polymerase chain reaction (PCR) and real-time quantitative PCR to detect VACV DNA. PCR-positive samples were subjected to virus isolation. VACV DNA was intermittently detected in milk in both phases and infectious viral particles could be detected only in phase 2, on the 69th, 73rd, 74th, 77th, 79th, and 81st days postinfection. Despite the possibility of propagation of VACV through milk, it is known that milk continues to be drawn and marketed normally during outbreaks of the disease. The detection of both VACV DNA and infectious particles in milk samples draws attention to the potential public health risk associated with the consumption of milk from BV outbreaks. Detection of VACV in the milk from noninfected teats demonstrated that VACV shedding in milk might be related to a systemic infection. Moreover, it was shown that VACV DNA and viral infectious particles could be detected in milk even after healing of the lesions, demonstrating that VACV may cause a persistent infection in cattle.

  18. Early detection of Toxoplasma gondii-infected cats by interferon-gamma release assay.

    PubMed

    Yin, Qing; El-Ashram, Saeed; Liu, Xian-Yong; Suo, Xun

    2015-10-01

    Felines, the only definitive hosts that shed the environmentally-durable oocysts, are the key in the transmission of Toxoplasma gondii to all warm-blooded animals. They seroconvert as late as the third week and begin to shed oocysts as early as 3-8 days after being fed tissue cysts. Early detection of Toxoplasma-infected cats is crucial to evaluate Toxoplasma-contaminated environment and potential risks to public health. Moreover, it is fundamental for Toxoplasma infection control. Interferon-gamma release assay (IGRA) is a blood-based test assessing the presence of IFN-γ released by the T-lymphocytes directed against specific antigens, which is an ideal assay for early detection of Toxoplasma-infected cats. Here, cats were orally infected with the tissue cysts and blood was collected for toxoplasmic antigen stimulation, and the released IFN-γ was measured by ELISA. Results showed that Toxoplasma-infection was detected by IGRA as early as 4 days post-infection (dpi); while serum Toxoplasma IgM and IgG were detected by ELISA at 10 dpi and 14 dpi, respectively. Our findings demonstrated that IGRA-positive and ELISA-negative samples revealed an early Toxoplasma infection in cats, indicating a new strategy for the early diagnosis of Toxoplasma infection by combining IGRA and ELISA. Therefore, IGRA could emerge as a reliable diagnostic tool for the exploration of cat toxoplasmosis prevalence and its potential risks to public health.

  19. Circulating Mycobacterium bovis peptides and host response proteins as biomarkers for unambiguous detection of subclinical infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Bovine tuberculosis remains one of the most damaging zoonotic diseases. A critical need exists for rapid and inexpensive diagnostics capable of detecting and differentiating M. bovis infection from other pathogenic and environmental mycobacteria at multiple surveillance levels. Method...

  20. Use of digital PCR to improve early detection of CLas infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Huanglongbing is a devastating disease of citrus caused by the bacterium Candidatus Liberibacter asiaticus. Huanglongbing has devastated the Florida citrus industry and is threatening citrus in Texas and California. Detection of Candidatus Liberibacter asiaticus infections as early as possible is ...

  1. Analysis of Wuchereria bancrofti infections in a village community in northern Nigeria: increased prevalence in individuals infected with Onchocerca volvulus.

    PubMed

    Engelbrecht, Fred; Oettl, Tobias; Herter, Ursula; Link, Claudia; Philipp, Diana; Edeghere, Henry; Kaliraj, P; Enwezor, Felicia

    2003-03-01

    Infections with Wuchereria bancrofti causing lymphatic filariasis still represent one of the major health problems in the tropics, with 120 million people infected and over 750 million exposed to this filarial parasite. We have studied lymphatic filariasis infections as part of a multi-parasite survey in a village community in the savannah of northern Nigeria. We analysed serum samples from 341 individuals aged 5-70 years, detecting a W. bancrofti circulating antigen using the commercially available ICT Filariasis card test. The prevalence of infections was 10% and clearly age-dependent, increasing from below 2% in children to over 20% in subjects older than 40 years. Measuring IgG4 antibodies against the recombinant W. bancrofti antigen SXP1 showed that 36% of all tested individuals had been at least exposed to the parasite. Antibody levels also increased very significantly with age. A further analysis measuring Onchocerca volvulus-specific IgG4 antibodies showed a very significant association between infections with O. volvulus and those with W. bancrofti. Our data show that infections with W. bancrofti in Nigeria are still a frequently occurring health problem, since they are more prevalent than previously reported, and that individuals with an O. volvulus infection are more often infected with W. bancrofti than expected statistically. PMID:12543143

  2. [Detection of viral infection pathogens in medicinal plants grown in Ukraine].

    PubMed

    Mishchenko, L T; Korenieva, A A; Molchanets', O V; Boĭko, A L

    2009-01-01

    Monitoring of viral infection on medicinal plant plantations is carried out. Panax ginseng C.A. Meyer, Valeriana officinalis L., Plantago major L. with symptoms of viral infection were revealed. Viral nature of symptoms was proved with biotesting method. Morphology and sizes of virus particles, detected in Panax ginseng method. Morphology and sizes of virus particles, detected in Panax ginseng C.A. Meyer, Valeriana officinalis L., Plantago major L., were determined with electron microscopy method. The paper is presented in Ukrainian.

  3. Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis.

    PubMed

    Burrell-Saward, Hollie; Ward, Theresa H

    2016-01-01

    Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites. Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection. Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches. PMID:27284970

  4. Infection.

    PubMed

    Saigal, Gaurav; Nagornaya, Natalya; Post, M Judith D

    2016-01-01

    Imaging is useful in the diagnosis and management of infections of the central nervous system. Typically, imaging findings at the outset of the disease are subtle and nonspecific, but they often evolve to more definite imaging patterns in a few days, with less rapidity than for stroke but faster than for neoplastic lesions. This timing is similar to that of noninfectious inflammatory brain disease, such as multiple sclerosis. Fortunately, imaging patterns help to distinguish the two kinds of processes. Other than for sarcoidosis, the meninges are seldom involved in noninfectious inflammation; in contrast, many infectious processes involve the meninges, which then enhance with contrast on computed tomography (CT) or magnetic resonance imaging (MRI). However, brain infection causes a vast array of imaging patterns. Although CT is useful when hemorrhage or calcification is suspected or bony detail needs to be determined, MRI is the imaging modality of choice in the investigation of intracranial infections. Imaging sequences such as diffusion-weighted imaging help in accurately depicting the location and characterizing pyogenic infections and are particularly useful in differentiating bacterial infections from other etiologies. Susceptibility-weighted imaging is extremely useful for the detection of hemorrhage. Although MR spectroscopy findings can frequently be nonspecific, certain conditions such as bacterial abscesses show a relatively specific spectral pattern and are useful in diagnosing and constituting immediate therapy. In this chapter we review first the imaging patterns associated with involvement of various brain structures, such as the epidural and subdural spaces, the meninges, the brain parenchyma, and the ventricles. Involvement of these regions is illustrated with bacterial infections. Next we illustrate the patterns associated with viral and prion diseases, followed by mycobacterial and fungal infections, to conclude with a review of imaging findings

  5. Relationship between airborne detection of influenza A virus and the number of infected pigs.

    PubMed

    Corzo, Cesar A; Romagosa, Anna; Dee, Scott A; Gramer, Marie R; Morrison, Robert B; Torremorell, Montserrat

    2013-05-01

    Influenza A virus infects a wide range of species including both birds and mammals (including humans). One of the key routes by which the virus can infect populations of animals is by aerosol transmission. This study explored the relationship between number of infected pigs and the probability of detecting influenza virus RNA in bioaerosols through the course of an acute infection. Bioaerosols were collected using a cyclonic collector in two groups of 7 week-old pigs that were experimentally infected by exposure with a contact infected pig (seeder pig). After contact exposure, individual pig nasal swab samples were collected daily and air samples were collected three times per day for 8 days. All samples were tested for influenza by real-time reverse transcriptase (RRT)-PCR targeting the influenza virus matrix gene. All pigs' nasal swabs became influenza virus RRT-PCR positive upon exposure to the infected seeder pig. Airborne influenza was detected in 28/43 (65%) air samples. The temporal dynamics of influenza virus detection in air samples was in close agreement with the nasal shedding pattern in the infected pigs. First detection of positive bioaerosols happened at 1 day post contact (DPC). Positive bioaerosols were consistently detected between 3 and 6 DPC, a time when most pigs were also shedding virus in nasal secretions. Overall, the odds of detecting a positive air sample increased 2.2 times for every additional nasal swab positive pig in the group. In summary, there was a strong relationship between the number of pigs shedding influenza virus in nasal secretions and the generation of bioaerosols during the course of an acute infection.

  6. Relationship between airborne detection of influenza A virus and the number of infected pigs

    PubMed Central

    Corzo, Cesar A.; Romagosa, Anna; Dee, Scott; Gramer, Marie; Morrison, Robert B; Torremorell, Montserrat

    2012-01-01

    Influenza A virus infects a wide range of species including both birds and mammals (including humans). One of the key routes by which the virus can infect populations of animals is by aerosol transmission. This study explored the relationship between number of infected pigs and the probability of detecting influenza virus RNA in bioaerosols through the course of an acute infection. Bioaerosols were collected using a cyclonic collector in two groups of 7 week-old pigs that were experimentally infected by exposure with a contact infected pig (seeder pig). After contact exposure, individual pig nasal swab samples were collected daily and air samples were collected three times per day for 8 days. All samples were tested for influenza by real-time reverse transcriptase (RRT)-PCR targeting the influenza virus matrix gene. All pigs' nasal swabs became influenza virus RRT-PCR positive upon exposure to the infected seeder pig. Airborne influenza was detected in 28/43 (65%) air samples. The temporal dynamics of influenza virus detection in air samples was in close agreement with the nasal shedding pattern in the infected pigs. First detection of positive bioaerosols happened at 1 day post contact (DPC). Positive bioaerosols were consistently detected between 3 and 6 DPC, a time when most pigs were also shedding virus in nasal secretions. Overall, the odds of detecting a positive air sample increased 2.2 times for every additional nasal swab positive pig in the group. In summary, there was a strong relationship between the number of pigs shedding influenza virus in nasal secretions and the generation of bioaerosols during the course of an acute infection. PMID:23164957

  7. Relationship between airborne detection of influenza A virus and the number of infected pigs.

    PubMed

    Corzo, Cesar A; Romagosa, Anna; Dee, Scott A; Gramer, Marie R; Morrison, Robert B; Torremorell, Montserrat

    2013-05-01

    Influenza A virus infects a wide range of species including both birds and mammals (including humans). One of the key routes by which the virus can infect populations of animals is by aerosol transmission. This study explored the relationship between number of infected pigs and the probability of detecting influenza virus RNA in bioaerosols through the course of an acute infection. Bioaerosols were collected using a cyclonic collector in two groups of 7 week-old pigs that were experimentally infected by exposure with a contact infected pig (seeder pig). After contact exposure, individual pig nasal swab samples were collected daily and air samples were collected three times per day for 8 days. All samples were tested for influenza by real-time reverse transcriptase (RRT)-PCR targeting the influenza virus matrix gene. All pigs' nasal swabs became influenza virus RRT-PCR positive upon exposure to the infected seeder pig. Airborne influenza was detected in 28/43 (65%) air samples. The temporal dynamics of influenza virus detection in air samples was in close agreement with the nasal shedding pattern in the infected pigs. First detection of positive bioaerosols happened at 1 day post contact (DPC). Positive bioaerosols were consistently detected between 3 and 6 DPC, a time when most pigs were also shedding virus in nasal secretions. Overall, the odds of detecting a positive air sample increased 2.2 times for every additional nasal swab positive pig in the group. In summary, there was a strong relationship between the number of pigs shedding influenza virus in nasal secretions and the generation of bioaerosols during the course of an acute infection. PMID:23164957

  8. Gedunin and photogedunin of Xylocarpus granatum possess antifilarial activity against human lymphatic filarial parasite Brugia malayi in experimental rodent host.

    PubMed

    Misra, Sweta; Verma, Meenakshi; Mishra, Sunil Kumar; Srivastava, Shishir; Lakshmi, Vijai; Misra-Bhattacharya, Shailja

    2011-11-01

    The present study is aimed to evaluate antifilarial activity of Xylocarpus granatum (fruit from Andaman) against human lymphatic filarial parasite Brugia malayi in vivo. The in vitro antifilarial activity has already been reported earlier for this mangrove plant which has traditionally been used against several ailments. Aqueous ethanolic crude extract, four fractions (ethyl acetate fraction, n-butanol fraction, water-soluble fraction and water-insoluble fraction) and pure molecule/s of X. granatum (fruit) were tested in vitro on adult worms and microfilariae (mf) of B. malayi and the active samples were further evaluated in vivo in B. malayi (intraperitoneally) i.p. transplanted in the jird model (Meriones unguiculatus) and Mastomys coucha subcutaneously infected with infective larvae (L3). The crude aqueous ethanolic extract was active in vitro (IC50: adult = 15.46 μg/ml; mf = 13.17 μg/ml) and demonstrated 52.8% and 62.7% adulticidal and embryostatic effect on B. malayi, respectively, in Mastomys at a dose of 5 × 50 mg/kg by oral route. The antifilarial activity was primarily localized in the ethyl acetate-soluble fraction which revealed IC50 of 8.5 and 6.9 μg/ml in adult and mf, respectively. This fraction possessed moderate adulticidal and embryostatic action in vivo in Mastomys. Out of eight pure molecules isolated from the active fraction, two compounds gedunin (IC50 = 0.239 μg/ml, CC50 = 212.5 μg/ml, SI = 889.1) and photogedunin (IC50 = 0.213 μg/ml, CC50 = 262.3 μg/ml, SI = 1231.4) at 5 × 100 mg/kg by subcutaneous route revealed excellent adulticidal efficacy resulting in to the death of 80% and 70% transplanted adult B. malayi in the peritoneal cavity of jirds respectively in addition to noticeable microfilaricidalo action on the day of autopsy. The findings reveal that the extract from the fruit X. granatum contains promising in vitro and in vivo antifilarial activity against human lymphatic filarial parasite B. malayi which could be attributed to

  9. Detection of occult hepatitis C virus among healthy spouses of patients with HCV infection.

    PubMed

    El Shazly, Yahia; Hemida, Khaled; Rafik, Mona; Al Swaff, Reham; Ali-Eldin, Zainab A; GadAllah, Shaimaa

    2015-03-01

    The criterion standard for the diagnosis of occult hepatitis C virus (HCV) infection is detection of HCV-RNA in liver cells. However, because of the invasive nature of liver biopsy, other methods have been studied. The present study aimed to identify subjects with occult HCV-4 infection among healthy sexual partners of patients with chronic HCV-4 infection by detecting HCV-RNA in peripheral blood mononuclear cells (PBMCs) using real-time polymerase chain reaction (PCR). Fifty healthy Egyptian spouses of patients with chronic HCV-4 infection were included in this study. Real-time PCR was used to detect HCV-RNA in PBMCs in all the study subjects. The prevalence of occult HCV-4 infection was 4%, and a statistically significant higher prevalence was found among patients with a history of sexually transmitted infection. The results of the present study indicate the importance of intra-spousal transmission of HCV-4 infection, especially in subjects with a history of sexually transmitted infection.

  10. Deep Whole-Genome Sequencing to Detect Mixed Infection of Mycobacterium tuberculosis

    PubMed Central

    Gan, Mingyu; Liu, Qingyun; Yang, Chongguang; Gao, Qian; Luo, Tao

    2016-01-01

    Mixed infection by multiple Mycobacterium tuberculosis (MTB) strains is associated with poor treatment outcome of tuberculosis (TB). Traditional genotyping methods have been used to detect mixed infections of MTB, however, their sensitivity and resolution are limited. Deep whole-genome sequencing (WGS) has been proved highly sensitive and discriminative for studying population heterogeneity of MTB. Here, we developed a phylogenetic-based method to detect MTB mixed infections using WGS data. We collected published WGS data of 782 global MTB strains from public database. We called homogeneous and heterogeneous single nucleotide variations (SNVs) of individual strains by mapping short reads to the ancestral MTB reference genome. We constructed a phylogenomic database based on 68,639 homogeneous SNVs of 652 MTB strains. Mixed infections were determined if multiple evolutionary paths were identified by mapping the SNVs of individual samples to the phylogenomic database. By simulation, our method could specifically detect mixed infections when the sequencing depth of minor strains was as low as 1× coverage, and when the genomic distance of two mixed strains was as small as 16 SNVs. By applying our methods to all 782 samples, we detected 47 mixed infections and 45 of them were caused by locally endemic strains. The results indicate that our method is highly sensitive and discriminative for identifying mixed infections from deep WGS data of MTB isolates. PMID:27391214

  11. Deep Whole-Genome Sequencing to Detect Mixed Infection of Mycobacterium tuberculosis.

    PubMed

    Gan, Mingyu; Liu, Qingyun; Yang, Chongguang; Gao, Qian; Luo, Tao

    2016-01-01

    Mixed infection by multiple Mycobacterium tuberculosis (MTB) strains is associated with poor treatment outcome of tuberculosis (TB). Traditional genotyping methods have been used to detect mixed infections of MTB, however, their sensitivity and resolution are limited. Deep whole-genome sequencing (WGS) has been proved highly sensitive and discriminative for studying population heterogeneity of MTB. Here, we developed a phylogenetic-based method to detect MTB mixed infections using WGS data. We collected published WGS data of 782 global MTB strains from public database. We called homogeneous and heterogeneous single nucleotide variations (SNVs) of individual strains by mapping short reads to the ancestral MTB reference genome. We constructed a phylogenomic database based on 68,639 homogeneous SNVs of 652 MTB strains. Mixed infections were determined if multiple evolutionary paths were identified by mapping the SNVs of individual samples to the phylogenomic database. By simulation, our method could specifically detect mixed infections when the sequencing depth of minor strains was as low as 1× coverage, and when the genomic distance of two mixed strains was as small as 16 SNVs. By applying our methods to all 782 samples, we detected 47 mixed infections and 45 of them were caused by locally endemic strains. The results indicate that our method is highly sensitive and discriminative for identifying mixed infections from deep WGS data of MTB isolates. PMID:27391214

  12. Detection of acute synthetic vascular graft infection with In-labeled leukocyte scanning: an animal study

    SciTech Connect

    Dries, D.J.; Alazraki, N.; Lawrence, P.F.; Murphy, K.M.; Kercher, J.; Datz, F.L.; Christian, P.; Taylor, A. Jr.

    1985-11-01

    Synthetic vascular graft infection is characterized by late diagnosis due to indolent and nonspecific symptoms. Indium- -labeled leukocyte imaging holds promise as a diagnostic tool to identify vascular graft infection, but reported data on its accuracy are somewhat sparse and conflicting. In this study, 13 mongrel dogs received Dacron aortic interposition grafts. Seven grafts were contaminated at the time of surgery by topical ATCC Staphylococcus aureus concentrated at 10(8) organisms/ml. Six control animals received no graft contamination. All infected animals were sacrificed on the second postoperative day after In leukocyte scanning. The results showed a sensitivity of 71%, specificity of 100%, and accuracy of 85% for the 111In leukocyte study in detecting early graft infections; false-positive leukocyte scans in the early postoperative period were not a problem as has been reported by others. These data indicate that leukocyte scanning for graft infection detection is likely to be clinically valuable.

  13. A Quality Improvement Project to Increase Early Detection of Syphilis Infection or Re-infection in HIV-infected Men Who Have Sex With Men.

    PubMed

    Cheeks, Miyesha A; Fransua, Mesfin; Stringer, Harold G; Silva, Susan; Relf, Michael

    2016-01-01

    Our quality improvement project evaluated whether testing for syphilis every 3 to 6 months with routine HIV laboratory monitoring had an effect on early detection of asymptomatic syphilis infection/re-infection in HIV-infected men who have sex with men. Retrospective analysis of syphilis testing and infections in a sample of this population (N = 245) was conducted after establishing a change-of-practice quality improvement initiative in a not-for-profit, community-based, grant-funded clinic. We compared the clinic's annual rates of syphilis before and after intervention implementation. The detection rate was 6.6% in the preintervention practice change group and 15.5% in the postintervention group. Increased testing identified 27 syphilis cases that would not otherwise have been identified until the annual comprehensive examination. Increased testing frequency led to earlier detection of syphilis, which was clinically significant, showing a potential to decrease the number of new syphilis and HIV infections and to decrease health care expenditures.

  14. LACK OF ASSOCIATION BETWEEN HERPESVIRUS DETECTION IN SALIVA AND GINGIVITIS IN HIV‑INFECTED CHILDREN.

    PubMed

    Otero, Renata A; Nascimento, Flávia N N; Souza, Ivete P R; Silva, Raquel C; Lima, Rodrigo S; Robaina, Tatiana F; Câmara, Fernando P; Santos, Norma; Castro, Gloria F

    2015-01-01

    The aims of this study were to compare the detection of human herpesviruses (HHVs) in the saliva of HIV-infected and healthy control children, and to evaluate associations between viral infection and gingivitis and immunodeficiency. Saliva samples were collected from 48 HIV-infected and 48 healthy control children. Clinical and laboratory data were collected during dental visits and from medical records. A trained dentist determined gingival indices and extension of gingivitis. Saliva samples were tested for herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV) by nested polymerase chain reaction assays. Thirty-five HIV-infected and 16 control children had gingivitis. Seventeen (35.4%) HIV-infected children and 13 (27%) control children were positive for HHVs. CMV was the most commonly detected HHV in both groups (HIV-infected, 25%; control, 12.5%), followed by HSV-1 (6.2% in both groups) and HSV-2 (HIV-infected, 4.2%; control, 8.3%). The presence of HHVs in saliva was not associated with the presence of gingivitis in HIV-1-infected children (p = 0.104) or healthy control children (p = 0.251), or with immunosuppression in HIV-infected individuals (p = 0.447). Gingivitis was correlated with HIV infection (p = 0.0001). These results suggest that asymptomatic salivary detection of HHVs is common in HIV-infected and healthy children, and that it is not associated with gingivitis.

  15. Individuals infected with JC polyomavirus do not present detectable JC virus DNA in oropharyngeal fluids.

    PubMed

    Matos, Ana; Duque, Vitor; Luxo, Cristina; Meliço-Silvestre, António; Major, Eugene O

    2012-04-01

    JC virus (JCV) is ubiquitous in the human population. Primary infection normally occurs during childhood and is followed by a lifelong persistent infection. The main mode of transmission remains unknown. Several authors have hypothesized that JCV transmission occurs through the respiratory route, and that respiratory secretions could represent a possible source of viral particles. The present study intended to evaluate oropharyngeal fluids from patients infected with JCV, in order to ascertain if respiratory secretions could indeed constitute a source of exposure to this polyomavirus. Oropharyngeal washing samples from 25 patients co-infected with JCV and human immunodeficiency virus type 1 were evaluated for the presence of JCV DNA. Regardless of the titre of antibodies or the presence of viral urinary excretion, JCV genome was not detected in oropharyngeal samples collected from any of the patients infected with JCV included in this study, which may suggest that oropharyngeal fluids are an unlikely source for JCV infection.

  16. Use of real-time quantitative PCR to detect Chlamydophila felis infection.

    PubMed

    Helps, C; Reeves, N; Tasker, S; Harbour, D

    2001-07-01

    A real-time PCR assay was developed to detect and quantify Chlamydophila felis infection of cats. The assay uses a molecular beacon to specifically identify the major outer membrane protein gene, is highly reproducible, and is able to detect fewer than 10 genomic copies.

  17. [Demand for and the Development of Detection Techniques for Source of Schistosome Infection in China].

    PubMed

    Wang, Shi-ping; He, Xin; Zhou, Yun-fei

    2015-12-01

    Schistosomiasis is a type of zoonotic parasitosis that severely impairs human health. Rapid detection of infection sources is a key to the control of schistosomiasis. With the effective control of schistosomiasis in China, the detection techniques for infection sources have also been developed. The rate and the intensity of infection among humans and livestocks have been significantly decreased in China, as the control program has entered the transmission control stage in most of the endemic areas. Under this situation, the traditional etiological diagnosing techniques and common immunological methods can not afford rapid detection of infection sources of schistosomiasis. Instead, we are calling for detection methods with higher sensitivity, specificity and stability while being less time-consuming, more convenient and less costing. In recent years, many improved or novel detection methods have been applied for the epidemiological surveillance of schistosomiasis, such as the automatic scanning microscopic image acquisition system, PCR-ELISA, immunosensors, loop-mediated isothermal amplification, etc. The development of new monitoring techniques can facilitate rapid detection of schistosome infection sources in endemic areas. PMID:27089776

  18. Radiotracers Used for the Scintigraphic Detection of Infection and Inflammation

    PubMed Central

    Tsopelas, Chris

    2015-01-01

    Over the last forty years, a small group of commercial radiopharmaceuticals have found their way into routine medical use, for the diagnostic imaging of patients with infection or inflammation. These molecular radiotracers usually participate in the immune response to an antigen, by tagging leukocytes or other molecules/cells that are endogenous to the process. Currently there is an advancing effort by researchers in the preclinical domain to design and develop new agents for this application. This review discusses radiopharmaceuticals used in the nuclear medicine clinic today, as well as those potential radiotracers that exploit an organism's defence mechanisms to an infectious or inflammatory event. PMID:25741532

  19. A Multiplex PCR Approach for Detecting Dual Infections and Recombinants Involving Major HIV Variants

    PubMed Central

    Cappy, Pierre; De Oliveira, Fabienne; Gueudin, Marie; Alessandri-Gradt, Elodie

    2016-01-01

    The cocirculation of different HIV types and groups can lead to dual infections and recombinants, which hinder diagnosis and therapeutic management. We designed two multiplex PCRs (mPCRs) coupled with capillary electrophoresis to facilitate the detection of such infections. The first, MMO2, targets three variants (HIV-1/M, HIV-1/O, and HIV-2), and the second, MMO, targets HIV-1/M and HIV-1/O. These mPCRs were validated on DNA and RNA extracts from 19 HIV-1/M, 12 HIV-1/O, and 13 HIV-2 cultures and from mixtures simulating dual infections. They were then assessed with DNA and RNA extracts from samples of 47 clinical monoinfections and HIV-1/M+O dual infections or infections with HIV-1/MO recombinants. Both mPCRs had excellent specificity. Sensitivities ranged from 80 to 100% for in vitro samples and from 58 to 100% for clinical samples, with the results obtained depending on the material used and the region of the genome concerned. Sensitivity was generally lower for DNA than for RNA and for amplifications of the integrase and matrix regions. In terms of global detection (at least one target gene for each strain), both mPCRs yielded a detection rate of 100% for in vitro samples. MMO2 detected 100% of the clinical strains from DNA and 97% from RNA, whereas MMO detected 100% of the strains from both materials. Thus, for in vitro and clinical samples, MMO2 was a useful tool for detecting dual infections with HIV-1 and HIV-2 (referred to as HIV-1+HIV-2) and HIV-1/M+O, and MMO was useful for detecting both MO dual infections and MO mosaic patterns. PMID:26912747

  20. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals.

    PubMed Central

    Leal-Klevezas, D S; Martínez-Vázquez, I O; López-Merino, A; Martínez-Soriano, J P

    1995-01-01

    A versatile method for the extraction of Brucella DNA and PCR are presented as reliable tools for the detection of Brucella spp. from body fluids of infected animals. Two oligonucleotides homologous to regions of the gene encoding for an outer membrane protein (omp-2) were designed to detect the pathogen from milk and/or blood of infected goats, bovines, and human patients. The sensitivity of our test and its ability to detect the pathogen in samples from the field reveal a promising advance in the diagnosis of brucellosis in animals and humans. PMID:8586678

  1. Detection of Trypanosoma cruzi infection in naturally-infected dogs and cats using serological, parasitological and molecular methods

    PubMed Central

    Enriquez, G.F.; Cardinal, M.V.; Orozco, M.M.; Schijman, A.G.; Gürtler, R.E.

    2013-01-01

    Domestic dogs and cats are major domestic reservoir hosts of Trypanosoma cruzi and a risk factor for parasite transmission. In this study we assessed the relative performance of a polymerase chain reaction assay targeted to minicircle DNA (kDNA-PCR) in reference to conventional serological tests, a rapid dipstick test and xenodiagnosis to detect T. cruzi infection in dogs and cats from an endemic rural area in northeastern Argentina. A total of 43 dogs and 13 cats seropositive for T. cruzi by an immunosorbent assay (ELISA) and an indirect hemagglutination assay (IHA), which had been examined by xenodiagnosis, were also tested by kDNA-PCR. kDNA-PCR was nearly as sensitive as xenodiagnosis for detecting T. cruzi- infectious dogs and cats. kDNA-PCR was slightly more sensitive than xenodiagnosis in seropositive dogs (91% versus 86%, respectively) and cats (77% against 54%, respectively), but failed to detect all of the seropositive individuals. ELISA and IHA detected all xenodiagnosis-positive dogs and both outcomes largely agreed (kappa coefficient, κ = 0.92), whereas both assays failed to detect all of the xenodiagnosis-positive cats and their agreement was moderate (κ = 0.68). In dogs, the sensitivity of the dipstick test was 95% and agreed closely with the outcome of conventional serological tests (κ = 0.82). The high sensitivity of kDNA-PCR to detect T. cruzi infections in naturally-infected dogs and cats supports its application as a diagnostic tool complementary to serology and may replace the use of xenodiagnosis or hemoculture. PMID:23499860

  2. Measurement of Circulating Filarial Antigen Levels in Human Blood with a Point-of-Care Test Strip and a Portable Spectrodensitometer.

    PubMed

    Chesnais, Cédric B; Vlaminck, Johnny; Kunyu-Shako, Billy; Pion, Sébastien D; Awaca-Uvon, Naomi-Pitchouna; Weil, Gary J; Mumba, Dieudonné; Boussinesq, Michel

    2016-06-01

    The Alere Filariasis Test Strip (FTS) is a qualitative, point-of-care diagnostic tool that detects Wuchereria bancrofti circulating filarial antigen (CFA) in human blood, serum, or plasma. The Global Program to Eliminate Lymphatic Filariasis employs the FTS for mapping filariasis-endemic areas and assessing the success of elimination efforts. The objective of this study was to explore the relationship between the intensity of positive test lines obtained by FTS with CFA levels as determined by enzyme-linked immunosorbent assay (ELISA) with blood and plasma samples from 188 individuals who live in a filariasis-endemic area. The intensity of the FTS test line was assessed visually to provide a semiquantitative score (visual Filariasis Test Strip [vFTS]), and line intensity was measured with a portable spectrodensitometer (quantitative Filariasis Test Strip [qFTS]). These results were compared with antigen levels measured by ELISA in plasma from the same subjects. qFTS measurements were highly correlated with vFTS scores (ρ = 0.94; P < 0.001) and with plasma CFA levels (ρ = 0.91; P < 0.001). Thus, qFTS assessment is a convenient method for quantifying W. bancrofti CFA levels in human blood, which are correlated with adult worm burdens. This tool may be useful for assessing the impact of treatment on adult filarial worms in individuals and communities. PMID:27114288

  3. Measurement of Circulating Filarial Antigen Levels in Human Blood with a Point-of-Care Test Strip and a Portable Spectrodensitometer

    PubMed Central

    Chesnais, Cédric B.; Vlaminck, Johnny; Kunyu-Shako, Billy; Pion, Sébastien D.; Awaca-Uvon, Naomi-Pitchouna; Weil, Gary J.; Mumba, Dieudonné; Boussinesq, Michel

    2016-01-01

    The Alere Filariasis Test Strip (FTS) is a qualitative, point-of-care diagnostic tool that detects Wuchereria bancrofti circulating filarial antigen (CFA) in human blood, serum, or plasma. The Global Program to Eliminate Lymphatic Filariasis employs the FTS for mapping filariasis-endemic areas and assessing the success of elimination efforts. The objective of this study was to explore the relationship between the intensity of positive test lines obtained by FTS with CFA levels as determined by enzyme-linked immunosorbent assay (ELISA) with blood and plasma samples from 188 individuals who live in a filariasis-endemic area. The intensity of the FTS test line was assessed visually to provide a semiquantitative score (visual Filariasis Test Strip [vFTS]), and line intensity was measured with a portable spectrodensitometer (quantitative Filariasis Test Strip [qFTS]). These results were compared with antigen levels measured by ELISA in plasma from the same subjects. qFTS measurements were highly correlated with vFTS scores (ρ = 0.94; P < 0.001) and with plasma CFA levels (ρ = 0.91; P < 0.001). Thus, qFTS assessment is a convenient method for quantifying W. bancrofti CFA levels in human blood, which are correlated with adult worm burdens. This tool may be useful for assessing the impact of treatment on adult filarial worms in individuals and communities. PMID:27114288

  4. Gamma Interferon Release Assays for Detection of Mycobacterium tuberculosis Infection

    PubMed Central

    Denkinger, Claudia M.; Kik, Sandra V.; Rangaka, Molebogeng X.; Zwerling, Alice; Oxlade, Olivia; Metcalfe, John Z.; Cattamanchi, Adithya; Dowdy, David W.; Dheda, Keertan; Banaei, Niaz

    2014-01-01

    SUMMARY Identification and treatment of latent tuberculosis infection (LTBI) can substantially reduce the risk of developing active disease. However, there is no diagnostic gold standard for LTBI. Two tests are available for identification of LTBI: the tuberculin skin test (TST) and the gamma interferon (IFN-γ) release assay (IGRA). Evidence suggests that both TST and IGRA are acceptable but imperfect tests. They represent indirect markers of Mycobacterium tuberculosis exposure and indicate a cellular immune response to M. tuberculosis. Neither test can accurately differentiate between LTBI and active TB, distinguish reactivation from reinfection, or resolve the various stages within the spectrum of M. tuberculosis infection. Both TST and IGRA have reduced sensitivity in immunocompromised patients and have low predictive value for progression to active TB. To maximize the positive predictive value of existing tests, LTBI screening should be reserved for those who are at sufficiently high risk of progressing to disease. Such high-risk individuals may be identifiable by using multivariable risk prediction models that incorporate test results with risk factors and using serial testing to resolve underlying phenotypes. In the longer term, basic research is necessary to identify highly predictive biomarkers. PMID:24396134

  5. Detection of acute synthetic vascular graft infection with IN-111 labeled leukocyte imaging

    SciTech Connect

    Alazraki, N.; Dries, D.; Lawrence, P.; Murphy, K.; Kercher, J.; Datz, F.; Christian, P.; Taylor, A.

    1985-05-01

    Synthetic vascular graft infection is characterized by late diagnosis due to indolent and nonspecific symptoms. Reported data on accuracy of In-111 labeled leukocyte imaging to identify vascular graft infection is sparse and conflicting. The purpose of this animal study was to clarify the accuracy of detection of early graft infection using a mixed population of In-111 labeled leukocytes. Twelve mongrel dogs received dacron aortic interposition grafts. Seven grafts were contaminated at surgery by topical ATCC S. aureus, 10/sup 8/ organisms per ml. Six control animals received no graft contamination Mixed population In-111 homologous leukocyte labeling was performed followed by imaging at 24 and 48 hours following intravenous injection of 250 ..mu..Ci In-111 leukocytes. Scans were done on Day 2 post-surgery. Infected dogs were sacrificed following Indium imaging; control dogs were rescanned at 3 weeks postop and sacrificed thereafter. Autopsy results were correlated with scans, yielding sensitivity 71%, specificity 100%, accuracy 85% for In-111 leukocyte imaging to detect early graft infection. False positive leukocyte imaging in the early postop period was not a problem. At autopsy all 5 dogs with infected grafts and positive scans had gross pus. The 2 dogs with false negative scans showed no gross pus at autopsy; cultures were positive for S. aureus in all 7 dogs. Scans at 2 days and 3 weeks post-surgery were true negatives in all 6 control dogs. These data suggest a high level of clinical reliability of leukocyte imaging for early graft infection detection.

  6. Detection of human herpesvirus 7 infection in young children presenting with exanthema subitum.

    PubMed

    Magalhães, Ivna de Melo; Martins, Rebeca Vazquez Novo; Vianna, Renata Oliveira; Moysés, Natalia; Afonso, Larissa Alves; Oliveira, Solange Artimos; Cavalcanti, Silvia Maria Baeta

    2011-05-01

    In this study, we assessed the prevalence of human herpesvirus-7 (HHV-7) in 141 serum samples from children less than four years of age with exanthematic disease. All samples were negative for measles, rubella, dengue fever and parvovirus B19 infection. Testing for the presence of human herpesvirus-6 (HHV-6)-specific high avidity IgG antibodies by indirect immunofluorescence assay (IFA) revealed two main groups: one composed of 57 patients with recent primary HHV-6 infection and another group of 68 patients showing signs of past HHV-6 infection. Another 16 samples had indeterminate primary HHV-6 infection, by both IgG IFA and IgM IFA. Serum samples were subjected to a nested polymerase chain reaction to detect the presence of HHV-7 DNA. Among patients with a recent primary HHV-6 infection, HHV-7 DNA was present in 1.7% of individuals; however, 5.8% of individuals tested positive for HHV-7 DNA in the group with past primary HHV-6 infection. Among the 16 samples with indeterminate diagnosis, 25% (4/16) had HHV-7 DNA (p < 0.002). We hypothesise that HHV-7 might be the agent that causes exanthema. However, a relationship between clinical manifestations and the detection of virus DNA does not always exist. Therefore, a careful interpretation is necessary to diagnose a primary infection or a virus-associated disease. In conclusion, we detected HHV-7 DNA in young children from the state of Rio de Janeiro, Brazil.

  7. Detection of infectious laryngotracheitis virus infected cells with cloned DNA probes.

    PubMed Central

    Nagy, E

    1992-01-01

    A genomic library of infectious laryngotracheitis virus (ILTV) DNA BamH1 fragments was prepared and two cloned fragments were evaluated for their potential as probes for the detection of ILTV infected cells. The virus was purified by a modified sucrose density gradient procedure for the isolation of pure ILTV DNA. A genomic library was constructed using BamH1-digested ILTV DNA and pGEM7 as a vector. A 1.1 kb cloned BamH1 fragment of ILTV DNA was tested in a slot or dot blot assay for the detection of ILTV infected cells. The limit of detection for this probe was at least 0.12 ng of pure ILTV DNA. The probe was able to identify both chicken embryo liver (CELi) cells and choriallantoic membranes infected with ILTV. Chicken embryo liver cells infected with several field isolates and a vaccine strain of ILTV were positive by dot blot analysis using this probe. Some qualitative differences in the degree of hybridization to cells infected by different ILTV isolates were observed. Uninfected cells and cells infected with fowlpox virus, turkey herpesvirus, Marek's disease virus or Newcastle disease virus were negative by the same assay. Compared with the 1.1 kb fragment, a larger 6 kb cloned BamH1 fragment of ILTV DNA showed a stronger hybridization signal to DNA from ILTV infected cells. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1316798

  8. Detection of human herpesvirus 7 infection in young children presenting with exanthema subitum.

    PubMed

    Magalhães, Ivna de Melo; Martins, Rebeca Vazquez Novo; Vianna, Renata Oliveira; Moysés, Natalia; Afonso, Larissa Alves; Oliveira, Solange Artimos; Cavalcanti, Silvia Maria Baeta

    2011-05-01

    In this study, we assessed the prevalence of human herpesvirus-7 (HHV-7) in 141 serum samples from children less than four years of age with exanthematic disease. All samples were negative for measles, rubella, dengue fever and parvovirus B19 infection. Testing for the presence of human herpesvirus-6 (HHV-6)-specific high avidity IgG antibodies by indirect immunofluorescence assay (IFA) revealed two main groups: one composed of 57 patients with recent primary HHV-6 infection and another group of 68 patients showing signs of past HHV-6 infection. Another 16 samples had indeterminate primary HHV-6 infection, by both IgG IFA and IgM IFA. Serum samples were subjected to a nested polymerase chain reaction to detect the presence of HHV-7 DNA. Among patients with a recent primary HHV-6 infection, HHV-7 DNA was present in 1.7% of individuals; however, 5.8% of individuals tested positive for HHV-7 DNA in the group with past primary HHV-6 infection. Among the 16 samples with indeterminate diagnosis, 25% (4/16) had HHV-7 DNA (p < 0.002). We hypothesise that HHV-7 might be the agent that causes exanthema. However, a relationship between clinical manifestations and the detection of virus DNA does not always exist. Therefore, a careful interpretation is necessary to diagnose a primary infection or a virus-associated disease. In conclusion, we detected HHV-7 DNA in young children from the state of Rio de Janeiro, Brazil. PMID:21655829

  9. Rapid Detection and Immune Characterization of Mycobacterium abscessus Infection in Cystic Fibrosis Patients

    PubMed Central

    Mayatepek, Ertan; Mackenzie, Colin R.; Schramm, Dirk; Jacobsen, Marc

    2015-01-01

    Cystic fibrosis patients are highly susceptible to infections with non-tuberculous mycobacteria. Especially Mycobacterium abscessus infections are common but reliable diagnosis is hampered by non-specific clinical symptoms and insensitive mycobacterial culture. In the present study we established novel methods for rapid detection and immune characterization of Mycobacterium abscessus infection in cystic fibrosis patients. We performed Mycobacterium abscessus specific DNA-strip- and quantitative PCR-based analyses of non-cultured sputum samples to detect and characterize Mycobacterium abscessus infections. Concomitantly in vitro T-cell reactivation with purified protein derivatives (PPDs) from different mycobacterial species was used to determine Mycobacterium abscessus specific T-cell cytokine expression of infected cystic fibrosis patients. Four of 35 cystic fibrosis patients (11.4%) were Mycobacterium abscessus culture positive and showed concordant DNA-strip-test results. Quantitative PCR revealed marked differences of mycobacterial burden between cystic fibrosis patients and during disease course. Tandem-repeat analysis classified distinct Mycobacterium abscessus strains of infected cystic fibrosis patients and excluded patient-to-patient transmission. Mycobacterium abscessus specific T-cells were detected in the blood of cystic fibrosis patients with confirmed chronic infection and a subgroup of patients without evidence of Mycobacterium abscessus infection. Comparison of cytokine expression and phenotypic markers revealed increased proportions of CD40L positive T-cells that lack Interleukin-2 expression as a marker for chronic Mycobacterium abscessus infections in cystic fibrosis patients. Direct sputum examination enabled rapid diagnosis and quantification of Mycobacterium abscessus in cystic fibrosis patients. T-cell in vitro reactivation and cytokine expression analyses may contribute to diagnosis of chronic Mycobacterium abscessus infection. PMID:25742660

  10. Rapid detection and immune characterization of Mycobacterium abscessus infection in cystic fibrosis patients.

    PubMed

    Steindor, Mathis; Nkwouano, Vanesa; Mayatepek, Ertan; Mackenzie, Colin R; Schramm, Dirk; Jacobsen, Marc

    2015-01-01

    Cystic fibrosis patients are highly susceptible to infections with non-tuberculous mycobacteria. Especially Mycobacterium abscessus infections are common but reliable diagnosis is hampered by non-specific clinical symptoms and insensitive mycobacterial culture. In the present study we established novel methods for rapid detection and immune characterization of Mycobacterium abscessus infection in cystic fibrosis patients. We performed Mycobacterium abscessus specific DNA-strip- and quantitative PCR-based analyses of non-cultured sputum samples to detect and characterize Mycobacterium abscessus infections. Concomitantly in vitro T-cell reactivation with purified protein derivatives (PPDs) from different mycobacterial species was used to determine Mycobacterium abscessus specific T-cell cytokine expression of infected cystic fibrosis patients. Four of 35 cystic fibrosis patients (11.4%) were Mycobacterium abscessus culture positive and showed concordant DNA-strip-test results. Quantitative PCR revealed marked differences of mycobacterial burden between cystic fibrosis patients and during disease course. Tandem-repeat analysis classified distinct Mycobacterium abscessus strains of infected cystic fibrosis patients and excluded patient-to-patient transmission. Mycobacterium abscessus specific T-cells were detected in the blood of cystic fibrosis patients with confirmed chronic infection and a subgroup of patients without evidence of Mycobacterium abscessus infection. Comparison of cytokine expression and phenotypic markers revealed increased proportions of CD40L positive T-cells that lack Interleukin-2 expression as a marker for chronic Mycobacterium abscessus infections in cystic fibrosis patients. Direct sputum examination enabled rapid diagnosis and quantification of Mycobacterium abscessus in cystic fibrosis patients. T-cell in vitro reactivation and cytokine expression analyses may contribute to diagnosis of chronic Mycobacterium abscessus infection. PMID:25742660

  11. Enhancing the detection and management of acute hepatitis C virus infection.

    PubMed

    Martinello, Marianne; Matthews, Gail V

    2015-10-01

    Acute HCV infection refers to the 6-month period following infection acquisition, although this definition is somewhat arbitrary. While spontaneous clearance occurs in approximately 25%, the majority will develop chronic HCV infection with the potential for development of cirrhosis, end stage liver disease and hepatocellular carcinoma. Detection of acute HCV infection has been hampered by its asymptomatic or non-specific presentation, lack of specific diagnostic tests and the inherent difficulties in identifying and following individuals at highest risk of transmitting and acquiring HCV infection, such as people who inject drugs (PWID). However, recognition of those with acute infection may have individual and population level benefits and could represent an ideal opportunity for intervention. Despite demonstration that HCV treatment is feasible and successful in PWID, treatment uptake remains low with multiple barriers to care at an individual and systems level. Given the burden of HCV-related disease among PWID, strategies to enhance HCV assessment, treatment and prevention in this group are urgently needed. As the therapeutic landscape of chronic HCV management is revolutionised by the advent of simple, highly effective directly-acting antiviral (DAA) therapy, similar opportunities may exist in acute infection. This review will discuss issues surrounding improving the detection and management of acute HCV infection, particularly in PWID. PMID:26254495

  12. Simultaneous detection of three lily-infecting viruses using a multiplex Luminex bead array.

    PubMed

    Lim, Mi Sang; Kim, Su Min; Choi, Sun Hee

    2016-05-01

    A Luminex bead array was applied to detect multiple-virus coinfection in lily plants exhibiting typical symptoms, and the efficiency of this detection system was assessed. Specific primer sets for the simultaneous detection of 4 targets in virus-infected lily plants were constructed and used for reverse transcription (RT)-polymerase chain reaction (PCR), and specific probes were used for Luminex-based assay. Each of the 4 targets was amplified, and the amplicons were used for Luminex bead array experiments. A Luminex bead array analysis of lily-infecting viruses was performed using the quadruplex RT-PCR products followed by hybridization between the biotinylated targets and anti-tagged microsphere beads. The hybridization products produced fluorescence signals that were detected by the Luminex system. Signal strengths were analyzed by their median fluorescence intensity (MFI) values. Detection of the different target elements was found to be very specific to the corresponding viruses in lilies, and coinfection with multiple viruses was specifically detected via the MFI signals. Therefore, the use of a Luminex bead array for the detection of co-infected multiple viruses in lily plants can be an improved system for screening and analyzing multiple-virus infection.

  13. Molecular approach for detecting early prepatent Schistosoma mansoni infection in Biomphalaria alexandrina snail host.

    PubMed

    Farghaly, Adel; Saleh, Ayman A; Mahdy, Soad; Abd El-Khalik, Dalia; Abd El-Aal, Naglaa F; Abdel-Rahman, Sara A; Salama, Marwa A

    2016-09-01

    The present study aimed to evaluate a polymerase chain reaction (PCR) assay used for detection of Schistosoma mansoni infection in Biomphalaria alexandrina snails in early prepatent period and to compare between it and the ordinary detection methods (shedding and crushing). Biomphalaria alexandrina snails are best known for their role as intermediate hosts of S. mansoni. DNA was extracted from infected snails in addition to non-infected "negative control" (to optimized the efficiency of PCR reaction) and subjected to PCR using primers specific to a partial sequence of S. mansoni fructose-1,6-bus phosphate aldolase (SMALDO). SMALDO gene was detected in the infected laboratory snails with 70, 85, and 100 % positivity at the 1st, 3rd, and 7th day of infection, respectively. In contrast, the ordinary method was not sensitive enough in detection of early prepatent infection even after 7 days of infection which showed only 25 % positivity. By comparing the sensitivity of the three methods, it was found that the average sensitivity of shedding method compared to PCR was 23.8 % and the average sensitivity of crushing method compared to PCR was 46.4 % while the sensitivity of PCR was 100 %. We conclude that PCR is superior to the conventional methods and can detect positive cases that were negative when examined by shedding or crushing methods. This can help in detection of the areas and times of high transmission which in turn will be very beneficial in planning of the exact timing of the proper control strategy.

  14. Molecular approach for detecting early prepatent Schistosoma mansoni infection in Biomphalaria alexandrina snail host.

    PubMed

    Farghaly, Adel; Saleh, Ayman A; Mahdy, Soad; Abd El-Khalik, Dalia; Abd El-Aal, Naglaa F; Abdel-Rahman, Sara A; Salama, Marwa A

    2016-09-01

    The present study aimed to evaluate a polymerase chain reaction (PCR) assay used for detection of Schistosoma mansoni infection in Biomphalaria alexandrina snails in early prepatent period and to compare between it and the ordinary detection methods (shedding and crushing). Biomphalaria alexandrina snails are best known for their role as intermediate hosts of S. mansoni. DNA was extracted from infected snails in addition to non-infected "negative control" (to optimized the efficiency of PCR reaction) and subjected to PCR using primers specific to a partial sequence of S. mansoni fructose-1,6-bus phosphate aldolase (SMALDO). SMALDO gene was detected in the infected laboratory snails with 70, 85, and 100 % positivity at the 1st, 3rd, and 7th day of infection, respectively. In contrast, the ordinary method was not sensitive enough in detection of early prepatent infection even after 7 days of infection which showed only 25 % positivity. By comparing the sensitivity of the three methods, it was found that the average sensitivity of shedding method compared to PCR was 23.8 % and the average sensitivity of crushing method compared to PCR was 46.4 % while the sensitivity of PCR was 100 %. We conclude that PCR is superior to the conventional methods and can detect positive cases that were negative when examined by shedding or crushing methods. This can help in detection of the areas and times of high transmission which in turn will be very beneficial in planning of the exact timing of the proper control strategy. PMID:27605788

  15. Detection of Sarcocystis spp. infection in bobcats (Lynx rufus).

    PubMed

    Verma, S K; Calero-Bernal, R; Lovallo, M J; Sweeny, A R; Grigg, M E; Dubey, J P

    2015-09-15

    The protozoan Sarcocystis neurona is an important cause of severe clinical disease of horses (called equine protozoal myeloencephalitis, EPM), marine mammals, companion animals, and several species of wildlife animals in the Americas. The Virginia opossum (Didelphis virginiana) is its definitive host in the USA and other animals act as intermediate or aberrant hosts. Samples of tongue and heart from 35 bobcats hunted for fur and food from Mississippi State, USA in February, 2014 were used for the present study. Muscles were examined for Sarcocystis infection by microscopic examination of either unfixed muscle squash preparations or pepsin digests, by histopathology of fixed samples, and by molecular methods. Sarcocystis-like bradyzoites were found in digests of 14 hearts and 10 tongues of 35 bobcats. In histological sections, sarcocysts were found in 26 of 35 bobcats; all appeared relatively thin-walled similar to S. felis sarcocysts under light microscope at 1000× magnification. S. neurona-like sarcocysts having thickened villar tips were seen in unstained muscle squash of tongue of two bobcats and PCR-DNA sequencing identified them definitively as S. neurona-like parasites. DNA extracted from bradyzoites obtained from tongue and heart muscle digests was analyzed by PCR-DNA sequencing at the ITS1 locus. Results indicated the presence of S. neurona-like parasite in 26 of 35 samples. ITS1 sequences identical to S. dasypi were identified in 3 bobcats, 2 of which were also co-infected with S. neurona-like parasite. The high prevalence of sarcocysts in bobcat tissues suggested an efficient sylvatic cycle of Sarcocystis spp. in the remote regions of Mississippi State with the bobcat as a relevant intermediate host.

  16. Detection of Sarcocystis spp. infection in bobcats (Lynx rufus).

    PubMed

    Verma, S K; Calero-Bernal, R; Lovallo, M J; Sweeny, A R; Grigg, M E; Dubey, J P

    2015-09-15

    The protozoan Sarcocystis neurona is an important cause of severe clinical disease of horses (called equine protozoal myeloencephalitis, EPM), marine mammals, companion animals, and several species of wildlife animals in the Americas. The Virginia opossum (Didelphis virginiana) is its definitive host in the USA and other animals act as intermediate or aberrant hosts. Samples of tongue and heart from 35 bobcats hunted for fur and food from Mississippi State, USA in February, 2014 were used for the present study. Muscles were examined for Sarcocystis infection by microscopic examination of either unfixed muscle squash preparations or pepsin digests, by histopathology of fixed samples, and by molecular methods. Sarcocystis-like bradyzoites were found in digests of 14 hearts and 10 tongues of 35 bobcats. In histological sections, sarcocysts were found in 26 of 35 bobcats; all appeared relatively thin-walled similar to S. felis sarcocysts under light microscope at 1000× magnification. S. neurona-like sarcocysts having thickened villar tips were seen in unstained muscle squash of tongue of two bobcats and PCR-DNA sequencing identified them definitively as S. neurona-like parasites. DNA extracted from bradyzoites obtained from tongue and heart muscle digests was analyzed by PCR-DNA sequencing at the ITS1 locus. Results indicated the presence of S. neurona-like parasite in 26 of 35 samples. ITS1 sequences identical to S. dasypi were identified in 3 bobcats, 2 of which were also co-infected with S. neurona-like parasite. The high prevalence of sarcocysts in bobcat tissues suggested an efficient sylvatic cycle of Sarcocystis spp. in the remote regions of Mississippi State with the bobcat as a relevant intermediate host. PMID:26138150

  17. Detection of Sarcocystis spp. infection in bobcats (Lynx rufus)

    PubMed Central

    Verma, S. K.; Calero-Bernal, R.; Lovallo, M. J.; Sweeny, A. R.; Grigg, M. E.; Dubey, J. P.

    2015-01-01

    The protozoan Sarcocystis neurona is an important cause of severe clinical disease of horses (called equine protozoal myeloencephalitis, EPM), marine mammals, companion animals, and several species of wildlife animals in the Americas. The Virginia opossum (Didelphis virginiana) is its definitive host in the USA and other animals act as intermediate or aberrant hosts. Samples of tongue and heart from 35 bobcats hunted for fur and food from Mississippi State, USA in February, 2014 were used for the present study. Muscles were examined for Sarcocystis infection by microscopic examination of either unfixed muscle squash preparations or pepsin digests, by histopathology of fixed samples, and by molecular methods. Sarcocystis-like bradyzoites were found in digests of 14 hearts and 10 tongues of 35 bobcats. In histological sections, sarcocysts were found in 26 of 35 bobcats; all appeared relatively thin-walled similar to S. felis sarcocysts under light microscope at 1000x magnification. S. neurona-like sarcocysts having thickened villar tips were seen in unstained muscle squash of tongue of two bobcats and PCR-DNA sequencing identified them definitively as S. neurona-like parasite. DNA extracted from bradyzoites obtained from tongue and heart muscle digests was analyzed by PCR-DNA sequencing at the ITS1 locus. Results indicated the presence of S. neurona-like parasite in 26 of 35 samples. ITS1 sequences identical to S. dayspi were identified in 3 bobcats, 2 of which were also co-infected with S. neurona-like parasite. The high prevalence of sarcocysts in bobcat tissues suggested an efficient sylvatic cycle of Sarcocystis spp. in the remote regions of Mississippi State with the bobcat as a relevant intermediate host. PMID:26138150

  18. Subcutaneously Administered Ultrafine PLGA Nanoparticles Containing Doxycycline Hydrochloride Target Lymphatic Filarial Parasites.

    PubMed

    Singh, Yuvraj; Srinivas, Adepu; Gangwar, Mamta; Meher, Jaya Gopal; Misra-Bhattacharya, Shailja; Chourasia, Manish K

    2016-06-01

    Systemic chemotherapeutic targeting of filarial parasites is unfocused due to their deep seated location in lymphatic vessels. This warrants a prolonged dosing regimen in high doses for an anthelmintic like doxycycline hydrochloride (DOX). In order to provide an alternative, we have constructed ultrafine PLGA nanoparticles of DOX (DPNPs), so as to exploit the peculiarity of lymphatic vasculature underneath the subcutaneous layer of skin, which preferentially allows entry of only 10-100 nm sized particles. DPNPs were constructed using a novel solvent diffusion method aided by probe sonication, which resulted in an average size 95.43 ± 0.8 nm as per DLS, PDI 0.168 ± 0.03, zeta potential -7.38 ± 0.32, entrapment efficiency 75.58 ± 1.94%, and refrigerator stability of 7 days with respect to size in the optimized batch. TEM further substantiated the spherical shape of DPNPs along with their actual nonhydrated size as being well below 100 nm. FTIR analysis of DOX, dummy nanoparticles, and freeze-dried DPNPs revealed that the formulation step did not induce prominent changes in the chemical nature of DOX. The drug release was significantly altered (p < 0.05) with 64.6 ± 1.67% release in 48 h from DPNPs and was dictated by Fickian diffusion. Pharmacokinetic studies in Wistar rats further revealed that DPNPs caused a 16-fold prolongation in attainment of plasma Tmax and a 2-fold extension of elimination half-life (28.569 ± 1.27 h) at a dose of 5 mg/kg when compared to native drug (DOX solution) of the same strength. Contrastingly the trend was reversed in regional lymph nodes where Cmax for DPNPs (820 ± 84 ng/mg) was 4-fold greater, and lymphatic Tmax was attained in one-fourth of what was required for DOX solution. This size based preferential lymphatic targeting resulted in significantly greater in vivo antifilarial activity of DPNPs when compared to DOX solution as gauged by several parameters in Brugia malayi infected Mastomys coucha. Interestingly, the

  19. Modelling environmental factors correlated with podoconiosis: a geospatial study of non-filarial elephantiasis

    PubMed Central

    2014-01-01

    Introduction The precise trigger of podoconiosis — endemic non-filarial elephantiasis of the lower legs — is unknown. Epidemiological and ecological studies have linked the disease with barefoot exposure to red clay soils of volcanic origin. Histopathology investigations have demonstrated that silicon, aluminium, magnesium and iron are present in the lower limb lymph node macrophages of both patients and non-patients living barefoot on these clays. We studied the spatial variation (variations across an area) in podoconiosis prevalence and the associated environmental factors with a goal to better understanding the pathogenesis of podoconiosis. Methods Fieldwork was conducted from June 2011 to February 2013 in 12 kebeles (administrative units) in northern Ethiopia. Geo-located prevalence data and soil samples were collected and analysed along with secondary geological, topographic, meteorological and elevation data. Soil data were analysed for chemical composition, mineralogy and particle size, and were interpolated to provide spatially continuous information. Exploratory, spatial, univariate and multivariate regression analyses of podoconiosis prevalence were conducted in relation to primary (soil) and secondary (elevation, precipitation, and geology) covariates. Results Podoconiosis distribution showed spatial correlation with variation in elevation and precipitation. Exploratory analysis identified that phyllosilicate minerals, particularly clay (smectite and kaolinite) and mica groups, quartz (crystalline silica), iron oxide, and zirconium were associated with podoconiosis prevalence. The final multivariate model showed that the quantities of smectite (RR = 2.76, 95% CI: 1.35, 5.73; p = 0.007), quartz (RR = 1.16, 95% CI: 1.06, 1.26; p = 0.001) and mica (RR = 1.09, 95% CI: 1.05, 1.13; p < 0.001) in the soil had positive associations with podoconiosis prevalence. Conclusions More quantities of smectite, mica and quartz within the soil

  20. Field evaluation of two diagnostic antigen tests for Wuchereria bancrofti infection among Indian expatriates in Saudi Arabia.

    PubMed

    Omar, M S; Sheikha, A K; Al-Amari, O M; Abdalla, S E; Musa, R A

    2000-06-01

    Two commercially available diagnostic tools (Tro-Bio ELISA and ICT card test) were used to detect circulating filarial antigen of Wuchereria bancrofti infections among Indian expatriate workers in Saudi Arabia. Daytime serum samples collected from 302 individuals (210 men and 92 women) were tested. Night blood surveys for microfilaraemia were restricted to those individuals who became positive for the trop-Bio assay test. The overall prevalence of filarial antigeaemia was 10.6% (32 individuals). Of these 32 antigen positive cases, microfilariae were found in 10 men (31.3%), with a mean microfilarial count of 105 mff/ml. No positive antigen results were found in control sera from 200 native healthy Saudis or from patients with helminthic infections (schistosomiasis, echinicoccosis, hookworm, ascariasis and trichuriasis). All 32 positive sera with the Trop-Bio ELISA showed a positive ICT card reaction (specificity and sensitivity 100%). It is concluded that, in Saudi Arabia and other Gulf states, where a continuous flow of south- and southeastern workers coming from areas endemic for bancroftian filariasis, the ICT card test may be useful in monitoring the potential risk of introducing bacncroftian filariasis to the host countries.

  1. A Novel Strategy for Live Detection of Viral Infection in Drosophila melanogaster

    PubMed Central

    Ekström, Jens-Ola; Hultmark, Dan

    2016-01-01

    We have created a transgenic reporter for virus infection, and used it to study Nora virus infection in Drosophila melanogaster. The transgenic construct, Munin, expresses the yeast transcription factor Gal4, tethered to a transmembrane anchor via a linker that can be cleaved by a viral protease. In infected cells, liberated Gal4 will then transcribe any gene that is linked to a promoter with a UAS motif, the target for Gal4 transcription. For instance, infected cells will glow red in the offspring of a cross between the Munin stock and flies with a UAS-RFPnls transgene (expressing a red fluorescent protein). In such flies we show that after natural infection, via the faecal-oral route, 5–15% of the midgut cells are infected, but there is little if any infection elsewhere. By contrast, we can detect infection in many other tissues after injection of virus into the body cavity. The same principle could be applied for other viruses and it could also be used to express or suppress any gene of interest in infected cells. PMID:27189868

  2. Detection of Plasmodium falciparum-infected red blood cells by optical stretching

    NASA Astrophysics Data System (ADS)

    Mauritz, Jakob M. A.; Tiffert, Teresa; Seear, Rachel; Lautenschläger, Franziska; Esposito, Alessandro; Lew, Virgilio L.; Guck, Jochen; Kaminski, Clemens F.

    2010-05-01

    We present the application of a microfluidic optical cell stretcher to measure the elasticity of malaria-infected red blood cells. The measurements confirm an increase in host cell rigidity during the maturation of the parasite Plasmodium falciparum. The device combines the selectivity and sensitivity of single-cell elasticity measurements with a throughput that is higher than conventional single-cell techniques. The method has potential to detect early stages of infection with excellent sensitivity and high speed.

  3. Detection of musculoskeletal infection with the indium-III leukocyte scan

    SciTech Connect

    Prchal, C.L.; Kahen, H.L.; Blend, M.J.; Barmada, R.

    1987-09-01

    Indium-111-labeled leukocyte scans were performed on 39 patients with suspected musculoskeletal infections to assess the usefulness of this study in detecting bone and joint infections. Results of these scans, as well as results of technetium-99m bone scans, were correlated with the patients' final diagnoses. The indium scan had an overall sensitivity of 77%, a specificity of 69%, and an accuracy of 72%. In 10 patients with a duration of symptoms of six weeks or less, the sensitivity was 100% and the specificity was 75%. In 29 patients with symptoms of greater than six weeks, the sensitivity and specificity were lower at 50% and 71% respectively. Technetium-99m bone scans were performed on 23 patients; sensitivity for infection was 100% while specificity was 60%. Our results suggest that the indium-111 leukocyte scan is a useful adjunct in the diagnosis of acute musculoskeletal infections, but may be inconclusive in chronic infections.

  4. Detection of Persistent West Nile Virus RNA in Experimentally and Naturally Infected Avian Hosts

    PubMed Central

    Wheeler, Sarah S.; Langevin, Stanley A.; Brault, Aaron C.; Woods, Leslie; Carroll, Brian D.; Reisen, William K.

    2012-01-01

    To determine whether West Nile virus (WNV) persistent infection in avian hosts may potentially serve as an overwintering mechanism, House Sparrows and House Finches, experimentally and naturally infected with several strains of WNV, and two naturally infected Western Scrub-Jays were held in mosquito-proof outdoor aviaries from 2007–March 2008. Overall, 94% (n = 36) of House Sparrows, 100% (n = 14) of House Finches and 2 Western Scrub-Jays remained WNV antibody positive. When combined by species, 37% of the House Sparrows, 50% of the House Finches, and 2 Western Scrub-Jays were WNV RNA positive at necropsy, up to 36 weeks post-infection. Infectious WNV was not detected. Our study supports the hypothesis that some avian hosts support the long-term persistence of WNV RNA, but it remains unresolved whether these infections relapse to restart an avian-arthropod transmission cycle and thereby serve as an overwintering mechanism for WNV. PMID:22826479

  5. A susceptible-infected model of early detection of respiratory infection outbreaks on a background of influenza.

    PubMed

    Mohtashemi, Mojdeh; Szolovits, Peter; Dunyak, James; Mandl, Kenneth D

    2006-08-21

    The threat of biological warfare and the emergence of new infectious agents spreading at a global scale have highlighted the need for major enhancements to the public health infrastructure. Early detection of epidemics of infectious diseases requires both real-time data and real-time interpretation of data. Despite moderate advancements in data acquisition, the state of the practice for real-time analysis of data remains inadequate. We present a nonlinear mathematical framework for modeling the transient dynamics of influenza, applied to historical data sets of patients with influenza-like illness. We estimate the vital time-varying epidemiological parameters of infections from historical data, representing normal epidemiological trends. We then introduce simulated outbreaks of different shapes and magnitudes into the historical data, and estimate the parameters representing the infection rates of anomalous deviations from normal trends. Finally, a dynamic threshold-based detection algorithm is devised to assess the timeliness and sensitivity of detecting the irregularities in the data, under a fixed low false-positive rate. We find that the detection algorithm can identify such designated abnormalities in the data with high sensitivity with specificity held at 97%, but more importantly, early during an outbreak. The proposed methodology can be applied to a broad range of influenza-like infectious diseases, whether naturally occurring or a result of bioterrorism, and thus can be an integral component of a real-time surveillance system. PMID:16556450

  6. The detection of the meq gene in chicken infected with Marek's disease virus serotype 1.

    PubMed

    Chang, Kyung-Soo; Lee, Sung-Il; Ohashi, Kazuhiko; Ibrahim, Ahmed; Onuma, Misao

    2002-05-01

    In the genome of strains of very virulent Marek's disease virus serotype 1(vvMDV1), such as Md5 and RB1B, the meq open reading frame (ORF) encoding a 339-amino-acid bZIP protein, is present, while a slightly longer meq ORF, termed as L-meq, in which a 180-bp sequence is inserted into the meq ORF is found in other strains of MDV1, such as CV1988/R6 and attenuated JM. When chickens were infected with vvMDV1 strains and the meq gene was amplified by nested polymerase chain reaction (PCR), the meq gene was detected throughout the experimental period for 7 weeks post inoculation (pi). However, the L-meq gene was also detected at 3 to 5 weeks and 3 to 4 weeks pi. in Md5-infected and RB1B-infected chickens, respectively. In the case of chickens infected with an attenuated MDV1, the JM strain, the L-meq gene was detected at 2 to 7 weeks pi., and the meq gene was also detected at 2 to 6 weeks pi. Both L-meq and meq genes were detected in chickens infected with an attenuated nononcogenic vaccine strain of MDV1 (CVI988/R6), throughout the experimental period. Though quantitative PCR was not performed, a larger amount of the PCR products corresponding to the L-meq than the meq gene was amplified from chickens infected with JM or CVI988/R6. These results suggest that a dynamic population shift between the MDV subpopulations displaying meq and L-meq genes occurs in chickens during the course of MDV infection. Since the MDV subpopulation that displays the L-meq gene only displays it during the latent phase, the L-meq and its gene product, if any, might contribute to the maintenance of the MDV latency.

  7. Nasopharyngeal Infection of Mice with Streptococcus pyogenes and In Vivo Detection of Superantigen Activity.

    PubMed

    Zeppa, Joseph J; Wakabayashi, Adrienne T; Kasper, Katherine J; Xu, Stacey X; Haeryfar, S M Mansour; McCormick, John K

    2016-01-01

    Streptococcus pyogenes is a globally prominent human-specific pathogen that is responsible for an enormous burden of infectious disease. Despite intensive experimental efforts to understand the molecular correlates that contribute to invasive infections, there has been less focus on S. pyogenes carriage and local infection of the nasopharynx. This chapter describes an acute nasopharyngeal infection model in mice that is utilized in our laboratory to study the role of superantigen toxins in the biology of S. pyogenes. We also describe a method to detect superantigen-specific T cell activation in vivo.

  8. Detection of mouse hepatitis virus infection by assay of anti-liver autoantibodies.

    PubMed

    Mathieu, Patricia A; Gómez, Karina A; Coutelier, Jean-Paul; Retegui, Lilia A

    2002-12-01

    The observation that mice infected with mouse hepatitis virus (MHV) develop autoantibodies directed mainly to liver fumarylacetoacetate hydrolase (FAH) enabled the development of an ELISA applicable to the detection of MHV-infection. The method, based on the titration of antibodies to semipurified FAH from rat liver, is easy, economical, and does not require the isolation of viral proteins from large MHV stocks. Furthermore, since sera from mice immunized with a purified fraction of the rat liver enzyme do react with its homologous protein, this antiserum can be used as a positive control avoiding the manipulation of samples from MHV-infected animals.

  9. Human papillomavirus detection in women with and without human immunodeficiency virus infection in Colombia

    PubMed Central

    2014-01-01

    Background HIV infection leads to a decreasing immune response, thereby facilitating the appearance of other infections, one of the most important ones being HPV. However, studies are needed for determining associations between immunodeficiency caused by HIV and/or the presence of HPV during the course of cervical lesions and their degree of malignancy. This study describes the cytological findings revealed by the Papanicolaou test, laboratory characteristics and HPV molecular profile in women with and without HIV infection. Methods A total of 216 HIV-positive and 1,159 HIV-negative women were invited to participate in the study; PCR was used for the molecular detection of HPV in cervical samples. Statistical analysis (such as percentages, Chi-square test and Fisher’s exact test when applicable) determined human papillomavirus (HPV) infection frequency (single and multiple) and the distribution of six types of high-risk-HPV in women with and without HIV infection. Likewise, a logistic regression model was run to evaluate the relationship between HIV-HPV infection and different risk factors. Results An association was found between the frequency of HPV infection and infection involving 2 or more HPV types (also known as multiple HPV infection) in HIV-positive women (69.0% and 54.2%, respectively); such frequency was greater than that found in HIV-negative women (44.3% and 22.7%, respectively). Statistically significant differences were observed between both groups (p = 0.001) regarding HPV presence (both in infection and multiple HPV infection). HPV-16 was the most prevalent type in the population being studied (p = 0.001); other viral types had variable distribution in both groups (HIV-positive and HIV-negative). HPV detection was associated with <500 cell/mm3 CD4-count (p = 0.004) and higher HIV-viral-load (p = 0.001). HPV-DNA detection, <200 cell/mm3 CD4-count (p = 0.001), and higher HIV-viral-load (p = 0.001) were associated with

  10. Detection of thoracic infections by nuclear medicine techniques in the acquired immunodeficiency syndrome

    SciTech Connect

    Kramer, E.L.; Sanger, J.J. )

    1989-11-01

    The challenge of the acquired immunodeficiency syndrome (AIDS) for nuclear medicine has been the early detection of related intrathoracic opportunistic infections, inflammatory conditions, and neoplasms. Gallium-67 citrate scanning has proved a sensitive test not only for Pneumocystis carinii pneumonia but for many of the other opportunistic infections and malignancies, including mycobacterial infections and lymphoma. Patterns and intensity of gallium uptake may suggest more specific diagnoses. Indium-111-labeled white blood cells may also be a valuable diagnostic tool in the AIDS patient.41 references.

  11. Early detection of dengue infections using cluster sampling around index cases.

    PubMed

    Beckett, Charmagne G; Kosasih, Herman; Faisal, Indra; Nurhayati; Tan, Ratna; Widjaja, Susana; Listiyaningsih, Erlin; Ma'roef, Chairin; Wuryadi, Suharyono; Bangs, Michael J; Samsi, Tatang K; Yuwono, Djoko; Hayes, Curtis G; Porter, Kevin R

    2005-06-01

    A two-year study using a cluster investigation method was conducted in West Jakarta, Indonesia to demonstrate the detection of dengue cases prior to onset of clinical illness. The clusters consisted of family members and neighbors of 53 hospitalized dengue index cases. Among 785 adult and child volunteers enrolled, 17 (2.2%) post-enrollment dengue (PED) infections were identified. Eight PED cases were asymptomatic and nine were symptomatic. Symptomatic cases included eight with dengue fever and one with dengue hemorrhagic fever (DHF) (grade II). Among the eight asymptomatic PED cases, viremia was detected in two. Eleven volunteers had acute dengue infections at the time of enrollment. Four of the 11 developed DHF, resulting in a total of five DHF cases detected during the investigation. This study design can serve as a benchmark for future investigations that seek to define early immunologic events following dengue infections that contribute to the development of DHF.

  12. Wolbachia Infections in the Cimicidae: Museum Specimens as an Untapped Resource for Endosymbiont Surveys

    PubMed Central

    Sakamoto, Joyce M.; Feinstein, Julie; Rasgon, Jason L.

    2006-01-01

    Wolbachia spp. are obligate maternally inherited endosymbiotic bacteria that infect diverse arthropods and filarial nematodes. Previous microscopic and molecular studies have identified Wolbachia in several bed bug species (Cimicidae), but little is known about how widespread Wolbachia infections are among the Cimicidae. Because cimicids of non-medical importance are not commonly collected, we hypothesized that preserved museum specimens could be assayed for Wolbachia infections. For the screening of museum specimens, we designed a set of primers that specifically amplify small diagnostic fragments (130 to 240 bp) of the Wolbachia 16S rRNA gene. Using these and other previously published primers, we screened 39 cimicid species (spanning 16 genera and all 6 recognized subfamilies) and 2 species of the sister family Polyctenidae for Wolbachia infections using museum and wild-caught material. Amplified fragments were sequenced to confirm that our primers were amplifying Wolbachia DNA. We identified 10 infections, 8 of which were previously undescribed. Infections in the F supergroup were common in the subfamily Cimicinae, while infections in the A supergroup were identified in the subfamilies Afrocimicinae and Haematosiphoninae. Even though specimens were degraded, we detected infections in over 23% of cimicid species. Our results indicate that Wolbachia infections may be common among cimicids and that archived museum material is a useful untapped resource for invertebrate endosymbiont surveys. The new screening primers listed in this report will be useful for other researchers conducting Wolbachia surveys with specimens with less-than-optimum DNA quality. PMID:16672453

  13. Wolbachia infections in the Cimicidae: museum specimens as an untapped resource for endosymbiont surveys.

    PubMed

    Sakamoto, Joyce M; Feinstein, Julie; Rasgon, Jason L

    2006-05-01

    Wolbachia spp. are obligate maternally inherited endosymbiotic bacteria that infect diverse arthropods and filarial nematodes. Previous microscopic and molecular studies have identified Wolbachia in several bed bug species (Cimicidae), but little is known about how widespread Wolbachia infections are among the Cimicidae. Because cimicids of non-medical importance are not commonly collected, we hypothesized that preserved museum specimens could be assayed for Wolbachia infections. For the screening of museum specimens, we designed a set of primers that specifically amplify small diagnostic fragments (130 to 240 bp) of the Wolbachia 16S rRNA gene. Using these and other previously published primers, we screened 39 cimicid species (spanning 16 genera and all 6 recognized subfamilies) and 2 species of the sister family Polyctenidae for Wolbachia infections using museum and wild-caught material. Amplified fragments were sequenced to confirm that our primers were amplifying Wolbachia DNA. We identified 10 infections, 8 of which were previously undescribed. Infections in the F supergroup were common in the subfamily Cimicinae, while infections in the A supergroup were identified in the subfamilies Afrocimicinae and Haematosiphoninae. Even though specimens were degraded, we detected infections in over 23% of cimicid species. Our results indicate that Wolbachia infections may be common among cimicids and that archived museum material is a useful untapped resource for invertebrate endosymbiont surveys. The new screening primers listed in this report will be useful for other researchers conducting Wolbachia surveys with specimens with less-than-optimum DNA quality.

  14. Enhancement of immunohistochemical detection of Salmonella in tissues of experimentally infected pigs.

    PubMed

    Rieger, J; Janczyk, P; Hünigen, H; Plendl, J

    2015-07-09

    Salmonella Typhimurium is one of the main pathogens compromising porcine and human health as well as food safety, because it is a prevailing source of foodborne infections due to contaminated pork. A prominent problem in the management of this bacteriosis is the number of subclinically infected carrier pigs. As very little is known concerning the mechanisms allowing Salmonella to persist in pigs, the objective of this study was to develop an immunohistochemical approach for the detection of salmonellae in tissue of pigs experimentally infected with Salmonella Typhimurium. Samples were obtained from a challenge trial in which piglets of the German Landrace were intragastrically infected with Salmonella enterica serovar Typhimurium DT104 (1.4-2.1x1010 CFU). Piglets were sacrificed on days 2 and 28 post infection. Tissue samples of jejunum, ileum, colon, ileocecal mesenteric lymph nodes (Lnn. ileocolici), and tonsils (Tonsilla veli palatini) were fixed in Zamboni's fixative and paraffin-embedded. Different immunohistochemical staining protocols were evaluated. Salmonella was detected in varying amounts in the tissues. Brown iron-containing pigments in the lymph nodes interfered with the identification of Salmonella if DAB was used as a staining reagent. Detergents like Triton X-100 or Saponin enhanced the sensitivity. It seems advisable not to use a detection system with brown staining for bacteria in an experimental setup involving intestinal damage including haemorrhage. The use of detergents appears to result in a higher sensitivity in the immunohistochemical detection of salmonellae.

  15. Coincidental detection of genomes of porcine parvoviruses and porcine circovirus type 2 infecting pigs in Japan.

    PubMed

    Saekhow, Prayuth; Kishizuka, Shingo; Sano, Natsuha; Mitsui, Hiroko; Akasaki, Hajime; Mawatari, Takahiro; Ikeda, Hidetoshi

    2016-01-01

    The infection status of 15 viruses in 120 pigs aged about 6 months was investigated based on tonsil specimens collected from a slaughterhouse. Only 5 species of porcine parvoviruses and porcine circovirus type 2 (PCV2) were detected at high frequencies; 67% for porcine parvovirus (PPV) (PPV-Kr or -NADL2 as the new abbreviation), 58% for PPV2 (CnP-PARV4), 39% for PPV3 (P-PARV4), 33% for PPV4 (PPV4), 55% for PBo-likeV (PBoV7) and 80% for PCV2. A phylogenetic analysis of PPV3 suggested that Japanese PPV3s showed a slight variation, and possibly, there were farms harboring homogeneous or heterogeneous PPV3s. Statistical analyses indicated that the detection of PCV2 was significantly coincidental with each detection of PPV, PPV2 and PPV3, and PPV and PPV4 were also coincidentally detected. The concurrent infection with PCV2 and porcine parvoviruses in the subclinically infected pigs may resemble the infection status of pigs with the clinical manifestations of porcine circovirus associated disease which occurs in 3-5 months old pigs and is thought to be primarily caused by the PCV2 infection. PMID:26166811

  16. Enhancement of immunohistochemical detection of Salmonella in tissues of experimentally infected pigs.

    PubMed

    Rieger, J; Janczyk, P; Hünigen, H; Plendl, J

    2015-01-01

    Salmonella Typhimurium is one of the main pathogens compromising porcine and human health as well as food safety, because it is a prevailing source of foodborne infections due to contaminated pork. A prominent problem in the management of this bacteriosis is the number of subclinically infected carrier pigs. As very little is known concerning the mechanisms allowing Salmonella to persist in pigs, the objective of this study was to develop an immunohistochemical approach for the detection of salmonellae in tissue of pigs experimentally infected with Salmonella Typhimurium. Samples were obtained from a challenge trial in which piglets of the German Landrace were intragastrically infected with Salmonella enterica serovar Typhimurium DT104 (1.4-2.1x1010 CFU). Piglets were sacrificed on days 2 and 28 post infection. Tissue samples of jejunum, ileum, colon, ileocecal mesenteric lymph nodes (Lnn. ileocolici), and tonsils (Tonsilla veli palatini) were fixed in Zamboni's fixative and paraffin-embedded. Different immunohistochemical staining protocols were evaluated. Salmonella was detected in varying amounts in the tissues. Brown iron-containing pigments in the lymph nodes interfered with the identification of Salmonella if DAB was used as a staining reagent. Detergents like Triton X-100 or Saponin enhanced the sensitivity. It seems advisable not to use a detection system with brown staining for bacteria in an experimental setup involving intestinal damage including haemorrhage. The use of detergents appears to result in a higher sensitivity in the immunohistochemical detection of salmonellae. PMID:26428884

  17. Enhancement of Immunohistochemical Detection of Salmonella in Tissues of Experimentally Infected Pigs

    PubMed Central

    Rieger, J.; Janczyk, P.; Hünigen, H.; Plendl, J.

    2015-01-01

    Salmonella Typhimurium is one of the main pathogens compromising porcine and human health as well as food safety, because it is a prevailing source of foodborne infections due to contaminated pork. A prominent problem in the management of this bacteriosis is the number of subclinically infected carrier pigs. As very little is known concerning the mechanisms allowing Salmonella to persist in pigs, the objective of this study was to develop an immunohistochemical approach for the detection of salmonellae in tissue of pigs experimentally infected with Salmonella Typhimurium. Samples were obtained from a challenge trial in which piglets of the German Landrace were intragastrically infected with Salmonella enterica serovar Typhimurium DT104 (1.4-2.1×1010 CFU). Piglets were sacrificed on days 2 and 28 post infection. Tissue samples of jejunum, ileum, colon, ileocecal mesenteric lymph nodes (Lnn. ileocolici), and tonsils (Tonsilla veli palatini) were fixed in Zamboni’s fixative and paraffin-embedded. Different immunohistochemical staining protocols were evaluated. Salmonella was detected in varying amounts in the tissues. Brown iron-containing pigments in the lymph nodes interfered with the identification of Salmonella if DAB was used as a staining reagent. Detergents like Triton X-100 or Saponin enhanced the sensitivity. It seems advisable not to use a detection system with brown staining for bacteria in an experimental setup involving intestinal damage including haemorrhage. The use of detergents appears to result in a higher sensitivity in the immunohistochemical detection of salmonellae. PMID:26428884

  18. A new filarial nematode (Onchocercidae) from warthogs (Phacochoerus aethiopicus) of the Kruger National Park.

    PubMed

    Palmieri, J R; Pletcher, J M; De Vos, V; Boomker, J

    1985-09-01

    Fifty-five warthogs [Phacochoerus aethiopicus (Suidae: Artiodactyla)] from the Kruger National Park, Republic of South Africa, were examined for parasites. Adult filarial nematodes were found in lymphatic vessels adjacent to peripheral and visceral lymph nodes, and microfilariae were found in lymph nodes and circulating blood. Both the adult parasite and the microfilaria are described. Specific identification is pending confirmation and recovery of intact adult specimens and microfilariae identical to those described herein. PMID:4067247

  19. Detection and monitoring of human bocavirus 1 infection by a new rapid antigen test

    PubMed Central

    Bruning, A.H.L.; Susi, P.; Toivola, H.; Christensen, A.; Söderlund-Venermo, M.; Hedman, K.; Aatola, H.; Zvirbliene, A.; Koskinen, J.O.

    2016-01-01

    Clinically relevant diagnosis of human bocavirus 1 (HBoV1) is challenging, as the virus is frequently detected in asymptomatic patients, and cofindings with other respiratory viruses are common. The clinical value of current diagnostic methods, such as PCR, is therefore low, and alternative diagnostic strategies are needed. We describe for the first time the use of an antigen detection assay for the rapid identification of HBoV1 in a paediatric patient with respiratory tract infection symptoms. We estimate the duration of active HBoV1 infection to be 6 days. PMID:27014463

  20. Detection and monitoring of human bocavirus 1 infection by a new rapid antigen test.

    PubMed

    Bruning, A H L; Susi, P; Toivola, H; Christensen, A; Söderlund-Venermo, M; Hedman, K; Aatola, H; Zvirbliene, A; Koskinen, J O

    2016-05-01

    Clinically relevant diagnosis of human bocavirus 1 (HBoV1) is challenging, as the virus is frequently detected in asymptomatic patients, and cofindings with other respiratory viruses are common. The clinical value of current diagnostic methods, such as PCR, is therefore low, and alternative diagnostic strategies are needed. We describe for the first time the use of an antigen detection assay for the rapid identification of HBoV1 in a paediatric patient with respiratory tract infection symptoms. We estimate the duration of active HBoV1 infection to be 6 days. PMID:27014463

  1. [Development of PCR methods for detection of EAV infection].

    PubMed

    Brunner; Santschi; Gerber; Burger; Zanoni

    2014-11-01

    The goal of this work was the development of suitable (real-time) RT-PCR techniques for fast and sensitive diagnosis of EAV and for molecular-epidemiological characterisation of viral strains, as an alternative to virus isolation. To this purpose two conventional RT-PCR methods and one real-time RT-PCR were adapted to detect the broadest possible spectrum of viral strains. Several dilutions with Bucyrus strain showed a 100-fold higher sensitivity of real-time RT-PCR and heminested RT-PCR compared to simple RT-PCR. Making use of 11 cell culture supernatants of different EAV isolates and 7 semen samples of positive stallions, the suitability of the techniques could be shown. Phylogenetic analysis of sequences of the newly analysed samples compared with known sequences indicated that more EAV-lineages exist than presently described.

  2. Clinical and Pathological Aspects of Filarial Lymphedema and Its Management

    PubMed Central

    2008-01-01

    Lymphatic filariasis, transmitted by mosquitoes is the commonest cause of lymphedema in endemic countries. Among 120 million infected people in 83 countries, up to 16 million have lymphedema. Microfilariae ingested by mosquitoes grow into infective larvae. These larvae entering humans after infected mosquito bites grow in the lymphatics to adult worms that cause damage to lymphatics resulting in dilatation of lymph vessels. This earliest pathology is demonstrated in adults as well as in children, by ultrasonography, lymphoscintigraphy and histopathology studies. Once established, this damage was thought to be irreversible. This lymphatic damage predisposes to bacterial infection that causes recurrent acute attacks of dermato-lymphangio-adenitis in the affected limbs. Bacteria, mainly streptococci gain entry into the lymphatics through 'entry lesions' in skin, like interdigital fungal infections, injuries, eczema or similar causes that disrupt integrity of skin. Attacks of dermato-lymphangio-adenitis aggravates lymphatic damage causing lymphedema, which gets worse with repeated acute attacks. Elephantiasis is a late manifestation of lymphatic filariasis, which apart from limbs may involve genitalia or breasts. Lymphedema management includes use of antifilarial drugs in early stages, treatment and prevention of acute attacks through 'limb-hygiene', antibiotics and antifungals where indicated, and physical measures to reduce the swelling. In selected cases surgery is helpful. PMID:18830049

  3. HTLV-1 viral RNA is detected rarely in plasma of HTLV-1 infected subjects.

    PubMed

    Demontis, Maria Antonietta; Sadiq, Maaz Tahir; Golz, Simon; Taylor, Graham P

    2015-12-01

    Plasma of patients infected with HTLV-1 is considered non-infectious but detection of HTLV-1 genomic RNA in plasma has been recently reported. The aim of this project was to detect and quantify HTLV-1 RNA in plasma and assess its potential value in diagnosis and prognosis. RNA from 1 ml of plasma from 65 subjects infected with HTLV-1 (27 asymptomatic carriers [AC]), 17 patients with HTLV-1-associated myelopathy (HAM/TSP), 14 with adult T-cell leukemia/lymphoma (ATLL), two co-infected with HIV, and five with other HTLV-1-associated disease, was extracted and reverse transcribed. HTLV-1 specific nested PCR was performed using primers to amplify the conserved Tax region. All samples were run in quadruplicate, nested PCR products were detected by gel electrophoresis. HTLV-1 RNA was detected in plasma from 18 (28%) patients, always at a very low copy number (3-13 copies viral cDNA per milliliter of plasma). Mean values of HTLV-1 proviral load did not differ between patients in whom HTLV-1 RNA was detected and patients in whom it was not possible to detect HTLV-1 RNA in plasma. HTLV-1 genomic RNA can be detected in the plasma of a minority of patients but not at a level or frequency to be useful clinically or diagnostically. Lack of transmission of HTLV-1 by plasma is due to the rare presence of HTLV-1 virions, regardless of any other factor.

  4. From synthetic DNA to PCR product: detection of fungal infections using SERS.

    PubMed

    Mabbott, Samuel; Thompson, David; Sirimuthu, Narayana; McNay, Graeme; Faulds, Karen; Graham, Duncan

    2016-06-23

    We report the use of silver hydroxylamine nanoparticles functionalised with single stranded monothiolated DNA for the detection of fungal infections. The four different species of fungi that were targeted were Candida albicans, Candida glabrata, Candida krusei and Aspergillus fumigatus. Rational design of synthetic targets and probes was carried out by carefully analysing the 2-D folding of the DNA and then by global alignment of the sequences to ensure specificity. The effects of varying the concentrations of the DNA and dye surrounding the nanoparticles on the resultant surface enhanced Raman scattering (SERS) signal were also investigated to ensure compatibility of the probes in a multiplexed environment. Using principal components analysis (PCA) it was possible to detect the individual presence of each target and group them accordingly. The move to detect the C. krusei single stranded PCR product (ssPCR) was significant to demonstrate that the methodology could be employed for the detection and diagnosis of invasive fungal infections (IFDs) within a clinical setting. Initially the PCR product was subjected to an alkali shock method in order to separate the strands ready for detection using the nanoparticle probes system. This time 18 base probes were employed to enhance hybridisation efficiency and dextran sulfate was found to have a vital role in ensuring that detection of the C. krusei target was achieved. This demonstrated the use of DNA functionalised silver nanoparticle for the detection of clinically relevant DNA relating to a specific fungal infection and offers significant promise for future diagnostic applications.

  5. From synthetic DNA to PCR product: detection of fungal infections using SERS.

    PubMed

    Mabbott, Samuel; Thompson, David; Sirimuthu, Narayana; McNay, Graeme; Faulds, Karen; Graham, Duncan

    2016-06-23

    We report the use of silver hydroxylamine nanoparticles functionalised with single stranded monothiolated DNA for the detection of fungal infections. The four different species of fungi that were targeted were Candida albicans, Candida glabrata, Candida krusei and Aspergillus fumigatus. Rational design of synthetic targets and probes was carried out by carefully analysing the 2-D folding of the DNA and then by global alignment of the sequences to ensure specificity. The effects of varying the concentrations of the DNA and dye surrounding the nanoparticles on the resultant surface enhanced Raman scattering (SERS) signal were also investigated to ensure compatibility of the probes in a multiplexed environment. Using principal components analysis (PCA) it was possible to detect the individual presence of each target and group them accordingly. The move to detect the C. krusei single stranded PCR product (ssPCR) was significant to demonstrate that the methodology could be employed for the detection and diagnosis of invasive fungal infections (IFDs) within a clinical setting. Initially the PCR product was subjected to an alkali shock method in order to separate the strands ready for detection using the nanoparticle probes system. This time 18 base probes were employed to enhance hybridisation efficiency and dextran sulfate was found to have a vital role in ensuring that detection of the C. krusei target was achieved. This demonstrated the use of DNA functionalised silver nanoparticle for the detection of clinically relevant DNA relating to a specific fungal infection and offers significant promise for future diagnostic applications. PMID:27034997

  6. Reliable Detection of Respiratory Syncytial Virus Infection in Children for Adequate Hospital Infection Control Management

    PubMed Central

    Abels, Susanne; Nadal, David; Stroehle, Angelika; Bossart, Walter

    2001-01-01

    By using a rapid test for respiratory syncytial virus (RSV) detection (Abbott TestPack RSV), a number of patients were observed, showing repeatedly positive results over a period of up to 10 weeks. A prospective study was initiated to compare the rapid test with an antigen capture enzyme immunoassay (EIA) and a nested reverse transcriptase PCR (RT-PCR) protocol for detection of RSV serotypes A and B. Only respiratory samples from children exhibiting the prolonged presence of RSV (≥5 days) as determined by the rapid test were considered. A total of 134 specimens from 24 children was investigated by antigen capture EIA and nested RT-PCR. Using RT-PCR as the reference method, we determined the RSV rapid test to have a specificity of 63% and a sensitivity of 66% and the antigen capture EIA to have a specificity of 96% and a sensitivity of 69% for acute-phase samples and the homologous virus serotype A. In 7 (29%) of 24 patients, the positive results of the RSV rapid test could not be confirmed by either nested RT-PCR or antigen capture EIA. In these seven patients a variety of other respiratory viruses were detected. For general screening the RSV rapid test was found to be a reasonable tool to get quick results. However, its lack of specificity in some patients requires confirmation by additional tests to rule out false-positive results and/or detection of other respiratory viruses. PMID:11526141

  7. Porous silicon photonic crystals for detection of infections

    NASA Astrophysics Data System (ADS)

    Gupta, B.; Guan, B.; Reece, P. J.; Gooding, J. J.

    2012-10-01

    In this paper we demonstrate the possibility of modifying porous silicon (PSi) particles with surface chemistry and immobilizing a biopolymer, gelatin for the detection of protease enzymes in solution. A rugate filter, a one-dimensional photonic crystal, is fabricated that exhibits a high-reflectivity optical resonance that is sensitive to small changes in the refractive index. To immobilize gelatin in the pores of the particles, the hydrogen-terminated silicon surface was first modified with an alkyne, 1,8-nonadiyne via hydrosilylation to protect the silicon surfaces from oxidation. This modification allows for further functionality to be added such as the coupling of gelatin. Exposure of the gelatin modified particles to the protease subtilisin in solution causes a change in the refractive index, resulting in a shift of the resonance to shorter wavelengths, indicating cleavage of organic material within the pores. The ability to monitor the spectroscopic properties of microparticles, and shifts in the optical signature due to changes in the refractive index of the material within the pore space, is demonstrated.

  8. [Systematics, differentiation, and detection of bacterial infections-- the family Mycobacteriaceae].

    PubMed

    Böttger, E C

    1991-10-01

    Comparative 16S rRNA sequencing allows to infer natural relationships among bacteria, to characterize and identify microorganisms at a molecular level and to develop DNA probes specific at any desired taxonomic level (e.g. family, genus, species). Probes targeted at ribosomal RNA are suitable for in situ hybridization of whole, intact bacterial cells as well as in polymerase-chain-reaction techniques for sensitive detection and identification of bacteria. Comparative 16S rRNA sequencing provided the basis for a systematic phylogenetic analysis of the genus Mycobacterium. Certain growth characteristics, i.e. thermotolerance and growth rate correspond to natural relationships among the mycobacteria. However, the phylogenetic relatedness within the slow-growing species did not reflect the Runyon classification of photochromogenic, scotochromogenic and nonphotochromogenic mycobacteria. The use of oligonucleotides targeted at highly or semi-conserved regions within the 16S rRNA molecule allows a universal procedure for amplification and rapid sequence determination of 16S rDNA-gene fragments from any virtually bacterial organism. This method of amplification of 16S rDNA-gene fragments was used to identify a novel, uncultured pathogen and opens new perspectives for other infectious diseases of unknown cause.

  9. Detection of elephant endotheliotropic herpesvirus infection among healthy Asian elephants (Elephas maximus) in South India.

    PubMed

    Stanton, Jeffrey J; Nofs, Sally A; Zachariah, Arun; Kalaivannan, N; Ling, Paul D

    2014-04-01

    Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in Asian (Elephas maximus) and African (Loxodonta africana) elephants. Of the seven known EEHV species, EEHV1 is recognized as the most common cause of hemorrhagic disease among Asian elephants in human care worldwide. Recent data collected from ex situ Asian elephants located in multiple North American and European institutions suggest that subclinical EEHV1 infection is common in this population of elephants. Although fatal EEHV1-associated hemorrhagic disease has been reported in range countries, data are lacking regarding the prevalence of subclinical EEHV infections among in situ Asian elephants. We used previously validated EEHV-specific quantitative real-time PCR assays to detect subclinical EEHV infection in three regionally distinct Asian elephant cohorts, totaling 46 in situ elephants in South India, during October and November 2011. Using DNA prepared from trunk washes, we detected EEHV1, EEHV3/4, and EEHV5 at frequencies of 7, 9, and 20% respectively. None of the trunk washes was positive for EEHV2 or 6. At least one EEHV species was detectable in 35% (16/46) of the samples that were screened. These data suggest that subclinical EEHV infection among in situ Asian elephants occurs and that Asian elephants may be natural hosts for EEHV1, EEHV3 or 4, and EEHV5, but not EEHV2 and EEHV6. The methodology described in this study provides a foundation for further studies to determine prevalences of EEHV infection in Asian elephants throughout the world.

  10. Photoacoustic detection of hemozoin in human mononuclear cells as an early indicator of malaria infection

    NASA Astrophysics Data System (ADS)

    Custer, Jonathan R.; Kariuki, Michael; Beerntsen, Brenda T.; Viator, John A.

    2010-02-01

    Malaria is a blood borne infection affecting hundreds of millions of people worldwide2. The parasites reproduce within the blood cells, eventually causing their death and lysis. This process releases the parasites into the blood, continuing the cycle of infection. Usually, malaria is diagnosed only after a patient presents symptoms, including high fever, nausea, and, in advanced cases, coma and death. While invading the bloodstream of a host, malaria parasites convert hemoglobin into an insoluble crystal, known as hemozoin. These crystals, approximately several hundred nanometers in size, are contained within red blood cells and white blood cells that ingest free hemozoin in the blood. Thus, infected red blood cells and white blood cells contain a unique optical absorber that can be detected in blood samples using static photoacoustic detection methods. We separated the white blood cells from malaria infected blood and tested it in a photoacoustic set up using a tunable laser system consisting of an optical parametric oscillator pumped by an Nd:YAG laser with pulse duration of 5 ns. Our threshold of detection was 10 infected white blood cells per microliter, which is more sensitive than current diagnosis methods using microscopic analysis of blood.

  11. Detection of murine cytomegalovirus DNA in circulating leukocytes harvested during acute infection of mice

    SciTech Connect

    Bale, J.F. Jr.; O'Neil, M.E. )

    1989-06-01

    The authors used virus assay and in situ hybridization with a cloned fragment of the murine cytomegalovirus (MCMV) genome to study MCMV infection of circulating leukocytes harvested from 3-week-old BALB/c, C57BL/6, and C3H mice infected with MCMV intraperitoneally. Infectious virus or MCMV DNA was detected in leukocytes on days 1 through 21 of infection in BALB/c mice and on days 3 through 7 in C57BL/6 mice. On days 5 and 7, MCMV DNA or infectious virus was detected in the leukocytes of 17 (94%) of 18 BALB/c mice and 10 (59%) of 17 C57BL/6 mice. In both strains infection peaked on days 5 and 7, when as many as 0.01 to 0.1% of the circulating leukocytes contained MCMV DNA. In C3H mice, however, infectious virus was rarely recovered from leukocyte fractions and MCMV DNA was detected in the circulating leukocytes of only one animal. Circulating leukocytes may have an important role in the dissemination of CMV infections in susceptible hosts.

  12. Anomaly Detection in Host Signaling Pathways for the Early Prognosis of Acute Infection

    PubMed Central

    O’Hern, Corey S.; Shattuck, Mark D.; Ogle, Serenity; Forero, Adriana; Morrison, Juliet; Slayden, Richard; Katze, Michael G.

    2016-01-01

    Clinical diagnosis of acute infectious diseases during the early stages of infection is critical to administering the appropriate treatment to improve the disease outcome. We present a data driven analysis of the human cellular response to respiratory viruses including influenza, respiratory syncytia virus, and human rhinovirus, and compared this with the response to the bacterial endotoxin, Lipopolysaccharides (LPS). Using an anomaly detection framework we identified pathways that clearly distinguish between asymptomatic and symptomatic patients infected with the four different respiratory viruses and that accurately diagnosed patients exposed to a bacterial infection. Connectivity pathway analysis comparing the viral and bacterial diagnostic signatures identified host cellular pathways that were unique to patients exposed to LPS endotoxin indicating this type of analysis could be used to identify host biomarkers that can differentiate clinical etiologies of acute infection. We applied the Multivariate State Estimation Technique (MSET) on two human influenza (H1N1 and H3N2) gene expression data sets to define host networks perturbed in the asymptomatic phase of infection. Our analysis identified pathways in the respiratory virus diagnostic signature as prognostic biomarkers that triggered prior to clinical presentation of acute symptoms. These early warning pathways correctly predicted that almost half of the subjects would become symptomatic in less than forty hours post-infection and that three of the 18 subjects would become symptomatic after only 8 hours. These results provide a proof-of-concept for utility of anomaly detection algorithms to classify host pathway signatures that can identify presymptomatic signatures of acute diseases and differentiate between etiologies of infection. On a global scale, acute respiratory infections cause a significant proportion of human co-morbidities and account for 4.25 million deaths annually. The development of clinical

  13. Anomaly Detection in Host Signaling Pathways for the Early Prognosis of Acute Infection.

    PubMed

    Wang, Kun; Langevin, Stanley; O'Hern, Corey S; Shattuck, Mark D; Ogle, Serenity; Forero, Adriana; Morrison, Juliet; Slayden, Richard; Katze, Michael G; Kirby, Michael

    2016-01-01

    Clinical diagnosis of acute infectious diseases during the early stages of infection is critical to administering the appropriate treatment to improve the disease outcome. We present a data driven analysis of the human cellular response to respiratory viruses including influenza, respiratory syncytia virus, and human rhinovirus, and compared this with the response to the bacterial endotoxin, Lipopolysaccharides (LPS). Using an anomaly detection framework we identified pathways that clearly distinguish between asymptomatic and symptomatic patients infected with the four different respiratory viruses and that accurately diagnosed patients exposed to a bacterial infection. Connectivity pathway analysis comparing the viral and bacterial diagnostic signatures identified host cellular pathways that were unique to patients exposed to LPS endotoxin indicating this type of analysis could be used to identify host biomarkers that can differentiate clinical etiologies of acute infection. We applied the Multivariate State Estimation Technique (MSET) on two human influenza (H1N1 and H3N2) gene expression data sets to define host networks perturbed in the asymptomatic phase of infection. Our analysis identified pathways in the respiratory virus diagnostic signature as prognostic biomarkers that triggered prior to clinical presentation of acute symptoms. These early warning pathways correctly predicted that almost half of the subjects would become symptomatic in less than forty hours post-infection and that three of the 18 subjects would become symptomatic after only 8 hours. These results provide a proof-of-concept for utility of anomaly detection algorithms to classify host pathway signatures that can identify presymptomatic signatures of acute diseases and differentiate between etiologies of infection. On a global scale, acute respiratory infections cause a significant proportion of human co-morbidities and account for 4.25 million deaths annually. The development of clinical

  14. A DNA microarray-based assay to detect dual infection with two dengue virus serotypes.

    PubMed

    Díaz-Badillo, Alvaro; Muñoz, María de Lourdes; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G; Martínez-Muñoz, Jorge P; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

    2014-01-01

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

  15. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    PubMed Central

    Díaz-Badillo, Alvaro; de Lourdes Muñoz, María; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

    2014-01-01

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

  16. Molecular detection of Entamoeba histolytica and Entamoeba dispar infection among wild rats in Kuala Lumpur, Malaysia.

    PubMed

    Lau, Y L; Jamaiah, I; Rohela, M; Fong, M Y; Siti, C O S; Siti, F A

    2014-12-01

    Entamoeba histolytica infection is the third-greatest parasitic disease responsible for death in the world. Wild rats harbouring E. histolytica can be the possible reservoir hosts for human amoebiasis. There were numerous studies on prevalence of intestinal parasites among wild rats in Malaysia but none has reported E. histolytica. Rats were captured from Sentul and Chow Kit areas, Kuala Lumpur, Malaysia. The preserved stool samples were used for microscopy examination and molecular analysis. Out of 137 samples collected, 12 were positive for E. histolytica / E. dispar / E. moshkovskii microscopically. Two E. histolytica (1.4%), 1 E. dispar (0.7%) and 6 mixed infections of E. histolytica and E. dispar (4.3%) were detected using PCR. This is the first report of molecular detection of E. histolytica/dispar infection among wild rats in Malaysia. This study provides useful information about the potential risks of zoonotic agents and the importance of developing control measures to prevent zoonotic transmission.

  17. A DNA microarray-based assay to detect dual infection with two dengue virus serotypes.

    PubMed

    Díaz-Badillo, Alvaro; Muñoz, María de Lourdes; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G; Martínez-Muñoz, Jorge P; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

    2014-01-01

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

  18. Evaluation of fast volatile analysis for detection of Botrytis cinerea infections in strawberry.

    PubMed

    Vandendriessche, Thomas; Keulemans, Johan; Geeraerd, Annemie; Nicolai, Bart M; Hertog, Maarten L A T M

    2012-12-01

    Grey mold (Botrytis cinerea) is one of the major phytopathogens causing serious losses during strawberry postharvest and storage. B. cinerea-host interaction affect emissions of volatile compounds during infection resulting in a characteristic earthy, mushroom odor. Therefore, the objective of this study was to evaluate two analytical techniques based on fast volatile analysis on their performance for monitoring evolution and early detection of B. cinerea infections in strawberry. In a first experiment headspace multi-capillary column-ion mobility spectrometry (HS MCC-IMS) has been successfully used to evaluate development of strawberry aroma during shelflife. In a second experiment the same technique has been used to detect the degree of B. cinerea infection through changes in the volatile profile. Additionally, these samples were analyzed with headspace solid-phase-microextraction fast GC-MS (HS SPME fast GC-MS). Both HS MCC-IMS and HS SPME fast GC-MS could determine the changes in volatile composition as a function of the degree of B. cinerea infection as determined by an enzyme-linked immunosorbent assay (ELISA) and could be used to follow the evolution of infection. According to the ELISA data, some fruit were infected even without any symptoms and volatiles produced by the fungus may be overshadowed by the fruit volatiles. Therefore, both analytical techniques could not be used for early detection of B. cinerea infections. After identification of the volatile compounds and multivariate data analysis, potential biomarkers specific for B. cinerea were highlighted, being 3-methylbutanal, cis-4-decenal, 2-methyl-1-butanol, 2-methyl-1-propanol, 1-octen-3-one and 1-octen-3-ol. PMID:22986207

  19. Identification and characterization of novel membrane-bound PRL protein tyrosine phosphatases from Setaria cervi, a bovine filarial parasite.

    PubMed

    Singh, Neetu; Yadav, Smita; Rathaur, Sushma

    2015-11-01

    A significant amount of protein tyrosine phosphatase (PTP) activity was detected in the detergent-soluble membrane-bound fraction of Setaria cervi, a bovine filarial parasite. The membrane-bound PTP activity was significantly inhibited when the adult parasites were exposed to compounds having antifilarial activity like aspirin and SK7 as well as phenylarsine oxide, a specific PTP inhibitor suggesting that this activity is stress regulated. Further, this enzyme was purified as a single protein of apparently 21 kDa using two different chromatographic techniques. The MALDI-MS/MS analysis of its peptides showed closest match with protein tyrosine phosphatase PRL (Aedes aegypti). This purified enzyme (named as PRL) showed maximum activity at pH 5.5/37 °C and hydrolysed para nitro phenyl phosphate (pNPP) at the highest rate followed by O-P-L-tyrosine and O-P-L-threonine. It showed significant inhibition by specific inhibitors of PTP such as sodium orthovanadate, phenylarsine oxide and ammonium molybdate and was activated by dithiothreitol (DTT). The active site modification studies suggested involvement of cysteine, arginine, histidine and aspartic acid in the catalytic activity of PRL. The activity of S. cervi PRL was also found to be resistant towards the external oxidative stress. Thus, S. cervi PRL could be taken as a potential target for the management of human lymphatic filariasis. PMID:26341797

  20. Detection of Mycobacterium tuberculosis in latently infected lungs by immunohistochemistry and confocal microscopy

    PubMed Central

    Eugenin, Eliseo; Kaplan, Gilla

    2014-01-01

    Detection of latent Mycobacterium tuberculosis is a challenge in the diagnosis of asymptomatic, subclinical tuberculosis. We report the development of an immunofluorescence technique to visualize and enumerate M. tuberculosis in latently infected rabbit lungs where no acid-fast–stained organisms were seen and no cultivable bacilli were obtained by the agar-plating method. PMID:25161200

  1. Efficacy of agar-plate culture in detection of Strongyloides stercoralis infection.

    PubMed

    Arakaki, T; Iwanaga, M; Kinjo, F; Saito, A; Asato, R; Ikeshiro, T

    1990-06-01

    Agar-plate culture of feces using a modified petri dish proved to be highly efficient in the detection of Strongyloides stercoralis infection. Furrows left by S. stercoralis on the agar plate were distinguished readily in size from those left by Necator americanus. PMID:2352073

  2. Detection of Neospora sp. from infected bovine tissues by PCR and probe hybridization.

    PubMed

    Ho, M S; Barr, B C; Rowe, J D; Anderson, M L; Sverlow, K W; Packham, A; Marsh, A E; Conrad, P A

    1997-06-01

    Neospora sp. can cause fetal abortion or neurological disease in congenitally infected calves. Latent tissue stages in infected cows may contribute to vertical transmission of Neospora sp. from dam to offspring in multiple pregnancies. In this investigation, the polymerase chain reaction (PCR) and Neospora-specific assay were employed to detect Neospora sp. by amplification of nuclear small-subunit rRNA gene sequences in infected cattle tissues. Tissues from 11 cattle, including 6 experimentally and 2 naturally infected cows, 1 naturally infected newborn calf, and 2 uninfected control cows, were evaluated in this study. Neospora-specific PCR products were amplified from DNAs of different bovine tissues, including brain, spinal cord, heart, lung, kidney, diaphragm, skeletal muscle, and placenta, as well as amniotic fluid samples of infected cattle. The PCR-based amplification and probe hybridization system proved useful in assessing the location of tissue-stage parasites in naturally and experimentally infected cattle, even when Neospora sp. antibody titers fall below normal cut-off values by an indirect immunofluorescent antibody test.

  3. Polyclonal antibody based coproantigen detection immunoassay for diagnosis of Oesophagostomum columbianum infection in goats.

    PubMed

    Jas, R; Ghosh, J D; Das, K

    2010-06-24

    A polyclonal antibody based coproantigen detection enzyme linked immunosorbent assay (cAg-ELISA) for diagnosis of experimental and natural Oesophagostomum columbianum infection in goats was developed and evaluated. Adult O. columbianum worms, collected from the caecum and colon of slaughtered goats, were triturated and cultured for obtaining infective third stage larvae (L(3)) and also used for preparation of excretory-secretory antigen (ESAg). Experimental goats were orally infected each with 600 L(3)/kg of the body weight. Filter sterilized faecal supernatant, i.e. the coproantigen (cAg) was harvested from the rectal faeces of all the infected goats, on alternate days from day-5 till day-31 after the infection. Hyperimmune serum (HIS) against ESAg of O. columbianum was raised in rabbits. Molecular and antigenic characterization of ES products of O. columbianum by HIS revealed that 50 and 39kDa polypeptides were immuno-dominant. Coproantigen detection ELISA was standardized by using the cAg as coating antigen and its subsequent binding with the HIS against ESAg of O. columbianum. The sensitivity, specificity and accuracy of the standardized assay were determined by evaluating the assay on the faecal supernatant of 96 slaughtered goats taking into consideration their recorded parasitological status in respect of the abomasal and the intestinal parasites. The cAg-ELISA detected the prepatent oesophagostomosis on 20-24-day-post-infection with a sensitivity, specificity and accuracy of 88, 89.13 and 88.54%, respectively. The assay is relatively easy to perform and would serve as a reliable tool for detection of caprine nodular oesophagostomosis.

  4. Dirofilarial infections in Europe.

    PubMed

    Genchi, Claudio; Kramer, Laura H; Rivasi, Francesco

    2011-10-01

    Nematodes of the genus Dirofilaria are currently considered emerging agents of parasitic zoonoses in Europe. Climatic changes and an increase in the movement of reservoirs (mostly infected dogs) have caused an increase in the geographical range of these parasites from the traditionally endemic/hyperendemic southern regions, and the risk for human infection has increased. In the last several years, forecast models have predicted that current summer temperatures are sufficient to facilitate extrinsic incubation of Dirofilaria in many areas of Europe. The global warming projected by the Intergovernmental Panel on Climate Change suggests that warm summers suitable for Dirofilaria transmission in Europe will be the rule in the future decades, and if the actual trend of temperature increase continues, filarial infection should spread into previously infection-free areas. Dirofilaria repens is currently the filarial species that is most commonly reported as spreading from southern to northern areas. This article reviews the zoonotic aspects, effects of climate, and other global drivers on Dirofilaria infections in Europe and the possible implications on the transmission and control of these mosquito-borne nematodes.

  5. Evaluation of Elecsys Syphilis Assay for Routine and Blood Screening and Detection of Early Infection

    PubMed Central

    Kremastinou, J.; Polymerou, V.; Lavranos, D.; Aranda Arrufat, A.; Harwood, J.; Martínez Lorenzo, M. J.; Ng, K. P.; Queiros, L.; Vereb, I.

    2016-01-01

    Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum. The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient populations, at seven independent laboratories. The samples included routine diagnostic samples, blood donation samples, samples from patients with confirmed HIV infections, samples from living organ or bone marrow donors, and banked samples, including samples previously confirmed as syphilis positive. This study also investigated the seroconversion sensitivity of the assay. With a total of 1,965 syphilis-negative routine diagnostic samples and 5,792 syphilis-negative samples collected from blood donations, the Elecsys Syphilis assay had specificity values of 99.85% and 99.86%, respectively. With 333 samples previously identified as syphilis positive, the sensitivity was 100% regardless of disease stage. The assay also showed 100% sensitivity and specificity with samples from 69 patients coinfected with HIV. The Elecsys Syphilis assay detected infection in the same bleed or earlier, compared with comparator assays, in a set of sequential samples from a patient with primary syphilis. In archived serial blood samples collected from 14 patients with direct diagnoses of primary syphilis, the Elecsys Syphilis assay detected T. pallidum antibodies for 3 patients for whom antibodies were not detected with the Architect Syphilis TP assay, indicating a trend for earlier detection of infection, which may have the potential to shorten the time between infection and reactive screening test results. PMID:27358468

  6. Evaluation of Elecsys Syphilis Assay for Routine and Blood Screening and Detection of Early Infection.

    PubMed

    Kremastinou, J; Polymerou, V; Lavranos, D; Aranda Arrufat, A; Harwood, J; Martínez Lorenzo, M J; Ng, K P; Queiros, L; Vereb, I; Cusini, M

    2016-09-01

    Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient populations, at seven independent laboratories. The samples included routine diagnostic samples, blood donation samples, samples from patients with confirmed HIV infections, samples from living organ or bone marrow donors, and banked samples, including samples previously confirmed as syphilis positive. This study also investigated the seroconversion sensitivity of the assay. With a total of 1,965 syphilis-negative routine diagnostic samples and 5,792 syphilis-negative samples collected from blood donations, the Elecsys Syphilis assay had specificity values of 99.85% and 99.86%, respectively. With 333 samples previously identified as syphilis positive, the sensitivity was 100% regardless of disease stage. The assay also showed 100% sensitivity and specificity with samples from 69 patients coinfected with HIV. The Elecsys Syphilis assay detected infection in the same bleed or earlier, compared with comparator assays, in a set of sequential samples from a patient with primary syphilis. In archived serial blood samples collected from 14 patients with direct diagnoses of primary syphilis, the Elecsys Syphilis assay detected T. pallidum antibodies for 3 patients for whom antibodies were not detected with the Architect Syphilis TP assay, indicating a trend for earlier detection of infection, which may have the potential to shorten the time between infection and reactive screening test results.

  7. Evaluation of Elecsys Syphilis Assay for Routine and Blood Screening and Detection of Early Infection.

    PubMed

    Kremastinou, J; Polymerou, V; Lavranos, D; Aranda Arrufat, A; Harwood, J; Martínez Lorenzo, M J; Ng, K P; Queiros, L; Vereb, I; Cusini, M

    2016-09-01

    Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient populations, at seven independent laboratories. The samples included routine diagnostic samples, blood donation samples, samples from patients with confirmed HIV infections, samples from living organ or bone marrow donors, and banked samples, including samples previously confirmed as syphilis positive. This study also investigated the seroconversion sensitivity of the assay. With a total of 1,965 syphilis-negative routine diagnostic samples and 5,792 syphilis-negative samples collected from blood donations, the Elecsys Syphilis assay had specificity values of 99.85% and 99.86%, respectively. With 333 samples previously identified as syphilis positive, the sensitivity was 100% regardless of disease stage. The assay also showed 100% sensitivity and specificity with samples from 69 patients coinfected with HIV. The Elecsys Syphilis assay detected infection in the same bleed or earlier, compared with comparator assays, in a set of sequential samples from a patient with primary syphilis. In archived serial blood samples collected from 14 patients with direct diagnoses of primary syphilis, the Elecsys Syphilis assay detected T. pallidum antibodies for 3 patients for whom antibodies were not detected with the Architect Syphilis TP assay, indicating a trend for earlier detection of infection, which may have the potential to shorten the time between infection and reactive screening test results. PMID:27358468

  8. Serological survey of Borrelia infection of dogs in Sapporo, Japan, where Borrelia garinii infection was previously detected

    PubMed Central

    UESAKA, Karin; MAEZAWA, Masaki; INOKUMA, Hisashi

    2015-01-01

    A serological survey of Borrelia infection of dogs was performed in Sapporo, Japan, where Borrelia garinii infection in dogs was detected in 2011. A total of 314 serum samples were collected from dogs that visited three animal hospitals in Sapporo from 2012 to 2014. The two-step evaluation method, involving screening ELISA followed by Western blot analysis, was used to detect antibodies against Borrelia species. A total of 34 samples were positive by ELISA. Among those 34 samples, 32 were positive for Borrelia spp. by Western blot. These findings suggest that the 32 dogs (10.2%) generated antibodies against Borrelia burgdorferi sensu lato, such as B. garinii or B. afzelii. Antibody positivity was 7.6% and 13.3% for dogs living in urban and rural areas, respectively. Dogs with a history of tick infestation showed a positive rate of 16.7%, which was higher, although not significantly, than the 6.7% among dogs without a history. PMID:26522809

  9. A bead-based multiplex assay for the detection of DNA viruses infecting laboratory rodents.

    PubMed

    Höfler, Daniela; Nicklas, Werner; Mauter, Petra; Pawlita, Michael; Schmitt, Markus

    2014-01-01

    The Federation of European Laboratory Animal Science Association (FELASA) recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals' physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.

  10. Increases in Human Papillomavirus Detection During Early HIV Infection Among Women in Zimbabwe

    PubMed Central

    Nowak, Rebecca G.; Morrison, Charles S.; Gange, Stephen J.; Kwok, Cynthia; Oliver, Amy E.; Howard, Roslyn; Van Der Pol, Barbara; Salata, Robert A.; Padian, Nancy S.; Chipato, Tsungai; Munjoma, Marshall; Celentano, David D.

    2011-01-01

    Background. Individuals who acquire human immunodeficiency virus (HIV) may experience an immediate disruption of genital tract immunity, altering the ability to mount a local and effective immune response. This study examined the impact of early HIV infection on new detection of human papillomavirus (HPV). Methods. One hundred fifty-five Zimbabwean women with observation periods before and after HIV acquisition and 486 HIV-uninfected women were selected from a cohort study evaluating hormonal contraceptive use and risk of HIV acquisition. Study visits occurred at 3-month intervals. Cervical swab samples available from up to 6 months before, at, and up to 6 months after the visit when HIV was first detected were typed for 37 HPV genotypes or subtypes. Results. We observed ∼5-fold higher odds of multiple (≥2) new HPV detections only after HIV acquisition, relative to HIV-negative women after adjusting for sexual behavior and concurrent genital tract infections. We also observed ∼2.5-fold higher odds of single new HPV detections at visits before and after HIV acquisition, relative to HIV-uninfected women in multivariable models. Conclusions. These findings suggest that HIV infection has an immediate impact on genital tract immunity, as evidenced by the high risk of multiple new HPV detections immediately after HIV acquisition. PMID:21451006

  11. Detection of Simkania negevensis by culture, PCR, and serology in respiratory tract infection in Cornwall, UK

    PubMed Central

    Friedman, M G; Kahane, S; Dvoskin, B; Hartley, J W

    2006-01-01

    Respiratory tract infections are often treated empirically without investigation to detect the aetiological agent, which may be a virus or a bacterium, including atypical pathogens such as Chlamydophila pneumoniae or Mycoplasma pneumoniae. Recently, several types Chlamydia‐like intracellular bacteria have been detected in environmental samples and clinical specimens. Little is known of their geographical distribution and potential pathogenicity. We describe the detection, by PCR and isolation in cell culture, of Simkania negevensis in nasopharyngeal aspirates of paediatric patients with bronchiolitis in Cornwall, UK. We also present serological evidence of exposure to the organism in 62% of adult patients and 46% of a sample of pregnant women. Empirical treatment of serious respiratory tract infection should consider the possible contribution of these organisms. PMID:16505289

  12. Towards optical fibre based Raman spectroscopy for the detection of surgical site infection

    NASA Astrophysics Data System (ADS)

    Thompson, Alex J.; Koziej, Lukasz; Williams, Huw D.; Elson, Daniel S.; Yang, Guang-Zhong

    2016-03-01

    Surgical site infections (SSIs) are common post-surgical complications that remain significant clinical problems, as they are associated with substantial mortality and morbidity. As such, there is significant interest in the development of minimally invasive techniques that permit early detection of SSIs. To this end, we are applying a compact, clinically deployable Raman spectrometer coupled to an optical fibre probe to the study of bacteria, with the long term goal of using Raman spectroscopy to detect infection in vivo. Our system comprises a 785 nm laser diode for excitation and a commercial (Ocean Optics, Inc.) Raman spectrometer for detection. Here we discuss the design, optimisation and validation of this system, and describe our first experiences interrogating bacterial cells (Escherichia coli) in vitro.

  13. Multi-epitope proteins for improved serological detection of Trypanosoma cruzi infection and Chagas Disease.

    PubMed

    Duthie, Malcolm S; Guderian, Jeffery A; Vallur, Aarthy C; Misquith, Ayesha; Liang, Hong; Mohamath, Raodoh; Luquetti, Alejandro O; Carter, Darrick; Tavares, Suelene N B; Reed, Steven G

    2016-03-01

    We previously reported that tandem repeat (TR) proteins from Trypanosoma cruzi could serve as targets of the antibody response and be useful as diagnostic indicators. To optimize reagents for detecting T. cruzi infection we evaluated individual TR proteins and identified several that were recognized by the majority of Chagas patient's sera collected from individuals form Brazil. We then produced novel, recombinant fusion proteins to combine the reactive TR proteins into a single diagnostic product. Direct comparison of the antibody response of serum samples that were readily detected by the established fusion antigen used in commercial detection of Chagas disease, TcF, revealed strong responses to TcF43 and TcF26 proteins. While the TcF43 and TcF26 antigens enhanced detection and strength of signal, they did not compromise the specificity of detection compared to that obtained with TcF. Finally, it was apparent by testing against a panel of 84 serum samples assembled on the basis of moderate or weak reactivity against TcF (mostly signal:noise <5) that TcF43 and TcF26 could more strongly detected by many of the sera that had low TcF antibody levels. Taken together, these data indicate that TcF43 and TcF26 could be used to enhance the detection of T. cruzi infection as well as supporting a diagnosis of Chagas disease.

  14. High Frequency of Chlamydia trachomatis Mixed Infections Detected by Microarray Assay in South American Samples

    PubMed Central

    Gallo Vaulet, Lucía; Entrocassi, Carolina; Portu, Ana I.; Castro, Erica; Di Bartolomeo, Susana; Ruettger, Anke; Sachse, Konrad; Rodriguez Fermepin, Marcelo

    2016-01-01

    Chlamydia trachomatis is one of the most common sexually transmitted infections worldwide. Based on sequence variation in the ompA gene encoding the major outer membrane protein, the genotyping scheme distinguishes 17 recognized genotypes, i.e. A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, K, L1, L2, and L3. Genotyping is an important tool for epidemiological tracking of C. trachomatis infections, including the revelation of transmission pathways and association with tissue tropism and pathogenicity. Moreover, genotyping can be useful for clinicians to establish the correct treatment when LGV strains are detected. Recently a microarray assay was described that offers several advantages, such as rapidity, ease of standardization and detection of mixed infections. The aim of this study was to evaluate the performance of the DNA microarray-based assay for C. trachomatis genotyping of clinical samples already typed by PCR-RFLP from South America. The agreement between both typing techniques was 90.05% and the overall genotype distribution obtained with both techniques was similar. Detection of mixed-genotype infections was significantly higher using the microarray assay (8.4% of cases) compared to PCR-RFLP (0.5%). Among 178 samples, the microarray assay identified 10 ompA genotypes, i.e. D, Da, E, F, G, H, I, J, K and L2. The most predominant type was genotype E, followed by D and F. PMID:27082962

  15. MAIT Cells Detect and Efficiently Lyse Bacterially-Infected Epithelial Cells

    PubMed Central

    Bohineust, Armelle; Bessoles, Stéphanie; Martin, Emmanuel; Premel, Virginie; Coré, Maxime; Sleurs, David; Serriari, Nacer-Eddine; Treiner, Emmanuel; Hivroz, Claire; Sansonetti, Philippe; Gougeon, Marie-Lise; Soudais, Claire; Lantz, Olivier

    2013-01-01

    Mucosal associated invariant T cells (MAIT) are innate T lymphocytes that detect a large variety of bacteria and yeasts. This recognition depends on the detection of microbial compounds presented by the evolutionarily conserved major-histocompatibility-complex (MHC) class I molecule, MR1. Here we show that MAIT cells display cytotoxic activity towards MR1 overexpressing non-hematopoietic cells cocultured with bacteria. The NK receptor, CD161, highly expressed by MAIT cells, modulated the cytokine but not the cytotoxic response triggered by bacteria infected cells. MAIT cells are also activated by and kill epithelial cells expressing endogenous levels of MRI after infection with the invasive bacteria Shigella flexneri. In contrast, MAIT cells were not activated by epithelial cells infected by Salmonella enterica Typhimurium. Finally, MAIT cells are activated in human volunteers receiving an attenuated strain of Shigella dysenteriae-1 tested as a potential vaccine. Thus, in humans, MAIT cells are the most abundant T cell subset able to detect and kill bacteria infected cells. PMID:24130485

  16. Virus-based assay for antigen detection using infective growth as signal transduction mechanism.

    PubMed

    Cheok, Hui Shan; Jaworski, Justyn

    2016-03-15

    Viruses have the ability to infect and thereby confer new phenotypes on host cells. E. coli, for example, if infected by viruses containing antibiotic resistance genes, can benefit by surviving in the presence of the corresponding antibiotics to grow into colonies observable by the naked eye. Using this concept as a signal transduction mechanism for our immunoassay, we have engineered ampicillin resistant virions to display a dimer of the z domain from Protein A. This zz-domain selectively binds to the conserved heavy domain of IgG across various species. As commercially available antibodies are in no short supply, this engineered virion can be used modularly with existing antibodies for converting the presence of target antigen into a visually detectable colony forming unit. Here we demonstrate that this scheme for zz-phage transfection and selective growth of infected E. coli can facilitate sub-nanomolar detection limits for target antigen. Moreover, this phage infectivity assay works over a range of concentrations competitive with existing ELISA techniques. Because this system is derived from self-regenerating components (i.e., virus and bacteria) and furthermore obviates the need for chromogenic substrates or spectroscopic equipment, we find it particularly suitable for use in regions where cost effective detection is a necessity.

  17. Aluminosilicates enhance the infectivity of cytomegalovirus in urine using centrifugation-enhanced antigen detection technology.

    PubMed

    Lee, S H; Biondo, F X; Teichberg, S; Lipson, S M

    1996-04-01

    Due to the inherent lability of CMV, necessary laboratory identification of this infectious agent is often compromised by a delay in specimen transport. Previous studies have addressed the phenomenon of infectivity enhancement/reduction in the rate of infectivity loss by the incorporation into various viral assay systems of trace concentrations of the adsorbents montmorillonite (bentonite [M]) or kaolinite (kaolin [K]). We extended these studies to the clinical setting to identify whether such aluminosilicates would effect an enhanced level of CMV infectivity. The shell vial assay-indirect immunofluorescent assay (SVA-IFA) was utilized in comparative testing throughout this study. The addition of trace concentrations of M or K to the SVA-IFA was found to enhance the infectivity of CMV in urine by 115 and 126%, respectively. The total CMV detection rate by SVA-IFA was 29% (30/105). Three of the 30 (10%) CMV positive specimens were detected only in shell vials which had been supplemented with K or M. Two specimens were isolation positive alone. The addition of K or M to shell vials immediately prior to the start of the SVA-IFA has the potential of (a), enhancing assay readability by increasing the number of fluorescent focus units per vial monolayer and (b), of detecting positive urine specimens with low viral titers which might otherwise not be identified using the conventional SVA-IFA procedure.

  18. High Frequency of Chlamydia trachomatis Mixed Infections Detected by Microarray Assay in South American Samples.

    PubMed

    Gallo Vaulet, Lucía; Entrocassi, Carolina; Portu, Ana I; Castro, Erica; Di Bartolomeo, Susana; Ruettger, Anke; Sachse, Konrad; Rodriguez Fermepin, Marcelo

    2016-01-01

    Chlamydia trachomatis is one of the most common sexually transmitted infections worldwide. Based on sequence variation in the ompA gene encoding the major outer membrane protein, the genotyping scheme distinguishes 17 recognized genotypes, i.e. A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, K, L1, L2, and L3. Genotyping is an important tool for epidemiological tracking of C. trachomatis infections, including the revelation of transmission pathways and association with tissue tropism and pathogenicity. Moreover, genotyping can be useful for clinicians to establish the correct treatment when LGV strains are detected. Recently a microarray assay was described that offers several advantages, such as rapidity, ease of standardization and detection of mixed infections. The aim of this study was to evaluate the performance of the DNA microarray-based assay for C. trachomatis genotyping of clinical samples already typed by PCR-RFLP from South America. The agreement between both typing techniques was 90.05% and the overall genotype distribution obtained with both techniques was similar. Detection of mixed-genotype infections was significantly higher using the microarray assay (8.4% of cases) compared to PCR-RFLP (0.5%). Among 178 samples, the microarray assay identified 10 ompA genotypes, i.e. D, Da, E, F, G, H, I, J, K and L2. The most predominant type was genotype E, followed by D and F.

  19. [HPV detection in the mouth and cervix of patients with histological diagnosis suggestive of genital infection].

    PubMed

    De Guglielmo, Z; Avila, M; Veitía, D; Fernández, A; Venegas, C; Correnti de Plata, M

    2012-01-01

    This work evaluated HPV infection in the oral cavity (using oroscopy and exfoliative oral cytology) and its relation to genital infection in women with cytological diagnosis suggestive of HPV infection. The sample consisted of 60 patients who underwent oroscopy, cytology and viral determination in mouth and cervix by PCR using generic primers MY09/MY11 and MPCR. HPV DNA was detected in oral and genital mucosa in 48.33% and 73.3% of patients, respectively, yielding a concordance of 44.2% (k=0.44, moderate agreement). The most common viral types were low risk, especially type 6, found in 86.2% of oral samples and 65.9% of cervical specimens, alone or in combination with other types of low (11) or high oncogenic risk (16, 18, 33), with a concordance of 10.45% (k = 0.1, insignificant agreement). However, in relation to type 6, there was a concordance of 75.86% (k=0.7, high agreement). The cytology of the oral cavity had a sensitivity of 3.5% and a specificity of 93.6%. For oroscopy, sensitivity was 27.6% and specificity was 74.2%. The results indicate that HPV infection in the oral cavity of patients with genital infection could be frequent. The low concordance between HPV types suggests that HPV infection in the mouth and cervix has a different biological behavior.

  20. Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells.

    PubMed

    Takahashi, Tadanobu; Agarikuchi, Takashi; Kurebayashi, Yuuki; Shibahara, Nona; Suzuki, Chihiro; Kishikawa, Akiko; Fukushima, Keijo; Takano, Maiko; Suzuki, Fumie; Wada, Hirohisa; Otsubo, Tadamune; Ikeda, Kiyoshi; Minami, Akira; Suzuki, Takashi

    2015-01-01

    Mumps viruses show diverse cytopathic effects (CPEs) of infected cells and viral plaque formation (no CPE or no plaque formation in some cases) depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study), even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.

  1. TLR and RLR Signaling Are Reprogrammed in Opposite Directions after Detection of Viral Infection.

    PubMed

    Hotz, Christian; Roetzer, Laurin C; Huber, Thomas; Sailer, Andreas; Oberson, Anne; Treinies, Marina; Heidegger, Simon; Herbst, Tina; Endres, Stefan; Bourquin, Carole

    2015-11-01

    Innate immune recognition of RNA is key for the initiation of immunity in response to viral infection. Although the factors controlling the detection of viral RNA by innate immune receptors in host cells are increasingly well understood, little is known about the dynamic changes in signaling after the initial triggering of these receptors. In this study, we report that preconditioning with the synthetic dsRNA polyinosinic-polycytidylic acid [poly(I:C)], a mimetic of viral RNA, rapidly reprograms murine APCs by simultaneously augmenting sensitivity of endosomal TLRs and inhibiting activation of RIG-I-like receptors (RLRs) in an IFN-β-dependent manner. These changes in receptor sensitivity were also seen in vivo after treatment of mice with poly(I:C). Mechanistically, the increased sensitivity of the TLR pathway was associated with elevated MAPK and NF-κB activity. The RLR response was inhibited downstream of TANK-binding kinase-1, resulting in decreased IFN regulatory factor 3 phosphorylation. Reprogramming of pattern-recognition receptor signaling also occurred after viral infection, because infection of host cells with Sendai virus or their exposure to supernatant from virus-infected cells induced the same changes in TLR and RLR sensitivity as poly(I:C). Thus, innate recognition of viral infection critically modifies responses to pattern-recognition receptor stimulation. These dynamic adaptations to infection may reinforce antiviral immunity and at the same time serve to limit pathological inflammation. PMID:26392465

  2. TLR and RLR Signaling Are Reprogrammed in Opposite Directions after Detection of Viral Infection.

    PubMed

    Hotz, Christian; Roetzer, Laurin C; Huber, Thomas; Sailer, Andreas; Oberson, Anne; Treinies, Marina; Heidegger, Simon; Herbst, Tina; Endres, Stefan; Bourquin, Carole

    2015-11-01

    Innate immune recognition of RNA is key for the initiation of immunity in response to viral infection. Although the factors controlling the detection of viral RNA by innate immune receptors in host cells are increasingly well understood, little is known about the dynamic changes in signaling after the initial triggering of these receptors. In this study, we report that preconditioning with the synthetic dsRNA polyinosinic-polycytidylic acid [poly(I:C)], a mimetic of viral RNA, rapidly reprograms murine APCs by simultaneously augmenting sensitivity of endosomal TLRs and inhibiting activation of RIG-I-like receptors (RLRs) in an IFN-β-dependent manner. These changes in receptor sensitivity were also seen in vivo after treatment of mice with poly(I:C). Mechanistically, the increased sensitivity of the TLR pathway was associated with elevated MAPK and NF-κB activity. The RLR response was inhibited downstream of TANK-binding kinase-1, resulting in decreased IFN regulatory factor 3 phosphorylation. Reprogramming of pattern-recognition receptor signaling also occurred after viral infection, because infection of host cells with Sendai virus or their exposure to supernatant from virus-infected cells induced the same changes in TLR and RLR sensitivity as poly(I:C). Thus, innate recognition of viral infection critically modifies responses to pattern-recognition receptor stimulation. These dynamic adaptations to infection may reinforce antiviral immunity and at the same time serve to limit pathological inflammation.

  3. Detection of infective Nosema ceranae (Microsporidia) spores in corbicular pollen of forager honeybees.

    PubMed

    Higes, Mariano; Martín-Hernández, Raquel; Garrido-Bailón, Encarna; García-Palencia, Pilar; Meana, Aránzazu

    2008-01-01

    Nosema ceranae is a Microsporidia recently described as a parasite in Apis mellifera honeybees in Europe. Due to the short time since its description, no epidemiological data are available. In this study, spore detection in both pollen baskets and pollen collected from commercial traps is described (PCM, TEM and PCR methods). Spore infectivity is shown after artificial infection of Nosema-free adult bees. The epidemiological consequences of the presence of Nosema spores in corbicular pollen require more study and must be considered in beekeeping practices.

  4. Computed tomographic detection of sinusitis responsible for intracranial and extracranial infections

    SciTech Connect

    Carter, B.L.; Bankoff, M.S.; Fisk, J.D.

    1983-06-01

    Computed tomography (CT) is now used extensively for the evaluation of orbital, facial, and intracranial infections. Nine patients are presented to illustrate the importance of detecting underlying and unsuspected sinusitis. Prompt treatment of the sinusitis is essential to minimize the morbidity and mortality associated with complications such as brain abscess, meningitis, orbital cellulitis, and osteomyelitis. A review of the literature documents the persistence of these complications despite the widespread use of antibiotic therapy. Recognition of the underlying sinusitis is now possible with CT if the region of the sinuses is included and bone-window settings are used during the examination of patients with orbital and intracranial infection.

  5. The detection of circulating antibody in human toxocara infections using the indirect fluorescent antibody test

    PubMed Central

    Bisseru, B.; Woodruff, A. W.

    1968-01-01

    The indirect fluorescent antibody test has been used to detect Toxocara antibodies quantitatively in human sera. The results obtained correlate well with those obtained with the toxocara skin test. Cross reactions have been found with Ascaris lumbricoides and about one in five normal sera give a significant fluorescent reaction. The specificity of the test has been established by Ascaris adsorption studies and by a lack of cross reaction with sera from other helminthic infections. The test may be especially easy to use and therefore particularly valuable in regions where A. lumbricoides infection is relatively rare. Images PMID:4880409

  6. An Innovative Method for Rapid Identification and Detection of Vibrio alginolyticus in Different Infection Models

    PubMed Central

    Fu, Kaifei; Li, Jun; Wang, Yuxiao; Liu, Jianfei; Yan, He; Shi, Lei; Zhou, Lijun

    2016-01-01

    Vibrio alginolyticus is one of the most common pathogenic marine Vibrio species, and has been found to cause serious seafood-poisoning or fatal extra-intestinal infections in humans, such as necrotizing soft-tissue infections, bacteremia, septic shock, and multiple organ failures. Delayed accurate diagnosis and treatment of most Vibrio infections usually result to high mortality rates. The objective of this study was to establish a rapid diagnostic method to detect and identify the presence of V. alginolyticus in different samples, so as to facilitate timely treatment. The widely employed conventional methods for detection of V. alginolyticus include biochemical identification and a variety of PCR methods. The former is of low specificity and time-consuming (2–3 days), while the latter has improved accuracy and processing time. Despite such advancements, these methods are still complicated, time-consuming, expensive, require expertise and advanced laboratory systems, and are not optimal for field use. With the goal of providing a simple and efficient way to detect V. alginolyticus, we established a rapid diagnostic method based on loop-mediated Isothermal amplification (LAMP) technology that is feasible to use in both experimental and field environments. Three primer pairs targeting the toxR gene of V. alginolyticus were designed, and amplification was carried out in an ESE tube scanner and Real-Time PCR device. We successfully identified 93 V. alginolyticus strains from a total of 105 different bacterial isolates and confirmed their identity by 16s rDNA sequencing. We also applied this method on infected mouse blood and contaminated scallop samples, and accurate results were both easily and rapidly (20–60 min) obtained. Therefore, the RT-LAMP assay we developed can be conveniently used to detect the presence of V. alginolyticus in different samples. Furthermore, this method will also fulfill the gap for real-time screening of V. alginolyticus infections

  7. Molecular systematics of filarial parasites, with an emphasis on groups of medical and veterinary importance, and its relevance for epidemiology.

    PubMed

    Morales-Hojas, Ramiro

    2009-09-01

    Filarial parasites are members of the Phylum Nemata that comprise several species of medical and veterinary importance. Among the human diseases caused by members of this group of nematodes are river blindness and lymphatic filariasis, which afflict millions of people in the tropics. These diseases not only have an impact on the health of the people affected but also bear a great socioeconomic burden. Despite their relevance, the systematics of the filarial parasites is not well understood yet, and additional molecular phylogenetic studies are required to comprehend the evolution of these parasites. Identifying the patterns of evolution of these parasites will be of relevance in preventing emerging zoonoses. The present review examines the information about the molecular systematics of filarial parasites available in the literature and evaluates the relevance of the different directions of future research. Furthermore, it is also intended to highlight the relevance of molecular systematic studies in the molecular epidemiology research area.

  8. Development of loop-mediated isothermal amplification (LAMP) for detection of Babesia gibsoni infection in dogs.

    PubMed

    Mandal, Mrityunjay; Banerjee, Partha S; Kumar, Saroj; Ram, Hira; Garg, Rajat; Pawde, Abhijit M

    2015-04-15

    Diagnosis of canine babesiosis, caused by Babesia gibsoni is difficult, especially in chronically infected dogs. A loop mediated isothermal amplification (LAMP) assay was developed and standardized by using four oligonucleotide primers targeting the hypervariable region of 18S rRNA gene (GenBank Acc. no. KC461261). The primers specifically amplified B. gibsoni DNA, while no amplification was detected with DNA from non-infected dogs as well as from dogs infected with Babesia canis vogeli, Hepatozoon canis, Ehrlichia canis and Trypanosoma evansi. The assay could detect 1.35 × 10(-7) parasitaemia and 10(-4) dilution of recombinant plasmid, equivalent to 12 pg of target DNA. All the samples were tested by nested PCR as well as LAMP assay. LAMP was found to be 10 times more sensitive than nested PCR targeting the same gene. Out of 75 suspected field samples, collected from different parts of the country, LAMP could detect B. gibsoni in 43 samples, while nested PCR and microscopy could detect 37 and 23 samples, respectively. High sensitivity, specificity and rapidity of LAMP assay may be exploited for screening large number of samples in a field setting. PMID:25749021

  9. Rapid Detection of Cyprinid Herpesvirus 3 in Latently Infected Koi by Recombinase Polymerase Amplification.

    PubMed

    Prescott, Meagan A; Reed, Aimee N; Jin, Ling; Pastey, Manoj K

    2016-09-01

    Since the emergence of cyprinid herpesvirus 3 (CyHV-3), outbreaks have been devastating to Common Carp Cyprinus carpio and koi (a variant of Common Carp), leading to high economic losses. Current diagnostics for detecting CyHV-3 are limited in sensitivity and are further complicated by latency. Here we describe the detection of CyHV-3 by recombinase polymerase amplification (RPA). The RPA assay can detect as low as 10 copies of the CyHV-3 genome by an isothermal reaction and yields results in approximately 20 min. Using the RPA assay, the CyHV-3 genome can be detected in the total DNA of white blood cells isolated from koi latently infected with CyHV-3, while less than 10% of the latently infected koi can be detected by a real-time PCR assay in the total DNA of white blood cells. In addition, RPA products can be detected in a lateral flow device that is cheap and fast and can be used outside of the diagnostic lab. The RPA assay and lateral flow device provide for the rapid, sensitive, and specific amplification of CyHV-3 that with future modifications for field use and validation could lead to enhanced surveillance and early diagnosis of CyHV-3 in the laboratory and field. Received September 14, 2015; accepted April 9, 2016. PMID:27485254

  10. Trans-sialidase inhibition assay detects Trypanosoma cruzi infection in different wild mammal species.

    PubMed

    Sartor, Paula A; Ceballos, Leonardo A; Orozco, Marcela M; Cardinal, Marta V; Gürtler, Ricardo E; Leguizamón, María S

    2013-08-01

    The detection of Trypanosoma cruzi infection in mammals is crucial for understanding the eco-epidemiological role of the different species involved in parasite transmission cycles. Xenodiagnosis (XD) and hemoculture (HC) are routinely used to detect T. cruzi in wild mammals. Serological methods are much more limited because they require the use of specific antibodies to immunoglobulins of each mammalian species susceptible to T. cruzi. In this study we detected T. cruzi infection by trans-sialidase (TS) inhibition assay (TIA). TIA is based on the antibody neutralization of a recombinant TS that avoids the use of anti-immunoglobulins. TS activity is not detected in the co-endemic protozoan parasites Leishmania spp and T. rangeli. In the current study, serum samples from 158 individuals of nine wild mammalian species, previously tested by XD, were evaluated by TIA. They were collected from two endemic areas in northern Argentina. The overall TIA versus XD co-reactivity was 98.7% (156/158). All 18 samples from XD-positive mammals were TIA-positive (co-positivity, 100%) and co-negativity was 98.5% (138/140). Two XD-negative samples from a marsupial (Didelphis albiventris) and an edentate (Dasypus novemcinctus) were detected by TIA. TIA could be used as a novel tool for serological detection of Trypanosoma cruzi in a wide variety of sylvatic reservoir hosts. PMID:23930975

  11. Rapid Detection of Cyprinid Herpesvirus 3 in Latently Infected Koi by Recombinase Polymerase Amplification.

    PubMed

    Prescott, Meagan A; Reed, Aimee N; Jin, Ling; Pastey, Manoj K

    2016-09-01

    Since the emergence of cyprinid herpesvirus 3 (CyHV-3), outbreaks have been devastating to Common Carp Cyprinus carpio and koi (a variant of Common Carp), leading to high economic losses. Current diagnostics for detecting CyHV-3 are limited in sensitivity and are further complicated by latency. Here we describe the detection of CyHV-3 by recombinase polymerase amplification (RPA). The RPA assay can detect as low as 10 copies of the CyHV-3 genome by an isothermal reaction and yields results in approximately 20 min. Using the RPA assay, the CyHV-3 genome can be detected in the total DNA of white blood cells isolated from koi latently infected with CyHV-3, while less than 10% of the latently infected koi can be detected by a real-time PCR assay in the total DNA of white blood cells. In addition, RPA products can be detected in a lateral flow device that is cheap and fast and can be used outside of the diagnostic lab. The RPA assay and lateral flow device provide for the rapid, sensitive, and specific amplification of CyHV-3 that with future modifications for field use and validation could lead to enhanced surveillance and early diagnosis of CyHV-3 in the laboratory and field. Received September 14, 2015; accepted April 9, 2016.

  12. Trans-sialidase inhibition assay detects Trypanosoma cruzi infection in different wild mammal species.

    PubMed

    Sartor, Paula A; Ceballos, Leonardo A; Orozco, Marcela M; Cardinal, Marta V; Gürtler, Ricardo E; Leguizamón, María S

    2013-08-01

    The detection of Trypanosoma cruzi infection in mammals is crucial for understanding the eco-epidemiological role of the different species involved in parasite transmission cycles. Xenodiagnosis (XD) and hemoculture (HC) are routinely used to detect T. cruzi in wild mammals. Serological methods are much more limited because they require the use of specific antibodies to immunoglobulins of each mammalian species susceptible to T. cruzi. In this study we detected T. cruzi infection by trans-sialidase (TS) inhibition assay (TIA). TIA is based on the antibody neutralization of a recombinant TS that avoids the use of anti-immunoglobulins. TS activity is not detected in the co-endemic protozoan parasites Leishmania spp and T. rangeli. In the current study, serum samples from 158 individuals of nine wild mammalian species, previously tested by XD, were evaluated by TIA. They were collected from two endemic areas in northern Argentina. The overall TIA versus XD co-reactivity was 98.7% (156/158). All 18 samples from XD-positive mammals were TIA-positive (co-positivity, 100%) and co-negativity was 98.5% (138/140). Two XD-negative samples from a marsupial (Didelphis albiventris) and an edentate (Dasypus novemcinctus) were detected by TIA. TIA could be used as a novel tool for serological detection of Trypanosoma cruzi in a wide variety of sylvatic reservoir hosts.

  13. The relationship between microfilaraemic and amicrofilaraemic loiasis involving co-infection with Mansonella perstans and clinical symptoms in an exposed population from Gabon.

    PubMed

    Bouyou Akotet, M K; Owono-Medang, M; Mawili-Mboumba, D P; Moussavou-Boussougou, M N; Nzenze Afène, S; Kendjo, E; Kombila, M

    2016-07-01

    The relationship between the frequency of loiasis objective symptoms and microfilaraemic or amicrofilaraemic infection was assessed in 1148 exposed patients also infected, or not, with Mansonella perstans. Filarial infections were detected by direct microscopy, leucoconcentration and serology, with prevalence values of 39.5% Loa loa, 5.6% M. perstans and 3.4% co-infection with both filarial species. Amicrofilaraemic or occult loiasis (OL) predominated among L. loa-infected individuals, with a prevalence of 58.2%. Hypermicrofilaraemia (>8000 microfilariae (mf)/ml) was found in 18.4% of L. loa microfilaraemic patients, with 25.7% of them harbouring more than 30,000 mf/ml. Up to 34% of patients with OL showed evidence of Calabar swelling, compared with 26.3% of microfilaraemic patients (P= 0.03). Overall 5.3% of patients presented with adult worm migration across the eye, representing 16.3% of microfilaraemic individuals and 11.4% of amicrofilaraemic patients (P= 0.13). This symptom was similarly found in patients with more than 30,000 mf/ml (22%), those with microfilaraemia between 8 and 30,000 mf/ml (15.4%) and also in individuals with low or without microfilaraemia (16.1%) (P= 0.7). Five (14.3%) hypermicrofilaraemic patients did not present any L. loa-specific objective symptoms, as well as all the patients with single M. perstans infection. The presence of adult eye worm migration as a strong predictor of high microfilaraemia density would obscure the real burden of L. loa hypermicrofilaraemia in exposed individuals. For epidemiological purposes and control strategies, the mapping of L. loa in endemic areas should also take into account the group of patients with occult loiasis. PMID:26268068

  14. Detection of Japanese eel endothelial cells-infecting virus in Anguilla japonica elvers

    PubMed Central

    OKAZAKI, Sachiko; YASUMOTO, Shinya; KOYAMA, Satoshi; TSUCHIAKA, Shinobu; NAOI, Yuki; OMATSU, Tsutomu; ONO, Shin-ichi; MIZUTANI, Tetsuya

    2015-01-01

    Japanese eel endothelial cells-infecting virus (JEECV) has spread in eel farms and caused serious economic loss. In this study, we examined the prevalence of JEECV infection in 100 wild Japanese eel (Anguilla japonica) elvers caught from Yamaguchi prefecture, Japan, using quantitative PCR and conventional PCR. Total genomic DNA was obtained from the cranial quarter of the body in 70 of 100 eels and from the gill in the remaining. Of 30 gill samples, 20 were analyzed after pooling with other samples, and the remaining 10 were analyzed separately. A single positive result for JEECV was detected following analysis of the 10 separately analyzed samples. This result constitutes the first report of JEECV infection in wild A. japonica elvers. PMID:26672624

  15. Detection of Japanese eel endothelial cells-infecting virus in Anguilla japonica elvers.

    PubMed

    Okazaki, Sachiko; Yasumoto, Shinya; Koyama, Satoshi; Tsuchiaka, Shinobu; Naoi, Yuki; Omatsu, Tsutomu; Ono, Shin-Ichi; Mizutani, Tetsuya

    2016-05-01

    Japanese eel endothelial cells-infecting virus (JEECV) has spread in eel farms and caused serious economic loss. In this study, we examined the prevalence of JEECV infection in 100 wild Japanese eel (Anguilla japonica) elvers caught from Yamaguchi prefecture, Japan, using quantitative PCR and conventional PCR. Total genomic DNA was obtained from the cranial quarter of the body in 70 of 100 eels and from the gill in the remaining. Of 30 gill samples, 20 were analyzed after pooling with other samples, and the remaining 10 were analyzed separately. A single positive result for JEECV was detected following analysis of the 10 separately analyzed samples. This result constitutes the first report of JEECV infection in wild A. japonica elvers.

  16. Comparison of serologic tests for detection of antigen in canine heartworm infections.

    PubMed

    Brunner, C J; Hendrix, C M; Blagburn, B L; Hanrahan, L A

    1988-05-15

    In 30 random-source dogs, we determined sensitivity and specificity of 5 serologic tests for detection of canine heartworm antigens. Seventeen of the dogs were infected naturally with adult Dirofilaria immitis, and 4 of the infected dogs were amicrofilaremic. The ability of the serologic tests to predict whether a dog was infected or uninfected (overall test accuracy) ranged from 73 to 97%. Sensitivity was not affected by circulating D immitis microfilariae, but was markedly influenced by the number of adult D immitis present. False-positive reactions were rare and were not associated with intestinal parasites or Dipetalonema reconditum microfilariae. Modifications of some of the test procedures were necessary to maximize test accuracy and reproducibility. These modifications and other technical details might limit the usefulness of some of the tests in a veterinary practice.

  17. Factors affecting response to medical management in patients of filarial chyluria: A prospective study

    PubMed Central

    Goyal, Neeraj Kumar; Goel, Apul; Sankhwar, Satyanarayan; Singh, Vishwajeet; Ali, Wahid; Natu, S. M.; Singh, Bhupendra Pal; Sinha, Rahul Janak; Dalela, Divakar

    2014-01-01

    Introduction: Filarial chyluria is a common problem in filarial endemic countries. Its management begins with medical therapy but some patients progress to require surgery. The present study aimed to determine factors affecting response to medical management in patients of filarial chyluria. Materials and Methods: This prospective study conducted between August 2008 and November 2012, included conservatively managed patients of chyluria. Demographic profile, clinical presentation, treatment history and urinary triglycerides (TGs) and cholesterol levels at baseline were compared between the responders and non-responders. Apart from the clinical grade of chyluria, hematuria was evaluated as an independent risk factor. Results: Out of the 222 patients (mean age, 37.99 ± 13.29 years, 129 males), 31 patients failed to respond while 35 had a recurrence after initial response; the overall success rate being 70.3% at a mean follow-up of 25 months. No difference was observed in demographics, clinical presentation, presence of hematuria, disease duration and mean urinary TGs loss between responders and non-responders. On multivariate analysis, patients with treatment failure were found to have a higher-grade disease (14.3% Grade-I, 36.6% Grades-II and 60% Grade-III), higher number of pretreatment courses (1.59 ± 1.08 vs. 1.02 ± 0.79) and heavier cholesterol (26.54 ± 23.46 vs. 8.81 ± 8.55 mg/dl) loss at baseline compared with responders (P < 0.05). Conclusion: Conservative management has a success rate in excess of 70%, not affected by the disease chronicity, previous episodes and recurrent nature. However, higher-grade disease, extensive pre-treatment with drugs and higher urinary cholesterol loss at baseline are the predictors of poor response. Hematuria is not an independent poor risk factor for conservative management. PMID:24497677

  18. Ex vivo bioluminescence detection of alcelaphine herpesvirus 1 infection during malignant catarrhal fever.

    PubMed

    Dewals, Benjamin; Myster, Françoise; Palmeira, Leonor; Gillet, Laurent; Ackermann, Mathias; Vanderplasschen, Alain

    2011-07-01

    Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest asymptomatically, causes malignant catarrhal fever (WD-MCF) when cross-species transmitted to a variety of susceptible species of the Artiodactyla order. Experimentally, WD-MCF can be reproduced in rabbits. WD-MCF is described as a combination of lymphoproliferation and degenerative lesions in virtually all organs and is caused by unknown mechanisms. Recently, we demonstrated that WD-MCF is associated with the proliferation of CD8(+) cells supporting a latent type of infection in lymphoid tissues. Here, we investigated the macroscopic distribution of AlHV-1 infection using ex vivo bioluminescence imaging in rabbit to determine whether it correlates with the distribution of lesions in lymphoid and nonlymphoid organs. To reach that goal, a recombinant AlHV-1 strain was produced by insertion of a luciferase expression cassette (luc) in an intergenic region. In vitro, the reconstituted AlHV-1 luc(+) strain replicated comparably to the parental strain, and luciferase activity was detected by bioluminescence imaging. In vivo, rabbits infected with the AlHV-1 luc(+) strain developed WD-MCF comparably to rabbits infected with the parental wild-type strain, with hyperthermia and increases of both CD8(+) T cell frequencies and viral genomic charge over time in peripheral blood mononuclear cells and in lymph nodes at time of euthanasia. Bioluminescent imaging revealed that AlHV-1 infection could be detected ex vivo in lymphoid organs but also in lung, liver, and kidney during WD-MCF, demonstrating that AlHV-1 infection is prevalent in tissue lesions. Finally, we show that the infiltrating mononuclear leukocytes in nonlymphoid organs are mainly CD8(+) T cells and that latency is predominant during WD-MCF.

  19. Early detection of Haemonchus contortus infection in sheep using three different faecal occult blood tests.

    PubMed

    Rodríguez, A V; Goldberg, V; Viotti, H; Ciappesoni, G

    2015-01-01

    Haemonchus contortus is a blood-sucking parasite causing the presence of faecal occult blood (FOB). The objective was to study three different FOB tests in order to have a new indicator of H. contortus infection in sheep that could be included in the genetic evaluation system as an alternative selection criterion to faecal worm egg count (FEC). A total of 29 Corriedale lambs were experimentally infected with 10.000 larvae of H. contortus. Stool samples were recorded for FEC and FOB tests (Hexagon, Hematest(®) and Multistix(®)), blood for packed cell volume (PCV), haemoglobin, white and red blood cell count (RBC), and FAMACHA(©) for scoring anaemia. At the end of the experiment lambs were slaughtered to worm burden count. Field infection was achieved in 309 Merino lambs under natural parasite challenge. FEC data were normalized through logarithmic transformation (LnFEC). Pearson correlation was estimated to examine the relationship between all traits. The three tests were able to detect the presence of FOB at day 11. FEC, PCV and RBC decreased to sub-normal values from day 18. FAMACHA(©) score 3 was considered to be indicative of anaemia. Most of the correlations were of high magnitude, with the exception of Multistix(®) test that was moderately correlated with haematological parameters, LnFEC and FEC. In field infection, most samples were negative to FOB tests and the correlations were lower than those calculated under experimental infection. In conclusion, FOB tests were able to detect haemonchosis earlier than FEC under high experimental parasite challenge. However, they were not able to detect FOB under natural mixed parasite challenge. FAMACHA(©) and PCV demonstrated to be good indicators of Haemonchosis, having moderate to high correlations with FEC.

  20. Detection of elephant endotheliotropic herpesvirus infection among healthy Asian elephants (Elephas maximus) in South India.

    PubMed

    Stanton, Jeffrey J; Nofs, Sally A; Zachariah, Arun; Kalaivannan, N; Ling, Paul D

    2014-04-01

    Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in Asian (Elephas maximus) and African (Loxodonta africana) elephants. Of the seven known EEHV species, EEHV1 is recognized as the most common cause of hemorrhagic disease among Asian elephants in human care worldwide. Recent data collected from ex situ Asian elephants located in multiple North American and European institutions suggest that subclinical EEHV1 infection is common in this population of elephants. Although fatal EEHV1-associated hemorrhagic disease has been reported in range countries, data are lacking regarding the prevalence of subclinical EEHV infections among in situ Asian elephants. We used previously validated EEHV-specific quantitative real-time PCR assays to detect subclinical EEHV infection in three regionally distinct Asian elephant cohorts, totaling 46 in situ elephants in South India, during October and November 2011. Using DNA prepared from trunk washes, we detected EEHV1, EEHV3/4, and EEHV5 at frequencies of 7, 9, and 20% respectively. None of the trunk washes was positive for EEHV2 or 6. At least one EEHV species was detectable in 35% (16/46) of the samples that were screened. These data suggest that subclinical EEHV infection among in situ Asian elephants occurs and that Asian elephants may be natural hosts for EEHV1, EEHV3 or 4, and EEHV5, but not EEHV2 and EEHV6. The methodology described in this study provides a foundation for further studies to determine prevalences of EEHV infection in Asian elephants throughout the world. PMID:24484479

  1. Early detection of Haemonchus contortus infection in sheep using three different faecal occult blood tests

    PubMed Central

    Rodríguez, A.V.; Goldberg, V.; Viotti, H.; Ciappesoni, G.

    2015-01-01

    Haemonchus contortus is a blood-sucking parasite causing the presence of faecal occult blood (FOB). The objective was to study three different FOB tests in order to have a new indicator of H. contortus infection in sheep that could be included in the genetic evaluation system as an alternative selection criterion to faecal worm egg count (FEC). A total of 29 Corriedale lambs were experimentally infected with 10.000 larvae of H. contortus. Stool samples were recorded for FEC and FOB tests (Hexagon, Hematest® and Multistix®), blood for packed cell volume (PCV), haemoglobin, white and red blood cell count (RBC), and FAMACHA© for scoring anaemia. At the end of the experiment lambs were slaughtered to worm burden count. Field infection was achieved in 309 Merino lambs under natural parasite challenge. FEC data were normalized through logarithmic transformation (LnFEC). Pearson correlation was estimated to examine the relationship between all traits. The three tests were able to detect the presence of FOB at day 11. FEC, PCV and RBC decreased to sub-normal values from day 18. FAMACHA© score 3 was considered to be indicative of anaemia. Most of the correlations were of high magnitude, with the exception of Multistix® test that was moderately correlated with haematological parameters, LnFEC and FEC. In field infection, most samples were negative to FOB tests and the correlations were lower than those calculated under experimental infection. In conclusion, FOB tests were able to detect haemonchosis earlier than FEC under high experimental parasite challenge. However, they were not able to detect FOB under natural mixed parasite challenge. FAMACHA© and PCV demonstrated to be good indicators of Haemonchosis, having moderate to high correlations with FEC. PMID:26623372

  2. Antibody determination in the diagnosis of Wuchereria bancrofti infection in man

    PubMed Central

    Dissanayake, S.; Ismail, M. M.

    1981-01-01

    The levels of IgG and IgE antibodies reacting with somatic antigens of adult Setaria digitata and Wuchereria bancrofti microfilariae were determined in sera of 90 patients with Bancroftian filariasis and 379 non-filarial subjects. Antibodies reacting with adult antigens and with soluble microfilarial antigens were seen in both microfilaraemic and amicrofilaraemic patients. Antibodies reacting with surface antigens of W. bancrofti microfilariae were seen only in amicrofilaraemic subjects. IgE antibodies were detected with the adult antigen only in both microfilaraemic and amicrofilaraemic patients. The absolute levels of IgG antibodies were significantly higher than those of IgE antibodies. It is concluded that the determination of serum antibodies reacting with adult antigens is suitable for the diagnosis of both the microfilaraemic and amicrofilaraemic phases of infection, and the determination of antibody to microfilarial surface antigens is applicable in patients with clinically evident disease. PMID:7032737

  3. Filarial parasites possess an antizyme but lack a functional ornithine decarboxylase.

    PubMed

    Kurosinski, Marc-André; Lüersen, Kai; Ndjonka, Dieudonne; Younis, Abuelhassan Elshazly; Brattig, Norbert W; Liebau, Eva

    2013-06-01

    In eukaryotes, the key player in polyamine metabolism is the ornithine decarboxylase (ODC) that catalyses the first and rate limiting step in cellular polyamine synthesis. The half life of ODC is strictly regulated by the antizyme (AZ), which promotes its degradation. Older reports on the polyamine situation in filarial parasites indicate a lack of ornithine decarboxylation activity and an increased uptake of polyamines. Our in silico analysis of the Brugia malayi genome revealed only an ODC-like protein that lacks essential residues. Consequently, the recombinant protein had no enzymatic ODC activity. Furthermore, only ODC-like genes were found in the available draft genomes of other filarial parasites. In this ODC-free scenario, we set out to investigate the AZ of O. volvulus (OvAZ). The expression of the recombinant protein allowed us to analyse the localization of OvAZ in different O. volvulus stages as well as to identify it as target for the human humoral immune response. Strong immunostaining was observed in the outer zone of the uterine epithelium as well as in the uterus lumen around the periphery of the developing parasite, indicating a potential role of the OvAZ in the control of polyamine levels during embryonic development. By employing a novel in vivo method using Caenorhabditis elegans, we postulate that the OvAZ enters the secretory pathway. Even though the ODCs are absent in filarial parasites, OvAZ has the ability to bind to various ODCs, thereby demonstrating the functionality of the conserved AZ-binding domains. Finally, pull-down assays show an interaction between B. malayi AZ and the B. malayi ODC-like protein, indicating that the B. malayi ODC-like protein might function as an AZI. Taken together, our results suggest that filarial species do not possess the ODC while retaining the ODC-regulatory proteins AZ and AZI. It is tempting to speculate that both proteins are retained for the regulation of polyamine transport systems. PMID:23474393

  4. Chitosan based substrates for wound infection detection based on increased lysozyme activity.

    PubMed

    Tegl, Gregor; Rollett, Alexandra; Dopplinger, Jasmin; Gamerith, Clemens; Guebitz, Georg M

    2016-10-20

    There is a strong need of point-of-care diagnostics for early detection of wound infection. In this study, substrates based on functionalized chitosan were developed for visual detection of elevated lysozyme activity, an infection biomarker in wound fluids. For efficient hydrolysis by lysozyme, N-acetyl chitosan with a final degree of acetylation of around 50% was synthesized. N-acetylated chitosan and a chitosan-starch composite were labeled with structurally different dyes resulting in lysozyme-responsive biomaterials. Incubation with lysozyme in buffer and artificial wound fluid lead to a release of colored hydrolysis products already after 2h incubation. Tests in human wound fluid from infected wounds indicated a clear visual color change after 2.5h compared to control samples. A higher degree of swelling of the chitosan/starch containing substrate led to faster hydrolysis by lysozyme. This study demonstrates the potential of the lysozyme-responsive materials for diagnosis of wound infection and provides different diagnostic substrates for potential incorporation in point-of-care devices. PMID:27474566

  5. Molecular detection and characterization of piroplasms infecting cervids and chamois in Northern Spain.

    PubMed

    García-Sanmartín, J; Aurtenetxe, O; Barral, M; Marco, I; Lavin, S; García-Pérez, A L; Hurtado, A

    2007-03-01

    Wildlife can act as reservoir of different tick-borne pathogens of veterinary and zoonotic importance. To investigate the role of wild ruminants as reservoir of piroplasm infection, 28 red deer, 69 roe deer and 38 chamois from Northern Spain were examined by reverse line blot (RLB) hybridization. The survey detected a prevalence of 85.7% in red deer, 62.3% in roe deer and 28.9% in chamois. Four different piroplasms were identified: Theileria sp. OT3 (previously described in sheep) as the most prevalent (85.7% in red deer, 46.4% in roe deer and 26.3% in chamois); Theileria sp. 3185/02 (previously described in a red deer in Central Spain) more abundant in red deer (53.6%) than in roe deer (10.1%) but absent from chamois; Babesia divergens detected in 6 roe deer; Theileria ovis present in 1 chamois. Mixed infections (Theileria sp. OT3 and Theileria sp. 3185/02) were only found in red and roe deer. Sequencing analysis of the 18S rRNA gene confirmed the RLB results and showed 99.7% identity between Theileria sp. 3185/02 and T. capreoli, suggesting that they are the same species. Tick distribution and contact of wild ruminants with domestic animals are discussed in terms of piroplasm infection. The results suggest that a considerable number of wildlife ruminants are asymptomatic carriers that may serve as reservoirs of the infection posing a serious concern in terms of piroplasmosis control.

  6. Detection of plant viruses in mixed infection by a macroarray-assisted method.

    PubMed

    Shimura, Hanako; Furuta, Kazuyoshi; Masuta, Chikara

    2015-01-01

    The protocol for a simple, sensitive, and specific method using a cDNA macroarray to detect multiple viruses is provided. The method can be used even at the production sites for crops, which need a reliable routine diagnosis for mixed infection of plant viruses. The method consists of three steps: RNA extraction, duplex RT-PCR, and "microtube hybridization" (MTH). Biotinylated cDNA probes are prepared using RT-PCR and used to hybridize a nylon membrane containing target viral cDNAs by MTH. Positive signals can be visualized by colorimetric reaction and judged by eyes. We here demonstrate this method to detect asparagus viruses (Asparagus virus 1 and Asparagus virus 2) from latently infected asparagus plants. PMID:25287491

  7. Rapid and Selective Detection of Pathogenic Bacteria in Bloodstream Infections with Aptamer-Based Recognition.

    PubMed

    Shen, Haijing; Wang, Jie; Liu, Haoyang; Li, Zhihao; Jiang, Fenglei; Wang, Fu-Bing; Yuan, Quan

    2016-08-01

    Sepsis and bacteremia are life-threatening clinical syndromes associated with significant patient morbidity and mortality. Rapid and sensitive detection of pathogenic bacteria is the key to improve patient survival rates. Herein, we have rationally constructed a simple aptamer-based capture platform to shorten the time needed for confirmation of bacterial bloodstream infection in clinical blood samples. This capture platform is made of a mesoporous TiO2-coated magnetic nanoparticle and is modified with target aptamer. It features excellent bacterial enrichment efficiency of about 80% even at low bacterial concentrations (10-2000 CFU mL(-1)). More importantly, the bacteria can be enriched within 2 h, and the time for bacterial identification is effectively shortened in comparison to the "gold standard" in clinical diagnosis of bloodstream infection. The aptamer-based capture platform may pave a way for the detection of biomarkers and find potential applications in disease diagnosis. PMID:27411775

  8. Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC.

    PubMed

    Muniroh, M S; Sariah, M; Zainal Abidin, M A; Lima, N; Paterson, R R M

    2014-05-01

    Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70°C and medium high power for 30s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations. PMID:24681306

  9. Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC.

    PubMed

    Muniroh, M S; Sariah, M; Zainal Abidin, M A; Lima, N; Paterson, R R M

    2014-05-01

    Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70°C and medium high power for 30s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations.

  10. ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo

    PubMed Central

    Rolle, Anna-Maria; Hasenberg, Mike; Thornton, Christopher R.; Solouk-Saran, Djamschid; Männ, Linda; Weski, Juliane; Maurer, Andreas; Fischer, Eliane; Spycher, Philipp R.; Schibli, Roger; Boschetti, Frederic; Stegemann-Koniszewski, Sabine; Bruder, Dunja; Severin, Gregory W.; Autenrieth, Stella E.; Krappmann, Sven; Davies, Genna; Pichler, Bernd J.; Gunzer, Matthias; Wiehr, Stefan

    2016-01-01

    Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [64Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [64Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [18F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation. PMID:26787852

  11. ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo.

    PubMed

    Rolle, Anna-Maria; Hasenberg, Mike; Thornton, Christopher R; Solouk-Saran, Djamschid; Männ, Linda; Weski, Juliane; Maurer, Andreas; Fischer, Eliane; Spycher, Philipp R; Schibli, Roger; Boschetti, Frederic; Stegemann-Koniszewski, Sabine; Bruder, Dunja; Severin, Gregory W; Autenrieth, Stella E; Krappmann, Sven; Davies, Genna; Pichler, Bernd J; Gunzer, Matthias; Wiehr, Stefan

    2016-02-23

    Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [(64)Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [(64)Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [(18)F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation. PMID:26787852

  12. New Diagnostic Strategies for Detection of Helicobacter pylori Infection in Pediatric Patients

    PubMed Central

    Gold, Benjamin D.; Gilger, Mark A.; Czinn, Steven J.

    2014-01-01

    Helicobacter pylori (H pylori) is a common chronic bacterial infection that is an important cause of peptic ulcer disease and gastroduodenal disease in children. H pylori is also associated with extragastric manifestations, including growth reduction, iron-deficiency anemia, and idiopathic thrombocytopenic purpura. Current guidelines recommend endoscopy with biopsy for the definitive demonstration of H pylori infection. In contrast to serology, the fecal antigen test and the urea breath test provide reliable, sensitive, and specific results for detecting active H pylori infection in children before and after treatment. The first-line treatment option for pediatric patients is triple therapy with a proton pump inhibitor and 2 antibiotics, which include amoxicillin and clarithromycin or metronidazole. Decreasing eradication rates and the emergence of antibiotic-resistant strains of H pylori have led to the use of other treatments, such as sequential therapy or triple therapy with newer antibiotics, particularly in geographic areas with high rates of antibiotic resistance. Patients should be tested after treatment to confirm eradication, as the absence of symptoms does not necessarily mean that H pylori is no longer present. This clinical roundtable monograph provides an overview of H pylori infection, as well as expert insight into the diagnosis and management of H pylori infection in children. PMID:26491414

  13. Detection of mycoplasma infection in circulating tumor cells in patients with hepatocellular carcinoma

    SciTech Connect

    Choi, Hong Seo; Lee, Hyun Min; Kim, Won-Tae; Kim, Min Kyu; Chang, Hee Jin; Lee, Hye Ran; Joh, Jae-Won; Kim, Dae Shick; Ryu, Chun Jeih

    2014-04-04

    Highlights: • This study generates a monoclonal antibody CA27 against the mycoplasmal p37 protein. • CA27 isolates circulating tumor cells (CTCs) from the blood of liver cancer patients. • Results show the first evidence for mycoplasma infected-CTCs in cancer patients. - Abstract: Many studies have shown that persistent infections of bacteria promote carcinogenesis and metastasis. Infectious agents and their products can modulate cancer progression through the induction of host inflammatory and immune responses. The presence of circulating tumor cells (CTCs) is considered as an important indicator in the metastatic cascade. We unintentionally produced a monoclonal antibody (MAb) CA27 against the mycoplasmal p37 protein in mycoplasma-infected cancer cells during the searching process of novel surface markers of CTCs. Mycoplasma-infected cells were enriched by CA27-conjugated magnetic beads in the peripheral blood mononuclear cells in patients with hepatocellular carcinoma (HCC) and analyzed by confocal microscopy with anti-CD45 and CA27 antibodies. CD45-negative and CA27-positive cells were readily detected in three out of seven patients (range 12–30/8.5 ml blood), indicating that they are mycoplasma-infected circulating epithelial cells. CA27-positive cells had larger size than CD45-positive hematological lineage cells, high nuclear to cytoplasmic ratios and irregular nuclear morphology, which identified them as CTCs. The results show for the first time the existence of mycoplasma-infected CTCs in patients with HCC and suggest a possible correlation between mycoplasma infection and the development of cancer metastasis.

  14. Smart central venous port for early detection of bacterial biofilm related infections.

    PubMed

    Paredes, J; Alonso-Arce, M; Schmidt, C; Valderas, D; Sedano, B; Legarda, J; Arizti, F; Gómez, E; Aguinaga, A; Del Pozo, J L; Arana, S

    2014-06-01

    Central venous catheters (CVC) are commonly used in clinical practice to improve a patient's quality of life. Unfortunately, there is an intrinsic risk of acquiring an infection related to microbial biofilm formation inside the catheter lumen. It has been estimated that 80 % of all human bacterial infections are biofilm-associated. Additionally, 50 % of all nosocomial infections are associated with indwelling devices. Bloodstream infections account for 30-40 % of all cases of severe sepsis and septic shock, and are major causes of morbidity and mortality. Diagnosis of bloodstream infections must be performed promptly so that adequate antimicrobial therapy can be started and patient outcome improved. An ideal diagnostic technology would identify the infecting organism(s) in a timely manner, so that appropriate pathogen-driven therapy could begin promptly. Unfortunately, despite the essential information it provides, blood culture, the gold standard, largely fails in this purpose because time is lost waiting for bacterial or fungal growth. This work presents a new design of a venous access port that allows the monitoring of the inner reservoir surface by means of an impedimetric biosensor. An ad-hoc electronic system was designed to manage the sensor and to allow communication with the external receiver. Historic data recorded and stored in the device was used as the reference value for the detection of bacterial biofilm. The RF communication system sends an alarm signal to the external receiver when a microbial colonization of the port occurs. The successful in vitro analysis of the biosensor, the electronics and the antenna of the new indwelling device prototype are shown. The experimental conditions were selected in each case as the closest to the clinical working conditions for the smart central venous catheter (SCVC) testing. The results of this work allow a new generation of this kind of device that could potentially provide more efficient treatments for

  15. Smart central venous port for early detection of bacterial biofilm related infections.

    PubMed

    Paredes, J; Alonso-Arce, M; Schmidt, C; Valderas, D; Sedano, B; Legarda, J; Arizti, F; Gómez, E; Aguinaga, A; Del Pozo, J L; Arana, S

    2014-06-01

    Central venous catheters (CVC) are commonly used in clinical practice to improve a patient's quality of life. Unfortunately, there is an intrinsic risk of acquiring an infection related to microbial biofilm formation inside the catheter lumen. It has been estimated that 80 % of all human bacterial infections are biofilm-associated. Additionally, 50 % of all nosocomial infections are associated with indwelling devices. Bloodstream infections account for 30-40 % of all cases of severe sepsis and septic shock, and are major causes of morbidity and mortality. Diagnosis of bloodstream infections must be performed promptly so that adequate antimicrobial therapy can be started and patient outcome improved. An ideal diagnostic technology would identify the infecting organism(s) in a timely manner, so that appropriate pathogen-driven therapy could begin promptly. Unfortunately, despite the essential information it provides, blood culture, the gold standard, largely fails in this purpose because time is lost waiting for bacterial or fungal growth. This work presents a new design of a venous access port that allows the monitoring of the inner reservoir surface by means of an impedimetric biosensor. An ad-hoc electronic system was designed to manage the sensor and to allow communication with the external receiver. Historic data recorded and stored in the device was used as the reference value for the detection of bacterial biofilm. The RF communication system sends an alarm signal to the external receiver when a microbial colonization of the port occurs. The successful in vitro analysis of the biosensor, the electronics and the antenna of the new indwelling device prototype are shown. The experimental conditions were selected in each case as the closest to the clinical working conditions for the smart central venous catheter (SCVC) testing. The results of this work allow a new generation of this kind of device that could potentially provide more efficient treatments for

  16. Detection and distribution of ostreid herpesvirus 1 in experimentally infected Pacific oyster spat.

    PubMed

    Segarra, Amélie; Baillon, Laury; Faury, Nicole; Tourbiez, Delphine; Renault, Tristan

    2016-01-01

    High mortality rates are reported in spat and larvae of Pacific oyster Crassostrea gigas and associated with ostreid herpesvirus 1 (OsHV-1) detection in France. Although the viral infection has been experimentally reproduced in oyster larvae and spat, little knowledge is currently available concerning the viral entry and its distribution in organs and tissues. This study compares OsHV-1 DNA and RNA detection and localization in experimentally infected oysters using two virus doses: a low dose that did not induce any mortality and a high dose inducing high mortality. Real time PCR demonstrated significant differences in terms of viral DNA amounts between the two virus doses. RNA transcripts were detected in oysters receiving the highest dose of viral suspension whereas no transcript was observed in oysters injected with the low dose. This study also allowed observing kinetics of viral DNA and RNA detection in different tissues of oyster spat. Finally, viral detection was significantly different in function of tissues (p<0.005), time (p<0.005) with an interaction between tissues and time (p<0.005) for each probe. PMID:26674009

  17. Molecular detection of Babesia spp. and other haemoparasitic infections of cattle in Maputo Province, Mozambique.

    PubMed

    Martins, Tiago M; Neves, Luís; Pedro, Olívia C; Fafetine, José M; Do Rosário, Virgílio E; Domingos, Ana

    2010-05-01

    Molecular detection of Babesia species in apparently healthy cattle within an endemic region was carried out in order to determine the prevalence of carriers and the geographical distribution of Babesia bigemina and Babesia bovis in Maputo Province, Mozambique. Samples from 477 animals at 5 localities were analysed using 2 techniques, the semi-nested hot-start PCR and the reverse line blot (RLB) assay. With the semi-nested hot-start PCR, detection of B. bigemina ranged between 30% and 89%, and of B. bovis between 27% and 83%. The RLB assay was comparatively less sensitive in this study and detection of B. bovis ranged from 0% to 17%, and B. bigemina was not detected at all by this technique. Analysis of new sequences of the 18S rRNA gene revealed that the current B. bigemina RLB probe is not specific for the identification of isolates in Mozambique. The RLB assay, however, resulted in the detection of 8 other haemoparasite species belonging to the genera Babesia, Theileria, Anaplasma and Ehrlichia. 18S rRNA gene sequences from the Theileria spp. were identified, and a phylogenic tree constructed with these sequences yielded a heterogeneous T. mutans-like group. In conclusion, infection with B. bigemina and B. bovis is endemic in Maputo Province, but rates of transmission vary. Furthermore, mixed infections with the haemoparasites responsible for several tick-borne diseases in cattle are common in Mozambique. PMID:20128941

  18. Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples.

    PubMed

    Losada-Barragán, Monica; Cavalcanti, Amanda; Umaña-Pérez, Adriana; Porrozzi, Renato; Cuervo-Escobar, Sergio; Vallejo, Andrés Felipe; Sánchez-Gómez, Myriam; Cuervo, Patricia

    2016-08-15

    Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL). VL is the most severe form of leishmaniasis and can be fatal if it is not properly treated. Although several PCR works are intended to detect L. infantum, in silico analysis of available primers and/or primer-probes reveals potential cross species amplification. Here, a TaqMan-based quantitative real time PCR (qPCR) assay was developed for specific detection and quantitation of L. infantum in tissue samples from experimentally or naturally infected animals, mice or dogs, respectively. For this assay, primers and probes were designed for the kinetoplast minicircle DNA of L. infantum. The qPCR assay achieved a detection limit of 0.01pg of parasite DNA, and allowed specific amplification of L. infantum in both asymptomatic and symptomatic naturally infected dogs with inter-assay variation coefficients between 0.05-0.11. There was no cross amplification with dog DNA or with L. braziliensis, L. donovani, L. major, L. tropica or Trypanosoma cruzi. In addition, our assay detected a significantly higher parasite load in symptomatic than in the asymptomatic animals (p<0.0001). We believe this approach will be a valuable tool for the specific detection of L. infantum in regions of sympatric transmission of VL-causing parasites.

  19. Human immunodeficiency virus (HIV) antigen testing to detect HIV infection in female sex workers in Singapore.

    PubMed

    Chan, R K; Ali, K; Thoe, S Y

    1995-07-01

    Human immunodeficiency virus (HIV) infection is characterised by seroconversion after a ¿window¿ period of 2 to 3 months. After this period antibodies are usually detectable by screening tests (enzyme immunoassay or particle agglutination) confirmed by Western blot analysis. We studied 1000 newly enrolled female sex workers who had not been previously tested for HIV to assess the usefulness of HIV antigen testing to improve the efficacy of HIV infection detection. Blood was taken at enrollment for HIV antigen and HIV antibody testing. The Abbott HIVAG-1 test was used to detect antigen; antibody detection was by the Abbott recombinant HIV-1/HIV-2 3rd generation enzyme immunoassay (EIA) test, the Fujirebio Serodia-HIV particle agglutination (PA) test for screening, and the Diagnostic Biotechnology HIV Blot 2.2 Western blot (WB) test for antibody confirmation. Of the 1000 samples, 26 were positive for HIV antibody testing (26/26 for EIA, 25/25 for PA, 26/26 for WB), giving a prevalence rate of 2.6%, Of these 26 seropositive samples 1 was positive on HIV antigen testing. There were no samples which were antigen-positive and antibody-negative. HIV antigen testing does not add to increased efficacy of HIV detection among female sex workers in Singapore.

  20. Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples.

    PubMed

    Losada-Barragán, Monica; Cavalcanti, Amanda; Umaña-Pérez, Adriana; Porrozzi, Renato; Cuervo-Escobar, Sergio; Vallejo, Andrés Felipe; Sánchez-Gómez, Myriam; Cuervo, Patricia

    2016-08-15

    Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL). VL is the most severe form of leishmaniasis and can be fatal if it is not properly treated. Although several PCR works are intended to detect L. infantum, in silico analysis of available primers and/or primer-probes reveals potential cross species amplification. Here, a TaqMan-based quantitative real time PCR (qPCR) assay was developed for specific detection and quantitation of L. infantum in tissue samples from experimentally or naturally infected animals, mice or dogs, respectively. For this assay, primers and probes were designed for the kinetoplast minicircle DNA of L. infantum. The qPCR assay achieved a detection limit of 0.01pg of parasite DNA, and allowed specific amplification of L. infantum in both asymptomatic and symptomatic naturally infected dogs with inter-assay variation coefficients between 0.05-0.11. There was no cross amplification with dog DNA or with L. braziliensis, L. donovani, L. major, L. tropica or Trypanosoma cruzi. In addition, our assay detected a significantly higher parasite load in symptomatic than in the asymptomatic animals (p<0.0001). We believe this approach will be a valuable tool for the specific detection of L. infantum in regions of sympatric transmission of VL-causing parasites. PMID:27514885

  1. Infection.

    PubMed

    Miclau, Theodore; Schmidt, Andrew H; Wenke, Joseph C; Webb, Lawrence X; Harro, Janette M; Prabhakara, Ranjani; Shirtliff, Mark E

    2010-09-01

    Musculoskeletal infection is a clinical problem with significant direct healthcare costs. The prevalence of infection after closed, elective surgery is frequently estimated to be less than 2%, but in severe injuries, posttraumatic infection rates have been reported as 10% or greater. Although clinical infections are found outside the realm of medical devices, it is clear that the enormous increase of infections associated with the use of implants presents a major challenge worldwide. This review summarizes recent advances in the understanding, diagnosis, and treatment of musculoskeletal infections.

  2. Early detection of infected ureteral obstruction after SWL employing C-reactive protein.

    PubMed

    Wolff, J M; Boeckmann, W; Mattelaer, P; Krämer, U; Jakse, G

    1996-12-01

    Infection of the obstructed upper urinary tract is a severe complication of stone disease. Early detection and therapy is crucial to prevent septicemia. The authors investigated prospectively whether C-reactive protein (CRP) might act as a marker for the early detection of infected upper urinary tract obstruction. The serum concentration of CRP was compared with the classic markers of inflammation--white blood cell (WBC) count, erythrocyte sedimentation rate (ESR), and temperature--in the follow-up of 85 patients undergoing 173 extracorporeal shockwave lithotripsy (SWL) treatments. Post-SWL, 69% of the patients showed dilation of the upper tract on ultrasonography and 16.5% became symptomatic and required intervention. In this group, the mean CRP value was increased 18-fold, whereas the WBC count, ESR, and temperature were not markedly raised. In contrast, patients with no need for additional treatment showed no more than a 2-fold increase in CRP. C-Reactive protein seems to be a useful indicator for the early detection of infected upper urinary tract obstruction.

  3. Integrated management of filarial lymphedema for rural communities.

    PubMed

    Narahari, S R; Ryan, T J; Mahadevan, P E; Bose, K S; Prasanna, K S

    2007-03-01

    The Global Alliance for the Elimination of Lymphatic Filariasis (GAELF) has recommended exploring local health traditions of skin care and a low cost treatment paradigm for rural communities has been proposed by Vaqas and Ryan. Our case study incorporates these promising treatments for use in treating filariasis in rural communities. Patients having lymphedema of one or both lower limbs (skin: normal, thickened or with trophic/warty changes) received treatment components from ayurveda, yoga and biomedicine simultaneously: including soap wash, phanta soaking, Indian manual lymph drainage (IMLD), pre- and post-IMLD yoga exercises, and compression using bandages for 194 days, along with diet restrictions and oral herbal medicines indicated for "elephantiasis" in Ayurveda. Entry points when infected were treated with biomedical drugs. The study was conducted in the reverse pharmacology design. 112 patients and 149 lower limbs completed 194 days of treatment during 2003-2006. Significant improvements were observed in the limb circumference measurements and the frequency of acute dermatolymphangioadenitis, use of preventive antibiotics, and reduction in the number of entry points were also improved. The objective to obtain significant benefit for a common problem using locally available, sustainable and affordable means has been achieved. It has not been our purpose to show that the regimen employed is better than another but the results do pose the question--"Are there components of Ayurvedic medicine that deserve further study?" It is important to understand that the regimen has been delivered mostly at home and that participants we have treated, representing a population suffering from a common problem, have not had access to effective conservative therapy that is culturally acceptable, safe, and efficacious. PMID:17539459

  4. Integrated management of filarial lymphedema for rural communities.

    PubMed

    Narahari, S R; Ryan, T J; Mahadevan, P E; Bose, K S; Prasanna, K S

    2007-03-01

    The Global Alliance for the Elimination of Lymphatic Filariasis (GAELF) has recommended exploring local health traditions of skin care and a low cost treatment paradigm for rural communities has been proposed by Vaqas and Ryan. Our case study incorporates these promising treatments for use in treating filariasis in rural communities. Patients having lymphedema of one or both lower limbs (skin: normal, thickened or with trophic/warty changes) received treatment components from ayurveda, yoga and biomedicine simultaneously: including soap wash, phanta soaking, Indian manual lymph drainage (IMLD), pre- and post-IMLD yoga exercises, and compression using bandages for 194 days, along with diet restrictions and oral herbal medicines indicated for "elephantiasis" in Ayurveda. Entry points when infected were treated with biomedical drugs. The study was conducted in the reverse pharmacology design. 112 patients and 149 lower limbs completed 194 days of treatment during 2003-2006. Significant improvements were observed in the limb circumference measurements and the frequency of acute dermatolymphangioadenitis, use of preventive antibiotics, and reduction in the number of entry points were also improved. The objective to obtain significant benefit for a common problem using locally available, sustainable and affordable means has been achieved. It has not been our purpose to show that the regimen employed is better than another but the results do pose the question--"Are there components of Ayurvedic medicine that deserve further study?" It is important to understand that the regimen has been delivered mostly at home and that participants we have treated, representing a population suffering from a common problem, have not had access to effective conservative therapy that is culturally acceptable, safe, and efficacious.

  5. Multiplex Antibody Detection for Noninvasive Genus-Level Diagnosis of Prosthetic Joint Infection

    PubMed Central

    Marmor, Simon; Bauer, Thomas; Desplaces, Nicole; Heym, Beate; Roux, Anne-Laure; Sol, Olivier; Rogé, Julie; Mahé, Florence; Désiré, Laurent; Aegerter, Philippe; Ghout, Idir; Ropers, Jacques; Gaillard, Jean-Louis

    2016-01-01

    We developed and evaluated a multiplex antibody detection-based immunoassay for the diagnosis of prosthetic joint infections (PJIs). Sixteen protein antigens from three Staphylococcus species (Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus lugdunensis) (8 antigens), Streptococcus agalactiae (4 antigens), and Propionibacterium acnes (4 antigens) were selected by comparative immunoproteomics using serum samples from PJI cases versus controls. A bead-based multiplex immunoassay that measured serum IgG against purified, recombinant forms of each of the 16 antigens was developed. We conducted a prospective study to evaluate the performance of the assay. A PJI was defined by the presence of a sinus tract and/or positive intraoperative sample cultures (at least one sample yielding a virulent organism or at least two samples yielding the same organism). A total of 455 consecutive patients undergoing revision or resection arthroplasty (hip, 66.3%; knee, 29.7%; shoulder, 4%) at two French reference centers for the management of PJI were included: 176 patients (38.7%) were infected and 279 (61.3%) were not. About 60% of the infections involved at least one of the species targeted by the assay. The sensitivity/specificity values were 72.3%/80.7% for targeted staphylococci, 75%/92.6% for S. agalactiae, and 38.5%/84.8% for P. acnes. The assay was more sensitive for infections occurring >3 months after arthroplasty and for patients with an elevated C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR). However, it detected 64.3% and 58.3% of targeted staphylococcal infections associated with normal CRP and ESR values, respectively. This new multiplex immunoassay approach is a novel noninvasive tool to evaluate patients suspected of having PJIs and provides information complementary to that from inflammatory marker values. PMID:26865683

  6. Detection of Bovine IgG Isotypes in a PPA-ELISA for Johne's Disease Diagnosis in Infected Herds

    PubMed Central

    Fernández, Bárbara; Gilardoni, Liliana Rosa; Jolly, Ana; Colavecchia, Silvia Beatriz; Paolicchi, Fernando Alberto; Mundo, Silvia Leonor

    2012-01-01

    Johne's Disease or Paratuberculosis is a chronic granulomatous enteritis disease affecting ruminants. Detection of subclinically infected animals is difficult, hampering the control of this disease. The aim of this work was to evaluate the performance of detection of IgG isotypes in a PPA-ELISA to improve the recognition of cattle naturally infected with Map in different stages. A total of 108 animals from Tuberculosis-free herds were grouped as follows: exposed (n = 30), subclinically infected (n = 26), clinically infected (n = 14), and healthy controls (n = 38). Receiver-operating characteristic (ROC) curves of isotypes/PPA-ELISAs were constructed and areas under the curves were compared to evaluate the performance of each test. Our study demonstrated that the conventional PPA-ELISA (detecting IgG) is the best to identify clinically infected animals with high sensitivity (92.9%) and specificity (100%). Meanwhile, IgG2/PPA-ELISA improved the number of subclinically infected cattle detected as compared with conventional IgG/PPA-ELISA (53.8 versus 23.1%). In addition, it had the maximum sensitivity (65.0%, taking into account all Map-infected cattle). In conclusion, the combination of IgG and IgG2/PPA-ELISAs may improve the identification of Map-infected cattle in different stages of disease. The usefulness of IgG2 detection in serological tests for Johne's Disease diagnosis should be further evaluated. PMID:22792511

  7. Bovine vaccinia, a systemic infection: evidence of fecal shedding, viremia and detection in lymphoid organs.

    PubMed

    Rivetti, Anselmo V; Guedes, Maria Isabel M C; Rehfeld, Izabelle S; Oliveira, Tércia M L; Matos, Ana Carolina D; Abrahão, Jônatas S; Kroon, Erna G; Lobato, Zélia I P

    2013-02-22

    Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV) that affects dairy cattle and milkers, causing economic losses and impacting animal and human health. Based on the clinical presentation, BV appears to be a localized disease, with lesions restricted to the skin of affected individuals. However, there are no studies on the pathogenesis of the disease in cows to determine if there is a systemic spread of the virus and if there are different ways of VACV shedding. The objective of this work was to study if there is a systemic spread of VACV in experimentally infected cows and to study the kinetics of VACV circulation in the blood and shedding in the feces of these animals. To this end, eight crossbred lactating cows were used. Three teats of each cow were inoculated with the GP2V strain of VACV. All animals were monitored daily, and blood and fecal samples were collected for 67 days post-infection (dpi). After this period, four of these previously infected cows were immunosuppressed using dexamethasone. Viral DNA was continuously detected and quantified in the blood and feces of these animals in an intermittent way, even after the resolution of the lesions. At slaughter, tissues were collected, and viral DNA was detected and quantified in the mesenteric and retromammary lymph nodes, ileum, spleen and liver. The detection of VACV DNA in the feces for a longer period (67 dpi) and in the lymphatic organs provides new evidence about VACV elimination and suggests that BV could be a systemic infection with a chronic course and viral shedding through the feces.

  8. Automated Extraction Improves Multiplex Molecular Detection of Infection in Septic Patients

    PubMed Central

    Regueiro, Benito J.; Varela-Ledo, Eduardo; Martinez-Lamas, Lucia; Rodriguez-Calviño, Javier; Aguilera, Antonio; Santos, Antonio; Gomez-Tato, Antonio; Alvarez-Escudero, Julian

    2010-01-01

    Sepsis is one of the leading causes of morbidity and mortality in hospitalized patients worldwide. Molecular technologies for rapid detection of microorganisms in patients with sepsis have only recently become available. LightCycler SeptiFast test Mgrade (Roche Diagnostics GmbH) is a multiplex PCR analysis able to detect DNA of the 25 most frequent pathogens in bloodstream infections. The time and labor saved while avoiding excessive laboratory manipulation is the rationale for selecting the automated MagNA Pure compact nucleic acid isolation kit-I (Roche Applied Science, GmbH) as an alternative to conventional SeptiFast extraction. For the purposes of this study, we evaluate extraction in order to demonstrate the feasibility of automation. Finally, a prospective observational study was done using 106 clinical samples obtained from 76 patients in our ICU. Both extraction methods were used in parallel to test the samples. When molecular detection test results using both manual and automated extraction were compared with the data from blood cultures obtained at the same time, the results show that SeptiFast with the alternative MagNA Pure compact extraction not only shortens the complete workflow to 3.57 hrs., but also increases sensitivity of the molecular assay for detecting infection as defined by positive blood culture confirmation. PMID:20967222

  9. Ligase chain reaction to detect Chlamydia trachomatis infection of the cervix.

    PubMed Central

    Schachter, J; Stamm, W E; Quinn, T C; Andrews, W W; Burczak, J D; Lee, H H

    1994-01-01

    We performed a multicenter evaluation of ligase chain reaction (LCR) in the diagnosis of Chlamydia trachomatis infection of the cervix. This LCR provides an amplification of target sequences within the chlamydial cryptic plasmid. The LCR results were compared with those of isolation in cell culture. Discrepant (tissue culture-negative and LCR-positive) test results were resolved by the application of a direct immunofluorescent-antibody test to detect chlamydial elementary bodies and by the use of alternate DNA primers that targeted the chlamydial major outer membrane protein gene. A total of 234 of 2,132 specimens (10.9%) could be confirmed as containing C. trachomatis. Of these, 152 were detected by isolation in cell culture and 221 were detected by LCR. The corresponding sensitivities were 94% for LCR and 65% for cell culture. There was greater variability among study site results for cell culture sensitivity (52 to 92%) than for LCR sensitivity (87 to 98%). The specificity of each test was greater than 99.9%. Thus, LCR offers a highly sensitive nonculture method for detecting chlamydial infection of the cervix. PMID:7814494

  10. Epidemiology of subpatent Plasmodium falciparum infection: implications for detection of hotspots with imperfect diagnostics

    PubMed Central

    2013-01-01

    Background At the local level, malaria transmission clusters in hotspots, which may be a group of households that experience higher than average exposure to infectious mosquitoes. Active case detection often relying on rapid diagnostic tests for mass screen and treat campaigns has been proposed as a method to detect and treat individuals in hotspots. Data from a cross-sectional survey conducted in north-western Tanzania were used to examine the spatial distribution of Plasmodium falciparum and the relationship between household exposure and parasite density. Methods Dried blood spots were collected from consenting individuals from four villages during a survey conducted in 2010. These were analysed by PCR for the presence of P. falciparum, with the parasite density of positive samples being estimated by quantitative PCR. Household exposure was estimated using the distance-weighted PCR prevalence of infection. Parasite density simulations were used to estimate the proportion of infections that would be treated using a screen and treat approach with rapid diagnostic tests (RDT) compared to targeted mass drug administration (tMDA) and Mass Drug Administration (MDA). Results Polymerase chain reaction PCR analysis revealed that of the 3,057 blood samples analysed, 1,078 were positive. Mean distance-weighted PCR prevalence per household was 34.5%. Parasite density was negatively associated with transmission intensity with the odds of an infection being subpatent increasing with household exposure (OR 1.09 per 1% increase in exposure). Parasite density was also related to age, being highest in children five to ten years old and lowest in those > 40 years. Simulations of different tMDA strategies showed that treating all individuals in households where RDT prevalence was above 20% increased the number of infections that would have been treated from 43 to 55%. However, even with this strategy, 45% of infections remained untreated. Conclusion The negative relationship

  11. Detection of Marek's disease virus serotype 1 (MDV1) glycoprotein D in MDV1-infected chick embryo fibroblasts.

    PubMed

    Ono, M; Jang, H K; Maeda, K; Kawaguchi, Y; Tohya, Y; Niikura, M; Mikami, T

    1996-08-01

    Chick embryo fibroblasts (CEFs) infected with three strains of Marek's disease virus serotype 1 (MDV1), GA, Md5 and JM, were subjected to indirect immunofluorescence assay with monoclonal antibodies (MAbs) against MDV1 homolog of glycoprotein D (MDV1 gD) of herpes simplex virus. By the MAbs, a number of MDV1 gD-positive cells were detected in CEFs infected with GA, whereas only a few and no positive cells were detected in CEFs infected with Md5 and JM, respectively. The MDV1 gD in GA-infected CEFs was recognized as the band of 64 kDa in immunoblot analysis using one of the MAbs. This is the first report that the MDV1 gD was detected in MDV1-infected cell cultures.

  12. [A new method for detection and erradication of Helicobacter pylori infection by stool antigens test].

    PubMed

    Amèndola, R; Doweck, J; Katz, J; Racca, J; Menendez, G; Schenone, L; Farìas, R; Barrantes, C; Quintanta, C; Zerbo, O; Kogan, Z; Valero, J; Bartellini, M A; Questa, U; Luna, P; Corti, R E

    2002-01-01

    Nowadays technics for Helicobacter pylori detection in stools like culture, and PCR, are expensive and difficult to perform. The aim of this study was to evaluate ELISA test efficacy for detection of H. Pylori antigens in stools comparing this results with standarized technics like histology (Giemsa), ureasa test and UBT C 14. 26 patients were evaluated in this study, ages between 15-75 with upper gastrointestinal symptoms; all of them required gastroduodenal endoscopy, status H. Pylori was determined with methods upon mentioned. 24 hours after endoscopy H. Pylori antigens in stools with the technique Premier Platinum Htsa, Elisa were determined. The detection of H. Pylori antigens in stools accurately identified active H. Pylori infection. The performance characteristics of this non-invasive method was similar in sensibility and specificity to conventional tests.

  13. Optofluidic analysis system for amplification-free, direct detection of Ebola infection

    NASA Astrophysics Data System (ADS)

    Cai, H.; Parks, J. W.; Wall, T. A.; Stott, M. A.; Stambaugh, A.; Alfson, K.; Griffiths, A.; Mathies, R. A.; Carrion, R.; Patterson, J. L.; Hawkins, A. R.; Schmidt, H.

    2015-09-01

    The massive outbreak of highly lethal Ebola hemorrhagic fever in West Africa illustrates the urgent need for diagnostic instruments that can identify and quantify infections rapidly, accurately, and with low complexity. Here, we report on-chip sample preparation, amplification-free detection and quantification of Ebola virus on clinical samples using hybrid optofluidic integration. Sample preparation and target preconcentration are implemented on a PDMS-based microfluidic chip (automaton), followed by single nucleic acid fluorescence detection in liquid-core optical waveguides on a silicon chip in under ten minutes. We demonstrate excellent specificity, a limit of detection of 0.2 pfu/mL and a dynamic range of thirteen orders of magnitude, far outperforming other amplification-free methods. This chip-scale approach and reduced complexity compared to gold standard RT-PCR methods is ideal for portable instruments that can provide immediate diagnosis and continued monitoring of infectious diseases at the point-of-care.

  14. Enzyme-linked immunosorbent assay for detection of respiratory syncytial virus infection: development and description.

    PubMed Central

    Hendry, R M; McIntosh, K

    1982-01-01

    An indirect enzyme-linked immunosorbent assay for detection of respiratory syncytial virus (RSV) antigens was developed, using commercially available antisera. Horse anti-RSV and calf antiserum to bovine RSV were used as capture and detector antibodies, respectively. The assay could detect as few as 50 PFU of unpurified RSV per ml in infected cell culture supernatant fluids and as little as 10 ng of affinity-purified RSV antigen per ml. No cross-reactions were observed with heterologous virus types. Freeze-thaw treatment had no effect on RSV enzyme-linked immunosorbent assay titers, but viral transport medium inhibited RSV enzyme-linked immunosorbent assay titers from 10- to 100-fold. The assay can be easily performed in 24 h and is a sensitive and specific method for the detection of RSV antigens. PMID:6749894

  15. Detection and analysis of gene expression during infection by in vivo expression technology.

    PubMed Central

    Merrell, D S; Camilli, A

    2000-01-01

    Many limitations associated with the use of in vitro models for study of bacterial pathogenesis can be overcome by the use of technologies that detect pathogen gene expression during the course of infection within an intact animal. In vivo expression technology (IVET) accomplishes this with versatility: it has been developed with a variety of reporter systems which allow for either in vivo selection or ex vivo screening. Selectable gene fusion systems generally allow for the complementation of a bacterial metabolic defect that is lethal in vivo, or for antibiotic resistance during the course of in vivo antibiotic challenge. In contrast, the screenable gene fusion system uses a site-specific DNA recombinase that, when expressed in vivo, excises a selectable gene cassette from the bacterial chromosome. Loss of this cassette can then be either screened or selected for ex vivo. The recombinase-based IVET can be used to detect genes that are transcriptionally induced during infection, including those expressed transiently or at low levels and, in addition, can be used to monitor the spatial and temporal expression of specific genes during the course of infection. PMID:10874732

  16. Detection of Mycolactone A/B in Mycobacterium ulcerans–Infected Human Tissue

    PubMed Central

    Sarfo, Fred Stephen; Phillips, Richard O.; Rangers, Brian; Mahrous, Engy A.; Lee, Richard E.; Tarelli, Edward; Asiedu, Kingsley B.; Small, Pamela L.; Wansbrough-Jones, Mark H.

    2010-01-01

    Background Mycobacterium ulcerans disease (Buruli ulcer) is a neglected tropical disease common amongst children in rural West Africa. Animal experiments have shown that tissue destruction is caused by a toxin called mycolactone. Methodology/Principal Findings A molecule was identified among acetone-soluble lipid extracts from M. ulcerans (Mu)-infected human lesions with chemical and biological properties of mycolactone A/B. On thin layer chromatography this molecule had a retention factor value of 0.23, MS analyses showed it had an m/z of 765.6 [M+Na+] and on MS:MS fragmented to produce the core lactone ring with m/z of 429.4 and the polyketide side chain of mycolactone A/B with m/z of 359.2. Acetone-soluble lipids from lesions demonstrated significant cytotoxic, pro-apoptotic and anti-inflammatory activities on cultured fibroblast and macrophage cell lines. Mycolactone A/B was detected in all of 10 tissue samples from patients with ulcerative and pre-ulcerative Mu disease. Conclusions/Significance Mycolactone can be detected in human tissue infected with Mu. This could have important implications for successful management of Mu infection by antibiotic treatment but further studies are needed to measure its concentration. PMID:20052267

  17. Rhinoviral infection and asthma: the detection and management of rhinoviruses by airway epithelial cells.

    PubMed

    Parker, L C; Stokes, C A; Sabroe, I

    2014-01-01

    Human rhinoviruses (HRV) have been linked to the development of childhood asthma and recurrent acute asthma exacerbations throughout life, and contribute considerably to the healthcare and economic burden of this disease. However, the ability of HRV infections to trigger exacerbations, and the link between allergic status and HRV responsiveness, remains incompletely understood. Whilst the receptors on human airway cells that detect and are utilized by most HRV group A and B, but not C serotypes are known, how endosomal pattern recognition receptors (PRRs) detect HRV replication products that are generated within the cytoplasm remains somewhat of an enigma. In this article, we explore a role for autophagy, a cellular homeostatic process that allows the cell to encapsulate its own cytosolic constituents, as the crucial mechanism controlling this process and regulating the innate immune response of airway epithelial cells to viral infection. We will also briefly describe some of the recent insights into the immune responses of the airway to HRV, focusing on neutrophilic inflammation that is a potentially unwanted feature of the acute response to viral infection, and the roles of IL-1 and Pellinos in the regulation of responses to HRV.

  18. Image-Processing Scheme to Detect Superficial Fungal Infections of the Skin

    PubMed Central

    Mäder, Ulf; Quiskamp, Niko; Wildenhain, Sören; Schmidts, Thomas; Mayser, Peter; Runkel, Frank; Fiebich, Martin

    2015-01-01

    The incidence of superficial fungal infections is assumed to be 20 to 25% of the global human population. Fluorescence microscopy of extracted skin samples is frequently used for a swift assessment of infections. To support the dermatologist, an image-analysis scheme has been developed that evaluates digital microscopic images to detect fungal hyphae. The aim of the study was to increase diagnostic quality and to shorten the time-to-diagnosis. The analysis, consisting of preprocessing, segmentation, parameterization, and classification of identified structures, was performed on digital microscopic images. A test dataset of hyphae and false-positive objects was created to evaluate the algorithm. Additionally, the performance for real clinical images was investigated using 415 images. The results show that the sensitivity for hyphae is 94% and 89% for singular and clustered hyphae, respectively. The mean exclusion rate is 91% for the false-positive objects. The sensitivity for clinical images was 83% and the specificity was 79%. Although the performance is lower for the clinical images than for the test dataset, a reliable and fast diagnosis can be achieved since it is not crucial to detect every hypha to conclude that a sample consisting of several images is infected. The proposed analysis therefore enables a high diagnostic quality and a fast sample assessment to be achieved. PMID:26649072

  19. Experimental infection and detection of necrotizing hepatopancreatitis bacterium in the American lobster Homarus americanus.

    PubMed

    Avila-Villa, Luz A; Gollas-Galván, Teresa; Martínez-Porchas, Marcel; Mendoza-Cano, Fernando; Hernández-López, Jorge

    2012-01-01

    Necrotizing hepatopancreatitis bacterium (NHPB) is an obligated intracellular bacteria causing severe hepatopancreatic damages and mass mortalities in penaeid shrimp. The worldwide distribution of penaeid shrimp as alien species threatens the life cycle of other crustacean species. The aim of the experiment was to evaluate the possibility of experimentally infecting the American lobster (Homarus americanus) with NHPB extracted from shrimp hepatopancreas. Homogenates from infected shrimp were fed by force to lobsters. Other group of lobsters was fed with homogenates of NHPB-free hepatopancreas. After the 15th day from initial inoculation, the presence of NHPB was detected by polymerase chain reaction in feces and hepatopancreas from lobsters inoculated with infected homogenates. Necrotized spots were observed in the surface of lobster hepatopancreas. In contrast, lobsters fed on NHPB-free homogenates resulted negative for NHPB. Evidence suggests the plasticity of NHPB which can infect crustacean from different species and inhabiting diverse latitudes. Considering the results, the American lobster could be a good candidate to maintain available NHPB in vivo.

  20. Fluorescence in situ hybridization rapidly detects three different pathogenic bacteria in urinary tract infection samples.

    PubMed

    Wu, Qing; Li, Yan; Wang, Ming; Pan, Xiao P; Tang, Yong F

    2010-11-01

    The detection of pathogenic bacteria in urine is an important criterion for diagnosing urinary tract infections (UTIs). By using fluorescence in situ hybridization (FISH) with rRNA-targeted, fluorescently labeled oligonucleotide probes, bacterial pathogens present in urine samples were identified within 3-4 h. In this study, three probes that are specific for Escherichia coli, Enterococcus faecalis and Staphylococcus aureus were designed based on the conserved 16S RNA sequences, whereas probe Eub338 broadly recognizes all bacteria. We collected a total of 1000 urine samples, and 325 of these samples tested positive for a UTI via traditional culturing techniques; additionally, all 325 of these samples tested positive with the Eub338 probe in FISH analysis. FISH analyses with species-specific probes were performed in parallel to the test the ability to differentiate among several pathogenic bacteria. The samples for these experiments included 76 E. coli infected samples, 32 E. faecalis infected samples and 9 S. aureus infected samples. Compared to conventional methods of bacterial identification, the FISH method produced positive results for >90% of the samples tested. FISH has the potential to become an extremely useful diagnostic tool for UTIs because it has a quick turnaround time and high accuracy.

  1. Detection of subclinical peste des petits ruminants virus infection in experimental cattle.

    PubMed

    Sen, A; Saravanan, P; Balamurugan, V; Bhanuprakash, V; Venkatesan, G; Sarkar, J; Rajak, K K; Ahuja, A; Yadav, V; Sudhakar, S B; Parida, S; Singh, R K

    2014-01-01

    The present study was undertaken to investigate the possible involvement of cattle in the epidemiology of peste des petits ruminants (PPR) as subclinical carriers. Cattle were exposed experimentally to PPR virus (PPRV) infection or placed in contact with PPR infected goats. Clinical samples including heparinized/EDTA blood, plasma, peripheral blood monocyte cells (PBMCs) and clotted blood (for serum) were collected periodically from 21 days post infection (dpi) to 397 dpi (21, 45, 50, 57, 65, 95, 111, 119, 148, 190, 203 and 397 dpi) and tested for PPRV antigen, nucleic acid and antibody. Exposed cattle seroconverted and maintained PPRV specific haemagglutinin antibodies and detectable PPRV antigen/nucleic acid in blood, plasma and PBMCs from 21 to 397 dpi. PPRV was recovered from blood and PBMC collected from experimental animals at 21 dpi, initially in B95a cells and then adapted to Vero cells. The study indicated that PPRV can infect cattle subclinically and PPRV antigen/nucleic acid persist in cattle for at least 397 days. PMID:25674614

  2. Detection of Vaccinia virus in blood and faeces of experimentally infected cows.

    PubMed

    Guedes, M I M C; Rehfeld, I S; de Oliveira, T M L; Assis, F L; Matos, A C D; Abrahão, J S; Kroon, E G; Lobato, Z I P

    2013-12-01

    Bovine vaccinia (BV), a zoonosis caused by Vaccinia virus (VACV), affects dairy cattle and milkers, causing economic, veterinary and human health impacts. Despite such impacts, there are no experimental studies about the pathogenesis of BV in cows to assess whether there is a systemic spread of the virus and whether there are different ways of VACV shedding. Trying to answer some of these questions, a study was proposed using experimental inoculation of VACV in cows. All experimentally infected cows developed lesions compatible with VACV infection in cattle. Two of the six animals presented VACV DNA in blood and faecal samples, starting at the 2nd and the 3rd day post-infection (d.p.i.), respectively, and lasting until the 36th d.p.i., in an intermittent way. This study provides new evidence that VACV can be detected in blood and faeces of infected cows, suggesting that BV could be a systemic disease, and also bringing new information about the epidemiology and pathogenesis of BV.

  3. A simple isothermal DNA amplification method to screen black flies for Onchocerca volvulus infection.

    PubMed

    Alhassan, Andy; Makepeace, Benjamin L; LaCourse, Elwyn James; Osei-Atweneboana, Mike Y; Carlow, Clotilde K S

    2014-01-01

    Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis. PMID:25299656

  4. A simple isothermal DNA amplification method to screen black flies for Onchocerca volvulus infection.

    PubMed

    Alhassan, Andy; Makepeace, Benjamin L; LaCourse, Elwyn James; Osei-Atweneboana, Mike Y; Carlow, Clotilde K S

    2014-01-01

    Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis.

  5. A Simple Isothermal DNA Amplification Method to Screen Black Flies for Onchocerca volvulus Infection

    PubMed Central

    Alhassan, Andy; Makepeace, Benjamin L.; LaCourse, Elwyn James; Osei-Atweneboana, Mike Y.; Carlow, Clotilde K. S.

    2014-01-01

    Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis. PMID:25299656

  6. Mycobacterium genotypes in pulmonary tuberculosis infections and their detection by trained African giant pouched rats.

    PubMed

    Mgode, Georgies F; Cohen-Bacrie, Stéphan; Bedotto, Marielle; Weetjens, Bart J; Cox, Christophe; Jubitana, Maureen; Kuipers, Dian; Machang'u, Robert S; Kazwala, Rudovick; Mfinanga, Sayoki G; Kaufmann, Stefan H E; Drancourt, Michel

    2015-02-01

    Tuberculosis (TB) diagnosis in low-income countries is mainly done by microscopy. Hence, little is known about the diversity of Mycobacterium spp. in TB infections. Different genotypes or lineages of Mycobacterium tuberculosis vary in virulence and induce different inflammatory and immune responses. Trained Cricetomys rats show a potential for rapid diagnosis of TB. They detect over 28 % of smear-negative, culture-positive TB. However, it is unknown whether these rats can equally detect sputa from patients infected with different genotypes of M. tuberculosis. A 4-month prospective study on diversity of Mycobacterium spp. was conducted in Dar es Salaam, Tanzania. 252 sputa from 161 subjects were cultured on Lowenstein-Jensen medium and thereafter tested by rats. Mycobacterial isolates were subjected to molecular identification and multispacer sequence typing (MST) to determine species and genotypes. A total of 34 Mycobacterium spp. isolates consisting of 32 M. tuberculosis, 1 M. avium subsp. hominissuis and 1 M. intracellulare were obtained. MST analyses of 26 M. tuberculosis isolates yielded 10 distinct MST genotypes, including 3 new genotypes with two clusters of related patterns not grouped by geographic areas. Genotype MST-67, shared by one-third of M. tuberculosis isolates, was associated with the Mwananyamala clinic. This study shows that diverse M. tuberculosis genotypes (n = 10) occur in Dar es Salaam and trained rats detect 80 % of the genotypes. Sputa with two M. tuberculosis genotypes (20 %), M. avium hominissuis and M. intracellulare were not detected. Therefore, rats detect sputa with different M. tuberculosis genotypes and can be used to detect TB in resource-poor countries.

  7. Detection of hemoplasma infection of goats by use of a quantitative polymerase chain reaction assay and risk factor analysis for infection.

    PubMed

    Johnson, Kathy A; do Nascimento, Naíla C; Bauer, Amy E; Weng, Hsin-Yi; Hammac, G Kenitra; Messick, Joanne B

    2016-08-01

    OBJECTIVE To develop and validate a real-time quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma ovis in goats and investigate the prevalence and risk factors for hemoplasma infection of goats located in Indiana. ANIMALS 362 adult female goats on 61 farms. PROCEDURES Primers were designed for amplification of a fragment of the dnaK gene of M ovis by use of a qPCR assay. Blood samples were collected into EDTA-containing tubes for use in total DNA extraction, blood film evaluation, and determination of PCV. Limit of detection, intra-assay variability, interassay variability, and specificity of the assay were determined. RESULTS Reaction efficiency of the qPCR assay was 94.45% (R(2), 0.99; slope, -3.4623), and the assay consistently detected as few as 10 copies of plasmid/reaction. Prevalence of infection in goats on the basis of results for the qPCR assay was 18.0% (95% confidence interval, 14% to 22%), with infected goats ranging from 1 to 14 years old, whereby 61% (95% confidence interval, 47% to 73%) of the farms had at least 1 infected goat. Bacterial load in goats infected with M ovis ranged from 1.05 × 10(3) target copies/mL of blood to 1.85 × 10(5) target copies/mL of blood; however, no bacteria were observed on blood films. Production use of a goat was the only risk factor significantly associated with hemoplasma infection. CONCLUSIONS AND CLINICAL RELEVANCE The qPCR assay was more sensitive for detecting hemoplasma infection than was evaluation of a blood film, and production use of a goat was a risk factor for infection. PMID:27463552

  8. [Comparison of methods of detection of bovine adenovirus serotype 3 in infected culture of calf kidney cells (MDBK)].

    PubMed

    Kaliuzhnaia, A N; Trifonov, V D; Zil'berman, M I; Zalmanzon, E S; Kaledin, A S

    1988-10-01

    The comparative study of the dynamics of the main antigen (hexon) and viral DNA of the bovine adenovirus type 3 accumulation in the established cell line MDBK under the conditions of single- or multistep cycle of infection has been undertaken. The quantitative immunoelectrophoresis and immunoenzyme assay detected the viral antigens on the late stages of infection in the period of cellular monolayer degradation. The immunofluorescence reaction and histochemical immunoenzyme method detected the antigen in the infected cells concurrently with the primary expression of the viral cytopathic effect. The reaction of the spot molecular hybridization with the [32P]-DNA probes detected the viral DNA considerably earlier than the antigen was detected by the immunological methods, before the appearance of degenerative changes in the infected cells. Preference of the immunoenzyme assay and DNA-probes in diagnosis of the virus are discussed.

  9. Influence of temperature on symptom expression, detection of host factors in virus infected Piper nigrum L.

    PubMed

    Umadevi, P; Bhat, A I; Krishnamurthy, K S; Anandaraj, M

    2016-05-01

    Expression of symptoms in black pepper plants (Piper nigrum) infected with Piper yellow mottle virus (PYMoV) vary depending on the season, being high during summer months. Here, we explored the influence of temperature on symptom expression in PYMoV infected P. nigrum. Our controlled environment study revealed increase in virus titer, total proteins, IAA and reducing sugars when exposed to temperature stress. There was change in the 2-D separated protein before and after exposure. The 2-D proteomics LC-MS identified host and viral proteins suggesting virus-host interaction during symptom expression. The analysis as well as detection of host biochemical compounds may help in understanding the detailed mechanisms underlying the viral replication and damage to the crop, and thereby plan management strategies.

  10. Influence of temperature on symptom expression, detection of host factors in virus infected Piper nigrum L.

    PubMed

    Umadevi, P; Bhat, A I; Krishnamurthy, K S; Anandaraj, M

    2016-05-01

    Expression of symptoms in black pepper plants (Piper nigrum) infected with Piper yellow mottle virus (PYMoV) vary depending on the season, being high during summer months. Here, we explored the influence of temperature on symptom expression in PYMoV infected P. nigrum. Our controlled environment study revealed increase in virus titer, total proteins, IAA and reducing sugars when exposed to temperature stress. There was change in the 2-D separated protein before and after exposure. The 2-D proteomics LC-MS identified host and viral proteins suggesting virus-host interaction during symptom expression. The analysis as well as detection of host biochemical compounds may help in understanding the detailed mechanisms underlying the viral replication and damage to the crop, and thereby plan management strategies. PMID:27319055

  11. Infectivity in chimpanzees (Pan troglodytes) of plasma collected before HCV RNA detectability by FDA-licensed assays: implications for transfusion safety and HCV infection outcomes

    PubMed Central

    Murthy, Krishna K.; Kleinman, Steven H.; Hirschkorn, Dale F.; Herring, Belinda L.; Delwart, Eric L.; Racanelli, Vito; Yoon, Joo Chun; Rehermann, Barbara; Alter, Harvey J.

    2012-01-01

    Serial plasma aliquots (50 mL) obtained from 10 commercial donors who converted from hepatitis C virus (HCV) RNA negative to positive were transfused into 2 chimpanzees to assess infectivity during early HCV infection. Plasma, obtained 4 days before HCV RNA detectability by licensed assays, transmitted HCV infection to chimpanzee X355. The infectious PCR-negative plasma was subsequently shown to be positive in 2 of 23 replicates using a sensitive transcription-mediated amplification (TMA) assay, and estimated to contain 1.2 HCV RNA copies/mL (60 copies/50 mL transfused). Plasma units obtained up to 8 weeks earlier were not infectious in a second susceptible chimp, even when from donors with low-level, intermittent HCV RNA detection. Chimp x355 developed acute viremia with subsequent seroconversion, but cleared both virus and Ab in 17 weeks. When rechallenged 38 months later with 6000 RNA copies/mL from the same donor, X355 was transiently reinfected and again rapidly lost all HCV markers. We conclude that: (1) transfusions can transmit HCV infection before RNA detection, but the interval of test-negative infectivity is very brief; (2) early “blips” of HCV RNA appear noninfectious and can be ignored when calculating residual transfusion risk; and (3) markers of HCV infection can be lost rapidly after exposure to low-dose inocula. PMID:22498743

  12. Innate immune detection of flagellin positively and negatively regulates salmonella infection.

    PubMed

    Lai, Marvin A; Quarles, Ellen K; López-Yglesias, Américo H; Zhao, Xiaodan; Hajjar, Adeline M; Smith, Kelly D

    2013-01-01

    Salmonella enterica serovar Typhimurium is a flagellated bacterium and one of the leading causes of gastroenteritis in humans. Bacterial flagellin is required for motility and also a prime target of the innate immune system. Innate immune recognition of flagellin is mediated by at least two independent pathways, TLR5 and Naip5-Naip6/NlrC4/Caspase-1. The functional significance of each of the two independent flagellin recognition systems for host defense against wild type Salmonella infection is complex, and innate immune detection of flagellin contributes to both protection and susceptibility. We hypothesized that efficient modulation of flagellin expression in vivo permits Salmonella to evade innate immune detection and limit the functional role of flagellin-specific host innate defenses. To test this hypothesis, we used Salmonella deficient in the anti-sigma factor flgM, which overproduce flagella and are attenuated in vivo. In this study we demonstrate that flagellin recognition by the innate immune system is responsible for the attenuation of flgM(-) S. Typhimurium, and dissect the contribution of each flagellin recognition pathway to bacterial clearance and inflammation. We demonstrate that caspase-1 controls mucosal and systemic infection of flgM(-) S. Typhimurium, and also limits intestinal inflammation and injury. In contrast, TLR5 paradoxically promotes bacterial colonization in the cecum and systemic infection, but attenuates intestinal inflammation. Our results indicate that Salmonella evasion of caspase-1 dependent flagellin recognition is critical for establishing infection and that evasion of TLR5 and caspase-1 dependent flagellin recognition helps Salmonella induce intestinal inflammation and establish a niche in the inflamed gut.

  13. Preparation and characterization of specific monoclonal antibodies for the detection of adult worm infections in onchocerciasis.

    PubMed

    Cho-Ngwa, Fidelis; Daggfeldt, Annika; Titanji, Vincent P K; Grönvik, Kjell-Olov

    2005-12-01

    Suitable molecular tests for monitoring the viability of adult worms of Onchocerca in vivo are required to accelerate the development of new macrofilaricides in river blindness (onchocerciasis). Hence, three monoclonal antibodies (MAbs) were prepared and evaluated in a sandwich enzyme-linked immunosorbent assay (ELISA) for their abilities to detect circulating adult worm antigens in onchocercal bovine and human sera. The MAbs did not cross-react with a number of control antigens, which included extracts of Ascaris suum, Loa loa, and O. ochengi microfilariae. They were all IgG1 molecules. Their targets in O. ochengi total extract were a set of the same 15 polypeptides with apparent molecular weights of 21-220 Kda. Immunohistochemical studies confirmed their adult worm specificity and showed their binding to the hypodermis of the adult worm. The ELISA could detect as little as 100 pg/mL of the affinity-purified target antigens. It also detected the antigens with 94.1% specificity in 50 out of 56 infected bovine sera (90% sensitivity) and in 21 out of 43 infected human sera (48.8% sensitivity, which could go up to 72.1% on elimination of two skewed control cases). We conclude that the MAbs could be field tested and used in responder populations as described herein or employed as components of more sensitive assays for the evaluation of novel Onchocerca macrofilaricides.

  14. Microscopic Examination of Gallbladder Stones Improves Rate of Detection of Clonorchis sinensis Infection

    PubMed Central

    Ma, Rui-hong; Luo, Xiao-bing; Zheng, Pei-ming; Luo, Zhen-liang; Yang, Liu-qing

    2013-01-01

    To improve the rate of detection of Clonorchis sinensis infection, we compared different specimens from patients with cholecystolithiasis. Feces, gallbladder bile, and gallbladder stones collected from 179 consecutive patients with cholecystolithiasis underwent microscopic examination, and according to the results, 30 egg-positive and 30 egg-negative fecal, gallbladder bile, and gallbladder stone specimens, respectively, underwent real-time fluorescent PCR. The detection rates of eggs in feces, bile, and gallbladder stones were 30.7%, 44.7%, and 69.8%, respectively, and the differences were statistically significant (P < 0.01). The PCR results confirmed that the eggs in the specimens were C. sinensis eggs. Eggs in the feces were “fresh” and in the gallbladder stones were “old.” Microscopic examination of gallbladder stones may improve the detection rates of C. sinensis infection, which is important for developing individualized treatments to prevent the recurrence of gallbladder stones and to prevent the occurrence of severe liver damage and cholangiocarcinoma. PMID:23698535

  15. IFN-γ response on T-cell based assays in HIV-infected patients for detection of tuberculosis infection

    PubMed Central

    2010-01-01

    Background Individuals infected with human immunodeficiency virus (HIV) have an increased risk of progression to active tuberculosis following Mycobacterium tuberculosis infection. The objective of the study was to determine IFN-γ responses for the detection of latent tuberculosis infection (LTBI) with QuantiFERON-TB GOLD In Tube (QFT-G-IT) and T-SPOT.TB in HIV patients, and evaluate the influence of CD4 cell count on tests performance. Methods We studied 75 HIV patients enrolled for ongoing studies of LTBI with T-SPOT.TB, QFN-G-IT and TST. Mean CD4 cell counts ± standard deviation was 461.29 ± 307.49 cells/μl. Eight patients had a BCG scar. Results T-SPOT.TB, QFN-G-IT and TST were positive in 7 (9.3%), 5 (6.7%) and 9 (12%) cases, respectively. Global agreement between QFN-G-IT and T-SPOT.TB was 89% (κ = 0.275). The overall agreement of T-SPOT.TB and QFN-G-IT with TST was 80.8% (κ = 0.019) and 89% (κ = 0.373), respectively. We have found negative IFN-γ assays results among 2 BCG-vaccinated HIV-infected individuals with a positive TST. In non BCG-vaccinated patients, QFN-G-IT and TST were positive in 5 cases (7.5%) and T-SPOT.TB in 7 (10.4%). In contrast, in BCG-vaccinated patients, only TST was positive in 4/8 (50%) of the cases. The differences obtained in the number of positive results between TST and both IFN-γ assays in BCG vaccinated patients were significant (95% CI 3-97%, p = 0.046), however, the confidence interval is very wide given the small number of patients. In patients with CD4< 200, we obtained only one (5%) positive result with T-SPOT.TB; however, QFN-G-IT and TST were negative in all cases. On the contrary, percentages of positive results in patients with CD4> 200 were 10.9% (6/55), 9.1% (5/55) and 16.4% (9/55) with T-SPOT.TB, QFN-G-IT and TST, respectively. Conclusions IFN-γ tests have the benefit over TST that are less influenced by BCG vaccination, consequently they are more specific than TST. Although our number of patients with

  16. Detection of arboviruses and other micro-organisms in experimentally infected mosquitoes using massively parallel sequencing.

    PubMed

    Hall-Mendelin, Sonja; Allcock, Richard; Kresoje, Nina; van den Hurk, Andrew F; Warrilow, David

    2013-01-01

    Human disease incidence attributed to arbovirus infection is increasing throughout the world, with effective control interventions limited by issues of sustainability, insecticide resistance and the lack of effective vaccines. Several promising control strategies are currently under development, such as the release of mosquitoes trans-infected with virus-blocking Wolbachia bacteria. Implementation of any control program is dependent on effective virus surveillance and a thorough understanding of virus-vector interactions. Massively parallel sequencing has enormous potential for providing comprehensive genomic information that can be used to assess many aspects of arbovirus ecology, as well as to evaluate novel control strategies. To demonstrate proof-of-principle, we analyzed Aedes aegypti or Aedes albopictus experimentally infected with dengue, yellow fever or chikungunya viruses. Random amplification was used to prepare sufficient template for sequencing on the Personal Genome Machine. Viral sequences were present in all infected mosquitoes. In addition, in most cases, we were also able to identify the mosquito species and mosquito micro-organisms, including the bacterial endosymbiont Wolbachia. Importantly, naturally occurring Wolbachia strains could be differentiated from strains that had been trans-infected into the mosquito. The method allowed us to assemble near full-length viral genomes and detect other micro-organisms without prior sequence knowledge, in a single reaction. This is a step toward the application of massively parallel sequencing as an arbovirus surveillance tool. It has the potential to provide insight into virus transmission dynamics, and has applicability to the post-release monitoring of Wolbachia in mosquito populations.

  17. Genome-Wide Detection of Fitness Genes in Uropathogenic Escherichia coli during Systemic Infection

    PubMed Central

    Subashchandrabose, Sargurunathan; Smith, Sara N.; Spurbeck, Rachel R.; Kole, Monica M.; Mobley, Harry L. T.

    2013-01-01

    Uropathogenic Escherichia coli (UPEC) is a leading etiological agent of bacteremia in humans. Virulence mechanisms of UPEC in the context of urinary tract infections have been subjected to extensive research. However, understanding of the fitness mechanisms used by UPEC during bacteremia and systemic infection is limited. A forward genetic screen was utilized to detect transposon insertion mutants with fitness defects during colonization of mouse spleens. An inoculum comprised of 360,000 transposon mutants in the UPEC strain CFT073, cultured from the blood of a patient with pyelonephritis, was used to inoculate mice intravenously. Transposon insertion sites in the inoculum (input) and bacteria colonizing the spleen (output) were identified using high-throughput sequencing of transposon-chromosome junctions. Using frequencies of representation of each insertion mutant in the input and output samples, 242 candidate fitness genes were identified. Co-infection experiments with each of 11 defined mutants and the wild-type strain demonstrated that 82% (9 of 11) of the tested candidate fitness genes were required for optimal fitness in a mouse model of systemic infection. Genes involved in biosynthesis of poly-N-acetyl glucosamine (pgaABCD), major and minor pilin of a type IV pilus (c2394 and c2395), oligopeptide uptake periplasmic-binding protein (oppA), sensitive to antimicrobial peptides (sapABCDF), putative outer membrane receptor (yddB), zinc metallopeptidase (pqqL), a shikimate pathway gene (c1220) and autotransporter serine proteases (pic and vat) were further characterized. Here, we report the first genome-wide identification of genes that contribute to fitness in UPEC during systemic infection in a mammalian host. These fitness factors may represent targets for developing novel therapeutics against UPEC. PMID:24339777

  18. Dirofilaria immitis infection of a snow leopard (Uncia uncia) in a Japanese zoo with mitochondrial DNA analysis.

    PubMed

    Murata, Koichi; Yanai, Tokuma; Agatsuma, Takeshi; Uni, Shigehiko

    2003-08-01

    Three dog heartworms (Dirofilaria immitis) were detected in the lumen of the right cardiac ventriculus and of the pulmonary artery of a captive female snow leopard (Uncia uncia) that died of pancreatic carcinoma at a zoo in Japan. Neither clinical respiratory nor circulatory symptoms caused by the heartworm infection were observed. The filarial worms were identified as D. immitis from the morphologic characteristics of the esophagus, the presence of faint longitudinal ridges on the cuticular surface, the situation of vulva posterior to the esophagus, and the measurements of the body. The heartworms from the snow leopard were identical to that of D. immitis from dogs in the sequence of the cytochrome oxidase I region in the mitochondrial DNA. This host record is the first of D. immitis in U. uncia.

  19. Comparison of specimens for detection of porcine reproductive and respiratory syndrome virus infection in boar studs.

    PubMed

    Pepin, B J; Kittawornrat, A; Liu, F; Gauger, P C; Harmon, K; Abate, S; Main, R; Garton, C; Hargrove, J; Rademacher, C; Ramirez, A; Zimmerman, J

    2015-06-01

    Porcine reproductive and respiratory syndrome virus (PRRSV)-contaminated semen from boars is a route of transmission to females, and early detection of PRRSV infection in boars is a key component in sow farm biosecurity. The purpose of this study was to determine the optimum diagnostic specimen(s) for the detection of acute PRRSV infection in boars. Individually housed boars (n = 15) were trained for semen and oral fluid collection and then vaccinated with a commercial PRRSV modified live virus vaccine. Starting on the day of vaccination and for 14 days thereafter, oral fluid specimens were collected daily from all boars. The 15 boars were subdivided into three groups of 5, and serum, blood swabs and 'frothy saliva' were collected at the time of semen collection on a 3-day rotation. Frothy saliva, derived from the submandibular salivary gland, is produced by aroused boars. Semen was centrifuged, and semen supernatant and cell fractions were tested separately. All samples were randomly ordered and then tested by PRRSV real-time quantitative reverse-transcription polymerase chain reaction assay (rRT-PCR) and PRRSV antibody ELISA. In this study, a comparison of serum, blood swab, and oral fluid rRT-PCR results found no statistically significant differences in the onset of detection or proportion of positives, but serum was numerically superior to oral fluids for early detection. Serum and oral fluid provided identical rRT-PCR results at ≥ 5 day post-vaccination. Likewise, the onset of detection of PRRSV antibody in serum, oral fluid and frothy saliva was statistically equivalent, with serum results again showing a numerical advantage. These results showed that the highest assurance of providing PRRSV-negative semen to sow farms should be based on rRT-PCR testing of serum collected at the time of semen collection. This approach can be augmented with oral fluid sampling from a random selection of uncollected boars to provide for statistically valid surveillance of the

  20. Detection of bacteria with molecular methods in prosthetic joint infection: sonication fluid better than periprosthetic tissue.

    PubMed

    Rak, Mitja; KavčIč, Martina; Trebše, Rihard; CőR, Andrej

    2016-08-01

    Background and purpose - The correct diagnosis of prosthetic joint infection (PJI) can be difficult because bacteria form a biofilm on the surface of the implant. The sensitivity of culture from sonication fluid is better than that from periprosthetic tissue, but no comparison studies using molecular methods on a large scale have been performed. We assessed whether periprosthetic tissue or sonication fluid should be used for molecular analysis. Patients and methods - Implant and tissue samples were retrieved from 87 patients who underwent revision operation of total knee or total hip arthroplasty. Both sample types were analyzed using broad-range (BR-) PCR targeting the 16S rRNA gene. The results were evaluated based on the definition of periprosthetic joint infection from the Workgroup of the Musculoskeletal Infection Society. Results - PJI was diagnosed in 29 patients, whereas aseptic failure was diagnosed in 58 patients. Analysis of sonication fluid using BR-PCR detected bacteria in 27 patients, whereas analysis of periprosthetic tissue by BR-PCR detected bacteria in 22 patients. In 6 of 7 patients in whom BR-PCR analysis of periprosthetic tissue was negative, low-virulence bacteria were present. The sensitivity and specificity values for periprosthetic tissue were 76% and 93%, respectively, and the sensitivity and specificity values for sonication fluid were 95% and 97%. Interpretation - Our results suggest that sonication fluid may be a more appropriate sample than periprosthetic tissue for BR-PCR analysis in patients with PJI. However, further investigation is required to improve detection of bacteria in patients with so-called aseptic failure. PMID:27123818

  1. Detection of bacteria with molecular methods in prosthetic joint infection: sonication fluid better than periprosthetic tissue.

    PubMed

    Rak, Mitja; KavčIč, Martina; Trebše, Rihard; CőR, Andrej

    2016-08-01

    Background and purpose - The correct diagnosis of prosthetic joint infection (PJI) can be difficult because bacteria form a biofilm on the surface of the implant. The sensitivity of culture from sonication fluid is better than that from periprosthetic tissue, but no comparison studies using molecular methods on a large scale have been performed. We assessed whether periprosthetic tissue or sonication fluid should be used for molecular analysis. Patients and methods - Implant and tissue samples were retrieved from 87 patients who underwent revision operation of total knee or total hip arthroplasty. Both sample types were analyzed using broad-range (BR-) PCR targeting the 16S rRNA gene. The results were evaluated based on the definition of periprosthetic joint infection from the Workgroup of the Musculoskeletal Infection Society. Results - PJI was diagnosed in 29 patients, whereas aseptic failure was diagnosed in 58 patients. Analysis of sonication fluid using BR-PCR detected bacteria in 27 patients, whereas analysis of periprosthetic tissue by BR-PCR detected bacteria in 22 patients. In 6 of 7 patients in whom BR-PCR analysis of periprosthetic tissue was negative, low-virulence bacteria were present. The sensitivity and specificity values for periprosthetic tissue were 76% and 93%, respectively, and the sensitivity and specificity values for sonication fluid were 95% and 97%. Interpretation - Our results suggest that sonication fluid may be a more appropriate sample than periprosthetic tissue for BR-PCR analysis in patients with PJI. However, further investigation is required to improve detection of bacteria in patients with so-called aseptic failure.

  2. Cerebrospinal fluid (CSF) neuronal biomarkers across the spectrum of HIV infection: hierarchy of injury and detection.

    PubMed

    Peterson, Julia; Gisslen, Magnus; Zetterberg, Henrik; Fuchs, Dietmar; Shacklett, Barbara L; Hagberg, Lars; Yiannoutsos, Constantin T; Spudich, Serena S; Price, Richard W

    2014-01-01

    The character of central nervous system (CNS) HIV infection and its effects on neuronal integrity vary with evolving systemic infection. Using a cross-sectional design and archived samples, we compared concentrations of cerebrospinal fluid (CSF) neuronal biomarkers in 143 samples from 8 HIV-infected subject groups representing a spectrum of untreated systemic HIV progression and viral suppression: primary infection; four groups of chronic HIV infection neuroasymptomatic (NA) subjects defined by blood CD4+ T cells of >350, 200-349, 50-199, and <50 cells/µL; HAD; treatment-induced viral suppression; and 'elite' controllers. Samples from 20 HIV-uninfected controls were also examined. The neuronal biomarkers included neurofilament light chain protein (NFL), total and phosphorylated tau (t-tau, p-tau), soluble amyloid precursor proteins alpha and beta (sAPPα, sAPPβ) and amyloid beta (Aβ) fragments 1-42, 1-40 and 1-38. Comparison of the biomarker changes showed a hierarchy of sensitivity in detection and suggested evolving mechanisms with progressive injury. NFL was the most sensitive neuronal biomarker. Its CSF concentration exceeded age-adjusted norms in all HAD patients, 75% of NA CD4<50, 40% of NA CD4 50-199, and 42% of primary infection, indicating common neuronal injury with untreated systemic HIV disease progression as well as transiently during early infection. By contrast, only 75% of HAD subjects had abnormal CSF t-tau levels, and there were no significant differences in t-tau levels among the remaining groups. sAPPα and β were also abnormal (decreased) in HAD, showed less marked change than NFL with CD4 decline in the absence of HAD, and were not decreased in PHI. The CSF Aβ peptides and p-tau concentrations did not differ among the groups, distinguishing the HIV CNS injury profile from Alzheimer's disease. These CSF biomarkers can serve as useful tools in selected research and clinical settings for patient classification, pathogenetic analysis

  3. Optical biosensor system for the quick and reliable detection of virus infections: VIROSENS

    NASA Astrophysics Data System (ADS)

    Proll, Günther; Hartjes, Anja; Sinclair, Alexander; Markovic, Goran; Pröll, Florian; Patel, Pranav; Niedrig, Matthias

    2014-10-01

    Viral infections are of special threat because they can induce severe courses of disease but only few medical treatments are available. Because of socio-economic and climate changes, increased worldwide mobility and population growth, the risk of newly occurring and quickly spreading viral pathogens has increased. A diagnosis of these diseases at an early stage is essential for a quick risk assessment and a proper health management as well as patient's treatment in an optimal way. Currently, the diagnosis of such diseases is based on time consuming and costly detection methods that can only be performed by specially trained personnel in laboratories at specific security levels. Aim of the project VIROSENS is the development of a biosensor platform that can specifically detect virus particles as well as virus-specific antibodies out of biological matrices like blood, serum, plasma and other body fluids. For this purpose, a disposable cartridge for such antibody- and virus-arrays is designed and developed within the project. The optical detection of viruses is performed with a portable device that will be benchmarked and evaluated concerning currently used standard detection methods in terms of its analytical performance. Within this project, a novel combination of serological tests and direct detection of virus particles will be developed, which will provide faster and more reliable results than presently available and used test systems.

  4. Metagenomic analysis for detecting pathogens in culture-negative infective endocarditis.

    PubMed

    Fukui, Yuto; Aoki, Kotaro; Okuma, Shinnosuke; Sato, Takahiro; Ishii, Yoshikazu; Tateda, Kazuhiro

    2015-12-01

    Pathogen identification is important for proper diagnosis and optimal treatment of infective endocarditis (IE). Blood and valve cultures are the gold standard for detecting pathogens responsible for IE. However, these tests only detect culturable pathogens, and have low sensitivity, especially in patients previously treated with antibiotics. Culture-negative IE is still a major clinical problem and a diagnostic challenge. Recently, metagenomic analysis using next generation sequencing has been used to detect pathogens directly from clinical samples. However, there are very few reports of the use of metagenomic analysis for pathogen identification in culture-negative IE cases and the usefulness of this new method is unknown. Here, we report a case of successful pathogen detection with metagenomic analysis in a patient of culture-negative IE. The patient underwent valve replacement surgery and received antibiotics for 5 weeks and survived. Using metagenomic analysis of resected vegetation, we detected Abiotrophia defectiva, which is often associated with culture-negative IE due to its fastidious growth. This method may be useful for pathogen identification in future cases of culture-negative IE.

  5. Validation of Urine Test for Detection of Helicobacter pylori Infection in Indonesian Population

    PubMed Central

    Syam, Ari Fahrial; Miftahussurur, Muhammad; Uwan, Willy Brodus; Simanjuntak, David; Uchida, Tomohisa; Yamaoka, Yoshio

    2015-01-01

    We measured the accuracy of the urine test (RAPIRUN) for detection of Helicobacter pylori infection in Indonesia (Jakarta, Pontianak, and Jayapura) using histology confirmed by immunohistochemistry and/or culture as gold standards. We also used immunohistochemistry to identify CagA phenotype and analyzed H. pylori CagA diversity in Indonesia. The overall prevalence of H. pylori infection in 88 consecutive dyspeptic patients based on the urine test was 15.9% (14/88), 38.1% for patients in Jayapura that had higher prevalence of H. pylori infection than that in Jakarta (9.7%, P = 0.02) and Pontianak (8.3%, P = 0.006). Overall sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of RAPIRUN were 83.3%, 94.7%, 71.4%, 97.3%, and 93.2%, respectively. All of the H. pylori-positive patients were immunoreactive for anti-CagA antibody but not immunoreactive for East Asian specific anti-CagA antibody in all H. pylori-positive subjects. We confirmed the high accuracy of RAPIRUN in Indonesian population. In general, we found less virulent type of H. pylori in Indonesia, which partly explained the low incidence gastric cancer in Indonesia. PMID:26824034

  6. Border disease virus shedding and detection in naturally infected Pyrenean chamois (Rupicapra pyrenaica).

    PubMed

    Cabezón, Oscar; Rosell, Rosa; Velarde, Roser; Mentaberre, Gregorio; Casas-Díaz, Encarna; Lavín, Santiago; Marco, Ignasi

    2010-09-01

    Pyrenean chamois (Rupicapra pyrenaica) populations of the central and eastern Pyrenees have been affected by severe outbreaks associated with Border disease virus (BDV) since 2001. Eight Pyrenean chamois (7 males and 1 female) from 1 to 8 years of age with clinical signs consistent with BDV infection were studied. At necropsy, whole blood, tissue samples (skin, brain, prescapular lymph node, thyroid gland, lung, liver, spleen, kidney, small intestine, bone marrow, and testicle), urine, and nasal, oral, and rectal swabs were obtained. The fetus from a pregnant female was also studied. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the virus in all samples, and virus isolation was performed. Sera and tissue samples were positive to RT-PCR, and the virus was isolated from all chamois. The nasal, oral, and rectal swabs and urine samples were RT-PCR positive in 100%, 85.71%, 71.43%, and 100% of chamois, respectively, confirming the excretion of the virus via these 4 routes. In addition, sera were tested for BDV antibodies using enzyme-linked immunosorbent assay and seroneutralization techniques, with negative results. Sequence analysis of the 5' untranslated region in 7 of the chamois confirmed that the virus is grouped into the BDV-4 genotype, the same BDV previously described in Pyrenean chamois. To the authors' knowledge, this is the first study of naturally infected Pyrenean chamois, providing evidence that infected animals shed BDV through nasal, oral, fecal, and urinary excretion routes.

  7. Scintigraphic detection of bone and joint infections with indium-111-labeled nonspecific polyclonal human immunoglobulin G

    SciTech Connect

    Oyen, W.J.; Claessens, R.A.; van Horn, J.R.; van der Meer, J.W.; Corstens, F.H. )

    1990-04-01

    The utility of indium-111-({sup 111}In) labeled immunoglobulin G (IgG) to detect infection of bone and adjacent tissues was investigated. Proof of infection was obtained by cultures taken at surgery. All 32 patients showed focally increased uptake on the technetium-99m- (99mTc) methylene diphosphonate (MDP) skeletal scintigraphies. Labeled immunoglobulin correctly identified presence, location, extent and soft-tissue involvement of the suspected inflammatory site. In these patients, focally increasing accumulation was noted over 48 hr. Discrimination between infection and sterile inflammatory lesions was not possible. Two fractures, 6-mo-old, and an aseptic loosening of a total-hip prosthesis were not visualized. Side effects after the immunoglobulin administration were not observed. Radiolabeled immunoglobulin is a new and safe radiopharmaceutical for the investigation of infectious bone and joint disease. The sensitivity of this agent appears at least as high as that of labeled leukocytes. However, labeled immunoglobulin can easily be prepared in every nuclear medicine department.

  8. Detection of Different Bovine Papillomavirus Types and Co-infection in Bloodstream of Cattle.

    PubMed

    Santos, E U D; Silva, M A R; Pontes, N E; Coutinho, L C A; Paiva, S S L; Castro, R S; Freitas, A C

    2016-02-01

    Bovine papillomavirus (BPV) is a diverse group of double-stranded DNA oncogenic viruses. BPVs are classically described as epitheliotropic, however, they have been detected in body fluids, such as blood and semen. The presence of BPV in these sites can have implications for the dissemination of BPV. The aim of this study was to verify the prevalence of BPV types in cattle blood. A total of 57 blood samples were analyzed by PCR using BPV type-specific primers to BPVs 1-6 and 8-10, and subsequent sequencing. Sequencing quality was determined using Staden package with Phred 20. Similarity analysis was performed with BioEdit and BLAST programs to assess the identity with known BPV types. Statistical analysis was performed by Fisher's exact test. The results showed seven different types of BPVs in the blood, with the exception of BPV 5 and 9. This is the first study that demonstrates BPVs 3, 6, 8 and 10 DNA in cattle blood. BPVs 1 and 2 were the viral types most frequent in blood, while BPVs 4 and 10 were the least frequent types. All the samples showed co-infection by at least two BPV types. These data suggest that several BPV types may infect blood cells at the same time and demonstrate the possibility that the BPV infection in non-epithelial tissue can occur without restriction to one or two viral types. These results can contribute to future studies aimed at the control and prevention of papillomaviruses.

  9. Usefulness of serological ELISA assay for Taenia saginata to detect naturally infected bovines.

    PubMed

    Paulan, Silvana de Cássia; Gonzáles, Rutilia Marisela Hernándes; Peralta, Laura Adalid; Vicentini-Oliveira, Josy Campanhã; Biondi, Germano Francisco; Conde, Edda Sciuto; Parkhouse, Robert Michael Evans; Nunes, Cáris Maroni

    2013-01-01

    Bovine cysticercosis, a cosmopolitan disease caused by Taenia saginata, leads to economic losses due to carcass devaluation at slaughter. Sanitary inspection at slaughterhouses, the routine diagnostic method in Brazil, lacks the necessary sensitivity to detect the mildly infected cattle that are typically encoutered in Brazil. In this study we have tested cattle sera from animals diagnosed as positive and negative by veterianry inspection for (1) anti-parasite antibodies using metacestodes antigens (T. solium vesicular fluid and T. saginata secretions) and (2) the HP10 secreted antigen of viable metacestodes. The cut-off values were calculated by ROC curve for intense and mild infections conditions, and by the classical method ( for negative samples). The sensitivity and specificity of these diagnostic tests were different depending on the assumed cut-off value and, importantly, whether the infection was mild or intense. In spite of these observations, however, such ELISA assays for serum antibodies and parasite antigens constitute an important tool for epidemiological porposes, and in establishing priorities for the control of bovine cysticercosis. PMID:23802239

  10. [Hepatitis B virus genotype E infection in Turkey: the detection of the first case].

    PubMed

    Sayan, Murat; Sanlıdağ, Tamer; Akçalı, Sinem; Arıkan, Ayşe

    2014-10-01

    Hepatitis B virus (HBV) infection is a global major health problem. Currently, 10 genotypes (A-J) of hepatitis B virus (HBV) are identified based on the nucleic acid sequence heterogeneity, and these genotypes have been shown to have distinct geographic distribution. Reports of the previous studies indicated that the genotype D is the predominant type among hepatitis B patients in different regions of Turkey. However, recent studies indicated that other HBV genotypes are also seen with an increasing rate. Although epidemiological and clinical information on genotype E infection is currently limited, it is known that genotype E infection is common in West and Central Africa. In this report, the first case of HBV genotype E infection in Turkey was presented. A 22-year-old Nigerian male employee who resided in Manisa for five years was admitted to Celal Bayar University Hospital Manisa, Turkey, for his routine check-up. Since HBsAg was found positive, other HBV markers were tested with a repeated serum sample. Laboratory findings were as follows; HBsAg (+), anti-HBs (-), HBeAg (-), anti-HBe (+), anti-HBc (+), anti-HCV (-), anti-HIV (-), ALT: 44 U/L and AST: 45 U/L. HBV-DNA level was detected as 700 IU/ml by real-time PCR (Artus HBV QS RGQ Qiagen, Germany). HBV-DNA isolated from the serum sample of the patient was amplified by PCR and polymerase gene segment of HBV was directly sequenced. UPGMA method was used for phylogenetic analysis and Inno-LIPA HBV genotyping method (Innogenetics, Belgium) was performed to determine multiple HBV genotype infection. On the basis of those methods the genotype of the virus was identified as genotype E. The partial sequences of the HBV polymerase gene were loaded to the international DNA data bank (GenBank) for contribution to the global HBV surveillance. This report emphasized that besides genotype D the other HBV genotypes could be found in Turkey. Since the patient was an inactive HBsAg carrier before his residence in Turkey, this

  11. [Hepatitis B virus genotype E infection in Turkey: the detection of the first case].

    PubMed

    Sayan, Murat; Sanlıdağ, Tamer; Akçalı, Sinem; Arıkan, Ayşe

    2014-10-01

    Hepatitis B virus (HBV) infection is a global major health problem. Currently, 10 genotypes (A-J) of hepatitis B virus (HBV) are identified based on the nucleic acid sequence heterogeneity, and these genotypes have been shown to have distinct geographic distribution. Reports of the previous studies indicated that the genotype D is the predominant type among hepatitis B patients in different regions of Turkey. However, recent studies indicated that other HBV genotypes are also seen with an increasing rate. Although epidemiological and clinical information on genotype E infection is currently limited, it is known that genotype E infection is common in West and Central Africa. In this report, the first case of HBV genotype E infection in Turkey was presented. A 22-year-old Nigerian male employee who resided in Manisa for five years was admitted to Celal Bayar University Hospital Manisa, Turkey, for his routine check-up. Since HBsAg was found positive, other HBV markers were tested with a repeated serum sample. Laboratory findings were as follows; HBsAg (+), anti-HBs (-), HBeAg (-), anti-HBe (+), anti-HBc (+), anti-HCV (-), anti-HIV (-), ALT: 44 U/L and AST: 45 U/L. HBV-DNA level was detected as 700 IU/ml by real-time PCR (Artus HBV QS RGQ Qiagen, Germany). HBV-DNA isolated from the serum sample of the patient was amplified by PCR and polymerase gene segment of HBV was directly sequenced. UPGMA method was used for phylogenetic analysis and Inno-LIPA HBV genotyping method (Innogenetics, Belgium) was performed to determine multiple HBV genotype infection. On the basis of those methods the genotype of the virus was identified as genotype E. The partial sequences of the HBV polymerase gene were loaded to the international DNA data bank (GenBank) for contribution to the global HBV surveillance. This report emphasized that besides genotype D the other HBV genotypes could be found in Turkey. Since the patient was an inactive HBsAg carrier before his residence in Turkey, this

  12. Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections.

    PubMed

    Bernotienė, Rasa; Palinauskas, Vaidas; Iezhova, Tatjana; Murauskaitė, Dovilė; Valkiūnas, Gediminas

    2016-04-01

    Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemosporida) predominate in wildlife, and such infections are particularly virulent. However, currently used polymerase chain reaction (PCR)-based detection methods often do not read mixed infections. Sensitivity of different PCR assays in detection of mixed infections has been insufficiently tested, but this knowledge is essential in studies addressing parasite diversity in wildlife. Here, we applied five different PCR assays, which are broadly used in wildlife avian haemosporidian research, and compared their sensitivity in detection of experimentally designed mixed infections of Haemoproteus and Plasmodium parasites. Three of these PCR assays use primer sets that amplify fragments of cytochrome b gene (cyt b), one of cytochrome oxidase subunit I (COI) gene, and one target apicoplast genome. We collected blood from wild-caught birds and, using microscopic and PCR-based methods applied in parallel, identified single infections of ten haemosporidian species with similar parasitemia. Then, we prepared 15 experimental mixes of different haemosporidian parasites, which often are present simultaneously in wild birds. Similar concentration of total DNA was used in each parasite lineage during preparation of mixes. Positive amplifications were sequenced, and the presence of mixed infections was reported by visualising double-base calling in sequence electropherograms. This study shows that the use of each single PCR assay markedly underestimates biodiversity of haemosporidian parasites. The application of at least 3 PCR assays in parallel detected the majority, but still not all lineages present in mixed infections. We determined preferences of different primers in detection of parasites belonging to different genera of haemosporidians during mixed infections.

  13. First detection of autochthonous Zika virus transmission in a HIV-infected patient in Rio de Janeiro, Brazil.

    PubMed

    Calvet, Guilherme A; Filippis, Ana Maria B; Mendonça, Marcos Cesar L; Sequeira, Patricia C; Siqueira, Andre M; Veloso, Valdilea G; Nogueira, Rita M; Brasil, Patrícia

    2016-01-01

    Since May 2015, Brazil's Ministry of Health has reported autochthonous transmission of Zika virus (ZIKV) in some states of the country. Simultaneous circulation of Dengue, Chikungunya and ZIKV in the country hinder both the diagnosis and the therapeutic approach of patients seeking care with acute febrile illnesses especially in patients with comorbidities. The association between HIV infection and endemic diseases has been described especially in tropical regions with varying levels of complications, although there has been no report of ZIKV in HIV-infected patients. We report the first autochthonous case of laboratory confirmed ZIKV infection in a HIV-infected patient in Rio de Janeiro, Brazil. He evolved with only mild symptoms and recovered well without major laboratory abnormalities. Phylogenetic analysis of the ZIKV detected in the patient sera clustered within the Asian clade. To the best of our knowledge, this is the first time that Zika virus co-infection is reported in a HIV-infected patient.

  14. Oral Human Papillomavirus Detection in Older Adults Who Have Human Immunodeficiency Virus Infection

    PubMed Central

    Fatahzadeh, Mahnaz; Schlecht, Nicolas F.; Chen, Zigui; Bottalico, Danielle; McKinney, Sharod; Ostoloza, Janae; Dunne, Anne; Burk, Robert D.

    2014-01-01

    Objective To evaluate reproducibility of oral rinse self-collection for HPV detection and investigate associations between oral HPV, oral lesions, immune and sociodemographic factors, we performed a cross-sectional study of older adults with HIV infection. Study Design We collected oral rinse samples from 52 subjects at two different times of day followed by an oral examination and interview. We identified HPV using PCR platforms optimized for detection of mucosal and cutaneous types. Results Eighty seven percent of individuals had oral HPV, of which 23% had oncogenic alpha, 40% had non-oncogenic alpha, and 46% had beta or gamma HPV. Paired oral specimens were concordant in all parameters tested. Significant associations observed for oral HPV with increased HIV viral load, hepatitis-C seropositivity, history of sexually transmitted diseases and lifetime number of sexual partners. Conclusions Oral cavity may be a reservoir of subclinical HPV in older adults who have HIV infection. Understanding natural history, transmission and potential implications of oral HPV warrants further investigations. PMID:23375488

  15. The lateral flow card test: an alternative method for the detection of Trichinella infection in swine.

    PubMed

    Patrascu, I; Gamble, H R; Sofronic-Milosavljevic, L; Radulescu, R; Andrei, A; Ionescu, V; Timoceanu, V; Boireau, P; Cuperlovic, K; Djordjevic, M; Murrell, K D; Noeckler, K; Pozio, E

    2001-06-01

    A novel lateral flow card (TS-Card pork) test was developed for the serological detection of Trichinella infected pigs. Based on extensive studies performed in Romania during 1999-2000 this test proved to be highly specific sensitive, rapid (3-12 minutes) and easy to use (no need for laboratory facilities). It can be used both for the detection of Trichinella infection in carcasses and for epizooliological studies using a variety of samples including whole or dried blood, serum, or tissue fluids. The TS-Card pork test, used as a screening test, can be the foundation of an on-farm or field based inspection system to significantly improve food safety in countries with a high prevalence of Trichinella in pigs or other food animal species. The results presented are also promising for application of the test in an on-line laboratory based inspection system since the speed of the test allows sufficient time to rail out suspected hog carcasses during the slaughter process. PMID:11484368

  16. Detection of multiple viral infections in cattle and buffalo with suspected vesicular disease in Brazil.

    PubMed

    Laguardia-Nascimento, Mateus; Sales, Érica Bravo; Gasparini, Marcela Ribeiro; de Souza, Natália Mendes; da Silva, Josiane Aparecida Gonçalina; Souza, Giovana Gonçalves; Carani, Fernanda Rezek; Dos Santos, Alyane Figueiredo; Rivetti Júnior, Anselmo Vasconcelos; Camargos, Marcelo Fernandes; Fonseca Júnior, Antônio Augusto

    2016-07-01

    Vesicular diseases are of high importance for livestock, primarily because of foot-and-mouth disease (FMD), which is a high-morbidity disease that generates direct losses caused by low milk production, weight loss, and indirect losses because of the need for sanitary barriers. Other vesicular diseases are also of importance for livestock because of direct impacts or because their clinical signs may be confused with those of FMD. We report herein the detection of multiple infections in cattle with suspected vesicular disease in the Brazilian states of Amazonas (AM), Mato Grosso (MT), and Roraima. Thirty-seven epithelial samples from cattle and 1 sample from a buffalo were sent to the laboratory for testing for FMDV and similar disease agents. All samples from MT were positive for parapoxvirus (Pseudocowpox virus and Bovine papular stomatitis virus). In addition, 3 samples were positive for Bluetongue virus, and 5 samples were positive for Bovine herpesvirus 1 Among these samples, 1 was positive for all of these 3 agents. Only 2 samples from AM were negative for parapoxvirus. The molecular tests conducted in this study detected multiple infections, with a high prevalence of parapoxvirus. PMID:27154321

  17. Updated guidelines for using Interferon Gamma Release Assays to detect Mycobacterium tuberculosis infection - United States, 2010.

    PubMed

    Mazurek, Gerald H; Jereb, John; Vernon, Andrew; LoBue, Phillip; Goldberg, Stefan; Castro, Kenneth

    2010-06-25

    n 2005, CDC published guidelines for using the QuantiFERON-TB Gold test (QFT-G) (Cellestis Limited, Carnegie, Victoria, Australia) (CDC. Guidelines for using the QuantiFERON-TB Gold test for detecting Mycobacterium tuberculosis infection, United States. MMWR;54[No. RR-15]:49-55). Subsequently, two new interferon gamma (IFN- gamma) release assays (IGRAs) were approved by the Food and Drug Administration (FDA) as aids in diagnosing M. tuberculosis infection, both latent infection and infection manifesting as active tuberculosis. These tests are the QuantiFERON-TB Gold In-Tube test (QFT-GIT) (Cellestis Limited, Carnegie, Victoria, Australia) and the T-SPOT.TB test (T-Spot) (Oxford Immunotec Limited, Abingdon, United Kingdom). The antigens, methods, and interpretation criteria for these assays differ from those for IGRAs approved previously by FDA. For assistance in developing recommendations related to IGRA use, CDC convened a group of experts to review the scientific evidence and provide opinions regarding use of IGRAs. Data submitted to FDA, published reports, and expert opinion related to IGRAs were used in preparing these guidelines. Results of studies examining sensitivity, specificity, and agreement for IGRAs and TST vary with respect to which test is better. Although data on the accuracy of IGRAs and their ability to predict subsequent active tuberculosis are limited, to date, no major deficiencies have been reported in studies involving various populations. This report provides guidance to U.S. public health officials, health-care providers, and laboratory workers for use of FDA-approved IGRAs in the diagnosis of M. tuberculosis infection in adults and children. In brief, TSTs and IGRAs (QFT-G, QFT-GIT, and T-Spot) may be used as aids in diagnosing M. tuberculosis infection. They may be used for surveillance purposes and to identify persons likely to benefit from treatment. Multiple additional recommendations are provided that address quality control, test

  18. Laboratory and field evaluation of medicinal plant extracts against filarial vector, Culex quinquefasciatus Say (Diptera: Culicidae).

    PubMed

    Kovendan, Kalimuthu; Murugan, Kadarkarai; Panneerselvam, Chellasamy; Mahesh Kumar, Palanisamy; Amerasan, Duraisamy; Subramaniam, Jayapal; Vincent, Savariar; Barnard, Donald R

    2012-06-01

    The present study explored the effects of Jatropha curcas, Hyptis suaveolens, Abutilon indicum, and Leucas aspera tested against third instar larvae of filarial vector, Culex quinquefasciatus. The dried plant materials were powdered by an electrical blender. From each sample, 500 g powder was macerated with 1.5 L of hexane, chloroform, ethyl acetate, and methanol 8h, using Soxhlet apparatus, and filtered. The extracts were concentrated at reduced temperature on a rotary evaporator and stored at a temperature of 4°C. The yield of crude extract was 11.4, 12.2, 10.6, and 13.5 g in hexane, chloroform, ethyl acetate, and methanol, respectively. The hexane, chloroform, ethyl acetate, and methanol extract of J. curcas with LC(50) values of 230.32, 212.85, 192.07, and 113.23 ppm; H. suaveolens with LC(50) values of 213.09, 217.64, 167.59, and 86.93 ppm; A. indicum with LC(50) values of 204.18, 155.53, 166.32, and 111.58 ppm; and L. aspera with LC(50) values of 152.18, 118.29, 111.43, and 107.73 ppm, respectively, against third instar larvae of C. quinquefasciatus. The larval mortality was observed after 24 h of exposure. Maximum larvicidal activity was observed in the methanolic extract followed by ethyl acetate, chloroform, and hexane extract. No mortality was observed in the control. The observed mortality were statistically significant at P < 0.05 level. L. aspera showed the highest mortality rate against the mosquito larvae in laboratory and field. The larval density was decreased after the treatment of plant extracts at the breeding sites (sewage water), and hence, these plant extracts of the suitable alternatives of synthetic insecticides for the mosquito vector management. The present results suggest that the medicinal plants extract was an excellent potential for controlling filarial vector, C. quinquefasciatus. PMID:22146997

  19. Detection of respiratory viruses and Bordetella bronchiseptica in dogs with acute respiratory tract infections.

    PubMed

    Schulz, B S; Kurz, S; Weber, K; Balzer, H-J; Hartmann, K

    2014-09-01

    Canine infectious respiratory disease (CIRD) is an acute, highly contagious disease complex caused by a variety of infectious agents. At present, the role of viral and bacterial components as primary or secondary pathogens in CIRD is not fully understood. The aim of this study was to investigate the prevalence of canine parainfluenza virus (CPIV), canine adenovirus type 2 (CAV-2), canine influenza virus (CIV), canine respiratory coronavirus (CRCoV), canine herpes virus-1 (CHV-1), canine distemper virus (CDV) and Bordetella bronchiseptica in dogs with CIRD and to compare the data with findings in healthy dogs. Sixty-one dogs with CIRD and 90 clinically healthy dogs from Southern Germany were prospectively enrolled in this study. Nasal and pharyngeal swabs were collected from all dogs and were analysed for CPIV, CAV-2, CIV, CRCoV, CHV-1, CDV, and B. bronchiseptica by real-time PCR. In dogs with acute respiratory signs, 37.7% tested positive for CPIV, 9.8% for CRCoV and 78.7% for B. bronchiseptica. Co-infections with more than one agent were detected in 47.9% of B. bronchiseptica-positive, 82.6% of CPIV-positive, and 100% of CRCoV-positive dogs. In clinically healthy dogs, 1.1% tested positive for CAV-2, 7.8% for CPIV and 45.6% for B. bronchiseptica. CPIV and B. bronchiseptica were detected significantly more often in dogs with CIRD than in clinically healthy dogs (P < 0.001 for each pathogen) and were the most common infectious agents in dogs with CIRD in Southern Germany. Mixed infections with several pathogens were common. In conclusion, clinically healthy dogs can carry respiratory pathogens and could act as sources of infection for susceptible dogs. PMID:24980809

  20. Detection of virulent Rhodococcus equi in exhaled air samples from naturally infected foals.

    PubMed

    Muscatello, G; Gilkerson, J R; Browning, G F

    2009-03-01

    Virulent Rhodococcus equi causes pyogranulomatous bronchopneumonia in foals. The route of infection of foals has been considered to be inhalation of aerosolized bacteria from soil that is contaminated with equine feces. Thus, disease caused by R. equi has been regarded as an opportunistic infection of environmental origin and not a contagious disease. In this study, we report the exhalation of virulent R. equi from the respiratory tract of naturally infected foals. A handheld air-monitoring system was used to recover virulent R. equi from the exhaled breath of foals, and the concentration of virulent R. equi organisms in exhaled air was compared to the concentration in environmental air samples taken from the holding pens and lane areas on farms. R. equi strains carrying the vapA gene of the virulence plasmid were detected by using colony blotting and DNA hybridization techniques in cultures of exhaled air from 67% (37/55) of foals tested. The concentration of virulent R. equi organisms in exhaled air from foals was significantly higher than that in environmental air (P<0.001). There were no significant differences in the median concentrations of virulent R. equi bacteria exhaled by clinically healthy or diseased foals. The high concentrations of virulent R. equi bacteria in exhaled air suggested that aerosol transmission between foals is possible and may have a significant impact on the prevalence of R. equi pneumonia on farms. The air sampling technique described is potentially useful as a noninvasive method for the detection and quantification of virulent R. equi in the respiratory tract of foals.

  1. Detection of bacterial infection of agave plants by laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    Cervantes-Martinez, Jesus; Flores-Hernandez, Ricardo; Rodriguez-Garay, Benjamin; Santacruz-Ruvalcaba, Fernando

    2002-05-01

    Greenhouse-grown plants of Agave tequilana Weber var. azul were inoculated with Erwinia carotovora, the causal agent of stem soft rot. We investigated the laser-induced fluorescence (LIF) of agave plants to determine whether LIF can be used as a noninvasive sensing tool for pathological studies. The LIF technique was also investigated as a means of detecting the effect of the polyamine biosynthesis inhibitor beta-hydroxyethylhydrazine as a bactericide against the pathogenic bacterium Erwinia carotovora. A He-Ne laser at 632.8 nm was used as the excitation source, and in vivo fluorescence emission spectra were recorded in the 660-790-range. Fluorescence maxima were at 690 and 740 nm. The infected plants that were untreated with the bactericide showed a definite increase in fluorescence intensity at both maxima within the first three days after infection. Beginning on the fifth day, a steady decrease in fluorescence intensity was observed, with a greater effect at 740 than at 690 nm. After 30 days there was no fluorescence. The infected plants that had been treated with the bactericide showed no significant change in fluorescence compared with that of the uninfected plants. The ratio of fluorescence intensities was determined to be F 690 nm/F 740 nm for all treatments. These studies indicate that LIF measurements of agave plants may be used for the early detection of certain types of disease and for determining the effect of a bactericide on bacteria. The results also showed that fluorescence intensity ratios can be used as a reliable indicator of the progress of disease.

  2. Detection of respiratory viruses and Bordetella bronchiseptica in dogs with acute respiratory tract infections.

    PubMed

    Schulz, B S; Kurz, S; Weber, K; Balzer, H-J; Hartmann, K

    2014-09-01

    Canine infectious respiratory disease (CIRD) is an acute, highly contagious disease complex caused by a variety of infectious agents. At present, the role of viral and bacterial components as primary or secondary pathogens in CIRD is not fully understood. The aim of this study was to investigate the prevalence of canine parainfluenza virus (CPIV), canine adenovirus type 2 (CAV-2), canine influenza virus (CIV), canine respiratory coronavirus (CRCoV), canine herpes virus-1 (CHV-1), canine distemper virus (CDV) and Bordetella bronchiseptica in dogs with CIRD and to compare the data with findings in healthy dogs. Sixty-one dogs with CIRD and 90 clinically healthy dogs from Southern Germany were prospectively enrolled in this study. Nasal and pharyngeal swabs were collected from all dogs and were analysed for CPIV, CAV-2, CIV, CRCoV, CHV-1, CDV, and B. bronchiseptica by real-time PCR. In dogs with acute respiratory signs, 37.7% tested positive for CPIV, 9.8% for CRCoV and 78.7% for B. bronchiseptica. Co-infections with more than one agent were detected in 47.9% of B. bronchiseptica-positive, 82.6% of CPIV-positive, and 100% of CRCoV-positive dogs. In clinically healthy dogs, 1.1% tested positive for CAV-2, 7.8% for CPIV and 45.6% for B. bronchiseptica. CPIV and B. bronchiseptica were detected significantly more often in dogs with CIRD than in clinically healthy dogs (P < 0.001 for each pathogen) and were the most common infectious agents in dogs with CIRD in Southern Germany. Mixed infections with several pathogens were common. In conclusion, clinically healthy dogs can carry respiratory pathogens and could act as sources of infection for susceptible dogs.

  3. Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs.

    PubMed

    Colombo, Fabio A; Odorizzi, Rosa M F N; Laurenti, Marcia D; Galati, Eunice A B; Canavez, Flavio; Pereira-Chioccola, Vera L

    2011-08-01

    The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination. PMID:21221638

  4. Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii

    PubMed Central

    Louis, M.; Guitard, J.; Jodar, M.; Ancelle, T.; Magne, D.; Lascols, O.

    2015-01-01

    Quantitative PCR (qPCR) is now a key diagnostic tool for Pneumocystis pneumonia. However, cutoffs to distinguish between infected and colonized patients according to their HIV status have not yet been determined. According to clinical, radiological, and biological data, we retrospectively classified bronchoalveolar lavage (BAL) samples subjected to qPCR over a 3-year period into four categories, i.e., definite PCP, probable PCP, Pneumocystis colonization, and no infection. Fungal burden was then analyzed according to the HIV status of the patients. Among 1,212 episodes of pneumonia screened in immunocompromised patients, 52 and 27 HIV-positive patients were diagnosed with a definite and probable PCP, whereas 4 and 22 HIV-negative patients had definite and probable PCP, respectively. Among patients with definite or a probable PCP, HIV-negative patients had a significantly lower burden than HIV-positive patients (P < 10−4). In both groups, the median fungal burden was significantly higher in patients with definite PCP than in colonized patients. A single cutoff at 1.5 × 104 copies/ml allowed to differentiate colonized and infected HIV-positive patients with 100% sensitivity and specificity. In HIV-negative patients, cutoff values of 2.87 × 104 and 3.39 × 103 copies/ml resulted in 100% specificity and sensitivity, respectively. Using cutoffs determined for the whole population would have led us to set aside the diagnosis of PCP in 9 HIV-negative patients with definite or probable PCP. qPCR appeared to be the most sensitive test to detect Pneumocystis in BAL samples. However, because of lower inocula in HIV-negative patients, different cutoffs must be used according to the HIV status to differentiate between colonized and infected patients. PMID:26468505

  5. Evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection.

    PubMed Central

    Rowe, C L; Baker, S C; Nathan, M J; Fleming, J O

    1997-01-01

    High-frequency RNA recombination has been proposed as an important mechanism for generating viral deletion variants of murine coronavirus. Indeed, a number of variants with deletions in the spike glycoprotein have been isolated from persistently infected animals. However, the significance of generating and potentially accumulating deletion variants in the persisting viral RNA population is unclear. To study this issue, we evaluated the evolution of spike variants by examining the population of spike RNA sequences detected in the brains and spinal cords of mice inoculated with coronavirus and sacrificed at 4, 42, or 100 days postinoculation. We focused on the S1 hypervariable region since previous investigators had shown that this region is subject to recombination and deletion. RNA isolated from the brains or spinal cords of infected mice was rescued by reverse transcription-PCR, and the amplified products were cloned and used in differential colony hybridizations to identify individual isolates with deletions. We found that 11 of 20 persistently infected mice harbored spike deletion variants (SDVs), indicating that deletions are common but not required for persistent infection. To determine if a specific type of SDV accumulated during persistence, we sequenced 106 of the deletion isolates. We identified 23 distinct patterns of SDVs, including 5 double-deletion variants. Furthermore, we found that each mouse harbored distinct variants in its central nervous system (CNS), suggesting that SDVs are generated during viral replication in the CNS. Interestingly, mice with the most severe and persisting neurological disease harbored the most prevalent and diverse quasispecies of SDVs. Overall, these findings illustrate the complexity of the population of persisting viral RNAs which may contribute to chronic disease. PMID:9060655

  6. Early detection of foot-and-mouth disease virus from infected cattle using a dry filter air sampling system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foot-and-mouth disease (FMD) is a highly contagious livestock disease of high economic impact. Early detection of FMD virus (FMDV) is fundamental for rapid outbreak control. Air sampling collection has been demonstrated as a useful technique for detection of FMDV RNA in infected animals, related to ...

  7. Bacteroides fragilis in biopsies of patients with major abscesses and diabetic foot infections: direct molecular versus culture-based detection.

    PubMed

    Stappers, Mark H T; Hagen, Ferry; Reimnitz, Peter; Mouton, Johan W; Meis, Jacques F; Gyssens, Inge C

    2016-06-01

    Direct determination by pathogen-specific real-time PCR assay for Bacteroides fragilis was compared to culture in major abscess and diabetic foot infection biopsy samples. Real-time PCR resulted in an increased detection rate of 12% for B. fragilis and could improve the detection of B. fragilis in clinical samples. PMID:27112830

  8. Molecular detection and characterization of Theileria infection in cattle and yaks from Tibet Plateau Region, China.

    PubMed

    Qin, Gege; Li, Youquan; Liu, Junlong; Liu, Zhijie; Yang, Jifei; Zhang, Lin; Liu, Guangyuan; Guan, Guiquan; Luo, Jianxun; Yin, Hong

    2016-07-01

    Theileriosis continues to threaten the livestock industry worldwide, but comprehensive epidemiological surveys for this disease have not been conducted in the Tibet Plateau Region, China. In this study, we screened 154 cattle blood samples from the Tibet Plateau Region (Lhasa, Lhoka, and Tianzhu), China, for detection of Theileria pathogens by polymerase chain reaction (PCR) with species-specific primers. The results revealed that the prevalence was 6.9 % (2/29) for Theileria orientalis and 27.6 % (8/29) for Theileria sinensis in Lhasa, 0 % (0/30) for T. orientalis and 26.7 % (8/30) for T. sinensis in Lhoka, and 0 % (0/95) for T. orientalis and 30.5 % (29/95) for T. sinensis in Tianzhu. Interestingly, Theileria luwenshuni, which was a previously reported pathogenic Theileria sp. in sheep and goats, was detected in blood samples from cattle and yaks for the first time, with a prevalence of 10 % (3/30) in Lhoka and 1.1 % (1/95) in Tianzhu. No other Theileria sp. was detected in these samples. T. sinensis and T. orientalis infections were detected in cattle and yaks, and T. luwenshuni was discovered for the first time in cattle and yaks in the Tibet Plateau Region, China. PMID:27000088

  9. A single polyprobe for detecting simultaneously eight pospiviroids infecting ornamentals and vegetables.

    PubMed

    Torchetti, Enza Maria; Navarro, Beatriz; Di Serio, Francesco

    2012-12-01

    The spread of viroids belonging to the genus Pospiviroid (family Pospiviroidae), recorded recently in ornamentals and vegetables in several European countries, calls for fast, efficient and sensitive detection methods. Based on bioinformatics analyses of sequence identity among all pospiviroids, a digoxigenin-labeled polyprobe (POSPIprobe) was developed that, when tested by dot-blot and Northern-blot hybridization, detected Potato spindle tuber viroid, Citrus exocortis viroid, Columnea latent viroid, Mexican papita viroid, Tomato planta macho viroid, Tomato apical stunt viroid, Pepper chat fruit viroid and Chrysanthemum stunt viroid. The end-point detection limits of the POSPIprobe ranged from 5(-2) to 5(-4), and from 5(-1) to 5(-3) for nucleic acid preparations obtained by phenol extraction and silica-capture, respectively, similar to those of single probes. Based on sequence identity, the POSPIprobe is expected to detect also the two pospiviroid species not tested in this study (Tomato chlorotic dwarf viroid and Iresine viroid-1). Dot-blot assays with the POSPIprobe were validated by testing 68 samples from tomato, chrysanthemum and argyranthemum infected by different pospiviroids as revealed by RT-PCR, thus confirming the potential of this polyprobe for quarantine, certification and survey programs. PMID:22935607

  10. Detection of infective poliovirus by a simple, rapid, and sensitive flow cytometry method based on fluorescence resonance energy transfer technology.

    PubMed

    Cantera, Jason L; Chen, Wilfred; Yates, Marylynn V

    2010-01-01

    The rapid and effective detection of virus infection is critical for clinical management and prevention of disease spread during an outbreak. Several methods have been developed for this purpose, of which classical serological and viral nucleic acid detection are the most common. We describe an alternative approach that utilizes engineered cells expressing fluorescent proteins undergoing fluorescence resonance energy transfer (FRET) upon cleavage by the viral 2A protease (2A(pro)) as an indication of infection. Quantification of the infectious-virus titers was resolved by using flow cytometry, and utility was demonstrated for the detection of poliovirus 1 (PV1) infection. Engineered buffalo green monkey kidney (BGMK) cells expressing the cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) substrate linked by a cleavage recognition site for PV1 2A(pro) were infected with different titers of PV1. After incubation at various time points, cells were harvested, washed, and subjected to flow cytometry analysis. The number of infected cells was determined by counting the number of cells with an increased CFP-to-YFP ratio. As early as 5 h postinfection, a significant number of infected cells (3%) was detected by flow cytometry, and cells infected with only 1 PFU were detected after 12 h postinfection. When applied to an environmental water sample spiked with PV1, the flow cytometry-based assay provided a level of sensitivity similar to that of the plaque assay for detecting and quantifying infectious virus particles. This approach, therefore, is more rapid than plaque assays and can be used to detect other viruses that frequently do not form clear plaques on cell cultures.

  11. E4 Antibodies Facilitate Detection and Type-Assignment of Active HPV Infection in Cervical Disease

    PubMed Central

    Marnane, Rebecca; Dewar, Vincent; Molijn, Anco; Quint, Wim; Van Hoof, Christine; Struyf, Frank; Colau, Brigitte; Jenkins, David; Doorbar, John

    2012-01-01

    High-risk human papillomavirus (HPV) infections are the cause of nearly all cases of cervical cancer. Although the detection of HPV DNA has proved useful in cervical diagnosis, it does not necessarily predict disease presence or severity, and cannot conclusively identify the causative type when multiple HPVs are present. Such limitations may be addressed using complementary approaches such as cytology, laser capture microscopy, and/or the use of infection biomarkers. One such infection biomarker is the HPV E4 protein, which is expressed at high level in cells that are supporting (or have supported) viral genome amplification. Its distribution in lesions has suggested a role in disease staging. Here we have examined whether type-specific E4 antibodies may also allow the identification and/or confirmation of causal HPV-type. To do this, type-specific polyclonal and monoclonal antibodies against three E4 proteins (HPV-16, -18, and -58) were generated and validated by ELISA and western blotting, and by immunohistochemistry (IHC) staining of epithelial rafts containing these individual HPV types. Type-specific detection of HPV and its associated disease was subsequently examined using formalin-fixed paraffin-embedded cervical intra-epithelial neoplasias (CIN, (n = 247)) and normal controls (n = 28). All koilocytotic CIN1 lesions showed type-specific E4 expression of their respective HPV types. Differences were noted amongst E4 expression patterns in CIN3. HPV-18 E4 was not detected in any of the 6 HPV-18 DNA-positive CIN3 lesions examined, whereas in HPV-16 and -58 CIN3, 28/37 (76%) and 5/9 (55.6%) expressed E4 respectively, usually in regions of epithelial differentiation. Our results demonstrate that type-specific E4 antibodies can be used to help establish causality, as may be required when multiple HPV types are detected. The unique characteristics of the E4 biomarker suggest a role in diagnosis and patient management particularly when used in combination

  12. Evaluation of three rapid diagnostic tests for the detection of human infections with Plasmodium knowlesi

    PubMed Central

    2014-01-01

    Background Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, infects humans and can cause fatal malaria. It is difficult to diagnose by microscopy because of morphological similarity to Plasmodium malariae. Nested PCR assay is the most accurate method to distinguish P. knowlesi from other Plasmodium species but is not cost effective in resource-poor settings. Rapid diagnostic tests (RDTs) are recommended for settings where malaria is prevalent. In this study, the effectiveness of three RDTs in detecting P. knowlesi from fresh and frozen patient blood samples was evaluated. Methods Forty malaria patients (28 P. knowlesi, ten P. vivax and two P. falciparum) diagnosed by microscopy were recruited in Sarawak, Malaysian Borneo during a 16-month period. Patient blood samples were used to determine parasitaemia by microscopy, confirm the Plasmodium species present by PCR and evaluate three RDTs: OptiMAL-IT, BinaxNOW® Malaria and Paramax-3. The RDTs were also evaluated using frozen blood samples from 41 knowlesi malaria patients. Results OptiMAL-IT was the most sensitive RDT, with a sensitivity of 71% (20/28; 95% CI = 54-88%) for fresh and 73% (30/41; 95% CI = 59-87%) for frozen knowlesi samples. However, it yielded predominantly falciparum-positive results due to cross-reactivity of the P. falciparum test reagent with P. knowlesi. BinaxNOW® Malaria correctly detected non-P. falciparum malaria in P. knowlesi samples but was the least sensitive, detecting only 29% (8/28; 95% CI = 12-46%) of fresh and 24% (10/41; 95% CI = 11-37%) of frozen samples. The Paramax-3 RDT tested positive for P. vivax with PCR-confirmed P. knowlesi samples with sensitivities of 40% (10/25; 95% CI = 21-59%) with fresh and 32% (13/41; 95% CI = 17-46%) with frozen samples. All RDTs correctly identified P. falciparum- and P. vivax-positive controls with parasitaemias above 2,000 parasites/μl blood. Conclusions The RDTs detected Plasmodium in P. knowlesi-infected blood samples with

  13. Evaluation and Field Validation of PCR Tests for Detection of Actinobacillus pleuropneumoniae in Subclinically Infected Pigs

    PubMed Central

    Fittipaldi, Nahuel; Broes, André; Harel, Josée; Kobisch, Marylène; Gottschalk, Marcelo

    2003-01-01

    Eight PCR tests were evaluated for their abilities to detect Actinobacillus pleuropneumoniae in swine tonsils. At first they were compared regarding their specificities by using A. pleuropneumoniae and related bacterial species and their analytical sensitivities by using tonsils experimentally infected in vitro. PCRs were carried out both directly with tonsil homogenates (direct PCR) and after culture of the sample (after-culture PCR). Most tests demonstrated good specificities; however, some tests gave false-positive results with some non-A. pleuropneumoniae species. High degrees of variation in the analytical sensitivities among the tests were observed for the direct PCRs (109 to 102 CFU/g of tonsil), whereas those of most of the after-culture PCRs were similar (102 CFU/g of tonsil). In a second phase, the effects of sample storage time and storage conditions were evaluated by using tonsils from experimentally infected animals. Storage at −20°C allowed the detection of the organism for at least 4 months. Finally, the omlA PCR test described by Savoye et al. (C. Savoye et al., Vet. Microbiol. 73:337-347, 2000) and the commercially available Adiavet App PCR test were further validated with field samples. Their effectiveness was compared to those of standard and immunomagnetic separation-based methods of bacterial isolation. In addition, a comparison of tonsil biopsy specimens (from living animals) and whole tonsils (collected at the slaughterhouse) was also conducted. A. pleuropneumoniae was neither isolated nor detected by PCR from a herd serologically negative for A. pleuropneumoniae. PCR was more sensitive than the standard isolation method with whole tonsils from three infected herds. After-culture PCR offered the highest degree of sensitivity (93 and 83% for the omlA and Adiavet App PCRs, respectively). The PCR detection rate was higher with whole tonsils than with tonsil biopsy specimens. Good agreement (κ = 0.65) was found between the presence of A

  14. Development of a PCR Assay for Detection of Yersinia ruckeri in Tissues of Inoculated and Naturally Infected Trout

    PubMed Central

    Gibello, A.; Blanco, M. M.; Moreno, M. A.; Cutuli, M. T.; Domenech, A.; Domínguez, L.; Fernández-Garayzábal, J. F.

    1999-01-01

    A PCR-based method was developed for the specific detection of Yersinia ruckeri in tissues of inoculated trout and naturally infected trout. No amplification products were obtained with other yersiniae, bacterial fish pathogens, or phylogenetically related bacteria (n = 34). The sensitivity of PCR detection was 60 to 65 bacterial cells per PCR tube, which was decreased to 10 to 20 cells by hybridization with a nonradioactive probe. The PCR assay proved to be as reliable as and faster than the conventional culture method for the detection of Y. ruckeri in infected trout tissues. PMID:9872807

  15. Evaluation of a Commercial Multiplex PCR for Rapid Detection of Multi Drug Resistant Gram Negative Infections

    PubMed Central

    Chavada, Ruchir; Maley, Michael

    2015-01-01

    Introduction: Community and healthcare associated infections caused by multi-drug resistant gram negative organisms (MDR GN) represent a worldwide threat. Nucleic Acid Detection tests are becoming more common for their detection; however they can be expensive requiring specialised equipment and local expertise. This study was done to evaluate the utility of a commercial multiplex tandem (MT) PCR for detection of MDR GN. Methods: The study was done on stored laboratory MDR GN isolates from sterile and non-sterile specimens (n=126, out of stored 567 organisms). Laboratory validation of the MT PCR was done to evaluate sensitivity, specificity and agreement with the current phenotypic methods used in the laboratory. Amplicon sequencing was also done on selected isolates for assessing performance characteristics. Workflow and cost implications of the MT PCR were evaluated. Results: The sensitivity and specificity of the MT PCR were calculated to be 95% and 96.7% respectively. Agreement with the phenotypic methods was 80%. Major lack of agreement was seen in detection of AmpC beta lactamase in enterobacteriaceae and carbapenemase in non-fermenters. Agreement of the MT PCR with another multiplex PCR was found to be 87%. Amplicon sequencing confirmed the genotype detected by MT PCR in 94.2 % of cases tested. Time to result was faster for the MT PCR but cost per test was higher. Conclusion: This study shows that with carefully chosen targets for detection of resistance genes in MDR GN, rapid and efficient identification is possible. MT PCR was sensitive and specific and likely more accurate than phenotypic methods. PMID:26464612

  16. The Heme Biosynthetic Pathway of the Obligate Wolbachia Endosymbiont of Brugia malayi as a Potential Anti-filarial Drug Target

    PubMed Central

    Wu, Bo; Novelli, Jacopo; Foster, Jeremy; Vaisvila, Romualdas; Conway, Leslie; Ingram, Jessica; Ganatra, Mehul; Rao, Anita U.; Hamza, Iqbal; Slatko, Barton

    2009-01-01

    Background Filarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti) are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole) can interrupt transmission predominantly by killing microfilariae (mf) larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs. Methods and Findings Genome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP), which targets ferrochelatase (FC, the last step). Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a ∼600-fold difference in drug sensitivities to succinyl acetone (SA) between Wolbachia and human 5

  17. Comparison between DNA Detection in Trigeminal Nerve Ganglia and Serology to Detect Cattle Infected with Bovine Herpesviruses Types 1 and 5.

    PubMed

    Puentes, Rodrigo; Campos, Fabrício Souza; Furtado, Agustin; Torres, Fabrício Dias; Franco, Ana Cláudia; Maisonnave, Jacqueline; Roehe, Paulo Michel

    2016-01-01

    Bovine herpesviruses (BoHVs) types 1 (BoHV-1) and 5 (BoHV-5) are alphaherpesviruses of major importance to the bovine production chain. Such viruses are capable of establishing latent infections in neuronal tissues. Infected animals tend to develop a serological response to infection; however, such response-usually investigated by antibody assays in serum-may eventually not be detected in laboratory assays. Nevertheless, serological tests such as virus neutralization (VN) and various enzyme-linked immunosorbent assays (ELISAs) are widely employed to check individual or herd status of BoHV infections. The correlation between detection of antibodies and the presence of viral nucleic acids as indicatives of infection in infected cattle has not been deeply examined. In order to investigate such correlation, 248 bovine serum samples were tested by VN to BoHV-1 and BoHV-5, as well as in a widely employed (though not type-differential) gB ELISA (IDEXX IBR gB X2 Ab Test) in search for antibodies to BoHVs. Immediately after blood withdrawal, cattle were slaughtered and trigeminal ganglia (TG) excised for DNA extraction and viral nucleic acid detection (NAD) by nested PCR. Neutralizing antibodies to BoHV-1 and/or BoHV-5 were detected in 44.8% (111/248) of sera, whereas the gB ELISA detected antibodies in 51.2% (127/248) of the samples. However, genomes of either BoHV-1, BoHV-5, or both, were detected in TGs of 85.9% (213/248) of the animals. These findings reveal that the assays designed to detect antibodies to BoHV-1 and/or BoHV-5 employed here may fail to detect a significant number of latently infected animals (in this study, 35.7%). From such data, it is clear that antibody assays are poorly correlated with detection of viral genomes in BoHV-1 and BoHV-5-infected animals.

  18. Comparison between DNA Detection in Trigeminal Nerve Ganglia and Serology to Detect Cattle Infected with Bovine Herpesviruses Types 1 and 5.

    PubMed

    Puentes, Rodrigo; Campos, Fabrício Souza; Furtado, Agustin; Torres, Fabrício Dias; Franco, Ana Cláudia; Maisonnave, Jacqueline; Roehe, Paulo Michel

    2016-01-01

    Bovine herpesviruses (BoHVs) types 1 (BoHV-1) and 5 (BoHV-5) are alphaherpesviruses of major importance to the bovine production chain. Such viruses are capable of establishing latent infections in neuronal tissues. Infected animals tend to develop a serological response to infection; however, such response-usually investigated by antibody assays in serum-may eventually not be detected in laboratory assays. Nevertheless, serological tests such as virus neutralization (VN) and various enzyme-linked immunosorbent assays (ELISAs) are widely employed to check individual or herd status of BoHV infections. The correlation between detection of antibodies and the presence of viral nucleic acids as indicatives of infection in infected cattle has not been deeply examined. In order to investigate such correlation, 248 bovine serum samples were tested by VN to BoHV-1 and BoHV-5, as well as in a widely employed (though not type-differential) gB ELISA (IDEXX IBR gB X2 Ab Test) in search for antibodies to BoHVs. Immediately after blood withdrawal, cattle were slaughtered and trigeminal ganglia (TG) excised for DNA extraction and viral nucleic acid detection (NAD) by nested PCR. Neutralizing antibodies to BoHV-1 and/or BoHV-5 were detected in 44.8% (111/248) of sera, whereas the gB ELISA detected antibodies in 51.2% (127/248) of the samples. However, genomes of either BoHV-1, BoHV-5, or both, were detected in TGs of 85.9% (213/248) of the animals. These findings reveal that the assays designed to detect antibodies to BoHV-1 and/or BoHV-5 employed here may fail to detect a significant number of latently infected animals (in this study, 35.7%). From such data, it is clear that antibody assays are poorly correlated with detection of viral genomes in BoHV-1 and BoHV-5-infected animals. PMID:27224314

  19. Comparison between DNA Detection in Trigeminal Nerve Ganglia and Serology to Detect Cattle Infected with Bovine Herpesviruses Types 1 and 5

    PubMed Central

    Furtado, Agustin; Torres, Fabrício Dias; Franco, Ana Cláudia; Maisonnave, Jacqueline; Roehe, Paulo Michel

    2016-01-01

    Bovine herpesviruses (BoHVs) types 1 (BoHV-1) and 5 (BoHV-5) are alphaherpesviruses of major importance to the bovine production chain. Such viruses are capable of establishing latent infections in neuronal tissues. Infected animals tend to develop a serological response to infection; however, such response—usually investigated by antibody assays in serum—may eventually not be detected in laboratory assays. Nevertheless, serological tests such as virus neutralization (VN) and various enzyme-linked immunosorbent assays (ELISAs) are widely employed to check individual or herd status of BoHV infections. The correlation between detection of antibodies and the presence of viral nucleic acids as indicatives of infection in infected cattle has not been deeply examined. In order to investigate such correlation, 248 bovine serum samples were tested by VN to BoHV-1 and BoHV-5, as well as in a widely employed (though not type-differential) gB ELISA (IDEXX IBR gB X2 Ab Test) in search for antibodies to BoHVs. Immediately after blood withdrawal, cattle were slaughtered and trigeminal ganglia (TG) excised for DNA extraction and viral nucleic acid detection (NAD) by nested PCR. Neutralizing antibodies to BoHV-1 and/or BoHV-5 were detected in 44.8% (111/248) of sera, whereas the gB ELISA detected antibodies in 51.2% (127/248) of the samples. However, genomes of either BoHV-1, BoHV-5, or both, were detected in TGs of 85.9% (213/248) of the animals. These findings reveal that the assays designed to detect antibodies to BoHV-1 and/or BoHV-5 employed here may fail to detect a significant number of latently infected animals (in this study, 35.7%). From such data, it is clear that antibody assays are poorly correlated with detection of viral genomes in BoHV-1 and BoHV-5-infected animals. PMID:27224314

  20. Detection of circulating trypanosomal antigens in Trypanosoma evansi-infected animals using a T. brucei group-specific monoclonal antibody.

    PubMed

    Nantulya, V M; Bajyana Songa, E; Hamers, R

    1989-09-01

    An antigen-detection enzyme immunoassay based on a T. brucei group-specific monoclonal antibody was used for the detection of circulating antigens in several animal species experimentally infected with T. evansi stocks from Sudan, Indonesia, Thailand and South America. Circulating antigens were detected as early as 6 days after infection, and they persisted throughout the observation period of up to 60 days postinfection. In an analysis of sera from naturally infected water buffaloes from Thailand, the test identified all the animals with positive parasitological findings, and 3 additional cases that had not been diagnosed by parasitological techniques. In an analysis of sera from pigs on a farm in Thailand suspected of a T. evansi outbreak, the assay detected "antigenaemia" in 66.7% of the animals, with antigen titres ranging from 1:2 to 1.512.

  1. Development of a Multiantigen Panel for Improved Detection of Borrelia burgdorferi Infection in Early Lyme Disease

    PubMed Central

    Panas, Michael W.; Mao, Rong; Delanoy, Michelle; Flanagan, John J.; Binder, Steven R.; Rebman, Alison W.; Montoya, Jose G.; Soloski, Mark J.; Steere, Allen C.; Dattwyler, Raymond J.; Arnaboldi, Paul M.; Aucott, John N.

    2015-01-01

    The current standard for laboratory diagnosis of Lyme disease in the United States is serologic detection of antibodies against Borrelia burgdorferi. The Centers for Disease Control and Prevention recommends a two-tiered testing algorithm; however, this scheme has limited sensitivity for detecting early Lyme disease. Thus, there is a need to improve diagnostics for Lyme disease at the early stage, when antibiotic treatment is highly efficacious. We examined novel and established antigen markers to develop a multiplex panel that identifies early infection using the combined sensitivity of multiple markers while simultaneously maintaining high specificity by requiring positive results for two markers to designate a positive test. Ten markers were selected from our initial analysis of 62 B. burgdorferi surface proteins and synthetic peptides by assessing binding of IgG and IgM to each in a training set of Lyme disease patient samples and controls. In a validation set, this 10-antigen panel identified a higher proportion of early-Lyme-disease patients as positive at the baseline or posttreatment visit than two-tiered testing (87.5% and 67.5%, respectively; P < 0.05). Equivalent specificities of 100% were observed in 26 healthy controls. Upon further analysis, positivity on the novel 10-antigen panel was associated with longer illness duration and multiple erythema migrans. The improved sensitivity and comparable specificity of our 10-antigen panel compared to two-tiered testing in detecting early B. burgdorferi infection indicates that multiplex analysis, featuring the next generation of markers, could advance diagnostic technology to better aid clinicians in diagnosing and treating early Lyme disease. PMID:26447113

  2. Economic and health implications from earlier detection of HIV infection in the United Kingdom

    PubMed Central

    Zah, Vladimir; Toumi, Mondher

    2016-01-01

    Purpose To model the budget and survival impact of implementing interventions to increase the proportion of HIV infections detected early in a given UK population. Patients and methods A Microsoft Excel decision model was designed to generate a set of outcomes for a defined population. Survival was modeled on the Collaboration of Observational HIV Epidemiological Research Europe (COHERE) study extrapolated to a 5-year horizon as a constant hazard. Hazard rates were specific to age, sex, and whether detection was early or late. The primary outcomes for each year up to 5 years were: annual costs, numbers of infected cases, hospital admissions, and surviving cases. Three locations in the UK were chosen to model outcomes across a range of HIV prevalence areas: Lambeth, Southwark, and Lewisham (LSL), Greater Manchester Cluster (GMC), and Kent and Medway (K&M). Results In LSL, the projected cumulative cost savings over 5 years were £3,210,206 or £5,290,206 when including the value of the 104 life-years saved. Savings were insensitive to transmission rates, but sensitive in direct proportion to the percentage shift from late to early detection. In GMC, savings were in a similar proportion to LSL, but the magnitude was smaller, as a consequence of the lower base-case HIV prevalence. In K&M, with a smaller population and lower HIV prevalence than GMC, savings were commensurately smaller (£733,202 cumulatively over 5 years). Conclusion The results strengthen the rationale for implementing increased testing in high prevalence areas. However, in areas of low prevalence, it is unlikely that costs will be returned over a 5-year period. PMID:27073328

  3. Development of a Multiantigen Panel for Improved Detection of Borrelia burgdorferi Infection in Early Lyme Disease.

    PubMed

    Lahey, Lauren J; Panas, Michael W; Mao, Rong; Delanoy, Michelle; Flanagan, John J; Binder, Steven R; Rebman, Alison W; Montoya, Jose G; Soloski, Mark J; Steere, Allen C; Dattwyler, Raymond J; Arnaboldi, Paul M; Aucott, John N; Robinson, William H

    2015-12-01

    The current standard for laboratory diagnosis of Lyme disease in the United States is serologic detection of antibodies against Borrelia burgdorferi. The Centers for Disease Control and Prevention recommends a two-tiered testing algorithm; however, this scheme has limited sensitivity for detecting early Lyme disease. Thus, there is a need to improve diagnostics for Lyme disease at the early stage, when antibiotic treatment is highly efficacious. We examined novel and established antigen markers to develop a multiplex panel that identifies early infection using the combined sensitivity of multiple markers while simultaneously maintaining high specificity by requiring positive results for two markers to designate a positive test. Ten markers were selected from our initial analysis of 62 B. burgdorferi surface proteins and synthetic peptides by assessing binding of IgG and IgM to each in a training set of Lyme disease patient samples and controls. In a validation set, this 10-antigen panel identified a higher proportion of early-Lyme-disease patients as positive at the baseline or posttreatment visit than two-tiered testing (87.5% and 67.5%, respectively; P < 0.05). Equivalent specificities of 100% were observed in 26 healthy controls. Upon further analysis, positivity on the novel 10-antigen panel was associated with longer illness duration and multiple erythema migrans. The improved sensitivity and comparable specificity of our 10-antigen panel compared to two-tiered testing in detecting early B. burgdorferi infection indicates that multiplex analysis, featuring the next generation of markers, could advance diagnostic technology to better aid clinicians in diagnosing and treating early Lyme disease.

  4. Detection of PrPsc in Blood from Sheep Infected with the Scrapie and Bovine Spongiform Encephalopathy Agents▿

    PubMed Central

    Terry, L. A.; Howells, L.; Hawthorn, J.; Edwards, J. C.; Moore, S. J.; Bellworthy, S. J.; Simmons, H.; Lizano, S.; Estey, L.; Leathers, V.; Everest, S. J.

    2009-01-01

    The role of blood in the iatrogenic transmission of transmissible spongiform encephalopathy (TSE) or prion disease has become an increasing concern since the reports of variant Creutzfeldt-Jakob disease (vCJD) transmission through blood transfusion from humans with subclinical infection. The development of highly sensitive rapid assays to screen for prion infection in blood is of high priority in order to facilitate the prevention of transmission via blood and blood products. In the present study we show that PrPsc, a surrogate marker for TSE infection, can be detected in cells isolated from the blood from naturally and experimentally infected sheep by using a rapid ligand-based immunoassay. In sheep with clinical disease, PrPsc was detected in the blood of 55% of scrapie agent-infected animals (n = 80) and 71% of animals with bovine spongiform encephalopathy (n = 7). PrPsc was also detected several months before the onset of clinical signs in a subset of scrapie agent-infected sheep, followed from 3 months of age to clinical disease. This study confirms that PrPsc is associated with the cellular component of blood and can be detected in preclinical sheep by an immunoassay in the absence of in vitro or in vivo amplification. PMID:19740979

  5. HPV genotypes detected in the oropharyngeal mucosa of HIV-infected men who have sex with men in Northern Italy.

    PubMed

    Martinelli, M; Mazza, F; Frati, E R; Fasolo, M M; Colzani, D; Bianchi, S; Fasoli, E; Amendola, A; Orlando, G; Tanzi, E

    2016-09-01

    The aim of this study was to investigate the epidemiological profile of HPV oropharyngeal infections in HIV-infected men who have sex with men. A total of 135 subjects were enrolled at the L. Sacco University Hospital (Milan, Italy) to evaluate their HPV oropharyngeal infection status at baseline and at a follow-up visit at least 12 months later. HPV DNA was detected from oropharyngeal swabs using an in-house nested PCR that amplifies a segment of the L1 gene. The PCR products were then sequenced and genotyped. A greater percentage of high-risk genotypes was identified compared to low-risk genotypes (13·7% vs. 6·9%, P < 0·05), and two uncommon alpha-HPV genotypes were detected, i.e. HPV-102 and HPV-114. HPV infection prevalence was 24·4% and the cumulative incidence was 24·1%. During the follow-up period, one case of HPV infection (HPV-33) persisted, while the overall rate of infection clearance was 58·3%. HPV oropharyngeal infection was widespread in the cohort examined, and most of the infections were transient and cleared within 12 months. These results may help to clarify the role of HPV in the oropharynx and may also improve our understanding of the need to implement preventive strategies in at-risk populations.

  6. Flow Cytometric Detection of Mycobacterium avium subsp. paratuberculosis-Specific Antibodies in Experimentally Infected and Naturally Exposed Calves

    PubMed Central

    Bridger, P. S.; Bulun, H.; Fischer, M.; Akineden, Ö.; Seeger, T.; Barth, S.; Henrich, M.; Doll, K.; Bülte, M.; Menge, C.; Bauerfeind, R.

    2013-01-01

    A desirable test to diagnose infections with Mycobacterium avium subsp. paratuberculosis facilitates identification of infected cattle prior to the state of M. avium subsp. paratuberculosis shedding. This study aimed at adjusting a flow cytometry (FC)-based assay, using intact M. avium subsp. paratuberculosis bacteria as the antigen, for diagnosis of M. avium subsp. paratuberculosis infections in calves. Serum samples were collected from experimentally infected (n = 12) and naturally exposed (n = 32) calves. Samples from five calves from positive dams were analyzed to determine the dynamics of maternal antibodies. Samples from adult cattle with defined infection status served as the standard (18 M. avium subsp. paratuberculosis shedders, 22 M. avium subsp. paratuberculosis free). After preadsorption with Mycobacterium phlei, sera were incubated with M. avium subsp. paratuberculosis and M. avium subsp. avium bacterial suspensions, respectively, followed by the separate detection of bovine IgG, IgG1, IgG2, and IgM attached to the bacterial surface. M. avium subsp. paratuberculosis-specific sample/positive (S/P) ratios were compared to enzyme-linked immunosorbent assay (ELISA) S/P ratios. In adult cattle, the FC assay for IgG1 had a sensitivity of 78% at a specificity of 100%. Maternally acquired antibodies could be detected in calves up to 121 days of life. While all but two sera taken at day 100 ± 10 postnatum from naturally exposed calves tested negative, elevated S/P ratios (IgG and IgG1) became detectable from 44 and 46 weeks postinoculation onwards in two calves infected experimentally. Even with the optimized FC assay, M. avium subsp. paratuberculosis-specific antibodies can only occasionally be detected in infected calves less than 12 months of age. The failure to detect such antibodies apparently reflects the distinct immunobiology of M. avium subsp. paratuberculosis infections rather than methodological constraints. PMID:23885032

  7. Long-term follow-up of treatment with diethylcarbamazine on anti-filarial IgG4: dosage, compliance, and differential patterns in adults and children.

    PubMed

    Terhell, A J; Haarbrink, M; van den Biggelaar, A; Mangali, A; Sartono, E; Yazdanbakhsh, M

    2003-01-01

    We have followed a population in an area endemic for Brugia malayi for three years after intensive treatment with diethylcarbamazine (DEC). Microfilariae were cleared from the circulation within four months in all eligible study participants (n = 60). There appeared to be a strong correlation between the maximum reduction in specific IgG4 and the number of days drug was taken under supervision (p = 0.41, P < 0.001), indicating that high total dosage of DEC is necessary for optimal reduction of active infection. In individuals with good compliance (at least 180 mg/kg of body weight, n = 34), we observed variable IgG4 patterns. All pre-treatment IgG4+ children (9-14 years old) and 40% of the IgG4+ adult population (> or = 15 years old) showed a gradual decrease in anti-filarial IgG4; 53% of these showed complete clearance of worm burden by the end of the study. In contrast, another group of male IgG4+ adults showed IgG4 patterns that started to increase between nine months and two years after treatment, indicating either a partial efficacy of DEC that allowed recovery of resident adult worms or reinfection. PMID:12556144

  8. Detection and survival of Toxoplasma gondii in milk and cheese from experimentally infected goats.

    PubMed

    Dubey, J P; Verma, S K; Ferreira, L R; Oliveira, S; Cassinelli, A B; Ying, Y; Kwok, O C H; Tuo, W; Chiesa, O A; Jones, J L

    2014-10-01

    The consumption of unpasteurized goat cheese and goat's milk has been suggested as a risk factor for toxoplasmosis in humans. In the present study, detection and survival of Toxoplasma gondii in milk and cheese was studied by bioassay in mice (milk) and in cats (cheese). Eight goats were inoculated orally with 300 to 10,000 oocysts of T. gondii strain TgGoatUS26. Milk samples were collected daily up to 30 days postinoculation and bioassayed in mice and cats. For mouse bioassay, 50 ml of milk samples were centrifuged, and the sediment was inoculated subcutaneously into mice. Mice were tested for T. gondii infection by seroconversion and by the demonstration of parasites. By mouse bioassay, T. gondii was detected in milk from all eight goats. The T. gondii excretion in milk was intermittent. For cat bioassay, 400 ml (100 ml or more from each goat) of milk from four goats from 6 to 27 days postinoculation were pooled daily, and cheese was made using rennin. Ten grams of cheese was fed daily to four cats, and cat feces were examined for oocyst shedding. One cat fed cheese shed oocysts 7 to 11 days after consuming cheese. Attempts were made to detect T. gondii DNA in milk of four goats; T. gondii was detected by PCR more consistently, but there was no correlation between detection of viable T. gondii by bioassay in mice and T. gondii DNA by PCR. Results indicate that T. gondii can be excreted in goat's milk and can survive in fresh cheese made by cold-enzyme treatment. To prevent transmission to humans or animals, milk should not be consumed raw. Raw fresh goat cheese made by cold-enzyme treatment of unpasteurized milk also should not be consumed. PMID:25285492

  9. [Rapid determination of food grain infection by fungi Fusarium and their species detection (part 1)].

    PubMed

    Minaeva, L P; Korotkevich, Iu V; Sheveleva, S A

    2013-01-01

    Currently contamination of food grains with Fusarium spp. is determined by conventional mycological methods and can last up to 30-40 days. The method specificity is highly dependent on the subjective evaluation of the researchers. The alternative to traditional mycological methods is detection by PCR. The purpose of the study was to improve the contamination analysis method of food grains infected by Fusarium for analysis time reducing and species detection specificity increasing. Investigations were carried out on food grains samples harvested in 2009-2011 from 5 federal regions of Russia. On the first stage, 100 grains were sowing on potato-sucrose medium and then incubated at 24 degrees C for 7-10 days. At the second stage, the Fusarium species composition grown from food grains was estimated by two methods. The first method was mycological for monospore isolates. The second one was PCR analysis of DNA extracts from the combined sample of Fusarium mycelium which grew on a foodgrains sample. Species-specific primers of such mycotoxins producers as F. graminearum, F. culmorum, F. sporotrchiodes, F. langsethiae, F. poae, F. avenaceum, F. tricinctum were used for PCR detection. The species composition of viable Fusarium spp. fungi revealed in foodgrains samples by mycological method completely concided with the results of PCR analysis. In result, the method that combines traditional mycological sowing for detection of viable species with PCR species detection of the mycelium in integrated sample has been developed. The method significantly reduces test duration (3-4 times) by excluding the sieving step in obtaining monospore fungi isolates for further species identification. The method also allows obtaining reliable data on the viable Fusarium species including producers of toxins in foodgrains. Thus, the idea of improving of the identification stage allowing reducing labor costs and increasing of the method specificity was realized.

  10. Detection and survival of Toxoplasma gondii in milk and cheese from experimentally infected goats.

    PubMed

    Dubey, J P; Verma, S K; Ferreira, L R; Oliveira, S; Cassinelli, A B; Ying, Y; Kwok, O C H; Tuo, W; Chiesa, O A; Jones, J L

    2014-10-01

    The consumption of unpasteurized goat cheese and goat's milk has been suggested as a risk factor for toxoplasmosis in humans. In the present study, detection and survival of Toxoplasma gondii in milk and cheese was studied by bioassay in mice (milk) and in cats (cheese). Eight goats were inoculated orally with 300 to 10,000 oocysts of T. gondii strain TgGoatUS26. Milk samples were collected daily up to 30 days postinoculation and bioassayed in mice and cats. For mouse bioassay, 50 ml of milk samples were centrifuged, and the sediment was inoculated subcutaneously into mice. Mice were tested for T. gondii infection by seroconversion and by the demonstration of parasites. By mouse bioassay, T. gondii was detected in milk from all eight goats. The T. gondii excretion in milk was intermittent. For cat bioassay, 400 ml (100 ml or more from each goat) of milk from four goats from 6 to 27 days postinoculation were pooled daily, and cheese was made using rennin. Ten grams of cheese was fed daily to four cats, and cat feces were examined for oocyst shedding. One cat fed cheese shed oocysts 7 to 11 days after consuming cheese. Attempts were made to detect T. gondii DNA in milk of four goats; T. gondii was detected by PCR more consistently, but there was no correlation between detection of viable T. gondii by bioassay in mice and T. gondii DNA by PCR. Results indicate that T. gondii can be excreted in goat's milk and can survive in fresh cheese made by cold-enzyme treatment. To prevent transmission to humans or animals, milk should not be consumed raw. Raw fresh goat cheese made by cold-enzyme treatment of unpasteurized milk also should not be consumed.

  11. The vectors of human infections by Wuchereria species in endemic areas and their biology*

    PubMed Central

    Raghavan, N. G. S.

    1961-01-01

    In this paper the author has compiled an up-to-date list of the principal natural vectors of human Wuchereria infections, arranged by zoogeographical regions and countries, and gives data on natural and experimental filarial infection rates in vectors in different parts of the world. As an aid to workers in areas where Wuchereria and Plasmodium infections co-exist, he also provides a list of anophelines as filarial vectors, including in this list the results of natural and experimental studies of Wuchereria infection both alone and in combination with malaria. Finally, the author suggests a number of aspects of the biology of infected and uninfected vectors on which further studies might to advantage be conducted. PMID:13739128

  12. Cost Analysis of Tests for the Detection of Schistosoma mansoni Infection in Children in Western Kenya

    PubMed Central

    Worrell, Caitlin M.; Bartoces, Monina; Karanja, Diana M. S.; Ochola, Elizabeth A.; Matete, Daniel O.; Mwinzi, Pauline N. M.; Montgomery, Susan P.; Secor, W. Evan

    2015-01-01

    Financial resources tend to be limited in schistosomiasis endemic areas, forcing program managers to balance financial and scientific considerations when selecting detection assays. Therefore, we compared the costs of using single stool Kato-Katz, triplicate stool Kato-Katz, and point-of-contact circulating cathodic antigen (POC-CCA) assays for the detection of Schistosoma mansoni infection. Economic and financial costs were estimated from the viewpoint of a schistosomiasis control program using the ingredients approach. Costs related to specimen collection, sample processing and analysis, and treatment delivery were considered. Analysis inputs and assumptions were tested using one-way and two-way sensitivity analysis. The total per-person cost of performing the single Kato-Katz, triplicate Kato-Katz, and POC-CCA was US$6.89, US$17.54, and US$7.26, respectively. Major cost drivers included labor, transportation, and supplies. In addition, we provide a costing tool to guide program managers in evaluating detection costs in specific settings, as costs may vary temporally and spatially. PMID:25870422

  13. Cost analysis of tests for the detection of Schistosoma mansoni infection in children in western Kenya.

    PubMed

    Worrell, Caitlin M; Bartoces, Monina; Karanja, Diana M S; Ochola, Elizabeth A; Matete, Daniel O; Mwinzi, Pauline N M; Montgomery, Susan P; Secor, W Evan

    2015-06-01

    Financial resources tend to be limited in schistosomiasis endemic areas, forcing program managers to balance financial and scientific considerations when selecting detection assays. Therefore, we compared the costs of using single stool Kato-Katz, triplicate stool Kato-Katz, and point-of-contact circulating cathodic antigen (POC-CCA) assays for the detection of Schistosoma mansoni infection. Economic and financial costs were estimated from the viewpoint of a schistosomiasis control program using the ingredients approach. Costs related to specimen collection, sample processing and analysis, and treatment delivery were considered. Analysis inputs and assumptions were tested using one-way and two-way sensitivity analysis. The total per-person cost of performing the single Kato-Katz, triplicate Kato-Katz, and POC-CCA was US$6.89, US$17.54, and US$7.26, respectively. Major cost drivers included labor, transportation, and supplies. In addition, we provide a costing tool to guide program managers in evaluating detection costs in specific settings, as costs may vary temporally and spatially.

  14. Molecular method for the detection of Andes hantavirus infection: validation for clinical diagnostics.

    PubMed

    Vial, Cecilia; Martinez-Valdebenito, Constanza; Rios, Susana; Martinez, Jessica; Vial, Pablo A; Ferres, Marcela; Rivera, Juan C; Perez, Ruth; Valdivieso, Francisca

    2016-01-01

    Hantavirus cardiopulmonary syndrome is a severe disease caused by exposure to New World hantaviruses. Early diagnosis is difficult due to the lack of specific initial symptoms. Antihantavirus antibodies are usually negative until late in the febrile prodrome or the beginning of cardiopulmonary phase, while Andes hantavirus (ANDV) RNA genome can be detected before symptoms onset. We analyzed the effectiveness of quantitative reverse transcription polymerase chain reaction (RT-qPCR) as a diagnostic tool detecting ANDV-Sout genome in peripheral blood cells from 78 confirmed hantavirus patients and 166 negative controls. Our results indicate that RT-qPCR had a low detection limit (~10 copies), with a specificity of 100% and a sensitivity of 94.9%. This suggests the potential for establishing RT-qPCR as the assay of choice for early diagnosis, promoting early effective care of patients, and improving other important aspects of ANDV infection management, such as compliance of biosafety recommendations for health personnel in order to avoid nosocomial transmission.

  15. Optofluidic analysis system for amplification-free, direct detection of Ebola infection

    PubMed Central

    Cai, H.; Parks, J. W.; Wall, T. A.; Stott, M. A.; Stambaugh, A.; Alfson, K.; Griffiths, A.; Mathies, R. A.; Carrion, R.; Patterson, J. L.; Hawkins, A. R.; Schmidt, H.

    2015-01-01

    The massive outbreak of highly lethal Ebola hemorrhagic fever in West Africa illustrates the urgent need for diagnostic instruments that can identify and quantify infections rapidly, accurately, and with low complexity. Here, we report on-chip sample preparation, amplification-free detection and quantification of Ebola virus on clinical samples using hybrid optofluidic integration. Sample preparation and target preconcentration are implemented on a PDMS-based microfluidic chip (automaton), followed by single nucleic acid fluorescence detection in liquid-core optical waveguides on a silicon chip in under ten minutes. We demonstrate excellent specificity, a limit of detection of 0.2 pfu/mL and a dynamic range of thirteen orders of magnitude, far outperforming other amplification-free methods. This chip-scale approach and reduced complexity compared to gold standard RT-PCR methods is ideal for portable instruments that can provide immediate diagnosis and continued monitoring of infectious diseases at the point-of-care. PMID:26404403

  16. Detection of infection or infectious agents by use of cytologic and histologic stains.

    PubMed Central

    Woods, G L; Walker, D H

    1996-01-01

    A wide variety of stains are useful for detection of different organisms or, for viruses, the cytopathologic changes they induce, in smears prepared directly from clinical specimens and in tissue sections. Other types of stains, such as hematoxylin and eosin, are used routinely to stain tissue sections and are most valuable for assessing the immunologic response of the host to the invading pathogen. In many cases, the pattern of inflammation provides important clues to diagnosis and helps to guide the selection of additional "special" stains used predominantly for diagnosis of infectious diseases. A stain may be nonspecific, allowing detection of a spectrum of organisms, as do the Papanicolaou stain and silver impregnation methods, or detection of only a limited group of organisms, as do the different acid-fast techniques. Some nonspecific stains, such as the Gram stain, are differential and provide valuable preliminary information concerning identification. Immunohistochemical stains, on the other hand, are specific for a particular organism, although in some cases cross-reactions with other organisms occur. Despite the wealth of information that can be gleaned from a stained smear or section of tissue, however, the specific etiology of an infection often cannot be determined on the basis of only the morphology of the organisms seen; culture data are essential and must be considered in the final diagnosis. PMID:8809467

  17. Added Value of Long-Term Cytokine Release Assays to Detect Mycobacterium tuberculosis Infection in HIV-Infected Subjects in Uganda

    PubMed Central

    Dirix, Violette; Schepers, Kinda; Massinga-Loembe, Marguerite; Worodria, William; Colebunders, Robert; Singh, Mahavir; Locht, Camille; Kestens, Luc

    2016-01-01

    Objectives: To investigate whether mycobacterial antigen–induced cytokine secretions are helpful in detecting Mycobacterium tuberculosis (Mtb) infection in a cohort of HIV-infected patients living in a country with a high burden of Mtb and HIV infections, and to determine their predictive value for the development of tuberculosis (TB)-associated immune reconstitution inflammatory syndrome. Design: A total of 352 HIV-infected patients (186 with active TB) were prospectively enrolled when initiating antiretroviral therapy (ART). Sequential blood samples were collected during the first 6 months of ART. Eighty-three HIV-uninfected subjects (39 with active TB) were enrolled as controls. Methods: The concentrations of 13 cytokines were measured in supernatants from blood mononuclear cells in vitro stimulated with purified protein derivative (PPD), heparin-binding hemagglutinin (HBHA) or early secreted antigen-6 (ESAT-6) and culture filtrate protein-10 (CFP-10), and results were compared with those of tuberculin skin tests (TST). Results: The best detection of Mtb infection was achieved by ESAT-6/CFP-10–induced interferon-γ concentrations, but results were often negative for patients with CD4+ T-cell counts <50 per cubic millimeters. Patients with active TB were identified by high ESAT-6/CFP-10–induced interleukin-6. Conversions of interferon-γ-release assays (IGRA) and TST occurred under ART, and combined TB and antiretroviral treatments of coinfected patients resulted in a decrease of ESAT-6/CFP-10–induced and an increase of HBHA-induced interferon-γ responses. No Mtb antigen–induced cytokines allowed us to predict TB–immune reconstitution inflammatory syndrome or ART-associated TB. Conclusions: In Uganda, ESAT-6/CFP-10–IGRA is better in detecting Mtb infection than TST and, when combined with an HBHA–IGRA, could help to evaluate anti-TB treatment success. PMID:27306506

  18. Decreased sensitivity of early imaging with In-111 oxine-labeled leukocytes in detection of occult infection: concise communication

    SciTech Connect

    Datz, F.L.; Jacobs, J.; Baker, W.; Landrum, W.; Alazraki, N.; Taylor, A. Jr.

    1984-03-01

    Imaging with leukocytes labeled with indium-111 oxine is a sensitive technique for detecting sites of occult infection. Traditionally, imaging is performed 24 hr after injection. The authors undertook a prospective study of 35 patients (40 studies) with possible occult infection to see whether a 24-hr delay in imaging is really necessary. Patients were imaged at 1-4 hr and again at 24 hr after injection. The early images had a sensitivity of only 33%, compared with 95% for the 24-hr images. Of the seven studies that were positive on both early and delayed images, 71% had more intense uptake at 24 hr. There were no false-positive early images. It was concluded that imaging 1-4 hr after injection with In-111 oxine-labeled leukocytes has a low sensitivity for detecting occult infection. However, a positive early image is specific for a site of infection.

  19. The Wolbachia endosymbiont of Brugia malayi has an active phosphoglycerate mutase: a candidate target for anti-filarial therapies.

    PubMed

    Foster, Jeremy M; Raverdy, Sylvine; Ganatra, Mehul B; Colussi, Paul A; Taron, Christopher H; Carlow, Clotilde K S

    2009-04-01

    Phosphoglycerate mutases (PGM) interconvert 2- and 3-phosphoglycerate in the glycolytic and gluconeogenic pathways. A putative cofactor-independent phosphoglycerate mutase gene (iPGM) was identified in the genome sequence of the Wolbachia endosymbiont from the filarial nematode, Brugia malayi (wBm). Since iPGM has no sequence or structural similarity to the cofactor-dependent phosphoglycerate mutase (dPGM) found in mammals, it may represent an attractive Wolbachia drug target. In the present study, wBm-iPGM cloned and expressed in Escherichia coli was mostly insoluble and inactive. However, the protein was successfully produced in the yeast Kluyveromyces lactis and the purified recombinant wBm-iPGM showed typical PGM activity. Our results provide a foundation for further development of wBm-iPGM as a promising new drug target for novel anti-filarial therapies that selectively target the endosymbiont.

  20. Reliability of non-lethal surveillance methods for detecting ranavirus infection.

    PubMed

    Gray, Matthew J; Miller, Debra L; Hoverman, Jason T

    2012-05-15

    Ranaviruses have been identified as the etiologic agent in many amphibian die-offs across the globe. Polymerase chain reaction (PCR) is commonly used to detect ranavirus infection in amphibian hosts, but the test results may vary between tissue samples obtained by lethal and non-lethal procedures. Testing liver samples for infection is a common lethal sampling technique to estimate ranavirus prevalence because the pathogen often targets this organ and the liver is easy to identify and collect. However, tail clips or swabs may be more practicable for ranavirus surveillance programs compared with collecting and euthanizing animals, especially for uncommon species. Using PCR results from liver samples for comparison, we defined false-positive test results as occurrences when a non-lethal technique indicated positive but the liver sample was negative. Similarly, we defined false-negative test results as occurrences when a non-lethal technique was negative but the liver sample was positive. Using these decision rules, we estimated false-negative and false-positive rates for tail clips and swabs. Our study was conducted in a controlled facility using American bullfrog Lithobates catesbeianus tadpoles; false-positive and false-negative rates were estimated after different periods of time following exposure to ranavirus. False-negative and false-positive rates were 20 and 6%, respectively, for tail samples, and 22 and 12%, respectively, for swabs. False-negative rates were constant over time, but false-positive rates decreased with post-exposure duration. Our results suggest that non-lethal sampling techniques can be useful for ranavirus surveillance, although the prevalence of infection may be underestimated when compared to results obtained with liver samples. PMID:22585297

  1. Reliability of non-lethal surveillance methods for detecting ranavirus infection.

    PubMed

    Gray, Matthew J; Miller, Debra L; Hoverman, Jason T

    2012-05-15

    Ranaviruses have been identified as the etiologic agent in many amphibian die-offs across the globe. Polymerase chain reaction (PCR) is commonly used to detect ranavirus infection in amphibian hosts, but the test results may vary between tissue samples obtained by lethal and non-lethal procedures. Testing liver samples for infection is a common lethal sampling technique to estimate ranavirus prevalence because the pathogen often targets this organ and the liver is easy to identify and collect. However, tail clips or swabs may be more practicable for ranavirus surveillance programs compared with collecting and euthanizing animals, especially for uncommon species. Using PCR results from liver samples for comparison, we defined false-positive test results as occurrences when a non-lethal technique indicated positive but the liver sample was negative. Similarly, we defined false-negative test results as occurrences when a non-lethal technique was negative but the liver sample was positive. Using these decision rules, we estimated false-negative and false-positive rates for tail clips and swabs. Our study was conducted in a controlled facility using American bullfrog Lithobates catesbeianus tadpoles; false-positive and false-negative rates were estimated after different periods of time following exposure to ranavirus. False-negative and false-positive rates were 20 and 6%, respectively, for tail samples, and 22 and 12%, respectively, for swabs. False-negative rates were constant over time, but false-positive rates decreased with post-exposure duration. Our results suggest that non-lethal sampling techniques can be useful for ranavirus surveillance, although the prevalence of infection may be underestimated when compared to results obtained with liver samples.

  2. EVALUATION OF A COMMERCIAL RAPID TEST KIT FOR DETECTION OF ACUTE DENGUE INFECTION.

    PubMed

    Krishnananthasivam, Shivankari; Fernando, Anira N; Tippalagama, Rashmi; Tennekoon, Rashika; De Man, Jeroen; Seneviratne, Dammika; Premawansa, Sunil; Premawansa, Gayani; De Silva, Aruna Dharshan

    2015-07-01

    Early diagnosis is important for clinical management of dengue disease. While classic laboratory tests are often tedious and time consuming, point of care devices offer a rapid, cost-effective and user-friendly alternative provided their accuracy is acceptable. This study evaluated the sensitivity, specificity and efficiency of SD BIOLINE Dengue Duo® rapid NS1, IgM and IgG test kit for diagnosis of acute dengue virus infection. Standard laboratory diagnostics, RT-PCR, IgM and IgG capture ELISAs were carried out on 143 suspected dengue patient samples obtained from a Sri Lankan population. Using the results of these standard laboratory tests as reference, the sensitivity and specificity of the SD Dengue Duo® NS1 test was 57% and 87%, respectively, and those of the IgM test was 50% and 84%, respectively. The combined sensitivity and specificity of the SD Dengue Duo® NS1/ IgM test was 72% and 80%, respectively. The SD Dengue Duo® NS1 test detected NS1 for up to 9 days from onset of fever. Primary and secondary dengue cases were classified according to the IgG test, of which the kit identified 88% and 26% of primary and of secondary infection, respectively. Although the SD Dengue Duo® kit was not as accurate as the standard tests, it still can serve the useful reference for initial screening of suspected dengue cases, especially in poor resource hospital settings and aid in clinical disease management of dengue infection. PMID:26867379

  3. Human papillomavirus detection from human immunodeficiency virus-infected Colombian women's paired urine and cervical samples.

    PubMed

    Munoz, Marina; Camargo, Milena; Soto-De Leon, Sara C; Sanchez, Ricardo; Parra, Diana; Pineda, Andrea C; Sussmann, Otto; Perez-Prados, Antonio; Patarroyo, Manuel E; Patarroyo, Manuel A

    2013-01-01

    Infection, coinfection and type-specific human papillomavirus (HPV) distribution was evaluated in human immunodeficiency virus (HIV)-positive women from paired cervical and urine samples. Paired cervical and urine samples (n = 204) were taken from HIV-positive women for identifying HPV-DNA presence by using polymerase chain reaction (PCR) with three generic primer sets (GP5+/6+, MY09/11 and pU1M/2R). HPV-positive samples were typed for six high-risk HPV (HR-HPV) (HPV-16, -18, -31, -33, -45 and -58) and two low-risk (LR-HPV) (HPV-6/11) types. Agreement between paired sample results and diagnostic performance was evaluated. HPV infection prevalence was 70.6% in cervical and 63.2% in urine samples. HPV-16 was the most prevalent HPV type in both types of sample (66.7% in cervical samples and 62.0% in urine) followed by HPV-31(47.2%) in cervical samples and HPV-58 (35.7%) in urine samples. There was 55.4% coinfection (infection by more than one type of HPV) in cervical samples and 40.2% in urine samples. Abnormal Papanicolau smears were observed in 25.3% of the women, presenting significant association with HPV-DNA being identified in urine samples. There was poor agreement of cervical and urine sample results in generic and type-specific detection of HPV. Urine samples provided the best diagnosis when taking cytological findings as reference. In conclusion including urine samples could be a good strategy for ensuring adherence to screening programs aimed at reducing the impact of cervical cancer, since this sample is easy to obtain and showed good diagnostic performance.

  4. A multiplexed nucleic acid microsystem for point-of-care detection of HIV co-infection with MTB and PCP.

    PubMed

    Xu, Lingjia; Kong, Jilie

    2013-12-15

    Many individuals infected with the human immunodeficiency virus (HIV), especially children in African countries, die of co-infections with Mycobacterium tuberculosis (MTB) (coinfection rate: 50%) or Pneumocystis carinii pneumonia (PCP) (coinfection rate: 81%). The present proposal describes a rapid, portable, low-cost, multiplexed point-of-care diagnostic technique for simultaneously detecting HIV, MTB, and PCP. This technique incorporates a creative micro-device (hardware) and a loop-mediated isothermal amplification strategy (software). PMID:24209377

  5. Prion amplification and hierarchical Bayesian modeling refine detection of prion infection.

    PubMed

    Wyckoff, A Christy; Galloway, Nathan; Meyerett-Reid, Crystal; Powers, Jenny; Spraker, Terry; Monello, Ryan J; Pulford, Bruce; Wild, Margaret; Antolin, Michael; VerCauteren, Kurt; Zabel, Mark

    2015-01-01

    Prions are unique infectious agents that replicate without a genome and cause neurodegenerative diseases that include chronic wasting disease (CWD) of cervids. Immunohistochemistry (IHC) is currently considered the gold standard for diagnosis of a prion infection but may be insensitive to early or sub-clinical CWD that are important to understanding CWD transmission and ecology. We assessed the potential of serial protein misfolding cyclic amplification (sPMCA) to improve detection of CWD prior to the onset of clinical signs. We analyzed tissue samples from free-ranging Rocky Mountain elk (Cervus elaphus nelsoni) and used hierarchical Bayesian analysis to estimate the specificity and sensitivity of IHC and sPMCA conditional on simultaneously estimated disease states. Sensitivity estimates were higher for sPMCA (99.51%, credible interval (CI) 97.15-100%) than IHC of obex (brain stem, 76.56%, CI 57.00-91.46%) or retropharyngeal lymph node (90.06%, CI 74.13-98.70%) tissues, or both (98.99%, CI 90.01-100%). Our hierarchical Bayesian model predicts the prevalence of prion infection in this elk population to be 18.90% (CI 15.50-32.72%), compared to previous estimates of 12.90%. Our data reveal a previously unidentified sub-clinical prion-positive portion of the elk population that could represent silent carriers capable of significantly impacting CWD ecology. PMID:25665713

  6. Associate investigations: detection of tuberculosis infections in children resulting in discovery of undiagnosed tuberculosis in adults.

    PubMed

    Moonan, Patrick K; Quitugua, Teresa; Cox, Rebecca A; Weis, Stephen E

    2002-07-01

    The authors present the design and implementation of associate investigations of young children with positive tuberculin skin test results. Case study analysis of an associate investigation was done using epidemiologic surveillance techniques, medical interviewing, sociogram mapping, tuberculin skin testing, radiographic evidence, and bacteriologic analysis. Deoxyribonucleic acid fingerprinting of the Mycobacterium tuberculosis isolates using a standardized IS6110-based restriction fragment length polymorphism analysis and IS6110-independent DNA spoligotyping methods was done to track and identify specific bacterial strains. Deoxyribonucleic acid fingerprinting and spoligotyping done on isolates obtained from family members demonstrated same-strain transmission of M. tuberculosis. Three adults with active pulmonary disease and six individuals with latent tuberculosis (TB) were discovered during this investigation. The arrival of a family member from Mexico who had the same strain suggests that the source case lives in Mexico. A child with positive tuberculin skin test results indicates recent and potentially ongoing transmission of TB in the community. Targeted tuberculin skin testing performed on high-risk groups by primary care physicians allows for detection of TB infections. When TB infections are discovered in children, associate investigations can result in the discovery of undiagnosed adult cases and prevent further transmission within the community.

  7. Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection.

    PubMed

    Arruda, Mauro Maciel de; Figueiredo, Fabiano Borges; Marcelino, Andreza Pain; Barbosa, José Ronaldo; Werneck, Guilherme Loureiro; Noronha, Elza Ferreira; Romero, Gustavo Adolfo Sierra

    2016-03-01

    In Brazil, human and canine visceral leishmaniasis (CVL) caused by Leishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL. PMID:26910354

  8. Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

    PubMed Central

    de Arruda, Mauro Maciel; Figueiredo, Fabiano Borges; Marcelino, Andreza Pain; Barbosa, José Ronaldo; Werneck, Guilherme Loureiro; Noronha, Elza Ferreira; Romero, Gustavo Adolfo Sierra

    2016-01-01

    In Brazil, human and canine visceral leishmaniasis (CVL) caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL. PMID:26910354

  9. Evolution of Drosophila resistance against different pathogens and infection routes entails no detectable maintenance costs.

    PubMed

    Faria, Vítor G; Martins, Nelson E; Paulo, Tânia; Teixeira, Luís; Sucena, Élio; Magalhães, Sara

    2015-11-01

    Pathogens exert a strong selective pressure on hosts, entailing host adaptation to infection. This adaptation often affects negatively other fitness-related traits. Such trade-offs may underlie the maintenance of genetic diversity for pathogen resistance. Trade-offs can be tested with experimental evolution of host populations adapting to parasites, using two approaches: (1) measuring changes in immunocompetence in relaxed-selection lines and (2) comparing life-history traits of evolved and control lines in pathogen-free environments. Here, we used both approaches to examine trade-offs in Drosophila melanogaster populations evolving for over 30 generations under infection with Drosophila C Virus or the bacterium Pseudomonas entomophila, the latter through different routes. We find that resistance is maintained after up to 30 generations of relaxed selection. Moreover, no differences in several classical life-history traits between control and evolved populations were found in pathogen-free environments, even under stresses such as desiccation, nutrient limitation, and high densities. Hence, we did not detect any maintenance costs associated with resistance to pathogens. We hypothesize that extremely high selection pressures commonly used lead to the disproportionate expression of costs relative to their actual occurrence in natural systems. Still, the maintenance of genetic variation for pathogen resistance calls for an explanation.

  10. Prion amplification and hierarchical Bayesian modeling refine detection of prion infection.

    PubMed

    Wyckoff, A Christy; Galloway, Nathan; Meyerett-Reid, Crystal; Powers, Jenny; Spraker, Terry; Monello, Ryan J; Pulford, Bruce; Wild, Margaret; Antolin, Michael; VerCauteren, Kurt; Zabel, Mark

    2015-02-10

    Prions are unique infectious agents that replicate without a genome and cause neurodegenerative diseases that include chronic wasting disease (CWD) of cervids. Immunohistochemistry (IHC) is currently considered the gold standard for diagnosis of a prion infection but may be insensitive to early or sub-clinical CWD that are important to understanding CWD transmission and ecology. We assessed the potential of serial protein misfolding cyclic amplification (sPMCA) to improve detection of CWD prior to the onset of clinical signs. We analyzed tissue samples from free-ranging Rocky Mountain elk (Cervus elaphus nelsoni) and used hierarchical Bayesian analysis to estimate the specificity and sensitivity of IHC and sPMCA conditional on simultaneously estimated disease states. Sensitivity estimates were higher for sPMCA (99.51%, credible interval (CI) 97.15-100%) than IHC of obex (brain stem, 76.56%, CI 57.00-91.46%) or retropharyngeal lymph node (90.06%, CI 74.13-98.70%) tissues, or both (98.99%, CI 90.01-100%). Our hierarchical Bayesian model predicts the prevalence of prion infection in this elk population to be 18.90% (CI 15.50-32.72%), compared to previous estimates of 12.90%. Our data reveal a previously unidentified sub-clinical prion-positive portion of the elk population that could represent silent carriers capable of significantly impacting CWD ecology.

  11. Prion Amplification and Hierarchical Bayesian Modeling Refine Detection of Prion Infection

    NASA Astrophysics Data System (ADS)

    Wyckoff, A. Christy; Galloway, Nathan; Meyerett-Reid, Crystal; Powers, Jenny; Spraker, Terry; Monello, Ryan J.; Pulford, Bruce; Wild, Margaret; Antolin, Michael; Vercauteren, Kurt; Zabel, Mark

    2015-02-01

    Prions are unique infectious agents that replicate without a genome and cause neurodegenerative diseases that include chronic wasting disease (CWD) of cervids. Immunohistochemistry (IHC) is currently considered the gold standard for diagnosis of a prion infection but may be insensitive to early or sub-clinical CWD that are important to understanding CWD transmission and ecology. We assessed the potential of serial protein misfolding cyclic amplification (sPMCA) to improve detection of CWD prior to the onset of clinical signs. We analyzed tissue samples from free-ranging Rocky Mountain elk (Cervus elaphus nelsoni) and used hierarchical Bayesian analysis to estimate the specificity and sensitivity of IHC and sPMCA conditional on simultaneously estimated disease states. Sensitivity estimates were higher for sPMCA (99.51%, credible interval (CI) 97.15-100%) than IHC of obex (brain stem, 76.56%, CI 57.00-91.46%) or retropharyngeal lymph node (90.06%, CI 74.13-98.70%) tissues, or both (98.99%, CI 90.01-100%). Our hierarchical Bayesian model predicts the prevalence of prion infection in this elk population to be 18.90% (CI 15.50-32.72%), compared to previous estimates of 12.90%. Our data reveal a previously unidentified sub-clinical prion-positive portion of the elk population that could represent silent carriers capable of significantly impacting CWD ecology.

  12. Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection.

    PubMed

    Arruda, Mauro Maciel de; Figueiredo, Fabiano Borges; Marcelino, Andreza Pain; Barbosa, José Ronaldo; Werneck, Guilherme Loureiro; Noronha, Elza Ferreira; Romero, Gustavo Adolfo Sierra

    2016-03-01

    In Brazil, human and canine visceral leishmaniasis (CVL) caused by Leishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.

  13. Molecular detection and genetic characterization of Toxoplasma gondii infection in sika deer (Cervus nippon) in China.

    PubMed

    Cong, Wei; Qin, Si-Yuan; Meng, Qing-Feng; Zou, Feng-Cai; Qian, Ai-Dong; Zhu, Xing-Quan

    2016-04-01

    The objective of the present study was to investigate the prevalence and genetic characterization of Toxoplasma gondii infection in sika deer in China. During August 2014 to November 2014, a total of 450 tissue samples coming from 150 sika deer were collected to detect the T. gondii B1 gene using a nested PCR, and the positive samples were genotyped at 11 genetic markers (SAG1, 5'- and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and Apico) using multilocus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. Seventeen of 150 sika deer (11.33%) were tested positive by nested PCR. Six DNA samples from the 17 positive samples were completely typed, in which 4 samples from lung tissues, and 2 from muscular tissues, were identified as ToxoDB Genotype #9 (http://toxodb.org/toxo/). The results of the present study revealed the existence of T. gondii infection in sika deer in China, which provided the information of T. gondii genetic diversity in this host species. This study also indicated that ToxoDB Genotype #9 has a wide distribution in sika deer that could be potential reservoirs for T. gondii transmission, which may pose a threat to human health. PMID:26772153

  14. A smart phone-based robust correction algorithm for the colorimetric detection of Urinary Tract Infection.

    PubMed

    Karlsen, Haakon; Tao Dong

    2015-08-01

    This paper presents the preliminary work of developing a smart phone based application for colorimetric detection of Urinary Tract Infection. The purpose is to make a smart phone function as a practical point-of-care device for nurses or medical personnel without access to strip readers. The main challenge is the constancy of camera color perception across different illuminations and devices, which is the first step towards a practical solution without additional equipment. A reported black and white reference correction and a comprehensive color image normalization have been utilized in this work. Comprehensive color image normalization appears to be quite effective at correcting the difference in perceived color due to different illumination, and is therefore a candidate for inclusion in the further work. PMID:26736494

  15. Utilization of an Eilat Virus-Based Chimera for Serological Detection of Chikungunya Infection.

    PubMed

    Erasmus, Jesse H; Needham, James; Raychaudhuri, Syamal; Diamond, Michael S; Beasley, David W C; Morkowski, Stan; Salje, Henrik; Fernandez Salas, Ildefonso; Kim, Dal Young; Frolov, Ilya; Nasar, Farooq; Weaver, Scott C

    2015-01-01

    In December of 2013, chikungunya virus (CHIKV), an alphavirus in the family Togaviridae, was introduced to the island of Saint Martin in the Caribbean, resulting in the first autochthonous cases reported in the Americas. As of January 2015, local and imported CHIKV has been reported in 50 American countries with over 1.1 million suspected cases. CHIKV causes a severe arthralgic disease for which there are no approved vaccines or therapeutics. Furthermore, the lack of a commercially available, sensitive, and affordable diagnostic assay limits surveillance and control efforts. To address this issue, we utilized an insect-specific alphavirus, Eilat virus (EILV), to develop a diagnostic antigen that does not require biosafety containment facilities to produce. We demonstrated that EILV/CHIKV replicates to high titers in insect cells and can be applied directly in enzyme-linked immunosorbent assays without inactivation, resulting in highly sensitive detection of recent and past CHIKV infection, and outperforming traditional antigen preparations. PMID:26492074

  16. ``Fatal Scream'' Of Bacteria Infected By Phages: Nanoscale Detection Of Bacteriophage Triggered Ion Cascade

    NASA Astrophysics Data System (ADS)

    King, Maria D.; Seo, Sungkyu; Kim, Jong; Cheng, Mosong; Young, Ryland; Biard, Robert J.; Bezrukov, Sergey M.; Granqvist, Claes-Goran; Kish, Laszlo B.

    2005-11-01

    A rapid, inexpensive and specific identification of arbitrary bacteria under field conditions is urgently needed. To this end, we have introduced and tested a new technology, called SEPTIC, SEnsing of Phage-Triggered Ion Cascade. In its prototype form based on a nanowell chip, SEPTIC has already been shown to be capable of unambiguous identification of live bacteria on a time scale of seconds to minutes, many times faster than any other system. The technology is based on using noise analysis to detect the massive ionic fluxes associated with the initial step of bacteriophage infection, the injection of the phage DNA into the cell. Here we show the results and pose a number of unsolved problems of noise. Ultimately, sensors based on this new technology would be able to save many lifes.

  17. Utilization of an Eilat Virus-Based Chimera for Serological Detection of Chikungunya Infection

    PubMed Central

    Erasmus, Jesse H.; Needham, James; Raychaudhuri, Syamal; Diamond, Michael S.; Beasley, David W. C.; Morkowski, Stan; Salje, Henrik; Fernandez Salas, Ildefonso; Kim, Dal Young; Frolov, Ilya; Nasar, Farooq; Weaver, Scott C.

    2015-01-01

    In December of 2013, chikungunya virus (CHIKV), an alphavirus in the family Togaviridae, was introduced to the island of Saint Martin in the Caribbean, resulting in the first autochthonous cases reported in the Americas. As of January 2015, local and imported CHIKV has been reported in 50 American countries with over 1.1 million suspected cases. CHIKV causes a severe arthralgic disease for which there are no approved vaccines or therapeutics. Furthermore, the lack of a commercially available, sensitive, and affordable diagnostic assay limits surveillance and control efforts. To address this issue, we utilized an insect-specific alphavirus, Eilat virus (EILV), to develop a diagnostic antigen that does not require biosafety containment facilities to produce. We demonstrated that EILV/CHIKV replicates to high titers in insect cells and can be applied directly in enzyme-linked immunosorbent assays without inactivation, resulting in highly sensitive detection of recent and past CHIKV infection, and outperforming traditional antigen preparations. PMID:26492074

  18. Early detection of Zika virus infection among travellers from areas of ongoing transmission in China.

    PubMed

    Zhang, Jiwen; Jin, Xia; Zhu, Zhaoyin; Huang, Li; Liang, Shaojun; Xu, Yuan; Liao, Ruyan; Zhou, Licheng; Zhang, Yan; Wilder-Smith, Annelies

    2016-05-01

    Nine imported Zika virus (ZIKV) infections (four through temperature monitoring and epidemiological investigation at entry and five by active surveillance tracking of index case contacts during follow-up; from Venezuela [n = 5], Samoa [n = 3] and both Samoa and Fiji [n = 1]) were detected in mainland China from February 1 to 29, 2016. The minimal incubation period lasted 5.2 days, with mean lag time to diagnosis of 2.6 days. Diagnosis relied on positive real-time reverse transcriptase polymerase chain reaction for ZIKV RNA in serum (n = 7), urine (n = 4) or saliva (n = 3), respectively. All cases recovered rapidly without serious complications. PMID:27378370

  19. Early detection of Zika virus infection among travellers from areas of ongoing transmission in China.

    PubMed

    Zhang, Jiwen; Jin, Xia; Zhu, Zhaoyin; Huang, Li; Liang, Shaojun; Xu, Yuan; Liao, Ruyan; Zhou, Licheng; Zhang, Yan; Wilder-Smith, Annelies

    2016-05-01

    Nine imported Zika virus (ZIKV) infections (four through temperature monitoring and epidemiological investigation at entry and five by active surveillance tracking of index case contacts during follow-up; from Venezuela [n = 5], Samoa [n = 3] and both Samoa and Fiji [n = 1]) were detected in mainland China from February 1 to 29, 2016. The minimal incubation period lasted 5.2 days, with mean lag time to diagnosis of 2.6 days. Diagnosis relied on positive real-time reverse transcriptase polymerase chain reaction for ZIKV RNA in serum (n = 7), urine (n = 4) or saliva (n = 3), respectively. All cases recovered rapidly without serious complications.

  20. The prevalence, distribution and severity of detectable pathological lesions in badgers naturally infected with Mycobacterium bovis.

    PubMed

    Jenkins, H E; Morrison, W I; Cox, D R; Donnelly, C A; Johnston, W T; Bourne, F J; Clifton-Hadley, R S; Gettinby, G; McInerney, J P; Watkins, G H; Woodroffe, R

    2008-10-01

    The Randomized Badger Culling Trial (RBCT) began in 1998 to determine the impact of badger culling in controlling bovine tuberculosis in cattle. A total of 1166 badgers (14% of total) proactively culled during the RBCT were found to be tuberculous, offering a unique opportunity to study the pathology caused by Mycobacterium bovis in a large sample of badgers. Of these, 39% of adults (approximately 6% of all adults culled) had visible lesions (detectable at necropsy) of bovine tuberculosis; cubs had a lower prevalence of infection (9%) but a higher percentage of tuberculous cubs (55.5%) had visible lesions. Only approximately 1% of adult badgers had extensive, severe pathology. Tuberculous badgers with recorded bite wounds (approximately 5%) had a higher prevalence of visible lesions and a different distribution of lesions, suggesting transmission via bite wounds. However, the predominance of lesions in the respiratory tract indicates that most transmission occurs by the respiratory route.

  1. Detection of rare and possibly carcinogenic human papillomavirus genotypes as single infections in invasive cervical cancer.

    PubMed

    Geraets, Daan; Alemany, Laia; Guimera, Nuria; de Sanjose, Silvia; de Koning, Maurits; Molijn, Anco; Jenkins, David; Bosch, Xavier; Quint, Wim

    2012-12-01

    The contribution of carcinogenic human papillomavirus (HPV) types to the burden of cervical cancer has been well established. However, the role and contribution of phylogenetically related HPV genotypes and rare variants remains uncertain. In a recent global study of 8977 HPV-positive invasive cervical carcinomas (ICCs), the genotype remained unidentified in 3.7% by the HPV SPF10 PCR-DEIA-LiPA25 (version 1) algorithm. The 331 ICC specimens with unknown genotype were analysed by a novel sequence methodology, using multiple selected short regions in L1. This demonstrated HPV genotypes that have infrequently or never been detected in ICC, ie HPV26, 30, 61, 67, 68, 69, 73 and 82, and rare variants of HPV16, 18, 26, 30, 34, 39, 56, 67, 68, 69, 82 and 91. These are not identified individually by LiPA25 and only to some extent by other HPV genotyping assays. Most identified genotypes have a close phylogenetic relationship with established carcinogenic HPVs and have been classified as possibly carcinogenic by IARC. Except for HPV85, all genotypes in α-species 5, 6, 7, 9 and 11 were encountered as single infections in ICCs. These species of established and possibly carcinogenic HPV types form an evolutionary clade. We have shown that the possibly carcinogenic types were detected only in squamous cell carcinomas, which were often keratinizing and diagnosed at a relatively higher mean age (55.3 years) than those associated with established carcinogenic types (50.9 years). The individual frequency of the possibly carcinogenic types in ICCs is low, but together they are associated with 2.25% of the 8338 included ICCs with a single HPV type. This fraction is greater than seven of the established carcinogenic types individually. This study provides evidence that possibly carcinogenic HPV types occur as single infections in invasive cervical cancer, strengthening the circumstantial evidence of a carcinogenic role.

  2. Detection of airborne influenza a virus in experimentally infected pigs with maternally derived antibodies.

    PubMed

    Corzo, C A; Allerson, M; Gramer, M; Morrison, R B; Torremorell, M

    2014-02-01

    This study assessed whether recently weaned piglets with maternally derived antibodies were able to generate infectious influenza aerosols. Three groups of piglets were assembled based on the vaccination status of the dam. Sows were either non-vaccinated (CTRL) or vaccinated with the same (VAC-HOM) strain or a different (VAC-HET) strain to the one used for challenge. Piglets acquired the maternally derived antibodies by directly suckling colostrum from their respective dams. At weaning, pigs were challenged with influenza virus by direct contact with an infected pig (seeder pig) and clinical signs evaluated. Air samples, collected using a liquid cyclonic air collector, and individual nasal swabs were collected daily for 10 days from each group and tested by matrix real-time reverse transcriptase polymerase chain reaction (RRT-PCR) assay. Virus isolation and titration were attempted for air samples on Madin-Darby canine kidney cells. All individual pigs from both VAC-HET and CTRL groups tested positive during the study but only one pig in the VAC-HOM group was positive by nasal swab RRT-PCR. Influenza virus could not be detected or isolated from air samples from the VAC-HOM group. Influenza A virus was isolated from 3.2% and 6.4% air samples from both the VAC-HET and CTRL groups, respectively. Positive RRT-PCR air samples were only detected in VAC-HET and CTRL groups on day 7 post-exposure. Overall, this study provides evidence that recently weaned pigs with maternally derived immunity without obvious clinical signs of influenza infection can generate influenza infectious aerosols which is relevant to the transmission and the ecology of influenza virus in pigs.

  3. Detection of airborne influenza a virus in experimentally infected pigs with maternally derived antibodies.

    PubMed

    Corzo, C A; Allerson, M; Gramer, M; Morrison, R B; Torremorell, M

    2014-02-01

    This study assessed whether recently weaned piglets with maternally derived antibodies were able to generate infectious influenza aerosols. Three groups of piglets were assembled based on the vaccination status of the dam. Sows were either non-vaccinated (CTRL) or vaccinated with the same (VAC-HOM) strain or a different (VAC-HET) strain to the one used for challenge. Piglets acquired the maternally derived antibodies by directly suckling colostrum from their respective dams. At weaning, pigs were challenged with influenza virus by direct contact with an infected pig (seeder pig) and clinical signs evaluated. Air samples, collected using a liquid cyclonic air collector, and individual nasal swabs were collected daily for 10 days from each group and tested by matrix real-time reverse transcriptase polymerase chain reaction (RRT-PCR) assay. Virus isolation and titration were attempted for air samples on Madin-Darby canine kidney cells. All individual pigs from both VAC-HET and CTRL groups tested positive during the study but only one pig in the VAC-HOM group was positive by nasal swab RRT-PCR. Influenza virus could not be detected or isolated from air samples from the VAC-HOM group. Influenza A virus was isolated from 3.2% and 6.4% air samples from both the VAC-HET and CTRL groups, respectively. Positive RRT-PCR air samples were only detected in VAC-HET and CTRL groups on day 7 post-exposure. Overall, this study provides evidence that recently weaned pigs with maternally derived immunity without obvious clinical signs of influenza infection can generate influenza infectious aerosols which is relevant to the transmission and the ecology of influenza virus in pigs. PMID:22827737

  4. Protein antigens of Chlamydia psittaci present in infected cells but not detected in the infectious elementary body.

    PubMed

    Rockey, D D; Rosquist, J L

    1994-01-01

    Ocular infection of guinea pigs with the guinea pig inclusion conjunctivitis (GPIC) strain of Chlamydia psittaci produces a clinical condition representative of acute chlamydial conjunctivitis in humans. Guinea pigs which had recovered from two challenges with GPIC were used as a source of sera for the identification of antigens present in GPIC-infected tissue culture cells but absent in the infectious elementary body (EB). Immunoblots of lysates of infected HeLa cells probed with the convalescent-phase sera identified protein antigens of 22, 34, and 52 kDa (p22, p34, and p52, respectively) that were not detected in lysates of purified EB or in uninfected HeLa cells. Protein p22 was also not detected in lysates of purified reticulate bodies. Immunoblotting of lysates of HeLa cells infected with other chlamydiae demonstrated that the antigenicity of p22 and p34 was subspecies specific. Immunoblotting was also used to detect p22 and p34 in lysates of the conjunctivae of infected guinea pigs. Adsorption of convalescent-phase sera with GPIC EB produced a reagent with dominant reactivity toward p22, p34, and a 28-kDa EB protein. Immunofluorescent staining of GPIC-infected HeLa cells demonstrated that these adsorbed sera labeled the inclusion and inclusion membrane, with no apparent reactivity toward EB or reticulate bodies. Collectively, these data identify non-EB chlamydial components which may be released into the inclusion during intracellular growth.

  5. The TcTASV proteins are novel promising antigens to detect active Trypanosoma cruzi infection in dogs.

    PubMed

    Floridia-Yapur, N; Monje Rumi, M; Ragone, P; Lauthier, J J; Tomasini, N; Alberti D'Amato, A; Diosque, P; Cimino, R; Marco, J D; Barroso, P; Sanchez, D O; Nasser, J R; Tekiel, V

    2016-09-01

    In regions where Chagas disease is endemic, canine Trypanosoma cruzi infection is highly correlated with the risk of transmission of the parasite to humans. Herein we evaluated the novel TcTASV protein family (subfamilies A, B, C), differentially expressed in bloodstream trypomastigotes, for the detection of naturally infected dogs. A gene of each TcTASV subfamily was cloned and expressed. Indirect enzyme-linked immunosorbent assays (ELISA) were developed using recombinant antigens individually or mixed together. Our results showed that dogs with active T. cruzi infection differentially reacted against the TcTASV-C subfamily. The use of both TcTASV-C plus TcTASV-A proteins (Mix A+C-ELISA) enhanced the reactivity of sera from dogs with active infection, detecting 94% of the evaluated samples. These findings agree with our previous observations, where the infected animals exhibited a quick anti-TcTASV-C antibody response, coincident with the beginning of parasitaemia, in a murine model of the disease. Results obtained in the present work prove that the Mix A+C-ELISA is a specific, simple and cheap technique to be applied in endemic areas in screening studies. The Mix A+C-ELISA could help to differentially detect canine hosts with active infection and therefore with high impact in the risk of transmission to humans. PMID:27173912

  6. The TcTASV proteins are novel promising antigens to detect active Trypanosoma cruzi infection in dogs.

    PubMed

    Floridia-Yapur, N; Monje Rumi, M; Ragone, P; Lauthier, J J; Tomasini, N; Alberti D'Amato, A; Diosque, P; Cimino, R; Marco, J D; Barroso, P; Sanchez, D O; Nasser, J R; Tekiel, V

    2016-09-01

    In regions where Chagas disease is endemic, canine Trypanosoma cruzi infection is highly correlated with the risk of transmission of the parasite to humans. Herein we evaluated the novel TcTASV protein family (subfamilies A, B, C), differentially expressed in bloodstream trypomastigotes, for the detection of naturally infected dogs. A gene of each TcTASV subfamily was cloned and expressed. Indirect enzyme-linked immunosorbent assays (ELISA) were developed using recombinant antigens individually or mixed together. Our results showed that dogs with active T. cruzi infection differentially reacted against the TcTASV-C subfamily. The use of both TcTASV-C plus TcTASV-A proteins (Mix A+C-ELISA) enhanced the reactivity of sera from dogs with active infection, detecting 94% of the evaluated samples. These findings agree with our previous observations, where the infected animals exhibited a quick anti-TcTASV-C antibody response, coincident with the beginning of parasitaemia, in a murine model of the disease. Results obtained in the present work prove that the Mix A+C-ELISA is a specific, simple and cheap technique to be applied in endemic areas in screening studies. The Mix A+C-ELISA could help to differentially detect canine hosts with active infection and therefore with high impact in the risk of transmission to humans.

  7. Community-Based Sexually Transmitted Infection Screening and Increased Detection of Pharyngeal and Urogenital Chlamydia trachomatis and Neisseria gonorrhoeae Infections in Female Sex Workers in Hong Kong

    PubMed Central

    Wong, Horas T.H.; Lee, Krystal C.K.; Chan, Denise P.C.

    2015-01-01

    Background Female sex workers (FSWs) are vulnerable to sexually transmitted infections (STIs) and are one of the key populations being infected most by Chlamydia trachomatis and Neisseria gonorrhoeae infections. In Hong Kong, limited data on the burden of chlamydial and gonococcal infections exist because regular screenings are not offered. This study aimed to investigate the prevalence of C. trachomatis and N. gonorrhoeae in FSWs and to assess predictors associated with unprotected fellatio. Methods A cross-sectional study was conduct on 340 FSWs attending a community organization for HIV/STI screening, and a questionnaire addressing sociodemographic and behavioral characteristics was administered to all FSWs. Results The prevalence of syphilis infection was 2.1%, and none was tested positive for HIV. The positivity for pharyngeal C. trachomatis and N. gonorrhoeae was 3.2% and 4.4%, respectively, whereas that for urogenital chlamydial and gonococcal infection was 10.6% and 0.9%, respectively. Of 313 FSWs offering fellatio, having unprotected fellatio with clients was significantly associated with the perceived low risk of contracting STI via fellatio (adjusted odds ratio [OR], 1.88), working in clubs (adjusted OR, 11.14), working on streets (adjusted OR, 3.28), recently started working in the sex industry for 1 year or less (adjusted OR, 3.05), and reporting group sex in the previous year (adjusted OR, 11.03). Conclusions The prevalence of HIV and syphilis infection remains low. This study reveals a relatively high prevalence of N. gonorrhoeae detected mostly in the pharynx. Offering pharyngeal screening for STI would facilitate early diagnosis and treatment of gonococcal infection in FSWs in Hong Kong. PMID:25768859

  8. Absence of the Filarial Endosymbiont Wolbachia in Seal Heartworm (Acanthocheilonema spirocauda) but Evidence of Ancient Lateral Gene Transfer.

    PubMed

    Keroack, Caroline D; Wurster, Jenna I; Decker, Caroline G; Williams, Kalani M; Slatko, Barton E; Foster, Jeremy M; Williams, Steven A

    2016-06-01

    The symbiotic relationship of Wolbachia spp. was first observed in insects and subsequently in many parasitic filarial nematodes. This bacterium is believed to provide metabolic and developmental assistance to filarial parasitic nematodes, although the exact nature of this relationship remains to be fully elucidated. While Wolbachia is present in most filarial nematodes in the family Onchocercidae, it is absent in several disparate species such as the human parasite Loa loa . All tested members of the genus Acanthocheilonema, such as Acanthocheilonema viteae, have been shown to lack Wolbachia. Consistent with this, we show that Wolbachia is absent from the seal heartworm (Acanthocheilonema spirocauda), but lateral gene transfer (LGT) of DNA sequences between Wolbachia and A. spirocauda has occurred, indicating a past evolutionary association. Seal heartworm is an important pathogen of phocid seals and understanding its basic biology is essential for conservation of the host. The findings presented here may allow for the development of future treatments or diagnostics for the disease and also aid in clarification of the complicated nematode-Wolbachia relationship. PMID:26859724

  9. A nonenzymatic optical immunoassay strategy for detection of Salmonella infection based on blue silica nanoparticles.

    PubMed

    Sun, Qian; Zhao, Guangying; Dou, Wenchao

    2015-10-22

    A novel nonenzymatic optical immunoassay strategy was for the first time designed and utilized for sensitive detection of antibody to Salmonella pullorum and Salmonella gallinarum (S. pullorum and S. gallinarum) in serum. The optical immunoassay strategy was based on blue silica nanoparticles (Blue-SiNps) and magnetic beads (MB). To construct such an optical immunoassay system, the Blue-SiNPs were first synthesized by inverse microemulsion method, characterized by SEM, Zeta potential and FTIR. Two nanostructures including Blue-SiNPs and MB were both functionalized with antibody against S. pullorum and S. gallinarum (anti-PG) without using enzyme labeled antibody. Anti-PG functionalized blue silica nanoparticles (IgG-Blue-SiNps) were used as signal transduction labels, while anti-PG functionalized magnetic beads (IgG-MB) were selected to separate and enrich the final sandwich immune complexes. In the process of detecting negative serum, a sandwich immunocomplex is formed between the IgG-MB and IgG-Blue-SiNPs. With the separation of the immunocomplex using an external magnetic field, the final plaque displayed bright blue color. While in the detection of infected serum, IgG-MB and anti-PG formed sandwich immunocomplexes, IgG-Blue-SiNPs were unable to bind to the limited sites of the antigen, and a light brown plaque was displayed in the bottom of microplate well. Stable results were obtained with an incubation time of 60 min at room temperature, and different colors corresponding to different results can be directly detected with naked eye. The reaction of IgG-Blue-SiNPs with S. pullorum was inhibited by 1:100 dilution of positive chicken serum. Such a simple immunoassay holds great potential as sensitive, selective and point-of-care (POC) tool for diagnosis of other biological molecules.

  10. Generation of Calves Persistently Infected with HoBi-Like Pestivirus and Comparison of Methods for Detection of These Persistent Infections

    PubMed Central

    Falkenberg, S. M.; Vander Ley, B.; Decaro, N.; Brodersen, B. W.; Harmon, A.; Hessman, B.; Flores, E. F.; Ridpath, J. F.

    2014-01-01

    The identification and elimination of persistently infected (PI) cattle are the most effective measures for controlling bovine pestiviruses, including bovine viral diarrhea virus (BVDV) and the emerging HoBi-like viruses. Here, colostrum-deprived calves persistently infected with HoBi-like pestivirus (HoBi-like PI calves) were generated and sampled (serum, buffy coat, and ear notches) on the day of birth (DOB) and weekly for 5 consecutive weeks. The samples were subjected to diagnostic tests for BVDV—two reverse transcriptase PCR (RT-PCR) assays, two commercial real-time RT quantitative PCR (RT-qPCR), two antigen capture enzyme-linked immunosorbent assays (ACE), and immunohistochemistry (IHC)—and to HoBi-like virus-specific RT-PCR and RT-qPCR assays. The rate of false negatives varied among the calves. The HoBi-like virus-specific RT-PCR detected HoBi-like virus in 83%, 75%, and 87% of the serum, buffy coat, and ear notch samples, respectively, while the HoBi-like RT-qPCR detected the virus in 83%, 96%, and 62%, respectively. In comparison, the BVDV RT-PCR test had a higher rate of false negatives in all tissue types, especially for the ear notch samples (missing detection in at least 68% of the samples). The commercial BVDV RT-qPCRs and IHC detected 100% of the ear notch samples as positive. While ACE based on the BVDV glycoprotein Erns detected infection in at least 87% of ear notches, no infections were detected using NS3-based ACE. The BVDV RT-qPCR, ACE, and IHC yielded higher levels of detection than the HoBi-like virus-specific assays, although the lack of differentiation between BVDV and HoBi-like viruses would make these tests of limited use for the control and/or surveillance of persistent HoBi-like virus infection. An improvement in HoBi-like virus tests is required before a reliable HoBi-like PI surveillance program can be designed. PMID:25122860

  11. Detection of hepatitis B virus DNA sequences in infected hepatocytes by in situ cytohybridisation

    SciTech Connect

    Gowans, E.J.; Burrell, C.J.; Jilbert, A.R.; Marmion, B.P.

    1981-01-01

    Plasmid pHBV 114 DNA, which contains 73% of the genome of hepatitis B virus (HBV), was radiolabelled with tritium to 1-2 X 10(8) dpm/microgram by nick translation and used as a radioactive probe to detect HBV DNA present in sections of infected liver tissue by in situ hybridisation followed by autoradiography. Factors affecting the sensitivity of the reaction were examined, including different methods of fixation, hybridisation time, temperature, and buffers. The specificity of the reaction for detecting viral DNA was carefully established by the use of unrelated DNA probes, pretreatment of sections with DNAase, and comparing the stability of the binding of DNA probe at different temperatures, with the melting curve of double-stranded DNA in solution. In the one liver studied in detail, cells containing large amounts of viral DNA were distributed in foci corresponding to areas containing morphologically damaged hepatocytes. This observation suggested a relationship between active viral replication and cell damage. Viral DNA was found mainly in the cytoplasm, although a minority of nuclei in these foci were also positive.

  12. Mitochondrial malate dehydrogenase and malic enzyme of a filarial worm Setaria digitata: some properties and effects of drugs and herbal extracts.

    PubMed

    Banu, M J; Nellaiappan, K; Dhandayuthapani, S

    1992-06-01

    Mitochondrial malate dehydrogenase (mMDH) and malic enzyme (mME) of a filarial worm Setaria digitata were studied. mMDH exhibited the highest activities in the oxidation and reduction reactions at pH 9.5 and pH 6.2, respectively, while mME did so in the malate decarboxylation reaction at pH 6.8. mME showed no detectable activity on the pyruvate carboxylation direction. The Km values for malate (1.7 mM) and oxaloacetate (0.17 mM) and the ratio of Vmax oxidation: Vmax reduction (2.73) tend to favor the oxaloacetate reduction by mMDH. mME showed a relatively high Km value of 8.3 mM, for malate decarboxylation. A drug, diethylcarbamazine citrate (DEC-C), did not change appreciably the activity of either mMDH or mME, while filarin (a drug of herbal origin) effectively inhibited mMDH. The leaf extracts of Ocimum sanctum, Lawsonia inermis and Calotropis gigantea and leaf and flower extracts of Azadirachta indica were, however, found to inhibit both mMDH and mME.

  13. Evaluation of a method to detect Mycobacterium bovis in air samples from infected Eurasian badgers (Meles meles) and their setts.

    PubMed

    Jones, R M; Ashford, R; Cork, J; Palmer, S; Wood, E; Spyvee, P; Parks, S; Bennett, A; Brewer, J; Delahay, R; Chambers, M; Sawyer, J

    2013-05-01

    Environmental air sampling was evaluated as a method to detect the presence of M. bovis in the vicinity of infected badgers and their setts. Airborne particles were collected on gelatine filters using a commercially available air sampling instrument and tested for the presence of M. bovis using bacteriological culture and real-time PCR. The sensitivity of bacteriological culture was broadly similar to that of real-time PCR when testing samples artificially spiked with M. bovis. Sampling was undertaken from directly under the muzzles of badgers which had been experimentally infected with M. bovis (37 samples), within enclosures housing the experimentally infected animals (50 samples), and in the vicinity of setts with resident infected wild badgers (52 samples). The methods employed did not detect M. bovis from either infected badgers or artificial or natural setts known to contain infected animals. However, samples taken at four of the six natural setts were positive for Mycobacterium gordonae.

  14. Detection of the meq gene in the T cell subsets from chickens infected with Marek's disease virus serotype 1.

    PubMed

    Chang, Kyung-Soo; Ohashi, Kazuhiko; Lee, Sung-Il; Takagi, Michihiro; Onum, Misao

    2005-08-01

    The meq gene was thought to be only detected in Marek's disease virus serotype 1 (MDV 1) including a very virulent strain, Md5, while L-meq, in which a 180-bp sequence is inserted into the meq open reading frame, is found in other strains of MDV 1, such as CVI 988/R6. However, both meq and L-meq were previously detected by PCR in chickens infected with MDV 1, suggesting that MDV 1 may consists of at least two subpopulations, one with meq, the other with L-meq. To further analyze these subpopulations, we analyzed the time course changes in distribution of these subpopulations among T cell subsets from chickens infected with MDV 1. Both meq and L-meq were detected in CD4+ and CD8+ T cells infected with strain Md5 or CVI 988/R6. The shift in MDV subpopulations from one displaying meq to the other displaying L-meq and/or the conversion from meq to L-meq occurred mainly in the CD8+ T cell subset from Md5-infected chickens. PCR products corresponding to L-meq rather than meq were frequently amplified from the CD8+ T cell subset from CVI 988/R 6 -infected chickens. These results suggest that a dominant subpopulation of MDV 1 changes depending on the T cell subsets, and that L-meq is dominantly present in the CD8+ T cells which play a role in the clearance of pathogenic agents.

  15. Role of Histological Findings and Pathologic Diagnosis for Detection of Human Papillomavirus Infection in Men

    PubMed Central

    Vyas, Nikki S.; Pierce Campbell, Christine M.; Mathew, Rahel; Abrahamsen, Martha; Van der Kooi, Kaisa; Jukic, Drazen M.; Stoler, Mark H.; Villa, Luisa L.; da Silva, Roberto Carvalho; Lazcano-Ponce, Eduardo; Quiterio, Manuel; Salmeron, Jorge; Sirak, Bradley A.; Ingles, Donna J.; Giuliano, Anna R.; Messina, Jane L.

    2016-01-01

    Early HPV infection in males is difficult to detect clinically and pathologically. This study assessed histopathology in diagnosing male genital HPV. External genital lesions (n = 352) were biopsied, diagnosed by a dermatopathologist, and HPV genotyped. A subset (n = 167) was diagnosed independently by a second dermatopathologist and also re-evaluated in detail, tabulating the presence of a set of histopathologic characteristics related to HPV infection. Cases that received discrepant diagnoses or HPV-related diagnoses were evaluated by a third dermatopathologist (n = 163). Across dermatopathologists, three-way concordance was fair (k = 0.30). Pairwise concordance for condyloma was fair to good (k = 0.30–0.67) and poor to moderate for penile intraepithelial neoplasia (k = −0.05 to 0.42). Diagnoses were 44–47% sensitive and 65–72% specific for HPV 6/ 11-containing lesions, and 20–37% sensitive and 98–99% specific for HPV 16/18. Presence of HPV 6/ 11 was 75–79% sensitive and 35% specific for predicting pathologic diagnosis of condyloma. For diagnosis of penile intraepithelial neoplasia, HPV 16/18 was 95–96% specific but only 40–64% sensitive. Rounded papillomatosis, hypergranulosis, and dilated vessels were significantly (P<0.05) associated with HPV 6/11. Dysplasia was significantly (P= 0.001) associated with HPV 16/18. Dermatopathologists’ diagnoses of early male genital HPV-related lesions appear discordant with low sensitivity, while genotyping may overestimate clinically significant HPV-related disease. Rounded papillomatosis, hypergranulosis, and dilated vessels may help establish diagnosis of early condyloma. PMID:25945468

  16. Role of histological findings and pathologic diagnosis for detection of human papillomavirus infection in men.

    PubMed

    Vyas, Nikki S; Pierce Campbell, Christine M; Mathew, Rahel; Abrahamsen, Martha; Van der Kooi, Kaisa; Jukic, Drazen M; Stoler, Mark H; Villa, Luisa L; da Silva, Roberto Carvalho; Lazcano-Ponce, Eduardo; Quiterio, Manuel; Salmeron, Jorge; Sirak, Bradley A; Ingles, Donna J; Giuliano, Anna R; Messina, Jane L

    2015-10-01

    Early HPV infection in males is difficult to detect clinically and pathologically. This study assessed histopathology in diagnosing male genital HPV. External genital lesions (n = 352) were biopsied, diagnosed by a dermatopathologist, and HPV genotyped. A subset (n = 167) was diagnosed independently by a second dermatopathologist and also re-evaluated in detail, tabulating the presence of a set of histopathologic characteristics related to HPV infection. Cases that received discrepant diagnoses or HPV-related diagnoses were evaluated by a third dermatopathologist (n = 163). Across dermatopathologists, three-way concordance was fair (k = 0.30). Pairwise concordance for condyloma was fair to good (k = 0.30-0.67) and poor to moderate for penile intraepithelial neoplasia (k = -0.05 to 0.42). Diagnoses were 44-47% sensitive and 65-72% specific for HPV 6/11-containing lesions, and 20-37% sensitive and 98-99% specific for HPV 16/18. Presence of HPV 6/11 was 75-79% sensitive and 35% specific for predicting pathologic diagnosis of condyloma. For diagnosis of penile intraepithelial neoplasia, HPV 16/18 was 95-96% specific but only 40-64% sensitive. Rounded papillomatosis, hypergranulosis, and dilated vessels were significantly (P < 0.05) associated with HPV 6/11. Dysplasia was significantly (P = 0.001) associated with HPV 16/18. Dermatopathologists' diagnoses of early male genital HPV-related lesions appear discordant with low sensitivity, while genotyping may overestimate clinically significant HPV-related disease. Rounded papillomatosis, hypergranulosis, and dilated vessels may help establish diagnosis of early condyloma.

  17. Detection and identification of Theileria infection in sika deer ( Cervus nippon ) in China.

    PubMed

    He, Lan; Khan, Muhanmad Kasib; Zhang, Wen-Jie; Zhang, Qing-Li; Zhou, Yan-Qin; Hu, Min; Zhao, Junlong

    2012-06-01

    The sika deer ( Cervus nippon ) is a first-grade state-protected animal in China and designated a threatened species by the World Conservation Union. To detect hemoparasite infection of sika deer, blood samples were collected from 24 animals in the Hubei Province Deer Center. Genomic DNA was extracted, and the V4 hypervariable region encoding 18S rRNA was analyzed by reverse line blot hybridization assay. PCR products hybridized with Babesia / Theileria genus-specific probes but failed to hybridize with any of the Babesia or Theileria species-specific probes, suggesting the presence of a novel, or variant, species. Here 18S rRNA and internal transcribed spacer (ITS) genes were amplified, cloned, and sequenced from 7 isolates. Alignment and BlastN of the cloned sequences revealed high similarities to the homologous 18S rRNA genes and ITS genes of Theileria cervi (AY735122), Theileria sp. CNY1A (AB012194), and Theileria sp. ex Yamaguchi (AF529272). Phylogenetic analysis based on the 18S rRNA gene and ITS sequences showed that all cloned sequences were grouped within the Theileria clade. Phylogeny based on the 18S rRNA gene divided the organisms into 2 groups. Group 1 was closest to Theileria sp. ex Yamaguchi (AF529272), and group 2 was distinct from all other identified Theileria and Babesia species. These results suggest the existence of Theileria sp. infection in sika deer in China. To our knowledge, this is the first report of cervine Theileria sp. in China.

  18. Xenodiagnosis to Detect Borrelia burgdorferi Infection: A First-in-Human Study

    PubMed Central

    Marques, Adriana; Telford, Sam R.; Turk, Siu-Ping; Chung, Erin; Williams, Carla; Dardick, Kenneth; Krause, Peter J.; Brandeburg, Christina; Crowder, Christopher D.; Carolan, Heather E.; Eshoo, Mark W.; Shaw, Pamela A.; Hu, Linden T.

    2014-01-01

    Background. Animal studies suggest that Borrelia burgdorferi, the agent of Lyme disease, may persist after antibiotic therapy and can be detected by various means including xenodiagnosis using the natural tick vector (Ixodes scapularis). No convincing evidence exists for the persistence of viable spirochetes after recommended courses of antibiotic therapy in humans. We determined the safety of using I. scapularis larvae for the xenodiagnosis of B. burgdorferi infection in humans. Methods. Laboratory-reared larval I. scapularis ticks were placed on 36 subjects and allowed to feed to repletion. Ticks were tested for B. burgdorferi by polymerase chain reaction (PCR), culture, and/or isothermal amplification followed by PCR and electrospray ionization mass spectroscopy. In addition, attempts were made to infect immunodeficient mice by tick bite or inoculation of tick contents. Xenodiagnosis was repeated in 7 individuals. Results. Xenodiagnosis was well tolerated with no severe adverse events. The most common adverse event was mild itching at the tick attachment site. Xenodiagnosis was negative in 16 patients with posttreatment Lyme disease syndrome (PTLDS) and/or high C6 antibody levels and in 5 patients after completing antibiotic therapy for erythema migrans. Xenodiagnosis was positive for B. burgdorferi DNA in a patient with erythema migrans early during therapy and in a patient with PTLDS. There is insufficient evidence, however, to conclude that viable spirochetes were present in either patient. Conclusions. Xenodiagnosis using Ixodes scapularis larvae was safe and well tolerated. Further studies are needed to determine the sensitivity of xenodiagnosis in patients with Lyme disease and the significance of a positive result. Clinical Trials Registration NCT01143558. PMID:24523212

  19. Role of histological findings and pathologic diagnosis for detection of human papillomavirus infection in men.

    PubMed

    Vyas, Nikki S; Pierce Campbell, Christine M; Mathew, Rahel; Abrahamsen, Martha; Van der Kooi, Kaisa; Jukic, Drazen M; Stoler, Mark H; Villa, Luisa L; da Silva, Roberto Carvalho; Lazcano-Ponce, Eduardo; Quiterio, Manuel; Salmeron, Jorge; Sirak, Bradley A; Ingles, Donna J; Giuliano, Anna R; Messina, Jane L

    2015-10-01

    Early HPV infection in males is difficult to detect clinically and pathologically. This study assessed histopathology in diagnosing male genital HPV. External genital lesions (n = 352) were biopsied, diagnosed by a dermatopathologist, and HPV genotyped. A subset (n = 167) was diagnosed independently by a second dermatopathologist and also re-evaluated in detail, tabulating the presence of a set of histopathologic characteristics related to HPV infection. Cases that received discrepant diagnoses or HPV-related diagnoses were evaluated by a third dermatopathologist (n = 163). Across dermatopathologists, three-way concordance was fair (k = 0.30). Pairwise concordance for condyloma was fair to good (k = 0.30-0.67) and poor to moderate for penile intraepithelial neoplasia (k = -0.05 to 0.42). Diagnoses were 44-47% sensitive and 65-72% specific for HPV 6/11-containing lesions, and 20-37% sensitive and 98-99% specific for HPV 16/18. Presence of HPV 6/11 was 75-79% sensitive and 35% specific for predicting pathologic diagnosis of condyloma. For diagnosis of penile intraepithelial neoplasia, HPV 16/18 was 95-96% specific but only 40-64% sensitive. Rounded papillomatosis, hypergranulosis, and dilated vessels were significantly (P < 0.05) associated with HPV 6/11. Dysplasia was significantly (P = 0.001) associated with HPV 16/18. Dermatopathologists' diagnoses of early male genital HPV-related lesions appear discordant with low sensitivity, while genotyping may overestimate clinically significant HPV-related disease. Rounded papillomatosis, hypergranulosis, and dilated vessels may help establish diagnosis of early condyloma. PMID:25945468

  20. Detection of tissue culture-adapted Theiler's virus RNA in spinal cord white matter cells throughout infection.

    PubMed

    Stroop, W G; Brahic, M; Baringer, J R

    1982-08-01

    The appearance of histological lesions and the localization of viral RNA in the central nervous system of mice infected with tissue culture-adapted Theiler's murine encephalomyelitis virus (WW strain) (TMEV-WW) was studied. Viral RNA was detected by autoradiography after in situ hybridization, using a (3)H-labeled DNA probe complementary to virion RNA, which was applied to deparaffinized sections of central nervous system tissues from infected mice. Subjacent histological sections of tissues were used to assess the location and extent of lesions. Lesions were first observed at 20 days post-inoculation and appeared to enlarge throughout infection. They consisted of infiltrates of mononuclear cells and lymphocytes in spinal cord white matter and leptomeninges; at 78 days post-inoculation severe necrotizing and demyelinative myelitis and gliosis were observed. In contrast to the pathogenesis of brain-derived TMEV-WW-infected mice, no lesions were found in the central nervous system gray matter of mice infected with tissue culture-adapted TMEV-WW at any time post-infection. Tissue culture-adapted viral RNA was found in the cells of spinal cord white matter throughout infection; only one neuron in close proximity to the injection site was found to contain viral RNA shortly after infection. At early times after infection, spinal cord white matter cells containing viral RNA were found before development of inflammatory lesions; at later days post-inoculation, positive cells were found within, at the periphery of, or at a distance from lesions. The number of infected cells and the amount of viral RNA per cell appeared to remain constant from 20 to 78 days post-inoculation despite the increasing intensity of the inflammatory response. The nearly exclusive spinal cord white matter tropism of tissue culture-adapted TMEV-WW appeared to directly correlate with the disease-inducing potential of this virus.

  1. Detection of Ehrlichia canis, Babesia vogeli, and Toxoplasma gondii DNA in the Brain of Dogs Naturally Infected with Leishmania infantum.

    PubMed

    Cardinot, Cinthya B; Silva, José E S; Yamatogi, Ricardo S; Nunes, Cáris M; Biondo, Alexander W; Vieira, Rafael F C; Junior, João P Araujo; Marcondes, Mary

    2016-04-01

    The aims of this study were to investigate the presence of Leishmania infantum and possible co-infection with Anaplasma platys , Babesia vogeli, Ehrlichia canis , and Toxoplasma gondii in the brain of 24 dogs naturally infected by L. infantum . A total of 24 mongrel adult dogs (22 clinically affected, 2 with neurological signs, and 2 subclinically infected) aged between 2 and 5 yr, naturally infected by visceral leishmaniasis, were selected. Fragments from meninges, frontal cortex, thalamus, cerebellum, and choroid plexus of the lateral ventricles and fourth ventricle were collected, mixed, and tested by real-time polymerase chain reaction (qPCR). Leishmania infantum DNA was detected in 95.8% (23/24) of the infected dogs, including the subclinically infected. A total of 14/24 (58.3%) dogs were co-infected by E. canis and L. infantum , 4/24 (16.7%) were co-infected by E. canis , B. vogeli, and L. infantum , 2/24 (8.3%) were co-infected by B. vogeli and L. infantum , and 1/24 (4.2%) dog was co-infected by E. canis , B. vogeli, T. gondii , and L. infantum . All 24 brain samples tested negative for A. platys . These results demonstrate that L. infantum is able to penetrate into the brain parenchyma, either alone or in association to other zoonotic pathogens. In addition, qPCR could be considered for adequate evaluation of Leishmania in the brain tissue of dogs with neurological signs that have died. PMID:26765523

  2. A microtitration agglutination test for detecting group E streptococcus infection in swine.

    PubMed

    Armstrong, C H; Wood, R L; Wessman, G E

    1982-04-01

    A microtitration agglutination test was developed and evaluated for detecting infection of swine with group E streptococci type IV, the most common causative agent of streptococcic lymphadenitis of swine. Whole cell agglutinogens representing group and type antigens of group E streptococci were tested in the microtitration agglutination test against reference antisera to Streptococcus groups A, B, C, D, E, F, G. H, K, L, M, N, O, P, Q, R, S and U, as well as specific antisera to types II, IV and V of group E. Group E specific agglutinogens were unsatisfactory in the microtitration agglutination test because of cross reactions with group P and U antisera and because of poor reproducibility of the test. Type specific agglutinogens of group E streptococci reacted only with their respective homologous antisera and not with any heterologous group antisera. None of the group E streptococci agglutinogens reacted with 52 normal swine sera. Agglutinogen made from group E streptococci type IV was selected for further evaluation in the microtitration agglutination test because group E streptococci types II and V are considered to be of minor importance in the etiology of streptococcic lymphadenitis of swine. Swine experimentally infected with a type IV strain developed significant titers in the microtitration agglutination test. All swine tested negative before exposure and seroconverted (titer >/=4) two to six weeks postexposure.The microtitration agglutination test was used by two different laboratories to test 187 duplicate samples of serum from infected swine. A total of 94.1% of the tests were read at either the same titer (48.1%) or a difference of not more than one dilution (46.0%) at the two laboratories. There was disagreement between the two laboratories in the test-positive test-negative status of 19 of the sera (10.2%). Titers of two of the sera differed by two dilutions (<4 at one laboratory and 8 at the other). The remaining 17 sera differed in titer by only one

  3. Use of siRNA molecular beacons to detect and attenuate mycobacterial infection in macrophages

    PubMed Central

    George, Remo; Cavalcante, Renata; Jr, Celso Carvalho; Marques, Elyana; Waugh, Jonathan B; Unlap, M Tino

    2015-01-01

    elucidating the roles of various genes mediating infectivity and survival in mycobacteria. Molecular beacons are a newer class of antisense RNA tagged with a fluorophore/quencher pair and their use for in vivo detection and knockdown of mRNA is rapidly gaining popularity. PMID:26309818

  4. Detection and genotyping of Toxoplasma gondii DNA in the blood and milk of naturally infected donkeys (Equus asinus)

    PubMed Central

    2014-01-01

    Background Toxoplasma gondii is a worldwide zoonotic protozoan. Consumption of raw milk from infected animals is considered a risk factor for acquiring toxoplasmosis in humans. Recently, donkey milk has been indicated for therapeutic and nutritional purposes and T. gondii infection is common in donkeys. The purpose of the present paper was to detect the presence of parasite DNA in milk of T. gondii positive donkeys. Findings Antibodies to T. gondii were found in 11 out of 44 healthy lactating donkeys by IFAT. T. gondii DNA was detected by PCR in blood of 6 and milk of 3 seropositive jennies. Results of limited RFLP-PCR genotyping indicated the presence of T. gondii genotype II or III, commonly found in Europe. Conclusions The occurrence of T. gondii DNA in milk suggests that the consumption of raw milk from seropositive donkeys could be a potential source of human infection. PMID:24708691

  5. First molecular detection of co-infection of honey bee viruses in asymptomatic Bombus atratus in South America.

    PubMed

    Reynaldi, F J; Sguazza, G H; Albicoro, F J; Pecoraro, M R; Galosi, C M

    2013-11-01

    Pollination is critical for food production and has the particularity of linking natural ecosystems with agricultural production systems. Recently, losses of bumblebee species have been reported worldwide. In this study, samples from a commercial exploitation of bumblebees of Argentina with a recent history of deaths were studied using a multiplex PCR for the detection of the honey bee viruses most frequently detected in South America. All samples analysed were positive for co-infections with Deformed wing virus, Black queen cell virus and Sacbrood virus. This is the first report of infection of Bombus atratus with honey bee viruses. A better understanding of viral infections in bumblebees and of the epidemiology of viruses could be of great importance as bumblebees can serve as possible viral reservoirs, resulting in pathogen spillover towards honey bees and native bumblebees. PMID:24789396

  6. Selected Pool of Peptides from ESAT-6 and CFP-10 Proteins for Detection of Mycobacterium tuberculosis Infection

    PubMed Central

    Scarpellini, Paolo; Tasca, Silvana; Galli, Laura; Beretta, Alberto; Lazzarin, Adriano; Fortis, Claudio

    2004-01-01

    We have validated a new test for detecting Mycobacterium tuberculosis infection. A pool of synthetic peptides derived from ESAT-6 and CFP-10 proteins was used to detect the number of specific gamma interferon-producing T cells by means of an enzyme-linked immunospot assay. Sixty-eight individuals positive for M. tuberculosis infection, either human immunodeficiency virus-seropositive or -seronegative, were studied. The test results were highly specific (87.5%) and sensitive (93.1%), more so than a classical lymphoproliferative assay (specificity and sensitivity of 77.27%), opening new possibilities for diagnosis and screening of tuberculosis. Moreover, the test allowed us to distinguish individuals infected with M. tuberculosis from those vaccinated with BCG. PMID:15297485

  7. Shaking the Tree: Multi-locus Sequence Typing Usurps Current Onchocercid (Filarial Nematode) Phylogeny.

    PubMed

    Lefoulon, Emilie; Bain, Odile; Bourret, Jérôme; Junker, Kerstin; Guerrero, Ricardo; Cañizales, Israel; Kuzmin, Yuriy; Satoto, Tri Baskoro T; Cardenas-Callirgos, Jorge Manuel; de Souza Lima, Sueli; Raccurt, Christian; Mutafchiev, Yasen; Gavotte, Laurent; Martin, Coralie

    2015-11-01

    During the past twenty years, a number of molecular analyses have been performed to determine the evolutionary relationships of Onchocercidae, a family of filarial nematodes encompassing several species of medical or veterinary importance. However, opportunities for broad taxonomic sampling have been scarce, and analyses were based mainly on 12S rDNA and coxI gene sequences. While being suitable for species differentiation, these mitochondrial genes cannot be used to infer phylogenetic hypotheses at higher taxonomic levels. In the present study, 48 species, representing seven of eight subfamilies within the Onchocercidae, were sampled and sequences of seven gene loci (nuclear and mitochondrial) analysed, resulting in the hitherto largest molecular phylogenetic investigation into this family. Although our data support the current hypothesis that the Oswaldofilariinae, Waltonellinae and Icosiellinae subfamilies separated early from the remaining onchocercids, Setariinae was recovered as a well separated clade. Dirofilaria, Loxodontofilaria and Onchocerca constituted a strongly supported clade despite belonging to different subfamilies (Onchocercinae and Dirofilariinae). Finally, the separation between Splendidofilariinae, Dirofilariinae and Onchocercinae will have to be reconsidered.

  8. Shaking the Tree: Multi-locus Sequence Typing Usurps Current Onchocercid (Filarial Nematode) Phylogeny

    PubMed Central

    Lefoulon, Emilie; Bourret, Jérôme; Junker, Kerstin; Guerrero, Ricardo; Cañizales, Israel; Kuzmin, Yuriy; Satoto, Tri Baskoro T.; Cardenas-Callirgos, Jorge Manuel; de Souza Lima, Sueli; Raccurt, Christian; Mutafchiev, Yasen; Gavotte, Laurent; Martin, Coralie

    2015-01-01

    During the past twenty years, a number of molecular analyses have been performed to determine the evolutionary relationships of Onchocercidae, a family of filarial nematodes encompassing several species of medical or veterinary importance. However, opportunities for broad taxonomic sampling have been scarce, and analyses were based mainly on 12S rDNA and coxI gene sequences. While being suitable for species differentiation, these mitochondrial genes cannot be used to infer phylogenetic hypotheses at higher taxonomic levels. In the present study, 48 species, representing seven of eight subfamilies within the Onchocercidae, were sampled and sequences of seven gene loci (nuclear and mitochondrial) analysed, resulting in the hitherto largest molecular phylogenetic investigation into this family. Although our data support the current hypothesis that the Oswaldofilariinae, Waltonellinae and Icosiellinae subfamilies separated early from the remaining onchocercids, Setariinae was recovered as a well separated clade. Dirofilaria, Loxodontofilaria and Onchocerca constituted a strongly supported clade despite belonging to different subfamilies (Onchocercinae and Dirofilariinae). Finally, the separation between Splendidofilariinae, Dirofilariinae and Onchocercinae will have to be reconsidered. PMID:26588229

  9. Shaking the Tree: Multi-locus Sequence Typing Usurps Current Onchocercid (Filarial Nematode) Phylogeny.

    PubMed

    Lefoulon, Emilie; Bain, Odile; Bourret, Jérôme; Junker, Kerstin; Guerrero, Ricardo; Cañizales, Israel; Kuzmin, Yuriy; Satoto, Tri Baskoro T; Cardenas-Callirgos, Jorge Manuel; de Souza Lima, Sueli; Raccurt, Christian; Mutafchiev, Yasen; Gavotte, Laurent; Martin, Coralie

    2015-11-01

    During the past twenty years, a number of molecular analyses have been performed to determine the evolutionary relationships of Onchocercidae, a family of filarial nematodes encompassing several species of medical or veterinary importance. However, opportunities for broad taxonomic sampling have been scarce, and analyses were based mainly on 12S rDNA and coxI gene sequences. While being suitable for species differentiation, these mitochondrial genes cannot be used to infer phylogenetic hypotheses at higher taxonomic levels. In the present study, 48 species, representing seven of eight subfamilies within the Onchocercidae, were sampled and sequences of seven gene loci (nuclear and mitochondrial) analysed, resulting in the hitherto largest molecular phylogenetic investigation into this family. Although our data support the current hypothesis that the Oswaldofilariinae, Waltonellinae and Icosiellinae subfamilies separated early from the remaining onchocercids, Setariinae was recovered as a well separated clade. Dirofilaria, Loxodontofilaria and Onchocerca constituted a strongly supported clade despite belonging to different subfamilies (Onchocercinae and Dirofilariinae). Finally, the separation between Splendidofilariinae, Dirofilariinae and Onchocercinae will have to be reconsidered. PMID:26588229

  10. Development of a PCR procedure for the detection of a herpes-like virus infecting oysters in France.

    PubMed

    Renault, T; Le Deuff, R M; Lipart, C; Delsert, C

    2000-07-01

    A PCR-based procedure for detecting a herpes-like virus that infects the Japanese oyster, Crassostrea gigas, in France was developed. Two primers were designed to provide specific amplification products ranging in size from 917 to 1001 bp when carried out on oyster herpes-like virus DNA. No amplification was observed of oyster genomic DNA nor of the DNA from vertebrate herpesviruses. Crude samples were prepared and submitted to nested PCR, allowing amplification of DNA fragments of the expected size when carried out on infected larval and spat samples. The procedure used to prepare the sample for PCR was found to be critical because of the presence of unidentified substances in oyster tissues that inhibit the PCR reaction. A rapid and convenient sample preparation using ground tissues allowed a sensitive detection of the herpes-like virus infected oysters. The ability of the defined PCR protocol to diagnose herpes-like virus infections in oysters was compared to the transmission electron microscopy technique using 15 C. gigas larval batches with or without mortalities. PCR amplification is as sensitive a diagnostic assay for herpes-like virus as transmission electron microscopy. However, the nested PCR protocol is more convenient and less time consuming. The relationship between reported mortalities among C. gigas oyster spat and herpes-like virus DNA detection by PCR was also investigated. Statistical analysis showed that virus detection and mortalities are correlated. This observation highlights the importance of studying the causative role of herpes-like virus in oyster spat mortalities.

  11. Detection of cucumber green mottle mosaic virus-infected watermelon seeds using short wave infrared (SWIR) hyperspectral imaging system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cucurbit diseases caused by cucumber green mottle mosaic virus (CGMMV) have led to a serious problem to growers and seed producers because it is difficult to prevent spreading through causal agent of seeds. Conventional detection methods for infected seed such as a biological, serological, and m...

  12. Sensitivity of IFN-γ Release Assay to Detect Latent Tuberculosis Infection Is Retained in HIV-Infected Patients but Dependent on HIV/AIDS Progression

    PubMed Central

    Karam, Farba; Mbow, Fatou; Fletcher, Helen; Senghor, Cheikh S.; Coulibaly, Koura D.; LeFevre, Andrea M.; Ngom Gueye, Ndeye F.; Dieye, Tandakha; Sow, Papa S.; Mboup, Souleymane; Lienhardt, Christian

    2008-01-01

    Background Detection and treatment of latent TB infection (LTBI) in HIV infected individuals is strongly recommended to decrease morbidity and mortality in countries with high levels of HIV. Objective To assess the validity of a newly developed in-house ELISPOT interferon-γ release assay (IGRA) for the detection of LTBI amongst HIV infected individuals, in comparison with the Tuberculin Skin Test (TST). Methodology/Principal Findings ESAT6/CFP10 (EC) ELISPOT assays were performed, together with a TST, in 285 HIV infected individuals recruited in HIV clinics in Dakar, Senegal, who had no signs of active TB at time of enrolment. Thirty eight of the subjects (13.3%) failed to respond to PHA stimulation and were excluded from the analysis. In the 247 remaining patients, response to PHA did not vary according to CD4 cell count categories (p = 0.51). EC ELISPOT was positive in 125 (50.6%) subjects, while 53 (21.5%) had a positive TST. Concordance between EC ELISPOT and TST was observed in 151 patients (61.1%) (kappa = 0.23). The proportion of subjects with a positive response to the EC ELISPOT assay decreased with declining CD4 counts (p trend = 0.001), but were consistently higher than the proportion of TST responders. In multivariate analysis, the risk of being EC-ELISPOT positive in HIV infected individuals was associated with age, CD4 count and HIV-1 strain. Conclusion Our study indicates that IGRAs using M. tuberculosis specific antigens are likely to retain their validity for the diagnosis of LTBI among HIV positive individuals, but may be impaired by T-cell anergy in severely immuno-suppressed individuals. PMID:18197251

  13. Early detection of hepatocellular carcinoma co-occurring with hepatitis C virus infection: A mathematical model

    PubMed Central

    Zekri, Abdel-Rahman Nabawy; Youssef, Amira Salah El-Din; Bakr, Yasser Mabrouk; Gabr, Reham Mohamed; Ahmed, Ola Sayed; Elberry, Mostafa Hamed; Mayla, Ahmed Mahmoud; Abouelhoda, Mohamed; Bahnassy, Abeer A

    2016-01-01

    AIM: To develop a mathematical model for the early detection of hepatocellular carcinoma (HCC) with a panel of serum proteins in combination with α-fetoprotein (AFP). METHODS: Serum levels of interleukin (IL)-8, soluble intercellular adhesion molecule-1 (sICAM-1), soluble tumor necrosis factor receptor II (sTNF-RII), proteasome, and β-catenin were measured in 479 subjects categorized into four groups: (1) HCC concurrent with hepatitis C virus (HCV) infection (n = 192); (2) HCV related liver cirrhosis (LC) (n = 96); (3) Chronic hepatitis C (CHC) (n = 96); and (4) Healthy controls (n = 95). The R package and different modules for binary and multi-class classifiers based on generalized linear models were used to model the data. Predictive power was used to evaluate the performance of the model. Receiver operating characteristic curve analysis over pairs of groups was used to identify the best cutoffs differentiating the different groups. RESULTS: We revealed mathematical models, based on a binary classifier, made up of a unique panel of serum proteins that improved the individual performance of AFP in discriminating HCC patients from patients with chronic liver disease either with or without cirrhosis. We discriminated the HCC group from the cirrhotic liver group using a mathematical model (-11.3 + 7.38 × Prot + 0.00108 × sICAM + 0.2574 × β-catenin + 0.01597 × AFP) with a cutoff of 0.6552, which achieved 98.8% specificity and 89.1% sensitivity. For the discrimination of the HCC group from the CHC group, we used a mathematical model [-10.40 + 1.416 × proteasome + 0.002024 × IL + 0.004096 × sICAM-1 + (4.251 × 10-4) × sTNF + 0.02567 × β-catenin + 0.02442 × AFP] with a cutoff 0.744 and achieved 96.8% specificity and 89.7% sensitivity. Additionally, we derived an algorithm, based on a binary classifier, for resolving the multi-class classification problem by using three successive mathematical model predictions of liver disease status. CONCLUSION: Our

  14. Canine Dirofilaria Infections in Two Uninvestigated Areas of Serbia: Epidemiological and Genetic Aspects

    PubMed Central

    Tasić, Aleksandar; Tasić-Otašević, Suzana; Gabrielli, Simona; Miladinović-Tasić, Nataša; Ignjatović, Aleksandra; Đorđević, Jovana; Dimitrijević, Sanda

    2012-01-01

    Abstract In 2009 canine filarial infections were investigated in two northern areas of Serbia (Pančevo and Veliko Gradište), applying morphometry, biochemical staining, and immunological kit to detect Dirofilaria immitis antigens, and two home-made ELISAs to detect antibodies to D. repens and D. immitis somatic/metabolic polyproteins. Moreover, molecular tools were applied to analyze the phylogenetic relationships of the isolates. The microfilariae