Resistance of Asian Cryptococcus neoformans Serotype A Is Confined to Few Microsatellite Genotypes

Contains fulltext : 109375.pdf (publisher's version ) (Open Access)BACKGROUND: Cryptococcus neoformans is a pathogenic yeast that causes cryptococcosis, a life threatening disease. The prevalence of cryptococcosis in Asia has been rising after the onset of the AIDS epidemic and estimates indicate more than 120 cases per 1,000 HIV-infected individuals per year. Almost all cryptococcal disease cases in both immunocompromised and immunocompetent patients in Asia are caused by C. neoformans var. grubii. Epidemiological studies on C. neoformans in pan-Asia have not been reported. The present work studies the genetic diversity of the fungus by microsatellite typing and susceptibility analysis of approximately 500 isolates from seven Asian countries. METHODOLOGY/PRINCIPAL FINDINGS: Genetic diversity of Asian isolates of C. neoformans was determined using microsatellite analysis with nine microsatellite markers. The analysis revealed eight microsatellite complexes (MCs) which showed different distributions among geographically defined populations. A correlation between MCs and HIV-status was observed. Microsatellite complex 2 was mainly associated with isolates from HIV-negative patients, whereas MC8 was associated with those from HIV-positive patients. Most isolates were susceptible to amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole, but 17 (3.4%) and 10 (2%) were found to be resistant to 5-flucytosine and fluconazole, respectively. Importantly, five Indonesian isolates (approximately 12.5% from all Indonesian isolates investigated and 1% from the total studied isolates) were resistant to both antifungals. The majority of 5-flucytosine resistant isolates belonged to MC17. CONCLUSIONS: The findings showed a different distribution of genotypes of C. neoformans var. grubii isolates from various countries in Asia, as well as a correlation of the microsatellite genotypes with the original source of the strains and resistance to 5-flucytosine

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Not AvailableThe present study is aimed at the development of inhibition ELISA (I-ELISA) exploring monoclonal antibodies (MAbs) and recombinant invariant surface glycoprotein. The extracellular domain (ED) of invariant surface glycoprotein (ISG-75) from Trypanosoma evasni has been heterologously expressed in Pichia pastoris (X-33). The recombinant ISG-75 (rISG-75ED) was characterized by immunoblot and ELISA, followed by the production of MAbs against rISG-75ED. The MAbs were characterized by immunoblot and then explored in the development of I-ELISA for the detection of surra. The diagnostic potential of the developed test has been evaluated using 1192 field sera sample including cattle, buffalo, donkey, horse and camel. The statistical analysis of the data showed optimum combination of diagnostic sensitivity and specificity at 98.8% and 99.2% respectively, with cut-off percentage inhibition (PI) value of >45. The Cohen's kappa coefficient of agreement was found to be 0.98. Hence, the diagnostic test developed in the present study can be exploited as a potential and reliable tool in the serodiagnosis and surveillance of surra in animals.Not Availabl

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Not AvailableBackground: Surra is caused by Trypanosoma evansi and is considered as one of the important disease in camels in India. Very often camel acts as the carrier of the infection with very low level of parasitaemia which cannot be detected by conventional blood smear examination. Methodology: In the present study, a pilot sero-epidemiological study was conducted among camels for trypanosomosis in Rajasthan using purified flagellar (FLA) antigen based indirect enzyme linked immunosorbent assay (I-ELISA) and compared with standard CATT/ T. evansi test. The diagnostic potentiality of purified FLA antigen based I-ELISA (FLA-I-ELISA) were evaluated using 230 camel sera samples from different districts of Rajasthan including Jaipur, Udaipur, Bikaner, Jodhpur, and Ajmer. Results: The test showed an overall 23. 04% sero positivity (S. P). In Jaipur, S. P was found to be 23. 07%, in Udaipur 25. 5%, in Bikaner 18. 18%, in Jodhpur 25. 3% and in Ajmer, S. P was found to be 23. 33%. Conclusion: Thus, epidemiological survey were done in camels with purified FLA-I-ELISA test in different districts of Rajasthan and revealed that, Udaipur showed the maximum sero positivity for trypanosomosis.Not Availabl

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Not AvailableBackground: Trypanosoma evansi causes trypanosomosis or ‘surra’ in domestic and wild animals and is transmitted by haematophagus tabanid fly. For successful control of the disease, it is important to detect the carrier animals followed by successive treatment. For this purpose a reliable serological test is useful for mass screening of the animals. Methodology: In the present study, emphasis was given to address the need for the development of a mass screening serological test by using purified flagellar antigen of T. evansi for the detection of antibody against surra. Also a pilot sero-epidemiological study for trypanosomosis in different species using purified flagellar (FLA) antigen based indirect enzyme linked immunosorbant assay (I-ELISA) was conducted and compared with standard CATT/T. evansi test. The diagnostic potentiality of purified FLA antigen based I-ELISA (FLA-I-ELISA) was evaluated using 197 sera samples from field animals including cattle, buffaloes, horses and donkeys. Results: The test showed an overall 14.72% sero positivity (S.P). Among cattle, S.P was found to be 18.03%, in buffalo 25%, in horse 6.25% and in donkeys 5.5%. Conclusion: Thus, epidemiological survey were done with purified FLA antigen based I-ELISA test in Karnataka reveals that, buffalo samples showed the maximum sero positivity for trypanosomosis.Not Availabl

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Not AvailableTrypanosoma evansi causes a disease known as 'surra' in wide range of domesticated and wild animals including cattle, buffaloes, horses, camels etc. The disease is transmitted through the bites of haematophagous tabanid flies and is characterized by undulating fever, chronic progressive weakness, and hypoglycemia leading to low productivity in animals. In the present study, monoclonal antibodies (MAbs) have been produced (IgG3 sub-type) against purified flagellar (FLA) protein of T. evansi and its immunoreactivity was evaluated by serological tests. MAb and purified protein were then exploited in the development of CI-ELISA and the diagnostic potentiality of the new ELISA test has been evaluated using 1230 sera samples from field animals including cattle, buffaloes, camels, horses and donkeys. The statistical analysis of the data showed optimum combination of sensitivity and specificity at 95.8 and 94.4, respectively. The positive-negative cut off percentage inhibition (PI) value was found to be >55, with a Cohen's Kappa value of 0.83. The study showed that the new assay has potential for application in sero-diagnosis as well as sero-surveillance of surra.Not Availabl

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Not AvailableTrypanosomosis, an endemic disease in Asia, America (central and south) and Africa causes havoc economical loss in livestock industry. The carrier animals which are symptomless and harbours low level of parasites can act as a source of infection. The level of parasitaemia fluctuates, especially during the latent infection; moreover the antibodies which are not found early in the infection may persit even after recovery or chemotherapy. The parasitological and/or serological tests always can not detect current infection or carrier animals. Hence, in the present study double antibody sandwitch antigen detection ELISA (Ag-ELISA) is developed to detect circulating trypanosomes. The new assay has been evaluated using 554 field samples comprising bovine and camel. The diagnostic sensitivity and specificity of the new assay was found to be 97.4% and 99.0% respectively, with a Cohen's kappa value of 0.96. The developed assay could detect 11.5 Trypanosoma evansi per mL from the experimentally infected blood, buffy coat and purified T. evansi samples. The findings revealed that the developed assay can be exploited as a potential diagnostic tool in the detection of active trypanosomal infection.Not Availabl

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Not AvailableSurra is caused by Trypanosoma evansi and is considered as one of the important disease in camels in India. Very often camel acts as the carrier of the infection with very low level of parasitaemia which cannot be detected by conventional blood smear examination. Methodology: In the present study, a pilot sero-epidemiological study was conducted among camels for trypanosomosis in Rajasthan using purified flagellar (FLA) antigen based indirect enzyme linked immunosorbent assay (I-ELISA) and compared with standard CATT/T. evansi test. The diagnostic potentiality of purified FLA antigen based I-ELISA (FLA-I-ELISA) were evaluated using 230 camel sera samples from different districts of Rajasthan including Jaipur, Udaipur, Bikaner, Jodhpur, and Ajmer. Results: The test showed an overall 23. 04% sero positivity (S. P). In Jaipur, S. P was found to be 23. 07%, in Udaipur 25. 5%, in Bikaner 18. 18%, in Jodhpur 25. 3% and in Ajmer, S. P was found to be 23. 33%. Conclusion: Thus, epidemiological survey were done in camels with purified FLA-I-ELISA test in different districts of Rajasthan and revealed that, Udaipur showed the maximum sero positivity for trypanosomosis.Not Availabl

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Not AvailableIn the present study, the variable surface glycoprotein (VSG) gene of Trypanosoma evansi was cloned and expressed in Pichia pastoris (X-33). The diagnostic potential of recombinant VSG (rVSG) in ELISA has been determined using 1818 field sera samples collected from different species across different states of India. The developed test was compared with the standard reference test such as, CATT/T. evansi; moreover, the new assay was also compared in ELISA using VSG RoTat 1.2 antigen. The diagnostic sensitivity and specificity of recombinant protein were found to be 95.4% and 93.8% respectively, with Cohen’s kappa value of 0.86. The epidemiological study revealed varied prevalence of surra in different species and across different geographical regions of India. Cattle experienced higher prevalence of surra with 42.2% seropositivity from eastern region of India, whereas camel showed 19.9% seropositivity from Rajasthan. Hence, the present study is useful asNot Availabl

Development of an enzyme immunoassay using recombinant invariant surface glycoprotein (rISG) 75 for serodiagnosis of bovine trypanosomosis

7-15Trypanosomosis or surra is caused by the haemoflagellate parasite, <i style="mso-bidi-font-style: normal">Trypanosoma evansi and is an important disease of animals, including domestic and wild herbivores and carnivores, in tropical countries. The invariant surface glycoproteins (ISGs) are blood stream stage specific and are uniformly distributed over the entire surface of the trypanosomes. In the present study, the extracellular domain (ED) region of ISG-75 from <i style="mso-bidi-font-style: normal">T. evansi, consisting of 1320 nt, encoding a polypeptide of 440 amino acids, has been heterologously expressed in Escherichia coli. Further, the immunoreactivity of recombinant ISG-75 (rISG-75) was characterized in immunoblot and ELISA using T. evansi hyper immune sera raised in experimental animals. The protein was found immunoreactive when compared with a panel of antigens (VSG RoTat 1.2 and whole cell lysate) using bovine serum samples from field. The diagnostic potential of rISG-75 was evaluated in ELISA with large number of bovine field serum samples. The optimum sensitivity and specificity were 98.47 and 99.1, respectively. The present finding showed that the expressed protein has potential use in the serodiagnosis of trypanosomosis. </span

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Not AvailableTrypanosoma (T) evansi causes a chronic wasting disease called “surra” in cattle, buffaloes, horses, canines, felines etc. In the present study T.evansi isolated from dog from Karnataka state in India was used to sequence invariant surface glycoprotein (ISG) gene. The sequence obtained was analyzed to elucidate its relationship with other isolates/ species. The ISG gene sequences obtained from four recombinant clones revealed the open reading frame (ORF) of 1521 nucleotide (nt) encoding a polypeptide of 506 amino acids (aa) and belongs ISG-75 gene family. Sequence analysis revealed respectively, 92-99% and 65-99% similarity at nucleotide and amino acid levels, with other isolates/ species. Hence, the present studied T.evansi isolate belongs to the RoTat 1.2 strain.Not Availabl