Last data update: May 13, 2024. (Total: 46773 publications since 2009)
Records 1-3 (of 3 Records) |
Query Trace: Koru O[original query] |
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Detection and Differentiation of Leishmania spp. in Clinical Specimens Using a SYBR Green-Based Real-Time PCR Assay.
de Almeida ME , Koru O , Steurer F , Herwaldt BL , da Silva AJ . J Clin Microbiol 2016 55 (1) 281-290 Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the Internal Transcribed Spacer 2 (ITS2) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by melting temperature (Tm) analysis of the amplicons on qPCR platforms (Mx3000P qPCR System [Stratagene-Agilent] and 7500 Real-Time PCR System [ABI-Life Technologies]). We initially evaluated the assay by testing reference Leishmania isolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to our laboratory for Leishmania diagnostic testing: specimens from 477 patients tested positive for Leishmania spp. with the LSG-qPCR assay, 465 of which also tested positive with the conventional ITS2-PCR approach, 10 of which had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of the Tm values of LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups of Leishmania parasites: the Viannia subgenus in aggregate; the L. (L.) donovani spp. complex in aggregate; the species L. (L.) tropica; and the spp. L. (L.) mexicana, L. (L.) amazonensis, L. (L.) major, and L. (L.) aethiopica in aggregate. |
Identification of Leishmania spp. by molecular amplification and DNA sequencing analysis of a fragment of rRNA internal transcribed spacer 2.
de Almeida ME , Steurer FJ , Koru O , Herwaldt BL , Pieniazek NJ , da Silva AJ . J Clin Microbiol 2011 49 (9) 3143-9 Isoenzyme analysis of cultured parasites is the conventional approach for Leishmania species identification. Molecular approaches have the potential to be more sensitive and rapid. We designed polymerase chain reaction (PCR) generic primers to amplify a segment of the rRNA Internal Transcribed Spacer 2 (ITS2) from multiple Leishmania species. To validate the selected ITS2 fragment, we tested clinical specimens and compared the species results obtained by the molecular approach (PCR, followed by DNA sequencing analysis) with those from the parasitologic approach (in vitro culture, followed by isoenzyme analysis). Among the 159 patients with clinical specimens positive by both approaches, a total of 8 Leishmania species were identified. The species results were concordant for all but two patients: for one patient, the results were L. (Viannia) guyanensis by the molecular approach versus L. (V.) braziliensis by the parasitologic approach; for the other patient, the results were L. (Leishmania) tropica versus L. (L.) major, respectively. ITS2 PCR, followed by sequencing analysis, can be used to detect and discriminate among Leishmania species. The results confirmed our hypothesis that a region of the ITS2 gene can complement the characterization of Leishmania parasites at the species level. The approach we developed can be used as a diagnostic tool in reference laboratories with adequate infrastructure to perform molecular characterization of pathogens. |
Human zoonotic enteropathogens in a constructed free-surface flow wetland
Graczyk TK , Lucy FE , Mashinsky Y , Andrew Thompson RC , Koru O , Dasilva AJ . Parasitol Res 2009 105 (2) 423-8 Effluents from a small-scale free-surface flow constructed wetland, used for polishing of secondary treated wastewater, contained significantly higher concentrations of potentially viable Giardia duodenalis cysts and Enterocytozoon bieneusi spores than did wetland influents consisting of secondary treated wastewater. Zoonotic Assemblage A of G. duodenalis cysts was identified in wetland inflows, while Assemblage A and two nonhuman infective Assemblages (i.e., C, and E) were present in wetland effluents. E. bieneusi spores represented genotype K based on DNA sequencing analysis of internal transcribed spacer. The study demonstrated that: (1) free-surface flow small-scale constructed wetlands may not provide sufficient remediation for human zoonotic protozoa and fungi present in secondary treated wastewater; (2) dogs and livestock can substantially contribute human-pathogenic protozoan and fungal microorganisms to engineered vegetated wetland systems; and (3) large volumes of wetland effluents can contribute to contamination of surface waters used for recreation and drinking water abstraction and therefore represent a serious public health threat. |
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