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Abstract Background Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites. Methods A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation

Background: Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites. Methods: A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay. Results: This new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients. Conclusions: Our assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management. [ABSTRACT FROM AUTHOR]

Abstract Background The point mutations in 23S rRNA gene of Mycoplasma pneumoniae (M. pneumoniae) can lead to high-level resistance to macrolides. This study aimed to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions A2063G and A2064G of 23S rRNA gene. Methods We detected 178 pharyngeal swab specimens and calculated the proportions of resistant and sensitive quasispecies using ASPCR assays. ASPCR assays can detect down to 10 copies of 23S rRNA gene and achieved sensitivities of

In 73 gDNA samples from Babesia gibsoni-infected dogs, the M121I variant population was measured by using allele-specific real-time PCR. Although the mechanism of atovaquone against B. gibsoni has not been clearly identified, it is reported that the mitochondria cytochrome b gene of the atovaquone-resistant B. gibsoni had a single-nucleotide substitution at nt363 (G to T), which resulted in the substitution of methionine with isoleucine (M121I). In this study, 3/73 samples showed over 5% M121I variant population. Although the M121I variant population is a low percentage, it runs the risk of spreading drug-resistant parasites. It is important to prevent the spread of drug-resistance, so we need to gather information about this at regular intervals. [ABSTRACT FROM AUTHOR]

Invasive aspergillosis caused by triazole-resistant strains of Aspergillus fumigatus is a growing public health concern, as is the occurrence of mixed infections with triazole-resistant and -susceptible A. fumigatus strains. Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of A. fumigatus , even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus In this work, we developed a robust, highly selective, and broad-range allele-specific TaqMan real-time PCR platform consisting of 7 simultaneous assays that detect TR 34 (a 34-bp tandem repeat in the promoter region), TR 46 , G54W (a change of G to W at position 54), G54R, L98H, Y121F, and M220I mutations in the cyp51A gene of A. fumigatus The method is based on the widely used TaqMan real-time PCR technology and combines allele-specific PCR with a blocking reagent (minor groove binder [MGB] oligonucleotide blocker) to suppress amplification of the wild-type cyp51A alleles. We used this method to detect triazole-resistant clinical strains of A. fumigatus with a variety of cyp51A gene mutations, as well as the triazole-resistant strains in mixtures of triazole-resistant and -susceptible strains of A. fumigatus The method had high efficiency and sensitivity (300 fg/well, corresponding to about 100 CFU per reaction mixture volume). It could promptly detect triazole resistance in a panel of 30 clinical strains of A. fumigatus within about 6 h. It could also detect cyp51A -associated resistance alleles, even in mixtures containing only 1% triazole-resistant A. fumigatus strains. These results suggest that this method is robustly able to detect cyp51A -associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus and that it should have important clinical applications., (Copyright © 2019 American Society for Microbiology.)