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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

The presence of activating somatic mutations in codons 542 and 545 of exon 9 ( p.E542K c.1624G>A and p.E545K c.1633G>A) and in codon 1047 of exon 20 ( p.H1047R c.3140A>G and p.H1047L c.3140A>T) of PIK3CA gene encoding catalytic p110α-subunit of phosphatidylinositol-3-kinase was studied in tumors of 473 breast cancer patients by multiplex allele-specific real-time PCR. Fifty-eight (12.3%) different mutations were found. An increase in the frequency of PIK3CA gene mutations with disease progression (from 2.4 to 28.7% with tumor progression from I-IIa to III-IV stage; p=0.0001) and a trend towards its increase in the tumors with unfavorable prognostic characteristics (high histological grade, triple negative phenotype) were demonstrated. The presence of the studied PIK3CA gene mutations in tumors significantly reduces relapse-free survival in the total group and in stage III cancer patients. [ABSTRACT FROM AUTHOR]

Background. Drug-resistant human immunodeficiency virus type 1 (HIV-1) minority variants (MVs) are present in some antiretroviral therapy (ART)–naive patients. They may result from de novo mutagenesis or transmission. To date, the latter has not been proven.Methods. MVs were quantified by allele-specific polymerase chain reaction in 204 acute or recent seroconverters from the Zurich Primary HIV Infection study and 382 ART-naive, chronically infected patients. Phylogenetic analyses identified transmission clusters.Results. Three lines of evidence were observed in support of transmission of MVs. First, potential transmitters were identified for 12 of 16 acute or recent seroconverters harboring M184V MVs. These variants were also detected in plasma and/or peripheral blood mononuclear cells at the estimated time of transmission in 3 of 4 potential transmitters who experienced virological failure accompanied by the selection of the M184V mutation before transmission. Second, prevalence between MVs harboring the frequent mutation M184V and the particularly uncommon integrase mutation N155H differed highly significantly in acute or recent seroconverters (8.2% vs 0.5%; P < .001). Third, the prevalence of less-fit M184V MVs is significantly higher in acutely or recently than in chronically HIV-1–infected patients (8.2% vs 2.5%; P = .004).Conclusions. Drug-resistant HIV-1 MVs can be transmitted. To what extent the origin—transmission vs sporadic appearance—of these variants determines their impact on ART needs to be further explored. [ABSTRACT FROM AUTHOR]

Identifying low-abundance mutations is important for the therapy and diagnose of cancer. Since the potential for tumor heterogeneity, the efficient detection of cancer-relevant mutations largely depends on the sensitivity of the methods employed. To confirm whether the mutation detection platforms affect the perceived prevalence of the BRAF and its correlation with clinicopathologic features in papillary thyroid carcinomas (PTC), we compared Sanger Sequencing (SS), Pyrosequencing (PS), and a newly built allele-specific real-time PCR (AS-qPCR) apparatus for the detection of BRAF in a Chinese cohort of conventional variant PTC. Accurate plasmid standards were built to assess the limit of detection of the three platforms. In this research, AS-qPCR has been found both the most sensitive and reliable at detecting mutation. The mutations detected by AS-qPCR which were not detected by SS or PS due to low abundance were confirmed by mutation enrichment platform COLD-PCR followed by SS. When analyzed by AS-qPCR, BRAF was associated with a more aggressive phenotype. Our results indicate that the reported prevalence of the BRAF mutations in PTC has been underestimated and more sensitive methods such as AS-qPCR should be applied in clinical settings. [ABSTRACT FROM AUTHOR]

Abstract: Background and objectives: Minority drug-resistant HIV-1 variants, undetected by conventional genotyping, may impair the outcome of antiretroviral therapy (ART). Thus, we retrospectively analyzed the prevalence of minority drug-resistant HIV-1 variants before ART in chronically HIV-1 infected patients initiating first-line therapy and assessed the impact on clinical outcome in the prospective German Truvada cohort. Study design: Samples from 146 antiretroviral treatment-naïve patients were collected between April 2005 and August 2006. K65R, K103N, and M184V variants at low frequencies were detected by allele-specific real-time PCR. Results: Minority drug-resistant HIV-1 variants were detected in 20/146 patients (13.7%): the M184V mutation in 12/146 patients (8.2%), the K103N mutation in 8/146 patients (5.5%), and the K65R mutation in 4/146 patients (2.7%). Four patients with the M184V mutation also harbored the K65R or the K103N mutation. The 12- and 24 months virological efficacy data revealed that the rate of treatment failure was not increased in the group of patients harboring minority drug-resistant HIV-1 variants prior to ART. Conclusions: Minority drug-resistant HIV-1 variants can be frequently detected in treatment-naïve, chronically HIV-1 infected patients. Despite the presence of those mutations as minority variants before initiating ART, most of the patients were successfully treated. [ABSTRACT FROM AUTHOR]

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]

Qol fungicides, inhibitors ofmitochondrial respiration at the Qo site of cytochrome b in the mitochondrial bc1 enzyme complex, are commonly applied in vineyards against Plasmopara viticola (Berk. & MA Curtis) Ben. & De Toni. Numerous treatments per year with Qol fungicides can lead to the selection of resistant strains in the pathogen population owing to the very specific and efficient mode of action. In order to evaluate the resistance risk and its development, two different methods, biological and molecular, were applied to measure the sensitivity of oospores differentiated in vineyards, both treated and untreated with azoxystrobin, from 2000 to 2004. Assays using oospores have the advantage of analysing the sensitivity of bulked samples randomly collected in vineyards, describing accurately the status of resistance at the end of the grapevine growing season. Both methods correlated well in describing the resistance situation in vineyards. Qol resistance was not observed in one vineyard never treated with Qol fungicides. In the vineyard where azoxystrobin had been used in mixture with folpet, the selection of Qol-resistant strains was lower, compared with using solely Qol. In vineyards where Qol treatments have been stopped, a decrease in resistance was generally observed. [ABSTRACT FROM AUTHOR]

The levels of expression of the HLA-class I molecules are critical for modulating T/NK lymphocytes effector functions. Among HLA molecules, HLA-C, the most recent developed form of class I antigens, is subjected to multiple post transcriptional level of regulation that affect its cell surface expression.We describe a new method of allele-specific real-time PCR that monitor the integrity/disruption of the binding site of the microRNA Hsa-miR-148a, a key factor associated to the levels of HLA-C expression in the Caucasian populations.

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.)