Molecular epidemiology of HIV-1 subtypes based on analysis of pol sequences in Slovenia, 1996-2005

Various studies have demonstrated the increasing prevalence of non-B HIV-1 subtypes in Western Europe. In contrast, knowledge about the molecular epidemiology of HIV-1 in Central and Eastern Europe is limited. The objective of present study was to investigate the HIV-1 molecular diversity as well as time trends in HIV-1 subtype distribution in Slovenia. A retrospective molecular epidemiological survey was conducted on a cohort representing 88% (131/149) of all HIV-1 infected patients diagnosed between January 1996 and June 2005. The study revealed that subtype B is a predominant HIV-1 subtype in Slovenia (110/131; 84%), although a relatively high proportion (21/131; 16%) of non-B subtypes was found. Among them, a high proportion of recombinant (10/21; 48%) and different unclassified strains (8/21; 38%) were identified. Non-B subtype viruses were predominant among heterosexuals (19/21; 90%) and subtype B viruses among men who have sex with men (84/110; 76%). Importantly, 86% (18/21) of patients infected with non-B subtypes were of Slovenian nationality. In contrast to Western European countries, a significant increase (P = 0.015) in the proportion of men who have sex with men was observed recently among newly diagnosed HIV-1 infected patients in Slovenia.     

Real-time polymerase chain reaction assay based on high-resolution melting analysis for the determination of the rs12979860 polymorphism involved in hepatitis C treatment response

Treatment of chronic hepatitis C is associated with varying success rates, substantial medical costs and serious side effects. Several host polymorphisms have been identified near the IL28B gene, of which the homozygous rs12979860 CC was found to be associated with significantly favourable treatment outcome. To determine accurately the presence of this variant, a real-time PCR assay with detection based on post-PCR high-resolution melting analysis (HRM) was developed. The assay, performed on a Roche LightCycler 480, was able to differentiate among all three rs12979860 variants (CC, TT, CT) across a dynamic range of four orders of magnitude (10³-10⁷). The sensitivity of the assay was determined at a 95% detection level to be 44.6 copies/reaction for the CC variant, 57.1 copies/reaction for the TT variant and 47.4 copies/reaction for the CT variant. Input DNA amount above 10³ copies/reaction is recommended for clinical samples to ensure optimal performance. Clinical performance was assessed on 60 clinical samples by comparative testing using the assay and Sanger sequencing. Concordant results were obtained for all 60 samples, showing high specificity of the assay. The novel assay can be easily added to the testing repertoire of virological laboratories, providing additional information for physicians treating chronic hepatitis C patients.     

Clinical Validation of the Fully Automated NeuMoDx HPV Assay for Cervical Cancer Screening

The NeuMoDx HPV assay is a novel fully automated, real-time PCR-based assay for the qualitative detection of high-risk human papillomavirus (HPV) DNA in cervical specimens. The assay specifically identifies HPV16 and HPV18 and concurrently detects 13 other high-risk HPV types at clinically relevant infection levels. Following the international guidelines, the clinical performance of the NeuMoDx HPV assay for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) against the reference standard Hybrid Capture 2, as well as intra- and inter-laboratory reproducibility were assessed on PreservCyt samples. The clinical accuracy of the assay was additionally evaluated against the clinically validated Alinity m HR HPV and COBAS 4800 HPV Test on PreservCyt samples, and against the clinically validated HPV-Risk assay on SurePath samples. The NeuMoDx HPV assay performance for CIN2+ was non-inferior to the reference methods on both sample types (all p < 0.05), and showed excellent intra- and inter-laboratory reproducibility (95.7%; 95% CI: 93.9–97.3; kappa value 0.90 (95% CI: 0.86–0.94); and 94.5%; 95% CI: 92.6–96.2; kappa value 0.87 (95% CI: 0.82–0.92), respectively). In conclusion, the NeuMoDx HPV assay meets international guideline criteria for cross-sectional accuracy and reproducibility, and performs equally well on cervical screening specimens collected in two widely used collection media. The NeuMoDx HPV assay fulfils the requirements to be used for primary cervical screening.     

Chromosomal microarray in postnatal diagnosis of congenital anomalies and neurodevelopmental disorders in Serbian patients

BackgroundArray‐based genomic analysis is a gold standard for the detection of copy number variations (CNVs) as an important source of benign as well as pathogenic variations in humans. The introduction of chromosomal microarray (CMA) has led to a significant leap in diagnostics of genetically caused congenital malformations and neurodevelopmental disorders, with an average diagnostic yield of 15%. Here, we present our experience from a single laboratory perspective in four years’ postnatal clinical CMA application.MethodsDNA samples of 430 patients with congenital anomalies and/or neurodevelopmental disorders were analyzed by comparative genome hybridization using oligonucleotide‐based microarray platforms. Interpretation of detected CNVs was performed according to current guidelines. The detection rate (DR) of clinically significant findings (pathogenic/likely pathogenic CNVs) was calculated for the whole cohort and isolated or combined phenotypic categories.ResultsA total of 140 non‐benign CNVs were detected in 113/430 patients (26.5%). In 70 patients at least one CNV was considered clinically significant thus reaching a diagnostic yield of 16.3%. The more complex the phenotype, including developmental delay/intellectual disability (DD/ID) as a prevailing feature, the higher the DR of clinically significant CNVs is obtained. Isolated congenital anomalies had the lowest, while the “dysmorphism plus” category had the highest diagnostic yield.ConclusionIn our study, CMA proved to be a very useful method in the diagnosis of genetically caused congenital anomalies and neurodevelopmental disorders. DD/ID and dysmorphism stand out as important phenotypic features that significantly increase the diagnostic yield of the analysis.     

Human papillomavirus DNA prevalence and type distribution in anal carcinomas worldwide

Knowledge about human papillomaviruses (HPV) types involved in anal cancers in some world regions is scanty. Here, we describe the HPV DNA prevalence and type distribution in a series of invasive anal cancers and anal intraepithelial neoplasias (AIN) grades 2/3 from 24 countries. We analyzed 43 AIN 2/3 cases and 496 anal cancers diagnosed from 1986 to 2011. After histopathological evaluation of formalin‐fixed paraffin‐embedded samples, HPV DNA detection and genotyping was performed using SPF‐10/DEIA/LiPA₂₅ system (version 1) . A subset of 116 cancers was further tested for p16^INK4a expression, a cellular surrogate marker for HPV‐associated transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance in the anal cancer data set. HPV DNA was detected in 88.3% of anal cancers (95% confidence interval [CI]: 85.1–91.0%) and in 95.3% of AIN 2/3 (95% CI: 84.2–99.4%). Among cancers, the highest prevalence was observed in warty–basaloid subtype of squamous cell carcinomas, in younger patients and in North American geographical region. There were no statistically significant differences in prevalence by gender. HPV16 was the most frequent HPV type detected in both cancers (80.7%) and AIN 2/3 lesions (75.4%). HPV18 was the second most common type in invasive cancers (3.6%). p16^INK4a overexpression was found in 95% of HPV DNA‐positive anal cancers. In view of the results of HPV DNA and high proportion of p16^INK4a overexpression, infection by HPV is most likely to be a necessary cause for anal cancers in both men and women. The large contribution of HPV16 reinforces the potential impact of HPV vaccines in the prevention of these lesions.     

An Improved Protocol for Comprehensive Etiological Characterization of Skin Warts and Determining Causative Human Papillomavirus Types in 128 Histologically Confirmed Common Warts

Human papillomaviruses (HPVs) are etiologically associated with various benign and malignant neoplasms of cutaneous and mucosal epithelia. We describe an improved diagnostic protocol for comprehensive characterization of causative HPV types in common warts, in which broad-spectrum PCRs followed by Sanger sequencing, two previously described and seven newly developed type-specific quantitative real-time PCRs (qPCRs) coupled with the human beta-globin qPCR were used for: (i) diagnosis of HPV infection in warts; (ii) estimation of cellular viral loads of all HPV types detected; and (iii) determination of their etiological role in 128 histologically confirmed fresh-frozen common wart tissue samples. A total of 12 different causative HPV types were determined in 122/126 (96.8%) HPV-positive warts, with HPV27 being most prevalent (27.0%), followed by HPV57 (26.2%), HPV4 (15.1%), HPV2 (13.5%), and HPV65 (7.9%). The cellular viral loads of HPV4 and HPV65 were estimated for the first time in common warts and were significantly higher than the viral loads of HPV2, HPV27, and HPV57. In addition, we showed for the first time that HPV65 is etiologically associated with the development of common warts in significantly older patients than HPV27 and HPV57, whereas HPV4-induced warts were significantly smaller than warts caused by HPV2, HPV27, HPV57, and HPV65.