Mario Poljak

Transmission of Drug-Resistant HIV-1 Is Stabilizing in Europe

The SPREAD Programme investigated prospectively the time trend from September 2002 through December 2005 of transmitted drug resistance (TDR) among 2793 patients in 20 European countries and in Israel with newly diagnosed human immunodeficiency virus type 1 (HIV-1) infection. The overall prevalence of TDR was 8.4% (225 of 2687 patients; 95% confidence interval [CI], 7.4%-9.5%), the prevalence of nucleoside reverse-transcriptase inhibitor (NRTI) resistance was 4.7% (125 of 2687 patients; 95% CI, 3.9%-5.5%), the prevalence of nonucleoside reverse-transcriptase inhibitor (NNRTI) resistance was 2.3% (62 of 2687 patients; 95% CI, 1.8%-2.9%), and the prevalence of protease inhibitor (PI) resistance was 2.9% (79 of 2687 patients; 95% CI, 2.4%-3.6%). There was no time trend in the overall TDR or in NRTI resistance, but there was a statistically significant decrease in PI resistance (P = .04) and in NNRTI resistance after an initial increase (P = .02). We found that TDR appears to be stabilizing in Europe, consistent with recent reports of decreasing drug resistance and improved viral suppression in patients treated for HIV-1 infection.

Hybrid Capture II HPV Test detects at least 15 human papillomavirus genotypes not included in its current high-risk probe cocktail

BACKGROUND Hybrid Capture II HPV Test (HCII) (Digene Corporation, Gaithersburg, MD) is a signal amplified hybridization microplate-based assay designated to detect 18 human papillomavirus (HPV) genotypes using two probe cocktails, for high-risk HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 and low-risk HPV genotypes 6, 11, 42, 43 and 44. At present, HCII is the only commercially available HPV assay with sufficient scientific data to support its performance in the clinical setting. OBJECTIVES To determine the specificity and accuracy of HCII high-risk probe cocktail for detection of 13 HPV genotypes included in the high-risk probe cocktail. STUDY DESIGN Cervical samples obtained from 325 women recognized as HPV positive using the HCII high-risk probe cocktail were included in the study. HPV genotypes were determined by restriction fragment analysis of PGMY09/PGMY11 polymerase chain reaction (PCR) products using seven restriction endonucleases. RESULTS A 450 bp fragment of HPV L1 gene was successfully amplified from 312 out of 325 samples. Of the 312 PCR-positive samples, 280 samples were associated with the expected high-risk HPV genotypes and 32 samples with the HPV genotypes not included in the HCII high-risk probe cocktail. Thus, HPV53 was detected in 8 samples, HPV66 in 4 samples, HPV54 in 3 samples, HPV6, HPV26, HPV70 each in 2 samples, and HPV11, HPV40, HPV42, HPV61, HPV73, HPV81, MM4, IS39, CP6108 each in 1 sample. In 2 samples, we were not able to determine the HPV genotype. CONCLUSIONS Our study shows that HCII high-risk probe cocktail detects at least 15 HPV genotypes not included in the current HCII high-risk probe cocktail. The potential impact of HCII high-risk probe cocktail cross-reactivity with phylogenetically related and unrelated HPV genotypes, including genotypes currently considered to be low-risk HPVs, remains to be determined.

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 p16INK4a 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%). p16INK4a overexpression was found in 95% of HPV DNA‐positive anal cancers. In view of the results of HPV DNA and high proportion of p16INK4a 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.

Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

Background Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. Methods and Findings We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Conclusions Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Which high-risk HPV assays fulfil criteria for use in primary cervical cancer screening?

Several countries are in the process of switching to high-risk human papillomavirus (hrHPV) testing for cervical cancer screening. Given the multitude of available tests, validated assays which assure high-quality screening need to be identified. A systematic review was conducted to answer the question which hrHPV tests fulfil the criteria defined by an international expert team in 2009, based on reproducibility and relative sensitivity and specificity compared to Hybrid Capture-2 or GP5+/6+ PCR-enzyme immunoassay. These latter two hrHPV DNA assays were validated in large randomized trials and cohorts with a follow-up duration of 8 years or more. Eligible studies citing the 2009 guideline were retrieved from Scopus (http://www.scopus.com) and from a meta-analysis assessing the relative accuracy of new hrHPV assays versus the standard comparator tests to detect high-grade cervical intraepithelial neoplasia or cancer in primary screening. The cobas 4800 HPV test and Abbott RealTime High Risk HPV test were consistently validated in two and three studies, respectively, whereas the PapilloCheck HPV-screening test, BD Onclarity HPV assay and the HPV-Risk assay were validated each in one study. Other tests which partially fulfil the 2009 guidelines are the following: Cervista HPV HR Test, GP5+/6+ PCR-LMNX, an in-house E6/E7 RT quantitative PCR and MALDI-TOF (matrix-assisted laser desorption-ionization time-of-flight). The APTIMA HPV assay targeting E6/E7 mRNA of hrHPV was also fully validated. However, the cross-sectional equivalency criteria of the 2009 guidelines were set up for HPV DNA assays. Demonstration of a low risk of CIN3+ after a negative APTIMA test over a longer period is awaited to inform us about its utility in cervical cancer screening at 5-year or longer intervals.

Commercially available assays for multiplex detection of alpha human papillomaviruses

Five main groups of commercial assays for the multiplex detection of alpha human papillomaviruses (HPVs) are currently available. DNA-based screening assays, which test for the presence of 13–14 HPVs without determination of HPV type, have been the standard for HPV detection in the last decade. Assays that combine testing for 14 HPVs and HPV-16 and HPV-18 genotyping are a potential future standard for HPV detection. The clinical value of HPV genotyping assays has still not been finally determined. Recently, one of the mRNA-based assays showed equal clinical sensitivity but higher clinical specificity for CIN2+/CIN3+ in comparison with the validated DNA-based assay. In situ hybridization assays are too laborious and have insufficient clinical sensitivity to be used in routine screening. Automation, price reduction and improvement of clinical specificity are the main goals for the future development of HPV assays.

Two- to four-year histological follow-up of gastric mucosa after Helicobacter pylori eradication

In a 2‐ to 4‐year prospective study, the reversibility of gastritis after Helicobacter pylori eradication was analysed. Sixty‐three H. pylori‐positive, chronic duodenal ulcer patients were studied after the successful eradication of bacteria in the period from 1990 to 1993. H. pylori eradication was obtained by triple antimicrobial regimens (colloidal bismuth subcitrate, amoxycillin, and metronidazole) applied for at least 14 days. The criteria for eradication were the absence of bacteria from two antral and two body of stomach biopsies stained with haematoxylin, eosin, and Warthin Starry, and a negative antral biopsy culture. The same diagnostic procedures were repeated, at regular follow‐up endoscopies, each year for up to 4 years. Neutrophil‐granulocyte infiltration of gastric mucosa disappeared in 2 months after bacterial eradication. Mononuclear cellular infiltration was disappearing with statistical significance up to the second year and normal mucosa was observed in the majority of patients in the fourth year of follow‐up. Degeneratively changed lymphoid aggregates were also present in the fourth year in the antrum (12·5 per cent of patients) and in the body of stomach (14 per cent of patients). There was no significant change in antral intestinal metaplasia during the 4 years of follow‐up. Antral atrophy declined significantly in the period from 1 to 3 years of follow‐up. In conclusion, 3–4 years are needed for gastric mucosa to become normal after H. pylori eradication, although some residual lymphoid aggregates persist even after that period. Copyright

Transmission of Mycobacterium tuberculosis from Human to Cattle

ABSTRACT We describe the first transmission of Mycobacterium tuberculosis from human to cattle confirmed by molecular typing of isolates involved in the transmission. IS6110-based restriction fragment length polymorphism analysis showed that the isolates from the cattle and farm worker who suffered from pulmonary tuberculosis 1 year prior to this case were the same strains.

HIV-1 subtype distribution and its demographic determinants in newly diagnosed patients in Europe suggest highly compartmentalized epidemics

BackgroundUnderstanding HIV-1 subtype distribution and epidemiology can assist preventive measures and clinical decisions. Sequence variation may affect antiviral drug resistance development, disease progression, evolutionary rates and transmission routes.ResultsWe investigated the subtype distribution of HIV-1 in Europe and Israel in a representative sample of patients diagnosed between 2002 and 2005 and related it to the demographic data available. 2793 PRO-RT sequences were subtyped either with the REGA Subtyping tool or by a manual procedure that included phylogenetic tree and recombination analysis. The most prevalent subtypes/CRFs in our dataset were subtype B (66.1%), followed by sub-subtype A1 (6.9%), subtype C (6.8%) and CRF02_AG (4.7%). Substantial differences in the proportion of new diagnoses with distinct subtypes were found between European countries: the lowest proportion of subtype B was found in Israel (27.9%) and Portugal (39.2%), while the highest was observed in Poland (96.2%) and Slovenia (93.6%). Other subtypes were significantly more diagnosed in immigrant populations. Subtype B was significantly more diagnosed in men than in women and in MSM > IDUs > heterosexuals. Furthermore, the subtype distribution according to continent of origin of the patients suggests they acquired their infection there or in Europe from compatriots.ConclusionsThe association of subtype with demographic parameters suggests highly compartmentalized epidemics, determined by social and behavioural characteristics of the patients.

Genetic analysis of wild-type Dobrava hantavirus in Slovenia: co-existence of two distinct genetic lineages within the same natural focus

Genetic analysis was performed of wild-type (wt) Dobrava hantavirus (DOB) strains from Slovenia, the country where the virus was first discovered and where it was found to cause haemorrhagic fever with renal syndrome (HFRS), with a fatality rate of 12%. Two hundred and sixty mice of the genus APODEMUS:, trapped in five natural foci of DOB-associated HFRS during 1990-1996, were screened for the presence of anti-hantavirus antibodies and 49 APODEMUS: flavicollis and four APODEMUS: agrarius were found to be positive. RT-PCR was used to recover partial sequences of the wt-DOB medium (M) and small (S) genome segments from nine A. flavicollis and one A. agrarius. Sequence comparison and phylogenetic analysis of the Slovenian wt-DOB strains revealed close relatedness of all A. flavicollis-derived virus sequences (nucleotide diversity up to 6% for the M segment and 5% for the S segment) and the geographical clustering of genetic variants. In contrast, the strain harboured by A. agrarius showed a high level of genetic diversity from other Slovenian DOB strains (14%) and clustered together on phylogenetic trees with other DOB strains harboured by A. agrarius from Russia, Estonia and Slovakia. These findings suggest that the DOB variants carried by the two species of APODEMUS: in Europerepresent two distinct genetic lineages.