[Show abstract][Hide abstract] ABSTRACT: Background:
The HIV-1 epidemic in Slovenia, a small Central European country, has some characteristics that make it an ideal model to study HIV-1 transmission. The epidemic is predominantly affecting men who have sex with men infected with subtype B (89% of all patients), has a low prevalence (less than 1/1000) and is growing slowly. The aim of the present study was to analyze in detail the evolutionary history and the determinants of transmission.
A total of 223 partial pol gene sequences from therapy naïve individuals were included, representing 52% of all patients newly diagnosed in 13 years (2000-2012) and analyzed together with genetically similar worldwide sequences, selected in a BLAST search.
Combined analysis (maximum likelihood and Bayesian) of HIV-1 transmission chains revealed 8 major clusters (n ≥ 10 patients), 1 group of 4 patients, 2 trios and 12 transmission pairs, thus leaving only 43 (19.3%) Slovenian patients infected with subtype B without a local epidemiological link, indicating a predominance of local transmission of HIV-1 infection. Bayesian analysis performed on a full set of sequences estimated several introductions of HIV-1 into Slovenia, with the most recent common ancestor (tMRCA) of the earliest Slovenian cluster dated to the late 1980s, although tMRCAs obtained from separate independent analysis of each cluster showed considerably more recent estimates. These findings indicate inconsistencies in molecular clock estimation, which we further explored. We hypothesize that these inconsistent dating estimates across the tree could be caused by an evolutionary rate acceleration of HIV-1 after entering the Slovenia epidemic that is not taken into account by the molecular clock model. It could be caused by a lower transmission rate in this setting, as demonstrated by the low epidemic growth rate estimated by Bayesian skyline plot analysis.
HIV-1 subtype B was introduced into Slovenia at several time points from the late 80s onward. The majority of patients had a local transmission link, indicating a closed HIV community. The observed slower epidemic rate suggests that individuals with a long-lasting infection are the driving force of the epidemic in this region.
[Show abstract][Hide abstract] ABSTRACT: Background:
Testing for high-risk HPV is more effective in primary cervical cancer screening than the cytological examination of a Pap smear. Separate genotyping may be useful for triage in both HPV-based and cytology-based screening. Only clinically validated tests should be used in clinical practice.
VALGENT is a study framework for test comparison and validation of HPV assays in general and HPV genotyping tests in particular according to clinically relevant outcomes and for clinical applications endorsed by scientific evidence.
VALGENT involves the collation of fresh or archived cervical cell specimen from women attending routine screening supplemented with cytologically abnormal samples. Multiple aliquots of residual material are sent from a central laboratory to participating laboratories for testing with novel HPV assays with limited, extended or full genotyping capacity. Outcomes are derived from screening and pathology registries. Each VALGENT panel includes an assay already validated for screening. A series of accuracy and concordance statistics were generated.
Currently, two VALGENT study rounds, originated from laboratories in Antwerp (Belgium) and Edinburgh (Scotland), were completed. Two new assays (G5+/6+ PCR-LMNX and Xpert HPV) were validated for screening by showing similar accuracy for cervical precancer as the standard comparator test. For two other tests (BD Onclarity, PapilloCheck) validation was confirmed. Inter-test agreement was high although certain type-specific discordances were observed which warrant further analysis.
VALGENT extends current guidelines for high-risk HPV test validation in cervical cancer screening and has produced a large study resource for test comparison. More robust procedures of sample selection and handling and integration with the global WHO reference laboratory network focusing on analytical accuracy, may result in the generation of an international standard and a formalized system for clinical validation of HPV assays and quality control in HPV-based screening.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 11/2015; DOI:10.1016/j.jcv.2015.09.014 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Commercial molecular tests for human papillomaviruses (HPV) are invaluable diagnostic tools in cervical carcinoma screening and management of women with cervical precancerous lesions as well as important research tools for epidemiological studies, vaccine development, and implementation and monitoring of vaccination programs. In this third inventory of commercial HPV tests, we identified 193 distinct commercial HPV tests and at least 127 test variants available on the market in 2015, which represents a 54% and 79% increase in the number of distinct HPV tests and variants, respectively, in comparison to our last inventory performed in 2012. Identified HPV tests were provisionally divided into eight main groups and several subgroups. Among the 193 commercial HPV tests, all but two target alpha-HPV types only. Although the number of commercial HPV tests with at least one published study in peer-reviewed literature has increased significantly in the last three years, several published performance evaluations are still not in line with agreed-upon standards in the HPV community. Manufacturers should invest greater effort into evaluating their products and publishing validation/evaluation results in peer-reviewed journals. To achieve this, more clinically oriented external quality-control panels and initiatives are required. For evaluating the analytical performance of the entire range of HPV tests currently on the market, more diverse and reliable external quality-control programs based on international standards for all important HPV types are indispensable. The performance of a wider range of HPV tests must be promptly evaluated on a variety of alternative clinical specimens. In addition, more complete HPV assays containing validated sample-extraction protocols and appropriate internal controls are urgently needed. Provision of a broader range of automated systems allowing large-scale HPV testing as well as the development of reliable, rapid, and affordable molecular point-of-care tests are priorities for the further improvement of HPV tests.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 11/2015; DOI:10.1016/j.jcv.2015.10.023 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Formalin-fixed, paraffin-embedded (FFPE) tissue specimens stored in pathology departments worldwide are an invaluable source for diagnostic purposes when fresh clinical material is unavailable as well as for retrospective molecular and epidemiological studies, especially when dealing with rare clinical conditions for which prospective collection is not feasible. Accurate detection of HPV infection in these specimens is particularly challenging because nucleic acids are often degraded and therefore, not suitable for amplification of larger fragments of the viral genome or viral gene transcripts. This review provides a brief summary of molecular methods for detecting alpha-HPV DNA/RNA in FFPE tissue specimens. We specifically address the key procedural and environmental factors that have the greatest impact on the quality of nucleic acids extracted from FFPE tissue specimens, and describe some solutions that can be used to increase their integrity and/or amplifiability. Moreover, commonly used methods for HPV DNA/RNA detection in FFPE tissue specimens are presented and discussed, focusing on studies using polymerase chain reaction as an HPV detection method and published after 1999. Finally, we briefly summarize our 22 years of experience with HPV detection in FFPE tissue specimens.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 10/2015; DOI:10.1016/j.jcv.2015.10.007 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction: Formalin-fixed, paraffin-embedded (FFPE) tissues represent an invaluable source for diagnostic purposes when fresh clinical material is unavailable, and also for molecular and epidemiological studies. The recovery of nucleic acids from FFPE tissues is particularly challenging, and several in-house methods have been developed for this purpose over the last three decades. Recently, several commercial kits specifically developed for DNA and/or RNA extraction from FFPE tissues have been introduced to the market, but their inventory is not available in peer-reviewed literature. Methods: This article provides the first comprehensive inventory of commercial FFPE DNA/RNA extraction kits currently available on the market and describes their basic characteristics and features. Results: A total of 69 commercial kits from 43 companies were identified. Thirty-five kits were developed specifically for DNA extraction, 22 for RNA extraction, and 12 for both DNA and RNA extraction. Only two commercial kits allow full automation of the entire nucleic acid extraction procedure. The tissue deparaffinization step is omitted in many protocols by melting paraffin directly in a tissue lysis buffer. Purification of the released nucleic acids is mainly based on silica or resin adsorption technology. A formalin reverse cross-linking step to increase the quality of extracted DNA and RNA is an intrinsic part of over half of the kits identified. Conclusions: It is hope that this comprehensive list of available commercial kits for extracting nucleic acids from FFPE will encourage researchers to strongly consider using them in diagnostic and research settings instead of old-fashioned, crude, and probably less effective in-house methods.
Acta dermatovenerologica Alpina, Panonica, et Adriatica 09/2015; 24(3). DOI:10.15570/actaapa.2015.12
[Show abstract][Hide abstract] ABSTRACT: The novel human papillomavirus type 199 (HPV199) was initially identified in a nasopharyngeal swab sample obtained from a 25 year-old immunocompetent male. The complete genome of HPV199 is 7,184 bp in length with a GC content of 36.5%. Comparative genomic characterization of HPV199 and its closest relatives showed the classical genomic organization of Gammapapillomaviruses (Gamma-PVs). HPV199 has seven major open reading frames (ORFs), encoding five early (E1, E2, E4, E6, and E7) and two late (L1 and L2) proteins, while lacking the E5 ORF. The long control region (LCR) of 513 bp is located between the L1 and E6 ORFs. Phylogenetic analysis additionally confirmed that HPV-199 clusters into the Gamma-PV genus, species Gamma-12, additionally containing HPV127, HV132, HPV148, HPV165, and three putative HPV types: KC5, CG2 and CG3. HPV199 is most closely related to HPV127 (nucleotide identity 77%). The complete viral genome sequence of additional HPV199 isolate was determined from anal canal swab sample. Two HPV199 complete viral sequences exhibit 99.4% nucleotide identity. To the best of our knowledge, this is the first member of Gamma-PV with complete nucleotide sequences determined from two independent clinical samples. To evaluate the tissue tropism of the novel HPV type, 916 clinical samples were tested using HPV199 type-specific real-time PCR: HPV199 was detected in 2/76 tissue samples of histologically confirmed common warts, 2/108 samples of eyebrow hair follicles, 2/137 anal canal swabs obtained from individuals with clinically evident anal pathology, 4/184 nasopharyngeal swabs and 3/411 cervical swabs obtained from women with normal cervical cytology. Although HPV199 was found in 1.4% of cutaneous and mucosal samples only, it exhibits dual tissue tropism. According to the results of our study and literature data, dual tropism of all Gamma-12 members is highly possible.
PLoS ONE 09/2015; 10(9):e0138628. DOI:10.1371/journal.pone.0138628 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human papillomavirus (HPV)-related screening technologies and HPV vaccination offer enormous potential for cancer prevention, notably prevention of cervical cancer. The effectiveness of these approaches is, however, suboptimal owing to limited implementation of screening programmes and restricted indications for HPV vaccination. Trials of HPV vaccination in women aged up to 55 years have shown almost 90% protection from cervical precancer caused by HPV16/18 among HPV16/18-DNA-negative women. We propose extending routine vaccination programmes to women of up to 30 years of age (and to the 45-50-year age groups in some settings), paired with at least one HPV-screening test at age 30 years or older. Expanding the indications for HPV vaccination and much greater use of HPV testing in screening programmes has the potential to accelerate the decline in cervical cancer incidence. Such a combined protocol would represent an attractive approach for many health-care systems, in particular, countries in Central and Eastern Europe, Latin America, Asia, and some more-developed parts of Africa. The role of vaccination in women aged >30 years and the optimal number of HPV-screening tests required in vaccinated women remain important research issues. Cost-effectiveness models will help determine the optimal combination of HPV vaccination and screening in public health programmes, and to estimate the effects of such approaches in different populations.
[Show abstract][Hide abstract] ABSTRACT: Background:
European guidelines recommend treatment of chronic hepatitis B virus infection (CHB) with the nucleos(t)ide analogs (NAs) entecavir or tenofovir. However, many European CHB patients have been exposed to other NAs, which are associated with therapy failure and resistance. The CAPRE study was performed to gain insight in prevalence and characteristics of NA resistance in Europe.
A survey was performed on genotypic resistance testing results acquired during routine monitoring of CHB patients with detectable serum hepatitis B virus DNA in European tertiary referral centers.
Data from 1568 patients were included. The majority (73.8%) were exposed to lamivudine monotherapy. Drug-resistant strains were detected in 52.7%. The most frequently encountered primary mutation was M204V/I (48.7%), followed by A181T/V (3.8%) and N236T (2.6%). In patients exposed to entecavir (n = 102), full resistance was present in 35.3%. Independent risk factors for resistance were age, viral load, and lamivudine exposure (P < .001).
These findings support resistance testing in cases of apparent NA therapy failure. This survey highlights the impact of exposure to lamivudine and adefovir on development of drug resistance and cross-resistance. Continued use of these NAs needs to be reconsidered at a pan-European level.
The Journal of Infectious Diseases 07/2015; DOI:10.1093/infdis/jiv363 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The majority of people infected with hepatitis C virus (HCV) are unaware of their infection. Assessment of the prevalence of HCV infection in the general population and in key populations at increased risk is needed for evidence-based testing policies. Our objectives were to estimate the prevalence of antibodies to HCV (anti-HCV), the prevalence of HCV viraemia (HCV RNA), and to describe HCV genotype distribution among pregnant women in Slovenia. Unlinked anonymous testing was performed on residual sera obtained from 31,849 pregnant women for routine syphilis screening during 1999, 2003, 2009, and 2013. Anti-HCV reactive specimens were tested for HCV RNA and HCV genotypes were determined. Annual prevalence of anti-HCV ranged between 0.09% (95% confidence interval (CI): 0.03–0.18) in 2009 and 0.21% (95% CI: 0.12–0.34) in 2003 and HCV RNA positivity between 0.06% (95% CI: 0.02–0.14) in 2009 and 0.14% (95% CI: 0.07–0.25) in 2003. We observed no statistically significant differences in anti-HCV or HCV RNA prevalence between age groups (<20, 20–29 and ≥30 years) in any year and no trend in time. Of 29 HCV active infections, 19 were with genotype 1 and 10 with genotype 3. HCV infection among pregnant women was rare suggesting a low burden in the Slovenian general population. Antenatal screening for HCV in Slovenia could not be recommended.
Eurosurveillance: bulletin europeen sur les maladies transmissibles = European communicable disease bulletin 06/2015; 20(22). DOI:10.2807/1560-7917.ES2015.20.22.21144 · 5.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human papillomaviruses (HPVs) are small, non-enveloped viruses with a circular double-stranded DNA genome, etiologically associated with various benign and malignant neoplasms of the skin and mucosa. As of May 30, 2015, 201 different HPV types had been completely sequenced and officially recognized and divided into five PV-genera: Alpha-, Beta-, Gamma-, Mu-, and Nupapillomavirus. The Mupapillomavirus genus currently consists of only two HPV types: HPV1 and HPV63, identified in 1980 and 1993, respectively, both associated with sporadic cases of cutaneous warts. In this preliminary study, we announce the complete genome sequence of a novel HPV type, now officially recognized as HPV204. Based on preliminary data, the genome of HPV204 comprises a total of 7,227 bp and contains five early open reading frames (E1, E2, E4, E6, and E7) and two late ORFs (L1 and L2). No E5 ORF could be identified. Preliminary HPV204 clusters to the Mu-PV genus, species Mu-3.
Acta dermatovenerologica Alpina, Panonica, et Adriatica 06/2015; 24(2):21-3. DOI:10.15570/actaapa.2015.7
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
PLoS Medicine 04/2015; 12(4):e1001810. DOI:10.1371/journal.pmed.1001845 · 14.43 Impact Factor