Molecular epidemiology and characterization of enteroviruses detected in cerebrospinal fluid and respiratory samples in Slovenia, 2014-2023.

Enterovirus (EV) infections have various symptoms and severe complications, including death. To determine EV prevalence and EV types in Slovenia, data on over 25 000 EV RNA tests for diagnostics and surveillance from 2014 to 2023 were analyzed. Altogether, 3733 cerebrospinal fluid (CSF) and 21 297 respiratory (sentinel and clinical) samples were tested for EV RNA. EV typing was performed on all residual EV-positive CSF samples and on subset of respiratory specimens. Altogether, 1238 samples tested positive for EV RNA: 238 (6.4%) CSF and 1000 (4.7%) respiratory samples. EV-positive patients were predominantly male (p < 0.001). Many EV-positive CSF samples were from infants under 3 months (33.1%), whereas most EV-positive respiratory samples were from children 1 to 2 years old (49.2%). Echovirus 30 (E-30) was most frequent in CSF (33.0%), followed by CV-B5 (13.8%) and E-6 (13.8%). CV-A6 was most frequent in respiratory samples (16.0%), followed by EV-D68 (7.6%) and CV-A5 (7.4%). EV types in CSF and respiratory samples show diverse dynamics, with some outbreaks indicated. A significant difference was found in the EV detection rate between CSF and respiratory samples by age. Various EV types were characterized, showing that some EV types are more neurotropic or cause more severe infections.

Prevalence of the Helicobacter pylori babA2 Gene in Children Mainly Depends on the PCR Primer Set Used.

Various polymerase chain reaction- (PCR-) based methods with varying positivity rates were designed to detect the Helicobacter pylori babA2 gene. To compare different primer sets, babA2 prevalence was determined in 279 H. pylori-positive pediatric samples using the 832 bp, 139 bp, and 271 bp PCR primer sets, resulting in 34.0%, 51.3%, and 79.6% prevalence of the babA2 gene, respectively. The babA2 status determined using the 832 bp and 139 bp PCR primer sets significantly correlated with bacterial density and activity of inflammation, whereas no such correlations were found using the 271 bp PCR primer set. The 139 and 832 bp PCR primer sets concordantly detected the babA2 gene in 93 cases; however, in comparison to the 832 bp PCR primer set, the 139 bp PCR primer set detected additional 50 babA2 cases, whereas only two 832 bp positive cases were missed. The 271 bp PCR primer set missed 32 babA2 cases that were 832 bp and/or 139 bp PCR positive, but tested solely positive in 109 cases. Interestingly, cloning of a subset of 271 bp PCR positive samples revealed amplification of the babA/B gene chimera. Hence, in our opinion, the 271 bp PCR protocol is not a reliable diagnostic tool for detecting the babA2 gene in children. Our results reaffirm previous observations that the use of certain babA2 PCR primer sets can significantly impact estimation of the prevalence and clinical relevance of the H. pylori babA2 gene in children, suggesting babA2 detection methods should be carefully selected.

Immune-escape mutations and stop-codons in HBsAg develop in a large proportion of patients with chronic HBV infection exposed to anti-HBV drugs in Europe.

BACKGROUND: HBsAg immune-escape mutations can favor HBV-transmission also in vaccinated individuals, promote immunosuppression-driven HBV-reactivation, and increase fitness of drug-resistant strains. Stop-codons can enhance HBV oncogenic-properties. Furthermore, as a consequence of the overlapping structure of HBV genome, some immune-escape mutations or stop-codons in HBsAg can derive from drug-resistance mutations in RT. This study is aimed at gaining insight in prevalence and characteristics of immune-associated escape mutations, and stop-codons in HBsAg in chronically HBV-infected patients experiencing nucleos(t)ide analogues (NA) in Europe. METHODS: This study analyzed 828 chronically HBV-infected European patients exposed to ≥ 1 NA, with detectable HBV-DNA and with an available HBsAg-sequence. The immune-associated escape mutations and the NA-induced immune-escape mutations sI195M, sI196S, and sE164D (resulting from drug-resistance mutation rtM204 V, rtM204I, and rtV173L) were retrieved from literature and examined. Mutations were defined as an aminoacid substitution with respect to a genotype A or D reference sequence. RESULTS: At least one immune-associated escape mutation was detected in 22.1% of patients with rising temporal-trend. By multivariable-analysis, genotype-D correlated with higher selection of ≥ 1 immune-associated escape mutation (OR[95%CI]:2.20[1.32-3.67], P = 0.002). In genotype-D, the presence of ≥ 1 immune-associated escape mutations was significantly higher in drug-exposed patients with drug-resistant strains than with wild-type virus (29.5% vs 20.3% P = 0.012). Result confirmed by analysing drug-naïve patients (29.5% vs 21.2%, P = 0.032). Strong correlation was observed between sP120T and rtM204I/V (P < 0.001), and their co-presence determined an increased HBV-DNA. At least one NA-induced immune-escape mutation occurred in 28.6% of patients, and their selection correlated with genotype-A (OR[95%CI]:2.03[1.32-3.10],P = 0.001). Finally, stop-codons are present in 8.4% of patients also at HBsAg-positions 172 and 182, described to enhance viral oncogenic-properties. CONCLUSIONS: Immune-escape mutations and stop-codons develop in a large fraction of NA-exposed patients from Europe. This may represent a potential threat for horizontal and vertical HBV transmission also to vaccinated persons, and fuel drug-resistance emergence.

A very high prevalence of hepatitis C virus infection among patients undergoing hemodialysis in Kosovo: a nationwide study.

BACKGROUND: Patients on hemodialysis are at high risk for hepatitis C virus (HCV) infection if measures for effective control of HCV infection in the hemodialysis environment are not implemented. Whereas in developed countries isolated small-scale outbreaks of HCV in hemodialysis units are occasionally reported, HCV transmission in the hemodialysis environment still represents a substantial problem in low-resource countries. This study systematically assessed the prevalence of HCV infection among all patients at all hemodialysis centers in Kosovo, determined the HCV genotype distribution, and reviewed the main risk factors associated with HCV infection in this group of patients. METHODS: From January to March 2013, blood samples from all patients undergoing hemodialysis at all seven hemodialysis centers in Kosovo were collected. The samples were screened for the presence of anti-HCV antibodies, and seropositive samples were also tested for HCV RNA. Genotyping was performed by sequencing the core region of the HCV genome. Subsequently, face-to-face interviews were conducted with consented patients attending hemodialysis in December 2015 and with the management of all hemodialysis centers in Kosovo. RESULTS: The overall seroprevalence of HCV infection among hemodialysis patients in Kosovo was 53.0% (354/668), ranging from 22.3 to 91.1% at different centers. HCV RNA was detected in 323/354 (91.2%) seropositive patients. The most frequent HCV genotype was genotype 1a (62.2%), followed by genotypes 4d (33.1%), 1b (4.0%), and 2c (0.7%). The duration of hemodialysis and receiving dialysis at more than one center were identified as independent significant predictors of anti-HCV positivity. Shortage of staff, lack of resources, and inconsistent use of hygienic precautions and/or isolation strategies were observed. CONCLUSIONS: The prevalence of HCV infection among hemodialysis patients in Kosovo is extremely high. The relatively low prevalence of HCV infection in the general population, predominance of two otherwise rare HCV genotypes among hemodialysis patients, and longer history of hemodialysis as a predictor of HCV infection all indicate nosocomial transmission due to inappropriate infection control practices as the main transmission route.

Combined Analysis of the Prevalence of Drug-Resistant Hepatitis B Virus in Antiviral Therapy-Experienced Patients in Europe (CAPRE).

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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Longitudinal trends of recent HIV-1 infections in Slovenia (1986-2012) determined using an incidence algorithm.

Resolving dilemma whether the rise in the number of HIV diagnoses represents an actual increase in HIV transmissions or is a result of improved HIV surveillance is crucial before implementing national HIV prevention strategies. Annual proportions of recent infections (RI) among newly diagnosed persons infected with HIV-1 in Slovenia during 27 years (1986-2012) were determined using an algorithm consisting of routine baseline CD4 and HIV viral load measurements and the Aware BED EIA HIV-1 Incidence Test (BED test). The study included the highest coverage of persons diagnosed with HIV during the entire duration of an HIV epidemic in a given country/region (71%). Out of 416 patients, 170 (40.9%) had a baseline CD4 cell count less than 200 cells/mm(3) and/or HIV-1 viral load less than 400 copies/ml and were characterized as having a long-standing infection (LSI). The remaining 246 patients were additionally tested using the BED test. Overall, 23% (97/416) of the patients were labeled RI. The characteristics significantly associated with RI were as follows: younger age, acute retroviral syndrome, CDC class A and other than C, no AIDS defining illnesses, HIV test performed in the past, a higher viral load, and a higher CD4 cell count. An interesting trend in the proportion of RI was observed, with a peak in 2005 (47% of RI) and the lowest point in 2008 (12%) in parallel with a rise in the numbers of new HIV diagnoses. This study could help promote the idea of introducing periodic HIV incidence monitoring using a simple and affordable algorithm.

Bridging epidemiology with population genetics in a low incidence MSM-driven HIV-1 subtype B epidemic in Central Europe.

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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Persistence of frequently transmitted drug-resistant HIV-1 variants can be explained by high viral replication capacity.

BACKGROUND: In approximately 10% of newly diagnosed individuals in Europe, HIV-1 variants harboring transmitted drug resistance mutations (TDRM) are detected. For some TDRM it has been shown that they revert to wild type while other mutations persist in the absence of therapy. To understand the mechanisms explaining persistence we investigated the in vivo evolution of frequently transmitted HIV-1 variants and their impact on in vitro replicative capacity. RESULTS: We selected 31 individuals infected with HIV-1 harboring frequently observed TDRM such as M41L or K103N in reverse transcriptase (RT) or M46L in protease. In all these samples, polymorphisms at non-TDRM positions were present at baseline (median protease: 5, RT: 6). Extensive analysis of viral evolution of protease and RT demonstrated that the majority of TDRM (51/55) persisted for at least a year and even up to eight years in the plasma. During follow-up only limited selection of additional polymorphisms was observed (median: 1).To investigate the impact of frequently observed TDRM on the replication capacity, mutant viruses were constructed with the most frequently encountered TDRM as site-directed mutants in the genetic background of the lab strain HXB2. In addition, viruses containing patient-derived protease or RT harboring similar TDRM were made. The replicative capacity of all viral variants was determined by infecting peripheral blood mononuclear cells and subsequently monitoring virus replication. The majority of site-directed mutations (M46I/M46L in protease and M41L, M41L + T215Y and K103N in RT) decreased viral replicative capacity; only protease mutation L90M did not hamper viral replication. Interestingly, most patient-derived viruses had a higher in vitro replicative capacity than the corresponding site-directed mutant viruses. CONCLUSIONS: We demonstrate limited in vivo evolution of protease and RT harbouring frequently observed TDRM in the plasma. This is in line with the high in vitro replication capacity of patient-derived viruses harbouring TDRM compared to site-directed mutant viruses harbouring TDRM. As site-directed mutant viruses have a lower replication capacity than the patient-derived viruses with similar mutational patterns, we propose that (baseline) polymorphisms function as compensatory mutations improving viral replication capacity.

Comparative performances of HIV-1 RNA load assays at low viral load levels: results of an international collaboration.

Low-level viremia during antiretroviral therapy and its accurate measurement are increasingly relevant. Here, we present an international collaboration of 4,221 paired blood plasma viral load (pVL) results from four commercial assays, emphasizing the data with low pVL. The assays compared were the Abbott RealTime assay, the Roche Amplicor assay, and the Roche TaqMan version 1 and version 2 assays. The correlation between the assays was 0.90 to 0.97. However, at a low pVL, the correlation fell to 0.45 to 0.85. The observed interassay concordance was higher when detectability was defined as 200 copies/ml than when it was defined as 50 copies/ml. A pVL of ∼100 to 125 copies/ml by the TaqMan version 1 and version 2 assays corresponded best to a 50-copies/ml threshold with the Amplicor assay. Correlation and concordance between the viral load assays were lower at a low pVL. Clear guidelines are needed on the clinical significance of low-level viremia.

Comparing COVID-19 severity in patients hospitalized for community-associated Delta, BA.1 and BA.4/5 variant infection.

Background: Despite decreasing COVID-19 disease severity during the Omicron waves, a proportion of patients still require hospitalization and intensive care. Objective: To compare demographic characteristics, comorbidities, vaccination status, and previous infections in patients hospitalized for community-associated COVID-19 (CAC) in predominantly Delta, Omicron BA.1 and BA.4/5 SARS-CoV-2 waves. Methods: Data were extracted from three national databases-the National COVID-19 Database, National Vaccination Registry and National Registry of Hospitalizations. Results: Among the hospitalized CAC patients analyzed in this study, 5,512 were infected with Delta, 1,120 with Omicron BA.1, and 1,143 with the Omicron BA.4/5 variant. The age and sex structure changed from Delta to BA.4/5, with the proportion of women (9.5% increase), children and adolescents (10.4% increase), and octa- and nonagenarians increasing significantly (24.5% increase). Significantly more patients had comorbidities (measured by the Charlson Comorbidity Index), 30.3% in Delta and 43% in BA.4/5 period. The need for non-invasive ventilatory support (NiVS), ICU admission, mechanical ventilation (MV), and in-hospital mortality (IHM) decreased from Delta to Omicron BA.4/5 period for 12.6, 13.5, 11.5, and 6.3%, respectively. Multivariate analysis revealed significantly lower odds for ICU admission (OR 0.68, CI 0.54-0.84, p < 0.001) and IHM (OR 0.74, CI 0.58-0.93, p = 0.011) during the Delta period in patients who had been fully vaccinated or boosted with a COVID-19 vaccine within the previous 6 months. In the BA.1 variant period, patients who had less than 6 months elapsed between the last vaccine dose and SARS-CoV-2 positivity had lower odds for MV (OR 0.38, CI 0.18-0.72, p = 0.005) and IHM (OR 0.56, CI 0.37- 0.83, p = 0.005), but not for NIVS or ICU admission. Conclusion: The likelihood of developing severe CAC in hospitalized patients was higher in those with the Delta and Omicron BA.1 variant compared to BA.4/5.

Sporadic Hepatitis A Virus PCR False-Positive Results Observed during Reflex Testing of Serum Samples Previously Tested for Anti-HAV Antibodies and Caused by Contamination with HAV RNA Present in the Reagents of the Commercial Anti-HAV Immunoassay.

Hepatitis A diagnosis relies on serology and occasionally on hepatitis A virus (HAV) RNA detection. For timely diagnosis and the avoidance of drawing additional blood, molecular testing is often performed as reflex testing by using blood specimens that were initially sent for anti-HAV serology. Reflex molecular testing is preferably performed from different sample aliquots, but, for limited sample quantities, it uses samples that have been preprocessed in an immunoassay analyzer. In 2012, we first observed sporadic HAV RNA-positive cases that were inconsistent with patients' serological profiles and/or medical histories, suggesting that occasional laboratory contamination was causing false-positive PCR results. Multiple external quality assurance (EQA) and laboratory surface contamination checks were performed, questionable specimens were tested with various HAV RNA tests, and follow-up serum/stool samples were collected. All contamination-check samples and samples from healthy individuals tested HAV RNA-negative, and the laboratory successfully passed all EQAs. The HAV RNA-positive results were reproducible with various HAV RNA assays. No patients seroconverted, and their follow-up samples were consistently HAV RNA-negative. Finally, a detailed review of testing protocols revealed a correlation between HAV RNA false positivity and preceding anti-HAV testing with the Cobas-e411 automated immunoassay analyzer. HAV RNA was detected in the Cobas-e411 anti-HAV reagents, with the HAV sequences matching those from the false-positive samples. Preceding anti-HAV testing using two other immunoassay analyzers did not result in subsequent HAV RNA false positivity during reflex testing. The Cobas-e411 pipetting procedure with a single pipette tip collecting samples and anti-HAV reagents contaminated the original sample with the HAV RNA that was present in the immunoassay's reagents, thereby resulting in HAV RNA false positivity during the reflex testing. IMPORTANCE We present the first report of sporadic HAV PCR false-positive results that have been observed during the reflex testing of serum samples that have previously been tested for anti-HAV antibodies and have been caused by contamination with HAV RNA that is present in the reagents of the commercial anti-HAV immunoassay, with potentially serious clinical consequences. Although HAV RNA was consistently detected in the anti-HAV reagents of all three automated immunoassay analyzers that were in use in our laboratory, only the use of one analyzer and the corresponding commercial anti-HAV immunoassay reagents resulted in contamination that led to false positive HAV RNA results, and this was due to a peculiar pipetting mode of action in which the analyzer uses a single pipette tip to collect both anti-HAV reagents and a sample, which consequently causes the permanent contamination of the original sample with HAV RNA. Manufacturers should strongly consider the occasional need for reflex molecular testing from preprocessed samples and design their analyzers in a way that prevents contamination.

HIV-1 subtype diversity and phylogenetic insight into non-B subtype transmission in Slovenia, 1989-2013.

INTRODUCTION: Disease progression, drug resistance mutations, and treatment strategies may vary by HIV-1 subtype. This study determined HIV-1 subtypes circulating in Slovenia, a Central European country with an HIV-1 epidemic driven by men who have sex with men, focusing on molecular epidemiology of non-B subtypes. METHODS: A total of 367 HIV-1 sequences were included. Subtype was assigned by employing eight different HIV subtyping tools coupled with maximum likelihood phylogenetic analyses. RESULTS: The subtyping tools COMET, jpHMM, and REGA 3.0 exhibited the best performance on the dataset studied. Phylogenetic analyses showed a 14.7% prevalence of non-B subtypes, with subtype A detected most frequently (4.9%), followed by CRF02_AG (2.4%), subtype C (1.1%), subtypes D, G, and CRF01_AE (0.8% each), and subtypes F and CRF22_01A1 (0.3% each). A subtype could not be assigned to 12 sequences (3.3%), indicating potential unique recombinant forms. Non-B subtypes were significantly associated with a heterosexual route of transmission and infection acquired in Eastern Europe, Africa, or Asia. CONCLUSION: In a country where subtype B is predominant, non-B subtypes were observed in one out of seven patients, a non-negligible proportion, which underlines the importance of systematic surveillance of HIV subtype diversity and the corresponding molecular epidemiology.

HIV-1 subtype B spread through cross-border clusters in the Balkans: a molecular analysis in view of incidence trends.

OBJECTIVES: To analyze phylogenetic relations and assess the role of cross-border clusters in the spread of HIV-1 subtype B across the Balkans, given the general trends of new HIV diagnoses in seven Balkan countries. DESIGN: Retrospective phylogenetic and trend analysis. METHODS: In-depth phylogenetic, phylodynamic and phylogeographic analysis performed on 2415 HIV-1 subtype B sequences from 1999 to 2019 using maximal likelihood and Bayesian methods. The joinpoint regression analysis of new HIV diagnoses by country and modes of transmission using 2004-2019 ECDC data. RESULTS: Ninety-three HIV-1 Subtype B transmission clusters (68% of studied sequences) were detected of which four cross-border clusters (11% of studied sequences). Phylodynamic analysis showed activity of cross-border clusters up until the mid-2000s, with a subsequent stationary growth phase. Phylogeography analyses revealed reciprocal spread patterns between Serbia, Slovenia and Montenegro and several introductions to Romania from these countries and Croatia. The joinpoint analysis revealed a reduction in new HIV diagnoses in Romania, Greece and Slovenia, whereas an increase in Serbia, Bulgaria, Croatia and Montenegro, predominantly among MSM. CONCLUSION: Differing trends of new HIV diagnoses in the Balkans mirror differences in preventive policies implemented in participating countries. Regional spread of HIV within the countries of former Yugoslavia has continued to play an important role even after country break-up, whereas the spread of subtype B through multiple introductions to Romania suggested the changing pattern of travel and migration linked to European integration of Balkan countries in the early 2000s.

Milder outcomes of SARS-CoV-2 genetically confirmed reinfections compared to primary infections with the delta variant: A retrospective case-control study.

Background: SARS-CoV-2 infection does not confer long immunity. However, studies suggest that prior infection is associated with lower risk of reinfection and milder outcomes of recurrent infections. The aims of this retrospective observational case-control study were to describe the clinical and molecular characteristics of genetically confirmed Delta reinfection cases and to assess the potential protective role of preceding infection on the severity of reinfection. Methods: We used next generation sequencing (NGS) to explore if cases with two positive real time RT-PCR tests > 90 days apart were infected with a different SARS-CoV-2 variant. Cases with confirmed reinfection between August 1st and October 31st, 2021 (the Delta wave) in Slovenia were matched 1:4 by age, sex and timeframe (week of positive test) with individuals with primary infection. Sociodemographic and epidemiologic data, vaccination status, and data on hospitalization and outcome of infection were retrieved from several centralized and standardized national databases. Additional epidemiologic surveys were performed on a limited number of cases and controls. Results: We identified 628 cases of genetically confirmed reinfection during the study period and matched them with 2,512 control subjects with Delta primary infection. Primary infections in individuals with reinfection were mainly caused by B.1.258.17 (51.1%), followed by B.1.1.7 (15.1%) and reinfection was detected on average 271 days after primary infection (range 101-477 days). Our results show a substantially lower probability of hospitalization in cases with reinfection compared with controls (OR: 0.21, p = 0.017), but no significant difference was observed in intensive care unit admission and deaths. We observed a significantly lower proportion of vaccinated individuals among cases compared to controls (4.5% vs. 28.2%), suggesting that hybrid immunity leads to lower probability of reinfection. Detailed analysis of the temporal distribution of variants, responsible for reinfections, showed no significant differences in reinfection potential. Conclusion: Reinfection with the SARS-CoV-2 Delta variant resulted in fewer hospitalizations compared to the primary Delta infection, suggesting that primary infection may, to some extent, produce at least short lasting protective immunity. This study provides additional insight into the reinfection dynamics that may allow appropriate public health measures to be taken in subsequent waves of the COVID-19 pandemic.

First Report of Phodopus sungorus Papillomavirus Type 1 Infection in Roborovski Hamsters (Phodopus roborovskii).

Papillomaviruses (PVs) are considered highly species-specific with cospeciation as the main driving force in their evolution. However, a recent increase in the available PV genome sequences has revealed inconsistencies in virus-host phylogenies, which could be explained by adaptive radiation, recombination, host-switching events and a broad PV host range. Unfortunately, with a relatively low number of animal PVs characterized, understanding these incongruities remains elusive. To improve knowledge of biology and the spread of animal PV, we collected 60 swabs of the anogenital and head and neck regions from a healthy colony of 30 Roborovski hamsters (Phodopus roborovskii) and detected PVs in 44/60 (73.3%) hamster samples. This is the first report of PV infection in Roborovski hamsters. Moreover, Phodopus sungorus papillomavirus type 1 (PsuPV1), previously characterized in Siberian hamsters (Phodopus sungorus), was the only PV detected in Roborovski hamsters. In addition, after a detailed literature search, review and summary of published evidence and construction of a tanglegram linking the cladograms of PVs and their hosts, our findings were discussed in the context of available knowledge on PVs described in at least two different host species.

HIV-1 Unique Recombinant Forms Identified in Slovenia and Their Characterization by Near Full-Length Genome Sequencing.

Surveillance of HIV circulating recombinant forms (CRFs) is important because HIV diversity can affect various aspects of HIV infection from prevention to diagnosis and patient management. A comprehensive collection of pol sequences obtained from individuals diagnosed with HIV-1 from 2000 to 2016 in Slovenia was subtyped to identify possible unique recombinant forms (URFs). Selected samples were subjected to near full-length genome (NFLG) sequencing and detailed recombination analyses. Discordant subtyping results were observed for 68/387 (17.6%) sequences and 20 sequences were identified as the most probable URFs and selected for NFLG characterization. Further, 11 NFLGs and two sequences of >7000 base pairs were obtained. Seven sequences were identified as "pure" subtypes or already characterized CRFs: subtype B (n = 5), sub-subtype A6 (n = 1), and CRF01_AE (n = 1). The remaining six sequences were determined to be URFs; four displayed a single recombination event and two exhibited a complex recombination pattern involving several subtypes or CRFs. Finally, three HIV strains were recognized as having epidemic potential and could be further characterized as new CRFs. Our study shows that the identification of new CRFs is possible, even in countries where HIV diversity is considered limited, emphasizing the importance of the surveillance of HIV recombinant forms.

Human papillomavirus genotype specificity of hybrid capture 2 low-risk probe cocktail.

A genotyping study of 285 Hybrid Capture 2 low-risk probe cocktail-positive specimens showed cross-reactivity with several untargeted human papillomavirus genotypes. Cross-reactivity was often clinically beneficial due to the detection of untargeted low-risk genotypes. A total of 8.4% of positive results, usually weak, were due to cross-reactivity with high-risk genotypes. Establishment of a gray zone is recommended.

Low prevalence of hepatitis B and C infections among HIV-infected individuals in Slovenia: a nation-wide study, 1986-2008.

BACKGROUND: With the increased lifespan of HIV-1 infected patients, mostly due to highly active antiretroviral therapy, hepatitis C virus (HCV) and hepatitis B virus (HBV) have recently emerged as important pathogens in these patients. HIV-1 infection has an important negative impact on the natural history of HCV and HBV infections, which has consequently caused increased liverassociated and overall morbidity and mortality in HIV-1 infected patients. Thus, liver disease is currently the second leading cause of death in HIV-infected persons in Europe. OBJECTIVE: To determine the prevalence of HBV and HCV infection in HIV-infected individuals in Slovenia. METHODS AND RESULTS: 356 out of 409 Slovenian individuals, confirmed as HIV positive by the end of 2008, were tested for the presence of HBV and HCV infection. Evidence of prior and current HBV infection was found in 77 (21.6%) and 14 (3.9%) of HIV-positive patients, respectively. 38 of 356 (10.7%) HIV-infected individuals were confirmed as anti-HCV positive, and 26 of them (68.4%) were also HCV RNA positive. Concomitant active HBV and HCV infection was found in only two HIV-positive individuals. CONCLUSION: In a study carried out on the highest proportion per entire population of HIV-infected individuals from a certain country or geographical region, Slovenia was identified as the country with the lowest prevalence of HCV infection among HIV-infected individuals.

Sequence ambiguity determined from routine pol sequencing is a reliable tool for real-time surveillance of HIV incidence trends.

Identifying individuals recently infected with HIV has been of great significance for close monitoring of HIV epidemic dynamics. Low HIV sequence ambiguity (SA) has been described as a promising marker of recent infection in previous studies. This study explores the utility of SA defined as a proportion of ambiguous nucleotides detected in baseline pol sequences as a tool for routine real-time surveillance of HIV incidence trends at a national level. A total of 353 partial HIV-1 pol sequences obtained from persons diagnosed with HIV infection in Slovenia from 2000 to 2012 were studied, and SA was reported as a percentage of ambiguous base calls. Patients were characterized as recently infected by examining anti-HIV serological patterns and/or using commercial HIV-1 incidence assays (BED and/or LAg-Avidity assay). A mean SA of 0.29%, 0.14%, and 0.19% was observed for infections classified as recent by BED, LAg, or anti-HIV serological results, respectively. Welch's t-test showed a significant difference in the SA of recent versus long-standing infections (p < 0.001). CD4+ T-cell counts ≤250 cells/mm3 significantly correlated with higher SA (p < 0.001), whereas the homo/bisexual transmission route significantly correlated with lower SA (p = 0.005). When the LAg-assay was used as an indicator of recent infection, a receiver operating characteristic curve with the largest area under the curve (0.896) was observed for SA (sensitivity and specificity of 79%), indicating the best correlation of the data. A reliable estimation of the trends of HIV incident infection could be inferred from measuring SA irrespective of the cutoff used; however, in Slovenia it seems that lower cutoffs are more appropriate. Our data suggest that SA could be used as a real-time surveillance tool for close monitoring of HIV incidence trends, especially in countries where baseline HIV resistance genotyping is performed routinely, rendering this approach cost-effective.

Analysis of HIV-1 diversity, primary drug resistance and transmission networks in Croatia.

Molecular epidemiology of HIV-1 infection in treatment-naive HIV-1 infected persons from Croatia was investigated. We included 403 persons, representing 92.4% of all HIV-positive individuals entering clinical care in Croatia in 2014-2017. Overall prevalence of transmitted drug resistance (TDR) was estimated at 16.4%. Resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside RTI (NNRTIs) and protease inhibitors (PIs) was found in 11.4%, 6.7% and 2.5% of persons, respectively. Triple-class resistance was determined in 2.2% of individuals. In addition, a single case (1.0%) of resistance to integrase strand-transfer inhibitors (InSTIs) was found. Deep sequencing was performed on 48 randomly selected samples and detected additional TDR mutations in 6 cases. Phylogenetic inference showed that 347/403 sequences (86.1%) were part of transmission clusters and identified forward transmission of resistance in Croatia, even that of triple-class resistance. The largest TDR cluster of 53 persons with T215S was estimated to originate in the year 1992. Our data show a continuing need for pre-treatment HIV resistance testing in Croatia. Even though a low prevalence of resistance to InSTI was observed, surveillance of TDR to InSTI should be continued.