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 transmitted drug resistance in Slovenia and its impact on predicted treatment effectiveness: 2011-2016 update.

HIV-positive individuals that have a detected transmitted drug resistance (TDR) at baseline have a higher risk of virological failure with antiretroviral therapy (ART). This study offers an update on the prevalence of TDR in Slovenia, looks for onward transmission of TDR, and reassesses the need for baseline drug resistance testing. Blinded questionnaires and partial pol sequences were obtained from 54.5% (168/308) of all of the patients diagnosed with HIV-1 from 2011 to 2016. Subtype B was detected in 82.7% (139/168) of patients, followed by subtype A (8.3%), subtype C (2.4%), and CRF01_AE (1.8%). Surveillance drug resistance mutations (SDRMs) were found in four individuals (2.4%), all of them men who have sex with men (MSM) and infected with subtype B. K103N was detected in two patients and T68D and T215D in one person each, corresponding to a prevalence of 0%, 1.2%, and 1.2% of TDR to protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs), and non-NRTIs (NNRTIs), respectively. The impact of mutations on drug susceptibility was found to be most pronounced for NNRTIs. No forward spread of TDR within the country was observed; however, phylogenetic analysis revealed several new introductions of HIV into Slovenia in recent years, possibly due to increased risky behavior by MSM. This was indirectly confirmed by a substantial increase in syphilis cases and HIV-1 non-B subtypes during the study period. A drug-resistant HIV variant with good transmission fitness is thus more likely to be imported into Slovenia in the near future, and so TDR should be closely monitored.

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.

Prevalence, genotype distribution, and risk factors for hepatitis C infection among HIV-infected individuals in Slovenia: a 1986-2013 update.

INTRODUCTION: Since the introduction of highly active antiretroviral therapy, chronic hepatitis C has become one of the leading causes of non-AIDS-related morbidity and mortality in patients with HIV infection. Two previous Slovenian nationwide studies published in 2002 and 2009 showed a very low prevalence of hepatitis C virus (HCV) infection among Slovenian HIV-infected individuals (14.5% and 10.7%, respectively). METHODS AND RESULTS: The presence of HCV infection was tested in 579/639 (90.6%) patients that were confirmed as HIV-positive in Slovenia by the end of 2013. Among them, 7.6% (44/579) of HIV-infected individuals were anti-HCV-positive, and 33/44 (75%) anti-HCV-positive patients were also HCV RNA-positive. HCV genotype 1 was most prevalent among HIV-infected patients (68%), followed by genotype 3 (20%), genotype 4 (8%), and genotype 2 (4%). Anti-HCV positivity was significantly higher in those that acquired HIV by the parenteral route (91.8%) than in those that acquired HIV by the sexual route (2.8%). DISCUSSION: Slovenia remains among the countries with the lowest prevalence of HCV infection in HIV-infected individuals. Because the burden of HIV among men who have sex with men in Slovenia is disproportionately high and increasing rapidly, the current favorable situation could change quickly and should be therefore monitored regularly.

Short communication: prevalence of HIV type 1 transmitted drug resistance in Slovenia: 2005-2010.

Slovenia is a small European country with a total of 547 HIV-infected individuals cumulatively reported by the end of 2011. However, the estimated incidence rate of HIV infections increased from 7.0 per million in 2003 to 26.8 per million in 2011. In this study, we assessed the prevalence of transmitted drug resistance (TDR) in the past 6 years (2005-2010) and analyzed the time trend of the proportion of men having sex with men (MSM) and HIV-1 subtype B among newly diagnosed individuals in a 15-year period (1996-2010) in Slovenia. Among 150 patients included in the study, representing 63% of HIV-1 newly diagnosed patients in 2005-2010, TDR was found in seven patients (4.7%). The prevalence of TDR to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors was 2% (3/150), 2% (3/150), and 0.7% (1/150), respectively. The majority of patients were infected with subtype B (134/150, 89%), while subtype A was detected in 6.0% (9/150), subtype D in 1.3% (2/150), and subtype G and CRF02_AG in 0.7% (one patient each). Three of 150 sequences could not be typed. Infection with subtype B was found to be significantly associated with male gender, Slovenia being reported as the country of the patient's nationality and origin of the virus, CDC class A, mode of transmission with homosexual/bisexual contact, sex with an anonymous person, and a higher CD4(+) count. Among patients carrying the subtype B virus, an MSM transmission route was reported in 87% of patients. Although the prevalence of TDR in Slovenia is still below the European average, active surveillance should be continued, especially among MSM, the most vulnerable population for HIV-1 infection in this part of Europe.

The 2,6-disubstituted naphthalene derivative FDDNP labeling reliably predicts Congo red birefringence of protein deposits in brain sections of selected human neurodegenerative diseases.

Deposition of conformationally altered proteins prominently characterizes pathogenesis and pathomorphology of a number of neurodegenerative disorders. 2-(1-{6-[(2-[F-18]fluoroethyl) (methyl)amino]-2-naphthyl} ethylidene) malononitrile ([F-18]FDDNP), a hydrophobic, viscosity-sensitive, solvent-sensitive, fluorescent imaging probe has been used with positron emission tomography to visualize brain pathology in the living brain of Alzheimer disease (AD) patients. Its non-radiofluorinated analog FDDNP was shown to label senile plaques and neurofibrillary tangles (NFTs) in brain tissue sections. This work aimed at evaluating FDDNP labeling of various protein deposits in fixed, paraffin-embedded brain tissue sections of selected neurodegenerative disorders: AD, cerebral amyloid angiopathy (CAA), transmissible spongiform encephalopathies, progressive supranuclear palsy (PSP), Pick disease (PiD), Parkinson disease, dementia with Lewy bodies, multiple system atrophy (MSA). Cerebral hypertensive vascular hyalinosis (HVH) was used as negative control. Significant agreement between amyloid histochemical properties and FDDNP labeling of the deposits was established. FDDNP labeling showed high positive predictive value for birefringence in senile plaques and NFTs in AD, prion plaques and amyloid deposits in CAA. No FDDNP labeled structures were observed in HVH, PSP, PiD or MSA tissue sections. Our findings may be of significant value for the detection of neuropathological aggregates with [F-18]FDDNP in some of these disorders in the living brain of human subjects.

Molecular-imaging probe 2-(1-[6-[(2-fluoroethyl)(methyl) amino]-2-naphthyl]ethylidene) malononitrile labels prion plaques in vitro.

The study aimed to evaluate the fluorescent molecular-imaging probe 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene)malononitrile (FDDNP) for its ability to selectively and reproducibly label prion plaques in fixed, paraffin-embedded cerebellar sections from patients of confirmed Gerstmann-Sträussler-Scheinker disease, sporadic Creutzfeldt-Jacob disease (CJD) with kuru plaques, and variant CJD (vCJD). FDDNP is a highly hydrophobic, viscosity-sensitive, solvent-sensitive, fluorescent substance, whose radiofluorinated analog [18F]FDDNP has recently been successfully used to label senile plaques and neurofibrillary tangles in the living brain of Alzheimer's disease patients with positron emission tomography. Our results show that FDDNP reliably identifies all prion plaques, including small cluster-plaques in vCJD. This finding may open new in vivo diagnostic possibilities for vCJD.