HIV-1 subtypes distribution and resistance to ART in HIV-infected persons in Slovakia (2019-2021).

AIM: The aim of the study was to describe the prevalence of HIV-1 subtypes and HIV-1 strains resistant to antiretroviral therapy (ART) in HIV-positive persons newly diagnosed in Slovakia in 2019-2021. MATERIALS AND METHODS: The study group consisted of 184 HIV-positive naïve patients newly diagnosed in Slovakia from 2019 to 2021. The viral HIV-1 RNA was isolated from plasma by the QIAamp Viral RNA Mini Kit (QIAGEN, Germany). For RT-PCR and sequencing of the HIV pol region, in-house procedures were used according to the ANRS AC11 protocol for RT (reverse transcriptase), PRO (protease), and IN (integrase) [ANRS AC11 Resistance Study Group, 2015]. Analysis of sequences was performed using Sequencing Analysis Software v5.3 (Applied Biosystems®). HIV sequences were manually edited using BioEdit (version 7.2.5), compared with consensus HIV-1 sequences in the Los Alamos Sequence Database (URL 2), aligned using CLUSTAL W [Labarga et al., 2007] and BioEdit software packages (version 7.2 .5) [Hall, 1999]. HIVDB Algorithm (version 9.0) of the Stanford HIV Drug resistance database (URL 1.) was used for sequence evaluation. For HIV-1 subtype analysis, the REGA HIV-1 Subtyping Tool [De Oliviera et al., 2005] and phylogenetic analysis MEGA X [Kumar et al., 2018] were used. RESULTS: Phylogenetic analyses performed in samples of 184 persons revealed the most prevalent subtype B (129/184, 70.11%), detected to the greatest extent in the population of men who have sex with men (MSM) (96/129 74.42%). Concerning non-B subtypes (55/184, 29.89%), subtype A was found with the highest prevalence (48/184, 26.09%) compared to subtype F (F1) (3; 1.63%), C (1; 0.54%) and circulating recombinant forms CRF02_AG (2; 1.09%), CRF01_AE (1; 0.54%). In 9.24% (17/184) of samples, 25 mutations clinically relevant and associated with HIV resistance ART were detected, of which 7.07% (13/184) to reverse transcriptase inhibitors, 1.66% (3/181) to protease inhibitors and 1.32% (2/151) to integrase inhibitors. In addition, multiclass resistance was present in 1.63% (3/184) of patients. Mutations associated with HIV resistance to ART were found in 9.30 % of persons infected with subtype B. CONCLUSION: Our study confirmed ongoing highest prevalence of subtype B with a slightly decreasing trend compared to last years. Detection of mutations causing HIV resistance to ART underlines the need for resistance testing in naïve patients even before the initiation of ART in Slovakia.

Validation study of a conventional enzyme immunoassay to detect HIV antibodies in oral fluid.

OBJECTIVES: The aim of our study was to validate the Genscreen HIV ½ version 2 (BIO-RAD) for detecting HIV antibodies in oral fluid specimens (OF). BACKGROUND: The advantage of assays to detect HIV infection in OF lies in the on-site easy access and noninvasive sample collection. METHODS: Paired serum and OF were collected from 496 subjects (263 HIV-positive and 233 HIV-negative) using the Oracol test kit (Oracle Diagnostics, Inc). The quality of OF was verified by measuring total IgGs using the Human IgG ELISA Quantitation Kit (Bethyl Lab.inc). All reactive OF samples were retested by Western blot HIV1/2 BLOT 2.2 (MP Biomedical, Singapore, China). RESULTS: Of 263 OF samples from participants with blood-based HIV-positive results, 259 were positive by Genscreen HIV ½ version 2 (98.48% sensitivity, 95% CI; 96.2-99.6). The 233 individuals who had a non-reactive HIV blood test were found negative on testing their OF by Genscreen HIV ½ version 2 (100% specificity, 95% CI; 98.4-100). NPV and PPV of the assay were 98.31% (95% CI; 95.74-99.34) and 100%, (95% CI; 98.53-100.00), respectively. CONCLUSION: Genscreen HIV ½ version 2 (Bio-Rad) is a prospective method for HIV surveillance studies in hard-to-reach populations with high risk behavior using non-invasive OF collection (Tab. 1, Fig. 1, Ref. 16).

Primary resistance to integrase strand-transfer inhibitors in Europe.

OBJECTIVES: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. RESULTS: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals. CONCLUSIONS: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.

Distribution of HIV-1 subtypes circulating in Slovakia (2009-2012).

Slovakia is one of the European countries reporting a low prevalence of human immunodeficiency virus 1 (HIV-1) infection. The HIV-1 subtype B was previously the most prevalent in Slovakia and mostly associated with men having sex with men (MSM) (Habekova et al., 2010). The aim of this study was to determine the distribution of individual HIV-1 subtypes among the virus-positive individuals diagnosed in 2009-2012 in Slovakia and to compare these data with those from 2004-2008. The HIV-1 subtype B exhibited the highest prevalence (86.1%) and was mostly associated with MSM as stated before. HIV-1 non-B subtypes were found in 13.9% of patients, led by CRF01_AE (6.3%) and followed by subtype C (2.8%), F1 (1.4%), CRF03_AB (1.4%), G (0.7%), CRF02_AG (0.7%), and CRF12_BF (0.7%). The incidence of non-B subtypes varied in 2009-2012, showing a maximum in 2012. MSM represented the main risk group for total HIV-1 (68.1%) as well as B subtype (63.9%) infections in Slovakia. In contrast, the heterosexual contact represented the main route of HIV-1 acquisition of the non-B subtype virus infection. Together with the increase in the number of patients infected with the viruses of non-B subtype also other changes were observed: infections with the viruses of non-B subtype became more common among MSM and not so strongly associated with heterosexual contact. All these findings may help to design the right strategy for retaining of HIV epidemic in Slovakia at low level.

Genetic subtypes of HIV type 1 circulating in Slovakia.

Slovakia belongs to the group of European countries with a low prevalence of HIV infection. The major proportion of HIV-positive cases in Slovakia is still represented by MSM, followed by heterosexuals infected through unprotected sexual intercourse. This study was conducted to update the description of HIV subtypes circulating in Slovakia. HIV-1 partial pol gene sequences from 143 individuals were prospectively collected from 2004 to 2008 and analyzed. Phylogenetic analysis based on HIV-1 partial pol gene sequences revealed the highest prevalence of HIV-1 B subtype (93.0 %), predominantly associated with the MSM group. Ten (7.0%) individuals were infected with HIV-1 non-B subtypes. The pure subtypes were more frequent (7; 4.9%) than CRFs (3; 2.1%) and their occurrence was as follows: subtype C (3; 2, 1%), subtype A (2; 1.4%), subtype F (2; 1.4%), CRF_01AE (1; 0.7%), CRF_02AG (1; 0.7%), and CRF08_BC (1; 0.7%). Data show slightly increasing HIV-1 subtype diversity, with HIV-1 subtype B still having the highest prevalence in the Slovak-infected population.

HIV bio-behavioural survey among men who have sex with men in Barcelona, Bratislava, Bucharest, Ljubljana, Prague and Verona, 2008-2009.

Data from 23 European countries show that the annual number of HIV diagnoses in men who have sex with men (MSM) increased by 86% between 2000 and 2006. This paper reports the main preliminary results of a bio-behavioural survey in MSM with a specific focus on HIV prevalence and use of United Nations General Assembly Special Session (UNGASS) indicators in six cities in Southern and Eastern Europe. Time-location sampling (TLS) was used. A total number of 2,356 questionnaires and 2,241 oral fluid samples were collected (invalid samples 4.1%). The data show different socio-demographic patterns across countries regarding age, level of education, living conditions, living area and self-identity. Southern European cities had the highest percentage of people who had tested for HIV and collected the result. More than 50% of respondents in the sample from Barcelona reported having used a condom last time they had anal sex (57.2%), whilst in all other cities this proportion was below 50%. The cities with the highest HIV prevalence in MSM were Barcelona (17.0%) and Verona (11.8%) whilst lower percentages were reported in Bratislava (6.1%), Bucharest (4.6%), Ljubljana (5.1%) and Prague (2.6%). The low prevalence in Eastern European cities is encouraging. However, with the level of high-risk sexual behaviour documented and the lower frequency of HIV test seeking behaviour, there is a clear risk of an increase in HIV transmission.

HIV prevalence and clinical care for HIV-positive pregnant women in Slovakia.

OBJECTIVE: To evaluate clinical care for HIV positive pregnant women who delivered in Slovakia from 1985 till 2008. BACKGROUND: National guidelines for the prevention of mother-to-child transmission of HIV have not yet been established. METHOD: Retrospective analysis of 14 HIV-infected pregnant women and their infants. Factors examined include maternal social, demographic, immunological and virological characteristics, method of HIV testing, antiretroviral therapy during pregnancy, delivery and puerperium, mode of delivery, mode of infant feeding, pregnancy outcomes and HIV status of infants. CONCLUSIONS: Due to the increase in cases of HIV-infected pregnant women within the last several years, it would be advisable to create National Guidelines for PMTCT (Prevention of Mother-to-Child Transmission) in order to consolidate the care management in all HIV/AIDS care centers around Slovakia (Fig. 4, Ref. 7).

Prevalence of a 32 bp deletion in the gene for human immunodeficiency virus 1 co-receptor CCR5 in Slovak population.

The chemokine (C-C motif) receptor 5 (CCR5) represents a major co-receptor for macrophagetropic Human immunodeficiency virus 1 (HIV-1) strains. A 32 bp deletion mutant allele in the CCR5 gene (CCR5-Δ32) provides to homozygotes a strong resistance to HIV-1 infection. In this study, the prevalence of CCR5-Δ32 in 200 HIV-1-negative and 162 HIV-1-positive individuals was determined. The presence of CCR5-Δ32 in blood samples was detected by PCR. CCR5-Δ32 was found to be homozygous in 1% and heterozygous in 17% of the HIV-1-negative individuals. In HIV-1-positive patients, no homozygous CCR5-Δ32 was found, while heterozygous CCR5-Δ32 occurred in 15.4% patients. Thus, no significant difference between HIV-1- negative and HIV-1-positive individuals was found, what is in accord with the findings obtained in other EU countries. The results of this study do not indicate that a relatively low incidence of HIV-1 infection in Slovakia could be caused by the CCR5-Δ32 mutation.

HIV infection and risk behaviour of commercial sex workers and intravenous drug users in Slovakia.

INTRODUCTION: Aim of the study was to determine risk behaviour and HIV prevalence among commercial sex workers (CSWs) and intravenous drug users (IDUs) in streets of Bratislava and B. Bystrica, SR. METHODS: HIV antibodies were tested from saliva using ELISA test. Anonymous questionnaire was completed. RESULTS: 121 persons (61 men and 60 women) were involved in the sociological study. Mean age of the participants was 21.9 years. 185.1% of subjects were from Bratislava. 108 participants were tested for the presence of HIV-antibodies, one was confirmed HIV-positive (0.82%). In the past 47.9% of participants and 22.3% of their partners were tested for the presence of HIV-antibodies. 10.8% of subjects proclaimed that they suffered from other sexually transmitted infection (STI) in the past HIV testing of participants significantly correlated with the testing for other STI (p<0.002) as well as with HBV/HCV (p<0.001). 58 participants were using tattooing (47.9%). 46.3% of all participants never used condoms with partners. 31.4% of respondents proclaimed disruption of condom during sexual intercourse. Significant correlation was found between testing of participants for other STI and usage of condoms with their partners (p<0.013). Women used condoms more often by sexual contacts with partners than men used condoms (p<0.094). They were also significantly more tested for other STI in the past (p<0.021) and they suffered from other STI more often than men (p<0.033). 26.5% of person--only women--were involved in commercial sex work. 93.5% of them were taking drugs as well, 21.8% suffered for other STI in the past They were working in sex business on average for 26 months. The average number of their clients per week was 12.3. CSWs used condoms more often with clients than with partners. 98.2% of all participants were taking drugs, 93% of them intravenously. 24.6% of IDUs always used new or their own needles and syringes, while 69.4% shared equipments with the other users. IDUs drug users used condoms significantly less often with their partners than did CSWs (p<0.006). CSWs were significantly more often tested for other STI (p<0.001) and they also more often suffered for other STI than IDUs (p<0.045). CONCLUSION: More effort should be done to decrease risk behaviour revealed in the groups of CSWs and drug users.

Sexually transmitted infections among prostitutes in Bratislava, Slovakia.

Sera from 18 prostitutes from Bratislava were examined for the presence of antibodies to several sexually transmitted pathogens, namely Herpes simplex virus 2 (HSV-2), Human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), Hepatitis B and C viruses (HBV and HCV), Chlamydia trachomatis, and Treponema pallidum. Results of this screening indicated that 11 prostitutes (61%) carried 1 or more sexually transmitted infections. The most prevalent antibodies were directed against HSV-2 (9 cases, i.e. 50%), which represents the most common sexually transmitted infection agent.

Pilot study of risk behaviour, voluntary HIV counselling and HIV antibody testing from saliva among inmates of prisons in Slovakia.

OBJECTIVE: To implement a pilot study of risk behaviour and HIV infection using HIV antibody testing from saliva to improve the situation as regards HIV/AIDS infection in prison institutions in the Slovak Republic. MATERIAL AND METHODS: The study comprised adult and juvenile males of grade one correction categories and prisoners from the prison for juveniles in Martin, as well as females prisoners in Nitra. Preventive activities were implemented in May 1998 in the form of discussions concerning topics related to HIV/AIDS infection. Saliva was collected for the presence of HIV antibodies and a questionnaire regarding sexual practice was completed. RESULTS: 32 persons [8 adult males (25%), 6 juvenile males (18.7%) and 18 females (56%)] were voluntarily tested for the presence of HIV antibodies in saliva. Nobody was HIV-positive. 75 persons (20 adult males, 30 juvenile males and 25 females) were involved in the study of risk behaviour. 40.8% participants had primary education, 28.2% secondary education, 2.8% were students of universities and 28.2% were apprenticies. 60% inmates (mostly females) were religious. Juvenile males reported the highest number of partners while females the smallest (p < 0.001). The more partners were reported by respondents, the lower was condom usage (p < 0.07). 47.6% females relied on credibility of partners, while 75% adult males and 50% adolescent males did not use protection. 0% females, 5% adult males and 10.3% juvenile males reported to have homosexual contacts outside prison while 19%, 5.6% and 8.3% in the prison, respectively. Paid sexual services were offered by 9.1% females, 15.8% adult males and 25% juvenile males. Outside prison adult and juvenile males used non-sterile used syringes as well as tattooing more often than females (p < 0.07 and p < 0.04, respectively). CONCLUSION: The present study provides information on the results of HIV-antibody testing in saliva. The results are based on a study of risk behaviour and difficulties linked with HIV/AIDS prevention among prisoners.

HIV infection and sexual behaviour among homosexual and bisexual men in Bratislava.

OBJECTIVE: To determine sexual behaviour and HIV prevalence in a sample of homosexual and bisexual men in Bratislava. PATIENTS AND METHODS: Participants were recruited at gay discotheque in Bratislava in February and June 1996. Saliva samples were collected for testing the presence of anti-HIV antibodies and a questionnaire regarding sexual practice was completed. RESULTS: In the study 170 men (1st discotheque) and 124 men (2nd discotheque) were enrolled. The prevalence of antibodies against HIV was 5.4% and 1.6%, respectively. A group of 119 attenders (mean age: 25 years) was voluntarily questioned about their sexual lifestyle and related behaviour. Sexual orientation of participants was following: 66.4% homosexuals, 20.2% bisexuals, 13.4% others. Most responders were single and did not ever been married (86.55% vs. 12.6%, respectively). The figures of sexual intercourse were: steady partners: 45.38%, non-steady partners: 49.58%. More individuals were using condoms (70.58% vs. 15.97%, respectively), while only 28.57% were using them by each chance sexual intercourse. 6.7% responders suffered from an other STD and 3.36% were intravenous drug users. CONCLUSION: The HIV prevalence (5.4% and 1.6%) was relatively low. Considering the high prevalence of unsafe sexual practice further spread of HIV infection among homosexual men in Slovakia is to be expected.

Prevalence of antibodies to herpes simplex virus 2 among homosexual men either positive or negative for human immunodeficiency viruses in Slovakia.

We determined the prevalence of antibodies to herpes simplex virus 2 (HSV-2, HSV-2 antibodies) in sera of homosexual men either positive for human immunodeficiency virus 1 (HIV-1, HIV+, a group of 27 sera) or negative for HIV-1 and HIV-2 (HIV-, a group of 52 sera) in Slovakia. Antibodies to HSV-2 glycoprotein G-2 (gG-2, gG-2 antibodies) were determined by a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and immunoblot analysis. We found that 40% of HIV+ and 23% of HIV- homosexual men were positive for the gG-2 antibodies, what is 3.6 and 2.1 times higher incidence, respectively, than that in the control heterosexual population (Bystrická et al., Acta Virol. 42, 319-324, 1998). Identification of individuals infected with genital herpes among HIV+ and HIV- homosexual men should be succeeded by antiviral therapy in order to prevent transmission of HSV-2 and HIV as well in this community.

Antibody responses to the herpes simplex virus type 2 glycoprotein G in sera of human immunodeficiency virus-infected patients in Slovakia.

Thirty sera of human immunodeficiency virus-positive (HIV+) and 37 sera of HIV-negative (HIV-) individuals in Slovakia were tested for the presence of antibodies to herpes simplex virus type 2 (HSV-2) glycoprotein G (gG). A notable difference between the prevalence of HSV-2-specific antibodies in HIV+ and that in HIV- individuals was found (37% vs. 11%) confirming and extending previous reports that HSV-2 infection is an important risk factor for HIV transmission. Efforts toward the detection of HSV-2 infection and its therapy by anti-HSV drugs should be considered an important factor in decreasing the risk of contracting and spreading of HIV in Slovakia.

Genetic polymorphism of factor B (Bf) and C3 component of complement in type 1 (insulin-dependent) diabetes mellitus: BFQO allele observed in a diabetic child.

C3 and Bf polymorphisms were studied in 215 and 192 children with type 1 diabetes mellitus (IDDM), respectively. No significant differences in C3 phenotypes and allele frequencies were found between IDDM patients and a healthy population. The rare allele BfF1 was found in 9.37% of diabetic patients but in only 0.35% of the general Slovak population (0.0468 vs. 0.0017). An increased frequency rate of BfSO.7 was also observed in 8.85% of IDDM patients compared with 3.57% of healthy controls (0.0442 vs. 0.0178). The relative risk was 28.83 for BfF1 and 2.55 for BfSO.7. One diabetic child was found to be heterozygous for a silent allele BfQO. This rare Bf allele was transmitted to the boy from his healthy mother.

Genetic polymorphism of the C4 component of human complement in the Slovak population.

The distribution of C4 phenotypes and gene frequencies were studied in 104 genetically unrelated persons of Slovakia using high-voltage agarose gel electrophoresis with subsequent immunofixation. Five C4A alleles and three C4B alleles were detected. The gene frequencies were as follows: A2 = 0.0576, A3 = 0.7644, A4 = 0.0336, A6 = 0.0625, AQ0 = 0.817, B1 = 0.7836, B2 = 0.1009, BQ0 = 0.1153. The C4AQ0 and C4BQ0 alleles established by densitometry appeared in the Slovak population in 16.34% and 23.07%, respectively.

Genetic polymorphism of factor B of the complement system (Bf) in the Slovak population.

The distribution of factor B (Bf) phenotypes and gene frequencies were investigated in 280 genetically unrelated persons of the Slovak population. Thin-layer agarose gel high-voltage electrophoresis and subsequent immunofixation were used. A low frequency of the "rare" allele BfFl was observed (BfFl = 0.0017). The frequencies of common Bf alleles BfS and BfF (BfS = 0.816, BfF = 0.1625) and a "rare" allele BfSO.7 (BfSO.7 = 0.0178) were inside the corresponding ranges of BfS, BfF and BfSO.7 found in European Caucasoids. No other variants were observed.

Pathogenesis of acute and persistent murine herpesvirus infection in mice.

Outbred laboratory mice were inoculated at the age of 5, 10 and 21 days by oral and/or intranasal routes with 2 different (a lethal and a nonlethal) doses of the murine herpesvirus isolate 68 (MHV-68). Severe exudative pneumonia with haematogenous dissemination of the virus to liver, heart muscle, and kidneys developed in the 5-day-old as well as in a part of the 10-day-old mice. Virus antigen was found by immunofluorescence (IF) in the alveolar lining of lungs, in heart muscle fibres, in spleen and thymic lymphocytes, in the tubular epithelium cells of kidneys, in the neurons of Gasserian ganglia and in the intima of large pulmonary vessels. Electron microscopy confirmed the transfer of virus particles through the capillary endothelium of the damaged alveolar septa. The surviving progeny and the mothers of animals, which had not succumbed to the lethal virus dose, were kept for 141-169 days when lungs and Gasserian ganglia were examined for virus presence. MHV-68 was recovered both by direct examination of the tissue homogenates as well as by the explantation technique. The results are suggestive for a dynamic persistence of MHV-68 rather than for static latency.

Experimental pathogenesis of murine herpesvirus in newborn mice.

Newborn white mice were susceptible to peroral (p.o.) infection with murine alphaherpesvirus isolated from free-living Clethrionomys glareolus. Death occurred within 6-8 days in animals infected with the higher virus dose of 4.8 log10 TCID50. Clinical symptoms also occurred in some animals infected with lower doses, while others developed inapparent infection as judged by presence of humoral antibodies at 60 days post-infection (p.i.). The virus was detected in the lungs, blood, liver, spleen, kidneys, heart muscle, brain and urinary bladder of sick animals. Necrotising pneumonia accompanied the replication of the virus in the epithelial cells of alveolar ducts and alveolar lining as confirmed by immunofluorescence and histological examination. Latent infection of Gasserian ganglia in the survivors was not necessarily related to the administered dose of infectious virus. Two of mother females, which had eaten their diseased offspring, became inapparently infected as proved by reisolation of the virus from trigeminal ganglion explants and by detection of specific antibodies at 60 days p.i.