Eveli Kallas

Association between TLR3 rs3775291 and resistance to HIV among highly exposed Caucasian intravenous drug users.

BACKGROUND TLR3 recognizes dsRNA and triggers immune responses against RNA and DNA viruses. A polymorphism in TLR3, rs3775291 (Leu412Phe), has been associated with the increased susceptibility to enteroviral myocarditis, protection against tick-borne encephalitis virus and HIV-1 infection. We investigated Caucasian intravenous drug users (IDUs) and blood donors in order to evaluate the associations between TLR3 genotypes and susceptibility to HIV infection. MATERIALS AND METHODS A total of 345 Caucasian IDUs were recruited, 50% of them were HIV positive, 89% HCV and 77% HBV positive. Based on their history of needle sharing, 20 of the HIV negative IDUs were classified as highly exposed HIV seronegatives (HESNs), 68 as non-HESNs and 85 as unexposed. The control group consisting of 497 blood donors tested negative for all three viruses. TLR3 rs3775291 were determined by using TaqMan Allelic Discrimination Assay. RESULTS The TLR3 rs3775291 T allele frequency was similar among the HIV negative and HIV positive IDUs and blood donors - 36%, 31% and 34%, respectively. The frequency of persons possessing at least one TLR3 rs3775291 T allele was significantly higher in HESNs compared with blood donors and HIV positive IDUs (80% vs. 55%; p=0.037 and 80% vs. 53%; p=0.031, respectively). In the univariate analysis, persons who possessed at least one T allele had reduced odds of being HIV seropositive (OR=0.29, 95% CI=0.09-0.90). This association remained significant (OR=0.25, 95% CI=0.07-0.87) after the adjustment for other co-variates (HCV, HBV serostatus and duration of intravenous drug use). CONCLUSIONS The TLR3 rs3775291 T allele has a protective effect against HIV infection among HESNs IDUs.

Influence of interleukin 10 polymorphisms -592 and -1082 to the HIV, HBV and HCV serostatus among intravenous drug users

BACKGROUND Interleukin 10 (IL-10) is a multifunctional cytokine produced by macrophages, monocytes, and T-helper cells. Two polymorphisms at positions -592 and -1082 have been associated with HIV susceptibility. However, their associations with susceptibility to HIV and its co-infections among intravenous drug users (IDUs) are largely unknown. METHODS A total of 345 IDUs were recruited. Of the 173 HIV negative IDUs, 20 were classified as highly exposed HIV seronegative subjects (HESNs). A control group consisted of 496 blood donors; all HIV, HCV, and HBV negative. The IL-10-592C/A and -1082A/G were determined using TaqMan allelic discrimination assay. RESULTS Of the IDUs, 50% were HIV positive, 89% HCV positive, 67% HBV positive and 41% had triple infection. IL-10-592C allele and -1082A allele were the most common and the -1082AG/-592CC was the most common genotype pair. All HESNs exhibited -1082A allele as compared to 81.4% of the HIV positive IDUs and 79% of donors (p=0.029 and p=0.019, respectively). None of HESNs had GG/CC genotype pair compared with 18.6% of HIV positive IDUs and 21.0% of donors (p=0.029 and p=0.019, respectively). The possession of -592AC and genotype pair AG/AC were associated with the decreased odds of HBV infection (OR=0.28; 95% CI 0.09-0.87; p=0.028 and OR=0.19; 95% CI 0.06-0.61; p=0.052, respectively). CONCLUSIONS The presence of low producing IL-10-1082A and -592A alleles and their containing genetic variants protect highly exposed IDUs against acquisition of HIV and HBV infections.

The prevalence and genotypes of GBV‐C and its associations with HIV infection among persons who inject drugs in Eastern Europe

We aimed to determine the rate of GBV‐C viremia, seropositivity, and genotypes among people who inject drugs (PWID) and healthy volunteers in Estonia and to evaluate associations between GBV‐C and sociodemographic factors, intravenous drug use, co‐infections. The study included 345 Caucasian PWID and 118 healthy volunteers. The presence of GBV‐C RNA (viremia) was determined by reverse transcriptase‐nested PCR in 5′ long terminal repeat. PCR products were sequenced and genotyped by phylogenetic analysis. GBV‐C seropositivity was determined by ELISA. One third of PWID (114/345) and 6% (7/118) of healthy volunteers (OR = 7.8, 95% CI = 3.5–20.5, P < 0.001) were GBV‐C viremic. In PWID group, 79% of sequences belonged to subtype 2a, 19% to subtype 2b, and two remained unclassified. In healthy volunteers, six out of seven sequences belonged to subtype 2a and one to subtype 2b. We found HIV+ PWID to have two times increased odds of being GBV‐C viremic compared to HIV− PWID (62% vs. 38%; OR = 2.13, 95% CI = 1.34–3.36, P = 0.001). In addition, odds of being GBV‐C viremic decreased with increasing age (OR = 0.94, 95% CI = 0.90–0.98, P = 0.001). HIV positivity remained associated with GBV‐C viremia in multivariate analysis after adjustment for age (OR = 2.23, 95% CI = 1.39–3.58, P = 0.001). GBV‐C seropositivity was similar among PWID and healthy volunteers (2.3% vs. 1.7%, respectively; OR = 1.4, 95% CI =0.3–13.5, P = 1). In an Eastern European country we demonstrated that GBV‐C viremia is common among PWID, but uncommon among healthy volunteers, and GBV‐C seropositivity is infrequent among both groups. Similarly to other European countries and USA, GBV‐C 2a is the most common genotype in Estonia. J. Med. Virol. 89:632–638, 2017.

A CCL5 Haplotype Is Associated with Low Seropositivity Rate of HCV Infection in People Who Inject Drugs.

Objective The role of CC chemokine receptor 5 (CCR5) and its ligand CCL5 on the pathogenesis of HIV infection has been well studied but not for HCV infection. Here, we investigated whether CCL5 haplotypes influence HIV and HCV seropositivity among 373 Caucasian people who inject drugs (PWID) from Estonia. Methods Study included 373 PWID; 56% were HIV seropositive, 44% HCV seropositive and 47% co-infected. Four CCL5 haplotypes (A-D) were derived from three CCL5 polymorphisms (rs2107538/rs2280788/rs2280789) typed by Taqman allelic discrimination assays. The data of CCR5 haplotypes were used from our previous study. The association between CCL5 haplotypes with HIV and/or HCV seropositivity was determined using logistic regression analysis. Results Possessing CCL5 haplotype D (defined by rs2107538A/rs2280788G/rs2280789C) decreased the odds of HCV seropositivity compared to those not possessing it (OR = 0.19; 95% CI 0.09–0.40), which remained significant after adjustment to co-variates (OR = 0.08; 95% CI 0.02–0.29). An association of this haplotype with HIV seropositivity was not found. In step-wise logistic regression with backward elimination CCL5 haplotype D and CCR5 HHG*1 had reduced odds for HCV seropositivity (OR = 0.28 95% CI 0.09–0.92; OR = 0.23 95% CI 0.08–0.68, respectively) compared to those who did not possess these haplotypes, respectively. Conclusions Our results suggest that among PWID CCL5 haplotype D and CCR5 HHG*1 independently protects against HCV. Our findings highlight the importance of CCL5 genetic variability and CCL5-CCR5 axis on the susceptibility to HCV.

Prevalence of drug resistance mutations in HAART patients infected with HIV-1 CRF06_cpx in Estonia.

HIV‐1 drug resistance mutations (DRMs) and substitutions were assessed after the failure of the first line non‐nucleoside reverse transcriptase inhibitors (NNRTIs) + 2 nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) treatment regimens (efavirenz [EFV] + lamivudine[3TC] + zidovudine [ZDV] vs. EFV + 3TC + ddI) among the HIV‐1 CRF06_cpx infected subjects in Estonia. HIV‐1 genomic RNA was sequenced; DRMs and amino acid substitutions were compared in 44 treatment naïve and 45 first‐line NNRTI + 2 NRTI treatment failed patients consisting of EFV + 3TC + ZDV (n = 17) and EFV + 3TC + didanosine[ddI] (n = 21) therapy failed sub‐populations. At least one DRM was found in 78% of treatment experienced patients. The most common NRTI mutations were M184V (80%), L74V (31%), L74I (17%), K219E (9%), and M184I (9%), NNRTI mutations were K103N (83%), P225H (14%), L100I (11%), and Y188L (11%), reflecting generally the similar pattern of DRMs to that seen in treatment failed subtype B viruses. Sub‐population analysis revealed that EFV + 3TC + ddI failed patients had more DRMs compared to EFV + 3TC + ZDV failed patients, especially the ddI DRM L74IV and several additional NNRTI DRMs. Additionally, CRF06_cpx specific mutation E179V and substitutions R32K, K122E, and V200AE were also detected in treatment experienced population. After the failure of the first‐line EFV + 3TC + ddI therapy HIV‐1 CRF06_cpx viruses develop additional NRTI and NNRTI mutations compared to EFV + 3TC + ZDV regimen. Therefore the usage of EFV + 3TC + ddI in this subtype decreases the options for next regimens containing abacavir, and NNRTI class agents. J. Med. Virol. 88:???–???, 2016.

Human T-lymphotropic virus types 1 and 2 are rare among intravenous drug users in Eastern Europe.

BACKGROUND In Europe, human T-lymphotropic virus (HTLV) type 2 mainly occurs among intravenous drug users (IDUs) with prevalence up to 15% and HTLV-1 among general population with prevalence <1%. However, there is no data regarding the prevalence of HTLV-1 or HTLV-2 in Eastern European IDUs population where HIV prevalence is relatively high. We aimed to determine the prevalence and genotypes of HTLV-1/HTLV-2 among IDUs and healthy volunteers in Estonia. METHODS The study included 345 IDUs and 138 healthy volunteers. The presence of HTLV-1/HTLV-2 was determined by nested PCR; positive and negative controls were used in every PCR run. RESULTS The analysed IDUs resembled the IDUs of HIV epidemic in Estonia: mainly male (79%) with median age of 30years (interquartile range [IQR] 25-34), and prolonged duration of intravenous drug usage (11years; IQR 7-14). The prevalence exposure to blood-borne viral infections was high - 50% were HIV positive, 88% hepatitis C positive, 67% hepatitis B positive. Of IDUs, 64% reported receptive needle sharing in the past and 18% at least once a month during last six months. None of the IDUs carried HTLV-1 but there was a case of HTLV-2 (prevalence 0.3%; 95% CI 0.1-1.6). All healthy volunteers were HTLV-1 and HTLV-2 PCR negative. CONCLUSION This is the first study investigating the prevalence of HTLV-1/HTLV-2 among high risk population and healthy volunteers in Eastern European region. Our results suggest that despite other widely spread blood-borne infections (e.g. HIV, HBV, HCV) HTLV-1 and HTLV-2 are rare among IDUs in Estonia.

Antiretroviral drug resistance and viral tropism in HIV-1 CRF06_cpx infected patients failing antiretroviral (ARV) therapy

While the prevalence of X4-tropic viruses in subtype B is relatively well studied the distribution of tropism in CRF06_cpx (causing Estonian epidemic) is less known, especially in the treatment experienced patients with established multidrug-resistance and who are candidates for therapy with CCR5 antagonist. Aim: to describe HIV-1 drug resistance mutations (DRMs) and co-receptor tropism in antiretroviral (ARV) treatment failing patients infected with HIV-1 CRF06_cpx and to establish their suitability to treatment with CCR5 receptor antagonists. Altogether 12 patients were studied – all with ARV therapy failure and considered to start CCR5 antagonist therapy. Genomic viral DNA was directly sequenced in pol region and V3 loop of gp120 in triplicates. HIV-1 DRMs and tropism were detected using Stanford University Drug Resistance Database and Geno2pheno[coreceptor] 2.5. algorithm, respectively. All sequence prediction results with a false-positivity rate above 10% were considered R5-tropic. Of 12 viruses 11 were successfully sequenced and analyzed. Patients have been diagnosed and ARV therapy initiated in 2002-2013. 8/11 cases had failure of the second ARV regimen, 4/11 of the third and in 2/11 of the fourth ARV regimen. Median viral load at the time of tropism testing was 53,326 (IQR 16,328-113,018) and median CD4+ count 184 (IQR 142-217) cells/cmm. The most commonly used regimens were EFV + 3TC plus AZT (5 cases) or plus ABC (4 cases). Of 11 viruses 10 had resistance against two ARV classes (NRTI + NNRTI or NNRTI + INI) and 1 had triple class resistance (NRTI + NNRTI + PI). In NRTI treated population the most common primary DRM was M184V (10/11), followed by L74V/IL/I and K70E/R/EKQ (both 4/11). In NNRTI treated population DRMs were as follows: K103N (5/11), P225H (4/411), G190A/S and K101H/E (both 3/11), others were seen in lower frequency. In PI treated patients two primary DRMs were represented – I54V, V82A (both in same case). In INI treated population one primary DRM was represented – Y143R. The majority of viruses (10/11) were R5-tropic and all determined as CRF06_cpx by phylogenetic analysis. The Estonian HIV epidemic has reached the stage where the first patients are considered to start treatment with CCR5 antagonists. Almost all highly resistant viruses were R5-tropic suggesting that CCR5 antagonists are an appropriate option for CRF06_cpx viruses resistant to other ARV agents.

Association of IFNλ4 rs12979860 polymorphism with the acquisition of HCV and HIV infections among people who inject drugs: JÕGEDA et al.

We investigated the presence of a single‐nucleotide polymorphism designated rs12979860 in the interferon λ4 (IFNλ4) gene among 345 people who inject drugs (PWID) and 495 blood donors to evaluate associations between the rs12979860 genotypes and human immunodeficiency virus/hepatitis C virus (HIV/HCV). The rs12979860 TT genotype was over‐represented among HIV+ PWID than HIV− PWID and blood donors (16% vs 8% and 10%, P = 0.03, respectively). PWID with TT genotype had approximately twice the probability of being HIV+ (odds ratio [OR], 2.19; 95% confidence interval [CI], 1.11 to 4.33) than PWID without TT. Every additional year of intravenous drug use (IVDU) decreased the OR 1.16 times (OR, 0.86; 95% CI, 0.75 to 0.98). This suggests that rs12979860 TT increases susceptibility to HIV and this impact decreases with increasing duration of IVDU.

HTLV-1/2 is rare among Eastern-European intravenous drug users (IDUs)

Background HTLV-1/2 and HIV likely share the same transmission routes and thus in Europe HTLV occurs mainly among IDUs, interests in clinical implications of HTLV-1/2 coinfection on HIV disease progression have risen. So far the available evidence suggests a protective role of HTLV-2 and adverse effect of HTLV-1 on HIV infection. In Europe, the overall prevalence of HTLV infection is up to 18% among IDUs and less than 1% among blood donors. However, there is no data regarding the overall prevalence of HTLV-1/2 in Eastern-European IDU population where the HIV epidemic mainly lies. We aimed to determine the prevalence of HTLV among IDUs in Estonia and compare it with the rate in healthy volunteers.

Full-length genome sequences of Estonian HIV-1 CRF06 and Eastern-European subtype A1 recombinant forms

“Eastern-European type” of HIV-1 epidemic started among Estonian intravenous drug users (IDU) population in 2000. Unlike in other former Soviet Union countries the Estonian HIV-1 epidemic was not caused by subtype A1 viruses but recombinant form CRF06_cpx and its next generation recombinants with subtype A1. A high variety of different recombinant forms has also been described in other IDU driven HIV-1 epidemics in other countries. Aim: to sequence and describe the subtypic structure of near-full genome Estonian HIV-1 inter-subtype recombinant forms in Estonian IDU population. The study included plasma samples from 10 HIV positive subjects collected in 2000-2010. Samples were selected based on discordant subtypes or recombinant sequences in different genomic regions in recent Estonian studies. Patients’ viral genomic RNA was reverse transcribed, amplified, cloned and Sanger sequenced in four overlapping genomic regions. Phylogenetic trees were constructed using the Maximum-Likelihood method and recombination pattern was assessed using Simplot software (similarity and bootscanning blots). Drug resistance mutations and tropism were determined by Stanford HIV Drug Resistance Database and geno2pheno analysis tool using a false positive rate of 10%. According to bootscanning analysis the majority of strains (80%) indicated unique recombination structure between Estonian CRF06_cpx and Russian subtypes A1 sequences, whereas prevailing proportions of regions belong to the CRF06_cpx clade. Phylogenetic tree analysis confirmed these results indicating monophyletic clustering with Estonian CRF06_cpx or Russian subtype A1 sequences in corresponding regions. Of 10 sequences in one NNRTI DRM K103N and V179E was found and in 9 INI accessory mutations L74I was found. Of seven gag region sequences in three cases protease inhibitor associated substitution L449F, and in one case V128I, P452S and maturation inhibitor substitutions V370M were present. Of 9 env sequences one strain was R4 tropic, the others were R5 tropic. Analyzed sequences revealed high recombinational diversity of Estonian inter-subtype HIV-1 recombinant strains, which is also characteristic to other IDU HIV-1 epidemics. Nearly full-length genome sequences generated by this study provide reference material for high-throughput sequencing and for tracking the molecular epidemics in different risk groups and in Estonia and neighboring countries.