Characterization of HIV-1 subtypes and drug resistance mutations in Slovakia: update 2017-2018.

OBJECTIVES: This study aimed to provide an overview on the HIV-1 subtypes circulating in Slovakia between 2017 and 2018 and to evaluate the risk of transmission of HIV­resistant strains. BACKGROUND: The HIV epidemic in Slovakia is characterised by low incidence of new and pre-existing infections and a slightly elevated level of strain heterogeneity. METHODS: Partial HIV pol gene sequences of 110 individuals newly diagnosed with HIV between 2017 and 2018 were analysed. RESULTS: The genotypic analysis revealed sporadic occurrence of mutations linked to HIV resistance to antiretroviral therapy (ART). The HIV-1 B subtype has been found as predominant (84.55 %) and primarily linked to men who have sex with men (MSM). A total of eighteen individuals (15.45 %) were found to be infected with HIV-1 non-B subtypes. CONCLUSION: The data suggest a minimal risk of a resistant HIV strain transmission and a marginal rise of HIV-1 subtypes´ diversity. The HIV-1 B subtype remains the most prevalent in the period 2017-2018 in Slovakia (Tab. 2, Fig. 2, Ref. 37).

Integration of community-based testing data into national HIV surveillance in Poland, Serbia and Slovakia within the framework of INTEGRATE project.

BACKGROUND: Community-based voluntary counselling and testing contributes to early HIV diagnoses among key populations. Testing data from such decentralized services is however often not standardized nor linked to national surveillance systems. This study aimed to support the integration of community testing data into respective national surveillance and monitoring and evaluation systems for those infections. We present results from three national pilots, focused on improved data collection and transfer. METHODS:  Within the Joint Action INTEGRATE different pilot activities were planned and implemented according to the local context. In Slovakia, standardised data collection tools were implemented in three community testing services. The data generated was used to calculate the proposed indicators. In Poland, positive test results from the community testing database were linked to the national case-based surveillance database using confirmatory test number, to improve the completeness of behavioural data in the national database. In Serbia, voluntary counselling and testing forms were improved enabling identification of community-based testing. A system to generate unique client identifiers was initiated in the National registry of HIV cases to monitor linkage to care. RESULTS: All three sites were able to estimate most of the agreed indicators. In Slovakia during the study period 675 people were tested for HIV, 410 for hepatitis C and 457 for syphilis, with reactivity rates of 0.4, 2.5 and 1.8%, respectively. For HIV, 66.7% of reactive cases were confirmed and linked to care. In Poland, 28.9% of the community testing sites' records were linked to the national surveillance database (and accounted for 14.3% of all new diagnoses registered here during 2017-2018). Reactivity rate ranged between 1.9% and 2.1%. In Serbia, 80 persons were tested at community sites, from which two had a reactive HIV test result. By linking unique client identifiers from voluntary counselling and testing and National Registry of HIV cases databases, linkage to care within a two-month period was observed for one of two people with reactive HIV test result. CONCLUSIONS:  Pilot activities in the three countries demonstrate that integration of community-based testing data into surveillance systems is feasible and can help improve national surveillance data by providing key information.

HIV and risk behaviour among men who have sex with men in Slovakia (2008-2009).

The HIV infection remains a major public health issue in the world and especially in those countries where there exists unprotected sexual intercourse between people of the same sex. It is namely the category of men having sex with men (MSM) that still represents a group at high risk of being infected with HIV or transmitting the virus. The aim of our study was to present HIV 2nd generation surveillance data on MSM in Slovakia in the period between the years 2008 and 2009. Time-location sampling (TLS) was used to recruit participants and oral fluid samples together with completed anonymous questionnaires were collected simultaneously. The oral fluids were tested with use of the Genscreen HIV ½ version 2, (Bio-Rad) and Western Blot (Genlabs) diagnostic kits. The data analysis was performed using Stata version 8. Saliva testing revealed HIV prevalence of 6.1% (21/349) among MSM in Slovakia. 75% of HIV-positive cases were undiagnosed. The high-risk behaviour of MSM in respect to the number of their sexual partners as well as drug and condom usage did not correspond with their relatively high knowledge about HIV/AIDS. People's attitude towards MSM was found to be the worst in the religious context. Though our results reflect a relative good knowledge of MSM about HIV/AIDS infection, new HIV-positive cases and high-risk behaviour still appear, suggesting the need for more effective HIV prevention among members of this high-risk group in Slovakia.

KNQ1, a Kluyveromyces lactis gene encoding a drug efflux permease.

Several transport systems play an important role in conferring multiple drug resistance, presumably due to their catalysis of the energy-dependent extrusion of a large number of structurally and functionally unrelated compounds out of the cells. In the present work, the gene named KNQ1 (encoding Kluyveromyces lactis membrane permease) was cloned by functional complementation of the cycloheximide-hypersensitivity phenotype of the Saccharomyces cerevisiae mutant strain lacking a functional PDR5 gene. The isolated gene exhibited 48.9% identity with the S. cerevisiae ATR1 gene conferring resistance to aminotriazole and 4-nitroquinoline- N-oxide and encoded a protein of 553 amino acids. When present in multicopy, it efficiently complemented the phenotype associated with the Delta pdr5 or Delta pdr1Delta pdr3 mutations in S. cerevisiae. Overexpression of the KNQ1 gene in K. lactis wild-type strains led to resistance against several cytotoxic compounds, like 4-nitroquinoline- N-oxide, 3-aminotriazole, bifonazole and ketoconazole. The gene was assigned to K. lactis chromosome III and its expression was found to be responsive to oxidative stress induced by hydrogen peroxide. Based on the phenotype of homologous and heterologous transformants, we propose that the gene encodes a membrane-associated component of the machinery responsible for decreasing the concentration of several toxic compounds in the cytoplasm of yeast cells.

Isolation, heterological cloning and sequencing of the RPL28 gene in Kluyveromyces lactis.

By virtue of heterologous functional complementation of the Saccharomyces cerevisiae Delta pdr5 mutant strain, using a Kluyveromyces lactis genomic library, three different K. lactis chromosomal inserts were obtained. Transformation of the S. cerevisiae Delta pdr1 Delta pdr3 mutant strain, hypersensitive to drugs, with isolated plasmids resulted in resistance to cycloheximide and fluconazole. Transformation of K. lactis host strains, using the cloned chromosomal fragments, led to an increased level of resistance to some mitochondrial inhibitors and azole antifungals. The nucleotide sequence of the cloned inserts revealed that two of them contain the drug efflux transporter gene Kl-PDR5 and the third contains a DNA segment homologous to chromosome VII of S. cerevisiae. Along with three novel ORFs, encoding two proteins of unknown molecular function and one putative hexose transporter, this segment also contained the Kl-RPL28 gene, found to be responsible for the cycloheximide resistance of heterologous transformants. This gene codes for the large subunit ribosomal protein (149 amino acids) that shares 89.9% identity with its S. cerevisiae counterpart. The coding region of Kl-RPL28 was found to be interrupted with one intron near the 5' end. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the accession number AF493565.