Development of a new comprehensive HIV-1 genotypic drug resistance assay for all commercially available reverse transcriptase, protease and integrase inhibitors in patients infected with group M HIV-1 strains.

Comprehensive PCR assays for the genotypic drug resistance analysis of all HIV-1 antiretroviral agents (reverse transcriptase, protease and integrase inhibitors) are increasingly in demand due to introduction of integrase inhibitors in the first line regimens and the increasing presence of non-B HIV-1 clades around the world. This study focused on the development and evaluation of a new PCR-based assay for the amplification and sequencing of the entire HIV-1 pol region of major circulating group M HIV-1 strains in Europe for genotypic drug resistance analysis. The comprehensive touchdown PCR assay developed in this study utilized HIV-1 RNA extracted from the plasma of blood samples of consenting HIV-1 infected patients in Cyprus, collected from 2017 to 2019. The HIV-1 pol region was amplified by touchdown PCR for both the primary RT-PCR and the secondary PCR steps. Successful PCR amplicons were determined by population DNA sequencing, using the Sanger method and the genotypic drug resistance analysis was performed with the Stanford University HIV Drug Resistance Database Program. The newly developed assay successfully amplified the entire HIV-1 pol region (2844 nucleotides long) of 141 out of 144 samples of group M HIV-1 subtypes and recombinant strains of the Cyprus HIV-1 Transmission Cohort Study (CHICS) isolated from 2017 to 2019 and genotypic analyses were conducted for all currently available HIV-1 reverse transcriptase, protease and integrase inhibitors. The drug resistance, epidemiological and demographic data of these study subjects will be expanded upon in the CHICS (L.G. Kostrikis et al., manuscript in preparation for publication). The newly developed HIV-1 genotypic drug resistance assay would benefit clinical settings, and research focusing on the world-wide spread of HIV-1 drug-resistant strains, especially in geographic regions characterized by polyphyletic HIV-1 infections.

HCV Phylogeography of the General Population and High-Risk Groups in Cyprus Identifies the Island as a Global Sink for and Source of Infection.

Hepatitis C virus (HCV) genotype and subtype distribution differs according to geographic origin and transmission risk category. Previous molecular epidemiology studies suggest the presence of multiple subtypes among Cypriot subjects. To investigate HCV genotype- and subtype-specific dissemination patterns, origins, and transmission in Cyprus, we analyzed HCV sequences encoding partial Core-E1 and NS5B regions. Analyzed populations comprised the general population and high-risk cohorts in Cyprus and a globally sampled dataset. Maximum-likelihood phylogeny reconstruction with bootstrap evaluation, character reconstruction using parsimony, and bootstrap trees estimated by ML were performed to identify the geographic origin of HCV subtypes and statistically significant dispersal pathways among geographic regions. Phylogeographic analyses traced the origin of subtypes in the general population and among PWID in Cyprus to unique and overlapping globally distributed regions. Phylogenetic analysis in Core-E1 revealed that most sequences from incarcerated populations in Cyprus clustered with the general population and PWID. We estimate that HCV infections in Cyprus originate from multiple global sources while most HCV transmissions among incarcerated individuals occur locally. This analysis is one of a few studies tracing HCV dispersal patterns using global datasets, and these practices and findings should inform how HCV epidemics are targeted by future prevention policies.

HIV-1 transmission networks across Cyprus (2010-2012).

A molecular epidemiology study of HIV-1 infection was conducted in one hundred diagnosed and untreated HIV-1-infected patients in Cyprus between 2010 and 2012, representing 65.4% of all the reported HIV-1 infections in Cyprus in this three-year period, using a previously defined enrolment strategy. Eighty-two patients were newly diagnosed (genotypic drug resistance testing within six months from diagnosis), and eighteen patients were HIV-1 diagnosed for a longer period or the diagnosis date was unknown. Phylogenetic trees of the pol sequences obtained in this study with reference sequences indicated that subtypes B and A1 were the most common subtypes present and accounted for 41.0 and 19.0% respectively, followed by subtype C (7.0%), F1 (8.0%), CRF02_AG (4.0%), A2 (2.0%), other circulating recombinant forms (CRFs) (7.0%) and unknown recombinant forms (URFs) (12%). Most of the newly-diagnosed study subjects were Cypriots (63%), males (78%) with median age 39 (Interquartile Range, IQR 33-48) reporting having sex with other men (MSM) (51%). A high rate of clustered transmission of subtype B drug-sensitive strains to reverse transcriptase and protease inhibitors was observed among MSM, twenty-eight out of forty-one MSM study subjects (68.0%) infected were implicated in five transmission clusters, two of which are sub-subtype A1 and three of which are subtype B strains. The two largest MSM subtype B clusters included nine and eight Cypriot men, respectively, living in all major cities in Cyprus. There were only three newly diagnosed patients with transmitted drug resistant HIV-1 strains, one study subject from the United Kingdom infected with subtype B strain and one from Romania with sub-subtype A2 strain, both with PI drug resistance mutation M46L and one from Greece with sub-subtype A1 with non-nucleoside reverse transcriptase inhibitors (NNRTI) drug resistance mutation K103N.

Cellular HIV type 1 DNA levels are equivalent among drug-sensitive and drug-resistant strains in newly diagnosed and antiretroviral naive patients.

The emergence of resistance against current antiretroviral drugs to human immunodeficiency virus type 1 (HIV-1) is an increasingly important concern to the continuous success of antiretroviral therapy to HIV-1-infected patients. In the past decade, a number of studies reported that the prevalence of transmitted drug resistance among newly diagnosed patients has reached an overall 9% prevalence worldwide. Also, a number of studies using longitudinal HIV-1 patient study cohorts demonstrated that the cellular HIV-1 DNA level in peripheral blood mononuclear cells (PBMCs) has a prognostic value for the progression of HIV-1 disease independently of plasma HIV-1 RNA load and CD4 count. Using a previously established molecular-beacon-based real-time PCR methodology, cellular HIV-1 DNA levels were quantified in newly diagnosed and antiretroviral-naive patients in Northern Greece recruited between 2009 and 2010 using a predefined enrolling strategy, in an effort to investigate whether there is any relationship between cellular HIV-1 DNA levels and HIV-1 transmitted drug resistance. As part of the same study, DNA sequences encoding the env (C2-C5 region of gp120) were also amplified from PBMC-extracted DNA in order to determine the genotypic coreceptor tropism and genetic subtype. Cellular HIV-1 DNA levels had a median of 3.309 log10 HIV-1 copies per 10(6) PBMCs and demonstrated no correlation between cellular HIV-1 DNA levels and HIV-1 transmitted drug resistance. An absence of association between cellular HIV-1 DNA levels with plasma viral HIV-1 RNA load and CD4 levels was also found reconfirming the previously published study. Genotypic analysis of coreceptor tropism indicated that 96% of samples, independently of the presence or not of genotypic drug resistance, were CCR5-tropic. Overall, the findings reconfirmed the previously proposed proposition that transmitted drug resistance does not have an impact on disease progression in HIV-1-infected individuals. Also, CCR5 coreceptor tropism dominance suggests that both drug-resistant and drug-sensitive strains behave similarly in early infection in newly diagnosed patients.

Molecular characterization of HIV type 1 strains from newly diagnosed patients in Cyprus (2007-2009) recovers multiple clades including unique recombinant strains and lack of transmitted drug resistance.

HIV-1 evolution generates substantial genetic diversity among isolates, the majority of which are represented in areas where multiple strains cocirculate. A heterogeneous genetic HIV-1 pool has been found in Cyprus, directing us to determine the dynamics of the local HIV-1 infection by characterizing strains isolated from 74 subjects during 2007-2009, representing 88% of the known-living HIV-1-infected population, of whom 53 are newly diagnosed therapy-naive patients and 21 are chronic patients, according to the European HIV Resistance guidelines. Near full-length genome sequences were amplified by RT-nested PCR using diluted RNA from all HIV-1 seropositives and sequenced using a newly designed assay. Resistant mutations were not found among the population of the newly diagnosed therapy-naive patients either to protease, reverse transcriptase, or integrase inhibitors. Phylogenetic analyses indicated subtype B as the main subtype (48.6%), followed by subtype A (18.9%), subtype C (10.8%), CRF02_AG (8.1%), CRF11_cpx (2.7%), and (sub)subtype F1 and CRF37_cpx (1.4% each). Six HIV-1 isolates (8.1%) were not classified in any pure (sub)subtype or circulating recombinant form (CRF). Complete phylogenetic and bootscanning analyses revealed that each isolate had a new, unique recombinant pattern and is distinct from all other CRFs or unique recombinant forms (URFs) reported so far. Two of the six isolates have the same mosaic pattern. Analogous to results of the earlier epidemiological studies, this study expands on the HIV-1 sequence database and reveals the high degree of diversity of HIV-1 infection in Cyprus.

Hepatitis C infection among intravenous drug users attending therapy programs in Cyprus.

The most high-risk population for HCV transmission worldwide today are intravenous drug users. HCV genotypes in the general population in Cyprus demonstrate a polyphyletic infection and include subtypes associated with intravenous drug users. The prevalence of HCV, HBV, and HIV infection, HCV genotypes and risk factors among intravenous drug users in Cyprus were investigated here for the first time. Blood samples and interviews were obtained from 40 consenting users in treatment centers, and were tested for HCV, HBV, and HIV antibodies. On the HCV-positive samples, viral RNA extraction, RT-PCR and sequencing were performed. Phylogenetic analysis determined subtype and any relationships with database sequences and statistical analysis determined any correlation of risk factors with HCV infection. The prevalence of HCV infection was 50%, but no HBV or HIV infections were found. Of the PCR-positive samples, eight (57%) were genotype 3a, and six (43%) were 1b. No other subtypes, recombinant strains or mixed infections were observed. The phylogenetic analysis of the injecting drug users' strains against database sequences observed no clustering, which does not allow determination of transmission route, possibly due to a limitation of sequences in the database. However, three clusters were discovered among the drug users' sequences, revealing small groups who possibly share injecting equipment. Statistical analysis showed the risk factor associated with HCV infection is drug use duration. Overall, the polyphyletic nature of HCV infection in Cyprus is confirmed, but the transmission route remains unknown. These findings highlight the need for harm-reduction strategies to reduce HCV transmission.

Use of molecular beacons and multi-allelic real-time PCR for detection of and discrimination between virulent Bacillus anthracis and other Bacillus isolates.

The awareness of the threat of Bacillus anthracis, the causative agent of the disease anthrax, as a biowarfare and bioterrorism weapon has revived the development of new technologies for rapid and accurate detection of virulent isolates in environmental and clinical samples. Here we explore the utility of molecular beacon real-time PCR technology for detection of virulent Bacillus anthracis strains. Molecular beacons are nucleic acid probes with high specificity, that act as switches by emitting fluorescence when bound to their nucleotide sequence targets by means of altering their conformation. In this study, five molecular beacons targeting Bacillus anthracis capA, capB, capC, lef, and pag alleles were designed and used in five uniplex assays. Another molecular beacon targeting the Bacillus group chromosomal 16s rRNA allele was designed for use in a duplex assay with an internal PCR amplification control. The molecular beacons were used in a real-time PCR assay for the detection of and differentiation between virulent B. anthracis and other members of the B. cereus group at the molecular level. Various B. anthracis samples as well as other bacterial and human samples were used to demonstrate the sensitivity and specificity of this assay. Use of the molecular beacon real-time PCR technology should accelerate current efforts to swiftly detect B. anthracis strains and its virulence plasmids in clinical and environmental samples and may extend to the development of additional molecular beacon-based assays for the identification of other pathogenic agents or the identification of B. anthracis directly from clinical samples.