Experiences of Stigma and Support Reported by Participants in a Network Intervention to Reduce HIV Transmission in Athens, Greece; Odessa, Ukraine; and Chicago, Illinois.

A growing body of evidence suggests that network-based interventions to reduce HIV transmission and/or improve HIV-related health outcomes have an important place in public health efforts to move towards 90-90-90 goals. However, the social processes involved in network-based recruitment may pose a risk to participants of increasing HIV-related stigma if network recruitment causes HIV status to be assumed, inferred, or disclosed. On the other hand, the social processes involved in network-based recruitment to HIV testing may also encourage HIV-related social support. Yet despite the relevance of these processes to both network-based interventions and to other more common interventions (e.g., partner services), there is a dearth of literature that directly examines them among participants of such interventions. Furthermore, both HIV-related stigma and social support may influence participants' willingness and ability to recruit their network members to the study. This paper examines (1) the extent to which stigma and support were experienced by participants in the Transmission Reduction Intervention Project (TRIP), a risk network-tracing intervention aimed at locating recently HIV-infected and/or undiagnosed HIV-infected people and linking them to care in Athens, Greece; Odessa, Ukraine; and Chicago, Illinois; and (2) whether stigma and support predicted participant engagement in the intervention. Overall, experiences of stigma were infrequent and experiences of support frequent, with significant variation between study sites. Experiences and perceptions of HIV-related stigma did not change significantly between baseline and six-month follow-up for the full TRIP sample, and significantly decreased during the course of the study at the Chicago site. Experiences of HIV-related support significantly increased among recently-HIV-infected participants at all sites, and among all participants at the Odessa site. Both stigma and support were found to predict participants' recruitment of network members to the study at the Athens site, and to predict participants' interviewer-rated enthusiasm for naming and recruiting their network members at both the Athens and Odessa sites. These findings suggest that network-based interventions like TRIP which aim to reduce HIV transmission likely do not increase stigma-related risks to participants, and may even encourage increased social support among network members. However, the present study is limited by its associational design and by some variation in implementation by study site. Future research should directly assess contextual differences to improve understanding of the implications of site-level variation in stigma and support for the implementation of network-based interventions, given the finding that these constructs predict participants' recruitment of network members and engagement in the intervention, and thereby could limit network-based interventions' abilities to reach those most in need of HIV testing and care.

Re-analysis of unassigned hepatitis C virus (HCV) strain CYHCV025: Evidence of a highly divergent lineage within genotype 1.

HCV global sequences have been classified into 7 genotypes, several subtypes and a number of unassigned sequences. Our aim was to perform an in depth investigation of the taxonomic relationships of the unclassified CYHCV025 strain by means of phylogenetic analysis. Phylogenetic tree reconstructions were performed using ML methods on full-length genomic and partial HCV alignments. Phylogenetic analysis of full-length sequences revealed that CYHCV025 clustered close to the root node of genotype 1, showing distant genetic relationships to all previously classified subtypes and unclassified sequences. Single section analysis using the SSE showed that the distances between the query and all subtypes were much higher than distances within and between subtypes of genotype 1 (p<0.05). Recombination analysis revealed no evidence for intersubtype or genetic mixing between the query and the references from different genotype 1 subtypes. Different analyses revealed that CYHCV025 is the most genetically divergent within genotype 1, showing no high- or low-level clustering with any of the previous subtypes or unclassified sequences. Identification of a single lineage within a genotype without any late branching can be explained by "genetic isolation" until the late stage of HCV epidemic spread.

Evaluation of the limiting antigen avidity EIA (LAg) in people who inject drugs in Greece.

This analysis assessed the utility of the limiting antigen avidity assay (LAg). Samples of people who inject drugs (PWID) in Greece with documented duration of HIV-1 infection were tested by LAg. A LAg-normalized optical density (ODn) ⩽1·5 corresponds to a recency window period of 130 days. The proportion true recent (PTR) and proportion false recent (PFR) were estimated in 28 seroconverters and in 366 samples collected >6 months after HIV diagnosis, respectively. The association between LAg ODn and HIV RNA level was evaluated in 232 persons. The PTR was 85·7%. The PFR was 20·8% but fell to 5·9% in samples from treatment-naive individuals with long-standing infection (>1 year), and to 0 in samples with the circulating recombinant form CRF35 AD. A LAg-based algorithm with a PFR of 3·3% estimated a similar incidence trend to that calculated by analyses based on HIV-1 seroconversions. In recently infected persons indicated by LAg, the median log10 HIV RNA level was high (5·30, interquartile range 4·56-5·90). LAg can help identify highly infectious HIV(+) individuals as it accurately identifies recent infections and is correlated with the HIV RNA level. It can also produce reliable estimates of HIV-1 incidence.

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.

Renal transplantation from hepatitis B surface antigen (HBsAg)-positive donors to HBsAg-negative recipients: a case of post-transplant fulminant hepatitis associated with an extensively mutated hepatitis B virus strain and review of the current literature.

PURPOSE: The purpose of this study was to present a fatal case of fulminant hepatitis B (FHB) that developed in a renal transplant recipient, immunized against hepatitis B, 1 year post transplantation. METHODS: Polymerase chain reaction amplification and full genome sequencing were performed to investigate whether specific mutations were associated with hepatitis B virus (HBV) transmission and FHB. RESULTS: Molecular analysis revealed multiple mutations in various open reading frames of HBV, the most important being the G145R escape mutation and a frameshift mutation-insertion (1838insA) within the pre-C/C reading frame. CONCLUSIONS: Our results highlight the possibility of developing FHB, despite previous immunization against HBV or administration of hyperimmune gammaglobulin, because of the selection of escape virus mutants. The current literature and guidelines regarding renal transplantation from hepatitis B surface antigen (HBsAg)-positive to HBsAg-negative patients were also reviewed.

Relating SARS-CoV-2 shedding rate in wastewater to daily positive tests data: A consistent model based approach.

During the COVID-19 pandemic, wastewater-based epidemiology (WBE) has been engaged to complement medical surveillance and in some cases to also act as an early diagnosis indicator of viral spreading in the community. Most efforts worldwide by the scientific community and commercial companies focus on the formulation of protocols for SARS-CoV-2 analysis in wastewater and approaches addressing the quantitative relationship between WBE and medical surveillance are lacking. In the present study, a mathematical model is developed which uses as input the number of daily positive medical tests together with the highly non-linear shedding rate curve of individuals to estimate the evolution of global virus shedding rate in wastewater along calendar days. A comprehensive parametric study by the model using as input actual medical surveillance and WBE data for the city of Thessaloniki (~700,000 inhabitants, North Greece) during the outbreak of November 2020 reveals the conditions under which WBE can be used as an early warning tool for predicting pandemic outbreaks. It is shown that early warning capacity is different along the days of an outbreak and depends strongly on the number of days apart between the day of maximum shedding rate of infected individuals in their disease cycle and the day of their medical testing. The present data indicate for Thessaloniki an average early warning capacity of around 2 days. Moreover, the data imply that there exists a proportion between unreported cases (asymptomatic persons with mild symptoms that do not seek medical advice) and reported cases. The proportion increases with the number of reported cases. The early detection capacity of WBE improves substantially in the presence of an increasing number of unreported cases. For Thessaloniki at the peak of the pandemic in mid-November 2020, the number of unreported cases reached a maximum around 4 times the number of reported cases.

HIV-1 outbreak among injecting drug users in Greece, 2011: a preliminary report.

A significant increase (more than 10-fold) in the number of newly diagnosed HIV-1 infections among injecting drug users (IDUs) was observed in Greece during the first seven months of 2011. Molecular epidemiology results revealed that a large proportion (96%) of HIV-1 sequences from IDUs sampled in 2011 fall within phylogenetic clusters suggesting high levels of transmission networking. Cases originated from diverse places outside Greece supporting the potential role of immigrant IDUs in the initiation of this outbreak.

Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event.

BACKGROUND: A novel coronavirus (2019-nCoV) associated with human to human transmission and severe human infection has been recently reported from the city of Wuhan in China. Our objectives were to characterize the genetic relationships of the 2019-nCoV and to search for putative recombination within the subgenus of sarbecovirus. METHODS: Putative recombination was investigated by RDP4 and Simplot v3.5.1 and discordant phylogenetic clustering in individual genomic fragments was confirmed by phylogenetic analysis using maximum likelihood and Bayesian methods. RESULTS: Our analysis suggests that the 2019-nCoV although closely related to BatCoV RaTG13 sequence throughout the genome (sequence similarity 96.3%), shows discordant clustering with the Bat_SARS-like coronavirus sequences. Specifically, in the 5'-part spanning the first 11,498 nucleotides and the last 3'-part spanning 24,341-30,696 positions, 2019-nCoV and RaTG13 formed a single cluster with Bat_SARS-like coronavirus sequences, whereas in the middle region spanning the 3'-end of ORF1a, the ORF1b and almost half of the spike regions, 2019-nCoV and RaTG13 grouped in a separate distant lineage within the sarbecovirus branch. CONCLUSIONS: The levels of genetic similarity between the 2019-nCoV and RaTG13 suggest that the latter does not provide the exact variant that caused the outbreak in humans, but the hypothesis that 2019-nCoV has originated from bats is very likely. We show evidence that the novel coronavirus (2019-nCov) is not-mosaic consisting in almost half of its genome of a distinct lineage within the betacoronavirus. These genomic features and their potential association with virus characteristics and virulence in humans need further attention.

Development and assessment of a multiplex real-time PCR assay for quantification of human immunodeficiency virus type 1 DNA.

Previous studies showed that high levels of human immunodeficiency virus type 1 (HIV-1) DNA are associated with a faster progression to AIDS, an increased risk of death, and a higher risk of HIV RNA rebound in patients on highly active antiretroviral therapy. Our objective was to develop and assess a highly sensitive real-time multiplex PCR assay for the quantification of HIV-1 DNA (RTMP-HIV) based on molecular beacons. HIV-1 DNA quantification was carried out by RTMP in a LightCycler 2.0 apparatus. HIV-1 DNA was quantified in parallel with CCR5 as a reference gene, and reported values are numbers of HIV-1 DNA copies/10(6) peripheral blood mononuclear cells (PBMCs). The clinical sensitivity of the assay was assessed for 115 newly diagnosed HIV-1-infected individuals. The analytical sensitivity was estimated to be 12.5 copies of HIV-1 DNA per 10(6) PBMCs, while the clinical sensitivity was 100%, with levels ranging from 1.23 to 4.25 log(10) HIV-1 DNA copies/10(6) PBMCs. In conclusion, we developed and assessed a new RTMP-HIV assay based on molecular beacons, using a LightCycler 2.0 instrument. This multiplex assay has comparable sensitivity, reproducibility, and accuracy to single real-time PCR assays.

Quantitative detection of the M204V hepatitis B virus minor variants by amplification refractory mutation system real-time PCR combined with molecular beacon technology.

Mutations in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif are frequently associated with resistance to antivirals and represent a major concern in the treatment of hepatitis B virus (HBV) infection. Conventional methods fail to detect minority populations of drug-resistant viral quasispecies if they represent less than 25% of the total sample virus population. The amplification refractory mutation system real-time PCR (ARMS RT-PCR) was combined with molecular beacon technology using the LightCycler system. The samples from HBV patients selected for assay evaluation included (i) 57 samples from treatment-naïve patients for biological discriminatory ability (cutoff) estimation, (ii) 12 samples from patients with treatment failure that were M204V positive by sequencing, and (iii) 13 samples from patients with treatment failure that were negative for mutation at codon 204 by sequencing. The discriminatory ability of the assay was 0.25% when tested with laboratory-synthesized DNA target sequences. The median mutant-to-wild-type ratio for samples from naive patients tested positive for the wild type and for mutant variants was 0.01% (5th and 95th percentiles = 0.0001 and 0.04%, respectively). A value of 0.04% was selected as the biological cutoff of the assay of clinical samples. In all samples M204V positive by sequencing (12/12), the mutant variant was detected as the predominant population (range, 82.76 to 99.43%). Interestingly, in 5 (38%) of 13 samples negative by sequencing, the M204V variant was detected at a ratio above the biological cutoff (0.05 to 28%). The assay represents an efficient technique for the early detection and quantification of M204V variants before mutant strains emerge to dominate the population.

The phylogenetic information profile of HIV-1 and the degradation effect of recombination.

HIV-1, while known to recombine frequently and evolve rapidly, is one of the most sequenced organisms. The availability of many and long sequences (almost full-length) renders HIV-1 as a good model for studying theoretical predictions linked to evolution and phylogenetic inferences. Here we study the effects of rapid and through-recombination evolution on phylogenetic information in order to confirm theoretical predictions of the characteristics of phylogenetic information on a real dataset. Firstly we study the fluctuation of the phylogenetic information along the HIV-1 genome showing that genomic regions such as the first part and the last part of the pol gene contain less phylogenetic information, while the vpr, vpu and the first exon of the tat gene contain more phylogenetic information compared to the rest of the genome. Moreover, we provide evidence that phylogenetic information is correlated to the sequence similarity of the dataset used and is degraded by the effect of recombination.

Analysing the evolutionary history of HCV: puzzle of ancient phylogenetic discordance.

Though recombination is an important evolutionary strategy in RNA viruses, only two cases of HCV recombinant strains have been reported. Our objective was to analyze the evolutionary history of the HCV genotypes aiming to obtain evidence of significant phylogenetic discordance due to either recombination or selective forces leading to convergent/divergent evolution. The data support an evolutionary preservation of the interferon-resistance related genomic region (ISDR) for the genotypes 1 and 4. On the other hand, there was no evidence that recombination has occurred in the past with the possible exception of genotype 4. Moreover, it is evidenced that genotypes 3 and 10 split more recently than genotypes 6-9 and 11. This analysis reverberates a commonly found pattern in rapidly evolving viruses, that is the strongly disturbed evolutionary history which deforms the uniform distribution of the phylogenetic relationships across the genome, and introduces a conservative inference framework for approaching this kind of data.

Prevalence of drug resistance among HIV-1 treatment-naive patients in Greece during 2003-2015: Transmitted drug resistance is due to onward transmissions.

BACKGROUND: The prevalence of HIV-1 drug resistance among treatment-naïve patients ranges between 8.3% and 15% in Europe and North America. Previous studies showed that subtypes A and B were the most prevalent in the Greek HIV-1 epidemic. Our aim was to estimate the prevalence of resistance among drug naïve patients in Greece and to investigate the levels of transmission networking among those carrying resistant strains. METHODS: HIV-1 sequences were determined from 3428 drug naïve HIV-1 patients, in Greece sampled during 01/01/2003-30/6/2015. Transmission clusters were estimated by means of phylogenetic analysis including as references sequences from patients failing antiretroviral treatment in Greece and sequences sampled globally. RESULTS: The proportion of sequences with SDRMs was 5.98% (n=205). The most prevalent SDRMs were found for NNRTIs (3.76%), followed by N(t)RTIs (2.28%) and PIs (1.02%). The resistance prevalence was 22.2% based on all mutations associated with resistance estimated using the HIVdb resistance interpretation algorithm. Resistance to NNRTIs was the most common (16.9%) followed by PIs (4.9%) and N(t)RTIs (2.8%). The most frequently observed NNRTI resistant mutations were E138A (7.7%), E138Q (4.0%), K103N (2.3%) and V179D (1.3%). The majority of subtype A sequences (89.7%; 245 out of 273) with the dominant NNRTI resistance mutations (E138A, K103N, E138Q, V179D) were found to belong to monophyletic clusters suggesting regional dispersal. For subtype B, 68.1% (139 out of 204) of resistant strains (E138A, K103N, E138Q V179D) belonged to clusters. For N(t)RTI-resistance, evidence for regional dispersal was found for 27.3% and 21.6% of subtype A and B sequences, respectively. CONCLUSIONS: The TDR rate based on the prevalence of SDRM is lower than the average rate in Europe. However, the prevalence of NNRTI resistance estimated using the HIVdb approach, is high in Greece and it is mostly due to onward transmissions among drug-naïve patients.

The application of HIV molecular epidemiology to public health.

HIV is responsible for one of the largest viral pandemics in human history. Despite a concerted global response for prevention and treatment, the virus persists. Thus, urgent public health action, utilizing novel interventions, is needed to prevent future transmission events, critical to eliminating HIV. For public health planning to prove effective and successful, we need to understand the dynamics of regional epidemics and to intervene appropriately. HIV molecular epidemiology tools as implemented in phylogenetic, phylodynamic and phylogeographic analyses have proven to be powerful tools in public health planning across many studies. Numerous applications with HIV suggest that molecular methods alone or in combination with mathematical modelling can provide inferences about the transmission dynamics, critical epidemiological parameters (prevalence, incidence, effective number of infections, Re, generation times, time between infection and diagnosis), or the spatiotemporal characteristics of epidemics. Molecular tools have been used to assess the impact of an intervention and outbreak investigation which are of great public health relevance. In some settings, molecular sequence data may be more readily available than HIV surveillance data, and can therefore allow for molecular analyses to be conducted more easily. Nonetheless, classic methods have an integral role in monitoring and evaluation of public health programmes, and should supplement emerging techniques from the field of molecular epidemiology. Importantly, molecular epidemiology remains a promising approach in responding to viral diseases.

Prevalence of resistance-associated mutations in newly diagnosed HIV-1 patients in Greece.

The prevalence of HIV-1 drug resistance mutations in naïve patients has been previously shown to differ greatly with the geographic origin. The purpose of this study was to prospectively estimate the prevalence of HIV-1 drug resistance in Greece by analyzing a representative sample of newly HIV-1 diagnosed patients, as part of the SPREAD collaborative study. Protease (PR) and partial reverse transcriptase (RT) sequences were determined from 101 newly diagnosed HIV-1 patients, in Greece, during the period September 2002--August 2003, representing one-third of the total newly diagnosed HIV-1 patients in the same time period. The prevalence of HIV-1 drug resistance was estimated according to the IAS-USA mutation table taking into account all mutations in RT and only major mutations in PR region. The overall prevalence of resistance was 9% [95% confidence interval (CI): 4.2--16.2%]. The prevalence of mutations associated with resistance to NRTIs was 5% (95% CI: 1.6--11.2%), for NNRTIs was 4% (95% CI: 1.1--9.8%), while no major resistance mutations were found in PR. No multi-class resistance was detected in the study population. The prevalence of resistant mutations in the recent seroconverters was 22%. For two individuals, there was clear evidence for transmitted resistance based on epidemiological information for a known source of HIV-1 transmission. The prevalence of the HIV-1 non-B subtypes and recombinants was 52%.

Analysis of complex HIV-1 intersubtype recombinants using a Bayesian scanning method.

The increased complexity of HIV-1 genetic heterogeneity raises the issue for reliable classification and analysis of these sequences. Until now, bootscanning analysis has been the main method used for the analysis of potential HIV-1 intersubtype recombinants. We show evidence that in some cases of complex recombinants, where three or more segments with discordant phylogenetic signal may exist in protease (PR) and partial reverse transcriptase (RT) region, Bayesian scanning provides a clearer picture than bootscanning plots about the boundaries of potential recombination. Thus, a recently developed Bayesian scanning tool can facilitate the analysis and classification of HIV-1 mosaic sequences.

Re-analysis of 34 full-length HIV-1 intersubtype recombinant sequences.

One of the main characteristics of the HIV-1 is its extensive genetic heterogeneity. Intersubtype recombination was first described in 1995 and since then a significant proportion of the HIV-1 isolates was found to comprise mosaic sequences. Re-analysis of 34 full-length HIV-1 intersubtype recombinants, including all "pure" HIV-1 subtypes revealed that 19 of the 34 analyzed mosaics consist of a more complex mosaic pattern than initially described. These findings indicate that the complexity of the HIV-1 recombinants is much greater than previously estimated.

HIV-1 genetic variants circulation in the North of Angola.

Few molecular epidemiological data on HIV-1 in Angola are available. In this study, we analysed 37 pol sequences from patients originated from Luanda and Cabinda in Angola. It was our objective to investigate the circulation of different HIV-1 subtypes in this country. We found a high HIV-1 genetic diversity. The predominant subtypes were C and F, while subtypes A, D, G and H were also detected. Three sequences were untypable and may possibly belong to new subtypes or recombinants of unknown subtypes. Moreover, 13 recombinant sequences were found, most of them with very complex patterns including untypable fragments.

Low-dose IFN-alpha monotherapy in treatment-naive individuals with HIV-1 infection: evidence of potent suppression of viral replication.

To evaluate the safety and antiviral action of interferon-alpha (IFN-alpha) in HIV-1 infection, we undertook a proof of concept study in 27 treatment-naive patients. Eligible patients comprised two groups: the IFN-alphaT group (n = 17), which received 5 MIU IFN-alpha s.c. daily for 32 consecutive days, and the IFN-alphaNT group (n = 10), which did not receive IFN-alpha prior to highly active antiretroviral therapy (HAART), which was commenced on day 28 in both groups. IFN-alphaTreatment was well tolerated in 14 of the 17 patients of the IFN-alphaT group who completed the study. The mean HIV RNA reduction in the IFN-alphaT group on day 14 was 1.1 log(10). Viral load suppression was inversely associated with baseline viral load (p = 0.031). Four weeks after initiation of HAART, IFN-alphaT and IFN-alphaNT group patients had 2.40 and 1.82 log(10) HIV RNA reduction from baseline, respectively (p < 0.001). There was no evidence of cross-resistance with existing antiretrovirals in patients with HIV-RNA rebound after initial plasma viral load decline > or = 1 log(10) during IFN-alpha monotherapy. Thus, low daily IFN-alpha exhibits potent anti-HIV-1 activity in vivo without serious adverse effects. These properties render IFN-alpha an attractive candidate for further assessment as a constituent of HAART.

Mutations associated with genotypic resistance to antiretroviral therapy in treatment naïve HIV-1 infected patients in Greece.

The widespread use of antiviral drugs against HIV has increased the prevalence of HIV-1 resistant strains among naïve individuals due to transmission of resistant strains. The purpose of this study was to investigate the presence of HIV-1 strains harboring resistance mutations in naïve patients in Greece. Blood samples were collected from 25 individuals. The DNA sequence of protease and partial reverse transcriptase regions (codons 41-223) were obtained by direct sequencing. Our results showed the absence of any primary resistance mutations in the study population. However, we were able to identify high prevalence of sequence polymorphisms at positions in reverse transcriptase region associated mainly with resistance to NNRTIs. Moreover, in protease region several secondary mutations were detected, suggesting the higher genetic variability of this region. The clinical significance of the polymorphisms associated with reduced susceptibility to NNRTIs remains to be clarified.