BG Recombinants Research Articles

Recent HIV-1 Outbreak Among Intravenous Drug Users in Romania: Evidence for Cocirculation of CRF14_BG and Subtype F1 Strains.

Since 2011, Romania has faced an HIV outbreak among injecting drug users (IDUs). Our aim was to identify and describe clinical and epidemiological patterns of this outbreak. A cross-sectional study enrolled 138 IDUs diagnosed with HIV infection between 2011 and 2013 with 58 sexually infected individuals included as the control group. The IDUs had a long history of heroin abuse (10 years) and a recent history of new psychostimulant injection (3-4 years). Classical epidemiological data and molecular techniques were used to describe the transmission dynamics. A high prevalence of hepatitis C virus (HCV) coinfection was noted (98.6%) compared to the control group (10.3%) (p<0.001). IDUs had initially been infected with HCV. HIV infection was more recent, linked to starting injecting stimulants. HIV subtype analysis showed a predominance of the local F1 strain in both IDUs and sexually infected patients; in IDUs it also identified 28 CRF14_BG recombinants and six unique recombinant forms (URFs) between F1 and CRF14_BG. A few patients from both risk groups were infected with subtype B. Among IDUs, CRF14_BG was associated with a lower CD4 cell count and more advanced stages of disease, which correlated with CXCR4 tropism. Phylogenetic analysis revealed the spread of HIV through three major IDU clusters of recent date. Among IDUs with CRF14_BG, some reported travel abroad (Spain, Greece). By identifying clusters of IDUs with related viruses, molecular epidemiologic methods provide valuable information on patterns of HIV transmission that can be useful in planning appropriate harm reduction interventions.

High frequency of antiviral drug resistance and non-b subtypes in HIV-1 patients failing antiviral therapy in Cuba.

IntroductionEmergence of HIV-1 drug resistance may limit the sustained benefits of antiretroviral therapy (ART) in settings with limited laboratory monitoring and drug options. The objective is to implement the surveillance of drug resistance and subtypes in HIV-1 patients failing ART in Cuba.MethodsThis study compiled clinical and genotypic drug resistance data 588 ART-experienced HIV-1 patients attending a clinical center in Havana in 2009–2013. Drug resistance testing was performed as part of routine clinical care. Drug resistance mutations and levels were determined using Rega version 8.0.2.ResultsEighty-three percent received solely ART containing at least three drugs. Patients from 2009 to 2010 were longer treated (median: 4.9 vs 2.7 years) and exposed to more ART regimens (median: 4 vs 2 regimens) compared to patients from 2011–2013. Nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside RTI (NNRTI) and PI mutations were present in 83.5, 77.4 and 52.0%. Full-class resistance (FCR) to NRTI, NNRTI, PI and multidrug resistance (MDR) were detected in 25.0, 33.7, 11.4 and 6.3%. FCR to NRTI, NNRTI, PI and MDR were present in 12.8, 28.7, 0 and 0% after first-line failure (164 patients) and in 23.1, 34.6, 3.8 and 3.1% after second-line failure (130 patients). Subtype B (32.5%), BG recombinants (19.6%) and CRF19_cpx (16.2%) were the most prevalent genetic forms. Subtype distribution did not change significantly between 2009–2010 and 2011–2013, except for BG recombinants that increased from 12.2 to 21.3% (p=0.002).ConclusionsOur study found a high prevalence of drug resistance and supports the need for appropriate laboratory monitoring in clinical practice and access to drug options in case of virological failure.

Recombination pattern reanalysis of some HIV-1 circulating recombination forms suggest the necessity and difficulty of revision.

BackgroundRecombination is one of the major mechanisms underlying the generation of HIV-1 variability. Currently 61 circulating recombinant forms of HIV-1 have been identified. With the development of recombination detection techniques and accumulation of HIV-1 reference stains, more accurate mosaic structures of circulating recombinant forms (CRFs), like CRF04 and CRF06, have undergone repeated analysis and upgrades. Such revisions may also be necessary for other CRFs. Unlike previous studies, whose results are based primarily on a single recombination detection program, the current study was based on multiple recombination analysis, which may have produced more impartial results.MethodsRepresentative references of 3 categories of intersubtype recombinants were selected, including BC recombinants (CRF07 and CRF08), BG recombinants (CRF23 and CRF24), and BF recombinants (CRF38 and CRF44). They were reanalyzed in detail using both the jumping profile hidden Markov model and RDP3.ResultsThe results indicate that revisions and upgrades are very necessary and the entire re-analysis suggested 2 types of revision: (i) length of inserted fragments; and (ii) number of inserted fragments. The reanalysis also indicated that determination of small regions of about 200 bases or fewer should be performed with more caution.ConclusionResults indicated that the involvement of multiple recombination detection programs is very necessary. Additionally, results suggested two major challenges, one involving the difficulty of accurately determining the locations of breakpoints and the second involving identification of small regions of about 200 bases or fewer with greater caution. Both indicate the complexity of HIV-1 recombination. The resolution would depend critically on development of a recombination analysis algorithm, accumulation of HIV-1 stains, and a higher sequencing quality. With the changes in recombination pattern, phylogenetic relationships of some CRFs may also change. All these results may be critical to understand the role of recombination in a complex and dynamic HIV evolution.

Phylogenetic and demographic characterization of HIV-1 transmission in Madrid, Spain

BackgroundThe combination of phylogenetic analyses of HIV sequences with patients’ demographic data allows us to understand local HIV transmission, a necessary knowledge for designing prevention strategies. The Community of Madrid represents a challenge for the control of HIV epidemic in Spain given its high HIV prevalence and increasing proportion of immigrant people among HIV-infected population. MethodsWe applied maximum likelihood methods and Bayesian Markov chain Monte Carlo (MCMC) inference using the program BEAST to a set of HIV-1 pol sequences from 1293 patients diagnosed in 1995–2010 in Madrid, Spain. ResultsTwo-hundred and thirty six patients (18.2% of the cohort) were included in 100 transmission chains using phylogenetic criteria, 67 (67%) belonging to HIV-1 subtype B and 33 (33%) to 11 different non-B strains, especially BG and BF recombinants. Most networks involved transmission between MSM (48/100). Half of non-B clusters (15/33) included at least one Spaniard. Sub-Saharan African patients presented a low linkage rate (9%) in contrast to Spanish (21%) and Latin American (25%) patients. Three clusters involving treatment-independent transmission of drug-resistance mutations were found. ConclusionsOne out of five HIV-infected patients in our cohort in Madrid was epidemically linked, mainly by transmission pairs. The inclusion in transmission networks was more likely for MSM, Spaniards and patients from Latin America. We found no evidence of self-sustained non-B epidemics due to the absence of large transmission chains with the exception of Cuban BG recombinants and CRF47_BF. However, the differences in transmission across variants are probably determined by the patient profile, especially the infection route.

Antiretroviral drug resistance in HIV‐1 therapy‐naïve patients in Cuba, 2006–2011

In 2009, genotypic drug resistance testing was introduced for HIV‐1 patients failing antiviral therapy in Cuba. The high prevalence of drug resistance in this population indicated the need for surveillance of transmitted drug resistance (TDR) in therapy‐naïve patients. Therefore, the objectives of this study were to analyze the level and patterns of TDR and subtype in therapy‐naïve HIV‐1 patients in Cuba from 2006 to 2011, and to compare it with reported data from 2004 that indicated 4% TDR, solely restricted to NRTI. 153 plasma from HIV‐1 therapy‐naïve patients were collected between June 2006 and December 2011 and subsequently extracted, amplified and sequenced. Drug resistance was interpreted according to HIVdb v.6.1.1 and WHO list for TDR surveillance (2009) using the CPR tool v.6.0. Phylogenetic analysis was performed using Neighbour Joining (Kimura 2) in Mega 4. The majority of patients was male (82.4%), MSM (68.6%) and originated from Havana province (68.1%). 8.4% were recent infections. Subtype B was the most prevalent subtype (31.3%) followed by CRF20‐23‐24_BG (28.1%), CRF19 (18.3%) and CRF18 (13.0%). The prevalence of subtype B declined from 43.7% in the 2004 study to 31.3% in the present study, whereas BG recombinants increased from 14.4% to 28.1%. Overall, 12.4% (19/154) had evidence of TDR. 3.9% carried at least one NRTI, 1.9% at least one NNRTI and 1.9% at least one PI mutation. Drug resistance mutations against both NRTI and NNRTI were observed in 3.9%, whereas triple class resistance was found in only 0.6%. The most frequent NRTI mutations were M184V (55.5%), T215F/Y/rev (16.6%) and K70R (16.6%). The most frequent NNRTI mutations were K103N (61.1%) and G190A (22.2%). The most common PI mutation was L90M (5.5%). From the 19 patients with TDR, 13 (68.4%) were diagnosed with a recent HIV‐1 infection. AZT/D4T + 3TC + NVP may be effective in 6 of the patients with TDR (31.5%), partially effective in 6 (31.5%) and ineffective in 7 (36.8%). AZT/D4T + 3TC + IDV would be effective in 9 of the patients with TDR (47.3%), partially effective in 8 (42.1%) and ineffective in 2 (10.5%). This analysis confirmed the further expansion of BG recombinants in Cuba and revealed that antiretroviral drug resistance in HIV‐1 therapy‐naïve patients has increased to 12.4% in 2006–2011. The current study emphasizes the need to perform surveillance studies for TDR in therapy‐naïve patients, as the extent of TDR might jeopardize the effectiveness of first‐line regimens prescribed in Cuba.

High frequency of antiviral drug resistance and non-B subtypes in HIV-1 patients failing antiviral therapy in Cuba

BackgroundEmergence of HIV-1 drug resistance may limit the sustained benefits of antiretroviral therapy (ART) in settings with limited laboratory monitoring and drug options. ObjectivesSurveillance of drug resistance and subtypes in HIV-1 patients failing ART in Cuba. Study designThis study compiled data of ART-experienced HIV-1 patients attending a clinical center in Havana in 2003 and 2009–2011. The first period included results of a cross-sectional study, whereas in the second period genotyping was performed as part of routine care. Drug resistance mutations and levels were determined using HIVdb version 6.0.9. ResultsSeventy-six percent received solely ART containing at least 3 drugs, of which 79.1% ever receiving unboosted protease inhibitors (PI). Patients from 2009 to 2011 were longer treated and exposed to more ART regimens. Subtype B (39%) and CRF19_cpx (18%) were the most prevalent genetic forms. Subtype distribution did not change significantly between both periods, except for BG recombinants that increased from 6% to 14%. Nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside RTI (NNRTI) and PI mutations were present in 69.5%, 54.8% and 44.4%. Full-class resistance (FCR) to NRTI, NNRTI, PI and multidrug resistance (MDR) were detected in 31.8%, 37.9%, 18.5% and 15.4%. FCR to NRTI, NNRTI, PI and MDR were present in 9.8%, 14.1%, 0%, 0% after first-line failure and in 19.8%, 20.8%, 2.9% and 2.9% after second-line failure. ConclusionsOur study found a high prevalence of drug resistance and supports the need for appropriate laboratory monitoring in clinical practice and access to drug options in case of virological failure.

HIV Type 1 Intersubtype Recombinants during the Evolution of a Dual Infection with Subtypes B and G

ABSTRACT The aim of this study was to characterize the HIV-1 intersubtype recombinant forms generated during the follow-up of a dual natural infection with subtypes B and G. Near full-length sequences from plasma and peripheral blood mononuclear cell (PBMC) compartments were analyzed and the biological characteristics of their derived primary isolates studied. Different mutations were detected in V1, V2, and V3 sequences from primary isolates but not in sequences from plasma RNA or PBMC DNA. The HIV-1 near full-length sequence from the first collected plasma was of subtype G and the presence of subpopulations of subtypes B and G was observed with subtype-specific primers for protease and reverse transcriptase segments. Subsequent sequences from plasma, PBMCs, and primary isolates were obtained during a follow-up of 6 years; all of them were BG recombinants and showed identical intersubtype breakpoints between subtypes B and G in pol and nef. The env sequence from all primary isolates harbored a unique insert of subtype B. Specific primers for the V3 loop identified fluctuating subtype B and/or subtype G sequences either from plasma RNA or PBMC DNA.

Identification of 3 Phylogenetically Related HIV-1 BG Intersubtype Circulating Recombinant Forms in Cuba

BG intersubtype recombinants represented 11.6% of HIV-1 isolates in a recent survey in Cuba based on pol sequences, most of them forming a single clade further subdivided into 3 subclades. Here, we analyze 8 near full-length genomes and 1 gag-pol sequence from epidemiologically unlinked Cuban BG recombinants from these 3 subclades (3 from each). Near full-length sequences were also obtained from 3 subtype G and 2 subtype B Cuban viruses. Phylogenetic relationships were estimated via maximum likelihood, and mosaic structures of the recombinants were inferred with the bootscanning, MaxChi, Genconv, and GARD methods. For the near full-length genomes, all recombinants formed a strongly supported clade further subdivided into the same subclades previously defined in pol. Mosaic structures were identical within each subclade and different among subclades, although 5 breakpoints were coincident among all recombinants. Individual phylogenetic trees for nonrecombinant fragments (concatenated B and G subtype segments) indicated a common ancestry for the parental viruses and their relationships to local subtype B and G strains. These results allow us to identify 3 new BG intersubtype circulating recombinant forms in Cuba derived from a common recombinant ancestor, which originated from B and G subtype parental strains circulating in Cuba.

Genotypic Resistance to Antiretroviral Drugs in Patients Infected with Several HIV Type 1 Genetic Forms in Cuba

The main objective of this study is to evaluate the prevalence of resistance-associated mutations to reverse transcriptase (RT) and protease (PR) inhibitors (I) 2 years after the introduction of antiretroviral treatment in Cuba, analyzing the mutations corresponding to different HIV-1 genetic forms circulating in Cuba. A total of 425 plasma samples were collected in 2003, corresponding to 175 (41.2%) subtype B and 250 (58.8%) non-B genetic forms, including 56 (22.4 %) non-B subtypes, 112 (44.8%) circulating recombinant forms (CRFs), and 82 (32.8%) unique RFs (URFs). Of these, 175 (41.2%) patients were under highly active antiretroviral therapy (HAART) and 250 (58.8%) were treatment-naive. The presence of RT and PR resistance-associated mutations was established by sequencing. Levels of resistance were evaluated according to the Stanford Database program (http://hivdb.stanford.edu). The prevalence of resistance to RTI was 52.2% among RTI-treated patients, 51.5% for subtype B, and 53.2% for non-B genetic forms, including CRF18_cpx, CRF19_cpx, subtype C, and BG URF. In treatment-naive patients it was 6.4% in subtype B and 4.2% in non-B subtypes and RFs. The prevalence of resistance to PRI was 30% among PRI-treated patients, 28% in subtype B and 31% in non-B genetic forms, and 3.2% among treatment-naive subjects, mostly BG recombinants. In conclusion, significant differences in the prevalence of resistance to RTI and PRI were not detected among the most frequent genetic forms from treated patients, suggesting that the genetic diversity of HIV-1 in Cuba does not play a main role in the development of resistance to antiretroviral drugs. The presence of transmitted resistance mutations supports the study of resistance at baseline of treatment.

HIV Type 1 Molecular Epidemiology in Cuba: High Genetic Diversity, Frequent Mosaicism, and Recent Expansion of BG Intersubtype Recombinant Forms

Highly diverse HIV-1 genetic forms are circulating in Cuba, including subtypes B and G and two recombinant forms of African origin (CRF18_cpx and CRF19_cpx). Here we phylogenetically analyze pol sequences from a large collection of recent samples from Cuba, corresponding to 425 individuals from all Cuban provinces, which represents approximately 12% of prevalent infections in the country. RNA from plasma was used to amplify a pol segment by reverse transcription-polymerase chain reaction; phylogenetic analyses were performed with neighbour-joining trees and bootscanning. The distribution of genetic forms was subtype B, 41.2%; CRF19_cpx, 18.4%; BG recombinants, 11.6%; CRF18_cpx, 7.1%; subtype C, 6.1%; subtype G, 3.8%; B/CRF18 recombinants, 2.6%; subtype H, 2.1%; B/CRF19 recombinants, 1.7%; and others, 5.4%. Seventy-five (17.6%) viruses were recombinant between genetic forms circulating in Cuba. In logistic regression analyses, adjusting by gender and region, subtype B was more prevalent (OR 5.0, 95% CI 2.0-12.3) and subtype G less prevalent (OR 0.1, 95% CI 0.0-0.5) among men who have sex with men (MSM) than among heterosexuals. Within the main genetic forms of Cuba there were phylogenetic subclusters, several of which correlated with risk exposure or region. BG recombinants formed three phylogenetically related subclusters, corresponding to three different mosaic structures; most of these recombinants were from MSM from Havana City, among whom they have expanded recently, reaching 31% HIV-1 infections diagnosed in 2003. This study confirms the high HIV-1 diversity and frequent recombination in Cuba and reveals the recent expansion of diverse related BG recombinant forms in this country.

Heterogeneous nature of HIV-1 recombinants spreading in Spain

HIV-1 infections due to non-B subtypes are increasing rapidly in number and spreading across Europe. The genetic nature of HIV-1 non-B variants containing subtype G sequences at the protease (PR)-coding region are described from 48 unrelated subjects living in Spain. Phylogenetic analyses of the HIV-1 reverse transcriptase (RT) and envelope (env) genes (including the V3 loop) were performed. Up to 32 (66.6%) of samples carried inter-subtype recombinant viruses. Although double recombinants were found most frequently (G/A in 20; G/B in 8; G/K in 2), two individuals harbored triple recombinant viruses (GPR/BRT/Aenv and GPR/KRT/Aenv, respectively). Only 33 (68.7%) and 9 (18.7%) sequences clustered with clade G when examining the RT and env genes, respectively. Nearly 70% of samples with pol sequences (PR/RT) belonging to subtype G harbored env sequences ascribed to other clades: A (55.6%), B (11.1%), or K (3.7%). Of note, most recombinant viruses clustered with CRF02_AG, although CRF14_BG recombinants were also found. This study demonstrates that most viruses circulating in Spain with clade G sequences at the pol-coding region are in fact inter-subtype recombinants, with CRF02_AG being the most prevalent virus.

Primary resistance mutations to fusion inhibitors and polymorphisms in gp41 sequences of HIV-1 non-B subtypes and recombinants.

Primary resistance mutations to fusion inhibitors and polymorphisms in gp41 sequences of non-B subtypes and recombinant HIV-1 isolates were analysed. L91H to RPR103611 was detected in one DGpol/Denv/Dgp41 recombinant; L9F and K144R, rarely reported previously, were frequent in the B region of CRF14_BG recombinants. V194I and V318A, not described in the G subtype, were detected in the G region of BG recombinants and in G subtype viruses that also show the rare mutations T115L, M118V and K90R.

Biological characteristics of newly described HIV-1 BG recombinants in Spanish individuals

Biological characteristics of newly described HIV-1 BG recombinants in Spanish individuals