non-B HIV-1 Strains Research Articles

Short Communication: HIV and Tuberculosis Co-Infection Among Migrants in Portugal: A Brief Study on Their Sociodemographic, Clinical, and Genomic Characteristics.

HIV and tuberculosis (TB) are among the global deadliest diseases. Migrant populations are particularly vulnerable to these infections. Yet, literature is still scarce on the epidemiology of HIV-TB co-infection among migrants. In this study, we characterized native and migrant HIV patients followed in Portuguese hospitals, who were diagnosed with TB, regarding their sociodemographic, clinical, and genomic characteristics. Among 67 patients with HIV and TB diagnoses, there were 24 migrants, most from sub-Saharan Africa. Most patients had CD4+ T cell counts below 350 cells/μL, and were diagnosed simultaneously for HIV and TB. When compared to natives, migrants presented a higher proportion of non-B HIV-1 infections. Patients infected with these non-B HIV-1 strains presented higher viral loads, which can have an important impact for the transmissibility and pathogenicity of both diseases. Future studies should investigate different HIV strains and consequences for TB and HIV transmission and disease outcomes, especially among vulnerable populations.

HIV-1 recombinant forms in immigrants regularly residing in Milan, northern Italy

To assess the HIV -1subtypes distribution in HIV-1 positive migrants living in Milan we studied 77 HIV-1 patients followed at the San Raffaele Hospital of Milan. Twenty subjects were born in Europe, 43 in the Americas, 10 in Africa and 4 in Asia. Unsafe heterosexual activity prevailed in migrants born in Africa and male homosexuality in those born in European, American and Asian countries (p = 0.05). The phylogeny showed that 38/77 (49.3%) subjects carried HIV-B subtype while the remaining strains were classified as not pure HIV-1 B subtypes 13/77 (16.9%) or recombinant forms 26/77 (33.8%). Female gender more frequently showed HIV-1 non-B strains and rarely HIV-1 B subtypes (12/39, 30.8% vs. 3/38, 7.9%, p = 0.02). Transmitted drug resistance was identified in 10/77 (13%) patients predominately with B subtype. Our data underscore a large heterogeneity in HIV-1 subtypes and a large proportion of recombinant forms.

Short Communication: High Natural Polymorphism in thegagGene Cleavage Sites of non-B HIV Type 1 Isolates from Gabon

The main goal of the present study was to determine the frequency of substitutions in the cleavage sites (CS) of gag gene among non-B HIV-1 isolates from Gabon. Fifty plasma specimens, collected in 2010-2011, from HIV-1-infected patients failing first-line antiretroviral (ARV) regimens (constituted of two nucleoside reverse transcriptase inhibitors+one nonnucleoside reverse transcriptase inhibitor) (n=38) and from HIV-1-infected individuals untreated with ARV (n=12) were analyzed in the gag and gag-pol cleavage sites. Compared to HXB2 reference sequence, the total median number of substitutions in gag and gag-pol CS was 10 (range, 5-18). The cleavage site p2/NC was the most variable of the four gag CS with 100% (50/50) isolates carrying at least 1 substitution (range, 1-9). The two gag-pol TFP/p6pol and p6pol/PR CS sites were also highly variable (at least one substitution, 50/50, 100% in both cases). Substitutions at position G381 (p2/NC), L449 (p1/p6gag), and K444 (TFP/p6pol) were significantly more frequent in CRF02_AG strains, compared to other non-B strains (30.4% vs. 3.7%, p=0.03; 87.0% vs. 59.3%, p=0.03; and 91.3% vs. 59.3%, p=0.01, respectively). Other non-B subtypes were significantly more likely to harbor substitutions at position N487 (p6pol) (70.4%) than CRF02_AG (39.1%) (p=0.02). In Gabon, gag and gag-pol cleavage sites were highly polymorphic in protease inhibitor-naive patients harboring non-B HIV-1 strains. In sub-Saharan Africa, further studies are definitively required to better understand the impact of gag mutations among subjects receiving second-line LPV/r-containing regimens (monotherapy or triple combinations).

Performance of the ViroSeq HIV-1 genotyping system v2.0 on HIV-1 strains circulating in Senegal

The objective of this study was to investigate the performance of the ViroSeq HIV-1 Genotyping System v2.0 on HIV-1 non-B strains identified in Senegalese patients. The study involved 150 patients, and genotyping was performed using the ViroSeq HIV-1 Genotyping System v2.0 or an in-house method developed by the French National Agency on AIDS Research AC11 when the ViroSeq HIV-1 Genotyping System v2.0 failed. The sequences were edited to assess the performance of sequencing primers at their presumed binding regions. The Polymorphism was studied in the regions between the sequences of Senegalese patients and the subtype B strains used as references. The phylogenetic analysis showed a predominance of CRF02_AG (88/150; 58.7%) and the circulation of 11 subtypes/CRFs, 16 unique recombinant forms (URFs) and one unclassified sample. The amplification and sequencing rates were 98% (147/150) and 96.6% (142/147), respectively. This study showed that only primer B exhibited 100% success, while the failure rate ranged from 1.4% to 71.4% for the other primers (D: 71.4%, A and H: 12.2%, F: 7.5%, G: 5.5% and C: 1.4%). These findings suggest the need for an alternative method or alternative primers for non-B strains that were not sequenced successfully using the ViroSeq HIV-1 Genotyping System v2.0.

English

Forty samples collected from HIV-1 AIDS and asymptomatic patients in 2002 and 2003 before the initiation of antiretroviral treatment (ART), most in poorly investigated regions of Ghana were retrospectively examined. For baseline data on genetic diversity before therapy, env, gagand pol phylogenetic analysis was generated. pol was further analysed for potential resistance to antiretroviral drugs. Plasma viral load was also quantified. HIV-1 subtypes A1 and G constituted a minority, while simple and/or complex recombinant strains dominated by CRF02_AG and including CRF06_cpx, CRF09 and CRF10 was observed. Major mutations,protease (PR) L33F and reverse transcriptase (RT) M41L, associated with high levels of phenotypic resistance or clinical evidence for reduced virological response were identified. In the protease-coding domain, positions 10 and 11 were mutated in 21% (5/24) of patients examined, carrying putative resistance to PIs. In the reverse transcriptase-coding domain, seven mutations were detected in 6/35 patients (17%). Of these, 8.5% (3/35) had either double or single mutations possibly affecting NRTIs or NNRTIs respectively. Within PR and RT regions, the mutation rate ranged between 2.6 and 7.0%. Position 10 had 57% mutation frequency. The mutational profile of our non-B HIV-1 strains were identical with subtype B strains at documented DR-related positions. Therefore, drug resistance (DR) pathways of non-B strains were presumed similar to subtype B viruses. Although antiretroviral therapy was expected to be successful, patients presenting resistance-related mutations prior to the initiation of ART could develop clinically relevant resistance earlier than wild-type strains.   Key words: HIV-1 genotyping, antiretroviral therapy, drug-naive HIV-1 patients, drug resistance.

Comparative Analysis of Drug Resistance Among B and the Most Prevalent Non-B HIV Type 1 Subtypes (C, F, and CRF02_AG) in Italy

In recent years, increasing numbers of patients infected with HIV-1 non-B subtypes have been treated with modern antiretroviral regimens. Therefore, a better knowledge of HIV drug resistance in non-B strains is crucial. Thus, we compared the mutational pathways involved in drug resistance among the most common non-B subtypes in Italy (F, C, and CRF02_AG) and the B subtype. In total, 2234 pol sequences from 1231 virologically failing patients from Central Italy were analyzed. The prevalence of resistance mutations in protease and reverse transcriptase between non-B and B subtypes has been evaluated. Among patients treated with nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) and with thymidine analogues (TA) experience, TAMs1 M41L and L210W were less prevalent in CRF02_AG, while TAMs2 T215F and K219E were more prevalent in the F subtype. In NRTI-treated patients having experience with abacavir, didanosine, tenofovir, or stavudine the K65R mutation was mostly prevalent in the C subtype. In non-NRTI (NNRTI)-treated patients infected by the C subtype the prevalence of K103N was lower than in patients infected with other subtypes, while the prevalence of Y181C and Y188L was higher compared to subtype B. The prevalence of Y181C was higher also in subtype F as compared to subtype B. In patients treated with protease inhibitors, L89V was predominantly found in CRF02_AG, while the TPV resistance mutation T74P was predominantly found in the C subtype. Some differences in the genotypic drug resistance have been found among patients infected with B, C, F, and CRF02_AG subtypes in relationship to treatment. These results may be useful for the therapeutic management of individuals infected with HIV-1 non-B strains.

Virological failure rates and HIV‐1 drug resistance patterns in patients on first‐line antiretroviral treatment in semirural and rural Gabon

IntroductionAs antiretroviral treatment (ART) continues to expand in resource-limited countries, the emergence of HIV drug resistance mutations (DRMs) is challenging in these settings. In Gabon (central Africa), no study has yet reported the virological effectiveness of initial ART given through routine HIV care.MethodsFollowing the World Health Organization (WHO) recommendations, a cross-sectional study with a one-time HIV-1 RNA viral load (VL) measurement was conducted in Gabon to assess virological failure (VF) defined by a VL result ≥1000 copies/ml and DRMs among adult patients living with non-B HIV-1 strains and receiving first-line non-nucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy for at least 12 months. Risk factors associated with VF and DRMs were assessed.ResultsBetween March 2010 and March 2011, a total of 375 patients were consecutively enrolled from two decentralized (one semirural and one rural) HIV care centres. Median time on ART was 33.6 months (range, 12–107). Overall, the rate of VF was 41.3% (36.4–46.4). Among viremic patients, 56.7% (80/141) had at least one DRM and 37.6% had dual-class resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and NNRTIs. The most frequent DRMs were K103N/S (46.1%) and M184V/I (37.6%). Thymidine analogue mutations were found in 10.6%. Independent risk factors associated with VF were being followed up at the semirural centre (P=0.033), having experienced unstructured treatment interruptions (P=0.0044), and having low CD4+ counts at enrolment (P<0.0001). A longer time on ART (P=0.0008) and being followed up at the rural centre (P=0.021) were risk factors for DRMs.ConclusionsThis is the first study conducted in Gabon providing VF rates and DRM patterns in adult patients receiving first-line ART. In sub-Saharan Africa, where NNRTI-based regimens are recommended as the standard for first-line ART, strengthening virological monitoring together with preventing unplanned treatment interruptions are a global public health priority.

High Failure Rate of the ViroSeq HIV-1 Genotyping System for Drug Resistance Testing in Cameroon, a Country with Broad HIV-1 Genetic Diversity

The ViroSeq HIV-1 genotyping system is used in many African countries for drug resistance testing. In this study, we used a panel of diverse HIV-1 group M isolates circulating in Cameroon to show that the performance of this assay can be altered by the sequence variation of non-B HIV-1 strains that predominate in African settings.

Expanded evaluation of blood donors in the United States for human immunodeficiency virus type 1 non‐B subtypes and antiretroviral drug–resistant strains: 2005 through 2007

In a previous study of 66 human immunodeficiency virus (HIV)-infected US blood donors from 1999 to 2005, HIV-1 non-B and antiretroviral drug-resistant strains accounted for 4.7 and 6.5% of HIV infections, respectively. This study was expanded to include an additional 11 recently acquired infections and 197 established infections collected from January 2005 through December 2007. HIV-infected donors were detected using FDA-licensed assays. Drug resistance profiles for protease and reverse transcriptase (RT) genes were determined using a genotyping system (ViroSeq, Celera Diagnostics); genetic subtype was determined by phylogenetic analysis of these sequences. Drug resistance profiles were obtained for 203 of 208 specimens; 9.9% had mutations that confer drug resistance. Ten showed resistance to a single drug class: nine to nonnucleoside RT inhibitors (NNRTIs) and one to nucleoside RT inhibitors (NRTIs). Eight showed two drug class resistance: five NRTI plus NNRTI, two NRTI plus protease inhibitor (PI), and one NNRTI plus PI. Two showed three drug class resistance. Non-B strains were identified in 2.5% of donors and consisted of subtypes A1 and D, CRF02_AG, CRF43-02G, and URF_BF. Data from this and the previous study show that antiretroviral drug-resistant HIV-1 is present in 9.1% of HIV-infected donors from 1999 through 2007; 9.3% of established infections and 6.9% of recent infections. Diverse HIV-1 non-B strains presently account for 3.0% of HIV infections in US donors.

A combined genotypic and phenotypic human immunodeficiency virus type 1 recombinant virus assay for the reverse transcriptase and integrase genes

With the approval of the first HIV-1 integrase inhibitor raltegravir and a second one in phase III clinical development (elvitegravir), genotypic and phenotypic resistance assays are required to guide antiretroviral therapy and to investigate treatment failure. In this study, a genotypic and phenotypic recombinant virus assay was validated for determining resistance against integrase inhibitors. The assays are based on the amplification of a region encompassing not only HIV-1 integrase, but also reverse transcriptase and RNAseH. The overall amplification success was 85% (433/513) and increased to 93% (120/129) for samples with a viral load above 3 log 10 copies/ml. Both B and non-B HIV-1 subtypes could be genotyped successfully (93%; 52/56 and 100%; 49/49, respectively) and reproducibly. The phenotypic assay showed a high success rate (96.5%; 139/144) for subtype B (100%; 19/19) and non-B subtypes (92%; 45/49), and was found to be accurate and reproducible as assessed using well-characterized integrase mutants. Using both assays, baseline resistance to raltegravir and elvitegravir in subtype B and non-B HIV-1 strains selected at random was not observed, although integrase polymorphisms were present at varying prevalence. Biological cutoff values were found to be 2.1 and 2.0 for raltegravir and elvitegravir, respectively. In summary, a genotypic and phenotypic integrase resistance assay was validated successfully for accuracy, reproducibility, analytical and clinical sensitivity, and dynamic range.

New and old complex recombinant HIV-1 strains among patients with primary infection in 1996–2006 in France: The French ANRS CO06 primo cohort study

BackgroundPrevalence of HIV-1 non-B subtypes has increased overtime in patients diagnosed at the time of primary infection (PHI) in France. Our objective was to characterize in detail non-B strains which could not be genetically classified into the known subtypes/Circulating Recombinant Forms (CRFs).MethodsAmong 744 patients enrolled in the ANRS PRIMO Cohort since 1996, 176 (23.7%) were infected with HIV-1 non-B strains. The subtype/CRF could not be identified in RT for 15 (2%). The V3-V5 env region was sequenced and 3 strains (04FR-KZS, 06FR-CRN, 04FR-AUK) were full-length sequenced. Phylogenetic and bootscan analyses were used to characterize the mosaic structures.ResultsAmong V3-V5 sequences, 6 were divergent A, 2 distantly related to E or D, 2 C, 1 B and 2 remained unclassified. 04FR-KZS, isolated in a Congolese woman infected in France, clustered with 2 previously described viruses from the Democratic Republic of Congo. They represent CRF27_cpx involving A/E/G/H/J/K/U subtypes. 06FR-CRN, isolated in a homosexual Caucasian patient, was a B/C/U recombinant involving a Brazilian C strain. 04FR-AUK, isolated in a Congolese patient infected in France, was a A/K/CRF09/U recombinant clustering from gag to vif with HIV-1 MAL. Others PHI were further observed in 2006–2007 with 1 KZS and 5 CRN-like viruses, suggesting their spread in France.ConclusionThis study illustrates the increasing HIV-1 diversity in France associating new (06FR-CRN) and old (CRF27_cpx and "MAL-like" 04FR-AUK) strains, which are rare in their region of origin but may have a possible founder effect in France. Our results strengthen the French guidelines recommending viro-epidemiological surveillance of HIV-1 diversity.

Gp41 Sequence Variability in HIV Type 1 Non-B Subtypes Infected Patients Undergoing Enfuvirtide Pressure

Enfuvirtide is the first of a new class of antiretroviral drugs that inhibits HIV entry. It is a 36 amino acid synthetic peptide that mimics the HR2 region of the HIV-1 gp41, preventing the fusion of viral and cellular membranes. Up to now, enfuvirtide was designed based on the HIV-1 B-subtype gp41, and resistance mutations to the fusion inhibitor have been investigated primarily in individuals infected with this subtype. To fill the gap, we analyzed the full length gp41 protein sequence of HIV-1 non-B strains from individuals receiving enfuvirtide-containing regimens. No primary resistance to the enfuvirtide binding domain (36-45 residues) was found. Resistance mutations were detected at follow-up visits and were comparable to those described among B-subtype HIV-1-infected patients; no sequence changes were detected in crucial HR1/HR2 gp41 sites such as the cytotoxic T lymphocyte epitope, cysteine loop, ectodomain, and 5-helix interaction and binding region.

Genetic Diversity of HIV-1 Non-B Strains in Sicily: Evidence of Intersubtype Recombinants by Sequence Analysis ofgag,pol, andenvGenes

The molecular epidemiology of HIV-1 strains in Sicily (Italy) was phylogenetically investigated by the analysis of HIV-1 gag, pol, and env gene sequences from 11 HIV-1 non-B strains from 408 HIV-1-seropositive patients observed from September 2001 to August 2006. Sequences suggestive of recombination were further investigated by bootscanning analysis of various fragments. Overall, we identified several second-generation recombinant (SGRs) strains, which contained genetic material of CRF02_AG in at least one gene. Notably, three individuals were found to be infected with subsubtype A3, and one of them showed genetic recombination with subsubtype A4. The current study emphasizes the genetic analysis of gag, pol, and env genes as a powerful tool to trace the spread of complex HIV-1 recombinant forms, and highlight the genetic diversity of HIV-1 non-B strains in Italy.

Introduction of Non-B Subtypes among Spaniards Newly Diagnosed with HIV Type 1 in the Canary Islands

The Spanish Canary Islands face the northwest coast of Africa, being one gate of entrance of African immigrants into Europe and of HIV-1 non-B strains, prevalent in most African regions. The presence and nature of HIV-1 non-B subtypes were examined in Spanish natives newly diagnosed with HIV-1 infection from 1998 to 2004 in a reference hospital located in one of the largest islands of the archipelago. Subtyping of HIV-1 pol genes was performed in 139 out of 429 individuals found to be HIV-1 positive during that period. Non-B strains infected 40 (28.8%) subjects, 12 (13.7%) out of 87 native Spaniards and 28 (53.8%) out of 52 immigrants (75% Africans) (p < 0.01). HIV-1 non-B subtypes, mostly recombinant forms, were present in Spaniards at least since 1998. HIV-1 non-B subtypes are frequently recognized in HIV+ Africans arriving in the Canary islands and continuously introduced into the newly diagnosed HIV-1+ native population living in the archipelago.

Natural polymorphism in protease and reverse transcriptase genes and in vitro antiretroviral drug susceptibilities of non-B HIV-1 strains from treatment-naive patients

Background Most studies on antiretroviral (ARV) resistance of human immunodeficiency virus (HIV) have been done on subtype B which only represent a limited proportion of infections worldwide. Objective Understand baseline susceptibilities to ARVs in non-B strains. Methods To explore in greater detail possible intrinsic resistance to antiretroviral drugs in non-B subtypes, phenotypic resistance was tested in 35 non-B (A, D, F, G, J; CRF02, 06, 09, 11, 13) HIV-1 isolates obtained from ARV treatment naive patients. The panel includes strains with an increasing number of minor mutations in the protease gene and/or with atypical mutations at positions associated with resistance in protease and RT. Results We detected phenotypic resistance (fold-change values equal or superior to biological test cut-offs (BCO) in 14 of the 35 strains, 4 strains had decreased in vitro susceptibility to more than one drug. However, it is important to note that in most cases the increased fold-changes were close to the BCOs. Phenotypic resistance was observed against each of the three ARV drug classes: ritonavir ( n = 3), nelfinavir ( n = 2), saquinavir ( n = 2), zidovudine ( n = 2), stavudine ( n = 1), didanosine ( n = 1); delavirdine ( n = 6), efavirenz ( n = 1) and nevirapine ( n = 1). Some mutations could be associated with decreased in vitro susceptibility: 1 of 3 strains only with mutations M46I/L in protease, 1/2 A98S, K101N, V108I, V179I, and P236L in reverse transcriptase. Interestingly, the presence of an increasing number of minor mutations in the protease gene was not associated with decreased in vitro susceptibility to protease inhibitors. Conclusion It is necessary to continue phenotypic studies on non-subtype B strains to identify the role of all polymorphisms present in protease and RT genes and to optimize interpretation algorithms. Data obtained from large, diverse populations of HIV-1 infected individuals is critical for defining and standardizing the quantification of resistance (phenotypic and genotypic testing).

Detection of HIV type 1 non-B subtypes in Sicily, Italy.

To evaluate the presence of HIV-1 non-B subtypes in Sicily, we sequenced and genotyped HIV-1 PR and RT regions of the pol gene using plasma from 169 HIV-1-infected adult patients. All samples were obtained from a study of antiretroviral-associated resistance mutations resulting in virological failure during highly active antiretroviral therapy (HAART). Eight (4.7%) patients had the non-B HIV-1 subtype including some circulating recombinant forms (CRFs). All of these individuals acquired the infection by heterosexual transmission. The detection of HIV-1 non-B strains was significantly associated with younger age of HIV-1 acquisition. Our findings indicate, for the first time, the presence of HIV-1 non-B subtypes in Sicily in patients who experienced virological failure during HAART, and highlight the need for implementing a network for the epidemiological surveillance of HIV-1 subtypes in Southern Europe.

Protease mutations in HIV-1 non-B strains infecting drug-naive villagers in Cameroon.

To describe the presence of protease inhibitor (PI) resistance-associated mutations and subtype distribution in drug-naive villagers of six provinces of Cameroon, we sequenced the protease (PR) gene (297 bp) of 128 viruses. Secondary PI resistance-associated mutations were identified at five sites: L10I/V (16%), K20R (8%), M36I (98%), L63P (13%), and V77I (6%). No primary mutation in the PR was identified. Of the 128 specimens analyzed, subtypes A (11%), C(2%), D (6%), F2 (3%), G (6%), H (0.8%), J (6%), and CRF02_AG (60%) were identified. The mutations identified were not characteristic to any particular subtype. The absence of primary mutations, in addition to the few secondary mutations, gives good perspectives for PI treatment interventions in these rural areas.