Nucleoside reverse transcriptase inhibitor resistance mutations associated with first-line stavudine-containing antiretroviral therapy: programmatic implications for countries phasing out stavudine.

BACKGROUND: The World Health Organization Antiretroviral Treatment Guidelines recommend phasing-out stavudine because of its risk of long-term toxicity. There are two mutational pathways of stavudine resistance with different implications for zidovudine and tenofovir cross-resistance, the primary candidates for replacing stavudine. However, because resistance testing is rarely available in resource-limited settings, it is critical to identify the cross-resistance patterns associated with first-line stavudine failure. METHODS: We analyzed HIV-1 resistance mutations following first-line stavudine failure from 35 publications comprising 1,825 individuals. We also assessed the influence of concomitant nevirapine vs. efavirenz, therapy duration, and HIV-1 subtype on the proportions of mutations associated with zidovudine vs. tenofovir cross-resistance. RESULTS: Mutations with preferential zidovudine activity, K65R or K70E, occurred in 5.3% of individuals. Mutations with preferential tenofovir activity, ≥ two thymidine analog mutations (TAMs) or Q151M, occurred in 22% of individuals. Nevirapine increased the risk of TAMs, K65R, and Q151M. Longer therapy increased the risk of TAMs and Q151M but not K65R. Subtype C and CRF01_AE increased the risk of K65R, but only CRF01_AE increased the risk of K65R without Q151M. CONCLUSIONS: Regardless of concomitant nevirapine vs. efavirenz, therapy duration, or subtype, tenofovir was more likely than zidovudine to retain antiviral activity following first-line d4T therapy.

Virological characterization of an infection with a dual-tropic, multidrug-resistant HIV-1 and further evolution on antiretroviral therapy.

We studied a case of recent infection with multidrug-resistant (MDR) HIV-1. Over 16 months off-therapy, the CD4 cell count decreased from 419 to 184 cells/mul. Antiretroviral therapy (ART) then led to an incomplete virological response but to an immunological benefit, concurrently with a shift to CCR5-only tropism and a reduction in replication capacity. ART, even if suboptimal, can be of interest in the case of MDR virus infection.

Resistance mutations in subtype C HIV type 1 isolates from Indian patients of Mumbai receiving NRTIs plus NNRTIs and experiencing a treatment failure: resistance to AR.

We present here the first data available on resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) in India. In these subtype C isolates, we have observed most of the mutations noted in reverse transcriptase (RT) for subtype B with some additional substitutions (at positions 98, 203, 208, and 221) that will warrant attention in the algorithms used.

Reverse transcriptase mutations in Cambodian CRF01_AE isolates after antiretroviral prophylaxis against HIV Type 1 perinatal transmission.

This study explores amino acid changes of the reverse transcriptase (rt) of CRF01_AE isolates from pregnant women naive to antiretroviral drugs before and 2, 6, and 52 weeks after exposure to single dose nevirapine (sdNVP). Results based on 51 observations showed that the proportion of isolates with nonnucleoside reverse transcriptase inhibitor (NNRTI) RMs in the group treated with sdNVP (n = 35) increased from 0% pre-NVP to 22.9% at week 2 postpartum (pp) and 22.9% at week 6 pp. In the group treated with zidovudine + sdNVP (n = 16), the proportion with RM was 31.3% and 18.8% at weeks 2 and 6 pp, respectively. Only a few RMs were still detected at week 52 pp. No apparent subtype-specific treatment-related mutations were detected. NNRTI RM occurrence in CRF01_AE strains is similar to subtype A, D, and CRF02_AG strains after exposure to antiretroviral drugs for PMTCT.

Characterization of HIV-1 isolates from antiretroviral drug-naive children in southern India.

Access to antiretroviral therapy has expanded in many developing countries, including India. The standard first-line regimens consist of a combination of two nucleoside reverse transcriptase inhibitors and a nonnucleoside reverse transcriptase inhibitor, in a fixed drug combination. Data regarding resistance to these drugs are scarce, especially in children. We evaluated the pattern of polymorphism and potential drug resistance mutations (DRMs) in HIV-1 isolates from 48 children naive to antiretroviral therapy attending the outpatient clinics of the Tuberculosis Research Center in Chennai. The samples were subjected to genotyping of reverse transcriptase (RT) and protease genes. All the samples showed significant polymorphisms in both RT and protease genes, but none had major DRMs. The currently recommended generic first-line antiretroviral drug combination is an appropriate treatment strategy for HIV-1-infected children in India.

HIV-1 antiretroviral drug resistance in recently infected patients in Abidjan, Côte d'Ivoire: A 4-year survey, 2002-2006.

We performed HIV-1 drug resistance genotypic analysis of viral isolates from 100 antiretroviral (ARV)-naive, recently HIV-1-infected (between 2002 and 2006) individuals from Abidjan (Côte d'Ivoire). The overall prevalence of HIV-1 variants with resistance mutations to reverse transcriptase, protease, or fusion inhibitors was 6%. The majority of isolates were CRF02_AG. Compared with a previous study carried out by our group in 2001-2002 in a similar population in Abidjan, our findings confirm the circulation and transmission of HIV-1 carrying key ARV drug resistance mutation.

High diversity of HIV type 1 in Algeria.

We have sequenced different genes of HIV-1 strains from infected individuals recruited in various geographic parts of Algeria; phylogenetic trees were constructed yielding molecular characterization of these strains. Subtype B accounts for 56% of the samples studied and is therefore the predominant subtype, particularly in the north part of the country; but there is a high diversity of the virus including CRF02_AG, CRF06_cpx, CRF02/CRF06 interrecombinants, and different other intersubtype and/or inter-CRF recombinants. The prevalence of these non-B viruses increases in the south part of Algeria that borders sub-Saharan African countries. The high diversity of HIV-1 in Algeria has implications for virological follow-up, resistance surveys, and vaccine design.

Susceptibility to antiretroviral drugs of CRF01_AE, CRF02_AG, and subtype C viruses from untreated patients of Africa and Asia: comparative genotypic and phenotypic data.

Non-B HIV-1 viruses are predominant in developing countries where access to antiretroviral drugs (ARVs) is progressively being intensified. It is important to obtain more data on the susceptibility of these viruses to available ARVs. CRF01_AE, CRF02_AG, and subtype C strains of HIV-1 obtained from untreated patients from Vietnam, Cote d'Ivoire, and India were analyzed for their in vitro susceptibility to NRTIs, NNRTIs, PIs, and an entry inhibitor (T-20) using a recombinant viral assay (PHENOSCRIPT). The corresponding viruses, which had been previously sequenced in reverse transcriptase (RT), protease (prot), plus envelope (env) C2/V3 genes and had therefore been fully characterized, were further sequenced in env HR1 + HR2 regions. CRF01_AE isolates are sensitive to NRTIs and NNRTIs with the exception of one isolate that exhibits a decreased susceptibility to NNRTIs associated with a I135T substitution in RT. CRF02_AG and subtype C viruses are sensitive to NRTIs and NNRTIs but some CRF02_AG isolates tend to be resistant to abacavir, potentially related to associated substitutions of RT at positions 123 (D123N) plus 135 (I135V). Whereas all but one CRF01_AE isolates are fully susceptible to PIs, some CRF02_AG and, more frequently, some subtype C isolates are resistant to atazanavir. The role of substitutions in prot at positions of secondary resistance mutations 20, 36, 63, and 82 is raised with a potentially crucial role of the V82I substitution. Finally, all viruses tested, regardless of the CRF or subtype, are fully susceptible to T-20.

Molecular characterization, phylogenetic relationships, and developmental expression patterns of prion genes in zebrafish (Danio rerio).

Prion diseases are characterized by the accumulation of a pathogenic misfolded form of a prion protein (PrP) encoded by the Prnp gene in humans. In the present study in zebrafish, two transcripts and the corresponding genes encoding prion proteins, PrP1 and PrP2, related to human PrP have been characterized with a relatively divergent deduced amino acid sequence, but a well preserved overall organization of structural prion protein motifs. Whole-mount in situ hybridization analysis performed during embryonic and larval development showed a high level of PrP1 mRNA spatially restricted to the anterior floor-plate of the central nervous system and in ganglia. Transcripts of prp2 were detected in embryonic cells from the mid-blastula transition to the end of the segmentation period. From 24 h postfertilization up to larval stages, prp2 transcripts were localized in distinct anatomical structures, including a major expression in the brain, eye, kidney, lateral line neuromasts, liver, heart, pectoral fins and posterior intestine. The observed differential developmental expression patterns of the two long PrP forms, prp1 and prp2, and the short PrP form prp3, a more divergent prion-related gene previously identified in zebrafish, should contribute to understanding of the phylogenetic and functional relationships of duplicated prion gene forms in the fish genome. Together, the complex history of prion-related genes, reflected in the deduced structural features, conserved amino acid sequence and repeat motifs of the corresponding proteins, and the presence of differential developmental expression patterns suggest possible acquisition or loss of prion protein functions during vertebrate evolution.

New insights in HTLV-I phylogeny by sequencing and analyzing the entire envelope gene.

The HTLV-I envelope plays a major role in the process of target cell infection. It is implied in the recognition of the viral receptor(s), penetration of the viral genetic material, and induction of host immunity to the virus. It is thus important to study the genetic variability of the viral env gene as well as its variation in terms of evolution. In a new approach to these features, we sequenced the entire env gene of 65 HTLV-I isolates originating from Gabon, French Guiana, West Indies, and Iran, such isolates representing all major HTLVI phylums but the Australo-Melanesian one. The sequences obtained and all PTLV-I (HTLV-I and STLV-I) env sequences available in the literature were analyzed. Phylogenetic studies using different algorithms (minimum evolution, neighbor joining, maximum parsimony, and maximum likelihood) gave the same clear-cut results. Newly sequenced HTLV-I isolates described in this report allocated in three well-defined subtypes: Cosmopolitan, Central African, and a new distinct one that we termed "Maroni" subtype (present in the Maroni Basin, French Guiana, and West Indies). Clearly, the most divergent PTLV-I strains present in Asia- Australo-Melanesia as well as African and Asian STLV-I derived from the same node in the phylogenetic tree as isolates of the Central African subtype. In addition, we showed that within each PTLV-I subtype, groups of isolates may be characterized by nonrandom and systematically associated mutations.

Characterization of mutations in HIV type 1 isolates from 144 Cambodian recently infected patients and pregnant women naive to antiretroviral drugs.

A baseline study has been conducted to determine the polymorphism of reverse transcriptase, protease, and envelope genes of HIV-1 isolates from 146 antiretroviral drug-naive Cambodian patients including 22 seroconverters and 124 pregnant women having been diagnosed HIV positive for less than 1 year. Amplification of at least one gene was successful for 144 isolates. All three genes were obtained for 136 isolates. Subtyping showed that CRF01_AE was predominant (130 cases). According to the ANRS September 2004 list, polymorphism substitutions (>50% versus the subtype B consensus) of CRF01_AE at drug resistance positions were observed only in protease: I13V (81%), E35D (87%), M36I (100%), R41K (96%), and H69K (100%). Two strains bore one major resistance mutation to PIs: M46I and N88D. Five other strains carried drug resistance mutations to RTIs: K70R (one strain), V75M (three strains), and K101E (one strain). Of the isolates 4.9% had drug resistance mutations to antiretroviral drugs.

Presence of CRF09_cpx and complex CRF02_AG/CRF09_cpx recombinant HIV type 1 strains in Côte d'Ivoire, West Africa.

Based on partial env and pol (protease and RT) subtyping, we recently documented that the majority (>80%) of the HIV-1 strains that circulate in Côte d'Ivoire were CRF02_AG and about 11% were recombinants or could not be clearly assigned to a known subtype or CRF. In order to determine in more detail the precise structure of these viruses we sequenced the full-length genomes for six such strains. Bootscan and phylogenetic tree analysis showed that four strains were complex and unique CRF02_AG/CRF09_cpx recombinants, one was a CRF02_AG/CRF06_cpx recombinant, and one was a pure CRF09_cpx. Reanalysis of the remaining recombinants asserted the predominance of CRF09_cpx within intersubtype recombinants and circulation of CRF09_cpx in Côte d'Ivoire. More detailed analysis of the CRF09_cpx strains revealed also that part of the pol gene belonged to subtype K. This is the first time that such recombinants are described.

Characterization of nevirapine (NVP) resistance mutations and HIV type 1 subtype in women from Abidjan (Côte d'Ivoire) after NVP single-dose prophylaxis of HIV type 1 mother-to-child transmission.

Nevirapine (NVP) single dose is widely used in developing countries to prevent HIV-1 mother-to-child transmission. However, this regimen selects key drug resistance mutations that can impair further HAART efficacy. We studied the HIV-1 reverse transcriptase genotype from 29 Ivoirian women 1 month after an NVP single-dose prophylaxis. NVP resistance mutations were observed in six (20.7%) women. The majority of the isolates were CRF02_AG. These results confirm previous studies and suggest the need for different prophylaxis regimens in this setting.

Virological, intracellular and plasma pharmacological parameters predicting response to lopinavir/ritonavir (KALEPHAR study).

OBJECTIVES: To assess the impact of HIV-1 protease mutations and intracellular and plasma lopinavir minimum concentrations (Cmin) on virological success or failure on lopinavir/ritonavir-containing highly active antiretroviral therapy (HAART). DESIGN: HIV-1-infected HAART-experienced patients included in an observational study, received lopinavir/ritonavir (400/100 mg twice a day) plus two to three nucleoside reverse transcriptase inhibitors (NRTI) or one NRTI plus one non-NRTI. A viral load less than 50 copies/ml at month 6 defined virological success. METHODS: Intracellular and plasma lopinavir concentrations were determined by high-pressure liquid chromatography with mass-spectrometry detection. Reverse transcriptase and protease genes were sequenced at baseline and the time of virological failure. RESULTS: When the 38 patients started the lopinavir/ritonavir-based regimen, baseline median (25-75th percentile) values were: CD4 cell count 218 cells/microl (133-477); plasma HIV-1-RNA load 5.3 log10 copies/ml (3.8-5.1); number of lopinavir mutations four per protease gene (two to six). Univariate analysis associated virological success or failure at month 6 (21/38 patients) with the number of baseline lopinavir mutations, intracellular and plasma lopinavir Cmin, and the genotype inhibitory quotient (GIQ) at months 1 and 6. Multivariate analysis showed that the number of baseline lopinavir mutations and intracellular and plasma lopinavir Cmin were independently associated with virological success or failure. We defined the most discriminating intracellular and plasma lopinavir Cmin efficacy thresholds (8 and 4 microg/ml, respectively) and GIQ thresholds (1 and 3, respectively). CONCLUSION: The monitoring of lopinavir/rironavir-based HAART efficacy should include the number of baseline lopinavir/ritonavir mutations, intracellular and plasma lopinavir Cmin and GIQ calculation.

Virologic response to nelfinavir-based regimens: pharmacokinetics and drug resistance mutations (VIRAPHAR study).

OBJECTIVE: To assess the impact of HIV-1 protease and reverse transcriptase (RT) mutations, and pharmacokinetic parameters on virological responses to nelfinavir (NFV)-containing highly active antiretroviral therapy. DESIGN: Naive or antiretroviral-experienced HIV-1-infected subjects were included in a non-randomized, observational cohort study and received two nucleoside RT inhibitors + NFV (750 mg three times per day or 1250 mg twice per day). Virologic success was defined as a virus load < 50 copies/ml for > 6 months. METHODS: RT and protease genes were sequenced at baseline and at the time of virological failure. Plasma NFV trough concentration (Cmin), maximum concentration (Cmax), and AUC0-tau at steady-state were subjected to population pharmacokinetic analysis. RESULTS: Patients (n = 154) enrolled between November 1998 and February 2000 started a twice per day (n = 84) or three times per day (n = 70) NFV-based regimen as first- (n = 48) or second-line therapy when protease inhibitor-naive (n = 64) or -experienced (n = 42). Median follow-up duration was 16 months. Virologic failure occurred in 88 patients. No significant differences were observed between twice per day and three times per day regimens. According to multivariate analysis, NFV Cmin and Cmax, CD4 cell count, number of baseline RT + protease gene mutations, D67N, M184V, T215F/Y in RT, and M36I in protease, were independent factors that were significantly predictive of failure. At failure, L10I, D30N, M36I, V77I, N88S/D or L90M protease mutations had emerged since baseline. Pharmacokinetic parameters were similar in patients with or without emergence of these neo-mutations. The more discriminating NFV Cmin efficacy-threshold was estimated to be 1 mg/l. CONCLUSIONS: Our data confirm the association among individual pharmacokinetic parameters, genotype pattern and virological response to NFV-containing regimens.

Molecular characterization of HIV type 1 isolates from untreated patients of Mumbai (Bombay), India, and detection of rare resistance mutations.

The molecular characterization of HIV-1 isolates in drug-naive cases in the early stages of HIV disease was studied in 128 cases from Mumbai (Bombay), India. Subtype C was largely predominant followed by A-C intersubtype recombinants, one subtype A and one CRF01 AE. Compared to subtype B, subtype C exhibited an important polymorphism; the percentages of substitutions could reach more than 90%. Two isolates showed M184V substitution the reverse transcriptase indicating resistance to 3TC.

Presence of key drug resistance mutations in isolates from untreated patients of Abidjan, Côte d'Ivoire: ANRS 1257 study.

A total of 107 HIV-1 isolates from untreated adult patients recruited in Abidjan, CMte d'Ivoire, in 2001 and 2002 were sequenced in the env, reverse transcriptase (RT), and protease genes. The results show that CRF02_AG is still predominant in this west African population; key mutations of resistance to antiretroviral drugs (NRTI, NNRTI, and PIs) were detected in 5.6% of the patients. We hypothesize that these resistant mutants have been acquired through horizontal transmission. Compared to a previous study carried out by our group in 1997-2000 in a similar population of Abidjan, it seems that there is a dynamic process of resistance and that a survey will be necessary.

HIV type 1 isolates from 200 untreated individuals in Ho Chi Minh City (Vietnam): ANRS 1257 Study. Large predominance of CRF01_AE and presence of major resistance mutations to antiretroviral drugs.

HIV-1 isolates from 200 untreated patients recruited in 2001 and 2002 in the south part of Vietnam and particularly in Ho Chi Minh City were sequenced in the RT, protease, and env genes. Out of 200 isolates 198 belonged to CRF01_AE while only one subtype B and one intersubtype (B-CRF01_AE) recombinant could be observed. Of the isolates 6.5% had major resistance mutations to antiretroviral drugs.

Molecular characterization of non-B HIV type 1 isolates from patients of a department of infectious diseases, University Hospital of Bordeaux, France, 1989-2009.

The molecular characterization of non-B HIV type 1 subtypes and the sociodemographic baseline characteristics have been studied for 114 non-B HIV-1-infected patients followed at the University Hospital of Bordeaux, France, and diagnosed as HIV infected between 1989 and 2009. Individuals enrolled in this study were mainly women with heterosexual transmission in West and Central Africa and who have been discovered to be HIV positive during pregnancy. Nevertheless, HIV acquisition among individuals born in France was significantly increasing. Recombinant form CRF02_AG was the most frequent subtype (38%) among a highly diverse viral background since 19 subtypes and CRFs have been characterized with a maximal diversity observed in the past decade.

Susceptibility to etravirine of HIV type 1 subtype C isolates from nevirapine/efavirenz-experienced patients: comparative interpretation of ANRS and STANFORD algorithms.

Abstract We analyzed subtype C HIV-1 isolates from patients at failure of a regimen including nevirapine or efavirenz for their susceptibility or resistance to etravirine according to the ANRS and STANFORD algorithms. Statistical analysis showed a consensus that more than 45% of these viral strains are potentially resistant to etravirine.