Walid Heneine

Drug Resistance Mutations for Surveillance of Transmitted HIV-1 Drug-Resistance: 2009 Update

Programs that monitor local, national, and regional levels of transmitted HIV-1 drug resistance inform treatment guidelines and provide feedback on the success of HIV-1 treatment and prevention programs. To accurately compare transmitted drug resistance rates across geographic regions and times, the World Health Organization has recommended the adoption of a consensus genotypic definition of transmitted HIV-1 drug resistance. In January 2007, we outlined criteria for developing a list of mutations for drug-resistance surveillance and compiled a list of 80 RT and protease mutations meeting these criteria (surveillance drug resistance mutations; SDRMs). Since January 2007, several new drugs have been approved and several new drug-resistance mutations have been identified. In this paper, we follow the same procedures described previously to develop an updated list of SDRMs that are likely to be useful for ongoing and future studies of transmitted drug resistance. The updated SDRM list has 93 mutations including 34 NRTI-resistance mutations at 15 RT positions, 19 NNRTI-resistance mutations at 10 RT positions, and 40 PI-resistance mutations at 18 protease positions.

No evidence of infection with porcine endogenous retrovirus in recipients of porcine islet-cell xenografts

BACKGROUND The study of whether porcine xenografts can lead to porcine endogenous retrovirus (PERV) infection of recipients is critical for evaluating the safety of pig-to-man xenotransplantation. PERV is carried in the pig germline, and all recipients of porcine tissues or organs will be exposed to the virus. METHODS We studied 10 diabetic patients who had received porcine fetal islets between 1990 and 1993, looking for evidence of PERV infection by using PCR serology, PCR, and reverse transcriptase assays. Prolonged xenograft survival (up to a year) was confirmed in five patients by porcine C-peptide excretion and detection of pig mitochondrial DNA (mtDNA) in serum. FINDINGS Despite the evidence for extended exposure to pig cells and despite concomitant immunosuppressive therapy, we were unable to detect markers of PERV infection in any patient. Screening for two PERV sequences in peripheral blood lymphocytes collected 4-7 years after the xenotransplantation was negative. Markers of PERV expression, including viral RNA and reverse transcriptase, were undetectable in sera from both early (day 3 to day 180) and late (4-7 years) time points. Western blot analysis for antibodies was consistently negative. INTERPRETATION These results suggested the absence of PERV infection in these patients. Also this study establishes a minimum standard for post-transplant surveillance of patients given porcine xenografts.

Emergence of unique primate T-lymphotropic viruses among central African bushmeat hunters.

The human T-lymphotropic viruses (HTLVs) types 1 and 2 originated independently and are related to distinct lineages of simian T-lymphotropic viruses (STLV-1 and STLV-2, respectively). These facts, along with the finding that HTLV-1 diversity appears to have resulted from multiple cross-species transmissions of STLV-1, suggest that contact between humans and infected nonhuman primates (NHPs) may result in HTLV emergence. We investigated the diversity of HTLV among central Africans reporting contact with NHP blood and body fluids through hunting, butchering, and keeping primate pets. We show that this population is infected with a wide variety of HTLVs, including two previously unknown retroviruses: HTLV-4 is a member of a phylogenetic lineage that is distinct from all known HTLVs and STLVs; HTLV-3 falls within the phylogenetic diversity of STLV-3, a group not previously seen in humans. We also document human infection with multiple STLV-1-like viruses. These results demonstrate greater HTLV diversity than previously recognized and suggest that NHP exposure contributes to HTLV emergence. Our discovery of unique and divergent HTLVs has implications for HTLV diagnosis, blood screening, and potential disease development in infected persons. The findings also indicate that cross-species transmission is not the rate-limiting step in pandemic retrovirus emergence and suggest that it may be possible to predict and prevent disease emergence by surveillance of populations exposed to animal reservoirs and interventions to decrease risk factors, such as primate hunting.

Naturally acquired simian retrovirus infections in central African hunters

BACKGROUND Hunting and butchering of wild non-human primates infected with simian immunodeficiency virus (SIV) is thought to have sparked the HIV pandemic. Although SIV and other primate retroviruses infect laboratory workers and zoo workers, zoonotic retrovirus transmission has not been documented in natural settings. We investigated zoonotic infection in individuals living in central Africa. METHODS We obtained behavioural data, plasma samples, and peripheral blood lymphocytes from individuals living in rural villages in Cameroon. We did serological testing, PCR, and sequence analysis to obtain evidence of retrovirus infection. FINDINGS Zoonotic infections with simian foamy virus (SFV), a retrovirus endemic in most Old World primates, were identified in people living in central African forests who reported direct contact with blood and body fluids of wild non-human primates. Ten (1%) of 1099 individuals had antibodies to SFV. Sequence analysis from these individuals revealed three geographically-independent human SFV infections, each of which was acquired from a distinct non-human primate lineage: De Brazzas guenon (Cercopithecus neglectus), mandrill (Mandrillus sphinx), and gorilla (Gorilla gorilla), two of which (De Brazzas guenon and mandrill) are naturally infected with SIV. INTERPRETATION Our findings show that retroviruses are actively crossing into human populations, and demonstrate that people in central Africa are currently infected with SFV. Contact with non-human primates, such as happens during hunting and butchering, can play a part in the emergence of human retroviruses and the reduction of primate bushmeat hunting has the potential to decrease the frequency of disease emergence.

Minority HIV-1 Drug Resistance Mutations Are Present in Antiretroviral Treatment–Naïve Populations and Associate with Reduced Treatment Efficacy

Background Transmitted HIV-1 drug resistance can compromise initial antiretroviral therapy (ART); therefore, its detection is important for patient management. The absence of drug-associated selection pressure in treatment-naïve persons can cause drug-resistant viruses to decline to levels undetectable by conventional bulk sequencing (minority drug-resistant variants). We used sensitive and simple tests to investigate evidence of transmitted drug resistance in antiretroviral drug-naïve persons and assess the clinical implications of minority drug-resistant variants. Methods and Findings We performed a cross-sectional analysis of transmitted HIV-1 drug resistance and a case-control study of the impact of minority drug resistance on treatment response. For the cross-sectional analysis, we examined viral RNA from newly diagnosed ART-naïve persons in the US and Canada who had no detectable (wild type, n = 205) or one or more resistance-related mutations (n = 303) by conventional sequencing. Eight validated real-time PCR-based assays were used to test for minority drug resistance mutations (protease L90M and reverse transcriptase M41L, K70R, K103N, Y181C, M184V, and T215F/Y) above naturally occurring frequencies. The sensitive real-time PCR testing identified one to three minority drug resistance mutation(s) in 34/205 (17%) newly diagnosed persons who had wild-type virus by conventional genotyping; four (2%) individuals had mutations associated with resistance to two drug classes. Among 30/303 (10%) samples with bulk genotype resistance mutations we found at least one minority variant with a different drug resistance mutation. For the case-control study, we assessed the impact of three treatment-relevant drug resistance mutations at baseline from a separate group of 316 previously ART-naïve persons with no evidence of drug resistance on bulk genotype testing who were placed on efavirenz-based regimens. We found that 7/95 (7%) persons who experienced virologic failure had minority drug resistance mutations at baseline; however, minority resistance was found in only 2/221 (0.9%) treatment successes (Fisher exact test, p = 0.0038). Conclusions These data suggest that a considerable proportion of transmitted HIV-1 drug resistance is undetected by conventional genotyping and that minority mutations can have clinical consequences. With no treatment history to help guide therapies for drug-naïve persons, the findings suggest an important role for sensitive baseline drug resistance testing.

Prevention of Rectal SHIV Transmission in Macaques by Daily or Intermittent Prophylaxis with Emtricitabine and Tenofovir

Background In the absence of an effective vaccine, HIV continues to spread globally, emphasizing the need for novel strategies to limit its transmission. Pre-exposure prophylaxis (PrEP) with antiretroviral drugs could prove to be an effective intervention strategy if highly efficacious and cost-effective PrEP modalities are identified. We evaluated daily and intermittent PrEP regimens of increasing antiviral activity in a macaque model that closely resembles human transmission. Methods and Findings We used a repeat-exposure macaque model with 14 weekly rectal virus challenges. Three drug treatments were given once daily, each to a different group of six rhesus macaques. Group 1 was treated subcutaneously with a human-equivalent dose of emtricitabine (FTC), group 2 received orally the human-equivalent dosing of both FTC and tenofovir-disoproxil fumarate (TDF), and group 3 received subcutaneously a similar dosing of FTC and a higher dose of tenofovir. A fourth group of six rhesus macaques (group 4) received intermittently a PrEP regimen similar to group 3 only 2 h before and 24 h after each weekly virus challenge. Results were compared to 18 control macaques that did not receive any drug treatment. The risk of infection in macaques treated in groups 1 and 2 was 3.8- and 7.8-fold lower than in untreated macaques (p = 0.02 and p = 0.008, respectively). All six macaques in group 3 were protected. Breakthrough infections had blunted acute viremias; drug resistance was seen in two of six animals. All six animals in group 4 that received intermittent PrEP were protected. Conclusions This model suggests that single drugs for daily PrEP can be protective but a combination of antiretroviral drugs may be required to increase the level of protection. Short but potent intermittent PrEP can provide protection comparable to that of daily PrEP in this SHIV/macaque model. These findings support PrEP trials for HIV prevention in humans and identify promising PrEP modalities.

The Epidemiology of Antiretroviral Drug Resistance among Drug-Naive HIV-1-Infected Persons in 10 US Cities

BACKGROUND The prevalence and characteristics of persons with newly diagnosed human immunodeficiency virus (HIV) infections with or without evidence of mutations associated with drug resistance have not been well described. METHODS Drug-naive persons in whom HIV had been diagnosed during the previous 12 months and who did not have acquired immune deficiency syndrome were sequentially enrolled from 39 clinics and testing sites in 10 US cities during 1997-2001. Genotyping was conducted from HIV-amplification products, by automated sequencing. For specimens identified as having mutations previously associated with reduced antiretroviral-drug susceptibility, phenotypic testing was performed. RESULTS Of 1311 eligible participants, 1082 (83%) were enrolled and successfully tested; 8.3% had reverse transcriptase or major protease mutations associated with reduced antiretroviral-drug susceptibility. The prevalence of these mutations was 11.6% among men who had sex with men but was only 6.1% and 4.7% among women and heterosexual men, respectively. The prevalence was 5.4% and 7.9% among African American and Hispanic participants, respectively, and was 13.0% among whites. Among persons whose sexual partners reportedly took antiretroviral medications, the prevalence was 15.2%. CONCLUSIONS Depending on the characteristics of the patients tested, HIV-genotype testing prior to the initiation of therapy would identify a substantial number of infected persons with mutations associated with reduced antiretroviral-drug susceptibility.

Emergence of Drug-Resistant HIV-1 after Intrapartum Administration of Single-Dose Nevirapine Is Substantially Underestimated

Conventional sequence analysis detects human immunodeficiency virus (HIV)-1 drug resistance mutations in approximately 40% of women shortly after they receive intrapartum single-dose nevirapine (SD-NVP). Using sensitive real-time polymerase chain reaction assays for the K103N and Y181C resistance mutations, we tested genotyped virus before and after SD-NVP in 50 South African women infected with HIV-1 subtype C. By sequence analysis, 40 women had no detectable resistance mutations, and an additional 6 women were negative for Y181C after SD-NVP. We found K103N in 16 (40%) of 40 women and Y181C in 5 (11%) of 46 women at 6-36 weeks postpartum. Clonal sequencing confirmed K103N in 5 of 5 representative samples and Y181C in 4 of 4 samples. Four of the 5 women with newly identified Y181C also had K103N. These findings indicate that resistance mutations emerged in at least 65% of the women after SD-NVP and emphasize the importance of further research to determine the clinical implications.

Ancient co-speciation of simian foamy viruses and primates

Although parasite–host co-speciation is a long-held hypothesis, convincing evidence for long-term co-speciation remains elusive, largely because of small numbers of hosts and parasites studied and uncertainty over rates of evolutionary change. Co-speciation is especially rare in RNA viruses, in which cross-species transfer is the dominant mode of evolution. Simian foamy viruses (SFVs) are ubiquitous, non-pathogenic retroviruses that infect all primates. Here we test the co-speciation hypothesis in SFVs and their primate hosts by comparing the phylogenies of SFV polymerase and mitochondrial cytochrome oxidase subunit II from African and Asian monkeys and apes. The phylogenetic trees were remarkably congruent in both branching order and divergence times, strongly supporting co-speciation. Molecular clock calibrations revealed an extremely low rate of SFV evolution, 1.7 × 10-8 substitutions per site per year, making it the slowest-evolving RNA virus documented so far. These results indicate that SFVs might have co-speciated with Old World primates for at least 30 million years, making them the oldest known vertebrate RNA viruses.

Prevalence of transmitted drug resistance associated mutations and HIV-1 subtypes in new HIV-1 diagnoses, U.S.-2006.

Objective:To determine the distribution of HIV-1 subtypes and the prevalence of transmitted drug resistance-associated mutations (TDRM) among persons newly diagnosed with HIV-1 infection in the United States. Methods:We used sequence data from Variant, Atypical, and Resistant HIV Surveillance (VARHS) collected from newly diagnosed persons in 10 states and 1 county health department in 2006. To evaluate TDRM, we used a mutation list for surveillance of TDRM appropriate for the primarily subtype B HIV epidemic in the United States. Results:Sequences were obtained from 2030 of 10 860 persons newly diagnosed with HIV in 11 surveillance areas. Mutations associated with transmitted drug resistance occurred in 292 (14.6%) persons; TDRM associated with a specific drug class occurred in 156 (7.8%) for non-nucleoside reverse transcriptase inhibitors, 111 (5.6%) for nucleoside reverse transcriptase inhibitors and 90 (4.5%) for protease inhibitors. There were no significant differences in prevalence of TDRM by demographic characteristic. The HIV-1 subtype B was the most prevalent subtype occurring in 1922 (96.2%) persons; subtype C (1.3%) was the most prevalent non-B subtype. Conclusion:We presented a clade B-optimized mutation list for evaluating surveillance of TDRM in the United States and analyzed the largest collection of sequence data obtained from individuals newly diagnosed with HIV. The prevalence of TDRM in persons newly diagnosed with HIV is higher than in previous U.S. studies; however, this is not necessarily a significant trend. Continued reporting of sequence data for public health purposes from all sources will improve representativeness and accuracy in analyzing trends in transmitted drug resistance and genetic diversity.