High Seroprevalence of IgG Antibodies to Multiple Arboviruses in People Living with HIV (PLWHIV) in Madagascar.

To estimate the prevalence of IgG antibodies against six arboviruses in people living with HIV-1 (PLWHIV) in Madagascar, we tested samples collected between January 2018 and June 2021. We used a Luminex-based serological assay to detect IgG antibodies against antigens from Dengue virus serotypes 1-4 (DENV1-4), Zika virus (ZIKV), West Nile virus (WNV), Usutu virus (USUV), Chikungunya virus (CHIKV), and O'nyong nyong virus (ONNV). Of the 1036 samples tested, IgG antibody prevalence was highest for ONNV (28.4%), CHIKV (26.7%), WNV-NS1 (27.1%), DENV1 (12.4%), USUV (9.9%), and DENV3 (8.9%). ZIKV (4.9%), DENV2 (4.6%), WNV-D3 (5.1%), and DENV4 (1.4%) were lower. These rates varied by province of origin, with the highest rates observed in Toamasina, on the eastern coast (50.5% and 56.8%, for CHIKV and ONNV, respectively). The seroprevalence increased with age for DENV1 and 3 (p = 0.006 and 0.038, respectively) and WNV DIII (p = 0.041). The prevalence of IgG antibodies against any given arborvirus varied over the year and significantly correlated with rainfalls in the different areas (r = 0.61, p = 0.036). Finally, we found a significant correlation between the seroprevalence of antibodies against CHIKV and ONNV and the HIV-1 RNA plasma viral load. Thus, PLWHIV in Madagascar are highly exposed to various arboviruses. Further studies are needed to explain some of our findings.

Natural Killer Repertoire Restoration in TB/HIV Co-Infected Individuals Experienced an Immune Reconstitution Syndrome (CAMELIA Trial, ANRS 12153).

IRIS is a common complication in HIV-infected patients treated for tuberculosis (TB) and cART. Our aim was to evaluate NK cell reconstitution in HIV-infected patients with TB-IRIS compared to those without IRIS. 147 HIV-infected patients with TB from the CAMELIA trial were enrolled. HIV+TB+ patients were followed for 32 weeks. The NK cell repertoire was assessed in whole blood at different time points. As CAMELIA has two arms (early and late cART initiation), we analysed them separately. At enrolment, individuals had low CD4 cell counts (27 cells/mm3) and high plasma viral loads (5.76 and 5.50 log/mL for IRIS and non-IRIS individuals, respectively). Thirty-seven people developed IRIS (in the early and late arms). In the early and late arms, we observed similar proportions of total NK and NK cell subsets in TB-IRIS and non-IRIS individuals during follow-up, except for the CD56dimCD16pos (both arms) and CD56dimCD16neg (late arm only) subsets, which were higher in TB-IRIS and non-IRIS individuals, respectively, after cART. Regarding the repertoire and markers of NK cells, significant differences (lower expression of NKp30, NKG2A (CD159a), NKG2D (CD314) were observed in TB-IRIS compared to non-IRIS individuals after the start of cART. In the late arm, some changes (increased expression of CD69, NKG2C, CD158i) were observed in TB-IRIS compared to non-IRIS individuals, but only before cART initiation (during TB treatment). KIR expression by NK cells (CD158a and CD158i) was similar in both groups. CD69 expression by NK cells decreased in all groups. Expression of the NCR repertoire (NKp30, NKp44, NKp46) has similar kinetics in TB-IRIS subjects compared to non-IRIS subjects regardless of the arm analysed. NK cell reconstitution appeared to be better in TB-IRIS subjects. Although NK cell reconstitution is impaired in HIV infection after cART, as previously reported, it does not appear to be affected by the development of IRIS in HIV and TB-infected individuals.

Positive Virological Outcomes of HIV-Infected Patients on Protease Inhibitor-Based Second-Line Regimen in Cambodia: The ANRS 12276 2PICAM Study.

BACKGROUND: Assessment of virological outcomes among HIV-infected patients receiving protease (PR) inhibitor-based second-line regimen are uncommon in Cambodia. The objective of this study is to assess the virological effectiveness of this regimen as well as impact of adherence boosting for patients experiencing virological failure. METHODS: The 2PICAM study (Clinicaltrial: NCT01801618) is a cross-sectional study of HIV-infected adults on PR inhibitor-based second-line regimen since at least 6 months, conducted in 13 representative sites, comprising more than 90% of the target population. Adults with HIV RNA above 250 copies/mL (threshold of the assay) at inclusion received boosted adherence counseling during 3 months followed by HIV RNA control. For confirmed virological failure, genotype resistance test was performed and expert committee used results for therapeutic decision. RESULTS: Among the 1,317 adults enrolled, the median duration of second-line regimen was 5 years. At inclusion, 1,182 (89.7%) patients achieved virological success (<250 copies/mL) and 135 (10.3%) experienced a virological failure (>250 copies/mL). In multivariable analysis, factors associated with virological success were: CD4 cell count between 201 and 350/mm3 (OR: 4.66, 95% CI: 2.57-8.47, p < 0.0001) and >350/mm3 (OR: 6.67, 95% CI: 4.02-11.06, p < 0.0001), duration of PI-based regimen >2 years (OR: 1.64, 95% CI: 1.03-2.62, p = 0.037), ATV-containing regimen (0R: 1.65, 95% CI: 1.04-2.63, p = 0.034) and high level of adherence (OR: 2.41, 95% CI: 1.07-5.41, p = 0.033). After adherence counseling, 63 (46.7%) patients were rescued while 72 (53.3%) were not. For the 54 patients with genotype resistance tests available, high or intermediate levels of resistance to lopinavir, atazanavir, and darunavir were reported for 13 (24%), 12 (22.2%), and 2 (3.7%) patients, respectively. Change to an alternative PR inhibitor-based regimen was recommended for 17 patients and to third-line regimen, including integrase inhibitors for 12. CONCLUSION: This study reports high rate of virological suppression of second-line regimen and importance of adherence boosting prior to deciding any change of ART regimen. Genotype resistance tests appear necessary to guide decisions. Such information was of great importance for National HIV Program to adapt guidelines and program needs for third-line regimen.

Adherence to PI-based 2nd-line regimens in Cambodia is not simply a question of individual behaviour: the ANRS 12276 2PICAM study.

OBJECTIVES: To investigate whether adherence to antiretroviral treatment (ART) can be explained not only by individual factors but also by health care facilities' characteristics, among a sample of people living with HIV (PLWH) treated with PI-based regimens in Cambodia. METHODS: The ANRS 12276 2PICAM cross-sectional survey was conducted between February 2013 and April 2014 among PLWH followed up in 13 health care facilities. The 1316 patients in this analysis corresponded to 90% of the total number of adult patients treated with 2nd-line PI-based regimens in Cambodia in the study period. A variable indicating whether patients were non-adherent (=1) or completely adherent (=0) was constructed. Health care facilities and individual characteristics were included in a two-level logistic model to investigate their influence on patients' adherence to ART. RESULTS: A total of 17% of participants did not adhere to ART. Patients in health care facilities outside the capital Phnom Penh were six times more likely to be non-adherent than those treated in health care facilities in the capital (OR: 6.15, 95% CI [1.47, 25.79]). Providing psychosocial care (provided by psychologist counsellors and/or full-time coaches) was found to be a structural facilitator of adherence, as the probability of non-adherence fell by 38.5% per each additional psychological worker present in health care facilities (OR: 0.62, 95% CI [0.43, 0.89]). Financial constraints were the main individual factor preventing adherence. CONCLUSIONS: Our results suggest that inefficiencies in health care delivery are detrimental to PLWH health and to the exceptional progress currently being made by Cambodia in response to HIV. Policy makers should focus on increasing the number of psychosocial workers, especially in areas outside the capital.

Zoonotic Transmission of Two New Strains of Human T-lymphotropic Virus Type 4 in Hunters Bitten by a Gorilla in Central Africa.

Molecular screening of 300 at-risk people from Central Africa identified 2 human T-lymphotropic virus (HTLV)-4-infected individuals. A zoonotic origin of infection was suggested, as both individuals reported being severely bitten by a gorilla during hunting activities. One strain was highly divergent and was designated as the HTLV-4 subtype-b prototype.

Molecular characterization of a new mosaic Simian Immunodeficiency Virus in a naturally infected tantalus monkey (Chlorocebus tantalus) from Cameroon: a challenge to the virus-host co-evolution of SIVagm in African green monkeys.

African green monkeys (AGMs) represent the most widely distributed non-human primates species in Africa. SIVagm naturally infects four of the 6 AGMs species at high prevalence in a species-specific manner. To date, only limited information is available on molecular characteristics of SIVagm infecting Chlorocebus tantalus. Here, we characterized the full-length genome of a virus infecting a naturally infected captive C. tantalus from Cameroon by amplifying and sequencing sub-genomic PCR fragments. The isolate (SIVagmTAN-CM545) is 9923bp long and contained all canonical genes of a functional SIV. SIVagmTAN-CM545 showed a mosaic structure, with gag, pol, nef and accessory genes closely related to SIVagmSAB infecting Chlorocebus sabaeus monkeys from west Africa, and the env gene, closely related to SIVagmTAN infecting tantalus monkeys from Central Africa. Thus SIVagmTAN-CM545 is SIVagmSAB/SIVagmTAN recombinant. These unexpected findings suggest that the evolution of SIVagm is more complex than previously thought and warrant further studies.

African great apes are naturally infected with roseoloviruses closely related to human herpesvirus 7.

UNLABELLED: Primates are naturally infected with herpesviruses. During the last 15 years, the search for homologues of human herpesviruses in nonhuman primates allowed the identification of numerous viruses belonging to the different herpesvirus subfamilies and genera. No simian homologue of human herpesvirus 7 (HHV7) has been reported to date. To investigate the putative existence of HHV7-like viruses in African great apes, we applied the consensus-degenerate hybrid oligonucleotide primers (CODEHOP) program-mediated PCR strategy to blood DNA samples from the four common chimpanzee subspecies (Pan troglodytes verus, P. t. ellioti, P. t. troglodytes, and P. t. schweinfurthii), pygmy chimpanzees (Pan paniscus), as well as lowland gorillas (Gorilla gorilla gorilla). This study led to the discovery of a novel roseolovirus close to HHV7 in each of these nonhuman primate species and subspecies. Generation of the partial glycoprotein B (1,111-bp) and full-length DNA polymerase (3,036/3,042-bp) gene sequences allowed the deciphering of their evolutionary relationships. Phylogenetic analyses revealed that HHV7 and its African great ape homologues formed well-supported monophyletic lineages whose topological resemblance to the host phylogeny is suggestive of virus-host codivergence. Notably, the evolutionary branching points that separate HHV7 from African great ape herpesvirus 7 are remarkably congruent with the dates of divergence of their hosts. Our study shows that African great apes are hosts of human herpesvirus homologues, including HHV7 homologues, and that the latter, like other DNA viruses that establish persistent infections, have cospeciated with their hosts. IMPORTANCE: Human herpesviruses are known to possess simian homologues. However, surprisingly, none has been identified to date for human herpesvirus 7 (HHV7). This study is the first to describe simian homologues of HHV7. The extensive search performed on almost all African great ape species and subspecies, i.e., common chimpanzees of the four subspecies, bonobos, and lowland gorillas, has allowed characterization of a specific virus in each. Genetic characterization of the partial glycoprotein B and full-length DNA polymerase gene sequences, followed by their phylogenetic analysis and estimation of divergence times, has shed light on the evolutionary relationships of these viruses. In this respect, we conclusively demonstrate the cospeciation between these new viruses and their hosts and report cases of cross-species transmission between two common chimpanzee subspecies in both directions.

Plasma concentrations, efficacy and safety of efavirenz in HIV-infected adults treated for tuberculosis in Cambodia (ANRS 1295-CIPRA KH001 CAMELIA trial).

OBJECTIVE: To assess efavirenz plasma concentrations and their association with treatment efficacy and tolerance of efavirenz 600 mg daily in HIV-tuberculosis co-infected patients. METHODS: HIV-infected adults with CD4+ T cell count ≤ 200/mm(3) received standard 6-month tuberculosis treatment and antiretroviral therapy including a daily-dose of 600 mg of efavirenz, irrespective of their body weight. Mid-dose blood samples were drawn both on tuberculosis treatment (week +2 and week +6 after antiretroviral therapy initiation, and week 22 of follow-up) and off tuberculosis treatment (week 50 of follow-up). Considered therapeutic range was 1,000 to 4,000 ng/mL. Multivariate analysis was performed to evaluate the association between efavirenz concentration below 1,000 ng/mL and virological failure. Linear regression was used to test the association between efavirenz exposure and CD4+ T cell gain. Severe side effects potentially related to efavirenz were described and their association with efavirenz exposure was tested by multivariate analysis. RESULTS: Efavirenz plasma concentrations were available in 540 patients. Median [interquartile range] efavirenz concentrations were 2,674 ng/mL [1,690-4,533], 2,667 ng/mL [1,753-4,494] and 2,799 ng/mL [1,804-4,744] at week +2, week +6, week 22, respectively, and 2,766 ng/mL [1,941-3,976] at week 50. Efavirenz concentrations were lower at week 50 (off rifampicin) compared to week 22 (on rifampicin) (p<0.001). Late attendance to study visit and low hemoglobinemia were the only factors associated with an increased risk of efavirenz concentration below 1,000 ng/mL. Efavirenz concentration below 1,000 ng/mL was not associated with treatment failure. Efavirenz concentration above 4,000 ng/mL was associated with higher risk of central nervous system side effects (p<0.001) and of hepatotoxicity (p<0.001). CONCLUSION: Body weight and tuberculosis treatment were not associated with low efavirenz concentrations or treatment failure, supporting the 600 mg daily-dose of efavirenz in HIV-tuberculosis co-infected patients. High efavirenz concentrations were related to a higher risk of central nervous system side effects and hepatotoxicity. TRIAL REGISTRATION: ClinicalTrials.gov NCT01300481.

[Tuberculosis and HIV co-infection: clinical trial under the coordination of the Institut Pasteur in Cambodia]. / Co-infection tuberculose et VIH: coordination d'un essai clinique randomisé par l'Institut Pasteur du Cambodge.

Tuberculosis is a major cause of death among adults infected by HIV. The CAMELIA (ANRS 1295/CIPRA KH001) randomized clinical trial aimed to determine the optimal timing of ARV initiation after tuberculosis treatment onset to reduce mortality. Here, we describe the trial implementation in five hospitals in Cambodia under the coordination of the Institut Pasteur in Cambodia, its conduct, the challenges and public health benefits in Cambodia and beyond.

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome after early initiation of antiretroviral therapy in a randomized clinical trial.

OBJECTIVE: To analyze cases of paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) in the CAMbodian Early versus Late Introduction of Antiretrovirals (CAMELIA) randomized trial designed to compare early (2 weeks) versus late (8 weeks) antiretroviral therapy (ART) initiation after tuberculosis treatment onset in Cambodia (NCT00226434). METHODS: ART-naive adults with CD4 cell count of 200 cells/µl or less, newly diagnosed tuberculosis, and at least one follow-up visit after ART initiation were included in this analysis. Each case of suspected TB-IRIS was systematically validated by two physicians not involved in patients' management. Factors associated with occurrence of TB-IRIS were identified using the Cox proportional hazard model. RESULTS: Among 597 patients, 26% experienced TB-IRIS with an incidence rate of 37.9 cases per 100 person-years [95% confidence interval (CI) 32.4-44.4]. Main clinical manifestations included new or worsening lymphadenopathy (77.4%) and fever (68.4%). Chest radiograph revealed new or worsening abnormalities in 53.4%. Symptoms resolved in 95.5% of patients. Six deaths were directly related to TB-IRIS. Initiating ART early increased the risk of TB-IRIS by 2.61 (95% CI 1.84-3.70). Extrapulmonary or disseminated tuberculosis, CD4 cell count of 100 cells/µl or less, and HIV RNA concentration more than 6 log10 copies/ml were also significantly associated with higher risk of TB-IRIS. CONCLUSION: Shortening the delay between tuberculosis treatment onset and ART initiation to 2 weeks was associated with an increased risk of developing TB-IRIS. However, TB-IRIS was generally easily manageable. Given the marked reported survival advantage of early ART initiation after tuberculosis treatment onset, these data indicate that fear of TB-IRIS should not be an impediment to early ART in adults with advanced immunodeficiency in resource-limited, high burden settings.

Increased risk of Q151M and K65R mutations in patients failing stavudine-containing first-line antiretroviral therapy in Cambodia.

BACKGROUND: Multi-nucleos(t)ide resistance (MNR) mutations including Q151M, K65R mutations, and insertion at codon 69 of HIV-1 reverse transcriptase coding region may confer resistance to all molecules of nucleos(t)ide reverse transcriptase inhibitors (NRTI). The presence of these mutations is an emerging problem compromising non-nucleoside reverse transcriptase inhibitors and protease inhibitors-based therapies. Furthermore, factors associated with selection of these mutations are still not well defined. The current study aimed to evaluate the frequency and to characterize factors associated with the occurrence of multi-nucleos(t)ide resistance mutations among HIV-1 infected patients failing recommended first-line antiretroviral regimens in Cambodia. METHODOLOGY/PRINCIPAL FINDING: This is a retrospective analysis of HIV-1 drug resistance genotyping of 520 HIV-1 infected patients in virological failure (viral load > 250 copies/mL) while on first-line antiretroviral therapy in Cambodia with at least one reverse transcriptase inhibitor resistance associated mutation. Among these 520 patients, a total of 66 subjects (66/520, 12.7%) presented ≥ 1 MNR mutation, including Q151M, K65R, and Insert69 for 59 (11.3%), 29 (5.6%), and 2 (0.4%) patients, respectively. In multivariate analysis, both Q151M (p = 0.039) and K65R (p = 0.029) mutations were independently associated with current stavudine- compared to zidovudine-use. CONCLUSION: Such selection of mutations by stavudine drastically limits the choice of antiretroviral molecules available for second-line therapy in resource-limited settings. This finding supports the World Health Organization's recommendation for stavudine phase-out.

Detection of cytomegalovirus DNA on dried blood spots collected from infants infected with HIV: an in-house method adaptable in resource-limited settings.

In countries with limited resources, infants infected with HIV are highly exposed to CMV co-infection which probably represents a major risk factor for disease progression in this population. This study aimed to evaluate the performance of a low cost CMV DNA extraction method from DBS and the feasibility of its implementation in laboratories of 4 countries with limited resources. DNA was extracted from DBS with a cationic resin (chelex 100) and amplified with an "in house" real time CMV PCR. Dilutions of a quantified whole blood sample were spotted on paper to evaluate the 95% detection limit. A DBS quality control panel was analyzed in all laboratories. CMV PCR was compared between DBS and liquid whole blood (gold standard) in 2 populations: 418 transplanted patients and 59 infants infected with HIV (median age of 2 months). The CMV PCR 95% detection limit in DBS was 3.87 log10 copies/mL. Its positive and negative predictive values for CMV diagnosis in infants infected with HIV were 100% and 87.5% respectively. Quality control panels gave consistent qualitative results in all laboratories. This assay had high predictive values for CMV diagnosis in infants infected with HIV and its implementation in resource-limited countries with limited resources is feasible.

Different selective pressures shape the evolution of Toll-like receptors in human and African great ape populations.

The study of the genetic and selective landscape of immunity genes across primates can provide insight into the existing differences in susceptibility to infection observed between human and non-human primates. Here, we explored how selection has driven the evolution of a key family of innate immunity receptors, the Toll-like receptors (TLRs), in African great ape species. We sequenced the 10 TLRs in various populations of chimpanzees and gorillas, and analysed these data jointly with a human data set. We found that purifying selection has been more pervasive in great apes than in humans. Furthermore, in chimpanzees and gorillas, purifying selection has targeted TLRs irrespectively of whether they are endosomal or cell surface, in contrast to humans where strong selective constraints are restricted to endosomal TLRs. These observations suggest important differences in the relative importance of TLR-mediated pathogen sensing, such as that of recognition of flagellated bacteria by TLR5, between humans and great apes. Lastly, we used a population genetics-phylogenetics method that jointly analyses polymorphism and divergence data to detect fine-scale variation in selection pressures at specific codons within TLR genes. We identified different codons at different TLRs as being under positive selection in each species, highlighting that functional variation at these genes has conferred a selective advantage in immunity to infection to specific primate species. Overall, this study showed that the degree of selection driving the evolution of TLRs has largely differed between human and non-human primates, increasing our knowledge on their respective biological contribution to host defence in the natural setting.

Nonhuman primate retroviruses from Cambodia: high simian foamy virus prevalence, identification of divergent STLV-1 strains and no evidence of SIV infection.

Nonhuman primates (NHPs) carry retroviruses such as simian immunodeficiency viruses (SIV), simian T-cell lymphotropic viruses (STLV) and simian foamy viruses (SFV). Here, we revisited NHPs from Cambodia to assess the prevalence and diversity of these retroviruses using updated viral detection tools. We screened blood from 118 NHPs consisting of six species (Macaca fascicularis (n=91), Macaca leonine (n=8), Presbytis cristata (n=3), Nycticebus coucang (n=1), Hylobates pileatus (n=14), and Pongo pygmaeus) (n=1) by using a Luminex-based multiplex serology assay that allows the detection of all known SIV/HIV and SFV lineages. We also used highly sensitive PCR assays to detect each simian retrovirus group. Positive PCR products were sequenced and phylogenetically analyzed to infer evolutionary histories. Fifty-three of 118 (44.9%) NHPs tested positive for SFV by serology and 8/52 (15.4%), all from M. fascicularis, were PCR-confirmed. The 8 novel SFV sequences formed a highly supported distinct lineage within a clade composed of other macaque SFV. We observed no serological or molecular evidence of SIV infection among the 118 NHP samples tested. Four of 118 (3.3%) NHPs were PCR-positive for STLV, including one M. fascicularis, one P. cristata, and two H. pileatus. Phylogenetic analyses revealed that the four novel STLV belonged to the PTLV-1 lineage, outside the African radiation of PTLV-1, like all Asian PTLV identified so far. Sequence analysis of the whole STLV-1 genome from a H. pileatus (C578_Hp) revealed a genetic structure characteristic of PTLV. Similarity analysis comparing the STLV-1 (C578_Hp) sequence with prototype PTLVs showed that C578_Hp is closer to PTLV-1s than to all other types across the entire genome. In conclusion, we showed a high frequency of SFV infection but found no evidence of SIV infection in NHPs from Cambodia. We identified for the first time STLV-1 in a P. cristata and in two H. pileatus.

Extensive drug resistance in HIV-infected Cambodian children who are undetected as failing first-line antiretroviral therapy by WHO 2010 guidelines.

Antiretroviral therapy in resource-limited settings is monitored clinically and immunologically according to WHO guidelines. Frequent misclassification of virologic failure is reported, mostly in adults, leading to early therapy switch or late failure diagnosis. Pediatric treatment monitoring and resistance data upon first-line failure are limited, particularly when the 2010-WHO pediatric guidelines are used without routine viral load monitoring. We previously reported high treatment failure misclassification rates by pediatric 2010 guidelines in Cambodian children on first-line therapy. Here we determine the extent and patterns of resistance, with yearly viral load and 6-monthly CD4. Drug resistance mutations were determined using the IAS-USA 2011 list. Predicted resistance interpretation was determined with Stanford Database tools. Fifty-one children with available genotypes met inclusion criteria. All but one (subtype B) were CRF01_AE. The most common regimen was stavudine, lamivudine, and nevirapine (96%), taken for a median of 2.2 years. Resistance was seen in 98%; 96% to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs); 51% with ≥4 mutations. The most common NRTI mutations were 184V/I and 67N and the most common NNRTI mutations were 181C/Y/I/V and 190A/S. A total of 22% had multiresistant mutations and 18% had predicted high-level resistance to subsequent therapy options didanosine, abacavir, etravirine, and tenofovir. In 98% of Cambodian children misclassified as nonfailing first-line therapy by 2010 guidelines, 51% had extensive drug resistance to current and 18% to subsequent antiretroviral therapy. Affordable routine viral load monitoring allowing for early and more accurate treatment failure diagnosis is desperately needed in resource-limited settings.

Prediction of treatment failure using 2010 World Health Organization Guidelines is associated with high misclassification rates and drug resistance among HIV-infected Cambodian children.

BACKGROUND: Antiretroviral therapy (ART) in resource-limited settings (RLSs) is monitored clinically and immunologically, according to World Health Organization (WHO) or national guidelines. Revised WHO pediatric guidelines were published in 2010, but their ability to accurately identify virological failure is unclear. METHODS: We evaluated performance of WHO 2010 guidelines and compared them with WHO 2006 and Cambodia 2011 guidelines among children on ≥6 months of first-line ART at Angkor Hospital for Children between January 2005 and September 2010. We determined sensitivity, specificity, positive and negative predictive values, and accuracy using bootstrap resampling to account for multiple tests per child. Human immunodeficiency virus (HIV) resistance was compared between those correctly and incorrectly identified by each guideline. RESULTS: Among 457 children with 1079 viral loads (VLs), 20% had >400 copies/mL. For children with WHO stage 1/2 HIV, misclassification as failure (met CD4 failure criteria, but VL undetectable) was 64% for WHO 2006 guidelines, 33% for WHO 2010 guidelines, and 81% for Cambodia 2011 guidelines; misclassification as success (did not meet CD4 failure, but VL detectable) was 11%, 12%, and 12%, respectively. For children with WHO stage 3/4 HIV, misclassification as failure was 35% for WHO 2006 guidelines, 40% for WHO 2010 guidelines, and 43% for Cambodia 2011 guidelines; misclassification as success was 13%, 24%, and 21%, respectively. Compared with WHO 2006 guidelines, WHO 2010 guidelines significantly increased the risk of misclassification as success in stage 3/4 HIV (P < .05). The WHO 2010 guidelines failed to identify 98% of children with extensive reverse-transcriptase resistance. CONCLUSIONS: In our cohort, lack of virological monitoring would result in unacceptable treatment failure misclassification, leading to premature ART switch and resistance accumulation. Affordable virological monitoring suitable for use in RLSs is desperately needed.

Genomic tools for evolution and conservation in the chimpanzee: Pan troglodytes ellioti is a genetically distinct population.

In spite of its evolutionary significance and conservation importance, the population structure of the common chimpanzee, Pan troglodytes, is still poorly understood. An issue of particular controversy is whether the proposed fourth subspecies of chimpanzee, Pan troglodytes ellioti, from parts of Nigeria and Cameroon, is genetically distinct. Although modern high-throughput SNP genotyping has had a major impact on our understanding of human population structure and demographic history, its application to ecological, demographic, or conservation questions in non-human species has been extremely limited. Here we apply these tools to chimpanzee population structure, using ∼700 autosomal SNPs derived from chimpanzee genomic data and a further ∼100 SNPs from targeted re-sequencing. We demonstrate conclusively the existence of P. t. ellioti as a genetically distinct subgroup. We show that there is clear differentiation between the verus, troglodytes, and ellioti populations at the SNP and haplotype level, on a scale that is greater than that separating continental human populations. Further, we show that only a small set of SNPs (10-20) is needed to successfully assign individuals to these populations. Tellingly, use of only mitochondrial DNA variation to classify individuals is erroneous in 4 of 54 cases, reinforcing the dangers of basing demographic inference on a single locus and implying that the demographic history of the species is more complicated than that suggested analyses based solely on mtDNA. In this study we demonstrate the feasibility of developing economical and robust tests of individual chimpanzee origin as well as in-depth studies of population structure. These findings have important implications for conservation strategies and our understanding of the evolution of chimpanzees. They also act as a proof-of-principle for the use of cheap high-throughput genomic methods for ecological questions.

Natural killer cell degranulation capacity predicts early onset of the immune reconstitution inflammatory syndrome (IRIS) in HIV-infected patients with tuberculosis.

Immune reconstitution inflammatory syndrome (IRIS) is a common and potentially serious complication occurring in HIV-infected patients being treated for tuberculosis (TB) using combined antiretroviral treatment. A role of adaptive immunity has been suggested in the onset of IRIS, whereas the role of natural killer (NK) cells has not yet been explored. The present study sought to examine the involvement of NK cells in the onset of IRIS in HIV-infected patients with TB and to identify predictive markers of IRIS. A total of 128 HIV-infected patients with TB from the Cambodian Early versus Late Introduction of Antiretroviral Drugs (CAMELIA) trial were enrolled in Cambodia. Thirty-seven of the 128 patients developed IRIS. At inclusion, patients had low CD4 cell counts (27 cells/mm(3)) and high plasma viral load (5.76 and 5.50 log/mL in IRIS and non-IRIS patients, respectively). At baseline, NK-cell degranulation capacity was significantly higher in IRIS patients than in non-IRIS patients (9.6% vs 6.38%, P < .005). At IRIS onset, degranulation capacity did not differ between patients, whereas activating receptor expression was lower in IRIS patients. Patients with degranulation levels > 10.84% had a higher risk of IRIS (P = .002 by log-rank test). Degranulation level at baseline was the most important IRIS predictor (hazard ratio = 4.41; 95% confidence interval, 1.60-12.16). We conclude that NK-degranulation levels identify higher IRIS risk in HIV-infected patients with TB.

Earlier versus later start of antiretroviral therapy in HIV-infected adults with tuberculosis.

BACKGROUND: Tuberculosis remains an important cause of death among patients infected with the human immunodeficiency virus (HIV). Robust data are lacking with regard to the timing for the initiation of antiretroviral therapy (ART) in relation to the start of antituberculosis therapy. METHODS: We tested the hypothesis that the timing of ART initiation would significantly affect mortality among adults not previously exposed to antiretroviral drugs who had newly diagnosed tuberculosis and CD4+ T-cell counts of 200 per cubic millimeter or lower. After beginning the standard, 6-month treatment for tuberculosis, patients were randomly assigned to either earlier treatment (2 weeks after beginning tuberculosis treatment) or later treatment (8 weeks after) with stavudine, lamivudine, and efavirenz. The primary end point was survival. RESULTS: A total of 661 patients were enrolled and were followed for a median of 25 months. The median CD4+ T-cell count was 25 per cubic millimeter, and the median viral load was 5.64 log(10) copies per milliliter. The risk of death was significantly reduced in the group that received ART earlier, with 59 deaths among 332 patients (18%), as compared with 90 deaths among 329 patients (27%) in the later-ART group (hazard ratio, 0.62; 95% confidence interval [CI]; 0.44 to 0.86; P=0.006). The risk of tuberculosis-associated immune reconstitution inflammatory syndrome was significantly increased in the earlier-ART group (hazard ratio, 2.51; 95% CI, 1.78 to 3.59; P<0.001). Irrespective of the study group, the median gain in the CD4+ T-cell count was 114 per cubic millimeter, and the viral load was undetectable at week 50 in 96.5% of the patients. CONCLUSIONS: Initiating ART 2 weeks after the start of tuberculosis treatment significantly improved survival among HIV-infected adults with CD4+ T-cell counts of 200 per cubic millimeter or lower. (Funded by the French National Agency for Research on AIDS and Viral Hepatitis and the National Institutes of Health; CAMELIA ClinicalTrials.gov number, NCT01300481.).

Very high concentrations of active intracellular phosphorylated emtricitabine in neonates (ANRS 12109 trial, step 2).

Our objective was to investigate neonatal emtricitabine (FTC) plasma and intracellular pharmacokinetics. The study was designed as a phase I/II prospective trial in two sequential steps evaluating the combination of tenofovir disoproxil fumarate (TDF) and FTC for the prevention of mother-to-child-transmission (PMTCT) of HIV. HIV-1-infected pregnant women received two tablets of TDF (300 mg) and FTC (200 mg) at onset of labor and then one tablet daily for 7 days postpartum. Based on the data obtained in the first part of the Tenofovir/Emtricitabine in Africa and Asia (TEmAA) Study, single doses of 2 mg/kg of FTC and 13 mg/kg of TDF were given to the neonates within 12 h after birth. A total of 540 FTC plasma concentrations and 44 active intracellular phosphorylated metabolite FTC-TP concentrations were taken from the 36 enrolled women and their neonates. Concentrations were measured by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and analyzed by a population approach. The proposed dose obtained by simulations based on plasma drug concentrations was confirmed. However, median FTC-TP exposures were, respectively, 5.9 and 6.8 times higher in the fetus and the neonate than in the adult. High FTC-TP concentrations were observed in the four children who had serious adverse events (SAEs), but the link between FTC-TP concentrations and SAEs in children was not formally identified. The exposure to the active form of FTC was high in neonates despite plasma drug concentrations equivalent to those in adults. Our results are similar to those obtained with zidovudine or lamivudine.