Optimized phylogenetic clustering of HIV-1 sequence data for public health applications.

Clusters of genetically similar infections suggest rapid transmission and may indicate priorities for public health action or reveal underlying epidemiological processes. However, clusters often require user-defined thresholds and are sensitive to non-epidemiological factors, such as non-random sampling. Consequently the ideal threshold for public health applications varies substantially across settings. Here, we show a method which selects optimal thresholds for phylogenetic (subset tree) clustering based on population. We evaluated this method on HIV-1 pol datasets (n = 14, 221 sequences) from four sites in USA (Tennessee, Washington), Canada (Northern Alberta) and China (Beijing). Clusters were defined by tips descending from an ancestral node (with a minimum bootstrap support of 95%) through a series of branches, each with a length below a given threshold. Next, we used pplacer to graft new cases to the fixed tree by maximum likelihood. We evaluated the effect of varying branch-length thresholds on cluster growth as a count outcome by fitting two Poisson regression models: a null model that predicts growth from cluster size, and an alternative model that includes mean collection date as an additional covariate. The alternative model was favoured by AIC across most thresholds, with optimal (greatest difference in AIC) thresholds ranging 0.007-0.013 across sites. The range of optimal thresholds was more variable when re-sampling 80% of the data by location (IQR 0.008 - 0.016, n = 100 replicates). Our results use prospective phylogenetic cluster growth and suggest that there is more variation in effective thresholds for public health than those typically used in clustering studies.

Public health in genetic spaces: a statistical framework to optimize cluster-based outbreak detection.

Genetic clustering is a popular method for characterizing variation in transmission rates for rapidly evolving viruses, and could potentially be used to detect outbreaks in 'near real time'. However, the statistical properties of clustering are poorly understood in this context, and there are no objective guidelines for setting clustering criteria. Here, we develop a new statistical framework to optimize a genetic clustering method based on the ability to forecast new cases. We analysed the pairwise Tamura-Nei (TN93) genetic distances for anonymized HIV-1 subtype B pol sequences from Seattle (n = 1,653) and Middle Tennessee, USA (n = 2,779), and northern Alberta, Canada (n = 809). Under varying TN93 thresholds, we fit two models to the distributions of new cases relative to clusters of known cases: 1, a null model that assumes cluster growth is strictly proportional to cluster size, i.e. no variation in transmission rates among individuals; and 2, a weighted model that incorporates individual-level covariates, such as recency of diagnosis. The optimal threshold maximizes the difference in information loss between models, where covariates are used most effectively. Optimal TN93 thresholds varied substantially between data sets, e.g. 0.0104 in Alberta and 0.016 in Seattle and Tennessee, such that the optimum for one population would potentially misdirect prevention efforts in another. For a given population, the range of thresholds where the weighted model conferred greater predictive accuracy tended to be narrow (±0.005 units), and the optimal threshold tended to be stable over time. Our framework also indicated that variation in the recency of HIV diagnosis among clusters was significantly more predictive of new cases than sample collection dates (ΔAIC > 50). These results suggest that one cannot rely on historical precedence or convention to configure genetic clustering methods for public health applications, especially when translating methods between settings of low-level and generalized epidemics. Our framework not only enables investigators to calibrate a clustering method to a specific public health setting, but also provides a variable selection procedure to evaluate different predictive models of cluster growth.

HIV-1 Transmission Clustering and Phylodynamics Highlight the Important Role of Young Men Who Have Sex with Men.

More persons living with HIV reside in the Southern United States than in any other region, yet little is known about HIV molecular epidemiology in the South. We used cluster and phylodynamic analyses to evaluate HIV transmission patterns in middle Tennessee. We performed cross-sectional analyses of HIV-1 pol sequences and clinical data collected from 2001 to 2015 among persons attending the Vanderbilt Comprehensive Care Clinic. Transmission clusters were identified using maximum likelihood phylogenetics and patristic distance differences. Demographic, risk behavior, and clinical factors were assessed evaluating "active" clusters (clusters including sequences sampled 2011-2015) and associations estimated with logistic regression. Transmission risk ratios for men who have sex with men (MSM) were estimated with phylodynamic models. Among 2915 persons (96% subtype-B sequences), 963 (33%) were members of 292 clusters (distance ≤1.5%, size range 2-39). Most clusters (62%, n = 690 persons) were active, either being newly identified (n = 80) or showing expansion on existing clusters (n = 101). Correlates of active clustering among persons with sequences collected during 2011-2015 included MSM risk and ≤30 years of age. Active clusters were significantly more concentrated in MSM and younger persons than historical clusters. Young MSM (YMSM) (≤26.4 years) had high estimated transmission risk [risk ratio = 4.04 (2.85-5.65) relative to older MSM] and were much more likely to transmit to YMSM. In this Tennessee cohort, transmission clusters over time were more concentrated by MSM and younger age, with high transmission risk among and between YMSM, highlighting the importance of interventions among this group. Detecting active clusters could help direct interventions to disrupt ongoing transmission chains.

Tracing the epidemic history of HIV-1 CRF01_AE clusters using near-complete genome sequences.

Human immunodeficiency virus (HIV) has a number of circulating recombinant forms that are the product of recombination between different HIV subtypes. The first circulating recombinant form of HIV-1 to be identified was CRF01_AE, which originated in Central Africa and is now most prevalent in Southeast and East Asia. In this study, we investigated the timescale, evolutionary history, and population genetics of the HIV-1 CRF01_AE strains primarily responsible for the epidemic in Asia. A further aim of our study was to define and standardize the nomenclature and provide well-characterized reference sequences for the phylogenetic transmission clusters of CRF01_AE. We analysed a data set of 334 near-complete genome sequences from various risk groups, sampled between 1990 and 2011 from nine countries. Phylogenetic analyses of these sequences were performed using maximum likelihood and Bayesian methods. Our study confirms that the diversity of HIV-1 CRF01_AE originated in Central Africa in the mid-1970s, was introduced into Thailand between 1979 and 1982, and began expanding there shortly afterwards (1982-1984). Subsequently, multiple clusters significantly contributed to China's HIV epidemic. A Bayesian skyline plot revealed the rapid expansion of CRF01_AE in China around 1999-2000. We identified at least eight different clusters of HIV-1 CRF01_AE formed by rapid expansion into different risk groups and geographic regions in China since the late 1980s.

Reconstructing the HIV-1 CRF02_AG and CRF06_cpx epidemics in Burkina Faso and West Africa using early samples.

BACKGROUND: HIV-1 circulating recombinant forms (CRFs) represent viral recombinant lineages that play a significant role in the global epidemic. Two of them dominate the epidemic in Burkina Faso: CRF06_cpx (first described in this country) and CRF02_AG. We reconstructed the phylodynamics of both recombinant viruses in Burkina Faso and throughout West Africa. METHODS: We analysed CRF06_cpx and CRF02_AG sequences (protease/gp41) from early samples collected in Burkina Faso in 1986 together with other GenBank sequences (1984-2013) in 4 datasets: African CRF06_cpx (210/60); down-sampled CRF06_cpx (146/45); Burkina Faso CRF02_AG (130/39) and West/Central African CRF02_AG (691/298). For each dataset, we analysed both protease and gp41 jointly using the BEAST multilocus analysis and conducted phylogeographic analysis to reconstruct the early migration routes between countries. RESULTS: The time to the most recent common ancestor (tMRCA) of CRF06_cpx was 1979 (1973-1983) for protease and 1981 (1978-1983) for gp41. The gp41 analysis inferred the origin of CRF06_cpx (or at least its parental subtype G lineage) in the Democratic Republic of Congo but migrated to Burkina Faso soon after (1982). Both genes showed that CRF06_cpx radiated to the rest of West Africa predominantly after around 1990. These results were robust to the oversampling of Burkina Faso sequences as they were confirmed in the down-sampled dataset. The tMRCA of the Burkina Faso CRF02_AG lineage was 1979 (1977-1983) for protease and 1980 (1978-1981) for gp41. However, we reconstructed its presence in West Africa much earlier (mid-1960s), with an initial origin in Cameroon and/or Nigeria, and its phylogeographic analysis revealed much interconnection within the region with a lack of country-specific phylogenetic patterns, which prevents tracking its exact migration routes. CONCLUSIONS: Burkina Faso presents a relatively young HIV epidemic, with the diversification of the current in-country CRF02_AG and CRF06_cpx lineages taking place around 1980. This country represents the main source of CRF06_cpx in West Africa. The CRF02_AG epidemic started at least a decade earlier and showed much interchange between West African countries (especially involving coastal countries) suggesting great population mobility and an extensive viral spread in the region.

Integrated prevention of mother-to-child HIV transmission services, antiretroviral therapy initiation, and maternal and infant retention in care in rural north-central Nigeria: a cluster-randomised controlled trial.

BACKGROUND: Antiretroviral therapy (ART) and retention in care are essential for the prevention of mother-to-child HIV transmission (PMTCT). We aimed to assess the effect of a family-focused, integrated PMTCT care package. METHODS: In this parallel, cluster-randomised controlled trial, we pair-matched 12 primary and secondary level health-care facilities located in rural north-central Nigeria. Clinic pairs were randomly assigned to intervention or standard of care (control) by computer-generated sequence. HIV-infected women (and their infants) presenting for antenatal care or delivery were included if they had unknown HIV status at presentation (there was no age limit for the study, but the youngest participant was 16 years old); history of antiretroviral prophylaxis or treatment, but not receiving these at presentation; or known HIV status but had never received treatment. Standard of care included health information, opt-out HIV testing, infant feeding counselling, referral for CD4 cell counts and treatment, home-based services, antiretroviral prophylaxis, and early infant diagnosis. The intervention package added task shifting, point-of-care CD4 testing, integrated mother and infant service provision, and male partner and community engagement. The primary outcomes were the proportion of eligible women who initiated ART and the proportion of women and their infants retained in care at 6 weeks and 12 weeks post partum (assessed by generalised linear mixed effects model with random effects for matched clinic pairs). The trial is registered with ClinicalTrials.gov, number NCT01805752. FINDINGS: Between April 1, 2013, and March 31, 2014, we enrolled 369 eligible women (172 intervention, 197 control), similar across groups for marital status, duration of HIV diagnosis, and distance to facility. Median CD4 count was 424 cells per µL (IQR 268-606) in the intervention group and 314 cells per µL (245-406) in the control group (p<0·0001). Of the 369 women included in the study, 363 (98%) had WHO clinical stage 1 disease, 364 (99%) had high functional status, and 353 (96%) delivered vaginally. Mothers in the intervention group were more likely to initiate ART (166 [97%] vs 77 [39%]; adjusted relative risk 3·3, 95% CI 1·4-7·8). Mother and infant pairs in the intervention group were more likely to be retained in care at 6 weeks (125 [83%] of 150 vs 15 [9%] of 170; adjusted relative risk 9·1, 5·2-15·9) and 12 weeks (112 [75%] of 150 vs 11 [7%] of 168 pairs; 10·3, 5·4-19·7) post partum. INTERPRETATION: This integrated, family-focused PMTCT service package improved maternal ART initiation and mother and infant retention in care. An effective approach to improve the quality of PMTCT service delivery will positively affect global goals for the elimination of mother-to-child HIV transmission. FUNDING: Eunice Kennedy Shriver National Institute of Child Health and Human Development and US National Institutes of Health.

Genomic characterization of two novel HIV-1 unique (CRF01_AE/B) recombinant forms among men who have sex with men in Beijing, China.

We report here two novel HIV-1 recombinant forms (CRF01_AE/B) isolated from two HIV-positive male subjects infected through homosexual contact in Beijing, China. Recombination contributes substantially to the genetic diversity of HIV-1, and is likely to occur in populations in which multiple subtypes circulate. Molecular epidemiological studies showed that subtype B, CRF01_AE, and CRF07_BC are currently cocirculating in parallel among men who have sex with men (MSM) in China, providing the opportunity for the emergence of new recombinants. Phylogenetic analysis of near full-length genome (NFLG) sequences showed that the unique recombinant forms (URFs) were composed of gene regions from CRF01_AE and subtype B. The CRF01_AE region of the recombinants clustered together with a previously described cluster 4 lineage of CRF01_AE. The B regions of both the recombinants clustered within the B strains. The two recombinants were quite similar with six breakpoints in common. These data highlight the importance of continuous surveillance of the dynamic change of HIV-1 subtypes and new recombinants among the MSM population.

Genomic characterization of two novel HIV-1 second-generation recombinant forms among men who have sex with men in Beijing, China.

We report two different unique HIV-1 recombinant viruses from two HIV-positive men who have sex with men (MSM) in Beijing, China. Phylogenetic analysis of near full-length genomes (NFLG) showed that the unique recombinant forms (URFs) were comprised of gene regions from two circulating recombinant forms, CRF01_AE and CRF07_BC, both common in China. The parental CRF01_AE region of the recombinants clustered together with a previously described cluster 4 lineage of CRF01_AE. The CRF07_BC regions of both the recombinants clustered within the CRF07_BC radiation, but were distinct from other CRF07_BC reference sequences. The two recombinant forms had two breakpoints in common. The emergence of the two URFs indicates the ongoing generation of recombinant viruses involving CRF01_AE and CRF07_BC, and may provide insight into our understanding of the dynamics and complexity of the HIV-1 epidemic in China.

Molecular epidemiology of HIV-1 in Jilin province, northeastern China: emergence of a new CRF07_BC transmission cluster and intersubtype recombinants.

OBJECTIVE: To investigate the HIV-1 molecular epidemiology among newly diagnosed HIV-1 infected persons living in the Jilin province of northeastern China. METHODS: Plasma samples from 189 newly diagnosed HIV-1 infected patients were collected between June 2010 and August 2011 from all nine cities of Jilin province. HIV-1 nucleotide sequences of gag P17-P24 and env C2-C4 gene regions were amplified using a multiplex RT-PCR method and sequenced. Phylogenetic and recombination analyses were used to determine the HIV-1 genotypes. RESULTS: Based on all sequences generated, the subtype/CFR distribution was as follows: CRF01_AE (58.1%), CRF07_BC (13.2%), subtype B' (13.2%), recombinant viruses (8.1%), subtype B (3.7%), CRF02_AG (2.9%), subtype C (0.7%). In addition to finding CRF01_AE strains from previously reported transmission clusters 1, 4 and 5, a new transmission cluster was described within the CRF07_BC radiation. Among 11 different recombinants identified, 10 contained portions of gene regions from the CRF01_AE lineage. CRF02_AG was found to form a transmission cluster of 4 in local Jilin residents. CONCLUSIONS: Our study presents a molecular epidemiologic investigation describing the complex structure of HIV-1 strains co-circulating in Jilin province. The results highlight the critical importance of continuous monitoring of HIV-infections, along with detailed socio-demographic data, in order to design appropriate prevention measures to limit the spread of new HIV infections.

HIV-1 subtype A gag variability and epitope evolution.

OBJECTIVE: The aim of this study was to examine the course of time-dependent evolution of HIV-1 subtype A on a global level, especially with respect to the dynamics of immunogenic HIV gag epitopes. METHODS: We used a total of 1,893 HIV-1 subtype A gag sequences representing a timeline from 1985 through 2010, and 19 different countries in Africa, Europe and Asia. The phylogenetic relationship of subtype A gag and its epidemic dynamics was analysed through a Maximum Likelihood tree and Bayesian Skyline plot, genomic variability was measured in terms of G → A substitutions and Shannon entropy, and the time-dependent evolution of HIV subtype A gag epitopes was examined. Finally, to confirm observations on globally reported HIV subtype A sequences, we analysed the gag epitope data from our Kenyan, Pakistani, and Afghan cohorts, where both cohort-specific gene epitope variability and HLA restriction profiles of gag epitopes were examined. RESULTS: The most recent common ancestor of the HIV subtype A epidemic was estimated to be 1956 ± 1. A period of exponential growth began about 1980 and lasted for approximately 7 years, stabilized for 15 years, declined for 2-3 years, then stabilized again from about 2004. During the course of evolution, a gradual increase in genomic variability was observed that peaked in 2005-2010. We observed that the number of point mutations and novel epitopes in gag also peaked concurrently during 2005-2010. CONCLUSION: It appears that as the HIV subtype A epidemic spread globally, changing population immunogenetic pressures may have played a role in steering immune-evolution of this subtype in new directions. This trend is apparent in the genomic variability and epitope diversity of HIV-1 subtype A gag sequences.

Recombinant viruses initiated the early HIV-1 epidemic in Burkina Faso.

We analyzed genetic diversity and phylogenetic relationships among 124 HIV-1 and 19 HIV-2 strains in sera collected in 1986 from patients of the state hospital in Ouagadougou, Burkina Faso. Phylogenetic analysis of the HIV-1 env gp41 region of 65 sequences characterized 37 (56.9%) as CRF06_cpx strains, 25 (38.5%) as CRF02_AG, 2 (3.1%) as CRF09_cpx, and 1 (1.5%) as subtype A. Similarly, phylogenetic analysis of the protease (PR) gene region of 73 sequences identified 52 (71.2%) as CRF06_cpx, 15 (20.5%) as CRF02_AG, 5 (6.8%) as subtype A, and 1 (1.4%) was a unique strain that clustered along the B/D lineage but basal to the node connecting the two lineages. HIV-2 PR or integrase (INT) groups A (n = 17 [89.5%]) and B (n = 2 [10.5%]) were found in both monotypic (n = 11) and heterotypic HIV-1/HIV-2 (n = 8) infections, with few HIV-2 group B infections. Based on limited available sampling, evidence suggests two recombinant viruses, CRF06_cpx and CRF02_AG, appear to have driven the beginning of the mid-1980s HIV-1 epidemic in Burkina Faso.

Optimizing PMTCT service delivery in rural North-Central Nigeria: protocol and design for a cluster randomized study.

Nigeria has more HIV-infected women who do not receive needed services for the prevention of mother-to-child transmission of HIV (PMTCT) than any other nation in the world. To meet the UNAIDS/WHO goal of eliminating mother-to-child HIV transmission by 2015, multiple interventions will be required to scale up PMTCT services, especially to lower-level, rural health facilities. To address this, we are conducting a cluster-randomized controlled study to evaluate the impact and cost-effectiveness of a novel, family-focused integrated package of PMTCT services. A systematic re-assignment of patient care responsibilities coupled with the adoption of point-of-care CD4 + cell count testing could facilitate the ability of lower-cadre health providers to manage PMTCT care, including the provision and scale-up of antiretroviral therapy (ART) to pregnant women in rural settings. Additionally, as influential community members, male partners could support their partners' uptake of and adherence to PMTCT care. We describe an innovative approach to scaling up PMTCT service provision that incorporates considerations of where and from whom women can access services (task-shifting), ease of obtaining a CD4 + cell count result (point-of-care testing), the degree of HIV service integration for HIV-infected women and their infants, and the level of family and community involvement (specifically male partner involvement). This systematic approach, if proven feasible and effective, could be scaled up in Nigeria and similar resource-limited settings as a means to accelerate progress toward eliminating mother-to-child transmission of HIV and help women with HIV infection take ART and live long, healthy lives (Trial registration: NCT01805752).

Estimating the rate of intersubtype recombination in early HIV-1 group M strains.

West Central Africa has been implicated as the epicenter of the HIV-1 epidemic, and almost all group M subtypes can be found there. Previous analysis of early HIV-1 group M sequences from Kinshasa in the Democratic Republic of Congo, formerly Zaire, revealed that isolates from a number of individuals fall in different positions in phylogenetic trees constructed from sequences from opposite ends of the genome as a result of recombination between viruses of different subtypes. Here, we use discrete ancestral trait mapping to develop a procedure for quantifying HIV-1 group M intersubtype recombination across phylogenies, using individuals' gag (p17) and env (gp41) subtypes. The method was applied to previously described HIV-1 group M sequences from samples obtained in Kinshasa early in the global radiation of HIV. Nine different p17 and gp41 intersubtype recombinant combinations were present in the data set. The mean number of excess ancestral subtype transitions (NEST) required to map individuals' p17 subtypes onto the gp14 phylogeny samples, compared to the number required to map them onto the p17 phylogenies, and vice versa, indicated that excess subtype transitions occurred at a rate of approximately 7 × 10(-3) to 8 × 10(-3) per lineage per year as a result of intersubtype recombination. Our results imply that intersubtype recombination may have occurred in approximately 20% of lineages evolving over a period of 30 years and confirm intersubtype recombination as a substantial force in generating HIV-1 group M diversity.

Prevalence of drug resistance-related polymorphisms in treatment-naive individuals infected with nonsubtype B HIV type 1 in Cameroon.

Mutations associated with the use of protease (PR) and reverse transcriptase (RT) inhibitors have been mostly mapped for HIV-1 subtype B. The prevalence of these mutations in drug-naive HIV-1 subtype B-infected individuals is low but occurs at high frequencies in treated individuals. To determine the prevalence of treatment-associated mutations in non-B viruses, we analyzed a 1613-bp pol region of specimens collected from 57 HIV-1-infected treatment-naive individuals from Cameroon. Of the 57 HIV-1 sequences, 43 belonged to CRF02-AG, two to CRF11-cpx, six to subtype A, one to subtype D, and five were unclassifiable. Of the 57 PR sequences, 100% contained at least one codon change giving substitutions at positions 10, 11, 16, 20, 33, 36, 60, 62, 64, 69, 77, and 89. These substitutions gave the following prevalence pattern, 36I/L (100%, 57/57) >89M/I (98%, 56/57)>69K/R (93%, 53/57)>20I/R (89%, 51/57)>16E (16%, 9/57)>64M (12%, 7/57)>10I (11%, 6/57)>11V (5%, 3/57)=62V (5%, 3/57)=77I (5%, 3/57)>233F/V (4%, 2/57)=60E (4%), which differed significantly from subtype B at positions 20, 36, 69, and 89. All but one (98%) of the 57 RT sequences (438 amino acid residues) carried substitutions located at codons 39A (7%), 43E (7%), 122E (7%), 312Q (2%), 333E (2%), 335C/D (89%), 356K (89%), 358K (14%), 365I (2%), 371V (81%), 376S (11%), or 399D (4%); the frequency of these substitutions ranged from <0.5% to 4% in RT of subtype B. The high prevalence of minor mutations associated with protease inhibitors (PI) and reverse transcriptase inhibitors (RTI) represents natural polymorphisms. HIV-1 PR and RT sequences from antiretroviral (ARV)-naive HIV-infected persons in Cameroon are important for monitoring the development of resistance to PIs and RTIs as such mutations could lead to treatment failures in individuals undergoing ARV therapy.

Practice of feeding premasticated food to infants: a potential risk factor for HIV transmission.

OBJECTIVES: Although some caregivers are known to premasticate food for infants, usually during the weaning period, HIV transmission has not been linked to this practice. We describe 3 cases of HIV transmission in the United States possibly related to this practice. PATIENTS AND METHODS: Three cases of HIV infection were diagnosed in children at ages 9, 15, and 39 months; clinical symptomatology prompted the testing. A thorough investigation to rule out alternative modes of transmission was conducted. In addition, phylogenetic comparisons of virus from cases and suspected sources were performed by using the C2V3C3 or gp41 region of env and the p17 coding region of gag. RESULTS: In 2 cases, the mothers were known to be infected with HIV, had not breastfed their children, and perinatal transmission of HIV had previously been ruled out following US HIV testing guidelines. In the third case, a great aunt who helped care for the child was infected with HIV, but the child's mother was not. All 3 children were fed food on multiple occasions that had been premasticated by a care provider infected with HIV; in 2 cases concurrent oral bleeding in the premasticating adult was described. Phylogenetic analyses supported the epidemiologic conclusion that the children were infected through exposure to premasticated food from a caregiver infected with HIV in 2 of the 3 cases. CONCLUSIONS: The reported cases provide compelling evidence linking premastication to HIV infection, a route of transmission not previously reported that has important global implications including being a possible explanation for some of the reported cases of "late" HIV transmission in infants, so far attributed to breastfeeding. Until the risk of premastication and modifying factors (eg, periodontal disease) are better understood, we recommend that health care providers routinely query children's caregivers and expecting parents who are infected with HIV or at risk of HIV infection about this feeding practice and direct them to safer, locally available, feeding options.

Coinfection with HIV-1 and simian foamy virus in West Central Africans.

Frequent infection with zoonotic simian foamy virus (SFV) has been reported among HIV-negative primate hunters in rural Cameroon. Plasma samples obtained from urban commercial sex workers (CSWs; n = 139), patients with sexually transmitted diseases (n = 41), and blood donors (n = 179) in the Democratic Republic of Congo [formerly known as Zaire] and Cameroon were tested for SFV and HIV-1 infection. One CSW and one blood donor were found to be seropositive for both SFV and HIV-1, thereby documenting what are, to our knowledge, the first reported cases of dual SFV and HIV infection. The findings of the present study suggest opportunities for bloodborne and sexual transmission of SFV and highlight the importance of defining the clinical consequences of dual infections.

Evaluation of dried blood spots for human immunodeficiency virus type 1 drug resistance testing.

Dried blood spots (DBS) are simpler to prepare, store, and transport than plasma or serum and may represent a good alternative for drug resistance genotyping, particularly in resource-limited settings. However, the utility of DBS for drug resistance testing is unknown. We investigated the efficiency of amplification of large human immunodeficiency virus type 1 (HIV-1) pol fragments (1,023 bp) from DBS stored at different temperatures, the type of amplified product(s) (RNA and/or DNA), and the similarity between plasma and DBS sequences. We evaluated two matched plasma/DBS panels stored for 5 to 6 years at several temperatures and 40 plasma/DBS specimens collected from untreated persons in Cameroon and stored for 2 to 3 years at -20 degrees C. The amplification of HIV-1 pol was done using an in-house reverse transcriptase-nested PCR assay. Reactions were done with and without reverse transcription to evaluate the contribution of HIV DNA to pol sequences from DBS. Amplification was successful for the DBS samples stored for 5 to 6 years at -20 degrees C or at -70 degrees C but not for those stored at room temperature. Thirty-seven of the 40 (92.5%) DBS from Cameroon were amplifiable, including 8/11 (72.7%) with plasma virus loads of <10,000 RNA copies/ml and all 29 with plasma virus loads of >10,000. Proviral DNA contributed significantly to DBS sequences in 24 of the 37 (65%) specimens from Cameroon. The overall similarity between plasma and DBS sequences was 98.1%. Our results demonstrate the feasibility of DBS for drug resistance testing and indicate that -20 degrees C is a suitable temperature for long-term storage of DBS. The amplification of proviral DNA from DBS highlights the need for a wider evaluation of the concordance of resistance genotypes between plasma and DBS.

Chimpanzee adenovirus antibodies in humans, sub-Saharan Africa.

Human sera from the United States, Thailand, and sub-Saharan Africa and chimpanzee sera were tested for neutralizing antibodies to 3 chimpanzee adenoviruses. Antibodies were more common in humans residing in sub-Saharan Africa than in humans living in the United States or Thailand. This finding suggests cross-species transmission of chimpanzee adenoviruses.

HIV genetic diversity in Cameroon: possible public health importance.

To monitor the evolving molecular epidemiology and genetic diversity of HIV in a country where many distinct strains cocirculate, we performed genetic analyses on sequences from 75 HIV-1-infected Cameroonians: 74 were group M and 1 was group O. Of the group M sequences, 74 were classified into the following env gp41 subtypes or recombinant forms: CRF02 (n = 54), CRF09 (n = 2), CRF13 (n = 2), A (n = 5), CRF11 (n = 4), CRF06 (n = 1), G (n = 2), F2 (n = 2), and E (n = 1, CRF01), and 1 was a JG recombinant. Comparison of phylogenies for 70 matched gp41 and protease sequences showed inconsistent classifications for 18 (26%) strains. Our data show that recombination is rampant in Cameroon with recombinant viruses continuing to recombine, adding to the complexity of circulating HIV strains. This expanding genetic diversity raises public health concerns for the ability of diagnostic assays to detect these unique HIV mosaic variants and for the development of broadly effective HIV vaccines.