In vivo and in vitro analysis of chloroquine resistance in Plasmodium falciparum isolates from Senegal

To determine the predictive value of chloroquine (CQ) resistance markers in Senegal, Plasmodium falciparum DNA polymorphisms in pfmdr1and pfcrt were examined in relation to clinical outcome. Despite CQ treatment, 17% of patients had parasitemia after 28 days. Examination of molecular markers of CQ resistance revealed that 64% of all isolates had the T76 resistant allele at the pfcrt locus, while 30% carried the Y86 resistant allele at the pfmdr1 locus. The pfcrt T76 allele was present not only in all in vivo resistant isolates, 89% of in vitro resistant isolates, but also in 35% of in vitro sensitive isolates. The pfmdr1 N86Y polymorphism did not correlate with in vitro or in vivo CQ resistance. Our data suggest that the pfcrt T76 allele alone is required but not a sufficient predictor for in vivo CQ resistance.     

Molecular evolution of human immunodeficiency virus type 1 subtype A in Senegal: 1988-1997

OBJECTIVE: Few studies have been able to track the genetic diversity of HIV-1 viruses in human populations over time. We analyzed the molecular evolution of subtype A over a 10-year period, in a cohort of female sex workers with a known time of infection. STUDY DESIGN/METHODS: We amplified and sequenced the C2-V3 region of the surface envelope glycoprotein from 73 HIV-1-infected women, infected between 1987-1997. RESULTS: Fifty-one patients were infected by subtype A viruses. The viruses demonstrated significant diversification (p < 0.001) with mean genetic distance increasing from 8.6% in 1989 to 15.9% in 1997. The slope of the fitted curve suggested a rate of diversification of 0.7% per year. The majority of subtype A viruses clustered with HIV-1 subtype A/G recombinant form (IbNG). CONCLUSION: The genetic diversity of HIV-1 subtype A infections doubled over the first 10 years of this high risk population's epidemic, suggesting that implementation of vaccines early in the epidemic may have a higher likelihood of success based on levels of genetic diversity. The A/G recombinant form (IbNG) has taken epidemic proportions in West Africa. This is of particular importance in understanding the epidemiology of HIV-1 subtypes in Africa and to further dissect the potential phenotypic and biological characteristics of these viruses.     

Associations between MHC class I and susceptibility to HIV-2 disease progression

OBJECTIVES: Human immunodeficiency virus type 2 (HIV-2) progression to disease is significantly slower than that of human immunodeficiency virus type 1 (HIV-1). Genetic determinants for susceptibility to disease progression were hypothesized to play a more significant role in this infection compared with HIV-1. We sought to identify common human lymphocyte antigen (HLA) alleles in the Senegalese population and to compare HLA profiles between HIV-2-infected individuals with low and high risk for disease progression. STUDY DESIGN/METHODS: We conducted a case-control study investigating possible associations between MHC class I genes and the risk of disease progression in HIV-2-infected individuals. The MHC class I genotype was molecularly defined using polymerase chain reaction with sequence specific primers (PCR-SSP) in 62 female sex workers from the Dakar, Senegal cohort. Lack of antibodies to the HIV-2 antigen p26 has been previously shown to predict disease progression and was used in this study as a surrogate marker. Twenty-one cases were identified lacking antibodies to p26, therefore at a higher risk of disease progression, and were compared with 41 p26 antibody-positive, randomly selected controls. RESULTS: Statistical analysis showed that HLA B35 was significantly associated with lack of p26 antibodies, and higher risk of disease progression ( < 0.05). The same association was found for the self-defined class I haplotypes B35-Cw4 and A23-Cw 7 ( < 0.05). The HLA B 53 allele was associated with slower disease progression; however, this association was not statistically significant. We observed a trend whereby heterozygotes were at lower risk for HIV-2 disease progression, as previously reported in HIV-1 disease. CONCLUSIONS: In this West African population, a distinct profile of HLA class I alleles was observed, and many of these appear to influence disease progression in HIV-2 infection.     

Role of the CCR5 delta 32 allele in resistance to HIV-1 infection in west Africa

OBJECTIVE: To determine the frequency of the mutant CCR5 delta 32 allele in high-risk HIV-seronegative Africans as compared with the general African population, and to assess its in vitro protective efficacy against HIV-1 infection. STUDY DESIGN: In the homozygous form, the CCR5 delta 32 allele confers resistance to macrophage-tropic (M-tropic) strains of HIV-1. Assuming that genetic characteristics favoring HIV resistance would prevail in a high-risk HIV-seronegative population, we examined the CCR5 genotypes of female commercial sex workers (CSWs) from Dakar, Senegal, who have remained uninfected for an elongated period. METHODS: The CCR5 genetic profile of study participants was determined by polymerase chain reaction (PCR) amplification of genomic DNA followed by sequencing. Peripheral blood mononuclear cells (PBMCs) were infected with different strains of HIV-1 and monitored by p24 enzyme-linked immunosorbent assay (ELISA). RESULTS: We confirmed the presence of two CCR5wt/delta 32 genotypes among 139 individuals (1.44%). PBMCs from these 2 heterozygous individuals were also found to be less susceptible to in vitro infection by an M-tropic HIV-1 primary isolate. CONCLUSIONS: Evidence was found of an increased prevalence of the CCR5wt/delta 32 genotype in a high-risk HIV-seronegative cohort in West Africa. Furthermore, reduced susceptibility to HIV-1 infection among heterozygous individuals supports a role for 32-bp CCR5 deletion in HIV-1 resistance.     

Absolute CD4 T-cell counting in resource-poor settings: direct volumetric measurements versus bead-based clinical flow cytometry instruments

Flow cytometry is an accurate but expensive method to determine absolute CD4 cell counts. We compared different methods to measure absolute CD4 counts in blood samples from HIV-infected and uninfected subjects using a research/clinical flow cytometer (FACScan); a dedicated clinical instrument (FACSCount); and a volumetric, mobile, open-system flow cytometer equipped with 3 fluorescence and 2 light scatter detectors (Cyflow SL blue). The FACScan and Cyflow were used as single-platform instruments, but they differ in running cost, which is a central factor for resource-poor settings. Direct volumetric and bead-based CD4 measurements on the Cyflow were compared with 2 bead-based single-platform CD4 measurements on the FACSCount and on FACScan (TruCount) in "Le Dantec" Hospital, Dakar, Senegal, using whole blood samples from 102 HIV+ and 28 HIV- subjects. The agreement between the various measurement methods was evaluated by Bland-Altman analysis. Volumetric CD4 measurements on the Cyflow using a no-lyse-no-wash (NLNW) procedure and a lyse-no-wash (LNW) procedure correlated well with each other (R2 = 0.98) and with CD4 measurements on the FACSCount (R2 = 0.97) and FACScan (R2 = 0.97), respectively. Red blood cell lysis had no negative effect on the accuracy of absolute CD4 counting on the Cyflow. An excellent correlation was observed between bead-based CD4 measurements on the Cyflow and CD4 measurements on the FACSCount (R2 = 0.99) and FACScan (R2 = 0.99). Rigid internal and external quality control monitoring and adequate training of technicians were considered essential to generate accurate volumetric CD4 measurements on the Cyflow.     

An association of HIV infection and noma in Niger

We report a case of noma having occurred in an adult female patient with HIV. The strain was characterized as HIV-1 group M subtype G. In order to explore the interactions between HIV/AIDS and this disease, we purpose systematic HIV screening for any case of noma, especially for adult patients.     

No difference in clinical progression between patients infected with the predominant human immunodeficiency virus type 1 circulating recombinant form (CRF) 02_AG strain and patients not infected with CRF02_AG,in Western and West-Central Africa: a four-year prospective multicenter study

To compare human immunodeficiency virus (HIV) type 1 disease progression in patients infected by the predominant strain circulating recombinant form (CRF) 02_AG in western and west-central Africa and in patients infected by other strains, a prospective multicenter cohort study was conducted in Cameroon and Senegal. Among the 335 patients, a broad HIV-1 group M subtype diversity was observed in the envelope V3-V5 region, but strain CRF02_AG predominated in both Cameroon and Senegal (61.2% and 62.9%, respectively; P<.8). Multivariate analyses showed no difference between patients infected by CRF02 strains and those infected by other strains in terms of survival (adjusted hazards ratio [HR], 1.16; 95% confidence interval [CI], 0.76-1.78; P=.5), clinical disease progression (HR, 0.79; 95% CI, 0.50-1.25; P=.3), or square root CD4 cell decline (regression coefficient, -0.01; 95% CI, -0.82 to 0.81; P=.9). This study suggests that the predominance of HIV-1 CRF02_AG strain in western and west-central Africa should have no major clinical consequences.     

HIV prevention among male clients of female sex workers in Kaolack, Senegal: results of a peer education program.

This article reports the results of a peer-led HIV prevention education and condom promotion program among transport workers in Kaolack, Senegal. As part of a 2-year longitudinal follow-up study, changes in men's AIDS-related knowledge, sexual behavior, condom use, and perceived barriers to condom use were evaluated by self-reports obtained from a systematic sample of transport workers interviewed before and after intervention. In addition to men's self-reports, preintervention and postintervention data on men's sexual and condom use behavior were gathered from a sample of licensed, commercial sex workers, who cited transport workers as their primary source of clients. Significant increases in men's HIV-related knowledge, previous use of condoms (from 30.4% to 53.5%), and consistent condom use with regular sex partners were documented over the study period, as were significant declines in perceived barriers to condom use. Though men reported significantly fewer sexual encounters with casual and commercial partners at follow-up compared to baseline, these data were unreliable. Women's postintervention reports indicate that a greater proportion of clients (including, but not limited to transport workers) "always" agree to use condoms (p < .01) compared with baseline and that fewer men offer more money for unprotected sex (p < .01). However, women also report taking greater initiative in the mechanics of condom use (supplying the condom, putting it on, and taking it off) than they did prior to the intervention, and significantly (p < .05) fewer women think that most of their clients know how to use a condom. The findings indicate that the peer-mediated intervention had a positive impact on several important outcomes measured and suggest that HIV prevention efforts need to focus on male client groups despite the logistical and methodological challenges.     

Metabolomic analysis of patient plasma yields evidence of plant-like α-linolenic acid metabolism in Plasmodium falciparum

Metabolomics offers a powerful means to investigate human malaria parasite biology and host-parasite interactions at the biochemical level, and to discover novel therapeutic targets and biomarkers of infection. Here, we used an approach based on liquid chromatography and mass spectrometry to perform an untargeted metabolomic analysis of metabolite extracts from Plasmodium falciparum-infected and uninfected patient plasma samples, and from an enriched population of in vitro cultured P. falciparum-infected and uninfected erythrocytes. Statistical modeling robustly segregated infected and uninfected samples based on metabolite species with significantly different abundances. Metabolites of the α-linolenic acid (ALA) pathway, known to exist in plants but not known to exist in P. falciparum until now, were enriched in infected plasma and erythrocyte samples. In vitro labeling with (13)C-ALA showed evidence of plant-like ALA pathway intermediates in P. falciparum. Ortholog searches using ALA pathway enzyme sequences from 8 available plant genomes identified several genes in the P. falciparum genome that were predicted to potentially encode the corresponding enzymes in the hitherto unannotated P. falciparum pathway. These data suggest that our approach can be used to discover novel facets of host/malaria parasite biology in a high-throughput manner.