Christelle Butel

African origin of the malaria parasite Plasmodium vivax

Plasmodium vivax is the leading cause of human malaria in Asia and Latin America but is absent from most of central Africa due to the near fixation of a mutation that inhibits the expression of its receptor, the Duffy antigen, on human erythrocytes. The emergence of this protective allele is not understood because P. vivax is believed to have originated in Asia. Here we show, using a non-invasive approach, that wild chimpanzees and gorillas throughout central Africa are endemically infected with parasites that are closely related to human P. vivax. Sequence analyses reveal that ape parasites lack host specificity and are much more diverse than human parasites, which form a monophyletic lineage within the ape parasite radiation. These findings indicate that human P. vivax is of African origin and likely selected for the Duffy-negative mutation. All extant human P. vivax parasites are derived from a single ancestor that escaped out of Africa.

Biological and genetic characteristics of HIV infections in Cameroon reveals dual group M and O infections and a correlation between SI-inducing phenotype of the predominant CRF02_AG variant and disease stage.

In Yaounde, Cameroon, HIV-1 group-specific V3 serology on 1469 HIV-positive samples collected between 1996 and 2001 revealed that group O infections remained constant around 1% for 6 years. Only one group N sample was identified and 4.3% reacted with group M and O peptides. Although the sensitivity of the group-specific polymerase chain reaction (PCR) in two genomic regions was not optimal, we confirmed, in at least 6 of 49 (12.2%) dual O/M seropositive samples and in 1 of 9 group O samples, dual infection with group O and M viruses (n = 4) or with group O or M virus and an intergroup recombinant virus (n = 3). Partial env (V3-V5) sequences on a subset of 295 samples showed that at least eight subtypes and five circulating recombinant forms (CRFs) of HIV-1 group M co-circulate; more than 60% were CRF02_AG and 11% had discordant subtype/CRF designations between env and gag. Similarly as for subtype B, the proportion of syncytium-inducing strains increased when CD4 counts were low in CRF02_AG-infected patients. The V3-loop charge was significantly lower for non-syncytium-inducing strains than for syncytium-inducing strains but cannot be used as an individual marker to predict phenotype. The two predominant HIV-1 variants in Africa, CRF02_AG and subtype C, thus have different biological characteristics.

The predominance of Human Immunodeficiency Virus type 1 (HIV-1) circulating recombinant form 02 (CRF02_AG) in West Central Africa may be related to its replicative fitness

BackgroundCRF02_AG is the predominant HIV strain circulating in West and West Central Africa. The aim of this study was to test whether this predominance is associated with a higher in vitro replicative fitness relative to parental subtype A and G viruses. Primary HIV-1 isolates (10 CRF02_AG, 5 subtype A and 5 subtype G) were obtained from a well-described Cameroonian cohort. Growth competition experiments were carried out at equal multiplicity of infection in activated T cells and monocyte-derived dendritic cells (MO-DC) in parallel.ResultsDual infection/competition experiments in activated T cells clearly indicated that CRF02_AG isolates had a significant replication advantage over the subtype A and subtype G viruses. The higher fitness of CRF02_AG was evident for isolates from patients with CD4+ T cell counts >200 cells/μL (non-AIDS) or CD4+ T cell counts <200 cells/μL (AIDS), and was independent of the co-receptor tropism. In MO-DC cultures, CRF02_AG isolates showed a slightly but not significantly higher replication advantage compared to subtype A or G isolates.ConclusionWe observed a higher ex vivo replicative fitness of CRF02_AG isolates compared to subtype A and G viruses from the same geographic region and showed that this was independent of the co-receptor tropism and irrespective of high or low CD4+ T cell count. This advantage in replicative fitness may contribute to the dominant spread of CRF02_AG over A and G subtypes in West and West Central Africa.

Inaccurate Diagnosis of HIV-1 Group M and O Is a Key Challenge for Ongoing Universal Access to Antiretroviral Treatment and HIV Prevention in Cameroon

Background Increased access to HIV testing is essential in working towards universal access to HIV prevention and treatment in resource-limited countries. We here evaluated currently used HIV diagnostic tests and algorithms in Cameroon for their ability to correctly identify HIV infections. Methods We estimated sensitivity, specificity, and positive and negative predictive values of 5 rapid/simple tests, of which 3 were used by the national program, and 2 fourth generation ELISAs. The reference panel included 500 locally collected samples; 187 HIV -1 M, 10 HIV-1 O, 259 HIV negative and 44 HIV indeterminate plasmas. Results None of the 5 rapid assays and only 1 ELISA reached the current WHO/UNAIDS recommendations on performance of HIV tests of at least 99% sensitivity and 98% specificity. Overall, sensitivities ranged between 94.1% and 100%, while specificities were 88.0% to 98.8%. The combination of all assays generated up to 9% of samples with indeterminate HIV status, because they reacted discordantly with at least one of the different tests. Including HIV indeterminate samples in test efficiency calculations significantly decreased specificities to a range from 77.9% to 98.0%. Finally, two rapid assays failed to detect all HIV-1 group O variants tested, with one rapid test detecting only 2 out of 10 group O specimens. Conclusion In the era of ART scaling-up in Africa, significant proportions of false positive but also false negative results are still observed with HIV screening tests commonly used in Africa, resulting in inadequate treatment and prevention strategies. Depending on tests or algorithms used, up to 6% of HIV-1 M and 80% of HIV-1 O infected patients in Cameroon do not receive ART and adequate counseling to prevent further transmission due to low sensitivities. Also, the use of tests with low specificities could imply inclusion of up to 12% HIV negative people in ART programs and increase budgets in addition to inconveniences caused to patients.

Scale-up of antiretroviral treatment in sub-Saharan Africa is accompanied by increasing HIV-1 drug resistance mutations in drug-naive patients.

Objectives:To evaluate the frequency and progression over time of the WHO-defined transmitted HIV-1 drug resistance mutations (DRMs) among antiretroviral treatment (ART)-naive HIV-1-infected patients in Cameroon. Design:We analyzed HIV-1 DRM data generated from 369 ART-naive individuals consecutively recruited between 1996 and 2007 in urban and rural areas in Cameroon. Methods:HIV-1 drug resistance genotyping was performed in the pol gene using plasma samples and surveillance DRMs were identified using the 2009 WHO-DRM list. Results:We observed in Yaounde, the capital city, an increasing prevalence of DRMs over time: 0.0% (none of 61 participants) in 1996–1999; 1.9% (one of 53 participants) in 2001; 4.1% (two of 49 participants) in 2002; and 12.3% (10 of 81 participants) in 2007. In the rural areas with more recently implemented ART programs, we found DRMs in six of 125 (4.8%) ART-naive individuals recruited in 2006–2007. DRMs identified in both areas included resistance mutations to protease inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs) and non-NRTIs (NNRTIs) that might impair the efficacy of available first-line and second-line treatments. Conclusion:This report showed an increase in transmitted DRMs in areas where antiretroviral drugs were introduced earlier, although other factors such as natural viral polymorphisms and acquired DRMs through exposure to antiretroviral cannot be totally excluded. Further surveillances are needed to confirm this evolution and inform public health policies on adequate actions to help limit the selection and transmission of drug-resistant HIV, while scaling up access to ART in developing countries.

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.

Effect of storage conditions of dried plasma and blood spots on HIV-1 RNA quantification and PCR amplification for drug resistance genotyping

OBJECTIVES Dried blood spots (DBS) and dried plasma spots (DPS) are easy to collect and store, and have been successfully tested as an alternative to plasma for performing virological analyses. Adequate storage conditions still need to be established and cell-associated proviral DNA in DBS can contribute to the amplified products. We evaluated these two parameters. METHODS Residual samples from 34 HIV-1-infected patients [mean viral load (VL) = 3.93 log(10) copies/mL] were used to prepare DPS and DBS, then stored at 20 degrees C and 37 degrees C. HIV-1 nucleic acids were extracted, with or without DNase treatments, to perform HIV-1 VL quantification and nested RT-PCR to amplify the reverse transcriptase gene (798 bp). RESULTS For DBS stored for 3 months at 20 degrees C, VL could be measured for all samples and results were comparable to plasma VL. At 37 degrees C, a slight decrease was observed after 2 and 3 months (0.16 and 0.37 log(10) copies/mL mean difference, respectively). For DPS, a significant decrease in VL (0.70 and 1.07 log(10) copies/mL after 1 and 2 months, respectively) was seen at 37 degrees C, but not at 20 degrees C. PCR amplifications from DPS were only successful for 50% of samples with an initial VL >10 000 copies/mL after 1 month at 20 degrees C. From DBS, PCR amplifications are possible until 3 months for samples with plasma VL >5000 copies/mL. VL and PCR results for DBS treated with DNase are close to results obtained for DPS. CONCLUSIONS Virological monitoring is still feasible for DBS after 3 months of storage at 37 degrees C when VL is >5000 copies/mL, but DNA contributes largely to the final results.

Identification of a New Circulating Recombinant Form of HIV Type 1, CRF11-cpx, Involving Subtypes A, G, J, and CRF01-AE, in Central Africa

In this study, we characterized three full-length genome sequences with a similar mosaic structure from epidemiologically unlinked individuals from Cameroon (97CM-MP818) and the Central African Republic (99CF-MP1298 and 99CF-MP1307). Phylogenetic and recombinant analysis confirmed that the three strains had a similar complex recombinant genome, which we can designate now as CRF11-cpx. This new CRF was composed of successive fragments of subtype A, G, J, and CRF01-AE. The previously reported GR17 virus from a Greek patient infected in the Democratic Republic of Congo (DRC) has a similar structure and should be considered as the prototype strain of CRF11-cpx. This new CRF circulates in Cameroon, Central African Republic, Gabon, and DRC, although the exact prevalences remain to be determined.

High HIV type 1 group M pol diversity and low rate of antiretroviral resistance mutations among the uniformed services in Kinshasa, Democratic Republic of the Congo.

For the first time the genetic diversity among the uniformed personnel in Kinshasa, the capital city of the Democratic Republic of Congo (DRC), a country that has experienced military conflicts since 1998 and in which the global HIV-1/M pandemic started, has now been documented. A total of 94 HIV-1-positive samples, collected in 2007 in Kinshasa garrison settings from informed consenting volunteers, were genetically characterized in the pol region (protease and RT). An extensive diversity was observed, with 51% of the strains corresponding to six pure subtypes (A 23%, C 13.8%, D, G, H, J, and untypable), 15% corresponding to nine different CRFs (01, 02, 11, 13, 25, 26, 37, 43, and 45), and 34% being unique recombinants with one-third being complex mosaic viruses involving three or more different subtypes/CRFs. Only one strain harbored a single mutation, I54V, associated with drug resistance to protease inhibitors. Due to their high mobility and potential risk behavior, HIV infections in military personnel can lead to an even more complex epidemic in the DRC and to a possible increase of subtype C.

Antiretroviral drug resistance and routine therapy, Cameroon.

Among 128 patients routinely receiving highly active antiretroviral therapy in an HIV/AIDS outpatient clinic in Cameroon, 16.4% had drug resistance after a median of 10 months. Of these, 12.5% had resistance to nucleoside reverse transcriptase inhibitors (NRTIs), 10.2% to non-NRTIs, and 2.3% to protease inhibitors.