Rapid, field-deployable method for genotyping and discovery of single-nucleotide polymorphisms associated with drug resistance in Plasmodium falciparum

Despite efforts to reduce malaria morbidity and mortality, drug-resistant parasites continue to evade control strategies. Recently, emphasis has shifted away from control and toward regional elimination and global eradication of malaria. Such a campaign requires tools to monitor genetic changes in the parasite that could compromise the effectiveness of antimalarial drugs and undermine eradication programs. These tools must be fast, sensitive, unambiguous, and cost-effective to offer real-time reports of parasite drug susceptibility status across the globe. We have developed and validated a set of genotyping assays using high-resolution melting (HRM) analysis to detect molecular biomarkers associated with drug resistance across six genes in Plasmodium falciparum. We improved on existing technical approaches by developing refinements and extensions of HRM, including the use of blocked probes (LunaProbes) and the mutant allele amplification bias (MAAB) technique. To validate the sensitivity and accuracy of our assays, we compared our findings to sequencing results in both culture-adapted lines and clinical isolates from Senegal. We demonstrate that our assays (i) identify both known and novel polymorphisms, (ii) detect multiple genotypes indicative of mixed infections, and (iii) distinguish between variants when multiple copies of a locus are present. These rapid and inexpensive assays can track drug resistance and detect emerging mutations in targeted genetic loci in P. falciparum. They provide tools for monitoring molecular changes associated with changes in drug response across populations and for determining whether parasites present after drug treatment are the result of recrudescence or reinfection in clinical settings.     

In vitro antiplasmodial properties of Flacourtia flavescens Willd. (Flacourtiaceae) and Rytigynia canthioides (Benth.) Robyns (Rubiaceae)

The present study was conducted to investigate the in vitro antimalarial activity of Flacourtia flavescens Willd. (Flacourtiaceae) and Rytigynia canthioides (Benth.) Robyns (Rubiaceae). These two plants are used in Benin folk medicine to treat malaria and fever. Antimalarial activity was assayed on fresh clinical isolates of chloroquine resistant Plasmodium falciparum using the in vitro semi-microtest. The results revealed that the IC(50) varied from 1.55 to 22.36μg/ml. F. flavescens hydro methanol extract was more active than R. canthioides. The study demonstrated scientific rationale behind the traditional usage of these plants, however further bioactivity guided phytochemical analyses are necessary to identify the active principles.     

Posterior sternoclavicular dislocations

The frequency of posterior sternoclavicular dislocations represents 0.019% of the shoulder injuries in the Centre of Traumatology and Orthopedics of Dakar. The posterior form is 0.033% compared to the anterior form. The authors report the cases of posterior sternoclavicular dislocations, occurred with seven men and one woman. Seven of these dislocations were located on the left side, including one case of polytraumatism, one associated with a brachial plexus compression, and one case with a fracture of the kneecap. The authors show the interest of the incidence of Heinig in the diagnosis. The open treatment following the technique of burrows which uses the tendon of the subclavius gives satisfactory results in both functional and anatomical respects. They discuss the orthopaedic methods and insist on their disadvantages, especially in the event of vascular lesion with a risk of haemorrhage of a clogged breach or source of instability, and the difficulty of radiological control after reduction.     

Preliminary study of human Herpesvirus type 8 infection in pregnant women in Dakar (Senegal)]

HHV8 was discovered in 1994 and few studies on this virus have been conducted in Africa. The virus is related to Kaposi sarcoma, an opportunistic affection occurring during HIV infection. No studies have been carried out on this subject in Senegal, a country known for its low KS prevalence even among people living with HIV/AIDS. Thus it will be interesting to explore this field. The aim of our study has been first, to demonstrate the presence of HHV8; second, to evaluate sero-prevalence of the infection in Senegal and third, to determine the specificities of HHV8 infection in our country. We performed our study on 407 pregnant women whose average age was 29.24 years, the majority of whom were Senegalese. HIV serology was done by dot blot for the screening and western blot for the confirmation. For the diagnosis of HHV8 infection, we used the indirect immunofluorescence kit of ABI. HIV infection was low among this study population; 0.5% and no HIV1 infection was mentioned. Among the 407 women, 58 or 14.3% were HHV8 positive and there was no HHV8/HIVco-infection. Regarding marital status, no significant difference was found between HHV8 positive and HHV8 negative among unmarried, monogamous or polygamous women. However, women having had 4 to 5 children were more likely to test positive for HHV8. The difference is significant and a relationship has been established with a p value of 0.02. Regarding pregnancy, HHV8 infection is more closely related to abortion: 17.2% of women who had aborted were HHV8 positive versus 4.9% seronegative. The odds ratio calculation shows a strong correlation with a p value of 0.01. No correlation was found between HHV8 infection of the mother and neonate mortality or Apgar score. However, a relationship did show up between HHV8 infection of the mother and low birth weight. 29.2% of seropositive women had had a child with a birth weight under 2600 g whereas only 16.3% of seronegative women had had babies with low birth weight. We determined that HHV8 is indeed present in Senegal. Further studies should focus on transmission routes as well as the molecular epidemiology of this virus and diseases related to HHV8 infection in Senegal.     

Efficacy, safety, and selection of molecular markers of drug resistance by two ACTs in Mali

We conducted a randomized single-blinded trial comparing the efficacy and safety of artesunate (AS) + amodiaquine (AQ, 3 days) versus AS (3 days) + sulfadoxine-pyrimethamine (SP, single dose) versus AS monotherapy (5 days) in Southern Mali. Uncomplicated malaria cases were followed for 28 days. Molecular markers of drug resistance were determined. After identification of recrudescences by genotyping, both artemisinin-based combination therapies (ACTs) reached nearly 100% efficacy at Day 14 and Day 28 versus 98.3% and 96.5% for AS, respectively (P > 0.05). AS + SP significantly selected DHFR and DHPS mutations associated with sulfadoxine and pyrimethamine resistance (P < 0.001), and AS + AQ equally selected PfCRT and PfMDR1 point mutations associated with chloroquine and AQ resistance (P < 0.001). No significant adverse event attributable to any of the study drugs was found. The ACTs were efficacious and safe, but the selection of markers for resistance to the partner drugs raises concerns over their lifespan in areas of intense malaria transmission.     

Determinants of adherence to highly active antiretroviral therapy among HIV-1-infected patients in Côte d'Ivoire

OBJECTIVE: To assess adherence to HAART and to determine factors associated with poor adherence among HIV-1-infected patients in Abidjan, C?te d'Ivoire. METHODS: A prospective observational study of 614 consecutive patients attending an HIV/AIDS outpatient clinic. Adherence was measured twice at 3-month intervals by self-report of missing doses over 4 days. An adherence level of less than 95% was defined as poor adherence. We used generalized estimating equation models for binomial distribution with repeated measures for data analysis. RESULTS: Of the 591 subjects who completed the study, 74.3% reported adherence levels of 95% or greater. Six factors were independently related to poor adherence: age less than 35 years [relative risk (RR) 1.45; 95% confidence interval (CI) 1.17-1.79], absence of social support (RR 1.66; 95% CI 1.24-2.24), number of daily pills 10 or more (RR 1.47; 95% CI 1.14-1.91), time of adherence assessment (first versus second time assessment RR 1.36; 95% CI 1.12-1.66), CD4 cell count of 250 cells/mul or greater (RR 1.43; 95% CI 1.10-1.88), and not being less worried about HIV infection now that treatments have improved (RR 1.26; 95% CI 1.01-1.58). Drug supply interruptions in the pharmacies were reported by 10.0% of the non-adherent patients as the reason for missing pills. CONCLUSION: Psychosocial factors were found to impact adherence and should be analysed in more detail by further studies. Scaling up antiretroviral therapy in sub-Saharan Africa should be preceded by reliable drug supply and distribution systems.     

Cellular human immunodeficiency virus (HIV)-protective factors: a comparison of HIV-exposed seronegative female sex workers and female blood donors in Abidjan,Côte d'Ivoire

Cellular factors that may protect against human immunodeficiency virus (HIV) infection were investigated in 27 HIV-exposed seronegative (ESN) female sex workers (FSWs) and 27 HIV-seronegative female blood donors. Compared with blood donors, ESN FSWs had significantly decreased expression levels of C-X-C chemokine receptor 4 (CXCR4), but not of C-C chemokine receptor 5, on both memory (P<.001) and naive (P=.041) CD4(+) T cells. CXCR4 down-regulation was associated with prolonged duration of commercial sex work by ESN FSWs. CD38 expression on CD8(+) T cells was significantly increased among ESN FSWs, compared with that among blood donors (P=.017). There were no differences in HLA-DR and CD62L expression between blood donors and ESN FSWs. Proportions of T cells producing the beta-chemokines RANTES (regulated on activation, normally T cell-expressed and -secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta or the cytokines interleukin (IL)-2, IL-4, interferon-gamma, and tumor necrosis factor-alpha, were similar in the 2 groups. These data indicate that ESN FSWs differ from HIV-seronegative female blood donors with respect to immunological factors that have no clear protective potential against HIV transmission.     

Measurement of HIV-1 CRF02_AG-specific T cell responses indicates the dominance of a p24gag epitope in blood donors in Abidjan, Cote d'Ivoire

Characterization of human immunodeficiency virus (HIV)-1-specific immune responses against subtypes circulating in areas where the virus is endemic is critical for the design of candidate vaccines. In Cote d'Ivoire, the most prevalent HIV-1 subtype is CRF02_AG. We detected T cell responses to CRF02_AG consensus p24(gag) or protease peptides in 81% of HIV-1- or HIV-1/2-infected blood donors in Abidjan, Cote d'Ivoire. Both the magnitude and the breadth of interferon- gamma enzyme-linked immunospot responses were inversely correlated with plasma viral load. One frequently recognized peptide in p24(gag) was mapped to the optimal epitope (TPQDLNMML). Further studies of this epitope may be important for the development of HIV-1 vaccines for West Africa and West-Central Africa.     

CCR5 receptor expression is down-regulated in HIV type 2 infection: implication for viral control and protection

HIV-2 is known to display an attenuated phenotype in vivo with prolonged time to disease and decreased rate of transmission. Observational studies in Senegal have demonstrated protection from HIV-1 infection, although the putative mechanism for immunoprotection remains undefined. We evaluated HIV-2-seropositive women from a cohort of commercial sex workers in Dakar, Senegal and identified individuals with very low surface CCR5 receptor expression on CD4+ T cells. In vitro up-regulation of the CCR5 receptor was readily achieved. Down-regulation of the CCR5 was not correlated with activation markers (HLA-DR), beta-chemokine levels, or plasma viral loads. A correlation was observed with HIV-2-specific CD8+ T cell activity as measured by intracellular cytokine production. We postulate that down-regulation of the CCR5 receptor in HIV-2 infection contributes to slower disease course and to the protective mechanism against HIV-1 superinfection, mediated in part by HIV-2-specific cellular immune responses.