Evaluation of a flow cytometry method for CD4 T cell enumeration based on volumetric primary CD4 gating using thermoresistant reagents

Laboratory follow-up of HIV patients in resource-limited settings requires appropriate instruments for CD4 T cell enumeration. In this study, we evaluated the application of a simplified, mobile and robust flow cytometry system, the Apogee Auto 40 analyzer (Auto40) using thermoresistant reagents, for CD4 T cell enumeration. We measured the absolute CD4 counts in fresh whole blood samples from 170 Senegalese subjects, including 129 HIV-positive (HIV+) patients and 41 HIV-negative (HIV-) controls. Based on volumetric primary CD4 gating, cells were stained with commercially available reagents (Easy MoAb CD4;Bio-D, Valenzano, Italy) and analyzed on the Auto40. The results were compared with those from the FACSCount system (Becton Dickinson, San Jose, USA). Repeatability analysis was performed on duplicate testing of 49 samples on both FACSCount and Auto40. The intra-run precision was measured by 10 replicates using 3 clinical blood samples with low, intermediate and high CD4 concentrations. The results from the two instruments were in good agreement. The percent similarity between the results of both instruments was 99%±relative standard deviation of 12.7%. The concordance correlation coefficient was 0.99. The absolute bias and limits of agreement (LOA) between the two instruments, calculated by Bland-Altman analysis, were clinically acceptable (bias: +4 cells/μl; LOA: -111 to +120 cells/μl). The clinical agreement between the two instruments at a cutoff of 200 CD4 cells/μl was 94%. The repeatability of measurements on the Auto40 was also similar to that observed with FACSCount system (bias +0.1 cells/μl, coefficient of variation 2.5% vs bias -1.1cells/μl, coefficient of variation 2.9% respectively). In conclusion, our results indicate that the Auto 40 system, using thermoresistant reagents, is suitable for CD4 T cell enumeration and will be a helpful tool to improve HIV laboratory monitoring in resource-limited settings.     

Short-term virological efficacy, immune reconstitution, tolerance, and adherence of once-daily dosing of didanosine, lamivudine, and efavirenz in HIV-1-infected African children: ANRS 12103 Burkiname

Access to antiretroviral therapy (ART) and routine laboratory monitoring are limited for HIV-1-infected children from sub-Saharan Africa. This trial conducted in Bobo-Dioulasso, Burkina Faso, aimed to describe the biological efficacy, tolerance, and adherence of the combination of didanosine, lamivudine, efavirenz in once-daily administration among eligible HIV-1-infected children. From February 2006 to November 2007, 51 HIV-1-infected children aged from 30 months to 15 years and eligible for ART were enrolled in a phase II open clinical trial with follow-up visits every 3 months. HIV-1 genotype testing was performed in children with plasma viral load (PVL) >1000 copies per milliliter after ART initiation. Children were followed for a median of 13.4 months [interquartile range (IQR) 12.8-14.2]. At enrollment, median CD4 count was 8% (IQR = 4.5-12). PVL was 341,032 (IQR = 127,838-761,539) copies per milliliter. At 12 months, median CD4 increased significantly by +15% (P < 10(-3)), and median PVL decreases significantly by -290,500 copies per milliliter (P < 10(-4)). Hemoglobin and platelets counts increased significantly by +1.05 g/dL (P < 10(-5)) and 108,500 cells per milliliter (P < 10(-3)), respectively. Based on pill count, mean yearly adherence was 97.3%, and 48% of the children had an adherence rate ≥ 95% at the four quarterly visits. Adherence was better for girls than for boys independently of other sociodemographic variables or markers of HIV disease progression. Drug-resistant mutations were found in 11 children (21.6%). This once-daily drug combination is associated with excellent virological efficacy, immune reconstitution, and good adherence. However, the high prevalence of drug resistance mutations is a matter of concern.     

Diagnostic value of five commercial tests for the rapid diagnosis of Clostridium difficile-associated disease

We compared five commercial immunoassays (Biostar OIA CdTOX AB, ImmunoCard Toxins A&B - Meridian, Xpect C. difficile toxin A/B -Remel, C. difficile toxin A test- Oxoid, and TOX A/B QUIK CHEK- Techlab) which allow a rapid diagnosis of C. difficile associated disease. The tests were performed directly on patient's stool specimen submitted for routine investigation of C. difficile infection from two University Hospitals in Brussels. The cell cytotoxicity assay was considered as the gold standard. Of the 100 stool specimens included in the study 23 were positive for C. difficile toxin. The sensitivity, specificity, positive and negative predictive values were respectively, 95.7%, 100%, 100% and 98.7% for TOX A/B QUIK CHEKTM, 91.3%, 100%, 100% and 97.5% for ImmunoCard Toxins A&B and for Xpect C. difficile toxin A/B, 87%, 100%, 100% and 96.3% for OIA CdTOX AB and 87%, 98.7%, 97.2% and 96.3% for C. difficile toxin A test. The differences were not statistically significant (p > 0.05). These data suggest that the tested immunoassays are acceptable for rapid detection of C. difficile toxin.     

Surveillance sentinelle du VIH chez les femmes enceintes en Mauritanie entre 2001 et 2007

According to the 2008 report on global AIDS epidemic, 33 millions of people are living with HIV/AIDS. Subsaharian Africa is the most affected part of the world. The first case of AIDS in Mauritania was reported in 1987. The national prevalence of HIV/AIDS in the country is estimated at less than 1%. The HIV serosurveillance among pregnant women started in country in 2001. This work has focused on HIV sentinel surveillance among pregnant women in antenatal clinics, attending health centres in different wilayas (regions) of the country in order to assess evolution of prevalence between 2001 and 2007. An anonymous and non-correlated method is used for this survey. A questionnaire was administered and venous sampling made for eligible women. Analyses were performed with an algorithm based on two screening tests (ELISA) and another test for confirmation (New Lav Blot). Despite some disparities between the sites considered, the results have shown a low prevalence rate (between 0.1 and 1.48). The average prevalence of HIV infection samples collected increased from 0.57% [0.34–0.80] in 2001 to 0.61% [0.40–0.82] in 2007 with 95% confidence interval. Statistical analysis showed no significant changes between 2001 and 2007 at all these sites. HIV1 is the most frequent type with a proportion of 93.5% in 2007. After several years of classic HIV sentinel surveillance, and to better understand disparities between sites, we recommend a second generation sentinel surveillance (behavioural and serological) approach.     

The absence of anti-Tat antibodies is associated with risk of disease progression in HIV-2 infection

The Tat protein of human immunodeficiency virus (HIV) is essential for viral replication and has extracellular pathogenic activity. We sought to determine whether the anti-Tat antibody response was predictive of disease progression in 144 HIV type 2 (HIV-2)-infected subjects observed longitudinally between 1985 and 2003. Sixty-eight percent of the subjects tested positive for anti-Tat antibodies, with reactivity notably established early after seroconversion and stably maintained over the course of infection. The risk and rate of progression to advanced HIV-2 AIDS was significantly higher in anti-Tat-negative subjects than in anti-Tat-positive subjects, extending the importance of this prognostic marker for HIV-2 AIDS.     

Low seroprevalence of herpes simplex virus type 2 among pregnant women in Senegal

Herpes simplex virus type 2 (HSV-2) is considered as a major co-factor of both sexual transmission and acquisition of the human immunodeficiency virus (HIV). The HIV epidemic in Senegal is characterized by a remarkable and stable low prevalence. Whether HSV-2 may also constitute a possible co-factor favouring the spreading of HIV epidemic in Senegal is yet unknown. This prompted us to evaluate the HSV-2 seroprevalence in the sentinel population of pregnant women in Senegal. Two hundred and sixty pregnant women attending Roi Baudouin maternity in the capital city Dakar (n = 14; 135) and the antenatal clinic in Kaolack (n = 125), the third city of Senegal, were prospectively recruited between March and August 2003. Fifty-six women (22%) were positive for HSV-2 serology. The prevalence of HSV-2 seropositivity was higher in women living in Dakar (26%) than in those living in Kaolack (16%) (P < 0.01). Only two women from Dakar and two other from Kaolack were found to be HIV-1-infected. Our observations suggest a seemingly low seroprevalence of HSV-2 infection in adult women Senegal, comparable with those usually reported in Western countries. Further, epidemiological surveys are needed to confirm these results in the general population.     

Sequence-based association and selection scans identify drug resistance loci in the Plasmodium falciparum malaria parasite

Through rapid genetic adaptation and natural selection, the Plasmodium falciparum parasite-the deadliest of those that cause malaria-is able to develop resistance to antimalarial drugs, thwarting present efforts to control it. Genome-wide association studies (GWAS) provide a critical hypothesis-generating tool for understanding how this occurs. However, in P. falciparum, the limited amount of linkage disequilibrium hinders the power of traditional array-based GWAS. Here, we demonstrate the feasibility and power improvements gained by using whole-genome sequencing for association studies. We analyzed data from 45 Senegalese parasites and identified genetic changes associated with the parasites' in vitro response to 12 different antimalarials. To further increase statistical power, we adapted a common test for natural selection, XP-EHH (cross-population extended haplotype homozygosity), and used it to identify genomic regions associated with resistance to drugs. Using this sequence-based approach and the combination of association and selection-based tests, we detected several loci associated with drug resistance. These loci included the previously known signals at pfcrt, dhfr, and pfmdr1, as well as many genes not previously implicated in drug-resistance roles, including genes in the ubiquitination pathway. Based on the success of the analysis presented in this study, and on the demonstrated shortcomings of array-based approaches, we argue for a complete transition to sequence-based GWAS for small, low linkage-disequilibrium genomes like that of P. falciparum.     

Human Antibody Response to Aedes aegypti Saliva in an Urban Population in Bolivia: A New Biomarker of Exposure to Dengue Vector Bites

Abstract. Aedes mosquitoes are important vectors of re-emerging diseases in developing countries, and increasing exposure to Aedes in the developed world is currently a source of concern. Given the limitations of current entomologic methods, there is a need for a new effective way for evaluating Aedes exposure. Our objective was to evaluate specific antibody responses to Aedes aegypti saliva as a biomarker for vector exposure in a dengue-endemic urban area. IgG responses to saliva were strong in young children and steadily waned with age. Specific IgG levels were significantly higher in persons living in sites with higher Ae. aegypti density, as measured by using entomologic parameters. Logistic regression showed a significant correlation between IgG to saliva and exposure level, independently of either age or sex. These results suggest that antibody responses to saliva could be used to monitor human exposure to Aedes bites.