Could pooled samples method affect SARS-CoV-2 diagnosis accuracy using BGI and Sansure-Biotech RT-PCR kits used in Gabon, Central Africa?

This study aims at establishing specimens pooling approach for the detection of SARS-CoV-2 using the RT-PCR BGI and Sansure-Biotech kits used in Gabon. To validate this approach, 14 positive samples, stored at -20°C for three to five weeks were analyzed individually (as gold standard) and in pools of five, eight and ten in the same plate. We created 14 pools of 5, 8 and 10 samples using 40 µL from each of the selected positive samples mixed with 4, 7 and 9 confirmed negative counterparts in a total volume of 200 µL, 320 µL and 400 µL for the pools of 5, 8 and 10 respectively. Both individual and pooled samples testing was conducted according to the BGI and Sansure-Biotech RT-PCR protocols used at the Professor Daniel Gahouma Laboratory (PDGL). Furthermore, the pooling method was also tested by comparing results of 470 unselected samples tested in 94 pools and individually. Results of our experiment showed that using a BGI single positive sample with cycle threshold (Ct) value of 28.42, confirmed by individual testing, detection occurred in all the pools. On the contrary samples with Ct >31 were not detected in pools of 10 and for these samples (Ct value as high as 37.17) their detection was possible in pool of 8. Regarding the Sansure-Biotech kit, positive samples were detected in all the pool sizes tested, irrespective of their Ct values. The specificity of the pooling method was 100% for the BGI and Sansure-Biotech RT-PCR assays. The present study found an increase in the Ct values with pool size for the BGI and Sansure-Biotech assays. This trend was statistically significant (Pearson's r = 0.978; p = 0,022) using the BGI method where the mean Ct values were 24.04±1.1, 26.74±1.3, 27.91±1.1 and 28.32±1.1 for the individual, pool of 5, 8 and 10 respectively. The testing of the 470 samples showed that one of the 94 pools had a positive test similar to the individual test using the BGI and Sansure-Biotech kits. The saving of time and economizing test reagents by using the pooling method were demonstrated in this study. Ultimately, the pooling method could be used for the diagnosis of SARS-CoV-2 without modifying the accuracy of results in Gabon. We recommend a maximum pool size of 8 for the BGI kit. For the Sansure-Biotech kit, a maximum pool size of 10 can be used without affecting its accuracy compared to the individual testing.

Longitudinal estimation of Plasmodium falciparum prevalence in relation to malaria prevention measures in six sub-Saharan African countries.

BACKGROUND: Plasmodium falciparum prevalence (PfPR) is a widely used metric for assessing malaria transmission intensity. This study was carried out concurrently with the RTS,S/AS01 candidate malaria vaccine Phase III trial and estimated PfPR over ≤ 4 standardized cross-sectional surveys. METHODS: This epidemiology study (NCT01190202) was conducted in 8 sites from 6 countries (Burkina Faso, Gabon, Ghana, Kenya, Malawi, and Tanzania), between March 2011 and December 2013. Participants were enrolled in a 2:1:1 ratio according to age category: 6 months-4 years, 5-19 years, and ≥ 20 years, respectively, per year and per centre. All sites carried out surveys 1-3 while survey 4 was conducted only in 3 sites. Surveys were usually performed during the peak malaria parasite transmission season, in one home visit, when medical history and malaria risk factors/prevention measures were collected, and a blood sample taken for rapid diagnostic test, microscopy, and haemoglobin measurement. PfPR was estimated by site and age category. RESULTS: Overall, 6401 (survey 1), 6411 (survey 2), 6400 (survey 3), and 2399 (survey 4) individuals were included in the analyses. In the 6 months-4 years age group, the lowest prevalence (assessed using microscopy) was observed in 2 Tanzanian centres (4.6% for Korogwe and 9.95% for Bagamoyo) and Lambaréné, Gabon (6.0%), while the highest PfPR was recorded for Nanoro, Burkina Faso (52.5%). PfPR significantly decreased over the 3 years in Agogo (Ghana), Kombewa (Kenya), Lilongwe (Malawi), and Bagamoyo (Tanzania), and a trend for increased PfPR was observed over the 4 surveys for Kintampo, Ghana. Over the 4 surveys, for all sites, PfPR was predominantly higher in the 5-19 years group than in the other age categories. Occurrence of fever and anaemia was associated with high P. falciparum parasitaemia. Univariate analyses showed a significant association of anti-malarial treatment in 4 surveys (odds ratios [ORs]: 0.52, 0.52, 0.68, 0.41) and bed net use in 2 surveys (ORs: 0.63, 0.68, 1.03, 1.78) with lower risk of malaria infection. CONCLUSION: Local PfPR differed substantially between sites and age groups. In children 6 months-4 years old, a significant decrease in prevalence over the 3 years was observed in 4 out of the 8 study sites. Trial registration Clinical Trials.gov identifier: NCT01190202:NCT. GSK Study ID numbers: 114001.

Toxicity studies of medicinal plants used in sub-Saharan Africa.

In sub-Saharan Africa, traditional medicine is widely used in rural and urban areas also. This is essentially due to the prohibitive cost of pharmaceutical-based medicine and the low incomes of a major part of the population. In addition, the efficacies of many of these traditional and plant-based medicines are proven, but the fact remains that certain plants used in traditional medicine have toxic effects. It is in this perspective that we investigated by bibliographic literature on the toxicity of plants used in traditional medicine. It is crucial to gain knowledge on these plant-based medicines prepared and prescribed by practitioners, particularly in terms of toxicity, composition, specific efficacy of disease and to advise practitioners of this alternative medicine on the protection and security of patients.

Alterations in peripheral blood B cell subsets and dynamics of B cell responses during human schistosomiasis.

Antibody responses are thought to play an important role in control of Schistosoma infections, yet little is known about the phenotype and function of B cells in human schistosomiasis. We set out to characterize B cell subsets and B cell responses to B cell receptor and Toll-like receptor 9 stimulation in Gabonese schoolchildren with Schistosoma haematobium infection. Frequencies of memory B cell (MBC) subsets were increased, whereas naive B cell frequencies were reduced in the schistosome-infected group. At the functional level, isolated B cells from schistosome-infected children showed higher expression of the activation marker CD23 upon stimulation, but lower proliferation and TNF-α production. Importantly, 6-months after 3 rounds of praziquantel treatment, frequencies of naive B cells were increased, MBC frequencies were decreased and with the exception of TNF-α production, B cell responsiveness was restored to what was seen in uninfected children. These data show that S. haematobium infection leads to significant changes in the B cell compartment, both at the phenotypic and functional level.

Induction of Plasmodium falciparum-specific CD4+ T cells and memory B cells in Gabonese children vaccinated with RTS,S/AS01(E) and RTS,S/AS02(D).

UNLABELLED: The recombinant circumsporozoite protein (CS) based vaccine, RTS,S, confers protection against Plasmodium falciparum infection in controlled challenge trials and in field studies. The RTS,S recombinant antigen has been formulated with two adjuvant systems, AS01 and AS02, which have both been shown to induce strong specific antibody responses and CD4 T cell responses in adults. As infants and young children are particularly susceptible to malaria infection and constitute the main target population for a malaria vaccine, we have evaluated the induction of adaptive immune responses in young children living in malaria endemic regions following vaccination with RTS,S/AS01(E) and RTS,S/AS02(D). Our data show that a CS-specific memory B cell response is induced one month after the second and third vaccine dose and that CS-specific antibodies and memory B cells persist up to 12 months after the last vaccine injection. Both formulations also induced low but significant amounts of CS-specific IL-2(+) CD4(+) T cells one month after the second and third vaccine dose, upon short-term in vitro stimulation of whole blood cells with peptides covering the entire CS derived sequence in RTS,S. These results provide evidence that both RTS,S/AS01(E) and RTS,S/AS02(D) induced adaptive immune responses including antibodies, circulating memory B cells and CD4(+) T cells directed against P. falciparum CS protein. TRIAL REGISTRATION: ClinicalTrials.gov NCT00307021.

Safety and immunogenicity of the malaria vaccine candidate GMZ2 in malaria-exposed, adult individuals from Lambaréné, Gabon.

Malaria is still one of the major public health threats in sub-Saharan Africa. An effective vaccine could be a sustainable control measure that can be integrated into existing health infrastructures. The malaria vaccine candidate GMZ2 is a recombinant fusion protein of conserved parts of Plasmodium falciparum Glutamate Rich Protein and Merozoite Surface Protein 3 adjuvanted with aluminium hydroxide. GMZ2 is immunogenic and well tolerated in malaria-naive adults from Germany. To assess safety and immunogenicity in malaria-exposed individuals, 40 adults from Lambaréné, Gabon were randomly assigned to receive either 100 µg GMZ2 or a rabies control vaccine three times in monthly intervals. Both vaccines were well tolerated. One month after a full course of vaccination, GMZ2-vaccinated individuals had 1.4-fold (95% confidence interval: [1.1, 1.7]) higher baseline-corrected anti-GMZ2 antibody levels and more GMZ2-specific memory B-cells compared to the rabies group (p=0.039), despite a high prevalence of GMZ2-specific immune reactivity due to previous intense exposure to P. falciparum.

Malaria antigen-mediated enhancement of interleukin-21 responses of peripheral blood mononuclear cells in African adults.

We recently showed that IL-21 is associated with high level of anti-EBA-175 IgG1 and IgG3. Here we have investigated the ability of two malarial antigens, Glutamate-rich protein and merozoite surface protein 3 to induce IL-21 production from PBMCs from malaria-exposed and non-exposed donors. We found that malaria-exposed donors produced significantly more IL-21 compared to non-exposed donors. These data suggest that IL-21 could be involved in the acquisition of immunity to malaria.

Markers of vascular endothelial cell damage and P. falciparum malaria: association between levels of both sE-selectin and thrombomodulin, and cytokines, hemoglobin and clinical presentation.

We investigated associations between markers of damage of vascular endothelial cells (MDVECs) and plasma cytokine levels, hemoglobin level and temperature in individuals with acute uncomplicated malaria, as well as healthy controls, using enzyme linked immunosorbent assay (ELISA) for the presence of soluble endothelial cell adhesion molecule-1 (sE-selectin), circulating granule membrane protein-140 (sP-selectin), circulating thrombomodulin (TM), circulating von Willebrand factor (VWf), interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Significant differences were observed between falciparum malaria patients and the healthy people in term of levels of both sE-selectin and TM. The serum levels of sP-selectin and VWf were comparable between the two groups. The levels of both sE-Selectin and TM correlated positively with temperature, levels of IFN-gamma and levels of TNF-alpha; and negatively with hemoglobin levels. Trends of positive correlations were observed between level of sP-selectin or VWf and temperature. Furthermore, sE-selectin levels correlated with vomiting. These data suggest that sE-selectin and TM might be useful markers of endothelium activation in in vivo studies. Moreover, our results highlight the use of both sE-selctin and TM as markers of anemia.

Interleukin-21 is associated with IgG1 and IgG3 antibodies to erythrocyte-binding antigen-175 peptide 4 of Plasmodium falciparum in Gabonese children with acute falciparum malaria.

Interleukin-21 (IL-21) is a newly described, typical, four-helix cytokine showing significant homology with IL-2, IL-4 and IL-15. It regulates IgG1 production and co-operates with IL-4 in the production of multiple antibody classes in vivo. IgG1 and IgG3 are critically involved in the development of clinical immunity to Plasmodium falciparum malaria. However, the mechanisms driving class-switch recombination towards these specific isotypes remain to be elucidated. Seventy-three children with P. falciparum-positive, thick blood smears were recruited from the pediatric wards of the Albert Schweitzer Hospital and the General Hospital in Lambaréné. Children were grouped into two categories according to age: group A (1 to 5 years old) and group B (6 to 16 years old). Patients with severe (severe anemia and/or hyperparasitemia) and mild malaria were enrolled. Prevalence and level of IL-21, total IgG and subclass (IgG1, IgG2, IgG3 and IgG4) titers were determined in plasma by enzyme-linked immunosorbent assay (ELISA). Plasma IL-21 levels correlated with IgG1 and IgG3 levels. Additionally, plasma IL-21 levels correlated with hemoglobin levels in younger children and with parasite density. Here we describe the relationship between IL-21 and antibodies for erythrocyte-binding antigen-175 (EBA-175) peptide 4, a malaria vaccine candidate in Gabonese children with acute falciparum malaria. This study provides new insights into the field of malaria.

A Duffy binding-like domain is involved in the NKp30-mediated recognition of Plasmodium falciparum-parasitized erythrocytes by natural killer cells.

The recent demonstration that purified natural killer (NK) cells lyse Plasmodium falciparum-parasitized red blood cells (Pf-pRBCs) suggests that innate immunity is important in malaria. NK cell killing--presumably an early host response to infection--requires intimate contact between NK natural cytotoxicity receptors (NCRs) and ligands expressed on the surface of Pf-pRBCs. We investigated whether the Duffy binding-like (DBL)-1 alpha domain of P. falciparum erythrocyte membrane protein-1 (PfEMP-1) expressed on parasitized erythrocytes rendered Pf-pRBCs susceptible to NK cell lysis. We showed that with NKp30-immunoglobulin and NKp46-immunoglobulin fusion proteins and DBL-1alpha peptides NCRs are involved in the NK cell-Pf-pRBC interaction. This interaction was direct, specific, and functional, leading to perforin production and granzyme B release. The prior treatment of NK cells with DBL-1 alpha peptides abolished both this interaction and killing activity, suggesting that DBL-1 alpha -NCRs interaction is the key recognition mechanism leading to parasite killing by NK cells.