C-reactive protein and high-sensitivity C-reactive protein levels in asymptomatic intestinal parasite carriers from urban and rural areas of Gabon.

BACKGROUND: Chronic carriage of intestinal parasitic infections (IPIs) can induce chronic inflammation and dysbiosis, which are risk factors for non-communicable diseases. The objective of this study was to determine the relationship between IPI carriage and inflammation in a population of volunteers living in Gabon. METHODOLOGY AND PRINCIPAL FINDINGS: A cross-sectional study was conducted from September 2020 to November 2021 in asymptomatic volunteers aged 18 years old and over, residing in different areas of Gabon: Libreville (urban area) and Koula-Moutou and Bitam (rural areas). The detection of IPIs was carried out using four common microscopic techniques. C-reactive protein (CRP), and high-sensitivity C-reactive protein (hsCRP) were measured and levels were compared according to the presence or absence of IPI. Overall, 518 participants were included, 64.5% (n = 334) of whom resided in urban area and 35.5% (n = 184) in rural areas. The median age was 35 years (27; 46). The prevalence of asymptomatic IPIs was 29.9% (n = 155), with a significantly higher frequency in rural areas than in urban area (adjusted OR 6.6 (CI 3.2-13.8), p < 0.01). Protozoa were more frequent than soil-transmitted helminths (STHs) in both areas: 81.6% (n = 40) in urban area and 69.8% (n = 74) in rural areas. STHs were predominant in rural areas (48.1% vs 22.4% in urban area. In case of IPI, the median values of CRP (15 (13-15) mg/L vs 13.0 (11.1-14.9) mg/L) and hsCRP (4.2 (1.4-13.0) mg/L vs 2.2(0.4-6.1) mg/L) were higher (p<0.01). Elevated hsCRP and CRP were significantly more frequent in parasitized individuals (for hsCRP: 22.6%, n = 35; for CRP: 52.9%, n = 82); in particular among STH carriers (for hsCRP: 65.9%, n = 27, for CRP: 36.6%, n = 15) (p < 0.01). CONCLUSIONS/SIGNIFICANCE: This first study showed that asymptomatic IPIs, particularly STH carriage are associated with high CRP and hsCRP levels. Further larger and longitudinal studies are needed to elucidate the global and specie-specific enteropathogens link with chronic inflammation.

Prevalence of cardiometabolic risk factors according to urbanization level, gender and age, in apparently healthy adults living in Gabon, Central Africa.

BACKGROUND: The prevalence of cardiometabolic risk factors (CMRFs) is increasing in sub-Saharan Africa and represents a serious public health issue. Accurate data are required to implement adapted prevention programs and healthcare strategies. Thus, the aim of this study was to estimate the prevalence rates of CMRFs according to the level of urbanization, age and gender in Gabon. METHODS: A cross-sectional study was conducted in northern (Bitam), western coast (Libreville, Melen) and southeast (Koulamoutou) areas of Gabon using the World Health Organization's (WHO) stepwise approach for the surveillance of chronic disease risk factors. Participants over 18 years of age, without known underlying disease, living in rural and urban areas of Gabon were included. Sociodemographic, biological, and behavioral data were collected. Univariate and multivariate analysis were used to identify the CMRFs. RESULTS: Of the 978 participants, 499 lived in urban and 479 in rural areas. Their median age was 38[28-50] years. Tobacco (26.1% vs 6.2%; p < 0.01) and excessive alcohol consumption (19.4% vs 9.6%; p < 0.01) predominated in rural than in urban areas, respectively. Urban dwellers had more often insufficient physical activity than rural people (29.5% vs 16.3%; p < 0.01). In total, 79.9% of participants aged under 54 years had a high blood pressure;10.6% of the younger participants had pre-hypertension. Metabolic syndrome was more frequent in women (21.7%) than in men (10.0%) (p < 0.01); 6.4% of men and 2.5% of women had a high Framingham score (p = 0.03). Finally, 54.0% of the participants had three or four CMRFs. The multivariate analysis showed that men were more likely to be smokers and to be at risk of pre-hypertension or high blood pressure (p < 0.01). Women were more likely to be obese or to have a metabolic syndrome (p < 0.01). Living in urban areas was also a risk factor for hypertension, diabetes, metabolic syndrome and high LDL cholesterol level. CONCLUSION: The prevalence of CMRFs was high in the study population. Disparities were observed according to urban and rural areas, gender and age. National prevention and healthcare strategies for cardiometabolic diseases in Gabon should consider these observed differences.

Low diagnostic performance of thick blood smears of 50 µl in comparison with direct examination of 10 µl blood and the leukoconcentration technique of 5ml blood among loiasis-suspected patients with low microfilaremia in Gabon, Central Africa, using the STARD-BLCM guidelines.

BACKGROUND: The aim of this study was to determine performance indicators of thick blood smears of 50 µl (TBS-50), following the Standards for the Reporting of Diagnostic Accuracy Studies-Bayesian Latent Class Model (STARD-BLCM) guidelines. TBS-50 was compared with two common parasitological techniques-direct examination of 10 µl blood and a leukoconcentration of 5 ml-for the diagnosis of microfilaremic loiasis. METHODS: The study population was recruited among patients of the Department of Parasitology-Mycology-Tropical Medicine over a period of 1 year. Age, sex, symptoms, and eosinophilia variables were recorded from laboratory registers and medical files. Direct examination of 10 µl of blood, TBS-50, and the leukoconcentration technique with 5 ml of blood were performed for each patient. The classical formula and BLCM were used to determine the diagnostic accuracy of the three techniques as well as the prevalence of microfilaremic loiasis. Three models were built within the framework of BLCM-the BLCM model I and alternative models II and III-for sensitivity analysis. RESULTS: In total, 191 patients consented to be included. The direct blood examination and TBS-50 yielded comparable qualitative and quantitative results. Hence, they are reported together. The prevalence of Loa loa microfilaremia was 9.4% (95% CI 5.7-14.5; n = 18/191) with direct blood examination/TBS-50 and 12.6% [8.2-18.1] (n = 24/191) for leukoconcentration. Comparing TBS-50 with the leukoconcentration method using the classical formula, the sensitivity was 75.0% [53.3-90.2], specificity was 100.0% [97.8-100.0], the positive predictive value was 100.0% [81.5-100.0], and the negative predictive value was 96.5% [92.6-98.7]. The prevalence of microfilaremic loiasis was estimated at 9.7% [6.2-13.7] using BLCM model I. The outputs of BLCM model I showed sensitivity of 78.9% [65.3-90.3], specificity of 100.0% [99.3-100.0], a positive predictive value of 99.1% [87.2-100.0], and a negative predictive value of 93.0% [87.3-97.7] for direct blood examination/TBS-50. CONCLUSIONS: TBS-50 demonstrates low sensitivity relative to two other techniques. In one in five cases, the result will be falsely declared negative using these methods. However, this method can be deployed with limited funds.

Evolution and spread of Plasmodium falciparum mutations associated with resistance to sulfadoxine-pyrimethamine in central Africa: a cross-sectional study.

BACKGROUND: Efficacy of sulfadoxine-pyrimethamine, the malaria chemoprophylaxis used in pregnant women, and in children when combined with amodiaquine, is threatened by the accumulation of mutations in the Plasmodium falciparum dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes. Data on the prevalence of resistant alleles in central Africa and the new pfdhps I431V mutation, particularly associated with other mutations to form the pfdhps vagKgs allele, are scarce. We explored the frequency and geographical distribution of pfdhps and pfdhfr mutations in central Africa in 2014-18, and assessed the evolutionary origin of the vagKgs allele. METHODS: Samples were collected at 18 health-care centres in seven countries (Angola, Cameroon, Central African Republic, Democratic Republic of the Congo, Gabon, Nigeria, and Republic of the Congo) from patients who showed possible symptoms of malaria between March 1, 2014, and Oct 31, 2018. Samples that were positive for P falciparum were transported to a laboratory in Toulouse, France, and genotyped. The frequency of pfdhfr and pfdhps mutations was studied in 1749 samples. Microsatellites in pfdhps flanking regions and whole-genome analysis compared with parasite genomes from the data-sharing network MalariaGEN were performed on samples carrying the vagKgs allele. FINDINGS: Mapping of the prevalence of single nucleotide polymorphisms and corresponding alleles of pfdhfr and pfdhps showed a substantial spread of alleles associated with sulfadoxine-pyrimethamine resistance in central Africa during the 2014-18 period, especially an increase going west to east in pfdhps alleles carrying the K540E and A581G mutations. A high prevalence of the pfdhps I431V mutation was observed in Cameroon (exceeding 50% in the northern region) and Nigeria. Genomic analysis showed a recent African emergence and a clonal expansion of the most frequent pfdhps vagKgs allele. INTERPRETATION: Reduced sulfadoxine-pyrimethamine efficacy due to increased resistance is a worrying situation, especially because the malaria transmission level is high in central Africa. Although the resistance phenotype remains to be confirmed, the emergence and spread of the vagKgs allele in west and central Africa could challenge the use of sulfadoxine-pyrimethamine. FUNDING: Toulouse Institute for Infectious and Inflammatory Diseases.

Sociodemographics, Clinical Factors, and Biological Factors Associated with Loiasis in Endemic Onchocerciasis Areas in Southern Gabon.

To implement the appropriate strategies for scale-up interventions to eliminate onchocerciasis without severe adverse events, clinical and biological factors associated with loiasis were analyzed in onchocerciasis-endemic areas. Blood was collected from volunteers after examination by a physician. Detection of microfilariae and measurement of Ov16 IgG4 were performed using direct microscopic examination of blood and onchocerciasis rapid test detection, respectively. Areas with sporadic, hypoendemic, and hyperendemic onchocerciasis endemicity were found. Participants with microfilaremia were considered microfilaremic, and those without microfilaremia were seen as amicrofilaremic. Of the 471 study participants, 40.5% (n = 191) had microfilariae. Among them, Mansonella spp. was the most common (78.2%, n = 147), followed by Loa loa (41.4%, n = 79). The association between the two species represented 18.3% (n = 35). The specific immunoglobulins of Onchocerca volvulus were detected in 24.2% of participants (n = 87/359). Overall prevalence of L. loa was 16.8%. Hypermicrofilaremia was found in 3% (N = 14), and one participant had more than 30,000 microfilaremiae per milliliter. The frequency of L. loa did not vary according to the level of onchocerciasis transmission. Pruritus was the most common clinical sign (60.5%, n = 285) reported, mainly in microfilaremic participants (72.2%, n = 138/191). The prevalence of L. loa microfilaria in the study population was below the threshold at risk for the occurrence of serious side effects due to ivermectin. Clinical manifestations frequently observed could be exacerbated by microfilaremia in areas where onchocerciasis transmission is high.

Drug Resistance Molecular Markers of Plasmodium falciparum and Severity of Malaria in Febrile Children in the Sentinel Site for Malaria Surveillance of Melen in Gabon: Additional Data from the Plasmodium Diversity Network African Network.

The objective of this study was to analyze the relationship between the frequency of artemisinin-based combination (ACT) drug resistance molecular markers and clinical forms of P. falciparum malaria and parasitemia. A cross-sectional study was carried out between January and April 2014 at the Operational Clinical Research Unit of Melen in febrile children aged 12 to 240 months with a Plasmodium sp. infection. A total of 3 mL of peripheral blood collected from an EDTA tube was used for leukocyte depletion. DNA mutation detection was performed by next generation sequencing (NGS). A total of 1075 patients were screened for malaria. Among them, 384 had a Plasmodium infection. P. falciparum mono-infection was found in 98.9% of the patients. Pfcrt-326T mutation was found in all isolates, while 37.9% had Pfmdr2-484I mutant allele. The highest median parasite densities were found in patients infected by parasites carrying the CVIET haplotype of the Pfcrt gene. The different genetic profiles found here, and their variations according to clinical and biological signs of severe malaria, are additional arguments for the surveillance of P. falciparum strains.

Randomized, open-label, phase 2a study to evaluate the contribution of artefenomel to the clinical and parasiticidal activity of artefenomel plus ferroquine in African patients with uncomplicated Plasmodium falciparum malaria.

BACKGROUND: The contribution of artefenomel to the clinical and parasiticidal activity of ferroquine and artefenomel in combination in uncomplicated Plasmodium falciparum malaria was investigated. METHODS: This Phase 2a, randomized, open-label, parallel-group study was conducted from 11th September 2018 to 6th November 2019 across seven centres in Benin, Burkina Faso, Gabon, Kenya, and Uganda. Patients aged ≥ 14-69 years with microscopically confirmed infection (≥ 3000 to ≤ 50,000 parasites/µL blood) were randomized 1:1:1:1 to 400 mg ferroquine, or 400 mg ferroquine plus artefenomel 300, 600, or 1000 mg, administered as a single oral dose. The primary efficacy analysis was a logistic regression evaluating the contribution of artefenomel exposure to Day 28 PCR-adjusted adequate clinical and parasitological response (ACPR). Safety was also evaluated. RESULTS: The randomized population included 140 patients. For the primary analysis in the pharmacokinetic/pharmacodynamic efficacy population (N = 121), the contribution of artefenomel AUC0-∞ to Day 28 PCR-adjusted ACPR was not demonstrated when accounting for ferroquine AUC0-d28, baseline parasitaemia, and other model covariates: odds ratio 1.1 (95% CI 0.98, 1.2; P = 0.245). In the per-protocol population, Day 28 PCR-adjusted ACPR was 80.8% (21/26; 95% CI 60.6, 93.4) with ferroquine alone and 90.3% (28/31; 95% CI 74.2, 98.0), 90.9% (30/33; 95% CI 75.7, 98.1) and 87.1% (27/31; 95% CI 70.2, 96.4) with 300, 600, and 1000 mg artefenomel, respectively. Median time to parasite clearance (Kaplan-Meier) was 56.1 h with ferroquine, more rapid with artefenomel, but similar for all doses (30.0 h). There were no deaths. Adverse events (AEs) of any cause occurred in 51.4% (18/35) of patients with ferroquine 400 mg alone, and 58.3% (21/36), 66.7% (24/36), and 72.7% (24/33) with 300, 600, and 1000 mg artefenomel, respectively. All AEs were of mild-to-moderate severity, and consistent with the known profiles of the compounds. Vomiting was the most reported AE. There were no cases of QTcF prolongation ≥ 500 ms or > 60 ms from baseline. CONCLUSION: The contribution of artefenomel exposure to the clinical and parasitological activity of ferroquine/artefenomel could not be demonstrated in this study. Parasite clearance was faster with ferroquine/artefenomel versus ferroquine alone. All treatments were well tolerated. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03660839 (7 September, 2018).

Circulating IL-6, IL-10, and TNF-alpha and IL-10/IL-6 and IL-10/TNF-alpha ratio profiles of polyparasitized individuals in rural and urban areas of gabon.

Malaria, blood-borne filarial worms and intestinal parasites are all endemic in Gabon. This geographical co-distribution leads to polyparasitism and, consequently, the possibility of immune-mediated interactions among different parasite species. Intestinal protozoa and helminths could modulate antimalarial immunity, for example, thereby potentially increasing or reducing susceptibility to malaria. The aim of the study was to compare the cytokine levels and cytokine ratios according to parasitic profiles of the population to determine the potential role of co-endemic parasites in the malaria susceptibility of populations. Blood and stool samples were collected during cross-sectional surveys in five provinces of Gabon. Parasitological diagnosis was performed to detect plasmodial parasites, Loa loa, Mansonella perstans, intestinal helminths (STHs) and protozoan parasites. Nested PCR was used to detect submicroscopic plasmodial infection in individuals with negative blood smears. A cytometric bead array was used to quantify interleukin (IL)-6, IL-10 and tumour necrosis factor (TNF)-α in the plasma of subjects with different parasitological profiles. Median IL-6 and IL-10 levels and the median IL-10/TNF-α ratio were all significantly higher among individuals with Plasmodium (P.) falciparum infection than among other participants (p<0.0001). The median TNF-α level and IL-10/IL-6 ratio were higher in subjects with STHs (p = 0.09) and P. falciparum-intestinal protozoa co-infection (p = 0.04), respectively. IL-6 (r = -0.37; P<0.01) and IL-10 (r = -0.37; P<0.01) levels and the IL-10/TNF-α ratio (r = -0.36; P<0.01) correlated negatively with age. Among children under five years old, the IL-10/TNF-α and IL-10/IL-6 ratios were higher in those with intestinal protozoan infections than in uninfected children. The IL-10/TNF-α ratio was also higher in children aged 5-15 years and in adults harbouring blood-borne filariae than in their control counterparts, whereas the IL-10/IL-6 ratio was lower in those aged 5-15 years with filariae and intestinal parasites but higher in adults with intestinal parasitic infections. Asymptomatic malaria is associated with a strong polarization towards a regulatory immune response, presenting high circulating levels of IL-10. P. falciparum/intestinal protozoa co-infections were associated with an enhanced IL-10 response. Immunity against malaria could differ according to age and carriage of other parasites. Helminths and intestinal protozoa can play a role in the high susceptibility to malaria currently observed in some areas of Gabon, but further investigations are necessary.

Plasmodium falciparum Allelic Diversity: A Comparison of DNA Extraction from Isolates Collected on Rapid Diagnostic Tests (Rdts) and Filter Paper.

BACKGROUND: To perform molecular epidemiologic studies based on large cohorts, material such as RDTs or filter papers are useful for biological sample collection and extraction of RNA or DNA of good quality. Thus, we aimed to assess the quality of DNA extracted from malaria rapid diagnostic tests (RDTs) stored at various temperatures for the analysis of Plasmodium falciparum genetic diversity. METHODS: Febrile patients benefitted from free malaria diagnosis using microscopy in a malaria sentinel site, at the Regional Hospital Estuaire-Melen, in Gabon, in 2015. P. falciparum isolates were collected onto one filter paper and 2 similar RDTs devices (Acon®) per patient. Nucleic acids were extracted with QiAmp Qiagen kit from paper and RDTs and the quality of the DNA was analyzed by msp1 gene amplification. RESULTS: Msp1gene amplification was achieved in nucleic acids extracted from all filter papers and RDTs devices (n = 45, 100%). K1 alleles were detected in 93.3% (n = 42/45) of the samples and Mad20 alleles in 73.3% (n = 33/45). The number and the intensity of K1 and/or Mad20 fragments were comparable according to the sample collection material and the storage conditions (room temperature vs -20°C) of the samples. The size of the fragments indicating allelic diversity was comparable in 80% (n=36) of the samples. CONCLUSION: These data show that RDTs are a valuable source of DNA for malaria parasite genetic polymorphism analysis. Storage conditions of the devices did not influence the quality of DNA extracted from RDTs device, although some alleles may be missed.

Comparative performance of four malaria rapid diagnostic tests, Vikia Malaria Pf/Pan, Meriline-Meriscreen Pf/Pv/Pan, Right Sign Malaria Pf/Pan, and Right Sign Malaria Pf, among febrile patients in Gabon.

INTRODUCTION: Rapid diagnostic tests (RDTs) are selected based on their performances. Here, we compared the diagnostic performance of different malaria RDTs. METHODS: Febrile patients were tested for malaria using Vikia Malaria Pf/Pan, Meriline-Meriscreen Pf/Pv/Pan, Right Sign Malaria Pf/Pan, and Right Sign Malaria Pf RDTs at Melen Regional Hospital in Gabon. RESULTS: In total, 120 of 274 tested children (43.8%) had malaria. The sensitivity was > 95% for all RDTs, while the specificity was > 85% for two tests. One test generated invalid tests (8%). CONCLUSIONS: Based on their performances, all tests except one may be recommended for malaria diagnosis.