Prevalence and risk factors for blood filariasis among HIV-infected adults in Gabon, Central Africa: a pilot study.

BACKGROUND: The level of blood filariasis parasitaemia as well as the frequency of and the relationship between cotrimoxazole prophylaxis (CTX-P), antiretroviral therapy (ART) intake and CD4 cell count among people living with human immunodeficiency virus (PLHIV) in rural areas of Gabon were being studied. METHODS: Sociodemographic data and recent biological tests of PLHIV and HIV-negative participants were collected. Loa loa and Mansonella perstans microfilaria were detected by direct microscopy examination and leucoconcentration. RESULTS: Overall, 209 HIV-positive and 148 HIV-negative subjects were enrolled. The overall prevalence of microfilaria was comparable between PLHIV (19.9% [n=41/206]) and HIV-negative participants (14.8% [n=22/148]) (p=0.2). The L. loa infection rate was comparable between HIV-positive (9.2%) and HIV-negative participants (6.8%) (p=0.2), while the M. perstans infection rate was 14-fold higher among PLHIV (p<0.01). L. loa parasitaemia was 6-fold lower in PLHIV receiving CTX-P (median 150 mf/mL [interquartile range {IQR} 125-350]) than in patients without (900 [550-2225]) (p<0.01). Among subjects with a CD4 cell count <200 cells/µL, the prevalence of M. perstans was 7-fold higher than that of L. loa (20.6% vs 2.9%). CONCLUSIONS: This study suggests a similar exposure to L. loa infection of PLHIV and HIV-negative patients while M. perstans is more frequently found in HIV-positive individuals, notably those with a CD4 count <200 cells/µL.

Anaemia in asymptomatic parasite carriers living in urban, rural and peri-urban settings of Gabon.

BACKGROUND: This cross-sectional study was carried out in different settlements of Gabon to determine the influence of single or multiple parasite carriage on haemoglobin (Hb) levels. METHODS: Between April 2015 and June 2016, healthy volunteers from urban, peri-urban and rural areas were screened for malaria, blood filariasis and intestinal parasitic infections using microscopic methods. Hb concentration was measured with a Hemocue analyser. The association between parasite carriage and anaemia was assessed. RESULTS: Among the 775 volunteers examined, 319 (41.2%) were from rural villages and 76.0% were adults. Filariasis, intestinal parasitic infections, Plasmodium falciparum and polyparasitism were detected in 15.6, 14.6, 9.5 and 6.8% of participants, respectively. Anaemia prevalence was 72.6%, with rates of mild, moderate and severe anaemia being 30.9, 61.1 and 8.0%, respectively. The median Hb level was lowest in the presence of hookworms (7.1 g/dl [interquartile range {IQR} 6.8-7.5]), Schistosoma intercalatum (6.9 g/dl), Trichuris trichiura (10.1 g/dl [IQR 8.9-11.5]) and Plasmodium falciparum (10.0 g/dl [IQR 9.1-11.2]) compared with filariaemia (12.1 g/dl [IQR 10.5-13.2]) (p=0.03). Moderate to severe anaemia predominated among those single-infected with P. falciparum (69.5%) or co-infected with intestinal parasitic infections and P. falciparum (76.2%), while it was found in only 23.2% of individuals with filariasis. All participants with soil-transmitted helminths and more than half with a Blastocystis sp. (68.8%) infection had moderate anaemia. CONCLUSIONS: The prevalence of anaemia is high. Asymptomatic parasite carriage is associated with anaemia in this surveyed population in Gabon.

High Frequency of Pfcrt-76T Allele in Plasmodium falciparum Isolates From Gabon and Ivory Coast After the Withdrawal of Chloroquine.

The aim of this study was to assess the proportion of Pfcrt-76T mutant allele four years after the adoption of new malaria control guidelines in Gabon and Ivory Coast. Frequency of K76T mutation of Pfcrt gene was compared between Plasmodium falciparum isolates from Gabon and Ivory Coast. Samples were collected in 2008 in Gabon and in 2009 in Ivory Coast. In total, 151 isolates were selected and analysed by nested-PCR-RFLP for Pfcrt-76 allele identification: 63 in Abobo (Ivory Coast) and 78 in Oyem (Gabon). The proportion of Pfcrt-76T mutant allele was higher in Oyem (70%) compared to Abobo (46%) (p=0.005). This allele was more frequently detected in patients less than 5 years old in Oyem (75 %) compared to Abobo (52%) (p<0.01). The present work showed different prevalence of Pfcrt-76T allele between both sites probably due to a different drug pressure on P. falciparum strains circulating in these areas. Moreover, a decrease of the mutation frequency is observed compared to data obtained in 2005. Therefore, a continuous monitoring of this allele and other drug resistance molecular markers is required in these countries.

Le but de cette étude était d'évaluer la proportion de l'allèle mutant Pfcrt-76T quatre ans après l'adoption de nouvelles stratégies de contrôle du paludisme au Gabon et en Côte d'Ivoire. La fréquence de la mutation K76T du gène Pfcrt a été comparée entre les isolats de P. falciparum du Gabon et de Côte d'Ivoire. Des échantillons ont été prélevés en 2008 au Gabon et en 2009 en Côte d'Ivoire. Au total, 151 isolats ont été sélectionnés et analysés par PCR-nichée-RFLP pour l'identification de l'allèle Pfcrt-76: 63 à Abobo (Côte d'Ivoire) et 78 à Oyem (Gabon). La proportion de l'allèle mutant Pfcrt-76T était plus élevée à Oyem (70 %) comparée à Abobo (46 %) (p = 0,005). Cet allèle était plus fréquemment détecté chez les patients de moins de 5 ans à Oyem (75 %) comparé à Abobo (52 %) (p < 0,01). Le présent travail a montré une prévalence différente de l'allèle Pfcrt-76T entre les deux sites, probablement en raison d'une pression médicamenteuse différente sur les souches de P. falciparum circulant dans ces zones. Une diminution de la fréquence de cette mutation est observée par rapport aux résultats de 2005. Par conséquent, un suivi continu de cet allèle et d'autres marqueurs moléculaires de la résistance aux antipaludiques est recommandé dans ces pays.

Submicroscopic Plasmodium falciparum parasitaemia in human immunodeficiency virus-infected adults living in Gabon (Central Africa)-a pilot study.

Background: Submicroscopic malaria infections contribute to malaria transmission. Describing the extent of the parasite reservoir is of importance. In people living with human immunodeficiency virus (HIV), the frequency of subpatent malaria infections is rarely reported. The aim of the present study was to determine the frequency of submicroscopic infections in people living with HIV in Gabon and its relationship with cotrimoxazole (CTX) use. Methods: A survey was conducted in two health care centres in rural areas (Koulamoutou and Oyem) and three in urban areas (Libreville) of Gabon from March 2015 to June 2016. Blood samples were collected from consenting people living with HIV with a negative blood smear. Information on CTX and antiretroviral therapy intake was recorded from the medical file of the patient and through an interview. For molecular analysis, the Plasmodium small subunit ribosomal RNA gene was amplified by nested polymerase chain reaction. Results: Submicroscopic infections were detected in 10.1% (n=12/119) of the people living with HIV, more frequently in those residing in rural areas (15.1%) compared with urban areas (2.1%) (p<0.01). The proportion of anaemic patients was 1.74-fold more frequent in malaria-infected patients, although not statistically significant. Submicroscopic infections frequency did not vary according to CTX intake (p=0.6). Conclusions: The present pilot study highlights a non-negligible frequency of submicroscopic malaria infections in people living with HIV from rural areas, but no relationship with CTX intake was found.

Spatial and temporal distribution of Pfmsp1 and Pfmsp2 alleles and genetic profile change of Plasmodium falciparum populations in Gabon.

Plasmodium population dynamics analysis may help to assess the impact of malaria control strategies deployment. In Gabon, new strategies have been introduced, but malaria is still a public health problem marked by a rebound of the prevalence in 2011. The aim of the study was to investigate the spatial and temporal distribution of P. falciparum strains in different areas in Gabon during a period of malaria transmission transition, between 2008 and 2011. A total of 109P. falciparum isolates were genotyped using nested-PCR of Pfmsp1 and Pfmsp2 genes. 3D7, FC27 and K1 allele frequencies were comparable between sites (p=0.9); those of Ro33 (93.6%; 44/47) and Mad20 (60%; 12/20) were significantly higher in isolates from Oyem (p<0.01) and Port-Gentil (p=0.02), respectively. The frequency of multiples infections (77%) and the complexity of infection (2.66±1.44) were the highest at Oyem. Pfmsp1 gene analysis highlighted a trend of a decreasing frequency of K1 family, in Libreville and Oyem between 2008 and 2011; while that of Ro33 (p<0.01) and Mad20 (p<0.01) increased. The prevalence of multiple infections was comparable between both periods in each site: 42.2% vs 47.6% (p=0.6) in Libreville and 57.7% vs 61.7% in Oyem (p=0.8). In contrast, in 2011, the COI tends to be higher in Libreville and did not vary in Oyem. These data confirm an extended genetic diversity of P. falciparum isolates over time and according to geographic location in Gabon. Nevertheless, the impact of the deployment of malaria control strategies on the parasites genetic profile is not clearly established here.

Utilisation des mesures préventives contre le paludisme au cours de la grossesse chez les femmes enceintes à Libreville au Gabon

Contexte : Depuis le déploiement des mesures de prévention contre le paludisme chez la femme enceinte au Gabon en 2005, les taux de couverture en Traitement Préventif Intermittent à la Sulfadoxine-Pyriméthamine (TPI-SP) et Moustiquaire Imprégnée d'Insecticides à Longue Durée d'Action (MILDA) demeurent faibles comparativement aux objectifs visés par l'OMS. Il s'avère donc important de rechercher les facteurs qui influenceraient l'adhésion des femmes enceintes au TPI-SP et à l'utilisation des MILDA. Patientes et méthodes : Il s'est agi d'une enquête transversale menée de juillet 2013 à septembre 2014 chez des femmes enceintes vues en consultation prénatale (CPN). Des informations concernant leurs connaissances et pratiques vis-à-vis des mesures de prévention ont été recueillies à l'aide d'un questionnaire. La couverture en TPI-SP a été déterminée chez celles vues en fin de grossesse. Résultats : Au total, 122 femmes ont été incluses. Elles avaient déjà entendu parler du paludisme et connaissaient la MILDA comme moyens de prévention du paludisme. Seulement un quart (n = 31) des gestantes avaient entendu parler du TPI-SP. Enfin de grossesse, près de la moitié (48,4% ; n = 15) d'entre elles a reçu une chimio-prévention correcte (≥ 3 doses de TPI-SP), contre 25,2% (n = 23) de celles qui ne connaissaient pas cette méthode de prévention (p < 0,01). Seulement 40% des femmes ayant eu au plus trois CPN avaient reçu au moins trois doses de SP alors que cette proportion était de 72,9% (n = 35) chez celles qui avaient eu au moins quatre CPN (p < 0,01). Les femmes suivies en structure publique ont reçu moins de doses de SP comparativement à celles suivies en clinique privée, en dépit de la gratuité de la SP. Par contre, elles utilisaient plus fréquemment la MILDA. Conclusion : L'utilisation du TPI-SP n'est pas encore optimale. Il existe un écart entre la connaissance et l'utilisation des mesures préventives du paludisme. La faible participation aux CPN des femmes, des connaissances insuffisantes sur le paludisme et le TPI-SP ont un impact sur la couverture en moustiquaire et TPI-SP

Low sensitivity of the ImmunocardSTAT® Crypto/Giardia Rapid Assay test for the detection of Giardia and Cryptosporidium in fecal samples from children living in Libreville, Central Africa.

Giardiasis and cryptosporidiosis are now recognized as neglected tropical parasitic diseases. The risk of their dissemination  in developing countries, such as Gabon, is increasing, due to urban crowding and poor sanitation. Accurate, simple and rapid diagnosis tools are thus necessary for the estimation of their real burden. The aim of this study was to evaluate the performances of the ImmunocardSTAT®Crypto/Giardia Rapid Assay test for the detection of Cryptosporidium (C.) spp. and Giardia (G.) duodenalis in children living in Libreville, Gabon. Stool samples of 173 healthy children were screened by routine microscopic using the merthiolate iodine formol concentration technique for Giardia, the modified Ziehl Neelsen (ZN) staining for Cryptosporidium and the ImmunocardSTAT® Crypto/Giardia RDT for the detection of Giardia and Cryptosporidium parasite forms and antigens respectively. G. duodenalis was detected with microscopy and the ImmunocardSTAT® Crypto/Giardia in 27 (15.6 %) and 22 (13.3 %) fecal samples respectively. C. spp. oocysts were found in 18 (10.4 %) ones, whereas only one sample was positive with the immunochromatographic assay. When microscopic examination was considered as the reference method, sensitivity and specificity of the ImmunocardSTAT® Crypto/Giardia Rapid Assay were found to be 63.0 %, 96.6 and 5.5 %, 99.3 % for G. duodenalis and C. spp. respectively. The prevalence of G. duodenalis and C. spp. carriage is high in children from Libreville. A low sensitivity of the ImmunocardSTAT® Crypto/Giardia for the detection of both parasites is observed. It is thus inappropriate as a diagnostic tool for detecting asymptomatic carriers.

Decrease of microscopic Plasmodium falciparum infection prevalence during pregnancy following IPTp-SP implementation in urban cities of Gabon.

BACKGROUND: Six years after the implementation of intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) in Gabon, its impact on placental malaria and pregnancy outcomes remains unknown. METHODS: Age, gestational data, use of IPTp-SP and birth weight were recorded during a hospital-based cross-sectional survey performed in 2011 in 387 women at the end of pregnancy. RESULTS: Malaria prevalence was 6.7 and 5.3% in peripheral and placental blood respectively. Overall, 59.0% women took at least two IPTp-SP doses which was associated with 50% reduction of Plasmodium; (P.) falciparum infection in primigravidae. Previous malaria treatment was a risk factor for peripheral P. falciparum infection, while uptake of IPTp-SP was associated with reduced parasitaemia. Anaemia prevalence was 38.0%, low birth weight and prematurity rates were 6.0 and 12.0% respectively. Young age was associated with a higher frequency of malaria, anaemia, low birth weight and preterm delivery (p<0.01). Birth weight significantly rose with increasing age (p<0.01), parity (p=0.03) and number of SP doses (p=0.03). A birth weight reduction of 230 g in case of peripheral parasitaemia (p=0.02) and of 210 g with placental parasitaemia (p=0.13) was observed. CONCLUSIONS: Microscopic P. falciparum prevalence during pregnancy significantly declined between 2005 and 2011, following IPTp-SP implementation in Gabon. Young women and paucigravidae remain the most susceptible to malaria and associated outcomes.

High Frequency of Trypanosoma congolense Savannah Type (Kinetoplastida: Trypanosomatidae) Among Tsetse Flies (Diptera: Glossinidae) in a Historic Trypanosoma Foci in North-Eastern Gabon: Preliminary Study [corrected].

Human African trypanosomiasis became a neglected disease after the 1960s, when case numbers dropped dramatically. It again became a public health problem in sub-Saharan Africa at the end of the 1990s, when new cases were reported, notably in Central Africa, and specifically in Gabon, where historic foci existed and new cases have been reported. Therefore, the present study reports on an entomological survey conducted in May 2012 to determine the pathogenic trypanosome infection rate in tsetse flies and characterize the diversity of Trypanosoma species in the Ivindo National Park (INP) in northeastern Gabon. Nine Vavoua traps were used to catch tsetse over a 7-days period. All tsetse flies captured were identified to species, dissected, and trypanosome species identified using polymerase chain reaction (PCR). In total, 160 tsetse flies were analyzed, including Glossina palpalis palpalis, Glossina fusca congolense, and Glossina tachinoïdes The trypanosome infection rate of the flies was 6.3 and 31.9% using microscopy and PCR, respectively. The species identified were Trypanosoma congolense savannah type, Trypanosoma brucei brucei, Trypanosoma brucei gambiense, Trypanosoma vivax, and Trypanosoma congolense forest type. Trypanosoma risk index was 0.75 and 7.05 for humans and for animals, respectively. This study illustrates the diversity of Trypanosoma species infecting the tsetse flies in the INP. The simultaneous occurrence of Trypanosoma and tsetse from the palpalis group may suggest that the reservoirs of African animal trypanosomiasis should be carefully monitored in this area.

The relationship between microfilaraemic and amicrofilaraemic loiasis involving co-infection with Mansonella perstans and clinical symptoms in an exposed population from Gabon.

The relationship between the frequency of loiasis objective symptoms and microfilaraemic or amicrofilaraemic infection was assessed in 1148 exposed patients also infected, or not, with Mansonella perstans. Filarial infections were detected by direct microscopy, leucoconcentration and serology, with prevalence values of 39.5% Loa loa, 5.6% M. perstans and 3.4% co-infection with both filarial species. Amicrofilaraemic or occult loiasis (OL) predominated among L. loa-infected individuals, with a prevalence of 58.2%. Hypermicrofilaraemia (>8000 microfilariae (mf)/ml) was found in 18.4% of L. loa microfilaraemic patients, with 25.7% of them harbouring more than 30,000 mf/ml. Up to 34% of patients with OL showed evidence of Calabar swelling, compared with 26.3% of microfilaraemic patients (P= 0.03). Overall 5.3% of patients presented with adult worm migration across the eye, representing 16.3% of microfilaraemic individuals and 11.4% of amicrofilaraemic patients (P= 0.13). This symptom was similarly found in patients with more than 30,000 mf/ml (22%), those with microfilaraemia between 8 and 30,000 mf/ml (15.4%) and also in individuals with low or without microfilaraemia (16.1%) (P= 0.7). Five (14.3%) hypermicrofilaraemic patients did not present any L. loa-specific objective symptoms, as well as all the patients with single M. perstans infection. The presence of adult eye worm migration as a strong predictor of high microfilaraemia density would obscure the real burden of L. loa hypermicrofilaraemia in exposed individuals. For epidemiological purposes and control strategies, the mapping of L. loa in endemic areas should also take into account the group of patients with occult loiasis.

Inventory of potential vectors of trypanosoma and infection rate of the Tsetse fly in the National Park of Ivindo, Gabon.

BACKGROUND: Trypanosoma's vectors distribution is poorly investigated in Gabon, where Trypanosomiasis historical foci exist. Thus, an active detection of Trypanosoma sp transmission needs to be assessed. OBJECTIVES: The present study aims to identify potential vectors of Trypanosoma sp and to evaluate the infection rate of the Tsetse fly in an area of Gabon. METHODS: An entomological survey was conducted in the National Park of Ivindo in May 2012 using Vavoua traps. All captured insects were identified. Tsetse were dissected and organs were microscopically observed to detect the presence of Trypanosoma sp. RESULTS: 247 biting flies known as vectors of Trypanosomiasis were caught including 189 tsetse flies, 32 Tabanid and 26 Stomoxys. Tsetse flies had the highest bulk densities per trap per day (ADT = 3 tsetse / trap / day), while the lowest density was found among Stomoxys (ADT= 0.41 Stomoxys / trap / day). The infection rate of flies was 6.3%. Infectious organs were midguts and to a lesser extent salivary glands and proboscis. CONCLUSION: The presence of Tsetse infected by Trypanosoma highlights an existing risk of trypanosomiasis infection in the National Park of Ivindo.

Pfcrt 76T and pfmdr1 86Y allele frequency in Plasmodium falciparum isolates and use of self-medication in a rural area of Gabon.

BACKGROUND: Studies showed that chloroquine resistance may revert to sensitivity after its withdrawal mainly detected by a significant decrease of Plasmodium falciparum pfcrt 76T and pfmdr1 86Y alleles. Besides, self-medication is considered as a key factor of antimalarial drug resistance expansion. Thus, pfcrt 76T and pfmdr1 86Y allele frequency and its relationship with antimalarial drug self-medication was analyzed in P. falciparum isolates collected in Gabon. METHODS: Samples were collected from febrile children screened for P. falciparum infection in 2005 and 2008 at the regional hospital of Oyem. Self-use of antimalarial drugs before the day of consultation was recorded. Polymorphic codons 76 and 86 of pfcrt and pfmdr1 genes were analyzed by PCR-RFLP. RESULTS: The frequency of pfcrt 76T mutant allele was greater than 70.0% in 2005 and 2008. Wild type isolates were 1.7-fold more prevalent in 2008. The prevalence of pfmdr1 86Y mutant allele was comparable between 2005 and 2008 (p=0.1); the proportion of wild type allele reached 20.5% in 2008. The frequency of wild type allele pfcrt K76 or pfmdr1 N86 was higher among patients without anti-malarial drug self-medication compared to those who used it. CONCLUSIONS: An increase of the frequency of P. falciparum wild type allele pfcrt 76K and pfmdr1 86N was observed within a short period after chloroquine withdrawal. The proportion of mutant genotypes is still high, mainly among patients using self-medication with antimalarial drugs.

Submicroscopic infections among children with adequate clinical and parasitological response (ACPR).

The aim of the study was to re-assess the treatment outcomes of Gabonese children, treated with sulfadoxine-pyrimethamine (SP) and artesunate-mefloquine (AM) and categorized by microscopy as adequate clinical and parasitological response (ACPR), using polymerase chain reaction (PCR). Dried blood spots were collected at day 0 and day 28 and stevor gene amplification was performed to detect Plasmodium falciparum infections. Plasmodial DNA was found in 27.5% (n=19/69) of the isolates collected at day 28; this proportion was 34.3% (n=12/35) in the SP group and 20.6% (n=7/34) in the AM group. This study underlines the need of an accurate and more appropriate technique such as PCR to evaluate antimalarial drug efficacy during clinical trials.

Molecular analysis of DHFR and DHPS genes in P. falciparum clinical isolates from the Haut--Ogooué region in Gabon.

The main objective of this work was to determine the prevalence of mutations in genes coding for the dihydropteroate synthase (DHPS) and the dihydrofolate reductase (DHFR) enzymes which are implicated in resistance of P. falciparum to antifolate (pyrimethamine-sulfadoxine (P/S)). In this study, 117 human blood samples were collected at Franceville located in the region of Haut-Ogooué (South-eastern Gabon). In this area, a relatively low level of sensitivity of Plasmodium falciparum to P/S has been reported with 18.2% of RII and 12.1% of RI resistance. A nested polymerase chain reaction was used to amplify a fragment of the DHFR gene containing codon 108, where a point mutation causing a Serine (wild type) to Asparagine or to a Threonine (resistant types) change occurs in pyrimethamine resistant parasites. Eleven DHFR fragments were sequenced and mutations occurring at codons 51, 59 and 108 were analysed. The DHPS gene was amplified by polymerase chain reaction (PCR) and sequenced directly or after cloning. Variant amino acid residues 436, 437, 540, 581, 613 associated with sulfadoxine resistance were analysed. The analysis of codon 108 of the DHFR gene was undertaken for 81 isolates. More than one DHFR P. falciparum genotype was present in 64% of the samples. We showed that 47% of 141 DHFR gene PCR products had Serine (wild phenotype), and 52% had Asparagine. We found one isolate with the Thr-108 confirmed by sequencing of the PCR product. Triple, double and single DHFR mutant at positions 51, 59 and 108 were found. Only codons 436 and 437 of the 38 analysed sequences of the DHPS gene revealed point mutations. These results have been compared with those reported from different sites in Africa, Asia or South-America.