Molecular and serological pattern of Toxoplasma gondii among rural population in four regions of Gabon

Toxoplasma gondii is an obligatory intracellular parasite that causes a zoonotic disease capable of infecting nearly all warm-blooded hosts, including humans. However, reports on the molecular prevalence of T. gondii in humans are rare in Gabon. The present study aimed to evaluate the serological and molecular prevalence of T. gondii among apparently healthy rural populations in four regions of Gabon. This study included six hundred blood samples from the Interdisciplinary Center for Medical Research (CIRMF) bank, including 300 women and 300 men living in 111 villages. Blood samples were screened using enzyme-linked fluorescent assay (ELFA), while buffy coat samples were analyzed using PCR analyses. Of the 600 samples screened, 548 (91.3%) showed IgG antibodies against T. gondii; 11 (2%) had both IgG and IgM. Among the 548 positive samples, 155 (28%) had higher IgG titers (>300 UI/ml), and 49 of them (31.6%) were detected with T. gondii DNA. The present findings on human toxoplasmosis in Gabon suggest that at an older age, reactivation of old infections seems more frequent than new infections, as indicated by the presence of T. gondii using PCR among elevated IgG subjects without IgM. Further studies should be performed to identify the genotypes of T. gondii that infect humans in Gabon.

The Human Filaria Loa loa: Update on Diagnostics and Immune Response.

Loa loa loiasis was considered an anecdotal disease 30 years ago. Its spread in Equatorial Africa and the side effects associated with mass drug administration programs against filariasis in co-endemic areas have drawn the attention of the international research community. Progress in research conducted to date has provided insight into the immunobiology of this parasite. An interesting finding reported in several studies is that 70% of individuals with loiasis do not carry microfilariae in their blood, and 30% are microfilaremic, suggesting the involvement of several immunological mechanisms, as shown by elevated specific IgG4 and IgE levels signifying a potential cross-linking mechanism between the two isotypes via L. loa antigen to prevent allergy. A mechanism of anergy in the appearance of microfilariae in the peripheral blood results in immunological unresponsiveness in individuals with microfilariae. There is an interaction between other pathogens (parasites, bacteria, viruses) in individuals co-infected with L. loa. The strong antigen cross-reactivity between L. loa and lymphatic filarial worms warrants a re-evaluation of the distribution of the latter in co-endemic regions. The mechanism of concomitant immunity observed in the elimination of microfilariae or infective larvae (third-stage larvae, L3) may be used for the conception of an immunoprophylactic strategy.

Comparison of six methods for Loa loa genomic DNA extraction.

OBJECTIVES: Good-quality and sufficient DNA is essential for diagnostics and vaccine development. We aimed to compare six DNA extraction techniques applied to Loa loa microfilariae in order to evaluate the purity and integrity of extracts in terms of quality and quantity. METHODS: The microfilariae were purified via a Percoll gradient procedure with blood from hyper-microfilaremic individuals (> 30,000 microfilaria [mf]/ml). DNA extraction was carried out in duplicate at a rate of 350,000 mf/tube for each technique: phenol/chloroform, commercial Qiagen kit, salting out, Tris-EDTA, methanol, and cetyltrimethylammonium bromide (CTAB). The integrity, purity, concentration, and quality of the DNA extracts were successively verified by agarose gel electrophoresis, spectrophotometry (A260/A280 and A260/A230 wavelength ratio), Qubit fluorometry, and endonuclease and polymerase activity. The six techniques were compared on the basis of the following parameters: concentration, purity, efficiency, effectiveness, integrity, safety of the technique, as well as cost and duration of the protocol. RESULTS: The ratios of the optical densities of the extracts A260/A280 and A260/A230 were, respectively: phenol/chloroform (1.82; 1.11), Qiagen (1.93; 1.36), salting-out (1.9; 2.04), Tris-EDTA (1.99; 1.183), methanol (2.126; 1.343), and CTAB (2.01; 2.426). The DNA yield was: phenol/chloroform (3.920 µg), Qiagen (10.280 µg), salting-out (10.390 µg), Tris-EDTA (0.5528 µg), methanol (0.1036 µg), and CTAB (1.115 µg). Endonuclease and polymerase activity was demonstrated by digestion of DNA and through amplicons obtained via polymerase chain reaction assays with phenol/chloroform, Qiagen, and salting-out extracts. CONCLUSION: The phenol/chloroform, Qiagen, and salting-out DNA extracts were all of good quality. Salting out had the best yield followed by Qiagen and then phenol/chloroform. Endonuclease and polymerase activity was effective in all three extracts despite the presence of some contaminants. These methods are therefore suitable for the extraction of DNA from Loa loa microfilariae. Tris-EDTA and methanol did not show adequate sensitivity, while CTAB was found to be unsuitable.

Loa loa infection detection using biomarkers: current perspectives.

Loa loa is originally a restricted filarial worm from central Africa and some west African countries. However, numerous imported cases are being reported throughout the world due to human movement. Traditionally, its diagnosis is based on identification of microfilariae in the peripheral blood or the passage of the adult worm under the conjunctiva. However, few patients have microfilariae in their peripheral blood, while the majority of infected people are amicrofilaremic (without microfilariae in their blood), despite clinical symptoms suggesting L. loa infection. This situation suggests that diagnoses based on the presence of microfilariae in the blood or the ocular passage of an adult worm, are not sensitive. Therefore, it seems necessary to search for biomarkers to remedy this situation. Furthermore, L. loa is a major obstacle in the control of other filarial worms in areas where these filariae are co-endemic. To develop a diagnostic tool based on a biomarker, several approaches have been considered using antibodies, antigens or nucleic acid detection. However, none of the diagnostic techniques in loiasis based on biomarkers has reached the point of care as have microscopic detection of microfilariae or observation of ocular passage of a worm.

Serological Evidence for the Circulation of Rift Valley Fever Virus in Domestic Small Ruminants in Southern Gabon.

Rift Valley fever (RVF) is a zoonotic disease, which caused several epidemics in humans in many countries of Africa. Using an inhibition enzyme-linked immunosorbent assay (ELISA), real-time reverse transcription PCR, and nested one-step reverse transcription PCR, we conducted a cross-sectional study in populations of sheep and goats from the Mongo County in 2014 to determine the circulation of the Rift Valley fever virus (RVFV) in small ruminants from this area. From a total of 201 small ruminants (95 sheep and 106 goats), the overall IgG seroprevalence against the RVFV was 6.47% (13/201). No RVFV RNA was detected in the animal plasmas. Logistic regression analysis showed that age, species, sex, and locality were not the significant risk factors. The findings of this study highlight the risk of RVF for domestic ruminants bred in this region and for the human rural population living in contact with these animals and they emphasize the need to develop adequate control measures to limit this threat.

In vitro proliferation and production of cytokine and IgG by human PBMCs stimulated with polysaccharide extract from plants endemic to Gabon.

Polysaccharides were extracted from seven plants endemic to Gabon to study their potential immunological activities. Peripheral blood mononuclear cell (PBMC) (5×105 cells/mL) proliferation, cytokine and immunoglobulin G (IgG) assays were performed after stimulation with different concentrations of polysaccharide fractions compared with lipopolysaccharides (LPS) and concanavalin A (ConA) from healthy volunteers. The culture supernatants were used for cytokine and IgG detection by enzyme-linked immunosorbent assay (ELISA). The results show that pectin and hemicellulose extracts from Uvaria klainei, Petersianthus macrocarpus, Trichoscypha addonii, Aphanocalyx microphyllus, Librevillea klaineana, Neochevalierodendron stephanii and Scorodophloeus zenkeri induced production levels that were variable from one individual to another for IL-12 (3-40 pg/mL), IL-10 (6-443 pg/mL), IL-6 (7-370 pg/mL), GM-CSF (3-170 pg/mL) and IFN-γ (5-80 pg/mL). Only hemicelluloses from Aphanocalyx microphyllus produce a small amount of IgG (OD=0.034), while the proliferation of cells stimulated with these polysaccharides increased up to 318% above the proliferation of unstimulated cells. However, this proliferation of PBMCs was abolished when the pectin of some of these plants was treated with endopolygalacturonase (p<0.05), but the trend of cytokine synthesis remained the same, both before and after enzymatic treatment or saponification. This study suggests that these polysaccharides stimulate cells in a structure-dependent manner. The rhamnogalacturonan-I (RGI) fragment alone was not able to induce the proliferation of PBMC.

Development of a highly sensitive loop-mediated isothermal amplification (LAMP) method for the detection of Loa loa.

The filarial parasite Loa loa, the causative agent of loiasis, is endemic in Central and Western Africa infecting 3-13 million people. L. loa has been associated with fatal encephalopathic reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. In endemic areas, the only diagnostic method routinely used is the microscopic examination of mid-day blood samples by thick blood film. Improved methods for detection of L. loa are needed in endemic regions with limited resources, where delayed diagnosis results in high mortality. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid, inexpensive, molecular diagnosis of loiasis. Primers for LAMP were designed from a species-specific repetitive DNA sequence from L. loa retrieved from GenBank. Genomic DNA of a L. loa adult worm was used to optimize the LAMP conditions using a thermocycler or a conventional heating block. Amplification of DNA in the LAMP mixture was visually inspected for turbidity as well as addition of fluorescent dye. LAMP specificity was evaluated using DNA from other parasites; sensitivity was evaluated using DNA from L. loa 10-fold serially diluted. Simulated human blood samples spiked with DNA from L. loa were also tested for sensitivity. Upon addition of fluorescent dye, all positive reactions turned green while the negative controls remained orange under ambient light. After electrophoresis on agarose gels, a ladder of multiple bands of different sizes could be observed in positive samples. The detection limit of the assay was found to be as little as 0.5 ag of L. loa genomic DNA when using a heating block. We have designed, for the first time, a highly sensitive LAMP assay for the detection of L. loa which is potentially adaptable for field diagnosis and disease surveillance in loiasis-endemic areas.

Epidemiology of concomitant infection due to Loa loa and Mansonella perstans in Gabon.

BACKGROUND: The filarial parasites Loa loa and Mansonnella perstans are endemic in the central and western African forest block. Loa loa is pathogenic and represents a major obstacle to the control of co-endemic filariae because its treatment can cause fatal complications such as encephalitis. METHODOLOGY/PRINCIPAL FINDINGS: 4392 individuals aged over 15 years were studied both by direct examination and a concentration technique. The overall prevalence rates were 22.4% for Loa loa microfilaremia, 10.2% for M. perstans microfilaremia, and 3.2% for mixed infection. The prevalence of both filariae was higher in the forest ecosystem than in savannah and lakeland (p<0.0001). The intensity of microfilariae (mf) was also higher in the forest ecosystem for both parasites. The prevalence and intensity of microfilaria were both influenced by age and gender. Correlations were found between the prevalence and intensity of Loa loa microfilariae (r = 0.215 p = 0.036), and between the prevalence of Loa loa and the prevalence of individuals with microfilaria >8000 mf/ml (r = 0.624; p<0.0001) and microfilariae >30 000 mf/ml (r = 0.319, p = 0.002). In contrast, the prevalence of pruritis and Calabar swellings correlated negatively with the prevalence of Loa loa microfilaria (r = -0.219, p = 0.032; r = -0.220; p = 0.031, respectively). Pruritis, Calabar swellings and eye worm were not associated with L. loa mf intensity (r = -0.144, p = 0.162; r-0.061, p = 0.558; and r = 0.051, p = 0.624, respectively), or with the prevalence or intensity of M. perstans microfilariae. CONCLUSIONS/SIGNIFICANCE: This map of the distribution of filariae in Gabon should prove helpful for control programs. Our findings confirm the spatial uniformity of the relationship between parasitological indices. Clinical manifestations point to a relationship between filariae and allergy.

Risk factors for Zaire ebolavirus--specific IgG in rural Gabonese populations.

BACKGROUND: In Gabon, several Ebolavirus outbreaks have occurred exclusively in the northeastern region. We conducted a large serosurvey to identify areas and populations at risk and potential demographic, clinical, and behavioral risk factors. METHODS: Blood samples and clinical and sociodemographic data were collected from 4349 adults and 362 children in a random sample of 220 villages in the 9 provinces of Gabon. An enzyme-linked immunosorbent assay was used to detect Zaire ebolavirus (ZEBOV)-specific IgG, and thin blood smears were used to detect parasites. Logistic regression was implemented using Stata software (Stata), and a probability level of <.05 was considered to be statistically significant. RESULTS: The prevalence of ZEBOV-specific IgG was 15.3% overall, increasing to 32.4% (P< .001) in forest areas. No sociodemographic risk factors were found, but the antibody prevalence increased linearly up to 20 years of age. Chronic arthralgia and amicrofilaremia were the only factors associated with ZEBOV seropositivity. CONCLUSIONS: These findings confirm the endemicity of ZEBOV in Gabon and its link to the ecosystem. Human antibody positivity would appear to be to the result of exposure to contaminated fruits.

Prevalence of Plasmodium falciparum infection in asymptomatic rural Gabonese populations.

BACKGROUND: Malaria may be perennial or epidemic in sub-Saharan Africa, and its transmission may be stable or unstable, depending on the region. The prevalence of asymptomatic Plasmodium falciparum carriage is poorly documented in Gabon. A large survey of P. falciparum infection was conducted in asymptomatic individuals living in rural Gabon. METHODS: Two hundred and twenty-two villages were randomly selected in the nine administrative regions. With the participants' informed consent, blood samples were collected for thick and thin blood film examination after 20% Giemsa staining. Prevalence rates were calculated per village, per region and per ecosystem, and nationwide. Demographic risk factors were identified with STATA software version 9.0. Significance was assumed at p < 0.05. RESULTS AND DISCUSSION: The prevalence of P. falciparum in adults was 6.2% (269/4342) nationwide, with a maximum of 37.2% in one village; a linear decrease was observed with increasing age (p = 0.045). Only 5% of the 399 children from forest areas tested positive. The prevalence was significantly higher in forest areas (7%) than in savannah (4%) and lakeland (2.5%). Within the forest region, the prevalence was significantly higher in forest grassland (10.9%) than in the mountain forest (3.5%), interior forest (6.8%) and north-eastern forest (4.5%). CONCLUSION: Plasmodium falciparum carriage remains high among adults in rural Gabon. Control measures must be adapted to the region and ecosystem. Routine treatment of asymptomatic individuals should be considered.

In vitro activities of plant extracts on human Loa loa isolates and cytotoxicity for eukaryotic cells.

Loa loa, a filarial worm, can cause fatal encephalitis in humans. In an attempt to find alternatives to the standard treatments (ivermectin and diethylcarbamazine citrate), we tested 12 methanolic extracts of nine traditional plant remedies. The extracts (100-0.09 microg/ml) were incubated with 20 Loa loa microfilariae isolated from patients at 37 degrees C with 5% CO(2) in modified Eagle's medium supplemented with 10% fetal serum and antibiotics. Activity was evaluated 120 h later by counting live microfilariae under a microscope. Cytotoxicity for eukaryotic cells was estimated by measuring 3-[4,5-dimethylthiazol-2-yl]-2-5 diphenyl tetrazolium bromide transformation to formazan at 450 nM in a spectrophotometer. The plants tested were Lophira alata, Greenwayodendron suaveolens, Uapaca togoensis, Zanthoxylum heitzii, Peperomia pellucida, Piptadeniastrum africanum, Petersianthus macrocarpus, Vernonia conferta, and Vernonia hymenolepis. Chemical screening showed that most of the extracts contained reducing sugars, tannin or polyphenols, sterols or triterpenes, saponosides, and alkaloids. None contained carotinoids and few contained flavonoids. The 50% lethal concentration ranged from 0.22 to 70.28 microg/ml, while the 50% inhibitory concentration for eukaryotic cells (IC(50)) ranged from 8.52 to 119.52 microg/ml. Extracts of P. macrocarpus (selectivity index = 72.16), P. africanum (13.69), Z. heitzii (12.11), and L. alata (9.26) were highly selective for L. loa.

Ethics committees in western and central Africa: concrete foundations.

The involvement of developing countries in international clinical trials is necessary for the development of appropriate medicines for local populations. However, the absence of appropriate structures for ethical review represents a barrier for certain countries. Currently there is very little information available on existing structures dedicated to ethics in western and central Africa. This article briefly describes historical milestones in the development of networks dedicated to capacity building in ethical review in these regions and outlines the major conclusions of two workshops on this issue, which were held in September and October 2002 in Libreville, Gabon, and Paris, France. The workshops were the culmination of collaboration between the African Malaria Network Trust (AMANET) and the Pan African Bioethics Initiative (PABIN). They produced an update on ethics organizations with regard to mission, function, activities, members, and contact people, in eight countries within the regions discussed. As a result of the commitment of mandated delegates, a further prominent outcome followed these workshops: the creation of national structures, where none existed before, dedicated to the ethical review of clinical trials.

Evidence against Wolbachia symbiosis in Loa loa.

BACKGROUND: The majority of filarial nematode species are host to Wolbachia bacterial endosymbionts, although a few including Acanthocheilonema viteae, Onchocerca flexuosa and Setaria equina have been shown to be free of infection. Comparisons of species with and without symbionts can provide important information on the role of Wolbachia symbiosis in the biology of the nematode hosts and the contribution of the bacteria to the development of disease. Previous studies by electron microscopy and PCR have failed to detect intracellular bacterial infection in Loa loa. Here we use molecular and immunohistological techniques to confirm this finding. METHODS: We have used a combination of PCR amplification of bacterial genes (16S ribosomal DNA [rDNA], ftsZ and Wolbachia surface protein [WSP]) on samples of L. loa adults, third-stage larvae (L3) and microfilariae (mf) and immunohistology on L. loa adults and mf derived from human volunteers to determine the presence or absence of Wolbachia endosymbionts. Samples used in the PCR analysis included 5 adult female worms, 4 adult male worms, 5 mf samples and 2 samples of L3. The quality and purity of nematode DNA was tested by PCR amplification of nematode 5S rDNA and with diagnostic primers from the target species and used to confirm the absence of contamination from Onchocerca sp., Mansonella perstans, M. streptocerca and Wuchereria bancrofti. Immunohistology was carried out by light and electron microscopy on L. loa adults and mf and sections were probed with rabbit antibodies raised to recombinant Brugia malayi Wolbachia WSP. Samples from nematodes known to be infected with Wolbachia (O. volvulus, O. ochengi, Litomosoides sigmodontis and B. malayi) were used as positive controls and A. viteae as a negative control. RESULTS: Single PCR analysis using primer sets for the bacterial genes 16S rDNA, ftsZ, and WSP were negative for all DNA samples from L. loa. Positive PCR reactions were obtained from DNA samples derived from species known to be infected with Wolbachia, which confirmed the suitability of the primers and PCR conditions. The quality and purity of nematode DNA samples was verified by PCR amplification of 5S rDNA and with nematode diagnostic primers. Additional analysis by 'long PCR' failed to produce any further evidence for Wolbachia symbiosis. Immunohistology of L. loa adults and mf confirmed the results of the PCR with no evidence for Wolbachia symbiosis. CONCLUSION: DNA analysis and immunohistology provided no evidence for Wolbachia symbiosis in L. loa.

Transmission intensity affects both antigen-specific and nonspecific T-cell proliferative responses in Loa loa infection.

T-cell proliferative responses were studied in two villages in Gabon with different levels of Loa loa transmission. The first village (Okoumbi) had an annual transmission potential (ATP) of approximately 9,000 infective larvae (L3)/person/year (high transmission village), while the second village (Ndjokaye) had an ATP of approximately 1,000 L3/person/year (low transmission village). Proliferation and cytokine assays were performed on peripheral blood mononuclear cells (PBMC) from individuals aged 18 years and over using either mitogens (concanavalin A or phytohemagglutinin), antigens (purified protein derivative [PPD], irrelevant antigen), or soluble extracts of L3, microfilariae, or adult L. loa. PBMC from individuals in the low transmission village responded better to stimulation with adult antigen and to PPD than did PBMC from individuals in the high transmission village (P = 0.0031 and P = 0.0012, respectively). These data suggest that high levels of transmission of L. loa depress both specific and nonspecific T-cell proliferative responses in infected humans.