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.

Peptostreptococcus faecalis sp. nov., new bacterial species isolated from healthy indigenous congolese volunteer.

The Microbial Culturomics Project aiming to discover several bacterial species made it possible to isolate the strain Marseille-P4308T from a stool sample of a healthy indigenous Congolese volunteer. Strain Marseille-P4308T is a Gram-positive coccus shaped bacterium that optimally grows at 37 °C. The 16S rRNA gene sequence of the strain has a 96.2% sequence similarity to Peptostreptococcus anaerobius strain NCTC 11460T (GenBank accession number: NR_042847.1). In addition, the average nucleotide identity of strain Marseille-P4308T with its closest related species was 71.1%, which was far below the recommended threshold (>95-96%). The genome of the strain Marseille-P4308T has a length of 2.14 Mbp with G + C content of 30.4 mol%. Based on phenotypic, biochemical, genomic and phylogenetic analysis, strain Marseille-P4308T (= CSUR P4308 = CECT 9960) clearly appears to be a new species for which the name Peptostreptococcus faecalis sp. nov., is proposed.

Bacterial Infections in Humans and Nonhuman Primates from Africa: Expanding the Knowledge.

The close phylogenetic relationship between humans and other primates creates exceptionally high potential for pathogen exchange. The surveillance of pathogens in primates plays an important role in anticipating possible outbreaks. In this study, we conducted a molecular investigation of pathogenic bacteria in feces from African nonhuman primates (NHPs). We also investigated the pathogens shared by the human population and gorillas living in the same territory in the Republic of Congo. In total, 93% of NHPs (n=176) and 95% (n=38) of humans were found to carry at least one bacterium. Non-pallidum Treponema spp. (including T. succinifaciens, T. berlinense, and several potential new species) were recovered from stools of 70% of great apes, 88% of monkeys, and 79% of humans. Non-tuberculosis Mycobacterium spp. were also common in almost all NHP species as well as in humans. In addition, Acinetobacter spp., members of the primate gut microbiota, were mainly prevalent in human and gorilla. Pathogenic Leptospira spp. were highly present in humans (82%) and gorillas (66%) stool samples in Congo, but were absent in the other NHPs, therefore suggesting a possible gorillas-humans exchange. Particular attention will be necessary for enteropathogenic bacteria detected in humans such as Helicobacter pylori, Salmonella spp. (including S. typhi/paratyphi), Staphyloccocus aureus, and Tropheryma whipplei, some of which were also present in gorillas in the same territory (S. aureus and T. whipplei). This study enhances our knowledge of pathogenic bacteria that threaten African NHPs and humans by using a non-invasive sampling technique. Contact between humans and NHPs results in an exchange of pathogens. Ongoing surveillance, prevention, and treatment strategies alone will limit the spread of these infectious agents.

Occurrence of Ten Protozoan Enteric Pathogens in Three Non-Human Primate Populations.

Non-human primate populations act as potential reservoirs for human pathogens, including viruses, bacteria and parasites, which can lead to zoonotic infections. Furthermore, intestinal microorganisms may be pathogenic organisms to both non-human primates and humans. It is, therefore, essential to study the prevalence of these infectious agents in captive and wild non-human primates. This study aimed at showing the prevalence of the most frequently encountered human enteric protozoa in non-human primate populations based on qPCR detection. The three populations studied were common chimpanzees (Pan troglodytes) in Senegal and gorillas (Gorilla gorilla) in the Republic of the Congo and in the Beauval Zoo (France). Blastocystis spp. were mainly found, with an occurrence close to 100%, followed by Balantidiumcoli (23.7%), Giardiaintestinalis (7.9%), Encephalitozoonintestinalis (1.3%) and Dientamoebafragilis (0.2%). None of the following protozoa were detected: Entamoebahistolytica, Enterocytozoonbieneusi, Cryptosporidiumparvum, C. hominis, Cyclosporacayetanensis or Cystoisosporabelli. As chimpanzees and gorillas are genetically close to humans, it is important to monitor them frequently against different pathogens to protect these endangered species and to assess potential zoonotic transmissions to humans.

Stool Serology: Development of a Non-Invasive Immunological Method for the Detection of Enterovirus-Specific Antibodies in Congo Gorilla Faeces.

BACKGROUND: The incidence of poliovirus has been significantly reduced by as much as 99.9% globally. Alongside this, however, vaccine-associated paralytic poliomyelitis has emerged. Previously, our team reported in the Lésio-Louna-Léfini Nature Reserve (Republic of Congo) the presence of a new Enterovirus C (Ibou002) in a male gorilla that was put away because of clinical symptoms of facial paralysis. This new virus, isolated was from the stool samples of this gorilla but also from the excrement of an eco-guardian, is very similar to Coxsackievirus (EV-C99) as well as poliovirus 1 and 2. We hypothesised that these symptoms might be due to poliovirus infection. To test our hypothesis, we developed and optimised a non-invasive immunoassay for the detection of Enterovirus-specific antibodies in gorilla faeces that could be useful for routine serosurveillance in such cases. METHODS: In order to assess the potential role of poliovirus infection, we have developed and optimised a protocol, based on the lyophilisation and solubilisation of small volumes of stool extracts from 16 gorilla and 3 humans, to detect specific antibodies by western blot and ELISA. RESULTS: First, total immunoglobulins were detected in the concentrated stool extracts. Specific antibodies were then detected in 4/16 gorilla samples and 2/3 human samples by western blot using both the polio vaccine antigen and the Ibou002 antigen and by ELISA using the polio vaccine antigen. Humoral responses were greater with the Ibou002 antigen. CONCLUSION: We therefore suggest that this recombinant virus could lead to a polio-like disease in the endangered western lowland gorilla. The development of a non-invasive approach to detect microorganism-specific immunoglobulins from faecal samples opens numerous prospects for application in zoonotic infectious diseases and could revolutionise the screening of animals for important emerging infections, such as Ebola fever, rabies and coronavirus infections.

Enteroviruses from Humans and Great Apes in the Republic of Congo: Recombination within Enterovirus C Serotypes.

Enteroviruses (EVs) are viruses of the family Picornaviridae that cause mild to severe infections in humans and in several animal species, including non-human primates (NHPs). We conducted a survey and characterization of enteroviruses circulating between humans and great apes in the Congo. Fecal samples (N = 24) of gorillas and chimpanzees living close to or distant from humans in three Congolese parks were collected, as well as from healthy humans (N = 38) living around and within these parks. Enteroviruses were detected in 29.4% of gorilla and 13.15% of human feces, including wild and human-habituated gorillas, local humans and eco-guards. Two identical strains were isolated from two humans coming from two remote regions. Their genomes were similar and all genes showed their close similarity to coxsackieviruses, except for the 3C, 3D and 5'-UTR regions, where they were most similar to poliovirus 1 and 2, suggesting recombination. Recombination events were found between these strains, poliovirus 1 and 2 and EV-C99. It is possible that the same EV-C species circulated in both humans and apes in different regions in the Congo, which must be confirmed in other investigations. In addition, other studies are needed to further investigate the circulation and genetic diversity of enteroviruses in the great ape population, to draw a definitive conclusion on the different species and types of enteroviruses circulating in the Republic of Congo.

Parasitic Infections in African Humans and Non-Human Primates.

Different protozoa and metazoa have been detected in great apes, monkeys and humans with possible interspecies exchanges. Some are either nonpathogenic or their detrimental effects on the host are not yet known. Others lead to serious diseases that can even be fatal. Their survey remains of great importance for public health and animal conservation. Fecal samples from gorillas (Gorilla gorilla) and humans living in same area in the Republic of Congo, chimpanzees (Pan troglodytes) from Senegal and one other from the Republic of Congo, Guinea baboons (Papio papio) from Senegal, hamadryas baboons (Papio hamadryas) from Djibouti and Barbary macaques (Macaca sylvanus) from Algeria, were collected. DNA was extracted and screened using specific qPCR assays for the presence of a large number of helminths and protozoa. Positive samples were then amplified in standard PCRs and sequenced when possible. Overall, infection rate was 36.5% in all non-human primates (NHPs) and 31.6% in humans. Great apes were more often infected (63.6%) than monkeys (7.3%). At least twelve parasite species, including ten nematodes and two protozoa were discovered in NHPs and five species, including four nematodes and a protozoan in humans. The prevalences of Giarida lamblia, Necator americanus, Enterobius vermicularis, Strongyloides stercoralis were similar between gorillas and human community co-habiting the same forest ecosystem in the Republic of Congo. In addition, human specific Mansonella perstans (5.1%) and other Mansonella spp. (5.1%) detected in these gorillas suggest a possible cross-species exchange. Low prevalence (2%) of Ascaris lumbricoides, Enterobius vermicularis, Strongyloides stercoralis were observed in chimpanzees, as well as a high prevalence of Abbreviata caucasica (57.1%), which should be considered carefully as this parasite can affect other NHPs, animals and humans. The Barbary macaques were less infected (7.2%) and Oesophagostomum muntiacum was the main parasite detected (5.8%). Finally, we report the presence of Pelodera sp. and an environmental Nematoda DNAs in chimpanzee feces, Nematoda sp. and Bodo sp. in gorillas, as well as DNA of uncharacterized Nematoda in apes and humans, but with a relatively lower prevalence in humans. Prevalence of extraintestinal parasites remains underestimated since feces are not the suitable sampling methods. Using non-invasive sampling (feces) we provide important information on helminths and protozoa that can infect African NHPs and human communities living around them. Public health and animal conservation authorities need to be aware of these infections, as parasites detected in African NHPs could affect both human and other animals' health.

Adenovirus Infections in African Humans and Wild Non-Human Primates: Great Diversity and Cross-Species Transmission.

Non-human primates (NHPs) are known hosts for adenoviruses (AdVs), so there is the possibility of the zoonotic or cross-species transmission of AdVs. As with humans, AdV infections in animals can cause diseases that range from asymptomatic to fatal. The aim of this study was to investigate the occurrence and diversity of AdVs in: (i) fecal samples of apes and monkeys from different African countries (Republic of Congo, Senegal, Djibouti and Algeria), (ii) stool of humans living near gorillas in the Republic of Congo, in order to explore the potential zoonotic risks. Samples were screened by real-time and standard PCRs, followed by the sequencing of the partial DNA polymerase gene in order to identify the AdV species. The prevalence was 3.3 folds higher in NHPs than in humans. More than 1/3 (35.8%) of the NHPs and 1/10 (10.5%) of the humans excreted AdVs in their feces. The positive rate was high in great apes (46%), with a maximum of 54.2% in chimpanzees (Pan troglodytes) and 35.9% in gorillas (Gorilla gorilla), followed by monkeys (25.6%), with 27.5% in Barbary macaques (Macaca sylvanus) and 23.1% in baboons (seven Papio papio and six Papio hamadryas). No green monkeys (Chlorocebus sabaeus) were found to be positive for AdVs. The AdVs detected in NHPs were members of Human mastadenovirus E (HAdV-E), HAdV-C or HAdV-B, and those in the humans belonged to HAdV-C or HAdV-D. HAdV-C members were detected in both gorillas and humans, with evidence of zoonotic transmission since phylogenetic analysis revealed that gorilla AdVs belonging to HAdV-C were genetically identical to strains detected in humans who had been living around gorillas, and, inversely, a HAdV-C member HAdV type was detected in gorillas. This confirms the gorilla-to-human transmission of adenovirus. which has been reported previously. In addition, HAdV-E members, the most often detected here, are widely distributed among NHP species regardless of their origin, i.e., HAdV-E members seem to lack host specificity. Virus isolation was successful from a human sample and the strain of the Mbo024 genome, of 35 kb, that was identified as belonging to HAdV-D, exhibited close identity to HAdV-D members for all genes. This study provides information on the AdVs that infect African NHPs and the human populations living nearby, with an evident zoonotic transmission. It is likely that AdVs crossed the species barrier between different NHP species (especially HAdV-E members), between NHPs and humans (especially HAdV-C), but also between humans, NHPs and other animal species.

High prevalence of Plasmodium falciparum antimalarial drug resistance markers in isolates from asymptomatic patients from the Republic of the Congo between 2010 and 2015.

OBJECTIVES: This study investigated the prevalence of haplotypes of the Pfdhps, Pfdhfr, Pfcrt, Pfmdr1 and PfK13 resistance markers in isolates from asymptomatic patients from the Republic of the Congo following implementation of artemisinin based-combination therapy (ACT). METHODS: Peripheral blood was collected from asymptomatic children in 2010 and 2015 from Brazzaville in the south and in 2013 in the north of the Congo. Genotypes of Pfmdr1, Pfcrt, Pfdhps, Pfdhfr and PfK13 were assessed by PCR. RESULTS: Children from 2010 were younger than those from 2015 (mean age 5.38 years vs. 8.67 years; P=0.003). The main Pfcrt haplotype was the wild-type CVMNK (84.85%) in 2010, whereas the mutant CVIET (61.64%) predominated in 2015 (P<0.001). In the north, 45.00% of samples were CVMNK and 10.00% were CVIET. Other samples harboured new haplotypes in the country or mixed alleles. No significant difference in Pfmdr1 haplotypes was observed in 2010 and 2015 and the main haplotypes were NYD and NFD (30.56% vs. 28.57% and 61.11% vs. 42.86% for 2010 and 2015, respectively). In the south, the Pfdhps haplotypes observed were AAKAA, AGKAA, SGKAA and SGEGA (87.50% vs. 0%, 12.50% vs. 33.33%, 0% vs. 33.33% and 0% vs. 33.33% for 2010 and 2015, respectively). For Pfdhfr, the IRNI haplotype was most prevalent (85.71% for 2010, 87.50% for 2013 and 100% for 2015). No PfK13 mutations were found. CONCLUSIONS: Monitoring the efficacy of ACT and intermittent preventive treatment with sulfadoxine-pyrimethamine is necessary to ensure an epidemiological survey of asymptomatic malaria.

Detection of novel RNA viruses from free-living gorillas, Republic of the Congo: genetic diversity of picobirnaviruses.

Most of the emerging infectious diseases reported so far originated in wildlife. Therefore, virological surveillance of animals and particularly great apes is of great interest to establish the repertory of viruses associated with healthy hosts. This will further help to identify the emergence of new viruses and predict the possibility of interspecies transmission. In this study, we performed shotgun viral metagenomics on stool samples collected from seventeen free-living wild gorillas from the Republic of the Congo. The analysis revealed the presence of novel RNA viruses (picobirnaviruses, partitivirus, and Picornavirales (posa-like and dicistrovirus-like viruses)). Among these, picobirnavirus-related sequences were abundantly covered in the stools. Based on genetic variations both in capsid and RdRp proteins of picobirnaviruses, at least 96 variants were identified and most of them were novel. Among the 96, 22 variants had a nearly complete genome or segment. A comprehensive sequence analysis identified a potential new genogroup/genetic cluster and the presence of a short linear amino acid motif (ExxRxNxxxE) in a hypothetical protein. The sequence analysis of posa-like virus and dicistrovirus showed that these two viruses were novel members in the respective viral families. In conclusion, the identification of novel RNA viruses and their genetic diversity increases our knowledge about viruses that are associated with stools of wild gorillas and contributes to the initiatives in the search for potential emerging zoonotic viruses.

Head Lice of Pygmies Reveal the Presence of Relapsing Fever Borreliae in the Republic of Congo.

BACKGROUND: Head lice, Pediculus humanus capitis, occur in four divergent mitochondrial clades (A, B, C and D), each having particular geographical distributions. Recent studies suggest that head lice, as is the case of body lice, can act as a vector for louse-borne diseases. Therefore, understanding the genetic diversity of lice worldwide is of critical importance to our understanding of the risk of louse-borne diseases. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the results of the first molecular screening of pygmies' head lice in the Republic of Congo for seven pathogens and an analysis of lice mitochondrial clades. We developed two duplex clade-specific real-time PCRs and identified three major mitochondrial clades: A, C, and D indicating high diversity among the head lice studied. We identified the presence of a dangerous human pathogen, Borrelia recurrentis, the causative agent of relapsing fever, in ten clade A head lice, which was not reported in the Republic of Congo, and B. theileri in one head louse. The results also show widespread infection among head lice with several species of Acinetobacter. A. junii was the most prevalent, followed by A. ursingii, A. baumannii, A. johnsonii, A. schindleri, A. lwoffii, A. nosocomialis and A. towneri. CONCLUSIONS/SIGNIFICANCE: Our study is the first to show the presence of B. recurrentis in African pygmies' head lice in the Republic of Congo. This study is also the first to report the presence of DNAs of B. theileri and several species of Acinetobacter in human head lice. Further studies are needed to determine whether the head lice can transmit these pathogenic bacteria from person to another.

Biomarkers of oxidative stress and personalized treatment of pulmonary tuberculosis: emerging role of gamma-glutamyltransferase.

Background. The objectives were (i) to evaluate the impact of acute pulmonary tuberculosis (PTB) and anti-TB therapy on the relationship between AST, ALT, and GGT levels in absence of conditions related to hepatotoxicity; (ii) to evaluate the rate and the time of alterations of AST, ALT, and GGT. Design and Methods. A prospective followup of 40 adults (21 males; mean age of 34.7 ± 5.8 years) with active PTB on initial phase and continuation phase anti-TB. Results. Only 3% (n = 1) developed a transient and benign ADR at day 30 without interruption of anti-TB treatment. Within normal ranges, GGT decreased significantly from day 0 to day 60, while AST and ALT increased significantly and respectively. During day 0-day 60, there was a significant, negative, and independent association between GGT and AST. Conclusion. The initial two months led to significant improvement of oxidative stress. Values of oxidative markers in normal ranges might predict low rate of ADR.

Multiple independent introductions of Plasmodium falciparum in South America.

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence--archeological and genetic--suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

African monkeys are infected by Plasmodium falciparum nonhuman primate-specific strains.

Recent molecular exploration of the Plasmodium species circulating in great apes in Africa has revealed the existence of a large and previously unknown diversity of Plasmodium. For instance, gorillas were found to be infected by parasites closely related to Plasmodium falciparum, suggesting that the human malignant malaria agent may have arisen after a transfer from gorillas. Although this scenario is likely in light of the data collected in great apes, it remained to be ascertained whether P. falciparum-related parasites may infect other nonhuman primates in Africa. Using molecular tools, we here explore the diversity of Plasmodium species infecting monkeys in Central Africa. In addition to previously described Hepatocystis and Plasmodium species (Plasmodium gonderi and Plasmodium sp DAJ-2004), we have found one African monkey to be infected by a P. falciparum-related parasite. Examination of the nuclear and mitochondrial genomes of this parasite reveals that it is specific of nonhuman primates, indicating that P. falciparum-related pathogens can naturally circulate in some monkey populations in Africa. We also show that at least two distinct genetic entities of P. falciparum infect nonhuman primates and humans, respectively. Our discoveries bring into question the proposed gorilla origin of human P. falciparum.

Increase of malaria attacks among children presenting concomitant infection by Schistosoma mansoni in Senegal.

Helminthic infections concomitant with malaria are common in inter-tropical areas. A recent study showed that mice co-infected with Schistosoma mansoni and Plasmodium chabaudi develop higher P. chabaudi parasitaemia and had a higher mortality rate. This important observation deserved to be further investigated among human populations.Malaria attacks were recorded in 512 children aged 6-15 years living in Richard Toll (Northern Senegal) among whom 336 were infected by S. mansoni, and 175 were not. The incidence rate of malaria attacks was significantly higher among S. mansoni-infected individuals, particularly those carrying the highest worm loads, as compared to uninfected subjects (26.6% versus 16,4 %). In contrast, the rate of malaria attacks was lower, without reaching significance, in medium grade S. mansoni infections. Thus, infection by S. mansoni affects susceptibility to malaria, but this can vary according to the intensity of parasite load. The immunological mechanisms underlying this dual effect need to be further explored.

Severe malaria attack is associated with high prevalence of Ascaris lumbricoides infection among children in rural Senegal.

In human populations, the concomitance of various parasitic infections can induce modifications of the specific immune response to each pathogen and thus induce changes in their clinical expression. Several studies, however, have produced conflicting results. To study the hypothesis that there is an association between helminthiasis and the occurrence of severe malaria a prospective case-control study was carried out in a rural zone of Senegal where 105 presumptive severe malaria attacks were studied in 2001 and 2002. Following parasitological control the cases were divided into two groups: A (severe malaria) with severe symptoms and parasite density >5000 parasites/microl (n = 64) and B (other causes) with severe symptoms and negative or weak parasite density (n = 41). In group A the prevalence of Ascaris lumbricoides infection was higher in cases of severe malaria than in controls, odds ratio (OR) = 9.95 (95% CI 3.03-32.69). Similar but not significantly different results were observed between patients in group B and their controls, OR = 2.47 (95% CI 0.95-6.38).