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Introduction: Plasmodium malariae is the most common non-falciparum species in sub-Saharan Africa. Despite this, data on its genetic diversity is scarce. Therefore, we aimed to establish a P. malariae genotyping approach based on size polymorphic regions that can be easily applied in molecular epidemiological studies. Methods: Four potential genotyping markers, Pm02, Pm09, P. malariae thrombospondin-related anonymous protein (pmtrap), and P. malariae merozoite surface protein fragment 2 (pmmsp1 F2) were amplified via nested PCR and analysed using automated capillary gel electrophoresis. Results: We observed the highest allelic diversity for pmtrap (MOI = 1.61) and pmmsp1 F2 (He = 0.81). Further applying the two markers pmtrap and pmmsp1 F2 on a different sample set of 21 P. malariae positive individuals followed up over one week, we saw a high consistency in their performance. The results show a large complexity and high dynamics of P. malariae infections in the asymptomatic Gabonese study population. Discussion: We successfully implemented a new genotyping panel for P. malariae consisting of only two markers: pmtrap and pmmsp1 F2. It can be easily applied in other settings to investigate the genotype diversity of P. malariae populations, providing further important data on the molecular epidemiology of this parasite species.
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Variación Genética , Genotipo , Malaria , Epidemiología Molecular , Plasmodium malariae , Proteínas Protozoarias , Plasmodium malariae/genética , Plasmodium malariae/aislamiento & purificación , Humanos , Malaria/epidemiología , Malaria/parasitología , Epidemiología Molecular/métodos , África del Sur del Sahara/epidemiología , Proteínas Protozoarias/genética , Técnicas de Genotipaje/métodos , Reacción en Cadena de la Polimerasa/métodos , ADN Protozoario/genética , Alelos , Gabón/epidemiología , Marcadores GenéticosRESUMEN
OBJECTIVES: We investigated the diversity and dynamics of Plasmodium infection in serially collected samples from asymptomatic participants of a clinical trial assessing the efficacy and safety of ivermectin in Gabon. We checked whether the baseline sample reflected the P. falciparum genotype and Plasmodium species diversity seen over 7 days of follow-up. METHODS: Blood samples were collected at inclusion, every 8 hours until hour 72, daily until day 7, and on day 14. Plasmodium species was determined by qPCR and pfmsp1 length polymorphism was assessed for P. falciparum genotyping. RESULTS: In 17/48 (35%) individuals, all pfmsp1 genotypes identified during the assessed period were detected at baseline; in 31/48 (65%), new genotypes were found during follow-up. Additional sampling at hour 24 allowed the identification of all genotypes seen over 7 days in 50% of the individuals. Ivermectin did not impact the genotype dynamics. Mixed Plasmodium spp. infections were detected in 28/49 (57%) individuals at baseline, and detection of non-falciparum infections during follow-up varied. CONCLUSIONS: Our results reveal complex intra-host dynamics of P. falciparum genotypes and Plasmodium species and underscore the importance of serial sampling in clinical trials for antimalarial drugs with asymptomatically P. falciparum-infected individuals. This might allow a more accurate identification of genotypes in multiple infections, impacting the assessment of drug efficacy.
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Infecciones Asintomáticas , Genotipo , Ivermectina , Malaria Falciparum , Humanos , Gabón/epidemiología , Infecciones Asintomáticas/epidemiología , Adulto , Malaria Falciparum/parasitología , Malaria Falciparum/epidemiología , Malaria Falciparum/tratamiento farmacológico , Masculino , Ivermectina/uso terapéutico , Femenino , Variación Genética , Plasmodium falciparum/genética , Plasmodium falciparum/efectos de los fármacos , Plasmodium/genética , Plasmodium/clasificación , Plasmodium/aislamiento & purificación , Plasmodium/efectos de los fármacos , Adulto JovenRESUMEN
Introduction: Mansonella species are filarial parasites that infect humans worldwide. Although these infections are common, knowledge of the pathology and diversity of the causative species is limited. Furthermore, the lack of sequencing data for Mansonella species, shows that their research is neglected. Apart from Mansonella perstans, a potential new species called Mansonella sp "DEUX" has been identified in Gabon, which is prevalent at high frequencies. We aimed to further determine if Mansonella sp "DEUX" is a genotype of M. perstans, or if these are two sympatric species. Methods: We screened individuals in the area of Fougamou, Gabon for Mansonella mono-infections and generated de novo assemblies from the respective samples. For evolutionary analysis, a phylogenetic tree was reconstructed, and the differences and divergence times are presented. In addition, mitogenomes were generated and phylogenies based on 12S rDNA and cox1 were created. Results: We successfully generated whole genomes for M. perstans and Mansonella sp "DEUX". Phylogenetic analysis based on annotated protein sequences, support the hypothesis of two distinct species. The inferred evolutionary analysis suggested, that M. perstans and Mansonella sp "DEUX" separated around 778,000 years ago. Analysis based on mitochondrial marker genes support our hypothesis of two sympatric human Mansonella species. Discussion: The results presented indicate that Mansonella sp "DEUX" is a new Mansonella species. These findings reflect the neglect of this research topic. And the availability of whole genome data will allow further investigations of these species.
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Mansonella , Simpatría , Animales , Humanos , Mansonella/genética , Filogenia , ADN Ribosómico , Secuencia de AminoácidosRESUMEN
[This corrects the article DOI: 10.1371/journal.pntd.0009511.].
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BACKGROUND: Schistosomiasis is highly prevalent in Africa. Praziquantel is effective against adult schistosomes but leaves prepatent stages unaffected-which is a limit to patient management and elimination. Given the large-scale use of praziquantel, development of drug resistance by Schistosoma is feared. Antimalarials are promising drugs for alternative treatment strategies of Schistosoma infections. Development of drugs with activity against both malaria and schistosomiasis is particularly appealing as schistosome infections often occur concomitantly with malaria parasites in sub-Saharan Africa. Therefore, antiplasmodial compounds were progressively tested against Schistosoma in vitro, in mice, and in a clinical study. RESULTS: Amongst 16 drugs and 1 control tested, pyronaridine, methylene blue and 5 other antimalarials were highly active in vitro against larval stage schistosomula with a 50% inhibitory concentration below 10 µM. Both drugs were lethal to ex vivo adult worms tested at 30 µM with methylene blue also active at 10 µM. Pyronaridine treatment of mice infected with S. mansoni at the prepatent stage reduced worm burden by 82% and cured 7 out of 12 animals, however in mice adult stages remained viable. In contrast, methylene blue inhibited adult worms by 60% but cure was not achieved. In an observational pilot trial in Gabon in children, the antimalarial drug combination pyronaridine-artesunate (Pyramax) reduced S. haematobium egg excretion from 10/10 ml urine to 0/10 ml urine, and 3 out of 4 children were cured. CONCLUSION: Pyronaridine and methylene blue warrant further investigation as candidates for schistosomiasis treatment. Both compounds are approved for human use and evidence for their potential as antischistosomal compounds can be obtained directly from clinical testing. Particularly, pyronaridine-artesunate, already available as an antimalarial drug, calls for further clinical evaluation. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT03201770.
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Antiprotozoarios/farmacología , Naftiridinas/farmacología , Schistosoma mansoni/efectos de los fármacos , Esquistosomiasis Urinaria/tratamiento farmacológico , Animales , Antimaláricos/farmacología , Artesunato/farmacología , Niño , Combinación de Medicamentos , Gabón , Humanos , Larva/efectos de los fármacos , Azul de Metileno/farmacología , Ratones , Schistosoma haematobium/efectos de los fármacos , Schistosoma mansoni/crecimiento & desarrollo , Esquistosomiasis mansoni/tratamiento farmacológicoRESUMEN
Immunization with Plasmodium falciparum (Pf) sporozoites under chemoprophylaxis (PfSPZ-CVac) is the most efficacious approach to malaria vaccination. Implementation is hampered by a complex chemoprophylaxis regimen and missing evidence for efficacy against heterologous infection. We report the results of a double-blinded, randomized, placebo-controlled trial of a simplified, condensed immunization regimen in malaria-naive volunteers (EudraCT-Nr: 2018-004523-36). Participants are immunized by direct venous inoculation of 1.1 × 105 aseptic, purified, cryopreserved PfSPZ (PfSPZ Challenge) of the PfNF54 strain or normal saline (placebo) on days 1, 6 and 29, with simultaneous oral administration of 10 mg/kg chloroquine base. Primary endpoints are vaccine efficacy tested by controlled human malaria infection (CHMI) using the highly divergent, heterologous strain Pf7G8 and safety. Twelve weeks following immunization, 10/13 participants in the vaccine group are sterilely protected against heterologous CHMI, while (5/5) participants receiving placebo develop parasitemia (risk difference: 77%, p = 0.004, Boschloo's test). Immunization is well tolerated with self-limiting grade 1-2 headaches, pyrexia and fatigue that diminish with each vaccination. Immunization induces 18-fold higher anti-Pf circumsporozoite protein (PfCSP) antibody levels in protected than in unprotected vaccinees (p = 0.028). In addition anti-PfMSP2 antibodies are strongly protection-associated by protein microarray assessment. This PfSPZ-CVac regimen is highly efficacious, simple, safe, well tolerated and highly immunogenic.
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Vacunas contra la Malaria/inmunología , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Vacunación/métodos , Vacunas Atenuadas/inmunología , Adulto , Antimaláricos/uso terapéutico , Línea Celular , Quimioprevención , Cloroquina/uso terapéutico , Femenino , Humanos , Inmunoglobulina G/inmunología , Vacunas contra la Malaria/efectos adversos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Masculino , Parasitemia/inmunología , Análisis por Matrices de Proteínas , Esporozoítos/inmunología , Vacunación/efectos adversos , Vacunas Atenuadas/efectos adversosRESUMEN
Mansonella perstans, a filarial nematode, infects large populations in Africa and Latin America. Recently, a potential new species, Mansonella sp "DEUX," was reported. Carriage of endosymbiotic Wolbachia opens treatment options for Mansonella infections. Within a cross-sectional study, we assessed the prevalence of filarial infections in 834 Gabonese individuals and the presence of the endosymbiont Wolbachia. Almost half of the participants (400/834 [48%]) were infected with filarial nematodes, with Mansonella sp "DEUX" being the most frequent (295/400 [74%]), followed by Loa loa (273/400 [68%]) and Mansonella perstans (82/400 [21%]). Being adult/elderly, male, and living in rural areas was associated with a higher risk of infection. Wolbachia carriage was confirmed in M. perstans and Mansonella sp "DEUX." In silico analysis revealed that Mansonella sp "DEUX" is not detected with currently published M. perstans-specific assays. Mansonella infections are highly prevalent in Gabon and might have been underreported, likely also beyond Gabon.
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Mansonella/clasificación , Mansonella/genética , Mansoneliasis/epidemiología , Mansoneliasis/parasitología , Animales , Portador Sano/parasitología , Estudios Transversales , Gabón/epidemiología , Humanos , Loa/genética , Masculino , Epidemiología Molecular , Reacción en Cadena de la Polimerasa , Población RuralRESUMEN
BACKGROUND: Plasmodium falciparum deficient for hrp2 and hrp3 genes are a threat to malaria management and elimination, since they escape widely used HRP2-based rapid diagnostic tests and treatment. Hrp2/hrp3 deletions are increasingly reported from all malaria endemic regions but are currently only identified by laborious methodologies. METHODS: We developed a novel hydrolysis probe-based, quantitative, real-time PCR (4plex qPCR) for detection and discrimination of P. falciparum infection (cytb) and hrp2 and hrp3 gene status, and to control assay validity (btub). A cross-sectional, diagnostic accuracy study was performed in Gabon for assay validation and deletion screening. FINDINGS: In parallel to identification of P. falciparum infection in samples down to 0.05 parasites/µl, the 4plex qPCR enabled specific and valid interrogation of the parasites´s hrp2 and hrp3 genes in one go - even in low parasitemic samples. The assay was precise and robust also when performed in a routine healthcare setting in Gabon. The risk of falsely identifying hrp2 or hrp3 deletion was reduced by 100-fold compared to conventional PCR. Evaluation against microscopy was performed on 200 blood samples collected in Gabon: sensitivity and specificity of 4plex qPCR (cytb) were 100% and 80%, respectively. Stringent testing revealed hrp2 deletion in 2 of 95 P. falciparum positive and validated samples. INTERPRETATION: The novel 4plex qPCR is sensitive, accurate and allows resource-efficient rapid screening. Monitoring and mapping of hrp2/hrp3 deletions is required to identify areas where control strategies may need to be adapted to ensure appropriate patient care and ultimately achieve malaria elimination. FUNDING: BMBF (03VP00402).