Genetic diversity of Plasmodium malariae in sub-Saharan Africa: a two-marker genotyping approach for molecular epidemiological studies.

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.

Malaria screening: what is the contribution of molecular biology?

INTRODUCTION: According to the World Health Organization, Microscopy is the gold standard for diagnosing malaria. However, the performance of this examination depends on the experience of the microscopist and the level of parasitemia. Thus, molecular biology detection of malaria could be an alternative technique. AIM: evaluate the contribution of molecular biology in detecting imported malaria. METHODS: This was a descriptive, prospective study, including all students, from the Monastir region, and foreigners, from countries endemic to malaria. The study period was from September 2020 to April 2021. Each subject was screened for malaria by three methods: direct microscopic detection of Plasmodium, detection of plasmodial antigens, and detection of plasmodial DNA by nested PCR. RESULTS: Among the 127 subjects screened, only one had a positive microscopic examination for Plasmodium falciparum. Among the 126 subjects with a negative microscopic examination, twelve students had a positive nested PCR result, i.e. 9.5%. Molecular sequencing allowed the identification of ten isolates of Plasmodium falciparum, one Plasmodium malariae and one Plasmodium ovale. Our study showed that the results of nested PCR agreed with those of microscopy in 90.6% of cases. CONCLUSION: Nested PCR seems more sensitive for the detection of low parasitemias. Hence the importance of including molecular biology as a malaria screening tool to ensure better detection of imported cases.

Ancient Plasmodium genomes shed light on the history of human malaria.

Malaria-causing protozoa of the genus Plasmodium have exerted one of the strongest selective pressures on the human genome, and resistance alleles provide biomolecular footprints that outline the historical reach of these species1. Nevertheless, debate persists over when and how malaria parasites emerged as human pathogens and spread around the globe1,2. To address these questions, we generated high-coverage ancient mitochondrial and nuclear genome-wide data from P. falciparum, P. vivax and P. malariae from 16 countries spanning around 5,500 years of human history. We identified P. vivax and P. falciparum across geographically disparate regions of Eurasia from as early as the fourth and first millennia BCE, respectively; for P. vivax, this evidence pre-dates textual references by several millennia3. Genomic analysis supports distinct disease histories for P. falciparum and P. vivax in the Americas: similarities between now-eliminated European and peri-contact South American strains indicate that European colonizers were the source of American P. vivax, whereas the trans-Atlantic slave trade probably introduced P. falciparum into the Americas. Our data underscore the role of cross-cultural contacts in the dissemination of malaria, laying the biomolecular foundation for future palaeo-epidemiological research into the impact of Plasmodium parasites on human history. Finally, our unexpected discovery of P. falciparum in the high-altitude Himalayas provides a rare case study in which individual mobility can be inferred from infection status, adding to our knowledge of cross-cultural connectivity in the region nearly three millennia ago.

Varied Prevalence of Antimalarial Drug Resistance Markers in Different Populations of Newly Arrived Refugees in Uganda.

Newly arrived refugees offer insights into malaria epidemiology in their countries of origin. We evaluated asymptomatic refugee children within 7 days of arrival in Uganda from South Sudan and the Democratic Republic of Congo (DRC) in 2022 for parasitemia, parasite species, and Plasmodium falciparum drug resistance markers. Asymptomatic P. falciparum infections were common in both populations. Coinfection with P. malariae was more common in DRC refugees. Prevalences of markers of aminoquinoline resistance (PfCRT K76T, PfMDR1 N86Y) were much higher in South Sudan refugees, of antifolate resistance (PfDHFR C59R and I164L, PfDHPS A437G, K540E, and A581G) much higher in DRC refugees, and of artemisinin partial resistance (ART-R; PfK13 C469Y and A675V) moderate in both populations. Prevalences of most mutations differed from those seen in Ugandans attending health centers near the refugee centers. Refugee evaluations yielded insights into varied malaria epidemiology and identified markers of ART-R in 2 previously little-studied countries.

Assessment of asymptomatic Plasmodium spp. infection by detection of parasite DNA in residents of an extra Amazonian region of Brazil

The hypotheses put forward to explain the malaria transmission cycle in extra-Amazonian Brazil, an area of very low malaria incidence, are based on either a zoonotic scenario involving simian malaria, or a scenario inwhich asymptomatic carriers play an important role. Objectives: To determine the incidence of asymptomatic infection by detecting Plasmodium spp. DNA and its role inresidual malaria transmission in a non-Amazonian region of Brazil. Methods: Upon the report of the first malaria case in 2010 in the Atlantic Forest region of the state of Espírito Santo, inhabitants within a 2 km radius were invited to participate in a follow-up study. After providing signed informed consent forms, inhabitants filled out a questionnaire and gave blood samples for PCR, and thick and thin smears. Follow up visits were performed every 3 months over a 21 month period, when new samples were collected and information was updated. Results: Ninety-two individuals were initially included for follow-up. At the first collection, all of them were clearly asymptomatic. One individual was positive for Plasmodium vivax, one for Plasmodium malariae and one for both P.vivax and P. malariae, corresponding to a prevalence of 3.4% (2.3% for each species). During follow-up, four new PCR positive cases (two for each species) were recorded, corresponding to an incidence of 2.5 infections per 100 person years or 1.25 infections per 100 person-years for each species...

Merozoite surface protein-1 genetic diversityin Plasmodium malariae and Plasmodium brasilianum from Brazil

The merozoite surface protein 1 (MSP1) gene encodes the major surface antigen of invasive forms of the Plasmodium erythrocytic stages and is considered a candidate vaccine antigen against malaria. Due to its polymorphisms, MSP1 is also useful for strain discrimination and consists of a good genetic marker. Sequence diversity in MSP1 has been analyzed in field isolates of three human parasites: P. falciparum, P. vivax, and P. ovale. However, the extent of variation in another human parasite, P. malariae, remains unknown. This parasite shows widespread, uneven distribution in tropical and subtropical regions throughout South America, Asia, and Africa. Interestingly, it is genetically indistinguishable from P. brasilianum, a parasite known to infect New World monkeys in Central and South America. Methods: Specific fragments (1 to 5) covering 60 % of the MSP1 gene (mainly the putatively polymorphic regions), were amplified by PCR in isolates of P. malariae and P. brasilianum from different geographic origin and hosts. Sequencing of the PCR-amplified products or cloned PCR fragments was performed and the sequences were used to construct a phylogenetic tree by the maximum likelihood method. Data were computed to give insights into the evolutionary and phylogenetic relationships of these parasites...

The genetic diversity of Plasmodium malariae and Plasmodium brasilianum from human, simian and mosquito hosts in Brazil

Plasmodium malariae is a protozoan parasite that causes malaria in humans and is genetically indistinguish able from Plasmodium brasilianum, a parasite infecting New World monkeys in Central and South America. P. malariae has a wide and patchy global distribution in tropical and subtropical regions, being found in South America, Asia, and Africa. However, little is known regarding the genetics of these parasites and the similarity between them could be because until now there are only a very few genomic sequences available from simian Plasmodium species. This study presents the first molecular epidemiological data for P. malariae and P. brasilianum from Brazil obtained from different hosts and uses them to explore the genetic diversity in relation to geographical origin and hosts. By using microsatellite genotyping, we discovered that of the 14 human samples obtained from areas of the Atlantic forest, 5 different multilocus genotypes were recorded, while in a sample from an infected mosquito from the same region a different haplo type was found. We also analyzed the longitudinal change of circulating plasmodial genetic profile in two untreated non-symptomatic patients during a 12-months interval...

A Case of Imported Plasmodium malariae Malaria

Malaria, the most common vector-borne parasite infection worldwide, results from infection by Plasmodium species. Approximately 80% of malaria cases are caused by P. vivax, which is broadly distributed from tropical to temperate regions; P. falciparum is the second most common infectious species. P. malariae and P. ovale are responsible for a relatively small proportion of malaria cases. Here, we report the case of a 23-yr-old Korean woman who acquired a P. malariae infection while visiting the Republic of Ghana in West Africa for business. She was diagnosed with P. malariae malaria on the basis of peripheral blood smear (PBS) and species-specific conventional and real-time PCR assays for 18S rRNA. She was treated with hydroxychloroquine, and the resulting PBS examination on day 2 suggested that negative conversion occurred. At her 1-month follow-up, however, both the PBS examination and molecular test for malaria demonstrated recurrent parasitemia. We started rescue therapy with mefloquine, and the patient recovered successfully. This is an important finding suggesting possible late recrudescence of a chloroquine-resistant P. malariae strain identified not only by its morphological features, but also by molecular tests.

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.

Diagnóstico molecular da malária em uma unidade de atenção terciária na Amazônia Brasileira / Molecular diagnosing of malaria in a tertiary care center in the Brazilian Amazon region

O exame de rotina para o diagnóstico da malária continua sendo a gota espessa, apesar da comprovada diminuição da sensibilidade e especificidade em situações de densidade parasitária baixa e infecções mistas. A reação em cadeia da polimerase vem sendo cada vez mais utilizada para a detecção molecular e identificação das espécies de plasmódio, por apresentar maior sensibilidade e especificidade. Foi realizada a nested-PCR em amostras de sangue total de 344 pacientes com síndrome febril aguda que se apresentaram para o diagnóstico de malária, em uma unidade terciária de saúde, em Manaus (Amazonas). Nenhum caso de malária por Plasmodium malariae foi diagnosticado à gota espessa ou PCR. Observou-se co-positividade de 96,7 por cento, co-negatividade de 62,2 por cento e coeficiente kappa de 0,44 entre PCR e gota espessa para Plasmodium falciparum. Para Plasmodium vivax, co-positividade de 100 por cento, co-negatividade de 78,1 por cento e coeficiente kappa de 0,56. Na detecção da malária mista, co-positividade de 100 por cento, co-negatividade de 84,9 por cento e coeficiente kappa de 0,26. A reação em cadeia da polimerase detectou alto número de infecções mistas nas amostras analisadas, mas seu uso rotineiro no diagnóstico da malária merece ainda ampla discussão.

The routine test for diagnosing malaria is still the thick blood smear, despite its known decreased sensitivity and specificity in situations of low parasite density and mixed infections. The polymerase chain reaction is increasingly being used for molecular detection and identification of Plasmodium species, due to its higher sensitivity and specificity. Nested PCR was performed on whole-blood samples from 344 patients with acute febrile syndrome who came to a tertiary healthcare center in Manaus (State of Amazonas) for diagnostic confirmation of malaria. No malaria cases caused by Plasmodium malariae were detected through the blood smear or PCR. Co-positivity of 96.7 percent, co-negativity of 62.2 percent and kappa coefficient of 0.44 were observed between PCR and thick blood smear for Plasmodium falciparum. For Plasmodium vivax, co-positivity of 100 percent, co-negativity of 78.1 percent and kappa coefficient of 0.56 were observed. For mixed infection, co-positivity of 100 percent, co-negativity of 84.9 percent and kappa coefficient of 0.26 were observed. Polymerase chain reaction detected a high number of mixed infections in the samples analyzed, but its routine use for diagnosing malaria still deserves further discussion.