BACKGROUND: 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.
OBJECTIVE: 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.
CONCLUSIONS: 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.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

BACKGROUND: Plasmodium falciparum is the dominant malaria species in the sub-Saharan Africa and the main cause of severe disease and death. Notwithstanding, severe malaria and death due to non-falciparum infections have been reported, but at much lower rates than P. falciparum infections. Following increasing use of molecular detection techniques in epidemiological studies, a higher prevalence of non-falciparum species has been reported in the region than previously thought. This article reviews the literature on the prevalence of non-falciparum malaria species in Uganda and the clinical figures of their severe diseases. It aims to elucidate the extent to which mono non-falciparum malaria infections in a highly malaria-endemic country contribute to malaria mortality and outline its policy implications on malaria case management.
METHODS: The available English-language published peer-reviewed literature up to March 2024 was sought via PubMed and Google Scholar. The keywords used were severe malaria, AND P. falciparum, P. malariae, P. vivax, P. ovale spp., mixed infections AND Uganda. The review encompassed 53 articles. Articles using molecular diagnosis methods were accounted for analysis.
RESULTS: The literature reported a substantial prevalence of non-falciparum infections in Uganda. Plasmodium malariae and Plasmodium ovale spp. were the second and third most prevalent reported malaria species respectively after P. falciparum as dominant species. Non-falciparum malaria infections often occur as mixed infections rather than mono-infections. Besides, molecular diagnostics revealed that 21% of initially reported mono-infections of P. falciparum were, in fact, mixed infections. No article was found on the prevalence of severe malaria or case fatality rate due to mixed or non-falciparum infections.
CONCLUSIONS: A critical knowledge gap exists regarding the impact of mixed and non-falciparum species on severe malaria and death in Uganda. Robust evidence on prevalence, recurrent parasitaemia, and severe clinical manifestations of mixed and non-falciparum malaria infections is crucial for evidence-based and effective policymaking regarding malaria case management.

Intracellular pathogens comprise a diverse group of pathogens that all share a required location in a host cell to infect, survive, and replicate. Intracellular location allows pathogens to hide from host immune responses, avoid competition with other pathogens, mediate host cellular functions, replicate safely, and cause infection that is difficult to target with therapeutics. All intracellular pathogens have varying routes of infiltration into host cells and different host cell preferences. For example, bacteria Mycobacterium tuberculosis chooses to invade antigen-presenting cells, which allows them to moderate host antigen presentation to memory cells, whereas rabies virus prefers to invade neurons because they have pre-existing innate immunity protection systems. Regardless of the pathway that each intracellular pathogen follows, all share the capacity to cause disease if they succeed in entering host cells. Here, we give an overview of selected intracellular pathogens and infections they cause, immune responses they induce, and intervention strategies used to treat and control them.

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.

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.

BACKGROUND: Recent studies point to the need to incorporate the detection of non-falciparum species into malaria surveillance activities in sub-Saharan Africa, where 95% of the world\'s malaria cases occur. Although malaria caused by infection with Plasmodium falciparum is typically more severe than malaria caused by the non-falciparum Plasmodium species P. malariae, P. ovale spp. and P. vivax, the latter may be more challenging to diagnose, treat, control and ultimately eliminate. The prevalence of non-falciparum species throughout sub-Saharan Africa is poorly defined. Tanzania has geographical heterogeneity in transmission levels but an overall high malaria burden.
METHODS: To estimate the prevalence of malaria species in Mainland Tanzania, we randomly selected 1428 samples from 6005 asymptomatic isolates collected in previous cross-sectional community surveys across four regions and analyzed these by quantitative PCR to detect and identify the Plasmodium species.
RESULTS: Plasmodium falciparum was the most prevalent species in all samples, with P. malariae and P. ovale spp. detected at a lower prevalence (< 5%) in all four regions; P. vivax was not detected in any sample.
CONCLUSIONS: The results of this study indicate that malaria elimination efforts in Tanzania will need to account for and enhance surveillance of these non-falciparum species.

BACKGROUND: Adults infected with Plasmodium spp. in endemic areas need to be re-evaluated in light of global malaria elimination goals. They potentially undermine malaria interventions but remain an overlooked aspect of public health strategies.
METHODS: This study aimed to estimate the prevalence of Plasmodium spp. infections, to identify underlying parasite species, and to assess predicting factors among adults residing in an endemic area from the Democratic Republic of Congo (DRC). A community-based cross-sectional survey in subjects aged 18 years and above was therefore carried out. Study participants were interviewed using a standard questionnaire and tested for Plasmodium spp. using a rapid diagnostic test and a nested polymerase chain reaction assay. Logistic regression models were fitted to assess the effect of potential predictive factors for infections with different Plasmodium spp.
RESULTS: Overall, 420 adults with an estimated prevalence of Plasmodium spp. infections of 60.2% [95% CI 55.5; 64.8] were included. Non-falciparum species infected 26.2% [95% CI 22.2; 30.5] of the study population. Among infected participants, three parasite species were identified, including Plasmodium falciparum (88.5%), Plasmodium malariae (39.9%), and Plasmodium ovale (7.5%) but no Plasmodium vivax. Mixed species accounted for 42.3% of infections while single-species infections predominated with P. falciparum (56.5%) among infected participants. All infected participants were asymptomatic at the time of the survey. Adults belonging to the \"most economically disadvantaged\" households had increased risks of infections with any Plasmodium spp. (adjusted odds ratio, aOR = 2.87 [95% CI 1.66, 20.07]; p < 0.001), compared to those from the \"less economically disadvantaged\" households. Conversely, each 1 year increase in age reduced the risk of infections with any Plasmodium spp. (aOR = 0.99 [95% CI 0.97, 0.99]; p = 0.048). Specifically for non-falciparum spp., males had increased risks of infection than females (aOR = 1.83 [95% CI 1.13, 2.96]; p = 0.014).
CONCLUSIONS: Adults infected with malaria constitute a potentially important latent reservoir for the transmission of the disease in the study setting. They should specifically be taken into account in public health measures and translational research.

This case report presents a perplexing case of Plasmodium malariae breakthrough infection despite prophylaxis with appropriate antimalarial prophylactic regimen of mefloquine in a compliant patient. A 78-year-old missionary who travels each year to the African subcontinent for multiple weeks to months, over 25 years, adheres to stringent antimalarial prophylaxis with Mefloquine as prescribed, starting prior to the trip and continuing after the return to the U.S.A. She gave no prior history of malaria during her 25 years of travel to Africa and back. Since she had no prior history of malaria and due to her excellent compliance with antimalarial regiment, despite her presentation which were suggestive of malaria, neither the patient nor her providers recognized the onset of malaria in this case. Infectious diseases physicians approached this case with an open mind, investigated appropriately, requested appropriate tests, found the presence of malarial parasite, identified as P. malariae species thereafter. She was started on antimalarial treatment in a timely fashion and showed an excellent response. This intriguing recovery of malarial parasite and response to treatment despite the patient being on antimalarial prophylaxis raised the possibility of mefloquine failure as an antimalarial prophylactic agent against P. malariae species.

BACKGROUND: Transfusion-transmitted malaria (TTM) is a public health problem in endemic and nonendemic areas. The Brazilian Ministry of Health (MH) requested the development of a nucleic acid amplification test (NAT) for the detection of Plasmodium spp. in public blood centers to increase blood safety.
METHODS: The new Brazilian NAT kit named NAT PLUS HIV/HBV/HCV/Malaria Bio-Manguinhos was first implemented in HEMORIO, a public blood center in the city of Rio de Janeiro. Since October 1, 2022, this blood center has been testing all its blood donations for malaria in a pool of six plasma samples to detect Plasmodium spp. by real-time polymerase chain reaction (PCR).
RESULTS: Since the implementation of the NAT PLUS platform until February 2023, HEMORIO has successfully received and tested 200,277 donations. The platform detected two asymptomatic donors in the city of Rio de Janeiro, which is a nonendemic region for malaria. Our analyses suggested a malaria from the Amazon region caused by Plasmodium vivax, in the first case, while an autochthonous transmission case by Plasmodium malariae was identified in the rural area of Rio de Janeiro state.
CONCLUSIONS: The NAT PLUS platform detects Plasmodium spp. in plasma samples with sensitivity capable of detecting subpatent infections. This is the first time worldwide that a group developed and implemented molecular diagnosis for Plasmodium spp. to be used by public blood centers to avoid TTM.