Biochemical Properties of Two Plasmodium malariae Cysteine Proteases, Malapain-2 and Malapain-4

Cysteine proteases belonging to the falcipain (FP) family play a pivotal role in the biology of malaria parasites and have been extensively investigated as potential antimalarial drug targets. Three paralogous FP-family cysteine proteases of Plasmodium malariae, termed malapains 2–4 (MP2–4), were identified in PlasmoDB. The three MPs share similar structural properties with the FP-2/FP-3 subfamily enzymes and exhibit a close phylogenetic lineage with vivapains (VXs) and knowpains (KPs), FP orthologues of P. vivax and P. knowlesi. Recombinant MP-2 and MP-4 were produced in a bacterial expression system, and their biochemical properties were characterized. Both recombinant MP-2 and MP-4 showed enzyme activity across a broad range of pH values with an optimum activity at pH 5.0 and relative stability at neutral pHs. Similar to the FP-2/FP-3 subfamily enzymes in other Plasmodium species, recombinant MP-2 and MP-4 effectively hydrolyzed hemoglobin at acidic pHs. They also degraded erythrocyte cytoskeletal proteins, such as spectrin and band 3, at a neutral pH. These results imply that MP-2 and MP-4 are redundant hemoglobinases of P. malariae and may also participate in merozoite egression by degrading erythrocyte cytoskeletal proteins. However, compared with other FP-2/FP-3 enzymes, MP-2 showed a strong preference for arginine at the P2 position. Meanwhile, MP-4 showed a primary preference for leucine at the P2 position but a partial preference for phenylalanine. These different substrate preferences of MPs underscore careful consideration in the design of optimized inhibitors targeting the FP-family cysteine proteases of human malaria parasites.

Molecular identification and anti-malarial drug resistance profile of Plasmodium falciparum from patients attending Kisoro Hospital, southwestern Uganda

Background The evolution of malaria infection has necessitated the development of highly sensitive diagnostic assays, as well as the use of dried blood spots (DBS) as a potential source of deoxyribonucleic acid (DNA) yield for polymerase chain reaction (PCR) assays. This study identified the different Plasmodium species in malaria-positive patients, and the anti-malarial drug resistance profile for Plasmodium falciparum using DBS samples collected from patients attending Kisoro Hospital in Kisoro district, Southwestern Uganda. Methods The blood samples were prospectively collected from patients diagnosed with malaria to make DBS, which were then used to extract DNA for real-time PCR and high-resolution melting (HRM) analysis. Plasmodium species were identified by comparing the control and test samples using HRM-PCR derivative curves. Plasmodium falciparum chloroquine (CQ) resistance transporter (pfcrt) and kelch13 to screen the samples for anti-malarial resistance markers. The HRM-PCR derivative curve was used to present a summary distribution of the different Plasmodium species as well as the anti-malarial drug profile. Results Of the 152 participants sampled, 98 (64.5%) were females. The average age of the participants was 34.9 years (range: 2 months–81 years). There were 134 samples that showed PCR amplification, confirming the species as Plasmodium. Plasmodium falciparum (N = 122), Plasmodium malariae (N = 6), Plasmodium ovale (N = 4), and Plasmodium vivax (N = 2) were the various Plasmodium species and their proportions. The results showed that 87 (71.3%) of the samples were sensitive strains/wild type (CVMNK), 4 (3.3%) were resistant haplotypes (SVMNT), and 31 (25.4%) were resistant haplotypes (CVIET). Kelch13 C580Y mutation was not detected. Conclusion The community served by Kisoro hospital has a high Plasmodium species burden, according to this study. Plasmodium falciparum was the dominant species, and it has shown that resistance to chloroquine is decreasing in the region. Based on this, molecular identification of Plasmodium species is critical for better clinical management. Besides, DBS is an appropriate medium for DNA preservation and storage for future epidemiological studies.

Neglected malaria parasites in hard-to-reach areas of Odisha, India: implications in elimination programme

Background Information on the foci of Plasmodium species infections is essential for any country heading towards elimination. Odisha, one of the malaria-endemic states of India is targeting elimination of malaria by 2030. To support decision-making regarding targeted intervention, the distribution of Plasmodium species infections was investigated in hard-to-reach areas where a special malaria elimination drive, namely Durgama Anchalare Malaria Nirakaran (DAMaN) began in 2017. Methods A cross-sectional survey was conducted in 2228 households during July to November 2019 in six districts, to evaluate the occurrence of Plasmodium species. The species were identified by polymerase chain reaction (PCR) followed by sequencing, in case of Plasmodium ovale. Results Of the 3557 blood specimens tested, malaria infection was detected in 282 (7.8%) specimens by PCR. Of the total positive samples, 14.1% were P. ovale spp. and 10.3% were Plasmodium malariae infections. The majority of P. ovale spp. (75.8%) infections were mixed with either Plasmodium falciparum and/or Plasmodium vivax and found to be distributed in three geophysical regions (Northern-plateau, Central Tableland and Eastern Ghat) of the State, while P. malariae has been found in Northern-plateau and Eastern Ghat regions. Speciation revealed occurrence of both Plasmodium ovale curtisi (classic type) and Plasmodium ovale wallikeri (variant type). Conclusions In the present study a considerable number of P. ovale spp. and P. malariae were detected in a wide geographical areas of Odisha State, which contributes around 40% of the country’s total malaria burden. For successful elimination of malaria within the framework of national programme, P. ovale spp. along with P. malariae needs to be incorporated in surveillance system, especially when P. falciparum and P. vivax spp. are in rapid decline.

Transfusion-Transmitted Malaria of Plasmodium malariae in Palermo, Sicily

Transfusion-transmitted malaria (TTM) is a rare occurrence with serious consequences for the recipient. In non-endemic areas, the incidence of transmission of malaria by transfusion is very low. We report a clinical case of transfusion-transmitted malaria due to Plasmodium malariae, which happened in a patient with acute hemorrhagic gastropathy. Case presentation: In April 2019, a 70-year-old Italian man with recurrent spiking fever for four days was diagnosed with a P. malariae infection, as confirmed using microscopy and real-time PCR. The patient had never been abroad, but about two months before, he had received a red blood cell transfusion for anemia. Regarding the donor, we revealed that they were a missionary priest who often went to tropical regions. Plasmodium spp PCR was also used on donor blood to confirm the causal link. Discussion and Conclusions: The donations of asymptomatic blood donors who are predominantly “semi-immune” with very low parasitic loads are an issue. The main problem is related to transfusion-transmitted malaria. Our case suggests that P. malariae infections in semi-immune asymptomatic donors are a threat to transfusion safety. Currently, microscopy is considered the gold standard for the diagnosis of malaria but has limited sensitivity to detect low levels of parasitemia. Screening using serological tests and molecular tests, combined with the donor’s questionnaire, should be used to reduce the cases of TTM.

Zoonotic Transmission and Host Switches of Malaria Parasites

Malaria is a deadly disease that affects the health of hundreds of millions of people annually. Five Plasmodium parasite species naturally infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi. These parasites can also infect various non-human primates. Parasites mainly infecting monkeys, such as Plasmodium cynomolgi and P. knowlesi, the latter of which was considered to be a monkey parasite for years, can also be transmitted to human hosts. Recently, many new Plasmodium species have been discovered in African apes, some of which may be transmitted to humans in the future. Here, we searched PubMed and the internet via Google and selected articles on the zoonotic transmission and evolution of selected malaria parasite species. We review current advances in the relevant topics, emphasizing the transmission of malaria parasites between humans and non-human primates. We also briefly discuss the transmission of some avian malaria parasites between wild birds and domestic fowls. Zoonotic malaria transmission is widespread, thus posing a threat to public health. More studies on parasite species, including their identification in non-human primates, transmission, and evolution, are needed to decrease or prevent the transmission of malaria parasites from non-human primates to humans.

Plasmodium malariae infections as a cause of febrile disease in an area of high Plasmodium falciparum transmission intensity in Eastern Uganda

Background Plasmodium falciparum is responsible for the vast majority of (severe) clinical malaria cases in most African settings. Other Plasmodium species often go undiagnosed but may still have clinical consequences. Case presentation Here, five cases of Plasmodium malariae infections from Eastern Uganda (aged 2–39 years) are presented. These infections were all initially mistaken for P. falciparum, but Plasmodium schizonts (up to 2080/µL) were identified by microscopy. Clinical signs included history of fever and mild anaemia. Conclusion These findings highlight the importance of considering non-falciparum species as the cause of clinical malaria. In areas of intense P. falciparum transmission, where rapid diagnostic tests that detect only P. falciparum antigens are commonly used, non-falciparum malaria cases may be missed.

Assessing Asymptomatic Malaria Carriage of Plasmodium falciparum and Non-falciparum Species in Children Resident in Nkolbisson, Yaoundé, Cameroon

Malaria is still a threat to public health as it remains the first endemic disease in the world. It is a pervasive parasitic disease in tropical and subtropical regions where asymptomatic malaria infection among humans serves as a significant reservoir for transmission. A rapid and correct diagnosis is considered to be an important strategy in the control of the disease especially in children, who are the most vulnerable group. This study assessed the prevalence of asymptomatic malaria in children at the Nkolbisson health area in Yaoundé, Cameroon. A cross-sectional study design and a convenience sampling plan were used. A total of 127 participants were recruited after informed and signed consent from parents and/or guardians. Blood samples were collected by finger-pricking and venipuncture from children aged 6 months to 10 years and then screened for asymptomatic parasitemia by a rapid diagnostic test (RDT), light microscopy (LM) staining with Giemsa and 18S rRNA polymerase chain reaction (PCR) for speciation. The data were analyzed using SPSS version 20 software. The study identified 85 children who were positive from the PCR, 95 positive from the RDT and 71 from the LM, revealing a malaria prevalence of 66.9%, 74.8% and 55.9%, respectively. The prevalence was not observed to be dependent on the sex and age group of the participants. Plasmodium falciparum was the predominant species followed by Plasmodium malariae and then Plasmodium ovale. The RDT and LM had the same sensitivity (90.6%) with a slight difference in their specificity (RDT: 57.1%; LM: 54.8%). The RDT also demonstrated higher positive and negative predictive values compared with those of the LM.