Non-falciparum Malaria Research Articles

LLIN Evaluation in Uganda Project (LLINEUP)–effects of a vector control trial on Plasmodium infection prevalence and genotypic markers of insecticide resistance in Anopheles vectors from 48 districts of Uganda

Pyrethroid bednets treated with the synergist piperonyl butoxide (PBO) offer the possibility of improved vector control in mosquito populations with metabolic resistance. In 2017–2019, we conducted a large-scale, cluster-randomised trial (LLINEUP) to evaluate long-lasting insecticidal nets (LLINs) treated with a pyrethroid insecticide plus PBO (PBO LLINs), as compared to conventional, pyrethroid-only LLINs across 104 health sub-districts (HSDs) in Uganda. In LLINEUP, and similar trials in Tanzania, PBO LLINs were found to provide greater protection against malaria than conventional LLINs, reducing parasitaemia and vector density. In the LLINEUP trial, we conducted cross-sectional household entomological surveys at baseline and then every 6 months for two years, which we use here to investigate longitudinal changes in mosquito infection rate and genetic markers of resistance. Overall, 5395 female Anopheles mosquitoes were collected from 5046 households. The proportion of mosquitoes infected (PCR-positive) with Plasmodium falciparum did not change significantly over time, while infection with non-falciparum malaria decreased in An. gambiae s.s., but not An. funestus. The frequency of genetic markers associated with pyrethroid resistance increased significantly over time, but the rate of change was not different between the two LLIN types. The knock-down resistance (kdr) mutation Vgsc-995S declined over time as Vgsc-995F, the alternative resistance mutation at this codon, increased. Vgsc-995F appears to be spreading into Uganda. Distribution of LLINs in Uganda was previously found to be associated with reductions in parasite prevalence and vector density, but here we show that the proportion of infective mosquitoes remained stable across both PBO and non-PBO LLINs, suggesting that the potential for transmission persisted. The increased frequency of markers of pyrethroid resistance indicates that LLIN distribution favoured the evolution of resistance within local vectors and highlights the potential benefits of resistance management strategies.Trial registration: This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395.

Prevalence of non-falciparum malaria infections among asymptomatic individuals in four regions of Mainland Tanzania

BackgroundRecent 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.ResultsPlasmodium 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.Graphical

Epidemiology of Plasmodium malariae and Plasmodium ovale spp. in Kinshasa Province, Democratic Republic of Congo

Reports suggest non-falciparum species are an underappreciated cause of malaria in sub-Saharan Africa but their epidemiology is ill-defined, particularly in highly malaria-endemic regions. We estimated incidence and prevalence of PCR-confirmed non-falciparum and Plasmodium falciparum malaria infections within a longitudinal study conducted in Kinshasa, Democratic Republic of Congo (DRC) between 2015-2017. Children and adults were sampled at biannual household surveys and routine clinic visits. Among 9,089 samples from 1,565 participants, incidences of P. malariae, P. ovale spp., and P. falciparum infections by 1-year were 7.8% (95% CI: 6.4%-9.1%), 4.8% (95% CI: 3.7%-5.9%) and 57.5% (95% CI: 54.4%-60.5%), respectively. Non-falciparum prevalences were higher in school-age children, rural and peri-urban sites, and P. falciparum co-infections. P. falciparum remains the primary driver of malaria in the DRC, though non-falciparum species also pose an infection risk. As P. falciparum interventions gain traction in high-burden settings, continued surveillance and improved understanding of non-falciparum infections are warranted.

Laboratory evaluation of the miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA), a simplified molecular diagnostic test for Plasmodium

BackgroundPoint-of-care diagnosis of malaria is currently based on microscopy and rapid diagnostic tests. However, both techniques have their constraints, including poor sensitivity for low parasitaemias. Hence, more accurate diagnostic tests for field use and routine clinical settings are warranted. The miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative, easy-to-use molecular assay for diagnosis of malaria in resource-limited settings. Unlike traditional molecular methods, mini-dbPCR-NALFIA does not require DNA extraction and makes use of a handheld, portable thermal cycler that can run on a solar-charged power pack. Result read-out is done using a rapid lateral flow strip enabling differentiation of Plasmodium falciparum and non-falciparum malaria infections. A laboratory evaluation was performed to assess the performance of the mini-dbPCR-NALFIA for diagnosis of pan-Plasmodium and P. falciparum infections in whole blood.MethodsDiagnostic accuracy of the mini-dbPCR-NALFIA was determined by testing a set of Plasmodium-positive blood samples from returned travellers (n = 29), and Plasmodium-negative blood samples from travellers with suspected malaria (n = 23), the Dutch Blood Bank (n = 19) and intensive care patients at the Amsterdam University Medical Centers (n = 16). Alethia Malaria (LAMP) with microscopy for species differentiation were used as reference. Limit of detection for P. falciparum was determined by 23 measurements of a dilution series of a P. falciparum culture. A fixed sample set was tested three times by the same operator to evaluate the repeatability, and once by five different operators to assess the reproducibility.ResultsOverall sensitivity and specificity of the mini-dbPCR-NALFIA were 96.6% (95% CI, 82.2%–99.9%) and 98.3% (95% CI, 90.8%–100%). Limit of detection for P. falciparum was 10 parasites per microlitre of blood. The repeatability of the assay was 93.7% (95% CI, 89.5%–97.8%) and reproducibility was 84.6% (95% CI, 79.5%–89.6%).ConclusionsMini-dbPCR-NALFIA is a sensitive, specific and robust method for molecular diagnosis of Plasmodium infections in whole blood and differentiation of P. falciparum. Incorporation of a miniature thermal cycler makes the assay well-adapted to resource-limited settings. A phase-3 field trial is currently being conducted to evaluate the potential implementation of this tool in different malaria transmission areas.

Non-falciparum malaria infection and IgG seroprevalence among children under 15 years in Nigeria, 2018

Plasmodium falciparum (Pf) is the dominant malaria parasite in Nigeria though P. vivax (Pv), P. ovale (Po), and P. malariae (Pm) are also endemic. Blood samples (n = 31,234) were collected from children aged 0-14 years during a 2018 nationwide HIV survey and assayed for Plasmodium antigenemia, Plasmodium DNA, and IgG against Plasmodium MSP1-19 antigens. Of all children, 6.6% were estimated to have Pm infection and 1.4% Po infection with no Pv infections detected. The highest household wealth quintile was strongly protective against infection with Pm (aOR: 0.11, 95% CI: 0.05–0.22) or Po (aOR= 0.01, 0.00–0.10). Overall Pm seroprevalence was 34.2% (95% CI: 33.3-35.2) with lower estimates for Po (12.1%, 11.6-12.5) and Pv (6.3%, 6.0-6.7). Pm seropositivity was detected throughout the country with several local government areas showing >50% seroprevalence. Serological and DNA indicators show widespread exposure of Nigerian children to Pm with lower rates to Po and Pv.

Prevalence and molecular identification of Plasmodium falciparum in symptomatic individuals in Nyala city, Sudan

Background: Malaria is one of the most important infectious diseases in tropical and subtropical regions, and 45% of the population living in areas that suffer from stable malaria transmission in Sudan. The prevalence of symptomatic Plasmodium infection based on microscopy and RDTs is poorly documented in Nyala city. This study was conducted to determine the prevalence of plasmodium infection and molecular identification of plasmodium species in Nyala city, Sudan.&#x0D; Methods: A cross-sectional study was conducted from July 2018 to December 2018 to determine the prevalence of malaria and molecular identification of plasmodium species in Nyala city, Sudan. A total of 300 suspected patients were enrolled in this study. Microscopy and RDTs were performed to estimate the prevalence and nPCR to detect the plasmodium genus and species.&#x0D; Results: Of 300 patients, 113 (37.7%) were positive for malaria. Plasmodium falciparum was the only prevalent species detected amongst the study population. The prevalence of plasmodium infection by microscopy and RDTs was 113 (37.7 %) and 106 (35.3 %), respectively. While PCR result showed prevalence of 47 (47%).&#x0D; Conclusions: This study showed that the only prevalent species identified was P. falciparum, the findings demonstrate no existence of non-falciparum malaria, and children less than five years old were the most infected patients. The diagnostic performance of microscopy was better than RDTs in the diagnosis of malaria infection.

Prevalence and molecular identification of Plasmodium falciparum

Background: Malaria is one of the most important infectious diseases in tropical and subtropical regions, and 45% of the population living in areas that suffer from stable malaria transmission in Sudan. The prevalence of symptomatic Plasmodium infection based on microscopy and RDTs is poorly documented in Nyala city. This study was conducted to determine the prevalence of plasmodium infection and molecular identification of plasmodium species in Nyala city, Sudan.&#x0D; Methods: A cross-sectional study was conducted from July 2018 to December 2018 to determine the prevalence of malaria and molecular identification of plasmodium species in Nyala city, Sudan. A total of 300 suspected patients were enrolled in this study. Microscopy and RDTs were performed to estimate the prevalence and nPCR to detect the plasmodium genus and species.&#x0D; Results: Of 300 patients, 113 (37.7%) were positive for malaria. Plasmodium falciparum was the only prevalent species detected amongst the study population. The prevalence of plasmodium infection by microscopy and RDTs was 113 (37.7 %) and 106 (35.3 %), respectively. While PCR result showed prevalence of 47 (47%).&#x0D; Conclusions: This study showed that the only prevalent species identified was P. falciparum, the findings demonstrate no existence of non-falciparum malaria, and children less than five years old were the most infected patients. The diagnostic performance of microscopy was better than RDTs in the diagnosis of malaria infection.

Non-falciparum species and submicroscopic infections in three epidemiological malaria facets in Cameroon

BackgroundThere are growing reports on the prevalence of non-falciparum species and submicroscopic infections in sub-Saharan African countries but little information is available from Cameroon.MethodsA hospital-based cross-sectional study was carried out in four towns (Douala, Maroua, Mayo-Oulo, and Pette) from three malaria epidemiological strata (Forest, Sahelian, and Soudanian) of Cameroon. Malaria parasites were detected by Giemsa light microscopy and polymerase chain reaction (PCR) assay. Non-falciparum isolates were characterized and their 18S gene sequences were BLASTed for confirmatory diagnosis.ResultsPCR assay detected malaria parasites in 82.4% (98/119) patients, among them 12.2% (12/98) were asymptomatic cases. Three Plasmodium species viz. P.falciparum, P.ovalecurtisi and P.vivax, and two co-infection types (P.falciparum + P.vivax and P.falciparum + P.ovalecurtisi) were found. The remaining infections were mono–infections with either P.falciparum or P.ovalecurtisi. All non–falciparum infections were symptomatic and microscopic. The overall proportion of submicroscopic infections was 11.8% (14/119). Most asymptomatic and submicroscopic infection cases were self-medicated with antimalarial drugs and/or medicinal plants. On analysis, P.ovalecurtisi sequences were found to be phylogenetically closer to sequences from India while P.vivax isolates appeared closer to those from Nigeria, India, and Cameroon. No G6PD-d case was found among non-falciparum infections.ConclusionsThis study confirms our previous work on circulation of P.vivax and P.ovalecurtisi and the absence of P.knowlesi in Cameroon. More studies are needed to address non-falciparum malaria along with submicroscopic infections for effective malaria management and control in Cameroon.

Diagnostic accuracy and limit of detection of ten malaria parasite lactate dehydrogenase-based rapid tests for Plasmodium knowlesi and P. falciparum.

Plasmodium knowlesi causes zoonotic malaria across Southeast Asia. First-line diagnostic microscopy cannot reliably differentiate P. knowlesi from other human malaria species. Rapid diagnostic tests (RDTs) designed for P. falciparum and P. vivax are used routinely in P. knowlesi co-endemic areas despite potential cross-reactivity for species-specific antibody targets. Ten RDTs were evaluated: nine to detect clinical P. knowlesi infections from Malaysia, and nine assessing limit of detection (LoD) for P. knowlesi (PkA1-H.1) and P. falciparum (Pf3D7) cultures. Targets included Plasmodium-genus parasite lactate dehydrogenase (pan-pLDH) and P. vivax (Pv)-pLDH. Samples were collected prior to antimalarial treatment from 127 patients with microscopy-positive PCR-confirmed P. knowlesi mono-infections. Median parasitaemia was 788/µL (IQR 247-5,565/µL). Pan-pLDH sensitivities ranged from 50.6% (95% CI 39.6-61.5) (SD BIOLINE) to 87.0% (95% CI 75.1-94.6) (First Response® and CareStart™ PAN) compared to reference PCR. Pv-pLDH RDTs detected P. knowlesi with up to 92.0% (95% CI 84.3-96.7%) sensitivity (Biocredit™). For parasite counts ≥200/µL, pan-pLDH (Standard Q) and Pv-pLDH RDTs exceeded 95% sensitivity. Specificity of RDTs against 26 PCR-confirmed negative controls was 100%. Sensitivity of six highest performing RDTs were not significantly different when comparing samples taken before and after (median 3 hours) antimalarial treatment. Parasite ring stages were present in 30% of pre-treatment samples, with ring stage proportions (mean 1.9%) demonstrating inverse correlation with test positivity of Biocredit™ and two CareStart™ RDTs.For cultured P. knowlesi, CareStart™ PAN demonstrated the lowest LoD at 25 parasites/µL; LoDs of other pan-pLDH ranged from 98 to >2000 parasites/µL. Pv-pLDH LoD for P. knowlesi was 49 parasites/µL. No false-positive results were observed in either P. falciparum-pLDH or histidine-rich-protein-2 channels. Selected RDTs demonstrate sufficient performance for detection of major human malaria species including P. knowlesi in co-endemic areas where microscopy is not available, particularly for higher parasite counts, although cannot reliably differentiate among non-falciparum malaria.

Cation ATPase (ATP4) Orthologue Replacement in the Malaria Parasite Plasmodium knowlesi Reveals Species-Specific Responses to ATP4-Targeting Drugs.

ABSTRACTSeveral unrelated classes of antimalarial compounds developed against Plasmodium falciparum target a parasite-specific P-type ATP-dependent Na+ pump, PfATP4. We have previously shown that other malaria parasite species infecting humans are less susceptible to these compounds. Here, we generated a series of transgenic Plasmodium knowlesi orthologue replacement (OR) lines in which the endogenous pkatp4 locus was replaced by a recodonized P. knowlesi atp4 (pkatp4) coding region or the orthologous coding region from P. falciparum, Plasmodium malariae, Plasmodium ovale subsp. curtisi, or Plasmodium vivax. Each OR transgenic line displayed a similar growth pattern to the parental P. knowlesi line. We found significant orthologue-specific differences in parasite susceptibility to three chemically unrelated ATP4 inhibitors, but not to comparator drugs, among the P. knowlesi OR lines. The PfATP4OR transgenic line of P. knowlesi was significantly more susceptible than our control PkATP4OR line to three ATP4 inhibitors: cipargamin, PA21A092, and SJ733. The PvATP4OR and PmATP4OR lines were similarly susceptible to the control PkATP4OR line, but the PocATP4OR line was significantly less susceptible to all ATP4 inhibitors than the PkATP4OR line. Cipargamin-induced inhibition of Na+ efflux was also significantly greater with the P. falciparum orthologue of ATP4. This confirms that species-specific susceptibility differences previously observed in ex vivo studies of human isolates are partly or wholly enshrined in the primary amino acid sequences of the respective ATP4 orthologues and highlights the need to monitor efficacy of investigational malaria drugs against multiple species. P. knowlesi is now established as an important in vitro model for studying drug susceptibility in non-falciparum malaria parasites.

Quando la malaria tarda a farsi riconoscere: un’infezione da Plasmodium ovale

Plasmodium ovale is a cause of non-falciparum malaria infection that is endemic in tropical Western Africa. The life cycle of Plasmodium ovale includes hypnozoites, which are dormant stages in the liver. These stages can be reactivated after weeks, months, or years from the initial infection, causing disease relapse. This paper reports the case of a female adolescent who presented with high fever and abdominal pain. The girl was referring to a single travel to Nigeria 4 years ago. On that occasion she showed short-term gastrointestinal symptoms, without fever. The blood test revealed thrombocytopenia (76,000/µl) and she was unexpectedly diagnosed with ma-laria through blood smear. Considering the geographical area of her previous travel, a parasitemia by Plasmodium ovale was suspected. The diagnosis was soon confirmed by the positivity of the PCR. The literature describes some cases of recrudescence of the infection from the first episode. The diagnosis of Plasmodium ovale malaria can be challenging because of its possible delayed presentation.

Effectiveness of pyronaridine-artesunate against Plasmodium malariae, Plasmodium ovale spp, and mixed-Plasmodium infections: a post-hoc analysis of the CANTAM-Pyramax trial

SummaryBackgroundHigh-quality evidence for the therapeutic efficacy and effectiveness of antimalarials for infections caused by Plasmodium malariae, Plasmodium ovale spp, and mixed-Plasmodium infections is scarce. In this study, we aimed to analyse the efficacy of pyronaridine–artesunate for the treatment of non-falciparum and mixed-species Plasmodium infections from a large phase 3b/4 clinical trial in central Africa.MethodsThis post-hoc analysis was done in a random subset of samples from two sites (in the Democratic Republic of the Congo and in Gabon) of the CANTAM-Pyramax trial assessing pyronaridine-artesunate therapy. We randomly selected paired dried blood spot samples from day 0 and day 28 (or unforeseen visit) and analysed them by quantitative PCR for mixed Plasmodium infections or non-falciparum mono-infections. Day 28 (or unforeseen visit) samples positive for non-falciparum malaria were re-assessed by microscopy to identify microscopic versus submicroscopic infections. Analyses were done on two sample sets: a per-protocol set and an intention-to-treat set.FindingsAmong 1502 randomly selected samples, 192 (12·8%) showed mixed-Plasmodium infections or non-falciparum mono-infections. We did not detect P vivax in the samples. For both the per-protocol and intention-to-treat sets, the overall day 28 cure rates for P malariae, P ovale curtisi, and P ovale wallikeri were 96·3% or higher (95% CIs from 81·0–99·9 to 95·7–100). Cure rates were consistently high in P malariae (99·2%, 95·7–100) and P ovale spp (97·9%, 88·7–99·9, for P ovale curtisi and 96·3%, 81·0–99·9, for P ovale wallikeri) infections.InterpretationThis post-hoc analysis provides important evidence supporting the high efficacy of pyronaridine-artesunate against mono-infections with P malariae, P ovale curtisi, or P ovale wallikeri and mixed-Plasmodium infections in a real-world setting.FundingMedicines for Malaria Venture.

Seasonality and transmissibility of Plasmodium ovale in Bagamoyo District, Tanzania

BackgroundPlasmodium ovale is a neglected malarial parasite that can form latent hypnozoites in the human liver. Over the last decade, molecular surveillance studies of non-falciparum malaria in Africa have highlighted that P. ovale is circulating below the radar, including areas where Plasmodium falciparum is in decline. To eliminate malaria where P. ovale is endemic, a better understanding of its epidemiology, asymptomatic carriage, and transmission biology is needed.MethodsWe performed a pilot study on P. ovale transmission as part of an ongoing study of human-to-mosquito transmission of P. falciparum from asymptomatic carriers. To characterize the malaria asymptomatic reservoir, cross-sectional qPCR surveys were conducted in Bagamoyo, Tanzania, over three transmission seasons. Positive individuals were enrolled in transmission studies of P. falciparum using direct skin feeding assays (DFAs) with Anopheles gambiae s.s. (IFAKARA strain) mosquitoes. For a subset of participants who screened positive for P. ovale on the day of DFA, we incubated blood-fed mosquitoes for 14 days to assess sporozoite development.ResultsMolecular surveillance of asymptomatic individuals revealed a P. ovale prevalence of 11% (300/2718), compared to 29% (780/2718) for P. falciparum. Prevalence for P. ovale was highest at the beginning of the long rainy season (15.5%, 128/826) in contrast to P. falciparum, which peaked later in both the long and short rainy seasons. Considering that these early-season P. ovale infections were low-density mono-infections (127/128), we speculate many were due to hypnozoite-induced relapse. Six of eight P. ovale-infected asymptomatic individuals who underwent DFAs successfully transmitted P. ovale parasites to A. gambiae.ConclusionsPlasmodium ovale is circulating at 4–15% prevalence among asymptomatic individuals in coastal Tanzania, largely invisible to field diagnostics. A different seasonal peak from co-endemic P. falciparum, the capacity to relapse, and efficient transmission to Anopheles vectors likely contribute to its persistence amid control efforts focused on P. falciparum.Graphical

Assessment of Complete Blood Count and D-dimer among Patients with Plasmodium falciparum Malaria

Background: The most virulent of the human malaria parasites is P. falciparum which is responsible for the bulk of the malaria-related morbidity and mortality. Changes in blood cell parameters are already a well-known feature of malarial infections&#x0D; Aim: The purpose of this research was to assess the total blood count and D-dimer levels in patients with P. falciparum malaria in Khartoum State, Sudan.&#x0D; Study Design: It is case- control study was conducted on blood cells prameters and plasma D-dimer for patients infected with Plasmdium falciparum malaria&#x0D; Place and Duration of Study: study was carried out in Yastabshiroon hospital, Khartoum state, from October 2020 to January 2021.&#x0D; Methodology: The study comprised fifty people who tested positive for P. falciparum using the National Public Health Laboratory's malaria diagnostic standard. The control group consisted of fifty healthy Khartoum locals with about equal gender and age distribution. To collect data from the study group's personal and medical information, such as name, gender, age, and medical condition, a structured questionnaire was developed.&#x0D; Result: D-dimer levels were considerably greater in P. falciparum malaria cases compared to non-falciparum malaria cases, neutrophil and lymphocyte counts were significantly lower, although white blood cells (WBCs) count was significantly lower in P. falciparum infection patients compared to non-falciparum (p≤0.05). Hemoglobin concentration and platelet count were similarly significantly lower (p≤0.05). Platelet counts and mean cell hemoglobin concentration (MCHC), on the other hand, were significantly lower in individuals with severe parasitemia compared to those with low and moderate parasitemia.&#x0D; Conclusion: The study concluded that P. falciparum infection causes considerable hematological alterations. The results of the current investigation revealed a significant increase in the D-dimer mean level, as well as a significant decrease in the count of hemoglobin, total white blood cells (TWBCs), lymphocytes, neutrophils, and platelets. It was also discovered that patients with high parasitemia had considerably lower platelet counts and mean cell hemoglobin concentration (MCHC) levels than those with low and moderate parasitemia.

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

BackgroundPlasmodium 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 presentationHere, 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.ConclusionThese 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.

A Review on Chemistry of Anti-malarials

Malaria remains an important public health concern in countries where transmission occurs regularly, as well as in temperate areas and it is a major global health problem, with an estimated 500 million clinical cases occurring annually. Malaria is a complex disease that varies widely in epidemiology and clinical manifestation in different parts of the world. The most prevalent and dangerous type of malaria is caused by Plasmodium falciparum. P. vivax is a common cause of malaria in Latin America, Asia, and Oceania, but not Africa. P. malaria and P. ovale are much less common. Antimalarials are used in three different ways: prophylaxis, treatment of falciparum malaria, and treatment of non-falciparum malaria. Prophylactic antimalarials are used almost exclusively by travelers from developed countries who are visiting malaria-endemic countries. Keywords: Malaria, antimalarial drugs, plasmodium.

Improving the quality of malaria diagnosis in southern Africa through the development of a regional malaria slide bank

BackgroundA malaria slide bank (MSB) is a useful asset for any malaria microscopy testing laboratory to have access to. However, it is not feasible for every country to have its own MSB. If countries are able to pool their resources, a regional MSB is a viable solution. This paper describes the methodology, costing and lessons learnt of establishing and maintaining an MSB over a 3-year period, for a Southern Africa Development Community region.MethodsA national reference laboratory in South Africa was granted funding for setting up the MSB; it possessed experienced staff and suitable resources. Two additional full-time personnel were employed to carry out the activities of this project. Strict protocols for donor/patient blood sample screening, smear preparation, mass staining, quality control and slide validation were followed. Slides from the MSB were used for training and proficiency testing purposes. The initial and recurrent yearly costs to set up and maintain the MSB were calculated.ResultsOver 35 months, 154 batches (26,623 slides) were prepared; the majority were Plasmodium falciparum. Ninety-two percent (141/154) of batches passed internal quality control, and 89% (93/104) passed external validation. From these slides, two training slide sets and six proficiency testing slide sets were sent out. The initial year’s cost to establish an MSB was calculated at approximately $165,000, and the recurrent year-on-year cost was $130,000.ConclusionsThe key components for maintaining a high-quality MSB are consistent funding, competent staff and adherence to standardized protocols. Travel to malaria-endemic areas for access to non-falciparum malaria species, and dilution of P. falciparum blood to desired parasite densities, are extremely useful to ensure variety. The MSB created here supported multiple laboratories in eight countries, and has the potential to expand.

Genetic Manipulation of Non-Falciparum Human Malaria Parasites

The development of genetic manipulation of Plasmodium falciparum in the 1980s was key to study malaria biology. Genetically modified parasites have been used to study several aspects of the disease, such as red blood cell invasion, drug resistance mechanisms, gametocyte development and mosquito transmission. However, biological and genetic differences between P. falciparum and the other human malaria parasites make P. falciparum a poor model to study different species. The lack of robust systems of long-term in vitro culture of P. vivax and the other human malaria parasites lagged the genetic manipulation of these species. Here we review the efforts to generate genetically modified non-falciparum human malaria parasites, in vivo and in vitro. Using in vivo models – infection of non-human primates such as rhesus macaques and saimiri monkeys – researchers were able to generate transgenic lines of P. knowlesi, P. cynomolgi, and P. vivax. The development of long-term in vitro culture of P. knowlesi in the 2000’s, using rhesus and human red blood cells, created a platform to genetically manipulate non-falciparum malaria parasites. Recently, the use of CRISPR/Cas9 technology to genome edit P. knowlesi provides another tool to non-falciparum malaria research, extending the possibilities and allowing researchers to study different aspects of the biology of these parasites and understand the differences between these species and P. falciparum.

Ex vivo susceptibility to new antimalarial agents differs among human-infecting Plasmodium species

Several promising antimalarial drugs are currently being tested in human trials, such as artefenomel, cipargamin, ferroquine and ganaplacide. Many of these compounds were identified using high throughput screens against a single species of human malaria, Plasmodium falciparum, under the assumption that effectiveness against all malaria species will be similar, as has been observed for other antimalarial drugs. However, using our in vitro adapted line, we demonstrated recently that P. knowlesi is significantly less susceptible than P. falciparum to some new antimalarial drugs (e.g., cipargamin and DSM265), and more susceptible to others (e.g., ganaplacide). There is, therefore, an urgent need to determine the susceptibility profile of all human malaria species to the current generation of antimalarial compounds. We obtained ex vivo malaria samples from travellers returning to the United Kingdom and, using the [3H]hypoxanthine incorporation method, compared susceptibility to select established and experimental antimalarial agents among all major human infective Plasmodium species. We demonstrate that P. malariae and P. ovale spp. are significantly less susceptible than P. falciparum to cipargamin, DSM265 and AN13762, but are more susceptible to ganaplacide. Preliminary ex vivo data from single isolates of P. knowlesi and P. vivax demonstrate a similar profile. Our findings highlight the need to ensure cross species susceptibility profiles are determined early in the drug development pipeline. Our data can also be used to inform further drug development, and illustrate the utility of the P. knowlesi in vitro model as a scalable approach for predicting the drug susceptibility of non-falciparum malaria species in general.

Occurrence and Distribution of Nonfalciparum Malaria Parasite Species Among Adolescents and Adults in Malawi.

Plasmodium falciparum malaria dominates throughout sub-Saharan Africa, but the prevalence of Plasmodium malariae, Plasmodium ovale spp., and Plasmodium vivax increasingly contribute to infection in countries that control malaria using P. falciparum-specific diagnostic and treatment strategies. We performed quantitative polymerase chain reaction (qPCR) on 2987 dried blood spots from the 2015-2016 Malawi Demographic and Health Survey to identify presence and distribution of nonfalciparum infection. Bivariate models were used to determine species-specific associations with demographic and environmental risk factors. Nonfalciparum infections had broad spatial distributions. Weighted prevalence was 0.025 (SE, 0.004) for P. malariae, 0.097 (SE, 0.008) for P. ovale spp., and 0.001 (SE, 0.0005) for P. vivax. Most infections (85.6%) had low-density parasitemias ≤ 10 parasites/µL, and 66.7% of P. malariae, 34.6% of P. ovale spp., and 40.0% of P. vivax infections were coinfected with P. falciparum. Risk factors for P. malariae were like those known for P. falciparum; however, there were few risk factors recognized for P. ovale spp. and P. vivax, perhaps due to the potential for relapsing episodes. The prevalence of any nonfalciparum infection was 11.7%, with infections distributed across Malawi. Continued monitoring of Plasmodium spp. becomes critical as nonfalciparum infections become important sources of ongoing transmission.