Infectivity of Symptomatic Malaria Patients to Anopheles farauti Colony Mosquitoes in Papua New Guinea

Plasmodium transmission from humans to mosquitoes is an understudied bottleneck in the transmission of malaria. Direct membrane feeding assays (DMFA) allow detailed malaria transmission studies from humans to mosquitoes. Especially for Plasmodium vivax, which cannot be cultured long-term under laboratory conditions, implementation of DMFAs requires proximity to P. vivax endemic areas. In this study, we investigated the infectivity of symptomatic Plasmodium infections to Anopheles farauti colony mosquitoes in Papua New Guinea (PNG). A total of 182 DMFAs were performed with venous blood collected from rapid diagnostic test (RDT) positive symptomatic malaria patients and subsequently analysed by light microscopy and quantitative real time polymerase chain reaction (qPCR). DMFAs resulted in mosquito infections in 20.9% (38/182) of cases. By light microscopy and qPCR, 10 – 11% of P. falciparum and 32 – 44% of P. vivax positive individuals infected An. farauti. Fifty-eight percent of P. vivax and 15% of P. falciparum gametocytaemic infections infected An farauti.

Marked reduction in antibiotic usage following intensive malaria control in a cohort of Ugandan children

Background Intensive malaria control may have additional benefits beyond reducing the incidence of symptomatic malaria. We compared antibiotic treatment of children before and after the implementation of highly effective malaria control interventions in Tororo, a historically high transmission area of Uganda. Methods Two successive cohorts of children, aged 0.5 to 10 years, were followed from September 2011 to October 2019 in a dedicated study clinic. Universal distribution of long-lasting insecticidal nets was conducted in 2013 and 2017. Sustained indoor residual spraying of insecticide (IRS) was initiated in December 2014. Generalized linear mixed-effects models were used to compare the incidence of antimalarial and antibiotic treatments before and after vector control measures were implemented. Results Comparing the period prior to the implementation of IRS to the period after IRS had been sustained for 4–5 years, the adjusted incidence of malaria treatments decreased from 2.68 to 0.05 per person-year (incidence rate ratio [IRR] = 0.02, 95% CI 0.01–0.03, p < 0.001), and the adjusted incidence of antibiotic treatments decreased from 4.14 to 1.26 per person-year (IRR = 0.30, 95% CI 0.27–0.34, p < 0.001). The reduction in antibiotic usage was primarily associated with fewer episodes of symptomatic malaria and fewer episodes of fever with sub-microscopic parasitemia, both of which were frequently treated with antibiotics. Conclusions In a historically high transmission setting, the implementation of highly effective vector control interventions was followed by a marked reduction in antibiotic treatment of children. This added benefit of malaria control could have important implications for antibiotic prescribing practices, efforts to curtail antimicrobial resistance, and health system costs.

Malaria parasite species composition of Plasmodium infections among asymptomatic and symptomatic school-age children in rural and urban areas of Kinshasa, Democratic Republic of Congo

Background Malaria remains a major public health concern in the Democratic Republic of Congo (DRC), and school-age children are relatively neglected in malaria prevalence surveys and may constitute a significant reservoir of transmission. This study aimed to understand the burden of malaria infections in school-age children in Kinshasa/DRC. Methods A total of 634 (427 asymptomatic and 207 symptomatic) blood samples collected from school-age children aged 6 to 14 years were analysed by microscopy, RDT and Nested-PCR. Results The overall prevalence of Plasmodium spp. by microscopy, RDT and PCR was 33%, 42% and 62% among asymptomatic children and 59%, 64% and 95% in symptomatic children, respectively. The prevalence of Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale spp. by PCR was 58%, 20% and 11% among asymptomatic and 93%, 13% and 16% in symptomatic children, respectively. Among P. ovale spp., P. ovale curtisi, P. ovale wallikeri and mixed P. ovale curtisi + P. ovale wallikeri accounted for 75%, 24% and 1% of infections, respectively. All Plasmodium species infections were significantly more prevalent in the rural area compared to the urban area in asymptomatic infections (p < 0.001). Living in a rural as opposed to an urban area was associated with a five-fold greater risk of asymptomatic malaria parasite carriage (p < 0.001). Amongst asymptomatic malaria parasite carriers, 43% and 16% of children harboured mixed Plasmodium with P. falciparum infections in the rural and the urban areas, respectively, whereas in symptomatic malaria infections, it was 22% and 26%, respectively. Few children carried single infections of P. malariae (2.2%) and P. ovale spp. (1.9%). Conclusion School-age children are at significant risk from both asymptomatic and symptomatic malaria infections. Continuous systematic screening and treatment of school-age children in high-transmission settings is needed.

Impact of asymptomatic Plasmodium falciparum infection on the risk of subsequent symptomatic malaria in a longitudinal cohort in Kenya

Background:Asymptomatic Plasmodium falciparum infections are common in sub-Saharan Africa, but their effect on subsequent symptomaticity is incompletely understood.Methods:In a 29-month cohort of 268 people in Western Kenya, we investigated the association between asymptomatic P. falciparum and subsequent symptomatic malaria with frailty Cox models.Results:Compared to being uninfected, asymptomatic infections were associated with an increased 1-month likelihood of symptomatic malaria [adjusted Hazard Ratio (aHR):2.61, 95%CI:2.05-3.33], and this association was modified by sex, with females [aHR:3.71, 95%CI:2.62-5.24] at higher risk for symptomaticity than males [aHR:1.76, 95%CI:1.24-2.50]. This increased symptomatic malaria risk was observed for asymptomatic infections of all densities and in people of all ages. Long-term risk was attenuated but still present in children under 5 [29-month aHR:1.38, 95%CI:1.05-1.81].Conclusions:In this high-transmission setting, asymptomatic P. falciparum can be quickly followed by symptoms and may be targeted to reduce the incidence of symptomatic illness.Funding:This work was supported by the National Institute of Allergy and Infectious Diseases (R21AI126024 to WPO, R01AI146849 to WPO and SMT).

Determinants of the varied profiles of Plasmodium falciparum infections among infants living in Kintampo, Ghana

Background Understanding why some infants tolerate infections, remaining asymptomatic while others succumb to repeated symptomatic malaria is beneficial for studies of naturally acquired immunity and can guide control interventions. This study compared demographic, host and maternal factors associated with being either parasite negative or having asymptomatic infections versus developing symptomatic malaria in the first year of life. Methods A birth cohort (n = 1264) was monitored longitudinally over two years for malaria infections in Kintampo, Ghana. Symptomatic and asymptomatic infections were detected actively through monthly home visits, complemented by passive case detection. Light microscopy was used to detect parasitaemia. Based on data from a minimum of eight monthly visits within the first year of life, infants were classified into one of four groups: “parasite negative”, “only-asymptomatic”, “only-symptomatic” or “alternating” i.e., sometimes symptomatic and other times asymptomatic. The host and maternal characteristics and demographic factors in relation to these four groups were compared. Results The parasite negative group formed 36% of the cohort, whilst the only-symptomatic were 35%. The alternating group were 22% and the only-asymptomatic were 7% of the cohort. There were significant associations between residence, socio-economic status (SES), parity, IPTp doses, delivery place of infant and having or not having malaria parasites. Maternal factors such as early commencement and frequency of ante-natal care (ANC) were significantly higher in the parasite negative group compared to all others. ITN use in pregnancy increased the odds of infant having only asymptomatic infections (“protected against disease”). Placental malaria was more common in the groups of infants with symptomatic malaria. Urban residence was significantly higher in the parasite negative group, while birth in the malaria transmission season were significantly more common in the alternating and parasite negative groups. Risk factors for infants with symptomatic malaria included low SES, birth in private maternity homes, sickle cell normal variant, lower MUAC, reported intake of anti-malarials and increased morbidity before the first microscopic infection was detected. Conclusion Strengthening ANC by encouraging early and regular attendance, the use of IPTp, maternal bed nets and improving the nourishment of infants help reduce the frequency of symptomatic malaria over the first year of life.

Prevalence of Plasmodium falciparum isolates lacking the histidine rich protein 2 gene among symptomatic malaria patients in Kwilu Province of the Democratic Republic of Congo

Background Malaria rapid diagnostic tests have become a primary and critical tool for malaria diagnosis in malaria-endemic countries where Plasmodium falciparum Histidine Rich Protein 2-based rapid diagnostic tests (PfHRP2-based RDTs) are widely used. However, in the last decade, the accuracy of PfHRP2-based RDTs has been challenged by the emergence of P. falciparum strains harbouring deletions of the P. falciparum histidine rich protein 2 (pfhrp2) gene, resulting in false-negative results. In the Democratic Republic of Congo (D.R. Congo), little is known about the prevalence of the pfhrp2 gene deletion among P. falciparum isolates infecting symptomatic patients, especially in low to moderate transmission areas where pfhrp2 deletion parasites are assumed to emerge and spread. Here we determine the local prevalence and factors associated with pfhrp2 gene deletions among symptomatic malaria patients in the Kwilu Province of the D.R. Congo. Methods We used secondary data from a prospective health facility-based cross-sectional study conducted in 2018. Blood was collected for microscopy, PfHRP2-RDT, and spotted onto Whatman filter paper for downstream genetic analysis. Genomic DNA was extracted and used to perform PCR assays for the detection and confirmation of pfhrp2 gene deletions. Fischer’s exact and the Kruskal–Wallis tests were applied to look for associations between potential explanatory variables and the pfhrp2 gene deletion with a level of statistical significance set at P < 0.05. Results Of the 684 enrolled symptomatic patients, 391 (57.7%) were female. The majority (87.7%) reported the presence of mosquito breeding sites within the household’s compound, and fever was the most reported symptom (81.6%). The overall prevalence of the pfhrp2 gene deletion was 9.2% (95% CI: 6.7%–12.1%). The deletion of the pfhrp2 gene was associated with health zone of origin (P = 0.012) and age (P = 0.019). Among false-negative PfHRP2-RDT results, only 9.9% were due to pfhrp2 gene deletion. Conclusions P. falciparum isolates with pfhrp2 gene deletions are relatively common among symptomatic patients in Kwilu province. Further investigations are needed to provide enough evidence for policy change. Meanwhile, the use of RDTs targeting PfHRP2 and parasite lactate dehydrogenase (pLDH) antigens could limit the spread of deleted isolates. Graphic Abstract

Analysis of false-negative rapid diagnostic tests for symptomatic malaria in the Democratic Republic of the Congo

The majority of Plasmodium falciparum malaria diagnoses in Africa are made using rapid diagnostic tests (RDTs) that detect histidine-rich protein 2. Increasing reports of false-negative RDT results due to parasites with deletions of the pfhrp2 and/or pfhrp3 genes (pfhrp2/3) raise concern about existing malaria diagnostic strategies. We previously identified pfhrp2-negative parasites among asymptomatic children in the Democratic Republic of the Congo (DRC), but their impact on diagnosis of symptomatic malaria is unknown. We performed a cross-sectional study of false-negative RDTs in symptomatic subjects in 2017. Parasites were characterized by microscopy; RDT; pfhrp2/3 genotyping and species-specific PCR assays; a bead-based immunoassay for Plasmodium antigens; and/or whole-genome sequencing. Among 3627 symptomatic subjects, 427 (11.8%) had RDT-/microscopy + results. Parasites from eight (0.2%) samples were initially classified as putative pfhrp2/3 deletions by PCR, but antigen testing and whole-genome sequencing confirmed the presence of intact genes. 56.8% of subjects had PCR-confirmed malaria. Non-falciparum co-infection with P. falciparum was common (13.2%). Agreement between PCR and HRP2-based RDTs was satisfactory (Cohen’s kappa = 0.66) and superior to microscopy (0.33). Symptomatic malaria due to pfhrp2/3-deleted P. falciparum was not observed. Ongoing HRP2-based RDT use is appropriate for the detection of falciparum malaria in the DRC.

HLA Alleles B*53:01 and C*06:02 Are Associated With Higher Risk of P. falciparum Parasitemia in a Cohort in Uganda

Variation within the HLA locus been shown to play an important role in the susceptibility to and outcomes of numerous infections, but its influence on immunity to P. falciparum malaria is unclear. Increasing evidence indicates that acquired immunity to P. falciparum is mediated in part by the cellular immune response, including NK cells, CD4 and CD8 T cells, and semi-invariant γδ T cells. HLA molecules expressed by these lymphocytes influence the epitopes recognized by P. falciparum-specific T cells, and class I HLA molecules also serve as ligands for inhibitory receptors including KIR. Here we assessed the relationship of HLA class I and II alleles to the risk of P. falciparum infection and symptomatic malaria in a cohort of 892 Ugandan children and adults followed prospectively via both active and passive surveillance. We identified two HLA class I alleles, HLA-B*53:01 and HLA-C*06:02, that were associated with a higher prevalence of P. falciparum infection. Notably, no class I or II HLA alleles were found to be associated with protection from P. falciparum parasitemia or symptomatic malaria. These findings suggest that class I HLA plays a role in the ability to restrict parasitemia, supporting an essential role for the cellular immune response in P. falciparum immunity. Our findings underscore the need for better tools to enable mechanistic studies of the T cell response to P. falciparum at the epitope level and suggest that further study of the role of HLA in regulating pre-erythrocytic stages of the P. falciparum life cycle is warranted.

Age-related Changes in Malaria Clinical Phenotypes During Infancy are Modified by Sickle Cell Trait

Background Young infants are protected against Plasmodium falciparum malaria. Mechanisms driving this protection remain unclear due to a poor understanding of malaria clinical phenotypes during infancy. Methods We enrolled a birth cohort of 678 infants in Busia, Uganda, an area of high malaria transmission. We followed infants through 12 months of age, and quantified protection against parasitemia and clinical disease. Results Symptomatic malaria incidence increased from 1.2 to 2.6 episodes per person year between 0-&lt;6 months and 6-12 months of age, while the monthly probability of asymptomatic parasitemia given infection decreased from 32% to 21%. Sickle cell trait (HbAS) was protective against symptomatic malaria (incidence rate ratio (IRR) = 0.57 comparing HbAS vs. HbAA, 95% CI 0.44-0.74, p&lt;0.001), but age modified this relationship (Pint=&lt;0.001), with non-linear protection that waned between 0-9 months of age before increasing. Increasing age was associated with higher parasite densities at the time of infection, and, in infants with HbAS, a reduced ability to tolerate high parasite densities without fever. Conclusions Age-dependent changes in HbAS protective efficacy in infancy were accompanied by differential loss of anti-parasite and anti-disease protection among HbAS and HbAA infants. This provides a framework for investigating mechanisms underlying infant protection against malaria.

Prevalence of Cardiovascular Complications in Malaria: A Systematic Review and Meta-Analysis

Recent studies have suggested that malaria may affect the cardiovascular system. The aim of this systematic review and meta-analysis was to determine the prevalence of cardiovascular complications in symptomatic malaria patients. We searched databases such as Pubmed, Embase, Cochrane, and Web of Science (January 1950–April 2020) for studies reporting on cardiovascular complications in adults and children with malaria. Cardiovascular complications were defined as abnormalities in electrocardiogram (ECG), cardiac biomarkers, and echocardiography on admission or during outpatient examination. Studies of patients with known heart disease or cardiovascular evaluation performed after the start of intravenous antimalarial medication were excluded. The study was registered in PROSPERO (No.: CRD42020167672). The literature search yielded 1,243 studies, and a total of 43 studies with symptomatic malaria patients were included. Clinical studies (n = 12 adults; n = 5 children) comprised 3,117 patients, of which a majority had Plasmodium falciparum (n = 15) and were diagnosed with severe malaria (n = 13). In random-effects models of adults, the pooled prevalence estimate for any cardiovascular complication was 7% (95% CI: 5–9). No meta-analysis was conducted in children, but the range of abnormal ECG was 0–8%, cardiac biomarkers 0–57%, and echocardiography 4–9%. We analyzed 33 cases (n = 10 postmortem), in which the most common cardiovascular pathologies were myocarditis and acute coronary syndrome. All histopathological studies found evidence of parasitized red blood cells in the myocardium. Cardiovascular complications are not uncommon in symptomatic adults and children with malaria. Additional studies investigating malaria and cardiovascular disease are encouraged.