Potent transmission-blocking monoclonal antibodies from naturally exposed individuals target a conserved epitope on Plasmodium falciparum Pfs230.

Pfs230 is essential for Plasmodium falciparum transmission to mosquitoes and is the protein targeted by the most advanced malaria-transmission-blocking vaccine candidate. Prior understanding of functional epitopes on Pfs230 is based on two monoclonal antibodies (mAbs) with moderate transmission-reducing activity (TRA), elicited from subunit immunization. Here, we screened the B cell repertoire of two naturally exposed individuals possessing serum TRA and identified five potent mAbs from sixteen Pfs230 domain-1-specific mAbs. Structures of three potent and three low-activity antibodies bound to Pfs230 domain 1 revealed four distinct epitopes. Highly potent mAbs from natural infection recognized a common conformational epitope that is highly conserved across P. falciparum field isolates, while antibodies with negligible TRA derived from natural infection or immunization recognized three distinct sites. Our study provides molecular blueprints describing P. falciparum TRA, informed by contrasting potent and non-functional epitopes elicited by natural exposure and vaccination.

Highly potent, naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes.

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that interrupt malaria parasite development in the mosquito, thereby blocking onward transmission, and provide a much-needed tool for malaria control and elimination. The parasite surface protein Pfs48/45 is a leading TBV candidate. Here, we isolated and characterized a panel of 81 human Pfs48/45-specific monoclonal antibodies (mAbs) from donors naturally exposed to Plasmodium parasites. Genetically diverse mAbs against each of the three domains (D1-D3) of Pfs48/45 were identified. The most potent mAbs targeted D1 and D3 and achieved >80% transmission-reducing activity in standard membrane-feeding assays, at 10 and 2 µg/mL, respectively. Co-crystal structures of D3 in complex with four different mAbs delineated two conserved protective epitopes. Altogether, these Pfs48/45-specific human mAbs provide important insight into protective and non-protective epitopes that can further our understanding of transmission and inform the design of refined malaria transmission-blocking vaccine candidates.

Population pharmacokinetics of primaquine and its metabolites in African males.

BACKGROUND: Primaquine (PQ) is the prototype 8-aminoquinoline drug, a class which targets gametocytes and hypnozoites. The World Health Organization (WHO) recommends adding a single low dose of primaquine to the standard artemisinin-based combination therapy (ACT) in order to block malaria transmission in regions with low malaria transmission. However, the haemolytic toxicity is a major adverse outcome of primaquine in glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects. This study aimed to characterize the pharmacokinetic properties of primaquine and its major metabolites in G6PD-deficient subjects. METHODS: A single low-dose of primaquine (0.4-0.5 mg/kg) was administered in twenty-eight African males. Venous and capillary plasma were sampled up to 24 h after the drug administration. Haemoglobin levels were observed up to 28 days after drug administration. Only PQ, carboxy-primaquine (CPQ), and primaquine carbamoyl-glucuronide (PQCG) were present in plasma samples and measured using liquid chromatography mass spectrometry. Drug and metabolites' pharmacokinetic properties were investigated using nonlinear mixed-effects modelling. RESULTS: Population pharmacokinetic properties of PQ, CPQ, and PQCG can be described by one-compartment disposition kinetics with a transit-absorption model. Body weight was implemented as an allometric function on the clearance and volume parameters for all compounds. None of the covariates significantly affected the pharmacokinetic parameters. No significant correlations were detected between the exposures of the measured compounds and the change in haemoglobin or methaemoglobin levels. There was no significant haemoglobin drop in the G6PD-deficient patients after administration of a single low dose of PQ. CONCLUSIONS: A single low-dose of PQ was haematologically safe in this population of G6PD-normal and G6PD-deficient African males without malaria. Trial registration NCT02535767.

Asymptomatic Plasmodium falciparum carriage at the end of the dry season is associated with subsequent infection and clinical malaria in Eastern Gambia.

BACKGROUND: Chronic carriage of asymptomatic low-density Plasmodium falciparum parasitaemia in the dry season may support maintenance of acquired immunity that protects against clinical malaria. However, the relationship between chronic low-density infections and subsequent risk of clinical malaria episodes remains unclear. METHODS: In a 2-years study (December 2014 to December 2016) in eastern Gambia, nine cross-sectional surveys using molecular parasite detection were performed in the dry and wet season. During the 2016 malaria transmission season, passive case detection identified episodes of clinical malaria. RESULTS: Among the 5256 samples collected, 444 (8.4%) were positive for P. falciparum. A multivariate model identified village of residence, male sex, age ≥ 5 years old, anaemia, and fever as independent factors associated with P. falciparum parasite carriage. Infections did not cluster over time within the same households or recurred among neighbouring households. Asymptomatic parasite carriage at the end of dry season was associated with a higher risk of infection (Hazard Ratio, HR = 3.0, p < 0.0001) and clinical malaria (HR = 1.561, p = 0.057) during the following transmission season. Age and village of residence were additional predictors of infection and clinical malaria during the transmission season. CONCLUSION: Chronic parasite carriage during the dry season is associated with an increased risk of malaria infection and clinical malaria. It is unclear whether this is due to environmental exposure or to other factors.

Immunity against sexual stage Plasmodium falciparum and Plasmodium vivax parasites.

The efficient spread of malaria from infected humans to mosquitoes is a major challenge for malaria elimination initiatives. Gametocytes are the only Plasmodium life stage infectious to mosquitoes. Here, we summarize evidence for naturally acquired anti-gametocyte immunity and the current state of transmission blocking vaccines (TBV). Although gametocytes are intra-erythrocytic when present in infected humans, developing Plasmodium falciparum gametocytes may express proteins on the surface of red blood cells that elicit immune responses in naturally exposed individuals. This immune response may reduce the burden of circulating gametocytes. For both P. falciparum and Plasmodium vivax, there is a solid evidence that antibodies against antigens present on the gametocyte surface, when co-ingested with gametocytes, can influence transmission to mosquitoes. Transmission reducing immunity, reducing the burden of infection in mosquitoes, is a well-acknowledged but poorly quantified phenomenon that forms the basis for the development of TBV. Transmission enhancing immunity, increasing the likelihood or intensity of transmission to mosquitoes, is more speculative in nature but is convincingly demonstrated for P. vivax. With the increased interest in malaria elimination, TBV and monoclonal antibodies have moved to the center stage of malaria vaccine development. Methodologies to prioritize and evaluate products are urgently needed.

Modeling the Impact of a Highly Potent Plasmodium falciparum Transmission-Blocking Monoclonal Antibody in Areas of Seasonal Malaria Transmission.

Transmission-blocking interventions can play an important role in combating malaria worldwide. Recently, a highly potent Plasmodium falciparum transmission-blocking monoclonal antibody (TB31F) was demonstrated to be safe and efficacious in malaria-naive volunteers. Here we predict the potential public health impact of large-scale implementation of TB31F alongside existing interventions. We developed a pharmaco-epidemiological model, tailored to 2 settings of differing transmission intensity with already established insecticide-treated nets and seasonal malaria chemoprevention interventions. Community-wide annual administration (at 80% coverage) of TB31F over a 3-year period was predicted to reduce clinical incidence by 54% (381 cases averted per 1000 people per year) in a high-transmission seasonal setting, and 74% (157 cases averted per 1000 people per year) in a low-transmission seasonal setting. Targeting school-aged children gave the largest reduction in terms of cases averted per dose. An annual administration of the transmission-blocking monoclonal antibody TB31F may be an effective intervention against malaria in seasonal malaria settings.

A Cohort Study on the Duration of Plasmodium falciparum Infections During the Dry Season in The Gambia.

BACKGROUND: In areas where Plasmodium falciparum malaria is seasonal, a dry season reservoir of blood-stage infection is essential for initiating transmission during the following wet season. METHODS: In The Gambia, a cohort of 42 individuals with quantitative polymerase chain reaction-positive P falciparum infections at the end of the transmission season (December) were followed monthly until the end of the dry season (May) to evaluate infection persistence. The influence of human host and parasitological factors was investigated. RESULTS: A large proportion of individuals infected at the end of the wet season had detectable infections until the end of the dry season (40.0%; 16 of 40). At the start of the dry season, the majority of these persistent infections (82%) had parasite densities >10 p/µL compared to only 5.9% of short-lived infections. Persistent infections (59%) were also more likely to be multiclonal than short-lived infections (5.9%) and were associated with individuals having higher levels of P falciparum-specific antibodies (P = .02). CONCLUSIONS: Asymptomatic persistent infections were multiclonal with higher parasite densities at the beginning of the dry season. Screening and treating asymptomatic infections during the dry season may reduce the human reservoir of malaria responsible for initiating transmission in the wet season.

Asymptomatic School-Aged Children Are Important Drivers of Malaria Transmission in a High Endemicity Setting in Uganda.

Achieving malaria elimination requires a better understanding of the transmissibility of human infections in different transmission settings. This study aimed to characterize the human infectious reservoir in a high endemicity setting in eastern Uganda, using gametocyte quantification and mosquito feeding assays. In asymptomatic infections, gametocyte densities were positively associated with the proportion of infected mosquitoes (ß = 1.60; 95% CI, 1.32-1.92; P < .0001). Combining transmissibility and abundance in the population, symptomatic and asymptomatic infections were estimated to contribute to 5.3% and 94.7% of the infectious reservoir, respectively. School-aged children (5-15 years old) contributed to 50.4% of transmission events and were important drivers of malaria transmission.

Efficacy of Single-Dose Primaquine With Artemisinin Combination Therapy on Plasmodium falciparum Gametocytes and Transmission: An Individual Patient Meta-Analysis.

BACKGROUND: Since the World Health Organization recommended single low-dose (0.25 mg/kg) primaquine (PQ) in combination with artemisinin-based combination therapies (ACTs) in areas of low transmission or artemisinin-resistant Plasmodium falciparum, several single-site studies have been conducted to assess efficacy. METHODS: An individual patient meta-analysis to assess gametocytocidal and transmission-blocking efficacy of PQ in combination with different ACTs was conducted. Random effects logistic regression was used to quantify PQ effect on (1) gametocyte carriage in the first 2 weeks post treatment; and (2) the probability of infecting at least 1 mosquito or of a mosquito becoming infected. RESULTS: In 2574 participants from 14 studies, PQ reduced PCR-determined gametocyte carriage on days 7 and 14, most apparently in patients presenting with gametocytemia on day 0 (odds ratio [OR], 0.22; 95% confidence interval [CI], .17-.28 and OR, 0.12; 95% CI, .08-.16, respectively). Rate of decline in gametocyte carriage was faster when PQ was combined with artemether-lumefantrine (AL) compared to dihydroartemisinin-piperaquine (DP) (P = .010 for day 7). Addition of 0.25 mg/kg PQ was associated with near complete prevention of transmission to mosquitoes. CONCLUSIONS: Transmission blocking is achieved with 0.25 mg/kg PQ. Gametocyte persistence and infectivity are lower when PQ is combined with AL compared to DP.

The potential impact of Anopheles stephensi establishment on the transmission of Plasmodium falciparum in Ethiopia and prospective control measures.

BACKGROUND: Sub-Saharan Africa has seen substantial reductions in cases and deaths due to malaria over the past two decades. While this reduction is primarily due to an increasing expansion of interventions, urbanisation has played its part as urban areas typically experience substantially less malaria transmission than rural areas. However, this may be partially lost with the invasion and establishment of Anopheles stephensi. A. stephensi, the primary urban malaria vector in Asia, was first detected in Africa in 2012 in Djibouti and was subsequently identified in Ethiopia in 2016, and later in Sudan and Somalia. In Djibouti, malaria cases have increased 30-fold from 2012 to 2019 though the impact in the wider region remains unclear. METHODS: Here, we have adapted an existing model of mechanistic malaria transmission to estimate the increase in vector density required to explain the trends in malaria cases seen in Djibouti. To account for the observed plasticity in An. stephensi behaviour, and the unknowns of how it will establish in a novel environment, we sample behavioural parameters in order to account for a wide range of uncertainty. This quantification is then applied to Ethiopia, considering temperature-dependent extrinsic incubation periods, pre-existing vector-control interventions and Plasmodium falciparum prevalence in order to assess the potential impact of An. stephensi establishment on P. falciparum transmission. Following this, we estimate the potential impact of scaling up ITN (insecticide-treated nets)/IRS (indoor residual spraying) and implementing piperonyl butoxide (PBO) ITNs and larval source management, as well as their economic costs. RESULTS: We estimate that annual P. falciparum malaria cases could increase by 50% (95% CI 14-90) if no additional interventions are implemented. The implementation of sufficient control measures to reduce malaria transmission to pre-stephensi levels will cost hundreds of millions of USD. CONCLUSIONS: Substantial heterogeneity across the country is predicted and large increases in vector control interventions could be needed to prevent a major public health emergency.

Safety of single-dose primaquine as a Plasmodium falciparum gametocytocide: a systematic review and meta-analysis of individual patient data.

BACKGROUND: In 2012, the World Health Organization (WHO) recommended single low-dose (SLD, 0.25 mg/kg) primaquine to be added as a Plasmodium (P.) falciparum gametocytocide to artemisinin-based combination therapy (ACT) without glucose-6-phosphate dehydrogenase (G6PD) testing, to accelerate malaria elimination efforts and avoid the spread of artemisinin resistance. Uptake of this recommendation has been relatively slow primarily due to safety concerns. METHODS: A systematic review and individual patient data (IPD) meta-analysis of single-dose (SD) primaquine studies for P. falciparum malaria were performed. Absolute and fractional changes in haemoglobin concentration within a week and adverse effects within 28 days of treatment initiation were characterised and compared between primaquine and no primaquine arms using random intercept models. RESULTS: Data comprised 20 studies that enrolled 6406 participants, of whom 5129 (80.1%) had received a single target dose of primaquine ranging between 0.0625 and 0.75 mg/kg. There was no effect of primaquine in G6PD-normal participants on haemoglobin concentrations. However, among 194 G6PD-deficient African participants, a 0.25 mg/kg primaquine target dose resulted in an additional 0.53 g/dL (95% CI 0.17-0.89) reduction in haemoglobin concentration by day 7, with a 0.27 (95% CI 0.19-0.34) g/dL haemoglobin drop estimated for every 0.1 mg/kg increase in primaquine dose. Baseline haemoglobin, young age, and hyperparasitaemia were the main determinants of becoming anaemic (Hb < 10 g/dL), with the nadir observed on ACT day 2 or 3, regardless of G6PD status and exposure to primaquine. Time to recovery from anaemia took longer in young children and those with baseline anaemia or hyperparasitaemia. Serious adverse haematological events after primaquine were few (9/3, 113, 0.3%) and transitory. One blood transfusion was reported in the primaquine arms, and there were no primaquine-related deaths. In controlled studies, the proportions with either haematological or any serious adverse event were similar between primaquine and no primaquine arms. CONCLUSIONS: Our results support the WHO recommendation to use 0.25 mg/kg of primaquine as a P. falciparum gametocytocide, including in G6PD-deficient individuals. Although primaquine is associated with a transient reduction in haemoglobin levels in G6PD-deficient individuals, haemoglobin levels at clinical presentation are the major determinants of anaemia in these patients. TRIAL REGISTRATION: PROSPERO, CRD42019128185.

Environmentally sustainable practices in global health research and higher education institutions: Lessons from consultation with the TropEd Global Health institutions.

OBJECTIVE: To examine how global health institutions are reducing the greenhouse gas emissions from their own operations and analyse the facilitators and barriers to achieving decarbonisation goals. METHODS: We reviewed the sustainability goals and implementation plans of 10 global health universities from the 'TropEd' network. We systematically collected information from institutional websites and annual reports. Through online interviews, 11 key informants validated the information from 9 of the institutions and shared their opinions regarding what factors are helping their institutions decarbonise and what factors are hindering progress. RESULTS: 4/10 institutions sampled have a sustainability strategy and implementation plan, only 3/10 have specific decarbonisation goals, and 3/10 are reporting on progress. 5/10 institutions reported that they are in the process of determining emission reduction targets. CONCLUSION: This paper identifies common success factors that facilitate decarbonisation as well as common challenges and how they are being tackled, and makes recommendations on sustainability efforts in academic institutions.

A Randomized Clinical Trial to Compare Plasmodium falciparum Gametocytemia and Infectivity After Blood-Stage or Mosquito Bite-Induced Controlled Malaria Infection.

BACKGROUND: For malaria elimination efforts, it is important to better understand parasite transmission to mosquitoes and develop models for early-clinical evaluation of transmission-blocking interventions. METHODS: In a randomized open-label trial, 24 participants were infected by bites from Plasmodium falciparum 3D7-infected mosquitoes (mosquito bite [MB]; n = 12) or by induced blood-stage malaria (IBSM) with the same parasite line (n = 12). After subcurative piperaquine treatment, asexual parasite and gametocytes kinetics were assessed, and mosquito feeding experiments were performed. RESULTS: Study procedures were well tolerated. The median peak gametocyte density was 1304/mL (interquartile range, 308-1607/mL) after IBSM, compared with 14/mL (10-64/mL) after MB inoculation (P < .001), despite similar peak asexual parasite densities (P = .48). Peak gametocyte density was correlated with preceding pfap2-g transcripts, indicative of gametocyte commitment (ρ = 0.62; P = .002). Direct feeding assays resulted in mosquito infections from 9 of 12 participants after IBSM versus 0 of 12 after MB inoculation (P < .001). CONCLUSIONS: We observed a striking effect of inoculation method on gametocyte production, suggesting higher gametocyte commitment after IBSM. Our direct comparison of MB and IBSM establishes the controlled human malaria infection transmission model, using intravenous administration of P. falciparum-infected erythrocytes as a model for early-clinical evaluation of interventions that aim to interrupt malaria transmission. CLINICAL TRIAL REGISTRATION: NCT03454048.

Plasmodium falciparum Gametocyte Density and Infectivity in Peripheral Blood and Skin Tissue of Naturally Infected Parasite Carriers in Burkina Faso.

BACKGROUND: Plasmodium falciparum transmission depends on mature gametocytes that can be ingested by mosquitoes taking a blood meal on human skin. Although gametocyte skin sequestration has long been hypothesized as important contributor to efficient malaria transmission, this has never been formally tested. METHODS: In naturally infected gametocyte carriers from Burkina Faso, we assessed infectivity to mosquitoes by direct skin feeding and membrane feeding. We directly quantified male and female gametocytes and asexual parasites in finger-prick and venous blood samples, skin biopsy samples, and in of mosquitoes that fed on venous blood or directly on skin. Gametocytes were visualized in skin tissue with confocal microscopy. RESULTS: Although more mosquitoes became infected when feeding directly on skin then when feeding on venous blood (odds ratio, 2.01; 95% confidence interval, 1.21-3.33; P = .007), concentrations of gametocytes were not higher in the subdermal skin vasculature than in other blood compartments; only sparse gametocytes were observed in skin tissue. DISCUSSION: Our data strongly suggest that there is no significant skin sequestration of P. falciparum gametocytes. Gametocyte densities in peripheral blood are thus informative for predicting onward transmission potential to mosquitoes and can be used to target and monitor malaria elimination initiatives.

Anopheles stephensi Mosquitoes as Vectors of Plasmodium vivax and falciparum, Horn of Africa, 2019.

Anopheles stephensi mosquitoes, efficient vectors in parts of Asia and Africa, were found in 75.3% of water sources surveyed and contributed to 80.9% of wild-caught Anopheles mosquitoes in Awash Sebat Kilo, Ethiopia. High susceptibility of these mosquitoes to Plasmodium falciparum and vivax infection presents a challenge for malaria control in the Horn of Africa.

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.

A portfolio of geographically distinct laboratory-adapted Plasmodium falciparum clones with consistent infection rates in Anopheles mosquitoes.

BACKGROUND: The ability to culture Plasmodium falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research. The portfolio of isolates has remained limited and research is still largely based on NF54 and its derived clone 3D7. Since 1978, isolates were collected and cryopreserved at Radboudumc from patients presenting at the hospital. Here, procedures are described for culture adaptation of asexual parasites, cloning and production of sexual stage parasites responsible for transmission (gametocytes) and production of oocysts in Anopheles mosquitoes. This study aimed to identify new culture-adapted transmissible P. falciparum isolates, originating from distinct geographical locations. METHODS: Out of a collection of 121 P. falciparum isolates stored in liquid nitrogen, 21 from different geographical origin were selected for initial testing. Isolates were evaluated for their ability to be asexually cultured in vitro, their gametocyte production capacity, and consistent generation of oocysts. RESULTS: Out of 21 isolates tested, twelve were excluded from further analysis due to lack of mature gametocyte production (n = 1) or generation of satisfactory numbers of oocysts in mosquitoes (n = 11). Nine isolates fulfilled selection criteria and were cloned by limiting dilution and retested. After cloning, one isolate was excluded for not showing transmission. The remaining eight isolates transmitted to Anopheles stephensi or Anopheles coluzzii mosquitoes and were categorized into two groups with a reproducible mean oocyst infection intensity above (n = 5) or below five (n = 3). CONCLUSIONS: These new P. falciparum culture-adapted isolates with reproducible transmission to Anopheles mosquitoes are a valuable addition to the malaria research tool box. They can aid in the development of malaria interventions and will be particularly useful for those studying malaria transmission.

Gametocyte carriage after seasonal malaria chemoprevention in Plasmodium falciparum infected asymptomatic children.

BACKGROUND: Treatment of clinical Plasmodium falciparum malaria with sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) is associated with increased post-treatment gametocyte carriage. The effect of seasonal malaria chemoprevention (SMC) with SP and AQ on gametocyte carriage was assessed in asymptomatic P. falciparum infected children. METHODS: The study was carried out in eastern Gambia. Asymptomatic P. falciparum malaria infected children aged 24-59 months old who were eligible to receive SMC (SMC group) and children 5-8 years that were not eligible to receive SMC (comparison group) were recruited. Gametocytaemia was determined by molecular methods before and after SMC administration. Gametocyte carriage between the groups was compared using the chi-squared test and within-person using conditional logistic regression. RESULTS: During the 2017 and 2018 malaria transmission seasons, 65 and 75 children were recruited in the SMC and comparison groups, respectively. Before SMC administration, gametocyte prevalence was 10.7% (7/65) in the SMC group and 13.3% (10/75) in the comparison group (p = 0.64). At day 13 (IQR 12, 13) after SMC administration, this was 9.4% (5/53) in children who received at least the first dose of SMC treatment and 12.7% (9/71) for those in the comparison group (p = 0.57). Similarly, there was no difference in prevalence of gametocytes between children that adhered to all 3-day doses of SMC treatment 15.6% (5/32) and those in the comparison group (p = 0.68). In the SMC group, within-group gametocyte carriage was similar before and after SMC administration in children that received at least the first dose of SMC treatment (OR 0.6, 95% CI 0.14-2.51; p = 0.48) and in those that adhered to all 3-day doses of SMC treatment (OR 1.0, 95% CI 0.20-4.95; p = 1.0). CONCLUSION: In this study with relative low gametocyte prevalence prior to SMC treatment, no evidence was observed that SMC treatment increased gametocyte carriage in asymptomatic P. falciparum malaria infected children.

The epidemiology and detectability of asymptomatic plasmodium vivax and plasmodium falciparum infections in low, moderate and high transmission settings in Ethiopia.

BACKGROUND: As countries move to malaria elimination, detecting and targeting asymptomatic malaria infections might be needed. Here, the epidemiology and detectability of asymptomatic Plasmodium falciparum and Plasmodium vivax infections were investigated in different transmission settings in Ethiopia. METHOD: A total of 1093 dried blood spot (DBS) samples were collected from afebrile and apparently healthy individuals across ten study sites in Ethiopia from 2016 to 2020. Of these, 862 were from community and 231 from school based cross-sectional surveys. Malaria infection status was determined by microscopy or rapid diagnostics tests (RDT) and 18S rRNA-based nested PCR (nPCR). The annual parasite index (API) was used to classify endemicity as low (API > 0 and < 5), moderate (API ≥ 5 and < 100) and high transmission (API ≥ 100) and detectability of infections was assessed in these settings. RESULTS: In community surveys, the overall prevalence of asymptomatic Plasmodium infections by microscopy/RDT, nPCR and all methods combined was 12.2% (105/860), 21.6% (183/846) and 24.1% (208/862), respectively. The proportion of nPCR positive infections that was detectable by microscopy/RDT was 48.7% (73/150) for P. falciparum and 4.6% (2/44) for P. vivax. Compared to low transmission settings, the likelihood of detecting infections by microscopy/RDT was increased in moderate (Adjusted odds ratio [AOR]: 3.4; 95% confidence interval [95% CI] 1.6-7.2, P = 0.002) and high endemic settings (AOR = 5.1; 95% CI 2.6-9.9, P < 0.001). After adjustment for site and correlation between observations from the same survey, the likelihood of detecting asymptomatic infections by microscopy/RDT (AOR per year increase = 0.95, 95% CI 0.9-1.0, P = 0.013) declined with age. CONCLUSIONS: Conventional diagnostics missed nearly half of the asymptomatic Plasmodium reservoir detected by nPCR. The detectability of infections was particularly low in older age groups and low transmission settings. These findings highlight the need for sensitive diagnostic tools to detect the entire parasite reservoir and potential infection transmitters.

Persistence of mRNA indicative of Plasmodium falciparum ring-stage parasites 42 days after artemisinin and non-artemisinin combination therapy in naturally infected Malians.

BACKGROUND: Malaria control in sub-Saharan Africa relies upon prompt case management with artemisinin-based combination therapy (ACT). Ring-stage parasite mRNA, measured by sbp1 quantitative reverse-transcriptase PCR (qRT-PCR), was previously reported to persist after ACT treatment and hypothesized to reflect temporary arrest of the growth of ring-stage parasites (dormancy) following exposure to artemisinins. Here, the persistence of ring-stage parasitaemia following ACT and non-ACT treatment was examined. METHODS: Samples were used from naturally infected Malian gametocyte carriers who received dihydroartemisinin-piperaquine (DP) or sulfadoxine-pyrimethamine (SP-AQ) with or without gametocytocidal drugs. Gametocytes and ring-stage parasites were quantified by qRT-PCR during 42 days of follow-up. RESULTS: At baseline, 89% (64/73) of participants had measurable ring-stage parasite mRNA. Following treatment, the proportion of ring-stage parasite-positive individuals and estimated densities declined for all four treatment groups. Ring-stage parasite prevalence and density was generally lower in arms that received DP compared to SP-AQ. This finding was most apparent days 1, 2, and 42 of follow-up (p < 0.01). Gametocytocidal drugs did not influence ring-stage parasite persistence. Ring-stage parasite density estimates on days 14 and 28 after initiation of treatment were higher among individuals who subsequently experienced recurrent parasitaemia compared to those who remained free of parasites until day 42 after initiation of treatment (pday 14 = 0.011 and pday 28 = 0.068). No association of ring-stage persistence with gametocyte carriage was observed. CONCLUSIONS: The current findings of lower ring-stage persistence after ACT without an effect of gametocytocidal partner drugs affirms the use of sbp1 as ring-stage marker. Lower persistence of ring-stage mRNA after ACT treatment suggests the marker may not reflect dormant parasites whilst it was predictive of re-appearance of parasitaemia.