Bone marrow reticulocytes: a Plasmodium vivax affair?

In this issue of Blood, Malleret and colleagues show the importance of the bone marrow in Plasmodium vivax biology by proving the preferential infection of young reticulocytes (generally restricted to the bone marrow), which then experience accelerated maturation postinvasion

"Resistance" to diagnostics: A serious biological challenge for malaria control and elimination

Delay in diagnosis and treatment is the leading cause of death in malaria patients. The recommendation issued in 2010 by the World Health Organization (WHO) to reserve malaria treatment to parasitologically confirmed malaria infections has boosted the use of malaria rapid diagnostic tests (RDTs), which have now become a critical component of management and surveillance of malaria. Indeed, it has been estimated that over 280 million RDTs are now used annually, at a cost of hundreds of millions of euros [1]. Beyond their use as a diagnostic tool for patients with suspected malaria, the detection of Plasmodium antigens in blood samples is also used in in vitro tests of sensitivity to antimalarial drugs, as a marker of clinical severity and to verify the elimination of the parasite after treatment, although the decay of parasite antigens may take longer than the clearance of parasitaemia.2 p

Malaria Parasites in the Asymptomatic: Looking for the Hay in the Haystack

With malaria elimination back on the international agenda, programs face the challenge of targeting all Plasmodium infections, not only symptomatic cases. As asymptomatic individuals are unlikely to seek treatment, they are missed by passive surveillance while remaining infectious to mosquitoes, thus acting as silent reservoirs of transmission. To estimate the risk of asymptomatic infections in various phases of malaria elimination, we need a deeper understanding of the underlying mechanisms favoring carriage over disease, which may involve both pathogen and host factors. Here we review our current knowledge on the determinants leading to Plasmodium falciparum symptomless infections. Understanding the host-pathogen interactions that are most likely to affect transitions between malaria disease states could guide the development of tools to tackle asymptomatic carriers in elimination settings

A humanized mouse model for sequestration of Plasmodium falciparum sexual stages and in vivo evaluation of gametocytidal drugs

The development of new drugs to disrupt malaria transmission requires the establishment of an in vivo model to address the biology of Plasmodium falciparum sexual stages (gametocytes). Herein we show that chemically immune-modulated NSG mice grafted with human erythrocytes support complete sexual development of P. falciparum parasites and generate high gametocytemia. Immunohistochemistry and RT-qPCR analyses indicate an enrichment of immature gametocytes in the bone marrow and the spleen, suggesting a sequestration mechanism reminiscent to that observed in humans. Upon primaquine treatment, elimination of gametocytes from peripheral blood and from sequestration sites was observed, providing a proof of concept that these mice can be used for testing drugs. Therefore, this model allows the investigation of P. falciparum sexual commitment, gametocyte interactions with the bone marrow and spleen and provides the missing link between current in vitro assays and Phase I trials in humans for testing new malaria gametocytidal drugs

Analytical sensitivity of current best-in-class malaria rapid diagnostic tests

BACKGROUND: Rapid diagnostic tests (RDTs) are today the most widely used method for malaria diagnosis and are recommended, alongside microscopy, for the confirmation of suspected cases before the administration of anti-malarial treatment. The diagnostic performance of RDTs, as compared to microscopy or PCR is well described but the actual analytical sensitivity of current best-in-class tests is poorly documented. This value is however a key performance indicator and a benchmark value needed to developed new RDTs of improved sensitivity. METHODS: Thirteen RDTs detecting either the Plasmodium falciparum histidine rich protein 2 (HRP2) or the plasmodial lactate dehydrogenase (pLDH) antigens were selected from the best performing RDTs according to the WHO-FIND product testing programme. The analytical sensitivity of these products was evaluated using a range of reference materials including P. falciparum and Plasmodium vivax whole parasite samples as well as recombinant proteins. RESULTS: The best performing HRP2-based RDTs could detect all P. falciparum cultured samples at concentrations as low as 0.8 ng/mL of HRP2. The limit of detection of the best performing pLDH-based RDT specifically detecting P. vivax was 25 ng/mL of pLDH. CONCLUSION: The analytical sensitivity of P. vivax and Pan pLDH-based RDTs appears to vary considerably from product to product, and improvement of the limit-of-detection for P. vivax detecting RDTs is needed to match the performance of HRP2 and Pf pLDH-based RDTs for P. falciparum. Different assays using different reference materials produce different values for antigen concentration in a given specimen, highlighting the need to establish universal reference assays

Molecular surveillance of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum isolates from Mozambique

BACKGROUND: Malaria programmes use Plasmodium falciparum histidine-rich protein-2 (PfHRP2) based rapid diagnostic tests (RDTs) for malaria diagnosis. The deletion of this target antigen could potentially lead to misdiagnosis, delayed treatment and continuation of active transmission. METHODS: Plasmodium falciparum isolates (n = 1162) collected in Southern Mozambique were assessed by RDTs, microscopy and/or 18SrRNA qPCR. pfhrp2 and pfhrp3 deletions were investigated in isolates from individuals who were negative by RDT but positive by microscopy and/or qPCR (n = 69) using gene-specific PCRs, with kelch13 PCR as the parasite DNA control. RESULTS: Lack of pfhrp2 PCR amplification was observed in one of the 69 isolates subjected to molecular analysis [1.45% (95% CI 0.3-7.8%)]. CONCLUSIONS: The low prevalence of pfhrp2 deletions suggests that RDTs will detect the vast majority of the P. falciparum infections. Nevertheless, active surveillance for changing deletion frequencies is required

Mosquitoes as a feasible sentinel group for anti-malarial resistance surveillance by Next Generation Sequencing of Plasmodium falciparum

Background: Plasmodium falciparum drug resistance surveillance is key to successful disease control and eradication. Contemporary methods that only allow determination of prevalence of resistance are expensive, time consuming and require ethical considerations. A newer method involving Next Generation Sequencing (NGS) permits obtaining frequency of resistance while allowing to detect minority variants in mixed infections. Here, NGS was tested for P. falciparum resistance marker detection in mosquito samples as a feasible and suitable alternative for molecular resistance surveillance. Anopheles funestus were collected in southern Mozambique using CDC light traps and manual collec‑ tions. DNA was extracted from either whole mosquito, head-thorax and abdomen separately or pools of fve mosqui‑ toes. These samples were screened for P. falciparum and if positive for k13, pfcrt, pfmdr1, pfdhps and pfdhfr mutations related to anti-malarial drug resistance with Sanger sequencing and NGS. Results: Among the 846 samples screened for P. falciparum, 122 were positive by 18S ssrDNA qPCR with an infection rate of 23.6%. No mutations were observed for k13 and pfcrt72-76 and almost zero for pfmdr86, but quintuple pfdhfr/ pfdhps mutations were near fxation and about half of the isolates contained the pfmdr184F polymorphism. Similar allele frequencies of resistance markers were estimated with NGS in comparison with the prevalence of markers obtained with the gold standard Sanger sequencing. Conclusions: Pooled deep sequencing of P. falciparum isolates extracted from mosquitoes is a promising, efcient and cost-efective method to quantify allele frequencies at population level which allows to detect known and unknown markers of resistance in single and mixed infections in a timelier manner. Using mosquitoes as sentinel group and focusing on allele frequency opposed to prevalence, permits active surveillance across a more homogene‑ ous geographical range

Dynamics of Afebrile Plasmodium falciparum Infections in Mozambican Men

Background: Afebrile Plasmodium falciparum infections usually remain undetected and untreated in the community and could potentially contribute to sustaining local malaria transmission in areas aiming for malaria elimination. Methods: Thirty-two men with afebrile P. falciparum infections detected with rapid diagnostic test (RDTs) were followed for 28 days. Kaplan-Meier estimates were computed to estimate probability of parasite positivity and of reducing parasitaemia by half of its initial level by day 28. Trends of parasite densities quantified by microscopy and qPCR were assessed using Poisson regression models, and the microscopy to qPCR positivity ratio was calculated at each time point. Three survival distributions (Gompertz, Weibull, and gamma) were used to evaluate their strength of fit to the data and to predict the median lifetime of infection. Results: The cumulative probability of parasite qPCR positivity by day 28 was 81% (95% CI 60.2-91.6). Geometric mean parasitemia at recruitment was 516.1 parasites/muL and fell to <100 parasites/muL by day 3, reaching 56.7 parasites/muL on day 28 (p-value<0.001). The ratio of P. falciparum positive samples by microscopy to qPCR decreased from 0.9 to 0.52 from recruitment to day 28. The best model fit to the data was obtained assuming a Gompertz distribution. Conclusions: Afebrile P.falciparum infections detectable by RDT in semi-immune adults fall and stabilize at low-density levels during the first four days since detection, suggesting a rapid decline of potential transmissibility in this hidden parasite reservoir

Community-informed research on malaria in pregnancy in Monrovia, Liberia: a grounded theory study

Background: Liberia is a West African country that needs substantial investment to strengthen its National Malaria Control Programme (NMCP), which was disrupted during the 2014–2016 Ebola epidemic. As elsewhere, Liberian pregnant women are especially vulnerable to malaria. Understanding prevention and treatment-seeking behaviours among the population is crucial to strategize context-specifc and women-centred actions, including locally-led malaria research, to improve women’s demand, access and use of NMCP strategies against malaria in pregnancy. Methods: In 2016, after the Ebola crisis, a qualitative inquiry was conducted in Monrovia to explore populations’ insights on the aetiology, prevention and therapeutics of malaria, as well as the community and health workers’ perceptions on the utility of malaria research for pregnant women. In-depth interviews and focus group discussions were conducted among pregnant women, traditional community representatives and hospital staf (n=38), using a feminist interpretation of grounded theory. Results: The narratives indicate that some Liberians believed in elements other than mosquito bites as causes of malaria; many had a low malaria risk perception and disliked current efective prevention methods, such as insecticide-treated nets; and some would resort to traditional medicine and spiritual care to cure malaria. Access to clinic-based malaria care for pregnant women was reportedly hindered by lack of fnancial means, by unofcial user fees requested by healthcare workers, and by male partners’ preference for traditional medicine. The participants suggested that malaria research in Liberia could help to design evidence-based education to change current malaria prevention, diagnostic and treatment-seeking attitudes, and to develop more acceptable prevention technologies. Conclusion: Poverty, insufcient education on malaria, corruption, and poor trust in healthcare establishment are structural factors that may play a greater role than local traditional beliefs in deterring Liberians from seeking, access‑ ing and using government-endorsed malaria control strategies. To increase access to and uptake of preventive and biomedical care by pregnant women, future malaria research must be informed by people’s expressed needs and constructed meanings and values on health, ill health and healthcare

Effects of HIV infection on maternal and neonatal health in southern Mozambique: A prospective cohort study after a decade of antiretroviral drugs roll out

INTRODUCTION: The HIV epidemic is concentrated in sub-Saharan Africa. However, limited information exists on its impact on women and infant's health since the introduction of antiretroviral drugs in this region, where health resources are often scarce. METHODS: The effect of HIV infection on maternal health, birth outcomes and infant health was analysed in two contemporary cohorts of HIV-uninfected and HIV-infected pregnant women from southern Mozambique. Pregnant women attending the first antenatal care visit were followed until one month after delivery. Antiretroviral therapy was administered based on CD4+T cell count and clinical stage. Maternal and neonatal morbidity and mortality, as well as pregnancy outcomes were assessed by mother's HIV status. RESULTS: A total of 1183 HIV-uninfected and 561 HIV-infected pregnant women were enrolled. HIV-infected women were more likely to have anaemia both at the first antenatal care visit and at delivery than HIV-uninfected women (71.5% versus 54.8% and 49.4% versus 40.6%, respectively, p<0.001). Incidence of hospital admissions during pregnancy was increased among HIV-infected women (RR, 2.04, [95%CI, 1.45; 2.86]; p<0.001). At delivery, 21% of HIV-infected women reported being on antiretroviral therapy, and 70% having received antiretroviral drugs for prevention of mother to child transmission of HIV. The risk of stillbirths was doubled in HIV-infected women (RR, 2.16 [95%CI 1.17; 3.96], p = 0.013). Foetal anaemia was also increased among infants born to HIV-infected women (10.6% versus 7.3%, p = 0.022). No differences were found in mean birth weight, malaria, prematurity and maternal and neonatal deaths between groups. CONCLUSIONS: HIV infection continues to be associated with significant maternal morbidity and poor neonatal health outcomes. Efforts should urgently be made to identify the barriers that impede improvements on the devastating effects of HIV in African women and their infants. TRIAL REGISTRATION: ClinicalTrials.gov NCT 00811421