In this report, patients living in a malaria-endemic area underwent trauma-related splenectomy. In these asymptomatic patients who were naturally infected with
Plasmodium falciparum
or
P. vivax,
the ...predominant biomass of intact, infected red cells was in the spleen.
A very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected with Plasmodium vivax. The mechanisms underlying this intense ...tropism are not clear. We hypothesised that immature reticulocytes, in which P. vivax develops, may display high densities in the spleen, thereby providing a niche for parasite survival.
We examined spleen tissue in 22 mostly untreated individuals naturally exposed to P. vivax and Plasmodium falciparum undergoing splenectomy for any clinical indication in malaria-endemic Papua, Indonesia (2015 to 2017). Infection, parasite and immature reticulocyte density, and splenic distribution were analysed by optical microscopy, flow cytometry, and molecular assays. Nine non-endemic control spleens from individuals undergoing spleno-pancreatectomy in France (2017 to 2020) were also examined for reticulocyte densities. There were no exclusion criteria or sample size considerations in both patient cohorts for this demanding approach. In Indonesia, 95.5% (21/22) of splenectomy patients had asymptomatic splenic Plasmodium infection (7 P. vivax, 13 P. falciparum, and 1 mixed infection). Significant splenic accumulation of immature CD71 intermediate- and high-expressing reticulocytes was seen, with concentrations 11 times greater than in peripheral blood. Accordingly, in France, reticulocyte concentrations in the splenic effluent were higher than in peripheral blood. Greater rigidity of reticulocytes in splenic than in peripheral blood, and their higher densities in splenic cords both suggest a mechanical retention process. Asexual-stage P. vivax-infected erythrocytes of all developmental stages accumulated in the spleen, with non-phagocytosed parasite densities 3,590 times (IQR: 2,600 to 4,130) higher than in circulating blood, and median total splenic parasite loads 81 (IQR: 14 to 205) times greater, accounting for 98.7% (IQR: 95.1% to 98.9%) of the estimated total-body P. vivax biomass. More reticulocytes were in contact with sinus lumen endothelial cells in P. vivax- than in P. falciparum-infected spleens. Histological analyses revealed 96% of P. vivax rings/trophozoites and 46% of schizonts colocalised with 92% of immature reticulocytes in the cords and sinus lumens of the red pulp. Larger splenic cohort studies and similar investigations in untreated symptomatic malaria are warranted.
Immature CD71+ reticulocytes and splenic P. vivax-infected erythrocytes of all asexual stages accumulate in the same splenic compartments, suggesting the existence of a cryptic endosplenic lifecycle in chronic P. vivax infection. Findings provide insight into P. vivax-specific adaptions that have evolved to maximise survival and replication in the spleen.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Within the human host, the malaria parasite Plasmodium falciparum is exposed to multiple selection pressures. The host environment changes dramatically in severe malaria, but the extent to which the ...parasite responds to-or is selected by-this environment remains unclear. From previous studies, the parasites that cause severe malaria appear to increase expression of a restricted but poorly defined subset of the PfEMP1 variant, surface antigens. PfEMP1s are major targets of protective immunity. Here, we used RNA sequencing (RNAseq) to analyse gene expression in 44 parasite isolates that caused severe and uncomplicated malaria in Papuan patients. The transcriptomes of 19 parasite isolates associated with severe malaria indicated that these parasites had decreased glycolysis without activation of compensatory pathways; altered chromatin structure and probably transcriptional regulation through decreased histone methylation; reduced surface expression of PfEMP1; and down-regulated expression of multiple chaperone proteins. Our RNAseq also identified novel associations between disease severity and PfEMP1 transcripts, domains, and smaller sequence segments and also confirmed all previously reported associations between expressed PfEMP1 sequences and severe disease. These findings will inform efforts to identify vaccine targets for severe malaria and also indicate how parasites adapt to-or are selected by-the host environment in severe malaria.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a diverse family of multidomain proteins expressed on the surface of malaria-infected erythrocytes, is an important target of protective ...immunity against malaria. Our group recently studied transcription of the
genes encoding PfEMP1 in individuals from Papua, Indonesia, with severe or uncomplicated malaria. We cloned and expressed domains from 32 PfEMP1s, including 22 that were upregulated in severe malaria and 10 that were upregulated in uncomplicated malaria, using a wheat germ cell-free expression system. We used Luminex technology to measure IgG antibodies to these 32 domains and control proteins in 63 individuals (11 children). At presentation to hospital, levels of antibodies to PfEMP1 domains were either higher in uncomplicated malaria or were not significantly different between groups. Using principal component analysis, antibodies to 3 of 32 domains were highly discriminatory between groups. These included two domains upregulated in severe malaria, a DBLβ13 domain and a CIDRα1.6 domain (which has been previously implicated in severe malaria pathogenesis), and a DBLδ domain that was upregulated in uncomplicated malaria. Antibody to control non-PfEMP1 antigens did not differ with disease severity. Antibodies to PfEMP1 domains differ with malaria severity. Lack of antibodies to locally expressed PfEMP1 types, including both domains previously associated with severe malaria and newly identified targets, may in part explain malaria severity in Papuan adults.
Submicroscopic Plasmodium infections are an important parasite reservoir, but their clinical relevance is poorly defined. A cross-sectional household survey was conducted in southern Papua, ...Indonesia, using cluster random sampling. Data were recorded using a standardized questionnaire. Blood samples were collected for haemoglobin measurement. Plasmodium parasitaemia was determined by blood film microscopy and PCR. Between April and July 2013, 800 households and 2,830 individuals were surveyed. Peripheral parasitaemia was detected in 37.7% (968/2,567) of individuals, 36.8% (357) of whom were identified by blood film examination. Overall the prevalence of P. falciparum parasitaemia was 15.4% (396/2567) and that of P. vivax 18.3% (471/2567). In parasitaemic individuals, submicroscopic infection was significantly more likely in adults (adjusted odds ratio (AOR): 3.82 95%CI: 2.49-5.86, p<0.001) compared to children, females (AOR = 1.41 1.07-1.86, p = 0.013), individuals not sleeping under a bednet (AOR = 1.4 1.0-1.8, p = 0.035), and being afebrile (AOR = 3.2 1.49-6.93, p = 0.003). The risk of anaemia (according to WHO guidelines) was 32.8% and significantly increased in those with asymptomatic parasitaemia (AOR 2.9 95% 2.1-4.0, p = 0.007), and submicroscopic P. falciparum infections (AOR 2.5 95% 1.7-3.6, p = 0.002). Asymptomatic and submicroscopic infections in this area co-endemic for P. falciparum and P. vivax constitute two thirds of detectable parasitaemia and are associated with a high risk of anaemia. Novel public health strategies are needed to detect and eliminate these parasite reservoirs, for the benefit both of the patient and the community.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Asia-Pacific region faces formidable challenges in achieving malaria elimination by the proposed target in 2030. Molecular surveillance of Plasmodium parasites can provide important information ...on malaria transmission and adaptation, which can inform national malaria control programmes (NMCPs) in decision-making processes. In November 2019 a parasite genotyping workshop was held in Jakarta, Indonesia, to review molecular approaches for parasite surveillance and explore ways in which these tools can be integrated into public health systems and inform policy. The meeting was attended by 70 participants from 8 malaria-endemic countries and partners of the Asia Pacific Malaria Elimination Network. The participants acknowledged the utility of multiple use cases for parasite genotyping including: quantifying the prevalence of drug resistant parasites, predicting risks of treatment failure, identifying major routes and reservoirs of infection, monitoring imported malaria and its contribution to local transmission, characterizing the origins and dynamics of malaria outbreaks, and estimating the frequency of Plasmodium vivax relapses. However, the priority of each use case varies with different endemic settings. Although a one-size-fits-all approach to molecular surveillance is unlikely to be applicable across the Asia-Pacific region, consensus on the spectrum of added-value activities will help support data sharing across national boundaries. Knowledge exchange is needed to establish local expertise in different laboratory-based methodologies and bioinformatics processes. Collaborative research involving local and international teams will help maximize the impact of analytical outputs on the operational needs of NMCPs. Research is also needed to explore the cost-effectiveness of genetic epidemiology for different use cases to help to leverage funding for wide-scale implementation. Engagement between NMCPs and local researchers will be critical throughout this process.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Dihydroartemisinin-piperaquine (DHP) has been the first-line treatment of uncomplicated malaria due to both
and
infections in Papua, Indonesia, since March 2006. The efficacy of DHP was reassessed to ...determine whether there had been any decline following almost a decade of its extensive use. An open-label drug efficacy study of DHP for uncomplicated
and
malaria was carried out between March 2015 and April 2016 in Timika, Papua, Indonesia. Patients with uncomplicated malaria were administered supervised DHP tablets once daily for 3 days. Clinical and laboratory data were collected daily until parasite clearance and then weekly for 6 weeks. Molecular analysis was undertaken for all patients with recurrent parasitemia. A total of 129 study patients were enrolled in the study. At day 42, the polymerase chain reaction-adjusted efficacy was 97.7% (95% confidence intervals CI: 87.4-99.9) in the 61 patients with
malaria, and 98.2% 95% CI: 90.3-100 in the 56 patients with
malaria. By day 2, 98% (56/57) of patients with
and 96.9% (63/65) of those with
had cleared their peripheral parasitemia; none of the patients were still parasitaemic on day 3. Molecular analysis of
parasites showed that none (0/61) had K13 mutations associated previously with artemisinin resistance or increased copy number of
2-3 (0/61). In the absence of artemisinin resistance, DHP has retained high efficacy for the treatment of uncomplicated malaria despite extensive drug pressure over a 9-year period.
In Papua (Indonesia), infants with P. falciparum and/or P. vivax malaria are at risk of severe anaemia and death. We hypothesized that in an area of high malaria transmission, intermittent screening ...and treatment of infants with malaria (ISTi) will reduce morbidity compared to passive case detection (PCDi).
We conducted a cluster randomised, open label, superiority trial. A total of 21 clusters of village health posts (VHP) were randomised 1:1 to either IST for infants coinciding with 4 routine immunisation visits or PCDi. Healthy term infants born to consenting mothers enrolled into a maternal malaria cluster randomised trial were included in the study and followed for 12 months. Point of care malaria rapid diagnostic tests were used to detect peripheral parasitaemia at 2, 3, 4 and 9 months old in all infants in ISTi clusters and when symptomatic in PCDi clusters. Infants with detected peripheral parasitaemia were treated with dihydroartemisinin-piperaquine. The co-primary outcomes were the incidence rate of clinical malaria in the first year of life and the prevalence of parasitaemia at age 12 months. The incidence rate ratio and prevalence ratio between ISTi and PCDi were estimated using mixed-effects Poisson and log-binomial regression modelling (accounting for clustering at VHP level).
Between May 2014 and February 2017, 757 infants were enrolled into the study, 313 into 10 ISTi clusters, and 444 into 11 PCDi clusters. Overall, 132 episodes of parasitaemia were detected, of whom 17 (12.9%) were in symptomatic infants. Over 12 months, the incidence rate (IR) of clinical malaria was 24 95% CI, 10-50 per 1000 children-years at risk in the ISTi arm and 19 95% CI, 8,38 per 1000 children-years in the PCDi arm (adjusted incidence rate ratio aIRR 1.77 95% CI, 0.62-5.01; p = 0.280). The prevalence of parasitaemia at 12 months was 13% (33/254) in the IST clusters and 15% (57/379) in the PCD clusters (adjusted prevalence ratio (aPR) = 0.92 (95% CI, 0.70-1.21), p = 0.55). There was no difference in the risk of anaemia between treatment arms.
In high malaria transmission area outside of Africa, our study suggests that compared to PCDi, ISTi offers no significant benefit in reducing the risk of clinical malaria in infants born to women receiving effective protection from malaria during pregnancy.
ClinicalTrials.gov NCT02001428 , registered on 20 Nov 2013.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The sensitivity of rapid diagnostic tests (RDTs) for malaria is inadequate for detecting low-density, often asymptomatic infections, such as those that can occur when screening pregnant women for ...malaria. The performance of the Alere™ Ultra-sensitive Malaria Ag Plasmodium falciparum RDT (uRDT) was assessed retrospectively in pregnant women in Indonesia.
The diagnostic performance of the uRDT and the CareStart™ Malaria HRP2/pLDH VOM (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) Combo RDT (csRDT) were assessed using 270 stored red blood cell pellets and plasma samples from asymptomatic pregnant women. These included 112 P. falciparum negative and 158 P. falciparum positive samples detected by a composite test (qPCR, LAMP, nPCR) as reference standard. Diagnostic indicators: sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), diagnostic odds ratio (DOR) and the level of agreement (kappa) were calculated for comparison.
Compared with the reference test, the uRDT had a sensitivity of 19.6% (95% CI 13.9-26.8) and specificity of 98.2% (93.1-99.7%). The csRDT was 22.8% (16.7-30.3) sensitive and 95.5% (89.4-98.3) specific for P. falciparum infections. Performance of the uRDT was non-significantly different to the csRDT (p = 0.169). RDT outcome was stratified by qPCR cycling threshold (Ct), and performance of the RDTs was found to be comparable across parasite loads.
The uRDT performed similarly to the currently used csRDTs in detecting P. falciparum infections in asymptomatic pregnant women. In these settings, molecular diagnostics are currently the most sensitive for malaria.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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