Estimating the sequestered load in "Plasmodium falciparum" malaria

Abstract

Plasmodium falciparum, the most highly virulent malaria parasite, still remains a big threat despite years of research and great advances in the field of science and technology. The particular virulence of this parasite is believed to be due to the ability of trophozoite and schizont stages of infected red blood cells to adhere to endothelial vasculature in various organs. The sequestration of these late stages of the parasite, which makes them difficult to detect in peripheral blood smears, is an important factor limiting performance of microscopy, the most widely used method for diagnosing malaria in endemic areas. A reliable technique for estimating the burden of sequestered parasites would also be an invaluable tool for investigations of pathogenesis and epidemiological field studies. The objective of this study was to develop methods for detecting sequestered loads and correlate the results obtained using these methods with estimates of sequestered loads obtained from a statistical model. Quantification of either cytoadherence or schizogony using parasite density or biochemical (host or parasite) markers could resolve current problems in estimating the total parasite burden in malaria patients. The current status of malaria diagnosis was addressed by reviewing the performance of the following available diagnostic methods: standard optical microscopy; histidine rich protein 2 (HRP2); parasite lactate dehydrogenase (pLDH); acridine orange; quantitative buffy coat. These methods are not 100% sensitive and are not reliable for detecting low density malaria infections. Biochemical assays were established for the detection of the stage specific release of parasite biochemical markers in vitro: HRP2; pLDH and Dlactate. These assays together with an assay detecting the host marker soluble tumour necrosis factor (sTNF-R75) were then analysed in a small group of 30 month old children to establish the performance of these assays in measuring markers in vivo. The disease severity was evaluated using the quantitative HRP2 method developed in 366 children aged 12 to 59 months. These children had different symptoms: 98 severe malaria, 92 mild malaria, 76 asymptomatic parasitaemia and 100 with no malaria. Having determined suitable candidate markers for determination of sequestered loads, we then evaluated a series of host markers: sTNF-R75, circulating host DNA, packed cell volume (PCV) and parasite markers: HRP2, pLDH, pigmented polymorphonucleated cells (PMNs) and pigmented monocytes, circulating parasite DNA (pDNA) in a series of 22 patients with severe Plasmodium falciparum malaria treated with quinine. We closely monitored the peripheral parasite density and used these to obtain estimates of sequestered loads from an established statistical model that inco-oporates the effects of quinine. The main findings were: i. HRP2 dipsticks perform better than microscopy in detecting P falciparum infections in clinical cases in endemic areas, while pLDH dipsticks are suitable for monitoring response to treatment. ii. P. falciparum releases stage specific products into culture supernatant with pLDH enzyme activity measured after schizont rupture, while HRP2 is released at trophozoite and during schizont rupture and the amounts at this later stage are greatly increased. Both these biochemical markers are good candidate markers of sequestration though their release is altered by antimalarial administration. Dlactate is released by all stages of the parasite life cycle. iii. The methods developed for HRP2, pLDH and a commercially available method that measures a host molecule sTNF-R75 are suitable for distinguishing children with severe and mild malaria from healthy controls with or without P. falciparum. iv. The quantitative HRP2 method together with peripheral parasite densities could provide a better way of determining malaria disease severity. v. On admission of young children the markers that appeared to be most useful as predictors of sequestered loads were peripheral parasite densities, circulating pDNA and sTNF-R75. However, these and all the other markers did not appear to be good indicators of changes in sequestered load during the course of a clinical episode. Though we were unable to estimate the sequestered load using host or parasite biochemical markers in partially immune patients, we were able to exclud some candidates: D-lactate; pigmented PMNs, pigmented monocytes, PCV and circulating host DNA. Though sTNF-R75 showed some correlation with sequestered loads its release is affected by host factors such as genetics and age making it unreliable as a marker of sequestration. The parasite markers HRP2, pLDH and circulating pDNA should be further evaluated as potential markers of sequestration. Analyses should consider the effects of antimalarials on release of these markers, as well as the kinetics of their release and clearance in vivo.