Estimating the sequestered load in "Plasmodium falciparum" malaria

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.