Molecular Markers for Sensitive Detection of Plasmodium falciparum Asexual Stage Parasites and their Application in a Malaria Clinical Trial

Abstract

Abstract.Plasmodium falciparum parasite life stages respond differently to antimalarial drugs. Sensitive stage-specific molecular assays may help to examine parasite dynamics at microscopically detectable and submicroscopic parasite densities in epidemiological and clinical studies. In this study, we compared the performance of skeleton-binding protein 1 (SBP1), ring-infected erythrocyte surface antigen, Hyp8, ring-exported protein 1 (REX1), and PHISTb mRNA for detecting ring-stage trophozoite-specific transcripts using quantitative reverse transcriptase polymerase chain reaction. Markers were tested on tightly synchronized in vitro parasites and clinical trial samples alongside established markers of parasite density (18S DNA and rRNA) and gametocyte density (Pfs25 mRNA). SBP1 was the most sensitive marker but showed low-level expression in mature gametocytes. Novel markers REX1 and PHISTb showed lower sensitivity but higher specificity for ring-stage trophozoites. Using in vivo clinical trial samples from gametocyte-negative patients, we observed evidence of persisting trophozoite transcripts for at least 14 days postinitiation of treatment. It is currently not clear if these transcripts represent viable parasites that may have implications for clinical treatment outcome or transmission potential.

P. falciparum gametocyte density and infectivity in peripheral blood and skin tissue of naturally infected parasite carriers

“…The blood content of individually fed mosquitoes was released into an RNA preservative 15 minutes after starting the feeding; RNA was then extracted and quantified from pools of 4 mosquitoes. We quantified asexual parasites by skeleton-binding protein 1 sbp1 qRT-PCR (31) and gametocytes ( Pfs25 and Pfmget qRT-PCR) in a median of 3 mosquito pools per participant, each containing 4 individual mosquitoes, from skin-feeding (range=2-3) and 4 pools per participant, each containing 4 individual mosquitoes, from membrane feeding (range=2-4). We observed strong correlations between parasite quantities in pools of mosquitoes that fed on skin or venous blood through artificial membranes for asexual ring-stage parasites (r=0.921, p<0.0001), male (r=0.790, p<0.0001) and female gametocytes (r=0.655, p=0.0001) (Figure 1B).…”

“…Nucleic acids from these 200μL mosquito pools and from 100μL venous and finger prick whole blood samples in RNAprotect Cell Reagent were isolated using the bead-based MagNAPure LC automatic extractor (Total Nucleic Acid Isolation Kit—High Performance, Roche Applied Science) and eluted in 50μL of water. In these samples, ring-stage asexual parasites, female gametocytes and male gametocytes were quantified by individual quantitative reverse-transcription PCR (qRT-PCR) assays targeting sbp1 (31); Pfs25 (45) and PfMGET (29), respectively. Skin biopsy samples were immediately stored in RNAlater solution after collection.…”

Persistence of P. Falciparum Ring-Stage Parasites 42 Days After Artemisinin and Non-Artemisinin Combination Therapy in Naturally Infected Malians

“…Ring-stage parasites were quanti ed by qRT-PCR targeting the sbp1 mRNA transcript using previously described methods (6). Male and female gametocyte densities were quanti ed by qRT-PCR, targeting male PfMGET and female Pfs25 mRNA transcripts as described elsewhere (14).…”

“…Post-treatment detection of parasite DNA may re ect both (remnants of) asexual parasites and gametocytes (6,7), the latter commonly persisting after ACT treatment (8). A study in travelers in Sweden (9) indicated that residual parasite DNA can be detected by qPCR for up to 42 days after successful treatment without evidence of viable asexual parasites or gametocytes.…”

“…A study in travelers in Sweden (9) indicated that residual parasite DNA can be detected by qPCR for up to 42 days after successful treatment without evidence of viable asexual parasites or gametocytes. Recently, mRNA transcripts speci c to ring-stage parasites (skeleton binding protein; sbp1) were reported following ACT treatment (6,10). This persistence of low-level asexual parasitemia after ACTs may be explained by the "dormancy theory" (11) which postulates that under artemisinin pressure, a subpopulation of young ring-stage parasites undergo developmental arrest where they remain metabolically inactive.…”