Estimation of parasite age and synchrony status in Plasmodium falciparum infections

Human malaria parasites have complex but poorly understood population dynamics inside their human host. In some but not all infections, parasites progress synchronously through the 48 h lifecycle following erythrocyte invasion, such that at any one time there is a limited spread of parasites at a particular time (hours) post-invasion. Patients presenting with older parasites, and with asynchronous infections, have been reported to have higher risks of fatal outcomes, associated with higher parasite biomass and multiplication rates respectively. However, practical tools to assess synchrony and estimate parasite age post-invasion in patient samples are lacking. We have developed a novel method based on three genes differentially expressed over the parasite intra-erythrocytic lifecycle, and applied it to samples from patients with uncomplicated malaria attending two health clinics in Ghana. We found that most patients presented with synchronous infections, and with parasites within 12 h of erythrocyte invasion. Finally we investigated if clinical features such as fever and parasite density could act as predictors of parasite age and synchrony. The new method is a simple and practicable approach to study parasite dynamics in naturally-infected patients, and is a significant improvement on the subjective microscopical methods for parasite staging in vivo, aiding patient management

An improved and highly sensitive microfluorimetric method for assessing susceptibility of Plasmodium falciparum to antimalarial drugs in vitro

BACKGROUND: The standard in vitro protocol currently in use for drug testing against Plasmodium falciparum, based on the incorporation of the purine [(3)H]-hypoxanthine, has two serious drawbacks. Firstly it is unsuitable for the testing of drugs that directly or indirectly impact on purine salvage or metabolism. Secondly, it relies on the use of expensive radiolabelled material, with added issues concerning detection, storage and waste disposal that make it unsuitable for use in many disease-endemic areas. Recently, the use of fluorochromes has been suggested as an alternative, but quenching of the fluorescence signal by the haemoglobin present in cultures of Plasmodium falciparum-infected erythrocytes severely limits the usefulness of this approach. METHODS: In order to resolve this problem, a new PicoGreen(®)-based procedure has been developed which incorporates additional steps to remove the interfering haemoglobin. The 50% inhibitory concentration (IC(50)) values of chloroquine and pyrimethamine against P. falciparum laboratory lines 3D7 and K1 were determined using the new protocol. RESULTS: The IC(50 )values of chloroquine and pyrimethamine against P. falciparum laboratory lines 3D7 and K1 determined with the new fluorescence-based protocol were statistically identical to those obtained using the traditional (3)H-hypoxanthine incorporation method, and consistent with literature values. CONCLUSION: The new method proved to be accurate, reproducible and sensitive, and has the advantage of being non-radioactive. The improved PicoGreen(® )method has the potential to replace traditional in vitro drug resistance assay techniques

Copy number variation of plasmepsins 2 and 3 genes in Plasmodium falciparum isolates and implication for dihydroartemisinin-piperaquine resistance in Ghana

Background: In 2008, dihydroartemisinin-piperaquine and artemether-lumefantrine were introduced to supplement artesunate-amodiaquine for the treatment of uncomplicated malaria in Ghana. Drug pressure over the years enhances the development of parasite resistance to drugs. The World Health Organization recommends the detection of copy number variations of plasmepsins 2 (PfPm2) and plasmepsins 3 (PfPm3) genes linked to dihydroartemisinin-piperaquine resistance in treatment efficacy studies.Objective: This study investigated the copy number variations of PfPm2 and PfPm3 genes in the malaria parasite population in Ghana.Methods: Overall, 313 blood samples from children ≤ 9 years presenting with uncomplicated malaria at three sentinel sites used for monitoring antimalarial drug efficacy and resistance in Ghana were used for genetic investigations. The samples were collected in the malaria transmission seasons of 2015 and 2016. Malaria parasite DNA extraction from the blood samples followed by real-time quantitative PCR was used to determine the copy number of the PfPm2 and PfPm3 genes. The gene copy number was calculated by the relative expression formula 2-ΔΔCt for quantification, where ΔΔ is the relative delta-delta, and Ct is the cycle threshold. ΔΔCt was calculated as (Ctβ-tubulin − Ctpfpm2/3) - (Ctβ-tubulin cal − Ctpfpm2/3 cal), where cal is the calibration control of genomic 3D7 DNA with one copy of both the β-tubulin endogenous control and pfpm2 and pfpm3. A change in Ct (ΔCt = Ct PfPm2/3 - Ct Pfβ-tubulin) where is the difference in Ct values for the target gene of interest PfPm2 and PfPm3 and the reference gene Pfβ-tubulin. Statistical significance was defined as p < 0.05.Results: Of the parasites analyzed, 79.2% (n = 228/288) and 80.5% (n = 227/282) had one gene copy for PfPm2 and PfPm3, respectively. For PfPm2, 14.9% (n = 43/288), 3.8% (n = 11/288), and 2.1% (n = 6/288) of the isolates had copy numbers 2, 3 and 4 respectively. For PfPm3, gene copies of 2, 3 and 4 were observed in 16.3% (n = 46/282), 2.1% (n = 6/282), and 1.1% (n = 3/282) of isolates. Analysis of the copy number variation across the three study sites in Cape-Coast, Begoro, and the Navrongo areas showed no significant difference for PfPm2 (p = 0.93) and PfPm3 (p = 0.94) genes.Conclusion: After over a decade of the use of dihydroartemisinin-piperaquine, the mutations associated with resistance to the drug have been observed in Ghanaian P. falciparum isolates. This serves as baseline data for further monitoring of this molecular marker extensively as part of ongoing surveillance of antimalarial drug efficacy studies in Ghana

Mutations in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance genes, and treatment outcomes in Ghanaian children with uncomplicated malaria.

The association between the clinical outcome of chloroquine treatment and mutations in the putative Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene at codon 76 and multidrug resistance gene 1 (Pf mdr1) at codon 86 were investigated among 406 children with uncomplicated malaria presenting at five sentinel health centres in Ghana. Presence of mutations in isolates taken at pre-treatment and on day of recurrence of parasites was detected using PCR followed by RFLP techniques. The prevalence of Pfcrt T76 mutants was 80% at Hohoe, 46% at Navrongo, 98% at Tarkwa, 61% at Sunyani and 46% at Yendi. The prevalence of the mutant Pfmdr1 at Hohoe, Navrongo, Tarkwa, Sunyani and Yendi were 78, 58, 95, 53 and 42%, respectively. Significant association between the Pfcrt mutation and treatment outcome was observed at Hohoe and Sunyani (p 0.05). Similarly, a statistical significant association between Pfmdr1 86 and treatment failures was observed at Hohoe and Sunyani (p < 0.05) but not at the other three sites. A positive correlation was found between mutant Pfcrt prevalence only and treatment failures with a Spearman's rho-value of 0.872 and a p-value = 0.027. All parasite isolates from samples taken at recrudescence from patients with chloroquine treatment failures were found to have both Pfcrt and Pfmdr mutations

Towards antimalarial drug discovery: purine transporters in Plasmodium falciparum as a possible drug target

No abstract available