A new method for detection of pfmdr1 mutations in Plasmodium falciparum DNA using real-time PCR

Background

The erythrocyte binding antigen-175 (EBA-175) on Plasmodium falciparum merozoites mediates sialic acid dependent binding to glycophorin A on host erythrocytes and, therefore, plays a crucial role in cell invasion. Dimorphic allele segments have been found in its encoding gene with a 342 bp segment present in FCR-3 strains (F-segment) and a 423 bp segment in CAMP strains (C-segment). Possible associations of the dimorphism with severe malaria have been analysed in a case-control study in northern Ghana.

Methods

Blood samples of 289 children with severe malaria and 289 matched parasitaemic but asymptomatic controls were screened for eba- 175 F- and C-segments by nested polymerase chain reaction.

Results

In children with severe malaria, prevalences of F-, C- and mixed F-/C-segments were 70%, 19%, and 11%, respectively. The C-segment was found more frequently in severe malaria cases whereas mixed infections were more common in controls. Infection with strains harbouring the C-segment significantly increased the risk of fatal outcome.

Conclusion

The results show that the C-segment is associated with fatal outcome in children with severe malaria in northern Ghana, suggesting that it may contribute to the virulence of the parasite.     

Origin and Evolution of Sulfadoxine Resistant Plasmodium falciparum

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.     

Ensuring quality and access for malaria diagnosis: how can it be achieved?

The replacement of conventional antimalarial drugs with high-cost, artemisinin-based alternatives has created a gap in the successful management of malaria. This gap reflects an increased need for accurate disease diagnosis that cannot be met by traditional microscopy techniques. The recent introduction of rapid diagnostic tests (RDTs) has the potential to meet this need, but successful RDT implementation has been curtailed by poor product performance, inadequate methods to determine the quality of products and a lack of emphasis and capacity to deal with these issues. Economics and a desire for improved case management will result in the rapid growth of RDT use in the coming years. However, for their potential to be realized, it is crucial that high-quality RDT products that perform reliably and accurately under field conditions are made available. In achieving this goal, the shift from symptom-based diagnosis to parasite-based management of malaria can bring significant improvements to tropical fever management, rather than represent a further burden on poor, malaria-endemic populations and their overstretched health services.     

Malaria drug-sensitivity testing: new assays,new perspectives

Over the past five decades, the drug resistance of Plasmodium falciparum has become an issue of utmost concern. At the same time, in vitro assays for assessing antimalarial drug sensitivity have become indispensable tools for the surveillance of drug resistance and the planning of therapeutic guidelines. Several new in vitro assays have been introduced, designed to be easier to handle than previous tests and allow a faster identification of drug-resistant parasites, as well as for simple evaluation of new drugs. This review examines the various new approaches to the in vitro assessment of malaria drug sensitivity and their limitations.     

Gametocytemia in Plasmodium vivax and Plasmodium falciparum infections

Two expert research microscopists, each blinded to the other's reports, diagnosed single-species malaria infections in 2,141 adults presenting at outpatient malaria clinics in Tak Province, Thailand, and Iquitos, Peru, in May-August 1998, May-July 1999, and May-June 2001. Plasmodium vivax patients with gametocytemia had higher fever and higher parasitemia than those without gametocytemia; temperature correlated with parasitemia in the patients with gametocytemia. Plasmodium falciparum patients with gametocytemia had lower fever than those without gametocytemia, but similar parasitemia; temperature correlated with parasitemia in the patients without gametocytemia. Hematologic data in Thailand in 2001 showed lower platelet counts in P. vivax patients with gametocytemia than in the P. vivax patients without gametocytemia, whereas P. falciparum patients with gametocytemia had similar platelet counts but lower red blood cell counts, hemoglobin levels, hematocrit levels, and higher lymphocyte counts than patients without gametocytemia.     

Rapid diagnostic techniques for malaria control.

The past decade was a milestone in the development of malaria diagnostic technology. Today, a variety of simplified and rapid malaria diagnostic devices, collectively referred to as 'dipsticks', is available. This paper discusses the potential roles of these devices, and obstacles to their use, in supporting malaria control strategies.     

Unusual plasmodium malariae-like parasites in southeast Asia

During malaria surveys in Myanmar, 2 peculiar forms of Plasmodium malariae-like parasites were found. The morphologies of their early trophozoite stages were distinct from that of the typical P. malariae, resembling instead that of Plasmodium vivax, var. minuta, reported by Emin, and Plasmodium tenue, reported by Stephens, both in 1914. Two polymerase chain reaction (PCR)-based diagnoses, which target the same regions in the small subunit ribosomal RNA (SSUrRNA) genes, indicated that these parasites were new variant forms of P. malariae and that they could be separated into 2 genetic types that correlated with the 2 morphological types. Sequence analysis of the SSUrRNA and the circumsporozoite protein genes revealed that they were distinct both from each other and from other known P. malariae isolates and that the P. tenue-like type was closer to a monkey quartan malaria parasite, Plasmodium brasilianum. These results illustrate that the microscopic appearance of human P. malariae parasites may be more varied than previously assumed and suggest the value of molecular tools in the evaluation of malaria morphological variants.     

Delayed Plasmodium falciparum clearance following artesunate-mefloquine combination therapy in Thailand, 1997-2007

ABSTRACT: BACKGROUND: There is concern that artesunate resistance is developing in Southeast Asia. The purpose of this study is to investigate the prevalence of parasitaemia in the few days following treatment with artesunate-mefloquine (AM), which is an indirect measure of decreased artesunate susceptibility. METHODS: This is a retrospective analysis of 31 therapeutic efficacy studies involving 1,327 patients treated with AM conducted by the Thai National Malaria Control Programme from 1997- 2007. RESULTS: The prevalence of patients with parasitaemia on day 2 was higher in the east compared to the west (east: 20 %, west: 9 %, OR 2.47, 95 % CI: 1.77, 3.45). In addition, the prevalence of day-2 parasitaemia increased over time (OR for each year = 1.10, 95 % CI: 1.03, 1.19). After controlling for initial parasitaemia and age, year and region remained important determinants of day-2 parasitaemia (OR for region = 3.98, 95%CI 2.63, 6.00; OR for year = 1.28, 95%CI: 1.17, 1.39). The presence of parasitaemia on day 2 and day 3 were specific, but not sensitive predictors of treatment failure. DISCUSSION: Delayed resolution of parasitaemia after AM treatment increased in eastern Thailand between 1997 and 2007, which may be an early manifestation of decreased artesunate susceptibility. However, clinical and parasitological treatment failure after 28 days is not changing over time (which is related to both mefloquine and artesunate decreased susceptibility). The presence of parasitaemia on day 2 is a poor indicator of AM 28-day treatment failure.