Declining Artesunate-Mefloquine Efficacy against Falciparum Malaria on the Cambodia–Thailand Border

Emerging resistance in Southeast Asia raises concern over possible spread or similar evolution of resistance to other artemisinin-based combination therapies in Africa

Key Knowledge Gaps for Plasmodium vivax Control and Elimination

There is inadequate understanding of the biology, pathology, transmission, and control of Plasmodium vivax, the geographically most widespread cause of human malaria. During the last decades, study of this species was neglected, in part due to the erroneous belief that it is intrinsically benign. In addition, many technical challenges in culturing the parasite also hampered understanding its fundamental biology and molecular and cellular responses to chemotherapeutics. Research on vivax malaria needs to be substantially expanded over the next decade to accelerate its elimination and eradication. This article summarizes key knowledge gaps identified by researchers, national malaria control programs, and other stakeholders assembled by the World Health Organization to develop strategies for controlling and eliminating vivax malaria. The priorities presented in this article emerged in these technical discussions, and were adopted by expert consensus of the authors. All involved understood the priority placed upon pragmatism in this research agenda, that is, focus upon tools delivering better prevention, diagnosis, treatment, and surveillance of P. vivax

pfmdr1 GENOTYPING AND IN VIVO MEFLOQUINE RESISTANCE ON THE THAI-MYANMAR BORDER

Molecular markers have been proposed as a method of monitoring malaria drug resistance and could potentially be used to prolong the life span of antimalarial drugs. Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum gene pfmdr1 and increased gene copy number have been associated with in vitro drug resistance but have not been well studied in vivo. In a prospective cohort study of malaria patients receiving mefloquine treatment on the Thai-Myanmar border, there was no significant association between either pfmdr1 SNPs or in vitro drug sensitivity and mefloquine resistance in vivo. Increased pfmdr1 gene copy number was significantly associated with recrudescence (relative risk 2.30, 95% CI 1.27–4.15). pfmdr1 gene copy number may be a useful surveillance tool for mefloquine-resistant falciparum malaria in Thailand

HEMATOLOGIC AND CLINICAL INDICES OF MALARIA IN A SEMI-IMMUNE POPULATION OF WESTERN THAILAND

This study examines hematologic profiles of persons with acute Plasmodium falciparum or P. vivax infection in Maesod on Thailand’s western border with Myanmar compared with febrile, non-parasitemic persons also reporting to malaria clinics. Nine hundred seventy-nine subjects were malaria-negative, 414 were infected with P. falciparum, and 646 were infected with P. vivax. Persons with patent parasitemia tended to have significantly lower white blood cell, red blood cell, platelet, and hemoglobin levels than those who were malaria-negative. For the first time, a parallel trend in thrombocytopenia with parasitemia was found to be associated with both P. falciparum, and P. vivax infection. Using logistic regression, persons with platelet counts < 150,000/µL were 12−15 times more likely to have malaria than persons with platelet counts ≥ 150,000/µL. This study supplements previous literature on the hematologic effects of malaria and helps define those alterations for a semi-immune population. Thrombocytopenia is identified as a key indicator of malaria in these febrile patients

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 (which is related to both mefloquine and artesunate decreased susceptibility) is not changing over time. The presence of parasitaemia on day 2 is a poor indicator of AM 28-day treatment failure

MEASURING ALLELIC HETEROGENEITY IN PLASMODIUM FALCIPARUM BY A HETERODUPLEX TRACKING ASSAY

We developed a novel Plasmodium falciparum genotyping strategy based on the heteroduplex tracking assay (HTA) method commonly used to genotype viruses. Because it can detect both sequence and size polymorphisms, we hypothesized that HTA is more sensitive than current methods. To test this hypothesis, we compared the ability of HTA and a nested polymerase chain reaction (PCR) to detect genetic diversity in 17 Thai samples. The HTA detected more MSP1 sequence variants in eight isolates (47%), less sequence variants in three isolates (18%), and an equal number of sequence variants in six isolates (35%), suggesting that HTA is equal to or more sensitive than the nested PCR. This study is a proof of concept that HTA is a sensitive allelic discrimination method able to determine genetic diversity in P. falciparum and warrants its use in studies of antimalarial drug efficacy

Molecular Surveillance for Multidrug-Resistant Plasmodium falciparum, Cambodia

We conducted surveillance for multidrug-resistant Plasmodium falciparum in Cambodia during 2004–2006 by assessing molecular changes in pfmdr1. The high prevalence of isolates with multiple pfmdr1 copies found in western Cambodia near the Thai border, where artesunate–mefloquine therapy failures occur, contrasts with isolates from eastern Cambodia, where this combination therapy remains highly effective

PFMDR1 AND IN VIVO RESISTANCE TO ARTESUNATE-MEFLOQUINE IN FALCIPARUM MALARIA ON THE CAMBODIAN–THAI BORDER

Artemisinin combination therapies (ACTs) have recently been adopted as first-line therapy for Plasmodium falciparum infections in most malaria-endemic countries. In this study, we estimated the association between artesunate-mefloquine therapy failure and genetic changes in the putative transporter, pfmdr1. Blood samples were acquired from 80 patients enrolled in an 2004 in vivo efficacy study in Pailin, Cambodia, and genotyped for pfmdr1 copy number and haplotype. Having parasites with three or more copies of pfmdr1 before treatment was strongly associated with recrudescence (hazard ratio [HR] = 8.30; 95% CI: 2.60–26.43). This relationship was maintained when controlling for initial parasite density and hematocrit (HR = 7.91; 95% CI: 2.38–26.29). Artesunate-mefloquine treatment selected for increased pfmdr1 copy number, because isolates from recurrent episodes had higher copy numbers than the paired enrollment samples (Wilcoxon rank test, P = 0.040). pfmdr1 copy number should be evaluated further as a surveillance tool for artesunate-mefloquine resistance in Cambodia

Misclassification of Drug Failures in Plasmodium falciparum Clinical Trials in Southeast Asia

Most trials of antimalarials occur in areas where reinfections are possible. For Plasmodium falciparum, reinfections are distinguished from recrudescences by PCR analysis of 3 polymorphic genes. However, the validity of this approach has never been rigorously tested. We tested for misclassification in 6 patients from clinical trials in Thailand and Cambodia who were classified as reinfected by the standard PCR protocol. Using heteroduplex tracking assays and direct DNA sequencing, we found that 5 of 6 (83%) patients were misclassified. Misclassification in this manner overestimates the efficacy of antimalarials and delays recognition of decreasing therapeutic efficacy, thus delaying potential policy changes

Production and validation of durable, high quality standardized malaria microscopy slides for teaching, testing and quality assurance during an era of declining diagnostic proficiency

Background: Sets of Giemsa-stained, blood smear slides with systematically verified composite diagnoses would contribute substantially to development of externally validated quality assurance systems for the microscopic diagnosis of malaria. Methods: whole blood from Plasmodium-positive donors in Cambodia and Indonesia and individuals with no history of risk for malaria was collected. Using standard operating procedures, technicians prepared Giemsastained thick and thin smears from each donor. One slide from each of the first 35 donations was distributed to each of 28 individuals acknowledged by reputation as having expertise in the microscopic diagnosis of malaria. These reference readers recorded presence or absence of Plasmodium species and parasite density. A composite diagnosis for each donation was determined based on microscopic findings and species-specific small subunit ribosomal RNA (ssrRNA) DNA polymerase chain reaction (PCR) amplification. Results: More than 12, 000 slides were generated from 124 donations. Reference readers correctly identified presence of parasites on 85% of slides with densities \u3c100 parasites/μl, which improved to 100% for densities \u3e350 parasites/μl. Percentages of agreement with composite diagnoses were highest for Plasmodium falciparum (99%), followed by Plasmodium vivax (86%). Conclusion: Herein, a standardized method for producing large numbers of consistently high quality, durable Giemsa-stained blood smears and validating composite diagnoses for the purpose of creating a malaria slide repository in support of initiatives to improve training and competency assessment amidst a background of variability in diagnosis is described