Malaria situation in the Greater Mekong Subregion.

The epidemiology of malaria in the Greater Mekong Subregion is complex and rapidly evolving. Malaria control and elimination efforts face a daunting array of challenges including multidrug-resistant parasites. This review presents secondary data collected by the national malaria control programs in the six countries between 1998 and 2010 and examines trends over the last decade. This data has a number of limitations: it is derived exclusively from public sector health facilities; falciparum-specific and then pan-specific rapid diagnostic tests were introduced during the period under review; and, recently there has been a massive increase in case detection capability as a result of increased funding. It therefore requires cautious interpretation. A series of maps are presented showing trends in incidence, mortality and proportion of cases caused by Plasmodium falciparum over the last decade. A brief overview of institutional and implementation arrangements, historical background, demographics and key issues affecting malaria epidemiology is provided for each country. National malaria statistics for 2010 are presented and their robustness discussed in terms of the public sector's share of cases and other influencing factors such as inter-country variations in risk stratification, changes in diagnostic approach and immigration. Targets are presented for malaria control and where appropriate for elimination. Each country's artemisinin resistance status is described. The epidemiological trends presented reflect the improvement in the malaria situation, however the true malaria burden is as yet unknown. There is a need for continuing strengthening and updating of surveillance and response systems.

Malaria control in the Greater Mekong Subregion: an overview of the current response and its limitations.

The malaria burden in the Greater Mekong Subregion has been dramatically reduced over the last 20 years but the disease remains an important public health issue in all six countries. This chapter introduces the standard tools for malaria control (long lasting insecticidal nets; indoor residual spraying; early diagnosis and appropriate treatment; epidemic surveillance and response; and, communication) and presents the evidence base supporting the use of each of these tools in the Subregion. Targeting approaches and delivery mechanisms for these tools are presented and discussed country by country. The technical limitations of these standard tools and delivery mechanisms are then discussed in the context of local variations in the epidemiology of the disease. The challenges presented by the feeding and resting habits of local vectors, by the characteristics and behavior of different human population groups, and by particular species and drug resistant strains of malaria parasites are considered. A range of innovative tools and delivery mechanism that have been developed to address these problems are presented and moves to bring these various innovations together to provide a comprehensive package of malaria control services for each risk group are discussed. Implementation arrangements are introduced and an overview of the stakeholder landscape at regional and country level is provided. Finally, remaining programmatic gaps (which include limited coverage, declining funds, drug resistance, weak surveillance and weak health systems) are highlighted and areas in need of further action (including the need for continued innovation) are discussed.

Sub-microscopic malaria cases and mixed malaria infection in a remote area of high malaria endemicity in Rattanakiri province, Cambodia: implication for malaria elimination.

BACKGROUND: Malaria microscopy and rapid diagnostic tests are insensitive for very low-density parasitaemia. This insensitivity may lead to missed asymptomatic sub-microscopic parasitaemia, a potential reservoir for infection. Similarly, mixed infections and interactions between Plasmodium species may be missed. The objectives were first to develop a rapid and sensitive PCR-based diagnostic method to detect low parasitaemia and mixed infections, and then to investigate the epidemiological importance of sub-microscopic and mixed infections in Rattanakiri Province, Cambodia. METHODS: A new malaria diagnostic method, using restriction fragment length polymorphism analysis of the cytochrome b genes of the four human Plasmodium species and denaturing high performance liquid chromatography, has been developed. The results of this RFLP-dHPLC method have been compared to 1) traditional nested PCR amplification of the 18S rRNA gene, 2) sequencing of the amplified fragments of the cytochrome b gene and 3) microscopy. Blood spots on filter paper and Giemsa-stained blood thick smears collected in 2001 from 1,356 inhabitants of eight villages of Rattanakiri Province have been analysed by the RFLP-dHPLC method and microscopy to assess the prevalence of sub-microscopic and mixed infections. RESULTS: The sensitivity and specificity of the new RFLP-dHPLC was similar to that of the other molecular methods. The RFLP-dHPLC method was more sensitive and specific than microscopy, particularly for detecting low-level parasitaemia and mixed infections. In Rattanakiri Province, the prevalences of Plasmodium falciparum and Plasmodium vivax were approximately two-fold and three-fold higher, respectively, by RFLP-dHPLC (59% and 15%, respectively) than by microscopy (28% and 5%, respectively). In addition, Plasmodium ovale and Plasmodium malariae were never detected by microscopy, while they were detected by RFLP-dHPLC, in 11.2% and 1.3% of the blood samples, respectively. Moreover, the proportion of mixed infections detected by RFLP-dHPLC was higher (23%) than with microscopy (8%). CONCLUSIONS: The rapid and sensitive molecular diagnosis method developed here could be considered for mass screening and ACT treatment of inhabitants of low-endemicity areas of Southeast Asia.

Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers.

BACKGROUND: Several strategies are currently deployed in many countries in the tropics to strengthen malaria control toward malaria elimination. To measure the impact of any intervention, there is a need to detect malaria properly. Mostly, decisions still rely on microscopy diagnosis. But sensitive diagnosis tools enabling to deal with a large number of samples are needed. The molecular detection approach offers a much higher sensitivity, and the flexibility to be automated and upgraded. METHODS: Two new molecular methods were developed: dot18S, a Plasmodium-specific nested PCR based on the 18S rRNA gene followed by dot-blot detection of species by using species-specific probes and CYTB, a Plasmodium-specific nested PCR based on cytochrome b gene followed by species detection using SNP analysis. The results were compared to those obtained with microscopic examination and the "standard" 18S rRNA gene based nested PCR using species specific primers. 337 samples were diagnosed. RESULTS: Compared to the microscopy the three molecular methods were more sensitive, greatly increasing the estimated prevalence of Plasmodium infection, including P. malariae and P. ovale. A high rate of mixed infections was uncovered with about one third of the villagers infected with more than one malaria parasite species. Dot18S and CYTB sensitivity outranged the "standard" nested PCR method, CYTB being the most sensitive. As a consequence, compared to the "standard" nested PCR method for the detection of Plasmodium spp., the sensitivity of dot18S and CYTB was respectively 95.3% and 97.3%. Consistent detection of Plasmodium spp. by the three molecular methods was obtained for 83% of tested isolates. Contradictory results were mostly related to detection of Plasmodium malariae and Plasmodium ovale in mixed infections, due to an "all-or-none" detection effect at low-level parasitaemia. CONCLUSION: A large reservoir of asymptomatic infections was uncovered using the molecular methods. Dot18S and CYTB, the new methods reported herein are highly sensitive, allow parasite DNA extraction as well as genus- and species-specific diagnosis of several hundreds of samples, and are amenable to high-throughput scaling up for larger sample sizes. Such methods provide novel information on malaria prevalence and epidemiology and are suited for active malaria detection. The usefulness of such sensitive malaria diagnosis tools, especially in low endemic areas where eradication plans are now on-going, is discussed in this paper.

Let's 'cut to the chase' on malaria elimination in the Greater Mekong Subregion.

The Greater Mekong Subregion has made remarkable progress towards eliminating malaria in recent years, but efforts are now faltering in some areas. The development of tools to control forest-based transmission is taking too long and efforts to control malaria among high-risk mobile people are failing. If countries are to meet their elimination targets and prevent the spread of multidrug-resistant falciparum malaria, urgent and radical changes will be required. This commentary proposes changes in the approach to the development and roll-out of new tools as well as changes to the management of elimination efforts targeting transmission in forests and forest-farms.

Large sequence heterogeneity of the small subunit ribosomal RNA gene of Plasmodium ovale in cambodia.

Plasmodium ovale malaria has been reported in various countries in southeast Asia, but never in Cambodia. Using a species-specific polymerase chain reaction (PCR) targeting the small subunit (SSU) ribosomal RNA (rRNA) gene, we detected P. ovale in nearly 4% of the inhabitants of a northeastern Cambodian village. Plasmodium ovale was associated with at least one other Plasmodium species, and two quadruple infections were detected. The diagnosis was confirmed by microscopy and by SSU rRNA PCR product sequencing. The sequences shared 96-99% identity with published sequences, and displayed a substantial heterogeneity with 2-4 different haplotypes per sample. Nine distinct SSU rRNA haplotypes were identified, including seven novel variants. Phylogenetic analysis showed two major genetic clusters, suggesting amplification of two distinct gene sets and/or P. ovale variants from each sample. Our data indicate that P. ovale was overlooked in Cambodia until now, and call for the implementation of larger prevalence surveys and accurate diagnosis methods in this country.

Efficacy and safety of dihydroartemisinin-piperaquine (Artekin) in Cambodian children and adults with uncomplicated falciparum malaria.

The safety and efficacy of a novel combination of dihydroartemisinin (DHA) and piperaquine, Artekin (Holleykin Pharmaceuticals), were assessed in 106 patients (76 children and 30 adults) with uncomplicated falciparum malaria from 2 remote areas in Cambodia. Age-based doses were given at 0, 8, 24, and 32 h. Mean total DHA and piperaquine doses were 9.1 and 73.9 mg/kg, respectively, for children and 6.6 and 52.9 mg/kg for adults. All patients became aparasitemic within 72 h. Excluding the results for 1 child who died on day 4, there was a 96.9% 28-day cure rate (98.6% in children and 92.3% in adults). Patients who had recrudescent infection received low doses of Artekin. Side effects were reported by 22 patients (21%) but did not necessitate premature cessation of therapy. Although Artekin is a promising and inexpensive option for antimalarial therapy, further efficacy and pharmacokinetic studies are needed, especially for its use in children.

Treatment of uncomplicated falciparum malaria in southern Vietnam: can chloroquine or sulfadoxine-pyrimethamine be reintroduced in combination with artesunate?

The effectiveness of chloroquine or sulfadoxine-pyrimethamine administered with artesunate for treating uncomplicated falciparum malaria was assessed in 2 Vietnamese provinces where the sensitivity of parasites in vitro to conventional therapies had increased with the removal of drug pressure. In the province of Dac Lac, where potential malaria exposure begins at birth, 57 subjects (mean age, 9.6 years) were randomized to receive artesunate-chloroquine (group 1) or artesunate-sulfadoxine-pyrimethamine (group 2). In the province of Binh Phuoc, 66 nonimmune workers and their relatives (mean age, 24.2 years) were similarly randomized. By day 28 of follow-up, >96% of Dac Lac patients and <52% of Binh Phuoc patients in group 1 and group 2 had an in vivo response that demonstrated sensitivity to therapy. PCR-confirmed cure rates paralleled in vivo results among patients in Binh Phuoc, but PCR results identified 9 (15.7%) of the Dac Lac patients as having experienced asymptomatic, submicroscopic recrudescences. In Dac Lac, pfcrt K76T was near fixation, but infection with parasites with this mutation predicted recrudescence among group 1 patients in Binh Phuoc. Common pfdhfr mutations did not predict outcome in group 2. The successful reintroduction of conventional therapies in combination with artesunate depends on epidemiological and/or parasitological factors.

Anopheline vectors and malaria transmission in eastern Afghanistan.

Anopheline vectors and malaria transmission were studied in 2 river-irrigated, rice-growing districts of eastern Afghanistan from May 1995 to December 1996. Clinical malaria was monitored in 12 rural villages (population 14,538) by passive case detection at local clinics. Adult mosquitoes were collected by space-spraying of living quarters and stables and by cattle bait catches. Mosquito head-thoraces (17,255 specimens) were tested for Plasmodium falciparum and P. vivax circumsporozoite protein (CSP) using enzyme-linked immunosorbent assay. The recorded incidence of P. vivax and P. falciparum was 199 and 41 episodes per 1000 person years, respectively. Twelve species of anopheline were recorded; Anopheles stephensi comprised 82% and A. culicifacies 5%. Eight species tested positive for CSP: A. stephensi, A. culicifacies, A. fluviatilus, A. annularis, A. pulcherrimus, A. maculatus, A. splendidus and A. superpictus. Among infected mosquitoes 46% were positive for P. falciparum, 45% for P. vivax VK-247, and 9% for P. vivax PV-210. Estimates of the feeding rates of infective vectors on humans indicated that A. stephensi would contribute 76% of infective bites, A. fluviatilis and A. pulcherrimus 7% each, and A. culicifacies and A. superpictus 3% each. The overall infective vector feeding rate correlated with the P. vivax incidence rate in the human population. The conventional view of A. culicifacies being the main rural vector and A. stephensi important only in urban settings needs to be reconsidered in western outreaches of the Indo-Pakistan subcontinent.

Evidence for a new conceptualization of semantic representation in the left and right cerebral hemispheres.

Two experiments are reported that examined qualitative differences in how semantic information is represented in the two hemispheres. In the first experiment, items that were associatively related but did not share semantic features or membership in semantic categories produced priming when delivered to the LH (RVF) but not to the RH (LVF). In the second experiment items that shared semantic features but were neither associates nor in the same category produced priming in the RH (LVF), but not in the LH (RVF). Together, the two experiments support the theory that, in the right hemisphere, semantic memories are represented within a distributed system, on the basis of semantic features, whereas, in the left hemisphere representations are, as in local models, relatively more holistic, and are connected via associative links.

Physiological evidence that a masked unrelated intervening item disrupts semantic priming: implications for theories of semantic representation and retrieval models of semantic priming.

Event-related potentials were recorded in a paradigm where an unrelated word was interposed between two related words. In one condition, the intervening item was masked and in another condition it was not. The N400 component indicated that priming of the related word was disrupted by the intervening item whether it was masked or not. The data are interpreted to be inconsistent with retrieval models of priming.

Population pharmacokinetics of piperaquine in adults and children with uncomplicated falciparum or vivax malaria.

AIMS: To study the population pharmacokinetics of piperaquine after co-administration with dihydroartemisinin in uncomplicated malaria. METHODS: The disposition of piperaquine was studied in 85 Cambodian patients with uncomplicated falciparum or vivax malaria treated with the piperaquine-dihydroartemisinin coformulation Artekin. All patients were given Artekin orally at 0, 6, 24 and 32 h with a total piperaquine dose of 32-35 mg base kg-1. Adults were given tablets while children received either tablets or a dispersible granule formulation. Patients underwent either intensive (17-19 samples) or sparse (2-5 samples) blood sampling schedules over 35 days and clinical/parasitological follow-up over > 28 days. Piperaquine in plasma was quantified by high performance liquid chromatography. RESULTS: All patients achieved fever clearance within 24 h and parasite clearance within 72 h. The 28-day cure rate was 97% in adults and 98% in children. A covariate-free two-compartment population model with first-order absorption and elimination gave the most robust representation of the plasma concentration-time data in both adults and children. In adults (n = 38), the median (interquartile range) derived pharmacokinetic descriptors CL/F, Vss/F and t1/2,z were 0.9 l h-1 kg-1 (0.79-1.02 l h-1 kg-1), 574 l kg-1(371-711 l kg-1) and 23 days (19-28 days), respectively. In children (n = 47), corresponding values were 1.8 l h-1 kg-1 (1.29-2.3 l h-1 kg-1), 614 l kg-1 (332-1205 l kg-1) and 14 days (10-18 days), respectively. CONCLUSIONS: Piperaquine is a highly lipid-soluble drug with a large Vss/F, long t1/2,z and a clearance that is markedly higher in children than in adults.

Comparison of three antigen detection methods for diagnosis and therapeutic monitoring of malaria: a field study from southern Vietnam.

OBJECTIVES: To compare the sensitivity, specificity and post-treatment persistence of three commonly used rapid antigen detection methods. METHOD: We studied 252 Vietnamese patients aged from 4 to 60 years, 157 with falciparum and 95 with vivax malaria and 160 healthy volunteers. An initial blood sample was taken for microscopy, and OptiMAL, immunochromatographic test (ICT) malaria P.f./P.v. and Paracheck-Pf tests. Patients with falciparum malaria were treated with an artesunate-based combination regimen and those with vivax malaria received chloroquine. Eighty-seven patients with falciparum malaria who were initially positive for one of the antigen tests and who remained blood smear-negative underwent follow-up testing over 28 days. RESULTS: Paracheck-Pf was the most sensitive test for Plasmodium falciparum (95.8% vs. 82.6% for ICT malaria P.f./P.v. and 49.7% for OptiMAL). Specificities were all 100%. For vivax malaria, OptiMAL performed better than ICT malaria P.f./P.v. (sensitivities 73.7% and 20.0%, respectively), with 100% specificity in both cases. All tests had low sensitivities (< or = 75.0%) at parasitaemias < 1000/microl regardless of malaria species. During follow-up, Paracheck-Pf remained positive in the greatest proportion of patients, especially at higher parasitaemias (> 10,000/microl). Residual OptiMAL positivity occurred only in a relatively small proportion of patients (< 10%) with parasitaemias > 10,000/microl during the first 2 weeks after treatment. CONCLUSIONS: Although microscopy remains the gold standard for malaria diagnosis, Paracheck-Pf may prove a useful adjunctive test in uncomplicated falciparum malaria in southern Vietnam. OptiMAL had the lowest sensitivity for P. falciparum but it might have a use in the diagnosis of vivax malaria and perhaps to monitor efficacy of treatment for falciparum malaria where microscopy is unavailable.

pfcrt polymorphism and chloroquine resistance in Plasmodium falciparum strains isolated in Cambodia.

Plasmodium falciparum chloroquine resistance was first detected in Cambodia in the early sixties. Treatment with chloroquine was abandoned 20 years ago. In vitro chloroquine sensitivity monitoring indicates that all eastern Cambodian isolates were sensitive to chloroquine, whereas most isolates collected from western provinces displayed reduced susceptibility to chloroquine. This indicates that the rate of chloroquine resistance remains high and stable in this region in the absence of chloroquine pressure. Characterization of codons 72 to 78 and 218 to 220 of pfcrt revealed six distinct haplotypes, four of which had never been described. The frequency of each haplotype depended on the geographical origin of the samples. The CVIETIF//ISS haplotype was detected in 92% of western Cambodian isolates and in 11% of isolates collected from the eastern province, where CVMNKIF//ISA and CVIDTIF//ISS predominate. The detection of an intermediate haplotype from a susceptible area with 76T/220A, suggests that acquisition of chloroquine resistance might be a stepwise process, during which accumulation of point mutations modulates the response to chloroquine. The association of the K76T mutation with chloroquine resistance was not clear. The mutation was detected in resistant and susceptible samples, suggesting that additional factors are involved in chloroquine resistance. By contrast, the pfcrt D/N75E mutation was strongly associated with the in vitro chloroquine resistance in Cambodian isolates. The N86 allelic form of pfmdr1 was detected in all isolates, consistent with a poor association with resistance to chloroquine. This indicates that in vitro resistance to chloroquine was associated with accumulation of point mutations in pfcrt.