Focused Screening and Treatment (FSAT): a PCR-based strategy to detect malaria parasite carriers and contain drug resistant P. falciparum, Pailin, Cambodia.

Recent studies have shown that Plasmodium falciparum malaria parasites in Pailin province, along the border between Thailand and Cambodia, have become resistant to artemisinin derivatives. To better define the epidemiology of P. falciparum populations and to assess the risk of the possible spread of these parasites outside Pailin, a new epidemiological tool named "Focused Screening and Treatment" (FSAT), based on active molecular detection of asymptomatic parasite carriers was introduced in 2010. Cross-sectional malariometric surveys using PCR were carried out in 20 out of 109 villages in Pailin province. Individuals detected as P. falciparum carriers were treated with atovaquone-proguanil combination plus a single dose of primaquine if the patient was non-G6PD deficient. Interviews were conducted to elicit history of cross-border travel that might contribute to the spread of artemisinin-resistant parasites. After directly observed treatment, patients were followed up and re-examined on day 7 and day 28. Among 6931 individuals screened, prevalence of P. falciparum carriers was less than 1%, of whom 96% were asymptomatic. Only 1.6% of the individuals had a travel history or plans to go outside Cambodia, with none of those tested being positive for P. falciparum. Retrospective analysis, using 2010 routine surveillance data, showed significant differences in the prevalence of asymptomatic carriers discovered by FSAT between villages classified as "high risk" and "low risk" based on malaria incidence data. All positive individuals treated and followed-up until day 28 were cured. No mutant-type allele related to atovaquone resistance was found. FSAT is a potentially useful tool to detect, treat and track clusters of asymptomatic carriers of P. falciparum along with providing valuable epidemiological information regarding cross-border movements of potential malaria parasite carriers and parasite gene flow.

Performance of "VIKIA Malaria Ag Pf/Pan" (IMACCESS®), a new malaria rapid diagnostic test for detection of symptomatic malaria infections.

BACKGROUND: Recently, IMACCESS® developed a new malaria test (VIKIA Malaria Ag Pf/Pan™), based on the detection of falciparum malaria (HRP-2) and non-falciparum malaria (aldolase). METHODS: The performance of this new malaria rapid diagnostic test (RDT) was assessed using 1,000 febrile patients seeking malaria treatment in four health centres in Cambodia from August to December 2011. The results of the VIKIA Malaria Ag Pf/Pan were compared with those obtained by microscopy, the CareStart Malaria™ RDT (AccessBio®) which is currently used in Cambodia, and real-time PCR (as "gold standard"). RESULTS: The best performances of the VIKIA Malaria Ag Pf/Pan™ test for detection of both Plasmodium falciparum and non-P. falciparum were with 20-30 min reading times (sensitivity of 93.4% for P. falciparum and 82.8% for non-P. falciparum and specificity of 98.6% for P. falciparum and 98.9% for non-P. falciparum) and were similar to those for the CareStart Malaria™ test. CONCLUSIONS: This new RDT performs similarly well as other commercially available tests (especially the CareStart Malaria™ test, used as comparator), and conforms to the World Health Organization's recommendations for RDT performance. It is a good alternative tool for the diagnosis of malaria in endemic areas.

Reduced impact of pyrimethamine drug pressure on Plasmodium malariae dihydrofolate reductase gene.

Molecular investigations performed following the emergence of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum have allowed the identification of the dihydrofolate reductase (DHFR) enzyme as the target of pyrimethamine. Although clinical cases of Plasmodium malariae are not usually treated with antifolate therapy, incorrect diagnosis and the high frequency of undetected mixed infections has probably exposed non-P. falciparum parasites to antifolate therapy in many areas. In this context, we aimed to assess the worldwide genetic diversity of the P. malariae dhfr gene in 123 samples collected in Africa and Asia, areas with different histories of SP use. Among the 10 polymorphic sites found, we have observed 7 new mutations (K55E, S58R, S59A, F168S, N194S, D207G, and T221A), which led us to describe 6 new DHFR proteins. All isolates from African countries were classified as wild type, while new mutations and haplotypes were recognized as exclusive to Madagascar (except for the double mutations at nucleotides 341 and 342 [S114N] found in one Cambodian isolate). Among these nonsynonymous mutations, two were likely related to pyrimethamine resistance: S58R (corresponding to C59R in P. falciparum and S58R in Plasmodium vivax; observed in one Malagasy sample) and S114N (corresponding to S108N in P. falciparum and S117N in P. vivax; observed in three Cambodian samples).

Performance of the CareStart™ G6PD deficiency screening test, a point-of-care diagnostic for primaquine therapy screening.

Development of reliable, easy-to-use, rapid diagnostic tests (RDTs) to detect glucose-6-phosphate dehydrogenase (G6PD) deficiency at point of care is essential to deploying primaquine therapies as part of malaria elimination strategies. We assessed a kit under research and development called CareStart™ G6PD deficiency screening test (Access Bio, New Jersey, USA) by comparing its performance to quantitative G6PD enzyme activity using a standardized spectrophotometric method ('gold standard'). Blood samples (n = 903) were collected from Cambodian adults living in Pailin province, western Cambodia. G6PD enzyme activities ranged from 0 to 20.5 U/g Hb (median 12.0 U/g Hg). Based on a normal haemoglobin concentration and wild-type G6PD gene, the normal values of G6PD enzymatic activity for this population was 3.6 to 20.5 U/g Hg (95(th) percentiles from 5.5 to 17.2 U/g Hg). Ninety-seven subjects (10.7%) had <3.6 U/g Hg and were classified as G6PD deficient. Prevalence of deficiency was 15.0% (64/425) among men and 6.9% (33/478) among women. Genotype was analyzed in 66 G6PD-deficient subjects and 63 of these exhibited findings consistent with Viangchang genotype. The sensitivity and specificity of the CareStart™ G6PD deficiency screening test was 0.68 and 1.0, respectively. Its detection threshold was <2.7 U/g Hg, well within the range of moderate and severe enzyme deficiencies. Thirteen subjects (1.4%, 12 males and 1 female) with G6PD enzyme activities <2 U/g Hg were falsely classified as "normal" by RDT. This experimental RDT test here evaluated outside of the laboratory for the first time shows real promise, but safe application of it will require lower rates of falsely "normal" results.