Application of the ParaSight-F dipstick test for malaria diagnosis in a district control program

A rapid test for the diagnosis of Plasmodium falciparum infections based on the detection of histidine-rich-protein II, the ParaSight-F test, was evaluated after introduction in a district malaria control program in Uganda. Suspected treatment failures, pregnant women and infants with clinical malaria and general fever cases were tested at health facilities in malaria hypo-, meso- and holoendemic areas. A total of 1326 tests were carried out by health unit staff, cross read by experienced laboratory staff and results compared with thick film microscopy as the standard. Rater agreement in reading the dipstick result between health unit staff and laboratory staff was high, kappa index 0.94 (0.88-0.99). Sensitivity was 99.6% (99.0-100) for parasite densities above 500/microl, 98.6% (97.7-99.6) for densities above 50/microl and 22.2% (8.6-42.3) for densities below 10/microl. With the applied testing strategies no differences were found between endemicity levels or patient categories. Specificity was 86.2% (83.3-88.8) overall, but significantly higher in general fever cases (92.7%) compared to the other patient groups (84.3%, P=0.009). At the given prevalences positive predictive values (ppv) were above 80% and negative predictive values (npv) above 90% in all cases except in pregnant women (ppv: 77.8%). We conclude that in certain situations this test is an alternative to microscopy to improve diagnostic facilities for case management in malaria control programs in endemic African countries.     

Population-based validation of dihydrofolate reductase gene mutations for the prediction of sulfadoxine-pyrimethamine resistance in Uganda

Mutations in the dihydrofolate reductase gene (dhfr) of Plasmodium falciparum have been proposed as molecular markers for the surveillance of sulfadoxine-pyrimethamine (SP)-resistant malaria, but such proposals have not been validated. At 7 Ugandan sites in 1999, we determined the population-based prevalence of infections with mutations and the mutant allele frequency of dhfr codons 108, 51, and 59 using a random sample of infected individuals aged 1-45 years. Sulfadoxine-pyrimethamine treatment failure was independently estimated by In vivo in 327 children aged 6-59 months with clinical malaria. The prevalence of infections with the single point mutations and the dhfr codons 108 and 51 mutant allele frequency were not correlated to SP treatment failure. However, the dhfr codon 59 mutant allele frequency was positively correlated to SP treatment failure (r= 0.72, P= 0.06). The ratio of the infections with the mutant to wild genotype (M/W) and that of the mutant to wild allele (MA/WA) had the same values. Both dhfr codon 59 M/W and MA/WA ratio were significantly and positively correlated to SP treatment failure (r= 0.73, P= 0.05). Moreover, the prevalence of infections with only 2 mutations (Asn-108 plus Ile-51) was significantly and inversely correlated to the prevalence of infections with 3 mutations (Asn-108 plus Ile-51 plus Arg-59) (r = 0.92, P = 0.004), suggesting the stepwise accumulation of the dhfr mutations is Asn-108 Ile-51 Arg-59 and further supporting the idea of using the dhfr codon 59 M/W ratio as a molecular index for the prediction of SP treatment failure. At the population level, the dhfr codon 59 M/W ratio is a simple and stable index for the estimation of SP treatment failure.     

Intensity of malaria transmission,antimalarial-drug use and resistance in Uganda: what is the relationship between these three factors?

We studied (in 1998 and 1999) some factors that may be linked to the spread of chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) resistance in 7 discrete communities in Uganda. Exposure to malaria infection was measured by parasitological surveys in children aged 1-9 years, drug use by community surveys and drug resistance by in-vivo tests on children aged 6-59 months with clinical malaria. CQ use was inversely related to parasite prevalence (r = -0.85, P = 0.01). CQ and SP treatment failure rates varied significantly according to parasite prevalence (P = 0.001 and 0.04 respectively). The highest CQ (42.4%, 43.8%) and SP (12.5%, 14.8%) treatment failure rates were observed in sites characterized by high parasite prevalence. Using areas with medium parasite prevalence as reference, the relative risk (RR) for CQ treatment failure was 3.2 (95% CI 1.6-6.4) in high parasite prevalence sites and 3.1 (95% CI 1.2-7.7) in low parasite prevalence sites. The RR for SP treatment failure was also higher in sites with high parasite prevalence but low in those with low parasite prevalence. According to our findings, drug resistance seems to spread faster in higher transmission areas, regardless of drug pressure. In low transmission areas, drug pressure seems to be the critical factor. A decrease in transmission coupled with rational use of drugs may delay the spread of resistance.     

Simultaneous quantification of losartan and active metabolite in human plasma by liquid chromatography–tandem mass spectrometry using irbesartan as internal standard

A simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method employing electronspray ionization was developed and validated for quantification of losartan and its carboxylic acid metabolite in human plasma using irbesartan as internal standard (IS). Following a simple pretreatment procedure, the analytes were separated using a gradient mobile phase on reverse phase C₁₈ column. Selected reaction monitoring was specific for losartan, losartan acid and irbesartan. The method validation demonstrated the specificity, lower limit of quantification, accuracy and precision of measurements. The assay exhibited a linear dynamic range of 2.0–400 ng/mL for losartan and 1.85–370 ng/mL for losartan acid. A run time of 3.5 min for each sample made it possible to analyze more than 200 samples per day. The validated method has been successfully used to analyze human plasma samples for application in bioavailability/bioequivalence studies.