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 tests 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. Atthe 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.

Malaria: Current Situation and Way Forward

Malaria remains one of the most serious causes of ill health in the world; affecting people in more than 100 countries (World Health Organisation 1993). WHO estimates that the global incidence is between 300-500 million cares annually and this results in 1.5-2.7 million deaths. Nearly 25of all childhood deaths in Africa and half of the fever episodes in African children are attributable to malaria (WHO/CTD 1996). In Uganda; malaria has remained the leading cause of morbidity and mortality. The proportional morbidity attributable to malaria has been on the increase in Uganda (HMIS reports; unpublished)

Reliability of malaria microscopy in epidemiological studies: results of quality control.

To assess the interrater reproducibility of malaria microscopy in epidemiological studies, 711 thick blood films from population-based surveys were randomly selected and reread by 4 experienced microscopists. Sample estimates of the prevalence of P. falciparum infection, geometric mean parasite density and the proportion of samples above various parasite density cut-off levels were almost identical in the routine and quality control readings. Differences were, however, encountered in the sample estimates for gametocyte ratio, proportion of mixed infection and average density index. In all three cases the quality control result was significantly higher than the routine evaluation. On the level of the individual slide there was good interrater agreement for the presence of P. falciparum infections (Kappa index kappa = 0.79) which was even better when parasite densities between 4 and 100/microl were excluded (kappa = 0.94). With respect to the assessment of parasite density, a high level of disagreement was found. While the mean difference between the two readings was not different from 0, the second reading was between 0.12 and 10 times that of the first. However, the level of disagreement significantly fell with increasing parasite densities. Thus malaria microscopy is very reliable for the estimation of parasite ratios and geometric mean parasite densities within and between studies as long as the same methodology is used, but tends to underestimate the gametocyte ratio and proportion of mixed infections. Care must be taken, however, when individual parasite density is related to other explanatory variables, due to the high degree of variability in the parasite enumeration.

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.