Estimating malaria parasite prevalence from community surveys in Uganda: a comparison of microscopy, rapid diagnostic tests and polymerase chain reaction.

BACKGROUND: Household surveys are important tools for monitoring the malaria disease burden and measuring impact of malaria control interventions with parasite prevalence as the primary metric. However, estimates of parasite prevalence are dependent on a number of factors including the method used to detect parasites, age of the population sampled, and level of immunity. To better understand the influence of diagnostics, age, and endemicity on estimates of parasite prevalence and how these change over time, community-based surveys were performed for two consecutive years in three settings and the sensitivities of microscopy and immunochromatographic rapid diagnostic tests (RDTs) were assessed, considering polymerase chain reaction (PCR) as the gold standard. METHODS: Surveys were conducted over the same two-month period in 2012 and 2013 in each of three sub-counties in Uganda: Nagongera in Tororo District (January-February), Walukuba in Jinja District (March-April), and Kihihi in Kanungu District (May-June). In each sub-county, 200 households were randomly enrolled and a household questionnaire capturing information on demographics, use of malaria prevention methods, and proxy indicators of wealth was administered to the head of the household. Finger-prick blood samples were obtained for RDTs, measurement of hemoglobin, thick and thin blood smears, and to store samples on filter paper. RESULTS: A total of 1200 households were surveyed and 4433 participants were included in the analysis. Compared to PCR, the sensitivity of microscopy was low (65.3% in Nagongera, 49.6% in Walukuba and 40.9% in Kihihi) and decreased with increasing age. The specificity of microscopy was over 98% at all sites and did not vary with age or year. Relative differences in parasite prevalence across different age groups, study sites, and years were similar for microscopy and PCR. The sensitivity of RDTs was similar across the three sites (range 77.2-82.8%), was consistently higher than microscopy (p < 0.001 for all pairwise comparisons), and decreased with increasing age. The specificity of RDTs was lower than microscopy (76.3% in Nagongera, 86.3% in Walukuba, and 83.5% in Kihihi) and varied significantly by year and age. Relative differences in parasite prevalence across age groups and study years differed for RDTs compared to microscopy and PCR. CONCLUSION: Malaria prevalence estimates varied with diagnostic test, age, and transmission intensity. It is important to consider the effects of these parameters when designing and interpreting community-based surveys

Intermittent preventive treatment with dihydroartemisinin-piperaquine in Ugandan schoolchildren selects for Plasmodium falciparum transporter polymorphisms that modify drug sensitivity.

Dihydroartemisinin-piperaquine (DP) offers prolonged protection against malaria, but its impact on Plasmodium falciparum drug sensitivity is uncertain. In a trial of intermittent preventive treatment in schoolchildren in Tororo, Uganda, in 2011 to 2012, monthly DP for 1 year decreased the incidence of malaria by 96% compared to placebo; DP once per school term offered protection primarily during the first month after therapy. To assess the impact of DP on selection of drug resistance, we compared the prevalence of key polymorphisms in isolates that emerged at different intervals after treatment with DP. Blood obtained monthly and at each episode of fever was assessed for P. falciparum parasitemia by microscopy. Samples from 160 symptomatic and 650 asymptomatic episodes of parasitemia were assessed at 4 loci (N86Y, Y184F, and D1246Y in pfmdr1 and K76T in pfcrt) that modulate sensitivity to aminoquinoline antimalarials, utilizing a ligase detection reaction-fluorescent microsphere assay. For pfmdr1 N86Y and pfcrt K76T, but not the other studied polymorphisms, the prevalences of mutant genotypes were significantly greater in children who had received DP within the past 30 days than in those not treated within 60 days (86Y, 18.0% versus 8.3% [P = 0.03]; 76T, 96.0% versus 86.1% [P = 0.05]), suggesting selective pressure of DP. Full sequencing of pfcrt in a subset of samples did not identify additional polymorphisms selected by DP. In summary, parasites that emerged soon after treatment with DP were more likely than parasites not under drug pressure to harbor pfmdr1 and pfcrt polymorphisms associated with decreased sensitivity to aminoquinoline antimalarials. (This study has been registered at ClinicalTrials.gov under no. NCT01231880.)

The impact of an intervention to introduce malaria rapid diagnostic tests on fever case management in a high transmission setting in Uganda: A mixed-methods cluster-randomized trial (PRIME).

Rapid diagnostic tests for malaria (mRDTs) have been scaled-up widely across Africa. The PRIME study evaluated an intervention aiming to improve fever case management using mRDTs at public health centers in Uganda. A cluster-randomized trial was conducted from 2010-13 in Tororo, a high malaria transmission setting. Twenty public health centers were randomized in a 1:1 ratio to intervention or control. The intervention included training in health center management, fever case management with mRDTs, and patient-centered services; plus provision of mRDTs and artemether-lumefantrine (AL) when stocks ran low. Three rounds of Interviews were conducted with caregivers of children under five years of age as they exited health centers (N = 1400); reference mRDTs were done in children with fever (N = 1336). Health worker perspectives on mRDTs were elicited through semi-structured questionnaires (N = 49) and in-depth interviews (N = 10). The primary outcome was inappropriate treatment of malaria, defined as the proportion of febrile children who were not treated according to guidelines based on the reference mRDT. There was no difference in inappropriate treatment of malaria between the intervention and control arms (24.0% versus 29.7%, adjusted risk ratio 0.81 95\% CI: 0.56, 1.17 p = 0.24). Most children (76.0\%) tested positive by reference mRDT, but many were not prescribed AL (22.5\% intervention versus 25.9\% control, p = 0.53). Inappropriate treatment of children testing negative by reference mRDT with AL was also common (31.3\% invention vs 42.4\% control, p = 0.29). Health workers appreciated mRDTs but felt that integrating testing into practice was challenging given constraints on time and infrastructure. The PRIME intervention did not have the desired impact on inappropriate treatment of malaria for children under five. In this high transmission setting, use of mRDTs did not lead to the reductions in antimalarial prescribing seen elsewhere. Broader investment in health systems, including infrastructure and staffing, will be required to improve fever case management

Impact of a Rapid Decline in Malaria Transmission on Antimalarial IgG Subclasses and Avidity

Understanding how immunity to malaria is affected by declining transmission is important to aid vaccine design and understand disease resurgence. Both IgG subclasses and avidity of antigen-specific responses are important components of an effective immune response. Using a multiplex bead array assay, we measured the total IgG, IgG subclasses, and avidity profiles of responses to 18 P. falciparum blood stage antigens in samples from 160 Ugandans collected at two time points during high malaria transmission and two time points following a dramatic reduction in transmission. Results demonstrated that, for the antigens tested, (i) the rate of decay of total IgG following infection declined with age and was driven consistently by the decrease in IgG3 and occasionally the decrease in IgG1; (ii) the proportion of IgG3 relative to IgG1 in the absence of infection increased with age; (iii) the increase in avidity index (the strength of association between the antibody and antigen) following infection was largely due to a rapid loss of non-avid compared to avid total IgG; and (iv) both avid and non-avid total IgG in the absence of infection increased with age. Further studies are required to understand the functional differences between IgG1 and IgG3 in order to determine their contribution to the longevity of protective immunity to malaria. Measuring changes in antibody avidity may be a better approach of detecting affinity maturation compared to avidity index due to the differential expansion and contraction of high and low avidity total IgG