Malaria rapid diagnostic tests (RDTs) have performed well in a variety of studies, but recent reports have described sensitivity for Plasmodium falciparum as significantly lower than that required for operational deployment. Exposure to high temperature has been suggested as an explanation. This study assessed the temperature stability of two different Plasmodium lactate dehydrogenase (pLDH)- and three histidine-rich protein 2 (HRP2)-detecting RDTs. One HRP2 test proved insufficiently sensitive for assessment. After incubation at 35, 45 and 60 degrees C, two RDTs detecting pLDH showed a substantial fall in percentage test line positivity over time, which was not seen with the remaining two HRP-2-based RDTs. For the particular products studied, variability was high, with the pLDH-based RDTs being less sensitive than HRP2-based RDTs against the sample of P. falciparum used and more susceptible to heat-induced damage, but the reasons for this are unclear. The performance of malaria RDTs can be adversely affected at the temperatures to which they will be exposed when transported to, and used in, the rural tropics.
BackgroundThe introduction of artemesinin-based treatment for falciparum malaria has led to a shift away from symptom-based diagnosis. Diagnosis may be achieved by using rapid non-microscopic diagnostic tests (RDTs), of which there are many available. Light microscopy, however, has a central role in parasite identification and quantification and remains the main method of parasite-based diagnosis in clinic and hospital settings and is necessary for monitoring the accuracy of RDTs. The World Health Organization has prepared a proficiency testing panel containing a range of malaria-positive blood samples of known parasitaemia, to be used for the assessment of commercially available malaria RDTs. Different blood film and counting methods may be used for this purpose, which raises questions regarding accuracy and reproducibility. A comparison was made of the established methods for parasitaemia estimation to determine which would give the least inter-rater and inter-method variationMethodsExperienced malaria microscopists counted asexual parasitaemia on different slides using three methods; the thin film method using the total erythrocyte count, the thick film method using the total white cell count and the Earle and Perez method. All the slides were stained using Giemsa pH 7.2. Analysis of variance (ANOVA) models were used to find the inter-rater reliability for the different methods. The paired t-test was used to assess any systematic bias between the two methods, and a regression analysis was used to see if there was a changing bias with parasite count level.ResultsThe thin blood film gave parasite counts around 30% higher than those obtained by the thick film and Earle and Perez methods, but exhibited a loss of sensitivity with low parasitaemia. The thick film and Earle and Perez methods showed little or no bias in counts between the two methods, however, estimated inter-rater reliability was slightly better for the thick film method.ConclusionThe thin film method gave results closer to the true parasite count but is not feasible at a parasitaemia below 500 parasites per microlitre. The thick film method was both reproducible and practical for this project. The determination of malarial parasitaemia must be applied by skilled operators using standardized techniques.
BackgroundThe detection of submicroscopic infections in low prevalence settings has become an increasingly important challenge for malaria elimination strategies. The current field rapid diagnostic tests (RDTs) for Plasmodium falciparum malaria are inadequate to detect low-density infections. Therefore, there is a need to develop more sensitive field diagnostic tools. In parallel, a highly sensitive laboratory reference assay will be essential to evaluate new diagnostic tools. Recently, the highly sensitive Alere™ Malaria Ag P.f ELISA (HS ELISA) was developed to detect P. falciparum histidine-rich protein 2 (HRP2) in clinical whole blood specimens. In this study, the analytical and clinical performance of the HS ELISA was determined using recombinant P. falciparum HRP2, P. falciparum native culture parasites, and archived highly pedigreed clinical whole blood specimens from Karen village, Myanmar and Nagongera, Uganda.ResultsThe HS ELISA has an analytical sensitivity of less than 25 pg/mL and shows strong specificity for P. falciparum HRP2 when tested against P. falciparum native culture strains with pfhrp2 and pfhrp3 gene deletions. Additionally, the Z′-factor statistic of 0.862 indicates the HS ELISA as an excellent, reproducible assay, and the coefficients of variation for inter- and intra-plate testing, 11.76% and 2.51%, were acceptable. Against clinical whole blood specimens with concordant microscopic and PCR results, the HS ELISA showed 100% (95% CI 96.4–100) diagnostic sensitivity and 97.9% (95% CI 94.8–99.4) diagnostic specificity. For P. falciparum positive specimens with HRP2 concentrations below 400 pg/mL, the sensitivity and specificity were 100% (95% CI 88.4–100) and 88.9% (95% CI 70.8–97.6), respectively. The overall sensitivity and specificity for all 352 samples were 100% (CI 95% 96–100%) and 97.3% (CI 95% 94–99%).ConclusionsThe HS ELISA is a robust and reproducible assay. The findings suggest that the HS ELISA may be a useful tool as an affordable reference assay for new ultra-sensitive HRP2-based RDTs.
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