External quality assurance of malaria nucleic acid testing for clinical trials and eradication surveillance

Abstract

Nucleic acid testing (NAT) for malaria parasites is an increasingly recommended diagnostic endpoint in clinical trials of vaccine and drug candidates and is also important in surveillance of malaria control and elimination efforts. A variety of reported NAT assays have been described, yet no formal external quality assurance (EQA) program provides validation for the assays in use. Here, we report results of an EQA exercise for malaria NAT assays. Among five centers conducting controlled human malaria infection trials, all centers achieved 100% specificity and demonstrated limits of detection consistent with each laboratory's pre-stated expectations. Quantitative bias of reported results compared to expected results was generally <0.5 log10 parasites/mL except for one laboratory where the EQA effort identified likely reasons for a general quantitative shift. The within-laboratory variation for all assays was low at <10% coefficient of variation across a range of parasite densities. Based on this study, we propose to create a Molecular Malaria Quality Assessment program that fulfills the need for EQA of malaria NAT assays worldwide.

Figure 1. Study data.

Results were plotted on a log₁₀ parasites/mL scale for the five participating laboratories; bars show the mean and 95% confidence interval. Nominal (expected) values for all samples are plotted as follows: high (300,000 parasites/mL or 5.48 log₁₀ parasites/mL); mid (6,000 parasites/mL or 3.78 log₁₀ parasites/mL); low (600 parasites/mL or 2.78 log₁₀ parasites/mL); very low (60 parasites/mL or 1.78 log₁₀ parasites/mL), trace (6 parasites/mL or 0.78 log₁₀ parasites/mL) and negative (no parasites). Samples with no parasites detected were plotted as 0.1 log₁₀ parasites/mL. Two-way ANOVA comparisons across all high, mid and low parasite density samples with quantitatively positive results showed non-statistically significant differences amongst all groups (p>0.05) except for RUMC vs. Oxford at high (p<0.0001), mid (p<0.01) and low (p≤0.05) parasite densities. *NIH quantities were generated by regression of C_T values to expected EQA values and are provided to visualize variation and qualitative agreement; quantitative statistical comparisons were not included. 60 of 120 representative NIH samples are displayed for consistency.