Operational accuracy and comparative persistent antigenicity of HRP2 rapid diagnostic tests for Plasmodium falciparum malaria in a hyperendemic region of Uganda

Abstract

Background: Parasite-based diagnosis of malaria by microscopy requires laboratory skills that are generally unavailable at peripheral health facilities. Rapid diagnostic tests (RDTs) require less expertise, but accuracy under operational conditions has not been fully evaluated in Uganda. There are also concerns about RDTs that use the antigen histidine-rich protein 2 (HRP2) to detect Plasmodium falciparum, because this antigen can persist after effective treatment, giving false positive test results in the absence of infection. An assessment of the accuracy of Malaria Pf immuno-chromatographic test (ICT) and description of persistent antigenicity of HRP2 RDTs was undertaken in a hyperendemic area of Uganda.

Methods: Using a cross-sectional design, a total of 357 febrile patients of all ages were tested using ICT, and compared to microscopy as the gold standard reference. Two independent RDT readings were used to assess accuracy and inter-observer reliability. With a longitudinal design to describe persistent antigenicity of ICT and Paracheck, 224 children aged 6-59 months were followed up at 7-day intervals until the HRP2 antigens where undetectable by the RDTs.

Results: Of the 357 patients tested during the cross-sectional component, 40% (139) had positive blood smears for asexual forms of P. falciparum. ICT had an overall sensitivity of 98%, a specificity of 72%, a negative predictive value (NPV) of 98% and a positive predictive value (PPV) of 69%. ICT showed a high inter-observer reliability under operational conditions, with 95% of readings having assigned the same results (kappa statistics 0.921, p < 0.001). In children followed up after successful antimalaria treatment, the mean duration of persistent antigenicity was 32 days, and this duration varied significantly depending on pre-treatment parasitaemia. In patients with parasite density >50,000/microl, the mean duration of persistent antigenicity was 37 days compared to 26 days for parasitaemia less than 1,000/microl (log rank 21.9, p < 0.001).

Conclusion: ICT is an accurate and appropriate test for operational use as a diagnostic tool where microscopy is unavailable. However, persistent antigenicity reduces the accuracy of this and other HRP2-based RDTs. The low specificity continues to be of concern, especially in children below five years of age. These pose limitations that need consideration, such as their use for diagnosis of patients returning with symptoms within two to four weeks of treatment. Good clinical skills are essential to interpret test results.

Figure 1

Longitudinal component patient profile. Longitudinal component patient profile showing screened patients, smear and rapid diagnostic test (RDT) results and the number that were followed up. At the beginning of the longitudinal component to evaluate persistent antigenicity of HRP2 RDTs, 259 children were enrolled on day-3. 224 remained in the study until an outcome was determined (at the end of the follow up when either the RDTs were negative or smear was positive due to recurrent parasitaemia).

Figure 2

Proportion of children with persistent antigen in Soroti, Uganda. Proportion of children with persistent antigen positive HRP2-RDT at clinic visits, stratified by day-zero parasite density at < 1,000/μl, 1,000–50,000/μl and > 50,000/μl. 224 children were followed up with censoring for those with recurrent parasitaemias. The log rank test statistics for equality of survival distributions was 21.93 (p < 0.001).