Background Malaria rapid diagnostic tests (RDTs) are an essential tool in managing febrile illnesses in endemic set- tings. However, persisting parasite antigen after treatment or spontaneous remission, which can be detected by histi- dine-rich protein 2 (HRP2)-based RDT or Plasmodium lactate dehydrogenase (pLDH)-based RDTs, could lead to misdi- agnosis. To overcome the latter, a diagnostic approach combining sequential interpretation of two-step malaria RDTs incorporating two parasite antigens detecting PfHRP2 and pLDH coupled with a direct-on-blood mini PCR-nucleic acid lateral flow immunoassay (dbPCR-NALFIA) has been evaluated.
Methods Febrile patients visiting two rural health facilities in Burkina Faso were enrolled. For each participant,
an HRP2-based RDT and blood slides for microscopy (gold standard) were performed. A capillary blood sample was also collected in an EDTA tube and transported to laboratory to perform the two-step malaria RDT detecting HRP2 and LDH, and dbPCR-NALFIA testing. Malaria diagnostic results were sequentially interpreted and reported as: (i) positive when pLDH line appears regardless of the HRP2 results (PfHRP2 + /pLDH + or PfHRP2-/pLDH +); (ii) negative when both lines are absent (PfHRP2-/pLDH-); and (iii) undetermined when only the HRP2 line appears (PfHRP2 + /pLDH-). Undetermined cases were subsequently confirmed by dbPCR-NALFIA test and reported as posi- tive or negative. Malaria microscopy was used as reference test of conclusive diagnostic results (PfHRP2 + /pLDH + , PfHRP2-/pLDH + or PfHRP2-/pLDH-) and qPCR for undetermined cases (PfHRP2 + /pLDH-).
Results Out of 438 blood samples analysed, 87.2% (382/438) of patients with conclusive sequential interpretation
of HRP2 and pLDH did not need confirmative testing with dbPCR-NALFIA. The sensitivity and specificity of these conclusive results were 98.8% and 95.3%, respectively. Following confirmation of undetermined sequential interpreta- tion with dbPCR-NALFIA, the sequential algorithm had a sensitivity of 97.9%, a specificity of 94.8%, a positive predic- tive value of 97.2%, and a negative predictive value of 96.1%. For single HRP2-based RDT, the sensitivity was 95.2%, the specificity 73.2%, the positive predictive value 85.1%, and the negative predictive value 90.4%.
Conclusions The sequential algorithm of the two-step RDTs combined with dbPCR-NALFIA on inconclusive results enhances the diagnosis of malaria in febrile patients.

Malaria diagnosis, Microscopy, RDTs, DbPCR-NALFIA, Sequential algorithm