Field evaluation of the miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) for the detection of Plasmodium falciparum in high seasonal malaria transmission setting in Burkina Faso

Introduction: Accurate diagnosis of malaria, caused by Plasmodium falciparum , remains challenging in endemic resource-limited settings. Molecular diagnostics, like PCR, offer a higher sensitivity, but are often impractical at the point-of-care. A relatively simple molecular diagnostic test has been developed to overcome the technological challenges frequently encountered during the implementation of molecular tools. We present here the results of the field evaluation of this novel mini direct-on-blood PCR platform with lateral flow readout (dbPCR-NALFIA), in a rural setting of Burkina Faso. Methods: A phase 3 diagnostic accuracy study was conducted over one year in a high P. falciparum transmission setting in Burkina Faso. Febrile patients of all ages (n=438) were screened using Pf HRP2-based RDT, microscopy, and the investigational dbPCR-NALFIA test. Reference diagnostic test was qPCR targeting the P. falciparum varATS gene. Diagnostic accuracy metrics (sensitivity, specificity, predictive values) and agreement (Cohen’s kappa) were calculated for each method. Results: Malaria prevalence by qPCR was 65.5% (287/438). A total of 99.2% (259/261) of P. falciparum microscopy-positive samples were detected by qPCR, 97.7% (253/259) detected with RDT and 99.6% (258/259) with the investigational dbPCR-NALFIA. Overall, 85.2% (149/177) of microscopy negative samples were also confirmed negative with qPCR. Among these qPCR negative samples, 80.5% (120/149) were concordantly negative by RDT, while 98.0% (146/149) were confirmed negative by dbPCR-NALFIA. Using qPCR as reference, the dbPCR-NALFIA demonstrated a sensitivity of 96.5% and specificity of 98.0%, comparable to RDT sensitivity (95.1%) and microscopy specificity (98.7%), but out-performed RDT specificity (80.8%) and microscopy sensitivity (90.2%). dbPCR-NALFIA detected 67.9% of sub microscopic qPCR-positive samples missed by the microscopy. Agreement with qPCR was the highest for dbPCR-NALFIA (kappa value = 0.90), compared to microscopy (kappa value = 0.80) and RDT (kappa value = 0.71). Conclusion: The dbPCR-NALFIA shows excellent diagnostic performance to detect P. falciparum under field conditions. It addresses key limitations of other diagnostics and could play a vital role in case detection.