Evaluation of the analytical performance of the BioFire FilmArray Tropical Fever Panel for detection of malaria in archived EDTA-blood samples

Rapid and reliable diagnosis of malaria is essential in non-endemic areas, where most analysed samples are negative and microscopy expertise can be difficult to maintain. Loop mediated isothermal amplification (LAMP) is sensitive, but the commercially available LAMP assay used in this study does not allow species identification. The BioFire FilmArray ® Tropical Fever (TF) Panel is a multiplex PCR assay designed to detect Plasmodium species and other tropical pathogens within 50 min. This study evaluated the analytical sensitivity of the TF Panel and its performance for species-level identification in a non-endemic area. To estimate the LoD of the TF Panel, tenfold serial dilutions of a clinical sample with known P. falciparum parasitaemia were tested and compared to the Alethia malaria LAMP assay. To further evaluate the performance of the TF Panel, 30 archived clinical samples that had previously tested positive with LAMP or RDT were analysed. In these samples, species identification was established by polymerase chain reaction (PCR) amplification and Oxford Nanopore sequencing of the cox III gene present in plasmodial mitochondrial DNA. To further challenge the capability of the TF Panel to identify Plasmodium spp., mixed-species samples were assembled and tested (N=3). Finally, five clinical samples that tested negative by the LAMP assay were included as negative controls. The TF Panel demonstrated high analytical sensitivity, detecting P. falciparum in 4/5 replicates at 0.05 parasites/µL, whereas LAMP detected 1/5 replicates at the same concentration. All P. falciparum infections were correctly detected. Non-falciparum infections were reported as P. vivax / P. ovale including samples containing P. knowlesi (N=1) and P. malariae (N=1) . For the assembled multi-species samples, the TF Panel correctly identified P. falciparum whereas non-falciparum species were not accurately differentiated. In one sample with two non-falciparum species the assay failed to detect the mixed infection. All negative controls were correctly reported as negative for Plasmodium . The TF Panel demonstrated high analytical sensitivity for P. falciparum detection, but microscopy remains necessary for assessment of parasitaemia and treatment monitoring . Although differentiation for non-falciparum species was limited, the assay may reduce the time to diagnosis and reliance on urgent microscopy in routine clinical practice.