Method validation for dried blood spot Plasmodium 18S rRNA RT-PCR on Roche cobas automated molecular diagnostic platform

Abstract

Malaria, caused by Plasmodium spp., is a major health burden worldwide. Plasmodium falciparum is responsible for the most severe cases and most human mortality in sub-Saharan Africa. Conventional diagnostic approaches like microscopy and rapid diagnostic tests (RDTs) provide point-of-care screening but lack sufficient sensitivity for low-density infections and surveillance in endemic regions. Molecular diagnostic platforms, particularly nucleic acid tests (NATs), which target highly abundant Plasmodium 18S ribosomal RNA (rRNA), have emerged as sensitive and specific platforms for malaria detection and quantification. The FDA approved Roche cobas® 6800 system, which is a fully automated, high-throughput molecular platform assay for the qualitative detection of Plasmodium spp. from whole blood samples by targeting the 18S rRNA.This study aimed (1) to adapt the cobas® malaria assay for off-label use with non-standard sample types, including 50 μL of whole blood and dried blood spot (DBS) samples, and (2) to generate a standard curve for absolute quantification of the parasites. To achieve this, asexual-stage P. falciparum cultures were established and diluted to generate a full panel of validation samples across clinically relevant densities (1 x 10⁸–1 x 10¹ parasites/mL). Fifty- microliter blood samples were spotted onto DBS cards. Together, whole blood liquid and DBS samples were processed then tested using the cobas® 6800 system. Plasmodium Armored RNA calibrators were used to generate standard curves for quantification. The study evaluated performance metrics parameters, that included pilot feasibility, standard curve assessment, linearity, limit of detection (LoD), precision, accuracy, carryover, DBS RNA stability under different storage conditions, calibrators-sample matrix matched assessment and extraction buffer elution assessment. The modified FDA-approved Roche cobas® 6800 malaria assay demonstrated successful adaptation for detecting and quantifying P. falciparum 18S rRNA in 50 µL whole blood samples. However, performance was reduced for DBS samples. Both whole blood and DBS sample formats achieved ≥95% detection at the 100 parasites/mL threshold. A matrix mismatch using liquid calibrators to quantify DBS samples resulted in systematic underestimation bias of +0.79 log₁₀ parasites/mL due to poor RNA recovery from DBS samples. The use of DBS-specific calibrators corrected the loss with a reduced bias of -0.07 log₁₀ parasites/mL, which enabled for the reliable quantification of P. falciparum concentration in the DBS samples. Low (100 parasites/mL) density DBS samples stored at room temperature (22–25 °C) and -80°C consistently maintained a 100% positivity hit rate across all the two timepoints (weeks 1 and 6). All MID (2000 parasites/mL) parasite density DBS samples exhibited 100% positivity across both time points and temperature storage conditions. The stability of DBS samples indicated reliable qualitative detection of P. falciparum RNA for DBS samples stored at 22-25°C or -80°C conditions, which supports their use in decentralized sample collection. Overall, these results suggest that the modified FDA- approved Roche cobas® 6800 malaria assay performs well with 50 μL liquid whole blood samples but improvements are needed to achieve optimal results for 50 μL DBS samples.