Monitoring the threatened utility of malaria rapid diagnostic tests by novel high-throughput detection of Plasmodium falciparum hrp2 and hrp3 deletions: A cross-sectional, diagnostic accuracy study

• Published in: EBioMedicine 2019-12, Vol.50, p.14-22
• Main Authors: Kreidenweiss, Andrea, Trauner, Franziska, Rodi, Miriam, Koehne, Erik, Held, Jana, Wyndorps, Lea, Manouana, Gédéon Prince, McCall, Matthew, Adegnika, Ayola Akim, Lalremruata, Albert, Kremsner, Peter G., Fendel, Rolf, Sandri, Thaisa Lucas
• Format: Article
• Language: English
• Subjects: Antigens, Protozoan - genetics; Cross-Sectional Studies; Electrophoresis, Capillary; High-Throughput Screening Assays - methods; High-Throughput Screening Assays - standards; Humans; Malaria, Falciparum - diagnosis; Malaria, Falciparum - epidemiology; Malaria, Falciparum - parasitology; Plasmodium falciparum - genetics; Protozoan Proteins - genetics; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Research paper; Sensitivity and Specificity
• ISSN: 2352-3964; 2352-3964
• DOI: 10.1016/j.ebiom.2019.10.048

Plasmodium falciparum deficient for hrp2 and hrp3 genes are a threat to malaria management and elimination, since they escape widely used HRP2-based rapid diagnostic tests and treatment. Hrp2/hrp3 deletions are increasingly reported from all malaria endemic regions but are currently only identified by laborious methodologies. We developed a novel hydrolysis probe-based, quantitative, real-time PCR (4plex qPCR) for detection and discrimination of P. falciparum infection (cytb) and hrp2 and hrp3 gene status, and to control assay validity (btub). A cross-sectional, diagnostic accuracy study was performed in Gabon for assay validation and deletion screening. In parallel to identification of P. falciparum infection in samples down to 0.05 parasites/µl, the 4plex qPCR enabled specific and valid interrogation of the parasites´s hrp2 and hrp3 genes in one go - even in low parasitemic samples. The assay was precise and robust also when performed in a routine healthcare setting in Gabon. The risk of falsely identifying hrp2 or hrp3 deletion was reduced by 100-fold compared to conventional PCR. Evaluation against microscopy was performed on 200 blood samples collected in Gabon: sensitivity and specificity of 4plex qPCR (cytb) were 100% and 80%, respectively. Stringent testing revealed hrp2 deletion in 2 of 95 P. falciparum positive and validated samples. The novel 4plex qPCR is sensitive, accurate and allows resource-efficient rapid screening. Monitoring and mapping of hrp2/hrp3 deletions is required to identify areas where control strategies may need to be adapted to ensure appropriate patient care and ultimately achieve malaria elimination. BMBF (03VP00402).