Multiplex-RT-PCR-ELISA panel for detecting mosquito-borne pathogens: Plasmodium sp. preserved and eluted from dried blood spots on sample cards

Abstract

AbstractBackgroundChildren are the most vulnerable group affected by malaria and other tropical, vector-borne diseases in low-resource countries. Infants presenting with acute onset fever represent a major sector of outpatient care in the Lake Victoria region. Misclassification and overuse of antibiotics and anti-malarial medications are consistent problems. Identifying the prevalent mosquito-borne pathogens in the region will reduce the prescription of non-indicated medicines.MethodsThe literature was reviewed focusing on the mosquito-borne pathogens most prevalent in sub-Saharan Africa. Accordingly, an assay comprised of a multiplex-reverse transcriptase-polymerase chain reaction and an enzyme-linked immunosorbent assay (multiplex-RT-PCR-ELISA) was designed and validated in its ability to identify and differentiate nine human mosquito-borne pathogens including eight arboviruses andPlasmodiumsp., the aetiologic agents of malaria. Blood samples obtained from 132 children suspected of having malaria were spotted and preserved on Whatman®903 protein sample cards. Multiplex-RT-PCR-ELISA analysis was assessed and compared to results obtained by blood smear microscopy and the malaria rapid diagnostic test (RDT).ResultsNine out of nine pathogens were amplified specifically by the multiplex-RT-PCR-ELISA panel. Twenty-seven out of 132 paediatric patients presenting with acute fever were infected withPlasmodiumsp., confirmed by multiplex-RT-PCR. The results of blood smear microscopy were only 40% sensitive and 92.8% specific. The malaria RDT, on the other hand, detected acutePlasmodiuminfections with 96.3% sensitivity and 98.1% specificity. The preservation ofPlasmodiumsp.in clinical sera and whole blood samples spotted on sample cards was evaluated. The duration of successful, sample card storage was 186 to 312 days.ConclusionsReliable, easy-to-use point of care diagnostic tests are a powerful alternative to laboratory-dependent gold standard tests. The multiplex-RT-PCR-ELISA amplified and identified nine vector-borne pathogens includingPlasmodiumsp.with great accuracy. Translation of improved diagnostic approaches, i.e., multiplex-RT-PCR-ELISA, into effective treatment options promises to reduce childhood mortality and non-indicated prescriptions.