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

Abstract

Background Children 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. Methods The 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 and Plasmodium sp., 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). Results Nine 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 with Plasmodium sp., 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 acute Plasmodium infections with 96.3% sensitivity and 98.1% specificity. The preservation of Plasmodium sp. 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. Conclusions Reliable, 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 including Plasmodium sp. 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.

Challenges Diagnosing and Treating Acute Febrile Children with Suspected Malaria at Healthcare Facilities in the Mwanza Region, Tanzania

“…In this regard, dried blood spots are a valuable method of transporting P. falciparum-positive samples. In a previous analysis of the present clinical data, a high concordance between sera samples and a long-lasting preservation capability was documented [43]. Importantly, the capacity to preserve arboviruses is limited.…”

“…The protocol for sample collection was described previous [43,45]. The NADAL® Malaria Pf/Pan Ag 4 Species Test (nal von minden GmbH, Regensburg, Germany) was performed and the results of additional testing, i.e., blood smear microscopy and mRDTs, were obtained at the healthcare facility indicated.…”

“…The samples were shipped to the University of Mainz, Department of Pediatric Immunology and Infectious Diseases according to IATA and ICAO regulations. The sample card extraction procedure, including standardized punching, prevention of carryover contamination and incubation with vortexing for 60 min at 72°C was established by Koliopoulos et al [43].…”

“…Multiplex-RT-PCR-ELISA A multiplex reverse-transcriptase-polymerase-chain-reaction (MPCR) -ELISA panel for malaria-like diseases was designed by the Laboratory of Pediatric Immunology and Infectious Diseases, Mainz, Germany [43]. It covers 9 mosquito-transmitted diseases and includes a highly sensitive primer set that targets Plasmodium spp.…”

“…Total nucleic acid was isolated using the High Pure Viral Nucleic Acid Kit according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Subsequent ampli cation and gel electrophoresis was performed according to Koliopoulos et al [43]. The PCR product was then incubated with 3′biotinylated capture probe and anti-digoxigenin-peroxidase (Roche Diagnostics) on streptavidin-coated microtiter plates.…”