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

TY - JOUR

T1 - Multiplex-RT-PCR-ELISA panel for detecting mosquito-borne pathogens: Plasmodium sp. preserved and eluted from dried blood spots on sample cards

AU - Koliopoulos, Philip

AU - Kayange, Neema Mathias

AU - Daniel, Tim

AU - Huth, Florian

AU - Gröndahl, Britta

AU - Medina-Montaño, Grey Carolina

AU - Pretsch, Leah

AU - Klüber, Julia

AU - Schmidt, Christian

AU - Züchner, Antke

AU - Ulbert, Sebastian

AU - Mshana, Steven E

AU - Addo, Marylyn

AU - Gehring, Stephan

PY - 2021/2/1

Y1 - 2021/2/1

N2 - 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.

AB - 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.

KW - Child

KW - Child, Preschool

KW - Diagnostic Tests, Routine/methods

KW - Dried Blood Spot Testing/methods

KW - Humans

KW - Infant

KW - Mosquito Vectors/parasitology

KW - Multiplex Polymerase Chain Reaction/methods

KW - Plasmodium/isolation & purification

KW - Reverse Transcriptase Polymerase Chain Reaction/methods

KW - Sensitivity and Specificity

KW - Tanzania

U2 - 10.1186/s12936-021-03595-4

DO - 10.1186/s12936-021-03595-4

M3 - SCORING: Journal article

C2 - 33526038

VL - 20

JO - MALARIA J

JF - MALARIA J

SN - 1475-2875

IS - 1

M1 - 66

ER -