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Updated: Nov 19, 2025
Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay
Published on: September 30, 2021
Multiplex-RT-PCR-ELISA panel for detecting mosquito-borne pathogens: Plasmodium sp. preserved and eluted from dried
Philip Koliopoulos1, Neema Mathias Kayange2, Tim Daniel1
1Center of Pediatric and Adolescent Medicine, University Medical Center, Mainz, Germany.
Insights
A new multiplex assay accurately identifies nine mosquito-borne pathogens, including Plasmodium, in children with fever. This diagnostic tool can reduce misdiagnosis and unnecessary medication in low-resource settings.
Area of Science:
- Tropical Medicine
- Molecular Diagnostics
- Public Health
Background:
- Children in low-resource countries are highly vulnerable to malaria and other vector-borne diseases.
- Feverish infants in the Lake Victoria region often receive misdiagnosed and overtreated conditions.
- Accurate identification of prevalent mosquito-borne pathogens is crucial to reduce unnecessary antibiotic and antimalarial prescriptions.
Purpose of the Study:
- To develop and validate a multiplex assay for identifying key mosquito-borne pathogens in pediatric patients.
- To compare the diagnostic performance of the multiplex assay against existing methods like blood smear microscopy and RDTs.
- To assess the stability of pathogen samples preserved on collection cards for potential field use.
Main Methods:
- A literature review identified prevalent mosquito-borne pathogens in sub-Saharan Africa.
- A multiplex-reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay (multiplex-RT-PCR-ELISA) was designed to detect eight arboviruses and Plasmodium sp.
- Blood samples from 132 febrile children were analyzed using the multiplex-RT-PCR-ELISA, blood smear microscopy, and RDTs.
Main Results:
- The multiplex-RT-PCR-ELISA successfully amplified all nine target mosquito-borne pathogens.
- Plasmodium sp. infection was confirmed in 27 out of 132 children by multiplex-RT-PCR.
- Malaria RDTs showed high sensitivity (96.3%) and specificity (98.1%), while blood smear microscopy had lower sensitivity (40%).
- Preserved samples on collection cards remained viable for 186 to 312 days.
Conclusions:
- The multiplex-RT-PCR-ELISA is a highly accurate diagnostic tool for identifying multiple vector-borne pathogens.
- This assay offers a reliable, point-of-care alternative to laboratory-dependent methods.
- Implementing advanced diagnostics like multiplex-RT-PCR-ELISA can significantly reduce childhood mortality and inappropriate medication use.
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.
