Evaluation of Diagnostic Performance of Multiplex Real Time PCR for the Diagnosis of Malaria in Malaria Elimination Targeted Settings of Ethiopia.
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Date
2021-09
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Addis Abeba University
Abstract
Background: Malaria incidence has declined in Ethiopia in the past ten years. Current malariadiagnostic tests, including light microscopy and antigen-detecting rapid tests (RDTs) cannotreliably detect low-density infections. Studies have shown that nucleic acid amplification testsare highly sensitive and specific in detecting malaria infection. Thus, this study took place withthe aim of evaluating the performance of multiplex real time PCR for the diagnosis of malariausing patient samples collected from health facilities located at malaria elimination targeted lowtransmission settings in Ethiopia.
Methods: A health facility based cross sectional survey was conducted in selected malariasentinel sites. Malaria suspected febrile outpatients referred to laboratory for malaria testingbetween December 2019 and March 2020 were enrolled into this study. Socio demographicinformation and capillary blood samples were collected from the study participants and tested atspot with RDTs. Additionally, five circles of dry blood sample (DBS) samples on Whatman filterpaper and thick and thin smear were prepared for molecular testing and microscopicexamination, respectively. Multiplex real time PCR assay was performed at EPHI malarialaboratory. The performance of multiplex real time PCR assay, microscopy and RDT for thediagnosis of malaria was compared and evaluated against each other.
Results: Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as P.falciparum infection, 16 as P. vivax and 3 as mixed infections. Of the total samples, lightmicroscopy detected 33 as Pf, 18 as PV and RDT detected 43 as Pf, 17 as PV, and one mixedinfection. Using light microscopy as reference test, the sensitivity and specificity of multiplexreal time PCR were 100% (95% CI [93-100]) and 83.2% (95% CI [77.6-87.9]), respectively.Using multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58%(95% CI [46.9-68.4] and 67% (95% CI [56.2-76.7]); and specificity of 100% (95% CI [98-100]and 98.9 (95% CI (96-99.9), respectively. Substantial level of agreement was reported betweenmicroscopy and multiplex real time PCR results with kappa value of 0.65.
Conclusions: Multiplex real time PCR had an advanced performance in parasite detection andspecies identification on febrile patients’ samples than did microscopy and RDT in low malaria transmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria elimination program, particularly for community based epidemiological samples. Althoughmicroscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinical facilities.
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Malaria elimination, Multiplex real time PCR, Diagnostic performance