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 malaria
diagnostic tests, including light microscopy and antigen-detecting rapid tests (RDTs) cannot
reliably detect low-density infections. Studies have shown that nucleic acid amplification tests
are highly sensitive and specific in detecting malaria infection. Thus, this study took place with
the aim of evaluating the performance of multiplex real time PCR for the diagnosis of malaria
using patient samples collected from health facilities located at malaria elimination targeted low
transmission settings in Ethiopia.
Methods: A health facility based cross sectional survey was conducted in selected malaria
sentinel sites. Malaria suspected febrile outpatients referred to laboratory for malaria testing
between December 2019 and March 2020 were enrolled into this study. Socio demographic
information and capillary blood samples were collected from the study participants and tested at
spot with RDTs. Additionally, five circles of dry blood sample (DBS) samples on Whatman filter
paper and thick and thin smear were prepared for molecular testing and microscopic
examination, respectively. Multiplex real time PCR assay was performed at EPHI malaria
laboratory. The performance of multiplex real time PCR assay, microscopy and RDT for the
diagnosis 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, light
microscopy detected 33 as Pf, 18 as PV and RDT detected 43 as Pf, 17 as PV, and one mixed
infection. Using light microscopy as reference test, the sensitivity and specificity of multiplex
real 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 between
microscopy and multiplex real time PCR results with kappa value of 0.65.
Conclusions: Multiplex real time PCR had an advanced performance in parasite detection and
species 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. Although
microscopy 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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Keywords
Malaria elimination, Multiplex real time PCR, Diagnostic performance