Evaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia

Abstract

Background Malaria incidence has declined in Ethiopia in the past 10 years. Current malaria diagnostic tests, including light microscopy and rapid antigen-detecting diagnostic 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. 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 was enrolled into this study. Sociodemographic information and capillary blood samples were collected from the study participants and tested at spot with RDTs. Additionally, five circles of dry blood spot (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 Ethiopian Public Health Institute (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 Plasmodium falciparum infection, 16 as Plasmodium vivax and 3 as mixed infections. Of the total samples, light microscopy detected 33 as P. falciparum, 18 as P. vivax, and RDT detected 43 as P. falciparum, 17 as P. vivax, 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 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 programme, 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.

Comparison of Rapid Diagnostic Test, Microscopy, and Polymerase Chain Reaction for the Detection of Plasmodium falciparum Malaria in a Low-Transmission Area, Jazan Region, Southwestern Saudi Arabia

“…This study showed that the performance of RDT and microscopy decreased as age increased, agreeing with previous studies [ 20 , 36 , 47 ]; this could be explained by age-dependent immunity from frequent exposure [ 20 , 62 ], which could be independent of parasite density [ 63 ]. Interestingly, this study showed that RDT and microscopy had significantly higher sensitivity among non-Saudi patients.…”

“…It also accurately detects mixed-species infections; thus, it has been widely used for diagnosis, epidemiological surveys, and drug efficacy trials [ 10 , 20 ]. Furthermore, several studies have used nested PCR as a reference method to assess the performance of malaria diagnostics in different settings [ 20 , 22 , 33 , 34 , 35 , 36 ]. In this study, PCR was superior to microscopy and RDT for diagnosing P. falciparum malaria.…”

Feasibility of community at-home dried blood spot collection combined with pooled reverse transcription PCR as a viable and convenient method for malaria epidemiology studies

“…Most previous studies that utilized DBS for parasite detection relied on quantitative PCR of the Plasmodium 18S rRNA coding genes [22][23][24][25][26][27][28][29][30][31], but DNA-based DBS tests are less sensitive than qRT-PCR for the highly expressed Plasmodium 18S rRNAs, which means more infections may be missed by standard DNA-based tests [32]. Ultrasensitive DNA-based tests, such as var-ATS as well as 18S qRT-PCR assays can detect low levels of parasites from DBS [32][33][34][35][36][37][38].…”

Field evaluation of the novel One Step Malaria Pf and Pf/Pv rapid diagnostic tests and the proportion of HRP-2 gene deletion identified on samples collected in the Pwani region, Tanzania

“…Thin and thick blood slides remained as gold standard for malaria diagnosis to date (Mathison and Pritt 2017 ) (Belachew et al 2022 ). The rapid diagnostic tests (RDTs) and microscope are the common tests that are routinely used for the management of malaria cases in Tanzania, although microscope remained to be the gold standard [11] [12].…”