Molecular detection of human Plasmodium species in Sabah using PlasmoNex™ multiplex PCR and hydrolysis probes real-time PCR.

Ping Chin Lee,Kek Heng Chua,Eric Tzyy Jiann Chong,Yvonne Al Lim,Ching Hoong Chew,Fread Anderios

doi:10.1186/s12936-015-0542-5

Ping Chin Lee, Kek Heng Chua + Show 4 more

Open Access

https://doi.org/10.1186/s12936-015-0542-5

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Abstract

BackgroundMalaria is a vector borne-parasitic disease transmitted through the bite of the infective female Anopheles mosquitoes. Five Plasmodium species have been recognized by World Health Organization (WHO) as the causative agents of human malaria. Generally, microscopic examination is the gold standard for routine malaria diagnosis. However, molecular PCR assays in many cases have shown improvement on the sensitivity and specificity over microscopic or other immunochromatographic assays.MethodsThe present study attempts to screen 207 suspected malaria samples from patients seeking treatment in clinics around Sabah state, Malaysia, using two panels of multiplex PCRs, conventional PCR system (PlasmoNex™) and real-time PCR based on hydrolysis probe technology. Discordance results between two PCR assays were further confirmed by sequencing using 18S ssu rRNA species-specific primers.ResultsOf the 207 malaria samples, Plasmodium knowlesi (73.4% vs 72.0%) was the most prevalent species based on two PCR assays, followed by Plasmodium falciparum (15.9% vs 17.9%), and Plasmodium vivax (9.7% vs 7.7%), respectively. Neither Plasmodium malariae nor Plasmodium ovale was detected in this study. Nine discrepant species identification based on both the PCR assays were further confirmed through DNA sequencing. Species-specific real-time PCR only accurately diagnosed 198 of 207 (95.7%) malaria samples up to species level in contrast to PlasmoNex™ assay which had 100% sensitivity and specificity based on sequencing results.ConclusionsMultiplex PCR accelerate the speed in the diagnosis of malaria. The PlasmoNex™ PCR assay seems to be more accurate than real-time PCR in the speciation of all five human malaria parasites. The present study also showed a significant increase of the potential fatal P. knowlesi infection in Sabah state as revealed by molecular PCR assays.

Highlights

Malaria is a vector borne-parasitic disease transmitted through the bite of the infective female Anopheles mosquitoes
Species-specific real-time PCR indicated that 202 malaria samples were caused by single-species infection, i.e., 16 (7.7%), 37 (17.9%), 149 (72.0%) by P. vivax, P. falciparum, and P. knowlesi, respectively, while determination up to species level based on species-specific primers and probes failed for the balance five samples
Two samples diagnosed as P. falciparum infection based on multiplex real-time PCR assay were infected with P. knowlesi based on sequencing results and BLAST data, which were in agreement with the results obtained from PlasmoNexTM assay

Summary

Introduction

Malaria is a vector borne-parasitic disease transmitted through the bite of the infective female Anopheles mosquitoes. Malaria is a mosquito-borne parasitic disease cause by the unicellular, eukaryotic protozoan parasites of the genus Plasmodium and the infective female Anopheles mosquitoes are the sole vector of human-to-human transmission. The malaria main focal regions in Malaysia include Sabah and Sarawak states situated on the Borneo Island and central interior regions of Peninsular Malaysia. These areas are the home for a majority of the isolated indigenous populations. Microscopy (parasite morphology identification), immunochromatographic-based rapid diagnostic test (antigen detection), and molecular PCR assays (parasite nucleic acid detection) are the three main malaria diagnostic methods and they target the parasites in the peripheral blood with wide ranges of sensitivity and specificity as reviewed by Moody [3]

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