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

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.

Molecular epidemiology of malaria parasite amongst patients in a displaced people’s camp in Sudan

“…Malaria is a fatal vector-borne tropical disease that remains one of the leading causes of death in many developing countries [1]. The disease continues to pose global public health challenges, and its related morbidity and mortality remain significantly high in endemic countries such as Sudan.…”

“…Thus, highly sensitive diagnostic methods are necessary. In recent years, several molecular methods have been developed and evaluated for the detection of Plasmodium species, and various sensitivities and specificities have been reported [1]. Amongst them, polymerase chain reaction (PCR) is the most frequently used method in the field [8].…”

“…Amongst them, polymerase chain reaction (PCR) is the most frequently used method in the field [8]. PCR has also been helpful in the differential detection of all malaria parasites up to species levels, thereby revealing the high prevalence of mixed infections [1,9]. The application of sensitive methods such as PCR to determine the prevalence of Plasmodium species will allow better documentation of malaria epidemiology [10] and overcome the lack of knowledge on the prevalence of malaria infection in the displaced population.…”

Development of a deep amplicon sequencing method to determine the proportional species composition of piroplasm haemoprotozoa as an aid in their control

“…Next generation genomic resources have potential applications in the diagnosis, surveillance, treatment and control of haemoprotozoan diseases, as well as in the evaluation of parasite population responses to drug treatments and other control strategies. Determination of sequence variations in the hyper-variable 18S rDNA cistron can discriminate between haemoprotozoan parasites (Gubbels et al , 1999) and overcome limitations of traditional gross parasitological methods for the diagnosis of haemoprotozoa at species level (Agudelo et al , 2013; Haanshuus et al , 2013; Lee et al , 2015; Lefterova et al , 2015; Mens et al , 2006; Rougemont et al , 2004; Steenkeste et al , 2009). Various PCR methods (reverse line blot (RLB)-PCR, quantitative (q)PCR, multiple PCR) have been described to amplify the 18S region for sequence determination, but these are low throughput, hence relatively expensive, and can be error-prone (Bilgic et al , 2013; Chaisi et al , 2013; Gubbels et al , 1999; Kundave et al , 2018).…”

A novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

“…High-throughput genomic resources have potential applications in the surveillance, diagnosis, control, and treatment of haemoprotozoan diseases, as well as to study the parasite biology (Chaudhry et al, 2019; Shaukat et al, 2019). Determination of sequence variations in the hypervariable 18S rDNA cistron can discriminate between Plasmodium species (Agudelo et al, 2013; Haanshuus et al, 2013; Lee et al, 2015; Lefterova et al, 2015), and overcome limitations of traditional microscopic and immunochromatographic methods for the diagnosis of this group of parasites at species level. Molecular methods including qPCR, species-specific PCR, nested PCR, and multiplex PCR have been described (Canier et al, 2013; Cunha et al, 2009; Das et al, 1995; Echeverry et al, 2016; Haanshuus et al, 2013; Steenkeste et al, 2009), but these are low throughput, hence relatively expensive (Chaudhry et al, 2019).…”