Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA

Abstract

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/l. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ϳ100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms.

Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood

“…Candidate primer sets targeting dhfr were designed for P. vivax, P. malariae, and P. ovale and were accepted if they had 100% alignment to all available sequences of the target species and reduced alignment to other species. The species-specific regions targeted for P. falciparum and P. knowlesi were based on prior reports (17,18), and the corresponding published primers were adopted with minor modifications (Table 1). Primer conditions were optimized using control nucleic acids for each species, and the expected melting temperature for each species amplicon was determined.…”

“…Primer sensitivities were evaluated in silico by querying with BLAST against all complete dhfr coding sequences available for the respective species in the NCBI nucleotide database: 19 sequences for P. vivax, 8 for P. ovale, and 9 for P. malariae. Species-specific primers targeting repetitive sequences in P. falciparum and P. knowlesi have been described previously (17,18) and were adopted with minor modifications (Table 1). The pan-Plasmodium primers target highly conserved sequences in the 18S rRNA gene and encompass a region of ϳ320 bp with internal sequence divergence that is sufficient to allow for species discrimination by sequencing.…”

“…The human beta actin primer set, which serves as an extraction control, has been described previously (19). a Species-specific primers targeting repetitive sequences in P. falciparum and P. knowlesi were described previously (17,18) and were adopted with minor modifications.…”

Molecular Testing for Plasmodium falciparum by Use of Serum or Plasma and Comparison with Microscopy and Rapid Diagnostic Testing in Febrile Nigerian Patients

“…This multiplex assay contains primers and probes for two possible malaria targets: the Pfr364 repetitive element, which is specific to P. falciparum, and a region of the 18S rRNA gene that is conserved across the five Plasmodium species known to cause human disease. Given that more copies of the Pfr364 element are present in the P. falciparum genome than in the 18S rRNA target, samples with detectable signal for Pfr364 were interpreted as positive for P. falciparum, even in the absence of signal for the 18S target (16). Samples with detectable signal only for the 18S target were considered positive for "Plasmodium, nonfalciparum."…”

<strong>A portable image-based cytometer for rapid malaria detection and quantification</strong>

“…Due to the recent advancements in technology, several malaria diagnostic techniques such as microarray [17], PCR [18], loop-mediated isothermal amplification (LAMP) [19], flow cytometry [20], hemozoin detection using automated hematology analyser [21] have been developed for efficient malaria diagnosis. Diagnostic methods leveraging PCR, 18s-rRNA detection [22], mitochondrial cytochrome b activity [23,24], PgMt19 and PfMT869 mitochondrial regions [25], and the Pvr47 and Pfr364 genes [26,27], have been used for detecting Plasmodium species. However, quantitative amplification of genes demands careful processing of blood to remove the inhibitors of amplification and thermal cycling, making it a cumbersome procedure.…”