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/μl. 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 ∼100% 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.

Fig. 1.

Schematic of diagnostic target screening and development pipeline. All genomic sequences for P. vivax and P. falciparum were downloaded from PlasmoDB. Data were mined for repeats using the RepeatScout algorithm to construct consensus repeat sequences (CRS) for each identified repeat family. CRS were then screened in parallel for tandem repeats, similarity to human sequences, and vector sequences. Any CRS failing these screens were removed from further consideration. CRS that were not species specific or less than 300 bp long were eliminated. Family copy numbers for the remaining candidates were determined via comparison of the CRS against the appropriate genome data. Candidate repeat families containing 6 or more copies separated by at least 100 bp were considered for further testing. For additional information and clinical sample validation, see Materials and Methods.

Fig. 2.

Spatial distribution of Pfr364 family members across the 14 P. falciparum chromosomes. Tick marks indicate 200 kb of sequence. Pfr364 family members occur in two proximal copies at most chromosome ends. Black lines represent the outermost copies (subfamily 1), and gray lines represent the innermost copies (subfamily 2). Chromosome 6 has three copies at its 3′ end (only two are shown). Circos 0.51 (http://mkweb.bcgsc.ca/circos/) was used to generate this map.

Fig. 3.

Alignments of Pfr364 and Pvr47 family members with PCR primers. (A) Pfr364 with primers. Arrows represent locations of PCR primers in context of the full alignment. The full alignment is 1,538 positions in length; here a partial alignment is shown. Vertical black lines indicate where the sequence alignment has been truncated to enable viewing of all 4 primer locations. The alignment shows two subfamilies within Pfr364. We have designated the upper 22 sequences subfamily 1 and the lower 19 sequences subfamily 2. Forward and reverse primer pairs used for multiplex and conventional PCR are, respectively, the last two sequence pairs in the alignment. (B) Pvr47 with primers. Arrows represent locations of PCR primers in context of the full alignment. The full alignment is 1,070 positions; here only positions 433 to 776 are shown. Vertical lines indicate where the sequence alignment has been truncated for easier viewing. Forward and reverse primers are, respectively, the last two sequences in the alignment.

Fig. 4.

Limits of detection for conventional PCR assays. Primers to novel targets P. falciparum Pfr364 (A) and P. vivax Pvr47 (B) were used to amplify parasite DNA of the appropriate species. DNA was quantified, and 10-fold serial dilutions from 10,000 parasites/μl (lane 1) to 0.01 parasites/μl (lane 7) were used to determine the limit of detection. A 100-bp standard ladder (L) and a no-template control (NTC) were included.

Fig. 5.

Evaluation of Pfr364 and Pvr47 primers on geographically diverse field isolates. (A) Pfr364 primers tested on various P. falciparum isolates. Lanes: 1, 3D7; 2, W2; 3, V1-S; 4, Dd2; 5, Hb3; 6, D6; 7, FCR3. A 100-bp standard ladder (L) and a no-template control (N) were included. (B) Pvr47 primers tested on various P. vivax field isolates. Lanes: 1, Thailand; 2, North Korea; 3,Vietnam; 4, India; 5, NAM/CDC; 6, Miami; 7, New Guinea; 8, Sal-1; 9, South Vietnam; 10, Brazil. The Pfr364 and Pvr47 primers clearly detect all of the tested isolates. Pfr364 primers detected an additional 91/91 (100%) P. falciparum isolates from Tanzania, and Pvr47 primers detected an additional 95/96 (98.9%) P. vivax isolates from Venezuela (not shown).

Fig. 6.

Multiplex PCR. The multiplex method clearly identified mock mixed P. falciparum and P. vivax infections (lane Pf/Pv). Single-species infections (lanes Pf and Pv) were also detected. The P. falciparum band appears at 220 bp and the P. vivax band at 333 bp. A 100-bp standard ladder (L) and a no-template control (NTC) were used.