Diagnosing infection levels of four human malaria parasite species by a polymerase chain reaction/ligase detection reaction fluorescent microsphere-based assay

Abstract

Improving strategies for diagnosing infection by the four human Plasmodium species parasites is important as field-based epidemiologic and clinical studies focused on malaria become more ambitious. Expectations for malaria diagnostic assays include rapid processing with minimal expertise, very high specificity and sensitivity, and quantitative evaluation of parasitemia to be delivered at a very low cost. Toward fulfilling many of these expectations, we have developed a post-polymerase chain reaction (PCR)/ligase detection reaction-fluorescent microsphere assay (LDR-FMA). This assay, which uses Luminex FlexMAP microspheres, provides simultaneous, semi-quantitative detection of infection by all four human malaria parasite species at a sensitivity and specificity equal to other PCR-based assays. In blinded studies using P. falciparum-infected blood from in vitro cultures, we identified infected and uninfected samples with 100% concordance. Additionally, in analyses of P. falciparum in vitro cultures and P. vivax-infected monkeys, comparisons between parasitemia and LDR-FMA signal intensity showed very strong positive correlations (r > 0.95). Application of this multiplex Plasmodium species LDR-FMA diagnostic assay will increase the speed, accuracy, and reliability of diagnosing human Plasmodium species infections in epidemiologic studies of complex malaria-endemic settings.

Figure 1

Post-polymerase chain reaction (PCR) ligase detection reaction-fluorescent microsphere assay (LDR-FMA) for diagnosis of human Plasmodium parasite species. A, Components of the Plasmodium species LDR-FMA. Species-specific TAG sequence-labeled primers (F = P. falciparum; V = P. vivax; M = P. malariae; and O = P. ovale) carry unique 5′ sequence extensions. These primers anneal to species-specific DNA sequence polymorphisms in the small subunit ribosomal RNA gene target. Conserved sequence primers are 5′ phosphorylated (P) and 3′ biotinylated. B, These primers anneal to a DNA sequence conserved among the human malaria parasite species. Species-specific anti-TAG probes hybridize to the unique 5′ TAG of the species-specific primers; probes are coupled to uniquely fluorescing microspheres represented by attached filled circles (Black = P. falciparum; white = P. vivax; diagonal stripe = P. malariae; horizontal strip = P. ovale). The orientation of species-specific and conserved sequence primers during ligase detection reactions is shown. Thermostable DNA ligase ligates (star) these primers when exact sequence hybridization occurs between the upstream and downstream primers. C, Hybridization of species-specific anti-TAG probes to LDR products and labeling of the 5′ end of these molecules with the fluorescent classification microsphere. D, Incubation of LDR products in buffer containing streptavidin-R-phycoerythrin (SA:PE) labels the 3′ end of these molecules with reporter fluorescence.

Figure 2

Semi-quantitative detection of Plasmodium species small subunit (ssu) ribosomal RNA (rRNA) polymerase chain reaction products by ligase detection reaction-fluorescent microsphere assay (LDR-FMA). Each experiment was performed on genomic DNA extracted from non-human primate blood samples individually infected with A, P. falciparum (5.0 × 10⁴ infected red blood cells [IRBCs]/μL), B, P. vivax (4.4 × 10⁴ IRBCs/μL), C, P. malariae (1.5 × 10⁴ IRBCs/μL), and D, P. ovale (3.6 × 10² IRBCs/μL). Three-fold serial dilutions were performed on these genomic DNA samples to represent parasitemias ranging between approximately 1,000 and 0.1 IRBCs/μL. Individual semi-quantitative detection assays were performed following PCR amplification (27 cycles) of single-species ssu rRNA template sequences. Each detection experiment was performed using multiplex LDR and FlexMAP™ anti-TAG probe hybridization master mixes. For these experiments, the Bio-Plex array reader was set to assay 75 microsphere plus phycoerythrin fluorescence (molecules doubly labeled with classification and reporter fluorescence); P. falciparum microsphere set 78, P. vivax microsphere set 37, P. malariae microsphere set 30, and P. ovale microsphere set 14. Highly significant linear fit between mean fluorescent intensity (MFI) (log₁₀ transformed) and the number of IRBCs/μL (log₁₀ transformed) were observed for each individual Plasmodium species experiment; R² = 0.91–0.96.

Figure 3

Comparison of semi-quantitative microscopy and post-polymerase chain reaction (PCR) detection of Plasmodium vivax infection in Aotus monkeys. Blood samples from 11 different Aotus monkeys infected with P. vivax (Sal I or Chesson strains) were collected at 4–9 time points. A, Linear relationship between blood smear microscopy and ligase detection reaction-fluorescent microsphere assay. MFI = mean fluorescent intensity. B, Linear relationship between blood smear microscopy and real-time PCR. Blood smear parasitemia ranged between 0 and 36,000 infected red blood cells (IRBCs)/μL of whole blood. Monkeys were not receiving antimalarial treatment during the time of these infections. RTPCR Ct = reverse transcription-PCR cycle number.