PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods

Plasmodium genomic DNAs of diverse geographic origin (MRA numbers, 102G, 149G, 150G, 152G, 153G, 157G, 159G, 160G, 161G, 163G, 165G, 167G, 168G, 176G, 200G, 201G, 202G, 273G, 274G, 275G, 276G, 340G, 341G) and four plasmid clones (MRA number 177–180) carrying a PCR insert of the ssrRNA gene amplified from either P. falciparum, Plasmodium vivax, Plasmodium malariae or Plasmodium ovale were obtained from the Malaria Research and Reference Reagent Resource Center (ATCC, Manassas, Virgnia, USA). Additional Plasmodium DNAs were kindly provided by Dr Debbie Nolder at the HPA Malaria Reference Laboratory, London School of Hygiene & Tropical Medicine, UK. To determine the sensitivity of each of the PCR methods a dilution series of three DNA samples for each of the four human Plasmodium species was used. For this, the DNAs were diluted to 20 ng/μl (as determined by absorption at 260 nm using a NanoDrop spectrophotometer, NanoDrop Technologies, Wilmington, DE) and then serial dilutions were made down to 1 in 1 × 10⁶ (20 fg/μl).

The PCR methods described in this study were tested in a blind trial using artificially infected Anopheles stephensi. For this mature P. falciparum 3D7 gametocytes were cultured in continuous culture apparatus as described previously [15] for periods of 14–18 days at 37°C under 3% O₂, 5% CO₂, 92% N₂ continuous gas flow. The initial haematocrit of cultures was 7% (v/v) RBC in RPMI medium (Gibco), containing 2 g/l NaHCO₃, 25 mM HEPES, 50 μg/ml hypoxanthine and 10% (v/v) human group 'AB' heat inactivated serum and the medium was changed every 12 hours. After 14 days, gametocyte quality and quantity was tested every 24 hours by giemsa smears and an exflagellation test. Fifty fields of a monolayer of infected erythrocytes were examined at 40 × magnification by phase-contrast optics and the numbers of centres of exflagellation following incubation at 24°C for 20 mins were recorded. Mature cultures showing high levels of exflagellation were identified, harvested and fed to An. stephensi mosquitoes. During this process, gametocyte cultures were spun at 500 g for 4 min, the supernatant removed and the pellet containing parasitized red blood cells was mixed with 700 μl of freshly washed and pre-warmed uninfected RBC. 1 ml of pre-warmed heat inactivated AB serum was then added to this mixture, which was then fed to pots of 50 mosquitoes (starved 5–6 hours pre-feed) via 500 μl mini-feeders, as described previously [16]. Mosquitoes were allowed to engorge on each blood feed for 20 min through a Parafilm membrane, warmed to 37°C using a water jacket. After feeding, mosquitoes were kept at 25–26°C, 80 % humidity with access to 5% glucose, 0.05 % para-aminobenzoic acid (PABA) solution. Individual mosquitoes were assessed for the presence of sporozoites by salivary gland dissection in TE buffer at 16 days post-feed. The dissected glands and remains of the head and thorax in 100 μl TE buffer were then frozen at -20°C until use. The trial plate consisted of eighty four mosquitoes that had been dissected and scored as either sporozoite positive (n = 36) or negative (n = 48). As low level infections can be easily missed by dissection [17] an additional ten mosquitoes that had no prior exposure to P. falciparum were also dissected in the same way and included in the trial as negative controls. Two buffer only wells were also included as additional negative controls. The samples were heated to 99°C for ten minutes to release the DNA from sporozoites then cooled on ice and five μl used as template in PCR.

PCR methods were subsequently tested with 483 field-collected mosquitoes collected from Equatorial Guinea, Ghana, Kenya, and Mozambique. These were morphologically identified as Anopheles gambiae sensu lato (s.l) or Anopheles funestus before storing on silica gel, in 70% ethanol, or in isopropanol. Mosquitoes from each storage condition were divided into two groups and then DNA extracted from the head and thorax of single mosquitoes using either the Livak or DNAzol methods [18, 19]. Mosquitoes were identified to species by allele-specific PCR [20, 21] or a new TaqMan protocol [22]. The trial of PCR methods using these samples comprised of 263 An. gambiae individuals, 184 An. funestus individuals and 36 An. arabiensis individuals and five water negative controls.

Agreement between the results of the TaqMan assay and the nested PCR 'gold standard' reference method was measured using Cohen's Kappa measure of test association [23].

Single and nested PCRs were carried out as described previously [9, 11].

Nucleotide alignment of the small subunit ribosomal RNA gene sequences of the four human infecting Plasmodium species available in the National Center for Biotechnology Information (NCBI) database revealed a region that contained two single nucleotide polymorphisms (SNPs) four base pairs apart specific to P. falciparum flanked by an area of conserved sequence. Two minor groove binding (MGB) probes (Applied Biosystems) that bind over both SNPs were designed using the Primer Express™ Software Version 2.0. The probe Falcip+ (5'-TCTGAATACGAATGTC-3') was labelled with 6-FAM at the 5' end for the detection of P. falciparum and the probe OVM+ (5'-CTGAATACAAATGCC-3') was labelled with VIC for the detection of either P. malariae, P. ovale or P. vivax. Each probe also carried a 3' nonfluorescent quencher and a minor groove binder at the 3' end. The minor groove binder provides more accurate allelic discrimination by increasing the T_M between matched and mis-matched probes [24]. Forward, PlasF (5'-GCTTAGTTACGATTAATAGGAGTAGCTTG-3') and reverse, PlasR (5'-GAAAATCTAAGAATTTCACCTCTGACA-3') primers that flank the probe binding site were also designed using the Primer Express software.

PCR reactions (20 μl) contained 1 μl of genomic DNA, 10 μl of SensiMix DNA kit (Quantace), 800 nM of each primer and 300 nM of probe PlasF and 200 nM of probe OVM+. Reactions were run on a Rotor-Gene 6000™ (Corbett Research) using the temperature cycling conditions of: 10 minutes at 95°C followed by 40 cycles of 95°C for 10 seconds and 60°C for 45 seconds. The increase in VIC and FAM fluorescence was monitored in real time by acquiring each cycle on the yellow (530 nm excitation and 555 nm emission) and green channel (470 nm excitation and 510 emission) of the Rotor-Gene respectively.

PCR was carried out as described previously [10] except for modification of the annealing temperature from 45°C to 50°C.

The nested and single PCRs described by Snounou et al [9, 11] were optimized using Plasmodium genomic DNA as template and the sensitivity was compared to the other assays using the same set of DNA dilutions of each of the four Plasmodium species (an example using dilutions of P. falciparum DNA is shown in Figure 1). The nested PCR protocol showed a limit of detection for P. falciparum and P. vivax, at a 1 :10,000 dilution representing a low limit detection of 2 picograms of DNA. The limit of detection for P. ovale was a 1:100,000 dilution (0.2 pg DNA) and for P. malaria a 1:100 (0.2 ng DNA) dilution. The single PCRs showed a low limit detection of 1:5,000 (4 pg DNA) for P. falciparum and P. vivax and 1: 1,000 (20 pg DNA) for P. ovale and 1:10 (2 ng DNA) for P. malariae. The two PCR methods were then tested in a blind trial of 96 samples that included mosquitoes artificially infected with P. falciparum. The results are shown in full in Additional file 1 and summarized in Table 1. Both methods scored the ten 'P. falciparum un-exposed negative' mosquito samples and the buffer blanks as negative. Of the 36 samples which had scored positive by dissection, 33 also scored positive by nested PCR and of the 48 samples which had scored negative by dissection 10 were scored positive by nested PCR. The single PCR approach failed to detect P. falciparum in any of the samples.

Both PCR methods were then tested using DNA extracted from wild-caught An. gambiae s.s., An. arabiensis and An. funestus specimens that had been stored on silica gel, in ethanol or in isopropanol (Additional file 2 and Table 2). The single PCR approach was unable to detect Plasmodium in the first 192 samples tested and therefore the decision was made to abandon this method due to its apparent lack of sensitivity. The nested PCR approach recorded 12 P. falciparum positive and 1 P. vivax positive specimens from the DNAs of the 269 silica-dried mosquitoes. However PCR using the 215 DNA samples derived from mosquitoes stored in ethanol or isopropanol produced a high number of non-specific PCR products when screening for P. falciparum (Figure 2) preventing unambiguous scoring of these samples which were therefore designated as 'failed reactions'.

After minimal optimisation the TaqMan assay was able to detect all four human Plasmodium species using genomic DNA as template. This assay uses two probes, the first labelled with 6FAM detects P. falciparum and the second, labelled with VIC, detects P. vivax/P. ovale/P. malariae. Thus, a substantial increase in FAM fluorescence during PCR indicates the presence of P. falciparum whilst a substantial increase in VIC fluorescence indicates the presence of P. vivax, P. ovale or P. malariae as shown in Figure 3. An increase in both dyes would indicate a mixed infection. To help with species assignment the Rotor-Gene software allows endpoint fluorescence values for the two dyes to be automatically corrected for background and plotted against each other in bi-directional scatter plots (for an example see Figure 4). Tests of the assay using a range of Plasmodium genomic DNA samples and plasmid clones carrying a PCR insert of the ssrRNA gene showed the new method to be specific with all samples correctly scored (n = 36). This also confirmed that the two SNP sites over which the probes were designed are conserved in P. falciparum from several geographic regions. The analytical sensitivity of the TaqMan method was examined using a dilution series of three DNA samples for each of the four Plasmodium species. The limit of detection for each species was a 1:100,000 dilution equivalent to 0.2 picograms of DNA which is approximately 10 parasite genomes (an example using dilutions of P. falciparum DNA is shown in Figure 3). The performance of the optimized TaqMan assay was assessed in a blind trial of 96 samples that included An. stephensi mosquitoes artificially infected with P. falciparum. The results are shown in full in Additional file 1 and summarized in Table 1. The TaqMan assay scored the ten 'non-exposed negative' mosquito samples and the buffer controls as negative. Of the 36 samples assessed as positive by dissection 35 scored positive by TaqMan and of the 48 samples scored negative by dissection 12 scored positive by TaqMan. Thus close agreement was seen between the results of TaqMan and the 'gold standard' nested PCR approach (κ = 0.858).

Finally the TaqMan assay was tested using DNA extracted from wild-caught An. gambiae s.s., An. arabiensis and An. funestus specimens that had been stored on silica gel or in ethanol or isopropanol. Of the 483 samples 31 (6%) tested positive for P. falciparum and one sample (0.2%) tested positive for P. vivax/P. ovale/P. malariae (see Additional file 2 and Table 2). Of the positive samples 18 were An. gambiae s.s., 7 were An. funestus and 7 were An. arabiensis. The assay did not appear to be affected by any of the storage methods. When the results using the TaqMan method were compared with the Snounou nested PCR reference they were shown to be highly concordant, κ = 0.925 (this comparison did not include the samples stored in isopropanol/ethanol due to the failure of these samples in nested PCR).

The PCR protocol of Tassanakajon et al [10] was optimized using Plasmodium genomic DNA as template. This method is designed to detect only P. falciparum and the sensitivity using dilutions of P. falciparum DNA showed the limit of detection to be a 1 in 10,000 dilution (Figure 1) representing a low limit detection of 2 picograms of DNA. The performance of the method was assessed in a blind trial of 96 samples that included mosquitoes that had been artificially infected with P. falciparum. The results are shown in full in Additional file 1 and summarized in Table 1. The Tassanakajon assay scored the ten 'non-exposed negative' mosquito samples and the buffer controls as negative. Of the 36 samples scored positive by dissection 30 scored positive by Tassanakajon PCR and of the 48 samples scored negative by dissection 8 scored positive by Tassanakajon PCR.

The results of this method to test DNA extracted from wild-caught An. gambiae s.s., An. arabiensis and An. funestus specimens that had been stored on silica gel or in ethanol/isopropanol are shown in Additional file 2 and summarized in Table 2. A total of 53 of the 483 samples (11%) tested positive for P. falciparum. However, some non-specific PCR products were present in some of the samples analysed (including those stored on silica gel) using this method (Figure 2) which made the results more difficult to interpret. Attempts were made to improve the specificity of the PCR by modifying the annealing temperature in the original protocol from 45°C to 50°C, however, this did not completely eliminate the problem. The difficulties in interpreting some of the PCRs may partially explain the low agreement between this method and the nested PCR reference (κ = 0.264). In addition, no Plasmodium was detected in any of the 93 DNA samples from mosquitoes stored in ethanol.