Multiplex, DNase-free one-step reverse transcription PCR for Plasmodium 18S rRNA and spliced gametocyte-specific mRNAs

mRNA expression fold-change from Lopez-Barragan et al. [28] was evaluated. Fold-change data was summed for asexual or gametocyte stages, sorted and filtered to remove mRNAs with total asexual expression ≥0 or total gametocyte expression ≤0 (all relative to ring stage expression). Single exon genes were eliminated as were those lacking data in http://​www.​plasmodb.​org and those representing redundant mRNA isoforms. Fold change data in [28] were based on RNASeq reads per kilobase of exon per million (RPKM). Since RPKM can originate from sense or antisense transcripts, the strand-specific fragments per kilobase of exon per million (FPKM) [28] were also inspected, and genes with schizont antisense FPKM equal to 50–100% of maximum were eliminated as were those with minimum asexual antisense FPKM within twofold of the maximum gametocyte antisense FPKM values. Remaining genes were sorted on stage V gametocyte expression, and the most highly expressed genes were evaluated for suitable intron-spanning splice RT-PCR designs. RPKM and FPKM data deposited in plasmodb.org from [28] for selected genes is in Additional file 1.

Primers and probes were designed to target exons using the web-based PrimerQuest tool (http://​www.​idtdna.​com/​Primerquest/​). Primers were commercially synthesized (IDT DNA, Coralville, IA, USA); primer set names (S1, S2, S3, etc.) were arbitrarily assigned by the IDT PrimerQuest software.

Asexual stage Plasmodium parasites were cultured as described [21]. Gametocytes were cultured as described [29]. Purity of cultures was confirmed by microscopy. Some asexual stage cultures were further confirmed gametocyte-free by pfs25 RT-PCR.

Leftover clinical whole blood specimens (50 μL) were preserved in 2 mL of NucliSens Lysis Buffer (bioMérieux, Marcy-l’Étoile, France). These samples were used under a protocol approved by the University of Washington Institutional Review Board (IRB) (protocol 47026, S. Murphy). Samples were included from a clinical trial of a novel chemical entity DSM265 approved under Fred Hutchinson Cancer Research Center IRB (protocol 8408, J. Kublin). Samples from a field study in Uganda were collected from consenting study participants or with the assent of children and consenting legal care givers within the context of studies approved by the relevant IRBs. Specimens from Nagongera, Tororo District, Uganda were collected under a study approved by the University of California San Francisco IRB 11-05995 and Uganda IRB 2011-0167. In Uganda, EDTA whole blood samples were collected from a previously described surveillance cohort [30]; all participants provided informed consent. Samples from Uganda were collected from May to October 2015.

Nucleic acids were extracted on a bioMérieux EasyMAG [21] or an Abbott m2000sp as described [22]. Briefly, on the bioMérieux instrument, 1 mL of the 2.05 mL lysed sample (50 µL whole blood plus 2 mL NucliSENS lysis buffer) was extracted for total nucleic acids and eluted into 40 µL of elution buffer. On the Abbott m2000sp instrument, 1 mL of the 2.05 mL lysed sample was extracted preferentially for RNA and eluted into 53 μL of elution buffer.

In initial studies of candidate primers, singleplex RT-PCR was performed using the AgPath mastermix kit as described by the manufacturer (Thermo Fisher Scientific, Waltham, MA) in the presence of LCGreen DNA-binding dye as described by the manufacturer (BioFire, Salt Lake City, UT). Some such RT-PCR products were evaluated by 1.5% agarose gel electrophoresis. For multiplex studies of spliced mRNA targets with 18S rRNA, pan-Plasmodium 18S rRNA RT-PCR was performed as described [31] with the addition of the candidate spliced gametocyte primers (200 nM) and probe (100 nM) on an Abbott m2000rt or Bio-Rad C1000 (Bio-Rad, Hercules, CA). Briefly, 15 µL of eluate was combined with 35 µL of mastermix as described in the SensiFAST LO-ROX one step RT-PCR kit product manual (Bioline, Tauton, MA). Cycling conditions were reverse transcription 10 min at 48 °C, 95 °C for 2 min followed by 45 cycles of 95 °C for 5 s followed by 50 °C for 35 s. For pfs25/18S rRNA multiplex RT-PCR, total nucleic acids were DNase treated (TURBO DNA-free Kit, Ambion/Life) and pfs25 and 18S rRNA RT-PCR performed using published cycling conditions and concentrations of Plasmodium 18S rRNA primers/probe [31] supplemented with pfs25 primers (400 nM) and probe (200 nM) [17]. Reagents for a gametocyte-specific mRNA from P. falciparum meiotic recombination protein DMC1-like protein gene (PF3D7_0816800) were as previously reported [26]: forward 5′-ATATCGGCAGCGAAAATGTGT-3′; reverse 5′-GACAATTCCCCTCTTCCACTGA-3′; probe 5′-(6-FAM)-TGCCCTTCTCGTAGTTGATTCGATTATT(BHQ1)-3′. Reagents for the early/mid- (PF3D7_1477700/PF14_0748) and mid/late-gametocyte expressed mRNAs (PF3D7_1438800/PF14_0367) were as previously reported [27]: PF3D7_1477700: forward 5′-CTTATGTGCTGAATTTTGTGTTATGGT-3′, reverse 5′-TTGGCCACACTGCTCTAGGA-3′, probe: 5′(VIC)-CACATAATGAATTCAAGGGTAG(MGBNFQ)-3′; PF3D7_1438800: forward 5′- GTTACATTTCGACCCAGCATAAATT-3′, reverse 5′-TCCCTGTGTTTTTGCTCATCTTC-3′, probe 5′-(VIC)-CAGTGCATATTGTTGCCTGT(MGBNFQ)-3′. All probes were 6-FAM-labelled and sourced from IDT (Coralville, IA) with the exceptions of the CAL Fluor orange 560-labeled pan-Plasmodium 18S rRNA hydrolysis probe (LCG Biosearch Technologies, Novato, CA) and the VIC-labeled PF3D7_1477700 and PF3D7_1438800 probes (Invitrogen). Where indicated, some reactions were tested at other annealing temperatures to investigate specificity. Quantification of parasites was achieved using an absolute 18S rRNA standard curve as described [22, 31], which allowed estimation of gametocyte-specific assay limits of detection (LOD). For gametocyte markers evaluated using whole parasite standard curves, C_T values up to 40.0 cycles were converted into quantitative values.

To evaluate unlysed sample stability, an unpaired t test was used to compare mean C_T values for 18S rRNA and gametocyte-specific markers and the difference in these C_T values. For sensitivity analyses, exact confidence intervals were calculated [32]. Statistical significance for both was P < 0.05.

One-thousand one-hundred twenty-nine genes evaluated previously by RNAseq [28] were filtered to remove those with increased asexual expression (sum of asexual fold change ≥0) and decreased gametocyte expression (sum of stage II and V gametocyte fold change ≤0). This resulted in 372 genes with gametocyte stage II and/or V expression and an absence of asexual-stage expression. The most well-known gametocyte target pfs25 (PF3D7_1031000) was one of the most highly expressed gametocyte-specific genes in this set (Additional file 1). Six genes were eliminated because they lacked data in plasmodb.org and all single exon genes were also eliminated. Two-hundred genes contained two or more exons. Since antisense transcripts are produced for many Plasmodium genes [28] and since such transcripts can lead to false positive results in primer-specific RT-PCRs [33‐36], antisense strand-specific FPKM data [28] were evaluated next. Genes with low or absent antisense transcript expression in non-gametocyte stages were retained (48 genes), sorted on stage V gametocyte expression and evaluated for intron-spanning primer/probe designs compatible with the Plasmodium 18S rRNA RT-PCR cycling temperatures. RT-PCR designs for 14 genes were ultimately selected using this strategy as were designs for four additional genes overexpressed in stage II/V gametocytes but not identified by published fold-change data in [28]; published RT-PCRs for pfs25 and three reported multi-exon gametocyte-specific genes [26, 27] were also included (Additional file 1). Based on recently published data [37], two genes were male-specific (PF3D7_1477700, PF3D7_1438800), one showed mixed expression (PF3D7_1020100) and the rest were female-specific (Additional file 1).

LCGreen RT-PCR was performed on 27 target regions of the 18 novel genes of interest using total nucleic acids obtained from either cultured mature P. falciparum gametocytes or from cultured synchronized ring-stage asexual parasites (Additional file 2). Although these RT-PCR assays do not provide absolute quantification, the difference (Δ) between C_T values for the 18S rRNA and the mRNA of interest provide an estimate of spliced mRNA abundance. In cultured samples enriched for mature gametocytes, gametocyte-specific spliced mRNAs were detected 6.9–25.2 cycles later than Plasmodium 18S rRNA (ΔC_{T Gam mRNA–18SrRNA}). In cultured samples containing only asexual-stage parasites, the ΔC_{T iRBC mRNA–18SrRNA} was typically larger than the aforementioned ΔC_{T Gam mRNA–18SrRNA} for each candidate mRNA, as expected for gametocyte-expressed mRNAs (Additional file 2). mRNAs with the smallest ΔC_{T Gam mRNA–18SrRNA} and the largest ΔC_{T iRBC mRNA–18SrRNA} were considered to represent the best balance between high and specific gametocyte expression. Gel electrophoresis was used to evaluate some RT-PCR products. For example, for PF3D7_0630000, a band of the expected size (141 bp) was only generated in the presence of gametocytes by RT-PCR (Fig. 1a). The probe for this target was designed to span an intron (Fig. 1b).

Four primer sets that specifically detected gametocytes by LCGreen, had RT-PCR efficiencies of 90–110% and had probe melting temperatures compatible with the 18S rRNA RT-PCR [PF3D7_0514500 (S1), PF3D7_0518800 (S1), PF3D7_0630000 (S3), PF3D7_1214500 (S1)] were further evaluated using hydrolysis probes (Table 1). These RT-PCRs detected gametocyte mRNA in gametocyte-containing samples (Fig. 2). However, PF3D7_1214500 (S1) reacted with some parasite-negative human blood samples and PF3D7_1214500 (S1) and PF3D7_0518800 (S1) occasionally gave false positives for blood from microscopically-pure cultured asexual-stage parasites, so further study of these targets was discontinued. Positive C_Ts for PF3D7_0630000 (S3) RT-PCR were ~2–4 cycles earlier than for PF3D7_0514500 (S2) so PF3D7_0630000 was prioritized for development.

PF3D7_0630000 (aliases MAL6P1.128 and PFF1455c) encodes a CPW-WPC family protein. CPW-WPC protein-coding mRNAs are expressed and post-transcriptionally regulated in gametocytes and later translated into surface proteins in ookinetes [38]. The PF3D7_0630000 (S3) reagents target exons 3–6 of the mature mRNA with the probe and reverse primer spanning introns 4 and 5, respectively (Fig. 1b). The spliced mRNA amplicon was 141 bp. Nucleic acids from a dilution series of mature gametocytes (Day 14 of culture) were tested using RT-PCRs for Plasmodium 18S rRNA, PF3D7_0630000 and pfs25 to generate a standard curve and evaluate assay efficiency and linearity (Fig. 3a; Table 2). Plasmodium 18S rRNA qRT-PCR was able to quantitatively detect as few as 30 parasites/mL of whole blood, consistent with the assay limit of detection of 20 parasites/mL; the C_T curve generated for the most dilute sample was below the assay limit of quantification. Probe-based qRT-PCR for pfs25 and PF3D7_0630000 detected gametocytes from 3 × 10⁷ to 3 × 10² parasites/mL, and pfs25 additionally detected parasites at the 3 × 10¹ parasites/mL dilution. PF3D7_0630000 mRNA was never detected in samples containing asexual-stage parasites (even at high parasite densities) confirming that this spliced target is absent from asexual stages. All reactions demonstrated high cycling efficiencies (Fig. 3b: 18S rRNA slope −3.14, efficiency 108%, y-intercept: 29.4 cycles; pfs25 slope −3.32, efficiency 100%, y-intercept 35.71 cycles; PF3D7_0630000 slope −3.59, efficiency 90%, y-intercept 42.67 cycles). Consistent with RNAseq data [28], PF3D7_0630000 was less abundant than pfs25, and both mRNAs were much less abundant than the highly expressed Plasmodium 18S rRNA.

To assess spliced mRNA target stability, intact gametocytes in whole blood were either preserved in lysis buffer immediately or held at room temperature for 3–7 days before processing into lysis buffer. For all samples (nominal densities 3 × 10²–3 × 10⁵ gametocytes/mL), mRNA (pfs25 and PF3D7_0630000) degradation was observed at 3 and 7 days of storage, whereas statistically significant 18S rRNA degradation was not observed until 7 days of storage (Fig. 4). PF3D7_0630000 degraded more rapidly than pfs25 at 3 days but this difference was not statistically different at 7 days. These data suggest, while samples for 18S rRNA testing can be stored for several days prior to sample processing, samples for gametocyte-specific pfs25 or PF3D7_0630000 mRNA testing should be processed immediately following collection.

To study the time course of PF3D7_0630000 expression, samples were taken from gametocyte cultures during Days 6–14 of culture and compared to expression of the Day 6 sample. This approach was used because on the sixth day of culture, a mix of residual asexual stages and immature gametocytes present, but after the tenth day of culture, most of the parasites were mature gametocytes with ring, trophozoite and schizont stages no longer observed. Unlike asexual-stage parasites that do not express the PF3D7_0630000 spliced mRNA, gametocytes express both pfs25 and PF3D7_630000 mRNAs from by the sixth day of culture. Expression of both targets increased as the cultures matured with pfs25 expression exceeding that of PF3D7_0630000 (Fig. 5). At peak expression (day 14), pfs25 was increased by >2000-fold compared to day 6 of culture while PF3D7_0630000 increased >25-fold.

To determine if the novel spliced gametocyte-specific mRNA RT-PCR could detect low level gametocytaemia from clinical specimens, samples were first evaluated from a clinical trial in a Seattle-based controlled human malaria infection trial evaluating the prophylactic activity of the drug DSM265. This placebo-controlled prophylactic drug trial was blinded at the time of this manuscript, and it was thus unknown whether subjects received the active compound or the placebo control. Subjects were challenged with 3200 PfSPZ (Sanaria, Inc., Rockville, MD) on Day 0. Final details of the ongoing DSM265 study will be published in a separate manuscript at a later date and do not affect the data described below. Plasmodium infection status was monitored by daily 18S rRNA RT-PCR. One subject developed a late onset infection with 18S rRNA qRT-PCR positivity starting on Day 23 (Fig. 6). The subject was symptomatic (grade 1–2 nausea, vomiting, headache) during this time and was thick blood smear positive on Day 25. The subject was treated on Day 25 with atovaquone–proguanil with rapid conversion to negative blood smears and resolution of symptoms. Plasmodium 18S rRNA concentrations dropped following treatment but did not reach undetectable levels. On Day 33 post-challenge, resurgence in Plasmodium 18S rRNA was noted although the subject was asymptomatic. Persistent asymptomatic positivity for Plasmodium 18S rRNA continued from days 33–42 post-challenge. Neither pfs25 nor PF3D7_0630000 mRNAs were detected during the symptomatic period but both were detected during the second wave of asymptomatic parasitaemia (Fig. 6). The patient was subsequently treated orally with 45 mg primaquine and was negative for all biomarkers at 56 days post-challenge.

In addition to PF3D7_0630000, three other spliced mRNA targets have been reported: the P. falciparum meiotic recombination protein DMC1-like protein gene (PF3D7_0816800) [26], an immature gametocyte marker PHISTa of unknown function (PF3D7_1477700/PF14_0748) [27] and a mature gametocyte marker of unknown function (PF3D7_1438800/PF14_0367) [27]. Each of these targets were individually multiplexed with the pan-Plasmodium 18S rRNA assay and tested against whole blood samples spiked with high (4 × 10⁵ parasites/mL) or low (8 × 10²) density pure cultured ring-stage samples or with day 14 cultured gametocytes (Table 3). The PF3D7_1477700 and PF3D7_1438800 markers [27] could not be successfully multiplexed with the pan-Plasmodium 18S rRNA RT-PCR because false positive results were obtained for asexual samples at annealing temperatures of 50 °C (standard for 18S rRNA RT-PCR) and 60 °C (as originally reported for these gametocyte markers [27]). False positive results for the PF3D7_1477700 and PF3D7_1438800 markers were also obtained when testing singleplex reactions against asexual stage parasites (data not shown). PF3D7_0630000 and pfs25, the spliced PF3D7_0816800 target were positive only in the presence of gametocytes (Table 3).

Multiplex qRT-PCRs combining Plasmodium 18S rRNA with either PF3D7_0816800, PF3D7_0630000 or pfs25 were evaluated against 74 Plasmodium-infected samples collected from asymptomatic human volunteers in a Ugandan field study. The parasite densities of these samples ranged from 25 to 2.8 × 10⁷ parasites/mL with estimated gametocyte densities ranging from 1 to 156,828 gametocytes/mL (based on pfs25 standard curves). The pfs25 multiplex assay detected gametocytes in 47/74 samples, PF3D7_0630000 in 50/74 samples and PF3D7_0816800 in 30/74 samples (Additional file 3). pfs25 and PF3D7_0630000 showed a high degree of agreement. Compared to the pfs25 gold standard, the sensitivity of PF3D7_0630000 for gametocyte detection was 97.9% (95% CI 88.7–100.0%) and specificity 85.2% (95% CI 66.3–95.8%). The sensitivity of PF3D7_0816800 qRT-PCR was 63.8% (95% CI 48.5–77.3%) and specificity 100% (87.2–100.0%). Whereas the PF3D7_0630000 reagents detected gametocytes across all pfs25-positive gametocyte densities, PF3D7_0816800 RT-PCR was less sensitive at densities <1000 gametocytes/mL but was positive in all samples with a density of >1000 gametocytes/mL (Table 4; Fig. 7).

Quantification of gametocytes in field samples based on pfs25 resulted in estimated densities 1.18 log₁₀ parasites/mL (95% CI 0.89–1.46 log₁₀ parasites/mL) lower on average than the paired estimates made with PF3D7_0630000. Lower pfs25-derived gametocyte quantification could not be explained by overall loss of total nucleic acids during DNase treatment since 35/39 evaluable paired samples (positive by pfs25 and PF3D7_0630000) actually showed earlier 18S rRNA C_Ts in the pfs25 sample compared to the PF3D7_0630000 sample (Additional file 3).