Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials

Abstract

To detect pre-patent parasitemia, we developed a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the asexual 18S ribosomal RNA (rRNAs) of Plasmodium falciparum. Total nucleic acids extracted from whole blood were combined with control RNA and tested by qRT-PCR. The assay quantified > 98.7% of parasite-containing samples to ±0.5 log(10) parasites/mL of the nominal value without false positives. The analytical sensitivity was ≥ 20 parasites/mL. The coefficient of variation was 0.6% and 1.8% within runs and 1.6% and 4.0% between runs for high and low parasitemia specimens, respectively. Using this assay, we determined that A-type 18S rRNAs are stably expressed at 1 × 10(4) copies per ring-stage parasite. When used to monitor experimental P. falciparum infection of human volunteers, the assay detected blood-stage infections 3.7 days earlier on average than thick blood smears. This validated, internally controlled qRT-PCR method also uses a small (50 μL) sample volume requiring minimal pre-analytical handling, making it useful for clinical trials.

Figure 1.

Alignment of Plasmodium falciparum 18S sexual- and asexual-type rRNAs and assay reagents. CLUSTAL-W alignments of the assay reagents to the four 18S ribosomal RNA (rRNA) genes of P. falciparum 3D7. Asterisks denote homology between assay reagents and all S- and A-type genes. The assay reagents are completely matched to the A-type 18S rRNAs (MAL5_18S and MAL7_18Sa). PF1473F18 and PL1679R18 (Rev. Comp.) refer to the forward primer and the reverse complement of the reverse primer; PFALF and PFALR refer to the donor and acceptor probes, respectively.

Figure 2.

Plasmodium falciparum real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) reagents. The full-length P. falciparum A-type 18S rRNA and in vitro transcribed (IVT) synthetic RNAs corresponding to the 18S ribosomal RNA (rRNA) amplicon and a competitor RNA with a mutated internal region can all be amplified with the same common PCR primers and all hybridize to the same FITC-labeled donor probe. An LC610-labeled acceptor probe binds to the native 18S rRNA sequence present in the full-length sequence and IVT RNA sequence, whereas a unique LC705-labeled acceptor probe for the competitor RNA is used to bind the mutated region. In this way, competitive amplification is achieved with minimal multiplexing.

Figure 3.

Sensitivity of multiplexed quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for Plasmodium falciparum A-type 18S ribosomal RNA (rRNA). Dilution series of samples containing a known concentration of IVT RNA in nucleic acids from whole blood (A and B) or a known concentration of whole parasites in whole blood (C and D). Analytical sensitivity was 1×103 copies (dark green curves) of IVT target RNA per reaction (A, representative CT analysis of IVT standards; B, representative melting curve analysis of IVT standards). Lower concentrations of IVT standards were detectable by melting curve analysis (purple curve, 5×102 copies/reaction), but are not quantitatively reportable. Using parasite-containing standards, samples containing 40 parasites/mL (pink/purple curve) were routinely detected (C, representative CT analysis of parasite-containing standards; C, representative melting curve analysis of parasite-containing standards). Numbers refer to RNA copies/reaction (A and B) or parasites/mL (C and D).

Figure 4.

Copy number and expression of the A-type 18S ribosomal RNA (rRNA) in cultured Plasmodium falciparum 3D7. (A) Box plot showing an average of 10,000 copies of the A-type 18S rRNA per 12-hr ring-stage parasite (mean 3.98 log₁₀ copies each). Parasites were cultured and synchronized by magnetic activated cell sorting (MACS) and sorbitol treatments, added to whole blood and quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (n = 51 samples ranging from 1% to 0.000002% parasitemia). (B) Expression of the A-type 18S rRNA by qRT-PCR in highly synchronized P. falciparum cultures sampled at the indicated time points after the invasion time point. RNA copies per parasite were normalized to the starting parasitemia determined at the 2-hr time point. Black boxes show mean log₁₀ RNA copies/parasite (n = 2, error bars 95% CI; representative experiment shown) and the dashed line shows the percentage change relative to the 2-hr time point throughout the lifecycle.

Figure 5.

Assay correlation and difference assessment. Correlation (left) of nominal (expected) and observed (measured) values (log₁₀ RNA copies/mL) showed strong correlation across samples with wide ranging parasitemias (r² = 0.9826, n = 63); the line of identity is also shown. A Bland-Altman difference plot (right) showed strong agreement between nominal and observed measurements [95% limits of agreement (UL, upper limit; LL, lower limit; dashed lines): + 0.50075 to −0.43184 log₁₀ RNA copies/mL, n = 63], with only one outlier. The overall assay bias was −0.0344 log₁₀ RNA copies/mL. Recovery was not concentration dependent (r² = 0.0165; p = 0.2548).

Figure 6.

Early detection of pre-patent parasitemia in six volunteers experimentally infected with Plasmodium falciparum. Sporozoites kinetics of parasite load determined by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) during challenge of six human volunteers with P. falciparum 3D7 sporozoites. For qRT-PCR, venous blood was collected twice daily starting on Day 5. The first qRT-PCR positive sample in each participant is denoted with a black arrow listing the day post-challenge. Thick blood smears were positive starting on Day 9 (participant 5), 11 (participants 1–4) and 14 (participant 6), as denoted with circles. Chloroquine treatment was initiated on the first detection of a positive thick smear. The limit of quantification (20 parasites/mL) is indicated with a horizontal line. Samples from subjects 1 and 2 that were positive by melting curve analysis but were below the limit of quantification were plotted at the “+” level on the parasitemia axis.

Figure 7.

Kaplan-Meier curves for time to detection and clearance of parasites by thick blood smears and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Kaplan-Meier curves for the time to detection (A) and post-treatment clearance (B) of parasites for thick blood smears (BS) and qRT-PCR. *No qRT-PCR testing was performed during this interval 6–15 days following treatment.