Edinburgh Research Explorer Quantification of female and male Plasmodium falciparum gametocytes by reverse transcriptase quantitative PCR

The transmission of malaria parasites depends on the presence of sexual stages (gametocytes) in the blood, making the ratio and densities of female and male gametocytes important determinants of parasite ﬁtness. This manuscript describes the development of reverse transcriptase quantitative PCR (RT-qPCR) assays to separately quantify mature female and male gametocytes of the human malaria parasite Plasmodium falciparum , and reveals that Pfs25 mRNA is expressed only in female gametocytes. The female ( Pfs25 ) and male ( Pfs230p ) gametocyte speciﬁc RT-qPCR assays have lower detection limits of 0.3 female and 1.8 male gametocytes per microlitre of blood, respectively, making them more sensitive than microscopy. Accurate quantiﬁcation of the ratio and densities of female and male gametocytes will increase under-standing of P. falciparum transmission and improve the evaluation of transmission blocking interventions.


a b s t r a c t
The transmission of malaria parasites depends on the presence of sexual stages (gametocytes) in the blood, making the ratio and densities of female and male gametocytes important determinants of parasite fitness. This manuscript describes the development of reverse transcriptase quantitative PCR (RT-qPCR) assays to separately quantify mature female and male gametocytes of the human malaria parasite Plasmodium falciparum, and reveals that Pfs25 mRNA is expressed only in female gametocytes. The female (Pfs25) and male (Pfs230p) gametocyte specific RT-qPCR assays have lower detection limits of 0.3 female and 1.8 male gametocytes per microlitre of blood, respectively, making them more sensitive than microscopy. Accurate quantification of the ratio and densities of female and male gametocytes will increase understanding of P. falciparum transmission and improve the evaluation of transmission blocking interventions. For malaria (Plasmodium) parasites, transmission between vertebrate hosts requires a mosquito vector. Throughout infections in the vertebrate host, Plasmodium produces female and male sexual stages (gametocytes). Upon ingestion by a blood-feeding mosquito, gametocytes differentiate into gametes, mate, and after several developmental transitions, the progeny are able to infect another vertebrate host. The densities of female and male gametocytes in the blood and the sex ratio (proportion of gametocytes that are male) influence infectivity to mosquitoes, and so, onwards transmission to new hosts [1][2][3].
Techniques to determine the sex of P. falciparum gametocytes are laborious. They include microscopical examination of Giemsastained thin blood smears and immunofluorescence assays using antibodies recognising female-and/or male-specific antigens [4]. Furthermore, the sensitivity of these methods is low: transmission to mosquitoes readily occurs at densities below the microscopic detection limit of 8-16 gametocytes/l [3,5]. This makes it very difficult to obtain accurate estimates of sex-specific gametocyte densities, and sex ratios, from as many as 50% of infections capable of transmitting to mosquitoes [3] because in some cases as few as 10 gametocytes are sexed [6]. However, separately following the densities of female and male gametocytes during natural infections is necessary to understand how their dynamics and sex ratios influence disease transmission and to evaluate transmission blocking interventions [2,7].
To facilitate the study of P. falciparum female and male gametocyte dynamics and gametocyte sex ratios, high-throughput, sensitive molecular methods for the quantification of mature  female and male gametocytes were developed. The female-specific assay quantifies mRNA of the gene encoding Pfs25 (PF3D7 1031000). Pfs25 is expressed in late stage gametocytes, shows limited polymorphism and is currently used to quantify gametocytes from field isolates [3,8].
The sex-specificity of Pfs25 mRNA expression is tested in this manuscript, and confirmed to be female-specific, like mRNA of its orthologue in the rodent malaria P. berghei (Pbs21) [9,10].
Only a few P. falciparum male-specific proteins have been described. The most well-known are alpha-tubulin II and the Pfs230 paralogue, Pfs230p [11,12]. Low levels of ␣-tubulin II protein have been demonstrated in asexual blood stages and early stage female gametocytes [12]. Therefore, the male-specific assay quantifies Pfs230p (PF3D7 0208900) mRNA [11], and its specific expression in late stage male gametocytes is verified. The design, optimisation, and validation (including the assessment of specificity and sensitivity) of assays to quantify female or male P. falciparum mature gametocytes, are presented below.
Primers and probe (Table 1) for the Pfs25 and Pfs230p assays are P. falciparum specific (BLAST). No significant single nucleotide polymorphisms were detected in the Pfs25 and Pfs230p primer and probe regions among almost 2000 P. falciparum samples collected from all over the world, (The Pf3K Project (2014): pilot data release 2. www.malariagen.net/data/pf3k-2) [13]. Therefore, the primers and probe are suitable for use on field samples that potentially contain multiple parasite genotypes. To provide large amounts of material from the target genes for assay optimisation and calibration, independent of the need to repeatedly culture P. falciparum parasites, constructs were made to produce DNA, RNA and cDNA in vitro (Supplementary data, section 1). Because the target genes have single exons, DNA sequences of the target regions (in vitroproduced DNA: ivDNA) and cDNA are identical, so ivDNA was used to optimise assay conditions (Table 1). PCR efficiencies for the Pfs25 and Pfs230p qPCRs were 93.6% with a standard error (se) of 1.5%, and 98.5 (se 2.4%), respectively. Amplification was not observed in the no-template controls, and the lower limit of detection was similar at 9 and 2 molecules/25 l reaction for the Pfs25 and Pfs230p qPCRs, respectively (Fig. 1). The concentration of Pfs25 and Pfs230p ivDNA was highly correlated to PCR quantification cycles (Cq; adjusted r 2 = 0.98, p < 0.001 for both assays) and the small standard errors indicate high repeatability between replicates ( Fig. 1; for details on statistical analysis see Supplementary data, section 2). In summary, the assays are sensitive and quantify their respective ivDNA accurately.
Next, the Pfs25 and Pfs230p assays were assessed for speciesspecificity. The Pfs25 and Pfs230p assays do not amplify nucleic acids from uninfected human blood samples that were collected from volunteer donors (n = 10, with written informed consent). Orthologues for the Pfs25 and Pfs230p genes are present in P. vivax, P. knowlesi, and in the rodent malaria species P. berghei and P. chabaudi (PlasmoDB.org). Sequence similarity between the P. falciparum sequences and the different orthologues is comparable (44-47% for Pfs25, 41-42% for Pfs230p). Therefore, and given the difficulty in obtaining samples for single-species infections of P. knowlesi or P. vivax, DNA from P. chabaudi (CW) and P. berghei (ANKA) (from experiments carried out in accordance with the UK Animals Scientific Procedures Act 1986) was used to empirically test P. falciparum species-specificity of the Pfs25 and Pfs230p qPCR assays. No amplicons were detected, confirming the in silico analysis.
For the assessment of stage-and sex-specificity and to determine sensitivity of the assays, a variety of P. falciparum samples were used. These samples include: (1) "Asexual stages": sorbitolsynchronised asexual ring stage parasites (clone 3D7), freed of gametocytes through magnetic separation [14,15]; (2) "Mixed sex gametocytes" free of asexual stages after treatment with N-acetyl glucosamine [15], including (a) sample 3D7-1: gametocytes of clone 3D7 at development stage IV, with a sex ratio (proportion of gametocytes that are males) of 0.05 as determined by morphological examination of Giemsa stained thin films; (b) sample 3D7-2: 3D7 stage IV gametocytes depleted of uninfected red blood cells by magnetic purification [14] with a sex ratio of 0.05 determined by morphological examination of Giemsa stained thin films; (c) sample NF54: stage IV-V gametocytes of isolate NF54. NF54 parasites cultured using the methods as described in [15] consistently have a sex ratio of 0.2 as determined by morphological examination of Giemsa stained thin films; (3) "Purified females": stage IV-V gametocytes of line p47GFP (derived from NF54) that expresses green fluorescent protein specifically in female gametocytes, purified using flow cytometry, free from asexual stages and containing 98% females (assessed by re-sorting aliquots by flow cytometry [16]); and (4) "Purified males": stage IV-V gametocytes of line Dyn-GFP (derived from NF54) that expresses green fluorescent protein specifically in male gametocytes, purified using flow cytometry, free of asexual stages and containing >98% males (assessed by flow cytometry) [16]. Samples were stored in RNA-preserving reagents (for details on sample processing see Supplementary data, section 3) and stored at −70 • C. RNA from all samples were extracted on a semi-automatic MagMAX TM Express Particle Processor (Life Technologies) using the MagMAX TM -96 total RNA isolation kit according to manufacturer's protocol, with the exception of RNA from the "Mixed sex gametocytes NF54" sample, which was processed in a different laboratory (Supplementary data, section 3) and was extracted using the MagNAPure system at protocol HS500 and the MagNAPure LC Total Nucleic Acid Kit-High Performance. All samples were DNAse I treated (RQ1, Promega) and confirmed to be DNA free by 18S qPCR [17] before cDNA was synthesised by reverse transcriptase PCR (High capacity cDNA Reverse Transcription Kit, Life Technologies). The female-specific Pfs25 qPCR followed by the male-specific Pfs230p qPCR are presented the following paragraphs.
First, to test whether Pfs25 mRNA expression is female gametocyte-specific, like mRNA of its orthologue in the rodent malaria P. berghei (Pbs21) [9,10], qPCR was performed using cDNA from "Asexual stages" (8.9 × 10 5 parasites/l blood) and the "Purified males" (6.7 × 10 3 gametocytes/l blood, <2% females). No amplicons were obtained with samples from "Asexual stages", confirming gametocyte-specificity. However, low levels of Pfs25 amplicons were detected from the "Purified males". The expression of Pfs25 mRNA for each gametocyte from the "Purified males" was significantly lower than in the "Purified females", and "Mixed sex gametocytes" samples 3D7-1 and 3D7-2 (F 3,178 = 253.11, p < 0.001). Pfs25 mRNA expression was 274-fold higher in the "Purified females", which is consistent with the "Purified males" (purity expected to be >98% males) containing a 0.36% contamination of female gametocytes. When the proportion of female gametocytes in each sample (0.36%, 95% and 98% for the "Purified males", "Purified females", "Mixed sex gametocytes" 3D7-1 and 3D7-2, respectively) is taken into account, there are no significant differences in Pfs25 mRNA expression per female gametocyte across these sex ratios (F 3,178 = 2.63, p = 0.052; Fig. 2A). Extremely low expression of Pfs25 mRNA in male gametocytes cannot be ruled out, but even in this case, the 274-fold lower mRNA expression in male versus female gametocytes will not significantly reduce the ability of the Pfs25 assay to accurately quantify female gametocytes. Therefore, the Pfs25 RT-qPCR, and the Pfs25 QT-NASBA currently used for field surveys [3], can be considered to quantify female gametocytes only.
In conclusion, both sex-specific RT-qPCRs are accurate and more sensitive than microscopy [5], and can quantify mature female and male P. falciparum gametocytes, including submicroscopic gametocyte densities as low as 0.3 female and 1.8 male gametocytes/l of blood. The female-specificity of the Pfs25 assay means that the Pfs25 QT-NASBA, currently used for field surveys, quantifies female gametocytes only [3]. Although this should be taken into account when interpreting data, female density reflects an important aspect of transmission because it places the upper limit on ookinete (offspring) numbers. The sex-specific RT-qPCRs facilitate the study of why the densities of mature female and male gametocytes, and the sex ratio (proportion of gametocytes that are male) vary during infections and between hosts, and their influence on transmission. Another application of the assays is the separate assessment of female and male gametocyte clearance after drug treatment. Male gametocytes are thought to be limiting for transmission in general [2] and this could be exacerbated after treatment with gametocytocidal drugs because males are more sensitive to a variety of drugs compared to female gametocytes [7,18].