An assay for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure gametocyte standards

Background Malaria transmission from humans to mosquitoes requires the presence of gametocytes in human peripheral circulation, and the dynamics of transmission are determined largely by the density and sex ratio of the gametocytes. Molecular methods are thus employed to measure gametocyte densities, particularly when assessing transmission epidemiology and the efficacy of transmission-blocking interventions. However accurate quantification of gametocytes with molecular methods requires pure male and female gametocytes as reference standards, which are not widely available. Methods qRT-PCR assays were used to quantify levels of sex-specific mRNA transcripts in Plasmodium falciparum female and male gametocytes ( pfs25 and pfMGET respectively) using synthetic cRNA standards and in-vitro cultured gametocytes. Assay were validated and assay performance was investigated using blood samples of clinical trial participants (ClinicalTrials.gov reference number NCT02431637 and NCT02431650) using these standards and compared to absolute quantification by droplet digital PCR (ddPCR). Results The number of transcript copies per gametocyte were determined to be 279.3 (95% CI 253.5 -307.6) for the female-specific transcript pfs25, and 12.5 (95% CI 10.6 - 14.9) for the male-specific transcript pfMGET . These numbers can be used to convert from transcript copies/mL to gametocyte/mL. The reportable range was determined to be 5.71x10 6 to 5.71 gametocytes/mL for pfs25, and 1.73x10 7 to 5.59 for pfMGET. The limit of detection was 3.9 (95% CI 2.5-8.2) gametocytes/mL for pfs25, and 26.9 (95% CI 19.3 - 51.7) gametocytes/mL for PfMGET . Both assays showed minimal intra-assay and inter-assay variability with coefficient of variation < 3%. No cross-reactivity was observed in both assays in uninfected human blood samples. Comparison of results from ddPCR to qRT-PCR assays on clinical blood samples indicated high level agreement (ICC=0.998 digestion using RNase-Free DNase set (Qiagen, Australia) to eliminate genomic DNA. 100 ∝l of RNA extract was eluted.

the estimation of gametocyte density in the absence of pure female and male gametocyte standards, and will facilitate clinical trials and epidemiological studies.

Background Successful transmission of Plasmodium infection from humans to mosquitoes requires that a female
Anopheles mosquito imbibes at least one mature male and one mature female gametocyte in a blood meal. The density and the ratio of female to male gametocytes in peripheral blood are therefore key determinants of the dynamics of transmission, transmission epidemiology in endemic settings, and for evaluating the efficacy of transmission blocking interventions, such as drugs and vaccines (1)(2)(3)(4)(5)(6).
Thus, techniques to accurately detect and quantify gametocytemia are required (7). Successful transmission has been reported to occur at submicroscopic gametocyte levels both in endemic areas during asymptomatic infections, and in volunteer infection studies (VIS), also referred to as controlled human malaria infection (CHMI)-transmission studies (6,8,9). Understanding the contribution of submicroscopic infections to the human infectious reservoir would greatly assist elimination efforts.
Gametocytemia has traditionally been measured by thick-or thin-film microscopy; however, both methods have limited sensitivity and cannot accurately measure gametocytemia below 10,000 gametocytes per mL (10). To overcome the limited sensitivity of microscopy, quantitative reverse transcription PCR (qRT-PCR) and quantitative nucleic acid sequence based amplification (NASBA) assays targeting gametocyte specific mRNA transcripts have been developed (6,(11)(12)(13)(14)(15). The highly conserved pfs25 gene transcript is present in abundance in female gametocytes, and is therefore commonly targeted for quantification by qRT-PCR (7,14). More recently, male-specific gametocyte mRNA transcripts have been described (5,16,17), and among them pfMGET (Pf3D7_1469900) shows an abundant transcription profile (5) making it an appropriate candidate for quantitation of male gametocytes. The use of both female-and male-specific assays allows evaluation of the gametocyte sex ratio, a parameter of interest in quantifying infectivity to mosquitoes, particularly at lower gametocyte densities (18). The Pfs25-PfMGET combination for sex ratio determination was recently validated by immunofluorescence assays on field samples from gametocyte donors (19).This ratio may be particularly important for assessment of gametocytocidal drug activity, as the less abundant male gametocytes may be more readily sterilised or killed by some antimalarial drugs (14,20,21).
qRT-PCR methods have superior sensitivity over microscopy, allowing the quantification of gametocyte densities across the epidemiologically relevant range (1). These assays require the use of reference standards to generate standard curves for quantification. These ideally consist of biological reference standards, namely pure populations of female and male gametocytes. However, preparation of such material requires laborious approaches to culture and FACS-sort gametocyte reporter lines that are not readily available to all laboratories (22). An alternative method for generation of standard curve material is the use of synthetic standards such as cRNA, which can be prepared by any laboratory. Although this approach permits quantification of the absolute number of mRNA transcripts, the assay does not allow the quantification of gametocyte numbers unless we know how many copies of the target mRNA transcript are expressed per gametocyte. As the number of mRNA transcripts expressed per gametocyte differs for each target (5,22), the expression level of each must be known in order to convert transcripts per mL to gametocytes per mL. By determining these conversation factors it would significantly improve the assessment of gametocyte density and sex ratios by qRT-PCR using synthetic cRNA standards.
Here, we describe the validation of two qRT-PCR assays that enable the accurate and sensitive detection and quantification of female (pfs25) and male (pfMGET) gametocytes in whole blood samples without the need for purified female and male gametocytes reference standards.

RNA extraction
250∝ l of packed red blood cells (pRBCs) from each clinical trial participant (see below) were stored (1:5) in RNA Protect Cell Reagent (Qiagen, Australia) at -80 °C until RNA extraction. Prior to RNA extraction, a known concentration of equine arteritis virus (EAV) culture was spiked into each sample as an internal control to monitor extraction efficiency and qRT-PCR inhibition (23). RNA extraction was performed using RNeasy Plus Mini Kit (Qiagen, Australia) following manufacturer's instructions with treatment of DNase on-column digestion using RNase-Free DNase set (Qiagen, Australia) to eliminate genomic DNA. 100 ∝l of RNA extract was eluted.
Female and male gametocyte qRT-PCR assays Sex-specific qRT-PCR assays were used to measure RNA transcripts specific to female and male gametocytes ( Table 1). The previously described female marker assay was designed to target the P. falciparum gametocyte surface protein (pfs25) mRNA (Genbank accession number AF154117) (11).
The male marker assay targeted the exon-exon junction of the P. falciparum PF3D7_1469900 mRNA transcript (Genbank accession number XM_001348805) recently characterised as male gametocyteenriched transcript (pfMGET) (5, 6). Table 1 List of oligonucleotides used in this study.  Dilution series were analysed in replicates to generate standard curves for transcript quantification.
The linear regression using an established external standard curve (pfs25 slope: -3.395, pfMGET slope: -3.236) was imported into all qRT-PCR runs within Rotorgene software with fixing to the highest standard to achieve final quantification of RNA copies.

Assay sensitivity
To determine assay sensitivity or limit of detection (LOD), neat cRNA was serial-diluted in uninfected human blood extracts to produce 14 concentrations of pfs25 cRNA (equivalent to the range of 5.71 × 10 6 to 0.18 gametocytes/mL) and 11 concentrations of pfMGET cRNAs (equivalent to the range of 1.73 × 10 7 to 5.59 gametocytes/mL), with focus on more dense dilutions in the lower range for more accurate determination of assay limits. Replicates of each dilution and a negative control (uninfected human blood extracts) were analysed on separate qRT-PCR runs (6 for pfs25 and 3 for pfMGET) on a Rotorgene Q instrument (Qiagen, Australia) with same batch of mastermix used.

Assay specificity
Analytical specificity was determined by comparing the sequence of the nucleic acid target (primer and probe sequences) to sequences available on publicly accessible databases using BLAST search tool (NCBI) to check its specificity to P. falciparum pfs25 and PF3D7_1469900 (pfMGET) transcripts. No cross reactions were observed for other malarial mRNA transcripts and human genomic DNA or mRNA transcripts. Blood from malaria-naïve volunteers enrolled in clinical trials (pfs25 n = 66, pfMGET n = 11) was assessed for assay specificity.
Female and male gametocyte ddPCR Since no "gold standards" were available for RNA quantification, ddPCR was utilised to generate absolute quantification of RNA copy numbers to confirm the accuracy of RNA estimates in both cRNA standards and infected blood. The female and male gametocyte qRT-PCR assays were adapted to the droplet digital PCR (RT-ddPCR) format. cRNA controls and RNA extracts from participants (see below) were analysed on QX200 ddPCR system (BioRad, Australia). The RT-ddPCR reactions were prepared gametocytes/mL) were stored frozen in RNA Protect Cell Reagent (Qiagen, Australia).
Gametocytes were thawed and RNA extracted using methods described above. A 10-fold dilution series of the sample was made ranging from 10 6 to 10 gametocytes/mL. Each sample dilution series was run in duplicate over three days (a total of 36 PCR reactions). The pfs25 and pfMGET mRNA transcript numbers were determined in these samples using the qRT-PCR assays with cRNA standards. The study design was a randomised block design with the dilution series as treatments, daily runs as blocks and technical replicates used to estimate intra-assay variability. An analysis of variance on the residual difference in log 10 copies per gametocyte between log 10 (copies/mL) and log 10 (gametocytes/mL) was used to obtain mean and 95% confidence intervals for each conversion factor to translate from copies/mL to gametocytes/mL. as the standard deviation for intra-assay variability, was estimated as the variability between technical replicates pooled across standards. The inter-assay variability was determined using standard curve data from historical records of 17 runs of 7 study cohorts for pfs25 and 29 runs from deviation estimated as the variability between assay runs pooled within cohorts. Relative variability was measured as the percent coefficient of variation (%CV) for each standard concentration. Linear regression was used to assess the relationship between log 10 concentration of standard relative to the highest standard and C q value. Accuracy was assessed using intraclass correlation coefficient (ICC), paired t-test and Passing-Bablok regression to examine differences in gametocyte per mL of whole blood between qRT-PCR and ddPCR using R Studio (ver. 1.1.442, R version 3.4.4).

Results
Estimating transcript numbers per gametocyte using purified in-vitro cultured female and male gametocytes A dilution series of purified female and male gametocytes of known concentration obtained from the PfDynGFP/PfP47mCherry reporter line were analysed by qRT-PCR, and used to calculate the number of copies of each mRNA target expressed per gametocyte for converting copies/mL to gametocytes/mL.

Female and male gametocyte qRT-PCR assay performance
Sex-specific qRT-PCR assays were validated to quantify mRNA transcript levels specific to female (pfs25) and male (pfMGET) gametocytes. Both assays showed reliable amplification across a large linear range with good precision, sensitivity and specificity. The reportable range was determined to be 5.71 × 10 6 to 5.71 gametocytes/mL for pfs25, and 1.73 × 10 7 to 5.59 for pfMGET. The relationship between log 10 concentration of standard relative to the highest standard and C q value for pfs25 had for pfMGET. The linearity of calibration across the lower dilutions in both series extended the reportable range and lower limit of quantification to 0.18 gametocytes/mL for pfs25 and to 5.59 gametocytes/mL for pfMGET. No amplification was observed in the negative control extracts (pfs25 n = 33, pfMGET n = 6) from uninfected human blood extracts.
The LOD 95% for these assays was determined to be 3.9 gametocytes/mL of whole blood (95% CI 2.5-8.2) for pfs25, and 26.9 gametocytes/mL of whole blood (95% CI 19.3-51.7) for pfMGET when a starting volume of 250∝ l of packed RBCs (equivalent to 500∝ l whole blood) is analysed.
There was a tendency for both intra-and inter-assay variability to increase at lower dilutions but the %CV was less than 3% for each standard in both assays. The standard deviation measure for intraassay variability pooled across all dilutions was 0.52 C q units for pfs25 and 0.51 C q units for pfMGET, indicating minimal variability between replicates pooled across standards (Tables 2 and 3). The overall standard deviation measures for inter-assay variability were 0.50 C q units for pfs25 and 0.46 C q units for pfMGET (Tables 4 and 5), which were higher than the pooled intra-assay variability at similar dilutions (0.30 for pfs25 and 0.18 for pfMGET.    No off target primer or probe interactions were identified by search of GenBank (BLAST, NCBI) indicating good analytical specificity of the two assays. Diagnostic specificity for both pfs25 and pfMGET was demonstrated using blood samples from malaria-naïve individuals (pfs25 n = 66, pfMGET n = 11), all being negative, to give a specificity of 100% with lower bounds of 95% confidence of 95.6% and 76.2%, respectively.

Absolute quantification by ddPCR verifies qRT-PCR quantification
To confirm the accuracy of the gametocyte-specific qRT-PCR assays, a collection of gametocytepositive blood samples from two clinical trials were used to compare the quantitative results of the qRT-PCR to those from droplet digital PCR technology. Only samples with gametocyte densities greater than one gametocyte/mL as measured by qRT-PCR were included in the analysis for pfs25 (n = 21) and pfMGET (n = 36

Discussion
A good understanding of the infectious reservoir of malaria and malaria transmission dynamics is required to inform the development and implementation of transmission-blocking interventions. To determine the relative contribution of submicroscopic gametocytemia to transmission (8,9), molecular assays able to accurately detect very low gametocyte densities have been developed (5,6,15). Here, we report the validation of qRT-PCR assays for female and male gametocytes using cRNA standards. Use of these standards overcomes previous limitations of qRT-PCR assays that required These two assays were validated and shown to be highly sensitive with their LOD for female gametocytes determined to be 3.9 gametocytes/mL, and 26.9 gametocytes/mL for male gametocytes.
In addition, the specificity, and reproducibility of these two assays was confirmed. The accuracy of our calculations is supported by the fact that absolute quantification of male and female gametocytes in clinical samples using ddPCR assay were very close to those derived from qRT-PCR assays in terms of agreement, paired, systematic or proportional differences. This supports our estimates of transcript abundance, as well as demonstrating that qRT-PCR is sufficiently accurate where ddPCR is not available. A significant advantage of these assays is that they are sufficiently sensitive to quantify submicroscopic female and male gametocyte levels and determine gametocyte sex ratios in natural infections or during transmission studies.
Determining the numbers of transcripts per gametocyte is challenging due to the need for pure Firstly, the variability in levels of transcription of the two chosen targets may vary with gametocyte age. Although we used a stage-V gametocyte culture, low-level contamination of different stage gametocytes cannot be ruled out, and copy numbers at different stages of gametocyte maturation was not evaluated. In addition, it is plausible that transcript levels may differ between in-vivo and in-vitro generated gametocytes. Likewise, it is unknown if there are differences in levels of these two transcripts in different strains of P. falciparum. However, in support of our calculations, the copy numbers per gametocyte were similar to those estimated from in-vivo samples during a CHMItransmission study (6).

Conclusions
We validated P. falciparum male and female gametocyte-specific qRT-PCR assays that can quantify gametocyte densities with excellent reproducibility, sensitivity, specificity and accuracy. Moreover, sex-specific mRNA transcript levels per gametocyte were determined, enabling accurate quantification of gametocyte densities in the absence of pure female and male gametocyte standards.
The methodology described here can enable the wider use of qRT-PCR assays for detecting and quantifying gametocytemia over a broad range of gametocyte densities, including submicroscopic gametocytemia. This will facilitate studies that either evaluate transmission-blocking interventions, or studies aiming to improve understanding of the infectious reservoir, which will be increasingly more valuable as we move towards elimination. The plots show log10 gametocytes/mL by ddPCR and qRT-PCR. The solid black line represents the fitted Passing-Bablok regression line. The 95% confidence bounds, in grey, were calculated using the bootstrap quantile method. The female pfs25 assay is shown to the left and the male pfMGET assay is shown to the right.