Selection of marker transcripts for assay I (sex-specific) and assay II (stage-specific)
The choice of targets for the first assay (Assay I), for the detection and quantification of P. falciparum asexual rings parasites together with the sexual stage male and female gametocytes included the ring stage marker SBP1 (PF3D7_0501300), male marker Pfs13 (PF3D7 1311100), and female marker CCp4 (PF3D7 0903800) which have all been previously described and validated in different studies (3, 8, 41, 42). For the second assay (Assay II), intended to identify and quantify P. falciparum gametocyte developmental stages, the choice of marker transcripts included early gametocyte stage PHISTa (PF3D7_1477700) (35, 43, 44), intermediate gametocyte stage Pfs48/45 (PF3D7_1346700) and mature gametocyte specific stage Pfs25 (PF3D7_1031000) (30, 31). Similarly, these have all previously been described and validated (31, 30, 35). We employed the transcript encoding PF3D7_1120200, a conserved Plasmodium protein, as an internal control which has been previously shown to be expressed in all stages of the parasites, at least in in vitro cultured parasites (35). A positive amplification of the internal control gene shows success in the extraction process thus serving as quality control in addition to the nanodrop readings that we check after every extraction. Primers and probes for assays I and II were designed using the PrimerQuest® tool (SciTools, Coralville, Iowa, USA) according to the suggested quantitative RT-qPCR primer and probe design criteria. To further reduce the likelihood of non-specific amplification, all primers and probes were examined for homology against Plasmodium or human homologous sequences using the PlasmoDB and NCBI Blast links on the PrimerQuest tool. Prior to placing an order for their purification, SerialCloner 2.6.1 was used to test each primer to confirm that it will result in the amplification of the respective target. Tests were not performed to determine whether these primers will recognize P. ovale spp or P. malariae.
Design of RT-qPCR multiplex assay I for gametocyte detection and quantification
We modified and created novel primers and probes with the help of PrimerQuest® tool that we used in a multiplex experiment to simultaneously target ring transcripts SBP1, gametocyte transcripts pf13 and CCp4, predominantly expressed in rings, male and female gametocyte respectively together with a parasite internal control (PF3D7_1120200) which is present in all parasites, in a single reaction. In both naturally occurring infections and parasites grown in culture (Dd2), various levels of amplification of rings and gametocyte transcripts from cDNA were seen (Figure 1). The limit of detection (LOD) and limit of quantification were ascertained using dilution series from synchronized rings and matured gametocyte generated from Dd2 (39). Figure 1 displays graphs of the internal control target (A), the three targets (B male gametocyte target, C ring target, and D female gametocyte target), and their normalized fluorescence representing assay I. Similarly, figure 2 displays graphs of the internal control (A), the three development gametocyte targets (B early gametocyte marker, C intermediate gametocyte marker and D mature gametocyte marker), and their normalized fluorescence representing assay II. The standards utilized to quantify the genes had efficiency and R2 values of 1.25, 0.97 for PF3D7_1120200, 1.08, 0.99 for SBP1, 1.60, 1.00 for pf13, and 0.59, 0.91 for CCp4 representing internal control gene, rings, male and female gametocyte markers respectively for assay I. Also, the standards utilized to quantify the genes in assay II had efficiency and R2 values of 1.25, 0.97 for PF3D7_1120200, 0.88, 0.96 for PHISTa, 1.09, 0.96 for pfs48/45, and 1.66, 0.99 for pfs25 representing internal control gene, early, mid and late gametocyte stages respectively.
Estimation of gametocyte and ring-stage parasite densities using cDNA standards from cultured Dd2 parasites
cDNA from cultured parasite controls that had been serially diluted was used to create standard curves. To remove genomic DNA contamination, RNAs from synchronized rings and various stages of gametocytes were treated with DNase digestion (RNase free DNase kit, Qiagen, Germany). The Qiagen RNeasy mini kit was then used to purify the cDNA (Qiagen, Germany). To reflect the same matrix specimen type as the clinical patient samples, five doses of the cDNA standards were created by producing tenfold serial dilutions (starting concentration for ring: 6.29x107 p/µL; gametocytes: 7.45x106 p/µL) in uninfected human whole blood samples. These five concentrations were tested in duplicate (n = 5) to create an "external standard curve" for each assay. These five concentrations make up the cDNA standard curves for each assay. During clinical validation, aliquots of the five concentrations for every standard curve were also run with every RT-qPCR test in singlet. This was accomplished by importing the linear regression model from the external standard curve (SBP1 slope: 3.145, Pf13 slope: 2.404, CCp4 slope: 4.993 for assay I and PHISTa slope: 3.659, Pfs48/45: 3.11, Pfs25: 2.351 for assay II) into each RT-qPCR run in the Mic Real Time PCR software, and then fixing the intercept to the Cq value of the standard with the highest parasitaemia, in order to determine the parasitaemia of cDNA in test samples.
Performance of RT-qPCR assay I and II on blood samples and bone marrow aspirates
A total of 20 paired samples from two study groups (adults and children) were used for the validation of the two assays, which had 10 paired samples of Day 0 (D0) and their corresponding Day 7 (D7) from children (GI study) and 10 paired samples of peripheral blood (PB) and corresponding bone marrow aspirate (BM) (BM study) which were collected largely from adults. The demographic characteristics of the samples available for the current analysis are presented in table 1.
Table 1: Demographics of gametocyte immunity (GI) and bone marrow studies’ participants at enrolment
Parameters
|
GI (n=10)
|
BM (n=10)
|
Sex
|
|
|
Male/Female
|
8/2
|
6/4
|
|
|
|
Age (years)
|
|
|
Median (IQR)
|
8
(10 .75– 3)
|
36 (46.5 – 34)
|
Min-Max
|
2 - 12
|
9 - 67
|
|
|
|
Temp (oC)
|
|
|
Median (IQR)
|
37.5 (37.9 – 36.4)
|
36.4 (36.6 – 36.3) *
|
Min-Max
|
36.3 – 39.6
|
35.8 – 36.9
|
|
|
|
Hb (g/dL)
|
|
|
Median (IQR)
|
11 (11.7 – 10.35)
|
10.7 (12.4 – 9.4) *
|
Min-Max
|
7.9 – 12.4
|
7.5 – 13.7
|
Using assay I, a total of 40% (4/10) of the samples were identified as P. falciparum rings parasite positive by microscopy, and SBP1 qPCR in D0 peripheral blood of the GI study. None were positive on day 7 post-treatment by either diagnostic method as expected. Similar analysis in the BM study revealed 30% (3/10) and 20% (2/10) P. falciparum rings parasite positives by microscopy in peripheral blood and bone marrow aspirate respectively, while the SBP1 target detected 60% (6/10) of samples being P. falciparum rings parasite positive in both peripheral blood and bone marrow aspirate respectively (Table 2).
Table 2: Asexual parasite and male/female gametocyte prevalence and densities in venous blood by microscopy and RT-qPCR
Parasites
|
Methods
|
|
%(Count/Total)
|
|
|
|
|
|
Total PD (p/uL)
|
|
|
|
|
GI Study
|
BM Study
|
|
|
D0
|
D7
|
PB
|
BMA
|
|
Microscopy
|
40% (4/10)
|
0
|
30% (3/10)
|
20% (2/10)
|
|
|
56000
|
-
|
25040
|
10200
|
Rings
|
|
|
|
|
|
|
|
|
|
|
|
|
RTqPCR (SPB1)
|
40% (4/10)
|
0
|
60% (6/10)
|
60% (6/10)
|
|
|
1507562.3
|
-
|
848902.8
|
167720.4
|
|
|
|
|
|
|
|
Microscopy
|
-
|
-
|
-
|
-
|
|
|
-
|
-
|
-
|
-
|
|
|
|
|
|
|
Gametocyte
|
RTqPCR_Pf13
|
20% (2/10)
|
10% (1/10)
|
30% (3/10)
|
10% (1/10)
|
|
|
1510.74
|
3.66
|
486.82
|
125.59
|
|
|
|
|
|
|
|
RTqPCR_CCp4
|
50% (5/10)
|
40% (4/10)
|
60% (6/10)
|
60% (6/10)
|
|
|
410005.1
|
245270.8
|
77431.82
|
126851.1
|
D0 - Day 0, D7 - Day 7; PB-Peripheral blood; BMA-Bone Marrow Aspirate; PD-Parasite Density; p/uL – parasite per microliter; “-” Not calculated because they were not parasite positivity.
In addition, Assay I identified 20% (2/10) and 50% (5/10) of the sample as male and female P. falciparum gametocyte parasite positive on pre-treatment day D0, whereas on treatment D7 it detected 10% (1/10) and 40% (4/10) of the samples as male and female P. falciparum gametocyte parasite positive, respectively (Table 2). In the BM study, Assay I detected 30% (3/10) and 60% (6/10) of the sample as male and female P. falciparum gametocyte parasite positives respectively in PB, 10% (1/10) and 60% (6/10) of the sample as male and female P. falciparum gametocyte parasite positive respectively in BMA. However, microscopy identified no samples as P. falciparum gametocyte parasite positive in both study groups (Table 2).
Finally, the true prevalence and densities of mature gametocytes was calculated by considering if a sample is positive for Pf13 and/or CCp4 from Assay I. The assay detected 40% (4/10) samples with total gametocyte density of 411,515.8 p/μL and 50% (5/10) samples with total gametocyte parasite density of 245,274.4 p/μL as mature P. falciparum gametocyte parasite positives respectively in D0 and D7 in the GI study. Similarly, the BM study identified 70% (7/10) samples with total gametocyte parasite density of 77,918.65 p/μL and 60% (6/10) samples with total gametocyte parasite density of 126,976.7 p/μL as mature P. falciparum gametocyte parasite positives in PB and BMA respectively.
Reducing/blocking malaria transmission is critical to malaria elimination and eradication. Blocking transmission requires tools for accurate assessment of various developmental stages of the malaria parasites, particularly those involved in transmission. In Assay II (stage-specific), 40% (4/10), 50% (5/10), and 30% (3/10) of the samples tested positive for early, intermediate, and mature stage of P. falciparum gametocytes, with total PD of 86,536.36, 7,826,740.4 and 10,270,216.42 respectively in peripheral blood (Table 3). Whereas similar analysis in the bone marrow aspirate identified parasite prevalence of 70% (7/10), 20% (2/10), and 30% (3/10) of the samples testing positive for early, intermediate, and mature stage of P. falciparum gametocytes, with the sum total PD of 76545.56, 2475685.26, and 8630142.54 p/ul respectively (Table 3).
Table 3: Gametocyte stages and densities in peripheral blood (PB) and bone marrow (BM) by RTqPCR assay II
Parasites
|
Methods
|
% (Count/Total)
Total PD (p/uL)
|
|
|
PB
|
BM
|
|
PHISTa
|
40% (4/10)
|
70% (7/10)
|
|
|
86536.46
|
76545.56
|
|
|
|
|
Gametocyte
|
Pfs48/45
|
50% (5/10)
|
20% (2/10)
|
parasites
|
|
7826740.40
|
2475685.26
|
|
|
|
|
|
Pfs25
|
30% (3/10)
|
30% (3/10)
|
|
|
12070216.42
|
8630142.54
|
|
|
|
|
D0 - Day 0, D7 - Day 7; PB-Peripheral blood; BMA-Bone Marrow Aspirate; PD-Parasite Density; p/uL – parasite per microliter
Estimation of P. falciparum gametocyte sex ratios
Malaria transmission to mosquitoes depends on the presence of gametocytes in the peripheral blood circulation of an infected human and the transmission dynamics are mainly determined by the density and sex ratio of the gametocytes (7). Therefore, molecular methods allow the measurement of Plasmodium infectivity and the determination of gametocyte sex ratio (male/female) when assessing transmission epidemiology and the efficacy of transmission-blocking interventions. Here, we adapted a method by (45) and (46), where sex ratio is defined as the proportion or percentage of all gametocytes that are male and this was determined in both study groups; GI had a sex ratio = (2/(2+7))*100 = 22.2% and BM had a sex ratio = (3/(2+7))*100 = 30.0%. Gametocyte detection in both studies were female-biased, consistent with previous findings (5, 7, 9, 47, 48) and the BM study had a slightly higher sex ratio (30.0%) compared GI study (22.2%).
Comparison of asexual parasite and gametocyte densities between the two studies by parasite targets
Overall, more ring-stages (60% in BM) and gametocytes (70% in both GI and BM) were detected by RT-qPCR compared to ring-stages (40% in GI and 30% in BM) by microscopy. There was slightly higher prevalence of parasites detected in the BM study for both the rings (6 vs 4) and the male (3 vs 2) target compared to the GI but the reverse was seen in their densities. However, the female target identified the same prevalence (70% (7/10)) in both study groups with GI having slightly higher parasite density (Figures 3 and 4).