Development of a novel real-time polymerase chain reaction assay for the sensitive detection of Schistosoma japonicum in human stool.

BACKGROUND: Elimination and control of Schistosoma japonicum, the most virulent of the schistosomiasis-causing blood flukes, requires the development of sensitive and specific diagnostic tools capable of providing an accurate measurement of the infection prevalence in endemic areas. Typically, detection of S. japonicum has occurred using the Kato-Katz technique, but this methodology, which requires skilled microscopists, has been shown to radically underestimate levels of infection. With the ever-improving capabilities of next-generation sequencing and bioinformatic analysis tools, identification of satellite sequences and other highly repetitive genomic elements for use as real-time PCR diagnostic targets is becoming increasingly common. Assays developed using these targets have the ability to improve the sensitivity and specificity of results for epidemiological studies that can in turn be used to inform mass drug administration and programmatic decision making. METHODOLOGY/PRINCIPAL
FINDINGS: Utilizing Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2, a cluster-based analysis of the S. japonicum genome was performed and a tandemly arranged genomic repeat, which we named SjTR1 (Schistosoma japonicum Tandem Repeat 1), was selected as the target for a real-time PCR diagnostic assay. Based on these analyses, a primer/probe set was designed and the assay was optimized. The resulting real-time PCR test was shown to reliably detect as little as 200 ag of S. japonicum genomic DNA and as little as 1 egg per gram of human stool. Based on these results, the index assay reported in this manuscript is more sensitive than previously published real-time PCR assays for the detection of S. japonicum.
CONCLUSIONS/SIGNIFICANCE: The extremely sensitive and specific diagnostic assay described in this manuscript will facilitate the accurate detection of S. japonicum, particularly in regions with low levels of endemicity. This assay will be useful in providing data to inform programmatic decision makers, aiding disease control and elimination efforts.

Introduction

Schistosomiasis is a debilitating Neglected Tropical Disease (NTD) estimated to infect more than 230 million people worldwide. Of the blood flukes causing this disease, Schistosoma japonicum, which is endemic in China, Indonesia, Taiwan (zoophilic strain), and the Philippines, is the most virulent species [1-3]. S. japonicum is known to successfully infect 46 mammalian hosts [4] with each adult female schistosome producing approximately 1,000 eggs per day [5]. The deposition of S. japonicum eggs can result in the formation of tissue granulomas, in turn giving rise to a severe immune response that frequently leads to cognitive impairment, growth stunting, diarrhea, rectal bleeding and abdominal pain, with some people developing severe hepatosplenic disease [1,6].

While S. japonicum infection in both children and adults can be controlled through periodic administration of the preventive chemotherapeutic praziquantel [7], until now S. japonicum has only been successfully eliminated from Japan [8]. S. japonicum’s wide range of animal hosts make its elimination highly challenging [9]. This pathogen’s complicated life cycle, involving the excretion of eggs into freshwater environments followed by further growth in Oncomelania hupensis, a freshwater snail that serves as an intermediate host [9], results in the exposure of many reservoir hosts—such as bovines, rodents, goats, and dogs—to infection [10-11]. Given these challenges, elimination will require accurate, efficient, and sensitive diagnostic tools that can be applied to screening humans, other definitive hosts, and snail vectors.

Typically, detection of S. japonicum eggs from human-derived stool samples has occurred using the quantitative, coprological Kato-Katz technique [12-13]. However, this technique, which requires skilled microscopists, has been shown to have low reproducibility in determining egg counts [14] and to have poor sensitivity in low endemicity/infection intensity settings [15]. In contrast, molecular techniques such as real-time PCR can detect extremely limited concentrations of parasite-derived DNA, exhibiting greater sensitivity than microscopy-based approaches [16]. While a small number of S. japonicum-targeting real-time PCR diagnostic assays can be found in the literature, each assay has targeted comparatively low copy number sequences such as the mitochondrial NADH dehydrogenase I gene [17], a putative DNA photo-lyase gene [18], and the mitochondrial DNA 16S rRNA gene [19]. While these assays have exhibited greater sensitivity than microscopy-based techniques, targeting coding sequences inevitably raises concerns about target specificity as such genes frequently exhibit greater conservation among closely related species [20]. In contrast, non-coding tandemly repeated genomic regions make ideal real-time PCR targets, often providing high sensitivity and species-level specificity [20-21]. Here we report the development of a highly sensitive, specific real-time PCR assay for the detection of S. japonicum in human stool by targeting an abundant, tandemly-repeated genomic DNA sequence, which we have named SjTR1 (chistosoma aponicum Tandem Repeat 1) (GenBank:MW631938).

Materials and methods

Ethics statement

Informed written consent was received from all human participants in the study and ethical approval was provided by the Ethics Committee of the Research Institute of Tropical Medicine (RITM), Manila, and the Queensland Institute of Medical Research (QIMR) Human Research Ethics Committee (Approval Number: H0309-058 (P524)).

Assay target identification

A library of raw, paired-end reads resulting from the next-generation sequencing of female S. japonicum-derived DNA (SRR6841388) was retrieved from the Sequence Read Archive at the National Center for Biotechnology Information (NCBI) website (www.ncbi.nlm.nih.gov). Following quality analysis using FASTQC, a random sampling of 500,000 sequences was analyzed using Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2 software as implemented on the Galaxy-based web server [22-26]. The repetitive elements of the genome were characterized using a graph-based technique, which forms clusters of reads that demonstrate 90% or greater identity over 55% or greater of the longer sequence length. Clusters containing threshold-exceeding numbers of shared paired-end reads were then aligned to form superclusters, resulting in the identification of multiple, high-coverage consensus sequences [24-26]. From these genomic elements, the most repetitive sequence (SjTR1) was chosen as a putative real-time PCR assay target.

Real-time PCR assay design

A set of forward and reverse primers and a probe (Table 1) were designed using PrimerQuest Tool software (Integrated DNA Technologies, Coralville, IA) to target the SjTR1 sequence (Fig 1). The primers and probes were analyzed with the Primer-Blast tool, available from the NCBI website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to screen for possible off-target amplification sites. The probe was labeled with a 56-FAM fluorophore at the 5’ end and was double quenched with ZEN and 3IABkFQ.

Table 1

S. japonicum assay primers and probe.

Forward Primer	5’-TGT CGT GCA CAA CCT TCT TC-3’
Reverse Primer	5’-ACA ACT CAT CAC CGC CAA TC-3’
Probe	5’-/56-FAM/ TGG CGA GAT / ZEN/ GTT GTG GGT GTA AGT / 3IABkFQ/-3’

Fig 1

The amplified region of the SjTR1 genomic sequence.

Locations of the S. japonicum assay primer and probe binding sites are indicated.

Nucleic acid extraction and whole genome amplification of S. japonicum, S. mansoni, S. haematobium, and S. mekongi genomic DNA

DNA was extracted from samples of S. japonicum (Hubei, China), S. mansoni (Senegal River Basin), and S. haematobium (Zanzibar Island, Tanzania) single male worms graciously provided by the Natural History Museum, London, UK [27]. These extractions were performed using the Isolate II Genomic DNA kit from Meridian Bioscience (Memphis, TN) and extracted DNA was whole genome amplified using the REPLI-g UltraFast Mini Kit (Qiagen, Germantown, MD) in accordance with manufacturer protocols. DNA from S. mekongi worms (Maintained at Applied Malacology Laboratory, Faculty of Tropical Medicine, Mahidol University, Thailand) was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD).

Real-time PCR assay optimization and limit of detection

Real-time PCR assay optimization experiments were performed as described in previous studies [26] using the StepOnePlus Real-Time PCR System (Thermofisher Scientific, Waltham, MA). Briefly, the optimal annealing/extension temperature for the assay was determined by testing a range of annealing temperatures from 52°C to 62°C. We initially tested six temperatures at 2°C increments from 52°C to 62°C and then narrowed the range, testing six temperatures at 0.5°C increments from 56.5°C to 59°C. The annealing temperature yielding the lowest Cq value was chosen. Optimal primer concentrations were determined using 7 μL reactions containing 3.5 μL of TaqPath ProAmp Master Mix (Thermofisher Scientific), and employing doubling dilutions of forward and reverse primers at concentrations ranging from 62.5 nM to 1000 nM in all possible combinations. The limit of detection was determined under optimized assay conditions by testing ten-fold serial dilutions of a S. japonicum genomic DNA stock with template masses ranging from 100 pg to 1 ag. Negative controls for all experiments were prepared using the same master mix without any template DNA added.

Specificity panel for the S. japonicum assay

Specificity of the assay was assessed by testing against a variety of non-target DNA templates. All testing occurred in 7 μL reactions containing 3.5 μL of TaqPath ProAmp Master Mix and optimized primer/probe concentrations. All templates were added at a mass of 200 pg/reaction and included isolated DNA from S. mansoni, S. haematobium, S. mekongi, Trichuris trichiura, Strongyloides stercoralis, Ancylostoma duodenale, Ascaris lumbricoides, Baylisascaris procyonis, Ancylostoma caninum, Parascaris univalens, Anisakis typica, Necator americanus, Taenia solium, Taenia crassiceps, Giardia intestinalis, Mock Microbial Community B(HM-277D) (BEI resources, Manassas,VA), Candida albicans (strain L26), Escherechia coli, and human DNA. Origins of samples are provided in S1 Table.

Generation of plasmid control containing the assay target sequence

SjTR1 qPCR forward and reverse primers were used to amplify the target sequence from genomic S. japonicum DNA using conventional PCR. The target sequence was size selected using agarose gel electrophoresis, purified from the gel using the Monarch Genomic DNA Purification Kit (New England Biolabs, Ipswich, MA), and cloned using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen, Carlsbad, CA) as previously described [26]. The resulting plasmid was used to transform NEB Express Competent E. coli cells (New England Biolabs), which were plated on LB-kanamycin plates and grown overnight at 37°C. Following the selection of colonies, PCR and sequencing were performed to identify a plasmid containing a single copy of the assay’s target sequence which was subsequently used as a positive control and to calculate the assay’s efficiency.

Real-time PCR assay efficiency

To determine assay efficiency, the plasmid concentration was quantified using a Qubit 2.0 Fluorometer (Thermofisher Scientific), and serial dilutions were created resulting in stocks spanning 10-fold serial dilutions ranging from 100 pg/μL to 100 ag/μL. The number of plasmid copies was calculated for each dilution with 100 ag of S. japonicum DNA corresponding to 26 copies of the target sequence. The log of plasmid copies was plotted against the mean Cq resulting from the amplification of each concentration of plasmid (11 replicates per concentration) and the slope of the linear line was used to calculate the assay’s efficiency.

Testing naive human stool spiked with S. japonicum eggs

A series of Lysing Matrix E tubes (MP Biomedicals, Santa Ana, CA) were prepared according to the manufacturer’s recommendations. Three samples each of 1, 3 and 10 S. japonicum eggs were transferred to their respective Lysing Matrix E tubes. Using a sterile loop, 50 mg of naive stool (BioIVT, Westbury, NY) was then added to each tube, resulting in three samples containing 20, 60, and 200 eggs per gram (epg), respectively. Three samples were prepared for each concentration of eggs. Following preparation, all samples were homogenized for 40 seconds using the FastPrep-24 5G Instrument (MP Biomedicals) on a speed setting of 6, and DNA extraction was performed as described previously using the FastDNA Spin Kit for Soil (MP Biomedicals) [26]. To test the assay’s ability to detect as little as 1 epg of stool, additional replicates were prepared by spiking a single egg into each of ten 1 g stool samples. The weight of the stool was measured using an accurate balance and the full 1 g of stool was extracted. For preparation of these samples, a smaller volume of sodium phosphate buffer (528 μL) was used to reserve room in the tube for the additional stool. These samples were homogenized for 80 seconds using the FastPrep Instrument on a speed setting of 6 and were then subjected to DNA extraction as described above.

Comparison of index and previously published real-time PCR assay performance

To assess comparative performance, the sensitivity of our real-time PCR index assay was compared to that of previously published reference assays [17-19]. Testing was done using S. japonicum gDNA as template, as well as DNA extracted from human stool spiked with S. japonicum eggs. When conducting assays targeting SjTR1, the mitochondrial NADH dehydrogenase I [17] or putative DNA photo-lyase [18] genes, testing was performed in 7 μL reaction volumes containing 3.5 μL of TaqPath ProAmp Master Mix. When targeting the mitochondrial DNA 16S rRNA gene [19], reactions were performed in 5 μL volumes containing 2.5 μL of PrimeTime Gene Expression Master Mix (Integrated DNA Technologies). Testing using all published assays utilized primer and probe concentrations and annealing temperatures described in the literature. Each assay was tested against 2 ng and 200 pg of gDNA template with five [17-18] or three [19] replicates respectively run for each assay and each template mass. When testing against spiked stool samples, three reaction replicates were run for each assay.

Validating the SjTR1 assay using clinical samples collected from endemic area

A panel of 100 human stool samples collected from an S. japonicum endemic area in the Philippines, of which 38 were positive and 62 were negative based on Kato-Katz data, was used to validate the performance of our assay on clinical samples [28]. DNA from these samples was extracted using the FastDNA Spin Kit for Soil (MP Biomedicals). To evaluate the efficiency of each extraction, an internal control IAC plasmid (100 pg) was spiked into each extracted sample prior to the DNA binding step [29]. The recovery of the IAC plasmid was tested via real-time PCR in duplicate 7 uL reactions with 125 nM primer/probe concentrations and an annealing temperature of 59°C. All samples with mean Cq values that were >3 standard deviations from the mean were re-extracted and re-tested. To assess the performance of our index assay in comparison to a published real-time PCR reference assay, we tested all samples using the published mitochondrial NADH dehydrogenase I gene-targeting assay [17]. This assay was chosen for comparison due to its performance during the spiking experiments described above. For both assays, samples that showed inconsistent amplification (amplifying in one of two replicates) as well as samples that had a standard deviation >3 between tested replicates underwent re-testing, again in duplicate, and retest results were reported. Each experiment was run for 40 cycles and a sample was considered positive if it had a Cq of 40 or less.

Results

Target identification and real-time PCR assay design

RepeatExplorer2 and TAREAN analyses resulted in 5.95% of the analyzed S. japonicum sequence reads mapping to putative satellite repeats yielding a total of 40 clusters and superclusters. From among the 24 clusters classified as “high confidence” satellites, the cluster comprising the highest proportion of the analyzed sequences (1.7%) was chosen as our assay target. The consensus sequence resulting from this cluster, SjTR1, had a length of 101 base pairs and contained no protein-coding domains. The primers and probe designed for the assay had a GC content of 50% and produced an amplicon of 80 nucleotides (Table 1 and Fig 1).

Real-time PCR assay validation and optimization

Optimization reactions resulted in an ideal annealing/extension temperature of 59°C (S2 Table). Primer limiting reactions determined that primers performed optimally with the forward primer at a concentration of 62.5 nM and the reverse primer at a concentration of 500 nM (S3 Table). Dilutions of genomic DNA showed the assay to be reliably sensitive at template masses as low as 200 ag (Tables 2 and S4). The assay did not amplify the DNA of any of the common gut parasites, S. mansoni, S. haematobium, the Mock Microbial Community, C. albicans, E. coli, or human DNA in the specificity panel. The assay did, however, amplify S. mekongi DNA (S5 Table).

Table 2

Analytical limits of detection for the SjTR1 assay.

Mass of S. japonicum DNA	Mean Cq +/- SD
200 pg	11.79 +/- 0.11
20 pg	14.93 +/- 0.08
2 pg	17.79 +/- 0.36
200 fg	21.52 +/- 0.21
20 fg	25.21+/- 0.62
2 fg	28.71 +/- 0.18
200 ag	32.14 +/-1.47
20 ag	N/A
2 ag	N/A

Using the size of the S. japonicum repeat-containing plasmid to estimate the number of target copies in successive 10-fold dilutions of the plasmid, a linear curve was constructed as described previously [26]. The slope of the line (-3.26) was used to calculate an assay efficiency of 102.7% with an amplification factor of 2. The R2 value of the linear curve was 0.983 (Fig 2).

Fig 2

Assay efficiency.

Ten-fold serial dilutions of the plasmid, ranging from 100 pg to 100 ag were prepared and the target copy number was estimated for each dilution (S6 Table). The log of the target copy number was plotted against the mean Cq of 11 replicates for each respective dilution. The slope of the line was used to determine the efficiency of the assay. Error bars are included but due to the small standard deviations resulting from each concentration of plasmid DNA tested, they are not distinguishable at most points.

Comparison of S. japonicum real-time PCR assay targets

Real-time PCR comparing the detection of S. japonicum assay targets using S. japonicum gDNA showed that the mean Cq values produced by the SjTR1-targeting assay were 16.45, 20.66, and 14.21 cycles lower than those produced by previously published assay targets when testing 2 ng of template DNA and 15.10, 19.92, and 19.48 cycles lower when testing 200 pg of template DNA (Fig 3 and S7 Table) [17-19]. Although comparisons made using genomic DNA as template cannot be used to evaluate the comparative clinical sensitivities of the examined assays, these sizable differences in mean Cq values strongly suggest that the SjTR1 is considerably more abundant in copy number within the S. japonicum genome than previously published targets [17-19,21]. When testing stool spiked with S. japonicum eggs, the mean Cq values produced by the SjTR1-targeting assay were 12.70, 17.86, and 12.09 cycles lower than values produced by previously published assays on samples containing 20 epg of stool; 12.78, 17.77, and 12.27 cycles lower on samples containing 60 epg of stool; and 12.24, 17.74, and 11.75 cycles lower on samples containing 200 epg of stool [17-19] (Tables 3 and S8). Most notably, when testing 1 gram stool samples spiked with a single egg, the SjTR1-targeting assay detected S. japonicum DNA in all tested samples, while the mitochondrial NADH dehydrogenase I gene target assay [17] detected DNA in only eight of the ten samples, with sporadic detection in four of these replicates. All other assays failed to detect DNA from any of the “one egg” samples (Tables 4 and S9). Ten negative controls were analyzed in parallel, containing 1 gram of naive stool and no S. japonicum eggs. No negative control samples were amplified by any of the assays.

Fig 3

Comparison of mean Cq values.

Results from gDNA testing with the SjTR1 real-time PCR assay and previously published S. japonicum assays.

Table 3

Comparative detection of DNA extracted from naive human stool spiked with S. japonicum eggs.

	1 egga = 20 EPGMean Cq [Range]	3 eggsa = 60 EPGMean Cq [Range]	10 eggsa = 200 EPGMean Cq [Range]
SjTR1	16.68 [15.93–17.19]	15.58 [14.89–15.99]	14.06 [13.60–14.34]
mitochondrial NADH dehydrogenase I gene [17]	29.38 [28.94–30.00]	28.36 [26.73–29.46]	26.30 [25.71–26.73]
putative DNA photo-lyase gene [18]	34.54 [33.38–35.96]	33.35 [32.36–33.88]	31.80 [31.31–32.25]]
mitochondrial 16S rRNA gene [19]	28.77 [28.44–29.17]	27.85 [26.42–28.95]	25.81[25.32–26.19]

Number of eggs spiked in a sample containing 0.05 g of stool.

Each concentration of eggs was tested using three biological replicates, and each replicate was analyzed by the SjTR1 assay in triplicate. The reported mean Cq value was calculated as a mean value of all component sample means. The reported range includes the smallest and greatest individual Cq values for each egg concentration. EPG = eggs per gram of stool.

Table 4

Comparative Detection of 1 gram stool samples spiked with an individual S. japonicum egg (positive samples are bolded; negative controls are in standard font).

	SjTR1*		mitochondrial NADH dehydrogenase I gene [17]*		putative DNA photo-lyase gene [18]*		mitochondrial 16S rRNA gene [19]*
	Mean Cq [Range]a	Total Detected	Mean Cq [Range]a	Total Detected	Mean Cq [Range]a	Total Detected	Mean Cq [Range]a	Total Detected
Sample 1	38.45 [36.96–39.95]	2/3	35.44	1/3	N/A	0/3	N/A	0/3
Sample 2	31.62 [31.42–31.95]	3/3	36.58	1/3	N/A	0/3	N/A	0/3
Sample 3	N/A	0/3	N/A	0/3	N/A	0/3	N/A	0/3
Sample 4	30.05 [29.93–30.25]	3/3	37.67 [35.02–39.31]	3/3	N/A	0/3	N/A	0/3
Sample 5	N/A	0/3	N/A	0/3	N/A	0/3	N/A	0/3
Sample 6	N/A	0/3	N/A	0/3	N/A	0/3	N/A	0/3
Sample 7	30.90 [30.55–31.19]	3/3	36.57 [36.27–36.86]	2/3	N/A	0/3	N/A	0/3
Sample 8	N/A	0/3	N/A	0/3	N/A	0/3	N/A	0/3
Sample 9	N/A	0/3	N/A	0/3	N/A	0/3	N/A	0/3
Sample 10	33.45 [33.26–33.72]	3/3	N/A	0/3	N/A	0/3	N/A	0/3
Sample 11	31.93 [31.49–32.27]	3/3	35.00 [33.42–36.20]	3/3	N/A	0/3	N/A	0/3
Sample 12	30.34 [30.08–30.49]	3/3	33.97 [33.58–34.40]	3/3	N/A	0/3	N/A	0/3
Sample 13	N/A	0/3	N/A	0/3	N/A	0/3	N/A	0/3
Sample 14	33.08 [32.87–33.45]	3/3	N/A	0/3	N/A	0/3	N/A	0/3
Sample 15	31.06 [30.85–31.34]	3/3	35.85	1/3	N/A	0/3	N/A	0/3
Sample 16	N/A	0/3	N/A	0/3	N/A	0/3	N/A	0/3
Sample 17	N/A	0/3	N/A	0/3	N/A	0/3	N/A	0/3
Sample 18	N/A	0/3	N/A	0/3	N/A	0/3	N/A	0/3
Sample 19	N/A	0/3	N/A	0/3	N/A	0/3	N/A	0/3
Sample 20	33.15 [32.81–33.34]	3/3	37.16	1/3	N/A	0/3	N/A	0/3

a. Each spiked sample was run in triplicate, and the mean Cq values are reported. The reported range includes the smallest and greatest individual Cq values for each individual sample. None of the negative control samples were amplified by any of the assays.

*The SjTR1 assay detected S. japonicum DNA in all 10 of the samples spiked with a single egg (amplification in three out of three replicates for 9 of the 10 samples and two out of three for one of the samples). The mitochondrial NADH dehydrogenase I real-time PCR assay [17] detected S. japonicum DNA in only eight of the ten samples tested, with sporadic detection (amplification of only one out of three replicates) in four of these samples. The real-time PCR assays targeting the putative DNA photo-lyase and mitochondrial 16S rRNA genes [18–19] did not detect S. japonicum DNA in any of the samples.

Validation of the assay on clinical samples

A panel of 100 field collected samples from endemic areas in the Philippines were blindly tested, of which 38 were positive and 62 were negative based on Kato-Katz data [28]. Testing of our SjTR1 real-time PCR assay resulted in 59 positive samples and 41 negative samples (S10 Table). On the other hand, testing using the NADH dehydrogenase I real-time PCR assay [17] resulted in 52 positive samples and 48 negative samples (S10 Table). Seven of the SjTR1 positive samples were not detected by the NADH dehydrogenase I assay while all 41 negatives by the SjTR1 assay were not detected by the NADH dehydrogenase I assay [17] (S10 Table). The same thirty-six of the thirty-eight Kato-Katz positive samples were amplified by both the SjTR1 and the NADH Dehydrogenase assays. The two unidentified Kato-Katz positive samples were not detected by any of the published real-time PCR assays discussed in this manuscript [17-19]. For the positive samples, the minimum, the median, the maximum, and the quartiles of mean Cq values are shown below for both assays (Fig 4). Among the positive samples, the mean Cq difference between the two assays was 10 cycles lower for the SjTR1 assay compared to the NADH dehydrogenase I assay (S10 Table).

Fig 4

Boxplots for mean Cq values of positive field collected samples.

Results for testing with the SjTR1 and mitochondrial NADH dehydrogenase I gene [17] assays.

Discussion

Endemic in 3 provinces in Indonesia, 28 provinces in the Philippines, in Taiwan (zoophilic strain), and in China, where its infection levels exceed 10% in high-risk populations, the diagnosis of S. japonicum has typically occurred using the Kato-Katz technique, a method which has been shown to underestimate infection levels by up to 70% [3,9,30-32]. Immunological tests, like antigen detection tests and enzyme-linked immunosorbent assays (ELISA), are another possible avenue for diagnosis of S. japonicum. These tests, however, often lack specificity and sensitivity [33-34]. Such detection methods may lead to insufficient intervention efforts, making the development of highly sensitive, specific molecular diagnostic tools imperative for the successful elimination of S. japonicum.

Within target areas, preventive chemotherapy efforts through praziquantel mass drug administration as well as eligibility of different age groups for treatment is determined based on prevalence of infection as assessed via positive parasitological diagnosis [35]. Thus, for the Western Pacific Region and South-East Asia, the WHO regional priorities for the 2012–2020 period included maintenance of high and/or regular coverage of preventive chemotherapy in the Philippines and Lao People’s Democratic Republic, intensification of preventive chemotherapy in Cambodia, China and Indonesia, and verification of status of elimination in Japan, Malaysia, Thailand, and India [35]. Of these areas, S. japonicum, whose elimination from its endemic areas is complicated by the presence of animal reservoir hosts, is endemic in the Philippines and China—the two countries with the highest proportion of people requiring treatment for schistosomiasis in the Western Pacific Region—and in Indonesia [9,35]. Furthermore, to determine the success of these treatment efforts, sensitive and specific diagnostic tools are needed [9,36-37].

The results reported here demonstrate that our repeat-targeting assay can reliably detect S. japonicum at concentrations as low as 1 egg per gram of human stool. Putting these results in context, S. japonicum infection intensity is defined by the World Health Organization as light (1–100 eggs per gram), moderate (101–400 eggs per gram), and heavy (more >400 eggs per gram) [38]. As such, this assay should greatly improve detection capabilities in areas of low infection intensity if used for determination of infection prevalence.

For the accurate estimation of parasite prevalence necessary to shape and guide mass treatment efforts, the specificity of a diagnostic method is also critical. Accordingly, one of our assay’s main limitations is its ability to detect the closely related Asian schistosome, S. mekongi. However, the range of S. mekongi is believed to be limited to select areas along the Mekong River Basin within the Lao People’s Democratic Republic and Cambodia [39-40]. In addition, S. japonicum and S. mekongi are only thought to overlap in a limited region in Myanmar [41]. Thus, in this area of possible co-endemicity, a positive test result from our highly sensitive assay would need to be confirmed using methods allowing for the molecular differentiation of S. mekongi and S. japonicum [42-43]. It is worth noting that our assay can reliably detect as little as 2 fg of S. mekongi gDNA, which provides evidence for the possible use of the SjTR1 assay for the detection of S. mekongi. The suitability of our assay for detection of S. mekongi in human stool as well as optimization of real-time PCR for differential detection of the two species will be explored in a future study. Further work will also test the SjTR1 assay against various S. japonicum and S. mekongi strains found in Asia. Although mass drug administration for all blood flukes causing schistosomiasis relies on the same drug, praziquantel, specificity of testing is nonetheless important for accurate surveillance of parasite endemicity and infection prevalence.

When compared to other published real-time PCR assays, testing of the SjTR1 real-time PCR assay described here demonstrated remarkable reductions in Cq values and therefore increased sensitivity. This suggests that the target of the assay, a putative tandem repeat, is more abundant in the S. japonicum genome than the target regions of the other qPCR assays tested. This provides additional support for improved analytical sensitivity via the use of satellite repeats instead of traditional targets such as ribosomal or mitochondrial DNA targets [20-21]. Evidence of the increased clinical sensitivity for our assay compared to previously published assays is supported by the results of the spiking study in which the SjTR1 assay was the only real-time PCR assay capable of reliably detecting 1 egg per gram in all samples (Table 4).

Providing further evidence of increased clinical sensitivity, our assay also showed superior performance when used to test field-collected clinical stool samples from an endemic area in the Philippines. Outperforming the most promising published real-time PCR assay through the detection of seven additional samples, our assay detected twenty-one samples more than the traditional Kato-Katz technique. We were not surprised to see that two Kato-Katz positive samples (containing 35 and 113.3 epg, respectively) were not detected by any of the four real-time PCR assays using various targets discussed in the study (S10 Table). Given the known specificity challenges associated with the Kato-Katz technique and the lack of amplification by four sensitive real-time PCR assays, we strongly suspect that those two samples represent Kato-Katz false positive results. Our assay was able to detect parasite DNA in clinical samples with as few as 3 epg, providing evidence that sensitivity of our assay seems like an unlikely cause for these results. Furthermore, the internal control results give us confidence that the quality of the extraction did not interfere with the results. Nonetheless, these two samples comprise an important avenue for further exploration and will be examined using next generation DNA sequencing in a future study. For the positive samples, the mean Cq difference between our assay and the NADH dehydrogenase assay [17] was 10 cycles, providing additional evidence for our assay’s superior sensitivity. Although these results provide strong evidence for the clinical utility of our assay, given S. japonicum’s wide range of hosts, further validation of the assay on stool samples from different endemic areas as well as multiple animal hosts will be important.

Given the acknowledged shortcomings of current coproscopic and immunological methods employed for schistosome detection, improved diagnostic methods for S. japonicum are urgently needed. Due to increased need for HIV, and more recently COVID-19, real-time PCR testing, the availability of updated PCR testing labs in endemic countries has greatly increased, facilitating the use of cost-effective, real-time PCR assays for detection of numerous infectious agents. In fact, it has been shown that the cost of PCR is comparable to that of Kato-Katz with both double-slide Kato-Katz and duplicate PCR testing of a sample costing around 2 US dollars [44-45]. In conclusion, this assay has the capacity to improve detection of S. japonicum, helping to guide programmatic decision-making efforts to control and eventually eliminate S. japonicum from endemic countries.

Checklist indicating where criteria for assessing potential study biases have been addressed.

(DOCX)

Click here for additional data file.

Supplier/origins of parasites tested in specificity panel.

(XLSX)

Click here for additional data file.

Cq values from annealing temperature optimization experiment.

(XLSX)

Click here for additional data file.

The results of primer optimization reactions for SjTR1 assay.

The Cq values are provided for each replicate of forward and reverse primers diluted at concentrations ranging from 62.5 nM to 1000 nM in all possible combinations.

(XLSX)

Click here for additional data file.

Cq values for each replicate for analytical limits of detection for the SjTR1 assay on S. japonicum gDNA, ranging from 200 pg to 2 ag.

(XLSX)

Click here for additional data file.

Cq values for each replicate for analytical limits of detection for the SjTR1 assay on S. mekongi gDNA, ranging from 200 pg to 20 ag.

(XLSX)

Click here for additional data file.

Cq values for each replicate of plasmid target copy number used to calculate the assay’s efficiency.

(XLSX)

Click here for additional data file.

Cq values for each replicate resulting from gDNA testing with the SjTR1 real-time PCR assay and published S. japonicum assays.

(XLSX)

Click here for additional data file.

Cq values for each replicate of comparative detection of DNA extracted from naive human stool spiked with S. japonicum eggs using SjTR1 and published assays.

(XLSX)

Click here for additional data file.

Cq values for each replicate of each sample from comparative detection of 1 gram stool samples spiked with an individual S. japonicum egg using SjTR1 and published assays.

(XLSX)

Click here for additional data file.

Cq values for each replicate for experimental data from testing SjTR1 and NADH dehydrogenase I assays on clinical stool samples, Kato-Katz data, and IAC control data.

(XLSX)

Click here for additional data file.

13 Apr 2021

Dear Ms. Halili,

Thank you very much for submitting your manuscript "Development of a novel quantitative polymerase chain reaction assay for the sensitive and species-specific detection of Schistosoma japonicum in human stool" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Cinzia Cantacessi

Deputy Editor

PLOS Neglected Tropical Diseases

Makedonka Mitreva

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The paper focuses on the analytical design and testing of a qPCR for S. japonicum. For Plos NTD the methods need more detail. Point details are shown below but a careful read and editing by the authors would help bring clarity to the methods and to provide the detail needed by the readers.

• Page 7 – first paragraph. For the samples from the NHM – were these from worms or miracidia? If from worms why was WGA done? Also the geographical origin would be useful.

• Validation and Optimization – last sentence – which, genomic DNA was used. ? S. japonicum.

• Specificity section page 7 and 8 – the origin / supplier of the samples / DNA tested needs to be added.

• Plasmid control – add some more detail on how the target sequence was generated. PCR and then clean up etc.? How many repeats were in the target cloned. Also, a little more detail to this. How the colonies were selected, screened and purified etc.

• Explain more on how you go from a plasmid to the working concentration. This will not be common knowledge for many readers.

• Testing naïve stool samples – where were the eggs from, state that they are S. japonicum and say how you counted / selected them. How was the weight of the stool measured and also I could not understand the addition of buffer to reserve room in the tube and was the whole 1g sample extracted?

• Figure 1 is confusing. You have highlighted the forward primer and the probe on the reverse strand. Although it is not incorrect it is confusing as I am not sure why you would not just highlight them on the forward strand and the reverse primer on the reverse strand? Also the two strands are not aligned so it does not make it easy to look at. Just have one strand in the forward direction and then mark the primers and probe and the sequence.

Reviewer #2: I feel that the number of species of gut microbes used in cross-reaction testing is insufficient. At a bare minimum, this must be tested against Schistosoma mekongi DNA. I understand that the authors have had difficulty obtaining this, but I encourage them to redouble their efforts. Researchers have published the transcriptome, are there at least in silico sequences that could be tested? I see this as a major deficiency in the paper.

Further cross-reaction testing against Giardia duodenalis, a series of other Enterobacteriaceae, Enterococcus spp., Taenia spp., Necator americanus, Clonorchis sinensis and Opisthorchis viverrini, should be performed. DNA of the former five species/groups are easily obtained, so I am surprised that this has not already been performed.

As this assay may well be used on animals by other researchers, if possible, it should be tested on animal schistosomes as well, particularly S. malayensis of rats and S. incognitum of rats, dogs and pigs, both of which may cause rare human zoonotic infections in Asia.

Where was the NHM S. japonicum DNA used from geographically? S. japonicum from Indonesia, China and the Philippines are quite genetically distinct. The Formosan (Taiwanese) strain of S. japonicum, which only infect animals, are genetically distant from other S. japnonicum. You must state the origin of your S. japonicum DNA and eggs used in this study.

Reviewer #3: This manuscript describes a newly designed species-specific real-time PCR for the detection of Schistosoma japonicum DNA in human stool. The authors claim that by cleverly designing the right target (in this case: Schistosoma japonicum Tandem Repeat 1), their PCR is more sensitive than previously published real-time PCR assays for the detection of S. japonicum.

I found the manuscript interesting to read and the PCR described seems to have great potential. My major criticism is that the authors did not use the opportunity to test their PCR on a collection of field samples. In my opinion this would have greatly benefitted the impact of the publication. I find it hard to imagine these authors have no access at all to a set of clinical stool samples, even if only a small one, to prove the diagnostic value of their PCR. A set of samples collected before and after treatment with praziquantel would have been ideal to illustrate the clinical value of their PCR.

Some minor points concerning methods:

Page 7: ” DNA was extracted from samples of S. japonicum, S. mansoni, and S. haematobium graciously provided by the Natural History Museum, London, UK” – more details about the original samples would have been informative.

Page 8 bottom: I would like to read more details of the source of the S. japonicum eggs. Do these eggs fully represent the eggs normally seen in freshly collected stool samples?

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results need some detail added and also need a careful read to make sure nothing is missing. Also as required by Plos NTD all qC values should be provided for all replicates and all samples, even the controls.

• Describe what happened at temperatures above and below 59.

• Was there a cut off Cq score for non-amplification used to test the LOD and the specificity? You do not mention S. mansoni or S. haematobium in the specificity section.

• It would be better to refer to the published targets that you tested as published targets rather than reference targets and make it clear that these are published and not designed as part of this study.

• You mention the negative controls in the results but all the negative controls and how they were set up need to be described in the methods. Negative controls also need to be described for the validation steps.

Reviewer #2: No specific comments

Reviewer #3: See above.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Although the discussion supports the data and the study it is very brief and does not address the wider context of the study. Suggestions for improvement are shown below.

Line three change “historically occurred” to “typically performed” as you should refer to what is typically done now.

• It would be good to provide some information on the interventions in S. japonicum areas. Is routine mass testing done or is it done on a case by case basis, is MDA performed or is it a test and treat scenario. The wider context for the use of qPCR as a test needs to be discussed particularly in relation to other tests such as serology and antigen. What would be the application of a qPCR test in these settings?

• Page 15 – when talking about the critical need for specificity you could include the recent Plod NTD paper by Katie Gass.

• When talking about the need to look at field samples you should also mention other variables such as would the presence of CFPD have an impact?

• You should also talk about the endemic setting and how such a qPCR could fit. For example in S. japonicum settings are labs available for the testing or are portable systems needed like in Africa and also some discussion around cost is needed. Does the test need to fit TPP’s of schisto?

• You should also talk about the diagnostic in terms of intensity analysis – limitations or if it can be used as a quantitative assay or not?

• Also, talk about the potential utility in animal and snail hosts.

Reviewer #2: The greatest issue with the discussion as it stands is the attempt to minimise the need for further validation of this assay in an endemic setting on both human and animal samples. This must be amended and the deficiencies of not having done this very clearly and openly acknowledged and explored.

Discussion first sentence: Also endemic in Taiwan (zoophilic Formosan strain)

If the authors do not test against the many genotypes of S. japonicum found in Asia, they must clearly state that their assay described here has not been validated against the multiple genotypes possible and that this work should be performed in the future.

Similarly, if the authors do not test against the S. japonicum from multiple animal hosts and multiple human hosts, they have not entirely validated their assay and they must state such testing is required to validate the assay before any clinical, public health and particularly veterinary use.

Page 16 first para. I do not believe that the spiking of S. japonicum eggs into naive human stool samples provides a close approximation of real samples since our mock samples. The point of such validations is to test multiple isolates from different hosts as well as to approximate its validity in the faecal matrix. Please significantly amend this sentence to reflect my comment here.

Page 16 para 1: Overall, this assay needs to be compared to Kato Katz and some of the other PCRs against a statistically valid number of human and animal samples in an endemic setting, preferably three endemic settings (one in Indonesia, one in China and one in Indonesia). I understand that this work may yet be performed, but I think it is important that the authors better acknowledge the need for such validation very clearly in the discussion, please amend the sentence addressing the need for field validation to better.

Line 15: I see the failure to test the assay against S. mekongi as a very significant limitation. You should remove the sentence saying that it is not and acknowledge that validation is not complete without such testing.

Reviewer #3: As mentioned above, I find the overall content of this manuscript rather limited. As the authors have mentioned, the focus is entirely on the technical characteristics of the PCR, without any clinical validation. This is a serious omission. One of the major advantages of S. japonicum is the fact that this parasite also affects various non-human hosts, such a cattle. So even if the authors have no access to human stool, they could have tested naturally infected animal samples.

Another limitation of the manuscript is that the authors suggest throughout the manuscript that PCR is the only diagnostic alternative to microscopic detection of S. japonicum eggs. In fact, detection of circulating antigen in serum or urine, a more field-friendly procedure for diagnosing an active S. japonicum infection, has also shown to be a promising alternative. See: Van Dam et al., (2015) Evaluation of banked urine samples for the detection of circulating anodic and cathodic antigens in Schistosoma mekongi and S. japonicum infections: a proof-of-concept study. Acta Trop. 2015 Jan;141(Pt B):198-203.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: A careful read and careful revision of terminology would help improve the paper. Some sections are hard to follow and could be written better and with more detail to help interpretation.

Reviewer #2: The results section is very "table heavy" – this could be remedied by changing table 3 into a figure.

page 12 first para - no space between genus and species name for "S. japonicum"

Page 15 final para - italicise S. mekongi

Reviewer #3: See above.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This is a very informative paper that describes the development and lab. testing of a S. japonicum qPCR assay to support diagnosis. This will be a very useful assay due to it superior sensitivity. The way the target was identified and used shows substantial progress in the design of molecular assays and the use of genomic data that is now becoming available. This will also support the design of assays for other species. The paper needs more detail and a more wider discussion to bring it up to the standard required for Plos NTD.

Some points on the author summary and intro are shown below.

Please add line number as this helps with reviewing.

• Author Summary – 230 million is for schisto total not for S. japonicum – revise the first sentence. This should also be made clear in the first line of the introduction.

• Page 4 last paragraph – add stool or faecal before samples. Line 5 state if this is egg derived DNA or CFPD or both.

Reviewer #2: This is a worthwhile assay but further validation against other common gut parasites found in S. japonicum endemic regions is needed.

Furthermore, if S. mekongi DNA cannot be obtained, or sequences analysed in silico, this must be acknowledged as a significant deficiency.

The authors tend to use terms such as "we do not feel this is a significant limitation" when describing deficiencies of their own work. This not only gets a reviewer's "back up" as it is not for the authors to decide what is an is not a significant limitation of their approach, it is also not good scientific writing. I suggest simply acknowledging your work's deficiencies without attempting to editorialize on how important or unimportant they are.

Reviewer #3: This manuscript is well written, the objectives are clear and the PCR described is potentially relevant for those working in the field of S. japonicum. Never the less, the study would benefit from some additional experimental work, in particular testing of real clinical samples.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

14 Sep 2021

Submitted filename: Response to Reviewers_09.12.2021 (2).docx

Click here for additional data file.

3 Oct 2021

Dear Ms. Halili,

Thank you very much for submitting your manuscript "Development of a novel real-time polymerase chain reaction assay for the sensitive detection of Schistosoma japonicum in human stool" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Cinzia Cantacessi

Deputy Editor

PLOS Neglected Tropical Diseases

Makedonka Mitreva

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #2: (No Response)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #2: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #2: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #2: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #2: The authors have now tested their assay against S. mekongi, which was my major concern upon review of the original manuscript. The have addressed my other comments well.

While the reviewers have now included the zoophilic S. japonicum from Taiwan, it has not been acknowledged as only infecting animals. I would like to suggest the authors include a sentence noting this. I think it is important, as otherwise an error may enter the literature with this article being cited by the uninformed as describing human infection from/in Taiwan.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

4 Oct 2021

Submitted filename: PNTD-D-21-00356R2_Response to Reviewers .docx

Click here for additional data file.

6 Oct 2021

Dear Ms. Halili,

We are pleased to inform you that your manuscript 'Development of a novel real-time polymerase chain reaction assay for the sensitive detection of Schistosoma japonicum in human stool' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Cinzia Cantacessi

Deputy Editor

PLOS Neglected Tropical Diseases

Makedonka Mitreva

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

21 Oct 2021

Dear Ms. Halili,

We are delighted to inform you that your manuscript, "Development of a novel real-time polymerase chain reaction assay for the sensitive detection of Schistosoma japonicum in human stool," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases