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Evaluation of a modified quantitative polymerase chain reaction assay for genus Schistosoma detection using stool and urine samples from schistosomiasis endemic areas in Kenya
Modified Schistosoma qPCR for urine and stools samples
https://orcid.org/0000-0003-2509-8692
Kanyi Henry Conceptualization Data curation Formal analysis Methodology Project administration Supervision Validation Writing – original draft Writing – review & editing 1 *
https://orcid.org/0000-0003-2512-1645
Kihoro Richelle W. Formal analysis Writing – original draft Writing – review & editing 1
Chieng Benard Formal analysis 1
Araka Sylvie Formal analysis 1
Emisiko Henry Formal analysis 1
Ramos Thierry Conceptualization 2
Nogaro Sarah Conceptualization Methodology Supervision Writing – original draft Writing – review & editing 2
Njenga Sammy M. Conceptualization Investigation Methodology Project administration Supervision Writing – original draft Writing – review & editing 1
1 Eastern and Southern Africa Centre of International Parasite Control (ESACIPAC), Kenya Medical Research Institute (KEMRI), Nairobi, Kenya
2 FIND, Geneva, Switzerland
Aboelhadid Shawky M. Editor
Beni Suef University Faculty of Veterinary Medicine, EGYPT
Competing Interests: The authors declare that they have no competing interests.

* E-mail: hkanyi@kemri.go.ke
20 9 2024
2024
19 9 e03101184 4 2024
26 8 2024
© 2024 Kanyi et al
2024
Kanyi et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Introduction

The microscopy-based Kato-Katz and urine filtration techniques have traditionally faced challenges in the detection of schistosomiasis in areas with low infection levels. A modified singleplex Schistosoma genus-specific quantitative real-time polymerase chain reaction (qPCR) assay was therefore evaluated as a sensitive and confirmatory schistosomiasis diagnostic test.

Methodology

The qPCR assay utilized primers and probe targeting internal transcribed spacer– 2 (ITS2) sequence of S. mansoni, S. haematobium and S. intercalatum. A plasmid (pDMD801, 100pg/ul) was used as an internal amplification control and its qPCR assays were run in parallel to the Schistosoma assays. This assay utilized samples collected from 774 participants and microscopically examined for three consecutive days. A total of 699 day-one samples (urine and stools) from two schistosomiasis endemic sites were analyzed. Similarly, 75 persons from a non-endemic control site provided both urine and stool samples that were also analyzed.

Results

Using microscopy, the proportion of positives in the two endemic regions altogether was 289/699 (41.3%). Using qPCR, 50.4% of the samples (352/699) were found to be positive for schistosome infection. The percentage of positive samples was slightly higher at 57.8% (203/351) in the S. mansoni endemic site compared with the S. haematobium site at 42.8% (149/348). Majority of the microscopy results were light infections at 26.8% (n = 94) and 26.1% (n = 91) while qPCR majority of the infections were high at 41.6% (n = 146) and 31.3% (n = 109) for the S. mansoni and S. haematobium sites, respectively. There were no positives detected by either microscopy or qPCR in the non-endemic site. Using Bayesian Latent Class Model, which does not use any technique as a gold standard, qPCR showed higher sensitivity (86.4% (PCI: 82.1–90.3)) compared to microscopy (75.6% (PCI: 71.1–80.0)).

Conclusions

This study documents a single day-one sample modified Schistosoma qPCR assay as a powerful improved molecular assay for the detection of schistosomiasis infection that utilize either stool or urine samples. The assay is therefore recommended for monitoring in areas with low infection levels to enable accurate determination of the disease’s control endpoint.

http://dx.doi.org/10.13039/100000865 Bill and Melinda Gates Foundation INV_024908 Nogaro Sarah This work was supported by the Bill and Melinda Gates Foundation (grant award: INV_024908) that was awarded to SH. The funder had no role in decision to publish, or preparation of the manuscript. Data AvailabilityKindly note that the consent forms utilized to collect data from participants explicitly stated that access to the data is limited to study staff, representatives from FIND and KEMRI, members of the ethics committee and the regulatory authorities. Therefore, access to anonymized and de-identified data can be obtained by emailing the Kenya Medical Research Institute (KEMRI) Scientific Ethics Review Unit (SERU) at seru@kemri.go.ke.
Data Availability

Kindly note that the consent forms utilized to collect data from participants explicitly stated that access to the data is limited to study staff, representatives from FIND and KEMRI, members of the ethics committee and the regulatory authorities. Therefore, access to anonymized and de-identified data can be obtained by emailing the Kenya Medical Research Institute (KEMRI) Scientific Ethics Review Unit (SERU) at seru@kemri.go.ke.
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pmcIntroduction

Schistosomiasis (also known as bilharzia) is a prevalent parasitic disease that often leads to chronic illness (if left untreated) especially amongst populations living in areas that lack adequate safe water and sanitation facilities [1]. The human infection is caused by 6 trematode species, namely Schistosoma guineensis, S. haematobium, S. intercalatum, S. mansoni, S. japonicum and S. mekongi. In Sub-Saharan Africa, the predominant species are S. haematobium and S. mansoni that cause urogenital and intestinal schistosomiasis, respectively [2]. The infection is prevalent in 78 countries, with about 240 million people infected globally, thus making it a major public health concern. Sub-Saharan Africa bears the greatest burden of the disease accounting for 93% of the global infection, of which children and young adults are the most affected groups [3, 4]. Humans are infected when they come into contact with fresh water infested with the larval forms of the parasite known as cercariae, which penetrates the skin. In the body, the larvae develop into adult schistosomes. The adult worms live in the veins that drain from the urinary tract and intestines [4]. If left untreated, schistosomiasis could lead to anemia, stunted growth, reduced mental ability and chronic inflammation of the organs, which can be fatal in the most serious instances [2].

The control of schistosomiasis has primarily been done through mass drug administration (MDA) campaigns of praziquantel which is the primary drug recommended by the World Health Organization for chemopreventive therapy [5]. The drug has been shown to be easily administrable, highly effective, tolerable and has minimal side effects [6]. Efforts led by the Kenya’s National School Based Deworming Programme (NSBDP), implemented in 2012, have shown some impact on the overall prevalence of schistosomiasis after repeated rounds of MDAs [7, 8].

Success of control and elimination of any infection is highly dependent on the availability of highly sensitive and specific diagnostic techniques. Diagnosis of schistosomiasis has traditionally relied on urine filtration microscopy and the Kato-Katz methods for the detection of urogenital and intestinal infections, respectively. These techniques are widely used especially in resource poor settings because they are relatively cheap, simple, specific and easily applicable in the field setting [9–12]. However, they have the disadvantage of being time-consuming, laborious, requiring well-trained and experienced personnel, and have poor sensitivity, especially in the context of low-intensity infection [4, 11]. While the search for accurate, widely adaptable and user-friendly new diagnostic tools for schistosomiasis is gaining momentum, the World Health Organization (WHO) target product profile (TPP) guidelines recommend that the ideal requirements for clinical sensitivity and clinical specificity of the test tools should be above 75% and 96.5% respectively for a sample size of 100 individuals [4]. Whilst the microscopy-based urine filtration and Kato-Katz techniques fulfil some of the attributes of the TPPs, they do not consistently meet the sensitivity requirements outlined in the TPP. These guidelines continue to guide the search for new tools to support monitoring and evaluation of schistosomiasis control efforts.

Lately, the development of tests that detect circulating cathodic antigen (CCA) and circulating anodic antigen (CAA) that are secreted by living schistosomes seems to be adding impetus to the development of field-friendly and easily applicable diagnostic tools [13, 14]. A test for CAA is available as a lab-based lateral flow format–known as the Up-Converting Phosphor Lateral Flow (UCP-LF) CAA assay. The UCP-LF CAA assay has shown its usefulness and high sensitivity for the diagnosis of S. haematobium and S. mansoni in low endemicity settings. However, this test is not field friendly and can only be deployed for use in a central laboratory. Additionally, being a commercially available kit, its adoption for use in a resource scarce setting in future may be limited by the associated costs and lot-to-lot variations [15, 16]. Customization of available tests for use in diverse environmental settings would be a boost to schistosomiasis elimination efforts especially in the low resource endemic countries.

The application of various polymerase chain reaction (PCR) techniques such as conventional PCR, real-time quantitative PCR (qPCR) as well as droplet digital PCR (ddPCR) have facilitated the diagnosis of schistosomiasis [17–19]. Real-time quantitative polymerase chain reaction (qPCR) assay for the detection of the Schistosoma deoxyribonucleic acids (DNA) in stool, urine and serum samples is also considered a highly sensitive and specific tool for monitoring of schistosomiasis control/elimination programmes. Several studies have demonstrated the superior performance of qPCR on single species of Schistosoma or while utilizing a single type of sample [9, 20–25]. Elsewhere, the technique has been advanced leading to the development of qPCR assay that simultaneously detect Schistosoma DNA and an internal control [25]. Unlike conventional PCR assays, the use of real-time qPCR has proven advantageous because it can quantitatively detect fewer copies of target DNA, and does not require running of the gel electrophoresis to visualize DNA thus is less laborious [24].

Given that the qPCR can quantify DNA, it is therefore possible to use its cycle threshold (Ct) value as a proxy indicator for parasite burden at a glance [21, 24]. Despite this superior performance, the use of qPCR is generally limited due to the requirement of highly skilled manpower, sophisticated equipment and costly reagents. However, in the context of low parasite burden in areas undergoing elimination, it remains one of the most ideal diagnostic tool for evaluating treatment end point alongside the other gold standard techniques [22, 23, 26–28].…

Unlike the previous PCR assays done for genus Schistosoma, the current assay varies in the following aspects. First, it was used to test both stool and urine samples from the respective schistosomiasis (SCH) endemic and non-endemic sites on a single platform. Secondly, the assay utilized a plasmid as an internal control for DNA extraction but the sample and the internal control assays were done separately (singleplex). Finally, the performance of the assay was validated using same person’s stool and urine samples collected from schistosomiasis non-endemic site. As a result, this modified singleplex Schistosoma genus-specific qPCR assay was therefore evaluated as a sensitive and confirmatory schistosomiasis diagnostic test for possible use in accurate determination of the disease’s control endpoint.

Materials and methods

Ethics statement

The study received ethical clearance from the Scientific and Ethics Review Unit (SERU) of KEMRI (KEMRI/SERU protocol No. 4057). Permission for sample collection was further sought and obtained from the respective county governments. Community mobilization and sensitization was done through meetings organized by the local administrators at the village level. The study participants provided written informed consent for sample collection which included that further molecular analysis would be conducted on the samples. For participants less than 18 years old, parental consent was obtained followed by assent from children (13-<18 years) before enrollment. Illiterate participants provided a thumbprint on the consent form and the consent form was also signed by an impartial witness.

Study design and study area

This was a comparative diagnostic study to evaluate a modified qPCR diagnostic test using stored samples. These samples were collected from two sites that had been identified based on schistosomiasis prevalence and species (Fig 1). The areas are also undergoing control interventions through MDA being steered by the Neglected Tropical Diseases (NTDs) programme in the Ministry of Health (MoH), Kenya. The first site was South Sakwa Ward in Bondo Sub-county, Siaya County which is endemic for S. mansoni [29, 30]. The village borders the shores of Lake Victoria in the western Kenya region. The main economic activity of this area is predominantly fishing and subsistence farming. The area is characterized by warm, dry and humid climatic conditions. The mean rainfall range is between 800–1600 mm annually. The second site was Mkongani Ward in Mwaluphamba location in Matuga Sub-county, Kwale County in the coastal region and endemic for S. haematobium [31–33]. Subsistence farming is the main economic activity of this area. The area is hot and humid all year round with annual mean temperatures range of 22°C—34°C, average relative humidity range of 70% - 80%, and annual rainfall range of 900–1500 mm. Altitude ranges from 0 to 462 meters above sea level. A third site that is non-endemic for schistosomiasis (acted as a control site) was involved in the current study [32]. This site was Kijabe Ward in Lari Sub-county, Kiambu County [34]. This site is largely mountainous and lies on the windward side of the Aberdare ranges in central Kenya. The main economic activity of this area is predominantly agriculture and subsistence farming. The area experiences relatively cold weather and is considerably rainy throughout the year with a mean annual rainfall of 1200mm.

10.1371/journal.pone.0310118.g001 Fig 1 The location of the three study sites.

Study population

Study population comprised of 351 and 348 participants from the S. mansoni and the S. haematobium endemic sites, respectively. Additionally, 76 participants were recruited from the schistosomiasis non-endemic site and each provided both urine and stool samples thus acting as the control site for both the S. mansoni and S. haematobium endemic sites (Fig 2). However, one enrolled individual from the non-endemic site, a young boy, who had travelled to the Western Kenya region where SCH is endemic was removed from the analysis.

10.1371/journal.pone.0310118.g002 Fig 2 Schematic diagram of the study population and procedures.

Study samples

This evaluation was done using microscopically confirmed samples collected from S. mansoni and S. haematobium endemic sites for three consecutive days in Kenya. In addition, both stool and urine samples from a Schistosoma non-endemic control site were similarly analyzed (Fig 2). Collection of the samples in the three study sites was done from February–June 2021. The stool samples were preserved in absolute ethanol at 1:3 mass to volume ratio, while 1 ml of the neat urine samples was aliquoted and stored for qPCR assay. These samples were stored at—20 ⁰C at a local laboratory in the field and later transported in dry ice to the Eastern and Southern Africa Centre of International Parasite Control (ESACIPAC) laboratory in KEMRI, Nairobi where they were stored in a—40 ⁰C freezer until DNA extraction and qPCR assay was done.

Microscopy

Duplicate Kato-Katz thick smears (41.7mg) were made from the same stool sample in laboratories identified in the field sites and the smears examined under the microscope at 100x magnification [12]. Schistosoma mansoni eggs were counted and intensity expressed as eggs per gram of stool (epg) by multiplying the arithmetic mean of egg counts from the six slides per participant by 24 and categorized as per the WHO guidelines (S1 Appendix). The urine samples were also collected for three consecutive days, filtered and examined for ova of S. haematobium. The intensity of infection was calculated as the arithmetic mean of the eggs per 10 ml of urine from the total slides per participant and expressed as the numbers of eggs per 10 ml of urine as per the WHO guidelines (S1 Appendix) [35–37]. For quality assurance purposes, a random examination of 10% of the slides were re-examined by a senior laboratory technologist.

DNA extraction

The DNA was isolated from all samples using QIAamp DNA mini kit (QIAGEN, Hilden, Germany) and as described by Aryeetey et al and Verweij et al with slight modifications [25, 38]. For stool, two hundred and fifty microlitres (250μl) of stool suspended in ethanol was added into 200μl of 2% polyvinylpolypyrrolidone (PVP) dissolved in 1X Phosphate Buffered Saline (PBS) solution. The mixture was homogenized using a vortex mixer for 1 minute and frozen overnight at -20°C. The sample was then incubated for 10 minutes at 100°C followed with a one-minute spin at 8000 rpm. After this, 200μl of tissue lysis buffer (ATL) containing 20μl of proteinase K was added and incubated for 2 hours at 55°C. As an internal DNA extraction control, 1 μl of pDMD801 plasmid at a concentration of 100 pg/μl was added to the samples as a means of verifying the success and consistency of DNA extraction [39, 40]. A negative extraction (nuclease free water only) was included in every extraction cycle. For the urine samples, a similar procedure of DNA extraction was performed but using 200μl of the specimen, no resuspension in PVP solution and without an overnight freezing incubation step. The DNA from both urine and stool were finally double eluted in a volume of 50μl elution (AE) buffer. The DNA extraction and amplification processes were performed in separate rooms to prevent contaminations.

Polymerase chain reaction

Parallel real-time qPCR assays were run for the Schistosoma genus and for the internal control. A Schistosoma qPCR was done using genus-specific primers amplifying a 77-basepair (bp) fragment of the internal transcribed spacer-2 (ITS2) subunit as described by Obeng and others with slight modification [41]. Schistosoma genus-specific primers Ssp48F (5’-GGT CTA GAT GAC TTG ATT GAG ATG CT-3’) and Ssp124R (5’-TCC CGA GCG TGT ATA ATG TCA TTA-3’) were used to amplify the 77-bp fragment of ITS2 and the amplification product detected by the probe, Ssp78T [FAM-5’-TGG GTT GTG CTC GAG TCG TGG C-3’-Black Hole Quencher] (Biolegio, Nijmegen, The Netherlands). The amplification was performed in a 7μl reaction mixture containing qPCR buffer (TaqPath ProAmp mastermix; Thermo Fisher), 12.5pmol of each Schistosoma genus-specific primer, 2.5pmol of the Schistosoma genus-specific double-labeled probe, and 2μL of the DNA sample using a StepOnePlus™ real-time qPCR System (Thermo Fisher Scientific Inc., USA). The thermocycler cycling conditions were as follows: 15 minutes at 95°C, followed by 50 cycles, each of 15 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C. The qPCR output was reported as cycle threshold (Ct) value which represents amplification cycle in which fluorescent level exceeded the background fluorescence. An internal control was also run per sample prior to the Schistosoma assay to test for successful DNA extraction and to ensure exclusion of any presence of qPCR inhibitors. Amplification of internal control (pDMD801 plasmid) was performed in a 7μl reaction mixture containing qPCR buffer (TaqPath ProAmp mastermix; Thermo Fisher), 12.5pmol of each pDMD801 plasmid primer (F: 5’-CTAACCTTCGTGATGAGCAATCG-3’, R: 5’-GATCAGCTACGTGAGGTCCTAC-3’, 2.5pmol of the pDMD801 plasmid double-labeled probe:—56FAM/AGCTAGTCG/ZEN/ATGCACTCCAGTCCTCCT/3IABkFQ/-3’), and 2μL of the DNA sample. The thermocycler used was set to give 15 minutes at 95°C, followed by 40 cycles, each of 15 seconds at 95°C, 30 seconds at 59°C, and 30 seconds at 72°C. Any sample that failed to amplify the internal control was re-extracted and the resulting DNA used as the template in the repeat qPCR. Preparation of master mix and addition of DNA template were done in separate laminar flow chambers which had previously been irradiated for 10 minutes using ultraviolet light and further sterilized using freshly made 10% bleach. The qPCR results were stratified into high (Ct < 30), moderate (30 ≤Ct ≥ 35), low (Ct > 35) and negative (No amplification Ct = 0) [25].

Data analysis

The Bayesian Latent Class Model (BLCM) was used for the data analysis. The first step was to generate a dichotomous variable “location” based on where the study participants were drawn from, that is, either the endemic or the non-endemic regions. The BLCM was then fit in Open Bugs version 3.2.2 software [42] and called from R software via B Rugs Package [43] to generate prevalence, sensitivity (Se), specificity (Sp) and predictive values of the two diagnostic assays for Schistosoma infection and species specific infections of S. haematobium and S. mansoni.

There are three key assumptions when constructing BLCM [44]. Firstly, the target population should consist of two or more sub-populations with different disease prevalence estimates. In this regard, the target population consisted of two separate subpopulations: endemic and non-endemic. The two sub-populations are either endemic for S. haematobium or S. mansoni or non-endemic [45]. Secondly, the sensitivity and specificity should be constant across sub-groups. Finally, the tests should be conditionally independent given the disease status. On this point, conditional independence was assumed.

In our analysis, each sub-population was treated as a separate population k with its own prevalence (Pk). Each sub-population was subjected to two diagnostic tests, i (i = 1,2) The test results are distributed according to a multinomial model. The multinomial probabilities are defined using the specific test characteristics, that is, sensitivity and specificity and prevalence for each sub-population.

From the two sub-populations, the BLCM contained six parameters, that is, the sensitivity and the specificity of the two tests and the prevalence in each sub-population. These six parameters were estimated from the six degrees of freedom obtained from each of the two sub-populations. As per available literature, the Se and Sp estimates of microscopy for S. mansoni fall within the ranges (74.1%-89.7%) and (72.8%-100%), respectively [11, 46, 47]. The Se and Sp estimates of microscopy for S. haematobium fall within the ranges (69.9%-98%) and (85.6%-100%), respectively [10, 48]. These test ranges were used to specify the uniform prior distribution for the microscopy test within the BLCM. Non-informative priors (beta (1,1)) were utilized for the remaining parameters since no prior information was available for qPCR because of the innovativeness of the assay. The diagnostic performance of the tests was further assessed in terms of positive predictive value (PPV) and negative predictive value (NPV). The test estimates of sensitivity and specificity and the posterior distribution of the prevalence of the sub-populations were reported as the median value with the associated posterior credible interval (PCI). A detailed description of the model is provided in the S2 Appendix.

Results

Demographics of study participants

Overall, a total of 849 samples were collected from schistosomiasis endemic and non-endemic (control site) sites. With regard to S. haematobium endemic site, a total of 348 samples were collected (Table 1). Regarding S. mansoni endemic site, 351 samples were collected. In terms of age in S. mansoni endemic population, nearly half of the participants 175 (49.9%) were aged between 5 to 15 years. The same trend was observed for S. haematobium endemic population at 179 (51.4%). A total of 150 samples were collected from the control site, majority of the participants 72 (48.0%) were between the age of 5 to 15 years. Gender distribution was balanced at 76 (50.7%) males and 74 (49.3%) females.

10.1371/journal.pone.0310118.t001 Table 1 Demographics of participants by study population.

Participant characteristics	Overall (N = 849) (%)	Endemic population	Control site	
S mansoni (n = 351) (%)	S haematobium (n = 348) (%)	(n = 150) (%)	
Age categories					
5–15 years	426 (50.2%)	175 (49.9%)	179 (51.4%)	72 (48.0%)	
16–36 years	135 (15.9%)	60 (17.1%)	63 (18.1%)	12 (8.0%)	
27–37 years	119 (14.0%)	61 (17.4%)	42 (12.1%)	16 (10.7%)	
38–48 years	78 (9.2%)	26 (7.4%)	30 (8.6%)	22 (14.7%)	
49–59 years	38 (4.5%)	13 (3.7%)	15 (4.3%)	10 (6.6%)	
60 and above years	53 (6.2%)	16 (4.6%)	19 (5.5%)	18 (12.0%)	
Gender					
Male	498 (58.7%)	145 (41.3%)	130 (37.4%)	76 (50.7%)	
Female	351 (41.3%)	206 (58.7%)	218 (62.6%)	74 (49.3%)	

Stool and urine examinations

The proportion of positive results by microscopy in the two endemic regions combined was 289/699 (41.3%). In terms of species-specific Schistosoma infection, the positivity for S. haematobium infection was 38.5% (134/348) and 44.2% (155/351) for S. mansoni infection. The overall intensity of Schistosoma infection by microscopy was predominantly light for both S. mansoni and S. haematobium. Out of the 155 cases with S. mansoni in the endemic population 26.8% (n = 94), 13.1% (n = 46) and 4.3% (n = 15) had light, moderate and heavy infections, respectively. With regard to S. haematobium in the endemic population, 26.1% (n = 91) had light infection and 12.4% (n = 43) had heavy infection. In the non-endemic population, all samples tested negative using microscopy for either S. mansoni or S. haematobium infection.

Using qPCR, in the two endemic sites altogether, 50.4% (352/699) were found to be positive. Regarding endemic sites, the positivity was 57.8% (203/351) and 42.8% (149/348) in the S. mansoni and S. haematobium sites, respectively. With regard to qPCR results for the endemic population, high intensity infections were 41.6% (n = 146) and 31.3% (n = 109), and low intensity infections were 6.3% (n = 22) and 5.2% (n = 18) for S. mansoni and S. haematobium sites respectively (Table 2).

10.1371/journal.pone.0310118.t002 Table 2 Positivity and intensity of infection categories of Schistosoma infection.

Diagnostic test and intensity category	Overall	Endemic	Non-endemic	
Microscopy–S. mansoni				
Negative	271 (63.6%)	196 (55.8%)	75 (100%)	
Light (1–99 EPG)	94 (22.1%)	94 (26.8%)	0	
Moderate (100–399 EPG)	46 (10.8%)	46 (13.1%)	0	
Heavy (≥400 EPG)	15 (3.5%)	15 (4.3%)	0	
Microscopy–S. haematobium				
Negative	289 (68.3%)	214 (61.5%)	75 (100%)	
Light (1–50 eggs/10ml of urine)	91 (21.5%)	91 (26.1%)	0	
Heavy (≥50 eggs/10ml of urine)	43 (10.2%)	43 (12.4%)	0	
qPCR–S. mansoni				
Negative (No amplification)	223 (52.3%)	148 (42.2%)	75 (100%)	
High (Ct < 30)	146 (34.3%)	146 (41.6%)	0	
Moderate (30 ≤Ct ≥ 35)	35 (8.2%)	35 (9.9%)	0	
Low (Ct > 35)	22 (5.2%)	22 (6.3%)	0	
qPCR–S. haematobium				
Negative (No amplification)	274 (64.8%)	199 (57.2%)	75 (100%)	
High (Ct < 30)	109 (25.8%)	109 (31.3%)	0	
Moderate (30 ≤Ct ≥ 35)	22 (5.2%)	22 (6.3%)	0	
Low (Ct > 35)	18 (4.2%)	18 (5.2%)	0	

Test outcomes of Schistosoma infection

Tables 3 and 4 display the cross-tabulated performance of qPCR and microscopy for the diagnosis of Schistosoma infection by population, sites and species. Out of 348 samples from the S. haematobium endemic site, 128 tested positive by both the qPCR and microscopy assays. Regarding S. mansoni infection, a total of 137 individuals out of 351 tested positive by both the qPCR and microscopy assays in the endemic population. For the combined sites, 265 samples were positive while 304 were negative by both qPCR and microscopy respectively.

10.1371/journal.pone.0310118.t003 Table 3 Cross-tabulated results for combination of qPCR and microscopy for the diagnosis of Schistosoma infection by population, site and species.

Population	Test outcome (qPCR; microscopy)	Total (%)	
(++)	(+-)	(-+)	(—)		
S. haematobium	
Endemic site	128	21	25	174	348 (41.0%)	
S. mansoni	
Endemic site	137	66	18	130	351 (41.3%)	
Non-endemic (Control site)	0	0	0	150	150 (17.7%)	
Total	265	87	43	454	849 (100%)	
+ Positive;—Negative

10.1371/journal.pone.0310118.t004 Table 4 Overall cross-tabulated results for qPCR and microscopy for the diagnosis of Schistosoma infection.

Population	Test outcome (qPCR; Microscopy)	Total (%)	
(++)	(+-)	(-+)	(—)		
Endemic site(s) (Combined)	265	87	43	304	699 (82.3%)	
Non-endemic (Control site)	0	0	0	150	150 (17.7%)	
Total	265	87	43	454	849 (100%)	
+ Positive;—Negative

Sensitivities and specificities of the diagnostic assays

In regards to S haematobium infection, the Se of microscopy (86.8% (PCI: 80.1, 93.1)) was higher than the Se of qPCR (84.5% (PCI: 77.8, 90.9)). As for the Sp, microscopy and qPCR were numerically similar at (99.1% (PCI: 95.2, 100.0)). On the other hand, for S mansoni, the Se of qPCR (88.0% (PCI: 83.5, 89.6)) was higher than the Se of microscopy (75.0% (PCI: 74.1, 78.1)). As for the Sp microscopy and qPCR estimates were (99.3% (PCI: 96.5, 100.0)) and (98.3% (PCI: 92.2, 99.9)) respectively (Table 5).

10.1371/journal.pone.0310118.t005 Table 5 Estimates of the sensitivity and specificity of PCR and microscopy and their respective 95% posterior credibility interval (PCI) and Youden indices for diagnosis of Schistosoma per site.

	S. haematobium	S. mansoni	
Test parameter 1	Estimate (95% PCI)2	Youden indices	Estimate (95% PCI)2	Youden indices	
SeqPCR	84.5 (77.8,90.9)	Youden indexqPCR 83.6	88.0 (83.5,89.6)	Youden indexqPCR 86.3	
SpqPCR	99.1 (95.2,100.0)	98.3 (92.2,99.9)	
SeM	86.8 (80.1,93.1)	Youden indexM 85.9	75.0 (74.1,78.1)	Youden indexM 74.3	
SpM	99.1 (95.2,100.0)	99.3 (96.5,100.0)	
1 Median estimate; 2 Posterior Credible Interval; M-Microscopy; Se- Sensitivity; Sp-Specificity

Overall sensitivities and specificities of the diagnostic assays

On evaluating the overall performance of the two techniques in the detection of genus Schistosoma, the Se of qPCR (86.4% (PCI: 82.1, 90.3)) was higher than the Se of microscopy (75.6% (PCI: 71.1, 80.0)). As for the Sp, microscopy and qPCR were numerically similar at (99.5% (PCI: 97.6, 100.0)). Strikingly, test parameters for specific sites were comparable to the overall performances except for sensitivity in which the overall sensitivity for microscopy technique was inferior to microscopy in the S. haematobium site.

Predictive values and prevalences

The true positivity of Schistosoma infection determined using the BLCM by species varied in the two populations (Table 6). The estimated true positivity of S haematobium infection in the endemic population was recorded at 49.8% (PCI: 43.6, 55.6) which is higher than in the non-endemic population which was recorded at 0.9% (PCI: 0.0, 4.9). In the endemic population, qPCR had a PPV of 98.9% and an NPV of 86.5%, while microscopy had a PPV of 98.9% and an NPV of 88.3%. Conversely, in the non-endemic population, qPCR had a PPV of 46.4% and an NPV of 99.9%, while microscopy had a PPV of 46.5% and an NPV of 99.9% (Table 6).

10.1371/journal.pone.0310118.t006 Table 6 Estimates of the predictive values of qPCR and microscopy and their respective 95% posterior credibility interval (PCI) for diagnosis of Schistosoma infection.

		S. haematobium	S. mansoni	
Population	Test Parameter 1	Estimate (95% PCI)2	Estimate (95% PCI)2	
		qPCR	Microscopy	qPCR	Microscopy	
Endemic	PPV	98.9% (94.0, 100.0.)	98.9%(94.2, 100.0)	98.9%(94.5, 100.0)	99.5%(97.3, 100.0)	
	NPV	86.5%(80.2, 92.5)	88.3%(82.0, 94.1)	82.2%(75.4, 86.4)	69.6%(63.9, 75.5)	
	Positivity	49.8%(43.6, 55.6)	63.5%(57.4, 69.1)	
Non-endemic	PPV	46.4%(2.2, 97.0)	46.5%(2.2, 97.1)	32.8% (1.3, 94.7)	50.5%(2.6, 97.6)	
	NPV	99.9%(99.2, 100.0)	99.9%(99.3, 100.0)	99.9%(99.3, 100.0)	99.8%(98.7, 100.0)	
	Positivity	0.9%(0.0, 4.9)	0.9%(0.0, 4.9)	
1 Median estimate; 2 Posterior Credible Interval; PPV- Positive Predictive Value; NPV- Negative Predictive Value

In the endemic population, the recorded true positivity of S. mansoni was 63.5% (PCI: 57.4, 69.1), compared to 0.9% (PCI: 0.0, 4.9) observed in the non-endemic population. Within the endemic population, qPCR had a PPV of 98.9% and an NPV of 82.2%, while microscopy had a PPV of 99.5% and an NPV of 69.6%. Conversely, in the non-endemic population, qPCR demonstrated a low PPV of 32.8% and a high NPV of 99.9%, while microscopy showed a PPV of 50.5% and an NPV of 99.8% (Table 6).

Discussion

This study presents the performance of microscopy test on urine and stool samples collected over three consecutive days versus genus-specific Schistosoma qPCR assay done on a day one sample only. The analysis of performance of the tests in this study was done using BLCM. This model is primarily based on the fact that there is no reference test and involves explicit definition of the conditions targeted by the tests being evaluated and the statistical model’s complexity [49]. Traditionally methods of estimating sensitivity and specificity are based on the premise that there is a gold standard or reference tests [50]. A drawback of this approach is that, given the imperfection of these reference tests the results generated may be prone to misclassification errors. On the other hand, BLCM allows estimation of the sensitivity and specificity without assumptions of the true disease status of each individual [49, 51]. Additionally, the model allows incorporation of prior information from literature about parameters such as sensitivity, specificity or prevalence enhancing the estimates generated. The defined conditions in the current study is that stool samples collected from S. mansoni endemic and urine samples from S. haematobium endemic areas were combined into one single group defined as endemic group while the paired (stool and urine) samples collected from the control sites defined as non-endemic group. Additionally, species-specific results were also reported.

This study shows a higher Genus Schistosoma detection rate by qPCR of 50.4% compared to microscopy rate that was 41.3% in endemic areas. These findings are despite the repeated microscopy in this study that was meant to cure microscopy’s documented low sensitivity which is possibly attributed to egg output variations and uneven distribution of eggs in the sample [52]. The superior performance of the qPCR is in agreement with other similar studies in different regions [20–23, 28]. This observation has previously led to qPCR assay being fronted as a powerful tool for accurate establishment of prevalence and intensity of Schistosoma infection especially in monitoring the effectiveness of programmatic directed treatment [25]. Besides the superior performance of the qPCR assay, the current study presents an additional advantageous platform in that it enables detection of the schistosomiasis infection without subtyping the amplicon to the species level, which is not necessary in the context of control programmes. Moreover, this assay has also simplified what would otherwise be a laborious process of analyzing a single type of sample, either stool or urine, separately using different protocols.

The current study shows specificity of 99.5% (PCI: 97.6–100.0) for qPCR and microscopy and sensitivity of 86.4% (PCI: 82.1–90.3) for qPCR, 75.6% (PCI: 71.1–80.0) for microscopy. According to the WHO TPP guidelines for schistosomiasis diagnostics, the ideal requirements for the clinical sensitivity and clinical specificity should be above 75% and 96.5% respectively for a sample size of 100 individuals [4]. In this study, the specificity target was exceedingly attained by both tests. However, overall sensitivity of qPCR was much higher while performance by the microscopy was close to cut-off of the WHO-recommended values. Interestingly microscopy showed higher sensitivity in S. haematobium compared to S. mansoni and in relations to the above WHO TPP. It is worth noting that urine filtration and urine PCR utilized 10mls and 1ml of neat urine samples respectively. Conversely, Kato-Katz utilized 41.7g of stool while stool PCR utilized sample preserved in absolute ethanol at 1:3 mass to volume ratio. This could probably have effects on the recovery of eggs and detection of the infection in favour of S. haematobium microscopy due to samples volumes compared to its PCR and both microscopy and PCR in S. mansoni.

The low sensitivity due to low detection limit associated with microscopy in S. mansoni has been observed elsewhere and shown to hamper its use in the settings with light intensity infections. This could potentially have an impact on control programmes by inhibiting confirmation of cure rate after successful treatment and underestimating true prevalence especially in low infection settings [37, 53, 54]. This provides additional justification for use of other sensitive diagnostic assays alongside microscopy in the S. mansoni settings. This study also shows an improvement in the performance of microscopy in comparison to another study by Aryeetey et al. [25]. Notably, the study by Aryeetey observed that the poor sensitivity by microscopy could have been caused by PCR sensitivity overestimate and thus recommended using BLCM, which does not use an apparent “true gold standard” for further work as was done in this study. Elsewhere, using this model, a study done on school children in Tanzania found out that Kato-Katz similarly had lower sensitivity of 89.7% compared to real-time PCR whose sensitivity was 98.7% [11]. However, the improved performance in this current study could probably be attributed to examination of samples over three consecutive days.

The results show discordance between the qPCR and microscopy. Eighty-seven samples were positive by qPCR but negative by microscopy. Similarly, there was increase in the numbers of samples with heavy infections category and overall reduction in number of negative individuals after analysis by qPCR. This could be an affirmation of the superior detection capacity of the qPCR assay even in the event of low egg count in the individuals [28, 55]. Also, this could be due to the fact that probably the qPCR systems can detect infection during larval stage or before eggs are found in stool [21]. Further, ability of the qPCR to at times to pick S. haematobium DNA present in stool samples may yield more positives [18]. On the flipside, 43 samples were negative by qPCR and positive by microscopy. A similar observation was observed in a study by Allam et al [55]. This could be as a result of diverse reasons. First, microscopy was done on samples collected on three consecutive days while qPCR was done on day one samples, a limitation acknowledged by this study due to associated cost and storage challenges that would accompany increased number of aliquots. This could have increased sensitivity in microscopy and not PCR in the event of variation in egg shedding across the three days. Secondly, detection of DNA could fail due to the presence of PCR inhibition. Similar observations have been documented in other studies [7, 38]. However, PCR inhibition could be ruled out in the current study since a separate and parallel assay for internal control was ran for all the samples. This notwithstanding, qPCR has additional advantages over microscopy in that its performance is not subject to methodological variability like microscopy quality and could be done in centralized facility for accurate monitoring diseases control activities [56]

Study limitations

The overarching limitation of this study is that qPCR assay was done on the samples collected on day one only which could potentially affect positivity in the context of varying egg shedding in the study participants. Further parallel analysis using microscopy and qPCR for samples collected at the identical time points over three consecutive days is recommended to enrich the body of knowledge on the performance of the two assays. Additionally, applying molecular techniques such as qPCR in community settings can be challenging due to potential limitations occasioned by high costs and significant resource requirements.

Conclusions

A modified genus Schistosoma assay qPCR is a overally sensitive technique for the detection of schistosomiasis compared to microscopy and thus suitable for use in areas with low infection due to control interventions. Additionally, this assay provides a common integrated platform for monitoring of the progress of elimination efforts in both the S. mansoni and S. haematobium endemic areas. It is therefore recommended for use by the national programmes for accurate determination elimination endpoints and disease surveillance to avert the re-emergence of the infection after effective control. However, to mitigate the cost, a subset of the sample size could be analyzed using the assay as a proxy indicator of the infection level.

Supporting information

S1 Appendix WHO intensity thresholds for light, moderate and heavy infections for Schistosoma.

(DOCX)

S2 Appendix BLCM used for data analysis.

(DOCX)

List of abbreviations

CAA Cathodic anodic antigen

CCA Circulating Cathodic Antigen

Ct Cycle threshold

DNA deoxyribonucleic acid

EPG Eggs per gram

ITS-2 Internal transcribed spacer– 2

KEMRI Kenya Medical Research Institute

M Microscopy

NPV Negative predictive values

qPCR Quantitative Polymerase Chain Reaction

PBS Phosphate Buffered Saline

PCI Posterior credible interval

PPV Positive predictive values

PVP Polyvinylpolypyrrolidone

Se Sensitivity

Sp Specificity

SERU Scientific and Ethics Review Unit

UCP-LF : Up-Converting Phosphor Lateral Flow

WHO World Health Organization

10.1371/journal.pone.0310118.r001
Decision Letter 0
Inocencio da Luz Raquel Academic Editor
© 2024 Raquel Inocencio da Luz
2024
Raquel Inocencio da Luz
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
31 May 2024

PONE-D-24-12369Evaluation of a modified quantitative polymerase chain reaction assay for genus Schistosoma detection using stool and urine samples from schistosomiasis endemic areas in KenyaPLOS ONE

Dear Dr. Kanyi,

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Reviewer #1: This is an interesting study pointing towards the need of sensitive diagnostic tools such as qPCR assays particularly in disease control and elimination process. Refer to the specific comments given within the attached manuscript embedded in Comments boxes, to improve the presented work further.

Reviewer #2: The manuscript by Kanyi et al., "Evaluation of a modified quantitative polymerase chain reaction assay for genus Schistosoma detection using stool and urine samples from schistosomiasis endemic areas in Kenya", describes the development of a modified singleplex Schistosoma genus-specific quantitative polymerase chain reaction (qPCR) assay for Schistosoma diagnosis. The manuscript is well written, and the results align with the conclusions. However, I have the following comments that need to be looked at:

1. It would be important for the authors to mention the chemotherapeutic strategies that have been deployed for schistosomiasis control and any successes that have been achieved using this strategy in the introduction section of the manuscript

2. In lines 86–92, where the authors highlight the limitations of current diagnostic tests, the requirement for well-trained and experienced personnel is mentioned. I am wondering if the qPCR test developed in this manuscript would solve this problem. In addition, how suitable is the developed test for low-resource settings? In the same light, the authors mention qPCR as being advantageous over conventional PCR (lines 117–119), but what about the sophistication and equipment?

3. Concerning the ethical considerations, the authors mention that participants provided written informed consent. Here are my concerns about this: Were all participants above the age of consent? Were all participants literate?

How was consent obtained from the illiterate and minors? More clarity is needed on this.

4. Concerning the study sites selected, the authors should provide any previous data on Schistosoma prevalence. In addition, the authors should provide consistent data on the different study sites (e.g., rainfall, economic activity, humidity, temperature, etc)

5. Figure 1 should read, "Map of Kenya...", and not "Kenyan map...". In addition, I am wondering if permission is needed from ESPEN for this map to be used. In addition, I am wondering if there is no prevalence data that is more recent, given that this map was produced 6 years ago.

6. It would be important for the authors to provide information on other diagnostic parameters, like likelihood ratios, % positive agreement, Youden's index, etc).

7. In lines 435–437, the authors should provide specific sensitivity information on the study being referred to.

8. Lastly, the manuscript needs to be carefully proofread again and grammatical errors corrected. DNA (line 112) is an acid, the full meaning of NTD (line 146) should be provided. A consistent style should be used to indicate temperatures

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Reviewer #1: No

Reviewer #2: Yes: Robert Adamu Shey

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Attachment Submitted filename: Schistosomiasis PCR Manuscript - REVIEW.docx

10.1371/journal.pone.0310118.r002
Author response to Decision Letter 0
Submission Version1
30 Jul 2024

Racquel Inocencio da Luz, PhD

Academic Editor

PLOS ONE

30th July 2024

Dear Dr. Racquel Inocencio da Luz,

Re: Revised version of manuscript reference PONE-D-24-12369

Thank you for giving us an opportunity to revise our manuscript entitled " Evaluation of a modified quantitative polymerase chain reaction assay for genus Schistosoma detection using stool and urine samples from schistosomiasis endemic areas in Kenya " for submission to PLOS ONE. We are grateful for the valuable feedback from both you and the reviewers, which has helped us to enhance the paper. We are pleased to resubmit the article for your further consideration, incorporating changes based on your insightful suggestions. We believe our revisions and the responses provided below adequately address the issues and concerns raised

For your review here is a point by point response to the comments and questions raised. The comments are indicated in bold while our responses are not bold.

Journal Requirements

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

The manuscript has been reformatted as per the requirement of PLOS ONE’s requirements

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The text in the “Funding Information” and “Financial Disclosure” sections have been rewritten to match as follows:

“This work was supported by the Bill and Melinda Gates Foundation (grant award: INV_024908) that was awarded to SH. The funder had no role in decision to publish, or preparation of the manuscript.”

3. In the online submission form, you indicated that "All relevant data are within the manuscript and its Supporting Information files. The raw data will be available upon request"

All PLOS journals now require all data underlying the findings described in their manuscript to be freely available to other researchers, either 1. In a public repository, 2. Within the manuscript itself, or 3. Uploaded as supplementary information.

This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If your data cannot be made publicly available for ethical or legal reasons (e.g., public availability would compromise patient privacy), please explain your reasons on resubmission and your exemption request will be escalated for approval.

Kindly note that the consent forms utilized to collect data from participants explicitly stated that access to the data is limited to study staff, representatives from FIND and KEMRI, members of the ethics committee and the regulatory authorities. Therefore, access to anonymized and de-identified data can be obtained by emailing the Kenya Medical Research Institute (KEMRI) Scientific Ethics Review Unit (SERU) at seru@kemri.go.ke

4. Please ensure that you refer to Figure 1 in your text as, if accepted, production will need this reference to link the reader to the figure.

Reference to Figure 1 has been provided in lines 159 – 161. The figure has been updated. The source has also been acknowledged and the access date provided accordingly.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly.

Thank you for this comment we have provided captions at the end of the manuscript for the supporting information and have updated in- text citations to match accordingly (Line 677 - 680) & (Line 184 - 193 ;Line 248 - line 281)

6. 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.

Based on the recommendations from the reviewers some other references have been introduced and captured accordingly.

Reviewer 1 Comments

1. It would be important for the authors to mention the chemotherapeutic strategies that have been deployed for schistosomiasis control and any successes that have been achieved using this strategy in the introduction section of the manuscript

Agreed, this has been added in the introduction section. A paragraph in the introduction section has been added to give context to the main chemotherapeutic strategy used for schistosomiasis through mass drug administration (MDA) of praziquantel. This strategy has mostly been implemented by the National School Based Deworming Programme (NSBDP) and has shown some impact on lowered the overall prevalence of schistosomiasis after repeated rounds of MDAs. The paragraph is on Line 64 - 70.

2. In lines 86–92, where the authors highlight the limitations of current diagnostic tests, the requirement for well-trained and experienced personnel is mentioned. I am wondering if the qPCR test developed in this manuscript would solve this problem. In addition, how suitable is the developed test for low-resource settings? In the same light, the authors mention qPCR as being advantageous over conventional PCR (lines 117–119), but what about the sophistication and equipment?

We thank Reviewer 1 for the above comments. Please note that our recommendations are as follows (Line 468 – 471):- It is therefore recommended for use by the national programmes for accurate determination elimination endpoints and disease surveillance to avert the re-emergence of the infection after effective control. However, to mitigate the cost, a subset of the sample size could be analyzed using the assay as a proxy indicator of the infection level.

We are cognizant that cost is a limiting factor. However, the recommended test is not for routine use but rather for determination of the diseases elimination endpoints. Further, we recommend that a subset can be selected based on the available funding. This will enable striking of the balance between cost and accurate determination of the disease elimination.

3. Concerning the ethical considerations, the authors mention that participants provided written informed consent. Here are my concerns about this: Were all participants above the age of consent? Were all participants literate? How was consent obtained from the illiterate and minors? More clarity is needed on this.

The points raised have valid concerns. We have added the following information in the ethics statement to clarify these matters. For participants less than 18 years old, parental consent was obtained, with assent from children (13- <18 years) before enrollment. Illiterate participants provided a thumbprint on the consent form and the consent form was also signed by an impartial witness. This information is provided in Line 133 - 136.

4. Concerning the study sites selected, the authors should provide any previous data on Schistosoma prevalence. In addition, the authors should provide consistent data on the different study sites (e.g., rainfall, economic activity, humidity, temperature, etc)

In response to the comments above we have done the following:-

i. The three study sites have been amended to provide consistent information on the general location, economic activities, general climatic conditions and rainfall. Line 140 - 157

ii. We have provided the data supporting prevalences in the endemic sites and the reference for classification of the control site as non-endemic sites by the Ministry of Health, Kenya (Line 154)

iii. We have also provided a Kenyan map with updated endemicity for Schistosomiasis. In addition to the endemicity of the sites, the maps provide the location of the sites. Line 159 - 161

5. Figure 1 should read, "Map of Kenya...", and not "Kenyan map...". In addition, I am wondering if permission is needed from ESPEN for this map to be used. In addition, I am wondering if there is no prevalence data that is more recent, given that this map was produced 6 years ago.

The most recent map (2022) has been prepared, renamed and acknowledged as per review comments. Line 159 -161. With regard to permission to use the map (Figure 1), we reached out to ESPEN and advised that no permission is required, but only to acknowledge the source and date accessed.

6. It would be important for the authors to provide information on other diagnostic parameters, like likelihood ratios, % positive agreement, Youden's index, etc).

Thank you for this comment. As added in the discussion section they are two main methods of estimating sensitivity and specificity 1) method that utilizes a gold standard or reference test known as frequentists statistics 2) methods that do not utilize a gold standard known as Bayesian methods this is in Line 382 to 388. Bayesian statistics has been utilized in the data analysis of the paper. Cohen Kappa Index is a test of agreement that utilizes frequentist statistics. This also applies to likelihood ratios, % positive agreement but we have added Youden's index in table 4 of the manuscript in line 342 - 344

7. In lines 435–437, the authors should provide specific sensitivity information on the study being referred to.

The specific sensitivity of the study being referred to has been added that is the Tanzanian study found a sensitivity for Kato-Katz at 89.7% and sensitivity for qPCR at 98.7%. This is in line 428 - 430

8. Lastly, the manuscript needs to be carefully proofread again and grammatical errors corrected. DNA (line 112) is an acid, the full meaning of NTD (line 146) should be provided. A consistent style should be used to indicate temperatures

We thank Reviewer 1 for this comment. We have undertaken proofreading of the manuscript and corrected various grammatical errors throughout the manuscript.

Reviewer 2 Comments

1. Abstract - Refer to the main text comments and apply the modifications here in to the abstract as well.

We have made the necessary changes to the entire abstract

2. Abstract Introduction- Move to methodology

This sentence has been moved to methodology (Line 9 -11 moved to 16 -17)

3. Abstract Methodology- Any particular reason why S. japonicum (being a species causing significant morbidity in SEA) was not looked in to?

This is because S. japonicum is not geographically distributed in the African continent and is predominantly found in Asia. There was therefore no site to collect site from in Kenya for comparison

4. Abstract Methodology - What types of samples?

The type of samples has been added in brackets for clarity. Please see line 17 – 18.

5. Abstract Results - Not clear how the denominator becomes 849, and not 699. This should focus exclusively on the endemic regions.

Agreed and revised in line 21 -22. Please note that the study had 3 sites (two endemic and one non-endemic site) where samples were collected as follows: - In S. mansoni endemic site, 351 stool samples were collected while in S. haematobium endemic site 348 urine samples were collected. This added up to 699 samples in the two endemic sites. However, in non-endemic site, each of the 75 participants provided both stool and urine samples. Therefore, 150 samples (75 participants x 2 samples each) were collected in the non-endemic site. Overally then, 699 and 150 samples (total of 849) were collected.

6. Abstract Results - Same as above

Agreed, this has been edited as indicated above

7. Abstract Results - Both microscopy and PCR is supposed to provide quantitative outcomes. A briefing of the comparison needs to come in here.

Comparison has been provided on the performance of the two techniques (Line 25 to 27).

8. Introduction - The diagnosis of schistosomiasis has been facilitated through the application of different PCR techniques, including conventional PCR, qPCR, as well as digital PCR (ddPCR). Suggest briefing with appropriate references.

Thank you for this important suggestion; a topic sentence has been added at the beginning of the paragraph to introduce the concept of PCR techniques in control of schistosomiasis. This is found in line 97 to 99.

9. Introduction- Specify the study objectives

Agreed, this has been done on the last paragraph of the introduction (Line 121 to 124)

10. Introduction- Comment on line 108 – 114 Shouldn’t be places in the Introduction, and instead can be incorporated in to the discussion. –

Thank you for this insightful comment. However, although the statement can also fit in the discussion, I strongly feel that it also fits here for two reasons. The first reason is that it lays basis for the reader to understand the background/general scope of the work reported in this manuscript. Secondly, it exemplifies the how the current PCR method was different from other PCR done on the schistosomiasis and specifically why it is referred to as “modified” under Line 116 - 121

11. Introduction- Not a abbreviation introduced above in the text

Defined – line 118

12. Materials and Methods- Study area- This section addresses both study type as well as the sites.

Agreed, the title of this section has been amended to reflect the information found in this section. This is in Line 138

13. Materials and Methods- Suggest citing the publication(s) arose from the primary study – Line 139 - 140

Although the samples utilized in this study are from stored, there is no publication that has been done on these samples. Therefore it is expected that this will be the first publication and that will be cited by any subsequent publications if any.

14. Materials and Methods- The figure appears to be from another publication/source. Need to cite the source with permissions.

Line 159 - 161. With regard to permission to use the map (Figure 1), we reached out to ESPEN and advised that no permission is required, but only to acknowledge the source. Same response provided to similar comment by reviewer 1 in comment 5. Thanks.

15. Materials and Methods- Not abbreviated previously

Agreed, the text containing this abbreviation has been rephrased in line 167 – 170. The abbreviation was done in line 118

16. Materials and Methods- Were these microscopic findings published previously? Please cite and link if done so.

As indicated earlier the microscopy results are not yet published and thus this is first publication and which compares both the microscopy and PCR results. Line 173

17. Materials and Methods- Either briefly describe or cite a reference to provide the methodological aspects.

A citation is in place that contains the Kato-Katz technique used for stool examination (Reference Number 12) in line 187

18. Materials and Methods- Outcome of two slides? How were the samples from three days considered (was it altogether six smears)? Specify

Thank you for this comment, this has been clarified as follows:-. The samples were considered altogether from the six smears. A sample was considered to be positive if any of the slide was positive while a negative was where it was negative by all the slides. In the case of S. mansoni, eggs were counted and intensity expressed as eggs per gram of stool (epg) by multiplying the arithmetic mean of egg counts from the six slides per participant by 24 and categorized as per the WHO guidelines. In the case of S. haematobium the intensity of infection was calculated as the arithmetic mean of the eggs per 10 ml of urine from the six slides per participant and expressed as the numbers of eggs per 10 ml of urine as per the WHO guidelines, this can be found on Line 187 -193.

19. Materials and Methods- Specify how the quality assurance was ensured in microscopic analysis

Thank you for raising an important point. For quality assurance purposes, a random examination of 10% of the slides were re-examined by senior technologists. This is in Line 193 to 194

20. Materials and Methods- Specify the ‘modification

Attachment Submitted filename: Response to Reviewers.docx

10.1371/journal.pone.0310118.r003
Decision Letter 1
Aboelhadid Shawky M Academic Editor
© 2024 Shawky M Aboelhadid
2024
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https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
26 Aug 2024

Evaluation of a modified quantitative polymerase chain reaction assay for genus Schistosoma detection using stool and urine samples from schistosomiasis endemic areas in Kenya

PONE-D-24-12369R1

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10.1371/journal.pone.0310118.r004
Acceptance letter
Aboelhadid Shawky M Academic Editor
© 2024 Shawky M Aboelhadid
2024
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https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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PONE-D-24-12369R1

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==== Refs
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