Informed and written consent was obtained from all participants prior to inclusion into the study. For minors, informed and written consent was obtained from their legal guardians and assent was obtained from the children. The Senegalese survey was part of a larger investigation on the epidemiology of schistosomiasis and innate immune responses (SCHISTOINIR) for which approval was obtained from the review board of the Institute of Tropical Medicine, the ethical committee of the Antwerp University Hospital and ‘Le Comité National d’Ethique de la Recherche en Santé’ of Senegal. All community members were offered praziquantel (40 mg/kg) and mebendazole (500 mg) treatment after the study according to WHO guidelines [19]. The Kenyan survey was performed within the framework of the Schistosomiasis Consortium for Operational Research and Evaluation (SCORE). Ethical clearance from this study was obtained from the Scientific Steering Committee of the Kenya Medical Research Institute (KEMRI-SSC no. 1768), the Kenyan Ethical Review Committee, and the Institutional Review Board of the Centers for Disease Control and Prevention in the USA. All children who were positive for Schistosoma infection were treated with praziquantel (40 mg/kg), and those positive for STHs were treated with albendazole (400mg).

Samples were derived from one community-wide study population from a S. mansoni and S. haematobium co-endemic area in northern Senegal with high S. mansoni transmission [20–22], and from a population of schoolchildren living in a S. mansoni mono-endemic area with comparatively low transmission in western Kenya [23]. The Senegalese survey was conducted in Ndieumeul and Diokhor Tack, two neighboring communities on the Nouk Pomo peninsula in Lac de Guiers (Guiers Lake). Details on this study area have been described elsewhere [20–22]. Stool and urine samples were collected between July and October 2009 and stool samples for PCR were stored for each participant. Stool samples from a subsample of 197 individuals with complete parasitological data were analyzed by PCR. The Kenyan survey was conducted in the Asembo division of the Rarieda district along the shores of Lake Victoria in western Kenya, within the framework of a larger study on the distribution of S. mansoni amongst school children. Eight to twelve-year-old children attending public primary schools within a 10km from the lake were included (12km wide transect). In this area, S. haematobium is virtually absent. Stool samples were collected between October 2010 and April 2011, preferentially from the lower prevalence zones [23], and PCR was performed in a subsample of 760 children from 40 schools with complete parasitological data (see also S1 STARD Checklist).

In Senegal, two stool and two urine samples were collected from each participant on consecutive days. From each stool sample, a duplicate 25 mg Kato-Katz slide was prepared for quantitative detection of Schistosoma spp. eggs and qualitative diagnosis of STHs Ascaris lumbricoides and Trichuris trichiura by microscopy [24–26]. Duplicate slides were examined by two different technicians >24h after preparation of the Kato-Katz smear, and for S. mansoni the average egg count was calculated. In addition, filtration of 10 ml of urine was performed using a 12 μm pore-size filter (Isopore, USA) according to standard procedures to detect S. haematobium eggs [25]. Urine filters were read by a single technician. In Kenya, three stool samples were collected on consecutive days, and from each sample, duplicate 42 mg Kato-Katz slides were prepared for microscopy. Schistosoma mansoni was diagnosed quantitatively at least 24h after slide preparation. STHs were diagnosed qualitatively: A. lumbricoides and T. trichiura at 24h after slide preparation, and hookworm within 1h of slide preparation. Each slide was examined by two independent microscopists and the average was recorded. Urine filtration was not performed in Kenya. In both countries, microscopy was performed blinded to previous results, and S. mansoni infection intensity was expressed as the number of eggs detected per gram of feces (epg). Egg-based microscopy results were compared to DNA-based PCR results.

Real-time PCR was performed blinded to previous results. During preparation of the first stool sample, an additional amount of fecal material (~0.7ml) was sieved and diluted in 2ml of 96% ethanol [12]. Samples were frozen, transported to the Netherlands, and stored for weeks to months until PCR analysis was performed at the Leiden University Medical Center. Washing of samples, DNA isolation and the setup of the PCR were performed with a custom-made automated liquid handling station (Hamilton, Bonaduz, Switzerland).

For DNA isolation, 200μl of feces suspension was centrifuged and the pellet was washed twice with 1ml of phosphate-buffered saline. After centrifugation, the pellet was resuspended in 200μl of 2% polyvinylpolypyrolidone (Sigma) suspension and heated for 10 min at 100°C. After sodiumdodecyl sulfate–proteinase K treatment (2h at 55°C), DNA was isolated using QIAamp DNA-easy 96-well plates (QIAgen, Limburg, the Netherlands). In each sample, 10³ PFU/mL Phocin Herpes Virus 1 (PhHV-1) was included within the isolation lysis buffer [27,28].

A Schistosoma multiplex real-time PCR (Schisto-PCR) was performed as described previously [29], with some minor modifications [30]. This PCR targets the Schistosoma-specific internal transcriber-spacer-2 (ITS2) sequence of S. mansoni, S. haematobium, and S. intercalatum, as well as PhHV-1 as an internal positive amplification control. The ITS2-based PCR has been validated extensively with a panel of well-defined DNA and stool sample controls and is virtually 100% specific [30]. Amplification was performed by heating samples for 15 minutes at 95°C, followed by 50 cycles, each of 15 seconds at 95°C and 60 seconds at 60°C. Another multiplex real-time PCR, the ANAS-PCR [31], was performed for the detection of STHs Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale and Strongyloides stercoralis. In contrast to the ANAS-PCR, the Schisto-PCR was not designed to differentiate between the different species tested.

Amplification, detection and data analysis were performed with the CFX96 Real-Time System version 1.1 (Bio-Rad, Hercules, CA) [29]. Negative and positive control samples were included in each PCR run. The PCR output from this system consisted of a cycle-threshold (C_t) value, representing the amplification cycle in which the level of fluorescent signal exceeded the background fluorescence. Hence, low C_t values correspond to high parasite-specific DNA loads in the sample tested, and vice versa. The maximum C_t value was 50, and indicated DNA-negative stool samples. The C_t values of the internal PhHV-1 control were within the expected range for all samples, indicating that there was no evidence of inhibition of amplification in any of these samples.

IBM SPSS 22.0 (SPSS, Inc.) was used for statistical analyses (see also S1 Dataset and S1 SPSS Syntax). Results were considered significant when the p-value was <0.05. Kappa (κ) values were calculated as follows to obtain the level of agreement between microscopy and PCR results beyond that which may be obtained by chance: κ=observed test agreement−expected test agreement1−expected test agreement

Standard cut-off values were used for egg-based infection categories [1]: Schistosoma mansoni infections with 1–99 epg were classified as light-intensity, those with 100–399 epg as moderate, and those with ≥400 epg as heavy-intensity infections. DNA loads as reflected by C_t-values were not normally distributed. Consequently, the Mann-Whitney U test was used to determine differences in DNA loads between S. mansoni egg-negative and S. mansoni egg-positive individuals, and the Kruskal-Wallis test to determine differences in DNA loads between the different egg-based infection categories. Spearman’s rank correlation coefficients were calculated to investigate the correlation between egg- and DNA-based infection intensities, which did not show a linear trend.

In the Senegalese study subjects, we investigated whether PCR outcomes were influenced by S. haematobium infection status. The Pearson Chi² test (with continuity correction) was used to compare PCR positivity between those with and without S. haematobium infection. The Mann-Whitney U test was used to compare DNA loads in stool samples between individuals with and without S. haematobium eggs in urine, as well as between individuals with single S. mansoni and with mixed Schistosoma infections stratified according to S. mansoni infection intensity.

For the analysis of the Kenyan data at the school level, only schools with data on ≥15 children were included (i.e. 24/40 schools). Pearson’s correlation coefficients were calculated to investigate the correlation between egg- and DNA-based infection prevalences in the different schools. Schools were classified into three groups according to their distance from the shore of Lake Victoria: A) the highest prevalence zone ≤1200m from the lake; B) moderate prevalence zone 1200-3800m from the lake; and C) lowest prevalence zone >3800m away.

When only the first stool sample was taken into account, microscopy detected S. mansoni infections in 57.4% of subjects in Senegal and in 19.2% of subjects in Kenya (Table 1) whilst PCR detected Schistosoma DNA in 72.6% and 32.4% of subjects, respectively. Thus, in Senegal, the Schisto-PCR detected 15.2% ((143–113)/197) more infections than microscopy, and in Kenya, 13.2% ((246–146)/760) more infections than microscopy. When two stool samples were taken into account, 68.5% and 25.9% S. mansoni-positives were detected by microscopy in Senegal and Kenya, respectively. When three stool samples were taken into account in Kenya, 29.5% S. mansoni-positives were detected by microscopy. While the percentages of S. mansoni-positives detected by microscopy increased with an increasing number of stool samples, they were still lower than those detected by Schisto-PCR in a single stool sample, in both countries.

When based on the first stool sample, egg- and DNA-based results corresponded in 76.6% (κ = 0.500) and 81.8% (κ = 0.536) of subjects in Senegal and Kenya, respectively (Table 2). When egg counts were based on all stool samples provided (2 samples in Senegal and 3 in Kenya), test agreement increased to 81.7% (κ = 0.561), and 86.3% (κ = 0.680), respectively. Differences in test agreement between countries were mainly due to the fact that in Senegal, egg-negatives were more often found positive in PCR than in Kenya (>twofold difference). Fig 1 demonstrates that mainly low-intensity infections were missed when egg counts were based on only one stool sample.

People that were classified as having heavy infections by microscopy were always PCR-positive. Percentages of PCR-positives varied from 97% to 83% in the moderate egg count group, and from 79% to 87% in the group with light intensity infections. Median DNA loads were very similar in both countries for the different Schistosoma infection categories (Fig 1).

In both countries, Spearman’s rank correlations between egg- and DNA-based infection intensities were statistically significant (p<0.001) with correlation coefficients ranging from -0.638 to -0.782. These correlations became stronger with the number of stool samples that were taken into account (ρ = -0.747 and ρ = -0.782 for 1 and 2 stool samples, respectively, in Senegal; ρ = -0.638, ρ = -0.708, and ρ = -0.738 for 1, 2 and 3 stool samples, respectively, in Kenya).

Based on standard microscopy on stool and urine, 80% (157/197) of the Senegalese subjects were infected with either Schistosoma spp. The majority of these infections (92/157) were mixed S. mansoni and S. haematobium infections. Single S. mansoni infections were found in 22%, and single S. haematobium infections in 11% of subjects (Table 3). Table 3 compares Schisto-PCR outcomes according to Schistosoma infection status (by microscopy). DNA-based infection frequencies were highest in those individuals with single S. mansoni and mixed infections and lowest in persons with single S. haematobium infections and those without any schistosome infection. As by definition, no Schistosoma eggs were observed in stools from uninfected people. In people with single S. haematobium infections, one would expect a similar (low) percentage of PCR-positives as in uninfected individuals. However, 59% of the single S. haematobium group was PCR positive, compared to 23% of the microscopy negatives (p = 0.009). C_t-values were comparable. Percentages of PCR-positives were similar in the single S. mansoni and mixed Schistosoma infection groups, but the mixed infection group showed significantly lower C_t-values (p = 0.003), indicative of a higher intensity of infection. No effect of the presence of S. haematobium on C_t-values in mixed as compared to single S. mansoni infections was observed after stratification for egg-based S. mansoni infection intensity (Table 4).

To explore the diagnostic value of PCR on stool samples in identifying high-risk schools and/or communities, Kenyan test results were analyzed at school level. Data for 24 schools with at least ≥15 children per school, representing 688 school children, were aggregated. The median sample size per school was 27 (range 15–47). Fig 2 indicates a strong, linear correlation between the percentage of microscopy- and PCR-positives per school (p<0.001). DNA-based infection frequencies were consistently higher than egg-based infection frequencies at the school level when both were based on the same stool samples, and PCR identified 25% (22/24 versus 16/24) more S. mansoni-positive schools than microscopy. When egg counts from all stool samples were taken into account, microscopy identified more S. mansoni-positive schools (20/24), and also more high-risk schools (infection frequencies ≥50% [1]), as compared to when only the first stool sample was taken into account (Fig 2). In those high-risk schools, egg-based infection frequencies calculated from three stool samples (six slides) were as high as, or higher than DNA-based infection frequencies. In low-risk schools on the other hand (infection frequencies <10%), PCR detected more infections than microscopy on three stool samples, and it detected more positive schools.

In addition to Schistosoma, we investigated the occurrence of STH infections by Kato-Katz and ANAS-PCR. In both study areas, microscopy indicated low prevalences of STH infections and this was confirmed by PCR (Table 5). The two techniques detected similar percentages of A. lumbricoides-positives in both countries. Hookworm was only present in Kenya, and the ANAS-PCR showed that these infections only involved N. americanus. Interestingly, PCR detected more than threefold the number of hookworm infections than microscopy.