Limited efficacy of repeated praziquantel treatment in Schistosoma mansoni infections as revealed by highly accurate diagnostics, PCR and UCP-LF CAA (RePST trial)

Pytsje T Hoekstra; Miriam Casacuberta-Partal; Lisette van Lieshout; Paul L A M Corstjens; Roula Tsonaka; Rufin K Assaré; Kigbafori D Silué; Eliézer K N’Goran; Yves K N’Gbesso; Eric A T Brienen; Meta Roestenberg; Stefanie Knopp; Jürg Utzinger; Jean T Coulibaly; Govert J van Dam

doi:10.1371/journal.pntd.0011008

. 2022 Dec 22;16(12):e0011008. doi: 10.1371/journal.pntd.0011008

Limited efficacy of repeated praziquantel treatment in Schistosoma mansoni infections as revealed by highly accurate diagnostics, PCR and UCP-LF CAA (RePST trial)

Pytsje T Hoekstra ^1,^*, Miriam Casacuberta-Partal ¹, Lisette van Lieshout ¹, Paul L A M Corstjens ², Roula Tsonaka ³, Rufin K Assaré ^4,^5,^6,⁷, Kigbafori D Silué ^4,⁵, Eliézer K N’Goran ^4,⁵, Yves K N’Gbesso ⁸, Eric A T Brienen ¹, Meta Roestenberg ^1,⁹, Stefanie Knopp ^6,⁷, Jürg Utzinger ^6,⁷, Jean T Coulibaly ^4,^5,^6,⁷, Govert J van Dam ¹

Editor: Aaron R Jex¹⁰

¹Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

²Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands

³Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, The Netherlands

⁴Centre Suisse de Recherches Scientifiques en Côte d’Ivoire, Abidjan, Côte d’Ivoire

⁵Unité de Formation et de Recherche Biosciences, Université Félix Houphouët-Boigny, Abidjan, Côte d’Ivoire

⁶Swiss Tropical and Public Health Institute, Allschwil, Switzerland

⁷University of Basel, Basel, Switzerland

⁸Département d’Agboville, Centre de Santé Urbain d’Azaguié, Azaguié, Côte d’Ivoire

⁹Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands

¹⁰Walter and Eliza Hall Institute of Medical Research, AUSTRALIA

The authors have declared that no competing interests exist.

^✉

* E-mail: pthoekstra@lumc.nl

Roles

Pytsje T Hoekstra: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing

Miriam Casacuberta-Partal: Data curation, Investigation, Project administration, Resources, Writing – review & editing

Lisette van Lieshout: Conceptualization, Supervision, Writing – review & editing

Paul L A M Corstjens: Writing – review & editing

Roula Tsonaka: Data curation, Formal analysis, Writing – review & editing

Rufin K Assaré: Investigation, Project administration, Resources, Writing – review & editing

Kigbafori D Silué: Investigation, Project administration, Resources, Writing – review & editing

Eliézer K N’Goran: Resources

Yves K N’Gbesso: Investigation

Eric A T Brienen: Investigation, Writing – review & editing

Meta Roestenberg: Conceptualization, Methodology, Writing – review & editing

Stefanie Knopp: Conceptualization, Methodology, Writing – review & editing

Jürg Utzinger: Conceptualization, Methodology, Writing – review & editing

Jean T Coulibaly: Conceptualization, Investigation, Methodology, Project administration, Resources, Writing – review & editing

Govert J van Dam: Conceptualization, Funding acquisition, Investigation, Methodology, Resources, Supervision, Validation, Writing – review & editing

Aaron R Jex: Editor

PMCID: PMC9822103 PMID: 36548444

Abstract

Background

Most studies assessing praziquantel (PZQ) efficacy have used relatively insensitive diagnostic methods, thereby overestimating cure rate (CR) and intensity reduction rate (IRR). To determine accurately PZQ efficacy, we employed more sensitive DNA and circulating antigen detection methods.

Methodology

A sub-analysis was performed based on a previously published trial conducted in children from Côte d’Ivoire with a confirmed Schistosoma mansoni infection, who were randomly assigned to a standard (single dose of PZQ) or intense treatment group (4 repeated doses of PZQ at 2-week intervals). CR and IRR were estimated based on PCR detecting DNA in a single stool sample and the up-converting particle lateral flow (UCP-LF) test detecting circulating anodic antigen (CAA) in a single urine sample, and compared with traditional Kato-Katz (KK) and point-of-care circulating cathodic antigen (POC-CCA).

Principal findings

Individuals positive by all diagnostic methods (i.e., KK, POC-CCA, PCR, and UCP-LF CAA) at baseline were included in the statistical analysis (n = 125). PCR showed a CR of 45% (95% confidence interval (CI) 32–59%) in the standard and 78% (95% CI 66–87%) in the intense treatment group, which is lower compared to the KK results (64%, 95% CI 52–75%) and 88%, 95% CI 78–93%). UCP-LF CAA showed a significantly lower CR in both groups, 16% (95% CI 11–24%) and 18% (95% CI 12–26%), even lower than observed by POC-CCA (31%, 95% CI 17–35% and 36%, 95% CI 26–47%). A substantial reduction in DNA and CAA-levels was observed after the first treatment, with no further decrease after additional treatment and no significant difference in IRR between treatment groups.

Conclusion/Significance

The efficacy of (repeated) PZQ treatment was overestimated when using egg-based diagnostics (i.e. KK and PCR). Quantitative worm-based diagnostics (i.e. POC-CCA and UCP-LF CAA) revealed that active Schistosoma infections are still present despite multiple treatments. These results stress the need for using accurate diagnostic tools to monitor different PZQ treatment strategies, in particular when moving toward elimination of schistosomiasis.

Clinical trial registration

www.clinicaltrials.gov, NCT02868385.

Author summary

Efficacy of praziquantel (PZQ) for the treatment of schistosomiasis is usually assessed by classical microscopic detection of parasite eggs in stool or urine. Due to low sensitivity, especially in case of low-intensity infections, the prevalence of infection is underestimated leading to an overestimated cure rate (CR) when using these methods. In a repeated treatment trial, the efficacy of one versus four repeated PZQ treatments, given at 2-week intervals, was investigated in school-aged children from Côte d’Ivoire by applying a range of diagnostic methods, including traditional microscopy as well as more sensitive DNA and circulating antigen detection methods. Our results demonstrate that PZQ efficacy measurements vary based on the diagnostic method used: while egg-based diagnostics (stool microscopy and DNA detection methods) show an improved CR after repeated treatment, the CR determined by worm-based diagnostics (urine circulating antigen detection methods) remained poor over time. Although all four diagnostic methods showed a significant reduction in intensity of infection already after a single treatment, more accurate antigen diagnostics revealed that, in most cases, worms remain present even after multiple treatments. Hence, using accurate diagnostic tools is essential to determine the true infection status and to monitor and evaluate treatment programs.

Introduction

Schistosomiasis remains a major public health problem in many sub-Saharan African countries. The cornerstone of schistosomiasis control programs is large-scale administration of the anthelmintic drug praziquantel (PZQ) [1]. Even though the World Health Organization (WHO) recommends using the intensity reduction rate (IRR) to measure the efficacy of PZQ treatment, the efficacy is still often expressed as cure rate (CR) [2]. Observed CRs in school-aged children range from 42% to 79% for Schistosoma mansoni and between 37% and 93% for S. haematobium (3). Repeating PZQ treatment can increase the CR up to 99% [3–5]. However, the majority of studies assessing the efficacy of PZQ treatment have used classical parasitologic methods, including Kato-Katz (KK) to determine the CR [6]. It is widely acknowledged that these methods lack sensitivity, especially in case of low intensity infections–as often observed after treatment [7–9]–thereby underestimating the prevalence of infection, and hence, overestimating the CR [10–11].

To determine the efficacy of PZQ more accurately, diagnostic methods with a higher sensitivity should be used. Highly specific and sensitive molecular PCR techniques detecting Schistosoma-specific DNA in stool and urine are available and could be suitable for monitoring PZQ efficacy [7,12]. Alternatively, as PZQ affects the adult worm, treatment efficacy could also be evaluated by measuring the worm burden. Schistosome worms regurgitate various antigens into the blood circulation, which are then excreted via the urine, for example the antigens ‘circulating cathodic antigen’ (CCA) and ‘circulating anodic antigen’ (CAA); two antigens that have been described and studied extensively [13,14]. Detection of CCA is done via the commercially available point-of-care (POC) CCA urine cassette test, which is currently being recommended by WHO as an alternative for KK for diagnosing intestinal schistosomiasis [15–18]. CAA is detected quantitatively using an ultra-sensitive reporter technology (up-converting particle, UCP), combined with common immunochromatography, lateral flow (LF) [19]. This UCP-LF CAA test has demonstrated high sensitivity and specificity for the main human schistosome species (S. haematobium, S. mansoni, S. japonicum, and S. mekongi) [19–22]. Combining egg-derived nucleic acid and worm-derived antigen detection methods is expected to provide a more accurate determination of the efficacy of PZQ, both in terms of parasite worm dynamics as well as fecundity.

Previous results from the Repeated Praziquantel for Schistosomiasis Treatment (RePST) study–an open-label randomized controlled trial evaluating the efficacy of repeated PZQ on parasite clearance in school-aged children with a confirmed S. mansoni infection–showed a significantly higher CR after four PZQ treatments compared to a single PZQ based on the traditional KK technique [23]. However, the CR based on POC-CCA was substantially lower than the CR based on KK, even after four repeated PZQ treatments, indicating that worms are most likely still present after repeated treatment. Here, we further evaluate the efficacy of PZQ by using two additional sensitive and highly specific diagnostic methods, i.e., stool PCR and the UCP-LF CAA urine test, to determine the CR as well as the IRR after single and four repeated treatments. The current study uniquely combines all diagnostic methods, and provides new insights into the post-treatment dynamics of S. mansoni infections.

Methods

Ethics statement

Ethical approval was granted in Côte d’Ivoire from both the Comité National d’Éthique des Sciences de la Vie et de la Santé de Côte d’Ivoire (no. 091-18/MSHP/CNESVS-km) and the Direction de la Pharmacie, du Médicament et des Laboratoires de Côte d’Ivoire (no. 99433/MSPH/DGS/DPML/DAR and ECCI00618), and in The Netherlands from the Ethics Committee of the Leiden University Medical Center (P16.254). Oral assent from school-aged children as well as signed informed consent from children’s parents or guardians was obtained before data and sample collection.

Study design and participants

Previously, an open-label, randomized controlled trial was conducted between October 2018 and January 2019 in the Taabo health district in south-central Côte d’Ivoire, results of which have been published elsewhere [23,24]. Briefly, after clinical and parasitologic assessment for eligibility, school-aged children (5–17 years) with a confirmed S. mansoni infection (i.e., positive by KK (>8 eggs per gram of stool, EPG) and POC-CCA (traces excluded)), were randomized into the ‘standard treatment’ or into the ‘intense treatment’ group [23]. At baseline, all participating children were given PZQ (40 mg/kg). Subsequently, children assigned to the standard group did not receive any further treatment during the trial period, while children assigned to the intense treatment group received additional PZQ treatment at 2, 4, and 6 weeks after the initial treatment [23].

Diagnostic procedures

A detailed description of all field and laboratory procedures can be found in the previously published study protocol [24]. In brief, urine and stool samples were collected from each participating child weekly and two-weekly, respectively, over a period of 10 weeks in total. In the field, urine samples were subjected to the POC-CCA test using the G-score method [25], while stool samples were processed and examined using the KK technique [23,24]. Aliquots of all available urine and stool (mixed with ethanol for storage and transport purposes [26]) samples were stored at -20°C. After the trial was completed, all available urine and stool aliquots were shipped to the Leiden University Medical Center (Leiden, The Netherlands) and stored at -20°C pending further testing.

The Schistosoma genus specific (ITS2) real-time PCR was used for the detection of Schistosoma DNA in stool samples, as described previously [27,28]. Besides negative and positive control samples included in each amplification run, phocin herpes virus-1 (PhHV-1) was added to the lysis buffer in each sample as an internal positive control. Virus-specific primers and detecting probe were included in each reaction mixture. Fifty PCR amplification cycles were run per sample, using the amplification cycle in which the level of fluorescent signal exceeded the background as the cycle-threshold (Ct)-value PCR output. Since its implementation, the LUMC-team scored 100% in sensitivity and specificity of their Schistosoma PCR at the annual international Helminths External Molecular Assessment Scheme (HEMQAS) provided by the Dutch Foundation for Quality Assessment in Medical Laboratories (SKML) [29].

Urine samples were subjected to the UCP-LF CAA test [19]. A set of reference standards with a known CAA concentration were included to quantify CAA-levels as well as to validate the cut-off (0.6 pg/ml for the urine UCAAhT417 test [19]). Samples were considered positive if the CAA concentration exceeded the cut-off, while samples below the cut-off were considered negative.

Statistical analysis

Only individuals with a positive baseline outcome in all diagnostic methods (i.e., KK, POC-CCA, PCR, and UCP-LF CAA) were included in the analysis. As the diagnostics used typically focus on direct detection of the presence of worms (by their metabolic excretion products, CCA and CAA) or indirectly by showing the presence of the eggs (KK and PCR), data were analyzed to compare both approaches. Descriptive statistics were performed using IBM Statistical Package for Social Sciences version 25 (SPSS Inc., Chicago, United States of America). Prevalence over time as well as CR and IRR based on PCR and UCP-LF CAA were determined using a mixed effects model taking into account the correlation between the different measurements from the same individual [30–32]. CR and IRR were determined by comparing baseline data to outcomes at 4 weeks after the last PZQ treatment, i.e., considering week 4 as the final time point for the standard treatment group and week 10 as the final time point for the intense treatment group. Ct-values were transformed into arbitrary units (AU) of copy numbers of Schistosoma DNA, as described before [33]. In short, to each low positive sample, i.e., a Ct-value between 35 and 50, an arbitrary value of 1 AU was assigned. Starting from Ct-value 35, for each PCR cycle reduction a duplication of AU was computed, assuming 100% efficacy of the DNA multiplication process.

To determine the prevalence over time as well as the CR and IRR (based on all diagnostic methods) mixed effects models were used, as described previously [23,31]. This model framework takes into account the zero inflation, the correlation between repeated measurements from the same individual and gives valid results under the missing at random assumption for the missing data, which is valid in this study. For the prevalence and CR, we used mixed effects logistic regression where prevalence was modeled as a function of time, treatment group, and their interaction. In the case of KK and PCR, the time variable was taken as categorical, while for POC-CCA and UCP-LF CAA we modeled progression over time using natural cubic splines with knots. For the IRR, the mean was modeled using the main effect of time (taken as categorical), the main effect of treatment and their interaction. KK data were analyzed in time using a zero-inflated mixed effects negative binomial model, while PCR and UCP-LF CAA data were analyzed in time using a two-part linear mixed model. The correlation between the repeated measurements of each individual was modeled using random effects (i.e., random intercepts). All analyses have been done in R (version 3.43) using the GLMMadaptive package. CR and IRR estimated from the model are given with their corresponding 95% pointwise confidence intervals (CIs).

In the absence of a true ‘gold’ standard, the sensitivity of the different diagnostic methods was determined by comparison against a composite reference standard (CRS) [34–36], which was based on a positive result by KK and/or PCR and/or UCP-LF CAA, all considered highly specific methods. Consequently, an individual was considered true positive if either KK, PCR, or UCP-LF CAA was positive.

Results

From the 153 school-aged children who participated in the original trial, in 19 cases no aliquots from baseline urine and/or stool samples were available for additional testing. Another nine school-aged children were omitted as they had a negative UCP-LF CAA (n = 7) or a negative PCR (n = 2) at baseline. In total, 125 school-aged children were included in the final analysis, 56 and 69 in the standard and intense treatment group, respectively (S1 Fig). The demographic and parasitologic baseline data for the school-aged children included in the current analysis are summarized in S1 Table.

Prevalence over time

In Fig 1, the percentage of S. mansoni positive samples over time based on PCR (Fig 1A) and UCP-LF CAA (Fig 1B) is shown, including previously published results based on KK and POC-CCA [23] (Fig 1C and 1D, respectively). Based on PCR, the total percentage of positives decreased to 55% (95% CI 41–68%) in the standard treatment group and to 22% (95% CI 13–34%) in the intense treatment group, both measured 4 weeks post-treatment. The corresponding CR for the standard and intense treatment group were 45% (95% CI 32–59%) and 78% (95% CI 66–87%), respectively (Tables 1 and S2). Based on UCP-LF CAA, the total percentage of positives decreased to 86% (95% CI 79–90%) in the standard treatment group and to 82% (95% CI 73–89%) in the intense treatment group, both measured 4 weeks post-treatment. The corresponding CR for the standard and intense treatment group were 16% (95% CI 11–24%) and 18% (95% CI 12–26%), respectively (Tables 1 and S2).

Table 1. Cure rate (CR) and intensity reduction rate (IRR) of a single (standard treatment group) and four (intense treatment group) repeated PZQ treatments in 125 school-aged children with a confirmed S. mansoni infection.

Data based on polymerase chain reaction (PCR) and up-converting particle circulating anodic antigen (UCP-LF CAA).

	Standard treatment group (n = 56)	Intense treatment group (n = 69)
PCR
Cured children 4 weeks post-treatment	25	52
CR (95% CI)^a	45.3% (32.3–58.8%)	78.1% (66.4–86.6%)
Median AU^b
Before treatment	32,768	16,384
4 weeks post-treatment	2,048	24
Arithmetic mean AU^a
Before treatment (95% CI)	5.6 x 10⁵ (2.3 x 10⁵–1.2 x 10⁶)	2.0 x 10⁵ (9.5 x 10⁴–4.2 x 10⁵)
4 weeks post-treatment (95% CI)	1.0 x 10⁴ (3.4 x 10³–2.8 x 10⁴)	9.9 x 10² (2.2 x 10²–4.3 x 10³)
IRR (95% CI) ^a	99.6% (98.6–99.9%)	99.5% (97.2–99.9%)
UCP-LF CAA
Cured children 4 weeks post-treatment	9	13
CR (95% CI)^a	16.1% (10.5–24.0%)	17.8% (11.5–26.3%)
Median urine CAA-level (pg/ml)^b
Before treatment	286	270
4 weeks post-treatment	61	14
Arithmetic mean urine CAA-level (pg/ml)^a
Before treatment (95% CI)	145.6 (93.3–217.8)	153.4 (102.5–219.3)
4 weeks post-treatment (95% CI)	40.6 (25.2–62.2)	15.2 (9.3–23.3)
IRR (95% CI)^a	72.1% (62.4–79.4%)	90.1% (86.9–92.5%)

Open in a new tab

Abbreviations: AU, arbitrary unit (see Methods for definition); CAA, circulating anodic antigen; CR, cure rate; IRR, intensity reduction rate; PCR, polymerase chain reaction; PZQ, praziquantel; UCP-LF, up-converting particle lateral flow.

a Calculated from the model

b Median of the positives

Intensity of infection over time

Fig 2 illustrates the infection intensity over time based on PCR (Fig 2A) and UCP-LF CAA (Fig 2B), including KK and POC-CCA results (Fig 2C and 2D, respectively). Based on PCR, most remaining infections after the first PZQ treatment were of low intensity. In the standard treatment group, the average PCR outcome (AU) decreased from 56 x 10⁴ (95% CI 23 x 10⁴–12 x 10⁵) to 10 x 10³ (95% CI 34 x 10²–28 x 10³), and in the intense treatment group from 20 x 10⁴ (95% CI 95 x 10³–42 x 10⁴) to 99 x 10¹ (95% CI 22 x 10¹–43 x 10²), both measured 4 weeks post-treatment. The corresponding IRR for the standard and intense treatment group were 99.6% (95% CI 98.6–99.9%) and 99.5% (95% CI 97.2–99.9%), respectively (Tables 1 and S2). Based on UCP-LF CAA, infection levels decreased from 146 pg/ml (95% CI 93–218) to 41 pg/ml (95% CI 25–62) in the standard treatment group, and from 153 pg/ml (95% CI 103–219) to 15 pg/ml (95% CI 9–23) in intense treatment group. The corresponding IRRs for the standard and the intense treatment group were 72.1% (95% CI 62.4–79.4%) and 90.1% (95% 86.9–92.5%), respectively (Tables 1 and S2).

Correlation between tests

In Fig 3, the baseline correlation between egg-based detection methods (Fig 3A) and worm-based detection methods (Fig 3B) is shown. The correlation between egg-based methods PCR (in AU) and KK (in EPG) at baseline was higher (Spearman’s rho 0.64, p<0.01) compared to the correlation between worm-based detection by UCP-LF CAA and POC-CCA (Spearman’s rho 0.37, p<0.01). The majority of individuals with ≥400 EPG (86%) had a POC-CCA G-score of 6 or higher (corresponding to a visual score of 2+ or higher) and a urine CAA-level of ≥100 pg/ml (S2 and S3 Figs).

Fig 4 shows the post-treatment correlation between egg-based detection methods (Fig 4A) and worm-based detection methods (Fig 4B). A similar correlation was observed between PCR and KK (Spearman’s rho 0.51, p<0.01) and between UCP-LF CAA and POC-CCA (Spearman’s rho 0.59, p<0.01) 4 weeks after treatment. The majority of egg- and PCR-positive individuals were observed in the standard treatment group, while UCP-LF CAA and POC-CCA positives were observed in both groups (S4 Fig).

Accuracy of tests

Sensitivity of egg-based diagnostic methods decreased over time after treatment; a higher reduction in sensitivity was observed in the intense treatment group compared to the standard treatment group (Table 2). While in both groups the sensitivity of POC-CCA fluctuated over time, the sensitivity of UCP-LF CAA remained high and stable, regardless of additional treatment.

Table 2. Sensitivity of polymerase chain reaction (PCR), Kato-Katz (KK), up-converting particle circulating anodic antigen (UCP-LF CAA), and point-of-care circulating cathodic antigen (POC-CCA) compared to a composite reference standard (CRS) over time.

Data shown for the standard group (n = 56) who received a single PZQ treatment at week 0 and the intense treatment group (n = 69) who received four repeated PZQ treatments at weeks 0, 2, 4, and 6. The composite reference standard (CRS) was based on KK, PCR, and UCP-LF CAA: an individual was considered positive if at least one of these tests was positive (i.e., test specificity assumed to be 100%).

			Number of individuals positive per diagnostic test and corresponding sensitivity (%) compared to the composite reference standard (CRS)
		N	CRS	Stool PCR		Kato-Katz		UCP-LF CAA		POC-CCA
Standard group	Baseline	56	56	56	100%	56	100%	56	100%	56	100%
	Week 2	54	48	25	52.0%	10	20.8%	46	95.8%	42	87.5%
	Week 4	55	50	30	60.0%	20	40.0%	46	92.0%	38	76.0%
	Week 6	55	49	32	65.3%	29	59.2%	46	93.9%	43	87.8%
	Week 10	53	51	34	66.7%	36	70.6%	44	86.3%	45	88.2%
Intense group	Baseline	69	69	69	100%	69	100%	69	100%	69	100%
Intense group	Week 2	68	61	29	47.5%	17	27.9%	58	95.1%	51	83.6%
	Week 4	69	57	16	28.1%	2	3.5%	55	96.5%	42	73.7%
	Week 6	63	49	11	22.4%	1	2.0%	46	93.9%	34	69.4%
	Week 10	66	55	14	25.5%	8	14.5%	54	98.2%	40	72.7%

Open in a new tab

Discussion

In this study, performing additional diagnostic analysis on samples previously collected at a multiple treatment trial, comparing single versus 4 treatments with PZQ in school-aged children from Côte d’Ivoire, we confirmed our prior conclusion that highly accurate diagnostic methods are important for determining PZQ efficacy [23].

Due to its high sensitivity compared to KK, while remaining intrinsically specific, the Schistosoma genus-specific ITS2 PCR detecting Schistosoma DNA in stool samples revealed more positive cases over time. Yet, the overall post-treatment dynamics were similar to KK results [23]. This supports the notion that both tests reflect the number of Schistosoma eggs in stool (as adult Schistosoma worms are located in the mesenteric blood vessels), although the ITS2 PCR target is considered to be present in all life-stages of the parasite. The good correlation between KK and PCR is consistent with previous studies [12,27,28,37,38] indicating the capability of the Schistosoma ITS2 PCR to determine infection intensity. PCR also showed a significant reduction in DNA levels after treatment, as observed previously [12,38], confirming its usefulness to monitor PZQ treatment efficacy. The latter is in contrast to other studies using a serum-based PCR that remained positive for months after treatment [39,40].

Turning to worm-based diagnostics, the highly accurate UCP-LF CAA test confirmed the already low CR observed previously by POC-CCA [23]. In 285 out of 483 (59%) post-treatment samples, CAA was detected in urine, while no eggs were observed in the stool. To a lesser extent, this was already shown by previously published POC-CCA positive but egg-negative individuals [23,41,42]. As both CAA and CCA are worm-derived antigens, the presence of either antigen indicates that worms are still metabolically active and that the infection is hence not fully cleared, indicating that the CR is even lower than generally assumed [3,6,43]. The opposite, egg-positive with no CAA in urine, was observed in only a few cases (15 out of 483, 3%). This apparent incongruence of absence of CAA despite presence of eggs could be due to variation in biological excretion, but administrative errors such as sample processing and/or labelling cannot be excluded.

Egg-based diagnostic methods (i.e., PCR and KK), showed an increased CR after repeated treatment, as also observed in other studies [3–5]. Contrastingly, the more sensitive worm-based methods, UCP-LF CAA and POC-CCA, demonstrated a poor CR, which did not significantly improve with additional treatments. Observed CRs should therefore be interpreted with care, keeping in mind that they depend on the type of diagnostic method used. In addition, the time point after treatment chosen for measurement of CRs also influences its value, e.g., for KK it is highest 2 weeks after treatment, and subsequently decreases in the next weeks if no additional treatment is given (S5A Fig). Using different diagnostics, our results confirm that the CR has limited value in assessing PZQ efficacy. Diagnostic sensitivity has a higher impact on the CR than on the IRR, as the detection of an egg, DNA, or circulating antigen makes the difference between cured and non-cured (S5 and S6 Figs).

A significant decrease in urine CAA-levels was observed already after a single treatment and no substantial differences were observed whether data were calculated as arithmetic or geometric mean or median (S7 Fig). By repeating the treatments at relatively short intervals, it was anticipated that non-PZQ-susceptible schistosomula would be targeted more effectively as with a few weeks they would have matured into PZQ-susceptible worms. Also, an additional effect on mature worms was expected, as the short metabolic half-life of PZQ might limit its effectiveness when given as single treatment. Our results indicate that even though a further reduction in egg output occurred with multiple treatments, worm numbers reflected by urine CAA-levels continued to decrease slightly and remained solidly low from 3 weeks onwards, with only very few cases becoming negative. The rapid rise in egg numbers after treatment would indicate that in this setting transmission continued which, for CAA, resulted in continuous low levels originating probably from young worms (2–4 weeks old) [14,19]. Alternatively or additionally, worms could reside in capillaries not reached by a single dose of PZQ, or worms could revitalize after the damage occurred by PZQ and the host immune attack, thus surviving the treatment. This would suggest that repeating treatment at 2-week intervals is not sufficient and that shorter intervals or perhaps a higher dose could be considered. Although generally rapidly cleared, it cannot be excluded that also a slow release of antigens, restrained in various tissues, contributed to the low CAA-levels after treatment. To further elucidate these mechanisms and to differentiate between surviving adult worms and establishing new infections (reinfection), research in endemic settings with various (seasonal) transmission patterns, in particular the absence of transmission is needed. Alternatively, experimental infections in animals or controlled human infection models would be helpful in unravelling the exact metabolic aspects of CAA excretion and immune-mediated clearance patterns. Due to limited sensitivity of KK for light intensity infections after treatment, eggs may also continue to be excreted but remain below the detection limit of microscopy. Eggs that may be trapped in the host tissue, and therefore not detected, could still result in a continued risk of pathology [44]. Further research is needed to determine to the impact of the continued presence of worms without the detection of eggs and to what extent the egg production and excretion is reconstituted over time.

Conclusion

A single PZQ treatment had a major impact on the egg output as well as on the worm burden measured by CAA-levels, but it appears difficult to get rid of the remaining worms. Even though additional treatment resulted in a further decrease in the number of eggs, the effect on the number of worms remaining in the host was limited, which indicates that–in this setting–(up to) four repeated treatments at a relatively short interval were not sufficient to clear all S. mansoni infections. In particular when moving toward interruption of transmission or even elimination of schistosomiasis, it is important to focus on complete clearance of infection, i.e., not only the absence of eggs, but more importantly the absence of worms. To determine the ideal schistosomiasis treatment schedule, still considering other important factors influencing post-treatment status such as transmission season, environmental conditions, and water contact behavior, the use of accurate egg- and worm-based diagnostic tools is essential.

Supporting information

S1 Fig. Trial profile.

(PNG)

Click here for additional data file.^{(310.2KB, png)}

S2 Fig

Correlation between worm-based detection methods including intensity categories based on Kato-Katz (eggs per gram of feces, EPG) at (a) baseline and (b) 4 weeks after (the last) treatment. Data based on point-of-care circulating cathodic antigen (POC-CCA) and up-converting particle circulating anodic antigen (UCP-LF CAA) in combination with Kato-Katz (KK), with colors indicating the EPG intensity (n = 125).

(TIF)

Click here for additional data file.^{(625.7KB, tif)}

S3 Fig. Baseline correlation between egg-detection methods and worm-based detection methods.

Data based on stool polymerase chain reaction (PCR) versus Kato-Katz (KK) (a-b) and point-of-care circulating cathodic antigen (POC-CCA) versus up-converting particle circulating anodic antigen (UCP-LF CAA) (c-f) at baseline (n = 125).

(TIF)

Click here for additional data file.^{(1MB, tif)}

S4 Fig. Post-treatment correlation between egg-detection methods and worm-based detection methods.

(TIF)

Click here for additional data file.^{(1.1MB, tif)}

S5 Fig

Cure rate (CR) over time determined by the different diagnostics in (a) the standard treatment group, who received a single dose of PZQ at week 0, and (b) the intense treatment group, who received four doses of PZQ at weeks 0, 2, 4, and 6. Data based on stool polymerase chain reaction (PCR), Kato-Katz (KK), urine up-converting particle circulating anodic antigen (UCP-LF CAA), and point-of-care circulating cathodic antigen (POC-CCA) (n = 125).

(TIF)

Click here for additional data file.^{(338.6KB, tif)}

S6 Fig

Intensity reduction rate (IRR) over time determined by the different diagnostics in (a) the standard group, who received a single dose of PZQ at week 0, and (b) the intense group, who received four doses of PZQ at weeks 0, 2, 4, and 6. Data based on stool polymerase chain reaction (PCR), Kato-Katz (KK), and urine up-converting particle circulating anodic antigen (UCP-LF CAA) (n = 125).

(TIF)

Click here for additional data file.^{(311.6KB, tif)}

S7 Fig

Circulating anodic antigen (CAA) levels over time in (a) the standard group, who received a single dose of PZQ at week 0, and (b) the intense group, who received four doses of PZQ at weeks 0, 2, 4, and 6. Data shown as arithmetic mean (AM), arithmetic mean of the positives, geometric mean (GM) of the positives, and median.

(TIF)

Click here for additional data file.^{(348.6KB, tif)}

S1 Table. Baseline characteristics of the standard treatment group and the intense treatment group.

Adapted from (24), for the current study based on data from 125 school-aged children.

(DOCX)

Click here for additional data file.^{(17.1KB, docx)}

S2 Table. Cure rate (CR) and intensity reduction rate (IRR) of a single (standard treatment group) and four (intense treatment group) repeated PZQ treatments in 125 school-aged children with a confirmed S. mansoni infection.

Data based on polymerase chain reaction (PCR), Kato-Katz (KK), up-converting particle circulating anodic antigen (UCP-LF CAA), and point-of-care circulating cathodic antigen (POC-CCA).

(DOCX)

Click here for additional data file.^{(18KB, docx)}

Acknowledgments

We would like to thank E. van Kaathoven for her support in performing the UCP-LF CAA and PCR assays. We thank all children for their enthusiastic participation and providing samples. We are grateful to the parents and guardians of children and the communities for allowing us to conduct this trial. We thank the technicians, nurses, volunteers, and drivers who assisted during the field and laboratory work. Special thanks are addressed to the directors and staff of Centre Suisse de Recherches Scientifiques en Côte d’Ivoire and the ‘Programme National de Lutte contre les Maladies Tropicales Négligées à Chimiothérapie Préventive’ for administrative support.

Data Availability

All relevant data are available within the manuscript and its supporting information file.

Funding Statement

This work received financial support from the Prof. Dr. Flu Foundation, based in The Netherlands and the Coalition for Operational Research on Neglected Tropical Diseases (COR-NTD) (no. NTDSC087G) to GJD, which is funded at The Task Force for Global Health, primarily by the Bill & Melinda Gates Foundation, by the United States Agency for International Development through its Neglected Tropical Disease Program, and with UK Aid from the British people. The use of the PCR internal control PhHV was supported by the European Virus Archive goes Global (EVAg) project that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 653316. The funders had no role in the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

References

1.WHO. Schistosomiasis: progress report 2001–2011 and strategic plan 2012–2020. Geneva: World Health Organization; 2013. [Google Scholar]
2.WHO. Assessing the efficacy of anthelminthic drugs against schistosomiasis and soil-transmitted helminthiases. Geneva: World Health Organization; 2013. [Google Scholar]
3.King CH, Olbrych SK, Soon M, Singer ME, Carter J, Colley DG. Utility of repeated praziquantel dosing in the treatment of schistosomiasis in high-risk communities in Africa: a systematic review. PLoS Negl Trop Dis. 2011;5:e1321. doi: 10.1371/journal.pntd.0001321 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Munisi DZ, Buza J, Mpolya EA, Angelo T, Kinung’hi SM. The efficacy of single-dose versus double-dose praziquantel treatments on Schistosoma mansoni infections: its implication on undernutrition and anaemia among primary schoolchildren in two on-shore communities, northwestern Tanzania. Biomed Res Int. 2017;2017:7035025. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Nalugwa A, Nuwaha F, Tukahebwa EM, Olsen A. Single versus double dose praziquantel comparison on efficacy and Schistosoma mansoni re-infection in preschool-age children in Uganda: a randomized controlled trial. PLoS Negl Trop Dis. 2015;9:e0003796. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Fukushige M, Chase-Topping M, Woolhouse MEJ, Mutapi F. Efficacy of praziquantel has been maintained over four decades (from 1977 to 2018): a systematic review and meta-analysis of factors influence its efficacy. PLoS Negl Trop Dis. 2021;15:e0009189. doi: 10.1371/journal.pntd.0009189 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Utzinger J, Becker SL, van Lieshout L, van Dam GJ, Knopp S. New diagnostic tools in schistosomiasis. Clin Microbiol Infect. 2015;21:529–42. doi: 10.1016/j.cmi.2015.03.014 [DOI] [PubMed] [Google Scholar]
8.Coulibaly JT, N’Gbesso YK, Knopp S, N’Guessan NA, Silué KD, van Dam GJ, et al. Accuracy of urine circulating cathodic antigen test for the diagnosis of Schistosoma mansoni in preschool-aged children before and after treatment. PLoS Negl Trop Dis. 2013;7:e2109. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Hoekstra PT, van Dam GJ, van Lieshout L. Context-specific procedures for the diagnosis of human schistosomiasis–a mini review. Front Trop Dis. 2021;2:722438. [Google Scholar]
10.Lamberton PH, Kabatereine NB, Oguttu DW, Fenwick A, Webster JP. Sensitivity and specificity of multiple Kato-Katz thick smears and a circulating cathodic antigen test for Schistosoma mansoni diagnosis pre- and post-repeated-praziquantel treatment. PLoS Negl Trop Dis. 2014;8:e3139. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Mwinzi PN, Kittur N, Ochola E, Cooper PJ, Campbell CH Jr., King CH, et al. Additional evaluation of the point-of-contact circulating cathodic antigen assay for Schistosoma mansoni infection. Front Public Health. 2015;3:48. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Vinkeles Melchers NV, van Dam GJ, Shaproski D, Kahama AI, Brienen EA, Vennervald BJ, et al. Diagnostic performance of Schistosoma real-time PCR in urine samples from Kenyan children infected with Schistosoma haematobium: day-to-day variation and follow-up after praziquantel treatment. PLoS Negl Trop Dis. 2014;8:e2807. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.van Dam GJ, Bergwerff AA, Thomas-Oates JE, Rotmans JP, Kamerling JP, Vliegenthart JF, et al. The immunologically reactive O-linked polysaccharide chains derived from circulating cathodic antigen isolated from the human blood fluke Schistosoma mansoni have Lewis x as repeating unit. Eur J Biochem. 1994;225:467–82. [DOI] [PubMed] [Google Scholar]
14.van Dam GJ, Bogitsh BJ, van Zeyl RJ, Rotmans JP, Deelder AM. Schistosoma mansoni: in vitro and in vivo excretion of CAA and CCA by developing schistosomula and adult worms. J Parasitol. 1996;82:557–64. [PubMed] [Google Scholar]
15.Bärenbold O, Garba A, Colley DG, Fleming FM, Haggag AA, Ramzy RMR, et al. Translating preventive chemotherapy prevalence thresholds for Schistosoma mansoni from the Kato-Katz technique into the point-of-care circulating cathodic antigen diagnostic test. PLoS Negl Trop Dis. 2018;12:e0006941. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.WHO. WHO guideline on control and elimination of human schistosomiasis. Geneva: World Health Organzation; 2022. [PubMed] [Google Scholar]
17.WHO. Schistosomiasis 2022 [Available from: https://www.who.int/en/news-room/fact-sheets/detail/schistosomiasis].
18.WHO. Report of the first meeting of the WHO diagnostic technical advisory group for neglected tropical diseases. Geneva: World Health Organization; 2019. [Google Scholar]
19.Corstjens P, de Dood CJ, Knopp S, Clements MN, Ortu G, Umulisa I, et al. Circulating anodic antigen (CAA): a highly sensitive diagnostic biomarker to detect active Schistosoma infections-improvement and use during SCORE. Am J Trop Med Hyg. 2020;103(1_Suppl):50–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.van Dam GJ, Odermatt P, Acosta L, Bergquist R, de Dood CJ, Kornelis D, et al. 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;141:198–203. [DOI] [PubMed] [Google Scholar]
21.van Dam GJ, Xu J, Bergquist R, de Dood CJ, Utzinger J, Qin ZQ, et al. An ultra-sensitive assay targeting the circulating anodic antigen for the diagnosis of Schistosoma japonicum in a low-endemic area, People’s Republic of China. Acta Trop. 2015;141:190–7. [DOI] [PubMed] [Google Scholar]
22.Vonghachack Y, Sayasone S, Khieu V, Bergquist R, van Dam GJ, Hoekstra PT, et al. Comparison of novel and standard diagnostic tools for the detection of Schistosoma mekongi infection in Lao People’s Democratic Republic and Cambodia. Infect Dis Poverty. 2017;6:127. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Hoekstra PT, Casacuberta-Partal M, van Lieshout L, Corstjens P, Tsonaka R, Assaré RK, et al. Efficacy of single versus four repeated doses of praziquantel against Schistosoma mansoni infection in school-aged children from Côte d’Ivoire based on Kato-Katz and POC-CCA: an open-label, randomised controlled trial (RePST). PLoS Negl Trop Dis. 2020;14:e0008189. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Hoekstra PT, Casacuberta Partal M, Amoah AS, van Lieshout L, Corstjens P, Tsonaka S, et al. Repeated doses of Praziquantel in Schistosomiasis Treatment (RePST)—single versus multiple praziquantel treatments in school-aged children in Côte d’Ivoire: a study protocol for an open-label, randomised controlled trial. BMC Infect Dis. 2018;18:662. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Casacuberta-Partal M, Hoekstra PT, Kornelis D, van Lieshout L, van Dam GJ. An innovative and user-friendly scoring system for standardised quantitative interpretation of the urine-based point-of-care strip test (POC-CCA) for the diagnosis of intestinal schistosomiasis: a proof-of-concept study. Acta Trop. 2019;199:105150. doi: 10.1016/j.actatropica.2019.105150 [DOI] [PubMed] [Google Scholar]
26.ten Hove RJ, Verweij JJ, Vereecken K, Polman K, Dieye L, van Lieshout L. Multiplex real-time PCR for the detection and quantification of Schistosoma mansoni and S. haematobium infection in stool samples collected in northern Senegal. Trans R Soc Trop Med Hyg. 2008;102:179–85. [DOI] [PubMed] [Google Scholar]
27.Meurs L, Brienen E, Mbow M, Ochola EA, Mboup S, Karanja DM, et al. Is PCR the next reference standard for the diagnosis of Schistosoma in stool? A comparison with microscopy in Senegal and Kenya. PLoS Negl Trop Dis. 2015;9:e0003959. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Obeng BB, Aryeetey YA, de Dood CJ, Amoah AS, Larbi IA, Deelder AM, et al. Application of a circulating-cathodic-antigen (CCA) strip test and real-time PCR, in comparison with microscopy, for the detection of Schistosoma haematobium in urine samples from Ghana. Ann Trop Med Parasitol. 2008;102:625–33. [DOI] [PubMed] [Google Scholar]
29.Cools P, van Lieshout L, Koelewijn R, Addiss D, Ajjampur SSR, Ayana M, et al. First international external quality assessment scheme of nucleic acid amplification tests for the detection of Schistosoma and soil-transmitted helminths, including Strongyloides: a pilot study. PLoS Negl Trop Dis. 2020;14:e0008231. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Walker M, Churcher TS, Basáñez MG. Models for measuring anthelmintic drug efficacy for parasitologists. Trends Parasitol. 2014;30:528–37. doi: 10.1016/j.pt.2014.08.004 [DOI] [PubMed] [Google Scholar]
31.Walker M, Mabud TS, Olliaro PL, Coulibaly JT, King CH, Raso G, et al. New approaches to measuring anthelminthic drug efficacy: parasitological responses of childhood schistosome infections to treatment with praziquantel. Parasit Vectors. 2016;9:41. doi: 10.1186/s13071-016-1312-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Olliaro PL, Vaillant M, Diawara A, Coulibaly JT, Garba A, Keiser J, et al. Toward measuring Schistosoma response to praziquantel treatment with appropriate descriptors of egg excretion. PLoS Negl Trop Dis. 2015;9:e0003821. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Pillay P, Taylor M, Zulu SG, Gundersen SG, Verweij JJ, Hoekstra P, et al. Real-time polymerase chain reaction for detection of Schistosoma DNA in small-volume urine samples reflects focal distribution of urogenital schistosomiasis in primary school girls in KwaZulu Natal, South Africa. Am J Trop Med Hyg. 2014;90:546–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Banoo S, Bell D, Bossuyt P, Herring A, Mabey D, Poole F, et al. Evaluation of diagnostic tests for infectious diseases: general principles. Nat Rev Microbiol. 2006;4(9 Suppl):S21–31. doi: 10.1038/nrmicro1523 [DOI] [PubMed] [Google Scholar]
35.Knopp S, Corstjens PL, Koukounari A, Cercamondi CI, Ame SM, Ali SM, et al. Sensitivity and specificity of a urine circulating anodic antigen test for the diagnosis of Schistosoma haematobium in low endemic settings. PLoS Negl Trop Dis. 2015;9:e0003752. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Alonzo TA, Pepe MS. Using a combination of reference tests to assess the accuracy of a new diagnostic test. Stat Med. 1999;18:2987–3003. doi: [DOI] [PubMed] [Google Scholar]
37.Aryeetey YA, Essien-Baidoo S, Larbi IA, Ahmed K, Amoah AS, Obeng BB, et al. Molecular diagnosis of Schistosoma infections in urine samples of school children in Ghana. Am J Trop Med Hyg. 2013;88:1028–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Hoekstra PT, Chernet A, de Dood CJ, Brienen EAT, Corstjens P, Labhardt ND, et al. Sensitive diagnosis and post-treatment follow-up of Schistosoma mansoni infections in asymptomatic Eritrean refugees by circulating anodic antigen detection and polymerase chain reaction. Am J Trop Med Hyg. 2022;106:1240–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Wichmann D, Panning M, Quack T, Kramme S, Burchard GD, Grevelding C, et al. Diagnosing schistosomiasis by detection of cell-free parasite DNA in human plasma. PLoS Negl Trop Dis. 2009;3:e422. doi: 10.1371/journal.pntd.0000422 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Cnops L, Soentjens P, Clerinx J, Van Esbroeck M. A Schistosoma haematobium-specific real-time PCR for diagnosis of urogenital schistosomiasis in serum samples of international travelers and migrants. PLoS Negl Trop Dis. 2013;7:e2413. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Clark J, Arinaitwe M, Nankasi A, Faust CL, Moses A, Ajambo D, et al. Reconciling egg- and antigen-based estimates of Schistosoma mansoni clearance and reinfection: a modelling study. Clin Infect Dis. 2022;74:1557–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Assaré RK, Tra-Bi MI, Coulibaly JT, Corstjens P, Ouattara M, Hürlimann E, et al. Accuracy of two circulating antigen tests for the diagnosis and surveillance of Schistosoma mansoni infection in low-endemicity settings of Côte d’Ivoire. Am J Trop Med Hyg. 2021;105:677–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Zwang J, Olliaro PL. Clinical efficacy and tolerability of praziquantel for intestinal and urinary schistosomiasis-a meta-analysis of comparative and non-comparative clinical trials. PLoS Negl Trop Dis. 2014;8:e3286. doi: 10.1371/journal.pntd.0003286 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.King CH. It’s time to dispel the myth of "asymptomatic" schistosomiasis. PLoS Negl Trop Dis. 2015;9:e0003504. doi: 10.1371/journal.pntd.0003504 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011008.r001

Decision Letter 0

Aaron R Jex

9 Oct 2022

Dear Mrs Hoekstra,

Thank you very much for submitting your manuscript "Limited efficacy of repeated praziquantel treatment in Schistosoma mansoni infections as revealed by highly accurate diagnostics, PCR and UCP-LF CAA (RePST trial)" 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.

Dear Dr Hoekstra,

Firstly, I'd like to apologise for the lengthy delay in processing your manuscript. I trust the journal has been in contact with you. This delay was in part due to, I think, major changes in the journals submission cite, which meant the article sat in limbo for some weeks before it actually appeared in my assignment feed. I then had some further significant delays with finding reviewers. I mention this not to make excuses for these delay but by way of acknowledging that the length of time it has taken to process your submission is not acceptable and I hope it will not be repeated in the future.

As you'll see from the reviewers comments, the reviews are quite split on your manuscript. Unfortunately, somehow, one of the reviewer's (who recommended rejection of the manuscript) comments were not uploaded or otherwise went missing. I can't say what has caused this, but I followed up with the review and they provided the following summary of their concerns.

"The manuscript "Limited efficacy of repeated praziquantel treatment in Schistosoma mansoni infections as revealed by highly accurate diagnostics, PCR and UCP-LF CAA (RePST trial)" aimed to evaluate PZQ efficacy using PCR and UCP-LF CAA in addition to the techniques already employed in this kind of study (KK and POC-CCA). However, the results did not show the relevance of the different methods used. Also, there is no relevant difference between the obtained data from KK and POC-CCA and PCR and UCP-LF CAA. Accordingly, I recommend rejecting the manuscript."

In an effort to prevent any further delays to processing your submission, I have informally reviewed the article myself, particularly in order to see if I could understand this second reviewer's concerns. I think it would be helpful to see the comparisons you undertook in your supplementaries featuring more prominently in your manuscript in order to address these concerns. I certainly won't tell you how to design your study or author your manuscript, but I think it might be helpful if you considered a main text figure and/or table (both perhaps?) clearly demonstrating:

1. The differences in sensitivity of the four methods you employed and their relative agreement with your surrogate 'gold-standard'. You mention in your text that the UPC-LC CAA method has been shown in prior study to have 'high' specificity and sensitivity, but I think demonstrating this using your control material is important for reader's in interpreting the PZQ CR results. I understand that this is in your supplementaries, but I think it might be better placed in the main text.

2. Similarly, you demonstrate in the supplementaries, as notable difference in the estimated CR based on the four methods you have used, but again, I think having this as a main figure and highlighting which of these differences are statistically significant would be helpful to the reader.

2. Finally, do you have data for the relative diagnostic specificity of each method? Particularly specificity for patent/live infections. With the antigen tests, is there any way to know for how long hosts will continue to shed worm antigens after the worms themselves have been killed through PZQ treatment? Similarly, is there any data that following the apparent cessation of egg production after PZQ (inferred by KK for example) that hosts still showing infection based on UPC-LC CAA or POC-CCA eventually resume egg-shedding (as evidence of the survival of any remaining worms and their return to reproductive fecundity). I presume such data would have to come from a hamster or similar model animal. I think this is particularly worth considering in the intense treatment group in whom you measured a substantial reduction in infection based on KK, but much less so with the other methods after week 2 or so. Could it be that either treatment has sterilised the surviving adults (as has been seen in, for example, canine heartworm treatment)? Again, is their data that can refute concerns that what you are seeing is prolonged antigen shedding from dead and decaying worms and not live adults?

Please address these and the other review comments in revision. If at all possible, I will attempt to review the revised manuscript without sending it out for further review in order to minimise any further delays.

Thank you

Aaron

In addition, I noted that you record in your supplementaries that you see a major decline in the estimated

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,

Aaron R. Jex

Section Editor

PLOS Neglected Tropical Diseases

Aaron Jex

Section Editor

PLOS Neglected Tropical Diseases

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

Dear Dr Hoekstra,

Thank you

Aaron

In addition, I noted that you record in your supplementaries that you see a major decline in the estimated

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: (No Response)

Reviewer #2: Yes, the proposed objectives are in accordance with the tested hypothesis. The study design is in accordance with the proposed objective. The sample used here originates from a previous publication and although it started from a very large group of schoolchildren and only a little more than 125 children were left for the analyses, even so this number is enough to carry out the statistical analyzes which support well the conclusions drawn.

The authors present the necessary approvals to the local ethics committee and the institution responsible for carrying out the exams.

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

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: (No Response)

Reviewer #2: The results are well described and in accordance with the proposed objectives. The amount of tables and figures is enough to clearly show what is proposed. Although there is no gold standard for comparison, the authors determined for comparison the composite reference standard and considered the individual who was positive in the four tests truly positive.

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

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: (No Response)

Reviewer #2: Yes, the conclusions are supported by the data presented. The data obtained make a great contribution to the question of eliminating schistosomiasis as proposed in the WHO guidelines. It is clear that the determination of the cure rate requires a more complex analysis. More sensitive methods certainly showed if, in fact, these worms were truly eliminated or not. It remained a curiosity to know if the analyzes had been extended for another 2 weeks how this data would behave. I consider that these data are relevant for Public Health.

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

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: (No Response)

Reviewer #2: Although the authors refer to previous works, it would be important to make reference in the discussion (or even in the methodology) about the issue of rejection or even the difficulty of treating children with PZQ pills, speak also if in the intensive treatment group how they dealt with the adverse reactions that routinely occur. Another curiosity is to know if with so many evaluations (sample collections) there was no loss of individuals for not delivering the samples. A comment would be in order. They are minor modifications.

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

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: (No Response)

Reviewer #2: The data presented clearly warns us that we need to rethink the treatment of schistosomiasis, not only in terms of the dose to be applied, but also in the search for new drugs.

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

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

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

PLoS Negl Trop Dis. 2022 Dec 22;16(12):e0011008. doi: 10.1371/journal.pntd.0011008.r002

Author response to Decision Letter 0

29 Nov 2022

Attachment

Submitted filename: 221121_RePST_diagnostics_Response to editor+reviewers.docx

Click here for additional data file.^{(26.3KB, docx)}

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011008.r003

Decision Letter 1

Aaron R Jex

6 Dec 2022

Dear Mrs Hoekstra,

We are pleased to inform you that your manuscript 'Limited efficacy of repeated praziquantel treatment in Schistosoma mansoni infections as revealed by highly accurate diagnostics, PCR and UCP-LF CAA (RePST trial)' 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,

Aaron R. Jex

Section Editor

PLOS Neglected Tropical Diseases

Aaron Jex

Section Editor

PLOS Neglected Tropical Diseases

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

Thank you to the authors' for their patience with this submision and again my apologies for the delays during review. My apologies also for the confusion with my review. I got confused when reading the intial submission and had the impression that part of the intent was to demonstrate that the worm-based antigen methods were more sensitive etc that egg-based (ie, prooving the method) rather than that this was already well established and rather the intent was to show that applying these methods provides a more accurate representation of the efficacy of a PZQ trial result. In reading through the authors' responses and revisiting the manuscript, I'm not sure how I got this confused. Nonetheless, I thank the authors' for their responses and I feel this is clear to me now. Regarding my comment about the reduction in the differences in infection reductions as inferred by egg or worm-based approaches, again, my apologies for my imprecise language. I was referring to the data shown in Figure 1 (% positives after treatment) in relation to my question about what remaining antigen was either dying worms or worms rendered senescent by treatment. I thank the authors for clarifying this in their response.

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011008.r004

Acceptance letter

Aaron R Jex

19 Dec 2022

Dear Mrs Hoekstra,

We are delighted to inform you that your manuscript, " Limited efficacy of repeated praziquantel treatment in Schistosoma mansoni infections as revealed by highly accurate diagnostics, PCR and UCP-LF CAA (RePST trial) ," 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

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Trial profile.

(PNG)

Click here for additional data file.^{(310.2KB, png)}

S2 Fig

(TIF)

Click here for additional data file.^{(625.7KB, tif)}

S3 Fig. Baseline correlation between egg-detection methods and worm-based detection methods.

(TIF)

Click here for additional data file.^{(1MB, tif)}

S4 Fig. Post-treatment correlation between egg-detection methods and worm-based detection methods.

(TIF)

Click here for additional data file.^{(1.1MB, tif)}

S5 Fig

(TIF)

Click here for additional data file.^{(338.6KB, tif)}

S6 Fig

(TIF)

Click here for additional data file.^{(311.6KB, tif)}

S7 Fig

(TIF)

Click here for additional data file.^{(348.6KB, tif)}

S1 Table. Baseline characteristics of the standard treatment group and the intense treatment group.

Adapted from (24), for the current study based on data from 125 school-aged children.

(DOCX)

Click here for additional data file.^{(17.1KB, docx)}

Data based on polymerase chain reaction (PCR), Kato-Katz (KK), up-converting particle circulating anodic antigen (UCP-LF CAA), and point-of-care circulating cathodic antigen (POC-CCA).

(DOCX)

Click here for additional data file.^{(18KB, docx)}

Attachment

Submitted filename: 221121_RePST_diagnostics_Response to editor+reviewers.docx

Click here for additional data file.^{(26.3KB, docx)}

Data Availability Statement

All relevant data are available within the manuscript and its supporting information file.

[pntd.0011008.ref001] 1.WHO. Schistosomiasis: progress report 2001–2011 and strategic plan 2012–2020. Geneva: World Health Organization; 2013. [Google Scholar]

[pntd.0011008.ref002] 2.WHO. Assessing the efficacy of anthelminthic drugs against schistosomiasis and soil-transmitted helminthiases. Geneva: World Health Organization; 2013. [Google Scholar]

[pntd.0011008.ref003] 3.King CH, Olbrych SK, Soon M, Singer ME, Carter J, Colley DG. Utility of repeated praziquantel dosing in the treatment of schistosomiasis in high-risk communities in Africa: a systematic review. PLoS Negl Trop Dis. 2011;5:e1321. doi: 10.1371/journal.pntd.0001321 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref004] 4.Munisi DZ, Buza J, Mpolya EA, Angelo T, Kinung’hi SM. The efficacy of single-dose versus double-dose praziquantel treatments on Schistosoma mansoni infections: its implication on undernutrition and anaemia among primary schoolchildren in two on-shore communities, northwestern Tanzania. Biomed Res Int. 2017;2017:7035025. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref005] 5.Nalugwa A, Nuwaha F, Tukahebwa EM, Olsen A. Single versus double dose praziquantel comparison on efficacy and Schistosoma mansoni re-infection in preschool-age children in Uganda: a randomized controlled trial. PLoS Negl Trop Dis. 2015;9:e0003796. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref006] 6.Fukushige M, Chase-Topping M, Woolhouse MEJ, Mutapi F. Efficacy of praziquantel has been maintained over four decades (from 1977 to 2018): a systematic review and meta-analysis of factors influence its efficacy. PLoS Negl Trop Dis. 2021;15:e0009189. doi: 10.1371/journal.pntd.0009189 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref007] 7.Utzinger J, Becker SL, van Lieshout L, van Dam GJ, Knopp S. New diagnostic tools in schistosomiasis. Clin Microbiol Infect. 2015;21:529–42. doi: 10.1016/j.cmi.2015.03.014 [DOI] [PubMed] [Google Scholar]

[pntd.0011008.ref008] 8.Coulibaly JT, N’Gbesso YK, Knopp S, N’Guessan NA, Silué KD, van Dam GJ, et al. Accuracy of urine circulating cathodic antigen test for the diagnosis of Schistosoma mansoni in preschool-aged children before and after treatment. PLoS Negl Trop Dis. 2013;7:e2109. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref009] 9.Hoekstra PT, van Dam GJ, van Lieshout L. Context-specific procedures for the diagnosis of human schistosomiasis–a mini review. Front Trop Dis. 2021;2:722438. [Google Scholar]

[pntd.0011008.ref010] 10.Lamberton PH, Kabatereine NB, Oguttu DW, Fenwick A, Webster JP. Sensitivity and specificity of multiple Kato-Katz thick smears and a circulating cathodic antigen test for Schistosoma mansoni diagnosis pre- and post-repeated-praziquantel treatment. PLoS Negl Trop Dis. 2014;8:e3139. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref011] 11.Mwinzi PN, Kittur N, Ochola E, Cooper PJ, Campbell CH Jr., King CH, et al. Additional evaluation of the point-of-contact circulating cathodic antigen assay for Schistosoma mansoni infection. Front Public Health. 2015;3:48. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref012] 12.Vinkeles Melchers NV, van Dam GJ, Shaproski D, Kahama AI, Brienen EA, Vennervald BJ, et al. Diagnostic performance of Schistosoma real-time PCR in urine samples from Kenyan children infected with Schistosoma haematobium: day-to-day variation and follow-up after praziquantel treatment. PLoS Negl Trop Dis. 2014;8:e2807. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref013] 13.van Dam GJ, Bergwerff AA, Thomas-Oates JE, Rotmans JP, Kamerling JP, Vliegenthart JF, et al. The immunologically reactive O-linked polysaccharide chains derived from circulating cathodic antigen isolated from the human blood fluke Schistosoma mansoni have Lewis x as repeating unit. Eur J Biochem. 1994;225:467–82. [DOI] [PubMed] [Google Scholar]

[pntd.0011008.ref014] 14.van Dam GJ, Bogitsh BJ, van Zeyl RJ, Rotmans JP, Deelder AM. Schistosoma mansoni: in vitro and in vivo excretion of CAA and CCA by developing schistosomula and adult worms. J Parasitol. 1996;82:557–64. [PubMed] [Google Scholar]

[pntd.0011008.ref015] 15.Bärenbold O, Garba A, Colley DG, Fleming FM, Haggag AA, Ramzy RMR, et al. Translating preventive chemotherapy prevalence thresholds for Schistosoma mansoni from the Kato-Katz technique into the point-of-care circulating cathodic antigen diagnostic test. PLoS Negl Trop Dis. 2018;12:e0006941. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref016] 16.WHO. WHO guideline on control and elimination of human schistosomiasis. Geneva: World Health Organzation; 2022. [PubMed] [Google Scholar]

[pntd.0011008.ref017] 17.WHO. Schistosomiasis 2022 [Available from: https://www.who.int/en/news-room/fact-sheets/detail/schistosomiasis].

[pntd.0011008.ref018] 18.WHO. Report of the first meeting of the WHO diagnostic technical advisory group for neglected tropical diseases. Geneva: World Health Organization; 2019. [Google Scholar]

[pntd.0011008.ref019] 19.Corstjens P, de Dood CJ, Knopp S, Clements MN, Ortu G, Umulisa I, et al. Circulating anodic antigen (CAA): a highly sensitive diagnostic biomarker to detect active Schistosoma infections-improvement and use during SCORE. Am J Trop Med Hyg. 2020;103(1_Suppl):50–7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref020] 20.van Dam GJ, Odermatt P, Acosta L, Bergquist R, de Dood CJ, Kornelis D, et al. 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;141:198–203. [DOI] [PubMed] [Google Scholar]

[pntd.0011008.ref021] 21.van Dam GJ, Xu J, Bergquist R, de Dood CJ, Utzinger J, Qin ZQ, et al. An ultra-sensitive assay targeting the circulating anodic antigen for the diagnosis of Schistosoma japonicum in a low-endemic area, People’s Republic of China. Acta Trop. 2015;141:190–7. [DOI] [PubMed] [Google Scholar]

[pntd.0011008.ref022] 22.Vonghachack Y, Sayasone S, Khieu V, Bergquist R, van Dam GJ, Hoekstra PT, et al. Comparison of novel and standard diagnostic tools for the detection of Schistosoma mekongi infection in Lao People’s Democratic Republic and Cambodia. Infect Dis Poverty. 2017;6:127. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref023] 23.Hoekstra PT, Casacuberta-Partal M, van Lieshout L, Corstjens P, Tsonaka R, Assaré RK, et al. Efficacy of single versus four repeated doses of praziquantel against Schistosoma mansoni infection in school-aged children from Côte d’Ivoire based on Kato-Katz and POC-CCA: an open-label, randomised controlled trial (RePST). PLoS Negl Trop Dis. 2020;14:e0008189. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref024] 24.Hoekstra PT, Casacuberta Partal M, Amoah AS, van Lieshout L, Corstjens P, Tsonaka S, et al. Repeated doses of Praziquantel in Schistosomiasis Treatment (RePST)—single versus multiple praziquantel treatments in school-aged children in Côte d’Ivoire: a study protocol for an open-label, randomised controlled trial. BMC Infect Dis. 2018;18:662. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref025] 25.Casacuberta-Partal M, Hoekstra PT, Kornelis D, van Lieshout L, van Dam GJ. An innovative and user-friendly scoring system for standardised quantitative interpretation of the urine-based point-of-care strip test (POC-CCA) for the diagnosis of intestinal schistosomiasis: a proof-of-concept study. Acta Trop. 2019;199:105150. doi: 10.1016/j.actatropica.2019.105150 [DOI] [PubMed] [Google Scholar]

[pntd.0011008.ref026] 26.ten Hove RJ, Verweij JJ, Vereecken K, Polman K, Dieye L, van Lieshout L. Multiplex real-time PCR for the detection and quantification of Schistosoma mansoni and S. haematobium infection in stool samples collected in northern Senegal. Trans R Soc Trop Med Hyg. 2008;102:179–85. [DOI] [PubMed] [Google Scholar]

[pntd.0011008.ref027] 27.Meurs L, Brienen E, Mbow M, Ochola EA, Mboup S, Karanja DM, et al. Is PCR the next reference standard for the diagnosis of Schistosoma in stool? A comparison with microscopy in Senegal and Kenya. PLoS Negl Trop Dis. 2015;9:e0003959. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref028] 28.Obeng BB, Aryeetey YA, de Dood CJ, Amoah AS, Larbi IA, Deelder AM, et al. Application of a circulating-cathodic-antigen (CCA) strip test and real-time PCR, in comparison with microscopy, for the detection of Schistosoma haematobium in urine samples from Ghana. Ann Trop Med Parasitol. 2008;102:625–33. [DOI] [PubMed] [Google Scholar]

[pntd.0011008.ref029] 29.Cools P, van Lieshout L, Koelewijn R, Addiss D, Ajjampur SSR, Ayana M, et al. First international external quality assessment scheme of nucleic acid amplification tests for the detection of Schistosoma and soil-transmitted helminths, including Strongyloides: a pilot study. PLoS Negl Trop Dis. 2020;14:e0008231. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref030] 30.Walker M, Churcher TS, Basáñez MG. Models for measuring anthelmintic drug efficacy for parasitologists. Trends Parasitol. 2014;30:528–37. doi: 10.1016/j.pt.2014.08.004 [DOI] [PubMed] [Google Scholar]

[pntd.0011008.ref031] 31.Walker M, Mabud TS, Olliaro PL, Coulibaly JT, King CH, Raso G, et al. New approaches to measuring anthelminthic drug efficacy: parasitological responses of childhood schistosome infections to treatment with praziquantel. Parasit Vectors. 2016;9:41. doi: 10.1186/s13071-016-1312-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref032] 32.Olliaro PL, Vaillant M, Diawara A, Coulibaly JT, Garba A, Keiser J, et al. Toward measuring Schistosoma response to praziquantel treatment with appropriate descriptors of egg excretion. PLoS Negl Trop Dis. 2015;9:e0003821. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref033] 33.Pillay P, Taylor M, Zulu SG, Gundersen SG, Verweij JJ, Hoekstra P, et al. Real-time polymerase chain reaction for detection of Schistosoma DNA in small-volume urine samples reflects focal distribution of urogenital schistosomiasis in primary school girls in KwaZulu Natal, South Africa. Am J Trop Med Hyg. 2014;90:546–52. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref034] 34.Banoo S, Bell D, Bossuyt P, Herring A, Mabey D, Poole F, et al. Evaluation of diagnostic tests for infectious diseases: general principles. Nat Rev Microbiol. 2006;4(9 Suppl):S21–31. doi: 10.1038/nrmicro1523 [DOI] [PubMed] [Google Scholar]

[pntd.0011008.ref035] 35.Knopp S, Corstjens PL, Koukounari A, Cercamondi CI, Ame SM, Ali SM, et al. Sensitivity and specificity of a urine circulating anodic antigen test for the diagnosis of Schistosoma haematobium in low endemic settings. PLoS Negl Trop Dis. 2015;9:e0003752. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref036] 36.Alonzo TA, Pepe MS. Using a combination of reference tests to assess the accuracy of a new diagnostic test. Stat Med. 1999;18:2987–3003. doi: [DOI] [PubMed] [Google Scholar]

[pntd.0011008.ref037] 37.Aryeetey YA, Essien-Baidoo S, Larbi IA, Ahmed K, Amoah AS, Obeng BB, et al. Molecular diagnosis of Schistosoma infections in urine samples of school children in Ghana. Am J Trop Med Hyg. 2013;88:1028–31. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref038] 38.Hoekstra PT, Chernet A, de Dood CJ, Brienen EAT, Corstjens P, Labhardt ND, et al. Sensitive diagnosis and post-treatment follow-up of Schistosoma mansoni infections in asymptomatic Eritrean refugees by circulating anodic antigen detection and polymerase chain reaction. Am J Trop Med Hyg. 2022;106:1240–6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref039] 39.Wichmann D, Panning M, Quack T, Kramme S, Burchard GD, Grevelding C, et al. Diagnosing schistosomiasis by detection of cell-free parasite DNA in human plasma. PLoS Negl Trop Dis. 2009;3:e422. doi: 10.1371/journal.pntd.0000422 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref040] 40.Cnops L, Soentjens P, Clerinx J, Van Esbroeck M. A Schistosoma haematobium-specific real-time PCR for diagnosis of urogenital schistosomiasis in serum samples of international travelers and migrants. PLoS Negl Trop Dis. 2013;7:e2413. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref041] 41.Clark J, Arinaitwe M, Nankasi A, Faust CL, Moses A, Ajambo D, et al. Reconciling egg- and antigen-based estimates of Schistosoma mansoni clearance and reinfection: a modelling study. Clin Infect Dis. 2022;74:1557–63. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref042] 42.Assaré RK, Tra-Bi MI, Coulibaly JT, Corstjens P, Ouattara M, Hürlimann E, et al. Accuracy of two circulating antigen tests for the diagnosis and surveillance of Schistosoma mansoni infection in low-endemicity settings of Côte d’Ivoire. Am J Trop Med Hyg. 2021;105:677–83. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref043] 43.Zwang J, Olliaro PL. Clinical efficacy and tolerability of praziquantel for intestinal and urinary schistosomiasis-a meta-analysis of comparative and non-comparative clinical trials. PLoS Negl Trop Dis. 2014;8:e3286. doi: 10.1371/journal.pntd.0003286 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pntd.0011008.ref044] 44.King CH. It’s time to dispel the myth of "asymptomatic" schistosomiasis. PLoS Negl Trop Dis. 2015;9:e0003504. doi: 10.1371/journal.pntd.0003504 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Limited efficacy of repeated praziquantel treatment in Schistosoma mansoni infections as revealed by highly accurate diagnostics, PCR and UCP-LF CAA (RePST trial)

Pytsje T Hoekstra

Miriam Casacuberta-Partal

Lisette van Lieshout

Paul L A M Corstjens

Roula Tsonaka

Rufin K Assaré

Kigbafori D Silué

Eliézer K N’Goran

Yves K N’Gbesso

Eric A T Brienen

Meta Roestenberg

Stefanie Knopp

Jürg Utzinger

Jean T Coulibaly

Govert J van Dam

Roles

Abstract

Background

Methodology

Principal findings

Conclusion/Significance

Clinical trial registration

Author summary

Introduction

Methods

Ethics statement

Study design and participants

Diagnostic procedures

Statistical analysis

Results

Prevalence over time

Fig 1. Percentage positive over time with corresponding 95% confidence intervals estimated from the mixed effects logistic regression model.

Table 1. Cure rate (CR) and intensity reduction rate (IRR) of a single (standard treatment group) and four (intense treatment group) repeated PZQ treatments in 125 school-aged children with a confirmed S. mansoni infection.

Intensity of infection over time

Fig 2. Average infection levels over time with corresponding 95% confidence intervals* estimated from the mixed effects logistic regression model.

Correlation between tests

Fig 3. Baseline correlation between (a) egg-detection methods and (b) worm-based detection methods.

Fig 4. Post-treatment correlation between (a) egg-detection methods and (b) worm-based detection methods.

Accuracy of tests

Table 2. Sensitivity of polymerase chain reaction (PCR), Kato-Katz (KK), up-converting particle circulating anodic antigen (UCP-LF CAA), and point-of-care circulating cathodic antigen (POC-CCA) compared to a composite reference standard (CRS) over time.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Aaron R Jex

Roles

Author response to Decision Letter 0

Decision Letter 1

Aaron R Jex

Roles

Acceptance letter

Aaron R Jex

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases