Two Kato-Katz thick smears (Kato-Katzs) from a single stool are currently recommended for diagnosing Schistosoma mansoni infections to map areas for intervention. This ‘gold standard’ has low sensitivity at low infection intensities. The urine point-of-care circulating cathodic antigen test (POC-CCA) is potentially more sensitive but how accurately they detect S. mansoni after repeated praziquantel treatments, their suitability for measuring drug efficacy and their correlation with egg counts remain to be fully understood. We compared the accuracies of one to six Kato-Katzs and one POC-CCA for the diagnosis of S. mansoni in primary-school children who have received zero to ten praziquantel treatments. We determined the impact each diagnostic approach may have on monitoring and evaluation (M&E) and drug-efficacy findings. In a high S. mansoni endemic area of Uganda, three days of consecutive stool samples were collected from primary school-aged children (six - 12 years) at five time-points in year one: baseline, one-week-post-, four-weeks-post-, six-months-post-, and six-months-one-week-post-praziquantel and three time-points in years two and three: pre-, one-week-post- and four-weeks-post-praziquantel-treatment/retreatment (n = 1065). Two Kato-Katzs were performed on each stool. In parallel, one urine sample was collected and a single POC-CCA evaluated per child at each time-point in year one (n = 367). At baseline, diagnosis by two Kato-Katzs (sensitivity = 98. 6%) or one POC-CCA (sensitivity = 91. 7%, specificity = 75. 0%) accurately predicted S. mansoni infections. However, one year later, a minimum of three Kato-Katzs, and two years later, five Kato-Katzs were required for accurate diagnosis (sensitivity >90%) and drug-efficacy evaluation. The POC-CCA was as sensitive as six Kato-Katzs four-weeks-post and six-months-post-treatment, if trace readings were classified as positive. Six Kato-Katzs (two/stool from three stools) and/or one POC-CCA are required for M&E or drug-efficacy studies. Although unable to measure egg reduction rates, one POC-CCA appears to be more sensitive than six Kato-Katzs at four-weeks-post-praziquantel (drug efficacy) and six-months-post-praziquantel (M&E).
Schistosomiasis remains a major public health concern despite praziquantel reaching over 30 million people in endemic areas in 2013 [1]. Goals to eliminate schistosomiasis by 2020 have been articulated by the World Health Organization' s (WHO) ‘Roadmap for Neglected Tropical Disease (NTD) Implementation’ [2], and the London Declaration of the NTD Coalition [3]. Accurate diagnostic techniques, recently highlighted by Gomes and colleagues [4], are essential for monitoring and evaluation (M&E) of mass drug administration (MDA) programs at all stages [5]–[9], and particularly when considering elimination [2], [10], [11] and/or drug-resistance pharmacovigilance [12], [13]. The WHO recommends two Kato-Katz thick smears (Kato-Katzs) from a single stool [14] for Schistosoma mansoni diagnosis to determine prevalence to map areas for control interventions [15]. Kato-Katzs have assumed 100% specificity, but large inter- and intra-specimen variation [16]–[18] and low sensitivity for the detection of low intensity infections have been reported [19]–[21]. In Brazil, where M&E programs use only one Kato-Katz, S. mansoni prevalence has been significantly underestimated in low intensity regions [22], [23]. This may be associated with overestimated cure rates (CRs) and praziquantel efficacy [24], potentially missing drug resistance. Conversely, using one Kato-Katz can overestimate infection intensities [22]. These differences in sensitivity for prevalence versus infection intensity highlight complex interactions with egg reduction rates (ERRs) as true intensities decrease. Inter-day variation of excreted egg numbers post-treatment remains poorly understood. Two Kato-Katzs are commonly used for annual M&E of program impact and/or drug-efficacy studies, without being rigorously tested in these scenarios. Detailed analyses of the sensitivity and specificity of single and multiple Kato-Katzs following praziquantel treatment is urgently required. It is not possible to directly measure S. mansoni adult worm numbers, due to their location in the mesenteric system, with eggs counted from Kato-Katzs used as a proxy for infection intensity. Immunodiagnostics for adult worm circulating cathodic antigens (CCA) or circulating anodic antigens (CAA) detect current infections and are potentially more sensitive for diagnosis of cases in low transmission areas [25]–[27]. Recent developments using innovative immunomagnetic separation with several target CCAs [28] and novel monoclonal antibody diagnostics for serum CCA [27] show high sensitivity even in low endemic areas. Serum CAA tests are more accurate than urine CAA tests [29], with advances made on a, not yet commercially available, point-of-care-CAA (POC-CAA) [30]. In contrast, urine CCA tests were more accurate than serum CCA tests [29]. Being easier to collect and more socially acceptable than stool or blood, urine POC-CCAs were developed for rapid, non-invasive diagnostics and proposed as alternatives to Kato-Katzs for S. mansoni prevalence mapping [31]–[37]. One urine POC-CCA is as sensitive as two Kato-Katzs for S. mansoni diagnosis for prevalence mapping [35] or two years post-treatment [36] and has now been used to map S. mansoni prevalence in low intensity areas in Uganda [37]. Major POC-CCA limitations are their ability to only semi-quantitatively measure infection intensity [32], [34], [38], inaccuracy for S. haematobium infection detection [39], [40], and not measuring soil-transmitted helminth (STH) infections. Intensity of infection measures are vital for control program M&E and drug-efficacy evaluation. Therefore knowledge on the ability of POC-CCAs to detect S. mansoni intensity reductions is essential [34]–[36], [41]. We compared the accuracy of the currently available POC-CCA and one to six Kato-Katzs (two smears per day from three consecutive stool samples) for S. mansoni diagnosis, in primary-school children in Mayuge District, Uganda, at baseline and after up to ten praziquantel treatments per child over three years (STARD Checklist S1 and Figure 1). We evaluated the epidemiological implications of diagnostic methods for control program M&E and praziquantel-efficacy studies. We inform on the number of Kato-Katzs required to accurately detect S. mansoni infections pre- and post-praziquantel treatment and whether POC-CCAs are suitable alternatives. Our detailed longitudinal design enabled novel investigations into individuals' recent versus total praziquantel treatments, aiding biological understanding of differences between Kato-Katz and POC-CCA results post-praziquantel treatment. We predicted that the accuracy of M&E and drug-efficacy findings are limited by Kato-Katz sensitivity at low infection intensities post-treatment. We also predicted that a single POC-CCA would have a higher sensitivity than multiple Kato-Katzs, and be more informative for prevalence monitoring as control programs progress.
Approvals were granted by the Uganda National Council of Science and Technology (Memorandum of Understanding: sections 1. 4,1. 5,1. 6) and the Imperial College Research Ethics Committee (EC NO: 03. 36. R&D No: 03/SB/033E). Verbal assent was given by every child before inclusion into this study and at school committee meetings comprising of parents, teachers, and community leaders before the onset of the study. Written consent for the children to participate in the study was attained from each head teacher. Participation was voluntary and children could withdraw or be withdrawn from the study at any time without obligation. Children were treated with 40 mg/kg praziquantel and 400 mg albendazole (active against STH infections) as detailed below. Samples were collected, between 2004 to 2006, from primary-school children, in a high S. mansoni-endemic area, in Mayuge district, Uganda from three schools on the shores of Lake Victoria: Bugoto Lake View, Bwondha, and Musubi Church of God. Children at Musubi were, to the authors' knowledge, praziquantel-naïve. Children at Bugoto and Bwondha had received 40 mg/kg praziquantel one year previously in 2003 [42]. Inclusion criteria were to have lived in the area since birth and to attend the schools sampled. In 2004, samples were collected at five time-points: baseline, one-week-post-, four-weeks-post-, six-months-post- and six-months-one-week-post-praziquantel treatment (Figure 1). In 2005 and 2006, samples were collected pre-, one-week-post-, and four-weeks-post-praziquantel re/treatment. On the third day of sampling, at baseline, six-months, one-year, and two-years all children were treated with 40 mg/kg praziquantel and 400 mg albendazole (active against STH infections). At one-week post-treatment, children with infections of >100 S. mansoni eggs per gram of stool (EPG) were retreated with 40 mg/kg praziquantel. At all other time-points all children with positive diagnoses for S. mansoni or STHs were retreated with 40 mg/kg praziquantel and 400 mg albendazole respectively. Cohort and sample collection are described elsewhere [43]. In brief, 110 children from Bugoto, 110 from Bwondha and 68 from Musubi were recruited in 2004 with an equal sex ratio, aged six to 12 years, without prior knowledge of infection status and/or symptoms of S. mansoni infection. In addition, at one- and two-years, 30 praziquantel-naïve six year old children were recruited at each school and followed up with the original cohorts at the time points described above. This enabled monitoring of the impact of MDA on untreated children entering the school system, assessing diagnostic accuracies for Kato-Katzs and POC-CCA, in praziquantel-naïve and praziquantel-exposed children, as control programs progress. Diagnostic accuracy increases with the number of Kato-Katzs, however, in Brazilian low intensity regions, the additional benefit of more than six Kato-Katzs from repeated stools was negligible [21], supporting our six Kato-Katzs ‘gold standard’. Stool samples, marked with unique child IDs, were collected on three consecutive days, between 10: 00 and 12: 00 hours. Two 41. 7 mg Kato-Katzs were prepared per stool and read onsite using a compound microscope with natural light source, by highly trained personnel from the Ugandan Vector Control Division, Ministry of Health. S. mansoni, hookworm, Ascaris lumbricoides, and Trichuris trichiura egg counts were recorded. Five percent of slides were reread after the study for S. mansoni, A. lumbricoides, and T. trichiura egg counts for quality control, but no significant differences were observed. One urine sample per child was collected between 10: 00 and 12: 00 hours on the first day. In the first year, at all five time-points, POC-CCAs (European Veterinary Laboratory, The Netherlands) were performed, according to the producer' s protocols, by the first author, blind of other test results. Microhematuria was tested for using Hemastix (Bayer, United Kingdom). SPSS version 19 (SPSS, Inc. , Chicago, IL, United States of America) was used for all statistical analyses. The double entered data were not normally distributed and could not be normalized by transformation, therefore non-parametric tests were used. Individuals without the full six Kato-Katzs were excluded from the study (Figure 1). Arithmetic mean infection intensities were categorized as by the WHO (S. mansoni: light = 1–99 EPG, moderate = 100–399 EPG and high ≥400 EPG; A. lumbricoides: light = 1–4999 EPG; Hookworm: light = 1–1,999 EPG; T. trichiura: light = 1–999 EPG) [15]. Exact confidence intervals (CIs) were calculated for prevalence measures and standard errors for EPGs.
Observed cure (measured at four-weeks-post-praziquantel treatment) and reinfection (measured at six-months) rates depended on sampling method and effort (Figures 4 and 5). Two Kato-Katzs underestimated S. mansoni reinfection whilst overestimating CRs (Figure 4A) (two Kato-Katzs CR = 81. 5%; six Kato-Katzs CR = 70. 4%). Cure rates determined with POC-CCA-t− were 47. 8% and 26. 1% for POC-CCA-t+. One-week-post-recent-praziquantel (from data at both one-week and six-months-one-week), prevalence was significantly lower when measured by POC-CCAs than by six Kato-Katzs (Figure 4B) (OR 0. 33 (95% CI: 0. 19,0. 59). Pre-re/treatment and at four-weeks-post-praziquantel-re/treatment in years zero, one, and two, the number of days of Kato-Katzs did not significantly affect the infection intensities (Figure 5) (all p>0. 05). However, at one-week-post-re/treatment (one-week, six-months-one-week, one-year-one-week and two-years-one-week), two Kato-Katzs and to a lesser extent four Kato-Katzs systematically, and significantly, overestimated the mean infection intensity (Friedman χ2 = 33. 08, d. f. = 2, p<0. 001). Infection intensity measured by the strength of the POC-CCA bands (graded from negative, + (inc. trace), ++ and +++) did not accurately predict the six Kato-Katzs infection intensity categories (negative, light, moderate, and heavy) (Table S1). However, strong positive correlations were seen between the ordinal POC-CCA band strengths and Kato-Katzs infection intensity categories (baseline: r = 0. 402, p = 0. 003; one-week: r = 0. 647, p<0. 001; four-weeks: r = 0. 389, p = 0. 001; six-months: r = 0. 413, p<0. 001; six-months-one-week: r = 0. 424, p<0. 001) as well as between POC-CCA band strengths and individual arithmetic mean EPG (Figures 6A to E). Pre-treatment, one POC-CCA-t+, and two Kato-Katzs had sensitivities above 90%, however the POC-CCA-t− and t+ NPVs were extremely low, similar to one Kato-Katz (Table 3). At one-week-post-praziquantel, one POC-CCA was less sensitive and had lower NPVs than one Kato-Katz, whilst two Kato-Katzs had an NPV of only 55. 6%. At four-weeks-post-praziquantel POC-CCA-t+ had high sensitivity and NPV (>80%), whilst POC-CCA-t− and one to six Kato-Katzs all had sensitivities and NPVs of <60%. Indeed, two Kato-Katzs only detected a quarter of the infections. At six-months-post-praziquantel POC-CCA-t+ was more sensitive than six Kato-Katzs, however all diagnostics showed NPV values below 15%. Some Bugoto and Bwondha children had been treated once before the study and new praziquantel-naïve cohorts were recruited each year. We therefore also analyzed our data along timelines specific for each individual child' s praziquantel exposure (Tables S2 and S3). Key results were not affected by re-analyzing the data in this manner. Pre-treatment, six-months, one-year and one-year-six-months POC-CCA-t+s showed high sensitivities but low NPVs throughout (Table S2). One-week-post-recent-praziquantel, three Kato-Katzs were required for >90% sensitivity in general, and four-weeks-post-recent-praziquantel four or five Kato-Katzs were required for >90% sensitivities (Table S3). An increased Kato-Katz sampling effort was required year on year to achieve sensitivities of >90% (Table S3), which was not clearly seen in the original M&E timeline (Figure 2, Table 2). Praziquantel-naïve children and children one-year-post-praziquantel required three Kato-Katzs for accurate S. mansoni diagnosis, whilst four Kato-Katzs were required at two-years, and five Kato-Katzs at three-years.
At least four Kato-Katzs (two smears per stool from two stools) are required for M&E, in the early years of a MDA program in a highly endemic area, increasing to six Kato-Katzs (two smears per stool from three stools) by year three. One POC-CCA is a suitable alternative to current prevalence M&E protocols, but they provide no information on STHs and limited intensity data post treatment, therefore we recommend their use for S. mansoni M&E with Kato-Katzs performed in a subset of schools. For drug-efficacy studies, at least six Kato-Katzs (two smears per stool from three stools) are required for accurate prevalence assessment four-weeks-post-praziquantel treatment. POC-CCAs may be a promising alternative with low specificity findings potentially due to low Kato-Katzs sensitivity, however further work is required to elucidate POC-CCA' s full potential for drug-efficacy studies. Further work on improved ‘gold standards’ is required to elucidate discordant POC-CCA and Kato-Katzs results. Data on multiple Kato-Katzs from a single stool post-treatment would ascertain if accuracies of multiple days of Kato-Katzs or POC-CCAs could be matched, minimizing logistical costs without overestimating M&E success and drug efficacy, whilst retaining vitally important intensity data.
Schistosomiasis is a parasitic disease infecting over 200 million people. It remains a major public health concern despite treatment of over 120 million people in sub-Saharan Africa alone. Accurate diagnostic methods are essential for monitoring drug efficacy and long-term control program success. The World Health Organization recommends two Kato-Katz thick smears (Kato-Katzs) from a single stool for Schistosoma mansoni diagnosis to map prevalence and areas for control interventions. Although highly specific, Kato-Katzs are thought to be insensitive at low egg counts. The recently refined urine point-of-care circulating cathodic antigen test (POC-CCA) has been proposed as a diagnostic alternative for mapping areas for interventions, and potentially for assessing drug efficacy. Over three years we assessed the accuracy of six Kato-Katzs and a single POC-CCA in detecting infections in Ugandan primary-school children at 11 time points with repeated praziquantel treatments. Our results demonstrate that two Kato-Katzs accurately detect S. mansoni infection pre-treatment, but at least three days of two Kato-Katzs per stool or one POC-CCA are required for annual monitoring and treatment evaluation and/or drug-efficacy studies. One POC-CCA may be more sensitive in measuring S. mansoni prevalence than six Kato-Katzs, but its accuracies for rigorous intensity measures are still to be proven.
helminth infections
schistosomiasis
plant science
medicine and health sciences
diagnostic medicine
epidemiology
disease surveillance
plant pathology
biology and life sciences
tropical diseases
neglected tropical diseases
soil-transmitted helminthiases
parasitic diseases
epidemiological methods and statistics
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