To evaluate the diagnostic performance of antigen detecting urine-CCA cassette test for the detection of Schistosoma mansoni infection in areas of moderate prevalence in Ethiopia.
To evaluate the diagnostic performance of antigen detecting urine-CCA cassette test for the detection of Schistosoma mansoni infection in areas of moderate prevalence in Ethiopia.
Stool specimens were collected from 620 schoolchildren on three consecutive days. The samples were microscopically examined using double Kato slides; midstream urine specimens were also collected for three consecutive days and tested for S. mansoni. The sensitivity of the urine-CCA cassette test was determined using combined results of six Kato–Katz thick smears and three urine-CCA cassette tests as gold standard. The specificity of the urine-CCA cassette test was evaluated in an area where schistosomiasis is not endemic.
Prevalence of S. mansoni infection as determined by single urine-CCA cassette test was 65.9%, by single Kato–Katz smear 37.3% and by six Kato–Katz thick smears 53.1% (P < 0.001). A single urine-CCA cassette test was significantly (P < 0.001) more sensitive (89.1%), had a lower negative predictive value (78.2%), was more accurate (92.6%) and agreed better with the gold standard (k = 0.83) than one or six Kato–Katz thick smears. However, both the Kato–Katz and urine-CCA cassette test showed 100% specificity in endemic settings.
In moderate and high prevalence areas, urine-CCA cassette test is more sensitive than the Kato–Katz method and can be used for screening and mapping of S. mansoni infection.
Estimer le risque de récurrence d'accouchements prématurés et estimer la mortalité périnatale dans les accouchements prématurés répétés.
Etude prospective en Tanzanie sur 18.176 femmes qui ont accouché d'un singleton entre 2000 et 2008 à l'hôpital KCMC. Les femmes ont été suivies jusqu'en 2010 pour les naissances consécutives. 3.359 femmes ont été identifiées avec un total de 3.867 accouchements ultérieurs, au cours de la période de suivi. Le risque de récurrence de prématurité et de mortalité périnatale a été estimé en utilisant la régression log-binomiale et en ajustant pour les facteurs confusionnels potentiels.
Pour les femmes ayant eu un accouchement prématuré précédent, le risque de naissance prématurée lors d'une grossesse ultérieure était de 17%. Ce risque de récurrence a été estimé à 2,7 fois (IC95%: 2,1–3,4) le risque chez les femmes ayant eu une naissance précédente à terme. La mortalité périnatale des bébés lors d'un second accouchement prématuré de la même mère était de 15%. Les bébés nés à terme qui avaient un frère/sœur plus âgé né avant terme, avaient une mortalité périnatale de 10%. Ceux nés à terme, qui avaient un frère/sœur plus âgé également né à terme, avaient une mortalité périnatale de 1,7%.
L'accouchement précédent d'un bébé prématuré est un prédicteur fort de futures naissances prématurées en Tanzanie. Les accouchements précédents ou répétés de prématurés augmentent substantiellement le risque de décès périnatal dans la grossesse suivante.
Calcular el riesgo de recurrencia de los partos pretérmino y la mortalidad en nacimientos prematuros repetidos.
Estudio prospectivo en Tanzania de 18,176 mujeres que dieron a luz un hijo único entre el 2000 y el 2008 en el hospital KCMC. A las mujeres se les siguió hasta el 2010 para partos consecutivos. Se identificaron 3,359 mujeres, con un total de 3,867 partos posteriores ocurridos durante el periodo de seguimiento. El riesgo de recurrencia de un parto pretérmino y de la mortalidad perinatal se calculó utilizando una regresión logística binomial, ajustada para posibles factores de confusión.
En mujeres con un parto pretérmino previo, el riesgo de un parto pretérmino durante un embarazo posterior era del 17%. Este riesgo de recurrencia se estimó que era 2.7-veces (IC 95%: 2.1–3.4) el riesgo de una mujer que previamente había tenido un embarazo a término. La mortalidad perinatal de los niños en un segundo parto pretérmino de la misma mujer era del 15%. Los bebés nacidos a término, con un hermano mayor que había nacido pretérmino, tenían una mortalidad perinatal del 10%. Los bebes nacidos a término, con un hermano mayor que también había nacido a término, tenían una mortalidad perinatal del 1.7%.
El haber tenido previamente un parto de un bebé prematuro es un vaticinador importante de futuros partos pretérmino en Tanzania. Los partos pretérmino previos o repetidos aumentan sustancialmente el riesgo de muerte perinatal en un embarazo posterior.
Schistosomiasis is a chronic, parasitic disease caused by blood flukes of the genus Schistosoma. It ranks second behind malaria among the parasitic diseases in terms of socio-economic and public health importance in tropical and subtropical areas (Waknine-Grinberg et al. 2010). The current strategy for the control of schistosomiasis is the reduction of morbidity through regular treatment of all people in at-risk groups with praziquantel (WHO 2012). Proper diagnosis of Schistosoma infection enables accurate prevalence estimation for deciding about the necessity of regular drug administration to communities at risk of infection with the parasite, evaluation of drug efficacy and control programs and better patient management (Sturrock 2001).
Currently, Kato–Katz is the method recommended for diagnosis of S. mansoni infection by WHO for its high specificity, handiness and cost effectiveness (WHO 2002). However, the Kato–Katz technique has limited sensitivity in situations where light intensity infection is common (Berhe et al. 2004), and the procedure is also relatively time consuming, requiring well-trained personnel and heavy equipment (Speich et al. 2010; Shane et al. 2011). Although its sensitivity can be improved by increasing the number of stool samples tested, getting stool specimens from individuals on different days would be challenging (Barreto et al. 1990). In addition, Kato–Katz method exposes laboratory workers to potentially harmful fresh stools which can contain infectious agents (Shane et al. 2011).
The concern that conventional Kato–Katz method misses Schistosoma mansoni infection in a proportion of young people in areas of low prevalence and intensity (Allam et al. 2009) has a very important implication, particularly when it is used in monitoring the impact of preventive chemotherapy. The few missed light infections constitute a potential source of continued schistosomiasis transmission.
The single urine-CCA cassette test is more sensitive than Kato–Katz thick smear (Standley et al. 2010; Coulibaly et al. 2011; Shane et al. 2011; Tchuem Tchuenté et al. 2012), but some investigators reported that urine-CCA cassette test has several limitations such as low sensitivity in cases of light intensity of infection (Stothard et al. 2006). Furthermore, high genetic variability of S. mansoni and its potential impact on cathodic antigens might contribute to differences in test diagnostic performance (Stothard et al. 2009). Hence, it is crucial to evaluate commercially available urine-CCA tests in different endemic regions (Stothard 2009) and at levels of endemicity.
The Schistosomiasis Consortium for Operational Research and Evaluation (SCORE) funded evaluation of the performance of point-of-care circulating cathodic antigen (POC/CCA) cassette test among school children in five African countries including Ethiopia (http://score.uga.edu/). This multi-country study has recommended the POC-urine-CCA cassette test for community-level prevalence mapping of S. mansoni infection (Colley et al.2013). As a part of this multicountry study, we evaluated the diagnostic performance and accuracy of the urine-CCA cassette test for the diagnosis of S. mansoni infection among school children in two areas of moderate prevalence in Ethiopia.
A cross-sectional study involving 320 school children from Jiga and 300 from Harbu Elementary Schools (ages ranged between 8 and 12) was conducted between 2010 and 2011. Jiga is a town located 375 km north-west of Addis Ababa on the road to Bahir Dar city. Jiga has been known to be endemic for Schistosomiasis mansoni, and recent survey showed that the prevalence of the disease was 34% (Teklemariam 2007). Harbu Town is located some 355 km to the north-east of Addis Ababa and is also known to be endemic for Schistosomiasis mansoni (Erko et al. 2002). The specificity of the test was evaluated in school children from Gedo Elementary School in western Ethiopia, an area where human schistosomiasis is not endemic.
Stool samples of about 2 g were collected from each child for three consecutive days. The specimens were processed using a 41.7 mg Kato–Katz template (WHO 1991), preparing duplicate slides per stool sample. The slides were quantitatively examined for intestinal schistosomiasis independently by two experienced laboratory technicians. The third technician checked the reading of the two technicians for discrepancy, and in case of discrepancies, the slides were re-examined. The results were recorded as eggs count per slide and multiplied by 24 to convert them into eggs per gram (epg) of stool before analysis.
Midstream urine specimens were collected for three consecutive days from the same children who provided stool specimens. The urine POC/CCA (point-of-care circulating cathodic antigen) cassette test was performed according to the protocol and procedures described by the manufacturer (http://www.rapid-diagnostics.com/downloads/RMD%20Pamphlet%202011_06_13%20.pdf).
The study was approved by the Institutional Review Board (IRB) of Aklilu Lemma Institute of Pathobiology, Addis Ababa University. Permission to conduct the study was also obtained from local government administration and school directors. The children were included in the study after obtaining informed consent from their parents/guardians. The objective of the study was also clearly explained to the children, and their assent was sought. Children found positive for intestinal schistosomiasis, and soil-transmitted helminthiasis were treated with a single dose of PZQ (40 mg/kg body weight) and a single dose of albendazole (400 mg) after the third day of specimen collection and examination.
Data were analysed using STATA (version 10). As there has been no perfect diagnostic technique for S. mansoni infection, we considered combined results of six Kato–Katz and triple urine-CCA cassette tests as a ‘gold standard’ in determining the sensitivities, specificities and negative predictive value (NPV) and accuracy of both urine-CCA cassette test and Kato–Katz thick smear. Accordingly, results which were positive in six Kato–Katz and/or triple urine-CCA cassette were considered as ‘true positive’. Results based on the six Kato–Katz method alone were also used as ‘gold’ standard to evaluate the performance of CCA cassette. Agreement of both Kato–Katz and urine-CCA cassette test with the gold standard was evaluated using Kappa statistics. Results of the urine-CCA cassette test as a categorical variable (weak and strong intensity of the urine-CCA cassette test band colour) and egg counts as a continuous explanatory variable were used in a logistic regression model to evaluate the association of S. mansoni intensity based on egg count and the intensity of the urine-CCA cassette test band colour. The urine-CCA cassette test band colour with very weak intensity results was considered as negative during analysis. McNemar's chi-squared test was used to evaluate differences in the prevalence of S. mansoni infection based on six Kato–Katz and one or triple urine-CCA cassette test. The arithmetic mean egg count was calculated as the average egg counts (epg) of six Kato–Katz thick smears, and classes of intensity of S. mansoni were determined as light (1–99 EPG), moderate (100–399 EPG) and heavy (≥400 EPG). Ninety-five percentage of confidence interval (CI) was estimated for each diagnostic performance parameter. Values were considered significant when P < 0.05.
Prevalence of S. mansoni infection as determined by Kato–Katz method and urine-CCA cassette test is summarised in Tables 1 and 2. A total of 620 school children (mean age in years ± SD = 9.9 ± 1.3 females = 306, males = 314) were examined for S. mansoni infection, of whom 73.4% tested positive based on combined six Kato–Katz method and three urine-CCA cassette tests. Prevalence of S. mansoni infection was significantly higher as diagnosed using six Kato–Katz thick smears than by double or single Kato–Katz thick smears (P < 0.001). Similarly, there was an increase in the prevalence of S. mansoni infection as diagnosed using three urine-CCA cassette tests compared to one urine-CCA cassette test. When trace positive results were included in the analysis, the prevalence of S. mansoni infection was 70.8% based on single urine-CCA cassette test and 81.1% based on triple urine-CCA cassette tests.
|Method||Number positive||Per cent positive|
|Number examined||Number positive (%) based on six Kato–Katz||Number positive (%) based on three CCA||P value||Number positive (%) based on combined six Kato–Katz and three CCA|
|5–9||237||124 (52.3)||166 (70.0)||P < 0.001||173 (73.0)|
|10–12||383||205 (53.5)||273 (71.2)||P < 0.001||282 (73.6)|
|Male||314||179 (58.9)||235 (77.3)||P < 0.001||239 (78.6)|
|Female||306||150 (47.5)||204 (64.6)||P < 0.001||216 (68.4)|
|P a||0.004||P < 0.00|
|Jiga||320||158 (49.4)||200 (62.5)||P < 0.001||205 (64.1)|
|Harbu||300||171 (57.0)||239 (79.7)||P < 0.001||250 (83.3)|
|Total||620||329 (53.1)||439 (70.8)||P < 0.001||455 (73.4)|
|P a||0.057||P < 0.001|
Of 329 children found positive for S. mansoni [mean egg per gram (epg) = 110.7 ± 187.8] using six Kato–Katz thick smears, 229 (69.6%), 80 (24.6%) and 18 (5.8%) showed light, moderate and heavy classes of intensity, respectively. Of 439 children found positive for S. mansoni using triple urine-CCA cassette test, 360 showed strong intensity of the urine-CCA cassette test band colour, while 79 showed weak intensity. In all children who had moderate and heavy intensity of infection, the urine-CCA cassette test band colour was intense. The odds of strong intensity of the urine-CCA cassette test band colour increased with an increase in 1 epg (OR = 1.07, 95% CI = 1.04, 1.10). There was also a significant increase in the prevalence of S. mansoni infection as determined using single or triple urine-CCA cassette tests with an increase in 1 epg (OR = 1.06, 95% CI = 1.04, 1.08). Prevalence of S. mansoni infection based on single urine-CCA cassette test was 90.8% (206/227), 97.5% (78/80) and 100% (18/18) among children who had light, moderate and heavy intensity of infection, respectively based on six Kato–Katz thick smear. Among children who had light (227), moderate (80) and heavy (18) S. mansoni infections based on estimates from six Kato–Katz thick smears, 93.4%, 98.8% and 100% were positive for the parasite based on triple urine-CCA cassette tests, respectively.
Results of stool examinations of school children in Gedo town showed that the prevalence of any soil-transmitted helminthiasis (ascariasis and trichuriasis only) was 50%. Of 100 urine specimens tested using urine-CCA cassette test, only one child tested positive (1%) for S. mansoni, the intensity of urine-CCA cassette colour band being weak. The specificity of the urine-CCA cassette test based on this result was 99%.
Information about performance of both the Kato–Katz method and urine-CCA cassette test in diagnosing S. mansoni infection using combined six Kato–Katz and three urine-CCA cassette test as a ‘gold standard’ is summarised in Tables 3. Six Kato–Katz thick smears showed higher sensitivity, NPV and accuracy compared to double and single Kato–Katz methods. The three urine-CCA cassette tests showed higher sensitivity, negative predictive value and accuracy for detecting S. mansoni infection than single urine-CCA cassette test, and the single urine-CCA cassette test was better in its performance than the six Kato–Katz thick smears. Both Kato–Katz (based on single, double or six slides) and urine-CCA cassette test (single or triple) showed 100% specificity for diagnosing S. mansoni infection. The agreement between Kato–Katz/urine-CCA cassette test and the ‘gold standard’ for detecting S. mansoni infection was higher in the case of six Kato–Katz than double or single Kato–Katz and three urine-CCA cassette tests than single urine-CCA cassette test. Single urine-CCA cassette test (weak and strong positive) agreed much more than six Kato–Katz with the ‘gold standard’ in diagnosing S. mansoni infection. Of 291 children tested negative for S. mansoni egg by six Kato–Katz method, 126 were found positive for S. mansoni antigen by urine-CCA cassette test (95 showed strong band colour and 31 showed weak band colour intensity). On the other hand, only 16 children (of 181) who tested negative by triple urine-CCA cassette test were diagnosed as positive by six Kato–Katz method.
|Methods||Six Kato–Katz as gold standard||Combined six Kato–Katz and three urine-CCA cassette test as gold standard|
|Sensitivity (95% CI)||70.2 (65.3, 75.1)||50.8 (46.2, 55.3)|
|Specificity (95% CI)||100||100|
|NPV(95% CI)||74.8 (70.4, 79.1)||42.4 (37.5, 47.3)|
|Accuracy (95% CI)||84.2 (81.1, 87.3)||63.9 (60.1, 67.7)|
|Kappa (95% CI)||0.69 (P < 0.001) (0.65, 0.70)||0.35 (0.32, 0.36)|
|Sensitivity (95% CI)||81.2 (76.9, 85.4)||58.7 (54.2, 63.2)|
|Specificity (95% CI)||100||100|
|NPV (95% CI)||82.4 (78.4, 86.3)||46.7 (40.5, 52.8)|
|Accuracy (95% CI)||90 (87.6, 92.3)||69.7 (66.1, 73.3)|
|Kappa (95% CI)||0.80 (0.77, 0.81)||0.43 (0.40, 0.44)|
|Sensitivity (95% CI)||N/A||N/A||72.3 (68.2, 76.4)|
|Specificity (95% CI)||N/A||N/A||100 (0.973, 1.00)|
|NPV (95% CI)||N/A||N/A||56.7 (51.0, 62.4)|
|Accuracy (95% CI)||N/A||N/A||79.7 (76.5, 82.9)|
|Kappa (95% CI)||N/A||N/A||0.58 (0.55, 0.59)|
|Sensitivity (95% CI)||93.0 (90.2, 95.7)||89.9 (87.1, 92.6)|
|Specificity (95% CI)||64.6 (59.1, 70.1)||100|
|NPV (95% CI)||89.1 (84.9, 93.3)||78.2 (72.6, 83.7)|
|Accuracy (95% CI)||79.7 (76.5, 82.9)||92.6 (90.5, 94.6)|
|Kappa (95% CI)||0.59 (0.52, 0.63)||0.83 (0.79, 0.84)|
|Sensitivity (95% CI)||95.1 (92.8, 97.4)||96.5 (94.8, 98.1)|
|Specificity (95% CI)||56.7 (51.0, 62.4)||100 (0.96, 1.00)|
|NPV (95% CI)||91.2 (87.1, 95.3)||91.2 (87.1, 95.3)|
|Accuracy (95% CI)||77.1 (73.8, 80.4)||97.4 (96.1, 98.7)|
|Kappa (95% CI)||0.53 (0.47, 0.57)||0.94 (0.89, 0.95)|
|Variable||Six Kato–Katz||Triplicate urine-CCA cassette test|
|Sensitivity (95% CI)||Specificity (95% CI)||NPV (95% CI)||Sensitivity (95% CI)||Specificity (95% CI)||NPV (95% CI)|
|5–9||71.7 (64.9, 78.4)||100||56.4 (47.6, 65.7)||95.9 (92.9, 98.8)||100||90.1 (83.1, 97.0)|
|10–12||72.7 (67.5, 77.8)||100||56.7 (49.4, 63.9)||96.8 (9, 4.7, 98.8)||100||91.8 (86.7, 96.9)|
|Female||69.4 (63.2, 75.5)||100||60.2 (52.7, 67.6)||94.4 (91.3, 97.4)||100||89.3 (83.6, 95.0)|
|Male||74.9 (69.4, 80.3)||100||52.0 (43.2, 60.7)||98.3 (96.7, 99.9)||94.2 (88.7, 99.7)|
|Jiga||77.1 (71.3, 82.8)||100||70.9 (63.9, 77.8)||97.6 (95.5, 99.6)||100||95.8 (92.2, 99.3)|
|Harbu||68.4 (62.6, 74.1)||100||38.7 (30.3, 47.1)||95.6 (93.0, 98.1)||100||81.9 (72.2, 91.5)|
This study was conducted to evaluate the performance and accuracy of urine-CCA cassette test for diagnosis of Schistosoma mansoni infection in relation to combined results of six Kato–Katz thick smears and three urine-CCA cassette tests as a ‘gold standard’. The prevalence of S. mansoni infection as determined by single or triple urine-CCA cassette test was greater than the prevalence determined by single and six Kato–Katz thick smears. According to WHO (2002), the outlined treatment strategies to guide preventive chemotherapy of schistosomiasis is based on prevalence of the diseases. In accordance with these strategies, the urine-CCA cassette test seems to be more appropriate candidate tool for screening S. mansoni infection for preventive chemotherapy in endemic communities. The observation of lower prevalence of S. mansoni infection among the study participants when determined even by six Kato–Katz compared to single urine-CCA test could be attributed to variations in the number of eggs released in stool (Berhe et al. 2004). Had the number of stool samples examined in the present study increased to 9, as in the study carried out by Coulibaly et al. (2011), comparable results would have been obtained for Kato–Katz and urine-CCA cassette test. In the present study, single urine-CCA cassette test also showed greater sensitivity, negative predictive value and accuracy in detecting S. mansoni infection than six, double or single Kato–Katz thick smears. Sensitivity, NPV and accuracy of both the Kato method and urine-CCA cassette rose with an increase in the number of samples tested.
Previous studies also reported better performance of single urine-CCA cassette test for diagnosis of S. mansoni infection compared to single or more Kato–Katz thick smears (Standley et al. 2010; Coulibaly et al. 2011; Shane et al. 2011; Stothard et al. 2011; Tchuem Tchuenté et al. 2012; Colley et al.2013). These previous studies have also indicated the possible use of urine-CCA cassette test for diagnosis and mapping of S. mansoni infection. Findings from the present study can also corroborate the previous recommendation that the urine-CCA cassette test (batch 32727) can be used for the mapping of schistosomiasis and guiding preventive chemotherapy.
Both Kato–Katz method and urine-CCA cassette test are claimed to be insensitive to assess low intensity of S. mansoni infection (Berhe et al. 2004; Stothard et al. 2006). The present study corroborates this previous claim to some degree. Of 291 children found negative for S. mansoni egg by six Kato–Katz thick smears, 126 tested positive for S. mansoni antigen by urine-CCA cassette test, of whom 95 showed strong band colour and 31 showed weak band colour intensity. On the other hand, only 16 children (of 181) who tested negative by triple urine-CCA cassette test were diagnosed as positive by six Kato–Katz thick smears. From among 16 children who tested positive for S. mansoni egg but negative for S. mansoni antigen, 15 had light infection intensity, and only one child had moderate infection intensity of S. mansoni. Hence, it is preferable to use a combination of both methods in areas of low S. mansoni infection prevalence.
Failure to detect eggs in stool by Kato method while children tested positive for S. mansoni by urine-CCA cassette test could be due to the reduction in the number of egg-laying females releasing eggs below the detection level by Kato–Katz method (Berhe et al. 2004) or the presence of light, single sex or pre-patent infections (Mutapi et al. 2011). Detection of S. mansoni by urine-CCA cassette test could also be false positive results that may arise due to non-specific cross reaction of the urine-CCA test with Lewis-X tri-saccharide epitopes of inflammatory biomarkers (van Dam et al. 1996). Nevertheless, the present study showed that of 100 urine specimens tested using urine-CCA cassette test in an area non-endemic for schistosomiasis, only one child tested positive for S. mansoni, giving the specificity of 99%. On the other hand, the reasons why urine-CCA cassette test revealed negative results when eggs are present in stool as confirmed by the Kato–Katz method is not clear.
The urine-CCA cassette test band colour was intense in all children who had moderate and heavy intensity of infection suggesting strong colour reaction in heavy intensity of S. mansoni infection. Previous studies also reported increased positive rate and band colour strength of urine-CCA cassette test with an increase in egg counts (Stothard et al. 2006; Legesse & Erko 2008; Standley et al. 2010; Coulibaly et al. 2011; Shane et al. 2011; Tchuem Tchuenté et al. 2012). This suggests that the urine-CCA cassette test could be used as an alternative to indicate the intensity of S. mansoni infection. However, the urine-CCA cassette test needs to be formulated for semi-quantitative detection of S. mansoni infection with limits representing classes of intensity.
The characteristic that circulating cathodic antigens (CCAs) are released by viable adult worms that are absent after treatment and the resolution of infection (van Lieshout et al. 1993; Agnew et al. 1995) would make the urine-CCA cassette test suitable for the evaluation of drug efficacy trials. However, the effectiveness of the urine-CCA cassette test needs to be evaluated after drug treatment before using it for large scale follow-up studies.
In conclusion, the urine-CCA cassette test showed much better sensitivity, NPV and accuracy compared to Kato–Katz in diagnosing S. mansoni infection in moderate and heavy intensity of infection. The test could be used for screening of S. mansoni infection in moderate and high prevalence area to guide preventive chemotherapy if its present cost of about 1.98 USD/casette test were substantially reduced. Evaluation of the performance of the test in areas of low prevalence and intensity of infection is indicated.
The study was funded by the Schistosomiasis Consortium for Operational Research and Evaluation (SCORE). We gratefully acknowledge the technical assistance by the technical staff of Medical Parasitology Research Unit of Aklilu Lemma Institute of Pathobiology. We are also grateful to district education and health bureaus, school directors and teachers, parents/guardians and school children for their cooperation.