Validity of urine-CCA cassette test and indirect haem-agglutination assay (IHA) in the detection of schistosomiasis-mansoni infection relative to microscopic examination

Abstract

Several immunodiagnostic assays have been commercially presented over the last years as easy diagnostic methods for schistosomiasis using serum or urine samples. The performance of immunochromatographic test (ICT) and indirect hemagglutination assay (IHA) was validated in the identification of active schistosomiasis infection. Detection of circulating cathodic antigen (CCA) of the parasite in urine samples and anti-Schistosoma antibodies in serum using ICT (Urine-CCA Cassette test) and IHA respectively. Proved diagnosis of Schistosoma mansoni infection was defined by the sum of positive results from microscopic examination (Gold standard) and Kato–Katz method. Out of 173 (mean age, 45 ± 10 years; 70 from Giza, 103 from different Egyptian governorates), 9 4 adult patients were infected. Urine-CCA cassette test despite showing high specificity (91.14%) it was of low sensitivity (23.40%). PPVs was 75.86% and NPV was 50.00% and diagnostic accuracy of 54.34%. The IHA showed a sensitivity of 57.45% and specificity of 48.10%. PPVs was 56.84% whereas NPVs was 48.72%. As for diagnostic accuracy, it was 53.18%. Urine-CCA Cassette test had lower sensitivity than expected for detection of circulating antigen and the IHA kit is generally more expensive than microscopic examination and Urine-CCA cassette test with low sensitivity and specificity. On the basis of this diagnostic performance none of the two tested immune-assays can be a sole tool in the principal diagnosis of active schistosomiasis infections.

Introduction

Schistosomiasis is one of the major parasitic diseases in the world following malaria, affects more than 250 million people worldwide and caused by blood-dwelling flukes (Engels et al. 1996; Asundi et al. 2019). Humans have been involved in the life cycle of five species of the Schistosomes, but 90% of all infections are produced by just three: S. mansoni (mostly in Africa and South America) and S. japonicum (predominantly in China, Indonesia, and the Philippines) produce intestinal schistosmiasis, while S. haematobium (in Africa and Middle East) causes urinary disease (Alemayehu et al. 2017; Mutapi et al. 2017; Tchuenté et al. 2017; WHO 2017).

In the definitive host (humans and other mammals), the parasite is distinguished into males and females and reproduces sexually where huge amounts of eggs are produced by adult females, that almost causes all symptoms. Asexual reproduction occurs in the intermediate host which is species-specific freshwater snails depending on the species of the Schistosomes (Nelwan 2019). Infection in humans is caused by cercariae released from the snails that reach the venous circulation through skin penetration. Young schistosomulum is the most vulnerable to immune damage. Retaining certain immune evasion mechanisms, the parasite becomes unrecognisable to the host immune defences. Accordingly, adult worms may survive for several years in the definitive host, even up to 40 years (Angeles et al. 2020).

Egg release starts approximately after six weeks post-infection in the capillaries of the supplying venous plexus to penetrate the intestinal wall in S. mansoni and S. japonicum or the bladder wall in S. haematobium to be excreted. Substantial amounts of eggs are not excreted but retain in the tissue triggering granuloma formation with consequent complications to the affected organs (Ross and Yuesheng 2017).

Cautious review of residence and travel history is critical for defining whether infection is possible and which species may be producing infection (Bottieau et al. 2006). It is of significant importance to remember that both S. mansoni and S. haematobium are prevalent in some regions in sub-Saharan Africa where patients with freshwater exposures should have both stool and urine examinations for eggs. However, testing of stool or urine specimens can be of constrained sensitivity, predominantly for travellers who may have lighter intensities of infection. To upgrade sensitivity of stool and urine microscopic examination, at least three successive samples are required on different days. For S. mansoni, presence of dysentery can suggest active infection but this test is more convenient for population studies in Africa but not adequately sensitive or specific for distinct patient diagnosis. Moreover, in light-intensity infections eggs are shed in low amounts and intermittently (Doenhoff et al. 1986).

In regards to previous research papers available rapid assays may involve reagent urine strip for the assessing of blood in the urine that seems to be beneficial in schistosomiasis haematobium-infected patients being simple and had shown to correlate with the parasitological analysis. In 2013, Adams and Wiredu deduced that in the precise clinical settings, the prevalence of eosinophils in the urinary sediments can be diagnostic of urinary schistosomiasis even if ova are not realised. However, they are not applicable for the detection of intestinal schistosomiasis infections. High eosinophilia has been shown to be due to concomitant parasitic infections in immigrants meanwhile in travellers it is due to acute schistosomiasis. However, Bierman et al. (2005) demonstrated that eosinophilia despite being indicative it is not a sufficient screening tool for the schistosomiasis infection.

Several immunodiagnostic tests based on mono-clonal antibodies (M-Abs) for the recognition of bilharzia antigens in the urine and serum of infected patients have been defined. Deelder et al. (1989, 1994) and De Jonge et al. (1988, 1989, 1990, 1991) used a murine M-Ab counter to the Circulating Cathodic Antigen (CCA) or the M antigen in numerous enzyme immunoassays (Van Ettenet al. 1994). However, the reliability of CCA detection test for schistosomiasis diagnosis in endemic areas remains a questionable point (Peralta and Cavalcanti 2018).

Serologic testing for anti-schistosomal antibody is specified for diagnosis of immigrants or travellers coming from endemic areas who have not been received appropriate anti-Schistosoma therapy in the past. Generally available serologic tests detect antibody production to the adult worm. For recent infections, serum samples tested should be obtained at least six to eight weeks after expected infections, to permit for full development and growth of the worms and antibody production to the adult stage (Maddison 1987; Noya et al. 1995; Certon et al. 1996).

Diagnosis of schistosomiasis infections in human is very essential to case-specific management and successful control programs, maintenance of research for progressing new diagnostic measures or authentication of a developed procedure is very critical. Hence, the purpose of the current study was to evaluate the diagnostic efficacy of a commercially available Urine-CCA cassette test and indirect hemagglutination assay (IHA) for the detection of schistosomiasis mansoni relative to microscopic methods.

Material and methods

The current study was evaluated and approved by Committee of Ethics, Kasralainy School of Medicine, Cairo University, Egypt after perceiving agreement from the local health authorities and consents of all contributors.

Study population and ethical consideration

This study included 173 subjects from endemic areas attended the Parasitology Department; Cairo University Hospitals, Egypt, which includes a large Out-Patient Laboratory Unit and procedures more than 27,000 stool, urine, serum samples per year for tropical parasites. The study was done during the period (January 2018–July 2020). The study subjects were haphazardly (randomly) selected irrespective of their age-group and both male and females were included. All patients included in the study were symptomatically positive for dysentery and tenesmus ± iron deficiency anaemia; in addition to a present habitat history in endemic areas. Microscopic examination, anti-schistosomal serological testing, and Urine-CCA detection were performed throughout the study period. The purpose of samples gathering was explained for all the studied population and their consents were attained.

Study design

The present research is a cross-sectional study intended to evaluate the diagnostic efficiency of the commercial “Urine Circulating Cathodic Antigen (CCA) cassette test” and indirect hemagglutination (IHA) test for the presumptive detection of an active Schistosomiasis-mansoni infection relative to microscopic investigation technique as the gold standard. We excluded patients treated with praziquantel in the last 30 days of data collection as well as patients who were seriously ill. To avoid cross-reactivity (El Saftawy et al. 2019), direct microscopy of all stool samples was performed for parasitological examination. Patients solely infected with schistosomiasis mansoni were involved in the present study.

Stool collection

Three to four consecutive stool specimens from each patient were collected in clean stool cups. Collection of samples was sometimes made easier for the study subjects using the delivery service on the account of the study. The stool samples were then transported to the Medical Parasitology lab. All specimens were screened for Schistosoma mansoni eggs (diagnostic stage) by both direct and formalin-ether concentration by sedimentation methods.

Kato–Katz technique

In the current study, to increase efficacy of microscopic examination (the gold standard) it was combined with Kato–Katz technique for all suspected individuals (Santos et al. 2005). Kato–Katz technique is recommended as a special method of concentration and copro-parasitological detection technique for helminthic infections in reference records (Kato and Miura 1954; Katz et al. 1972; Feldmeier and Poggensee, 1993; WHO 1993; Calvopina et al. 2018) and we considered the sum of all positive results as proven infection. Faeces was pressed through a mesh and an amount of filtered stool (20 ± 50 mg restrained by a template) is relocated and spread on a glass slide. Then slides were allowed to dry in room temperature. A piece of glycerine-soaked cellophane is compelled on the stool specimen; the glycerine clears stool debris, increasing the realisation of ova (Peters et al. 1980).

The urine-CCA cassette test

Patients brought mid-stream urine samples in clean cups, labelled with the needed information and issued to the participating individuals whose informed consent was sought earlier. This was carried out between 10.00 am and 2.00 pm when the ova excretion of S. haematobium is expected to be at its peak as the study included only schistosomaisis mansoni infections from endemic areas. The test is based on immunochromatography and full steps are defined in Fig. 1. Invalid test without C- or T-line, or with only T-line were excluded out and repeated. Haematuria or pyuria were excluded to avoid false positive results according to the manufacturer’s instructions.

Fig. 1.

Steps of the Urine-CCA cassette test. Four drops of the suspension are added in the appropriate area on the cartridge. The device test is incubated at room temperature for 20 min. Appearance of only control (C)-line indicates negative test. Interpretation of positive test based on the presence of C-line and a pink band in test (T) line

Microbiological examination

Stool samples were held on Remel Cary Blair transport medium and transported to the Bacteriology Unit, Clinical Pathology Department, Kasralainy School of Medicine. Bacterial analyses were performed using standard culture procedures to exclude Salmonella spp., Shigella spp., Aeromonas spp., Campylobacter jejuni, and Yersinia enterocolitica (Old and Duguid 1970). Endemic viral infections such as Rota-virus and Adeno-virus, were ruled out using immunochromatography technique (ICT) rapid test for the dual detection of rota-virus and adeno-virus in a single stool test in watery and loose specimens.

Urine cultures in CLED Agar/MacConkey-II Agar bi-plate for isolating and enumerating bacteria in urine and differentiating gram-negative rods in the collected urinary specimens were performed to avoid cross-reactivity in consistent with the manufacturer’s instructions of Urine-CCA test.

Serology assay

Serum was separated by centrifugation of 1 ml of blood sample and collected for performing Indirect haem-agglutination test (IHA) using the commercially available Fumouze Diagnostics-France on 94-positive S.mansoni samples and 79-negative samples for participants matching the test samples in age and gender. In accordance with the manufacturer’s instructions reagents of Fumouze IHA kit and samples were allowed to be at room temperature (22–27 °C) prior to testing procedure. The full steps are described in Fig. 2. Titre less than (1:160) is not significant of an infection and may correspond with a former or an eventually treated infection, while titre ≥ 1:160 is a significant reaction.

Fig. 2.

Steps of IHA test. a 1.95 ml of buffer solution plus 0.05 ml of test serum are delivered in a disposable tube to obtain 1/40 stock dilution of test serum. b By means of a micro pipettor, buffer solution (50 μl) was delivered into 7 successive wells. c 50 μl of serum-stock-dilution was delivered in the first well, mixed well with the phosphate buffer saline (PBS) buffer and transferred to the 2nd well then to the third and so on until the end of the 6 wells (titer1/2560) and finally 50 μl was discarded from the sixth well. d Microplate reaction: opened ring is positive while closed ring is negative

Statistical methods

Data were coded and entered using the statistical package for the Social Sciences (SPSS) version 26 (IBM Corp., Armonk, NY, USA). Data was summarized using mean, standard deviation, maximum minimum, and median in quantitative data and using frequency (count) and relative frequency (percentage) for categorical data. For comparing categorical data, Chi square (χ²) test was performed. Exact test was used instead when the expected frequency is less than 5 (Chan 2003). Standard diagnostic indices including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic efficacy were calculated as described by (Galen 1980). p values less than 0.05 were considered as statistically significant.

Result

We included 173 patients with a mean age of 45 years (SD ± 10) years. Fifty-one percent were male, 70 patients were from Giza and 103 from different Egyptian governorates. All suspected individuals were confirmed for schistosomiasis mansoni infection by direct egg detection using microscopic/Kato–Katz technique (Fig. 3). Results of microscopic examination and Kato–Katz technique despite being of insignificant differences; it was beneficial to clarify and confirm diagnostic stages in several cases.

Fig. 3.

Microscopic examination. a S. mansoni eggs [115–180 µm (length) × 50–70 µm (width)], each contains a mature miracidium (M) and is characterized with a prominent lateral spine (S) nearby the posterior end and the anterior end is slightly curved and tapered. b Schistosome eggs were occasionally difficult to be recognized within the faecal debris but were more obvious and confirmed in Kato–Katz test preparations with-in 24 h as it is in c

We excluded and repeated three patients with CCA-cassette tests classified as invalid. Parasitological diagnosis in a total of 173 individuals revealed S. mansoni infection in 94 patients (54.3%). All the infected subjects were symptomatic, almost with dysentery and tenesmus. A total of 79 individuals (45.7%) were parasitologically and microbiologically diagnosed as being non-infected for other intestinal pathogens and age and gender matched to the schistosomiasis-infected patients.

The number of patients positive by each assay is shown in Table 1. Of the 173 individual sera evaluated by the Urine-CCA Test, 22 (23.4%) were positive for S. mansoni antigens and for anti-Schistosoma antibody test by the IHA assay, 54 (57.4%) were positive. There was significant difference between positive and negative results for the infected patients using Urine-CCA Test (p value 0.011). Conversely, in concordance with the IHA difference between positive and negative results was insignificant.

Table 1.

Accuracy of S. mansoni infection using Urine-CCA Test and Indirect hemagglutination (IHA) compared to microscopic examination (gold-standard method) plus Kato–Katz technique

	Microscopic gold standard plus Kato–Katz technique				p value
	Positive		Negative
	Count	%	Count	%
Urine-CCA test
Positive	22	23.4	7	8.9	0.011
Negative	72	76.6	72	91.1
Indirect hemagglutination (IHA)
Positive	54	57.4	41	51.9	0.465
Negative	40	42.6	38	48.1

Performance of different assays for the diagnosis of schistosomiasis infection is detailed in Table 2 which showed the results of schistosomiasis incidence detected by different diagnostics approaches. The 95% confidence intervals, 95% CI are shown. The specificity of active schistosomiasis detection was high in Urine-CCA Test, reaching 91.14% in diagnostic setting. For the overall two immune-assays, the sensitivity ranged from 23.40% with Urine-CCA Test to 57.45% with IHA test. PPV varied between study tested assays, with a higher PPV value of 75.86 (95% CI, 58.64% to 87.45%) for Urine-CCA test and a lower value of 56.84% (95% CI, 50.02% to 63.41%) for IHA. The NPV was 50.00% for Urine-CCA Test and 48.72% for IHA test.

Table 2.

Performance of Urine-CCA antigen and Indirect hemagglutination (IHA) tests for the diagnosis of active schistosomiasis mansoni infection

Assay	Statistics	Sensitivity (%)	Specificity (%)	Positive likelihood ratio	Negative likelihood ratio	Positive predictive value(%)	Negative predictive value (%)	Accuracy (%)
Urine-CCA test	Value	23.40	91.14	2.64	0.84	75.86	50.00	54.34
	95% CI	15.29–33.26	82.59–96.36	1.19–5.86	0.74–0.96	58.64–87.45	46.72–53.28	46.60–61.92
Indirect hemagglutination (IHA)	Value	57.45	48.10	1.11	0.88	56.84	48.72	53.18
	95% CI	46.82–67.59	36.71–59.64	0.84–1.46	0.64–1.23	50.02–63.41	40.63–56.88	45.46–60.79

Discussion

Qualitative detection of the Schistosoma antigen, Circulating Cathodic Antigen (CCA) in urine is a non-invasive test for diagnosis of bilharziasis. Schistosomes have blinded gastro-intestinal tracts (a cul-de-sac) and the parasites have to regurgitate at consistent intervals the undigested elements as well as the parasitic gut related glycoproteins. One of the chief regurgitated antigens by the parasites are the CCA. Although miracidium in Bilharzia eggs also produce CCA antigen it is in miniature quantities. The foremost source of CCA originates from the living adult helminths (Van Dam et al. 1996).

In the present study, the Urine-CCA Test developed on the basis of an IgG2a MAb for the detection of circulating Schistosoma antigen excreted in urine. The procedure in spite of being rapid of total 20 min, simple, and highly specific (91.14%), it was of low sensitivity (23.40%) compared with microscopic examination. Validity of the assay could therefore be questionable in the field of mass screening programs.

Van Etten et al. (1997), Standley et al. (2010) and Beltrame et al. (2017) deduced similar overall high diagnostic specificity and negative predictive value of the CCA detection in urine, in contrary to our results the same authors deduced higher sensitivities. Van Etten et al. (1997) postulated their results post-treatment to assess therapeutic efficacy, Standley et al. (2010) interpreted traces as positive readings, while Beltrame et al. (2017) study subjects were almost asylum seekers or African migrants from highly endemic areas.

Coulibaly et al. (2011) deduced the low sensitivity of CCA that precludes its use for the diagnosis S. mansoni infections. Casacuberta et al. (2016) recommended optimization of Urine-CCA cassette test by assessing using computer software to quantify the colour intensity of the Test lines in the strip of the ICT.

Several authors speculated that efficacy of CCA detection test may be indeterminate in areas of multiple Schistosomiasis infections or S. haematobium mono-infection (Obeng et al. 2008; Ashton et al. 2011; Sanneh et al. 2017). In contrary, Van Etten et al. (1997) presented the assay as being applicable for the evaluation of treatment in schistosomiasis patients. Beltrame et al. (2017) voted ICT for the screening of human bilharziasis in refugees and immigrants from African countries. Moreover, Adriko et al. (2014) demonstrated CCA test to replace the Kato–Katz sampling within the control programs.

This study showed low sensitivity of IHA despite being almost more expensive if compared with microscopic examination for schistosomiasis infection, similar findings were deduced by Yameny (2019). Also, the current study reported the low specificity of IHA, Jones et al. (1992) reported that immunodiagnostic methods almost detect IgE, IgM, or IgG against soluble antigens of the adult worm or crude antigens of eggs, however, seroconversion mostly occurs within 4–8 weeks post-infection and in some cases seronegative interval or window may extend up to 22 weeks.

However, CCA detection in urine for S. mansoni exhibited contradictory results depending on the studied area. Shiff (2015), Clements et al. (2018), Grenfell et al. (2018) and Oliveira et al. (2018) deduced that in low endemic areas where no ova-excretion or low infection intensity predominates, Urine-CCA test was unsatisfactory as a solitary diagnostic tool for active infections. Standley et al. (2010) and Bezerra et al. (2018) indicated that in low endemic settings, the Urine-CCA test revealed a significant increase in positive cases compared with Kato–Katz technique, when trace results were assumed as positive, however, the exact significance of trace results remains not clear. The same authors demonstrated the need to associate different tools to reinforce the accuracy of schistosomiasis detection in patients with low parasite loads. Interestingly, Fuss et al. (2018) postulated that sensitivity of Urine-CCA cassette test is higher than Kato–Katz method in high and moderate prevalence areas.

Patient schistosome infection is highly immunogenic, and anti-schistosome antibodies can be readily detected using a wide range of immunodiagnostic techniques (Doenhoff et al. 2004). Queiroz et al. (2013) reported that antibody titter increases 15-days post-infection in murine models. Serological testing may not be precise for defining an active infection as antibody titre may remain increased in the first 6 to 12 months or a fail to reduce even after 3 years that don’t inevitably justify re-treatment (Rabello et al. 1997; Mott and Dixon 1998; Yong et al. 2010). They can also cross-react with other helminths and are not easily applicable under field conditions (Rabello 1997). However, such assays are important, for diagnosis in travellers, migrants, and other occasionally exposed people (Bottieau et al. 2006). In this accordance van Gool et al. (2002) necessitated the combination of IHA Test with other diagnostic tests to prove infection with S. mansoni.

We conclude that the novel monoclonal Urine-CCA cassette test (ICT) is less expensive than other laboratorial methods and accepted by the patients, however, its diagnostic performance seems not to recommend its use solely in the principal assessment of active schistosomiasis infection. The commercially available kit for IHA test which is widely used in the Egyptian medical laboratories and was also used in this study is more costly than other diagnostic methods and of lower specificity and sensitivity compared to microscopic examination in active S.mansoni infection. We recommend collaboration of different diagnostic methods to increase accuracy of detection for intestinal schistosomiasis in active stage.

Compliance with ethical standards

Conflict of interest

The author declares that there is no conflict of interest.