Evaluation of Circulating Cathodic Antigen (CCA) Urine-Tests for Diagnosis of Schistosoma mansoni Infection in Cameroon

Background

The Kato-Katz is the most common diagnostic method for Schistosoma mansoni infection. However, the day-to-day variability in host egg-excretion and its low detection sensitivity are major limits for its use in low transmission zones and after widespread chemotherapy. We evaluated the accuracy of circulating cathodic antigen (CCA) urine-assay as a diagnostic tool of S. mansoni. In comparison, a low sensitive CCA test (CCA-L) was assessed.

Methodology

The study was conducted in three settings: two foci with single S. mansoni infections (settings A and B), and one mixed S. mansoni – S. haematobium focus (setting C). Stool and urine samples were collected from school-children on three consecutive days. Triplicate Kato-Katz readings were performed per stool sample. Each urine sample was tested with one CCA and only the first urine sample was subjected to CCA-L. Urine samples were also examined for S. haematobium eggs using the filtration method and for microhaematuria using urine reagent strips. Overall, 625 children provided three stool and three urine samples.

Principal Findings

Considering nine Kato-Katz thick smears as ‘reference’ diagnostic test, the prevalence of S. mansoni was 36.2%, 71.8% and 64.0% in settings A, B and C, respectively. The prevalence of S. haematobium in setting C was 12.0%. The sensitivities of single Kato-Katz, CCA and CCA-L from the first stool or urine samples were 58%, 82% and 46% in setting A, 56.8%, 82.4% and 68.8% in setting B, and 49.0%, 87.7% and 55.5% in setting C. The respective specificities were 100%, 64.7% and 100%; 100%, 62.3% and 91.3%; and 100%, 42.5% and 92.0%. Mixed infection with S. haematobium did not influence the CCA test results for S. mansoni diagnosis.

Conclusions/Significance

Urine CCA revealed higher sensitivity than CCA-L and triplicate Kato-Katz, and produced similar prevalence as nine Kato-Katz. It seems an attractive method for S. mansoni diagnosis.     

Accuracy of Urine Circulating Cathodic Antigen Test for the Diagnosis of Schistosoma mansoni in Preschool-Aged Children before and after Treatment

Background

The Kato-Katz technique is widely used for the diagnosis of Schistosoma mansoni, but shows low sensitivity in light-intensity infections. We assessed the accuracy of a commercially available point-of-care circulating cathodic antigen (POC-CCA) cassette test for the diagnosis of S. mansoni in preschool-aged children before and after praziquantel administration.

Methodology

A 3-week longitudinal survey with a treatment intervention was conducted in Azaguié, south Côte d''Ivoire. Overall, 242 preschoolers (age range: 2 months to 5.5 years) submitted two stool and two urine samples before praziquantel administration, and 86 individuals were followed-up posttreatment. Stool samples were examined with duplicate Kato-Katz thick smears for S. mansoni. Urine samples were subjected to POC-CCA cassette test for S. mansoni, and a filtration method for S. haematobium diagnosis.

Principal Findings

Before treatment, the prevalence of S. mansoni, as determined by quadruplicate Kato-Katz, single CCA considering ‘trace’ as negative (t−), and single CCA with ‘trace’ as positive (t+), was 23.1%, 34.3% and 64.5%, respectively. Using the combined results (i.e., four Kato-Katz and duplicate CCA(t−)) as diagnostic ‘gold’ standard, the sensitivity of a single Kato-Katz, a single CCA(t−) or CCA(t+) was 28.3%, 69.7% and 89.1%, respectively. Three weeks posttreatment, the sensitivity of a single Kato-Katz, single CCA(t−) and CCA(t+) was 4.0%, 80.0% and 84.0%, respectively. The intensity of the POC-CCA test band reaction was correlated with S. mansoni egg burden (odds ratio = 1.2, p = 0.04).

Conclusions/Significance

A single POC-CCA cassette test appears to be more sensitive than multiple Kato-Katz thick smears for the diagnosis of S. mansoni in preschool-aged children before and after praziquantel administration. The POC-CCA cassette test can be recommended for the rapid identification of S. mansoni infections before treatment. Additional studies are warranted to determine the usefulness of POC-CCA for assessing drug efficacy and monitoring the impact of control interventions.     

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

Background

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.

Method/Principle 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.

Conclusions/Significance

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).     

Sensitivity and Specificity of a Urine Circulating Anodic Antigen Test for the Diagnosis of Schistosoma haematobium in Low Endemic Settings

Background

Elimination of schistosomiasis as a public health problem and interruption of transmission in selected areas are key goals of the World Health Organization for 2025. Conventional parasitological methods are insensitive for the detection of light-intensity infections. Techniques with high sensitivity and specificity are required for an accurate diagnosis in low-transmission settings and verification of elimination. We determined the accuracy of a urine-based up-converting phosphor-lateral flow circulating anodic antigen (UCP-LF CAA) assay for Schistosoma haematobium diagnosis in low-prevalence settings in Zanzibar, Tanzania.

Methodology

A total of 1,740 urine samples were collected in 2013 from children on Pemba Island, from schools where the S. haematobium prevalence was <2%, 2–5%, and 5–10%, based on a single urine filtration. On the day of collection, all samples were tested for microhematuria with reagent strips and for the presence of S. haematobium eggs with microscopy. Eight months later, 1.5 ml of urine from each of 1,200 samples stored at -20°C were analyzed by UCP-LF CAA assay, while urine filtration slides were subjected to quality control (QCUF). In the absence of a true ‘gold’ standard, the diagnostic performance was calculated using latent class analyses (LCA).

Principal Findings

The ‘empirical’ S. haematobium prevalence revealed by UCP-LF CAA, QCUF, and reagent strips was 14%, 5%, and 4%, respectively. LCA revealed a sensitivity of the UCP-LF CAA, QCUF, and reagent strips of 97% (95% confidence interval (CI): 91–100%), 86% (95% CI: 72–99%), and 67% (95% CI: 52–81%), respectively. Test specificities were consistently above 90%.

Conclusions/Significance

The UCP-LF CAA assay shows high sensitivity for the diagnosis of S. haematobium in low-endemicity settings. Empirically, it detects a considerably higher number of infections than microscopy. Hence, the UCP-LF CAA employed in combination with QCUF, is a promising tool for monitoring and surveillance of urogenital schistosomiasis in low-transmission settings targeted for elimination.     

Schistosoma mansoni:The Circulating Cathodic Antigen Forms an Abundant Product of 41/42 kDa in the Urine of Infected Patients

Abdeen, H. H., Attallah, A.-F. M., El-Mohamady, H. I., Harrison, R. A., and Mansour, M. M. 1998.Schistosoma mansoni: The circulating cathodic antigen forms an abundant product of 41/42 kDa in the urine of infected patients.Experimental Parasitology90, 286–289.     

Circulating antigens levels in different clinical forms of the Schistosoma mansoni infection

With the aim to evaluate the circulating cathodic antigen (CCA) levels in relation to the different clinical phases of Schistosoma sp. infection a sandwich ELISA using monoclonal antibody 5H11 was performed. The sera of three groups of 25 Brazilian patients with acute, intestinal and hepatosplenic forms of S. mansoni infection were tested and compared to a non-infected control group. Patients and control groups were matched for age and sex and the number of eggs per gram of feces was equally distributed among the three patient groups. Sensitivity of 100%, 72%, 52% of the assay was observed for the intestinal, hepatosplenic and acute toxemic groups respectively. The specificity was 100%. Intestinal and hepatosplenic groups presented CCA levels significantly higher in comparison to those observed for acute patients (F-ratio = 2,524; p = 0.000 and F-ratio = 6,314; p = 0.015 respectively). There was no significant difference of CCA serum levels between hepatosplenic and intestinal groups (F-ratio = 1,026; p = 0.316).     

A Latent Markov Modelling Approach to the Evaluation of Circulating Cathodic Antigen Strips for Schistosomiasis Diagnosis Pre- and Post-Praziquantel Treatment in Uganda

Regular treatment with praziquantel (PZQ) is the strategy for human schistosomiasis control aiming to prevent morbidity in later life. With the recent resolution on schistosomiasis elimination by the 65th World Health Assembly, appropriate diagnostic tools to inform interventions are keys to their success. We present a discrete Markov chains modelling framework that deals with the longitudinal study design and the measurement error in the diagnostic methods under study. A longitudinal detailed dataset from Uganda, in which one or two doses of PZQ treatment were provided, was analyzed through Latent Markov Models (LMMs). The aim was to evaluate the diagnostic accuracy of Circulating Cathodic Antigen (CCA) and of double Kato-Katz (KK) faecal slides over three consecutive days for Schistosoma mansoni infection simultaneously by age group at baseline and at two follow-up times post treatment. Diagnostic test sensitivities and specificities and the true underlying infection prevalence over time as well as the probabilities of transitions between infected and uninfected states are provided. The estimated transition probability matrices provide parsimonious yet important insights into the re-infection and cure rates in the two age groups. We show that the CCA diagnostic performance remained constant after PZQ treatment and that this test was overall more sensitive but less specific than single-day double KK for the diagnosis of S. mansoni infection. The probability of clearing infection from baseline to 9 weeks was higher among those who received two PZQ doses compared to one PZQ dose for both age groups, with much higher re-infection rates among children compared to adolescents and adults. We recommend LMMs as a useful methodology for monitoring and evaluation and treatment decision research as well as CCA for mapping surveys of S. mansoni infection, although additional diagnostic tools should be incorporated in schistosomiasis elimination programs.     

Exposure versus Susceptibility as Alternative Bases for New Approaches to Surveillance for Schistosoma japonicum in Low Transmission Environments

Currently, schistosomiasis in China provides an excellent example of many of the challenges of moving from low transmission to the elimination of transmission for infectious diseases generally. In response to the surveillance dimension of these challenges, we here explore two strategic approaches to inform priorities for the development of improved methods addressed specifically to schistosomiasis in the low transmission environment. We utilize an individually-based model and the exposure data used earlier to explore surveillance strategies, one focused on exposure assessment and the second on our estimates of variability in individual susceptibility in the practical context of the current situation in China and the theoretical context of the behavior of transmission dynamics near the zero state. Our findings suggest that individual susceptibility is the major single determinant of infection intensity in both the low and medium risk environments. We conclude that there is considerable motivation to search for a biomarker of susceptibility to infection in humans, but that there would also be value in a method for monitoring surface waters for the free-swimming forms of the parasite in endemic or formerly endemic environments as an early warning of infection risk.     

New foci of transmission of Schistosoma mansoni in the state of Pará

Two new foci of transmission of Schistosoma mansoni in the state of Pará are recorded, with the finding of naturally infected Biomphalaria glabrata in the municipalities of Viseu and Belém. Uninfected specimens of Biomphalaria straminea, as well as the planorbid species Biomphalaria schrammi, Drepanotrema lucidum and D. anatinum, were found in the same area.     

Newly Established Monoclonal Antibody Diagnostic Assays for Schistosoma mansoni Direct Detection in Areas of Low Endemicity

Background

Current available methods for diagnosis of schistosomiasis mansoni lack sufficient sensitivity, which results in underreporting of infectious in areas of low endemicity.

Methodology/Principal Findings

We developed three novel diagnostic methodologies for the direct detection of schistosome infection in serum samples. These three new methods were evaluated with positive patients from a low endemicity area in southeast Brazil. The basis of the assay was the production of monoclonal antibodies against the protein backbone of heavily glycosylated Circulating Cathodic Antigen (CCA). The antibodies were also selected for having no specificity to repeating poly-Lewis x units. Assays based on the detection CCA-protein should not encounter a limitation in sensitivity due to a biological background of this particular epitope. Three diagnostic methodologies were developed and validated, (i) Immunomagnetic Separation based on improved incubation steps of non-diluted serum, (ii) Direct Enzyme-linked Immunosorbent Assay and (iii) Fluorescent Microscopy Analysis as a qualitative assay. The two quantitative assays presented high sensitivity (94% and 92%, respectively) and specificity (100%), equivalent to the analysis of 3 stool samples and 16 slides by Kato-Katz, showing promising results on the determination of cure.

Conclusions/Significance

The Immunomagnetic Separation technique showed excellent correlation with parasite burden by Cohen coefficient. The qualitative method detected 47 positive individuals out of 50 with the analysis of 3 slides. This easy-to-do method was capable of discriminating positive from negative cases, even for patients with low parasite burden.     

Different levels of immunity to Schistosoma mansoni in the mouse: the role of variant cercariae.

Very variable levels of immunity to a second infection with Schistosoma mansoni were recorded in 7 strains of mice, 12--15 weeks following a small primary infection. When 2 or more strains of mice were assayed at the same time, less variation occurred within the experiment than between different experiments. This evidence suggested variation between pools of cercariae as the main cause of variability in immunity. In direct experiments in one strain of mouse, 2 different pools of cercariae stimulated widely different levels of immunity to the same challenge. Conversely, challenge infections drawn from different pools showed different susceptibility to immunity stimulated by the same primary infection. Individual clones of cercariae, from snails infected with single miracidia, showed a high level of susceptibility to immunity stimulated by a small bisexual infection, or were not susceptible at all. Antigenic polymorphism is the most likely explanation for the differences observed between clones of cercariae. However, indirect immunofluorescence showed the presence of at least 1 common antigen on the surface of schistosomula derived from different clones of cercariae and clone-specific antigens have not been detected.     

Schistosoma mansoni: the basis for the antischistosomal effect of cyclosporine A

A potent antiparasitic effect against a broad range of microorganisms is now known to follow the administration of cyclosporine A (CSA) to a variety of mammalian species and birds. Even though in the case of Schistosoma mansoni a direct antischistosomal effect by CSA has been in principle ruled out, this possible mechanism of action has not been previously explored in depth. In this paper we show that (a) concentrations of CSA of as low as 1 microgram/ml are schistosomulicidal in vitro as determined by morphological criteria, (b) mice receiving antischistosomal regimens in vivo exhibit serum concentrations of CSA that are within the range of toxic doses in vitro, (c) schistosomulicidal concentrations of CSA are maintained in vivo for sufficiently long periods of time to secure parasite death, (d) the antischistosomal activity of heat-inactivated mouse sera closely correlates with their levels of CSA, and (e) independent of the site of administration, CSA offers protection against a schistosomal infection in vivo only when present in the serum. The data strongly support the contention that CSA has a direct effect on the early forms of Schistosoma mansoni, and suggest that interference with a survival mechanism, perhaps common to several parasites, represents one likely mode of action.     

Antigen presentation by CD1d contributes to the amplification of Th2 responses to Schistosoma mansoni glycoconjugates in mice

During murine schistosomiasis, there is a gradual switch from a predominant Th1 cytokine response to a Th2-dominated response after egg laying, an event that favors the formation of granuloma around viable eggs. Egg-derived glycoconjugates, including glycolipids, may play a crucial role in this phenomenon. In this study, we used a model of dendritic cell sensitization to study the role of egg glycoconjugates in the induction of specific immune response to soluble egg Ag (SEA) and to investigate the possibility that CD1d, a molecule implicated in glycolipid presentation, may be involved in such a phenomenon. We show that, when captured, processed, and presented to naive T lymphocytes by dendritic cells, egg, but not larval, Ag skew the immune response toward a Th2 response. Periodate treatment reversed this effect, indicating that the sugar moiety of SEA is important in this phenomenon. Using DC treated ex vivo with a neutralizing anti-CD1d Ab or isolated from CD1d knockout mice, we show that CD1d is crucial in the priming of SEA-specific Th2 lymphocytes. We then evaluated the contribution of CD1d on the development of the SEA-specific immune response and on the formation of the egg-induced liver granuloma during murine schistosomiasis. We find that CD1d knockout mice have a reduced Th2 response after egg laying and develop a less marked fibrotic pathology compared with wild-type mice. Altogether, our results suggest that Ag presentation of parasite glycoconjugates to CD1d-restricted T cells may be important in the early events leading to the induction of Th2 responses and to egg-induced pathology during murine schistosomiasis.     

The interaction of human LDL with the tegument of adult Schistosoma mansoni

The occurrence of a receptor for human LDL was investigated in the tegument of adult Schistosoma mansoni employing several approaches. Binding of LDL to SDS-PAGE fractionated tegument proteins was measured directly on nitro-cellulose membranes and visualised by an anti-human LDL antibody. Proteins with an Mr of 60, 35 and 14 kDa were evidenced. Affinity chromatography of ¹²⁵ I-labelled tegument proteins on a LDL-Sepharose column, revealed the same pattern of proteins observed in the immunoblot experiments. Finally, the binding of human LDL to the intact tegument was measured by microcalorimetry. Binding was shown to be an exothermic reaction, releasing approximately 2500 kcal/mol, it was saturable, and reproducibly displayed a biphasic curve suggesting that binding of LDL to S. mansoni might occur through a two step process, initiated by a nonspecific hydrophobic interaction followed by a specific high affinity ligand-receptor reaction. Pre-treatment of the tegument with trypsin reduced the binding of LDL to the tegument. Furthermore, albumin, which is an abundant lipid carrier protein in the serum and thus a potential ligand, failed to release any measurable heat when incubated with the tegument.     

The effect of treatment on the age-antibody relationship in children infected with Schistosoma mansoni and Schistosoma haematobium

The effect of praziquantel treatment on the age-antibody relationship was studied in 174 children aged between 6 and 17 years from a schistosome endemic area in Zimbabwe. The children were co-infected with Schistosoma mansoni and S. haematobium with infection prevalences of 74% and 53% respectively. Antibody levels for the isotypes IgA, IgE, IgM, IgG1, IgG2, IgG3 and IgG4, directed against soluble egg antigen were measured using an indirect ELISA assay. Treatment resulted in a significant increase in levels of IgG2 and IgG3 while levels of IgA decreased significantly. In untreated children there were significant decreases in levels of IgG4. Treatment also resulted in significant alteration in the age-antibody profiles for the isotypes IgE, IgM, IgG1 and IgG2 in treated children but not in untreated children. The results are discussed in the context of factors believed to give rise to the age-antibody relationship; i.e. age-related exposure patterns, age-related development of acquired immunity, age-related hormonal changes and age-related changes in innate susceptibility to infection.