Association between cervical dysplasia and female genital schistosomiasis diagnosed by genital PCR in Zambian women.

BACKGROUND: Female genital schistosomiasis (FGS) is a neglected tropical gynaecological disease that affects millions of women in sub-Saharan Africa (SSA). FGS is caused by Schistosoma haematobium, a parasitic carcinogen involved in the pathogenesis of squamous cell carcinoma of the bladder. Cervical cancer incidence and mortality are highest in SSA, where pre-cancerous cervical dysplasia is often detected on screening with visual inspection with acetic acid (VIA). There are no studies evaluating the association between VIA positivity and FGS diagnosed by genital PCR. METHODS: Women were recruited from the Bilharzia and HIV (BILHIV) study in Zambia a community-based study comparing genital self-sampling to provider obtained cervicovaginal-lavage for the diagnosis of FGS in women aged 18-31. FGS was defined as positive Schistosoma DNA from any genital PCR. Urogenital schistosomiasis diagnostics included urine circulating anodic antigen, urine microscopy and portable colposcopy. Participants were offered cervical cancer screening using VIA at Livingstone Central Hospital. Associations of PCR confirmed FGS and other diagnostics with VIA positivity were assessed using multivariable logistic regression. RESULTS: VIA results were available from 237 BILHIV participants. A positive Schistosoma PCR in any genital specimen was detected in 14 women (5.9%), 28.6% (4/14) of these women had positive VIA compared to 9.0% without PCR evidence of schistosome infection (20/223). Schistosoma PCR positivity in any genital specimen was strongly associated with VIA positivity (OR: 6.08, 95% CI: 1.58-23.37, P = 0.02). CONCLUSIONS: This is the first study to find an association between FGS and positive VIA, a relationship that may be causal. Further longitudinal studies are needed.

How can schistosome circulating antigen assays be best applied for diagnosing male genital schistosomiasis (MGS): an appraisal using exemplar MGS cases from a longitudinal cohort study among fishermen on the south shoreline of Lake Malawi.

We provide an update on diagnostic methods for the detection of urogenital schistosomiasis (UGS) in men and highlight that satisfactory urine-antigen diagnostics for UGS lag much behind that for intestinal schistosomiasis, where application of a urine-based point-of-care strip assay, the circulating cathodic antigen (CCA) test, is now advocated. Making specific reference to male genital schistosomiasis (MGS), we place greater emphasis on parasitological detection methods and clinical assessment of internal genitalia with ultrasonography. Unlike the advances made in defining a clinical standard protocol for female genital schistosomiasis, MGS remains inadequately defined. Whilst urine filtration with microscopic examination for ova of Schistosoma haematobium is a convenient but error-prone proxy of MGS, we describe a novel low-cost sampling and direct visualization method for the enumeration of ova in semen. Using exemplar clinical cases of MGS from our longitudinal cohort study among fishermen along the shoreline of Lake Malawi, the portfolio of diagnostic needs is appraised including: the use of symptomatology questionnaires, urine analysis (egg count and CCA measurement), semen analysis (egg count, circulating anodic antigen measurement and real-time polymerase chain reaction analysis) alongside clinical assessment with portable ultrasonography.

Haemostatic changes in urogenital schistosomiasis haematobium: a case-control study in Gabonese schoolchildren.

In many tropical areas schistosomiasis is a major health problem causing hepatosplenic, intestinal or urogenital complaints. Hepatosplenic schistosomiasis mansoni is also characterized by blood coagulation abnormalities. Liver pathology plays a role in the development of haemostatic changes and the parasitic infection may directly affect coagulation. However, these contributing factors cannot be studied separately in hepatosplenic schistosomiasis infections. This pilot study provides insight in haemostatic changes in urinary schistosomiasis by studying coagulation parameters in schistosomiasis haematobium-infected Gabonese schoolchildren. Selection on urinary schistosomiasis patients without hepatosplenic complaints allows for the investigation of the direct effects of the parasite on haemostasis. Levels of von Willebrand Factor (VWF) antigen, active VWF and osteoprotegerin were elevated, indicating inflammation-mediated endothelial activation. In contrast to hepatosplenic schistosomiasis, thrombin-antithrombin complex and D-dimer levels were not affected. Despite its small sample size, this study clearly indicates that Schistosoma haematobium directly alters the activation status of the endothelium, without initiation of coagulation.

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.

BACKGROUND: Large scale administration of the anthelminthic drug praziquantel (PZQ) to at-risk populations is the cornerstone of schistosomiasis control, although persisting high prevalence of infections in some areas and growing concerns of PZQ resistance have revealed the limitations of this strategy. Most studies assessing PZQ efficacy have used relatively insensitive parasitological diagnostics, such as the Kato-Katz (KK) and urine-filtration methods, thereby overestimating cure rates (CRs). This study aims to determine the efficacy of repeated PZQ treatments against Schistosoma mansoni infection in school-aged children in Côte d'Ivoire using the traditional KK technique, as well as more sensitive antigen- and DNA-detection methods. METHODS: An open-label, randomised controlled trial will be conducted in school-aged children (5 to 18 years) from the region of Taabo, Côte d'Ivoire, an area endemic for S. mansoni. This 8-week trial includes four two-weekly standard doses of PZQ in the "intense treatment" intervention group and one standard dose of PZQ in the "standard treatment" control group. The efficacy of PZQ will be evaluated in stool samples using the KK technique and real-time PCR as well as in urine using the point-of-care circulating cathodic antigen test and the up-converting phosphor, lateral flow, circulating anodic antigen assay. The primary outcome of the study will be the difference in CR of intense versus standard treatment with PZQ on individuals with a confirmed S. mansoni infection measured by KK. Secondary outcomes include the difference in CR and intensity reduction rate between the intense and standard treatment groups as measured by the other diagnostic tests, as well as the accuracy of the different diagnostic tests, and the safety of PZQ. DISCUSSION: This study will provide data on the efficacy of repeated PZQ treatment on the clearance of S. mansoni as measured by several diagnostic techniques. These findings will inform future mass drug administration policy and shed light on position of novel diagnostic tools to evaluate schistosomiasis control strategies. TRIAL REGISTRATION: The study is registered at EudraCT (2016-003017-10, date of registration: 22 July 2016) and ( NCT02868385 , date of registration: 16 August 2016).

Candidate gene family-based and case-control studies of susceptibility to high Schistosoma mansoni worm burden in African children: a protocol.

Background: Approximately 25% of the risk of Schistosoma mansoni is associated with host genetic variation. We will test 24 candidate genes, mainly in the T h2 and T h17 pathways, for association with S. mansoni infection intensity in four African countries, using family based and case-control approaches. Methods: Children aged 5-15 years will be recruited in S. mansoni endemic areas of Ivory Coast, Cameroon, Uganda and the Democratic Republic of Congo (DRC). We will use family based (study 1) and case-control (study 2) designs. Study 1 will take place in Ivory Coast, Cameroon, Uganda and the DRC. We aim to recruit 100 high worm burden families from each country except Uganda, where a previous study recruited at least 40 families. For phenotyping, cases will be defined as the 20% of children in each community with heaviest worm burdens as measured by the circulating cathodic antigen (CCA) assay. Study 2 will take place in Uganda. We will recruit 500 children in a highly endemic community. For phenotyping, cases will be defined as the 20% of children with heaviest worm burdens as measured by the CAA assay, while controls will be the 20% of infected children with the lightest worm burdens. Deoxyribonucleic acid (DNA) will be genotyped on the Illumina H3Africa SNP (single nucleotide polymorphisms) chip and genotypes will be converted to sets of haplotypes that span the gene region for analysis. We have selected 24 genes for genotyping that are mainly in the Th2 and Th17 pathways and that have variants that have been demonstrated to be or could be associated with Schistosoma infection intensity.   Analysis: In the family-based design, we will identify SNP haplotypes disproportionately transmitted to children with high worm burden. Case-control analysis will detect overrepresentation of haplotypes in extreme phenotypes with correction for relatedness by using whole genome principal components.

Localization and characterization of the carbohydrate-binding site of the porcine lymphocyte mannan-binding protein.

Mannan-binding proteins found in the liver and serum of several vertebrate species are supposed to play an important role in the intracellular transport of glycoproteins, as well as in several protective reactions including complement activation and elimination of various pathogens. To study these protective functions at molecular level it is necessary to understand the fine oligosaccharide specificity and mutual relation among various forms of these soluble lectins. We have isolated mannan-binding protein as peripheral membrane proteins of porcine lymphocytes. This lectin was purified to homogeneity and shown to possess many properties in common with the well studied rat liver proteins (mol. mass, subunit composition and general organization of the molecule). Binding studies performed with three series of defined oligosaccharides (high mannose, hybrid type, and complex) on native lectin molecules as well as isolated carbohydrate-binding domains revealed distinctive features of this mannan-binding protein, including its impaired ability to bind the oligosaccharide ligand after reduction and decyclization at core N-acetyl-D-glucosamine 1.

Estimation of the avidity of antibodies in polyclonal antisera against Streptococcus pneumoniae type 3 by inhibition ELISA.

The reliability of the determination of antibody avidity in polyclonal sera by indirect sandwich ELISA was studied. Binding of IgM and IgG (sub)classes in unpurified serum to Streptococcus pneumoniae type 3 capsular polysaccharide, which was coated onto ELISA plates, was inhibited with different inhibitors. The inhibitor concn at which 50% inhibition of antibody binding to the ELISA coat was achieved, was used as a measure for antibody avidity. As this 50% inhibition value is dependent upon the dilution of the serum and thus upon the initial amount of free antibody, it is necessary to define (a narrow range of) final ELISA absorbance values to which the dilutions of non-inhibited sera have to be adjusted. The shapes of the serum dilution curves have a good correlation with the numerical 50% inhibition values of the antibody avidity. The inhibition ELISA is suitable to compare the avidity values of the different antibody isotypes, but two remarks should be made: (1) antibody heterogeneity should be considered to influence the results and prevent the accurate measurement of absolute numerical avidity values. Because in the ELISA system merely antibody "activity" is measured, comparison of the efficacy of vaccines by means of the 50% inhibition (avidity) value of various antibody (sub)classes can still be performed in a reliable way; (2) results of the determination of the 50% inhibition values of the different antibody (sub)classes showed them to be dependent on the molecular ratio between antibody (sub)class levels. More aspects of the determination should be taken into account, like shapes of simple dilution curves, influences of various inhibitor concns in the diluent and whole (extended) inhibition curves.

New diagnostic tools in schistosomiasis.

Schistosomiasis is a water-based parasitic disease that affects over 250 million people. Control efforts have long been in vain, which is one reason why schistosomiasis is considered a neglected tropical disease. However, since the new millennium, interventions against schistosomiasis are escalating. The initial impetus stems from a 2001 World Health Assembly resolution, urging member states to scale-up deworming of school-aged children with the anthelminthic drug praziquantel. Because praziquantel is safe, efficacious and inexpensive when delivered through the school platform, diagnosis before drug intervention was deemed unnecessary and not cost-effective. Hence, there was little interest in research and development of novel diagnostic tools. With the recent publication of the World Health Organization (WHO) Roadmap to overcome the impact of neglected tropical diseases in 2020, we have entered a new era. Elimination of schistosomiasis has become the buzzword and this has important ramifications for diagnostic tools. Indeed, measuring progress towards the WHO Roadmap and whether local elimination has been achieved requires highly accurate diagnostic assays. Here, we introduce target product profiles for diagnostic tools that are required for different stages of a schistosomiasis control programme. We provide an update of the latest developments in schistosomiasis diagnosis, including microscopic techniques, rapid diagnostic tests for antigen detection, polymerase chain reaction (PCR) assays and proxy markers for morbidity assessments. Particular emphasis is placed on challenges and solutions for new technologies to enter clinical practice.

Measurement of the humoral immune response against Streptococcus pneumoniae type 14-derived antigens by an ELISA and ELISPOT assay based on biotin-avidin technology.

A Streptococcus pneumoniae type 14-specific ELISA and ELISPOT assay have been developed based on the use of biotinylated type 14 capsular polysaccharide (S14PS-biotin). A major advantage of this application over other methods is the use of 10-100-fold less antigen than that reported in the literature for other similar assays. Moreover, the prepared biotinylated polysaccharides are very stable and it is possible to use the same procedures for other pneumococcal polysaccharide antigens (e.g., S6BPS) with no major changes necessary in the ELISA and ELISPOT protocols. Furthermore, a simple thin layer chromatography method has been developed as a method for quality control of the biotinylated polysaccharide. Immunization with the thymus-independent antigen S14PS resulted in the induction of IgM spot-forming cells (SFC) and antibodies while S14PS-protein conjugates induced a thymus-dependent response. The immune response to the conjugates was enhanced by the addition of the adjuvant Quil A resulting in high levels of both IgG SFC and antibodies at day 14 after immunization. The developed assays are reliable and reproducible tools for studying the humoral immune response against Streptococcus pneumoniae type 14 capsular polysaccharide derived antigens.

Application of the FITC-anti-FITC-gold system to ultrastructural localization of antigens.

We report the application of a fluorescein isothiocyanate (FITC)-anti-FITC method to localize antigens at the ultrastructural level. In the systems studied, the anti-FITC-based detection method displays high specificity and sensitivity. These observations, combined with ease of production and with availability of FITC-protein conjugates, suggest that the FITC-anti-FITC method is a good alternative to presently used methods and is widely applicable to immunochemical and immunocytochemical procedures. The same preparation and protocol can be used for light and electron microscopic studies, thereby reducing possible artifacts introduced if different procedures are used. In the present study, two systems were used to test the method. One system used an FITC-labeled monoclonal antibody (MAb) to schistosome circulating cathodic antigen. In this system, the label was detected in the gut of adult Schistosoma mansoni by an anti-FITC MAb conjugated to 10-nm gold particles. The second system used human IgM antibodies pooled from patients infected with Schistosoma mansoni. In this system detection was accomplished using an anti-human IgM-FITC conjugate followed by the anti-FITC-Au antibody conjugate.

Epidemiologic application of circulating antigen detection in a recent Schistosoma mansoni focus in northern Senegal.

Quantitative enzyme-linked immunosorbent assays (ELISAs) for the detection of circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) in serum and urine were applied as an epidemiologic tool in a recent, intense focus of Schistosoma mansoni in Senegal. Both CAA and CCA in serum and CCA in urine were found in 94%, 83%, and 95%, respectively, of the population, of which 91% were positive on stool examination. Circulating antigens were also detectable in sera and urines of most egg-negative individuals. The sensitivities of the urine CCA and serum CAA ELISA were substantially higher than that of a single egg count, and increased with egg output. The CAA and CCA levels correlated well with egg counts and with each other. The age-related evolution of antigen levels followed a similar pattern as egg counts, providing supplementary evidence for a genuine reduction of worm burdens in adults in spite of the supposed absence of acquired immunity in this recently exposed community. The antigen:egg ratios decreased in adults, suggesting lower worm fecundity in children. This would be compatible with a density-dependent reduction of fecundity, but not with anti-fecundity immunity in adults that perhaps has not yet developed in this new focus.

The contribution of host-related factors to low cure rates of praziquantel for the treatment of Schistosoma mansoni in Senegal.

Surprisingly low cure rates were repeatedly observed after treatment with a standard dosage of praziquantel in a recently established Schistosoma mansoni focus in northern Senegal. In 4 discrete cohorts from the same population, cure rates were 18-36% and egg count reduction rates were 77-88%. Data and material of 920 compliant subjects from all 4 cohorts were further analyzed to identify possible host-related factors associated with low cure rates. The lowest cure rates were found in the highest egg count groups. However, in low and moderate egg count groups, drug efficacy was also below normal values. Cure rates were similar in males and females, showed no seasonal variation, and were independent of previous praziquantel treatment. They were significantly higher in adults than in children, also after allowing for intensity of infection. Individual water contact behavior and specific humoral immune responses were examined in 2 extreme subgroups, either without significant egg count reduction or showing complete parasitologic cure. There was no significant difference in frequency and duration of water contact between those individuals with complete cure and those that showed little effect of praziquantel treatment. Levels of IgG, IgG1, IgG3, IgG4, IgM, and IgE against adult worm antigen were not different between the 2 subgroups. Thus, the abnormally frequent failure of treatment observed in this focus could not be associated with any host-related factor, other than age and pretreatment egg counts.

The dynamics of a soluble egg antigen of Schistosoma haematobium in relation to egg counts, circulating anodic and cathodic antigens and pathology markers before and after chemotherapy.

A cohort of Schistosoma haematobium infected schoolchildren from Cameroon (n = 146) was studied for urine circulating soluble egg antigen (SEA), in comparison to other urine infection parameters: the circulating adult worm-derived antigens, circulating anodic and cathodic antigens (CAA and CCA), egg counts and the reagent strip index (RSI). Before treatment, SEA prevalence was 90%, while 89% and 65% of the children were positive for CCA and CAA respectively. The children were treated with 2 doses of praziquantel (2 x 40 mg/kg bodyweight) at an interval of 10 days and followed-up at 1, 2, 3, 5 and 12 months after treatment. Urine SEA correlated significantly with egg counts and RSI; levels of CAA and CCA were also significantly correlated with those of SEA. The levels of SEA showed a better correlation to both egg counts and RSI than did the levels of CAA and CCA. SEA levels dropped sharply 1 month after treatment, with few children excreting any SEA whereas egg counts decreased less rapidly. The prevalence and levels of SEA remained low during the whole post-treatment period whereas egg counts, RSI and CCA in urine rose progressively in the post-treatment period with a final egg count prevalence of 78%. The results of the present study indicate that for S. haematobium infections, measurement of SEA in urine is a valuable additional diagnostic parameter.

The fibre-web blood sampling technique applied to serological diagnosis of schistosomiasis mansoni.

The fibre-web technique for sampling, storing and transport of venous or capillary blood has been evaluated, in 84 schoolchildren from the Mwanza region of Tanzania, with regard to diagnostic efficacy for determination of the schistosome circulating anodic antigen (CAA) under conditions similar to those prevailing in the field. Although the average concentrations determined in fibre-web eluates were only about half of those determined in serum, the prevalences of CAA-positive individuals for the 2 sample materials were approximately the same. The average coefficient of variation calculated on determination of CAA in venous-blood fibre-web eluates amounted to 7%. The study shows that the fibre-web technique is well suited for use under field conditions.

Detection of the schistosome circulating cathodic antigen by enzyme immunoassay using biotinylated monoclonal antibodies.

We have developed an enzyme immunoassay (ELISA) for the quantification of the schistosome circulating cathodic antigen (CCA), a glycoprotein associated with the syncitium lining the gut of the parasite. A mouse monoclonal antibody of IgG3 isotype was used as coating (antigen-capture) antibody, while a biotinylated mouse monoclonal IgM was used as second (antigen-detecting) antibody. Streptavidin-alkaline phosphatase was used as enzyme label. The lower detection limit of the assay was 1.0 ng of the trichloroacetic acid soluble fraction of adult worm antigen (AWA-TCA) per ml, which corresponds to approximately 0.2 ng CCA per ml. The ELISA showed a linear range from 1.0 to 62.5 ng AWA-TCA per ml. Serum and urine samples of 16 individuals infected with Schistosoma mansoni (egg counts ranging from 5 to 4820 eggs per gram of faeces) were tested in the assay. Antigen titres ranged from less than 4-8192. This assay represents a considerable advantage in diagnosis of Schistosoma infections as it allows the detection and quantification of CCA in serum and urine in even lightly infected individuals.

Variations in the immune response to natural Schistosoma mattheei infections in calves born to infected mothers.

During previous work Schistosoma antibodies and circulating antigens were detected at birth in the serum from some calves born to Schistosoma mattheei infected mothers. The objectives of the present survey were: (1) to investigate the proportion of calves, born to cows infected with S. mattheei, which have specific antibodies and circulating schistosome antigens present in their serum at birth and (2) to investigate whether the presence or absence of these specific antibodies and/or circulating antigens at birth may affect the pattern of a natural S. mattheei infection in calves from 4 to 5 months of age, when the colostral antibodies are thought to be of negligible importance. A total of 28 calves born to infected mothers were randomly selected. Faeces, serum and colostrum samples were collected from the cows at calving, serum samples were collected from the calves at birth (day 0), after intake of colostrum (day 1) and monthly thereafter up to the age of 10 months. Both serum and colostrum samples were analysed for IgG(H+L) against SWAP mattheei and schistosome circulating anodic antigen (CAA) levels. The calves were exposed to a natural challenge from the age of 4-5 months. Faecal samples were collected from the calves monthly, starting at an age of 5 months up to 10 months, and were examined for faecal egg counts. Nine (group 1) out of the 28 calves were found to have specific antibodies in their serum at birth, in 5 of them CAA levels were also detected. In the other 19 calves (group 2) no IgG(H+L) or CAA were detected. At the end of the study faecal egg counts and CAA levels were significantly lower in calves from group 1 compared to group 2. Results confirm earlier work that specific antibodies and circulating antigens may be present in serum from calves at birth, and show that these calves have lower faecal egg counts and CAA levels after exposure to a natural challenge.

Schistosoma mansoni: in vitro and in vivo excretion of CAA and CCA by developing schistosomula and adult worms.

In this study we describe the excretion patterns of circulating anodic (CAA) and cathodic antigen (CCA) by freshly transformed and developing Schistosoma mansoni schistosomula and adult worms. In vitro, CAA and CCA were excreted by the parasites immediately after transformation. During the first days of development CAA and CCA levels were similar, but after 1 wk more CCA was excreted. Neither feeding the schistosomula with red blood cells nor addition of colchicine influenced the rates of antigen excretion. Female worms produced more antigen than males. In heavily infected mice CCA was the first antigen detectable from the third week of infection onward. A few days later, CAA showed a steep increase, becoming the predominant antigen during the course of infection. In urine samples, obtained at the time of perfusion (7 wk), CCA was the predominant antigen. In conclusion, although CAA and CCA levels in serum and urine generally correlate well with worm burden (as determined by egg output), the present study and a literature review show that the actual quantities produced by the worms and detected in the host circulation or excreta may depend on many factors, e.g., host and parasite species, clearance rates, or duration and intensity of infection.

Diagnostic evaluation of dot-binding assays for circulating cathodic antigen (CCA) and anti-CCA determinations in schistosomiasis japonica using defined biotinylated conjugates.

Using an affinity purified circulating cathodic antigen (CCA) preparation and an anti-CCA monoclonal IgM antibody, we modified dot-binding assays for anti-CCA and CCA detections with biotinylated conjugates. A study on 3 groups of 138 schistosomiasis japonica patients and 105 healthy individuals demonstrated a high predictive rate of over 95% in both assays, highly comparable to those of circumoral precipitin test (COPT). A blind test in 342 samples of various groups revealed higher detection rates in Ab-binding assay with both acute and chronic case groups (over 90%), but comparatively lower rates in Ag-binding assay with chronic groups (50%-76%). Distinct reductions of either GMRT and dot-indexes were found in 48 praziquantel treated patients whose sera were collected 9 months after chemotherapy. The major target molecules detected by the two binding assays were proved to be the protein incorporated moieties readily precipitated by trichloroacetic acid (TCA), ammonium sulphate and higher concentration of polyethylene glycol (PEG) suggestive of pathological, specific immunoglobulins in the free or complex forms. Non-specific dot reactions were found in some acute and chronic patients with non-relevant bovine serum albumin (BSA) conjugated peroxidase and streptavidin-PO controls, and also in normal sera with the McAb peroxidase conjugate or when working concentration of the biotinylated McAb was not properly titrated. The reliability and proper management of the dot binding assay as an immunodiagnostic tool were discussed.

Isolation and specific detection of two major schistosoma gut-associated circulating antigens.

OBJECTIVES: To investigate the nature of the common epitopes of Schistosoma japonicum (S. japonicum) circulating anodic (CAA) and circulating cathodic antigen (CCA) and to try to obtain sufficient purified material to set up a standard series for quantitative determinations. METHODS: Isolation of the two worm fractions from a trichloroacetic acid (TCA) soluble preparation of S. japonicum adult worm antigen (AWAj-TCA) via Mono-Q anion exchange chromatography was performed and analysis of specific reactivity of the eluted fractions was done by antigen-capture Enzyme Linked Immuno Sorbent Assay (ELISA) specific for CAA or CCA with reference to affinity purified preparations of S. mansoni CAA and CCA. RESULTS: When an ionic strength gradient was used, CCA was eluted in two major peaks, an unbound fraction CCA-1, and a major bound fraction, CCA-2. Two additional minor peaks, CCA-3 and CCA-4, were eluted at higher ionic strengths. CAA was only detected in the bound fraction, partly overlapping with CCA-3. In the CCA-1 and CCA-2 fractions, reactivity was only found in the antigen-capture ELISA using anti-CCA McAbs both for capture and detection. The CAA fraction was predominantly found to be positive in the antigen-capture ELISA using anti-CAA McAbs both for capture and detection. However, in ELISA using combined anti-CAA and anti-CAA McAbs for capture and detection, this fraction showed some reactivity. CONCLUSION: The two CCA fractions contain molecules which bear at least two CCA-epitopes; the CAA fraction contains molecules which contain at least two CAA-epitopes, and one CCA-epitope.