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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.

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.

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.

Rapid diagnostic tests are needed for the implementation and monitoring of national schistosomiasis control programmes. The field applicability of the circulating cathodic antigen (CCA) urine reagent strip for the diagnosis of Schistosoma haematobium infection was evaluated among 265 pre- and primary schoolchildren aged 2-19 years in a rural area of Zimbabwe. The CCA strip was compared with egg detection before and six weeks after treatment with praziquantel. Pre-treatment prevalence (overall 40.4%) and intensity of infection, as determined by egg counts, increased with age. CCA and parasitological results were significantly correlated (P

In the detection of parasitic infection, the traditional methods based on microscopy often have low sensitivity and/or specificity compared with the newer, molecular tests. An assay based on real-time PCR and a reagent strip test for detecting circulating cathodic antigen (CCA) have both now been compared with urine filtration and microscopy, in the detection of Schistosoma haematobium infections. Urine samples, obtained from 74 'cases' in areas of Ghana with endemic S. haematobium and 79 'controls' from non-endemic areas, were each checked using the three methods. With the results of the filtration and microscopy taken as the 'gold standard', real-time PCR was found to be 100% specific and 89% sensitive whereas the CCA strips were 91% specific and 41% sensitive. With the samples found to contain > or =50 eggs/10 ml (indicating relatively intense infections), the sensitivities of the PCR and CCA were higher, at 100% and 62%, respectively. As expected, egg counts were negatively correlated with the number of amplification cycles needed, in the PCR, to give a signal that exceeded the background (r=-0.38; P

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.

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 × 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.

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.

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.

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.

The development of the humoral anti-glycan immune response of chimpanzees, either or not vaccinated with radiation-attenuated Schistosoma mansoni cercariae, was followed during 1 year after infection with S. mansoni. During the acute phase of infection both the vaccinated and the control chimpanzees produce high levels of immunoglobulin G (IgG) antibodies against carbohydrate structures that are characteristic for schistosomes carrying the Fucalpha1-3GalNAc and Fucalpha1-2Fucalpha1-3GlcNAc motifs, but not to the more widespread occurring structures GalNAcbeta1-4GlcNAc, GalNAcbeta1-4(Fucalpha1-3)GlcNAc, and Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis(x)). In addition, high levels of IgM antibodies were found against the trimeric Lewis(x) epitope. Apparently, the schistosome-characteristic carbohydrate structures are dominant epitopes in the anti-glycan humoral immune response of the chimpanzees. All chimpanzees showed an increase in the level of antibodies against most of the carbohydrate structures tested directly after vaccination, peaking at challenge time and during the acute phase of infection. With the exception of anti-F-LDN antibody responses, the anti-carbohydrate antibody responses upon schistosome infection of the vaccinated animals were muted in comparison to the control animals.

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.

The present work investigated the transplacental passage of circulating anodic schistosome antigens (CAA) and the production of foetal antibodies in response to antigenic stimulation in Schistosoma mattheei infected cows. Three groups were available: six calves born to non-infected cows received colostrum from a pool from non-infected cows (group 1), six calves born to non-infected cows (group 2) and six calves born to infected cows (group 3) received colostrum from a pool from infected cows. Schistosoma-specific IgG (1) antibody and CAA levels were measured in the colostrum pools, the sera collected from the cows, and the sera collected from the calves at birth, after intake of colostrum and at day 30. The specific IgG (1) antibody levels were significantly higher in the sera from cows of group 3. In four cows of group 3 high CAA levels were detected. The specific IgG (1) antibody levels were 0.646 and 0.176 OD for the infected and non-infected colostrum pool, respectively, and the CAA levels were 5667 and 2557 pg CAA/mL, respectively. At birth high levels of specific IgG (1) antibody and CAA were detected in 4 calves of group 3; levels in the other two calves were negligible. After intake of colostrum, specific IgG (1) antibody levels of group 1 increased slightly at day 1 to become again insignificant at day 30. In group 2 specific IgG (1) antibody levels increased significantly between days 0 and 1, to decrease, although not significantly, at day 30. Finally, in group 3 the delta OD values increased at day 1 and remained high until day 30. After intake of colostrum the CAA level increased very slightly for groups 1 and 2 to become again undetectable at day 30. In group 3 a nonsignificant decrease in CAA levels was observed at day 1 followed by a further significant decrease to reach low levels at day 30. The suggested intrauterine antigenic stimulation may be important not only for generating immune responses to natural early infections, but also for enhancing the immunogenicity and efficacy of vaccines administered to newborns.

Gut-associated glycoproteins constitute a major group of the circulating excretory antigens produced by human Schistosoma species. The O-glycans of the relatively abundant circulating anodic antigen (CAA) from S. mansoni carry long stretches of unique --6(GlcA beta 1--3)GalNAc beta 1--repeats. Specific anti-carbohydrate monoclonal antibodies (mAbs) are essential tools for the immunodiagnostic detection of CAA in the serum or urine of Schistosoma-infected subjects. In order to define the epitopes recognised by these anti-CAA mAbs, we screened a series of protein-coupled synthetic di- to pentasaccharide building blocks of the CAA polysaccharide for immunoreactivity, using ELISA and surface plasmon resonance spectroscopy. It was shown that anti-CAA IgM mAbs preferentially recognise --6(GlcA beta 1--3)GalNAc beta 1--disaccharide units. Interestingly, no mouse anti-CAA mAbs of the IgG class were found that bind to the synthetic epitopes, although many of the IgG mAbs tested do recognise native CAA in a carbohydrate-dependent manner. In addition, both IgM and IgG class antibodies could be detected in human infection sera using the synthetic CAA fragments. These synthetic schistosome glycan epitopes and their matching set of specific mAbs are useful tools that further the development of diagnostic methods and are helpful in defining the immunological responses of the mammalian hosts to schistosome glycoconjugates.

Few studies have examined the interaction between schistosomiasis and infection with human immunodeficiency virus (HIV). The overlap between the two infections, and the effect of HIV infection on the egg output and worm load of individuals co-infected with Schistosoma mansoni, were therefore investigated in a sugar estate in central Ethiopia. The 1239 subjects were selected by stratified sampling of residents aged 15-54 years. The intensities of infection with S. mansoni were measured as egg output in stools (all subjects) and as the concentration of circulating cathodic antigen (CCA) in urine (a proxy for worm load, measured in 287 subjects). Schistosome infection was detected in 358 subjects [adjusted prevalence (AP) = 31.4%] and HIV infection in 52 (AP = 3.1%). The two infections clustered into different populations of the estate: the schistosome infections were predominantly found in the camps, and primarily affected young people (aged < 20 years) and those working in the field, whereas the HIV epidemic was found in the main village, primarily affecting those aged > 20 years and those who had recently arrived on the estate. Schistosome infection was detected in 348 of the 1187 HIV-negatives (AP = 31.6%) and 10 of the 52 HIV-positives (AP = 25.1%; P > 0.05). Schistosoma mansoni egg output was significantly lower in the HIV-positives than in the HIV-negatives (Mann-Whitney test; P = 0.03; ratio of geometric means = 0.74), and remained so after controlling for potential confounders (gender, age, and residence). However, CCA concentrations (i.e. worm loads) were found to be similar for these two groups, after controlling for potential confounders (age, gender, residence, and duration of residence)

Specific IgG4 and IgE responses and polyclonal cytokine profiles were studied in 110 Schistosoma haematobium-infected persons before and 5 weeks after chemotherapy. Pretreatment IgG4 responses to soluble egg antigen (SEA) correlated with intensity of infection. After chemotherapy, a significant decrease in egg output and circulating anodic antigen was associated with a substantial drop in the IgG4 response to SEA (IgG4-SEA) in adults and children, suggesting that egg laying is a major stimulus for IgG4-SEA. After chemotherapy, IgG4 and IgE to adult worm antigen and IgE to SEA increased in children but remained unchanged in adults. This indicates that the immunoregulatory mechanisms operative in S. haematobium-infected adults differ from those in infected children. The effect of treatment on cytokine profiles was determined following stimulation of whole blood with anti-CD3 antibodies. A significant decrease in interleukin-4 production after treatment indicated that reduction in helminth load may lead to a reduced number of Th2-type cells.

SUMMARYUsing spleen cells of mice infected or immunized respectively with cercariae or antigen preparations ofSchistosoma mansoni, S. haematobiumorS. japonicummonoclonal antibodies (mAbs) were produced against the schistosome gut-associated antigens CAA (circulating anodic antigen) and CCA (circulating cathodic antigen). Fusions nearly exclusively produced either anti-CAA (n= 25) or anti-CCA mAbs (n= 55) with a strong isotype restriction (IgM, IgG1 and IgG3) against both antigens, the majority of anti-CAA mAbs being IgG1 and the majority of anti-CCA mAbs being IgM. The mAbs, which on the basis of their selection were reactive with multiple carbohydrate epitopes of CAA or CCA, were applied in different immunological techniques including immunofluorescence, a dot immunobinding assay and immuno-electrophoresis to study the epitope repertoire. Anti-CAA mAbs were found to be reactive with 5 different epitopes, none of which occurred as multiple epitopes on eggs. Anti-CCA mAbs, on the other hand, recognized at least 10 different epitopes, while 44% of anti-CCA mAbs recognized epitopes common to the adult worm and the egg. Both CAA-and CCA-epitopes were found to be developmentally expressed at the level of the tegument in cercariae, schistosomula and 5-day-old lung worms, but in the adult worm were primarily found in the gut. Thus, the production of panels of mAbs has not only resulted in the selection of reagents optimally performing in diagnostic immunoassays, but also allowed a more detailed study of the epitope repertoire of these important schistosome antigens.

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.