Govert J. van Dam

Up-Converting Phosphor Technology-Based Lateral Flow Assay for Detection of Schistosoma Circulating Anodic Antigen in Serum

ABSTRACT Schistosoma sp. circular anodic antigen (CAA) serum concentrations reflect actual worm burden in a patient and are a valuable tool for population screening and epidemiological research. However, for the diagnosis of individual imported schistosomiasis cases, the current enzyme-linked immunosorbent assay (ELISA) lacks sensitivity and robustness. Therefore, a lateral flow (LF) assay was developed to test CAA in serum for individual diagnosis of imported active schistosome infections. Application of fluorescent submicron-sized up-converting phosphor technology (UPT) reporter particles increased analytical sensitivity compared to that of the standard ELISA method. Evaluation of the UPT-LF test with a selection of 40 characterized epidemiologic samples indicated a good correlation between signal intensity and infection intensity. Subsequently, the UPT-LF assay was applied to 166 serum samples of Dutch residents (immigrants and travelers) suspected of schistosomiasis, a case in which group routine antibody detection frequently fails straightforward diagnosis. The UPT-LF assay identified 36 CAA-positive samples, compared to 15 detected by CAA-ELISA. In conclusion, the UPT-LF assay is a low-complexity test with higher sensitivity than the CAA-ELISA, well suited for laboratory diagnosis of individual active Schistosoma infections.

Schistosomiasis and HIV-1 Infection in Rural Zimbabwe: Effect of Treatment of Schistosomiasis on CD4 Cell Count and Plasma HIV-1 RNA Load

To determine whether treatment of schistosomiasis has an effect on the course of human immunodeficiency virus type 1 (HIV-1) infection, individuals with schistosomiasis and with or without HIV-1 infection were randomized to receive praziquantel treatment at inclusion or after a delay of 3 months; 287 participants were included in the study, and 227 (79%) were followed up. Among the 130 participants who were coinfected, those who received early treatment (n=64) had a significantly lower increase in plasma HIV-1 RNA load than did those who received delayed treatment (n=66) (P<.05); this difference was associated with no change in plasma HIV-1 RNA load in the early intervention group (P=.99) and an increase in plasma HIV-1 RNA load in the delayed intervention group (P<.01). Among the 227 participants who were followed up, those who received early treatment (n=105) had an increase in CD4 cell count, whereas those who received delayed treatment (n=122) did not (P<.05); this effect did not differ between participants when stratified by HIV-1 infection status (P=.17). The present study suggests that treatment of schistosomiasis can reduce the rate of viral replication and increase CD4 cell count in the coinfected host.

An evaluation of urine-CCA strip test and fingerprick blood SEA-ELISA for detection of urinary schistosomiasis in schoolchildren in Zanzibar

To develop better monitoring protocols for detection of urinary schistosomiasis during ongoing control interventions, two commercially available diagnostic tests - the urine-circulating cathodic antigen (CCA) strip and the soluble egg antigen enzyme-linked immunosorbent assay (SEA-ELISA) - were evaluated for detection of Schistosoma haematobium infections in 150 schoolchildren from Zanzibar. The children originated from five primary schools representative of different levels of disease endemicity across the island; using standard urine filtration assessment with microscopy, mean prevalence of S. haematobium was 30.7% (95% confidence interval (CI)=23.4-38.7%) and a total of 35.3% (95% CI=27.7-43.5%) and 8.0% (95% CI=4.2-13.6%) children presented with micro- and macro-haematuria, respectively. Diagnostic scores of the urine-CCA strip were not satisfactory, a very poor sensitivity of 9% (95% CI=2-21%) was observed, precluding any further consideration. By contrast, the performance of the SEA-ELISA using sera from fingerprick blood was good; a sensitivity of 89% (95% CI=76-96%), a specificity of 70% (95% CI=60-79%), a positive predictive value of 57% (95% CI=45-69%) and a negative predictive value of 90% (95% CI=86-98%) were found. At the unit of the school, a positive linear association between prevalence inferred from parasitological examination and SEA-ELISA methods was found. The SEA-ELISA holds promise as a complementary field-based method for monitoring infection dynamics in schoolchildren over and above standard parasitological methods.

Profiles of immunoglobulin M (IgM) and IgG antibodies against defined carbohydrate epitopes in sera of Schistosoma-infected individuals determined by surface plasmon resonance.

ABSTRACT We report here that sera of children and adults infected withSchistosoma mansoni, S. haematobium, or S. japonicum contain antibodies against GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF) and to a lesser extent to Galβ1-4(Fucα1-3)GlcNAc (Lewisx) and GalNAcβ1-4GlcNAc (LDN). Surface plasmon resonance (SPR) spectroscopy was used to monitor the presence of serum antibodies to neoglycoconjugates containing these carbohydrate epitopes and to define the immunoglobulin M (IgM) and IgG subclass distribution of the antibodies. The serum levels of antibodies to LDN-DF are high related to LDN and Lewisx for all examined groups ofSchistosoma-infected individuals. A higher antibody response to the LDN-DF epitope was found in sera of infected children than in sera of infected adults regardless of the schistosome species. With respect to the subclasses, we found surprisingly that individuals infected with S. japonicum have predominantly IgG antibodies, while individuals infected with S. mansonimainly show an IgM response; high levels of both isotypes were measured in sera of individuals infected with S. haematobium. These data provide new insights in the human humoral immune response to schistosome-derived glycans.

Cellular and humoral immune responses and protection against schistosomes induced by a radiation-attenuated vaccine in chimpanzees.

ABSTRACT The radiation-attenuated Schistosoma mansoni vaccine is highly effective in rodents and primates but has never been tested in humans, primarily for safety reasons. To strengthen its status as a paradigm for a human recombinant antigen vaccine, we have undertaken a small-scale vaccination and challenge experiment in chimpanzees (Pan troglodytes). Immunological, clinical, and parasitological parameters were measured in three animals after multiple vaccinations, together with three controls, during the acute and chronic stages of challenge infection up to chemotherapeutic cure. Vaccination induced a strong in vitro proliferative response and early gamma interferon production, but type 2 cytokines were dominant by the time of challenge. The controls showed little response to challenge infection before the acute stage of the disease, initiated by egg deposition. In contrast, the responses of vaccinated animals were muted throughout the challenge period. Vaccination also induced parasite-specific immunoglobulin M (IgM) and IgG, which reached high levels at the time of challenge, while in control animals levels did not rise markedly before egg deposition. The protective effects of vaccination were manifested as an amelioration of acute disease and overall morbidity, revealed by differences in gamma-glutamyl transferase level, leukocytosis, eosinophilia, and hematocrit. Moreover, vaccinated chimpanzees had a 46% lower level of circulating cathodic antigen and a 38% reduction in fecal egg output, compared to controls, during the chronic phase of infection.

A robust dry reagent lateral flow assay for diagnosis of active schistosomiasis by detection of Schistosoma circulating anodic antigen.

UNLABELLED An earlier reported laboratory assay, performed in The Netherlands, to diagnose Schistosoma infections by detection of the parasite antigen CAA in serum was converted to a more user-friendly format with dry reagents. The improved assay requires less equipment and allows storage and worldwide shipping at ambient temperature. Evaluation of the new assay format was carried out by local staff at Ampath Laboratories, South Africa. The lateral flow (LF) based assay utilized fluorescent ultrasensitive up-converting phosphor (UCP) reporter particles, to be read by a portable reader (UPlink) that was also provided to the laboratory. Over a period of 18 months, about 2000 clinical samples were analyzed prospectively in parallel with a routinely carried out CAA-ELISA. LF test results and ELISA data correlated very well at CAA concentrations above 300 pg/mL serum. At lower concentrations the UCP-LF test indicates a better performance than the ELISA. The UCP-LF strips can be stored as a permanent record as the UCP label does not fade. At the end of the 18 months testing period, LF strips were shipped back to The Netherlands where scan results obtained in South Africa were validated with different UCP scanning equipment including a novel, custom developed, small lightweight UCP strip reader (UCP-Quant), well suited for testing in low resource settings. CONCLUSION The dry format UCP-LF assay was shown to provide a robust and easy to use format for rapid testing of CAA antigen in serum. It performed at least as good as the ELISA with respect to sensitivity and specificity, and was found to be superior with respect to speed and simplicity of use. Worldwide shipping at ambient temperature of the assay reagents, and the availability of small scanners to analyze the CAA UCP-LF strip, are two major steps towards point-of-care (POC) applications in remote and resource poor environments to accurately identify low (30 pg CAA/mL serum; equivalent to about 10 worm pairs) to heavy Schistosoma infections.

Tools for diagnosis, monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels

The potential of various quantitative lateral flow (LF) based assays utilizing up-converting phosphor (UCP) reporters for the diagnosis of schistosomiasis is reviewed including recent developments. Active infections are demonstrated by screening for the presence of regurgitated worm antigens (genus specific polysaccharides), whereas anti-Schistosoma antibodies may indicate ongoing as well as past infections. The circulating anodic antigen (CAA) in serum or urine (and potentially also saliva) is identified as the marker that may allow detection of single-worm infections. Quantitation of antigen levels is a reliable method to study effects of drug administration, worm burden and anti-fecundity mechanisms. Moreover, the ratio of CAA and circulating cathodic antigen (CCA) is postulated to facilitate identification of either Schistosoma mansoni or Schistosoma haematobium infections. The UCP-LF assays allow simultaneous detection of multiple targets on a single strip, a valuable feature for antibody detection assays. Although antibody detection in endemic regions is not a useful tool to diagnose active infections, it gains potential when the ratio of different classes of antibody specific for the parasite/disease can be determined. The UCP-LF antibody assay format allows this type of multiplexing, including testing a linear array of up to 20 different targets. Multiple test spots would allow detection of specific antibodies, e.g. against different Schistosoma species or other pathogens as soil-transmitted helminths. Concluding, the different UCP-LF based assays for diagnosis of schistosomiasis provide a collection of tests with relatively low complexity and high sensitivity, covering the full range of diagnostics needed in control programmes for mapping, screening and monitoring.

Schistosomiasis and HIV-1 Infection in Rural Zimbabwe: Implications of Coinfection for Excretion of Eggs

BACKGROUND Stunted development and reduced fecundity of Schistosoma parasites in immunodeficient mice and the impaired ability of human immunodeficiency virus 1 (HIV-1)-infected humans to excrete schistosome eggs have been described. This study explores the effect that HIV-1-associated immunodeficiency has on the excretion of schistosome eggs in a large cohort of coinfected individuals. METHODS In a cross-sectional survey, urine and stool samples were obtained from and HIV-1 status was determined for 1545 individuals. More extensive data, including quantitative measures of intensity of infection in schistosomiasis and immunodeficiency, were collected in the Mupfure schistosomiasis and HIV longitudinal cohort, composed of 379 participants of whom 154 were coinfected with HIV-1 and Schistosoma parasites. RESULTS In the cross-sectional survey, the overall prevalence of schistosomiasis was 43.4%, and 26.3% of the participants were infected with HIV-1. Schistosome infections were due to Schistosoma haematobium in 63.6% of cases, S. mansoni in 18.1% of cases, and dual infections in 18.4% of cases. Intensities of Schistosoma infections, measured by the number of eggs excreted and by the level of circulating anodic antigens, did not differ between HIV-1-negative and HIV-1-positive participants coinfected with S. haematobium, S. mansoni, or both. CD4 cell counts were significantly lower in HIV-1-positive participants and in S. mansoni-infected HIV-1-negative participants than in other participants. CONCLUSION The present study suggests that adult HIV-1-related immunodeficiency does not impair the ability to excrete eggs in low-intensity infection with S. haematobium, S. mansoni, or both and that infection with HIV-1 may not have major implications for diagnosis and surveillance of schistosomiasis.

Association of schistosomiasis and HIV infection in Tanzania

Animal and human studies suggest that Schistosoma mansoni infection may increase risk of human immunodeficiency virus (HIV) acquisition. Therefore, we tested 345 reproductive age women in rural Tanzanian villages near Lake Victoria, where S. mansoni is hyperendemic, for sexually transmitted infections (STIs) and schistosomiasis by circulating anodic antigen (CAA) serum assay. Over one-half (54%) had an active schistosome infection; 6% were HIV-seropositive. By univariate analysis, only schistosome infection predicted HIV infection (odds ratio [OR] = 3.9, 95% confidence interval = [1.3-12.0], P = 0.015) and remained significant using multivariate analysis to control for age, STIs, and distance from the lake (OR = 6.2 [1.7-22.9], P = 0.006). HIV prevalence was higher among women with more intense schistosome infections (P = 0.005), and the median schistosome intensity was higher in HIV-infected than -uninfected women (400 versus 15 pg CAA/mL, P = 0.01). This finding suggests that S. mansoni infection may be a modifiable HIV risk factor that places millions of people worldwide at increased risk of HIV acquisition.

Parameters of the attenuated schistosome vaccine evaluated in the olive baboon.

ABSTRACT Five exposures of baboons to the attenuated schistosome vaccine gave greater protection than three exposures, but this attenuation was not sustained when challenge was delayed. Within the scope of the data collected, fecal egg counts and circulating antigen levels did not accurately predict the observed worm burdens. Levels of immunoglobulin G at challenge correlated best with protection, but there was little evidence of a recall response.