A rapid one-step method of EIA for detection of circulating antigen of Schistosoma japonicum

Objective To establish a highly sensitive serologic method for detecting the circulating antigen of Schistosoma japonicum for the diagnosis of this disease and the evaluation of drug therapy effect.Methods A set of complex support made of poly vinyl chloride (PVC) film slide, adsorbed with specific antibody, was used to test serum samples collected from cases with schistosomiasis japonica, by adding specific enzyme conjugate and substrate tetra methyl benzidine (TMB) with 3% H 2O 2 in 10∶0.1, developed or prepared in our laboratory. Tests were carried out with the set on cases with malaria, paragonimiasis, clonorchiasis, visceral leishmaniasis and cysticercosis as well as normal individuals as control. Serum sample of 50 μl was used in each test and reacted with reagents at room temperature for 30 minutes. The color of positive reaction was blue while negative reaction was colorless.Results Positive rate among cases with schistosomiasis japonica was 100% (45/45) in acute stage, 94.8% (530/559) in early chronic stage and 52.4% (66/126) in late stage. False positive reaction was found neither among all normal individuals (0/513), nor among cases of 155 with malaria, 120 with clonorchiasis, 24 with visceral leishmaniasis and 110 with cysticercosis, except one among 29 cases with parasgonimiasis (1/29).Conclusions The rapid one step enzyme Immunoassay (EIA) to detect circulating schistosome antigen (CSA) has been well established in our laboratory with high sensitivity and good specificity as well as reproducibility. The reaction result can be read easily even in field conditions without power supply. Moreover, the assay is time saving, simple to handle and suitable for the diagnosis of shistosomiasis japonica and evaluation of drug therapeutic effect in practice in the control program of the disease.

Accuracy and precision of dried urine spot method for the detection of Schistosoma mansoni circulating cathodic antigens in resource-limited settings

Background The World Health Organization recommends the use of Schisto point-of-care circulating cathodic anti-gens(Schisto POC-CCA)for screening of Schistosoma mansoni as it offers better sensitivity than microscopy.However,there are limitation facing the use of this method including timely availability of the test cassettes.The aim of this study was to determine the reliability of dried urine spot(DUS)method for collection of urine and detection of S.mansoni using Schisto POC-CCA cassettes in a resource-limited settings.Methods A cross-sectional study was conducted between October and November 2022 among 250 primary school children in Sengerema District,northwestern Tanzania.S.mansoni CCA was detected in filter paper-based DUSs,liquid urine using DUS Schisto POC-CCA(index),and direct urine Schisto POC-CCA(comparator)methods respectively.S.mansoni eggs in stool were detected using duplicate Kato-Katz(KK)method.The measures of accuracy were com-puted and compared between the index and comparator methods.The strength of agreement between inter-raters precisions was tested using Cohen's kappa(k).Results This study revealed S.mansoni prevalence rates of 28.8%,54.0%and 50.8%by duplicate KK,direct urine Schisto POC-CCA and DUS Schisto POC-CCA methods respectively.The mean intensity of infection among infected participants Was 86.3 eggs per gram of stool(EPG)ranging from 12.0 EPG to 824.0 EPG.The sensitivity of DUS Schisto POC-CCA and direct urine Schisto POC-CCA Was 94.44%(95%CI:89.15-99.74%)and 97.22%(95%CI:93.43-100.00%)respectively.The DUS Schisto POC-CCA method had slightly higher specificity(66.85%)than direct urine Schisto POC-CCA method(63.48%).The accuracy of the DUS Schisto POC-CCA Was found to be slightly high(74.80%,95%CI:68.94-79.06%)compared to that of direct urine Schisto POC-CCA(73.20%,95%CI:67.25-78.59%).There was good agreement between two laboratory technologists who performed the DUS Schisto POC-CCA method on similar samples(k=0.80,95%CI:0.59-0.95).Conclusions The DUS Schisto POC-CCA method had comparable S.mansoni detection accuracy to direct urine Schisto POC-CCA.This suggests that the method could be a potential alternative to direct urine Schisto POC-CCA for screening S.mansoni in resource-limited situations.

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-CCA 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.

Utilizing the ultrasensitive Schistosoma up-converting phosphor lateral flow circulating anodic antigen(UCP-LF CAA)assay for sample pooling-strategies

Background:Methodological applications of the high sensitivity genus-specific Schistosoma CAA strip test,allowing detection of single worm active infections(ultimate sensitivity),are discussed for efficient utilization in sample pooling strategies.Besides relevant cost reduction,pooling of samples rather than individual testing can provide valuable data for large scale mapping,surveillance,and monitoring.Method:The laboratory-based CAA strip test utilizes luminescent quantitative up-converting phosphor(UCP)reporter particles and a rapid user-friendly lateral flow(LF)assay format.The test includes a sample preparation step that permits virtually unlimited sample concentration with urine,reaching ultimate sensitivity(single worm detection)at 100%specificity.This facilitates testing large urine pools from many individuals with minimal loss of sensitivity and specificity.The test determines the average CAA level of the individuals in the pool thus indicating overall worm burden and prevalence.When requiring test results at the individual level,smaller pools need to be analysed with the pool-size based on expected prevalence or when unknown,on the average CAA level of a larger group;CAA negative pools do not require individual test results and thus reduce the number of tests.Results:Straightforward pooling strategies indicate that at sub-population level the CAA strip test is an efficient assay for general mapping,identification of hotspots,determination of stratified infection levels,and accurate monitoring of mass drug administrations(MDA).At the individual level,the number of tests can be reduced i.e.in low endemic settings as the pool size can be increased as opposed to prevalence decrease.Conclusions:At the sub-population level,average CAA concentrations determined in urine pools can be an appropriate measure indicating worm burden.Pooling strategies allowing this type of large scale testing are feasible with the various CAA strip test formats and do not affect sensitivity and specificity.It allows cost efficient stratified testing and monitoring of worm burden at the sub-population level,ideally for large-scale surveillance generating hard data for performance of MDA programs and strategic planning when moving towards transmission-stop and elimination.

Schistosomiasis is more prevalent than previously thought:what does it mean for public health goals,policies,strategies,guidelines and intervention programs?

Mapping and diagnosis of infections by the three major schistosome species(Schistosoma haematobium,S.mansoni and S.japonicum)has been done with assays that are known to be specific but increasingly insensitive as prevalence declines or in areas with already low prevalence of infection.This becomes a true challenge to achieving the goal of elimination of schistosomiasis because the multiplicative portion of the life-cycle of schistosomes,in the snail vector,favors continued transmission as long as even a few people maintain low numbers of worms that pass eggs in their excreta.New mapping tools based on detection of worm antigens(circulating cathodic antigen-CCA;circulating anodic antigen-CAA)in urine of those infected are highly sensitive and the CAA assay is reported to be highly specific.Using these tools in areas of low prevalence of all three of these species of schistosomes has demonstrated that more people harbor adult worms than are regularly excreting eggs at a level detectable by the usual stool assay(Kato-Katz)or by urine filtration.In very low prevalence areas this is sometimes 6-to10-fold more.Faced with what appears to be a sizable population of“egg-negative/worm-positive schistosomiasis”especially in areas of very low prevalence,national NTD programs are confounded about what guidelines and strategies they should enact if they are to proceed toward a goal of elimination.There is a critical need for continued evaluation of the assays involved and to understand the contribution of this“egg-negative/worm-positive schistosomiasis”condition to both individual morbidity and community transmission.There is also a critical need for new guidelines based on the use of these more sensitive assays for those national NTD programs that wish to move forward to strategies designed for elimination.

Comparison of novel and standard diagnostic tools for the detection of Schistosoma mekongi infection in Lao People’s Democratic Republic and Cambodia

Background:Given the restricted distribution of Schistosoma mekongi in one province in Lao People’s Democratic Republic(Lao PDR)and two provinces in Cambodia,together with progress of the national control programmes aimed at reducing morbidity and infection prevalence,the elimination of schistosomiasis mekongi seems feasible.However,sensitive diagnostic tools will be required to determine whether elimination has been achieved.We compared several standard and novel diagnostic tools in S.mekongi-endemic areas.Methods:The prevalence and infection intensity of S.mekongi were evaluated in 377 study participants from four villages in the endemic areas in Lao PDR and Cambodia using Kato-Katz stool examination,antibody detection based on an enzyme-linked immunosorbent assay(ELISA)and schistosome circulating antigen detection by lateral-flow tests.Two highly sensitive test systems for the detection of cathodic and anodic circulating antigens(CCA,CAA)in urine and serum were utilized.Results:Stool microscopy revealed an overall prevalence of S.mekongi of 6.4%(one case in Cambodia and 23 cases in Lao PDR),while that of Opisthorchis viverrini,hookworm,Trichuris trichiura,Ascaris lumbricoides and Taenia spp.were 50.4%,28.1%,3.5%,0.3%and 1.9%,respectively.In the urine samples,the tests for CCA and CAA detected S.mekongi infections in 21.0%and 38.7%of the study participants,respectively.In the serum samples,the CAA assay revealed a prevalence of 32.4%,while a combination of the CAA assay in serum and in urine revealed a prevalence of 43.2%.There was a difference between the two study locations with a higher prevalence reached in the samples from Lao PDR.Conclusions:The CCA,CAA and ELISA results showed substantially higher prevalence estimates for S.mekongi compared to Kato-Katz thick smears.Active schistosomiasis mekongi in Lao PDR and Cambodia might thus have been considerably underestimated previously.Hence,sustained control efforts are still needed to break transmission of S.mekongi.The pivotal role of highly sensitive diagnostic assays in areas targeting elimination cannot be overemphasised.

Burden and factors associated with schistosomiasis and soil-transmitted helminth infections among school-age children in Huambo,Uige and Zaire provinces,Angola

Background:Schistosomiasis and soil-transmitted helminths(STHs)contribute high disease burdens amongst the neglected tropical diseases(NTDs)and are public health problems in Angola.This study reports the prevalence,intensity and risk factors for schistosomiasis and STH infection in Huambo,Uige and Zaire provinces,Angola,to inform a school-based preventive chemotherapy program.Methods:A two-stage cluster design was used to select schools and schoolchildren to participate in parasitological and water,sanitation and hygiene(WASH)surveys across Huambo,Uige,and Zaire provinces.Point-of-care circulating cathodic antigen and urinalysis rapid diagnostic tests(RDTs)were used to determine the prevalence of Schistosoma mansoni and S.haematobium,respectively.Kato-Katz was used to identify and quantify STH species and quantify and compare with RDTs for S.mansoni.Urine filtration was used to quantify and compare with RDTs for S.haematobium.Descriptive statistics were used for prevalence and infection intensity of schistosomiasis and STH infection.Performance of RDTs was assessed through specificity and Cohen’s Kappa agreement with microscopy.A multivariate regression analysis was used to determine demographic and WASH factors associated with schistosomiasis and STH infection.Results:A total 575 schools and 17,093 schoolchildren participated in the schistosomiasis survey,of which 121 schools and 3649 schoolchildren participated in the STH survey.Overall prevalence of S.mansoni was 21.2%(municipality range 0.9–74.8%)and S.haematobium 13.6%(range 0–31.2%),with an overall prevalence of schistosomiasis of 31.4%(range 5.9–77.3%).Overall prevalence of Ascaris lumbricoides was 25.1%(range 0–89.7%),hookworm 5.2%(range 0–42.6%),and Trichuris trichiura 3.6%(range 0–24.2%),with an overall prevalence of STH infection of 29.5%(range 0.8–89.7%).Ecological zone and ethnicity were factors associated with schistosomiasis and STH infection,with older age and female sex additional risk factors for S.haematobium.Conclusions:Most municipalities met World Health Organization defined prevalence thresholds for a schistosomiasis preventive chemotherapy program.A STH preventive chemotherapy program is indicated for nearly all municipalities in Uige and select municipalities in Huambo and Zaire.The association between ecological zone and ethnicity with schistosomiasis and STH infection necessitates further evaluation of home and school environmental,sociodemographic and behavioural factors to inform targeted control strategies to complement preventive chemotherapy programs.

Selecting accurate post-elimination monitoring tools to prevent reemergence of urogenital schistosomiasis in Morocco:a pilot study

Background:After alleged stop of transmission of schistosomiasis and further down the line in post elimination settings,sensitive tools are required to monitor infection status to prevent potential re-emergence.In Rahala,where transmission cycle of Schistosoma haematobium is interrupted since 2004 but where 30%of snails are still infected by S.bovis,potential human S.bovis infection can’t be excluded.As methods based on egg-counts do not provide the required sensitivity,antibody or antigen assays are envisaged as the most appropriate tools for this type of monitoring.Methods:In this pilot study,the performances of three assays were compared:two commercially available antibody tests(ELISA and haemagglutination format)indicating exposure,and an antigen test(lateral flow strip format)demonstrating active infection.All 37 recruited study participants resided in Rahala(Akka,province Tata,Morocco).Participants had been diagnosed and cured from schistosomiasis in the period between 1983 and 2003.In 2015 these asymptomatic participants provided fresh clinical samples(blood and urine)for analysis with the aforementioned diagnostics tests.Results:No eggs were identified in the urine of the 37 participants.The haemagglutination test indicated 6 antibody positives whereas the ELISA indicated 28 antibody positives,one indecisive and one false positive.ELISA and haemagglutination results matched for 18 individuals,amongst which 5 out of 6 haemagglutination positives.With the antigen test(performed on paired serum and urine samples),serum from two participants(cured 21 and 32 years ago)indicated the presence of low levels of the highly specific Schistosoma circulating anodic antigen(CAA),demonstrating low worm level infections(less than 5 pg/ml corresponding to probably single worm pair).One tested also CAA positive with urine.ELISA indicated the presence of human anti-Schistosoma antibodies in these two CAA positive cases,haemagglutination results were negative.Conclusions:To prevent reemergence of schistosomiasis in Morocco current monitoring programs require specific protocols that include testing of antibody positives for active infection by the UCP-LF CAA test,the appropriate diagnostic tool to identify Schistosoma low grade infections in travelers,immigrants and assumed cured cases.The test is genus specific will also identify infections related to S.bovis.