| Schistosomiasis is a zoonosis that severely affects human health and hinders social-economic development in endemic countries.Nowadays,it is estimated that 207 million people are suffering from schistosomiasis while 779 million people are still at risk of infection in 76 countries and regions all over the world.Owing to the application of praziquantel,a highly efficacious and safe drug for treatment of schistosomiasis since 1980s and the comprehensive control approaches implemented,the prevalence and intensity of schistosomiasis have been decreased remarkably in China.It is reported that there are still 671.3 thousand schistosomiasis cases in the endemic areas of China in 2006.Thus,schistosomiasis remains one of major problems of public health in China as well as in the world.Diagnosis of schistosomiasis is always critical in the control programs. It provides not only criteria for endemic area classification but also indispensable information and scientific basis for the implementation of schistosomiasis control approaches.Diagnosis has been applied to target -subject selection for treatment and evaluation of treatment efficacy at the individual level,as well as to monitor dynamic change of endemicity,to evaluate epidemic situation and to assess control efficacy,etc.,at the population level.There are three diagnostic methods widely applied along with the schistosomiasis control program in China,i.e.,modified quantitative Kato-Katz thick smear technique(Kato-Katz method),Indirect Hemagglutination Assay(IHA)and Enzyme-Linked Immunosorbent Assay (ELISA). Kato-Katz method is a direct pathogen finding technique that quantitatively detect parasite egg and thus provides information of infection status at the investigation(time)"point".So far,definitive diagnosis of schistosomiasis still relies on parasitological techniques,mainly through the identification of eggs in stool by Kato-Katz method.Moreover,despite the fact that Kato-Katz method is known to have a low sensitivity especially in the endemic areas with low intensity,it is currently used as the unique approach for endemic area classification and true prevalence estimation in endemic fields in China because of its value as the definitive diagnostic method.Nowadays,in China,both prevalence and infection intensity have been drastically reduced due to national schistosomiasis control program and large scale population-based chemotherapy implemented over the past three decades.Thus,the prevalence of S. japonicum infection is surely to be underestimated in schistosomiasis endemic areas with low intensity in China,by routinely applied Kato-Katz method.Immuo-diagnostic methods including IHA and ELISA are indirect approaches based on detection of antibody in serum,which merely provide information of infection status for a period of time at the individuals or the population level.Therefore,the qualitative results detected by these approaches,in general,are not able to discriminate active and previous infection or re-infection,and also can not provide accurate epidemiological information of true prevalence or transmission intensity.Although no currently available immuno-diagnostic methods are good enough to take place of parasitological methods due to their limitation in specificity,in practice,these methods have been used as a compensation for diagnosis taking the advantage of their high sensitivity and hence heighten potential prospect of their application,especially under the conditions of safe and easy delivered chemotherapy available and of reducing prevalence as the control target.As a matter of fact,both kinds of diagnostic approaches,Kato-Katz and immuno-diagnostic methods(either IHA or ELISA),have their own advantages and disadvantages or limitation in their widespread application for control activities and thus lead to the confusion in understanding the results by the two types of diagnostic approaches.Therefore,the interpretation of results often varies due to the subjectivity of researchers or investigators.Sometimes,misinterpretation of the results even leads to contradictions or erroneous conclusion and more difficulties in decision-making by policymakers.China is now in the process of achieving the goals of "The national medium-and long-term planning schemes for schistosomiasis prevention and control(2004-2015)".Professionals engaging in schistosomiasis control and the policymakers of disease control are more eager to acquire accurate endemic information than they were at any time before so as to follow up the changing epidemic situation and figure out guidance to control activities.Hence,it is vital to apply and illustrate the results of diagnostic tests in a scientific and rational way in order to provide precise information.The subjective of this study is to closely meet these requiremnets to resolve some issues of diagnostic tests existing in application of schistosomiasis control from viewpoint of getting close to practical control activities.This study is designed to focus on the scientific analysis and interpretation of three diagnostic testes,namely,Kato-Katz, IHA and ELISA,which are being the most widely used in current schistosomiasis control program.The main contents and results of the study are as follows.1.Analysis and evaluation on the underestimation of prevalence of Schistosomiasis japonica in human population by routine Kato-Katz method in endemic areasFour villages with low intensity of infection and different infection rates(belonging to Class 1,2 3 endemic areas,respectively)in China were selected as the study sites.The method of Kato-Katz with 2 stool samples and 6 thick smears each was adopted to implement parasitological examination.The cumulative results of "2 samples and 6 smears" were used as the gold standard of diagnosis for estimating the prevalence of S. japonicum infection and the results were compared with theose based on fewer Kato-Katz thick smear readings to assess the degree of underestimation in different endemic areas.The results showed that,in Class 1 and Class 2 endemic areas,single Kato-Katz thick smear readings showed the rate of underestimation as high as over 50%.When increasing to three Kato-Katz thick smears,a typical diagnostic approach used in the national schistosomiasis control programme,still remain 17.0%～25.3%of positive rate was underestimated,about 22.5%～33.6%cases with low infection intensity (EPG≤40)were undetected.In Class 3 endemic areas,the rate of underestimation by single Kato-Katz thick smear readings was even higher than 70%,while with three Kato-Katz thick smears,33.7%of underestimation rate and 42.5%of overlook rate for cases with low infection intensity(EPG≤40)were defined.The result also indicated that at population level,accurate detection of infection status mainly depended on the number of thick smears examined rather than the number of stool samples when adopting same number of Kato-Katz thick smears.Moreover, it was found that the number of Kato-Katz thick smears required to secure detection of S.japonicum infection was close related with the intensity of infection.The number of Kato-Katz thick smears needed to be increased as the infection intensity decreased.In the endemic areas with similar intensity of infection,it was similar in the underestimation rate of different number of Kato-Katz thick smears and the number of Kato-Katz thick smears required to secure detection of S.japonicum infection in different endemic areas with various infection rates(Class 1 and Class 2 endemic region).EPG(the intensity in egg-positive subjects)is probably a key index to determine the degree of underestimation and proper number of Kato-Katz thick smears.In conclusion,our results indicate that the prevalence of S.japonicum infection based on the routine Kato-Katz method with "1 sample and 3 smears" in a community is generally considerably underestimated.The degree of underestimated rate increased as the prevalence decreased in endemic areas.Moreover,our findings lead to a better understanding of the influence by different factors such as number of stool samples,number of Kato-Katz thick smears and infection intensity and thus provide a benchmark for proper application of the Kato-Katz technique and for rational evaluation of epidemic situation,as well as a scientific basis for constructing a mathematic diagnostic model. 2.Analysis and evaluation of IHA and ELISA for the diagnosis of S.japonicum infection in an endemic areaQuantitative sero-epidemiological study was carried out in a Class 1 endemic village.Residents were simultaneously examined by Kato-Katz technique for parasitological stool examination,as well as by immuno-diagnostic techniques IHA and ELISA for detection of IgG antibody against soluble egg antigen for two consecutive years.The results of examination were compared on the performance characteristics IHA and ELISA with Kato-Katz.With respect to superiority of quantitative analysis, the study focused on exploring the correlation of population characteristics of sero-reactivity with quantitative antibody based-IHA and ELISA and transmission parameters,such as epidemic situation,transmission status or infection trend in population.We were also striving toward interpreting scientifically their epidemiological signification of serologic data and evaluating comprehensively the utility and potential of IHA and ELISA in field application of schistosomiasis control.When Kato-Katz technique based on 2 stool samples,each read in 3 thick smears,was used as the reference,the overall sensitivity of IHA and ELISA was higher than 80%with a relatively poor specificity of lower than 60%,the NPV of IHA and ELISA was excellent of higher than 93% (ranged from 93.1%to 96.8%).Especially to those egg positive or negative in two consecutive years,IHA and ELISA showed its sensitivity and NPV as high as 94.6%and 99.2%.The specificity of IHA and ELISA decreased with the increase of the age in different age-groups population,showing its hightest among the younger less than aged 15 years.The distribution trends of positive rate of antibody in different age groups by IHA and ELISA showed similar to that of egg positive detected by Kato-Katz,but a big gap was observed when the results of IHA and ELISA(antibody positive)were compared to Kato-Katz in terms of Kappa indices(less than 0.24 ranged from 0.15 to 0.24).This showed that a higher false positive(range from 42.8%to 61.1%)and a certain false negative(ranged from 12.9%to 21.8%) existed in IHA and ELISA.The positive rate of antibody decreased slowly among the individuals with S.japonicum infection,who received treatment. The results also suggested that IHA and ELISA was not useful for determining the intensity of infection of individuals or populations at investigation(time)point,but the changes in antibody levels could be used as the changes of infection status with S.japonicum in different populations.We conclude,under the current epidemic situation in China,IHA and ELISA are valuable in their utility of epidemiologic surveys.We suggest that it should be further deliberation when applying IHA and ELISA as the screening approaches for identification of target individuals for treatment or determination of infection rate in community(combined with parasitological examination).It is necessary to investigate the baseline of antibody level in non-infected population in different endemic areas for formulating an assessment scheme of immuno-diagnosis technique,which is suitable to evaluate diagnostic assays for field use.Some proposes for further sero-epidemiological studies are put forward,such as,to establish a system of quantative antibody-based immuological assays for better evaluation and surveillance of epidemic situation as well as estimation of prevalence at the population level,and to set up the models to clarify the correlation of population characteristics of sero-reactivity with quantitative antibody based-IHA and ELISA and epidemic situation,transmission status or infection trend in population and so on.This study further interpreted test results and the utility in field application of antibody-based IHA and ELISA.Meanwhile,our results provided scientific basis for developing a system of quantative antibodybased immuological assays.3.Evaluation on estimation of prevalence of S.japonicum infection by IHA screening methodIn a Class 2 endemic village,the residents were simultanously examined by Kato-Katz technique with "2 samples and 12 smears" for parasitological examination and by IHA quantitative immunodiagnostic techniques for antibody detection.Taking results of 12 Kato-Katz thick smears as the gold standard,diagnostic yield of current routine Kato-Katz method(1 stool samples and each read in 3 thick smears)and IHA,as well as the correlation of the intensity of infection and IHA were analyzed systematically,so as to evaluate the reliability and creditability of IHA screening method for estimating the prevalence of S.japonicum infection in endemic areas.The results showed that the average underestimated rate of triplet Kato-Katz thick smears was high to 32.3%.The increasing rate of the positive derived from duplicate stool sample was 30.2%compared with the results of one samples.The sensitivity and specificity of IHA were 69.6%and 88.4%,respectively.The predicted value of the negatives was 96.8%but 36.8%in the positives.The missing rate of IHA(false negative, i.e.egg positive but sero-negative)and false positive rate(egg-negatives but sero-positives)were up to 30.4%and 11.6%,respectively. Consequently,the missing rate of IHA screening method was estimated to be 35.8%according to the formula recommended by MOH(Ministry of Health).The results confirmed that IHA screening method considerably underestimated actual prevalence of schistosomiasis in endemic areas due to the missing of infected cases by both IHA and Kato-Katz.Moreover, there were false negative and false positive cases by IHA.Therefore,IHA could only be used as an auxiliary diagnostic method of schistosomiasis. Therefore,we suggest that marked caution be taken in assessing the utility of IHA as an initial screening tool applied before stool examination.Because of great heterogeneity regarding to the type of endemic areas in China,together with the geographic distribution and prevalence of S. japonicum infection,it is our expectation that the results from this study are to be served as the reference for application of similar endemic areas with our pilots or evaluation of epidemic situation and formulation of control measures,also merit further verification of investigations.
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