【作者】 林丹丹；

【导师】 吴观陵；

【作者基本信息】 南京医科大学 ， 病原生物学， 2008， 博士

【摘要】 血吸虫病是一种严重危害人类健康和阻碍疫区社会经济发展的寄生虫病,目前全球仍约有2.07亿人受感染,7.79亿人受威胁。上世纪80年代以来,高效、安全的血吸虫病化疗药物吡喹酮的应用加之其它防治措施的有效实施,我国疫区日本血吸虫感染率和感染度大幅度下降,但目前我国仍有血吸虫病病人67.13万。血吸虫病至今仍然是世界和我国主要公共卫生问题之一。诊断在血吸虫病防治活动中始终处于中心位置,它不仅为疫区分类提供标准,还为防治活动的各环节提供必不可少的信息和科学依据,包括在个体水平上确定化疗对象、考核化疗效果,更重要的是在社区(群体)水平上监测疫情变化、评估流行态势和考核疾病控制效果等。改良加藤厚涂片粪检法(Kato-Katz法)、间接红血球凝集试验(IndirectHemagglutination Assay,IHA)和酶联免疫吸附试验(Enzyme-LinkedImmunosorbent Assay,ELISA)是目前我国血吸虫病防治实践中最常用的三种诊断方法。基于病原检查的Kato-Katz法是直接诊断方法,它提供调查时“点”的感染状态信息。迄今为止,病原学检查仍是血吸虫病确定诊断的唯一途径和手段。虽然在低流行水平状态下,病原学检查的敏感性受到质疑,但因其不可替代的“确定诊断”价值,依然是我国目前疫区分类及评价疫区真实流行状态的唯一技术途径。目前,我国绝大多数血吸虫病疫区处于低流行状态,这种情况下,采用常规Kato-Katz法(1检3片)确诊病人,极易漏检,评估疫情,明显低估。基于抗体检出的IHA和ELISA是间接诊断方法,是所谓“期”的感染提示,其检测的定性结果不能区别现症感染和既往感染,在作为流行病学用途时,也难以提供真实的感染率或传播强度信息。虽然目前还没有一项免疫学诊断试验可以真正“替代”病原学诊断技术,但其敏感性提示的诊断意义,在一定程度上弥补了病原学诊断敏感性方面的不足,并赋予了其强劲的发展动力,在简便、安全的化疗措施易于实施和追求以疫情控制为主要目标的条件下,尤其如此。实际上,这两类诊断途径普遍地共同应用于我国当前血吸虫病防治活动中,因各自都具有特定的优缺点和局限性,使得结果解释在相当程度上带有主观色彩,甚至产生一定的矛盾和混乱,从而影响决策者的取舍。目前,我国正处在实现《全国预防控制血吸虫病中长期规划纲要(2004～2015年)》目标的进程中,防治工作者,尤其是疾病控制管理者与决策者比以往任何时候都迫切地希冀获得较为“精确”的疫区流行信息,以及时跟进不断变化的血吸虫病流行态势,指导血吸虫病防治工作。因此,作为提供信息的关键工具—诊断方法的科学、合理应用显得尤为重要。本项研究正是应对这一需求,以更加贴近防治活动的视角,针对当前血吸虫病诊断在防治活动中应用的若干问题加以研究和讨论。本研究设计涉及的诊断试验为目前我国血吸虫病防治中最常用的三种方法:Kato-Katz法、IHA和ELISA,研究关注的重点置于诊断试验结果的科学分析和解释。主要进行了以下三方面的研究:一、常规改良加藤厚涂片粪检法(Kato-Katz法)低估日本血吸虫病疫区人群血吸虫感染率的分析和评价在4个低感染度、不同流行程度(分别为一、二、三类疫区村)的血吸虫病疫区村,采用“2检6片”Kato-Katz法(2次粪样,每次粪样制作3张Kato-Katz片)对疫区常住居民进行整群调查,并以“2检6片”结果为金标准,分析、评估在我国当前血吸虫病流行状态下,Kato-Katz法对不同流行区疾病流行性的低估程度。结果显示:在一、二类疫区(试区1、2、3),1张Kato-Katz片对人群血吸虫感染率的平均低估率高达50%以上,即使增加到3张Kato-Katz片(目前大多数疫区为减少漏检而采用的片数)亦低估了17.0%～25.3%,有22.5%～33.6%的低感染度者(EPG≤40)不能检出;在三类疫区(试区4),1张Kato-Katz片平均低估率高达70%以上;3张Kato-Katz片达33.7%,有42.5%的低感染度者(EPG≤40)漏检。在群体水平上,采用相同Kato-Katz片数情况下,阳性检出率主要取决于Kato-Katz片数而不是粪检次数。感染度可能是Kato-Katz法低估疫区人群血吸虫感染率、确定Kato-Katz法合适应用片数的关键因素,病人感染度越低,得到正确诊断所需的Kato-Katz片数越多;而且,在感染度类似的疫区,尽管居民的感染率相差较大(一或二类疫区),但不同Kato-Katz片数对感染率的低估率及不同感染度病人确诊所需Kato-Katz片数类似。所获结果提示现行的常规“1检3片”Kato-Katz法,无论在哪类疫区应用,均严重低估了疫区感染率,其低估程度随着疫区流行程度的降低而增加。而且结果有助于了解目前疫情状态下,粪检次数、Kato-Katz片数及感染度等诸多因素对Kato-Katz法评估感染率的影响;并且对今后现场防治中正确应用Kato-Katz法、合理评估疫情及在此基础上确定防治措施有一定的指导意义;同时为建立日本血吸虫卵计量模型提供科学依据。二、IHA和ELISA现场应用价值的分析与评价在上述之一的一类血吸虫病疫区村(试区1),同时采用Kato-Katz法、IHA和ELISA3种方法对人群进行连续2年的纵向调查,开展定量血清流行病学研究。着重从定量方面,探讨血清学检测结果的群体特征与疫区疫情、传播状态或人群感染趋势的关系,科学解释抗体阳性率、抗体水平变化在血吸虫病流行病学调查中的意义和作用。结果显示:IHA和ELISA在现场实际应用时,以“2检6片”Kato-Katz法为金标准,它们的敏感度均较高(＞80%),特异度低(＜60%),阴性预测值好(93.1%～96.8%)。其中,对连续2年Kato-Katz法检查结果均为阳性和阴性的人群,IHA、ELISA的敏感度和阴性预测值则更高,分别达94.6%和99.2%。IHA、ELISA的特异度随着人群年龄的增加而降低,在低年龄组人群(＜15岁人群)的特异度最高。IHA和ELISA的人群抗体阳性率远高于粪检阳性率,但其年龄分布趋势均与Kato-Katz法粪检阳性率的年龄分布一致。IHA和ELISA的定性结果(阴性/阳性)与Kato-Katz法结果一致性较差,其Kappa值低于0.24(0.15～0.24)。而且IHA和ELISA的假阳性率较高(42.8%～61.1%),并存在假阴性(12.9%～21.8%)。感染者(虫卵阳性)治疗后,IHA和ELISA的抗体阳性率下降均缓慢。在某一时点上,IHA和ELISA的抗体水平难以反映个体或人群感染度,但人群抗体水平变化可反映人群感染状态的变化。结果提示在我国目前疫情状态下,IHA和ELISA在群体水平上更具应用价值;而应用抗体检测“筛查”抗体阳性,以确定疫区定期常规化疗的对象,或“抗体筛查法”(人群抗体检测后,抗体阳性再做病原学检查)确定疫区人群感染率,均有必要进一步探讨。我们认为,应开展各类疫区非感染人群的血清学研究,掌握疫区人群抗体水平的基线数据,以建立真正适于现场应用评估的“免疫学诊断技术评估方案”。今后血吸虫病血清流行病学研究值得探索的方向应包括:建立血吸虫病疫区血清学定量检测系统,发展和完善血吸虫病疫情评估和监测体系,并根据人群抗体水平特征构建血清学诊断试验确定疫区化疗人群、反映疫区流行状态及监测疫情变化和防治效果的模型。本研究结果进一步诠释了抗体检测法的检测结果及其应用价值,同时为发展血清学定量抗体检测试验系统提供了理论依据。三、IHA筛查法确定日本血吸虫病疫区人群感染率的评价选择1个二类血吸虫病疫区村(试区5),采用“2检12片”Kato-Katz法和IHA对人群同时进行调查。以“2检12片”结果为金标准,通过分析目前常规“1检3片”Kato-Katz法的阳性检出率与低估率、IHA的诊断效率及IHA与Kato-Katz法结果的相关性,从而评价目前我国疫情监测点普遍采用的IHA筛查法确定血吸虫病疫区人群感染率方法的可信性和科学性。结果显示,以“2检12片”结果为金标准,目前常规“1检3片”Kato-Katz法对人群血吸虫感染率的平均低估率高达32.3%;增加粪检次数可提高阳性检出率,2次粪检较1次粪检增加的检出率为30.2%。IHA的敏感度和特异度分别为69.6%和88.40%,阴性预测值为96.8%,但阳性预测值较低(36.8%),假阴性高达30.4%,假阳性为11.6%。由于IHA和“1检3片”Kato-Katz法均存在漏检,IHA筛查法对试点区人群感染率估算的漏检率达35.8%。结果提示,应用IHA筛查法确定疫区人群感染率与人群实际感染状况有较大的偏差。而且,由于IHA存在着明显的假阴性与假阳性,使用该方法诊断某一个体是否感染时的真实性较小,将其作为粪检前初筛工具的价值同样需要谨慎评价。由于我国血吸虫病疫区类型、分布范围、流行程度差异较大,本项研究对三种常用诊断方法的评价和分析结果,在作为类似疫区现场应用、疫情评估及防治措施拟订的参考信息的同时,有待在今后进一步的防治研究工作中获得验证。更多还原

【Abstract】 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.更多还原