消灭丝虫病地区病原学监测方法的研究

为筛选适宜在消灭丝虫病地区应用的简易、经济、实用的病原学监测方法,对单克隆抗体（McAb）-ELISA检测丝虫特异IgG4和快速免疫色谱技术（ICT）检测血清和血浆中的班氏丝虫抗原诊断丝虫病的效果进行对比研究。结果检测班氏微丝蚴血症者59例,丝虫特异IgG4阳性57例,阳性率96.61%;ICT丝虫抗原阳性56例,阳性率为94.92%。两种方法均具有较高的敏感性,差异无显著性（P＞0.05）。同时分别检测非流行区健康者40人,囊虫病患者30例及25例华支睾吸虫病患者血清,两种方法均呈阴性,未出现交叉反应,特异性为100%。现场研究两种方法分别检测基本消灭丝虫病后出生儿童302人,消灭丝虫病地区人群372人,晚期丝虫病患者55例（乳糜尿41例,象皮肿14例）,原微丝蚴血症转阴者60例。结果仅消灭丝虫病地区人群中1例（以往血检阴性）丝虫特异IgG4强阳性,ICT丝虫抗原弱阳性,但反复镜检未发现微丝蚴。其余均为阴性。研究表明,两种方法均具有较高的敏感性和特异性,均可用于消灭丝虫病地区病原学的监测。McAb-ELISA检测丝虫特异IgG4可大面积用于消灭丝虫病地区的病原学监测。而ICT检测班氏丝虫抗原更适用于个案病例的检测。     

Field trial of the ICT filariasis for diagnosis of Wuchereria bancrofti infections in an endemic population of Thailand

The ICT Filariasis, a rapid card test format, which is based on qualitative detection by monoclonal antibody of the circulating antigen of Wuchereria bancrofti adult worm, is a new diagnostic test of choice for determining the infections under field conditions. By using clinical and recall techniques and microscopy (thick smear and capillary tube technique) as reference, we assessed the efficiency of the ICT card test in sera from 225 subjects living in W. bancrofti-endemic villages of Tak Province, Thailand, who were recruited during a cross-sectional community survey. The ICT card test gave a 20% antigen positive rate, while other tests gave lower positive rates of the same 5.8% by clinical and recall techniques and thick smear, and 5.3% by capillary tube technique, respectively. The ICT card test had a specificity of 100% when sera from microfilaremic subjects were positive, as when sera from W. bancrofti non-endemic subjects either with Brugia malayi microfilaremia or with other parasites, and those from normal controls were all negative by the test. When done in W. bancrofti microfilaremia sera, the ICT card test had a sensitivity of 100% using a microscopy as reference, and 84.6% when using clinical and recall techniques. However, the ICT card test was more sensitive than the others when done in endemic normal sera (14% positive). Such findings compared well with findings in endemic area of South America, suggested its usefulness to detect W. bancrofti infections in endemic area, especially on the Thai-Myanmar border.     

Use of Brugia Rapid dipstick and ICT test to map distribution of lymphatic filariasis in the Democratic Republic of Timor-Leste

The newly-introduced Brugia Rapid dipstick for filarial antibodies and ICT filarial antigen card test were used to confirm historical data on the distribution of lymphatic filariasis in the Republic of Timor-Leste. Twelve out of thirteen districts were confirmed as being endemic. Brugian filariasis predominates, with an average prevalence of 11.6%. The average prevalence of Bancroftian filariasis was 1.1%. The study demonstrated that the Brugia Rapid test can provide useful information about the distribution of Brugian filariasis in circumstances where it is difficult or impossible to obtain night blood samples for microfilariae.     

免疫色谱技术用于班式丝虫病诊断及丝虫病防治后期监测的实验研究

目的：评价免疫色谱技术（ＩＣＴ）用于班氏丝虫病诊断及丝虫病防治后期监测的效果。方法：丝虫病ＩＣＴ系采用单克隆抗体检测血中班氏丝虫抗原。结果：在１１６例班氏微丝蚴血症者中有１１１例ＩＣＴ阳性，敏感性为９５．７％。1２例马来微丝蚴血症者、３３例蛔虫感染者、２０例血吸虫感染者及６例旋毛虫感染者血清，ＩＣＴ均为阴性，表明其特异性 强；７３例晚期丝虫病患者有１８例ＩＣＴ阳性，为活动性感染，其中１６例伴有微丝蚴血症；用伊维菌素治疗３０例班氏微丝蚴血症者，治疗前ＩＣＴ阳性２９例（９６．７％）。治疗后８ｄ—１４ｄ微丝蚴全部转阴，追踪观察６个月及１２个月，分别有５例及７例微丝蚴复现，治疗后６个月用ＩＣＴ测试，除５例ＩＣＴ和微丝蚴均阳性外，８例微丝蚴阴性ＩＣＴ阳性者于疗后１２个月有５例查见微丝蚴；ＩＣＴ及常规血检法同步用于河南省柘城县候大村丝虫病防治后期监测，血检１３２人，９例微丝蚴阳性者ＩＣＴ均为阳性，血检阴性的１２３人中ＩＣＴ阳性者１例。结论：实验研究证实，ＩＣＴ法快速、简便、敏感性高和特异性强，可用于班氏丝虫病诊断、疗效考核及丝虫病防治后期监测。     

Rapid assessment for lymphatic filariasis in central Nigeria: a comparison of the immunochromatographic card test and hydrocele rates in an area of high endemicity

The rapid immunochromatographic card test (ICT) for Wuchereria bancrofti circulating filarial antigen is being used to map areas endemic for lymphatic filariasis. However, the ICT is expensive; thus, surveys based on this test must be relatively limited. Our study was conducted to determine if village-based hydrocele surveys could be used to supplement the ICT surveys in the mapping activities. We compared in 144 Nigerian villages the two assessment methods, ICT and examination for clinical hydrocele, in random samples of 30 adults selected using a procedure that obtained 15 younger males (reported age = 16-39 years old) and 15 older males (> or = 40 years), based on the assumption that hydrocele rates may be more prevalent in older age groups. The men were asked if they had scrotal swelling, then examined and tested by the ICT. We found a weakly positive correlation between village prevalence determined by the ICT and hydrocele (r = 0.041, P < 0.001). Only villages with hydrocele rates of 20% or greater were also consistently classified as having endemic filariasis by the ICT. There was no correlation between an individual's ICT positivity and clinical presence of hydrocele, and questioning about scrotal swelling was not predictive for presence of hydrocele. More research is needed to determine if community level hydrocele prevalence surveys can offer an economical and broadly applicable supplement to the ICT for determining the endemicity of filariasis.     

Detection of filarial antigen using antibodies raised against Wuchereria bancrofti microfilarial SDS soluble antigen.

Polyclonal antibodies raised in mouse ascitic fluid against Wuchereria bancrofti microfilarial antigens (Wb Mf SDS S Ag) were studied for their diagnostic use in bancroftian filariasis using a dip stick, enzyme-linked immunosorbent assay. In sandwich ELISA, 100% of microfilaremic sera (30 out of 30) 53% of acute filarial sera (7/13), 40% of subacute filarial sera (6 out of 15), 13% of chronic filarial sera (2/15) and 20% of endemic area normal sera (3/15) showed the presence of filarial antigen. Determination of filarial antigen titer in microfilaremic sera showed an apparent positive correlation between microfilarial density and antigen titer. The antibody raised against Wb Mf SDS S Ag was found to be cross reactive with phosphorylcholine epitopes. The filarial antigen detected by anti Wb Mf SDS S Ag antibodies in sandwich ELISA is possibly associated with the active stage (microfilaremia) of infection.     

Detection of circulating parasite antigen in bancroftian filariasis by counterimmunoelectrophoresis

We used counterimmunoelectrophoresis (CIEP) with rabbit antibodies to Dirofilaria immitis and Brugia malayi to detect soluble filarial antigen in sera collected in a Wuchereria bancrofti-endemic area in South India. Filarial antigen was detected in 38 of 38 sera from microfilaremic patients, 3 of 48 sera from amicrofilaremic patients with lymphatic pathology, and 3 of 5 sera from former microfilaria carriers with negative blood examinations 6 months or more after diethylcarbamazine therapy. One of 32 endemic control sera, 0 of 35 nonendemic sera, and 0 of 20 B. malayi sera were positive. Antigenemia was equally detectable in sera collected at night or during the day (when microfilariae are absent from the blood). Parasite antigen was also detected in the urine of patients with positive serum tests. Antibodies to circulating filarial antigen (also detected by CIEP) were absent in all but 2 antigen-positive sera but present in 22 of 45 antigen-negative sera from clinical filariasis patients and in 9 of 31 antigen-negative sera from endemic controls. Parasite antigen detection by CIEP appears to be a sensitive, specific, and practical diagnostic test for active W. bancrofti infection.     

Evaluation of Og4C3 antigen ELISA as a tool for detection of bancroftian filariasis under lymphatic filariasis elimination programme

Diagnosis of Lymphatic Filariasis by microscopic examination of thick blood films (TBF) collected between 8.30 pm to 12 midnight, though highly specific is operationally problematic. We evaluated the TropBio Og4C3 serum ELISA as a tool for detection of W. bancrofti microfilaria carriers using Dried Blood Spots (DBS). The study was carried out in two parts (i) to test the sensitivity and specificity of the ELISA test for detection of circulating filarial antigen (CFA) in microfilaria (Mf) carriers vis-à-vis the conventional thick blood film (TBF) microscopy and its persistence in different categories of individuals during the course of disease viz., Endemic normals (n=51), microfilaria (Mf) carriers (n=27), acute cases (n=27), chronic cases (n=50) and a control group of non-endemic normals (n=48) using sera samples and ii) to study the utility of finger prick Dried Blood Spots (DBS) collected on filter paper for detection of Mf carriers and its comparison with another antigen detection assay, the Immunochromatographic test (ICT).Considering the non-endemic normals and microfilaria carriers, the ELISA test was found to have 100% sensitivity and 94.12% specificity for detection of Mf carriers in sera samples. The CFA was absent in majority of the subjects tested under other categories with a positivity of 7.8% among endemic normals, 11.12% among acute cases, 7.84% among chronic cases and 6.25% among nonendemic normals. Comparison of finger prick DBS and sera samples by ELISA vis-à-vis the ICT, carried out on Mf carriers (n=91) and endemic normals (n=97), showed a positivity of 88 (96.7%) in DBS as against 86 (94.5%) in sera samples and 88 (96.7%) by ICT, amongst Mf carriers, with a statistically significant correlation in antigen units between sera and DBS samples (r = 0.959, p = 0.000) amongst the microfilaria carriers. Out of 97 endemic normals, 19 (19.6%) sera and 17 (17.5%) DBS samples tested positive by ELISA while as 12(12.4%) tested positive by ICT, again with a statistically significant correlation between the antigen units in sera and DBS samples (r = 0.942, p = 0.000). DBS prepared from 25 microl of blood were found to be as sensitive as 50 microl for antigen detection. Antigen positivity detected from DBS collected during day and night from known microfilaria carriers (n=27) showed a statistically insignificant difference (p = 0.125) and a significant correlation in antigen units (r = 0.820 and p = 0.013).In view of the comparable results of ELISA, ICT and TBF microscopy, it is concluded that the TropBio Og4C3 ELISA using finger prick DBS can be used as an alternate to TBF microscopy for detection of bancroftian Filariasis under the LFE programme.     

Filarial antigen in circulating immune complexes from patients with Wuchereria bancrofti filariasis

Detection of filarial antigen in circulating immune complexes from patient sera was performed by an enzyme immunoassay in which the immune complexes were precipitated in the cold with polyethylene glycol and then dissociated in an acid pH buffer before being added to an ELISA plate. The dissociated antigen bound to the plate where it could be detected by peroxidase-labeled polyclonal rabbit antifilarial antiserum. Control sera used for defining the specificity of the assay included sera with immune complexes not related to parasite infection with and without free parasite antigen added prior to polyethylene glycol precipitation as well as sera from normal individuals. Filarial antigen was detected in the circulating immune complexes from 10 of 28 patients with bancroftian filariasis residing in either the Cook Islands (subperiodic Wuchereria bancrofti) or India (periodic W. bancrofti). By immunoblotting, the most frequently identified filarial antigen in these complexes was an approximately equal to 200 kDa circulating antigen.     

Comparative sensitivity and specificity of ELISA and IHA for serodiagnosis of strongyloidiasis with larval antigens

Sera from 68 patients with parasitologically proven strongyloidiasis were tested by the ELISA and IHA tests using larval antigens prepared from Strongyloides stercoralis and Strongyloides ratti. The ELISA using the S. stercoralis antigen detected the greatest number of sero-reactors (83.8%), whereas the IHA using the S. ratti antigen detected the fewest (55.9%). In addition, the S. stercoralis antigen had higher geometric mean titers than the S. ratti antigen in both the ELISA and the IHA tests. Sera from 37 presumed normal individuals also were tested by IHA and ELISA and nonspecific reactions were seen only with the IHA test. When sera from patients with parasitic infections other than strongyloidiasis were tested, the only consistent cross-reactions were with those sera from patients who had occult filariasis and acute schistosomiasis.     

Differential recognition of microfilarial antigens by sera from immigrants into an area endemic for brugian filariasis

Studies in animal models indicate that antibodies to surface antigens of microfilariae participate in the control of parasitaemia resulting from infections with lymphatic filarial nematodes. In an attempt to identify parasite antigens that elicit such 'protective' host responses, we compared the antigen recognition patterns of persons who remained amicrofilaraemic after 3-6 years of exposure to Brugia malayi with those of individuals who developed patent filariasis during the same period. IgG antibodies in sera from immigrants identified between 0 and 25 microfilarial antigens on Western blots. The highest degree of reactivity was observed with antigens in the 65-75 kD and 20-30 kD ranges, and with a group of high mol. wt antigens (greater than 180 kD). Sera from amicrofilaraemic donors preferentially reacted with 70/75 kD microfilarial antigens. A proportion of such sera inhibited the binding of monoclonal antibody MF1 to its target epitope; eight of nine inhibitory sera were from patients with active infections, evidenced by the presence of microfilariae or filarial antigens in the donors' blood, but who were amicrofilaraemic. These results indicate that some amicrofilaraemic residents of areas where brugian filariasis is endemic develop immune reactions to a microfilarial stage-specific antigen that was previously identified as a potentially 'protective' parasite antigen in animal models of lymphatic filariasis.     

免疫色谱技术用于班氏丝虫病诊断及丝虫病防治后期监测的实验研究

目的：评价免疫色谱技术（ＩＣＴ）用于班氏丝虫病诊断及丝虫病防治后期监测的效果。方法：丝虫病ＩＣＴ系采用单克隆抗体检测血中班氏丝虫抗原。结果：在１１６例班氏微丝蚴血症者中有１１１例ＩＣＴ阳性，敏感性为９５．７％。１２例马来微丝蚴血症者、３３例蛔虫感染者、２０例血吸虫感染者及６例旋毛虫感染者血清，ＩＣＴ均为阴性，表明其特异性强；７３例晚期丝虫病患者有１８例ＩＣＴ阳性，为活动性感染，其中１６例伴有微丝蚴血症；用伊维菌素治疗３０例班氏微丝蚴血症者，治疗前ＩＣＴ阳性２９例（９６．７％）。治疗后８ｄ—１４ｄ微丝蚴全部转阴，追踪观察６个月及１２个月，分别有５例及７例微丝蚴复现，治疗后６个月用ＩＣＴ测试，除５例ＩＣＴ和微丝蚴均阳性外，８例微丝蚴阴性ＩＣＴ阳性者于疗后１２个月有５例查见微丝蚴；ＩＣＴ及常规血检法同步用于河南省柘城县候大村丝虫病防治后期监测，血检１３２人，９例微丝蚴阳性者ＩＣＴ均为阳性，血检阴性的１２３人中ＩＣＴ阳性者１例。结论：实验研究证实，ＩＣＴ法快速、简便、敏感性高和特异性强，可用于班氏丝虫病诊断、疗效考核及丝虫病防治后期监测。     

Symptoms reported after mass drug administration for lymphatic filariasis in Leogane, Haiti

Mass drug administration (MDA) for lymphatic filariasis (LF) can cause adverse reactions from microfilarial and adult worm death. Symptoms after the fifth annual MDA in Leogane, Haiti, were studied to determine whether they resulted from parasite death. Persons reporting post-MDA systemic symptoms at 5 of 148 drug distribution posts and men reporting scrotal pain at any post were interviewed. Participants were tested with immunochromatographic tests (ICTs), and men with scrotal symptoms were examined. At the five posts, 3,781 persons took anti-filarial medication. Of these, 314 (8%) returned with symptoms; the most common were headache (36%) and gastrointestinal complaints (28%). Of the 294 (94%) who consented to ICT testing, 47 (16%) were positive. Of 69 men with scrotal symptoms who consented to ICT testing, 18 (26.1%) were positive. After Leogane's fifth MDA, most symptomatic persons had undetectable levels of filarial antigen by ICT. Free symptomatic treatment may motivate some people to report symptoms and seek care.     

Evaluation of fractionated circulating filarial antigen in diagnosis of bancroftian filariasis.

Circulating filarial antigen (CFA) isolated from the plasma of microfilaraemic patients was fractionated on an Ultrogel ACA 34 column. The second protein peak (CFA2) showing filarial antigen was further fractionated by DEAE-cellulose column chromatography into two fractions (CFA2 DE1 and DE2). CFA2 DE1 fraction, showing antigenic activity, was further evaluated in an ELISA for its diagnostic use in bancroftian filariasis. Studies with CFA2 DE1 and anti-CFA2 DE1 antibody showed that they were highly active in the detection of filarial antibody and antigen in asymptomatic microfilaraemia sera and thus obviate the need for the tedious night blood collection and examination. Fractionated filarial plasma can be another candidate antigen for immunodiagnosis of bancroftian filariasis.     

Antigenemia is associated with low antibody response to carbohydrate determinants of a filarial surface antigen

The IgG response to a filarial surface antigen (DssdI) was evaluated in circulating antigen (Og4C3 test) positive and negative individuals from the Wuchereria bancrofti endemic region of Orissa, India. Circulating filarial antigen (CFA) positive individuals exhibited depressed antibody levels to DssdI and individuals with high antibody levels were invariably CFA negative. Low IgG levels to DssdI are associated with CFA positivity irrespective of microfilaraemia and clinical status. Thus asymptomatic microfilaraemic individuals with antigen positivity exhibited low IgG levels similar to symptomatic (chronic filariasis with elephantiasis, hydrocele) or asymptomatic amicrofilaraemic individuals with antigenemia. Western blot analysis revealed a band of approximately 210 kDa reactive with CFA-negative, but not with CFA-positive sera. DssdI was subjected to mild periodate oxidation to investigate the role of carbohydrate epitopes. The treatment considerably reduced the antigenic recognition of DssdI, stressing the immunodominance of carbohydrate residues of DssdI in infection-free individuals. In contrast, individuals with active infection failed to produce such antibodies to filarial surface carbohydrates.     

Human infection with Wuchereria bancrofti in Matara,Sri Lanka: the use,in parallel,of an ELISA to detect filaria-specific IgG4 in urine and of ICT card tests to detect filarial antigen in whole blood

The ICT card test to detect circulating filarial antigen and an ELISA that detects filaria-specific urinary IgG(4) were each used to screen 473 subjects from a community in Sri Lanka where Wuchereria bancrofti is endemic. When the ICT test was used as the gold standard, the ELISA was found to have a sensitivity of 91.2%. However, far more of the subjects were found ELISA-positive than ICT-positive (76.5% v. 31.1%). The youngest children studied (aged 1-10 years) were similar to the adult subjects in terms of the prevalence of antigenaemia (33.8%) and the prevalence (72.1%) and concentration of filaria-specific IgG(4) in their urine. Therefore, especially as urine samples are easier, less painful and safer to collect than blood samples, the ELISA may be particularly useful to screen very young and school-age children, to estimate current levels of transmission in a particular area.     

河南省基本消除丝虫病后残存微丝蚴血症者传播作用的研究

目的探讨基本消除丝虫病后残存微丝蚴血症者在不采取任何病原防治措施下的传播作用。方法选择柘城县张庄村为观察点,病原学监测和蚊媒监测采取常规方法,血清学检测采用IFAT、ELISA、Dot-ELISA(查抗体)和ICT(查循环抗原)方法,同时进行居民防蚊情况调查。结果1990年残存微丝蚴血症者39人,人群微丝蚴率2.01%(39/2040),2000年微丝蚴血症者全部转阴,人群微丝蚴率降为0;1990、1991和1994年检测人群丝虫抗体阳性率和抗体阳性者的几何平均滴度均逐渐下降,1998年检测92名12岁以下儿童血中丝虫循环抗原均为阴性;淡色库蚊幼丝虫自然感染率逐年下降,自1997年未再发现感染蚊;1990~2002年当地居民经济收人逐渐增加,普遍采取防蚊措施。结论基本消除丝虫病后,随居民经济和文化水平的逐渐提高,防病意识的不断增强以及防蚊措施的加强,即使未采取任何干预措施,残存微丝蚴血症者也不能引发丝虫病流行,丝虫病传播已经被阻断。     

A monoclonal antibody-based enzyme immunoassay for detecting parasite antigenemia in bancroftian filariasis

We evaluated a monoclonal antibody-based enzyme immunoassay for detecting soluble parasite antigen in sera collected in an area in South India endemic for Wuchereria bancrofti. Filarial antigen was detected in sera from 56 of 57 microfilaremic patients, 9 of 64 aminofilaremic patients with clinical filariasis, and 11 of 70 endemic controls. Antigen was not detected in sera from patients from nonendemic areas who had a variety of other filarial and nonfilarial helminth infections. Parasite antigen titers were significantly correlated with microfilarial counts in night blood smears (r = .64, P less than .01). Negative antigen tests in patients with clinical filariasis may be explained in part by antibody-mediated clearance of circulating antigen. Antibodies to circulating W. bancrofti antigen were detected in 41 of 55 antigen-negative sera from patients with clinical filariasis. Despite this limitation, detecting parasite antigen by enzyme immunoassay provides significant advantages over previously available methods for diagnosing active W. bancrofti infection.     

华支睾吸虫病金标诊断试剂盒的研制和现场初步实验

目的　研制华支睾吸虫病金标快速免疫诊断试剂盒 ,评价其敏感性、特异性和现场试用效果。　方法　以华支睾吸虫成虫水溶性抗原、分泌排泄抗原和重组抗原磷酸甘油酸激酶 (PGK)作为诊断试剂 ,制备胶体金免疫层析检测 (Im-munochromatography test,ICT)试剂盒 ,检测患者血清和唾液中抗华支睾吸虫 Ig G,并与常规 EL ISA血清学检测方法和粪便虫卵检查法相比较。　结果　实验室检测临床确诊的华支睾吸虫病人血清中特异的 Ig G,3种抗原的敏感性均为10 0 %。天然抗原除与慢性血吸虫病人血清有较强的交叉反应外 ,与囊虫、包虫、弓形虫病人血清没有交叉反应。重组PGK抗原同慢性血吸虫病人血清也没有交叉反应。用分泌排泄抗原制备的 ICT检测试剂盒在流行区现场检测被调查者的血清和唾液 ,总符合率为 92 .31% ;ICT与 EL ISA血清检测的总符合率为 81.5 4 %。现场获取了 9人的粪便样本 ,其中 4人检出华支睾吸虫卵 ,其血清 ICT和 EL ISA检测均呈强阳性 ,另外未检出虫卵的 5人中 ,1人血清 ICT和 EL ISA均呈阳性 ,另 1人仅血清 EL ISA呈弱阳性。　结论　诊断华支睾吸虫感染的 ICT抗体检测技术 ,尤其是无创性唾液检测技术 ,简便、快速、准确、安全 ,优于血清 EL ISA检测和粪便虫卵检测 ,适用于临床检验和现场大规模流行病     

检测特异IgG4用于丝虫病监测的研究

应用细胞融合技术建立抗人ＩｇＧ４ＭｃＡｂ杂交瘤细胞，生产ＭｃＡｂ。以此为探针，应用ＥＬＩＳＡ方法检测班氏微丝蚴血症血清中特异ＩｇＧ４的阳性率为９５．９１％（４７／４９）；治疗后的原微丝蚴血症ＩｇＧ４阳性率为１．４５％（１／６９）；７７例晚期丝虫病症状者及５０份非流行区健康人血清均为阴性；肠道线虫感染、囊虫感染及华支睾吸虫感染者血清均未出现交叉反应；检测原班氏丝虫病高、中度流行区正常人群滤纸干血的特异ＩｇＧ４阳性率分别为０．７９％（６／７６０）和０．２７％（３／１０９５）。显示特异ＩｇＧ４检测具有较高的敏感性与特异性。是诊断丝虫感染的有效方法，可取代微丝蚴血检用于现场的人群丝虫病监测。