Co-agglutination test for the detection of circulating antigen in amebic liver abscess.

We report here a simple and economical slide agglutination test, the co-agglutination (Co-A) test, for the detection of circulating amebic antigen in sera for the diagnosis of amebic liver abscess. Fifty serum specimens from cases of amebic liver abscess, 25 from other individuals with parasitic and miscellaneous infections, and 25 from healthy controls were tested for the presence of serum antigen by the Co-A test. Forty-five (90%) amebic liver abscess sera were found to be amebic-antigen positive by the Co-A test. None of 25 sera from healthy controls were positive for the antigen. However, false-positive results were seen with two sera from those with other parasitic and miscellaneous infection controls. These results show that the Co-A test can be used as a sensitive and specific rapid slide agglutination test for the detection of amebic antigen in the sera for diagnosis of cases of amebic liver abscess in a routine parasitology laboratory.     

Antigenic stability and immunodominance of the Gal/GalNAc adherence lectin of Entamoeba histolytica

Immunoprecipitation of Entamoeba histolytica proteins was performed with the sera of patients recovered from amebic liver abscess and colitis. The patients' amebic infection had been acquired in diverse areas of the world. The amebic galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin was the major amebic antigen immunoprecipitated. The adherence lectin was recognized by all of the patients' sera tested regardless of the site (liver abscess vs. colitis) or geographic region that the amebic infection had occurred.     

用聚合酶链反应检测血清标本诊断阿米巴肝脓肿的价值

用聚合酶链反应（ Ｐ Ｃ Ｒ）检测阿米巴肝脓肿患者血清标本中的溶组织内阿米巴 ３０ ０００ 蛋白基因,结果４２ 例患者中有３５ 例呈阳性反应,阳性率为８３．３％ ,低于脓标本 Ｐ Ｃ Ｒ阳性率（１００％ ）（ Ｐ＜ ０．０１）。３ 例细菌性肝脓肿、１ 例肝癌及１０ 例其它部位脓肿患者的血清和脓对照标本 Ｐ Ｃ Ｒ均呈阴性反应。对 Ｐ Ｃ Ｒ检测血标本诊断阿米巴肝脓肿的价值进行了讨论。     

Comparison of serologic tests for the diagnosis and follow-up of alveolar hydatid disease

Alveolar hydatid disease is a serious and often fatal condition caused by infection with the metacestode form of Echinococcus multilocularis. Sera of 21 patients with histologically confirmed disease were tested by an enzyme-linked immunosorbent assay (ELISA) using a semi-purified E. multilocularis antigen fraction (Em2) and by indirect hemagglutination (IHA) and double diffusion (DD5) tests using antigens prepared from E. granulosus cyst fluid. At diagnosis, sera from all 21 patients were positive by Em2 ELISA, 18 (86%) by IHA, and 5 (24%) by DD5. Em2 ELISA detected an antibody response earlier than IHA in 4 of 9 patients from whom sera were available before diagnosis. Following complete surgical resection, Em2 ELISA converted from positive to negative in serum of 2 of 3 patients, while IHA results did not change. Following incomplete resection, 14 of 15 patients tested remained positive by Em2 ELISA, while 12 remained positive by IHA. Of sera from 361 healthy persons from regions free of E. multilocularis, none were positive by Em2 ELISA, while 8% were positive by IHA. Of sera from 59 patients with non-echinococcal parasitic infections, none were positive by Em2 ELISA, while 31% were positive by IHA. Thus, in comparison with tests using E. granulosus antigens, Em2 ELISA appears to be more sensitive and specific for diagnosing AHD, useful on follow-up of resected patients, and positive earlier in the course of disease.     

Indirect hemagglutination antibody titers for Entamoeba Histolytica in dried filter paper blood and sera

Serum and elutates from dried filter paper bloods from 404 patients with and without amebic liver abscess (ALA) were tested for Entamoeba histolytica antibodies by the indirect hemagglutination test. Eighty-nine percent of the sera from patients with ALA were positive (titers greater than or equal to 1 : 128) and 94% of those without ALA negative. Fifty-five percent of the filter paper bloods from ALA patients were positive. Overall agreement was 80% for positives and negatives and the correlation coefficient between the methods showed a linear relationship (r = 0.77). Although the quantity of sera available from dried filter paper bloods is limited and some antibody activity may be lost, the method should be of value in seroepidemiologic studies where venous bloods are not easily obtained.     

Combination hepatitis C virus antigen and antibody immunoassay as a new tool for early diagnosis of infection

Reduction of the window period of hepatitis C virus (HCV) infection represents an important goal in the transfusional and diagnostic setting. A prototype assay designed to simultaneously detect circulating HCV antigen and anti-HCV, has been developed. Aim of this study was to evaluate the performance of this new assay in terms of specificity and sensitivity and to compare its efficacy with commercial assays. To evaluate the specificity of the assay, 400 samples from the general population and 100 'difficult' sera, negative for anti-HCV, were tested. To assess sensitivity, the new test was used on 76 PCR-positive and anti-HCV negative sera, seven natural or commercial seroconversion panels that included 17 RNA-positive and anti-HCV negative sera and 31 anti-HCV positive sera, 20 weak anti-HCV positive sera, 80 viraemic and anti-HCV-positive sera from patients infected with different subtypes and 10 sera from patients with HBV-HCV or HIV-HCV co-infections. Of 500 anti-HCV negative samples, 499 (99.8%) were negative with a cut-off index <0.5, while one sample was within the grey zone. Of the 93 HCV-RNA positive and anti-HCV negative sera from patients and panels, 85 (91.4%) resulted positive, and one had the cut-off index in the grey zone. The reduction in the diagnostic window period observed with the new test and HCV-RNA assays were equal, on average, to 24 and 34.4 days respectively. All anti-HCV positive sera were positive. The new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or HCV-RNA detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible because of costs, organization, emergency and/or logistic difficulties.     

HIV-1 infection in HIV-1 enzyme-linked immunoassay seronegative patients in Kinshasa, Zaire

Serum samples of 62 African patients who had clinical manifestations of HIV-1 infection but were seronegative for HIV-1 by ELISA (Organon) were subsequently further tested by another HIV-1 ELISA test (Wellcozyme), HIV-1 IgG Western blot, HIV-1 antigen detection and HIV-2 ELISA. Patients' lymphocytes were cultured for HIV-1 and 2. Because of limited quantities of serum available all tests were not performed on all samples. Seven (26%) of 27 sera of patients meeting the WHO clinical case definition of AIDS were Western-blot-positive. In contrast, of 35 patients' sera with possible HIV related disease, only one (3%) was Western blot positive (P = 0.02) and none of 75 sera from HIV-1 ELISA (Organon) seronegative blood donors (P less than 0.01) were Western blot positive. Of 30 HIV-1 ELISA (Organon) seronegative patients tested with the HIV-1 ELISA Wellcozyme assay only one was seropositive (this patient's serum was also Western blot positive). Of 17 HIV-1 ELISA (Organon) seronegative patients tested, HIV-1 antigen was found in 1 case (6%) (this patient's serum was Western blot negative). None of the 34 patients tested by HIV-2 serology was HIV-2 seropositive. HIV-1 was isolated by culture in 3 (21%) of 14 HIV-1 ELISA seronegative patients (sera of the 3 patients were Western blot negative). In total, 12 (19%) of 62 HIV-1 ELISA (Organon) seronegative patients were found to be positive for HIV, either by Western blot HIV antigen testing or viral culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Appropriate use of rapid diagnostic testing for influenza

To determine a more timely acquisition of accurate results for influenza patients, a rapid diagnostic testing for influenza were studied on 877 pediatric patients performed during the 2002-2003 flu season in our hospital. Of these, 337 patients were finally diagnosed as influenza based on the test results and treated with antiviral agents, amantadine or oseltamivir. Ten (29%) of the 34 patients whose tests were negative within 12 hours after onset became positive over 12 hours after onset. On the other hand, diagnoses based on antigen tests over 12 hours after onset were reliable because all 13 patients first confirmed negative were unchanged when tested afterward. These 10 patients missed the opportunity to take antivirals early, which possibly caused them to have significantly longer (p = 0.0003) febrile duration and higher frequency of admission (p < 0.0001) than the 106 patients first confirmed positive within 12 hours after onset. Days from onset until starting antivirals (mean 1.4 days), the febrile duration (mean 2.7 days) and frequency of hospitalization (20.5%) of the 219 patients who tested positive over 12 hours after onset were significantly worse (p < 0.0001, p < 0.0001 and p = 0.0406, respectively) than those of patients testing positive within 12 hours after onset (mean 0.2 days, mean 1.7 days and 11.3%, respectively). The febrile duration (mean 2.3 days) of the patients confirmed positive even over 12 hours, but within 48 hours, of onset was tolerable but significantly longer (p < 0.0001) than that of patients confirmed positive within 12 hours after onset. The frequency (19.6%) of hospitalization of the patients confirmed positive even over 12 hours, but within 48 hours, of onset was not significantly different from that of patients confirmed positive within 12 hours after onset. These results suggested that over 12 hours but within 48 hours after onset of illness is the best period for the rapid diagnosis to correctly determine whether a patient should be treated with antiviral agents based on the result.     

The Ausria Test: Critical Evaluation of Sensitivity and Specificity

Ausria was shown to be considerably moresensitive than CEP by comparing titers ofHBAg+ sera and by testing selected proficiency panels, sera from hepatitis patients, and sera from experimentally infected rhesus monkeys; CEP+, Ausria-sera were never encountered. However, intesting NIH donors and personnel, 19 of 20Ausria+, CEP-sera were shown byneutralization studies to be nonspecific forHBAg, i.e., false positives. Nonspecificitywas confirmed by an alternate radioimmunoassay procedure and, in oneserum, by density-gradiant ultracentrifugation. Testing of 35 additional Ausria+,CEP-sera from New Jersey donorsshowed 13 specific and 22 (63%) nonspecific reactions. It is mandatory thatserum from every Ausria+, CEP-donorundergo specificity testing. In addition tothe 14 specific Ausria+, CEP-sera, twosera, subsequently shown to be CEP+,were undetected on the initial CEP test(one was transfused and resulted inhepatitis). These sera were strongly positive by Ausria; sera subject to reader errorin CEP are unequivocally positive byAusria. Ausria tests on CEP-donors suspected of transmitting HBAg failed to implicate a donor in each of eight cases.Some HBAg+ hepatitis will continue tooccur despite universal application ofAusria or other tests of equal sensitivity.

Submitted on June 13, 1973 Revised on July 20, 1973 Accepted on July 25, 1973     

Evaluation of a Malaria Antibody ELISA and Its Value in Reducing Potential Wastage of Red Cell Donations from Blood Donors Exposed to Malaria, with a Note on a Case of Transfusion-Transmitted Malaria

Background and objectives: Blood donations are often wasted for lack of a satisfactory procedure to evaluate donors potentially exposed to malaria. Materials and methods: We evaluated a commercial ELISA for the detection of antibodies to malaria and compared it with an immunofluorescent antibody test (IFAT). Results: When 5,311 sera from routine non-exposed donors were tested, 24 (0.45%) were positive by the ELISA, using a Plasmodium falciparum antigen. Seventeen were subjected to confirmatory testing but none were positive by IFAT. Of 1,000 donors potentially exposed in endemic areas 15 (1.5%) were repeatably reactive by ELISA. 10 of these were tested by IFAT and 2 were positive. When 150 patients attending the Hospital for Tropical Diseases in London with acute malaria were tested, 73% of those infected with P. falciparum were repeatably reactive for malarial antibodies by ELISA and 56% with Plasmodium vivax. Of 88 stored clinical sera tested by both IFAT and ELISA 56 were positive by IFAT and of these 52 (93°/0) were positive by ELISA. Conclusion: The ELISA is sufficiently sensitive and specific to screen at-risk donors. Its use could safely retrieve 40,000 red cell units currently discarded each year in Great Britain.     

Amebic liver abscess: a therapeutic approach

The clinical presentation of 48 patients with amebic liver abscess was no different than that reported in earlier studies. However, most patients were from countries endemic for parasitic disease. Failure to consider this diagnosis resulted in potentially avoidable surgery for six patients. Although metronidazole was successful primary therapy in 85% of 41 patients so treated, four of seven ruptured abscesses occurred in cases where metronidazole treatment failed. For assessment of factors that might predict metronidazole treatment failures, multiple parameters were analyzed. Of the factors evaluated, only timing of clinical response correlated with successful therapy. Ninety-four percent of metronidazole responders showed dramatic clinical improvement within 72 hours of initiation of therapy, whereas only 33% nonresponders had improved modestly during this time (P = .0014). Therefore, early diagnosis of amebic liver abscess in patients from endemic areas and treatment with metronidazole will result in successful therapy in 85% of cases. Surgical intervention or alternative medical therapy is indicated for those patients who do not respond after 72 hours of metronidazole therapy.     

Use of an enzyme-linked immunosorbent assay to detect anti-adherence protein antibodies in sera of patients with invasive amebiasis in Cairo, Egypt.

We used enzyme-linked immunosorbent assay (ELISA) to detect IgG antibodies to the Entamoeba histolytica galactose-inhibitable adherence protein in the sera of 50 uninfected controls, 50 cases with asymptomatic cyst passage, 100 patients with amebic colitis, and six patients with amebic liver abscess from Cairo, Egypt, and in 50 healthy controls from the United States. When the mean + 3 SD value above that of the controls from the United States was used as a criterion for a positive ELISA result, 100% of those with invasive amebiasis, 80% of those with asymptomatic infection, and 64% of the Egyptian controls had anti-adherence protein antibodies. However, when the mean + 2 SD value of Egyptian control sera (optical density = 0.094) was used as the criterion for positivity, 33 (89%) of 37 sera from individuals with invasive amebiasis having symptoms for at least one week were antibody positive, in contrast to only 12% of asymptomatic cyst passers (P < 0.01). In a highly endemic area such as Cairo, Egypt, detection of serum anti-adherence protein antibodies by ELISA may have greatest diagnostic use in patients with symptomatic invasive amebiasis of greater than one week duration.     

Determination of hepatitis B virus subtypes by an enzyme immunoassay method using monoclonal antibodies to type-specific epitopes of HBsAg

We described a Hepatitis B surface antigen (HBsAg) subtyping method based on a commercial enzyme immunoassay (EIA) for detection of HBsAg in which the procedure was modified to include the use of monoclonal antibodies with restricted anti-HBs specificities. This method, which was able to classify HBsAg as: ayw1, ayw2, ayw3, ayw3 * (intermediate between ayw3 and ayw4 ), ayw4, ayr, adw2, adw4 and adr , was compared to counter electrophoresis procedure (CEP) by testing HBsAg positive sera from blood donors included in a prospective national epidemiological survey. Among the 256 HBsAg positive samples tested with both techniques, 111 (43.3%) could not be subtyped with CEP vs 10 (3.9%) with our modified EIA. This difference was related to the serum HBsAg concentration which must be greater than 3000 ng/mL and 100 ng/mL for CEP and EIA, respectively. The results obtained from 145 sera with both methods were concordant. Seventeen out of 18 samples partially classified as ay with CEP were completely determined with EIA.
This reliable procedure, derived from commercially available reagents, can be easily used in several applications such as large epidemiologic studies and as a substitute for nucleotide sequencing genotyping which is not adapted for large-scale screening and not applicable on samples from nonviremic hepatitis B virus (HBV) carriers.     

Protein A-antibody mediated hemagglutination assay for serodiagnosis of amebic liver abscess

A successful modification of the indirect hemagglutination test to demonstrate antibodies for serodiagnosis of amebic liver abscess has been described in the present study. In the modified test, the protein A-IMA, Staphylococcus aureus (Cowan's strain I) bearing protein A (SAPA) cells were used to enhance hemagglutination of sensitized red cells. Use of SAPA cells markedly enhanced sensitivity of the test and greatly increased the titers obtained with most of the sera. At a diagnostic antibody titer of 1:128 and above, the protein A-IHA could detect 72 (100%) of amebic liver abscess (ALA) cases. Amongst the controls, no false positive reaction was observed in non-amebic liver disease controls. However 1(2%) of sera demonstrated false positive reactions from healthy controls. The protein A-IHA was highly sensitive when compared with that of the indirect hemagglutination (IHA) for serodiagnosis of amebic liver abscess. The novel immunoassay is simple, inexpensive and requires little technical skill. It has the potential for wide application in serodiagnosis of amebic liver abscess.     

'Shelf life' of Trichomonas vaginalis

The diagnosis of Trichomonas vaginalis (TV) infection in women may be made by observing motile trichomonads in wet-mount preparations of vaginal discharge. The duration for which these organisms can be identified in such samples is unknown. We sought to assess this by performing wet-mount microscopy on samples from female patients immediately, and then in positive samples, every 10 minutes until motile trichomonads could no longer be identified. Of the 65 wet-mount specimens positive for TV, a cumulative total of 13 (20%) samples had become negative by 10 minutes, 23 (35%) samples by 30 minutes and 51 samples (78%) by two hours, with the remainder exceeding two hours. We conclude that one-fifth of wet-mount vaginal preparations initially positive for motile TV become negative within 10 minutes of the initial, immediate reading. In order to maximize the sensitivity of this widely used test it is recommended that all specimens be examined immediately after they are taken.     

旋毛虫幼虫排泄分泌抗原用于诊断人旋毛虫病的可行性探讨

作者将自行分离制备的旋毛虫肌肉期幼虫２４小时排泄分泌抗原，用间接ＥＬＩＳＡ共检测３４份旋毛虫病人血清，３２份为阳性，阳性率９４％．而８２份正常人血清均为阴性反应。且不与蛔虫病人、囊虫病人血清发生交叉反应，但与肺吸虫及血吸虫病人血清发生交叉反应。经用ＳＤＳ－ＰＡＧＥ及铵银染色后显示，该抗原含有１１种蛋白组份，分子量介于９６ｋＤ－１７．６ｋＤ之间。有３条主带，分子量分别为４０、４８、５３ｋＤ。酶联免疫印迹试验表明旋毛虫病人血清能特异识别分子量为４８ｋＤ蛋白。而肺吸虫及血吸虫病人血清所能识别的区带均小于４０ｋＤ。该结果表明４８ｋＤ蛋白抗原组分在人旋毛虫病上具有潜在诊断价值，而酶联免疫印迹试验不失为一种可行的人旋毛虫病的诊断方法。     

Gold immunoblot analysis of IgM-specific antibody in the diagnosis of human leptospirosis.

An immunoblot for the detection of leptospirosis was developed in our laboratory. Antigen prepared from Leptospira interrogans serovar bataviae was dotted onto nitrocellulose paper and blocked with skim milk. Test and control sera diluted 1:20 were applied to the dot, incubated, and washed. Anti-human IgM colloidal gold conjugate was added and the dots were washed. A positive reaction was shown by the development of a pink dot against a white background. The test was performed on 62 sera that tested positive for leptospirosis by a microagglutination (MA) test, on 40 sera that were positive by an indirect hemagglutination (IHA) test, and on sera from forty healthy blood donors. Four sera from the blood donors showed a faint pink dot, but the remainder showed a colorless reaction. All 62 sera that tested positive by MA were positive by this new test, while 95% of the 20 sera that tested positive by IHA were positive. Tests for IgG antibody were performed on 20 sera positive by MA using protein A-colloidal gold conjugate, and all showed weak reactivity. The results confirmed previous findings that most antibodies present in leptospirosis patients are of the IgM type. The ELISA takes three hours to perform, but the gold immunoblot can be completed in 30 min. In addition, the test blot can be kept as a permanent record, and is a significant improvement over existing tests.     

Mac ELISA测定乙脑患者血清和脑脊液中特异性IgM的评价

用抗体捕捉ELISA法(Mac ELISA)检测临床疑似流行性乙型脑炎(下称乙脑)病人脑脊液88份,阳性51份,阳性率57.95％;血清91份,阳性56份,阳性率为61.53％,两者无显著性差异(X~2=0.238 P>0.05);包括取自同一患者的脑脊液和血清各72份,阳性率分别为61.11％和68.05％;而分析其中14例IgG呈4倍以上增高,乙脑患者脑脊液IgM阳性13例,而血清IgM阳性却只有8例,提示在疫区流行季节内仅仅血清学IgM阳性有时可能为乙脑隐性感染的同时还伴其它有关病毒感染。     

Occult hepatitis B virus among chronic liver disease patients in Yemen

ObjectiveTo estimate the rate of occult hepatitis B virus (HBV) among patients with chronic liver disease (CLD).MethodsAfter an informed consent, sera samples were collected during April 2004 to April 2005 from 280 patients (200 male and 80 female). They were previously diagnosed with CLD based on history and ultrasound and were investigated for occult HBV infection. Sera were first screened for HBsAg and those which showed negative were tested for anti-HBc. The anti-HBc positive sera were further tested for anti-HBs to identify sera with isolated anti-HBc which in turn were subjected to HBV–DNA testing using PCR to determine the rate of occult HBV infection. Moreover, sera with occult HBV were tested for Anti-HCV and HCV-RNA using RT-PCR.ResultsHBsAg was detected in 44 of 280 (15.7%). Of 236 HBsAg negative sera anti-HBc was detected in 22 (9.3%). All anti-HBc positive sera were found to be anti-HBs negative. HBV–DNA was detected in 11 of 22 (50.0%) sera with isolated anti-HBc indicating occult HBV in 4.3% of all sera. None of the sera with occult HBV had anti-HCV or HCV-RNA.ConclusionsOccult HBV infection does exist among CLD patients in Yemen and the mechanism of its occurrence merits further investigation.