The Fluorescent Antibody Technique for the Detection of Salmonella in Routine Use

S ummary : The direct and indirect fluorescent antibody technique (FAT) were compared with cultural methods for detecting salmonellae in meat products, animal feedingstuffs, poultry carcase swabs, giblets and poultry plant and equipment swabs. Salmonellae were not isolated from meat products and fluorescent cells were not seen on slides prepared by either FAT. The indirect and direct FAT recorded 13% and 9% respectively, false positive results, with samples of animal feedingstuffs, but the direct FAT recorded a single false negative result. Salmonellae were not isolated from poultry carcase swabs but 3% and 4·5% respectively, of false positive results were obtained with the indirect and direct FAT. Salmonellae were isolated from both giblet samples and poultry plant swabs and both gave rise to false negative FAT results. Preliminary studies of the efficacy of the FAT for screening animal faecal material for salmonellae indicated that no single combination of enrichment broth and FAT gives unequivocal results, but the staining of smears from tetrathionate broth by either FAT gives rise to a high percentage of false negative results.     

Detection of Vibrio cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture methods.

Vibrio cholerae O1 in plankton samples collected from ponds and rivers between February 1987 and January 1990 in Matlab, Bangladesh, was detected by the fluorescent-monoclonal antibody (FA) technique. Samples were collected at sites which were monitored fortnightly (fixed sites) as well as at sites that were part of a case-control study. FA results were compared with those obtained by conventional culture methods (CM). A total of 876 samples were collected; V. cholerae O1 was detected in 563 samples (64.27%) by the FA method and in 3 samples (0.34%) by CM. Of the fixed-site plankton samples, 439 (63.62%) were positive by FA and none were positive by CM. Of the 93 case sites sampled on the day after the occurrence of a case of cholera, 73 (78.49%) were positive for V. cholerae O1 by FA and 3 (3.2%) were positive by CM. In comparison, of the 93 first-day sample collections at control sites at the time a case of cholera occurred, only 51 (54.83%) were positive by FA and none were positive by CM. From the data, it is concluded that V. cholerae O1 is present throughout the year in the ponds and rivers of Bangladesh that were examined in this study and that V. cholerae can be detected by FA but not always by CM. The FA procedure was found to be very useful in detecting V. cholerae in plankton, with which it was associated and often occurred in large numbers in the nonculturable stage. Thus, studies investigating the significance of the role of environmental factors in the epidemiology of cholera can be performed effectively by using FA. Such studies are in progress.     

Detection of Vibrio cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture methods

Vibrio cholerae O1 in plankton samples collected from ponds and rivers between February 1987 and January 1990 in Matlab, Bangladesh, was detected by the fluorescent-monoclonal antibody (FA) technique. Samples were collected at sites which were monitored fortnightly (fixed sites) as well as at sites that were part of a case-control study. FA results were compared with those obtained by conventional culture methods (CM). A total of 876 samples were collected; V. cholerae O1 was detected in 563 samples (64.27%) by the FA method and in 3 samples (0.34%) by CM. Of the fixed-site plankton samples, 439 (63.62%) were positive by FA and none were positive by CM. Of the 93 case sites sampled on the day after the occurrence of a case of cholera, 73 (78.49%) were positive for V. cholerae O1 by FA and 3 (3.2%) were positive by CM. In comparison, of the 93 first-day sample collections at control sites at the time a case of cholera occurred, only 51 (54.83%) were positive by FA and none were positive by CM. From the data, it is concluded that V. cholerae O1 is present throughout the year in the ponds and rivers of Bangladesh that were examined in this study and that V. cholerae can be detected by FA but not always by CM. The FA procedure was found to be very useful in detecting V. cholerae in plankton, with which it was associated and often occurred in large numbers in the nonculturable stage. Thus, studies investigating the significance of the role of environmental factors in the epidemiology of cholera can be performed effectively by using FA. Such studies are in progress.     

反向点杂交法快速检测HPV基因型的临床应用

应用反向点杂交法(RDB)的原理,针对HPV 6B, 11, 16, 18, 31, 33和35设计了7条序列作为未标记的特异性寡核苷酸(SSO)探针,分别固定在尼龙膜条上,形成7个点,再与经PCR扩增的样品DNA序列杂交,即可在一个膜条上分辨出这7型HPV中的任一型.此法快速简便,特异性高,不存在假阳性;且因PCR灵敏度高,亦不易出现假阴性.用PCR-RDB法检测保存的宫颈癌组织石蜡包埋标本32例,结果:HPV16阳性22例(68.8%),HPV18阳性5例(15.6%),HPV16/18双重感染2例(6.3%),阴性仅3例(9.3%).     

Flow cytometric prescreening of cervical smears.

One-parameter (nuclear DNA) and two-parameter (nuclear DNA and protein or cellular light scatter) measurements of cervical smears were performed using an ICP 11 and a cytofluorograf 4800 respectively. A total of about 1000 cases was analyzed. For the estimation of nuclear DNA alone two fluorochromes were tested (ethidium bromide (EB) and mithramycin (MMC)) combined with three different methods of cell preparation. For the two-parameter measurements cells were double stained with EB and fluorescein isothiocyanate (FITC). Red fluorescence (EB) versus green fluorescence (FITC) or red fluorescence versus scatter were recorded. A computer analysis of the one-parameter histograms was performed using discriminant analysis and the results were compared with the cytodiagnosis of microscopic specimens stained with the Papanicolaou technique. The error rates of the flow cytometric (FCM) data were as follows: (a) standard EB staining, 11% false negative, 26% false positive, 6% unsatisfactory results; (b) pepsination of vital cells and EB staining, 12% false negative, 14% false positive and 4% unsatisfactory results; (c) MMC staining, 10% false negative, 65% false positive and 5% unsatisfactory results. Our two-parameter measurements prove that, as confirmed by cell sorting, red fluorescence versus scatter allows separation of at least three subpopulations in most analyzed samples: (a) anucleated cells; (b) leukocytes; and (c) intermediate and superficial cells.     

Evaluation of a Fluorescent Antibody-Enrichment Serology Combination Procedure for the Detection of Salmonellae in Condiments, Food Products, Food By-Products, and Animal Feeds

The reliability of the enrichment serology (ES), fluorescent antibody (FA), and a combination of the FA and ES procedures for the detection of salmonellae were compared to the Salmonella cultural procedure outlined in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). A total of 126 subsamples from 22 different products were analyzed. By utilizing the BAM procedure as the reference standard, a total of 66 samples were positive for salmonellae. Within 44 h approximately 65% of the Salmonella-negative samples could be cleared by the FA test. At the end of 50 h 97% of the Salmonella-negative samples could be cleared by the combination FA-ES test. The FA procedure detected all 66 BAM positives but exhibited a high incidence of presumptive positives which were cultural negatives. The ES procedure detected 64 of the 66 BAM positives but exhibited a low incidence of presumptive positives which were cultural negatives. Incorporating positive FA and positive ES results in a combination FA-ES technique revealed that FA-ES positives were statistically equivalent to BAM positives.     

A rapid immunochromatographic assay for the detection of Mycobacterium tuberculosis antigens in pulmonary samples from HIV seropositive patients and its comparison with conventional methods

As tuberculosis generates a highly heterogeneous antibody repertoire, its diagnosis requires tests based on cocktails of antigens. We describe a new, rapid method called rapid immunochromatographic assay (RICA) for cocktail-based diagnosis, which can detect Mycobacterial antigens in sputum specimens. Six antigenic fractions of pathogenic Mycobacterium tuberculosis were used in combination as the capture antigens in the control line of the flow-through assay. Antigen detection of 200 sputum samples from HIV seropositive patients by RICA assay gave a sensitivity of 97.9%, specificity of 99.0%, positive predictive value of 98.9%, negative predictive value of 98.0%, false positive rate of 0.9%, false negative rate of 2.0%, prevalence rate of 49%, likelihood ratio for positive results 97 and likelihood ratio for negative results 0.02. The combination of RICA and AFB staining gave a sensitivity of 100%, specificity of 100%, positive predictive value of 100%, negative predictive value of 100%, false positive rate of 0%, false negative rate of 0%, likelihood ratio for negative results 0. The assay was simple, rapid and economical for the detection of M. tuberculosis infection and suitable for large scale screening of samples in endemic areas without any sophisticated equipment. The results of the assay proved to be superior to conventional methods and combined with clinical data, could form the basis for starting an earlier course of treatment.     

An interlaboratory comparison for the detection of Mycoplasma pneumoniae in respiratory samples by the polymerase chain reaction

A panel of 78 respiratory samples collected from 43 patients was analyzed in three different Centers for the presence of Mycoplasma pneumoniae DNA by polymerase chain reaction (PCR). One Center collected the samples and extracted the DNA by two different methods. DNA extracted according to the first method was amplified using primers targetting the 16 S rRNA gene. DNA extracted according to the second method was amplified using the same primers in a semi-nested format and was sent to the two other Centers. The latter Centers both used the same primers targetting the P1 gene but with a different detection format. Thirty-nine samples (50%) from 19 patients were positive by at least two PCR assays. None of the laboratories were free of false positive or false negative PCR results. Calculated specificities of the individual PCR assays ranged from 97.4% to 87.2% and sensitivities ranged from 97.4% to 89.2%. Complement fixation was done on sera of 33 patients. The calculated specificity and sensitivity of serology was 100% and 58.8%, respectively. Several aspects concerning false positive and false negative results with PCR are discussed.     

Direct Immunofluorescence Test for the Diagnosis of Genital Herpesvirus Infections

Herpetic lesions of the genitalia may be confused clinically with other ulcerative, genital lesions. Direct immunofluorescence (FA) provides a rapid method of diagnosis, and the utility of this method for the diagnosis of genital ulcers was examined. One hundred and ten patients with genital lesions were examined by darkfield for syphilis and by FA and culture for herpes simplex virus (HSV) infections. Satisfactory samples were obtained from 102 patients, of which 81 were clinically suspected cases of HSV. Acetone-fixed slides of scrapings of ulcerative lesions were stained with conjugated antiserum prepared in rabbits against HSV type 2. HSV was isolated from 73% of specimens of suspected herpetic lesions, and 77% of these specimens were positive by FA. Nine percent were positive by FA only and these were not thought to represent false positives. Five percent were positive by culture only. A comparison of clinical diagnoses with laboratory findings revealed that 4% of the cases were misdiagnosed when only the clinical evaluation was considered. The data suggest that the inclusion of a diagnostic FA test for HSV along with the darkfield examination may be useful for differentiating the etiological agents of ulcerative, genital lesions.     

18F-FDG PET 和治疗量131I 全身扫描对分化型甲 状腺癌根治术后转移灶检测的临床价值评估

目的:探讨18F-FDG PET显像和131I-全身扫描(131I-WBS)SPECT显像对分化型甲状腺癌(DTC)术后转移灶的临床诊断价值。方法:对57例外科术后拟行131I治疗的DTC患者行18F-FDG PET全身显像和131I-WBS扫描,观察和记录在糖代谢和碘代谢中DTC转移灶的定位及数量变化,并同时测定甲状腺球蛋白(Tg),促甲状腺激素释放激素(TSH)等实验室检查项目。结果:57例DTC患者18F-FDG PET显像发现真阳性20例、假阳性3例、真阴性31例、假阴性3例,其灵敏度为87.0%,特异性为91.2%。而131I-WBS扫描发现真阳性13例、假阳性2例、真阴性34例、假阴性8例,其灵敏度为61.9%,特异性为94.4%。PET显像和131I-WBS扫描共检出阳性病灶73个,其中淋巴结32个,肺5个,纵隔6个,骨26个,其他部位4个。PET显像发现43个阳性病灶(58.9%),而131I-WBS检出30个(41.1%)。当Tg水平>10μg/L时,随着Tg在血清含量的增高,两种显像方法的对DTC转移灶的阳性检出率亦随之升高。结论:两种检查对DTC术后转移灶的监测和131I的治疗具有良好的互补性,18F-FDG PET显像在Tg阳性和131I-WBS阴性的患者的转移灶检出上更具有优势,有重要的临床指导意义。     

BioPEPR: a system for the automatic prescreening of cervical smears.

A feasibility study has indicated that a Prescion Encoding and Pattern Recognition (PEPR) cathode ray tube prescreening system for cervical smears can be both accurate and fast. Smears are prepared using a syringing technique and are stained with a Feulgen-type nuclear stain and a protein counter-stain. The use of film as an intermediate step between the cells and Bio PEPR allows the scanning of fields as large as 8 x 8 mm. The morphological features of the cells are measured as directed by a hierarchical decision strategy. Additional programs detect artifacts, overlaps, and leukocytes. For clean samples, false positive and false negative rates on the cell level have been obtained that will allow acceptable smear level rates (10% false positive, 1% false negative). These rates have been reached without compromising the required speed goals of 120 to 180 smears per hr. The efficiency of the system is dependent on the quality of the smears. Measurements on a set of 192 routinely prepared smears indicate acceptable false negative rates and a false positive rate of about 18%. A reduction of this rate is expected with small improvements in cell preparation and measuring software, leading to the overall system efficiency required for commercial feasibility.     

Further Evaluation of a Rapid Immunofluorescence Technique for Detecting Salmonellae in Meat and Poultry

A rapid 18–24 h immunofluorescence technique detected 14 of 15 positive samples in tests on 706 routine samples, which included 656 home produced raw beef samples. The rapid technique also recorded 49 false positive results, i.e. samples which proved negative in subsequent cultural tests. The immunofluorescence technique could be used as a presumptive screening test aimed at the rapid detection of negative samples. In this way salmonella free raw materials should usually be cleared for production within 1 day of sampling.     

ELISA法检测HBeAg假性结果原因分析

目的:探讨ELISA法检测HBeAg假性结果原因方法:用ELISA法检测乙肝血清标志物,对HBsAg阳性而HBeAg阴性的标本以及HBeAg阳性的标本用ELISA法和电化学发光法复查。结果:136例HBsAg阳性而HBeAg阴性的标本经稀释复查后检出10例HBeAg阳性标本。23例溶血标本引起HBeAg假阳性。结论:钩状效应和标本溶血是引起HBeAg假阴性和假阳性的重要原因。必要时应加以复查,以减少HBeAg的错检和漏检。     

Rapid Identification and Typing of Herpes Simplex Virus Types 1 and 2 by a Direct Immunofluorescence Technique

An immunofluorescence (FA) technique has been developed which can identify herpes simplex virus (HSV) in clinical specimens and also type the virus directly as type 1 or type 2. This test, first applied to cervicovaginal specimens obtained from 80 mice genitally inoculated with HSV, indicated a sensitivity approaching 80% in comparison to standard viral isolation methods. A similar sensitivity was found when the test was applied to 185 clinical specimens with adequate cells for staining, which were obtained from a variety of sites of patients with suspect herpetic infection. In only 1 of 6 specimens positive by both FA and culture methods was the HSV type wrongly identified by the FA technique. There were also six specimens which were negative by culture methods but positive by the FA test, indicating a specificity of 91%. It is likely that these are not instances of false-positive tests but of other factors which may have resulted in negative viral isolations by culture methods. As more specific reagents become available, it is anticipated that the FA technique will have wider usage in diagnostic laboratories for the identification and typing of HSV types 1 and 2.     

手工和仪器两种血培养结果差异性分析

目的对手工法双相血培养瓶和BACTEC9120全自动血培养仪的阳性率作回顾性分析。方法将血液标本同时接种双相血培养基和BACTEC9120全自动血培养仪配套血瓶中,将阳性结果移种血平板,如为阴性再移种巧克力平板。结果370例血培养,双相血培养瓶阳性25例,阳性率为6．76％（25／370）,树脂需氧（儿童）瓶BACTEC9120报警显示阳性59例,阳性率为15．9％（59／370）,阳性标本移种到血平板及巧克力平板阳性54例,阳性率为14．6％（54／370）,假阳性5例,假阳性率为1．4％（5／370）,共有29例树脂需氧（儿童）瓶阳性,而双相血培养瓶为阴性,P〈0．001。结论BACTEC9120全自动血培养仪提高阳性率,缩短阳性的报告时间优于传统的双相血培养基。     

Fine needle aspiration cytology of breast fibroadenoma. A cytohistologic correlation study of 405 cases.

OBJECTIVE: To evaluate the cytohistologic correlation of breast fibroadenoma (FA) in order to assess the value of cytology in the conservative management of this lesion. STUDY DESIGN: A retrospective analysis of all aspirates diagnosed as FA or fibroadenomatous lesion (n = 1,549) for which a histologic follow-up study was available (n = 362). Forty-three aspirates, including 14 nonrepresentative smears, from histologically proven FAs with a different cytologic report were also included in the study. RESULTS: Cytohistologic agreement was present in 287 of the 362 cytodiagnoses. Lack of correlation was observed in 75 cases. Most diagnostic errors accumulated in the older patient group. The sensitivity of the cytologic diagnosis of FA was 86.9% (90.8% excluding nonrepresentative cases), with a positive predictive value of 79.3%. In 43 cases a histologic diagnosis of FA was given after previous erroneous (n = 29) or nonrepresentative cytodiagnoses (n = 14). The specificity of the cytologic diagnosis of FA reached 93.8%, with a negative predictive value of 96.3% (97.5% excluding nonrepresentative cases). Regarding malignancy, five tumors were diagnosed as FA and were malignant. No false positive diagnoses of malignancy were given, but nine aspirates were included in the category "suspicious for carcinoma." CONCLUSION: FA of the breast remains a diagnostic challenge for the cytopathologist. A considerable amount of benign breast lesions can mimic FA on cytology, and such diagnostic categories as "fibroadenomatous lesion" or "consistent with FA" are associated with low diagnostic accuracy. While the cytologic requisites for entering a program of conservative management of FA are established, it seems that strict diagnostic criteria should be applied even at the expense of diminishing sensitivity.     

HIV antibody assay that gave false negative results: multicentre collaborative study.

OBJECTIVE: To identify false negative results arising from the use of a commercial kit to detect antibody to HIV-1 and HIV-2 between July 1995 and March 1996. DESIGN: The 56 laboratories in the United Kingdom that were using the assay were asked to retrieve and retest specimens with an alternative assay for HIV-1 and HIV-2. Details of false negative results were obtained and these serum samples further investigated. SUBJECTS: 24,181 patients tested with the assay who were reported as being negative for HIV antibody. An additional 497 patients were confirmed as HIV positive with the assay. RESULTS: Serum samples of 20,973 of the patients were retested, and four patients were found to have had false negative results with the kit; three further patients were found to have had false negative results in the course of other laboratory testing. The seven patients with false negative results with the kit were of diverse risk group and HIV-1 subtype. Four had evidence of recent HIV infection. CONCLUSION: The commercial kit had a sensitivity of 99.2% (497/501), or less if the additional three patients with false negative results were taken into account.     

18F-FDG PET和治疗量131I全身扫描对分化型甲状腺癌根治术后转移灶检测的临床价值评估

目的：探讨18F-FDG PET显像和131I-全身扫描（131I-WBS）SPECT显像对分化型甲状腺癌（DTC）术后转移灶的临床诊断价值。方法：对57例外科术后拟行131I治疗的DTC患者行18F-FDG PET全身显像和131I-WBS扫描,观察和记录在糖代谢和碘代谢中DTC转移灶的定位及数量变化,并同时测定甲状腺球蛋白（Tg）,促甲状腺激素释放激素（TSH）等实验室检查项目。结果：57例DTC患者18F-FDG PET显像发现真阳性20例、假阳性3例、真阴性31例、假阴性3例,其灵敏度为87.0%,特异性为91.2%。而131I-WBS扫描发现真阳性13例、假阳性2例、真阴性34例、假阴性8例,其灵敏度为61.9%,特异性为94.4%。PET显像和131I-WBS扫描共检出阳性病灶73个,其中淋巴结32个,肺5个,纵隔6个,骨26个,其他部位4个。PET显像发现43个阳性病灶（58.9%）,而131I-WBS检出30个（41.1%）。当Tg水平〉10μg/L时,随着Tg在血清含量的增高,两种显像方法的对DTC转移灶的阳性检出率亦随之升高。结论：两种检查对DTC术后转移灶的监测和131I的治疗具有良好的互补性,18F-FDG PET显像在Tg阳性和131I-WBS阴性的患者的转移灶检出上更具有优势,有重要的临床指导意义。     

Value of fine needle aspiration in the diagnosis of breast lesions.

OBJECTIVE: To evaluate the accuracy values of 276 fine needle aspriations (FNA) of breast lesions with a subsequent excisional biopsy diagnosis and to make a comparison between 25 studies of the literature using the same criteria to calculate those values. STUDY DESIGN: Cytologic findings were compared with the histologic diagnosis of each mass. The correlation of results was analyzed by a decision-analysis approach, and the following values concerning diagnostic accuracy were calculated in the present study and in 25 other reports: sensitivity, specificity, positive predictive value, negative predictive value, false positive fraction and false negative fraction. To calculate those values, we eliminated unsatisfactory results and assumed that suspicious and positive cytologic findings represented carcinoma of the breast. RESULTS: Comparing our results with the means in the literature (numbers in parenthesis), FNA detected cancer with a sensitivity of 92.1% (87.7%), specificity of 98.6% (94.7%), positive predictive value of 99.4% (92.8%), negative predictive value of 82.1% (90.7%), false positive fraction of 0.6% (7.1%) and false negative fraction of 17.9% (13.4%); in 6.2% of cases the material was unsatisfactory (13.4%). CONCLUSION: All the rates varied enormously between the studies and during the past 13 years. It seems that false positive and false negative fractions tended to diminish and stabilize in more recent years, and specificity and sensitivity underwent a slight increase. The differences between the rates of those studies suggest that FNA of the breast has some unavoidable limitations.     

无创产前筛查(NIPT)的"不准确性"与阳性结果的验证

无创产前筛查(Non-Invasive Prenatal Testing, NIPT)通过检测孕妇外周血中的游离胎儿DNA来筛查胎儿常见非整倍体,已成为产前筛查中重要的一项技术,甚至可作为高龄孕妇初步筛查的首选方式。但因为难免会出现假阴性和假阳性,所以其阴性结果也并不能总是保证胎儿正常。而对于阳性结果,需通过有创产前诊断进行验证。目前,我国临床主要采用的有创产前诊断方法有绒毛活检(Chorionic Villous Sampling, CVS)、羊膜腔穿刺(Amniocentesis, AC)和脐血穿刺。绒毛活检和羊膜腔穿刺术是NIPT阳性结果验证的主要方式。本文主要对造成NIPT假阳性和假阴性结果的原因及其阳性结果的验证进行综述。