Combined Immunomagnetic Separation and Detection of Salmonella enteritidis in Food Samples

A model system for immunochemical detection of Salmonella enteritidis has been developed, using immunomagnetic separation (IMS) with both commercially‐available and laboratory‐prepared antibodies, followed by an enrichment stage and end‐point detection by ELISA. IMS alone gave an average of 77% recovery of cells artificially inoculated into the food, and a range of 20–110% recovery, depending on food type. The combined model system (IMS‐ELISA) enables detection of either < 10 cells m^?1 whole egg extract (using overnight enrichment after IMS), or > 10 cells ml^?1 (3 h enrichment after IMS). Cross‐reactivity of antibodies with Citrobacter was decreased by using two different immunochemical steps: IMS with monoclonal antibody‐coated beads, and a ‘sandwich’ ELISA with polyclonal capture antibody and monoclonal detector antibody. Discussion is presented on the best food preparation method, optimal substrate type for the ELISA and on the potential of IMS.     

Combined PCR-oligonucleotide ligation assay for rapid detection of Salmonella serovars.

We have developed a rapid and sensitive assay for the detection of Salmonella serovars in veterinary clinical specimens. This method utilizes a short cultivation period followed by PCR. For detection of the amplified product, an enzyme-linked immunosorbent assay (ELISA)-based oligonucleotide ligation assay (OLA) was used. In this study, the PCR-OLA technique was compared with conventional culture and membrane hybridization for the detection of Salmonella bacteria. In evaluating the PCR-OLA with Salmonella serovars and non-Salmonella strains of bacteria, A490 readings for 51 Salmonella strains, representing 28 serovars, were significantly higher (P < 0.05) than those for 25 non-Salmonella bacteria. With serial 10-fold dilutions of Salmonella CFU or with known concentrations of purified chromosomal DNA from Salmonella typhimurium ATCC 29946, the PCR-OLA was able to detect > or = 20 CFU per assay or > or = 80 fg of chromosomal DNA (corresponding to 160 molecules of DNA). Of 102 suspect clinical specimens screened, 15 were positive for Salmonella bacteria by both culture and the PCR-OLA procedure (100% sensitivity), and 3 samples were positive only by PCR-OLA (96.6% specificity), indicating a positive predictive value of 83.3% and a negative predictive value of 100%. In all experiments, the PCR-OLA was as sensitive as membrane hybridization. These results indicate that a limited enrichment cultivation and PCR-OLA could be used as a presumptive screening test for the detection of Salmonella serovars from any sample that currently requires extensive cultivation and that this assay would be adaptable to automation.     

Specific detection of Salmonella enterica and Escherichia coli strains by using ELISA with bacteriophages as recognition agents

The use of bacteriophages, instead of antibodies, in the ELISA-based detection of bacterial strains was tested. This procedure appeared to be efficient, and specific strains of Salmonella enterica and Escherichia coli could be detected. The sensitivity of the assay was about 10⁵ bacterial cells/well (10⁶/ml), which is comparable with or outperforms other ELISA tests detecting intact bacterial cells without an enrichment step. The specificity of the assay depends on the kind of bacteriophage used. We conclude that the use of bacteriophages in the detection and identification of bacteria by an ELISA-based method can be an alternative to the use of specific antibodies. The advantages of the use of bacteriophages are their environmental abundance (and, thus, a possibility to isolate various phages with different specificities) and the availability of methods for obtaining large amounts of phage lysates, which are simple, rapid, cheap, and easy.     

Reliability of rapid diagnostic tests for HIV variant infection

The sensitivity of one ELISA method, six HIV-1/HIV-2 rapid screening tests, and one confirmatory test was evaluated in comparison with a third-generation EIA method (taken as the 'gold standard') and Western blot on well-characterized panels of sera. HIV diversity was represented by 50 HIV-1 group M subtype A to H, nine HIV-1 group O, 12 HIV-2, two HIV1+2 positive and six indeterminate Western blot profiles. Sensitivity during HIV-1 seroconversion was studied on 39 serial samples collected from six patients during early primary infection. Serial samples obtained from two primates during experimental primary SIV infection were used to mimic HIV-2 seroconversion samples. The sensitivity ranged from 100 to 94.6% according to the test. During seroconversion, rapid tests became positive 2-8 days later than the third-generation EIA. This reveals a major limitation of rapid tests, which are being recommended for use in developing countries. The lack of sensitivity seen during early HIV-1 seroconversion and/or limited specificity in some of the evaluated tests present serious limitations to their use in countries with high HIV incidence and variability. It is suggested that, as soon as possible, less sensitive rapid tests for blood bank screening should be abandoned in favor of highly sensitive rapid tests and/or more robust, more sensitive and cheaper ELISAs. These results stress the need for better screening tools and specific local evaluations.     

Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay.

In order to make a rapid and definite diagnosis of Salmonella enteritis in children, an enrichment broth culture-multiplex PCR combination assay was devised to identify Salmonella serovars directly from fecal samples. Two pairs of oligonucleotide primers were prepared according to the sequences of the chromosomal invA and plasmid spvC genes. PCR with these two primers would produce either one amplicon (from the invA gene) or two amplicons (from the invA and spvC genes), depending on whether or not the Salmonella bacteria contained a virulence plasmid. The fecal sample was diluted 10- to 20-fold into gram-negative enrichment broth and incubated to eliminate inhibitory compounds and also to allow selective enrichment of the bacteria. One or two amplicons were obtained, the expected result if Salmonella bacteria were present. The detection limit of this PCR was about 200 bacteria per reaction mixture. The primers were specific, as no amplification products were obtained with 18 species and 22 isolates of non-Salmonella bacteria tested which could be present in the feces or cause contamination. In contrast, when 23 commonly seen Salmonella serovars (38 isolates) were tested, all were shown to carry the invA gene and seven concomitantly harbored the spvC gene of the virulence plasmid. This assay was applied to the diagnosis of Salmonella enteritis in 57 children who were suffering from mucoid and/or bloody diarrhea. Of the 57 children, 38 were PCR positive and 22 were culture positive. There were two culture-positive samples that were not detected by PCR. Thus, this PCR assay showed an efficiency of 95% (38 of 40), which is much higher than the 60% (24 of 40) by culture alone. Not only is this method more sensitive, rapid, and efficient but it will cause only an incremental increase in the cost of stool processing, since enrichment cultivation of fecal samples from diarrheal patients using gram-negative enrichment broth is a routine practice for identification in many diagnostic microbiology laboratories. This PCR method, therefore, has clinical application.     

Two rapid and simple enzyme immunoassays for human antibodies to Entamoeba histolytica

Two rapid and simple enzyme immunoassays (EIA) for antibodies to E. histolytica the protozoa causing ambiasis, are described. In the rapid dot EIA, a qualitative procedure, antigens were dried as a small dot (3 mm in diameter) on a thin white opaque polystyrene strip and serum samples were assayed undiluted. The assay required 3 incubation periods, 1 to 3 minutes each, and was completed in 9 minutes, with a positive reaction revealed as a blue color (precipitate) on the antigen dot and negative as colorless. The developed color is stable for permanent record. In the Microwell EIA, a quantitative procedure, antigens were dried in the Microwells. The assay also consisted 3 incubation periods of 15 minutes each, and was completed in 50 minutes. The results in absorbance values were normalized to EIA units (EU). Both tests had good reproducibility, sensitivity and specificity; and highly correlated with 3 other serologic tests. Their reagents can be stored for more than a year. Both tests could be suitable for small and physicians' office laboratories, especially in developing countries.     

Enrichment culture coagglutination test for rapid, low-cost diagnosis of salmonellosis.

Specific diagnosis of salmonellosis by conventional culture and identification methods usually requires 2 to 4 days. Since Salmonella may be disseminated from infected individuals during this period, this amount of time required for diagnosis may be too slow to aid in epidemic control. To obtain earlier diagnoses of salmonellosis, a coagglutination test was used for rapid, simplified detection of Salmonella oranienburg antigens in enrichment broth cultures of fecal specimens from infants involved in a nursery outbreak. Two selective enrichment broths were used, selenite cystine and dulcitol selenite. These were compared in parallel for efficiency by subculture on deoxycholate lactose sucrose, MacConkey, xylose lysine deoxycholate, and tryptic soy lactose teepol agars. These overnight enrichment broth cultures of stool specimens were also examined by a coagglutination slide test with stabilized protein A-containing staphylococci sensitized with antisera for Salmonella antigens C1, E, and Vi. Of 113 diarrhea stool specimens tested, 86 were positive by conventional culture, 82 were positive by dulcitol selenite-coagglutination, and 55 were positive by selenite cystine-coagglutination. All these tests were negative on 50 stool specimens from infants in a noninfected nursery. Salmonellae were specifically detected in stool cultures within 20 h by the coagglutination technique. This early detection of Salmonella antigens provided a useful adjunct to culture for rapid diagnosis of salmonellosis.     

Asthma screening of high school athletes: identifying the undiagnosed and poorly controlled.

BACKGROUND: It is believed that there are many high school-age athletes who have undiagnosed asthma or exercise-induced asthma (EIA). The screening of these athletes for EIA will allow them to be identified and treated. OBJECTIVES: 1) To obtain reliable peak expiratory flow rate (PEFR) measurements and administer questionnaires to high school-age athletes to evaluate their asthma risk. 2) To identify high-risk athletes for having EIA or asthma by a free run challenge test. 3) To evaluate whether an athlete's present asthma control is adequate. 4) To evaluate these tools for their value as screening tools for asthma or EIA. METHODS: Eight hundred one student athletes from 10 suburban Pittsburgh schools were screened for more than 18 months for asthma as part of their preparticipation sports physicals. The screening included all athletes from all high school sports. The athletes were given a brief questionnaire, had PEFR measured, and then participated in a free running exercise challenge. RESULTS: Forty-six of 801 athletes had asthma or EIA, Of the remaining 755 athletes, 49 athletes were identified as having undiagnosed asthma. In the previously unrecognized athletes with EIA, the positive and negative predictive value of the questionnaire was 42% and 97%, respectively. Eighty-five percent (39 of 46) of the known asthmatic athletes, using their recommended medication, failed their free running test by a >15% drop of their PEFR. CONCLUSIONS: The free running test is a good test for identifying and assessing the athlete with EIA. The PEFR meter is not a good screening tool for EIA in the high school athlete. A questionnaire may be a good negative screening tool, but further development is needed before it can be used for widespread screening.     

实时荧光定量PCR技术在致病菌检测中的应用

目的本文将实时荧光定量PCR(Real-time Q-PCR)法检查致病菌与细菌培养法作比较,探讨对指导临床诊断治疗的意义。方法采用Real-time Q-PCR法和细菌培养法对100份标本进行6个微生物项目的检测,并比较两种方法的灵敏度、特异性和准确性。结果 Real-time Q-PCR法检测结果与细菌培养法,除完全符合的19例标本之外,另有8例用细菌培养法为阴性的标本,用Real-time Q-PCR法检测为阳性。结论 Real-time Q-PCR法较细菌培养法具有准确性好、灵敏度高、特异性强和检测快速等优点,是快速筛查致病菌的理想检测方法之一。     

Binding of porcine ficolin-alpha to lipopolysaccharides from Gram-negative bacteria and lipoteichoic acids from Gram-positive bacteria

Protein(s) reactive with N-acetyl-D-glucosamine (GlcNAc) was isolated from porcine nonimmune serum. The molecular weight of the purified protein was found to be mainly 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The N-terminal 10 amino acid sequence of the purified protein were found to be identical to that of porcine ficolin-alpha reported previously. In enzyme-linked immunosorbent assay, the purified protein was found to react with lipopolysaccharides (LPS) from different Gram-negative bacteria such as Esherichia coli, Salmonella typhimurium, Salmonella enteritidis, Salmonella abortus equi, Pseudomonas aeruginosa, Shigella flexeneri, and Serratia marcescens and with lipoteichoic acid (LTA) from Gram-positive bacteria such as Streptococcus sanguis, Bacillus subtilis, Streptococcus pyogenes, and Staphylococcus aureus. The purified protein also reacted with E. coli O26 isolated from food poisoning and bovine feces and heat-treated Gram-positive bacteria such as S. aureus, B. cereus, B. subtilis, Enterococcus faecium, and Corynebacterum bovis. On the other hand, porcine IgG isolated from nonimmune serum showed different reactivity with these LPS, LTA, and heat-treated bacterial cells. From the present findings, purified porcine serum protein reactive with GlcNAc is concluded to be ficolin-alpha playing an important role(s) in innate immunity against microbial infection with Gram-positive and -negative bacteria.     

Evaluation of the Magnetic Immuno PCR assay for rapid detection ofSalmonella

A new technique, the Magnetic Immuno PCR Assay (MIPA), has been developed for the detection ofSalmonella. The assay utilizes magnetic particles coated with monoclonal antibodies againstSalmonella to extract these bacteria from the sample. Trapped bacteria are lysed, and the supernatant, which contains bacterial DNA, is then subjected to the polymerase chain reaction (PCR) using primers from theSalmonella typhimurium origin of DNA replication to amplify a 163 bp region. The specificity of the primer set was tested in the PCR; amplification occurred with all 25Salmonella strains tested but not with 19 other species ofEnterobacteriaceae tested. A sensitivity of 100 cfuSalmonella typhimurium was achieved for the MIPA by visualization of the amplified products by ethidiumbromide stained agarose gel electrophoresis. A tenfold higher sensitivity was obtained by Southern blotting of the amplified products. The presence of 10⁷ cfuEscherichia coli did not interfere with these detection levels. The MIPA thus specifically detected 100 cfu ofSalmonella within 5 h and may be potentially useful for rapid detection ofSalmonella in clinical specimens and food.     

Detection of Salmonella typhimurium in raw meats using in-house prepared monoclonal antibody coated magnetic beads and PCR assay of the fimA gene

A method for detection of Salmonella Typhimurium in meat samples that uses in-house monoclonal antibody (MAb) coated magnetic beads for immunomagnetic separation (IMS) associated with PCR amplification of the gene fimA was developed. An internal amplification control (IAC) of the PCR reaction was constructed. The fimA PCR has shown 100% sensitivity and specificity when tested with various bacteria. The detection limit of the IMS-PCR method, using a post-enrichment in BHI broth for 6 h between IMS and PCR, was 1-10 CFU/mL. The method proved to be rapid (27 hrs), highly sensitive (1-10 CFU/25 g), and specific for detection of S. Typhimurium from experimentally contaminated pork and chicken meat samples.     

Evaluation of the Wellcolex Colour Salmonella Test for detection of Salmonella spp. in enrichment broths.

The Wellcolex Colour Salmonella Test was evaluated for detection of Salmonella spp. in enrichment broths of 1,010 stool samples. In 39 specimens, Salmonella spp. could be isolated from the selenite F broth (SF). Wellcolex agglutination indicative of the presence of Salmonella spp. was noted with the SF in 36 cases, 34 of which were in agreement with the subculture results. Therefore, relative to subculture, the sensitivity and specificity of the Wellcolex-selenite F procedure were 87 and 99%, respectively. Five false-negative results were noted. The gram-negative broth (GN) subculture revealed only 23 Salmonella spp. (59% sensitivity). The Wellcolex agglutination procedure applied to the GN indicated Salmonella spp. for 21 samples; its sensitivity was 70% and its specificity was 99% compared with GN subcultures. The Wellcolex agglutination procedure applied to the SF performed better than the agglutination of GN or direct plating procedures and detected 17 of the 39 Salmonella spp. at least 24 h earlier than did culture.     

Enzyme immunoassay for diagnosis of gonorrhea.

An enzyme immunoassay (EIA; Gonozyme [Abbott Laboratories]) for gonococcal antigen was assessed for the rapid diagnosis of gonorrhea. Patients attending two sexually transmitted disease clinics were tested by EIA and culture on Thayer-Martin medium. EIA was highly effective in detecting gonococcal infection among symptomatic men, with 70 of 75 (93.3%) culture-positive men having positive tests and no false-positive reactions. The performance of the test was not as good in detecting cervical gonorrhea; the best result obtained was a sensitivity of 87% (33 of 38) for EIA compared with culture. EIA false-positives occurred at a relatively low rate for women, with the test having a specificity of ca. 97%. The test clearly is capable of detecting gonococcal antigen in cervical and urethral specimens, but its role in routine diagnosis is not clear. Its performance seems equal to that of the Gram stain for men, but it seems to be less sensitive than culture for cervical gonorrhea--a drawback in high-risk populations. The low false-positive rate could be an important issue in screening low-prevalence populations.     

ImmunoCard STAT! cartridge antigen detection assay compared to microplate enzyme immunoassay and modified Kinyoun's acid-fast staining technique for detection of Cryptosporidium in fecal specimens

Cryptosporidium species infect humans and a wide range of animals worldwide; outbreaks of cryptosporidiosis have been reported in several countries. Routine diagnostic methods may be insufficient to demonstrate the presence of these organisms. The study assessed the diagnostic accuracy of the antigen detection immuno-cartridge test, ImmunoCard STAT! (Meridian Bioscience Inc., Cincinnati, OH, USA), compared to the combined gold standard: modified Kinyoun's acid-fast technique confirmed with the microplate enzyme immunoassay (EIA) for the detection of Cryptosporidium in fecal specimens. Three hundred fifteen formalin-fixed stool specimens were submitted for testing. The Kinyoun's acid-fast-stained smear revealed 24 positive samples for Cryptosporidium (of which 23 specimens were confirmed by the EIA) and 291 negative samples (of which 289 were negative by EIA). Agreement between the three used tests was shown in 22 positive and 288 negative samples for Cryptosporidium. Kappa score of agreement between the immuno-cartridge test and EIA was 0.957, p?=?0.000. The sensitivity of the immuno-cartridge test was 96% (95% confidence interval (CI), 87% to 104%) and the total accuracy of the test was 97% (95% CI, 93-103). The ImmunoCard STAT! Cryptosporidium cartridge assay is easy to use and does not require specialized training or equipment and is useful in routine diagnosis and screening for Cryptosporidium especially where rapid, point of care testing is needed or where other reliable tests are unfeasible with a performance comparable to the EIA and acid-fast technique.     

Screening tests for antibodies to cytomegalovirus: an evaluation of five commercial products.

Four hundred and ninety two samples of serum from blood donors were screened for the presence of antibodies specific to cytomegalovirus using radioimmunoassay, a modified complement fixation test, and five commercially available tests: the Cetus CMV IHA, Abbott CMV total AB EIA, Cytomegalisa Stat EIA, Enzygnost EIA, and Virenz G-CMV EIA. A wide variation in results was found, with only 53.5% of the sera giving total concordance by all methods. Rates of seropositivity in the different tests ranged from 34.9% to 59.3%, with sensitivities ranging from 75.2% to 99.1% compared with the radioimmunoassay. Of 211 sera which gave positive results with four or more of the tests, none was negative by the radioimmunoassay and Abbott EIA, three were negative in Cetus IHA and Enzygnost EIA, and 11 were negative in the modified complement fixation test. Virenz G and Cytomegalisa Stat EIAs, however, gave 40 (19%) and 49 (23.2%), respectively, as negative. The results confirmed the reliability of the radioimmunoassay for the detection of the antibody status to CMV, but this test is too elaborate for a screening procedure. The Abbott EIA and Cetus IHA were found to be the most suitable for this purpose in spite of high false positive rates.     

Microbiology of the intestinal lymph follicle: a clue to elucidate causative microbial agent(s) in Crohn's disease

It has been suggested that microbial agent(s) are involved in the onset of Crohn's disease. None of the candidates, however, has been unequivocally demonstrated to be a causative agent. The macroscopically earliest lesion takes place in the lymph follicle, irrespective of the initial attack or relapse in Crohn's disease. Human leucocyte antigen-DR (HLA-DR) antigens are expressed on the epithelium around the lymph follicle even in areas endoscopically uninvolved in Crohn's disease. These observations make the lymph follicle critical in the onset of Crohn's disease. The lymph follicle is a port of entry of a variety of microbial agent(s), leading to the speculation that microbial agent(s) exist in the lymph follicle. Polymerase chain reaction (PCR) using universal primers designed from conserved regions of bacterial ribosomal RNA or techniques such as representational difference analysis, may well identify microbial agent(s) in the lymph follicle that are specific to Crohn's disease. The existence of bacteria in the lymph follicle is here indicated by preliminary studies.     

Evaluation of an indigenously manufactured rapid immunochromatographic test for detection of HBsAg

A 'rapid' one step immunochromatographic, visually read, antigen capture assay--the "HEPACARD" (J Mitra & Co. Ltd., New Delhi, India) used for rapid screening of HBsAg was evaluated. Thousand consecutive sera sent to our laboratory for the purpose of HBsAg screening were tested by this device and by a third generation enzyme immunoassay (EIA) (Auszyme Monoclonal, Abbott Laboratories, Chicago, Illinois) or an automated Axsym microparticle enzyme immunoassay (MEIA) (Axsym HBsAg V2, Abbott Laboratories, Abbott Park, Chicago, Illinois, ISA). Hepacard showed a sensitivity of 79% (CU: 57.3-92%) and specificity of 98.9% (CI: 97.9-99.4%) when compared to the findings by the third generation EIA assays. This study suggested that this particular rapid HBsAg test results have to be confirmed by either an EIA or MEIA where the facility exists. The test may be used only in a small hospital setting where the facilities for enzyme immuno assays do not exist.     

Physicochemical studies of equine infectious anemia virus IV. Determination of the nucleic acid type in the virus

Summary The nucleic acid type in equine infectious anemia (EIA) virus was determined using isotope technique. In the present investigation, EIA virus grown in cultured horse leukocytes in the presence of³H-thymidine or³H-uridine was examined to see which precursor would be incorporated. The virus was purified from each infected culture by the combined procedure of ultracentrifugation, diethylaminoethyl cellulose chromatography and cesium chloride equilibrium density gradient centrifugation.³H-uridine was found to be incorporated into EIA virus, whereas³H-thymidine was not detected in the virus. Furthermore, purified³Huridine labeled EIA virus was fractionated by the Schmidt-Thannhauser-Schneider procedure. Radioactivity incorporated into the virus was mainly found in the fraction of ribonucleic acid (RNA). These results indicate that EIA virus contains RNA as the essential nucleic acid.Physicochemical and biological characteristics of EIA virus which have thus far been established seem to closely resemble those of RNA containing tumor viruses, and visna and maedi viruses.     

Phylogenetic analysis of Salmonella,Shigella, and Escherichia coli strains on the basis of the gyrB gene sequence

Phylogenetic analysis of about 200 strains of Salmonella, Shigella, and Escherichia coli was carried out using the nucleotide sequence of the gene for DNA gyrase B (gyrB), which was determined by directly sequencing PCR fragments. The results establish a new phylogenetic tree for the classification of Salmonella, Shigella, and Escherichia coli in which Salmonella forms a cluster separate from but closely related to Shigella and E. coli. In comparison with 16S rRNA analysis, the gyrB sequences indicated a greater evolutionary divergence for the bacteria. Thus, in screening for the presence of bacteria, the gyrB gene might be a useful tool for differentiating between closely related species of bacteria such as Shigella spp. and E. coli. At present, 16S rRNA sequence analysis is an accurate and rapid method for identifying most unknown bacteria to the genus level because the highly conserved 16S rRNA region is easy to amplify; however, analysis of the more variable gyrB sequence region can identify unknown bacteria to the species level. In summary, we have shown that gyrB sequence analysis is a useful alternative to 16S rRNA analysis for constructing the phylogenetic relationships of bacteria, in particular for the classification of closely related bacterial species.