Comparative evaluation of detection assays for Chlamydia trachomatis.

The performances of a commercial nucleic acid hybridization test (Gen-Probe Pace 2 Chlamydia trachomatis) and two commercial enzyme immunoassays (EIAs) (Abbott Chlamydiazyme and Pharmacia Chlamydia EIA) were evaluated against cell culture for the detection of C. trachomatis infection, with cervical swabs obtained from 1,037 women visiting a public sexual health center. The positivity rate by cell culture was 4.7%. Sensitivity and specificity for each test were as follows: Gen-Probe, 95.8 and 98.3%; Chlamydiazyme, 80.4 and 99.3%; Pharmacia EIA, 80.8 and 99.1%. Analysis of discrepant results with probe confirmation assay (Gen-Probe) and direct immunofluorescence (Syva Microtrak) revealed 12 cases of C. trachomatis infection for which culture was negative, resulting in the definition of a true-positive case as opposed to a culture positive. The positivity rate by true-positive definition was 5.9%, and sensitivity and specificity for each test were as follows: Gen-Probe, 96.7 and 99.6%; Chlamydiazyme, 77.5 and 100%; Pharmacia EIA, 77.0 and 100%; cell culture, 80.0 and 100%. We conclude that the Gen-Probe Pace 2 C. trachomatis test is a sensitive and specific alternative to cell culture for the detection of C. trachomatis.     

Personal experience in testing for E. coli enterotoxigenicity]

When testing the production of the thermolabile enterotoxin by strains of E. coli, using six different methods, the highest positivity (100%) was recorded when using the modified test of oedema on mouse paws and the lowest one (5%) when using the Biken test. The strains were isolated from diarrhoeal infections in young children.     

Single phage-typing set for differentiating salmonellae.

A phage-typing system is described for characterizing commonly isolated salmonellae. Fifty-eight serovars representative of groups A, B, C1, C2, D, E1, E2, E3, and E4 were delineated by using a single set of 50 phages isolated from sewage. All of the 735 cultures used in this effort were typable and were distinguished and differentiated on the basis of the 347 phage patterns observed. All results were reproducible. Characteristic phage patterns were produced by a variety of Salmonella serovars isolated from a campus incident and a number of hospital and family outbreaks to indicate an existing epidemiological relationship.     

New semisolid agar for the detection of motile salmonellae.

A semisolid selective motility agar based on selenite broth and designated semisolid selenite fecal (SSF) agar was developed for recovery of motile salmonellae from large numbers of rodent fecal specimens. The medium is easily prepared and inoculated; results are readily interpreted. When combined with selenite or gram-negative broth enrichment, SSF agar yielded significantly better Salmonella isolation from experimentally infected rodents than a standard battery of media. Only 14 non-Salmonella isolates from 1,002 Salmonella-free rodent fecal specimens migrated on SSF agar.     

Reduced set of phages for typing salmonellae.

A set composed of 27 phages is described for differentiating Salmonella spp. representative of groups A, B, C1, C2, D1, D2, E1, E2, E3, E4, G1, K, and N. All of the 1,245 cultures used in this effort were typable and were differentiated on the basis of the 420 phage patterns observed. All results were reproducible. Characteristic phage patterns were produced by a variety of Salmonella serovars isolated from campus incidents and a number of hospital, family, restaurant, and processing plant outbreaks to indicate an existing epidemiological relationship.     

Patterns of loss of enterotoxigenicity by Escherichia coli isolated from adults with diarrhea: suggestive evidence for an interrelationship with serotype.

Enterotoxigenic Escherichia coli isolates obtained in Mexico from adult subjects with diarrhea and from healthy controls were examined for the production of heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) after serial passage in the laboratory. Isolates were found to be either stable for the production of ST and LT or unstable with respect to ST, LT, or both. Unilateral loss of either ST or LT production allowed classification of E. coli isolates into four groups according to stability/instability of enterotoxin production. Fewer serotypes, with more representative isolates, were in group I (stable) than in group IV (completely unstable). Isolates from Dacca, Bangladesh, could be similarly classified into stability groups. There is an apparent relationship between serotype, stability of enterotoxin production, particularly LT, and isolation from diarrhea cases as opposed to isolation from healthy controls.     

Synthesis and biological assays of peptides from a tuberculin-active protein.

The octapeptide Asp-Gly-Gly-Ser-Glu-Ser-Glu-Gly and the hexadecapeptide Asp-Gly-Gly-Ser-Glu-Ser-Glu-Gly-Lys-Asn-Gly-Ser-Gln-Met-Arg-Leu, part of a tuberculin-active intracellular mycobacterial protein, were synthesized. The synthetic peptides were shown to possess tuberculin activity by their ability to elicit a delayed-type allergic reaction in skin tests on Mycobacterium tuberculosis-sensitized guinea pigs. Purified protein derivative, the complex mixture of proteins of unknown composition which are excreted into the culture medium by M. tuberculosis and which is in wide use as a tuberculin-active preparation, was shown to cross-react weakly in the radioimmunoassays with the synthetic octapeptide when the 125I-labeled octapeptide and an anti-octapeptide antiserum were used.     

Treponema-specific tests for serodiagnosis of syphilis: comparative evaluation of seven assays

The diagnosis of syphilis is challenging and often relies on serologic tests to detect treponemal or nontreponemal antibodies. Recently, the Centers for Disease Control and Prevention and the Association of Public Health Laboratories proposed an update to the syphilis serology testing algorithm, in which serum samples are first tested using a treponema-specific test and positive samples are analyzed with a nontreponemal assay. The goal of this study was to compare the performance of seven treponemal assays (BioPlex 2200 syphilis IgG [Bio-Rad, Hercules, CA], fluorescent treponemal antibody [FTA] assay [Zeus Scientific, Raritan, NJ], Treponema pallidum particle agglutination [TP-PA; Fujirebio Diagnostics, Malvern, PA], Trep-Sure enzyme immunoassay [EIA; Phoenix Biotech, Oakville, Ontario, Canada], Trep-Chek EIA [Phoenix Biotech], Trep-ID EIA [Phoenix Biotech], and Treponema ViraBlot IgG [Viramed Biotech AG, Planegg, Germany]) using serum samples (n = 303) submitted to our reference laboratory. In addition to testing with these 7 assays, all samples were tested by a rapid plasma reagin (RPR) assay and a treponemal IgM Western blot assay (Viramed ViraBlot). Compared to the FTA assay as the gold standard, the evaluated treponemal tests demonstrated comparable levels of performance, with percent agreement ranging from 95.4% (95% confidence interval, 92.3 to 97.3) for the Trep-Sure EIA to 98.4% (96.1 to 99.4) for the Trep-ID EIA. Compared to a "consensus of the test panel" (defined as at least 4 of 7 treponemal tests being in agreement), the percent agreement ranged from 95.7% (92.7 to 97.5) for Trep-Sure to 99.3% (97.5 to 99.9) for Trep-ID. These data may assist clinical laboratories that are considering implementing a treponemal test for screening or confirmatory purposes.     

Prevalence of enterotoxigenicity in human and nonhuman isolates of Yersinia enterocolitica.

A total of 414 cultures of Yersinia enterocolitica isolated from human and nonhuman sources were examined for heat-stable enterotoxin (ST) production to determine whether enterotoxigenicity was related to the source of isolation, serotype, or biochemical characteristics of the organism. A total of 65% of all cultures were found to produce ST. Enterotoxin production was much more prevalent in strains isolated from humans (218/232) than in those isolated from animals (17/34), water (9/49), raw milk (14/44), and food (10/55). Strains belonging to the serotypes O:3; 8; 5,27; 6,30; 9, often isolated from human infections, were almost always enterotoxigenic (191/196), although ST production was also highly prevalent among a few other serotypes. The most significant difference was observed between the groups that differed in the ability to ferment rhamnose; only 13 of 130 rhamnose-positive isolates produced ST (10%) compared to 255 of 284 rhamnose-negative cultures (90%). These results suggest that ST production is ubiquitous in Y. enterocolitica, with the highest prevalence among strains associated with human infections.     

Diagnostic performance of phospholipid-specific assays for the evaluation of antiphospholipid syndrome

The diagnostic performance of commercially available nonstandard antiphospholipid (aPL) assays for the evaluation of antiphospholipid syndrome (APS) is unknown. In 62 patients with APS, 88 with recurrent pregnancy loss, 50 healthy blood donors, and 24 women with one or more successful pregnancies, we measured antiphosphatidic acid (aPA), antiphosphatidyl-choline (aPC), antiphosphatidylethanolamine (aPE), antiphosphatidylglycerol (aPG), antiphosphatidylinositol (aPI), and antiphosphatidyl-serine (aPS) IgG and IgM antibodies from 2 manufacturers. We computed the areas under the curve (AUC), sensitivities, specificities, positive and negative predictive values, and 95% confidence intervals to assess diagnostic performance. The AUC analyses of the IgM assays demonstrated significant differences (P < .01) for all markers except aPC, whereas the IgG markers showed comparable performance for most assays with the exception of aPE (P < .01) and aPS (P = .02) antibodies. Overall, the combined sensitivity of the aPL assays differed significantly between manufacturers and did not improve the diagnostic yield for APS.     

Design and evaluation of consensus PCR assays for henipaviruses

Henipaviruses were first discovered in the 1990s, and their potential threat to public health is of increasing concern with increasing knowledge. Old-world fruit bats are the reservoir hosts for these viruses, and spill-over events cause lethal infections in a wide range of mammalian species, including humans. In anticipation of these spill-over events, and to investigate further the geographical range of these genetically diverse viruses, assays for detection of known and potentially novel strains of henipaviruses are required.The development of multiple consensus PCR assays for the detection of henipaviruses, including both SYBR Green and TaqMan real-time PCRs and a conventional heminested PCR is described. The assays are highly sensitive and have defined specificity. In addition to being useful tools for detection of known and novel henipaviruses, evaluation of assay efficiency and sensitivity across both biological and synthetic templates has provided valuable insight into consensus PCR design and use.     

Synthesis and biological assays of a peptide from a tuberculin-active protein.

The heptapeptide Asn-Gly-Ser-Gln-Met-Arg-Leu, part of a tuberculin-active intracellular mycobacterial protein and described in the literature as having residual tuberculin activity, has been synthesized. Biological assays of the synthetic peptide showed it to be recognized as an antigen of mycobacterial origin by its ability to elicit an early allergic reaction in Mycobacterium bovis BCG-infected mice. The synthetic peptide was shown to be devoid of any tuberculin activity in BCG-infected mice and in skin tests on Mycobacterium tuberculosis-sensitized guinea pigs. Purified protein derivative, complex mixture of proteins of unknown composition which is excreted into the culture medium by M. tuberculosis and is in wide use as a tuberculin-active preparation, was shown to weakly cross-react in radioimmunoassays with the synthetic heptapeptide when 125I-labeled heptapeptide and an anti-heptapeptide antiserum were used.     

Comparison of selective media for the isolation of salmonellae

A study of the selective action of four fluid media used for the isolation of salmonellae is described. When incubated in the presence of three competing organisms, test strains were recovered most frequently from Rappaport medium but mannitol selenite broth proved superior for the isolation of S. typhi.     

Functional activities of synthetic anaphylatoxic peptides in widely used biological assays.

A comparison study was carried out between the modern ATP release assay (ARA) with guinea-pig platelets and the traditional guinea-pig ileum contraction assay (ICA). The biological activities of the anaphylatoxin C3a and synthetic C3a analogue peptides were determined in both assays. In dose-response curves with C3a, a human C3a peptide with the last 21 amino acids of the C terminus (C3a 56-77) and a peptide with 13 amino acids which was acylated N-terminal with the aromatic fluorenylmethoxycarbonyl group and an aminohexanoyl group (Fmoc-Ahx YRRGRAAALGLAR) were tested. The ARA turned out to be 100 times more sensitive than the ICA. In contrast to previous reports the 21 amino acid long C3a analogue peptide did not exhibit full C3a activity but only 7% (ARA) or 12% (ICA). The potentiation of biological activity in the ARA by coupling non-peptide acyl-residues N terminal to peptide C3a analogues could be confirmed with Fmoc-Ahx-YRRGRAAALGLAR in the ICA. In addition, the tri-peptide Fmoc-Ahx-LAR displayed C3a specific activity in the ICA demonstrated by desensitization experiments.     

Relationships among in vitro mutagenicity assays: Quantitative vs. qualitative test results

In previous investigations, we studied the relationships between the profiles of the qualitative responses of in vitro short-term tests (mutation in Salmonella typhimurium, chromosomal aberrations in CHO cells, sister chromatid exchanges in CHO cells, and mutation in mouse lymphoma cells) and common sets of chemicals. In this paper, we address the study of the quantitative responses (potency). We show that two analyses point to similar patterns of relationships: the mutation in mouse lymphoma cells assay is most similar to the CHO sister chromatid exchange assay, and the Salmonella assay is most similar to the CHO chromosomal aberrations assay. © 1995 Wiley-Liss, Inc.     

Transepithelial signaling to neutrophils by salmonellae: a novel virulence mechanism for gastroenteritis.

Salmonella serotypes which elicit human enteritis cannot be distinguished from those that do not on the basis of their in vitro interactions with eukaryotic cells. We have recently reported that an enteritis-producing strain of Salmonella typhimurium signals intact intestinal epithelium to recruit subepithelial neutrophils to migrate across the epithelial (B. A. McCormick, S. P. Colgan, C. D. Archer, S. I. Miller, and J. L. Madara, J. Cell Biol. 123:895-907, 1993). We now utilize a cell culture model of human intestinal epithelium (with T84 cells) to examine whether such transepithelial signaling to neutrophils by salmonellae is predictive of potential to elicit gastroenteritis. Various Salmonella serotypes, including S. typhimurium, S. enteritidis, S. pullorum, S. arizonae, S. typhi, and S. paratyphi, as well as invasion-defective mutants of S. typhimurium, were studied. Strains or serotypes which elicit diffuse enteritis in humans (defined histologically as transepithelial migration of neutrophils) exhibited transepithelial signaling to neutorphils across epithelial cell monolayers, while those which do not elicit diffuse enteritis in humans did not display transepithelial signaling. In contrast, the ability to enter the apical surface of T84 cells did not differentiate strains or serotypes which induce diffuse enteritis from those which do not. These results strongly suggest that the ability of salmonellae to elicit transepithelial signaling to neutrophils is a key virulence mechanism underlying Salmonella-elicited enteritis.     

Comparative evaluation of real-time PCR assays for detection of the human metapneumovirus

The human metapneumovirus (hMPV) is a new member of the Paramyxoviridae family associated with acute respiratory tract infections in humans. The objective of this study was to compare the sensitivity of real-time RT-PCR assays performed in a LightCycler instrument and designed to amplify the viral nucleoprotein (N), matrix (M), fusion (F), phosphoprotein (P), and polymerase (L) genes. In a first evaluation of 20 viral cultures with characteristics compatible with hMPV cytopathic effect, the PCR positivity rates were 100, 90, 75, 60, and 55% using primers for the N, L, M, P, and F genes. In a second evaluation of 10 nasopharyngeal aspirates from children with bronchiolitis and found to be positive for the hMPV N gene, the PCR positivity rates for the L, M, P, and F genes were 90, 60, 30, and 80%, respectively. The analytic sensitivity of the real-time RT-PCR assay for the hMPV N gene was 100 copies using a transcribed viral plasmid. In conclusion, real-time PCR assays aimed at amplifying the N and L genes which are coding for two internal viral proteins appear particularly suitable for hMPV diagnostic.     

Comparative evaluation of 15 serological assays for the detection of syphilis infection

Fifteen commercial syphilis kits were assessed against the same moderately sized specimen panel that included 114 serum and plasma specimens from syphilis cases and 249 specimens from unselected blood donors. The 114 specimens from syphilis cases comprised 40 from cases of primary syphilis, 43 from cases of secondary syphilis, 19 from cases of early latent syphilis, and 12 from cases of late latent syphilis. Of the 15 kits, ten were enzyme immunoassays, four were Treponema pallidum haemagglutination assays, and one was a T. pallidum particle agglutination assay. Thirteen of the 15 kits gave final specificities of 100%; the other two kits were repeatedly reactive with one to two specimens. Initial sensitivities ranged from 93.9 to 99.1%. Most variation between kits was observed in results for the groups with untreated primary and treated late latent disease, although the differences were not statistically significant. The comparative data on kit performance derived from this study is useful for examining syphilis testing guidelines and for making informed purchasing decisions.