Correlation of Treponemal Immunoassay Signal Strength Values with Reactivity of Confirmatory Treponemal Testing.

Automated treponemal immunoassays are used for syphilis screening with the reverse-sequence algorithm; discordant results (e.g., enzyme immunoassay [EIA] reactive and reactive plasma reagin [RPR] nonreactive) are resolved with a second treponemal test. We conducted a study to determine automated immunoassay signal strength values consistently correlating with reactive confirmatory treponemal testing. We conducted a cross-sectional analysis of four automated immunoassays (BioPlex 2200 microbead immunoassay [MBIA], Liaison chemiluminescence immunoassay [CIA], Advia-Centaur CIA, and Trep-Sure EIA) and three manual assays (Treponema pallidum particle agglutination [TP-PA], fluorescent treponemal antibody absorption [FTA-ABS] test, and Inno-LIA line immunoassay). We compared signal strength values of automated immunoassays and positive and negative agreement. Among 1,995 specimens, 908 (45.5%) were true positives (≥4/7 tests reactive) and 1,087 (54.5%) were true negatives (≥4/7 tests nonreactive). Positive agreement ranged from 86.1% (83.7 to 88.2%) for FTA-ABS to 99.7% (99.0 to 99.9%) for Advia-Centaur CIA; negative agreement ranged from 86.3% (84.1 to 88.2%) for Trep-Sure EIA to 100% for TP-PA (99.6 to 100%). Increasing signal strength values correlated with increasing reactivity of confirmatory testing (P < 0.0001 for all automated immunoassays by Cochran-Armitage test for trend). All automated immunoassays had signal strength cutoffs corresponding to ≥4/7 reactive treponemal tests. BioPlex MBIA and Liaison CIA had signal strength cutoffs correlating with ≥99% and 100% TP-PA reactivity, respectively. The Advia-Centaur CIA and Trep-Sure EIA had signal strength cutoffs correlating with at least 95% TP-PA reactivity. All automated immunoassays had signal strength cutoffs correlating with at least 95% FTA-ABS reactivity. Assuming that a 95% level of confirmation is adequate, these signal strength values can be used in lieu of confirmatory testing with TP-PA and FTA-ABS.

Laboratory diagnosis of Clostridium difficile infections: there is light at the end of the colon.

Single molecular or multistep assays (glutamate dehydrogenase, toxin A/B, ± molecular) are recommended for the diagnosis of CDI in patients with clinically significant diarrhea. Rapid and accurate tests can improve resource allocations and improve patient care. Enzyme immunoassay (EIA) for toxins A/B is too insensitive for use as a stand-alone assay. This guideline will examine the use of molecular tests and multitest algorithms for the diagnosis of Clostridium difficile infection (CDI). These new tests, alone or in a multistep algorithm consisting of >1 assay, are more expensive than the older EIA assays; however, rapid and accurate testing can save money overall by initiating appropriate treatment and infection control protocols sooner and by possibly reducing length of hospital stay. We recommend testing only unformed stool in patients with clinically significant diarrhea by a molecular method or by a 2- to 3-step algorithm.

Clostridium difficile ribotype diversity at six health care institutions in the United States.

Capillary-based PCR ribotyping was used to quantify the presence/absence and relative abundance of 98 Clostridium difficile ribotypes from clinical cases of disease at health care institutions in six states of the United States. Regionally important ribotypes were identified, and institutions in close proximity did not necessarily share more ribotype diversity than institutions that were farther apart.

Evaluation of the Cepheid Xpert MTB/RIF assay for direct detection of Mycobacterium tuberculosis complex in respiratory specimens.

A total of 217 specimens submitted for routine smear and culture from three different sites within the western United States were used to evaluate the GeneXpert MTB/RIF assay (for research use only) (Cepheid, Sunnyvale, CA). Overall agreement compared to culture was 89% (98% for smear positives and 72% for smear negatives) for detection of Mycobacterium tuberculosis.

Comparison of premier CAMPY enzyme immunoassay (EIA), ProSpecT Campylobacter EIA, and ImmunoCard STAT! CAMPY tests with culture for laboratory diagnosis of Campylobacter enteric infections.

Campylobacter enteritis is a food-borne or waterborne illness caused almost exclusively by Campylobacter jejuni and, to a lesser extent, by Campylobacter coli. These organisms produce indistinguishable clinical diseases and together represent the second most common cause of bacterial diarrhea in the United States and the leading cause of enteric infection throughout the world. The conventional approach to the laboratory diagnosis of Campylobacter enteritis is based on the recovery of the organism from a stool specimen, which requires the use of a specialized medium incubated at 42°C for several days in an artificially created microaerophilic environment. Recently, several commercially available enzyme immunoassays (EIAs) have been developed for the direct detection of C. jejuni and C. coli in stool specimens. This study compared conventional culture with three EIA methods, the Premier CAMPY EIA (Meridian Bioscience, Cincinnati, OH), the ProSpecT Campylobacter EIA (Remel, Lenexa, KS), and the ImmunoCard STAT! CAMPY test (Meridian Bioscience, Cincinnati, OH), for the detection of C. jejuni and C. coli in 485 patient stool samples. Discordant results were arbitrated by using an in-house, real-time PCR assay that was developed and validated by a public health reference laboratory. Following analyses of the discrepant specimens by PCR, the sensitivity and specificity of both the Premier CAMPY and ProSpecT Campylobacter EIAs were 99.3% and 98%, respectively, while the ImmunoCard STAT! CAMPY test had a sensitivity of 98.5% and a specificity of 98.2%. By use of the PCR test as the reference standard, culture detected 127 of 135 Campylobacter-positive stool specimens, yielding a sensitivity of 94.1%. These results showed that the three EIAs evaluated in this study provide a rapid and reliable alternative for the laboratory diagnosis of enteric infections with C. jejuni and C. coli and that conventional culture may no longer be recognized as the "gold standard" for diagnosis.

Clostridium difficile testing in the clinical laboratory by use of multiple testing algorithms.

The incidence of Clostridium difficile infection (CDI) has risen almost 3-fold in the United States over the past decade, emphasizing the need for rapid and accurate tests for CDI. The Cepheid Xpert C. difficile assay is an integrated, closed, nucleic acid amplification system that automates sample preparation and real-time PCR detection of the toxin B gene (tcdB). A total of 432 stool specimens from symptomatic patients were tested by a glutamate dehydrogenase (GDH) assay, a toxin A and B enzyme immunoassay (EIA), the Xpert C. difficile assay, and a cell culture cytotoxicity neutralization assay (CCCN). The results of these methods, used individually and in combination, were compared to those of toxigenic culture. Results for the Xpert C. difficile assay alone showed a sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of 94.4, 96.3, 84.0, and 98.8%, while the EIA alone gave corresponding values of 58.3, 94.7, 68.9, and 91.9%, respectively. An algorithm using the GDH assay and the EIA (plus the CCCN if the EIA was negative) showed corresponding values of 83.1, 96.7, 83.1, and 96.1%. The Xpert C. difficile assay was statistically superior to the EIA (P, <0.001 by Fisher's exact test) and to the GDH-EIA-CCCN algorithm (P, 0.0363). Combining the GDH and Xpert C. difficile assays lowered both the sensitivity and the NPV of the Xpert assay. The GDH-EIA-CCCN procedure required, on average, 2 days to complete testing on GDH-positive results, while testing by the Xpert C. difficile assay was completed, on average, in less than 1 h. Xpert C. difficile testing yielded the highest sensitivity and NPV, in the least amount of time, of the individual- and multiple-test algorithms evaluated in this study.

Trimethoprim in vitro antibacterial activity is not increased by adding sulfamethoxazole for pediatric Escherichia coli urinary tract infection.

PURPOSE: The combination of trimethoprim/sulfamethoxazole is often used to treat uncomplicated urinary tract infections in children. The rationale for combining trimethoprim and sulfamethoxazole is that they may act synergistically to increase antibacterial activity. However, approximately 3% of patients show allergic reactions to sulfamethoxazole, of which some are serious (liver failure and Stevens-Johnson syndrome). We determined whether adding sulfamethoxazole is necessary to increase in vitro antibacterial activity for pediatric urinary tract infection compared to that of trimethoprim alone. MATERIALS AND METHODS: We prospectively identified 1,298 children with urinary tract infection (greater than 100,000 cfu/ml Escherichia coli) from a total of 4 American regions. In vitro susceptibility of bacterial isolates to sulfamethoxazole, trimethoprim and trimethoprim/sulfamethoxazole was determined using disk diffusion. Ampicillin susceptibility was tested at 2 sites. At 1 site all uropathogens from consecutive urinary isolates were evaluated. RESULTS: E. coli susceptibility to trimethoprim was 70%, comparable to the 70% of trimethoprim/sulfamethoxazole (p = 0.9) and higher than the 56.9% of sulfamethoxazole (p <0.05). This susceptibility pattern was without regional differences. At 2 sites susceptibility to trimethoprim was significantly higher than to ampicillin. At 1 site the susceptibility of other uropathogens to trimethoprim and trimethoprim/sulfamethoxazole was similar to that of E. coli. CONCLUSIONS: In children with urinary tract infection in vitro susceptibility to trimethoprim was comparable to that to trimethoprim/sulfamethoxazole and significantly higher than to sulfamethoxazole. This finding was similar at all sites. Adding sulfamethoxazole appears unnecessary and may represent a risk to patients. Trimethoprim can be used as an alternative to trimethoprim/sulfamethoxazole based on in vitro antibacterial susceptibility. Routine trimethoprim/sulfamethoxazole use for urinary tract infection should be carefully reevaluated.

Rapid group B streptococci screening using a real-time polymerase chain reaction assay.

OBJECTIVE: To estimate the clinical performance characteristics of a real-time polymerase chain reaction (PCR) assay using vaginal/rectal swabs from antepartum (35-37 weeks of gestation) and intrapartum women. METHODS: The assay evaluated is a qualitative, automated, real-time PCR test for the detection of group B streptococci, with results available in approximately 75 minutes. Enrollment in this multicenter clinical study occurred between October 2005 and January 2006. Vaginal/rectal swabs were analyzed by nursing personnel (intrapartum tests) or by laboratory technologists (all others). Polymerase chain reaction assay results were compared with culture using standard methods, including selective broth medium, and to a predicate nucleic acid amplification test. RESULTS: Of 1,028 enrolled women, 234 were deemed ineligible, and 10 had unresolved test results. Of the 784 remaining women, valid PCR assay results were obtained on the first test attempt for 93.0%. Performance characteristics relative to culture were sensitivity 91.1%, specificity 96.0%, positive predictive value 87.8%, negative predictive value 97.1%, and accuracy 94.8%. These results exceeded those obtained using the predicate nucleic acid amplification test. CONCLUSION: Performance characteristics of the PCR assay exceed the threshold recommended by the Centers for Disease Control and Prevention when compared with culture. The test is sufficiently robust to be performed for intrapartum patients in a point-of-care setting by medical professionals. LEVEL OF EVIDENCE: II.

Performance of a cytomegalovirus IgG enzyme immunoassay kit modified to measure avidity.

The measurement of cytomegalovirus (CMV) IgG avidity accurately discriminates recent and past CMV infections. We sought to determine if the Wampole Laboratories CMV IgG enzyme immunoassay (EIA) could be modified to measure avidity. The evaluation panel consisted of 156 serum samples we used in 2002 to validate a laboratory-developed EIA, in which 78 serum samples exhibited low avidity, 7 exhibited intermediate avidity, and 71 exhibited high avidity. The qualitative agreement between the two avidity assays was 94% (147/156); all 9 sera with discordant results exhibited intermediate avidity in one of the assays. The avidity index (AI) values in the two assays showed excellent correlation (r = 0.96, P < 0.0001). The definition of high avidity was verified for the Wampole assay by demonstrating high avidity in 91/93 (98%) recently collected CMV IgG-positive/IgM-negative serum samples. The performance of the Wampole avidity assay in a reference laboratory setting was assessed using 470 consecutive serum samples submitted for CMV IgG avidity testing. Surprisingly, 101 serum samples were negative when screened for CMV IgG using the Wampole kit per the package insert; 98 of these 101 serum samples were tested using a CMV IgG chemiluminescent immunoassay, and only 5 were positive. Of the 369 CMV IgG-positive samples, 6% exhibited low IgG avidity, 6% exhibited intermediate avidity, and 88% exhibited high avidity; CMV IgM detection rates were inversely related to AI levels. These findings show that (i) the Wampole CMV IgG EIA can be modified to measure CMV IgG avidity, (ii) many samples are apparently submitted for avidity testing without knowledge of their CMV IgG status, and (iii) most CMV IgG-positive sera submitted for avidity testing exhibit high avidity.

Comparison of the premier toxin A and B assay and the TOX A/B II assay for diagnosis of Clostridium difficile infection.

Clostridium difficile causes nosocomial diarrhea and is responsible for complications such as pseudomembranous colitis, megacolon, and perforation. Using 442 stool specimens, we compared the sensitivities and specificities of the Premier toxin A and B (Meridian Bioscience, Inc.) and C. difficile TOX A/B II (TechLab, Inc., Blacksburg, VA) immunoassays in the Virology Department of the Kaiser Permanente Regional Reference Laboratories. The Premier toxin A and B assay demonstrated a higher sensitivity (97.44%) and a higher positive predictive value (79.17%) than the C. difficile TOX A/B II assay (87.18% and 75.56%, respectively), while assay specificities and negative predictive values were similar. We also performed experiments using serially diluted, purified toxin A and B antigens to understand the basis for assay differences. The two assays' toxin A antibodies detected toxin A at comparable levels. Preliminary results indicated that the toxin B antibody in the Premier toxin A and B assay could detect toxin B at a concentration of 125 pg/100 microl, while the toxin B antibody in the C. difficile TOX A/B II assay could not detect toxin B below a concentration of 250 pg/100 microl. Therefore, the Premier toxin A and B assay provides greater sensitivity than the C. difficile TOX A/B II assay, perhaps due to a superior detection ability of its toxin B antibody.