Susan M. Novak-Weekley

Clostridium difficile Testing in the Clinical Laboratory by Use of Multiple Testing Algorithms

ABSTRACT 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 Fishers 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.

Evaluation of the Cepheid Xpert MTB/RIF Assay for Direct Detection of Mycobacterium tuberculosis Complex in Respiratory Specimens

ABSTRACT 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.

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.

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

Comparison of Premier CAMPY Enzyme Immunoassay (EIA), ProSpecT Campylobacter EIA, and ImmunoCard STAT! CAMPY Tests with Culture for Laboratory Diagnosis of Campylobacter Enteric Infections

ABSTRACT 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 Ribotype Diversity at Six Health Care Institutions in the United States

ABSTRACT 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.

Multicenter Evaluation of a New Shortened Peptide Nucleic Acid Fluorescence In Situ Hybridization Procedure for Species Identification of Select Gram-Negative Bacilli from Blood Cultures

ABSTRACT A shortened protocol for two peptide nucleic acid fluorescence in situ hybridization (PNA FISH) assays for the detection of Gram-negative bacilli from positive blood cultures was evaluated in a multicenter trial. There was 100% concordance between the two protocols for each assay (368 of 368 and 370 of 370 results) and 99.7% (367 of 368 and 369 of 370 results) agreement with routine laboratory techniques.

The role of the clinical laboratory in the future of health care: lean microbiology.

ABSTRACT This commentary will introduce lean concepts into the clinical microbiology laboratory. The practice of lean in the clinical microbiology laboratory can remove waste, increase efficiency, and reduce costs. Lean, Six Sigma, and other such management initiatives are useful tools and can provide dividends but must be accompanied by organizational leadership commitment to sustaining the lean culture in the laboratory setting and providing resources and time to work through the process.

Enhancing the Function of Clinical Microbiology Laboratories: Can We Navigate the Road Less Traveled?

This is a very exciting and dynamic period for clinical microbiology: not only are we seeing changes in the way we practice, but we also have a very sophisticated array of tools that are increasingly at our disposal. These tools, and the changes they impart, can substantially improve the quality and

Performance of the cobas HPV Test for the Triage of Atypical Squamous Cells of Undetermined Significance Cytology in Cervical Specimens Collected in SurePath

Objectives Determine performance of the cobas human papillomavirus (HPV) test for triage of atypical squamous cells of undetermined significance (ASC-US) in SurePath. Methods Women presenting for routine screening had cervical specimens collected in SurePath and specimen transport medium (STM); those with ASC-US cytology underwent colposcopy. Performance of cobas HPV in SurePath specimens that had undergone a preanalytic procedure to reverse possible cross-linking of HPV DNA was compared with Hybrid Capture 2 (hc2) specimens in STM. Results Among 856 women, HPV prevalence was 45.8%; HPV 16 and HPV 18 prevalences were lower than expected in the 21- to 29-year-old group in this highly vaccinated population. cobas HPV performance in SurePath was comparable to hc2 in STM. Sensitivity and specificity for detection of cervical intraepithelial neoplasia grade 3 or worse were 87.5% (95% confidence interval [CI], 71.9%-95.2%) and 55.5% (95% CI, 52.1%-58.9%) for cobas and 85.3% (95% CI, 69.9%-93.6%) and 54.7% (95% CI, 51.4%-57.9%) for hc2. Sensitivity was negatively affected by random biopsies performed at colposcopy; comparable sensitivities were achieved in the nonvaccinated and vaccinated populations with disease determined by directed biopsy only. Conclusions Performance of cobas HPV for ASC-US triage in pretreated SurePath specimens meets criteria for validation. Preliminary data indicate reliable performance of HPV testing in a highly vaccinated population.