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ABSTRACTWe compared the performance characteristics of a real-time PCR method, the LightCycler vanA/vanBdetection assay (Roche Diagnostics Corporation, Indianapolis, Ind.) to that of Enterococcosel agar (BBL, Sparks, Md.) for direct detection of vancomycin-resistant enterococci (VRE) from 894 perianal stool swabs. For 421 of 894 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing vancomycin at 6 µg/ml; for the remaining 473 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing 8 µg/ml vancomycin. The LightCycler method produced considerably more positive results than either the Enterococcosel plate containing vancomycin at 6 µg/ml (n= 25 versus n= 11; sensitivity, 100%; specificity, 97%; positive predictive value [PPV], 42%; negative predictive value [NPV], 100%) or the Enterococcosel plate containing vancomycin at 8 µg/ml (n= 31 versus n= 10; sensitivity, 100%; specificity, 95%; PPV, 32%; NPV, 100%). When possible, additional testing, including culture, LightCycler PCR, and/or a conventional PCR method (PCR-restriction fragment length polymorphism assay), were performed on either the original specimens or original cultures or subsequent specimens for cases in which the original specimen was positive by LightCycler PCR but the Enterococcosel plate was negative. This additional testing demonstrated positive results for 7 of 14 (50%) evaluable discordant specimens which initially tested as LightCycler PCR positive but culture negative using the Enterococcosel plate containing vancomycin at 6 µg/ml and 12 of 17 (71%) evaluable discordant specimens which initially tested as LightCycler positive but culture negative using the Enterococcosel plate containing vancomycin at (8 µg/ml). These results demonstrate that the LightCycler VRE detection assay is considerably more sensitive than the standard culture method for detecting VRE directly from perianal swab specimens. The LightCycler assay also provides results much faster than culture (~3.5 versus =72 h). The use of this test could have important implications for the effective control and prevention of nosocomial outbreaks of VRE.

ABSTRACTThe prevalence of microsporidiosis is likely underestimated due to the labor-intensive, insensitive, and nonspecific clinical laboratory methods used for the diagnosis of this disease. A real-time PCR assay was designed to assess DNA extraction methods and to detect three Encephalitozoonspecies in feces. Modifications of the MagNA Pure LC DNA isolation kit protocol (Roche Applied Sciences, Indianapolis, Ind.) were compared by using the automated MagNA Pure LC instrument (Roche) and fecal specimens spiked with Encephalitozoonintestinalisspores. Extracted DNA was amplified by the LightCycler (Roche) PCR assay. Assay sensitivity, reproducibility, and efficiency were assessed by comparing threshold crossover values achieved with different extraction and storage conditions (fresh, refrigerated, frozen, and preserved specimens). Optimal extraction conditions were achieved by using a commercial buffer, tissue lysis buffer (Roche), as the specimen diluent. LightCycler PCR results were compared to those obtained from routine stool microscopy with trichrome blue stain. The lower limit of detection for the LightCycler PCR assay varied by storage conditions from 102to 104spores/ml of feces, a value which represented a significant improvement over that achieved by staining (=1.0 × 106spores/ml). Melting temperature analysis of the amplicons allowed for the differentiation of three Encephalitozoonspecies (E. intestinalis, E. cuniculi, and E. hellem). The assay is readily adaptable to the clinical laboratory and represents the first real-time PCR assay designed to detect Encephalitozoonspecies.

The oxygen tolerance and sensitivity of 57 freshly isolated anaerobic bacteria from clinical specimens was studied. All the organisms tolerated 8 h or more of exposure to oxygen in room air. Growth of the isolates in increasing oxygen concentrations demonstrated that the 57 isolates varied in oxygen sensitivity from strict to aerotolerant anaerobes. Comparison of the oxygen tolerance and sensitivity showed that the most tolerant organisms (best survival after prolonged exposure) included anaerobes capable of growth at only 0.4% or less O2 (strict) as well as those able to grow in as much as 10% O2. The least tolerant were predominately strict anaerobes. Decrease in the inoculum size from a concentration of 10(8) to 10(6) colony-forming units per ml had only a minor effect. The data indicate that the brief oxygen exposure with bench techniques in clinical laboratories would not be deleterious to the anaerobic bacteria present in clinical specimens.

During a 2-year period, 1,892 patients underwent biliary tract surgery at the Mayo Clinic. Both aerobic and anaerobic cultures of bile were performed in 371 of these patients. Sixty-nine percent of the cultures were positive, and 41% (117) of these grew anaerobes, although they were present in pure culture only twice. Mixed cultures most commonly contained four different organisms (three aerobes and one anaerobe). Bacteroides fragilis was the single most commonly isolated anaerobe and ranked fourth in terms of overall isolates behind Escherichia coli, group D streptococci, and Klebsiella B. fragilis accounted for 7.0% of the total group D streptococci, and Klebsiella. B. fragilis accounted for 7.0% of the total aerobic and anaerobic isolates and was present in 21% of all positive cultures. Pseudomonas aeruginosa and Clostridium perfringens ranked fifth and sixth, providing 6.5 and 5.9% of all isolates, respectively. This study demonstrates the frequent presence of anaerobes in patients with bactibilia and suggests that they be considered in the formulation of antimicrobial therapy for infections involving human biliary tracts.

In a new method of anaerobic culture, a transparent, gas-impermeable bag is used and the anaerobic environment is established with copper sulfate-saturated steel wool. An Alka-Seltzer tablet generates carbon dioxide. The agar plate surface can be inspected through the bag at any time without interrupting the anaerobic atmosphere or disturbing other specimens. Methylene blue indicator strips are completely reduced by 4 h after the bag is set up and have remained reduced for as long as 3 weeks. Growth of 16 different stock culture anaerobes was generally equivalent by the bag and GasPak jar methods. Yield and growth of anaerobic isolates also were equivalent with 7 of 10 clinical specimens; from the other 3 specimens, 13 isolates were recovered, 5 by both the bag and jar methods and the rest by one method or the other. No consistent differences were found between the anaerobic bag and GasPak jar methods in the yield of anaerobes from clinical specimens. Early growth (24 h of incubation) of anaerobes from one specimen was detected with the bag method.

ABSTRACTDetection of GiardiaandCryptosporidiumin clinical stool specimens using the ColorPAC and ProSpecT enzyme immunoassays revealed 98.7% agreement forGiardiadetection and 98.1% agreement forCryptosporidiumdetection. Sensitivities were uniformly 100%. The specificities of the ColorPAC immunoassay forGiardiaand Cryptosporidiumdetection were 100 and 99.5%, respectively, and those for the ProSpecT assay were 98.4 and 98.6%, respectively. The false-positive reactions with the ProSpecT assay occurred with specimens that were grossly bloody.

ABSTRACTRecently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of =0.650 and =0.900) were retested; if the second analysis produced an index of >0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidummicrohemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a reliable method for syphilis testing and that personnel performing tests which require subjective interpretation, like the FTA-ABS test, may be biased by RPR test results.