Cockerill, Franklin R.

ABSTRACT Using data from 23,313 patients, we assessed whether two blood culture sets of three bottles per set would detect more pathogens than two sets of two bottles per set and achieve similar sensitivity to collecting three sets of two bottles per set. We also compared the yield of aerobic and anaerobic bottles. Thirty milliliters of blood was distributed to one anaerobic and two aerobic bottles. Among 26,855 collections of ≥60 ml within 30 min, 1,379 (5.1%) were positive for a pathogen not requiring detection in more than one set to be considered a pathogen, with 72 additional distinct pathogens detected using two 30-ml compared to two 20-ml sets of one aerobic and one anaerobic bottle (increased yield, 7.9%; 95% confidence interval [CI], 6.2 to 9.8%). For conditional pathogens requiring detection in at least two positive blood cultures for classification as pathogens (i.e., otherwise classified as contaminants), there were 162 positive detections with two 30-ml sets, of which 16 would not have been detected by two 20-ml sets (increased yield, 11.0% [95% CI, 6.4 to 17.2%]). Among 134 subjects who had three sets of 30 ml each within a 30-min interval, there was complete concordance between 60 ml of blood drawn in the first two sets of 30 ml and three 20-ml sets ( P = 1.0). One aerobic bottle plus one anaerobic bottle yielded more pathogens than two aerobic bottles for organisms requiring a single ( P < 0.001) and two ( P = 0.04) positive sets to be defined as pathogens. In conclusion, we showed that collection of two aerobic and one anaerobic blood culture bottles per set results in improved yield compared to two bottles per set. We also confirmed that an anaerobic bottle should be included in blood culture sets.

ABSTRACT Since some strains of Brucella species may require carbon dioxide for growth, a multilaboratory study was conducted to compare broth microdilution susceptibility results using ambient air (AA) and 5% CO 2 incubation conditions. Six antimicrobial agents were tested against 39 Brucella isolates. Aminoglycoside MICs tended to be 1 log 2 dilution higher in CO 2 than in AA; tetracycline-class MICs to be 1 log 2 dilution lower in CO 2 .

ABSTRACT The acquisition of β-lactamases in members of the Enterobacteriaceae family poses a challenge to antimicrobial susceptibility testing in the clinical laboratory. We correlated the distribution of the MICs for Klebsiella spp. and Escherichia coli with the presence of extended-spectrum β-lactamase (ESBL) and plasmid-mediated AmpC β-lactamase (pAmpC) genes. A total of 264 isolates were subjected to cefazolin, ceftriaxone, cefotaxime, ceftazidime, cefepime, and aztreonam agar dilution MIC determination; ESBL screening and confirmatory testing by the methods of the Clinical and Laboratory Standards Institute (CLSI); and for isolates for which the MICs of extended-spectrum cephalosporins were ≥1 μg/ml or the MICs of cefpodoxime were >4 μg/ml, PCR amplification and sequencing of the ESBL and pAmpC genes. PCR was positive for 73/81 isolates (45 isolates with an ESBL gene alone, 24 isolates with a pAmpC gene alone, with 4 isolates with both genes). Compared to PCR, confirmatory testing by the CLSI method yielded a sensitivity and a specificity of 98.0 and 96.3%, respectively; there were six false-positive results and one false-negative result. No distinction in the MIC distribution was apparent between isolates with the ESBL gene and isolates with the pAmpC gene. A substantial percentage of the isolates with PCR-confirmed ESBL and/or pAmpC genes fell within the current CLSI susceptible category. For a ceftazidime, ceftriaxone, or cefotaxime MIC of ≥2 μg/ml, a dichotomy existed between isolates with and without ESBL and pAmpC genes in most cases. This suggests that the presence of the ESBL and the pAmpC enzymes may yield similar MICs of extended-spectrum cephalosporins, many of which fall within the current nonresistant categories. Lowering of the current CLSI breakpoints for cephalosporins appears to be warranted.

ABSTRACT Prosthetic valve endocarditis is an uncommon manifestation of infection with Bartonella species. Herein, we report a case of Bartonella henselae endocarditis involving prosthetic mitral and aortic valves. The patient had a favorable outcome with combined medical and surgical therapy. Concomitant crescentic glomerulonephritis led to an initial mistaken diagnosis of Wegener's granulomatosis.

Abstract Rationale Although the serial dilution technique for quantitative culture of bronchoalveolar fluid is considered to be the gold standard for the diagnosis of ventilator-associated pneumonia, it is more labor intensive than the calibrated loop technique. Objective We sought to determine the agreement between the calibrated loop and serial dilution techniques in the diagnosis of ventilator-associated pneumonia. Methods We prospectively measured bacterial colony counts by the serial dilution and calibrated loop techniques in 121 bronchoalveolar lavage samples of 104 patients with suspected ventilator-associated pneumonia. Measurements and Main Results At the time of bronchoscopy, patients had received mechanical ventilation for a median of 8 d. Patients were receiving antibiotics when 90 of the 121 (74.4%) bronchoalveolar samples were obtained. The colony counts of 13 bacterial isolates were too numerous to count by the calibrated loop technique; by serial dilution technique, their counts ranged from 4.70 to 6.74 log10 cfu/ml. Fifty other bacteria had paired colony counts measured by each of the two techniques: the bias (95% confidence interval) between the two techniques was −0.380 (−0.665 to −0.095) log10 cfu/ml, with precision of 1.002 log10 cfu/ml and 95% limits of agreement of −2.344 to 1.584 log10 cfu/ml. Using the threshold of 4 log10 cfu/ml as a criterion for the diagnosis of ventilator-associated pneumonia, there was discordance only for one bacterial organism between the two techniques. Conclusions The calibrated loop technique can be used for the diagnosis of ventilator-associated pneumonia using bronchoalveolar lavage fluid.

ABSTRACT Explanted orthopedic implants from 54 patients with aseptic failure and 24 patients with prosthetic knee or hip infection were sonicated in polyethylene bags. The sensitivities of periprosthetic tissue and sonicate fluid cultures for the diagnosis of prosthetic joint infection were 54% and 75%, whereas the specificities were 98% and 87%, respectively. Sonication in bags improved bacterial recovery from the surface of orthopedic implants; however, it lacked specificity, due to bag leakage.

SUMMARY Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

ABSTRACT The results for a real-time PCR assay, using the LightCycler Strep B analyte-specific reagents (Roche Diagnostics Corporation, Indianapolis, Ind.), were compared to a direct plate method combined with a broth enrichment culture method for detection of group B streptococcus colonization in pregnant women. Two separate evaluations were conducted using two different automated nucleic extraction instruments, the MagNA Pure LC instrument (Roche Diagnostics Corporation) and the lower-capacity MagNA Pure Compact instrument (Roche Diagnostics Corporation). The sensitivities, specificities, and positive and negative predictive values for the different evaluation methods were as follow: for the LightCycler Strep B assay with MagNA Pure LC, 100, 97, 90, and 100%, respectively; for the LightCycler Strep B assay with MagNA Pure Compact, 92.5, 99, 97, and 97.5%, respectively. The LightCycler Strep B assay combined with either MagNA Pure LC or MagNA Pure Compact extraction is a suitable method for detecting group B streptococcus colonization in pregnant women. An advantage of the LightCycler assay over culture is the considerably reduced turnaround time for results.

ABSTRACT A real-time PCR method using the LightCycler (Roche Applied Science, Indianapolis, IN) was compared to a conventional PCR assay for the detection of Tropheryma whipplei in 321 clinical specimens. The LightCycler method had sensitivity and specificity comparable to the conventional method but required considerably less labor and time (3 h versus 3 to 5 days).