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On behalf of the Clinical and Laboratory Standards Institute (CLSI), the Expert Panel on Microbiology would like to respond to the recent commentary by Kirby and colleagues voicing concerns related to verification of commercial antimicrobial susceptibility testing (AST) for new drugs that are introduced into the clinical laboratory (1).…

The FecalSwab system (Copan Italia, Brescia, Italy) is a convenient alternative to bulk stool for the diagnosis of enteric pathogens. Although the U.S. Food and Drug Administration (FDA) approved for transport and culture of enteric bacterial pathogens, the FecalSwab has not been well assessed for its suitability with molecular platforms. In this study, we evaluated the FecalSwab as a specimen type for the BD Max system using the viral and bacterial enteric panels (BD Diagnostics, Baltimore, MD, USA)., The FecalSwab system (Copan Italia, Brescia, Italy) is a convenient alternative to bulk stool for the diagnosis of enteric pathogens. Although the U.S. Food and Drug Administration (FDA) approved for transport and culture of enteric bacterial pathogens, the FecalSwab has not been well assessed for its suitability with molecular platforms. In this study, we evaluated the FecalSwab as a specimen type for the BD Max system using the viral and bacterial enteric panels (BD Diagnostics, Baltimore, MD, USA). A total of 186 unpreserved stool specimens were collected and used to prepare matched bulk stool and FecalSwab samples. Performance was equivalent (P > 0.48) to bulk stool for all targets when 50 μl of FecalSwab specimen was loaded onto the BD Max assays. As stool specimens are often collected off-site from the clinical microbiology laboratory and require transport, we assessed the stability of stool specimens stored for up to 14 days at 4°C, 22°C, or 35°C to account for varying transportation conditions. Molecular detection for the majority of viral targets (excluding astrovirus) was unaffected (change in cycle threshold [ΔCT] ≤ 1) by sample storage temperature over the 2-week period; however, detection of enteric bacteria was variable if specimens were not refrigerated (22°C or 35°C). By demonstrating equivalent performance to matched bulk stool and maintaining molecular detection sensitivity when stored at 4°C, we suggest that the FecalSwab is a suitable specimen type for enteropathogen diagnostics on the BD Max system.

Urinary tract infections are one of the most common reasons for health care visits. Diagnosis and optimal treatment often require a urine culture, which takes an average of 1.5 to 2 days from urine collection to results, delaying optimal therapy. Faster, but accurate, alternatives are needed. Light scatter technology has been proposed for several years as a rapid screening tool, whereby negative specimens are excluded from culture. A commercially available light scatter device, BacterioScan 216Dx (BacterioScan, Inc.), has recently been advertised for this application. Paired use of mass spectrometry (MS) for bacterial identification and automated-system-based susceptibility testing straight from the light scatter suspension might provide dramatic improvement in times to a result. The present study prospectively evaluated the BacterioScan device, with culture as the reference standard. Positive light scatter specimens were used for downstream rapid matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS organism identification and automated-system-based antimicrobial susceptibility testing. Prospective evaluation of 439 urine samples showed a sensitivity of 96.5%, a specificity of 71.4%, and positive and negative predictive values of 45.1% and 98.8%, respectively. MALDI-TOF MS analysis of the suspension after density-based selection yielded a sensitivity of 72.1% and a specificity of 96.9%. Antimicrobial susceptibility testing of the samples identified by MALDI-TOF MS produced an overall categorical agreement of 99.2%. Given the high sensitivity and negative predictive value of results obtained, BacterioScan 216Dx is a reasonable approach for urine screening and might produce negative results in as few as 3 h, with no downstream workup. Paired rapid identification and susceptibility testing might be useful when MALDI-TOF MS results in an organism identification, and it might decrease the time to a result by more than 24 h.

Chlamydia trachomatis and Neisseria gonorrhoeae are the two most common causes of sexually transmitted disease in the United States. Studies in adults, mostly in men who have sex with men, have shown that the prevalence of C. trachomatis and N. gonorrhoeae infections is much higher in extragenital sources compared to urogenital sources. A similar large sample of data on the burden of C. trachomatis and N. gonorrhoeae infections by anatomic site is lacking in children. We retrospectively analyzed data from 655 patients tested for C. trachomatis (887 specimens) and N. gonorrhoeae (890 specimens) at the Children’s Hospital of Philadelphia. We restricted the analysis to include patients between 2 and 17 years of age that had all three sources (urine, oropharynx, and rectum) collected at the same visit. The final data set included specimens from all three sources from 148 and 154 patients for C. trachomatis and N. gonorrhoeae, respectively. Specimens were tested for C. trachomatis and N. gonorrhoeae using a Gen-Probe Aptima Combo 2 assay. The burden of C. trachomatis and N. gonorrhoeae infection was significantly higher in the 14- to 17-year age group (24.7%, P = 0.041; 25.8%; P = 0.001) compared to the 10- to 13-year (5.9%; 5.6%), 6- to 9-year (4.6%; 4.6%), and 2- to 5-year (8.3%; 0%) age groups, respectively. The positivity rate for C. trachomatis was highest for rectal (16.2%), followed by urine (5.4%) and oropharyngeal (0.7%) sites. The positivity rate for N. gonorrhoeae was highest for rectal sites (10.4%), followed by oropharyngeal (9.7%) and urine (1.9%) sites. The source with highest diagnostic yield is rectum for C. trachomatis and rectum and oropharynx for N. gonorrhoeae. Hence, extragenital screening is critical for the comprehensive detection of C. trachomatis and N. gonorrhoeae in the pediatric population.

Chlamydia trachomatis and Neisseria gonorrhoeae are the two most common causes of sexually transmitted disease in the United States. Studies in adults, mostly in men who have sex with men, have shown that the prevalence of C. trachomatis and N. gonorrhoeae infections is much higher in extragenital sources compared to urogenital sources. A similar large sample of data on the burden of C. trachomatis and N. gonorrhoeae infections by anatomic site is lacking in children. We retrospectively analyzed data from 655 patients tested for C. trachomatis (887 specimens) and N. gonorrhoeae (890 specimens) at the Children’s Hospital of Philadelphia. We restricted the analysis to include patients between 2 and 17 years of age that had all three sources (urine, oropharynx, and rectum) collected at the same visit. The final data set included specimens from all three sources from 148 and 154 patients for C. trachomatis and N. gonorrhoeae, respectively. Specimens were tested for C. trachomatis and N. gonorrhoeae using a Gen-Probe Aptima Combo 2 assay. The burden of C. trachomatis and N. gonorrhoeae infection was significantly higher in the 14- to 17-year age group (24.7%, P = 0.041; 25.8%; P = 0.001) compared to the 10- to 13-year (5.9%; 5.6%), 6- to 9-year (4.6%; 4.6%), and 2- to 5-year (8.3%; 0%) age groups, respectively. The positivity rate for C. trachomatis was highest for rectal (16.2%), followed by urine (5.4%) and oropharyngeal (0.7%) sites. The positivity rate for N. gonorrhoeae was highest for rectal sites (10.4%), followed by oropharyngeal (9.7%) and urine (1.9%) sites. The source with highest diagnostic yield is rectum for C. trachomatis and rectum and oropharynx for N. gonorrhoeae. Hence, extragenital screening is critical for the comprehensive detection of C. trachomatis and N. gonorrhoeae in the pediatric population.

Rapid identification (ID) and antimicrobial susceptibility testing (AST) techniques promise to improve the management of patients with blood stream infections (BSI).…

Mycobacterium llatzerense was cultured from a subdiaphragmatic abscess. To our knowledge, this is the first report of isolation of this rapidly growing mycobacterium from a human. Growth characteristics and antimicrobial susceptibilities different from those previously reported for environmental isolates were observed. Copyright © 2014, American Society for Microbiology. All Rights Reserved., M.G. and J.L. thank the Spanish MINECO for financial support through projects CGL2011-24318 and Consolider CSD2009-00006. M.G. was supported by a postdoctoral contract from the University of the Balearic Islands, with funds from Spanish Ministry of Education, Culture and Sports through the International Excellence Campus

Mixed-population (heterogeneous) enterococcal bacteremia (MEB) is rarely reported. Based on one occasion in which Vitek2 missed a vancomycin-resistant subpopulation isolated from a patient, we developed a simple method to detect this subpopulation and determined MEB frequency. The four patients presented here had either Enterococcus faecium or Enterococcus faecalis bacteremia caused by both vancomycin-resistant enterococci (VRE) and vancomycin-susceptible enterococci (VSE). No prior common antibiotic therapy was observed, and bacteremia resolved with daptomycin, gentamicin, and/or linezolid treatment. In two cases, VRE presence was missed by Vitek2. To detect the VRE subpopulation, tryptic soy broth was inoculated from positive blood cultures and a saline suspension was inoculated to a vancomycin (6-μg/ml) (V6) plate. Two isolates from each patient were studied further. Relatedness was assessed by multilocus sequence typing, fitness was evaluated by growth curve and competition assays, and vanA presence was determined by PCR. MEB represented ∼5% of all enterococcal bacteremias. All VRE subpopulations grew on V6 plates but were missed in two instances by Vitek2. VRE and VSE isolates from each patient were closely related and did not differ in overall fitness. All four VRE isolates and 2/4 VSE isolates were vanA positive. MEBs occur regardless of prior antimicrobial therapy, are relatively common in our hospital, and are important to detect. As far as we know, this study is the first to report heterogeneous E. faecalis bacteremia. There is a simple method to detect VRE subpopulations that may be missed by Vitek2.

In order to overcome some of the limitations of conventional microbiological techniques in the diagnosis of human brucellosis, a simple PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed. After amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein specific for the Brucella genus (BCSP31), the digoxigenin-labeled amplified product was hybridized with a biotinylated capture probe which was complementary to the inner part of the amplicon. The hybrid was captured on streptavidin-coated microtiter plates and detected by using an antidigoxigenin Fab-peroxidase conjugate. The detection limit of the PCR-ELISA in a background of 3.5 μg of human genomic DNA was 10 fg (two bacterial cells). The PCR-ELISA showed an analytical sensitivity higher than that of ethidium bromide staining and equal to that obtained by conventional PCR followed by dot blot hybridization. In 59 peripheral blood samples from 57 consecutive patients with active brucellosis and 113 control samples, the PCR-ELISA was found to be 94.9% sensitive and 96.5% specific, whereas the sensitivity of the blood culture was only 70.1%. Since the assay can be performed in 1 day, is very reproducible, is easily standardized, and avoids the risk of infection in laboratory workers, this PCR-ELISA seems to be a practical and reliable tool for the diagnosis of human brucellosis.