Citations

ABSTRACT The BACTEC MYCO/F Lytic blood culture bottle (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) is designed to optimize the recovery of fungi and mycobacteria; however, this bottle also supports the growth of most aerobic bacteria. We compared the MYCO/F Lytic bottle with two other BACTEC bottles and the Isolator system for the recovery of bacteria as well as fungi and mycobacteria from blood. A total of 6,108 blood culture sets were inoculated with blood obtained from adult patients. Twenty-five to 28 ml of blood collected by a phlebotomy team for each blood culture set was randomly distributed into each of four blood culture receptacles: the Isolator tube (Wampole Laboratories, Cranbury, N.J.) and three BACTEC bottles: the MYCO/F Lytic bottle, the BACTEC Plus Aerobic/F bottle, and the BACTEC Anaerobic Lytic/10 bottle. The sediment from the Isolator tube was inoculated onto chocolate agar (CA), brain heart infusion agar (BHI), and Sabouraud dextrose agar (SDA) and into a BACTEC 13A bottle. Incubation durations were as follows: MYCO/F Lytic bottle, 42 days; Plus Aerobic/F bottle, 5 days; Anaerobic Lytic/10 bottle, 5 days; sediment from Isolator tube on CA, 3 days; sediment from Isolator tube on BHI, 30 days; sediment from Isolator tube on SDA, 30 days; and sediment from Isolator tube in a BACTEC 13A bottle, 42 days. Two isolates of Histoplasma capsulatum were recovered from the Isolator tube only. Three isolates of Mycobacterium tuberculosis complex were recovered: two isolates from the MYCO/F Lytic bottle only and one isolate from the Isolator tube (whose sediment was inoculated into the BACTEC 13A bottle) only. Two isolates of Cryptococcus neoformans were recovered: one from the MYCO/F Lytic bottle only and the other from the MYCO/F Lytic bottle and the Isolator tube (whose sediment was inoculated into the BACTEC 13A bottle). For potential pathogens overall, there was a statistical difference in recovery that favored the Isolator system over the MYCO/F Lytic bottle ( P = 0.0015), including statistically significant differences for Staphylococcus aureus ( P = 0.0001) and Streptococcus pneumoniae ( P = 0.0313). However, there was no statistically significant difference between the two blood culture systems when detection of bloodstream infection was considered. The time to detection for all potential pathogens combined was less for the MYCO/F Lytic bottle than for the Isolator system ( P = 0.0004). Overall, the potential pathogen recovery was greater for the BACTEC Plus Aerobic/F bottle than for either the Isolator system ( P = 0.0003) or the MYCO/F Lytic bottle ( P = 0.0001). However, the BACTEC Plus Aerobic/F bottle did not recover M. tuberculosis , H. capsulatum , or C. neoformans isolates. The combination of the Isolator system and MYCO/F Lytic bottle may be useful as a selective blood culture method to optimize the recovery of fungi and mycobacteria from blood. Compared with the manual Isolator system, the MYCO/F Lytic system has the advantage of less preanalytic processing and continuous automated monitoring of bottles for growth by the BACTEC 9240 instrument.

ABSTRACT In a previous study which evaluated the BACTEC 9240 automated blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), we noted a 1.3% “instrument false-positive” rate. That is, the BACTEC system signaled that a bottle (BACTEC Plus Aerobic/F bottle or BACTEC Anaerobic Lytic/10 bottle) culture was positive but a Gram stain was negative and there was no growth of bacteria or yeasts on subculture to chocolate agar. Furthermore, from the same sample of blood, cultures for fungi using the Isolator blood culture system (Wampole Laboratories, Cranbury, N.J.) were negative for growth. For the present study, we evaluated 76 instrument false-positive samples for the presence of 16S ribosomal DNA using the MicroSeq 500 kit (PE Biosystems, Foster City, Calif.). These samples also were negative for fungi by the Isolator method. This kit has a PCR module and sequencing module for the amplification and sequencing of the 16S RNA gene and provides a database for sequence alignment and identification of bacteria. To optimize the assay, we evaluated the effect of adding 0.5% bovine serum albumin to the sample from blood culture bottles and found that it decreased the effects of inhibitors on the PCR. Two control groups of blood culture specimens were also evaluated. One group ( n = 45) were “instrument true positives”; the instrument signaled positive, and subsequent Gram stains were positive and subcultures on chocolate agar grew bacteria. The other group ( n = 20) were “instrument true negatives”; the instrument signaled negative, the Gram stain was negative, and subcultures on chocolate agar and from the Isolator tube on fungal media showed no growth. None of the 76 instrument false-positive samples had evidence for 16S rRNA gene sequences. All of the instrument true-positive samples and all of the instrument true-negative specimens were positive and negative, respectively, using the MicroSeq 500 kit. Total peripheral white blood cell counts were statistically significantly higher for patients who had instrument false-positive results than for patients who had instrument true-positive or true-negative results ( P = 0.001). We conclude that instrument false positives signaled by the BACTEC 9240 system are not due to bacteria in the blood culture samples.

ABSTRACT Recent reports associate Staphylococcus lugdunensis with severe infection in humans. The frequency of this microorganism in urine cultures is unknown. Five hundred isolates of coagulase-negative staphylococci (CoNS) were recovered from 4,652 consecutive urine specimens submitted for culture to the Mayo Clinic Microbiology Laboratory. Thirty-one (6%) of 500 isolates of CoNS were identified as S. lugdunensis . In no case was S. lugdunensis isolated in pure culture; 29 (94%) of 31 S. lugdunensis isolates were part of mixed nonpathogenic flora. Medical records were reviewed for 30 of the 31 patients from whom these 31 isolates were isolated. Twenty-one (70%) of the 30 evaluable patients were not treated with antibiotics; the remaining 9 (30%) of 30 patients were treated with antibiotics that may be effective against S. lugdunensis. S. lugdunensis may be an unrecognized yet infrequent cause of urinary tract infection.

ABSTRACT A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS 481 and IS 1001 of B. pertussis and B. parapertussis , respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9 B. pertussis and 3 B. parapertussis ) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS 481 ) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens ( n = 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different ( P = 0.0654). The insertion sequence IS 481 is also present in Bordetella holmesii ; specimens containing B . holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of B . pertussis and B . parapertussis in nasopharyngeal specimens.