Minimizing the workup of blood culture contaminants: implementation and evaluation of a laboratory-based algorithm.

An algorithm was implemented in the clinical microbiology laboratory to assess the clinical significance of organisms that are often considered contaminants (coagulase-negative staphylococci, aerobic and anaerobic diphtheroids, Micrococcus spp., Bacillus spp., and viridans group streptococci) when isolated from blood cultures. From 25 August 1999 through 30 April 2000, 12,374 blood cultures were submitted to the University of Iowa Clinical Microbiology Laboratory. Potential contaminants were recovered from 495 of 1,040 positive blood cultures. If one or more additional blood cultures were obtained within +/-48 h and all were negative, the isolate was considered a contaminant. Antimicrobial susceptibility testing (AST) of these probable contaminants was not performed unless requested. If no additional blood cultures were submitted or there were additional positive blood cultures (within +/-48 h), a pathology resident gathered patient clinical information and made a judgment regarding the isolate's significance. To evaluate the accuracy of these algorithm-based assignments, a nurse epidemiologist in approximately 60% of the cases performed a retrospective chart review. Agreement between the findings of the retrospective chart review and the automatic classification of the isolates with additional negative blood cultures as probable contaminants occurred among 85.8% of 225 isolates. In response to physician requests, AST had been performed on 15 of the 32 isolates with additional negative cultures considered significant by retrospective chart review. Agreement of pathology resident assignment with the retrospective chart review occurred among 74.6% of 71 isolates. The laboratory-based algorithm provided an acceptably accurate means for assessing the clinical significance of potential contaminants recovered from blood cultures.

Comparison of porphyrin-based, growth factor-based, and biochemical-based testing methods for identification of Haemophilus influenzae.

The accurate identification of Haemophilus spp. is essential for optimizing the role of the clinical microbiology laboratory in the diagnosis and management of Haemophilus infections. One laboratory-prepared medium and eight commercially available test systems were examined in parallel as a means of identifying 378 clinical isolates of Haemophilus spp. as either Haemophilus influenzae or non- Haemophilus influenzae spp. At least one discordant result was noted with 187 (49.5%) of the isolates tested. Discordant results were resolved either by majority rule for isolates with less than three discordant test results or by confirming the identity using conventional biochemical tests for isolates with three or more discordant test results ( n=20). Among these 20 isolates, 2 were judged not to belong to the Haemophilus genus. Comparisons of three porphyrin-based methods, three growth factor-based methods (1 of which also incorporates a porphyrin testing component), and three biochemical-based methods revealed varying discrepancy rates within each testing method. In general, porphyrin-based methods, with overall discrepancy rates of 1.3% or less, outperformed other testing methods. One important exception was the performance of the porphyrin testing component of the Haemophilus Identification Test Kit (Remel, USA), which produced an overall discrepancy rate of 28.5% and a false-negative rate of 52.2% with non- Haemophilus influenzae isolates. Growth factor-based methods yielded overall discrepancy rates ranging from 1.6% ( Haemophilus Identification Agar Quad; Remel) to 10.4% (hemin and nicotinamide adenine dinucleotide disk component of the Haemophilus Identification Test Kit). Biochemical-based assays produced overall discrepancy rates ranging from 4.5% (API NH; bioMérieux Vitek, USA) to 10.1% ( Neisseria Haemophilus Identification Card; bioMérieux Vitek). Collectively, these results suggest that porphyrin-based testing methods represent the most reliable means for identifying Haemophilus spp.

Trends in post-operative infections by Gram-positive bacteria.

Surgical patients are now more prone than ever to have a post-operative infection. On average, they are now older and more have chronic disease histories with reduced immunocompetence or iatrogenic immunosuppression, and many undergo more aggressive, more complex surgical procedures. Moreover, the infectious agents have changed. A comparison of data collected by the SENTRY Antimicrobial Surveillance Program for the years 1988 and 1998 from North and Latin America and Europe shows important shifts in the nature of the infectious agents. Among the Gram-positive agents, Staphylococcus aureus was the most frequent isolate in both years, but its share has more than doubled. Beta-hemolytic streptococci increased their share from 3 to 5% while enterococci fell from 13 to 8%. Perhaps more important than the shifts in incidence are dramatic changes in the antimicrobial resistance patterns of these agents. Data from the past several years show increasing resistance for the drugs that were previously considered 'first line' treatment for post-surgical infections. The majority of S. aureus and coagulase-negative staphylococci are now resistant to most classes of antibiotics. Antimicrobial resistance is beginning to be detected in beta-hemolytic streptococci, and vancomycin-resistant enterococci, which were not even reported in 1987-1988, now represent 17% of all enterococci isolated in the USA and Canada. To stay ahead in the fight against surgical infections, we must react in a combination of ways, using disinfection, prophylaxis, new antibiotics and, above all, we must practice superb hospital infection control and world-wide antimicrobial epidemiology studies.

Colony morphology of Candida spp. as a guide to species identification.

The presence of colony projections, often referred to as "feet," have typically been considered a characteristic of Candida albicans. In the current study that examined the colony morphology of numerous different species of Candida, several clinical isolates of Candida tropicalis and Candida krusei were also noted to produce "feet." The medium and growth conditions under which colony projections were produced by these species were characterized.

New Pathogens and Rapid Diagnostic Methods -Clinical Microbiology in the 21st Century-

This presentation is actually a predictive opinion piece from the author's experience. No one really knows what new pathogens are awaiting us around the year of 2000 corner but, in 1985, the author was asked by the NCCLS (USA) to predict the emerging pathogens for the 1990's in the U.S. and hit on 80% of them. Thus, using the experience of this decade, we can predict what will happen in the U.S. in the first decade of the new century with 80-90% accuracy of those we predict. In the next millennium we will see a marked increase in the infection rate of the atypical (non-TB) mycobacteria, the environmental fungi, and even greater problems with the "water organisms". All of this will be described in detail with the preliminary data that indicates these trends for the years "2000 and beyond". In the area of what will be new in rapid testing, whether manual or automated, the future is even more cloudy. Manufacturers will not discuss with consumers what their long-range plans are for new products. Even long-time associations with Becton-Dickinson, Gen-Probe, Vitek, etc., has yielded little if any indication of their company's plans for the years "2000 and beyond". One can, however, conceive and get into their minds to some degree based on their previous track records. We think these companies are working very hard on current tests or instruments to upgrade and improve their products yielding tests with greater sensitivity and specificity, while decreasing the technicians "hands-on time". We anticipate the increased utilization of chromogenic detectors rather than nucleotides due to the inherent problem of disposal of radioactive compounds regardless of how few actual "micro-curies" involved. The use of ligand assay technology to supplement or replace currently employed PCR technology is totally predictable and will occur. One must remember that industry faces the same problems as we do with the "new pathogens" of the future. They will need to develop detection and identification systems for organisms that at this time don't even have names because we haven't encountered them yet. Additionally there are all those organisms out there that environmental microbiologists may be familiar with, but not clinical microbiologists. Industry will respond to need, they always have, they always will, but it is hard to respond to the unknown organism at this time. That problem relates to the philosophical nature and brevity of this abstract, that is "the unknown". If we knew for sure what the future was, it would be the past.

Accuracy of the Vitek system for antimicrobial susceptibility testing Enterobacteriaceae bloodstream infection isolates: use of "direct" inoculation from Bactec 9240 blood culture bottles.

A recent investigation indicates that rapid antimicrobial susceptibility tests (AST) can affect patient therapy leading to reductions in health-care costs for some patient populations. However, there is little information relative to the often performed direct inoculation of positive blood culture bottles into rapid AST systems. AST results of direct inoculated Vitek (bioMerieux Vitek, Hazelwood, MO, USA) GNS cards were compared to those inoculated per package insert recommendations and a reference broth microdilution test using 50 consecutive Enterobacteriaceae bloodstream infection isolates. Escherichia coli (44% of isolates), Klebsiella ssp. (30%), and six other members of this family were tested against 15 antimicrobial agents. The direct inoculation method produced only two false-susceptible (0.3%), seven false-resistant (0.9%; six different drugs), and 48 minor errors (6.4%). The GNS cards inoculated in the usual, recommended manner had no very major error, and 7.5% combined major and minor errors. If the results of the urinary infection-specific drugs (nitrofurantoin, trimethoprim/sulfamethoxazole; not appropriate for bacteremia therapy) and ampicillin/sulbactam were deleted, both Vitek inoculation methods yielded results well within acceptable limits (< or = 4.5% overall error). These results indicate that the direct inoculation method of Vitek GNS cards from Enterobacteriaceae bloodstream infections (detected by Bactec 9240, Becton-Dickinson, Cockeysville, MD, USA) performed as well as the NCCLS broth microdilution test. Thus, a procedural modification of this type could further accelerate rapid access to accurate AST data.

Polymerase chain reaction typing of nonviable Mycobacterium tuberculosis isolates.

Repetitive element polymerase chain reaction (PCR) typing was applied to two Mycobacterium tuberculosis isolates for which both viable and nonviable cultures were available. DNA extracted from the nonviable cultures and from fresh subcultures of the viable cultures was amplified with primers directed against the insertion sequence IS6110 and the polymorphic GC-rich repetitive sequence. For both isolates, the nonviable cultures generated PCR-banding patterns identical to those generated by the fresh subcultures.

The approach to stool examination for parasites.

This article presents the sequences followed in order to maximize the yield of laboratory assistive diagnosis of parasitic infections in the physiologic passage and/or collection of specimens for those parasites producing pathologic disease in the gastrointestinal tract and liver. The advantages and disadvantages of the many methods available to the clinician and laboratorian in the examination of those specimens are presented in a work-flow mode. The controversy relative to the current practice of controlling unwarranted specimen submission and the reasons for their rejection of limited examination and interpretation are discussed. Quality assurance and safety practices are also addressed.

Vitek GPS card susceptibility testing accuracy using direct inoculation from BACTEC 9240 blood culture bottles.

The emergent need for antimicrobial susceptibility testing (AST) data for the therapy of bacteremic patients has led to the development of rapid methods and local procedure modification of some commercial AST products such as the direct inoculation from blood culture systems. We compared the Vitek GPS card results using direct and standardized inoculation with a reference broth microdilution method for 112 consecutive staphylococcal bloodstream infections (seven drugs). Among the 28 Staphylococcus aureus strains, 0%-3.6% total error/drug was observed with both Vitek inoculation procedures. However, the only oxacillin-resistant strain was not detected (100% true very-major error). For 84 coagulase-negative staphylococci (CNS), the direct inoculation procedure had an 11.9% very-major error rate for oxacillin, ampicillin-sulbactam, and cephalothin, plus 4.8% very-major error rate for ciprofloxacin and trimethoprim-sulfamethoxazole (total error rate 4.8%-16.7% for five of seven drugs compared). The Vitek direct inoculation procedure routinely missed 20.4% of oxacillin-resistant CNS strains. The use of Vitek direct inoculation procedures for staphylococcal bloodstream infection isolates (from BACTEC 9240 cultures) produced serious false-susceptible results; this procedure should be avoided in favor of routine package insert-recommended Vitek procedures or other reference-quality overnight incubation susceptibility tests.

Bacterial contamination rates following processing of bone marrow and peripheral blood progenitor cell preparations.

BACKGROUND: The performance of cultures to assess possible bacterial contamination of bone marrow and peripheral blood progenitor cell preparations is required by the standards of the American Association of Blood Banks. STUDY DESIGN AND METHODS: Consecutive (n = 893) bone marrow and peripheral blood progenitor cell preparations were cultured for assessment of possible contamination by microorganisms. RESULTS: Consecutive bone marrow and peripheral blood progenitor cell preparations (n = 893) were cultured; the overall positive rate detected was 2.5 percent (22/893). The isolates predominantly were skin contaminants (gram-positive cocci) and so-called water-borne organisms (gram-negative rods). The 6.0-percent rate of positivity in 317 bone marrow preparations was higher than the 0.5-percent rate in 576 peripheral blood progenitor cell preparations (p < 10(-6)). Culture-positive preparations were transfused to 16 patients at this institution; however, none of these transfusions led to documented sepsis with the contaminating organism. CONCLUSION: The culture method described here complies with the standards of the American Association of Blood Banks. Contamination can be detected in both bone marrow and peripheral blood progenitor cell preparations. When contaminated preparations are transfused, there are few complications that can be attributed to the contamination.

Evaluation of BACTEC 9240 blood culture system by using high-volume aerobic resin media.

The BACTEC 9240 blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) is one of three automated, continuous-monitoring systems that is widely used in clinical laboratories. The BACTEC 9240 was compared with the BACTEC NR 660 for the detection of organisms and bacteremic episodes; time to detection of positive cultures; number of false-positive and false-negative cultures; and time needed to load, process, and perform quality control functions by using high-volume aerobic media. Blood specimens (5,282) were inoculated in equal volumes (5 to 10 ml per bottle) into BACTEC Plus Aerobic/F (9240 system) and BACTEC Plus NR26 (660 system) bottles. Clinically significant isolates were detected in 6.6% of cultures, representing 348 microorganisms and 216 bacteremic episodes. Two hundred forty-eight microorganisms were detected by both systems, 48 by the 9240 only and 52 by the 660 only (P = not significant). Of the bacteremic episodes, 158 were detected by both systems, 27 by the 9240 only and 31 by the 660 only (P = not significant). Analysis of data by month revealed equivalent recovery rates for both systems, with the exception of a 30-day period at one study site during which the 660 system detected significantly more microorganisms. Following a proprietary hardware design retrofit of the 9240 instrument, detection rates were again equivalent for the remaining three months at this study site. Positive cultures detected by both systems were detected an average of 4.3 h faster by the 9240 system (21 versus 25.3 h). The numbers of false-positive cultures for the 9240 and 660 systems were 40 (1.0%) and 9 ( < 1.0%), respectively. The numbers of false-negative cultures were five for the 9240 system and three for the 660 system. The 9240 system required 23 s less technologist time per bottle to operate during the 5-day protocol. In conclusion, the BACTEC 9240 used with high-volume Aerobic/F medium is equivalent to the BACTEC 660 used with high volume NR26 medium for the detection of microorganisms and bacteremic episodes. In addition, the 9240 system detects positive cultures more rapidly than the 660 system but requires further evaluation to ensure reliability of instrument components.

Etest for routine clinical antimicrobial susceptibility testing of rapid-growing mycobacteria isolates.

The Etest has become a widely accepted alternative susceptibility-testing method for difficult-to-assess organisms, including rapid-growing Mycobacterium spp. Following an internal validation and literature reviews, the Etest was applied as the routine method for testing Mycobacterium chelonae and Mycobacterium fortuitum isolates. Results from testing 31 strains confirmed the utility of the Etest and the simplicity of the procedure. Mycobacterium chelonae were generally more resistant to all drugs except amikacin (MIC90, 32 micrograms/ml), compared with M. fortuitum strains that were inhibited (MIC50 in the susceptible range) by amikacin (1 microgram/ml), ciprofloxacin (0.032 microgram/ml), doxycycline (0.125 microgram/ml), and trimethoprim-sulfamethoxazole (0.032 microgram/ml). The polymyxin-B disk used as an identification method was confirmed (> or = 10 mm = M. fortuitum). The Etest provides a simple and accurate method for selecting appropriate therapy for infections caused by rapid-growing mycobacteria (a typical case report is presented).

Disseminated Mycobacterium genavense infection in two patients with AIDS.

Mycobacterium genavense is a recently defined fastidious organism that has been identified as a cause of disseminated infection in patients with AIDS. We report the cases of two patients who had advanced AIDS and a clinical syndrome of fever, anorexia, abdominal pain, diarrhea, and weight loss. In addition, splenomegaly and lymphadenopathy were prominent in both cases, and in one patient's case radiographic findings were suggestive of splenic abscesses. Mycobacteria isolated from specimens of blood and bone marrow grew in liquid media but not on solid media. The results of DNA probe tests for Mycobacterium tuberculosis and Mycobacterium avium complex were false-positive for both patients. After treatment of the broth cultures to lyse red blood cells, the results of DNA probe tests were negative for these pathogens. Amplification and sequencing of 16S rRNA with use of the polymerase chain reaction indicated that the mycobacterial isolates from both patients had sequences identical to those previously reported for M. genavense. One patient survived 5 months after diagnosis, the other 2 months after diagnosis; only one patient responded (transiently) to antimycobacterial chemotherapy.

Comparison of identification systems for Staphylococcus epidermidis and other coagulase-negative Staphylococcus species.

Three commercially available systems (API Staph-Trac, API 20GP, and Vitek GPI), used to identify coagulase-negative staphylococci, were evaluated against 277 bloodstream isolates, including 94 isolates of Staphylococcus epidermidis and 183 isolates of other coagulase-negative Staphylococcus species. The conventional method of Kloos and Schleifer served as the reference method. Controls included 14 ATCC type culture strains of coagulase-negative staphylococci. The API Staph-Trac system showed the highest rate of agreement with reference method, correctly identifying 73% of the isolates. The Vitek GPI System had an overall rate of agreement of 67% and the API 20GP system correctly identified 61%. The API Staph-Trac system correctly identified 94% of the isolates of S. epidermidis compared with 64% by both Vitek GPI and API 20GP. The most common error for both Vitek GPI and API 20GP systems was the failure to identify organisms contained within the database of the systems. Because none of the tested commercial identification systems identified "non-epidermidis" coagulase-negative Staphylococcus species with a high degree of accuracy, the systems need to be markedly improved or new systems developed.

Controlled comparison of the BACTEC high-blood-volume fungal medium, BACTEC Plus 26 aerobic blood culture bottle, and 10-milliliter isolator blood culture system for detection of fungemia and bacteremia.

The BACTEC high-blood-volume fungal medium (HBV-FM) (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) was compared with the Isolator (IS) tube and the BACTEC Plus 26 (BP26) blood culture bottle for the ability to recover fungi from the blood of adult patients suspected of having fungemia. A total of 6,836 blood culture sets that fulfilled criteria for inclusion in the study were received. Three separate comparisons were performed: 4,907 HBV-FM versus IS, 4,886 BP26 versus HBV-FM, and 4,949 BP26 versus IS. For the HBV-FM versus IS comparison, 218 isolates were recovered: 125 (57.3%) were bacteria and 93 (42.7%) were fungi. HBV-FM was comparable to IS for recovery of yeasts, but IS was superior for recovery of Histoplasma capsulatum (25 versus 0 isolates recovered [P < 0.001]). Growth of Torulopsis glabrata was detected earlier (P < 0.05) in HBV-FM bottles. For the BP26 versus HBV-FM comparison, 229 isolates were recovered: 161 (70.3%) were bacteria, and 68 (29.7%) were fungi. HBV-FM was superior for recovery of T. glabrata (P < 0.025) and all fungi combined (P < 0.025). There were no statistically significant differences in the speed of detection of microbial growth. For the BP26 versus IS comparison, 251 isolates were recovered: 165 (65.7%) were bacteria, and 86 (34.2%) were fungi. IS was superior for recovery of H. capsulatum (P < 0.001), T. glabrata (P < 0.05), and fungi other than H. capsulatum (P < 0.025). BP26 was superior for recovery of all bacteria combined (P < 0.001) and viridans group streptococci (P < 0.01). Growth of T. glabrata (P < 0.05) was detected earlier in IS tubes. Growth of Staphylococcus aureus (P < 0.01), viridans group streptococci (P < 0.01), Pseudomonas aeruginosa (P < 0.05), and all microorganisms combined (P < 0.05) was detected earlier in BP26 bottles. For yeast, 57 of 59 (96.6%), 79 of 80 (98.7%), and 64 of 67(95.5%) were recovered from BP26 bottles, HBV-FM bottles, and IS tubes, respectively, by day 14; for H. capsulatum, 14 of 36 (38%) isolates were recovered from IS tubes by day 14. Mean times of recovery were similar for BACTEC bottles and IS. We conclude that (i) for recovery of fungi from blood cultures, HBV-FM is equivalent to IS (with the exception of H. capsulatum); (ii) for recovery of bacteria, BP26 is superior to IS; (iii) BP26 bottles are inferior to both HBV-FM bottles and IS tubes for recovery of T. glabrata; and (iv) HBV-FM bottles must be paired with another blood culture bottle or system to optimize detection of bacteremia.

Cefmetazole and trospectomycin in vitro susceptibility testing interpretive criteria and quality control guidelines for Neisseria gonorrheae.

Cefmetazole and trospectomycin were tested in a multilaboratory trial to establish Neisseria gonorrhoeae susceptibility testing criteria and quality control (QC) guidelines. Cefmetazole was active against the penicillinase-producing isolates and has an MIC90 of 16 micrograms/ml, the breakpoint MIC previously used for nonfastidious species. However, a single-dose gonorrhea regimen (1 g i.m.) would require a lower less than or equal to 2 micrograms/ml breakpoint with a correlate zone (greater than or equal to 33 mm) consistent with similarly used cephamycins (cefoxitin and cefotetan). An intermediate category was proposed for MICs greater than 2-4 micrograms/m (28-32 mm) pending more clinical experience with higher and/or prolonged cefmetazole dosing regimens. Trospectomycin was active (MIC90, 8 micrograms/ml) against all spectinomycin-susceptible gonococci. A susceptible breakpoint MIC of less than or equal to 16 micrograms trospectomycin per milliliter was proposed with a correlate zone diameter of greater than or equal to 17 mm. An intermediate category was also suggested for trospectomycin at 32 micrograms/ml (14-16 mm). QC guidelines were established for 30-micrograms cefmetazole and 30-micrograms trospectomycin disk diffusion tests and the GC agar base MICs using a multilaboratory study design consistent with National Committee for Clinical Laboratory Standards (NCCLS) M23-T guidelines. Both drugs were stable in GC agar plates for 21 days stored at 2 degrees-5 degrees C.

Cefdinir (FK482), an orally administered cephalosporin in vitro activity comparison against recent clinical isolates from five medical centers and determination of MIC quality control guidelines.

Cefdinir, a new oral cephalosporin, was compared to cefaclor, cefadroxil, cefixime, and cefuroxime against greater than 5000 recent aerobic clinical isolates. This multicenter study revealed broad-spectrum cefdinir activity against all Enterobacteriaceae (MIC50s, 0.06-2 micrograms/ml) except Enterobacter cloacae, Morganella morganii, Proteus vulgaris, and Serratia marcescens (MIC50s, greater than or equal to 4 micrograms/ml). Oxacillin-susceptible staphylococci (MIC90s, 0.5-2 micrograms/ml), beta-hemolytic Streptococcus group B (MIC90, 0.06 micrograms/ml), and Acinetobacter lwoffii were also susceptible to cefdinir. The activity of cefdinir was similar to that of cefixime and cefuroxime against Gram-negative organisms and superior to all tested oral cephems when tested against Gram-positive cocci. None of the cephalosporins were active against oxacillin-resistant Staphylococcus spp., enterococci, Pseudomonas spp., or Xanthomonas maltophilia. MIC quality control range guidelines were established for the strains recommended by the National Committee for Clinical Laboratory Standards documents.

MIC and disk diffusion quality control guidelines for Neisseria gonorrhoeae susceptibility tests of cefdinir, cefetamet, CI-960, fleroxacin, lomefloxacin, and temafloxacin.

Cefdinir (FK482), cefetamet (Ro 15-8074), CI-960, fleroxacin, lomefloxacin, and temafloxacin have potent activities against Neisseria gonorrhoeae. They were tested in a multilaboratory study to establish quality control guidelines. Quality control ranges for N. gonorrhoeae ATCC 49226 were determined by using multiple GC agar lots, three disk lots, and a number of test replicates consistent with the M23-T guidelines of the National Committee for Clinical Laboratory Standards. The MIC ranges included 2 to 4 log2 dilution steps. The recommended inhibition zone diameter ranges were generally 7 to 8 mm and included greater than or equal to 91.3% of all recorded study results.

Disk diffusion quality control guidelines for Haemophilus susceptibility tests using cefdinir, CI-960, fleroxacin, temafloxacin, and trospectomycin.

A multilaboratory study to determine disk diffusion quality control ranges for Haemophilus influenzae ATCC 49247 and five investigational drugs was performed. Multiple lots of Haemophilus Test Medium and antibiotic disks were used for replicate testing in conformance with the recommendations of the National Committee for Clinical Laboratory Standards. Quality control disk zone diameter ranges were proposed for cefdinir, CI-960, fleroxacin, temafloxacin, and trospectomycin.