Practical Guidance for Clinical Microbiology Laboratories: A Comprehensive Update on the Problem of Blood Culture Contamination and a Discussion of Methods for Addressing the Problem

In this review, we present a comprehensive discussion of matters related to the problem of blood culture contamination. Issues addressed include the scope and magnitude of the problem, the bacteria most often recognized as contaminants, the impact of blood culture contamination on clinical microbiology laboratory function, the economic and clinical ramifications of contamination, and, perhaps most importantly, a systematic discussion of solutions to the problem. We conclude by providing a series of unanswered questions that pertain to this important issue.

Changes in pneumococcal serotypes and antimicrobial resistance after introduction of the 13-valent conjugate vaccine in the United States.

Ongoing surveillance for Streptococcus pneumoniae is needed to assess the impact of the pneumococcal conjugate vaccine introduced in 2010 (PCV13). Forty-two U.S. centers submitted S. pneumoniae isolates between 1 October 2012 and 31 March 2013. Susceptibility testing was performed by use of a broth dilution method as recommended by the Clinical and Laboratory Standards Institute. Serotyping was performed by multiplex PCR and the Quellung reaction. Multidrug resistance (MDR) was defined as nonsusceptibility to penicillin (PNSP; MIC ≥ 0.12 µg/ml) combined with resistance to ≥2 non-ß-lactam antimicrobials. Penicillin-resistant S. pneumoniae (PRSP) was defined as a penicillin MIC of ≥2 µg/ml. For the 1,498 isolates collected during 2012-13, the PRSP and MDR rates were 14.2 and 21.0%, respectively. These percentages were lower than rates obtained in a surveillance study conducted 4 years earlier in 2008-09 (17.0 and 26.6%, respectively). The most common serotypes identified in 2012-13 were 3, 35B, and 19A, each representing 9 to 10% of all isolates. The largest percentage of PNSP in 2012-13 were found in serotypes 35B (24.8%), 19A (23.5%), and 15A (10.3%). Predominant PRSP serotypes were 19A (54.5%), 35B (28.2%), and 19F (7.0%). Major MDR serotypes were 19A (38.5%), 15A (16.9%), 6C (8.3%), and 35B (6.4%). The change in prevalence of PCV13 serotypes (43.4 to 27.1%) was primarily due to a decrease in serotype 19A strains, i.e., 22% of all strains in 2008-09 to 10% of all strains in 2012-13. Among the PNSP subset, serotypes showing a proportional increase were 35B, 15B, and 23B. Among MDR strains, the largest proportional increases were observed in serotypes 35B, 15B, and 23A.

Continued emergence of USA300 methicillin-resistant Staphylococcus aureus in the United States: results from a nationwide surveillance study.

BACKGROUND: The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is changing, with USA300 emerging first in community and then in healthcare settings. We performed nationwide surveillance to assess recent trends in the molecular epidemiology of MRSA. METHODS: One hundred consecutive unique clinically significant S. aureus isolates were recovered from patients at each of 43 US centers between July 1, 2011, and December 31, 2011. Susceptibility testing, pulsed-field gel electrophoresis (PFGE), staphylococcal protein A gene (spa) and staphylococcal cassette chromosome mec typing, and Panton-Valentine leukocidin detection were performed on all MRSA isolates. RESULTS: Of 4,131 isolates collected, 2,093 (51%) were MRSA. Specimen sources of MRSA isolates included wound or abscess (54%), blood (24%), lower respiratory tract (11%), and other sterile site (10%). Thirty percent were isolated more than 48 hours after hospital admission (ie, were associated with nosocomial acquisition of infection). USA300 was the most common PFGE type (1,269 isolates; 61%), overall and in all regions, followed by USA100 (368 isolates; 18%). Among 173 spa types found, the most common were t008 (51%) and t002 (18%); no other spa type accounted for more than 2% of isolates. One strain type (USA300/t008/IV) constituted almost half of all MRSA isolates (1,005 isolates; 48%) and was the most common at all body sites, causing 37% of MRSA bloodstream infections (BSIs) and 38% of nosocomial MRSA infections. Multidrug-resistant phenotypes were found among 34 USA300 isolates (3%) from 18 states. CONCLUSIONS: The USA300 PFGE type continues to advance nationwide. A single strain type (USA300/t008/IV) predominates in all regions and infection sites and is now more common than USA100 as a cause of MRSA BSI and nosocomial infections. Although most USA300 retain typical susceptibility profiles, multidrug-resistant phenotypes are emerging.

Activities of vancomycin, ceftaroline, and mupirocin against Staphylococcus aureus isolates collected in a 2011 national surveillance study in the United States.

Forty-two medical centers from throughout the United States participating in a longitudinal surveillance program were asked to submit 100 consecutive Staphylococcus aureus isolates during July to December 2011. Susceptibility testing using CLSI broth microdilution and mecA detection by PCR analysis was performed on the 4,131 isolates collected. Methods employing Etest glycopeptide resistance detection (GRD; bioMérieux) and brain heart infusion agar containing 4 µg/ml vancomycin (BHIV) were used to screen methicillin-resistant S. aureus (MRSA) isolates for heterogeneous intermediate-level resistance to vancomycin (hVISA). Isolates with positive hVISA screen results were confirmed by population analysis profiling-area under the curve (PAP-AUC) determinations. The genetic relatedness of hVISA, ceftaroline-nonsusceptible, or high-level (HL) mupirocin resistance MRSA isolates was assessed by pulsed-field gel electrophoresis (PFGE). Among 2,093 MRSA isolates, the hVISA screen results were positive with 47 isolates by Etest GRD and 30 isolates by BHIV agar screen. Twenty-five of the GRD- or BHIV screen-positive isolates were confirmed as hVISA by PAP-AUC testing. Results of the current study were compared to results obtained from prior surveillance performed in 2009. The prevalence of hVISA among MRSA isolates was higher in 2011 than in 2009 (1.2% versus 0.4%, P = 0.003), especially for isolates with a vancomycin MIC of 2 (45.4% versus 14.3%, P = 0.01). The overall rate of ceftaroline susceptibility in the current study was 99.4% (one hVISA isolate had an intermediate ceftaroline MIC). HL mupirocin resistance increased from 2.2% in 2009 to 3.2% in 2011 (P = 0.006). Although overall rates of hVISA and HL mupirocin resistance are low, they have increased since 2009.

Evaluation of pneumococcal serotyping by multiplex PCR and quellung reactions.

Screening of 1,750 pneumococcal isolates for common serotypes by PCR was followed by Quellung reaction analysis of PCR-negative isolates with a comparison to the conventional (Quellung reaction only) approach. PCR agreed with Quellung reaction results for 99% of isolates. The sequential PCR/Quellung reaction algorithm is accurate and more cost-effective than the conventional approach.

Antimicrobial stewardship programs.

The development of antimicrobial agents represents one of the most significant achievements in medicine during the past century. However, the emergence of antimicrobial resistance combined with the downturn in the development of new antimicrobial agents in the pharmaceutical industry poses unanticipated challenges in the effective management of infection. The issue of how we can most effectively utilize these invaluable resources, antimicrobials, in the face of infections that are ever more difficult to treat arises. This issue serves as the fundamental basis for the concept of antimicrobial stewardship, the topic of this minireview.

Pneumococcal serotypes before and after introduction of conjugate vaccines, United States, 1999-2011(1.).

Serotyping data for pneumococci causing invasive and noninvasive disease in 2008-2009 and 2010-2011 from >43 US centers were compared with data from preconjugate vaccine (1999-2000) and postconjugate vaccine (2004-2005) periods. Prevalence of 7-valent pneumococcal conjugate vaccine serotypes decreased from 64% of invasive and 50% of noninvasive isolates in 1999-2000 to 3.8% and 4.2%, respectively, in 2010-2011. Increases in serotype 19A stopped after introduction of 13-valent pneumococcal vaccine (PCV13) in 2010. Prevalences of other predominant serotypes included in or related to PCV13 (3, 6C, 7F) also remained similar for 2008-2009 and 2010-2011. The only major serotype that increased from 2008-2009 to 2010-2011 was nonvaccine serotype 35B. These data show that introduction of the 7-valent vaccine has dramatically decreased prevalence of its serotypes and that addition of serotypes in PCV13 could provide coverage of 39% of isolates that continue to cause disease.

Safety and immunological outcomes following human inoculation with nontypeable Haemophilus influenzae.

BACKGROUND: Nontypeable Haemophilus influenzae (NTHi) exclusively infects humans, causing significant numbers of upper respiratory tract infections. The goal of this study was to develop a safe experimental human model of NTHi nasopharyngeal colonization. METHODS: A novel streptomycin-resistant strain of NTHi was developed, and 15 subjects were inoculated in an adaptive-design phase I trial to rapidly identify colonizing doses of NTHi. Bayesian analysis was used to estimate the human colonizing dose 50 and 90 (HCD50 and HCD90, respectively). Side effects and immunological responses to whole-cell sialylated NTHi were measured. RESULTS: Nine subjects were colonized and tolerated colonization well. Immunological analyses demonstrated that 7 colonized subjects and 0 noncolonized subjects had a 4-fold rise in serum levels of immunoglobulin A, immunoglobulin M, or immunoglobulin G. Preexisting immunity to whole-cell NTHi did not predict success or failure of colonization. CONCLUSIONS: The statistical design incorporated a slow escalation to higher dose levels. HCD50 and HCD90 Bayesian estimates were identified as approximately 2000 and 150 000 colony-forming units, respectively; credible interval estimates were broad. This study provides a potential platform for early proof of concept studies for NTHi vaccines, as well as a way to evaluate bacterial factors associated with colonization.

In vitro activity of ceftaroline against clinical isolates of Streptococcus pneumoniae recovered in 43 U.S. medical centers during 2010-2011.

The in vitro activity of ceftaroline, a recently introduced parenteral cephalosporin, was assessed versus 1,750 isolates of Streptococcus pneumoniae recovered from patients with a variety of pneumococcal infections in 43 U.S. medical centers during 2010-2011. Using a breakpoint of ≤ 0.5 µg/ml for susceptibility, all of the isolates were found to be susceptible to ceftaroline. Ceftaroline MICs were consistently 16-fold lower than ceftriaxone MICs. Among the isolates characterized in this investigation, 38.9% were found to be nonsusceptible to penicillin (oral penicillin breakpoints) and 9.1% were nonsusceptible to ceftriaxone (nonmeningitis breakpoints).

Detection of Staphylococcus aureus isolates with heterogeneous intermediate-level resistance to vancomycin in the United States.

The prevalence of heterogeneous intermediate-level resistance to vancomycin (hVISA) in Staphylococcus aureus was assessed by screening a large collection of recent isolates. Susceptibility testing by the Clinical and Laboratory Standards Institute broth microdilution method and the Etest GRD (glycopeptide resistance detection) method (bioMérieux) was performed on 4,210 clinically significant S. aureus isolates obtained in 2009 from 43 U.S. centers. Isolates with Etest GRD-positive results for hVISA were evaluated further by repeat GRD testing and population analysis profiling-area under the curve (PAP-AUC) analysis. No VISA (vancomycin MIC, 4 to 8 µg/ml) or vancomycin-resistant (MIC ≥ 16 µg/ml) strains were detected. The Etest GRD screen for hVISA was initially positive for 68 isolates (1.6%; all by teicoplanin MIC ≥ 8 µg/ml at 24 or 48 h). Among those 68 isolates, 45 were reproducibly GRD positive. PAP-AUC testing confirmed only 11 isolates as hVISA (all had reproducible GRD-positive results). The 11 hVISA isolates were from nine medical centers and appeared genetically diverse (ten different PFGE types). The rates of resistance (including intermediate) for hVISA were as follows: oxacillin, 82%; erythromycin, 82%; clindamycin, 73%; levofloxacin, 73%; trimethoprim-sulfamethoxazole, 9%; and daptomycin, 9%. All hVISA isolates were susceptible to linezolid, tigecycline, and ceftaroline. Our data suggest that the overall prevalence of hVISA in the United States is low (0.3%). The hVISA isolates represented 10.5% of isolates with vancomycin MICs of 2 µg/ml and 0.1% of isolates with vancomycin MICs of 1 µg/ml. The positive predictive value of GRD Etest for hVISA was 16.2% for initial screen positive and 24.4% for reproducibly positive results.

Activity of ceftaroline and epidemiologic trends in Staphylococcus aureus isolates collected from 43 medical centers in the United States in 2009.

A Staphylococcus aureus surveillance program was initiated in the United States to examine the in vitro activity of ceftaroline and epidemiologic trends. Susceptibility testing by Clinical and Laboratory Standards Institute broth microdilution was performed on 4,210 clinically significant isolates collected in 2009 from 43 medical centers. All isolates were screened for mecA by PCR and evaluated by pulsed-field gel electrophoresis. Methicillin-resistant S. aureus (MRSA) were analyzed for Panton-Valentine leukocidin (PVL) genes and the staphylococcal cassette chromosome mec (SCCmec) type. All isolates had ceftaroline MICs of ≤2 µg/ml with an MIC(50) of 0.5 and an MIC(90) of 1 µg/ml. The overall resistance rates, expressed as the percentages of isolates that were intermediate and resistant (or nonsusceptible), were as follows: ceftaroline, 1.0%; clindamycin, 30.2% (17.4% MIC ≥ 4 µg/ml; 12.8% inducible); daptomycin, 0.2%; erythromycin, 65.5%; levofloxacin, 39.9%; linezolid, 0.02%; oxacillin, 53.4%; tetracycline, 4.4%; tigecycline, 0%; trimethoprim-sulfamethoxazole, 1.6%; vancomycin, 0%; and high-level mupirocin, 2.2%. The mecA PCR was positive for 53.4% of the isolates. The ceftaroline MIC(90)s were 0.25 µg/ml for methicillin-susceptible S. aureus and 1 µg/ml for MRSA. Among the 2,247 MRSA isolates, 51% were USA300 (96.9% PVL positive, 99.7% SCCmec type IV) and 17% were USA100 (93.4% SCCmec type II). The resistance rates for the 1,137 USA300 MRSA isolates were as follows: erythromycin, 90.9%; levofloxacin, 49.1%; clindamycin, 7.6% (6.2% MIC ≥ 4 µg/ml; 1.4% inducible); tetracycline, 3.3%; trimethoprim-sulfamethoxazole, 0.8%; high-level mupirocin, 2.7%; daptomycin, 0.4%; and ceftaroline and linezolid, 0%. USA300 is the dominant clone causing MRSA infections in the United States. Ceftaroline demonstrated potent in vitro activity against recent S. aureus clinical isolates, including MRSA, daptomycin-nonsusceptible, and linezolid-resistant strains.

Antibiotic resistance in sputum isolates of Streptococcus pneumoniae in chronic obstructive pulmonary disease is related to antibiotic exposure.

Streptococcus pneumoniae (S. pneumoniae) is recovered from sputum of patients with chronic obstructive pulmonary disease (COPD) during stable disease and exacerbations. In patients with community acquired pneumonia, antibiotic exposure in the prior 3-6 months is associated with recovery of antibiotic resistant isolates of S. pneumoniae. Whether the same relationship is seen in COPD is not known. From April 1994 to June 2004, 127 adults with COPD were enrolled in a prospective longitudinal study. Sputum isolates of S. pneumoniae were characterized with susceptibility testing and pulsed-field gel electrophoresis (PFGE). The relationship between antibiotic use in the previous 3 and 6 months with either new acquisition of a resistant pneumococcal isolate or development of resistance (4-fold increase in MIC) in a pre-existing colonizing pneumococcal strain was determined. A total of 194 pneumococcal isolates were recovered from 38 patients. Among 71 newly acquired and 4 resistance-emergent strains analyzed further, rates of resistance to penicillin (MIC ≥2), erythromycin (MIC ≥1), tetracycline (MIC ≥8) and trimethoprim/sulfamethoxazole (MIC ≥4) were 8%, 24%, 17% and 16% respectively. Flouroquinolone resistance was not seen. Among strains isolated from patients exposed to a macrolide within 6 months, 53.6% displayed erythromycin resistance vs. 14% of strains without such exposure (p = 0.00085). Similar associations were not seen for other antibiotics. Macrolide use in the previous 6 months is associated with macrolide resistance in sputum isolates of S. pneumoniae. Recent antibiotic exposure may help in determining appropriate antibiotic treatment in these patients.

Community-associated methicillin-resistant Staphylococcus aureus, Iowa, USA.

We performed antimicrobial drug susceptibility testing and molecular typing on invasive methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 1,666) submitted to the University of Iowa Hygienic Laboratory during 1999-2006 as part of a statewide surveillance system. All USA300 and USA400 isolates were resistant to

BD Phoenix and Vitek 2 detection of mecA-mediated resistance in Staphylococcus aureus with cefoxitin.

The BD Phoenix (BD Diagnostics, Sparks, MD) and Vitek 2 (bioMérieux, Durham, NC) automated susceptibility testing systems have implemented the use of cefoxitin to enhance the detection of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). To assess the impact of this change, 620 clinically significant S. aureus isolates were tested in parallel on Phoenix PMIC/ID-102 panels and Vitek 2 AST-GP66 cards. The results for oxacillin and cefoxitin generated by the automated systems were compared to those generated by two reference methods: mecA gene detection and MICs of oxacillin previously determined by broth microdilution according to CLSI guidelines. Testing of isolates with discordant results was repeated to attain a majority or consensus final result. There was 100% final agreement between the results of the two reference methods. For the 448 MRSA and 172 methicillin-susceptible S. aureus isolates tested, the rates of categorical agreement of the results obtained with the automated systems with those obtained by the reference methods were 99.8% for the Phoenix panels and 99.7% for the Vitek 2 cards. A single very major error occurred on each instrument (0.2%) with different MRSA isolates. The only major error was attributed to the Vitek 2 system overcalling oxacillin resistance. In 16 instances (9 on the Phoenix system, 7 on the Vitek 2 system), an oxacillin MIC in the susceptible range was correctly changed to resistant by the expert system on the basis of the cefoxitin result. The inclusion of cefoxitin in the Phoenix and Vitek 2 panels has optimized the detection of MRSA by both systems.

Comparison of the Bactec 9240 and BacT/Alert blood culture systems for evaluation of placental cord blood for transfusion in neonates.

The Bactec 9240 and the BacT/Alert blood culture systems were compared as a means for detection of bacterial contaminants in whole blood, concentrated red cells, and plasma preparations prepared from umbilical cord blood (UCB) samples. Ninety-two UCB units seeded with low levels of various bacteria were evaluated. In more than 50% of cases, growth was not detected in plasma using either system (P < 0.001). When concentrated red cells and whole blood were compared, the Bactec system detected bacterial growth consistently sooner than the BacT/Alert system in all seeded bacteria except Staphylococcus species in whole blood. The median lengths of time to detection (LTD) for whole blood and concentrated cells in BacT/Alert were 18.7 h and 18.5 h, respectively. The median LTD for the same blood fractions using the Bactec system were 16.05 h and 15.64 h. These differences in LTD by blood culture system and sample type were statistically significant (whole blood, P = 0.0449; concentrated cells, P = 0.0037). Based on the results of our study, we recommend the use of either concentrated red cells or whole blood for sterility testing in UCB samples. In our laboratory, the Bactec system compared to the BacT/Alert system was the superior method for rapid detection of bacterial contaminants in cord blood.

Patterns and density of early tracheal colonization in intensive care unit patients.

OBJECTIVE: The study aimed to describe the patterns and density of early tracheal colonization among intubated patients and to correlate colonization status with levels of antimicrobial peptides and inflammatory cytokines. DESIGN: The was a prospective cohort study. SETTING: The study was conducted in medical and cardiovascular intensive care units of a tertiary referral hospital. PATIENTS: Seventy-four adult patients admitted between March 2003 and May 2006 were recruited for the study. INTERVENTIONS: Tracheal aspirates were collected daily for the first 4 days of intubation using standardized, sterile technique and sent for quantitative culture and cytokines, lactoferrin and lysozyme measurements. MEASUREMENTS AND MAIN RESULTS: The mean acute physiology and chronic health evaluation (APACHE II) score in this cohort was 24 +/- 7. Proportion of subjects colonized by any microorganism increased over the first 4 days of intubation (47%, 60%, 70%, 70%, P = .08), but density of colonization for bacteria or yeast did not change significantly. No known risk factors predicted tracheal colonization on day 1 of intubation. Several patterns of colonization were observed (persistent, transient, new colonization, and clearance of initial colonization).The most common organisms cultured were Candida albicans and coagulase-negative Staphylococcus. Levels of cytokines, lactoferrin, or lysozyme did not change over time and were not correlated with tracheal colonization status. Four subjects (6%) had ventilator-associated pneumonia. CONCLUSIONS: The density of tracheal colonization did not change significantly over the first 4 days of intubation in medical intensive care unit patients. There was no correlation between tracheal colonization and the levels of antimicrobial peptides or cytokines. Several different patterns of colonization may have to be considered while planning interventions to reduce airway colonization.