Practical Guidance for Clinical Microbiology Laboratories: Implementing a Quality Management System in the Medical Microbiology Laboratory

This document outlines a comprehensive practical approach to a laboratory quality management system (QMS) by describing how to operationalize the management and technical requirements described in the ISO 15189 international standard. It provides a crosswalk of the ISO requirements for quality and competence for medical laboratories to the 12 quality system essentials delineated by the Clinical and Laboratory Standards Institute. The quality principles are organized under three main categories: quality infrastructure, laboratory operations, and quality assurance and continual improvement. The roles and responsibilities to establish and sustain a QMS are outlined for microbiology laboratory staff, laboratory management personnel, and the institution's leadership. Examples and forms are included to assist in the real-world implementation of this system and to allow the adaptation of the system for each laboratory's unique environment. Errors and nonconforming events are acknowledged and embraced as an opportunity to improve the quality of the laboratory, a culture shift from blaming individuals. An effective QMS encourages "systems thinking" by providing a process to think globally of the effects of any type of change. Ultimately, a successful QMS is achieved when its principles are adopted as part of daily practice throughout the total testing process continuum.

Pleuritis and suppurative pneumonia associated with a hypermucoviscosity phenotype of Klebsiella pneumoniae in California sea lions (Zalophus californianus).

The aim of this study is to document the isolation of a hypermucoviscosity (HMV) phenotype of Klebsiella pneumoniae from 25 cases of suppurative pneumonia and pleuritis and two cases of abscesses in California sea lions (Zalophus californianus) from the central California coast, representing the first report of this zoonotic pathogen from the marine environment and only the second report in non-humans. Animals died 2h to 4 days after first being observed sick on beaches. Clinical signs varied from dyspnoea to coma. Gross post-mortem examination of 25 cases revealed fibrinous pleuritis, copious pus in the pleural cavity and suppurative bronchopneumonia. K. pneumoniae isolates obtained from lung and pleural swabs and the hepatic and subcuticular abscesses were highly mucoid on blood agar culture media and were positive to the "string test". Twenty-one of the 27 isolates were examined by PCR and all were positive for rmpA and K2wyz and negative for K1magA genes. Although pneumonia and pleuritis have previously commonly been observed in marine mammals, their association with pure cultures of a zoonotic bacteria, K. pneumoniae HMV phenotype, has not. This report provides further evidence of the role marine mammals play as sentinels of health risks to humans from coastal waters.

Accuracy of commercial and reference susceptibility testing methods for detecting vancomycin-intermediate Staphylococcus aureus.

We compared the results obtained with six commercial MIC test systems (Etest, MicroScan, Phoenix, Sensititre, Vitek Legacy, and Vitek 2 systems) and three reference methods (agar dilution, disk diffusion, and vancomycin [VA] agar screen [VScr]) with the results obtained by the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference method for the detection of VA-intermediate Staphylococcus aureus (VISA). A total of 129 S. aureus isolates (VA MICs by previous BMD tests,

Characterization of Staphylococcus aureus isolates from nasal cultures collected from individuals in the United States in 2001 to 2004.

This study characterizes methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates recovered from nasal cultures of noninstitutionalized individuals in the United States obtained in 2001 to 2004 as part of the National Health and Nutrition Examination Survey. Every tenth MSSA isolate and all MRSA isolates were typed by pulsed-field gel electrophoresis (PFGE), screened for multiple toxin genes, and tested for susceptibility to 14 antimicrobial agents. USA200, USA600, and USA900 were the predominant PFGE types among MSSA isolates in both the 2001 to 2002 and the 2003 to 2004 time periods, although they accounted for only 51.3% of 316 MSSA isolates typed in 2001 and 2002 and only 43.4% of 237 MSSA isolates typed in 2003 and 2004. In contrast, USA100, USA800, and USA700 accounted for 80.0% of the 75 MRSA isolates typed in 2001 and 2002, while USA100, USA800, and USA300 accounted for 78.4% of 134 MRSA isolates typed in 2003 and 2004. The proportion of MRSA isolates that were USA300 increased significantly from the first to the second time period (P = 0.03). Most USA200 isolates (both MSSA and MRSA) carried the gene for toxic shock syndrome toxin; however, carriage of the genes encoding Panton-Valentine leukocidin, while common among MRSA of PFGE type USA300, was rare among MSSA USA300 in both time periods. Most MSSA isolates remained susceptible to all antimicrobial agents except erythromycin (79.1 and 76.0% susceptibilities in the 2001 to 2002 and the 2003 to 2004 periods, respectively). In contrast, the proportions of MRSA isolates that were susceptible to chloramphenicol, clindamycin, and erythromycin were lower in 2003 and 2004 than in 2001 and 2002, although none of these differences was statistically significant.

Invasive methicillin-resistant Staphylococcus aureus infections in the United States.

CONTEXT: As the epidemiology of infections with methicillin-resistant Staphylococcus aureus (MRSA) changes, accurate information on the scope and magnitude of MRSA infections in the US population is needed. OBJECTIVES: To describe the incidence and distribution of invasive MRSA disease in 9 US communities and to estimate the burden of invasive MRSA infections in the United States in 2005. DESIGN AND SETTING: Active, population-based surveillance for invasive MRSA in 9 sites participating in the Active Bacterial Core surveillance (ABCs)/Emerging Infections Program Network from July 2004 through December 2005. Reports of MRSA were investigated and classified as either health care-associated (either hospital-onset or community-onset) or community-associated (patients without established health care risk factors for MRSA). MAIN OUTCOME MEASURES: Incidence rates and estimated number of invasive MRSA infections and in-hospital deaths among patients with MRSA in the United States in 2005; interval estimates of incidence excluding 1 site that appeared to be an outlier with the highest incidence; molecular characterization of infecting strains. RESULTS: There were 8987 observed cases of invasive MRSA reported during the surveillance period. Most MRSA infections were health care-associated: 5250 (58.4%) were community-onset infections, 2389 (26.6%) were hospital-onset infections; 1234 (13.7%) were community-associated infections, and 114 (1.3%) could not be classified. In 2005, the standardized incidence rate of invasive MRSA was 31.8 per 100,000 (interval estimate, 24.4-35.2). Incidence rates were highest among persons 65 years and older (127.7 per 100,000; interval estimate, 92.6-156.9), blacks (66.5 per 100,000; interval estimate, 43.5-63.1), and males (37.5 per 100,000; interval estimate, 26.8-39.5). There were 1598 in-hospital deaths among patients with MRSA infection during the surveillance period. In 2005, the standardized mortality rate was 6.3 per 100,000 (interval estimate, 3.3-7.5). Molecular testing identified strains historically associated with community-associated disease outbreaks recovered from cultures in both hospital-onset and community-onset health care-associated infections in all surveillance areas. CONCLUSIONS: Invasive MRSA infection affects certain populations disproportionately. It is a major public health problem primarily related to health care but no longer confined to intensive care units, acute care hospitals, or any health care institution.

Accuracy of six antimicrobial susceptibility methods for testing linezolid against staphylococci and enterococci.

A challenge panel of enterococci (n = 50) and staphylococci (n = 50), including 17 and 15 isolates that were nonsusceptible to linezolid, respectively, were tested with the Clinical and Laboratory Standards Institute broth microdilution and disk diffusion reference methods. In addition, all 100 isolates were tested in parallel by Etest (AB Biodisk, Solna, Sweden), MicroScan WalkAway (Dade, West Sacramento, CA), BD Phoenix (BD Diagnostic Systems, Sparks, MD), VITEK (bioMérieux, Durham, NC), and VITEK 2 (bioMérieux) by using the manufacturers' protocols. Compared to the results of the broth microdilution method for detecting linezolid-nonsusceptible staphylococci and enterococci, MicroScan results showed the highest category agreement (96.0%). The overall categorical agreement levels for VITEK 2, Etest, Phoenix, disk diffusion, and VITEK were 93.0%, 90.0%, 89.6%, 88.0%, and 85.9%, respectively. The essential agreement levels (results within +/-1 doubling dilution of the MIC determined by the reference method) for MicroScan, Phoenix, VITEK 2, Etest, and VITEK were 99.0%, 95.8%, 92.0%, 92.0%, and 85.9%, respectively. The very major error rates for staphylococci were the highest for VITEK (35.7%), Etest (40.0%), and disk diffusion (53.3%), although the total number of resistant isolates tested was small. The very major error rate for enterococci with VITEK was 20.0%. Three systems (MicroScan, VITEK, and VITEK 2) provided no interpretations of nonsusceptible results for staphylococci. These data, from a challenge panel of isolates, illustrate that the recent emergence of linezolid-nonsusceptible staphylococci and enterococci is providing a challenge for many susceptibility testing systems.

Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing.

Detecting beta-lactamase-mediated carbapenem resistance among Klebsiella pneumoniae isolates and other Enterobacteriaceae is an emerging problem. In this study, 15 blaKPC-positive Klebsiella pneumoniae that showed discrepant results for imipenem and meropenem from 4 New York City hospitals were characterized by isoelectric focusing; broth microdilution (BMD); disk diffusion (DD); and MicroScan, Phoenix, Sensititre, VITEK, and VITEK 2 automated systems. All 15 isolates were either intermediate or resistant to imipenem and meropenem by BMD; 1 was susceptible to imipenem by DD. MicroScan and Phoenix reported 1 (6.7%) and 2 (13.3%) isolates, respectively, as imipenem susceptible. VITEK and VITEK 2 reported 10 (67%) and 5 (33%) isolates, respectively, as imipenem susceptible. By Sensititre, 13 (87%) isolates were susceptible to imipenem, and 12 (80%) were susceptible to meropenem. The VITEK 2 Advanced Expert System changed 2 imipenem MIC results from >16 ?g/mL to <2 ?g/mL but kept the interpretation as resistant. The recognition of carbapenem-resistant K. pneumoniae continues to challenge automated susceptibility systems.

Methicillin-resistant S. aureus infections among patients in the emergency department.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is increasingly recognized in infections among persons in the community without established risk factors for MRSA. METHODS: We enrolled adult patients with acute, purulent skin and soft-tissue infections presenting to 11 university-affiliated emergency departments during the month of August 2004. Cultures were obtained, and clinical information was collected. Available S. aureus isolates were characterized by antimicrobial-susceptibility testing, pulsed-field gel electrophoresis, and detection of toxin genes. On MRSA isolates, we performed typing of the staphylococcal cassette chromosome mec (SCCmec), the genetic element that carries the mecA gene encoding methicillin resistance. RESULTS: S. aureus was isolated from 320 of 422 patients with skin and soft-tissue infections (76 percent). The prevalence of MRSA was 59 percent overall and ranged from 15 to 74 percent. Pulsed-field type USA300 isolates accounted for 97 percent of MRSA isolates; 74 percent of these were a single strain (USA300-0114). SCCmec type IV and the Panton-Valentine leukocidin toxin gene were detected in 98 percent of MRSA isolates. Other toxin genes were detected rarely. Among the MRSA isolates, 95 percent were susceptible to clindamycin, 6 percent to erythromycin, 60 percent to fluoroquinolones, 100 percent to rifampin and trimethoprim-sulfamethoxazole, and 92 percent to tetracycline. Antibiotic therapy was not concordant with the results of susceptibility testing in 100 of 175 patients with MRSA infection who received antibiotics (57 percent). Among methicillin-susceptible S. aureus isolates, 31 percent were USA300 and 42 percent contained pvl genes. CONCLUSIONS: MRSA is the most common identifiable cause of skin and soft-tissue infections among patients presenting to emergency departments in 11 U.S. cities. When antimicrobial therapy is indicated for the treatment of skin and soft-tissue infections, clinicians should consider obtaining cultures and modifying empirical therapy to provide MRSA coverage.

Investigation of inducible clindamycin and telithromycin resistance in isolates of beta-hemolytic streptococci.

We evaluated the accuracy of an erythromycin-clindamycin double-disk test (D-zone test) and an erythromycin-telithromycin D-zone test for detection of inducible resistance in isolates of beta-hemolytic streptococci with erythromycin resistance. The results of these tests were compared to results of a broth microdilution (BMD) induction test using combinations of erythromycin and either clindamycin or telithromycin. Of 29 erythromycin-resistant, clindamycin-susceptible isolates, 16 were positive by the erythromycin-clindamycin D-zone test; all of these demonstrated inducible clindamycin resistance by BMD. Twelve isolates were D-zone test-negative, did not demonstrate inducible resistance by BMD, and were positive for a mef determinant. Of 39 erythromycin-resistant, telithromycin-susceptible isolates, 13 were erythromycin-telithromycin D-zone test-positive, 19 questionably positive (unclear blunting of the zone), and 7 were D-zone test-negative. The erythromycin-telithromycin D-zone test result did not correlate with inducible resistance by BMD or the presence of an erm or mef gene. These results demonstrate that the erythromycin-clindamycin D-zone and BMD induction tests accurately detect inducible clindamycin resistance, but the erythromycin-telithromycin D-zone test is not reliable for detecting inducible telithromycin resistance.

Potassium sorbate reduces gastric colonization in patients receiving mechanical ventilation.

OBJECTIVES: Tube feeding might increase gastric burden of pathogenic bacteria and predispose patients to ventilator-associated pneumonia. We sought to determine whether a tube feeding formula acidified using potassium sorbate could reduce gastric burden of potentially pathogenic bacteria. DESIGN: Prospective, randomized, double-blind trial. SETTING: RML Specialty Hospital, a facility with expertise in weaning patients from prolonged mechanical ventilation. PATIENTS: Thirty patients recovering from prolonged mechanical ventilation. INTERVENTION: Patients were randomized to receive either a standard tube feeding formula (n=14) or a formula acidified using potassium sorbate to a pH of 4.25 (n=16). MEASUREMENTS AND RESULTS: Weekly quantitative cultures of gastric aspirates. The number of colony-forming units (CFUs) per patient was higher in the control than in the treatment group (53%+/-11% vs 9%+/-3.4%, threshold of >or=100,000 CFU/mL fluid, P=.003). The number of organisms isolated in each patient per week was higher among patients receiving standard tube feeding formula than among patients receiving acidified formula (0.91 +/- 0.20 vs 0.13 +/- 0.05 organisms per patient per week, threshold of >or=100,000 CFU/mL fluid, P=.0014). There was no difference in the incidence of gastrointestinal bleeding or ventilator-associated pneumonia between study groups. CONCLUSION: Tube feeding formula acidified using potassium sorbate was well tolerated and reduced gastric bacterial burden in patients recovering from prolonged mechanical ventilation.

Tuberculous tenosynovitis.

Tuberculous tenosynovitis is rare and may be overlooked as a cause of chronic tenosynovitis. This report presents a case of a young woman with tuberculosis tenosynovitis of the wrist, and highlights the clinical, imaging, histological, and laboratory features most commonly seen in this disease.

Fatal hemorrhagic pneumonia concomitant with Chlamydia pneumoniae and parainfluenza virus 4 infection.

CONTEXT: Cases of fatal hemorrhagic pneumonia need to be investigated for highly contagious viral causes. While not all hemorrhagic pneumonias are caused by very contagious agents, the etiology must be correctly determined in order to administer appropriate patient care. OBJECTIVE: To determine whether chlamydia, paramyxovirus, or mycoplasma was the causative agent in a case of fatal hemorrhagic pneumonia, and to evaluate the possibility that this was the first case of hantavirus pulmonary syndrome in Illinois. DESIGN: Nonroutine virological and molecular analyses were performed on lung tissue taken during an unrestricted autopsy of a patient who died in 2002. SETTING AND PATIENT: An elderly, male, Chicago-area resident with a 3-week history of nonspecific, mild upper respiratory tract infection was admitted for hospital treatment of the respiratory infection and viral myositis without cardiac involvement. The patient became febrile, hypoxic, developed hemorrhagic pneumonia, and died. Because he had proven exposure to mice and had developed hemorrhagic pneumonia, hantavirus pulmonary syndrome was suspected as the cause of death. Mice known to carry hantaviruses live in Illinois, including the Chicago area. INTERVENTIONS: Gatifloxacin and heparin anticoagulation were initiated because community-acquired pneumonia and pulmonary embolism were considered likely etiologies for an acute exacerbation of hypoxemia. RESULTS: Two respiratory pathogens were isolated and identified: Chlamydia pneumoniae and human parainfluenza virus 4a. CONCLUSIONS: A mixed (polymicrobial) infection contributed to the patient's death. Hemorrhage was likely a result of anticoagulation therapy superimposed on lung tissues damaged by pneumonia. The uncommon nature of this case and the pathogens involved underscore the challenges in infection control and clinical evaluation that hospitals will face when confronted with possibly new and potentially deadly communicable diseases.

Staphylococcus epidermidis adherence to human amniotic membrane and to human, rabbit, and cat conjunctiva.

PURPOSE: To evaluate Staphylococcus epidermidis adherence to human amniotic membrane (HAM) and compare it with S epidermidis adherence to human, rabbit, and cat conjunctiva in vitro. SETTING: Research laboratory, Loyola University Medical Center, Maywood, Illinois, USA. METHODS: Commercially available HAM (N = 3) was used. Conjunctival specimens from humans, rabbits, and cats (n = 3 each) were processed similarly to HAM. The tissues were exposed to S epidermidis (3 x 10(8) colony-forming units per milliliter) for 0, 5, 30, and 90 minutes, rinsed in sterile saline, and processed for light, scanning (SEM), and transmission (TEM) electron microscopy. Scanning electron microscopy (x2000) was used to quantify adherent bacteria/mm(2) of tissue (SEM photographs = 144). RESULTS: The following mean levels (+/- SD) of adherent S epidermidis/mm(2) were found at 0, 5, 30, and 90 minutes: HAM, 3833 +/- 1570, 9060 +/- 2512, 15,431 +/- 10,752, and 30,315 +/- 14,803, respectively; human conjunctiva, 1493 +/- 672, 7218 +/- 3179, 17,273 +/- 7168, and 19,861 +/- 9624, respectively; rabbit conjunctiva, 3385 +/- 5074, 14,386 +/- 14,569, 15,283 +/- 13,679, and 20,113 +/- 24,016, respectively; and cat conjunctiva, 4032 +/- 2240, 12,345 +/- 3413, 8512 +/- 4032, and 19,214 +/- 5584, respectively. No statistically significant differences were found at any time point (P>.16). CONCLUSION: There was no statistically significant difference in the adherence of S epidermidis to HAM and to human, rabbit, and cat conjunctiva. Bacterial adherence to HAM may be clinically significant.

In vitro study of bacterial adherence to processed dura mater, processed pericardium, pericardium in saline, and human sclera.

PURPOSE: To study bacterial adherence to processed dura mater, processed pericardium, pericardium in saline, and human sclera and the difference in bacterial adherence to these tissues. SETTING: Research Laboratory, Loyola University Medical Center, Maywood, Illinois, USA. METHODS: Specimens of processed dura mater, processed human pericardium, pericardium in saline, and human sclera (N = 32) were exposed to Staphylococcus epidermidis (concentration 3 x 10(8)) for 10, 20, 40, and 60 minutes, washed for 5 seconds, fixed, and processed for scanning electron microscopy (SEM). Each bacterial count represents an average of 50 random SEM fields at x5,000 magnification. After SEM, selected specimens were processed for transmission electron microscopy. RESULTS: The mean number of bacteria/mm(2) +/- SD adhering to the tissues at 10, 20, 40, and 60 minutes, respectively, were dura mater, 107,833 +/- 65,410, 104,500 +/- 13,471, 96,067 +/- 113,884, and 204,267 +/- 153,697; processed pericardium, 131,550 +/- 86,194, 100,900 +/- 20,031, 144,683 +/- 51,730, and 176,933 +/- 111,818; pericardium in saline, 7,925 +/- 1,520, 33,933 +/- 32,085, 1,217 +/- 1,287, and 21,550 +/- 16,436; and human sclera, 4,850 +/- 2,121, 23,700 +/- 17,961, 5,150 +/- 1,273, and 8,175 +/- 8,450. A 2-way analysis of variance showed significant differences among groups (P =.001) and no significant difference in sample time (P =.929). CONCLUSIONS: Bacterial adherence to processed dura mater, processed pericardium, pericardium in saline, and human sclera should be considered when these materials are used clinically during ophthalmic surgery and other surgical specialties. Adequate broad-spectrum antibiotic coverage is needed to prevent infection and subsequent complications in patients.