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Context.—Bacterial cultures of bronchoscopic samples require 1 to 2 days for results to be available for use in clinical decisions. We developed a rapid diagnostic testing strategy that is highly sensitive for screening bacteria in bronchoalveolar lavage (BAL) samples, with results available within hours of collection. Objective.—To validate the ability of a bacterial adenosine triphosphate (ATP) assay and routine Gram stain microscopy to detect significant bacterial counts in BAL samples. Design.—Four hundred seventy-seven BAL samples from 319 patients suspected of having pneumonia were tested using a rapid diagnostic strategy, consisting of Gram stain and a bacterial ATP assay. Rapid results were compared with quantitative cultures with a positive cutoff of 104 CFU/mL or higher. Results.—Significant bacterial counts were identified in 107 samples (22%). The most common etiologic agents were Staphylococcus aureus (25%), Haemophilus influenzae (17%), and Streptococcus pneumoniae (12%). The rapid test results were false negative in 5 cases (S aureus in 2, both Klebsiella pneumoniae and S aureus in 1, and Stenotrophomonas maltophilia and S pneumoniae in 1 case each). The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the rapid diagnostic strategy were 95.3%, 54.9%, 37.9%, 97.6%, and 63.9%, respectively. Conclusion.—A negative result with this rapid diagnostic testing strategy rules out significant bacterial counts in BAL samples with a high degree of certainty and may allow use of narrow-spectrum antimicrobial agents or withholding of empiric antimicrobial therapy in patients suspected of having ventilator-associated pneumonia.

Context.—Surface swab cultures have attracted attention as a potential alternative to biopsy histology or quantitative culture methods for microbiological burn wound monitoring. To our knowledge, the utility of adding a Gram-stained slide in this context has not been evaluated previously. Objective.—To determine the degree of correlation of Gram stain with culture for the microbiological analysis of burn wound surfaces. Design.—Prospective laboratory analysis. Setting.—Urban health region/centralized diagnostic microbiology laboratory. Patients.—Burn patients hospitalized in any Calgary Health Region burn center from November 2000 to September 2001. Intervention.—Gram stain plus culture of burn wound surface swab specimens obtained during routine dressing changes or based on clinical signs of infection. Main Outcome Measures.—Degree of correlation (complete, high, partial, none), including weighted κ statistic (κw), of Gram stain with culture based on quantitative microscopy and degree of culture growth. Results.—A total of 375 specimens from 50 burn patients were evaluated. Of these, 239 were negative by culture and Gram stain, 7 were positive by Gram stain only, 89 were positive by culture only, and 40 were positive by both methods. The degree of complete, high, partial, and no correlation of Gram stain with culture was 70.9% (266/375), 1.1% (4/375), 2.4% (9/375), and 25.6% (96/375), respectively. The degree of correlation for all 375 specimens, as expressed by the weighted κ statistic, was found to be fair (κw = 0.32). Conclusion.—The Gram stain is not suitable for the microbiological analysis of burn wound surfaces.

Context.—A rapid, real-time, duplex, fluorescent molecular beacon probe–based polymerase chain reaction (PCR) assay was recently developed for the detection of methicillin-resistant Staphylococcus aureus. Objective.—To describe the development and validation of this unique assay. Design.—Prospective laboratory analysis. Setting.—Urban health region/centralized diagnostic microbiology laboratory. Bacterial Strains.—One hundred eighty-one previously characterized clinical and American Type Culture Collection isolates, including 50 strains each of methicillin-resistant and methicillin-sensitive S aureus, plus 50 strains of coagulase-negative staphylococci and 31 nonstaphylococcal isolates to ensure assay specificity. Intervention.—Assays were performed on purified genomic DNA extracted from growing bacterial colonies. Two sets of oligonucleotide primers were used to specifically amplify the mecA and nuc genes, followed by detection of amplicons using fluorophore-labeled molecular beacon probes. Assays were performed on the Mx4000 Multiplex Quantitative PCR System (Stratagene Inc, La Jolla, Calif). Main Outcome Measures.—(1) Assay sensitivity and specificity, and (2) analytical sensitivity. Results.—The assay demonstrated 100% sensitivity and 100% specificity, and accurately characterized isolates as methicillin-resistant S aureus, methicillin-sensitive S aureus, or methicillin-resistant coagulase-negative staphylococci, with test results available in 2.5 hours. The analytical sensitivity of the assay was determined to be between 6 and 60 genomic equivalents. Conclusions.—This assay is rapid, accurate, easy to perform, and is compatible with other real-time PCR instruments, making it a suitable alternative to conventional PCR methodologies.

Context.—A regional centralized laboratory service for the rapid diagnosis of malaria was implemented 3 years ago in May 1999 within the Division of Microbiology, Calgary Laboratory Services. Objective.—To describe the design and performance of this unique microbiology laboratory service. Design.—Blood specimens must arrive at the central laboratory within 2 hours of collection. Thin blood smears are read and reported from suspected acute cases within 1 hour of receipt, 24 hours per day, 7 days a week, by trained and experienced microbiology technologists. All positive malaria smears are reviewed by a medical microbiologist and confirmed by polymerase chain reaction at a reference laboratory. Setting.—Calgary Laboratory Services provides integrated laboratory services to the Calgary Health Region, an urban area of more than 1 million people. Main Outcome Measures.—Performance of the service has been continuously monitored by measuring preanalytic and analytic test turnaround times, test accuracy, clinical relevance, and the results of proficiency testing. Results.—More than 90% of blood specimens for malaria from community locations have consistently arrived within 2 hours of collection, and hospitals have reached this target within the past year. Although polymerase chain reaction was more sensitive at detecting the presence of malaria, the expert microscopists were as accurate at determining the type of Plasmodium infection. More than 95% of all positive smear results are consistently reported within 2 hours of receipt of a blood specimen. Conclusions.—Implementation of a regional centralized microbiology service has improved our ability to make a rapid and accurate diagnosis of malaria in this region.