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EBM 7/17/09, Legionnaires’ Disease Citation: Diederen BM, Kluytmans JA, Vandenbroucke-Grauls CM, and Peeters MF. 2008. Utility of Real-Time PCR for Diagnosis of Legionnaires' Disease in Routine Clinical Practice. J Clin Microbiol 46:671-677. Study Summary: Objectives: To determine the value of PCR in the diagnosis of Legionnaires’ disease in routine clinical practice. Design: Retrospective data collection. Setting: Data obtained from the Regional Public Health Laboratory, in St. Elisabeth Hospital, the Netherlands. Specimens tested were those submitted for routine diagnosis of pneumonia from December 2002 to November 2005. Patients: All hospitalized patients, had symptoms compatible with pneumonia, and showed radiological signs of infiltration. To be included in the analysis, specimens had to have included PCR and either culture for Legionella spp. or urinary antigen. Of the included patients (n=151), 61% were male with a mean age of 57 years (range 23-74 years). Mean age of the female participants was 60 years (range 25-85 years). Methods: A case of confirmed Legionella pneumonia (ie the gold standard) was defined according to the European Working Group on Legionella Infections criteria. These consider an “LD-positive” patient as one with a positive Legionella spp. cultured from a respiratory sample, or a positive Legionella urinary antigen. At the time of collection, respiratory samples were sent to the molecular biology lab for PCR, and simultaneously cultured. PCR was targeted at two specific regions: 16S rRNA gene and the mip gene. All positive PCR results were confirmed with a second PCR run on a second area of DNA isolated from the respiratory sample – if negative, the sample as a whole was considered LD negative by PCR. “Discrepant analysis” was performed on specimens for which PCR and the gold standard gave different results. In an attempt to determine which one was correct, additional testing was performed to try and confirm the PCR positivity, given the imperfection of the gold standard. A different PCR assay was used, a different laboratory performed the same PCR (16S rRNA or mip) or sequencing analysis. If any of these approaches confirmed a true positive, the specimen was considered “LD positive”. Validity Criteria: Are the results of this diagnostic study valid? Was there an independent, blind comparison with a reference (“gold”) standard of diagnosis? Was the diagnostic test evaluated in an appropriate spectrum of patients (like Yes, although the “gold standard” itself is imperfect in this case. Reported culture sensitivity varies from 41%-91%. Urinary antigen testing has sensitivities reported from 60-100%, but only detects L. pneumophila serogroup 1. Yes, for inpatient care. those in whom it would be used in practice)? Was the reference standard applied Yes regardless of the diagnostic test result? Was the test (or cluster of tests) validated No (only one group) in a second, independent group of patients? Results: Of the 151 specimens included in the study, 37 of these patients (25%) fulfilled the EWGLI criteria as an “LD positive” case. Of these 37: 21 were culture positive 35 were urinary Ag positive 19 were culture and Ag positive For PCR, two samples had inhibitors that could not be removed. Therefore, 149 patients were included in the final calculations. 40 out of the 149 patients (27%) tested positive for Legionella based on PCR diagnostic criteria 36 were positive for L. pneumophila, and 4 for non-pneumophila Legionella spp. 37 tested positive by the 16S rRNA PCR (5 false positives and 5 false negatives compared to GS) 36 tested positive by mip PCR (2 false positives and 3 false negatives compared to GS) 33 were positive for both the mip and 16S rRNA PCRs 3 were positive for mip only Using discrepant analysis, 2/5 false positives in 16S rRNA group would actually be true positives, and 2/2 in mip group. **My calculations are based on original data, not the discrepant analysis reclassification.** Blank 2x2 table for your own calculation purposes (mip pcr): Totals Totals Sensitivity = a/(a+c) = 34/37 = 92% Specificity = d/(b+d) = 110/112 = 98% Likelihood ratio for a positive test result = LR+ = sens/(1-spec) = 92%/2% = 46 Likelihood ratio for a negative test result = LR - = (1-sens)/spec = 8%/98% = 0.08 Positive Predictive Value = a/(a+b) = 34/36 = 94% Negative Predictive Value = d/(c+d) = 110/113 = 97% Pre-test probability (prevalence using this target population – those hospitalized and diagnosed with pna) = (a+c)/(a+b+c+d) = 37/149 = 25% Pre-test odds = prevalence/(1-prevalence) = 25%/75% = 0.33 Post-test odds = pre-test odds LR = 0.33 x 46 = 15.33 Post-test probability = post-test odds/(post-test odds +1) = 15.33/16.33 = 94% Interpretation/Conclusions: Can you apply this valid, important evidence about a diagnostic test in caring for your patient? Is the diagnostic test available, affordable, accurate, and precise in your setting? Can you generate a clinically sensible estimate of your patient’s pre-test probability (from personal experience, prevalence statistics, practice databases, or primary studies)? Are the study patients similar to your own? Is it unlikely that the disease possibilities or probabilities have changed since the evidence was gathered? Will the resulting post-test probabilities affect your management and help your patient? Could it move you across a testtreatment threshold? Would your patient be a willing partner in carrying it out? Would the consequences of the test help your patient? No, we do not carry Legionella PCR testing in-house Pre-test probability: Yes (Responsible for 1-5% of CAP) Study patients: All from the Netherlands, where there may be different strains of Legionella spp. But overall, likely similar enough. Changes: No Would consider testing if high suspicion with negative urinary antigen, and particularly in cases of possible outbreaks. (Head to head analysis of urinary Ag vs. PCR was relatively equivalent, but combination of Ag and mip PCR had some additive effect, p=0.7) Likely – simple blood draw Minimally. Empiric treatment for CAP usually is either levofloxacin or Zithromax, both of which treat Legionella. However, if a patient diagnosed with Legionella were not improving, could alter treatment to include a tetracycline or erythromycin, which have been shown to have better outcomes in some studies. Additional thoughts/Questions: Possible there is a somewhat decreased specificity secondary to an imperfect gold standard All retrospective analysis Discrepant analysis approach introduces bias by the selective use of confirmation testing, and reclassification of “true” and “false” negatives and positives Entire study population was biased to those in whom they felt the need to check for Legionella rather than just treat empirically Is there a significant number of non-penumophila Legionella spp causing clinically significant pneumonia (only 1 case in 151 tested in this study)