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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Pediatr Infect Dis J. Author manuscript; available in PMC 2014 March 1.
Published in final edited form as:
PMCID: PMC3618625
NIHMSID: NIHMS420730

Species-Specific PCR Improves Detection of Bacterial Pathogens in Parapneumonic Empyema Compared with 16S PCR and Culture

To the Editors

Parapneumonic empyema (PPE) is a serious complication of bacterial pneumonia in children that is increasingly reported in the U.S. 1. Identification of pathogens causing PPE has generally relied on culture, however in up to 70%of cases pleural fluid cultures are negative24. The high rate of culture-negative disease complicates clinical care and selection of appropriate antibiotics.

Culture-independent techniques such as 16S polymerase chain reaction (PCR) have been used to determine the etiology of PPE25. However, some studies have shown limitations to its sensitivity 3, 5. Our previously published studies demonstrated the efficacy of genus and species-specific assays to detect pathogens causing PPE4. Here we directly compared the sensitivity of pathogen detection using genus and species-specific PCR to that obtained withbroad-range16SPCR.

This study was approved by the Children’s Hospital of Philadelphia (CHOP) Committees for the Protection of Human Subjects and was exempt from IRB oversight at the University of Utah. Samples were collected at CHOP to assess the diagnostic accuracy of broad-range 16S PCR5. Patient selection, sample collection methods and 16S PCR methodology have been previously described 5. Residual frozen pleural fluid samples were transferred to the University of Utah and Idaho Technology, Inc. (now BioFire Diagnostics, Inc.)for analysis by genus and species-specific PCR as previously described 4. Results of conventional culture were compared to results of 16S and species-specific PCR using McNemar’s test.

Twenty-five pleural fluid samples underwent testing with both 16S and genus/species-specific PCR. Culture of pleural fluid and/or blood identified a pathogen in 6 (24%)children and16S PCR of pleural fluid identified a pathogen in 3 (12%). Genus or species-specific PCR identified at least one bacterial pathogen in pleural fluid samples from 22of 25children (88%; p < 0.001 for comparison to both culture and 16S PCR). Species-specific assays most commonly detected and identified S. pneumoniae (56% of cases). MRSA was detected in one pleural fluid specimen (4%) that was also culture positive. In two cases polymicrobial infection was detected by species-specific PCR. Detailed results are shown in the Table, Supplemental Digital Content 1, http://links.lww.com/INF/B385. This table shows the detailed comparison, by organism, of species-specific PCR vs. 16S PCR and conventional culture of pleural fluid and blood.

This study directly compares pathogen detection using 16S PCR to PCR specifically targeting pneumonia-causing bacteria in pleural fluid from children with complicated pneumonia. 16S PCR did not improve detection of bacteria, however species-specific PCR significantly increased pathogen detection with a positivity rate of 88%. S. pneumoniae was the most common pathogen identified. MRSA was detected only once, in a culture-positive specimen.

The significantly improved yield of species-specific PCR indicates that PCR methodology is an important consideration for molecular detection of pathogens in pneumonia. While outside the scope of this study, further analysis of results at the patient-level could inform testing algorithms regarding the utility of broad range vs. specific PCR in certain populations. As testing costs decrease and multiplex testing is more often available, the feasibility and cost of performing multiple specific PCR tests on a single sample will improve.

Supplementary Material

1

Supplemental Digital Content 1. Bacterial detection by culture and PCR methodology: Comparison of species-specific PCR, 16S rRNA PCR and culture for 25 patients This table shows the detailed comparison, by organism, of species-specific PCR vs. 16S PCR and conventional culture of pleural fluid and blood.

Acknowledgments

Sources of Support: This work was supported by the National Institute of Allergy and Infectious Diseases at the National Institutes of Health [1K23AI079401 to AJB, K01AI73729 to SSS, 1U01AI082184-01 to AJB and ATP, 1R01 AI089489-01 to ATP]; the National Institute of Child Health and Development at the National Institutes of Health [K24HD047249-01 to CLB]; the Centers for Disease Control [1U18IP000303-01 to AJB, KKA, CLB, ATP]; the McCabe Fund Advisory Committee [to SSS], and the University Research Foundation of the University of Pennsylvania [to SSS]. The project described was supported by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health [UL1RR025764 to CLB and AJB, UL1RR024134 to SSS]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Footnotes

Conflict of Interest: CH was an employee of Idaho Technology, Inc. (now BioFire Diagnostics, Inc.) at the time of the study. AJB, CLB, ATP and KA collaborate with BioFire Diagnostics, Inc., on several NIH and CDC-funded projects (see sources of support above).

References

1. Grijalva CG, Nuorti JP, Zhu Y, Griffin MR. Increasing incidence of empyema complicating childhood community-acquired pneumonia in the United States. Clin Infect Dis. 2010;50:805–813. [PubMed]
2. Le Monnier A, Carbonnelle E, Zahar JR, et al. Microbiological diagnosis of empyema in children: comparative evaluations by culture, polymerase chain reaction, and pneumococcal antigen detection in pleural fluids. Clin Infect Dis. 2006;42:1135–1140. [PubMed]
3. Saglani S, Harris KA, Wallis C, Hartley JC. Empyema: the use of broad range 16S rDNA PCR for pathogen detection. Arch Dis Child. 2005;90:70–73. [PMC free article] [PubMed]
4. Blaschke AJ, Heyrend C, Byington CL, et al. Molecular analysis improves pathogen identification and epidemiologic study of pediatric parapneumonic empyema. Pediatr Infect Dis J. 2011;30:289–294. [PMC free article] [PubMed]
5. Gollomp K, Rankin SC, White C, et al. Broad-range bacterial polymerase chain reaction in the microbiologic diagnosis of complicated pneumonia. J Hosp Med. 2012;7:8–13.