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Comparative Study
. 2016 Mar 2:15:3.
doi: 10.1186/s12952-016-0047-y.

Comparison of serological methods with PCR-based methods for the diagnosis of community-acquired pneumonia caused by atypical bacteria

Mariana Herrera  1   2 , Yudy Alexandra Aguilar  3   4 , Zulma Vanessa Rueda  5 , Carlos Muskus  6 , Lázaro Agustín Vélez  7   8
Affiliations
Comparative Study

Comparison of serological methods with PCR-based methods for the diagnosis of community-acquired pneumonia caused by atypical bacteria

Mariana Herrera et al. J Negat Results Biomed. .

Abstract

Background: The diagnosis of community-acquired pneumonia (CAP) caused by Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae is traditionally based on cultures and serology, which have special requirements, are time-consuming, and offer delayed results that limit their clinical usefulness of these techniques. We sought to develop a multiplex PCR (mPCR) method to diagnosis these bacterial infections in CAP patients and to compare the diagnostic yields obtained from mPCR of nasopharyngeal aspirates (NPAs), nasopharyngeal swabs (NPSs), and induced sputum (IS) with those obtained with specific PCR commercial kits, paired serology, and urinary antigen.

Results: A total of 225 persons were included. Of these, 10 patients showed serological evidence of L. pneumophila infection, 30 of M. pneumoniae, and 18 of C. pneumoniae; 20 individuals showed no CAP. The sensitivities were mPCR-NPS = 23.1%, mPCR-IS = 57.1%, Seeplex®-IS = 52.4%, and Speed-oligo®-NPA/NPS = 11.1%, and the specificities were mPCR-NPS = 97.1%, mPCR-IS = 77.8%, Seeplex®-IS = 92.6%, and Speed-oligo®-NPA/NPS = 96.1%. The concordance between tests was poor (kappa <0.4), except for the concordance between mPCR and the commercial kit in IS (0.67). In individuals with no evidence of CAP, positive reactions were observed in paired serology and in all PCRs.

Conclusions: All PCRs had good specificity but low sensitivity in nasopharyngeal samples. The sensitivity of mPCR and Seeplex® in IS was approximately 60%; thus, better diagnostic techniques for these three bacteria are required.

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Figures

Fig. 1
Fig. 1
Analytical sensitivity of mPCR using 1,500; 750; 375; and 187 copies of L. pneumophila mip genes, p1 of M. pneumoniae, and Pstl of C. pneumoniae MW: 100 bp molecular weight marker; NC: negative control; Lines marked with arrows correspond to the amplicons from 375 copies of each gene
Fig. 2
Fig. 2
Analytical specificity of mPCR. 1. Molecular weight marker 100 bp; 2. Negative control; 3. Positive control (487 bp L. pneumophila, 360 bp M. pneumoniae, and 283 bp C. pneumoniae); Bacteria: 4. Streptococcus pneumoniae; 5. Haemophilus influenzae; 6. Klebsiella pneumoniae; 7. Escherichia coli; 8. Pseudomonas aeruginosa; 9. Staphylococcus aureus; 10 . Nocardia spp.; 11. Enterobacter cloacae; Fungi: 12. Histoplasma capsulatum; 13. Aspergillus terreus; 14. Cryptococcus neoformans; 15. Candida tropicalis; 16. Candida albicans; 17. Candida guilliermondii; 18. Candida glabrata; 19. Paracoccidioides brasiliensis; 20. Mycobacterium tuberculosis (bacteria); 21. Human DNA
Fig. 3
Fig. 3
Concordance (kappa index) between in-house (mPCR) and commercial PCR for Mycoplasma pneumoniae. NPS: Nasopharyngeal swab; NPA: Nasopharyngeal aspirate; IS: Induced sputum
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