Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples

• Published in: APMIS : acta pathologica, microbiologica et immunologica Scandinavica Vol. 113; no. 2; pp. 99 - 111
• Main Authors: STRÅLIN, KRISTOFFER, BÄCKMAN, ANDERS, HOLMBERG, HANS, FREDLUND, HANS, OLCÉN, PER
• Format: Journal Article
• Language: English
• Published: Oxford, UK Munksgaard International Publishers 01.02.2005; Blackwell
• Subjects: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Bacteriology; Biological and medical sciences; Child; Chlamydophila pneumoniae; Chlamydophila pneumoniae - genetics; Chlamydophila pneumoniae - isolation & purification; False Negative Reactions; False Positive Reactions; Fundamental and applied biological sciences. Psychology; Haemophilus influenzae; Haemophilus influenzae - genetics; Haemophilus influenzae - isolation & purification; Humans; Infectious diseases; Medical sciences; MEDICIN; MEDICINE; Microbiology; Middle Aged; Miscellaneous; Multiplex PCR; Mycoplasma pneumoniae; Mycoplasma pneumoniae - genetics; Mycoplasma pneumoniae - isolation & purification; Pilot Projects; Polymerase Chain Reaction - methods; Respiratory Tract Infections - diagnosis; Respiratory Tract Infections - microbiology; Sensitivity and Specificity; Sputum - microbiology; Streptococcus pneumoniae; Streptococcus pneumoniae - genetics; Streptococcus pneumoniae - isolation & purification; Chlamydia pneumoniae; Chlamydiaceae; Microbiology; Multiplex polymerase chain reaction; Mycoplasma pneumoniae; Mycoplasmataceae; Haemophilus influenzae; Streptococcus pneumoniae; Mollicutes; Pasteurellaceae; Mycoplasmatales; Streptococcaceae; Multiplex PCR; Immunology; Sputum; Streptococcus A; Chlamydiales; Bacteria; Micrococcales; Chlamydophila pneumoniae
• ISSN: 0903-4641; 1600-0463; 1600-0463
• DOI: 10.1111/j.1600-0463.2005.apm1130203.x

A multiplex PCR (mPCR) was developed for simultaneous detection of specific genes for Streptococcus pneumoniae (lytA), Mycoplasma pneumoniae (P1), Chlamydophila pneumoniae (ompA), and Haemophilus influenzae (16S rRNA, with verification PCR for P6). When the protocol was tested on 257 bacterial strains belonging to 37 different species, no false negatives and only one false positive were noted. One Streptococcus mitis out of thirty was positive for lytA. In a pilot application study of 81 sputum samples from different patients with suspected lower respiratory tract infection (LRTI), mPCR identified S. pneumoniae in 25 samples, H. influenzae in 29, M. pneumoniae in 3, and C. pneumoniae in 1. All samples culture positive for S. pneumoniae (n=15) and H. influenzae (n=15) were mPCR positive for the same bacteria. In a pilot control study with nasopharyngeal swabs and aspirates from 10 healthy adults, both culture and mPCR were negative. No PCR inhibition was found in any of the mPCR‐negative sputum or nasopharyngeal samples. Whether all samples identified as positive by mPCR are truly positive in an aetiological perspective regarding LRTI remains to be evaluated in a well‐defined patient material. In conclusion, the mPCR appears to be a promising tool in the aetiological diagnostics of LRTI.