Comparison of pathogens from 90 MEF samples detected by culture and PCR

Assay resultNo. (%) of samples
Moraxella catarrhalisHaemophilus influenzaeStreptococcus pneumoniaeAlloiococcusotitidisStaphylococcus aureusPseudomonas aeruginosa
Culture +, PCR +8 (8.9)16 (18)15 (17)01 (1.1)0
Culture +, PCR −01 (1.1)a0000
Culture −, PCR +34 (38)14 (16)12 (13)6 (6.7)4 (4.4)1 (1.1)
Culture −, PCR −48 (53)59 (66)63 (70)84 (93)85 (94)89 (99)
  • a This instance of PCR negativity of a sample containing H. influenzae was a consequence of the sequence of the forward primer. Some strains of H. influenzae are not detectable by the primer used in this study (16, 24, 32). The presence of H. influenzae in a high quantity was verified by sequencing the V3-to-V5 region of the bacterial 16S rRNA gene using generic primers. To reinforce the finding, and to amend the design of the detection primer, we then retrieved available sequences of the 16S region of H. influenzae from GenBank, aligned them, removed the ambiguous sequences, and assessed the primer annealing site in the 557 remaining sequences. The H. influenzae-specific forward primer designed by Holder et al. (16) and used here annealed with no mismatches to 401 of 557 retrieved sequences. Two other prevalent motifs were related to the forward primer annealing site: the first one differed by only two nucleotides in the 5′-proximal portion of the primer, so safe detection by the original primer may be reasonably expected; it was present in 53 of 557 retrieved sequences. The other motif, however, differed by eight nucleotides, including those in the 3′-distal end of the original primer; such a motif was noted in 58 of 557 retrieved sequences. Because isolates of this sequence could not be intercepted by the original primer, we amplified the sample in question using a novel forward primer, H_infl_Fv2 (5′-CGTAGTGTCGAGAGACGAAAGG-3′), and a generic reverse panbacterial 16S primer flanking the V5 region. Sequencing of the amplicon then confirmed the presence of a high quantity of H. influenzae in this sample. None of the remaining samples was positive for this sequence variant.