Fast and accurate quantitative detection of Helicobacter pylori and identification of clarithromycin resistance mutations in H. pylori isolates from gastric biopsy specimens by real-time PCR.
Rapid identification of patients infected with clarithromycin-resistant Helicobacter pylori without the need for culture can help to avoid useless prescriptions of clarithromycin. We developed and tested a routine real-time quantitative PCR assay dedicated to that purpose. One hundred ninety-six consecutive gastric biopsy specimens were examined by culture, histology performed by a trained physician, and rapid PCR with the LightCycler apparatus. Infection was defined as (i) positivity of culture, (ii) positivity of histology, or (iii) positivity of PCR if confirmed by positivity of a concomitant indirect test (serology or urea breath test). Susceptibility to clarithromycin was tested by E-test and PCR. The prevalence of infection was 33.7% (66 of 196 samples). The sensitivities of culture, histology, and PCR were 90.9% (60 of 66 samples), 87.9% (58 of 66 samples), and 97.0% (64 of 66 samples), respectively. The specificity of PCR was 94.6% (123 of 130 samples). The linearity of the PCR results was achieved over a 6-log range of input DNA, and we were able to accurately quantify as few as 300 bacteria and to qualitatively detect as few as 30 bacteria per DNA sample. For clarithromycin susceptibility testing, there was 98.2% (55 of 56 samples) concordance between E-test and PCR. Forty-eight strains were clarithromycin susceptible, and 9 strains were clarithromycin resistant. The single discrepancy concerned a culture which was a mixture of mutant and wild type, with a susceptible-to-resistant ratio of 11.5: the resistant population was detected by E-test but not by PCR. Our PCR assay is accurate for fast detection of H. pylori as well as of clarithromycin resistance and is also able to objectively determine bacterial density.
Lascols
Christine
C
Laboratoire de Bactériologie, Centre Hospitalo-Universitaire Henri-Mondor, Assistance Publique-Hôpitaux de Paris, Université Paris XII, Créteil, France.
Lamarque
Dominique
D
Costa
Jean-Marc
JM
Copie-Bergman
Christiane
C
Le Glaunec
Jeanne-Marie
JM
Deforges
Lionel
L
Soussy
Claude-James
CJ
Petit
Jean-Claude
JC
Delchier
Jean-Charles
JC
Tankovic
Jacques
J
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001706:Biopsy; D017291:Clarithromycin / Q000494:pharmacology*; D003470:Culture Media; D004275:DNA, Ribosomal / Q000032:analysis; D024881:Drug Resistance, Bacterial / Q000235:genetics; D016481:Helicobacter Infections / Q000382:microbiology*; D016480:Helicobacter pylori / Q000187:drug effects / Q000254:growth & development / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D013270:Stomach / Q000382:microbiology*; D013997:Time Factors
D000900:Anti-Bacterial Agents; D003470:Culture Media; D004275:DNA, Ribosomal; D012338:RNA, Ribosomal, 23S; D017291:Clarithromycin