Pyrosequencing assay to rapidly detect clarithromycin resistance mutations in Canadian Helicobacter pylori isolates.
BACKGROUND
Mutations at positions 2142 or 2143 in the twocopy 23S ribosomal RNA gene of Helicobacter pylori are highly predictive of in vitro clarithromycin resistance and failure of clarithromycin-containing treatment regimens.


OBJECTIVE
To design an assay to rapidly detect these mutations using rapid polymerase chain reaction and pyrosequencing, a novel method of 'sequencing by synthesis', and to test this assay with a collection of Canadian H pylori isolates.


METHODS
Forty-two H pylori isolates (24 clarithromycin-resistant, 18 clarithromycin-susceptible) were studied. A target region in the 23S gene was rapidly amplified and sequenced by pyrosequencing.


RESULTS
Mutations at one of the two positions studied were present in 20 of the 24 (83%) clarithromycin-resistant isolates; 13 had double- copy A2143G mutations, four had double-copy A2142G mutations and three had single-copy A2143G mutations. There were no mutations in 17 of the 18 (94%) susceptible isolates. A single-copy A2142G mutation was detected in one susceptible isolate.


CONCLUSIONS
The pyrosequencing assay developed was able to detect and differentiate mutations at positions 2142 and 2143 in either one or both copies of the H pylori 23S ribosdomal RNA gene. Further study is needed to determine whether this pyrosequencing assay can be used to determine H pylori susceptibility to clarithromycin from clinical specimens such as stools or gastric biopsies.
Slinger
R
R
Children's Hospital of Eastern Ontario, University of Ottawa, Ottawa, ON, Canada. slinger@cheo.on.ca
Yan
L
L
Chan
F
F
Forward
K
K
Cooper-Lesins
G
G
Best
L
L
Haldane
D
D
Veldhuyzen van Zanten
S
S
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002170:Canada; D017291:Clarithromycin / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D020459:Genes, rRNA / Q000235:genetics; D016481:Helicobacter Infections / Q000235:genetics / Q000382:microbiology; D016480:Helicobacter pylori / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012329:RNA, Bacterial / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D017291:Clarithromycin