Helicobacter pylori DNA isolation in the stool: an essential pre-requisite for bacterial noninvasive molecular analysis.
OBJECTIVE
Real-time polymerase chain reaction (RT-PCR) is a widely used technique for bacterial and viral infection diagnosis. Herein, we report our preliminary experience in retrieving H. pylori genetic sequences in stools and analyzing genotypic clarithromycin resistance by RT-PCR (noninvasive), with the aim of comparing this procedure with that performed on biopsy samples (invasive).


METHODS
After 'in vitro' demonstration of H. pylori DNA detection from pure and stool-mixed bacteria, 52 consecutive patients at the first diagnosis of infection were investigated. DNA was extracted from biopsy tissue and stool samples (THD®Fecal Test, Italy). RT-PCR was performed to detect 23S rRNA encoding bacterial subunit gene and search A2143G, A2142C, A2142G point mutations for clarithromycin resistance assessment.


RESULTS
RT-PCR showed H. pylori positive DNA in all infected patients with full concordance between tissue and stool detection (100%). We found A2143G mutation in 10 (19.2%), A2142G in 4 (7.7%) and A2142C in 5 (9.6%) patients; there was a full agreement between biopsy and fecal samples. A2143G was found in all the four A2142G positive cases and in three out of the five A2142C positive strains. Overall clarithromycin resistance rate in our series was 23%.


CONCLUSIONS
Despite the need of confirmation on large sample, stool RT-PCR analysis could represent a feasible tool to detect H. pylori DNA sequences and antibiotic resistance point mutations. As compared to tissue molecular analysis, this technique is noninvasive, with potential advantages such as improvement of patient compliance, reduction of diagnostic procedure time/cost and improvement of therapeutic outcome.
a Section of Gastroenterology, Department of Emergency and Organ Transplantation , University of Bari , Bari , Italy.
Floriana
F
Giorgio
a Section of Gastroenterology, Department of Emergency and Organ Transplantation , University of Bari , Bari , Italy.
Enzo
E
Ierardi
a Section of Gastroenterology, Department of Emergency and Organ Transplantation , University of Bari , Bari , Italy.
Claudia
C
Sorrentino
a Section of Gastroenterology, Department of Emergency and Organ Transplantation , University of Bari , Bari , Italy.
Mariabeatrice
M
Principi
a Section of Gastroenterology, Department of Emergency and Organ Transplantation , University of Bari , Bari , Italy.
Michele
M
Barone
a Section of Gastroenterology, Department of Emergency and Organ Transplantation , University of Bari , Bari , Italy.
Giuseppe
G
Losurdo
a Section of Gastroenterology, Department of Emergency and Organ Transplantation , University of Bari , Bari , Italy.
Andrea
A
Iannone
a Section of Gastroenterology, Department of Emergency and Organ Transplantation , University of Bari , Bari , Italy.
Antonio
A
Giangaspero
b Section of Microbiology, Department of Basic Medical Science, Neuroscience and Sense Organs , University of Bari , Bari , Italy.
Rosa
R
Monno
a Section of Gastroenterology, Department of Emergency and Organ Transplantation , University of Bari , Bari , Italy.
Alfredo
A
Di Leo
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000900:Anti-Bacterial Agents / Q000627:therapeutic use*; D001706:Biopsy; D017291:Clarithromycin / Q000627:therapeutic use*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005243:Feces / Q000382:microbiology; D005260:Female; D016481:Helicobacter Infections / Q000175:diagnosis* / Q000188:drug therapy; D016480:Helicobacter pylori; D006801:Humans; D007558:Italy; D008297:Male; D008826:Microbial Sensitivity Tests; D008875:Middle Aged; D017354:Point Mutation; D060888:Real-Time Polymerase Chain Reaction; D055815:Young Adult
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D017291:Clarithromycin
Helicobacter pylori; THD® Fecal Test; antibiotic resistance; noninvasive method; real-time polymerase chain reaction; stool