What's new in helicobacter pylori testing?
Expires: March 22, 2024
Robin Patel, M.D.
Vice Chair of Education
Elizabeth P. and Robert E. Allen
Professor of Individualized Medicine
Professor of Medicine and Microbiology
Division of Clinical Microbiology
Mayo Clinic, Rochester, Minnesota
Hi, this presentation is entitled “What's new with Helicobacter pylori testing.” My name is Robin Patel, and I'm the vice chair of Education in the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, Minnesota, where I'm also the Elizabeth P. and Robert E. Allen Professor of Individualized Medicine, Professor of Medicine, and Professor of Microbiology.
My disclosures are shown here.
This bacterium is a slender, curved gram-negative bacillus that is strongly urease positive. It lives in the mucosa of the stomach and it's challenging to grow in culture. It grows under microaerophilic conditions and it grows pretty slowly. The World Health Organization (WHO) has classified Helicobacter pylori as a Group 1 carcinogen as a result of its association with gastric carcinoma. The clinical presentation of infection with Helicobacter pylori can be varied from asymptomatic to peptic ulcer disease, either gastric or duodenal to nonulcer dyspepsia, to gastric carcinoma and gastric mucosa-associated lymphoid tissue lymphoma or MALT lymphoma, MALToma, or marginal zone B-cell lymphoma of MALT type.
Testing for Helicobacter pylori is indicated in those with active peptic ulcer disease or a history of peptic ulcer disease (unless eradication of Helicobacter pylori has been confirmed), low-grade gastric mucosa-associated lymphoid tissue lymphoma, or a history of endoscopic resection of early gastric cancer, uninvestigated dyspepsia, long-term NSAID or aspirin use, unexplained iron-deficiency anemia after evaluation for other causes, and immune thrombocytopenia in adults.
Treatment for Helicobacter pylori has become more complex in the era of antimicrobial resistance. Typically, Helicobacter pylori is treated with combinations of two to three antibiotics along with a proton pump inhibitor (PPI). It's been said that the initial course of eradication therapy generally offers the greatest likelihood of treatment success. Therefore, careful attention must be paid to selection of the most appropriate first-line eradication therapy for an individual patient. The main determinants of successful Helicobacter pylori eradication are the choice of regimen, the patient's adherence to the regimen, and the susceptibility of the Helicobacter pylori isolates to the combination of antibiotics administered.
We took a look at susceptibility of Helicobacter pylori to antibiotics several years ago, in this publication from Antimicrobial Agents and Chemotherapy from four years ago. We looked at 413 Helicobacter pylori isolates that were tested at Mayo Clinic Laboratories. We recovered these in culture at Mayo Clinic Laboratories and performed susceptibility testing. What we found was that 70% of the isolates were resistant to clarithromycin. Now this is an extremely high rate, but of course, this is a very biased sample collection. And I'm here today to share with you some new data about clarithromycin resistance.
In that publication back in 2017, we also look at molecular mechanisms of resistance to clarithromycin. We took 111 Helicobacter pylori isolates. We performed susceptibility testing to clarithromycin phenotypically. But we also sequenced the 23S ribosomal RNA gene to look for clarithromycin resistance associated mutations. What we found is that there were 3 mutations in the 23S ribosomal RNA gene, A213G, A2142G, and A2142C, that were associated with clarithromycin resistance. The presence of any one of these three SNPs or mutations predicted resistance or susceptibility to clarithromycin in these isolates in 95% of cases, which is really remarkable genotypic prediction of phenotypic susceptibility.
The diagnosis of Helicobacter pylori is based on either non-invasive tests or invasive tests. Non-invasive tests include the one that I'm going to talk about today, stool PCR. And the PCR assay I will talk about not only detects Helicobacter pylori, but also predicts clarithromycin susceptibility or resistance based on those three mutations that I just covered, stool antigen testing, and the Helicobacter pylori urea breath test. Invasive tests require a biopsy of the stomach or duodenum with a rapid urease test, stain of the tissue, or ideally to recover the organism and perform susceptibility testing, and culture. This provides an isolate on which we can perform amoxicillin, ciprofloxacin, clarithromycin, metronidazole, and tetracycline susceptibility testing.
This new test is Helicobacter pylori with Clarithromycin Resistance Prediction, Molecular Detection, PCR, Feces. We did two studies to look at the performance of this assay. First, we took 535 fecal samples that were submitted to Mayo Clinic Laboratories for Helicobacter pylori stool antigen testing. We analyzed for 524 that did not have inhibition, and we found that compared to antigen testing, the PCR assay had a sensitivity of 89% and a specificity of 97%, and positive and negative predictive values of 89% and 97% respectively.
We also looked at molecular prediction of clarithromycin susceptibility. We took 113 PCR positive samples. 68% of them had wild-type 23S ribosomal RNA gene sequences at the points that I talked about predict clarithromycin resistance. And 32% had one of the three clarithromycin resistance associated SNPs. This was confirmed by Sanger sequencing of the amplified PCR products with 100% agreement with what the assay predicted. So, this might suggest that 32% of stool that harbors Helicobacter pylori harbors clarithromycin resistance. But I'm going to show you some more detailed and nuanced data about that point.
We did a second study, also reported in the same publication, where we looked at 223 fecal samples from Mayo Clinic patients. These were recovered between 2015 and 2018, and all of these patients were stool antigen positive. It involved 197 unique Mayo Clinic patients. We also reviewed the electronic medical records of these patients looking for demographic information, information about prior macrolide use, and longitudinal data pertaining to diagnosis, treatment, follow-up testing, and treatment outcome. We documented treatment success or failure when the test was performed as a test of eradication. And we defined failure as a positive result and success as a negative result. We split this patient population into two. In one group, the testing was used for initial diagnosis. Mostly this was treatment naive patients being tested for diagnosis of Helicobacter pylori, but there were a small number that had had Helicobacter pylori infections in the past that were successfully treated and now were presenting with a new episode. The second group was the test of eradication group. And those were patients being tested for follow-up proof of eradication by antigen test after receiving antibiotic therapy for an initial diagnosis of Helicobacter pylori.
The patients ranged in age from 3 to 91 years with most being 18 years of age. 139 were female and 84 male. The most commonly prescribed regimen at Mayo Clinic was clarithromycin triple therapy. This was prescribed in two-thirds of cases, followed by other regiments including bismuth, quadruple therapy, and other combinations.
So, here's what we found overall. Again, we looked at 223 total cases and we split them into an initial diagnosis group and a test of eradication group that contained 169 and 54 patients respectively. First of all, we found that the vast majority of those patients, all of whom recall tested positive with the antigen test, tested positive by PCR, 93% of them. Also, and very importantly, in the test eradication group 53 and 54, who tested positive with the antigen assay, tested positive with PCR as well, for sensitivity of 98% as a test of eradication. And this is very important because it's the first demonstration that PCR can be used as a test of eradication. We also looked at clarithromycin resistance-associated SNPs. In the larger group of 223 cases, we found 207 were positive by PCR. And of those 207, 81 or 39% harbored SNPs associated with clarithromycin resistance. When we focus just on the initial diagnosis group, there were 154 PCR positive specimens and 47 harbored clarithromycin resistance associated SNPs, for a 31% rate of predicted clarithromycin resistance at initial diagnosis in this Mayo Clinic population. I think this is very important because it gives us an estimate of what the rate of clarithromycin resistance is in patients being tested in the first place for Helicobacter pylori at Mayo Clinic.
We also looked at initial diagnosis test group treatment outcomes and Helicobacter pylori PCR results. So here we looked at 92 cases in whom clarithromycin resistance was assayed by PCR. And we split these into those treated with clarithromycin based triple therapy and those treated with other therapy regiments. There were 69 who were treated with clarithromycin-based triple therapy regiments. When we looked at the cases overall, there were 92 cases, and treatment was successful in 63 and failed in 29 of these cases. When we detected clarithromycin resistance using the PCR assay, the success rate was 13 out of 27 or 48% with this predicted resistance. When resistance was not predicted, the success rate was 43 out of 58 or 74%, difference that was statistically significantly different. This is very important because it suggests that when you detect clarithromycin resistance, that predicts treatment failure. But what was more interesting to look at was the specific group that received clarithromycin-based triple therapy. In that group, shown in the middle here, the success rate was 9 out of 22, or 41%, when resistance was predicted by PCR, versus 30 out of 43, or 70%, when no resistance was predicted. A statistically significant difference with a p-value of 0.03.
We also looked at the test of eradication group. So here we have 54 total cases of which 35 had been previously treated with the clarithromycin-based triple therapy regimen. Of the total cases, 54 PCR predicted clarithromycin resistance in 64% of these cases, suggesting a very high rate of clarithromycin resistance in the treatment failure cases. In those who specifically received clarithromycin-based triple therapy, PCR detected resistance in 69% of these cases.
We've worked with our gastroenterologists, infectious diseases specialists, and microbiologists to come up with a diagnostic algorithm for Helicobacter pylori. This is a complicated algorithm, especially given the problems that I've highlighted with antimicrobial resistance. But this algorithm incorporates the use of this new assay Helicobacter pylori Detection with Clarithromycin Resistance Prediction directly from stool to provide a new option, to very early on, not just recognize that a patient is infected with this organism but provide some guidance on how to treat or not treat that patient based on prediction of clarithromycin resistance or susceptibility. We also showed as demonstrated, that this test can be used as a test of eradication with equivalent performance to antigen tests.
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