Advantage of 16S rRNA amplicon sequencing in Helicobacter pylori diagnosis

Abstract

Background 16S rRNA amplicon sequencing is an accurate method of detecting microbial infection without culture. It is unclear if sequencing has additional benefits over routine diagnostic methods for Helicobacter pylori testing. Methods We enrolled Mongolian volunteers with dyspepsia. Using routine diagnostic methods, positive H. pylori was defined as positive results on histology/immunohistochemistry, culture, rapid urease test, or serology; negative H. pylori was defined by negative results from all these tests. We performed 16S rRNA sequencing on gastric biopsy specimens and calculated cutoffs for operational taxonomic units (OTUs) and relative abundance (RA) to define positive results using ROC curves. Results We examined 161 individuals with a mean age of 43.6 years, and 64.6% were women. Using routine diagnostic methods, 122 (75.8%) participants were H. pylori positive, the sensitivity and specificity for 16S rRNA sequencing were 94.3% and 82.1% or 93.4% and 82.1% when cutoff values were set to 1113 (OTU number) or 4.4% RA, respectively (both p < .001). When combining the validated values, the concordance rate was high (91.1%); however, 16S rRNA sequencing had additional positive yield in 9 cases (5.6%) compared with routine diagnostic methods, and much greater additional positive yield compared to histopathology/IHC, culture, RUT, serology separately with 12 (7.4%), 37 (23.0%) and 43 (26.7%). Conclusion 16S rRNA amplicon sequencing detects potentially important proportion of H. pylori‐positive cases that test negative with routine diagnostic methods. The quantitative number of H. pylori can help to understand how it can be changing by diseases and RA give opportunity to understand how H. pylori communicate with other microbiota.

Application of molecular techniques inHelicobacter pyloridetection: limitations and improvements

“…19 This limitation also applies to 16S rRNA sequencing: single gene sequencing with limited depth is difficult to resolve taxonomic diversity at species level, that is, separating H. pylori from other Helicobacter species or even Campylobacter species. 23 Another NGS method, metagenomic sequencing covering whole genome, may solve this problem with deeper sequencing if we can lower the cost, improve the efficiency and reduce human DNA background in gastric biopsies. 22 One concern of 16S rRNA sequencing, raised by the study of Gantuya et al, 23 is that because the samples with low-intensity infection are negative by conventional tests, it is difficult to create a "real" control group of subjects who are unequivocally negative for H. pylori due to the limited sensitivity of current detection methods.…”

“…25,26 With the increased sensitivity, molecular methods can detect cases of low-density infection, which are usually false-negative with conventional tests. 23 The first identification of "occult" infection was reported in 2006 by Bik et al, detecting H. pylori in conventionally undiagnosed biopsies by using 16S rRNA sequencing. 27 Real-time PCR, dd-PCR, and NGS were also reported to identify low-grade H. pylori infection in a subgroup of patients diagnosed as negative by conventional tests.…”

“…27 Real-time PCR, dd-PCR, and NGS were also reported to identify low-grade H. pylori infection in a subgroup of patients diagnosed as negative by conventional tests. 18,19,23 Detecting low bacterial load is difficult to achieve but important for us to ensure successful management while avoiding unnecessary treatments. It is meaningful as low-density infection is harmful in some clinical settings, and it may be linked to antibiotic-resistant or more virulent H. pylori strains with certain vacA-cagA genotypes in gastric biopsies.…”

“…42 Similar to dd-PCR, 16S rRNA sequencing measures the quantitative number of H. pylori as additional information that shows bacterial load variation during disease development. 23 With quantitative microbiome data generated by NGS, we are able to check microbiota shifts (dysbiosis and restoration) in H. pylorinegative pre-infection, 43 active infection, 44 and successful bacterial eradication. 45 Sometimes long-term microbiome monitoring is needed in the cases with persistent dysbiosis after eradication.…”

40 Years of Helicobacter pylori: A Revolution in Biomedical Thought

“…[54] However, among the virulence agents of H. pylori is the antigenic protein cytotoxin-associated protein A (CagA, molecular weight 125-140 kDa). CagA is an immunomodulatory extracellular protein of some strains of H. pylori (Figure 6) [55,56]. After 1997, the cag region of microbial DNA was identified, which was designated as cag pathogenicity island-cag-PAI (dimension~40 kbp, composed by~30 genes).…”