Review of current diagnostic methods and advances in Helicobacter pylori diagnostics in the era of next generation sequencing

Table 5 A PubMed, MEDLINE and EMBASE search using the terms “Helicobacter pylori AND next generation sequencing” yielded 19 original research studies

Study	Main finding	Method	Sequencing	Ref.
1	Objective	DNA extraction from gastric biopsy specimens	Targeted 16S rRNA sequencing on an Ion S5XLplatform (Thermo Fisher Scientific, United States)	Han et al[117]
	Assessment of the correlation between the microbial gut community composition and the degree of inflammatory cell infiltration, endoscopic findings and the gastrointestinal disorders symptom severity index (PAGI-SYM)
	Main finding
	Histological and endoscopic gastritis was associated with the abundance of H. pylori and that of commensal bacteria in the stomach. The abundances of Variovorax paradoxus and Porphyromonas gingivalis were correlated with histological gastritis, but not with endoscopic or symptomatic gastritis. The total PAGI-SYM score showed a stronger correlation with the duodenal microbiota (Prevotella nanceiensis and Alloprevotella rava) than with the gastric microbiota (H. pylori, Neisseria elongate, and Corynebacterium segmentosum)
2	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina, United States)	Miftahussurur et al[128]
	Resistance to metronidazole rifaximin, rifabutin, furazolidone, garenoxacin and sitafloxacin was investigated in Indonesian H. pylori strains
	Main finding
	Furazolidone-, rifabutin-, and sitafloxacin-based therapies might be considered as alternative regimens to eradicate metronidazole and clarithromycin resistant H. pylori in Indonesian patients. Moreover, sitafloxacin but not garenoxacin should be considered for eradication of levofloxacin-resistant H. pylori strains
3	Objective	DNA extraction from gastric FFPE tissue blocks	16S rRNA targeted sequencing on an Ion Torrent (Thermo Fischer) platform	Nezami et al[132]
	Detection of H. pylori mutations that are known to confer resistance to clarithromycin, levofloxacin, and tetracycline directly from formalin-fixed paraffin-embedded (FFPE) gastric biopsy specimens using next generation sequencing
	Main finding
	Therapy failure correlated with the number of mutated genes: no failure in cases with no mutations (0/15), 19% (5/27) failure in cases with one gene mutation, and 69% (11/16) failure in cases with more than one mutated gene. Common 23S rRNA mutations (A2146G or A241G) were present in 88% (14/16) of failed cases as opposed to only 10% (4/42) of eradicated cases (P < 0.001). NGS can be used on clinical specimens collected during standard of care testing to detect mutations that correlate with increased risk of treatment failure
4	Objective	DNA extraction from gastric biopsy specimens	16S rRNA targeted sequencing on an Illumina HiSeq 2500 platform (Illumina; 2 × 250 bp)	Yu et al[118]
	Assessment of the changes in the microbial esophagal community composition in Chinese patients with reflux esophagitis and healthy volunteers using metagenomic high-throughput DNA sequencing
	Main finding
	Moderate changes in the microbial community composition were found in patients with reflux esophagitis and compared with the healthy volunteers. At the phylum level, only Bacteroidetes differed between the groups, being less abundant in the reflux esophagitis group. The overall number and diversity of species tended to be lower in reflux esophagitis patients, but there were no significant differences between the groups. Three genera, Prevotella, Helicobacter and Moraxella, were obviously depleted in reflux esophagitis patients
5	Objective	DNA extraction from gastric biopsy specimens	16S rRNA targeted sequencing on an Illumina MiSeq platform (Illumina)	Zhao et al[119]
	Characterization of H. pylori-induced alterations in the gastric and tongue coating microbiota and evaluation of potential impacts on human health in patients with chronic gastritis
	Main finding
	Significant alterations of the gastric microbiota were found in H. pylori-positive (cagA-positive) samples represented by a decrease in bacterial diversity, a reduced abundance of Roseburia and increased abundances of Helicobacter and Haemophilus. At the community level, functions involved in biofilm formation, mobile element content, and facultative anaerobiosis were significantly decreased in the microbial community in H. pylori-positive subjects. Presence of CagA was linked to an increased proportion of Gram-negative bacteria in the stomach, thereby contributing to an up regulation of lipopolysaccharide biosynthesis
6	Objective	DNA extraction from gastric biopsy specimens or surgical specimens of non-neoplastic gastric mucosa adjacent to the tumor	16S rRNA targeted sequencing on an Ion PGM Torrent platform (Thermo Fischer Scientific)	Ferreira et al[138]
	Characterization of the microbial community in patients suffering from gastritis and gastric cancer
	Main finding
	The gastric carcinoma microbiota was characterized by reduced microbial diversity, decreased abundance of H. pylori and the enrichment of other bacterial genera, mostly represented by intestinal commensals. The combination of these taxa into a microbial dysbiosis index revealed that dysbiosis can be used to discriminate between gastritis and gastric carcinoma. Analysis of the functional features of the microbiota was compatible with the presence of a nitrosating microbial community in carcinoma
7	Objective	DNA extraction from stool specimens	16S rRNA targeted sequencing on an Illumina MiSeq platform (Illumina)	Gotoda et al[142]
	Assessment of the changes in the gut microbiome after H. pylori eradication therapy in teenagers
	Main finding
	Alpha diversity revealed that both species richness and evenness were recovered to pre-treatment levels at 2 mo after H. pylori eradication therapy. Although H. pylori eradication therapy caused short-term dysbiosis, microbial diversity was restored in healthy teenagers
8	Objective	DNA extraction from stool specimens	16S rRNA targeted sequencing on an Illumina MiSeq platform (Illumina; 2 × 300bp)	Iino et al[120]
	Assessment of the association between H. pylori infection and the abundance of Lactobacillus species in the gut microbial community in Japanese patients
	Main finding
	The relative abundance of Lactobacillus in H. pylori-infected subjects with severe atrophic gastritis was higher compared with patients with mild atrophic gastritis and without atrophic gastritis (P < 0.001) and non-infected subjects (P < 0.001). The proportion of Lactobacillus salivarius was high in H. pylori-infected subjects while that of Lactobacillus acidophilus was high in non-infected subjects
9	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina)	Aftab et al[103]
	Determination of the sequences of virulence genes (cagA and vacA) and seven housekeeping genes by next generation sequencing
	Main finding
	All H. pylori strains were considered Western-type, and 73.2% of them carried cagA. Patients infected with cagA-positive strains had more severe histological scores than patients infected with cagA-negative strains. Thus, the low incidence of gastric cancer in Bangladesh might be attributable to the high proportion of less-virulent H. pylori genotypes
10	Objective	DNA extraction from gastric biopsy specimens	16S rRNA targeted sequencing on an Illumina MiSeq platform (Illumina; 1 × 500 bp)	Klymiuk et al[139]
	Assessment of the bacterial microbiome in a total of 30 homogenized and frozen gastric biopsy samples from eight geographic locations.
	Main finding
	H. pylori infection of the gastric habitat dominates the gastric microbiota in most patients and is associated with a significant decrease of the microbial alpha diversity. Moreover, some bacterial genera like Actinomyces, Granulicatella, Veillonella, Fusobacterium, Neisseria, Helicobacter, Streptococcus, and Prevotella were associated with the presence of H. pylori
11	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina)	Miftahussurur et al[124]
	Characterization of levofloxacin, metronidazole, clarithromycin, amoxicillin and tetracycline resistance in H. pylori isolated from 158 dyspeptic patients in Santo Domingo
	Main finding
	Clarithromycin and amoxicillin resistance were low (3.1% and 1.6%), and no resistance to tetracycline was found. In contrast, metronidazole and levofloxacin resistance were high (82.8% and 35.9%). Most levofloxacin-resistant H. pylori strains had an amino acid substitution at codon 87 or 91 in the gyrA gene. Many different rdxA and frxA mutations in metronidazole-resistant H. pylori strains were found without synergistic effects. Novel mutations in dppA, dppB, fdxA and fdxB, irrespective of rdxA and frxA mutations were associated with different levels of metronidazole resistance in H. pylori
12	Objective	DNA extraction from stool specimens	16S rRNA targeted sequencing on an Illumina MiSeq platform (Illumina; 2 × 300 bp)	Yanagi et al[121]
	Assessment of the influence of antimicrobials on both, the gut microbiota community composition and the plasma ghrelin level in H. pylori-infected patients, who underwent eradication therapy (amoxicillin, clarithromycin and proton-pump inhibitors)
	Main finding
	The Bacteroidetes:Firmicutes (B:F) ratio was significantly increased 3 months after than before antibiotic treatment (P < 0.01). A significant decrease in the concentration of active ghrelin (P < 0.01) in the plasma was observed before and 3 mo after antibiotic therapy
13	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina HiSeq 2000 and MiSeq platform (Illumina; 2 × 150 bp and 2 × 300 bp)	Hashinaga et al[100]
	Comparison of cagA and vacA sequences of H. pylori strains isolated from patients with gastric cancer and MALT lymphoma in Japan
	Main finding
	Conventional genotyping of cagA and vacA showed no significant difference between patients with gastric cancer and MALT lymphoma. When comparing full protein sequences of CagA and VacA, four novel loci were found on CagA, and three loci were detected on VacA. Significant differences were observed at one CagA locus between gastritis and MALT lymphoma H. pylori strains, and at one VacA locus between gastritis and gastric cancer H. pylori strains
14	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina)	Miftahussurur et al[126]
	Characterization of levofloxacin, metronidazole, clarithromycin, amoxicillin and tetracycline resistance in H. pylori isolates from Indonesia
	Main finding
	Clarithromycin, amoxicillin and tetracycline resistance were low (9.1%, 5.2% and 2.6%). In contrast, high resistance rates to metronidazole (46.7%) and levofloxacin (31.2%) were found. Metronidazole resistant H. pylori strains showed different rdxA amino acid substitutions, and the 23S rRNA A2147G mutation occurred in clarithromycin resistant H. pylori. However, one clarithromycin resistant H. pylori strain had a novel mutation in rpl22 without an A2147G mutation. Amino acid exchanges at N87 and/or D91 of gyrA were associated with levofloxacin-resistance
15	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina)	Miftahussurur et al[127]
	Characterization of H. pylori strains isolated from 146 patients in Kathmandu, Nepal
	Main finding
	H. pylori was not resistant to amoxicillin and tetracycline. In contrast, metronidazole resistance was extremely high (88.1%), while clarithromycin resistance was modestly high (21.4%). Most of metronidazole resistant H. pylori strains had diverse rdxA and frxA mutations. Novel mutations in dppA (A212, Q382 and I485) and dapF (L145, T168, E117, V121, R221) aside from missense mutations in rdxA were found in metronidazole resistant H. pylori strains. Amino acid exchanges at N87 and/or D91 in gyrA were predominantly found in levofloxacin-resistant H. pylori strains
16	Objective	A metronidazole-resistant strain was cultured from the metronidazole-susceptible H. pylori reference strain 26695 by exposure to low concentrations of metronidazole	Sequencing on an Illumina HiSeq 2000 platform (Illumina; 2 × 90 bp)	Binh et al[129]
	Characterization of wildtype and metronidazole resistant H. pylori reference strain 26695 in order to elucidate the molecular basis of metronidazole resistance and the involved genes in H. pylori
	Main finding
	Mutated sequences in rdxA were successfully transformed into the H. pylori reference strain 26695, and the transformants showed resistance to metronidazole. Transformed H. pylori isolates containing a single mutation in rdxA showed a low MIC (16 mg/L), while those containing mutations in both rdxA and frxA showed a higher MIC (48 mg/L). Moreover, mutations in rpsU may play a role in metronidazole resistance
17	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina platform (Illumina)	Furuta et al[104]
	H. pylori strains were isolated from the members of five families to investigate the microevolution and adaptation of the H. pylori genome using next generation sequencing and multi-locus sequence typing
	Main finding
	Detection of nucleotide substitutions revealed likely transmission pathways involving children. Nonsynonymous mutations were found in virulence-related genes (cag, vacA, hcpDX, tnfα, ggt, htrA and the collagenase gene), outer membrane protein (OMP) genes and other cell surface-related protein genes, signal transduction genes and restriction-modification genes
18	Objective	H. pylori strain UM032 was grown on non-selective agar medium	Sequencing on a RS instrument (Pacific Biosciences, United States; yielding > 300 × average genome coverage) and on an Illumina MiSeq platform (Illumina; 2 × 300 bp)	Lee et al[109]
	Elucidating the biological significance of the restriction-modification (R-M) system in the physiology and pathogenesis of H. pylori
	Main finding
	Strain UM032 contains a relatively large number of R-M systems, including some MTase activities with novel specificities. Specifically, 17 methylated sequence motifs corresponding to 1 Type I and 16 Type II R-M systems were found
19	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina HiSeq 2000 platform (Illumina; 2 × 90bp)	Miftahussurur et al[122]
	Assessment of the prevalence of H. pylori infection and evaluation of human migration patterns in the remote areas of North Sulawesi using next-generation sequencing and multi-locus sequence typing
	Main finding
	H. pylori prevalence was low (14.3% in adults and 3.8% in children). One H. pylori strain carried East Asian type cagA (ABD type), vacA s1c-m1b, iceA1, jhp0562-positive/β-(1,3), oipA “status-on”. Phylogenetic analyses showed that the strain belonged to hspMaori type, a major type observed in native Taiwanese and Maori tribes

Their objective, employed sequencing method and main finding is briefly described in the table. H. pylori: Helicobacter pylori.