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

Table 6 A PubMed, MEDLINE and EMBASE search using the terms “Helicobacter pylori AND whole genome sequencing” yielded 15 original research studies

Study	Main finding	Method	Sequencing	Ref.
1	Objective	DNA extraction from biopsy specimens	Sequencing on an Illumina HiSeq 2500 platform (Illumina, United States; 2 × 50 bp)	Ailloud et al[123]
	Investigation of H. pylori evolution during infection and population dynamics inside the gastric environment
	Main finding
	Phylogenetic analyses suggested location-specific evolution and bacterial migration between gastric regions. Migration was significantly more frequent between the corpus and the fundus than with the antrum, suggesting that physiological differences between antral and oxyntic mucosa contribute to spatial partitioning of H. pylori populations. Associations between H. pylori gene polymorphisms and stomach niches suggested that chemotaxis, regulatory functions and outer membrane proteins contribute to the specific adaptation to the antral and oxyntic mucosa
2	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 150 bp)	Lauener et al[125]
	Single nucleotide polymorphisms (SNPs) were detected in H. pylori isolates by whole genome sequencing and their correlation with phenotypic resistance to clarithromycin, metronidazole, tetracycline, levofloxacin and rifampicin was assessed
	Main finding
	Overall, there was high congruence of > 99% between phenotypic drug susceptibility testing results for clarithromycin, levofloxacin, and rifampicin and SNPs identified in the 23S rRNA, gyrA and rpoB genes. However, it was not possible to infer a resistance phenotype for metronidazole based on the occurrence of distinct SNPs in rdxA and/or frxA
3	Objective	H. pylori strains grown on solid non-selective agar media	Sequencing on an Illumina HiSeq platform (Illumina; 2 × 150 bp)	Chen et al[133]
	Characterization of polymorphisms in Clarithromycin resistant and susceptible H. pylori strains using whole genome sequencing
	Main finding
	No mutations known to be associated with clarithromycin resistance, except for the controversial T2182C mutation, were detected. Single nucleotide variants (SNVs) in multidrug efflux transporter genes and HP0605 were significantly different between clarithromycin resistant and susceptible H. pylori strains. No significant difference in SNVs of membrane proteins of the RND family or MFS (HP1181) family were found
4	Objective	H. pylori strains isolated from patients with abdominal pain, gastritis, gastric or duodenal ulcers	Sequencing on an Illumina MiSeq platform (Illumina)	Quintana-Hayashi et al[151]
	Characterization of the binding ability, adhesion modes, and growth of H. pylori strains isolated from pediatric patients with abdominal pain, gastritis, gastric or duodenal ulcers
	Main finding
	Increased adhesion capacity of pediatric peptic ulcer disease (PUD) H. pylori strains to human gastric mucins compared to the non-ulcer dyspepsia (NUD) strains both at neutral and acidic pH, regardless if the mucins were positive for Lewis b (Leb), Sialyl-Lewis × (SLex) or LacdiNAc. In addition to babA positive strains being more common among PUD associated strains, H. pylori babA positive strains bound more avidly to gastric mucins than NUD babA positive strains at acidic pH. Binding to Leb was higher among babA positive PUD H. pylori strains compared to NUD strains at neutral, but not acidic, pH. PUD derived babA-knockout mutants had attenuated binding to mucins and Leb at acidic and neutral pH, and to SLex and DNA at acidic pH
5	Objective	H. pylori strain B128 isolated from a gastric biopsy of a patient with gastric ulceration was challenged with low/high salt concentrations	Sequencing on an Illumina MiSeq platform (Illumina)	Noto et al[99]
	H. pylori was continuously cultured in vitro under low iron or high salt conditions to characterize fur genetic variation. Moreover, fur sequence variation was assessed in 339 clinical H. pylori strains
	Main finding
	Exposure to low iron or high salt selected for a specific single nucleotide polymorphism in the fur gene (FurR88H) in H. pylori. Among the isolates examined, 17% of H. pylori strains isolated from patients with premalignant lesions harbored the FurR88H variant, compared to only 6% of H. pylori strains from patients with non-atrophic gastritis. These results indicate that specific genetic variation arises within H. pylori strains during in vivo adaptation to conditions conducive for gastric carcinogenesis
6	Objective	H. pylori strains were cultured on solid non-selective agar media	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 300 bp)	Zhang et al[152]
	Comparison of homogenization vs enzymatic digestion protocols for DNA extraction from gastric, esophageal and colorectal biopsies and survey of the microbial content and composition using whole genome sequencing
	Main finding
	Neither method demonstrated preferential extraction of any particular clade of bacteria, nor significantly altered the detection of Gram-positive or Gram-negative organisms. However, although the overall microbial community composition remained very similar and the most prevalent bacteria could be detected effectively using either method, the precise community structure and microbial abundances between the two methods were different. The homogenization extraction method provided higher microbial DNA content and higher read counts from human tissue biopsy samples of the gastrointestinal tract
7	Objective	H. pylori strains isolated from gastric biopsies of patients suffering from dyspepsia	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 300 bp)	Kumar et al[153]
	Whole genome sequencing and comparative analysis of three H. pylori strains isolated from three Arab patients
	Main finding
	The three genomes clustered along with HpEurope strains in the phylogenetic tree comprising various H. pylori lineages. The three genomes possessed a complete cag-pathogenicity island with an AB-C type EPIYA motif
8	Objective	H. pylori strains were isolated from gastric biopsy specimens of patients with chronic gastritis, gastric ulcer, duodenal ulcer and gastric cancer	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 300 bp)	Ogawa et al[101]
	Characterization of cagL and cagI in H. pylori isolated from patients in Southeast Asia with chronic gastritis, gastric ulcer, duodenal ulcer and gastric cancer
	Main finding
	CagL motifs were highly conserved among the H. pylori isolates. CagL E59 and I234 in the C-terminal motif were more common in H. pylori isolates from gastric cancer patients. The CagI C-terminal motif was completely conserved across all H. pylori isolates
9	Objective	H. pylori strains were isolated from gastric biopsy specimens of patients with non-ulcer dyspepsia, gastric ulcer and duodenal ulcer	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 150 bp)	Silva et al[102]
	Characterization of the expression of virB genes, encoding parts of the type-IV secretion system (T4SS)/Cag-pathogenicity island, in H. pylori strains isolated from Western patients with different gastrointestinal malignancies
	Main finding
	The region spanning from virB2 to virB10 constitutes an operon, whose expression is increased in the adherent fraction of bacteria during infection, as well as in both adherent and nonadherent fractions at acidic conditions.
10	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina HiScanSQ platform (Illumina; 2 × 100 bp)	Thorell et al[154]
	Characterization of H. pylori strains isolated in Nicaragua
	Main finding
	The Nicaraguan isolates showed a phylogenetic relationship with West African H. pylori isolates in whole genome sequence comparison and with Western and urban South- and Central-American isolates using multi-locus sequence analysis. A majority (77%) of the isolates carried the cancer-associated virulence gene cagA and also the s1/i1/m1 allele of the vacuolating cytotoxin gene that is linked to increased severity of disease. Moreover, it was found that Nicaraguan isolates have a blood group-binding adhesin (babA) variant highly similar to previously reported babA sequences from Latin America H. pylori isolates
11	Objective	H. pylori reference strain 26695, cagA and Cag-pathogenicity island deletion mutants were cultured	Sequencing on an Illumina MiSeq platform (Illumina)	Wong et al[105]
	Characterization of genes associated with biofilm formation in H. pylori
	Main finding
	Genes identified to be associated with biofilm formation in H. pylori included alpha (1,3)-fucosyltransferase, flagellar protein, 3 hypothetical proteins, outer membrane proteins and a Cag-pathogenicity island protein (CagPAI). These genes play a role in bacterial motility, lipopolysaccharide synthesis, Lewis antigen synthesis, adhesion and/or the type-IV secretion system (T4SS). Deletion of cagA and CagPAI confirmed that CagA and T4SS were involved in H. pylori biofilm formation
12	Objective	H. pylori was isolated from a gastroscopic antral biopsy specimen of a patient with chronic gastritis	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 150 bp)	Cao et al[155]
	H. pylori was isolated from a gastroscopic antral biopsy specimen of a 53-year-old male patient with chronic gastritis. Whole genome sequencing was applied to these isolates, and bioinformatic tools were used to investigate the within host evolution of H. pylori isolates
	Main finding
	The H. pylori genomes fall into two clades, reflecting colonization of the stomach by two distinct strains. The lineages have accumulated diversity during an estimated 2.8 and 4.2 yr of evolution. Around 150 clear recombination events between the two clades were found. Imputed ancestral sequences also showed evidence of recombination between the two strains prior to their diversification, and it was estimated that they have both been infecting the same host for at least 12 yr
13	Objective	Nineteen H. pylori clinical isolates were isolated from gastric epithelium biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 300 bp)	Iwamoto et al[131]
	Whole genome sequencing of 12 clarithromycin resistant and 7 susceptible H. pylori strains was done to identify novel genetic factors that reduce susceptibility to clarithromycin
	Main finding
	In clarithromycin resistant H. pylori strains specific point mutations in the 23S rRNA gene were found. In addition, genetic variants of four gene clusters (hp0605-hp0607, hp0971-hp0969, hp1327-hp1329, and hp1489-hp1487) of efflux pumps homologues, which have been previously implicated in multi-drug resistance, were found
14	Objective	H. pylori reference strain 26695 was used as amoxicillin-sensitive reference strain and as parental strain to create in vitro resistant H. pylori isolates	Sequencing on an Illumina Genome Analyzer (Illumina)	Qureshi et al[130]
	Investigation of the occurrence of genetic mutations that contribute to amoxicillin resistance in H. pylori when exposed to increasing concentrations of amoxicillin in vitro
	Main finding
	Using a whole genome sequencing approach, mutations in a number of genes were identified, notably pbp1, pbp2, hefC, hopC and hofH. Mutations in pbp1, hefC, hopC, hofH, and possibly pbp2, contributed to H. pylori high-level amoxicillin resistance
15	Objective	H. pylori reference strain 26695 and H. pylori strain J99 were grown on solid non-selective agar media	Sequencing on a PGM (Ion Torrent, Thermo Fischer Scientific, United States) and an Illumina MiSeq platform (Illumina)	Perkins et al[156]
	Next generation sequencing of the H. pylori reference strains J99 and 26695 and bioinformatic analysis of the sequencing data using publicly available algorithms. The accuracy of the coding sequence assemblies was compared to the originally published sequences
	Main finding
	With the Ion Torrent PGM, an inherently high-error rate in the raw sequencing data was found. With the Illumina MiSeq, significantly more non-covered nucleotides were found when using Illumina Nextera XT compared to the Illumina Nextera library preparation method. The most accurate de novo assemblies were found when using the Nextera technology. However, extracting an accurate multi-locus sequence type was inconsistent compared to the Ion Torrent PGM. The Cag-pathogenicity island failed to assemble onto a single contig in all technologies but was more accurate using the Nextera technology. The Illumina MiSeq Nextera method was found as the most accurate method for whole genome sequencing of H. pylori and de novo assembly of its genome

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