Daniel Pohl, Peter M Keller, Valentine Bordier, Karoline Wagner

Abstract Helicobacter pylori (H. pylori) infection is highly prevalent in the human population and may lead to severe gastrointestinal pathology including gastric and duodenal ulcers, mucosa associated tissue lymphoma and gastric adenocarcinoma. In recent years, an alarming increase in antimicrobial resistance and subsequently failing empiric H. pylori eradication therapies have been noted worldwide, also in many European countries. Therefore, rapid and accurate determination of H. pylori's antibiotic susceptibility prior to the administration of eradication regimens becomes ever more important. Traditionally, detection of H.pylori and its antimicrobial resistance is done by culture and phenotypic drug susceptibility testing that are cumbersome with a long turn-around-time. Recent advances in diagnostics provide new tools, like real-time polymerase chain reaction (PCR) and line probe assays, to diagnose H. pylori infection and antimicrobial resistance to certain antibiotics, directly from clinical specimens.Moreover, high-throughput whole genome sequencing technologies allow the rapid analysis of the pathogen's genome, thereby allowing identification of resistance mutations and associated antibiotic resistance. In the first part of this review, we will give an overview on currently available diagnostic methods for detection of H. pylori and its drug resistance and their implementation in H. pylori management. The second part of the review focusses on the use of next generation sequencing technology in H. pylori research. To this end, we conducted a literature search for original research articles in English using the terms “Helicobacter”, “transcriptomic”, “transcriptome”, “next generation sequencing” and “whole genome sequencing”. This review is aimed to bridge the gap between current diagnostic practice (histology, rapid urease test, H. pylori culture, PCR and line probe assays) and new sequencing technologies and their potential implementation in diagnostic laboratory settings in order to complement the currently recommended H. pylori management guidelines and subsequently improve public health.

Key words:Helicobacter pylori; Advances in diagnostics; Next generation sequencing;Whole genome sequencing; Clinical management

HELICOBACTER PYLORI PREVALENCE, EPIDEMIOLOGY AND ANTIBIOTIC RESISTANCE

Current Helicobacter pylori prevalence and epidemiology

Initial acquisition of Helicobacter pylori (H. pylori) occurs primarily during childhood and may persist throughout life[1]. Infection with H. pylori occurs worldwide, but there are substantial geographic differences in the prevalence of infection between countries[2]. Multiple studies have demonstrated that socioeconomic status and ethnic origin of the population are strongly associated with prevalence of H. pylori infection[3-5]. In Central and Northern Europe, H. pylori prevalence, excluding non-European immigrants, was found to be around 24% to 32%[6-10]. Studies conducted in Switzerland revealed a H. pylori prevalence of 12%-20% in patients born in Switzerland and a prevalence of 27% in immigrants[5,11]. H. pylori can be divided into relatively distinct populations that are specific for large geographical areas:HpEurope, hpSahul, hpEastAsia, hpAsia2, hpNEAfrica, hpAfrica1 and hpAfrica2[12-14].The most prevalent H. pylori populations in Europe are hpEurope and hpNEAfrica[15].

Management of Helicobacter pylori infections

In most patients, H. pylori infection stays asymptomatic, but it can progress to various gastrointestinal diseases including chronic active gastritis, peptic or duodenal ulcers,gastric adenocarcinoma and mucosa associated tissue lymphoma[16]. Consequently, it is a challenge for physicians to decide who should be tested for H. pylori infection and who should be treated. In general, treatment is recommended in case of detection of H. pylori infection, even in patients with asymptomatic H. pylori gastritis[17,18]. This practice is supported by results from a systematic review of six randomized trials evaluating H. pylori eradication therapy to prevent gastric cancer in healthy asymptomatic individuals that found a significant reduction in the incidence of gastric cancer[19]. However, this conclusion is mostly based on results of one interventional,placebo-controlled trial that was conducted in China[20], a high incidence country for gastric cancer. Therefore, further studies are needed in countries with low prevalence of gastric cancer to evaluate the long-term cost-effectiveness of such interventions. In the United Kingdom, two placebo-controlled trials conducted a H. pylori screening and treatment program in the general population that reduced dyspepsia in patients,who receive eradication therapy[21,22]. Though, they concluded that targeted eradication strategies in dyspeptic patients may be preferable. Therefore, the main question in clinical practice remains: Who should be tested and consequently treated?Based on recent research, current guidelines (i.e., fifth Maastricht/Florence consensus report[23]) recommend testing for H. pylori infection in situations described in Table 1.

Adult patients in industrial countries that have been successfully treated for H.pylori infection rarely show reinfection (reinfection rate of 2%)[24]. Therefore,adequate treatment promises high eradication efficacy (see next chapter for antimicrobial therapy options) without recurrence of H. pylori infection. However,there are major challenges in the treatment of H. pylori infection including increasing resistance to antibiotics, which will be discussed in detail in the next section, and compliance to therapy. A study performed in Switzerland showed that approximately 89% of the patients treated were considered as “good compliers”, meaning that they consumed more than 85% of the prescribed doses[25]. In this study, H. pylori eradication was inversely associated with poor compliance (P = 0.029) and the major reason mentioned by the patients not complying with the treatment was side effects.Antibiotic therapy indeed has non-negligible, short-term side effects such as diarrhea,nausea, vomiting, bloating and/or abdominal pain. Moreover, it has been shown, and also received media attention, that antibiotic treatment can alter the gut microbiota richness and diversity[26,27], possibly deferring health-conscious patients from following through with antibiotic treatment.

Antibiotic resistance in Helicobacter pylori

The only currently available efficient treatment against H. pylori infection includes the use of antibiotics. Main mechanisms of antibiotic resistance development in H. pylori include mutations that impair the capability of antibiotics to bind the ribosomes and interfere with protein synthesis; mutations that affect DNA replication and transcription; mutations that modify penicillin binding proteins, involved in peptidoglycan biosynthesis[28]. As H. pylori easily develops drug resistance to single antibiotics, combination therapy of several antibiotics is recommended. Combination of antibiotics used in therapy should depend on local drug resistance rates estimated in the respective country. Primary and acquired resistance to clarithromycin and metronidazole has increased globally in the last years, diminishing the effectiveness of conventional first-line treatment regimens and increasing treatment failures due to drug-resistant H. pylori strains[29-33]. In particular, clarithromycin resistance increased rapidly in several countries, to reach 30% in Japan and up to 50% in China[32]. Also in Europe, an increasing trend of clarithromycin resistance in H. pylori can be observed with an overall primary clarithromycin resistance rate of 17%[34]. However, prevalence of clarithromycin resistance varies from 21% in Austria to 6% in Finland and the Netherlands[10,35]. This shows that clarithromycin resistance is strongly variable between neighboring European countries emphasizing the need to examine drug resistance separately in each geographic region to better guide empiric treatment regimens. Metronidazole resistance, although not as important as clarithromycin resistance, also significantly reduces treatment efficiency of the standard triple regimen[36]. Overall, metronidazole resistance rates have been increasing in many European countries[34,37-40], ranging from 14% to 33% in Europe[41-46]. The general trend towards increasing resistance to first-line antibiotics in H. pylori has urged treating physicians to prescribe alternative treatment regimens including tetracycline with a PPI and a bismuth salt or the use of levofloxacin or rifabutin-based treatment regimens[23,47,48]. However, these regimens require high patient compliance as antibiotic therapy consist of many tablets that have to be taken daily for 10 to 14 d[49]. Incomplete patient adherence to antibiotic therapy is directly associated with further resistance development in H. pylori. Although levofloxacin resistance has not been studied as extensively as clarithromycin and metronidazole resistance, there is also a trend towards high primary and secondary levofloxacin resistance in H. pylori[35,38-40,50]. In contrast, resistance to amoxicillin and tetracycline seems to be negligible in H. pylori (0 to 2%) in European countries[35,40,51-53]. Though tetracycline resistance does not seem alarming yet in Western Europe, high resistance rates ranging from 5% to 19% were found in Eastern European and Asian countries[54,55], emphasizing the need to prevent further resistance spread in H. pylori. Therefore, similar as for first-line H. pylori eradication regimens, administration of alternative antimicrobial therapy, especially when levofloxacin based, should be guided by the regional and patient-specific antimicrobial resistance patterns and knowledge about their local effectiveness.

CURRENT HELICOBACTER PYLORI DIAGNOSTICS

Several diagnostic methods are available for detecting H. pylori infections. They can be broadly classified as invasive and non-invasive methods depending on the need to retrieve a gastric biopsy from the patient. For H. pylori detection, endoscopy is employed in combination with histology and/or culture from the gastric biopsy specimen. The major limitation of endoscopic examination is its relative invasiveness and that only a small portion of the gastric mucosa can be explored. Therefore,assessment of multiple gastric biopsy specimens is necessary to provide a global picture of H. pylori infection in the stomach[56,57]. When an endoscopy is indicated,H.pylori can be detected by histology, rapid urease test (RUT), culture and polymerase chain reaction (PCR)-based tests using gastric biopsy specimens[58,59]. The accuracy of histology depends on a number of factors like the pathologist's experience, density of H. pylori colonization in the gastric mucosa, the quality and quantity of the clinical specimen and subjective assessment of tissue changes.

Table 1 Who to test, summary of the recommendations from the fifth Maastricht/Florence consensus report[23]

The RUT is based on detecting urea produced by H. pylori, and results are obtained within minutes to hours. The RUT is a cheap, rapid and generally highly specific assay, but its sensitivity is affected if less than 104bacterial cells are present in the gastric biopsy, most probably leading to false-negative results. In some instances,RUT specificity may be negatively affected by the presence of other urease producing bacteria like Staphylococcus capitis urealiticum in the stomach that can lead to falsepositive test results[60]. Commercially available RUTs (e.g., HpFast, CLOTest, HpOne)have reported specificities from 95% to 100%, but their sensitivity is moderate (85% to 95%)[17,61,62].

Successful isolation and cultivation of H. pylori from gastric biopsy specimens is a challenging task that is affected by a number of factors like the quality of the clinical specimen, occurrence of microbial commensal flora in clinical specimens, time interval between sampling and culture and inappropriate transport conditions (temperature,duration of air exposure, etc.). Furthermore, H. pylori culture requires highly trained laboratory personnel and takes up to 7 d until samples can be reported as negative and up to 2 wk until H. pylori has grown and an antibiogram can be provided to the treating physician. H. pylori culture from gastric biopsy specimens typically has a sensitivity greater 90% and a specificity of 100%, when performed under optimal conditions[63]. H. pylori culture from clinical specimens obtained by non-invasive procedures, such as gastric juice, saliva and stool, is challenging and hampered by low sensitivity[64-66], and therefore not recommended in routine diagnostics[67]. With the global emergence of antibiotic resistant H. pylori isolates and subsequently increasingly failing empiric first-line therapies, bacterial culture and phenotypic drug susceptibility testing (DST) remains a crucial diagnostic method for antibiotic resistance surveillance and management of antibiotic treatment failures. However, it is not recommended to do a full phenotypic DST before administration of first-line treatment as: (1) An invasive endoscopy is required to obtain gastric biopsy specimens from the patient; (2) It is time consuming and costly; and (3) Less invasive,molecular based methods are also able to detect clarithromycin resistance that is momentarily the main cause of empiric treatment failure in European countries.

Due to these drawbacks, numerous attempts have been made to develop noninvasive diagnostic methods that avoid endoscopy. Classically, non-invasive tests for H. pylori detection include stool antigen assays, serology and the frequently used urea breath test (UBT)[68,69]. Antigen tests have been widely used for H. pylori detection in clinical specimens like gastric juice, saliva, urine and stool[70-72]. However, antigen detection methods, may suffer from poor specificity and sensitivity[70,71,73]. Different stool antigen tests have been developed to detect H. pylori in stool specimens with a sensitivity and specificity of 85% to 95%[17]. The UBT is the most frequently used point-of-care test in the clinic with a sensitivity and specificity of 85% to 95%[17,74].

One limitation of the beforehand presented non-invasive diagnostic methods is that they can solely detect H. pylori but do not provide information on the drug susceptibility of the bacterium. With increasing clarithromycin resistance rates in H.pylori, rapid and accurate methods that can simultaneously detect H. pylori and assess its clarithromycin susceptibility offer high added value[10,44,75]. Clarithromycin resistance in H. pylori is attributable, in a majority of cases, to three single point mutations (A2146C, A2146G and A2147G) in the 23S rRNA gene that can be accurately detected by PCR[76-79]. At the moment, there are a number of molecular assays commercially available for H. pylori and clarithromycin resistance detection,such as the H. pylori ClariRes (Ingenetix, Vienna, Austria), the Allplex H. pylori and ClariR (Seegene, Korea), the Lightmix?H. pylori (TIBMolbiol, Germany) and the H.pylori Taqman?real-time PCR assay (Meridian Bioscience, United States). These assays mostly combine real-time PCR with melting curve analysis and are highly specific and rapid (＜ 2 h) molecular methods that can be applied to gastric biopsy and stool specimens[77,78]. Moreover, they can distinguish the three most common point mutations (A2146G, A2147G and A2146C) in the 23S rRNA gene, which allows to genotypically distinguish low- and high-level clarithromycin resistance[77]. However,several studies found rather low sensitivity (ranging from 63% to 84%) of H. pylori detection from stool specimens using the ClariRes assay when compared to stool antigen test and H. pylori culture from gastric biopsy specimens[80-82]. Another study validating the H. pylori Taqman?real-time PCR assay in stool specimens reported higher sensitivity of 93.8%[79]. Therefore, H. pylori and clarithromycin resistance detection directly from stool specimens may strongly depend on the DNA extraction method and the PCR assay used. Consequently, no general statement on the diagnostic performance of PCR from stool can be made. One limitation of PCR assays is, however, that they can just provide resistance information for clarithromycin. At the moment, there is only one line probe assay (the Genotype HelicoDR assay; Hain Life Sciences, Germany) commercially available that enables the detection of the most common point mutations in the 23S rRNA (A2146G, A2147G and A2146C) and the gyrA gene (N87K, D91G, D91N, D91Y) to determine clarithromycin and levofloxacin susceptibility, respectively. The Genotype HelicoDR assay has been reported to accurately detect H. pylori and clarithromycin resistance from gastric biopsy specimens[83], but low concordance between H. pylori and clarithromycin resistance detection from biopsy and stool specimens was found[84]. Moreover, the Genotype HelicoDR assay has a long turn-around-time of 6 h compared to real-time PCR assays.

In sum, non-invasive molecular testing from stool would have the following advantages: (1) No invasive endoscopy is required; (2) Specimens can be stored longer and do not require immediate processing; (3) Batching of specimens is possible; (4)H.pylori detection and genotypic clarithromycin susceptibility screening can be done within one working day (＜ 4 h); (5) Detection of hetero-resistance in specimens is achievable when more than one H. pylori strain is present in a clinical specimen; (6)Automated DNA extraction and real-time PCR analysis offers a high degree of standardization and reproducibility. However, further studies are needed that assess the diagnostic performance of optimized DNA extraction procedures and noninvasive stool PCRs (ideally targeting the 23S rRNA and gyrA gene) in comparison to H. pylori culture based phenotypic DST from gastric biopsy specimens.

HELICOBACTER PYLORI TREATMENT AND PROPHYLAXIS

As H. pylori antibiotic therapy is mostly based on clarithromycin, clarithromycin resistance is the major determinant of antimicrobial treatment: In countries with low clarithromycin resistance (i.e., ＜ 15%), current first-line standard regimens for H. pylori eradication are a proton pump inhibitor (PPI)-based triple therapy (with clarithromycin in combination with metronidazole or amoxicillin) or a bismuth quadruple therapy[23,85,86]. The second line therapy should then be the bismuthquadruple (if not used as first-line therapy) or a triple therapy containing fluoroquinolones. Choice of third-line therapy should be guided by phenotypic DST or genotypic determination of drug resistance (associated costs for antibiotic therapy are listed in Tables 2 and 3; approximate drug prices from Germany).

In countries with high clarithromycin resistance (i.e., ＞ 15%), metronidazole resistance, although clinically less relevant, should be considered. If metronidazole resistance is low, a triple therapy with PPI, amoxicillin and metronidazole can be applied. If the dual resistance for clarithromycin and metronidazole is low, a bismuth quadruple or a concomitant non-bismuth quadruple therapy should be used.However, if the dual resistance is high, bismuth containing quadruple therapies should be used[23].

Vaccines against H. pylori have only recently been given serious consideration. In animal models, initial vaccination tried oral immunization with H. pylori bacterial lysate and cholera toxin as adjuvant[87]. Later on, intranasal and rectal delivery systems allowed reducing the required amount of purified antigen compared to oral immunization. Given the proposed mechanism of action involving the cellular immune system[88], parenteral immunization with H. pylori antigens has been shown to result in a significant protection from infection in mice models[89]. However,although clinical trials in human on immunization with H. pylori proteins have shown adaptive immune mechanisms, they have since failed to consistently reduce bacterial load[90]. More studies are needed in this area.

Table 2 Standard clarithromycin-based triple regimens using metronidazole or amoxicillin and associated costs

Due to increasing antibiotic resistances and side effects of antibiotics, alternative therapies are of great interest. Probiotics have been shown to have positive effects on eradication rates, prevention of adverse reactions and antibiotic-associated diarrhea when combined to eradication therapies. A recent systematic review and metaanalysis on probiotics as adjunct therapy found 19 randomized controlled trials, all showing positive effects on at least one of the above-mentioned aspects[91]. However, it appears that the number of meta-analysis on the topic exceeds the number of original publications, that, even in randomized controlled fashion show large detail variance[92]. Interestingly, a large and well performed meta-analysis showed that probiotic dose, duration, number of strains and duration of antibiotic treatment did not affect the benefits conferred by probiotic adjunction[93], reducing the scientific plausibility of this intervention based on current publications. The fifth Maastricht/Florence consensus rapport acknowledges probiotics as beneficial in its report, but evaluates the level of evidence as low to moderate with weak grade of recommendation. That being said, probiotics alone, not in combination with antibiotics, have not been shown to efficiently eradicate H. pylori[94]. We as others conclude that “more data are definitely needed to assess the direct efficacy of probiotics against H. pylori”[23].

Licorice root is a botanical product frequently used in Chinese medicine. It has detoxifying, antiulcer, anti-inflammatory, anti-viral and anticarcinogenic properties[95].A randomized controlled trial on 120 H. pylori positive dyspeptic patients (with or without peptic ulcer) assessed the effect of licorice in addition to clarithromycin-based triple regimen. They showed that treatment response was 83.3% in the licorice-group compared to 62.5% in the control group (P = 0.018). When distinguishing between peptic ulcer disease and non-ulcer dyspepsia, significantly better response to treatment was only observed in patients with peptic ulcer (P = 0.034)[96].

Several other plant-based products are used for the treatment of gastrointestinal disorders. Some of them have been mentioned as influencing H. pylori infections such as garlic, cranberry juice, oregano or broccoli sprouts (non-exhaustive list)[97].However, few studies have identified the active ingredient or its mechanism of action and dose/response or exposure level are not understood. Possible safety issues as well as impact of resistance on efficacy of phyto-therapeutic agents has to be addressed. Moreover, one review article mentioned the possibility of phage therapy against H. pylori[98].

NEXT GENERATION SEQUENCING AND HELICOBACTER PYLORI

In order to get an overview on original research studies that focused on the characterization of H. pylori by next generation sequencing (NGS) a PubMed MEDLINE and EMBASE literature search was conducted without any time constrains. Inclusion criteria: (1) Original research manuscripts; (2) Characterization of clinical human H. pylori isolates; (3) Use of second and/or third generation sequencing technologies. Exclusion criteria: (1) Reviews, case reports, comments,letters; (2) Characterization of non-human H. pylori isolates; (3) Original research manuscripts that that did not use second or third generation sequencing technology.First, the terms “Helicobacter pylori AND transcriptome OR transcriptomic” were searched and yielded 134 results, of which 12 were original research articles meeting the inclusion criteria (Table 4). Second, a PubMed, MEDLINE and EMBASE search using the terms “Helicobacter pylori AND next generation sequencing” was done that yielded 102 results, of which 19 met the inclusion criteria (Table 5). And finally, a PubMed, MEDLINE and EMBASE search with the terms “Helicobacter pylori AND whole genome sequencing” was done that yielded 89 results, of which 15 met the inclusion criteria (Table 6).

Table 3 Alternative antibiotic Helicobacter pylori eradication therapy using quadruple or levofloxacin-based regimens and associated costs

The literature search mostly yielded basic research studies investigating a broad spectrum of fundamental mechanism in H. pylori like the presence of genes associated with bacterial virulence[99-104]and biofilm formation[105,106], the characterization of methylases[107], nudix hydrolases[108], restriction-modification (R-M) systems[109], exoand endo-ribonucleases[110]; and the transcriptional response of H. pylori to exposition to different salt concentrations[111], different pH conditions[112], heat shock[113]and chemical agents like bismuth[114]and nickel[115]. Most of these studies initially depleted ribosomal RNA, either using RiboZero eukaryotic rRNA depletion treatment(Epicentre, Illumina)[112,113,116]or a rRNA modified capture hybridization approach[110],and performed sequencing on an Illumina platform (Illumina, United States). More applied studies focused on two main topics: The composition of the gut microbial community in patients with H. pylori associated gastrointestinal pathology[117-123]or the association between genotypic and phenotypic drug resistance in H. pylori[124-133].

The assessment of the human gut microbiome in health and disease is a hot topic in medical sciences. Changes in the gut microbial composition after H. pylori infection may induce pathogenesis and various disorders. However, studies investigating changes in the microbial community composition after H. pylori infection have generated conflicting results. While some studies could not detect any significant changes in the taxonomic composition of the gut microbiota[134-136], studies using NGS technology have reported increased abundance of the families Xanthomonadaceae and

Enterobacteriaceae, and the genera Spirochaetae, Streptococcus, Lactobacillus,Granulicatella, Prevotella and Veillonella in response to H. pylori infection[137-139].Moreover, some studies showed that antibiotic use to eradicate H. pylori affected the proliferation of the gut microbial community and altered microbial diversity[140-142],though; species diversity recovered to pre-treatment levels upon long-term followup[141,142]. This encourages the calculation of a microbial dysbiosis index based on the abundances of certain gut microbial taxa in H. pylori infected patients[143]. The dysbiosis index enables to distinguish patients with different H. pylori associated gastrointestinal pathologies[138], and may potentially help to guide patient management.

Table 4 A PubMed, MEDLlNE and EMBASE search using the terms “Helicobacter pylori AND transcriptome OR transcriptomic” yielded 12 original research studies

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

One limitation of using DNA based techniques for the determination of bacterial abundances and the calculation of a dysbiosis index is that all microorganisms, which are present in the human gut, are detected, including dead bacteria and DNA contaminations (e.g., from incautious sampling, unsterile laboratory procedures or from extraction and sequencing chemicals). In contrast, using meta-transcriptomics allows to investigate only the viable and transcriptionally active proportion of the human gut microbial community. A recently conducted study by Thorell et al[116]nicely demonstrated that meta-transcriptomics is a very sensitive method that can be applied directly on gastric biopsy specimens. They found that gastric biopsies from patients initially classified as H. pylori uninfected by conventional methods, contained actively replicating H. pylori, though in lower numbers than biopsy specimens initially scored as H. pylori positive. This suggests that H. pylori may be present in low abundance in individuals, in whom conventional methods have failed to detect H.pylori. It would be interesting to investigate if these patients develop H. pylori infection at a later stage and which circumstances may trigger or reinforce infection.Moreover, it would be interesting to investigate if probiotic treatment in these patients would reduce the transcriptionally active H. pylori population to undetectable levels,potentially preventing disease development.

The other research studies identified in our literature review focused on using NGS technology for the detection of drug resistance mutations in H. pylori and their corrleation with phenotypic drug resistance[124-133]. Collectively, these studies show that clarithromycin resistance is based on point mutations at nucleotide positions A2146 and A2147 in the 23S rRNA gene[125-127,133]. Additional mutations in rpl22 and infB were reported in clarithromycin-resistant H. pylori strains without 23S rRNA mutations[126,127]. However, some methodological concerns should be considered. First,multiple gastric biopsy specimens should be used for H. pylori culture in order to enhance sensitivity and detect H. pylori sub-populations. Second, multiple colonies should be picked from agar plates for DNA extraction and library preparation to not miss drug resistant sub-populations. Third, H. pylori strains should be sequenced with sufficient coverage to detect hetero-resistance. Fourth, multiple susceptible and resistant H. pylori strains should be sequenced to distinguish naturally occurring polymorphisms from mutations potentially conferring drug resistance.

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

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Levofloxacin resistance in H. pylori has been found associated with amino acid exchanges at codon 87 and/or 91 in the gyrA gene[124-127]. No synergistic effect on levofloxacin resistance has been found in H. pylori strains that carried additional mutations in gyrB. Rifampicin resistance has been reported to be associated with amino acid exchanges in the rifampicin resistance determining region of the rpoB gene[125]. In contrast, the prediction of metronidazole resistance based on genotypic information remains challenging for H. pylori. Most metronidazole resistant H. pylori strains have been reported to carry multiple rdxA and frxA mutations[124,125,127]; though,also metronidazole resistant strains without mutations in rdxA and/or frxA were reported[125]. Moreover, frameshift and resistance mutations in rdxA and frxA have also been reported in metronidazole susceptible H. pylori strains[125]. Collectively, this suggests that more studies are needed investigating the association between polymorphisms detected in rdxA, frxA, mdaB, omp11 and rpsU and other genes and phenotypic metronidazole resistance. NGS may also be used to detect novel mutations associated with metronidazole resistance in genes such as dapF, dppA, dppB,fdxA, and fdxB[124,127].

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

Another area of applicationf of NGS technology is the investigation of mutations associated with resistance against new antibiotic agents like the rifamycins rifabutin and rifaximin, the quinolones garenoxacin and sitafloxacin and the nitrofuran antimicrobial agent furazolidone[128]. Analysis of rpoB sequences of rifaximin resistant H. pylori strains showed amino acid exchanges at I837, A2414, K2068, Q2079 in rpoB,while amino acid exchanges at N87 and D91 in gyrA were associated with high levofloxacin resistance in H. pylori. No H. pylori strain was phenotypically resistant to garenoxacin or sitafloxacin, suggesting a higher genetic barrier to resistance development, and that multiple mutations or synergistic effects may be required to infer resistance against these antibiotics. NGS can further be employed for the identification of putative candidate mutations in phenotypically resistant H. pylori strains without known resistance mutations[129,130]; and to identify efflux pump genes that may be involved in the development of drug resistance in H. pylori[131].

CONCLUSION AND POTENTIAL FUTURE DIRECTIONS

During the last years, antibiotic resistance in H. pylori has continuously increased, also in Western and Central Europe, where antibiotic resistance has been traditionally considered low. This alarming trend leads to question the usefulness of the currently employed “test-and-treat” strategy and to considered determining H. pylori's antibiotic resistance prior to eradication therapy in order to achieve better treatment efficiency. When considering the current costs for H. pylori eradication regimens(Table 2 and Table 3; approximate drug prices from Germany), depending on local resistance rates, initial molecular determination of H. pylori drug susceptibility may be cost efficient, especially, when considering that costs for PCR assays (＜ 20 EUR) and WGS (＜ 100 EUR) have consistently decreased over the last years. In contrast,endoscopy (100-250 EUR) and H. pylori culture-based phenotypic DST (80-100 EUR)remains costly.

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

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

However, in order to determine drug resistance phenotypes prior to the administration of antibiotics, resistance information must be more rapidly available,ideally with non-invasive methods that do not require endoscopy. Diagnostic methods, like line probe assays or culture based phenotypic DST, that provide drug resistance information have long turn-around-times and require a gastric biopsy that can just be obtained by invasive endoscopy. In contrast, currently available, noninvasive diagnostic methods can only detect resistance mutations in the 23S rRNA gene of H. pylori (e.g., PCR from stool). This may be insufficient in areas with high metronidazole resistance or if levofloxacin- or rifampicin-based regimens have to be administered to patients. Our literature search yielded studies that focused on the prediction of drug resistance phenotypes based on the presence of certain point mutations in the H. pylori genome. However, all of these studies used culture H. pylori isolates or DNA extraction from gastric biopsy specimens. In an effort to decrease turn-around-times and apply diagnostic workflows that do not require endoscopy,future studies should aim at detecting H. pylori and associated resistance mutations directly from clinical specimens (gastric biopsies or stool) using meta-genomic and/or meta-transcriptomic sequencing. Our literature search yielded primary research articles that have successfully applied WGS directly on gastric biopsies for the detection of H. pylori[116,132,144,145]. One major limitation for the cost-effectiveness and feasibility of clinical meta-genomic and meta-transcriptomic sequencing has always been the rather big amounts of RNA or DNA required for subsequent library preparation and high human DNA background requiring deep sequencing. There has been a tremendous development in this area, and in-house developed[146,147]and commercial protocols [e.g., RiboZero (Illumina), RiboGold (Illumina), MICROBExpress (Ambion, Invitrogen)] are becoming available for the depletion of human DNA or the enrichment of bacterial DNA prior to performing WGS, thereby increasing the efficiency and cost-effectiveness of NGS due to less human DNA background in clinical specimens.

In conclusion, NGS technology has opened up new avenues for the characterization of complex microbial communities, including those associated with H. pylori associated gastrointestinal disease. Particularly exciting is the promise of cultureindependent approaches to H. pylori detection and assessment of antibiotic resistance.In the diagnostic laboratory, NGS may enable the implementation of rapid and accurate genotypic DST prior to the administration of antimicrobial therapy for H.pylori eradication.