Rapid Non-Invasive Techniques for Detecting Antibiotic Resistance in Helicobacter pylori: An Update and Clinical Applications

Abstract

The global prevalence of Helicobacter pylori infection remains high, posing a significant health challenge worldwide. The efficacy of anti-H. pylori antibiotic-based regimens is compromised by the increasing antibiotic resistance in H. pylori. Thus, it is important to detect antibiotic resistance in H. pylori to ensure appropriate and effective treatment strategies. Currently, conventional culture-based methods are used for detecting antibiotic resistance (so-called phenotypic resistance) in H. pylori, but these methods are tedious and time-consuming (at least 72 h) and rely on the successful culture of H. pylori. Over the past decade, emerging genotypic or molecular techniques based on polymerase chain reaction or gene sequencing of DNA extracted from cultured H. pylori cells or H. pylori-containing specimens, such as gastric biopsy, stool, or saliva, have been developed to detect antibiotic resistance (so-called genotypic resistance) in H. pylori. These methods are rapid (usually within 4 h), non- or minimally invasive, cost-effective, and highly reproducible. Moreover, they can detect heteroresistant strains, enabling tailored therapy. The development and implementation of molecular techniques have significantly improved the accuracy and speed of identifying antibiotic resistance in H. pylori, allowing for more effective and personalized treatment strategies.

1. Introduction

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach, affecting approximately half of the global population [1]. H. pylori infection is not only a major cause of various gastrointestinal diseases, including gastritis, peptic ulcers, mucosa-associated lymphoid tissue lymphoma, and gastric cancer but also associated with various extra-gastrointestinal diseases, such as diabetes mellitus, idiopathic thrombocytopenic purpura, etc. [2]. It has been established that eradication of H. pylori infection is crucial for the treatment and prevention of these diseases. Currently recommended H. pylori eradication regimens include triple therapies containing a proton pump inhibitor such as omeprazole/esomeprazole, lansoprazole or pantoprazole, or vonoprazan and two of the following antibiotics including metronidazole, amoxicillin, clarithromycin, tetracycline, levofloxacin, or quadruple therapies containing a proton pump inhibitor, a bismuth salt, and two of the above-mentioned antibiotics [3]. Clinically, these regimens usually initially achieved eradication rates of over 85% [4]. However, the emergence and spread of antibiotic resistance among H. pylori strains significantly affects the clinical efficacy of these triple or quadruple regimens, thus becoming major challenges in achieving successful eradication. Although vonoprazan appears to regain the efficacy of triple therapies, vonoprazan-containing triple therapies are reported to contribute to antibiotic resistance if treatment fails [5, 6]. Therefore, identifying antibiotic resistance in H. pylori is important for specifically selecting antibiotics for the eradication of H. pylori infection.

Currently, conventional culture-based microbiological methods are commonly used to determine the susceptibility of H. pylori strains to various recommended antibiotics [7, 8, 9, 10, 11]. However, these methods are tedious, time-consuming, costly, and, more importantly, invasive. In addition, culture of H. pylori from gastric biopsies is not always successful due to technical difficulty, and thus susceptibility testing results may not be available for some patients with H. pylori infection. Therefore, a rapid, non-invasive, and accurate technique for the determination of susceptibility of H. pylori to antibiotics is required for specific targeting treatment of the infection. At present, several rapid, mostly non-invasive, techniques for determining the susceptibility of H. pylori to antibiotics have been explored [12, 13, 14, 15, 16, 17, 18, 19, 20]. This chapter aims to emphasize the importance of detecting antibiotic resistance in H. pylori, describe currently used conventional methods and particularly emerging rapid non-invasive techniques for detecting antibiotic resistance in H. pylori and their clinical implications, and propose future research directions.

2. Importance of detecting antibiotic resistance in H. pylori

Antibiotic resistance refers to the ability of bacteria to survive and multiply in the presence of antibiotics which would normally inhibit or kill them. The development of antibiotic resistance in H. pylori is primarily attributed to the overuse and misuse of antibiotics in clinical practice.

The clinical efficacy of currently recommended eradication treatment regimens heavily relies on the susceptibility of H. pylori strains to antibiotics. Early studies have demonstrated that primary or secondary (acquired) resistance leads to treatment failure [21, 22] and the persistence or recrudescence of H. pylori infection is associated with chronic or recurrent diseases associated with H. pylori infection [23]. The impact of antibiotic resistance on the success rate of eradication therapies has been confirmed by recent studies. For instance, Wong et al. showed that metronidazole resistance was associated with a decreased response to metronidazole-containing regimens; the eradication rate was 87.2 and 67.6% (P = 0.001) for patients with metronidazole sensitive and resistant isolates, respectively [24]. Similarly, Arenas et al. found that clarithromycin resistance significantly reduced the eradication rate of H. pylori when using clarithromycin-based regimens (OR: 0.13; 95% confidence interval, 0.04–0.49) [25]. The effects of antibiotic resistance on the clinical efficacy of H. pylori eradication regimens are not limited to these two antibiotics. Resistance to other commonly used antibiotics, such as amoxicillin, levofloxacin, and tetracycline, has also been reported to compromise the success of eradication therapies [26, 27, 28]. Thus, H. pylori eradication should be guided by antibiotic susceptibility testing (AST), preferably by genotypic methods. Indeed, a recent meta-analysis enrolled 12 qualified randomized controlled trials containing 3940 patients clearly demonstrated that the pooled eradication rates of tailored therapy based on the detection of genotypic resistance were consistently higher than those in the empirical treatment group [29].

Therefore, to ensure the efficacy of H. pylori eradication therapies, it is essential to detect antibiotic resistance in H. pylori before initiating treatment. By identifying the antibiotic susceptibility profile of H. pylori, clinicians can tailor the treatment regimens to the individual patients.

3. Conventional phenotypic culture-based methods for detecting antibiotic resistance in H. pylori and their limitations

The conventional microbiological methods are mainly based on the culture of the gastric biopsies taken during upper endoscopy, subculture of H. pylori colonies, and observation of the ability of the H. pylori isolates to grow on agar plates containing various concentrations of antibiotics, and measurements of the minimal inhibitory concentrations (MIC) or the minimal diameter of the inhibitory zone (MID) [7, 8]; the cut-off MICs and MIDs of commonly used antibiotics are listed in Table 1. Thus, these methods directly determine the phenotypic pattern of antibiotic susceptibility of H. pylori and are thus considered to be accurate in detecting antibiotic resistance (or specifically phenotypic antibiotic resistance) in H. pylori.

Antibiotic	The cut-off MIC (μg/mL) differentiating susceptibility and resistance	The cut-off MID (cm) differentiating susceptibility and resistance
Metronidazole	>8.0 [7, 30, 31, 32, 33, 34]	<20 (5 μg/disk) [7]
Clarithromycin	>0.5 [31, 32, 33, 34, 35] or 1.0 [19] or 2.0 [36]	<30 (15 μg/disk) [36]
Tetracycline	>1.0 [31, 32, 33, 34, 35]
Amoxicillin	>0.125 [31, 33, 34], 0.25 [32] or 0.5 [35]
Levofloxacin	>0.5 [35] or 1.0 [31, 32, 33, 34]
Rifampicin	>1.0 [31, 32, 34]
Rifabutin	>1.0 [33]
Furazolidone	>2.0 [32]	<21 (100 μg/disk) [31, 37]

Table 1.

The cut-off minimal inhibitory concentrations and the minimal diameter of the inhibitory zones differentiating susceptibility and resistance in Helicobacter pylori to various commonly used antibiotics.

The cut-off criteria are mainly derived from EUCAST [34].

MIC, the minimal inhibitory concentration as detected by the agar dilution method, or the Epsilometer test (or the E-test); MID, the minimal diameter of the inhibitory zone as detected by the disk diffusion test.

Currently, four phenotypic methods including the agar dilution method, broth microdilution method, disk diffusion test, and the Epsilometer test (or the E-test) are commonly used in clinical practice [7, 8, 9].

• Agar dilution method

  The agar dilution method is considered the gold standard for detecting bacterial susceptibility to antibiotics and is recommended by the Clinical and Laboratory Standard Institute (CLSI). Briefly, bacterial suspension (2.0 McFarland) of each of multiple H. pylori strains or isolates is spot-inoculated on Mueller-Hinton or chocolate agar in a 150 mm plate; the agar medium is supplemented with 5 to 10% sheep or horse blood and contains one of 2-fold dilution concentrations of an antibiotic. The agar plates are incubated at 35–37°C in a microaerophilic environment and read for bacterial growth after 72 h of incubation. The lowest antibiotic concentration at which there is no growth is defined as the MIC of the antibiotic for the tested H. pylori strain or isolate [7, 8].

• Broth microdilution method

  Similar to the agar dilution method, this method involves the preparation of serial dilutions of antibiotics in a liquid growth medium. H. pylori suspension is added to each well containing one of the antibiotic dilutions, and the MIC is determined as the lowest concentration of the antibiotic that inhibits bacterial growth. This method is now increasingly used in epidemiological studies and clinical practice [9].

• Disk diffusion method

  The disk diffusion method, or Kirby-Bauer test, is commonly used in epidemiological studies. Briefly, a standardized inoculum is swabbed onto the surface of Mueller-Hinton or chocolate agar in a 150 mm plate. Then, filter paper disks impregnated with standardized concentrations of a few different antibiotics are placed on the surface of predefined areas. The plates are incubated as described for the agar dilution method, and the size of the zone of inhibition around each of the disks is measured after 72 h [7, 10]. However, this method has a labor-intensive nature stemming from manual measurements and data documentation, and mainly relies on investigator skill, leading to potential variations in results [38, 39].

• E-test

  This method is also known as the Epsilometer test. It employs a bacterial solution calibrated to the McFarland standard of 3.0 on Muller-Hinton agar supplemented with 5–10% sheep blood. Under microaerophilic circumstances, an E-test strip impregnated with escalating quantities of antibiotics is placed on inoculation plates and incubated for 72 hours at a temperature of 37°C. The MIC is provided by an elliptical zone of inhibition. When compared to the agar dilution or broth dilution procedures, the E-test is straightforward to execute, and the results have been found to correlate closely with results obtained by the gold standard (agar dilution), except for metronidazole, for which recent studies have revealed inconsistency in the results [11]. The E-test possesses all the disadvantages that the disk diffusion method does [7, 10, 11].

The above-described culture-based conventional phenotypic AST methods provide valuable and accurate information on the susceptibility of H. pylori to antibiotics. However, they have several limitations. First, the commonly used specimens for isolation and culture of H. pylori are gastric biopsies taken during the upper endoscopy. Thus, upper endoscopic examination is a prerequisite for most cases. Noticeably, upper endoscopy is an invasive procedure that does not apply to the general population. Second, isolation and subculture of H. pylori are difficult due to the finicky nature of the bacterium and the high possibility of contamination with commensals, and thus, AST of H. pylori can be carried out only in well-equipped laboratories with well-trained technicians. In addition, sufficient live H. pylori bacterial cells in the specimen are required for successful isolation of the bacterium. Inadequate or delayed specimen handling and processing and recent exposure to antibiotics or proton pump inhibitors (PPIs) would reduce the number of live H. pylori and thus decrease the success rates [40, 41]. Indeed, the success rates for isolation of H. pylori from gastric biopsies range from 67.9 to 96.0% in clinical practice [41, 42]. Finally, all culture-based conventional phenotypic methods are tedious and time-consuming as they all require a long (at least 72 h) cultural procedure and rely on the successful isolation and subculture of the H. pylori isolate.

4. Genotypic or molecular-based methods for detecting antibiotic resistance in H. pylori and their clinical applications

Given the limitations of conventional phenotypic methods, there is a pressing need for rapid non-invasive techniques for detecting antibiotic resistance in H. pylori. It has been revealed that antibiotic resistance in H. pylori is primarily attributed to point mutations in specific genes; the point mutations rendering the resistance in H. pylori to antibiotics commonly used in clinical practice are summarized in Table 2. Therefore, genotypic or molecular methods that are based on polymerase chain reaction (PCR) or gene sequencing of DNA extracted from purely cultured H. pylori cells or H. pylori-containing specimens, such as gastric biopsy, stool, or saliva, have emerged to detect antibiotic resistance (or specifically genotypic resistance) in H. pylori. These methods are non- or minimally invasive, more rapid (usually within 4 h), cost-effective, highly reproducible, and less affected by the modulation of bacterial burden caused by the recent use of antibiotics and PPIs [12, 13, 14, 15, 16, 17, 18, 19, 20, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75]. Moreover, they can detect heteroresistant strains, which enables tailored therapy [41, 73, 74, 76, 77]. Therefore, genotypic methods can be used as an alternative to culture-based methods for detecting the resistance in H. pylori to various antibiotics.

Antibiotic	Drug class	Resistance mechanism	Mutation position
Metronidazole [43, 44]	Nitroimidazole	Insertions/deletions,; frameshift mutations or; missense and premature; truncations in rdxA gene
Clarithromycin [43, 45, 46]	Macrolide	The V domain of 23S rRNA	A2142G, A2143G, A2142C
Tetracycline [47, 48, 49]	Tetracycline	Triple base pair substitutions of 16S rRNA	Positions 926–928
Amoxicillin [50, 51]	β-lactam	Mutations in pbp1A gene
Levofloxacin [52, 53]	Fluoroquinolone	The QRDR within gyrA gene	Codons 87 and 91
Rifampicin [54, 55]	Rifamycin	Point mutations in the RRDR of rpoB gene	Codons 525 to 545, 547, and 586

Table 2.

The genetic mechanisms rendering the resistance in Helicobacter pylori to various antibiotics commonly used in clinical practice.

Several molecular-based methods, mainly based on PCR and sequencing, including allele-specific PCR (AS-PCR), real-time quantitative PCR (RT-qPCR), multiplex PCR, nested PCR, digital PCR (dPCR), and next-generation sequencing (NGS) have been developed to detect genotypic resistance in H. pylori [12, 13, 14, 15, 16, 17, 18, 19, 20, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75]. The methods detect mutations either by polymorphic or mutant allele-directed specific analysis using specific primers or melting curve analysis [78]. In addition, the fluorescence in situ hybridization (FISH) technique has also been applied to detect antibiotic heteroresistant strains of H. pylori [73, 74].

4.1 PCR-based methods

4.1.1 Allele-specific PCR (AS-PCR)

This method involves designing specific primers that target known resistance-associated mutations in genes responsible for antibiotic resistance. By amplifying the target region, the presence or absence of specific mutations can be determined. AS-PCR is applied by performing PCR using primers specific to the target mutation(s) and analyzing the resulting PCR products to identify the presence of resistance-associated mutations [12].

4.2 Real-time quantitative PCR (RT-qPCR)

This technique allows for the quantification of DNA in real-time during the amplification process. It can be used to detect the presence of specific antibiotic resistance genes or mutations in H. pylori. RT-qPCR is applied by designing primers and a fluorescent probe specific to the target gene or mutation. The amplification is monitored in real-time using a fluorescent signal, and the presence or absence of the target sequence is determined based on the amplification curve [13, 17, 18, 19]. Currently, a few commercial RT-qPCR kits that detect clarithromycin resistance in H. pylori, namely, ClariRes RT-PCR, Lightmix RT-PCR, and TaqMan RT-PCR, are available [56, 57, 58, 59, 60, 61]. These methods are reported to produce high concordance or accuracy rates (>90%), compared with the E-test [44].

4.3 Multiplex PCR

This method enables the simultaneous amplification of multiple target genes or regions in a single reaction. It can be used to detect multiple antibiotic-resistance genes or mutations in H. pylori. Multiplex PCR is applied by designing primers specific to each target gene or mutation and optimizing the reaction conditions to ensure efficient amplification of all targets in a single reaction [14, 15, 16].

4.4 Nested PCR

This method involves two rounds of PCR amplification, with the second round using primers that are specific to the product of the first round. It can be used to increase the sensitivity and specificity of detecting antibiotic-resistance genes or mutations in H. pylori. Nested PCR is applied by performing an initial PCR using outer primers that amplify the target region, followed by a second PCR using inner primers that specifically amplify the product from the first round. The resulting PCR products are then analyzed to detect the presence of resistance-associated genes or mutations [20].

4.5 Digital PCR

Digital PCR partitions the PCR reaction into thousands of individual reactions, allowing for the absolute quantification of target DNA molecules. This technique has been explored to detect and quantify low-abundance antibiotic resistance mutations in H. pylori [62]. Specifically, a droplet digital PCR assay was established to detect H. pylori clarithromycin resistance-related 23S rRNA alleles in gastric and stool samples. Of the gastric samples of 46 patients, 22 (48%) had only wild-type (clarithromycin susceptible) alleles, and 24 (52%) had clarithromycin resistance alleles. Of the 24 with clarithromycin resistance alleles, 11 had clarithromycin resistance alleles only, and 13 had a mixed population of clarithromycin resistance and wild-type alleles, suggesting heteroresistance. Of the stool samples of 45 patients, 17 (38%) had only wild-type (clarithromycin susceptible) alleles, and 28 (62%) had clarithromycin resistance alleles. Of the 28 with clarithromycin resistance alleles, 11 had only clarithromycin resistance alleles, and 17 had a mixed population of clarithromycin resistance and wild-type alleles. The dPCR results were highly concordant with that of culture E-test results [62].

4.5.1 Sequencing-based methods

Next-generation sequencing (NGS) techniques have evolved into potent, quick (turnaround time of 24–72 h), and cost-effective methods for predicting antibiotic resistance in H. pylori [63]. They can evaluate several genes at the same time. NGS techniques are highly sensitive for detecting the presence of mutations and resistance genes, compared with PCR-based techniques which only detect specific target genes, and thus can lead to false negatives. The commonly used NGS technique is whole-genome sequencing (WGS), which allows the determination of the entire DNA sequence of an organism, and thus the presence or absence of certain genes and single-nucleotide polymorphisms (SNPs) that are associated with or responsible for antibiotic resistance [64, 65, 66, 67]. This method can be used to directly guide the treatment for individual patients and to indirectly establish treatment guidelines according to the local pattern of antibiotic resistance [64]. At present, the WGS technique has already been applied directly to H. pylori resistance to antibiotics not only with cultured H. pylori bacterial cells but also with clinical specimens, providing an attractive option [68, 69].

Another NGS technique that has been explored to detect antibiotic resistance in H. pylori is ChIP-sequencing, a gene chip technique. Based on the principle of complementary pairing of bases, this technique is reported to have high sensitivity, specificity, and high-throughput capacity for detecting antibiotic resistance of H. pylori directly from gastric biopsies. Yin et al. used ChIP-sequencing to rapidly diagnose H. pylori infection and detect antibiotic-resistant H. pylori in children. They found that the sensitivity, specificity, and accuracy of the gene chip technology for diagnosing H. pylori infection were 96.1, 85.0, and 93.6%, respectively. Moreover, they identified several main H. pylori resistance gene loci using the gene chip; the main mutation loci were 2143A/G for clarithromycin, GyrA 91 and GyrA 87 for levofloxacin, and PBP1 556ser for amoxicillin. Concordance rates between the gene chip technique and DNA sequencing were greater than 95% for VacA-S/M, 16S rRNA, 23S rRNA, and GyrA, and greater than 82% for PBP1 [70].

Other NGS techniques, including whole exome sequencing, methyl sequencing, targeted resequencing, de novo sequencing, and RNA sequencing [71, 72], may be also applied in the detection of antibiotic resistance in H. pylori although their values have not yet been explored.

4.5.2 FISH

This technique has also been applied to detect antibiotic heteroresistant strains of H. pylori [73, 74]. Kocsmár et al. used an rRNA-targeted clarithromycin resistance FISH test for the antral and corpus biopsy specimens of 305 H. pylori-infected patients. They detected clarithromycin-resistant H. pylori in 73 (23.9%) cases, which consisted of 35 (11.5%) homoresistant and 38 (12.5%) heteroresistant cases [73]. They further used the same technique to investigate the proportion of resistant bacteria in the bacterial population (R-fraction) and its predictive role for the use of clarithromycin-based therapies in patients with clarithromycin-heteroresistant H. pylori infection. They observed that the R-fraction was significantly increased in patients with multiple previous eradication attempts, including at least one clarithromycin-containing therapy than in those with only one previous eradication attempt. In addition, multivariable regression analysis showed that the therapeutic outcome using clarithromycin-based regimens depended on the bacterial density, not a high R-fraction [74], suggesting the potential re-use of clarithromycin-containing regimens in patients with clarithromycin-heteroresistant H. pylori infection. However, a high bacterial density is prone to the acquisition of antibiotic resistance during the treatment, which is likely to be the actual cause of the treatment failure [75].

4.5.3 Clinical applications of genotypic or molecular-based methods

The use of molecular-based methods for detecting antibiotic resistance in H. pylori has revolutionized the management approach for patients with H. pylori infection. By providing rapid and accurate results, these methods enable clinicians to prescribe the most effective antibiotic regimen, thereby increasing the chances of successful eradication and reducing the spread of resistant strains. In two large randomized controlled trials of 880 patients (560 eligible treatment-naive and 320 with refractory H pylori infection), Chen et al. demonstrated that clinical efficacy as represented by the eradication rate was comparable between the molecular method-guided therapy and culture-based method-guided therapy when used as the first-line therapy and rescue treatment of H. pylori infection, hence supporting the use of molecular method-guided therapy for H. pylori eradication [79]. More recently, Li et al. reported that genotypic antibiotic resistance-tailored bismuth-containing quadruple therapy resulted in eradication rates of 87.1 and 95.8% by intention-to-treat (ITT) analysis and per-protocol (PP) analysis, respectively [80]. Lin et al. reported that, in a meta-analysis of 4626 patients, the pooled eradication rates of the first-line and rescue genotypic antibiotic resistance-tailored therapies were 86.6 and 85.1% by ITT analysis and 92.0 and 87.9% by PP analysis, respectively. When tailored rescue therapy was based on the genotypic resistance to at least four antibiotics, the pooled eradication rates reached 89.4% by ITT analysis and 92.1% by PP analysis. For genotype-susceptible strains, the pooled eradication rate with targeted antibiotics was 93.1% (95% CI:91.3–94.9%) [81]. Moreover, a recent meta-analysis demonstrated that molecular method-guided therapy is more efficacious than empirical therapy when used as triple therapy (not as quadruple therapy) [29].

Despite numerous advantages, molecular-based methods have limitations. PCR- or NGS-based techniques are incapable of determining MIC, and the contribution of each mutation to the MIC is not always obvious. Moreover, genomic prediction of resistance and phenotypic culture-based susceptibility testing do not always correspond. In addition, molecular-based methods can also underestimate resistance caused by non-genetic mechanisms. Therefore, there is a need for additional research to determine which genes or gene involvement patterns correlate most strongly with phenotypic outcomes. To overcome the diminished predictive value that could be the result of DNA fragmentation in formalin-fixed paraffin-embedded tissue samples or associated contamination in stool samples, excellent DNA extraction methods need to be developed. Consequently, studies with broader gene coverages, larger sample sizes, or multicenter designs from various geographic regions are necessary. Moreover, standardized and user-friendly computational software and tools must be developed so that data obtained from molecular-based methods, especially NGS, can be easily analyzed and implemented in routine clinical settings.

Taken together, molecular-based methods have emerged as valuable tools for detecting antibiotic resistance in H. pylori. They offer rapid and accurate results, enabling effective patient management. However, continuous research is needed to improve these methods and overcome their limitations.

4.5.4 Novel techniques for collecting specimens for genotypic-based methods for detecting antibiotic resistance in H. pylori

Specimens currently used for detecting genotypic resistance of H. pylori include gastric biopsy, stool, or saliva [57, 61, 76]. Recently, Han et al. evaluated a string-test kit for the collection of gastric material, on which RT-qPCR was performed for the detection of H. pylori infection and genotypic resistance to clarithromycin and levofloxacin. They demonstrated that there was a concordance rate of 95.9% between such a string-qPCR and ¹³C-urea breath test, an accurate non-invasive method widely used in clinical practice for diagnosis of H. pylori infection. Moreover, they reported a high eradication rate of 91.8% with genotypic resistance-tailored bismuth quadruple therapy [82]. The value of the string test coupled with RT-qPCR for detecting H. pylori resistance to clarithromycin and levofloxacin has also been validated in urban China in a recent multicenter, cross-sectional study [83]. Therefore, the string-qPCR test is a feasible method that can facilitate the detection of genotypic antibiotic resistance in H. pylori, and thus further improve the clinical practice in the treatment of H. pylori infection. The detailed procedures of the string-qPCR test are presented as a video demonstration in a recent publication [84]. In addition, Tang et al. evaluated surface-enhanced Raman spectroscopy (SERS) in combination with machine learning algorithms for the detection of H. pylori infection in human gastric fluid samples collected with the string test. They found that this novel technique achieved a prediction accuracy of 82.2% in the detection of H. pylori infection [85]; however, the value of SERS coupled with machine learning algorithms in detecting genotypic antibiotic resistance in H. pylori needs to be further investigated.

5. Future research directions

The global prevalence of H. pylori infection remains high, posing a significant health burden. However, current antibiotic treatment strategies are not entirely effective due to increasing antibiotic resistance. Therefore, there is an urgent need to optimize antibiotic-based therapies or to explore novel non-antibiotic therapeutic approaches.

First, future research should focus on the optimization of antibiotic-based therapies. On the one hand, antibiotic resistance-tailored therapy should be applied where feasible. The recent Maastricht VI Consensus recommends that second-line and rescue therapies be guided by local resistance patterns as determined by susceptibility testing and monitoring eradication rates to maximize treatment success [86]. With the advent of molecular techniques, rapid, non- or minimally invasive, accurate detection of antibiotic resistance in H. pylori will be readily available soon. Well-designed randomized controlled trials that include a large number of cases and compare AST-guided rescue therapy with empirical rescue therapy should be carried out to achieve optimized antibiotic-based therapies. On the other hand, further research is needed to evaluate the efficacy of newly claimed promising empirical medicines, such as vonoprazan-based therapies [4, 5, 87], with susceptibility-guided therapy.

Besides antibiotic therapeutic targets, future research should also focus on understanding the molecular mechanisms of H. pylori pathogenesis and host immune response, which could lead to the development of targeted therapies and vaccines. For example, a recent animal study by Liu et al. explored a nanoparticle-based sonodynamic therapy, that effectively neutralizes vacuolating cytotoxin A, a key virulence factor secreted by H. pylori, and generates reactive oxygen species. It was observed that this non-antibiotic approach reduced H. pylori infection in mice without disrupting gut microbiota, and thus could be an alternative to antibiotic-based therapies for H. pylori infection [88]. However, its clinical relevance needs to be verified.