Eradication success and change in antibiotic resistance after the inclusion of PCR in the approach to Helicobacter pylori infection

INTRODUCTION

Infection by Helicobacter pylori (HP) is widely distributed worldwide. The global prevalence of infection is estimated at 40% to 45%, with a slightly decreasing trend in the past decade.^1-3 In children, it is usually asymptomatic,  and there is no evidence of an association between infection by HP with chronic abdominal pain compatible with functional disorders.^4-7 In patients with symptoms suggestive of an organic rather than a functional disorder, performance of upper endoscopy with collection of a biopsy sample for direct testing should be considered,^8,9 as this infection can cause complications such as gastric or duodenal ulcers or mucosa-assisted lymphoid tissue (MALT) lymphoma.^10,11

The infection can be confirmed by means of isolation from culture or positive histopathological identification, in addition to other direct tests, such as polymerase chain reaction (PCR) or the urease test.^12-14 The most recent guidelines of the ESPGHAN/NASPGHAN, published in 2023, consider culture and PCR equally valid for diagnosis, emphasizing the need to target initial therapy based on the results of macrolide susceptibility testing and questioning the usefulness of metronidazole susceptibility testing in culture.¹⁵

Culture offers a high specificity nearing 100% but a variable sensitivity that ranges from 50 to 90%.^16,17 The advent of molecular techniques has opened new paths and improved diagnostic yield on account of its high sensitivity and specificity (>95%), while offering the opportunity to increase eradication rates thanks to a greater sensitivity in the detection of macrolide resistance.^18-21

Antibiotic resistance is the leading cause of eradication therapy failure.^9,22 The young age of some patients and difficulties in completing treatment may lead to treatment failure due to abandonment. This, combined with the use of nontargeted “test and treat” strategies or the massive use of antimicrobials in the general population could increase antimicrobial resistance and the frequency of ineffective treatments.^22-26 Therefore, it is essential to conduct antibiotic susceptibility testing before starting eradication therapy to select an appropriate regimen.^13-16 Considering that the costs of culture and PCR are similar,¹⁸ it is important to determine which strategy achieves the best eradication outcomes to ensure rational use of health care resources.

The main objective of our study was to compare eradication success in patients with active HP infection after treatment during 3 different periods, depending on the strategy implemented for diagnosis (culture; culture + PCR or PCR), analyzing the usefulness of direct microbiological diagnostic tests as well as the economic impact of each strategy through a cost-effectiveness study. As a secondary objective, we analyzed local longitudinal trends in HP antibiotic resistance profiles in our area between 2013 and 2023.

MATERIAL AND METHODS

We conducted a cross-sectional, retrospective, observational, and inferential study in pediatric patients aged less than 16 years in whom eradication therapy was initiated after detection of active HP infection between 2013 and 2023. We compared eradication rates in relation to the use of 3 diagnostic strategies, defined according to the ordered microbiological tests (period 1 [2013-2016]: culture; period 2 [2017-2019]: culture + PCR; period 3 [2020-2023]: PCR), and analyzed the cost-effectiveness ratio (CER) in each period.

In all cases, two samples were collected for histopathological examination (one from the antrum and one from the corpus), in addition to another gastric sample for microbiological testing. We defined confirmed infection as positive culture or positive histopathology + positive PCR results. Eradication was defined as a negative result in an indirect test 8 weeks after completion of treatment. In the cost-effectiveness study, we analyzed the costs of the direct microbiological tests (cost of culture: €1 per plate, €24.63 per antimicrobial susceptibility test; cost of PCR: €25) in relation to the number of patients with successful eradication in each period.

We also analyzed local trends in HP antimicrobial resistance between 2013 and 2023 in patients in whom HP was isolated by direct microbiology testing, regardless of whether treatment was initiated.

Patients who received empiric treatment prior to the upper GI endoscopy, patients who do not complete the treatment and patients in whom eradication was not verified with an indirect test were excluded from the analysis of eradication. These patients were, however, included in the analysis of antibiotic resistance.

We summarized qualitative variables as absolute frequencies and percentages, and quantitative variables using the mean and standard deviation. Qualitative and ordinal quantitative variables were compared with the chi-square test or the Fisher exact test, while continuous quantitative variables were compared with the Student t test. The statistical analysis was performed with the software SPSS, version 15.

RESULTS

A total of 288 patients were included, of who 98 were diagnosed by culture (2013-2016), 94 by culture + PCR (2017-2019) and 96 by PCR (2020-2023).

Antibiotherapy was initiated in 228 patients: 90 in period 1 (ESPGHAN 2011 guidelines, sequential therapy), 63 in the period 2 and 75 in period 3 (ESPGHAN 2017 guidelines, treatment based on susceptibility testing results, 14 days’ duration). In every case involving strains susceptible to macrolides or metronidazole, the patient received triple therapy with omeprazole, clarithromycin and amoxicillin.

We excluded 7.7% of patients (n = 7) in period 1, 7.9% (n = 5) in period 2 and 3.9% (n = 3) in period 3. The percentages of successful eradication were 67.5% (n = 56) between 2013 and 2016, 86.2% (n = 50) between 2017 and 2019 and 95.8% (n = 69) between 2020 and 2023 in the per-protocol analysis. We found statistically significant differences in the comparison of periods 1 and 2 (p <0.05), and of periods 1 and 3 (p <0.001), but not when we compared periods 2 and 3 (p = 0.087).

In the cost-effectiveness analysis, PCR testing alone (strategy 3) was the most cost-effective strategy, with a CER of 26.74, compared to the other two options (strategy 1 CER, 37.98; strategy 2 CER, 58.73) (Table 1).

Table 1. Percentage of successful eradication and cost-effectiveness ratio for each strategy
Strategy	Eradicated/treated cases	Cost (euro)	Cost-effectiveness ratio
Culture	56/83 (67.5%)	2127.29	37.98
Culture + PCR	50/58 (86.2%)	2936.54	58.73
PCR	69/72 (95.8%)	1845.36	26.74

With regard to the method used for microbiological detection of HP, 100% of the cases were detected by culture in period 1 and 100% by PCR in period 3. In period 2, 96.8% (91/94) of the cases were detected by PCR and 71.2% (67/94) by culture, with the following distribution: 64 with positive culture and positive PCR, 27 with positive PCR and negative culture, 3 with positive culture and negative PCR, and 13 with negative culture and negative PCR. The sensitivity of culture and PCR in this period was 71.2% and 96.8%, respectively, with a specificity of 100% for both techniques.

With respect to macrolide resistance,16.3% (16/98) of isolates were resistant in the 2013-2016 period. In the 2017-2019 period, the percentage rose to 53.2% (50/94), with resistance detected by PCR in 98% of cases (49/50) and by culture in 58% (29/50). The degree of concordance in the detection of macrolide resistance between PCR and culture was of 55% (27/49). In the 2020-2023 period, 61.4% (59/96) of patients had macrolide resistance. Table 2 presents the changes in the frequency of macrolide resistance in relation to whether or not PCR was ordered in each period, extrapolating the level of concordance observed between 2017 and 2019. On the other hand, the frequency distribution of resistance to other drugs remained stable (Table 3).

Table 2. Resistance to macrolide with/without PCR by study period
Strategy	Macrolide resistance without PCR	Macrolide resistance with PCR	Statistical significance
Culture	16.3% (16/98)	16.3% (16/98)	p = 1
Culture + PCR	30.8% (29/94)	53.2% (50/94)	p <0.001
PCR	34.3% (33/96)	61.4% (59/96)	p <0.001

Table 3. Antibiotic resistance by study period
Strategy	Metronidazole	Dual resistance (macrolides + metronidazole)	Ampicillin	Fluoroquinolones
Culture	7% (7/98)	3% (3/98)	1% (1/98)	7% (7/98)
Culture + PCR	6.3% (6/94)	2% (2/94)	1% (1/94)	7.5% (7/94)
PCR	-	-	-	8.3% (8/96)

DISCUSSION

In the evaluation of an active HP infection, it is key to identify those patients in whom eradication therapy can achieve significant clinical improvement.^12,13 The high prevalence of functional gastrointestinal disorders in the pediatric population may lead to overdiagnosis and overtreatment of HP infection, since in many cases HP detection is only indicative of colonization and the bacterium is not the cause of the patient’s symptoms.^5-7

Performance of upper endoscopy, when indicated, makes it possible to directly confirm the presence of the bacterium in the stomach, to assess the degree of damage to the gastric mucosa associated with the infection by means of histopathology and to carry out susceptibility testing in order to guide the selection of targeted therapy.^8,9

Given that the increase in antibiotic resistance is one of the main causes of eradication failure^22-25 and that primary failure may increase the probability of secondary resistance,^9,23 it is essential to set up a detection system that can identify these resistances as reliably as possible.

The introduction of molecular techniques for direct diagnosis opens up new opportunities due to a greater sensitivity and specificity in diagnosis in addition to a greater capacity to detect macrolide resistance.^18-20

It is worth noting that in period 1, prior to the introduction of molecular diagnostic techniques, the rate of eradication was low, far from the 90% target set by the ESPGHAN. This could be due to the use of culture alone, which has a lower sensitivity for diagnosis and a lower capacity to detect macrolide resistance, as well as the use of treatments that were not guided by susceptibility testing (sequential therapy).

In period 2, when both tests were ordered together, eradication rates increased significantly due to the higher sensitivity of PCR over culture for diagnosis of infection (96.8% vs 71.2%) in addition to a greater capacity to detect macrolide resistance. If PCR had not been available during this period, 28.7% of HP infections (27/94) would not have been diagnosed (corresponding to patients with positive PCR and negative culture). Furthermore, the frequency of macrolide resistance would have been 30.8% (29/94), and macrolide resistance would have not been identified in 22.4% of cases, in which, as a result, eradication may have failed since clarithromycin-based regimens were used in every case in which resistance was not identified.

In period 3, the rate of eradication increased to 95.8%, with macrolide resistance detected in 61.4% of isolates. Extrapolating the sensitivity data for diagnostic tests during period 2, the frequency of macrolide resistance, without the use of PCR, would have been of 34.3% (33/96), and it is more than likely that the frequency of successful eradication would have also been lower.

The cost-effectiveness analysis showed that the highest eradication rate was achieved with the PCR-only strategy. The culture-only strategy proved to be an inferior option. In period 3, not ordering culture did not result in a decrease in successful eradication compared to period 2, while there was a significant reduction in the use of health care resources.

It should be taken into account that the PCR-only strategy could result in initiation of inappropriate treatment in cases with dual resistance to macrolides and metronidazole.^24-26. In our area, we found a prevalence of dual resistance of 2.5% between 2013 and 2019,¹⁸ which could translate to prescribing of inappropriate treatment to two patients. However, after the 2023 update of the ESPGHAN/NASPGHAN guidelines, which question the usefulness of metronidazole resistance detected by culture, recommends tailored eradication therapy based on susceptibility testing results for macrolides, which further enhances the utility of PCR.¹⁵

CONCLUSIONS

We may conclude that the introduction of PCR offers a great opportunity to improve the management and treatment of active HP infection due to its greater diagnostic sensitivity and capacity to detect macrolide resistance. The marked increase in antibiotic resistant strains in the past few decades, together with the risk of resistance going undetected with the exclusive use of culture, may result in the initiation of ineffective treatments, which in turn leads to an increase in secondary resistance and a decrease in eradication rates. The PCR-only strategy proved to be the most cost-effective approach. Table 4 presents the main conclusions of the study.

Table 4. Overall results of the study
Strategy	Identified infections	Treated infections	Excluded patients (analysis of eradication)	Percentage of successful eradication	Cost-effectiveness ratio (CER)	Macrolide resistance with PCR	Macrolide resistance without PCR
Culture (2013-2016)	98	90	7 (7.7%)	56/83 (67.5%)	37.98	16.3% (16/98)	16.3% (16/98)
Culture + PCR (2017-2019)	94	63	5 (7.9%)	50/58 (86.2%)	58.73	53.2% (50/94)	30.8% (29/98)
PCR (2020-2023)	96	75	3 (3.9%)	69/72 (95.8%)	26.74	61.4% (59/96)	34.3% (33/96)

CONFLICTS OF INTEREST

The authors have no conflicts of interest to declare in relation to the preparation and publication of this article.

AUTHORSHIP

Author contributions: original concept, data collection and data analysis (MGP and ELOI), contribution of relevant ideas and development of secondary objectives (SMC, CRG, ALV and PGP).

ABBREVIATIONS

CER: cost-effectiveness ratio · HP: Helicobacter pylori · PCR: polymerase chain reaction.

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