Quantitative PCR of string- test collected gastric material: A feasible approach to detect Helicobacter pylori and its resistance against clarithromycin and levofloxacin for susceptibility- guided therapy

Background: As the reduced eradication rate of Helicobacter pylori ( H. pylori ), we introduced string- test and quantitative PCR (qPCR) for susceptibility- guided therapy innovatively. The practicality of the string test was evaluated. Methods: It was an open- label, non- randomized, parallel, single- center study, in which subjects tested by 13 C- urea breath test (UBT) and string- qPCR were enrolled. Based on the results of string- qPCR, we calculated clarithromycin and levofloxacin resistance rates and gave 13 C- UBT positive patients 14 days susceptibility- guided bismuth quadruple therapy. In the empirical therapy group, we retrospectively analyzed the treatment results of 13 C- UBT positive patients also treated with bismuth quadruple at Shenzhen Luohu People's Hospital from January 2021 to May 2022. The eradication rate was compared between susceptibility- guided therapy and empirical therapy groups. Results: The diagnosis of H. pylori infection using the string- qPCR had an overall concordance rate of 95.9% with the 13 C- UBT results. Based on the results of string-qPCR, the clarithromycin and levofloxacin resistance rates were 26.1% and 31.8%, respectively. The patients who were given 14 days susceptibility- guided bismuth- based quadruple therapy achieved a high H. pylori eradication rate of 91.8%. Retrospective analysis of patient treatment data from January 2021 to May 2022 available in the hospital database revealed an overall success rate of 82.3% for those who received empirical bismuth- based quadruple therapies, which is marginally significantly lower than that of the string- qPCR susceptibility- guided group ( p = 0.084). Conclusion: The high treatment success rate of 91.8% indicates that the string


| INTRODUC TI ON
Helicobacter pylori infects nearly half of the world's population. 1 It is known to cause acute and chronic gastritis, peptic ulcer disease, and almost 90% of non-cardia gastric cancer. 2,3 Attributed to the important role of H. pylori in the pathophysiology of gastroduodenal diseases and more importantly, to reduce the risk of gastric cancer development, there is a broad consensus that all H. pylori-infected individuals should be offered eradication therapy, which usually consists of a proton pump inhibitor (PPI/P) and two different classes of antibiotics with or without bismuth for 10-14 days. 4 Over the past two decades, H. pylori has been gaining resistance against several commonly used antibiotics including metronidazole, clarithromycin, and levofloxacin, resulting in a suboptimal treatment success rate. 5 In the Asia-Pacific region, while the overall mean resistance rates for metronidazole, clarithromycin, and levofloxacin were 44%, 17%, and 18%, respectively, 6 higher primary resistance rates of 78%, 34%, and 35% were reported in China according to a recent meta-analysis conducted by Chen and colleagues. 7 These values far exceed the currently recommended threshold of 15% for a drug to be considered effective in the treatment of H. pylori infection. 8 Further, the H. pylori clinical isolates in southern China patients who failed first-line therapy were highly resistant to metronidazole, clarithromycin, and levofloxacin, at 93.7%, 34.3%, and 39.8%, respectively. 9 Therefore, it is logical that the antibiotic susceptibility-guided treatment strategy should be adopted to avoid the use of ineffective antibiotics when treating potentially resistant organisms, thereby improving the treatment success rate. 10 While the phenotypic agar dilution method and Epsilometer test (E-test) remain the current gold standards for determining H. pylori antibiotic resistance profiles, their invasive, skill-dependent, and time-consuming natures attributed to the need of gastric biopsy specimens for the isolation and culturing of this fastidious and slow-growing organism hinder the wide-spread application of susceptibility-guided therapy in actual clinical settings. 11 Consequently, different molecular genetic testing methods such as the use of qPCR to detect 23S rRNA and gyrA mutations conferring clarithromycin and levofloxacin resistance, respectively, had been developed. [12][13][14] These experimentally validated mutation hotspots include A2143G, A2142G, and A2142C in the 23S rRNA gene for clarithromycin resistance, 15,16 and the missense changes at codon positions 87 and 91 in the gyrA gene leading to resistance against quinolones including levofloxacin. 17 Further, it has been reported that there is a high concordance between the phenotypic antibiotic susceptibility testing results for clarithromycin and levofloxacin, and the above-mentioned mutations, 18 indicating that the testing of H. pylori for both clarithromycin and levofloxacin resistance can now be readily achieved using the faster and more sensitive PCR molecular technique than the conventional culture-based testing method.
Next, as we considered how H. pylori-containing gastric samples could be obtained from infected individuals in a quick and minimally invasive manner for molecular testing, there had been studies reporting the use of string test that involves swallowing an encapsulated string which is then withdrawn orally to collect gastric material for the detection of H. pylori by PCR or culturing methods. 19

| String-test followed by qPCR (String-qPCR) to determine H. pylori infection status and antibiotic resistance
The string-test kit manufactured by Shenzhen Hongmed-Infagen Co. Ltd. was used to collect a patient's gastric sample. In brief, patients fasted overnight and swallowed a gelatine capsule containing an absorbent cotton string with water after taping one end of the string that protrudes from the capsule to the cheek. After 60 min, the string was retrieved and cut with a pair of sterile scissors at the

| Susceptibility-guided treatment
In this study, patients who tested H. pylori-positive by qPCR were assigned to the susceptibility-guided treatment group and received bismuth-based quadruple therapy for 14 days to eradicate H. pylori.
The recommended dosages of bismuth, PPI and antibiotics were available in The Fifth Chinese National Consensus Report on the management of Helicobacter pylori infection. 22 Briefly, the quadruple therapy contained bismuth 220 mg bid, PPI (rabeprazole 10 mg/omeprazole 20 mg/esomeprazole 20 mg/ilaprazole 5 mg/pantoprazole 40 mg/lansoprazole 30 mg) bid, and depending on the antibiotic susceptibility outcomes, two antibiotics were selected from followings: amoxicillin (A) 1000 mg bid, clarithromycin (C) 500 mg bid, levofloxacin (L) 500 mg qd and furazolidone (F) 100 mg bid. A follow-up 13 C-UBT was performed to confirm H. pylori eradication at least 6 weeks after treatment completion.

| Empiric treatment
We retrospectively evaluated the H. pylori eradication rate of Shenzhen Luohu People's Hospital patients by reviewing the 13 C-UBT and treatment records from January 2021 to May 2022. In addition to the exclusion criteria as earlier stated, further criteria were applied to exclude patients who without both pre-and posttreatment UBT results, did not receive and complete a full course of bismuth-based quadruple therapy and without past treatment history. The treatment regimens were then succinctly reviewed to identify the most frequently used empirical formulas for further statistical comparisons.

| Statistical analysis
Statistical analysis was performed using SPSS 23.0. To assess the differences between continuous and categorical variables, the independent sample t-test and Fisher's exact test were used, respectively. Only differences with a two-sided p < 0.05 were considered statistically significant.

| Characteristics of string-qPCR subjects
As shown in Figure 1, a total of 146 eligible subjects with 13  Meanwhile, all UBT-negative subjects were tested H. pylori-negative via qPCR. The overall concordance rate between the UBT and string-qPCR results was 95.9% (140/146) ( Table 1).
Next, the 88 subjects who were tested H. pylori-positive via string-qPCR, among which 50 and 38 were treatment-naive and previously treated for H. pylori infection, respectively, were assigned to the susceptibility-guided therapy group with their clarithromycin and levofloxacin resistance profiles being examined. The overall prevalence of clarithromycin and levofloxacin resistance was 26.1% (23/88) and 31.8% (28/88), respectively. Dual drug resistance was found in 11.4% (10/88) of subjects whilst 55.7% (49/88) showed no resistance to either antibiotic (Table 2). However, it is important to note that a significantly elevated clarithromycin resistance rate was observed in the previously treated subjects than that of the treatment-naive group (44.7% vs. 12.0%, p = 0.001). In addition, resistance to both clarithromycin and levofloxacin or neither antibiotic, was significantly higher (21.1% vs. 4.0%, p = 0.017) or lower (42.1% vs. 66.0%, p = 0.032), respectively, in the previously treated subjects than those who had no past treatment history.

| Susceptibility-guided H. pylori eradication therapy outcomes
Depending on the antibiotic susceptibility outcomes, six different bismuth-based quadruple therapies including PBAC, PBAL, PBAF, PBCL, PBCF, and PBLF were prescribed to eradicate H. pylori infection in 82 subjects ( Figure 1). However, five subjects did not complete the entire treatment course and another 16 subjects were lost to follow-up, leaving 61 subjects for the examination of treatment success rates. A treatment success rate of 91.8% (56/61) was achieved in the susceptibility-guided therapy group.
In the treatment-naive group, a high treatment success rate of 96.9% (31/32) was achieved. While a lower treatment success rate of 86.2% (25/29) was observed among the previously treated subjects, no significant difference was tested when compared to that of the treatment-naive group (Table 3).

| Empiric H. pylori eradication therapy outcomes
To investigate if susceptibility-guided therapy based on string-qPCR approach would improve the H. pylori eradication rate based on empiric treatments in our center, with ethics approval to access the hospital database, we reviewed the information of patients who underwent 13 C-UBT from January 2021 to May 2022. As shown in Figure 2 The demographic information is available in Table 4. The empiric treatment success rates are summarized in Table 5. In general, the treatment success rate of the empiric treatment group was 82.3% (251/305). Between the treatment-naive and previously treated groups, PBAC had achieved a significantly higher eradication rate in the former group as compared to the latter (91.2% vs. 73.7%, p = 0.037). While no significant treatment success rate differences were seen in the previously treated group, in the treat-naive group, both PBAC and PBAT were markedly more effective (p < 0.001) in eradicating H. pylori

| Comparison of treatment success rates between susceptibility-guided and empiric therapies
As shown in

| DISCUSS ION
With the gradual increase of H. pylori antibiotic resistance rates, there is a growing demand for routine pre-treatment antibiotic susceptibility testing to help improve the treatment success rate. To present, H. pylori antibiotic susceptibility testing via the phenotypic or molecular approach relies heavily on the availability of gastric biopsies which can be obtained only through the invasive, costly, and often long-wait-time endoscopy procedure in established tertiary hospitals, prompting a search for less invasive and cheaper alternatives. 23 The string test, invented nearly 50 years ago, has originally been used to sample human duodenal contents in a minimally invasive manner for the diagnosis of enteric parasites. 24 Since then, this method has also been proposed for other applications including its use to extract gastric material for culturing and PCR detection of H. pylori bacteria. [25][26][27] In this study, the gastric material collected by string test was innovatively used for qPCR detection of clarithromycin and levofloxacin resistance mutations, which were used for susceptibility-guided treatment. The results showed that the H. pylori eradication rate of susceptibility-guided treatment was 91.8%, which was higher than that of empirical treatment (82.3%), proving its feasibility and reliability in formulating appropriate treatment regimens and thus improving patient treatment outcomes. However, the difference between both treatment success TA B L E 2 Characteristics of the susceptibility-guided treatment group.

All (N = 88) Treatment-naive (n = 50) Previously treated (n = 38) p-value a
Age, year (mean ± SD) 40. Note: The independent sample t-test and Fisher's exact test were used to assess the statistical significance of the continuous and categorical differences between groups, respectively.
Abbreviations: A, amoxicillin; B, colloidal bismuth tartrate; C, clarithromycin; F, furazolidone; L, levofloxacin; P, proton pump inhibitor. a Statistical comparison between the treatment-naive and previously treated subjects.

TA B L E 3 Eradication success rates of susceptibility-guided
therapies between treatment-naive and previously treated subjects.

Treatmentnaive
Previously treated

| CON CLUS ION
In the present study, we discovered that the string-qPCR test is a practical, convenient, and cost-effective approach that can reliably detect H. pylori and its resistance to both clarithromycin and levofloxacin antibiotics, resulting in a high susceptibility-guided treatment success rate of 91.8%. Even among those who had past but failed H. pylori eradication treatment, an optimal treatment success rate of 86.2% could be achieved. It can be readily adopted for routine H. pylori antibiotic susceptibility testing in clinical settings to help overcome the increasing resistance to both clarithromycin and levofloxacin which are the primary drugs used to eradicate this gastric pathogen. In the events where susceptibility testing is not available, both PBAC and PBATet could be adopted as the first-line treatments based on the results in this study but may be suitable only for the Shenzhen population. Therefore, it is important that clinicians should always be aware of the local H. pylori antibiotic resistance profiles and their patient's medication history when formulating the treatment plan.

ACK N OWLED G M ENTS
We greatly thank all the doctors and patients who participated in this study. We would also like to express our gratitude to Shenzhen

CO N FLI C T O F I NTE R E S T S TATE M E NT
BJM is the co-founder for Shenzhen Hongmed-Infagen Co. Ltd.
which provides the string-qPCR testing service in this study. CYT is the honorary co-founder for Shenzhen Hongmed-Infagen Co. Ltd.
which provides the string-qPCR testing service in this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated and analyzed in this study are included in Table S1.