Helicobacter pylori (H. pylori) is known to play a major contributory role in the pathogenesiss of chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma1. Evidence has shown that H. pylori eradicationcan reduce the risks of above conditions although the data is less consistent regarding nonulcer dyspepsia¹. Clarithromycin-based standard triple therapy, which has been considered as first line therapy to treat H. pylori infection should be avoided except in areas of known low clarithromycin resistance². In patients with clarithromycin resistant H. pylori infection, risk of developing failed eradication rises to seven-fold as compared to patients with clarithromycin susceptible strains³. Bismuth-containing quadruple therapy or non-bismuth quadruple therapy is used as first-line empirical treatment in areas of high clarithromycin resistance². An earlier study conducted in our institute found lower efficacy (eradication rate – 67%) of bismuth containing drug regimen against H. pylori infection⁴. Bismuth containing quadruple therapy has not been widely used due to restricted availability of bismuth and tetracycline, complicated dosing schedule and adverse effects⁵. Even efficacy of concomitant therapy was also shown to be suboptimal with 70% intention to treat (ITT) eradication rate in our recently published study⁶. Lower efficacy of above treatment regimen may be explained by the development of drug resistance against H. pylori infection. Antimicrobial susceptibility testing against H. pylori infection is an effective method to select antibiotics, which could improve the treatment response.However, this could not be widely performed because the culture of H. pylori is time-consuming and costly. Overall, a worldwide increase in H. pylori resistance to metronidazole, clarithromycin has been observed, causing a decrease in the efficacy of the classic regimen⁷. In India, this decrease occurred unevenly due to the size of the country and socioeconomic differences between regions⁷. Many Indian studies assessed the prevalence of drug resistance against H. pylori infection. Resistance of H. pylori to clarithromycin varies from 11.8% to as high as 76%^8-10. Most of the Indian studies showed high prevalence of metronidazole resistance to H. pylori infection which varies from 81% to as high as 100%^8-10. Even, combined clarithromycin and metronidazole resistant H. pylori strain has been found in 41% to 57% patients^8-10. In view of increasing H. pylori drug resistance pattern which is associated with decreasing effcacy of H. pylori eradication over time, there is need to evaluate the other treatment options. One such treatment regimen of interest is levofloxacin containing regimen.

Levofloxacin-based triple therapy has been used as second-line and third-line rescue regimens for those who failed standard treatment, with an eradication rate ranging from 75% to 90%^1,15.  There has been a controversy regarding use of levofloxacin containing regimen as first line therapy against H. pylori infection.Toronto and Maastricht V/Florence guideline has not recommended levofloxacin containing regimen as a first line therapy contrary to American College of Gastroenterology (ACG) guideline which permitted levofloxacin to be used as first line therapy². There are few studies in India that assessed the efficacy of levofloxacin containing regimen as a first line therapy against H. pylori infection. Aim of this study was to evaluate the efficacy and tolerability of 2-week course of levofloxacin, amoxicillin and proton pump inhibitor (PAL regimen) as first-line treatment against H. pylori infection.

This prospective, single-center, open-labeled study for the treatment of H. pylori-positive patients was conducted between November 2018 and January 2020. This study included consecutive patients with dyspeptic symptoms who underwent upper gastro-intestinal endoscopy (UGIE). Those patients who underwent UGIE for other indication and found to have mucosal hyperemia, erosions or ulcers in stomach or duodenal bulb incidentally were also evaluated for H. pylori infection. Informed written consent was taken from each patient. Patients with history of prior H. pylori therapy, allergy to drugs used in treatment of H. pylori infection, use of  PPI, antibiotics or anticoagulant drugs within 1 month of enrolment, concomitant significant co-morbidities including malignancy, pregnant or lactating women or patients with age younger than eighteen years or older than eighty years were excluded from the study. Patients who refused to give consent to participate in study were also excluded. Study was approved by institute ethics committee and conformed to the declaration of Helsinki.

Detailed baseline demographic, clinical and endoscopic features were evaluated. UGIE was done after eight hours fasting. Endoscopic findings were described as ulcer, gastritis or duodenitis. Gastritis or duodenitis was defined as localized or diffuse mucosal hyperaemia or erosions seen endoscopically in stomach or duodenum respectively. Ulcer was defined as breach in mucosal continuity of size 5 mm or more with apparent depth.

Before enrolment, H. pylori infection was assessed by using rapid urease test (RUT), gastric biopsy with histological examination or stool antigen assay. Post-treatment H. pylori eradication was assessed after 4 to 6 weeks of completion of therapy by using stool antigen assay or histological evidence of H. pylori infection in gastric biopsy specimen. We did not use urea breath test to diagnose H. pylori infection as facility of this test is not available in our institute. In RUT test, one biopsy each was taken from corpus and antrum of stomach. Biopsied material along with one drop of distilled water was introduced into yellow media of RUT kit which contains urea and pH indicator. We used RUT DRY test kit (Gastro Cure System, Kolkata, India) for RUT testing. Urease enzyme released from H. pylori hydrolyzed urea in RUT kit to produce ammonia. Ammonia raised the pH of media resulting in change in color of media from yellow to red. If color of media remains unchanged even after 3 hours of introducing biopsied material into media, patients were considered negative for H. pylori infection. In histological examination to detect H. pylori infection, multiple biopsy materials were taken from mid body and mid antrum along greater and lesser curvature and from incisura and were submitted to hematoxylin and eosin or Giemsa staining. In stool antigen assay, bacterial antigen in stool was detected by immunochromatography method.

Patients with H. pylori infection were treated with amoxicillin (1000 mg twice daily after breakfast and supper), levofloxacin (500 mg once daily after breakfast) and esomeprazole (40 mg twice daily before breakfast and supper) for 14 days. 

All patients were explained regarding the drug regimen and possible adverse effects. Treatment compliance and adverse effects were evaluated during treatment period telephonically and after completion of therapy in outpatient clinic. Patients were asked to bring used strips of medicine at follow up to check for compliance.Compliance was assessed by pill count. Patients were said to be compliant if pill intake was more than 80% of total pills. Individuals poorly complying were not taken into account in the per-protocol (PP) analysis. Adverse events were determined by asking open-ended questions using patient self-reports and physical examinations, and grouped into the mild (no interference with daily routine), moderate (limited effects on daily routine) and severe (marked effects on daily routine and medication discontinuation).

The primary outcome was eradication rates of H. pylori infection. Secondary outcomes included adverse event and compliance rates. 

We assumed 85% efficacy of H. pylori eradication and 10% probability of loss of follow up16. The final sample size was calculated as 136 patients. Descriptive analyses were presented as mean±standard deviation (SD) for quantitative variables and absolute numbers or percentages for qualitative variables. Eradication rates and 95% confidence interval (CI) were determined. Analysis of H. pylori eradication was performed on ITT and on PP basis. ITT analysis included all patients, who was administered at least one drug dose and PP analysis included patients with complete adhesion to the trial protocol. Univariate analysis was carried out to determine factors predicting H. pylori eradication. Multivariate logistic regression analysis was planned to perform for variables found statistically significant in univariate analysis. p-value < 0.05 was considered statistically significant. SPSS version 20 was used for all statistical analyses.

140 consecutive patients, positive for H. pylori infection by RUT method were included. Main indications for UGIE were dyspepsia (n=113), hematemesis or melena (n=10), epigastric pain (n=7), recurrent vomiting (n=5), anemia (n=3) and heart burn (n=2). Five patients were excluded from study. Causes of exclusion were as follows: 

1) Gastric resection found during endoscopy in one patient, 2) Gastric adenocarcinoma diagnosed in biopsy sample of one patient with endoscopic feature of gastric ulcer, 

3) History of prior H. pylori treatment elicited in one patient, 4) One patient was allergic to fluoroquinolones and 5) One patient did not give consent for drug regimen after endoscopy. Therefore, PAL regimen was started to 135 H. pylori positive patients. One patient discontinued treatment due to development of severe dizziness. One patient was lost to follow-up. Therefore, 133 patients completed 14 days course of PAL regimen. Two patients did not turn up for follow-up endoscopy or stool antigen assay to assess eradication. H. pylori eradication was confirmed by either stool antigen assay (n=69) or histological evidence of H. pylori (n=62). Therefore, 131 patients entered into study for final analysis as shown in flow chart (Figure 1).

Table 2 shows the eradication rate of H. Pylori infection by using PAL regimen. In PP analysis, 110 (83.97%, 95% CI:78-90) patients achieved H. pylori eradication. We did ITT analysis which included patients who were lost to follow-up or who discontinued treatment due to development of adverse effects and considered them as treatment failure. In ITT analysis, 81.48%, 95% CI:75 - 88 (110 out of 135) patients achieved H. pylori eradication. On univariate analysis to assess the factors predicting H. pylori eradication,we did not find correlation of H. pylori eradication with age (p=0.34), sex (p=0.92), duodenal (p=0.16) or gastric lesion (p=0.43), ulcerated (0.77) or nonulcerated lesion, smoking (p=0.90), alcohol (0.35) or diabetic status (0.46).   

Treatment regimen was generally well tolerated and 98.5% patients were compliant to therapy. Twenty-four patients reported with adverse effects. Most adverse effects were mild in nature. Mild abdominal cramp, nausea and mild diarrhea were seen in seven, five and four patients respectively. One patient developed mild skin rash and responded to antihistaminic drug. Three patients developed mild abdominal cramp and diarrhea.Anorexia and bloating were seen in three patients. One patient discontinued treatment due to development of severe dizziness.

H. pylori is an infectious disease that is typically treated with combinations of 2-3 antibiotics along with a PPI, taken concomitantly or sequentially, for periods ranging from 7 to 14 days¹. First line therapy, which is referred to as the initial course of eradication therapy, usually provides the greatest likelihood of H. pylori eradication. Thus, it is important to select the most appropriate first-line eradication therapy for an individual patient. In recent years, H. pylori eradication has been increasingly difficult, mostly due to increased antibiotic resistance especially to clarithromycin and metronidazole. Bismuth quadruple or non-bismuth quadruple concomitant therapies have been recommended in areas of high (>15%) clarithromycin resistance^1,17. In regions of high dual clarithromycin (>15%) and metronidazole resistance (>40%), bismuth-containing quadruple therapies are the treatment of choice^1,17. In clinical practice, bismuth-based quadruple therapy has not been widely used because of limited availability of bismuth salt and tetracycline and suboptimal compliance due to complex dosing schedule. Presence of high dual drug resistance to metronidazole and clarithromycin has restricted the use of concomitant, sequential and hybrid therapy.

Levofloxacin-based triple therapy has been shown to be effective as second-line and third-line rescue regimens. There is scanty data in India that assessed the efficacy of levofloxacin containing regimen as a first line therapy against H. pylori infection. In our study, PAL therapy as a first line therapeutic regimen showed good efficacy against H. pylori infection. H. pylori eradication was seen in about 84% and 81.5% patients in per PP and ITT analysis respectively. This may be considered as an effective regimen against H. pylori infection especially in populations with high dual resistance to clarithromycin and metronidazole. A meta-analysis of seven randomized controlled trials (RCTs) comparing levofloxacin-based therapy with standard triple therapy for 7 days and found 79% ITT eradication rate in levofloxacin-based therapy. Out of these seven RCTs, four trials contained levofloxacin, amoxicillin and PPI as therapeutic regimen with ITT eradication rate ranging from 65% to 85%16. In addition, many non RCT studies also showed 75% to 96% H. pylori eradication rate^14,18-20. We found three studies in India that assessed the efficacy of levofloxacin based triple therapy to eradicate H. pylori infection. Two RCTs from south India that used PAL regimen showed H. pylori eradication in 67.6% and 80% patients respectively in ITT analysis^21,22. An open label study from north India that used levofloxacin, tinidazole and PPI as therapeutic regimen showed H. pylori eradication in 91.8% and 85.5% patients in PP and ITT analysis respectively²³. Most of the previous studies used seven days course of levofloxacin-based therapy which is contrary to our study in which we have given 14 days course. A study comparing 10 days versus 14 days course of levofloxacin containing regimen for H. pylori eradication, showed better eradication with 14 days course (86% vs. 68% in ITT analysis, p=0.002)²⁴. The duration of H. pylori therapy was shown to be an independent risk factor for H. pylori eradication24. Variation in efficacy of PAL therapy could be due to development of drug resistance used for eradication of H. pylori infection. Data on levofloxacin resistance is limited in India. A study from western India showed that 13.8% H. pylori strains were resistant to levofloxacin⁹. On the other hand,73.2% and 54.9% of H. pylori strain from north and south India developed levofloxacin resistance^7,25. Over last few years, many studies have been done worldwide which showed levofloxacin resistance to H. pylori infection ranges from 19% to 45%²⁶. Efficacy of levofloxacin triple regimen was significantly higher for levofloxacin-susceptible strains than for resistant strains (eradication rate: 81.1% vs 36.3%) as shown in a meta-analysis by Chen et al²⁷. Efficacy of PAL regimen may be improved by adding bismuth salt to the regimen. Recently a study found that addition of bismuth salt to PAL therapy improved the H. pylori eradication rate by about 9%²⁸. Bismuth salt decreases the bacterial load and hence increases the eradication rate²⁹. It possesses antibacterial effects by inhibiting various enzymes produced by H. pylori including urease, catalase, and phospholipase, preventing adhesion of bacteria to surface epithelial cells, inhibiting ATP synthesis and some membrane functions in H. pylori²⁸.

We used the combination of levofloxacin, amoxicillin and esomeprazole (PAL regimen) as first-line therapy to eradicate H. pylori infection. This regimen is found to be effective, compliant, well tolerated and could be used as an alternative first line regimen especially in area of high dual clarithromycin and metronidazole resistance.