Modified sequential H. pylori therapy: PPI and amoxicillin for 14 days with clarithromycin and metronidazole added as a quadruple (hybrid) therapy for the final 7 days

Abstract

Background

Ten day sequential therapy with a proton pump inhibitor (PPI) and amoxicillin followed by a PPI, clarithromycin, and an imidazole typically achieves Helicobacter pylori eradication rates of 90 to 94% (Grade B success).

Aims

We tested whether prolonging treatment and continuing amoxicillin throughout the 14-day treatment period would produce a ≥95% result.

Methods

This was a multi-center pilot study in which H pylori infected patients received a 14-day sequential-concomitant hybrid therapy (esomeprazole and amoxicillin for 7 days followed by esomeprazole, amoxicillin clarithromycin, and metronidazole for 7 days). H pylori status was examined 8 weeks after therapy. Success was defined as achieving ≥95% eradication by per-protocol analysis.

Results

117 subjects received hybrid therapy. The eradication rate was 99.1% (95% CI, 97.3%–100.0%) by per-protocol analysis and 97.4% by intention-to-treat analysis (95% CI, 94.5–100.0%). Adverse events were seen in 14.5%; drug compliance was 94.9%.

Conclusions

14-day hybrid sequential-concomitant therapy achieved greater than 95% H. pylori eradication (Grade A result). Further studies are needed (i) in regions with different patterns and frequencies of resistance to confirm these findings and (ii) to examine whether Grade A success is maintained with hybrid therapy shorter than 14 days.

Introduction

Until recently triple regimen with a proton pump inhibitor (PPI) plus clarithromycin and amoxicillin or metronidazole administered for 7 to 14 days was often regarded as the gold standard for H pylori eradication [1,2]. It has become recognized that treatment success with this regimen has generally declined to unacceptable levels (i.e., 80% or less) [ 3] and that the fall in efficacy is primarily related to an increase in the prevalence of clarithromycin-resistance [4,5].

The decline in treatment success with triple therapy resulted in the introduction of new regimens such as a 10-day sequential regimen consisting of a dual therapy (PPI and amoxicillin for 5 days), followed by triple therapy with a PPI, clarithromycin and tinidazole (or metronidazole) triple therapy for 5 days [6,7] The mean H pylori cure rate of the new therapy has been reported to be 91.3% by intention-to-treat (ITT) analysis [6] and recent studies with antimicrobial susceptibility testing have confirmed that the superiority of sequential therapy over standard triple therapy is primarily due to an improved outcome with clarithromycin-resistant strains [6,7] However, sequential therapy has not reliably achieved 95% or greater treatment success or Grade A success based on grading treatment success as ordered categories (by PP analysis) of ≥95% = A, 90–94% = B, 86–89% = C and ≤ 85% = F) [8–10]. In contrast, treatment success of common bacterial infections is not typically based on a comparison to another regimen but rather acceptable or unacceptableness is based on absolute outcome. [11,12].

The factors that affect outcome of sequential therapy such as dose, duration etc [12] were chosen empirically and the regimen has not yet been optimized. It has been suggested that sequential might be improved by increasing the duration of therapy (e.g., to more than 10 days) [9,10,13]. Here we used the recently describe three stage approach to identifying a regimen with high treatment success [14]. These steps include: 1) pilot studies to identify tentative highly successful regimens, 2) further pilot studies to simplify and refine the regimen while maintaining the efficacy of 95% or greater, and 3) large scale multicenter trials to confirm that the optimal therapy is successful in different regions with different patterns of disease, and antimicrobial susceptibility.

Concomitant therapy is another regimen proven successful in the presence of clarithromycin resistance and superior to triple therapy [15]. Concomitant therapy is a 4-drug non-bismuth containing regimen containing the same drugs as does sequential therapy but they are all given for the entire duration of therapy instead of sequentially. A head-to-head non-inferiority trial of sequential and concomitant therapy showed they were equivalent, however, in that trial neither achieved 95% treatment success [13]. It has previously been suggested that sequential might be improved by continuing the amoxicillin throughout the entire treatment period which would produce a sequential-concomitant quadruple therapy (hybrid therapy) [9,10,13]. In this study, we report the results the initial pilot study to investigate whether increasing the duration of sequential therapy to 14-days and continuing the amoxicillin throughout the 14-day treatment period resulted in ≥95% treatment success. The second pilot study tested whether increasing the duration of sequential therapy to 14 days (7+7) would produce treatment success to 95% or greater. The results can be found in the companion paper [16].

Methods

Setting and Participants

The study was designed as two concomitantly conducted but separate pilot studies [14,17]. Patients were randomized into the two pilot study in order to eliminate selection bias. The details of that study are found in the companion paper [16]. Patients who visited the gastroenterology clinics of the Kaohsiung Veterans General Hospital, Kaohsiung Medical University, and Kaosiung Municipal Hsiao-Kang Hospital between August 2008 and January 2010 for dyspeptic symptoms were surveyed for H pylori infection. Those with H pylori infection were recruited. Pre-enrollment procedures included biopsy of gastric mucosa where the presence of H pylori was assessed by rapid urease test and histological examination. Active H pylori infection was defined as positive results of both rapid urease test and histology. Blood samples were taken for routine laboratory tests including complete blood count, renal function and liver biochemical tests to ascertain that there were no abnormal tests that would preclude entry into the trial.

Exclusion criteria included (a) previous attempted H pylori-eradication therapy, (b) ingestion of antibiotics, bismuth, or PPIs within the prior 4 weeks, (c) patients with allergic history to the medications used, (d) patients with previous gastric surgery, (e) the coexistence of serious concomitant illness (for example, decompensated liver cirrhosis, uremia), (f) pregnancy or lactation, and (g) presence of serous medical condition(s) precluding participation or endoscopy with biopsy.

Intervention

Eligible patients received a sequential-concomitant hybrid therapy (hybrid therapy consisting of esomeprazole 40 mg and amoxicillin 1 g twice daily for 7 days followed by esomeprazole 40 mg, amoxicillin 1 g, clarithromycin 500 mg and metronidazole 500 mg twice daily for 7 days). All drugs were taken one hour before breakfast and dinner. Patients were given a written instruction on how to take the medications correctly.

A trained interviewer used a standard questionnaire to obtain demographic data and medical history of the patients. Patients were asked to return two weeks after the start of drug administration to assess drug compliance and adverse events. Drug compliance was assessed via pill counts. Compliance was defined as good defined as taking more than 90% of the total medication or poor (i.e., taking less) by counting unused medication. As is the standard practice locally, patients with peptic ulcers at the initial endoscopy received an additional three weeks of monotherapy with esomeprazole 40 mg orally once daily whereas patients with H. pylori gastritis without peptic ulcer received three-weeks of antacid (aluminum hydroxide 334 mg, Lederscon®, Taiwan, q.d.s.) following eradication therapy.

Eradication efficacy was assessed by a follow-up endoscopy with rapid urease test and histological examination eight weeks after the end of anti-H. pylori therapy. If patients declined follow-up endoscopy, ¹³C-urea breath tests were conducted to assess H. pylori status. Eradication was defined as either negative results of both rapid urease test and histology, or a negative result of the urea breath test.

All participants gave written informed consent. The Medical Committee of the Kaohsiung Veterans General Hospital approved the trial (VGHKS97-CT6-08). The study was registered on ClinialTrials.gov; registrations # NCT01085786.

Questionnaires

A complete medical history and demographic data were obtained from each patient, including age, sex, medical history, history of smoking, and alcohol, coffee and tea consumption. Adverse events were prospectively evaluated. The adverse events were assessed according to a 4-point scale system: none; mild (discomfort annoying but not interfering with daily life); moderate (discomfort sufficient to interfere with daily life); and severe (discomfort resulting in discontinuation of eradication therapy) [18]. All participants gave written informed consent. The Medical Committee of the Kaohsiung Veterans General Hospital approved the trial (VGHKS97-CT6-08). The study was registered on ClinialTrials.gov; registrations # NCT01085786.

Rapid urease test

The rapid urease test was performed according to our previous studies [19]. A biopsy specimen taken from the antrum was placed immediately in 1 mL of a 10% solution of urea in deionized water (pH 6.8) to which two drops of 1% phenol red solution was added and incubated at 37°C for up to 24 hours. If the yellowish color around the area of inserted specimen changed to bright pink within the 24-hour limit, the urease test was considered positive. In our laboratory, the sensitivity and specificity of rapid urease test were 96% and 91% respectively [19].

Histological examination

Gastric specimens were taken one specimen from the lesser curvature site of the antrum and another from the lesser curvature site of the corpus for histological examination, fixed in 10% 10% buffered formalin, embedded in paraffin, and sectioned. The sections, 4-μm thick, were stained with a haematoxylin and eosin stain and a modified Giemsa stain to observe the presence of curved rod shape bacteria on the mucosal surface. Biopsy specimens were assessed by histopathologists, blinded to patient status and the results of other laboratory tests.

Urea breath test

The urea breath test was performed according to our previous studies [20]. The cutoff value was set at 4.8‰ of δ¹³CO₂. The staff who were blind to the H. pylori status performed the tests.

Culture and antimicrobial resistance

Culture of H pylori was performed in the patients recruited from the Kaohsiung Veterans General Hospital and the Kaohsiung Medical University as in both sites culture facilities were available. One antral gastric biopsy specimen was obtained for isolation of H pylori, using previously described culture methods [21]. The organisms were identified as H pylori by Gram staining, colony morphology, and positive oxidase, catalase, and urease reactions.

The antibiotic susceptibility was tested by E test (AB Biodisk, Solna, Sweden). H pylori subculturing was done by rubbing the specimens on the surface of a Campy-BAP agar plate (Brucella agar; Difco, Sparks, Maryland) + IsoVitalex (Gibco, Grand Island, New York) + 10% whole sheep blood) followed by incubation at 37°C under microaerobic conditions (5% O2, 10% CO2, and 85% N2) for 4–5 days. H pylori strains were tested for clarithromycin, amoxicillin, and metronidazole susceptibility using the E-test (AB Biodisk, Solna, Sweden). H pylori strains with a minimal inhibitory concentration value >1 μg/mL >0.5 μg/mL, and >8 μg/mL, were considered to be resistant to clarithromycin, amoxicillin, and metronidazole, respectively [22].

Statistical analysis

The study was designed as a pilot study based on the three stage approach to identify a new effective therapy [14, 17]. The requirement for treatment success of ≥95% was based on the concept that H pylori is an infectious disease and the results should be similar to other infectious diseases [3,11,12,14]. With infectious diseases such as H. pylori infection there is essentially no placebo effect (i.e., treatment success with placebo is zero) and results less than 99–100% can almost always be explained in terms of resistance, or failure to use the dosages or duration of therapy. As such there is no need for, or advantage to use of, a comparator regimen as the treatment results can be judged based on achieving an eradication rate equal to or greater than a prespecified outcome (e.g., 100% success) [12]. Based on the results of our prior study, [13] we expected the eradication rate when used as an empiric therapy to be greater than 90% (i.e., to fall between 93 and 99%).

Success was prespecified as a cure rate ≥95% (i.e., Grade A) by per-protocol (PP) analysis and failure as treatment success lower than 95% [14]. The primary analysis was PP. Of note, intention-to-treat (ITT) analyses have been described as most suitable for pragmatic trials of effectiveness rather than for explanatory investigations of efficacy [23].

Sample sizes for pilot studies of new therapies are generally somewhat arbitrary (i.e., there is no null hypothesis and the goal is usually to obtain an estimate of the effectiveness so as to design new studies more effectively). Here, success was defined as being able to eradicate at least 95% of infections. The choice of a sample size has both practical issues (e.g., cost, availability of subjects) and the level of confidence in the results one desires. The 95% confidence interval (CI) decreases as sample size increases. Because of the expectation of a very high success rate only the lower 95% CI was of interest (Figure 1). From experience we knew we could enter approximately 250 patients over an 18-month period. As we wished to do two completely independent pilot studies, we chose approximately 120 per group based on the facts that the sample size was within our capability and that the lower boundary of the 95% CI was less than 7% at any sample size of greater than approximately 80 (i.e., any sample size greater than 75–80 would have been suitable) (Figure 1). Approximately 120 patients were chosen because it would give a fairly reliable estimate of the true outcome (i.e., relative narrow 95% CI) and if the true treatment success was ≥95%, that fact would not likely be missed.

Figure 1.

The lower 95% confidence interval is shown for different sample sizes achieving a 98% treatment success.

A computer-generated randomization list was used to generate a ‘random sequence and potential subjects were randomized by an independent staff member and were assigned the treatments according to consecutive numbers using sealed envelopes. This study was an open-labeled trial and patients were not blinded. The two studies were otherwise completely independent.

Secondary outcomes were proportions of adverse events and compliance. To determine the independent factors affecting the treatment response, clinical, endoscopic and bacterial factors are analyzed by Chi-square test with or without Yates correction for continuity and Fisher’s exact test using the SPSS program (version 10.1, Chicago, Illinois, USA). A P value ≤0.05 was considered statistically significant. These variables include the following: age (<60 or ≥60 years), gender, history of smoking (<1 pack/week or ≥1 pack/week), history of alcohol consumption (<80 g/day or ≥80 g/day), ingestion of coffee (<1 cup/day or ≥1 cup/day), ingestion of tea (<1 cup/day or ≥1 cup/day), coexistence of a systemic disease (yes or no), clinical disease (peptic ulcer or functional dyspepsia), adverse event (presence or absence), drug compliance (good or poor), and antibiotic resistance (presence or absence). Because case number of the patients with eradication failure in each subgroup was too small, examination by regression analysis to identify independent factors influencing eradication outcome was not performed.

Results

Characteristics of the study groups

A total of 117 H. pylori-infected patients received hybrid therapy. All randomized subjects were included in the ITT analysis for H. pylori eradication. Data regarding the clinical characteristics of patients at entry are summarized in Table 1. Among the subjects, six with poor compliance and two with incomplete follow-up were excluded from PP analysis for H. pylori eradication.

Table 1.

Demographic data of the patients

Characteristics	Hybrid therapy (n = 117)
Age (yr) (mean ± SD)	54.3 ± 11.4
Gender (male / female)	59/58
Smoking	19 (16%)
Alcohol consumption	5 (4%)
Ingestion of coffee	26 (22%)
Ingestion of tea	46 (39%)
NSAID user	7 (6%)
Underlying diseases	32 (27%)
Endoscopic Findings
Gastritis	33 (28%)
Gastric ulcer	29 (25%)
Duodenal ulcer	33 (28%)
Gastric ulcer and duodenal ulcer	22 (19%)
Antibiotic sensitivity a
Clarithromycin (susceptible/resistance)	53/4
Amoxicillin (susceptible/resistance)	56/1
Metronidazole (susceptible/resistance)	25/32

^aFifty-seven strains were isolated.

Antibiotic resistance

H. pylori strains were successfully isolated from 57 (80.3%) of the 71 patients recruited in the Kaohsiung Veterans General Hospital and the Kaohsiung Medical University. The rates of resistance were: clarithromycin - 7% (4/57); amoxicillin - 2% (1/57), and metronidazole - 56% (32/57) of the patients, respectively.

Eradication of H. pylori

Table 2 lists the therapeutic outcomes. The eradication rate according to the PP analyses, H. pylori infection was eradicated in (108/109 or 99.1% (95% CI, 98.9% – 100.0%) of those receiving the hybrid therapy. By ITT analysis treatment success was 114/117 or 97.4% (95% CI, 94.5% – 100.0%) in hybrid therapy. Hybrid therapy successfully achieved a grade A eradication outcome using either the PP or the ITT criteria.

Table 2.

The major outcomes of 14-day hybrid therapy

Parameters of Outcomes	Hybrid therapy (n = 117)
Eradication rate
Per-protocol	99.1% (108/109) (97.3% – 100.0%)
Intention-to-treat	97.4% (114/117) (94.5% – 100.0%)
Adverse events	14.5% (17/117) (8.1% – 20.9%)
Compliance	94.9% (111/117) (91.0% – 98.8%)

^a95% confidence interval

Adverse events and compliances

All of the patients received at least one dose of eradication medication and were included in the adverse event analysis. In total, 14.5% (17/117) of those treated with hybrid therapy reported at least one adverse event during eradication therapy. The profiles and frequencies of adverse events are listed in Table 3. Five patients discontinued treatment owing to adverse events during eradication therapy (diarrhea: 1 patient; dizziness: 1 patient; nausea: 1 patient; vomiting: 1 patient; skin rash: 1 patient).

Table 3.

Adverse events of 14-day hybrid therapy

Adverse Events	Hybrid therapy (n = 117)
Abdominal pain	4 (1/2/1)
Constipation	0 (0/0/0)
Diarrhea	8 (6/1/1)
Dizziness	6 (4/1/1)
Taste perversion	8 (7/1/0)
Headache	5 (4/1/0)
Anorexia	1 (1/0/0)
Nausea	3 (0/1/2)
Vomiting	2 (0/1/1)
Skin rash	1 (0/0/1)
Fatigue	2 (2/0/0)
Others	4 (2/1/1)

^aNumbers of patients who suffered from mild, moderate and severe adverse events.

The percentage of total medication taken by the subjects was 10.7% – 100.0%. Five patients took less than 90% of the assigned tablets because of severe adverse events, and one patient forgot to take more than 10% of medicines during anti-H pylori therapy. Overall, six patients had poor drug compliance (compliance rate 94.9%).

Factors influence efficacy of eradication with 14-day sequential-concomitant hybrid therapy

Univariate analysis for the clinical and bacterial factors did not identify any risk factors associated with treatment failure by sequential-concomitant hybrid therapy (Table 4). The proportion with H. pylori eradication were similar between those with gastritis only and those with peptic ulcer subjects (97.0% [32/33] vs. 97.6% [82/84], P = 1.00). Additionally, all those with amoxicillin-, clarithromycin- and metronidazole-susceptible and resistant strains (i.e., 32/32 or 100% had H. pylori eradication both by ITT and PP analyses.

Table 4.

Univariate Analysis of the Clinical Factors Influencing the Efficacy of 14-day hybrid Therapy

Principle parameter	No of patients	Eradication rate	P value
Age			0.55
< 60 y	74	98.6% (73/74)
≥ 60 y	43	95.3% (41/43)
Sex			1.00
Female	59	96.6% (57/59)
Male	58	98.3% (57/58)
Smoking			1.00
(−)	98	96.9% (95/98)
(+)	19	100.0% (19/19)
Alcohol consumption			1.00
(−)	112	97.3% (109/112)
(+)	5	100.0% (5/5)
Ingestion of coffee			1.00
(−)	91	96.7% (88/91)
(+)	26	100.0% (26/26)
Ingestion of tea			0.28
(−)	71	96% (68/71)
(+)	46	100.0% (46/46)
NSAID user			1.00
(−)	110	97.3% (107/110)
(+)	7	100.0%
Underlying diseases			0.18
(−)	85	98.8% (84/85)
(+)	32	93.8% (30/32)
Gastroduodenal diseases			1.00
Non-ulcer dyspepsia	33	97.0% (32/33)
Peptic ulcer	84	97.6% (82/84)
Adverse event			0.38
(−)	100	98.0% (98/100)
(+)	17	94.1% (16/17)
Compliance			1.00
Good	111	97.3% (88/111)
Poor	6	100.0% (6/6)
Antibiotic resistance
Clarithromycin resistance			—
susceptible	53	100.0% (53/53)
resistant	4	100.0% (4/4)
Amoxicillin			—
susceptible	56	100.0% (56/56)
resistant	1	100.0% (1/1)
Metronidazole resistance			—
susceptible	25	100.0% (25/25)
resistant	32	100.0% (32/32)
Presence of any resistant strain			—
(−)	25	100.0% (25/25)
(+)	32	100.0% (32/32)
Dual resistance			—
(−)	53	100.0% (53/53)
(+)	4	100.0% (4/4)
Triple resistance			—
(−)	57	100.0% (57/57)
(+)	0	—

^aFifty seven strains were isolated.

Discussion

The fall in H pylori eradication rates with standard triple therapies resulted in a search for novel therapies for H pylori infections [3]. Here, we tested the hypotheses that extending the duration of sequential therapy and continuing the amoxicillin for the full 14 days of therapy could improve treatment outcome from the typical 90% to 94% to 95% or greater (i.e., from Grade B to Grade A). We found that the 14-day sequential-concomitant hybrid therapy produced 99.1% treatment success by PP analysis. The eradication rate by ITT analysis was 97.4% (95% CI, 94.5–100%). In the companion paper, a 14-day sequential therapy achieved an eradication rate of 93.9% by PP analysis (Grade B success) and 91.9% by ITT analysis. Taken together, extending sequential therapy to 14-days did not result in a Grade A result. However, the novel 14-day hybrid therapy could achieve a Grade A success.

In this study, we found no risk factors were associated with treatment failure by univariate analysis. Specifically, hybrid therapy achieved a high eradication rate for both peptic ulcer patients and the patients with non-ulcer dyspepsia (97.6% and 97.0%, respectively; P = 1.00). According to our analysis, there were also no significant relationships in antibiotic resistance rates between H. pylori strains from NUD and PU patients. However, the numbers of cases were probably too small to avoid type II error

One limitation of the current study is that although it was done in three-centers, they were all in a single country and region of that country and the results will need to be confirmed in regions where different patterns of resistant are present. Secondly, the numbers of cases with eradication failure were too small and precluded further regression analyses. Nonetheless, we confirmed the hypothesis that continuing the amoxicillin through the entire 14 days resulted in a Grade A result. We are currently investigating whether Grade A success can be maintained by the hybrid therapies with less than a 14 day treatment duration using 3 pilot studies evaluating therapies of 10 days (3+7), 12 days (5+7) and 14 days (7+7).

In conclusion, a 14-day sequential-concomitant hybrid therapy achieved Grade A treatment success for H. pylori eradication. Larger studies and studies in different regions are needed to confirm this finding and to examine the optimal treatment duration of hybrid therapy.

Abbreviations used in this paper

CI

confidence interval

ITT

intention to treat

H pylori

Helicobacter pylori

PP

per protocol

PPI

proton pump inhibitor