Clinical trial NCT02978157
Conditions: Helicobacter Pylori Infection
Keywords: Helicobacter pylori Eradication rate Rescue treatment
Sponsor: Kaohsiung Veterans General Hospital.
Start date: 2015-02-01; Completion date: 2017-09-01
Enrollment: 102 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
From the profiles of antibiotic susceptibility data following eradication therapy, tetracycline, amoxicillin and levofloxacin are all good candidates of antibiotics used in the rescue treatment.

Detailed description:
The H pylori-infected adult patients with failure of standard triple therapy and H pylori-infected adult patients with failure of non-bismuth quadruple therapy are randomly assigned to either EAL (esomeprazole 40 mg b.d., amoxicillin 500 mg q.d.s., and levofloxacin 500 mg o.d.) or EBTL (esomeprazole 40 mg b.d., bismuth 500 mg q.d.s., tetracycline 500 mg q.d.s., and levofloxacin 500 mg o.d.) therapy for 10 days. Repeated endoscopy with rapid urease test, histological examination and culture or urea breath tests is performed at six weeks after the end of anti-H pylori therapy.

Arms: arm 1: esomeprazole 40 mg b.d., amoxicillin 500 mg q.d.s., and levofloxacin 500 mg o.d. arm 2: esomeprazole 40 mg b.d., bismuth 120 mg q.d.s., tetracycline 500 mg q.d.s., and levofloxacin 500 mg o.d.
Interventions: intervention 1: esomeprazole+amox+levo intervention 2: esomeprazole+bismuth+tetra+levo
Intervention descriptions: intervention 1: esomeprazole 40 mg b.d., amoxicillin 500 mg q.d.s., levofloxacin 500 mg o.d. intervention 2: esomeprazole 40 mg b.d., bismuth 120 mg q.d.s., tetracycline 500 mg q.d.s., and levofloxacin 500 mg o.d.

Protocol documents:
Prot_SAP_000.pdf: 
VGHKS104-062
Institution/Department：Kaohsiung Veterans General Hospital
Principal Investigator (PI)：Prof. Ping-I Hsu 
Research Project Title：A prospective randomized trial of levofloxacin-amoxicillin 
triple therapy vs. levofloxacin-tetracycline quadruple therapy 
in second-line Helicobacter pylori treatment
NCT Number：NCT02978157 
Date of the document：2017/09/30 
ABSTRACT
Background: Although proton pump inhibitor（PPI）-amoxicillin-levofloxacin triple therapy has 
been recommended by the Maastricht IV/Florence Consensus Report for recue treatment of both 
standard triple therapy and non-bismuth quadruple therapy, the eradication rates of the rescue 
treatment for  standard triple therapy and non-bismuth quadruple therapy by intention-to-treat 
analysis are only 66.9% and 77.5%, respectively. It is therefore mandatory and urgent to develop a 
highly effective rescue therapy for second-line treatment of H pylori infection. From the profiles of 
antibiotic  susceptibility  data  following  eradication  therapy,  tetracycline,  amoxicillin  and 
levofloxacin are all good candidates of antibiotics used in the rescue treatment for both standard 
triple therapy and non-bismuth quadruple therapy. Our recent study also revealed that PPI- bismuth-
tetracycline-levofloxacin rescue treatment might achieve a very high eradication rate 96% for H 
pylori infection after failure of non-bismuth quadruple therapy.
Aims: To investigate (1) the efficacy of PPI- bismuth -levofloxain-tetracycline quadruple therapy 
and PPI-amoxicillin-levofloxacin triple therapy in rescue treatment for H pylori infection, and (2) 
the bacterial and host factors determining the efficacy of PPI-bismuth-levofloxain-tetracycline 
quadruple therapy and PPI-amoxicillin-levofloxacin triple therapy. 
Methods: Consecutive 164 H pylori-infected adult patients with failure of first-line therapies are 
randomly assigned to either EBLT (esomeprazole 40 mg b.d., bismuth 120 mg q.d.s., levofloxacin 
500 mg o.d., and tetracycline 500 mg q.d.s.) therapy or EAL (esomeprazole 40 mg b.d., amoxicillin 
1


VGHKS104-062
500 mg q.d.s., and levofloxacin 500 mg o.d.) for 10 days. Additionally, antibiotic susceptibility of 
H pylori and CYP2C19 polymorphism of the hosts are examined. Urea breath test is performed at 
six weeks after the end of anti-H pylori  therapy. The rates of eradication, adverse events and 
compliance will be compared between EAL and EBTL group by chi-square test. Besides, the host 
and bacterial factors influencing the efficacy of above two salvage regimens are assessed.
INTRODUCTION
Helicobacter pylori (H pylori) infect more than 50% of humans globally. It is the 
principal cause of chronic gastritis, gastric ulcer, duodenal ulcer, gastric adenocarcinoma and 
gastric mucosa-associated lymphoid tissue lymphoma (MALToma) [1,2]. H pylori eradication 
has become the standard and most widely adopted therapy to cure peptic ulcer disease [3]. This 
therapy is also strongly recommended in the treatment of H pylori-related MALToma [4]. In 
regions with high incidence of gastric adenocarcinoma, eradication of H pylori is advocated as 
a preventative measure [5].
Standard triple therapy has been recommended as first-line regimen for H pylori infection 
in main international guidelines [6,7]. However, several large clinical trials and meta-analyses 
have  shown  that  the  eradication  rate  of the  standard  therapy  has  generally  declined  to 
unacceptable levels (i.e., 80% or less) recently [8,9]. In some European countries, the success 
rates are disappointingly low with values only 25-60% [10,11]. Therefore, several novel first-
line therapies including sequential therapy, concomitant therapy and hybrid therapy have 
emerged to treat naive H pylori infection [12-14]. 
The Maastricht IV/Florence Consensus Report [15] has recommended treatment of  H 
pylori infection  according  to  antibiotic  resistance  rates  in  local  areas  recently.  In  some 
countries with low clarithromycin resistance (< 15%) of H pylori, standard triple therapy is 
still the best option for first-line anti-H pylori therapy, but bismuth-containing quadruple 
therapies such as sequential therapy and concomitant therapy are the preferred option in 
countries with clarithromycin resistance >20%. 
2


VGHKS104-062
In clinical practice, the regimen for second-line therapy of H pylori infection depends on 
the regimen used in first-line therapy. After failure of a standard triple therapy, either a 
bismuth-containing  quadruple  therapy  or  PPI-amoxcillin-levofloxacin  triple  therapy  is 
recommended [15]. Bismuth–containing quadruple therapy regimen fails in 5-63% of patients 
with an average eradication rate of 76% on the basis of a pooled analysis [16-18]. PPI-
amoxcillin-levofloxacin has been recommended as an encouraging strategy for second-line 
therapy  [19].  A  meta-analysis  by  Saad  et  al.  [19]  showed  that  a  10-day  regimen  of 
levofloxacin-based triple therapy was superior to 7-day bismuth-based quadruple therapy. 
However, levofloxin-based triple therapies seem less effective in Asia. Our recent study 
showed that PPI-amoxicillin-levofloxacin triple therapy only achieved a 69.9% eradication rate 
for rescue treatment of standard triple therapy in Taiwan [20].
Currently, the best second-line therapy after failure of non-bismuth quadruple therapies 
(such as sequential therapy, concomitant therapy or hybrid therapy) remains unclear. Although 
PPI-amoxicillin-levofloxacin triple therapy also has been recommended by the Maastricht 
IV/Florence Consensus Report as a rescue treatment for sequential therapy [15], the mean 
eradication rate of the rescue regimen is suboptimal (mean, 77.5% [79/102]; range: 50% [3/6] 
to 100% [7/7]) [21-25]. 
Antibiotic resistance is the key factor determining the outcome of rescue treatment for 
anti-H pylori  therapy. According to our previous reports [20,26], the drug resistant rates to 
tetracycline, levofloxacin, amoxicillin, clarithromycin and metronidazole were 0%, 21%, 6%, 
59% and 57% respectively following standard triple therapy [20] and 0%, 25%, 0%, 75% and 
75%  respectively  following  sequential  therapy  [26].  The  data  indicate  that  tetracycline, 
amoxicillin and levofloxacin are good candidates of antibiotics used in the rescue treatment for 
both standard triple therapy and non-bismuth quadruple therapy (such as sequential therapy, 
concomitant therapy and hybrid therapy). 
Although  PPI-amoxicillin-levofloxacin  triple  therapy has  been  recommended  by the 
Maastricht IV/Florence Consensus Report for recue treatment of both standard triple therapy 
3


VGHKS104-062
and non-bismuth quadruple therapy [15], the eradication rates of the rescue treatment for 
standard triple therapy and  sequential therapy by intention-to-treat analysis are only 66.9% 
[20]  and  77.5%  [23],  respectively.  Our  recent  study  also  revealed  that  PPI- bismuth-
tetracycline-levofloxacin rescue treatment might achieve a very high eradication rate 96%) for 
H pylori infection after failure of non-bismuth quadruple therapy [26]. We therefore design the 
prospective, multicenter, randomized controlled trial to investigate (1) the efficacy of PPI-
bismuth -levofloxain-tetracycline quadruple therapy and PPI-amoxicillin-levofloxacin triple 
therapy in  rescue  treatment  for  standard  triple  therapy,  (2)  the  efficacy of  PPI-bismuth 
-levofloxain-tetracycline quadruple therapy and PPI-amoxicillin-levofloxacin triple therapy in 
rescue treatment for  nonbismuth-containing quadruple therapy (such as sequential therapy, 
concomitant therapy and hybrid therapy), and (3) the bacterial and host factors determining the 
efficacy of PPI-  bismuth- levofloxain-tetracycline quadruple therapy and PPI-amoxicillin-
levofloxacin triple therapy. 
REFERENCES
1. Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Med 2002;347: 1175–86.
2. Malfertheiner P, Chan FK, McColl KE. Peptic ulcer disease. Lancet 2009;374:1449–61.
3. Sung JJY, Chung SCS, Ling TKW, et al. Antibacterial treatment of gastric ulcer associated 
with Helicobacter pylori. N Eng J Med 1995;332:139-42.
4. Zucca  E
 
 ,  Dreyling  M;  ESMO  Guidelines  Working  Group.  Gastric  marginal  zone 
lymphoma of MALT type: ESMO clinical recommendations for diagnosis, treatment and 
follow-up. Ann Oncol 2009;20(Suppl 4):113-4.
5. Asaka M, Kato M, Takahashi S, et al, Guidelines for the management of Helicobacter 
pylori infection in Japan: 2009 revised edition. Helicobacter 2010;15:1–20.
6. Malfertheiner P, Megraud F, O'Morain C, et al. Current concepts in the management of 
Helicobacter pylori infection: the Maastricht III Consensus Report. Gut 2007;56:772-81.
7. Fock KM, Katelaris P, Sugano K, et al.  Second Asia-Pacific Consensus Guidelines for 
4


VGHKS104-062
Helicobacter pylori infection. J Gastroenterol Hepatol 2009;24:1587-600.
8. Graham DY, Fischbach L. Helicobacter pylori treatment in the era of increasing antibiotic 
resistance. Gut 2010; 59: 1143-53. 
9. Gumurdulu Y, Serin E, Ozer B, et al. Low eradication rate of Helicobacter pylori with 
triple 7-14 days and quadruple therapy in Turkey. World J Gastroenterol 2004;10:668-71. 
10. Bigard MA
 
 , Delchier JC, Riachi G, Thibault P, Barthelemy P. One-week triple therapy 
using omeprazole, amoxycillin and clarithromycin for the eradication of Helicobacter 
pylori in patients  with non-ulcer dyspepsia: influence of dosage of omeprazole and 
clarithromycin. Aliment Pharmacol Ther 1998;12:383-8. 
11. Gatta L , Vakil N , Leandro G, et al. Sequential therapy or triple therapy for Helicobacter 
pylori infection: systematic review and meta-analysis of randomized controlled trials in 
adults and children. Am J Gastroenterol 2009;104:3069–79. 
12. Chuah SK, Tsay FW, Hsu PI, Wu DC. A new look at anti-Helicobacter pylori therapy. 
World J Gastroenterol 2011;17:3971-5.
13. Wu DC, Hsu PI, Wu JY et al.  Sequential and concomitant therapies with 4 drugs are 
equally  effective  for  eradication  of  H  pylori Infection.  Clin  Gastroenterol  Hepatol 
2010;8:36-41.
14. Hsu PI, Wu DC, Wu JY, Graham DY. Modified sequential Helicobacter pylori therapy: 
proton pump inhibitor and amoxicillin for 14 days with clarithromycin and metronidazole 
added as a quadruple (hybrid) therapy for the final 7 days. Helicobacter 2011;16:139-45.
15. Malfertheiner P, Megraud F, O’Morain CA, et al. Management of Helicobacter pylori 
infection — The Maastricht IV/ Florence Consensus Report. Gut 2012;61:646-64.
16. Wu DC
 
 , Hsu PI, Tseng HH, et al. Helicobacter pylori Infection: A randomized, controlled 
study comparing 2 rescue therapies after failure of standard triple therapies.  Medicine 
(Baltimore) 2011；90：180-5. 
17. Gisbert JP. "Rescue" regimens after  Helicobacter pylori treatment failure.  World. J.. 
Gastroenterol 2008；14：5385-402. 
5


VGHKS104-062
18. Hojo M, Miwa H, Nagahara A, et al. Pooled analysis for Helicobacter pylori infection. 
Scand J Gastroenterol 2001；36：690-700.
19. Saad RJ, Schoenfeld P, Kim HM, et al. Levofloxacin-based triple therapy versus bismuth-
based quadruple therapy for persistent Helicobacter pylori infection: a meta-analysis. Am. 
J. Gastroenterol 2006；101：488-96. 
20. Kuo CH
 
 .,  Hu HM,  Kuo FC, et al.  Efficacy of levofloxacin-based rescue therapy for 
Helicobacter pylori infection after standard triple therapy: a randomized controlled trial. J. 
Antimicrob. Chemother 2009；63：1017-24.
21. Pontone S
 
 ,  Standoli M,  Angelini R,  et al. Efficacy of  H. pylori  eradication with a 
sequential regimen followed by rescue therapy in clinical practice. Dig Liver Dis 2010；
42：541-3.
22. Manfredi M, Bizzarri B, De’angelis GL. Helicobacter pylori Infection: Sequential therapy 
followed by levofloxacin-containing triple therapy provides a good cumulative eradication 
rate. Helicobacter 2012;17:246–53.
23. Perna F, Zullo A, Ricci C, et al. Levofloxacin-based triple therapy for Helicobacter pylori 
retreatment: role of bacterial resistance. Dig Liver Dis 2007；39：1001–5.
24. Zullo  A,  De  Francesco  V,  Hassan  C.  Second-line  treatment  for  Helicobacter  pylori 
eradication after sequential therapy failure: a pilot study. Therapy 2006；3：251–4.
25. Gisbert JP, Molina-Infante J, Marin AC, et al. Second-line rescue triple  therapy with 
levofloxacin  after  failure  of  non-bismuth  quadruple  "sequential"  or  "concomitant" 
treatment to eradicate H. pylori infection. Scand J Gastroenterol 2013；48:652-6. doi: 
10.3109/00365521.2013.786132.
26. Hsu PI, Chen WC, Tsay FW, et al. Ten-Day Quadruple Therapy Comprising Proton-Pump 
Inhibitor, Bismuth, Tetracycline, and Levofloxacin Achieves a High Eradication Rate for 
Helicobacter pylori Infection after Failure of Sequential Therapy. Helicobacter. 2013；
doi: 10.1111/hel.12085.
27. Hsu PI, Lai KH, Tseng HH, et al. Correlation of serum immunoglobulin G Helicobacter 
6


VGHKS104-062
pylori antibody titers with histologic and endoscopic findings in patients with dyspepsia. J 
Clin Gastroenterol 1997；25：587-91.
28. Hsu PI, Lai KH, Chien EJ, et al. Impact of bacterial eradication on the cell proliferation 
and p53 protein accumulation in Helicobacter pylori-associated gastritis. Anticancer Res 
2000；20：1221-8.
29. Peng NJ, Lai KH, Liu RS, et al.  Endoscopic 13C-urea breath test for the diagnosis of 
Helicobacter pylori infection. Dig Liver Dis 2003；35：73-7.
30. Peng NJ, Lai KH, Liu RS, et al. Clinical significance of oral urease in the diagnosis of H. 
pylori infection by 13C-urea breath test. Dig Dis Sci 2001；46：1773-8.
31. Kita T, Sakaeda T, Aoyama N, et al. Optimal dose of omeprazole for CYP2C19 extensive 
metabolizers in anti-Helicobacter pylori therapy: pharmacokinetic considerations.  Biol 
Pharm Bull 2002；25：923-7.
PATIENTS AND METHODS
Patients
    Consecutive 164 H pylori-infected adult patients ( 20 years old) with failure of first-line therapy 
for H pylori infection will be enrolled for this study after giving informed consent. The presence of 
H pylori after a previous eradication therapy is defined as (1) positive results of both rapid urease 
test and histology, (2) a positive result of 13C urea breath test, or (3) a positive result of culture. 
Criteria for exclusion included (a) ingestion of antibiotics, bismuth, or PPI within the prior 4 weeks, 
(b) patients with allergic history to the medications used, (c) patients with previous gastric surgery, 
(d) the coexistence of serious concomitant illness (for example, decompensated liver cirrhosis, 
uremia), and (e) pregnant women.
Methods
Using a computer-generated number sequence, the eligible patients are randomized to 
either EBTL (esomeprazole 40 mg b.d.,  bismuth 120 mg q.d.s., levofloxacin 500 mg o.d., and 
7


VGHKS104-062
tetracycline 500 mg q.d.s.) therapy or EAL (esomeprazole 40 mg b.d., amoxicillin 500 mg q.d.s., 
and levofloxacin 500 mg o.d.) for 10 days. All drugs are taken one hour before meals or night sleep. 
Patients are asked to return at the 2nd week to assess drug compliance and adverse events. 
To assess eradication efficacy, 13C urea breath tests are conducted to assess H. pylori status. 
Demographic data
    A complete medical history and demographic data are obtained from each patient, including age, 
sex, medical history, history of smoking, alcohol, coffee and tea consumption. Smoking was defined 
as consumption of cigarettes 1 pack or more per week. Coffee or tea consumption is defined as 
drinking 1 cup or more per day.
    Adverse events are prospectively evaluated. The adverse events are 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)  [26,27].  Compliance  is  checked  by  counting  unused 
medication at the completion of treatment. Poor compliance is defined as taking less than 80% of 
the total medication. 
Histological examination
    Two biopsy specimens are taken from the lesser curvature sites of the antrum and the corpus, 
respectively. They are fixed in 10% buffered formalin, embedded in paraffin, and sectioned. The 
sections, 4-μm thick, are stained with a haematoxylin and eosion stain and a modified Giemsa stain 
to observe the presence of curved rod shape bacteria on the mucosal surface [28]. Biopsy specimens 
are assessed by a histopathologist (HH Tseng), blinded to patient status and the results of other 
laboratory tests.
Urea breath test
    The urea breath test is performed according to our previous studies [29,30]. The staffs who are 
8


VGHKS104-062
blind to the H pylori status perform the tests.
Culture and antimicrobial resistance
    One antral gastric biopsy specimen is obtained for isolation of  H pylori, using previously 
described culture methods [14]. All stock cultures are maintained at –80C in Brucella broth (Difco. 
Detroit, MI) supplemented with 20% glycerol (Sigma Chem. Co. St. Louis, MO).  The organisms 
are identified as H pylori by Gram staining, colony morphology, and positive oxidase, catalase, and 
urease reactions. As previously described in more detail [8], the antibiotic susceptibility is tested by 
E test (AB Biodisck, Solna, Sweden). H pylori strains with a MIC value  > 0.5 g/mL, > 1 g/mL, 
> 4 g/mL, > 8 g/mL and > 1 g/mL are considered to be resistant to amoxicillin, levofloxacin, 
tetracycline, metronidazole and clarithromycin, respectively [14,16].     
Genotyping of CYP2C19
    Blood sampling for genotyping of CYP2C19 is carried out before endoscopy for the subjects who 
provide informed consent for genetic study. The  CYP2C19 genotype is determined using the 
polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) [31].  A two 
single-tube  tetra-primer  PCR  assays  is  applied  to  detect  the  CYP2C19*2  and  the  *3  allele, 
respectively. To genotype the CYP2C19*2 polymorphism, four primers (Table 1) are combined in 
one tetra-primer PCR assay. Amplification of  CYP2C19  exon 5 with primers Ex5U and Ex5L 
produced a 321-bp product that served as internal control for the quality of the PCR amplification 
and as template for the allele-specific amplification (ASA). ASA produced the 229-bp PCR product 
specific for the *2 allele and the 127-bp PCR product specific for the wild-type allele. The tetra-
primer PCR produced amplification of the 309-bp CYP2C19 exon 4 region (Ex4U and Ex4L), and 
ASA (*3mutU and *3wtL) produced the 110-bp PCR product for the *3 allele and the 228-bp PCR 
product for the wild-type allele. The following reaction mixture was prepared for the tetra-primer 
PCR assay to genotype CYP2C19*2: 16.4 mL of water, 2.5 mL of buffer 1 (1.5 mM MgCl2), 0.2 
mL of Gold Taq (5 U/mL), 0.5 mL of dNTP mixture (10 mM), 0.4 mL of Ex5U (10 mM), 0.5 mL of 
Ex5L (10 mM), 0.75 mL of *2mutU (10 mM), 0.75 mL of *2wtL (10 mM), and 3.0 mL of genomic 
9


VGHKS104-062
DNA (;50 ng/mL). The following cycling conditions were used: 10 min at 94 °C; 44 cycles of 94 °C 
for 30 s, 50 °C for 30 s, and 72 °C for 60 s; and a final extension of 7 min at 72 °C. Separation by 
2% agarose gel electrophoresis and ethidium bromide staining allowed identification of the three 
PCR products and the interpretation of the CYP2C19*2 genotype. 
Genotypes  are  classified  into  three  groups:  homEM  (CYP2C19*1/CYP2C19*1);  hetEM 
(CYP2C19*1/CYP2C19*2  and
 CYP2C19*1/CYP2C19*3);  PM  (CYP2C19*2/CYP2C19*2, 
CYP2C19*2/CYP2C19*3, and CYP2C19*3/CYP2C19*3).
Statistical analysis
   The primary outcome variable is eradication rate. The secondary outcome variables are the rate of 
adverse events and compliance. Chi-square test with or without Yates correction for continuity and 
Fisher’s exact test are used when appropriate to compare the major outcomes between groups using 
the SPSS program (version 10.1, Chicago, Illinois, USA). A P value less than 0.05 is considered 
statistically significant. The sample size is estimated 164 (82 subjects per group) in order to detect a 
difference of 10% in the eradication rate between the EAL therapy (assumed eradication rate of 
70% and the EBTL therapy (assumed eradication rate of 80%) for rescue treatment.  
    Eradication rates are evaluated by ITT and per-protocol (PP) analyses. ITT analysis includes all 
randomized patients who have taken at least one dose of study medication. Patients whose infection 
status is unknown following treatment are considered treatment failures for the purposes of ITT 
analysis. The PP analysis excludes the patients with unknown H. pylori status following therapy and 
those with major protocol violations. 
    To determine the independent factors affecting the treatment response, Host and bacterial 
10


VGHKS104-062
parameters are analyzed by univariate analysis. These variables include the following: age (<60 or 
60 years), gender, history of smoking, 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), previous history of peptic ulcer disease, endoscopic 
appearance (ulcer or gastritis), CYP2C19 polymorphism, drug compliance (good or poor), and 
antibiotic  susceptibility.  Those  variables  found  to  be  significant  by  univariate  analysis  are 
subsequently assessed by a stepwise logistic regression method to identify independent factors for 
eradication outcome.
11