Volker Strenger

Tanja Gschliesser

Andrea Grisold

Gernot Zarfel

Gebhard Feierl

Lilian Masoud

Martin Hoenigl

Bernhard Resch

Wilhelm Müller

Berndt Urlesberger

Orally administered

Keywords:

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Introduction

Extended-spectrum -lactamase-producing Enterobacteriaceae (ESBL-E) are of growing importance both in community and healthcare settings. As well as being resistant to penicillins and cephalosporins, these bacteria often show additional antimicrobial resistances (including aminoglycoside resistance) and have been associated with increased morbidity and mortality, especially in critically ill patients [START_REF] Paterson | Extended-spectrum -lactamases: a clinical update[END_REF]. Intestinal colonisation with bacteria, including Enterobacteriaceae, is believed to be a co-factor in the pathogenesis of neonatal necrotising enterocolitis (NEC) [START_REF] Bury | Enteral antibiotics for preventing necrotizing enterocolitis in low birthweight or preterm infants[END_REF][START_REF] Alfaleh | Probiotics for prevention of necrotizing enterocolitis in preterm infants[END_REF], and ESBL-producing Klebsiella pneumoniae has been implicated in at least one outbreak of NEC [START_REF] Gregersen | Klebsiella pneumoniae with extended spectrum -lactamase activity associated with a necrotizing enterocolitis outbreak[END_REF]. Enteral aminoglycosides [START_REF] Alfaleh | Probiotics for prevention of necrotizing enterocolitis in preterm infants[END_REF] are used in some institutions as prophylaxis against NEC. However, the increasing prevalence of ESBL-E that are resistant to aminoglycosides means that the utility of other antibiotic classes requires further evaluation. Colistin has been reported to be effective in the treatment of infections with ESBL-E in adults and children [START_REF] Falagas | Colistin: the revival of polymyxins for the management of multidrug-resistant Gram-negative bacterial infections[END_REF][START_REF] Falagas | Colistin therapy for microbiologically documented multidrug-resistant Gram-negative bacterial infections: a retrospective cohort study of 258 patients[END_REF][START_REF] Falagas | Systemic colistin use in children without cystic fibrosis: a systematic review of the literature[END_REF], in selective digestive decontamination (SDD) [START_REF] De Smet | Decontamination of the digestive tract and oropharynx in ICU patients[END_REF] and in NEC prophylaxis [START_REF] Grylack | Effects of oral antibiotics on stool flora and overall sensitivity patterns in an intensive care nursery[END_REF].

During three outbreaks of colonisation with ESBL-E in the Neonatal Intensive Care Unit (NICU) of the University Hospital Graz (Graz, Austria), oral colistin was used in place of gentamicin as prophylaxis against NEC. Here we describe a retrospective analysis of the impact of this intervention.

A c c e p t e d M a n u s c r i p t 5

Methods

Setting and study design

At the NICU of the University Hospital Graz, a combination of gentamicin [7.5 mg/kg every 12 h (q12h) orally], nystatin [10 000 IU/kg every 6 h (q6h)] and enteral probiotics [Lactobacillus rhamnosus GG 0.5 g (0.5  10 9 colony-forming units) q12h] for all hospitalised neonates during the whole stay is the local standard of care for preventing NEC [START_REF] Schmolzer | Multi-modal approach to prophylaxis of necrotizing enterocolitis: clinical report and review of literature[END_REF]. Furthermore, stool cultures from all NICU patients are routinely taken twice a week for active surveillance of intestinal colonisation. Patients colonised with ESBL-E are cohorted with dedicated staff, and hygienic precautions are intensified by wearing gloves and gowns. Since the majority of isolated ESBL-E were observed to be resistant to gentamicin, NEC prophylaxis was modified from May 2005 to September 2007; oral gentamicin was replaced by oral colistin (2 mg/kg q6h) in all NICU patients as long as ESBL-E colonisation was detected in at least one NICU patient.

To evaluate the ability of orally administered colistin to prevent colonisation with ESBL-E, all neonates colonised with ESBL-E during the study period were analysed retrospectively. Standards Institute (CLSI) [START_REF]Performance standards for antimicrobial susceptibility testing[END_REF] by means of a semi-automated system (VITEK II instrument; bioMérieux) as well as by the disk diffusion test. ESBL production was detected by the resistance phenotype, the CLSI double-disk synergy test and Etest using ESBL confirmation strips. Susceptibility testing for colistin and gentamicin of ESBL-E strains was additionally performed by Etest (AB BIODISK, Solna, Sweden).

Laboratory examinations

For colistin, a minimum inhibitory concentration (MIC) breakpoint of ≤2 mg/L was applied for susceptibility [START_REF]Performance standards for antimicrobial susceptibility testing[END_REF][START_REF] Galani | Colistin susceptibility testing by Etest and disk diffusion methods[END_REF].

To assess clonality, molecular typing of ESBL-E isolates was performed by semiautomated repetitive sequence-based polymerase chain reaction (rep-PCR) on a DiversiLab system (bioMérieux) as described previously [START_REF] Grisold | Use of automated repetitive-sequence-based PCR for rapid laboratory confirmation of nosocomial outbreaks[END_REF]. Clonality was defined as similarity >95%.

Statistical analyses

Data are presented as median and range or as counts and proportions. Fisher's exact test was used to compare proportions. All but two isolates (one ESBL-Ko in the gentamicin group and one ESBL-Kp in the colistin group) were gentamicin-resistant (Table 1).

Results

From

Four different clones of ESBL-Kp (clusters of ten and six isolates as well as two single isolates) and three different clones of ESBL-Ko (clusters of four and two isolates as well as one single isolate) were identified, indicating the occurrence of patient-to-patient transmission in the majority of patients. Five isolates (three ESBL-Kp and two ESBL-Ko) were not available for molecular typing. Interestingly, within the two major clones of ESBL-Kp, colistin-susceptible (six and two isolates, respectively) and colistin-resistant (four isolates each) isolates were detected.

Discussion

Owing to the high rate of gentamicin resistance among ESBL-E, colistin was administered instead of gentamicin during ESBL-E outbreaks. However, nearly onehalf of analysed patients were colonised with ESBL-E despite receiving colistin prophylaxis. Therefore, colistin was abandoned as part of NEC prophylaxis after September 2007.

The percentage of colistin resistance in isolated ESBL-Kp was significantly higher in the colistin group compared with the gentamicin group. Oral administration of colistin might therefore select colistin-resistant isolates or induce colistin resistance during the short period between intestinal colonisation and detection of ESBL-E [START_REF] Falagas | Colistin: the revival of polymyxins for the management of multidrug-resistant Gram-negative bacterial infections[END_REF]. This might explain the detection of colistin-susceptible as well as colistin-resistant ESBL-Kp isolates within the same clone. On the other hand, in four patients colistinsusceptible ESBL-E (three ESBL-Kp and one ESBL-Ko) were isolated despite colistin administration. Because colistin susceptibility testing of consecutive isolates after ESBL-E colonisation was not routinely performed, it was not possible to evaluate the development of resistance in primarily susceptible strains. There are two main possible explanations for colistin failing to prevent colonisation with colistinsusceptible ESBL-E. First, the faecal concentration of colistin might be too low to prevent colonisation effectively. Second, colistin might have been inactivated by stool and therefore lack effectiveness [START_REF] Van Saene | Influence of faeces on the activity of antimicrobial agents used for decontamination of the alimentary canal[END_REF]. There are two main limitations of this retrospective study that must be taken into account when interpreting the results.

A c c e p t e d M a n u s c r i p t 9 First, as reported in the literature [START_REF] Paterson | Extended-spectrum -lactamases: a clinical update[END_REF], patient-to-patient transmission was the major mode of ESBL-E acquisition. Since colistin was only used during outbreaks, patients in the colistin group were certainly at higher risk of colonisation with ESBL-E by nosocomial transmission. Second, the same dose of colistin was administered in all patients. It might well be that higher colistin dosages and concentrations are more effective in preventing ESBL-E colonisation and avoiding the induction of resistance.

Conclusion

At the dosage used, oral administration of either gentamicin or colistin does not reliably prevent colonisation with ESBL-E in NICU patients. Moreover, it appears to induce antibiotic resistance or to select antibiotic-resistant bacteria. Randomised studies and further analyses are needed to assess the usefulness of colistin for SDD or NEC prophylaxis in settings with increased ESBL-E occurrence. Furthermore, development of resistance has to be taken into account when analysing the benefits and drawbacks of the widespread use of enteral antibiotics for prophylactic purposes.

*

  Corresponding author. Tel.: +43 316 385 2605; fax: +43 316 385 3300.E-mail address: volker.strenger@medunigraz.at (V. Strenger).

A c c e p t e d M a n u s c r i p t 3 ABSTRACT

 3 Colonisation and infection with extended-spectrum -lactamase-producingEnterobacteriaceae (ESBL-E) is an emerging problem. The aim of this study was to investigate whether colistin, which is reported to be effective against multiresistant enterobacteria, prevents ESBL-E colonisation in neonates. For prophylaxis of necrotizing enterocolitis, oral gentamicin (15 mg/kg/day) is routinely used in all neonates hospitalised at the Neonatal Intensive Care Unit of University Hospital Graz (Austria). During the study period from May 2005 to September 2007, three ESBL-E outbreaks (total duration 18 months) occurred. During these outbreaks, gentamicin was immediately replaced by oral colistin (8 mg/kg/day) in all hospitalised neonates. All neonates colonised with ESBL-E during the study period were retrospectively analysed with regard to the influence of colistin on ESBL-E colonisation. Genetic relatedness of isolates was assessed by repetitive sequence-based polymerase chain reaction (rep-PCR). During the study period, 30 (4.5%) of 667 neonates were colonised with ESBL-E. Twelve of twenty-one patients colonised with Klebsiella pneumoniae (ESBL-Kp) and one of nine patients colonised with Klebsiella oxytoca (ESBL-Ko) had received oral colistin at time of colonisation with ESBL-E. Among ESBL-Kp, the rate of colistin resistance was significantly higher in the colistin group (P = 0.0075). Four different clones of ESBL-Kp and three different clones of ESBL-Ko were isolated, indicating the occurrence of patient-to-patient transmission. Colistinresistant as well as colistin-susceptible isolates were detected within the same clones, indicating induction of resistance. At the dosage used, oral colistin did not prevent colonisation with ESBL-E and appeared to select colistin-resistant strains or to induce colistin resistance.

Faecal

  samples were inoculated into brain-heart infusion broth (Becton Dickinson, Cockeysville, MD) and were incubated at 35 C overnight and then streaked onto three plates, including blood agar, MacConkey agar and chromID ESBL agar (bioMérieux, Marcy l'Etoile, France). Identification of all Gram-negative bacilli was A c c e p t e d M a n u s c r i p t 6 performed by standard methods, and susceptibility testing for antimicrobial agents was performed and interpreted according to the criteria of the Clinical and Laboratory

  May 2005 to September 2007, three outbreaks with clustered occurrence of intestinal ESBL-E colonisation and subsequent replacement of gentamicin by colistin as part of NEC prophylaxis were observed (May-September 2005, April-December 2006 and June-September 2007). No ESBL-E colonisation was observed beyondA c c e p t e d M a n u s c r i p t 7 these outbreaks. During the outbreaks, 30 term and preterm born neonates (gestational age 24-40 weeks, median 31 weeks; birth weight 540-3700 g, median 1780 g; 14 female; age at colonisation 3-102 days, median 22 days) among 667 hospitalised patients (4.5%) had been colonised with ESBL-producing K. pneumoniae (ESBL-Kp) (n = 21) or Klebsiella oxytoca (ESBL-Ko) (n = 9). Twelve of twenty-one patients colonised with ESBL-Kp and one of nine patients colonised with ESBL-Ko had received colistin orally prior to colonisation with ESBL-E (colistin group, n = 13) instead of gentamicin (gentamicin group, n = 17). In one patient in the colistin group gentamicin had been switched to colistin 3 days before the patient was colonised with ESBL-Kp; all other patients in the colistin group received colistin from the first day of their hospitalisation. All ESBL-Ko and 11 (52.4%) of 21 ESBL-Kp isolates were colistin-susceptible. Moreover, 3 (25%) of 12 ESBL-Kp isolates in the colistin group and 8 (88.9%) of 9 ESBL-Kp isolates in the gentamicin-group were susceptible to colistin (P = 0.0075).

A c c e p t e d M a n u s c r i p t 8 In

 8 the analysed period, no infections clearly attributable to ESBL-E and no adverse effects of colistin were observed.

A

  c c e p t e d M a n u s c r i p t

Funding

None.

Competing interests

None declared.

Ethical approval

Not required. Colistin-resistant 0 (0) 0 (0) 0 (0)

Gentamicin-resistant 1 (100) 7 (87.5) 8 (88.9) * P = 0.0075, Fisher's exact test.

Edited Table 1


Pirkko Kostamo
email: pirkko.kostamo@gmail.com

Lea Veijola

Aino Oksanen

Seppo Sarna

Hilpi Rautelin

Recent trends in primary antimicrobial resistance of Helicobacter pylori in Finland

Keywords:

A c c e p t e d M a n u s c r i p t

Introduction

Helicobacter pylori is a major causative agent of gastroduodenal diseases such as chronic gastritis, peptic ulcer disease, gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma [START_REF] Dixon | Patterns of inflammation linked to ulcer disease[END_REF][START_REF] Parsonnet | Helicobacter pylori infection and gastric lymphoma[END_REF][START_REF] Suerbaum | Helicobacter pylori infection[END_REF]. The infection is typically acquired in childhood or early adolescence [START_REF] Malaty | Transmission of Helicobacter pylori infection. Studies in families of healthy individuals[END_REF]. According to the latest European recommendations, eradication therapy is recommended in peptic ulcer disease, MALT lymphoma, atrophic gastritis, unexplained iron deficiency anaemia and chronic idiopathic thrombocytopenic purpura as well as in first-degree relatives of patients with gastric cancer [START_REF] Malfertheiner | Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report[END_REF]. Standard eradication therapy is based on a combination of a proton pump inhibitor and two antimicrobials, with clarithromycin being recognised as the key component [START_REF] Mégraud | H pylori antibiotic resistance: prevalence, importance, and advances in testing[END_REF]. However, an increasing number of H. pylori isolates worldwide show resistance to clarithromycin, which is considered the main cause of eradication failure [START_REF] Horiki | Annual change of primary resistance to clarithromycin among Helicobacter pylori isolates from 1996 through 2008 in Japan[END_REF][START_REF] De Francesco | Prevalence of primary clarithromycin resistance in Helicobacter pylori strains over a 15 year period in Italy[END_REF]. If the local primary resistance rates to clarithromycin exceed 15-20%, empirical treatment with this agent is no longer recommended [START_REF] Malfertheiner | Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report[END_REF]. Metronidazole resistance is even more common, although metronidazole resistance in vitro does not always predict treatment failure [START_REF] Rautelin | Role of metronidazole resistance in therapy of Helicobacter pylori infections[END_REF].

Resistance to amoxicillin and tetracycline, two further components useful in combined therapy, has remained at a low level, whereas recent studies suggest an increasing trend in primary fluoroquinolone resistance [START_REF] Zullo | Primary antibiotic resistance in Helicobacter pylori strains isolated in northern and central Italy[END_REF][START_REF] Hung | Prevalence of primary fluoroquinolone resistance among clinical isolates of Helicobacter pylori at a university hospital in Southern Taiwan[END_REF]. However, so far the fluoroquinolones, such as levofloxacin, have been successfully used in H. pylori salvage therapy in patients with previous eradication failure [START_REF] Wolle | Treatment of Helicobacter pylori[END_REF][START_REF] Egan | Treatment of Helicobacter pylori[END_REF][START_REF] Coelho | New once-daily, highly effective rescue triple therapy after multiple Helicobacter pylori treatment failures: a pilot study[END_REF][START_REF] Wong | Lansoprazole, levofloxacin and amoxicillin triple therapy vs. quadruple therapy as second-line treatment of resistant Helicobacter pylori infection[END_REF].

A c c e p t e d M a n u s c r i p t 5 The antimicrobial susceptibility of H. pylori is an important predictor of the success of eradication therapy. Choosing the most effective first-line therapy for H. pylori infection is critical, as higher eradication rates are usually obtained in the first attempt to treat this chronic infection whereas the following therapies are hampered by problems caused by secondary resistance [START_REF] Mégraud | H pylori antibiotic resistance: prevalence, importance, and advances in testing[END_REF]. Antimicrobial susceptibility tests are rarely available to aid decisions regarding first-line therapy. Even according to the latest guidelines, antimicrobial susceptibility testing is first recommended after therapy failure [START_REF] Malfertheiner | Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report[END_REF]. This emphasises the need for knowledge of the local primary antimicrobial resistance situation. However, clinicians are usually unaware of the prevalence of antimicrobial resistance among H. pylori isolates in their region, resulting in poorer cure rates. Local resistance rates also appear to change dynamically owing to changes in annual antibiotic consumption [START_REF] Perez Aldana | The relationship between consumption of antimicrobial agents[END_REF] and may even vary with geographical, gender and age differences and therefore require constant monitoring [START_REF] Mégraud | H pylori antibiotic resistance: prevalence, importance, and advances in testing[END_REF][START_REF] Kobayashi | Changing antimicrobial susceptibility epidemiology of Helicobacter pylori strains in Japan between 2002 and 2005[END_REF].

In Finland, the prevalence of primary clarithromycin resistance (before any eradication attempts) was as low as 2% in 2000-2001, whereas metronidazole resistance was 38% [START_REF] Koivisto | Primary Helicobacter pylori resistance to metronidazole and clarithromycin in the Finnish population[END_REF], this latter figure being in line with some early reports [START_REF] Rautelin | Role of metronidazole resistance in therapy of Helicobacter pylori infections[END_REF]. The aim of this study was to evaluate the recent possible changes in primary antimicrobial resistance of H. pylori isolated from Finnish patients during a 9-year period to determine whether increasing resistance trends reported from elsewhere could be detected. during the study period, 1037 were H. pylori-positive. In order to assess primary antimicrobial resistance, patients previously treated for H. pylori were excluded from the study; thus, 505 patients with H. pylori isolates available were enrolled. The median age of patients was 62 years (range 18-92 years), 45% of patients were male and 16% were of foreign origin. Permission to explore the patient files was allowed by the authorities of Helsinki Health Center.

Bacterial isolates and antimicrobial susceptibility testing

Helicobacter pylori isolates had originally been identified on the basis of colony appearance, Gram staining and positive reactions in catalase, oxidase and urease tests. Bacterial isolates were kept frozen at -70 C before analysis. Only one isolate per patient was included in the study. Antimicrobial susceptibility testing was performed by Etest (AB BIODISK, Solna, Sweden). Minimal inhibitory concentrations (MICs) were determined for clarithromycin, metronidazole, levofloxacin, tetracycline and amoxicillin.

The inoculum was prepared from a 2-day-old or 3-day-old culture grown on a blood agar plate, and the bacterial suspension in Brucella broth was adjusted to match the turbidity metronidazole, >8 g/mL; levofloxacin, >1 g/mL; tetracycline, >1 g/mL; and amoxicillin, >0.5 g/mL.

Antimicrobial consumption

Data on the regional consumption of antimicrobial drugs were obtained from the Finnish Medicines Agency FIMEA. Sales statistics were based on total sales, including both hospitals and pharmacies, on an annual basis. Antimicrobial consumption was expressed as defined daily doses per 1000 inhabitants per day.

Statistical methods

Differences in demographic data were analysed with the 2 test using GraphPad Software (GraphPad QuickCalcs Online Calculators for Scientists; http://www.graphpad.com/quickcalcs/index.cfm). Associations between consumption of

Results

All H. pylori isolates were susceptible to amoxicillin [MIC for 90% of the isolates (MIC 90 ) <0.016 g/mL], and only one tetracycline-resistant isolate was observed (MIC = 1.5 g/mL). The overall prevalence of clarithromycin resistance was 8% during the 9-year study period, ranging from 0% (in 2000) to 16% (in 2003). The increase in clarithromycin resistance did not reach significance in the study period. Levofloxacin resistance varied between 0% and 12% (overall 7%), and metronidazole resistance varied between 29% and 59% (overall 41%) (Fig. 1.) Resistance to metronidazole was significantly more common in women than in men (49% vs. 31%, respectively; P < 0.001). Women were also more likely than men to harbour clarithromycin-resistant H. pylori (10.2% vs. 5.2%, respectively; P = 0.035). No significant age-or origin-related differences in resistance rates were found.

The overall prevalence of resistance to at least two antimicrobials was 7% (33/505); the most common type of multiresistance detected was to clarithromycin and metronidazole (48%; 16/33), followed by levofloxacin and metronidazole (33%; 11/33). Resistance to three different antimicrobial agents was also observed, although only in two isolates.

A c c e p t e d M a n u s c r i p t 9 Fig. 2 shows the annual consumption of macrolides, fluoroquinolones and metronidazole in the hospital district of Helsinki and Uusimaa during the study period. Annual consumption of macrolides including clarithromycin showed a significant decrease in the study area during the 9-year period (P = 0.007) but there was no significant correlation between macrolide consumption and resistance to clarithromycin. However, there was a positive correlation between consumption of fluoroquinolones and resistance to clarithromycin (P = 0.001). During the 9-year study period there was a significant increase in the consumption of metronidazole (P = 0.013), however despite this no significant increase in metronidazole resistance was detected.

Discussion

In contrast to many reports from other countries, primary clarithromycin resistance in H. pylori has not become a problem in the study area despite the rapidly increasing resistance rates during 2002-2003. After 2006, primary clarithromycin resistance rates have even been decreasing (Fig. 1). Levofloxacin resistance has been fluctuating close to the 10% level during the latest study years.

In the present study, the prevalence of primary metronidazole resistance was at a high level and was significantly higher in women than in men. The latter finding is in accordance with many earlier studies [START_REF] Mégraud | H pylori antibiotic resistance: prevalence, importance, and advances in testing[END_REF][START_REF] Koivisto | Primary Helicobacter pylori resistance to metronidazole and clarithromycin in the Finnish population[END_REF]. Resistance to levofloxacin did not show any gender differences in this study, whereas clarithromycin resistance was significantly A c c e p t e d M a n u s c r i p t 10 more common in women than in men. Some recent studies have also revealed this kind of gender difference with regard to clarithromycin resistance [START_REF] De Francesco | Prevalence of primary clarithromycin resistance in Helicobacter pylori strains over a 15 year period in Italy[END_REF][START_REF] Kobayashi | Changing antimicrobial susceptibility epidemiology of Helicobacter pylori strains in Japan between 2002 and 2005[END_REF][START_REF] Osato | Pattern of primary resistance of Helicobacter pylori to metronidazole or clarithromycin in the United States[END_REF].

During the 9-year observation in the study area, macrolide consumption decreased significantly, but there was no significant correlation between consumption of macrolides and resistance to clarithromycin. A recent study showed that rates of resistance in H. pylori to clarithromycin and metronidazole decreased following government policy to restrict the use of antimicrobial agents for infectious diseases [START_REF] Poon | Prevalence of antimicrobial resistance in Helicobacter pylori isolates in Taiwan in relation to consumption of antimicrobial agents[END_REF]. Earlier in Finland, a significant decline in the frequency of erythromycin resistance among group A streptococci was observed following nationwide reductions in the use of macrolide antibiotics for outpatient therapy [START_REF] Seppälä | The effect of changes in the consumption of macrolide antibiotics on erythromycin resistance in group A streptococci in Finland. Finnish Study Group for Antimicrobial Resistance[END_REF]. A statistically significant association also existed between regional erythromycin resistance in Streptococcus pyogenes and consumption of macrolides [START_REF] Bergman | Finnish Study Group for Antimicrobial Resistance (FiRe Network). Effect of macrolide consumption on erythromycin resistance in Streptococcus pyogenes in Finland in 1997-2001[END_REF]. However, the recent decrease in antimicrobial consumption may not necessarily be reflected in the susceptibility of an H. pylori isolate that has infected the gastric mucosa for decades.

Consumption of fluoroquinolones remained at a stable level, although in this study the primary resistance rates to fluoroquinolones showed an increasing tendency. However, primary levofloxacin resistance of H. pylori in Finland is still at a reasonably low level.

The breakpoints used for resistance to clarithromycin have slightly varied in different studies, ranging from >0.5 g/mL to >2 g/mL [START_REF] Perez Aldana | The relationship between consumption of antimicrobial agents[END_REF][START_REF] Osato | Pattern of primary resistance of Helicobacter pylori to metronidazole or clarithromycin in the United States[END_REF][START_REF] Poon | Prevalence of antimicrobial resistance in Helicobacter pylori isolates in Taiwan in relation to consumption of antimicrobial agents[END_REF][START_REF] Glupczynski | European multicentre survey of in vitro antimicrobial resistance in Helicobacter pylori[END_REF][START_REF] Storskrubb | Antimicrobial susceptibility of Helicobacter pylori strains in a random adult Swedish population[END_REF][START_REF] Raymond | High level of antimicrobial resistance in French Helicobacter pylori isolates[END_REF]. In this study, the breakpoint for resistance to clarithromycin was set at >1 g/mL and the separation A c c e p t e d M a n u s c r i p t 11 between clarithromycin-susceptible and -resistant isolates was quite clear-cut as there were only 9 isolates (2%) (distributed evenly in the study period) with clarithromycin MICs between 0.75 g/mL and 2 g/mL.

Effective therapy for H. pylori should achieve an intention-to-treat eradication rate of 80% [START_REF] Graham | New concepts of resistance in the treatment of Helicobacter pylori infections[END_REF]. However, during the past years increasing antimicrobial resistance has complicated therapy and the usual cure rates have been lower than those considered acceptable for other serious, treatable bacterial infections [START_REF] Graham | New concepts of resistance in the treatment of Helicobacter pylori infections[END_REF]. Therefore, empirical use of eradication therapies should be based on a knowledge of antimicrobial drug resistance. Maastricht guidelines recommend local reference centres to monitor antimicrobial resistance and eradication rates within countries in order to determine the best treatment regimens [START_REF] Malfertheiner | Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report[END_REF]. However, considerable fluctuation in primary antimicrobial resistance from year to year, as also shown in the present study, should be taken into account when results from cross-sectional studies form the basis for treatment guidelines.

In conclusion, this 9-year study showed a slight increasing tendency of primary clarithromycin resistance of H. pylori in Finland; however, the resistance rates were still at the level where clarithromycin-containing triple therapies could be used empirically. Furthermore, during the past 2 years clarithromycin resistance rates have even been declining. Metronidazole resistance has remained approximately at the same level as in early 2000 [START_REF] Koivisto | Primary Helicobacter pylori resistance to metronidazole and clarithromycin in the Finnish population[END_REF][START_REF] Glupczynski | European multicentre survey of in vitro antimicrobial resistance in Helicobacter pylori[END_REF]. There are no earlier studies regarding primary fluoroquinolone resistance of H. pylori in Finland, but according to the present findings the mean

  of Helicobacter pylori is an important predictor of the success of eradication therapy. To evaluate recent changes in primary antimicrobial resistance of H. pylori isolated from Finnish patients, the clinical records of H. pyloripositive patients referred for endoscopy to Herttoniemi Hospital (Helsinki, Finland) during 2000-2008 were investigated retrospectively. Stored H. pylori strains from 505 patients without previous eradication therapy were tested for clarithromycin, metronidazole, levofloxacin, tetracycline and amoxicillin susceptibility by Etest. Data on local consumption of antimicrobials were collected and correlations between consumption and resistance were calculated. During the 9-year study period, metronidazole resistance was high (range 29-59%, overall 41%). After an initial increase in clarithromycin resistance (0% in 2000 to 16% in 2003), resistance to clarithromycin decreased to 4% in 2008. No significant correlation was detected between consumption of macrolides and resistance of clarithromycin. Resistance to levofloxacin varied between 0% and 12%. Primary metronidazole resistance in H. pyloriis at a high level, however levofloxacin and clarithromycin resistances are still at a reasonable level. Thus, primary clarithromycin resistance in H. pylori in Finland has not become such a problem as in many other countries. Primary resistance to the antimicrobials studied varied considerably from year to year.

  Clinical records of consecutive H. pylori-positive patients referred for endoscopy on clinical indications to Herttoniemi Hospital (Helsinki, Finland) during 2000-2008 were investigated retrospectively. Of 3045 H. pylori cultures taken in 6774 gastroscopies

A c c e p t e d M a n u s c r i p t 7 equivalent

 7 to a McFarland 3 standard. Freshly prepared plates (BBL TM Mueller Hinton II Agar; BD, Le Pont de Claix, France) supplemented with 10% horse blood and used within 7 days of preparation were inoculated with the suspension and then Etest strips (one strip per agar plate) were added. The agar plates were incubated at 35 C in a microaerobic atmosphere (BBL TM CampyPak TM Plus microaerophilic system; BD, Sparks, MD) for 72 h. MIC values were determined according to the manufacturer's instructions and were read where the inhibition zone ellipse intersected the strip. The following interpretive criteria for resistance were used: clarithromycin, >1 g/mL;

  resistance of H. pylori to the different antimicrobials were analysed using Pearson correlation coefficients, and time trends were analysed using linear regression models. Data were analysed using SPSS software version 17.0 (SPSS Inc., Chicago, IL). Statistical significance was set at level of 0.05 (two-tailed).

Fig. 1 .

 1 Fig. 1. Antimicrobial resistance of Helicobacter pylori isolates during the study period.

Fig. 2 .

 2 Fig. 2. Annual consumption of macrolides, fluoroquinolones and metronidazole in the

Acknowledgments

The authors warmly thank researcher Tinna Voipio, BSc (Pharm) from FIMEA for the sales statistics. The skilful technical assistance of Mr Marko Haverinen is gratefully acknowledged. The isolates from 2008 were tested as part of the third European multicentre study on antibiotic susceptibility of Helicobacter pylori. For these particular isolates, Etests were kindly provided by AB BIODISK (Solna, Sweden). Part of this study was presented at the XXIInd International Workshop on Helicobacter and Related Bacteria in Chronic Digestive Inflammation and Gastric Cancer, Porto, Portugal, 17-19 September 2009.

Funding

None.

resistance rate (7%) was lower than that observed in many other countries, with values ranging from 14.4% up to 22.1% [START_REF] Bogaerts | Prevalence and mechanisms of resistance to fluoroquinolones in Helicobacter pylori strains from patients living in Belgium[END_REF]. In the near future, regional treatment regimens based on local antimicrobial resistance may improve eradication results for H. pylori.

Competing interests

None declared.

Ethical approval

Permission to explore the patient files was allowed by the authorities of Helsinki Health Center.


MD Edoardo Savarino
email: edoardo.savarino@unige.it

MD Lorenzo Gemignani

MD Daniel Pohl

MD Patrizia Zentilin

MD Pietro Dulbecco

MD Lorenzo Assandri

MD Elisa Marabotto

MD Daria Bonfanti

MD Simona Inferrera

MD Valentina Fazio

MD Alberto Malesci

MD Radu Tutuian

MD Vincenzo Savarino

Oesophageal Motility And Bolus Transit Abnormalities Increase In Parallel With The Severity Of Gastro-Oesophageal Reflux Disease

Keywords: Diagnostic tests < Topics, Functional Oesophageal motility, Gastro-Oesophageal Reflux, Functional Heartburn, Manometry, Bolus transit

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Limited data are available regarding the frequency of oesophageal motility and bolus transit abnormalities in subgroups of patients with gastro-oesophageal reflux disease (GORD).

Aim:

To assess oesophageal motility and bolus transit in endoscopically-defined GORD subgroups.

Methods:

Patients (N=755) with typical reflux symptoms underwent upper endoscopy, conventional or impedance oesophageal manometry and/or impedance-pH testing. They were divided into: erosive oesophagitis (EO; N=340), Barrett Oesophagus (BO; N=106), non-erosive reflux disease (NERD; endoscopy-, abnormal pH and/or SAP/SI+; N=239) and functional heartburn (FH; endoscopy-, normal pH and SAP/SI-; N=70). Manometric patterns and bolus transit were defined according to previously published criteria.

Results:

Increasing GORD severity was associated with decreased lower oesophageal sphincter resting pressure (p<0.05) and distal oesophageal amplitude (p<0.01), higher prevalence of hiatal hernia (p<0.01) and increased prevalence of ineffective oesophageal motility (p<0.01).

Patients with EO and BO had a significantly lower percentage of complete bolus transit compared to NERD and FH (p<0.01). Overall, abnormal bolus transit (ABT) for liquid swallows was found in 12% of FH, 20% of NERD, 54% of EO and 56% of BO (p<0.01).

Combined impedance-manometry showed abnormal oesophageal function in 4% of FH, 4% of NERD, 22% of EO and 21% of BO patients with normal oesophageal manometry.

Conclusions:

Oesophageal motility abnormalities increases in parallel with the severity of GORD from NERD to EO and BO. Bolus transit abnormalities in severe reflux disease underscores the importance of impaired oesophageal function in the development of mucosal injury. Gastro-oesophageal reflux disease (GORD) is a chronic relapsing disorder including a variety of conditions ranging from non-erosive reflux disease (NERD), erosive oesophagitis (EO), peptic stricture, Barrett's oesophagus (BO) and adenocarcinoma [START_REF] Kahrilas | Gastroesophageal reflux disease[END_REF]. Recent studies documented that up to 70% of reflux patients have typical reflux symptoms in the absence of endoscopically visible oesophageal mucosal injuries, making NERD the most common form of reflux disease [START_REF] Fass | Erosive esophagitis and nonerosive reflux disease (NERD): comparison of epidemiologic, physiologic, and therapeutic characteristics[END_REF][START_REF] Smout | Endoscopy-negative acid reflux disease[END_REF]. While EO and BO are objectively identifiable with certain, NERD patients are characterized by normal endoscopy and incorporate subgroups which differ significantly in terms of clinical manifestations, pathophysiological characteristics and response to therapy. We have recently proposed to identify endoscopy-negative patients with reflux from those with a functional disorder on the basis of oesophageal 24-hour impedance combined with pH-metry (MII-pH) and symptom association analysis [START_REF] Savarino | The role of non-acid reflux in NERD -Lessons learned from impedance-pH monitoring in 150 patients off therapy[END_REF][START_REF] Savarino | Functional Heartburn has more in common with Functional Dyspepsia than with Non-Erosive Reflux Disease[END_REF].

Several factors have been implicated in the protection against abnormal reflux: the His angle, the competence of Lower Oesophageal Sphincter (LOS), the crural diaphragm, a normal thorax-abdomen pressure gradient and an adequate oesophageal motility. Impairment of one or more of these mechanisms favours the reflux of stomach and/or duodenal contents into the oesophagus leading to symptoms and/or lesions [START_REF] Orlando | Overview of the mechanisms of gastroesophageal reflux[END_REF]. Previous studies have shown that impaired oesophageal motility leads to abnormal oesophageal clearance of the refluxed gastric content [START_REF] Kahrilas | Esophageal peristaltic dysfunction in peptic esophagitis[END_REF][START_REF] Kahrilas | Effect of peristaltic dysfunction on esophageal volume clearance[END_REF]. Ineffective oesophageal motility (IOM), which is characterized by an increased number of low-amplitude contractions, is one of the most common types of dysmotility [START_REF] Spechler | Classification of oesophageal motility abnormalities[END_REF]. In GORD patients, IOM has been associated with severe reflux episodes, worse symptoms and impaired oesophageal clearance with consequent prolonged acid exposure of the distal oesophagus [START_REF] Diener | Esophageal dysmotility and Gastroesophageal reflux disease[END_REF]. It has been reported that NERD patients have less severe reflux episodes and a much lower incidence of motility abnormalities than patients with erosive oesophagitis (EO) [START_REF] Frazzoni | Pathophysiological characteristics of patients with non-erosive reflux disease differ from those of patients with functional heartburn[END_REF][START_REF] Wu | Distinct Clinical Characteristics Between Patients With NERD and Those With Reflux Esophagitis[END_REF]; however, other studies did not confirm the existence of significant differences between the two groups, regarding both reflux patterns and motility abnormalities [START_REF] Dickman | Comparison of esophageal acid exposure distribution along the esophagus among the different gastroesophageal reflux disease (GERD) groups[END_REF][START_REF] Martínek | Non-erosive and erosive gastroesophageal reflux diseases: No difference with regard to reflux pattern and motility abnormalities[END_REF][START_REF] Ouatu-Lascar | Reflux esophagitis: effect of position, nocturnal acid reflux (upright versus supine reflux in gastroesophageal reflux disease)[END_REF][START_REF] Lemme | Ineffective oesophageal motility in gastro-oesophageal erosive reflux disease and in non-erosive reflux disease: are they different?[END_REF][START_REF] Chen | Differences in Oesophageal Bolus Transit Between Patients with and without erosive reflux disease[END_REF].

Therefore, data comparing motility abnormalities in patients with NERD and ERD are not conclusive. We suspect that these discrepancies are due to the difficulty to identify NERD patients and those with functional heartburn among the pool of endoscopy-negative patients. Moreover, there are few studies comparing the main manometric features and motility patterns in large groups of patients presenting all phenotypic manifestations of GORD.

Combined multichannel intraluminal impedance and oesophageal manometry (MII-EM) is considered an advanced tool for oesophageal function testing, since it provides simultaneous evaluation of oesophageal contractions and bolus transit and this reveals the functional aspects of oesophageal motility [START_REF] Savarino | Combined multichannel intraluminal impedance and manometry testing[END_REF]. Normal values for oesophageal clearance assessed by combined impedance-manometry have been previously reported by Tutuian et al. [START_REF] Tutuian | Esophageal function testing using combined multichannel intraluminal impedance and manometry. Multicenter study of healthy volunteers[END_REF]. Given the recent application of this technique in clinical setting, information about the characteristics of bolus transport in patients with various forms of GORD are still lacking [START_REF] Chen | Differences in Oesophageal Bolus Transit Between Patients with and without erosive reflux disease[END_REF][START_REF] Domingues | Characteristics of oesophageal bolus transport in patients with mild oesophagitis[END_REF].

The primary aim of the present study was to compare the manometric features and motility patterns in well-defined subgroups of GORD, subclassified by means of endoscopy and/or impedance-pH monitoring. A secondary aim was to assess whether differences exist in terms of functional aspects of oesophageal motility among the various subsets of reflux patients by means of MII-EM.

Subjects

In this retrospective analysis of prospectively collected data we included patients with typical GORD symptoms (e.g., heartburn and regurgitation), lasting for more than 6 months and occurring at least three times weekly, presenting to the GI motility centre at the University Hospital of Genoa, Italy. Exclusion criteria were: history of thoracic, oesophageal, or gastric surgery; primary or secondary severe oesophageal motility disorders (e.g., achalasia, scleroderma, diabetes mellitus, autonomic or peripheral neuropathy, myopathy); underlying psychiatric illness; history of alcohol or drug abuse; evidence of erosive oesophagitis at previous (5 years) endoscopy in case of patients with NERD.

Previously collected data in 48 healthy volunteers [22 men, mean age 44 years (range 22-77), mean BMI 23 Kg/m² [START_REF] Lemme | Ineffective oesophageal motility in gastro-oesophageal erosive reflux disease and in non-erosive reflux disease: are they different?[END_REF][START_REF] Chen | Differences in Oesophageal Bolus Transit Between Patients with and without erosive reflux disease[END_REF][START_REF] Savarino | Combined multichannel intraluminal impedance and manometry testing[END_REF][START_REF] Tutuian | Esophageal function testing using combined multichannel intraluminal impedance and manometry. Multicenter study of healthy volunteers[END_REF][START_REF] Domingues | Characteristics of oesophageal bolus transport in patients with mild oesophagitis[END_REF][START_REF] Lundell | Endoscopic assessment of esophagitis: clinical and functional correlates and further validation of Los Angeles classification[END_REF][START_REF] Vakil | The Montreal definition and classification of gastroesophageal reflux disease: a global evidence-based consensus[END_REF][START_REF] Savarino | Characteristics of reflux episodes and symptom association in patients with erosive esophagitis and non-erosive reflux disease: study using combined impedance-pH off therapy[END_REF][START_REF] Zentilin | Comparison of the main oesophageal pathophysiological characteristics between short-and long-segment Barrett's oesophagus[END_REF][START_REF] Frazzoni | Pathophysiological characteristics of the various forms of gastroesophageal reflux disease. Spectrum disease or distinct phenotypic presentations?[END_REF][START_REF] Tutuian | Clarification of the esophageal function defect in patients with manometric IEM -studies using combined impedance-manometry[END_REF][START_REF] Savarino | Characteristics of gastro-esophageal reflux episodes in Barrett's esophagus, erosive esophagitis and healthy volunteers[END_REF][START_REF] Zentilin | Normal values of 24-h ambulatory intraluminal impedance combined with pH-metry in subjects eating a Mediterranean diet[END_REF][START_REF] Galmiche | Functional esophageal disorders[END_REF][START_REF] Herschcovici | Nonerosive Reflux Disease (NERD) -An Update[END_REF][START_REF] Savarino | The added value of impedance-pH monitoringto Rome III criteria in distinguishing functional heartburn from nonerosive reflux disease[END_REF][START_REF] Savarino | Functional heartburn and non-erosive reflux disease[END_REF][START_REF] Iwakiri | Defective triggering of secondary peristalsis in patients with non-erosive reflux disease[END_REF][START_REF] Jenkinson | Compliance measurement of lower oesophagealsphincter and oesophageal body in achalasia and gastro-oesophageal reflux disease[END_REF]] without any type of digestive and systemic symptoms were used as normal values. The study protocol was approved by the local Ethics Committee and performed according to the Declaration of Helsinki. All patients provided written informed consent to use collected data before their data were used in this study.

Study Protocol

All subjects underwent physical and clinical examination and a detailed medical history (including current medication, height and weight for the calculation of their Body Mass Index (BMI), tobacco use, alcohol and coffee consumption), oesophageal manometry and upper GI endoscopy to assess the presence or not of oesophageal mucosal injury. Based on the results of upper endoscopy, patients were then subdivided into three major groups: a) EO, in case of presence of visible lesions of the oesophageal mucosa and graded according to Los Angeles Classification [START_REF] Lundell | Endoscopic assessment of esophagitis: clinical and functional correlates and further validation of Los Angeles classification[END_REF]; enrolled patients with erosive oesophagitis underwent a further upper endoscopy after 8-14 weeks of high dose proton pump inhibitor (PPI) therapy to exclude the presence of intestinal metaplasia under their ulcerated mucosa; b) BO in case of histological detection of specialized intestinal metaplasia on four-quadrant biopsy specimens taken at 1-cm intervals above the gastro-oesophageal junction or the proximal margin of gastric folds in case of hiatal hernia (5); c) endoscopy-negative in case of absence of oesophageal mucosal breaks, on the basis of Montreal Classification [START_REF] Vakil | The Montreal definition and classification of gastroesophageal reflux disease: a global evidence-based consensus[END_REF]. Within 1-5 days (median 3 days) of upper endoscopy, endoscopy-negative patients underwent 24-hour oesophageal MII-pH monitoring off-PPI therapy. Patients treated with antisecretory drugs had been asked to discontinue acid suppressive therapy at least 30 days before the examination. needed basis for the relief of heartburn. Endoscopy-negative patients were classified based on the results of impedance-pH testing with symptom association probability analysis (SAP), as follows: patients were considered to have NERD in case of abnormal oesophageal acid exposure time (AET) and in case of normal AET with positive SAP (≥ 95%) and SI (≥ 50%) for acid and/or non-acid refluxes [START_REF] Savarino | Characteristics of reflux episodes and symptom association in patients with erosive esophagitis and non-erosive reflux disease: study using combined impedance-pH off therapy[END_REF]; patients were classified as having functional heartburn (FH) if they had normal endoscopy, normal distal oesophageal acid exposure on 24-h impedance-pH monitoring and negative symptom association analysis [START_REF] Savarino | The role of non-acid reflux in NERD -Lessons learned from impedance-pH monitoring in 150 patients off therapy[END_REF][START_REF] Savarino | Functional Heartburn has more in common with Functional Dyspepsia than with Non-Erosive Reflux Disease[END_REF]. NERD patients examined with oesophageal pH-metry only were excluded from the study.

Oesophageal Conventional and Impedance Manometry testing

Patients were asked to discontinue any medication that would influence oesophageal [START_REF] Zentilin | Comparison of the main oesophageal pathophysiological characteristics between short-and long-segment Barrett's oesophagus[END_REF][START_REF] Frazzoni | Pathophysiological characteristics of the various forms of gastroesophageal reflux disease. Spectrum disease or distinct phenotypic presentations?[END_REF][START_REF] Tutuian | Clarification of the esophageal function defect in patients with manometric IEM -studies using combined impedance-manometry[END_REF][START_REF] Savarino | Characteristics of gastro-esophageal reflux episodes in Barrett's esophagus, erosive esophagitis and healthy volunteers[END_REF]. Manometric parameters used to characterize swallows included: (1) contraction amplitude 5 and 10 cm above the LOS, (2) distal oesophageal amplitude (DEA) as the average of contraction amplitude 5 and 10 cm above the LOS, and (3) onset velocity of oesophageal contractions in the distal part of the oesophagus (i.e., contraction velocity between 10 and 5 cm above the LOS). Mid-respiratory resting pressure and LOS residual pressure during swallowing were used to assess LOS function. The MII parameters we analyzed included bolus entry at each specific level obtained at the 50% point between 3-sec pre-swallow impedance baseline and impedance nadir during bolus presence and bolus exit determined as return to this 50% point on the impedance recovery curve [START_REF] Tutuian | Esophageal function testing using combined multichannel intraluminal impedance and manometry. Multicenter study of healthy volunteers[END_REF].

Swallows were classified by manometry as: (1) normal if contraction amplitudes 5 and 10 cm above the LOS were each ≥30 mm Hg and distal onset velocity was <8 cm/s, ( ineffective if either of the contraction amplitudes 5 and 10 cm above the LOS was <30 mm Hg, or (3) simultaneous if contraction amplitudes 5 and 10 cm above the LOS were each ≥30 mm Hg and distal onset velocity was >8 cm/s. Diagnoses of manometric motility abnormalities were established by using international criteria and considering only saline swallows [START_REF] Spechler | Classification of oesophageal motility abnormalities[END_REF] as follows: a) Ineffective Oesophageal Motility (IOM) was defined as ≥30% saline swallows with contraction amplitude <30 mm Hg in either 1 of the 2 distal sites located 5 and 10 cm above the LOS; b) Diffuse Oesophageal Spasm (DOS) defined as 20% or more simultaneous contractions; c) Nutcracker Oesophagus (NO) defined as normal peristalsis of the oesophageal body with an average DEA exceeding 180 mmHg; d) Normal Peristalsis (NP), defined as not more than 20% ineffective and not more than 10% simultaneous swallows with DEA < 180mmHg and with normal LOS resting and residual pressures.

Swallows were classified by MII as showing: (1) complete bolus transit if bolus entry occurred at the most proximal site (20 cm above the LOS) and bolus exit points were recorded in all 3 distal impedance measuring sites (i.e., 15, 10, and 5 cm above the LOS), and (2) incomplete bolus transit if bolus exit was not identified at any of the 3 distal impedance measuring sites. Overall diagnosis of oesophageal transit abnormalities was defined as normal liquid transit if at least 80% of liquid swallows had complete bolus transit and normal viscous transit if at least 70% of viscous swallows had complete bolus transit. Conversely, abnormal liquid transit is defined as ≥30% liquid swallows having incomplete bolus transit and abnormal viscous transit as ≥40% viscous swallows having incomplete viscous swallows [START_REF] Tutuian | Esophageal function testing using combined multichannel intraluminal impedance and manometry. Multicenter study of healthy volunteers[END_REF].

Oesophageal impedance and pH monitoring

Oesophageal impedance-pH monitoring was performed using an ambulatory multichannel intraluminal impedance and pH monitoring system (Sleuth ® , Sandhill Scientific, Inc.; Highland Ranch, CO). The system included a portable data logger with impedance-pH amplifiers and a catheter with one antimony pH electrode and eight impedance electrodes at 2, 4, 6, 8, 10, 14, 16, and 18 cm from the tip of the catheter. Each pair of adjacent electrodes represented an impedance-measuring segment (2 cm length) corresponding to one recording channel. The six impedance and one pH signals were recorded at 50 Hz on a 128 MB CompactFlash (SanDisk, Milpitas, California, US). The methodology of probe calibration, catheter placement, patient instruction and performance has been previously described [START_REF] Savarino | The role of non-acid reflux in NERD -Lessons learned from impedance-pH monitoring in 150 patients off therapy[END_REF][START_REF] Savarino | Functional Heartburn has more in common with Functional Dyspepsia than with Non-Erosive Reflux Disease[END_REF].

On the monitoring day, each subject ate three standard meals of a Mediterranean diet, as previously reported [START_REF] Zentilin | Normal values of 24-h ambulatory intraluminal impedance combined with pH-metry in subjects eating a Mediterranean diet[END_REF]. Definitions of reflux episodes, parameters of gastro-oesophageal reflux and symptom association analysis are described in previous publications [START_REF] Savarino | The role of non-acid reflux in NERD -Lessons learned from impedance-pH monitoring in 150 patients off therapy[END_REF][START_REF] Savarino | Characteristics of reflux episodes and symptom association in patients with erosive esophagitis and non-erosive reflux disease: study using combined impedance-pH off therapy[END_REF].

Statistical analysis

Differences in proportions were compared using the chi-square or Fisher's exact test, depending on the sample size. Unless otherwise specified, data are presented as mean and range values. Since data were not normally distributed, differences between groups were compared using Kruskal-Wallis and/or Mann-Whitney tests. Multi-nominal logistic regression using gender and endoscopic finding as factors and age as covariant was employed to test the likelihood of potential confounders on the differences in manometric abnormalities.

Differences were considered statistically significant when p<0.05. During the impedance-pH monitoring period of endoscopy-negative patients, 309 (186 F, mean age 49, range 18-78) reported at least one type of typical gastroesophageal reflux symptom (i.e., heartburn and regurgitation) and were included in the final analysis. Among them, 213 were classified as having NERD, whereas 96 patients were identified as having FH.

Finally, 48 healthy volunteers (27F, mean age 44, range 22-77) were enrolled, and underwent oesophageal manometry and 24h pH-MII testing, as well as upper endoscopy.

Detailed demographic features of all patients and HVs are shown in Table 1. Patients with BO and EO were more frequently male (62% vs.50% vs. 41% Vs. 38%, P<0.01) and had a higher mean body mass index (BMI) compared to those with NERD and FH (27 vs. 26,4 vs. 25.2% Vs. 23%, P<0.01). Moreover, patients with BO were more frequently smokers than those of other subgroup (38% vs. 22% vs. 19% vs. 26%, P<0.01) and they tended to consume alcohol more often (55% vs. 41% vs. 43% vs. 38%, P=NS) but statistical significance was not reached. No differences among these groups were found with regard to mean age and coffee consumption. The HVs control group had similar demographic (27 females; mean age 44 years; range 22-71 years) and body mass index (BMI 23 kg/m 2 ; range 18-34 kg/m 2 ) characteristics compared to reflux patients.

OESOPHAGEAL PERISTALSIS EVALUATED BY OESOPHAGEAL MANOMETRY

Manometric findings regarding mean LOS resting pressure, distal oesophageal waves amplitude (DEA) and prevalence of hiatal hernia, are shown in Table 2. Mean LOS resting pressure was not different between the controls and patients with FH, but decreased in relation to the progressive severity of GORD, with EO and BO patients having the lowest values (p <0.01). Also the mean DEA was similar in patients with FH and HVs, but decreased significantly in patients with NERD (p<0.05) and in those with EO and BO (p<0.01). Hiatal hernia prevalence was higher in patients with BO compared to the other subgroups (p>0.01).

The same was true for patients with EO compared to NERD, FH and HVs (p<0.01). Finally, patients with NERD had more frequently a hiatal hernia than patients with FH and HVs In the present study, the retrospective analysis of prospectively collected data in a large group of unselected GORD patients, accurately classified by means of upper endoscopy, oesophageal biopsies and impedance-pH findings, supports earlier results [START_REF] Frazzoni | Pathophysiological characteristics of patients with non-erosive reflux disease differ from those of patients with functional heartburn[END_REF][START_REF] Frazzoni | Pathophysiological characteristics of the various forms of gastroesophageal reflux disease. Spectrum disease or distinct phenotypic presentations?[END_REF] that with increasing severity of mucosal damage there is an increase of manometric abnormalities findings. However, in contrast with previous investigations (14-17), we observed that there is an increase of IOM prevalence as the degree of mucosal lesions become worse. Indeed, we found that IOM was the most common motility abnormality in all patients with GORD and its prevalence gradually increased on passing from NERD to EO and BO, while patients with FH and HVs had a much lower frequency of this alteration, thus supporting the exclusion of FH patients from the realm of GORD in agreement with Rome III criteria [START_REF] Galmiche | Functional esophageal disorders[END_REF]. We have hypothesized that the so far inconclusive data in previous studies regarding this issue were mainly due to the difficulty to clearly separate patients with NERD from the functional population [START_REF] Martínek | Non-erosive and erosive gastroesophageal reflux diseases: No difference with regard to reflux pattern and motility abnormalities[END_REF][START_REF] Ouatu-Lascar | Reflux esophagitis: effect of position, nocturnal acid reflux (upright versus supine reflux in gastroesophageal reflux disease)[END_REF][START_REF] Lemme | Ineffective oesophageal motility in gastro-oesophageal erosive reflux disease and in non-erosive reflux disease: are they different?[END_REF][START_REF] Chen | Differences in Oesophageal Bolus Transit Between Patients with and without erosive reflux disease[END_REF]. Moreover, as novel finding, we observed that oesophageal clearing function, assessed by means of MII-EM, decreased as the severity of mucosal damage increased, with EO and BO patients having the greatest prevalence of abnormal bolus transit pattern. Interestingly, in contrast with NERD and FH patients, abnormal bolus transit was found also in case of normal motility and not only in case of IOM or DOS/NO, as one could expect. Therefore, combined impedance manometry permits to observe an impairment of oesophageal function in patients with GORD, despite a normal peristaltic profile reported by conventional manometry. This suggests that in severe reflux disease patients the impaired oesophageal clearance may be due to failure of primary peristalsis -IOM -or reduced capacity to transport along the oesophagus -abnormal bolus transit -.

In the current study, we confirmed data obtained by Frazzoni et al [START_REF] Frazzoni | Pathophysiological characteristics of the various forms of gastroesophageal reflux disease. Spectrum disease or distinct phenotypic presentations?[END_REF], who demonstrated that patients with reflux disease have a greater prevalence of manometric abnormalities in terms of lower LOS resting pressure, lower distal oesophageal wave amplitude and higher prevalence of hiatal hernia than those with FH and controls. In agreement with Wu et al. [START_REF] Wu | Distinct Clinical Characteristics Between Patients With NERD and Those With Reflux Esophagitis[END_REF], the frequency of these abnormalities gradually increases from NERD to EO and also to BO [START_REF] Zentilin | Comparison of the main oesophageal pathophysiological characteristics between short-and long-segment Barrett's oesophagus[END_REF], while there was no difference between EO and BO. In contrast, our manometric findings are at variance with those observed in recent investigations [START_REF] Martínek | Non-erosive and erosive gastroesophageal reflux diseases: No difference with regard to reflux pattern and motility abnormalities[END_REF][START_REF] Ouatu-Lascar | Reflux esophagitis: effect of position, nocturnal acid reflux (upright versus supine reflux in gastroesophageal reflux disease)[END_REF][START_REF] Lemme | Ineffective oesophageal motility in gastro-oesophageal erosive reflux disease and in non-erosive reflux disease: are they different?[END_REF], where no significant differences where found between NERD and EO. However, the above Authors defined NERD patients on the sole basis of a negative upper endoscopy and abnormal pH-testing or presence of symptom-reflux relationship in patients with abnormal pH-metry in order to exclude FH patients. This carries the risk of ruling out patients with normal pH and positive symptom-reflux association to acid and non-acid reflux, who represent a significant part of NERD, as shown by several studies [START_REF] Savarino | The role of non-acid reflux in NERD -Lessons learned from impedance-pH monitoring in 150 patients off therapy[END_REF][START_REF] Savarino | Functional Heartburn has more in common with Functional Dyspepsia than with Non-Erosive Reflux Disease[END_REF][START_REF] Herschcovici | Nonerosive Reflux Disease (NERD) -An Update[END_REF][START_REF] Savarino | The added value of impedance-pH monitoringto Rome III criteria in distinguishing functional heartburn from nonerosive reflux disease[END_REF][START_REF] Savarino | Functional heartburn and non-erosive reflux disease[END_REF]. Moreover, according to Wu et al. [START_REF] Wu | Distinct Clinical Characteristics Between Patients With NERD and Those With Reflux Esophagitis[END_REF] and in contrast with other investigations [START_REF] Lemme | Ineffective oesophageal motility in gastro-oesophageal erosive reflux disease and in non-erosive reflux disease: are they different?[END_REF][START_REF] Chen | Differences in Oesophageal Bolus Transit Between Patients with and without erosive reflux disease[END_REF] we found that patients with reflux disease have a greater prevalence of IOM than those with FH and controls, and that IOM gradually increases from NERD to EO to BO. Again the discrepancies of these results were mainly due to the unclear separation among the different entities of GORD, with the possible inclusion of patients with FH and exclusion of patients with Non-Acid Reflux Disease.

As to the assessment of bolus transit patterns in patients with various degree of reflux disease, to the best of our knowledge, only two studies have been published [START_REF] Chen | Differences in Oesophageal Bolus Transit Between Patients with and without erosive reflux disease[END_REF][START_REF] Domingues | Characteristics of oesophageal bolus transport in patients with mild oesophagitis[END_REF] Several hypotheses may explain the increased prevalence of IOM and the abnormal bolus transit patterns in patients with more severe reflux disease. First, it could be partly related to a severe oesophageal mucosal inflammation induced by increased gastrooesophageal reflux, which determines a reduction in oesophageal compliance and an increased resistance during bolus movement with a consequent delayed bolus transport.

Moreover, it has been recently demonstrated that the rate of triggering secondary peristalsis in patients with NERD is significantly lower than in normal subjects [START_REF] Iwakiri | Defective triggering of secondary peristalsis in patients with non-erosive reflux disease[END_REF]. The same phenomenon may be also present in patients with severe reflux disease causing a delay or stop during bolus transport. Different patterns could be also explained by the difference in oesophageal tone noted in severe GORD patients compared to those with mild GORD [START_REF] Jenkinson | Compliance measurement of lower oesophagealsphincter and oesophageal body in achalasia and gastro-oesophageal reflux disease[END_REF].

Also oesophageal abnormalities represented by different traction force or oesophageal shortening have been taken into consideration [START_REF] Pouderoux | Timing, propagation, coordination, and effect of esophageal shortening during peristalsis[END_REF][START_REF] Kahrilas | Attenuation of esophageal shortening during peristalsis with hiatal hernia[END_REF][START_REF] Williams | Diminished oesophageal traction forces with swallowing in gastrooesophageal reflux disease and in functional dysphagia[END_REF]. Anyway, our results suggest that the peristaltic amplitude alone does not explain the rate of incomplete bolus transit and that other mechanisms are involved in determining incomplete bolus transit in patients with oesophageal mucosal injuries.

As to the limitations of our study, the 30 day washout period before the upper endoscopy in patients using acid suppressive medication could be considered as too short for lesions to develop. Still, in our experience, this represents the maximum we can ask to patients in order to obtain good compliance and without them dropping out from the study.

Moreover, the incidence of recurring erosive lesions after a shorter period of PPI cessation remains unknown.

In conclusion, this study shows that identifying with accuracy -i.e. upper endoscopy and impedance-pH monitoring -GORD patients from those with functional disease leads to show relevant differences in terms of oesophageal manometric features, motility and bolus transit patterns, therefore clarifying the discrepancies observed in earlier studies. We demonstrate that increasing degrees of oesophageal mucosal damage is associated with a progressively more severe deflection of oesophageal function, expressed by an increased frequency of IOM and abnormal bolus transit patterns. This seems to reveal a novel piece of the puzzle related to the development of oesophageal mucosal injury.

The authors declared no conflict of interest.

F

  motor function at least one week before performing tests of oesophageal function. From 2003 to 2007 each patient underwent oesophageal function testing by means of conventional manometry using an eight-lumen, water-perfused, oesophageal manometry catheter (Mui Scientific, Canada) assembly consisting of four radial ports and four lateral ports spaced 5 cm apart and radially orientated 120° with respect to each other. Moreover, from 2007 to 2010, each patient underwent oesophageal function testing by means of MII-EM using a Koenigsberg 9-channel probe (Sandhill oesophageal function testing [EFT] catheter; Sandhill Scientific Inc, Highlands Ranch, CO). The 4.5-mm diameter catheter design has 2 circumferential solid-state pressure sensors at 5 and 10 cm from the tip and 3 unidirectional solid-state pressure sensors at 15, 20, and 25 cm. Impedance measuring segments, consisting of 2 rings placed 2 cm apart, centered 10, 15, 20, and 25 cm from the tip. The methodology of catheter placement, patient instruction and performance has been previously described for both techniques

  Seven hundred and ninety-eight consecutive patients (412 female, mean age 51, range 18-88) with typical symptoms of GORD met the enrolment criteria and entered the study. During upper endoscopy, we identified 340 patients (171 F, mean age 49, range 21-87) with EO, 352 without mucosal breaks (201 F, mean age 49, range 18-78) and 106 (40 F, mean age 58, 21-87) patients had histologically confirmed Barrett oesophagus. In the EO group, 199 patients had grade A and 82 had grade B, 39 had grade C and 20 had grade D oesophagitis.

  Gemignani L et al. -Prevalence of esophageal motility abnormalities in patients with EE, BE, NERD and FH 10 (p<0.01). No differences have been observed regarding onset velocity of contractions, LOS relaxation duration and UES pressure (p=ns).

Figure 1

 1 Figure1illustrates the different motility patterns among the various groups of patients. For the purpose of this study DES and Nutcracker Oesophagus were considered together (DES/NE). Moreover, the final manometric diagnosis for each patient was based on the liquid swallows to remain consistent with current tradition. Prevalence of IOM was statistically higher (p<0.01) in patients with mucosal lesions than in the other groups, occurring in 42% of patients with BO and in 38% of patients with EO, compared to 19% of NERD patients, 8% of FH and 4% of HVs. Patients with NERD had more frequently IOM than those with FH and HVs (p<0.01). Normal peristalsis was found more in HVs and FH than the other subgroups (p<0.01). No differences were found in terms of DES/NE prevalence among the various subgroups (p=ns).

  Gemignani L et al. -Prevalence of esophageal motility abnormalities in patients with EE, BE, NERD and FH 13

  regarding this issue. Domingues et al.[START_REF] Domingues | Characteristics of oesophageal bolus transport in patients with mild oesophagitis[END_REF] evaluated 25 patients with mild GORD -22 with grade A-B EO and 3 with NERD -and they found that GORD patients had a delayed bolus transport compared to controls, although normal bolus transport was observed in the majority of the swallows (91% of liquid and 81% of viscous swallows). Similarly, Chen et al.[START_REF] Chen | Differences in Oesophageal Bolus Transit Between Patients with and without erosive reflux disease[END_REF] noted no difference in the prevalence of complete bolus transport in 20 NERD patients compared with controls, while 20 patients with EO exhibited an increase in the frequency of incomplete bolus transit, which was also accompanied by more prolonged bolus transit. However, these studies have several limitations, mainly related to the small sample size, the enrolment of patients with only EO or NERD and finally the correct identification of NERD patients. Indeed, Chen et al. defined NERD only by means of endoscopically undetectable mucosal lesions with the consequent risk to include patients with FH in this group, while Domingues et al. included only 3 NERD patients and identified them only on the basis of an abnormal oesophageal acid exposure. Instead, in the current study evaluating a large population of well defined GORD patients using MII-pH, we found that the oesophageal clearing function decreased as the severity of mucosal damage increased, with EO and BO patients having the greatest prevalence of abnormal bolus transit pattern. This impairment was evident in case of both liquid and viscous swallows. Moreover, they had a significant delayed bolus transport compared to NERD and FH. Interestingly, patients with more severe reflux disease presented an abnormal bolus transit pattern also in case of normal motility and not only in case of IOM or DOS/NO, as observed in patients with NERD and FH. Therefore, in these patients the defect of oesophageal motility is more severe than the simple peristaltic profile one can assess by conventional manometry, thus underlying the importance of oesophageal clearance evaluation by means of MII-EM in them.

Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 2345 Figure 2. Abnormal Bolus Transit Diagnosis in the various subgroups of GORD patients (n=260) for liquid swallows (2a) and viscous swallows (2b)

  Bolus transit for liquid swallows in relation to the manometric findings is shown in Figure3. Combined MII-EM information for liquid swallows revealed that out of all patients

	COMBINED IMPEDANCE-MANOMETRY FINDINGS
	with IOM, 63% of EO patients and 67% of BO patients had ABT versus 50% of NERD and
	29% of FH (p<0.05). Of all patients with DOS, the frequency of ABT was similar in all
	subgroups (60% EO vs. 57% BO vs. 46% NERD vs. 50% FH, p=ns). Considering all patients
	with normal peristalsis, 45% of EO and 47% of BO patients had ABT versus 5% of NERD
	and 5% of FH (p<0.01). Similar results were observed evaluating bolus transit for viscous
	swallows, as illustrated in Figure 4. Of all patients with IOM, 67% of EO patients and 57% of
	BO patients had ABT versus 41% of NERD and 25% of FH (p<0.05). Of all patients with F DOS, the frequency of ABT was similar in all subgroups (50% EO vs. 60% BO vs. 56% NERD vs. 67% FH, p=ns). Considering all patients with normal peristalsis, 50% of EO and o r 56% of BO patients had ABT versus 7% of NERD and 8% of FH (p<0.01). Overall,
	P
	e
	e r
	R Six patients with FH (6/48), 23 with NERD (23/113), 35 with EO (35/65) and 19 (19/34) with e BO had abnormal bolus transit (ABT) with liquid swallows (p<0.01), while 5 with FH (5/31), 15 with NERD (15/66), 18 with EO (18/33) and 12 (12/21) with BO had ABT with viscous swallows (p<0.01). In addition, patients with EO and BO had a significant increase in TBTT v i e
	(8.7 s, liquid; 8.9 s, viscous and 9.1 s, liquid; 9.6 s, viscous, respectively) compared to NERD (6.8 s, liquid; 7.7 s, viscous) and FH controls (6.8 s, liquid; 7.3 s, viscous) (p< 0.05) with both w
	liquid and viscous swallows.
	The multi-nominal regression analysis found no significant (p=0.196) effect of gender
	distribution but a significant (p<0.001) co-variation of age on the distribution of manometric
	abnormalities in patients with various endoscopic findings. Of note is that age didn't differ
	across various endoscopic findings (Table 1) and the age difference between patients with
	normal manometry (47.0 ± 0.7 years), ineffective oesophageal motility (54.7 ± 0.9 years) and
	distal oesophageal spasms (51.9 ± 1.4 years), albeit being statistically significant (p<0.001), is
	most likely clinically non-relevant.

combined impedance manometry showed an oesophageal motility abnormality in 4% of FH, 4% of NERD, 22% of EO and 21% of BO patients in whom conventional manometry alone would have reported a normal motility peristaltic profile (Figure

5

).

Table 1 .

 1 Demographic parameters of overall GORD patients (n=755) and HV (n=48)

	o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 .

 2 Manometric features in GORD patients (n=755) and HV (n=48) Figure 1. Manometric patterns in GORD patients (n=755) and HV (n=48) Savarino E, Gemignani L et al. -Prevalence of esophageal motility abnormalities in patients with EE, BE, NERD and FH 22

	Population	Mean LOS Tone, mmHg (range)	Mean DOA, mmHg (range)	Hiatal Hernia, n (%)
	Healthy Volunteers (n=48)	27.6 (12-44) #	101.6 (24.2-211.8) #	7 (15%) #
	Functional Heartburn (n=96)	26.1 (9-42) #	94.4 (20.1-198) #	32 (33%) #
	NERD (n =213)	20 (3-44) &	79 (22.1-193.1) &	124 (58%) &
	Erosive Oesophagitis (n=340)	15.4 (1-43) *	49.2 (14.1-188-3) *	238 (70%) *
	Barrett Oesophagus (n=106)	13 (1-39) *	41.2 (12-231.8) *	87 (82%) *
	# p<0.05 vs. NERD, EO and BO
	& p<0.05 vs. BO, EO, FH and HV
	* p<0.05 vs. NERD, FH and HV

STATEMENTS:

A: Guarantor of this paper: Edoardo Savarino, MD, PhD; Vincenzo Savarino