Wulf Dominiek

MD Jan Tack

MODULATION OF THE POSTPRANDIAL ACID AND BILE POCKETS AT THE GASTRO-OESOPHAGEAL JUNCTION BY DRUGS THAT AFFECT GASTRIC MOTILITY

Keywords:

Objectives: Previous studies have established the presence of a postprandial acid pocket at the gastro-oesophageal junction (GOJ). We investigated whether altering gastric motility would affect the location and the extent of postprandial acid pockets in healthy volunteers. In addition, we studied the presence of bile in this pocket.

Methods: 16 healthy volunteers underwent pH and Bilitec® probe stepwise pullthrough to measure regional differences in pH and bile absorbance before and 10, 30 and 50 min after a liquid meal. At the start of the meal, saline, erythromycin or sumatriptan was administered. Results: After saline, ingestion of a meal induced an acid pocket, with a mean pH drop of 2.26 (compared to 0.25 before the meal, p<0.05) and a nadir pH of 2.71. The acid pocket persisted 30 and 50 minutes postprandially (pH drops 1.59 and 1.68 and nadir pH 3.17 and 2.52 respectively). Compared to saline, erythromycin significantly suppressed the pH drop and nadir pH (on average 0.66 and 3.4 at 10 min; comparable patterns at 30 and 50 min). After sumatriptan a significantly lower nadir pH was observed at 30 and 50 min (respectively 2.02 and 1.74, p<0.05 compared to saline). A postprandial bile pocket was noted in 50% of pull-throughs after saline, compared to 78% after erythromycin and 31% after sumatriptan. Conclusions: The presence of a bile pocket in a subset of the subjects confirms the heterogeneity of postprandial intragastric contents. Erythromycin disrupts the acid pocket but increases the presence of bile, while sumatriptan has opposite effects.

INTRODUCTION

Gastro-oesophageal reflux disease (GORD) is present when the reflux of gastric contents causes typical lesions or symptoms such as heartburn, regurgitation and noncardiac chest pain [START_REF] Vakil | The Montreal definition and classification of gastroesophageal reflux disease: a global evidencebased consensus[END_REF]. GERD is one of the most common disorders affecting the gastrointestinal tract [START_REF] Locke Gr 3rd | Prevalence and clinical spectrum of gastroesophageal reflux: a populationbases study in Olmsted County[END_REF]. Although acid reflux is classically accepted to be the cause of these symptoms, the relationship between acid and heartburn is far from clear [START_REF] Tack | Review article: the role of bile and pepsin in the pathophysiology and treatment of gastroesophageal reflux disease[END_REF].

While many GORD patients achieve symptom relief and healing of endoscopic lesions with proton pump inhibitors (PPIs) , others have persistent reflux symptoms despite PPI therapy [START_REF] Tack | Review article: the role of bile and pepsin in the pathophysiology and treatment of gastroesophageal reflux disease[END_REF][START_REF] Bardhan | The role of proton pomp inhibitors in the treatment of gastroesophageal reflux disease[END_REF][START_REF] Tack | Gastroesophageal reflux disease poorly responsive to single-dose proton pump inhibitors in patients without Barrett's esophagus: acid reflux, bile reflux or both?[END_REF]. These remaining symptoms are due to either acid or nonacid gastro-oesophageal reflux episodes or are not related to reflux at all [START_REF] Tack | Weakly acidic reflux[END_REF][START_REF] Blondeau | Pro: impedance testing is useful in the management of GERD[END_REF].

Simultaneous pH and Bilitec® monitoring is a valuable tool for characterizing the composition of the refluxate [START_REF] Tack | Gastroesophageal reflux disease poorly responsive to single-dose proton pump inhibitors in patients without Barrett's esophagus: acid reflux, bile reflux or both?[END_REF][START_REF] Vaezi | Role of acid and duodenogastroesophageal reflux disease[END_REF]. In previous studies combined pH and Bilitec® monitoring has been used to establish that reflux often consists of a mixture of acid and bile [START_REF] Vaezi | Role of acid and duodenogastroesophageal reflux disease[END_REF][START_REF] Koek | The role of acid and duodenal gastroesophageal reflux in symptomatic GERD[END_REF][START_REF] Koek | Multivariate analysis of the association of acid and duodeno-gastro-oesophageal reflux exposure with the presence of oesophagitis, the severity of oesophagitis and Barrett's oesophagus[END_REF]. In addition, duodenogastric reflux, also referred to as bile reflux, has been implicated in the pathogenesis of oesophageal lesions in GORD [START_REF] Koek | Multivariate analysis of the association of acid and duodeno-gastro-oesophageal reflux exposure with the presence of oesophagitis, the severity of oesophagitis and Barrett's oesophagus[END_REF] and in the generation of symptoms that persist in spite of PPI therapy [START_REF] Tack | Gastroesophageal reflux disease poorly responsive to single-dose proton pump inhibitors in patients without Barrett's esophagus: acid reflux, bile reflux or both?[END_REF].

Fletcher et al descriped a postprandial highly acidic unbuffered gastric juice or acid pocket at the gastro-oesophageal junction (GOJ) [START_REF] Fletcher | Unbuffered highly acidic gastric juice exists at the gastro-oesophageal junction after a meal[END_REF]. Postprandial exposure of the most distal oesophagus to acid has been implicated in the pathogenesis of GERD lesions in the distal esophagus [START_REF] Fletcher | Unbuffered highly acidic gastric juice exists at the gastro-oesophageal junction after a meal[END_REF][START_REF] Fletcher | Studies of acid exposure immediately above the gastro-oesophageal squamocolumnar junction: evidence of short segment reflux[END_REF]. More recent imaging studies have confirmed the presence of a liquid acid pocket, near the junction, and have shownits position relative to the squamocolumnar junction represents a major risk factor for the occurrence of acidic reflux into the distal oesophagus in healthy subjects and GORD patients [START_REF] Goetze | The effect of gastric secretion on gastric physiology and emptying in the fasted and fed state assessed by magnetic resonance imaging[END_REF][START_REF] Beaumont | The position of the acid pocket as a major risk factor for acidic reflux in healthy subjects and patients with GORD[END_REF]. Conversely, changing the extent or the position of the acid pocket relative to the gastro-esophageal junction might be relevant for GORD treatment. As outlined above, duodenogastric reflux is an additional important factor in the pathogenesis of GORD [START_REF] Tack | Gastroesophageal reflux disease poorly responsive to single-dose proton pump inhibitors in patients without Barrett's esophagus: acid reflux, bile reflux or both?[END_REF][START_REF] Vaezi | Role of acid and duodenogastroesophageal reflux disease[END_REF][START_REF] Koek | The role of acid and duodenal gastroesophageal reflux in symptomatic GERD[END_REF][START_REF] Koek | Multivariate analysis of the association of acid and duodeno-gastro-oesophageal reflux exposure with the presence of oesophagitis, the severity of oesophagitis and Barrett's oesophagus[END_REF], but whether bile is also present in the acid pocket just distal to the gastro-oesophageal squamocolumnar junction still remains unclear. The aims of this study therefore were to investigate 1) whether the postprandial acid pocket is associated with a bile pocket in healthy volunteers and 2) whether altering gastric motility would affect the location and the extent of postprandial acid and bile pockets in healthy volunteers.

MATERIALS AND METHODS

Subjects

The study was performed in 16 healthy volunteers (HV) (9 women and 7 men, mean age 27.8 (range 21-39)). All HV were free of any gastrointestinal symptoms and none of them had a history of reflux disease or surgery to the upper gastrointestinal tract or thorax. Volunteers were asked not to smoke on the morning of the test and not to eat at least 12 hours prior to the test. None of them took any medication.

Exclusion criteria were peptic ulcer disease; previous oesophageal, gastric or biliary surgery; previous abdominal or thoracic radiotherapy; active gastrointestinal bleeding; presence of oesophageal or fundic varices; oesophageal or upper small intestinal Crohn's disease; infectious oesophagitis or active neoplastic disease. All volunteers gave written informed consent for the study, which was approved by the Ethical Committee of the hospital.

Study protocol

The volunteers underwent acid and bile pocket studies on 3 different occasions, at least one week apart. Probes for assessing acid and duodeno-gastro-oesophageal reflux were introduced via the mouth into the oesophagus 5cm proximal to the lower oesophageal sphincter (LOS), defined by preceding pull-through manometry.

Combined pH and Bilitec® monitoring was performed by stepwise pull-through before and 10, 30 and 50 minutes after a liquid meal (200 ml Nutridrink®, 300 kcal). At the start of the meal, placebo (saline 100 ml IV), the gastrokinetic erythromycin (Abbott, 200 mg IV in 100 ml of saline) or the gastric relaxant sumatriptan (GlaxoSmithKline, 6 mg SC) was administered (15--18) in a random fashion, with the sequence determined by drawing envelopes. The setup of the study protocol is shown in Figure 1. A combined pH and Bilitec® monitoring was performed using a two-channel antimony pH catheter with separate skin reference electrode attached to a fiberoptic spectrophotometer Bilitec® 2000 (Synectics Medical, Stockholm, Sweden). Before each study, the pH probe was calibrated in buffer solutions of pH 1.0 and 7.0 while Bilitec® probe was calibrated in water [START_REF] Tack | Gastroesophageal reflux disease poorly responsive to single-dose proton pump inhibitors in patients without Barrett's esophagus: acid reflux, bile reflux or both?[END_REF][START_REF] Koek | The role of acid and duodenal gastroesophageal reflux in symptomatic GERD[END_REF][START_REF] Koek | Multivariate analysis of the association of acid and duodeno-gastro-oesophageal reflux exposure with the presence of oesophagitis, the severity of oesophagitis and Barrett's oesophagus[END_REF]. A stepwise pull-through was performed pulling back 1cm every minute from 10 cm below the LOS until 5 cm above the LOS. Pull-through was performed pre-and postprandially (10, 30 and 50 minutes after drinking a liquid meal of 200 ml, ie 300 kcal) in semi-recumbent position.

pH and Bilitec® monitoring

Data analysis

Analysis was performed on average preprandial and postprandial pH and bilirubin absorbances in the gastric body. In the range of 10 cm below to 5 cm above the LOS, we studied the presence and persistence of an acid pocket at 10, 30 and 50 minutes postprandially.

Intragastric pH was defined as the mean pH between 10 cm below and 5 cm above the LOS. Delta pH was defined as the difference between the intragastric pH and the nadir pH, with nadir pH being the lowest pH between 2 cm below and 2 cm above the LOS (ie the lowest pH at the level of LOS). The presence of an acid pocket was defined as a segmentary (> 4 cm) drop in pH of more than one unit compared to intragastric pH. The extent of the acid pocket was defined as the distance between the upper and lower limit of the acid pocket, and the latter were expressed as the distance from the LOS where the presence of the pH-drop started and ended. We also studied the presence of a "bile pocket" defined as a segmentary rise (> 4 cm) in Bilitec® absorbance ≥ 0.14. This cut-off level is the one used in esophageal bilirubin absorbance studies and has also been applied to intragastric measurements [START_REF] Fletcher | Unbuffered highly acidic gastric juice exists at the gastro-oesophageal junction after a meal[END_REF][START_REF] Annese | Erythromycin accelerates gastric emptying in man by inducing powerful antral contractions and an improved gastroduodenal coordination[END_REF].

Although in vitro studies showed that fiberoptic bilirubin measurements are influenced by an acidic pH, the impact in in vivo measurements seems neglibile [START_REF] Caldwell | An ambulatory bile reflux monitoring system: an in vitro appraisal[END_REF][START_REF] Cuomo | Analysis of ambulatory duodenogastroesophageal reflux monitoring[END_REF].

Statistical analysis was performed using Student's t test.

Formatted: Font: Arial Formatted: Font: Arial Formatted: Font: Arial Deleted: , a segment defined as a length of ≥ 4 to 15cm [START_REF] Fletcher | Unbuffered highly acidic gastric juice exists at the gastro-oesophageal junction after a meal[END_REF][START_REF] Caldwell | An ambulatory bile reflux monitoring system: an in vitro appraisal[END_REF][START_REF] Cool | Characteristics and clinical relevance of proximal esophageal pH monitoring[END_REF]..

RESULTS

Acid pocket

In the placebo studies, preprandial (T0) intragastric pH was significantly lower than the oesophageal pH (1.62 ± 0.39 versus 4.85 ± 0.59, p<0.0001). Ingestion of a meal caused a buffering of intra-gastric pH at 10, 30 and 50 min (Table 1 and2). Ingestion of the meal was also associated with the appearance of an acid pocket at 10, 30 and 50 minutes postprandially (Figure 2). The extent of the acid pocket is summarized in Table 3 and was generally between 2 cm below and 3 cm above the GOJ. Significant (p<0.05) differences in delta pH (mean 2.26 versus 0.25 before meal) and nadir pH (mean 2.71 versus 1.26 before meal) were present between LOS -2 and +2cm, 10 minutes after the meal. A similar pattern was found after 30 and 50 minutes with respectively mean delta pH 1.59 and 1.68 and nadir pH 3.17 and 2.52 (Figure 2).

"Bile" pocket

Preprandially the averaged Bilitec® pull-throughs did not show a significant variance in the proximal stomach and bilirubin absorbance values were low. Postprandially, the intragastric bile content was significantly elevated compared to preprandially (Table 4). An example of a bile pocket is shown in Figure 3. A postprandial bile pocket was noted in 50% of pull-throughs in the saline group after 50 minutes (Figure 4).

Influence of erythromycin

The intragastric pH at 50 minutes was significantly higher after erythromycin compared to saline (4.19 ± 2.01 (SEM 0.50) versus 5.40 ± 1.15 (SEM 0.30), p<0.05).

Compared to saline, the pH at the LOS after the meal was significantly higher after erythromycin. No obvious postprandial acid pocket was observed in most of the subjects at the LOS after erythromycin. There was a significant suppression of the delta pH (mean 0.66) and nadir pH (mean 3.43) after 10 minutes, and comparable patterns at 30 and 50 minutes (Figure 2). These values indicate suppression or elimination of the acid pocket in most of the patients, but when still present, the extent of the acid pocket after erythromycin did not seem to differ significantly (Table 3).

After erythromycin, peak bilirubin absorbance was significantly higher after 30 and 50 minutes in comparison to placebo (Table 4). Analyses of individual postprandial tracings on 45 successful Bilitec® pull-throughs after erythromycin showed the presence of a bile pocket in 11 subjects (73,3%) after 10 and 30 minutes and in 13 subjects (86,7%) after 50 minutes. A postprandial bile pocket was present in 77,8% of pull-throughs after erythromycin, compared to 45,2% in the saline group (p<0.005).

(Figure 4). The majority of the bile pockets (55%) coincided with the acid pocket, 27% were situated immediately proximal to the acid pocket and 18% were situated immediately distal to the acid pocket.

Influence of sumatriptan

The intragastric pH did not differ significantly after administration of sumatriptan, and the delta pH and nadir pH after 10 minutes was not altered (Tables 1 and2). However, after 30 and 50 min, nadir pH (respectively 2.02 and 1.74 versus 3.17 and 2.52 after saline) was lower compared to saline (Table 2). In addition, sumatriptan tended to increase the extent of the acid pocket at 30 and 50 minutes postprandially (Figure 2), both for the intragastric extent as for the extent above the LES (Table 3).

A postprandial bile pocket occurred in 31% of the subjects (Figure 4). [START_REF] Holloway | Provocation of transient lower esophageal sphincter relaxations by meals in patients with symptomatic gastroesophageal reflux[END_REF] and is most pronounced in the distal oesophagus [START_REF] Cool | Characteristics and clinical relevance of proximal esophageal pH monitoring[END_REF]. Ingestion of a meal is expected to increase the intragastric pH by the meal's buffering capacity, but it was previously established that a pocket of unbuffered, highly acidic juice is present at the gastro-oesophageal junction (GOJ) after intake of a solid meal in healthy volunteers, a so-called acid pocket [START_REF] Fletcher | Unbuffered highly acidic gastric juice exists at the gastro-oesophageal junction after a meal[END_REF][START_REF] Fletcher | Studies of acid exposure immediately above the gastro-oesophageal squamocolumnar junction: evidence of short segment reflux[END_REF][START_REF] Goetze | The effect of gastric secretion on gastric physiology and emptying in the fasted and fed state assessed by magnetic resonance imaging[END_REF][START_REF] Beaumont | The position of the acid pocket as a major risk factor for acidic reflux in healthy subjects and patients with GORD[END_REF]. On the other hand, intragastric Bilitec® studies have shown that duodeno-gastric bile reflux is a physiological postprandial event, which occurs in every healthy volunteer, raising the possibility that bile may also be present at the GOJ in some subjects [START_REF] Koek | Mechanisms underlying duodeno-gastric reflux in man[END_REF], thereby potentially contributing to the pathogenesis of GORD-related lesions and symptoms. A close correlation exists between acid and duodenogastric reflux exposure of the distal esophagus, and both acid and duodenogastro-oesophageal reflux occurred simultaneously in the majority of the reflux episodes in patients with GORD who were not treated with acid-suppressive therapy, suggesting that both are simultaneously present at the GOJ (8,9,230).. Factors that determine the location and the extent of the acid and bile pockets have not been identified yet, but gastric motility is likely to play a role, as it is a strong determinant of movement and location of gastric contents. This is further supported by observations that gastric motility may play a role in symptom generation in some non-erosive reflux disease (NERD) patients [START_REF] Kamiya | Impaired gastric motility and its relationship to reflux symptoms in patients with nonerosive gastroesophageal reflux disease[END_REF]. The fiberoptic spectrophotometric probe Bilitec® 2000 was developed to quantify duodeno-gastro-oesophageal reflux by evaluation of bilirubin concentration [START_REF] Bechi | Long-term ambulatory enterogastric reflux monitoring. Validation of a new fiberoptic technique[END_REF]. Owing to its construction, the Bilitec® probe is prone to solid food impaction causing absorbance and measurement artifacts [START_REF] Bechi | Long-term ambulatory enterogastric reflux monitoring. Validation of a new fiberoptic technique[END_REF]. Previously, we have shown that a liquid meal (Nutridrink® 200ml, 300Kcal) does not interfere with Bilitec® measurements [START_REF] Tack | Dietary restrictions during ambulatory monitoring of duodenogastroesophageal reflux[END_REF]. The present study established that this meal induces an acid pocket in the stomach in all subjects and a bile pocket in a subset of subjects. After saline administration, a significant drop in pH, indicative of the acid pocket, was present at the gastro-esophageal junction at all time points studied (10, 30 and 50 minutes after the meal) in spite of the relatively low volume and low caloric contents of the liquid meal we used. Previously we have shown that the gastrokinetic erythromycin is a strong stimulant of gastric contractility [START_REF] Annese | Erythromycin accelerates gastric emptying in man by inducing powerful antral contractions and an improved gastroduodenal coordination[END_REF][START_REF] Piessevaux | Perception of changes in wall tension of the proximal stomach in man[END_REF][START_REF] Janssens | Improvement of gastric emptying in diabetic gastroparesis by erythromycin. Preliminary studies[END_REF][START_REF] Tack | Dose-related effect of erythromycin on gastroduodenal motility and plasma motilin levels. Studies in healthy volunteers and in patients with diabetic gastroparesis[END_REF].

Erythromycin induces faster clearance of duodeno-gastric reflux from the stomach, which depends on enhanced antral contractile activity rather than a premature phase III [START_REF] Koek | Mechanisms underlying duodeno-gastric reflux in man[END_REF]. Erythromycin increases lower oesophageal sphincter pressure in man in the fasting and fed state, but has no effect on oesophageal body peristalsis [START_REF] Chaussade | Motilin agonist erythromycin increases human lower esophageal sphincter pressure by stimulation of cholinergic nerves[END_REF]. In the present study, erythromycin rapidly suppressed and eliminated the acid pocket, which was shown numerically as a significant suppression of the delta pH and nadir pH at all time points. Furthermore, the postprandial intragastric pH was significantly elevated and was more rapidly neutralized after administration of erythromycin, which may reflect a better mixing of the gastric acid contents and a more rapid emptying. The lack of simultaneous measurement of gastric contractility in the proximal stomach is a clear limitation of the present experimental set-up, which should be addressed in follow-up studies.

However, motility of the proximal stomach cannot be reliably assessed by manometry catheters, and barostat measurements would interfere with the physiological location of the acid pocket. It is conceivable that magnetic resonance imaging may provide the most suitable tool for such studies [START_REF] Goetze | The effect of gastric secretion on gastric physiology and emptying in the fasted and fed state assessed by magnetic resonance imaging[END_REF]. Acid suppression by erythromycin as a contributing mechanism cannot entirely be excluded, and the effect of erythromycin on gastric acid secretion in man has not been well studied. However, the increased presence of bile at the gastro-oesophageal junction after erythromycin is supportive of a mixing effect rather than an acid suppressive action. A

The 5HT 1 agonist sumatriptan on the other hand, is a strong inhibitor of gastric contractility which reduces fasting fundic tone, prolongs the meal-induced fundic relaxation and significantly delays gastric emptying (17,18,30,). In a previous study by our group, we demonstrated that sumatriptan prevented the decay in rate of transient LES relaxations that occurs after a meal and favored the occurrence of gastroesophageal reflux despite increasing LES pressure [START_REF] Sifrim | Effect of sumatriptan, a 5-HT1 agonist, on the frequency of transient lower esophageal sphincter relaxations and gastro-oesophageal reflux in healthy subjects[END_REF]. The sustained postprandial high rate of transient LES relaxations after sumatriptan may be a consequence of a prolonged fundus relaxation and retention of meal in the proximal stomach [START_REF] Coulie | Sumatriptan, a selective 5-HT1 receptor agonist, induces a lag phase for gastric emptying of liquids in man[END_REF][START_REF] Tack | Influence of sumatriptan on gastric fundus tone and on the perception of gastric distention on man[END_REF]. In the current study, a significantly lower nadir pH was observed 30 and 50 minutes after sumitriptan administration, suggestive of a longer persistence of the acid pocket. In addition to confirming the presence of an acid pocket after a liquid meal and its modulation by motility-modifying drugs, we also demonstrated the presence of a bile pocket in a subset of the subjects (Figure 3). Besides confirming the heterogeneous nature of intragastric contents shortly after the meal, this observation is also supportive of potential synergistic actions of acid and bile in the pathogenesis of GORD related distal oesophageal lesions. It remains to be investigated whether this bile pocket is different in patients with severe reflux oesophagitis or Barrett's oesophagus [START_REF] Blondeau | Pro: impedance testing is useful in the management of GERD[END_REF][START_REF] Beaumont | The position of the acid pocket as a major risk factor for acidic reflux in healthy subjects and patients with GORD[END_REF]. The current study is limited in that respect, as only healthy volunteers were included, and future studies should address whether these findings can be confirmed in GERD patients too.

In general, altering the gastric motility affects the location and the extent of postprandial acid and bile pockets in healthy volunteers. We have demonstrated in the present study that enhanced motility by erythromycin disrupt the acid pocket but increases the presence of a bile pocket at the GOJ, while motor inhibition by sumatriptan has opposite effects. These studies confirm that gastric motility modifying drugs could be a target for the treatment of GORD, through their actions on the presence and extent of caustic material at the GOJ. prandially. An acid pocket (segmentary pH < 4) can be observed at all time points after saline administration (arrows). The acid pocket is absent after erythromycin administration but more pronounced after sumatriptan administration. The order of the drugs and placebo was randomized, by drawing envelopes. This has been added in the method section of the manuscript, with some additional references.

F

The cut-off to define a bile acid pocket seems arbitrary -what was this based upon?

The cut-of value is based on previous literature evaluating both intra-esophageal and intra-gastric bilirubin monitoring. Although Bilitec® monitoring has been reported to underestimate bilirubin concentrations in an acid environment, this seems limited in an in vivo setting. This has been added to the methods section. The anatomical location was similar to the one for the acid pocket. The paper is well written and consice. The inclusion only of healthy volunteers impacts on the potential relevance of this work.

F

Studies regarding the postprandial acid pocket in patients with gastro-oesophageal reflux disease are currently ongoing in our centre to assess the characteristics of the acid pocket in these patient group.

The method focuses exclusively on the acid/bile pocket and does not attempt to explain the mechanism of effect. This study is a proof-of-concept study to establish that altering the location and extent of the acid pocket can be achieved by stimulating or inhibiting gastric motility. We think the findings confirm that such an effect was observed. We agree that we did not simultaneously assess gastric motility, and that this is a limitation. On the other hand, the effects of erythromycin and of sumatriptan on gastric motility have been extensively documented in the literature and are remarkably reproducible. This has been added to the manuscript, with some additional references, as suggested by the reviewer..

This study shows that the prokinetic increases gastric emptying and mixing of the acid layer with the meal and duodenal secretions so neutralising the acid.

Conversely the antikinetic slows emptying and decreases gastric mixing with the opposite effect. These are novel observations; however the proposed mechanism of effect is not documented. This study is a proof-of-concept study to establish that altering the location and extent of the acid pocket can be achieved by stimulating or inhibiting gastric motility. We think the findings confirm that such an effect was observed. We agree that we did not simultaneously assess gastric motility, and that this is a limitation. On the other hand, the effects of erythromycin and of sumatriptan on gastric motility have been extensively documented in the literature and are remarkably reproducible. This has been added to the manuscript, with some additional references, as suggested by the reviewer.

In the absence of patient data the clinical relevance of these findings is difficult to assess.

Studies regarding the postprandial acid pocket in patients with gastro-oesophageal reflux disease are currently ongoing in our centre to assess the characteristics of the acid pocket in these patient group. We have acknowledged this limitation in the discussion section.

Formatted

Formatted

  reflux occurs mainly during the postprandial period through increased triggering of transient lower oesophageal sphincter relaxations (TLOSR)

Figure 1 :

 1 Figure 1: Design of the study. Probes for assessing acid and duodeno-gastrooesophageal reflux were introduced via the mouth. The lower oesophageal sphincter (LOS) was defined by preceding pull-through manometry. Dual pH and Bilitec® monitoring was performed by stepwise pull-through before and 10, 30 and 50 minutes after a liquid meal (200 ml Nutridrink®, 300 kcal). At the start of the meal, saline 100 ml, erythromycin 200 mg IV or sumatriptan 6 mg SC was administered.

Figure 2 :

 2 Figure 2: pH monitoring. Average pH in stomach, LOS and oesophagus is shown in a time dependent fashion (pre-prandial, 10, 30 and 50 minutes postprandial). The X-axis represents the distance (from -10 cm intragastric to 5 cm in the oesophagus), while pH values are depicted on the Y-axis. No differences were noticed pre-

Figure 3 :

 3 Figure 3: Example of a bile pocket. Pull-through profiles with a pH and a Bilitec probe before (upper panel) and after (low panel) a liquid meal. Note the neutralisation and pH drop (acid pocket, black arrow) at the level of the lower esophageal sphincter in the lower panel pH trace, and the presence of a rise in bilirubin absorbance and an absorbance peak (grey arrow) below the level of the lower esophageal sphincter in the lower panel.

Figure 4 :

 4 Figure 4: Bilitec monitoring. In healthy volunteers a bile pocket is present at all time points. The number is increased after erythromycin administration but decreased after sumatriptan administration.

Table 1 .

 1 Mean intra-gastric pH values, before and after the meal, with administration of saline, sumatriptan or erythromycin. *p<0.05 compared to saline. + p<0..05 for sumatriptan compared to erythromycin.

	o r
	P
	e
	e r
	R
	e
	v i e
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Table 2 .

 2 Mean intra-oesophageal pH, mean nadir pH in LOS and mean nadir pH in the acid pocket, before and after the meal, with administration of saline, sumatriptan or erythromycin. *p<0.05 compared to saline. ‡ p<0.05 compared to erythromycin.

Table 3 .

 3 Extent of the acid pocket, after the meal, with administration of saline, sumatriptan or erythromycin. *: only 2 subjects with an acid pocket; **:only 1 subject with an acid pocket

Table 4 .

 4 Mean bilirubin absorbance in the stomach, the esophagus and the acid pocket, before and after the meal, with administration of saline, sumatriptan or erythromycin. *p<0.05 compared to saline. ‡ p<0.05 compared to erythromycin.

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

Table 1 .

 1 Mean intra-gastric pH values, before and after the meal, with administration of saline, sumatriptan or erythromycin. *p<0.05 compared to saline. ‡ p<0.05 compared to erythromycin. †p<0.05 compared to postprandial values

	TABLES
		mean intra-gastric pH
		preprandial	10 min	30 min 50 min
	Saline	1.51 †	4.97	4.77	4.19
	Erythromycin	1.81 †	4.09	5.17	5.40*
	Sumatriptan	1.45 †	4.20	3.65 ‡	3.34 ‡

Table 2 .

 2 Mean intra-oesophageal pH, mean nadir pH in LOS and mean nadir pH in the acid pocket, before and after the meal, with administration of saline, sumatriptan or erythromycin. *p<0.05 compared to saline. ‡ p<0.05 compared to erythromycin.

		mean intra-oesophageal pH
		preprandial	10 min	30 min 50 min
	Saline	4.73	4.61	4.57	4.05
	Erythromycin	4.89	4.82	5.22	5.32*
	Sumatriptan	5.31	3.94	3.93 ‡	4.20 ‡
		mean nadir pH in LOS
		preprandial	10 min	30 min 50 min
	Saline	1.26	2.71	3.17	2.52
	Erythromycin	1.58	3.43*	4.20*	4.13*
	Sumatriptan	1.54	2.16 ‡	2.02 ‡	1.74 ‡
		mean nadir pH in pocket
		preprandial	10 min	30 min 50 min
	Saline	/	2.09	3.43	1.89
	Erythromycin	0.96	3.66*	4.24	2.84
	Sumatriptan	0.96	2.06 ‡	1.53* ‡	1.76

Table 3 .

 3 Extent of the acid pocket, after the meal, with administration of saline, sumatriptan or erythromycin. *: only 2 subjects with an acid pocket; **:only 1 subject with an acid pocket

			Average	Upper limit	Lower	Average
			pH AP	(cm)	limit (cm)	extent (cm)
	Saline	10 min	3,2	3,2	-2,4	5,6
		30 min	3,5	2,8	-1,4	4,2
		50 min	3,4	3,6	-2,4	6,0
	Erythromycin 10 min	-	-	-	-
		30 min*	4,9	2,0	-3,5	5,5
		50
		min**	4,6	3,0	-3,0	6,0
	Sumatriptan 10 min	2,8	3,5	-3,3	6,8
		30 min	2,7	2,3	-3,3	5,5
		50 min	3,1	4,0	-2,3	6,3

Table 4 .

 4 Mean bilirubin absorbance in the stomach, the esophagus and the acid pocket, before and after the meal, with administration of saline, sumatriptan or erythromycin. *p<0.05 compared to saline. ‡ p<0.05 compared to erythromycin. Comments for Transmission to the Authors I am unclear if the order of drugs/placebo was randomized (and by what method of randomization)? Or if not, how was this done in an unbiased fashion?

		mean intra-gastric bilirubin absorbance
		preprandial	10 min	30 min 50 min
	Saline	0.13	0.17	0.11	0.25
	Erythromycin	0.08	0.10	0.16	0.28*
	Sumatriptan	0.04	0.06 ‡	0.13	0.11 ‡
		mean intra-oesophageal bilirubin
		absorbance
		preprandial	10 min	30 min 50 min
	Saline	0.09	0.13	0.14	0.15
	Erythromycin	0.09	0.16	0.19	0.19*
	Sumatriptan	0.06	0.10 ‡	0.12 ‡	0.14
		peak bilirubin absorbance in pocket
		preprandial	10 min	30 min 50 min
	Saline	0.31	0.22	0.19	0.21
	Erythromycin	/	0.25	0.30*	0.37*
	Sumatriptan	/	0.18 ‡	0.19 ‡	0.19 ‡

  Comments for Transmission to the Authors Boecxstaens et al. assess the effects of erythromycin, a prokinetic, and sumatriptan, an antikinetic, on the composition of the "acid / bile pocket" present at the GOJ in healthy volunteers. The acid / bile pocket almost certainly represents the layer of gastric secretions that collects on the surface of the meal.1 Anatomical changes and / or increased secretion in reflux patients has been shown to increase the size and acidity of this "pocket" and this is thought to promote reflux in this group.[START_REF] Locke Gr 3rd | Prevalence and clinical spectrum of gastroesophageal reflux: a populationbases study in Olmsted County[END_REF][START_REF] Tack | Review article: the role of bile and pepsin in the pathophysiology and treatment of gastroesophageal reflux disease[END_REF][START_REF] Bardhan | The role of proton pomp inhibitors in the treatment of gastroesophageal reflux disease[END_REF]

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  1. Goetze O, Treier R, Fox M, Steingoetter A, Fried M, Boesiger P, et al. The effect of gastric secretion on gastric physiology and emptying in the fasted and fed state assessed by magnetic resonance imaging. Neurogastroenterol Motil 2009;21(7):725-42. Added to article. 2. Fletcher J, Wirz A, Young J, Vallance R, McColl KE. Unbuffered highly acidic gastric juice exists at the gastroesophageal junction after a meal. Gastroenterology 2001;121(4):775-83. 3. Fletcher J, Wirz A, Henry E, McColl KE. Studies of acid exposure immediately above the gastro-oesophageal squamocolumnar junction: evidence of short segment reflux. Gut 2004;53(2):168-73. Added to article. 4. Beaumont H, Bennink RJ, de Jong J, Boeckxstaens GE. The position of the acid pocket as a major risk factor for acidic reflux in healthy subjects and patients with GORD. Gut 2010;59(4):441-51.

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ACKNOWLEDGMENTS AND DISCLOSURES

KB is a postdoctoral research fellow of the Flanders Research Foundation (FWO),

Belgium. This study is supported by a Methusalem grant from the University of Leuven to Prof. J. Tack. DD and RV performed the research. JT designed the study. VB, KB, RB, DD, JT and RV analysed the data. VB, KB and JT wrote the paper.

Competing Interests: the authors have no competing interests

Deleted: 3 Deleted: ¶


P A F G Bruggeman

A J C Wolffs

Brule

Anne J M Loonen

Arjan R Jansz

Herman Kreeftenberg

Cathrien A Bruggeman

Petra F G Wolffs

Dr Adriaan J C Van Den Brule
email: a.van.den.brule@pamm.nl

Acceleration of direct identification of Staphylococcus aureus versus Coagulase Negative Staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods

Keywords: Blood culture material, bacterial DNA isolation, real-time PCR, Staphylococci

    Acceleration of the direct identification of versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods

Introduction

S.aureus is a pathogen which can cause both hospital-and community associated infectious diseases, ranging from minor skin infections to endocarditis, bacteremia, sepsis and septic shock [START_REF] Lowy | Staphylococcus aureus infections[END_REF]. Sepsis can result in high morbidity and mortality. In the United States, blood stream infections develop in approximately 250.000 people yearly [START_REF] Cdc | Report of mortality statistics[END_REF]. In the Netherlands, the incidence of patients admitted to the ICU with severe sepsis ranges around 8643 ± 929 per year [START_REF] Van Gestel | Prevalence and incidence of severe sepsis in dutch intensive care units[END_REF]. Currently, blood culture is the gold standard for identification of pathogens from suspected bacterial sepsis patients.

Unfortunately, blood culture is time-consuming taking at least 24-72 hours for final determination of the bacteria causing the disease. Staphylococci are number one of Gram positive organisms in blood cultures. Differentiating S.aureus from Coagulase Negative Staphylococci (CNS) is important, because sepsis with S.aureus is common and virulent with mortality rates in the range of 20-30% [START_REF] Saginur | Staphylococcus aureus bacteraemia of unknown primary source: Where do we stand?[END_REF]. CNS are often considered as being contaminants in blood cultures due to the fact that these species are members of the normal skin flora and mucous membranes and can contaminate the sample when it is taken. However, it is known that CNS infections are increasingly recognized as clinically relevant infections and confirmation on the presence of these species in blood culture is therefore important (reviewed in [START_REF] Von Eiff | Pathogenesis of infections due to coagulase-negative staphylococci[END_REF][START_REF] Piette | Role of coagulase-negative staphylococci in human disease[END_REF][START_REF] Huebner | Coagulase-negative staphylococci: Role as pathogens[END_REF][START_REF] Kloos | Update on clinical significance of coagulase-negative staphylococci[END_REF]).

Several molecular methods for rapid accurate detection of bacteria, from positive blood culture material have been described, including (commercial) real-time PCR based diagnostic tests [START_REF] Wellinghausen | Algorithm for the identification of bacterial pathogens in positive blood cultures by real-time lightcycler polymerase chain reaction (pcr) with sequence-specific probes[END_REF][START_REF] Sakai | Simultaneous detection of staphylococcus aureus and coagulase-negative staphylococci in positive blood cultures by real-time pcr with two fluorescence resonance energy transfer probe sets[END_REF][START_REF] Granger | Multiplex pcr-ligation detection reaction assay for the simultaneous detection of drug resistance and toxin genes from staphylococcus aureus, enterococcus faecalis and enterococcus faecium[END_REF], fluorescence in situ hybridization [START_REF] Hartmann | Rapid identification of staphylococcus aureus in blood cultures by a combination of fluorescence in situ hybridization using peptide nucleic acid probes and flow cytometry[END_REF][START_REF] Peters | Rapid identification of pathogens in blood cultures with a modified fluorescence in situ hybridization assay[END_REF], MALDI-TOF-MS [START_REF] Stevenson | Rapid identification of bacteria in positive blood culture broths by maldi-tof mass spectrometry[END_REF], but also DNA micro arrays [START_REF] Yoo | DNA microarray-based identification of bacterial and fungal pathogens in bloodstream infections[END_REF][START_REF] Cleven | Identification and characterization of bacterial pathogens causing bloodstream infections by DNA microarray[END_REF]. However, all these techniques are used on positive blood culture material. Reduction in the time to result can be achieved by applying molecular methods either directly on whole blood or on blood culture material with reduced incubation times. Ideally usage of whole blood is preferred but the techniques that are now available are often, clinically, not sensitive enough, as has been shown by others investigating a commercial real-time PCR test available [START_REF] Avolio | Molecular identification of bloodstream pathogens in patients presenting to the emergency department with suspected sepsis[END_REF][START_REF] Dierkes | Clinical impact of a commercially available multiplex pcr system for rapid detection of pathogens in patients with presumed sepsis[END_REF][START_REF] Wallet | Preliminary clinical study using a multiplex real-time pcr test for the detection of bacterial and fungal DNA directly in blood[END_REF]. Blood culture materials are known to contain inhibiting factors which can reduce detection in a sensitive real-time PCR [START_REF] Wilson | Inhibition and facilitation of nucleic acid amplification[END_REF][START_REF] Wa | Purification and characterization of pcr-inhibitory components in blood cells[END_REF][22][23]. It is therefore important to include a good isolation method, in the molecular diagnostic strategy, which is able to efficiently remove inhibiting factors and one which still allows sensitive DNA detection by PCR. In this study we compared 6 different, both manual and automated, bacterial DNA isolation methods for 2 commonly used blood culture systems, i.e. BACTEC (Becton Dickinson) and Bact/ALERT (bioMérieux), to be able to find the most sensitive bacterial DNA isolation method. Additionally, we investigated the occurrence of inhibition in PCR amplification after DNA isolation. A sensitive real-time PCR assay was designed to be able to detect staphylococci and to differentiate S.aureus from CNS [Loonen A.J.M. et al., manuscript submitted]. Subsequently, this real-time PCR was used in combination with the optimal DNA isolation method to investigate the level of time reduction to identify staphylococci from blood culture material. Results were compared with conventional blood culture techniques used in diagnostic laboratories.

Materials and Methods

Culture methods: Negatively cultured blood culture bottles, derived from routine diagnostics of both BACTEC (Becton Dickinson, the Netherlands) and Bact/ALERT (bioMérieux, the Netherlands) systems, were used in this study for spiking experiments (bottles have been cultured for 1 week and remained negative).

Additionally, healthy volunteers donated 10 ml blood as input for the Bact/ALERT bottles. Bottles were incubated and handled according to manufactures protocol.

DNA isolation methods: Methicillin-resistant S.aureus ATCC 33592 was cultured overnight on bloodagar. Fresh plates were used to make a 2 McF solution which was subsequently serially diluted (1:10) in negative blood culture material from both systems (BACTEC and Bact/ALERT). One hundred µl was plated on bloodagar plates to check CFU per sample. Three different isolation protocols on the NucliSens EasyMAG (bioMérieux) were tested (generic, specific A and specific B protocol: main difference between protocols is the number of washing steps, with least steps in the generic protocol and most washing steps in the specific B protocol), 1 isolation kit (LC Microbiology Kit M Grade ) on the MagNA Pure LC (Roche Diagnostics), the MolYsis Plus kit (Molzym GmBh, Bremen, Germany) was used according to Molzym instructions and in combination with the EasyMAG. When MolYsis was used in combination with the EasyMAG, manufacturer's instructions (Molzym) were followed until the Buglysis step and subsequently bacterial DNA was extracted according to the specific B protocol on the EasyMAG. Input volume in each isolation was 200µl, only for the MagNA Pure LC 100µl was used, and samples were always eluted in 100µl elution buffer provided with each isolation method. Each extraction was performed at least 2-3 times in independent experiments. Phocine herpes virus type 1 (PhHV-1) was added to each sample eluate as an amplification and inhibition control. Samples were analyzed with the LightCycler assay described below.

Results

Evaluation of different bacterial DNA extraction methods

In table 1 the results are presented of the comparison of 6 different bacterial DNA extraction methods on S.aureus spiked blood culture material. The MagNA Pure LC (Roche Diagnostics) resulted in a detection limit of 10 4 CFU/ml for both culture systems. On the EasyMAG 3 protocols were evaluated to isolate bacterial DNA from spiked clinical material, generic, specific A and specific B. We tested all 3 protocols and usage of the specific B protocol resulted in the best detection limit of 10 2 CFU/ml of S.aureus in Bact/ALERT material. Although MolYsis Plus did not result in a low detection limit, we tested this manual kit in combination with the specific B protocol on the EasyMAG. By combining both methods all human DNA was removed and bacteria were lysed by the MolYsis Plus kit, and subsequently the EasyMAG was used for optimal washing and elution of bacterial DNA. It was possible to reduce hands-on time and avoid possible contamination due to skipping of manual handlings. This combination of methods resulted in the most sensitive detection limit, i.e.10 CFU/ml, in Bact/ALERT material. Time to result for this combined method followed by real-time PCR is approximately 4-5 hours, but strongly depending on the number of samples. No inhibition was observed in the real-time PCR, all samples and controls showed similar Cp values in one run (Cp±0.5). All experiments were performed at least 2-3 times.

Reduced blood culture times

To investigate the effect of reduced blood culture incubation for pathogen detection by PCR a combination between the sensitive tuf LightCycler assay and the sensitive isolation method, the MolYsis Plus Kit in combination with the specific B protocol on the EasyMAG, was analyzed on S.aureus (ATCC 33592) spiked Bact/ALERT blood culture bottles containing fresh blood of healthy volunteers. First it was investigated how many CFU S.aureus per ml needed to be present to be able to detect a signal with the LightCycler assay, therefore 1000, 100 and 10 CFU were spiked in different bottles, both SA (Standard Aerobic) and SN (Standard Anaerobic) Bact/ALERT bottles. BACTEC bottles were not investigated due to poorer results obtained with all isolation methods analysed. The results of this experiment are depicted in figure 2 and indicate that 1000 CFU of S.aureus (100 CFU/ml blood) could be detected by Clinical strains of S.aureus and S.epidermidis were also spiked in Bact/ALERT blood culture bottles (aerobe (SA) and anaerobe (SN)) containing blood of healthy volunteers. The results of these experiments were similar as the results presented for the S.aureus ATCC 33592 strain. With conventional detection methods, results of the bottles containing 10 CFU S.aureus were available after at least 28 hours.

Discussion

In this study, 6 different bacterial DNA extraction protocols were evaluated, by using a sensitive and specific tuf gene based real-time PCR detection assay, which enabled the differentiation between S.aureus and CNS. From this study it can be concluded that the MolYsis Plus kit (Molzym) in combination with the specific B protocol on the EasyMAG (bioMérieux) resulted in the most optimal isolation of bacterial DNA from Bact/ALERT blood culture material. A detection limit of 10 CFU/ml was obtained in the tuf LightCycler assay for Bact/ALERT material. By using the tuf LightCycler assay 1 CFU/ml S.aureus or CNS could be detected from Bact/ALERT blood culture material when an incubation step of a maximum of 5 hours was applied. By using this reduced incubation time it was possible to identify S.aureus versus CNS within one working day.

Six different bacterial DNA extraction methods were compared on two commonly used blood culture systems (BACTEC and Bact/ALERT). The specific B protocol on the EasyMAG contains most washing steps compared to the generic and specific A protocol, possibly explaining the better results obtained with this isolation protocol.

The bacterial DNA isolation performed with the MolYsis Plus kit resulted in a low detection limit, for both culture systems. Gebert et al., published a study in 2008 in which they show that the MolYsis Plus kit performs well in their hands [START_REF] Gebert | Rapid detection of pathogens in blood culture bottles by real-time pcr in conjunction with the pre-analytic tool molysis[END_REF]. They used 500µl input material, and were able to conclude that PCR analysis in combination with MolYsis Plus DNA isolation resulted in accelerated detection of pathogens in blood culture samples. Inhibiting factors were not found to be present in the samples in the study of Gebert et al [START_REF] Gebert | Rapid detection of pathogens in blood culture bottles by real-time pcr in conjunction with the pre-analytic tool molysis[END_REF]. In our study, no PCR inhibition was observed (all samples and controls showed similar Cp±0,5 values in one run). The detection limit in the LightCycler assay, after MolYsis Plus isolation, was low i.e., 10 4 CFU/ml. The MolYsis Plus kit eliminates human background DNA from the samples and enrichment of bacterial DNA takes place. Complete isolation with the MolYsis Plus kit takes 3 hrs, of which 2 hours hands-on time. By using the EasyMAG specific B protocol after completion of the MolYsis Plus Buglysis step, the total isolation time is not significantly reduced. However, hands-on time and the chance for contamination are drastically reduced, due to the fact that column handling is skipped. A comparison between both culture systems resulted in a difference in bacterial DNA isolation efficiency. Usage of Bact/ALERT blood culture material leaded in most isolation methods to better results compared to BACTEC material.

We experienced difficulties in the bacterial DNA isolations due to the beads present in BACTEC bottles, but we can not conclude that the beads leaded to the weaker performance of BACTEC material compared to Bact/ALERT material. However, due to this finding all experiments were continued with Bact/ALERT material.

By applying a 5 hour incubation step for blood culture bottles containing approximately 10 CFU of S.aureus or CNS it was possible to detect these bacteria by the tuf LightCycler assay. These results could be produced within 1 working day when starting early in the morning. Compared to conventional culture techniques for staphylococci, results were available at least 19hrs faster. Gebert et al. [START_REF] Gebert | Rapid detection of pathogens in blood culture bottles by real-time pcr in conjunction with the pre-analytic tool molysis[END_REF], also showed in their paper that by combining a sensitive real-time PCR and a sensitive isolation method, culture times can be reduced. They showed a 5.0-8.7 hours time gain in spiking experiments and an average time gain of 10.7 hours was obtained when the method was applied on blood culture bottles of septic patients. Maximum time gain can be reached when starting either immediately in the morning or when the microbiology laboratory has 24 hours service. Laboratories with a 24 hours setup will always benefit from this method.

The experiments described in this paper all focus on accelerated detection of staphylococci, especially on detection of S.aureus. Staphylococci were chosen as a model system because Gram positive bacteria are frequently detected in blood culture bottles, making it a relevant group of micro-organisms for such a study [START_REF] Alberti | Epidemiology of sepsis and infection in icu patients from an international multicentre cohort study[END_REF][START_REF] Annane | Septic shock[END_REF][START_REF] Martin | The epidemiology of sepsis in the united states from 1979 through 2000[END_REF]. Infections are, however, caused by a variety of pathogens. Staphylococci are fast growing bacteria and in this study a proof-of-principle is shown. Accelerated detection, of other pathogens causing blood stream infections (i.e. E.coli), directly from blood culture material in combination with reduced culture time and with this sensitive isolation method should be investigated in future experiments. It is of clinical importance in the future to focus especially on fastidious pathogens or fungi, since those pathogens are difficult to culture and for these pathogens time to result can than hopefully be drastically reduced. A similar approach as presented in this study can be applied to investigate reduced incubation times for other pathogens.

It is known from literature that staphylococci are the most frequently causative agent of medical device related and surgical-site infections [START_REF] Huebner | Coagulase-negative staphylococci: Role as pathogens[END_REF],. Therefore it is interesting to apply the tuf LightCycler duplex assay in these patient groups using whole blood samples. Preliminary results showed that the MolYsis Complete kit (Molzym) has already proven to be successful in our hands (data not shown) as well as in the hands of others [START_REF] Hansen | Evaluation of new preanalysis sample treatment tools and DNA isolation protocols to improve bacterial pathogen detection in whole blood[END_REF].

In conclusion, by combining the MolYsis Plus kit and the specific B protocol on the EasyMAG 10 CFU/ml could be detected from S.aureus spiked Bact/ALERT blood culture material. By applying this isolation method followed by the sensitive tuf LightCycler duplex assay, using 5 hours incubated spiked blood culture material, 1 CFU S.aureus or CNS per 1 ml blood could be detected. Future studies are needed to address the clinical application of this approach. Table 1.

Figures and Tables

  LightCycler assay: A duplex real-time PCR, detecting the tuf gene of both staphylococci and S.aureus specifically, is used as described in detail elsewhere [Loonen A.J.M. et al., submitted]. In brief, the positions of the primers and probes were derived from the S. aureus tuf gene sequence (GenBank accession no. AF298796). The sequences of the primers and probes were as follows; Staphylococcus spp. forward primer ccaatgccacaaactcgtga (position 32-51), Staphylococcus spp. reverse primer cacgaccagtgattgagaatacg (position 371-349), S.aureus forward primer tcctggttcaattacaccacatactg (position 583-608), S.aureus reverse primer ggaaatagaattgtggacgatagtttga (position 698-671), Staphylococcus spp. probe VIC-ccattcatgatgccagttg-BHQ1 (position 326-344) , S.aureus probe FAMtgataatacgtatacttctgc-BHQ1 (position 637-617). Oligonucleotide primers were obtained from Eurogentec (Liège, Belgium), Staphylococcus XS probe and the S.aureus XS probe were obtained from Biolegio (Nijmegen, the Netherlands). PhHV-1 primers and VIC labeled probe [24] were obtained from Applied Biosystems (Nieuwerkerk aan den IJssel, the Netherlands). PCR mixtures consisted of 5x LightCycler TaqMan Master mixture (Roche Diagnostics), 0.9µM concentration of each primer, 0.2µM concentration of each probe, and aquadest in a volume of 15µl to which 5µl DNA sample was added, leading to a final reaction volume of 20µl in each capillary. Primers and probe targeting PhHV had a concentration of 0.1µM each and were used in a separate single real-time PCR. All samples were amplified on the LightCycler v2.0 (Roche Diagnostics) according to the following cycling parameters: an initial incubation step for 10 min at 95ºC for enzyme activation and DNA denaturation, subsequently 45 cycli of 15 sec 95 ºC followed by 1 min 60ºC. Results were analyzed with LightCycler Software version 4.05 (Roche Diagnostics).

  PCR immediately after spiking indicating that no incubation step was required. The bottles containing 100 CFU of S.aureus (10 CFU/ml blood) resulted in positive realtime PCR signals after 1 hour incubation in the Bact/ALERT 3D. The 10 CFU bottles (1 CFU/ml blood) needed a maximum of 5 hours incubation in the Bact/ALERT3D to be able to detect the S.aureus bacteria present using a subsequent PCR. The experiment with 10 CFU S.aureus per bottle was repeated 8 times with both aerobe and anaerobe bottles and showed reproducible results. Standard deviations (SD) of the Cp values were calculated for the 10 CFU/bottle experiments (aureus probe). Three hour time point (2/8 positive) showed SD of 3.44, 4hr timepoint (5/8 positive) indicated SD of 1.09, and the 5 hr time point (8/8 positive) resulted in SD of 1.09.

  Evaluation of 6 different DNA isolation protocols on blood culture material spiked with 10-fold dilution series of S.aureus * BACTEC; + Bact/ALERT; -not detected +

Acknowledgements

We thank Wendy Hansen, Jitske Stalpers, Marielle Bierma, Christel van Herk, Paul van Kaathoven, Gitta Stienen and Jan Michielse for technical support.

Conflict of interest

The authors declare that they have no conflict of interest.


Emilie Morvant
email: emilie.morvant@lif.univ-mrs.fr

Amaury Habrard
email: amaury.habrard@lif.univ-mrs.fr

Stéphane Ayache
email: stephane.ayache@lif.univ-mrs.fr

Flexible Domain Adaptation for Multimedia Indexing

Different Distribution P T

For example:

• We have labeled images from a Web image corpus, i.e. ∼P S • Is there a Person in unlabeled images from a Video corpus, i.e. ∼D T ?

Person no Person

?

Is there a Person ? P S = P T ⇒ The Learning distribution is different from the Testing distribution ⇒ How can we learn, from the source domain, a low-error classifier on the target domain ?

Domain Adaptation

Theorem 1 ( [START_REF] Ben-David | A theory of learning from different domains[END_REF]). Let H an hypothesis space. If D S and D T are respectively the marginal distributions of source and target instances, then for all δ ∈ ]0, 1], with probability at least 1 -δ, for every h ∈ H:

err T (h) ≤ err S (h) + 1 2 d H∆H (D S , D T ) + ν,
+ +++ + + + + + + + + - - - -- - - - --- + + + + + + + + + + + + - - - -- - - - - --

Far domains Close domains

Idea

Build a new projection space to move closer the domains.

Learning with Good Similarity Functions φ R (.) = K(., x 1 ), . . . , K(., x d u )

Definition 1 ([1]). K : X × X → [-1; 1] is an ( ,γ
(Notation: H SF the hypothesis space of such classifiers)

Domain Adaptation of Linear Classifiers based on Good Similarity Functions

Building a new projection φ R new to move closer D S and D T with

C ST a pair set (x s , x t ) ∈ U S ∼ D S × U T ∼ D T such that the deviation of losses of x s and x t is small   1-y d u j=1 α j K(x s , x j )   + -   1-y d u j=1 α j K(x t , x j )   + ≤ ( t φ R (x s ) -t φ R (x t )) diag(α) 1 t φ R new (x s ) -t φ R new (x t ) 1 =⇒ φ R new (.) = α 1 K(., x 1 ) K new (., x 1 ) , . . . , α d u K(., x d u ) K new (., x d u ) Our global optimization problem With {(x i , y i )} d l i=1 i.i.d. from P S , R = {x j } d u
j=1 and C ST a set of pairs (x s , x t ) ∼ D S × D T . At iteration l, we build the φ R l+1 space with the help of α l inferred by

min α l (SF) 1 d l d l i=1   1 -y i d u j=1 α l j K l (x i , x j )   + + λ α l 1 +β (x s ,x t )∈C ST ( t φ R l (x s ) -t φ R l (x t )) diag(α l ) 1 (DASF)
Some little results

• Sparsity Analysis. With B R = min

x j ∈R max (x s ,x t )∈C ST |K(x s , x j ) -K(x t , x j )| , α * 1 ≤ 1 βB R + λ • Generalization bound.
Following the robustness notion of Xu&Mannor [START_REF] Xu | Robustness and generalization[END_REF], the Problem (DASF) is (2M η , N η βB R +λ ) robust on P S , with η > 0, M η is the η-covering number of X and

N η = max x a ,x b ∼D S ρ(x a ,x b )≤η t φ R (x a )-t φ R (x b ) ∞ .
Thus for any h ∈ H SF , for any δ > 0, with probability at least 1-δ,

err T (h) ≤ err S (h) + N η βB R + λ + 4M η ln 2 + 2 ln 1 δ d l + 1 2 d H∆H (D S , D T ) + ν.

Experiments on Multimedia Indexing

Experimental Setup

• Similarity function:

K Gaussian kernel, K * (.,x j )= K(.,x j )-µ x j σ x j ∈[-1,1]
• Reverse Validation (hyper-parameters tuning) 0.18 0.27 0.11 0.12 0.34 0.20 0.21 0.10 0.11 0.10 0.18 0.24 0.12 0.17 0.46 0.34 0.13 0.12 0.13 0.20 Reas. 237 203 233 212 185 178 241 139 239 253 200 247 203 243 226 178 236 128 224 202 TSVM 0.14 0.14 0.11 0.16 0.37 0.14 0.22 0.13 0.12 0.13 0.22 0.17 0.12 0.12 0.44 0.18 0.10 0.12 0.15 0.19 SV 814 704 718 445 631 779 864 390 888 515 704 828 861 861 1111 585 406 474 866 652 DASVM 0.16 0.22 0.11 0.14 0.37 0.20 0.23 0.14 0.11 0.15 0.22 0.23 0.12 0.14 0.55 0.30 0.12 0.13 0.17 0.28

+ + + + + h h r 1

••

  Toy problem "inter-twinning moons" • 1 P S , 8 P T according to 8 rotations • Image Indexing (according to F-measure)• Visual descriptors: Percepts Annotated blocs (15 classes) Low-level features representation -Color moments -Edge histogram Discretisation and concatenation of Nb_blocs x Nb_Classes classifiers prediction scores in [0,1] P S : PascalVOC'07 ratio +/-= 1/3 • P T : +/-= 1/3: PascalVOC'07 Test +/-= 1/3: TrecVid'07 Image Indexing: PascalVOC'07 Vs PascalVOC'07

SV

  922 223 295 421 866 10111418 706 335 536 180 802 668 841 303 356 1434 246 486 407 DASF 0.200.32 0.12 0.170.380.230.260.160.16 0.16 0.250.32 0.16 0.18 0.58 0.350.150.200.180.42 Reas. 50 184 78 94 51 378 229 192 203 372 391 384 287 239 6 181 293 153 167 75

  Conc. bird boat bottle bus car cat chair cycle cow dining dog horsemonitormotorpersonplaneplantsheep sofa train

	SV	867 351 587 476 1096 882 1195 392 681 534 436 761 698 670 951 428 428 261 631 510
	SF

* table

* bike * SVM 0.18 0.29 0.01 0.16 0.28 0.23 0.24 0.10 0.15 0.15 0.24 0.31 0.16 0.17 0.56 0.34 0.12 0.16 0.16 0.36

  0.46 0.50 0.34 0.45 0.54 Reas. 214 224 176 246 226 178 TSVM 0.56 0.48 0.52 0.37 0.46 0.61 SV 498 535 631 741 1024 259 DASVM 0.52 0.46 0.55 0.30 0.54 0.52 0.570.490.55 0.42 0.57 0.66 Reas. 120 130 254 151 19 7

	Conc. boat bus car monitorpersonplane * * * * * *	Conc. Average
			Reas.	211
			TSVM	0.50
			SV	615
			DASVM 0.48
	SV	202 222 627 523 274 450	SV	383
			DASF	0.54

SVM 0.56 0.25 0.43 0.19 0.52 0.32 SV 351 476 1096 698 951 428 SF 0.49 DASF SVM 0.38 SV 667 SF 0.46 Reas.