- Email: [email protected]
655 The Epidemiology of Clostridium difficile Infection in Children: A Population-Based Study
complete medical diagnostic coding and opportunity to describe trends in CDI risk of postinfectious FGD. Methods: Data from the Defense Medical Encounter Database were extracted from 2000 to 2010 to analyze trends. Diagnosis of CDI was based on a first medical encounter with an ICD-9 diagnostic code of 008.45. Incidence rates were estimated for the active duty population overall, and stratified by age, service branch, and gender. Additional data from a retrospective cohort study will be reported describing the risk of FGD following infection. Results: Between 2000 and 2010 there was an average of 1.39 million active duty service members followed annually. During this time period, the incidence of CDI was observed to increase from 5.6 per 100K in 2000 to 49.3 per 100K in 2010, an average yearly increase of 4 cases/100K/year. Highest rates of increase were among the over 40 age strata (increase of 9.4 incident cases per 100K each year). Overall, rates were 2 times higher in females compared to males (41.4 vs 23.6 per 100K), greater among Air Force (30.9 per 100K) and Marines (29.0 per 100K), and similar across race categories (Caucasians: 27.6 per 100K, African-American 24.4, other 20.5). The incidence of FGD among this population will be described and compared to a referent study population. Conclusions: Incidence of CDI diagnosis in the US military is increasing, particularly among the older age population. While difficult to compare to other populations this does provide robust measures of incidence in a large population. Service specific differences were noted and the risks of long-term health sequelae need to be explored.
information may underestimate the incidence and overestimate the percentage of severe or complicated CDI in children.
LPA/LPA5 Receptor Stimulation of NHE3 Involves Two Separate Events: Stimulation of NHE3 Exocytosis via EGFR and ERK and Dynamic Dissociation of NHE3 From the Microvillar (MV) Cytoskeleton/NHERF2 via PLC and a Novel PKC Mark Donowitz, Boyoung Cha, Tian-e Chen, Rafiquel Sarker, Daniel Raben, Olga Kovbasnjuk BACKGROUND: The BB Na/H exchanger NHE3 accounts for the majority of intestinal Na absorption. NHE3 is inhibited early in digestion to spread digestive enzymes over the absorbing surface and is then stimulated to prevent dehydration. NHE3 regulation occurs primarily by changes in trafficking. However, NHE3 has very limited basal MV mobility bringing into question how can a protein that is fixed to the MV actin cytoskeleton be regulated by trafficking. LPA acting by apical LPA5 receptors stimulates NHE3 activity in polarized epithelial cells. HYPOTHESIS: a) The NHE3 association with MV cytoskeleton is dynamically altered as part of LPA stimulation to allow the exocytosed NHE3 to distribute over the MV. b) Signaling that controls NHE3-MV cytoskeleton association is separate from that controlling NHE3 trafficking. METHODS: NHE3-eGFP and 3HA-NHE3 plus LPA5 receptor cDNAs were transiently transfected into polarized renal proximal tubule OK/ NHERF2 cells. NHE3 activity was determined fluorometrically (BCECF). NHE3 mobile fraction (Mf) in the OK BB was determined by FRAP (fluorescence recovery after photobleaching) using NHE3-eGFP and apical NHE3 studied via a LSM510 confocal microscope. RESULTS: a) Apical LPA (3 uM) stimulated NHE3 activity starting within 5 min and continued for at least 60 min and increased surface NHE3. b) LPA stimulation of NHE3 was prevented by inhibiting EGFR (AG1478) and ERK (PD98059) and by omitting NHERF2; but not by BAPTAM, inhibiting PLC (U73122), PKC (general PKC inhibitor Gf109203X), or Rho kinase (Y27632). c) The MV Mf of NHE3-eGFP was ~30% under basal conditions and transiently increased to ~80% starting 20 min after LPA, became maximum by 30 min and returned to baseline by 60 min. d) The LPA increase in NHE3 Mf was prevented by U71322, Gf109203X,the conventional and novel PKC inhibitor Go6983 and partially by BAPTAM. OAG and PMA duplicated the LPA increase in NHE3 Mf in a non-additive manner with LPA. e) NHE3-NHERF2 co-IP occurred under basal conditions, decreased 30 min after LPA exposure, and was reestablished by 60 min. Summary/ Conclusions: 1) LPA stimulates BB NHE3 activity in polarized epithelial cells by increasing surface expression by a NHERF2 dependent mechanism involving BB LPA5 receptors, apical EGFR transactivation and ERK. 2) This LPA stimulation is associated with a dynamic increase in NHE3 MV mobility. 3) The dynamic increase in Mf correlates with a dynamic decrease in NHE3-NHERF2 association, was dependent on PLC and elevated Ca2+ and involved a novel PKC, none of which were involved in rapid LPA stimulation of NHE3 activity. 4) LPA/LPA5 receptor stimulates NHE3 by increasing its exocytosis and uses separate signaling to dynamically dissociate NHE3NHERF2-MV cytoskeleton to spread NHE3 over the microvillus.
654 Use of Rectal Swab to Test for Clostridium difficile Infection by Real Time-PCR Faiz Shakir, David M. Thompson, Richard Marlar, Tauseef Ali Background: Clostridium difficile infection (CDI) is the leading identifiable cause of antibiotic associated diarrhea and one of the most common healthcare-associated infections. Testing for CDI is usually performed by real time polymerase chain reaction (RT-PCR) assay on patient provided stool specimen for detection of genes encoding the cytotoxin C. difficile toxin B (tcdB). However, stool specimens are not always easily obtainable, especially in the ambulatory setting. Aim: To evaluate whether a rectal swab can be used for collection of a stool specimen compared to the usual collection of patient provided stool specimen, for testing of CDI by RT-PCR. Methods: Patients hospitalized at a single Veterans Affairs Hospital were enrolled if they were tested for CDI by the usual method (RT-PCR on patient provided stool specimen). After consent was obtained for the rectal swab method, a Copan double swab was inserted briefly into the rectum, then removed and placed in the collection device. Testing by RT-PCR was performed according to the manufacturer's instructions within 4 hours with either method. Results: A total of 22 hospitalized patients were eligible for the study. RT-PCR testing by the usual method determined that 10 patients had toxin B while 12 were negative. The average length of time for the usual method, measured from the order placed for RT-PCR testing to the test result, was 10.3 hours (range 0.917-33.8 hours). 100% accuracy (95% CI: 84.6%-100%) was observed with 100% sensitivity (95% CI: 69.2%100%) and 100% specificity (95% CI: 73.5%-100%) between the usual method and the rectal swab method. In addition, there was 100% concordance between three samples that tested positive for Binary toxin and tCdC deletion. Discussion: Rectal swabs can be used for RT-PCR testing of CDI in the clinical setting. Minimal additional cost from the use of rectal swabs was incurred but this will be outweighed by the rapidity of obtaining a test result, compared to the traditional method of stool specimen collection. In turn, the use of rectal swab will allow for rapid clinical diagnosis and reduced length of hospital stay and indirect healthcare costs.
750 Phosphodiesterase Inhibitors Stimulate Intestinal CA Activated Cl Channels (CaCCS) by an Epac1-Mediated Mechanism: an Example of cAMP-CA+2 CrossTalk in the Intestine Ming Tse
655
Background: The recently identified TMEM16 family of CaCCs is believed to be involved in intestinal Cl- secretion in addition to CFTR. While it has been considered that CFTR and CaCCs are activated via separate pathways, we recently identified an intestinal cAMP-Ca+2 cross-talk signaling pathway that is initiated by elevation of [cAMP]i and elevates Ca+2 via the exchange protein activated by cAMP (Epac). This showed that both CFTR and CaCCs are targets of elevated [cAMP]i. Since local cAMP concentrations are modulated by phosphodiesterases (PDEs) In Vivo, cAMP signaling is compartmentalized in cells. Therefore, we hypothesized that CFTR and CaCCs could be differentially regulated by different PDEs and PDE inhibitors (PDEis) could be used to separate CFTR and non-CFTR activities in response to local cAMP signaling. Methods: Ussing Chambers/voltage clamps were used to determine the effects of apically added PDEis on the Isc response of T84 cells. Results: a) The PDEis, cilostazol (PDE3i, 50μM) and dipyridamole (20μM) increased the Isc in T84 cells. 8MMIBMX (PDE1 inhibitor), EHNA (PDE2 inhibitor) and rolipram (PDE4 inhibitor) had no effect. b) The cilostazol stimulated Isc was transient while the dipyridamole stimulated Isc was sustained and their effects were additive. c) Cl- channel blockers had different effects on PDEi stimulated Isc: DIDS had no effect on dipyridamole stimulated Isc but inhibited cilostazol stimulated Isc (40±5%↓). Glibenclamide (100μM) inhibited the dipyridamole stimulated Isc (30±6%↓), but had no effect on cilostazol stimulated Isc. However, both Iscs were completely inhibited by a CaCC inhibitor, tannic acid (100μM). d) To link the effects of PDEis to the activation of CaCCs, the effects of H89 (5μM, PKA inhibitor) and BAPTAAM (25μM, Ca chelator) on PDEi stimulated Isc were determined. Both the cilostazol and dipyridamole stimulated Isc were inhibited by BAPTA-AM, but not by H89. e) Epac1 might act as a “switch” of cAMP signaling into Ca+2. It was found that cilostazol and dipyridamole stimulated Isc were significantly reduced in T84 Epac1 knock-down cells (75±5%↓ and 84±4%↓, respectively). f) Dipyridamole stimulated fluid accumulation in a murine jejunal closed loop model and this fluid accumulation was reduced by tannic acid (32±19%↓). Summary: 1) Cilostazol and dipyridamole, but not other PDEis stimulate intestinal Clsecretion. 2) Cilostazol and dipyridamole stimulate different Cl- channels, both of which are likely to be CaCCs that have different pharmacological properties. 3) Epac1 mediates PDEi stimulation of intestinal Cl- secretion. Conclusions: CaCCs, but not CFTR are stimulated by PDEis in intestinal epithelial cells and multiple CaCCs are involved in apical Cl- secretion in murine intestine.
The Epidemiology of Clostridium difficile Infection in Children: A PopulationBased Study Sahil Khanna, Larry Baddour, W. Charles Huskins, Patricia P. Kammer, William S. Harmsen, Alan R. Zinsmeister, Darrell S. Pardi Background: Recent hospital-based studies have shown an increasing incidence of Clostridium difficile infection (CDI) in children. The results of these studies are potentially influenced by referral or hospitalization bias. There is limited information on the population-based incidence of CDI in children and adolescents. Aim: To study the epidemiology and clinical features of CDI in children in a well-defined population-based cohort. Methods: A computerized diagnostic index, which includes diagnoses and laboratory test results from all medical encounters for all residents of Olmsted County, MN, was used to identify subjects ≤ 18 years of age with CDI from 1991-2009. CDI was defined with ≥ 3 loose stools / 24 hours with a positive Clostridium difficile stool assay. Medical records were reviewed to confirm diagnoses , document demographic data, and assess risk factors & outcomes. Community acquired CDI was defined as infection in the outpatient setting (without recent hospitalization), or within 48 hours of hospital admission. Patients with a hospitalization in the previous 4 weeks or documentation of CDI >48 hours after admission were considered to have hospital-acquired infection. Severe CDI was defined as a leukocyte count ≥ 15,000 / ul or creatinine rise of ≥ 50% from baseline. Severe-complicated CDI was defined as ICU admission, surgery or death associated with CDI. Results: There were 92 CDI cases in children and adolescents identified from 1991 - 2009. The median age at symptom onset was 2.3 years (1 month - 17.6 years) with 47% occurring in age < 1 year; 46% cases were female. The majority of cases (75%) were community-acquired. The overall CDI incidence was 13.8 per 100,000 persons and increased from 2.6 (from 1991 - 1997) to 32.6 per 100,000 (from 2004 - 2009) over the study period (p<0.0001). The incidence of community-acquired CDI was 10.3 per 100,000 persons and increased significantly from 2.2 (1991 - 1997) to 23.4 per 100,000 (2004 - 2009), (p<0.0001). Exposure to antibiotics occurred in 70 (76%) cases and gastric acid suppression medications occurred in 18 (20%) cases. Severe CDI, severecomplicated CDI and death occurred in 9%, 3%, and 1% of cases respectively; 20% developed recurrent CDI. Metronidazole was the initial treatment in 82% of cases, with 18% of cases resulting in treatment failure. Oral vancomycin was the initial treatment in 8% of cases with no treatment failures. Conclusions: In this population-based cohort, the incidence of CDI in children increased significantly from 1991-2009, although severe and severe complicated cases were rare. CDI occurred mostly in children < 1 year of age and was more frequently community-acquired. Estimates of the incidence of CDI that rely on hospital dismissal
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information may underestimate the incidence and overestimate the percentage of severe or complicated CDI in children.
LPA/LPA5 Receptor Stimulation of NHE3 Involves Two Separate Events: Stimulation of NHE3 Exocytosis via EGFR and ERK and Dynamic Dissociation of NHE3 From the Microvillar (MV) Cytoskeleton/NHERF2 via PLC and a Novel PKC Mark Donowitz, Boyoung Cha, Tian-e Chen, Rafiquel Sarker, Daniel Raben, Olga Kovbasnjuk BACKGROUND: The BB Na/H exchanger NHE3 accounts for the majority of intestinal Na absorption. NHE3 is inhibited early in digestion to spread digestive enzymes over the absorbing surface and is then stimulated to prevent dehydration. NHE3 regulation occurs primarily by changes in trafficking. However, NHE3 has very limited basal MV mobility bringing into question how can a protein that is fixed to the MV actin cytoskeleton be regulated by trafficking. LPA acting by apical LPA5 receptors stimulates NHE3 activity in polarized epithelial cells. HYPOTHESIS: a) The NHE3 association with MV cytoskeleton is dynamically altered as part of LPA stimulation to allow the exocytosed NHE3 to distribute over the MV. b) Signaling that controls NHE3-MV cytoskeleton association is separate from that controlling NHE3 trafficking. METHODS: NHE3-eGFP and 3HA-NHE3 plus LPA5 receptor cDNAs were transiently transfected into polarized renal proximal tubule OK/ NHERF2 cells. NHE3 activity was determined fluorometrically (BCECF). NHE3 mobile fraction (Mf) in the OK BB was determined by FRAP (fluorescence recovery after photobleaching) using NHE3-eGFP and apical NHE3 studied via a LSM510 confocal microscope. RESULTS: a) Apical LPA (3 uM) stimulated NHE3 activity starting within 5 min and continued for at least 60 min and increased surface NHE3. b) LPA stimulation of NHE3 was prevented by inhibiting EGFR (AG1478) and ERK (PD98059) and by omitting NHERF2; but not by BAPTAM, inhibiting PLC (U73122), PKC (general PKC inhibitor Gf109203X), or Rho kinase (Y27632). c) The MV Mf of NHE3-eGFP was ~30% under basal conditions and transiently increased to ~80% starting 20 min after LPA, became maximum by 30 min and returned to baseline by 60 min. d) The LPA increase in NHE3 Mf was prevented by U71322, Gf109203X,the conventional and novel PKC inhibitor Go6983 and partially by BAPTAM. OAG and PMA duplicated the LPA increase in NHE3 Mf in a non-additive manner with LPA. e) NHE3-NHERF2 co-IP occurred under basal conditions, decreased 30 min after LPA exposure, and was reestablished by 60 min. Summary/ Conclusions: 1) LPA stimulates BB NHE3 activity in polarized epithelial cells by increasing surface expression by a NHERF2 dependent mechanism involving BB LPA5 receptors, apical EGFR transactivation and ERK. 2) This LPA stimulation is associated with a dynamic increase in NHE3 MV mobility. 3) The dynamic increase in Mf correlates with a dynamic decrease in NHE3-NHERF2 association, was dependent on PLC and elevated Ca2+ and involved a novel PKC, none of which were involved in rapid LPA stimulation of NHE3 activity. 4) LPA/LPA5 receptor stimulates NHE3 by increasing its exocytosis and uses separate signaling to dynamically dissociate NHE3NHERF2-MV cytoskeleton to spread NHE3 over the microvillus.
654 Use of Rectal Swab to Test for Clostridium difficile Infection by Real Time-PCR Faiz Shakir, David M. Thompson, Richard Marlar, Tauseef Ali Background: Clostridium difficile infection (CDI) is the leading identifiable cause of antibiotic associated diarrhea and one of the most common healthcare-associated infections. Testing for CDI is usually performed by real time polymerase chain reaction (RT-PCR) assay on patient provided stool specimen for detection of genes encoding the cytotoxin C. difficile toxin B (tcdB). However, stool specimens are not always easily obtainable, especially in the ambulatory setting. Aim: To evaluate whether a rectal swab can be used for collection of a stool specimen compared to the usual collection of patient provided stool specimen, for testing of CDI by RT-PCR. Methods: Patients hospitalized at a single Veterans Affairs Hospital were enrolled if they were tested for CDI by the usual method (RT-PCR on patient provided stool specimen). After consent was obtained for the rectal swab method, a Copan double swab was inserted briefly into the rectum, then removed and placed in the collection device. Testing by RT-PCR was performed according to the manufacturer's instructions within 4 hours with either method. Results: A total of 22 hospitalized patients were eligible for the study. RT-PCR testing by the usual method determined that 10 patients had toxin B while 12 were negative. The average length of time for the usual method, measured from the order placed for RT-PCR testing to the test result, was 10.3 hours (range 0.917-33.8 hours). 100% accuracy (95% CI: 84.6%-100%) was observed with 100% sensitivity (95% CI: 69.2%100%) and 100% specificity (95% CI: 73.5%-100%) between the usual method and the rectal swab method. In addition, there was 100% concordance between three samples that tested positive for Binary toxin and tCdC deletion. Discussion: Rectal swabs can be used for RT-PCR testing of CDI in the clinical setting. Minimal additional cost from the use of rectal swabs was incurred but this will be outweighed by the rapidity of obtaining a test result, compared to the traditional method of stool specimen collection. In turn, the use of rectal swab will allow for rapid clinical diagnosis and reduced length of hospital stay and indirect healthcare costs.
750 Phosphodiesterase Inhibitors Stimulate Intestinal CA Activated Cl Channels (CaCCS) by an Epac1-Mediated Mechanism: an Example of cAMP-CA+2 CrossTalk in the Intestine Ming Tse
655
Background: The recently identified TMEM16 family of CaCCs is believed to be involved in intestinal Cl- secretion in addition to CFTR. While it has been considered that CFTR and CaCCs are activated via separate pathways, we recently identified an intestinal cAMP-Ca+2 cross-talk signaling pathway that is initiated by elevation of [cAMP]i and elevates Ca+2 via the exchange protein activated by cAMP (Epac). This showed that both CFTR and CaCCs are targets of elevated [cAMP]i. Since local cAMP concentrations are modulated by phosphodiesterases (PDEs) In Vivo, cAMP signaling is compartmentalized in cells. Therefore, we hypothesized that CFTR and CaCCs could be differentially regulated by different PDEs and PDE inhibitors (PDEis) could be used to separate CFTR and non-CFTR activities in response to local cAMP signaling. Methods: Ussing Chambers/voltage clamps were used to determine the effects of apically added PDEis on the Isc response of T84 cells. Results: a) The PDEis, cilostazol (PDE3i, 50μM) and dipyridamole (20μM) increased the Isc in T84 cells. 8MMIBMX (PDE1 inhibitor), EHNA (PDE2 inhibitor) and rolipram (PDE4 inhibitor) had no effect. b) The cilostazol stimulated Isc was transient while the dipyridamole stimulated Isc was sustained and their effects were additive. c) Cl- channel blockers had different effects on PDEi stimulated Isc: DIDS had no effect on dipyridamole stimulated Isc but inhibited cilostazol stimulated Isc (40±5%↓). Glibenclamide (100μM) inhibited the dipyridamole stimulated Isc (30±6%↓), but had no effect on cilostazol stimulated Isc. However, both Iscs were completely inhibited by a CaCC inhibitor, tannic acid (100μM). d) To link the effects of PDEis to the activation of CaCCs, the effects of H89 (5μM, PKA inhibitor) and BAPTAAM (25μM, Ca chelator) on PDEi stimulated Isc were determined. Both the cilostazol and dipyridamole stimulated Isc were inhibited by BAPTA-AM, but not by H89. e) Epac1 might act as a “switch” of cAMP signaling into Ca+2. It was found that cilostazol and dipyridamole stimulated Isc were significantly reduced in T84 Epac1 knock-down cells (75±5%↓ and 84±4%↓, respectively). f) Dipyridamole stimulated fluid accumulation in a murine jejunal closed loop model and this fluid accumulation was reduced by tannic acid (32±19%↓). Summary: 1) Cilostazol and dipyridamole, but not other PDEis stimulate intestinal Clsecretion. 2) Cilostazol and dipyridamole stimulate different Cl- channels, both of which are likely to be CaCCs that have different pharmacological properties. 3) Epac1 mediates PDEi stimulation of intestinal Cl- secretion. Conclusions: CaCCs, but not CFTR are stimulated by PDEis in intestinal epithelial cells and multiple CaCCs are involved in apical Cl- secretion in murine intestine.
The Epidemiology of Clostridium difficile Infection in Children: A PopulationBased Study Sahil Khanna, Larry Baddour, W. Charles Huskins, Patricia P. Kammer, William S. Harmsen, Alan R. Zinsmeister, Darrell S. Pardi Background: Recent hospital-based studies have shown an increasing incidence of Clostridium difficile infection (CDI) in children. The results of these studies are potentially influenced by referral or hospitalization bias. There is limited information on the population-based incidence of CDI in children and adolescents. Aim: To study the epidemiology and clinical features of CDI in children in a well-defined population-based cohort. Methods: A computerized diagnostic index, which includes diagnoses and laboratory test results from all medical encounters for all residents of Olmsted County, MN, was used to identify subjects ≤ 18 years of age with CDI from 1991-2009. CDI was defined with ≥ 3 loose stools / 24 hours with a positive Clostridium difficile stool assay. Medical records were reviewed to confirm diagnoses , document demographic data, and assess risk factors & outcomes. Community acquired CDI was defined as infection in the outpatient setting (without recent hospitalization), or within 48 hours of hospital admission. Patients with a hospitalization in the previous 4 weeks or documentation of CDI >48 hours after admission were considered to have hospital-acquired infection. Severe CDI was defined as a leukocyte count ≥ 15,000 / ul or creatinine rise of ≥ 50% from baseline. Severe-complicated CDI was defined as ICU admission, surgery or death associated with CDI. Results: There were 92 CDI cases in children and adolescents identified from 1991 - 2009. The median age at symptom onset was 2.3 years (1 month - 17.6 years) with 47% occurring in age < 1 year; 46% cases were female. The majority of cases (75%) were community-acquired. The overall CDI incidence was 13.8 per 100,000 persons and increased from 2.6 (from 1991 - 1997) to 32.6 per 100,000 (from 2004 - 2009) over the study period (p<0.0001). The incidence of community-acquired CDI was 10.3 per 100,000 persons and increased significantly from 2.2 (1991 - 1997) to 23.4 per 100,000 (2004 - 2009), (p<0.0001). Exposure to antibiotics occurred in 70 (76%) cases and gastric acid suppression medications occurred in 18 (20%) cases. Severe CDI, severecomplicated CDI and death occurred in 9%, 3%, and 1% of cases respectively; 20% developed recurrent CDI. Metronidazole was the initial treatment in 82% of cases, with 18% of cases resulting in treatment failure. Oral vancomycin was the initial treatment in 8% of cases with no treatment failures. Conclusions: In this population-based cohort, the incidence of CDI in children increased significantly from 1991-2009, although severe and severe complicated cases were rare. CDI occurred mostly in children < 1 year of age and was more frequently community-acquired. Estimates of the incidence of CDI that rely on hospital dismissal
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