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Evaluation of Multiplex PCR for Diagnosis of Gastrointestinal Tuberculosis
AGA Abstracts
mucosal surface of the Sigirr -/-mouse. While p19A WT infection caused only minimal pathology on its own, it dramatically worsened the course of DSS colitis, in concert with deep penetration of the damaged colonic mucosa. Notably, intestinal tissue histology shows that the p19A hemolysin deletion mutant was severely attenuated in its ability to promote DSS colitis in Sigirr -/- mice. Conclusion Our findings provide evidence that UC associated phylogroup B2 E. coli strains p19A WT can readily and persistently colonize the intestines of susceptible hosts, and significantly worsen the course of colitis. The intestinal tissue damages in DSS colitis Sigirr -/- mice colonized with p19A hemolysin deletion mutant were severely attenuated, which indicates that p19A E. coli harbouring two alpha-hemolysin gene, might be responsible for barrier dysfunction and intestinal tissue damages in susceptible hosts. This model thus facilitates research into the role played by UC associated E. coli pathobionts in colitis pathogenesis.
2015 to November 2016, adult cases of clinically suspected GITB were recruited. All patients underwent mantoux test, contrast enhanced computed tomography of abdomen, esophagogastroduodenoscopy or colonoscopy as indicated. Multiple biopsies for tissue diagnosis were taken from lesions by a single observer. All specimens were subjected to Ziehl Neelsen staining for detection of acid fast bacilli. Tissue obtained were analysed by H&E by two independent pathologists. Multiplex PCR on tissue specimens using specific primers against IS6110, MPB64 and Protein b was also performed. The performance of the assay was assessed using a composite gold standard for diagnosis of tuberculosis, which comprised a combination of clinical characteristics, microbiology smear showing acid fast bacilli, histopathology showing caseous necrosis with granulomatous inflammation and response to antitubercular therapy. Results : A total of 55 cases of clinically suspected GITB were recruited. A final diagnosis of tuberculosis was made in 32 cases (duodenal n = 4, ileocolic n = 28) and rest 23 acted as control group. The mean age, haemoglobin and erythrocyte sedimentation rate of patients with tuberculosis were 36.41 + 14.6 years, 10.2 + 2.4 g/dl, 37.8 + 15.3 mm/hr respectively. The most common endoscopic findings were ulcerations (75%), nodularity (46.8), distorted ileocecal valve (28%) and strictures (21.8%). The most common radiological findings were mural wall thickening (65.6%), mesenteric lymphadenopathy (56%) and strictures (40%). The sensitivity, specificity, positive predictive value and negative predictive value of histopathology (H&E) for diagnosis of GITB was 28.12%, 100%, 100% and 48.89% respectively (Table 1). The sensitivity, specificity, positive predictive value and negative predictive value of multiplex PCR for diagnosis of GITB was 86.33 %, 100%, 100% and 86.2% respectively (Table 1). Four patients underwent surgery for subacute intestinal obstruction while on treatment; rest twenty eight patients completed nine months of antitubercular therapy and improved. Conclusion: Multiplex PCR using specific primers against IS6110, MPB64 and Protein b has a higher sensitivity compared to conventional techniques for diagnosis of GITB Sensitivity, Specificity, Positive predictive value and negative predictive value of diagnostic tests for GITB
Mo1922 GAMMA-GLUTAMYLTRANSPEPTIDASE EXPRESSION BY HELICOBACTER SAGUINI, A NOVEL ENTEROHEPATIC HELICOBACTER SPECIES ISOLATED FROM COTTON TOP TAMARINS WITH CHRONIC COLITIS Anthony Mannion, Zeli Shen, Yan Feng, Stephen C. Artim, Ravindra Kodihalli, Zhongming Ge, James G. Fox Helicobacter saguini is a novel enterohepatic Helicobacter species (EHS) commonly isolated from the colon and feces of captive cotton top tamarins with chronic colitis and colon cancer. Mono-associated H. saguini infection in germfree IL-10-/- mice causes inflammatory bowel disease (IBD) and pro-carcinogenic changes to the large intestine. H. saguini infection can also be naturally transmitted over sequential generations of germfree IL-10-/- mice, resulting in IBD. While H. saguini and other EHS are associated with gastrointestinal diseases, the mechanism by which these species cause infection and inflammation are poorly understood. Genomic and biochemical characterization suggests that H. saguini expresses gammaglutamyltranspeptidase (GGT), a constitutively expressed periplasmic enzyme that metabolizes extracellular glutamine and glutathione. GGT activity by other pathogenic Helicobacter spp. and Campylobacter spp., including H. pylori, exhibits virulence properties including inhibition of cellular proliferation, immunomodulation, host colonization persistence, and promotion of inflammatory-mediated pathology. In this study, we used molecular biology, biochemical, and cell culture methods to demonstrate that H. saguini expresses an enzymatically active GGT with virulence properties. Multi-sequence alignments indicated that the ggt gene from H. saguini is most homologous to the virulent ggt gene expressed by H. bilis. H. saguini and its recombinant GGT protein exhibited enzymatic GGT activity that was ablated by pre-treatment with the GGT-selective inhibitor acivicin. A viable isogenic GGT knockout mutant strain of H. saguini was created that lacked GGT activity. Using in vitro cell culture methods, sonicates from wild-type H. saguini, but not the isogenic knockout mutant, inhibited HT-29 colon epithelial cells and Jurkat T lymphocyte proliferation without evidence of cell death. Additionally, recombinant GGT protein treatment exhibited antiproliferative effects against HT-29 cells and Jurkat T lymphocytes as well as induced proinflammatory gene expression in HT-29 cells. This data suggests H. saguini utilizes GGT to elicit chronic gastrointestinal inflammation in vitro and in vivo.
Mo1925 THE EFFECT OF METHANE ON CALCIUM SIGNALING OF ILEAL SMOOTH MUSCLE OF GUINEA PIG Yoo Mi Park, Zahid Hussain, Young Ju Lee, Hyojin Park Background/Aims: Methane (CH4) has been associated with constipation-predominant irritable bowel syndrome, slowing intestinal transit time by augmenting contractile activity. However, the precise mechanism remains unclear. Therefore, the aim of this study is to investigate mechanisms underlying the aforementioned actions of CH4. Methods: Ileal muscle strips of guinea pig were placed in the tissue bath with force/tension transducers (BIOPAC TSD 105; BIOPAC system, Inc., Santa Barbara, CA, USA). Amplitudes of contraction measured in response to electrical field stimulation (EFS; 1, 2, 8, 16 Hz) following CH4 infusion in the presence of tetradotoxin (TTX), atropine, guanethidine, or GR 113808. Then, based on the results of the tissue bath study, we performed experiments to identify alterations in calcium signaling of ileal smooth muscle. We used a confocal inverted microscope (Nikon Eclipse T 1; Nikon Instruments Inc., Melville, NY) with Oregon Green 488 BAPTA-1 AM in order to visualize changes in calcium fluorescence in response to EFS following CH4 infusion in the presence of TTX, atropine, or a high K+ solution. A high K+ solution excites both nerves and muscles. Results: CH4 significantly increased amplitudes of contraction (P < 0.05, N=8), while treatment with TTX abolished such contraction. CH4 significantly increased calcium fluorescence, though this increase was attenuated following atropine infusion at lower frequencies (1 and 2 Hz) when EFS was applied (P < 0.05, N=6) (Fig 1). Although calcium fluorescence was increased by the high K+ solution under pre-treatment with TTX (P < 0.05), the intensity of the fluorescence was unchanged after CH4 infusion (P =0.229, N=6) (Fig 2). Conclusions: Given the decreased calcium fluorescence of ileal muscle following methane infusion in the presence of TTX and atropine, it is suggested CH4 is mediated by neural pathways, rather than directly on the muscle. The change of calcium fluorescence after a high K+ solution further supports the notion that CH4 acts on the enteric neurons. In conclusion, the actions of CH4 on the intestine are affected by the cholinergic pathway of the enteric nervous system, and the results of this study support the classification of CH4 as a gasotransmitter.
Mo1923 RIFAXIMIN DECREASES VIRULENCE OF CROHNS DISEASE ASSOCIATED E. COLI AND EPITHELIAL INFLAMMATORY RESPONSES Belgin Dogan, Jing Fu, Shiying Zhang, Ellen Scherl, Kenneth W. Simpson Adherent-invasive Escherichia coli (AIEC) is implicated in the pathogenesis of Crohn's disease (CD). Rifaximin, a non-absorbable rifampicin derivative, improves symptoms in mild-tomoderate CD. It is unclear if this effect is due to the antimicrobial or anti-inflammatory activities of rifaximin. The impact of resistance to rifaximin deserves to be clarified. The aims of this study were to examine the effects of rifaximin on the growth and virulence of CD-associated AIEC that are sensitive and resistant to rifaximin and intestinal epithelial inflammatory responses. Seven well-characterized CD-associated E. coli strains (6 AIEC, 1 non-AIEC; 4 rifaximin resistant MIC of >1024 µg/ml, 3 sensitive) were evaluated. The effects of rifaximin on growth, virulence gene expression, motility, epithelial cell adhesion and invasion, macrophage survival of CD-associated E. coli and intestinal epithelial inflammatory responses were determined. In vitro rifaximin exerted a dose-dependent effect on growth of sensitive strains but did not affect the growth of resistant strains. Rifaximin reduced virulence gene expression, motility, adhesion and invasion of CD-associated E. coli in a manner that was independent of its antimicrobial effect. Furthermore, rifaximin reduced IL-8 secretion from PXR-expressing T84 colonic epithelial cells. The effect of rifaximin on adhesion was largely attributable to its action on bacteria, whereas decreases in invasion and cytokine secretion were due to effects on the epithelium. Rifaximin has a multifaceted impact on AIEC virulence in vitro that is largely independent of its antimicrobial effect. Further study is required to determine the relationship of these effects to clinical responses in patients with CD.
Mo1924 EVALUATION OF MULTIPLEX PCR FOR DIAGNOSIS OF GASTROINTESTINAL TUBERCULOSIS Sarthak Malik, kusum sharma, Rakesh Kochhar, Kim Vaiphei, parul sarwal, Narendra Dhaka, Saroj Sinha Introduction: Prompt and accurate diagnosis of gastrointestinal tuberculosis (GITB) is challenging due to pauci-bacillary nature of disease. Current conventional techniques like smear, histopathology and culture lack sensitivity and are time consuming and have limited sensitivity. New rapid diagnostic methods like multiplex PCR seem to have a promising role. Aims and objectives: Aim of current study was to evaluate the role of multiplex PCR using specific primers against IS6110, MPB64 and Protein b for the diagnosis of GITB and compare it with histopathology. Method and materials : In a prospective study conducted from July
AGA Abstracts
S-822
mucosal surface of the Sigirr -/-mouse. While p19A WT infection caused only minimal pathology on its own, it dramatically worsened the course of DSS colitis, in concert with deep penetration of the damaged colonic mucosa. Notably, intestinal tissue histology shows that the p19A hemolysin deletion mutant was severely attenuated in its ability to promote DSS colitis in Sigirr -/- mice. Conclusion Our findings provide evidence that UC associated phylogroup B2 E. coli strains p19A WT can readily and persistently colonize the intestines of susceptible hosts, and significantly worsen the course of colitis. The intestinal tissue damages in DSS colitis Sigirr -/- mice colonized with p19A hemolysin deletion mutant were severely attenuated, which indicates that p19A E. coli harbouring two alpha-hemolysin gene, might be responsible for barrier dysfunction and intestinal tissue damages in susceptible hosts. This model thus facilitates research into the role played by UC associated E. coli pathobionts in colitis pathogenesis.
2015 to November 2016, adult cases of clinically suspected GITB were recruited. All patients underwent mantoux test, contrast enhanced computed tomography of abdomen, esophagogastroduodenoscopy or colonoscopy as indicated. Multiple biopsies for tissue diagnosis were taken from lesions by a single observer. All specimens were subjected to Ziehl Neelsen staining for detection of acid fast bacilli. Tissue obtained were analysed by H&E by two independent pathologists. Multiplex PCR on tissue specimens using specific primers against IS6110, MPB64 and Protein b was also performed. The performance of the assay was assessed using a composite gold standard for diagnosis of tuberculosis, which comprised a combination of clinical characteristics, microbiology smear showing acid fast bacilli, histopathology showing caseous necrosis with granulomatous inflammation and response to antitubercular therapy. Results : A total of 55 cases of clinically suspected GITB were recruited. A final diagnosis of tuberculosis was made in 32 cases (duodenal n = 4, ileocolic n = 28) and rest 23 acted as control group. The mean age, haemoglobin and erythrocyte sedimentation rate of patients with tuberculosis were 36.41 + 14.6 years, 10.2 + 2.4 g/dl, 37.8 + 15.3 mm/hr respectively. The most common endoscopic findings were ulcerations (75%), nodularity (46.8), distorted ileocecal valve (28%) and strictures (21.8%). The most common radiological findings were mural wall thickening (65.6%), mesenteric lymphadenopathy (56%) and strictures (40%). The sensitivity, specificity, positive predictive value and negative predictive value of histopathology (H&E) for diagnosis of GITB was 28.12%, 100%, 100% and 48.89% respectively (Table 1). The sensitivity, specificity, positive predictive value and negative predictive value of multiplex PCR for diagnosis of GITB was 86.33 %, 100%, 100% and 86.2% respectively (Table 1). Four patients underwent surgery for subacute intestinal obstruction while on treatment; rest twenty eight patients completed nine months of antitubercular therapy and improved. Conclusion: Multiplex PCR using specific primers against IS6110, MPB64 and Protein b has a higher sensitivity compared to conventional techniques for diagnosis of GITB Sensitivity, Specificity, Positive predictive value and negative predictive value of diagnostic tests for GITB
Mo1922 GAMMA-GLUTAMYLTRANSPEPTIDASE EXPRESSION BY HELICOBACTER SAGUINI, A NOVEL ENTEROHEPATIC HELICOBACTER SPECIES ISOLATED FROM COTTON TOP TAMARINS WITH CHRONIC COLITIS Anthony Mannion, Zeli Shen, Yan Feng, Stephen C. Artim, Ravindra Kodihalli, Zhongming Ge, James G. Fox Helicobacter saguini is a novel enterohepatic Helicobacter species (EHS) commonly isolated from the colon and feces of captive cotton top tamarins with chronic colitis and colon cancer. Mono-associated H. saguini infection in germfree IL-10-/- mice causes inflammatory bowel disease (IBD) and pro-carcinogenic changes to the large intestine. H. saguini infection can also be naturally transmitted over sequential generations of germfree IL-10-/- mice, resulting in IBD. While H. saguini and other EHS are associated with gastrointestinal diseases, the mechanism by which these species cause infection and inflammation are poorly understood. Genomic and biochemical characterization suggests that H. saguini expresses gammaglutamyltranspeptidase (GGT), a constitutively expressed periplasmic enzyme that metabolizes extracellular glutamine and glutathione. GGT activity by other pathogenic Helicobacter spp. and Campylobacter spp., including H. pylori, exhibits virulence properties including inhibition of cellular proliferation, immunomodulation, host colonization persistence, and promotion of inflammatory-mediated pathology. In this study, we used molecular biology, biochemical, and cell culture methods to demonstrate that H. saguini expresses an enzymatically active GGT with virulence properties. Multi-sequence alignments indicated that the ggt gene from H. saguini is most homologous to the virulent ggt gene expressed by H. bilis. H. saguini and its recombinant GGT protein exhibited enzymatic GGT activity that was ablated by pre-treatment with the GGT-selective inhibitor acivicin. A viable isogenic GGT knockout mutant strain of H. saguini was created that lacked GGT activity. Using in vitro cell culture methods, sonicates from wild-type H. saguini, but not the isogenic knockout mutant, inhibited HT-29 colon epithelial cells and Jurkat T lymphocyte proliferation without evidence of cell death. Additionally, recombinant GGT protein treatment exhibited antiproliferative effects against HT-29 cells and Jurkat T lymphocytes as well as induced proinflammatory gene expression in HT-29 cells. This data suggests H. saguini utilizes GGT to elicit chronic gastrointestinal inflammation in vitro and in vivo.
Mo1925 THE EFFECT OF METHANE ON CALCIUM SIGNALING OF ILEAL SMOOTH MUSCLE OF GUINEA PIG Yoo Mi Park, Zahid Hussain, Young Ju Lee, Hyojin Park Background/Aims: Methane (CH4) has been associated with constipation-predominant irritable bowel syndrome, slowing intestinal transit time by augmenting contractile activity. However, the precise mechanism remains unclear. Therefore, the aim of this study is to investigate mechanisms underlying the aforementioned actions of CH4. Methods: Ileal muscle strips of guinea pig were placed in the tissue bath with force/tension transducers (BIOPAC TSD 105; BIOPAC system, Inc., Santa Barbara, CA, USA). Amplitudes of contraction measured in response to electrical field stimulation (EFS; 1, 2, 8, 16 Hz) following CH4 infusion in the presence of tetradotoxin (TTX), atropine, guanethidine, or GR 113808. Then, based on the results of the tissue bath study, we performed experiments to identify alterations in calcium signaling of ileal smooth muscle. We used a confocal inverted microscope (Nikon Eclipse T 1; Nikon Instruments Inc., Melville, NY) with Oregon Green 488 BAPTA-1 AM in order to visualize changes in calcium fluorescence in response to EFS following CH4 infusion in the presence of TTX, atropine, or a high K+ solution. A high K+ solution excites both nerves and muscles. Results: CH4 significantly increased amplitudes of contraction (P < 0.05, N=8), while treatment with TTX abolished such contraction. CH4 significantly increased calcium fluorescence, though this increase was attenuated following atropine infusion at lower frequencies (1 and 2 Hz) when EFS was applied (P < 0.05, N=6) (Fig 1). Although calcium fluorescence was increased by the high K+ solution under pre-treatment with TTX (P < 0.05), the intensity of the fluorescence was unchanged after CH4 infusion (P =0.229, N=6) (Fig 2). Conclusions: Given the decreased calcium fluorescence of ileal muscle following methane infusion in the presence of TTX and atropine, it is suggested CH4 is mediated by neural pathways, rather than directly on the muscle. The change of calcium fluorescence after a high K+ solution further supports the notion that CH4 acts on the enteric neurons. In conclusion, the actions of CH4 on the intestine are affected by the cholinergic pathway of the enteric nervous system, and the results of this study support the classification of CH4 as a gasotransmitter.
Mo1923 RIFAXIMIN DECREASES VIRULENCE OF CROHNS DISEASE ASSOCIATED E. COLI AND EPITHELIAL INFLAMMATORY RESPONSES Belgin Dogan, Jing Fu, Shiying Zhang, Ellen Scherl, Kenneth W. Simpson Adherent-invasive Escherichia coli (AIEC) is implicated in the pathogenesis of Crohn's disease (CD). Rifaximin, a non-absorbable rifampicin derivative, improves symptoms in mild-tomoderate CD. It is unclear if this effect is due to the antimicrobial or anti-inflammatory activities of rifaximin. The impact of resistance to rifaximin deserves to be clarified. The aims of this study were to examine the effects of rifaximin on the growth and virulence of CD-associated AIEC that are sensitive and resistant to rifaximin and intestinal epithelial inflammatory responses. Seven well-characterized CD-associated E. coli strains (6 AIEC, 1 non-AIEC; 4 rifaximin resistant MIC of >1024 µg/ml, 3 sensitive) were evaluated. The effects of rifaximin on growth, virulence gene expression, motility, epithelial cell adhesion and invasion, macrophage survival of CD-associated E. coli and intestinal epithelial inflammatory responses were determined. In vitro rifaximin exerted a dose-dependent effect on growth of sensitive strains but did not affect the growth of resistant strains. Rifaximin reduced virulence gene expression, motility, adhesion and invasion of CD-associated E. coli in a manner that was independent of its antimicrobial effect. Furthermore, rifaximin reduced IL-8 secretion from PXR-expressing T84 colonic epithelial cells. The effect of rifaximin on adhesion was largely attributable to its action on bacteria, whereas decreases in invasion and cytokine secretion were due to effects on the epithelium. Rifaximin has a multifaceted impact on AIEC virulence in vitro that is largely independent of its antimicrobial effect. Further study is required to determine the relationship of these effects to clinical responses in patients with CD.
Mo1924 EVALUATION OF MULTIPLEX PCR FOR DIAGNOSIS OF GASTROINTESTINAL TUBERCULOSIS Sarthak Malik, kusum sharma, Rakesh Kochhar, Kim Vaiphei, parul sarwal, Narendra Dhaka, Saroj Sinha Introduction: Prompt and accurate diagnosis of gastrointestinal tuberculosis (GITB) is challenging due to pauci-bacillary nature of disease. Current conventional techniques like smear, histopathology and culture lack sensitivity and are time consuming and have limited sensitivity. New rapid diagnostic methods like multiplex PCR seem to have a promising role. Aims and objectives: Aim of current study was to evaluate the role of multiplex PCR using specific primers against IS6110, MPB64 and Protein b for the diagnosis of GITB and compare it with histopathology. Method and materials : In a prospective study conducted from July
AGA Abstracts
S-822