Gastrointestinal Infections and Laboratory Diagnosis Parameth Thiennimitr, M.D., Ph.D.

Transcription

1 Gastrointestinal Infections and Laboratory Diagnosis Parameth Thiennimitr, M.D., Ph.D. Department of Microbiology Faculty of Medicine, Chiang Mai University March 2, 2016

2 Learning Objectives After this lecture, student should be able to 1. Differentiate types of diarrhea in human (watery vs. inflammatory diarrhea) 2. Explain the differences in pathophysiology of each type of acute diarrhea 3. Give examples of medically important enteropathogens that cause acute diarrhea in human 4. Understand and recall principles of major laboratory investigations in acute diarrhea patient 5. Apply the fundamental microbiology and immunology knowledge of GI infection to proper approach in clinical cases.

3 Pathogenesis of GI infections: Host & Microbial Factors Host factors: human host - Physical barriers: stomach acidity, peristalsis - Gut mucosal immunity (both innate & adaptive) e.g. antimicrobial peptides, siga - Age: Too young, too old - Medications: proton-pump inhibitor, antibiotics enteropathogens Microbial factors: - Acid tolerance (survive in stomach environment) - Toxin production: enterotoxin, cytotoxin, neurotoxin - Host cell attachment - Host cell invasion - Immune evasion: e.g. capsule in S. typhi

4 Pathogenesis of GI infections: Microbial Toxin Productions (1) Enterotoxin - Cause watery (secretory) diarrhea, electrolyte and fluid loss - Cholera toxin of Vibrio cholerae increases camp microbial toxins host cell (enterocyte) - Non-cholera enterotoxin: ETEC, Bacillus cereus, Clostridium difficile toxin A, NSP4 protein of rotavirus (2) Cytotoxin - Cause inflammatory diarrhea, induce host innate immune response: PMNs (polymorphonuclear cell); neutrophil infiltration - WBC in stool, bloody mucus stool - Shigella dysenteriae, EHEC, Clostridium difficile (3) Neurotoxin - Cause nausea and vomiting, Food poisoning - Preformed toxin produced by microbes before ingestion - Bacillus cereus, Staphylococcus aureus, Clostridium botulinum

5 Pathogenesis of GI infections: Mechanism of Watery Diarrhea from Cholera Toxin Watery diarrhea cholera toxin Vibrio cholerae A subunit B subunits Electrolytes & Water loss into gut lumen GM1-ganglioside receptor Na + K + Cl - HCO 3 - H 2 O Adenylate cyclase Na + K + Cl - HCO 3 - H 2 O ATP camp Rice water stool cytoplasm nucleus cell membrane gut epithelial cell

6 Pathogenesis of GI infections: Host Cell Attachment (Attaching and Effacing (A/E) lesion) Enteropathogenic Escherichia coli (EPEC) Attachment by pili Effacing by EPEC effector protein pedestal formation SEM showing attaching and effacing lesion on human epithelial cells infected with EPEC (Knutton, S, EMBO, 1998)

7 Pathogenesis of GI infections: Host Cell Invasion Salmonella microvilli effector protein Salmonella-containing vacuole (endosome) colonic epithelium Shigella microvilli effector protein Shigella invade to adjacent cells apoptotic cell death colonic epithelium

8 3 Major Clinical Syndromes of GI Infection Organisms Sign, Symptoms & Stool examination No or low-grade fever (no fecal leukocytes) - Vibrio cholerae - ETEC - EAggEC - EPEC - EHEC - Rotavirus - Norovirus Watery (secretory) diarrhea Diarrhea (gastroenteritis) Fever Fecal PMNs (neutrophils) - Non-typhoidal Salmonella - Shigella spp. - Campylobacter jejuni - EIEC Inflammatory diarrhea Febrile illness High-grade fever Fecal mononuclear cells - Typhoidal Salmonella (S. Typhi) - Enteropathogenic Yersinia spp. - Brucella spp. Enteric fever Tsolis R, et al. Nat. Rev Microbiology, 2008

9 3 Major Clinical Syndromes of GI Infection Watery (Secretory) Diarrhea Pathogens: - Non-invasive - Not trigger host innate immune response (no fecal WBC) - Produce enterotoxin enterotoxin A subunit GM1- ganglioside receptor B subunits Adenylate cyclase ATP camp Watery diarrhea Electrolytes & Water loss into gut lumen Na + K + Cl - HCO 3 - Na + K + Cl - HCO 3 - H 2 O H 2 O Pathogens grow in host gut lumen cytoplasm nucleus microbial toxins B. cereus Heat-labile enterotoxin (> 1 day) (diarrheal form) cell membrane gut epithelial cell Bacillus cereus G+ spore forming rod bacteria Heat-stable enterotoxin produced outside host GI tract 1-6 h Nausea, vomiting and abdominal cramp or fulminant liver failure (emetic form) or Food Poisoning

10 3 Major Clinical Syndromes of GI Infection Inflammatory Diarrhea Pathogens: gut lumen Inflammatory diarrhea - Invasive - Do trigger host innate immune response typical Salmonella to bacterial infection (fecal WBC; PMNs) - Produce effector proteins that allow invasion - Non-typhoidal Salmonella: Salmonella Toll-like receptors (TLRs) invasion neutrophil serum protein Typhimurium - Shigella spp. - Campylobacter jejuni - EIEC (Enteroinvasive E. coli) innate immunity activation cell damage cell damage neutrophil infiltration Pathogens grow in host gut lumen and induce host innate immune response: Macrophage, dendritic cell proinflammatory cytokines Temperature setting in brain Increased vascular permeability gut lamina propria Fever

11 Watery VS. Inflammatory Diarrhea gut lumen Watery (secretory) diarrhea Inflammatory diarrhea (Exudative inflammation) Salmonella Vibrio cholerae Electrolytes and water invasion by virulence factors: Type III secretion system proteins serum protein cholera toxin TLR neutrophil colonic epithelium camp innate immunity activation cell damage lamina propria No inflammatory response from innate immunity macrophage or dendritic cell proinflammatory cytokines Inflammatory response from innate immunity Increased vascular permeability

12 3 Major Clinical Syndromes of GI Infection Febrile Illness Mononuclear cell Febrile illness patient (enteric fever) - Prolonged high-grade fever with right lower quadrant (RLQ) abdominal pain - Absence of diarrhea Pathogens: - Invasive, Intracellular organism, Systemic spreading - Not trigger host innate immune response typical to bacterial infection (fecal WBC; PMNs), but trigger host innate immune response resembles viral infection (mononuclear cell infiltration) - Cause Interstitial inflammation instead of exudative inflammation found in inflammatory diarrhea

13 Interstitial VS. Exudative Inflammation Gut Interstitial Inflammation (found in enteric fever) Gut Exudative Inflammation (found in inflammatory diarrhea) Predominant monocyte infiltration Predominant neutrophil infiltration Thiennimitr P, et al. PNAS, 2011

14 Acute Diarrheal Patient Approaches History taking: (1) History taking (2) Physical Examination (3) Laboratory investigation - Duration after consumption of the suspected food - Short duration (1-6 h): Food poisoning h: viral gastroenteritis h: bacterial gastroenteritis - Medications: antacid drug, antibiotics - Age: Infant/ young children rotavirus, - Habitat: endemic/epidemic area: cholera Physical Examination: - Vital signs (blood pressure, respiratory rate, heart rate and pulse rate - Abdomen (increased bowel sound), gaurding Laboratory Investigation: - Direct detection for enteropathogens - Stool culture

15 Laboratory Investigations in Diarrheal Patient Stool microscopic examination Direct detection of pathogen in stool Antigen-antibody reaction: enzyme immunoassays (EIA) Laboratory Investigations in diarrheal patient Stool culture Molecular biological techniques: PCR Selective media for bacterial culture

16 Laboratory Investigations in Diarrheal Patient: Stool Microscopic Examination fresh stool Stool wet mount examination: % NaCl drop on slide + fresh stool - Covered with cover glass - 10X (low power lens) - Then, 40X (high power lens) - WBC, RBC/ HPF (High power field) - Staining: Wright, Gram Wright s stain of stool from Shigellosis patient demonstrating predominate polymorphonuclear cells (Betty A. Forbes et al, Bailey & Scott s Diagnostic Microbiology, 12 th ed, 2007, p882)

17 Laboratory Investigations in Diarrheal Patient: Antigen-Antibody Reaction: ELISA Shiga toxin-producing E. coli (STEC) Shiga toxins Detect Shiga toxin in stool add sample add detecting antibody labeled with enzyme coated plate with a capture antibody (anti-shiga toxin) Shiga toxin in sample bind to a capture antibody Shiga toxin concentration compare to standard curve E add substrate absorbance at specific wavelength 96-well ELISA plate (ELISA = Enzyme-linked immunosorbent assay) E S S

18 Laboratory Investigations in Diarrheal Patient: Antigen-Antibody Reaction: Immunochromatography Immuno-Probe Co. Ltd., Japan (-) 10% stool suspension Rotavirus (major cause of infant watery diarrhea) (+) Control Test diffusion Eiken Rota kit (+) (-) Activate enteric nervous system Increased paracellular permeability Control line : Antibody against mouse immunoglobulin Test line : Polyclonal antibodies to rotavirus proteins Reaction Zone : Monoclonal antibody to rotavirus protein conjugated with gold-colloid

19 Laboratory Investigations in Diarrheal Patient: Molecular Biological Techniques: RT-PCR Rotavirus RNA virus Detect rotavirus in stool by Reverse Transcription Polymerase Chain Reaction (RT-PCR) Reverse transcription Khamrin P., J of Virological Methods, 2011 PCR for amplification cdna (complementary DNA) Detection DNA by loading on agarose gel electrophoresis

20 Laboratory Investigations in Diarrheal Patient: Stool Bacterial Culture Important human enteropathogenic bacteria fresh stool, rectal swab EPEC (Enteropatho genic E. coli) Salmonella spp. Shigella spp. Vibrio cholerae Gram stain Negative Negative Negative Negative Lactose Fermentation Occurs Does not occur Does not occur Occurs slowly H 2 S production No Yes No No Transport media e.g. Cary-Blair, Amie s, Stuart s transport media Other special transport media - Alkaline peptone water (APW) for Vibrio cholera (halophilic organism) - Buffered glycerol saline (BGS) for Shigella spp.

21 Laboratory Investigations in Diarrheal Patient: Stool Bacterial Culture: MacConkey Agar MacConkey agar: Peptone + Lactose + Bile salt + Crystal Violet+ Neutral red Lactose Lactose fermenter Inhibit G+ Lactic acid (decrease ph) ph 6.8 ph > 8.0 E. coli is a lactose fermenter and show pink to rose-red colonies on MacConkey agar

22 Stool Bacterial Culture: Salmonella/Shigella (SS) Agar Salmonella/Shigella (SS) agar: - Peptone - Lactose - Bile salts - Sodium citrate - Brilliant green - Ferric citrate - Sodium Thiosulfate - Neutral red cannot ferment lactose inhibit G+ colorless colonies Detection of H 2 S production ph 6.8 ph > 8.0 pink (lactose fermenter) black colorless Colonies with black centers Colonies with no black centers Salmonella spp. (non-lactose fermenter, H 2 S-producing ) Shigella spp. (non-lactose fermenter, not H 2 S-producing )

23 Stool Bacterial Culture: Thiosulfate Citrate-Bile Salt (TCBS) Agar TCBS agar Thiosulfate Citrate-Bile Salt (TCBS) agar: - Peptone - Sucrose - Oxgall (bile salt) - Sodium cholate inhibit G+ - Ferric citrate - Sodium Thiosulfate - Thymol blue - Bromthymol blue - NaCl - Strong alkaline ph Vibrio cholera Halophilic organism (2-4% NaCl) fermetable sugar by Vibrio ph 6.0 ph > 7.6 Selective for Vibrio spp. Large yellow colonies of V. cholera on TCBS agar Blue to green colonies of V. parahaemolyticus on TCBS agar Seafood-borne diarrhea

24 SUMMARY GI Infection Patient Approaches Watery (secretory) diarrhea Diarrhea >= 3 diarrheas/ day Acute: < 14 days Persistent: days Chronic: >= 30 days Acute Diarrhea WBC (neutrophil) in stool no yes - Diarrheal virus (rotavirus, norovirus) - Non-invasive bacterial pathogens: e.g. V. cholera, EPEC, EAggEC, Aeromonas, C. perfringens - Food poisoning: B. cereus, S. aureus, C. botulinum Inflammatory diarrhea (exudative inflammation) - Invasive bacterial pathogens: Salmonella, Shigella, EIEC, Campylobacter jejuni Febrile illness abdominal pain and absence of diarrhea Enteric infection (interstitial inflammation) - Invasive atypical bacterial pathogens: Salmonella typhi, Yersinia enterocolitica, Brucella spp.

25 SUMMARY: Laboratory Investigations in Diarrheal Patient Stool microscopic examination Laboratory Investigations in diarrheal patient Direct detection of pathogen in stool Rotavirus antigen detection Fecal WBC in Salmonella, Shigella Antigen-antibody reaction: enzyme immunoassays (EIA) Selective media for bacterial culture MacConkey: E. coli SS agar: Salmonella, Shigella TCBS agar: Vibrio cholera, V. parahaemolyticus Stool culture Molecular biological techniques: PCR Rotavirus antigen detection

26 References: 1. DuPont HL. Acute infectious diarrhea in immunocompetent adults. The New England journal of medicine. 2014;370(16): Tsolis RM, Young GM, Solnick JV, Baumler AJ. From bench to bedside: stealth of enteroinvasive pathogens. Nature reviews Microbiology. 2008;6(12): Thiennimitr P, Winter SE, Winter MG, Xavier MN, Tolstikov V, Huseby DL, et al. Intestinal inflammation allows Salmonella to use ethanolamine to compete with the microbiota. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(42): Khamrin P, Okame M, Thongprachum A, Nantachit N, Nishimura S, Okitsu S, et al. A single-tube multiplex PCR for rapid detection in feces of 10 viruses causing diarrhea. Journal of virological methods. 2011;173(2): Ross AG, Cripps AW. Enteropathogens and chronic illness in returning travelers. The New England journal of medicine. 2013;369(8): Betty A. Forbes, Daniel F. Sahm, Alice S. Weissfeld. Bailey & Scott s Diagnostic Microbiology. Mosby Elsevier International Edition 12 th. ISBN Connie R. Mahon, George Manuselis. Textbook of Diagnostic Microbiology. W.B. Saunders Company Edtion 2 nd. ISBN X

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