Laboratory Evaluation of Intestinal Infections

1
Laboratory Evaluation of Intestinal Infections

• Dr. John R. Warren
• Department of Pathology
• Northwestern University
• Feinberg School of Medicine
• June 2007

2
Intestinal Infections

• Pathophysiology, clinical features, and
  microbiology
• Laboratory detection of enteric pathogens
• Guidelines for laboratory evaluation of
  intestinal infections

3
Pathophysiology, Clinical Features, and
Microbiology

• Acute dysentery
• Enteric fever
• Mesenteric adenitis
• Antibiotic-associated colitis

4
Acute dysentery

• Frequent bowel movements accompanied by visible
  blood, mucus, fecal leukocytes, and tenesmus
• Pathologic changes of intestinal mucosa include
  moderate to intense exudative (neutrophilic)
  inflammation, often hemorrhage, and formation of
  superficial ulcers

5
Acute dysentery

• Shigella
• Salmonella
• Campylobacter jejuni
• Enterohemorrhagic Escherichia coli
• Yersinia enterocolitica

6
Shigella

• Fermentative, non-motile, small gram-negative
  bacilli
• Facultative intracellular parasite (colonic
  epithelial cell)
• Four species S. dysenteriae (serogroup A), S.
  flexneri (serogroup B), S. boydii (serogroup C),
  and S. sonnei (serogroup D)
• Elaborates potent exotoxin (Shiga toxin) with
  enterotoxic, cytotoxic, and neurotoxic properties
• Invades colonic epithelial cells provoking
  neutrophilic inflammatory response
• Infection produces widespread, shallow colonic
  mucosal ulcers 3-7 mm in diameter with intense
  inflammatory exudate

7
Shigellosis

• Incubation period from 6 hours to 9 days, usually
  less than 3 days, after person to person
  transmission by fecal-oral route
• Acute bloody dysentery with high fever, abdominal
  pain, malaise, headache, occasionally meningismus
  and fever
• Most common cause of bloody diarrhea in children
• Syndrome particularly severe in malnourished
  children

8
Complications of Shigellosis

• Untreated diarrhea lasts 1 day to 1 month with
  average of 7 days
• Severe dehydration
• Febrile seizures
• Intestinal obstruction (3)
• Hemolytic-uremic syndrome (Shiga toxin-producing
  S. dysenterieae 1)
• Post-infectious arthritis (2-5 weeks after acute
  dysentery) associated with the histocompatibility
  antigen HLA-B27 (S. flexneri)
• Bacteremia and disseminated infection rare

9
Salmonella

• Fermentative, non-spore forming, motile
  gram-negative bacilli
• Facultative intracellular parasite (intestinal
  epithelial cells, macrophages)
• Single species (Salmonella enterica) with 1,454
  serotypes in genetic subspecies 1 (Group I)
  having O-antigen groups A, B, C1, C2, D, E
• Subspecies 1 strains usually isolated from humans
  and other warm-blooded animals
• Nomenclature Salmonella serotype Dublin
• Salmonella serotype Typhimurium and S. serotype
  Enteritidis most frequent cause of salmonellosis

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Salmonellosis

• Fever, cramping, abdominal pain, and diarrhea
  within 8-48 hours after ingestion of infective
  dose (contaminated poultry, shell eggs, dairy
  products, beef, exotic pets such as reptiles)
• Inflammatory (neutrophilic) enteritis most
  typically involving the small bowel mucosa,
  occasional cause of colitis with crypt abscesses
  and erosive ulceration of colonic mucosa
  (Salmonella serotype Typhimurium)
• Moderate number of fecal neutrophils, usually
  fewer than in shigellosis except colitis with
  blood and pus in stool

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Complications of Salmonellosis

• Diarrhea usually self-limited (3-7 days), if
  persists gt10 days another microbial etiology
  likely
• Occasional dehydration requiring hospitalization
• Bacteremia (1-4 immunocompetent cases)
  (persistent bacteremia suggests endovascular
  infection site such as atherosclerotic plaques
  and aneurysms)
• After resolution of diarrhea mean duration of
  carriage in stool is 4-5 weeks

12
Campylobacter jejuni

• Microaerophilic, thermophilic, motile, S-shaped
  gram-negative bacilli
• Adhere to and invade intestinal epithelial cells
• Optimal growth at 42oC in ambient gas mixture of
  5 O2, 10 CO2, and 85 N2

13
Campylobacteriosis

• Severe abdominal pain with fever and diarrhea 1-7
  days after infectious dose (poultry, raw milk,
  contact with infected pets, fecal-oral route)
• Campylobacter jejuni multiplies in human bile,
  facilitating initial colonization of small
  intestine
• Tissue injury with invasion includes jejunum,
  ileum, and colon, provoking a mixed
  neutrophilic-mononuclear cell inflammatory
  response with erosive mucosal ulceration

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Complications of Campylobacteriosis

• Enteritis usually self-limiting (1 day to 1 week
  or longer)
• Guillain-Barré syndrome (structural homology of
  LPS O-antigen with human nerve gangliosides)
• Post-infectious reactive arthritis (associated
  with HLA-B27)
• Bacteremia (rate of 1.5/1,000 intestinal
  infections)

15
Enterohemorrhagic Escherichia coli

• Non-sorbitol fermenting Escherichia coli
  (Escherichia coli 94 for sorbitol
  fermentation)
• Majority of enterohemorrhagic strains positive
  for somatic O157 and flagellar H7 antigens (O104
  and O111 strains have caused outbreaks in the US)
• Bacteriophage-mediated production of Shiga-like
  toxin (Stx1 or Stx2) which are cytotoxic
  (verotoxin)
• Accounts for 15 to 36 of cases of bloody
  diarrhea

16
Enterohemorrhagic Escherichia coli Diarrhea

• Abdominal cramps and watery diarrhea 3 to 8 days
  following ingestion of contaminated food
  (undercooked beef, raw milk, fresh produce) or
  water
• Shiga toxin absorbed from intestine and damages
  vascular endothelial cells (intestinal mucosa and
  kidney)
• Watery diarrhea followed by grossly bloody
  diarrhea
• Uncomplicated illness lasts 1 to 12 days
• Use of antibiotics contraindicated
  (phage-mediated production of Shiga toxin
  enhanced by ampicillin, norfloxacin, and other
  antibiotics)

17
Complications of Hemorrhagic Escherichia coli
Colitis

• Fever and neutrophilic leukocytosis herald
  hemolytic uremic syndrome (HUS)
  (thrombocytopenia, oliguria, hematuria,
  microangiopathic hemolytic anemia)
• HUS in 8 of infections in children with a 3 to
  5 mortality

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Yersinia enterocolitica

• Fermentative, rod-shaped or coccoid gram-negative
  bacteria, non-motile and metabolically inactive
  at 37oC but motile and metabolically active at
  22-30oC
• Enteropathogenic strains cytotoxic by
  penetratating human epithelial cells
• Infection results in inflammatory ileitis
  (generally) and colitis (occasionally involving
  ascending colon) with mixed neutrophilic and
  mononuclear cell response
• Necrosis of Peyers patches, mesenteric lymph
  node enlargement, and in severe cases thrombosis
  of mesenteric blood vessels with intestinal
  necrosis and hemorrhage

19
Yersinosis

• Febrile diarrhea with abdominal pain 16 to 48
  hours following ingestion of an infectious
  inoculum (undercooked pork, chitterlings1)
• Duration of illness ranges from 1 day to a
  prolonged diarrhea of 4 weeks
• 1Chitterlings (chitlins) intestines of young
  pigs, cleaned and stewed, and then frequently
  battered and fried

20
Complications of Yersinosis

• Can simulate acute appendicitis (mesenteric
  lymphadenitis)
• Bacteremic dissemination with hepatic and splenic
  abscess formation
• Reactive arthritis associated with HLA-B27
  histocompatibility antigen (10-30)
• Exudative pharyngitis (8 of infections
  accompanied by fever but no diarrhea)

21
Infective Dose of Enteric Pathogens

• Shigella 10 to 102
• Campylobacter jejuni 102 to 106
• Salmonella 105
• Escherichia coli 108
• Yersinia 109

22
Enteric Infections United States 2002 (CDC)1

• Salmonella 16.10
• Campylobacter 13.17
• Shigella 10.34
• Escherichia coli O157H7 1.73
• Yersinia 0.44
• 1Incidence per 100,000 population

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Enteric Infections United States 2004 (CDC)1,2

• Salmonella 14.7
• Campylobacter 12.9
• Shigella 5.1
• Yersinia 3.9
• Escherichia coli O157H7 0.9
• 1CDC FoodNet (10 States)
• 2Incidence per 100,000 population

24
Mortality of Enteric Infections 2001 (Notifiable
Diseases, CDC)

• Cases Deaths ()
• Salmonellosis 40,495 40 (0.1)
• Shigellosis 20,221 2 (0.01)
• Hemorrhagic E. coli1 3,287 6 (0.2)
• HUS 202 35 (17.3)
• 1O157H7

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Enteric Fever

• Bacterial pathogens ingested (facultative
  intracellular parasites)
• Pathogens penetrate small intestinal mucosa,
  infect and multiply in intestinal lymphatic
  tissue (7-14 days), then disseminate by the
  lymphatic and hematogenous routes
• Infection of the mononuclear phagocyte system
  (reticuloendothelial system), including lymph
  nodes, spleen, liver and bone marrow
• Bacteremia secondary to infection of the
  mononuclear phagocyte system

26
Causes of Enteric Fever

• Prototype Salmonella serotype Typhi (typhoid
  fever)
• Other salmonellae S. serotype Paratyphi A, S.
  serotype Schottmuelleri (formerly Paratyphi B),
  S. serotype Hirschfeldii (formerly Paratyphi C)
  (paratyphoid fever) S. serotype Cholerasuis
• Non-salmonellae Yersinia enterocolitica, Y.
  pseudotuberculosis, and Campylobacter fetus

27
Typhoid and Paratyphoid Fever

• Ingestion of infectious inoculum (fecal
  contaminated food or water) followed within 5 to
  21 days by remittent fever that becomes sustained
  (occasional enterocolitis with fever for 3-4 days
  that resolves before fever)
• Patients acutely ill with fever, chills,
  diaphoresis, dull frontal headache, anorexia,
  weakness, muscle pains
• Leukopenia
• Bradycardia (lt50)
• Cutaneous rose spots (blanching erythematous
  maculopapular lesions) (30 typhoid fever, 3
  paratyphoid fever)

28
Typhoid Fever

• Typhoid fever in the United States associated
  with international travel
• During the period 1985-1994, of 2,445 reported
  typhoid cases, 72 associated with international
  travel, most frequently to Mexico, India, the
  Philippines, and Pakistan in descending order

29
Enteric Fever-Like Syndromes Due to
Non-Salmonellae

• Fever and headache as in typhoid and paratyphoid
  fever
• Fever with Yersinia intermittent not sustained as
  in Salmonella
• Leukopenia infrequent
• Acute diarrhea prominent feature of Yersinia
  enterocolitica
• Salmonella enteric fever in individuals lt30 years
  of age, non-Salmonella gt40 years
• Salmonella enteric fever associated with travel
  to developing countries, Yersinia to Northern
  Europe or no travel (acquisition in the United
  States), Campylobacter fetus not associated with
  foreign travel
• Enteric fever with Yersinia and Campylobacter
  fetus associated with underlying disease
  especially cirrhosis
• Campylobacter fetus has propensity for infection
  of vascular sites and is associated with
  thrombophlebitis

30
Complications of Typhoid Fever

• Intestinal perforation third-fourth week of
  infection in untreated patients, due to
  hyperplasia, ulceration, and necrosis of
  ileocecal lymphoid tissue (3-10)
• Aerobic and anaerobic bacteremia secondary to
  intestinal perforation
• Localized infections endocarditis, pericarditis,
  pneumonia, orchitis, ovarian abscess formation

31
Mesenteric Adenitis

• Variant of enteric fever with prominence of
  mesenteric lymph node infection (mesenteric
  lymphadenitis)
• Fever and right lower quadrant pain mimicking
  acute appendicitis accompanied by diarrhea
• Most common cause Yersinia enterocolitica and Y.
  pseudotuberculosis
• More prevalent in Europe (especially Belgium)
  than the United States
• Self-limited in vast majority of cases
• Iatrogenic complication inappropriate surgery
  for acute appendicitis (avoid by helical
  appendiceal CT scan)

32
Toxin-Producing Clostridium difficile

• Obligate anaerobic gram-positive bacilli
• Pathogenic Clostridium difficile produces two
  exotoxins toxin A (enterotoxin) (MW308-kd) and
  toxin B (cytotoxin) (MW269-kd)
• Toxin A and toxin B inactivate Rho proteins
  (GTP-binding proteins that regulate the actin
  cytoskeleton) in intestinal epithelial cells
  which results in apoptosis
• Toxin A if infused in ligated loops of rabbit
  small intestine stimulates fluid accumulation
  with the efficiency of cholera toxin (toxin B
  lacks this activity)
• Stains of C. difficile lacking toxin A
  demonstrate full potency in producing human
  colitis

33
Clostridium difficile Colitis

• Disruption of colonic microbiota (Bacteroides,
  Prevotella predominant) by antibiotics
  (clindamycin, cephalosporins especially
  third-generation agents, ampicillin and
  amoxicillin, other penicillins, erythromycin and
  other macrolides, trimethoprim-sulfamethoxazole,
  tetracyclines) or antineoplastic drugs
  doxorubicin, cisplatin, cyclophosphamide,
  5-fluorouracil, chlorambucil, and methotrexate)
• Colonization of colon by toxin-producing strain
  of Clostridium difficile (exogenous or endogenous
  source)
• Colonic mucosal injury and inflammation
  (pseudomembranous colitis)

34
Clostridium difficile Colitis

• Onset of diarrhea after 5 to 10 days of
  antibiotic treatment (nosocomial)
• Diarrhea ranges from brief and self-limited to
  cholera-like with more than 20 stools per day
• Fever (30 to 50)
• Neutrophilic leukocytosis (50 to 60) (mean
  count of 15,000-16,000/mm3)
• Abdominal pain or cramping (20 to 33)

35
Complications of Clostridium difficile Colitis

• Toxic megacolon (mortality of 60)
• Colonic perforation
• Transverse volvulus
• Protein-losing enteropathy
• Recurrent C. difficile associated diarrhea (20)

36
Recurrent Clostridium difficile Colitis

• Persistent disruption of colonic microbiota by
  metronidazole or vancomycin
• Poor antibody response to toxin
• Lack of antibiotic activity against bacterial
  spores

37
Laboratory Detection of Salmonella and Shigella

• Sheep blood agar
• MacConkey or eosin-methylene blue agar
• Triple-sugar iron slant
• Xylose-lysine-deoxycholate (XLD), Hektoen
  enteric, or Salmonella-Shigella agar
• Enrichment broth (selenite or gram-negative)

38
Laboratory Detection of Yersinia

• Same as for Salmonella and Shigella and in
  addition
• Cold enrichment of stool specimens at
  refrigerator temperatures
• Use of cefsulodin-irgasan-novobiocin (CIN) agar
  (Yersinia selective agar) Most enteric organisms
  inhibited (selective), and mannitol fermented by
  Yersinia which forms red bulls eye colonies (pH
  indicator neutral red) (differential)

39
NMH Short Series for Salmonella and Shigella

• Cytochrome oxidase (OXI)
• Lactose fermentation on MacConkey (MAC)
• Motility (MOT)
• Triple sugar iron agar (TSI)
• Indole (IND)
• Hydrogen sulfide (H2S)
• Lysine-iron agar (LYI)
• Urease (URE)
• ONPG (ONP)

40
NMH Short Series for Clinically Important
Salmonella Serotypes1

• OXI MAC MOT TSI IND H2S
  LYI URE ONP
• Most nlf k/a2
  
• Typhi nlf k/a3
  4
• Para A nlf k/a2
  
• 1R/O Salmonella indole, ONPT
• 2Gas production
• 3No gas production
• 4Whisp of H2S below the slant

41
Series for Shigella and Other Enteric Pathogens1

• OXI MAC MOT
  TSI IND H2S LYI URE ONP
• Shigella k/a4
  /6 /7
• Yersinia2 3
  a/a4 /
• Vibrio
  a/a4
• Aeromonas
  a/a5 /
• Plesiomonas k/a4
  
• 1R/O Shigella motility, a/a TSI, lysine,
  H2S, urea
• R/O Yersinia lysine, H2S
• 2Y. enterocolitica
• 3At 37oC motile at 25oC
• 4No gas production
• 5Gas production
• 6Serogroup A-C (25-500, serogroup D )
• 7Serogroup A-C , serogroup D

42
Laboratory Detection of Enterohemorrhagic E. coli

• Same as for Salmonella and Shigella and in
  addition
• Sorbitol-containing MacConkey agar (SMAC) for
  detection of colorless sorbitol-negative colonies
  of Escherichia coli O157H7
• Latex agglutination testing of colorless colonies
  (SMAC) for the somatic antigen O157
• Direct immunoassay of stool specimens for Shiga
  toxin 1 and 2

43
Laboratory Detection of Campylobacter jejuni

• Selective media containing antimicrobial agents
  to inhibit enteric bacterial flora
  (cefoperazone-containing media)
• Microaerophilic ambient gas for incubation (5
  O2, 10 CO2, 85 N2)
• Incubation temperature (42o C) for thermophilic
  growth

44
Laboratory Detection of Toxigenic Clostridium
difficile

• Direct testing of stool specimens for presence of
  toxin A and toxin B (fibroblast cell-culture for
  cytotoxin, EIA for toxin A and B)
• CCFA (cycloserine-cefoxitin-fructose agar) for
  detection for colonies with a yellow color
  (oxidative decarboxylation of peptones results in
  an alkaline pH, C. difficile non-fermentative for
  fructose, neutral red the pH indicator)
  (cycloserine inhibits gram-negatives especially
  E. coli, cefoxitin has broad activity against
  gram-negatives and gram-positives)
• Testing of broth supernatant for presence of
  toxin A and B

45
Guidelines for Laboratory Evaluation of
Infectious Diarrhea

• Community-acquired or travelers diarrhea
• Nosocomial diarrhea (onset gt3 days after
  hospitalization)
• Persistent diarrhea (gt7 days)
• Immunocompromised patient (especially if HIV )
• Thielman and Guerrant, Acute Infectious Diarrhea,
  New England Journal of Medicine,35038-47 (2004)

46
Community-acquired or travelers diarrhea

• Culture for Salmonella, Shigella, and
  Campylobacter (if history of bloody diarrhea or
  hemolytic-uremic syndrome)
• Culture for E. coli O157H7 and test for Shiga
  toxin (if recent antibiotics, chemotherapy, or
  hospitalization)
• Culture and/or test for Clostridium difficile
  toxins A and B

47
Nosocomial diarrhea (onset gt3 days after
hospitalization)

• Culture and/or test for C. difficile toxins A and
  B (if hospital outbreak of diarrhea or patient
  gt65 yr of age with coexisting conditions,
  immunocompromise, or neutropenia, or if systemic
  infection suscpeted) culture for Salmonella,
  Shigella, and Campylobacter (with blood cultures
  if systemic infection suspected) (if bloody
  diarrhea) culture for E. coli O157H7 and test
  for Shiga toxins

48
Persistent diarrhea (gt7 days)

• Consider protozoa
• Giardia
• Cryptosporidium
• Cyclospora
• Isospora bell

49
Immunocompromised patient (especially if HIV )

• Add
• Microsporidium
• Mycobacterium avium complex
• Cytomegalovirus

50
Recommended Reading

• Mandell, G.L., Bennett, J.E., and Dolin, R.
• (Eds.). Mandell, Douglas, and Bennetts
• Principles and Practice of Infectious
• Diseases, Sixth Edition, Elsevier Churchill
• Livingstone, 2005
• Guerrant, R.L., and Steiner, T.S. Chapter 89.
  Gastrointestinal Infections and Food Poisoning.
• Thielman, N.M., and Wilson, K.H. Chapter 92.
  Antibiotic-Associated Colitis.
• Guerrant, R.L., and Lima, A. A. M. Chapter 93.
  Inflammatory Enteritides.
• Thielman, N.M., and Guerrant, R.L. Chapter 94.
  Enteric Fever and Other Causes of Abdominal
  Symptoms With Fever.