Transcript Lab Diagnosis of GIT Infections

‫به‬
‫نام‬
‫خدا‬
Dr f.Hajmanoochehri
93/3
2nd leading cause of death
Infectious diarrhoea is the most common cause
of diarrhoea
Nosocomial diarrhea: patients, personnel
usually a self limiting condition/ only some require diagnostic
studies
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Gastric acidity
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Normal peristaltic movement
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Normal bacterial flora
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Esophagitis : Candia (C.albicans),HSV, CMV
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Gastritis :H.pylori
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Proctitis :Chlamydia T., HSV, T.Pallidum
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Enteric infections
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miscellaneous :Crohn,s dis.: Mycobacteria
Homosexuality : Mycobacterium avium
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Pathogenesis - General mechanism
21/12/08
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Bacteria
◦ Vibrio cholerae
◦ Enterotoxigenic E. coli
(ETEC)
◦ Enteropathogenic E. coli
(EPEC)
◦ Enterohemorrhagic E. coli
(EHEC)
◦ Campylobacter jejeuni (most
common in US)
◦ C.difficile
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Viruses
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Rotavirus
Corona viruses
Norwalk virus
Adenovirus
Calicivirus
Parasites
◦ Giardia lamblia
◦ Cyclospora and
Cryptosporidium
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Alternative water sources, consumption of raw
or undercooked meat, raw seafood (Vibrio), un
pasteurised milk (E coli, Salmonella, campylobacter)
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Travel to undeveloped area (wide range pathogens)
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Unwell patient contacts (Shigella, E coli, Salmonella,
Campylobacter, Giardia)
Recent antibiotic use
Outbreaks
Bloody diarrhea
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Dr Ekta, Microbiology
21/12/08
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Pharmacologic action of bacterial toxins, local or
distant to site of infection e.g. cholera, staphylococcal
food poisoning
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Local inflammation in response to superficial microbial
invasion e.g. shigellosis, amebiasis
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Deep invasion to blood or lymphatics; dissemination to
other body sites e.g. enteric fever, hepatitis A
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Perforation of mucosal epithelium after infection,
surgery or accidental trauma e.g. peritonitis, intraabdominal abscesses.
Toxin production
Enterotoxin
Vibrio cholerae / NCV
Enterotoxigenic E coli
Clostridium difficile (toxin A)
Campylobacter jejuni
Cytotoxin
Shigella spp
Enterohemorrhagic E coli
Clostridium difficile (toxin B)
Neurotoxin
Clostridium botulinum
Staphylococcus aureus
Bacillus cerues
Specific investigations are not required for most patients.
A laboratory diagnosis is useful for people who:
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may have an infection that could benefit from specific
therapy,
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are at risk of severe complications,
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at risk of spreading infection, or
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are involved in an outbreak.
 Feces, and in some cases rectal swabs or
Duodenal aspirate
 clean container with a tight lid , not be
contaminated
 delivered to lab within 1 hr, examined within
2 hours of collection
 Unpreserved stool specimens should be
maintained at refrigerator temperature during
storage and transport
As
the stool specimen cools, the drop in pH will
inhibit the growth of most Shigella spp. and some
Salmonella spp.
Rectal swabs should be placed in a tube transport
system
:Carry-Blair
(most
pathogen,
incl.
Campylobacter,Vibrio), Buffered glycerol (shigella)
It is becoming standard practice to reject stool
specimens for bacterial culture and parasite
examination from patients who have been hospitalized
longer than 3 days . For such patients, examination for
the toxins produced by Clostridium difficile is recommended.
Direct wet mount
Ova & parasites
Fecal leukocytes
Lactoferrin : sn & sp=WBC
Gram stain
Campylobacter
Vibrio
Modified Acid fast
Cryptosporidium
Isospora
Trichrome stain
Parasites
General priciple of agglutination :
 Tow phase reaction :
1-Binding of antigen an antibody
2- Lattice formation ( clumping)
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bacterial serological tests
Antigen detection test
Fluorescent antibody
stain
ELISA / LA
E coli 0157:H7
E coli 0157:H7
Campylobacter Spp.
Shiga toxin : EHEC
PCR
Shiga toxin, C. difficle
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Culture : anerobbic; at least 96hrs
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Tissue culture cytotoxicity assay : gold st. 48hrs
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Rapid Elisa : few hours- C.Diffiicile toxin A, or A&B
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Latex assay for GD : low specifity –for screening
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New Kit : GDH & A/B toxin
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Latex GD + PCR : high sen & sp
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Colonoscpy : pseudomembranous colitis
Culture
BA / MAC / XLD / SS /
TCBS / Selenite F broth
Identification tests
Antibiotic susceptibility test – if needed
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Supportive Medium : BA( TSA+5%sh.Blood):
yeast, Staph ,GNB
Slightly selective : MacConkey /EMB : Lactose
Neg: vibrio and most pathogen enterobacteriacea,
Moderately selective :inhibit most
enterobacteeriacea other than S & Sh : XLD, HE
Highly selective : inhibit some S &Sh: bismuth
sulfite, deoxycholate, brilliant green
Enrichment broth: salmonella, campylobacter,
Y.enterocolitica : GNB, SF , Campy-thioglycollate :
campylobacter.
Selenite F broth is an enrichment medium for the isolation of salmonella
species and some shigella species from faecal or urine specimens.
Ingredients :
Sodium hydrogen Selenite
Peptone
Disodium hydrogen
phosphate
sodium dihydrogen
phosphate
Lactose
Sodium Selenite
inhibits the growth of Gram-positive bacteria and many
Gram-negative bacteria, whereas the salmonellae are not affected.
Sodium selenite is highly toxic at near-neutral pH.
Buffer salts
are present to help maintain the pH which may rise as the
toxicity decreases . A rise in pH decreases selective activity of Selenite.
A fermentable carbohydrate ( lactose) is also present to provide acid to
neutralize the alkali produced when the selenite is reduced by bacteria.
Gram Negative (GN) broth is
selective enrichment medium used
for the cultivation of enteric
pathogens including shigella and
salmonella It is especially useful
when the salmonella or shigella
are present in low numbers.
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Requires a high microbial load for infection
Culture
◦ Look for LN on MAC
◦ Look for blue green colonies with blk centers on HE
◦ Look for red colonies with blk centers on XLD
Fecal wbc lab test
◦ See rbc’s and wbc’s in stool
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Salmonella on MacConkey
inhibits contaminating
Gram-positive flora.
Xylose is fermented by
practically all coliforms
bacteria and Salmonella,
except for Shigella
Salmonella
decarboxylate
lysine,causing
the pH to
H2S producing bacteria.
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Lactose negative
Negative for indole, VP, phenylalanine
deaminase, and urease
Most produce H2S
Do not grow in potassium cyanide
Large and complex group of organisms; grouped
by O, H, and Vi (for virulence) antigens
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Requires a low microbial load
Fecal WBC lab test
◦ Observe blood, WBCs, pus
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Culture
◦ Colorless colonies(NLF) on MAC
◦ Blue green colonies of HE
◦ Red/colorless on XLD
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Characteristics
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Non-motile
Do not produce gas from glucose
Do not hydrolyze urea
Do not produce H2S on TSI
Lysine decarboxylase negative
ONPG positive (delayed lactose +)
Possess O and some have K antigens
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Closely related to the Escherichia
All species cause bacillary dysentery
S. dysenteriae (Group A)
S. flexneri (Group B)
S. boydii (Group C)
S. sonnei (Group D)
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Campylobacter jejuni
◦ Most common cause of bacterial diarrhea in the
world
◦ Inadequately cooked poultry, untreated water,
unpasteurized milk, and exposure to animals with
diarrhea
◦ Self-limiting, antibiotics not needed
◦ Manifests with fever, diarrhea and abdominal
cramping
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Grows best at 42 degrees C
Microaerophilic conditions (An atmosphere with
reduced O2 (5% O2) with added CO2 (10% CO2)
Several selective media can be used (eg,
Skirrow’s medium)
Gram-negative curved rods, “seagull wings”
◦ Gastrointestinal Infections
 Enteropathogenic (EPEC) – primarily in infants and
children; outbreaks in hospital nurseries and day care
centers; stool has mucous but not blood; identified by
serotyping
 Enterotoxigenic (ETEC) – “traveler’s diarrhea”; watery
diarrhea without blood; self-limiting; usually not
identified
 Enteroinvasive (EIEC) – produce dysentery with bowel
penetration, watery diarrhea with blood; do NOT
ferment lactose; identified via DNA probes
 Enterohemorrhagic (EHEC serotype 0157:H7) –
associated with hemorrhagic diarrhea and hemolyticuremic syndrome (HUS), especially in young children;
undercooked hamburger, unpasteurized milk and apple
cider have spread the infection; does NOT ferment
sucrose; identified by serotyping
 Enteroaggregative (EaggEC) – cause diarrhea by adhering
to the mucosal surface of the intestine; watery diarrhea;
symptoms may persist for over two weeks
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Characteristics
◦ Dry, pink (lactose positive) colony with surrounding
pink area on MacConkey
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Ferments glucose, lactose, trehalose, & xylose
Positive indole and methyl red tests
Does NOT produce H2S or phenylalanine deaminase
Simmons citrate negative
Usually motile
Voges-Proskauer test negative
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Vibrio spp.
Plesiomonas shigelloides
Aeromonas hydrophilia
Yersinia enterocolita
Vibrio, Aeromonas, Plesiomonas
A. General characteristics
1. glucose-ferm, grow on Mac ,Different from enterics:
Oxidase-positive,
B. Laboratory diagnosis
1. Specimen collection, transport, processing
a. Vibrio should only be transported in Cary-Blair, Enrichment
:peptonated water : because glycerol in buffered glycerol saline
is toxic to vibrios
2. Direct detection methods
a. Vibrios: gnr, slightly curved, rapid darting movement
b. Aeromonas: gnr or coccobacilli, straight with round ends
c. Plesiomonas: gnr, single, pairs, short chains, or long filaments
D. Laboratory diagnosis
3. Culture and identification
a. Media
i. all grow on blood and Mac
ii. may be LFs or NLFs
iii. Thiosulfate citrate bile salts sucrose (TCBS);
selective media for vibrio: ferments sucrose and
makes yellow colonies on blue-green plate.
b. Colony appearance
i. look like gnrs, Aeromonas may be beta-heme
ii. all are OXIDASE-POSITIVE; we look for ox+ positive
that are not Pseudomonas
TCBS agar for the recovery of Vibrio sp.
l
Gram stain of Vibrio sp.
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Most common form of Yersinia
Found worldwide
Found in pigs, cats and dogs
Human also infected by ingestion of contaminated food or
water
Some infections result from eating contaminated market meat
and vacuum-packed beef
Is able to survive refrigerator temperatures (can use “cold
enrichment” to isolate)
Mainly causes acute gastroenteritis with fever
CIN agar (Cefsulodin-Irgasan
–Nevobiocin agar )
Report & Interpretations
Simple diarrhea
Antibiotic rarely beneficial
Antibiotics indicated if evidence of invasion
C difficile: stop antibiotics and start on metro / vanco
Isolation of pathogens in stool samples must be notified to
public health authority