LabM 421 Zoonoses and GI infection

National lab

• specialized characterization of organisms

• bioforensic

• handling select agent

Reference lab

• investigate and referral of high risk environmental/clinical samples

• train and guide Sentinel lab

Sentinel lab

any lab that can access or refer potentially pathogenic specimen

• routine diagnostic services

• rule-out

• referral

• capability of BSL-2 and package/shipping in category A substance

• participate in CAP LPX survey (program that helps laboratories practice identifying, handling, and shipping biothreat agents)

Category A bioterrorism agents

highest priority

Risk to national security because:

• easily disseminated

• high mortality rate

• cause public panic and social disruption

• require special action for public health preparedness

Category B bioterrorism agents

2nd highest priority

moderately easy to disseminate

moderate mortality rate

require specific enhancements of CDC’s diagnostic capacity and enhanced disease survelliance

Category C bioterrorism agents

3rd highest priority

pathogens that can be engineered to cause mass dissemination becuase

• high availability

• easily produce and disseminate

• potential high mortality rate

if suspect/can’t rule out a select agent

work in BSC-2

alert healthcare leader

secure culture and specimen

keep track of lab tech who handle specimens

communicate with Public health staff for guidance

DON’T Delay

MALIDI-TOF library is NOT available for

Select Agents because it can aersolized organism increase risk for lab tech

Rule out with biochemical test first

Tube extraction is the SAFEST way for GNR

Bacillus anthracis

large GPR in chains

“medusa-head”, ground-glass, non-hemolytic on BA

4 forms:

1. cutaneous

2. inhalational

3. injection

4. ingestion

Fulminant暴发性的 systemic disease: edema, hemorrhage, death

• caused by anthrax toxins

catalase pos

may have spores

Bacillus cereus Biovar Anthracis appear ONLY in

animals

• Rule-out ONLY if has compatible travel history

  • Look for beta-hemolysis < 24 hrs

Yersinia pestis

GN plump rod of Enteriobacteriaceae

NLF

endemic in rodents

characteristic lymphadenopathy

cause intravascular coagulation and endotoxic shock

bipolar staining or “Safety-pin” looking

better growth at 25-28C

“fried egg“ appearance on BA at 96hrs

Y. pestis lifecycle

1. wild rodents are natural reservoir

2. pass on to flea

3. flea infect domestic rodent

4. infective flea, wild rodent, and domestic rodent can all passed it on to human via direct contact

5. start as Bubonic plague

6. can progress into Secondary Plague Pneumonia and can now be transmitted person-to-person

biochemical tests for Y. pestis

catalase pos

oxidase neg

indole neg

urease neg at 35C

Francisella tularensis

tiny GNCB

carried by rodentia (esp. rabbits)

start as lesions at entry sites, move to lymph nodes

slow-growing on BA, CA, BCYE agar

catalase weakly pos/neg

oxidase neg

beta-lactmase pos

Brucella spp.

Facultatively intracellular: can live both inside or outside host cell

tiny, faintly stained GNCB

commonly obtain from unpasteurized dairy

GI & mucous membranes -> lymph nodes -> bacteremia

symptoms: Periodic fevers (FUO), night sweats, lymphadenopathy

Brucella spp. biochemical test

Catalase pos

Oxidase pos

Urea pos

Who decides the nomenclature of bacteria species

International Code of Nomenclature of Prokaryotes (ICNP) posted by International Committee on Systematics of Prokaryotes (ICSP)

Burkholderia mallei (AKA “Glanders”)

aerobic

NLF

slightly curved GNR/GNCB

poor growth on all media

Nonmotile

Severe equine马科 infections that are rarely transmitted to humans

• Nodular lesions in lungs, ulcerations of mucous membranes in respiratory tract

• can developed into Septicemia and died

no odor

Burkholderia mallei biochemical tests

catalase pos

Indole neg

Polymyxin B resistant

Amoxicillin-clavulante sensitive

penicillin resistant

Burkholderia pseudomallei

Aerobic

NLF

GNR

restricted to Australia and Southeast Asia

acquired through inhalation or direct inoculation to wounds/mucous membrane

poor growth at 24 hrs, but smooth and creamy colonies at 48 hrs

strong musty霉味的/earthy odor

grow on MacConkey

Burkholderia pseudomallei biochemical test

catalase pos

Oxidase pos

Indole neg

Polymyxin B resistant

non-hemolytic

Clostridium botulinum

Anaerobic

spore forming

GPR

Produces botulinum toxin (infant botulism from ingesting contaminated honey)

• ONLY place to treat infant botulism is at California Department of Public Health’s IBTPP program

difficult to culture

Hemorrhagic Fever Viruses

Many spread through vectors, animals

Limit lab testing, need to refer to specialized lab

Bartonella henselae

Short, gram-negative, pleomorphic rods

Intracellular

fastidious

Humans typically exposed by cat scratches/bites/licks

Diagnose for Bartonella henselae

IFA to detect antibodies

Warthin Starry stain: can be tricky to interpret

PCR

Leptospira

Motile

helical GNR

hard to culture (not worthy)

Distributed worldwide, Favors tropical/humid areas

Humans are dead end hosts

Classic biphasic disease

Diagnose for Leptospira

serology (IgM, IgG)

PCR (when acute)

Coxiella burnetii

Obligate intracellular pathogen

Q fever (from goats, sheep, livestock’s milk and farm aerosols)

from Asymptomatic/chronic to pneumoniae/endocarditis

Two forms of Coxiella burnetii

1. Small colony variants: survive in environment

2. Large colony variants: Replicates in host phagocytes

Exists in two antigenic phases:

1. Phase I – extremely infectious

2. Phase II – after passage in cell culture, less infectious

Coxiella burnetii –Serology (Hard to interpret)

Prior infection: Roughly equivalent phase I and II titers

Acute infection:

• 4x in IgG to phase I

• Antibody response to phase II is greater than phase I

Chronic infection:

• phase I IgG titer that may be higher than phase II IgG

• Endocarditis: suggested by a single phase I IgG titer of >1:800

Streptobacillus moniliformis- Rat Bite Fever

Characterized by fever (90%) rash (75%), arthritis (50%)

Haverhill Fever when ingested

Identification and treatment for Streptobacillus moniliformis

Culture might work, but isn’t great

• Fastidious: requires microaerophilic; enriched with 20% blood, serum, or ascetic fluid

• sensitive to Sodium polyanethol sulfonate (SPS)-added to blood culture bottles as an anticoagulant

Typically identified by molecular methods: PCR, mNGS

Treatment is just penicillin

Pathogenic Protozoa

single-cell

Can multiply in humans

transmission of Pathogenic Protozoa

If GI inhabitant: usually fecal-oral (e.g., contaminated food/water or person-to-person)

If blood/tissue inhabitant: usually via an arthropod vector

Four categories of pathogenic protozoa based on their mode of movement

1. Amoebozoa – the ameba, e.g., Entamoeba

2. Mastigophora – the flagellates, e.g., Giardia, Leishmania

3. Ciliophora – the ciliates, e.g., Balantidium

4. Sporozoa – organisms whose adult stage is not motile e.g., Plasmodium, Cryptosporidium

helminths蠕虫

Cestodes:

• Taenia (tapeworms)

• T. saginata (beef) & T. solium (pig)

• Echinococcus spp.

• Diphyllobothriid worms (fish tapeworm)

• Hymenolepis nana

Nematodes:

• Enterobius vermicularis

• Geohelminths (eggs/larvae need the soil!)

• Trichuris trichiura (GI)

• Ascaris lumbricoides (GI)

• Hookworms (GI)

• Strongyloides stercoralis (systemic)

Parasite Diagnosis

some PCR

Often through wet mounts

GI Viruses

Reoviridae

• Rotavirus (notable for LIVE vaccination减毒活疫苗)

Caliciviruses

• Norovirus, Sapovirus

Adenoviridae

• Adenovirus Group F (40/41)

Astroviridae

Major Bacterial Pathogens

• Salmonella spp.

• Shigella spp.

• Various strains of E. coli

• Yersinia enterocolitica

• Campylobacter spp.

• Plesiomonas shigelloides

• Aeromonas spp.

• Vibrio spp.

• Helicobacter pylori

• Clostridioides difficile

• Listeria monocytogenes

• Enterotoxins due to S. aureus, C. perfringens, B. cereus

Types of Gastrointestinal Infections

1. noninflammatory: Upsetting of fluid and electrolyte balance

   1. Watery diarrhea, no fecal leukocytes, no fever

   2. E.g. Vibrio cholerae, rotavirus, norovirus, enterotoxigenic E. coli (ETEC)

2. inflammatory: Invasion, +/- cytotoxin production

   1. Diarrhea with mucus, blood, white cells, fecal leukocytes; Fever

   2. E.g. Shigella, Salmonella, enteroinvasive E. coli

3. Penetration of mucosa with access to bloodstream

   1. Systemic symptoms, Fever

   2. E.g. Salmonella enterica serotype Typhi, Yersinia enterocolitica

Salmonella

NLF

GNR

H2S producing

2 species: S. enterica & S. bongori

• S. enterica subsp. enterica serovar Typhi only in humans

• Non-Typhoidal disease: Self limiting diarrhea, fever and stomach pains

  • 5% can become systemic

LIA: K/A or A/A

Phenylalanine neg

Salmonella Typhi Pathophysiology

1. Multiply in small intestine, invade intestinal mucosa of ileum through M cells

2. Subvert function of M cells to invade Peyer’s patches, where they are phagocytized

3. Then carried to bloodstream and lymphatics -> systemic disease (commonly a rash, intestinal hemorrhage)

1-3% become chronic carriers

Salmonella diagnosis

MALDI-TOF with further Serotyping

Serotype’s targeted Antigens:

• O antigen (“somatic” antigen) found on LPS

• H antigen (“flagellar” antigen) found on flagella

• Vi antigen (capsular antigen)

  • present in some strains, esp. Salmonella enterica serovar Typhi

Escherichia coli

Very difficult to differentiate commensal vs pathogenic

Cattle is the major reservoir of Shiga Toxin producing E. coli (STEC)

• bloody diarrhea

• DON’T treat with Antibiotics, increases chances of hemolytic uremic syndrome溶血性尿毒症综合征

MALDI-TOF CANNOT differentiate E. coli from Shigella

Shigella

LOW infectious dose (10-100 CFU)

Watery diarrhea to mucoid/bloody stools, fever, abdominal pain

Production of Shiga toxin (increased risk for hemolytic uremic syndrome溶血性尿毒症综合征)

MALDI-TOF CANNOT differentiate Shigella from E. coli

NLF

Vibrio spp.

afebrile无热, painless, watery diarrhea

Aymptomatic carriage observed

grow on TCBS agar: tolerate high salt

Oxidase pos

Vibrio parahaemolyticus

Most common cause of foodborne vibrio disease

Preferred Warm, brackish半咸 waters

ONLY those that produced thermostable hemolysin are pathogenic

Aeromonas spp.

common in fresh/brackish water environments

found as normal flora in medicinal leeches医用水蛭: Hirudo medicinalis

Acute watery diarrhea or dysentary

Beta-hemolytic

lactose variable

Oxidase pos

Plesiomonas shigelloides

transmitted by Seafood, or water contaminated with sewage

the ONLY oxidase positive member of the Enterobacterales

possibly only pathogen when coinfection occurs

Campylobacter

“gulled-wing“ GNR found in GI tract of livestock

Fastidious

best diagnosed via PCR

May lead to Guillain-Barre syndrome (0.1%)

grow in CAMPY agar

catalase pos

oxidase pos

Yersinia spp.

2 species are enteropathogenic:

• Y. enterocolitica: ranges from self limiting diarrhea to severe disease; last months long

• Y. pseudotuberculosis: commonly cause appendicitis阑尾炎 and may cause severe septicemia (high mortality)

Bullseye on CIN

NLF

Urease pos

Clostridium difficile

Anaerobic

spore forming GPR

long term antibiotic use depletes normal flora and increases risk of infection

Recurrence common

Toxins in Clostridium difficile

tcdA (enterotoxin) & tcdB (cytotoxin): inactivate GTP binding proteins, interfering with

cytoskeletal arrangement and leading to apoptosis

ONLY pathogenic when toxins are present

<3 years old are colonized with toxigenic C. difficile

Diagnosis of Clostridium difficile

EIA (enzyme immunoassay):

• Targets both organism and expression of toxins

• Potentially too insensitive

PCR:

• May target both organism and toxin gene targets

• Potentially too sensitive

CAN’T use solid stool for diagnosis

Listeria monocytogenes

beta-hemolytic

GPR

adapted for growth at refrigerator temps

Immunocompromised hosts at risk: esp. Pregnancy

Helicobacter pylori

Fastidious

Gram negative

helical

Urease is its most abundant protein: Allows penetration through mucous

Class I carcinogen (0.5-2%)

Helicobacter pylori - Diagnosis

Alarming symptoms -> Endoscopy & Biopsy

if no sympotoms:

• PCR detection in feces

• Urease Breath Test

Clostridium perfringens

anaerobic GPR

requires a relatively high infectious dose to cause infection

undercooked food: Spores survive and multiply and produce Enterotoxin

Bacillus cereus

beta-hemolytic

GPR

spores resistant to extreme temperature

• Germinate in improperly stored food

forms:

• Emetic引起呕吐的: ingestion of heat stable toxin (cereulide) leads to nausea/vomiting

• Diarrheal: Ingestion of vegetative cells/spores with Heat labile enterotoxins

Staphylococcus aureus

caused by ingestion of heat stable enterotoxin (Commonly encoded by a phage)

rapid onset food poisioning

Nausea, vomiting, abd pain, NO fever

Lab Diagnosis of Gastrointestinal Illness

1. collection: during acute illness, <5 days

   1. EIA

   2. PCR

   3. Culture

Collection and Transportation

~5mL liquid stool or ~1 gram soft stool or a swab in Cary-Blair (a media used to preserve fecal specimens)
Stability:

• Room temperature, Unpreserved specimens <2 hrs

• 4-6°C, Unpreserved specimens <24 hours

• Preserved specimens <48 hours

• Frozen usually okay for PCR only

• Cary-Blair media (limited nutrients + osmotic balance → no overgrowth)

Causes for rejection stool specimen

• Contaminated with Urine

• Containing Castor Oil, Bismuth, Barium

• >1 specimen per day

• Patient has been hospitalized >3 days unless immunocompromised / epidemic investigation

• Diapers, paper cups, dry rectal swabs

• Hard/solid stools

• Formalin/PVA preserved stools

Direct Detection: Gram Stains

generally NOT recommended
performed on request when high suspicion for Campylobacter (Poor Gram staining)

can be useful for quantifying WBCs (useDirect Detection: EIAinstead)

Direct Detection: EIA

Fast, cheap, insensitive

Limited to a 1/2 pathogens per kit

Commonly used when specific risk factors at play

Direct Detection: Rapid PCR

• Fast, sensitive, and expensive

• Risk of carryover contamination

• Panels typically include viruses, bacteria, parasites

• Good for severely ill patients, immunocompromised individuals

• NOT suitable for mild or routine illness

• ex. BioFire FilmArray

• PCR tests are NOT a test of cure! = a positive PCR result after treatment does not necessarily mean the patient still has an active infection

Key Stool Culture Media

• Blood Agar Plate (BAP)

• MacConkey Agar (MAC)

• MacConkey Sorbitol Agar (SMAC)

• Hektoen (HK) or Xylose-Lysine Desocycholate (XLD)

• Campy-CVA Agar (+/- Charcoal): for Campylobacter spp.

• Yersinia Selective Agar (CIN)

• Thiosulfate-citrate-bile-sucrose (TCBS)

Thiosulfate-citrate-bile-sucrose (TCBS)

• used for the isolation of Vibrio species

• High concentrations of sodium thiosulfate and sodium citrate, along with oxgall and bile salts inhibit non-Vibrios

• Color by virtue of sucrose fermentation

  • Yellow: V. cholerae, V. fluvialis, V. furnissii

  • Blue: V. parahaemolyticus, V. mimicus, V. hollisae

• V. cholerae report to Public Health

Yersinia Selective Agar (CIN)

Selective through sodium deoxycholate & crystal violate, antibiotics

Differentiates mannitol fermentation

Yersinia appears as red “bulls-eye”

• Y. enterocolitica – VP +

• Y. pseudotuberculosis - VP -

  • Both Urease positive

Interfering flora as colorless colonies

Campylobacter Agar

microaerophilic; 40-42°C for 48-72 hours

Campylobacter species are nonhemolytic & mucoid colonies

Hektoen (HK) or Xylose-Lysine Desocycholate (XLD)

• Selective via bile salts and sodium deoxycholate respectively

• differential for Salmonella/Shigella

  • report to Public Health

• Lactose fermenters (normal flora) turn yellow/orange

• H2S producers turn black → Salmonella

MacConkey Sorbitol Agar (SMAC):

• Screen for E. coli O157:H7

  • followed with latex serotyping kit for O157:H7

Bismuth Sulfite media

Useful for S. typhi outbreaks

Chromogenic STEC agar

useful for non-O:157 strains of E. coli

Inositol-Brilliant Green-Bile Salt agar

Differential for Plesiomonas

Selenite or Gram-Negative Broth

Useful for enrichment of Salmonella/Shigella from PCR positive specimens

Incubation conditions

All media incubated @ 35-37°C for 24hr

CIN – 25°C for 48hr (because yersinia grow best at room temp)

CAMPY – microaerophilic environment at 40-42°C for 48-72 hours

Oxidase sweep pos on BA

Aeromonas

Plesiomonas shigelloides

Vibrio

Culture Can’t Always Catch the Cause

Enterotoxigenic Bacteroides fragilis

Edwardsiella tarda

Listeria monocytogenes

Cronobacter sakazakii

EPEC, EIEC, ETEC

Overall Reporting Strategy

Report identities of pathogens and status of further workup

Specify which pathogens were included in screen

Include notes of unusual patterns

Susceptibility Testing is done if

Disseminated Infection

S. typhi, Shigella

Salmonella in <1 year or >65 years

Immunocompromised patient