BASIC CLINICAL MICROBIOLOGY - SUSTAINING THE FORCE FORWARD

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BASIC CLINICAL MICROBIOLOGY - SUSTAINING THE
FORCE FORWARD

• DAVID CRAFT, PhD D(ABMM)
• Commander
• 9th Area Medical Laboratory

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The preservation of the soldiers health should
be the commanders first and greatest
care. Regulations for Order and Discipline of
the Troops, 1779
Caring Beyond the Call of Duty
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OBJECTIVES

• Match the specimen and / or specimen associated
  rules to the appropriate bacteriology bench.
• Identify the common media, assays and algorithms
  used to provide diagnostic microbiology support
  in the patient care environment.
• Identify the common pathogenic microorganisms
  associated with each bench in the microbiology
  laboratory to include the most common bacterial,
  fungal, viral, and parasitologic agents causing
  disease.

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Laboratory Organization

• Clinical Microbiology
• Bacteriology - ID and Susceptibility
• Virology - Culture and/or antigen detection
• Mycobacteriology/Mycology - ID/Susc(?)
• Parasitology - Microscopy or antigen detection
• Infectious Disease Serology
• Hepatitis, other viral, autoimmune, Lyme, etc.
• Molecular Microbiology - n.a. amplification

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Microbiology - Bacteriology

• Smear - Gram stain, fluorescent, direct
• Culture -
• artificial media, incubation, isolation, ID
• Rapid detection -
• Group A Strep (pharyngitis), C. difficile (AAC)
• Susceptibility -
• Predicting resistance or susceptibility

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Cytospin for body fluids ... CSF
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Gram stains of CSF

• S. pneumoniae

H. influenzae
N. meningitidis
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Plating onto media
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Acute pharyngitis

• Template

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Rapid Detection / Screening Tests
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Antimicrobic Susceptibilty Testing (AST)
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Specimen Collection

• Sterile Containers
• urine, sputum, tissue, stool, BAL, CSF
• Blood Cultures - broth bottles
• Swabs and appropriate transport media
• TISSUE IS BEST!!

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Organization- Bacteriology Benches

• Blood/Sterile Fluids - critical values
• Urine - (sterile) separate bench, inc volume
• Wounds - tissue, OR, skin
• Respiratory- sputum, trach asp, BAL, throat
• Stools

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Bacterial Endocarditis

• Splinter hemorrahges
• Oslers Nodes

• Vegetation on Heart Valve

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Blood Culture Bench

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Blood Culture Bench - RULES

• Volume - gt 30 mls total
• 2 different sites, ie two cultures - rule out
  contaminants
• Automation - continuous monitoring
• Critical values - call Gram stain result
• ID and susceptibility

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Blood Culture Contamination

• Growing bugs
• Pseudobacteremia
• Phlebotomy
• Skin Prep (CHG/EtOH)
• Skin Contaminants OR
• Endocarditis

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Blood Cx Bench-Common Organisms

• Organisms most often associated with clinical
  bacteremia
• GPCs in Clusters
• GPCs in pairs and chains
• GNRs, short, fat
• GNCB
• GP, large oval, too large to be bacteria!

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Urine Bench - RULES

• Quantitation
• gt 100,000 cfu/ml - full ID and Susceptibility
• gt 10,000 cfu/ml - partial ID and Susceptibility
• lt 10,000 cfu/ml - no work up
• MUF - mixed urogenital flora, 3 or more organisms
  cultured from same urine Contamination!
• MEF - mixed enteric flora Contamination!

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E. coli, E. coli, E. coli
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Urine Bench - Common Organisms

• Organisms most often associated with clinical
  UTI?
• E. coli, E. coli, E. coli (80)
• Other Gram negative enterics
• Klebsiella, Enterobacter, Citrobacter
• Enterococcus/Streptococcus (GBS)
• S. aureus, CNS

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Wounds Bench

• No Rules
• TISSUE
• Skin contaminants
• ID and Susceptibility

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Wounds Bench - Common Organisms

• Organisms most often associated with clinical
  wound infections?
• S. aureus, S. aureus
• Mixed anaerobes - polymicrobic
• Skin contaminants
• Nosocomial organisms
• MRSA, VRE
• Pseudomonas aeruginosa
• Gram negative enteric bacilli
• Acinetobacter

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Clostridium perfringens

• Gas gangrene
• Myonecrosis
• -boxcar shape
• -double zone B
• hemolysis

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Respiratory
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Respiratory Bench - RULES

• lt 10 SECs / gt25 PMNs per lpf
• SPIT VS
  SPUTUM

10X
10X
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100X Oil immersion
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Respiratory Bench - RULES

• lt 10 SECs / gt25 PMNs per lpf
• Exception - AFB (M.tb), Fungal, Virology
• Sputum - expectorated or induced
• Tracheal aspirates - infection vs colonization
• BALs, Bronchial washes, brushes
• ID and Susceptibility

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Respiratory Bench - Common Organisms

• Organisms most often associated with clinical
  respiratory infections?
• Upper Respiratory -
• Otitis media - Streptococcus pneumoniae,
  Haemophilus influenzae, Moraxella catarrhalis
• Pharyngitis - Streptococcus pyogenes (GAS)
• Viral (respiratory, EBV, Coxsackie) GrpCG
  Strep, C. M. pneumoniae, A. haemolyticum, HIV
  (acute)

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Respiratory Bench - Common Organisms - Cont.

• Lower Respiratory -
• Viral - RSV, Influenza, Parainfluenza, Adenovirus
• Bacterial - community acquired
• S. pneumoniae, M. pneumoniae, C. pneumoniae
• Bacterial - nosocomial
• Pseudomonas, Gram negative enterics, Legionella
• Bacterial - aspiration
• Anaerobes
• Gram negative enterics

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Stool Bench - 3 DAY RULE

• Inpatient - gt3 days
• On antibiotics - antibiotic associated colitis -
  etiology?
• Outpatient or Inpatient lt 3 days
• Bacteria - ???
• Parasites - ???

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Clostridium difficile

• Antibiotic associated pseudomembranous colitis
• Nosocomially acquired
• Plaques on Colonoscopy
• Treat w/ metranidazole or vancomycin (oral)

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Non-fermenters(look for lack of color)

• Template

Hektoen
MacConkey
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Stool bench - 3 DAY RULE

• Inpatient - gt3 days
• On antibiotics - antibiotic associated colitis
  associated with Clostridium difficile
• Outpatient, Clinic, Inpatient lt 3 days
• Bacteria - Gram negative bacilli such as
  Salmonella, Shigella, Yersinia, Campylobacter, E.
  coli O157H7
• Parasites - Giardia, Cryptosporidium, Entamoeba
  histolytica worms

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Bacterial Gastroenteritis

• Campylobacter sp.
• Curved GN rods
• gull wing
• Oxidase POS
• Most common cause of community acquired bacterial
  gastroenteritis
• Microaerophilic, grown on selective media

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Cytotoxin mediated disease physically damages
the intestinal epithelium. These infections are
sometimes called invasive or inflammatory. Bacte
ria that produce cytotoxins include Shigella
species (A-D) Salmonella species (1400
serotypes) Campylobacter jejuni Yersinia
enterocolitica Clostridium difficile cytotoxin
B, this organism also produces an enterotoxin
A Vibrio species, not cholera Enteroinvasive E.
coli (EIEC) Enterohemorrhagic E. coli (EHEC), an
example is E. coli O157H7 Enteropathogenic E.
coli (EPEC) Normal flora Facultative anaerobes
Enterobacteriaceae E. coli, Klebsiella sp.,
Enterobacter sp., Proteus sp. Gram-positive
cocci S. aureus, Enterococcus,
Streptococci Anaerobes Gram-negative
Bacteroides fragilis, Fusobacterium
Gram-positive Lactobacillus, Clostridium,
Peptostreptococcus
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Enterotoxin mediated disease causes physiologic
change to the intestinal epithelium resulting in
fluid and electrolyte secretion. These
infections are sometimes called malabsorptive
or non-inflammatory. Bacteria that produce
enterotoxins include Vibrio cholerae Enterotox
igenic E. coli (ETEC) Staphylococcus aureus
toxin mediated, rapid symptoms Bacillus cereus -
toxin mediated, rapid symptoms Clostridium
perfringens - toxin mediated, rapid symptoms The
latter three are primarily toxin mediated not
requiring growth of the organism in the host to
cause disease. Other pathogens such as
Cryptosporidium parvum, Giardia lamblia,
rotavirus, calicivirus and norwalk agent (virus)
also manifest with similar symptoms. Clostridium
botulinum produces neurotoxin.
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Case Study 1

• A 44-year-old female with ESRD fromdiabetes on
  chronic HD was admitted with a3-week history of
  fever, fatigue, weight loss and weakness.
• Her temperature was 38 C, and she had a 2/6 SEM.
  A partially healed 2x3 ulcer on her left heal
  was noted.
• WBC 17,000/mL with 81 N, 7 B, 12 L

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Case Study 1 (cont.)

• The HD catheter site was unremarkable.
• A TTE was negative, but a TEE revealed a large
  vegetation on the mitral valve.
• Two sets of blood cultures were drawn.

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Case Study 1 (cont.)

• Vancomycin and gentamicin were given in single
  doses pending the results of blood cultures.
• What initial microbiology data do you want to
  know in order to refine the antibiotic treatment?

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Blood Culture

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Initial Microbiological Data

• BLOOD CULTURE POS (4/4 bottles)
• Gram Stain

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Case Study 1 (cont.)

• Patient develops a lower respiratory tract
  infection.
• A sputum or tracheal aspirate is obtained.
• What initial microbiology data do you want?

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100X oil immersion
10X
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Virology Bench

• Obligate intracellular parasites/no artificial
  media
• Rapid detection
• Microscopy - Tzanck, Fluorescent Antibody
• Detection by membrane bound Antibody
• Grow in tissue culture and ID by specific
  cytopathic effect (CPE)
• Electron microscopy
• Molecular techniques

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Mycobacteriology and Mycology Bench

• Mycobacteriology - respiratory, tissue
• Mycobacterium tuberculosis (M. tb)
• Mycobacterium other than tb (MOTT)
• Slow growth, slower susceptibilities
• Smear, culture, molecular methods
• Mycology - tissue, body fluids
• Yeast - Candida species, Cryptococcus
• Mold - Aspergillus, dimorphs, dermatophytes

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Smears fluorescent and acid-fast
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Tissue preparation for fungal elements
KOH digestion
Lactophenol Cotton Blue
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Yeasts
Cryptococcus

• Candida spp
• Candida albicans
• Cryptococcus spp
• Cryptococcus neoformans
• Torulopsis glabrata
• Trichosporon spp
• Geotrichum spp
• Malassezia furfur

Candida
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Parasitology Bench

• Blood parasites - Malaria, Trypanosomes
• Stool
• 3 day rule for full work-up of stool O P
• Stool parapak - 2 preservatives
• PVA - small things (100x) such as protozoa seen
  on permanently stained slide
• Formalin - somewhat larger things (40x) such as
  eggs, larvae, adult worms seen on wet mount

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Who is He?
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Molecular Diagnostics in the Microbiology
Laboratory
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Polymerase Chain Reaction
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PCR

• PCR is a powerful tool in molecular diagnostic
  pathology.
• Patented technology by Roche
• PCR can synthesize and exponentially amplify
  nucleic acid sequences in vitro (10 7 copies in
  minutes.)
• Can detect low concentrations of target with high
  specificity
• Goal To replicate a target sequence of 100-500
  bp that is unique to the organism

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PCR Amplifies a Targeted Sequence
Target Sequence
DNA Strand
Double Helix DNA Strand
Supercoiled DNA Strand
Chromosome
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Roche Cobas Amplicor Analyzer

• PCR or rtPCR for GC / Ct or HIV viral load
• Automated amplification and detection on a single
  instrument.
• Combines five instruments into one (thermal
  cycler, automatic pipettor, incubator, washer and
  reader).

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Real Time PCR

• Double-labeled fluorescent probe whose signal is
  diminished in its intact form, but when detached
  by Taq polymerase, emits a fluorescence signal.
• Intensity of fluorescence proportional to conc.
  of template.
• Quantitative PCR assay that is rapid,
  reproducible, specific
• Closed tube system that reduces the incidence of
  contamination.

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Bacillus anthracis
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Plague

• Yersinia pestis
• Inguinal buboe

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Gen-ProbeTMA Transcription Mediated
Amplification

• DTS 800
• TIGRIS

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Sexually Transmitted Diseases

• Gonorrhea (GC)
• Gram stain
• Cervicitis
• Dissimenated

GC or Chlamydia (Asympto.)
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TMA Transcription Mediated Amplification

• Used for mTB, Chlamydia and GC testing, GenProbe
• TMA is isothermal. No thermal cycler needed
• TMA can amplify either DNA or RNA, and produces
  RNA amplicon.
• TMA is an RNA transcription amplification system
  using 2 enzymes
• - Reverse transcriptase
• - RNA polymerase

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Transcription-Mediated Amplification (TMA)
TM
A Family of Three Technologies
Target Capture
Dual Kinetic Assay (DKA)
CT GC
CT
GC
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SDA Components
Target DNA
Polymerase
Primers
Restriction Enzyme
Detector Probe
SDA
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BDProbeTecET Workflow
Samples processed into Sample Diluent
LYSING RACK / LYSING HEATER Incubate samples 30
min
LYSING RACK Cool specimens 15 min at Room
Temperature
PRIMING PLATE Transfer 150 ?L sample to Priming
Wells Sit at Room Temp at least 20 min (can sit
up to 6 hours)
Place PRIMING PLATE and AMPLIFICATION PLATE in
Priming/Warming Heater. Incubate 10 min
Immediately place AMPLIFICATION PLATE into
instrument Press start key Testing complete in
60 min
Transfer 100 ?L to AMPLIFICATION PLATE Seal Plate
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Strand Displacement Amplification
SDA Primer
Target DNA
Restriction Enzyme Recognition Site (Bsob I)
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Strand Displacement Amplification
Elongation of DNA strands by polymerase
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Strand Displacement Amplification
Restriction enzyme nicks one strand
Unable to cut through both strands due to
modified dCTP
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Strand Displacement Amplification
New strand is displaced as elongation proceeds
Polymerase begins elongation again
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Strand Displacement Amplification
New copies can serve as another template
Nick and elongation process repeats
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BDProbeTec ETFluorescence Energy Transfer

• Detection System

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Fluorescent Energy Transfer
BsoB I recognition sequence
Hairpin secondary structure
Target specific hybridization area
Two fluorescent dyes Fluorescein Rhodamine
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Fluorescence Energy Transfer

• Close proximity of rhodamine masks fluorescein
  emission
• Energy is said to transfer to rhodamine
• During SDA, detector probe opens as target is
  elongated
• Restriction enzyme site is cut by BsoB I
• Fluorescein emits signal as rhodamine distance is
  increased

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HPV DNA Testing Greater Protection Against
Cervical Disease andImproved Patient Management
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Hybrid Capture 2 SystemA nucleic acid
hybridization assay with signal amplification
that utilizes chemiluminescent detection.
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Specimen Collection Strategies

• Cytyc ThinPrep System Preservcyt solution
• Reflex testing performed w/in 3 weeks from day of
  collection
• Digene Cervical Sampler (cervical brush)
• Storage and shipment up to 2 weeks at 2-30º C
• Remaining storage at 2-8º C for up to 1 week
• Long term storage at - 20º C for up to 3 months
• Cervical Biopsies
• Collected in the Digene Specimen Transport
  Medium
• Shipped overnight at 2-30º C
• Stored at -20º C for up to 3 months

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Human Papillomavirus

• Primary cause of cervical cancer
• Journal of National Cancer Institute
• WHO, 1996
• NIH Consensus Conference, 1996
• Over 70 site-specific types
• 5-10 of women gt35 are persistent carriers of HPV

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Limitations of Conventional Pap Smear

• Does not test for the cause of cervical cancer
• Subjectivity and variability
• Sensitivity and specificity
• Sampling errors
• Result is often inconclusive ASCUS
• Atypical squamous cells of undetermined
  significance

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ASCUS Management The Dilemma
Reflex with hc2 HPV Test
Reflex with hc2 HPV Test
Falsely Tagged/ No Colposcopy Required
How does a healthcare provider reliably
distinguish between these two groups?
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Step 1
Digenes Hybrid Capture 2
Step 2
Step 3
Release and denature nucleic acids
Hybridize RNA probe with target DNA
Capture RNADNA hybrids to solid phase
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Step 4
Step 5
React captured hybrids with multiple antibody
conjugates
Detect amplified chemiluminescent signal
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The Road Ahead ? Host Gene Expression Bio
Agent Profiles
Compare Gene Expression Profiles with library of
known Bio Threat Agents/Pathogens
Determine Gene Expression pattern Exposure
extent, Susceptibility, Prognosis, Bio
agent(s)/Contaminants
Rapid Exposure Analysis and Early Treatment
Dispels Chaos, Restores Confidence Continue
Monitoring - Asymptomatic Individuals, Follow-on
Episodes

• Collect samples
• Known/Suspected Exposed persons
• Environmental

Rapid Testing Limited May/May not determine
Agent(s) Delays Defensive Measures, Treatment
Implement Protection Measures and Treatment by
Pattern Analysis Perform Database Review -
Forensic Analysis
Ref M. Jett, et. al. WRAIR
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DOD Joint Biological Agent IDentification System
(JBAIDS) Concept
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JBAIDS System Description

• Block I System Components
• JBAIDS Platform (Modified COTS)
• Real-time polymerase chain reaction (PCR)
  analyzer
• Laptop computer with software
• Storage/shipping case
• On-board spares and supplies
• Assay Kits (PCR reagents)
• Specific for 16 molecular targets
• Sample Preparation Protocols and Kits

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David Lettermans Desert Top 10How to become MAJ
Steve Mahlen

• 10. MEDIA ! / Reagents supply lines
• 9. MEDIA ! Plate Media substitutes
• 8. MEDIA ! Shelf life
• 7. MEDIA ! Heat
• 6. MEDIA ! Dust

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David Lettermans Desert Top 10How to become MAJ
Steve Mahlen

• 5. Clinical support (ID)
• 4. Physician rotation (6 month)
• 3. Maintenance (why we chose Microscan)
• 2. Technical skills
• 1. ????????.........

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David Lettermans Top 10How to become MAJ Steve
Mahlen
Jacob Mahlen

• 1. Become a
• FATHER for
• the first time !

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Colonel David W. Craft
PhD D(ABMM) Commander 9th Area Medical
Laboratory E5158 Blackhawk Road
Comm [phone] Aberdeen
Proving Grd-EA, MD 21010 DSN 584-7143
Email
David.Craft_at_us.army.mil FAX (410)
436-1019 Secure David.Craft1_at_us.army.smil.mil

Questions?