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BASIC CLINICAL MICROBIOLOGY - SUSTAINING THE FORCE FORWARD

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Title: BASIC CLINICAL MICROBIOLOGY - SUSTAINING THE FORCE FORWARD


1
BASIC CLINICAL MICROBIOLOGY - SUSTAINING THE
FORCE FORWARD
  • DAVID CRAFT, PhD D(ABMM)
  • Commander
  • 9th Area Medical Laboratory

2
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
3
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.

4
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

5
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

6
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7
Cytospin for body fluids ... CSF
8
Gram stains of CSF
  • S. pneumoniae

H. influenzae
N. meningitidis
9
Plating onto media
10
Acute pharyngitis
  • Template

11
Rapid Detection / Screening Tests
12
Antimicrobic Susceptibilty Testing (AST)
13
Specimen Collection
  • Sterile Containers
  • urine, sputum, tissue, stool, BAL, CSF
  • Blood Cultures - broth bottles
  • Swabs and appropriate transport media
  • TISSUE IS BEST!!

14
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

15
Bacterial Endocarditis
  • Splinter hemorrahges
  • Oslers Nodes
  • Vegetation on Heart Valve

16
Blood Culture Bench

17
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

18
Blood Culture Contamination
  • Growing bugs
  • Pseudobacteremia
  • Phlebotomy
  • Skin Prep (CHG/EtOH)
  • Skin Contaminants OR
  • Endocarditis

19
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!

20
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!

21
E. coli, E. coli, E. coli
22
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

23
Wounds Bench
  • No Rules
  • TISSUE
  • Skin contaminants
  • ID and Susceptibility

24
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

25
Clostridium perfringens
  • Gas gangrene
  • Myonecrosis
  • -boxcar shape
  • -double zone B
  • hemolysis

26
Respiratory
27
Respiratory Bench - RULES
  • lt 10 SECs / gt25 PMNs per lpf
  • SPIT VS
    SPUTUM

10X
10X
28
100X Oil immersion
29
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

30
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)

31
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

32
Stool Bench - 3 DAY RULE
  • Inpatient - gt3 days
  • On antibiotics - antibiotic associated colitis -
    etiology?
  • Outpatient or Inpatient lt 3 days
  • Bacteria - ???
  • Parasites - ???

33
Clostridium difficile
  • Antibiotic associated pseudomembranous colitis
  • Nosocomially acquired
  • Plaques on Colonoscopy
  • Treat w/ metranidazole or vancomycin (oral)

34
Non-fermenters (look for lack of color)
  • Template

Hektoen
MacConkey
35
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

36
Bacterial Gastroenteritis
  • Campylobacter sp.
  • Curved GN rods
  • gull wing
  • Oxidase POS
  • Most common cause of community acquired bacterial
    gastroenteritis
  • Microaerophilic, grown on selective media

37
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
38
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.
39
Case Study 1
  • A 44-year-old female with ESRD from diabetes on
    chronic HD was admitted with a 3-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

40
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.

41
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42
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?

43
Blood Culture

44
Initial Microbiological Data
  • BLOOD CULTURE POS (4/4 bottles)
  • Gram Stain

45
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?

46
100X oil immersion
10X
47
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48
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

49
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54
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

55
Smears fluorescent and acid-fast
56
Tissue preparation for fungal elements
KOH digestion
Lactophenol Cotton Blue
57
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59
Yeasts
Cryptococcus
  • Candida spp
  • Candida albicans
  • Cryptococcus spp
  • Cryptococcus neoformans
  • Torulopsis glabrata
  • Trichosporon spp
  • Geotrichum spp
  • Malassezia furfur

Candida
60
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

61
Who is He?
62
Molecular Diagnostics in the Microbiology
Laboratory
63
Polymerase Chain Reaction
64
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

65
PCR Amplifies a Targeted Sequence
Target Sequence
DNA Strand
Double Helix DNA Strand
Supercoiled DNA Strand
Chromosome
66
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).

67
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.

68
Bacillus anthracis
69
Plague
  • Yersinia pestis
  • Inguinal buboe

70
Gen-Probe TMA Transcription Mediated
Amplification
  • DTS 800
  • TIGRIS

71
Sexually Transmitted Diseases
  • Gonorrhea (GC)
  • Gram stain
  • Cervicitis
  • Dissimenated

GC or Chlamydia (Asympto.)
72
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

73
Transcription-Mediated Amplification (TMA)
TM
A Family of Three Technologies
Target Capture
Dual Kinetic Assay (DKA)
CT GC
CT
GC
74
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75
SDA Components
Target DNA
Polymerase
Primers
Restriction Enzyme
Detector Probe
SDA
76
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
77
Strand Displacement Amplification
SDA Primer
Target DNA
Restriction Enzyme Recognition Site (Bsob I)
78
Strand Displacement Amplification
Elongation of DNA strands by polymerase
79
Strand Displacement Amplification
Restriction enzyme nicks one strand
Unable to cut through both strands due to
modified dCTP
80
Strand Displacement Amplification
New strand is displaced as elongation proceeds
Polymerase begins elongation again
81
Strand Displacement Amplification
New copies can serve as another template
Nick and elongation process repeats
82
BDProbeTec ET Fluorescence Energy Transfer
  • Detection System

83
Fluorescent Energy Transfer
BsoB I recognition sequence
Hairpin secondary structure
Target specific hybridization area
Two fluorescent dyes Fluorescein Rhodamine
84
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

85
HPV DNA Testing Greater Protection Against
Cervical Disease and Improved Patient Management
86
Hybrid Capture 2 System A nucleic acid
hybridization assay with signal amplification
that utilizes chemiluminescent detection.
87
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

88
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

89
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

90
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?
91
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
92
Step 4
Step 5
React captured hybrids with multiple antibody
conjugates
Detect amplified chemiluminescent signal
93
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
94
DOD Joint Biological Agent IDentification System
(JBAIDS) Concept
95
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

96
David Lettermans Desert Top 10 How 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

97
David Lettermans Desert Top 10 How to become MAJ
Steve Mahlen
  • 5. Clinical support (ID)
  • 4. Physician rotation (6 month)
  • 3. Maintenance (why we chose Microscan)
  • 2. Technical skills
  • 1. ????????.........

98
David Lettermans Top 10 How to become MAJ Steve
Mahlen
Jacob Mahlen
  • 1. Become a
  • FATHER for
  • the first time !

99
Colonel David W. Craft
PhD D(ABMM) Commander 9th Area Medical
Laboratory E5158 Blackhawk Road
Comm (410) 436-7143 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

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