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STD LABORATORY METHODS

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STD LABORATORY METHODS. Disclaimer. This is not a lab course. ... Solve problems for persons with some laboratory experience. Better understanding of lab results ... – PowerPoint PPT presentation

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Title: STD LABORATORY METHODS


1
STD LABORATORY METHODS
2
Disclaimer This is not a lab course. The
participant will not be trained as a lab
technician in this course.
3
Importance of Information
  • Answer questions
  • Solve problems for persons with some laboratory
    experience
  • Better understanding of lab results
  • Limitations
  • Interpertations

4
How Good is a Test?
  • Sensitivity
  • Specificity

5
CHD Testing
  • Central Laboratory
  • 600 GC culture / mo
  • 475 GC DNA Probe / mo
  • 350 GC Amplified / mo
  • 850 Chlamydia DNA / mo
  • 350 Chlamydia Amp / mo
  • 700 VDRL / mo
  • 500 HIV / mo

Clinic Laboratory 6800 Gramstains / yr 2800 Wet
Preps / yr 200 Darkfields / yr
6
N. gonorrhea
7
Gonorrhea Methods
Sensitivity Female Male
  • Gram stain 50 95 - 98
  • Culture 80 - 90 95 - 98
  • Direct detection 80 - 90 90 - 95
  • Amplified direct 98.5 97
    or molecular technology

8
Gram Stain
  • History
  • Classic microbiology tool
  • How does it work?

9
Exudate Gram Stain
  • Specimen collection
  • Smear preparation
  • Fixation
  • Staining

10
Application to GC Diagnosis
  • Uro-genital specimens only
  • Primarily male
  • Female - purulent exudate from cervical OS or
    urethral orifice

11
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12
Photomicrograph
13
GonorrheaCriteria for Positive
  • Gram negative diplococci
  • Typical cellular morphology
  • Intracellular PMN
  • Uro-genital source

14
Gonorrhea Culture
15
Gonorrhea Inoculation
  • TM / MTM / ML plates
  • N / Z pattern
  • CO2 requirement
  • Pre incubation _at_ 35º

16
Inoculation Picture
17
Gonorrhea
  • Incubation conditions
  • Identification

18
Gonorrhea
Oxidase Positive
19
Culture Gram Stain
20
Gonorrhea
  • Incubation conditions
  • Identification
  • Confirmation

21
GC Confirmation
  • Neisseria most common

Uro - genital Throat Rectal
N. gonorrhea 98 N. meningitidis N.
meningitidis N. meningitidis N.
lactamica N. lactamica N. lactamica
B. catarrhalis N. gonorrhea
N. gonorrhea
22
GonorrheaConfirmatory Methods
  • Sugar fermentation
  • Rapid sugars
  • Co-agglutination
  • DNA probe

23
GonorrheaConfirmatory Methods
GonoGen
Gonocheck II
Remel NET
API Quad Firm
24
N.gonorrhea N.meningitidis
Rapid Sugar
25
Gonorrhea
  • GISP Antibiotic Resistance
  • 26 sites
  • Monthly isolates
  • Resistance trends

26
GonorrheaDirect Detection Assays
  • ELISA
  • DNA probe

27
GonorrheaDirect Detection
  • Specimen sources
  • Instrumentation/ automation
  • Confirmation

28
Gonorrhea Molecular Technologies
  • Signal Amplified Assays
  • Digene

29
Gonorrhea Molecular Technologies
Target Amplified Assays
  • PCR
  • TMA
  • SDA

30
Amplified Specimens
  • PCR-female swab, male swab urine
  • TMA-female urine swab, male urine swab,
    vaginal swab
  • SDA-female urine swab, male urine swab

31
Summary
  • Expensive vs. greater sensitivity
  • Amplified technology not for medical legal (yet)
  • Only one confirmatory procedure
  • Approved specimens vary by manufacturer
  • False positive potential
  • Amplification inhibition

32
Biology of Chlamydia
33
Chlamydia Life Cycle
34
ChlamydiaSensitivity by Method
Method Percent
  • Rapid tests 52 - 85 Male
  • 65 - 85 Female
  • Tissue culture 70 - 85
  • EIA (ELISA) 80 - 92
  • male urine 76
  • DNA probe 80 - 96
  • DFA 80 - 96

35
ChlamydiaEB Detection Limits
  • Molecular technology 1 - 10
  • Tissue culture 5 - 100
  • DFA 10 - 500
  • Direct probe 500 - 10,000
  • EIA 5,000 - 100,000
  • Rapid tests 100,000

36
ChlamydiaCulture
  • Methods
  • tissue culture vials
  • shell vials
  • Incubation - 3-7 days
  • Experienced personnel
    required
  • Confirmation

37
ChlamydiaTissue Culture
Shell vial
38
ChlamydiaMolecular Technologies
  • Signal Amplified Assays
  • Digene

39
ChlamydiaMolecular Technologies
Target Amplified Assays
  • PCR Plasmid DNA
  • TMA Ribosomal RNA
  • SDA Plasmid DNA

40
PCR by Roche (cont.)
  • Sensitivity
  • Female urine 86
  • Female swab 95
  • Male urine 88
  • Male swab 97

41
TMA by Gen Probe (cont.)
  • Sensitivity
  • Female urine 83
  • Female swab 99
  • Male urine 93
  • Male swab 98

42
SDA by Becton Dickinson (cont.)
  • Sensitivity
  • Female urine 80
  • Female swab 92
  • Male urine 90
  • Male swab 94

43
Amplified Specimens
  • PCR-female swab, male swab urine
  • TMA-female urine swab, male urine swab,
    vaginal swab
  • SDA-female urine swab, male urine swab

44
Summary
  • Expensive vs. greater sensitivity
  • Amplified technology not for medical legal (yet)
  • Only one confirmatory procedure
  • Approved specimens vary by manufacturer
  • False positive potential
  • Amplification inhibition

45
Future Discussion Points
  • Negative gray zone testing
  • Pooling

46
Vaginosis
47
Prevalence and Recognition
  • Incidence range 5 in general population up to
    64 in patients visiting STD Clinics
  • Only 3 of women recognize classic symptoms of
    BV
  • Nyirijesy, Paul MD Contemporary OB / GYN , Nov
    2000
  • Ferris, DG, J. Fam. Pract. 1996 42595-600

48
Testing Methods
  • POL Tests
  • Culture
  • Microscopy
  • DNA Probe

49
POL Tests
  • G. vaginalis peptidase activity slide test
  • ( also Mobiluncus spp.)
  • pH and Amine detection ( pH gt 4.7 )
  • BV Blue test for sialidase activity gt7.8 U
  • ( Gardnerella, Bacteroides, Prevotella,
    Mobiluncus )

50
Culture
  • Yeast
  • Trichomonas
  • In Pouch
  • Trichosel
  • BV Not Recommended

51
Wet Mount
52
Wet Mount
  • Specimen collection handling

53
Wet Mount
  • Organisms of interest
  • Yeast
  • Clue cells (Gardnerella etc)
  • Trichomonas

54
Wet Mount
  • Microscopy
  • Focus
  • Selecting a field
  • Lighting
  • Brightfield, Darkfield, Phase

55
Wet Mount
  • Yeast
  • Budding
  • Pseudohyphae
  • Culture

56
Wet Mount
  • Clue Cells
  • Gardnerella vaginalis
  • Stippled epithelial cells
  • whiff test using KOH
  • amine odor

57
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58
Wet Mount
  • Trichomonas
  • Motility
  • Temperature
  • pH sensitive
  • Culturable

59
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60
Wet Mount
  • Motility needed for diagnosis

61
DNA Probe
  • BD Affirm
  • All three Organisms of interest

62
Regulatory Issues
  • CLIA
  • EPA Infectious waste
  • OSHA Bloodborne pathogens

63
STDLaboratory Methods
  • For More Information
  • Cincinnati STD/HIV
  • Prevention Training Center
  • 1-800-459-2820

64
STD LABORATORY METHODS
Has been brought to you by Cincinnati STD/HIV
Prevention Training Center
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