STD LABORATORY METHODS

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STD LABORATORY METHODS
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Disclaimer This is not a lab course. The
participant will not be trained as a lab
technician in this course.
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Importance of Information

• Answer questions
• Solve problems for persons with some laboratory
  experience
• Better understanding of lab results
• Limitations
• Interpertations

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How Good is a Test?

• Sensitivity
• Specificity

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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
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N. gonorrhea
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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

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Gram Stain

• History
• Classic microbiology tool
• How does it work?

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Exudate Gram Stain

• Specimen collection
• Smear preparation
• Fixation
• Staining

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Application to GC Diagnosis

• Uro-genital specimens only
• Primarily male
• Female - purulent exudate from cervical OS or
  urethral orifice

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Photomicrograph
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GonorrheaCriteria for Positive

• Gram negative diplococci
• Typical cellular morphology
• Intracellular PMN
• Uro-genital source

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Gonorrhea Culture
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Gonorrhea Inoculation

• TM / MTM / ML plates
• N / Z pattern
• CO2 requirement
• Pre incubation _at_ 35º

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Inoculation Picture
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Gonorrhea

• Incubation conditions
• Identification

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Gonorrhea
Oxidase Positive
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Culture Gram Stain
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Gonorrhea

• Incubation conditions
• Identification
• Confirmation

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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
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GonorrheaConfirmatory Methods

• Sugar fermentation
• Rapid sugars
• Co-agglutination
• DNA probe

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GonorrheaConfirmatory Methods
GonoGen
Gonocheck II
Remel NET
API Quad Firm
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N.gonorrhea N.meningitidis
Rapid Sugar
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Gonorrhea

• GISP Antibiotic Resistance
• 26 sites
• Monthly isolates
• Resistance trends

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GonorrheaDirect Detection Assays

• ELISA
• DNA probe

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GonorrheaDirect Detection

• Specimen sources
• Instrumentation/ automation
• Confirmation

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Gonorrhea Molecular Technologies

• Signal Amplified Assays
• Digene

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Gonorrhea Molecular Technologies
Target Amplified Assays

• PCR
• TMA
• SDA

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Amplified Specimens

• PCR-female swab, male swab urine
• TMA-female urine swab, male urine swab,
  vaginal swab
• SDA-female urine swab, male urine swab

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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

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Biology of Chlamydia
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Chlamydia Life Cycle
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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

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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

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ChlamydiaCulture

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

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ChlamydiaTissue Culture
Shell vial
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ChlamydiaMolecular Technologies

• Signal Amplified Assays

• Digene

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ChlamydiaMolecular Technologies
Target Amplified Assays

• PCR Plasmid DNA
• TMA Ribosomal RNA
• SDA Plasmid DNA

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PCR by Roche (cont.)

• Sensitivity
• Female urine 86
• Female swab 95
• Male urine 88
• Male swab 97

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TMA by Gen Probe (cont.)

• Sensitivity
• Female urine 83
• Female swab 99
• Male urine 93
• Male swab 98

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SDA by Becton Dickinson (cont.)

• Sensitivity
• Female urine 80
• Female swab 92
• Male urine 90
• Male swab 94

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Amplified Specimens

• PCR-female swab, male swab urine
• TMA-female urine swab, male urine swab,
  vaginal swab
• SDA-female urine swab, male urine swab

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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

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Future Discussion Points

• Negative gray zone testing
• Pooling

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Vaginosis
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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

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Testing Methods

• POL Tests
• Culture
• Microscopy
• DNA Probe

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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 )

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Culture

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

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Wet Mount
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Wet Mount

• Specimen collection handling

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Wet Mount

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

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Wet Mount

• Microscopy
• Focus
• Selecting a field
• Lighting
• Brightfield, Darkfield, Phase

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Wet Mount

• Yeast
• Budding
• Pseudohyphae
• Culture

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Wet Mount

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

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Wet Mount

• Trichomonas
• Motility
• Temperature
• pH sensitive
• Culturable

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Wet Mount

• Motility needed for diagnosis

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DNA Probe

• BD Affirm
• All three Organisms of interest

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Regulatory Issues

• CLIA
• EPA Infectious waste
• OSHA Bloodborne pathogens

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STDLaboratory Methods

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

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STD LABORATORY METHODS
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