Current Methods for Syphilis Herpes and HIV Testing

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Current Methods for Syphilis Herpes and HIV
Testing

• Jim Reynolds BS
• Health Laboratory Director
• Cincinnati Health Dept
• June 10, 2009

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Syphilis

• 40,920 Cases reported in US 2007 ( all
  stages )
• Increase of 10.7 over 2006
• Peak Post WWII 1991 gt 120,000

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A multiple Technology Survey

• Microscopy
• Serology
• Hemagglutination
• Enzyme Immunoassay
• PCR
• Western Blot

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vs. Non Treponemal
Treponemal
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Non Treponemal Tests

• VDRL 1940s
• RPR Late 1950s
• EIA 1987
• Erythrocyte Adherence 1997

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Non Treponemal

• Target - Reagin antibodies
• Require a human immune response
• Non specific
• Most are quantitative / semi quant.

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False Positives

• Other Treponemal diseases
• Pinta / Yaws / Bejel
• Tissue damage diseases acute / chronic
• Autoimmune diseases arthritis, lupus
• Febrile diseases measles, chicken pox, malaria,
  lyme, other viral infections
• IV Drug users

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VDRL

• Microscopic flocculation test
• For large numbers of samples in batch
• Antigen must be prepared daily
• Serum requires heat inactivation
• Paraffin ring slides
• Only CSF method

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VDRL
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RPR

• Macroscopic flocculation
• Charcoal trapped in Ag / Ab lattice
• For Stat Labs performing single tests
• Rapid 8 Min.
• On card titer

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RPR
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Titer

• Determines concentration of antibodies
• Serial dilution in tubes or on slide
• Each dilution dil is ½ the previous resulting
  in titers of 2, 4, 8, 16, 32 etc.
• Read to the highest dil still reactive

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Prozone

• False Negative Serology
• Antibody excess blocks antigen-antibody reaction
• Dilution of sample resolves 16X
• Undiluted may appear rough
• Can be caused by improper technique

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Other Tests

• EIA
• Erythrocyte Adherence

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Treponemal Tests

• Microscopy
• Hemagglutination
• Enzyme Immuno Assay
• PCR

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Microscopy

• Darkfield 1923
• DFA 1964
• FTA-ABS 1957

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Darkfield

• Direct Lesion specimens
• Specialized microscope
• Skill
• Rapid

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Darkfield pitfalls

• Productive Lesion
• Sensitivity ??
• Syphilis / Pinta / Yaws
• Not for Oral Lesions

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Direct Fluorescence DFA

• Direct Lesion Specimen
• Increased Specificity vs. Darkfield
• Higher skill level

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DFA pitfalls

• Fluorescent Scope
• Tedious staining
• Sensitivity ???
• Syphilis / Pinta / Yaws

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Fluorescent Treponemal Antibody Absorption

• FTA-ABS
• Serum or CSF
• Antibodies bind to Treponemal layer
• Titered by serum dilution
• Fluorescent Microscope w/ Darkfield

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FTA pitfalls

• Lifelong positive
• Cant distinguish current from past
• Syphilis / Pinta / Yaws
• Tedious stain / Scope work
• False positives elderly, 1 population

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Particle AgglutinationTP-PA

• Micro Well Format
• Antigen bound to Gelatin
• Sensitivity FTA gt TPHA
• Procedure 4 hrs ( or overnight )
• Replacing FTA and TPHA for Confirmation

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MicrohemagglutinationMHA-TP

• New Manufacturer TPHA
• Micro well plate format
• Sheep erythrocytes
• Visually read
• Two fold titer dilution
• Sensitivity 69-90 Specificity 98

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Treponemal EIA

• 9 or 10 available
• Visuwell Syphilis
• Captia Syphilis G
• Captia Syphilis M
• Spirotek Syphilis
• Trep Chek

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Future / Other Treponemal

• Erythrocyte adherence
• Western Blot
• PCR
• Rapid test cards

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Summary

• Treponemal vs non treponemal
• Visually read
• Instrument read
• Microscopic
• Test pitfalls

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Herpes
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Herpes

• Herpes simplex HSV 1 HSV 2
• Lifelong Infection
• 50 Million Genital Infections in US
• 1 Million New Infections per Year

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Type Prevalence

• 60 of US Population HSV 1 Pos
• 50 of these are genital
• 20 of US Population HSV 2 Pos
• Recurrence Rate tied to Site of Infection

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Technologies

• Antigen Staining
• Tissue Culture
• Serology
• Nucleic Acid Amplified Testing

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Antigen Staining

• Inexpensive
• Sensitivity lt 50
• Not Type Specific
• Tzank, PAP, IPA

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Tissue Culture

• High Sensitivity if Fresh Lesion 100
• Propagation is Amplification
• Expensive Tedious
• Gold Standard

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Tissue Culture
Shell vial
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Culture Methods

• Wide Choice of Cell Lines
• Variety of Vessels
• Confirm with FA or Cytostain
• Viral Transport media M4 and others

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Serology

• Rapid Tests
• Sure View / Herpes Select / etc.
• EIA
• EIA IGM
• Western Blot

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EIA

• Sensitivity good 80
• Automation, less Difficult
• Type Specific / Non-type specific
• Glycoprotein G1 / G2
• Window Period for Ab Detection

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EIA IgM

• Not a good Predictor of Primary Infection
• Positive with Recurrent Episodes
• Cross Reactions between HSV1 HSV2
• Not for Asymptomatic Screening
• May be useful in Late Term Pregnancy

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Nucleic Acid Amplified Test

• RT PCR Technology
• Very Sensitive and Specific
• Expensive and for Low Volume
• Future Will replace Culture when cost comes down

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Discussion

• High Prevalence in Population
• Asymptomatic Screening not Recommended
• Type Specific EIA
• NAAT in the Future

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HIV
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Human Immunodeficiency Virus

• 1 Million Estimated US Infections
• 25 Undiagnosed

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Test Methods

• Screening
• Confirmatory
• Treatment Monitoring

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Screening

• Test Choice based on
• Earliest Detection
• Cost
• Patient Volume
• Laboratory Capability and Equipment

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EIA, Lateral Flow Immunodiffusion

• 1st , 2nd , 3rd Generation Assays
• 1st 2nd Detect IgG Antibody
• 3rd Detects IgG IgM Antibodies
• 4th Generation Detects HIV Antigen p24
• ( Future )

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Lateral Flow Rapid Tests

• 6 FDA Approved tests in US
• Ora Quick, Reveal, Multi Spot, Clearview (2) and
  Uni Gold
• Most Detect HIV 1 HIV 2
• Uni Gold is only 3rd Generation Test
• Detect at 3-4 Weeks post Infection
• Owen et al., J. Clin. Micro May 2008
• Confirm with Western Blot / Repeat Reactives

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EIA

• Several Available
• Automated/Semi Automated Platforms
• Detect _at_ 3-6 Weeks
• Confirm w/ Western Blot / Repeat Reactives

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HIV RNA Pooling

• Earliest Detection Mode
• Pooling cuts RT-PCR Cost
• Not for High Volume

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Chemiluminescent Assays

• Ortho Vitros / Advia Centaur
• Large Platform Instruments
• Reference Laboratories Blood Banks

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Confirmation

• Western Blot
• Capsid, Envelope, Polymerase
• Criteria Vary
• Less Sensitive than EIA or Rapids

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Confirmation

• HIV 1 RNA
• Qualitative FDA Approved
• Expensive
• Will Resolve Antibody / Wb Neg.
• No Approved HIV 2 RNA
• Alternate Rapid Test

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Treatment / Disease Monitoring

• p24 Capsid Protein
• CD4 Flow Cytometry
• Viral Load - Quantitative HIV 1 RNA
• Gene Sequencing Codons for Resistance to Anti
  Retroviral Agents

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Discussion

• Screening Detection 10 days-7 weeks
• Rapid Tests while Patient Waits
• Wb Confirmation Sens. lt Screening Tests
• Greater Sens. ( HIV-RNA, p24 ) gt Cost
• Future 4th Generation tests ( RNA p24 )

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

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• Select Lecture title and follow instructions
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