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New Technologies for STDNew Technologies for STDLaboratory TestingLaboratory Testing
Richard Buller Ph.D.
Clinical LaboratoriesDepartment of Pediatrics
Washington University School of Medicine(No Disclosures)
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Conventional STD TestsConventional STD Tests
Culture for C. trachomatis andN.
gonorrhoeaeMethods vary from lab to lab
Specimen transport must maintain viability
EIAExist only for C. trachomatis
Laboratory tests vs. point-of-care tests Direct fluorescent antibody (DFA) stain
Exists for C. trachomatis only
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Conventional STD TestsConventional STD Tests
Gram stain forN. gonorrhoeaeSymptomatic males only
Herpes simplex virusCulture
Serology
Trichomonas vaginalisWet mount
Culture
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Nucleic Acid Amplification Tests (NAATs)Nucleic Acid Amplification Tests (NAATs)
forfor C. trachomatisC. trachomatis andandN. gonorrhoeaeN. gonorrhoeae
Offer substantial increase in sensitivity,especially for C. trachomatis, relative to
other methods Allow for detection of both agents
Allow for testing wider variety of specimens
Allow for relaxed specimen transport
conditions
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GenGen--Probe Aptima Combo 2Probe Aptima Combo 2
Introduced 2001
Detects rRNA from C. trachomatis
and/orN. gonorrhoeae
Specimens
Male: urethral swabs, urine
Female: endocervical swabs, urine,
self-collected vaginal swabs
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GenGen--Probe Aptima Combo 2Probe Aptima Combo 2
Utilizes target capturePrior to amplification, target rRNA
molecules are specifically isolated from
specimen by a hybridization step
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Prevalence of Inhibitory UrinesPrevalence of Inhibitory Urines
Assay % Total InhibitionPCR 4.9
LCR 2.6
TMA 7.5
Mahony et al. 1998 n=101 pregnant, 287 non-pregnant
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Prevalence of Inhibitory UrinesPrevalence of Inhibitory Urines
Assay % Total InhibitionAptima Combo 2 0.48
LCR 13
Chong et al. 2003 n=190 pregnant, 225 non-pregnant
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CDC NAAT ScreeningCDC NAAT Screening
RecommendationsRecommendations
All positive results should be consideredpresumptive evidence of infection.
False positive result can have adverse medical,social, and psychological impacts on patients.
An additional test should be considered after apositive screening result
Patients should be counseled about prompttreatment after a positive screening resultbecause an additional test might be falselynegative
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Confirming Positive Results ofConfirming Positive Results of
NAATs forNAATs for C. trachomatisC. trachomatis: All: AllNAATs are not Created EqualNAATs are not Created Equal
CDC recommends confirming positivescreening results when PPV are
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Vaginal Swabs Detect More ChlamydialVaginal Swabs Detect More Chlamydial
Infections Than Do First Catch UrineInfections Than Do First Catch UrineSpecimensSpecimens
Collected FCU, 2 Cx swabs, and patientcollected vaginal (PCV) swab from 1464
women attending STD, family planningand OB/Gyn clinics
FCU and 1 Cx swab tested by AC2 and
FCU and 2nd Cx swab by BDProbe Tec
PCV tested by AC2
Schachter and Chernesky APHL Poster 2005
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Vaginal Swabs.....(Cont.)Vaginal Swabs.....(Cont.)
AC2 more sensitive than BD
Detected 20% more positives with Cx swab
Detected 19% more positive with FCU
Testing of PCV with AC2 identified as manypositive women as did testing of Cx swabs and
more positives than did FCU
VS should be considered specimen of choice for
screening of women for chlamydia
Schachter and Chernesky APHL Poster 2005
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Swabs
Chernesky et al. JCM 2006Chernesky et al. JCM 2006
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Use of Flocked Swabs and a Universal TransportUse of Flocked Swabs and a Universal Transport
Medium to Enhance Molecular Detection ofMedium to Enhance Molecular Detection ofC.C.trachomatistrachomatis andandN. gonorrhoeaeN. gonorrhoeae
Prepared mock specimens in CopanUniversal Transport Medium (UTM)
Placed kit swab (KS) or Copan flocked
swab (FS) into UTM and then tested by 3assays
Found FS enhanced the analyticalsensitivity of each assay for both C.
trachomatis andN. gonorrhoeae
Chernesky et al. JCM 2006
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Methods to Reduce Costs ofMethods to Reduce Costs of
NAATsNAATs Pooling Specimens
Pool urine specimens
If pool is negative, all specimens reported as
negative If pool is positive, go back and test individual
specimens to determine which is (are) positive
Use of the leukocyte esterase test (LET)to select for NAAT testing for C.
trachomatis andN. gonorrhoeae
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RealReal--TimeTime PCRPCR
PCR product is detected during
amplification
Rapid Cycling Times
Quantitation capability
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LightCycler Reaction Capillaries
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LightCycler PCR Instrument
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LightCycler Hybridization Probes
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LightCycler Negative Results
Li h C l P i i R l
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LightCycler Positive Results
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Conventional Versus RealConventional Versus Real--TimeTime
PCRPCR
Conventional LightCycler
Extraction 2 Hours 2 Hours
PCR 2 Hours 1 Hour
Detection 1.5 Hours -
Total 5.5 Hours 3 Hours
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LightCycler Detection of HerpesLightCycler Detection of Herpes
Simplex Virus DNA in Skin LesionsSimplex Virus DNA in Skin Lesions Prospectively tested 103 skin, oral and
genital specimens submitted to the SLCHvirology laboratory to rule out herpes
simplex virus (HSV).
For each specimen, the laboratorys
standard herpes culture was performed.
In addition, DNA was extracted from each
specimen and tested for the presence of
HSV DNA using a LightCycler assay.
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LightCycler Detection of HerpesLightCycler Detection of Herpes
Simplex Virus DNA in SkinSimplex Virus DNA in Skin
LesionsLesionsLightCycler
Positive Negative
Positive 28 0
Culture
Negative 16 59
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SLCH LightCycler HSV PCRSLCH LightCycler HSV PCR
ExperienceExperience
176 oral or genital specimens tested by
LightCycler HSV PCR
76 (43%) HSV DNA Positive35 (46%) Type I
20 (57%) oral; 15 (43%) genital26 (34%) Type II
23 (88%) genital; 3 (12%) oral
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HSV TypeHSV Type--specific Serologic Testsspecific Serologic Tests
Culture/PCR tests for active viral
infection have been available for severalyearsculture/PCR performed on clinical lesions
culture cannot be performed if no lesionspresent
1999: FDA-approval of type-specificserologic tests for HSVallows detection of antibody to HSV-1 and
HSV-2 to document prior infection
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TypeType--specific HSV Serologic Testsspecific HSV Serologic Tests
Reference lab tests Focus enzyme immunoassay (HSV-1, HSV-2)
Focus immunoblot (HSV-1, HSV-2)
Point of care test Diagnology POC-kit rapid tests (HSV-2 only)
Tests provide information on presence or
absence of HSV antibody, but are notdiagnostic of clinical ulcer etiology
Allow clinicians to counsel seropositive patients
about transmission risk
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Clinical Utility of HSV SerologyClinical Utility of HSV Serology
Serodiscordant couplesone person +
one person -
Pregnant women, esp. seronegative
counsel against acquiring HSV during
pregnancy
counsel about transmissionrisk}
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HerpSelect Immunoblot
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Trichomonas vaginalisTrichomonas vaginalis
Wet mount examination picks up onlytwo-thirds of all infections
Culture with Diamonds medium is more
sensitive, but also more expensive andcumbersome in clinical settings
Bedside test for trichomonas culture isnow available
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Trichomonas vaginalisTrichomonas vaginalis
In-Pouch test kit
small pouch with modified Diamonds
medium
bedside inoculation of pouchincubation at 37o C
read daily for 5 days - look for motiletrichomonads
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InPouch T. vaginalis Culture System
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Trichomonas vaginalisTrichomonas vaginalis
In-Pouch increases yield by 30-50% infemales
May be performed on males
urethral swab specimenscentrifuged first-catch urine samples
Inexpensive: $2-3 per test kit (butrequires substantial staff time inincubating and reading specimens)
Viability ofViability of T. VaginalisT. Vaginalis in Urine:in Urine:
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Viability ofViability ofT. VaginalisT. Vaginalis in Urine:in Urine:
Epidemiological and ClinicalEpidemiological and ClinicalImplicationsImplications Studied viability ofT. vaginalis in urine
specimens
Found T. vaginalis rapidly lost viability in
urine, especially at room temp relative to37C
Recommend storing urine at 37 andinoculating InPouch within 30 min
Shafir and Sorvillo JCM 10/2006
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Trichomonas VaginalisTrichomonas Vaginalis
Xenostrip-Tv
Qualitative immunochromatographic assay
Company claims:
Sensitivity compared to culture of 99-100%
Specificity compared to culture of >98%
Results ready in 10 minutes
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XenostripXenostrip--TvTv
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Performance of a New Rapid AssayPerformance of a New Rapid Assay
for Detection offor Detection ofTrichomonas vaginalisTrichomonas vaginalis
Tested 936 women attending STD Clinic
Using InPouch culture system as gold
standard, compared Xenostrip and wet
mount for detection ofT. vaginalis Overall prevalence was 14.4%
Kurth et al. JCM 2004
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ResultsResults
Sensitivity Specificity
Xenostrip tv 78.5% 98.6%
Wet Mount 72.4% 100%
Kurth et al. JCM 2004
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ConclusionsConclusions Xenostrip performed as well or better
than wet mount
Sensitivity did not differ between
symptomatic and asymptomatic women Xenostrip is rapid does not require
specialized training or equipment
Kurth et al. JCM 2004
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SummarySummary New STD testing methods continue to
appear on the commercial marketplace Clinical utility of newer tests depends
on independent validation in real-worldclinical settings
Continuing research is required to fully
clarify the role of emerging STDproducts
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SummarySummary NAATs offer significant increases in
sensitivity with acceptable specificity
Some NAATs appear to be significantly
more sensitive than others Patient-collected vaginal swabs may be
the specimen of choice for screeningwomen for chlamydial infections usingthe Gen-Probe Aptima Combo 2 assay
SummarySummary
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SummarySummary
NAATs tests are not perfect and physiciansshould be aware of test limitationsFalse positives
False negatives
Specimen requirements
Effect on test performance of prevalence Serological tests can now accurately
discriminate between prior infection with
Herpes simples virus types 1 and 2 Convenient inexpensive new tests exist for
Trichomonas vaginalis