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7/29/2019 New+STD+Technologies+01 07

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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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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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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%



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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



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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

new+std+technologies+01 07

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