New Technologies for STD Laboratory Testing

Richard Buller Ph.D. Clinical Laboratories Department of Pediatrics Washington University School of Medicine
(No Disclosures)

Conventional STD Tests

Culture for C. trachomatis and N. gonorrhoeae
Methods vary from lab to lab Specimen transport must maintain viability

EIA
Exist only for C. trachomatis Laboratory tests vs. point-of-care tests

Direct fluorescent antibody (DFA) stain

Exists for C. trachomatis only

Conventional STD Tests

Gram stain for N. gonorrhoeae
Symptomatic males only

Herpes simplex virus

Culture Serology

Trichomonas vaginalis
Wet mount Culture

Nucleic Acid Amplification Tests (NAATs) for C. trachomatis and N. 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

Gen-Probe Aptima Combo 2

Introduced Detects Specimens Male: Female: 2001 rRNA from C. trachomatis and/or N. gonorrhoeae urethral swabs, urine endocervical swabs, urine, self-collected vaginal swabs

Gen-Probe Aptima Combo 2

Utilizes target capture
Prior to amplification, target rRNA molecules are specifically isolated from specimen by a hybridization step

Prevalence of Inhibitory Urines

Assay PCR LCR TMA
Mahony et al. 1998

% Total Inhibition 4.9 2.6 7.5

n=101 pregnant, 287 non-pregnant

Prevalence of Inhibitory Urines

Assay Aptima Combo 2 LCR % Total Inhibition 0.48 13

Chong et al. 2003

n=190 pregnant, 225 non-pregnant

CDC NAAT Screening Recommendations

All positive results should be considered presumptive evidence of infection. False positive result can have adverse medical, social, and psychological impacts on patients. An additional test should be considered after a positive screening result Patients should be counseled about prompt treatment after a positive screening result because an additional test might be falsely negative

Confirming Positive Results of NAATs for C. trachomatis: All NAATs are not Created Equal
CDC recommends confirming positive screening results when PPV are <90% Tested a variety of specimens from ~2700 men and women using BD and AC2 assays AC2 significantly more sensitive than BD Using BD to confirm AC2 would result in incorrectly reporting 15% of confirmable positive results as negative
Schachter et al. JCM 2005

Vaginal Swabs Detect More Chlamydial Infections Than Do First Catch Urine Specimens Collected FCU, 2 Cx swabs, and patient collected vaginal (PCV) swab from 1464 women attending STD, family planning and 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

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 many positive 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

Swabs

Chernesky et al. JCM 2006 Chernesky et al. JCM

2006

Use of Flocked Swabs and a Universal Transport Medium to Enhance Molecular Detection of C. trachomatis and N. gonorrhoeae

Prepared mock specimens in Copan Universal Transport Medium (UTM) Placed kit swab (KS) or Copan flocked swab (FS) into UTM and then tested by 3 assays Found FS enhanced the analytical sensitivity of each assay for both C. trachomatis and N. gonorrhoeae
Chernesky et al. JCM 2006

Methods to Reduce Costs of NAATs

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 and N. gonorrhoeae

Real-Time PCR
PCR product is detected during amplification Rapid Cycling Times Quantitation capability

LightCycler Reaction Capillaries

LightCycler PCR Instrument

LightCycler Hybridization Probes

LightCycler Negative Results

LightCycler Positive Results

Conventional Versus Real-Time PCR

Conventional Extraction PCR Detection Total 2 Hours 2 Hours 1.5 Hours 5.5 Hours LightCycler 2 Hours 1 Hour 3 Hours

LightCycler Detection of Herpes Simplex Virus DNA in Skin Lesions

Prospectively tested 103 skin, oral and genital specimens submitted to the SLCH virology 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.

LightCycler Detection of Herpes Simplex Virus DNA in Skin Lesions

LightCycler Positive Positive Culture Negative 16 59 28 Negative 0

SLCH LightCycler HSV PCR Experience

176 oral or genital specimens tested by LightCycler HSV PCR 76 (43%) HSV DNA Positive
35 (46%) Type I
20 (57%) oral; 15 (43%) genital

26 (34%) Type II
23 (88%) genital; 3 (12%) oral

HSV Type-specific Serologic Tests

Culture/PCR tests for active viral infection have been available for several years
culture/PCR performed on clinical lesions culture cannot be performed if no lesions present

1999: FDA-approval of type-specific serologic tests for HSV

allows detection of antibody to HSV-1 and HSV-2 to document prior infection

Type-specific 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 not diagnostic of clinical ulcer etiology Allow clinicians to counsel seropositive patients about transmission risk

Clinical Utility of HSV Serology

Serodiscordant couples
one person + one person -

counsel about transmission risk

Pregnant women, esp. seronegative

counsel against acquiring HSV during pregnancy

HerpSelect Immunoblot

Trichomonas vaginalis
Wet mount examination picks up only two-thirds of all infections Culture with Diamonds medium is more sensitive, but also more expensive and cumbersome in clinical settings Bedside test for trichomonas culture is now available

Trichomonas vaginalis
In-Pouch test kit
small pouch with modified Diamonds medium bedside inoculation of pouch incubation at 37o C read daily for 5 days - look for motile trichomonads

InPouch T. vaginalis Culture System

Trichomonas vaginalis
In-Pouch increases yield by 30-50% in females May be performed on males
urethral swab specimens centrifuged first-catch urine samples

Inexpensive: $2-3 per test kit (but requires substantial staff time in incubating and reading specimens)

Viability of T. Vaginalis in Urine: Epidemiological and Clinical Implications

Studied viability of T. vaginalis in urine specimens Found T. vaginalis rapidly lost viability in urine, especially at room temp relative to 37C Recommend storing urine at 37 and inoculating InPouch within 30 min
Shafir and Sorvillo JCM 10/2006

Trichomonas 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

Xenostrip-Tv

Performance of a New Rapid Assay for Detection of Trichomonas vaginalis

Tested 936 women attending STD Clinic
Using InPouch culture system as gold standard, compared Xenostrip and wet mount for detection of T. vaginalis

Overall prevalence was 14.4%

Kurth et al. JCM 2004

Results
Sensitivity Specificity Xenostrip tv Wet Mount 78.5% 72.4% 98.6% 100%

Kurth et al. JCM 2004

Conclusions
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

Summary
New STD testing methods continue to appear on the commercial marketplace Clinical utility of newer tests depends on independent validation in real-world clinical settings Continuing research is required to fully clarify the role of emerging STD products

Summary
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 screening women for chlamydial infections using the Gen-Probe Aptima Combo 2 assay

Summary
NAATs tests are not perfect and physicians should be aware of test limitations
False 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