PACE 2CPACE 2CGenetic probe screening for C.

Trachomatis & N. Gonorrhoeae in Endocervical and Male urethral

specimens

Genetic probe screening for C. Trachomatis & N. Gonorrhoeae in

Endocervical and Male urethral specimens

Heidi Philips and Michael S. Joseph

OutlineOutline

Biological background on Chlamydia Trachomatis and Neisseria Gonorrhoeae

Flow chart Biology of the PACE 2C Assay Applications Assay Components Interpretation of the assay

Biological background on Chlamydia Trachomatis and Neisseria Gonorrhoeae

Flow chart Biology of the PACE 2C Assay Applications Assay Components Interpretation of the assay

Why test for this disease?Why test for this disease?

Gonorrhea in the US, nearly 392,200 cases

Public health measures requires surveillance by monitoring and screening as a preventative and allocation of public funds.

Gonorrhea in the US, nearly 392,200 cases

Public health measures requires surveillance by monitoring and screening as a preventative and allocation of public funds.

Background: Neisseria GonorrhoeaeBackground: Neisseria Gonorrhoeae Gonorrhea is a very common sexually transmitted disease

(STD). The disease is caused by the gonococcus bacteria that can

multiply and grow in moist, warm areas of the body. In women, gonorrhea can infect the cervix (opening to the

uterus, or womb), uterus, and fallopian tube. It can also infect the urethra (urine canal) in men and

women, as well as the mouth, throat, and rectum. Gonorrhea can be treated and cured by antibiotics.

Gonorrhea is a very common sexually transmitted disease (STD).

The disease is caused by the gonococcus bacteria that can multiply and grow in moist, warm areas of the body.

In women, gonorrhea can infect the cervix (opening to the uterus, or womb), uterus, and fallopian tube.

It can also infect the urethra (urine canal) in men and women, as well as the mouth, throat, and rectum.

Gonorrhea can be treated and cured by antibiotics.

Background: Chlamydia TrachomatisBackground: Chlamydia Trachomatis Chlamydia is caused by the bacterium Chlamydia

trachomatis. It persists at body sites that are inaccessible to

phagocytes, T-cells, and B-cells. Endemic trachoma leads to blindness, whereas inclusion

conjunctivitis is associated with the sexually transmitted form and does not lead to blindness.

The surface of chlamydia does not contain proteins that are distinctive enough to induce a full immune response.

The organism is also extremely temperature sensitive and must be refrigerated at 4 C as soon as a sample is obtained.

Chlamydia is caused by the bacterium Chlamydia trachomatis.

It persists at body sites that are inaccessible to phagocytes, T-cells, and B-cells.

Endemic trachoma leads to blindness, whereas inclusion conjunctivitis is associated with the sexually transmitted form and does not lead to blindness.

The surface of chlamydia does not contain proteins that are distinctive enough to induce a full immune response.

The organism is also extremely temperature sensitive and must be refrigerated at 4 C as soon as a sample is obtained.

What is PACE 2C Assay?What is PACE 2C Assay?

Probe Activated Chemiluminescent Enhancement 2C

Direct Genetic probe DNA-RNA hybridization With Chemiluminescent probe

Acridinium ester

Direct assay with qualitative results

Probe Activated Chemiluminescent Enhancement 2C

Direct Genetic probe DNA-RNA hybridization With Chemiluminescent probe

Acridinium ester

Direct assay with qualitative results

Steps in PACESteps in PACE

One swab, one tube, two test convince.

1. Sample Preparation2. Hybridization

-incubation3. Separation

-incubation-magnetic separation

4. Detection

One swab, one tube, two test convince.

1. Sample Preparation2. Hybridization

-incubation3. Separation

-incubation-magnetic separation

4. Detection

Flow ChartFlow Chart

Obtain SampleObtain Sample

Reagent PreparationReagent PreparationSample PreparationSample PreparationReconstitute PrimerReconstitute Primer

HybridizationHybridization

SeparationSeparation

DetectionDetection

- Samples must be obtained using the PACE Specimen Collection Kits.

- All reagents aside from the probe reagent must reach room temperature prior to use.

-The solid phase must be reconstituted in the selection reagent prior to use.

- Sample must reach room temperature prior to use.

-Temperature dependent

- Incubation

- Forceful wash

- Incubation

- Magnetic separation unit

- Chemiluminescent probes

- Leader Illuminometer

What makes it attractive What makes it attractive PACE 2C is a qualitative assay for both C.

Trachomatis and N. Gonorrheae. Detects rRNA of the bacteria Initial screening High specificity of DNA Probe Enhanced by targeting rRNA, which is highly

abundant w/o amplification Free from false negative, due to amplification inhibition.

Hybridization protocol with addition of Chemiluminescent label detection.

PACE 2C is a qualitative assay for both C. Trachomatis and N. Gonorrheae.

Detects rRNA of the bacteria Initial screening High specificity of DNA Probe Enhanced by targeting rRNA, which is highly

abundant w/o amplification Free from false negative, due to amplification inhibition.

Hybridization protocol with addition of Chemiluminescent label detection.

What makes it attractive cont.What makes it attractive cont. Cost-effective, do not need amplification Increased efficiency and lower costs by

testing CT and GC from single specimen

Simple workflow high tolerance for various specimen

quality

Cost-effective, do not need amplification Increased efficiency and lower costs by

testing CT and GC from single specimen

Simple workflow high tolerance for various specimen

quality

SamplesSamples

Endocervical for female and male urethral swab specimens collected with the Gen-Probe PACE Specimen Collection Kits

Endocervical for female and male urethral swab specimens collected with the Gen-Probe PACE Specimen Collection Kits

Samples cont.Samples cont.

The kit consists of single tube assay There is no transfer

sample from collection container to assay tube.

Avoiding transportation of sample But once in the

specimen collection kit,it can last for 7 days.

The kit consists of single tube assay There is no transfer

sample from collection container to assay tube.

Avoiding transportation of sample But once in the

specimen collection kit,it can last for 7 days.

Additional Products & ReagentsAdditional Products & Reagents GEN-PROBE® PACE Specimen Collection Kits GEN-PROBE® Detection Reagent Kit GEN-PROBE® Leader® Luminometer GEN-PROBE® Magnetic Separation Unit Vortex Mixer Covered water bath (60°±1° C) Micropipettes (100 µL) Pipettes capable of delivering 1-25 mL Absorbent paper

GEN-PROBE® PACE Specimen Collection Kits GEN-PROBE® Detection Reagent Kit GEN-PROBE® Leader® Luminometer GEN-PROBE® Magnetic Separation Unit Vortex Mixer Covered water bath (60°±1° C) Micropipettes (100 µL) Pipettes capable of delivering 1-25 mL Absorbent paper

Economics of the PACE 2CEconomics of the PACE 2C

Luminometer $29,550 Separation Unit $450 PACE 2A kit $1000

100 test per kit

Specimen Collection Kit $500 50 tubes per kit

Luminometer $29,550 Separation Unit $450 PACE 2A kit $1000

100 test per kit

Specimen Collection Kit $500 50 tubes per kit

Sample LimitationsSample Limitations

ONLY sampled from the endocervical area and the male urethra.

Performance with other samples has not been tested.

Only to be used with swabs transported and stored in the GEN-PROBE transport medium.

Transport and store sample at 2º-25º until use if before 7 days.

If longer storage is needed prepare sample for freezing at -20º to -70ºC.

ONLY sampled from the endocervical area and the male urethra.

Performance with other samples has not been tested.

Only to be used with swabs transported and stored in the GEN-PROBE transport medium.

Transport and store sample at 2º-25º until use if before 7 days.

If longer storage is needed prepare sample for freezing at -20º to -70ºC.

Detection SchemeDetection Scheme

Specificity rapid DNA probe test which utilizes the nucleic

acid hybridization to screen for the presence of Chlamydia trachomatis and/or Neisseria gonorrhoeae

System uses single-stranded DNA probes with chemiluminescent labels

complementary to the ribosomal RNA of the target organism.

Specificity rapid DNA probe test which utilizes the nucleic

acid hybridization to screen for the presence of Chlamydia trachomatis and/or Neisseria gonorrhoeae

System uses single-stranded DNA probes with chemiluminescent labels

complementary to the ribosomal RNA of the target organism.

Target CaptureTarget Capture Magnetic particles (1m in

diameter), with synthetic oligonucleotides/ antibody specific/ charge specific for target nucleic acid are bound to the surface

The beads are used to capture target nucleic acid

target nucleic acid is the DNA/rRNA probe

Magnetic particles (1m in diameter), with synthetic oligonucleotides/ antibody specific/ charge specific for target nucleic acid are bound to the surface

The beads are used to capture target nucleic acid

target nucleic acid is the DNA/rRNA probe

Detection Detection

It confers specificity because the relationship between the DNA/rRNA hybrid molecule and the magnetic bead

It is selective because a minimal amount of non amplified rRNA is need to have quantifiable amount.

It confers specificity because the relationship between the DNA/rRNA hybrid molecule and the magnetic bead

It is selective because a minimal amount of non amplified rRNA is need to have quantifiable amount.

Detection: specificity cont.Detection: specificity cont.

Specificity- complementary base pairing- Precise hybridization conditions- Lyophilized probeThe use of single-strand DNA probes with chemiluminescent labels that are complementary to the ribosomal RNA of the target organism allows the DNA probes to combine with the rRNA of the target organism to form stable DNA:RNA hybrids.

Specificity- complementary base pairing- Precise hybridization conditions- Lyophilized probeThe use of single-strand DNA probes with chemiluminescent labels that are complementary to the ribosomal RNA of the target organism allows the DNA probes to combine with the rRNA of the target organism to form stable DNA:RNA hybrids.

Wash/ SeparationWash/ Separation

Washing/ Separation – Labeled DNA:RNA hybrids are separated and removed from the non-hybridized probes.

Incubation with separation suspension buffer Magnetic separation with stringent/ forceful

washes

Washing/ Separation – Labeled DNA:RNA hybrids are separated and removed from the non-hybridized probes.

Incubation with separation suspension buffer Magnetic separation with stringent/ forceful

washes

VisualizationVisualization

The luminometer measures the amount of chemiluminescent emission from reactive patient samples (PACE assays)

light emitted from these reactions is detected by a photomultiplier tube

It is converted into a digital signal The final measurable signal is directly proportional to

the number of photons released by the respective

reaction.

The luminometer measures the amount of chemiluminescent emission from reactive patient samples (PACE assays)

light emitted from these reactions is detected by a photomultiplier tube

It is converted into a digital signal The final measurable signal is directly proportional to

the number of photons released by the respective

reaction.

Visualization: chemiluminescenceVisualization: chemiluminescence Acridinium ester made pre-attached to the probe.

The probe binds to the target rRNA of the

bacterium. Selection reagent is added which will hydrolyze

acridinium ester of the single stranded probe. However, the probes that have hybridized

DNA/rRNA probe, the acridinium ester is protected in the double helix of the DNA RNA probe.

Detection reagent is added and no light is emitted from the ss probes, because the acradidium ester has been hydrolyzed.

In the hybridized probe, the acradidium ester is protected and can emit light, and the light can be quantified in the luminometer as Relative Light Unites.

Acridinium ester made pre-attached to the probe.

The probe binds to the target rRNA of the bacterium.

Selection reagent is added which will hydrolyze acridinium ester of the single stranded probe.

However, the probes that have hybridized DNA/rRNA probe, the acridinium ester is protected in the double helix of the DNA RNA probe.

Detection reagent is added and no light is emitted from the ss probes, because the acradidium ester has been hydrolyzed.

In the hybridized probe, the acradidium ester is protected and can emit light, and the light can be quantified in the luminometer as Relative Light Unites.

SensitivitySensitivity

Relatively large number of organisms in a typical sample collected in swab. No amplification is need

Targeting of rRNA Background Reduction

-Separation- Forceful washing: Angle wash to front sides of tube with the appearance of foam head to ensure adequate wash procedure.

Relatively large number of organisms in a typical sample collected in swab. No amplification is need

Targeting of rRNA Background Reduction

-Separation- Forceful washing: Angle wash to front sides of tube with the appearance of foam head to ensure adequate wash procedure.

ControlsControls

The Negative Reference and Positive Controls provided, control for the PACE 2C assay ONLY.

Negative Reference control- Non-infectious nucleic acid in a buffered solution containing <5% detergent. Provides a measure of the assay background and is used to calculate the run cut-off.

Chlamydia trachomatis Positive Control- Non-infectious C. trachomatis nucleic acid in a buffered solution containing <5% detergent

Neisseria gonorrhoeae Positive Control- Non-infectious N. gonorrhoeae nucleic acid in a buffered solution containing <5% detergent

The Negative Reference and Positive Controls provided, control for the PACE 2C assay ONLY.

Negative Reference control- Non-infectious nucleic acid in a buffered solution containing <5% detergent. Provides a measure of the assay background and is used to calculate the run cut-off.

Chlamydia trachomatis Positive Control- Non-infectious C. trachomatis nucleic acid in a buffered solution containing <5% detergent

Neisseria gonorrhoeae Positive Control- Non-infectious N. gonorrhoeae nucleic acid in a buffered solution containing <5% detergent

Results calculation:Results calculation:

Result is calculated based on the difference the Response Light Units of the specimen and the mean of the negative reference.

Mean of the Negative Reference = (65 RLU + 71 RLU + 80 RLU)/3 = 72 RLU

Assigned Cut-off = 300 RLU Calculated Cut-off = 300 RLU + 72 RLU = 372

RLU Negative Specimen Response = 900 RLU Positive The limit RLU = 600 to 3200

Result is calculated based on the difference the Response Light Units of the specimen and the mean of the negative reference.

Mean of the Negative Reference = (65 RLU + 71 RLU + 80 RLU)/3 = 72 RLU

Assigned Cut-off = 300 RLU Calculated Cut-off = 300 RLU + 72 RLU = 372

RLU Negative Specimen Response = 900 RLU Positive The limit RLU = 600 to 3200

InterpretationInterpretation

A negative test does not exclude the possibility that the numbers of C. trachomatis and/or N. gonorrhoeae organisms may be below the level of detection of the assay.

Second test is required Lack of forceful washing may result in false

positive

A negative test does not exclude the possibility that the numbers of C. trachomatis and/or N. gonorrhoeae organisms may be below the level of detection of the assay.

Second test is required Lack of forceful washing may result in false

positive

Detection Cont.Detection Cont.C. trachomatis N. gonorrhoeae

Sample Positive Control Positive Control

Number of Replicates 69 69

Mean Response (RLU) 1837 2165

Standard Deviation (RLU) 329 425

Coefficient of Variance 5.6% 5.1%

C. trachomatis N. gonorrhoeae

Sample Positive Control Positive Control

Number of Replicates 69 69

Mean Response (RLU) 1837 2165

Standard Deviation (RLU) 329 425

Coefficient of Variance 5.6% 5.1%

Critical ParametersCritical Parameters

Hybridization and separation temperature

- Temperature Dependent

- Uniform equilibration of reaction tubes needed Background Reduction

-Separation- Forceful washing: Angle wash to front sides of tube with the appearance of foam head to ensure adequate wash procedure.

Hybridization and separation temperature

- Temperature Dependent

- Uniform equilibration of reaction tubes needed Background Reduction

-Separation- Forceful washing: Angle wash to front sides of tube with the appearance of foam head to ensure adequate wash procedure.

ConclusionConclusion

Results form the PACE 2C system should be used in conjunction with other data. This is a screening test only.

Detection is based on luminometer RLU unites Positive 600-3200 RLU Negative 372 or less

Results form the PACE 2C system should be used in conjunction with other data. This is a screening test only.

Detection is based on luminometer RLU unites Positive 600-3200 RLU Negative 372 or less

ReferenceReference

1. http://www.gen-probe.com/prod_serv/std_pace.asp

2. http://www.4woman.gov/faq/stdgonor.htm

3. Iwen, P.C., Walker, P.A., Warren, K.L., Kelly, D.M., Hinrichs, S.H., and Linder, J. Evaluation of Nucleic Acid-Based Test (PACE 2C) for Simultaneous Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Endocervical Specimens. 1995, Journal of Clinical Microbiology, Vol. 33, No.10; 2587-2591

1. http://www.gen-probe.com/prod_serv/std_pace.asp

2. http://www.4woman.gov/faq/stdgonor.htm

3. Iwen, P.C., Walker, P.A., Warren, K.L., Kelly, D.M., Hinrichs, S.H., and Linder, J. Evaluation of Nucleic Acid-Based Test (PACE 2C) for Simultaneous Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Endocervical Specimens. 1995, Journal of Clinical Microbiology, Vol. 33, No.10; 2587-2591