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The Affirm™ VPIII Microbial Identification Test is a DNA probe test intended for use in the detection and identification of Candida species, Gardnerella vaginalis and Trichomonas vaginalis nucleic acid in vaginal fluid specimens from patients with symptoms of vaginitis/vaginosis.

Vaginitis, one of the most common problems in clinical medicine, accounts for more than 10 million office visits each year.¹ The three main categories of vaginitis are bacterial vaginosis (BV), yeast vaginitis (candidiasis) and T. vaginalis vaginitis (trichomoniasis). BV is the most common vaginal infection, and accounts for 15 to 50% of vaginitis/vaginosis depending upon the patient population.^2,3 While G. vaginalis is no longer thought to be the only etiologic agent of BV, it is still considered to be one of the major bacteria contributing to the infection which involves an increase in anaerobic bacteria and reduction in the normal Lactobacillus flora. The complications of BV can be especially significant in pregnant women, resulting in increased risk of adverse pregnancy outcome,^4,5 including pre-term labor⁶ and birth.^7,8 In addition, recent data suggest BV-associated bacteria in the endometrium may be etiologic agents of endometritis and pelvic inflammatory disease, independent of Neisseria gonorrhoeae and Chlamydia trachomatis infection.⁹ BV is also a risk factor for the development of post-hysterectomy cuff cellulitis.¹⁰ Vaginal candidiasis is the second most common form of vaginal infection seen in varied clinical settings.³ Three quarters of all adult women will experience at least one episode of vaginal candidiasis during their lifetime, with 40 to 50% experiencing a second episode. Approximately 5% of the adult female population suffers from recurrent, often intractable yeast infection.³ Trichomoniasis, a non-reportable sexually transmitted disease, has been estimated to affect 180 million annually worldwide.¹¹ In the United States, an estimated 3 million women contract trichomoniasis each year.¹² Pregnant women positive for T. vaginalis are more likely to have pre-term rupture of membranes,⁷ as well as pre-term labor and birth.¹³ T. vaginalis is a risk factor for the development of post-surgical gynecologic infections.^14,15 In addition, T. vaginalis is a risk factor for the development of post-hysterectomy cuff cellulitis.¹⁶

Laboratory methods for the identification of these organisms include microscopic evaluation, amine test, Gram stain, pH and culture.

The Affirm VPIII Microbial Identification Test includes two internal controls on each PAC: a Positive Control bead and a Negative Control bead. These control beads are tested simultaneously with each patient specimen, ensuring the proper performance of PAC, Reagent Cassette (RC) and Processor. The Positive Control also ensures the absence of specimen interference. The Negative Control also ensures the absence of non-specific binding from the specimen.

In a properly functioning test, the Positive Control bead will be blue and the Negative Control bead remains colorless (i.e., absence of blue color) after processing. If the Positive Control does not turn blue, and/or the Negative Control does not stay colorless, the test results are invalid and patient results should not be reported.

Each reagent lot must be tested for adequate sample lysis and release of target nucleic acid using a swab streak of fresh indicator culture (18-24 h growth) or commercially prepared swab of Candida albicans (ATCC™ 18804, 14053, 10231 or 60193). Since Trichomonas vaginalis and Gardnerella vaginalis lyse more readily than Candida species, it is only necessary to test Candida species to assure adequate sample lysis. The adequacy of the sample lysis process is confirmed if testing of Candida albicans results in a blue Candida bead, a colorless Gardnerella bead, a colorless Trichomonas bead and acceptable results for the internal controls (i.e., blue Positive Control bead and colorless Negative Control bead).

To further verify test performance, quality control testing with C. albicans (ATCC 10231), T. vaginalis (ATCC 30001) and G. vaginalis (ATCC 14018) may be conducted using fresh indicator cultures (18-24 h growth) or commercially prepared swabs.

If quality control (QC) testing with all three organisms, ensure that the results for the internal controls are both acceptable (i.e., blue Positive Control bead and colorless Negative Control bead) and interpret results as follows:

1. If all three organism beads turn blue, all patient results can be reported.

2. If the Candida bead does not turn blue, the entire QC run is invalid. The QC failure must be investigated and no patient results can be reported. Contact Technical Services for assistance.

3. If the Candida and Gardnerella beads turn blue, but the Trichomonas bead does not, the QC run is valid for Candida and Gardnerella. Patient results may be reported for Candida and Gardnerella only. The QC failure must be investigated. Contact Technical Services for assistance.

4. If the Candida and Trichomonas beads turn blue, but the Gardnerella bead does not, the QC run is valid for Candida and Trichomonas. Patient results may be reported for Candida and Trichomonas only. The QC failure must be investigated. Contact Technical Services for assistance.

Quality control requirements must be performed in accordance with applicable local, state and/or federal regulations or accreditation requirements and your laboratory's standard Quality Control procedures. It is recommended that the user refer to pertinent CLSI (previously NCCLS) guidance and CLIA regulations for appropriate Quality Control practices.

Materials Provided

Probe Analysis Cards (PAC) (24 or 120 tests): Individually packaged cards, wrapped in an absorbent paper towel moistened with a solution containing sodium azide (0.1%, w/v) as a preservative. Each card contains the following five beads: Negative Control, Trichomonas, Gardnerella, Candida, and Positive Control.

Reagent Cassettes (RC) (24 or 120 tests): Reagents are sealed in multi-well, foil-covered cassettes. Each cassette has seven wells. From front to back the wells contain:

Well No. 1 Patient Sample Reservoir, supplied empty

Well No. 2 Hybridization Solution, 350 μL: Color development probe, Formamide, Buffered chaotropic solution

Well No. 3 Wash Solution, 750 μL: Detergent, Buffer solution, Preservative (Proclin™)

Well No. 4 Conjugate, 500 μL: Enzyme conjugate, Preservative (Proclin)

Well No. 5 Wash Solution, 750 μL: Detergent, Buffer solution, Preservative (Proclin)

Well No. 6 Wash Solution, 750 μL: Detergent, Buffer solution, Preservative (Proclin)

Well No. 7 Substrate Buffer, 500 μL: Buffered peroxide solution

Substrate Solution (S) (Red Cap, 3.4 mL for 24 tests; Bottle, 12 mL for 120 tests): Individually packaged solution in foil pouch; Indicator substrate, Stabilizing agent, Alcohol.

Lysis Solution (L) (Blue Cap, 10.8 mL for 24 tests; Bottle, 48 mL for 120 tests): Detergent, Buffer solution, Preservative (Proclin).

Buffer Solution (B) (Green Cap, 15 mL for 24 tests; Bottle, 72 mL for 120 tests): Buffered chaotropic solution, Formamide.

Filter Tips (FT) (24 or 120 tests)

Sample Collection Caps (SCC) (24 tests)*

Sample Collection Tubes (SCT) (24 tests)*

Individually wrapped, pre-scored, sterile swabs (24 tests)*

*Not included with 120 test kit.

Materials Required But Not Provided

Available from BD:
- Affirm VPIII Ambient Temperature Transport System
- Affirm VPIII Sample Collection Sets
- BD MicroProbe Processor
- BD MicroProbe Lysis Block
- Thermometer
- Suitable Pipette for dispensing 120 test Lysis Solution and Buffer Solution

For in vitro Diagnostic Use.

Read all instructions carefully before use.

For specimen collection, use only the Affirm VPIII Ambient Temperature Transport System, the Affirm VPIII Sample Collection Set or the swabs provided in the Affirm VPIII Microbial Identification Test Kit. Use only vaginal fluid specimens from patients with symptoms of vaginitis/vaginosis. With each test run, monitor the temperature of Lysis Block, 85 ± 5°C and verify that the testing environment temperature is between 22 and 28°C.

Substrate Solution (S): Substance contains alcohol and is combustible. Keep away from heat, sparks and flame. Keep container tightly closed to prevent evaporation.

Paper Towel surrounding PAC: Towel is moistened with sodium azide (0.1%, w/v). Sodium azide is very toxic by inhalation, in contact with skin and if swallowed. Contact with acids liberates very toxic gas. After contact with skin, wash immediately with plenty of water.

Reagents contain ingredients that could be irritating or caustic if allowed to come in contact with skin, eyes or mucous membranes. Wear gloves, safety glasses and lab coat, and use standard laboratory precautions when handling. If swallowed, call a physician. In case of skin or eye contact, flush with copious amounts of water.

Pathogenic microorganisms including hepatitis viruses and Human Immunodeficiency Virus, may be present in clinical specimens. "Standard Precautions"^17-20 and institutional guidelines should be followed in handling all items contaminated with blood and other body fluids.

Proper handling and disposal methods should be established. Wipe up spillage of patient specimens immediately and disinfect with an appropriate disinfectant. Treat the cleaning materials as biohazardous waste.

The sterile swab should not be used if the packaging is open or damaged. Avoid touching the beads. Avoid contaminating tips of dropper bottles. Do not use a reagent after its expiration date.

The swab is for single use only; reuse may cause a risk of infection and/or inaccurate results.