BD Viper™ System with XTR™ Technology
CLSI Laboratory Procedure*
The BD ProbeTec™Herpes Simplex Viruses (HSV 1 & 2) Qx Amplified DNA Assays, when tested with the BD Viper™ System in Extracted Mode, use Strand Displacement Amplification technology for the direct, qualitative detection and differentiation ofHerpes Simplex virus type 1 (HSV1) and Herpes Simplex virus type 2 (HSV2) DNA in clinician-collected external anogenital lesion specimens. The assaysare indicated for use with symptomatic individuals to aid in the diagnosis of anogenital HSV1 and HSV2 infections.
II.SUMMARY AND EXPLANATION
Herpes Simplex virus type 1 (HSV1) and type 2 (HSV2) are ubiquitous double-stranded neurotropic DNA viruses of the Herpesviridae family that cause incurable lifelong infections and which result from inoculation of the virus through abraded skin or mucous membranes. Both viruses may remain latent for long periods and cause recurrent episodes of symptomatic disease.
The primary modes of transmission for HSV1 are via oral secretions and non-genital contact resulting in a predominance of infections of the oropharynx, face, eyes and central nervous system. In recent years the frequency of genital HSV1 infections has increased due to a lower rate of oral infection during prepubescent childhood that renders individuals susceptible to genital infection in later life and a rise in frequency of oro-genital contact.1,2Nevertheless, seroprevalence studies show that up to 80% of children infected with HSV1 by adulthood, with the highest rates among those in poor socioeconomic groups.3
In contrast with HSV1, infection with HSV2 is usually the result of sexual transmission. In the United States, 20-25% of the population has antibodies to HSV2 by the age of 40 and overall there are at least 50 million people with genital herpes.3 In the majority of cases, symptoms are mild or unrecognized and the infection remains undiagnosed, although intermittent shedding of infectious virus into the genital tract still occurs. As a result, most transmission of genital herpes occurs through sexual contact by persons who are asymptomatic or are unaware that they are infected. Infection with HSV2 is more common in women than men and is linked to an increased risk of sexually transmitted Human Immunodeficiency Virus (HIV).4While transmission of herpes to neonates in utero or intrapartum is rare; the consequences of such infections are severe and frequently fatal.
Classical primary genital herpes infection is preceded by localized pain or tingling, frequently accompanied by fever, malaise and inquinal lymphadenopathy. Within days, vesicles appear on the labia minora, introitus and urethra meatus of women and on the shaft and glans of the penis in men. The perineum and perianal areas may also be affected, as may the upper thighs and buttocks. Cervical lesions also occur frequently in women.
* This “Sample Procedure” is not indicated as a substitute for your facility procedure manual, instrument manual, or reagent labeling/package insert. This “Sample Procedure” is intended as a model for use by your facility to be customized to meet the needs of your laboratory.
For use with Package Insert: BD ProbeTec Herpes Simplex Viruses (HSV 1 & 2) Qx Amplified DNA Assays [8086121(01) 2012-10]
Primary infection with HSV1 cannot be distinguished from that caused by HSV2 on clinical grounds and the lesions may also be confused with those caused by other sexually transmitted diseases. As a consequence, laboratory testing is required for definitive diagnosis to reduce symptoms and hasten the healing of lesions. In addition, because the recurrence of HSV1 infections and subclinical shedding are less frequent than for HSV2, determination of the etiology of infection and typing of the virus is useful in the assessment of prognosis and counseling.1,2,6 The preferred method of diagnosis of herpes infection has historically been viral isolation in tissue culture followed by type-specific immunofluorescent detection; however, the enhanced sensitivity, robustness and rapid time-to-results of amplified methods for the detection of viral DNA are leading to their increasingly widespread adoption.1,3,6
When used with the BD Viper System in extracted mode, the BD ProbeTec HSVQxAmplified DNA Assays involve automated non-specific extraction of DNA from clinical specimens using chemical lysis of cells, followed by binding of DNA to magnetic particles, washing of the bound nucleic acid and elution in an amplification-compatible buffer. When present, HSV1 and/or HSV2 is detected by Strand Displacement Amplification (SDA) of type-specific target sequences in the presence of a fluorescently-labeled detector probe.7,8
III.PRINCIPLES OF PROCEDURE
The BD ProbeTecHSVQx Amplified DNA Assays are designed for use with the BDQxSwab Diluent and BD Universal Viral Transport (UVT) [or identical Copan manufactured media formulations-see COLLECTION KITS PROVIDED SEPARATELY section] specimen collection and transport devices, applicable reagents, theBD Viper System and BD Fox Extraction. Swab specimens are collected and transported in the prescribed transport devices which preserve the integrity of HSV DNA over the specified ranges of temperature and time.
To maintain a consistent workflow with other specimen types that are processed on the BD Viper System, the HSV specimens undergo a pre-warm step in the BD Viper Lysing Heater. After cooling, the specimens are loaded onto the BD Viper System which then performs all the steps involved in extraction and amplification of the target DNA, without further user intervention. The specimen is transferred to an Extraction Tube that contains ferric oxide particles in a dissolvable film and dried Extraction Control. A high pH is used to lyse the viruses to liberate their DNA into solution. Acid is then added to lower the pH and induce a positive charge on the ferric oxide, which in turn binds the negatively charged DNA. The particles and bound DNA are then pulled to the sides of the Extraction Tube by magnets and the treated specimen is aspirated to waste. The particles are washed and a high pH Elution Buffer is added to recover the purified DNA. Finally, a Neutralization Buffer is used to bring the pH of the extracted solution to the optimum for amplification of the target.
The BD ProbeTec HSV QxAmplified DNA Assays are based on the simultaneous amplification and detection of target DNA using amplification primers and a fluorescently labeled detector probe.7,8 The reagents for SDA are dried down in four separate disposable microwells: the Priming Microwells contain the assay specific amplification primers, fluorescently labeled detector probe, nucleotides and other reagents necessary for amplification, while the assay specific Amplification Microwells each contain the two enzymes (a DNA polymerase and a restriction endonuclease) that are required for SDA. The BD Viper System pipettes portions of the purified DNA solution from each Extraction Tube into two separate Priming Microwells to rehydrate their contents, one well corresponding to HSV1 and the other to HSV2. After a brief incubation, the reaction mixtures are transferred to corresponding, pre-warmed Amplification Microwells which are sealed to prevent contamination and then incubated in one of the two thermally controlled fluorescent readers. The presence or absence of HSV DNA is determined by calculating the peak fluorescence (Maximum Relative Fluorescence Units [MaxRFU]) over the course of the amplification process and by comparing this measurement to a predetermined threshold value.
In addition to the fluorescent probe used to detect amplified HSV target DNA, a second labeled oligonucleotide is incorporated in each reaction. The Extraction Control (EC) oligonucleotide is labeled with a different dye than that used for detection of the HSV specific target and is used to confirm the validity of the extraction process. The EC is dried in the Extraction Tubes and is rehydrated upon addition of the specimen and extraction reagents. At the end of the extraction process, the EC fluorescence is monitored by the BD Viper instrument and an automated algorithm is applied to both the EC and HSV specific signals to report specimen results as positive, negative, or EC failure.
Each BD ProbeTec HSV Qx Reagent Pack contains:
- HSV1Qx Amplified DNA Assay Priming Microwells, 3 pouches of 32 microwells each: each Priming Microwell contains approximately 108 pmol oligonucleotides, 27 pmol fluorescently-labeled detector probe, 150 nmol dNTPs, with stabilizers and buffer components.
- HSV1 Qx Amplified DNA Assay Amplification Microwells, 3 pouches of 32 microwells each: each Amplification Microwell contains approximately 100 units of DNA polymerase and 500 units restriction enzyme, with stabilizers and buffer components.
- HSV2 Qx Amplified DNA Assay Priming Microwells, 3 pouches of 32 microwells each: each Priming Microwell contains approximately 105 pmol oligonucleotides, 36 pmol fluorescently-labeled detector probe, 120 nmol dNTPs, with stabilizers and buffer components.
- HSV Qx Amplified DNA Assay Amplification Microwells, 3 pouches of 32 microwells each: each Amplification Microwell contains approximately 35 units of DNA polymerase and 500 units restriction enzyme, with stabilizers and buffer components.
NOTE: Each microwell pouch contains one desiccant bag.
MATERIALS PROVIDED SEPARATELY
Control Set for the BD ProbeTec Herpes Simplex Viruses (HSV 1 & 2) Qx Amplified DNA Assays: 24 HSVQx Positive Control Tubes containing approximately15,000 copies of pHSV1 and 16,500 copies of pHSV2 linearized plasmids in carrier nucleic acid, and 24 HSVQx Negative Control Tubes containing carrier nucleic acid alone. The concentrations of the pHSV1 and pHSV2 plasmids are determined by UV spectrophotometry.
Swab Diluent for the BD ProbeTec Qx Amplified DNA Assays: 48 tubes, each containing approximately 2mL of potassium phosphate/potassium hydroxide buffer with DMSO and preservative.
BD FOXExtraction Tubes for the BD ProbeTec Qx Amplified DNA Assays: 48 strips of 8 tubes, each containing approximately 10 mg of iron oxide in a dissolvable film and approximately 240 pmol fluorescently-labeled Extraction Control oligonucleotide.
Extraction Reagent and Lysis Trough for the BD ProbeTec Qx Amplified DNA Assays: 12 Reagent and 12 Lysis troughs, each 4-cavity Extraction Reagent trough contains approximately 16.5 mL Binding Acid, 117 mL Wash Buffer, 35 mL Elution Buffer, and 29 mL Neutralization Buffer with preservative; each Lysis Trough contains approximately 11.5 mL Lysis Reagent.
V. EQUIPMENT/SUPPLIES PROVIDED SEPARATELY
BD Viper Instrument, BD Viper Instrument Plates, BD Viper Pipette Tips, BD Viper Tip Waste Boxes, BD Viper Amplification Plate Sealers (Black), BD Viper Lysing Heater, BD Viper Lysing Rack, BD Viper Neutralization Pouches, and Pierceable Caps for use on the BD ViperSystem (Extracted Mode),BD Viper System Accessories.
COLLECTION KITS PROVIDED SEPARATELY
BD ProbeTecQxCollection Kit for Endocervical and Lesion Specimens for use on the BD Viper System in Extracted Mode, BD Universal Viral Transport Medium (UVT) (3 mL) with polyester-fiber-tip swab collection kit (Cat. No. 220221 [kit], 220220 [UVT medium] / 220239 [collection swab]) or identical Copan Universal Transport Medium* (UTM-RT) and polyester-fiber-tip swab collection kit (Cat. No. 302C.LC,302C, 330C, 340C or 321C).
*The collection device and collection medium in the BD UVT kit are identical to the Copan UTM-RT medium and collection device in the catalog numbers listed.
MATERIALS REQUIRED BUT NOT PROVIDED
Nitrile gloves, 1% (v/v) sodium hypochlorite*, pipettes capable of transferring 0.5 mL, phosphate buffered saline (PBS).
*Mix 200 mL of bleach with 800 mL of water. Prepare fresh daily.
Storage and Handling Requirements: Reagents may be stored at 2–33ºC. Unopened Reagent Packs are stable until the expiration date. Once a pouch is opened, the microwells are stable for 8 weeks if properly sealed or until the expiration date, whichever comes first. Do not freeze.
VI. SPECIMEN COLLECTION AND TRANSPORT
The BD ProbeTec HSV Qx Assays on the BD Viper System in Extracted Mode are designed to detect the presence of Herpes Simplex virus type 1 or Herpes Simplex virus type 2 from external anogenital lesion specimens.
The devices that can be used to collect lesion specimens for testing on the BD Viper System in Extracted Mode are:
- BD ProbeTecQxCollection Kit for Endocervical or Lesion Specimens for use with the BD Viper System in Extracted Mode.
- BD Universal Viral Transport Medium (UVT)-3 mL fill volume and regular sized polyester-fiber-tip swab with plastic shaft (Cat. No. 220221).
- Identical Copan Universal Transport Medium (UTM-RT) and polyester-fiber-tip swab collection kit (Cat. No. 302C.LC, 302C, 330C, 340C or 321C) may also be used.
For U.S. and international shipments, specimens should be labeled in compliance with applicable state, federal, and international regulations covering the transport of clinical specimens and etiologic agents/infectious substances. Time and temperature conditions for storage must be maintained during transport. Once pre-warmed, Qx swab Diluent and diluted UVT specimens may be stored for up to 120 days at -20ºC prior to testing on the BD Viper System.
VII. LESION SWAB SPECIMEN COLLECTION, STORAGE AND TRANSPORT
The external anogenital lesion specimens for the BD ProbeTec HSV QxAssays are collected with either the BD ProbeTecQx Swab Collection Kit for Endocervical or Lesion Specimens or the UVT collection device (polyester-tipped swab with plastic shaft used with 3 mL fill volume). A 0.5 mL volume of the UVT specimen must be added to a Qx Swab Diluent Tube during processing and thus has additional stability claims. Stability information is provided for each specimen type (Tables 1, 2A, 2B and 3).
External Anogenital Swab Specimen Collection using BD ProbeTecCollection Kit for Endocervical or Lesion Specimens for use with the BD ProbeTec HSV Qx Amplified DNA Assays
NOTE: All specimens should be obtained from the patient by appropriately trained individuals.
- Open the inner Qx Swab packaging and dispose of the swab with the white shaft.
- Remove the pink collection swab from the packaging.
- Swab the base of the exposed lesion firmly to absorb exudates and cellular material with the pink swab.
- Uncap the Qx Swab Diluent tube.
- Fully insert the pink collection swab into the Qx Swab Diluent tube.
- Break the shaft of the swab at the score mark. Use care to avoid splashing of contents.
- Tightly recap the tube.
- Label the tube with patient information and date/time collected.
- Transport to the laboratory.
BD ProbeTec Qx Swab Lesion Specimen Storage and Transport
The Qx Swab must be stored and transported to the laboratory and/or test site at either 2-30C for testing within 14 days of collection, or -20C within 120 days of collection.
Table 1: Stability of Anogenital Lesion Specimens Collected with the BD Qx Swab Collection KitBD ProbeTec Qx Collection Kit for Endocervical or Lesion Specimens
Temperature Condition for Transport to Test Site and Storage / 2 - 30C / -20C
Process Specimen According to Instructions / 14 days of collection / 120 days of collection
External Anogenital Swab Specimen Collection using Universal Viral Transport (UVT)Collection Kit (or equivalent Copan collection kit) for use with the BD ProbeTec HSV Qx Amplified DNA Assays
NOTE: All specimens should be obtained from the patient by appropriately trained individuals.
- Remove the cap from the transport medium provided in the transport kit.
- Expose the base of the lesion.
- Open the swab packaging and dispose of the second swab if present (either the second swab with the plastic shaft or the metal shaft minitip swab).
- Using only one plastic shaft, polyester-fiber-tip swab provided in the UVT transport kit, swab the lesion firmly to absorb exudates and cellular material at the base of the lesion.
- Immediately place the swab used to collect the lesion specimen into the transport medium.
- Break swab shaft by bending it against the vial wall evenly at the pre-scored line.
- Replace the cap on the vial and close tightly.
- Label the vial with patient information and date/time collected.
- Transport to the laboratory.
UVT Lesion Specimen Storage and Transport
If the UVT specimen is collected, stored, and transported prior to transfer to the Qx Swab Diluent, the UVT specimen may be stored under the conditions in Table 2A.
Table2A: Storage and Transport of Anogenital Lesion Specimens Collected with UVT Collection Kit (3 mL fill vial with polyester-fiber-tip swab with plastic shaft)UVT Specimen
Temperature Condition for Transport to Test Site and Storage / 20 - 35C / 2 - 8C / -70C
Process Specimen According to Instructions / Aliquot and prewarm within 48 h of collection / Aliquot and prewarm within 14 days of collection / Aliquot and prewarm within 120 days of collection
UVT specimen matrix can be stored and transported in the UVT vial within the following conditions:
- Up to 48 h at 20 - 25C, or
- Up to 14 days at 2 - 8C, or
- Up to 120 days at -70C.
Once the UVT specimen is aliquoted into Qx Swab Diluent, the specimen must be prewarmed.
NOTE: Wear clean gloves when handling the anogenital lesion specimen. If gloves come in contact with specimen, immediately change gloves to prevent contamination of other specimens.
- All UVT specimens should be at room temperature prior to processing.
- Verify that the UVT contains a swab with a white shaft.
- Remove a pre-filled Qx Swab Diluent Tube from the packaging.
- Label the pre-filled Qx Swab Diluent Tube with the patient identification and date/time collected on the UVT specimen.
- Vortex the UVT specimen for 5 – 10 s.
- Remove the black pierceable cap from the Qx Swab Diluent Tube.
- Uncap the UVT specimen transport vial. Avoid touching the swab. Using a pipette, aseptically transfer 0.5 mL of UVT specimen into the Qx Swab Diluent Tube.
- Discard the pipette. NOTE: The pipette is intended for use with a single specimen.
- Tightly recap the Qx Swab Diluent Tube containing 0.5 mL of UVT with a black pierceable cap and invert 3 -4 times to ensure that specimen and diluent are well mixed.
- The Qx Swab Diluent Tube containing 0.5 mL of UVT specimen is ready to be pre-warmed.
- Re-cap the UVT vial using the capture cap containing the swab and store at2 - 8C or - 70C.
- Change gloves before proceeding to avoid contamination.
- Repeat steps 1 – 10 for additional UVT external anogenital lesion specimens.
UVT Specimen Storage and Transportin Qx Swab Diluent
If the UVT specimen is collected and immediately transferred into Qx Swab Diluent, specimens may be stored under the conditions in Table 2B prior to the pre-warm procedure.
Table 2B: Storage and Transport of UVT Anogenital Lesion Specimens in Qx Swab Diluent