HIV-1 Quantitative NAAT
The Aptima® HIV-1 Quant Dx assay is an in vitro nucleic acid amplification test (NAAT) for the detection and quantitation of human immunodeficiency virus type 1 (HIV-1) on the fully automated Panther® system. It is intended to be used as an aid in diagnosis for HIV-1 infection using appropriate HIV testing algorithms. The presence of HIV-1 nucleic acid in the plasma or serum of individuals without antibodies to HIV-1 is indicative of acute or primary infection.
The Aptima HIV-1 Quant Dx assay may also be used as a supplemental test, when it is reactive, to confirm HIV-1 infection in an individual whose plasma or serum specimen is reactive with an approved assay with an indication as an aid in the diagnosis of HIV-1 infection.
The Aptima HIV-1 Quant Dx assay is intended for use in conjunction with clinical presentation and other laboratory markers for disease prognosis and for use as an aid in monitoring the effects of antiretroviral treatment, as measured by changes in plasma HIV-1 RNA levels. The Aptima HIV-1 Quant Dx assay quantitates HIV-1 RNA groups M, N, and O over the range of 30 to 10,000,000 copies/ mL. One international unit is equivalent to 0.35 copies of HIV-1 RNA for the 3rd HIV-1 WHO International Standard (subtype B, NIBSC code: 10/152).
This assay is not intended to be used as a donor screening test for HIV-1. Performance of this test has not been evaluated for use in pregnant women or in a pediatric population.
About HIV-1
Epidemiological studies identified human immunodeficiency virus type 1 (HIV-1) as the etiological agent of acquired immunodeficiency syndrome (AIDS) (-). HIV can be transmitted by sexual contact, exposure to infected blood or blood products, or through mother-to-child transmission.8 Within 3 to 6 weeks of exposure to HIV, infected individuals generally develop a brief, acute syndrome characterized by flu-like symptoms, and is associated with high levels of viremia in the peripheral blood.9-12 In most infected individuals, this early phase is followed by an HIV-specific immune response and a decline of plasma viremia, usually within 4 to 6 weeks of the onset of symptoms.13-14 After seroconversion, infected individuals typically enter a clinically stable, asymptomatic phase that can last for years.15-17 The asymptomatic period is characterized by persistent, low-level plasma viremia18 and a gradual depletion of CD4+ T lymphocytes. This depletion leads to severe immunodeficiency, multiple opportunistic infections, malignancies, and death.19 Although levels of virus in the peripheral blood are relatively low during the asymptomatic phase of the infection, virus replication and clearance appear to be dynamic processes in which high rates of virus production and infection of CD4+ cells are balanced by equally high rates of virus clearance, death of infected cells, and replenishment of CD4+ cells, resulting in relatively stable levels of both plasma viremia and CD4+ cells.20-22
Quantitative measurements of HIV in the peripheral blood have shown that higher virus levels may be correlated with increased risk of clinical progression of HIV-associated disease, and shown that reductions in plasma virus levels may be associated with decreased risk of clinical progression.23-25 Virus levels in the peripheral blood can be quantitated by measurement of the HIV p24 antigen in serum, by quantitative culture of HIV from plasma, or by direct measurement of viral RNA in plasma using nucleic acid amplification or signal amplification technologies.26-30
Molecular techniques such as transcription mediated amplification (TMA) have been widely used to amplify nucleic acids.31 TMA uses specific target capture and isothermal amplification to detect nucleic acids in multiple infectious diseases, including CT, NG, HPV, Trich, and HIV/HCV/HBV for blood donor testing.32
The Aptima HIV-1 Quant Dx assay, through TMA, utilizes multiple, long primers that target several regions of the HIV-1 genome in order to compensate for the high mutation rate of HIV-1. The assay includes dual-target amplification and detection systems, targeting two regions of the HIV-1 genome (pol and LTR) independently. The assay software does not average the signals from the two systems.
The Aptima dual-target system is designed to increase the chances of accurately detecting and quantitating samples.
ProcedureThe Aptima HIV-1 Quant Dx assay involves three main steps, which all take place in a single tube on the Panther system*: target capture, target amplification by transcription- Mediated amplification (TMA), and detection of the amplification products (amplicon) by the fluorescent labeled probes (torches).
During target capture, viral nucleic acids are isolated from specimens. The specimen is treated with a detergent to solubilize the viral envelope, denature proteins, and release viral genomic RNA. Capture oligonucleotides hybridize to highly conserved regions of the HIV-1 genome, if present, in the test specimen. The hybridized target is then captured onto magnetic microparticles that are separated from the specimen in a magnetic field. Wash steps remove extraneous components from the reaction tube.
Target amplification occurs via TMA, which is a transcription-mediated nucleic acid amplification method that utilizes two enzymes, Moloney murine leukemia virus (MMLV) reverse transcriptase and T7 RNA polymerase. The reverse transcriptase is used to generate a DNA copy (containing a promoter sequence for T7 RNA polymerase) of the target sequence. T7 RNA polymerase produces multiple copies of RNA amplicon from the DNA copy template. The Aptima HIV-1 Quant Dx assay utilizes the TMA method to amplify two regions of HIV-1 RNA (pol and LTR). An independent signal is generated from amplification of each region using specific primers, which are designed to amplify HIV-1 groups M, N, and O.
Detection is achieved using single-stranded nucleic acid torches that are present during the amplification of each target that hybridize specifically to the amplicons in real-time. Each torch has a fluorophore and a quencher. When the torch is not hybridized to the amplicon, the quencher is in close proximity of the fluorophore and suppresses the fluorescence. When the torch binds to the amplicon, the quencher is moved farther away from the fluorophore and it will emit a signal at a specific wavelength when excited by a light source. As more torches hybridize to amplicon a higher fluorescent signal is generated. The time taken for the fluorescent signal of each target to reach a specified threshold (“tTime”) is proportional to the starting HIV-1 concentration. Each reaction has an internal calibrator/internal control (IC) that controls for potential variations in specimen processing, amplification, and detection. The concentration of a sample is then calculated using the tTimes for both pol and LTR amplification by the Panther system software. Through its algorithm and comparison with stored calibration information, the Panther system software returns a single, clinically validated result for each sample.
Aptima® HIV-1 Quant is a product of Hologic® and is FDA approved for in vitro diagnostic use. See package insert for more information.
* All references to the Panther system in this document are applicable to the Panther system and the Panther Fusion system. There are no changes to the indications for use, labeling, and principles of operation for the Aptima HIV-1 Quant Dx Assay on the Panther system as a result of the add-on Panther Fusion Module.
Specificity of the Aptima HIV-1 Quant Dx assay was determined using 120 fresh and 510 frozen HIV-1 negative EDTA plasma specimens. HIV-1 RNA was not detected in all 630 samples (specificity of 100%; 95% CI: 99.4-100%).
Potential cross-reactivity to pathogens listed in Table 11 of the package insert was evaluated in the Aptima HIV-1 Quant Dx assay in the presence or absence of 3 log10 copies/mL HIV-1 RNA in HIV-1 negative human EDTA plasma. No interference in the performance of the assay was observed in the presence of the pathogens.
7 business days from receipt of specimen.
| Specimen Type | Test Code | CPT Code | NY Approved | Volume | Assay Range | Special Instructions |
|---|---|---|---|---|---|---|
| plasma | 33259 | 87536 | Yes | 2 mL (700 µL) |
30 to 10,000,000 copies/mL |
|
Ship Monday through Friday. Friday shipments must be labeled for Saturday delivery. All specimens must be labeled with patient's name and collection date. A Eurofins Viracor test requisition form must accompany each specimen. Multiple tests can be run on one specimen. Ship specimens FedEx Priority Overnight® to: Viracor Eurofins, 18000 W 99th St. Ste, #10, Lenexa, KS 66219.
Causes for RejectionSpecimens beyond their acceptable length of time from collection as listed in the specimen handling or specimen types other than those listed.
Specimens are approved for testing in New York only when indicated in the Specimen Information field above. The CPT codes provided are based on Eurofins Viracor’s interpretation of the American Medical Association's Current Procedural Terminology (CPT) codes and are provided for informational purposes only. CPT coding is the sole responsibility of the billing party. Questions regarding coding should be addressed to your local Medicare carrier. Eurofins Viracor assumes no responsibility for billing errors due to reliance on the CPT codes illustrated in this material.
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