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Laboratory Methods for Quantitating HIV RNA

Recent studies have shown that a very rapid turnover of HIV RNA occurs in the plasma of infected patients, with approximately 30% of the total virus population in the plasma being replenished daily.^1,2 Continual viral replication and rapid CD4 cell turnover are thought to play a central role in the pathogenesis of HIV infection; additionally, high plasma HIV RNA levels have been shown to be a strong predictor of rapid progression to AIDS after HIV seroconversion, independent of CD4 cell count.³ These findings have led to increased interest in quantitating HIV RNA in patients for prognostic purposes. Perhaps HIV RNA viral load measurements will prove useful in the management of HIV-infected patients, both in predicting rate of progression and monitoring response to antiretroviral therapy. However, an exact role for quantitative measures of viral load has not been determined. Moreover, reliable measures are currently available only in research laboratories.

Formerly, quantitation of virus from a clinical specimen required very expensive, labor-intensive, and difficult-to-reproduce culture techniques. Recently, however, quantitation of HIV viral load in plasma specimens has been accomplished with a variety of techniques that measure virion RNA and are less expensive and easier to perform.^4,5 Three commercial assays that have not been approved by the FDA are available on an investigational basis: 1) Amplicor HIV Monitor Test, which couples reverse transcription to quantitative polymerase chain reaction (PCR) (Roche Molecular Systems); 2) Nucleic Acid Sequence-Based Amplification (NASBA) (Organon Technika); and 3) Quantiplex HIV RNA assay, a branched DNA (bDNA) technology (Chiron Corporation).

Amplicor HIV Monitor Test

This multistep test includes specimen preparation, reverse transcription and PCR, and nonradioactive detection.⁶ Plasma samples (200 microliters) are treated with guanidinium isothiocyanate to lyse the viral particles and release RNA; isopropanol is then added to precipitate the RNA.⁶ This first step renders the sample noninfectious and safe for laboratory workers. Plasma specimens for this assay can be anticoagulated with EDTA or acid citrate dextrose (ACD), but samples collected in ACD tubes yield approximately 20% lower results because of the larger volume of anticoagulant.⁷ Therefore, if serial HIV RNA determinations are performed on a patient, blood specimens should be collected in the same anticoagulant each time. Heparinized plasma samples are not suitable for this assay.

Since HIV is an RNA virus, the genome must be reverse-transcribed into a double-stranded DNA molecule before it can serve as a template in a PCR reaction (Fig 1). Once the double-stranded DNA template is synthesized, large numbers of DNA copies are produced by PCR. PCR assays involve three steps per cycle, although sometimes steps two and three can occur together. In the first step, heat denaturation separates the two strands of the target DNA molecule; in step two, a primer pair is annealed to the single-stranded DNA; in step three, a thermostable polymerase enzyme extends each primer by adding nucleotides complementary to the target DNA. Through multiple cycles of these three steps, PCR results in a millionfold amplification of the target sequence.

The PCR products are quantitated using an internal standard that has the same primer binding sites as the HIV RNA template; however, a region of the sequence has been rearranged to allow for independent detection. The internal-quantitation-standard RNA, which is added to the plasma during specimen preparation, monitors the efficiency of sample processing and amplification. After reverse transcription and PCR amplification, the patient's HIV DNA and the laboratory's internal standard DNA are each detectable separately on colorimetric ELISA. Since a known number of copies of the internal standard are used, the number of HIV RNA copies in the patient sample can be calculated from the ratio of the HIV RNA signal to the internal standard signal.

The Amplicor HIV Monitor Test has a three-log dynamic range; its lower limit of sensitivity is about 500 copies of HIV RNA/ml. The assay is reproducible and fivefold differences in HIV RNA copy number are easily discernible. Using archived samples, it is possible to detect the high levels of HIV RNA that occur in acute infection and the suppressed levels of circulating virus that follow seroconversion.⁶

PCR is able to produce about a million copies of DNA from a single HIV RNA template molecule, creating the potential for one amplified sequence to contaminate pre-amplification specimens. The Amplicor HIV Monitor Test prevents false positive results by using a method that greatly reduces the likelihood that PCR products are reamplified in subsequent PCR reactions.

Nucleic Acid Sequence-Based Amplification (NASBA)

Like the Amplicor test, the NASBA assay is a nucleic acid amplification method, but it amplifies RNA rather than DNA. The NASBA assay includes nucleic acid isolation, amplification, and nonradioactive detection of the RNA product.⁵ Nucleic acid is extracted from the clinical specimen using the chaotropic agent guanidinium thiocyanate and acidified silica.⁸ This is more time-consuming than the extraction procedure used in the Amplicor HIV Monitor Test, but has several advantages: 1) it is applicable to many body fluids, including plasma, serum, whole blood, CSF, semen, cells, and urine; 2) it permits plasma specimens to be anticoagulated with EDTA, ACD, or heparin; 3) it removes substances that may inhibit subsequent amplification reactions; and 4) it requires only 100µl of plasma. As with the Amplicor test, the plasma is rendered noninfectious by the initial step of the extraction procedure.

RNA amplification by the nasba assay involves the coordinated, isothermal activities of the enzymes reverse transcriptase (RT), RNase H, and T7 RNA polymerase (Fig 2). Initially, an oligonucleotide primer (containing the T7 RNA polymerase promoter) is annealed to HIV RNA. RT then generates a single-strand DNA copy of the HIV RNA. Next, RNase H eliminates the RNA portion of the RNA-DNA hybrid, allowing a second oligonucleotide primer to anneal to the RNA strand. The DNA-dependent DNA polymerase activity of RT then extends from the second primer, producing a double-stranded DNA copy of the original RNA target with an intact T7 RNA polymerase promoter at one end. This promoter is then recognized by the T7 RNA polymerase, which initiates the transcription of a large number of copies of single-stranded RNA. This RNA can serve as template for repeated cycles of the same process, thus producing many copies of single-strand RNA.^5,9

With the NASBA assay, quantitation of HIV viral RNA is based on co-amplification of the specimen target and internal calibrators that are identical to the HIV target except for a small sequence rearrangement used for detection. The calibrators are added to the specimen before extraction of nucleic acid so that any loss of specimen viral RNA is normalized against a corresponding loss of the calibrator RNA. The specimen and calibrator amplificates are separately detected with a semiautomatic electro-chemiluminescence (ECL) detection instrument. The amount of initial HIV RNA in the sample can be calculated from the ratio of the HIV RNA signal to the internal calibrator signal. With this method of detection the assay is interpretable over a four-log range.^5,9

The NASBA assay can generate up to 10⁹ copies of RNA in 90 minutes, and has a clinical sensitivity of 400 molecules. It is reproducible, allowing reliable measurements of threefold differences in HIV RNA viral load.⁵ Since the NASBA assay is a target amplification method, potential exists for carryover contamination of amplified RNA. Since RNA is less stable than DNA, carryover contamination may theoretically be less likely with NASBA than with PCR. As with the other methods described, careful adherence to proper laboratory technique during specimen preparation is essential if contamination is to be avoided.

Quantiplex HIV RNA Assay (bDNA)

The bDNA assay is a signal, rather than target, amplification method, allowing for direct measurement of HIV RNA from plasma specimens.¹⁰ This assay requires one ml of plasma and includes specimen preparation, hybridization of probes and branch DNA (bDNA) amplifier molecules, and detection. EDTA is the preferred anticoagulant; ACD can be used but dilutes the plasma with its larger volume of anticoagulant. Heparin collection tubes are not acceptable.

The virus in the plasma specimen is concentrated by high speed centrifugation, then lysed to release the RNA. The virus remains infectious until after lysis. The RNA is then added to a microtiter plate coated with specific probes that bind the HIV RNA and adhere it to the plate. Through a series of hybridization events, multiple bDNA and alkaline phosphatase-labeled oligonucleotide molecules are bound to the immobilized HIV RNA molecule. With the intricate hybridization scheme a single molecule of HIV RNA may bind up to 1,755 alkaline phosphatase molecules, which greatly enhances the detection signal produced by the molecule of RNA. The RNA-probe complex is detected with the use of a chemiluminescent substrate; light emission is proportional to the amount of RNA target captured in the microwell.

The concentration of HIV RNA is determined from a standard curve run in parallel with the patient specimens. The bDNA assay has a dynamic range of 10⁴ to 1.6 x 10⁶ HIV RNA equivalents/ ml. (An RNA equivalent is the relative luminescence corresponding to one copy of an HIV RNA standard.)^10,11

The bDNA assay reliably measures decreases in plasma RNA levels after the initiation of antiretroviral therapy^1,2,12 as well as increases in plasma RNA levels after therapy is discontinued.12

The limit of detection of the bDNA assay is 10,000 RNA equivalents/ml, about 20-fold less sensitive than the other two assays described. The ability of the bDNA assay to detect HIV RNA in clinical specimens is related to CD4 cell count. In one study, the highest detection (95%) of RNA equivalents was seen in specimens from patients with CD4 cell counts of less than 200/mm³; a 67% detection rate was seen for patients with CD4 cell counts of 200 to 500, and the rate was 38% when the CD4 count was over 500.¹⁰ However, the bDNA assay has been reported to reliably detect two- to threefold changes in HIV RNA levels,¹¹ which is perhaps somewhat more reproducible than the other assays. Recently modifications in the bDNA assay increase the limit of sensitivity to about 500 equivalents/ml.¹

Since the bDNA assay is not a target amplification method, carryover contamination of amplified template is not a problem. However, careful laboratory technique is required to prevent contamination between patient specimens.

Summary

The methods described above have revolutionized the quantitation of viral nucleic acid from clinical specimens. They are applicable not only to HIV but also to other viruses. In addition, all three of these assays could potentially be adapted from the research laboratory to the clinical laboratory since specimens can be prepared easily and the methods of detection are nonradioactive. Further investigation may reveal how they can best be deployed in the management of HIV-infected patients.

If the decision is made to follow viral load in a patient, several issues should be addressed:

• A single viral load value is difficult to interpret. Viral load measures may best be used to follow changes in viral load over time, or after the initiation of specific therapies.
  
• Biological fluctuations in viral load probably occur from day to day, as is the case with CD4 cells. This, and unavoidable variations in laboratory testing, suggest that a viral load change should be three- to fourfold in magnitude to be considered significant.
  
• It is very important that physicians understand the limits of these assays, so that small changes in viral load measurements are not overinterpreted.
  
• When following viral load measurements it is essential that a single method be used for all determinations. The three assays, Amplicor HIV Monitor, NASBA, and bDNA, measure very different end points; comparisons of results from one technique to results from another technique are not valid.
  
• The assays described in this article are currently performed in research laboratories that are not set up to test individual patient samples; in these labs the assays are quality-controlled for clinical trials and other research purposes. Commercial laboratories perform the bDNA assay and various types of reverse transcription PCRs on individual patient samples, but these assays are not licensed by the FDA as diagnostic tests; they may or may not be insurance-reimbursable. They lack regulation and there are no standardized quality controls regulating their use.
  

— Angela M. Caliendo, MD, PhD

Dr. Caliendo is assistant director of the Massachusetts General Hospital microbiology laboratory and instructor in pathology, Harvard Medical School.

Published in AIDS Clinical Care November 1, 1995

Citation(s):

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2. Wei X et al. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 1995 373 117-122.

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3. Mellors JW et al. Quantitation of HIV-1 RNA in plasma predicts outcome after seroconversion. Ann Intern Med 1995 122 573-579.

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4. Lin HJ et al. Multicenter evaluation of quantification methods for plasma human immunodeficiency virus type 1 RNA. J Infect Dis 1994 170 553-562.

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5. Van Gemen B et al. A one tube quantitative HIV-1 RNA NASBA nucleic acid amplification assay using electrochemiluminescent (ECL) labelled probes. J Virol Methods 1994 49 157-167.

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6. Mulder J et al. Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 in plasma: application to acute retroviral infection. J Clin Microbiol 1994 32 292-300.

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7. Dreyer K et al. Analysis of variation in plasma HIV RNA levels in infected, asymptomatic patients. 2nd National Conference on Human Retroviruses and Related Infections. Washington, DC 1995 Abstract 342 .

8. Boom R et al. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990 28 495-503.

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9. Van Gemen B et al. Qualitative and quantitative detection of HIV-1 RNA by nucleic acid sequence-based amplification. AIDS 1993 7 Suppl 2 107-110.

10. Pachl C et al. Rapid and precise quantification of HIV-1 RNA in plasma using a branched DNA signal amplification assay. J Acquir Immune Defic Syndr Hum Retrovirol 1995 8 446-454.

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11. Todd J et al. Quantitation of human immunodeficiency virus plasma RNA by branched DNA and reverse transcription coupled polymerase chain reaction assay methods: a critical evaluation of accuracy and reproducibility. Serodiagn Immunother Infect Disease 1994 6 233-239.

12. Cao Y et al. Clinical evaluation of branched DNA signal amplification for quantifying HIV type 1 in human plasma. AIDS Res Hum Retroviruses 1995 11 353-361.

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