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April 2003
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New Test ANNOUNCEMENT
A
Mayo
Refer ence
Ser vices
Publication
HIV-1 Quantitation with Reflex to HIV-1 Genotyping
#13035
Profile
Information
The following test is reflexed when indicated:
Unit Code
Reporting Name
Available Separately
82951
HIV Genotyping, P
Yes
Clinical
HIV is an RNA virus that infects cells and is then converted to DNA (cDNA) by the action of viral reverse
transcriptase. HIV is the causative agent of acquired immunodeficiency syndrome (AIDS), a severe, lifethreatening condition.
Viral load is monitored before and during therapy in order to assess efficacy of antiviral treatments.
Studies have identified a number of mutations associated with antiviral resistance. Genotypic analysis allows
identification of nucleotide changes associated with HIV drug resistance. When combination therapy fails,
genotyping for drug resistance mutations may help direct appropriate changes in antiretroviral therapy and
may result in at least short-term benefit, as evidenced by viral load decrease.
Useful For
• Quantifying HIV-1 viral load and, if appropriate, automatically determining key HIV mutations (genotype)
associated with drug resistance
• As a guide for initiating or changing antiviral treatment regimens
Interpretation
HIV-1 viral load is determined. Because of the inherent variability in the assay, as well as biologic variation
in the host, less than a 2-fold change in viral load may not be significant.
If the viral load is ≥1,000 copies/mL, genotypic analysis is performed. Sequence data of the patient's viral
strain is compared with those in a database of known drug resistance mutations. A printout of mutations
detected is provided that highlights those base changes with specific drug-associated resistance. Mutations are
categorized and reported (see #82951 Human Immunodeficiency Virus Type 1 (HIV-1) Genotyping, Plasma).
Cautions
• The HIV-1 quantitative and genotypic tests are not to be used for diagnostic purposes.
• A single viral load test should not be used as the only criteria to form a clinical conclusion; results should
be correlated with serologic tests, patient symptoms, and clinical presentation.
• Negative HIV results do not indicate absence of disease. Inhibitory substances may be present in the
specimen. Inadequate specimen collection or storage may invalidate test results.
• Specimens collected using ACD will have quantitative values approximately 15% lower than specimens
collected using EDTA.
Supportive
Data
Our clinical microbiology laboratory employed HIV-1 genotyping using TRUGENE since May 1, 2001. From
May to November 2001, we performed 337 HIV-1 genotyping tests and documented a 16% failure rate of
HIV-1 genotyping during routine clinical use. Specimens with low delta values/poor resolution on first-time
sequencing were tested using the Roche Amplicor HIV-1 Monitor assay prior to repeat genotyping.
Specimens demonstrating viral load results of <1,000 copies/mL (c/mL) were reported as “unable to
genotype.” All other specimens underwent repeat genotyping. Specimens with viral load results of 1,00010,000 c/mL were concentrated during re-extraction prior to retesting. Thirty specimens had an HIV-1 viral
load ≤1,000 c/mL. For specimens containing >1,000 c/mL, the success rate upon first-time testing was
246/307 (80%); the success rate following repeat testing was 277/307 (90%). The success rates, as correlated
with viral load ranges, were as follows: <1,000 c/mL, 6/30 (20%); 1,000-4,000 c/mL, 41/51 (80%); 4,00110,000 c/mL, 33/38 (87%); 10,001-50,000 c/mL, 74/79 (94%); 50,001-100,000 c/mL, 59/62 (95%); >100,000 c/mL,
70/77 (91%). Higher genotyping failure rates were seen with specimens containing 1,000-10,000 c/mL, versus
specimens containing >10,001 c/mL (P=0.01) HIV-1 RNA. Our clients have been advised that the HIV-1 viral
load must be ≥1,000 copies/mL (c/mL) for HIV-1 genotyping to be successful.
Test Title:
HIV-1 Quantitation with Reflex to HIV-1 Genotyping
#13035
Reference
Hirsch MS, Brun-Vezinet F, D’Aquila RT, et al: Antiretroviral drug resistance testing in adult HIV-1 infection:
recommendations of an International AIDS Society-USA Panel. JAMA 2000 May;283(18):2417-2426
Method
HIV-1 RNA quantitation is performed by PCR using the Roche Amplicor System. Plasma is chemically
extracted and the viral RNA is precipitated with isopropanol. A known amount of a standard synthetic RNA
molecule is added to each specimen to permit quantitation of HIV RNA by a comparison of resulting data
following PCR amplification and detection. A 155 base pair sequence in the gag gene of HIV is amplified by
reverse transcription-polymerase chain reaction (RT-PCR) in a single reaction. The amplified DNA is detected
in separate wells of a microwell plate coated with specific DNA probes. The bound DNA is quantified with
an avidin-horseradish peroxidase conjugate and the resulting colorimetric reaction. The HIV RNA copy
number is then calculated by comparing the known input copy number of the standard RNA and the
resulting optical densities for several dilutions of the specimens and control samples. Sensitivity of the assay
is 400 HIV-1 RNA copies/mL. The linear range of the assay is 400 copies/mL to 750,000 copies/mL.
(Aschbacher R, Monari P, Lolli S, et al: Evaluation of three different commercial procedures for quantifying
human immunodeficiency virus type-1 RNA levels. New Microbiologica 1999;22:1-9)
HIV-1 genotyping is performed using the TrueGene HIV-1 assay, developed by Visible Genetics, Inc, which
determines the nucleotide bases by simultaneous bidirectional sequencing (1.9 kb total) of the viral reverse
transcriptase (1,600 nucleotide) and protease (300 nucleotide) genes of HIV-1 in a blood specimen and
identifies any mutations. An automated DNA sequencing system (OpenGene computer software) compares
the specimen genotype to the known resistance mutations and generates a list of the mutations present and
the antiviral drugs to which the mutations confer resistance. (Package insert: TrueGene HIV-1 Genotyping
Assay, Visible Genetics, Inc, Toronto, Ontario, Canada)
Specimen Required: Draw blood in a lavender-top (EDTA) tube(s) (GREEN TOP [HEPARIN] TUBE IS NOT ACCEPTABLE).
Aseptically spin down, remove plasma from cells within 4 hours, and send 3.0 mL of EDTA plasma frozen in
a screw-capped, sterile, plastic vial. Maintain sterility and forward promptly. See “Infectious Material” in
Special Instructions.
NOTE: 1. This test is intended to be used to monitor known HIV positive infections. It is not intended
for primary detection of HIV infections.
2. The following should be avoided:
a. Freeze/thaw cycles
b. Obvious microbial contamination
c. Prolonged ambient temperature exposure
d. Specimen-to-specimen contamination
3. EDTA is the preferred anticoagulant. ACD specimen is also acceptable. ACD anticoagulated
specimens yield test results that are approximately 15% lower than results obtained from EDTA
anticoagulated specimens. This is due to the dilution effect of 1.5 mL of ACD present in the
blood collection tube. If ACD is used, label specimen “ACD plasma.”
4. Please complete a ”Microbiology Request Form” (Supply T244) or a “MayoConnect Additional
Test Information Form” (Supply T357) and forward it with the specimen.
Reference Values:
Analytic Time:
Days Set Up:
Fee:
CPT Code:
HIV 1 RNA by PCR, QN
No HIV-1 RNA detected
HIV-1 GENOTYPING
No HIV-1 drug resistance/mutations detected
3-4 days
HIV 1 RNA by PCR, QN: Monday through Friday, Sunday
HIV-1 Genotyping: Varies; test will be performed in batches of 5
$301.00
87536/HIV 1 RNA by PCR, QN
87901/HIV-1 Genotyping (if appropriate)