Do not use heparin tubes
Plasma must be separated within 2 hours of collection.
If the specimen is being sent from outside Auckland Healthcare, send the plasma frozen.
It is essential that the blood specimen be collected while the patient is still receiving antiretroviral drug therapy, otherwise wild-type virus will outgrow resistant variants and mutations will not be detected.
Results available in 5 weeks.
The assay performed in this laboratory requires the amplification of HIV IN sequences covering majority of the IN gene. The first round product is 1347 bp in length. This is then nested using 2 nested reactions producing products of 579 and 604 bp each. The 2 nested products from each HIV isolate are then purified and sequenced. Sequence data is then analysed using DNAstar software and amino acid changes recorded. A report is then generated based on the sequence database at Stanford University.
Drug resistance is a major factor contributing to the failure of antiretroviral therapy. The high error rate of reverse transcriptase and the rapid turnover of the HIV-1 population contribute to the prompt emergence of resistant variants in the setting of incomplete suppression of virus replication. Data from retrospective and prospective studies suggest that resistance testing can be helpful in guiding the choice of antiretroviral therapy in patients failing their current regimen. Drug resistance evolves through a stepwise accumulation of mutations; early-stage mutants are often only partially resistant and tend to be less fit than wild-type virus in the absence of therapy. Resistance to antiretroviral drugs is determined by mutations in the genes that encode the protease (PR) and reverse transcriptase (RT) genes. Primary mutations are those that alter binding of the drug to its target and result in an increase in the amount of drug necessary to inhibit the enzyme. Secondary mutations increase the level of resistance by improving the fitness of viruses carrying primary mutations. Usually secondary mutations have little or no effect on the level of resistance in the absence of primary mutations.
Resistance mutations are described by the number representing the specific position of the mutant codon, prefixed by a letter indicating the relevant amino acid found at the position in the consensus (?wild-type?) sequence, and followed by a letter indicating the amino acid found at that position in the mutant virus. Thus, the mutation characteristic of 3TC resistance is described M184V, indicating that at codon 184 Valine (V) has been substituted for Methionine (M).
Specimens requiring HIV drug resistance analysis have usually been submitted for HIV viral load determination. If there has been a significant and sustained rise in viral load for no obvious reason the ID physician may request drug resistance analysis. This can be performed on the stored plasma remaining from the HIV viral load assay.
The assay is performed by amplifying HIV-1 reverse transcriptase (RT), protease (PR) and integrase genes using The DeepChek-HIV Genotyping Assay , ABL-TherapyEdge. Samples are sequenced using Massively Parallel Sequencing (MPS) and analysed by DeepCheck -HIV (CE-IVD marked, ABL_TherapyEdge SA) diagnostic software. Mutations over a 5% threshold are assessed for contribution to resistance. A comprehensive PDF report is available on request.
References
1. Gunthard HF, Wong JK, Ignacio CC, Guatelli JC, Riggs NL, Havlir DV and Richman DD. Human Immunodeficiency Virus replication and genotypic resistance in blood and lymph nodes after a year of potent antiretroviral therapy. J Virol. 1998; 72:2422-2428.
2. Birk m and Sonnerborg A. Variations in HIV-1 pol gene associated with reduced sensitivity to antiretroviral drugs in treatment-naive patients. AIDS 1998; 12:2369-2375.
3. de Jong JJ, Goudsmit J, Lukashov VV, Hillebrand ME, Baan E, Huismans R, Danner SA, ten Veen JH, de Wolf F and Jurriaans S. Insertion of two amino acids combined with changes in reverse transcriptase containing tyrosine-215 of HIV-1 resistant to multiple nucleoside analogs. AIDS 1999; 13:75-80.
4. Hirsch MS, Brun-Vezinet F, DAquila RT, Hammer SM, Johnson VA, Kuritzkes DR, Loveday C, Mellors JW, Clotet B, Conway B, Demeter LM, Vella S, Jacobsen DM and Richman DD. Antiretroviral drug resistance testing in adult HIV-1 infection: Recommendations of an international AIDS society-USA panel. JAMA 2000; 283:2418-2425.
For further information contact the laboratory (contact via Lablink: 22000 or (09) 307-8995 or 0800 522 7587) ,or:
the Virology team
virology@adhb.govt.nz
Plasma must be separated within 2 hours of collection and sent frozen to ACH.