Contents
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Introduction
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Automated Direct Sequencing
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DNA Chip Technology
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Line Probe Assay (LiPA)
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Limitations of Genotypic
Assays
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References
The HIV-1 Polgene encodes for both the reverse
transcriptase and protease enzymes. HIV drug resistance develops when there
are specific mutations within this gene. More than 80 different mutations
that contribute to various levels of drug resistance to the 13 FDA licensed
antiretroviral agents, and also to agents in clinical trials, have been
characterized. It was recognized early on that rapid determination of the
viral gene sequence of clinical HIV isolates might help identify drug-resistant
strains and assist clinicians in choosing appropriate antiretroviral therapy
for individual patients. Over the last three years, the diagnostic field
has been trying to develop accurate and fast HIV genotypic assays to do
just that. Unfortunately, the first generation of genotypic assays were
brought to market in the absence of adequate physician education which
is needed to help guide interpretation of results and clinical use of the
tests.
The first generation of HIV genotypic assays (which employ different
methodologies) detect changes in HIV gene sequences which are known to
be associated with drug resistance. These assays first require the isolation
of HIV RNA from plasma. This is done in a similar fashion to the licensed
viral load tests. In the next step, RT-PCR is usually performed to generate
a PCR product that contains copies of the desired gene sequence. This PCR
product is then analyzed for mutations. Most assays focus on the core drug
resistance mutations. Core resistance mutations are those which, when inserted
into the Pol gene of wild-type HIV, cause a decrease in drug
susceptibility.
Other viral gene changes that are found in HIV clinical isolates - accessory
mutations and natural polymorphisms - may not significantly decrease drug
susceptibility by themselves but can augment the effects of core mutations.
Many genotypic methods for determining antiretroviral drug resistance
have made it to the clinic. A partial list includes: automated direct
sequencing,
"DNA chip" sequencing (also known as oligonucleotide hybridization and
GeneChip), point mutation assays and the line probe assay (LiPA). The
historical
gold standard for determining the sequence of nucleotide bases (A, adenine;
C, cytosine; G, guanine; T, thymine) within a gene is direct dideoxynucleotide
sequencing based on the Sanger method, and all new genotyping methods are
compared for accuracy to this gold standard.
Automated Direct Sequencing
Automated direct DNA sequencing is the most common method used in sequencing
labs today. The development of DNA sequencing instruments that utilize
laser-induced fluorescence has greatly advanced the speed and accuracy
of DNA sequencing. Nonetheless,the accuracy of this method is quite variable
depending on the lab and the chemistry used. A study which examined the
variation of sequencing performance among 80 DNA sequencing laboratories
found the error rate one can expect to obtain with a 400-500 base pair
dye-primer DNA sequencing run is 1-2% [1]. Sequencing the >1,000
bases needed to fully analyze the desired region of an HIV Pol gene
can take over 6 sequencing reactions per sample to generate accurate double
stranded sequence information. Thus, even in the most experienced hands,
automated direct DNA sequencing is at best 98-99% accurate.
DNAChip Technology
DNA chip technology, also called solution hybridization technology,
is a new method for DNA sequencing. This method employs high density
oligonucleotide
arrays mounted on silicone chips (hence "DNA chip" and the trade name Gene
Chip) and sequencing-by-hybridization technology. The overall accuracy
of this methodology is comparable to automated sequencing at ~98-99% (for
an explanation of the technology see Kozal et al, 1996 [2]). Automated
sequencing and DNA Chip technology have intrinsic methodologic differences,
and the common mistakes (base calling errors) involved in automated sequencing
(gel compression, insertions, deletions and substitutions) are by nature
not inherent in the sequencing-by-hybridization DNA chip technique. The
most common problems associated with DNA chip technology are due to
cross-hybridization
reactions and the inability to detect unsuspected base insertions or deletions.
Thus the areas within the viral gene sequence where errors occur usually
do not overlap for DNA chip sequencing and standard DNA sequencing. A recent
study by Gunthard and colleagues comparing DNA chip technology with
conventional
automated sequencing found a high concordance rate (~98-99%) between the
methods depending on the sample tested (HIV clones or clinical samples)
[3].
Line Probe Assay (LiPA)
The Line Probe Assay (LiPA) is a rapid method for detecting individual
point mutations using DNA probes on nitrocellulose strips. Point mutation
assays were the first such technology to be utilized extensively in many
labs. This technique detects the presence of specific base changes by employing
PCR primer specificity and amplification. This technique is rapid and can
detect mixtures of bases in a patient sample. The major limitation for
this technique is that at least two separate PCR reactions are required
for each mutation you are trying to detect, and given that there are over
80 mutations associated with drug resistance it is not practical to design
PCR-based assays for individual mutations. (Click her
e for Dr. Mayers' review of two recent papers on LiPA).
Limitations of Genotypic Assays
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Current assays are unable to detect minority populations of drug-resistant
virus
One problem with the genotypic assays is their inability to detect minority
populations of drug-resistant variants within the dominant circulating
viral population. This is a valid concern which will need to be addressed
with improved assays. One way to address this problem is to combine viral
load testing with genotypic resistance assays. For example, if the sensitivity
of the genotypic assays could reach to <20 copies of HIV RNA, the
mathematical
probability of missing a resistant viral variant that is rapidly emerging
during therapy would be less than with the current assays (most assays
currently need ~1,000 copies of HIV RNA to be able to generate a product
for sequencing). Active research is ongoing in this area, but for the time
being one must remember the limitation of these assays.
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Current assays can not measure the potential interactions between
mutations
This problem can be overcome by continuing to amass large databases to
establish genotype- phenotype relationships. Researchers are trying to
establish these links and to develop software to allow for the prediction
of drug-resistant and drug-susceptible phenotypes based on the genotype.
I believe this is the most important tool currently needed by the clinicians
who use these assays (most of us have trouble remembering the most common
mutation which causes cystic fibrosis, let alone all of the mutations
associated
with HIV drug resistance). Many of us thought that after medical school
we would never again be faced with the difficult task of interpreting DNA
sequences (this was to be left for geneticists). But just as the complexity
of antiretroviral therapy has reached the echelons of cancer chemotherapy,
the advancement of molecular tools for detecting HIV drug resistance will
bring the HIV clinician to a level of proficiency in molecular biology
that surpasses most other specialties (at least transiently, as genotypic
assays will be commonplace in all of medicine within the next decade).
Some of the companies and institutions offering or selling the tests
are Stanford University (ABI sequencing; phone 650-723-5706), Specialty
Labs (GenotypR Plus; phone 800-323-9100), Abbott Diagnostics/Murex (LiPA;
phone 800-334-9332), LabCorp (VircoGen, phone 800-533-0567), Applied Sciences
(Genotyping test; phone 770-734-9872), Visible Genetics Inc. (TruGene HIV
Kit; phone 416-813-3217); Affymetrix (GeneChip HIV PRT assay; phone
408-731-5000).
Excellent reviews of genotypic and phenotypic assays for determining
antiretroviral drug resistance are available from HIVresist
ance.com
, the International
Association of Physicians in AIDS Care (IAPAC) and Project
Inform
Related HIVresistanceWeb Articles:
The Line Probe Assay (LiPA): An
Alternative to Direct Genotypic Sequencing
(Douglas Mayers, February/March 1999)
