Review

Molecular diagnosis of HIV

John R Clarke

The development of molecular techniques that access viral load and the development of
genotypic resistance have revolutionized the treatment of HIV disease. Commercially
available viral load assays use a number of different approaches from reverse transcriptase
PCR to amplification of branched chain DNA. The drawbacks of the assay are that there is no
international standard that allows comparision of viral load between assays and the diversity
of different clades of HIV results in the under or the nondetection of some patients samples.
New real-time PCR assays are under development, including LightCycler- and TaqMan-based
CONTENTS tests. The development of sequence-based genotyping assays for the detection of mutations
associated with the developement of the resistance to the 17 licensed drugs targeted against
HIV viral load assays
the pol gene of HIV have added to the improvements in patient management. However, next-
Comparison of viral
generation assays must extend detection to include the gp41 fusion region and the integrase
load assays
region of the genome as compounds directed against these targets move from clinical trails
Viral load assays under
into licensed drugs. Also, genotypic assays must improve detection of minor species and
development
detection of sequences from patients with low viral load number. Real-time sequence based-
Monitoring the development
diagnostics remains a realistic target within the next 5 years.
of antiretroviral resistance
Expert opinion & Expert Rev. Mol. Diagn. 2(3), 233–239 (2002)
five-year view
Key issues
The molecular diagnosis of HIV in the Western viral replication in infected individuals is para-
References world involves two major strategies. Firstly, the mount to the rate of progression to AIDS and
Affiliation monitoring of plasma HIV viral load in con- death (FIGURE 1). There is a significant relation-
junction with CD4 T-lymphocyte cell numbers ship between between HIV RNA viral load in
as surrogate markers for disease progression. plasma and progression to AIDS [1]. Patients
Secondly, the detection of specific point muta- with low viral load (<4530 copies/ml)
tions in the HIV-1 pol gene that when present progress to AIDS at a slower rate than those
confer resistance to specific antiretroviral drugs. with a high viral load (>36,270 copies/ml) [8].
HIV-1 RNA viral load assays have revolu- This led to the belief that the most effective
tionised patient management and current way to treat HIV-1 may be to 'hit' the virus as
thinking on HIV pathogenesis. Quantitative hard as possible for as long as possible [9] in the
measures of HIV-1 RNA have been used in the hope that this may lead to the eradication of
study of HIV-1 viral dynamics and as a marker the virus from the host. It has become standard
Department of Genitourinary
Medicine, Division of Medicine,
for clinical end-points in studies of new treat- practise to monitor HIV-1 RNA viral load in
Imperial College of Science, ment regimens [1]. Studies on the dynamics of order to assess the effectiveness of treatment
Technology and Medicine, HIV-1 replication using HIV-1 viral load and the likelihood of clinical progression.
St Mary’s Campus, Paddington, assays demonstrated that the virus is very
London W2 1PG, UK active during the long clinically asymptomatic HIV viral load assays
Tel.: +44 207 886 6738
Fax: +44 207 886 6123
stage of the disease [2–4]. As many as one hun- There are three commercially available HIV
[email protected] dred million virus particles are produced each RNA viral load assays in common use. Nucleic
day by HIV-1 infected host cells and approxi- acid sequence-based amplification (NASBA),
KEYWORDS: mately 30% of the virus is replaced each day. branched chain DNA amplification (bDNA)
genotypic resistance assays, HIV
vial load, molecular diagnosis of The lymph nodes appear to be the most active and reverse transcription PCR (RT-PCR). The
HIV, real-time PCR sites for HIV replication [5–7] and the rate of NASBA assay (Organon Technica) utilizes the

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Clarke

Comparison of viral load assays

<4530 copies/ml
4531-13020 copies/ml A number of studies have compared the performance of these
13021-36270 copies/ml assays in clinical situations [12-18]. Schuurman et al. showed that
1.2 >36270 copies/ml
1 interlaboratory reproducibility of all three assays was very good
Survival

0.8 [12]. Other authors have shown that each of the assays have rela-
0.6 tively low intra- and interassay variability [13,19–20]. The biologi-
0.4 cal variation of HIV-1 RNA in plasma in clinically stable individ-
0.2 uals is approximately 3-fold (0.5 log) and this variation was not
0
0 2 4 6 8 10 associated with diurnal fluctuations [14]. The anticoagulant used
Time (years) is important for optimal results and the preferred anticoagulant
Figure 1. The effect of viral load on the survival of infected for bDNA is EDTA, while citrate or EDTA may be used for
individuals (adapted from [8]). NASBA and RT-PCR. Heparin interferes with reagents used in
the assays and should be avoided. There is also some question
Boom method for the extraction of total RNA from a plasma over its effectiveness as a preservative for RNA [15].
sample. This method produces sufficient high quality RNA that One study from Belgium showed no significant differences in
can be used for other investigations, such as HIV genotyping for HIV-1 RNA copy number between the three assays for baseline
the determination of point mutations associated with resistance to and sequential samples [21]. Other authors found some samples
antiretroviral drugs. The major difficulty with NASBA is the gave a >0.5 log variation between NASBA and RT-PCR [17]. The
cumbersome nature of the RNA extraction procedure and the low differences were attributed to mismatches in the primer
throughput of the method. Automation of the extraction proce- sequences with the gag target sequences from the HIV-1 clinical
dure has, in part, addressed these difficulties but not eliminated strain. One of the biggest concerns of molecular diagnosis of
the problem altogether. The NASBA assay utilises three enzymes HIV-1 is that all commercially available kit-based assays have
that operate at low annealing temperatures and this could result in been optimized against HIV-1 clade B viruses, which predomi-
nonspecific interactions of the primers. The absence of tempera- nate in North America and Europe but represent only 3% of
ture-dependent cycling steps in NASBA reduces the extent to infections worldwide [22]. With the continued evolution of the
which the amplification process can be externally controlled and HIV-1 epidemic, increasing numbers of nonB infections are
may lead to variation in amplification efficiency. becoming identified in Europe and the USA. The challenge for
RT-PCR (Roche Amplicor) remains the most sensitive HIV commercial nucleic acid-based tests is to detect and quantify
viral load detection system. The new Ampliprep system pro- accurately very divergent strains of HIV. Under detection or
vides the first fully automated system for the determination of complete lack of detection of some strains of HIV remains a
HIV-1 viral load and this will eliminate potential variation in major concern [22]. Recombination between distantly related
assay results introduced by human error. The Ampliprep system strains may further contribute to the emergence of HIV-1 viral
is also barcoded and is optimised for high-throughput of sam- variants that escape detection or are under detected by current
ples. While Ampliprep is a closed system reducing its versatility, molecular tests. Therefore, it will become increasingly important
it may be used for other Roche applications, such as detection to determine the clade of HIV-1 present in a patients sample and
and quantification of Hepatitis C viral load. A major drawback this may be done during sequencing for resistance. Comparison
of PCR-based approaches is the possibility of introduction of of the performance of bDNA and RT-PCR assays for the quanti-
contaminating RNA which may then be amplified resulting in fication of viral load from patients infected with nonclade B
false-positive results. Another drawback is the reliance on one strains of HIV-1 have shown greater than 1.0 log variation in the
set of primers derived from the gag gene of HIV-1 may mean detection of some strains [23]. Other authors have shown that
that some quasi-species of HIV with multiple mutations in the NASBA fails to detect viral load in some African patients, while
3´ primer attachment sequences may be inefficiently amplified bDNA under-reported viral load [24]. The choice of method for
leading to under-detection or not being amplified at all, leading quantification is dictated by throughput of samples, the genetic
to false-negative results. make-up of the patient cohort under study and other operational
The bDNA method (Bayer Diagnostics) differs from the other needs of the centers carrying out the tests. However, there is an
two commercially available viral load assays because it amplifies urgent requirement for an international standard to enable qual-
signal and therefore it does not alter the number of target mole- ity control of viral load measurements [25]. Indeed, there may be a
cules present which reduces the effect that introduction of con- requirement for more than international standard to ensure the
tamination into a sample may have [10]. The assay is well-suited accurate quantification of nonclade B viruses.
to high-throughput of plasma specimens with as many as 168
samples being processed per assay run in the semi-automated 340 Viral load assays under development
machine. A major drawback of the bDNA assay is that there is Real-time PCR methods for the determination of HIV-1 viral
no internal control within the sample and therefore, there is no load are being developed [26,27]. The method uses LightCycler
way of discerning if amplification was successful or whether the instrumentation. The procedure has increased speed in detec-
test was inhibited [11]. tion over conventional PCR methods due to the removal of

234 Expert Rev. Mol. Diagn. 2(3), (2002)

 Molecular diagnosis of HIV

manipulations required for the detection of the PCR amplicon using nitroblue tetrazolium and 5'-bromo-4-chloro-3-indoyl
and the incorporation of rapid amplification coupled to fluo- phosphate, which results in a brown precipitate. The Inno-
rescence detection [28,29]. The HIV-1 TaqMan 5´ nuclease PCR LiPA strips allow the detection of limited mutations in RT and
is also under development and may have application to the protease. The assay has been used to detect specific mutations
large throughput applications, such as the screening of plasma in clinical trials [34] and in the detection of mutations that con-
from blood donors [27]. Use of an internal control in each reac- fer resistance to zidovudine and lamivudine when given to
tion is essential to monitor the sensitivity of each reaction. The pregnant women [35].
optimization of new quantitative assays should take into consider- The major drawback of LiPA and other hybridisation assays is
ation the diversity of HIV-1 and ensures the assay efficiently that they rely on the exact hybridization of primers to the target
amplifies field isolates from all clades of HIV-1. sequence – HIV-1 is extremely heterogenous and this results in
Other screening assays currently in use in the molecular inhibition of the assay, in some cases as many as 40% of samples
diagnosis of HIV-1 include the Roche Amplicor HIV-1 test, may fail to yield results [36], which reduces the clinical benefit of
which is an qualitatitive assay that utilises PCR and nucleic the assay in the routine detection of resistance.
acid hybridization for the detection of HIV DNA from The gold standard approach to the detection of mutations
whole blood. Again, the assay was optimised for clade B that confer resistance to antiretroviral drugs is DNA sequencing
viruses but there are additional primers which fascilitate the using dideoxynucleotide chain termination technology.
detection of nonclade B viruses. The assay has application for Sequencing has two main drawbacks, firstly, it is time consum-
the detection of HIV in recently infected individuals (sero- ing and requires well-trained personnel and secondly, it is rela-
converters) prior to the production of antibodies [30]. The tively insensitive – the mutant strain must constitute at least
method may also be adapted to detect HIV DNA in patients 25% of the sample. However, automated sequencing machines
where antibody tests may be ineffective – such as infants have made it commercially viable to develop sequencing kits,
born to HIV-seropositive mothers. such as the Visible Genetics Trugene or the Applied Biosystems
Viroseq version 2 systems. The advantage of sequencing is that
Monitoring the development of antiretroviral resistance it permits comparison of genetic sequences in patient samples
There are currently 17 drugs licensed for use against HIV-1 and with those in the now extensive gene banks and the same clinical
they are shown in TABLE 1. The compounds are active against samples can be used for detection of mutations to all classes of
the viral protease (protease inhibitors) and reverse transcriptase antiretroviral agents. Not only are known mutations detected
(nucleoside, nucleotide and non-nucleoside analogs) enzymes. this way but new mutations arising with novel combinations of
Other drugs in development include compounds directed therapy will also be detected. Of particular concern is the emer-
against other viral targets, such as integrase inhibitors and gence of new mutations that may confer cross-resistance to all
fusion inhibitors. Resistance is defined as the growth of the compounds in a class. For example, the Q151M mutation
virus in the presence of the drug and can be measured pheno- affecting the reverse transcriptase gene confers cross-resistance to
typically and genotypically. The mutations that confer resist- all six nucleoside analogs in clinical use [37]. One drawback of
ance to antiretroviral drugs are well-characterized (FIGURE 2) and current sequencing protocols is that they require at least 1000
there are a number of PCR-based assays for the specific detec- copies of HIV RNA per ml of plasma. However, a new nested
tion of known point mutations. Initially, allele-specific PCR PCR method is under development by ABI that should increase
assays were developed by incorporating the specific point- the sensitivity of the assay and in our hands sequences may be
mutation conferring resistance into the final codon of the PCR obtained from samples containing a plasma viral load of 100
oligonucleotide primer and using stringent annealing tempera- copies/ml [38]. This in turn would allow earlier detection of
tures to inhibit PCR amplification unless a perfect match was treatment failure and improve patient management. The con-
obtained [31]. cern with sequencing kits is that they may miss some clades of
The disadvantages of this approach were that low levels of non- HIV, although two studies have shown that the assays can detect
specific amplification could not be ruled out and that each muta- some viruses from clades A–G [39]. Indeed, VG and ABI
tion required a specific primer pair. An alternative approach for detected the same mutations in 91% of samples tested although
characterizing mutations was to use point-mutation assays some reduction in the detection of mixtures of wild type/mutant
(PMA), which captured PCR products containing either wild was observed in the ABI system due to the limitations of the
type or mutant codons using oligonucleotides containing the software [40]. Viroseq was able to sequence 97% of samples from
codon of interest [32]. nonclade-B HIV-1 strains from Uganda [41].
The Inno-LiPA HIV-1 resistance assay is based on reverse One major consideration in interpreting sequencing results is
hybridization technology [33]. The test procedure involves that the clinical history of the patient must be taken into consider-
extraction of HIV RNA from plasma, reverse transcription to ation. This may be complex in the case of transmission of resistant
obtain cDNA and PCR amplification of the DNA using bioti- virus either from a partner who has been extensively treated with
nylated primers. The DNA is denatured and incubated with antiretroviral drugs or as the result of the casual transmission of
the LiPA strips under very stringent hybridization conditions that resistant virus from an unnamed source. A useful tool in interpret-
can detect one base change. The captured DNA is visualized ing mutations in HIV sequences and for subtyping HIV clade is

www.future-drugs.com 235
Clarke

A. Mutations in the protease gene selected by protease inhibitors

10 20 24 32 46 54 63 71 73 82 84 90
Indinavir ________________________________________________________________ ________ -
20 32 3336 46 54 63 71 82 84 90
Ritonavir ________________________________________________________________ _________
10 48 63 71 73 82 84 90
Saquinavir ________________________________________________________________ _______
30 36 46 63 71 77 8488 90
Nelfinavir _______________________________________________________________ ________
10 20 32 46 47 50 54 82 84
Amprenavir _______________________________________________________________ _______
10 32 46 47 84 91
Lopinavir ________________________________________________________________ ________
82 84 90
Tipranavir ________________________________________________________________ ________

B. Mutations in the reverse transcriptase gene selected by RT inhibitors

41 67 70 210 215 219

Zidovudine ________________________________________________________________ _______

65 74 184
Didanosine ________________________________________________________________ _______

65 69 74 184
Zalcitabine ________________________________________________________________ _______

184
Lamivudine ________________________________________________________________ _______
41 67 70 75 210 215 219
Stavudine ________________________________________________________________ ________
65 74 115 184
Abacavir ________________________________________________________________ _________

Multinucleoside 62 69S-S 75 77 116 151

Resistance _____________________________________________________ ___________________

C. Nucleotide inhibitors
65 210
Tenofovir ________________________________________________________________ _________

Non-nucleoside inhibitors
98 100 103 106 108 181 188 190
Nevirapine _______________________________________________________________ _______
103 181 236
Delavirdine ________________________________________________________________ _______
98 100 103 108 179 188 190 225
Efavirenz _______________________________________________________________ __________

Figure 2. Known point mutations associated with genotypic resistance of HIV-1 to antiretroviral compounds. Major mutations are in bold accessory or
secondary mutations are in normal type (Figure was modified and updated [37]).

236 Expert Rev. Mol. Diagn. 2(3), (2002)

 Molecular diagnosis of HIV

resistant mutants. Improvements in the current viral load assays

Table 1. Drugs available for use in combination that would allow more uniform detection of all clades of HIV-1
antiretroviral therapy. would be welcomed. The development of international standards
Nucleoside Non-nucleoside Nucleotide Protease would also reduce the discrepancies observed between the current
analogs analogs inhibitors inhibitors commercial viral load assays. LightCycler technology will further
reduce the cost of carrying out viral load detection.
Zidovudine Nevirapine Tenofovir Saquinavir
Didanosine Delavudine Indinavir
Key issues
Stavudine Efavirenz Ritonavir
Lamivudine Nelfinavir • The diversity of HIV-1 makes the uniform detection of all
clades of the virus extremely difficult. The implementation of
Abacavir Amprenavir
an international standard will improve numerical differences
Zalcitabine Tipranavir in the quantification of virus between assays.
Lopinavir • However, an international standard should be developed that
normalises the detection of nonclade B viruses that account
for 97% of HIV-1 circulating in the world.
the University of Stanford database which may be accessed
• More sensitive sequencing assays are required that enable
through www.hivdb.stanford.edu. HIV subtype may also be
the detection of HIV-1 from patients with low viral loads and
determined via the National Centre for Biotechnology
that will enable the more reliable detection of minor species
Information website at [101].
in mixtures of wild type and mutant virus.
Expert opinion & five-year view • Improvements in the chemistry of sequencing kits and
In the next 5-years major efforts will be made to lessen the impact machinery will fascilitate the increase in throughput of
of HIV-1 in the Developing world. The reduced costs of antiretro- samples that will undoubtedly be required as the pandemic
viral to these countries should be extended to cut-price viral load increases.
assays and sequencing machinery and kits. This would enable these • Reduction in the costs of assays in line with the recent
countries to at least begin to monitor HIV-1 infections in the concessions by drug companies supplying antiretroviral
growing number of infected individuals within these countries. drugs to Africa would be welcome and would improve the
More antiretrovirals will become available and these drugs maybe diagnosis and monitoring of viral load and resistance in low
from new classes of drugs, such as fusion inhibitors and integrase income countries which are facing the brunt of new
inhibitors, which may result in even more complex detection of infections.

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Am. Med. Assoc. 279, 1984–1991 Human immunodeficiency virus Type 1 Affiliation
(1998). drug resistance testing: a comparison of • John R Clarke
• Review of all the clinically relevant three sequence based methods. J.Clin. Department of Genitourinary Medicine,
mutations associated with HIV-1 drug Microbiol. 39, 2157–2165 (2001). Division of Medicine,
resistance. Imperial College of Science,
• Describe the versatility of sequence based
Technology and Medicine,
38 Elbeik T, Hoo B, Campodonico M et al. assays for sequencing difficult samples. St Mary’s Campus, Paddington,
Detection of newly emerging primary 41 Mracna M, Becker-Pergola G, Dileans J London W2 1PG, UK
genotypic mutations at less than 50 et al. Performance of Applied Biosystems Tel.: +44 0207 886 6738
copies/ml with maintained presence at Viroseq HIV-1 genotyping system for Fax: +44 0207 886-6123
breakthrough in longitudinal plasma sequence based analysis of non-subtype B [email protected]
samples from patients on triple

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