Bridging the Gap: Conference Record [Abstract book, International Conference on AIDS (12th: 1998: Geneva, Switzerland)]

12th World AIDS Conference

Abstracts 41224-41228 787

been designed to detect mutations of some of these codons (41, 68, 70, 74, 75,
184, 214 & 215). The application of this assay on routine clinical specimens has
been reported. Extensive clinical trials on the validation of the LiPA assay have
been carried out in multiple centres around the world. Here we describe some of
the reported novel clinical applications of LiPA HIV-1 RT from nine retrovirology
centres.
Methods: Investigation of the usefulness of LiPA HIV-1 RT for the pre-treatment monitoring of drug naive HIV-1 infected patients; monitoring of patients on
long term antiviral therapy including pregnant women; follow up of needle-stick
incidents; and epidemiology. In addition one centre compared the genotyping
results obtained by LiPA HIV-1 RT with phenotypic analysis.
Results: a) Longitudinal Samples - detection of mutants correlated with rise
in viral load. b) Good correlation with phenotyping. c) LiPA HIV-1 RT can indicate drug compliance and can also predict an increase in viral load in pregnant
women. d) Demonstration of transmission of resistant virus. e) LiPA HIV-1 RT
has demonstrated different patterns of mutations in the viral population in CSF
compared to plasma, f) LiPA HIV-1 RT was used to demonstrate the presence
of resistant virus following needle-stick injury, g) 'Baseline' testing prior to clinical
trial entry.
Conclusions: LiPA HIV-1 RT provides information on mutations at key codons
on the RT gene which aids clinical decision making in different clinical settings
41224 Emergence of drug-resistant HIV-1 strains from patients
on long-term ddl therapy
Stephen Delaney12, N.N. Zheng2, P.W. McQueen3, D.A. Cooper3. 1P&S
Sciwrite, PO Box 748 Pacific Palms, NSW 2428; 2Dept Biotechnology University
of NSW Sydney; 3NCHECR University of New South Wales Sydney, Australia
Background: Drug-resistance is a critical factor contributing to the gradual loss
of clinical benefit. Therefore the evolution of resistance to ZDV and ddl was
monitored in 17 patients who had been on the Alpha ddl trial (ddl monotherapy
following ZDV monotherapy) for more than 48 weeks.
Methods: The RT gene of sequential HIV-1 isolates from these patients was
sequenced and susceptibility (ICso) to ZDV and ddl determined. Mutant viral
clones containing the G196E mutation, alone or in combination with the ZDVR
mutations, were constructed.
Results: ZDV resistance remained stable while ddl resistance increased. The
ZDVR mutation, T215Y, when present in isolates at the start of therapy was usually
maintained (7/8 patients), while other ZDVR mutations (M41L, D67N/V, K70R and
K219Q), present in 53% of patients' baseline isolates, fluctuated during therapy.
The ddlR mutations, K65R and L74V, were present in only 20% of isolates, while
mutations, V75T and M184V, were not detected. A change at codon 196 (G-- E)
was found in 30% of isolates from 7 patients. Two isolates with this mutation, but
none of the known ddlR mutations, were highly resistant to ddl (IC50 > 100 mM).
Constructs showed that, on its own, the G196E mutation had no effect on ddl
resistance but in combination with different ZDVR mutations it caused high levels
of ddl resistance. This would account for the persistence of the ZDVR mutations
in the absence of ZDV therapy.
Conclusion: These results have implications for sequential antiviral treatment
strategies and may help explain why antiviral naive patients often respond better
than experienced patients to a particular antiviral regimen.
41225 Semi-quantitative HIV-1 mutation analysis using the
Murex-lnnogenetics Lipa HIV-1 RT assay
Fran Sheridan1, D. Parker1, R. Shuurman2, L. De Graaf2, J. Tijnagel2,
C. Boucher2. 1Murex Biotech Limited, Central Road, Dartford, Kent, England;
2Eijkman-Winkler Institute, University Hospital Utrecht, The Nertherlands
Background: A line probe assay has been developed for the simultaneous detection of the common mutations of the reverse transriptase gene of HIV-1 which
have been associated with resistance to zidovudine (AZT), lamivudine (3TC),
didanosine (ddl) and zalcitabine (ddC). The assay detects the presence of both
consensus wild type and resistant genotypes at codons 41, 69, 70, 74, 75, 184
& 215. The test is based on the amplification of the reverse transcriptase gene
using biotinylated primers followed by reverse hybridisation of the amplicon with
a series of complementary probes bound to a nitro-cellulose strip. Signal generation is accomplished using a streptavidin-alkaline phosphatase conjugate and
BCIP/NBT substrate. We assessed the ability of LiPA HIV-1 RT to determine if an
increase in the proportion of resistant virus for a particular codon correlates with
increase in viral load.
Methods: Plasma samples from 19 patients were tested using the Murex-lnnogenetics HIV-1 RT LiPA assay and Roche Amplicor HIV-1 Monitor viral load test. A
semi-quantitative estimate of LiPA signal intensity at each codon was determined.
Results: The results show a correlation between an increasing viral load and
the emergence of mutations which are associated with resistance to AZT. LiPA
analysis illustrates the presence of a mixture of mutant and wild type consensus
sequence in the plasma virus population. The subsequent outgrowth of mutant
virus as the apparently dominant quasi-species as measured by relative LiPA

signal intensity correlated with an increase in the viral load.
Conclusions: Our results suggest that the signal intensity of both wild type and
resistant lines on the LiPA strip may be used to determine the relative proportions
of wild type and mutant virus present in the total virus population. The relative
increase in signal intensity of the mutant probe compared to the wild type probe
correlates with outgrowth of the resistant species which in turn accounts for an
increase in viral load.

141226 Susceptibility profile (AntivirograTM ) of 945 clinical HIV-1
isolates to abacavir
Rudi Pauwels1, K. Hertogs1, F. Peeters1, R. Lanier2, N. Graham2, J. Mellors3,
P. Stoffels1. Virco NV + Tibotec, NV, General de Wittelaan L 11B4, Intercity
Busines Park, 2800 Mechelen, Belgium; 2Glaxo Wellcome Inc, Raleigh Durham,
NC; 3University of Pittsburgh, Pittsburgh, PA, USA
Objectives: To determine the abacavir (1592U89) susceptibility of 945 recombinant abacavir-naive patient isolates displaying different patterns of resistance to
approved nucleoside RTI's by using the Antivirogram M method.
Methods and Results: Generation of recombinant viruses and subsequent
susceptibility testing was performed using the Antivirogram'" method (Hertogs et
al. Antimicrob. Agents Chemother. 1998 (42) 2: 269-276). Seven concentrations
of each drug were tested in 8 replicate cultures for each isolate. Results are
reported as % of isolates showing >4-fold or >8-fold increase in IC5o (compared
with recombinant control HIV-1/LAI) among the groups of isolates below showing
>10-fold to D4T, DDI and other new NRTI's.

Not Resist. to -4 Fold Resistant to:
DDIorD4T DDI D4T DDI + D4T
Number (0-3) of other
NRTIs having resistance 0 1-3 0 1-3 0 1-3 0 1-3
Abacavir Susceptibility
Susceptible n 368 390 4 15 6 16 0 2
(54-fold) (%) (100) (86) (100) (38) (100) (38) (7)
Intermediate n 58 18 22 11
(4-to 8 fold) (%) (13) (46) (52) (37)
Resistant n 8 6 4 17
(-8-fold) (%) (1) (16) (10) (56)

Total
801
(85)
109
(12)
35
(3)

Conclusions: All isolates that were resistant to D4T alone or DDI alone, remained susceptible to abacavir. Isolates resistant to D4T + DDI, and 1 to 3 other
nRTI's showed reduced susceptibility to abacavir. Clinical studies indicate that
abacavir susceptibility testing is useful in predicting the response to abacavir
therapy. Baseline susceptibility testing by the Antivirogram" can identify patients
who are most likely to benefit from abacavir therapy.
41227 First study on resistance profile in a group of drug naive
patients in Argentina
Horacio E. Salomon, Carlos M. Rossi, S.E. Pampuro, G.H. Kijak, M.B. Lasala,
G. Reboredo, O. Libonatti, M.T. France Fernandez. University of Buenos Aires,
Paraguay 2155 Piso 11, Buenos Aires, Argentina
Introduction: Since 1987 nucleoside analogs have been used in Argentina, first
as monotherapy and then combining 2 or 3 different drugs including protease
inhibitors. Nevertheless, drug resistance studies have not yet been done.
Objective: To analyze the existence of HIV pol gene mutations related to
antiviral resistance in drug naive HIV positive individuals.
Methods: Ten HIV-1 seropositive asymptomatic individuals were studied. Peripheral blood lymphocytes were separated and DNA was extracted. A region
of the pol gene of the proviral DNA was amplified by nested PCR. Consensus nucleotide sequences were determined by the fmol method (Promega) on
PCR-amplified DNA. The nucleotide sequences obtained were translated and the
amino acid sequences were aligned using the PC Gene software (IntelliGenetics
Inc.). Two pol genomic regions of the amplified DNA were sequenced: nucleotides
82 to 396 of the RT coding region (amino acids 28 to 132) and nucleotides 469
to 732 (amino acids 157 to 244). HIV-1 pol consensus B sequence was used as
a reference from Los Alamos GenBank (USA).
Results: None of the previous characterized drug resistance mutations were
found in the samples studied. However some extent of variability within the studied region was found. A range from 3 to 8 amino acid changes was observed in
the 10 sequences. None of these mutations were associated to drug resistance
Conclusion: It is of great interest to carry on drug resistance surveys with
larger number of patients to get information from different countries, specially
those like Argentina where antiretroviral therapy is widely used.
41228 Effect of HAART +/- IL-2 on viral kinetics and T cell
reconstitution in lymphoid tissues of asymptomatic HIV-1
infected patients
Jan Van Lunzen1, K. Tenner-Racs2, P. Racz2, F.T. Hufert3, J. Lauer4,
H.J. Stellbrink4. 1Universitat Hamburg, UKE Medizinsche Universitats-und
Poliklinik; 2Bernhard-Nocht-lnst. for Tropical Medicine Hamburg; 3lnst. of
Virology, University of Freiburg, Freiburg; 4 University Hospital Eppendorf, Med.
Dept. Hamburg, Germany
Objective: 1. To study viral kinetics and the development of T cell subsets in
lymph nodes (LN) during HAART. II: To evaluate the effect of additional 11-2
therapy on these parameters.
Patients and Methods: Fourteen antiretroviral naive pts. with stable asymptomatic HIV infection (CD4 > 350//pl) were treated either with AZT + ddC + hdSQV
(n = 10) or AZT + 3TC + SQV - SGC and additional 11-2 9 MU s.c. for 5d in an
interval of 6 weeks (n = 4). Axillary LN biopsies obtained at baseline and after 1
mth. or 3 mths. of therapy were analysed by flowcytometry, in situ- hybridisation
(ISH), immunehistochemistry (ICH) and DNA-PCR of FACS-sorted CD4+ T cells.
T cell subsets and viral kinetics in peripheral blood (PB) were studied in parallel.