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

786 Abstracts 41219-41223

12th World AIDS Conference

decrease in PMPA susceptibility to HIV and SIV. Importantly, in a study investigating PMPA for the treatment of SIV, the K65R mutation developed in 4/4
treated monkeys soon after therapy began, yet at up to 3 years post infection,
animals who are still receiving PMPA therapy continue to show a durable suppression of viral replication. Recently, 28 patients received 1 month of PMPA
Prodrug monotherapy and none of the 16 patients analyzed to date developed
any genotypic changes in RT from baseline.
Conclusions: Based on in vitro data, mutations which arose during Preveon
therapy would not be expected to affect subsequent response to PMPA treatment. Additionally, no clinically significant PMPA resistance has developed in
SIV-infected monkeys and no genotypic changes have been observed in RT of
HIV-infected patients as a result of PMPA therapy.
S41219 Line Probe assay compared with clonal and
population-based sequencing for detection of resistance
mutations in HIV-1 RT
Marlene M. Rayner1, J. Meek2, R. Bidwell2, K. Abremski1, G. Hollis1,
S. Erickson-Viitanen1, L. Bacheler'. ' The Dupont Merck Pharmaceutical Co.;
Wilmington, DE 19880 Molecular Biology/Virology; Experimental Station
E400/5255 DE; 2Dupont Merck Pharmaceutical Co Ex Station 336/8B,
Wilmington DE, USA
Objectives: To compare the results of the Line Probe Assay (INNO-LiPA HIV-1
RT'"; Murex Diagnostics, Inc., USA) with clonal and population-based sequencing methods for detection of drug-selected mutations in the HIV-1 RT (reverse
transcripase) gene from HIV positive clinical samples.
Design: Retrospective examination of patient plasma samples from CPCRA
007 NuCombo trial (AZT vs AZT/ddl or AZT/ddC, Wilmington Medical Center;
Wilmington, DE)
Methods: Plasma samples were initially collected and clonal sequencing of
multiple-independent RT-PCR products was performed; patient CD4 and viral
loads were also determined. Retrospectively, these same samples were examined for drug-selected mutations using the Line Probe Assay (detection by
hybridization of RT PCR/PCR products with the specific probes for HIV-1 RT mutations immobilized on strips) and population-based sequencing. A comparison
of all three methods was made.
Results: Line Probe Assay (LiPAT') detection of drug-resistant mutations compared very favorably (>90% agreement for mutations on the strips) with clonal
and population-based sequencing methods, as shown earlier by Stuyver et al.
[Antimicrob. Agents and Chemother. (1997) 41:284-291].
Conclusions: Line Probe Assay (INNO-LiPA HIV-1 RT'"; Murex Diagnostics,
Inc. USA) appeared to be an adequate method (>90% agreement with clonal and
population-based sequencing methods) for detection of known resistance mutations in HIV-1 RT Because the LiPA technology is rapid, sensitive, and simple,
this method may prove useful for assessing HIV patients, before and during HIV
therapy, and evaluation of future choices for therapy.
41220 Assessment of the Line Probe assay in the detection of
nucleoside analogue resistance in a HIV-infected
paedriatric cohort
Marek Slomka1, F. Sheridan1, S. Muckelt1, D. Parker1, S.E. Read2, S. Cassol3,
S. King2. 1Murex Biotech Limited, Central Road, Dartford, England; 2Hospital for
Sick Children, Toronto; 3University of Ottawa, Ottawa, Canada
Background: Fifty-four HIV infected paediatric patients (ages: 6 months-18
years) were referred on the basis of symptoms or maternal HIV screening. Antiretroviral therapy was not administered to six of these patients. Sixteen patients
enrolled between November 1989 and December 1995 received AZT monotherapy and were changed to AZT/3TC dual therapy in early 1996. Thirty-two patients
enrolled in 1996 received dual therapy from the outset. From April 1997 onwards,
therapy of these 48 treated patients was reconsidered: Dual therapy was either
(i) continued or (ii) discontinued and the patients changed to a triple regimen of
3TC/D4T/protease inhibitor.
Methods: Viral load, CD4 count and clinical status were noted. The Line Probe
Assay (LiPA) was used to identify individuals infected with drug-resistant HIV due
to mutations in the HIV reverse transcriptase (RT). LiPA involves reverse hybridisation to identify specific RT mutations which confer resistance to AZT, 3TC, ddl
and ddC. DNA sequencing of the amplicons verified the presence of nucleoside
analogue resistant mutations. Resistance to AZT was also investigated by in vitro
culture of isolated virus. HIV subtypes were identified from sequence data which
was analysed by the PHYLIP molecular phylogeny software package.
Results: Identification of drug-resistant mutations by DNA sequencing correlated well with their recognition by LiPA. This was further confirmed by phenotypic
testing of HIV isolates by in vitro culture. Resistance to 3TC was observed more
frequently than resistance to AZT. Viral load and CD4 levels will be presented
and correlated with the patients' clinical status. Phylogenetic analyses revealed
a number of these paediatric patients to be infected with non-B HIV-1 subtypes.
Conclusions: The results demonstrate (i) a good correlation between LiPA

and DNA sequencing in the identification of specific drug resistant mutations, (ii)
anticipation of emerging resistance and (iii) the possibility that this may influence
the choice of alternative therapy combinations. The efficacy of LiPA among non-B
HIV infected patients was also noted.

I41221 Pattern of drug resistance in HIV population
Paola Corsi1, M. Pozzil, M. Zazzi2, G. Buffini1, L. Incandela1, E. Savalli1,
F. Leoncini1. 1U.O. Malattie infective-Careggi, Firenze, I; 2Dip. Bioloia Molecolare
Univ. Siena, Siena, Italy
Objectives: to evaluate the prevalence of drug resistance (R) in HIV patients.
Patients and methods: we retrospectively evaluated 240 HIV+ pts (22 naive,
218 experienced) between Jan 1996 and Dec 1997 in order to detect mutations
responsible for in Vitro R to reverse transcriptase inhibitors (RTI) (225 pts) and/or
protease inhibitors (89 pts). Two HIV-1 genome regions coding for aa 30-230
and 1-110 of RT and protease (PRO), respectively, were amplified by nested
PCR and directly sequenced with a Licor 4000L automated infrared sequencing
system. Drug R was estimated on the basis of mutations known to confer in vitro
R to different RT and PRO inhibitors.
Results: Drug R was present in 163 (67.9%) pts out of 240: RTI R was identified in 161/225 pts (71.5%) (group A), while protease inhibitors (PI) R in 11/89
(12.3%) (group B). We identified 4 out of 22 (18.2%) naive pts resistant to RTI.
One hundred and fifty nine out of 218 (72.9%) experienced pts had one or more
drug R. A multidrug R was evident in 52 out of 240 (21.7%) pts. In group A, high
frequency of R to ZDV (151/161, 95.6%) and 3TC (39/161, 24.2%) was present,
while R to ddl only in 6/161 (3.7%), ddC in 12/161 (7.4%), d4T in 1/161 (0.7%)
and NVP in 9/161 (5.5%) was present. In group B SQV R was evident in 4/11 pts
(36.3%), RTV R in 6/11 (54.5%), IDV R in 6/11 (54.5%) and NFV R in 1/11 (9%).
ZDV R was present in 146 (69%) out of the 212 ZDV treated pts, ddl R in 4/139
(2.9%), ddC R in 6/75 (8%), 3TC 30/73 (41.1%), d4T 1/38 (2.6%), NVP 4/5 (80%),
SQV 3/44 (6.8%), RTV 6/25 (24%) and IDV 1/20 (5%). No previous experience of
the drug was present in 8/154 (5.2%) for ZVD resistant pts, 2/6 (33.3%) for ddl,
6/12 (50%) for ddC, 9/39 (27%) for 3TC, none for d4T, 5/9 (55.5%) for NVP, 1/4
(25%) for SQV, none for RTV, 5/6 (83.3%) for IDV. Most frequent mutations were
at codon 215 (90/225), 70 (85/225), 41 (70/225), 67 (46/225), 210 (43/225), 184
(39/225), 219 (24/225). More than one mutation was present in 123 (75.4%) out
of 163 resistant pts.
Conclusions: our data show low prevalence of genotipic R to ddl and to d4T
and high prevalence of genotipic R to ZDV and 3TC. Otherwise pts treated with
ddl, d4T and 3TC presented shorter mean treatment time; ZDV was frequently
used in monotherapy and for a long period and 3TC was mostly added to previous inefficacy drug therapy in a period in which there were no alternatives. Low
prevalence of R was present for PI.
|41222 Impact of protease gene mutations on virological
response to nelfinavir
Eva Wolf', S. Mauss1, R. Pascucci1, A. Immelmann2, U. Dietrich3
E. Jaegel-Guedes1, H. Jaeger1. 1KIS, Curatorium for Immunedeficiency
Mozartstr. 3, D-80336 Munich; 2Analysis GmbH, Frankfurt;
3Georg-Speyer-Haus, Frankfurt, Germany
Rationale: Recent data support the correlation of phenotype drug resistance to a
protease inhibitor (PI) and the accumulation of so-called primary mutations within
the active site of the HIV protease distinct for the different inhibitors along with
the accumulation of secondary mutations.
Objective: To evaluate the predictive value of resistance mutations within the
protease gene concerning virological response to a subsequent antiretroviral
treatment with nelfinavir (NFV).
Methods: Retrospective analysis of genotype resistance pattern prior to a nelfinavir containing regimen with respect to virological outcome (nadir of viral load
(VL) reduction) within 8 weeks of treatment with NFV. Efficacy parameter was
the change in VL with responders (R) being defined by a VL reduction of _>0.5
log or a level below the limit of detection (0.5 KEq/ml). The presence of primary
mutations had been assessed for the codons 30, 48, 82, 84 and 90, secondary
mutations had been assessed for codons 46, 63 and 71.
Results: Of N = 21 pts 19 received a triple therapy with NFV, 2 a quadruple
2-PI containing regimen. 13 pts were R with a median VL reduction of -1.2 log as
compared to -0.01 log in the non-responders (NR). Difference in baseline CD4
count and VL was statistically not significant with median values of 260/Il and
11.2 KEq/ml in NR vs. 200//pl and 7.2 KEq/ml in R. 62.5% of the NR and 31.8% of
the R had at least one primary mut. prior to NFV. 4 of 5 NR with primary mutations
had a mutation at codon 90. 4 pts of each group had primary and secondary mut.
at the same time. Only 1 NR had a mutation at codon 30. 62.5% (5/8) of the NR
had a prior exposure to more than 1 PI over 11.0 months (med.) compared to
10.5 m. of PI therapy in 46% (6/13) of the R.
Conclusion: There is a trend towards more primary mutations in patients who
do not sufficiently respond to a treatment with NFV. However the existence of
primary mutations is not automatically associated with failure of a subsequent
therapy with nelfinavir. Possibly other factors than prior PI exposure or resistance
pattern have an impact on outcome.
|41223 Novel clinical applications of the LiPA HIV-1 RT mutation
detection assay

Colin Evans1, D. Parker1, F. Sheridan1, J. Scott1, G. Wouters2, L. Styuver2,
A. van den Abeele2. 1Murex Biotech Limited Central Road, Dartford, Dent,
England; 2lnnogenetics BV, Canadastraat, Zwijndecht, Antwerp, Belgium
Background: Development of HIV-1 species resistant to nuceloside analogues
have been well reported. Mutations at key codons have been associated with
resistance to specific antiviral drugs. Murex-lnnogenetics LiPA HIV-1 RT has