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

770 Abstracts 41138-41142

12th World AIDS Conference

Results: The TaqManTM-based PERT showed the same sensitivity as the
PERT assay using ELISA for product measurement. 20 plasma samples could
be tested in duplicate within 5 hours. The cut-off was determined by analysing
100 healthy blood donor plasma samples. Measurement of particle associated
RT from 70 HIV-positive plasma samples by TaqManTM-based PERT showed a
strong correlation with particle-associated viral RNA (P < 0.0001 with r = 0.832).
Moreover, the individual treatment-induced changes in viral RNA and RT of 15
patients showed a similar course.
Conclusions: The combination of sample pretreatment by Eppendorf centrifugation and TaqMan'm-based PERT assay yields a high throughput assay for
RT-based virus load assessment and for detection of retroviruses in general.
41138 1 Evaluation of Vacutainer~ Brand PPTM plasma
preparation tubes for HIV viral load testing
Lynne Rainen1, K. Kuramoto2, E. Bonney2, T. Alcorn3. 'Becton Dickinson
Vacutainer Systems 1 Becton Drive Franklin Lakes NJ 07417; 2Sacramento
Center for Blood Research Sacramento CA; 3Laboratory Corp. of America Res.
Triangle Park NC, USA
Background: The objective of this study was to evaluate the performance of a
new blood collection tube for HIV viral load determinations.
Methods: Venous blood samples were drawn from forty (40) consenting HIVpositive patients. Four tubes for the preparation of plasma were drawn from
each patient: two Vacutainer" Brand Plus'" K2 EDTA and two Vacutainer> Brand
PPT~" tubes (PPT), plastic, gel-separator blood collection tubes spray coated with
K2EDTA. Blood samples were centrifuged in a swing-out bucket rotor at 1100 x g
within two hours of collection. Plasma from PLUS K2EDTA tubes was withdrawn
from the tubes immediately after centrifugation. Plasma in PPT~" tubes was stored
in situ. For each patient, one spun PPT tube and plasma from one PLUS K2EDTA
tube were stored at ambient temperature prior to sample extraction. The second
set of plasma and PPT tubes was frozen, stored at -70 C and shipped on dry ice.
On the day of testing, paired (PPT/PLUS K2EDTA) samples or extracted pellets
were thawed at ambient temperature and tested using the Roche Amplicor HIV-1
Monitor0" Test.
Results: Analysis of variance, correlation plots, and corresponding regression
analyses were used to compare viral load results between the PPT tube and
the EDTA PLUS control. For PLUS K2EDTA/PPT paired samples extracted from
fresh plasma and frozen plasma, the p-values were 0.2260 and 0.2300 respectively. For the same paired samples, correlation estimates were 0.967 and 0.968
respectively. These results indicate that the differences in HIV viral load between
the PLUS K2EDTA and PPT tubes are not significant when testing is performed
either on extracts of fresh plasma or on plasma stored and shipped in the PPT
tube at -70"C.
Conclusions: The results of this study indicate that HIV viral load obtained from
PPT tube plasma is equivalent to standard EDTA plasma. In addition, plasma
can be stored and shipped in spun PPT tubes making them safer and more
convenient than regular EDTA tubes.
41139 Ultrasensitive HIV detection and quantitation by
multi-target reverse transcription (RT)-linked polymerase
chain reaction (PCR)
Wei Lu1, L. Cao2, J. Yuan2, J.M. Andrieu2. 1 Unite de Virologie Moleculaire,
Hopital Laennec, 42 Rue de Sevres, Paris 750074; 2Hopital Laennec, Paris,
France
Viral load measurement has been demonstrated to be critical for therapy monitoring and disease prognosis. Due to the broad variation of HIV genomes, only a
limited number of HIV variants are so far detected and/or quantitated by conventional RT-PCR using a single primer pair and an internal standard. Recently, we
have developed a new assay that combines multi-target RT-PCR using multiple
primers pairs for high-sensitivity detection and a universal calibration system
including an external standard and an internal control. A prospective study was
conducted to determine whether this new assay is more sensitive and accurate
than the conventional one for the viral load measurement.
A total of 234 plasma samples from HIV-1 seropositive patients were tested
in parallel with the new assay and a commercial kit (Amplicor HIV- 1 Monitor0,
Roche Diagnostic System, Somerville, NJ, USA) which uses the conventional
quantitative RT-PCR. The detection limit of Monitor'" was 200 HIV RNA equivalent (eq) copies/ml, while our new assay attained a detection limit of 10 HIV
RNA eq copies/ml. Of 234 samples tested, 206 (88%) were positive for the new
assay and only 143 (61%) were positive for Monitor0t. Out of the 91 samples
undetectable by Monitor'", 63 (69%) were still detected by the new assay and
ranged widely from 10 to 10.000 eq copies/ml. None of the 28 samples undetectable by the new assay was detected by Monitor~'. Among 143 double positive
results, in 11 cases the viral load assayed by Monitor'" was >2 logio lower than
that assayed by the new assay; in the remaining 132 cases a linear correlation
between the two assays (r2 = 0.715) was observed with no difference >1 logio.
The improved clinical sensitivity and quantitation accuracy of this ultrasensi
tive assay based on the use of multi-target RT-PCR calibrated with an external
standard and an internal control represent a significant advance in molecular
diagnosis of HIV.

I41140 HIV-1 proviral DNA and plasma RNA changes in advanced
patients after 6 months of triple therapy including
indinavir
Lidia Ruiz1, C. Christopherson2, W.A. O'Brien3, T. Puigt, M.A. Martinez De La
Seirra1, J. Sninsky2, B. Clotet'. 'Fundacio IRST- CAIXA. Hosp. Germans Trias
1 Pujol, Badalona, Spain; 2Roche Molecular System, Alameda, CA;
3Department of Medicine, University of Texas Branch, Galveston, TX, USA
Objective: To evaluate plasma HIV-1 RNA and proviral DNA changes in advanced
HIV infected patients on HAART after 6 months.
Patients and Methods: Sixteen patients, in whom indinavir was added to the
previous reverse transcriptase (RT) nucleoside analogue therapy, were included
in the study. Overall, median initial CD4 counts were: 31 cells/mm3 (range:
0-80). Plasma HIV-1 RNA levels were determined by the Amplicor HIV-1 (Roche;
Madrid, Spain) at baseline and at weeks 2, 4, 12 and 24. HIV-1 proviral DNA
in PBMC's was quantified at baseline and at wk 24 using a prototype assay
involving DNA extraction with a proteinase K solution and amplification with rtTh
DNA polymerase. Detection was performed using the Amplicor HIV-1 Monitor Test
format. Patients were classified according to the decrease in plasma HIV-1 RNA
level as follows: i) Responders (R): an initial decrease in plasma viral load >1.0
log and sustained response >0.5 log from baseline (n = 7); ii) Non-Responders
(NR): either no decrease in viral load >1.0 log at any time point after treatment or
a transient decrease with a rebound above 0.5 log from baseline (n = 9). In both
groups, mean initial plasma HIV-1 RNA was 5.5 log (range:2.6-6.2).
Results: In R patients there was a decrease in plasma RNA of 1.5 logs at wk
24 compared with the baseline value. However, in the NR group no changes in
plasma RNA were observed. Total mean DNA copy number was 1,386/105 PBMC
before indinavir treatment. We observed that R patients had a lower initial proviral
DNA (421 copies/105 cells) compared with the NR patients (2,138 copies/105
cells). Since baseline HIV RNA levels were the same, the HIV RNA/DNA ratio was
approximately 4-fold higher in R patients. After 6 months, proviral DNA diminished
97 copies/106 and 405 copies/105 PBMC in R and NR groups respectively.
Conclusions: HIV-1 proviral DNA decreased modestly in total PBMC during
indinavir treatment in HIV advanced patients. A better virological response was
associated with higher initial HIV RNA/DNA ratios.
411411 Response of HIV viral load to antiretroviral mono and
combination therapy
George Kamkamidze, T.K. Shrtava, Georgia Tbilisi, T.N. Tsertsvadze. Georgian
AIDS & Clin. Immun. Center 16, Al Kazbegi Av Tbilisi 380060, Georgia
Objectives: Study of dynamics of HIV viral load and CD4+ cell counts in response
to mono-zidovudine (AZT) and triple (AZT + lamivudine + saquinavir) antiretroviral
therapy.
Design: Prospective follow-up study
Methods: 24 patients with HIV infection were included in prospective follow-up
study. The baseline average level of viral load was 86000 copies/ml and of CD4+
cell count was 146 cells/mI. The monitoring of viral load and CD4+ cells count
were performed at 2nd, 4th, 12th, 20th and 24th weeks after initiation of the
treatment. Two groups were separated (12 patients in each one) with different
regimens of treatment. The drug regimen in 1st group was as follows: AZT 600
mg daily and in 2nd group: AZT 600 mg daily, lamivudine 300 mg daily, saquinavir
1800 mg daily. Viral load was measured by quantitative PCR method and for
determining of CD4+ cell count the Immunofluorescence Assay was used.
Results: Results of laboratory investigations are given in the table:
Weeks after initiation of the treatment: 2 4 12 20 24
Mean viral load decrease (log) 1st group 0.5 0.7 0.6 0.2 0.1
2nd group 1.5 2 2.5 2.7 2.7
Mean CD4+ increase (cells/ml) 1st group 20 40 20 10 0
2nd group 100 105 110 100 110
Conclusion: Combination therapy has been shown to provide greater and
more sustained increases in CD4+ cell counts (p < 0.001) and decreases in HIV
viral load (p < 0.001) than monotherapy.
289*/41142 Assessment of viral load and CD4/CD8 ratio in
blood and lymph nodes in patients treated with
d4T, ddl ~ hydroxyurea (HU)
Sabine Yerly1, O. Rutschmann2, F. Marchal3, M. Opravil4, B. Hirschel2,
L. Perrint. Swiss HIV Cohort Study; tLaboratory of Virology, University Hospital
1211, Geneva 14; 2Div. Infectious Diseases, Geneva; 30RL Clinic, Geneva; 4Div.
Infectious Diseases, Zurich, Switzerland
Background: Lymphoid tissue represents the main site of viral replication. We
evaluated viral load and CD4/CD8 ratio in blood and lymph nodes (LN) in 18 pts
(15 drug-naive) after 6 to 14 months' treatment (5 pts ddl/d4T, 13 pts ddl/d4T/HU).

Methods: Viremia (detection limit, 1.3 log RNA/ml) and lymph node cells (LNC)
or peripheral blood mononuclear cells (PBMC) associated HIV RNA (detection
limit, 1.3 log RNA/106 CD4) were assessed using modified Amplicor HIV Monitor
assays.
Results: Baseline viremia: 4.25, CD4: 352. Mean viremia decrease from baseline to the time of LN biopsy was -2.21 (-3.60 to -1.16). Only 3 pts had viremia
<20 copies/ml. We have found correlations between viremia and PBMC RNA/106
CD4 (P = 0.006) and LNC RNA/106 CD4 (P = 0.007), and between CD4/CD8