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

12th World AIDS Conference

Abstracts 41133-41137 769

Teknika); Branched DNA (bDNA) Quantiplex 2.0 HIV-RNA (Chiron Diagnostics).
We analysed 100 consecutive samples from HIV1-infected patients representing
all stages of infection and whose viral load had to be evaluated for proper clinical
management. Uncoagulated samples (EDTA) were centrifugated at 4000 x g for
10 minutes and the resulting plasma fraction was collected, divided into aliquotes
and stored at -70 C until processing. With regard to the reproducibility of results,
2 serum samples with, respectively, 1000 and 5000 HIV-RNA copies/ml when
assayed in the NASBA test were selected for the intra-assay (8 replicates per
sample) and inter assay (5 runs) precision tests. Out of the quantitative results
obtained with the new method on 100 assayed specimens, 93% were in the range
of 0.3 logic when compared with HIV-RNA values obtained with the NASBA and
the bDNA assays. Such percentage decreased to 71% when HIV-RNA values
were compared with those of the Amplicor RT-PCR. Coefficients of variability
(CVs) in the intra-assay reproducibility tests were constantly <15% for both the
new method and the 3 reference assays. In the inter-assay reproducibility tests,
CVs ranged between 10% and 21%. Moreover, the specificity of HC-II HIV-RNA
test was evaluated by analysing 50 sera collected from HIV-negative subjects
and resulted equal to 98% (49/50 negative results).
The new test for HIV-RNA quantitation supplied results that are comparable
with those obtained by other commonly used methods, and proved to be rapid,
easy to perform and therefore useful in order to monitor patients undergoing
anti-retroviral therapy
41133 Highly sensitive HIV RNA quantitation from plasma,
peripheral blood mononuclear cells and lymphoid tissues
Marek Fischer, W. Huber, A. Kallivroussis, P. Ott, M. Opravil, R. Weber,
R.W. Cone. University Hospital Zurich Div Inf Dis Ramistrasse 100 8091 Zurich,
Switzerland
Objectives: To establish sensitive methods to monitor levels of HIV RNA in
plasma, peripheral blood mononuclear cells (PBMC) and lymphoid tissues from
HIV-infected subjects.
Design: Modified forms of the Amplicor HIV-1 Monitor test (Roche Diagnostic
Systems) was used to examine specimens from plasma, PBMCs and lymphoid
tissue from patients on potent antiretroviral therapy.
Methods: Nucleic acids from plasma were extracted in the presence of a
HIV-derived synthetic RNA (QS RNA) provided as quantitation standard in the
Amplicor test. Cellular RNA from PBMCs and lymphoid tissue was purified on
silica columns (Qiagen) in the absence of QS-RNA, treated with DNAsel, reextracted, monitored for total RNA yields by quantitative densitometry and mixed
with QS RNA. Extracted RNA was subjected to RT PCR using the Amplicor HIV-1
Monitorm" test.
Results: HIV RNA in plasma was measured with detection limits ranging from
4 to 20 copies HIV RNA per mL. Cellular HIV RNA was measured with detection
limits as low as 3 copies of HIV transcripts per million cells. HIV RNA in plasma
was determined with sensitivities comparable to the so called ultrasensitive protocol suggested by Roche. In our modification, since QS RNA is added at the
very beginning, we can control all the steps of the procedure from the extraction
to the end.
Conclusion: Adapting the Amplicor HIV-1 MonitorTM test to be used with specimens purified with silica matrices enabled us to monitor extremely low levels of
HIV RNA in either cells or plasma.
41134 1 HIV viral load measurement by Amplicor HIV-1 Monitor:
Analysis of within-lot and between-lot variation
Christopher Sherlock1, K.J.B. Craib2, P.G.A. Cornelisse2, J.S.G. Montaner2,
M.V. O'Shaugnessy2. 'Diagnostic Virology & Ref. Laboratory 1081 Burrard St.,
Vancouver, B.C. V6Z 1 Y6; 2B.C. Centre for Excellence in HIV/AIDS, Vancouver,
BC, Canada
Objectives: To measure the within-lot and between-lot analytic variation for the
Amplicor HIV-1 Monitor test for viral load measurement.
Methods: Anticoagulated blood samples were collected from patients known
to have HIV-1 infection. Plasma was separated from cells within 6 hours. All
samples were aliquotted and frozen at -20"C immediately after separation. Sample acquisition, handling and storage as well as test performance were rigidly
standardized according to the manufacturer's instructions. Within-lot variation
was measured by repeated testing, in consecutive runs, on high-volume samples
from 2 patients. Between-lot variation was measured by using 3 different lots (X to
Z) for this testing. Between-lot variation was also measured by testing a panel of
36 samples using 4 consecutive kit lots. ANOVA was used to assess between-lot
variation after adjustment for differences in viral load levels between samples.
Scheffe's test was used for pairwise comparison of lots. Within-lot variation was
assessed by calculating coefficients of variation (CV). Loglo transformations were
used for all calculations.
Results: High volume sample 1 was tested 19 times with lot X (CV = 23%),
70 times with lot Y (CV = 35.72%), 92 times with lot Z (CV = 28.9%). Sample 2
was tested 18 times with lot X (CV = 28.9), 69 times with lot Y (CV = 24.9%), 84

times with lot Z (CV = 31.44%). Scheffe's test demonstrated significant variation
between some lots (range 0.14 loglo [p < 0.05] to 0.29 loglo [p < 0.05]) but not
between others (range.0291oglo to.068 logio; p > 0.05).
Conclusions: It is safe to assume that lab variation is consistent in this study
so that we can conclude that the significant variation noted between kit lots is
due to the manufacturing process. It is important to quantify this before setting
standards for changes in viral load requiring medical intervention. The greatest

lot-to-lot variation seen in this study (0.29 loglo) is below the level usually set
for clinical significance (0.5 loglo). Within-lot CVs were consistent with other
published figures.
165*/41135 A community-based study of HIV-2 RNA load in
pregnant women attending health centres, in West
Africa
Pa Tamba Ngom', S.A. Alabi', K. Ariyoshi', N. Berry2, D. Odonovan1,
H.C. Whittle'. 'MRC Laboratories, PO Box 273, Banjul the Gambia, West Africa,
The Gambia; 2Dept of Virology University College, UK, London, UK
Objective: To investigate plasma HIV-2 RNA levels in an unbiased population.
Materials and Methods: 60 HIV-2 positive pregnant women were randomly
selected from 220 HIV-2 positive women identified in a community-based HIV
transmission study. RNA was extracted from 200 /l of plasma using a described
method (Boom 1990). Reverse-transcription and PCR amplification were separately carried out with primers targeting the conserved regions of the U5/R HIV-2
LTR. The lower limit of detectability by the nested PCR was estimated to be
250-500 copies/ml. The cDNA was quantified by the enzyme linked oligonucleotide assay. The number of RNA copies/ml was estimated using external
standard controls where the number of RNA copies/mi were determined by
limiting dilution analysis.
Results: HIV-2 RNA was detected in 61.7% (37/60) plasma samples. The
median RNA load among the RNA positive samples was 2588 copies/ml with
considerable variation (range 500-70,000 copies/ml). There was a significant
inverse correlation between RNA load and CD4 count (p = 0.03 by Spearman).
There was no evidence that active syphilis co-infection increased HIV-2 RNA load.
Conclusions:The majority of HIV-2 pregnant women have very low levels of
virus in plasma. Therefore the low rate of HIV-2 replication may be responsible
for better prognosis and low rate of transmission in HIV-2 infection. Comparison
with HIV-1 RNA level in the same population is in progress.
141136 A competitive RT-PCR method for the quantitation of
HIV-2 viral load in plasma
Perpetua Gomes', N. Taveira1, R. Antunes', K. Mansinho2, F. Antunes3,
M.H. Lourenco1. 1Faculdade Farmacia Universidade Lisboa, Av. Forcas
Armadas 1600, Lisboa; 2Hospital Egas Moniz, Lisboa; 3Hospital Santa Maria,
Lisboa, Portugal
Objectives: Development of a quantitative competitive RT-PCR assay for the
detection of HIV-2 RNA and quantitation of plasma viremia in HIV-2 infected
individuals.
Methods: Thirty HIV-2 seropositive individuals were included in this study, of
which 24 were asymptomatic and 6 symptomatic. CD4 + T cell number was determined by FACS analysis. Viral load in PBMCs was determined by performing
end-point-dilution cultures. Plasma viremia was determined by using a quantitative-competitive-RT-PCR assay with ELISA detection of the amplified products.
In this assay a dilution series of a competitor RNA of known concentration as
well as HIV-2 RNA from the test specimens were reverse transcribed, and PCR
amplified with biotin labelled primers. Amplicons were captured into streptavidincoated microplates and hybridized with digoxigenin-labelled probes. Hybridization
was detected by a colorimetric reaction with an alkaline phosphatase conjugated
anti-digoxigenin antibody. The relative amounts of HIV-2RNA versus competitor
were calculated by plotting the optical density (OD) for competitor /OD wild-type
against competitor RNA copy number.
Results: The dynamic range of the hassay was 3 logo with a lower detection
limit of 100 copies of HIV-2 RNA. Plasma viral load could be determined by
RT-PCR for only 33% (10) of the patients. Virus isolation was achieved for 12
subjects with titers ranging from 0.5 up to 500 TCID/106 cells. Viral load correlated inversely with the number of CD4+ T cells. In one patient plasma viral load
decreased by two orders of magnitude after one week of triple drug therapy.
Conclusions: The quantitative-competitive RT-PCR assay is highly sensitive
for the detection of HIV-2 RNA. Cellular viral load is low in HIV-2 infected individuals as compared to HIV-1 individuals. In contrast to infection by HIV-1,
the majority of HIV-2 infected individuals has undetectable levels of virus in the
plasma. Quantitation of plasma viral load with our RT-PCR assay may be usefull
to monitor the efficacy of antiviral therapy.
|141137 Quantitation of HIV-1 virus load by a TaqMan'"-based
product enhanced reverse transcriptase assay
Urs Hummel, J. Boni, J. Schupbach. National Center for Retroviruses,
Moussonstr. 13 Ch-8044 Zurich, Switzerland
Objectives: The ultrasensitive Product Enhanced Reverse Transkriptase (PERT)
assay (Pyra et al., PNAS, 1994, 91, 1544) can detect 3-11 retrovirus particles
based on their content of RT. The test is based on reverse transcription of MS2
phage RNA by the sample's RT and amplification of the cDNA by PCR. The aim

of this study was to simplify the method in order to enable testing of larger sample
numbers.
Methods: Filtered plasma was diluted with PBS and the virus was pelleted for
1 hour with a microcentrifuge. The pellet was resuspended in PBS. The PERT
assay was adapted to the TaqMan'" technology using a Fam/Tamra labeled probe
and the reference dye Rox during the PCR step. RNA was determined by the
Amplicor HIV Monitor Kit.