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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 91, Num. 6, 1996
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 91(6),
Nov./Dec. 1996,
RESEARCH NOTE
HIV-1 Isolation from Plasma Specimens
CI Costa/+, MG Morgado, VGV Santos*, HEC/FIOCRUZ AIDS Clinical
Research Group*, V Bongertz
Laboratorio de AIDS e Imunologia Molecular, Departamento de
Imunologia *Hospital Evandro Chagas, Instituto Oswaldo Cruz,
Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil
Financial support by the Integrated Programme of AIDS (PIAF)
of the FIOCRUZ/MS and the Brazilian National Council for
Research (CNPq).
*B Grinszteijn, MCG Galhardo, MRC Guimaraes, S Cavalcante, VC
Rolla.
+Corresponding author. Fax: +55-21-280.1589
Received 29 January 1996, Accepted 11 July 1996
Code Number: OC96135
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Key words: HIV-1 - AIDS - isolation - plasma
The human immunodeficiency virus type 1 (HIV-1), ethiologic
agent of AIDS, can be detected within weeks to months of
exposure. HIV-1 can be isolated from peripheral blood
mononuclear cells (PBMC) of HIV-1 infected patients (RW
Coombs et al. 1993 J Clin Microbiol 31: 1980-1986).
The data accumulated up to now indicate that plasma virus
levels may be useful to indicate the rate of viral replication
in infected people and thus may possibly be of value for
prognostic analyses. During acute HIV-1 infection, the
frequency of virus isolation and the plasma virus titers are
high (ES Daar et al. 1991 N Engl J Med 324: 961-964, DA
Cooper et al. 1995 Lancet 1: 537-540). Following
initial viremia, however, the viral load in plasma decreases
up to more than a 100 fold (Daar et al. loc. cit.). In
many cases this reduction in viremia occurs within a period of
several weeks and is correlated most probably with the
antiviral immune response.
In designing a collaborative project between laboratories and
hospitals in different states of Brazil, the difficulty of
shipment of fresh blood was discussed. As there is minimal
published data on the stability of HIV-1 load markers with
respect to sampling, processing and storing conditions, an
attempt to isolate virus from frozen plasma aliquotes was
carried out.
For the present study, nine adult women, belonging to a
cohort of the Evandro Chagas Hospital, were included. All
women were infected heterosexually, at between 24 and 37 years
of age (mean age: 26.5). Some additional clinical information
was available for these patients (see Table). Sample
collection dates varied between 2 months and 15 days before
starting of the experiment. On receival of blood samples, PBMC
were separated by gradient centrifugation and plasma was
aliquoted and stored at -20 C. Surplus PBMC were
cryopreserved.
Isolation of HIV-1 from fresh or frozen PBMCs was carried out
using the traditional co-culture method with
phytohemmaglutinin (PHA) stimulated normal donor PBMCs (H
Ruebsamen-Waigmann et al. 1994 AIDS Res Hum Retroviruses
11: 1401-1408). For isolation of HIV-1 from plasma
specimens, the sediment of 2ml of plasma (ultracentrifuged
40,000 x g/60 min), resuspended in 0.5 ml of culture medium
(RPMI 1640, Sigma, St Louis, USA) was used to infect 5x106
PHA-stimulated donor PBMCs. The cells were gently resuspended
in 0.5 ml of medium and incubated with the samples at 37 C/5%
CO2/30 min. At the end of the incubation period, 4 ml of
culture medium were added. Thereafter, the cultures were
maintained just as routine co-cultures (Ruebsamen-Waigmann et
al. loc. cit.). Isolation success was evaluated by
HIV-1 p24 antigen determination (HIV-1 p24 Ag detection kit,
ELISA, DuPont, Wilmington, DE) in supernatants of the 7th and
14th day of co-culture.
HIV-1 isolation from plasma was positive from all samples
from which HIV-1 had been originally isolated from fresh PBMC
(6/6). Isolation of HIV-1 from frozen PBMC was positive for
only three out of the four attempts carried out. Three samples
from which HIV-1 isolation from fresh PBMC had been negative
were also included. Isolation of HIV-1 from frozen PBMC was
successful in the attempt carried out, while isolation from
plasma samples from three patients were positive in two
attempts. These results indicate that a second attempt of
HIV-1 isolation from stored materials is worthwhile, and that
no significant difference between HIV-1 isolation from plasma
or cryopreserved PBMC could be noted.
A number of studies have reported the successful isolation of
HIV from cell-free body fluids, including plasma and serum (RW
Coombs et al. 1989 N Engl J Med 321: 1626-1630). The
frequency obtained of plasma isolation reported was higher in
individuals with more advanced disease as indicated by lower
(<200) concentrations of CD4 positive lymphocytes in the
peripheral blood (C Rouzioux et al. 1992 AIDS 6:
373-377, LD Robin et al. 1992 J Acquir Immune Defic Syndr
5: 822-828). In this study, only two women had AIDS, but
overall CD4 concentrations were quite low. However, our
results indicate no specific correlation between phase of
disease and of HIV-1 isolation from plasma.
In conlusion, a similar efficiency of recovery of virus from
PBMCs (both fresh and frozen) and from plasma was observed.
Therefore, it is possible to isolate HIV-1 even if local
laboratory facilities prepared for routine procedures in cell
culture are not available. An important difference observed
between positive isolation of HIV-1 from plasma and PBMCs was
the culture period needed for attaining positivity; probably
reflecting lower concentration of infectious virus in plasma
than in CD4 positive PBMCs. The efficient isolation of HIV-1
from plasma samples described here should be of importance for
studies envolving frozen plasma samples stored at sites were
efficient collection and storage of PBMCs is not feasable.
Acknowledgements: to Dr Carlos Bonecker, Santa Catarina
Center for Haemophiliacs, Rio de Janeiro, RJ, Brazil for
availability of blood from HIV-1 seronegative blood donors.
TABLE: Information on number of patients (No.), date of
sampling (Date), clinical classification (Clin. class -
according to CDC 1986), period of time between sampling and
first positive serologic diagnosis (Time post inf.), number of
CD4+ cells (No. CD4/mm3), and antiretroviral treatment
(Chemotherapy) is indicated for each patient in paralell to
viral detection (Days of co-culture)
Day of p24 detection
--------------------
No./Date Clin. Time No.CD4/ Chemo- Fresh Frozen Plasma
class post-inf. mm3 therapy PBMC PBMC
-------------------------------------------------------------
1 Mar 95 II >9m 83 Y - ND 14
2 Feb 95 II >5m NA Y 7 ND 14
3 Feb 95 III >3y 600 Y 7 14 14
4 Feb 95 IV >2y 87 Y 7 ND 14
5 Feb 95 IV >5m 312 N 7 14 14
6 Feb 95 III >2y 356 N 14 - 14
7 Mar 95 II >2y NA N 7 7 14
8 Apr 95 NA >7m NA Y - 14 -
9 Apr 95 II >1m 308 N - ND 14
NA: not available; ND: not done. Viral antigen detected using
HIV-1 p24 ELISA kit (DuPont, Wilmington, DE); N: no
treatment; Y: treatment; PBMC: peripheral blood mononuclear
cells.
Copyright 1996 Fundacao Oswaldo Cruz
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