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Journal of Clinical Microbiology, March 1998, p. 809-811, Vol. 36, No. 3
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Highly Sensitive Method for Amplification of Human
Immunodeficiency Virus Type 2 DNA
Florence
Damond,1,*
Ibtissam
Loussert-Ajaka,1
Cristian
Apetrei,1,2
Diane
Descamps,1
Sandrine
Souquière,1
Annie
Leprêtre,3
Sophie
Matheron,3
Françoise
Brun-Vézinet,1 and
François
Simon1
Laboratoire de
Virologie1 and
Service des Maladies
Infectieuses et Tropicales,3 Hôpital
Bichat-Claude Bernard, 75018 Paris, France, and
Virus
Laboratory, Microbiology Department, School of Medicine, "Gr. T. Popa" University, 6600 Iasi, Romania2
Received 30 July 1997/Returned for modification 27 October
1997/Accepted 10 December 1997
 |
ABSTRACT |
We evaluated a new human immunodeficiency virus type 2 (HIV-2) DNA
amplification strategy based on peripheral blood mononuclear cell long
PCR (XL PCR) followed by nested PCR amplification. The primers used
were located in the highly conserved long terminal repeat and in the
pol regions of the genome. Five primer pairs corresponding
to different regions of the HIV-2 env gene were used in the
nested step. Samples from 42 patients were tested, which yielded
positive amplification with at least two primer pairs in 40 (95%)
samples. A primer pair (EB2/EB5) located on the V3 region succeeded in
amplifying proviral DNA in 40 samples.
| |
TEXT |
The worldwide spread of human
immunodeficiency virus type 1 (HIV-1) contrasts with the limited
circulation of HIV-2, which is mainly restricted to West Africa
(5). Most cases of HIV-2 infection diagnosed outside Africa
occurred in France and Portugal (8). The vertical and
heterosexual HIV-2 transmission rates are lower than those for HIV-1
(10, 12). The incubation period of HIV-2 is longer than that
of HIV-1 (1). The HIV-2 reduced replicative capacity
supports these epidemiological and clinical pieces of evidence that
HIV-2 is less pathogenic than HIV-1 (15), even at the AIDS
stage (11). Phylogenetic studies have shown a relatively
wide diversity of HIV-2 lentiviruses, and six viral subtypes have been
described (4, 6).
Together with the low replicative capacity, viral variability
contributes to the inefficiency of direct virological diagnosis. HIV-2
is rarely isolated from peripheral blood mononuclear cells (PBMCs)
(15), and the detection rates of proviral DNA by PCR amplification vary between 50 and 80% (7, 9) according to clinical and immunological status. A highly sensitive test is badly
needed for diagnosis, mainly in HIV-2 vertical infection screening and
dual HIV-1-HIV-2 seropositivity differentiation. We describe a new PCR
strategy based on long PCR (XL PCR) followed by different nesting steps
to improve the sensitivity of HIV-2 DNA detection.
Patients.
The study group was composed of 41 patients included
in the French National HIV-2 Cohort and one recent HIV-2 seroconverter. These patients (20 males and 22 females) originated from various countries in West Africa (Burkina Faso, n = 1; Cape
Verde Islands, n = 4; Congo, n = 2;
Gambia, n = 1; Guinea-Bissau, n = 5;
Ivory Coast, n = 10; Mali, n = 6;
Senegal, n = 4), North Africa (Morocco, n = 1), and Europe (France, n = 7;
Poland, n = 1). According to the Centers for Disease
Control and Prevention (CDC) classification (3), 34 patients
were in stage A and 8 were in stage C. Thirteen patients had
CD4+ cell counts above 500 × 106/liter,
14 patients had counts between 200 × 106 and 500 × 106/liter, and 15 patients had counts below 200 × 106/liter. Ten patients received monotherapy, 5 were on
bitherapy, and 1 was on tritherapy.
Samples.
Whole blood was collected from all of the patients,
and PBMCs were isolated by Ficoll-Hypaque density gradient
centrifugation. DNA was extracted with phenol-chloroform, precipitated
with ethanol, and quantified spectrophotometrically. Cellular and
plasma viral cultures were carried out with each sample, and
quantitative cellular viremia and plasma viremia were determined as
previously described (15).
XL PCR.
HIV-2 DNA in fresh PBMCs was amplified by nested PCR
with first-round XL PCR. The Gene Amp XL PCR kit is optimized to
produce high yields of long (XL) PCR products. The enzyme is designated to amplify target DNA sequences ranging from 5 kb to more than 40 kb.
The assay was performed according to the manufacturer's instructions
(Perkin-Elmer, Applied Biosystems, Alameda, Calif.). We designed the
following primer pairs for use in the first amplification step: PFD1
(5')/long terminal repeat (LTR9574) (3') and LTR1 (5')/LTR2 (3') (Table
1). One microgram of genomic DNA was subjected to the first-round
amplification. The PCR conditions consisted of initial denaturation at
94°C for 1 min, followed by 16 cycles of denaturation at 94°C for
15 s, annealing at 55°C for 1 min, and extension at 68°C for 5 min. The subsequent 24 cycles were carried out under the same
conditions with incremental lengthening of the extension time (15 s per
cycle) and a last extension cycle at 68°C for 10 min. HLA-DQ
was
routinely amplified as a PCR efficacy control.
Nested PCR.
Different pairs of primers were used for nested
PCR (Table 1). The nested PCR procedure
consisted of amplification of 5 µl of the first XL PCR product in a
final volume of 100 µl with 10 µl of PCR buffer, 200 µM (each)
deoxynucleoside triphosphates, 30 pmol of internal primers, and 2 U of
Taq polymerase. The PCR conditions were as follows: 40 amplification cycles with denaturation at 94°C for 5 min, followed by
38 cycles (30 s at 94°C, 30 s at 55°C, and 30 s at
72°C), and a last extension cycle at 72°C for 7 min. The five
primer pairs used in the nested PCR were EB0/EB6, EB2/EB5,
EB7/ENVE, ENVA/ENVB, and ENVF/ENVG (Table 1). DNA from negative cells (PBMCs from healthy blood donors) was introduced into
each reaction. Nested PCR products were analyzed on a 1.4% agarose gel
with ethidium bromide staining.
The virus was isolated from PBMCs of 25 of the 42 patients (60%). The
frequency of virus isolation from PBMCs correlated negatively
(
P < 0.05) with the CD4
+ cell count.
Cultures were positive for 2 of 13 (15%) patients
with
CD4
+ counts of >500 × 10
6/liter and for
none of those receiving antiretroviral treatment.
For the 14 patients
with CD4
+ counts ranging from 200 × 10
6
to 500 × 10
6/liter, cultures were positive for 11 (78%) patients, 2 of whom
were receiving antiretroviral treatment.
Cultures were positive
for 12 (80%) of the 15 patients with
CD4
+ cell counts below 200 × 10
6/liter,
all of whom were receiving either one (
n = 8) or two
(
n = 4) antiretroviral drugs. Of the 25 patients with a
positive
HIV-2 culture, 19 were symptom free, and 6 were in stage C of
the CDC classification (Table
2).
The mean CD4
+ count in the 17 patients with negative
cultures was 637 × 10
6/liter (range, 139 × 10
6 to 1,515 × 10
6/liter); 15 patients were symptom free (stage A), and 2 were in
stage C. Fifteen of
the 17 patients with negative cultures had
never received
antiretroviral therapy. The virus was isolated
from the plasma of 4 of
41 (10%) patients, all of whom had CD4
+ cell counts below
200 × 10
6/liter (mean, 75 × 10
6/liter; range, 13 × 10
6 to 155 × 10
6/liter). Two of these patients were symptom free (stage
A3), and
the other two were at stage C. All four patients were on
monotherapy
(Table
2).
The PCR strategy consisted of first-round amplification with primer
pair PFD1/LTR9574. When the nested PCR was negative with
these primers,
a new XL PCR procedure was performed with LTR1/LTR2.
The results of nested PCR with the different primer pairs are presented
in Tables
2 and
3. The test was positive
with at
least one primer pair for 40 of 42 (95%) patients. The results
of proviral DNA amplification are presented in Table
2 according
to
clinical and virological data. Twenty-nine samples, of which
7 were
from culture-negative patients, were positive with all
five primer
pairs. Five samples, of which three were from culture-negative
patients, were amplified by four primer pairs. Four samples, one
of
which was from a culture-negative patient, were positive with
three
primer pairs. Finally, two samples, both from culture-negative
patients, were amplified by only two primer pairs (Table
3).
None of the primer pairs gave a positive reaction with samples from two
patients. Both patients were symptom free (stage A)
and previously
untreated. Both patients were negative for cellular
and plasma
viremias, and the CD4
+ cell counts were 705 × 10
6 and 700 × 10
6/liter.
Positive amplification of HLA-DQ
confirmed the accuracy of the PCR
approach.
HIV-2 genetic diversity and the reduced replicative capacity of the
virus, reflected by low cellular and plasma virus loads
(
2,
15), pose problems for proviral DNA detection in PCR-based
assays. To improve the capacity of PCR approaches for detection
of
HIV-2 DNA, we established a new strategy based on the use of
XL PCR,
followed by a nested PCR step with several primer pairs.
This strategy
allowed us to bypass HIV-2 diversity, because the
primers used in first
amplification step correspond to apparently
conserved regions in the
LTR and
pol genes (
13). The use of
different
primer pairs in the nested PCR step also improved the
detection rate
and increased the amount of proviral DNA, which
could be very small in
clinical samples. With this strategy, we
obtained positive results for
95% of the patients (40 of 42).
For 73% of the patients (29 of 40),
all five primer pairs used
in the nested PCR step yielded positive
amplification (Table
3).
The primers we designed for use in the nested PCR step were located in
different regions of the
env gene (
13) (Table
1).
The best results were obtained by using the EB2/EB5 primer pair
located
in the V3 region of the
env gene, which gave positive
results for 40 of 42 patients. These results confirm that the
HIV-2 V3
region is highly conserved, contrary to the HIV-1 V3
region
(
13). The ENVA/ENVB primer pair (
6) amplified
proviral
DNA in 38 of 42 samples. These primers are located in the
conserved
gp36 coding region of
env. EB0/EB6, based on
sequences located
in the V1 to V2 region of the
env gene,
was the least efficient
primer pair (32 of 42 samples), probably
because of the known
variability of the V1-to-V2 region of the HIV-2
env gene.
Grankvist et al. (
7) suggested that the low efficiency of
PCR amplification of the HIV-2 genome relative to HIV-1 was due
to the
presence of fewer than 1 infected cell per 100,000. In
their study,
Grankvist et al. obtained positive nested PCR amplification
in 83.7%
of samples by using primers originating in the
pol gene
(
7), a value similar to that obtained by other study groups
(i.e., 84% [
14] and 88% [
9]). Our
strategy, based on the
use of XL PCR in the first step and nested PCR
with primers from
the
env gene, succeeded in amplifying the
proviral DNA in 95%
of samples and avoided the use of radioactive
probes for the nested
step. Furthermore, direct amplification of HIV-2
DNA from fresh
PBMCs makes this method widely accessible. Our
amplification approach
should be useful for HIV-2 diagnosis and
follow-up of vertically
infected children. This strategy might equally
improve the efficiency
of the type differentiation in case of dual
serological reactivities.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Virologie, Hôpital Bichat-Claude Bernard, 46 Rue Henri Huchard,
75018 Paris, France. Phone: 33/1 40 25 88 94. Fax: 33/1 46 27 02 08. E-mail: francois.simon{at}bch.ap-hop-paris.fr.
| |
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Journal of Clinical Microbiology, March 1998, p. 809-811, Vol. 36, No. 3
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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