2
.
7
. Statistical analysis Plasma isolation data positivenegative, SINSI
phenotype were tabled by plasma HIV-1 RNA copy number median and geometric mean,
CD4 + count
and treatment
status on
treatment vs. no treatment at the time of sam- pling. Comparison of continuous variables was
performed using Wilcoxon’s two-sample test. Probabilities to be isolation-negative according to
treatment
status were
expressed by
relative risk plus 95 confidence interval. In order to
evaluate the effect of treatment on plasma isola- tion controlling for its effect on RNA, we used a
logistic regression model which considered as out- come variable a positive plasma isolation and as
explicative variables RNA dichotomised,
B 100 000 or ] 100 000 copies per ml, CD4 count
and treatment status. Statistical analyses were made using the statistical software EPI-INFO,
version 6.04 Centers For Disease Control Pre- vention,
USA; World
Health Organization,
Geneva, Switzerland, 1997 and the statistical package SAS SAS Inc., Cary, NC, USA, version
6.12.
3. Results
3
.
1
. Plasma isolation and HIV-
1
RNA copy number
Plasma isolation
qualitative coculture
of plasma from HIV-positive patients with unin-
fected PHA-stimulated PBMC from uninfected donor was performed on 124 samples from 57
patients. Forty-eight
samples 39
tested positive
and 76
negative 61.
Thirty-six patients 63 had at least one isolation-positive
sample. Data on both HIV-1 RNA copy number and
isolation results were available for 119 samples: a positive plasma isolation, in almost all cases 43
45 and irrespective of treatment status, was asso- ciated with an HIV viral load higher than 100 000
copies per ml Fig. 1. A negative isolation result, however, did not exclude high RNA levels Fig.
2. Overall, isolation-positive samples had higher plasma HIV-1 RNA values compared with isola-
tion-negative samples copies per ml: mean, 546 175; median 710 000 versus: mean, 13 643;
median 37 500; P B 0.000001, Wilcoxon’s two
Fig. 1. HIV plasma copy number and CD4 cell count associated to isolation-positive samples.
Fig. 2. HIV plasma copy number and CD4 cell count associated to isolation-negative samples. Table 1
HIV-1 RNA copy number and CD4 count by isolation results positivenegative CD4 counts per mm
3
HIV-1 RNA copies per ml N
Mean Median
Range N
Mean S.D.
Median Range
55 068 120 000
399–5 700 000 119
All samples 142.1
119 107.4
131 3–472
45 37.8 546 175
Isolation-positive 710 000
5300–5 700 000 46 38.7
92.3 88.3
62.5 3–330
74 62.2 13 643
37 500 399–1 370 000
73 61.3 173.5
107.0 171
Isolation-negative 9–472
3
.
3
. Isolation-positi6e samples
:
HIV-
1
RNA copy number and CD
4
cell count according to SINSI phenotype
Data on SINSI status were available for 45 of the 48 isolation-positive samples. SI and NSI
strains were not different in terms of concomitant HIV-1 RNA copy number mean, 475 480; me-
dian 470 000 for SI vs. mean 585 372; median, 790 000 for NSI; P = 0.33. SI strains, compared
with NSI strains, tended to be associated with lower CD4 counts at sampling mean, 69mm
3
, median 27mm
3
for SI vs. mean 104mm
3
, median 84mm
3
for NSI; P = 0.11, Table 2. sample test. HIV-1 RNA data in relation to
isolation results are shown in Table 1.
3
.
2
. Plasma isolation and CD
4
cell count Data on both CD4 counts and isolation
results were available for 119 samples. Overall, isolation-positive samples had lower CD4 counts
compared with
isolation-negative samples
mean, 92.3mm
3
, median 62.5mm
3
vs. mean 173.5mm
3
, median
171mm
3
; P = 0.000034,
Wilcoxon’s two-sample test. Results are sum- marised in Table 1.
3
.
4
. Effect of treatment
3
.
4
.
1
. RNA On-treatment samples were collected between
week 24 and 48 of treatment. Pre-treatment sam- ples had a lower probability to be isolation-negative
with respect to on-treatment samples 2357 vs. 5367, relative risk = 0.51, 95 CI 0.36 – 0.72. As
expected, on-treatment samples had overall lower RNA values mean, 8954; median, 9400 compared
with pre-treatment samples mean, 424 912; me- dian, 575 000; P B 0.000001. Isolation-positive
samples were characterised by high RNA values in both on-treatment and pre-treatment samples, al-
though values were lower for the samples collected on-treatment mean and median, 238 166 and
180 000, respectively, compared with pretreatment samples, 738 593 and 810 000, respectively; P =
0.026. The results of the logistic regression analy- sis, performed to estimate the strength of the
association between a positive plasma isolation and high RNA values and to assess the role of treatment
controlling for its effect on RNA, showed that only HIV-1 RNA was associated significantly with an
increased risk of a positive isolation result OR for HIV copy number \ 100 000 compared with a
value below 100 000: 41.3, CI 95 9.1 – 186.8, P B 0.0001, with treatment status and CD4 count
not associated with an increased risk of a positive plasma isolation odds ratios 1.32 and 1.00; P =
0.63 and 0.65, respectively. RNA and CD4 data by treatment status and isolation outcome are
summarised in Table 3.
3
.
4
.
2
. CD
4
On-treatment samples n = 63 had higher CD4 counts compared with pre-treatment n = 59 sam-
ples mean, 189.7mm
3
, median 194mm
3
vs. 88.6 and 62.5mm
3
, respectively. In contrast with the RNA results, however, the difference between iso-
lation-positive samples and isolation-negative re- sults observed in the whole group i.e. not
considering treatment status was evident only among pre-treatment samples mean, 63.9mm
3
, median 33mm
3
vs. 124 and 139mm
3
, respectively, with on treatment samples showing only a minor
difference between isolation-positive and isolation- negative groups mean, 164.2mm
3
, median 162 mm
3
vs. 196.4 and 196mm
3
, respectively Table 3.
3
.
4
.
3
. HIV-isolation results At baseline, 34 patients were isolation-positive
and 23 isolation-negative. Follow-up isolation re- sults were available for 28 out of 34 isolation-pos-
itive patients and for 19 of 23 isolation-negative patients. During treatment, most of isolation-posi-
tive patients became isolation-negative, with a trend for a higher negativisation rate among pa-
tients receiving the triple combination total, 20; triple combination 13 vs. double combination 7.
Most of the patients who were isolation-negative at baseline remained isolation-negative during follow-
up 17 out of 19 with available data. Only two patients who were isolation-negative at baseline
became isolation-positive during treatment. Both had very high baseline viral load 640 000 and
1 370 000 copies per ml, respectively.
3
.
4
.
4
. SINSI phenotytpe At baseline, among the 34 isolation-positive
patients, 11 had an SI phenotype and 23 an NSI phenotype. Follow-up data were available for nine
SI and 19 NSI patients. During follow-up, 15 patients with NSI baseline phenotype and five
Table 2 HIV-1 RNA copy number and CD4 count by SINSI phenotype
CD4 counts per mm
3
HIV-1 RNA copies per ml Mean
Mean N
Median S.D.
N Range
Range Median
3–330 45
546 175 710 000
Isolation-positive 5300–5 700 000 46
92.3 88.3
62.5 SI+NSI
27 78.8
69.9 16 34.8
77,000–4 900 000 470 000
475 480 3–229
15 33.3 SI phenotype
30 66.7 585 372
790 000 5300–5 700 000 30 65.2
104.2 92.0
NSI phenotype 84
3–330
S .
Vella et
al .
Anti 6
iral Research
47 2000
189 –
198
195 Table 3
HIV-1 RNA copy number and CD4 count by isolation results and treatment status HIV-1 RNA, copies per ml
CD4 counts per mm
3
Mean S.D.
Median Range
N N
Range Median
Mean 399–5 700 000
119 142.1
107.4 131
3–472 55 068
All samples 120 000
119 92.3
88.3 62.5
3–330 46 38.7
45 37.8 Isolation-positive total
5300–5 700 000 710 000
546 175 73 61.3
74 62.2 173.5
107.0 171
9–472 13 643
37 500 399–1 370 000
Isolation-negative total 56 47.1
56 47.1 88.5
84.1 62.5
3–376 424 912
575 000 Before treatment total
7700–5 700 000 189.7
103.9 194
9–472 63 52.9
On treatment total 399–2 200 000
9400 8954
63 52.9 124.0
97.0 139
9–376 Before treatment isolation-negative
23 41.1 192 209
230 000 7700–1 370 000
23 41.1 63.9
64.5 33
3–231 33 58.9
Before treatment isolation-positive 77 000–5 700 000
810 000 738 593
33 58.9 399–220 000
50 79.4 196.3
104.5 196
16–472 On treatment isolation-negative
51 81.0 4138
840 164.2
101.6 162
9–330 13 20.6
238 166 On treatment isolation-positive
12 19.0 5300–2 200 000
180 000
patients with SI baseline phenotype became isola- tion-negative. Five patients remained isolation-
positive maintaining their baseline phenotype NSI, 3; SI, 2 and three patients remained isola-
tion-positive switching to the opposite phenotype 1 switch NSI-SI, 2 SI-NSI.
4. Discussion
