A total of 374 patients, 160 HCV-monoinfected and 214 HCV/HIV coinfected, were included in the study. Overall, the mean age was 50.8 years (95% CI 50.0-51.6), and 70.6% were men. Patients were treated with diverse DAA regimens and were evaluated at baseline and at the 1st, 3rd, 6th, 12th, and 24th month afterwards, although 85 patients did not complete the final evaluation.
Table 1 shows the demographic, HCV, laboratory and fibrosis parameters of the patients as a whole, as well as those of the mono- and coinfected patients separately. It can be appreciated that there were statistically significant differences between mono- and coinfected patients in HCV genotype, leukocyte counts, ALT, total proteins, urea, creatinine, and HDL-cholesterol.
Table 1. Demographic, HCV-related, laboratory and fibrosis parameters in mono- and coinfected patients
|
|
All
(n=374)
|
Monoinfected
(n=160)
|
Coinfected
(n=214)
|
P value
|
Demography
|
|
|
|
|
|
Gender
|
male
|
264 (70.6%)
|
116 72.5(%)
|
148 (69.2%)
|
0.5
|
female
|
110 (29.4%)
|
44 (27.5%)
|
66 (30.8%)
|
|
|
|
|
|
|
|
Age
|
years
|
50.81 (50.02-51.59)
|
51.33 (49.96-52.70)
|
50.42 (49.50-51.33)
|
0.3
|
HCV-related parameters
|
|
|
|
|
|
HCV viral load at baseline
|
log copies/mL
|
5.896 (5.767-6.025)
|
5.880 (5.726-6.034)
|
5.908 (5.713-6.102)
|
0.8
|
HCV genotype
|
1
|
205 (75.6%)
|
81 (81.8%)
|
124 (72.1%)
|
0.035
|
2
|
2 (0.7%)
|
1 (1.0%)
|
1 (0.6%)
|
|
3
|
41 (15.1%)
|
15 (15.2%)
|
26 (15.1%)
|
|
4
|
23 (8.5%)
|
2 (2.0%)
|
21 (12.2%)
|
|
HCV treatment
|
|
|
|
|
|
Time on DAA therapy
|
weeks
|
13.09 (12.66-13.52)
|
12.60 (12.04-13.17)
|
13.45 (12.84-14.07)
|
0.054
|
Sofosbuvir
|
Yes
|
325 (86.9%)
|
137 (85.6%)
|
188 (87.9%)
|
0.53
|
No
|
49 (13.1%)
|
23 (14.4%)
|
26 (12.1%)
|
|
Drug combinations
|
Sofosb + Velpatasvir
|
149 (39.8%)
|
62 (38.8%)
|
87 (40.7%)
|
0.7
|
Sofosb + Ledipasvir
|
141 (37.7%)
|
59 (36.9%)
|
82 (38.3%)
|
|
Other combinations
|
84 (22.5%)
|
39 (24.4)
|
45 (21.0%)
|
|
HCV eradicated
|
Yes
|
363 (98.4%)
|
153 (98.1%)
|
210 (98.6%)
|
0.7
|
No
|
6 (1.6%)
|
3 (1.9%)
|
3 (1.4%)
|
|
Laboratory blood parameters at baseline
|
|
|
|
|
Hemoglobin
|
g/dL
|
14.72 (14.55-14.89)
|
14.84 (14.61-15.07)
|
14.63 (14.39-14.87)
|
0.2
|
Leukocytes
|
cells/ mL
|
6612 (6366-6859)
|
6916 (6557-7276)
|
6383 (6047-6719)
|
0.035
|
Platelets
|
x1000/mL
|
178.2 (171.0-185.5)
|
183.5 (171.6-195.3)
|
174.3 (165.3-183.4)
|
0.2
|
Aspartate aminotransferase
|
U/L
|
69.2 (62.8-75.6)
|
70.9 (62.0-79.9)
|
67.6 (58.4-76.9)
|
0.6
|
Alanine aminotransferase
|
U/L
|
76.8 (69.4-84.3)
|
88.0 (74.7-101.3)
|
68.4 (60.1-76.7)
|
0.01
|
Alkaline phosphatase
|
U/L
|
90.7 (87.1-94.4)
|
90.4 (84.6-96.1)
|
91.0 (86.2-95.8)
|
0.9
|
g-glutamyl transferase
|
U/L
|
145.6 (112.3-178.8)
|
128.7 (104.1-153.4)
|
159.3 (102.2-216.5)
|
0.4
|
Total bilirubin
|
mg/dL
|
0.959 (0.909-1.009)
|
0.943 (0.864-1.022)
|
0.971 (0.905-1.037)
|
0.6
|
Total proteins
|
g/dL
|
7.617 (7.520-7.714)
|
7.428 (7.306-7.550)
|
7.705 (7.576-7.835)
|
0.008
|
Albumin
|
g/dL
|
4.279 (4.230-4.329)
|
4.320 (4.246-4.395)
|
4.247 (4.181-4.313)
|
0.14
|
Fibrinogen
|
mg/dL
|
366.9 (352.9-381.0)
|
354.4 (334.4-374.4)
|
374.0 (355.1-393.0)
|
0.19
|
INR
|
|
1.066 (1.041-1.090)
|
1.084 (1.037-1.131)
|
1.050 (1.028-1.072)
|
0.18
|
Glucose
|
mg/dL
|
104.8 (100.3-109.2)
|
108.0 (99.6-116.4)
|
102.7 (97.7-107.6)
|
0.3
|
Urea
|
mg/dL
|
34.8 (33.5-36.0)
|
33.2 (31.3-35.0)
|
35.8 (34.1-37.5)
|
0.045
|
Creatinine
|
mg/dL
|
0.849 (0.829-0.870)
|
0.796 (0.767-0.826)
|
0.890 (0.863-0.917)
|
<0.0001
|
Total cholesterol
|
mg/dL
|
169.5 (165.2-173.8)
|
167.9 (161.3-174.5)
|
170.8 (165.0-176.5)
|
0.5
|
HDL cholesterol
|
mg/dL
|
49.6 (47.5-51.8)
|
52.2 (48.6-55.7)
|
47.6 (45.1-50.2)
|
0.037
|
LDL cholesterol
|
mg/dL
|
95.2 (91.0-99.4)
|
92.7 (86.8-98.6)
|
97.2 (91.2-103.2)
|
0.3
|
HIV viral load
|
log copies/mL
|
-
|
-
|
0.677 (0.476-0.878)
|
-
|
CD4 lymphocytes
|
cells/mL
|
-
|
-
|
614.1 (566.0-662.3)
|
-
|
CD4 lymphocytes
|
%
|
-
|
-
|
30.73 (29.13-32.33)
|
-
|
Fibrosis parameters at baseline
|
|
|
|
|
Transient elastometry
|
kPa
|
10.42 (9.69-11.22)
|
11.73 (10.44-13.17)
|
9.55 (8.70-10.49)
|
0.006
|
APRI index
|
|
0.841 (0.678-1.044)
|
0.973 (0.839-1.129)
|
0.948 (0.828-1.085)
|
0.8
|
Forns index
|
|
6.030 (5.500-6.611)
|
6.050 (5.700-6.421)
|
6.186 (5.869-6.521)
|
0.6
|
FIB-4 index
|
|
1.927 (1.602-2.317)
|
2.086 (1.859-2.341)
|
2.142 (1.922-2.387)
|
0.7
|
Degree of liver fibrosis (measured by TE)
|
F0-F1
|
125 (33.5%)
|
41 (25.8%)
|
84 (39.3%)
|
0.03
|
F2
|
76 (20.4%)
|
36 (22.6%)
|
40 (18.7%)
|
|
F3
|
61 (16.4%)
|
25 (15.7%)
|
36 (16.8%)
|
|
F4
|
111 (29.8%)
|
57 (35.8%)
|
54 (25.2%)
|
|
Change in fibrosis parameters after 24 months
|
|
|
|
|
Absolute change in TE †
|
kPa
|
-5.29 (-6.76, -3.81)
|
-6.876 (-9.49, -4.26)
|
-4.33 (-6.10, -2.56)
|
0.1
|
Absolute change in APRI*
|
|
-1.13 (-1.40, -0.86)
|
-1.28 (-1.65, -0.92)
|
-0.99 (-1.40, -0.59)
|
0.3
|
Absolute change in Forns
|
|
-1.32 (-1.49, -1.14)
|
-1.26 (-1.58, -0.93)
|
-1.36 (-1.56, -1.16)
|
0.6
|
Absolute change in FIB-4*
|
|
-1.03 (-1.33, -0.72)
|
-1.26 (-1.72, -0.79)
|
-0.83 (-1.23, -0.43)
|
0.16
|
Relative improvement in TE§
|
%
|
28.03% (23.5-32.5)
|
35.4% (28.3-42.6)
|
23.6% (17.8-29.3)
|
0.01
|
Relative improv. in APRI‡
|
%
|
44.2% (30.9-57.5)
|
52% (35.4-68.6)
|
37.2% (16.5-57.8)
|
0.3
|
Relative improv. in Forns‡
|
%
|
19.1% (16.2-21.9)
|
17.2% (12.0-22.4)
|
20.4% (17.2-23.7)
|
0.3
|
Relative improv. in FIB-4‡
|
%
|
19.0% (11.1-27.0)
|
21.6% (7.9-35.3)
|
16.9% (7.6-26.1)
|
0.6
|
Values are expressed as mean (95% CI) or % as appropriate.
DAA denotes direct acting antivirals, HCV hepatitis C virus, INR international normalized ratio, HDL high density lipoproteins, LDL low density lipoproteins, HIV human immunodeficiency virus, TE transient elastometry
† Difference and ‡ratio between 24-month and baseline intrasubject measurements
Regarding fibrosis parameters, monoinfected patients had significantly higher degrees of fibrosis at baseline, as evaluated by TE, and also experienced higher degrees of improvement at 24 months following anti-HCV-therapy, differences that were not observed in the other fibrosis indexes evaluated.
A logistic regression model was elaborated using the variables with a P value <0.1 in the univariate analyses to identify the factors independently associated with mono/coinfection. This model, which adequately fitted the data according to the Hosmer–Lemeshow goodness-of-fit test, revealed that lower baseline measurements of TE (OR 0.901, 95% CI 0.846-0.960, P=0.001), higher serum total proteins (OR 6.194, 95% CI 1.892-20.275, P=0.003), and lower serum HDL-cholesterol (OR 0.959, 95% CI 0.930-0.989, P=0.008) were independently predictive of HIV-HCV-coinfection.
Course of fibrosis parameters following treatment
Figure 1 depicts the evolution over time of the four fibrosis parameters evaluated at each time-point. The curves show that TE measurements decreased quite steadily until the end of follow-up. On the contrary, the other three indexes experienced a marked decrease during the first month, reaching a plateau afterwards. The paired, intra-subject comparison of baseline vs 24-month values yielded statistically significant decreases for all indexes (P<0.0001 for each).
Figure 2 shows the comparative course over time of the four fibrosis indexes in mono- and coinfected patients. The degree of fibrosis at each time point was very similar in both groups of patients for APRI, Forns, and FIB-4 indexes, but not for TE, as monoinfected individuals had significantly higher values than coinfected patients in all but the 24-month measurements. Therefore, only TE discriminates between the two patient groups.
Figure 3 displays the proportional improvement in fibrosis indexes of the 24-month vs the baseline measurements, according to the baseline fibrosis stage. The figure reveals that the more advanced the initial fibrosis stage, the greater intra-subject improvement in fibrosis following therapy for TE (P<0.0001), FIB-4 (P<0.0001), APRI (P=0.025), but not for Forns (P=0.5). No significant differences in improvement existed between mono- and co-infected patients according to the initial fibrosis stage (data not shown).
Multivariate analyses revealed that the strongest predictor for both absolute and relative improvements in fibrosis at 24 months as compared to baseline was a more advanced initial fibrosis stage (P<0.0001). The other baseline factors significantly associated with these endpoints were higher gamma-glutamyl transferase GGT levels (P=0.006) for absolute, and higher aspartate aminotransferase AST levels (P=0.008) for relative improvements in fibrosis. These models accounted for 40.2% and 22.4% of the total absolute and relative variability in the improvement, respectively.
HCV treatment
We also compared the evolution of fibrosis depending on the presence or absence of sofosbuvir in the DAA regimen. Patients treated with sofosbuvir had higher degrees of fibrosis at baseline than those receiving other regimens in each of the four fibrosis parameters evaluated: TE 10.75 kPa (95% CI 9.92-11.65) vs. 8.52 (7.22-10.05), respectively, P=0.03, APRI 1.010 (0.906-1.125) vs. 0.717 (0.560-0.918), P=0.02, Forns 6.25 (5.98-6.53) vs. 5.50 (5.06-5.98), P=0.02, and FIB-4 2.203 (2.019-2.403) vs. 1.678 (1.421-1.981), P=0.01.
The follow-up evaluations revealed that the fibrosis curves over time of sofosbuvir and non-sofosbuvir regimens were remarkably parallel (Fig.4), maintaining, therefore, the initial differences, and evidencing that the fibrosis response was the same in both groups.
Matrix metalloproteinases and tissue inhibitors
A subset of 61 patients (30 monoinfected and 31 coinfected) underwent sequential measurements of plasma MMP-2, MMP-8, MMP-9 and TIMP-1.
Table 2 describes the correlations among MMPs, TIMP-1 and the four fibrosis indexes. There were highly significant correlations among the diverse MMPs, as well as among the different fibrosis indexes among them, but no MMP correlated with any of the fibrosis indexes. On the contrary, TIMP-1 significantly correlated with each of the four fibrosis parameters.
Table 2. Correlations between MMPs, TIMP-1 and fibrosis parameters †
|
MMP-8
|
MMP-9
|
TIMP-1
|
TE
|
APRI
|
Forns
|
FIB-4
|
MMP-2
|
0.34 (0.008)
|
0.51 (<0.0001)
|
0.21 (0.11)
|
-0.08 (0.5)
|
-0.02 (0.9)
|
0.10 (0.5)
|
0.05 (0.7)
|
MMP-8
|
|
0.78 (<0.0001)
|
-0.11 (0.4)
|
0.03 (0.8)
|
-0.02 (0.9)
|
0.06 (0.7)
|
0.03 (0.8)
|
MMP-9
|
|
|
-0.11 (0.4)
|
-0.14 (0.3)
|
0.04 (0.8)
|
0.10 (0.5)
|
0.01 (0.9)
|
TIMP-1
|
|
|
|
0.35 (0.006)
|
0.31 (0.02)
|
0.40 (0.006)
|
0.42 (0.001)
|
TE
|
|
|
|
|
0.62 (<0.0001)
|
0.60 (<0.0001)
|
0.64 (<0.0001)
|
APRI
|
|
|
|
|
|
0.74 (<0.0001)
|
0.90 (<0.0001)
|
Forns
|
|
|
|
|
|
|
0.82 (<0.0001)
|
† r (P value)
TE denotes transient elastometry
Figure 5 shows the course over time of MMP-2, -8, -9 and TIMP-1. All of the 4 curves experienced a downward progression. The paired, intra-individual comparison of baseline vs 24-month values revealed statistically significant decreases of MMP-2 (P=0.02), MMP-8 (P=0.009), MMP-9 (P=0.0004) and TIMP-1 (P=0.0001).
Figure 6 depicts the course over time of the three MMPs and TIMP-1 in monoinfected and coinfected patients. Coinfected individuals had substantially higher levels at baseline of each MMP than monoinfected patients, but lower levels in the case of the inhibitor TIMP-1, an opposite pattern that persisted during the 24 months of follow-up.
Single nucleotide polymorphisms
There were no significant differences among the different genotypes of the SNPs studied in 319 patients according to the mono- or coinfected status of the patients: MMP-2 (-1306C/T) (P=0.7), MMP-2 (-735C/T) (P=0.4), MMP-8 (-799C/T ) (P=0.3), MMP-13 (- 77A/G) (P=0.4), and TIMP-1 (9830T/G) (P=0.6).
Most of the SNPs evaluated did not show any significant association with the diverse fibrosis parameters. However, the mutant homozygous TT genotype of the MMP-2 (-1306C/T) SNP was associated with somewhat higher degrees of fibrosis, both at baseline and during the follow-up, whereas the wild CC and heterozygous CT genotypes had almost identical degrees of fibrosis (supplementary Fig. S1 online). Nevertheless, the reduced number of patients with the mutant TT genotype precluded the detection of statistically significant differences, with the exception of the 6-month measurement, at which time point such differences were observed in each of the four fibrosis parameters: TE (P=0.006), APRI (P=0.001), Forns (P=0.04) and FIB-4 (P=0.01).