Hepatitis C virus NS5A region mutation in chronic hepatitis C genotype 1 patients who are non-responders to two or more treatments and its relationship with response to a new treatment

Table 1 NS5A primers used in the current study

Application	Direction	Sequence
First PCR	Sense	5´-ACAACTCCCATGCGAGCCCGAGCCGGA-3´
RT and first PCR	Antisense	5´-GAGGGGGAGCCGGGGGATCCCGATCTC-3´
Second PCR	Sense	5´-TGCTCACCGACCCATCCCACATTACAGCAGA-3´
	Antisense	5´-ATGCCCCCTCTCGAGGGGGAGCCGGG-3´

Table 2 Influence of the type of previous response on the sustained virological response to a subsequent treatment n (%)

Previous response	NVR, n = 46	SVR, n = 32	OR (95%CI)	P value
Relapse	7 (30)	16 (70)	6.09 (2.0-18.4)	0.006
Partial response viral	7 (64)	4 (36)	1.5 (0.3-6.1)	n.s.
Null response	32 (73)	12 (27)	1

This analysis excluded previous treatments for which the reason for non-response was unknown (n = 51). NVR: No virological response; SVR: Sustained virological response.

Table 3 Type of response according to the type of treatment received n (%)

	Total, n = 129	NVR, n = 81	SVR, n = 48	OR (95%CI)	P value
IFN	16 (12)	14 (88)	2 (12)	0.05 (0.01-0.3)	0.001
IFN + RBV	40 (31)	27 (68)	13 (32)	0.2 (0.06-0.5)	0.001
pegIFN + RBV	43 (34)	32 (74)	11 (26)	0.12 (0.04-0.4)	0.00
DAAs	30 (23)	8 (27)	22 (73)	1

This analysis excluded the first 72 treatments, which presented statistical bias, because one of the inclusion criteria was that these should be non-responders. DAAs: Direct-acting antivirals; IFN: Interferon; NVR: No virological response; RBV: Ribavirin; SVR: Sustained virological response.

Table 4 Number of mutations, according to the response to antiviral treatment

Treatments according to response, n = 134	SVR, n = 46	R, n = 29	pVR, n = 10	NR, n = 49	P value1
PKRBD mutations	5.82 ± 3.06	5.1 ± 2.3	4.9 ± 1.8	5.12 ± 2.2	n.s.
ISDR mutations	2.65 ± 2.5	1.38 ± 1.82	1.5 ± 1.3	1.6 ± 1.7	0.029

¹Kruskal-Wallis;

²SVR vs R: P = 0.025, Mann-Whitney U. This analysis excluded treatments for which the NVR type was unknown and samples that were not amplified (n = 134). NR: Null responders; NVR: No virological response; pVR: Partial virological response; SVR: Sustained virological response; PKRBD: PKR binding domain; ISDR: Interferon-sensitivity-determining region.

Table 5 Variation in the viral population of the PKR binding domain and interferon-sensitivity-determining region regions of NS5A over time and its relationship with the response to treatment n (%)

PKRBD (n = 119)	NVR, n = 74	SVR, n = 45	P value1	ISDR, n = 122	NVR, n = 77	SVR, n = 45	P value1
Mutations increase by ≥ 2, n = 24	10 (42)	14 (58)	0.02	Mutations increase by ≥ 2, n = 22	7 (32)	15 (68)	0.001
No such increase2, n = 95	64 (67)	31 (33)		No such increase2, n = 100	70 (70)	30 (30)

¹χ² test.

²Genetic variability increases when the number of mutations increases by 2 or more (22, 23, 30), while the viral population does not vary if the number of mutations remains unchanged, increases by only 1, or decreases. NVR: No virological response; SVR: Sustained virological response; PKRBD: PKR binding domain; ISDR: Interferon-sensitivity-determining region.

Table 6 Interaction between baseline viral load and the viral variability of the interferon-sensitivity-determining region

	Viral > 600000 IU/mL	Viral ≤ 600000 IU/mL
Decrease or no increase in mutations	1	1
≥ 2 increase in mutations	OR = 9.21 (2.5-33.3)	OR = 0.76 (0.13-4.3)
	P = 0.001	P = 0.75