Characteristic of Subjects
From 2017 to 2020, 450 HIV-infected cases receiving antiviral therapy for more than one year were confirmed to be ineffective (viral load≥1000copies/ml) in Suzhou city. Among these ART failure patients, a total of 398 viral pol gene sequences were successfully sequenced. Epidemiological features of ART failure HIV-infected cases in this study were summarized in Table 1. There was no significant difference in sex composition ratio, subtype distribution or viral load between group 2017-2018 and group 2019-2020. Males and females accounted for 92.96% (370/398) and 7.04% (28/398), respectively. A variety of HIV-1 virus circulating recombinant forms (CRFs) are prevalent in Suzhou City. The dominant subtype is CRF01_AE (57.29%, 228/398), followed by CRF07_BC (17.34%, 69/398), subtype B (7.54%, 30/398) and CRF08_BC (6.53%, 26/398). Other subtypes mainly include subtype CRF67_01B (3.02%, 12/398), CRF55_01B (2.51%, 10/398) and other CRFs (6.03%, 24/398). Among those who failed in antiviral treatment, the proportion of HIV-1 viral load in the range of 5000-100000 copies/ml was the largest, accounting for 62.56% (249/398).
Table 1. The features of subjects receiving drug-resistance mutation detection in Suzhou City
Item
|
Total
|
2017-2018
|
2019-2020
|
x2
|
p
|
N (%)
|
N (%)
|
Gender
|
|
|
|
1.80
|
0.17
|
Male
|
370
|
194
|
176
|
Female
|
28
|
11
|
17
|
Age (years)
|
|
|
|
2.086
|
0.35
|
﹤30
|
107
|
61
|
46
|
30-50
|
214
|
108
|
106
|
≥50
|
77
|
36
|
41
|
Subtypes/CRFs
|
|
|
|
2.34
|
0.89
|
CRF01_AE
|
228
|
118
|
110
|
CRF07_BC
|
69
|
38
|
31
|
Subtype B
|
30
|
13
|
17
|
CRF08_BC
|
26
|
11
|
15
|
CRF67_01B
|
12
|
7
|
5
|
CRF55_01B
|
10
|
5
|
5
|
others
|
24
|
13
|
11
|
Viral load(copies/ml)
|
|
|
|
3.84
|
0.15
|
1000-5000
|
110
|
48
|
62
|
5000-100000
|
249
|
135
|
114
|
>100000
|
39
|
22
|
17
|
Drug-resistance Mutations
According to the analysis of HIVdb Program, 257 strains were resistant to at least one drug of NRTIs, NNRTIs, or PIs, with an overall resistant rate of 64.57% (257/398). Frequency of drug-resistance associated mutations among HAART failure individuals was shown in Figure 1. The drug resistant mutations associated with NRTIs, NNRTIs, and PIs were 45.48% (181/398), 63.32% (252/398) and 3.02% (12/398), respectively. Among the PIs resistant HIV strains, four strains possessed PI-related major mutations (M46I/L, I54V, V82F) and the other 8 viruses had 3 accessory mutations (L33, FL10F and Q58E) in PR region. Respectively, 17 and 16 loci in RT region were found emerging NRTIs and NNRTIs resistance-associated substitutes. M184V/I (40.70%, 162/398), D67N/G /S/H (13.82%, 55/398) and K65R/N (12.81%, 51/398) were the three most common resistance-related mutations regarding to NRTIs, while V106M/I/A (21.86%, 87/398), K103N/S (21.11%, 84/398), V179D/E/T/L (20.35%, 81/398), G190A/S/Q/V/E (16.58%, 66/398) and Y181C/V/I (15.58%, 62/398) were the first five mutations associated with NNRTIs-resistance.
Resistance level to antiviral drugs
We analyzed the effects of mutations on drug resistance based on Stanford University HIV drug resistance database (https://hivdb.stanford.edu/). No other HIV strains with high-level resistance to PIs were detected except 2020-SZ-83050 strain. Three samples (2017-SZ-08303, 2018-SZ-08066 and 2020-XC-00107) with M46I/L mutation were resistant to ATV, FPV, IDV, LPV, NFV and SQV at a potential low level or intermediate. Besides, six samples were simultaneously resistant to FPV, NFV and TPV at a potential low level caused by L33F mutation, and one sample with L10LF mutation was resistant to NFV and FPV at low level. Resistance levels of different drugs among ART failure individuals were shown in figure 2. For NRTIs, the resistant frequency to ABC, AZT, D4T, DDI, FTC, 3TC and TDF were 45.48% (181/398), 15.08% (60/398), 31.66% (126/398), 45.98% (183/398), 45.23% (180/398), 45.23% (180/398) and 28.14% (112/398), respectively. The high-level of NNRTIs associated resistance was accounting for 20.10% (80/398), 51.26% (204/398), 56.28% (224/398), 5.28% (21/398), and 21.86% (87/398) for DOR, EFV, NVP, ETR, and RPV, respectively.
Analyses for NFLG sequences
Ten near full-length HIV-1 genomes (NFLG) covering from gag to nef genes were obtained by using next-generation sequencing (NGS). The Neighbor-joining phylogenetic tree of NFLG of HIV-1 viruses was shown in Figure 3. According to the results of jpHMM-HIV software (Figure 4), six sequences (2017SZ-83245, 2017SZ-0319, 2017SZ-1093, 2017SZ-1942, 2020SZ-83068 and 2020SZ-83463) were classified as recombinants of CRF 01_AE and subtype B. They were also supported by phylogenetic analysis (Figure 3), because these sequences were closer to CRF 01_AE than subtype B. They were inter-subtype of small fragments of subtype B inserted in CRF 01_AE gene sequence, but the recombination breakpoints of every sample were different (Figure 4). 2017SZ-703 and 2017SZ-1982 were identified as recombinants of CRF 01_AE, subtype B and subtype C. Sequences 2017SZ-703 and 2017SZ-1982 were closer to C subtype than others, indicating that they were recombinant viruses with subtype C virus as the backbone while CRF 01_AE and subtype B gene fragments as internal insertions. 2017SZ-0981 and 2020-SZXQ-256 sequences were recombinants of CRF 01_AE and subtype C.