HIV-1 Genotypic Resistance Testing Using Sanger and Next-Generation Sequencing in Adults with Low-Level Viremia in China

Figures & data

Table 1 Demographic Characteristics of the Study Participants

Variable	Patients (n=80)
Age (years)
<30	26 (32.5%)
30–50	45 (56.2%)
>50	9 (11.3%)
Gender
Male	72 (90%)
Female	8 (10%)
HIV transmission route
Homosexual	67 (83.8%)
Heterosexual	11 (13.8%)
Others	2 (2.5%)
CD4 count, cells/µL, at baseline, median (25th–75th)	263 (62–402)
HIV-1 RNA copies/mL at baseline, median (25th–75th)	297,107 (43,652–424,165)
Plasma viral load at LLV, copies/mL
50–99	43 (53.7%)
100–199	14 (17.5%)
200–399	12 (15%)
400–999	11 (13.8%)
ART regimen
3TC+TDF+EFV	63 (78.8%)
3TC+TDF+LPV/r	5 (6.3%)
TAF+EVG/c/FTC	3 (3.8%)
AZT+3TC+EFV	2 (2.5%)
3TC+TDF+DTG	2 (2.5%)
Others	5 (6.3%)
Type of LLV
iLLV	50 (62.5%)
pLLV	30 (37.5%)

Abbreviations: 3TC, lamivudine; TDF, tenofovir; EFV, efavirenz; LPV/r, lopinavir/ritonavir; TAF, tenofovir alafenamide; EVG/c/FTC, elvitegravir; FTC, emtricitabine; AZT, zidovudine; DTG, dolutegravir; LLV, low-level viremia; iLLV, intermittent low-level viremia; pLLV, persistent low-level viremia.

Figure 1 Genotyping success rates of different viremia categories. (A) Genotyping success rates of different viremia categories at protease and reverse transcriptase regions. (B) Genotyping success rates of different viremia categories at integrase region.

Figure 2 Patterns of DRMs in HIV-infected patients with LLV.

Figure 3 Different levels of drug resistance in HIV-infected patients with LLV.

Table 2 Drug Resistance Mutations in 8 Patients with Intermittent LLV

Case ID	Gender	Age	TR	VL	Subtype	ART at the Time of DRM Test	Drug-Resistant Mutation
							NNRTIs	NRTIs	PIs	INSTIs
F3821	Male	32	Heterosexual	53	CRF07_BC	3TC+TDF+EFV	–	–	Q58E	–
F4301	Male	27	Homosexual	60	A	3TC+TDF+DTG	E138K, F227L, V179L	M184V	–	–
F8115	Male	33	Homosexual	70	CRF55_01B	3TC+TDF+EFV	V179E	–	–	–
F8805	Male	27	Homosexual	73	B	3TC+TDF+EFV	V106I	–	–	–
F8830	Male	29	Homosexual	517	B	3TC+TDF+LPV/r	K103N	–	–	–
F9271	Male	23	Homosexual	82	CRF01_AE	3TC+AZT+LPV/r	Y188L	M184I, V75M	–	–
g1793	Female	31	Heterosexual	63	CRF01_AE	3TC+TDF+EFV	G190T	A62AV, K65R	–	–

Abbreviations: VL, viral load; TR, transmission route; NRTIs, nucleoside reverse transcriptase inhibitors; NNRTIs, non-nucleoside reverse transcriptase inhibitors; PIs, protease inhibitors; INSTIs, integrase inhibitors.

Table 3 Drug Resistance Mutations in 18 Patients with Persistent LLV

Case ID	Gender	Age	TR	VL	Subtype	ART at the Time of DRM Test	Drug-Resistant Mutation
							NNRTIs	NRTIs	PIs	INSTIs
F3848	Male	35	Homosexual	142	CRF01_AE	3TC+TDF+EFV	–	–	M46MI	–
F4947	Male	45	Homosexual	124	CRF07_BC	3TC+TDF+EFV	E138G	–	–	–
F5603	Male	28	Homosexual	71	CRF07_BC	3TC+TDF+EFV	V179D	–	–	–
F5756	Male	50	Homosexual	342	CRF01_AE	3TC+TDF+EFV	–	D67DN	–	–
F6332	Male	31	Heterosexual	443	CRF01_AE	3TC+TDF+EFV	K101E, G190S	K65R	–	–
F6370	Male	30	Homosexual	85	A	3TC+TDF+EFV	K101E, G190S, Y181C, M184I	A62V, K65R, M184I	–	–
F6856	Male	28	Homosexual	388	B	3TC+TDF+EFV	A98G, K103N	M41L, T215Y, M184V	–	–
F7282	Male	50	Homosexual	867	CRF01_AE	3TC+TDF+EFV	Y181C	L74V, M184V	I54V, V82A	–
F7343	Male	36	Homosexual	811	CRF55_01B	3TC+TDF+EFV	Y181YC, G190A, H221HY, V179E	L74LI, M184V, Y115YF	–	–
F7492	Male	35	Homosexual	66	B	3TC+TDF+EFV	K103N, Y181YC, V108I	K65R, M184V	–	–
F7570	Male	56	Homosexual	371	CRF07_BC	3TC+TDF+EFV	A98G, K103N, V179E	M184V	–	E138EAKT, Q148R
F8103	Male	26	Heterosexual	111	CRF01_AE	3TC+TDF+EFV	V106I, Y188L, V179VL	K70KQ, M184V	–	–
F8355	Male	28	Homosexual	805	CRF01_AE	3TC+TDF+EFV	V106M, K103R, V179D	M184V	–	–
F9710	Male	23	Homosexual	323	CRF01_AE	3TC+TDF+EFV	K103N	–	–	–
g622	Male	44	Homosexual	223	CRF01_AE	3TC+TDF+EFV	V108I	–	–	–
g3789	Male	28	Heterosexual	180	CRF07_BC	3TC+TDF+EFV	G190E, E138A	K65R, K219N	–	–
g3914	Male	20	Homosexual	166	CRF01_AE	3TC+TDF+EFV	V108I, K103N	K65R, M184V	–	–
g4391	Male	30	Homosexual	301	CRF07_BC	3TC+TDF+EFV	–	K103N	–	–

Abbreviations: VL, viral load; TR, transmission route; NRTIs, nucleoside reverse transcriptase inhibitors; NNRTIs, non-nucleoside reverse transcriptase inhibitors; PIs, protease inhibitors; INSTIs, integrase inhibitors.

Table 4 DRMs Detected by NGS but Missed by SS at the Threshold of 5%

Case ID	LLV	VL	Sanger			NGS
			PIs	NRTIs	NNRTIs	PIs	NRTIs	NNRTIs
f6462	pLLV	95	None	None	None	N88S (5.1)	None	None
f6332	pLLV	443	None	K65R	K101E, G190S	None	K65R (70.31)	K101E (69.52), G190S (69.72), Y181C (7.82),
f7282	pLLV	867	I54V, V82A	L74V, M184V	Y181C	M46I (15.15), I54V (98.66), V82A (97.71)	L74V (98.19), M184V (98.71)	Y181C (97.99), E138K (6.81), P236L (5.36)
G682	pLLV	89	None	None	None	None	None	E138G (97.95)
G1128	Blip	986	None	None	None	I47V (6.29)	None	None
GMg-3789	pLLV	180	None	K65R, K219N	G190E, E138A	None	K65R (98.44), K219N (98.48), D67G (98.81)	G190E (98.76), E138A (97.62)
GMg-3914	pLLV	166	None	K65R, M184V	V108I, K103N	None	K65R (98.94), M184V (98.12), K219E (41.44)	V108I (96.25), K103N (97.52)
GMg-4391	pLLV	301	None	None	K103N	None	None	K103N (97.84), K101E (33.39)

Note: The red-colored text represents DRMs detected by NGS but missed by SS at the threshold of 5%.

Abbreviations: LLV, low-level viremia; VL, viral load; NRTIs, nucleoside reverse transcriptase inhibitors; NNRTIs, non-nucleoside reverse transcriptase inhibitors; PI, protease inhibitors.

Table 5 DRMs Detected by SS but Missed by NGS at the Threshold of 5%

Case ID	LLV	VL	Sanger			NGS
			PIs	NRTIs	NNRTIs	PIs	NRTIs	NNRTIs
f3848	pLLV	142	M46MI	None	None	None	None	None
f4947	pLLV	124	None	None	E138G	None	None	None
f5756	pLLV	342	None	D67DN	None	None	None	None
f8103	pLLV	111	None	K70KQ, M184V	Y188L, V106I, V179VL	None	M184V (98.09)	Y188L (97.81)
f8355	pLLV	805	None	M184V	V106M, V179D, K103R,	None	M184V (98.22)	V106M (97.49), V179D (96.68)
f8805	Blip	73	None	None	V106I	None	None	None

Note: The red-colored text represents DRMs detected by SS but missed by NGS at the threshold of 5%.

Abbreviations: LLV, low-level viremia; VL, viral load; NRTIs, nucleoside reverse transcriptase inhibitors; NNRTIs, non-nucleoside reverse transcriptase inhibitors; PIs, protease inhibitors.

Figure 4 Comparison of drug resistance interpretation between SS and NGS.