HIV-1C in-House RNA-Based Genotyping Assay for Detection of Drug Resistance Mutations in Samples with Low-Level Viral Loads

Figures & data

Table 1 Rescue Primers Used When Samples Failed to Amplify

Primer Name	Sequence (5’-3’)	Purpose
PRTM-F1**	F1a-TGAARGAITGYACTGARAGRCAGGCTAAT F1b-ACTGARAGRCAGGCTAATTTTTTAG	Mixture of 2 primers for RT-PCR
RT-R1	ATCCCTGCATAAATCTGACTTGC	RT-PCR
PRT-F2	CTTTARCTTCCCTCARATCACTCT	Nested PCR
RT-R2	CTTCTGTATGTCATTGACAGTCC	Nested PCR
SeqF3	AGTCCTATTGARACTGTRCCAG	Sequencing
SeqR3	TTTYTCTTCTGTCAATGGCCA	Sequencing
SeqF4	CAGTACTGGATGTGGGRGAYG	Sequencing
SeqR4	TACTAGGTATGGTAAATGCAGT	Sequencing

Note: **A mixture of primers F1a and F1b.

Table 2 Baseline Demographics and Clinical Characteristics for Study Participants

Variable	Total n=96 (%)	LLV (401–999 Copies/mL) n=50 (%)	VL>1000 Copies/mL n=46 (%)	P-values
Gender, n (%)
Female	69 (72)	38 (76)	31 (67)	0.37^a
Male	27 (28)	12 (24)	15 (37)
Median Age in years (Q1, Q3)	33.5 (26.4, 41)	35.7 (27, 40.8)	31.8 (25.1, 42.7)	0.43
ART Status
Experienced	64 (67)	18 (36)	46 (100)	<0.01^a
Naïve	32 (33)	32 (64)	0
Median Viral Load in copies/mL (Q1, Q3)	982 (669–12,422)	699 (528–872)	14,004 (2561–47,521)	<0.01

Notes: ^aP-values were obtained by Fishers’ test while other p-values were from Wilcoxon rank sum test.

Abbreviations: ART, antiretroviral therapy; LLV, low level viremia; n, number; VL, viral load; Q1, first quartile; Q3, third quartile.

Figure 1 Genotyping success of in-house RNA-based HIV genotyping assay, overall and stratified by VL group.

Abbreviations: LLV, low-level viremia (VL 401–999 copies/mL); VL, viral load.

Figure 2 Association of genotyping outcomes with viral load measurement.

Abbreviations: Q1, first quartile; Q3, third quartile; VL, viral load.

Figure 3 Maximum likelihood phylogenetic tree constructed using 1000 bootstrap values showing the uniqueness of the sequences and that there is no clustering of sequences by their VL group. The light-blue dot represents standard HIV reference strain.

Abbreviation: HXB2, standard HIV reference strain.

Table 3 Prevalence of HIV DRM, Overall and by VL Groups

Resistance Measure	Total n=76	VL ≥ 1000 Copies/mL n=40	LLV (401–999 Copies/mL) n=36	p-value
Any drug resistance mutation, n^a (%)	18 (24%)	12 (30%)	6 (17%)	0.18
	95% CI:15–35	95% CI:17–46	95% CI: 6–33
Resistance category, n^b (%)
NNRTI	21 (28%)	15 (38%)	6 (17%)	0.04
NRTI	18 (24%)	12 (30%)	6 (17%)	0.18

Notes: ^aNumber of participants with at least 1 mutation associated with either of 2 drug classes (NNRTI or NRTI). ^bNumber of participants with at least 1 mutation in any specific drug resistance class. A comparison of proportion test was used to test the difference in the prevalence of DRM between 2 VL groups. Proportions with 95% Confidence Intervals (CI) were estimated using the Binomial exact method. Note that the sum of the number of persons with individual classes of mutations may exceed the number of persons with any mutation, as some people had more than 1 class of DRM. Note that the prevalence of PI-associated resistance mutations was not predicted.

Abbreviations: CI, 95% confidence intervals; LLV, low-level viremia (VL 401–999 copies/mL) in both ART-naïve and ART-experienced individuals; VL, viral load; NRTI, nucleoside reverse transcriptase inhibitor-associated mutations; NNRTI, non-nucleoside reverse transcriptase inhibitor-associated mutations.

Figure 4 HIV DRM classified by NRTI and NNRTI resistance classes (N=76) using an in-house RNA-based HIV genotyping assay with detectable plasma HIV-1 RNA > 400 copies/mL.

Abbreviations: DRM, drug resistance mutations; NRTI, nucleotide reverse transcriptase inhibitors; NNRTI, non-nucleotide reverse transcriptase inhibitors.

Figure 5 NRTI-associated resistance mutations stratified by VL groups.

Abbreviations: DRM, drug resistance mutations; LLV, low-level viremia; NRTI, nucleoside reverse transcriptase inhibitors; VL, viral load.

Figure 6 NNRTI-associated resistance mutations are stratified by VL groups.

Abbreviations: DRM, Drug resistance mutations; LLV, low level viremia; NNRTI, non-nucleoside reverse transcriptase inhibitors; VL, viral load.