Short Communication: Discordance in Drug Resistance Mutations Between Blood Plasma and Semen or Rectal Secretions Among Newly Diagnosed HIV-1-Infected Thai Men Who Have Sex with Men

Human immunodeficiency virus (HIV) infection is commonly transmitted across mucosal surfaces. ¹ Although blood plasma HIV-1 RNA levels of source partners correlated with HIV transmission, ¹ discordance between blood plasma and anogenital compartments in HIV-1 RNA levels and drug resistance-associated mutations (DRAMs) have been observed, raising concern that blood plasma may not be a full representative of DRAMs in other compartments, and transmitted drug resistance (TDR) can occur unnoticed. ² In Thailand, the TDR prevalence among Thai men who have sex with men (MSM) was 7%. ³ This study examined discordances in genotypic DRAMs between blood plasma and important vectors—semen or rectal secretions—among newly diagnosed, antiretroviral therapy (ART)-naive, HIV-1-infected MSM enrolled from the Test and Treat cohort in Thailand.

Previously unknown HIV-1 status Thai MSM aged ≥18 years were enrolled into the Test and Treat cohort in Thailand between 2012 and 2013. We studied those who were HIV-1 positive and had at least one genotypic DRAM in blood plasma at baseline. Genotypic drug resistance assay was done on semen and rectal secretion samples collected at baseline. From 811 participants enrolled, 7 participants fulfilled the criteria and were included into this analysis. The study was approved by the institutional review board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (IRB No. 307/55), and all participants gave informed consent.

Seminal plasma was prepared from semen by centrifugation at 2,330 g for 10 min at room temperature. One milliliter supernatant was aliquoted and incubated until complete liquefaction at 56°C for 30 min. Rectal secretions were collected using a sponge (Merocel^® Schindler Ear Packing; Meditronic Xomed, Inc.). One milliliter extraction buffer was added to the sponge, followed by 15 min incubation and 5 min centrifugation at 2,300 g at 4°C for three times. Blood contamination was excluded using Hema-Screen^® Lab Pack (Stanbio Laboratory, EKF Diagnostics). HIV-1 RNA levels in all compartments were quantified by Abbott RealTime HIV-1 (Abbott Molecular, Inc.). The assay's lower limit of detection was 40 copies/mL. Because integrase strand transferase inhibitors were not implemented into Thailand's national guideline until 2017, HIV-1 genotyping was performed to detect only mutations in reverse transcriptase (RT) and protease (PR) genes using a validated in-house method before ART initiation. ⁴ Genotype sequences were processed through the Stanford University Drug Resistance Database ⁵ and mutations were subsequently categorized according to the World Health Organization surveillance drug resistance mutation list. ⁶ Each mutation was classified as conferring resistance to nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs), non-nucleoside analogue reverse transcriptase inhibitors (NNRTIs), or protease inhibitors (PIs). Correlation of detectable HIV-1 RNA between blood plasma and each anogenital compartment was performed with Stata version 13 (StataCorp LP, College Station, TX) using the Exact McNemar's test with significance set at p-value <.05.

Seven HIV-1-infected MSM with a median (interquartile range) CD4⁺ cell count of 464 (322–496) cells/mL and blood plasma HIV-1 RNA of 5.3 log₁₀ (range 1.0 log₁₀–5.7 log₁₀) copies/mL were included in this analysis. Three participants had detectable HIV-1 RNA in all compartments, whereas three had undetectable HIV-1 RNA levels in semen and one had undetectable HIV-1 RNA levels in rectal secretions. In anogenital samples, HIV-1 RNA could be fully amplified for DRAMs assessment in semen from three participants (participants 3, 6, and 7; Table 1) and in rectal secretions from four participants (participants 1, 2, 4, and 5; Table 1). Rectal secretions from participant 6 (Table 1) could only be amplified in RT genes.

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Table 1.

Drug Resistance-Associated Mutations Detected in Each Anatomical Compartment—Blood Plasma, Semen, and Rectal Secretions—of Enrolled Participants

		Resistance mutations
		PI				NRTI				NNRTI
Participant	Source	V11	I47	I54	T74	K70	L74	V75	Y115	M184	K219	V90	K103	V108	E138	V179	G190	H221
1	Blood plasma	—	—	—	—	—	—	—	—	—	—	—	—	—	—	D	—	—
	Rectal secretions	—	IM	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
2	Blood plasma	—	—	—	S	—	—	—	—	—	—	—	—	—	—	—	—	—
	Rectal secretions	—	—	—	—	N	I	—	F	V	—	—	N	I	—	—	—	Y
3	Blood plasma	—	—	—	—	—	—	—	—	—	—	I	N	—	—	—	—	—
	Semen	—	—	—	—	—	—	—	—	—	—	I	N	—	—	—	—	—
4	Blood plasma	—	—	—	—	—	—	—	—	—	—	—	—	—	A	—	—	—
	Rectal secretions	—	—	—	—	—	—	—	—	—	—	—	—	—	A	—	—	—
5	Blood plasma	I	—	—	—	—	—	—	—	—	—	—	—	—	—	—	A	—
	Rectal secretions	I	—	—	—	—	—	—	—	—	—	—	—	—	—	—	A	—
6	Blood plasma	—	—	—	—	—	—	FLV	—	—	—	—	—	—	—	—	—	—
	Semen	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
	Rectal secretions	Nonamplifiable for PR genes				—	—	—	—	—	—	—	—	—	—	—	—	—
7	Blood plasma	—	—	—	—	—	—	—	—	—	KQ	—	—	—	—	V	—	—
	Semen	—	—	FI	—	—	—	—	—	—	—	—	—	—	—	DV	—	—

NRTIs, nucleoside/nucleotide analogue reverse transcriptase inhibitors; NNRTIs, non-nucleoside analogue reverse transcriptase inhibitors; PI, protease inhibitor; PR, protease.

Three participants (participants 3, 4, and 5; Table 1) had concordant DRAMs between blood plasma and amplifiable anogenital samples. One participant (participant 6; Table 1) had a DRAM that was detected in blood plasma but not in anogenital samples.

Three participants had DRAMs in anogenital compartments that were not detected in blood plasma. One participant had a DRAM detected only in semen (participant 7, I54FI; Table 1) and two had DRAMs detected only in rectal secretions (participant 1, I47IM; participant 2, K70N, L74I, Y115F, M184V, K103N, V108I, and H221Y; Table 1).

More participants had NNRTI-associated mutation (five of seven in blood plasma, two of three in semen, and three of five in rectal secretions) than patients with NRTI- (two of seven in blood plasma, none in semen, and one of five in rectal secretions) or PI-associated mutations (two of seven in blood plasma, one of three in semen, and two of four in rectal secretions) across all three compartments.

There was no significant concordance between HIV-1 RNA detection in blood plasma and in semen (57%, p = .08) or in rectal secretions (86%, p = .32). One participant had cross-compartment discordance in HIV-1 subtype (Patient 1).

The results from our study show that discordance in DRAMs between blood plasma and anogenital compartments is not uncommon, particularly those with DRAMs only in anogenital compartments, among ART-naive HIV-1-infected Thai MSM.

Discordance in sequences between blood plasma and semen among our ART-naive participants supports the concept that majority of seminal HIV may arise from an isolated reservoir of infection that may function independently in the pathobiology of HIV disease. ⁷ Discordance in sequences between blood plasma and rectal compartment has been reported using rectal biopsy samples. ^8,9 Although this may not completely represent the sequences of HIV in mucosal secretions that the insertive partner will be exposed to, our result also showed discordance between the two compartments.

The finding of distinct mutation patterns in anogenital compartments but not in blood plasma is intriguing as these compartments may represent the reservoir for viral transmission. Discordance seen in these vectors may also have implication on TDR in the community. Further studies on the pharmacokinetics of ARV in different anatomical compartments may aid our understanding of the emergence and the prevention of drug resistance development.

DRAMs profile of one participant (participant 2; Table 1) showed multiple DRAMs in rectal secretions that were not detected in blood plasma. The participant had concordant HIV-1 subtype (CRF01_AE) in both compartments. Because the participant is ART naive and the fact that CRF01_AE is the most common HIV-1 subtype in Thailand, ^3,10 we hypothesized that this could be a recent superinfection that is currently limited to the anorectal compartment.

NNRTI is the most common drug class associated with drug resistance in each compartment. The finding is consistent with previous reports in Thailand and reflects the local guideline that recommends NNRTI-based therapy as the first-line regimen. PI-associated mutations found in this study are most likely polymorphism and does not imply clinical significances. ⁶

Because our analysis was based on participants with DRAMs in blood plasma, we were unable to evaluate potential cases with DRAMs isolated in anogenital compartments. Low sample size and inability to obtain genotype from both anogenital compartments are also the limitations of this study. Besides rectal secretions from participant 6, in which improper binding of primers to PR genes was the likely cause, all nonamplifiable samples were due to low HIV-1 RNA levels. Despite achieving the objective of determining discordances in DRAMs between blood plasma and anogenital compartments, larger studies, including enrolling participants without DRAMs in blood plasma, are warranted to validate the findings from our study.

In conclusion, discordance in DRAMs between blood plasma and anogenital compartments is not uncommon among newly diagnosed, ART-naive, HIV-1-infected Thai MSM. Additional monitoring of drug resistance profiles in anogenital compartments may aid in the planning of prevention programs particularly as pre-exposure prophylaxis and treatment as prevention are among the mainstay HIV prevention strategies to end the AIDS epidemic.