Abstract
Genotypic drug-resistance testing for HIV-1 with low viral loads (VLs) (<1,000 copies/mL) is clinically important but technically challenging. Due to the overlapping physical size characteristics of HIV-1 viral particles (100–120 nm) and exosomes (40–150 nm), simultaneous enrichment of both using size separation technology may offer a new strategy to improve the success rate of genotypic resistance testing in low VL samples. To evaluate the application of exosome enrichment technology in genotypic drug resistance testing for HIV-1 in low VL samples. We conducted a study with the following design. Whole blood samples were collected from HIV/AIDS patients at the Guiyang Public Health Treatment Center, who had been receiving antiretroviral therapy for over 6 months, with HIV-1 RNA levels ranging from 20 to 1,000 copies/mL, between June 2023 and November 2024. Plasma was separated, and HIV-1 RNA genotypic resistance testing was performed both directly on the plasma and after exosome enrichment. The amplification success rates of HIV-1 genotypic resistance testing before and after exosome enrichment were compared, and resistance mutation sites were analyzed. Among the 26 participants, 22 were male (84.62%) and 4 were female (15.38%), with a median age of 36.5 years. Exosome enrichment technology achieved amplification success rates for the HIV-1 genotypic resistance testing in the RT & PR regions and the INSTI region of 65.38% (17/26) and 42.31% (11/26), respectively, significantly higher than the pre-enrichment success rates of 19.23% (5/26) and 15.38% (4/26). These differences were statistically significant (χ2 = 11.34, p = 0.001; χ2 = 4.62, p = 0.032). Genotypic sequencing revealed K103N and L74M resistance mutations in two samples. These findings indicate that exosome enrichment technology enhances the amplification success rate of HIV-1 genotypic resistance testing in low VL samples and identifies clinically relevant resistance mutation sites. This approach may provide an innovative solution for resistance testing in low VL samples.
Introduction
In recent years, antiretroviral therapy (ART) has been widely used in HIV/AIDS patients, significantly reducing both infection and mortality rates.1,2 Most patients can achieve long-term viral suppression with continuous treatment, bringing their viral load (VL) below the detection limit. However, some patients still experience low-level viremia (LLV), where the HIV VL remains above the method’s lowest detection limit but below 1,000 copies/mL.3,4 The prevalence of LLV varies across different regions, with studies showing that approximately 8.6% of HIV-1 patients in China exhibit LLV, 5 and these patients are at a higher risk for resistance mutations.6,7 Therefore, it is crucial to identify LLV patients early and perform genotypic resistance testing.
However, because the samples from LLV patients are considered low VL samples (VL <1,000 copies/mL), and HIV-1 genotypic resistance testing typically requires a VL above this threshold,8,9 increasing the success rate of genotypic resistance testing in low VL samples is the central focus of this study.
Genotypic resistance testing generally involves RT-PCR amplification of the target gene segment, followed by sequencing (either Sanger or deep sequencing) to obtain the sequence of the relevant gene. This sequence is then compared to wild-type sequences and resistant strains to analyze resistance-related mutations. A genotypic resistance interpretation system is used to determine whether resistance is present and, if so, to assess its extent. 10 At low VLs, amplification failure rates are higher due to insufficient viral template, which is why genotypic resistance testing typically requires a higher VL. 8 The key to addressing this issue lies in increasing the viral template amount in the sample, thereby improving the success rate of genotypic resistance testing. HIV-1 viral particles typically have a diameter of 100–120 nm, 11 while exosomes range from 40 to 150 nm,12,13 with some overlap in size. Due to this characteristic, exosome separation technology, based on size separation principles, could theoretically enrich both exosomes and viral particles in plasma simultaneously, thus improving the amplification success rate and the effectiveness of resistance testing in low VL samples.
This study aims to apply exosome enrichment technology to enhance the amplification success rate of HIV-1 genotypic resistance testing in low VL samples from Guiyang, providing crucial information for optimizing and adjusting ART regimens.
Subjects and Methods
Subjects
Study participants were HIV/AIDS patients who received ART at the Guiyang Public Health Treatment Center from June 2023 to October 2024. The inclusion criteria were: (1) confirmation of HIV-1 infection through initial screening and supplementary tests; (2) ART duration of at least 6 months; (3) HIV-1 RNA quantification between 20 and 1,000 copies/mL; and (4) voluntary signing of the informed consent form. This study was approved by the Medical Ethics Committee of the Guiyang Public Health Treatment Center (Approval No. [2024] Paper No. [07]).
Methods
Sample collection
Peripheral venous blood (10 mL) was collected from each participant using an Ethylenediaminetetraacetic Acid Dipotassium Salt (EDTA K2) anticoagulant tube. The blood was centrifuged at 500 × g for 10 min to separate the plasma. A 750 µL aliquot of plasma was used for exosome enrichment, and the remaining sample was stored at −20°C in an EP tube for future use.
Exosome enrichment
Exosome enrichment was performed using an exosome extraction device (Supbio Biotechnology Co., Ltd., Guangdong-Guangzhou Medical Device Filing Number: 20170482). The procedure was as follows: 750 µL of plasma was placed into a centrifuge tube, and 375 µL of extraction solution A and 37.5 µL of extraction solution B were added. The mixture was vortexed for 15 sec and incubated at room temperature for 10 min. After incubation, the entire sample was transferred to the upper filter chamber of an Exo column and centrifuged at 1,000 × g for 20 min. This process was repeated at room temperature (1,000 × g for 20 min) until the volume of retained fluid in the middle filter chamber reached approximately 300 µL. The retained fluid was then collected.
HIV-1 RNA genotypic resistance testing and genotyping
Both the separated plasma and the final product after exosome enrichment (with a sample volume of 300 µL each) were sent to the Dongguan Institute for Microscale and Precision Medical Measurement for HIV-1 RNA genotypic resistance testing using an in-house method. The main steps included: (a) HIV-1 RNA extraction; (b) Reverse Transcription-Polymerase Chain Reaction (RT-PCR) combined with nested PCR was used to amplify the reverse transcriptase (RT) and protease (PR) genes in the HIV-1 pol region (covering 99 amino acids in the protease region and at least 259 amino acids in the reverse transcriptase region, with an amplicon length of approximately 1.1 kb), as well as the integrase (IN) gene; (c) The amplification products were sequenced by Sanger sequencing; (d) The sequences were submitted to GenBank (accession numbers PV169322-PV169358); (e) The HIV-1 viral sequences were submitted to the Stanford University HIV drug resistance database (https://hivdb.stanford.edu) for genotyping and comprehensive resistance mutation analysis. Resistance levels were systematically classified according to the HIVDB algorithm into four categories: sensitive (S), potential resistance (P), low-level resistance (L), and intermediate resistance (I).
HIV-1 RNA quantification
HIV-1 RNA was measured in the separated plasma samples. The procedure was as follows: (1) HIV-1 RNA was extracted from the plasma; (2) quantification of HIV-1 RNA in the plasma was performed using fluorescent quantitative RT-PCR.
Data collection
Data were collected on the participants’ gender, age, duration of initial ART, and ART regimen.
Statistical analysis
Statistical analysis was performed using SPSS version 24.0. Age was described using the median, while categorical data were expressed as frequencies or percentages. The difference in amplification success rates before and after exosome enrichment was evaluated using the chi-square test for independence. A p value of <.05 was considered statistically significant.
Results
Basic characteristics
A total of 26 participants were included in this study, all of whom had received at least 6 months of ART at the Guiyang Public Health Treatment Center. Of these, 22 were male (84.62%) and 4 were female (15.38%). The median age of the participants was 36.5 years.
Amplification success rates before and after enrichment
In the 26 plasma samples, exosome enrichment significantly improved the amplification success rates for HIV-1 genotypic resistance testing in the RT and PR regions and the INSTI region. In the RT & PR regions, the amplification success rate before enrichment was 19.23% (5/26), which increased significantly to 65.38% (17/26) after enrichment. This difference was statistically significant (χ2 = 11.34, p = 0.001). For the INSTI region, the amplification success rate before enrichment was 15.38% (4/26), which increased significantly to 42.31% (11/26) following enrichment. This difference was also statistically significant (χ2 = 4.62, p = 0.032). Notably, all samples that successfully amplified before enrichment continued to amplify successfully after enrichment (Table 1).
Comparison of Amplification Success Rates Before and After Enrichment
Distribution of resistance mutations in successfully amplified samples
Among the 17 successfully amplified samples after exosome enrichment, the predominant viral subtype was CRF07_BC, accounting for 12 cases (70.59%). This was followed by CRF01_AE (4 cases, 23.53%) and CRF08_BC (1 case, 5.82%). A total of two resistance mutation sites were detected: L74M and K103N (Table 2).
Distribution of Viral Load, Viral Subtypes, and Resistance Mutations in 26 Samples
“/” indicates unsuccessful amplification; “N” indicates no resistance mutations detected.
Discussion
HIV-1 genotypic resistance testing is the primary method used in clinical practice to assess drug resistance. 14 However, conventional methods generally require a VL of ≥1,000 copies/mL for reliable amplification results. 9 In contrast, patients with LLV present a VL above the minimum detection threshold for standard testing methods but below 1,000 copies/mL. 4 This creates a technical bottleneck in the management of LLV patients. To address this issue, this study utilized exosome enrichment technology to concentrate HIV-1 RNA from low VL samples, which significantly increased the amplification success rate for genotypic resistance testing: the success rate for the RT & PR region rose from 19.23% (5/26) to 65.38% (17/26) (χ2 = 11.34, p = 0.001), and for the INSTI region, it increased from 15.38% (4/26) to 42.31% (11/26) (χ2 = 4.62, p = 0.032). To further investigate why exosome enrichment significantly improved the amplification success rate for low VL samples, we analyzed the physical similarities between HIV-1 viral particles and exosomes, as well as the operational principle of the enrichment device used.
HIV-1 viral particles typically have a diameter of 100–120 nm, 11 while exosomes range in size from 40 to 150 nm.12,13 This overlap in size at the nanoscale allows exosome separation techniques, which are based on size exclusion, to enrich both exosomes and viral particles simultaneously. As a result, this approach improves the success rate of genotypic resistance testing. The exosome separation kit used in this study employs ultrafiltration and requires only low-speed centrifugation (1,000× g), which can be efficiently performed using a standard centrifuge to extract exosomes from plasma. The recovery rate exceeds 95%, with over 80% of the exosome particles falling within the 20–140 nm range. Thus, this method effectively enriches HIV-1 viral particles in plasma while removing large molecular impurities, thereby increasing the concentration of HIV-1 RNA templates in low VL samples. This enrichment process effectively overcomes the limitations of traditional methods, where amplification success rates are low for low VL samples, providing a solid technical foundation for subsequent gene amplification.
Among the 17 successfully amplified samples in this study, the predominant HIV-1 subtype was CRF07_BC, accounting for 12 samples (70.59%), followed by CRF01_AE (4 samples, 23.53%) and CRF08_BC (1 sample, 5.88%). This distribution of subtypes is consistent with the dominant circulating strains in Guiyang, China. 15 Resistance mutation testing revealed that two resistance gene mutations were detected in the 17 successfully amplified samples, with mutation sites identified as K103N and L74M. The K103N mutation results in high resistance to efavirenz and nevirapine (H), while the L74M mutation confers potential resistance to cabotegravir (P). These findings suggest that genotypic resistance testing in low VL specimens can detect resistance mutations, thereby informing the adjustment of treatment regimens.
Despite the significant improvements observed in amplification success rates for low VL samples using exosome enrichment technology in this study, several limitations should be noted. First, the sample size in this study was small, with only 26 patients, which may limit the generalizability of the findings. In addition, studies from various regions in China have reported a relatively high incidence of drug resistance mutations (DRMs) in LLV patients, with reported rates ranging from 37.30% to 59.21%.6,16,17 The DRMs rate observed in this study was 11.76% (2/17), which is lower than the previously reported range. This difference may be attributed to the primary goal of this study, which was to explore methods for improving the success rate of genotypic resistance testing amplification in low VL samples. At the time of patient enrollment, VL testing was conducted only once on the observed samples, and these samples may have been from patients experiencing transient viral blips, 14 rather than representing typical LLV patients.
Furthermore, the stability of the exosome enrichment technology was not systematically assessed in this study. This characteristic is crucial for the technology’s reliability and applicability under different laboratory conditions. Future studies should aim to expand the sample size and implement multi-center, multi-batch experimental designs to further evaluate the stability of this technology and its applicability across various laboratory environments and protocols, ensuring its widespread clinical value in drug resistance testing for low VL samples.
In conclusion, the application of exosome enrichment technology in the preprocessing of low VL specimens significantly enhanced the success rate of genotypic resistance amplification, providing a new technical approach for the diagnosis and treatment of LLV patients.
Authors’ Contributions
J.P. and Y.S. contributed equally to the conceptualization, methodology, data curation, formal analysis, and writing of the original draft. Z.C. provided resources, conducted investigations, and acquired funding. L.X. was involved in data collection, while L.G. contributed to the methodology. X.X. handled data analysis. Y.F. and H.L. were both involved in investigation and resource provision. Y.F. and K.H. contributed equally to funding acquisition, writing—review and editing, conceptualization, supervision, and project administration.
Footnotes
Author Disclosure Statement
No competing financial interests exist.
Funding Information
This study was funded by The 2023 National Clinical Key Specialty Major Scientific Research Project from the Health Commission of Hunan Province (project number: Z2023111); The 2024 Hubei Provincial Natural Science Foundation General Program (project number: 2024AFB1059). The authors appreciate the financial support from these funding sources. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.