Abstract
In this study, we characterized HIV-1 RNA and HIV-1 DNA genotyping drug resistance detection in patients with low-level viremia (LLV) in Liangshan, China. Whole blood samples were collected from HIV/AIDS patients who had received antiretroviral therapy (ART) for ≥6 months and whose HIV-1 RNA loads were 50–1,000 copies/mL for two consecutive times at least 1-month apart. The patients were enrolled from a county in Liangshan Yi Autonomous Prefecture, Sichuan Province, between May 2021 and May 2022. Plasma and blood cells were separated. Plasma samples were tested for HIV-1 RNA genotyping drug resistance, while blood cell samples were tested for HIV-1 DNA genotyping drug resistance. Then, HIV-1 RNA and HIV-1 DNA genotyping drug resistance outcomes were compared. Among the 32 participants, 16 were males, while 16 were females, with the median age of 34.5 years. The main HIV-1 infection route was heterosexual transmission. The median ART duration was 3.9 years. Two types of nucleoside reverse transcriptase inhibitors (NRTIs) + one non-nucleoside reverse transcriptase inhibitor (NNRTI) were the main antiviral therapeutic options. Pol region genes for 28 HIV-1 DNA samples and 10 HIV-1 RNA samples were successfully amplified. The success rate of pol region gene amplification for HIV-1 DNA was significantly higher than that of HIV-1 RNA (χ 2 = 20.988, p < .05). In HIV-1 RNA and HIV-1 DNA samples, M184 (4/8) and K103 (3/8) were the most frequent drug resistance mutation sites. Among the NNRTIs, the rates of drug resistance were highest to efavirenz (EFV) (6/8) and nevirapine (NVP) (6/8), while among the NRTIs, the rates of drug resistance were highest to abacavir (ABC) (4/8), emtricitabine (FTC) (4/8), and lamivudine (3TC) (4/8). In conclusion, detection of HIV-1 RNA genotyping drug resistance combined with HIV-1 DNA genotyping drug resistance can improve the success rate of drug resistance detection in patients with LLV.
Introduction
AIDS is a serious immunodeficiency syndrome caused by the HIV infection. Antiretroviral therapy (ART) can inhibit HIV-1 replication, promote immune function recovery, reduce AIDS-associated mortality rate, and improve the quality of life for patients. 1,2 At present, relevant domestic and foreign treatment guidelines state that the goal of ART is to achieve and maintain HIV-1 RNA loads below the detection limit for long term. However, despite the effectiveness of ART, HIV-1 RNA loads in some patients remain persistently low, but detectable. 3 Low-level viremia (LLV) is defined as persistent continuous HIV-1 RNA loads between 50 and 1,000 copies/mL after ART. 4,5 The potential risks posed by persistent LLV include ART failure, increased drug resistance, fewer subsequent treatment options, and increased risk of HIV-1 sexual transmission. 4
HIV-1 drug resistance is one of the main reasons for ART failure. 2 The HIV-1 drug resistance detection is classified into phenotypic and genotyping drug resistance detection. Due to complex requirements, high costs, and slow reporting time of phenotypic drug resistance detection, genotyping drug resistance detection is currently the commonly used method. 2 For optimal HIV-1 RNA genotyping drug resistance detection, the viral load must reach a certain minimum level. When the viral load is <500 to 1,000 copies/mL, the failure rate of HIV-1 RNA amplification is high, and drug resistance detection cannot be performed. 6 Currently, the Department of Health and Human Services (DHHS, USA) guidelines recommend HIV-1 DNA genotyping drug resistance detection when the viral load is below the detection limit or at a low level. 7 However, there are no relevant recommendations in China.
The Liangshan Yi Autonomous Prefecture is located in southwestern China. Due to the unique geographical and cultural environments, the local AIDS epidemic is very serious, and has become one of the most serious public health events that endanger the local people. There are no systematic studies on HIV-1 RNA and HIV-1 DNA genotyping drug resistance characteristics among patients with LLV in Liangshan, China. This study is based on our previous exploratory research reported in the Chinese Journal of AIDS and STD. 8 In this study, we extended the enrollment time, expanded the sample size, and reanalyzed all data in depth to provide a theoretical basis and technical support for formulation and adjustment of local ART.
Materials and Methods
Study participants
All participants were HIV/AIDS patients on ART. They were independently enrolled from a county in Liangshan Yi Autonomous Prefecture, Sichuan Province, between May 2021 and May 2022. The inclusion criteria were as follows: (1) patients whose HIV-1 infection status had been confirmed by primary screening test and supplementary test; (2) patients who had been on ART ≥6 months; (3) patients whose HIV-1 RNA loads were between 50 and 1,000 copies/mL for two consecutive times at least 1-month apart; and (4) patients who had voluntarily signed the informed consent. The patients with incomplete clinical data were excluded from this study. The Clinical Trial Ethics Committee of the First People's Hospital of Yuexi County approved this study (No. 202105-01).
Sample collection
A total of 10 mL of peripheral venous whole blood samples was collected into ethylenediaminetetraacetic acid K2 anticoagulation tubes and centrifuged at 500 × g for 10 min. Plasma and blood cells were separated and stored in a −80℃ ultralow-temperature freezer.
Detection of HIV-1 RNA and HIV-1 DNA genotyping drug resistance
Plasma and blood cell samples were sent to Dongguan Micro-Precision Medicine Laboratory for detection of HIV-1 RNA and HIV-1 DNA genotyping drug resistance. The main analytic steps were as follows: (1) plasma HIV-1 RNA and blood cell HIV-1 DNA extraction; (2) PCR amplification of HIV-1 pol region genes (including reverse-transcriptase region, protease region and integrase region); (3) Sanger sequencing was performed to sequence the amplified products; and (4) the Sequencer 5.4.6 sequence splicing software was used for sequence splicing, after which the spliced sequences were exported and input into the HIV Drug Resistance Database (
Analysis of consistency between gene sequence and drug resistance mutation sites
The main steps in this assay were as follows: (1) the automatic mode of MAFFT software was used to carry out sequence comparisons between the protease and reverse transcriptase (PR + RT) region of patients with simultaneous detection of HIV-1 RNA and HIV-1 DNA drug resistance regions and reference sequence of the HIV-1 PR + RT region, respectively. Then, sequence alignment files for the PR + RT region were obtained; (2) the built-in Model Finder was used to find the best nucleotide replacement model for sequence alignment files, while the maximum likelihood (ML) method in important quartet (IQ)-Tree software was used to construct the phylogenetic tree. Finally, the interactive tree of life (iTOL) online tool was used to group and beautify the phylogenetic tree.
Data collection
Data on gender, age, HIV-1 transmission route, ART intake duration, and ART regimens for the patients were retrieved from the Database of Sichuan AIDS Treatment Information Management System. Data in the database were from patients' follow-up information.
Statistical analysis
Data were sorted using the Excel 2019 software (Microsoft Corp., Redmond, WA, USA). All analyses were performed using SPSS version 24.0 software (IBM Corp., Armonk, NY, USA). Age and ART duration were expressed as medians, while enumeration data were expressed as frequencies or percentages. Comparison of data between groups was performed using the chi-square test. Two-sided p-values were calculated, and p < .05 was the threshold for statistical significance.
Results
Basic information
A total of 32 participants (16 were males and 16 were females; median age was 34.5 years [range: 6–57]) were enrolled in this study. In terms of HIV-1 transmission routes, 20 cases were infected through heterosexual sex, 4 cases were infected through intravenous drug use (IDU), while 8 cases were infected through mother-to-child transmission. The median duration of ART administration was 3.9 years, of which 3 cases were 0.5 to <2 years, 13 cases were 2 to <4 years, 9 cases were 4 to <6 years, and 7 cases were ≥6 years. For the ART regimen, there were 27 cases using 2 NRTIs +1 NNRTI, and 5 cases using 2 NRTIs +1 protease inhibitor (PI) (Table 1).
Basic Information of Participants
ART, antiretroviral therapy; IDU, intravenous drug use; NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; PI, protease inhibitor.
HIV-1 RNA and HIV-1 DNA pol region gene amplification
Among the 32 HIV-1 RNA samples, the HIV-1 pol region genes were successfully amplified in 10 samples. The minimum HIV-1 RNA loads of samples with successful amplification of HIV-1 pol region genes were 188 copies/mL. Among the 32 HIV-1 DNA samples, HIV-1 pol region genes were successfully amplified in 28 samples, and the minimum HIV-1 RNA loads of samples with successful amplification of HIV-1 pol region genes were 50 copies/mL. The success rate of gene amplification in the pol region of HIV-1 DNA was significantly higher than that of HIV-1 RNA (χ 2 = 20.988, p < .05). In the HIV-1 RNA and HIV-1 DNA samples, the number of drug-resistant mutation sites increased with increasing HIV-1 viral loads (Table 2).
Comparisons of HIV-1 RNA and HIV-1 DNA Genotyping Drug Resistance Detection
HIV-1 subtypes
Analysis of 30 samples with successfully amplified HIV-1 pol region genes revealed successful amplification in 2 HIV-1 RNA samples, 20 HIV-1 DNA samples, and 8 successful amplifications in both HIV-1 RNA and HIV-1 DNA samples. There were 4 HIV-1 subtypes in the 30 samples, among which the CRF07_BC recombinant type was the most dominant, followed by the B + C recombinant type (Table 3).
HIV-1 Subtypes
Comparisons of HIV-1 RNA and HIV-1 DNA drug resistance mutation sites
Among the 10 HIV-1 RNA samples whose HIV-1 pol region genes were successfully amplified, 7 cases had drug resistance mutation sites against NNRTIs, while 4 cases had drug resistance mutation sites against NRTIs, and no PI and integrase inhibitor (INSTI) mutation sites were found. Among the 28 HIV-1 DNA samples with successfully amplified HIV-1 pol region genes, 14 cases had drug resistance mutation sites against NNRTIs, 8 cases had drug resistance mutation sites against NRTIs, 1 case had drug resistance mutation sites against PIs, and 3 cases had drug resistance mutation sites against INSTIs (Table 4).
Comparisons of HIV-1 RNA and HIV-1 DNA Drug Resistance Mutation Sites
“—” indicates that no drug resistance mutation site was found; “/” indicates that the sequencing primer failed to amplify the target band.
INSTI, integrase inhibitor.
Comparisons of HIV-1 RNA and HIV-1 DNA drug resistance
Among the 10 HIV-1 RNA samples whose HIV-1 pol region genes were successfully amplified, 4 cases were resistant to both NRTIs and NNRTIs, while 3 cases were resistant to NNRTIs only. Among the 28 HIV-1 DNA samples with successful HIV-1 pol region gene amplification, 7 cases were resistant to both NRTIs and NNRTIs, 6 cases were resistant to NNRTIs only, while 1 case was resistant to both NNRTIs and PIs. There was no resistance to INSTIs in both HIV-1 RNA and HIV-1 DNA samples (Table 5).
Comparisons of HIV-1 RNA and HIV-1 DNA Drug Resistance
“ND” indicates that no drug resistance was found.
3TC, lamivudine; ABC, abacavir; AZT, zidovudine; d4T, stavudine; ddI, dideoxyinosine; DOR, dorivirine; EFV, efavirenz; ETR, etravirine; FTC, emtricitabine; H, high resistance; I, intermediate resistance; L, low resistance; NFV, nelfinavir; NVP, nevirapine; RPV, rilpivirine; TDF, tenofovir.
Comparisons of consistency between HIV-1 RNA and HIV-1 DNA drug resistance mutation sites and drug resistance
Analysis of 8 samples with successfully amplified HIV-1 pol region genes in both HIV-1 RNA and HIV-1 DNA revealed 6 samples with drug resistance mutation sites and drug resistance. There were 4 samples with the same drug resistance mutation sites and drug resistance in both HIV-1 RNA and HIV-1 DNA. There was 1 sample with one more drug resistance mutation site and drug resistance in HIV-1 RNA, while there was another sample with one more drug resistance mutation site and drug resistance in HIV-1 DNA. Based on successful detection of HIV-1 RNA and HIV-1 DNA genotyping drug resistance in patient IDs12, 16, 17, 19, 21, 22, and 30, the detected HIV-1 RNA and HIV-1 DNA sequences in the same case were on the same branch (Fig. 1).
The phylogenetic tree.
These results implied that drug resistance mutation sites and drug resistance in HIV-1 RNA and HIV-1 DNA samples were relatively consistent. It was found that M184 (4/8) and K103 (3/8) were the most frequent drug resistance mutation sites. Among the NNRTIs, drug resistance rates were highest against efavirenz (EFV) (6/8) and nevirapine (NVP) (6/8) while among the NRTIs, drug resistance rates were highest against abacavir (ABC) (4/8), emtricitabine (FTC) (4/8) and lamivudine (3TC) (4/8).
Discussion
When HIV-1 enters the host cell, the viral genome is reverse-transcribed into DNA, and transported into the nucleus, where it is integrated into the host cell DNA to form a “provirus.” 2 When the provirus is activated to transcribe itself, the viral DNA is transcribed to form RNA, and then progeny viruses are formed via translation, assembly, and budding. 2 Due to the high replication and mutation rates of HIV-1, coupled with host's immunity and drug pressures, the virus strains have developed drug resistance over time. 9 Currently, amplification of HIV-1 pol region genes is the main method for detecting HIV-1 genotyping drug resistance. 10
In this study, the success rate of gene amplification in the pol region of HIV-1 DNA was higher than that of HIV-1 RNA. This is because free HIV-1 RNA in plasma of patients with LLV is relatively low, while the HIV-1 DNA genotyping drug resistance detection extracts the DNA in blood cells, which is not affected by the amount of free HIV-1 RNA in plasma, thereby improving the success rate of gene amplification. 11,12 This shows that detection of HIV-1 DNA genotyping drug resistance is more successful than HIV-1 RNA genotyping drug resistance detection in patients with LLV. 13 Raymond et al. found that when HIV-1 RNA loads are low, drug resistance detection against HIV-1 DNA can detect virological failure in LLV or guide simplified treatment. 14 Currently, DHHS guidelines also recommend HIV-1 DNA genotyping drug resistance detection for patients with LLV. 7
For the 32 patients with LLV, the CRF07_BC recombinant was the main subtype, followed by the B + C subtype. HIV-1 was mainly found to be present in blood, semen, and vaginal secretions of HIV-1-infected patients, and therefore, IDU and unsafe sexual behaviors were the main transmission routes. Since Liangshan was one of the important passages through which drugs enter China's inland areas, the proportion of local IDUs was relatively high. This implies that most of the local HIV-1 infections are transmitted via IDU, 15 and the CRF07_BC recombinant type was the most prevalent subtype in the local area. 16,17 We also found that for the 32 patients with LLV, heterosexual transmission was the main route of HIV-1 infection, while only two cases were infected by IDU.
Due to the vigorous crackdown on drugs in Liangshan in recent years, the number of IDUs has been steadily decreasing, and HIV-1 infection has begun to shift from the high-risk group of IDUs to the general population. Therefore, there is a need to strengthen public awareness and knowledge of AIDS prevention and control in the general population.
In this study, resistance mutation sites and drug resistance of HIV-1 RNA and HIV-1 DNA samples were relatively consistent. Based on genetic distance, the HIV-1 DNA drug resistance detection sequences for 4 patients (IDs16, 17, 19, and 21) were the same as those detected in HIV-1 RNA samples, proving that the sequences detected by HIV-1 DNA and HIV-1 RNA drug resistance analyses were consistent. In 3 patients (IDs12, 22, and 30), the difference between the genetic sequence distance detected by HIV-1 DNA drug resistance and the genetic sequence distance detected by HIV-1 RNA drug resistance was <10−. 6
These results proved that sequences for HIV-1 RNA and HIV-1 DNA resistance regions in all cases were highly comparable. This is because HIV-1 genotyping drug resistance initially occurs at the HIV-1 DNA level with drug resistance mutation sites, and HIV-1 DNA containing drug resistance mutation sites replicate and proliferate, resulting in the emergence of drug-resistant viruses. 18 However, the HIV-1 DNA may lack the ability to replicate, due to defects in the genome. If the drug-resistant mutation site occurs in the defective virus that lacks the ability to replicate, it is impossible to produce a drug-resistant virus. In this study, the most frequent drug-resistant mutation sites and the most frequent resistant drugs were consistent with the results of Cai et al. 10
This study had some limitations. First, this was a single-center study with a small sample size, and there may be some bias in the analysis of drug resistance results. Second, although Sanger sequencing is the most widely used sequencing method in clinics, it has a relatively limited sensitivity. When the proportion of resistant strains is low, their presence may not be detected. 6 Third, all participants were not subjected to baseline HIV-1 genotyping drug resistance detection, and therefore, it was impossible to determine whether drug resistance was pretreatment drug resistance or acquired drug resistance. Finally, since HIV-1 DNA may contain defective viruses that result in abortive replications, it was not determined whether HIV-1 DNA drug resistance will subsequently develop into HIV-1 RNA drug resistance, which needs further observation and study.
Conclusions
Detection of HIV-1 DNA genotyping drug resistance in patients with LLV is more successful than detection of HIV-1 RNA genotyping drug resistance. Therefore, detection of HIV-1 RNA genotyping drug resistance combined with HIV-1 DNA genotyping drug resistance can improve the success rate of drug resistance detection in patients with LLV. Given the drug resistance mutation sites found in HIV-1 DNA genotyping drug resistance detection in patients with LLV in Liangshan, there is a need to increase the frequency of HIV-1 RNA detection so as to identify patients with failed treatment outcomes in a timely manner.
Footnotes
Authors' Contributions
Data collection was conducted by T.Y., P.D., and X.Z. Data analysis and interpretation were conducted by B.C., M.L., T.J., and Q.Y. Drafting the article was conducted by B.C. Revising the article was conducted by F.H., Y.H., and J.J. Study design and final approval of the article were conducted by J.J.
Availability of Data and Materials
The data collected in this study will be available to external investigators interested in collaboration upon submission and approval of a data analysis plan. Requests for data should be submitted to
Author Disclosure Statement
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding Information
This work was supported by the Department of Science and Technology of Sichuan Province (No. 2020YFS0514) and the Health Commission of Sichuan Province (No. 20PJ139).