Artigos

1. Introduction
According to current estimates, there are more than 38 million HIV-positive people worldwide, 60% of whom reside in sub-Saharan Africa, which has been reported an increase in infectious cases in the North and Middle East Africa in the last decade 1. In this context, despite having effective therapy and prevention measures, it persists as a
relevant public health problem in the 21st century, considering that HIV/aids has already claimed the lives of more than 25 million people 2.

HIV is categorized into types, groups, subtypes, circulating recombinant forms (CRFs), and unique recombinant forms (URFs) based on phylogenetic study of multiple isolates acquired from individuals living in various geographical locations 3. HIV can be classified into types 1 and 2 (HIV-1 and HIV-2). Such subtypes have the same action in the human body, but HIV-2 produces fewer viral particles than HIV-1 and has restricted geographic circulation 2.
Recombinant forms, including Circulating recombinant forms (CRF) and unique recombinant forms (URF), emerge where multiple subtypes co-circulate. Because HIV produces between 10¹?–10¹¹ virions per day in untreated individuals, every possible mutation arises frequently, making combination therapy essential 4. These "hybrid populations" are the end result of viral recombination processes 5 are the two categories of recombinant forms that have been discovered. Hence, HIV-1 group M has a large variety of CRFs and URFs 6.
Every conceivable mutation and numerous double mutations are probably produced in every untreated person every day as a result of the high rates of replication, mutation, and recombination 7. Thus, the need for combination treatment for HIV infection is based on the reality that drug-resistant mutations (DRMs) are present in all infected individuals before the start of therapy 8.
Men who have Sex with Men (MSM) experience disproportionately high HIV incidence—around 20 times greater than the general population 9,10. Results of a recent study conducted in Brazil using surveillance data for HIV and aids show that there were about 41.000 new HIV infections in 2019, with 70% of cases resulting from male intercourse, 51.6% from homo- or bisexual exposure, and 31.3% from heterosexual contact
11. This image mirrors the pattern in other nations where, in contrast to other population groups, the chance of HIV infection among MSM has remained elevated in recent years 12. Epidemiological research on the HIV pandemic among MSM is rare in Belém, Pará,
Brazil 13. Studying molecular epidemiology and epidemiological characteristics can help identify actions and measures that should be taken in this particular illness. In order to better understand it in this community, this research examined the risk factors for this infection and the genetic variety of HIV isolates, which was drawn from the MSM population in the city of Belém. Then, this work is also part of a broader context of research into HIV and aids in Brazil and around the world. Understanding circulating variants and resistance profiles supports prevention and treatment strategies and helps address knowledge gaps regarding HIV dynamics in this key population 14,15.

2. Methods

2.1. Study Design and Logistics

This is a cross-sectional and retrospective study that analyzed epidemiological factors, prevalence of infections, and genetic diversity of HIV-1 in a cohort of 350 men who have sex with men (MSM) recruited in Belém-PA, in 2016, as part of a national survey in 12 capital cities 12,16–21. The Brazilian Ministry of Health decided on the sample size. MSM recruitment network in Belém, Pará, Brazil, 2016 has already been reported in another study of the same cohort but with different analyzes by Carneiro et al. (2023) 20. The recommendations of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) tool were followed 22.
Given that possible participants were familiar with the health unit, the Centro de Saúde Escola do Marco (CSE-Marco) was chosen as the site for field activities once the planning and conduction procedures of the study phases were established. The chosen

medical institution was open from 5 pm to 11 pm, when the majority of participants were available to participate, and it was easily accessible by public transit for our research population.


2.2. Exams and Laboratory procedures
The interviewee was offered to participate in pre-test counseling if they agreed. After the interview, all participants were invited to undergo rapid testing for HIV, syphilis, hepatitis B, and C, following the guidelines of the Department of Sexually Transmitted Infections and Viral Hepatitis (DIAHV) of the Brazilian Ministry of Health. Reactive tests were repeated (TR1 and TR2) according to the Ministry of Health's protocols. Five mL of blood were collected in ethylenediamine tetraacetic acid (EDTA) and eight mL in a dry tube for confirmatory tests, viral load, avidity, and genotyping. Reactive or discordant samples were sent to the Adolfo Lutz Institute (SP) for confirmation, transported under dry ice according to National Health Surveillance Agency (ANVISA) regulations.
In the Adolfo Lutz Institute, HIV-1 RNA was extracted from 500 ?L of participant’s plasma sample using the QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany). Primers from protease (PR) and reverse transcriptase (RT) regions of the HIV-1 polymerase gene (pol) were designed with Primer3Plus and they were specified on Supplementary Material S1 23. Genbank accession numbers: MN971800-MN972432. Platinum Taq DNA Polymerase, DNA-free (Invitrogen®, Thermo Fisher Scientific Corporation, Waltham, Massachusetts, USA) was used in the thermocycler 24. The mix used for amplification was in reaction with a final volume of 50 ?L, containing 1.5 mM MgCl2, 0.05 mM dNTPs, 0.25 ?M of the primers, 1 U Taq polymerase and 2 ?L of the extracted material.
In the thermocycler, the following primer amplification conditions were followed: denaturation step at 95°C for 1 min; annealing temperature step for 30 sec; extension step at 72°C for 1 min. The amplified products were electrophoresed in a 2% agarose gel with
3.0 µL using Sybr Safe (Invitrogen®, Thermo Fisher Scientific Corporation, Waltham, Massachusetts, USA) in order to see the amplified DNA fragments in a photodocumentation device.
The PCR products were purified in accordance with the manufacturer's instructions using the EasyPure PCR Purification Kit (TransGen Biotech Co.®, Beijing, Beijing, China). In summary, the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems Foster City, CA) was used to amplify and sequence an HIV genome fragment including the reverse transcriptase and protease genes in accordance with the manufacturer's instructions. The results of this reaction were then investigated using an automated sequencer, the ABI3500 (Applied Biosystems).

2.3. HIV-1 subtyping determination and transmitted drug resistance analysis
SeqMan (DNAStar, Madison, Wisconsin, USA) and BioEdit v7.0.9 25 were the two programs used to edit and assemble the nucleotide sequences of the same sample that were acquired via sequencing. The sequences were edited, aligned with GenBank references, and classified using the REGA v3.46 and COMET algorithms 26,27. Consequently, using the list provided by Tzou et al., 2020, the nucleotide sequences were submitted to the
Calibrated Population Resistance (CPR) analysis tool (https://hivdb.stanford.edu/cpr/) in order to determine the proportions of people with overall NRTI, NNRTI, PI, and INSTI-associated pretreatment drug resistance (PDR) 28.
Through submitting the FASTA files of the created strains to the online drug resistance interpretation tool accessible at the Stanford HIV resistance database website

(http://hivdb.stanford.edu/), the drug susceptibility level was deducted. Next, each mutation is given a drug resistance score using the Stanford HIV database algorithm.
These scores correspond to five categories of predicted antiretroviral resistance: susceptible, possible low-level, low-level, intermediate, and high-level resistance 29. HIV Database for Transmitted DRM-TDRM (CPR Tool version 9.0) and DRM (HIVdb Program version 6.3.1) for patients who are naive and those who have received treatment, respectively 30,31.

2.4. Statistical analysis
Data analysis incorporated RDS weighting using RDS Analyst®, ensuring proper adjustment for network-based sampling. Descriptive statistics included frequency distributions for categorical variables and measures of central tendency for continuous variables. Associations between HIV status and explanatory factors (sociodemographic, behavioral, and clinical) were tested using Chi-square or Fisher’s Exact test for categorical variables. G tests of independence followed by adjusted residual analysis were applied when appropriate.
For multivariable modeling, binary logistic regression was performed to identify independent predictors of HIV infection. The model included variables such as ART status, viral load, age group, education, ethnicity, sexual behaviors, cohabitation, income, health insurance, drug/alcohol use, co-infections, and resistance profiles. Model fit was evaluated using Deviance, Akaike Information Criterion (AIC), Pseudo R, McFadden’s R², and Nagelkerke’s R². Statistical significance was set at p<0.05. Analyses were conducted using SPSS® v26.0 (IBM Corp., Armonk, NY, USA).

2.5. Ethical considerations
The study was approved by the Research Ethics Committee of the Federal University of Ceará (COMEPE/UFC nº 1.024.053) and was carried out in the city of Belém in the state of Pará. Informed consent was obtained from all MSM individuals involved in the study.

3. Results
From 350 initial participants, 349 MSM were included. The analysis of the networks of this RDS showing the recruitment of participants was already presented in a previous study by our research group in which the same cohort was used but for the purposes of differentiated analyses in relation to epidemiological data 20. The descriptive analysis of sociodemographic and economic variables using absolute and relative numbers and their possible associations with the risk of HIV infection, Syphilis and Hepatitis B and Hepatitis C in the cohort of MSM from Belém-PA in 2016 was characterized in Table of the Supplementary Material S2. No Hepatitis C cases were detected. In bivariate analyses, ethnicity, living alone, illicit drug use, income and health insurance were not associated with HIV or Sexually Transmitted Infections outcomes (p>0.05).
For HIV, age ?48 years correlated with not undergoing confirmatory testing (p= 0.019). Marital status showed an association: married participants were more likely not to have completed testing (p= 0.028). Household size influenced HIV negativity in those living with ?4 or ?9 individuals (p= 0.016). Syphilis was associated with younger age (18–27) (p= 0.026). Hepatitis B was associated with nightclub attendance (p= 0.049) and gay-bar frequency (p= 0.034).
Regarding individual income and Hepatitis C, earning 2 to 3 minimum wages at the time of the study was statistically present in the groups with a negative diagnosis or those who had not taken any tests, while receiving more than 4 minimum wages was correlated

with not having been tested for the infection (p= 0.022). Hepatitis C was associated with unprotected commercial sex in the last 6 months, being related to a negative diagnostic element and not having a commercial partner (p= 0.047). Furthermore, the number of partners was a risk factor for Hepatitis C, as those who had up to 5 partners tested negative for this HCV infection (p= 0.034).
Only 42 HIV-positive individuals provided samples for sequencing. In multivariate analysis, HIV infection was associated with: unprotected sex with stable or casual partners in the last 6 months, being single, nightclub and gay-bar attendance, not responding about ART status, alcohol use (2–4×/month), and earning 1–2 minimum wages (Table 1).

Tab.1

A descriptive analysis was conducted using absolute and relative values of the viral load of HIV-infected individuals (N= 52), where it was possible to obtain viral load data, to the detriment of the ART status on this cohort in Table 2. Among 52 individuals with viral-load data, 44.23% of those with high viral load did not report ART status (p<0.001). Viremia did not correlate with other infections.

Tab.2

A description of absolute, relative and associative data was carried out on the detection of HIV-1 subtypes and presence/absence of mutations in HIV viral proteins in infected individuals (N= 42), in relation to ART status on this cohort in Tables 3 and 4, respectively. Other subtypes were characterized by C (n=1; 2.38%), F (n=2; 4.76%), BC (n=4; 9.52%) and BF (n=2; 4.76%). The ART status of these individuals did not show a

statistically significant difference between the groups with subtype B and the other non-B subtypes (p>0.05). The presence of mutations in HIV-1 Subtypes were not statistically different between the groups of infections analyzed. According to HIV-1 protease and reverse transcriptase portions, subtype B was the most found (n=33; 78.57%). Regarding the mutations under analysis, there was a predominance of absence of mutations in both the Stanford and CPR analyses for the group of individuals who reported adequately administering ART (p>0.05).

Tab.3

Tab. 4

Regarding mutations, for Stanford analysis, approximately 95% of the cohort was absent of PR-type mutation, while approximately 87% was absent of NRTI and approximately 75% was absent of NNRTI. Regarding Stanford evaluation, PR mutations found were: G48R, G73S (n=1; 2.4%); M46ML (n=1; 2.4%). For NRTI, we obtained findings of A62AV (n=1; 2.4%); M184I (n=1; 2.4%); M184V (n=1; 2.4%); Q151QLPR
(n=1; 2.4%); and T69D (n=1; 2.4%). For NNRTI, the following were found: E138A (n=4; 9.5%); G190R, M230I (n=1; 2.4%); K103KN (n=1; 2.4%); K103N (n=1; 2.4%); V179D
(n=1; 2.4%); Y188L (n=1; 2.4%). Regarding CPR analysis, approximately 98% of MSM individuals did not have a PR type mutation, 95% had no NRTI and 93% had no NNRTI. The CPR-PR mutations found were: M46L (n=1; 2.4%). For NRTI, we found the T69D (n=1; 2.4%) and M184V (n=1; 2.4%) mutations. For NNRTI, the K103N (n=2; 4.8%) and Y188L (n=1; 2.4%) mutations were present.
Table 5 describes the analysis of presence of the mutations in viral proteins and their statistical associations for the detection of Syphilis, Hepatitis B and C infections in relation to ART performed by the cohort. The category of not having responded to the ART questionnaire (skipping jumps) was the category with the highest frequency of individuals

with the prospect of mutations for all diseases, with the highest presence of all mutations found in individuals diagnosed positive for syphilis while negative for Hepatitis B and C.

Tab.5


4. Discussion

Research estimates a high HIV prevalence of around 12 to 18% in Brazilian MSM populations in recent years 16,21. HIV prevalence in the cohort exceeded 15%, much higher than Brazil’s general prevalence (0.47%) 32–35. According also to previous RDS studies, MSM who fit this profile—that is, those who have lower incomes, average levels of education, and ages close to 25—are more likely to contract HIV 36–38. Risk behaviors such as unprotected sex with stable or casual partners were major predictors of infection, consistent with global evidence. Socioeconomic markers such as lower income have been previously associated with increased HIV vulnerability among MSM and corroborate our data 39,40.
Subtype B predominated, in agreement with patterns seen across the Americas and Brazil 41,42. High HIV mutation rates facilitate emergence of resistant variants. In this study, significant proportions exhibited non-nucleoside reverse transcriptase inhibitors (NNRTI)-related mutations, which can compromise antiretroviral treatment (ART) effectiveness. Early detection of resistance mutations is essential to guide the choice of the most effective antiretroviral drugs, ensuring appropriate treatment and improving clinical outcomes for infected patients 43. Mutations such as K103N and M184V, frequently reported in Brazil, were also detected 44.
In a study carried out in the prison population in the Central region of Brazil, the same K103N mutation was detected in 13.1% of individuals, where they had no history of treatment 45. Leal et al. (2020) carried out a study in a state in the Brazilian Northeast and identified the high frequency of subtype B, in addition to a high level of antiretroviral resistance associated with L100I, G190A and K103N for NNRTIs and M41L, K65R, D67N, K70R, M184V, T215FY and L210W for NRTIs 46.
In the Southern Region of Brazil, some studies have demonstrated the prevalence of subtypes B and C of HIV-1, corroborating what was found here 47. Bahls et al ., (2019)

again identified the K103N mutation as the most frequent in the population of the state of Paraná 48. In southeastern Brazil, a study characterized the mutations detected in strains isolated in the population of Rio de Janeiro, and as in the present study, the M184V mutation was detected, which is related to reduced susceptibility to antiretroviral drugs used in treatment 49. In São Paulo, the resistance rate in treated patients was extremely high, being associated with the M184V mutation for NRTI and K103N for NNRTI, where the most common subtype was B 50. In Amazonas, a state in the north of the country, just like the study described here, participants showed a high prevalence of mutations associated with NRTI and NNRTI, where the most common mutation is K103N, being detected in 71.8%
51.
In a study carried out in countries in Eastern Europe and Russia, the most frequent viral mutations found in infected patients were identified. The most common polymorphic mutation found was E138A, which is quite common in the A6 subtype, but does not seem to affect the response to treatment. Furthermore, a 93% prevalence of the L74I mutation was detected in Ukrainian strains of the A6 subtype, which is directly associated with the HIV epidemic that occurred in Europe 52. Oluniyi et al. (2022) detected the Q148R mutation in the Nigerian population, being non-polymorphic, where it was identified in patients undergoing treatment with raltegravir and elvitegravir, presenting failure in monotherapy
53.
In this present study, the mutations associated with epidemics that occurred in the studies mentioned above were not identified. However, the K103N mutation for NNRTI was identified. Zhao et al. (2020) identified this same mutation in treatment-naive patients and detected the association of this mutation with high resistance to retroviral drugs used in treatment, such as Efavirenz and Nevirapine 54.
The presence of mutations in HIV-1 proteins can lead to greater susceptibility to other infections, such as syphilis and hepatitis B and C, due to the decrease in immune function caused by the progressive destruction of CD4+ lymphocytes by HIV-1. The reduction in CD4+ lymphocytes make people infected with HIV more vulnerable to opportunistic infections and other infectious diseases, including syphilis and hepatitis B. Then, the presence of mutations in HIV-1 proteins can compromise the individual's immune system, increasing the risk of contracting others infections 55. Despite expectations, mutations were not associated with coinfections such as syphilis or hepatitis B/C in this cohort. The concomitant presence of resistance mutations and STI co-infections is a concern, as it may lead to poorer clinical outcomes. These conditions may interact synergistically, accelerating disease progression and complicating treatment 56,57.
Furthermore, analysis of the genetic diversity of HIV-1 in different geographical regions reveals that genetic barriers vary significantly. In populations where multiple subtypes are present, as in some areas of Latin America and Africa, the risk of transmission of resistant variants increases 58–60. Understanding the genetic barrier is fundamental to developing effective treatment and prevention strategies. This implies the need for continuous monitoring of viral variants and emerging mutations in order to adjust ART regimens as necessary 61.
In methodological terms, the use of RDS made it possible to access a diverse social network, but it also imposes important limitations, such as dependence on the structure of the networks and potential recruitment biases. Self-reporting of sensitive behaviors is another limitation, especially considering stigma and discrimination that can lead to underreporting. Representativeness is also limited to individuals accessible through the networks formed during recruitment, and cannot be extrapolated to the entire MSM population of the region 62–64. Since the sample is drawn from specific social networks, the results cannot be extrapolated to all key populations in different geographical or

sociocultural contexts. Finally, the lack of longitudinal and comparative studies limits the ability to assess the consistency of RDS-derived results over time. Future research should consider triangulating RDS with other methodologies and employing mixed-method approaches to validate and expand upon the evidence presented here 65–67.
Despite the limitations, this study contributes by providing unprecedented epidemiological and genetic data for a little-studied Amazonian capital. The identification of mutations in individuals with unknown ART status highlights gaps in care and reinforces the need for continuous surveillance. While many studies focus on large urban centers or general populations, this study focuses on a specific cohort that may present unique characteristics in relation to HIV transmission and resistance 48–50.
Future research should integrate next-generation sequencing for greater sensitivity in detecting minority variants, as well as longitudinal approaches that allow monitoring the evolution of resistance and its impact on transmission networks 68. Strengthening combined prevention, coupled with the systematic incorporation of genomic monitoring, will be essential to guide therapeutic decisions and contain the circulation of resistant variants in key populations such as MSM 69.

5. Conclusions

A cohort of MSM from Belém-PA, Northern Brazil, was evaluated in relation to its epidemiological and genetic evaluation data for ART resistance mutations. Even though monitoring the emergence and spread of HIV drug resistance mutations at the population level has advanced significantly; in order to stop the spread of aids, it is still necessary to effectively detect and preferentially eradicate the mutated and resistant strains that reappear due to drug-resistance. induced selective pressure. The combination of epidemiological and genomic surveillance is essential to understand the dynamics of HIV infection and drug resistance, allowing the implementation of more effective interventions.


Availability of data and materials: All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests: The authors declare that they have no competing interests.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.



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