Intestinal Microbiota Changes in Individuals Living with HIV and the Effect of Antiretroviral Therapy

Intestinal Microbiota Changes in Individuals Living with HIV and the Effect of Antiretroviral Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Intestinal Microbiota Changes in Individuals Living with HIV and the Effect of Antiretroviral Therapy Yakup Demir, Mustafa Kemal Celen, Elif Seren Tanriverdi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7878027/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract HIV is one of the chronic infectious diseases that can be treated with the use of new generation antiretroviral therapy (HAART). HIV is closely associated with gastrointestinal diseases, changes in intestinal microbiota and inflammation. Therefore, we aimed to investigate changes in the gut microbiota in HIV-infected and uninfected populations living in Turkey. The study included 15 patients living with HIV and 10 healthy volunteers. Participants living with HIV were studied at baseline and again at least 3 months after treatment. Both blood and fecal samples were collected from all participants. For intestinal microbiota analysis, 16S rRNA gene sequence was analyzed. There was a significant difference between individuals living with HIV and healthy volunteers in terms of intestinal microbiota. While there was a significant decrease in the number of alpha diversity, Firmicutes, Proteobacteria in individuals living with HIV; Faecalibacterium, Prevotella, Streptococcus, Lactobacillus, Ruminococcus increased. Post-treatment improvements in intestinal microbiota were observed in individuals living with HIV receiving HAART with an increase in the number of Phascolarctobacterium and Blautia compared to the pretreatment period (naive patients). Improvement in intestinal microbiota was partially observed after treatment. Microbiota HIV Antiretroviral Therapy Dysbiosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The gut microbiota plays a critical role in maintaining host immune homeostasis. Recent studies have found that alterations in microbial diversity are associated with many health problems, including inflammatory bowel diseases, cardiovascular pathologies and metabolic disorders [ 1 , 2 ]. HIV has been transformed from a fatal disease to a chronic infectious disease with HAART. However, despite HAART, persistent inflammation in individuals living with HIV has been an important determinant of mortality and morbidity [ 3 , 4 ]. Despite suppression of viral load and increase in CD4 + T lymphocyte count, it has been reported in the literature that chronic inflammation persists due to immune damage in the mucosal area [ 5 ]. The gut is one of the leading immunologic organs, harboring approximately 70% of the immune system. HIV disrupts the integrity of the epithelial barrier by damaging the gut-associated lymphoid tissue (GALT) in the early stages, which leads to the circulation of microorganisms and microbial products. This condition, defined as “leaky gut”, is shown as one of the important causes of HIV related systemic inflammation [ 6 , 7 ]. In particular, the loss of Th17 cells and the resulting impaired barrier integrity exacerbate chronic inflammation by facilitating the translocation of microbial products such as lipopolysaccharides (LPS) [ 8 ]. These immunologic changes occurring in HIV/ directly affect the distribution and content of the gut microbiota. Many studies have reported a decrease in bacterial diversity, especially changes in the Prevotella/Bacteroides ratio and an increase in facultative anaerobes belonging to the Proteobacteria family in individuals living with HIV [ 9 – 10 ]. These changes are thought to trigger an inflammatory response and lead to metabolic complications. For example, it has been suggested that a decrease in Bacteroides species may be a factor that impairs immune regulation [ 11 ]. In HIV patients, despite virologic suppression achieved with antiretroviral therapy, it has been reported that these changes in the gut microbiota are not fully recovered and contribute to the continuation of inflammatory processes. The persistence of persistent dysbiosis in the gut microbial ecosystem in HIV-positive individuals under HAART is considered as one of the important causes of immune activation and chronic inflammation [ 12 , 13 ]. In this context, it is suggested that inflammation in individuals living with HIV is caused not only by viral replication but also by microbial translocation and disruptions in the gut microbiota. Findings in the literature suggest that these alterations in the gut microbiota may play a central role in the pathogenesis of HIV and the persistence of inflammatory responses [ 14 – 15 ]. The aim of our study was to investigate the changes in intestinal microbiota and the effects of antiretroviral therapies on these changes in individuals living with HIV, and to reveal the relationship between HIV RNA levels and CD4+/CD8 + lymphocyte counts, which are markers of inflammation, and microbiota distribution. Material and Methods Case Selection After the inclusion and exclusion criteria (Supplementary Material S1), 15 people living with HIV and 10 healthy volunteers admitted to the outpatient clinics and clinics of Dicle University Medical Faculty Hospital between December 2021 and May 2022 were included in the study (Clinical data is shown in Table 1). Participants residing in Diyarbakır, in the south-east of Turkey, were included in the study. The region has limited socio-economic resources, and participants generally maintain a diet low in fibre and vegetables but rich in fat. These living conditions and dietary habits represent environmental and lifestyle factors that could significantly affect the gut microbiota. Informed consent forms were obtained from all participants. The study research protocol was approved by the Ethics Committee of Dicle University Faculty of Medicine (Decision No. 251 dated 22.04.2021). Inclusion and Exclusion Criteria The study included people living with HIV (PLWH) aged 18–65 years with a normal BMI, who were on stable antiretroviral therapy (ART), without chronic infections, recent significant weight changes, or recent use of antibiotics, probiotics, or other microbiota-modifying agents. Healthy controls were age- and BMI-matched individuals without HIV infection or chronic conditions affecting gut microbiota. Exclusion criteria for both groups included inability to provide informed consent, pregnancy or breastfeeding, co-infections (e.g., hepatitis B/C, STDs), significant comorbidities, or irregular dietary habits. Sample collection Two tubes of blood samples (approximately 5ml) from PLWHA and 1 tube of blood samples (approximately 2.5ml) from healthy volunteers were collected and analyzed in the laboratory on the same day. Fresh stool samples (approximately 5g) were collected from all participants and delivered to the laboratory under anaerobic conditions at + 4°C. Stool samples were mixed with phosphate buffered saline containing 20% glycerol and frozen at -80°C under nitrogen and stored until the time of analysis. Lysozyme and acromopeptidase enzymatic lysis methods were used for DNA isolation. Measurement Methods Bacterial genomic DNA (gDNA) isolation from fecal samples was performed using kits. The quality of the samples was confirmed by gel electrophoresis, spectrophotometry and fluorometric methods. DNA samples for PCR analysis were stored at -20°C and the V4 region of the 16S rRNA genes was sequenced with the Illumina system. DNA was amplified by amplicon PCR using universal primers, the products obtained were purified and DNA amounts were measured by fluorometric methods. The purified DNA libraries were pooled and sequenced using the Illumina Miseq system and the data obtained were evaluated by bioinformatics analysis. Statistical Analysis Demographic and clinical data of the participants were evaluated on variables such as age, gender, HIV RNA levels, CD4 + and CD8 + lymphocyte counts, and antiretroviral therapy use. Mean, median, standard deviation, minimum and maximum values were calculated for numerical variables. Frequency and percentage values were presented for categorical variables. Mann-Whitney U and Student's t-test were used to evaluate intergroup differences. Chi-square and Fisher's exact test were applied for categorical variables. Various bioinformatics methods were used for microbial diversity and composition analyses. Shannon, Simpson and Chao1 diversity indices were calculated to assess microbial alpha diversity. Principal Coordinates Analysis (PCoA) and Principal Component Analysis (PCA) methods were used for beta diversity analyses to visualize similarities and differences between groups. Krona plot, Relative Abundance Analysis and Taxonomic Assignment analyses were performed to determine the microbial community composition. PERMANOVA (Permutational Multivariate Analysis of Variance) and ANOSIM (Analysis of Similarities) tests were applied to determine the differences between groups. Linear Discriminant Analysis Effect Size (LEfSe) method was used to determine microbial differences. All statistical analyses were performed using QIIME2, R and SPSS 26 programs and significance level was accepted as p < 0.05. PCR amplification and Illumina sequencing Variable regions of the 16S rRNA gene were amplified by PCR to determine the composition of intestinal microbiota. In this study, the V4 region of the 16S rRNA gene was targeted to obtain bacterial diversity at high resolution. Commonly used primer pairs were preferred for PCR amplification: Forward primer 341F: CCTACGGGNGGCWGCAG and Reverse primer 805R: GACTACHVGGGTATCTAATCC. PCR products were library prepared according to the manufacturer's (Illumina, San Diego, CA, USA) instructions and sequenced on the Illumina MiSeq platform using paired-end sequencing with a read length of 2x300 bp. The raw sequences were subjected to quality control, filtering and chimera removal, and then processed using QIIME2 (Quantitative Insights Into Microbial Ecology) software. Results Demographic Characteristics A total of 15 patients diagnosed with HIV and 10 healthy volunteers were included in the study. Of the participants, 84% (n = 21) were male and 16% (n = 4) were female. The mean age of the participants was 35.4 ± 9.1 years and the age range was 21–50 years. Treatment Options and Patient Compliance Patients were treated with Bictegravir/Emtricitabine/Tenofovir alafenamide fumarate, Emtricitabine/Tenofovir disoproxil fumarate/Dolutegravir, Lamivudine/Dolutegravir regimens. Treatment adherence was 100% and no drug-related long-term side effects were detected. There was no statistically significant relationship between CD4 + T cell changes and HIV RNA levels. Demographic data are summarized in Table-1. Faecal bacterial diversity in patients with HIV In microbiota analysis, alpha diversity indicates microbial richness or diversity within a group, while beta diversity reflects microbial community differences or similarities in different groups. is used to measure microbial species richness and diversity. We used 4 parameters for alpha diversity (Simpson and Shannon index). In our study, alpha diversity was significantly decreased in HIV group compared to healthy volunteers. Two parameters (PCA and PCoA) were used for beta diversity comparison. Again, a significant difference was observed between HIV group and healthy control group in beta diversity analysis. Comparative analysis of the 3 groups is shown in Fig. 1. Faecal bacterial composition in patients with HIV We compared different groups in terms of community between HIV and healthy volunteers. Bacteroidetes, Firmicutes, Proteobacteria constituted the majority of the phylum. There was an increase in the number of Bacteroidetes and Proteobacteria in the HIV naive group compared to the healthy control group, while there was a decrease in the Firmicutes group. It is presented in Fig. 2. We aimed to find the specific bacterial diversity by using LEfSe (Linear Discriminant Analysis Effect Size) in the study analysis. LEfSe is a method that combines classifier statistical tests, effect size estimation and linear discriminant analysis to determine differences between groups in microbiome data. According to the significance of LDA score, Clostridium was found to be high in the healthy control group, while Proteobacteria and Spirochaetes were found to be high in the HIV patient group. It is presented graphically in Fig. 3. Microbiota Changes Before and After Treatment A total of 15 patients with HIV were re-sampled and evaluated at least 3 months after HAART initiation. This group was compared with healthy volunteers and a newly treated HIV naive group. Post-treatment alpha diversity was increased compared to the HIV naive group. Especially Firmicutes and Elusimicrobia. At taxon level it was Ruminococcaceae. Fecal microbiota did not fully recover despite effective HAART treatment. Details are shown in Fig. 4. Discussion This is the first study in Turkey to compare intestinal microbiota in HIV with healthy volunteers. This study showed that microbial diversity was reduced in the HIV group. At the same time, it was found that although the diversity partially improved with the initiation of HARRT; the improvement was not complete. Thanks to HAART, HIV infection is now among the manageable chronic diseases and the life expectancy of HIV-positive individuals is approaching that of the general population. However, quality of life is affected by many factors such as age, gender, socioeconomic status, CD4 + T lymphocyte count and HIV RNA level [ 16 – 17 ]. In recent years, it has been emphasized that chronic inflammation is one of the most important determinants of morbidity and mortality. In this process, gut microbiota plays a critical role. HIV-induced immune activation and inflammation accelerate immune aging, leading to earlier onset of comorbidities. Damage to the intestinal epithelial barrier, especially in the early period of infection, increases permeability, leading to microbial translocation and contributes to disease progression by exacerbating systemic inflammation [ 18 – 19 ]. Many studies have emphasized that alpha diversity decreases in treatment-naive patients with HIV. Some studies have shown an increase in microbial diversity after HAART. In our study, a decrease in alpha diversity was observed in accordance with the literature and a partial improvement was found after treatment. However, as a contradictory finding with the literature, the number of beneficial bacteria was lower in both HIV untreated and treated groups and in healthy volunteers [ 20 ]. The possible reason for this may be related to the fact that Diyarbakır, where the study was conducted, is a city located in the east of Turkey, where carbohydrate consumption is common in the diet and consumption of fibrous foods, vegetables and fruits is lower. Some studies also emphasize the change in intestinal microbiota with food consumption. In our study, changes in intestinal microbiota in HIV-infected individuals before and after treatment were evaluated and the effects of treatment on microbial diversity and composition were analyzed. While a partial increase in Firmicutes level was observed after treatment, a significant decrease was found compared to healthy volunteers, indicating that the effects of antiretroviral therapy on gut microbiota may be limited. Previous studies suggest that dysbiosis in the gut microbiota of HIV-infected individuals is associated with changes in Actinobacteria and that the increase in this group may be associated with proinflammatory responses [ 21 , 22 ]. While these findings support the positive effects of antiretroviral therapy on the gut microbiota in HIV patients, they suggest that complete restoration is not achieved. Future long-term follow-up studies will be important to understand how antiretroviral therapy drives recovery of the gut microbiota and the effects of different treatment regimens on the microbiota [ 23 ]. Some limitations of this study should be considered. First, the generalizability of the results may be limited due to the relatively small number of participants. Furthermore, HIV infection itself may affect host-microbe interactions, which could have a potential impact on the findings. In addition, due to insufficient data on transmission routes, we were not able to assess microbial changes associated with different routes of transmission. These limitations should be taken into account when interpreting the results. In conclusion, HIV infection has been shown to cause marked alterations in the gut microbiota, reducing microbial diversity, which is associated with inflammation, immune response and disease progression. Although antiretroviral therapy can partially correct dysbiosis, it has been found that microbial structure and function are not fully normalized. Therefore, the gut microbiota is important not only in immune regulation but also in metabolic processes and long-term health outcomes. Future long-term, multicenter studies will be critical for integrating microbiota-based therapeutic approaches into HIV management and evaluating the potential benefits of probiotic, prebiotic or diet-based interventions. Declarations Ethics approval and consent to participate All participants provided written informed consent prior to participation in this study. The study protocol was reviewed and approved by the appropriate institutional ethics committee. Funding This study was supported by DUBAP for the microbiota analysis. No additional external funding was received from public, commercial, or not-for-profit sectors. Author Contribution We declare that all the listed authors have participated actively in the study and all meet the authorship requirements. YD and MC designed the study and wrote the protocol. YD, MC, and ST acquired the manuscript. YD and ST analyzed the data. YD and ST wrote the first draft of the manuscript, and MC mainly revised it. All authors approved the final version of the manuscript. References Bretto E, Urpì-Ferreruela M, Casanova GR, González-Suárez B. The Role of Gut Microbiota in Gastrointestinal Immune Homeostasis and Inflammation: Implications for Inflammatory Bowel Disease. Biomedicines. 2025;13(8):1807. Trøseid M, Andersen GØ, Broch K, Hov JR. (2020). The gut microbiome in coronary artery disease and heart failure: Current knowledge and future directions. EBioMedicine , 52 . Lin L, Zhang J. Role of intestinal microbiota and metabolites on gut homeostasis and human diseases. BMC Immunol. 2017;18(1):2. Choshi J, et al. A systematic review assessing the association of inflammatory markers with kidney dysfunction in people living with HIV on highly active antiretroviral therapy. BMC Infect Dis. 2024;24(1):776. Mazzuti L, Turriziani O, Mezzaroma I. The many faces of immune activation in HIV-1 infection: a multifactorial interconnection. Biomedicines. 2023;11(1):159. Gáspár Zsófia et al. Gut Microbiome Alteration in HIV/AIDS and the role of antiretroviral Therapy—A scoping review. Microorganisms 12.11 (2024): 2221. Brenchley JM, Serrano-Villar S. From dysbiosis to defense: harnessing the gut microbiome in HIV/SIV therapy. Microbiome. 2024;12(1):113. Pan Z, Wu N, Jin C. Intestinal Microbiota Dysbiosis Promotes Mucosal Barrier Damage and Immune Injury in HIV-Infected Patients. Can J Infect Dis Med Microbiol. 2023;2023(1):3080969. Rocafort, M., Gootenberg, D. B., Luévano Jr, J. M., Paer, J. M., Hayward, M. R., Bramante,J. T., … Kwon, D. S. (2024). HIV-associated gut microbial alterations are dependent on host and geographic context. Nature communications, 15(1), 1055.. Aluthge, N., Adams, S., Davila, C. A., Gocchi Carrasco, N. R., Chiou, K. S., Abadie,R., … Fernando, S. C. (2024). Gut microbiota profiling in injection drug users with and without HIV-1 infection in Puerto Rico. Frontiers in Microbiology, 15, 1470037.. Fakharian F, Thirugnanam S, Welsh DA, Kim WK, Rappaport J, Bittinger K, Rout N. The role of gut dysbiosis in the loss of intestinal immune cell functions and viral pathogenesis. Microorganisms. 2023;11(7):1849. Ishizaka A, Koga M, Mizutani T, Suzuki Y, Matano T, Yotsuyanagi H. Sustained gut dysbiosis and intestinal inflammation show correlation with weight gain in person with chronic HIV infection on antiretroviral therapy. BMC Microbiol. 2024;24(1):274. Shi Y, Hu M, Wu J, Liu T, Qi Y, Li A. Association between gut microbiota in HIV-infected patients and immune reconstitution following antiretroviral therapy (ART). BMC Infect Dis. 2025;25(1):666. Zhang, Y., Xie, Z., Zhou, J., Li, Y., Ning, C., Su, Q., … Huang, J. (2023). The altered metabolites contributed by dysbiosis of gut microbiota are associated with microbial translocation and immune activation during HIV infection. Frontiers in immunology, 13, 1020822.. Mac Cann, R., Newman, E., Devane, D., Sabin, C., Cotter, A. G., Landay, A., … Mallon,P. W. (2024). HIV, the gut microbiome and clinical outcomes, a systematic review.PloS one, 19(12), e0308859.. Portilla-Tamarit I, Rubio-Aparicio M, Fuster-RuizdeApodaca MJ, Portilla-Tamarit J, Reus S, Portilla J. Health-Related Quality of Life in People with Advanced HIV Disease, from 1996 to 2021: Systematic Review and Meta-analysis. AIDS Behav. 2024;28(6):1978–98. Vo LT, Nguyen PH, Nguyen HTN, Phan DQ, Vo XTT, Vo LY, Nguyen YNT, Huynh G. Health-Related Quality of Life Among Patients Living with HIV/AIDS in Vietnam: A Cross-Sectional Study. Patient Prefer Adherence. 2025;19:1197–210. Ouyang J, Yan J, Zhou X, Isnard S, Harypursat V, Cui H, Routy J-P, Chen Y. Relevance of biomarkers indicating gut damage and microbial translocation in people living with HIV. Front Immunol. 2023;14:1173956. Tincati C, Bono V, Cannizzo ES, Tosi D, Savi F, Falcinella C, Casabianca A, Orlandi C, Luigiano C, Augello M, Rusconi S, Muscatello A, Bandera A, Calcagno A, Gori A, Nozza S, Marchetti G. Primary HIV infection features colonic damage and neutrophil inflammation yet containment of microbial translocation. AIDS. 2024;38(5):623–32. Dillon, S. M., Lee, E. J., Kotter, C. V., Austin, G. L., Dong, Z., Hecht, F. M.,… Lynch, S. V. (2014). An Altered Intestinal Microbiome in HIV-1-Infected Individuals.Gut Microbes, 5(2), 209–218. Vazquez-Castellanos, J. F., Hernandez-Ramirez, G. F., Lopez-Gatell, H., Roldan-Fajardo,A., Juarez-Figueroa, L., Ruiz-Matus, C., … Geller, R. (2018). Changes in the gut microbiome in patients with HIV-1 infection before and after antiretroviral therapy. AIDS Research and Human Retroviruses, 34(1), 101–110. Gori A, Tincati C, d'Arminio Monforte A, Cislaghi A. HIV infection and the gut microbiota: a new frontier in the pathogenesis and treatment of HIV-related disorders. Expert Rev Anti-infective Therapy. 2014;12(12):1481–92. Ananthan A, Gopinath B, Uthayakumar M, Venkatesan M. Impact of highly active antiretroviral therapy on the gut microbiome of HIV-infected individuals: a systematic review and meta-analysis. Arch Microbiol. 2020;202(6):1187–98. Tables Table 1 Clinical data of each group Clinical data CON (n=10) HIV/AIDS (n=15) P-value Age (year) 35.4 ± 9.1 0.456 Gender (Male/Female) 8/2 13/2 0.376 BMI (kg/m 2 ) 24.8 ± 2.6 25.1 ± 3.1 0.346 Smoking 3 (30%) 5 (33.3%) 0.247 Drinking 2 (20%) 4 (26.6%) 0.534 Transmission Route Not Applicable 12 (80%) 2 (13.3%) 1 (6.6%) Not Applicable Heteroseksual Behaviour Homosexual Behaviour Intravenous drug injection HAART tretament (yes/no) Not Applicable Not Applicable Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial1S1.Inclusionandexclusioncriteria.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7878027","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":538558077,"identity":"9d172e09-f021-460d-85e4-ecf1b7dc76b1","order_by":0,"name":"Yakup Demir","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYFACNghmY2A/+OADiM1OvBaeZMMZIDYzsVqAwEyYB0QR0mJw/FiadEGZTTSf9IE0Zptf2+T5mBkYP3zMwaPlTNox6Rnn0nLb+BKPPc7tu23YxszALDlzGx4tB9LbpHnbDue28TCkG+f23GYEamFj5sWn5fxzkJb/IC1m0pY9t+0Ja7kBdBhv2wGIFoYftxMJapG88SzZesa5ZKAWYCD3NtxObmNmbMbrF77zaYa3C8rscuf3AKPyx5/btvPbmw9++IhHi8IB5IhgbAOTDbjVA4F8A0rc/cGreBSMglEwCkYoAADGvE8Kwg3/TQAAAABJRU5ErkJggg==","orcid":"","institution":"Memorial Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yakup","middleName":"","lastName":"Demir","suffix":""},{"id":538558078,"identity":"c0f18805-a654-416f-a6a5-efefc84b5d26","order_by":1,"name":"Mustafa Kemal Celen","email":"","orcid":"","institution":"Dicle Üniversitesi Hastaneleri","correspondingAuthor":false,"prefix":"","firstName":"Mustafa","middleName":"Kemal","lastName":"Celen","suffix":""},{"id":538558079,"identity":"155ddc1b-017e-4b04-b638-8bc7c0f6d8bb","order_by":2,"name":"Elif Seren Tanriverdi","email":"","orcid":"","institution":"Inonu University","correspondingAuthor":false,"prefix":"","firstName":"Elif","middleName":"Seren","lastName":"Tanriverdi","suffix":""}],"badges":[],"createdAt":"2025-10-16 13:08:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7878027/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7878027/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94988823,"identity":"a6ff5e23-0df0-4eac-a4db-f218675ce2b4","added_by":"auto","created_at":"2025-11-03 07:11:04","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26879,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/0aece95b7719d8e7a7b12478.docx"},{"id":94980515,"identity":"b392518e-736a-4327-81c3-7c17cf838cee","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4921,"visible":true,"origin":"","legend":"","description":"","filename":"b7fbf96031184c81ae18d6afac0c84eb.json","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/b3255af3a75e9207aae56ca8.json"},{"id":94980520,"identity":"e8c1c901-db2c-4e71-aeaf-43a6847217ae","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2754195,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1Shannonindex01.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/a797a34f2d3b268d970daa3e.jpg"},{"id":94980517,"identity":"47bf74de-4c72-46ba-b758-925f1faac603","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1512122,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2PcoA01.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/9945a04792a8d6f18b73c010.jpg"},{"id":94980519,"identity":"8c49b98b-3c8c-4967-9b9d-6fc917036088","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3578401,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3treatmentgenus01.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/1555abd2f022ca1e95e34378.jpg"},{"id":94980529,"identity":"f424779c-dfe6-4eb9-bd8a-bbb2d6db6d99","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3042813,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4lefse.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/8a7b8f0a83660d1d1ce1c71f.jpg"},{"id":94989312,"identity":"aac8b625-2eb9-482d-a4d2-b2990dd1d202","added_by":"auto","created_at":"2025-11-03 07:12:35","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2065828,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5Phylum01.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/052b9cc5c5cfbd3428f399d8.jpg"},{"id":94989094,"identity":"be84c294-53af-40a9-ab6e-f1227bfca820","added_by":"auto","created_at":"2025-11-03 07:12:05","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17493,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial1S1.Inclusionandexclusioncriteria.docx","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/ff73f97ef0bafed73478b284.docx"},{"id":94980525,"identity":"fbdef58a-e6a8-494f-84ae-d014df76cfb1","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":16415,"visible":true,"origin":"","legend":"","description":"","filename":"Table1Clinicaldataofeachgroup.docx","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/3fdc44beed1b8e009906b13c.docx"},{"id":94980518,"identity":"ede483b3-e68d-4ed2-bdbf-091570f30aff","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55647,"visible":true,"origin":"","legend":"","description":"","filename":"b7fbf96031184c81ae18d6afac0c84eb1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/c0cd149d1753f992c0ade9e0.xml"},{"id":94980523,"identity":"3a28aa6b-1b82-4833-94c2-9942ba10d164","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"xml","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52782,"visible":true,"origin":"","legend":"","description":"","filename":"b7fbf96031184c81ae18d6afac0c84eb1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/4d3b20f86617ea3a434895bd.xml"},{"id":94980527,"identity":"437e2b85-6867-49b1-89a7-72ece853581f","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61365,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/8518063b88633f9fdfd5026a.html"},{"id":94988636,"identity":"10d5fec8-3924-4cdd-8363-33ff627e8da5","added_by":"auto","created_at":"2025-11-03 07:10:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2754195,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure1Shannonindex01.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/507a8cd7ea21ef03a06b2b0f.jpg"},{"id":94980516,"identity":"63a5595c-b270-4a53-a03b-191277434b30","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1512122,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure2PcoA01.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/95dfe62fcdcedb73f578695b.jpg"},{"id":94980521,"identity":"46142610-acc6-42d1-853b-9c84a1fcfe32","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3578401,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure3treatmentgenus01.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/3b2eaba464a358240468bcd2.jpg"},{"id":94980528,"identity":"cbe4d9ad-e98d-4b63-8d65-b19ec2d933be","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3042813,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure4lefse.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/0831fe985a0dd961562ca216.jpg"},{"id":94980524,"identity":"e854c929-9bd0-42eb-8ce3-70189925f9fb","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2065828,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure5Phylum01.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/9f5c03ebfb776d9df918c5fb.jpg"},{"id":96362742,"identity":"b0b9da12-fb9e-4f3a-992e-9e1c256e9917","added_by":"auto","created_at":"2025-11-20 09:47:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13497187,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/5557edcb-6daa-4f21-a41f-12e5966591e9.pdf"},{"id":94980512,"identity":"858db3c2-a7da-4c04-9ed6-137cf0085faf","added_by":"auto","created_at":"2025-11-03 05:21:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17493,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial1S1.Inclusionandexclusioncriteria.docx","url":"https://assets-eu.researchsquare.com/files/rs-7878027/v1/da4548f38adb966cf6940f00.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intestinal Microbiota Changes in Individuals Living with HIV and the Effect of Antiretroviral Therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe gut microbiota plays a critical role in maintaining host immune homeostasis. Recent studies have found that alterations in microbial diversity are associated with many health problems, including inflammatory bowel diseases, cardiovascular pathologies and metabolic disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. HIV has been transformed from a fatal disease to a chronic infectious disease with HAART. However, despite HAART, persistent inflammation in individuals living with HIV has been an important determinant of mortality and morbidity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite suppression of viral load and increase in CD4\u0026thinsp;+\u0026thinsp;T lymphocyte count, it has been reported in the literature that chronic inflammation persists due to immune damage in the mucosal area [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe gut is one of the leading immunologic organs, harboring approximately 70% of the immune system. HIV disrupts the integrity of the epithelial barrier by damaging the gut-associated lymphoid tissue (GALT) in the early stages, which leads to the circulation of microorganisms and microbial products. This condition, defined as \u0026ldquo;leaky gut\u0026rdquo;, is shown as one of the important causes of HIV related systemic inflammation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In particular, the loss of Th17 cells and the resulting impaired barrier integrity exacerbate chronic inflammation by facilitating the translocation of microbial products such as lipopolysaccharides (LPS) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These immunologic changes occurring in HIV/ directly affect the distribution and content of the gut microbiota. Many studies have reported a decrease in bacterial diversity, especially changes in the Prevotella/Bacteroides ratio and an increase in facultative anaerobes belonging to the Proteobacteria family in individuals living with HIV [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These changes are thought to trigger an inflammatory response and lead to metabolic complications. For example, it has been suggested that a decrease in Bacteroides species may be a factor that impairs immune regulation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn HIV patients, despite virologic suppression achieved with antiretroviral therapy, it has been reported that these changes in the gut microbiota are not fully recovered and contribute to the continuation of inflammatory processes. The persistence of persistent dysbiosis in the gut microbial ecosystem in HIV-positive individuals under HAART is considered as one of the important causes of immune activation and chronic inflammation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In this context, it is suggested that inflammation in individuals living with HIV is caused not only by viral replication but also by microbial translocation and disruptions in the gut microbiota. Findings in the literature suggest that these alterations in the gut microbiota may play a central role in the pathogenesis of HIV and the persistence of inflammatory responses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe aim of our study was to investigate the changes in intestinal microbiota and the effects of antiretroviral therapies on these changes in individuals living with HIV, and to reveal the relationship between HIV RNA levels and CD4+/CD8\u0026thinsp;+\u0026thinsp;lymphocyte counts, which are markers of inflammation, and microbiota distribution.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCase Selection\u003c/h2\u003e\u003cp\u003eAfter the inclusion and exclusion criteria (Supplementary Material S1), 15 people living with HIV and 10 healthy volunteers admitted to the outpatient clinics and clinics of Dicle University Medical Faculty Hospital between December 2021 and May 2022 were included in the study (Clinical data is shown in Table\u0026nbsp;1). Participants residing in Diyarbakır, in the south-east of Turkey, were included in the study. The region has limited socio-economic resources, and participants generally maintain a diet low in fibre and vegetables but rich in fat. These living conditions and dietary habits represent environmental and lifestyle factors that could significantly affect the gut microbiota. Informed consent forms were obtained from all participants. The study research protocol was approved by the Ethics Committee of Dicle University Faculty of Medicine (Decision No. 251 dated 22.04.2021).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInclusion and Exclusion Criteria\u003c/h3\u003e\n\u003cp\u003eThe study included people living with HIV (PLWH) aged 18\u0026ndash;65 years with a normal BMI, who were on stable antiretroviral therapy (ART), without chronic infections, recent significant weight changes, or recent use of antibiotics, probiotics, or other microbiota-modifying agents. Healthy controls were age- and BMI-matched individuals without HIV infection or chronic conditions affecting gut microbiota. Exclusion criteria for both groups included inability to provide informed consent, pregnancy or breastfeeding, co-infections (e.g., hepatitis B/C, STDs), significant comorbidities, or irregular dietary habits.\u003c/p\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003eTwo tubes of blood samples (approximately 5ml) from PLWHA and 1 tube of blood samples (approximately 2.5ml) from healthy volunteers were collected and analyzed in the laboratory on the same day. Fresh stool samples (approximately 5g) were collected from all participants and delivered to the laboratory under anaerobic conditions at +\u0026thinsp;4\u0026deg;C. Stool samples were mixed with phosphate buffered saline containing 20% glycerol and frozen at -80\u0026deg;C under nitrogen and stored until the time of analysis. Lysozyme and acromopeptidase enzymatic lysis methods were used for DNA isolation.\u003c/p\u003e\n\u003ch3\u003eMeasurement Methods\u003c/h3\u003e\n\u003cp\u003eBacterial genomic DNA (gDNA) isolation from fecal samples was performed using kits. The quality of the samples was confirmed by gel electrophoresis, spectrophotometry and fluorometric methods. DNA samples for PCR analysis were stored at -20\u0026deg;C and the V4 region of the 16S rRNA genes was sequenced with the Illumina system. DNA was amplified by amplicon PCR using universal primers, the products obtained were purified and DNA amounts were measured by fluorometric methods. The purified DNA libraries were pooled and sequenced using the Illumina Miseq system and the data obtained were evaluated by bioinformatics analysis.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eDemographic and clinical data of the participants were evaluated on variables such as age, gender, HIV RNA levels, CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;lymphocyte counts, and antiretroviral therapy use. Mean, median, standard deviation, minimum and maximum values were calculated for numerical variables. Frequency and percentage values were presented for categorical variables. Mann-Whitney U and Student's t-test were used to evaluate intergroup differences. Chi-square and Fisher's exact test were applied for categorical variables. Various bioinformatics methods were used for microbial diversity and composition analyses. Shannon, Simpson and Chao1 diversity indices were calculated to assess microbial alpha diversity. Principal Coordinates Analysis (PCoA) and Principal Component Analysis (PCA) methods were used for beta diversity analyses to visualize similarities and differences between groups. Krona plot, Relative Abundance Analysis and Taxonomic Assignment analyses were performed to determine the microbial community composition. PERMANOVA (Permutational Multivariate Analysis of Variance) and ANOSIM (Analysis of Similarities) tests were applied to determine the differences between groups. Linear Discriminant Analysis Effect Size (LEfSe) method was used to determine microbial differences. All statistical analyses were performed using QIIME2, R and SPSS 26 programs and significance level was accepted as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePCR amplification and Illumina sequencing\u003c/h2\u003e\u003cp\u003eVariable regions of the 16S rRNA gene were amplified by PCR to determine the composition of intestinal microbiota. In this study, the V4 region of the 16S rRNA gene was targeted to obtain bacterial diversity at high resolution. Commonly used primer pairs were preferred for PCR amplification: Forward primer 341F: CCTACGGGNGGCWGCAG and Reverse primer 805R: GACTACHVGGGTATCTAATCC. PCR products were library prepared according to the manufacturer's (Illumina, San Diego, CA, USA) instructions and sequenced on the Illumina MiSeq platform using paired-end sequencing with a read length of 2x300 bp. The raw sequences were subjected to quality control, filtering and chimera removal, and then processed using QIIME2 (Quantitative Insights Into Microbial Ecology) software.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eDemographic Characteristics\u003c/h2\u003e\u003cp\u003eA total of 15 patients diagnosed with HIV and 10 healthy volunteers were included in the study. Of the participants, 84% (n\u0026thinsp;=\u0026thinsp;21) were male and 16% (n\u0026thinsp;=\u0026thinsp;4) were female. The mean age of the participants was 35.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.1 years and the age range was 21\u0026ndash;50 years. Treatment Options and Patient Compliance Patients were treated with Bictegravir/Emtricitabine/Tenofovir alafenamide fumarate, Emtricitabine/Tenofovir disoproxil fumarate/Dolutegravir, Lamivudine/Dolutegravir regimens. Treatment adherence was 100% and no drug-related long-term side effects were detected. There was no statistically significant relationship between CD4\u0026thinsp;+\u0026thinsp;T cell changes and HIV RNA levels. Demographic data are summarized in Table-1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eFaecal bacterial diversity in patients with HIV\u003c/h2\u003e\u003cp\u003eIn microbiota analysis, alpha diversity indicates microbial richness or diversity within a group, while beta diversity reflects microbial community differences or similarities in different groups. is used to measure microbial species richness and diversity. We used 4 parameters for alpha diversity (Simpson and Shannon index). In our study, alpha diversity was significantly decreased in HIV group compared to healthy volunteers. Two parameters (PCA and PCoA) were used for beta diversity comparison. Again, a significant difference was observed between HIV group and healthy control group in beta diversity analysis. Comparative analysis of the 3 groups is shown in Fig.\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eFaecal bacterial composition in patients with HIV\u003c/h2\u003e\u003cp\u003eWe compared different groups in terms of community between HIV and healthy volunteers. Bacteroidetes, Firmicutes, Proteobacteria constituted the majority of the phylum. There was an increase in the number of Bacteroidetes and Proteobacteria in the HIV naive group compared to the healthy control group, while there was a decrease in the Firmicutes group. It is presented in Fig.\u0026nbsp;2.\u003c/p\u003e\u003cp\u003eWe aimed to find the specific bacterial diversity by using LEfSe (Linear Discriminant Analysis Effect Size) in the study analysis. LEfSe is a method that combines classifier statistical tests, effect size estimation and linear discriminant analysis to determine differences between groups in microbiome data. According to the significance of LDA score, Clostridium was found to be high in the healthy control group, while Proteobacteria and Spirochaetes were found to be high in the HIV patient group. It is presented graphically in Fig.\u0026nbsp;3.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eMicrobiota Changes Before and After Treatment\u003c/h2\u003e\u003cp\u003eA total of 15 patients with HIV were re-sampled and evaluated at least 3 months after HAART initiation. This group was compared with healthy volunteers and a newly treated HIV naive group. Post-treatment alpha diversity was increased compared to the HIV naive group. Especially Firmicutes and Elusimicrobia. At taxon level it was Ruminococcaceae. Fecal microbiota did not fully recover despite effective HAART treatment. Details are shown in Fig.\u0026nbsp;4.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis is the first study in Turkey to compare intestinal microbiota in HIV with healthy volunteers. This study showed that microbial diversity was reduced in the HIV group. At the same time, it was found that although the diversity partially improved with the initiation of HARRT; the improvement was not complete.\u003c/p\u003e\u003cp\u003eThanks to HAART, HIV infection is now among the manageable chronic diseases and the life expectancy of HIV-positive individuals is approaching that of the general population. However, quality of life is affected by many factors such as age, gender, socioeconomic status, CD4\u0026thinsp;+\u0026thinsp;T lymphocyte count and HIV RNA level [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In recent years, it has been emphasized that chronic inflammation is one of the most important determinants of morbidity and mortality. In this process, gut microbiota plays a critical role. HIV-induced immune activation and inflammation accelerate immune aging, leading to earlier onset of comorbidities. Damage to the intestinal epithelial barrier, especially in the early period of infection, increases permeability, leading to microbial translocation and contributes to disease progression by exacerbating systemic inflammation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMany studies have emphasized that alpha diversity decreases in treatment-naive patients with HIV. Some studies have shown an increase in microbial diversity after HAART. In our study, a decrease in alpha diversity was observed in accordance with the literature and a partial improvement was found after treatment. However, as a contradictory finding with the literature, the number of beneficial bacteria was lower in both HIV untreated and treated groups and in healthy volunteers [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The possible reason for this may be related to the fact that Diyarbakır, where the study was conducted, is a city located in the east of Turkey, where carbohydrate consumption is common in the diet and consumption of fibrous foods, vegetables and fruits is lower. Some studies also emphasize the change in intestinal microbiota with food consumption.\u003c/p\u003e\u003cp\u003eIn our study, changes in intestinal microbiota in HIV-infected individuals before and after treatment were evaluated and the effects of treatment on microbial diversity and composition were analyzed. While a partial increase in Firmicutes level was observed after treatment, a significant decrease was found compared to healthy volunteers, indicating that the effects of antiretroviral therapy on gut microbiota may be limited. Previous studies suggest that dysbiosis in the gut microbiota of HIV-infected individuals is associated with changes in Actinobacteria and that the increase in this group may be associated with proinflammatory responses [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. While these findings support the positive effects of antiretroviral therapy on the gut microbiota in HIV patients, they suggest that complete restoration is not achieved. Future long-term follow-up studies will be important to understand how antiretroviral therapy drives recovery of the gut microbiota and the effects of different treatment regimens on the microbiota [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSome limitations of this study should be considered. First, the generalizability of the results may be limited due to the relatively small number of participants. Furthermore, HIV infection itself may affect host-microbe interactions, which could have a potential impact on the findings. In addition, due to insufficient data on transmission routes, we were not able to assess microbial changes associated with different routes of transmission. These limitations should be taken into account when interpreting the results.\u003c/p\u003e\u003cp\u003eIn conclusion, HIV infection has been shown to cause marked alterations in the gut microbiota, reducing microbial diversity, which is associated with inflammation, immune response and disease progression. Although antiretroviral therapy can partially correct dysbiosis, it has been found that microbial structure and function are not fully normalized. Therefore, the gut microbiota is important not only in immune regulation but also in metabolic processes and long-term health outcomes. Future long-term, multicenter studies will be critical for integrating microbiota-based therapeutic approaches into HIV management and evaluating the potential benefits of probiotic, prebiotic or diet-based interventions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003e\u003cem\u003e All participants provided written informed consent prior to participation in this study. The study protocol was reviewed and approved by the appropriate institutional ethics committee.\u003c/em\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003e\u003cem\u003eThis study was supported by DUBAP for the microbiota analysis. No additional external funding was received from public, commercial, or not-for-profit sectors.\u003c/em\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWe declare that all the listed authors have participated actively in the study and all meet the authorship requirements. YD and MC designed the study and wrote the protocol. YD, MC, and ST acquired the manuscript. YD and ST analyzed the data. YD and ST wrote the first draft of the manuscript, and MC mainly revised it. All authors approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBretto E, Urp\u0026igrave;-Ferreruela M, Casanova GR, Gonz\u0026aacute;lez-Su\u0026aacute;rez B. The Role of Gut Microbiota in Gastrointestinal Immune Homeostasis and Inflammation: Implications for Inflammatory Bowel Disease. Biomedicines. 2025;13(8):1807.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTr\u0026oslash;seid M, Andersen G\u0026Oslash;, Broch K, Hov JR. (2020). The gut microbiome in coronary artery disease and heart failure: Current knowledge and future directions. \u003cem\u003eEBioMedicine\u003c/em\u003e, \u003cem\u003e52\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin L, Zhang J. Role of intestinal microbiota and metabolites on gut homeostasis and human diseases. BMC Immunol. 2017;18(1):2.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoshi J, et al. A systematic review assessing the association of inflammatory markers with kidney dysfunction in people living with HIV on highly active antiretroviral therapy. BMC Infect Dis. 2024;24(1):776.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMazzuti L, Turriziani O, Mezzaroma I. The many faces of immune activation in HIV-1 infection: a multifactorial interconnection. Biomedicines. 2023;11(1):159.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eG\u0026aacute;sp\u0026aacute;r Zs\u0026oacute;fia et al. Gut Microbiome Alteration in HIV/AIDS and the role of antiretroviral Therapy\u0026mdash;A scoping review. \u003cem\u003eMicroorganisms\u003c/em\u003e 12.11 (2024): 2221.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrenchley JM, Serrano-Villar S. From dysbiosis to defense: harnessing the gut microbiome in HIV/SIV therapy. Microbiome. 2024;12(1):113.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePan Z, Wu N, Jin C. Intestinal Microbiota Dysbiosis Promotes Mucosal Barrier Damage and Immune Injury in HIV-Infected Patients. Can J Infect Dis Med Microbiol. 2023;2023(1):3080969.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRocafort, M., Gootenberg, D. B., Lu\u0026eacute;vano Jr, J. M., Paer, J. M., Hayward, M. R., Bramante,J. T., \u0026hellip; Kwon, D. S. (2024). HIV-associated gut microbial alterations are dependent on host and geographic context. Nature communications, 15(1), 1055..\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAluthge, N., Adams, S., Davila, C. A., Gocchi Carrasco, N. R., Chiou, K. S., Abadie,R., \u0026hellip; Fernando, S. C. (2024). Gut microbiota profiling in injection drug users with and without HIV-1 infection in Puerto Rico. Frontiers in Microbiology, 15, 1470037..\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFakharian F, Thirugnanam S, Welsh DA, Kim WK, Rappaport J, Bittinger K, Rout N. The role of gut dysbiosis in the loss of intestinal immune cell functions and viral pathogenesis. Microorganisms. 2023;11(7):1849.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIshizaka A, Koga M, Mizutani T, Suzuki Y, Matano T, Yotsuyanagi H. Sustained gut dysbiosis and intestinal inflammation show correlation with weight gain in person with chronic HIV infection on antiretroviral therapy. BMC Microbiol. 2024;24(1):274.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi Y, Hu M, Wu J, Liu T, Qi Y, Li A. Association between gut microbiota in HIV-infected patients and immune reconstitution following antiretroviral therapy (ART). BMC Infect Dis. 2025;25(1):666.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang, Y., Xie, Z., Zhou, J., Li, Y., Ning, C., Su, Q., \u0026hellip; Huang, J. (2023). The altered metabolites contributed by dysbiosis of gut microbiota are associated with microbial translocation and immune activation during HIV infection. Frontiers in immunology, 13, 1020822..\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMac Cann, R., Newman, E., Devane, D., Sabin, C., Cotter, A. G., Landay, A., \u0026hellip; Mallon,P. W. (2024). HIV, the gut microbiome and clinical outcomes, a systematic review.PloS one, 19(12), e0308859..\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePortilla-Tamarit I, Rubio-Aparicio M, Fuster-RuizdeApodaca MJ, Portilla-Tamarit J, Reus S, Portilla J. Health-Related Quality of Life in People with Advanced HIV Disease, from 1996 to 2021: Systematic Review and Meta-analysis. AIDS Behav. 2024;28(6):1978\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVo LT, Nguyen PH, Nguyen HTN, Phan DQ, Vo XTT, Vo LY, Nguyen YNT, Huynh G. Health-Related Quality of Life Among Patients Living with HIV/AIDS in Vietnam: A Cross-Sectional Study. Patient Prefer Adherence. 2025;19:1197\u0026ndash;210.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOuyang J, Yan J, Zhou X, Isnard S, Harypursat V, Cui H, Routy J-P, Chen Y. Relevance of biomarkers indicating gut damage and microbial translocation in people living with HIV. Front Immunol. 2023;14:1173956.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTincati C, Bono V, Cannizzo ES, Tosi D, Savi F, Falcinella C, Casabianca A, Orlandi C, Luigiano C, Augello M, Rusconi S, Muscatello A, Bandera A, Calcagno A, Gori A, Nozza S, Marchetti G. Primary HIV infection features colonic damage and neutrophil inflammation yet containment of microbial translocation. AIDS. 2024;38(5):623\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDillon, S. M., Lee, E. J., Kotter, C. V., Austin, G. L., Dong, Z., Hecht, F. M.,\u0026hellip; Lynch, S. V. (2014). An Altered Intestinal Microbiome in HIV-1-Infected Individuals.Gut Microbes, 5(2), 209\u0026ndash;218.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVazquez-Castellanos, J. F., Hernandez-Ramirez, G. F., Lopez-Gatell, H., Roldan-Fajardo,A., Juarez-Figueroa, L., Ruiz-Matus, C., \u0026hellip; Geller, R. (2018). Changes in the gut microbiome in patients with HIV-1 infection before and after antiretroviral therapy. AIDS Research and Human Retroviruses, 34(1), 101\u0026ndash;110.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGori A, Tincati C, d'Arminio Monforte A, Cislaghi A. HIV infection and the gut microbiota: a new frontier in the pathogenesis and treatment of HIV-related disorders. Expert Rev Anti-infective Therapy. 2014;12(12):1481\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnanthan A, Gopinath B, Uthayakumar M, Venkatesan M. Impact of highly active antiretroviral therapy on the gut microbiome of HIV-infected individuals: a systematic review and meta-analysis. Arch Microbiol. 2020;202(6):1187\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 Clinical data of each group\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinical data\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCON (n=10)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHIV/AIDS (n=15)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (year)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e35.4 \u0026plusmn; 9.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.456\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGender (Male/Female)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e8/2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e13/2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.376\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e24.8 \u0026plusmn; 2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e25.1 \u0026plusmn; 3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.346\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSmoking\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e3 (30%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e5 (33.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.247\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDrinking\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e2 (20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e4 (26.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.534\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTransmission Route\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eNot Applicable\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e12 (80%)\u003c/p\u003e\n \u003cp\u003e2 (13.3%)\u003c/p\u003e\n \u003cp\u003e1 (6.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eNot Applicable\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eHeteroseksual Behaviour\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eHomosexual Behaviour\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eIntravenous drug injection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eHAART tretament (yes/no)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eNot Applicable\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eNot Applicable\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Microbiota, HIV, Antiretroviral Therapy, Dysbiosis","lastPublishedDoi":"10.21203/rs.3.rs-7878027/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7878027/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHIV is one of the chronic infectious diseases that can be treated with the use of new generation antiretroviral therapy (HAART). HIV is closely associated with gastrointestinal diseases, changes in intestinal microbiota and inflammation. Therefore, we aimed to investigate changes in the gut microbiota in HIV-infected and uninfected populations living in Turkey. The study included 15 patients living with HIV and 10 healthy volunteers. Participants living with HIV were studied at baseline and again at least 3 months after treatment. Both blood and fecal samples were collected from all participants. For intestinal microbiota analysis, 16S rRNA gene sequence was analyzed. There was a significant difference between individuals living with HIV and healthy volunteers in terms of intestinal microbiota. While there was a significant decrease in the number of alpha diversity, Firmicutes, Proteobacteria in individuals living with HIV; Faecalibacterium, Prevotella, Streptococcus, Lactobacillus, Ruminococcus increased. Post-treatment improvements in intestinal microbiota were observed in individuals living with HIV receiving HAART with an increase in the number of Phascolarctobacterium and Blautia compared to the pretreatment period (naive patients). Improvement in intestinal microbiota was partially observed after treatment.\u003c/p\u003e","manuscriptTitle":"Intestinal Microbiota Changes in Individuals Living with HIV and the Effect of Antiretroviral Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-03 05:21:04","doi":"10.21203/rs.3.rs-7878027/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6b8644a5-1f4d-473b-a911-f2413604e25c","owner":[],"postedDate":"November 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-14T07:24:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-03 05:21:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7878027","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7878027","identity":"rs-7878027","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}