Oncogenic human papillomavirus and anal microbiota in men who have sex with men and are living with HIV in Northern Taiwan

Shu-Hsing Cheng; Yu-Chen Yang; Cheng-Pin Chen; Hui-Ting Hsieh; Yi-Chun Lin; Chien-Yu Cheng; Kuo-Sheng Liao; Fang-Yeh Chu; Yun-Ru Liu

doi:10.1371/journal.pone.0304045

. 2024 Dec 31;19(12):e0304045. doi: 10.1371/journal.pone.0304045

Oncogenic human papillomavirus and anal microbiota in men who have sex with men and are living with HIV in Northern Taiwan

Shu-Hsing Cheng ^1,^2,^‡, Yu-Chen Yang ^3,^‡, Cheng-Pin Chen ^1,⁴, Hui-Ting Hsieh ¹, Yi-Chun Lin ^1,⁵, Chien-Yu Cheng ^1,⁶, Kuo-Sheng Liao ⁷, Fang-Yeh Chu ^8,^9,^10,^‡,^*, Yun-Ru Liu ^3,^‡,^*

Editor: Ivan Sabol¹¹

¹Department of Infectious Diseases, Taoyuan General Hospital, Ministry of Health and Welfare, Taoyuan, Taiwan

²School of Public Health, Taipei Medical University, Taipei, Taiwan

³Joint Biobank, Office of Human Research, Taipei Medical University, Taipei, Taiwan

⁴Institute of Clinical Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan

⁵Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

⁶Institute of Public Health, National Yang Ming Chiao Tung University, Taipei, Taiwan

⁷Department of Pathology, Taoyuan General Hospital, Ministry of Health and Welfare, Taoyuan, Taiwan

⁸Department of Clinical Pathology, Far Eastern Memorial Hospital, New Taipei City, Taiwan

⁹Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan City, Taiwan

¹⁰School of Medical Laboratory Science and Biotechnology, Taipei Medical University, Taipei, Taiwan

¹¹Ruđer Bošković Institute, CROATIA

Competing Interests: The authors have declared that no competing interests exist.

‡ SHC and YCY contributed equally as 1st authors. FYC and YRL contributed equally as corresponding authors.

^✉

* E-mail: jacpha@mail.femh.org.tw (FYC); d90444002@tmu.edu.tw (YRL)

Roles

Shu-Hsing Cheng: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Validation, Writing – original draft, Writing – review & editing

Yu-Chen Yang: Formal analysis, Software, Validation, Visualization, Writing – review & editing

Cheng-Pin Chen: Data curation, Validation, Writing – review & editing

Hui-Ting Hsieh: Investigation, Writing – review & editing

Yi-Chun Lin: Data curation, Writing – review & editing

Chien-Yu Cheng: Data curation, Writing – review & editing

Kuo-Sheng Liao: Formal analysis

Fang-Yeh Chu: Conceptualization, Methodology, Supervision, Writing – review & editing

Yun-Ru Liu: Conceptualization, Methodology, Supervision, Validation, Writing – review & editing

Ivan Sabol: Editor

PMCID: PMC11687658 PMID: 39739827

Abstract

Few studies have demonstrated the interplay between human immunodeficiency virus (HIV), anal human papillomavirus (HPV), and anal microbiota, especially in persons living with HIV who are men who have sex with men. We, therefore, explored these interrelationships in a cohort of persons living with HIV, mainly comprising men who have sex with men. HPV genotyping using a commercial genotyping kit and ThinPrep cytology interpreted by Bethesda systems was performed on samples from 291 patients. Samples were characterized by high-throughput sequencing of dual-index barcoded 16s rRNA (V3–4). Bacterial diversity was diminished in individuals living with HIV with CD4+ T cells <500 cells/μL and anal cytology yielding atypical squamous cells of undetermined significance or higher grades (ASCUS+) with detectable HPV 16/18 compared with those with CD4+ T cells ≥500 cells/μL with ASCUS+ and HPV 16/18 and those with normal anal cytology or inflammation without HPV 16/18. Enterobacteriaceae, Ruminococcus, and Bacilli were significantly abundant in persons living with HIV with CD4+ T cells <500 cells/μL with ASCUS+ and HPV 16/18. Bacterial diversity, composition, and homogeneity of dispersion were different in individuals living with HIV with low CD4+ T cells with ASCUS+ and HPV 16/18, and understanding the interaction among immunocompromised hosts, oncogenic HPVs, and microbiota is essential, and the contribution of these factors to anal precancerous lesions needs more in-depth exploration.

Introduction

Human papillomavirus (HPV) is associated with cancer, including cervical, head and neck, and genital cancers, and anal squamous cell carcinoma [1, 2]. Certain populations have been noted to have a higher risk of developing anal cancer, namely persons living with human immunodeficiency virus (HIV) and men who have sex with men (MSM) [3]. Persistent oncogenic HPV infections were noted in persons living with HIV (PLWH), especially with immunocompromised status (CD4 + T cell counts less than 200 μL/mL) [4]. Furthermore, the incidence of HPV-related cancer continues to increase in the era of highly active antiretroviral therapy (HAART) in PLWH [2, 5].

Recently, there have been many studies on the symbiotic relationship between the host and indigenous microbiota in establishing viral infections and the progression of virus-related diseases. Changes in commensal microbiota may affect the prognosis of influenza, norovirus, and COVID-19 [6–8]. Dysbiosis has been implicated in carcinogenic pathways, including the progression from chronic hepatitis B or C to hepatocellular carcinoma [9–11], and the association of HPV with cervical carcinogenesis in women [12, 13]. Intestinal dysbiosis and chronic immune activation interact in PLWH [14, 15]. Peptostreptococcus and Fusobacteria have been implicated in the pathogenesis of colorectal cancers [16, 17] and are associated with poor prognosis [18].

There are fruitful studies on the interaction between the microbiota, HPV acquisition and persistence, and the development of cervical cancer [13, 19, 20]. Women with cervical intraepithelial neoplasia exhibit a microbiota profile with increased bacterial diversity and a reduced abundance of Lactobacillus spp. Ravel et al. [21] described bacterial vaginosis-associated bacteria (community state type IV) as a heterogeneous group characterized by the depletion of Lactobacillus spp. and the presence of anaerobic species like Gardnerella, Megasphaera, Sneathia, and Prevotella. Lactobacilli provide broad-spectrum protection in the vagina [22]. An imbalance, such as a depletion of Lactobacillus and an increase in other microbiota, can lead to histological alterations in the vaginal mucosa and cervical epithelium [23, 24]. A previous meta-analysis showed that bacterial vaginosis is associated with higher rates of HPV infection (odds ratio 1.43, 95% CI 1.11–1.84) [25], suggesting that a Lactobacillus-depleted microbiome may contribute to HPV persistence in the cervix. In addition, bacterial vaginosis may involve reactive oxygen species that cause DNA damage, facilitating HPV integration and malignant transformation [23]. Women living with HIV (PLWH) were found to have cervical lesions with increased bacterial diversity and reduced Lactobacillus spp. [26, 27], similar to that in women without HIV [13, 19]. However, studies on anal lesions, HPVs, and anal mucosa microbiota are limited [20, 28, 29]. In this study, we explored the interrelationships between HIV, anal HPV, anal precancerous lesions, and anal microbiota in a PLWH cohort comprising MSM (MSM-LWH).

Materials and methods

Study participants

Between Jun 1, 2018, and Dec 31, 2021, MSM-LWH visited one of the outpatient clinics of Taoyuan General Hospital, Taiwan, voluntarily enrolled in this study. Taoyuan General Hospital is a 1,020-bed referral hospital in northern Taiwan serving 2.3 million people, more than 4,300 of whom live with HIV [30]. After providing written informed consent, the participants completed a self-administered questionnaire (S1 File) that addressed their education level; marital status; sexual behavior (heterosexuality or homosexuality, lifetime number of sexual partners, number of new sexual partners within the previous 6 months, frequency of receptive anal sex (always, often, occasionally, seldom, or never), frequency of condom use during anal sex (always, often, occasionally, seldom, or never), frequency of chemsex (always, often, occasionally, seldom, or never); self-reported sexually transmitted infections (STIs) within the previous 6 months (syphilis, gonorrhea, chlamydial urethritis, condyloma acuminata, amebic colitis/liver abscess, or other clinical diagnoses of STIs), and history of HPV vaccination. Saline-wetted Dacron swabs (Amplicor STD Swab Specimen Collection and Transport Set; Roche Molecular Systems, Branchburg, NJ, USA) were inserted approximately 5 cm above the anal verge. Rectal swabs were immediately rinsed in vials of PreservCyt solution (Hologic Inc., Marlborough, MA, USA) and Omnigene collection tubes (OMR-130; DNA Genotek Inc., Stittsville, ON, Canada).

This study was approved by the Institutional Review Board of Taoyuan General Hospital (IRB#: TYGH106042, TYGH10743 and TYGH108050).

Anal Pap smears

Anal cytology samples were prepared using thin-preparation Pap smears (ThinPrep; Hologic Inc., Marlborough, MA, USA) and sent to a certified laboratory for blinded interpretation by two cytopathology technicians and two pathologists. The results were classified according to the 2001 Bethesda System [31]. We considered the following as anal cellular dysplasia: atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs), and atypical squamous cells that could not exclude HSIL (ASC-H). The cells were preserved in PreservCyt solution and stored at −70°C for DNA testing.

Human papillomavirus detection

HPV detection was performed using polymerase chain reaction (PCR) (Cobas HPV; Roche Molecular Systems, Branchburg, NJ, USA). Fourteen types of HPV have been detected, including oncogenic types 16 and 18, as well as pooled results of types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.

Targeted amplicon library preparation and sequencing

16S rRNA was purified from anal samples by QIAamp Fast DNA Stool Mini Kit (QIAGEN, Germany). The 16S rRNA gene amplification and library construction were performed according to Illumina’s recommended protocols [32]. Briefly, the universal primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) containing Illumina overhang adapter sequences in the forward (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′) and reverse (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′) primers were used to amplify the V3–V4 region of bacterial 16S rRNA using a limited cycle PCR. A Nextera XT Index kit (Illumina Inc., San Diego, CA, USA) was used to add Illumina sequencing adapters and dual-index barcodes to the amplicon targets. Quantification and quality of the sequencing libraries were checked using a QSep100 Analyzer (BiOptic Inc., New Taipei City, Taiwan). Finally, the libraries were normalized and pooled in an equimolar ratio by Nextera XT DNA Library Preparation Kit (Illumina) and sequenced using an Illumina Miseq instrument MiSeq Reagent Kit V3 (600 cycle) (Illumina Inc., San Diego, CA, USA).

16S rRNA gene sequence analysis

Universal primer sequences and low-quality reads were trimmed using Cutadapt (v1.18) [33] and then processed and analyzed using the DADA2/phyloseq workflow in the R environment. Briefly, filtering, trimming, dereplication, and denoising of forward and reverse reads were performed using DADA2 (v1.10.0) [34]. Batch effects were compensated using the DESeq2 package in R, with a maximum of 96 indexes per run [35]. Processed reads were merged, and chimeras were subsequently removed from the cleaned full-length amplicons. Taxonomic assignment of the inferred amplicon sequence variants was performed using the SILVA reference database (v138) [36] with a minimum bootstrap confidence of 80. Multiple sequence alignments of ASVs were performed using DECIPHER (v2.8.1) [37], and a phylogenetic tree was constructed using RaxML (v8.2.11) [38]. The frequency table, taxonomy, and phylogenetic tree information were used to create a phyloseq object for downstream bacterial community analyses using phyloseq (v1.30.0) [39]. Alpha diversity was measured, which indicated the richness and evenness of bacterial taxa within a community, calculated by using the estimate_richness function from the phyloseq package with observed, chao index, Shannon, and Simpson methods; Beta diversity, which reflects the extent of variation in microbial community composition between the two groups, using Principal Co-ordinates Analysis (PcoA) on unweighted Unique Fraction (UniFrac) and weighted UniFrac, PcoA on variance-adjusted weighted UniFrac; and PcoA on Generalized Unique Fraction (GUniFrac) with alpha 0.5. UniFrac distances were calculated using the GUniFrac package (v1.1) to assess the community dissimilarity between groups [40]. Non-metric Multidimensional Scaling (NMDs) on Bray–Curtis distance (VST), Adonis tests (to examine whether composition between groups was similar), and the Betadisper test (to test the homogeneity of dispersion between groups, which is an assumption of Adonis) was calculated using DESeq2 package (V1.26.0) [33]. Microbiota enrichment analysis was conducted using the Linear Discriminant Analysis (LDA) effect size (LefSe) method, applying the Wilcoxon–Mann–Whitney test (α = 0.05) and logarithmic LDA score > 2 [41]. The results were visualized as a cladogram using GraPhlAn [42].

Statistical analyses

Demographic data are presented as mean ± standard deviation (SD) for continuous variables, median (25^th, 75^th percentile), and percentiles for discrete variables. The distribution of the cytology grading was calculated, and the HPV genotype results were analyzed. Chi-square tests were used to compare categorical variables, while Wilcoxon–Mann–Whitney tests were used to compare continuous variables. Anal samples were divided into six groups; group 1: samples from PLWH whose CD4+ T cell counts were < 500 cells/μL with anal cytology yielding ASCUS or higher grades (ASCUS+) and detectable HPV 16/18; group 2: samples from PLWH whose CD4+ T cell counts were ≥ 500 cells/μL with anal cytology yielding ASCUS+ with detectable HPV 16/18; group 3: samples from PLWH who had anal cytology yielding ASCUS+ without HPV 16/18 detection, group 4: samples from PLWH whose CD4+ T cell counts were < 500 cells/μL and who had normal anal cytology or inflammation with detectable HPV 16/18; group 5: samples from PLWH whose CD4+ T cell counts ≥ 500 cells/μL and who had normal anal cytology or inflammation with detectable HPV 16/18; and group 6: samples from PLWH who had normal anal cytology or inflammation without HPV 16/18 detection. Each group (groups 2, 3, 4, 5, and 6) was compared to group 1. P < 0.05 was considered statistically significant. All statistical analyses were conducted using SAS 9.3 software (SAS Institute, Inc., Cary, NC, USA).

Results

In total, 338 anal samples were collected. However, 10 (2.9%) failed cytology interpretation, 28 (8.3%) failed HPV detection tests, and 9 (2.7%) were from heterosexual PLWH. Finally, 291 (86.1%) samples collected from MSM-LWH were analyzed (Table 1).

Table 1. Demographic characteristics, behavior risks, and immunologic factors for the study cohort of men who have sex with men and live with HIV in northern Taiwan.

Characteristics	Patients N = 291	Percentage, (SD), or (25, 75^th quartile)
Age, mean (SD)	39.26	(10.06)
Median (25^th, 75^th quartile)	37	(32, 47)
Years in school
6 to 9	14	4.8%
>9 to 12	80	27.5%
>12	197	67.7%
Marriage
Single	271	93.1%
Others	20	6.9%
HIV years, mean (SD)	8.55	(5.55)
Median (25^th, 75^th quartile)	8	(4, 11)
Smoking	115	39.5%
Drinking	116	39.9%
Betelnut	9	3.1%
Lifetime sexual partner, mean (SD)	21.62	(50.74)
Median (25^th, 75^th quartile)	10	(5;20)
Sexual partner in 1/2 y, mean (SD)	2.17	(4.33)
Median (25^th, 75^th quartile)	1	(1,2)
Condom use during anal sex
Every time, often	156	53.6%
Occasional	114	39.2%
Rare, never	21	7.2%
Met sexual partners on the web	196	67.4%
STDs in 1/2 y	85	29.2%
Circumcision	52	17.9%
HPV vaccination	62	21.3%
Substance use in 1/2 y	42	14.4%
Ever participated in chemsex	95	32.6%
On cART	291	100%
Current CD4+ T cell count, mean (SD)	607.62	(267.11)
Median (25^th, 75^th quartile)	598.5	(437.75;759.75)
Undetectable viral load (VL<50)	267	91.8%

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Abbreviations: cART: combinational antiretroviral therapy; HIV: human immunodeficiency virus; HPV: human papillomavirus; SD: standard deviation; STD: sexually transmitted diseases.

Among the anal samples from the 291 participants, 199 (68.4%) had detectable oncogenic HPVs, 92 (31.6%) did not, 68 (23.4%) were positive for HPV type 16/18, 36 (12.4%) yielded HPV 16, 38 (13.1%) yielded HPV 18, and 190 (65.3%) yielded pooled results for 12 other oncogenic HPVs (Table 2).

Table 2. Oncogenic HPV distribution among 291 participants who are MSM-LWH.

	Total N = 291(100%)	Normal or inflammation N = 158 (100%)	ASCUS+ N = 133(100%)	P value
Oncogenic HPV detection	199 (68.4%)	89 (56.3%)	110 (82.7%)	<0.00001
HPV16	36 (12.4%)	12 (7.6%)	24 (18.0%)	0.007
HPV18	38 (13.1%)	11 (6.9%)	27 (20.3%)	0.0007
HPV16 and/or 18	68 (23.3%)	22 (13.9%)	46 (34.6%)	0.00003
Pooled 12 other oncogenic HPVs^*	190 (65.3%)	85 (53.8%)	105 (78.9%)	<0.00001

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* HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.

Abbreviations: ASCUS+: included atypical squamous cells of undetermined significance, low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions (HSIL), and atypical squamous cells that could not exclude HSIL; HIV: human immunodeficiency virus; HPV: human papillomavirus, MSM: men who have sex with men; MSM-LWH: MSM who are living with HIV.

Among the anal samples from the 291 participants, 133 (45.7%) yielded cytology interpretations with ASCUS+, including 70 (24.1%) ASCUS, 49 (16.8%) LSIL, 2(0.7%) HSIL, and 12 (4.1%) ASC-H, and 158 (52.3%) with normal anal cytology or inflammation. The anal samples (n = 291) were divided into six groups according to HPV16/18 detection, cytology results, and participants’ CD4+ T cell counts (Fig 1).

Fig 1 — ASCUS, atypical squamous cells of undetermined significance; Gr, group; HPV, human papillomavirus; MSM-LWH, men who have sex with men and live with HIV.

We did not find a clear difference in alpha diversity metrics between samples from group 1 and samples from other groups (observed p = 0.033 for group 1 vs. group 2; p = 0.022 for group 1 vs. group 6; Chao1 p = 0.053 for group 1 vs. group 6; all by Wilcoxon–Mann–Whitney tests, Fig 2).

Fig 2 — Observed p = 0.033 for group 1 vs. group 2, p = 0.022 for group 1 vs. group 6; Chao1 p = 0.053 for group 1 vs. group 6, all by Wilcoxon–Mann–Whitney tests. Note: group 1: PLWH whose CD4+ T cell counts were < 500 cells/μL with anal cytology yielding ASCUS+ and detectable HPV 16/18 (n = 21); group 2: PLWH whose CD4+ T cell counts were ≥ 500 cells/μL with anal cytology yielding ASCUS+ with detectable HPV 16/18 (n = 25); group 3: PLWH who had anal cytology yielding ASCUS+ without HPV 16/18 detection (n = 87), group 4: PLWH whose CD4+ T cell counts were < 500 cells/μL and who had normal anal cytology or inflammation with detectable HPV 16/18 (n = 13); group 5: PLWH whose CD4+ T cell counts were ≥ 500 cells/μL and who had normal anal cytology or inflammation with detectable HPV 16/18 (n = 9); and group 6: PLWH who had normal anal cytology or inflammation without HPV 16/18 detection (n = 136). ASCUS, atypical squamous cells of undetermined significance; Gr, group; HPV, human papillomavirus; PLWH, persons living with HIV.

We also examined beta diversity. We found differences at this level between samples from group 1 and those from the other groups (S1 Table, Fig 3).

We used the LefSe discovery tool to identify the predictors of abnormal anal cytology. This method addresses the issue of identifying microorganisms that consistently explain the differences between ≥2 microbial communities. When comparing groups 1 and 2, LefSe showed abundant Enterobacterales, Enterobacteriaceae, and Mycoplasma in group 1 and abundant Finegoldia in group 2 (Fig 4A). When comparing groups 1 and 3, LefSe showed abundant Enterobacterales and Enterobacteriaceae in group 1, and abundant Alloprevotella and Erysipelatoclostridiaceae in group 3 (Fig 4B). When comparing groups 1 and 4, LefSe showed abundant Ruminococcus and Sutterella in group 1 and abundant Porphyromonadaceae, Porphyromonas, and Fenollaria in group 4 (Fig 4C). When comparing groups 1 and 5, LefSe did not show significant findings. When comparing groups 1 and 6, LefSe showed abundant Enterobacterales, Enterobacteriaceae, and Bacilli in group 1, and abundant Bacteroidota, Bacteroidia, Bacteroidales, and Clostridia in group 6 (Fig 4D).

Fig 4 — 4a: group 1 vs. group 2; 4b: group 1 vs. group 3; 4c: group 1 vs. group 4, 4d: group 1 vs. group 6. Note: group 1: PLWH whose CD4+ T cell counts were < 500 cells/μL with anal cytology yielding ASCUS+ and detectable HPV 16/18 (n = 21); group 2: PLWH whose CD4+ T cell counts were ≥ 500 cells/μL with anal cytology yielding ASCUS+ with detectable HPV 16/18 (n = 25); group 3: PLWH who had anal cytology yielding ASCUS+ without HPV 16/18 detection (n = 87), group 4: PLWH whose CD4+ T cell counts were < 500 cells/μL and who had normal anal cytology or inflammation with detectable HPV 16/18 (n = 13); group 5: PLWH whose CD4+ T cell counts were ≥ 500 cells/μL and who had normal anal cytology or inflammation with detectable HPV 16/18 (n = 9); and group 6: PLWH who had normal anal cytology or inflammation without HPV 16/18 detection (n = 136). ASCUS, atypical squamous cells of undetermined significance; Gr, group; HPV, human papillomavirus; PLWH, persons living with HIV.

Discussion

To the best of our knowledge, this is the first research focusing on the presentations of anal microbiota among Taiwanese MSM-LWH with different stages of HIV-related diseases and levels of cellular immunity. Depletion of certain Lactobacillus and an increase in the richness of microbiota may contribute to HPV persistence [25], and subsequent histological alterations of the vaginal mucosa [21] and the cervical epithelium (squamous intraepithelial neoplasm) [23, 24]. This biology could be also applied to the anal mucosal environment of MSM-LWH [43]. A small study of 42 MSM-LWH revealed that Peptostreptococcus, Campylobacter, and Gardnerella were associated with anal precancerous lesions [28]. Another study involving 113 Nigerian MSM found that a higher proportion of MSM with prevalent HPV16 was associated with a cluster enriched in Sneathia from the family Fusobacteriaceae [29]. However, there were only six PLWH treated with HAART with viral suppression in the study by Nowak et al. [29]. Another study showed that the Ruminococcaceae NK4A214 group, Alloprevotella genus, Prevotella melanonigenica, and Ruminococcaceae UCG-014 were the most predictive of biopsy-proven HSIL [44].

In the present study, diminished richness of bacterial diversity was noted in group 1 compared with other groups, i.e., group 2 and group 6. The variability within each group also differed, indicating differences in the spread or dispersion of microbial communities between the groups. Enterobacteriaceae, Proteus, Morganellaceae, Mycoplasma, Sutterella, Ruminococcus, and Bacilli were significantly more abundant in group 1 than in groups 2, 3, 4, and 6. In contrast, Bacteroidales and Clostridia were abundant in group 6.

Dinh et al. studied 21 PLWH and found the relative abundance of intestinal Proteobacteria, Gammaproteobacteria, Enterobacterales, and Enterobacteriaceae was significantly enriched compared to HIV-negative controls [45]. Dubourg et al. studied 31 PLWH and showed that PLWH has an increased relative abundance of gut Gammaproteobacteria compared to HIV-negative cases [46]. Previous researchers have conducted comprehensive reviews highlighting increased inflammatory biomarkers, including CD14, IL-6, and CD38+HLA-DR+CD8+ T cells in the gut, as well as increases in traditionally pathogenic bacteria such as Enterobacteriaceae, including Morganellaceae and Proteus [46, 47]. Similar to the current study, we also demonstrated a higher relative abundance of Enterobacterales and Enterobacteriaceae in group 1 compared to other groups. However, these previous studies did not examine the role of oncogenic HPVs or the differences in anal cytology findings. Mycoplasma is the smallest intracellular bacterium that establishes persistent intracellular infections that can lead to inflammatory cytokine-mediated tissue injury. In addition, Mycoplasma infection allows direct interaction with HPV during co-infection of epithelial cells [48], an observation consistent with our findings. Sutterella is a gram-negative, non-spore-forming rod grown under anaerobic or microaerophilic conditions. It belongs to the family Betaproteobacteria, is proinflammatory [49], and could explain our findings regarding the abundance of Sutterella in the anal samples of group 1 patients. Ruminococcus, a gram-positive, spore-forming, anaerobic rod, is a polyphyletic genus with species belonging to two Firmicutes families, Lachnospiraceae and Ruminococcaceae, capable of degrading cellulose. Ron et al. studied 128 MSM, of whom 47 (36.7%) had biopsy-proven HSIL, and found that Ruminococcus is a predictor of HSIL [44]. Although the richness of this anerobic commensal, Ruminococcus, was found to decrease in the gut samples of PLWH [50], caution should be exercised when it is found in abundance at anal sites, particularly the Ruminococcaceae NK4A214 group [44]. Bacilli have been reported to correlate with the progression of HPV-related cervical lesions [13] and may therefore deserve attention with regard to the progression of anal lesions. Previous studies have mentioned the contribution of Fusobacterium and Sneathia to HPV-related lesions [29, 43], which could not be proven in this study. Ethnic or environmental factors may contribute to these different findings [13, 29]. The above-mentioned anal bacterial ecology changes with the presence of oncogenic HPVs and the transformation of the anal epithelium.

The strength of this study lies in its interpretation of the anal microbial compositions of a large cohort of MSM-LWH that had different stages of HPV-related disease using high-throughput sequencing. The limitations of this study include its cross-sectional design. Hence, we could not conclude a causal relationship between the anal microbiota and HPV infection or precancerous anal cytology. Second, nearly 100% of the study cohort received HAART; therefore, the findings of this study cannot be generalized to PLWH without HAART. Third, the participants in this study were from a single urban tertiary care hospital, and the results cannot be generalized to different populations. Finally, although in this study we strongly recommended high-resolution anoscopy to obtain the histology results for patients with HPV 16/18 or cytology yielding ASCUS+, less than 20% of the patients actually visited proctologists. The patients’ reluctance to undergo anoscopy motivated us to explore non-invasive biomarkers.

The study showed that MSM-LWH with CD4+ T cell counts <500 cells/μL, anal ASCUS+, and detectable HPV16/18 have low bacterial diversity and a relative abundance of Enterobacteriaceae, Ruminococcus, and Bacilli compared to other participant groups. We concluded that understanding the interaction among immunocompromised hosts, oncogenic HPVs, and microbiota is essential, and the contribution of these factors to anal precancerous lesions needs more in-depth exploration. Furthermore, the anal swab signature microbiota profiles, such as Lactobacillus in the cervix, warrant further exploration.

Supporting information

S1 Table. Results of the permutational multivariate analysis of variance (adonis function) and the multivariate homogeneity of group dispersions analysis (betadisper function).

(DOCX)

pone.0304045.s001.docx^{(20.3KB, docx)}

S1 File. Questionnaire for enrolled participants.

(PDF)

pone.0304045.s002.pdf^{(61.8KB, pdf)}

Acknowledgments

We acknowledge the Core Instrument Center of the Taipei Medical University for providing the next generation sequence service. We also thank the patients and the care team of Comprehensive AIDS Care Center at Taoyuan General Hospital, Ministry of Health and Welfare.

Data Availability

The data that support the findings of this study are available in the Figshare database, doi: 10.6084/m9.figshare.25375087.

Funding Statement

SHC received funding for this work from Taoyuan General Hospital [grant numbers: PTH10715, PTH10805, PTH10915, and PTH11009]. https://www.tygh.mohw.gov.tw/ The sponsors or funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.zur Hausen HA. The role of papillomaviruses in anogenital cancer. Scand J Infect Dis Suppl. 1990;69: 107–111. [PubMed] [Google Scholar]
2.de Martel C, Georges D, Bray F, Ferlay J, Clifford GM. Global burden of cancer attributable to infections in 2018: a worldwide incidence analysis. Lancet Glob Health. 2020;8: e180–e190. doi: 10.1016/S2214-109X(19)30488-7 [DOI] [PubMed] [Google Scholar]
3.Clifford GM, Georges D, Shiels MS, Engels EA, Albuquerque A, Poynten IM, et al. A meta-analysis of anal cancer incidence by risk group: toward a unified anal cancer risk scale. Int J Cancer. 2021;148: 38–47. doi: 10.1002/ijc.33185 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Cheng SH, Chu FY, Lin YS, Hsueh YM. Influence of Age and CD4+ T cell counts on the prevalence of genital human papillomavirus infection among HIV-seropositive men who have sex with men in Taiwan. J Med Virol. 2012;84: 1876–1883. doi: 10.1002/jmv.23413 [DOI] [PubMed] [Google Scholar]
5.Jin F, Vajdic CM, Law M, Amin J, van Leeuwen M, McGregor S, et al. Incidence and time trends of anal cancer among people living with HIV in Australia. AIDS. 2019;33: 1361–1368. doi: 10.1097/QAD.0000000000002218 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Jones MK, Watanabe M, Zhu S, Graves CL, Keyes LR, Grau KR, et al. Enteric bacteria promote human and mouse Norovirus infection of B cells. Science. 2014;346: 755–759. doi: 10.1126/science.1257147 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Kaul D, Rathnasinghe R, Ferres M, Tan GS, Barrera A, Pickett BE, et al. Microbiome disturbance and resilience dynamics of the upper respiratory tract during influenza A virus infection. Nat Commun. 2020;11: 2537. Erratum in: Nat Commun. 2020;11: 3132. doi: 10.1038/s41467-020-16429-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Mizutani T, Ishizaka A, Koga M, Ikeuchi K, Saito M, Adachi E, et al. Correlation analysis between gut microbiota alterations and the cytokine response in patients with coronavirus disease during hospitalization. Microbiol Spectr. 2022;10: e0168921. doi: 10.1128/spectrum.01689-21 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Komiyama S, Yamada T, Takemura N, Kokudo N, Hase K, Kawamura YI. Profiling of tumour-associated microbiota in human hepatocellular carcinoma. Sci Rep. 2021;11: 10589. doi: 10.1038/s41598-021-89963-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Liu Q, Li F, Zhuang Y, Xu J, Wang J, Mao X, et al. Alteration in gut microbiota associated with hepatitis B and non-hepatitis virus related hepatocellular carcinoma. Gut Pathog. 2019;11: 1. doi: 10.1186/s13099-018-0281-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Inoue T, Nakayama J, Moriya K, Kawaratani H, Momoda R, Ito K, et al. Gut dysbiosis associated with hepatitis C virus infection. Clin Infect Dis. 2018;67: 869–877. doi: 10.1093/cid/ciy205 [DOI] [PubMed] [Google Scholar]
12.So KA, Yang EJ, Kim NR, Hong SR, Lee JH, Hwang CS, et al. Changes of vaginal microbiota during cervical carcinogenesis in women with human papillomavirus infection. PLOS ONE. 2020;15: e0238705. doi: 10.1371/journal.pone.0238705 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Chen Y, Qiu X, Wang W, Li D, Wu A, Hong Z, et al. Human papillomavirus infection and cervical intraepithelial neoplasia progression are associated with increased vaginal microbiome diversity in a Chinese cohort. BMC Infect Dis. 2020;20: 629. doi: 10.1186/s12879-020-05324-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Neff CP, Krueger O, Xiong K, Arif S, Nusbacher N, Schneider JM, et al. Fecal microbiota composition drives immune activation in HIV-infected individuals. EBiomedicine. 2018;30: 192–202. doi: 10.1016/j.ebiom.2018.03.024 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Cook RR, Fulcher JA, Tobin NH, Li F, Lee D, Javanbakht M, et al. Effects of HIV viremia on the gastrointestinal microbiome of young MSM. AIDS. 2019;33: 793–804. doi: 10.1097/QAD.0000000000002132 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Castellarin M, Warren RL, Freeman JD, Dreolini L, Krzywinski M, Strauss J, et al. Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Res. 2012;22: 299–306. doi: 10.1101/gr.126516.111 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Yu J, Feng Q, Wong SH, Zhang D, Liang QY, Qin Y, et al. Metagenomic analysis of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer. Gut. 2017;66: 70–78. doi: 10.1136/gutjnl-2015-309800 [DOI] [PubMed] [Google Scholar]
18.Mima K, Nishihara R, Qian ZR, Cao Y, Sukawa Y, Nowak JA, et al. Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis. Gut. 2016;65: 1973–1980. doi: 10.1136/gutjnl-2015-310101 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Nieves-Ramírez ME, Partida-Rodríguez O, Moran P, Serrano-Vázquez A, Pérez-Juárez H, Pérez-Rodríguez ME, et al. Cervical squamous intraepithelial lesions are associated with differences in the vaginal microbiota of Mexican women. Microbiol Spectr. 2021;9: e0014321. doi: 10.1128/Spectrum.00143-21 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Kyrgiou M, Mitra A, Moscicki AB. Does the vaginal microbiota play a role in the development of cervical cancer? Transl Res. 2017;179: 168–182. doi: 10.1016/j.trsl.2016.07.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SSK, McCulle SL, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A. 2011;108;Supplement 1: 4680–4687. doi: 10.1073/pnas.1002611107 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Łaniewski P, Cui H, Roe DJ, Barnes D, Goulder A, Monk BJ, et al. Features of the cervicovaginal microenvironment drive cancer biomarker signatures in patients across cervical carcinogenesis. Sci Rep. 2019;9: 7333. doi: 10.1038/s41598-019-43849-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Williams VM, Filippova M, Filippov V, Payne KJ, Duerksen-Hughes P. Human papillomavirus type 16 E6* induces oxidative stress and DNA damage. J Virol. 2014;88: 6751–6761. doi: 10.1128/JVI.03355-13 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Piyathilake CJ, Ollberding NJ, Kumar R, Macaluso M, Alvarez RD, Morrow CD. Cervical microbiota associated with higher grade cervical intraepithelial neoplasia in women infected with high-risk human papillomaviruses. Cancer Prev Res (Phila). 2016;9: 357–366. doi: 10.1158/1940-6207.CAPR-15-0350 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Gillet E, Meys JF, Verstraelen H, Bosire C, De Sutter P, Temmerman M, et al. Bacterial vaginosis is associated with uterine cervical human papillomavirus infection: a meta-analysis. BMC Infect Dis. 2011;11: 10. doi: 10.1186/1471-2334-11-10 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Curty G, Costa RL, Siqueira JD, Meyrelles AI, Machado ES, Soares EA, et al. Analysis of the cervical microbiome and potential biomarkers from postpartum HIV-positive women displaying cervical intraepithelial lesions. Sci Rep. 2017;7: 17364. doi: 10.1038/s41598-017-17351-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Chávez-Torres M, Gómez-Palacio-Schjetnan M, Reyes-Terán G, Briceño O, Ávila-Ríos S, Romero-Mora KA, et al. The vaginal microbiota of women living with HIV on suppressive antiretroviral therapy and its relation to high-risk human papillomavirus infection. BMC Microbiol. 2023;23: 21. doi: 10.1186/s12866-023-02769-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Serrano-Villar S, Vásquez-Domínguez E, Pérez-Molina JA, Sainz T, de Benito A, Latorre A, et al. HIV, HPV, and microbiota: partners in crime? AIDS. 2017;31: 591–594. doi: 10.1097/QAD.0000000000001352 [DOI] [PubMed] [Google Scholar]
29.Nowak RG, Bentzen SM, Ravel J, Crowell TA, Dauda W, Ma B, et al. Anal microbial patterns and oncogenic human papillomavirus in a pilot study of Nigerian men who have sex with men at risk for or living with HIV. AIDS Res Hum Retroviruses. 2019;35: 267–275. doi: 10.1089/AID.2018.0158 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Taiwan Centers for Disease Control. Statistics of communicable diseases and surveillance reports in Taiwan area; 2023. Taiwan: Ministry of Health and Welfare. p. 2017. [Cited 31 December 2023]. Available from: https://www.cdc.gov.tw/Category/Page/rCV9N1rGUz9wNr8lggsh2Q. [Google Scholar]
31.Solomon D, Davey D, Kurman R, Moriarty A, O’Connor D, Prey M, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA. 2002;287: 2114–2119. doi: 10.1001/jama.287.16.2114 [DOI] [PubMed] [Google Scholar]
32.Illumina support center. 16S Metagenomic Sequencing Library preparation; 2013. San Diego: Illumina Inc. [Cited 31 December 2023] Available from: https://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.html. [Google Scholar]
33.Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15: 550. doi: 10.1186/s13059-014-0550-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal. 2011;17: 10–12. doi: 10.14806/ej.17.1.200 [DOI] [Google Scholar]
35.Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13: 581–583. doi: 10.1038/nmeth.3869 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41: D590–D596. doi: 10.1093/nar/gks1219 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Wright ES. DECIPHER: harnessing local sequence context to improve protein multiple sequence alignment. BMC Bioinformatics. 2015;16: 322. doi: 10.1186/s12859-015-0749-z [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30: 1312–1313. doi: 10.1093/bioinformatics/btu033 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.McMurdie PJ, Holmes S. Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLOS ONE. 2013;8: e61217. doi: 10.1371/journal.pone.0061217 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Chen J, Bittinger K, Charlson ES, Hoffmann C, Lewis J, Wu GD, et al. Associating microbiome composition with environmental covariates using generalized UniFrac distances. Bioinformatics. 2012;28: 2106–2113. doi: 10.1093/bioinformatics/bts342 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, et al. Metagenomic biomarker discovery and explanation. Genome Biol. 2011;12: R60. doi: 10.1186/gb-2011-12-6-r60 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Asnicar F, Weingart G, Tickle TL, Huttenhower C, Segata N. Compact graphical representation of phylogenetic data and metadata with GraPhlAn. PeerJ. 2015;3: e1029. doi: 10.7717/peerj.1029 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Lee JE, Lee S, Lee H, Song YM, Lee K, Han MJ, et al. Association of the vaginal microbiota with human papillomavirus infection in a Korean twin cohort. PLOS ONE. 2013;8: e63514. doi: 10.1371/journal.pone.0063514 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Ron R, Cabello A, Gosalbes MJ, Sánchez-Conde M, Talavera-Rodríguez A, Zamora J, et al. Exploiting the microbiota for the diagnosis of anal precancerous lesions in men who have sex with men. J Infect Dis. 2021;224: 1247–1256. doi: 10.1093/infdis/jiab068 [DOI] [PubMed] [Google Scholar]
45.Gootenberg DB, Paer JM, Luevano JM, Kwon DS. HIV-associated changes in the enteric microbial community: potential role in loss of homeostasis and development of systemic inflammation. Curr Opin Infect Dis. 2017;30: 31–43. doi: 10.1097/QCO.0000000000000341 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Dinh DM, Volpe GE, Duffalo C, Bhalchandra S, Tai AK, Kane AV, et al. Intestinal microbiota, microbial translocation, and systemic inflammation in chronic HIV infection. J Infect Dis. 2015;211: 19–27. doi: 10.1093/infdis/jiu409 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Dubourg G, Lagier JC, Hüe S, Surenaud M, Bachar D, Robert C, et al. Gut microbiota associated with HIV infection is significantly enriched in bacteria tolerant to oxygen. BMJ Open Gastroenterol. 2016;3: e000080. doi: 10.1136/bmjgast-2016-000080 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Klein C, Samwel K, Kahesa C, Mwaiselage J, West JT, Wood C, et al. Mycoplasma co-infection is associated with cervical cancer risk. Cancers (Basel). 2020;12: 1093. doi: 10.3390/cancers12051093 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Hiippala K, Kainulainen V, Kalliomäki M, Arkkila P, Satokari R. Mucosal prevalence and interactions with the epithelium indicate commensalism of Sutterella spp. Front Microbiol. 2016;7: 1706. doi: 10.3389/fmicb.2016.01706 [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Meng J, Tao J, Abu Y, Sussman DA, Girotra M, Franceschi D, et al. HIV-positive patients on antiretroviral therapy have an altered mucosal intestinal but not oral microbiome. Microbiol Spectr. 2023;11: e0247222. doi: 10.1128/spectrum.02472-22 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0304045.r001

Decision Letter 0

Ivan Sabol

10 Jul 2024

PONE-D-24-17510

Oncogenic human papillomavirus and anal microbiota in men who have sex with men and are living with HIV in Northern Taiwan

PLOS ONE

Dear Dr. Chu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please note that some of the comments (Reviewer 1) were provided in a separate attachment and the attachment should be accompanying the email as per the note at the end..

Critically, additional clarity in the methods section is necessary and the data should be presented more concisely. Some of the statistical methodology might also be suboptimal.

Please submit your revised manuscript by Aug 23 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Ivan Sabol

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

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2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

https://academic.oup.com/jid/article/224/7/1247/6129308?login=false

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

Additional Editor Comments:

Please also take a look these more technical issues in addition to the reviewer’s comments:

Page 4 Line 79 listing inclusion and exclusion criteria for invitation to participate explicitly might be helpful. Currently the Line 79 implies MSM was an inclusion criteria but 9 participants failed this (P8L166)?

P6 L123– 124 how many indexes were used in one run? If all 291 samples were assessed by 16rRNA seq this means that batches were made. Was batch effect investigated and how was it addressed?

P6 L127 which flow cell and reagents (cartridge) were used (v2/v3)?

P7 L129 are the raw sequences deposited in a sequence repository?

P7 L138 was some preprocessing / filtering done on the pyloseq object or during preceeding steps? Ie normalization or filtering of taxa?

P7 L140-144 last two paragraphs lack version and or references for the packages used.

P7 L150 testing every combination with t-test is inappropriate without multiple testing correction. Why was Deseq not used since phyloseq is supposed to be analysed with deseq for differential abundance http://joey711.github.io/phyloseq-extensions/DESeq2.html?

P7 L151 6 groups for analysis seems excessive? Possibly this level of detail is appropriate for the supplement tables. It is difficult to confirm but the Figure 1 groups appear to be not mutually exclusive (Groups 3 and 6 appear to have the most participants and those 2 groups do not specify the CD4 cell counts unlike all other groups where the cutoff of 5000 cells was employed.)

P8 L165 it might be more informative if failed cytology and failed HPV exclusions were shown separately

P8 Table 1 “educational year” is worded sub optimally

P8 Table 1 it might be necessary to specify what smoking and drinking represent? Were some guidelines placed in the questionnaire that might be important for interpreting what “yes” answers mean?

P9 Table 1Was the normality of numerical variables tested? Can all reliably be summarized with mean(SD)?

P9 Table 1 were there missing answers in the questionnaires?

P8-9 Table 1 contains excessive white space.

P10 data regarding HPV/cytology and other laboratory measures should be provided in a table for the groups (plus total) discussed instead of text only.

P10 L195 the materials and methods do not adequately introduce the alpha and beta diversity metrics used in the results section. Figure 1 shows some numbers but no context is provided what 500 or 1000 means

P11 L199-201 using t-test for the pairwise comparison of 6 groups is inappropriate. If the data is normally distributed then an ANOVA test with suitable post test should be done.

P11 L201/201 total number of participants in each group should be shown.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Chu et al describes the results of a study that performed 16S analysis in anal samples collected from MSM-LWH, characterized in terms of cytology and HPV infection. This is a quite underexplored field of research. The study seems to be well designed and well conducted, although histology is missing and only cytology results are available.

Reviewer #2: In this study “Oncogenic human papillomavirus and anal microbiota in men who have sex with men and are living with HIV in Northern Taiwan”, HPV DNA tests and citology results in anal samples collected Jun 2018 and Dec 2021 in 291 persons living with HIV (PLWH), mainly MSM were analyzed and 16S sequencing was performed to characterize the anal microbiome. Data on the interaction of HPV, HIV and resident commensal bacteria are needed and may be useful in determining the relative risk of lesion progression to anal cancer precursors. Despite the merits of the study, a clearer presentation of the results, additional information and more in-depth discussion are needed in a revised version.

Specific points

Method:

A paragraph on the Bioinformatic analysis methods should be added to explain the tests that are listed in Results: e.g. UniFrac, PcoA on GuniFrac, Non-metric Multidimensional Scaling (NMDs), Bray-Curtis distance (VST), Adonis tests and the Betadisper test.

The paragraph HPV genotyping should be renamed to HPV detection and it should be more clearly stated that individual results for HPV 16 and HPV 18 are given, along with a simultaneous, pooled result for the other oncogenic HPVs.

Was high-resolution anoscopy (HRA) performed after an ASCUS+ result?

Results

The presentation of results should be improved. For example, the distribution of samples in the six groups could be presented in a table.

The individual results for HPV 16, HPV 18 and other oncogenic HPVs should be reported in a table or text and whether positivity was statistically associated with ASCUS+ results. Moreover, how many patients were diagnosed with anal SIL should be reported.

Discussion:

Lines 281-282: “The interaction between microbiota, HPV acquisition and persistence, and the development of cervical cancer in women has been researched in detail.” I would suggest to modify this sentence in: the vaginal microbiota has been studied in detail, but the interactions with HPV acquisition and persistence and cervical cancer risk are still not fully understood. Accordingly, I would suggest to shorten the description of the vaginal microbiota community state types (CSTs) that are well known (lines 283-295) and to focus on studies (if any) about the anal mucosa microbiota in women.

Lines 306-308: “In the pathophysiology of infectious diseases, pathogenic colonization and infection involve dynamic interactions between microbes and the microbiome, host, and environmental factors [39]. This biology is also applicable to PLWH who are MSM” The sentences “This biology is also applicable to PLWH who are MSM” should be rephrased, e.g. in This biology also applies to the anal mucosal environment of PLWH who are MSM.

At line 322: “homogeneity of the dispersion also differed between the groups...” This should be rephrased for a better comprehension of this concept.

At lines 323-325: “Enterobacteriaceae, Proteus, Morganellaceae, Mycoplasma, Sutterella, Ruminococcus, and Bacilli were significantly abundant in PLWH whose CD4+ T cell counts were < 500 cells/µL and who had anal cytology yielding ASCUS or higher grades with detectable HPV 16/18 (group 1).” As this sentence implies a comparison, other group(s) should be mentioned.

At lines 328-331: “HIV infection also frequently correlates with increased tissue and circulating measures of inflammation, such as CD14, IL-6, and CD38+HLA-DR+CD8+ T cells in the gut [41], as well as increases in traditionally pathogenic bacteria such as Enterobacteriaceae, including Morganellaceae and Proteus [42,43].” The sentence should be reworded to more clearly relate the results of the references mentioned here [41-43] to the results of this study.

The same applies to the sentences at lines 342-343: “Our findings are consistent with those of Ron et al., who described Ruminococcus-predicting HSIL [40]. In contrast, a decreased abundance of Ruminococcus was previously noted in the guts of PLWH [46].”

and to the sentences at lines 353-354 (limitations): “Third, previous studies have mentioned the contribution of Fusobacterium and Sneathia to HPV-related lesions [22,39], which could not be proven in this study” Ref 39 (Pathogens, microbiome and the host: emergence of the ecological Koch's postulates) is a review; hence, the specific study reporting about Fusobacterium and/or Sneathia should be specified and discussed.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2024 Dec 31;19(12):e0304045. doi: 10.1371/journal.pone.0304045.r002

Author response to Decision Letter 0

15 Aug 2024

Response to reviewers uploaded at Attach Files step.

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PLoS One. doi: 10.1371/journal.pone.0304045.r003

Decision Letter 1

Ivan Sabol

17 Sep 2024

PONE-D-24-17510R1Oncogenic human papillomavirus and anal microbiota in men who have sex with men and are living with HIV in Northern TaiwanPLOS ONE

Dear Dr. Chu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. The reviewer comments were sufficiently addressed. However, PLOS One publication criteria emphasize the need for high standard and rigor in data analysis and statistics.Therefore the problems with potentially inappropriately performed statistics and uncertain p values derived from this analysis must be addressed.

Please submit your revised manuscript by Nov 01 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Ivan Sabol

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

The material and methods revisions are appreciated and improved the clarity of the manuscript

Some small methodological problems remain

P7 Line 143-144 grammatically incorrect and possibly misplaced. Batch effect would be compensated after trimming and denoising.

Page 8 Line 173 statistical analysis section

The authors reply “Line 170: We used the Wilcoxon test, considering it as a better alternative to the ttest when the normal distribution of the differences between paired individuals cannot be assumed.“

However, there are several issues with this revision.

1)The revision deleted the mention of t-test from Statistical analysis section at line 177 leaving only chi-square test mentioned

2) The Figure 2 p values, the Figure 2 legend, or the text at P12 L227 remains unchanged suggesting that it still shows the old t-test individual p values. The test used to obtain the p values is undeclared and inconsistent with the paragraph Statistical analysis

3) The comparisons between the six groups presented at Figure 2 cannot be considered paired individuals and Wilcoxon test cannot be considered as appropriate for these comparisons

P12 Table 2 footer would benefit from listing the 12 other HPV types as a footnote

Supplementary table 1 was uploaded twice. (once as Table_S1.docx and once as S1_table.docx) on pages 40 and 41 of the combined PDF

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

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Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

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Reviewer #2: (No Response)

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PLoS One. 2024 Dec 31;19(12):e0304045. doi: 10.1371/journal.pone.0304045.r004

Author response to Decision Letter 1

3 Oct 2024

Response to reviewers

Comment: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Reply: We have reviewed and checked the reference list.

Comment: P7 Line 143-144 grammatically incorrect and possibly misplaced. Batch effect would be compensated after trimming and denoising.

Reply: We have corrected the grammar and revised the sentence to “Batch effects were compensated after trimming and denoising” and moved it to an appropriate location on page 7, line 150.

Comment: Page 8 Line 173 statistical analysis section

However, there are several issues with this revision.

1)The revision deleted the mention of t-test from Statistical analysis section at line 177 leaving only chi-square test mentioned

3) The comparisons between the six groups presented at Figure 2 cannot be considered paired individuals and Wilcoxon test cannot be considered as appropriate for these comparisons.

Reply: We apologize for the confusion regarding the statistical methods. To clarify:

1) The p-values in the figures were derived from Wilcoxon tests (the default method in the analysis package). Student t-tests were never used in this study. We have now explicitly mentioned the use of the Wilcoxon–Mann–Whitney test at both line 173 (in the "16s rRNA gene sequence analysis" section) and line 170 of the Statistical analysis section.

2) We have corrected Figure 2, its legend, and the text at P12 L227 to reflect the use of Wilcoxon tests. The statistical methods are now explicitly stated in the Figure 2 legend and at P13 L243.

3) In Figure 2, we have shown a comparison of group 1 (PLWH with CD4+ T cell counts < 500 cells/µL, ASCUS+, and detectable HPV 16/18) with each of the other groups individually using the Wilcoxon test, rather than comparing all six groups simultaneously. The Kruskal–Wallis test was not used.

Comment: P12 Table 2 footer would benefit from listing the 12 other HPV types as a footnote

Reply: We have added the 12 additional HPV types as a footnote in Table 2.

Comment: Supplementary table 1 was uploaded twice. (once as Table_S1.docx and once as S1_table.docx) on pages 40 and 41 of the combined PDF

Reply: Thank you for pointing this out. We have corrected the duplication of Supplementary Table 1.

Attachment

Submitted filename: Response to reviewers.docx

pone.0304045.s005.docx^{(21.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0304045.r005

Decision Letter 2

Ivan Sabol

8 Oct 2024

Oncogenic human papillomavirus and anal microbiota in men who have sex with men and are living with HIV in Northern Taiwan

PONE-D-24-17510R2

Dear Dr. Chu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Ivan Sabol

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Results of the permutational multivariate analysis of variance (adonis function) and the multivariate homogeneity of group dispersions analysis (betadisper function).

(DOCX)

pone.0304045.s001.docx^{(20.3KB, docx)}

S1 File. Questionnaire for enrolled participants.

(PDF)

pone.0304045.s002.pdf^{(61.8KB, pdf)}

Attachment

Submitted filename: gabri.DOCX

pone.0304045.s003.DOCX^{(15.6KB, DOCX)}

Attachment

Submitted filename: Response_to_Reviewers.docx

pone.0304045.s004.docx^{(45.9KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

pone.0304045.s005.docx^{(21.2KB, docx)}

Data Availability Statement

The data that support the findings of this study are available in the Figshare database, doi: 10.6084/m9.figshare.25375087.

[pone.0304045.ref001] 1.zur Hausen HA. The role of papillomaviruses in anogenital cancer. Scand J Infect Dis Suppl. 1990;69: 107–111. [PubMed] [Google Scholar]

[pone.0304045.ref002] 2.de Martel C, Georges D, Bray F, Ferlay J, Clifford GM. Global burden of cancer attributable to infections in 2018: a worldwide incidence analysis. Lancet Glob Health. 2020;8: e180–e190. doi: 10.1016/S2214-109X(19)30488-7 [DOI] [PubMed] [Google Scholar]

[pone.0304045.ref003] 3.Clifford GM, Georges D, Shiels MS, Engels EA, Albuquerque A, Poynten IM, et al. A meta-analysis of anal cancer incidence by risk group: toward a unified anal cancer risk scale. Int J Cancer. 2021;148: 38–47. doi: 10.1002/ijc.33185 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref004] 4.Cheng SH, Chu FY, Lin YS, Hsueh YM. Influence of Age and CD4+ T cell counts on the prevalence of genital human papillomavirus infection among HIV-seropositive men who have sex with men in Taiwan. J Med Virol. 2012;84: 1876–1883. doi: 10.1002/jmv.23413 [DOI] [PubMed] [Google Scholar]

[pone.0304045.ref005] 5.Jin F, Vajdic CM, Law M, Amin J, van Leeuwen M, McGregor S, et al. Incidence and time trends of anal cancer among people living with HIV in Australia. AIDS. 2019;33: 1361–1368. doi: 10.1097/QAD.0000000000002218 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref006] 6.Jones MK, Watanabe M, Zhu S, Graves CL, Keyes LR, Grau KR, et al. Enteric bacteria promote human and mouse Norovirus infection of B cells. Science. 2014;346: 755–759. doi: 10.1126/science.1257147 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref007] 7.Kaul D, Rathnasinghe R, Ferres M, Tan GS, Barrera A, Pickett BE, et al. Microbiome disturbance and resilience dynamics of the upper respiratory tract during influenza A virus infection. Nat Commun. 2020;11: 2537. Erratum in: Nat Commun. 2020;11: 3132. doi: 10.1038/s41467-020-16429-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref008] 8.Mizutani T, Ishizaka A, Koga M, Ikeuchi K, Saito M, Adachi E, et al. Correlation analysis between gut microbiota alterations and the cytokine response in patients with coronavirus disease during hospitalization. Microbiol Spectr. 2022;10: e0168921. doi: 10.1128/spectrum.01689-21 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref009] 9.Komiyama S, Yamada T, Takemura N, Kokudo N, Hase K, Kawamura YI. Profiling of tumour-associated microbiota in human hepatocellular carcinoma. Sci Rep. 2021;11: 10589. doi: 10.1038/s41598-021-89963-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref010] 10.Liu Q, Li F, Zhuang Y, Xu J, Wang J, Mao X, et al. Alteration in gut microbiota associated with hepatitis B and non-hepatitis virus related hepatocellular carcinoma. Gut Pathog. 2019;11: 1. doi: 10.1186/s13099-018-0281-6 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref011] 11.Inoue T, Nakayama J, Moriya K, Kawaratani H, Momoda R, Ito K, et al. Gut dysbiosis associated with hepatitis C virus infection. Clin Infect Dis. 2018;67: 869–877. doi: 10.1093/cid/ciy205 [DOI] [PubMed] [Google Scholar]

[pone.0304045.ref012] 12.So KA, Yang EJ, Kim NR, Hong SR, Lee JH, Hwang CS, et al. Changes of vaginal microbiota during cervical carcinogenesis in women with human papillomavirus infection. PLOS ONE. 2020;15: e0238705. doi: 10.1371/journal.pone.0238705 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref013] 13.Chen Y, Qiu X, Wang W, Li D, Wu A, Hong Z, et al. Human papillomavirus infection and cervical intraepithelial neoplasia progression are associated with increased vaginal microbiome diversity in a Chinese cohort. BMC Infect Dis. 2020;20: 629. doi: 10.1186/s12879-020-05324-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref014] 14.Neff CP, Krueger O, Xiong K, Arif S, Nusbacher N, Schneider JM, et al. Fecal microbiota composition drives immune activation in HIV-infected individuals. EBiomedicine. 2018;30: 192–202. doi: 10.1016/j.ebiom.2018.03.024 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref015] 15.Cook RR, Fulcher JA, Tobin NH, Li F, Lee D, Javanbakht M, et al. Effects of HIV viremia on the gastrointestinal microbiome of young MSM. AIDS. 2019;33: 793–804. doi: 10.1097/QAD.0000000000002132 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref016] 16.Castellarin M, Warren RL, Freeman JD, Dreolini L, Krzywinski M, Strauss J, et al. Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Res. 2012;22: 299–306. doi: 10.1101/gr.126516.111 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref017] 17.Yu J, Feng Q, Wong SH, Zhang D, Liang QY, Qin Y, et al. Metagenomic analysis of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer. Gut. 2017;66: 70–78. doi: 10.1136/gutjnl-2015-309800 [DOI] [PubMed] [Google Scholar]

[pone.0304045.ref018] 18.Mima K, Nishihara R, Qian ZR, Cao Y, Sukawa Y, Nowak JA, et al. Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis. Gut. 2016;65: 1973–1980. doi: 10.1136/gutjnl-2015-310101 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref019] 19.Nieves-Ramírez ME, Partida-Rodríguez O, Moran P, Serrano-Vázquez A, Pérez-Juárez H, Pérez-Rodríguez ME, et al. Cervical squamous intraepithelial lesions are associated with differences in the vaginal microbiota of Mexican women. Microbiol Spectr. 2021;9: e0014321. doi: 10.1128/Spectrum.00143-21 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref020] 20.Kyrgiou M, Mitra A, Moscicki AB. Does the vaginal microbiota play a role in the development of cervical cancer? Transl Res. 2017;179: 168–182. doi: 10.1016/j.trsl.2016.07.004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref021] 21.Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SSK, McCulle SL, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A. 2011;108;Supplement 1: 4680–4687. doi: 10.1073/pnas.1002611107 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref022] 22.Łaniewski P, Cui H, Roe DJ, Barnes D, Goulder A, Monk BJ, et al. Features of the cervicovaginal microenvironment drive cancer biomarker signatures in patients across cervical carcinogenesis. Sci Rep. 2019;9: 7333. doi: 10.1038/s41598-019-43849-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref023] 23.Williams VM, Filippova M, Filippov V, Payne KJ, Duerksen-Hughes P. Human papillomavirus type 16 E6* induces oxidative stress and DNA damage. J Virol. 2014;88: 6751–6761. doi: 10.1128/JVI.03355-13 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref024] 24.Piyathilake CJ, Ollberding NJ, Kumar R, Macaluso M, Alvarez RD, Morrow CD. Cervical microbiota associated with higher grade cervical intraepithelial neoplasia in women infected with high-risk human papillomaviruses. Cancer Prev Res (Phila). 2016;9: 357–366. doi: 10.1158/1940-6207.CAPR-15-0350 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref025] 25.Gillet E, Meys JF, Verstraelen H, Bosire C, De Sutter P, Temmerman M, et al. Bacterial vaginosis is associated with uterine cervical human papillomavirus infection: a meta-analysis. BMC Infect Dis. 2011;11: 10. doi: 10.1186/1471-2334-11-10 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref026] 26.Curty G, Costa RL, Siqueira JD, Meyrelles AI, Machado ES, Soares EA, et al. Analysis of the cervical microbiome and potential biomarkers from postpartum HIV-positive women displaying cervical intraepithelial lesions. Sci Rep. 2017;7: 17364. doi: 10.1038/s41598-017-17351-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref027] 27.Chávez-Torres M, Gómez-Palacio-Schjetnan M, Reyes-Terán G, Briceño O, Ávila-Ríos S, Romero-Mora KA, et al. The vaginal microbiota of women living with HIV on suppressive antiretroviral therapy and its relation to high-risk human papillomavirus infection. BMC Microbiol. 2023;23: 21. doi: 10.1186/s12866-023-02769-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref028] 28.Serrano-Villar S, Vásquez-Domínguez E, Pérez-Molina JA, Sainz T, de Benito A, Latorre A, et al. HIV, HPV, and microbiota: partners in crime? AIDS. 2017;31: 591–594. doi: 10.1097/QAD.0000000000001352 [DOI] [PubMed] [Google Scholar]

[pone.0304045.ref029] 29.Nowak RG, Bentzen SM, Ravel J, Crowell TA, Dauda W, Ma B, et al. Anal microbial patterns and oncogenic human papillomavirus in a pilot study of Nigerian men who have sex with men at risk for or living with HIV. AIDS Res Hum Retroviruses. 2019;35: 267–275. doi: 10.1089/AID.2018.0158 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref030] 30.Taiwan Centers for Disease Control. Statistics of communicable diseases and surveillance reports in Taiwan area; 2023. Taiwan: Ministry of Health and Welfare. p. 2017. [Cited 31 December 2023]. Available from: https://www.cdc.gov.tw/Category/Page/rCV9N1rGUz9wNr8lggsh2Q. [Google Scholar]

[pone.0304045.ref031] 31.Solomon D, Davey D, Kurman R, Moriarty A, O’Connor D, Prey M, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA. 2002;287: 2114–2119. doi: 10.1001/jama.287.16.2114 [DOI] [PubMed] [Google Scholar]

[pone.0304045.ref032] 32.Illumina support center. 16S Metagenomic Sequencing Library preparation; 2013. San Diego: Illumina Inc. [Cited 31 December 2023] Available from: https://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.html. [Google Scholar]

[pone.0304045.ref033] 33.Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15: 550. doi: 10.1186/s13059-014-0550-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref034] 34.Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal. 2011;17: 10–12. doi: 10.14806/ej.17.1.200 [DOI] [Google Scholar]

[pone.0304045.ref035] 35.Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13: 581–583. doi: 10.1038/nmeth.3869 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref036] 36.Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41: D590–D596. doi: 10.1093/nar/gks1219 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref037] 37.Wright ES. DECIPHER: harnessing local sequence context to improve protein multiple sequence alignment. BMC Bioinformatics. 2015;16: 322. doi: 10.1186/s12859-015-0749-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref038] 38.Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30: 1312–1313. doi: 10.1093/bioinformatics/btu033 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref039] 39.McMurdie PJ, Holmes S. Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLOS ONE. 2013;8: e61217. doi: 10.1371/journal.pone.0061217 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref040] 40.Chen J, Bittinger K, Charlson ES, Hoffmann C, Lewis J, Wu GD, et al. Associating microbiome composition with environmental covariates using generalized UniFrac distances. Bioinformatics. 2012;28: 2106–2113. doi: 10.1093/bioinformatics/bts342 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref041] 41.Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, et al. Metagenomic biomarker discovery and explanation. Genome Biol. 2011;12: R60. doi: 10.1186/gb-2011-12-6-r60 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref042] 42.Asnicar F, Weingart G, Tickle TL, Huttenhower C, Segata N. Compact graphical representation of phylogenetic data and metadata with GraPhlAn. PeerJ. 2015;3: e1029. doi: 10.7717/peerj.1029 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref043] 43.Lee JE, Lee S, Lee H, Song YM, Lee K, Han MJ, et al. Association of the vaginal microbiota with human papillomavirus infection in a Korean twin cohort. PLOS ONE. 2013;8: e63514. doi: 10.1371/journal.pone.0063514 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref044] 44.Ron R, Cabello A, Gosalbes MJ, Sánchez-Conde M, Talavera-Rodríguez A, Zamora J, et al. Exploiting the microbiota for the diagnosis of anal precancerous lesions in men who have sex with men. J Infect Dis. 2021;224: 1247–1256. doi: 10.1093/infdis/jiab068 [DOI] [PubMed] [Google Scholar]

[pone.0304045.ref045] 45.Gootenberg DB, Paer JM, Luevano JM, Kwon DS. HIV-associated changes in the enteric microbial community: potential role in loss of homeostasis and development of systemic inflammation. Curr Opin Infect Dis. 2017;30: 31–43. doi: 10.1097/QCO.0000000000000341 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref046] 46.Dinh DM, Volpe GE, Duffalo C, Bhalchandra S, Tai AK, Kane AV, et al. Intestinal microbiota, microbial translocation, and systemic inflammation in chronic HIV infection. J Infect Dis. 2015;211: 19–27. doi: 10.1093/infdis/jiu409 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref047] 47.Dubourg G, Lagier JC, Hüe S, Surenaud M, Bachar D, Robert C, et al. Gut microbiota associated with HIV infection is significantly enriched in bacteria tolerant to oxygen. BMJ Open Gastroenterol. 2016;3: e000080. doi: 10.1136/bmjgast-2016-000080 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref048] 48.Klein C, Samwel K, Kahesa C, Mwaiselage J, West JT, Wood C, et al. Mycoplasma co-infection is associated with cervical cancer risk. Cancers (Basel). 2020;12: 1093. doi: 10.3390/cancers12051093 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref049] 49.Hiippala K, Kainulainen V, Kalliomäki M, Arkkila P, Satokari R. Mucosal prevalence and interactions with the epithelium indicate commensalism of Sutterella spp. Front Microbiol. 2016;7: 1706. doi: 10.3389/fmicb.2016.01706 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0304045.ref050] 50.Meng J, Tao J, Abu Y, Sussman DA, Girotra M, Franceschi D, et al. HIV-positive patients on antiretroviral therapy have an altered mucosal intestinal but not oral microbiome. Microbiol Spectr. 2023;11: e0247222. doi: 10.1128/spectrum.02472-22 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Oncogenic human papillomavirus and anal microbiota in men who have sex with men and are living with HIV in Northern Taiwan

Shu-Hsing Cheng

Yu-Chen Yang

Cheng-Pin Chen

Hui-Ting Hsieh

Yi-Chun Lin

Chien-Yu Cheng

Kuo-Sheng Liao

Fang-Yeh Chu

Yun-Ru Liu

Roles

Abstract

Introduction

Materials and methods

Study participants

Anal Pap smears

Human papillomavirus detection

Targeted amplicon library preparation and sequencing

16S rRNA gene sequence analysis

Statistical analyses

Results

Table 1. Demographic characteristics, behavior risks, and immunologic factors for the study cohort of men who have sex with men and live with HIV in northern Taiwan.

Table 2. Oncogenic HPV distribution among 291 participants who are MSM-LWH.

Fig 1. Grouping of anal samples according to cytology, HPV 16/18, and CD4+ T cell counts.

Fig 2. Alpha diversity comparing group 1 and groups 2, 3, 4, 5, and 6.

Fig 3. Beta diversity comparing group 1 and groups 2, 3, 4, 5, and 6.

Fig 4. Linear discriminant analysis effect size comparing group 1 and groups 2, 3, 4, 5, and 6 and cladogram.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Ivan Sabol

Roles

Author response to Decision Letter 0

Decision Letter 1

Ivan Sabol

Roles

Author response to Decision Letter 1

Decision Letter 2

Ivan Sabol

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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