Altered Oral Salivary Microbiome In The Early Stage Of HIV Infections

HIV continues to be a major global public health issue.Opportunistic oral infections are the earliest symptoms of HIV infections,and can be as an indicator for dentists to suspect HIV infection clinically.Previous studies have revealed that after infection with HIV,both the immune system and the saliva microbiome experience specific alterations that,in turn,exacerbate oral infections.Several blood indices are the important parameters for the measurement of immune function,the guidance of clinical medication,the determination of disease progression and the evaluation of the curative effect.However,blood tests are invasive and dangerous which are not easy to accept by most patients.Hence,it is indispensable and meaningful to find a biomarker that is easily and noninvasively accessable.Saliva is a diversified biological oral fluid with an abundance of human oral microbes,is easy and noninvasive to collect.Therefore,we plan to explore the dynamic alterations in salivary microbiome before and after antiretroviral therapy（ART）of HIV-infected individuals using high-throughput sequencing technology,and then to analyze the correlation between the salivary microbiome and immunologic factors of the blood.Finally,we aim to find an easily and noninvasively salivary biomarker that can indicate the immune status and predict the prognosis of HIV infections.Part 1.Comparative analysis of salivary microbiome between HIV infections before ART and HIV-uninfected individualsObjective: we aimed to detect the differences of salivary microbiome composition and structure between HIV infections and HIV-uninfected individuals,and to find salivary biomarker for HIV infections.Methods: 20 HIV newly infected patients before the initiation of ART and 20 ageand gender-paired healthy Chinese people were enrolled.Samples of unstimulated whole saliva were collected and total bacterial DNA from saliva samples wasextracted.The genomic DNA was PCR amplified using bacteria 16 S r RNA gene primers targeting the V3–V4 hypervariable regions and then run on the Illumina Mi Seq platform.Finally,we analysed the alterations of salivary microbiome between HIV-infected and HIV-uninfected individuals at different taxonomic levels.Results: 1.A total of 1,845,904 high-quality sequences was generated after quality checks and 27 phyla,47 classes,89 orders,155 families,367 genera,and 671 species were identified from the filtered sequences.2.Firmicutes,Bacteroidetes,Proteobacteria,Actinobacteria,Fusobacteria followed by Saccharibacteria and Spirochaetes constituted over 98% of the total salivary microbiota.3.The alpha diversity of salivary microbiome in HIV infections did not show differences from the HIV uninfected individuals.4.Comparative analysis showed that at the phylum level,Spirochetes increased significantly（P=0.01829）and the Proteobacteria significantly decreased（P=0.006218）in HIV-infected individuals;at the genus level,Streptococcus was significantly higher in HIV-infected individuals（P=0.00114）,while Neisseria was significantly reduced（P=0.001959）.5.LEf Se demonstrated that Streptococcus,Prevotella₁,Filifactor,Olsenella,Acholeplasma and Fretibacterium were enriched in HIV-infected,whereas Neisseria and Bosea was enriched in the healthy controls.Conclusions: Salivary microbiome dysbiosis occured in the early stage of HIV infections,Streptococcus increased and Neisseria decreased.Streptococcus and Neisseria are potential salivary biomarkers for HIV infections.Part 2.Explore the dynamic alterations in salivary microbiome of HIV infections before and after ART and the correlation between salivary microbiome and blood immunologic factorsObjective: we aimed to explore the dynamic alterations in salivary microbiome of HIV infections before and after ART and the correlation between salivary microbiome and blood immunologic factors,and then to find an easily and noninvasively salivary biomarker that can indicate the immune status and predict the prognosis of HIV infections.Methods: A longitudinal study was applied to explore the dynamic alterations in salivary microbiomes of the HIV infections at three and six months after the initiation of the ART.Samples of unstimulated whole saliva were collected and total bacterial DNA from saliva samples was extracted.The genomic DNA was PCR amplified using bacteria 16 S r RNA gene primers targeting the V3–V4 hypervariable regions and then run on the Illumina Mi Seq platform.We analyzed the alterations of salivary microbiome before and after ART in HIV infections as well as the HIV-uninfected individuals at different taxonomic levels.We also explored the correlation between salivary microbiome and CD4+ T cell count and VL.Results: 1.A total of 3,440,992 high-quality sequences was generated after quality checks and 31 phyla,56 classes,113 orders,189 families,451 genera,and 797 species were identified from the filtered sequences.2.After ART for three months,the alpha diversity of salivary microbiome in HIV infections did not show difference but was reduced after six months under ART treatment.3.Comparative analysis showed there was no obvious alteration in the salivary microbiome after ART for three months.However,after ART for six months the salivary microbiome changed significantly compared with the HIVinfectios before ART.At the phylum level,Proteobacteria increased significantly（P=0.045）and the Bacteroidetes significantly decreased（P=0.045）in HIV-infected individuals;at the genus level,nornank_fCaulobacterace was significantly increased（P=0.0006728）,while Actinomyces and Alloprevotella were significantly reduced（P=0.03389,P=0.01079）.4.After ART,most of the salivary microbiome in HIV-infected patients recovered gradually,but not completely restored.5.After effective ART,CD4+ T cell count increased and VL decreased.In addition,we found that Provotella₇,Neisseria and Haemophilus were correlated negatively with CD4+ T cell count（r =-0.4797,P=0.03233;r =-0.4572,P=0.04272;r =-0.4889,P=0.02877）,while Neisseria wascorrelated positively with VL（r = 0.4511,P=0.04588）.Conclusions: Salivary microbiome dysbiosis occured in the early stage of HIV infections,Streptococcus increased and Neisseria decreased.Streptococcus and Neisseria are potential salivary biomarkers for HIV infections.