Female EcologiXTM Vaginal Health & Microbiome Profile Phylo Bioscience Laboratory

INTERPRETIVE GUIDE

DISCLAIMER: THIS INFORMATION IS PROVIDED FOR THE USE OF PHYSICIANS AND OTHER LICENSED HEALTH CARE PRACTITIONERS ONLY. THIS INFORMATION IS NOT FOR USE BY CONSUMERS. THE INFORMATION AND OR PRODUCTS ARE NOT INTENDED FOR USE BY CONSUMERS OR PHYSICIANS AS A MEANS TO CURE, TREAT, PREVENT, DIAGNOSE OR MITIGATE ANY DISEASE OR OTHER MEDICAL CONDITION. THE INFORMATION CONTAINED IN THIS DOCUMENT IS IN NO WAY TO BE TAKEN AS PRESCRIPTIVE NOR TO REPLACE THE PHYSICIANS DUTY OF CARE AND PERSONALISED CARE PRACTICES. INTERPRETIVE GUIDE

Female EcologiX™

INTRODUCTION

Due to recent advancements in culture-independent molecular techniques, it is now possible to measure the composition of the human microbiota. One key microbiota site is the vaginal tract, which hosts over 200 species of microorganisms1. The vaginal microbiota is a dynamic and complex ecosystem comprised of fluctuating populations that modulate host immune responses and maintain homeostasis. Disruption of microbiota composition and functions, termed dysbiosis, has been linked to a multitude of disorders including (BV), premature delivery in pregnant women, infertility, miscarriages and increased risk of sexually transmitted infections (STIs). Analysis and monitoring of the vaginal microbiota is therefore extremely important for health and can be used to inform treatment plans and lifestyle factors.

To meet this need, Phylobioscience have developed Female EcologiXTM Vaginal Health and Microbiome Profile, a ground breaking tool for analysis of vaginal microbiota composition and host immune responses. Using innovative microbial culture- independent technologies, including quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assays (ELISA), the profile provides an accurate, reliable and quantifiable measurement of microbiota abundance and markers for host immune responses.

For microbiota composition analysis, the technology detects: • Abundance of four key species (markers for vaginal health) • Abundance of strictly anaerobic (associated with dysbiosis) • Abundance of enteric bacteria including Escherichia coli and Staphylococcus aureus (associated with aerobic ) • Presence of STI pathogen trachomatis • Presence of STI viral pathogens type 1 (HSV-1) and HSV-2 (genital herpes) • Abundance of BV-associated bacteria including Gardenella, and Megasphaera species

For host biomarker analysis, the technology detects: • I L-1 β (proinflammatory cytokine)

Dependent on microbiota composition and host biomarkers, the profile will report three different states: • Healthy vaginal microbiota (homeostasis) • Vaginal dysbiosis (microbiota profile and/or host marker profile) • STI pathogen detected (Chlamydia, HSV-1, HSV-2)

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GLOSSARY

Term Description References Commensal Microorganism (i.e. bacteria, fungi) that lives in symbiosis with host when residing within 2 its specific environment Pathogen Microorganism (e.g. bacteria, fungi, virus) that may cause disease 3 Pathobiont Potential pathogen that lives in symbiosis under normal conditions 4 Homeostasis Ability to maintain internal stability in an organism despite environmental changes

Dysbiosis Imbalance or disturbance in the human microbiota 5 Microbiota Collective ecosystem of microorganisms that inhabit the human body 1 Culture-independent Experimental scientific techniques that do not require cultivation of microbes (i.e. PCR, 6 techniques DNA sequencing) Community-state type Vaginal microbiota profile based on species ofLactobacillus that dominates 1 Lipopolysaccharide (LPS) LPS, also known as endotoxin is a component of the outer membrane of Gram- 7 negative bacteria Toll-like receptors (TLRs) Toll-like receptors are pattern recognition receptors (PRRs) that sense invading 8 pathogens or endogenous damage signals Aerobic vaginitis Aerobic vaginitis (AV) is a state of vaginal dysbiosis that is distinct from bacterial 9 vaginosis (BV) Cytolytic vaginosis Lactobacillus overgrowth syndrome resulting in lysis of vaginal epithelial cells 10 Quantitative real-time Molecular technique that is used to quantify the number of copies of the gene of 11 PCR interest in a community sample (i.e. vaginal, gut) Enzyme-linked Immunological assay used to measure antibodies, proteins, antigens and glycoproteins 12 immunosorbent assays in biological samples (ELISA) IL-1β Pro-inflammatory cytokine associated with stress,injury, and infection 14 Bacteriocins Antimicrobial compounds produced by bacteria that kill or inhibit other bacteria 15

Table 1: Glossary of terms

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BACKGROUND

Vaginal Microbiota The human body is colonised by a multitude of microorganisms collectively referred to as the human microbiota. In females, one of the main microbiota sites is the genital tract that hosts over 200 bacterial species. Unlike other microbiota sites, the vagina is extremely dynamic and is affected by numerous host factors including age, ethnic background, sexual intercourse, changes in hormone levels during pregnancy, menstruation and menopause, genital infections and personal hygiene16. Due to its extensive interaction and cross-talk with host immune responses, small fluctuations in the vaginal microbiota can impact vaginal health significantly. The disruption of homeostasis, termed dysbiosis, has been linked to a multitude of conditions, including bacterial vaginosis (BV), miscarriages, preterm delivery, pelvic inflammatory disease (PID) and increased risk of sexually transmitted infections (STIs).

Homeostasis Detailed composition of the vaginal microbiota has been defined through high-throughput 16S rRNA gene sequencing. A cross-sectional study using 394 healthy women from four ethnic groups classified the vaginal microbiota into five microbial communities, termed community state types (CST), which are generally dominated by Lactobacillus spp.1. Briefly, CST I, II, III and V are dominated by Lactobacillus species (, Lactobacillus gasseri, and Lactobacillus jensenii, respectively) and CST IV has no dominant species and is classified as the diverse group, with higher numbers of strictly anaerobic bacteria and BV-associated bacteria such as Prevotella, Gardnerella, Aerococcus, Finegoldia, and Mobiluncus 1.

Lactobacilli are extremely important for vaginal health due to their protective and antimicrobial functions. Lactobacilli produce lactic acid, creating an acidic environment (pH 2.8–4.2) that is inhospitable to many non-Lactobacillus commensals and potential vaginal pathogens17,18. For example, reduced infectivity of by L. crispatus, via lactic acid production and acidic pH, has been reported in vitro19. Lactic acid also induces autophagy in epithelial cells to degrade intracellular microorganisms and promote homeostasis20. In addition, some Lactobacillus species also produce antimicrobial compounds such as bacteriocins that inhibit growth of pathogenic microorganisms such as Candida albicans, Prevotella bivia and E. coli21.

In addition to their direct role in regulating vaginal health, Lactobacilli also modulate the host immune response, mediated through vaginal epithelial cells22,23. Lactobacilli are tolerated by vaginal epithelial cells and inhibit induction of proinflammatory cytokines such as IL-6, IL-1β and TNF-α24. Vaginal lactic acid has also been shown to mediate an antiinflammatory response in the presence of inflammatory inducing pathogens25. Dominance of Lactobacillus spp. is therefore a good marker for vaginal health and indicates functional host-microbial interactions.

Dysbiosis Deviations from a healthy microbiota composition or homeostasis, termed dysbiosis, has been linked to pelvic inflammatory disease26 preterm labour27 and miscarriages28. Vaginal dysbiosis is often defined as a prolonged deviation from a low-diversity, Lactobacilli-dominated vaginal microbiota29. The most common dysbiosis of the vaginal microbiota is bacterial vaginosis (BV), an anaerobic polymicrobial dysbiosis29,30. BV-associated dysbiosis typically includes increased Gardnerella vaginalis, Atopobium vaginae and other anaerobes including Megasphaera spp., Dialister spp., Prevotella spp., Mobiluncus spp. with, or without, a low relative abundance of L. iners31,32. CST IV microbiota profiles show increased dominance of these BV-associated anaerobic bacteria. However, as the CST IV profile is detected in healthy women, recent studies now support a role for polymicrobial interactions, biofilm formation and host immune responses in the aetiology of BV5,33.

Clinical signs of BV include malodour, a creamy grey discharge and an elevated vaginal pH, linked to reduced lactic acid- production31. Malodour, has been linked to increased vaginal biogenic amines (BAs), including putrescine, cadaverine, and trimethylamine34, that are often produced by BV-associated anaerobic bacteria. In addition, BV associated bacteria produce little lactic acid, but produce several immunomodulatory substances, including succinate, sialydases and proteases, as well as proinflammatory substances such as lipopolysaccharides (LPS) and IL-835. These microbial products induce inflammation

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through Toll-like receptors (TLR), that are present on immune cells and other cells in the female lower genital tract. In addition, BV is also associated with increased risk of infection by STI pathogens, including Chlamydia trachomatis and Neisseria gonorrhoeae36, increased risk of HIV infection, PID and preterm delivery in pregnant women37,38. Typically BV is treated with broad-spectrum antibiotics, such as metronidazole; however, studies with extended follow-up show that recurrence rates in excess of 50% occur within 6–12 months5.

Other types of dysbiosis linked to perturbed Lactobacilli populations include aerobic vaginosis (AV), an inflammatory type of dysbiosis associated with growth of one or two commensal enteric bacteria including Streptococcus agalactiae, Staphylocuccus aureus, or Escherichia coli with reduced Lactobacilli39. Cytolytic vaginosis (CV), also termed Lactobacillus overgrowth syndrome, is an additional type of dysbiosis and cause of . Clinical symptoms include pruritus, and vulvar dysuria. Interestingly, CV patients show extremely high levels of Lactobacillus spp. compared to healthy women, alongside absence of Trichomonas spp., Gardnerella vaginalis, Atopobium vaginae, Megasphaera spp., Sneathia spp., and Prevotella spp.10. Furthermore, CV patients also show vaginal pH of <3.8 and increased prevalence of L. crispatus.10

Figure 1: Dysbiosis in the vaginal microbiota. Disruption of vaginal Lactobacillus species may result in colonisation and overgrowth of strictly anaerobic bacteria including Prevotella bivia and Gardnerella. Overgrowth of strict anaerobes results in increased levels of proinflammatory cytokines, reduced production of lactic acid, reduced production of antimicrobial peptides and thus, pathogen colonisation and epithelial injury. Adapted from Aldunate et al (2015)24.

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METHODOLOGY

The Phylobioscience methodology is comprised of two key culture-independent techniques: quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assays (ELISA). These techniques are used to quantify abundance of vaginal microbiota species and host immune biomarkers. The selected microbiota species and host biomarkers are based on clinically relevant research. qRT-PCR FOR QUANTIFICATION OF VAGINAL MICROBIOTA SPECIES

The Female EcologiX™ profile utilises quantitative real-time PCR (qRT-PCR) for analysis of vaginal microbiota populations. qRT-PCR is used to quantify the number of copies of the gene of interest in a community sample (i.e. vaginal, gut). qRT-PCR reactions are performed using Taqman technology.

ELISA ASSAY FOR MEASUREMENT OF PROINFLAMMATORY MARKERS

The Phylobioscience Female EcologiX™ vaginal microbiome kit utilises enzyme-linked immunosorbent assays (ELISA) for quantification of host proteins. The ELISA assay is an immunological assay used to measure antibodies, proteins, antigens and glycoproteins in biological samples. ELISA plates are coated with a capture antibody that is raised against the antigen of interest (e. g. I L-1 β). A standard curve of known concentrations of specific antigen is used to accurately quantify the concentration of antigen present in the biological sample.

METHODOLOGY FLOWCHART

Vaginal Microbiome Testing Kit Methodology. Samples are processed for preparation of DNA and supernatant for qRT-PCR and ELISA respectively.

Vaginal sample

→ →

Supernatant DNA extraction preparation → →

qRT-PCR ELISA → →

Microbiota Host immune composition and response profile abundance

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BIOMARKERS

BIOMARKER TYPE CLASSIFICATION

HOST IMMUNE MARKERS IL-1β Proinflammatory cytokine N/A pH Vaginal pH N/A

MICROBIOTA Commensals Lactobacillus crispatus Bacteria Gram-positive Lactobacillus iners Bacteria Gram-positive Lactobacillus jensenii Bacteria Gram-positive Lactobacillus gasseri Bacteria Gram-positive Atopobium vaginae Bacteria Gram-positive Prevotella bivia Bacteria Gram-positive Megasphera 1 Bacteria Gram-positive Megasphera 2 Bacteria Gram-positive Gardnerella vaginalis Bacteria Gram-variable Mobiluncus mulieris Bacteria Gram-variable Mobiluncus curtisii Bacteria Gram-variable Bacteria Gram-negative BVAB2 Bacteria Unculturable bacteria

Pathobionts Streptococcus agalactiae (group B) Bacteria Gram-positive Staphylococcus aureus Bacteria Gram-positive Enterococcus faecalis Bacteria Gram-positive Bacteroides fragilis Bacteria Gram-negative Escherichia coli Bacteria Gram-negative

STI Pathogens HSV-1 Virus N/A HSV-2 Virus N/A Chlamydia trachomatis Bacteria Gram-negative Mycoplasma genitalium Bacteria Not visible by Gram-staining N/A

Opportunistic Fungal Pathogens Candida albicans Fungi N/A Candida krusei Fungi N/A Candida parapsilosis Fungi N/A Candida tropicalis Fungi N/A

Table 2: Vaginal kit biomarkers. Commensals: Species that are normally associated with the healthy vaginal microbiota. Pathobionts: Enteric bacteria that have potential to cause pathogenesis in the vagina. STI Pathogens: Causative agents of sexually transmitted infections and pathogenesis in the genital tract. Opportunistic Fungal Pathogens: Commensal fungal species that can cause pathogenesis if provided with opportunity (overgrowth, elevated pH, dysbiosis).

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INTERPRETATION OF DATA

Vaginal pH

Vaginal pH HEALTHY: pH 3.8-4.5 DYSBIOSIS: pH >4.5

The healthy range for vaginal pH is between 3.8-4.5. Low vaginal pH indicates high levels of lactic acid production by Lactobacillus species and therefore healthy vaginal microbiota and minimal dysbiosis. Low vaginal pH reduces growth of BV-associated bacteria and STI pathogens (e.g. Chlamydia trachomatis) and favours dominance by Lactobacilli. High vaginal pH (>4.5) is indicative of overgrowth of BV-associated bacteria and vaginal dysbiosis. Please review the host biomarkers and the microbiota composition to assess the level of dysbiosis.

Host Biomarkers

IL-1β HEALTHY: <220 pg/ml HIGH: >220 pg/ml I L-1 β is a key mediator of the inflammatory response and a master cytokine that regulates induction of other cytokines, including I L- 8 37. Produced by innate immune cells, it is crucial for host responses against infection and injury. IL-1β is synthesised in response to inflammatory stimuli from pathogens, stress conditions, and other danger signals. Elevated IL-1β has been reported in women with BV-associated dysbiosis36,38, Candida albicans-associated vulvovaginal disease31 and following sexual intercourse and contraceptive use38. Chlamydia was not reported to affect vaginal cytokines significantly in a study of pregnant women35. Ranges are 178.8 pg/mL for AV, 71.2 pg/mL for BV, 5.0 pg/mL for normal.

Microbiota Profiles

Marker of vaginal health Lactobacillus crispatus COMMENSAL BACTERIA (+ Low vaginal pH)

Gram-positive, aerotolerant anaerobe with high tolerance of low pH. Reported to produce very high amounts of lactic acid and antimicrobial peptides termed bacteriocins39. High levels are associated with very acidic vaginal pH. L.crispatus has been reported to reduce infectivity of Chlamydia trachomatis in vitro5. Dominance of this species is associated with CST I, reported in 26.2% of women2.

Marker of vaginal health Lactobacillus gasseri COMMENSAL BACTERIA (+ Low vaginal pH)

Gram-positive, aerotolerant anaerobe with high tolerance of low pH. Reported to produce high amounts of lactic acid and antimicrobial peptides termed bacteriocins51. L. gasseri produces a bacteriocin called gassericin E, that inhibits growth of BV- associated bacteria including Prevotella bivia, Gardnerella vaginalis, Atopobium vaginae, Mobiluncus mulieris and pathobionts E. coli, Staphylococcus aureus and Streptococcus agalactiae, in vitro15. Dominance of L. gasseri is associated with CST II, reported in 6.3% of women2.

Marker of vaginal health Lactobacillus jensenii COMMENSAL BACTERIA (+ Low vaginal pH)

Gram-positive, aerotolerant anaerobe with high tolerance of low pH. Reported to produce moderate-high amounts of lactic acid52, which maintains low pH, and produces antimicrobial peptides termed bacteriocins51. L. jensenii has been shown to reduce growth and adherence of Gardnenella vaginalis and Prevotella bivia in vitro52. L. jensenii has been shown to inhibit growth and hyphae formation of Candida albicans in vitro50. Dominance of L. jensenii is associated with CST V, reported in 5.3% of women1.

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Marker of vaginal health Lactobacillus iners COMMENSAL BACTERIA (+ Low vaginal pH)

Lactobacillus iners has a unique cell morphology and genome size compared to other Lactobacillus species. Its very small genome is evidence of rapid evolutionary changes, resulting in extensive gene loss. Compared to other Lactobacilli, it is also reported to produce moderate levels of lactic acid53. Dominance of L. iners is associated with CST III, reported in 34% of women1. High levels of L. iners has been detected in women with BV, but also in healthy women54. High levels of L. iners has also been detected in women infected with Chlamydia trachomatis55.

Atopobium vaginae COMMENSAL BACTERIA High levels associated with BV

Gram-positive, facilitative anaerobe associated with BV. A vaginae has been detected by PCR in 96% of women with BV compared to in 12-19% without BV56. It is highly associated with recurrent BV infections after treatment with metronidazole and has been reported in 80-90% of cases of relapse57. Ranges around 0.6% is considered normal, while 1.5% is considered as intermediate and 7% as BV11. A. vaginae is reported to form biofilms in BV58 including polymicrobial biofilms with Gardnerella vaginalis59. It also triggers an inflammatory and innate immune response60.

Prevotella bivia COMMENSAL BACTERIA High levels associated with BV

Gram-negative, anaerobic bacteria that is commonly detected in the vaginal tract and occasionally in the oral flora. Due to its ability to grow well in the presence of oestrogens, P. bivia is traditionally implicated in vaginal tract infections, such as , and pelvic inflammatory disease61. P. bivia is significantly associated with BV. In a study of women with BV, P. bivia and other Prevotella spp. represented 44% of all anaerobes isolated in the BV-positive group62. P. bivia has also been reported to produce collagenases and fibrinolysins, which can degrade the mucosal surface of the vaginal epithelium and detach epithelial cells63. Clinically, P. bivia is associated with high cervovaginal levels of IL-8 and IL-1β64. It is also associated with the most proinflammatory (highest levels of IL-1α, I L-1 β, and TNF-α) vaginal community type in South African women65.

Megasphaera 1 and 2 COMMENSAL BACTERIA High levels associated with BV

Recently identified member of the vaginal microbiota. Megasphaera spp. are anaerobic Gram-negative cocci that cannot be cultured and are detected by culture-independent techniques (e.g. PCR). Megasphaera are detected in women with and without BV, but show significantly higher concentrations in BV66,67. This species is also associated with spontaneous preterm birth27,68.

Gardnerella vaginalis COMMENSAL BACTERIA High levels associated with BV

Gardnerella vaginalis is the most studied BV-associated bacteria. Gram-variable, fastidious anaerobe that shows increased abundance in BV69, but is also a member of the healthy vaginal microbiota. G. vaginalis is reported in 97% of women without BV70 and is associated with CST III71. Due to its biofilm forming abilities and ability to adhere to vaginal epithelial cells, G.vaginalis is proposed to function as a scaffold for other species and initiator of polymicrobial biofilms, that are associated with BV58. Colonisation of this bacterium is associated with increased production of proinflammatory cytokines (e.g. IL-6, IL-8, IL-1β) in vivo72. G. vaginalis produces cytolysic, vaginolysin that causes cell death of vaginal epithelial cells73. This bacterium also produces sialidase, prolidase, and putrescine, which may play a role in degrading mucins and contribute to the shedding of vaginal epithelial cells74. Urinary tract exposure to vaginal G. vaginalis was shown to trigger dormant E. coli in the bladder to cause active urinary tract infection75. Higher levels of this bacterium is associated with pelvic inflammatory disease, bacteraemia, and preterm birth.

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Mobiluncus spp. COMMENSAL BACTERIA High levels associated with BV (M. mulieris, M. curtisii)

Gram-negative rods that comprise members of the vaginal microbiota. Mobiluncus spp. produce malic acid and trimethylamine, associated with vaginal irritation and malodour and with BV. Mobiluncus was detected by PCR in 84.5% of women with BV and in 38% of women without BV76. M. curtisii was detected in 65.3% of women with BV77. Predominance and persistence of M. curtisii is associated with recurrent BV infections78. Higher levels of Mobiluncus spp. have also been linked to preterm birth68.

Associated with BV and gynaecological Ureaplasma urealyticum COMMENSAL BACTERIA diseases

Ureaplasma are commensal bacteria detected in the vaginal microbiota of 40–80% of women of reproductive age79. Ureaplasma has fourteen known serotypes and is divided into two biovars - Ureaplasma parvum and Ureaplasma urealyticum. Despite this, Ureaplasma has been implicated in several conditions like non-gonococcal (NGU), , gynaecological diseases and infertility. Contradicting results on the association of U. urealyticum with BV have been reported. For example, prevalence of U. urealyticum was 78% in healthy pregnant women and was significantly higher, at 92%, in pregnant women with BV80. However, a review showed that U. urealyticum is detected in women with and without BV, with no significant difference81. Despite this, the bacterium induces proinflammatory cytokine production in vaginal and cervical epithelial cells82.

Associated with BV and gynaecological Streptococcus agalactiae (group B) PATHOBIONT diseases

Group B Streptococcus agalactiae (GBS) are Gram-positive, cocci-shaped bacteria that can inhabit the human gastrointestinal tract as a member of the normal gut flora. Through the faecal-vaginal route, GBS can colonise the vaginal tract. Vaginal colonisation by GBS is transient and intermittent, and likely associated with vaginal pH, microbiota composition, pregnancy, and oestrogen concentrations83. GBS colonisation is associated with BV, AV and infection-induced inflammation of the upper female genital tract29. GBS colonisation of the vagina or rectum occurs in an estimated 10-30% of pregnant women84. GBS has been associated with preterm labour, prematurely ruptured membranes and puerperal and foetal infections, such as meningitis. About 30–70% of colonised mothers deliver GBS colonised newborns, and 1–2% of these develop early-onset infections, with heavily colonised mothers are more likely to transmit GBS to their offspring85.

Associated with BV and gynaecological Staphylococcus aureus PATHOBIONT diseases

Gram-positive, cocci-shaped bacteria that typically colonises the gastrointestinal tract, nasal tract and skin. It may also inhabit the genital tract in women and has been detected carrying antibiotic resistance-determinants in approximately 18% of pregnant women86. Colonisation of the vagina by S. aureus is associated with AV87. A subpopulation of S. aureus is able to induce toxic shock syndrome (TSS), through production of TSS toxin-1 (TSST-1). Menstrual TSS is rare, but is associated with menstruation and tampon use in previously healthy women colonised by S. aureus producing TSST- 188. However, host factors and immune responses are of clear importance considering vaginal colonisation by toxin- producing S. aureus has been reported in 1% to 4% of women, but TSS affects only one to three in 100,000 women89.

Enterococcus faecalis PATHOBIONT Enteric pathobiont associated with AV

Gram-positive, cocci shaped bacteria which is a normal inhabitant of the oral and intestinal microbiota. Vaginal colonisation by E. faecalis is associated with aerobic vaginitis87 and pelvic inflammatory disease. In addition, vaginal colonisation by E. faecalis is associated with preterm birth, very low birth weight delivery, and puerperal sepsis, which cause substantial morbidity and mortality in Sub-Saharan Africa90. E. faecalis can also invade the urinary tract and are a common cause of urinary tract infections91.

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Enteric pathobiont associated with Bacteroides fragillis PATHOBIONT pelvic infections and

Gram-negative, rod-shaped bacteria that inhabits the human gastrointestinal tract as a commensal member of the flora. Bacteroides spp. are not part of the normal vaginal microbiota but are occasionally isolated from vaginal cultures. The rates of vaginal carriage of Bacteroides in healthy women (both pregnant and non-pregnant) are estimated to be between 0 and 6%92. This increased to 16% in women in labour and to 27 to 28% in patients with . Pelvic infections associated with B. fragillis are often characterised with presence of an abscess93.

Enteric pathobiont associated with AV Escherichia coli PATHOBIONT and UTIs

Gram-negative, rod-shaped bacteria that inhabits the human gastrointestinal tract and is a member of the normal gut flora. Escherichia coli are reported as one of the most common organisms found in the genital tract of non-pregnant (9–28%) and pregnant women (24–31%)94. However, unlike commensal intestinal E. coli, vaginal strains typically carry virulence factors that allow them to colonise, invade and evade immune responses95. Carriage of vaginal E. coli is associated with increased production of proinflammatory cytokines and damage to the epithelial barrier96. Colonisation of the vagina by E. coli is associated with aerobic vaginitis87. Vaginal E. coli strains are considered to be a reservoir for vaginal and/or endocervical colonisation in pregnant women, and an important step in the development of urinary tract, intra-amniotic and puerperal infections through ‘faecal- vaginal-urinary/neonatal’ transmission.

Candida spp. Enteric pathobiont associated with AV (C. albicans, C. krusei, C. parapsilosis, OPPORTUNISTIC FUNGAL PATHOGENS and UTIs C. tropicalis)

Vulvovaginal candidiasis (VVC) is the second most common vaginal infection. Candida spp. are part of the normal vaginal flora, but its imbalance in the host leads to vulvovaginal candidiasis (VVC). Candida albicans is the predominant aetiological agent, causing 85–95 % of these infections97. C. albicans is an opportunistic pathogen, which can colonise approximately 20% of asymptomatic women97. Progression to VVC requires overgrowth of Candida spp., expression of virulence factors, and transition from the yeast growth mode to the hyphal growth mode98. Transition to the hyphal growth mode is induced by increased oestrogen levels, increased vaginal pH (>pH 7) and dysbiosis. Low vaginal pH has been reported to inhibit yeast-hyphae transition99. C. albicans also forms biofilms on vaginal epithelial cells100 and is associated with increased production of IL-8 in HIV-infected women12. Other Candida spp. associated with VVC include Candida krusei (lipase activity), Candida parapsilosis (strongest biofilm formation), and Candida tropicalis (highest phospholipase, lipase haemolytic activity).

Herpes infection present if detected HSV-1 & HSV-2 STI PATHOGEN, VIRUS (may be latent)

Genital herpes is a common sexually transmitted infection, passed through vaginal, oral and anal sex. It is caused by type 2 (HSV-2) and less commonly by HSV-1, which are large, double-stranded DNA . Although HSV genital infections are common, most people are asymptomatic. Primary infection may result in appearance of herpes lesions (small, painful blisters) on the vulva, labia and vagina. After primary infection, the majority of infected people do not experience further symptoms as the virus moves into a latent phase and resides in the central nervous system. HSV-2 has been shown to facilitate infection by HIV-1101.

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Detection of C. trachomatis requires Chlamydia trachomatis STI PATHOGEN, BACTERIA antimicrobial treatment

Chlamydia trachomatis is a causative agent for the common sexually transmitted infection, chlamydia. C. trachomatis is an obligate intracellular pathogen. It replicates within host epithelial cells in the genital tract and persists within specialised vacuoles. Infection with chlamydia can cause a wide range of symptoms including malodorous discharge; however, most infected women are asymptomatic. Chronic and untreated infection is associated with , endometritis102, pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility103. In addition, infection with this pathogen facilitates the transmission of HIV and is associated with cervical cancer103. C. trachomatis infection is associated with a very modest proinflammatory response involving IL-6 and TNF-α.

Detection of M. genitalium requires Mycoplasma genitalium STI PATHOGEN, BACTERIA antimicrobial treatment

Mycoplasma genitalium is a sexually transmitted bacterial infection that infects both men and women. It is the smallest known bacterium and is very difficult to cultivate. After chlamydia, M. genitalium is the second most common STI in the British population and shows increasing levels of antibiotic resistance104. If detected, antimicrobials are used for treatment, however doxycycline has a low cure rate of 30-40% while has a cure rate of 85-95% in macrolide susceptible infections. There is increasing concern about M. genitalium, as a cause of pelvic inflammatory disease105,106, tubal factor infertility107, ectopic pregnancy in women108,109 and increased HIV acquisition and transmission110.

Detection of T. vaginalis requires Trichomonas vaginalis STI PATHOGEN, PARASITE antimicrobial treatment

Trichomonas vaginalis is a flagellated, parasitic protozoan that typically infects the squamous epithelium of the lower genital tract111. T. vaginalis is spread through sexual intercourse and is estimated to be the most common non-viral STI globally. The majority of women infected with T. vaginalis (85%) are asymptomatic112. Infection is associated with increased risk of BV, candidiasis and infection by HSV-1, HSV-2, chlamydia, gonorrhoea, and syphilis113. T. vaginalis is also associated with preterm delivery, pelvic inflammatory disease, and premature rupture of membranes114. Infection requires treatment with antibiotics including metronidazole.

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Biomarker name Type Associated with Infertility Dybiosis PID STI Bacterial Vulvovaginal Pregnancy Aerobic Cervical UTIs CST IV Herpes Vaginosis candidiasis complications vaginits cancer

IL-1β Pro-inflammatory Stress, danger signals ↑ ↑* ↑ ↑ KEY Cytokines cytokine Lactobacillus spp. Bacteria (Gram-positive) Vaginal health ↓ ↑ Increased levels detected in specific condition Lactobacillus Bacteria (Gram-positive) Vaginal health ↓ ↑* Increased levels detected crispatus in specific condition with exceptions (i.e. chlamydia) Lactobacillus iners Bacteria (Gram-positive) Vaginal health ↑ ↓ ↑ x Causative agent for specific condition Lactobacillus Bacteria (Gram-positive) Vaginal health ↓ Increased Increased risk of jensenii risk developing condition Lactobacillus gasseri Bacteria (Gram-positive) Vaginal health ↓

Atopobium vaginae Bacteria (Gram-positive) x ↓ ↑ Increased risk Increased risk ↑

Prevotella bivia Bacteria (Gram-positive) x ↓ ↑ Increased risk Increased risk ↑ Commensals Megasphera 1 Bacteria (Gram-positive) BV, PID, Adverse ↓ ↑ Increased risk Increased risk ↑ pregnancy complications Megasphera 2 Bacteria (Gram-positive) BV ↓ ↑ Increased risk Increased risk ↑

Gardnerella vaginalis Bacteria (Gram-variable) BV ↓ ↑ Increased risk xx ↑

Mobiluncus mulieris Bacteria Vulvo-vaginal ↑ Increased risk ↑ abscesses Mobiluncus curtisii Bacteria ↑ Increased risk ↑

BVAB2 Bacteria ↑ Increased risk ↑

Ureaplasma Bacteria ↑ Increased risk x Increased risk

Streptococcus Bacteria (Gram-positive) GBS sepsis ↑ ↑ Increased risk Increased risk Increased risk agalactiae (group B) Staphylococcus Bacteria (Gram-positive) ↑ Increased risk Increased risk increased risk aureus Enterococcus Bacteria (Gram-positive) ↑ Increased risk Increased risk Increased risk faecalis Bacteroides fragilis Bacteria (Gram-negative) Bartholin's , ↑ Increased risk Increased risk Pathobionts as well as abscesses in the ovaries or fallopian tubes, bacteraemia. Risk factors for intrauterine growth retardation Escherichia coli Bacteria (Gram-negative) UTI ↑ Increased ↑ Increased risk Increased risk Increased risk Increased risk risk Mycoplasma Bacteria Causative agent for Increased risk x Increased risk genitalium Mycoplasma STI Chlamydia Bacteria (Gram-negative) Causative agent for Increased risk Increased Increased risk x Increased risk trachomatis chlamydia STI risk Trichomonas Protozoa Trichomonas STI x Sexually vaginalis transmitted infections HSV-1 Virus Genital herpes x x (infection may be asymptomatic) HSV-2 Virus Genital herpes x x (infection may be asymptomatic) Candida albicans Fungi Vulvocandidasis ↑ x

Opportunistic Candida krusei Fungi Vulvocandidasis ↑ x fungal pathogens Candida parapsilosis Fungi Vulvocandidasis ↑ x

Candida tropicalis Fungi Vulvocandidasis ↑ x

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Biomarker name Type Associated with Endometriosis Infertility Dybiosis PID STI Bacterial Vulvovaginal Pregnancy Aerobic Cervical UTIs CST IV Herpes Vaginosis candidiasis complications vaginits cancer IL1-beta Pro-inflammatory Stress, danger signals ↑ ↑* ↑ ↑ KEY cytokine Cytokines IL-8 Chemoattractant Pro-inflammatory ↑ ↑* ↑ ↑ ↓ Decreased levels cytokine responses detected in specific condition Lactobacillus spp. Bacteria (Gram-positive) Vaginal health ↓ ↑ Increased levels detected in specific condition Lactobacillus Bacteria (Gram-positive) Vaginal health ↓ ↑* Increased levels crispatus detected in specific condition with exceptions (i.e. chlamydia) Lactobacillus iners Bacteria (Gram-positive) Vaginal health ↑ ↓ ↑ x Causative agent for specific condition Lactobacillus Bacteria (Gram-positive) Vaginal health ↓ Increased Increased risk jensenii risk of developing condition Lactobacillus gasseri Bacteria (Gram-positive) Vaginal health ↓

Atopobium vaginae Bacteria (Gram-positive) x ↓ ↑ Increased risk Increased ↑ risk

Commensals Prevotella bivia Bacteria (Gram-positive) x ↓ ↑ Increased risk Increased ↑ risk Megasphera 1 Bacteria (Gram-positive) BV, PID, Adverse ↓ ↑ Increased Increased risk ↑ pregnancy risk complications Megasphera 2 Bacteria (Gram-positive) BV ↓ ↑ Increased Increased risk ↑ risk Gardnerella vaginalis Bacteria (Gram-variable) BV ↓ ↑ Increased x ↑ risk Mobiluncus mulieris Bacteria vulvo-vaginal ↑ Increased ↑ abscesses risk Mobiluncus curtisii Bacteria ↑ Increased ↑ risk BVAB2 Bacteria ↑ Increased ↑ risk Ureaplasma Bacteria ↑ Increased risk x Increased risk Streptococcus Bacteria (Gram-positive) GBS sepsis ↑ ↑ Increased risk Increased risk Increased agalactiae (group B) risk Staphylococcus Bacteria (Gram-positive) ↑ Increased risk Increased risk increased aureus risk Enterococcus Bacteria (Gram-positive) ↑ Increased risk Increased Increased risk faecalis risk Bacteroides fragilis Bacteria (Gram-negative) Bartholin's abscess, ↑ Increased risk Increased Pathobionts as well as abscesses risk in the ovaries or fallopian tubes, bacteraemia. Risk factors for intrauterine growth retardation Escherichia coli Bacteria (Gram-negative) UTI ↑ Increased ↑ Increased risk Increased risk Increased Increased risk risk risk

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COMMUNITY STATE TYPE CLASSIFICATION

Community States Types Recent work has classified the vaginal microbiota into five core Community State Types (CST). These groups are distinguished on whether communities are dominated by Lactobacillus spp., and by the species of Lactobacillus present. CST characterisation is extremely dynamic with healthy women showing temporal CST changes and fluctuations with menstruation, sexual development and sexual intercourse1.

Community State Type I, II, III, V (Lactobacillus dominated) CST I, II, III and V are dominated by Lactobacillus species and are therefore considered low risk1. CST I, II, III and V are dominated by L. crispatus, L. gasseri, L. iners and L. jensenii respectively. CST I, III and V are associated with low vaginal pH 1.

Community State Type IV (Increased risk of BV) CST IV has no specific dominant species and is termed the 'diverse group'. CST IV is associated with higher vaginal pH. CST IV is characterised by higher levels of anaerobic bacteria and BV-associated bacteria, including Prevotella, Dialister, Atopobium, Gardnerella, Megasphaera, Aerococcus, Finegoldia and Mobiluncus1.

CST IV is further divided into two sub-categories: CST IV-A and CST IV-B115. CST IV-A is characterised by presence of L. iners or other Lactobacillus spp., together with lower proportions of strictly anaerobic bacteria such as Anaerococcus, Corynebacterium, Finegoldia, or Streptococcus115.

CST IV-B is characterised by higher proportions of Atopobium, in addition to Prevotella, Parvimonas, Gardnerella, Mobiluncus, or Peptoniphilus and several other taxa and BV associated bacteria (BVAB)115.

Community State Type Classification and Ethnicity CST classification and community composition are influenced by a multitude of host factors including menstruation, hygiene and sexual intercourse. In a study of 394 women from four ethnic groups, ethnicity was shown to strongly influence CST classification1. For example, CST I (L. crispatus dominated) is more frequently detected in White females (41.9%), compared to Asian (22.9%), Black (21.9%) and Hispanic (13.3%) females. CST IV (diverse group comprised of anaerobic bacteria) was more frequently detected in Black females (38.99%) and Hispanic females (34.3%), compared to White (9.3%) and Asian females (17.6%).

Community State Type classification is influenced by ethnicity. Modified from Ravel et al., 2011 and Ravel and Forney, 2013.

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SUMMARY

The Phylobioscience Female EcologiXTM Vaginal Health and Microbiome Profile, is an innovative tool for analysis of the vaginal microbiota composition and host immune responses. Phylobioscience recommend that the panel is used on an annual basis to monitor changes in vaginal health. The profile may also be used as a diagnostic tool in symptomatic patients.

For effective analysis of results, the interpretive guide should be used in combination with Invivo Healthcare's Female Microbiome Clinical Considerations and the patient’s symptomology and results.

The Female Microbiome Clinical Considerations provides further information on lifestyle and environmental factors that may influence the urinary microbiotas. The Female Microbiome Clinical Considerations also provides recommendations for treatment and management plans for results indicative of dysbiosis, inflammation, and infection.

If you have any queries on patient results or clinical considerations, contact the Clinical Education team at support@invivohealthcare. com

RECOMMENDED READING

Vaginal microbiome of reproductive-age women Ravel, Jacques, Pawel Gajer, Zaid Abdo, G. Maria Schneider, Sara SK Koenig, Stacey L. McCulle, Shara Karlebach et al. Proceedings of the National Academy of Sciences 108, no. Supplement 1 (2011): 4680-4687.

Composition of the vaginal microbiota in women of reproductive age–sensitive and specific molecular diagnosis of bacterial vaginosis is possible? Shipitsyna, Elena, Annika Roos, Raluca Datcu, Anders Hallén, Hans Fredlund, Jørgen S. Jensen, Lars Engstrand, and Magnus Unemo. PloS one 8, no. 4 (2013): e60670.

Association of bacterial vaginosis with chlamydia and among women in the US Army Bautista, C.T., Wurapa, E.K., Sateren, W.B., Morris, S.M., Hollingsworth, B.P. and Sanchez, J.L., 2017.American journal of preventive medicine, 52(5), pp.632-639.

Temporal and spatial variation of the human microbiota during pregnancy DiGiulio, Daniel B., Benjamin J. Callahan, Paul J. McMurdie, Elizabeth K. Costello, Deirdre J. Lyell, Anna Robaczewska, Christine L. Sun et al. Proceedings of the National Academy of Sciences 112, no. 35 (2015): 11060-11065.

Unveiling the role of Gardnerella vaginalis in polymicrobial bacterial vaginosis biofilms: the impact of other vaginal pathogens living as neighbors. Castro, Joana, Daniela Machado, and Nuno Cerca. The ISME journal (2019): 1.

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REFERENCES

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