Female Genital Tract Infections Involving Anaerobes

Female genital tract infections involving anaerobes

Elisabeth Nagy MD, PhD, DSc

Institute of Clinical© by Microbiology, author University of Szeged Anaerobe Reference Laboratory for Hungary ESCMID Online Lecture Library

ESCMID Postgraduate Course on Anaerobes 2014, Groningen Normal flora of the female genital tract

Vaginal fluid of 79 control women Culture method (48h CO2) investigated by NGS

~90%

E. Shipitsyna et al : PLOSone 2013 Summary of 63 articles dealing with vaginal microbiome studies on health and BV.

Many different molecular techniques were used during the last 10 years: NGS,© by author Sequencing of colonies ESCMID OnlineFingerprinting, Lecture Library qPCR Infections of the female genital tract involving anaerobes

 Anaerobes of the normal flora are involved (mixed infections)  Bacterial vaginosis (infection??)  Postoperative infections  Non-puerperal and postpartum endometritis  PID (salpingitis)© by author  Chorioamnionitis ESCMID Premature Online rupture Lecture of membranes Library  Premature labour  IUD related infections

History of bacterial vaginosis

 Non-specific vaginitis (Krönig 1892)  Haemophilus vaginitis (Gardner and Dukes 1954)  Corynebacterium vaginitis (Zimmerman and Turner 1969)  Gardnerella vaginitis (Greenwood and Pickett 1980)  Anaerobic vaginosis© by (1982) author  Non-specific vaginosis (1984) ESCMID Bacterial vaginosis Online (Spigel Lecture and Roberts 1984) Library  Signal bacterium of BV: Gardnerella vaginalis versus Atopobium vaginae Bacterial vaginosis (BV)

 Flora change (dysbiosis) characterized by disappearance of the lactobacillus flora and predominance of a mixed bacterial (anaerobic) flora  Endogenous infection?  Reservoire of some BV associated bacteria in the gut (Holst E, JCM 1990)© by author  G. vaginalis in the oral biofilm ((Marrazzo JM et al.: JID 2012)

ESCMID Changed ‘normal Online flora’? Lecture (Mard PA. Infect Library Dis Obstet Gynecol 1997; Witkin SS. CID 2007)  STD/STI? Epidemiology of bacterial vaginosis

 In the industrialized world among symptomatic women: BV > candidiasis or trichomoniasis

 BV is common in the developing world too (Rakai, Uganda 1997) Prevalence of among patients with symptoms BV 51% trichomoniasis © by 24%author yeast <5%

ESCMID Prevalence of BV Online among pregnant Lecture women in the Library USA blacks > hispanic > whites (22%) (16%) (8%) Epidemiology of bacterial vaginosis

 BV is more prevalent among  women attending an STD clinic  IUD users  women who are using regular douching  frequent Candida infection  sexually experienced© by womenauthor  female sex workers ESCMID lesbians Online(extremely highLecture rates of BV) Library  women who practice oral sex

Berg 1984; Eschenbach 1988; Hillier 1990; Skinner 1996; Schmid 1999; CDC 2000; Fethers 2000; Joesoef 2001; Morris 2001; August 2004; Vaginal ecosystem

Normal versus Bacterial vaginosis

- Lactobacillus predominance - Few H2O2 producing Lactobacillus - Usually 107 CFU/g - 109 CFU/g - Ratio of anaerobes/aerobes - Ratio of anaerobes/aerobes 2-5:1 © by author 100 -1000 : 1 - G. vaginalis (5-60%) - G. vaginalis (95%) - MobiluncusESCMID (0-5%) Online Lecture- Mobiluncus Library(50-70%) - M. hominis (15-30%) - M. hominis (60-75%) E. Shipitsyna et al : PLOSone 2013

Categories were determined by relative abundance of taxa measured by NGS and bacterial load measured by qPCR

NORMAL BV   Lactobacillus    

Increasing pH

Gardnerella  Increasing symptomes  © by author    Anaerobes

NormalESCMID conditions Possible Online reasons LectureBacterial vaginosis Library intercorse (Amsel’s criteria) use of spermicids hormonal effects use of vaginal dushes use of IUD other infections Pathogenesis of bacterial vaginosis

32% of vaginal lactobacilli has lytic phages

(C. Spiegel: Reviews Med. Microbiol. 2002) New concept in the pathogenesis of BV

 S.S. Witkin et al.: Hypothesis of an altered immunity for the development of symptomatic bacterial vaginosis (CID; 2007)

 Components of innate immunity  Toll-like receptors (TLRs)  Inducible heat shock© proteinby author (70-kDs) (hsp70)  Mannose-binding lectin (MBL) ESCMID Online Lecture Library  BV develops as a result of inhibition of TLRs, decrease in hsp70 production, and inadequate MBL function.

Possibilities of the diagnosis

 Diagnostics:  Amsel’s criteria - clinical symptoms  Nugent’s scores - based on Gram-staining  Culture in an anaerobic environment (48-72h)  Detection of signalling bacterium: Gardnerella vaginalis vs. Atopobium vaginae © by author  Recently 16S rRNA-based amplification, sequencing, followed by broad-range clone library analyses of vaginal ESCMID flora Online Lecture Library Fredricks DN, et al: N. Eng. J. Med (2005) Hyman RW, et al: Proc. Natl.Acad. Sci. USA (2005) Shioitsyna E, et al: PLOSone (2013)

Amsel`s criteria (1983)

 A homogenous discharge (not always seen by the clinicians)  pH >4.5 (only if the sample is taken from the proper place of the vagina)  Positive amine „whiff” test in the presence of 10% KOH (may be positive also in trichomoniasis)  Fishy vaginal odor  during menstruation  after intercourse © by author  Presence of clue cells (WBC can be also present) ESCMIDMinimal itching Online or irritation Lecture Library Detection of „clue” cells (Gram)

Lactobacillus dominanted vaginal flora

Points scored per morphotype* Bacterial morphotype None 1+ 2+ 3+ 4+

Large gram-positive rods 4 3 2 1 0

Small gram-neg/var. rods 0 1 2 3 4

Curved gram-neg/var.© rodbys author0 1 2 3 4

*Score 0-3 points – Normal ESCMID 4-6 points Online – Intermediate Lecture Library 7-10 points – Bacterial vaginosis

Nugent RP, et al.:: Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991, 29:297-301.

Culture - based diagnosis of BV

 Media:

 Human blood agar (48 h in CO2 / anaerobically) Gardrenella vaginalis

 Columbia blood agar (24 h in CO2) lactobacilli, aerobic bacteria © by author  Columbia blood agar with vitamin K1 and hemine ESCMID(4-5 days Onlinein an anaerobic Lecture environment Library)

Frequently isolated and identified anaerobic (aerobic) bacteria: Gardnerella vaginalis Mobiluncus mulieris (long curved rod) Mobiluncus curtisii (short curved rod) Bacteroides urealyticus and other spp Prevotella bivia and other spp Prophyromonas spp Fusobacterium nucleatum Peptostreptococcus© by spp author Streptococcus morbillorumBV flora after incubation for 96 h in an anaerobic environment ESCMIDAtopobium Online vaginae Lecture Library Mycoplasma hominis etc. G. vaginalis

 Gram-labil coccobacillus, „surface pathogen”

 Produce hemolysin – cytolytic activity

 Synergism with P. bivia*

 Production of the biofilm!!! © by author *P. bivia utilizes aminoacids and produce ammonia and Gardrenella utilizesESCMID ammonia to produceOnline amino Lecture Library acids

Mobiluncus spp

 Gram-variable, anaerobic, mobile, curved rods  Produce trimethylamine - responsible for the fishy odor  They are involved in intra- and extra-genital infections  The reservoir is the gastrointestinal tact  M. mulieris (long curved© byrod) author  M. curtisii- (short curved rod) – more virulent ESCMID the IgG titre is higherOnline Lecture Library  „escape” from phagocytosis  Variable resistance to metronidazole Atopobium vaginale

 Newly detected species (Rodriguez Jovita M. et al. 1999)

 Gram-positive elliptic cocci in pairs or short chains

 Anaerobic (however it is growing slowly in CO2)

 55% of BV patients had a positive signal in the vaginal fluid using 16S rRNA© PCRby +author sequencing (Ferris MJ et al. 2004)  Some isolates are highly resistant to metronidazole, but all ESCMIDsusceptible to clindamycin Online (BackerLecture E.D. 2006) Library During research on bacterial vaginosis

 The results are influenced by „what is compared with what?”  Clinical symptoms (Amsel criteria) with molecular testing  Gram-staining (Nugent score) with molecular testing © by author  What about culturing the samples in anaerobic ESCMIDenvironment Online for 4 -Lecture5 days? MALDI Library-TOF MS will provide identification in real-time. (2013)

Amsel criteria versus NGS and 16 species/genus specific RT-PCR

Conclusion: Amsel criteria can be used by clinicens for diagnosis of BV  16S rRNA gene based amplification, sequencing, followed by broad-range clone library analyses of vaginal flora is used. (Dara compared with Nugent scores) © by author  Quantitative PCR assay was used to detect the metronidazole- resistant, newly described anaerobic bacterium Atopodium ESCMIDvaginae Online Lecture Library *Fredricks DN., et al.: N. Eng. J. Med (2005) *Hyman RW., et al.: Proc. Natl.Acad. Sci. USA (2005) Six patients with BV were treated with metronidazole and the vaginal flora was analysed before and after treatment (one month) by PCR-based 16S rRNS gene sequencing

Before treatment After treatment

Before After metronidazole treatment

Treatment of BV

 Main goal is to  re-establish normal vaginal lactobacillus flora  decrease the pH of the vagina  Antibiotics  Local or systemic metronidazole Cure rate 75-85%  Local or systemic clindamycin  Other methods © by author  Lyophilized H2O2 producing Lactobacillus locally with or without antibiotics ESCMID To destroy extracellularOnline DNALecture (biofilm) Libraryby DNAse (Hymes et al 2013)  Synthetic retrocyclin (defensins) inhibits biofilm formation of G. vaginalis (Hooven et al 2012)

Consequences of BV

 Bacterial vaginosis may cause  adverse pregnancy outcomes (McGregor JA. et al. 2000)  pelvic infection (Sweet RL. 2000)  biofilm formation on the surface of IUDs (Pál Z. et al. 2005)  post surgical© infection by author (Guaschino S. 2002)  acquisition of sexually transmitted microorganisms ESCMIDincluding Online HIV (Sewankombo Lecture N. 1997) Library

2.5x108 CFU/ml Scenning electron microscopy A. viscosus A. naeslundii Bifidobacterium sp Fingoldia magna Anaerococcus prevotii © by author Prevotella disiens P. ESCMIDasacharolyticus Online Lecture Library B. urealyicus Mobiluncus sp. Questions still to be answered

 BV flora is a stable or transient flora?  Can BV flora considered STI?  Transmission to the male partner?  Can BV associated bacteria be involved in chronic prostatitis or male infertility?  What about the parallel treatment of the partners?  What is the ideal© antibiotic,by author which can lead to the re- establishment of the Lactobacillus flora? ESCMID ?L. chrispatus Online - good Lecture/ L. iners – bad? Library  Is there any connection between BV flora and the parodontitis flora? Pelvic inflammatory disease

 Distribution of the pathogens  Often associated with BV  Mostly clinical diagnosis  In case of surgery anaerobe culturing is mandatory !!!© by author ESCMID Online Lecture Library

! Thank you for your attention!!

• Ny. V. 5 and half year old girl, • recurrent problems, complants: • Itching, discharge Culture results E. coli Enterococcus faecalis B. fragilis B. caccae, B. merdae Clostridium sp. © by author Bifidobacterium sp. Eubacterium sp. SutterrellaESCMID sp. Online Lecture Library Porphyromonas sp.

Széklet flóra!!! Hyginic problems