The Bacterial Vaginosis Syndrome.

BD Meeting, Oxford, 2015

The vagina is the Rodney Dangerfield of the body-

..”it don’t get no respect”

Srinivasan, 2008

What is Abnormal Vaginal Flora?

Ask a microbiologist?

Abnormal Vaginal Flora

• Introduction of abnormal organism not part of normal flora

– Sexually transmitted

– Non-Sexually transmitted

• Haemophilus influenzae

• Increased virulence of existing organisms (Esch. coli)

• Imbalance of normal bacteria

– Lactobacilli reduced in quality and quantity

– pH rises

– 1000-fold increase in other organisms (anaerobes, mycoplasmas, Gardnerella vaginalis, mobiluncus, etc)

– BACTERIAL VAGINOSIS

History and nomenclature

• Name of condition

– Non-specific vaginitis

– Haemophilus vaginitis

– Corynebacterium vaginitis

– Gardnerella vaginitis

– Anaerobic vaginosis

– Non-specific vaginosis

– Bacterial vaginosis

•Organism thought responsible

– Anaerobic Strep.

– Haemophilus vaginalis

– Corynebacterium species

– Gardnerella vaginalis

– Anaerobic species

– Polymicrobial

– Polymicrobial

? VAGINAL BACTERIOSIS?

“The Bacterial Vaginosis Syndrome”

Background: Non-specific Vaginitis (NSV)

Staphylococci

Streptococci

Coliforms

Micrococci

Diphtheroids

Gardner and Dukes 1955

Abnormal Vaginal Flora

Same, small, pleomorphic, Gram

negative bacilli in majority of smears

• Dukes task:Large variety of highly enriched media at different atmospheric conditions

• 48 hours, 10% sheep blood agar, low O2 tension

• Smears from those colonies showed bacilli similar to smears

• Same in next 13 of 14 patients

• Mono-etiologic agent of non-specific vaginitis (now known as bacterial vaginosis)

• – Haemophilus vaginalis , now known as Gardnerella vaginalis

Koch’s Postulates

1. “The bacterium must be observed in every case of the disease”

2. “The bacterium must be isolated and grown in pure culture”

3. “The bacterium in pure culture, must, when inoculated into a

susceptible animal give rise to the disease.”

4. “The bacterium must be observed in and recovered from the

experimentally diseased animal”

Robert Koch 1890

Evidence of Pathogenicity

STAGE 1

•13 volunteers free of disease inoculated vaginally with pure culture of G.vaginalis

• 10 failed to develop clinical evidence/positive culture

• 2 patients culture positive for 2 and 3 months but no signs

• One developed clinical signs and organisms recovered in pure culture completely replacing existing vaginal flora

• Sufficient to satisfy Koch’s 3rd postulate

STAGE 2

•15 volunteers

•Successfully inoculated vagina with vaginal secretions (not pure culture) from donors with BV

• Donors were screened for sexually transmitted disease

•11 developed BV

Gardner and Dukes 1955

Bacterial vaginosis

Prevalence of BV

6%

9%

11%

15%

16%

30%

30-50%

75% Age and Race

Is BV a serious condition?

OBSTETRICS

• Spontaneous abortion – Early

– Late

– Recurrent

• Preterm Birth

• Preterm prelabour rupture of the

membranes

• Post partum endometritis

• Chorioamnionitis

GYNAECOLOGY

• Urinary tract infection

• Post abortal sepsis

• Pelvic inflammatory disease

• Post Hysterectomy vaginal cuff

infection

• Infertility

• Acquisition of sexually transmitted

infections – Bacterial

– Protozoal

– Viral

• Herpes Simplex (HSV)

• Human Papillomal (HPV)

• HIV

Gardner and Dukes 1955

Is BV an Important Condition? US FDA Gain Act, 2012 (Generating Antibiotic Innovation Now)

Preterm Birth: a serious adverse outcome

of pregnancy

• Babies born 22-26 weeks gestation

– 65% die in delivery room or in neonatal intensive care

• Of the survivors at 2.5 years of age

– 50% disabled

• 50% of these the disability is severe

• At the age of 2.5 years

– Only 12-13% are alive and intact

• Annual Cost of PTB in USA (IOM)- $26 billion

• Cost of hospital re-admissions first 5-10 years

– 20x more costly <28 vs >37 weeks

Infection as a Cause of Spontaneous

Early Preterm Labour (SPTL)

• Bacterial products added to amnion cells in vitro results in a significant

increase in PGE2 production

– (Lamont et al, Lancet, 1985)

• Between 26 and 34 wks GA, women in SPTL compared to women not

in SPTL, are significantly more likely to have:

– Abnormal vaginal flora (47% v 15%)

– Neonatal infection

– Chorioamnionitis (56% v 10%)

– (Lamont et al, BJOG, 1986)

• Between 26 and 34 wks GA, women in SPTL compared to women not

in SPTL, are significantly more likely to be colonised by high numbers

Mycoplasmas and Ureaplasmas (18% v 0%)

– (Lamont et al, J Med Microbiol, 1987)

Late Miscarriage (<24w) and Early PTB (<34w) According to Grade of

Vaginal Flora before 16 weeks

16 weeks

16.7% 3.4%

Odds ratio = 5.35 (2.73 – 10.5) Relative risk = 3.12 (2.23 – 4.37) P-value = 0.000001

Hay PE, Lamont RF, Taylor-Robinson D et al, BMJ, 1994

It would appear logical to

consider using antibiotics to

prevent PTB of infectious

etiology

Antibiotics: Problems with antibiotic studies

‘Different studies have used different diagnostic methods, with different outcome parameters or differing definitions of success, to treat women of differing risks, with different susceptibilities and hence different host response, with different degrees of abnormal genital tract flora, at different gestational ages, using different antibiotics in different dose regimens by different routes of administration and not surprisingly DIFFERENT results’

Lamont 2001

Systematic Reviews/Meta-Analysis of Antibiotics

used Prophylactically for the Prevention of PTB

2001 Guise 2002 Koumans

2003 Leitich 2004 Klein

2004 Riggs 2005 Okun

2006 Varma 2007 Simcox

2007 McDonald 2008 Hutzal

2008 Swadpanich 2013 Brocklehurst

So why the confusion?

No Systematic review/meta-analysis has simultaneously addressed:

• optimal choice of agent

• optimal choice of patient

• optimal choice of timing of administration

“The Right Antibiotics in the Right

Women at the Right Time”

Lamont RF, Nhan-Chang C-L, Sobel JD, Workowski K, Conde-Aguledo A, Romero R

Perinatology Research Branch, NICHD,NIH,DHHS

Wayne State University, Detroit, MI

CDC, GA

Am J Obstet Gynecol. 2011 Sep;205(3):177-90.

Hypothesis

The conclusions of individual studies/systematic reviews/meta-analyses

on the use of antibiotics used prophylactically for the prevention of

PTB are flawed by the fact that undue reliance is placed on:

Studies with suboptimal choice of antibiotics (mainly metronidazole)

Used too late in pregnancy to influence outcome (23-27 weeks)

In women whose risk of PTB was not due to BV

(previous PTB, Low BMI, FFN, Ureaplasmas, GBS, TV, etc.)

Conversely, that antibiotics active against BV related organisms, used in

women whose risk of PTB is due to abnormal flora, and used early

in pregnancy before irreversible inflammatory damage occurs, can

reduce the rate of PTB

Lamont et al, Am J Obstet Gynecol. 2011 Sep;205(3):177-90.

Preterm Birth 24-36 completed weeks of

gestation

(5 Studies – Fixed Effects Model)

Lamont et al, Am J Obstet Gynecol. 2011 Sep;205(3):177-90.

Preterm Birth 24-36 completed weeks of

gestation

(5 Studies – Fixed Effects Model)

Lamont et al, Am J Obstet Gynecol. 2011 Sep;205(3):177-90.

Late miscarriage 16-23 completed

weeks of gestation(two studies)

Lamont et al, Am J Obstet Gynecol. 2011 Sep;205(3):177-90.

Late miscarriage 16-23 completed

weeks of gestation(two studies)

Lamont et al, Am J Obstet Gynecol. 2011 Sep;205(3):177-90.

Results Summary

• Preterm birth significantly reduced by 40%

• Late miscarriage significantly reduced by 80%

• Of those infants born preterm:

• Antibiotics: babies <2500g = 20%

• No treatment: babies <2500g = 80% (p<0.009)

• Antibiotics versus no treatment • 32.5 day difference in mean gestation age in favor of antibiotics (p<0.024)

• Delivery before 33 completed weeks of gestation

• Statistically significant 86% reduction for those who received antibiotics

• Women with Barn Door BV

• Late Miscarriage & Preterm Birth • Antibiotics = 5.4%

• Placebo = 35.7%

Lamont et al, Am J Obstet Gynecol. 2011 Sep;205(3):177-90.

0

10

20

30

40

50

60

70

80

1-y

ear

su

rviv

al ra

te (

%)

23 24 25 26 Completed gestation (weeks)

Finnstrom 1997

Each day of delay associated with 3% increase in survival

Results: Secondary Outcomes

Kekki 2001

Late Misc/PTB OR 95% CI

Persistent BV 28%

2.9

1.3-5.2

Cured BV 10%

Cured but

recurrent BV

15%

9.3

1.6-53.5

Cured BV/ no

recurrence

2%

Results: Secondary Outcomes

Kekki 2001

Late Misc/PTB OR 95% CI

Persistent BV 28%

2.9

1.3-5.2

Cured BV 10%

Cured but

recurrent BV

15%

9.3

1.6-53.5

Cured BV/ no

recurrence

2%

Results: Secondary Outcomes

Kekki 2001

Late Misc/PTB OR 95% CI

Persistent BV 28%

2.9

1.3-5.2

Cured BV 10%

Cured but

recurrent BV

15%

9.3

1.6-53.5

Cured BV/ no

recurrence

2%

Conclusions

• The appropriate antibiotics

– (clindamycin)

• Given to the appropriate women

– (those with objective evidence of abnormal genital tract

flora)

• At the appropriate time in pregnancy to prevent infection

and inflammatory tissue damage

– (<22 weeks)

• Significantly reduces the risk of LM (80%) & PTB (40%)

Lamont et al, Am J Obstet Gynecol. 2011 Sep;205(3):177-90.

Why is BV Poorly Understood?

• Etiology unknown

• Microbiology inconsistent

• Diagnosis difficult

– Quantitative not qualitative

• Inconsistent response to antibiotics

– Failure of treatment

– Recurrence

• Inconsistent outcomes

• Cynical view

– Fresh-faced board certified OB/GYNs

• Malodorous vaginal discharge

– Inconsistent cause/diagnosis/treatment/recurrence/outcome

• Boys Toys

– Robotic minimal access endoscopic surgery

Who Will Come to our Rescue and Make Us Understand?

Human Microbiome Project

(HMP)

HMP Background

•Human Genome Project

•100,000 genes predicted

•23,000 protein-coding genes found

•Human Supra-organism

•Human genome+ bacterial microbiome

•Bacterial cells 10x human somatic cells

•Human microbiome = collective genome of symbionts

•Bacterial genome provides traits that humans did not need to evolve

•Human Genome + Microbiome > 1,000,000 genes

• NIH investing $100,000,000 roadmap for medical research

i) Molecular microbiologists

ii) Bio-informatics & Computational statisticians

Relative abundance of vaginal

microbial communities using

culture-independent, molecular-

based techniques

Unpublished data pertaining to PPROM, Marian Kacerovsky, Czech Republic

Relative Abundance in Normal Flora (molecular techniques)

Lactobacillus crispatus dominated

Lactobacillus crispatus

Lactobacillus jensenii

Lactobacillus gasseri

Lactobacillus iners

Normal vaginal flora dominated by one, or at

most two of these four Lactobacillus species

Minimal diversity!

Relative Abundance in Abnormal Flora (molecular techniques)

Lactobacillus iners dominated

?abnormal

Definitely

abnormal

Maximal diversity?

Relative Abundance in Abnormal Flora (molecular techniques)

Maximal diversity!

What have we learned about BV

using molecular based techniques? • Diversity greater than in normal flora

• Not a single entity • Number of different sub-types of BV

• Anaerobe dominated

• L.iners dominated

• G. vaginalis + A. vaginae dominated

• Racial differences

• Likely to be different etiologies

• sexually transmitted

• Likely to induce different host responses

• Possibly different phenotypic outcomes

• Likely to respond differently to different antibiotics

• Explains a lot:

•Etiology,

•Bacteriology,

•Diagnosis,

•Treatment

•The “Bacterial Vaginosis Syndrome”

Aetiology of BV Lactobacilli

Potentially Pathogenic

Bacteria

• Hormonal Changes

• Phage Viruses

• Antibiotics

• Spermicides

• Douches

Potentially Pathogenic

Bacteria

Lactobacilli

• Vitamin D Deficiency

– “antibiotic vitamin”

– immunomodulator

• Genetic Factors

• race

• Sexual Transmission

• Is BV a sexually

transmitted infection?

Sexually Transmitted?

The Case For

• Sexually associated:

• Younger age of 1st sexual

experience

• Greater number of lifetime

sexual partners

• Change of partner

• Anecdotal evidence

• Gardner and Dukes (1955)

The Case Against

• Present in virgo intacta

• 30% prevalence in

women who have sex

with women

• No benefit of treating

male partner

There is probably at least one sub-type of BV which is sexually transmitted

“The Preterm Parturition Syndrome”

Nomenclature

• “The Preterm Parturition Syndrome”

• “The Bacterial Vaginosis Syndrome”

The Bacterial Vaginosis Syndrome

Sociodemographic Variables

Predisposing

factors

Etiological

factors

Mechanism

Condition

Phenotype

Antibiotics

Spermicides

Douches

Smoking Sexual Activity STIs Genetic

Factors

Vitamin D

Deficiency

Hormonal Changes Phage Viruses Sexual Transmission

Lactobacilli

Potentially Pathogen

Bacteria

Potentially Pathogen

Bacteria

Lactobacilli

Bacterial Vaginosis

Adverse OB Sequelae Adverse GYN Sequelae

PTL PET PPROM Late Misc. Infertility Early Misc. STI UTI PID Post

abortal

Sepsis

Post

Surgical

Infections

Thank you for your attention:

Clue cell? Galaxy? Universe? G.vaginalis?