Bacterial Vaginosis, Vulvovaginal
Candidiasis, and Trichomoniasis
Society of Armed Forces Medical Laboratory Scientists
(SAFMLS)
March 28-31, 2011
New Orleans, Louisiana
Richard Steece, Ph.D., D(ABMM)
DrRSteece@aol.com
Objectives
• To describe the etiology and epidemiology of
bacterial vaginosis (BV), trichomoniasis, and
vulvovaginal candidiasis
• To discuss the new STD Laboratory Treatment
Guidelines related to of bacterial vaginosis (BV),
trichomoniasis, and vulvovaginal candidiasis
• To provide a summary of the current clinical and
laboratory diagnostic procedures for bacterial
vaginosis (BV), trichomoniasis, and vulvovaginal
candidiasis
Vaginitis
• Vaginitis – vaginal irritation and discharge
– >10 million clinic visits annually
– >$500 million in health care annually
• Causes of Vaginitis
– Mechanical
– Chemical
– Infections
• >90% of cases caused by 3 “agents”
• >3 million clinic visits annually
Vaginitis
• Causes of Infectious Vaginitis
– Bacterial Vaginosis (40%-45%)
• Polymicrobial infection
– Prevotella sp., Mobiluncus sp., Ureaplasma,
Mycoplasma, and Gardnerella vaginalis
• G. vaginalis as a marker, not sole etiology
– Candida albicans (20%-25%)
– Trichomonas vaginalis (15%-20%)
Vaginitis Differentiation
Normal
Symptom
presentation
Vaginal discharge
Clear to
white
Clinical findings
Trichomoniasis
Candidiasis
Bacterial
Vaginosis
Itch, discharge, 50%
asymptomatic
Itch, discomfort,
dysuria, thick
discharge
Odor, discharge,
itch
Frothy, gray or yellowgreen; malodorous
Thick, clumpy, white
“cottage cheese”
Homogenous,
adherent, thin,
milky white;
malodorous “foul
fishy”
Cervical petechiae
“strawberry cervix”
Inflammation and
erythema
Vaginal pH
3.8 - 4.2
> 4.5
Usually < 4.5
> 4.5
KOH “whiff” test
Negative
Often positive
Negative
Positive
Lacto-bacilli
Motile flagellated
protozoa, many
WBCs
Few WBCs
Clue cells (> 20%),
no/few WBCs
Saline wet mount
KOH wet mount
Pseudohyphae or
spores if nonalbicans species
Bacterial Vaginosis (BV)
• Characterized by:
– Vaginal discharge
• Grey, thin homogeneous discharge
– Vulvar itching
– Irritation
– Odor
• Fishy amine odor when KOH is added to
discharge: “Whiff test”
Bacterial Vaginosis (BV)
• Caused by: overgrowth of bacterial species
normally present in the vagina with anaerobic
bacteria
• BV correlates with a decrease or loss of
protective lactobacilli:
• Vaginal acid pH normally maintained by lactobacilli through
metabolism of glucose/glycogen – produce lactic acid
• Hydrogen peroxide (H2O2) is produced by some
Lactobacilli,sp.
• H2O2 helps maintain a low pH, which inhibits bacteria
overgrowth
• Loss of protective lactobacilli may lead to BV
Bacterial Vaginosis - Epidemiology
• Most common cause of vaginitis
• Prevalence varies by population:
– 5%-25% among college students
– 12%-61% among STD patients
• Widely distributed
Bacterial Vaginosis - Epidemiology
• Linked to premature rupture of
membranes, premature delivery and low
birth-weight delivery, acquisition of HIV,
development of PID, and post-operative
infections after gynecological procedures
• Organisms do not persist in the male
urethra
Bacterial Vaginosis - Epidemiology
• Risk Factors
– African American
– Two or more sex partners in previous six
months/new sex partner
– Douching (prior 6 months)
– Absence of or decrease in lactobacilli
– Lack of H2O2-producing lactobacilli
Bacterial Vaginosis - Epidemiology
• Transmission
– Currently not considered a sexually
transmitted disease, but acquisition
appears to be related to sexual activity
• 15% of women with no history of sexual
intercourse were + for BV
Bacterial Vaginosis – Clinical
Presentation and Symptoms
• 50% asymptomatic
• Signs/symptoms when present:
– 50% report malodorous (fishy smelling)
vaginal discharge
– Reported more commonly after vaginal
intercourse and after completion of
menses
Bacterial Vaginosis
Associated Medical Complications
• Pregnancy
– Miscarriage
– Premature labor and delivery
– Post-caesarean delivery endometritis
• Non-pregnant
– PID
– Increased risk of other STD’s (HIV)
– Endometritis
Bacterial Vaginosis – Treatment
CDC - Recommended Regimens:
• Metronidazole 500 mg orally twice a day for
7 days, OR
• Metronidazole gel 0.75%, one full applicator
(5 grams) intravaginally, once a day for 5
days, OR
• Clindamycin cream 2%, one full applicator
(5 grams) intravaginally at bedtime for 7
days
Bacterial Vaginosis – Treatment
Alternative regimens:
• Tinidazole 2 g orally once daily for 2 days,
OR
• Tinidazole 1 g orally once daily for 5 days,
OR
• Clindamycin 300 mg orally twice a day for 7
days, OR
• Clindamycin ovules 100 g intravaginally
once at bedtime for 3 days
Bacterial Vaginosis
Treatment in Pregnancy
• Pregnant women with symptomatic disease should
be treated with
– Metronidazole 500 mg orally twice a day for 7 days, OR
– Metronidazole 250 mg orally three times a day for 7
days, OR
– Clindamycin 300 mg orally twice a day for 7 days
• Asymptomatic high-risk women (those who have
previously delivered a premature infant)
– Insufficient information to make recommendations at
this time
Bacterial Vaginosis - Diagnosis
• Clinical Criteria (Amsel’s Diagnostic Criteria)
 Vaginal pH >4.5
 Presence of >20% per HPF
of "clue cells" on wet mount
examination
Amsel Criteria:
Must have at least
three of the following  Positive amine or "whiff"
test
findings:
 Homogeneous, nonviscous, milky-white
discharge adherent to the
vaginal walls
Bacterial Vaginosis - Diagnosis
• Wet Prep
• pH
• KOH
• Gram Stain (Gold Standard)
– Nugent Score
• Lactobacilli (i.e., long Gram-positive rods), Gramnegative and Gram-variable rods and cocci (i.e., G.
vaginalis, Prevotella, Porphyromonas and
peptostreptococci), and curved Gram-negative rods
(i.e., Mobiluncus)
Organism Scoring Per Field and
Interpretation (Nugent Criteria)
•
•
•
•
•
•
Type
Number seen/OPF
None <1 1-5 5-30 >30
Lac
4
3
2
1
0
GVC 0
1
2
3
4
Mob
0
1
2
3
4
BV Scored Gram Stain Method
(Nugent NP. 1991. JCM. 29;297
• 0-3 = Normal
• 4-6 = Intermediate
– may indicate
trichomoniasis, GC or CT
– abnormal gram stain, but
not consistent with BV
• 7-10 = Consistent with
Bacterial Vaginosis
• Reports suggest a 89%
sensitivity for BV and an
83% specificity using
the scored gram stain
Bacterial Vaginosis - Diagnosis
• Pip Activity Test Card – Quidel
• OSOM BVBlue test
• Does not require Microscope
• 88% sens; 95% spec vs. Nugent
• 88% sens and 91% spec vs Amsel
– Bradshaw, Cs et al. JCM 2005; 43: 1304-8.
• Affirm VP III – Becton Dickinson
– DNA Probe
Vulvovaginal Candidiasis (VVC)
• Characterized by:
– Vulvar pruritis is most common symptom
– Thick, white, curdy vaginal discharge ("cottage
cheese-like“)
– Erythema, irritation, occasional erythematous
"satellite" lesion
– External dysuria and dyspareunia
– Symptoms are not specific for VVC
Source: Health Canada, Sexual Health and STI Section, Clinical Slide Gallery
Vulvovaginal Candidiasis (VVC)
• Etiologic agent
– Candida species are normal flora of the skin
and vagina
– VVC is caused by overgrowth of C. albicans
and other non-albicans species
– Yeast grows as oval budding yeast cells or as
a chain of cells (pseudohyphae)
– Symptomatic clinical infection occurs with
excessive growth of yeast
– Disruption of normal vaginal ecology or host
immunity can predispose to vaginal yeast
infections
Vulvovaginal Candidiasis (VVC)
• Candida species are normal flora of skin
and vagina and are not considered to be
sexually transmitted pathogens
Candidiasis - Epidemiology
• Affects most females during lifetime
– Estimated 75% of women will have >1
episode per lifetime, 40%-45% >2
• Most cases caused by C. albicans (85%90%)
• Second most common cause of vaginitis
(20%-25%)
• Estimated cost: $1 billion annually in the
U.S.
Candidiasis - Treatment
•
Recommended regimens
•
Over-the-Counter Intravaginal Agents:
–
–
–
–
–
–
–
–
–
•
Prescription Intravaginal Agents
–
–
–
–
–
•
Butoconazole 2% cream, 5 g intravaginally for 3 days
Clotrimazole 1% cream 5 g intravaginally for 7-14 days
Clotrimazole 2% cream 5 g intravaginally for 3 days
Miconazole 2% cream 5 g intravaginally for 7 days
Miconazole 4% cream 5 g intravaginally for 3 days
Miconazole 100 mg vaginal suppository, 1 suppository for 7 days
Miconazole 200 mg vaginal suppository, 1 suppository for 3 days
Miconazole 1,200 mg vaginal suppository, 1 suppository for 1 day
Tioconazole 6.5% ointment 5 g intravaginally in a single application
Butoconazole, 2% cream(single dose bioadhesive product), 5 g intravaginally for 1 day
Nystatin, 100,000 unit vaginal tablet, one tablet for 14 days
Terconazole 0.4% cream 5 g intravaginally for 7 days
Terconazole 0.8% cream 5 g intravaginally for 3 days
Terconazole 80 mg vaginal suppository, 1 suppository for 3 days
Oral agent:
–
Fluconazole 150 mg oral tablet, 1 tablet in a single dose
Note: The creams and suppositories in this regimen are oil-based and may weaken latex condoms and diaphragms.
Candidiasis - Diagnosis
• Clinically
– History, signs and symptoms
• Direct Observation
– Visualization of pseudohyphae (mycelia) and/or
budding yeast (conidia) on KOH or saline wet
mount
– pH normal (4.0 to 4.5)
• If pH > 4.5, consider concurrent BV or Trichomonas
– Cultures may be useful in symptomatic women
with negative wet mount
– Gram Stain
PMNs and Yeast Buds
Saline: 40X objective
Folded squamous
epithelial cells
PMNs
Yeast
buds
Source: Seattle STD/HIV Prevention Training Center at the University of Washington
Yeast Pseudohyphae
10% KOH: 10X objective
Masses of yeast
pseudohyphae
Lysed
squamous
epithelial cell
Source: Seattle STD/HIV Prevention Training Center at the University of Washington
Candidiasis - Diagnosis
• Clinically
– History, signs and symptoms
• Direct Observation
– Visualization of pseudohyphae (mycelia) and/or
budding yeast (conidia) on KOH or saline wet
mount
– pH normal (4.0 to 4.5)
• If pH > 4.5, consider concurrent BV or Trichomonas
– Cultures may be useful in symptomatic women with
negative wet mount
– Gram Stain
• DNA Probe
• BD Affirm VPIII Microbial ID Test
Trichomonas vaginalis
• Characterized by:
– May be asymptomatic in women
– Vaginitis
•
•
•
•
Frothy gray or yellow-green vaginal discharge
Strong odor
Pruritus
Cervical petechiae ("strawberry cervix") classic presentation, occurs in minority of
cases
• May also infect Skene's glands and urethra,
where the organisms may not be susceptible
to topical therapy
Source: Claire E. Stevens/Seattle STD/HIV Prevention Training Center at the University of Washington
Trichomonas vaginalis
• Etiologic agent
– Trichomonas vaginalis - flagellated anaerobic
protozoa
– Only protozoan that infects the genital tract
• Possible association with
– Pre-term rupture of membranes and pre-term
delivery
– Increased risk of HIV acquisition
Trichomonas vaginalis -Epidemiology
• Most common treatable STD
• Estimated 7.4 million cases annually in the U.S.
at a medical cost of $375 million
• Estimated prevalence:
– 2%-3% in the general female population
– 50%-60% in female prison inmates and commercial
sex workers
– 18%-50% in females with vaginal complaints
Trichomonas vaginalis -Epidemiology
• Risk Factors
– Multiple sexual partners
– Lower socioeconomic status
– History of STDs
– Lack of condom use
Trichomonas vaginalis -Epidemiology
• Transmission
– Almost always sexually transmitted
– T. vaginalis may persist for months to
years in epithelial crypts and
periglandular areas
– Transmission between female sex
partners has been documented
Trichomonas vaginalis -Epidemiology
• Clinical Manifestations
– May cause up to 11%-13% of nongonococcal
urethritis in males
– Urethral trichomoniasis has been associated
with increased shedding of HIV in HIVinfected men
– Frequently asymptomatic
Trichomonas vaginalis - Treatment
• Recommended Regimens
– Metronidazole 2 g orally in a single dose
OR
– Tinidazole 2 g orally in a single dose
Trichomonas vaginalis - Diagnosis
• Direct Observation
• Vaginal pH >4.5 often present
• Wet Mount (50%-90%)
• KOH “Wiff” Prep
Trichomonas
Trichomonas*
Trichomonas vaginalis - Diagnosis
Culture (Gold Standard – 85%-90% Sensitive)
• Diamond’s, others
• In-Pouch (BioMed Diagnostics)
Trichomonas vaginalis - Diagnosis
Point of Care Tests (POCTs)
• OSOM Trichomonas Rapid Test (Genzyme)
– Compared to wet mount sensitivity (94.7%), specificity
(100%), positive predictive value (100%), negative
predictive value (99.9%)
• Campbell, et. al., J. Clin. Micro. 2008. Oct;46(10):3467-9
• XenoStrip – Tv (Xenotope Diagnostics)
– Compared to wet mount 6% positive vs. 8.4% XenoStrip
• Pillay et. al., J.Clin. Micro. 2004. Aug;42(8):3853-6
Trichomonas vaginalis - Diagnosis
Nucleic Acid Amplification Tests (NAATs)
• Gen-Probe Trichomonas Test
• PCR Tests available (research sites)
Trichomonas vaginalis - Diagnosis
DNA Probe
• BD Affirm VPIII Microbial ID Test
• Trichomonas
• Candida species
• Gardnerella
AFFIRM PROBE TECHNOLOGY
•
•
Sample Lysis & Preparation:
10 minutes
Two distinct nucleic acid
probes for each organism
capture probe (PAC)
PAC goes in well #1 with
sample
color development probe
(well #2)
33 minutes on instrument
•
Candida sp.
– 1 x 10 (4) CFU/ml
•
Trichomonas vaginalis
– 5 x 10 (3) trichomonads/ml
•
Gardnerella vaginalis
– 2 x 10 (5) CFU/ml
Reagent Casette
Place Reagent Cassette
On Processor & Press Run
5
3
1
2
4
6
AFFIRM VPIII vs Wet Mount
N=425
AFFIRM VPIII
Number POS
Wet Mount
Number POS
Trichomonas
30 (7%)
23 (5%)
Candida
45 (11%)
31 (7%)
BV
190 (45%)
58 (14%)
Brown HL et al. ID OBGYN 2004. 12:17-21
Why Use A Probe For Detection Of Vaginitis?
• Advantages
–
–
–
–
Rapidity of results
Increased Sensitivity
Multiple infections detected
May be used in outpatient
facilities
– Reproducibility of testing
– Less expensive than
NAATs
• Disadvantages
– Cannot run assay for only
one analyte; but we
probably should look for
co-infections
– Few requests made to
clinical laboratories: need
to educate
– Cost as compared to what
is now done in most
laboratories
Summary
• Infectious vaginitis is most often caused by 3 entities:
– T. vaginalis
– Candida sp.
– Bacterial vaginosis – polymicrobial
• Sexually Transmitted Diseases Treatment Guidelines,
2010
– Recommendations for treatment
– http://www.cdc.gov/std/treatment/2010/default.htm
• Various clinical observations/tests are available
– BD Affirm is an economical, sensitive, and specific
test which is able to detect all three causes of
infectious vaginitis