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Leukorrhea Panel

What is Leukorrhea?

Leukorrhea means increased vaginal discharge; however,


the term is often used loosely to include any abnormal
vaginal discharge, even blood-tinged.

A symptom rather than a disease, leukorrhea is one of the


most common complaints for which a patient seeks help,
particularly if the discharge is accompanied by itching and
burning.

Microscopic examination revealing pus cells (>10 White


Blood Cells/ High power field) can confirm the diagnosis
of leukorrhea (1).

There are two classifications of Leukorrhea:


Pathologic leukorrhea is usually due to infections
of the upper and lower female genital tract. The most
common sexually transmitted pathogens associated
with leukorrhea are Chlamydia trachomatis, Neisseria
gonorrhoeae, and Trichomonas vaginalis. Leukorrhea
may be the only presenting sign in women infected with
these pathogens (1-3).

Physiologic leukorrhea is caused by congestion


of the vaginal mucosal membranes due to hormonal
stimulation. This may occur during ovulation and
pregnancy.

Medical Diagnostic Laboratories, L.L.C. (MDL), is currently


the only clinical laboratory in the United States that offers
this panel of testing which not only detects these pathogens
but also provides reflex antibiotic resistance testing at no
additional charge.

Test 121 Leukorrhea Panel (Neisseria gonorrhoeae,


Chlamydia trachomatis, Trichomonas vaginalis) by
Real-Time PCR (Reflex to antimicrobial resistance
Profile)
Includes:

Chlamydia trachomatis

Azithromycin

Neisseria gonorrhoeae
Azithromycin
Ciprofloxacin
Cephalosporin
Penicillin
Spectinomycin
Tetracycline

Trichomonas vaginalis
Metronidazole

This testing is currently available utilizing the OneSwab,


UroSwab (males and females), and ThinPrep specimen
collection platforms.

Test 105: Chlamydia trachomatis by Real-Time PCR


(Reflex to azithromycin resistance by Pyrosequencing).
This assay detects a single nucleotide polymorphism
(SNP) associated with azithromycin resistance utilizing
pyrosequencing. This technology can accurately detect the
presence or absence of this SNP, thus providing additional
molecular evidence for resistance to azithromycin. This
reflex test is performed at no additional charge on all
OneSwab,UroSwab and ThinPrep specimens that test
positive for C. trachomatis.

Test 167: Neisseria gonorrhoeae by Real-Time


PCR (Reflex to antibiotic resistance by Molecular
Analysis). This assay provides a simple method of
determining gonorrhea infections and the assessment of
N. gonorrhoeae-specific genetic markers of resistance to
six classes of antibiotics. This assay does not involve the
isolation of live bacterial cells from the specimen. Instead,
it screens for N. gonorrhoeae specific genes and mutations
from DNA extracted from the OneSwab, UroSwab and
ThinPrep collection systems. This reflex test is performed
at no additional charge on all OneSwab, UroSwab and
ThinPrep specimens that test positive for N. gonorrhoeae.

Test 111: Trichomonas vaginalis by Real-Time


PCR (Reflex to metronidazole resistance). Although
metronidazole treatment is reported to be 85%-95%
effective, recent reports suggest that between 2.5%
and 10% of clinical T. vaginalis isolates exhibit some
degree of metronidazole-resistance. MDL can now detect
metronidazole resistance in a subset of T. vaginalis
specimens by Real-Time PCR. Our current assay detects
a mutation that encodes a K80STOP change in the Tvntr6
protein, and has 40% sensitivity, 96% specificity, and a
91% positive predictive value (PPV) for the detection of
T. vaginalis metronidazole resistance. This reflex test
is performed at no additional charge on all OneSwab,
UroSwab and ThinPrep specimens that test positive for
T. vaginalis.

REFERENCES:
1. Elkabbakh GT, Elkabbakh GD, Broekhuizen F, Griner
BT. 1995. Value of wet mount and cervical cultures at the
time of cervical cytology in asymptomatic women. Obstet
Gynecol 85(4): 449-503.
2. Hakakka MM. 2002. Leukorrhea and bacterial vaginosis
as in-office predictors of cervical infection in high-risk
women. Obstet Gynecol 100(4): 808-812.
3. Marrazzo JM, Handsfield HH, Whittington WLH. 2002.
Predicting chlamydial and gonococcal cervical infection:
implications for management of cervicitis. Obstet Gynecol
100: 57984.

Medical Diagnostic Laboratories, L.L.C. www.mdlab.com 877.269.0090

Upd: 3/2016

Chlamydia trachomatis

by Real-time PCR (Reflex to azithromycin resistance by Pyrosequencing)

The sensitivity and specificity of the nucleic acid amplification


tests (NAATs) are clearly the highest of any of the test
platforms for the diagnosis of chlamydial and gonococcal
infections. Since accurate diagnosis is the goal, there is no
justification for the ongoing use of other technologies(1, 2). Centers for Disease Control and Prevention (CDC)
MDL provides detection of C. trachomatis by Real-Time PCR,
one of the most powerful and sensitive gene analysis techniques
available.
Sensitivity and specificity up to 99%.
Test results are typically available within 24-48 hours.
This test has been validated for detection of C. trachomatis using
the OneSwab, UroSwab (males and females), and ThinPrep.

Molecular Microbiology

C. trachomatis has a genome that consists of 1,042,519


nucleotide base pairs and has approximately 894 likely
protein coding sequences. C. trachomatis strains have an
extrachromosomal plasmid, which was sequenced to be a
7,493 base pair plasmid (4). There are 15 distinct serovariants.
Serovariants A-C are associated with Trachoma, D-K with
oculo-urogenital disease, and L1-3 with LGV.
Human C. trachomatis isolates are highly conserved with one
another, having a reported 1% variation in their nucleotide
sequences.
In 2006, a spontaneous variant of the cryptic plasmid was
discovered in Sweden in the serovar E, designated nvCT (6).
The change consisted of a 377 base pair deletion within the
coding sequence of CDS 1.
The target DNA of the MDL Real-Time PCR for C. trachomatis
Assay is the ORF8 of the cryptic plasmid pLGV440 which is
found in all 15 serovariants (Accession numbers: DQ06813
to DQ63827; GI: 73544092 to 7354418). The MDL assay is
capable of identifying all 15 serovariants, including the recently
discovered Swedish variant, nvCT (based upon an analysis of
the published genomic information).
Azithromycin resistance- A single nucleotide polymorphism
(SNP) was identified in domain V of the 23s rRNA of C.
trachomatis consistently associated with resistance to
azithromycin. This substitution of thymine to guanine (TG)
occurs at the position 2611 (T2611G) (12,13). MDL has
developed a test to detect this SNP utilizing pyrosequencing
which can accurately discover the presence or absence of
this SNP, thus providing additional molecular evidence for
resistance to azithromycin. This is provided as a reflex test
at no additional charge. Currently, MDL is the only medical
laboratory in the United States offering this service.

Annual screening of all sexually active women aged 25 years


is recommended, as is screening of older women with risk
factors (2).
All pregnant women should be routinely screened for C.
trachomatis during their first prenatal visit. Women aged 25
years and those at increased risk for chlamydia (e.g., women
who have a new or more than one sex partner) should also
be retested during the third trimester to prevent maternal
postnatal complications and chlamydial infection in the infant.
Women found to have a chlamydial infection during the first
trimester should be retested within approximately 36 months,
preferably in the third trimester.
The screening of sexually active young men should also be
considered in clinical settings with a high prevalence of C.
trachomatis, such as adolescent clinics, correctional facilities,
and STD clinics (2).
The World Health Organization has reported that infections
with C. trachomatis are responsible for about 3.6% of cases of
blindness in the world (3).

Clinical Significance
C. trachomatis is transmitted through infected secretions
only. It infects mainly mucosal membranes, such as the
cervix, rectum, urethra, throat, and conjunctiva. It is primarily
spread via sexual contact and manifests as a sexually
transmitted disease. Symptoms and physical findings are
usually nonspecific.

Up to 50% of men with chlamydial urethral infections, and
up to 75% of women with cervicitis, are asymptomatic. The
history may be crucial for the risk assessment of exposure.
However, a number of clinical syndromes require further
evaluation for C. trachomatis infection.

Definitive diagnosis of C. trachomatis infection for all
conditions is obtained with nucleic acid amplification tests.

Persons who are diagnosed with C. trachomatis infection
should be tested for other STDs, including Neisseria
gonorrhoeae.
Table 1: Summary of Clinical Manifestations.

Clinical condition
Cervicitis
Women

Epidemiology

Urogenital infections with C. trachomatis are amongst the most


common sexually transmitted reportable diseases in the United
States and the world. In women, the most serious complications
are Pelvic Inflammatory Disease (PID), ectopic pregnancy,
and infertility (2). In the United States, 1,244,180 cases of
C. trachomatis urogenital infection were reported to the CDC
in 2009 (3). However, many infections are not detected, and
an estimated 2.8 million infections occur in the United States
annually (3).

Men

Salpingitis (PID)
Urethritis (Urethral
Syndrome)
Nongonococcal
Urethritis (NGU)
Postgonococcal
Urethritis (PGU)
Epidydimitis Orchiitis
Proctitis

Adults

Neonates

Conjunctivitis
Conjunctivitis
Pneumonia

Signs and Symptoms

75% asymptomatic, mucopurulent discharge,


bleeding.
Adnexal , lower abdominal pain on direct
palpation and cervical motion tenderness.
Dysuria, urgency, frequency, pyuria, no
hematuria, Reiters syndrome.
Dysuria, urgency, frequency, pyuria, Reiters
syndrome.
Same as NGU.
Pain tenderness, swelling, fever presence of
NGU.
Rectal pain, bleeding, discharge.
Ocular pain, redness, discharge in association
with urogenital C. trachomatis infection.
Consider in all neonates with conjunctivitis
aged 30 days, especially if the mother has a
history of untreated C. trachomatis infection.
Staccato cough, lung hyperinflation,
eosinophilia.

Medical Diagnostic Laboratories, L.L.C. www.mdlab.com 877.269.0090

Table 2. Comparison of Multiple Assay Systems for the Detection of Chlamydia trachomatis.
Test

Prevalence (%)

Sensitivity (%)

Specificity (%)

PPV (%)

NPV (%)

References

PCR

1000

12.9

98

100

100

100

(14)

Amplicor

2254

7.5

96.9

98.6

84.9

99.7

(15)

Aptima Combo 2

1389

15.0

94.2

97.6

87.4

99.0

(16)

BD Probe Tec

1419

9.9

98.7

97.8

84.8

99.1

(17)

3.9

75.5

97.0

50.5

99.0

(18)

GEN-PROBE
940
(Pace 2)
= Unless otherwise noted, all specimens are swabs
= Calculated data

Treatment
Table 3. Current Recommendations from the CDC for Uncomplicated
C. trachomatis Infection of the Genito-Urinary Tract (19).

Table 4. Current Recommendations from the CDC for C. trachomatis Infection


in pregnant* women (2).

Recommended Regimens-Adults & Adolescents

Recommended Regimens

Azithromycin 1 g orally in a single dose OR

Azithromycinf 1 g orally in a single dose

Doxycycline a 100 mg orally twice a day for 7 days

Alternative Regimens

Alternative Regimens

Amoxicillin 500 mg orally three times a day for 7 days OR

Erythromycin base 500 mg orally four times a day for 7 days


OR
Erythromycin ethylsuccinatec 800 mg orally four times a day for
7 days OR

Erythromycin baseb,e 500 mg orally four times a day for 7 days


OR
Erythromycin base 250 mg orally four times a day for 14
days OR
Erythromycin
ethylsuccinate 800 mg orally four times a day for
Levofloxacind 500 mg orally once daily for 7 days OR
7 days OR
Erythromycin ethylsuccinate 400 mg orally four times a day for
Ofloxacine 300 mg orally twice a day for 7 days
14 days
a
Should not be administered during pregnancy, lactation, or to children <8 years of age.
b
If patient cannot tolerate high-dose erythromycin base schedules, change to 250 mg 4x/day for 14 days.
c
If patient cannot tolerate high-dose erythromycin ethylsuccinate schedules, change to 400 mg orally 4 times a day for 14 days.
d
Contraindicated for pregnant or lactating women.
e
Clinical experience and published studies suggest that azithromycin is safe and effective.
f
Erythromycin estolate is contraindicated during pregnancy.
* Complete treatment guidelines are available from the CDC http://www.cdc.gov/std/tg2015/default.htm (Accessed August 26, 2015).

Indicates update from the 2010 CDC Guidelines for the Treatment of Sexually Transmitted Diseases.
b

References:

1. APHL. 2009. Laboratory diagnostic testing for Chlamydia trachomatis and


Neisseria gonorrhoeae. http://www.aphl.org/aphlprograms/infectious/std/
Documents/CTGCLabGuidelinesMeetingReport.pdf. Date accessed: March
30, 2012.
2. Centers for Disease Control and Prevention (CDC). 2010. Sexually
Transmitted Disease Guidelines. MMWR 59: RR-12.
3. CDC. 2011. Grand Rounds: Chlamydia prevention: challenges and strategies
for reducing disease burden and sequelae. MMWR 60(12): 370-373.
4. Resnikoff S, Pascolini D, Etyaale D, et. al. 2004. Global data on visual
impairment in the year 2002. Bull World Health Org(82)11:844-51.
5. Stevens RS, Kalman S, Lammel C, et. al. 1998. Genome sequence of an
obligate intracellular pathogen of humans: Chlamydia trachomatis. Science
282(5389):754-9.
6. Unemo M, Seth-Smith HM, Cutcliffe LT, et. al. 2010. The Swedish new
variant of Chlamydia trachomatis: genome sequence, morphology, cell
tropism and phenotypic characterization. Microbiol 156: 1394-1404.
7. Darville T, Hiltke TJ. 2010. Pathogenesis of genital tract disease due to
Chlamydia trachomatis. JID 201(S2):S114S125.
8. Jolly M, Curran JJ. 2004. Chlamydial infection preceding the development
of rheumatoid arthritis: a brief report. Clin Rheumatol 5:453-5.
9. Bertshe A. 2008. An unusual manifestation of a neonatal Chlamydial
Infection. J Child Neurol 23: 948.
10. Gaydos C. 2012. in Harrisons Principles of Internal Medicine 18th Edition.
Chapter 176. U.S.A.: McGraw-Hill.
11. Stamm W. 2009. in Principles and Practices in Infectious Diseases, 7th
Edition. Chapter 180. Philadelphia: Churchill Livingstone.

12. Misyurina OY, Chipitsyna EV, Finashutina YP, et. al. 2004. Mutations in
a 23srRNA gene of Chlamydia trachomatis associated with resistance to
macrolides. Antimicrob Agents Chemother 48(4): 1347-1349.
13. Zhu H, Wang HP, Jiang Y, et. al. 2010. Mutations in 23srRNA and ribosomal
protein L4 account for resistance in Chlamydia trachomatis strains selected
in vitro by macrolide passage. Andrologia 42: 274-280.
14. Pasternack R, Vuorinen P, Pitkakarvi T, et al. 1997. Comparison of
manual Amplicor PCR, Cobas Amplicor PCR, and LCx assays for detection
of Chlamydia trachomatis infection in women by using urine specimens. J
Clin Microbiol 35(2):402-5.
15. 510(k) Summary, http://www.fda.gov/cdrh/pdf/k973707.pdf Device name:
Roche Amplicor CT/NG Test For Chlamydia trachomatis. Date accessed
06/2007.
16. Gaydos CA, Quinn TC, Willis D, et al. 2003. Performance of the APTIMA
Combo 2 assay for detection of Chlamydia trachomatis and Neisseria
gonorrhoeae in female urine and endocervical swab specimens. J Clin
Microbiol 41(1):304-9.
17. Van Der Pol B, Ferrero DV, Buck-Barrington L, et al. 2001. Multicenter
evaluation of the BDProbeTec ET System for detection of Chlamydia
trachomatis and Neisseria gonorrhoeae in urine specimens, female
endocervical swabs, and male urethral swabs. J Clin Microbiol 39(3):100816.
18. Blanding J, Hirsch L, Stranton L, et al. 1993. Comparison of the Clearview
Chlamydia, the PACE 2 assay, and culture for detection of Chlamydia
trachomatis from cervical specimens in a low-prevalence population. J Clin
Microbiol 31(6):1622-5.
19. CDC. 2015. Sexually Transmitted Diseases, Treatment Guidelines, 2015.
MMWR 64:72-75.

Medical Diagnostic Laboratories, L.L.C. www.mdlab.com 877.269.0090

Neisseria gonorrhoeae

by Real-time PCR (Reflex to antibiotic resistance by Molecular Analysis)

The sensitivity and specificity of the nucleic acid amplification


tests (NAATs) are clearly the highest of any of the test
platforms for the diagnosis of chlamydial and gonococcal
infections. Since accurate diagnosis is the goal, there is no
justification for the ongoing use of other technologies(1, 2).
- Centers for Disease Control and Prevention (CDC)



MDL provides detection of Neisseria gonorrhoeae by RealTime PCR, one of the most powerful and sensitive gene
analysis techniques available.
Sensitivity and specificity up to 99%.
Test results are typically available within 24-48 hours.
This test has been validated for detection of N. gonorrhoeae
using the OneSwab, UroSwab (males and females), and
ThinPrep.

Epidemiology

Gonorrhea is the second most commonly reported bacterial


STD in the United States with an estimated 700,000 new N.
gonorrhoeae infections occurring each year (7).
Due to the fact that gonococcal infections among women are
frequently asymptomatic, targeted screening of young women
at increased risk for infection is a primary component of
gonorrhea control in the United States (7).

Laboratory Diagnosis

Pathogenesis

Neisseria gonorrhoeae, a Gram-negative diplococci, is the


causative agent of gonorrhea.
Due to its affinity for columnar or pseudo stratified epithelium, it
is most commonly detected in the genital tract with the primary
site of involvement being the endocervical canal and transition
zone of the cervix.
N. gonorrhoeaes unique ability to alter surface structures
allows increased pathogenicity, facilitates epithelial surface
attachment, and enables evasion of the hosts immune
response.
Transmission of N. gonorrhoeae occurs almost exclusively
through sexual contact, though it can also be transmitted via
the passage of a neonate through an infected mothers birth
canal or via autoinoculation from the hands of an infected
person to their eye.
Incubation time for this infection is typically 3-5 days and
transmission more frequently occurs from male to female.
Some risks factors for infection include: low socioeconomic
status, early onset of sexual activity, unmarried status, a
history of previous gonorrhea infection, illicit drug abuse, and
prostitution.

chorioamnionitis, premature birth, intrauterine growth retardation,


neonatal sepsis, and postpartum endometritis.
During vaginal delivery with an infected mother, 30% to 35%
of neonates will acquire Neisseria gonorrhoeae which, if left
untreated, can progress to corneal ulceration and scarring, as
well as blindness called gonorrheal ophthalmia neonatorum.

Diagnosis of infections with N. gonorrhoeae has traditionally


relied upon Gram stain, culture, and immunochemical
techniques. Although culture techniques may be highly
specific, sensitivity is greatly impacted by the adequacy of the
clinical specimen and transport conditions, particularly when
transporting to off-site facilities.
Due to the genome plasticity of N. gonorrhoeae strains
circulating in the population, this bacterium has developed
resistance to multiple classes of antimicrobial agents, resulting
in decreased efficacy for gonorrhea therapy. An increase of
ceftriaxone-resistant N. gonorrhoeae demonstrated a similar
pattern to previous reports in Japan and Southeast Asia that
prompted the CDC to remove ciprofloxacin from the treatment
guidelines as a primary antibiotic (1-3).
In August 2012, the CDC called for Ceph-R NG surveillance
through N. gonorrhoeae antibiotic susceptibility testing for
patients that have failed treatment (3). Although susceptibility
testing by culture remains the standard for antibiotic
susceptibility determination in clinical microbiology, there are
inherent growth-related issues that can delay results by as
much as three days or more.
Known mechanisms of antibiotic resistance in N. gonorrhoeae
are linked to mutations in the chromosomal DNA as well as
the presence of plasmid-borne genes. Surveillance of genetic
markers of antibiotic resistance is important for the prediction of
clinical resistance as the antibiotic susceptibility signatures of
individual N. gonorrhoeae strains differ.

Clinical Significance

Clinical manifestation in men usually includes symptomatic


urethritis; however, pharyngeal, anorectal, and disseminated
infections are also possible.
In women, infections are often asymptomatic; however, when
manifested, symptoms may include: vaginal discharge, dysuria,
intermenstrual bleeding, menorrhagia, pelvic discomfort,
infection of the periurethral glands, Bartholin glands, and
anorectum.
Due to the fact that gonorrhea can have serious consequences
for both mother and neonate, it is crucial to screen pregnant
women for infection who reportedly have an incidence of
gonorrhea during pregnancy as high as 10%.
Complications that can occur during pregnancy include: amniotic
infection syndrome, premature rupture of the membranes,

NOTE: PenR = penicillinase producing Neisseria gonorrhoeae and chromosomally mediated


penicillin-resistant N. gonorrhoeae; TetR = chromosomally and plasmid mediated tetracyclineresistant N. gonorrhoeae; and QRNG = quinolone-resistant N. gonorrhoeae.

Figure 1: Gonococcal isolate surveillance project (GISP)-penicillin,


tetracycline, and ciprofloxacin resistance among GISP isolates, 2010 (7).

Test 167 Neisseria gonorrhoeae by Real-Time PCR (Reflex


to antibiotic resistance by Molecular Analysis) developed by
MDL, offers a valuable diagnostic tool for the reliable detection
of genetic determinants of antibiotic resistance, thereby
predicting antibiotic susceptibility of individual N. gonorrhoeae
strains in a given clinical specimen. This test addresses the
problem of genetic variability in N. gonorrhoeae and delivers
a prognostic recommendation for antibiotic therapy in a
personalized manner.

Medical Diagnostic Laboratories, L.L.C. www.mdlab.com 877.269.0090

Table 1: Comparison of Multiple Assay Systems for the Detection of Neisseria gonorrhoeae.
Test

Prevalence (%)

Sensitivity (%)

Specificity (%)

PPV (%)

NPV (%)

References

PCR

100

7.8

100

99.4

93.4

100

(16)

Amplicor

2238

5.2

96.3

98.7

80.2

99.8

(17)

Aptima Combo 2

1479

8.6

99.2

98.7

88.1

99.9

(18)

BD Probe Tec

1411

8.1

97.2

99.4

91.6

99.6

(19)

GEN-PROBE
(Pace 2)

1750

8.7

97.1

99.1

90.6

99.8

(20)

Culture

866

4.5

50.0

97.1

40.0

98.0

(21)

= Unless otherwise noted, all specimens are swabs.


= Calculated data.

Screening

Table 2: Summary of screening for N. gonorrhoeae infection by nucleic

acid amplification testing (NAAT) (derived from 7).


Women

Annual routine screening for all sexually active women at risk


for infection.
Screening at the first prenatal visit for all pregnant women at risk
or living in a high prevalence area.
In women with cervicitis via either vaginal, cervical, or urine
samples.

Men who have sex with men (MSM)

Screening for urethral infection via nucleic acid amplification


testing (NAAT) of urine in all men who have had insertive
intercourse the preceding year regardless of condom use.
Screening for rectal infection via nucleic acid amplification testing
(NAAT) of a rectal swab in all men who have had receptive anal
intercourse during the preceding year.
Screening for pharyngeal infection via nucleic acid amplification
testing (NAAT) in all men who have had receptive oral intercourse
during the preceding year.

Both Men and Women

Newly diagnosed HIV infection.

Treatment
Table 3: Current Recommendations from the CDC for adults, adolescents

& children >45 kg: uncomplicated infection of the cervix, urethra and
rectum (16).
Recommended Regimens
Ceftriaxone 250 mg IM in a single dose PLUS
Azithromycin a 1 g orally in a single dose
Alternative Regimens: If ceftriaxone is not available
Cefixime 400 mg orally in a single dose PLUS
Azithromycin b 1 g orally in a single dose
Alternative Regimens: If cephalosporin allergy
Gemifloxacin 320 mg orally in a single dose PLUS
Azithromycin 2 g orally in a single dose OR

a
b

Gentamicin 240 mg IM single dose PLUS


Azithromycin 2 g orally in a single dose

Effectiveness of erythromycin treatment is approximately 80%; a second course of


therapy may be required.
Clinical experience and published studies suggest that azithromycin is safe and effective.

Due to the concerns for developing patterns of antimicrobial


resistance, most current recommendations for treatment
should be followed. Guidance can be obtained from the CDC
website (http://www.cdc.gov/std/gisp) and state and local
health departments.
If treatment is still unsuccessful, contact the CDC for a
consultation.
Complete treatment guidelines are available from the CDC

References:
1. Miller WC, Ford CA, Morris M, et al. 2004. Prevalence of Chlamydia
Infections Among Young Adults in the United States. JAMA 291:22292236.
2. Monif GR, Baker DA. Infectious Diseases in Obstetrics and
Gynecology, Fifth Edition. New York, NY: The Parthenon Publishing
Group; 2004:222-233.
3. Mandell, GL, Bennett JE, Dolin R. Mandell, Douglas, and Bennetts
Principles and Practices of Infectious Diseases, Vol 2. Philadelphia:
Churchhill Livingstone; 2000:2242-2256.
4. Faro S, Soper DE. Infectious Diseases in Women. Philadelphia: W.B.
Saunders Company; 2001:435-450.
5. Girdner JL, Cullen AP, Salama TG, et al. (1998). ASM Meeting.
6. Donders GF, VanGerven V, de Wet HG, et al. 1996. Rapid antigen
tests for Neisseria gonorrhoeae and Chlamydia trachomatis are not
accurate for screening women with disturbed vaginal lactobacillary flora
Scan J Infect Dis. 28:559-562.
7. CDC. 2010. Sexually Transmitted Disease Guidelines. MMWR Vol. 59,
no. RR-12.
8. Girdner JL, Cullen AP, Salama TG, et al. 1998. ASM Meeting.
9. Vlaspolder F, Mustaers JA, Blog F, et al. 1993. Value of a DNA probe
assay (Gen-Probe) compared with that of culture for diagnosis of
gonococcal infection. J Clin Microbiol 31:107-110.
10. Cullen A, He L, Arthur P, et al. 1998. ASM Meeting.
11. Donders GF, VanGerven V, de Wet HG, et al. 1996. Scan J Infect Dis
28:559-562.
12. Johnson RE, Newhall WJ, Papp JR, et al. 2002. Screening
tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae
infections--2002.
13. CDC. 2002. MMWR Recomm Rep 2002 18;51(RR-15):1-38.
14. van Doornum GJ, Schouls LM, Pijl A, et al. 2001. Comparison
between the LCx Probe System and the COBAS AMPLICOR System
for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae
Infections in Patients Attending a Clinic for Treatment of Sexually
Transmitted Diseases in Amsterdam, The Netherlands. J Clin Microbiol
39(3):829-835.
15. Palmer HM, Mallinson H, Wood RL, Herring AJ. 2003. Evaluation of
the Specificities of Five DNA Amplification Methods for the Detection of
Neisseria gonorrheae. J Clin Microbiol 41(2):835-837.
16. Gaydos CA, et al. 2003. Performance of the APTIMA Combo 2 assay
for detection of Chlamydia trachomatis and Neisseria gonorrhoeae
in female urine and endocervical swab specimens. J Clin Microbiol
41(1):304-9.
17. Van Der Pol B, et al. 2001. Multicenter evaluation of the BDProbeTec
ET System for detection of Chlamydia trachomatis and Neisseria
gonorrhoeae in urine specimens, female endocervical swabs, and male
urethral swabs. J Clin Microbiol 39(3):1008-16.
18. Crotchfelt KA, et al. 1997. Detection of Neisseria gonorrhoeae and
Chlamydia trachomatis in genitourinary specimens from men and
women by a coamplification PCR assay. J Clin Microbiol 35(6):1536-40.
19. 510(k) Summary, http://www.fda.gov/cdrh/pdf/k974503. pdf. Device
name: Roche Amplicor CT/NG Test For Neisseria gonorrhoeae.
Accessed 06/2007.
20. Vlaspolder F, et al. 1993. Value of a DNA probe assay (Gen-Probe)
compared wh that of culture for diagnosis of gonococcal infection. J Clin
Microbiol 31(1):107-10.
21. Danders GG, et al. 1996. Rapid antigen tests for Neisseria gonorrhoeae
and Chlamydia trachomatis are not accurate for screening women with
disturbed vaginal lactobacillary flora. Scand J Infect Dis 28(6):559-62.
22. CDC. 2015. Sexually Transmitted Diseases, Treatment Guidelines,
2015. MMWR 64:72-75.

http://www.cdc.gov/std/tg2015/default.htm (Accessed August 26, 2015).

Medical Diagnostic Laboratories, L.L.C. www.mdlab.com 877.269.0090

Trichomonas vaginalis

by Real-time PCR (Reflex to metronidazole resistance)

T. vaginalis is a flagellated, anaerobic protozoan and is the most


common non-viral sexually transmitted pathogen. Approximately
half of female T. vaginalis infections are asymptomatic, as are
most male infections (1). Symptomatic infections manifest as
Trichomoniasis with symptoms of discharge (yellow, green or gray,
sometimes frothy), odor, itching, and pain during urination and/
or intercourse. Signs of infection include small red ulcerations on
the vagina and/or cervix, positive amine (whiff) test and elevated
pH. Wet-mount microscopy of a vaginal swab often reveals white
blood cells and rapidly motile trichomonads. However, detection of
trichomonads by microscopy has a sensitivity of only 60%-75%,
whereas, polymerase chain reaction (PCR) can detect T. vaginalis
with a sensitivity of 85%-100% (2,3). Trichomoniasis is associated
with a number of serious clinical complications, as pregnant
women with Trichomoniasis are at increased risk for pre-term
labor and delivery of low birth weight neonates (4,5). In addition,
Trichomoniasis is associated with HIV transmission (6, 7). Patients
are normally treated with a single oral dose of metronidazole, an
antibiotic used to treat infections caused by anaerobic bacteria and
parasites. Although generally effective, some T. vaginalis strains
are resistant to metronidazole. If metronidazole treatment fails, the
only other approved treatment for Trichomoniasis is the related
drug tinidazole. Therefore, identifying Trichomoniasis resistance
to metronidazole can help guide clinicians in prescribing effective
therapy for Trichomoniasis patients.

Epidemiology

There are more than seven million cases of Trichomoniasis


each year in the United States (3).
The overall prevalence of T. vaginalis among American
women is 3.2%, but varies dramatically by race, from 1.3%
for non-hispanic white women to 13.3% for non-hispanic
black women (8).
Most sexually-transmitted infections are more prevalent
among adolescents and young adults; however,
Trichomoniasis has a similar prevalence among sexually
active women of different age groups (3).
Although metronidazole treatment is reported to be 85%95% effective, recent reports suggest that between 2.5% and
10% of clinical T. vaginalis isolates exhibit some degree of
metronidazole-resistance (9-11).

Laboratory Diagnosis

Clinical Benefits of Testing


T. vaginalis attaches to the vaginal epithelium. Several T.


vaginalis adhesins, substances that enable the attachment
to epithelial surfaces, have been identified that mediate this
binding (12).
After binding, T. vaginalis triggers detachment of cells
through proteolytic activity, cytotoxicity and apoptosis (3).
Patients infected with T. vaginalis produce circulating (IgG)
and secreted (IgA) antibodies that recognize adhesins
and prevent parasite adhesion; however, protection is only
short-term as re-infection rates as high as 30% have been
observed (3).

This testing is currently available utilizing the OneSwab,


UroSwab (males and females), and ThinPrep specimen
collection platforms for the detection of T. vaginalis and associated
metronidazole resistance in cervico-vaginal specimens.
Detection of metronidazole resistance can assist clinicians in
administering effective treatment for Trichomoniasis patients.

Treatment Considerations
Table 2. Current Recommendations from the CDC for persistant or recurrent

T. vaginalis Infection (15).

Pathogenesis

A cervico-vaginal specimen can be submitted for laboratory


testing to detect T. vaginalis. Detection of trichomonads by PCR
has a sensitivity of 85%-100% (3).
Currently, only the Centers for Disease Control and Prevention
(CDC) can determine metronidazole susceptibility for T.
vaginalis. A viable culture of T. vaginalis must be received, using
a specialized collection and transport device.
Medical Diagnostic Laboratories, L.L.C. (MDL), can now detect
metronidazole resistance in a subset of T. vaginalis specimens
by Real-Time PCR. Our current assay detects a mutation that
encodes a K80STOP change in the Tvntr6 protein and has 40%
sensitivity, 96% specificity, and a 91% positive predictive value
(PPV) for the detection of T. vaginalis metronidazole resistance.
This test was developed using 100 well-characterized T.
vaginalis isolates from the CDC.
Test 111 Trichomonas vaginalis by Real-Time PCR (Reflex
to metronidazole resistance) developed by MDL, offers a
valuable diagnostic tool for the reliable detection of genetic
determinants of antibiotic resistance, thereby predicting antibiotic
susceptibility of T. vaginalis in a given clinical specimen. This
test delivers a prognostic recommendation for antibiotic therapy
in a personalized manner.
Currently, MDL is the only medical laboratory in the United
States to offer a reflex assay for metronidazole resistance at no
additional charge.

a
b

Recommended Regimens
Metronidazole a 2 g orally in a single dose OR
Tinidazole b 2 g orally in a single dose
Alternative Regimens
Metronidazole a 500 mg orally twice a day for 7 days
If This Regimen Fails
Metronidazole 2g orally twice a day for 7 days OR
Tinidazole 2 g orally for 7 days

Pregnant patients can be treated with 2 g single dose.


Randomized controlled trials comparing single 2 g doses of metronidazole and tinidazole
suggest that tinidazole is equivalent to,or superior to, metronidazole in achieving
parasitologic cure and resolution of symptoms.

Medical Diagnostic Laboratories, L.L.C. www.mdlab.com 877.269.0090

Patients should avoid alcohol during metronidazole or


tinidazole treatment, as well as for 24 hours after the end
of metronidazole treatment and 72 hours after the end of
tinidazole treatment.
In asymptomatic pregnant women, clinicians should
counsel patients regarding the potential risks and benefits
of treatment and communicate the option of therapy deferral
until after 37 weeks gestation
All symptomatic pregnant women should not only be
considered for treatment regardless of pregnancy stage,
but be provided careful counseling regarding condom use
and the continued risk of sexual transmission.
If treatment is still unsuccessful, contact the CDC for a
consultation.
The CDC recently reported an increase in treatment
success for women with Trichomoniasis that previously
failed metronidazole therapy by utilizing susceptibility
testing to tailor subsequent treatment (16).
All T. vaginalis positive results for specimens collected using
the MDL OneSwab, UroSwab and ThinPrep platforms are
further tested for metronidazole resistance at no additional
charge. This additional assay also serves to confirm the
initial positive result. This information assists clinicians
in administering an effective diagnosis and treatment for
their patients and is especially useful for those patients
presenting with recurring trichomoniasis. Information about
how the assay is performed, assay interpretation, and the
CDC 2015 STD Treatment Guidelines are distributed (1517).
Complete treatment guidelines for Trichomoniasis
are available from the CDC http://www.cdc.gov/std/
treatment/2010/vaginal-discharge.htm. (Accessed March
28, 2012). The guidelines include alternative treatment
therapy for cases of metronidazole treatment failure.

Frequently Asked Questions (FAQ)


What does a positive result mean for the detection of


the Tvntr6 K80STOP mutation?
A positive result indicates a >90% likelihood that the T.
vaginalis present in the specimen exhibits some degree of
resistance to metronidazole. It is not known if this level of
resistance is associated with clinical failure to metronidazole
treatment.

What does a negative result mean for the detection of


the Tvntr6 K80STOP mutation?
As our current assay only detects 40% of resistant T.
vaginalis isolates; a negative result is inconclusive. It does
not mean that the T. vaginalis in question is susceptible or
resistant to metronidazole.

References
1. Satterwhite CL, Torrone E, Meites E, et al. 2013.Sexually
transmitted infections among US women and men: prevalence
and incidence estimates, 2008. Sex Transm Dis 40:18793.
2. Krieger JN, Tam MR, Stevens CE, et al. 1988. Diagnosis
of trichomoniasis. Comparison of conventional wet-mount
examination with cytologic studies, cultures, and monoclonal
antibody staining of direct specimens. JAMA 259:1223-7.
3. Schwebke JR, Burgess D. 2004. Trichomoniasis. Clin
Microbiol Rev 17:794-803, table of contents.
4. Cotch MF, Pastorek JG 2nd, Nugent RP, et al.1997.
Trichomonas vaginalis associated with low birth weight and
preterm delivery. The Vaginal Infections and Prematurity
Study Group. Sex Transm Dis 24:353-60.
5. Minkoff H, Grunebaum AN, Schwarz RH, et al. 1984. Risk
factors for prematurity and premature rupture of membranes:
a prospective study of the vaginal flora in pregnancy. Am J
Obstet Gynecol 150:965-72.
6. Laga M, Manoka A, Kivuvu M, et al. 1993. Non-ulcerative
sexually transmitted diseases as risk factors for HIV-1
transmission in women: results from a cohort study. AIDS
7:95-102.
7. Wang CC, McClelland RS, Reilly M, et al. 2001. The effect
of treatment of vaginal infections on shedding of human
immunodeficiency virus type 1. J Infect Dis 183:1017-22.
8. Sutton M, Sternberg M, Koumans EH, et al. 2007. The
prevalence of Trichomonas vaginalis infection among
reproductive-age women in the United States, 2001-2004.
Clin Infect Dis 45:1319-26.
9. Lossick JG. 1990. Treatment of sexually transmitted
vaginosis/vaginitis. Rev Infect Dis 12 Suppl 6:S665-81.
10. Schmid G, Narcisi E, Mosure D, et al. 2001. Prevalence
of metronidazole-resistant Trichomonas vaginalis in a
gynecology clinic. J Reprod Med 46:545-9.
11. Schwebke JR, Barrientes FJ. 2006. Prevalence
of Trichomonas vaginalis isolates with resistance to
metronidazole and tinidazole. Antimicrob Agents Chemother
50:4209-10.
12. Hirt RP, Noel CJ, Sicheritz-Ponten T, et al. 2007.
Trichomonas vaginalis surface proteins: a view from the
genome. Trends Parasitol 23:540-7.
13. Paterson BA, Tabrizi SN, Garland SM, et al. 1998. The
tampon test for trichomoniasis: a comparison between
conventional methods and a polymerase chain reaction
for Trichomonas vaginalis in women. Sex Transm Infect
74(2):136-9.
14. Andrea SB, Chapin KC. 2011. Comparison of Aptima
Trichomonas vaginalis Transcription-Mediated Amplification
Assay and BD Affirm VPIII for detection of T. vaginalis
in symptomatic women: performance parameters and
epidemiological implications. J Clin Microbiol 49: 866-9.
15. CDC. 2015. Sexually Transmitted Diseases, Treatment
Guidelines, 2015. MMWR 64:72-75.
16. Bosserman EA, Helms DJ, Mosure DJ, et al. 2011. Utility of
antimicrobial susceptibility testing in Trichomonas vaginalisinfected women with clinical treatment failure Sex Trans Dis
38(10):983-987.
17. Paulish-Miller TE, Augostini P, Schuyler JA, et al. 2014.
Antimicrob Agents Chemother 59(5):2938-2943.

Medical Diagnostic Laboratories, L.L.C. www.mdlab.com 877.269.0090

MDL-developed molecular assays undergo a rigorous validation process to assure tests of the highest quality. We believe that
using molecular methods such as PCR to both detect the pathogen and identify mutations and genes strongly associated with
antibiotic resistance in difficult-to-culture organisms such as Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas
vaginalis is important. Additionally, our reflex antibiotic susceptibility genotyping assays provide confirmation of the initial positive
PCR result. This tailored medicine molecular approach of pathogen detection and reflex antibiotic susceptibility genotyping
provides clinicians with additional information in addition to a significant decrease in turn-around-time compared to culture-based
antibiotic susceptibility testing, so that they can make their decision regarding the best course of treatment in a timely manner.

MDL-developed molecular methods to detect mutations and genes with references from published scientific
literature used to support assay design:
Test 105: Chlamydia trachomatis by Real-Time PCR (Reflex to azithromycin resistance by Pyrosequencing)
The direct and indirect costs of treatment of the more than 3 million infections with Chlamydia trachomatis per 1. Horner PJ. 2012. Sex Transm Infect
year, have stretched into billions of dollars annually (1).
88(3):154-6.
The incidence of azithromycin treatment failures have been on the rise and the current prevalence rate is 2. Misyurina OY, Chipitsyna EV,
>5% in all patients, about 8% in women, and 23% in male non-gonococcal urethritis infections (1).
Finashutina YP, et al.. 2004. Antimicrob
Agents Chemother 48(4):1347-49.
MDL has developed an assay which:
Can identify a A2058C mutation within the 23S ribosomal RNA sequence through molecular sequencing 3. Zhu H, Wang HP, Jiang Y, et al. 2010.
that has specifically been associated with azithromycin resistance in Chlamydia trachomatis (2,3).
Andrologia 42:274-80.
Is performed at no additional charge on all positive Chlamydia trachomatis tests ordered on the
OneSwab, UroSwab and ThinPrep platforms.
Confirms the initial Chlamydia trachomatis positive result.
Test 167: Neisseria gonorrhoeae by Real-Time PCR (Reflex to antibiotic resistance by Molecular Analysis)
An increase of ceftriaxone-resistant N. gonorrhoeae (Ceph-R NG) demonstrated a similar pattern to previous
reports in Japan and Southeast Asia that prompted the CDC to remove ciprofloxacin from the treatment
guidelines as a primary antibiotic (1-3).
In August 2012, the CDC called for Ceph-R NG surveillance through N. gonorrhoeae antibiotic susceptibility
testing for patients that have failed treatment (3).
MDL developed an assay which:
Examines 31 genetic markers strongly associated with antibiotic resistance in the N. gonorrhoeae
chromosomal and plasmid genes.
Provides antibiotic susceptibility information for six antibiotics which can be used for Ceph-R NG
infections including: cefixime, penicillin, ciprofloxacin, tetracycline, azithromycin, and spectinomycin.
Does not require the bacterium to be viable.
Includes genetic markers associated with ceftriaxone non-susceptibility (5).
Is performed at no additional charge on all positive N. gonorrhoeae tests ordered on the OneSwab,
UroSwab and ThinPrep platforms.
Will greatly improve the clinicians ability to more easily comply with this CDC Public Health Response
Plan.
Provide physicians with the ability to identify effective treatment alternatives (4-7).
Has been submitted as a patent application to the United States Patent & Trademark Office.
Is described in an article that has been published in a high-impact peer-reviewed journal (8).

1.
2.
3.

4.

5.

6.

7.

8.

CDC. 2010. MMWR 59:49-55.


CDC. 2012. MMWR 61(31):590-4.
CDC. Cephalosporin-resistant N.
gonorrhoeae public health response plan.
August 2012.
Ito M, Yasude M, Yokoi S, et al. 2004.
Antimicrob Agents Chemother 48:31853187.
Ohnishi M, Golparian D, Shimuta K, et
al. 2011. Antimicrob Agents Chemother
55:3538-3545.
Whiley DM, Tapsall JW, Sloots TP. 2006.
J Mol Diagnostics 8:3-15.
Workowski KA. 2008. Ann Intern Med
148:606-613.
Balashov S, Mordechai E, Adelson ME,
et al. 2013. J Mol Diagnos 15(1):116-129.

Test 111: Trichomonas vaginalis by Real Time PCR (Reflex to metronidazole resistance)
Although metronidazole treatment is thought to be 90% to 95% effective, recent reports suggest that between
2.4% and 9.6% of Trichomonas vaginalis isolates exhibit metronidazole resistance (1-5).
The CDC recently reported an increase in treatment success for women with Trichomoniasis that previously
failed metronidazole therapy by utilizing susceptibility testing to tailor subsequent treatment (1).
MDL has developed an assay which:
Was developed in conjunction with the CDC.
Can identify T. vaginalis resistance to metronidazole.
Is performed at no additional charge on all positive T. vaginalis tests ordered on the OneSwab,
UroSwab and ThinPrep platforms.
Confirms the initial T. vaginalis positive result.
Assists clinicians in administering an effective diagnosis and treatment to their patients and is especially
useful for patients that present with recurringTrichomoniasis.
Has been submitted as a patent application to the United States Patent & Trademark Office.
Is described in an article that has been published in a high-impact peer-reviewed journal (6).

Bosserman EA, Helms DJ, Mosure DJ,


et al. 2011. Sex Trans Dis 38(10):983987.
2. Krashin JW, Koumans EH, BradshawSydnor AC, et al. 2010. Sex Trans Dis
37(7):440-444.
3. Schmid G, Narcisi E, Mosure D, et al.
2001. J Reprod Med 46:545-549.
4. Schwebke J, Barrientes F.. 2006.
Antimicrob Agents Chemother
50(12):4209-4210.
5. Sobel J, Nagappan V, Nyirjesy P. 1999.
N Engl J Med 341:292-293.
6. Paulish-Miller TE, Augostini P, Schuyler
JA, et al. 2014. Antimicrob Agents
Chemother 59(5):2938-2943.
1.

Medical Diagnostic Laboratories, L.L.C. www.mdlab.com 877.269.0090