MEDICAL MICROBIOLOGY II

Lesson 12

Laboratory Detection of Sexually

Transmitted Diseases

Sexually Transmitted Diseases

Sexually transmitted diseases (STDs) are
transmitted primarily through sexual
intercourse or other intimate contact
STDs can be caused by bacteria, protozoa,
fungi, or viruses
As sexual practices change, many
microorganisms once limited to the
genitourinary tract are found in other sites in
the body

Sexually Transmitted Diseases

Test for STDs should not be limited to
specimens from the genitourinary tract
For example, in suspected cases of gonorrhea,
urethral, rectal, and pharyngeal swabs can be
collected

Signs and Symptoms

STDs can have long-lasting effects in both
males and females
In males, STDs usually produce symptoms
such as:
Painful urination (often the first symptom)
Fever
Cloudy urine
Abnormal discharge from the penis
Crusting at the tip of the penis

Signs and Symptoms

Sores, lumps, blisters, rashes, or warts on or
around the genitals
Pain, swelling, or tenderness in the scrotum
(epididymitis)
Itching, tingling, pain, or burning of the
genitals
Deep pelvic ache (prostatitis)

Signs and Symptoms

In females, STDs usually produce symptoms
such as:
Change in vaginal discharge (thicker,
discoloured, or foul-smelling) over a period of
several days to two weeks
Pain, burning, or itching while urinating that
lasts more than 24 hours
Pain during sexual intercourse
Pain or a feeling of heaviness in the pelvic or
lower abdominal area

Signs and Symptoms

Itching, tingling, burning, or pain in the genitals
Sores, lumps, blisters, rashes, or warts on or
around the genitals
Vaginal spotting or bleeding after sexual
intercourse
General symptoms of an infection, such as fever
and fatigue or lack of energy

Females can be asymptomatic and go untreated

for some STDs, a factor important in disease
transmission and in infertility for many women

Signs and Symptoms

One main complication is pelvic inflammatory
disease (PID), a serious condition where a
womans reproductive organs are inflamed or
infected
This disease is serious because it may lead to
infertility, an ectopic pregnancy, pelvic abscess
or chronic pelvic pain

Signs and Symptoms

STDs are even more dangerous in pregnant
women, as they could cause miscarriage, low
birth weight baby, premature delivery or
infections in the newborn baby, such as
pneumonia, eye infections, or nervous system
problems
These infections may threaten the life of the
baby or cause serious long-term problems or
disabilities.

Detection of STDs in Females

Female patients may be tested for STDs
because of findings during a routine
examination and reporting of symptoms, or
because a partner has tested positive for STDs
Many STDs cause vaginitis, an infection or
inflammation of the vagina
Vaginitis can be caused by bacteria, fungi, or
protozoa

Detection of STDs in Females

Bacteria and yeast infections may occur
because of changes in the vaginal pH or
alteration in normal flora
Some of the most common organisms are
Gardnerella vaginalis (formerly Haemophilus
vaginalis), Mobiluncus species, Streptococcus
group B, Chlamydia trachomatis, Neisseria
gonorrhoeae, and Candida albicans

Detection of STDs in Females

Detection methods include Gram stain,
culture, wet mounts, serum antibody tests,
immunoassays, and DNA probes

I.

The Three-Slide Test for Vaginitis

The 3-slide tests is an important part of

discovering the cause of vaginitis
Relatively simple procedure
Components of the 3-slide test include:
1. Saline wet mount preparation of vaginal
secretions for Trichomonas and clue cells
2. KOH (potassium hydroxide) preparation of
vaginal secretions for fungi and yeasts
3. Gram satin of endocervical secretions for
bacteria and yeasts

I.

The Three-Slide Test for Vaginitis

Specimens for other tests can be collected at

the same time, such as swab for N.
gonorrhoeae culture, or swab for C.
trachomatis or N. gonorrhoeae DNA probe tests
If suspected herpes lesions are present, a
specimen can be collected from the lesions and
sent to the reference laboratory in viral
transport medium

I.

The Three-Slide Test for Vaginitis

1. Saline Wet Preparation

Prepared by mixing a drop of vaginal
specimen with a drop of 0.85% saline on a
microscope slide and adding a coverslip
A depression slide can also be used
It must be examined within 30 minutes of
collection to detect Trichomonas vaginalis, a
parasitic protozoan which causes
trichomoniasis

I.

The Three-Slide Test for Vaginitis

If Gardnerella vaginalis is present, clue cells

which are vaginal epithelial cells covered with
bacteria may also be seen

I.

The Three-Slide Test for Vaginitis

2. KOH Preparation
A drop of vaginal material is mixed with 1 or 2
drops of 10% KOH solution on a microscope
slide or in a depression slide to look for fungi
that can cause vaginitis such as Candida
albicans
The KOH destroys structures such as epithelial
cells and white blood cells
Any fungi present will appear as tangled
masses resembling hairs or threads

Candida infection

I.

The Three-Slide Test for Vaginitis

3. Gram Stain
The swab used to inoculate the media for
Neisseria culture can be used to prepare the
smear for Gram stain
The smear is Gram-stained and examined for
the presence of bacteria and pus cells (white
blood cells) using the oil immersion objective
of the microscope

I.

The Three-Slide Test for Vaginitis

Smears from uninfected females should have a

moderate amount of Lactobacillus (Gram
positive rods), few or no white blood cells, and
few epithelial cells
The smear is examined for the presence of N.
gonorrhoeae, a Gram negative, kidney beanshaped diplococcus that causes gonorrhea
The organism can be found both inside
neutrophilic leukocytes (intracellular) and also
extracellular on smears from infected patients

I.

The Three-Slide Test for Vaginitis

G. vaginalis is a Gram-variable microorganism,

which means it may stain either Gram negative
or Gram positive
If present on the smear, the tiny organisms will
be scattered over the other constituents on
the smear, especially the vaginal epithelial cells
If yeast cells are present, they will stain dark
purple

II. Neisseria gonorrhoeae Culture

A sterile rayon or Dacron swab is used to
collect the specimen for culture of N.
gonorrhoeae (causes gonorrhea)
The swab is used to streak the modified
Thayer-Martin (MTM) agar in the shape of a Z
or a W
The culture must be transported to the
laboratory where the plate is cross-streaked
using a sterile loop

II. Neisseria gonorrhoeae Culture

The culture plate is immediately placed in an
increased carbon dioxide (CO2) environment
and incubated at 35 - 37 C to prevent loss of
viability of any organisms present

III. Rapid Tests for Vaginitis and Vaginosis

Many rapid diagnostic tests are available for
detection of organisms responsible for vaginosis
and vaginitis
The tests are performed directly from the
specimen swab
e.g., the Genzyme OSOM BV BLUE kit for
bacterial vaginosis / vaginitis (BV) can detect
Gardnerella vaginalis, Bacteroides spp.,
Prevotela, and Mobiluncus spp. in less than 10
minutes

III. Rapid Tests for Vaginitis and Vaginosis

The Quidel Quick Vue ADVANCE is specific for
G. vaginalis
Quidel also has a kit for vaginal pH and
presence of amines
One instrument, the BD Affirm VPIII, uses RNA
probe technology to detect organisms causing
BV
This method can detect Candida sp., G.
vaginalis, and T. vaginalis from the same
specimen swab

Detection of STDs in Males

Male STD patients usually have no symptoms of
urethritis, an inflammation of the urethra
The major symptoms are a burning sensation
on urination or the presence of a penile
discharge
Tests may include a urinalysis and culture, a
Gram satin, a culture or DNA probe test for N.
gonorrhoeae, immunoassay or DNA probe for C.
trachomatis, and serology tests for herpes
simplex virus (HSV), HIV, and syphilis

Detection of STDs in Males

Males also sometimes develop a non-specific
or non-gonococcal urethritis in which the
organisms causing the condition cannot be
identified
In these cases, the patient is treated for the
standard microorganisms usually implicated in
urethritis

I.

Urinalysis and Urine Culture

If symptoms indicate possible STDs in male

patient, the specimens for those tests should
be collected before the patient collects the
urine sample
The results of the urinalysis can help
determine if the symptoms are due to urinary
tract infection (UTI) or STD

I.

Urinalysis and Urine Culture

If midstream urine specimen contains red

blood cells, bacteria, white blood cells, or
protein, the patient may have a UTI instead of
an STD
The results of urine culture can be used to
confirm the urine microscopic findings
If urine culture results are negative, the
problems may be due to an STD

II. Urethral Culture

The urethral discharge is collected on a
urogenital swab and cultured for N. gonorrhoeae
It is inoculated onto an MTM plate by touching
the urogenital swab to the plate five or six times
The plate is transported to the laboratory
immediately. Where it is cross-streaked using a
sterile loop and placed into a 35 - 37 C incubator
in an increased CO2 environment

III. Urethral Gram Stain

A smear of the urethral discharge is made
using the swab used for the culture
The smear is Gram stained in the laboratory
and examined microscopically
The entire smear is searched for the evidence
of bacteria, especially N. gonorrhoeae, other
microorganisms, and white blood cells
The presence of many white blood cells is a
strong indicator of bacterial infection

Identification of Neisseria
1. Appearance of colonies
After overnight incubation, the MTM plate is
examined for bacterial growth
N. gonorrhoeae appear as tiny, shiny, grayish
colonies growing along the streak pattern
If scant or no growth is present, the plate is
incubated for an additional 24 hrs
Other bacteria and yeasts can also grow on
MTM

Identification of Neisseria
2. Confirmatory tests from culture
The oxidase test can be used to aid in identifying
colonies of N. gonorrhoeae, an oxidase-positive
organism
A purple-black colour forms when oxidasepositive colonies are exposed to the oxidase
reagent
If a suspected Neisseria colony gives a positive
oxidase reaction, a smear and a Gram stain
should be performed on one of the oxidasepositive colonies

Identification of Neisseria
The presence of Gram negative diplococci on
the smear is presumptive for N. gonorrhoeae
and a confirmatory test must be performed
Several systems are available to confirm
identification of N. gonorrhoeae
e.g. are Duponts Gonochek II that identifies N.
gonorrhoeae, N. lactima, and N. meningitidis,
and GonoGen (Becton Dickinson Microbiology
Systems), a 15 minute agglutination test
DNA and RNA probe methods are also used to
identify Neisseria species

Identification of Neisseria
3. Nucleic acid methods for detection of
Neisseria gonorrhoeae
Several manufacturers have test methods for
detecting organisms using RNA or DNA
probes or PCR
PCR is more specific and sensitive but
technically more difficult

Identification of Neisseria
The BD ProbeTec ET System is a DNA
amplification assay - reduces the time required
for sample handling and amplification,
produces rapid results in the detection of N.
gonorrhoeae

Tests for Other STDs

Many types of tests are available for detecting
other STDs such as herpes, hepatitis, syphilis,
and HIV and chlamydial infections
Methods include negative staining, electron
microscopy, fluorescent antibody techniques,
enzyme immunoassays (EIAs), serological
tests, monoclonal antibody agglutination, and
nucleic acid (DNA and RNA) probes

I.

Tests for Herpes Infection

2 herpes viruses commonly cause disease in

humans
Herpes simplex virus, type 1 (HSV-1) which
causes oral herpes and herpes simplex virus,
type 2 (HSV-2) which causes genital herpes
HSV infections have chronic, painful, recurring
episodes

I.

Tests for Herpes Infection

2 methods of testing for herpes infection are

cell culture and detection of serum antibody
levels of anti-HSV
If herpes lesion is present, a vesicle can be
broken with a sterile swab or needle and the
vesicle fluid collected using another sterile
swab
The swab is then inserted into viral transport
medium and sent to a reference laboratory for
culture

I.

Tests for Herpes Infection

Cell culture results are available in 24 - 48 hrs

A sample of the patients blood can also be
sent to a reference laboratory for
measurement of serum titers of IgG and IgM
to HSV-1 or HSV-2
The titers indicate whether the patient has
active herpes or has ever had herpes type 1 or
type 2 in the past

I.

Tests for Herpes Infection

Rapid diagnostic tests are available to test for

antibodies to HSV-2
The Fisher Sure-Vue test uses a few drops of
the patients blood
The result can be read on the test cassette in 7
- 10 minutes

II. Tests for Chlamydia Infection

Chlamydia trachomatis infection can be
detected by EIA, cell culture, fluorescent
antibody (FA), and DNA probes
In the female patient, C. trachomatis infection
may cause the cervix to bleed easily just from
the touch of a swab during examination cervix is said to friable
C. trachomatis infection is a common cause of
urethritis in males, especially in young adults

II. Tests for Chlamydia Infection

Since urination temporarily flushes out any
microorganisms inhabiting the urethra, for
best chances of detection, the patient should
not have urinated in the 1 - 2 hrs before the
specimen collection
There are several tests for C. trachomatis
In some DNA probe tests, the same patient
swab can be used to test both Chlamydia and
N. gonorrhoeae

II. Tests for Chlamydia Infection

Immunoassay tests are also available for C.
trachomatis
2 of these are the Premier Chlamydia by
Meridian and CLEARVIEW Chlamydia from
Wampole Laboratories
These tests have built-in positive and negative
controls

II. Tests for Chlamydia Infection

The tests can be used to screen urines for
Chlamydia in male patients, but their lower
urine test sensitivity means negative results
should be confirmed by other method
The PathoDX Chlamydia trachomatis FA direct
test is a fluorescent antibody technique from
Remel. This test has the advantage of
detecting the organism directly from urethral
and endocervical swab specimens

II. Tests for Chlamydia Infection

Each of the various test has its own instructions
and supplies - follow manufacturers
instruction

III. Tests for Syphilis

Syphilis is a venereal disease caused by
Treponema pallidum, a spirochete or spiral
bacterium
Early (primary) syphilis is characterised by skin
lesions
Organ or tissue damage occurs in secondary
stage
Late stage syphilis can affect the
cardiovascular and CNS

III. Tests for Syphilis

It is difficult to grow Treponema in culture,
therefore the screening method of choice has
been detection of serum antibody
The Venereal Disease Research Laboratory
(VDRL) test and the rapid plasma reagin (RPR)
test are screening tests for syphilis

III. Tests for Syphilis

1. Venereal Disease Research Laboratory Test
Patients infected with T. pallidum produce a
non-specific, antibody-like substance called
reagin
When the VDRL antigen mixture, made of
cardiolipin, cholesterol, and lecithin, is reacted
with serum containing reagin, a visible reaction
- flocculation occurs
The test is reactive in 70 - 90% of primary and
secondary syphilis cases, but is usually nonreactive in tertiary cases

III. Tests for Syphilis

2. The Rapid Plasma Reagin Tests
The RPR test has a carbon-containing
cardiolipin antigen that reacts with the
antibody-like substance (reagin) produced in
response to syphilis and some other
conditions
The RPR is also a flocculation test, with the
carbon causing black clumps on a white
background in a reactive test

III. Tests for Syphilis

Test results are reported as reactive or nonreactive
A reactive result is not diagnostic of syphilis,
but only indicates the presence of these nonspecific antibodies

III. Tests for Syphilis

3. Confirmation of a Reactive Syphilis Screening
Test
A reactive result in a serum VDRL or RPR
screening test must be confirmed by a more
specific test method, since biologic false
positives (BFPs) can occur
Several non-syphilitic conditions can cause
BFPs, including tuberculosis, hepatitis,
pneumonia, pregnancy and rheumatoid
arthritis

III. Tests for Syphilis

The fluorescent treponemal antibodyabsorption (FTA-ABS) test is a specific test that
uses Treponema antigen to detect patient
serum Treponema antibodies
The T. pallidum microhaemagglutination assay
(MHA-TP) also detects serum antibodies to the
syphilis organism
Both the FTA-ABS and the MHA-TP are specific
treponemal antigen tests that are used to
confirm a reactive RPR or VDRL test

III. Tests for Syphilis

Newer test methods use enzyme-linked
immunosorbent assays (ELISAs)

IV. Tests for HIV

HIV is transmitted sexually and by contact
with infectious body fluids
The main approach to HIV testing is to assay
for anti-HIV in the patients serum
Since this antibody usually does not appear
until several months to a year after exposure,
a negative test must be followed by another
test in about 6 months

IV. Tests for HIV

Since saliva and urine from HIBV-infected
patients contain HIV antibodies, tests have
been developed to detect HIV antibodies in
saliva, urine, and blood
One rapid blood test for antibodies to HIV-1 is
the Trinity Biotech Uni-Gold Recombigen HIV
test
The test can be performed on whole blood,
plasma, or serum

IV. Tests for HIV

The sample is added to the sample well in the
test cassette , and a wash solution is added
Ten minutes later, the result is shown in the
result window
As HIV infections progresses, the virus
multiplies and can be cultured and isolated
Some tests detect viral components such as
p24 antigen

IV. Tests for HIV

The p24 antigen can be detected very early
after HIV infection, then disappears, and
cannot be detected again until the late stages
of the disease

THE END