Transcript Lab Diagnosis of Bacteria causing STDs

Neisseria gonorrhea
Specimen:
- In females: cervical swab in acute and chronic
infection.
- In males: urethral discharge in acute infection and
morning drops in chronic infection.
- Other specimens that may be used:
i- throat swab.
ii- anorectal swab
iii- conjunctival swab in case of neonatal conjunctivitis.
1- gram stain: gm-ve kidney-shaped diplococci that
are seen extracellularly and intracellularly inside
PNL.
2- Culture on Thayer-Marten agar (chocolate agar that
contains vancomycin, nystatin, colistin and
trimethoprim) to suppress normal flora..
Why chocolate agar and not blood agar?
Blood agar contains substances which inhibit the
growth of Neisseria as trace metals and fatty acids.
Heating blood to 80°C (chocolate agar) inactivates
these inhibitors.
- Incubation conditions: aerobically at 36ºC for 24-48
hours in presence of 5% CO2.
Suspected colonies are identified by:
a- morphology of the colonies on the culture plates.
b- gram stain from the colonies
c- BR:
- oxidase test: Oxidase +ve (i.e. produce cytochrome
oxidase) which is demonstrated by using filter paper
impregnated with a dye (tetramethyl phenylenediamin)
that on oxidation changes color to dark purple.
- Sugar fermentation:
Ferment glucose only. Does not ferment maltose or
sucrose.
3- Gonococcal antigens in the specimen can be
detected by ELISA.
4- DNA probe to detect gonococcal ribosomal genes.
Serological diagnosis:
- detection of specific antibodies to gonococcal
pilin and outer proteins by ELISA.
Gm-stained smear of pathological discharge
of case of gonorrhea showing gm-ve
diplococci inside & outside pus cells (PNLs)
Morphology of N. gonorrhea on modified
chocolate agar (Thayer-Marten agar)
Gm stain from colonies of N.
gonorrhea
Gm-ve kidney-shaped diplococci
glucose maltose sucrose
Oxidase test
N. meningitidis
+
+
-
N. gonorrhea
+
-
-
N. flavescens
-
-
-
N. Sicca
+
+
+
Sugar fermentation can be used
to differentiate pathogenic from
commensal neisseria.
Treponema pallidum
Syphilis
I- Detection of T. pallidum in lesions:
Serous exudate from lesions of 1ry and 2ry stages is
examined by:
1- wet film for dark ground microscopy to detect the
darting movement of the spiral T. pallidum.
2- IFT using fluorescein-labelled antitreponemal
antibodies.
3- staining with silver impregnation technique
(Fontana stain).
Dark-ground microscopy
showing long slender spiral
bacteria
Treponema pallidum
IF staining showing the spiral
Treponema pallidum
Treponema pallidum smear Stained
by silver impregnation Technique
(Fontana Stain) showing the spiral
morphology
II- Serological diagnosis:
A- non-treponemal antigen tests:
- detect the reagin antibody that react with a nonspecific antigen, cardiolipin, which is an alcohol
extract of beef heart muscle supplemented with
lecithin and cholesterol.
- Reagin is a mixture of IgG and IgM that appear 2-3
weeks in the patient serum and 4-8 weeks in the
CSF after exposure to infection.
1-Flocculation tests: VDRL and RPR (rapid plasma
reagin) tests.
2- CFT, complement fixation test (Wasserman test).
non-treponemal antigen tests are characterized by:
- Non-specific and can lead to false positive results.
- Become negative 6-18 months after effective
treatment, so can be used to follow up the effect of
treatment.
- They are mainly used for screening and
epidemiological studies because they are sheep,
rapid and simple.
The rapid plasma reagin (RPR) test
- The upper, left well shows a negative test result, the
carbon particles have remained unclumped.
- The clumped appearance of the carbon particles in the
right well indicate a positive test result, illustrating the
flocculation of the cardiolipin-based antigen by antibodies
in the test serum.
B- Treponemal antigen tests
- Highly specific and sensitive tests as they use
T. pallidum as the antigen.
- But they are complex and expensive, so used
mainly for confirmation of diagnosis.
-They remain positive for life, so cannot be used to
judge the efficacy of treatment.
1- Fluorescent treponema antibody test (FTA).
The presence of IgM FTA in the blood of a new-borne
is good evidence of in-utero infection.
2-Treponema pallidum hemagglutination (TPHA).
3- T. pallidum Particle Agglutination (TPPA).
1- Fluorescent treponema
antibody test (FTA).
2- TPHA
Lymphogranuloma venerium
Chlamydia trachomatis serotypes L1-L3
Specimen: Smears from the lesion and aspirate from
the LN
1- stained by Giemsa or IF to see the intracytoplasmic
inclusions.
2- Direct detection of chlamydial antigens in the specimen
using specific monoclonal antibodies by ELISA or IFT.
3- Direct detection of nucleic acid by PCR and DNA probes.
4- Isolation in tissue culture.
Serological diagnosis
to detect specific IgM or high titer of IgG by ELISA or IFT.
Chlamydia trachomatis serotypes D-K
Non-gonococcal urethritis
Specimen:
- In female: endocervical swab
- In male: urethral dicharge.
- Conjunctival swab in case of neonatal conjunctivitis.
Procedures:
As in case of LGV
Hemophilus ducreyi
Chancroid
Specimen:
Scraping from the lesion or aspirate from the enlarged
lymph nodes:a- gram stained smear: Gm-ve coccobacilli
b- culture on chocolate agar.
c- Subculture on nutrient agar containing 2 discs for X
and V factors. Growth is enhanced around X factor
only.
2- Serological diagnosis
Gm-ve coccobacilli
Ex: H. ducreyi
Growth apprears around
X factor
No growth around V factor
Growth on chocolate agar
Mycoplasma hominis
Specimen:
- female: endocervical swab
- Male: urethral discharge
1-Culture of the specimen on specific media→ fried
egg appearance.
2- Direct detection of Mycoplasma antigen in the
specimens by IFT and PCR.
3- Serological diagnosis: detection of specific IgM or
rising titer of IgG in the patient serum.
Fried egg appearance of mycoplasma
colonies on the specific medium
Candida vulvovaginitis
Mycotic vulvovaginitis
Specimen:
Swab from the curd like white vaginal discharge
1- gram stain
many epithelial cells and strongly gm+ve budding
yeast cells and pseudohypha.
2- culture on Sabouraud dextrose agar
Large white colonies.
Gm stain of vaginal discharge
In case of candida vaginitis→
Many epithelial cells and
gm+ve budding candida
yeast and pseudohyphae
Colonies of Candida albicans on
Sabouraud dextrose agar