This document provides information on laboratory tests for diagnosing several common STIs/RTIs. It discusses the basic terminology used in laboratory testing and then focuses on specific organisms and the tests available to detect each one. For syphilis, it describes tests to directly identify Treponema pallidum such as darkfield microscopy as well as non-treponemal and treponemal serological tests. Similar information is provided on tests for gonorrhea, chlamydia, chancroid, herpes, donovanosis, lymphogranuloma venereum, and causes of genital discharge. Common laboratory techniques mentioned include microscopy, culture, PCR and various antigen or antibody detection methods.

1. LAB DIAGNOSIS OF STI/RTI

2. Basic terminology of laboratory test
• Antigen : A molecule which is recognized by the immune system
and induces an immune reaction(the organism itself).
• Antibody : A class of serum proteins which are induced
following contact with the antigen(an infectious organism).
• False positive : uninfected people diagnosed as positive.
• False negative : infected people diagnosed as negative
• Sensitivity :
– Indicates how good a test is at identifying people who are
infected
– The higher the sensitivity,the lower the rate of false
negatives(missed infections)

3. • Specificity
– Indicates how good a test is at identifying people
who are infected
– The higher the specificity,the lower the rate of
false positives

4. SYPHILIS
• Caused by a spirochaete bacterium,Treponema Pallidum subspecies Pallidum.
Fritz Richard Schaudinn
Zoologist
Paul Erich Hoffmann
Dermatologist
Treponema pallidum, causative agent of syphilis, was discovered by Schaudinn and
Hoffmann (1905) in the chancres and inguinal lymph nodes of syphilitic patients

5. • Treponema Pallidum subspecies Pallidum is a slender
,thread-like ,motile,flexible ,uncapsulated,coiled
structure with tapering ends.There are about 8-24 coils
placed at regular intervals.
• About 6-20 µm in length and 0.10 -0.18 µm in diameter.
Electron Microscopic view of Treponema pallidum: Regular spiral coils 1 µm apart
SYPHILIS

6. Tests available for detecting syphilis
Direct identification of T.pallidum Serological tests
Direct microscopic identification of
T.pallidum by dark field microscopy
Direct fluorescent antibody-
T.pallidum
(DFA-TP)
Nucleoside amplification
techniques (PCR)
Non Treponemal tests (VDRL,RPR)
Treponemal tests (TPHA,FTA-ABS)
SYPHILIS

7. Dark Field Microscopy
T.pallidum in dark field microscopy is
identified by its typical morphology and
characteristic movements
T.pallidum is differentiated from the other
treponemes by the tightness of spirals and
characteristic cork screw movements
T pallidum
Has 6-14 regularly wounded coils
Shows a slow, deliberate forward & backward
movement,compression-expansion,bending
or angling on its long axis, burrowing or cork-
screw,looping,buckling and undulation.
Other non pathogenic treponemes
Irregularly wounded coils
And may be longer & thicker
Lack a characteristic mobility
DIRECT IDENTIFICATION OF T.PALLIDUM
SYPHILIS

8. DIRECT IDENTIFICATION OF T.PALLIDUM
• Direct fluorescent antibody-T.pallidum
(DFA-TP)
– It is a practical alternative to dark field examination and it
is more sensitive and specific than dark field microscopy.
– Samples from oral mucosa can also be examined .
– The smear is stained with fluorescein- labeled anti T.
pallidum globulin and examined under the fluorescent
microscope.
• Immunostaining
– Direct fluorescent antibody or silver stain
• Polymerase Chain Reaction (PCR)
– Not commercial available
SYPHILIS

9. SEROLOGICAL TESTS
• Useful in the latent stage of the disease as
treponemes are not readily sustainable in
culture and lesions are usually absent
• An ideal serological test
1. should have high specificity & sensitivity
2. should be suitable for treatment monitoring
3. should give a negative result on successful
therapy to give a clear cut diagnosis of
reinfection
SYPHILIS

10. Non-Treponemal test Treponemal test
Flocculation Tests
Rapid Plasma Reagin
(RPR)
 Venereal Disease Research
Laboratory(VDRL)
Treponema Hemaglutination
Absorption(TPHA)
Fluorescent treponemal antibody
absorption (FTA-ABS)
NON TREPONEMAL TESTS
Principle:
T. pallidum infection leads to the production of
reagin
Reagin – Antibodies to substances released from cells damaged
by T. pallidum
Reagin reacts with cardiolipin
Cardiolipin – a phospholipid component of certain eukaryotic and
prokaryotic membranes
SEROLOGICAL TESTSSYPHILIS

11. • VDRL
– Venereal Disease Research Laboratory
(VDRL) Test is a slide flocculation test
employed in the diagnosis of syphilis.
– The VDRL can be used for qualitative and quantitative
measurements and is recommended when a patient
suspected of having syphilis has a negative dark field
microscopy result or when atypical lesions are present.
– Routine part of prenatal care during pregnancy .
– Specimen for the test is SERUM
– Screening test is most likely to be positive in the secondary
and latent stages of syphilis
– Test is not accurate in Early and Tertiary stage.
NON TREPONEMAL TESTSSYPHILIS

12. Every Pregnant women Needs
Screening
SYPHILIS

13. NON TREPONEMAL TESTS
RPR
• RPR test allows to confirm diagnosis and begin
treatment.
• Similar test to the VDRL.
• Detection present of non-specific antibody.
• Secondary syphilis is one of the most accurate stage to
perform RPR
• FTA-ABS is confirmatory test.
• Large clumps (Positive)
• NO clumps (Negative)
SYPHILIS

14. TREPONEMAL TESTS
Fluorescent treponemal antibody absorption (FTA-ABS)
• Uses antibodies specific to T.palllidum
• Such tests are more specific than non-treponemal testing
• Positive earlier and remains positive longer than VDRL
• Should always be followed to confirm a
positive RPR and/or VDRL test for syphilis
• Highly sensitive but time consuming
• With fluorescence: REACTIVE;
• without fluorescence: NONREACTIVE
SYPHILIS

15. • A qualitative haemagglutination test using tanned
formalinised sheep RBC’s as the carrier for T.pallidum
antigen (sensitized cells)
• Patients serum is diluted in absorbing diluent which
contains:
1. Sonicated sheep & bovine red cell stroma
2. Normal rabbit testicular extract
3. Reiter treponemal sonicate
4. Normal rabbit serum
5. Stabilizers
TREPONEMAL TEST
SYPHILIS
Treponema pallidum Haemagglutination Assay (TPHA)

16. • Serum is tested with both sensitised and non
sensitised RBC’s
• Preliminary results are available in 3-4 hrs, final
results in 18 hrs
• REACTIVE : if agglutination occur in a dilution of 1:80
or more
• Reactivity Is positive around 4th-5th week of infection

17. CHANCROID
Causative agent - haemophilus ducreyi
GRAM STATINING
Pleomorphic gram negative coccobacilli
(H.ducreyi) arranged in a parallel
chains of two’s or four described as “school of
fish”

18. Cultureis now the accepted standard
for chancroid diagnosis in most areas.
selective artificial media used are
gonococcal agar base with 2% bovine
Hemoglobin and 5% fetal calf serum and
Mueller-Hinton agar with 5% chocolatized
horse blood.media are made by addition of vancomycin.culutres are
incubated at 350C in a candle jar.
Small,non-mucoid ,yellow-gray,semi opaque colonies appear 2 to 4 days
after inoculation.
Recent developments in the diagnosis of chancroid are Polymerase
Chain Reaction (PCR) and Indirect Immunofluorescence
using monoclonal antibodies.
CHANCROID

19. HERPES GENITALIS
• Caused by a DNA virus,Herpes Simples
Virus.
• HSV has been classified into
two distinct categories, HSV-1 and HSV-2.
STRUCTURE OF HERPES VIRUS

20. • Vesicles  painful ulcerations  crusting
• Recurrence a potential
HERPES GENITALIS

21. Diagnosis
– Histopathology – rarely done.the presence of ballon cells and
multinucleate giant cells will confirm the diagnosis.
Tzanck smear(Giemsa stain) -shows multinucleate giant cells
HERPES GENITALIS

22. – Viral Culture – cultures are usually positive in
primary or first episode infections and are
frequently negative from dry erosions or crusts.
– Serology – following tests detect antibodies to the
herpes simplex virus in the blood
• ELISA
• Complement fixation test
• Western blot
– PCR

23. Granuloma Inguinale (Donovanosis)
• Direct microscopy
Showing intracellular
donovan bodies
caused by Klebsiella granulomatis

24. • Biopsy – less frequent and is indicated in chronic
ulcers with suspicion of malignancy .
• Serology – complement fixing test Culture : yolk
sac of chick embryo,human peripheral blood monocytes
and Hep-2 cells
• PCR
• Recently colorimetric detection system for
calymmatobacterium granulomatis has been developed.
Granuloma Inguinale (Donovanosis)

25. Lympho granuloma
Venereum
caused by Chlamydia trachomatis

26. Lympho granuloma Venereum
• Laboratory diagnosis of LGV can be made by
– A positive serology(complement fixation ) for
C.trachomatis.
– Isolation of C.trachomatis from infected tissue or
bubo pus using mouse brain,yolk sac or tissue
culture.
– Identification of C trachomatis in tissues or bubo
pus by using special stains such as Giemsa –
Romanowsky,iodine or Macchiavello stains.

27. STD’S CAUSES GENITAL DISCHARGE
–Gonorrhea
–Chlamydia
–Bacterial vaginosis
–Trichomonasis
–Candidiasis
–PID

28. GONORRHOEA
Caused by Neisseria Gonorrhoeae,identified by
Albert Neisser in 1879
The infection affects the urethra in both the sexes,but it may
spread to paraurethral glands,cervix,fallopian tubes and
peritoneum in females

29. Microscopy
Showing gram negative intracellular diplococci
GONORRHOEA

30. • Culture – commonly used selective media for
N.gonorrhoes are modified Thayer Martin,Chacko Nayar
Medium,Martin Lewis media and New York City Media
• Recently Polymerase chain reaction(PCR),Ligase chain
reaction(LCR) and other nucleic acid amplification techniques
are also being developed to diagnose gonorrhoea
• Serology – the complement fixation,latex agglutination
immunofluorescence and anti-surface pili assays,ELISA and
immunoblotting can be used to detect serum antibody against
N.gonorrhoeae.
GONORRHOEA

31. Chlamydia
• Tissue culture has been the standard
– Specificity approaching 100%
– Sensitivity ranges from 60% to 90%
• Non-amplified tests
– Enzyme Immunoassay (EIA), e.g. Chlamydiazyme
• sensitivity and specificity of 85% and 97% respectively
• useful for high volume screening
• false positives

32. – Nucleic Acid Hybridization (NA Probe), e.g. Gen-Probe
Pace-2
• sensitivities ranging from 75% to 100%; specificities greater than
95%
• detects chlamydial ribosomal RNA
• able to detect gonorrhea and chlamydia from one swab
• need for large amounts of sample DNA
• DNA amplification assays
– polymerase chain reaction (PCR)
– ligase chain reaction (LCR)
• Sensitivities with PCR and LCR 95% and 85-98% respectively;
specificity approaches 100%
• LCR ability to detect chlamydia in first void urine
Chlamydia

33. Bacterial vaginosis
• BACTERIAL VAGINOSIS (BV) is the most common cause of
abnormal vaginal discharge in women of childbearing age.
• Condition first described by Gardner and Dukes
• in 1955.
• characterized by a foul smelling vaginal discharge,
loss or reduction of the normal vaginal Lactobacilli,
and overgrowth of other anaerobic bacteria.
• The causative organisms for this condition is
GARDNERELLA VAGINALIS.

34. • In a patient suspected of BV,diagnosis can be made using
Amsel’s criteria
(introduced 1984)
(3 out of 4 criteria below required to establish the diagnosis)
– Nonviscous homogenous white uniformly adherent vaginal dischagre.
– High pH
– Clue cells –vaginal squmaous cells covered by bacterial rods which blur
the border of squamous cells
– Whiff test – adding 10% KOH to vaginal secretions produces an amine
odour.
Bacterial vaginosis

35. Introduced
1991

36. Gram’s stain of vaginal secretions showing
clue cells

37. Trichomoniasis
• Caused by Trichomonas vaginalis
Structure of Trichomonas Vaginalis

38. Trichomonas vaginalis is a
flagellated protozoan.
Trophozite is the only stage
present in the life cycle that is
infective stage of the
parasite.no cystic stage.
It is 7-30 µm long by 5-10 µm
wide.
Trophozite is pear shaped &
shows “TWITCHING TYPE”
Of motility due to presence
of 5 number of flagellae

39. Symptoms in women
• In females there is urethritis,vaginitis&cervicitis.
• Pain during intercourse and urination.
yellow-green, itchy, frothy foul-smelling ("fishy" smell)
in vaginal discharge in rare cases, lower abdominal
pain and inflammation of the external genitals can
occur.
• Symptoms usually appear in women
within 5 to 28 days of exposure.
Trichomoniasis

40. Symptoms in mens
• Urethritis and Prostatitis
• Asymptomatic,Dysurea,
• Non purulent discharge ( fish-like odour)
• irritation inside the penis or slight burning
after urination or ejaculation
• painful intercourse, and inflammation of the
external genitals.
Trichomoniasis

41. • A purulent yellow–green vaginal discharge is
characteristic of trichomoniasis.
• Diagnosis of trichomoniasis
depends on the identification
of T.vaginalis by wet mount,
stains(Gram ,Giemsa ,
neutral red stains)
culture
(modified diamond medium). Wet mount showingTrichomonas vaginalis

42. Pelvic Inflammatory Disease (PID)
PID is an infection of-Uterus(endometritis)
Fallopian tubes(salpingitis)
Ovaries(oophoritis)
Pelvis peritonitis
Tubo-ovarian abscess
• It is a common and serious complication of
STD’s
• Bacteria causing PID-
– Chlamydia trachomatis
– Neisseria gonorrhoea

43. symptoms
• Lower abdominal pain
• Pain during or after sex
• Bleeding between periods or after sex
• Lower back pain
• Sense of pressure or swelling in the lower abdomen
• Fever
• Abnormal vaginal discharge
• Nausea,vomiting and dizzinss
• Dysuria,frequently urination
Pelvic Inflammatory Disease (PID)

44. • CDC minimal criteria
– uterine adnexal tenderness, cervical motion tenderness
• Other symptoms include
– endocervical discharge, fever, lower abd. Pain
• Gram staining – specimens may be obatined by endocervical
swab,endometrial aspiration.
• Culture and DNA-PCR based test for N.gonorrhoeae and
chlamydia.
• USG and MRI
– Identification of tubo-ovarian or pelvic abscess.
– Increased tubal diameter,intratubal fluid ,tubal wall
thickening in case of salpingitis
Pelvic Inflammatory Disease (PID)

45. • Most cases caused by C. albicans (85%-90%)
• Second most common cause of vaginitis
• Candida species are normal flora of the skin and vagina
• 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
• Vulvar pruritis is most common symptom
• Thick, white, curdy vaginal discharge ("cottage cheese-like")
• Erythema, irritation, occasional erythematous "satellite" lesion
• External dysuria and dyspareunia
candidiasis

46. Vulvovaginal Candidiasis

47. • Diagnosis depends upon the demonstration of the yeast by
simple microscope examination of vaginal secretions in 10%
KOH or by gram staining
• A wet mount or saline preparation
should rountinely be done not
only to identify yeast cells and
mycelia but also to exclude the
presence of clue cells
• Vaginal secretion culture on
sabouraud’s agar should be performed in
the presence of negative microscopic
findings
candidiasis
Yeast
pseudohyphae
Squamous epithelial
cells
Yeas
t
buds
Yeast Pseudohyphae

48. Life at Risk with Sexually Transmitted Infections
Best Choice Play safe