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BASICS IN DIAGNOSIS
Dr.T.V.Rao MD
Dr.T.V.Rao MD
In North America
The most commonly submitted sample is urine from women with acute or recurrent urinary tract infection. The most common cause of urinary tract infections in women is recent sexual activity.
Tests for sexually transmitted infections are the second most commonly submitted samples send to medical laboratories.
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Parasites
Trichomonas Vaginalis Entamoeba histolytica
Viruses
Herpes simplex II Hepatitis B Hepatitis C HIV Papillomavirus
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F.H.C.-Hepatitis
Acute tenosynovitis
Sepsis
P.I.D.
Gonorrhea
Urethral Strictures
Chronic Arthritis
Infertility
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P.I.D.
Chlamydia trachomatis
Chronic Arthritis
Infertility
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Lymphadenopathy
Cardiovascular Gumma
Neurosyphilis
Meningitis Tabes dorsalis General paresis
Chancres
HIV Transmission.
Syphilis
Congenital Transmission
Chronic Arthritis
Hepatitis
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Chlamydia trachomatis
DFA EIA PCR Culture
Treponema pallidum
Serological tests for Syphilis VDRL, RPR, FTA-abs, MHA-tp
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Diagnosis of Syphilis
The nontreponemal tests, VDRL and rapid plasma reagent (RPR), are antilipoidal antibodies seen in other disease states, pregnancy, and occasionally after vaccination. They are nonspecific and cannot rule in disease. These tests have sensitivities approaching 80% in patients with symptomatic primary syphilis and virtually 100% in patients with secondary syphilis.
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Laboratory Diagnosis
Identification of Treponema pallidum in lesions
Darkfield microscopy Direct fluorescent antibody - T. pallidum (DFATP)
Serologic tests
Nontreponemal tests Treponemal tests
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Diagnosis
Advantages:
Rapid and inexpensive Easy to perform and can be done in clinic or office Quantitative Used to follow response to therapy Can be used to evaluate possible reinfection
Disadvantages:
May be insensitive in certain stages False-positive reactions may occur Prozone effect may cause a falsenegative reaction (rare)
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Diagnosis
Patients with a reactive VDRL or RPR should have the result confirmed by specific treponemal testing. FTA-ABS and or EIA. Tertiary syphilis Serology is used in the diagnosis. Evaluation of neurosyphilis requires a lumbar puncture (LP) and evaluation of the CSF. The CDC currently recommends LP only if the patient is seroreactive and HIV positive, has symptoms of neurosyphilis
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Tests to Confirm
Syphilis may be confirmed either via blood tests or direct visualization using microscopy. Typical diagnosis is with blood tests using nontreponemal and/or treponemal tests. Nontreponemal test are used initially and include venereal disease research laboratory (VDRL) and rapid plasma regain however as these test occasionally are falsely positive confirmation is required with a treponemal test such as
treponemal pallidum particle agglutination (TPHA) or fluorescent treponemal antibody absorption test (FTAAbs)
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VDRL - Background
The Venereal Disease Research Laboratory (VDRL) test is one of two variations of flocculation procedures used for serological testing of syphilis, the other being the Rapid Plasma Reagin (RPR). Flocculation testing is based on antibody detection with the interaction of soluble antigen with an antibody that results in a precipitate formation of fine particles.
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RPR test
The RPR test is a nontreponemal testing procedure for the serologic detection of syphilis.
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Principle of RPR
When a specimen such as serum or plasma contains antibody, flocculation occurs with the resulting aggregation of the carbon particles. The flocculation appears as black clumps against the white background of the plastic coated card.
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Principle of RPR
Antibodies associated with syphilis begin to appear in the blood 4 to 6 weeks after infection. Nontreponemal tests determine the presence of reagin. Reagin is a nontreponemal autoantibody directed against cardiolipin antigens.
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The controls which are strongly reactive, moderately reactive, and non-reactive are contained on the control card in a dried form.
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Specimen Collection
Unheated Plasma specimen should be collected with an anticoagulant such as EDTA or heparin, plasma must be stored at 2C to 8C. Plasma must be tested within in 24 hrs. of collection
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Specimen Processing
*The addition of choline
chloride, which inactivates complement enables the serum to be tested without prior heating.
Unheated serumcentrifuge for sedimentation of cellular elements, serum may be frozen until time of testing. Heated Serum- transfer serum to clean tube and place in 56C water bath for 30 minutes
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Reactions of Controls
The following reactions should be observed to compare against the test results: Reactive control characteristic strong clumping. Reactive moderate control moderate clumping. Non-reactive control smooth, grayish appearance of unclumped particles
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Explanation of Results
A negative RPR test may indicate one of the
following:
1. The patient does not have syphilis. 2. The infection is too recent for antibodies to be produced. (Repeated tests should be administered at 1 week, 1 month, and 3 month intervals to establish presence or absence of disease). 3. The syphilis is latent or inactive 4. Faulty immunodefense mechanism 5. Faulty lab techniques
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Explanation of Results
A positive reaction is not conclusive for syphilis. Several conditions produce biologic false positive results for syphilis. (False positive means that the test revealed a positive reaction when it was actually negative). False positives may reveal the presence of other serious diseases.
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TREPONEMAL TESTS
FTA-ABS Used as a confirmatory tests. Sensitivity and specificity high.
85% of patients with primary syphilis are reactive 99% with secondary syphilis > 95% with late syphilis (It may be the only test with a positive result for patients with cardiovascular or neurologic syphilis).
Only 15-25 % of those treated for primary syphilis may turn negative by 23 yrs.
False positive in other treponemal diseases (pinta, yaws..) and other spirochete diseases (Lyme, leptospirosis)
MHA-TP test (microhemagglutination assay for T. pallidum; agglutination
of RBCs to which T. pallidum antigens have been fixed is the basis).
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For Routine Testing a Combination of VDRL or RPR and TPHA is highly preferred
TPHA Test is a sensitive passive haemagglutination test, that detects specific Treponema pallidum antibodies in serum within one hour. Used in combination, the VDRL or RPR and TPHA Tests provide accurate and reliable confirmation of active syphilis infection. No specialized equipment is required and results are clearly visible and easily interpreted.
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Microhemagglutination assay
The MHA-TP and TPHA are used to confirm a syphilis infection after another method tests positive for the syphilis bacteria. The MHA-TP and TPHA tests detect antibodies to the bacteria that cause syphilis and can be used to detect syphilis in all stages, except during the first 3 to 4 weeks when antibody levels are too low. These tests are also suitable for use as a screening procedure. Neither of these tests is suitable for use on cerebrospinal fluid (CSF).
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Syphilis
Stage of Disease (Percent Positive [Range])
Test
VDRL RPR FTA-ABS* Treponemal Agglutination* EIA
Primary
78 (74-87) 86 (77-99) 84 (70-100) 76 (69-90) 93
Secondary
100 100 100 100 100
Latent
95 (88-100) 98 (95-100) 100 97 (97-100) 100
Tertiary
71 (37-94) 73 96 94
*FTA-ABS and TP-PA are generally considered equally sensitive in the primary stage of disease.
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Diagnosis
Age
Autoimmune Diseases Cardiovascular Disease Dermatologic Diseases Drug Abuse Febrile Illness Glucosamine/chondroitin sulfate Leprosy Lyme disease Malaria Pinta, Yaws Pregnancy Recent Immunizations STD other than Syphilis
Yes
Yes Yes Yes Yes Yes --
*May cause increase in titer in women previously successfully treated for syphilis Source: Syphilis Reference Guide, CDC/National Center for Infectious Diseases, 2002
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Gonorrhea
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Drips
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Drips
Gonorrhea
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Drips
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Suggestive diagnosis
Suggestive diagnosis is defined by the presence of: A mucopurulent endocervical or urethral exudate on physical examination and sexual exposure to a person infected with N. gonorrhoea.
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Presumptive diagnosis of gonorrhoea is made on the basis of one of the following three criteria:
Typical gram-negative intracellular diplococci on microscopic examination of a smear of urethral exudate from men or endocervical secretions from women*; Growth of a gram-negative, oxidase-positive diplococcus, from the urethra (men) or endocervix (women), on a selective culture medium, and demonstration of typical colonial morphology, positive oxidase reaction, and typical gram- negative morphology;
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Drips
Nongonococcal Urethritis
Etiology:
20-40% C. trachomatis 20-30% genital mycoplasmas (Ureaplasma urealyticum, Mycoplasma genitalium) Occasional Trichomonas vaginalis, HSV Unknown in ~50% cases
Sx: Mild dysuria, mucoid discharge Dx: Urethral smear 5 PMNs (usually 15)/OI field Urine microscopic 10 PMNs/HPF Leukocyte esterase (+) 69
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Chlamydial Infections
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Drips
Chlamydial Infections
More than three million new cases annually Responsible for causing cervicitis, urethritis, Proctitis, lymphogranuloma venereum, and pelvic inflammatory disease Direct and indirect cost of chlamydial infections run into billions of dollars Potential to transmit to newborn during delivery
Conjunctivitis, pneumonia
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Drips
Non-amplified tests
Enzyme Immunoassay (EIA), e.g. Chlamydiazyme
sensitivity and specificity of 85% and 97% respectively useful for high volume screening false positives
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Drips
Sensitivities with PCR and LCR 95% and 85-98% respectively; specificity approaches 100% LCR ability to detect chlamydia in first void urine
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Drips
Complications:
Infertility: 15%-24% with 1 episode PID secondary to GC or chlamydia 7X risk of ectopic pregnancy with 1 episode PID chronic pelvic pain in 18%
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Chlamydia serology
Chlamydia serology (complement fixation titers >1:64) can support the diagnosis of LGV in the appropriate clinical context. Comparative data between types of serologic tests are lacking, and the diagnostic utility of serologic methods other than complement fixation and some micro immunofluorescence procedures has not been established.
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Chancroid
The combination of a painful genital ulcer and tender Suppurative inguinal adenopathy suggests the diagnosis of Chancroid A probable diagnosis of Chancroid, for both clinical and surveillance purposes, can be made if all of the following criteria are met: 1) the patient has one or more painful genital ulcers; 2) the patient has no evidence of T. pallidum infection by dark field examination of ulcer exudate or by a serologic test for syphilis performed at least 7 days after onset of ulcers;
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Chancroid
3) the clinical presentation, appearance of genital ulcers and, if present, regional lymphadenopathy are typical for Chancroid; and 4) a test for HSV performed on the ulcer exudate is negative.
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Chancroid
A definitive diagnosis of Chancroid requires the identification of H. ducreyi on special culture media that is not widely available from commercial sources; even when these media are used, sensitivity is <80% (145).
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Granuloma Inguinale
Clinically, the disease is commonly characterized as painless, slowly progressive ulcerative lesions on the genitals or perineum without regional lymphadenopathy; subcutaneous granulomas (pseudoboboes) might also occur. The lesions are highly vascular (i.e., beefy red appearance) and bleed easily on contact.
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Granuloma Inguinale
The causative organism is difficult to culture, and diagnosis requires visualization of darkstaining Donovan bodies on tissue crush preparation or biopsy. No FDA-cleared molecular tests for the detection of K. granulomatis DNA exist, but such an assay might be useful when undertaken by laboratories that have conducted a CLIA verification study.
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An estimated 5 million new cases occur each year in women and men. Occurs in vagina of women so may be sexually transmitted to men using infected washcloths and towels. It is transmitted to the baby during delivery. It also can occur in the urethra (carries urine to penis) in men, doesnt have symptoms usually. SYMPTOMS: Appear within 5 to 28 days of exposure Women usually have a vaginal discharge that FEMALE SYMPTOMS: Itching and burning at the outside of the opening of the vagina and vulva. Painful and frequent urination Heavy, unpleasant smelling greenish, yellow discharge MALE SYMPTOMS: Usually nothing, or discomfort in urethra, inflamed head of the penis.
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Trichomoniasis
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Background
Bacterial Vaginitis
Controversy: STD - yes or no Need for treatment
1980: only if patient complains 2002: increased risk of:
Preterm birth / premature rupture of membranes Amniotic fluid infection Chorioamnionitis / Postpartum endometritis Pelvic inflammatory disease Postsurgical infection Cervical intraepithelial neoplasia Mucopurulent cervicitis Acquisition of HIV infection
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Non-Curable STDs
HIV (Human Immunodeficiency Virus)
HIV is the virus that causes AIDS. You can get HIV if you do any of the following:
Have sex with someone who has HIV. It can be spread by having vaginal, anal, or oral sex. Your partner could have HIV and not know it. Share needles with someone who has HIV.
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