The Rapid Plasma Reagin Test and the Venereal Disease Research Laboratory Assay (VDRL)

1. The Rapid Plasma Reagin Test and the Venereal Disease Research Laboratory Assay (VDRL) Serological Diagnostic Screening Tests for Syphilis

2. Syphilis: Causative Agent and Disease Description • Treponema pallidum - the causative agent for the disease known as syphilis. • Order: Spirochaetales Family: Treponemataceae. • Genus: Treponema, a slender, tightly coiled, pointed-ended, 6-10 axial filamentous gram negative rod.

3. Causative Agent • T. pallidum- a microaerophilic, obligate parasite that requires mammalian cells for survival. Covered in a sheath, aids this organism in it’s pathogenicity by reducing or limiting antigenic stimulation of the host’s immune system. • Members of this genus contain of a number of species that can exist as either commensal flora or human pathogens.

4. Treponema Speciation • Nonpathogenic-exists as commensal flora of the oral cavity, gastrointestinal and urogenital tract. Are also considered to be strict anaerobes. • Pathogenic-transmitted by person-to-person contact. The microaerophilic subspecies of pallidum are associated with various Treponema-Related Diseases in humans

5. T. pallidum subspecies pallidum - syphilis T. pallidum subspecies pertenue - yaws T. pallidum subspecies endemicum - non-venereal (endemic) syphilis/bejel T. carateum - pinta Species- indistinguishable serologically and morphologically. Differentiation- based on clinical & epidemiological characteristics. Treponema pallidum sub speciation

6. Disease State • Untreated, syphilis is a chronic infectious disease with many clinical manifestations that occur in distinct stages consisting of subacute symptomatic periods separated by asymptomatic intervals. • Initially, T. pallidum penetrates intact mucous membranes or enter the body through tiny defects in the epithelium. Upon entrance, the microorganism is carried by the circulatory system to every organ of the body.

7. Clinical Manifestation • The progression of untreated syphilis is generally divided into into four stages: Primary, Secondary, Latent and Tertiary (Late) Syphilis, with Tertiary Syphilis being further divided into Neurosyphilis and Congenital Syphilis. • Spirochetemia occurs very early in infections before the appearance of the first lesion or reactive blood test described as clinical or serological detection. In this state, syphilis appears to be in an incubating state that can range from 10 to 90 days.

8. Primary Syphilis • Small, solitary nodule at the point of initial inoculation. • Primary inflammatory lesion enlarges and ulcerates developing into a characteristic painless ulcer or chancre that heals spontaneously. • Chancre are located on vaginal mucosa or cervix of the female and external genitalia of the male.

9. Secondary Syphilis • Systemic: within 2-8 weeks of visible chancre. • Generalized illness being with viral-like symptoms: headache, sore throat, low-grade fever and occasionally a nasal discharge. • Generalized rash of skin and mucous membranes.

10. Secondary Syphilis • Development of lymphadenopathy and lesions of the skin and mucous membranes. • Lesions contain a large number of spirochetes and are highly contagious when exposed on the skin surface. • Macular lesion and/or condylomata lata-flat lesions resembling warts in moist areas of the body (around the anus or vagina).

11. Latent Syphilis • Latency- exhibit no clinical symptoms, a noninfectious state which diagnosis can be made only by serologic methods. • During the first 2-4 years, patient may exhibit one or more mucocutaneous relapses of secondary syphilis manifestation. • During these relapses, patient is highly infectious and may transmit the disease.

12. Tertiary Syphilis • Characterized by the presence of destructive granulomas that produce lesions resembling segments of circles that heal with superficial scarring. • Exhibited 3-10 years after primary infection. • Skeletal system involvement • Cardiovascular system-involves the aortic endothelium, weakening the blood vessels of the aortic arch known as syphilitic aneurysm.

13. Other Clinical Manifestationsof Tertiary Syphilis • Neurosyphilis: Asymptomatic involvement of the CNS and detected only by examination of cerebrospinal fluid (CSF). • Congenital Syphilis: Caused by maternal spirochetemia and transplacental transmission of microorganism.

14. Neurosyphilis • Acute syphilitic meningitis • Meningovascular syphilis manifested as a seizure. • Cerebrovascular accident - stroke • General paresis, personality changes, dementia and delusional states. • Tabes dorsalis- involvement of the posterior column and dorsal root, characterized by broad- based gait with impotence and bladder dysfunction.

15. Congenital Syphilis • Hutchinsonian triad: Hutchinson’s teeth, interstitial keratitis, and nerve deafness. • Other characteristics- fissuring around the mouth and anus, skeletal lesions, perforation of the plate and collapse of nasal bones to produce a saddle-nose deformity.

16. Immunological Manifestations Specific antibodies- produced against T. pallidum are IgM and IgG antibodies. • Detected by Treponemal serological procedures Nonspecific antibodies-produced against the protein antigen group common to pathogenic spirochetes formed and are known as nontreponemal antibodies. • Detected by nontreponemal serological procedures

17. Diagnostic Evaluation • The diagnosis of syphilis is based on the clinical picture in conjunction with demonstration of microorganisms in a lesion and serologic testing. • Darkfield Microscopy • Nontreponemal Methods- Venereal Disease Research Laboratory (VDRL) and the Rapid Plasma Reagin (RPR) procedures are used to screen patient’s serum. • Treponemal Methods-Fluorescent Treponema pallidum-absorbed (FTA-ABS) and the microhemagglutination Treponema pallidum [MHA-TP]are used for confirmation.

18. Clinical Limitations Associated with Serological Testing • Infectious disease - measles, chicken pox, hepatitis, infectious mononucleosis, leptospirosis, malaria, leprosy, rickettsial disease, trypanosomiasis, lymphogranuloma venereum (LGV) • Noninfectious conditions - autoimmune disorders, drug addiction, old age, pregnancy and recent immunization

19. RPR test The RPR test is a nontreponemal testing procedure for the serologic detection of syphilis.

20. Principle of RPR • The RPR Card antigen suspension is a carbon particle cardiolipin antigen that detects reagin. • Reagin is an antibody like substance present in serum or plasma from individuals with syphilis. • The reagin binds to the test antigen which consists of cardiolipin-lecithin coated particles that cause macroscopic flocculation.

21. 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.

22. 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.

23. Materials for RPR • RPR Test Cards • RPR Control Cards • RPR Antigen • Distilled Water • Dispenstirs • Rotator

24. RPR Test Background • The RPR test uses a white plastic coated card that consist of several circles that are 18 mm in diameter. • The controls which are strongly reactive, moderately reactive, and non-reactive are contained on the control card in a dried form.

26. Procedure for Controls • A. Use Dispenstir to draw up distilled water • B. Drop 1 drop on the card test circle for each patient sample. • C. Invert Dipenstir and spread the water in the circle until the dried control is completely reconstituted. • D. Add antigen as described for the patients • E. Rotate for 8 minutes at 100 rpm

28. Reactions for 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

33. Specimen Collection *The addition of choline chloride, which inactivates complement enables the serum to be tested without prior heating. Unheated serum- centrifuge for sedimentation of cellular elements, serum may be frozen until time of testing. Heated Serum- transfer serum to clean tube and place in 56°C water bath for 30 minutes Specimen Collection

34. Specimen Collection Unheated Plasma - specimen should be collected with an anticoagulant such as EDTA or heparin, plasma must be stored at 2°C to 8°C. Plasma must be tested within in 24 hrs of collection. Specimen Collection

35. Procedure for Test • Label rings on test card with numbers of samples to be tested • Use Dispenstir to draw up serum sample. • Hold Dispenstir in a perpendicular position directly over the test circle to which the specimen is to be delivered. • Squeeze Dispenstir to allow 1 drop to fall on to each circle

38. Procedure for Test • Invert Dispenstir,and using the sealed end spread the specimen in the confines of the circle. • Reconstitute the antigen bottle, by shaking. Holding the bottle in a straight vertical position drop one or two drops in the upper corner of each test circle, then place one “free falling” drop on each test area.

39. Procedure for Test • Rotate card for 8 minutes on a mechanical rotator at 100 rpm. The test card she also be covered with a humidifier cover. • After rotating mechanically, the test card should be rotated manually by hand 3 to four rotations and then read immediately macroscopically in the “wet” state under a high intensity lamp.

41. Results of Test • The test results should be reported as reactive (even if minimally reactive) or non-reactive. • All test results that are nonreactive should be quantitated.

43. 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

44. 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.

45. Malaria Leprosy Relapsing fever Infectious Mononucleosis Atypical pneumonia Viral pneumonia Lupus erythematosus Measles pregnancy drug abuse Non-syphilitic Conditions Giving Biologic False-Positive Results

46. Resolving False Positive RPR Tests • False positive RPR tests may be resolved by testing the patient’s serum with a specific treponemal antigen test.

47. Explanation of Results • Diagnosis of syphilis requires correlation of geographical area or country, patient history, physical findings, and results of syphilis antibody tests. • T. pallidum is diagnosed when both thescreening and confirmatory test are reactive.

48. Explanation of Results • Treponemal tests are more specific than non-treponemal tests • Treponemal tests confirm syphilis when a reactive or positive non-treponemal result is obtained.

49. Confirmatory Tests for Syphilis • I.FTA-ABS flourescent treponemal antibodytest • II. TP-PA particle aggluntination T. pallidum test • III. MHA-TP Microhemagglutination assay - T. pallidum

50. Confirmatory Tests for Syphilis • Treponemal tests are used to confirm reactive non-treponemal procedures. • A positive FTA-ABS test almost always remains positive and therefore is not recommended for monitoring therapy.