SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES (Syphilis & other spirochetes) Flashcards
memorization
The causative agent of syphilis:
Treponema pallidum subspecies pallidum
Antigens in syphilis:
- Wassermann antigen
- Treponemal antigen
Wassermann antigen is also known as:
Cardiolipin (fat)
note: used in screening tests
Syphilis antigen that is used for confirmatory tests:
Treponemal antigens
Specific anti treponemal antibodies in early or untreated early latent syphilis are predominantly _______ antibodies.
predominantly M (IgM) antibodies
In syphilis, the early immune response to infection is rapidly followed by the appearance of _____ antibodies.
IgG antibodies
The greatest elevation in IgG concentration is seen in _______.
secondary syphilis
Treponemal antibodies are specific for:
- Outer membrane protein
- Endoflagellar protein
AKA reagin antibodies; antibodies that attack the cardiolipin
Non treponemal antibodies
A hard, painless firm lesion that is present in primary syphilis:
Chancre
In primary syphilis, the serum in ____% of cases become serologically active after 1 week and ____% become reactive after 3 weeks
In primary syphilis, the serum in 30% of cases become serologically active after 1 week and 90% become reactive after 3 weeks
Which test is more sensitive, and is equal to FTA ABS (in terms of sensitivity) for primary syphilis
RPR (rapid plasma reagin)
note: RPR is more sensitive than VDRL and equally sensitive with FTA ABS
The most contagious syphilis stage
Secondary syphilis
A wart-like lesion in moist areas of the body that is present in secondary syhpilis:
Condylomata lata
In secondary syphilis, all serologic tests detect the infection
True or False
True
Syphilis stage where the patient is asymptomatic; patients in this stage are noninfectious except for pregnant women
Latent syphilis
Duration of infection in early latent syphilis:
Early latent = <1 year of infection
Duration of infection in late latent syphilis:
Late latent = >1 year of infection
Most common complication in tertiary stage syphilis but can occur after primary stage:
Neurosyphilis
Degeneration of the lower spinal cord, and general paresis or chronic progressive dementia
TABES DORSALIS
Areas of granulomatous inflammation that are most often found on bones, skin or subcutaneous tissue
Gummas (granulomas)
What type of microscope is used for detecting spirochetes?
Dark-Field Microscope
What stages of syphilis can be diagnosed by dark-field microscopy?
Primary and Secondary syphilis can be diagnosed by demonstrating the presence of T. pallidum in exudates from skin lesions
Characteristic of treponemes in dark-field microscopy:
Corkscrew morphology
Flexing motility
A direct fluorescent antibody testing method that uses a fluorescent-labeled antibody conjugate to T. pallidum
FAD FTP
(Fluorescent Antibody Dark-Field T. pallidum)
An indirect fluorescent antibody testing method that uses antibodies specific for T. pallidum. A second labeled anti-immunoglobulin antibody
FTA ABS (Fluorescein Treponemal Antibody Absorption)
Advantage of FTA ABS (Indirect method):
FTA ABS (Indirect method) does not require the use of live specimens
Disadvantage of FTA ABS (Indirect method)
Cross-reacts with other T. pallidum subspecies
Non treponemal tests:
- Venereal Disease Research Laboratory (VDRL)
- Rapid Plasma Reagin (RPR)
Recommended specimen for neurosyphilis:
VDRL
Components of the VDRL antigen:
- Cardiolipin
- Lecithin
- Cholesterol
What component of the VDRL antigen serves as the antigen:
Cardiolipin (diphosphatidyl glycerol)
What component of the VDRL antigen helps neutralize anticomplementary properties of cardiolipin and enhances the sensitivity of the reaction?
Lecithin
What component of the VDRL antigen increases the effective reaction surface and complement-fixing capacity of cardiolipin with reagin?
Cholesterol
VDRL
Patient serum
- Inactivate complement by heating at _____
- Reinactivation is done at ____ for ____ minutes when more than 4 hours has elapsed
- When serum is used: test slides used should be _____ mm
- When CSF is used, the diameter of the ring should be ____ mm
VDRL
Patient serum
- Inactivate complement by heating at 56C for 30 minutes
- Reinactivation is done at 56C for 10 minutes when more than 4 hours have elapsed
- When serum is used: test slides used should be 14 mm
- When CSF is used, the diameter of the ring should be 16 mm
Qualitative serum VDRL
Gauge:
Bevel:
Drops:
Qualitative serum VDRL
Gauge: 18
Bevel: none
Drops: 60/mL
Quantitative serum VDRL
Gauge:
Bevel:
Drops:
Quantitative serum VDRL
Gauge: 19
Bevel: none
Drops: 75/mL
OR
Gauge: 23
Bevel: with or without bevel
Drops: 100/mL
CSF VDRL
Gauge:
Bevel:
Drops:
CSF VDRL
Gauge: 21/22
Bevel: none
Drops: 100/mL
Rotation (centrifugation requirements)
Serum:
CSF:
Rotation (centrifugation requirements)
Serum: 180 rpm, 4 minutes
CSF: 180 rpm, 8 minutes
VDRL microscopic examination is done at what objective?
LPO (low power objective)
Reporting in microscopic examination
Non-reactive:
Weakly reactive:
Reactive:
Reporting in microscopic examination
Non-reactive: No clumps
Weakly reactive: Small clumps
Reactive: Medium to large clumps
Test principle of VDRL:
Rapid slide microflocculation
Test principle of rapid plasma reagin (RPR)
Flocculation/ charcoal agglutination
Advantage of RPR over VDRL
RPR doesn’t need microscope
Components of the modified VDRL antigen:
- Original VDRL antigen
- Disodium salt of EDTA
- Charcoal
- Phosphates
- Thimerosal
- Choline chloride
Component of the modified VDRL antigen that inactivates complement:
Choline chloride
Visualizing agent of the modified VDRL antigen:
Charcoal
Modified VDRL antigen component that prevents oxidation of lipids:
Disodium salt of EDTA (versene)
component of the modified VDRL that acts as the preservative:
Thimerosal
Rapid Plasma Reagin (RPR)
Slide used:
Rotation:
Delivery needle: ____ gauge needle without bevel, ____ drops per 1 mL
Rapid Plasma Reagin (RPR)
Slide used: 18 mm circle RPR cards
Rotation: 100 rpm, 8 minutes
Delivery needle: 20 gauge needle without bevel, 60 drops per 1 mL
Treponemal tests:
- Fluorescent treponemal antibody absorption test (FTA ABS)
- Hemagglutination tests
- Particle agglutination (Serodia-TP-PA test)
In FTA ABS, it consist of an extract of nonpathogenic treponemes (reiter stain) that is incubated with the heat-inactivated patient serum:
Sorbent
What is the purpose of the sorbent in FTA ABS?
Removes cross-reactivity with treponemes other than T. pallidum
Nichols strain is a well-characterized strain of:
A) Treponema pallidum subspecies pallidum
B) Neisseria gonorrhoeae
C) Chlamydia trachomatis
D) Haemophilus ducreyi
A) Treponema pallidum subspecies pallidum
Why shouldn’t FTA ABS be used to monitor response to therapy?
Once a patient is reactive, that individual remains so for life, therefore it shouldn’t be used for monitoring response to therapy
In FTA ABS, the intensity of the green color is reported on a scale of 0 to 4+. What scale is considered to be reactive?
2+ and above
In FTA ABS, a result of 1+ means that the specimen was minimally reactive. What should the health provider do next? Should the lab tech report the result?
The test must be repeated with a second specimen drawn in 1 to 2 weeks.
Note: The test must be repeated after 1 to 2 weeks so that the patient will have enough time to synthesize more antibodies.
What animal RBC is used in Treponemal pallidum hemagglutination test (TPHA)?
Turkey erythrocytes
What animal RBC is used in Microhemagglutination Assay for Treponema pallidum (MHA-TP)?
Tanned sheep erythrocytes
What animal RBC is used in Hemagglutination Treponemal Tests for SY (HATTS)?
Glutaraldehyde stabilized TURKEY erythrocytes
A hemagglutination test that does not use animal RBC:
Serodia-TP-PA Test (particle agglutination)
Positive result of Serodia-TP-PA test:
Formation of a lattice-like structure that produces a smooth mat
False-positive nontreponemal tests for syphilis may occur because of which of the following?
a. Infectious mononucleosis
b. Systemic lupus
c. Pregnancy
d. All of the above
d. All of the above
False-positive nontreponemal tests (e.g., VDRL, RPR) for syphilis can occur due to:
Conditions causing false positives
1. Infectious mononucleosis (a)
2. Systemic lupus erythematosus (b)
3. Pregnancy (c)
4. Viral infections (e.g., HIV, hepatitis)
5. Autoimmune disorders (e.g., rheumatoid arthritis)
6. Malaria
7. Chronic diseases (e.g., liver disease)
Why false positives occur
1. Unspecific antibody reactions
2. Cross-reactivity with cardiolipin antigens
3. Immune complex formation
Confirmatory treponemal tests (e.g., FTA-ABS, TP-PA) help rule out false positives.
In the fluorescent treponemal antibody absorption (FTA-ABS) test, what is the purpose of absorption with Reiter treponemes?
a. It removes reactivity with lupus antibody.
b. It prevents cross-reactivity with antibody to other T. pallidum subspecies.
c. It prevents cross-reactivity with antibody to nonpathogenic treponemes.
d. All of the above.
c. It prevents cross-reactivity with antibody to nonpathogenic treponemes.
FTA-ABS Test:
Purpose of Absorption with Reiter Treponemes
1. Removes non-specific antibodies
2. Reduces cross-reactivity with antibodies against non-pathogenic treponemes (e.g., commensal treponemes)
3. Enhances specificity for Treponema pallidum subspecies pallidum (syphilis-causing strain)
How it Works
1. Patient serum is mixed with Reiter treponemes (non-pathogenic).
2. Non-specific antibodies bind to Reiter treponemes.
3. Absorbed serum is then tested against pathogenic T. pallidum.
4. Specific antibodies against T. pallidum are detected.
Options Clarification
a. Lupus antibody removal: incorrect (lupus antibody removal isn’t primary purpose).
b. Cross-reactivity with other T. pallidum subspecies: incorrect (test targets T. pallidum subsp. pallidum).
d. All of the above: incorrect (only option c is correct).
Which test is recommended for testing cerebrospinal fluid for detection of neurosyphilis?
a. RPR
b. VDRL
c. FTA-ABS
d. Enzyme immunoassay
b. VDRL (Venereal Disease Research Laboratory)
Recommended Tests for Neurosyphilis Diagnosis:
Primary Tests
1. VDRL (Venereal Disease Research Laboratory): CSF-VDRL is the standard test for diagnosing neurosyphilis.
2. CSF-TPPA (Treponema pallidum particle agglutination) or CSF-FTA-ABS (Fluorescent Treponemal Antibody Absorption): confirmatory tests.
Why VDRL?
1. High specificity (98-100%)
2. Sensitive for neurosyphilis (75-100%)
3. Quantifiable results
4. Can monitor treatment response
Limitations of Other Options
1. RPR (Rapid Plasma Reagin): not recommended for CSF testing.
2. FTA-ABS: sensitive but non-quantifiable; often used for confirmatory testing.
3. Enzyme Immunoassay (EIA): not standardized for CSF testing.
Additional Diagnostic Criteria
1. Elevated CSF protein
2. Pleocytosis (increased white blood cells)
3. Clinical presentation
Advantages of direct fluorescent antibody testing to T. pallidum include all of the following except
a. reading is less subjective than with dark-field testing.
b. monoclonal antibody makes the reaction very specific.
c. slides can be prepared for later reading.
d. careful specimen collection is less important than in dark-field testing.
d. Careful specimen collection is less important than in dark-field testing.
Direct Fluorescent Antibody (DFA) Testing Advantages:
1. Objective reading (a)
2. High specificity due to monoclonal antibodies (b)
3. Slides can be stored for later reading (c)
4. Rapid results
5. Sensitivity (70-100%)
However:
d. Careful specimen collection remains crucial for DFA testing, just like dark-field microscopy.
Key Considerations:
1. Specimen quality affects test accuracy.
2. Proper collection, handling, and storage ensure reliable results.
Which of the following is true of nontreponemal antibodies?
a. They can be detected in all patients with primary syphilis.
b. These antibodies are directed against cardiolipin.
c. Nontreponemal tests remain positive after successful treatment.
d. The antibodies are only found in patients with syphilis
b. These antibodies are directed against cardiolipin.
Nontreponemal Antibodies Characteristics:
Key Points
1. Directed against cardiolipin (b)
2. Detected in 70-80% of primary syphilis patients (not all, contradicting a)
3. Typically become negative after successful treatment (contradicting c)
4. Can occur in non-syphilitic conditions (e.g., lupus, pregnancy, viral infections), making (d) incorrect
Nontreponemal Tests Examples:
1. Rapid Plasma Reagin (RPR)
2. Venereal Disease Research Laboratory (VDRL)
These tests detect antibodies against cardiolipin, released from damaged host cells and Treponema pallidum.
Which syphilis test detects specific treponemal antibodies?
a. RPR
b. VDRL
c. FTA-ABS
d. Dark-field exam
c. FTA-ABS (Fluorescent Treponemal Antibody-Absorption)
FTA-ABS detects specific antibodies against Treponema pallidum, confirming syphilis infection.
Other options:
1. RPR (Rapid Plasma Reagin) and VDRL (Venereal Disease Research Laboratory): nontreponemal tests, detecting cardiolipin antibodies.
2. Dark-field exam: direct microscopic visualization of Treponema pallidum, not antibody detection.
Confirmatory Treponemal Tests:
1. FTA-ABS
2. TP-PA (Treponema pallidum Particle Agglutination)
3. EIA (Enzyme Immunoassay)
4. Western Blot
Which of the following is true of treponemal tests for syphilis?
a. They are usually negative in the primary stage.
b. Titers decrease with successful treatment.
c. In large-volume testing, they are often used as screening tests.
d. They are subject to a greater number of false positives than nontreponemal tests.
c. In large-volume testing, they are often used as screening tests.
Treponemal Tests Characteristics:
Key Points
1. Sensitive and specific: Detect antibodies against Treponema pallidum.
2. Used as screening tests: In large-volume testing (c).
3. Remain positive for life: Even after successful treatment (contradicting b).
4. May detect past infections: Not distinguishing current from past infections.
Examples of Treponemal Tests:
1. FTA-ABS (Fluorescent Treponemal Antibody-Absorption)
2. TP-PA (Treponema pallidum Particle Agglutination)
3. EIA (Enzyme Immunoassay)
4. Western Blot
Limitations
1. False positives: Less common than nontreponemal tests, but still possible (contradicting d).
2. Cannot distinguish: Between current and past infections.
Clinical Use
1. Confirmatory testing: Following positive nontreponemal tests.
2. Diagnosing late-stage syphilis: When nontreponemal tests may be negative.
An RPR test done on a 19-year-old woman as part of a prenatal workup was negative but exhibited a rough appearance. What should the technologist do next?
a. Report the result out as negative.
b. Do a VDRL test.
c. Send the sample for confirmatory testing.
d. Make serial dilutions and do a titer.
d. Make serial dilutions and do a titer.
RPR Test Protocol:
1. Rough or “rough-looking” results indicate potential false negatives or non-specific reactions.
2. Serial dilutions and titration help clarify results.
Procedure:
1. Dilute sample (1:1, 1:2, 1:4, etc.).
2. Repeat RPR testing.
3. Record endpoint titer.
This ensures accurate results, distinguishing:
1. False negatives
2. Non-specific reactions
3. True positives
Treponemal EIA tests for syphilis are characterized by all of the following except
a. they are adaptable to automation.
b. they are useful in monitoring antibody titers in syphilis patients undergoing therapy.
c. subjectivity in reading is eliminated.
d. they can be used to distinguish between IgG and IgM antibodies.
b. They are useful in monitoring antibody titers in syphilis patients undergoing therapy.
Treponemal EIA (Enzyme Immunoassay) Characteristics:
Advantages
1. Adaptable to automation (a)
2. Objective results (c)
3. Distinguish IgG/IgM antibodies (d)
Limitations
1. Not suitable for monitoring treatment response: Titers remain positive despite successful treatment.
2. Cannot differentiate between past and current infections.
Alternative Monitoring Methods:
1. Nontreponemal tests (RPR, VDRL)
2. Dark-field microscopy
Which of the following tests is the most specific during the early phase of Lyme disease?
a. IFA
b. EIA
c. Immunoblotting
d. Detection of B. burgdorferi DNA by PCR
d. Detection of B. burgdorferi DNA by PCR.
Early-phase Lyme disease (0-4 weeks) testing:
Most Specific Tests
1. PCR (Polymerase Chain Reaction): Detects Borrelia burgdorferi DNA in blood, CSF, or tissue.
2. Immunoblotting (Western Blot): IgM and IgG antibody detection.
Less Specific Tests
1. IFA (Indirect Fluorescent Antibody): May yield false positives.
2. EIA (Enzyme Immunoassay): Lower specificity, potential cross-reactivity.
CDC Recommendations:
1. PCR for early diagnosis.
2. Two-tier testing (EIA + Immunoblotting) for later stages.
False-positive serological tests for Lyme disease may be caused by all of the following except
a. shared antigens between Borrelia groups.
b. cross-reactivity of antibodies.
c. resemblance of flagellar antigen to that of Treponema organisms.
d. a patient in the early stage of the disease.
d. A patient in the early stage of the disease.
False-Positive Serological Tests for Lyme Disease Causes:
- Shared antigens between Borrelia groups (a)
- Cross-reactivity of antibodies (b)
- Resemblance of flagellar antigen to Treponema organisms (c)
Not typically causing false positives:
1. Early-stage patients (d): False negatives more common due to:
Factors contributing to false negatives
1. Low antibody levels
2. Immune response delay
3. Limited antigen exposure
Common causes of false positives:
1. Other Borrelia infections
2. Syphilis (Treponema)
3. Autoimmune diseases
4. Viral infections
A 24-year-old man who had just recovered from infectious
mononucleosis had evidence of a genital lesion. His RPR test was positive. What should the technologist do next?
a. Report out as false positive.
b. Do a confirmatory treponemal test.
c. Do a VDRL.
d. Have the patient return in 2 weeks for a repeat test.
b. Do a confirmatory treponemal test.
RPR (Rapid Plasma Reagin) Positive Follow-up:
Considerations
1. False positives: Associated with infectious mononucleosis, lupus, pregnancy, and viral infections.
2. Confirmatory testing necessary.
Next Steps
1. Perform treponemal test (e.g., FTA-ABS, TP-PA, EIA).
2. Differentiate between syphilis and non-syphilitic antibodies.
Rationale for Incorrect Options
1. a. Report as false positive: Premature without confirmation.
2. c. VDRL: Similar limitations as RPR; confirmatory testing needed.
3. d. Repeat test in 2 weeks: Delayed diagnosis may compromise treatment.
A 15-year-old girl returned from a camping trip. Approximately a week after her return, she discovered a small red area on her leg that had a larger red ring around it. Her physician had her tested for Lyme disease, but the serological test was negative. What is the best explanation for these results?
a. She definitely does not have Lyme disease.
b. The test was not performed correctly.
c. Antibody response is often below the level of detection in early stages.
d. Too much antibody was present, causing a false negative.
c. Antibody response is often below the level of detection in early stages.
Early-stage Lyme disease (0-2 weeks) characteristics:
Clinical Presentation
1. Erythema migrans (red ring-shaped rash)
2. Fever
3. Headache
4. Fatigue
Diagnostic Challenges
1. Serological tests (e.g., EIA, IFA) may yield false negatives due to:
- Low antibody levels
- Immune response delay
- Limited antigen exposure
Best Course of Action
1. Clinical diagnosis based on symptoms and exposure history.
2. Repeat serological testing in 2-4 weeks.
3. Consider PCR (Polymerase Chain Reaction) testing for direct detection.
Incorrect Options:
a. Definitive exclusion requires repeated testing and clinical evaluation.
b. Test correctness depends on laboratory quality control.
d. Prozone phenomenon (excessive antibody) rarely occurs in Lyme disease testing.
The reverse screening algorithm for syphilis testing
a. is the CDC-preferred algorithm.
b. is more labor intensive than the “traditional” method.
c. has a significant number of false positives that must be resolved by doing a TP-PA test.
d. is more prone to transcription errors in reporting.
c. has a significant number of false positives that must be resolved by doing a TP-PA test.
Reverse Screening Algorithm for Syphilis:
Characteristics:
1. Initial treponemal test (e.g., EIA, chemiluminescence).
2. Positive results confirmed with nontreponemal test (RPR/VDRL).
3. Discordant results resolved with TP-PA (Treponema pallidum Particle Agglutination).
Advantages:
1. Improved sensitivity
2. Reduced false negatives
Limitations:
1. Increased false positives (c)
2. Additional confirmatory testing required
CDC Recommendations:
1. Reverse algorithm acceptable, but traditional algorithm (nontreponemal + treponemal) still valid.
All of the following statements are true for leptospirosis except
a. It is caused by tightly coiled spirochetes under the genus
Leptospira.
b. Infection is zoonotic in nature.
c. Leptospires cannot be cultured on artificial culture media.
d. It is one of the nationally reportable diseases.
c. Leptospires cannot be cultured on artificial culture media.
Leptospirosis Facts:
1. Caused by Leptospira genus spirochetes (a).
2. Zoonotic infection, primarily spread through animal contact (b).
3. Nationally reportable disease in the United States (d).
Leptospira Cultivation:
1. Can be cultured on artificial media, such as:
- Fletcher medium
- Stuart medium
- EMJH (Ellinghausen-McCullough-Johnson-Harris) medium
1. Requires specific conditions (e.g., temperature, pH).
Which of the following specimens provides the most reliable means of detecting leptospires during the first week of infection?
a. Urine
b. Liver biopsy
c. Blood
d. Conjunctival swab
c. Blood.
Early-Stage Leptospirosis Detection (0-7 days):
Most Reliable Specimens:
1. Blood (c): Leptospira bacteremia occurs during early infection.
2. CSF (cerebrospinal fluid): For neurological manifestations.
Less Reliable Specimens (early stage):
1. Urine (a): Leptospira appears in urine after 7-10 days.
2. Liver biopsy (b): Invasive, rarely performed.
3. Conjunctival swab (d): Not commonly used.
Optimal Diagnostic Methods:
1. PCR (Polymerase Chain Reaction)
2. Culture
3. Serology (MAT/ELISA)
Which of the following statements regarding transmission of leptospirosis is not true?
a. Infection can be acquired by coming into contact with urine-contaminated soil or water.
b. People working in rat-infested areas run a high risk of contracting the infection.
c. High-risk activities include swimming or wading in water bodies contaminated with animal urine.
d. Person-to-person contact is a significant means of contracting the infection.
d. Person-to-person contact is a significant means of contracting the infection.
Leptospirosis Transmission:
True Statements:
1. Contact with urine-contaminated soil/water (a).
2. Occupational risk for those working around rodents (b).
3. High-risk activities: swimming, wading, or water sports (c).
False Statement:
d. Person-to-person transmission is rare, except:
1. Vertical transmission (mother-to-child).
2. Rare cases through intimate contact.
Primary Transmission Routes:
1. Direct contact with infected animal urine.
2. Indirect contact through contaminated environments.
The gold standard method for diagnosis of leptospirosis is
a. culturing the specimen on Fletcher semisolid or Stuart liquid media.
b. enzyme-linked immunosorbent assay (ELISA).
c. microscopic agglutination test (MAT).
d. dark-field or phase contrast microscopy.
c. Microscopic Agglutination Test (MAT).
Gold Standard for Leptospirosis Diagnosis:
MAT Characteristics
1. High specificity and sensitivity
2. Detects IgM and IgG antibodies
3. Differentiates between Leptospira serovars
4. Considered definitive diagnosis
Other Options:
1. a. Culturing (Fletcher/Stuart media): Difficult, time-consuming, and less sensitive.
2. b. ELISA: Useful for screening, but lower specificity.
3. d. Dark-field/phase contrast microscopy: Limited sensitivity, requires expertise.
