CHAPTER 18 IMMUNOLOGY & SEROLOGY
SYPHILIS (V. 1.4)
(BOOK, LECTURE NOTES n’ RECORDINGS)
HISTORY
 15th & 16th centuries indicates an epidemic that swept
across Europe. It was believed that Columbus and his
sailors brought the disease to Europe from the Americas.
Others believe that the organism was already in Europe
but underwent spontaneous mutation.
 The organism was identified in 1905 as Spirochaeta
pallida (old name).
GENERAL INFORMATION
 Treponema pallidum is causative agent of human
syphilis.
 A spirochete bacterium of the family Spirochaetaceae –
causing venereal disease (STD).
 Can only be viewed using
o Dark field microscopy – direct examination of the
movement of spirochetes
o Direct fluorescent antibody staining and
detecting antibodies in serum or CSF.
 it contains 8-24 coils and 6 -15 um long.
 contains trilaminar outer membrane similar to g (-) bacteria.
 Wasserman test (obsolete)- the first diagnostic blood test
for syphilis (1906)
 Penicillin is a drug of choice.
 Cannot be cultured on artificial media – but can be
inoculated using experimental animals (rabbit)
 NOTE: Treponema remains viable up to 5 days in tissue
specimen (from diseased host or from cryoprotected
specimens).
 Other names
o Syphilis
o Great Pox
o Evil Pox
o Spanish Disease
o French Disease
o Italian Disease
 Medically important Treponema (genus) includes the ff.
TRANSMISSION
(1) DIRECT CONTACT
Kissing – if the person has an active oral lesion (chancre)
Sexual contact- especially male having sex with male.
 Men is easier to get infected than women
o You know what’s the reason
Sharing of bed
 NOTE: It penetrates (1) intact mucous membrane or (2) or
through tiny defects (e.g. wound) in skin.
 multiplies locally and causes an ulcerative lesion known as
chancre (highly contagious stage). It also disseminates to
local regional lymph nodes, enter the lymphatic system
and then the circulatory system.
 Spirochetemia occurs is when T. pallidum enters the
circulatory system.
NOTE: Chancre plays an important role in the
transmission.
(2) PERINATAL ROUTE
*Especially during the last trimester of pregnancy.
NOTE: Congenital transfer is very evident in the latent
stage of syphilis. Newborn using cord cells are screened
for Ab of syphilis and representation of the current state of
infection of the mother.
(3) BLOOD CONTAMINATION (rare)
 Because it has a short survival period in blood.
 Can’t survive in citrated blood at 4 *C for no longer
than 72 hours.
(4) NOTE: Transmission can also be due to:
1. Migration or travel – ex. SARS/MERS-CoV
2. Mutation
SIGNS/SYMPTOMS
 Untreated syphilis is a chronic disease with subacute
symptomatic periods separated by asymptomatic
intervals, during which the diagnosis can be made
serologically.
 It penetrates intact mucous membrane or through tiny
defects (e.g. wound) in skin.
 Spirochetemia occurs is when T. pallidum enters the
circulatory system.
 Incubation time: ~3 Weeks (10-90 Days)
STAGES
1. Primary
 It occurs at the end of incubation period
 Appearance of transient, painless firm chancre 3 weeks
after initial infection.
o Develops papule then become ulcerated which
consequently develop as chancre (3 weeks after
infection)
o Some appear as early as 10 days or as late
as 3-4 months after infection
o May appear almost everywhere in the body.
o Regional adenopathy (swelling of lymph
nodes can be seen for those infected.
o It depends where the chancre is
predominantly located.
o If the chancre is predominantly seen on
genitalia, probably the patient will develop
inguinal adenopathy.
Dx note: Can only be detected using Microscopic Meningeal syphilis (>1 yr. of infection)
evaluation. If fluid is spread on a glass slide and examined  Brain & spinal cord
with a dark field microscope, the spirochete can be seen. Meningovascular syphilis (5-10 y of infection)
 Pia mater & arachnoid inflammation
2. Secondary Parenchymatous syphilis
 It occurs after the appearance of chancre (2-8 wks.)  Manifests general paresis, joint degeneration and tabes
 A generalized illness (e.g. fever, sore throat, nasal dorsalis (demyelination of dorsal root and ganglia
 Tabes dorsalis is characterized by a gait disturbance
discharge, increased WBC count) and bladder symptoms.
 The most infective stage
 It also progresses to generalized adenopathy. Dx note: if RPR is negative but with hi-suspicion of
 Macular lesions are now common Neurosyphilis, perform FTA-abs
 Appearance of condylomata lata.  For CSF, perform VDRL
 Palms and soles are the prominent sites of rashes IMMUNOLOGIC MANIFESTATIONS
unlike chickenpox which can’t invade the said area.
 If you’re immunocompetent enough, it will resolve Antigen may be observed in human syphilis
within 2-6 wks. (self-limiting) (1) Antigen restricted to t. pallidum
Dx: Screening test : RPR and VDRL (2) Antigen shared by different spirochetes
Confirmatory test: TP-PA Antibodies may be observed in human syphilis
(1) Specific Antibody for T. pallidum
3. Latent (hidden stage) a. IgM- rises on untreated early latent syphilis
b. IgG- rises on the subsequent stages of syphilis
 If the body can’t resolve the disease, it will progress to
(2) Nontreponemal antibodies (Reagin antibody)
latent stage.
 Note: It is a non-infectious stage  Not specific because it can be falsely
increase in
 Note: Sign & Symptoms may be insidious
(a) infectious diseases
 Can be detected only in serological methods.
 Measles
 Still, some of the patient develop relapses (or simply
 Chickenpox
they have a manifestation of secondary syphilis) for
 Hepatitis
the first 2- 4 years.
 infectious mononucleosis
o Relapses are extremely rare after 4
 leprosy
years of latency.
 tuberculosis
 All the stages are infectious except the latent stage  leptospirosis
but not in the case if patient develop relapses.  malaria
 It is during this time that the disease can be  rickettsial disease
transferred from the mother to the fetus.  trypanosomiasis
Dx note: serologic test is the only indication due to low abs-ag
 lymphogranuloma venereum
concentration
(b) noninfectious conditions
4. Tertiary (late syphilis)  Autoimmune disorders
 Occurs 3 to 10 years after the primary infection.  Drug addiction old age
 about 1/3 of the patient enters latent stage will  Pregnancy
progress to tertiary syphilis.  Recent immunization
 15% of untreated individual develops benign syphilis,  Blood transfusion
characterized by destructive granulomas or gummas  Connective tissue disorder
resembling segments of circles.
 Skeletal system is the most frequently affected. Food for thought….
 10% develops cardiac manifestation (aortic arch) Note: T. pallidum is not just the accountable of the diseases
 8% involves CNS disease e.g. Neurosyphilis manifestation of syphilis but the effect of the immune system
o CSF –Specimen choice itself, -as our body fights the bacteria, there will be a collateral
 OTHER SIGNIFICANT disorder cause by Syphilis damage may happen or simply our healthy cells will be
destroyed by our immune system (pathophysiologic response)
(1) Congenital syphilis due to the substance secreted by our leukocyte which is
 Can be prevented by early detection (30 supposedly intended only to syphilis bacteria. That is why
days b4 delivery) immunosuppression of cell mediated immunity is
 Early stage children (<2 yo) manifestations important to prolong the survival of infected patient.
include: anemia, condyloma latum and *Why cell mediated immunity?
hepatosplenomegaly. It’s because cell-mediated immunity is not specific for destroying
foreign substances that’s why collateral damage occurs like….
 Late stage children (>2yo)
 granulomatous infection (gumma) --it is the
 8th nerve deafness, keratitis and
effect of delayed hypersensitivity in the
Hutchinson’s teeth-collectively known as
immune host not from syphilis; syphilis is the
Hutchinson’s triad.
one who triggers it.
 Glomerulonephritis
(2) Neurosyphilis
It includes….
 Charity Rojas. | L.M Malaria, Systemic lupus erythematous, rheumatoid
LABORATORY METHODS arthritis, pregnancy (add’l list above)
Book: includes HIV, HSV, leprosy
DIRECT OBSERVATION Dx note: Requires inactivation of complement by heating in
(1) Dark field microscopy- primary syphilis test of order to reduce the interfering substances.
choice
(2) Direct/indirect fluorescent microscopy- alternative TREPONEMAL METHOD (More SPECIFIC)
choice because it doesn’t need to require live
(1) Fluorescent Treponemal Antibody Absorption
specimen.
Test (FTA-ABS)
 PRINCIPLE: Indirect Fluorescent Immunoassay
NON-TREPONEMAL METHOD (Less SPECIFIC)  REAGENT ANTIGEN: Nichol’s strain dried and fixed on
Determines the presence of Reagin slide
Reagin is an antibody-like protein that is secreted by the damaged  ABSORBENT: Reiter Treponemes (nonpathogenic
cells. It is composed of cardiolipin, cholesterol and lecithin.
strains T. pallidum)
(1) Rapid plasma Reagin (RPR) / Carbogen test
 PRINCIPLE: Flocculation with coagglutination of the NOTE: Source of Ab – patient’s serum
carbon particles Source of Ag – Nichol’s strain (nonviable strain of T.
 SERUM: undiluted, not heated pallidum from testicular tissue of the rabbits.
 REAGENT: Cardiolipin Ag, colorless alcoholic solution
containing cardiolipin, lecithin, charcoal, choline chloride & (1) Patient’s serum (diluted in absorbent coming from Reiter
Treponemes)
thimerosal1
(2) Then it is layered over into the slide – Slide is already fixed
 ROTATOR: 100 rpm for 8 min. with T. pallidum
 RING DIAMETER: 18 mm (3) If the patient is positive (+) – Abs bind to the bacteria
 ANTIGEN DELIVERY NEEDLE: gauge 20 needle, 60 (4) Abs will coat Treponema and add fluorescein
drops/mL Isothiocyanite (FITC), a labeled anti-human immunoglobin –
 A qualitative test in which undiluted serum is mixed with this combines with patient’s IgG (untreated infection) & IgM
cardiolipin antigen and microscopic charcoal particles on a (current infection) Abs that are adhering to T. pallidum and
cardboard slide. After a mechanical rotation for an results in a visible test reaction when examined by
appropriate length of time, the test is examined for fluorescence microscopy.
macroscopic agglutination of the charcoal particles. (5) POSITIVE PATIENT: Presence of fluorescence
 It measures IgG and IgM
(2) Hemagglutination Treponemal Test for Syphilis
 RPR is more sensitive than VDRL
(HATTS)
 Note: Flocculation is the reaction with the soluble Ag with the
soluble Ab forming fine particles
o PRINCIPLE: Hemagglutination
o REAGENT ANTIGEN: Glutaraldehyde stabilized
Undiluted serum (From patient) turkey RBC coated with Treponemal antigen.
+ Cardiolipin Ag (found in reagent)
+ Charcoal particle (found in kit)
------------(then swirl or mechanical rotation) -----------
(3) Microhemagglutination T. pallidum Test (MHA-
 formation of AGGLUTINATION/CLUMPING (+)
*Read microscopically
TP)
o PRINCIPLE: Hemagglutination
o REAGENT ANTIGEN: Tanned formalin sheep RBC
(2) Venereal Disease Research Laboratory (VDRL) coated with Treponemal antigen
 PRINCIPLE: Flocculation NOTE: Both are following the hemagglutination principle specifically
 SAMPLE: serum or CSF with the RBC clumping but they differ with the source.

 SERUM: Heated serum (50uL serum (56°C for 30 min)
 REAGENT: cardiolipin, cholesterol, lecithin
 ROTATOR: 180rpm for 4 min (serum VDRL)
 RING DIAMETER: 14 mm (serum VDRL)
 ANTIGEN DELIVERY NEEDLE:
o Qualitative serum VDRL – gauge 18 needle
without bevel that will deliver 60 drops of
antigen suspension/mL
o Quantitative serum VDRL – gauge 19 needle
that will deliver 75 drops of antigen
suspension per mL or gauge 23 needle
with/without bevel that will deliver 100 drops
of saline/mL.
o CSF VDRL – gauge 21 0r 22 needle that will
deliver 100 drops/mL
o REPORTING
Nonreactive – no clumps
Weakly reactive – small clumps
Reactive – medium or large clumps
FALSE (+) includes
(4) T. pallidum Immobilization Test (TPI)
o Most specific for syphilis (Gold Standard)
o PRINCIPLE: Ab produced against T. pallidum plus
complement can immobilize the live Treponemes.
o (+): >50% immobilized Treponemes
o SOURCES: 1. Non-pathogenic Reiter stain
2. Pathogenic Nichol’s strain
LABORATORY *Imunochromatographic method
 Add 10uL of plasma or serum (20uL of blood)
 Add 4 drops of assay diluent into the assay diluent well.
INTERPRETATION:
Interpret test results in 5-20 minutes after adding the assay
diluent. Do not read test results after 20 minutes. Reading too
late can give false results.
Charity Rojas. | L.M