Enclonar, Kimberly / MLS 3A

o Fever
Spirochetes o Pharyngitis
March 4, 2021 o Rash on the skin (generalized, or in palms and
Lean Kristin Ugdang, RMT soles of the feet) and mucus membranes.
Contains large amounts of treponemes.
Spirochetes
• Presence of papillomatous or wart-like lesions:
• Long, slender, helically coiled bacteria containing axial Condyloma lata
filaments or periplasmic flagella which wind around
• Last for a few days up to 8 weeks.
the bacterial cell wall and are enclosed by an other
• Spontaneously heals.
sheath
• Exhibit a corkscrew or flexion motility Latent Syphilis
• 2 families: Spirochetaceae (Borellia and Treponema) • Follows after the disappearance of the secondary
and Leptospiraceae (Leptospira) syphilis
• Lack or absence of clinical symptoms
Treponema
• No lesion
• The treponemes are thin, spiral organisms that are • Patients: non-infectious
about 0.1-0.2um in thickness and 6-20um in length
• Pregnant women - can pass the disease to the fetus
• Gram negative bacteria in terms of structure, but are even when they have no symptoms
difficult to stain because they are thin
• If still not treated, it can progress to tertiary syphilis.
• Visualized using dark-field microscope
• Pathogenic treponemes are under the species: Tertiary Syphilis
Treponema pallidum due to their morphological and
• Occurs 10-30 years after the disappearance of the
antigenic similarity. secondary syphilis
• Subspecies: • Characterized by:
o Treponema pallidum subspecies pallidum - o Gummatous syphilis (localized area of
venereal syphilis granulomatous lesions)
o Treponema pallidum subspecies pertenue - yaws
▪ Bones, skin, subcutaneous tissue
o Treponema pallidum subspecies endemicum -
▪ May heal spontaneously or may remain
non-venereal endemic syphilis destructive of chronic inflammation
o Treponema pallidum subspecies carateum - Pinta
▪ Little to no presence of treponemes
o CVD
Syphilis o Neurosyphilis
• Most commonly acquired spirochete disease in the US
• In the PH (2018), the disease accounted for 964 or Congenital Syphilis
0.16% of total deaths • Occurs when a woman with early syphilis or early
• Transmission: latent syphilis transmits the organism to the fetus
o Unprotected sexual contact
• Symptoms:
o Congenital transmission o Clear or hemorrhagic rhinitis or runny nose
o Parenteral transmission (needles or blood) o Skin eruptions in the form of rash prominent
o Transmitted through transfusion of "fresh around the mount, palms, and soles
blood" o Generalized lymphadenopathy
• Causative agent: Treponema pallidum subspecies o Splenomegaly or hepatosplenomegaly
pallidum o Jaundice, anemia
• Invades through breaks in the skin or penetrate o Painful limbs and bone abnormalities
directly
• 3 main stages Laboratory Diagnosis
o Primary syphilis
Direct Detection
o Secondary syphilis
• Dark field microscopy
o Tertiary syphilis o Use to diagnose primary and secondary syphilis
o Demonstrates the presence of live T. Pallidum
Primary Syphilis subspecies pallidum from skin lesion
• Characterized by the presence of chancre o Identified on the basis of characteristic
• Develops between 10-90 days (average 21 days) after corkscrew motility or flexing motility
exposure to the organism
• Painless, solitary lesion, characterized by raised, and Serologic Detection
well-defined borders
• Useful in diagnosing
• Contains large amounts of treponemes o Patients with no active lesion
• In men - occur on the outside of the penis o Patients who are in the secondary or tertiary
• In women - may appear in the vagina or endocervix syphilis
• Other possible locations: lips, tongue, anus • Not useful in primary syphilis
• Lasts from 1-6 weeks, lesions heal spontaneously • Non-treponemal Serologic Tests (NTT)
(disappears even without treatment) - self-limiting o Detects reagin - patient antibodies against
cardiolipin
Secondary Syphilis o Screening. Suggestive but not conclusive
• Characterized by systemic dissemination of organism • Treponemal Serologic Test (TT)
• Observed about 1-2 months after the disappearance o Detects patient antibodies against Treponema
of the chancre pallidum (any subspecies)
• Symptoms:
o Lymphadenopathy
o Malaise
 Enclonar, Kimberly / MLS 3A
Cardiolipin o Additional:
• Important component of the inner mitochondrial ▪ EDTA, thimerosal, and choline chloride -
membrane of mammalian cells, including humans stabilize antigen and inactivated
• Constitutes 20% of the total lipid composition of the complement
mitochondrial membrane ▪ Cardiolipin is bound to charcoal to facilitate
o "Cardiolipin" is derived from the fact that it was macroscopic reading
first found in animal hearts. It was isolated from • Specimen preparation
beef heart. o Serum does not require preheating
• Released from damaged cells due to infections • Procedure
(bacterial, viral, fungal, parasitic) or other disease o On the RPR-Carbon card, designate one reaction
o A cryptic antigen that can activate the immune circle each to contain 0.05mL of:
response and trigger antibody production ▪ Patient's serum
▪ Reactive (+) control - bottle with red cap
Reagin ▪ Non-reactive (-) control - bottle with blue
• Antibody against cardiolipin cap
• Found in the sera of patients with: o In each designated circle, add one drop of RPR
o Syphilis reagent (botte with white cap)
o Other disease states o Mix with sterile stirrer
• Serves as the unknown analyte in NTT o Rotate on the VDRL rotator for 8 minutes
@100rpm
Non-treponemal Tests (NTT) o Observe for macroscopic flocculation
VDRL test
• Designed by the Venereal Disease Research
Laboratory
• A flocculation test - type of precipitation which
involves antigens (cardiolipin - known) of very fine
particles
• Performed using ceramic glass slides with rings
• Reagents:
o Antigen suspension: Treponemal Tests (TT)
▪ CLC reagent: alcoholic solution of 0.03% • Treponema pallidum immobilization test (TPI)
cardiolipin, 0.21% lecithin, and 0.9% • Treponema pallidum hemagglutination test (TPHA)
cholesterol • Microhemagglutination - Treponema pallid (MHA-TP_
▪ Delivered using a Hamilton syringe • Hemagglutination Treponemal Test for Syphilis
• Specimen preparation: (HATTS)
o Serum is preheated at 56C for 30 minutes to • Fluorescent Treponemal Antibody Absorption Test
inactivate complement (FTA-Abs)
• Procedure: • Treponema pallidum particle agglutination Test (TP-
o 0.05mL of patient's serum, non-reactive control, PA)
minimally reactive control, and reactive control
o Using a stirrer, spread patient's serum and Reading Assignments
control to fill the entire ring Comprehensively describe what are Nichol's and Reiter's
o 1 drop of VDRL reagent is added to each ring and strains and their applications in the serologic tests for
mix using a stirrer syphilis.
o Slide is put on the VDRL rotator shaker and
In the fluorescent treponemal antibody absorption (FTA-
rotated for 4 minutes @180rpm
ABS) test, Reiter’s strains are non-pathogenic treponemes
o Read microscopically to determine presence of
which cross-react with the patient’s serum to remove
flocculation or small clumps
antibodies other than to those Treponema pallidum antigens.
Reactive Medium to large clumps
This enhances specificity towards Treponema pallidum. On
Weakly reactive Small clumps the other hand, Nichol’s strain contains dead T. pallidum
Nonreactive No clumps or slight roughness organisms embedded on the slide. This cross-reacts with the
patient’s antibodies towards T. pallidum.

Comprehensively discuss the kinetics of the immune

response for syphilis and correlate it with the stages of
syphilis infections as well as the methods of diagnosis (dark
field or serology) for each stage
Since treponemal invasion targets the skin and mucosal
membranes, the main responders are the T cells and
Rapid Plasma Reagin (RPR) macrophages in the area. This is the reason why dark-field
• Most commonly used NTT microscopy or the isolation of the organism is preferred in the
• Flocculation test early weeks of primary stage since antibodies develop later on
• A modified VDRL within the 1st and 3rd week after the exposure. Dark field
• Reagents: microscopy can also be used to identify live organisms on the
o Antigen suspension:
secondary stage due to the development of generalized rash.
▪ CLC reagent: alcoholic solution of 0.03%
However, serologic tests are invariably positive at this point.
cardiolipin, 0.21% lecithin, and 0.9%
The chronic nature of the disease shows that the organism is
cholesterol
 Enclonar, Kimberly / MLS 3A
able to evade the immune response of the person. Thus, This spirochete can be transmitted via an arthropod
tertiary manifestations include cardiovascular and vector. The Ixodes ticks. The main reservoir host is the white-
neurosyphilis complications. Serologic tests are the method of footed mouse however, it can also be found on dusky-footed
choice in the latent and tertiary stage. woodrat. The organism divides by binary fission
approximately every 12 hours. Nymphs and adult ticks can
Comprehensively discuss the principle of dark field transmit the disease. After transmission, the clinical
microscopy symptoms occur in three different stages. In the first stage, a
Dark field microscopy allows the direct detection of localized rash known as erythema migrans (EM) appears
spirochetes through the visualization of the treponemes between 2 days and 2 weeks after the tick bite. In the second
themselves. This is useful in primary and secondary syphilis stage, early dissemination in the bloodstream occurs. The
wherein there is the presence of active lesions. A serous fluid most prevalent neurologic sign is the facial palsy. The last
is collected from the skin lesions and viewed under the stage, the late Lyme disease, develops after months or years
microscope. The field condenser is responsible in keeping all of untreated infection. Arthritis, peripheral neuropathy, and
the incidental light out except those captured by the encephalomyelitis is evident in this stage. Lyme disease
organism. The corkscrew morphology or flexing motility is occurs despite high levels of circulating antibody and cellular
used to identify the organism. response.
Diagnosis of the disease is a clinical one, with
laboratory testing used as supporting evidence. Direct
Comprehensively discuss the principle and interpretation of
isolation of the organism is possible however, yield is low. A
the following treponemal tests:
two-tiered approach should be used in providing laboratory
Treponema pallidum Immobilization Test (TPI)
support for the diagnosis of Lyme disease.
This test is the reference and standard test used in
Immunofluorescence (IFE) or Enzyme Immunoassay (EIA)
the laboratory. This relies on the principle that live spirochete
should be used as a screening test and if positive, Western
are immobilized in the presence of antibodies and
blotting is used for the supplemental testing. PCR can also
complement in the serum. Thus, this utilizes a suspension of
now be used in the diagnosis of the disease.
motile T. pallidum as the reagent and immobilization of >50%
treponemes indicate a positive result.
Leptospirosis
Leptospirosis is a disease caused by the bacteria of
Treponema Pallidum hemagglutination Test (TPHA) genus Leptospira. Without treatment, this can cause damage
TPHA is a diagnostic test used to detect the amount
to the kidneys, central nervous system, liver, and even death.
of antibodies in the patient’s serum towards T. pallidum. In
These bacteria can be transmitted to humans via the contact
this test, sheep RBCs are sensitized with antigens from T.
with urine from infected animals. The most known vector are
pallidum. Agglutination occurs if these cells are exposed to
the rodents. Just like with other diseases, diagnosis can be
antibodies in the patient’s serum.
made through the help of serologic tests. Microscopic
Agglutination Test detects serovar-specific antibodies.
Micro hemagglutination – Treponema pallidum (MHA-TP)
Leptospira immunoglobulin M ELISA can also be used.
This is an agglutination test which used sheep RBCs
coated with T. pallidum antigens to cross-react with the
Relapsing Fever
patient’s antibody. The presence of agglutination shows a
Borrelia miyamotoi is a newly discovered organism,
positive reaction. Micro reactions can be seen in this test.
similar to the relapsing fever Borrelia group. This causes
similar clinical features with Lyme disease and also has at least
Hemagglutination Treponemal Test for Syphilis (HATTS)
one mammalian host. Compared to Lyme disease, no rash can
HATTS is another hemagglutination technique used
be seen in this disease. The infectious period of the disease
to detect the presence of antibodies towards the T. pallidum. also appears to be restricted to the bacteremia stage. Since
Unlike the TPHA which uses formalinized tanned sheep RBCs,
studies are still on-going about this infection, only a few
this test uses glutaraldehyde-stabilized turkey RBCs.
generalized laboratory methods to diagnose the disease are
present. Direct detection can be made by culture or PCR and
Treponema pallidum particle agglutination Test (TP-PA)
antibody detection against G1pQ proteins can also be made.
This is a semiquantitative test useful in diagnosing
infection in patients with a positive screening test and has
atypical signs of syphilis. This test utilizes colored gelatin
particles coated with treponemal antigens to form complexes
with antibodies from the patient’s serum. The presence of T.
pallidum antibodies is indicated by an agglutination which
forms a smooth mat covering in the well. A negative result
shows a compact button which settles at the bottom.

Comprehensively discuss the pathophysiology and serologic

testing of the following:
Lyme Disease
Lyme disease is transmitted via a spirochete, Borrelia
burgdorferi, which was named after Willy Burgdorfer. This
was known to be an arthritis-causing disease, but it has been
found out that it is a multisystem illness which affects the skin,
nervous system, heart, and joints.