LABORATORY Flashcards
Specimens: SYPHILIS
• Tissue fluid expressed from [?] and [?] for microscopy
• [?] or [?] for serologic testing
• A small section of [?] for testing congenital syphilis
ulcers and lesions
Blood (serum) or CSF
• For detection of T. pallidum in primary and secondary lesions
It will show brightly lit motile, long, coiled or spiral treponemes (in white arrows) against a dark backdrop
Darkfield microscopy for
T. pallidum
• For detection of T. pallidum in primary and secondary lesions
It visualizes specimens on slides with fluorescein-labeled antitreponemal
antibodies by fluorescence microscopy
Direct fluorescent antibody test
for T. pallidum (DFA-TP)
• For detection of T. pallidum in primary and secondary lesions
(Levaditi’s, Warthin-Starry, Dieterle,
or Fontana-Tribondeau method)
Silver-impregnation technique
T. pallidum are so thin and they do not stain well with a\
aniline dyes
T. pallidum are NOT cultured on
artificial media
used to confirm evidence of secondary and tertiary syphilis, and the only means to diagnose latent syphilis
Serological Test for syphilis (STS)
Standard these tests include the following:
i. VDRL (for Venereal Disease Research Laboratory)
ii. Rapid plasma reagin (RPR)
iii. Unheated serum reagin (USR)
iv. Toluidine red unheated serum (TRUST)
Nontreponemal tests
The nontreponemal tests detect serum antibodies, known as reagin, released from cells damaged by treponemes. They are based on the reaction of reagin with a phospholipid antigen, [?], which is usually extracted from beef heart to produce a visible clumping, or flocculation, within the serum.
cardiolipin
card showing nonreactive, weakly reactive, and strongly reactive serum samples (wells 1 to 3) with their respective agglutination patterns
Rapid plasmin reagin test
As a consequence, they are not specific for Treponema but have been used traditionally to screen for disease. However, they are useful only after the body has a sufficient amount of time to generate a detectable amount of reagin. Antibodies detected by these tests generally develop in 4-5 weeks of the initial infection (or, 1-4 weeks after the appearance of the primary chancre). There are many other diseases that result in [?] and false positives are common.
anti-cardiolipin antibodies
Nontreponemal tests are also used to monitor the course of the disease after
treatment. Positive tests revert to negative, often in [?] months In treated patients, and generally 3 years after.
6-18 mons
These tests include the following:
i. T pallidum-particle agglutination (TP-PA)
ii. T pallidum hemagglutination (TPHA)
iii. Microhemagglutination T pallidum (MHA-TP)
iv. Fluorescent treponemal antibody absorbed test (FTA-ABS)
v. Enzyme immunoassay (EIA)
Treponemal testsv
Treponemal tests are based on detecting the presence of [?] against T. pallidum surface antigens.
“specific” serum antibodies
They have traditionally been used to confirm a [?], or to confirm infection in the face of a negative nontreponemal test in late or latent disease stages. Treponemal-specific antibody develops 2-3 weeks of initial infection.
positive nontreponemal screening test
However, they cannot be used to monitor the response to therapy, or to detect relapse or reinfection in previously treated patients. [?] remain reactive for years, regardless of treatment status.
Antitreponemal antibodies
are available in some reference laboratories. It may be useful in the diagnosis of primary infection, when serologic tests are often still negative, and has been advocated for the diagnosis of neurosyphilis, particularly in AIDS patients, although some studies have not demonstrated advantages over currently used methods.
Molecular method (Nucleic Acid Amplification Test) = Polymerase Chain Reaction (PCR)
• Blood, obtained during febrile episode, for direct microscopy
• Blood (plasma), spinal fluid, synovial fluid or macerated tissue biopsy for
culture
RELAPSING FEVER (Borrelia recurrentis)
Microscopic examination of peripheral blood samples obtained during febrile episodes
RELAPSING FEVER (Borrelia recurrentis)
Borrelia species are stained well by aniline dyes, and reveal large, loosely coiled spirochetes.
Wright’s or Giemsa’s stains of fixed thin and thick blood smears
In blood mixed with equal parts of sterile, nonbacteriostatic saline, Borrelia
species move rapidly, often pushing the red blood cells around.
Dark- or brightfield microscopy of wet preparations of peripheral blood
The organism is extremely difficult to culture
Borrelia recurrentis
Borrelia recurrentis uses [?] medium, a nutritionally-rich fluid medium containing blood, serum, or tissue; incubate at 30°C to 34°C for up to 12 weeks under microaerophilic conditions.
Modified Kelly’s
are performed weekly from the lower portion of the broth to fresh media, and the cultures are examined by dark-field microscopy or by fluorescence microscopy after staining with acridine orange for the presence of spirochetes
Blind subcultures (0.1 mL)
Because of the long incubation time and low sensitivity associated with cultivation,
isolation is not a practical diagnostic test in most clinical laboratories. is often confined to reference or research laboratories.
Cultivation
are inoculated intraperitoneally with blood. Stained films of tail blood are examined for spirochetes 2–4 days later
White mice or young rats
There is no serological test for Borrelia causing relapsing fever because the organisms undergo during the course of disease.
antigenic variations
Specimens
• Blood
• CSF or joint fluid
LYME DISEASE
readily stain with aniline dyes and by silver impregnation techniques
B. burgdorferi
Microscopic visualization of [?] in tissues is too insensitive for most
stages of the disease because:
- Rare numbers of spirochetes are unevenly distributed in clinical
samples.
- Spirochete morphology is also variable and may be difficult to distinguish from host tissues in histologic sections.
- Highly nonspecific and may detect any type of bacterium.
B. burgdorferi
is highly fastidious, growing in tissue culture (not bacteriological)
media, but impractical because of organism’s extremely slow growth. The
vast majority of body fluid or tissue samples from patients with Lyme disease
do not yield spirochetes on culture
B. burgdorferi
has been isolated in a cell-free modification of Kelly’s medium
for spirochetes that was developed by Barbour. Although various
modifications to the medium have been made over time.
B. burgdorferi
Although various modifications to the medium have been made over time. Current media preparations such as [?] demonstrate better growth and recovery of the spirochete at lower inocula in a shorter time. The cultures are incubated at 34°C to 37°C in the dark. After incubation for 2 to 3 weeks, cultures are examined by darkfield microscopy or by fluorescence microscopy using either the fluorochrome dye acridine orange or a specific fluorescence-labeled antibody.
Barbour–Stoenner–Kelly II (BSK II)
remain the primary test method to confirm a clinical diagnosis of Lyme disease.
Antibody detection methods
• It is measured by enzyme-linked immunosorbent assay/immunofluorescence
assay and immunoblot.
Serology
Polymerase Chain Reaction (PCR)
Molecular method (Nucleic Acid Amplification Test)
Blood or CSF (during the bacteremic phase, in the first 7-10 days of infection)
for microscopic examination and culture
L. interrogans
(during the immune phase beginning the second week of illness up to 30 days) should be collected using great care to avoid contamination
Urine
is collected for agglutination testsv
Serum
are only faintly stained by aniline dyes
L. interrogans
Detection of motile leptospires in the specimens is optimized after centrifuging at
1500 g for 30 minutes
is initially spun at 500 g for 15 minutes to remove blood cells
sodium oxalate
heparin-treated blood
L. interrogans is so delicate that in
the darkfield microscopy, it may
appear only as a chain of minute
cocci
Most commonly used commercial culture media for L. interrogans are:
Fletcher’s semisolid medium
Ellinghausen–McCullough–Johnson–Harris (EMJH) medium
is an enriched medium containing rabbit serum and fatty acids required in the leptospiral growth. A low concentration of agar helps in detecting motility. When
supplemented with 5-fluorouracil, the medium is recommended for urine and other specimens containing mixed microbial flora to provide selective inhibition of bacterial contaminants without inhibiting the growth of leptospires
Fletcher’s semisolid medium
semisolid bovine serum albumin–Tween 80 (oleic acid) medium that contains 200 µg of 5-fluorouracil
Ellinghausen–McCullough–Johnson–Harris (EMJH) medium
Cultures are incubated in the dark under aerobic conditions at 25–30°C. Cultures should be held for up to [?] before discarding as negative
6 weeks
Examine tubes for growth every 5-7 days. Growth occurs as a band, known as [?],1-3 cm below the surface of the semisolid medium.
Dinger’s ring
Material collected from a few centimeters below the surface of cultures should be examined weekly by darkfield microscopy for the presence of leptospires with
corkscrew motility.
Microcolonies can be fixed with methanol and stained with [?] to show rod forms
Giemsa stain
They are the most readily culturable of the pathogenic spirochetes; but this is not routine and diagnosis is usually by serology
L. interrogans
The main diagnostic method for L. interrogans
Serology
is the gold standard serologic test based on the detection of antibody. Antibody production begins toward the end of the initial 7-day illness.
Microagglutination test (MAT)
Polymerase Chain Reaction (PCR)
Molecular method (Nucleic Acid Amplification Test)
