LABORATORY Flashcards
Specimens • Throat swabs • Nasopharyngeal swabs • Throat washings • Sputum • Tracheal and transtracheal aspirate • Bronchoscopy, BAL, and lung tissue specimens
Mycoplasma pneumoniae
In the early stages of [?], diagnosis is made on clinical grounds. However, as the infection progresses several laboratory tests are available.
M. pneumoniae infection
Gram staining in not useful in the detection of M. pneumoniae because of the absence of the [?]. It is of value only to eliminate other possible
pathogens.
cell wall
is used as a fluorescent staining method used to detect mycoplasma.
Acridine Orange (AO) staining
is a fluorochrome dye that binds nucleic acid. At a low pH under UV light, bacterial and fungal nucleic acid fluoresces orange whereas background mammalian nucleic acid fluoresces green
Acridine orange
Mycoplasmas are very susceptible to adverse environmental conditions, so specimens should be placed into appropriate [?] as soon after collection as possible
transport or growth media
are supplemented with Mycoplasma Supplement because Mycoplasma spp. are fastidious in their growth requirements
base media (PPLO agar and PPLO broth)
Media for the isolation of M. pneumoniae are buffered at an initial pH of about
7.8
, or both can be added to the base medium at the time of use to make it
selective against gram-positive and gram-negative bacteria that usually accompany mycoplasmas in
clinical materials
Thallium acetate or penicillin
The base media may be supplemented with
glucose and phenol red indicator
Inoculation and incubation of Mycoplasma pneumoniae
Inoculate the
complete medium (agar or broth)
Inoculation and incubation of Mycoplasma pneumoniae
Incubate plates at 35 ± 2°C in a moist aerobic (or containing [?]) atmosphere
5% carbon dioxide
Inoculation and incubation of Mycoplasma pneumoniae
Inspect the cultures daily for subtle changes because the organisms die rapidly once growth occurs and the substrates are utilized. If a potentially positive broth culture is detected visually, it is subcultured onto a [?] medium
solid agar
Inoculation and incubation of Mycoplasma pneumoniae
It may take 2 -3 weeks to get a positive identification. Media for isolation of M. pneumoniae should be incubated for up to [?] before a final culture report is made
4 weeks
M. pneumoniae colonies are round with a
dense center and a less dense periphery,
giving a [?] appearance on PPLO
(Mycoplasma) Agar. Vacuoles, large bodies characteristic of Mycoplasma spp., are seen in the periphery
“fried egg”
In a medium containing glucose and phenol red, M. pneumoniae grows and produces acid, causing the color
of the medium to change from [?].
purple to yellow
Culture of M. pneumoniae is a highly specialized test and is almost never done to diagnose mycoplasma infection. It is of little use to the clinician because recovery by culture and identification of the mycoplasmas take several weeks. Clinical diagnosis confirmed by
serology or PCR
consists of examining serum samples for antibodies
Serodiagnosis
autoantibodies agglutinating red blood cells at 4 oC; may be present in about 65% of patients after 1 to 2 weeks, with the maximum reached in the third or fourth week after onset. Titers greater
than 1:32 (i.e., titer of 1:54 or higher) are generally considered positive for M. pneumoniae; not specific because they are also seen in chronic lymphocytic leukemia (CLL), and infectious mononucleosis caused by Epstein-Barr virus.
Cold agglutinins
There is a rise in specific antibodies to M. pneumoniae that is demonstrable by CF tests; acute and convalescent phase sera are necessary to demonstrate a fourfold rise in the CF antibodies. CF test for antibodies to M. pneumoniae is more specific.
Complement fixation (CF) test
this test to detect IgM and IgG can be highly sensitive and specific and are considered more sensitive than CF tests
Enzyme-linked immunoassay (EIA)
Several molecular methods, including PCR assay, have been developed since for the detection of M. pneumoniae in a variety of specimen type.
Molecular diagnosis
Mucopurulent cervicitis
Endocervical swab, urine
Acute urethral syndrome (women)
Urethral swab, urine
Acute endometritis
Endometrial aspirate
Acute salpingitis
Fallopian tube biopsy
Nongonococcal urethritis (men)
Urethral swab, urine
Inclusion conjunctivitis
Conjunctival scrapings/swab
Trachoma
Conjunctival scrapings/swab
Lymphogranuloma venereum
Lymph node aspirate, biopsy of ulcerated lesion, serum
Pneumonitis (infants)
Serum, tracheobronchial aspirate, nasopharyngeal swab
is the standard
method for demonstrating cytoplasmic inclusions.
Giemsa staining technique
[?], which contains carbolfuchsin, is
preferred because the inclusions are colored well,
but the Giemsa stain is more generally available.
Gimenez stain
are round and vacuolar, found in the cytoplasm
of epithelial cell, and often have a perinuclear location.
C. trachomatis inclusions
Chlamydiae were named for the word [?], the ancient Greek term for the short
cloak worn by Greek military men draped around their upper shoulders and secured with a brooch on the right shoulder.
chlamys
The cytoplasmic inclusion of C. trachomatis was named [?] when they were
discovered in the conjunctiva of the infected eye in an experiment conducted by German dermatologist and radiologist Ludwig Halberstädter and Austrian bacteriologist Stanislaus von Prowazek in 1907.
‘Halberstädter-Prowazek bodies’
It is believed that the chlamydiae were named thus because the [?] formed by this agent inside host cells cluster around (are ‘draped’ around) the nucleus of the cell.
intracytoplasmic inclusions
are commercially available for diagnosis.
Direct fluorescent antibody (DFA) and enzyme-linked immunoassay (EIA) kits
uses monoclonal antibodies directed against a species-specific antigen on the chlamydial MOMP.
Direct fluorescent antibody (DFA)
detects the presence of genus-specific LPS antigens extracted from EBs in the specimen. To be positive, the clinical specimen must contain a brightly fluorescing mass, in general morphology similar to Giemsa-stained inclusions, in the cytoplasm of epithelial cell, preferably adjacent to the nucleus.
enzyme-linked immunoassay (EIA)
Specimens are added to cultures of susceptible cells,
most often McCoy cells treated with cycloheximide to inhibit metabolism and enhance sensitivity. After
incubation for 48-72 hours, the monolayers of infected
cells are stained with iodine and examined for the
presence of brown inclusions. Iodine stains glycogen in the inclusions. The presence of iodine-staining inclusions is specific for C. trachomatis since the inclusions of the
other species of chlamydia do not contain glycogen.
Chlamydia trachomatis
from infected humans are used to detect anti-Chlamydia antibodies by the complement fixation or microimmunofluorescence tests (an indirect fluorescent antibody technique).
Sera and tears
for diagnosis are of limited value in adults, since the tests do not distinguish between current and past infections.
Serological tests
Detection of [?] is indicative of a recent infection. Detection of IgM antibodies in neonatal infection is useful.
high titer IgM antibodies
are the tests of choice for the diagnosis of C trachomatis infections because they have increased sensitivity compared to other test methods.
Nucleic acid amplification tests—NAATs
The most commonly used molecular tests at present use
PCR, strand displacement, and transcription-mediated amplification (TMA)
