Diagnostika

Diagnostika

Complicated dg.

• Delayed onset of symptoms
• Similarity of symptoms to other pulmonary conditions
• Non-pathogenic mycoplasmas present in the respiratory tract are mistaken for M. pneumoniae
• Complete medical evaluation

Aglutinins

• Diagnosis of M. pneumoniae infections was historicaly made based on the presence of cold agglutinins [4]
• Ability of the infected material to reduce tetrazolium [4]

Bronchoscopy a BAL

Serum cold agglutination

• Nonspecific test for M pneumoniae
• Positive in only 50-70% of patients after 7-10 days of infection [6]
• Negative result does not exclude infection
• May be affected by cross-reactions with other pathogens
• Adenovirus [6]
• Epstein-Barr virus [6]
• Measles viruses [6]
• A quick bedside test
• Purple-top tube with blood and placing it in ice
• "grains of sand" (aglutinace +)
• Cold agglutinins agglutinate erythrocytes in vitro at 4oC [8]
• Over 50% of patients developing M. pneumoniae pneumonia [8]

HR CT

• Mosaic perfusion [8]
• Bronchiectasis [8]

CT hrudníku

• More sensitive than RTG
• Tree-in-bud pattern
• Centrilobular nodular opacities
• Patchy distribution
• Ground glass opacities
• Consolidation
• Pleural effusion in 15-20% [6]

Diagnosis and characterization of bacterial pneumonic infections

• Immunoblotting
• Immunofluorescent staining
• Hemadsorption tests
• Tetrazolium reduction
• Metabolic inhibition tests

Bacteriological + immunological methods + PCR

Complete blood count (CBC)

• Elevated erythrocyte sedimentation rates (ESR) may be present [6]
• White blood cell (WBC) count may be normal or elevated [6]
• Hemolytic anemia is rare [6]
• CRP - nemusí být zvýšené [8]

Blood cultures

• Rarely used

Kultivace

• Pathogenic mycoplasmas generally require intracellular conditions for growth
• Cannot be easily duplicated in culture

Serological testing

• Difficult because of the possibility of suppression of immune responses
• Sequestering of pathogenic mycoplasma antigens inside cells away from the immune system

Polymerase chain reaction (PCR) for detection of Mycoplasma species

• Zlatý standard
• www.scirp.org/journal/paperinformation.aspx?paperid=95720

Kultivace na agaru

• Colonies with the dense partly immersed in the agar center and delicate periphery,
• The morphology is known as “fried-egg”
• These colonies that further we will call typical colonies (TCs)
• Grow up to the full size within 48-72 h
• Have a diameter ranges from about 75 to 800 µm
• Mycoplasma colonies might be seen with the naked eye
• As very small dots
• Usually TCs are observed with light microscopy
• Multiple studies demonstrated that Mycoplasma colonies consist of
• Variable elements including spheres
• Strings of different electronic density
• Lay separately
• Form structures of a higher order
• Mycoplasma cells range in size from 200 to 800 nm
• With the smallest cells of about 100-125 nm
• Under laboratory conditions
• Generation rates are about 1,6-2 h
• For colony formation to take about 48-72 h
• Isolates obtained from clinical samples
• These time intervals increase

https://www.biorxiv.org/content/10.1101/535559v1.full

Polymerase chain reaction (PCR)

• The most rapid and effective
• Does not indicate the activity or viability [5]
• Accurately diagnose atypical pneumonia
• Becoming the criterion standard confirmatory test for M pneumoniae
• Epidemiologic studies
• Currently used in most clinical settings [6]
• Real-time PCR
• Promising test
• Detection of M pneumoniae DNA in all phases of infection
• Including early periods when the serum may be negative for antibody [6]

A radiolabeled DNA probe

• Detects M pneumoniae ribosomal RNA in respiratory secretions [6]
• With 90% sensitivity [6]

Rapid pharyngeal culture for M pneumoniae

• Has shown promising results [6]

Sérologické vyšetření protilátek [1]

• Průkaz M. pneumoniae
• 4-fold or greater increase / decrease in paired sera titers
• Single titer greater than / equal to 1:32
• Supports the diagnosis of mycoplasmal pneumonia
• Enzyme-linked immunoassay - EIA
• Most common method
• Low cost
• Relatively short testing time [5]
• Complement fixation [6]
• Indirect hemagglutination [6]
• IgM antibodies
• First to be produced
• Rise for a short time period and then decline
• Often remaining detectable in the blood for several months [7]
• IgG follows IgM production
• Rising over time, and then stabilizing
• Some measurable amount for the rest of their life
• IgG test 2-4 týdny later as a convalescent sample [7]
• Increases in IgG, without IgM, can also be seen with a re-infection [7]

RTG

• Zhodnocení nálezu
• Pneumonia
• Bronchitis
• Another respiratory infection [3]
• Abnormalities usually more striking than the findings upon physical examination
• Bronchopneumonia
• Patchy consolidation
• Single lower lobe
• Lobar consolidation is rare
• Unilateral or bilateral
• Platelike atelectasis
• Thin, flat areas of collapsed lung
• Often seen on a lateral image of the chest [6]
• Pleural effusions
• In less than 20%
• Can be seen on lateral decubitus films [6]
• Reticulonodular or interstitial infiltrates
• Primarily in the lower lobes
• May resemble other diseases with granulomatous pathology
• Tuberculosis
• Mycoses
• Sarcoidosis [6]
• Hilar adenopathy
• Sometimes mistaken for malignancy [6]
• Nonspecific bilateral infiltrates and bronchial thickness [8]
• Parapneumonic pleural effusion complicate M. pneumoniae infections in 4%-20% of the patients [8]
• Often of small volume and on the side
• Massive and bilateral effusion has been reported in patients with sickle-cell disease [8]

Serum

• Usually is free of Mycoplasma spp
• Failed to observed TCs in PCR-positive samples within standard cultivation time.
• Prolonging incubation time of immune- and/or PCR-positive serum samples up to 7-9 days or sometimes 12 days
• Revealed very small colonies of non-round morphology
• Colonies were highly likely to defects on the agar surface
• Meantime, they were not.
• These colonies, which are referred below as minicolonies (MCs)
• Could be re-plated from 2-10 times and up to 3 years
• Gave a positive signal in PCR in the absence of visible TCs
• Interacted with Mycoplasma specific antibodies
• Could be washed out from the agar surface to get samples for checking out in Western blotting (see below), proteomic (see elsewhere) and genomic (see elsewhere) assays
• Such type colonies have not been described yet
• www.biorxiv.org/content/10.1101/535559v1.full

Sputum Gram stains and cultures

• Usually not helpful because M pneumoniae
• Lacks a cell wall and cannot be stained
• Difficult to culture
• Requires special culture media
• Needs 7-21 days to grow
• Routine culturing is successful in only 40-90% of cases
• Does not provide information to guide patient management [6]
• Rarely used [3]

Open lung biopsy

• Only done in very serious illnesses
• When the diagnosis cannot be made from other sources [3]