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Chapter: Microbiology and Immunology: Bacteriology: Mycoplasma and Ureaplasma

Laboratory Diagnosis - Mycoplasma pneumonia Infections

Initial treatment of M. pneumoniae infection is based on clinical diagnosis of the condition. The definite diagnosis of the condi-tion usually takes 3–4 weeks. Hence, treatment is started with-out waiting for the result of laboratory tests.

Laboratory Diagnosis

Initial treatment of M. pneumoniae infection is based on clinical diagnosis of the condition. The definite diagnosis of the condi-tion usually takes 3–4 weeks. Hence, treatment is started with-out waiting for the result of laboratory tests.


Respiratory specimens include throat washings, bronchial washings, and expectorated sputum. Tracheal washings are more useful than sputum specimens, because most patients with respiratory tract infections do not produce any sputum as they have a dry and nonproductive cough. The specimens are collected and transported to the laboratory immediately.

      If delay is anticipated, they are usually inoculated in suitable transport media, such as SP4 transport medium to prevent desiccation.

      The specimens can be stored at 270°C if these cannot be sent immediately to the laboratory after collection.


Microscopy is of no value in diagnosis of M. pneumoniae infec-tions. Mycoplasmas are stained poorly, because they lack cell wall.

 Direct antigen detection

Antigen capture immunoassay has been used for direct detec-tion of M. pneumoniae in sputum specimen with high sensitivity and specificity. Direct immunofluorescence, counter-current immunoelectrophoresis, and immunoblotting with monoclo-nal antibodies are the tests used for detection of antigen in clinical specimens.


Bacterial culture is of little practical value because of its fastidious growth requirements and need for 3–4 weeks for culture. Isolation of M. pneumoniae from clinical specimens by culture confirms the diagnosis of M. pneumoniae respiratory ill-ness. The specimens are inoculated into mycoplasma medium, such as PPLO agar supplemented with serum (source for ste-rols), glucose, pH indicator, yeast extract (source for nucleic acid precursor), and antibiotics and antifungal agents (to inhibit bacteria and fungi). On this medium, M. pneumoniaegrows very slowly and colonies are demonstrated at 37°C. The growth is facilitated by incubation in the presence of 95% N2 and 5% of CO2. The bacteria produce small colonies with homogenous appearance, typically described as mulberry-shape colonies.

 Identification of bacteria

The identifying features of M. pneumoniae colonies are summa-rized. They are identified by the following tests.

Color change: The colonies are identified by noting a colorchange from red to yellow of phenol red due to fermentation of glucose resulting in the production of acid, which makes pH of the media acidic.

Diene test: In this method, the Diene stain (diluted 1:10 withdistilled water) is added directly to the plate containing sus-pected colonies of Mycoplasma. The plate is then immediately rinsed with distilled water to remove the stain. This is followed by decolorizing the medium by adding 1 mL of 95% ethanol and keeping it for 1 minute. The plate is rewashed with dis-tilled water and is allowed to dry. After drying, the colonies are observed under low power of the microscope.

      By Diene staining, the fried-egg appearance colonies of Mycoplasma appear highly granular, with the center of thecolonies stained dark blue and the periphery of the colonies staining light blue. The agar in the medium appears clear or slightly violet. Mycoplasma other than M. pneumoniae becomes colourless after a period of time because it reduces the methy-lene blue. The colonies identified are confirmed by inhibition of their growth with specific M. pneumoniae antisera.

Hemadsorption test: The test is performed by floodingM.pneumoniae colonies grown on surface of agar with 2 mL of0.2–0.4% suspension of guinea pig erythrocytes in Mycoplasmagrowth medium. The plate is incubated at 35°C for 35 minutes followed by washing with 3 mL of Mycoplasma growth medium by gently rotating the plate. The washing fluid is gently removed by aspiration with a pipette. M. pneumoniae colonies adsorb guinea pig erythrocytes, which is facilitated at 37°C. M.pneumoniae colonies adsorbing erythrocytes on their surface aredemonstrated under the microscope. The colonies are observed under 403 magnification.

Tetrazolium reduction test: The test is based on the principlethat M. pneumoniae has the ability to reduce triphenyl tetra-zolium, a colorless compound, to formazan, a red-colored compound. This test is performed by flooding the colonies of M. pneumoniae on agar with a solution of 2-p-iodophenyl-3-nitrophenyl-5-phenyltetrazolium chloride and incubating it at 35°C for an hour. In a positive test, M. pneumoniae colonies appear reddish after 1 hour and may appear purple to black after 3–4 hours of incubation.


Serodiagnosis is based on demonstration of specific antibodies in serum using Mycoplasma antigens. M. pneumoniae glycolipid antigen extracted by chloroform and methanol is also widely used for serodiagnosis of atypical pneumonia. Complement fixation test and ELISA are the tests frequently used.

Complement fixation test: A fourfold rise in complement-fixing antibody titer or a single titer of 1:64 or more is sug-gestive of recent infection. The complement-fixing antibodies appear after 7–10 days of infection and reach the peak after 4–6 weeks of infection. The complement-fixing antibodies are demonstrated in approximately 80% of the cases.

Enzyme-linked immunosorbent assay: IgM ELISA is mostfrequently used test. IgM ELISA is used to detect specific IgM antibodies in a single serum specimen. This test has a specific-ity of 99% and sensitivity of 97%. Recently, a quantitative, rapid, single-specimen, membrane-based ELISA has been evaluated as a rapid diagnostic method for demonstration of either IgM or IgG antibodies. The results for this test can be obtained within 30 minutes of performing the test.

Nonspecific serological tests: These tests are callednonspecific because they do not use specific Mycoplasma antigen; instead they use cross-reacting and nonspecific anti-gens. These tests include Streptococcus MG agglutination test and cold agglutination test.

·           Streptococcus MG agglutination test:In this test, a heat-killed suspension of Streptococcus MG is used as antigen. The antigen is mixed with serial dilution of patient’s unheated serum. The agglutination is observed after overnight incu-bation at 37°C. An antibody titer of 1:20 or more is sugges-tive of M. pneumoniae infection.

·           Cold agglutination test:In this test, human O group eryth-rocytes are used as antigen. This is based on the principle that autoantibodies that agglutinate human O group cells at low temperatures appear in most of the cases of atypical pneumonia. The test is performed by collecting patient’s blood, which should never be refrigerated before separa-tion of serum because agglutinins are readily absorbed by homologous RBCs at low temperatures. The patient’s serum is mixed with equal volume of 0.2% washed suspension of human O erythrocytes. The suspension is incubated at 4°C overnight and observed for clumping. The clumping is dis-associated at 37°C. The titer of 1:32 or more is suggestive of M. pneumoniae infection.

      The cold agglutinins usually appear in more than 50% of the cases by the second week of infection and reach a peak at 4–5 weeks. The antibody titer thereafter declines rapidly, and test becomes negative in about 5 month. A fourfold rise in cold agglutinin titer of the paired serum and convalescent sera, or a single titer of 1:32 is suggestive of M. pneumoniaeinfection. The noted dis-advantage of this test is that this is a nonspecific test, because the cold agglutinins are also demonstrated in sera of other diseases, such as rubella, infectious mononucleosis, adenovirus infections, psittacosis, tropi-cal eosinophilia, trypanosomiasis, cirrhosis of liver, and hemolytic anemia.

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