Laboratory diagnosis, Treatment and Prevention of Mycoplasma pneumoniae

Mycoplasma pneumoniae is a very small bacterium that belongs to the class Mollicutes. It is a human pathogen that causes the disease mycoplasma pneumonia, a form of atypical bacterial pneumonia related to cold agglutinin disease. M. pneumoniae is characterized by the absence of a peptidoglycan cell wall and resulting resistance to many antibacterial agents.

Mycoplasma pneumoniae bacteria commonly cause mild infections of the respiratory system (the parts of the body involved in breathing). Sometimes these bacteria can cause more serious lung infections that require care in a hospital. Mycoplasma pneumonia spreads quickly through contact with respiratory fluids in crowded areas, like schools, college campuses, and nursing homes. When someone coughs or sneezes, moisture containing the M. pneumoniae bacteria is released into the air, and others around them can easily breathe the bacteria in. Once inside the body, the bacterium can attach itself to your lung tissue and multiply until a full infection develops.

Laboratory diagnosis of Mycoplasma pneumoniae infection can be challenging because of the slow growth and fastidious nature of the organism. The diagnosis can be made by various methods, such as microscopy, culture, serology, antigen detection, and molecular techniques. However, each method has its own advantages and limitations. Therefore, a combination of methods may be required for a definitive diagnosis. In this section, we will briefly review each method and its application in clinical practice.

The choice of specimen for the diagnosis of Mycoplasma pneumoniae infection depends on the type and severity of the respiratory illness, as well as the availability and performance of the diagnostic methods. The most commonly used specimens are throat swabs and nasopharyngeal aspirates, which can be collected noninvasively and are suitable for molecular detection and culture of M. pneumoniae . Lung biopsies and expectorated sputum may also be used, but they are more invasive and less reliable, respectively . Bronchoalveolar lavage (BAL) fluid and pleural fluid are rarely obtained, but they may provide valuable information in severe or complicated cases .

Specimens should be collected as soon as possible following the onset of symptoms, preferably within the first week of illness, when the bacterial load is highest . Specimens should be labeled with the patient`s full name, date of collection, and one other unique identifier, such as the patient`s date of birth or health card number . Specimens should be stored at 2-8°C following collection and shipped to the laboratory on ice packs within 24 hours . If transport is delayed, specimens should be frozen at -70°C or lower and shipped on dry ice .

Microscopy is not a useful method for the detection of Mycoplasma pneumoniae because of its small size and lack of a cell wall. M. pneumoniae cells are only 1 to 2 μm long and 0.1 to 0.2 μm wide, which makes them invisible to light microscopy . Moreover, M. pneumoniae does not stain with conventional reagents because it does not have a peptidoglycan layer in its cell membrane . Therefore, microscopy cannot provide reliable information on the presence or absence of M. pneumoniae in clinical specimens.

Some specialized microscopy techniques, such as scanning-beam electron microscopy and confocal laser scanning microscopy, have been used to observe the morphology and intracellular location of M. pneumoniae in vitro . However, these techniques are not widely available or practical for routine diagnosis of M. pneumoniae infections in clinical laboratories.

Culture is one of the methods to diagnose Mycoplasma pneumoniae infections, but it has some limitations and challenges. M. pneumoniae is a slow-growing bacterium that requires complex and specialized media for growth. It does not have a cell wall and does not stain with conventional reagents. It also does not produce visible turbidity in liquid media, so visual confirmation of growth is difficult.

Some of the media used for culture of M. pneumoniae are:

• Standard solid medium: This contains PPLO (pleuropneumonia-like organism) agar with yeast extract, horse serum and penicillin. The agar provides a solid surface for the bacteria to grow on.
• Standard liquid medium: This contains PPLO broth, glucose and penicillin. It also has phenol red as an indicator of pH change. M. pneumoniae growth is detected by a color change from red to yellow due to fermentation of glucose.
• SP-4 (Sugar-phosphate) medium: This is a more complex liquid medium that contains fetal bovine serum. It is more suitable for large-scale cultivation of M. pneumoniae.

The organisms grow slowly in culture, with a generation time of 6 hours and can grow under both aerobic and anaerobic conditions. The colonies of M. pneumoniae are small and have a homogeneous granular appearance ("mulberry shaped"). They can be examined by a hand lens or Dienes` staining.

Some of the methods to identify M. pneumoniae colonies are:

• Hemolysis test: Most strains of M. pneumoniae produce hemolytic colonies that can be seen on blood agar plates.
• Hemadsorption test: M. pneumoniae agglutinates guinea pig red blood cells (RBC) and the colonies on agar adsorb RBCs to their surface. This can be observed under a microscope.
• Tetrazolium reduction test: M. pneumoniae colonies reduce the colorless tetrazolium compound to red colored formazan. This can be seen as red spots on the agar surface.
• Growth inhibition test: The growth of M. pneumoniae is inhibited by adding specific antisera to the culture medium. This can be used as a serotyping method.

Culture is a sensitive and specific method for diagnosis of M. pneumoniae infections, but it has some drawbacks, such as:

• It is time-consuming and labor-intensive, as it may take up to 4 weeks to obtain results.
• It requires special equipment and expertise to handle and maintain the cultures.
• It may be contaminated by other microorganisms or fungi that can interfere with the growth or identification of M. pneumoniae.
• It may not detect all cases of infection, as some patients may have low bacterial load or may have received antibiotics prior to specimen collection.

Therefore, culture is not widely used in clinical laboratories and is mostly reserved for research or reference purposes. Alternative methods, such as serology or molecular techniques, are more commonly used for diagnosis of M. pneumoniae infections.

Mycoplasma pneumoniae (Mp) colonies are small and have a homogeneous granular appearance, resembling a "fried egg" on agar plates . They can be examined by a hand lens or Dienes` staining, which reveals a dark central zone surrounded by a clear halo. Most strains of Mp produce hemolytic colonies, which can be observed by the presence of a clear zone around the colonies on blood agar . Hemolysis is caused by the production of hydrogen peroxide by Mp, which damages the red blood cells.

Another characteristic feature of Mp colonies is their ability to adsorb guinea pig red blood cells (RBCs) to their surface, which can be detected by the hemadsorption test . This test involves adding guinea pig RBCs to the culture medium and observing the colonies under a microscope. The RBCs adhere to the colonies and form a red fringe around them. The hemadsorption phenomenon is mediated by a surface protein called P1 adhesin, which binds to sialic acid residues on the RBC membrane.

A third method to identify Mp colonies is the tetrazolium reduction test, which exploits the ability of Mp to reduce the colorless tetrazolium compound to red colored formazan . This test involves adding tetrazolium salt to the culture medium and observing the color change of the colonies. The colonies that reduce tetrazolium appear red or pink, while those that do not remain colorless. The tetrazolium reduction test is useful for differentiating Mp from other mycoplasmas that do not reduce tetrazolium, such as Mycoplasma hominis and Mycoplasma genitalium.

A fourth method to identify Mp colonies is the growth inhibition test, which relies on the use of specific antisera to inhibit the growth of Mp . This test involves adding antisera against Mp to the culture medium and observing the growth of the colonies. The colonies that are sensitive to the antisera will show reduced or no growth, while those that are resistant will grow normally. The growth inhibition test is useful for confirming the identity of Mp and for detecting antigenic variations among different strains.

These methods can help in the laboratory diagnosis of Mp infection, but they are time-consuming, labor-intensive, and require specialized equipment and reagents. Therefore, they are not widely used in routine clinical practice. Instead, more rapid and sensitive methods based on antibody detection, antigen detection, or molecular techniques are preferred.

Chest radiography is a useful tool for diagnosing and assessing the severity of Mycoplasma pneumoniae pneumonia. However, the radiographic features are highly variable and not specific for this infection. The most common patterns of presentation on chest radiography are :

• peribronchial and perivascular interstitial infiltrates: these are reticular densities that may be patchy, segmental or non-segmental, and usually involve the lower lobes
• airspace consolidation: this is a homogeneous opacity that obscures the underlying vessels and may have a lobular distribution
• reticulonodular opacification: this is a combination of interstitial and nodular densities that may be diffuse or focal
• nodular or mass-like opacification: this is a discrete lesion that may be solitary or multiple, and may mimic a tumor or an abscess

Other less common findings include :

• bilateral lesions: these may be symmetrical or asymmetrical, and may indicate a more severe infection
• pleural effusion: this is an accumulation of fluid in the pleural space, and may be associated with complications such as empyema or bronchopleural fistula
• hilar lymphadenopathy: this is an enlargement of the lymph nodes in the hilum of the lung, and may be a sign of co-infection with other pathogens

Chest radiography may also show some extrapulmonary manifestations of Mycoplasma pneumoniae infection, such as pericarditis, myocarditis, meningoencephalitis, transverse myelitis, Guillain-Barre syndrome, Stevens-Johnson syndrome, or immune hemolytic anemia.

Chest computed tomography (CT) can provide more detailed information about the pulmonary lesions and their distribution. CT can also detect some findings that may be missed or subtle on chest radiography, such as :

• ground-glass attenuation: this is a hazy opacity that does not obscure the underlying vessels, and may indicate alveolar inflammation or edema
• intrapulmonary nodules: these are small lesions that are usually centrilobular and may coalesce to form larger nodules or masses
• thickening of the bronchovascular bundles: this is an increase in the caliber or density of the bronchi and vessels within the lung parenchyma, and may reflect peribronchial inflammation or fibrosis
• bronchiectasis: this is an irreversible dilation of the bronchi due to chronic inflammation or infection
• Swyer-James syndrome: this is a rare complication of Mycoplasma pneumoniae infection that causes unilateral hyperlucent lung with air-trapping and reduced vascularity

The radiographic findings of Mycoplasma pneumoniae pneumonia may change over time and may persist for weeks to months after clinical resolution. Therefore, chest radiography and CT should be interpreted in conjunction with the clinical history, physical examination, laboratory tests, and microbiological results.

Antibody detection methods are based on measuring the immune system`s response to Mycoplasma pneumoniae infection. There are two types of antibodies, IgM and IgG, that can be measured in the blood to detect and diagnose the infection . IgM antibodies are usually the first to appear and indicate a recent or current infection, while IgG antibodies persist for a longer time and indicate a past exposure or immunity .

Different scientific methods can be used to detect these antibodies, such as immunofluorescence, ELISA, and western blot . These methods vary in their sensitivity, specificity, cost, and availability. The level and change of these antibodies can indicate the stage and recurrence of the infection . However, antibody detection methods have some limitations, such as:

• They may not be able to distinguish between active and latent infections .
• They may cross-react with other pathogens or self-antigens and cause false-positive results .
• They may not detect low levels of antibodies or late seroconversion and cause false-negative results .
• They may require acute and convalescent phase sera to demonstrate a fourfold rise in antibody titers, which may delay the diagnosis .

Therefore, antibody detection methods are useful for screening for recent or past exposure to Mycoplasma pneumoniae, but they should not be used as a sole diagnostic criterion. They should be interpreted in conjunction with clinical symptoms, radiographic findings, and other laboratory tests . The preferred method of diagnosis of acute Mycoplasma pneumoniae infection is by molecular detection.

Antigen detection methods are based on the identification of Mycoplasma pneumoniae antigens directly in the clinical specimens, such as throat swabs, nasopharyngeal aspirates, sputum, or lung biopsies. These methods can provide a rapid and specific diagnosis of M. pneumoniae infection, especially in the early stages of the disease when antibodies are not yet detectable. However, antigen detection methods may have lower sensitivity than molecular techniques and may require confirmation by other methods.

There are two main types of antigen detection methods: direct and indirect immunofluorescence assays (IFA) and enzyme immunoassays (EIA).

• Direct and indirect IFA: These methods use fluorescent-labeled antibodies to bind and visualize M. pneumoniae antigens under a fluorescence microscope. Direct IFA uses monoclonal antibodies that are specific for M. pneumoniae antigens, while indirect IFA uses polyclonal antibodies that are produced by immunizing animals with M. pneumoniae antigens. Both methods have similar sensitivity, but indirect IFA is more convenient and less expensive.
• EIA: These methods use enzyme-labeled antibodies to bind and detect M. pneumoniae antigens by a colorimetric or chemiluminescent reaction. EIA can be performed in a direct or indirect format, similar to IFA, or in a capture format, where M. pneumoniae antigens are captured by immobilized antibodies on a solid phase and then detected by another antibody. EIA can also use monoclonal or polyclonal antibodies, or a combination of both. EIA is more sensitive than IFA and can be automated for high-throughput testing.

Some examples of commercially available antigen detection kits for M. pneumoniae are:

• Mycoplasma Direct Test (Meridian Bioscience): A direct EIA kit that uses monoclonal antibodies against P1 adhesin antigen of M. pneumoniae and can detect the antigen in 15 minutes.
• Mycoplasma pneumoniae Antigen ELISA Kit (Abnova): A capture EIA kit that uses polyclonal antibodies against whole-cell M. pneumoniae antigens and can detect the antigen in 3 hours.
• Mycoplasma pneumoniae DFA Test Kit (Zeus Scientific): A direct IFA kit that uses monoclonal antibodies against P1 adhesin antigen of M. pneumoniae and can detect the antigen in 30 minutes.

Antigen detection methods are useful for the diagnosis of M. pneumoniae infection, but they have some limitations, such as:

• The need for fresh and adequate specimens to avoid false-negative results.
• The possibility of cross-reactivity with other bacteria or host cells that may cause false-positive results.
• The dependence on the quality and specificity of the antibodies used in the assays.
• The lack of standardization and validation among different kits and laboratories.

Therefore, antigen detection methods should be interpreted with caution and in conjunction with other clinical and laboratory findings.

Molecular techniques are based on the detection of specific nucleic acid sequences of M. pneumoniae in clinical specimens, such as throat swabs, sputum, or bronchoalveolar lavage fluid. These techniques offer several advantages over conventional methods, such as high sensitivity, specificity, speed, and ability to detect multiple pathogens simultaneously. However, they also have some limitations, such as the need for specialized equipment and trained personnel, the risk of contamination and false-positive results, and the inability to provide information on antibiotic susceptibility.

The most widely used molecular technique for the diagnosis of M. pneumoniae infection is polymerase chain reaction (PCR), which amplifies a target DNA segment using specific primers and a DNA polymerase enzyme. PCR can detect very low amounts of M. pneumoniae DNA in a single specimen and can be positive earlier in the course of illness than serological tests. PCR targets M. pneumoniae specific 16S rRNA gene and P1 adhesin gene, which are highly conserved and unique to this organism. PCR can also be used to identify macrolide resistance-associated mutations within the V region of 23S rRNA gene of M. pneumoniae genome.

Several variations of PCR have been developed to improve its performance or convenience, such as real-time PCR, multiplex PCR, nested PCR, reverse transcription PCR (RT-PCR), loop-mediated isothermal amplification (LAMP), and droplet digital PCR (ddPCR). These methods differ in their detection mechanisms, amplification conditions, signal generation, and quantification methods. Some of these methods are commercially available as kits that can detect multiple respiratory pathogens, including M. pneumoniae.

Molecular techniques are useful tools for the rapid and accurate diagnosis of M. pneumoniae infection, but they should be interpreted in conjunction with clinical and epidemiological data. They should also be validated and standardized according to quality control guidelines to ensure their reliability and comparability.

Mycoplasma pneumoniae infections are generally mild and self-limiting, but some people may develop pneumonia or other complications that require antibiotic therapy. The choice of antibiotics depends on several factors, such as the severity of the infection, the patient`s age, medical history, and allergies, and the local prevalence of antibiotic resistance.

The most commonly used antibiotics for M. pneumoniae infections are macrolides (such as erythromycin, clarithromycin, and azithromycin), tetracyclines (such as doxycycline), and fluoroquinolones (such as levofloxacin, moxifloxacin, and gemifloxacin) . These antibiotics are effective because they target the protein synthesis or DNA replication of M. pneumoniae, which lacks a cell wall and is therefore resistant to beta-lactam antibiotics (such as penicillin).

Macrolides are usually the first-line treatment for M. pneumoniae infections, especially in children and pregnant women . They have a good safety profile and can be taken orally or intravenously. However, some strains of M. pneumoniae have developed resistance to macrolides, which may reduce their efficacy and increase the risk of treatment failure . Therefore, it is important to monitor the local patterns of macrolide resistance and to perform susceptibility testing when possible.

Tetracyclines are another option for M. pneumoniae infections, especially in adults who are not pregnant or breastfeeding . They have a broad spectrum of activity and can be taken orally or intravenously. However, they have some side effects, such as photosensitivity, gastrointestinal disturbances, and tooth discoloration in children. They are also contraindicated in patients with renal or hepatic impairment.

Fluoroquinolones are usually reserved for second-line treatment of M. pneumoniae infections, when macrolides or tetracyclines are not effective or tolerated . They have a high potency and a wide spectrum of activity against M. pneumoniae and other respiratory pathogens. They can be taken orally or intravenously. However, they have some serious side effects, such as tendon rupture, cardiac arrhythmias, central nervous system toxicity, and hypersensitivity reactions. They are also not recommended for children or pregnant women due to potential adverse effects on bone and cartilage development.

The duration of antibiotic treatment for M. pneumoniae infections varies depending on the type and severity of the infection, the patient`s response to therapy, and the presence of any complications. Generally, a 7- to 14-day course of antibiotics is sufficient for uncomplicated cases of pneumonia or bronchitis caused by M. pneumoniae . However, longer courses may be needed for more severe or complicated cases, such as those involving extrapulmonary manifestations or co-infections with other pathogens .

In addition to antibiotic therapy, supportive care is also important for M. pneumoniae infections. This may include hydration, fever control, pain relief, oxygen therapy, and mechanical ventilation if needed . Patients should also avoid smoking and exposure to irritants that may worsen their respiratory symptoms .

Mycoplasma pneumoniae infections can be prevented by practicing good hygiene measures, such as washing hands frequently, covering coughs and sneezes, avoiding close contact with sick people, and staying home when ill . There is no vaccine available for M. pneumoniae at this time.

We are Compiling this Section. Thanks for your understanding.