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Reference Test
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Specimen Information


Respiratory specimen: sputum, lung washes, tracheal aspirates, nasopharyngeal swab, pleural fluid, bronchoalveolar lavage (BAL), or bronchial brushings in M4 media


Respiratory Specimen: 2.0 mL
Cerebrospinal fluid: 1.0 mL

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Fasting Required?
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Reference Range

See Report


Polymerase Chain Reaction (PCR)

Clinical Significance

Mycoplasmas are the smallest self-replicating organisms. Because they possess a small genome and lack a cell wall, they are by necessity obligate intracellular parasites dependent upon their host for a supply of nutrients. Their fastidious nature makes them difficult to cultivate in vitro. Mycoplasma infections tend to be more chronic and indistinguishable on the basis of clinical symptoms alone. Thus, in many clinical settings, laboratory diagnosis is important for management. Mycoplasma pneumoniae is a frequent cause of upper and lower respiratory tract infections. Overall, M. pneumoniae accounts for approximately 15-20 percent of all cases of pneumonia with higher rates reported among school children. Only two to five percent of patients require hospitalization, contrasting to the 16 percent of patients with pneumonia caused by other infective agents. Infection tends to spread slowly among family members and throughout communities. The clinical features associated with M. pneumoniae are oftentimes indistinguishable from a variety of other viral and "atypical" bacterial pathogens. In general, patients may initially complain of malaise, myalgias, sore throat, headache (retro-orbital), ear pain, and fever. Chills, chest pain, nausea, vomiting, and diarrhea may follow in those patients who progress to pneumonia. Dry, nonproductive cough occurs three to five days after the onset of the initial nonspecific symptoms. Later, the cough may become productive of mucopurulent sputum. Patients usually seek medical attention on day 5-7 because the cough may become paroxysmal and nocturnal. The cough may persist for several weeks following resolution of the constitutional symptoms. Patients with pneumonia may have crackles or wheezes on auscultation of the chest. Chest radiograph findings may consist of patchy, unilateral infiltrates or a diffuse bilateral interstitial process. Pleural effusions are uncommon (2%) even in hospitalized patients. Other routine laboratory studies may likewise be nonspecific. The white blood cell count may be normal or slightly elevated with mild neutrophilia. Hypoxemia is absent or mild compared to other etiologies of community-acquired bacterial pneumonia. Complications of M. pneumoniae infections can be divided into those syndromes that are probably immunologically mediated such as skin rashes, anemia, thrombocytopenia, and Guillain-Barré syndrome and those caused by the spread of the organism such as bullous hemorrhagic otitis, arthritis, ARDS, myocarditis, and encephalitis/meningitis. Since pneumonia caused by M. pneumoniae may be indistinguishable from viral causes and other atypical pathogens such as Chlamydia pneumoniae, Bordetella pertussis, Legionella, and others, laboratory confirmation is appropriate for patient management. These are two definitive methods for diagnosis of Mycoplasma pneumoniae, culture and serology, neither of which are ideal. Culture requires specialized nutrient media such as SP4 and may require up to 4 weeks for recovery of the organism. Culture is also relatively insensitive (64 percent). These two factors considered together (delayed detection and insensitivity) make culture less than ideal and impractical for the management of the acutely ill patient with pneumonia. Serology is considered by many to be the "gold standard." Several types of assays are available. Complement fixation testing has both poor sensitivity and specificity, the latter because of cross-reactivity with other antigens. A single titer of ł 1:32 or a fourfold increase in titer is considered indicative of acute infection. Enzyme immunoassays and microparticle agglutination tests are more sensitive and specific than complement fixation and require paired sera to demonstrate rises in titers. A DNA-probe assay for direct detection of M. pneumoniae in clinical samples was marketed by Gen-Probe, Inc. However, the assay was found to be insensitive with clinical samples compared to its performance in simulated samples. One hypothesis for this has been attributed to loss of rRNA target in respiratory secretions during natural infection. The limitations of current diagnostic tests for M. pneumoniae have lead to an interest in amplification technologies. Reported PCR targets include 16 sRNA, P1 cytadhesin gene, the tuf gene encoding for an elongation factor, Tu, and finally a DNA sequence specific for M. pneumoniae selected from a genomic library. Kai, et al using M. pneumoniae 16S ribosomal RNA gene sequences as the amplification target region, found that PCR performed on throat swabs from children detected 22/30 culture-positive samples. Poor recovery of DNA or nonspecific reaction in the colorimetric media were offered as possible explanations for the false negative results. In addition, PCR was able to detect two children who were symptomatic and culture negative, as well as three additional positives from children whose throat cultures were indeterminate because of bacterial contamination. These were confirmed as "true positives" by three-primer PCR. None of the specimens from 33 healthy controls were PCR positive. The authors concluded that this was a useful assay for the direct detection of M. pneumoniae in clinical samples. Most published reports on PCR have used primers within the gene encoding for P1 protein. This is an external surface adhesin that is associated with pathogenicity, as it allows the organism to attach to respiratory epithelial cells. Multiple copies of this gene exist in the cell. Buck, et al demonstrated a sensitivity of PCR between one and 10 organisms in simulated specimens prepared from throat swabs. In a subsequent clinical study of 13 patients with clinical symptoms compatible with mycoplasma infection, 11 had positive PCR assays using the same target sequence. Several of the children had delayed serological responses. Other investigations have reported improved sensitivity compared to culture and serology for detection of M. pneumoniae in clinical samples when using primers within the P1 cytadhesin gene. Blackmore, et al noted no inhibition and a PCR sensitivity of 97 percent on throat swabs obtained from 99 hospitalized adults and children. Two of the investigators found PCR inhibition occurred more often in sputum, BAL, or nasopharyngeal aspirates than with throat swabs and they recommend the latter as the specimen of choice for M. pneumoniae PCR. Clinical validation of the in-house, tower-nested Mycoplasma pneumoniae qualitative assay was done by reference to culture and an EIA-based assay using alternative primers within the P1 attachment protein gene.


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