The laboratory diagnosis of mycoplasma infections is extremely challenging because of complex and time consuming culture requirements and the lack of reliable, widely available rapid diagnostic tests. Accurate, rapid diagnosis for M. pneumoniae is highly desired, because penicillin and other β-lactam agents are ineffective treatments. The laboratory diagnosis of the mycoplasmas well recognized as able to cause human disease (i.e., M. pneumoniae, U. urealyticum, M. hominis, and M. genitalium) is addressed.

SPECIMEN COLLECTION, TRANSPORT, AND PROCESSING

Various specimens are appropriate for the diagnosis of mycoplasma infections by culture or other means of detection. Acceptable specimens include body fluids (e.g., blood, joint fluid, amniotic fluid, urine, prostatic secretions, semen, pleural secretions, sputum, bronchoalveolar lavage specimens), tissues, wound aspirates and swabs of wounds, the throat, nasopharynx, urethra, cervix, or vagina. Blood for culture of genital mycoplasmas should be collected without anticoagulants and immediately inoculated into an appropriate broth culture medium. Mycoplasmas are inhibited by sodium polyanethol sulfonate (SPS), the anticoagulant typically found in commercial blood culture media. This may be overcome by the addition of 1% wt/vol of gelatin; however, commercial blood culture media and automated instruments are not adequate for the detection of Mycoplasma spp. Swab specimens should be obtained without the application of any disinfectants, analgesics, or lubricant; Dacron or polyester swabs on aluminum or plastic shafts should be used. Care must be taken to collect urine samples to avoid contamination with lubricants and antiseptics used during gynecologic examination.

Because mycoplasmas have no cell wall, they are highly susceptible to drying; therefore, transport media are necessary, particularly when specimens are collected on swabs. Liquid specimens such as body fluids do not require transport media if inoculated to appropriate media within 1 hour of collection. Tissues should be kept moist; if a delay in processing is anticipated, they should also be placed in transport media. Specific media for the isolation of Mycoplasma spp. include those containing 10% heat-inactivated calf serum containing 0.2 M sucrose in a 0.02 M phosphate buffer, pH 7.2, such as SP4, Shepard’s 10B broth or 2 SP. Additional commercial media available for cultivation of these organisms include Stu art’s medium, trypticase soy broth supplemented with 0.5% bovine serum albumin, Mycotrans (Irvine Scientific, Irvine, California), and A3B broth (Remel, Inc.). Excessive delays in processing can result in decreased viability and recovery of organisms from clinical specimens. If the storage time is expected to exceed 24 hours prior to cultivation, the samples should be placed in transport media and frozen at −80°C. Frozen samples should be thawed in a hot water bath at 37°C. Transport and storage conditions of various types of specimens are summarized in Table 1.

Table1. Transport and Storage Conditions for Mycoplasma pneumoniae, Ureaplasma urealyticum, and M. hominis

DIRECT DETECTION METHODS

 At present, no direct methods for identifying M. pneumoniae, Ureaplasmas spp., or other Mycoplasma spp. in clinical samples are recommended, although some methods have been described, such as immunoblotting and indirect immunofluorescence. Direct detection by gram staining may rule out the presence of other infectious organisms, but it will not stain cell wall-deficient mycoplasmas and ureaplasmas. Acridine orange or a flurochrome stain may be useful to visualize organisms. However, these are nonspecific stains that will stain nucleic acids in bacteria as well as human cells.

Molecular Diagnostics

 Several amplification methods, such as polymerase chain reaction (PCR), have been developed for the detection of the clinically relevant Mycoplasma and Ureaplasma species. Various targets including 16srRNA sequences, insertion sequences, and organism specific genes have been used in the development of these assays. As a result of the fast turnaround time, specificity, and lack of need to cultivate fastidious organisms, PCR amplification for the diagnosis of these organisms is particularly attractive. When considering the use of molecular amplification methods for the detection of infectious diseases, it is important to note that although an organism is detectable, the patient’s signs and symptoms must be correlated with the identified agent. It is possible to detect an organism by one method and not another—in other words, a patient may be PCR positive but culture negative or serologically negative for a Mycoplasma based on the patient’s response to infection and current disease manifestation. Chapter 8 provides a more detailed description of the advantages, limitations, and methods used in the development of amplification assays. Multiplexed real-time PCR assays that detect M. pneumoniae as well as other atypical respiratory tract pathogens such as Chlamydophila pneumoniae and Legionella pneumoniae have been developed.

Because there is no reliable medium for its isolation, M. genitalium has been directly detected by PCR targeting its attachment protein in urine and urethral swabs in men. In women, vaginal or cervical swabs are used.

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مواضيع ذات صلة

Cultivation of Mycoplasma and Ureaplasma

Epidemiology and Pathogenesis of Mycoplasma and Ureaplasma

Tropheryma whipplei

Mycoplasma Pneumonia and Atypical Pneumonias

Leptospira and Leptospirosis

Borrelia Species and Relapsing Fever

Treponema Pallidum and Syphilis

Bartonella henselae and Bartonella quintana

Bartonella bacilliformis

Legionella pneumophila and other Legionellae

Pathogenesis of Anaerobic Infections

Coxiella