SPECIMEN COLLECTION, TRANSPORT, AND PROCESSING

Appropriate specimens should be collected aseptically from affected areas. For the most part, no special requirements are needed for specimen collection, transport, or processing of the organisms discussed in this chapter . When nocardiosis is clinically suspected, multiple specimens should be submitted for culture, because smears and cultures are simultaneously positive in only a third of the cases. The significance of random isolation of Nocardia spp. from the respiratory tract is questionable, because these organisms are so widely distributed in nature. Some of the actinomycetes tend to grow as a microcolony in tissues, leading to the formation of granules. Most commonly, these granules are formed in actinomycetomas, such as those caused by Nocardia, Streptomyces, Nocardiopsis, and Actinomadura spp. Therefore, material from draining sinus tracts is an excellent specimen for direct examination and culture.

DIRECT DETECTION METHODS

 Direct microscopic examination of Gram-stained preparations of clinical specimens is of utmost importance in the diagnosis of infections caused by the aerobic actinomycetes. Often, the demonstration of gram-positive, branching or partially branching beaded filaments pro vides the first clue to the presence of an aerobic actinomycete (Figure 1). Unfortunately, the actinomycetes do not always exhibit such characteristic morphology; many times these organisms are not seen at all or appear as gram-positive cocci, rods, or short filaments. Never the less, if gram-positive, branching or partially branching organisms are observed, a modified acid-fast stain should be performed (i.e., 1% sulfuric acid rather than 3% hydrochloric acid as the decolorizing agent) (see Procedure 1 on the Evolve site). The modified acid-fast stain is positive in only about half of these smears showing gram-positive beaded, branching filaments subsequently confirmed as Nocardia sp. Histopathologic examination of tissue specimens using various histologic stains, such as Gomori’s methenamine-silver (GMS) stain, can also detect the presence of actinomycetes.

Fig1. A, Gram stain of sputum obtained from a patient with pulmonary nocardiosis caused by Nocardia asteroides. B, The same  sputum stained with a modified acid-fast stain. The organism is indicated by the arrow. 

PROCEDURE 1

It is important to examine any biopsy or drainage material from actinomycetomas for the presence of gran ules. If observed, the granules are washed in saline, emulsified in 10% potassium hydroxide or crushed between two slides, Gram stained, and examined microscopically for the presence of filaments.

MOLECULAR DIAGNOSTICS

Amplification techniques (i.e., polymerase chain reaction [PCR]) involving the 16srRNA sequence have been used to examine the relatedness among the genera and species within the non–acid-fast aerobic actinomycetes and thermophilic actinomycetes. When the MicroSeq System was used for identification , almost 15% of isolates were identified as Nocardia spp., but no definitive species were given. PCR paired with restriction endonuclease analysis has been used to identify commonly isolated Nocardia spp. Housekeeping heat shock protein genes coupled with the 16srRNA sequence are used in this assay. DNA sequencing of several genes, including the 16srRNA, a heat shock protein gene, and a housekeeping gene are referred to as secA1. These methods currently are not available in the clinical laboratory; they are predominantly used for taxonomic, epidemiologic, and research studies.

CULTIVATION

 Many of the aerobic actinomycetes do not have complex growth requirements; they are able to grow on routine laboratory media, such as sheep blood, chocolate, Sabouraud dextrose, and brain-heart infusion agar. However, because many of the aerobic actinomycetes grow slowly, they may be overgrown by other normal flora present in contaminated specimens. This is particularly true for the nocardiae that require a minimum of 48 to 72 hours of incubation before colonies become visible. Because of their slow growth and the possibility of being overgrown with contaminating flora, various selective media have been used to recover nocardiae. A solid medium using paraffin as the sole source of carbon has been effective for isolating Nocardia spp. and rapidly growing mycobacteria from contaminated clinical specimens. Selective media formulated for the isolation of Legionella spp. from contaminated specimens, such as buffered charcoal-yeast extract medium with polymyxin, anisomycin, and vancomycin, have been successful in the recovery of nocardiae from contaminated specimens. Martin Lewis and colistin nalidixic acid media also have been used. Nocardia spp. grow well on Sabouraud dextrose agar and on fungal media containing cycloheximide, such as Mycosel. Because Nocardia organisms are able to withstand the decontamination procedures used to isolate mycobacteria, isolates may be identified on mycobacterial culture media.

If other aerobic actinomycetes are considered, a selective medium, such as brain-heart infusion agar with chloramphenicol and cycloheximide, is recommended in addition to routine agar to enhance isolation from contaminated specimens. Although most aerobic actinomycetes grow at 35°C, recovery is increased at 30°C. Therefore, selective and nonselective agars should be incubated at 35°C and 30°C. Plates should be incubated for 2 to 3 weeks. The typical Gram-stain morphology and colonial appearance of the aerobic actinomycetes are summarized in Table 1. Examples of Gram stains and cultures of different aerobic actinomycetes are shown in Figures 2 and 3.

Table1. Typical Gram-Stain Morphology and Colonial Appearance

Fig2. Gram stains of different aerobic actinomycetes. A, Nocardia asteroides grown on Löwenstein-Jensen medium. The arrows  indicate branching rods. B, Rhodococcus equi from broth. C, R. equi grown on chocolate agar. D, Streptomyces spp. grown on Sabouraud  dextrose agar. 

Fig3. Aerobic actinomycetes grown on solid media. A, Nocardia asteroides grown on Löwenstein-Jensen medium. B, Rhodococcus equi grown on chocolate agar. 

Clinical laboratories are rarely asked to diagnose hypersensitivity pneumonitis caused by the thermophilic actinomycetes. These organisms grow rapidly on trypticase soy agar with 1% yeast extract. The ability to grow at temperatures of 50°C or greater is a characteristic of all thermophilic actinomycetes. Differentiation of the various agents is based on microscopic and macroscopic morphologies.

APPROACH TO IDENTIFICATION

 If Gram-stain morphology or colonial morphology suggests a possible actinomycetes (see Table 1), an acid fast stain should be performed first to rule out rapidly growing mycobacteria , followed by a modified acid-fast stain (see Procedure 1). If the modified acid-fast stain results are positive, the isolate is a probable partially acid-fast aerobic actinomycete (i.e., Nocardia, Rhodococcus, Tsukamurella, or Gordonia sp). If the acid-fast stain result is negative, these organ isms still are not completely ruled out because of the variability of acid-fastness among isolates belonging to this group. Aerobic actinomycetes can be initially placed into major groupings by considering the following:

• Gram-stain morphology (see Figures 1 and Figure 2)

 • Modified acid-fast stain results

• Presence or absence of aerial hyphae when grown on tap water agar

 • Growth or no growth in nutrient broth containing lysozyme (250 µg/mL (Figure 4) (see Procedure 2 )

 • Other tests: urea hydrolysis, nitrate reduction, and ability to grow anaerobically

Fig4. Lysozyme (A) and glycerol (B) broths. The lysozyme  broth demonstrates enhanced growth, which is typical of Nocardia asteroides. 

PROCEDURE 2

Table2.Preliminary Grouping of the Clinically Relevant Aerobic Actinomycetes

Accurate identification of Nocardia to the species level is important, because differences among the species have emerged in terms of virulence, antibiotic susceptibility, and epidemiology. However, identification of the pathogenic nocardiae to the species level can be problematic, because no single method can identify all Nocardia isolates, and the methods used are time-consuming, often requiring 2 weeks. Useful phenotypic tests include the use of casein, xanthine, and tyrosine hydrolysis; growth at 45°C; acid production from rhamnose; gelatin hydrolysis; opacification of Middlebrook agar; and antimicrobial susceptibility patterns. Some of these reactions with the nocardial pathogens are summarized in Table 3.

Table3. Key Tests for Differentiation of the Pathogenic Nocardia spp.

Many tests are needed to confirm the identification of the other actinomycetes at the level of speciation; these are beyond the capabilities of the routine clinical micro biology laboratory, and such cases therefore should be referred to a reference laboratory.

SERODIAGNOSIS

 Currently, no reliable serodiagnostic tests are available to help identify patients with active nocardiosis; such tests are used only to augment culture results. Infections caused by other aerobic actinomycetes currently cannot be diagnosed serologically.

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مواضيع ذات صلة

Epidemiology and Pathogenesis of Nocardia, Streptomyces, Rhodococcus, and Similar Organisms

General Characteristics of Nocardia, Streptomyces, Rhodococcus, and Similar Organisms

Pathogenesis of Streptococcus pneumoniae

Treatment and Prevention of Staphylococcus aureus

Pathogenesis of Staphylococcus aureus

Direct Detection Methods for Erysipelothrix, Lactobacillus, and Similar Organisms

Pathogenesis and Spectrum of Disease Erysipelothrix, Lactobacillus, and Similar Organisms

Antimicrobial Susceptibility Testing and Therapy for Listeria, Corynebacterium, and Similar Organisms

Approach to Identification for Listeria, Corynebacterium, and Similar Organisms

GROWTH AND METABOLISM

SPORES AND SPORULATION

Susceptibility Testing