FINAL DIAGNOSIS (LUNG):
BILATERAL PNEUMONIA WITH NOCARDIA
Since Nocardia spp. are ubiquitous soil saprophytes; route of infection can be either by inhalation or by direct cutaneous inoculation.
Most human infections (90%) are caused by inhalation of members of the N. asteroides group, which includes three subgroups:
N. brasiliensis, N. otitidiscaviarum, and N. transvalensis represent the remaining 10% of infections. Of these, N. brasiliensis is the most important in tropical areas; it is most often seen as a cutaneous infection that can affect individuals with normal immune function (although 70% of cases of N. brasiliensis are seen in immunocompromised individuals).
In general, nocardiosis is a disease that affects primarily the cell- or humorally-immunocompromised population: transplant recipients, patients on high-dose steroids, and patients with cancer, AIDS, or other leukocyte deficiencies. Nocardia may also colonize the respiratory tract of immunocompetent individuals with compromised pulmonary function, such as those with asthma or chronic obstructive pulmonary disease (COPD). Once established, nocardial pneumonia will usually disseminate hematologically, with kidney, skin, GI tract, and brain being common targets; brain abscess (33% of cases) is the most common presentation.
Sulfonamides, alone or in combination with trimethoprim, are the most effective first line agents against nocardiosis, and should be continued for several months to prevent a relapse, especially in immunocompromised patients. Recommended second-line agents (minocycline, imipenem, or an aminoglycoside in combination with a third-generation cephalosporin) are indicated in cases of intolerance to sulfonamides. Supplemental agents (amikacin, ceftriaxone, cefotaxime or imipenen) might help in cases of widely disseminated nocardiosis.
Nocardiosis should be included in the differential of any chronic pneumonia that does not respond to empirical or common antimicrobial regimens.
Growth and identification of Nocardia
Nocardia may be difficult to culture in clinical microbiology laboratories
Nocardia species are classically gram-positive, strictly aerobic, filamentous, branching, weakly acid-fast bacilli. They may be isolated on routine bacterial, fungal, and mycobacterial media. Colonies may appear within 4 days, but may require up to 2-4 weeks of culture. Pre-treament of the patient with antibiotics that slow but do not kill Nocardia will most often increase the time required to grow Nocardia from clinical isolates. If nocardiosis is suspected clinically, the bacteriology laboratory should be informed and cultures should be kept longer than usual. Nocardia can also be difficult to isolate by culture because of overgrowth by faster-growing nonpathogenic colonizers that may mask its presence.
Nocardia colonies may be smooth and moist, or have a "mold-like" verrucous grey-white waxy or powdery appearance from aerial hyphae. They have a very distinct, strong mildew odor that allows experienced microbiologists to suspect their presence. (The Actinomycetes genera including Nocardia are responsible for the musty "mildew" odor of the average basement). The colonies of N. brasiliensis (left) and N. caviae (right) pictured below show the wide range of colony appearance, even within the same species:
Nocardia have variable (and sometimes unreliable) staining properties
Nocardia are classically Gram-variable, with a ‘beaded’ appearance of alternating Gram-positive and Gram-negative segments along a filament:
However, Nocardia grown under suboptimal conditions sometimes appear uniformly Gram-negative; therefore, negative Gram-staining cannot be used to rule out Nocardia.
Likewise, with a modified Ziehl-Neelsen or Kinyoun acid-fast stain (for cultured organisms) or a Fite-Faraco modified acid-fast stain (for histology sections), Nocardia organisms are classically weakly acid-fast. This characteristic may help to distinguish Nocardia from negative Actinomyces (which is modified-acid-fast-negative). However, tests based on acid-fastness alone are not reliable for such a distinction. As seen in this case study, Nocardia can occasionally appear modified-acid-fast-stain negative.
Nocardia grown under suboptimal conditions (too many passages in culture, lipid-poor media, or presence of inhibitory antibiotics) will have retarded synthesis of mycolic acids in their cell wall that, in turn, compromises the ability of the organisms to retain Gram stain or modified-acid-fast stains.
In this patient, the administration of antibiotics piperacillin and cefuroxime (neither first-line agents for treatment of Nocardia) may have generated a "sub-optimal condition" for Nocardia growth that led to unexpectedly negative Gram stain and negative modified-acid-fast results.
Differentiation of Nocardia species
Histologically, Nocardia has delicate (< 2 microns in thickness) filaments with pronounced branching. Actinomyces in contrast has a similar filamentous appearance, albeit with slightly thicker, straighter, and less-branched filaments. Since the histologic appearance of Nocardia is similar to other Actinomycetes family members, culture and biochemical testing is necessary for definitive diagnosis/identification.
The UPMC Presbyterian Mycology Laboratory performs petri-dish cultures with tap water agar (1%) to differentiate the Nocardias and other aerobic Actinomycete genera from the rapid growing mycobacteria. (The trace organic material found in tap water provides sufficient nutrients to support growth). On tap-water agar, Nocardia species have recursively branching hyphae with aerial hyphae. Preliminary biochemical tests for differentiation and speciation of Nocardia from the other Actinomycetes genera require 14 days, and include the presence of the following biochemical characteristics: (1) hydrolysis of casein, tyrosine, and/or xanthine, (2) presence of urease, (3) utilization of rhamnose, and (4) positive resistance to lysozyme.
|Casein hydrolysis||L-tyrosine hydrolysis||Xanthine hydrolysis|
|N. asteroides complex,
N. farcinica, or N. nova
|Streptomyces or Nocardiopsis||+||+||+|
Hydrolysis test agar is made by adding casein, crystalline L-tyrosine, or crystalline xanthine to plate media. If a Nocardia species can hydrolyze the substrate, the substrate disappears as seen by a zone of clearance around the colony.
In the pictures below, observe the hydrolysis patterns of the yellowish N. brasiliensis (left petri dish, below) versus N. caviae (right petri dish, below). In each petri dish, casein media is in the lower left quadrant, followed clockwise by L-tyrosine (upper left quadrant) and xanthine (upper right quadrant).
If the organism is determined by hydrolysis pattern to be one of the members of the N. asteroides group (N. asteroides complex; N. farcinica; or N. nova), then an antibiotic susceptibility test may be performed for a more precise identification. The test uses the antibiotic disc diffusion method and requires 72 hours to complete:
|Tobramycin||Cefamandole or Cefotaxime||Erythromycin|
|N. asteroides complex||Variable Resistance||Sensitive||Resistant|
Contributed by James R Davie, MD PhD, Octavia Graur MD PhD, and John Sheaffer, MS