FINAL DIAGNOSIS: Nocardia veterana
Nocardiosis is infection by one of the members of the genus Nocardia, a group of slow growing ubiquitous soil-dwelling organisms that generally lead a saprophytic existence. They will generally grow within a few days to a few weeks on routine bacterial isolation media as an adherent, white to tan-yellow colony with a propensity to be "rolled up" and "chased" around the plate by a loop, and frequently smell of "moldy basement" or "freshly turned earth." They do especially well on enriched media like chocolate agar and buffered charcoal yeast extract agar. , Nocardia can be found in infected wounds in immunocompetent patients (mycetoma being somewhat common for certain strains that favor Latin America), and generally cause more severe manifestations of disease (pulmonary and CNS infection) in immunocompromised patients. Pulmonary infections can take on a wide range of clinical and radiographic forms, but bilateral opacities on x-ray representing necrotizing granulomatous inflammation histologically are most common. The organisms can be visualized by silver stains, Fite's modified acid fast stains (some modifications are available that are better for detecting Nocardia), and as a weakly positive filamentous organism with a characteristic "beaded" appearance on tissue gram stain. CNS infection is particularly feared, as Nocardia show marked predilection for brain abscess. Mortality for CNS infection, even without brain abscess, is disturbingly high. Infected wounds frequently need little more than trimethoprim/sulfamethoxazole unless in an especially vulnerable area, such as the eye. Pulmonary and CNS infections, particularly in immunocompromised patients, require multiple agents administered, usually for several months.
Nocardia veterana was first described in 2001 after its isolation from the bronchoalveolar lavage fluid of a 78 year old man with a history of tuberculosis and bilateral upper lobe lesions at the Austin and Repatriation Medical Centre in Heidelberg, Australia1. In the description of the initial type strain DSM 44445T, N. veterana was found to be most similar to N. vaccinii based on consenus phylogeny from 16S rRNA gene sequencing. Subsequent case reports and series have described N. veterana in association primarily with pneumonia in patients with some degree of underlying immunocompromise, although a mycetoma attributed to this species has also been described2,3,4,5. Several of these authors have described the difficulty in phenotypic identification of N. veterana, particularly from its relatively close relative N. nova, with Conville at el. reporting difficulty in the separation of these two species by 16S rRNA sequencing or restriction fragment length polymorphism analysis of an amplified region of the 65-kDa heat shock protein2. However, a few reliable, and relatively simple, phenotypic tests can differentiate N. nova and N. veterana. The latter will show equal and robust growth on suitably rich media at 25, 35, 37 and 42 degrees Celsius, while N. nova shows unequal, and particularly slow or no growth at 42 degrees Celsius. N. nova possesses a robustly active arylsulfatase, and will be strongly positive at 3 days and 14 days, while no isolate of N. veterana in the literature has positive arylsulfatase. Conversely, N. veterana will hydrolyze esculin and bile esculin, while N. nova will be unable to handle either.
Speciation of Nocardia is important. Although the genus, as a whole, is susceptible to trimethoprim/sulfamethoxazole, the second empiric agent for severe infections (such as CNS infection or pulmonary infection in an immunocompromised patient) is frequently driven by the known susceptibility patterns of the various pathogenic Nocardia species. In patients allergic to trimethoprim/sulfamethoxazole, such as this patient is, speciation for determination of empiric therapy is even more critical. This empiric therapy is frequently tailored to an isolate's individual susceptibility based on growth in the presence of a variety of antimicrobial agents; this testing is technically difficult and tedious due to the intrinsically slow growth of the genus and is usually done at specialized reference laboratories. N. veterana does not have a standard susceptibility profile attached to its name at present; however, most isolates in the literature have been susceptible to amikacin, imipenem, and trimethoprim-sulfamethoxazole in high doses2,3. Of particular note, Pottumarthy et al. reported production of a beta-lactamase by N. veterana detectable only in the presence of clavunate3.
Limited susceptibility testing (usually to gentamicin, tobramycin, amikacin, and erythromycin) and routine use of a few phenotypic identifiers are frequently, but not always, able to speciate Nocardia. Molecular methods, such as RFLP of heat shock protein genes, and more commonly 16S rRNA sequencing, have become an important tool for verifying Nocardia identification. However, as the report by Conville et al. showed, even molecular identification methods alone may not be sufficient to accurately identify some of the more closely related members of the Nocardia genus-in particular, N. veterana. Many of the available databases of bacterial sequences are not currently refereed for accuracy and reproducibility of source organism identification and sequence accuracy (although some, like RIDOM, are). Identifications made by comparison of sequences from clinical isolates to un-refereed databases such as GenBank must therefore be taken in careful consideration of the whole of the clinical presentation and any phenotypic data that are available for that isolate. In this case, initial routine phenotypic identification of the presumed Nocardial isolate suggested N. nova, while 16S rRNA sequencing showed strong and close association to N. veterana. Additional phenotypic testing with 14 day arylsulfatase and three days growth on chocolate agar at 35, 37 and 42 degrees Celsius showed no arylsulfatase activity and strong and equal growth at all tested temperatures, confirming the speciation of N. veterana in this elderly transplant patient with pulmonary nocardiosis.
Contributed by Joey Oakley, MD and William Pasculle, ScD