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Nocardia species (Nocardiosis)

Authors: Melissa Bell, M.D., Michael M. McNeil, M.D. , June M. Brown, M.D.

Previous authors: Michael M. McNeil, M.D. , June M. Brown, M.D.

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Microbiology Nocardiosis is difficult to diagnose clinically, radiologically, and

Epidemiology
histopathologically. A definitive diagnosis depends on the isolation and the
Clinical Manifestations
Laboratory Diagnosis identification of Nocardia species. Making the diagnosis may often involve
Pathogenesis
Susceptibility In Vitro and In
performing invasive techniques on the patient and may take up to 2 or 3
Vivo
Antimicrobial Therapy
weeks. Data derived from modern taxonomic methods have changed the
Adjunctive Therapy taxonomy of the genus Nocardia (27). Major
Endpoints for Monitoring
Therapy pathogenic Nocardia species, Nocardia farcinica, N. nova, N.
Vaccines
Prevention or Infection
cyriacigeorgica , and N. pseudobrasiliensis, have been
Control Measures
Comments
characterized. Nocardia cyriacigeorgica, N. farcinica, and N. nova were
separated from the Nocardia asteroides complex
and Nocardia pseudobrasiliensis from Nocardia brasiliensis. These species
were validated primarily on the basis of DNA-DNA hybridization, 16S rRNA
gene sequence analysis, antimicrobial susceptibility and biochemical
profiles, and, to a lesser degree, on high-performance liquid
chromatography (22, 54, 55, 69). The use of molecular technology for
identification and epidemiologic subtyping of the Nocardia species has
been limited by the lack of simple and rapid assays. Rapid molecular
identification and typing methods that may be useful include 16S rRNA
gene sequencing, random amplified polymorphic DNA and a combination
polymerase chain reaction-restriction fragment length polymorphism
analysis (39, 116).

MICROBIOLOGY
The genus Nocardia is currently composed of 87 validly described species:
46 of these species are medically relevant (16, 27, 53). However, despite
recent taxonomic changes, there is evidence that this genus is still
underspeciated. The distinguishing phenotypic characteristics of the five
major clinically relevant species are given in Table 1.
Nocardia microorganisms are filamentous rods that show right-angled
branching both in culture and in tissues. For culture they require aerobic
conditions, but growth on blood agar may be slow, and incubation for
periods longer than 48 h is usually necessary. As cultures enter the
stationary phase, the filaments tend to fragment into coccobacillary forms.
Although the organisms are gram positive, many strains give a faint beaded
appearance with alternating positive and negative areas.
Since Nocardia are weakly acid fast, the most useful acid-fast stain is the
modified Kinyoun method (33, 55). The Brown-Brenn modification of Gram
stain and the Gomori methenamine stain are useful in demonstrating the
organisms in histopathologic preparations from tissue (20). The presence of
aerial filaments, decomposition of substrates, and acid production from or
carbon utilization of carbohydrates, as previously described, are used to
differentiate the members of the genus Nocardia (8, 27, 55).

Of importance, the former N. asteroides complex, responsible for the

majority of invasive human infections, has been separated into six
susceptibility patterns: N. abscessus (formerly N. asteroides type I), N.
brevicatena/paucivorans complex (type II), N. cyriacigeorgica (type VI), N.
farcinica (type V), N. nova complex that includes N. africana, N.
kruczakiae, N. nova, and N. veterana (type III), and N. wallacei (type IV)
(16). N. farcinica is particularly important to distinguish since it has
increased virulence and differs in its antimicrobial susceptibility test results
and its epidemiology (16, 126). In addition to susceptibility studies, tests
used to separate the four most commonly isolated N. asteroides complex
and N. brasiliensisisolates further as N. abscessus, N. cyriacigeorgica, N.
farcinica, and N. nova include growth on tryptone glucose yeast agar at
45oC for 1 day, production of: 14-day arylsulfatase, nitrate reductase, and
urease; hydrolysis of adenine, casein, esculin, hypoxanthine, tyrosine, and
xanthine; and utilization of acetamide, citrate, L-rhamnose and D-sorbitol
(16); these same tests are used to separate N. pseudobrasiliensis from N.
brasiliensis (16). Since Nocardia species infections are very often sporadic,
information from randomized clinical trials comparing the clinical efficacy of
specific antimicrobial agents is lacking. Reports have been limited to
antimicrobial susceptibility test results of clinical isolates, usually from
reference laboratories, animal studies and case summaries (19, 78).
Interpretation of these data may be complicated by several factors. When
data are from a reference laboratory that is likely to receive referral isolates
from patients who are intolerant of therapy or for whom therapy has failed,
there is a potential for bias in their interpretation. Also, often it is difficult to
compare in vitro susceptibility results with data reported by other
investigators because of differences in methodology, e.g., lack of
interlaboratory standardization of inocula (e.g.,104 versus102 CFU/ml), and
use of a broth microdilution method versus agar dilution or disk diffusion
methods. The introduction in 2000 by the National Committee for Clinical
Laboratory Standards (NCCLS) of a tentative standard for susceptibility
testing of Nocardia spp. and other actinomycetes using the broth
microdilution method alleviated some of these problems (M24-T2) (96);
these standards were updated in 2011 as document M24-A2 (29). In
addition to differences in methodology, the results of some reported studies
may vary because no distinction was made between N. nova and the
former N. nova complex strains drug pattern type III (now includes N.
africana, N, kruczakiae, and N. veterana) (88) or between N.
brasiliensis and N. pseudobrasiliensis (27, 88, 105, 127). The typical in
vitro susceptibility profiles of the clinical important Nocardia are given in
Table 2.

16S rRNA gene sequencing

Currently, accurate identification of aerobic actinomycetes requires the use
of molecular methods, specifically, gene sequencing and analysis. 16S
rRNA gene sequence analysis can provide genus-level, and for most
genera species-level, identification of clinically relevant aerobic
actinomycetes (27, 28). Based on recommendations from Tindall et al.
(120), nearly complete high quality gene sequences are necessary to
maintain accurate genus/species assignment based on sequence similarity
values as defined by Clinical and Laboratory Standards Institute (120).
Speciation of isolates suspected of being an aerobic actinomycete requires
a 16S rRNA gene sequence with a minimum length of 1440 bp. A BLAST
analysis of a high quality gene sequence against a DNA sequence
database such as GenBank [www.ncbi.nlm.nih.gov/Genbank] will allow for
genus or species assignment based on sequence similarity results
compared to a reference strain. Limitations to identifying aerobic
actinomycetes by molecular methods include: lack of availability of
technology and expertise; difficulty in generating high quality sequence data
of sufficient length; inherent limitations of large public databases (25).

Many clinical laboratories rely on pyrosequencing to generate short (~500

bp) 16S rRNA gene sequences for identification of clinical isolates;
however, it has been shown that some pathogenic aerobic actinomycetes
cannot be accurately differentiated by short 16S sequences (26). It is
recommended to send clinical isolates to a reference laboratory for
confirmation of identification when adequate sequencing technology and
analysis are unavailable.

EPIDEMIOLOGY
Nocardia species are widely geographically distributed soil bacteria that
usually cause chronic, progressive infections (108). These infections may
be localized or disseminated and are more common and generally more
serious in severely immunocompromised and debilitated patients.
Invasive Nocardia infections may be an important cause of death and
infectious disease in immunocompromised solid organ transplant recipients
(23, 79, 85). Severe infections with Nocardia species have also been
reported to affect patients with human immunodeficiency virus (HIV)
infection (65, 66, 70, 81, 82, 117). Cases may go undiagnosed, either
because there is a delay in performing necessary diagnostic tests (invasive
biopsies) for seriously ill patients or because the infection partially or
successfully responds when prophylactic broad-spectrum antimicrobial
therapy is prescribed (85). In vitro and in vivo studies, clinical observations,
and taxonomic developments indicate that antimicrobial therapy must be
adjusted to the particular species of Nocardia present, to individual strain
antimicrobial susceptibility patterns, and to the site and type of infection (6,
23, 135).

CLINICAL MANIFESTATIONS

Pulmonary Nocardiosis
Pulmonary nocardiosis may be associated with nonspecific clinical findings;
however, immunocompetent patients may have a chronic course, as
opposed to the progressive, disseminated, and life-threatening infection
seen in severely immunocompromised patients. The most frequent clinical
presentation may be as a subacute or chronic, often necrotizing
pneumonia, which is frequently associated with cavitation (42, 85, 92).
Local complications of invasive Nocardia spp. pulmonary infections include
pleural effusion, empyema, pericarditis, mediastinitis, superior vena cava
obstruction, and rarely, development of local chest wall and neck
abscesses. Metastatic infective foci may be present but unrecognized at the
time of the patient's initial presentation with pulmonary nocardiosis, and
infection in these sites may not become clinically evident until after the
patient has begun receiving antimicrobial therapy.

Disseminated Nocardiosis
Disseminated nocardiosis is often a late-presenting and potentially life-
threatening infection. It is most frequently endogenous (i.e., secondary to
bloodstream spread) from a primary pulmonary infection (85, 92, 137).
However, very rarely, it may result from a primary nonpulmonary
(cutaneous) infection site (114). In patients with primary pulmonary
nocardiosis, the development of disseminated infection may result in brain
and skin lesions and invariably has a significant adverse effect on the
patient's prognosis. Disseminated nocardiosis has a mortality rate of 7% to
44% (85, 138). In severely immunocompromised patients the mortality may
be greater than 85% (85). Disseminated infection in susceptible patients
may be caused by any of the Nocardia spp. identified as causing invasive
pulmonary and cutaneous infections. As seen with pulmonary nocardiosis,
the patients at highest risk for developing disseminated infections are
severely immunocompromised patients. The brain is the most frequent
nonpulmonary site involved in disseminated nocardiosis, and cerebral
nocardiosis is an important cause of cerebral space occupying lesions.
However, the infection may also involve multiple other deep organs
including the kidney, spleen, liver, and rarely, bone, skin, and joints (85). In
the brain and other organs, abscess formation is a particularly common
pathologic manifestation of disseminated infection (85). Patients with
cerebral nocardiosis may present acutely with signs of sepsis and
intracranial mass effects (85). However, severely immunocompromised
patients with nocardial cerebral abscess may frequently be asymptomatic
initially, and demonstate a prolonged latency (up to 3 years) before this type
of clinical presentation in infected patients following the commencement of
immunosuppression (85). There may be clinical evidence of pulmonary
nocardial infection in about one third of the cases (12), and blood cultures
may also be positive for Nocardia spp. in these patients (12). Computed
tomographic (CT) scanning is an extremely useful technique for making the
diagnosis and may also be used to monitor the patient's response to
treatment (83). However, a definitive diagnosis may only be established in
the patient following the performance of a brain biopsy that yields clinical
specimens, which are positive for Nocardia spp. on microbiologic culture
and/or show morphologically compatible microorganisms on histopathologic
examination. Specific investigations to detect cerebral involvement are
recommended in all cases of pulmonary and invasive nocardiosis since a
brain abscess may be a common serious complication in these patients,
and early lesions may be asymptomatic (12).

Cutaneous Nocardiosis
Cutaneous nocardiosis may be subdivided into four clinical types:
mycetoma, lymphocutaneous infection, superficial skin infection (abscess or
cellulitis), and secondary cutaneous involvement with disseminated
disease. In North and South America, Mexico, and Australia, N.
brasiliensis is the chief cause of actinomycetoma;
whereas Actinomadura madurae, A. pelletieri,
and Streptomyces somaliensis predominate in India and the African
continent (85). These infections most commonly affect patients in rural
areas in developing countries. Patients with these chronic infections may
give a history of specific minor localized traumatic injury. The foot is the
commonest site of involvement; however, the hand, face, and neck may
also be affected (85). Nocardia brasiliensis has been associated particularly
with subcutaneous infections and is predominant in tropical countries (113,
114, 134). Frequently, there may be spread beyond the initial cutaneous
focus to involve the regional lymphatics and, in one-third of cases, the
disease may progress to form lymphatic abscesses (85). When regional
lymph node involvement occurs, this form of the disease is referred to as
the lymphocutaneous syndrome or the sporotrichoid form of cutaneous
nocardiosis because of its striking resemblance to the disease due to the
fungus Sporothrix schenckii (85, 113, 114).

N. cyriacigeorgica (formerly Nocardia asteroides type VI

susceptibility pattern and N. asteroides sensu stricto)
Of the previously established N. asteroides drug patterns, type VI is the
most commonly isolated drug pattern type in areas where actinomycotic
mycetoma is not endemic (16, 27). In 2012, in a study on sulfonamide
resistance in clinical Nocardiaisolates, Brown-Elliott et al. reported testing
552 total isolates from six major U.S. medical or referral centers received
from 2005-2011. Of these, 136 (25%) were identified as N.
cyriacigeorgica making this group the most numerous among all species
reported in the study (15). The Centers for Disease Control and Prevention
have reported N. cyriacigeorgica as the third most prevalent species
identified among clinical Nocardia isolates referred to the Special
Bacteriology Reference Laboratory for identification over the last 10 years
(unpublished data). In addition, Kageyama et al., in 2005, reported N.
cyriacigeorgica as a significant pathogen in Japan and Thailand (68). There
are also reports of N. cyriacigeorgica as the causative agent of
disseminated nocardiosis in Canada, France, Greece, The Netherlands,
and Turkey (27). Molecular methodologies including 16S rRNA, heat shock
protein, and secA1 gene sequencing have allowed more accurate
assignment of clinical isolates to this group. In 2001, Yassin et al.
described N. cyriacigeorgica from bronchial secretions of a patient with
chronic bronchitis and with this publication successfully validated the
establishment of the species (144). Nocardiacyriacigeorgica is often a
cause of pulmonary nocardiosis but there are also several reports of brain
abscess, and disseminated disease. A 2014 case report from Brazil
described disseminated disease in an immunocompetent patient who
required a total of 28 months treatment before resolution
(97). Nocardia cyriacigeorgica was isolated from this patient’s ulcerative
cutaneous lesions and pleural fluid; symptoms included high fever,
respiratory distress, arthritis, peripheric neuropathy, and reoccurrence of
new nodules. Evolution of antimicrobial therapy was as follows: initial
treatment was with ceftizoxime and clindamycin then switched to TMP-SMX
and imipenem. Due to intolerance of TMP-SMX, the combination therapy
was changed to imipenem, amikacin, and doxycycline. Imipenem was
removed and amikacin and doxycycline continued which resulted in
reoccurrence. Imipenem, amikacin, and vancomycin were then given for
two months with doxycycline monotherapy given as maintenance. After six
months, there was a second reoccurrence and ceftriaxone, amikacin, and
doxycycline were given for two months. With no change in disease
progression, linezolid with doxycycline were given and clinical cure
achieved. Linezolid had to be stopped after 3 months due to adverse effects
without complete eradication of the organism. Final resolution and cure was
attained only after treatment of relapsing episodes of acute cutaneous
lesions with TMP-SMX for 10 months. This case is an example the
persistence of nocardiosis and challenges in finding effective therapy, even
when the identity of the species is known.

N. farcinica
Nocardia farcinica may cause a variety of clinical presentations, including
cerebral abscess, keratitis, bacteremia, and pulmonary, kidney, and
cutaneous infections (91, 111, 132). There is a clear importance in
differentiating between N. farcinica and
other Nocardia species. Nocardia farcinica has a high degree of resistance
to various antibiotics, especially to third-generation cephalosporins, which
may make treatment of the infection difficult (109, 132). Mouse
pathogenicity studies have demonstrated that this may be a more virulent
species than the others (85). Nocardia farcinica occurs more frequently
than was previously recognized (109, 132). This can be attributed to recent
developments in diagnostic methods and possibly also to a change in
spectrum of human nocardiosis in countries such as Germany where N.
farcinica is the prevailing species (109). Other reports from France,
Germany, and the United States have implicated N. farcinica as the cause
of postoperative wound infections in patients undergoing cardiac and other
vascular surgeries (10, 39, 136).

N. nova
The clinical diseases associated with N. nova isolates are similar to those
previously described for diseases due to N. farcinica and other former N.
asteroides complex microorganisms. The reasons for identifying these
microorganisms include their susceptibility to erythromycin and third-
generation cephalosporins and resistance to amoxicillin-clavulanate (128).
Also, as suggested for N. farcinica infections, infection with N. nova may be
more common than is currently suspected; however, the successful
detection of these newly recognized species is dependent upon the
performance of appropriate isolation and characterization techniques as
well as increased clinical and microbiological awareness (128, 141).

N. transvalensis
Infections with N. transvalensis have been reviewed by McNeil et al. (86).
Initially recognized as a cause of mycetoma, N. transvalensis infections
have also been reported to cause life-threatening invasive pulmonary and
disseminated infections in severely immunocompromised patients (85, 86).
Importantly, clinical isolates of this unusual species may demonstrate a high
level of inherent resistance to amikacin and aminoglycosides in general. In
addition, therapy with trimethoprim-sulfamethoxazole (TMP-SMX) may not
always be effective for this infection (85). In a study by Wilson et al.
(139), N. transvalensis complex isolates showed a difference in geographic
distribution of the designated subgroups. No isolates of the former N.
asteroides complex type IV (now considered a member of the N.
transvalensis complex) were identified among the clinical isolates identified
in Queensland, Australia;however, the majority (75%) of N.
transvalensis isolates of the new taxon were from that location (139).

Four Nocardia species, N. abscessus, N. africana, N. paucivorans, and N.

veterana, have only rarely been encountered as agents of disease
(57, 60, 142, 143). N. abscessus has been isolated from patients with
abscesses of knee joint, fibula, and leg (143); N. africana has been isolated
from patients with pulmonary infections (61); N. paucivorans has been
isolated from the sputa and bronchial secretions of a patient with chronic
lung disease (142); and N. veterana has been isolated from bronchial
lavage of a patient with upper lobe lesions (the strain was thought not to be
of medical importance) (57).

LABORATORY DIAGNOSIS
In patients with suspected nocardial infection and a compatible clinical
picture, a definitive diagnosis usually depends on the demonstration of the
organisms in smears or sections examined microscopically together with
isolation and identification by microbiologic culture. The importance of direct
microscopic examination of stained preparations of clinical specimens in
the diagnosis of aerobic actinomycotic infections cannot be
overemphasized. The specimens most frequently received in the clinical
microbiology laboratory for evaluation include sputum, bronchial lavage
fluid, exudate, or CSF. If possible, the material should be spread out in a
petri dish and observed for clumps of the microorganisms, which may
resemble granules. If clumps or granules are present, they should be
selectively removed and crushed between two glass microscope slides for
microscopic examination. In addition, duplicate direct smears of the clinical
material should be always be prepared for staining, and one smear stained
with Gram's and the other with the modified Kinyoun acid-fast method. On
Gram-stained smears, gram-positive branched filamentous hyphae are
seen that are similar to the appearance of nocardiae in cultures: they
measure from 0.5 to 1 µm in diameter and as much as 20 µm in length. To
be of diagnostic value the hyphae must branch at right angles. Although the
hyphae of nocardiae may resemble those of Actinomyces species in width,
they are usually much greater in length and more widely scattered
throughout purulent material and in the walls of the abscesses. In
mycetoma, compact granules are formed, similar to those observed for the
anaerobic actinomycetes (134). Very rarely, clubbing has been seen with N.
asteroides, but often granules with clubbing are seen with N.
brasiliensis and N. otitidiscaviarum (85). The hematoxylin and eosin stain is
very useful for staining the tissue reaction and the granules but does not
stain the individual filaments (20). A tissue Gram stain such as the modified
Brown and Brenn procedure is recommended for demonstration of the
gram-positive filaments of nocardiae (20). The Gomori methenamine silver
stain may also be useful. However, in both of these procedures, the
filaments may not stain uniformly. Acid-fast stains are also of value in the
histopathologic diagnosis of infections caused by all Nocardia species.
These species are frequently, but not always, acid fast in tissue sections
stained with both the modified Kinyoun or the Fite-Faraco staining methods
(85). Actinomyces and related species are usually not acid-fast. These
examinations provide a rapid presumptive diagnosis of the patient's
infection and the information they yield may critically influence the clinician's
choice of initial antimicrobial therapy.

PATHOGENESIS
Nocardia species have been shown to act as facultative intracellular
organisms within macrophages (4, 31), where they inhibit the fusion of
phagosomes with the lysosomes. In addition, human neutrophils and
monocytes have been shown not to kill these organisms (40). Therefore, for
optimal therapy for these intracellular microorganisms, it may be important
to choose antimicrobial agents that are able to penetrate the cell. However,
a demonstrated ability of a drug to enter cells does not guarantee activity.
The microenvironment and intracellular distribution of the organisms and
antimicrobial agent, and interactions between antimicrobial agent,
pathogenic organism, and host cell all contribute to the determination of the
therapeutic result (34).

SUSCEPTIBILITY IN VITRO AND IN VIVO

Six major classes of antimicrobial compounds are currently in clinical use:
the sulfonamides, the aminoglycosides, the β-lactams (penicillins,
carbapenems, and cephalosporins) and the b-lactam/b-lactamase
inhibitors, the quinolones, the macrolides, and the tetracyclines (Table 3).

Single Drug
General Drug of Choice
Sulfamethoxazole inhibits bacterial synthesis of dihydrofolic acid by
competing with para-aminobenzoic acid. Trimethoprim blocks the
production of tetrahydrofolic acid from dihydrofolic acid by binding to and
reversibly inhibiting the essential enzyme, dihydrofolate reductase. Thus,
trimethoprim-sulfamethoxazole (TMP-SMX) blocks two consecutive steps in
the biosynthesis of nucleic acids and proteins (Table 3). Sulfamethoxazole
and trimethoprim have intermediate and high intracellular penetration of
human polymorphonuclear leukocytes (PMNs), respectively (44, 61)
(Table 3). For trimethoprim, in particular, this may be connected to its
efficacy in combination with sulfamethoxazole in treating intracellular
pathogens (61). Sulfonamides (or the combination TMP-SMX) are the
therapy of choice for nocardiosis (47,80, 114, 130).
Certain Nocardia isolates may be susceptible to sulfonamides or TMP-
SMX, and response to treatment may be attained in 90% or more of cases
if the infection is confined to pleuropneumonia (114). However, in patients
with disseminated disease to the central nervous system, or with
depressed cell-mediated immunity such as occurs with renal transplant
recipients and HIV-infected patients, several factors may complicate
therapy (6, 45, 51, 66, 70, 81, 93, 109, 115, 148).

One factor is the frequent occurrence of patient intolerance or side effects

with the most commonly used drug combination, TMP-SMX; this occurs in
HIV-infected patients with
either Pneumocystis carinii (now Pneumocystis jiroveci) (56, 70)
orNocardia species infections (117), and in renal transplant recipients with
nocardiosis (2). In these patient populations, adverse reactions such as
skin rash, fever, and neutropenia have been reported in 44% to 80% of
cases (56, 70). Hepatic toxicity, reported rarely with TMP-SMX, has
occurred in 20% of patients with acquired immunodeficiency syndrome
(AIDS) and the probability of toxicity is further increased by its prolonged
use as a prophylactic therapy (56).

Another factor is resistance of the infecting microorganism to drug therapy

(TMP-SMX, and alternative agents or drug therapy combinations)
(24, 45, 51, 72, 110, 115). In a review of nocardiosis in AIDS, TMP-SMX
was used as therapy for 50% of patients; however, 90% of these patients
were nonresponsive and died (81). The need for prolonged antimicrobial
therapy (6-12 months routinely) to prevent recurrences of the infection and
lifetime prophylactic therapy in AIDS patients further increases the
possibility of the development of drug resistance (53). In a review of 19
patients on TMP-SMX for P. carinii (P. jiroveci) prophylaxis, two patients
developed Nocardia infections (66). Also, an infection caused by a TMP-
SMX-resistant N. nova strain has been reported in a leukemic child placed
on TMP-SMX prophylaxis for P. carinii (P. jiroveci) infection (93).

A third factor is the lack of information on newer oral alternative

antimicrobial agents or combinations that might improve the outcome of
patients with Nocardia species infection. Table 4 contains the results of our
susceptibility study using a microdilutional technique to test 98 patient