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The cytopathology of Actinomyces , Nocardia , and their mimickers: MCHUGH

et al .

Article  in  Diagnostic Cytopathology · September 2017

DOI: 10.1002/dc.23816

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DOI: 10.1002/dc.23816

TIMELY REVIEWS

The cytopathology of Actinomyces, Nocardia, and their

mimickers

Kelsey E. McHugh, MD1 | Charles D. Sturgis, MD1 | Gary W. Procop, MD1 |

Daniel D. Rhoads, MD2,3

1
Department of Laboratory Medicine, Nocardia species and Actinomyces species are 2 of the most commonly diagnosed filamentous bac-
Cleveland Clinic, 9500 Euclid Avenue,
teria in routine cytopathology practice. These genera share many overlapping cytomorphologic
Cleveland, Ohio 44195
2 features, including their thin, beaded, branching, Gram-positive, GMS-positive filamentous struc-
Department of Pathology, Case Western
Reserve University, 10900 Euclid Ave, tures that fragment at their peripheries into bacillary- and coccoid-appearing forms. Features that
Cleveland, Ohio 44106 help distinguish between these 2 microorganisms include the width of their filamentous structures,
3
Department of Pathology, University the angles at which they branch, and their ability or lack thereof to retain a modified acid-fast
Hospitals Cleveland Medical Center, 11100 stain. In addition to cytomorphologic overlap, overlap in clinical presentation is frequent with pul-
Euclid Ave, Cleveland, Ohio 44106
monary and mucocutaneous presentations seen in both. Differentiating between Nocardia and
Correspondence Actinomyces is essential because patients with these infections require different approaches to
Kelsey E. McHugh, MD, Robert J. Tomsich medical management. Both antibiotic susceptibilities and the need for early surgical intervention
Pathology & Laboratory Medicine Institute,
as part of the treatment plan vary greatly among these 2 groups. This review focuses on the clini-
Cleveland Clinic, 9500 Euclid Avenue, L25,
Cleveland, OH 44195.
cal presentation, cytomorphology and staining characteristics that can be useful in identifying and
Email: mchughk3@ccf.org distinguishing between Nocardia and Actinomyces infections, as well as their mimickers.

KEYWORDS
Actinomyces, aerobic Actinomyces infection, cytology, fine needle aspiration biopsy, Nocardia

1 | INTRODUCTION
The most common clinically relevant filamentous bacteria include Acti-
nomyces, Nocardia, and their related genera. Although these organisms
may appear morphologically similar, their means of infection, disease
courses, and medical management are often very different. Cytology,
both exfoliative and fine-needle aspiration (FNA) based, serves as a
useful tool in the rapid identification and characterization of these
infections, allowing for early optimization of therapeutic management.
Both Nocardia and Actinomyces are bacteria that belong to the
order Actinomycetales.1 Nocardia species are ubiquitous, saprophytic
soil microorganisms.2 Actinomyces species are endogenous mucous
3,4
membrane microbiota that colonize healthy humans. Although both
genera are capable of infecting either immunocompetent or immuno-
compromised hosts, Nocardia are more commonly opportunistic patho-
gens affecting the immunocompromised, whereas Actinomyces are
typically seen in the setting of polymicrobial infections secondary to
mucous membrane or physical barrier breakdown.3,5
With either group of bacteria, the potential for serious infections
exists, and these microorganisms often demonstrate overlap in their
clinical presentations.6 Both can present as infections within the lung,
soft tissue, bone, joint, or central nervous system (CNS) as well as a
number of less frequent anatomic sites including the nipple and the
skin.7–12 Additionally, both are capable of systemic dissemination, typi-
cally through hematogenous routes.2,3
There have been significant taxonomic changes in the Nocardia
genera over the past decade, as the causative agents of infection have
been more accurately characterized by DNA sequencing techniques. In
the past, many infections were categorized as Nocardia asteroides com-
plex because of limitations in the ability to further characterize these
organisms. Important and invasive Nocardia species, formerly charac-
terized as members of the N. asteroides complex, include organisms
such as N. cyriacigeorgica, N. farcinica, N. abscessus, and members of the
N. nova complex, among others. An association between these Nocar-
dia species and clinical presentation or disease pattern has never been
elucidated.6 In contrast, N. brasiliensis is a Nocardia species commonly
associated with mycetoma. Identification of Nocardia to the species
level is important as antimicrobial susceptibilities vary per species.13
In immunocompetent hosts, environmental exposure to Nocardia leads
to cutaneous infections such as mycetomas, lymphocutaneous infections,

Diagnostic Cytopathology. 2017;1–11. wileyonlinelibrary.com/journal/dc V

C 2017 Wiley Periodicals, Inc. | 1
2 |
and superficial skin infections (abscess or cellulitis).13–17 In immunocompro-
mised hosts, with a frequency among solid organ transplant patients rang-
ing from 0.6 to 3.0%, Nocardia usually presents as an invasive pulmonary
infection.2,18 Hematogenous dissemination with a tropism for the brain has
2,19
also been well described in this patient population.
Unlike Nocardia, Actinomyces species are common endogenous
microbiota of the healthy human oropharynx, gastrointestinal tract,
respiratory tract, and vaginal vault. The principal infectious species in
humans is A. israelii. Infections associated with Actinomyces species are
typically polymicrobial, including otherwise commensal organisms that
have compromised an epithelial barrier or are associated with a foreign
body. Sinus tracts commonly form, and macroscopically visible sulfur
granules may drain from these tracts. On imaging, actinomycosis may
appear as a lesion that is directly spreading through tissues without
respect for physical planes or barriers.12 Because of this radiological
appearance, Actinomyces infections may be clinically mistaken for
malignancy.20 Surgical debridement in addition to medical therapy is
often necessary for complete resolution of actinomycosis.
Exfoliative and FNA cytology preparations can serve as rapid, accu-
rate, minimally invasive, and inexpensive means of diagnosing both
Nocardia and Actinomyces infections. Exfoliative samples, including effu-
sion fluid, cervicovaginal preparations, sputum, urine, and respiratory
lavage samples, often have a low concentration of the material of inter-
est and require centrifugation prior to slide preparation. In contrast, the
material obtained via FNA targeting a mass lesion is cellular enough to
be directly smeared onto glass slides. A portion of sample collected by
FNA can be used for microbiologic culture as long as it is unfixed. Anaer-
obic cultures for Actinomyces are not recommended on exfoliative cytol-
ogy specimens collected from mucous membranes because Actinomyces
colonization in healthy individuals is common. Nocardia is not a com-
mensal organism, so recovery of Nocardia by culture from any specimen
site may be due to infection. Specimens collected by exfoliation or FNA
are used to generate liquid based cytology preparations, which can
include a cell button and a cell block for microscopic examination. The
cell button is an alcohol-fixed concentrated suspension of cells, whereas
the cell block is a formalin- or alcohol-fixed, paraffin-embedded aggre-
gate of cellular and acellular material from the specimen. If ThinPrep
(Hologic, Inc) processing is employed, a proprietary filtration and vacuum
suction step may result in decreased accompanying debris, necrosis,
and/or proteinaceous exudate resulting in altered slide backgrounds. A
variety of stains are routinely used in cytology preparations, and these
stains can help to detect filamentous bacteria: Papanicolaou and Roma-
nowsky type (eg, Diff-Quik) stains on direct smears, Papanicolaou stains
on cell buttons, and hematoxylin & eosin (H&E) stains on cell blocks. On
liquid based cytology cell buttons and cell blocks, special stains can also
be performed to further characterize microorganisms.
2 | MORPHOLOGY AND ROUTINE
STAINING CHARACTERISTICS
Nocardia and Actinomyces are thin, filamentous, beaded, branching
bacteria that may present with peripheral clubbing and/or
MCHUGH ET AL.
peripheral fragmentation into bacillary and coccoid non-motile ele-
ments.4 Both microorganisms are thinner in diameter than true fun-
gal hyphae and lack septations, which are useful diagnostic
features (Figure 1).21 Although not distinct enough to be a useful
discriminator, it is worthwhile to note that Nocardia is described as
more delicate in appearance than Actinomyces due to its narrower
diameter. Nocardia species are typically 0.5 to 1.0 micrometer in
diameter, while Actinomyces species range from 1.0 to 1.5 Mm.21
These organisms also have different branching patterns, with Acti-
nomyces branching at a more acute angle than the right angle
branching of Nocardia.21
Nocardia and Actinomyces infections typically elicit an acute
inflammatory response in human tissue.4 Classically, Nocardia infec-
tions are associated with an intense neutrophilic reaction whereas
Actinomyces infections have a neutrophilic response associated with
a peripheral granulomatous, fibrosing reaction. With either infection,
the inflammation can be pyogenic and/or granulomatous depending
on the duration of disease and the immune status of the host. In
immunosuppressed patients, in whom Nocardia and Actinomyces
infections are able to persist, there is a transition from an initial neu-
trophilic response to a predominantly granulomatous tissue reaction.
Associated lymphoplasmacytic inflammation can be seen intermixed
with the histiocytes.1,4,14 Cytopathologic examination of Nocardia-
associated purulent and granulomatous samples typically reveals
small “grains” (granules) composed of amorphous eosinophilic matrix
material containing loose clumps of delicate, branching filaments that
fragment into bacillary and coccoid forms at their periphery (Figure
2). Nocardia “grains” are typically small in size, averaging <1 mm in
greatest diameter. A classic feature of an Actinomyces infection is the
macroscopically visible “sulfur granule” comprised of Actinomyces,
other bacterial species, and proteinaceous material. Microscopically,
acute and granulomatous inflammation may surround the granule in
situ (Figure 2).5 When not associated with a neutrophilic infiltrate or
a “sulfur granule,” Actinomyces contamination rather than a true
infection of actinomycosis should be considered.1,4
Both Actinomyces and Nocardia are associated with the Splendore-
Hoeppli phenomenon, which is frequently appreciable in cytologic
preparations, especially when granules are present.11,22,23 In this phe-
nomenon, eosinophilic, pseudomycotic structures composed of
necrotic debris and immunoglobulins form rings around the grains or
granules of microorganism (Figures 2 and 3).24
When lacking grain or granule formation, both bacteria may be dif-
ficult to detect using H&E because they stain lightly. Romanowsky
staining is preferred for detecting Nocardia, and the filamentous bacte-
ria appear as pale blue tangles or clusters (Figure 4).21 On Papanicolaou
stained slides, Nocardia appears pale pink and may be easily obscured
by associated inflammation.25,26 Conversely, Actinomyces that are caus-
ing infection are often associated with large aggregates of material that
are easily visualized using Papanicolaou stained preparations. These
actinomycotic infections often appear as fuzzy purple aggregates at
low power, with a reddish hue appreciable on high power examination
of individual organisms.27,28
MCHUGH ET AL. | 3

FIGURE 1 A side-by-side comparison of the width of filamentous bacteria versus true fungal hyphae at the same magnification. (A) Gomori
Methenamine Silver positive cluster of thin, branching, filamentous Actinomyces bacteria, Gomori Methenamine Silver 6003; (B) Gomori
Methenamine Silver positive cluster of Aspergillus sp. hyphae, Gomori Methenamine Silver 6003

3 | CYTOPATHOLOGY OF NOCARDIA AND projections that lack a dense central core. These structures do not con-
ACTINOMYCES ON EXFOLIATIVE tain microorganisms and thus are negative by Gomori Methenamine
SPECIMENS Silver and modified acid fast special staining.31 Background morphol-
ogy in specimens from patients with Actinomyces ranges from clean,
3.1 | Cervicovaginal specimens with little or no inflammation, to extensively obscured with substantial

The presence of Actinomyces in cervicovaginal preparations is unusual numbers of neutrophils and histiocytes.26

unless the patient is harboring a foreign body, most typically an intrau- Nocardia, unlike Actinomyces, is not typically found in cervicovagi-

terine device (IUD). In patients with IUDs, Actinomyces is identified in nal gynecologic cytology preparations.

approximately 5%-10% of gynecological preparations.26,27,29 When

fuzzy aggregates or sulfur granules are identified microscopically, care
must be taken to confirm the presence of filamentous Actinomyces
within these granules because non-filamentous bacteria are capable of
26,27
forming similar structures. The non-filamentous, non-Actinomyces
bacteria that contribute to the formation of these granules have been
described as “dust bunnies.”30 “Crystalline bodies,” thought to be the
cytology preparation analogue to “pseudoactinomycotic radiate gran-
ules” (PAMRAG) seen in histology, also serve as sulfur granule mimics.
PAMRAGs and “crystalline bodies” are identical radiate, refractile-
appearing structures that consist of irregular, broad, club-like
3.2 | Respiratory specimens
Actinomyces is a common commensal organism in the alimentary canal
and may contaminate and cause infection in the nearby lungs. Nocardia,
however, is not part of the normal microbiota of the body, although it
is a well-known cause of pulmonary infections.
A total of >40% of reported Nocardia infections present as pulmo-
nary disease.32 Rapid diagnosis of nocardiosis can be achieved on
expectorated sputum samples, bronchial washings, and bronchoalveolar
lavage fluid. Importantly, granules are not formed in pulmonary
4 | MCHUGH ET AL.

F I G U R E 2 Cytologic preparations from multiple patients. (A and B) Loose aggregate of thin, filamentous Nocardia bacteria (grains) with
branching and beading appreciable upon high-power examination, Modified Ziehl-Neelsen stain 1003 (A), 10003 (B). (C and D) Fuzzy
masses (sulfur granules) of Actinomyces bacteria with ringing neutrophils and clinging eosinophilic debris consistent with Splendore-Hoeppli
phenomenon, ThinPrep 4003

nocardiosis. Branching filamentous bacteria associated with a neutro- 3.3 | CNS specimens
philic response is the usual finding in these instances.
Central nervous system involvement by Nocardia or Actinomyces typically
Approximately 15% of actinomycosis involves the thorax.33 The
occurs after systemic dissemination of the infectious organism.2,3 Nocardia
diagnosis of actinomycosis on expectorated sputum or bronchoscopic
infections have a particularly impressive propensity for cerebral involve-
aspirates is challenging.34 In addition to being morphologically similar to
Nocardia, the presence of Actinomyces can also represent contamination ment, with the CNS being the most common extrapulmonary site of infec-

with endogenous flora rather than a true invasive pathogen. If Actinomy- tion, involved in 15%-44% of cases.2,36–38 Within the CNS, Nocardia

ces bacteria are not associated with neutrophils, careful consideration of
contamination from extrapulmonary sites such as the tonsillar crypts
should be considered rather than true infection. 34
Occasionally, sulfur
granules can be identified on exfoliative cytopathology preparations,
which are indicative although not diagnostic of actinomycosis.34,35
Additionally, Actinomyces can also be incidentally identified colonizing
debris associated with neoplasms or foreign bodies.
Culture can be especially helpful in corroborating cytology. How-
ever, Nocardia may require extended incubation periods before suc-
cessful growth in culture, and some Actinomyces are obligate
anaerobes, which are not recovered in routine aerobic respiratory
10,32
cultures.
presents most commonly as cerebral abscess, accounting for approximately
2% of cerebral abscesses in the United States. It can also present as cerebral
meningitis with or without abscess formation; the associated mortality is
high.2 In the setting of cerebral abscess, lumbar puncture for cerebrospinal
fluid (CSF) collection may increase the patient’s to risk of herniation.39
Cerebral involvement occurs in 5%-10% of all Actinomyces infec-
tions and can occur either through hematogenous spread or via direct
extension from cervicofacial sources.8,37–39 Primary actinomycosis of
the CNS is exceedingly rare.37 Similar to Nocardia, the most common
presentation of CNS actinomycosis is cerebral abscess formation, but
meningeal presentations with or without associated abscess are also
reported.37,38,40,41
MCHUGH ET AL.
F I G U R E 3 (A and C) Cluster of Actinomyces bacteria on cell block with mild peripheral Splendore-Hoeppli phenomenon with associated
neutrophilic and eosinophilic inflammation. Splendore-Hoeppli phenomenon circled in part C, H&E 2003 (A), 4003 (B), 10003 (C)
The CSF in patients with cerebral Nocardia is typically clear to
cloudy, whereas the CSF in patients with cerebral Actinomyces is classi-
cally described as extremely purulent, resembling frank pus.37,38 In
both settings, the CSF most typically demonstrates low glucose levels,
2,36,40,41
elevated protein levels, and a neutrophilic pleocytosis. Both
nocardial and actinomycotic infections can present as subacute or
chronic meningitides. In indolent infections, the CSF demonstrates a
persistent neutrophilic predominance for weeks, despite appropriate
37,38
antimicrobial therapy. In chronic infections, a lymphocytic predomi-
nance may be observed. Visualization of the filamentous bacteria in
cytologic preparations is not typical. This may be due to either their
low concentration in the CSF or the lack of continuity between the cer-
ebral abscess and the subarachnoid space. Of note, using concentrated
CSF may improve likelihood of recovery by culture.37,38 Culture of Acti-
nomyces is ideally performed under anaerobic conditions, which are not
routinely used for CSF specimens.
3.4 | Body cavity fluids
Both Actinomyces and Nocardia infections are rarely associated with
body cavity effusions (ie, thoracic, pericardial, peritoneal).42–46
| 5
Typically, these effusions present secondary to associated mass lesions
or pseudotumors. Rare reports of spontaneous peritonitis due to Acti-
nomyces without preceding abscess formation or inciting event have
been described, and in the single report with cytologic preparations,
sulfur granules were microscopically identified.40–48 On macroscopic
examination, these effusions range from serosanguinous to densely
purulent, and cytology may not always detect the bacteria.43 Microbio-
logic cultures may be confirmatory, although for Actinomyces, only a
minority of cultures return positive results.9,42,49
3.5 | Urinary tract specimens
Approximately 20%-30% of actinomycotic infections involve the pel-
vis.50 However, primary vesical actinomycosis is an extremely rare
entity with a total of 10 cases reported in the literature to date.49,51–61
More frequently, actinomycosis involving the urinary bladder occurs
secondary to direct extension from a primary infectious source in a
neighboring pelvic organ.49,53 Jang et al. reported a single patient with
the initial diagnosis of primary vesical actinomycosis made on routine
urine cytology.51 In this case, the patient presented with a urinary blad-
der wall ‘tumor’ suspicious for malignancy and routine urine cytology
6 | MCHUGH ET AL.

FIGURE 4 53 year old male renal transplant patient presented with a left axillary lesion. (A) PET scan demonstrating a left axillary PET-
avid lesion clinically concerning for malignancy; (B) FNA of left axillary lesion showing beaded, branching, filamentous bacteria, Diff-Quik
10003; (C) Modified acid-fast positive beaded, branching, filamentous bacteria consistent with Nocardia in a background of acute inflamma-
tion, Modified Ziehl-Neelsen stain 10003

demonstrated “dusty islands surrounded by dense populations of neu- for both Nocardia and Actinomyces infections, are accessible via endo-
trophils.” Within the islands, thin, delicate, filamentous bacterial struc- bronchial ultrasound guided (EBUS) FNA. Perirectal and intraabdominal
tures were identified, resulting in a cytologic diagnosis of lesions are accessible via endoscopic ultrasound guided (EUS) FNA.
actinomycosis. This diagnosis was confirmed via routine histology in a Aspiration of lumps and bumps on regions of the head and neck,
post-surgical excision.51 breast, genitalia, and general skin/soft tissue can be performed via
Unlike Actinomyces, there are no reports of Nocardia identified on palpation.22,72
routine urine cytology. The cytomorphology of aspirates depends less on the site aspi-
rated and more on the type of infection (Nocardia versus Actinomyces),
its duration, and the immunologic status of the host. In patients with
4 | FINE NEEDLE ASPIRATION OF intact immunity, the typical appearance of Nocardia or Actinomyces on
NOCARDIA AND ACTINOMYCES FNA is clusters of thin, filamentous organisms dispersed among clumps
or sheets of neutrophils admixed with macrophages and set within a
Nocardial and actinomycotic infections diagnosed via FNA are rela- degenerative proteinaceous or necrotic background.32,33,63 Nocardia
tively rare, but FNA has great utility in this setting as these infections will form granules if a mycetoma is present; otherwise, filamentous
often clinically and radiographically mimic malignancy.62 Case reports bacteria without granule formation may be seen. The material often
describing the utility of FNA for identifying these filamentous bacteria appears purulent at the time of aspiration.66 In subacute or more clini-
include the following sites of infection: lung, cerebrum, perirectal soft cally indolent presentations, predominantly granulomatous inflamma-
tissue, common bile duct, parotid, neck, eye, breast, vulva, and skin/ tion with or without associated necrosis may be seen on
9,10,12,28,38,63–71
subcutaneous tissue. Pulmonary lesions, a common site cytopathology. Of note, the differential diagnosis for granulomatous
MCHUGH ET AL. | 7

T AB LE 1 Morphology, staining characteristics, and culture isolate findings in Nocardia and Actinomyces infections as well as their mimickers

Empiric Expected
Modified acid gram background and Culture Common
fast (Fite) stain surrounding recovery infection
Morphology results findings materials conditions locations

Nocardia Bacilli and/or Positive or Positive 1/2 Pyogenic and/or No anaerobic Lung, soft tissue,
thin, branching Negative beading granulomatous growth (obligate brain
filaments inflammation aerobe)

Actinomyces Bacilli and/or Negative Positive 1/2 Pyogenic and/or Anaerobic growth Breached mucus
thin, branching beading granulomatous preferred, but membranes
filaments inflammation; some are
multiple co-infectious aerotolerant
bacterial species/
morphologies;
Splendore-Hoeppli
phenomenon or its
proteinaceous remnants

Mold Hyphae (wider Negative Positive or Pyogenic and/or Requires special Lung, soft tissue
than bacterial negative granulomatous media and
filaments) incubation
conditions

Mycobacteria Bacilli Positive Positive 1/2 Granulomatous Requires special Lung, soft tissue
beading or 1/2 neutrophils media
non-staining and incubation
“ghost bacilli” conditions

inflammation is broad and includes Mycobacterium, fungal etiologies,
sarcoidosis, granulomatosis with polyangiitis, Crohn’s disease, foreign
body reaction, idiopathic etiology, and Langerhans cell histiocyto-
sis.73,74 Reports of Nocardia and Actinomyces infections in immunocom-
promised patients detail an initial neutrophilic response not
significantly different from that seen in the immunocompetent.75 The
ability of these infections to persist and disseminate within immuno-
compromised patients may result in prolonged disease courses, with an
76
increased likelihood of subacute and chronic infectious processes.
Filamentous bacteria may not be observed with routine staining as
they have a very light staining quality and are thin in width.10,32,70,71
The presence of inflammation and/or acellular material that are com-
monly associated with Nocardia and Actinomyces should prompt further
investigation with ancillary histochemical stains. The use of special
stains is helpful in both identifying organisms and distinguishing
between the 2 microorganisms (Table 1). Additionally, confirmation of
the diagnosis should be attempted via culture or molecular
63,71,77
methodologies.
5 | ANCILLARY DIAGNOSTIC TECHNIQUES
Delay in establishing the correct diagnosis often occurs in Nocardia and
Actinomyces infections as both have non-specific clinical presenta-
tions.2,3 Histochemical stains, culture, and several new techniques are
available to assist in establishing a definitive diagnosis.4 Notably, serol-
ogy plays no role in the diagnosis of either infection.38,78
stains. Examples of morphologic mimics include true fungal hyphae,
proteinaceous stranding of acellular material (especially in CNS prepa-
rations), and non-filamentous bacteria that exhibit abnormal morpholo-
gies secondary to antibiotic effects. Both Nocardia and Actinomyces
stain positively with Gram and Gomori Methenamine Silver stains,
whereas their non-bacterial mimics are negative (Figure 5).18 It’s impor-
tant to note that weak or patchy positivity by either method falls
within the spectrum of true positive staining for both microorganisms.2
Periodic Acid Schiff (PAS) stain helps to distinguish Nocardia and
Actinomyces from their fungal mimickers. Although bacterial granules
or grains may appear as PAS-positive structures, examination of the
individual bacterial filaments reveals that both Actinomyces and Nocar-
dia are typically PAS-negative microorganisms whereas true fungal
hyphae are PAS-positive.18,27,28
When faced with gram-positive, Gomori Methenamine Silver-
positive filamentous bacteria, the use of a modified acid fast stain is an
excellent method for distinguishing between Nocardia and Actinomyces
species. Nocardia are typically modified acid fast positive (Figure 4)
whereas Actinomyces are not (Table 1).79 Important to note, modified
acid-fast positivity in Nocardia may be weak and/or patchy.2 Examples
of modified acid fast methodologies include modified Ziehl-Neelsen
staining as well as the Fite-Faraco method, both of which use a weak
1% sulphuric acid as the decolorizer.15,80 Both Nocardia and Actinomy-
ces are acid fast negative when classic, non-modified acid fast stains
(eg, Ziehl Neelsen) are used (ie, methodologies that use a strong acid
for decolorization).

5.1 | Histochemical stains 5.2 | Culture

Detection, characterization, and differentiation from other microorgan- Specialized media are not required for culture of either Nocardia or
isms, as well as morphologic mimics, are facilitated via histochemical Actinomyces. However, Thayer Martin media or selective buffer
8 | MCHUGH ET AL.

FIGURE 5 (A) Cytology studies of a right upper lobe pulmonary lesion reveal fuzzy granules including proteinaceous strands and thin,
filamentous, branching bacteria fragmenting into bacillary-like and coccoid-like elements (proven to be Actinomyces by culture) within a
background of acute inflammation, Papanicolaou (Pap) 4003; (B) Clustered thin, filamentous, beaded, branching Actinomyces bacteria, Diff-
Quik 10003; (C) gram-positive grouped, beaded, branching Actinomyces bacteria in a background of neutrophils, Gram Stain 10003; (D)
Gomori Methenamine Silver positive clusters of thin, branching, filamentous Actinomyces bacteria, Gomori Methenamine Silver 10003

charcoal yeast extract (sBCYE) agar can aid in recovering Nocardia in
specimens containing mixed bacteria.81 Anaerobic bacterial cultures
should be performed to recover Actinomyces.2,3,82,83 Recovery of both
microorganisms is best achieved with specimens obtained via invasive
procedures (ie, FNA) rather than those elicited via exfoliative method-
ologies.2 While growth of either microorganism by culture can take up
to 3 weeks, colonies are typically observed within 3–5 days.79
5.3 | Nucleic acid techniques
Molecular methods can be useful tools in the identification of Nocardia
and Actinomyces species because the turnaround time can be more
rapid than microbiologic culture.18 Molecular testing involves nucleic
acid amplification techniques (NAAT).84 Two frequently used method-
ologies for organism identification are real time polymerase chain reac-
tion (RT-PCR) and 16S ribosomal RNA (rRNA) amplification with
subsequent product sequencing.80,85–89 NAAT methods may be espe-
cially useful in instances wherein filamentous bacteria are unexpectedly
identified by cytology and no specimen was submitted for culture. In
these cases, fixed specimens can be used for NAAT in order to identify
the taxon of the filamentous bacterium.
5.4 | Antimicrobial susceptibility testing
Identification of Nocardia and Actinomyces microorganisms to the species
level is important for appropriate patient management, as species vary in
their pathogenic potential within human hosts as well as in their antimi-
crobial susceptibility profiles.90–92 Minimal inhibitory concentrations of
antimicrobials and their associated interpretative breakpoints can be
found in CLSI M24 (Nocardia) and M100 (Actinomyces) documents.
In conclusion, Actinomyces and Nocardia species are 2 of the most
commonly encountered filamentous bacteria in routine cytology speci-
mens, and they share abundant cytomorphologic overlap. Distinguish-
ing between these 2 organisms is clinically important as they require
different therapeutic management strategies. Combining cytopathology
findings with knowledge of these organisms’ predilection for infecting
MCHUGH ET AL.
specific sites and the use of ancillary testing techniques allow for
prompt and accurate diagnosis of both actinomycosis and nocardiosis.
Cytopathology can play a vital role in the medical management of
patients afflicted with actinomycosis and nocardiosis.
C ONFLICT OF INT E RE ST
There are no conflicts of interest to report for any of the authors.
OR CID
Kelsey E. McHugh http://orcid.org/0000-0002-2389-9851
Charles D. Sturgis http://orcid.org/0000-0003-0153-498X
R E FER E NCE S
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[3] Sullivan DC, Chapman SW. Bacteria that masquerade as Fungi: acti-
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[5] Graybill JR, Silverman BD. Sulfur granules: second thoughts. Arch
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How to cite this article: McHugh KE, Sturgis CD, Procop GW,
Rhoads DD. The cytopathology of Actinomyces, Nocardia, and
their mimickers. Diagnostic Cytopathology. 2017;00:1–11.
https://doi.org/10.1002/dc.23816