Nocardia: Nocardiosis, Modified Acid-Fast Staining, Diagnosis, vs Actinomyces

A man who had a kidney transplant a few months ago, and takes drugs to suppress his immune system, comes in with a cough, fever, and weight loss over several weeks. A chest scan shows a lung nodule with a cavity. He is treated for tuberculosis, but nothing improves. Then he develops a headache and one-sided weakness, and a brain scan shows an abscess. The clue that ties the lung and the brain together is his suppressed immunity and an organism that looks a little like TB on the slide but is not TB. A modified acid-fast stain of his sputum shows thin, branching, partly red-staining filaments. This is nocardiosis, caused by Nocardia.

Introduction

Nocardia species are gram-positive, branching, filamentous bacteria that are strictly aerobic and partially (modified) acid-fast. They belong to the aerobic actinomycetes. Nocardia forms branching filaments that grow along the surface (substrate hyphae) and into the air (aerial hyphae), which is why the colonies can look fuzzy or chalky. Four features together define the organism and explain almost everything about it: it is aerobic, it branches, it is partially acid-fast, and it mainly attacks people with weakened cell-mediated immunity.

Figure: Filamentous Nocardia spp. in Kinyoun acid fast stain(Image source; CDC/Phil Health)

Nocardia lives in soil and water and reaches humans from the environment, not from other people. There is no person-to-person spread. Several species cause human disease, called nocardiosis. Traditionally, Nocardia asteroides was said to cause most invasive infections and Nocardia brasiliensis most skin infections.

A note on naming: what older textbooks call N. asteroides is now known to be a group of several distinct species (including N. farcinica, N. nova, and N. cyriacigeorgica), separated by molecular methods. Exams and standard textbooks still use N. asteroides, so this article keeps that term, but be aware that modern laboratories identify the specific species, which matters for treatment.

Transmission

People acquire Nocardia by two routes, and the route decides the disease. Inhalation of the organism from the environment leads to lung infection, which can then spread through the blood, most importantly to the brain. Traumatic inoculation through the skin (for example, a thorn or soil-contaminated wound) leads to localized skin disease such as mycetoma.

The host matters as much as the route. In a person with normal immunity, Nocardia usually causes localized skin disease. In a person with weakened cell-mediated immunity (transplant recipients, people on long-term steroids, people with advanced HIV, or those with lymphoma), it causes invasive lung disease that can disseminate. This is why nocardiosis is often called an opportunistic infection.

How Nocardia causes disease

Nocardia is not highly aggressive in a healthy person. Its ability to cause serious disease comes from a small set of tricks that let it survive the very defense meant to kill it: the macrophage.

What it does: when a macrophage engulfs Nocardia, the organism resists being killed inside the cell.

Why it matters: Nocardia blocks the fusion of the phagosome with the lysosome, resists the acid environment inside the phagosome, and produces enzymes (catalase and superoxide dismutase) that neutralize the toxic oxygen products the macrophage uses as weapons. Because the macrophage cannot kill it, the organism survives and multiplies. Clearing Nocardia therefore depends on strong cell-mediated immunity (activated T cells helping macrophages). This one fact explains the disease pattern: when cell-mediated immunity is weak, the macrophages cannot finish the job, and the infection spreads from the lung to the blood and the brain.

The mycolic acids in its cell wall are the same feature that makes it partially acid-fast on staining, so the property you see in the laboratory is directly connected to the cell wall that helps it survive.

Clinical infections

In people with normal immunity, Nocardia (especially N. brasiliensis) causes localized skin infections after inoculation:

• Mycetoma (actinomycetoma): a chronic, localized, painless, swelling of subcutaneous tissue, usually on the foot or hand after inoculation. It slowly forms nodules, draining sinuses, and grains (granules) discharged from the sinuses. When caused by a bacterium such as Nocardia, it is called actinomycetoma; when caused by a fungus, it is called eumycetoma.
• Lymphocutaneous infections
• Skin abscesses or cellulitis

In people with weakened cell-mediated immunity, Nocardia (classically N. asteroides) causes invasive lung disease: pneumonia, lung abscess with cavity formation, lung nodules, or empyema. Because these look like tuberculosis, fungal infection, or cancer on imaging, nocardiosis is often missed at first, exactly the situation in the clinical scenario above.

From the lung, the organism spreads through the blood to other organs. The brain is the most important target, where it causes brain abscess. This lung-to-brain spread is a hallmark of disseminated nocardiosis and carries a poor prognosis. Because of this, patients with pulmonary nocardiosis are often imaged for silent brain abscesses even without neurological symptoms.

Laboratory Diagnosis

Sample

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 choice of the sample depends on the site affected. Depending on the clinical presentations, samples include sputum, pus from an abscess, and granules.

As Nocardia spp. are ubiquitous, isolation do not necessarily mean infection. It can possibly be the colonization of skin or upper respiratory tract or rarely laboratory contamination.

Direct Microscopy

If granules are seen in the actinomycetomas sample, the granules should be washed in saline, emulsified in 10% KOH, or crushed between two slides and gram stained to check for the presence of gram-positive filamentous bacilli.

Gram-staining (Brown-Brenn modification)

Presence of gram-positive branching or partially branching, filamentous bacilli often with beaded appearance suggest the presence of actinomycetes.

Figure: Nocardia spp in tissue section stained using the Brown & Brenn method (Image source: CDC/Phil Health)

Modified acid-fast staining (Kinyoun method)

Nocardia is partially (modified) acid-fast. On a modified acid-fast stain, which uses a weaker acid (1% sulfuric acid) as the decolorizer, Nocardia holds the red stain and appears as red, branching, filamentous rods. The reason is the mycolic acid in its cell wall, similar to but less than that of mycobacteria, so it resists weak-acid decolorization but not the strong acid used in the standard Ziehl-Neelsen stain. This "partial" acid-fastness is a key point: it separates Nocardia (partially acid-fast, branching) from both Mycobacterium (strongly acid-fast, not branching) and Actinomyces (not acid-fast, branching).

Histopathology (H and E stain)

H and E staining of the granules show multilobulated with sunray appearance.

Culture

Nocardia spp. do not have complex growth requirements and can grow on various media such as brain heart infusion agar, sheep blood agar, chocolate agar, and Sabouraud dextrose agar (SDA).

Colonies are slow to appear, usually taking several days (often 3 to 5 days or longer) at 35 to 37°C, so it is important to hold cultures long enough and to tell the laboratory that nocardiosis is suspected. Because it grows slowly, faster contaminating bacteria can overgrow it on routine media. To overcome this problem various selective media have been in use to isolate Nocardia which are:

• Buffered yeast extract containing polymyxin and vancomycin.
• Sabouraud dextrose agar with chloramphenicol.
• Paraffin bait technique: Media using paraffin as the sole carbon source
• Buffered charcoal yeast extract agar with polymyxin, anisomycin, and vancomycin.
• Brain-heart infusion agar with chloramphenicol and cycloheximide.
• Lowenstein-Jensen medium: Produces moist glabrous colonies (differentiates from mycobacteria)

Colonies are creamy, wrinkled, pigmented (orange or pink-colored due to carotenoid-like pigments), and adhere firmly to the medium. Some colonies possess abundant aerial growth and have a cotton wool ball appearance.

Biochemical Identification

Nocardia species are non-motile, catalase-positive, and aerobic. The catalase (and superoxide dismutase) they produce is the same enzyme system that helps them survive inside macrophages, as described in the pathogenesis section. Various biochemical tests are done for species identification such as:

1. Decomposition of casein, hypoxanthine, tyrosine
2. Growth in lysozyme
3. Acetamide utilization
4. Growth at 45°C (helps separate some species, for example N. farcinica)
5. Acid from rhamnose
6. Gelatin hydrolysis
7. Opacification of Middlebrook agar

Serology

Currently, no serological tests are available to diagnose nocardiosis.

Nocardia vs Actinomyces: how to tell them apart

Nocardia and Actinomyces are both gram-positive branching filamentous bacteria, so on a Gram stain they can look alike. They are separated by a few decisive features. The fastest discriminators are oxygen requirement and the modified acid-fast stain.

The two-line memory version: Nocardia is the aerobic, acid-fast one that comes from abroad (the environment) and hits the immunocompromised lung and brain. Actinomyces is the anaerobic, non-acid-fast one that is already at home in your mouth and causes lumpy jaw. For the full account of the anaerobic side of this pair, see the article on Actinomyces.

Treatment of nocardiosis

The drug of choice is a sulfonamide, usually trimethoprim-sulfamethoxazole (co-trimoxazole). This is worth remembering because it is also the point where treatment separates Nocardia from Actinomyces: Nocardia responds to sulfonamides, while Actinomyces responds to penicillin.

Serious or disseminated disease is treated with combinations of antibiotics, and the choice depends on the species and susceptibility testing, because species such as N. farcinica can be resistant to several agents. Treatment is prolonged, often for many months, especially when the brain is involved, and brain abscesses may also need surgical drainage. Because relapse is common if treatment is stopped early, completing the full long course is essential.

How to Remember

Key exam facts in one table

Where Students Get Confused

References and further readings

• Tille, P. M. (2022). Bailey & Scott's Diagnostic Microbiology (15th ed.). Elsevier.
• Procop, G. W., & Koneman, E. W. (2017). Koneman's Color Atlas and Textbook of Diagnostic Microbiology (7th ed.). Wolters Kluwer.
• Carroll, K. C., Pfaller, M. A., et al. (2020). Murray's Medical Microbiology (9th ed.). Elsevier.
• Brown-Elliott, B. A., Brown, J. M., Conville, P. S., & Wallace, R. J. (2006). Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy. Clinical Microbiology Reviews, 19(2), 259–282.