Final Diagnosis -- Mycobacterium kansasii with "ghost Mycobacteria" found on Gram-stain


Mycobacterium kansasii was first described by Buhler and Pollak in 1953.1 It was a very uncommon human pathogen prior to the HIV epidemic, with a prevalence of only 0.33 / 100,000 in the United States from 1981 to 1983.2 Although it is still an uncommon pathogen even among immunocompromised patients, it is second only to Mycobacterium avium intracellulare as a cause of non-tuberculous mycobacterial disease.3 Its prevalence rose sharply during the early years of the HIV epidemic, but fell again with the advent of highly active antiretroviral therapy (HAART) in the mid 1990s.4 The cases of disease show a geographic pattern, being much more common in the central and southern United States, and in England, Wales and continental Europe, especially in mining areas.2

Clinical Presentation

M. kansasii infection most commonly manifests as a chronic pulmonary disease mimicking classic tuberculosis. Radiographically, there is often an upper lobe cavitating lesion. Less commonly, the organism causes extrapulmonary disease, such as scrofula-like lymphadenitis, sporotrichosis-like cutaneous infection, osteomyelitis, and tenosynovitis. It rarely causes disease in non-immunocompromised patients, and only very rarely causes disseminated disease in patients who are not severely immunocompromised.5

Laboratory Diagnosis

Specimen collection
When a patient is suspected of having a mycobacterial respiratory infection, the specimen must be collected and handled in a special way.3 First, it is important to collect three consecutive daily samples (i.e. sputum, tracheal aspirates, or bronchoalveolar lavage fluid). Harvell, et al6 demonstrated that this number is necessary even with the increased sensitivity of the newer radiometric broth culture systems. They retrospectively analyzed 430 respiratory specimens from 143 patients who had positive M. tuberculosis cultures. Only 46 (32%) of the patients had positive AFB smears (44 on the first smear and 2 more on the second smear). Cultures were positive on the first specimen for 117 (82%) of the 143 patients, the second specimen for 14 patients (10%; cumulative rate 92%), and the third specimen for 12 patients (8%; cumulative rate 100%).

Specimen preparation
Mycobacteria grow slowly and require a long incubation, and therefore other organisms may overgrow the specimen, especially from a non-sterile site such as sputum. Before any staining or cultures are done, the specimen must first be decontaminated. This is done through a series of steps to liquefy and homogenize the sample while also decontaminating it. A common method is to treat the sample with a combination of sodium hydroxide (a decontaminant) and N-acetyl-L-cysteine (NALC, a mucolytic), along with a buffer to control the pH.3

A second problem is that the Mycobacteria may only be present in small numbers, so the specimen must be concentrated. The decontaminated specimen is centrifuged for 15 minutes at no less than 3000g. The supernatant is decanted, and the remaining concentrated specimen is used for culture and for the AFB smear.

Routine gram stain and culture are not adequate due to the unique architecture of the bacteria. The heavy lipid content of the mycobacterial cell wall resists the aqueous crystal violet and safranin in the Gram stain. Acid-fast stains use phenol to allow dyes to penetrate the waxy outer layers of the bacilli, and the mycolic acid retains the stain after exposure to acid alcohol or other strong mineral acids; hence the name "acid-fast". Typically, a fluorescent based stain, such as auramine-rhodamine is done first, because it is more sensitive and allows for faster screening. If this stain is positive, a carbol-fuschin based stain such as Ziehl-Neelsen is done to confirm the positivity ( Figure 4).3

In this patient, the amount of Mycobacteria in the sputum was so heavy that a routine gram stain did show evidence of the organisms. Figure 3 shows the gram stain with serpentine areas of clearing representing Mycobacteria that did not take up the gram stain. These are sometimes called "ghost Mycobacteria."7-10 These could easily be missed if there were only small numbers of bacteria.

Specimen culture
Mycobacteria can be cultured in two separate media. The first is a radiometric broth culture system. The bottle is inoculated with the concentrated sample and monitored for growth in a machine in a manner analogous to a routine blood culture. The second is a solid medium. Mycobacteria show "breadcrumb like growth" on these "slants" (so called because the nutrient gel is contained in a tube at a slant to increase surface area). This medium is a useful addition to the liquid medium, because the colony morphology can be used to provide a preliminary species identification. Figure 5 shows colonies of M. kansasii. M. kansasii is a photochromogen, that is, it produces a yellow pigment after exposure to light.5 Figure 6 shows colonies of the photochromogen before and after exposure to light. M. tuberculosis does not produce this pigment. Since M. kansasii and M. tuberculosis both have similar clinical presentations, this difference in morphology can alert the lab and the clinician that they are dealing with an atypical species, and not tuberculosis. This difference may not only help guide treatment until a definitive diagnosis is made, it may also help guide which diagnostic test to choose, as will be shown below.

Species identification
Traditionally, Mycobacteria were speciated biochemically. M. kansasii shows hydrolysis of Tween, reduction of nitrate to nitrite, and a positive catalase reaction.3 These tests can take weeks, however, and laboratories are increasingly relying on nucleic acid probes for speciation. Commercial DNA probes are now available for M. tuberculosis, M. avium intracellulare, M. gordonae, and M. kansasii.11 These probes are specific for the 16S rRNA gene sequence, and are reported to be highly sensitive and specific. Richter, et al report a sensitivity of 97.4% and a specificity of 100% with the newest DNA probes for M. kansasii.7 There are two important caveats concerning DNA probes. First, a negative test only rules out the specific species that is being probed. To rule out other species, other probes must be run. This may be prohibitively expensive for some labs, and is one reason why colony morphology may be helpful to guide the choice of probes. In practice, however, most labs that have this tool at their disposal will probably choose to run all of the probes at once for thoroughness and to save time. The second caveat is that a negative probe simply means that the DNA was not found. It does not necessarily mean that it was not present. The tests are not 100% sensitive and may miss the target.


The isolate from the sputum culture was DNA probe negative in our laboratory for M. tuberculosis, M. avium intracellulare, and M. gordonae. The isolate was sent to a reference laboratory where it was probe positive for M. kansasii. At this point there is still no quick way to test for drug susceptibility in these atypical species, so the isolate was sent to another reference lab for this purpose. It was resistant to kanamycin and capreomycin, as well as isoniazid at the 0.2ug/ml concentration, but sensitive at the 1.0ug/ml concentration. It was sensitive to rifampin, ethambutol, ethionamide, streptomycin, amikacin, cycloserine, and PAS. The patient was begun on empiric treatment for M. kansasii, and her anti-retroviral medications were temporarily discontinued to avoid drug interactions. These antiretroviral medications were restarted after a follow-up clinic appointment. Laboratory studies 6 months later showed an HIV viral titer of 225,000 copies / milliliter, and she appeared to have been poorly compliant with her, admittedly difficult, medication regimens.


  1. Buhler VB, Pollak A. Human infection with atypical acid-fast organisms: report of two cases with pathologic findings. Am J Clin Pathol. 23:363-74, 1953.
  2. Canueto-Quintero J, Caballero-Granado FJ, Herrero-Romero M, et al. Epidemiological, clinical, and prognostic differences between the diseases caused by Mycobacterium kansasii and Mycobacterium tuberculosis in patients infected with human immunodeficiency virus: a multicenter study. Clin Infect Dis. 37(4):584-90, 2003.
  3. Pfyffer GE, Brown-Elliott BA, Wallace Jr. RJ. Mycobacterium : general characteristics, isolation, and staining procedures. In Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, eds. Manual of Clinical Microbiology, 8th ed. ASM Press, Washington. 532-559, 2003.
  4. Manfredi R, Nanetti A, Valentini R, et al. Epidemiological, clinical and therapeutic features of AIDS-related Mycobacterium kansasii infection during the HIV pandemic: an 11-year follow-up study. HIV Medicine. 5(6):431-6, 2004.
  5. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology, 5th ed. Lippincott, Philadelphia. 893-952, 1997.
  6. Harvell JD, Hadley WK, Ng VL. Increased sensitivity of the BACTEC 460 mycobacterial radiometric broth culture system does not decrease the number of respiratory specimens required for a definitive diagnosis of pulmonary tuberculosis. J Clin Microbiol. 38(10):3608-11, 2000.
  7. Hinson JM Jr, Bradsher RW, Bodner SJ. Gram-stain neutrality of Mycobacterium tuberculosis. Am Rev Respir Dis. 123(4):365-6, 1981.
  8. Maygarden SJ, Flanders EL. Mycobacteria can be seen as "negative images" in cytology smears from patients with acquired immunodeficiency syndrome. Mod Path. 2(3):239-43, 1989.
  9. Trifiro S, Bourgault AM, Lebel F, Rene P. Ghost mycobacteria on Gram stain. J Clin Microbiol. 28(1):146-7, 1990.
  10. Fisher JF. Ganapathy M. Edwards BH. Newman CL. Utility of Gram's and Giemsa stains in the diagnosis of pulmonary tuberculosis. Am Rev of Respir Dis. 141(2):511-3, 1990.
  11. Richter E, Niemann S, Rusch-Gerdes S, Hoffner S. Identification of Mycobacterium kansasii by using a DNA probe (AccuProbe) and molecular techniques. J Clin Microbiol. 37(4):964-70, 1999.

Contributed by Andrew Walls, MD and A. William Pasculle, ScD

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