Vol. 28, No. 1
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1990,
p. 146-147 0095-1137/90/010146-02$02.00/0 Copyright © 1990, American Society for Microbiology
Ghost Mycobacteria on Gram Stain SILVANA TRIFIRO,"2 ANNE-MARIE BOURGAULT,3 FRANÇOIS LEBEL,' AND PIERRE RENÉ2,4* Department of Microbiology, Montreal General Hospital,' Department of Medicine, McGill University,2 Service de Microbiologie et Maladies Infectieuses, Hôpital Saint-Luc, and Département de Microbiologie et Immunologie, Université de Montréal,3 Montreal, Quebec, and Department of Microbiology, Royal Victoria Hospital, Montreal Quebec H3A lAJ,4* Canada Received 22 June 1989/Accepted 11 September 1989
The Gram stain is a key tool in diagnostic microbiology. Its usefulness with respect to mycobacteria is undefined. The neutrality of mycobacteria other than Mycobacterium tuberculosis on Gram staining of various clinical specimens is described.
For more than a century, the Gram stain has provided a reliable, quick, and reproducible method for rapid identification of different microorganisms in clinical specimens. It is widely believed that the Gram stain can identify most bacteria, including mycobacteria. Its role in the detection of mycobacteria, however, is highly questionable, as acid-fast stains are normally used for that purpose. Recently, two groups have reported that specimens containing many mycobacteria resulted in a remarkable gram-neutral (neither positive nor negative) or gram ghost appearance of the mycobacteria (4, 6); this phenomenon was observed in a sputum containing Mycobacterium tuberculosis (4) and in the bone marrow of two patients infected with M. leprae (6). We report herein the finding of gram ghost (i.e., gramneutral) mycobacteria from six Gram-stained specimens. The mycobacteria isolated were later identified as M. haemophilum, M. scrofulaceum, and M. tuberculosis. A 56-year-old man diagnosed as having acquired immunodeficiency syndrome was admitted to a hospital with fever, progressive cachexia, and multiple skin lesions. He complained of a cough productive of yellow sputum but no hemoptysis. An erythematous fluctuant mass located on the right thigh was aspirated, yielding purulent fluid. A Gram stain of this fluid revealed an abundance of leukocytes but no bacteria. Numerous unstained bacillus footprints or ghost bacilli were noted (Fig. 1), A Ziehl-Neelsen stain of the same specimen is shown in Fig. 2. An auramine O-rhodamine stain was done before the Ziehl-Neelsen stain, and both confirmed the presence of acid-fast bacilli. Later, cultures of the pus yielded M. haemophilum. Treatment with various combinations of isoniazid, rifampin, ethambutol, and pyrazinamide was unsuccessful. Gram staining of premortem sputum specimens and autopsy specimens of lung and kidney tissues revealed neutral footprints of mycobacteria. Again, auramine O-rhodamine and Ziehl-Neelsen staining confirmed the presence of acidfast bacilli. M. scrofulaceum was later isolated from lung tissue.
*
Two other patients with cavitary lung lesions provided an abundance of ghost bacilli on a Gram stain. Later, M. tuberculosis was identified, thus confirming the previous observations of Hinson et al. (4). The Gram-staining properties of mycobacteria are little commented upon or even ignored in most microbiology textbooks. Some authors have stated that although mycobacteria are not readily stainable because of their relative resistance to aniline dyes, they are usually considered gram positive (3, 9-11). Kretschmer addressed the question of the Gram-staining property of Koch bacilli in 1934 (5). He showed that cold aniline with gentian violet caused staining of tubercle bacilli in a gram-positive manner, but he concluded that the tubercle bacillus did not possess true gram positivity, since iodine was not necessary as a mordant and normal, not anilinefortified, crystal violet led to no appreciable staining of sputum which revealed
FIG. 1. Pus containing M. haemophilum ghost bacilli (Gram stain; magnification, x 1,000).
Corresponding author. 146
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and read by at least three observers. Regardless of the fixation technique, all observers reported the presence of ghost bacilli. Several mycobacterial species are considered important pathogens both in patients with acquired immunodeficiency syndrome and in other immunocompromised hosts. M. haemophilum causing skin infections is rare, but cases have been described in immunocompromised and normal hosts (1, 7), as well as in a patient with acquired immunodeficiency syndrome (8). For any clinical specimens found to be purulent in nature but lacking microorganisms on Gram staining, the microbiologic doctrine of staining for acid-fast bacilli holds true. However, observation of ghost bacilli on the initial Gram stain provides an early and helpful diagnostic clue before confirmation of mycobacteria is made by more specific stains. LITERATURE CITED 1. Feldman, R. A., and E. Hershfield. 1974. Mycobacterial skin infection by an unidentified species. A report of 29 cases. Ann. Intern. Med. 80:445-452. 2. Finegold, S. M., and W. J. Martin. 1982. Cultures, media, stains, reagents and tests, p. 655. In D. E. Ladig (ed.), Bailey and Scott's diagnostic microbiology, 6th ed. The C.V. Mosby
Co., St. Louis.
FIG. 2. Pus containing M. haemophilum (Ziehl-Neelsen stain; magnification, x 1,000).
bacilli. Hinson et al. described the gram neutrality of bacilli in a sputum specimen which eventually yielded M. tuberculosis on culture (4). In addition, Lawrence and Schreiber described M. leprae ghost footprints in histiocytes when the Fite stain was used on bone marrow aspirates (6). The entity of gram neutrality or ghost footprints of nontuberculous mycobacteria in clinical specimens has never been described. The preparation of slides was similar to the procedure described by Hinson et al. (4); i.e., they were heat fixed, but in addition, slides were fixed with methanol and Formalin. The slides were stained by the Hucker modification of the Gram stain technique (2). All slides were done in triplicate
3. Harris, H. W., and J. H. McClement. 1972. Tuberculosis, p. 351-378. In P. D. Hoeprich (ed.), Infectious diseases, lst ed. Harper & Row, Publishers, Inc., New York. 4. Hinson, J. M., R. W. Bradsher, and S. J. Bodner. 1981. Gram stain neutrality of Mycobacterium tuberculosis. Am. Rev. Respir. Dis. 123: 365-366. 5. Kretschmer, O. S. 1934. The Gram property of the acid-fast form of the tubercle bacillus. J. Lab. Clin. Med. 19:350-358. 6. Lawrence, C., and A. J. Schreiber. 1979. Leprosy footprints in bone marrow histiocytes. N. Engl. J. Med. 300:834-835. 7. Lomvardias, S., and G. E. Madge. 1972. Chaetoconidium and acid fast bacilli in skin ulcers. Arch. Dermatol. 106:875-876. 8. Males, B. M., T. E. West, and W. R. Bartholomew. 1987. Mycobacterium haemophilum infection in a patient with acquired immune deficiency syndrome. J. Clin. Microbiol. 25: 186-190. 9. Murohashi, T., and K. Toshida. 1965. Effect of ultraviolet irradiation on the acid-fastness and Gram positivity of mycobacteria. Am. Rev. Respir. Dis. 92:812-817. 10. Wayne, L. G., and G. P. Kubica. 1986. The mycobacteria, p. 1435-1457. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. 11. Wolinsky, E. 1980. Mycobacteria, p. 724-742. In D. Davis, R. Dulbecco, H. N. Eisen, and H. S. Ginsberg (ed.), Microbiology, 3rd ed. Harper & Row, Publishers, Inc., New York.
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1990,
p. 146-147 0095-1137/90/010146-02$02.00/0 Copyright © 1990, American Society for Microbiology
Ghost Mycobacteria on Gram Stain SILVANA TRIFIRO,"2 ANNE-MARIE BOURGAULT,3 FRANÇOIS LEBEL,' AND PIERRE RENÉ2,4* Department of Microbiology, Montreal General Hospital,' Department of Medicine, McGill University,2 Service de Microbiologie et Maladies Infectieuses, Hôpital Saint-Luc, and Département de Microbiologie et Immunologie, Université de Montréal,3 Montreal, Quebec, and Department of Microbiology, Royal Victoria Hospital, Montreal Quebec H3A lAJ,4* Canada Received 22 June 1989/Accepted 11 September 1989
The Gram stain is a key tool in diagnostic microbiology. Its usefulness with respect to mycobacteria is undefined. The neutrality of mycobacteria other than Mycobacterium tuberculosis on Gram staining of various clinical specimens is described.
For more than a century, the Gram stain has provided a reliable, quick, and reproducible method for rapid identification of different microorganisms in clinical specimens. It is widely believed that the Gram stain can identify most bacteria, including mycobacteria. Its role in the detection of mycobacteria, however, is highly questionable, as acid-fast stains are normally used for that purpose. Recently, two groups have reported that specimens containing many mycobacteria resulted in a remarkable gram-neutral (neither positive nor negative) or gram ghost appearance of the mycobacteria (4, 6); this phenomenon was observed in a sputum containing Mycobacterium tuberculosis (4) and in the bone marrow of two patients infected with M. leprae (6). We report herein the finding of gram ghost (i.e., gramneutral) mycobacteria from six Gram-stained specimens. The mycobacteria isolated were later identified as M. haemophilum, M. scrofulaceum, and M. tuberculosis. A 56-year-old man diagnosed as having acquired immunodeficiency syndrome was admitted to a hospital with fever, progressive cachexia, and multiple skin lesions. He complained of a cough productive of yellow sputum but no hemoptysis. An erythematous fluctuant mass located on the right thigh was aspirated, yielding purulent fluid. A Gram stain of this fluid revealed an abundance of leukocytes but no bacteria. Numerous unstained bacillus footprints or ghost bacilli were noted (Fig. 1), A Ziehl-Neelsen stain of the same specimen is shown in Fig. 2. An auramine O-rhodamine stain was done before the Ziehl-Neelsen stain, and both confirmed the presence of acid-fast bacilli. Later, cultures of the pus yielded M. haemophilum. Treatment with various combinations of isoniazid, rifampin, ethambutol, and pyrazinamide was unsuccessful. Gram staining of premortem sputum specimens and autopsy specimens of lung and kidney tissues revealed neutral footprints of mycobacteria. Again, auramine O-rhodamine and Ziehl-Neelsen staining confirmed the presence of acidfast bacilli. M. scrofulaceum was later isolated from lung tissue.
*
Two other patients with cavitary lung lesions provided an abundance of ghost bacilli on a Gram stain. Later, M. tuberculosis was identified, thus confirming the previous observations of Hinson et al. (4). The Gram-staining properties of mycobacteria are little commented upon or even ignored in most microbiology textbooks. Some authors have stated that although mycobacteria are not readily stainable because of their relative resistance to aniline dyes, they are usually considered gram positive (3, 9-11). Kretschmer addressed the question of the Gram-staining property of Koch bacilli in 1934 (5). He showed that cold aniline with gentian violet caused staining of tubercle bacilli in a gram-positive manner, but he concluded that the tubercle bacillus did not possess true gram positivity, since iodine was not necessary as a mordant and normal, not anilinefortified, crystal violet led to no appreciable staining of sputum which revealed
FIG. 1. Pus containing M. haemophilum ghost bacilli (Gram stain; magnification, x 1,000).
Corresponding author. 146
VOL. 28, 1990
NOTES
e_ 1
r-il
3
e
147
and read by at least three observers. Regardless of the fixation technique, all observers reported the presence of ghost bacilli. Several mycobacterial species are considered important pathogens both in patients with acquired immunodeficiency syndrome and in other immunocompromised hosts. M. haemophilum causing skin infections is rare, but cases have been described in immunocompromised and normal hosts (1, 7), as well as in a patient with acquired immunodeficiency syndrome (8). For any clinical specimens found to be purulent in nature but lacking microorganisms on Gram staining, the microbiologic doctrine of staining for acid-fast bacilli holds true. However, observation of ghost bacilli on the initial Gram stain provides an early and helpful diagnostic clue before confirmation of mycobacteria is made by more specific stains. LITERATURE CITED 1. Feldman, R. A., and E. Hershfield. 1974. Mycobacterial skin infection by an unidentified species. A report of 29 cases. Ann. Intern. Med. 80:445-452. 2. Finegold, S. M., and W. J. Martin. 1982. Cultures, media, stains, reagents and tests, p. 655. In D. E. Ladig (ed.), Bailey and Scott's diagnostic microbiology, 6th ed. The C.V. Mosby
Co., St. Louis.
FIG. 2. Pus containing M. haemophilum (Ziehl-Neelsen stain; magnification, x 1,000).
bacilli. Hinson et al. described the gram neutrality of bacilli in a sputum specimen which eventually yielded M. tuberculosis on culture (4). In addition, Lawrence and Schreiber described M. leprae ghost footprints in histiocytes when the Fite stain was used on bone marrow aspirates (6). The entity of gram neutrality or ghost footprints of nontuberculous mycobacteria in clinical specimens has never been described. The preparation of slides was similar to the procedure described by Hinson et al. (4); i.e., they were heat fixed, but in addition, slides were fixed with methanol and Formalin. The slides were stained by the Hucker modification of the Gram stain technique (2). All slides were done in triplicate
3. Harris, H. W., and J. H. McClement. 1972. Tuberculosis, p. 351-378. In P. D. Hoeprich (ed.), Infectious diseases, lst ed. Harper & Row, Publishers, Inc., New York. 4. Hinson, J. M., R. W. Bradsher, and S. J. Bodner. 1981. Gram stain neutrality of Mycobacterium tuberculosis. Am. Rev. Respir. Dis. 123: 365-366. 5. Kretschmer, O. S. 1934. The Gram property of the acid-fast form of the tubercle bacillus. J. Lab. Clin. Med. 19:350-358. 6. Lawrence, C., and A. J. Schreiber. 1979. Leprosy footprints in bone marrow histiocytes. N. Engl. J. Med. 300:834-835. 7. Lomvardias, S., and G. E. Madge. 1972. Chaetoconidium and acid fast bacilli in skin ulcers. Arch. Dermatol. 106:875-876. 8. Males, B. M., T. E. West, and W. R. Bartholomew. 1987. Mycobacterium haemophilum infection in a patient with acquired immune deficiency syndrome. J. Clin. Microbiol. 25: 186-190. 9. Murohashi, T., and K. Toshida. 1965. Effect of ultraviolet irradiation on the acid-fastness and Gram positivity of mycobacteria. Am. Rev. Respir. Dis. 92:812-817. 10. Wayne, L. G., and G. P. Kubica. 1986. The mycobacteria, p. 1435-1457. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. 11. Wolinsky, E. 1980. Mycobacteria, p. 724-742. In D. Davis, R. Dulbecco, H. N. Eisen, and H. S. Ginsberg (ed.), Microbiology, 3rd ed. Harper & Row, Publishers, Inc., New York.
