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Disseminated nocardiosis in a nonimmunocompromised host
5. If the specimen appears to be mucoid, add 10 drops of 10% KOH to the sediment and vortex until homogenous. One drop of 10% KOH can be added to the sediment on the slide and allowed to react for 1 minute before the slide is placed on the heating block (see step 7). 6. If 10% KOH is added to the sediment in the tube, repeat steps 2 through 4. 7. Place slides on a 70°C heating block for 10 minutes. 8. Proceed to staining.
Modified Acid-Fast (1) 1. Place slide on staining rack, and flood slide with carbolfuchsin. 2. Gently heat slide to steaming with Bunsen burner flame (DO NOT BOIL). 3. Allow stain to react for 5 minutes. If slide dries, add more stain without additional heatingl 4. Rinse slide with tap or distilled water. 5. Decolorize with 5% H2SO 4 solution for 30 seconds (thick smears may require a longer time, but it is important not to over-decolorize). 6. Rinse with tap or distilled water. Drain. 7. Flood slide with methylene blue counterstain, allow to react for 1 minute. 8. Rinse with tap or distilled water, drain, and air dry.
Ziehl-Neelsen (3) 1. Place slide on staining rack, and flood the slide with carbolfuchsin. 2. Gently heat slide to steaming with Bunsen burner flame (DO NOT BOIL).
3. Allow stain to react for 20 to 30 minutes. If slide dries, add more stain without additional heating. 4. Rinse slides with tap or distilled water. 5. Decolorize with acid-alcohol from 30 seconds to 1 minute (thick smears may require a longer time, but it is important not to over-decolorize). 6. Rinse with tap or distilled water. Drain. 7. Flood slide with methylene blue counterstain, allow to react for 1 minute. 8. Rinse with tap or distilled water, drain, and air dry. Note: The destain times indicated are optimal for the type of fecal specimen received. The color of the organisms ranges from deep red to pink, depending on the destain time; however, they are easily seen. Internal morphology can be seen using both the Giemsa and acid-fast stains. When only a few organisms are in the fecal material, use of the acid-fast technique may permit detection of the organism. Use of both techniques is recommended for optimal recovery and organism identification.
B. Preparation of Stool Specimen for Cryptosporidium Examination from Polyvinyl Alcohol Preserved Material To detect Cryptosporidium in polyvinyl alcohol (PVA) preserved stools, using acid-fast and/or Giemsa stains, specimens are processed as follows: I. Place I ml of PVA/stool mixture in a centrifuge tube. 2. Add 10 ml of D'Antoni's iodine (strong tea color).
3. Vortex and allow to stand for 5 minutes. 4. Centrifuge 2 minutes at 300 × g (tabletop centrifuge is adequate). 5. Decant supernatant fluid. 6. Resuspend stool material in 10 ml of 70% alcohol. 7. Vortex, and allow to stand 5 minutes. 8. Decant supernatant fluid. 9. Continue with Part A, step 5. In our experience smears prepared in this manner were indistinguishable from those prepared directly from stool preserved in 10% formalin. In order to shorten the procedure time, formalin preservation is recommended over PVA preservation. Of the many techniques available for detecting Cryptosporidium in stool samples, the combination of Giemsa and acid-fast permanent stained smears provides excellent results. Because of the increased importance of diagnosing cryptosporidiosis in immunosuppressed patients, laboratories need to provide this service.
References 1. Bailey, W. R. and E. G. Scott. 1966. Diagnostic microbiology, 2nd. ed. C.V. Mosby Co, St. Louis, Missouri. 2. Garcia, L. S. et al. 1982. Clinical laboratory diagnosis of Cryptosporidium from human fecal specimens. Clin. Microbiol. Newsletter 4: 136-137. 3. Vestal, A. L. 1975. Procedures for the isolation and identification of mycobacteria, U.S. Dept. Health, Education, and Welfare, (CDC) 77-8230, Atlanta, Georgia.
i
Case Report I
Disseminated Nocardiosis in a N o n i m m u n o c o m p r o m i s e d Host Lauren C. Roberts N4adeline Ducate, MS, MPH University of Illinois Chicago, Illhzois
Malcolm Dean, M.D. Infectious Disease Specialist West Lake Hospital Melrose Park, Illinois
A 35-year-old airline mechanic experienced the onset of fever, arthralgias, and malaise in March 1980. His symptoms improved over a two- to three-week period, but at the end of September of that year he noticed de-
creased appetite and weight loss. He became febrile and was admitted to a hospital in October. He had a right middle lobe pneumonia, and physical examination revealed a clubbing of his fingers. His temperature was 99 ° to 100°F; his blood-chemistry values, hematology studies, and routine urinalysis were normal. A bronchoscopy yielded rt~gative cytology and cultures. On admission he was treated with ampicillin and was discharged on erythromycin, which he took until November of 1980. In March 1981, the patient's symptoms recurred, with swelling of his ankles and feet. In June 1981, he noticed a left-sided neck mass and was again admitted to the hospital for evaluation. His symptoms included fever, malaise, and arthralgias of his knees and elbows. His clinical, occupational, and travel history were unremarkable, except that he smoked approximately two packs of cigarettes per day. The neck abscess was incised and drained on July 14, 1981. Four days later, he had a major motor seizure and was placed on Dilantin and phenobarbitol. CT and brain scans showed a defect with ring enhancement in the right parietal area. His chest x-ray showed a persistent right middle lobe infiltrate with widening of the upper mediastinum. The neck abscess drainage obtained at surgery was cultured for routine bacterial pathogens, mycobacteria, and fungi. Direct smears were negative. After three days of incubation, the routine bacterial culture had no growth. Approximately eight to ten days after inoculation, small pinpoint colonies, which were thought to be mycobacteria, grew on the Loewenstein-Jensen medium. The organisms were catalase positive, and an acid-fast stain revealed branching filaments that slightly retained carbolfuchsin. On Gram stain, the organism was found to be a branching, gram-positive bacillus.
Eventually, the organism grew on Sabouraud agar and with age, became yellow-orange. Casein, xanthine, tyrosine, and gelatin tests were negative after two weeks, indicating that the organism was Nocardia asteroides. Identification was confirmed by the Centers for Disease Control. This nonimmunocompromised patient was diagnosed as having disseminated nocardiosis with pulmonary, central nervous system, and cutaneous involvement and was placed on trimethoprim-sulfamethoxazole (TMP/ SXT) therapy for a total of five weeks. Later, because of a decreasing white blood celt and platelet count, he was placed on minocycline, and the TMP/ SXT was stopped. He was discharged from the hospital in August and returned to work in October 1981. Nocardiosis is a localized or disseminated infection caused by an aerobic gram-positive actinomycete. In 1888, Nocard first isolated this organism from cattle on the island of Guadeloupe (1). Nocardia occur worldwide, primarily as saprophytes in the soil. Human infections occur primarily through inhalation, but skin inoculation can also produce infection (1). The pulmonary process in humans may be subclinical, acute, or chronic. Dissemination from the lung may involve any organ or tissue but particularly the nervous system and soft tissues (3). Nocardiosis commonly occurs as an opportunistic infection in patients with diminished host defenses; however, there are many reported cases in patients without other discernible diseases.
In the United States, N. asteroidcs is the predominant Nocardia species isolated from humans (1-3). Although a chronic suppurative primary cutaneous infection can be produced by this species, the lung is the primary site in over 80% of infections (2). Microscopic examination of patient specimens reveals gram-positive branching.
filaments with a tendency to-fragment into bacillary and coccoid forms. The organisms are partially acid-fast if stained smears are decolorized with 1% sulfuric acid (modified Kinyoun method). Nocardia can be isolated on Sabouraud agar, blood agar, and media used for mycobacteria culture. Growth may occur within 72 hours, but may take up to 2 weeks. The optimum temperature is 37°C, and growth is enhanced by 5 to 10% CO2. Colony characteristics vary--colonies may be glabrous and waxy, pigmented, raised or heaped, or densely mycelial and moldlike. The clinical manifestations of nocardiosis include anorexia, weight loss, cough with production of thick green sputum, and dyspnea (3). Treatment is with sulfonamides. Duration of therapy is uncertain; however, treatment must -be continued for many months in order to prevent relapse (1, 3). In certain cases, surgical drainage of abscesses is necessary. The case of nocardiosis reported here was an interesting challenge for the laboratory because Nocardia is not often encountered. For this reason, a reference laboratory should be used to confirm the identification of all suspected isolates. Because of good communication between the laboratory and physician, treatment was initiated as soon as growth appeared.
References
I. Hoeprieh, P. D. 1977. Nocardiosis, pp. 356-363. h~ P. D. Hoeprich (ed.), Infectious diseases. Harper and Row, Hagerstown, Maryland. 2. Kahn, F. W. et al. 1981. Primary cutaneous Nocardia asteroides infection with dissemination. Am. J. Med. 70:859-863. 3. Lerner, P. I. 1979. Nocardia species, pp. 1962-1968. hz G. L. Mandell, R. G. Douglas, and J. E. Bennett (eds.), Principles and practice of infectious diseases, Vol. II. John Wiley and Sons, New York.
Modified Acid-Fast (1) 1. Place slide on staining rack, and flood slide with carbolfuchsin. 2. Gently heat slide to steaming with Bunsen burner flame (DO NOT BOIL). 3. Allow stain to react for 5 minutes. If slide dries, add more stain without additional heatingl 4. Rinse slide with tap or distilled water. 5. Decolorize with 5% H2SO 4 solution for 30 seconds (thick smears may require a longer time, but it is important not to over-decolorize). 6. Rinse with tap or distilled water. Drain. 7. Flood slide with methylene blue counterstain, allow to react for 1 minute. 8. Rinse with tap or distilled water, drain, and air dry.
Ziehl-Neelsen (3) 1. Place slide on staining rack, and flood the slide with carbolfuchsin. 2. Gently heat slide to steaming with Bunsen burner flame (DO NOT BOIL).
3. Allow stain to react for 20 to 30 minutes. If slide dries, add more stain without additional heating. 4. Rinse slides with tap or distilled water. 5. Decolorize with acid-alcohol from 30 seconds to 1 minute (thick smears may require a longer time, but it is important not to over-decolorize). 6. Rinse with tap or distilled water. Drain. 7. Flood slide with methylene blue counterstain, allow to react for 1 minute. 8. Rinse with tap or distilled water, drain, and air dry. Note: The destain times indicated are optimal for the type of fecal specimen received. The color of the organisms ranges from deep red to pink, depending on the destain time; however, they are easily seen. Internal morphology can be seen using both the Giemsa and acid-fast stains. When only a few organisms are in the fecal material, use of the acid-fast technique may permit detection of the organism. Use of both techniques is recommended for optimal recovery and organism identification.
B. Preparation of Stool Specimen for Cryptosporidium Examination from Polyvinyl Alcohol Preserved Material To detect Cryptosporidium in polyvinyl alcohol (PVA) preserved stools, using acid-fast and/or Giemsa stains, specimens are processed as follows: I. Place I ml of PVA/stool mixture in a centrifuge tube. 2. Add 10 ml of D'Antoni's iodine (strong tea color).
3. Vortex and allow to stand for 5 minutes. 4. Centrifuge 2 minutes at 300 × g (tabletop centrifuge is adequate). 5. Decant supernatant fluid. 6. Resuspend stool material in 10 ml of 70% alcohol. 7. Vortex, and allow to stand 5 minutes. 8. Decant supernatant fluid. 9. Continue with Part A, step 5. In our experience smears prepared in this manner were indistinguishable from those prepared directly from stool preserved in 10% formalin. In order to shorten the procedure time, formalin preservation is recommended over PVA preservation. Of the many techniques available for detecting Cryptosporidium in stool samples, the combination of Giemsa and acid-fast permanent stained smears provides excellent results. Because of the increased importance of diagnosing cryptosporidiosis in immunosuppressed patients, laboratories need to provide this service.
References 1. Bailey, W. R. and E. G. Scott. 1966. Diagnostic microbiology, 2nd. ed. C.V. Mosby Co, St. Louis, Missouri. 2. Garcia, L. S. et al. 1982. Clinical laboratory diagnosis of Cryptosporidium from human fecal specimens. Clin. Microbiol. Newsletter 4: 136-137. 3. Vestal, A. L. 1975. Procedures for the isolation and identification of mycobacteria, U.S. Dept. Health, Education, and Welfare, (CDC) 77-8230, Atlanta, Georgia.
i
Case Report I
Disseminated Nocardiosis in a N o n i m m u n o c o m p r o m i s e d Host Lauren C. Roberts N4adeline Ducate, MS, MPH University of Illinois Chicago, Illhzois
Malcolm Dean, M.D. Infectious Disease Specialist West Lake Hospital Melrose Park, Illinois
A 35-year-old airline mechanic experienced the onset of fever, arthralgias, and malaise in March 1980. His symptoms improved over a two- to three-week period, but at the end of September of that year he noticed de-
creased appetite and weight loss. He became febrile and was admitted to a hospital in October. He had a right middle lobe pneumonia, and physical examination revealed a clubbing of his fingers. His temperature was 99 ° to 100°F; his blood-chemistry values, hematology studies, and routine urinalysis were normal. A bronchoscopy yielded rt~gative cytology and cultures. On admission he was treated with ampicillin and was discharged on erythromycin, which he took until November of 1980. In March 1981, the patient's symptoms recurred, with swelling of his ankles and feet. In June 1981, he noticed a left-sided neck mass and was again admitted to the hospital for evaluation. His symptoms included fever, malaise, and arthralgias of his knees and elbows. His clinical, occupational, and travel history were unremarkable, except that he smoked approximately two packs of cigarettes per day. The neck abscess was incised and drained on July 14, 1981. Four days later, he had a major motor seizure and was placed on Dilantin and phenobarbitol. CT and brain scans showed a defect with ring enhancement in the right parietal area. His chest x-ray showed a persistent right middle lobe infiltrate with widening of the upper mediastinum. The neck abscess drainage obtained at surgery was cultured for routine bacterial pathogens, mycobacteria, and fungi. Direct smears were negative. After three days of incubation, the routine bacterial culture had no growth. Approximately eight to ten days after inoculation, small pinpoint colonies, which were thought to be mycobacteria, grew on the Loewenstein-Jensen medium. The organisms were catalase positive, and an acid-fast stain revealed branching filaments that slightly retained carbolfuchsin. On Gram stain, the organism was found to be a branching, gram-positive bacillus.
Eventually, the organism grew on Sabouraud agar and with age, became yellow-orange. Casein, xanthine, tyrosine, and gelatin tests were negative after two weeks, indicating that the organism was Nocardia asteroides. Identification was confirmed by the Centers for Disease Control. This nonimmunocompromised patient was diagnosed as having disseminated nocardiosis with pulmonary, central nervous system, and cutaneous involvement and was placed on trimethoprim-sulfamethoxazole (TMP/ SXT) therapy for a total of five weeks. Later, because of a decreasing white blood celt and platelet count, he was placed on minocycline, and the TMP/ SXT was stopped. He was discharged from the hospital in August and returned to work in October 1981. Nocardiosis is a localized or disseminated infection caused by an aerobic gram-positive actinomycete. In 1888, Nocard first isolated this organism from cattle on the island of Guadeloupe (1). Nocardia occur worldwide, primarily as saprophytes in the soil. Human infections occur primarily through inhalation, but skin inoculation can also produce infection (1). The pulmonary process in humans may be subclinical, acute, or chronic. Dissemination from the lung may involve any organ or tissue but particularly the nervous system and soft tissues (3). Nocardiosis commonly occurs as an opportunistic infection in patients with diminished host defenses; however, there are many reported cases in patients without other discernible diseases.
In the United States, N. asteroidcs is the predominant Nocardia species isolated from humans (1-3). Although a chronic suppurative primary cutaneous infection can be produced by this species, the lung is the primary site in over 80% of infections (2). Microscopic examination of patient specimens reveals gram-positive branching.
filaments with a tendency to-fragment into bacillary and coccoid forms. The organisms are partially acid-fast if stained smears are decolorized with 1% sulfuric acid (modified Kinyoun method). Nocardia can be isolated on Sabouraud agar, blood agar, and media used for mycobacteria culture. Growth may occur within 72 hours, but may take up to 2 weeks. The optimum temperature is 37°C, and growth is enhanced by 5 to 10% CO2. Colony characteristics vary--colonies may be glabrous and waxy, pigmented, raised or heaped, or densely mycelial and moldlike. The clinical manifestations of nocardiosis include anorexia, weight loss, cough with production of thick green sputum, and dyspnea (3). Treatment is with sulfonamides. Duration of therapy is uncertain; however, treatment must -be continued for many months in order to prevent relapse (1, 3). In certain cases, surgical drainage of abscesses is necessary. The case of nocardiosis reported here was an interesting challenge for the laboratory because Nocardia is not often encountered. For this reason, a reference laboratory should be used to confirm the identification of all suspected isolates. Because of good communication between the laboratory and physician, treatment was initiated as soon as growth appeared.
References
I. Hoeprieh, P. D. 1977. Nocardiosis, pp. 356-363. h~ P. D. Hoeprich (ed.), Infectious diseases. Harper and Row, Hagerstown, Maryland. 2. Kahn, F. W. et al. 1981. Primary cutaneous Nocardia asteroides infection with dissemination. Am. J. Med. 70:859-863. 3. Lerner, P. I. 1979. Nocardia species, pp. 1962-1968. hz G. L. Mandell, R. G. Douglas, and J. E. Bennett (eds.), Principles and practice of infectious diseases, Vol. II. John Wiley and Sons, New York.