Methods

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Mycobacterium tuberculosis was isolated on Lowenstein-Jensen slants and identified by the AccuProbe System (Gen-Probe, Inc., San Diego, California). Susceptibility testing was done by the New York City Department of Health Bureau of Laboratories.

For DNA fingerprinting, mycobacterial growth was washed from the Lowenstein-Jensen slants, and the cells were lysed by beating with siliconized zirconium beads and chloroform in a Mickle apparatus (H. Mickle, Gromshall, Surrey, United Kingdom). Mixed-linker PCR was done as previously described [8] at the Centers for Disease Control and Prevention.

Results

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A review of microbiology laboratory data from December 1990 to November 1992 showed 250 patients with tuberculosis identified by our institution. Three patients with RISE-resistant tuberculosis had paired isolates with markedly discordant susceptibility profiles, and DNA fingerprinting of the original isolates by mixed-linker PCR showed unique patterns that differed from the common pattern of the nosocomial superinfecting RISE-resistant M. tuberculosis isolates (Figure 1). The three patients are described below.

View larger version (158K): [in this window] [in a new window]   Figure 1. Mixed-linker fingerprints of Mycobacterium tuberculosis isolates. Polymerase chain reaction products were separated by gel electrophoresis on an 8% polyacrylamide gel and visualized under ultraviolet light by staining with 0.5 µg/mL ethidium bromide. Lanes 1 to 3 are original patient isolates and corresponding isolate after suprainfection with the RISE-resistant strain; lane C indicates negative controls for sample processing and PCR; lane M shows 100-base pair DNA ladder-size marker (BRL, Life Technologies, Inc., Gaithersburg, Maryland). RISE = rifampin-isoniazid-streptomycin-ethambutol.

Case 1

A 32-year-old man with human immunodeficiency virus (HIV) infection presented in August 1991 with Staphylococcus aureus endocarditis. He received 4 weeks of therapy in an AIDS inpatient ward, which was the epicenter of an outbreak of RISE-resistant tuberculosis [9].

The patient was discharged but then recalled when a culture of bronchoalveolar lavage fluid showed M. tuberculosis. He received isoniazid, rifampin, and pyrazinamide for drug-susceptible tuberculosis.

In July 1993, the patient was readmitted because of weight loss, fever, and cough. The CD4 lymphocyte count was 91 cells/µL. A sputum culture subsequently grew M. tuberculosis resistant to RISE, pyrazinamide, kanamycin, and ethionamide.

Case 2

A 28-year-old woman with HIV infection was prescribed isoniazid in June 1991 because of a positive tuberculin skin-test result. Two months later, she was hospitalized with fever, headache, and cough. The CD4 count was 172 cells/µL. Examination of cerebrospinal fluid showed lymphocytic meningitis, and first-line antituberculous agents were administered.

She was readmitted in December 1991 because of a generalized tonic-clonic seizure. Sputum and cerebrospinal fluid cultures grew M. tuberculosis that was resistant to isoniazid and susceptible to rifampin, pyrazinamide, ethambutol, and streptomycin. The patient improved and was discharged in March 1992. During her hospitalizations, she was exposed to RISE-resistant tuberculosis.

In December 1992, the patient was readmitted; she died 4 weeks later. Mycobacterium tuberculosis subsequently grew in culture and was resistant to RISE and pyrazinamide.

Case 3

A 28-year-old man with HIV infection had a positive tuberculin skin-test result in 1987. He presented in November 1991 with fever, productive cough, and a CD4 lymphocyte count of 3 cells/µL. Bronchoscopy showed Pneumocystis carinii. Sputum cultures grew drug-susceptible M. tuberculosis. Isoniazid, rifampin, pyrazinamide, and ethambutol were administered.

The patient was readmitted in March 1992. During his clinic visits and hospitalizations, the patient was exposed to many patients with RISE-resistant tuberculosis. Mycobacterium avium complex was identified in the sputum, and ciprofloxacin and amikacin were added to his drug regimen. He died 4 months later. A sputum culture subsequently grew M. tuberculosis that was resistant to RISE, ethionamide, and kanamycin and was susceptible to capreomycin, cycloserine, and ciprofloxacin.

Discussion

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Superinfection with RISE-resistant tuberculosis occurred in these three patients with AIDS despite therapy for concurrent drug-susceptible tuberculosis. Because the initial isolates of M. tuberculosis were no longer viable on subculture, standard DNA fingerprinting was not possible. The use of a new technique, DNA fingerprint analysis by mixed-linker PCR, facilitated the rapid recognition of the outbreak strain and enabled us to unambiguously differentiate the original isolates. Mixed-linker PCR is a modified single-site PCR technique that specifically amplifies HhaI restriction fragments containing one end of IS6110 and the adjacent flanking segment. The resulting fingerprint pattern is compatible with that obtained by the standard restriction fragment-length polymorphism technique, consisting of restriction with PvuII, Southern blotting, and hybridization with an IS6110-specific probe [10].

The waning protective immunity associated with AIDS may facilitate superinfection or exogenous reinfection in patients previously effectively treated or receiving therapy for tuberculosis. Our findings strongly argue against placing more than one patient with tuberculosis in one room. Population-based studies are required to quantify the incidence of exogenous reinfection or superinfection to determine if it poses a substantial public health threat. Mixed-linker PCR should prove to be a powerful tool in doing the necessary epidemiologic studies.