Misidentification and Diagnostic Delay Caused by a False-Positive Amplified Mycobacterium tuberculosis Direct Test in an Immunocompetent Patient with a Mycobacterium celatum Infection

Abstract

The Gen-Probe amplified Mycobacterium tuberculosis direct test can give discrepant results directly in respiratory or cultured samples from patients infected with Mycobacterium celatum, leading to inappropriate therapy for, in our case, an immunocompetent patient.

Mycobacterium celatum was first described in 1993 (3), and since then, sporadic reports have been published on the isolation of this mycobacterium from immunocompromised patients (1, 5, 6, 8, 11, 12; R. A. Bonomo, J. M. Briggs, W. Gross, M. Hassan, R. C. Graham, W. R. Butler, and R. A. Salata, Letter, Clin. Infect. Dis. 26:243–245, 1998; C. Piersimoni, E. Tortoli, and G. De Sio, Letter, Lancet 344:332, 1994), a child with lymphadenitis (G. Haase, H. Skopnik, S. Bätge, and E. C. Böttger, Letter, Lancet 344:1020–1021, 1994), and a fatal pulmonary infection in an apparently healthy adult (4). Biochemically, the organism is indistinguishable from the Mycobacterium avium complex, and mycolic acid high-pressure liquid chromatography analysis or genetic analysis is required for proper identification (3). For identification, cross-reactivity with nontuberculous mycobacteria has been described with the AccuProbe culture confirmation test (Gen-Probe, San Diego, Calif.) for the M. tuberculosis complex (2, 10). This note describes the initial misidentification and interpretation of a positive amplified M. tuberculosis direct (AMTD) test (Gen-Probe) result for a respiratory sample derived from an immunocompetent patient with an M. celatum infection.

A 61-year-old man was referred on 5 March 1999 by a general practitioner to the outpatient respiratory disease clinic because of general malaise, a productive cough, and an unexplained 16-kg weight loss in the past 3 years. The chest radiograph showed a pulmonary infiltrate in the right upper lobe, with extensive cavities identified by computed tomography. A bronchial lavage was performed and showed on average more than one acid-fast rod per high-power field (magnification, ×1,000. The AMTD test was positive with a value of 1,000,000 relative light units (RLU). Tuberculostatic drug therapy was started with 300 mg of isoniazid, 600 mg of rifampin, and 2,000 mg of pyrazinamide. Liquid culture (MB/BacT; Organon Teknika, Boxtel, The Netherlands) was positive on 25 March 1999 and was subcultured on Löwenstein Jensen agar. The subculture grew very small smooth colonies with a pale yellow pigment and was sent to the Mycobacterium section of the National Institute of Public Health and the Environment (RIVM, Bilthoven, The Netherlands) for drug susceptibility testing by the agar proportion method and species identification. The AccuProbe culture confirmation tests for M. tuberculosis, M. avium complex, Mycobacterium kansasii, and Mycobacterium gordonae were negative. Because of the discrepancy between the AMTD test as performed by the Medical Microbiology department at the University Hospital Maastricht and the AccuProbe results of the RIVM, a culture was sent again to the RIVM. This material again yielded a positive result (509,094 RLU) in the AMTD test in Maastricht and again yielded a negative result in the AccuProbe test at the RIVM. Therefore, it was concluded that the AMTD result was probably a false positive. Drug susceptibility data were only obtained on 26 June 1999 (Table 1) because of the slow growth and the initial suspicion that we were dealing with a multiple-strain isolate. Sequence analysis was complicated because of the possible identification of two different 16S ribosomal DNAs (rDNAs), both of which were from nontuberculous mycobacteria. Finally, identification of M. celatum was only available on 2 January 2000. The two strains subcultured by the RIVM were sent to our laboratory and yielded a positive result in the AMTD test (1,709,951 and 2,045,959 RLU, respectively). The original therapy was stopped on 30 June 2000 because of side effects. Therapy for nontuberculous mycobacteria was not started because the patient did not feel ill anymore and a new bronchial aspirate showed a reduction of the number of acid-fast microorganisms. In addition, eradication of nontuberculous mycobacteria from lungs with extensive damage is extremely difficult, and after a period of treatment it is difficult to differentiate between colonization and active infection. In the first few months, no progression occurred; thereafter, the infiltrate in the right thorax progressed and a new left-side infiltrate developed. After therapy with ethambutol, clarithromycin, and rifabutin was initiated, the left-side infiltrate disappeared and progression on the right side stopped. Despite clinical improvement, sputum was still positive 1 year after initiation of therapy.

TABLE 1.

Drug susceptibility data, obtained by the agar proportion method, for the M. celatum isolate

Drug	MIC (mg/liter)	Interpretation
Isoniazid	1	Intermediate
Rifampin	>5	Resistant
Ethambutol	5	Susceptible
Streptomycin	≤1	Susceptible
Pyrazinamide	>100	Resistant
Amikacin	5	Susceptible
Cycloserine	10	Susceptible
Protionamide	≤1	Susceptible
Ciprofloxacin	4	Resistant
Clofazimine	≤0.5	Susceptible
Clarithromycin	≤0.2	Susceptible
Rifabutin	2	Susceptible

M. celatum is an uncommon Mycobacterium species, especially in immunocompetent patients. The misidentification by the AccuProbe has been investigated in two studies by testing cultured M. celatum type 1 strains. This is due to the similarity of the 16S rDNAs of the two species in the probe region (2). Our report shows that the AMTD test from the same company can also have problems when it is used directly on clinical specimens or cultured material containing M. celatum. Although it has not been published, this problem is acknowledged by the company and the package leaflet states that cross-reactions can occur if more than 30 CFU of M. celatum or Mycobacterium terrae-like organisms are present in clinical material. Similar problems in testing samples from patients with pulmonary M. kansasii and M. avium infections have been described (7). The latter study was performed with the first-generation test, and we used the second-generation test. In the second-generation test, the selection procedure, during which nonhybridized acridine ester probe is hydrolyzed, is extended from 10 to 15 min at 60°C. This would prevent cross-reaction of the probe with amplified 16S rRNA of M. kansasii; the problems with M. avium are not acknowledged by the company.

The misidentification led in our case to the initiation of inappropriate therapy. The difficulty of making an identification by 16S rDNA sequencing can be explained by the existence of two different copies of the gene in M. celatum strains (9). The results obtained at the RIVM also suggested the existence of two different copies, but they were misinterpreted as being derived from different isolates which could not be timely separated by subculture.

The early appearance of yellow-pigmented colonies and the susceptibility data are more in agreement with those of Piersimoni et al. (8) than with the original description (3). The complete resistance to rifampin and the full susceptibility to rifabutin are typical. However, the resistance to ciprofloxacin is unusual (1, 3, 6, 8, 11).

In summary, the AMTD test can misidentify M. celatum as M. tuberculosis. This can lead to delay of appropriate treatment and even misidentification of the isolate as a multiresistant M. tuberculosis isolate. However, it probably remains a minor problem of the AMTD test because of the low rate of M. celatum isolation (2). One should think of M. celatum if a positive AMTD test is followed by culture of an isolate with a nontuberculous appearance and with resistance data more indicative of nontuberculous mycobacteria. Another clue may be the problems encountered in 16S rDNA sequencing due to the existence of two different copies of the gene.