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Journal of Clinical Microbiology, April 2003, p. 1783-1784, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1783-1784.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Use of Equivocal Zone in Interpretation of Results of the Amplified Mycobacterium Tuberculosis Direct Test for Diagnosis of Tuberculosis

A. Kerleguer,1* J.-L. Koeck,1 M. Fabre,2 P. Gérôme,3 R. Teyssou,1 and V. Hervé2

HIA Val de GrÂce, 75005 Paris,1 HIA Percy, 92140 Clamart,2 HIA Desgenettes, 69003 Lyon, France3

Received 8 October 2002/ Returned for modification 17 November 2002/ Accepted 7 January 2003


   ABSTRACT
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The sensitivity and specificity of the Amplified Mycobacterium Tuberculosis Direct (AMTD) test, evaluated with 1,363 respiratory samples (128 from tuberculous patients), were 92.97 and 98.7%, respectively. When an equivocal zone (30,000 to 1,000,000 relative light units [RLU]) was used instead of a 30,000-RLU cutoff, the sensitivity and specificity of the AMTD test were 92.97 and 100%, respectively.


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The Gen-Probe Amplified Mycobacterium Tuberculosis Direct (AMTD) test (Gen-Probe Incorporated) uses transcription-mediated amplification to isothermally amplify target rRNA via DNA intermediates, followed by chemiluminescent detection with acridinium ester-labeled DNA probes. The AMTD test has been shown to be a sensitive and specific method for detecting Mycobacterium tuberculosis in respiratory specimens (2, 4). In the European package insert, the cutoff value is set at 30,000 relative light units (RLU). We studied the use of an equivocal zone of 30,000 to 1,000,000 RLU in which the sample must be retested (1).

From May 1998 to September 2001, 1,363 respiratory samples were collected from 683 patients from France and Africa. The specimens were decontaminated by using the N-acetyl-L-cysteine-sodium hydroxide procedure. Each sample was separated into two equal parts. The first one was used for microscopy and culture, and the second one was used for the AMTD test, inhibition, and storage. Equal aliquots of the processed sediment were inoculated onto two solid slants (Löwenstein-Jensen and Coletsos) and onto a Mycobacterial Growth Indicator Tube (Becton Dickinson) (5). Isolates of mycobacteria were identified by DNA probes (AccuProbe; Gen-Probe Incorporated) for the M. tuberculosis complex (MTBC) strains and the most usual nontuberculous mycobacterial (NTM) species strains and by conventional biochemical tests.

AMTD test. The cutoff value for a positive AMTD test result was set at 30,000 RLU. Each run included a negative amplification control (APC) and a positive APC. The APC was prepared from a 10-4 to 10-5 dilution of a 1 McFarland suspension of M. tuberculosis. The run was validated when the negative cell control value was <20,000 RLU and the APC was >=500,000 RLU. Specimens that were smear positive or that grew MTBC strains but were AMTD test negative were analyzed for the presence of inhibitors of the amplification reaction by addition of an aliquot of the APC to the sample. A value of >500,000 RLU was considered negative for inhibitory substances, and a value of <500,000 RLU was considered to show the presence of inhibitors. In this case, a 1:5 dilution was used to remove the inhibitors and the sample was tested afterward. Between 30,000 and 1,000,000 RLU, the sample was retested and considered to be negative for a value of <30,000 RLU, positive for a value of >=1,000,000 RLU, and undetermined for a value of >=30,000 RLU and <1,000,000 RLU. In case of a discrepancy between culture and AMTD test results, the patient's clinical, biological, and radiological data were considered to be the "gold standard" for the diagnosis of tuberculosis.

We collected 1,363 samples (660 bronchial aspirate, 513 bronchoalveolar lavage fluid, 108 gastric aspirate, and 82 sputum samples) from 683 patients from France (n = 660) and Djibouti (n = 23). Out of the 1,363 samples, 73 were smear positive, 102 (7.5%) grew MTBC strains (M. tuberculosis, 98%; M. canetti, 2%), and 128 other strains (9.4%) grew NTM strains. Out of the 73 smear-positive samples, 7 (9.6%) were positive for the presence of inhibitory substances. After 1:5 dilution, three samples in which inhibitors persisted were excluded from the study. According to the defined gold standard, 128 samples came from tuberculous patients; out of these samples, 119 were AMTD test positive. The nine false-negative results could not be related to the presence of amplification inhibitors. Therefore, the sensitivity was 92.97%. Out of the 1,232 samples from nontuberculous patients, 1,216 were AMTD test negative. None of the 16 false-positive samples grew NTM strains (3). The corresponding specificity was 98.7%. Table 1 shows the classification of culture, gold standard, and repeat AMTD test results according to the RLU range of the first AMTD test results. With the use of an equivocal zone between 30,000 and 1,000,000 RLU, the sensitivity and specificity were 92.97 and 100%, respectively (Table 2).


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  		TABLE 1. Classification of culture, gold standard, and repeat AMTD test results according to the RLU range of the first AMTD test results

 

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  		TABLE 2. Statistical evaluation of AMTD test used with and without an equivocal zone

 
Conclusion. This study shows that the use of an equivocal zone of 30,000 to 1,000,000 RLU improves the specificity of the AMTD test without diminishing its sensitivity.


   FOOTNOTES
 
* Corresponding author. Mailing address: HIA Val de GrÂce, 74 Blvd. Port Royal, 75005 Paris, France. Phone: 33 1 40 51 46 33. Fax: 33 1 40 51 42 98. E-mail: alexandrakerleguer{at}net-up.com. Back


   REFERENCES
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  1. Alcala, L., M. J. Ruiz-Serrano, S. Hernangomez, M. Marin, D. G. de Viedma, R. San Juan, and E. Bouza. 2001. Evaluation of the upgraded amplified Mycobacterium Tuberculosis Direct test (Gen-Probe) for direct detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens. Diagn. Microbiol. Infect. Dis. 41(1-2):51-56.[CrossRef][Medline]
  2. Jonas, V., M. J. Alden, J. I. Curry, K. Kasimango, C. A. Knott, R. Lankford, J. M. Wolfe, and D. F. Moore. 1993. Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA. J. Clin. Microbiol. 31:2410-2416.[Abstract/Free Full Text]
  3. Jorgensen, J. H., J. R. Salinas, R. Paxson, K. Magnon, J. E. Patterson, and T. F. Patterson. 1999. False-positive Gen-Probe Direct Mycobacterium tuberculosis amplification test results for patients with pulmonary M. kansasii and M. avium infections. J. Clin. Microbiol. 37:175-178.[Abstract/Free Full Text]
  4. Pfyffer G. E., P. Kissling, R. Wirth, and R. Weber. 1994. Direct detection of Mycobacterium tuberculosis complex in respiratory specimens by a target-amplified test system. J. Clin. Microbiol. 32:918-923.[Abstract/Free Full Text]
  5. Tortoli, E., P. Cichero, C. Piersimoni, M. T. Simonetti, G. Gesu, and D. Nista. 1999. Use of BACTEC MGIT 960 for recovery of mycobacteria from clinical specimens: multicenter study. J. Clin. Microbiol. 37:3578-3582.[Abstract/Free Full Text]

Journal of Clinical Microbiology, April 2003, p. 1783-1784, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1783-1784.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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