Direct identification of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare from amplified primary cultures in BACTEC media using DNA probes

Abstract

San Diego, Calif.) directed at the Mycobacterium tuberculosis complex and Mycobacterium avium-M. intracellulare complex were used to identify acid-fast bacilli directly from specimens grown in BACTEC 12B bottles (Becton Dickinson and Co., Towson, Md.). Clinical specimens were inoculated directly or after decontamination into a BACTEC 12B bottle, Middlebrook 7H11 agar, and Lowenstein-Jensen medium. Conventional media were incubated at 37°C in 5% C02 and examined weekly for 6 weeks. Identification of isolates grown on conventional media by standard biochemicals, morphology, and growth characteristics served as the reference method for identification. BACTEC bottles were incubated at 37°C, and a growth index was taken twice a week. When a growth index of .-100 was reached, 1 ml of BACTEC 12B medium was put into each of three microfuge tubes which were centrifuged for 15 min at 15,000 X g. Pellets were used in hybridization reactions with an M. tuberculosis complex probe, an M. avium probe, and an M. intracellulare probe. The results of the hybridizations of the three probes with the same sample were compared, and the highest percent hybridization was divided by the average of the two lower hybridization values. If this value, the derived patient ratio (DPR), was-3, then the specimen was considered positive for the organism giving the highest percent hybridization. Of the 1,988 specimens cultured, the results of conventional tests for the 190 conventional culture-positive specimens were 64 M. tuberculosis, 61 M. avium, 14 M. intracellulare, 30 other Mycobacterium spp., and 25 non-acid-fast bacilli. There were four cultures that each contained two different Mycobacterium spp. Directly probing the BACTEC 12B sediment, at a DPR of-3 the M. tuberculosis probe identified 83% (53 of 64) of M. tuberculosis isolates, the M. avium probe identified 92% (56 of 61) M. avium isolates, and the M. intracellulare probe identified 86% (12 of 14) of M. intracellulare isolates. There were no false-positive results at this DPR level. The false-negative results from probing the sediment from the BACTEC 12B bottle could not solely be attributed to the number of organisms present, the growth index, or antimicrobial therapy.

Identification of Clinical isolates of Mycobacterium spp. by sequence analysis of the 16S ribosomal RNA gene

“…gordonae, and M . avium complex (MAC) which is composed of M. avium and M. intracellulare (Gen-Probe Inc., San Diego, CA) (10,14,16,17,19,33). The results of the sequence analysis were unknown when these conventional identifications were performed.…”

Examination of specimens for mycobacteria in clinical laboratories in 21 countries: a 10-year review of the UK National Quality Assessment Scheme for Mycobacteria Culture

“…Accurate laboratory diagnosis is essential to ensure effective clinical management of infected patients [10][11][12][13][14]. The performance of participating laboratories with MOTT has improved over time.…”

“…Increasingly, nucleic acid probes are being used for the identification of mycobacteria from positive cultures [10,11,[13][14][15][16]. The methods survey indicated that only 20% of respondents used molecular techniques to identify isolates of mycobacteria.…”

The Genomic Heterogeneity among Mycobacterium terrae Complex Displayed by Sequencing of 16S rRNA and hsp65 Genes

“…Disseminated M. terrae infection in a patient with advanced human immunodeficiency virus disease was recently reported (2).The identification of pathogenic versus nonpathogenic species is important for patient management, since treatment regimens for infection caused by one Mycobacterium species are often not effective against another.Conventional methods for species identification of mycobacteria mainly depend on morphological, cultural, and biochemical tests. These methods require diverse techniques and complex procedures that take a long time and yet the efficiency of identification is still limited (11,15). Other methods such as thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are more powerful, but are cumbersome, expensive and limited by the need for standardized growth conditions.…”

“…Conventional methods for species identification of mycobacteria mainly depend on morphological, cultural, and biochemical tests. These methods require diverse techniques and complex procedures that take a long time and yet the efficiency of identification is still limited (11,15). Other methods such as thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are more powerful, but are cumbersome, expensive and limited by the need for standardized growth conditions.…”