Conventional Methods versus 16S Ribosomal DNA Sequencing for Identification of Nontuberculous Mycobacteria: Cost Analysis

Abstract

The clinical profile of nontuberculous mycobacteria (NTM) has been raised by the human immunodeficiency virus and AIDS pandemic. Different laboratory techniques, often molecular based, are available to facilitate the rapid and accurate identification of NTM. The expense of these advanced techniques has been questioned. At the National Reference Center for Mycobacteriology and the Health Sciences Center, University of Manitoba, in Winnipeg, Canada, we performed a direct cost analysis of laboratory techniques for commercial DNA probe-negative (Gen-Probe, Inc., San Diego, Calif.), difficult-to-identify NTM. We compared the costs associated with conventional phenotypic methodology (biochemical testing, pigment production, growth, and colony characteristics) and genotypic methodology (16S ribosomal DNA [rDNA] sequence-based identification). We revealed a higher cost per sample with conventional methods, and this cost varied with organism characteristics: $80.93 for slowly growing, biochemically active NTM; $173.23 for slowly growing, biochemically inert NTM; and $129.40 for rapidly growing NTM. The cost per sample using 16S rDNA sequencing was $47.91 irrespective of organism characteristics, less than one-third of the expense associated with phenotypic identification of biochemically inert, slow growers. Starting with a pure culture, the turnaround time to species identification is 1 to 2 days for 16S rDNA sequencing compared to 2 to 6 weeks for biochemical testing. The accuracy of results comparing both methodologies is briefly discussed. 16S rDNA sequencing provides a cost-effective alternative in the identification of clinically relevant forms of probe-negative NTM. This concept is not only useful in mycobacteriology but also is highly applicable in other areas of clinical microbiology.

Identificación bacteriana mediante secuenciación del ARNr 16S: fundamento, metodología y aplicaciones en microbiología clínica

“…Además, la interpretación de los resultados exige experiencia, y está limitada por la baja especificidad y la subjetividad. Por ello, la secuenciación del ADNr 16S se está imponiendo como método rápido y preciso para la identificación tanto de micobacterias previamente reconocidas como de nuevas especies 13,15,[27][28][29] . La identificación molecular puede completarse 1 o 2 días después del aislado en cultivo puro, y puede incluso realizarla personal sin experiencia en biología molecular.…”

Novel Real-Time Simultaneous Amplification and Testing Method To Accurately and Rapidly Detect Mycobacterium tuberculosis Complex

“…For SAT-TB-positive samples which were negative by both the real-time PCR assay and Bactec MGIT 960 culture, the SAT-TB cDNA amplification products were sequenced using the primer with the sequence 5=-AATTTAATACGACTCACTATAG-3=. For Bactec MGIT 960 culture-positive specimens that were SAT-TB negative and real-time PCR negative, genomic DNA was extracted from the culture, and the 16S rRNA fragment was amplified and sequenced as previously described (4).…”

Phenotypic and Molecular Typing of Tuberculous and Nontuberculous Mycobacterium Species from Slaughtered Pigs in Egypt

“…10,28 Molecular identification of Mycobacterium species has 2 primary advantages compared with phenotypic identification: rapid turnaround time and improved accuracy. 4,26 Sequencedependent identification has been shown to be an especially effective molecular tool that provides rapid and accurate differential identification of Mycobacterium species. 3,18 Most molecular approaches have focused on the conserved 16S small subunit ribosomal DNA (rDNA) sequence.…”