An evaluation of the MicroSeq 500 microbial identification system by nucleic acid sequencing and the Mayo Clinic experience with its integration into a routine clinical laboratory setting are described. Evaluation of the MicroSeq 500 microbial identification system was accomplished with 59 American Type Culture Collection (ATCC) strains and 328 clinical isolates of mycobacteria identified by conventional and 16S ribosomal DNA sequencing by using the MicroSeq 500 microbial identification system. Nucleic acid sequencing identified 58 of 59 (98.3%) ATCC strains to the species level or to the correct group or complex level. The identification results for 219 of 243 clinical isolates (90.1%) with a distance score of <1% were concordant with the identifications made by phenotypic methods. The remaining 85 isolates had distance scores of >1%; 35 (41.1%) were identified to the appropriate species level or group or complex level; 13 (15.3%) were identified to the species level. All 85 isolates were determined to be mycobacterial species, either novel species or species that exhibited significant genotypic divergence from an organism in the database with the closest match. Integration of nucleic acid sequencing into the routine mycobacteriology laboratory and use of the MicroSeq 500 microbial identification system and Mayo Clinic databases containing additional genotypes of common species and added species significantly reduced the number of organisms that could not be identified by phenotypic methods. The turnaround time was shortened to 24 h, and results were reported much earlier. A limited number of species could not be differentiated from one another by 16S ribosomal DNA sequencing; however, the method provides for the identification of unusual species and more accurate identifications and offers the promise of being the most accurate method available.During the past two decades, clinical mycobacteriology has enjoyed several important advances that have improved the means for the detection and identification of mycobacteria from clinical specimens. Ultimately, all have improved patient care by shortening turnaround times in the laboratory, and the advances continue. For many years traditional phenotypic identification methods have been used to identify mycobacteria. All are slow, many provide equivocal results, and identifications are not always timely and accurate. High-performance liquid chromatography (HPLC) was used by state laboratories and some reference laboratories. This provided for the rapid identification of an expanded list of species of mycobacteria. Gas-liquid chromatography was also developed and proved to be a useful identification method; however, the number of species that could be identified by gas-liquid chromatography was less compared to the number that could be identified by HPLC. The development of nucleic acid probes for confirmation of the identity of the isolate had a major impact on the work flow in many laboratories. Probes were developed for the identification of members of the Mycobacte...

BackgroundThe Philippines has an extremely high rate of tuberculosis but little is known about M. tuberculosis genotypes and transmission dynamics in this country. The aim of this study was to determine the proportion of household contacts who develop active TB due to direct transmission from an index case in that household.MethodsMycobacterium tuberculosis isolates from household contacts of tuberculosis patients in the Philippines were characterized using restriction-fragment-length polymorphism analysis, spoligotyping, and mycobacterial interspersed repetitive units – variable number tandem repeats typing (12-loci) to determine their utility in elucidating transmission in an area of high tuberculosis prevalence. Drug susceptibility patterns for these isolates were also determined.ResultsSpoligotyping and MIRU-VNTR typing results matched in 10 (62.5%) of 16 index patient-household contact pairs while IS6110 fingerprints matched in only six (37.5%) pairs. Only 3/16 (18.8%) index patient-household contact pairs had identical drug susceptibility results.ConclusionsStrain typing of M. tuberculosis isolates from household contacts in the Philippines indicates that transmission of strains does not necessarily occur directly from the index patient living in close proximity in the same household but rather that community-based transmission also frequently occurs. Accurate susceptibility testing of all isolates is necessary to insure optimal care of both the index patients and any culture-positive household contacts.

Mycobacterial cultures are historically performed using a liquid medium and a solid agar medium with an incubation period of up to 60 days. We performed a retrospective analysis of 21,494 mycobacterial and aerobic actinomycetes cultures performed over 10 months to determine whether two medium types remain necessary and to investigate whether culture incubation length can be shortened. Specimens were cultured using Bactec MGIT liquid medium and Middlebrook 7H11/S7H11 solid medium with incubation periods of 42 and 60 days, respectively. Time-to-positivity and the identity of isolates recovered from each medium were evaluated. A total of 1,205/21,494 cultures (6%) were positive on at least one medium. Of the 1,353 isolates recovered, 1,110 (82%) were nontuberculous mycobacteria, 145 (11%) were aerobic actinomycetes, and 98 (7%) were Mycobacterium tuberculosis complex. Assessing medium types, 1,121 isolates were recovered from solid medium cultures, 922 isolates were recovered from liquid medium cultures, and 690 isolates were recovered on both media. Liquid cultures were positive an average of 10 days before solid cultures when the two medium types were positive (P < 0.0001). Isolates detected on solid medium after 6 weeks of incubation included 65 (5%) nontuberculous mycobacteria, 4 (0.3%) aerobic actinomycetes, and 2 (0.2%) isolates from the M. tuberculosis complex. Medical chart review suggested that most of these later-growing isolates were insignificant, as the diagnosis was already known, or they were considered colonizers/contaminants. This study reaffirms the need for both liquid medium and solid medium for mycobacterial and aerobic actinomycetes culture and demonstrates that solid medium incubation times may be reduced to 6 weeks without significantly impacting sensitivity.T raditionally, mycobacterial and aerobic actinomycetes cultures are performed using both liquid and solid media with extended incubation times of up to 60 days recommended for optimal recovery (1). The use of two types of media is recommended by the College of American Pathologists (CAP) accreditation program (MIC.32250) and the Clinical and Laboratory Standards Institute (CLSI) (1, 2).Many of the studies that support these recommendations were performed 10 to 20 years ago (3-6). Since that time, much has changed in mycobacteriology, including the transition of many laboratories from the use of radiometric liquid medium to mycobacterial growth indicator tubes (MGIT; Becton Dickinson, Franklin Lakes, NJ) or VersaTREK (TREK Diagnostics/Thermo Fisher, Oakwood Village, OH) medium and the description of many new species of mycobacteria (7,8).As laboratories enter into an era with a significant emphasis on cost-effective health care, it becomes increasingly important to examine traditional laboratory practices to ensure that they meet the needs of a changing health care environment and to look for cost-savings opportunities. The purpose of this study was to determine whether one type of medium can be eliminated or if incubation times can be s...

Thtep-nitro-a-acetylamino-pi-hydroxypropiophenone (NAP) differential test for the identification ofMycobacterium tuberculosis recovered from clinical specimens was evaluated by two laboratories and found to be a rapid and accurate procedure with a specificity exceeding 99%.

We recently completed verification studies comparing the MGIT 960 to the BACTEC 460 for antimicrobial susceptibility testing (AST) of Mycobacterium tuberculosis complex. A review of the literature revealed that most authors tested an overall large number of drug-susceptible isolates but only a small and arbitrary number of resistant isolates. The median numbers of resistant isolates of each drug used in published evaluations including streptomycin, isoniazid, rifampin, ethambutol, and pyrazinamide were 14 (range, 2 to 26), 25 (range, 17 to 77), 13 (range, 0 to 23), 12 (range, 4 to 15), and 11 (range, 6 to 13), respectively.We [18]) and showed a very major error rate of 1.8% and a 98.7% correlation between methods. It is our feeling that the use of 35 resistant isolates to test each antimicrobial is both optimal and achievable in the clinical laboratory.We endorse the MGIT 960 for AST and specifically advocate that Ն35 isolates resistant to each drug be used for the verification of AST for mycobacteriology. Using this number would allow for the detection of a low but significant very major error rate. It would be difficult for a clinical laboratory to acquire and test for a larger number of isolates resistant to each drug. We encourage CLSI to endorse the recommendation to ensure that adequate numbers of resistant isolates be used to verify AST assays for M. tuberculosis and other mycobacteria and to endorse an acceptable very major error rate of Յ3.0% to ensure that test performance provides clinically valid information to clinicians. We also advocate that those agencies that accredit clinical laboratories and recommend that manufacturer's data or published reports can be used as verification data allow this only if an adequate number of organisms was used in the documentation. It is our hope that the information presented here will allow laboratories to better and more accurately verify AST comparisons for M. tuberculosis and other mycobacteria as newer methods are developed.