Mycobacterium and Aerobic Actinomycete Culture: Are Two Medium Types and Extended Incubation Times Necessary?

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%) wereMycobacterium tuberculosiscomplex. 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 theM. tuberculosiscomplex. 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.

Genotypic characteristics of Mycobacterium tuberculosis isolated from household contacts of tuberculosis patients in the Philippines.

BACKGROUND: The 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. METHODS: Mycobacterium 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. RESULTS: Spoligotyping 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. CONCLUSIONS: Strain 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.

Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory.

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.