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.

Description, validation, and review of a decade of experience with a laboratory-developed PCR test for detection of Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens.

Rapid detection of Mycobacterium tuberculosis complex directly from clinical specimens is critical for patient care. Mycobacterial culture requires days to weeks for results and therefore many laboratories employ rapid molecular methods for the diagnosis of tuberculosis. There are two FDA-cleared molecular assays for the detection of M. tuberculosis complex in the United States and both are cleared for testing of respiratory specimens only. The detection of M. tuberculosis complex in extrapulmonary specimens is often done using laboratory-developed PCR methods. In this work, the verification and subsequent validation of test performance over a decade is detailed for a laboratory-developed PCR assay (MTBRP) that detects M. tuberculosis complex from respiratory and non-respiratory specimens. The assay also provides information about potential isoniazid resistance. The performance of the MTBRP PCR assay was compared to the Cepheid Xpert MTB/RIF assay in acid-fast smear positive and smear negative specimens and mycobacterial culture for acid-fast smear positive specimens. The MTBRP assay demonstrated 99% correlation with the Xpert MTB/RIF assay using 499 respiratory specimens. The performance of the MTBRP PCR assay compared with mycobacterial culture for 867 AFB smear positive respiratory and non-respiratory specimens demonstrated a sensitivity of 100% and a specificity of 99.1%. This work provides longitudinal evidence using real-world clinical laboratory conditions and specimens to demonstrate that laboratory-developed PCR assays such as the MTBRP can provide a rapid and sensitive method for detection of pulmonary and extra-pulmonary tuberculosis from a wide-variety of smear positive specimen sources.

Candida auris Direct Detection from Surveillance Swabs, Blood, and Urine Using a Laboratory-Developed PCR Method.

Candida auris is an emerging fungal pathogen with cases reported in countries around the world and in 19 states within the United States as of August 2020. The CDC has recommended that hospitals perform active surveillance upon admission for patients with the appropriate risk factors. Currently, active surveillance requires that local hospitals send surveillance swabs to a public health laboratory for analysis. In this work, a real-time PCR assay was developed for the specific detection of C. auris from surveillance swabs, blood, and urine to enable rapid detection of this pathogen. The assay uses commercially available primers and reporter probes and it was verified on the LightCycler 480 PCR platform. Contrived specimens and prospectively collected composite groin/axilla surveillance swabs were used to validate the assay. The performance of the PCR assay on surveillance swabs was also compared to a second PCR assay targeting C. auris that was performed at the Minnesota Department of Health-Public Health Laboratory (MDH-PHL). Our PCR assay is able to detect and differentiate C. auris from closely related Candida species such as C. duobushaemulonii, C. haemulonii, and C. pseudohaemulonii on the basis of melting curve temperature differences.