Implementation of a Quantitative Real-Time PCR Assay for the Detection of Mycobacterium immunogenum in Metalworking Fluids

Abstract

The bacterium Mycobacterium immunogenum has been implicated in causing the lung condition hypersensitivity pneumonitis (HP) in factory workers exposed to colonized metalworking fluids (MWFs). M. immunogenum-specific, real-time quantitative PCR detection technique (MiRT-qPCR) was implemented on a large scale to 363 MWFs of varying types, originating from the United States and Europe, that had been in use for between 30 days and 1 year. In MWFs that contained between 10³ and 10⁹ culturable general heterotrophs mL⁻¹ the technique detected between 5 and 2 × 10⁶ mL⁻¹ M. immunogenum cell equivalents (CE) in 12.2% (23 of 189) of U.S. samples and between 8 and 6 × 10⁵ mL⁻¹ CE in 39.1% (68 of 174) of samples from Europe. In contrast, only three cultured presumptive mycobacterial isolates across all samples were confirmed as M. immunogenum. Implementation of the assay demonstrated its practicality and further emphasized the limitations of using cultivation alone. Interestingly, no M. immunogenum were detected in mineral oil-based Bio-Concept MWFs from the United States, while it was more commonly detected in used MWFs based on formaldehyde-releasing biocides than in MWFs free of formaldehyde-depot biocides.

Keywords:

• hypersensitivity pneumonitis
• metalworking fluids
• Mycobacterium immunogenum
• real-time quantitative PCR

ACKNOWLEDGMENTS

The authors wish to express their gratitude to Blaser Swisslube AG for their continued support of this research.

Notes

^A nd = not done.

^B The three isolates that were shown to be M. immunogenum by real-time PCR originated in these samples (two from vegetable oil-based U.S. samples and one from a semisynthetic amine-based sample from Europe).

^C Due to incomplete product information, identification was not possible.