Factors that contribute to the success of primary isolation of Mycoplasma genitalium from clinical samples.

Introduction. Mycoplasma genitalium, a small and slow-growing bacterium, has gained notoriety due to rapidly increasing rates of antibiotic resistance.Gap Statement. M. genitalium is difficult to culture, limiting efforts to understand its biology and the mechanisms of antimicrobial resistance.Aim. To understand factors that influence success in primary isolation of M. genitalium from clinical samples.Methodology. Neat urine or swabs (high vaginal and anal, in universal transport medium) were collected from patients with confirmed or suspected M. genitalium infections attending the Melbourne Sexual Health Centre. The specimens were stored at -80 °C prior to laboratory analysis. Initial diagnosis was by transcription-mediated amplification (TMA) assay, and samples subsequently testing positive by quantitative PCR (qPCR) were washed twice and inoculated into Vero cell monolayers with a selective antibiotic mixture (cycloheximide and Thayer-Martin Medium I). Cultures were incubated at 37 °C with 5% CO(2) for 8 weeks and observed daily, with qPCR used to monitor growth.Results. In total, 127 TMA-positive samples were subjected to qPCR, and M. genitalium genomic DNA (gDNA) was detected in 53.5% (68/127) of these samples. An isolate was obtained from 26.5% (18/68) of the gDNA-positive samples following co-culture with Vero cells. The isolation rate varied between sample types, with growth detected in 12.5% (3/24) of the high vaginal swabs and 37.5% (15/40) of the urine samples. No isolates were obtained from anal swabs. The proportion with a successful culture was influenced by the initial M. genitalium load in the sample, which translated into the inoculum size for the Vero cell monolayer. Isolation was unsuccessful with low inoculum (<2,000 genome equivalents, geq), partially successful (13.3%) with a moderate inoculum (2,500-9,500 geq) and highly successful (100%) in samples with a high inoculum (>10,000 geq).Conclusion. The initial bacterial load emerged as a critical determinant of isolation success. This emphasizes the importance of optimizing sample collection and M. genitalium isolation procedures.