Background Chlamydia trachomatis (Ct) is the most prevalent bacterial sexually transmitted microorganism worldwide. Many researchers conveniently use stored samples for Ct research. We assessed the impact of four different temperature conditions, six different types of medium and five increasing lengths of duration of storage, on Ct DNA detection.

Methods Phosphate buffered saline (PBS), 2-sucrose-phosphate (2-SP) medium, COBAS Amplicor medium and urine samples were spiked with the same amount of Ct serovar D elementary bodies and were stored in at room temperature (RT), 4ºC, –20ºC and –80ºC. Clinical Ct positive urine samples and Ct positive swabs in COBAS Amplicor medium were collected, pooled and stored at the same 4 temperatures. Samples (136 clinical and 287 spiked samples) were tested in triplicate on day 0 and subsequently after 1, 7, 14 and 30 days of storage and two years hereafter for the presence of Ct DNA. Approximately 3000 plasmids were available per PCR reaction and each sample was thawed only once. Cycle thresholds were analysed using generalised linear models, controlling for repeated measurements.

Results Ct could be detected in all clinical samples and spiked media and cycle thresholds were stable over time with few exceptions. For Ct DNA detection in spiked COBAS Amplicor medium, cycle thresholds increased within the first month at –20ºC and –80ºC (both p < 0.01), but decreased in the samples frozen after two years of storage. In spiked urine and pooled clinical urine samples, the cycle threshold decreased within the first month (p < 0.01), including all but one (4ºC, p = 0.09) of the studied temperatures, but increased after two years in the frozen samples.

Conclusion Our results demonstrate that storage conditions and duration hardly affect Ct DNA detection by PCR in a negative manner, although frozen urine samples, stored for prolonged periods (more than two years), could become Ct negative.