MATERIALS AND METHODS

Study populations

For evaluation of the new CTM CT Test v2.0 to LightMix 480HT PCR (see below), 1059 consecutive PCR samples, urine (n=857), cervical (n=107), pharyngeal (n=40), vaginal (n=14), urine/cervical (n=13), rectal (n=12), urethral (n=3), and unspecified (n=13) samples, from 1 to 18 April, 2008 were analysed. These represented 966 patients, 614 females and 352 males.

For investigation of the nvCT proportion, all C trachomatis NAAT (LightMix) positive samples identified in routine diagnostics at Örebro University Hospital, Sweden from February 1 to December 31, 2007 (n=1084, one sample per consecutive patient), i.e. urine (n=1023), cervical (n=45), urethral (n=4), urethral/cervical (n=2), rectum (n=2), pharyngeal (n=1), conjunctival (n=1), unspecified (n=6) specimens, were examined. These represented 550 females, 533 males, and one with unspecified gender. Additionally, all consecutive culture positive C trachomatis samples (n=116, one sample per patient), cervical (n=106), conjunctival (n=3), urethral/cervical (n=2), urethral (n=1) and unspecified (n=4) samples, from August 1 to December 31, 2007 were investigated. Finally, in order to perform a later one-month sentinel surveillance, all consecutive NAAT positive samples, urine (n=61) and cervical (n=7), and all consecutive culture positive samples, cervical (n=12), unspecified (n=2), urethral (n=1) and urethral/cervical (n=1), during June 2008 were investigated.

All swab samples were collected in 2SP medium, including gentamicin, vancomycin, and amphotericin B. All samples were derived from symptomatic or asymptomatic patients with suspected C trachomatis infection, or were screening samples from women.

Cell culture

Swab samples (in 2SP medium including antibiotics, see above) were cultured using McCoy cell culture and Phadebact Chlamydia IF test (Bactus AB, Huddinge, Sweden).

DNA isolation

Of all culture positive samples, 0.5 ml were initially centrifuged at 20,000´g in 15 minutes and, subsequently, resuspended in 250 ml NaCl. Two-hundred ml of each processed culture or NAAT sample was used for DNA isolation in MagNA Pure instrument using MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics GmbH, Mannheim, Germany), and an elution volume of 100 ml, according to the manufacturer’s instructions and as previously evaluated.[7][8] The preparations were stored at 4°C prior to PCR, which was performed within one day.

Real-time PCR diagnosis of C trachomatis

COBAS TaqMan CT Test v2.0

CTM CT Test v2.0 ((Roche Molecular Systems, Branchburg, NJ, USA) on a COBAS TaqMan48 Analyzer (Roche Molecular Systems) was performed according to the manufacturer’s instructions. However, in the present study the CE Mark-certified protocol for the assay was slightly modified, i.e. i) 2SP medium (including antibiotics, see above) was used for collection of swab samples and ii) DNA was isolated using automation, instead of manual preparation.[7][8]

LightMix 480HT PCR

LightMix 480HT PCR (TIB MOLBIOL GmbH, Berlin, Germany) on a LightCycler 480 (Roche Diagnostics) was performed as previously described.[4]

All LightMix and culture positive samples were examined using a nvCT-specific real-time PCR as previously depicted,[2] on a LightCycler 1.2 instrument (Roche Diagnostics) using FastStart DNA master HybeProbe reagents (Roche Diagnostics), according to the manufacturer’s instructions.

Interpretation of true positive samples in the evaluation of CTM CT v2.0

A sample was regarded as true positive if it was positive in both LightMix and CTM CT v2.0. All samples that showed discrepant results using these assays after repeated testing were subsequently analyzed utilising the BD ProbeTec ET (Becton Dickinson Diagnostic Systems, Sparks, MD, USA) and the new Abbott m2000rt RealTime CT II (Abbott Molecular Diagnostics, Des Plaines, USA), according to the manufacturers’s instructions. If ³1 of these assays was positive, the sample was also regarded as true positive.

Determination of the detection limit (analytical sensitivity) of CTM CT v2.0

The detection limit of CTM CT v2.0 was determined (in genome copies per reaction) using Chlamydia trachomatis DNA control (VIRCELL, Santa Fe, Spain) in dilution series repeated five times.

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