The Journal of Molecular Diagnostics, Vol. 16, No. 1, January 2014

Sensitive and Rapid Detection of Chlamydia trachomatis by Recombinase Polymerase Amplification Directly from Urine Samples Katrin Krõlov,* Jekaterina Frolova,* Oana Tudoran,*y Julia Suhorutsenko,* Taavi Lehto,* Hiljar Sibul,* Imre Mäger,* Made Laanpere,z Indrek Tulp,x{ and Ülo Langel*k From the Institutes of Technology* and Chemistry,x University of Tartu, Tartu, Estonia; the Department of Functional Genomics and Experimental Pathology Cluj-Napoca,y I. Chiricuta Cancer Institute, Cluj-Napoca, Romania; the Tartu University Hospital’s Women’s Clinic and Tartu Sexual Health Clinique,z Tartu, Estonia; Selfdiagnostics OÜ,{ Tallinn, Estonia; and the Department of Neurochemistry,k Stockholm University, Stockholm, Sweden Accepted for publication August 9, 2013. Address correspondence to Katrin Krõlov, M.Sc., University of Tartu, Institute of Technology, Laboratory of Molecular Biotechnology, Nooruse 1a, Tartu 50411, Estonia. E-mail: katrin. [email protected].

Chlamydia trachomatis is the most common sexually transmitted human pathogen. Infection results in minimal to no symptoms in approximately two-thirds of women and therefore often goes undiagnosed. C. trachomatis infections are a major public health concern because of the potential severe long-term consequences, including an increased risk of ectopic pregnancy, chronic pelvic pain, and infertility. To date, several point-of-care tests have been developed for C. trachomatis diagnostics. Although many of them are fast and specific, they lack the required sensitivity for large-scale application. We describe a rapid and sensitive form of detection directly from urine samples. The assay uses recombinase polymerase amplification and has a minimum detection limit of 5 to 12 pathogens per test. Furthermore, it enables detection within 20 minutes directly from urine samples without DNA purification before the amplification reaction. Initial analysis of the assay from clinical patient samples had a specificity of 100% (95% CI, 92%e100%) and a sensitivity of 83% (95% CI, 51%e97%). The whole procedure is fairly simple and does not require specific machinery, making it potentially applicable in point-of-care settings. (J Mol Diagn 2014, 16: 127e135;

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen, affecting a mean of 5% to 10% of the population, with the highest incident rates (up to 40%) among patients attending sexually transmitted disease clinics.1 Infection is associated with nongonococcal urethritis in men and several inflammatory reproductive tract syndromes, such as cervicitis and pelvic inflammatory disease, in women.2 One of the main reasons for the high prevalence rates of C. trachomatis is that in most cases (75% of women and 50% of men) the infection remains asymptomatic. Untreated, the infection increases the risk of ectopic pregnancy and is one of the leading causes of female infertility worldwide.3,4 C. trachomatis is particularly prevalent among young adults (up to 25 years old) and therefore poses a major threat to the reproductive health of the population.5,6 As such, prompt diagnosis and treatment of the infection are of particular importance. Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Currently, PCR-based techniques are widely applied for clinical C. trachomatis diagnostics (eg, Abbot RealTime CT/NG assay and Roche Cobas Amplicor CT/NG assay). These techniques use amplification of the specific DNA region of the multicopy cryptic plasmid characteristic of C. trachomatis. Although PCR-based detection is highly sensitive and specific, it is only suitable for centralized hospital facilities, requiring trained personnel and use of expensive automated sample preparation machinery and real-time thermocyclers. Supported by grant EU32415 from Enterprise Estonia (European Regional Development Fund) and grants SF0140031Bs09 (I.T.) and SF0180027s08 (J.S., H.S., T.L., I.M., and Ü.L.) from Estonian Ministry of Education and Research and funding from Selfdiagnostics OÜ. Disclosure: I.T. is an employee and board member of Selfdiagnostics OÜ, which has no commercial interest regarding the test described in the article.

Krõlov et al Several point-of-care (POC) C. trachomatis tests have been developed that allow rapid on-site diagnostics and a costefficient strategy for large-scale screenings. These are immunoassays that detect the antigen (major outer membrane protein) or lipopolysaccharide specific to C. trachomatis. The major advantage of POC tests is that they yield a result at the time of the initial patient visit and do not require patient followup. Studies have found that up to 50% of patients never return to get the diagnostic result or required treatment.7 The available POC tests have good specificity (96% to 99%) but lack the sensitivity that nucleic acid amplificationebased techniques offer.8 The mean reported sensitivities of C. trachomatis POC tests are in the range of 40% to 60% compared with nucleic acid amplificationebased techniques.9e12 However, recent evaluations have found alarmingly poor performance, with sensitivity of just 10% to 30%, depending on the test.8 The low sensitivity of the available POC tests has limited their wider use, and there is a clear requirement for more sensitive and cost-effective diagnostic platforms. Isothermal nucleic acid amplification assays provide a good alternative to PCR-based diagnostics in laboratories with limited resources and POC settings. Recombinase polymerase amplification (RPA) is a recently described nucleic acid amplification technique that uses a recombinase complex from T4 bacteriophage to introduce primers to specific DNA sites to initiate an amplification reaction by the strand displacing DNA polymerase.13 Thus, RPA does not require template denaturation and can operate at a relatively low and constant temperature (38 C to 42 C). The sensitivity of the RPA is comparable to PCR with as few as 10 template copies required for amplification of the specific product.14e16 Furthermore, recent studies have indicated that even at near detection limit template concentrations, RPA is able to produce a detectable amplification signal within just 10 minutes.14e16 Thus, RPA represents a good basis for a POC diagnostics platform. We have developed an RPA-based POC assay for rapid and highly sensitive detection of C. trachomatis directly from urine samples. The assay takes

Sensitive and rapid detection of Chlamydia trachomatis by recombinase polymerase amplification directly from urine samples.

Chlamydia trachomatis is the most common sexually transmitted human pathogen. Infection results in minimal to no symptoms in approximately two-thirds ...
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