Sensitive and Rapid Detection of Chlamydia trachomatis by Recombinase Polymerase Amplification Directly from Urine Samples

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

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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; http://dx.doi.org/10.1016/j.jmoldx.2013.08.003)

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. http://dx.doi.org/10.1016/j.jmoldx.2013.08.003

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 <20 minutes and includes sample pretreatment, isothermal target amplification, and immunochromatographic product detection. The developed assay does not require DNA purification before the amplification reaction, is relatively simple to perform, and could therefore be applied in POC settings. Initial clinical evaluation of the assay has found a specificity of 100% (95% CI, 92%e100%) and a sensitivity of 83% (95% CI, 51%e97%). It has the potential to offer high-sensitivity testing in POC settings.

For assay sensitivity determination, a pGL3-CDS2 plasmid template was constructed as follows. A C. trachomatis CDS2

gene fragment was cloned into a pGL3-Promoter vector (Promega, Madison, WI) between the XhoI restriction site using a Pfu proofreading PCR with forward primer 50 CATCTCGAGCTATATTAGAGCCAGCTT-30 and reverse primer 50 -GTACTCGAGATGGGTAAAGGGATTTTA-30 . The pGL3-CDS2 clone was verified using PCR and restriction analysis. For the quantitative molecular standard, the pGL3CDS2 plasmid was propagated in Escherichia coli. Highly pure RNA-free plasmid stock was obtained using a GeneJET Plasmid Midiprep Kit (Thermo Scientific, Waltham, MA). The purity of the plasmid DNA was verified using the A260/A280 absorbance ratio (A260/A280 Z 1.87) and 1.2% 0.5 Tris-acetate-EDTA (TAE) agarose gel electrophoresis. The concentration of the pGL3-CDS2 plasmid stock was estimated at 0.354 mg/mL from absorbance at 260 nm and 96 nmol/L from a molecular weight of 3686648.04 g/mol (estimated for the 5968-bp pGL3CDS2 plasmid using the DNA Molecular Weight calculation tool, http://www.bioinformatics.org/sms2/dna_mw. html, last accessed April 5, 2012). The plasmid and accordingly the CDS2 gene molecule (copy) number per microliter was estimated from the plasmid stock molar concentration as 57.8  109 molecules/mL using the Avogadro constant. A series of amplification template dilutions were performed to estimate the analytical sensitivity of the RPA assay. This was determined as the lowest template (pGL3-CDS2) molecule copy number required for amplification product formation detected in at least three independent experiments. C. trachomatis genomic DNA (gDNA) was obtained from the ATCC (Manassas, VA). C. trachomatisespecific RPA assay sensitivity was additionally analyzed using the dilution series of the ATCC-obtained C. trachomatis gDNA. The lowest detectable template amount (in picograms) was determined in at least three independent experiments. Human GAPDH geneespecific RPA assay sensitivity was analyzed using RNA-free human gDNA purified from cultured HeLa cells using the NucleoSpin Tissue Kit (Macherey-Nagel GmbH, Düren, Germany). The purity of the human gDNA template was assessed using the A260/ A280 absorbance ratio and 1.2% 0.5 TAE agarose gel electrophoresis. The concentration of human gDNA was estimated from absorbance at 260 nm. The GAPDH gene copy number per RPA reaction was calculated taking into account that diploid human female nuclei in the G1 phase of the cell cycle contain 6.550 pg of the DNA.17 For RPA specificity determination, the assay was performed using 20 pg of C. trachomatis strain UW-36/Cx gDNA (ATCC VR-886D), 20 pg of Mycoplasma genitalium gDNA (ATCC 33530), 100 pg of Neisseria gonorrhoeae gDNA (ATCC-53422D), 20 pg of Ureaplasma urealyticum (NCTC 10177) gDNA (51-0177, Minerva Biolabs GmbH, Berlin, Germany), 100 pg of E. coli MG16.55 gDNA (kindly provided by Rya Ero), and 1 ng of human gDNA (extracted as described above).

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Materials and Methods Quantitative Molecular DNA Standards for Assay Sensitivity and Specificity Determination

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Detection of C. trachomatis

RPA Primer Design and Selection C. trachomatis CDS2 gene and Homo sapiens GAPDH genes were sought for highly conserved sequence regions using the Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov, last accessed January 28, 2013). All of the sequences of CDS2 present in the GenBank database were used for BLAST analysis to exclude the possibility that some C. trachomatis serovars/strains would remain unrecognized by the assay. The conservation of the C. trachomatis CDS2 amplicon among 11 C. trachomatis strains from a lymphogranuloma venereum biovar (serovars L1, L2, L2b, and L3) and 16 strains from a genital biovar (serovars D, E, F, G, Ia, J, and K) is shown in Supplemental Figure S1. Amplification primers were designed to overlap the conserved regions so that the oligonucleotide size was between 30 and 35 nt and the product size was in the range of 100 to 200 bp. In addition, long tracks of guanines at the 50 end of the oligonucleotides (the first three to five nucleotides) were avoided, whereas pyrimidines were preferred to encourage the formation of the recombinase filaments. Guanines and cytosines were preferred at the 30 end of the primer (the last three nucleotides) because they tend to provide a more stable clamped target for the polymerase, improving the performance of the assay (TwistDX, Cambridge, UK). Oligonucleotides that contain sequence elements that promote secondary structures and primer-primer interactions or hairpins were avoided (analyzed using the OligoAnalyser tool, http://www.idtdna.com/analyzer/Applications/OligoAnalyzer, last accessed April 5, 2012). Oligonucleotides were ordered from Microsynth AG (Balgach, Switzerland). Four forward and four reverse primers were designed for the CDS2 target, giving 16 possible combinations. Three forward and three reverse primers were designed for the GAPDH target, giving nine possible combinations. The primers were screened for highest target DNA detection sensitivity (lowest copy number of target DNA detected) and for absence of the nonspecific background signal on lateral flow strips. The latter appeared in some primer pairs, probably due to primer heterodimer formation. The best performing primer pairs were selected; their sequences are indicated in Table 1.

RPA Amplification and Product Detection The RPA pellet (TwistDX) was resuspended with 47.5 mL of the reaction buffer prepared by mixing the following Table 1

components: 2.1 mL of 10 mmol/L forward primer; 2.1 mL of 10 mmol/L reverse primer (the final concentration of each primer in the reaction being 0.42 mmol/L), 29.5 mL of rehydration buffer (TwistDX), 1.2 mL of template DNA, and 12.6 mL of double-distilled H2O (or 5 mL of urine and 8.8 mL of double-distilled H2O). The RPA reaction was initiated by adding 2.5 mL of 280 mmol/L magnesium acetate. The reaction was incubated at 38 C for 30 minutes (unless indicated otherwise). After 4 minutes of incubation, the reaction was mixed by flicking the tube. To determine the minimum reaction time required for product formation, the reaction was terminated after the indicated time via 10 minutes of incubation at 50 C. All of the RPA forward primers were labeled with biotin and the reverse primers with 6-carboxyfluorescein (FAM), enabling biotin-FAM double-labeled product detection on the lateral flow strips. Reaction products were diluted 500 times in a lateral flow strip dilution buffer and analyzed on PCRD-2 strips (Forsite Diagnostics, York, UK). For product size and intensity analysis, RPA reaction products were purified using an Invitek MSB Spin PCRapace Kit (Stratec Molecular GmbH, Berlin, Germany) and resolved on 1.8% 0.5 TAE agarose gel. For product specificity analysis, a restriction digest was performed with FastDigest PmlI restrictase (Thermo Scientific) for the C. trachomatis amplification product and with HphI restrictase (Thermo Scientific) for the H. sapiens GAPDH amplification product, according to the manufacturer protocol. The amplified product was considered specific if the signal on the PCRD-2 disappeared after treatment with restrictase.

Collection of the Clinical Samples and Their Preparation for the RPA Analysis First-void urine samples were collected from 70 patients (51 females and 19 males) attending the sexual health clinic (in Tartu, Estonia) in April and May 2013. Patients 18 to 25 years old (mean age, 21.5 years) who were directed by the clinician to test for sexually transmitted diseases were asked to participate in the study voluntarily by donating a selfcollected morning first-void urine sample. The criteria for patient selection by clinician were change of sexual partner, multiple sexual partners, unprotected sexual intercourse, sexually transmitted infection of partner, or symptoms of sexually transmitted disease (such as increased or abnormal vaginal discharge, abdominal pain, or painful urination). Approval for the study was obtained from the Research

C. trachomatise and H. sapienseSpecific Amplification Targets and Primer Sequences

Target organism and region

Amplification product size (bp)

C. trachomatis CDS2 (cryptic plasmid) H. sapiens GAPDH

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50 Modification

Sequence

CDS2-FW CDS2-RV GAPDH-FW GAPDH-RV

Biotin FAM Biotin FAM

50 -CCTTCATTATGTCGGAGTCTGAGCACCCTAGGC-30 50 -CTCTCAAGCAGGACTACAAGCTGCAATCCCTT-30 50 -AAGTCAGGTGGAGCGAGGCTAGCTGGCCCGATT-30 50 -CACCATGCCACAGCCACCACACCTCTGCGGGGA-30

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Figure 1 Assay sensitivity determined as the lowest detectable template amount and assay specificity determined as cross-reactivity with gDNA from other species. A: Assay sensitivity was analyzed by performing C. trachomatis CDS2specific RPA with a pGL3-CDS2 template at the indicated copy number per reaction, with the C. trachomatis gDNA (CT gDNA) template at the indicated amount per reaction and with total DNA purified from the indicated amount of C. trachomatisepositive urine (total DNA CT pos I or II). B: Human GAPDH-specific RPA sensitivity analysis was performed using pure human gDNA (HS gDNA) at the indicated amount per reaction and with total DNA purified from the indicated amount of C. trachomatisepositive urine. C: C. trachomatis CDS2 and human GAPDH detection assay specificity was analyzed using 20 pg of M. genitalium, 20 pg of C. trachomatis, 20 pg of U. urealyticum, 100 pg of N. gonorrhoeae, 100 pg of E. coli, and 1 ng of H. sapiens gDNA. All amplification reactions were performed at 38 C for 30 minutes. The reaction products were analyzed on PCRD-2 lateral flow strips, where T indicates the test band or presence of the biotin-FAMelabeled reaction product and C indicates the lateral flow control band.

Ethics Committee of the University of Tartu. Confidentiality was addressed on the patient information sheet. The first-void morning urine samples were self-collected in clean polypropylene containers without preservatives, with a mean sample volume of 25 to 35 mL (varying from 15 to 45 mL). The samples were stored at þ4 C and tested by RPA within 1 day of collection (regularly within 6 hours of collection). The lysates used for the RPA reaction were prepared by heating 5 mL of clinical sample for 5 minutes at 90 C. Total DNA from clinical samples was extracted using a QIAamp Viral RNA Mini Kit (Qiagen). The volume of the purified total DNA was adjusted to the volume of the sample subjected to purification. Therefore, 1 mL of purified DNA corresponds to the amount of total DNA present in 1 mL of a urine sample. A Roche Cobas Amplicor C. trachomatis test (HoffmanLa Roche, Basel, Switzerland) was performed according to the manufacturer instructions by the United Laboratories of Tartu University Hospital on the second day of sample collection but no longer than 72 hours after collection. Of the 12 C. trachomatisepositive patient samples, three patients had symptoms. Two of the three C. trachomatise positive males had painful urination, and one of the nine C. trachomatisepositive females had abdominal pain. The other nine patients were asymptomatic. Thus, on average, 25% of the infections were symptomatic. Of the 58 C. trachomatisenegative patients, 15 (26%) also had

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symptoms that could be associated with C. trachomatis infection. One of these tested positive for N. gonorrhoeae and M. genitalium. Others were diagnosed as having bladder inflammation (two patients), bacterial vaginosis (five patients), yeast infection (four patients), or abdominal pain of nongynecologic origins (three patients). Specificity, sensitivity, and 95% CIs were calculated using the calculator from VassarStats (http://www.vassar stats.net/clin1.html, last accessed August 8, 2013).

Results Sensitivity and Specificity of C. trachomatis Detection Assay We have developed a C. trachomatisespecific RPA-based assay for detection of CDS2 of the C. trachomatis cryptic plasmid. The plasmid is present in multiple copies per C. trachomatis genome,18,19 thus enabling higher detection sensitivity to be achieved in the diagnostics assay. CDS2 is also highly conserved between different serovars and strains of C. trachomatis, including Swedish variants (Supplemental Figure S1).19 For intrinsic positive control of the diagnostic assay, we used the human housekeeping gene GAPDH because healthy patient urine contains up to 105/mL human cells.20 Selected targets were amplified using a biotinlabeled forward primer and a FAM-labeled reverse primer (Table 1), enabling immunochromotographic detection of the

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Detection of C. trachomatis biotin-FAM double-labeled product using lateral flow strips (Figure 1). The RPA method, like other nucleic acid amplificatione based techniques, requires only a few pathogens for product amplification, thus offering highly sensitive detection compared with conventional immunoassays. The analytical sensitivity of the RPA depends on the target and selected primers. We have evaluated the sensitivity of the C. trachomatis RPA assay using dilutions of the pGL3-CDS2 plasmid template, C. trachomatis gDNA, and total DNA purified from C. trachomatisepositive patients (Figure 1A). As few as 50 copies of the plasmid template or 0.2 pg of the C. trachomatis gDNA were sufficient to detect the CDS2-specific RPA product (Figure 1A). By using purified DNA from two C. trachomatisepositive urine samples, we found that 0.05 to 0.5 mL of urine alone could contain a sufficient amount of DNA to obtain a positive diagnostic signal (Figure 1A). Human GAPDH-targeted RPA produced a detectable product from minimum 80 pg of human gDNA, which translates to approximately 25 genome copies (Figure 1B). When total DNA purified from a urine sample was used as a template, GAPDH could be detected from a dilution corresponding to 0.05 mL of urine. The amount of human DNA in the tested urine samples appeared to be the same for two positive and four negative tested samples (data not shown). Next we analyzed the specificity of the RPA reaction using excess gDNA from human and other bacterial agents that could be potentially present in the analyzed urine sample, such as M. genitalium, N. gonorrhoeae, U. urealyticum, and E. coli. The C. trachomatis CDS2-specific RPA gave a positive result on lateral flow strips only with C. trachomatis gDNA not with 1 ng of human gDNA, 20 pg of M. genitalium gDNA, 20 pg of U. urealyticum gDNA, 100 pg of N. gonorrhoeae gDNA, or 100 pg of E. coli gDNA (Figure 1C). The GAPDH-specific RPA gave a positive signal only with H. sapiens gDNA, indicating that RPA amplification reactions were specific to selected targets. C. trachomatis assay specificity was assessed further with total DNA purified from four Chlamydia-negative urine samples, all of which tested negative for CDS2 and positive for GAPDH (data not shown). Often POC tests are applied on site during patient visits to the clinic. Thus, a short time to obtain the result is of particular importance for POC tests; anything beyond 30 minutes would probably result in a patient follow-up being needed. We estimated the minimal time to product formation for the C. trachomatis assay. Our method required only 10 minutes to produce a detectable amount of CDS2-specific product when using as few as 100 copies of template per reaction (Figure 2). This enables a reduction in the assay amplification time from 30 minutes to 10 minutes, without losing sensitivity even at template concentrations near the detection limit. These results are in accordance with those previously reported by other research groups14,15 and indicate that RPA is a sufficiently rapid isothermal amplification method for on-site applications.

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Figure 2 Minimum reaction time required for product formation at template concentration near detection limit. C. trachomatisespecific assay was performed using 500 or 100 pGL3-CDS2 template copies per reaction. The reaction was terminated after 0, 5, 10, 15, and 20 minutes, respectively, using 10 minutes of incubation at 50 C. The reaction products were analyzed on PCRD-2 lateral flow strips, where T indicates the test band or presence of the biotin-FAMelabeled reaction product and C indicates the control band.

C. trachomatis Detection Directly from Urine Samples Although urine contains several PCR inhibitors that can abolish amplification altogether, some amplification systems (such as loop-mediated isothermal amplification) are known to be able to detect DNA directly from patient samples.21e23 Because RPA uses recombination and replication components from biological systems, we hypothesized that it could be robust enough to detect the infection directly from urine samples without prior DNA extraction and purification. To study this, we performed RPA assay targeting CDS2 directly from heat-treated C. trachomatis-positive urine samples and were able to successfully detect C. trachomatis specific product formation, whereas no amplification was detected using C. trachomatis-negative urine samples (Figure 3A). Analysis of RPA assay analytical sensitivity in the C. trachomatis-negative urine background showed that the addition of 5 mL of urine to the RPA reaction did not significantly affect its sensitivity (Supplemental Figure S2). In the presence of 5 mL of urine the RPA was still able to detect 0.2 pg of C. trachomatis gDNA as in the absence of urine (Figure 1A and Supplemental Figure S2). However, in the presence of urine the amplification signal was somewhat weaker for template DNA concentrations near to the lowest

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Figure 3 C. trachomatis detection directly from heat-treated urine samples. A: CDS2 detection from 5 mL of C. trachomatisepositive or enegative urine samples subjected to 5 minutes of incubation at 90 C. B: The sensitivity of the assay was established as a specific detection of CDS2 and GAPDH using a dilution series of C. trachomatisepositive heat-treated urine (sample 1) as a template. The reaction products were analyzed on PCRD-2 lateral flow strips, where T indicates the test band or presence of the biotin-FAMelabeled reaction product and C indicates the lateral-flow control band. C: General layout of C. trachomatis detection assay with indicated steps, required time, and temperature.

amount detectable e 0.2 pg and 0.5 pg of C. trachomatis gDNA (Supplemental Figure S2). The addition of 10 mL of urine to the RPA reaction affected amplification efficiency significantly, reducing RPA sensitivity at least 10-fold (Supplemental Figure S2). Thus up to 5 mL of a patient’s urine sample can be used for RPA assay without significant inhibition of amplification efficiency. Next we assessed C. trachomatis assay sensitivity with heat-treated urine samples (Figure 3B). Both CDS2 and GAPDH targets required a minimum 0.05 mL of heattreated urine for specific product amplification. Therefore for both targets the detection limit using heat-treated urine was in the same range as for purified total DNA (Figure 1A and 3B). These results show that the C. trachomatis detection assay does not require DNA purification before amplification and that it is compatible with robust sample lysate preparations, such as heating. Five minutes of incubation at 90 C is sufficient to release DNA from cells that could be subsequently used as a template in the RPA amplification reaction. Furthermore, the sensitivity of the C. trachomatis detection assay is not significantly affected when 5 mL of crude urine lysates are used as an amplification template. The final layout of the C. trachomatis detection assay is shown in Figure 3C. The whole process takes <20 minutes and requires only brief incubation (first at 90 C for 5 minutes for sample preparation and then at 38 C for 10 minutes for product amplification). No thermocycling or expensive machinery is required for assay performance. C. trachomatis-specific product is visualized within minutes using lateral flow strips (Figure 3C). The whole procedure is fairly simple yet enables highly specific and sensitive C. trachomatis detection starting from five pathogens per reaction.

*Total DNA was purified from patient urine sample using a QIAamp Viral RNA Mini Kit (Qiagen). DNA purified from 23.3 mL of urine was subjected to RPA reaction. y A total of 5 mL of urine sample was incubated at 90 C for 5 minutes and then subjected to RPA amplification.

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Clinical Evaluation of the Developed C. trachomatis Detection Assay 70 self-collected first-void morning urine samples from young adults were tested in parallel using RPA and Roche Cobas Amplicor C. trachomatis assays. Nineteen males and 51 females were recruited for the study and the overall prevalence of C. trachomatis among the group was 17% (16% among males and 18% among females) according to the Roche Cobas Amplicor assay results (Table 2). All 58 C. trachomatis-negative samples tested negative in the RPA assay (Table 2). As no false negatives were detected, the clinical specificity of the C. trachomatis RPA assay could be estimated at 100% (95% CI, 92%e100%). Of the 12 C. trachomatis-positive urine samples, 10 tested positive and two tested negative in the RPA reaction using 5 mL of heat-treated urine as an amplification template (Table 2). Based on these results, the clinical sensitivity of the C. trachomatis assay can be estimated at 83% (95% CI, 51%e97%). When purified total DNA was used, all 12 Table 2 C. trachomatis Detection in 70 First-Void Urine Samples with the Roche Cobas Amplicor CT Assay and RPA C. trachomatis Assay RPA C. trachomatis assay Urine lysatesy

Roche Cobas Amplicor CT assay

Pure DNA* Positive

Negative

Positive

Negative

Positive Negative

12 0

0 58

10 0

2 58

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Detection of C. trachomatis C. trachomatis-positive samples tested positive in the RPA assay (Table 2).

Discussion Although clinical C. trachomatis diagnostic techniques enable highly specific and sensitive pathogen detection, they do not represent a cost-efficient on-site screening option, for which POC tests are better suited. Currently there are no C. trachomatis POC tests that offer sensitivity comparable to laboratory nucleic acid amplification-based techniques. In fact, recent independent studies have shown alarmingly poor pathogen detection sensitivities (10% to 40%) of POC tests, which has hindered their large-scale application.8,10 However, the need for an applicable on-site C. trachomatis test that offers reasonably sensitive detection of the pathogen remains. Here we describe a novel assay for C. trachomatis detection that is based on recombinase polymerase amplification (RPA). RPA is a recently described isothermal amplification technique that offers highly sensitive and specific DNA detection without thermocycling of the reaction. The C. trachomatis RPA assay can be performed on pure DNA as well as minimally processed samples, such as heat-treated urine, without a significant loss in sensitivity. Furthermore, the C. trachomatis RPA assay takes only 20 minutes until the final readout of the results and consequently has a potential to be applied as a POC test (Figure 3C). Due to on-site application, a short waiting time for results is highly important for POC tests. Other isothermal amplification assays (such as loop-mediated isothermal amplification or helicase-dependent amplification assay) regularly require 30 to 60 minutes for detectable amplification product formation.22,24,25 The developed assay enables detection of C. trachomatis within just 10 minutes of amplification at 38 C. Thus, the RPA-based amplification assay offers a significant advantage for on-site application compared with other isothermal amplification methods. C. trachomatis carries a small genome of an approximately 1-Mb chromosome and 7.5-kb multicopy cryptic plasmid.26 The C. trachomatis plasmid has been widely used as an amplification target for pathogen diagnostics. Although some plasmid-free C. trachomatis isolates have been described, their virulence is significantly reduced compared with plasmid-carrying strains.27 The Chlamydia plasmid carries eight major coding sequences. For development of the C. trachomatisespecific diagnostic method, we chose CDS2 because of its high conservation among different sexually transmitted Chlamydia strains19 (Supplemental Figure S1). The C. trachomatis assay developed here was able to detect at least 50 copies of the CDS2 target. C. trachomatis harbors, on average, between 4 and 10 copies of the plasmid per elementary body, depending on the strain and development stage.18,19 The lowest detectable amount of the C. trachomatis RPA assay can therefore be translated to 5 to 12

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pathogens per reaction and is in the same range as other nucleic acid amplificationebased techniques. Addition of urine did not affect significantly analytical sensitivity of the RPA, and the lowest amount detectable was in the same range for purified DNA as for heat-treated urine samples (Figures 1A and 3B). Thus, the assay could detect target DNA directly from minimally processed patient samples. However, up to 5 mL of urine could be used in the reaction without significant inhibition of the assay sensitivity. The theoretical minimal C. trachomatis count required to obtain a positive result in the RPA assay would therefore be 1000 to 2400 cells/mL of urine. Different patient samples are known to have a varying number of C. trachomatis cells in urine.28e30 Relatively common counts are 1  103 to 106 of C. trachomatis per mL of urine, present in 65% to 85% of patient samples.28,30 Evaluation of our C. trachomatis RPA assay using 70 clinical samples showed its sensitivity and specificity to be 83% (95% CI, 51%e97%) and 100% (95% CI, 92%e100%), respectively, compared with the Roche Cobas Amplicor C. trachomatis assay. Of the 12 C. trachomatisepositive patients, 10 tested positive with the RPA assay when 5 mL of heated urine was used as a template. The established clinical sensitivity (83%; 95% CI, 51%e97%) correlates well with the expected assay sensitivity of 65% to 85%, calculated on the basis of the analytical sensitivity of the assay and reported C. trachomatis counts in urine. Thus, the C. trachomatis RPA assay described here presents a significant advantage of increased sensitivity of detection compared with C. trachomatis POC immunoassays while displaying the same levels of high specificity. The reported sensitivity values for DNA amplification (eg, PCR)ebased C. trachomatis assays are 96%,31 which is somewhat higher than that established for the RPA amplificationebased C. trachomatis assay. However, they use purified and concentrated total DNA samples; when the same was performed for the RPA C. trachomatis assay, sensitivity increased to 100% (95% CI, 70%e100%) in the reported clinical evaluation (all of the C. trachomatise positive samples were recognized as positive) (Table 2). The sensitivity of the developed C. trachomatis RPA assay is limited by the amount of the urine sample (5 mL) that can be applied for analysis in one reaction. Studies have found that the first 5 mL of the collected urine sample is particularly rich in C. trachomatis genomic material. Using specially designed devices for first-void urine collection could increase C. trachomatis counts five to eight times compared with regular sample collection.30 Thus, by optimizing sample collection, the sensitivity of the C. trachomatis RPA assay could be further increased to levels comparable with laboratory-use diagnostics assays. The simplicity and speed of the developed C. trachomatis diagnostics assay enable its application in POC settings. The assay does not require the purification of total DNA from the sample. Robust sample preparation, such as heating of the sample for 5 minutes at 90 C, is enough to release a sufficient amount of the amplification target for diagnosis of

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Krõlov et al pathogen presence. Elimination of the DNA purification step before amplification makes the procedure significantly less laborious, less time-consuming, and therefore less expensive. The amplification is a one-step procedure that requires just 10 minutes of incubation at 38 C. Result visualization is performed using lateral flow detection strips that enable an answer to be obtained in a few minutes. The whole procedure therefore takes only 15 to 20 minutes and does not require expensive machinery, which makes it potentially applicable in POC settings. At the same time, the assay enables highly specific C. trachomatis detection with sensitivity levels significantly improved compared with currently available C. trachomatis POC assays.

Acknowledgments We thank the Sexual Health Clinique for collaboration during the clinical study, in particular Pille Veskilt and Bianka Peetson; Raili Randoja and Eva Reinmaa (Tartu University Hospital United Laboratories); Rya Ero (Institute of Molecular and Cell Biology, University of Tartu) who kindly provided E. coli gDNA; and Tatjana Brilene (University of Tartu) for microbiology advice and consultations.

Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.jmoldx.2013.08.003.

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