Rapid Determination of Lymphogranuloma Venereum Serovars of Chlamydia. trachomatis by quantitative High-Resolution Melt Analysis (HRMA)

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1 JCM Accepts, published online ahead of print on 29 August 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 Rapid Determination of Lymphogranuloma Venereum Serovars of Chlamydia trachomatis by quantitative High-Resolution Melt Analysis (HRMA) 3 Authors: Jimmy Twin 1,2*, Matthew P. Stevens 1,2, Suzanne M. Garland 1,2,3,4, Angelo M. Zaia 5, and Sepehr N. Tabrizi 1,2,3 Department of Microbiology and Infectious Diseases, The Royal Women s Hospital, Melbourne, Australia 1, Murdoch Childrens Research Institute, Melbourne, Australia 2, Department of Obstetrics and Gynaecology, University of Melbourne, Australia 3, Department of Microbiology, The Royal Children s Hospital, Melbourne, Australia 4, Microbiological Diagnostic Unit, Public Health Laboratory, University of Melbourne, Melbourne, Australia 5 * Corresponding author. Mailing address: Department of Microbiology & Infectious Diseases, The Royal Women s Hospital, Locked Bag 300, Parkville, Vic. 3052, Australia. Phone: (61-3) Fax: (61-3) jimmy.twin@mcri.edu.au Running Title: HRMA determination of C. trachomatis LGV serovars Key Words: Chlamydia trachomatis; HRMA; lymphogranuloma venereum 1

2 19 ABSTRACT A quantitative high resolution melt analysis (HRMA) assay was developed to differentiate lymphogranuloma venereum causing serovars of Chlamydia trachomatis (L1-3) from other C. trachomatis serovars (D-K). The detection limit of this assay is approximately 10 copies per reaction, comparable to other qpcr based methodologies. Downloaded from on April 12, 2019 by guest 2

3 25 Short-Form Paper Chlamydia trachomatis is an obligate intracellular pathogenic bacterium consisting of 15 serovars responsible for conditions such as trachoma (serovars A-C), genital tract infections (serovars D-K), as well as the more invasive genital infection lymphogranuloma venereum (LGV) (serovars L1-3). The less common LGV results in painful regional inguinal lymphadenopathy, which may coalesce to form buboes: without treatment systemic spread and involvement of other systems can occur (12, 15). Since 2003 there have reports of sporadic outbreaks of LGV associated proctitis in men who have sex with men (MSM), particularly those who are HIV-positive (5, 14). In general, LGV infections require a more prolonged and intensive treatment than that for C. trachomatis infections caused by serovars D-K. It is therefore, clinically important to differentiate LGV cases from other chlamydia infections. Sequence based genotyping methodologies have been described in order to distinguish LGV serovars of C. trachomatis from non-lgv serovars (9, 16). While these methods are effective, they tend to be laborious and more expensive than high-throughput screening, and rely on a nested-pcr step for low copy templates. Real-time PCR assays, utilizing fluorescent probes, have also been described which offer a more rapid discrimination of LGV causing serovars (2-4, 8, 11). High-resolution melt analysis (HRMA) is a more economical alternative to probe-based real-time PCR which consists of comparing nucleotide sequences which result in differences in melting of PCR products when such analysis is incorporated at the end of real-time PCR assays. To discriminate between LGV and non-lgv C. trachomatis serovars, HRMA assays based on the pmph gene (1) and the omp1 gene (6) have recently been described. Although both of these assays are capable of detecting LGV serovars, they require an additional nested PCR step to increase each assay s sensitivity. In this study, we describe a one-step quantitative HRMA assay that is able to 3

4 49 50 rapidly discriminate between LGV and non-lgv serovars of C. trachomatis in clinical samples The assay targets a 61bp PCR fragment of the omp1 gene, where LGV serovars carry a guanine at position 30 and non-lgv serovars an adenine, using primers CTHRM-F (5 -TGA AAA AAC TCT TGA AAT CGG T-3 ) and CTHRM-R (5 -TGC AAG GAG GAA GCA GAA-3 ) (Figure 1). This nucleotide change was noted through a recent phylogenetic study of C. trachomatis genotypes (13), and confirmed by aligning all known omp1 sequences on Genbank at the time of submission of this manuscript. The real-time PCR and HRMA were performed on the LightCycler 480 real-time instrument (Roche Diagnostics, Indianapolis USA) using the following conditions: an initial denaturation of template and activation of enzyme at 95 C for ten minutes followed by 55 cycles of 95 C for ten seconds, 55 C for twenty seconds, and 72 C for forty seconds. Following this was a melting schedule of 95 C for one minute, 40 C for one minute, and an increment from 65 C to 95 C at a ramp rate of 0.02 C/second with 25 acquisitions taken per C. The reaction mix consisted of 1x LightCycler 480 High Resolution Melting Master mix (Roche Diagnostics) and 200nM each of primers CTHRM-F and CTHRM-R, plus 5µl template DNA in a total reaction volume of 20µl. Validation of the assay was performed using Roche MagNA Pure extracted DNA (13) from C. trachomatis culture obtained from the ATCC Global Bioresource Center (A-Har-13 [VR- 571B]; B-HAR-36 [VR-573]; Ba-Apache-2 [VR-347]; C-TW-3 [VR-1477]; D-UW-3/Cx [VR-885]; E-Bour [VR-348B]; F-IC-Cal-3 [VR-346]; G-UW-57/Cx [VR-878]; H-UW-43/Cx [VR-879]; I-UW-12/Ur [VR-880]; J-UW-36/Cx [VR-886]; K-UW-31/Cx [VR-887]; L1-440 [VR-901B]; L2-434 [VR-902B]; and L3-404 [VR-903]). The DNA from a total of 66 clinical C. trachomatis positive rectal swab samples (LGV=22 ; non-lgv=44) were screened 4

5 against this assay. This consisted of 45 samples (LGV=1; non-lgv=44) from a study investigating C. trachomatis in men who have sex with men (13) and 21 LGV samples sourced from the Microbiological Diagnostic Unit (University of Melbourne). Of these samples, 47 (71.2%) were able to be quantified by the qpcr method described by Stevens et al. (11), with the remaining 19 requiring a nested PCR step. Standard curves for the HRMA assay were created using quantified D-UW-3/Cx [VR-885] and L2-434 [VR-902B] DNA at concentrations of 1x10 5 serially diluted to extinction, to also determine the limits of detection. The C. trachomatis HRMA assay was able to differentiate between all LGV and non-lgv causing serovars down to 10 copies per reaction using purified DNA run in triplicate (Figure 2). The average Tm for the LGV samples was C (SD=0.28 C; 95% CI to 75.71; n=57) and for the non-lgv samples was C (SD=0.21 C; 95% CI to 74.67; n=58). To investigate whether mixed LGV/non-LGV infections are detectable using this assay, DNA from D-UW-3/Cx [VR-885] and L2-434 [VR-902B] were screened at two concentrations: 1x10 5 copies of and 1x10 2 copies per reaction. When equal concentrations were used, a clearly separate melting profile was achieved; however the melting profiles matched that of the higher concentration when mixed in unequal quantities. In light of this, further work is warranted to investigate whether mixed LGV/non-LGV infections can be identified from clinical samples Table 1 summarises the screening results of the 66 clinical samples. Of the 47 C. trachomatis samples able to be quantified by the Stevens et al. method, 44 (sensitivity=0.9362; 95%CI: ) could also be typed with the HRMA assay according to their LGV/non-LGV status. The three quantified samples unable to be typed with the HRMA 5

6 assays possessed concentrations of 9, 46 and 69 copies per reaction according to the qpcr methodology. The HRMA assay was able to however, characterise five samples that previously required a nested qpcr step. The concentrations of these samples were found by the HRMA assay to be 15, 17, 22, 40 and 101 copies per reaction. There was no significant difference found between the concentrations calculated for the 44 samples by both the HRMA (average=2100; SD=6513) and qpcr (average=1367; SD=3437) methodologies (p=0.4118), and calculating the concordance correlation coefficient (7) using Stata 12 (10) showed a strong correlation of the results given by these two assays (Figure 3). The HRMA assay described is the first single step HRMA to identify LGV causing serovars of C. trachomatis and can potentially identify mixed LGV/non-LGV infections. Although this assay was shown to possess high analytical sensitivity, it was primarily designed to differentiate the LGV status of known C. trachomatis positives, and as with other HRMA assays, are not suitable as a primary diagnostic test. Acknowledgements We would like to thank the staff and students at the Women s Centre for Infectious Diseases (Royal Women s Hospital, Victoria, Australia) for their assistance in this study, which was also supported by the Victorian Government's Operational Infrastructure Support Program

7 117 FIGURE 1. Region of ompa gene targeted in HRMA assay The C. trachomatis HRMA assay amplifies a 61bp region of the ompa gene from positions 2 to 62. Discrimination between LGV and non-lgv serovars is possible due to a SNP at position 30 where LGV serovars carry a guanine and non-lgv serovars an adenine residue FIGURE 2. Melt curves and difference plot for C. trachomatis HRMA Typical results showing the discrimination of LGV serovars (i. depicted in blue) and non- LGV serovars (ii. depicted in red) for both normalized and shifted melting curves (A) and normalized and temperature-shifted different plots (B). Mixed LGV and non-lgv serovar infections are able to be detected (iii. depicted in green) when the concentrations of both types are roughly equal. FIGURE 3. Normal plot of differences between the C. trachomatis HRMA and qpcr methods A concordance correlation coefficient calculation was used to determine any differences between the C. trachomatis HRMA assay and the qpcr assay by Stevens et al (11). The correlation between difference and mean was found to be and no significant difference was observed between these two methods (Bradley-Blackwood F test = 0.887; p= ). 7

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