Infection DOI 10.1007/s15010-015-0787-5
BRIEF REPORT
Chlamydia trachomatis genotypes in school adolescents, Italy Paola Stefanelli1 · Giorgia Sulis2 · Giovanna Renna1 · Franco Gargiulo3 · Paola Zanotti2 · Michela Capelli4 · Maria Antonia De Francesco5 · Francesco Donato4 · Sergio Pecorelli6 · Alberto Matteelli2
Received: 24 February 2015 / Accepted: 25 April 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Chlamydia trachomatis genogroups using ompA and multilocus sequence typing (MLST) were determined in consecutive isolates from school students aged 18 or older in the district of Brescia, Italy, 2012–2013. Among 40 samples, 4 ompA genovars and 18 STs were identified. Genovar E predominated (70 %) including five STs derived from ST59 (29 % of all isolates). This study, combining ompA and MLST typing of C. trachomatis school teenagers, suggests limited mixing and sexual interchange in this population. Keywords Chlamydia trachomatis · ompA gene · MLST · Survey · Typing
* Paola Stefanelli [email protected] 1
Department of Infectious, Parasitic and Immuno‑mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
2
University Division of Infectious and Tropical Diseases, Spedali Civili General Hospital, University of Brescia, Brescia, Italy
3
Laboratory of Microbiology, Spedali Civili General Hospital, Brescia, Italy
4
Unit of Hygiene, Epidemiology and Public Health, University of Brescia, Brescia, Italy
5
Department of Molecular and Translational Medicine, Institute of Microbiology, University of Brescia, Brescia, Italy
6
Clinic of Obstetrics and Gynaecology, Spedali Civili General Hospital, University of Brescia, Brescia, Italy
Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection (STI) in Europe, affecting almost 2 % of sexually active young adults [1]. In 2012, 384,105 cases of chlamydia were reported in 25 EU/EEA Member States, with an incidence rate of 199.4 per 100,000 inhabitants, compared to 469 cases in Italy [1]. Several typing schemes for C. trachomatis have been described. The most frequently used are based on the characterization of the highly variable ompA gene and on the sequencing of part of housekeeping genes by multilocus sequence typing (MLST) [2–5]. Nineteen serovars have been described: A, B, Ba, C (mainly observed among patients with trachoma), D, Da, E, F, G, Ga, H, I, Ia, J, Ja, K, (related to urogenital infections) and L1, L2, L2a and L3 (causing lymphogranuloma venereum, LGV), [6, 7]. Molecular typing of C. trachomatis permits to: (a) group bacterial strains for epidemiological purposes, (b) evaluate sexual networks, (c) differentiate among persistent infections and treatment failure from a re-infection and (d) monitor the spread of specific genotypes. We conducted a population-based cross-sectional study of C. trachomatis strains isolated from high school students aged 18 or older during a population-based epidemiological survey in the province of Brescia, North Italy. We report here the distribution of ompA genovars and sequence types obtained by MLST among C. trachomatis-positive patients. Briefly, a total of 4960 students attending 13th grade classes were contacted in 2012–2013; 4368 met the eligibility criteria and 2718 (67 %) signed a written informed consent, provided a valid urine sample, fully completed the socio-behavioural questionnaire and constituted our study sample. Adolescents with C. trachomatis infection received standard treatment with oral azithromycin and were further followed up to 1 year.
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P. Stefanelli et al. Table 1 Multilocus sequence typing (MLST) within each genovar (D–G) identified among 40 Chlamydia trachomatis DNAs
Genovar
E
D
G F
Total
N°
%
9 5 3 3 2 2 2 1 1 2 1 1 1 2 2 1 1 1
70
40
100
10
10 10
STsa
Alleles hctB
CT058
CT144
CT172
pbpB
1 7 7 1 64 5 71 9 35 10 5 9 5 10 12 5 5 5
19 19 19 2 19 19 2 2 19 8 2 2 19 8 5 19 19 19
7 14 7 6 7 7 7 7 7 1 5 7 7 1 11 7 7 1
2 2 2 2 2 2 2 2 2 4 2 2 1 4 9 1 15 1
1 1 1 2 1 2 2 2 1 17 4 2 4 5 5 4 4 4
56 16 59 3 334 237 342 262 153 35 188 262 12 128 296 12 238 90
a
Sequence types (STs) of C. trachomatis detected by MLST
The presence of C. trachomatis was assayed on 2 ml overnight urine using the Versant CT/GC assay (Siemens) on a Versant kPCR molecular system (Siemens). The extracted DNA was then amplified and sequenced for ompA gene to define the genovar [6] and for MLST with a modified version of the original Klint MLST scheme [3]. Sequencing was carried out bidirectionally with the appropriate sequencing primers [3]. Allele numbers were assigned by comparing the sequence at each locus to all known corresponding alleles available in the MLST database (http://mlstdb.bmc.uu.se). Allele profiles are expressed as sequence type (ST). DNA sequences were available for 40 of the 42 adolescents who had a positive result for C. trachomatis infection. As shown in Table 1, 4 ompA genovars and 18 STs were detected. Genovar E predominated (70 % of all isolates), subdivided into nine STs; five of them belonged to a cluster probably derived from ST59. Genovars D, G, and F were distinguished into four, two, and three STs, respectively. ST12 and ST262 were found in two different genovars. Overall, through MLST analysis, the highest genetic variability was observed in the hctB locus represented by ten different alleles. Among the 18 STs, 7 were found in both genders, 7 in females and the remaining 2 in males. All individuals within nine heterosexual couples shared the same genovar,
13
according to ompA sequencing. However, the members of four out of nine pairs did not share the same STs as shown by MLST analysis (ST56-ST3; ST56-ST237; ST56-ST59; ST59-ST16). All the 40 adolescents, with a confirmed C. trachomatis infection, were re-tested 3 months after supervised treatment with a single 1 g oral dose of azithromycin and three individuals were persistently infected. In particular, in one case a different ST was found (consistent with a re-infection from a new partner met after treatment completion); the other two showed the same ST (consistent with either re-infection or treatment failure). This is the first report of a molecular investigation of C. trachomatis in an unselected school population in Italy. Our study shows that genovar E is predominant in this young heterosexual population, consistent with the findings of a previous Italian analysis [7]. Moreover, genovar E is the most prevalent in other regions of the world and responsible for 40 % of urogenital infection in heterosexual patients [8–10]. The MLST system was more discriminatory than the ompA sequencing for C. trachomatis characterization, as already reported [11]. Within serovar E, one cluster of five different STs was identified. The existence of a “genogroup” including five STs, as established by the minimum spanning tree, probably reflects limited mixing and sexual interchange among our study population. The ST56 was
Chlamydia trachomatis genotypes in school…
first detected in this study among Italian teenagers and this result is consistent with that recently reported by others in Norway in an adolescent population [11]. We did not identify any isolate belonging to the ST55 genotype, generally linked to the deletion of 377 bp in the cryptic plasmid, which represents the target of some molecular diagnostic tests [12]. The detection of different STs among partners of four heterosexual couples suggests that the individuals had acquired the infection independently. Molecular analysis allowed us to establish that one of the three persistent infections was due to re-infection during the follow-up, since a different ST had been detected. Our study is limited by the small number of C. trachomatis-positive samples available. However, its strength is the systematic inclusion of consecutive samples from an unselected population of school adolescents. In conclusion, this is the first study combining ompA and MLST characterization of C. trachomatis isolates from teenagers in Italy, demonstrating the existence of a main genogroup circulating in the study area. Molecular typing was more useful compared to ompA in this populationbased epidemiological investigation on C. trachomatis. Acknowledgments The authors would like to thank all the adolescents who participated in the study and the research team of the Clamigon Study Group. Partial financial support was provided by Copan Italia S.p.A. and Siemens Healthcare Diagnostic S.p.A. Conflict of interest None. Ethics standard The study was approved by the Ethics Committee of both the Brescia Local Health Authority and the Spedali Civili General Hospital.
References 1. Annual epidemiological report. Sexually transmitted infections, including HIV and blood-borne viruses. 2014. ECDC. http:// www.ecdc.europa.eu/en/publications/Publications/sexuallytransmited-infections-HIV-AIDS-blood-borne-annual-epireport-2014.pdf. 2. Dean D, Bruno WJ, Ran R, Gomes JP, Devignot S, Mehari T, de Vries HJ, Morré SA, Myers G, Read TD, Spratt BG. Predicting
phenotype and emerging strains among Chlamydia trachomatis infections. Emerg Infect Dis. 2009;15:1385–94. doi:10.3201/ eid1509.090272. 3. Klint M, Fuxclius H-H, Goldkuhl RR, Skarin H, Rutemark C, Andersson SG, Persson K, Herrmann B. High-resolution genotyping of Chlamydia trachomatis strains by multilocus sequence analysis. J Clin Microbiol. 2007;45:1410–4. 4. Jurstrand M, Christerson L, Klint M, Fredlund H, Unemo M, Herrmann B. Characterization of Chlamydia trachomatis by ompA sequencing and multilocus sequence typing (MLST) in a Swedish county before and after identification of the new variant. Sex Transm Infect. 2010;86:56–60. doi:10.1136/ sti.2009.037572. 5. Lysén M, Osterlund A, Rubin C, Persson T, Persson I, Herrmann B. Characterization of ompA genotypes by sequence analysis of DNA from all detected cases of Chlamydia trachomatis infections during 1 year of contact tracing in a Swedish county. J Clin Microbiol. 2004;42:1641–7. 6. Bom RJ, Christerson L, Schim van der Loeff M, Coutinho RA, Herrmann B, Bruisten SM. Evaluation of high-resolution typing methods for Chlamydia trachomatis in samples from heterosexual couples. J Clin Microbiol. 2011;49:2844–53. doi:10.11.28/ JCM.00128-11. 7. Marangoni A, Foschi C, Nardini P, D’Antuono A, Banzola N, Di Francesco A, Ostanello F, Russo I, Donati M, Cevenini R. Chlamydia trachomatis serovar distribution and other sexually transmitted coinfections in subjects attending an STD outpatients clinic in Italy. New Microbiol. 2012;35:215–9. 8. Lysén M, Osterlund A, Rubin CJ, Persson T, Persson I, Herrmann B. Characterization of ompA genotypes by sequence analysis of DNA from all detected cases of Chlamydia trachomatis infections during 1 year of contact tracing in a Swedish County. J Clin Microbiol. 2004;42:1641–7. 9. Gharsallah H, Frikha-Gargouri O, Sellami H, Besbes F, Znazen A, Hammami A. Chlamydia trachomatis genovar distribution in clinical urogenital specimens from Tunisian patients: high prevalence of C. trachomatis genovar E and mixed infections. BMC Infect Dis. 2012;12:333. doi:10.1186/1471-2334-12-333. 10. Gita S, Suneeta M, Anjana S, Niranjan N, Sujata M, Pandey RM. C. trachomatis in female reproductive tract infections and RFLP-based genotyping: a 16-year study from a tertiary care hospital. Infect Dis Obstet Gynecol. 2011;2011:548219. doi:10.1155/2011/548219. 11. Gravningen K, Christerson L, Furberg AS, Simonsen GS, Ödman K, Ståhlsten A, Herrmann B. Multilocus sequence typing of genital Chlamydia trachomatis in Norway reveals multiple new sequence types and a large genetic diversity. PLoS One. 2012;7:e34452. doi:10.1371/journal.pone.0034452. 12. Ripa T, Nilsson PA. A Chlamydia trachomatis strain with a 377bp deletion in the cryptic plasmid causing false-negative nucleic acid amplification tests. Sex Transm Dis. 2007;34:255–6.
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BRIEF REPORT
Chlamydia trachomatis genotypes in school adolescents, Italy Paola Stefanelli1 · Giorgia Sulis2 · Giovanna Renna1 · Franco Gargiulo3 · Paola Zanotti2 · Michela Capelli4 · Maria Antonia De Francesco5 · Francesco Donato4 · Sergio Pecorelli6 · Alberto Matteelli2
Received: 24 February 2015 / Accepted: 25 April 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Chlamydia trachomatis genogroups using ompA and multilocus sequence typing (MLST) were determined in consecutive isolates from school students aged 18 or older in the district of Brescia, Italy, 2012–2013. Among 40 samples, 4 ompA genovars and 18 STs were identified. Genovar E predominated (70 %) including five STs derived from ST59 (29 % of all isolates). This study, combining ompA and MLST typing of C. trachomatis school teenagers, suggests limited mixing and sexual interchange in this population. Keywords Chlamydia trachomatis · ompA gene · MLST · Survey · Typing
* Paola Stefanelli [email protected] 1
Department of Infectious, Parasitic and Immuno‑mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
2
University Division of Infectious and Tropical Diseases, Spedali Civili General Hospital, University of Brescia, Brescia, Italy
3
Laboratory of Microbiology, Spedali Civili General Hospital, Brescia, Italy
4
Unit of Hygiene, Epidemiology and Public Health, University of Brescia, Brescia, Italy
5
Department of Molecular and Translational Medicine, Institute of Microbiology, University of Brescia, Brescia, Italy
6
Clinic of Obstetrics and Gynaecology, Spedali Civili General Hospital, University of Brescia, Brescia, Italy
Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection (STI) in Europe, affecting almost 2 % of sexually active young adults [1]. In 2012, 384,105 cases of chlamydia were reported in 25 EU/EEA Member States, with an incidence rate of 199.4 per 100,000 inhabitants, compared to 469 cases in Italy [1]. Several typing schemes for C. trachomatis have been described. The most frequently used are based on the characterization of the highly variable ompA gene and on the sequencing of part of housekeeping genes by multilocus sequence typing (MLST) [2–5]. Nineteen serovars have been described: A, B, Ba, C (mainly observed among patients with trachoma), D, Da, E, F, G, Ga, H, I, Ia, J, Ja, K, (related to urogenital infections) and L1, L2, L2a and L3 (causing lymphogranuloma venereum, LGV), [6, 7]. Molecular typing of C. trachomatis permits to: (a) group bacterial strains for epidemiological purposes, (b) evaluate sexual networks, (c) differentiate among persistent infections and treatment failure from a re-infection and (d) monitor the spread of specific genotypes. We conducted a population-based cross-sectional study of C. trachomatis strains isolated from high school students aged 18 or older during a population-based epidemiological survey in the province of Brescia, North Italy. We report here the distribution of ompA genovars and sequence types obtained by MLST among C. trachomatis-positive patients. Briefly, a total of 4960 students attending 13th grade classes were contacted in 2012–2013; 4368 met the eligibility criteria and 2718 (67 %) signed a written informed consent, provided a valid urine sample, fully completed the socio-behavioural questionnaire and constituted our study sample. Adolescents with C. trachomatis infection received standard treatment with oral azithromycin and were further followed up to 1 year.
13
P. Stefanelli et al. Table 1 Multilocus sequence typing (MLST) within each genovar (D–G) identified among 40 Chlamydia trachomatis DNAs
Genovar
E
D
G F
Total
N°
%
9 5 3 3 2 2 2 1 1 2 1 1 1 2 2 1 1 1
70
40
100
10
10 10
STsa
Alleles hctB
CT058
CT144
CT172
pbpB
1 7 7 1 64 5 71 9 35 10 5 9 5 10 12 5 5 5
19 19 19 2 19 19 2 2 19 8 2 2 19 8 5 19 19 19
7 14 7 6 7 7 7 7 7 1 5 7 7 1 11 7 7 1
2 2 2 2 2 2 2 2 2 4 2 2 1 4 9 1 15 1
1 1 1 2 1 2 2 2 1 17 4 2 4 5 5 4 4 4
56 16 59 3 334 237 342 262 153 35 188 262 12 128 296 12 238 90
a
Sequence types (STs) of C. trachomatis detected by MLST
The presence of C. trachomatis was assayed on 2 ml overnight urine using the Versant CT/GC assay (Siemens) on a Versant kPCR molecular system (Siemens). The extracted DNA was then amplified and sequenced for ompA gene to define the genovar [6] and for MLST with a modified version of the original Klint MLST scheme [3]. Sequencing was carried out bidirectionally with the appropriate sequencing primers [3]. Allele numbers were assigned by comparing the sequence at each locus to all known corresponding alleles available in the MLST database (http://mlstdb.bmc.uu.se). Allele profiles are expressed as sequence type (ST). DNA sequences were available for 40 of the 42 adolescents who had a positive result for C. trachomatis infection. As shown in Table 1, 4 ompA genovars and 18 STs were detected. Genovar E predominated (70 % of all isolates), subdivided into nine STs; five of them belonged to a cluster probably derived from ST59. Genovars D, G, and F were distinguished into four, two, and three STs, respectively. ST12 and ST262 were found in two different genovars. Overall, through MLST analysis, the highest genetic variability was observed in the hctB locus represented by ten different alleles. Among the 18 STs, 7 were found in both genders, 7 in females and the remaining 2 in males. All individuals within nine heterosexual couples shared the same genovar,
13
according to ompA sequencing. However, the members of four out of nine pairs did not share the same STs as shown by MLST analysis (ST56-ST3; ST56-ST237; ST56-ST59; ST59-ST16). All the 40 adolescents, with a confirmed C. trachomatis infection, were re-tested 3 months after supervised treatment with a single 1 g oral dose of azithromycin and three individuals were persistently infected. In particular, in one case a different ST was found (consistent with a re-infection from a new partner met after treatment completion); the other two showed the same ST (consistent with either re-infection or treatment failure). This is the first report of a molecular investigation of C. trachomatis in an unselected school population in Italy. Our study shows that genovar E is predominant in this young heterosexual population, consistent with the findings of a previous Italian analysis [7]. Moreover, genovar E is the most prevalent in other regions of the world and responsible for 40 % of urogenital infection in heterosexual patients [8–10]. The MLST system was more discriminatory than the ompA sequencing for C. trachomatis characterization, as already reported [11]. Within serovar E, one cluster of five different STs was identified. The existence of a “genogroup” including five STs, as established by the minimum spanning tree, probably reflects limited mixing and sexual interchange among our study population. The ST56 was
Chlamydia trachomatis genotypes in school…
first detected in this study among Italian teenagers and this result is consistent with that recently reported by others in Norway in an adolescent population [11]. We did not identify any isolate belonging to the ST55 genotype, generally linked to the deletion of 377 bp in the cryptic plasmid, which represents the target of some molecular diagnostic tests [12]. The detection of different STs among partners of four heterosexual couples suggests that the individuals had acquired the infection independently. Molecular analysis allowed us to establish that one of the three persistent infections was due to re-infection during the follow-up, since a different ST had been detected. Our study is limited by the small number of C. trachomatis-positive samples available. However, its strength is the systematic inclusion of consecutive samples from an unselected population of school adolescents. In conclusion, this is the first study combining ompA and MLST characterization of C. trachomatis isolates from teenagers in Italy, demonstrating the existence of a main genogroup circulating in the study area. Molecular typing was more useful compared to ompA in this populationbased epidemiological investigation on C. trachomatis. Acknowledgments The authors would like to thank all the adolescents who participated in the study and the research team of the Clamigon Study Group. Partial financial support was provided by Copan Italia S.p.A. and Siemens Healthcare Diagnostic S.p.A. Conflict of interest None. Ethics standard The study was approved by the Ethics Committee of both the Brescia Local Health Authority and the Spedali Civili General Hospital.
References 1. Annual epidemiological report. Sexually transmitted infections, including HIV and blood-borne viruses. 2014. ECDC. http:// www.ecdc.europa.eu/en/publications/Publications/sexuallytransmited-infections-HIV-AIDS-blood-borne-annual-epireport-2014.pdf. 2. Dean D, Bruno WJ, Ran R, Gomes JP, Devignot S, Mehari T, de Vries HJ, Morré SA, Myers G, Read TD, Spratt BG. Predicting
phenotype and emerging strains among Chlamydia trachomatis infections. Emerg Infect Dis. 2009;15:1385–94. doi:10.3201/ eid1509.090272. 3. Klint M, Fuxclius H-H, Goldkuhl RR, Skarin H, Rutemark C, Andersson SG, Persson K, Herrmann B. High-resolution genotyping of Chlamydia trachomatis strains by multilocus sequence analysis. J Clin Microbiol. 2007;45:1410–4. 4. Jurstrand M, Christerson L, Klint M, Fredlund H, Unemo M, Herrmann B. Characterization of Chlamydia trachomatis by ompA sequencing and multilocus sequence typing (MLST) in a Swedish county before and after identification of the new variant. Sex Transm Infect. 2010;86:56–60. doi:10.1136/ sti.2009.037572. 5. Lysén M, Osterlund A, Rubin C, Persson T, Persson I, Herrmann B. Characterization of ompA genotypes by sequence analysis of DNA from all detected cases of Chlamydia trachomatis infections during 1 year of contact tracing in a Swedish county. J Clin Microbiol. 2004;42:1641–7. 6. Bom RJ, Christerson L, Schim van der Loeff M, Coutinho RA, Herrmann B, Bruisten SM. Evaluation of high-resolution typing methods for Chlamydia trachomatis in samples from heterosexual couples. J Clin Microbiol. 2011;49:2844–53. doi:10.11.28/ JCM.00128-11. 7. Marangoni A, Foschi C, Nardini P, D’Antuono A, Banzola N, Di Francesco A, Ostanello F, Russo I, Donati M, Cevenini R. Chlamydia trachomatis serovar distribution and other sexually transmitted coinfections in subjects attending an STD outpatients clinic in Italy. New Microbiol. 2012;35:215–9. 8. Lysén M, Osterlund A, Rubin CJ, Persson T, Persson I, Herrmann B. Characterization of ompA genotypes by sequence analysis of DNA from all detected cases of Chlamydia trachomatis infections during 1 year of contact tracing in a Swedish County. J Clin Microbiol. 2004;42:1641–7. 9. Gharsallah H, Frikha-Gargouri O, Sellami H, Besbes F, Znazen A, Hammami A. Chlamydia trachomatis genovar distribution in clinical urogenital specimens from Tunisian patients: high prevalence of C. trachomatis genovar E and mixed infections. BMC Infect Dis. 2012;12:333. doi:10.1186/1471-2334-12-333. 10. Gita S, Suneeta M, Anjana S, Niranjan N, Sujata M, Pandey RM. C. trachomatis in female reproductive tract infections and RFLP-based genotyping: a 16-year study from a tertiary care hospital. Infect Dis Obstet Gynecol. 2011;2011:548219. doi:10.1155/2011/548219. 11. Gravningen K, Christerson L, Furberg AS, Simonsen GS, Ödman K, Ståhlsten A, Herrmann B. Multilocus sequence typing of genital Chlamydia trachomatis in Norway reveals multiple new sequence types and a large genetic diversity. PLoS One. 2012;7:e34452. doi:10.1371/journal.pone.0034452. 12. Ripa T, Nilsson PA. A Chlamydia trachomatis strain with a 377bp deletion in the cryptic plasmid causing false-negative nucleic acid amplification tests. Sex Transm Dis. 2007;34:255–6.
13
