Infection (2014) 42:905–912 DOI 10.1007/s15010-014-0665-6
ORIGINAL PAPER
Minimum spread of the new Swedish variant of Chlamydia trachomatis and distribution of C. trachomatis ompA genotypes in three geographically distant areas of Spain, 2011–2012 L. Pin˜eiro • S. Bernal • A. Bordes • J. C. Palomares R. Gilarranz • M. A. von Wichmann • G. Cilla
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Received: 3 February 2014 / Accepted: 8 July 2014 / Published online: 24 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose The aim of this study was to determine the presence of the new Swedish Chlamydia trachomatis (C. trachomatis) variant (nvCT) and the distribution of C. trachomatis ompA genotypes in three geographically distant regions of Spain. Methods The genotypes of strains causing 624 episodes of infection (January 2011–September 2012) were studied using a nested PCR that amplifies a fragment of the ompA gene, followed by sequencing. To detect nvCT, a real-time PCR was used that amplifies a fragment of the cryptic plasmid with a 377 base pair deletion, which identifies the nvCT.
L. Pin˜eiro G. Cilla (&) Microbiology Department, Hospital Universitario DonostiaInstituto de Investigacio´n Biodonostia, Paseo Dr Beguiristain s/n, 20014 San Sebastia´n, Spain e-mail:
[email protected] S. Bernal J. C. Palomares Clinical Unit of Infectious Diseases and Microbiology (UCEIM), Hospital Universitario Valme, Avda de Bellavista s/n, 41014 Sevilla, Spain A. Bordes R. Gilarranz Microbiology Department, Hospital Universitario Dr. Negrı´n, C/ Barranco de la Ballena s/n, 35010 Las Palmas de Gran Canaria, Spain M. A. von Wichmann Infectious Diseases Unit, Hospital Universitario DonostiaInstituto BioDonostia, Paseo Dr Beguiristain s/n, 20014 San Sebastia´n, Spain G. Cilla Biomedical Research Centre Network for Respiratory Diseases (CIBERES), San Sebastia´n, Spain
Results and conclusion The ompA genotype was identified in 565 (90.5 %) episodes. Eleven genotypes were detected, of which nine were found in all three regions. Only one nvCT strain was detected (0.4 %), despite the predominance of genotype E (41 %). Other frequent genotypes were genotypes D (19 %), F (13 %), G (11 %), and J (7 %). Genotype L2b, causing lymphogranuloma venereum, was detected in men who have sex with men (MSM) in all three regions. Genotypes E and F were more frequent in women and heterosexual men, and genotypes D, G, J and L2b in MSM. In men, the main factor causing differences in the distribution of C. trachomatis was sexual behavior (MSM versus heterosexual men), while the distribution of C. trachomatis genotypes was similar in women and heterosexual men. Keywords Chlamydia trachomatis New variant of Chlamydia trachomatis Genotyping ompA gene Genotype distribution Lymphogranuloma venereum
Introduction Chlamydia trachomatis (C. trachomatis) is the main bacterial cause of sexually transmitted infections (STI), mainly urethritis and vaginitis/cervicitis. This bacterium is an increasingly important public health problem, with an estimated 105.7 million new infections each year [1]. The infection may be in more than 50 % of cases asymptomatic and consequently its real incidence is probably higher than reported [2]. Untreated infections can lead to complications such as pelvic inflammatory disease or ectopic pregnancy in women, epididymitis in men, and infertility in both sexes. Moreover, in C. trachomatis-infected individuals, there is a greater risk of acquiring and, in the case of coinfection, of transmitting other STI.
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The main international agencies involved in STI surveillance recommend strengthening control of C. trachomatis infection through strategies that allow correct diagnosis and improved epidemiological surveillance [3, 4]. One strategy that can enhance knowledge of the epidemiology of C. trachomatis infection is identification of the serotype or genotype of circulating C. trachomatis strains through analysis of the major outer membrane protein (MOMP) or of the gene codifying MOMP (ompA), respectively. Such analyses have allowed C. trachomatis to be classified into 14 major serotypes/genotypes (A–C, D– K, L1–L3). This number increases if the distinct genovariants identified in some of these serotypes/genotypes are included (B/Ba, D/Da, G/Ga, I/Ia, J/Ja, L2/L2a/L2b, etc.). Moreover, in 2006 a new variant of C. trachomatis (nvCT) containing a 377-base pair (bp) deletion in the cryptic plasmid was reported in Sweden and spread rapidly in that country [5]. The aims of this study were to determine the presence of the nvCT and to analyze circulating C. trachomatis ompA genotypes in three geographically distant areas of Spain, given the scarcity of information on the molecular epidemiology of C. trachomatis and the high number of tourists visiting Spain from Scandinavian countries.
Materials and methods Patient samples Chlamydia trachomatis-positive samples consecutively detected in 2011 and 2012 in three geographically distant areas of Spain—Gipuzkoa (Basque Country, northern Spain), Seville (Andalusia, southern Spain), and Gran Canaria (Canary islands)—were frozen at -40 °C and sent to the Donostia University Hospital (Gipuzkoa) for genotyping. A total of 744 samples were processed from 616 patients (342 men and 274 women) with a mean age of 29.6 ± 8.4 years (median 28 years, range 0–77 years), in whom there were 624 episodes of C. trachomatis infection. The samples were obtained consecutively during a routine healthcare checkup from symptomatic patients, persons with risk exposures or pregnant women, attending STI clinics (46.7 %), general practitioner practices (29.3 %) or medical specialty offices (24 %). Samples obtained from a single patient within 6 months were considered to correspond to a single episode of C. trachomatis infection, except when the patient experienced two symptomatic episodes separated by at least one negative sample or when there were two positive samples with distinct genotypes (reinfection). Type of sample and the following clinical– epidemiological data were gathered: sex, age, origin (Spanish or immigrant), geographical area of residence,
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sexual behavior in men [classified as men who have sex with men (MSM) or heterosexual (HTS)], and symptoms (Table 1). For reasons of confidentiality, data collection was incomplete in some patients. The study was approved by the Ethics Committee for Clinical Research of the Donostia University Hospital. DNA extraction, amplification and sequencing techniques All the samples included in this study were C. trachomatispositive with the CobasÒ 4800 CT/NG test (Roche Diagnostics, Branchburg, NJ, USA), which targets both the cryptic plasmid and the chromosomal ompA gene, detecting both wild-type C. trachomatis (wtCT) and nvCT as well as plasmid-free C. trachomatis mutants. For genotyping, amplification of a 990 bp fragment of the ompA gene was performed with the remnant DNA from the commercial procedure, by using previously described methods [6]. When amplicons were not obtained, nucleic acids were re-extracted directly from the original samples using another method (NucliSENS Easy-Mag, Bio-Me`rieux, Marcy l’E´toile, France). Amplicons were sequenced through the use of the MOMP87 primer in the 3130XL Genetic Analyzer (Applied-Biosystems, Foster City, CA, USA). Sequences obtained were analyzed in the Basic Local Alignment and Search Tool (http://www.ncbi.nlm. nih.gov/blast/Blast.cgi). In samples with genotype E, a real-time PCR was performed that amplifies a fragment of the cryptic plasmid with the 377 bp deletion, which identifies the nvCT; primers and conditions were those described by Ripa et al. [7], but instead of FRET hybridization probes we used SYBR Green I (Roche Diagnostics, GmbH, Mannheim, Germany) and melting curve analysis in a Lightcycler 2.0 instrument (Roche), which allowed wtCT (melting temperature 80.5 °C) to be easily distinguished from nvCT (78.3 °C). For confirmation, the amplicons of strains suspicious for nvCT were sequenced. Purified DNA from the previously genome-sequenced nvCT (Sweden2) was included as a positive control in all PCRs [8]. Statistical analyses The Chi-square test was applied to compare categorical variables with application of Fisher’s corrections (two tailed) when required. The Mann–Whitney U test or analysis of variance was used to compare the means of continuous variables. To analyze associations between genotype distribution and the clinico-epidemiological variables of the study, those variables that obtained a significance of p \ 0.20 in univariate analyses were assessed
Spread of the new Swedish variant of C. trachomatis in Spain Table 1 Main characteristics of the 624 episodes (616 patients) of Chlamydia trachomatis infection included in this study and distribution by region
907
Total
Gipuzkoa
Seville
Gran Canaria
Statistical differences (p)*
No. of episodes
624
286
220
118
Age mean ± SDa
29.6 ± 8.4
30.7 ± 8.3
28.9 ± 8.3
28.3 ± 8.8
0.01** \0.01
Sex (%) Men
345 (55.3)
117 (40.9)
150 (68.2)
78 (66.1)
Women
279 (44.7)
169 (59.1)
70 (31.8)
40 (33.9)
Origin (%)
594 known
272 known
220 known
102 known
Spanish
478 (80.5)
181 (66.5)
205 (93.2)
92 (90.2)
Immigrant
116 (19.5)
91 (33.5)
15 (6.8)
10 (9.8)
232 known
86 known
144 known
2 known
92 (39.7)
23 (26.7)
67 (46.5)
2
HTSb Symptoms (%)
140 (60.3) 563 known
63 (73.3) 280 known
77 (53.5) 211 known
– 72 known
Symptomatic
321 (57.0)
180 (64.3)
72 (34.1)
69 (95.8)
Asymptomatic
242 (43.0)
100 (35.7)
139 (65.9)
3 (4.2)
Sexual behavior in men (%) MSMb
* Chi-square test unless otherwise specified ** Analysis of variance a
Excluding infants \1 year (n = 9) and patients of unknown age (n = 3)
\0.01
\0.01
\0.01
Type of sample (%)c Cervix/vagina
207 (33.2)
107 (37.4)
66 (30.0)
34 (28.8)
\0.01
Urine
164 (26.3)
63 (22.0)
94 (42.7)
7 (5.9)
\0.01
b
Urethra
164 (26.3)
86 (30.1)
11 (5.0)
67 (56.8)
\0.01
Rectal
64 (10.3)
14 (4.9)
48 (21.8)
2 (1.7)
\0.01
c
Conjunctival
14 (2.2)
6 (2.1)
0
8 (6.8)
NDd
Pharyngeal swab
10 (1.6)
9 (3.1)
1 (0.5)
0
NDd
Inguinal lymphadenopathy
1 (0.2)
1 (0.3)
0
0
NDd
MSM men who have sex with men, HTS heterosexual men Sample in which the genotype was obtained
d
ND: not done (due to the small number of episodes)
using a multivariate analysis through multinomial logistic regression. Because of the small number of episodes caused by some genotypes, genotypes A, B, I, K, H and L were excluded from this analysis. A p value of \0.05 was considered statistically significant. Analyses were performed with SPSS software (SPSS Statistics 20 Command, IBM, Chicago, IL, USA).
Results Genotypes detected and nvCT detection The C. trachomatis ompA genotype was obtained in 90.5 % (565/624) of the episodes [88.6 % of the samples (659/744)]. The percentage of genotyped episodes was 84.1 % (138/164) in urine samples, 90.9 % (149/164) in urethral samples, 93.8 % (60/64) in rectal samples, and 94.2 % (195/207) in cervical/vaginal samples (p = 0.008). Genotype determination was achieved in 100 % (175/175) of the episodes with positive samples with a cycle threshold \30 in the detection technique (CobasÒ 4800 CT/NG test), in 96.7 % (267/276) of those with a cycle threshold of 30–34 and in 71.1 % (123/173) of those with a cycle threshold C35 (p \ 0.001). Of the
565 genotyped episodes, 11 different genotypes were identified, the most frequent being genotype E (40.5 %) (Table 2). One nvCT strain in a urine sample from a MSM from Seville with multiple partners was identified (Fig. 1) and was confirmed through sequencing that yielded 100 % homology to published sequences of the nvCT (C. trachomatis plasmid pSW2, strain Sweden2, serovar E). Genotype E was followed in frequency by genotypes D (19.5 %), F (12.6 %), G (10.8 %), and J (7.3 %). The remaining genotypes (I, K, L2, H, A and B) were detected less frequently (0.2–3.0 %). Nine genotypes, among them L2, were detected in the three regions studied. In urogenital samples, two unusual strains of genotype A (a man and a woman without symptoms), and one of genotype B (a woman with a tubo-ovarian abscess and pelvic inflammatory disease) were detected [GenBank accession numbers: KF154357 (A), KF154358 (A) and KF154359 (B)]. During the study period, there were seven repeat episodes, of which five showed reinfection with a distinct genotype. Coinfection with Neisseria gonorrhoeae was documented in 9.5 % of the episodes (59/624), and was more frequent in patients attending STI clinics or primary care centers [11.4 % (54/474)] than in those attending specialized care [3.3 % (5/150)].
123
123
555
Total
249
565
Women
Total
435
Total
516
195
138
149
60
23
Cervix/vagina
Urine
Urethra
Rectum
Other
Sample
9 (39.1)
11 (18.3)
73 (49.0)
50 (36.2)
86 (44.1)
202 (39.1)
215
Total
82 (38.1)
301
Asymptomatic
120 (39.9)
218 (40.4)
539
Symptomatic
Symptoms
43 (41.3)
104
175 (40.2)
64 (29.2)
219
Total
Origin Spanish
Immigrant
53 (39.6)
85
134
HTSc
11 (12.9)
229 (40.5)
113 (45.4)
116 (36.7)
224 (40.4)
91 (37.9)
MSMc
Sexual behavior
316
Men
Sex
315
240
16–29
C30
133 (42.2)
229 (40.5)
565
Age(years)b
Total
76 (36.5) 100 (39.5)
208 253
Seville Gipuzkoa
53 (51.0)
104
Gran Canaria
Region
No. (%)
Feature
Total
E
Genotype
0.037
0.31
0.046