Objectives Lymphogranuloma venereum (LGV) infections caused by Chlamydia trachomatis L types have recently emerged in Europe among HIV-positive men having sex with men. Our aim was to introduce a genotyping strategy suitable for a diagnostic laboratory using nucleic acid amplification tests (NAATs) for detection of C trachomatis and to investigate the prevalence of LGV types in rectal and pharyngeal specimens in Finland.
Methods Aptima Combo 2 (Gen-Probe) was used to detect C trachomatis in swabs. Altogether 140 C trachomatis NAAT-positive rectal and pharyngeal samples were genotyped by pmpH and ompA real-time PCR.
Results Of the 140 NAAT-positive rectal and pharyngeal specimens, 114 (81%) were successfully typed by pmpH PCR. One hundred and four samples contained non-LGV, nine samples LGV and one sample both non-LGV and LGV C trachomatis types. The C trachomatis LGV types were mainly found in rectal samples. Six of the L types were confirmed to be genotype L2b and two were L2 with ompA PCR and sequencing.
Conclusions Our experience suggests that genotyping C trachomatis by pmpH PCR can be introduced as a function of a diagnostic laboratory already using NAAT for detection of C trachomatis. The data show that LGV infections occur also in Finland. LGV should be taken into account when considering treatment and management of rectal C trachomatis infections.
- Chlamydia trachomatis infections
- lymphogranuloma venereum
- chlamydia infection
- molecular epidemiology
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- Chlamydia trachomatis infections
- lymphogranuloma venereum
- chlamydia infection
- molecular epidemiology
Lymphogranuloma venereum (LGV) is a sexually transmitted infection of the lymphatic system caused by three types (L1, L2 and L3) of the bacterium C trachomatis. After transmission, inguinal lymph nodes become swollen and painful and can break down into large ulcers. If untreated, this can lead to lymphatic obstruction and strictures. Classic LGV infections have been uncommon in Western countries, but since 2003 outbreaks of proctitis due to C trachomatis LGV types have been reported in Europe, North America and Australia, mainly among HIV-positive men who have sex with men (MSM).1 ,2 LGV infection has been a notifiable infection in Finland since 1932, but classic LGV infections have virtually been non-existent for decades. In the 1930s, there were 100–200 cases of LGV per year in Finland.3 The prevalence of LGV infections causing proctitis in Finland has not recently been investigated.
Based on the differences in the sequence of the ompA gene coding the major outer membrane protein, C trachomatis strains can be divided into different genotypes.4 Types D–K cause mucosal urogenital infections but types L1–L3 cause invasive LGV infections. In Finland and elsewhere, the detection of C trachomatis is almost exclusively based on sensitive and specific nucleic acid amplification tests (NAATs). These widely used commercially available tests do not allow the differentiation between genotypes or the identification of LGV genotypes. However, LGV infections can also be diagnosed using rapid, sensitive and specific, mainly in-house, real-time PCR tests.5
The aim of this study was to set up a method for rapid detection of LGV and non-LGV types in C trachomatis-positive samples for a diagnostic laboratory that uses a commercially available NAAT for large-scale C trachomatis testing. The other aim was to analyse the prevalence of LGV types among rectal and pharyngeal specimens in Finland. Additionally, we wanted to compare the genotype distribution of C trachomatis strains in rectal and pharyngeal specimens to that of the urogenital specimens in Finland6 and to those of the rectal and pharyngeal strains genotyped elsewhere.
Clinical samples and DNA extraction
Patients attending the outpatient sexually transmitted infection (STI) clinic of Helsinki University Central Hospital, Helsinki, Finland, because of symptoms, for a check-up or because notified by an infected partner are tested for genital C trachomatis and Neisseria gonorrhoeae. In women and men reporting oral sex, also pharyngeal swabs are taken. In heterosexual females reporting anal sex and in MSM as well as bisexual males, also anorectal swabs are taken. In MSM, also anorectal and pharyngeal swabs are collected for N gonorrhoeae testing and sera for HIV and syphilis screening. The specimens collected are sent to HUSLAB, the diagnostic laboratory of the Helsinki University Central Hospital serving mainly the capital area of approximately 1 million people. In HUSLAB, as part of a clinical service, the specimens are tested for C trachomatis ribosomal RNA by Aptima Combo 2 Assay (Gen-Probe). From February 2009 to August 2011, C trachomatis-positive specimens were anonymised and referred to genotyping. Study permits were obtained from Helsinki University Central Hospital, Laboratory Division (HUSLAB). The Ethics Committee of the Department of Medicine, Hospital District of Helsinki and Uusimaa evaluated the research plan.
DNA was extracted from Aptima Swab Specimen buffer (400 μl) (Gen-Probe) with MagNA Pure Compact instrument (Roche) using MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche) with DNA Bacteria protocol. The concentration of total DNA was measured with a NanoDrop spectrophotometer (Thermo Scientific).
Real-time PCR for genotyping C trachomatis
For LGV genotyping, we used a polymorphic membrane protein H gene (pmpH) PCR developed by Chen et al.7 The genotyping is based on a unique 36 base pair deletion in the sequence of the pmpH gene, which is present only in LGV strains.8 In this method, two different TaqMan probes are used to detect LGV and non-LGV types. More detailed genotyping was performed according to a method described previously by Jalal et al.9 The method is based on the sequence variation of the ompA gene, and it has two primer sets and 11 genotype-specific TaqMan probes for types D–K and L1–L3. In this study, we used the non-nested version of the method. As controls, DNA extracted from C trachomatis reference strains (types A–K and L2) originally from American Type Culture Collection (Manassas, VA, USA) propagated in McCoy cells (mouse fibroblast cells) was used.10
The primers and probes used in this study were purchased from Applied Biosystems, Oligomer and TAG Copenhagen A/S. The ompA PCRs were performed in a 25 μl volume containing 250 nM primers, 100 nM probes and either 12.5 μl Maxima Probe qPCR Master Mix (Fermentas Life Sciences, Leon-Rot, Germany) or 12.5 μl Platinum Quantitative PCR Supermix-UDG (Invitrogen Life Technologies). Real-time PCR analyses were performed with an ABI 7500 instrument and Sequence Detection Software version 1.3.1 (Applied Biosystems, Foster City, CA, USA). The pmpH PCRs were performed in a 25 μl volume containing 600 nM primers, 100 nM probes and 12.5 μl of master mix (as above). Real-time PCR analyses were performed with an ABI 7500 instrument and Sequence Detection Software version 1.3.1 (Applied Biosystems) or Rotor-Gene 6000 (Qiagen, Hilden, Germany) equipment. Thermal cycling conditions were as described above for the ompA PCR. Template volume was 2 μl, and each sample was amplified in duplicate. If no amplification was detected, 5 μl of template was amplified in a duplicate.
From February 2009 to August 2011, 1444 rectal and 5314 pharyngeal samples were tested for the presence of C trachomatis ribosomal RNA by Aptima Combo 2 Assay. Of the rectal swabs, 128 were from heterosexual women and 1316 from MSM or bisexual males. One hundred and thirteen rectal (7.8%) and 76 pharyngeal (1.4%) specimens were positive for C trachomatis. Of these specimens, 140 (97 from men and 43 from women) were available for genotyping. This included 84 rectal (70 from men and 14 from women) and 56 pharyngeal swabs (27 from men and 29 from women).
The C trachomatis-positive samples were first analysed with pmpH PCR. Of them, 114 (81%) specimens (85% of the rectal, 77% of the pharyngeal swabs) could be genotyped by pmpH PCR. Of the 114 samples, 104 samples (91%) contained non-LGV, nine samples (8%) LGV and one sample (1%) both non-LGV and LGV C trachomatis types. LGV DNA was detected in nine rectal swabs and in one pharyngeal swab. One patient was positive both at the rectal and pharyngeal sites, so altogether, nine patients with LGV infection were identified. Twenty-six specimens (19%) remained negative by pmpH PCR and could not be typed with this method. The genotyping results of pmpH PCR are presented in table 1.
ompA genotyping and sequencing
To more precisely type the C trachomatis strains, the specimens were analysed by ompA PCR.6 Of the 140 samples, 104 (74%) (71% of the rectal and 79% of the pharyngeal swabs) could be genotyped with the method based on amplification of the ompA gene. The distribution of genotypes per sampling site is presented in figure 1. In MSM, genotypes G, D, E and L2 were the most frequent genotypes detected in rectal swabs, while in heterosexual females, genotypes E and D were the most frequent. In pharyngeal swabs, genotypes E and D were most detected in men and genotypes E and F in women. Thirty-six samples (26%) could not be typed with ompA PCR, most likely due to the insufficient amount of bacterial DNA present in the sample. All 10 samples with genotype LGV by pmpH PCR were genotyped as L2 by ompA PCR.
The samples containing L2 DNA were also sequenced. Primers P1 and P29 were used to amplify the sequenced part (459 base pairs) of the ompA gene harbouring the nucleotide mismatches between the different L2 types (L2, L2a and L2b).11 This portion of the ompA gene was identical to the ompA sequence of the genotype L2b in six patients and identical to the ompA sequence of genotype L2 in two patients. We were not able to sequence L2 present in one rectal sample because of a L2-J mixed infection.
Characteristics of the patients with LGV infection
All nine patients with LGV genotypes at rectal sites were MSM, and all except one were HIV positive. Other sexually transmitted infections, including gonorrhoea (3/9) and syphilis (4/9), were also detected among these patients. The mean age of the LGV patients was 37 (range 22–52) and that of the male patients that had had a rectal swab taken (MSM and bisexuals; N=1316) was 34 years (range 13–77), whereas urogenital C trachomatis infections are most often detected in younger (20–24 years of age) patients in Finland.12 Moreover, C trachomatis was not detected in first-void urine or urethral swabs taken at the same visit in any of the patients with LGV in the rectal swab. In addition, pharyngeal swabs from two patients (No. 1 and 9) tested positive for C trachomatis by NAAT, but only one of those could be genotyped by ompA PCR (No. 9). Five of the nine LGV patients had had sexual contacts in other European countries, and half of the patients reported more than 10 sexual partners during the last 12 months. The characteristics of the LGV-positive patients are presented in table 2.
Although highly sensitive and specific NAATs are widely used for rapid detection of C trachomatis, specific diagnosis of LGV infections can often be delayed because of the lack of routine diagnostic methods for LGV types and unawareness of the disease. Recently discovered outbreaks of LGV infections (usually mild proctitis) in many European countries13 warrant for prompt specific diagnosis and for treatment of LGV to stop transmission. To further refine the routine laboratory diagnosis of C trachomatis infections and to explore the occurrence of LGV genotypes in an outpatient STI clinic in Finland, we set up and evaluated a novel strategy to genotype rectal and pharyngeal specimens positive by NAAT. In a majority of specimens, enough chlamydial DNA could be extracted from the NAAT specimen collection buffer and used as a template in real-time amplification reactions. In our earlier study, 90% of the C trachomatis-positive urogenital specimens could be typed.6 Here, 81% and 74% of the C trachomatis-positive rectal and pharyngeal specimens could be typed by the pmpH PCR and the ompA PCR, respectively. The observed difference in typing efficiency is most likely due to the scanty amounts of C trachomatis DNA, differential sensitivity of the assays (rRNA vs DNA detection) and/or potential inhibitory factors present in rectal and pharyngeal samples.
In our earlier study, no LGV genotypes were detected in the urogenital samples investigated by ompA PCR,6 and hence, we studied here rectal and pharyngeal specimens only. During the collection period between February 2009 and August 2011, 7.8% of rectal specimens tested positive for C trachomatis by Aptima Combo 2 Assay and 13% of the genotyped specimens contained LGV DNA. The C trachomatis positivity in rectal specimens is quite similar to that reported from UK. In the period 2006–2007, 6% of the 6778 rectal samples contained non-LGV and 0.9% LGV DNA.14 In the pharyngeal specimens, the occurrence of LGV genotypes was rare (2%, 1/43) in our material.
Of the nine patients with LGV, all were shown to harbour L2 as judged by ompA PCR. Six were confirmed by sequencing to be infected with genotype L2b and two with genotype L2. This reflects well the situation elsewhere in Europe.2 In general, the symptomatology of LGV infection can be rather misleading: only one of the patients with L2 had significant lymphadenopathy, whereas most patients with L2b had either proctitis that often was rather mild or were asymptomatic. In contrast, in the UK study, the majority of rectal LGV infections were symptomatic (95%).14 Based on clinical findings, it was impossible to differentiate infections caused by types L2 and L2b. In accordance with previous studies,13 also in our study, the patients with rectal LGV were MSM and most of them also had HIV and other STIs.
The genotype distribution in rectal specimens differed from that of the pharyngeal (this study) and urogenital swabs,6 although the rectal specimens included in this study were from both heterosexual women and MSM and the pharyngeal specimens were from both heterosexual men and MSM as well as from women. The four most common genotypes among rectal samples from MSM were G (29%), D (27%), E (17%) and L2 (17%). The observed genotype distribution is in good accordance with data from previous studies on MSM individuals.15–20 Genotypes E and D were most frequently (67%) detected in rectal swabs from women, but the numbers are low. Types G, D and E were also most frequent among rectal swabs from women and men in Hague, the Netherlands.15 In a previous study from the USA, women were more often infected with genotype E and less often with genotype G than MSM at rectal sites.20 Similarly, here the most prevalent genotype in rectal samples from women was E (N=5/12). In our study, the rectal swabs from heterosexual females tested more often positive for C trachomatis (13%) than the rectal swabs taken from MSM men (7%). This suggests that to prevent transmission and reduce morbidity, rectal C trachomatis infection should be sought for in heterosexual females with history of anal intercourse and not only in MSM. Recently, the first case of C trachomatis L2b proctitis in a woman was reported.21 In our material, we detected only non-LGV genotypes in rectal swabs taken in women. The four most common genotypes in pharyngeal samples were E (45%), D (16%), F (14%) and G (9%), whereas in the urogenital specimens, the most commonly detected genotypes seen were E (40%), F (28%), G (13%) and D (8%).6 Several explanations for the differences in the genotype distribution between rectal and urogenital specimens have been proposed: different transmission dynamics, core group-associated factors or tissue tropism. Indeed, it has been suggested that polymorphisms in certain open reading frame sequences correlate with rectal tropism of serovar G isolates.22 Sexual behaviour is also likely to contribute to the observed difference in genotype distribution as the genotype G is often found in male rectal specimens, but the genotype E is frequently detected in female rectal swabs.20
We do not know if the first LGV case truly emerged in Finland in 2009 or earlier as the clinical laboratories have used NAAT for diagnostics, and chlamydial culture (and typing methods) are practically no longer available. The first two LGV cases in Sweden, our neighbouring country, were reported in 2004.23 We were not able to perform a retrospective analysis on rectal and pharyngeal samples in Finland, but such analysis in Sweden 2004–2005 revealed no additional cases of LGV.17 Until 2010, almost 60 cases of LGV have been reported in Sweden.24 These infections in Sweden seem to be sporadic imported cases from other European countries with major outbreaks. This is probably the case also in Finland.
The LGV genotyping strategy using pmpH PCR utilised in this study is applicable to diagnostic laboratories, including those with no facilities to culture C trachomatis. Although the current NAATs for C trachomatis are not formally approved for analysis of rectal and pharyngeal samples, their performance has proven to be superior compared with C trachomatis culture.25 ,26 In this study, we detected both L2b and L2 genotypes that were differentiated by sequencing after pmpH PCR. However, recently a L2b-specific real-time PCR-based method was developed.27 The specific and prompt diagnosis of LGV infection is important because of therapeutic aspects28 ,29 to prevent transmission and for epidemiological surveillance. The typing strategy proposed here can be implemented in clinical laboratories and will assist in timely detection of infections.
C trachomatis identified in specimens sent for NAAT can be genotyped to facilitate prompt detection of LGV infection. The most common C trachomatis types identified in rectal specimens were G, D, E and L2. L2 were only detected in MSM.
A strategy of genotyping of C trachomatis by PCR in a diagnostic laboratory is feasible to obtain specific and prompt diagnosis of LGV.
All LGV types detected were L2 or L2b, and all patients were men who have sex with men (MSM).
Testing rectal sites among MSM is especially important to LGV detection.
We thank HUSLAB, Department of Virology and Immunology, Chlamydia Laboratory, for help in collecting the samples to be genotyped and Teija Tekkala for the statistical data.
Presented in part: 12th International Symposium on Human Chlamydial Infections, Hof bei Salzburg, Austria, June 2010 (abstract pp. 405–408).
Funding This study was supported by an R&D grant from Helsinki University Central Hospital, Laboratory Division (HUSLAB), MLE82TK013, by The Finnish Society against Sexually Transmitted Diseases and by the Academy of Finland in the frame of the ERA-NET PathoGenoMics, #217554/ECIBUG and #130043/ChlamyTrans.
Competing interests None.
Ethics approval The Ethics Committee, Department of Medicine at Hospital District of Helsinki and Uusimaa.
Provenance and peer review Not commissioned; externally peer reviewed.