Genital Chlamydia trachomatis infections

Abstract

Chlamydia trachomatis infections affect young, sexually active persons. Risk factors include multiple partners and failure to use condoms. The incidence of infection has increased in the past 10 years. Untreated C. trachomatis infections are responsible for a large proportion of salpingitis, ectopic pregnancy, infertility and, to a lesser extent, epididymitis. Screening is a possible intervention to control the infection, which is often asymptomatic. The emergence of lymphogranuloma venereum proctitis in men who have sex with men, in Europe, and of a variant with a deletion in the cryptic plasmid, in Sweden, are new features of C. trachomatis infections in the last years. A diagnosis is best made by using nucleic acid amplification tests, because they perform well and do not require invasive procedures for specimen collection. Single-dose therapy has been a significant development for treatment of an uncomplicated infection of the patient and his or her sexual partner.

Introduction

Chlamydia trachomatis is an obligate intracellular bacterium. During its unique developmental cycle, two different forms are observed, elementary bodies (EBs), which are infectious but not able to divide, and reticulate bodies (RBs), which are metabolically active and able to multiply. Persistent forms can also be present under particular conditions [1].

C. trachomatis is the most common bacterium responsible for sexually transmitted infections. Most of these infections are asymptomatic and, if not treated, can lead to severe complications, mainly in young women. Advances in diagnostic techniques and methods of specimen collection make easier the detection, treatment and prevention of these infections of global public health significance.

C. trachomatis, a bacterium specifically found in humans, is currently divided into 19 serovars, according to the specificity of major outer membrane protein (MOMP) epitopes [2]. Serovars A, B, Ba and C are the agents of trachoma, a major cause of blindness in Africa, the Middle East, Asia and South America. Serovars D–K, including D, Da, E, F, G, Ga, H, I, Ia, J and K, are the most common sexually transmitted bacteria, and serovars L1, L2, L2a and L3 are the agents of transmission of lymphogranuloma venereum (LGV).

Epidemiology

With the exception of LGV, chlamydial infections are widely diffused among the general population, affecting mainly young people between 16 and 24 years of age. Risk factors include high frequency of partner change, multiple partners, unprotected sex, and being unmarried [3].

In the USA in 2006, more than one million cases of chlamydial infection, which is a notifiable disease, were reported to the CDC, corresponding to a rate of 347.8 cases/100 000, an increase of 5.6% as compared with the rate in 2005 (http://www.cdc.gov/std/stats/chlamydia.htm).

In Europe also, the incidence of chlamydial infections has increased in the past 10 years. In 2005, over 200 000 cases were reported in 17 European countries, (http://www.ecdc.europa.eu/en/Health_Topics/chlamydia_infection/aer_07.aspx), and this is probably an underestimate. Prevalence rates have been shown to range from 2% to 17% in asymptomatic women, depending on the setting, population and country.

In Denmark, the overall prevalence rate of infection was 456 cases/100 000 in 2007. In the UK, it has been reported that 10.3% of women and 13.3% of men <25 years of age are infected [3]. In comparison with other countries, the prevalence is lower in Switzerland, ranging from 2.8% in women [4] to 1.2% in men [5]. In France, where chlamydial infection is not a notifiable disease, screening studies showed large differences according to the population tested, ranging from 6–11% in individuals attending family planning centres [6] to 1–3% in individuals attending preventive medical centres of universities [7]. In 2005, the overall prevalence of C. trachomatis in the French population was 1.5% in the general population and 3% among 18–24-year-old individuals (6th Meeting of the European Society for Chlamydia Research, abstract P71, Goulet V, 2008).

LGV, endemic in tropical regions, was rare in industrialized countries until 2003. It presented as a genital ulcer with secondary lymphoid proliferation. In 2004, a cluster of cases presenting with proctitis was reported in Rotterdam [8,9]; these were cases of men who had sex with men, most being human immunodeficiency virus-seropositive. Subsequent reports from other European cities, e.g. Hamburg, Paris [10], London, Stockholm, Vienna and Zurich, and from North America and Australia, indicated the emergence of a new outbreak in this high-risk group. This outbreak was dominated by the C. trachomatis variant L2b, first described in patients from Amsterdam [11] and subsequently found in France [12], Germany, Canada and Australia.

Surveillance systems were established in different countries. In the UK, through February 2006, 327 cases of LGV (96% with proctitis) were reported [13]. In France, between 2002 and 2007, among 784 C. trachomatis-positive rectal specimens, 551 (71%) were from cases of LGV and 29% were positive for non-LGV serovars. Despite the information available, the number of LGV cases increased every year (Fig. 1). In 11 cases, LGV strains were isolated from non-rectal samples [14].

Figure 1.  Chlamydia trachomatis proctitis in France, 2002–2007 (lymphogranuloma venereum (LGV) and non-LGV isolates) (personal data).

Clinical Manifestations of C. Trachomatis Infections

Chlamydial infection can cause cervicitis in women and urethritis in men (Table 1). However, these infections produce few or no symptoms in approximately 70% of women and 50% of men [15] and thus remain undetected.

Table 1.   Clinical manifestations of Chlamydia trachomatis infections

Serovar	Clinical manifestation	Complication
A–C	Keratoconjunctivitis	Scarring trachoma, blindness
D–K	Males: urethritis, proctitis	Epididymitis
	Females: cervicitis, urethritis, proctitis	Endometritis, salpingitis, pelvic pain, ectopic pregnancy, perihepatitis (Fitz-Hugh–Curtis syndrome), infertility
	Males and females: conjunctivitis	Reiter’s syndrome, reactive arthritis
L1–L3	Lymphogranuloma venereum: inguinal syndrome, proctitis	Fibrosis, rectal stricture

Infections in men

C. trachomatis is the major cause of non-gonococcal urethritis and post-gonococcal urethritis. Urethritis can be complicated by acute epididymitis in young men. After 7–21 days of incubation, the symptoms include dysuria, and a moderate clear or whitish urethral discharge [16]. Acute proctitis can be associated with oculo-genital serovars, but is usually milder than that associated with LGV serovars. There is no evidence of the role of C. trachomatis in prostatitis [17], and chlamydial infection does not significantly contribute to male infertility [18].

Reiter’s syndrome (urethritis, conjunctivitis, arthritis and mucocutaneous lesions) or reactive arthritis have also been associated with genital C. trachomatis infections, with a high male/female ratio [17].

Infections in women

Women with cervicitis can be asymptomatic or may complain of mucopurulent vaginal discharge or postcoital bleeding. Oedema, congestion and bleeding of the cervix have been observed. Urethral infection can be associated with cervicitis. A culture-negative leucocyturia finding is suggestive of C. trachomatis infection.

Ascending infections can result from cervicitis. Endometritis is frequently associated with this and may produce irregular uterine bleeding. Salpingitis or pelvic inflammatory disease (PID) is often subclinical. It seems possible that, in Europe, C. trachomatis is the cause of at least 60% of cases of acute PID [19]. Salpingitis may lead to tubal scarring and severe reproductive complications. Two-thirds of all cases of tubal-factor infertility and one-third of all cases of ectopic pregnancy could be due to chlamydial infection [16,20]. Chronic pelvic pain linked to the presence of peritoneal adhesions may occur in more than 15% of women with previous episodes of PID [19].

Fitz-Hugh–Curtis syndrome, a perihepatitis observed after or in conjunction with salpingitis, is more commonly associated with chlamydial than with gonococcal infections.

There is little evidence, and this is conflicting, to implicate C. trachomatis in chorioamnionitis and adverse pregnancy outcome [19]. Postpartum endometritis occurs in 30% of women with antenatal chlamydial infection. In both men and women, C. trachomatis may be involved in conjunctivitis by auto-inoculation from the genital tract.

Neonatal infections

Infants of mothers with chlamydial infections can be infected at delivery. The transmission rate via infected vaginal secretions is high (50–70%). Approximately 30–50% of infants of infected mothers will have conjunctivitis 5–10 days after delivery. At least 50% of infants with conjunctivitis will have nasopharyngeal infection [16]. Chlamydial pneumonia develops in c. 30% of these cases, after 2–3 weeks of incubation. The untreated infection acquired at birth can persist for months or years [21,22].

LGV

LGV is associated with L serovars, which are more invasive than D–K serovars, affecting submucosal connective tissue layers, and being able to disseminate to locoregional lymph nodes. LGV proctitis can be misdiagnosed as inflammatory bowel disease [3], and lead to rectal stricture.

The persistence of LGV cases that may contribute to the transmission of human immunodeficiency virus infection highlights the importance of the need to control this infection.

Pathogenesis

Chlamydiae exhibit a unique biphasic developmental cycle consisting of the conversion of EBs to RBs, the division of RBs, and the reorganization of RBs back into EBs. The persistent cycle seems to be the norm.

Chlamydial persistence [23] has been described as a long-term association between chlamydiae and their host cells in which these bacteria remain in a viable but culture-negative state [24].

Characteristically, C. trachomatis infection is frequently low-grade or asymptomatic, and repeated infection is common, indicating that natural immunity is limited. The major sequelae arise as a result of inflammation and fibrosis. A key question is whether persistent forms of chlamydiae play a role in the immunopathology of disease. In vitro, some factors inducing the development of aberrant persistent forms of chlamydiae, e.g. nutrient depletion, antibiotics and cytokines, have been identified. Chlamydial interaction with the cytokine system of the host is likely to be central to disease, as the inflammation following chlamydial infection and exacerbated by re-infection leads to tissue damage and scarring.

Moreover, continued chlamydial Hsp60 expression secondary to the action of interferon-γ produced by the cell-mediated immune response might ultimately drive chronic inflammatory responses associated with the severe sequelae of chlamydial infection [25]. The presence of Chlamydia-specific anti-Hsp antibodies has been proposed as a marker of chronic C. trachomatis infection. Antibody response to the surface antigen, MOMP, is an important mediator of immunity. Antigenic variation can arise in response to antimicrobial and/or immune pressure, and may play a role in persistence and disease pathogenesis [26].

A cytotoxin and a type III secretion system have been described as virulence factors, but very little is known about this, due to the absence of genetic tools [27].

Direct Diagnosis

There have been major developments during the past 30 years. As C. trachomatis is an obligate intracellular bacterium, cell culture remains a reference method, but many commercial non-culture-based assays are now available for diagnosis (Table 2).

Table 2.   Direct detection of Chlamydia trachomatis

Method	Turn-around time	Advantages	Limits
DFA, direct fluorescent staining with monoclonal antibodies; EIA, enzyme immunoassay; NAAT, nucleic acid amplification test; SDA, strand displacement amplification; TMA, transcription-mediated amplification; NASBA, nucleic acid sequence-based amplification.
Cell culture	72 h	Specificity, strain	Sensitivity 80–85%
Antigen detection
DFA	45 min	Simple, unit test	Sensitivity 75–80% Subjective reading
EIA	4 h	Automation	Sensitivity 75–80% Low specificity (confirmatory test)
Point of care	30 min	Low cost, unit test
Molecular methods
DNA probing	2 h	Easy to perform	Sensitivity 75–80%
Hybrid capture	4 h	Sensitivity 95% Specificity 99%	Only for cervical specimens (FDA)
NAAT (real-time PCR, SDA, TMA, NASBA)	2–4 h	Sensitivity >95% Specificity 99%	Contamination, costly processing of specimen

Specimens

The type of specimen depends on the clinical picture, the diagnosis conditions, and the laboratory technique used for detection, with the conditions of transport and storage being adapted to the particular technique.

Invasive specimens include urethral swabs in men, and endocervical or urethral swabs, and specimens taken from the upper genital tract, in women (liquid from Douglas’s pouch, endometrium and tubal specimens). Other sites include the conjunctiva, nasopharynx or deeper respiratory tract.

Non-invasive self-collected specimens include first-void urine (FVU), vulvovaginal swabs, anal swabs and penile swabs (Table 3). The bacterial load of these specimens is a major aspect of their suitability for the diagnosis, which can be made only by using nucleic acid amplification tests (NAATs) [28]. Self-collected vaginal swabs have a lower bacterial load than endocervical swabs, but a higher load than FVU, and are very well adapted to screening programmes [29]. FVU is a suitable sample type for men [30]. The sensitivity of the results obtained with penile swabs is lower than that with FVU, and the results are not reproducible in our experience (6th Meeting of the European Society for Chlamydia Research, abstract P10, Barbeyrac de B, 2008).

Table 3.   Advantages and limits of main urogenital specimens

Sex	Specimen	Advantages	Limits	Usable technique
NAAT, nucleic acid amplification test; EIA, enzyme immunoassay. ^aOnly in association with cervical swabs to improve the diagnosis of infection.
Men	Urethral swab	High sensitivity	Invasive	All tests
	Urine	Non-invasive		NAATs
		Self-collected		Some EIA tests
	Penile swab	Non-invasive	Low sensitivity	NAATs
		Self-collected
Women	Cervical swab	High sensitivity	Invasive	All tests
	Urethral swab^a	High sensitivity	Invasive	All tests
	Urine	Non-invasive	Low sensitivity	NAATs
		Self-collected
	Vaginal swab	Non-invasive		NAATs
		Self-collected

Cell culture

Cell culture has near 100% specificity. However, it is not recommended for routine use, because of its lack of sensitivity, its technical complexity, the long turn-around time, the requirements concerning transport and storage of specimens, and the limited number of appropriate specimens [31,32]. Owing to the detection of only viable organisms, it remains the method of choice in medico-legal situations and for antibiotic susceptibility testing [33].

Antigen-based detection methods (direct fluorescent staining with monoclonal antibodies (DFA) and enzyme immunoassay (EIA))

DFA is rapid to perform and specific, but is subjective, and not suitable for a large number of specimens [32].

EIA tests can be automated. They are more reproducible than DFA, and the sensitivity of the best EIA is comparable to that of culture and lower than that of NAATs. They can give false-positive results due to cross-reactions with the lipopolysaccharide (LPS) of other microorganisms, and all positive EIA results must be confirmed.

Rapid or ‘point-of-care’ tests are proposed for patients who are unlikely to return for test results. They are not suitable for non-invasive specimens, have moderate sensitivity, and are not recommended for laboratory settings. A rapid test for diagnosis of chlamydial infection, recently developed by the Wellcome Trust [34,35], is based on a second-generation signal amplification EIA for chlamydial LPS in a dipstick-type format. The initial results are promising.

Nucleic acid hybridization tests

DNA probing (with Pace 2, Gen  Probe) was the first molecular DNA test for C. trachomatis, and was largely used before the advent of NAATs. The performance of these tests is comparable to that of the better antigen detection and cell culture methods. Pace 2 can be used with endocervical or urethral swabs, but is not recommended for use with non-invasive specimens [29].

The Digene Hybrid Capture II test is a nucleic acid hybridization test that is signal amplification-based. Its sensitivity is substantially higher than that of the Pace 2 test and is comparable to that of PCR [36].

NAATs

Because of their high sensitivity and specificity, and their possible use for a large range of sample types, including vulvovaginal swabs and FVU, NAATs are the tests of choice for the diagnosis of C. trachomatis genital infections.

Several commercial NAATs are available [36], and make use of different technologies: PCR and real-time PCR (Roche Diagnostics, Abbott, IL, USA); strand displacement amplification (Becton Dickinson, NJ, USA); transcription-mediated amplification (Gen  Probe); and nucleic acid sequence-based amplification (bioMerieux, Nancy L’Etoile, France). The major targets for amplification-based tests are generally multiple-copy genes, e.g. those carried by the cryptic plasmid of C. trachomatis, or gene products such as rRNAs.

These assays are automated and can be used for screening programmes and for the detection of C. trachomatis and Neisseria gonorrhoeae in the same specimen. Their primary disadvantage is the cost, which could be reduced by pooling specimens. Another drawback is the presence of inhibitors in specimens, which can be overcome by different procedures. Their specificity is very high. The necessity of confirmatory testing of positive specimens, previously recommended in low-prevalence populations, is controversial [37,38].

In 2006, a new C. trachomatis variant belonging to serovar E, with a 377-bp deletion in the cryptic plasmid, was described in Sweden [39], where it was reported in high proportions (10–65%) of the infected patients. There is currently no evidence that the variant has spread widely across Europe [40–43]. This new variant can obviously not be detected by amplification tests targeting the deleted area, but can be detected by amplification targeting a chromosomal gene, e.g. ompA or a rRNA gene. New versions of the COBAS Taqman v2.0 test and of the Abbott test allow simultaneous detection of the cryptic plasmid and of ompA, and simultaneous detection of two different regions of the cryptic plasmid, respectively.

The goal for the future is to improve the diagnosis of sexually transmitted infections by using multiplex tests, in particular DNA microarray technology.

Typing Systems

C. trachomatis strains are discriminated by serotyping based on the antigenic differences among the major MOMP epitopes. Genotypes corresponding to serovars can be separated by omp1 PCR–restriction fragment length polymorphism typing [44,45], which can be used directly on specimens. Sequencing of the omp1 gene can be more discriminating. Recently, genotyping methods exploiting genome variations, e.g. multilocus sequence [46] and variable-number tandem repeat [47] analysis, have been used to discriminate among strains.

Serology

Serology is useful only in some cases of C. trachomatis infection and in seroepidemiological studies [48]. It suffers from several drawbacks, including the serological cross-reactivity between C. trachomatis and Chlamydophila species, and the persistence of antibodies, which prevents a distinction being made between past and present infection. Although it is not recommended for the diagnosis of lower genital tract infections, or for screening in asymptomatic patients, serological testing may be useful for diagnosing LGV, neonatal pneumonia, and upper genital tract infections, and for the evaluation of tubal-factor infertility.

The serological methods available are complement fixation, microimmunofluorescence and EIA. The latter two allow the distinction among IgG, IgA and IgM.

The microimmunofluorescence method, which is species- and serovar-specific, and which is considered to be the reference method, was a complex technology in its original form. EIAs, which can make use of synthetic peptides from the variable domains of the MOMP or recombinant LPS, can be automated.

Screening

Screening programmes must be cost-effective and must be made acceptable to patients by using non-invasive procedures that allow sample collection at the patient’s home.

There are two main approaches to the design of screening programmes: proactive, consisting of screening the entire target population; and opportunistic, targeting individuals who attend a healthcare setting [49]. The opportunistic approach, targeting sexually active individuals under 25 years of age within a variety of healthcare settings, is used in most Chlamydia screening programmes in the USA, the UK and France. At this time, research studies are needed to establish the benefits and the disadvantages of Chlamydia screening programmes [50].

Treatment

Antimicrobial susceptibility

Evaluation of in vitro susceptibility is not currently performed, because a standardized method is lacking and MIC results are not consistently reproducible [51].

In vitro, the most active drug is rifampin, with the lowest MIC, followed by tetracyclines, macrolides, and some fluoroquinolones (ofloxacin and the newer compounds).

The potential of C. trachomatis to develop antimicrobial resistance has not been well studied, although some case reports [52–54] suggest resistance as a cause of treatment failure. However, mutants resistant to fluoroquinolones and to rifampin have been produced in vitro [55,56], and four clinical isolates that demonstrated in vitro resistance to macrolides were shown to carry mutations in a 23S rRNA gene [57].

Heterotypic resistance can be observed when cells are inoculated with a large number of organisms. It affects only a small proportion of organisms that are difficult to propagate and eventually lost through continued cell culture [58], suggesting that they may be ‘less fit’. Acquired antimicrobial resistance seems to be exceptional.

Management of infections

Both patients and their sexual partners must be treated. For treatment of uncomplicated lower genital tract infections in adults, major progress has been made in the use of single-dose therapy with azithromycin (1 g) [59]. A 7-day course of doxycycline gives comparable results, but with lower rates of compliance [60].

Guidelines have been proposed in different countries for the treatment of upper genital tract infections [61]. These require a longer treatment period (14–21 days), and the combination of several antibiotics to control other bacteria involved. The same duration of treatment is proposed for LGV. The possibility of persistence of the infection after treatment may justify the use of a test of cure (5 weeks post-treatment) in cases of presumptive treatment failure or during pregnancy.

In conclusion, the available opportunities for diagnosis of C. trachomatis infections of the genital tract using high-performance NAATs and specimens obtained through non-invasive procedures, in conjunction with single-dose antimicrobial therapy, should reduce the consequence of untreated infections.

Transparency Declaration

C. Bébéar and B. de Barbeyrac declare no conflicts of interest.