Chlamydiae are Gram-negative bacteria which can cause a wide range of diseases in humans. Chlamydia trachomatis is the most common STI and usually results in asymptomatic infection and re-infection. The ever increasing number of infected people highlights the need for an efficient, rapid and cost effective screening program which is essential to control and prevent the spread of infection. Recently, nucleic acid amplification technology (NAAT) has been commonly used in the UK because of its high sensitivity and specificity. However, the current preferred detection method is the Chlamydia rapid test which allows for rapid screening and instant results.
Chlamydiae are Gram-negative obligate intracellular bacteria which only grow within cells. There are three species of Chlamydia, Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumonia. Chlamydia psittaci and Chlamydia pneumonia have been classified into another genus, Chlamydophila psittaci and Chlamydophila pneumonia, which infect the respiratory tract (Mims et al., 2004). Chlamydophila psittaci, causing Psittacosis (pneumonia), infects birds and mammals and is transmitted in humans via bird feces. Clinically, the different species of Chlamydia can cause a wide variety of infections in humans. Chlamydophila pneumonia can cause pharyngitis, bronchitis, and pneumonia in humans and is transmitted from person to person. C. trachomatis only infects humans and can cause blindness, infertility and ectopic pregnancy and is commonly sexual transmitted (Levinson, 2004). Furthermore, a pregnant woman infected with C. trachomatis can transmit the infection to her infant during delivery and which may lead to neonatal ophthalmia and pneumonia (2009b). According to the National Health Service (NHS), there were over 110,000 new cases of Chlamydia diagnosed in England and Wales in 2006. It has been proposed that sexually active women under 25 years old, men between 20 to 24 years old, people who have more than one partner, and people who do not have protective intercourse are high risk factors to develop C. trachomatis infection (2009a).
Chlamydia trachomatis infection
C. trachomatis is a worldwide sexually transmitted infection (STI) which usually causes re-infection of the genital tract in women (Roan and Starnbach, 2008). C. trachomatis species are divided into different serotypes. Serotypes A-C can cause trachoma, also known as conjunctivitis, infection of the conjunctive epithelium. Serotypes D-K can lead to genital infection and are STIs which mainly affect women and may result in chronic pelvic inflammation, infertility and ectopic pregnancy. Serotypes L1-L3 can lead to the development of urogenital tract infection and systemic disease such as lymphogranuloma venereum (Roan and Starnbach, 2008, Mims et al., 2004).
Although different C. trachomatis species can infect different types of tissue (Table 1), they mainly cause genital tract epithelial infection in women. The bacteria have a complicated replicative cycle and are present in two forms, the elementary body (EB) and reticulate body (RB) (Fig 1.). The cycle starts from the EB, which is an infectious form and is present in the extracellular environment. When the EB encounters a host cell, it enters into the cell via endocytosis and differentiates into a metabolically active RB, which is a non-infectious form, in two hours. The RB begins to replicate via binary fission within an expanding cytoplasmic inclusion. After 18 hours, RB starts to convert back to the EB form towards the end stage of development. About 36 to 48 hours, the EB lyses and is released out from the host cell into the extracellular space where it can then spread to the adjacent cells (Roan and Starnbach, 2008, Mims et al., 2004).
Currently, C. trachomatis infection can be treated using antibiotics such as azithromycin or doxycycline. However, the bacteria can cause asymptomatic infection resulting in re-infection (Roan and Starnbach, 2008). According to the report from the Centers for Disease Control and Prevention (CDC), from 1989 through to 2008, chlamydial infection has increased from 102.5 to 401.3 cases per 100,000 of the population. In addition, the reported C. trachomatis cases among women are highest in the age range of 15 to 19 years old and 20 to 24 years old. Infection in men is much lower than women, C. trachomatis infection in men is highest between 20 to 24 years old (2009b). These indicate inefficient Chlamydia screening and a large number of people who are high risk and have not been tested. Thus, efficient diagnosis of C. trachomatis infection and better control of the spread of infection is essential. This review mainly focuses on laboratory diagnosis of C. trachomatis infection with emphasis on nucleic acid amplification technology (NAAT).
Laboratory diagnosis of C. trachomatis infection
Sample collection and preparation
Sample collection differs for women and men. Urethral discharge or urine samples are usually collected form men, and endocervical swabs are commonly collected from women as C. trachomatis infection is frequently asymptomatic and localized in the endocervix in women (Herring et al., 2006).
In order to obtain high validity of test results, avoiding sample contamination is essential. For instance, vaginal swabs should be collected before cervical swabs as cervical discharge can contaminate the vaginal canal (Herring et al., 2006). Furthermore, two or three specimens should be collected for further detection, and samples from women should not be taken during menstruation. (Herring et al., 2006).
Invasive samples such as urethral discharge and endocervical swabs can be painful and cause embarrassment in patients. Non-invasive methods are available, for example, urine samples from men and women are acceptable for nucleic acid based methods. Furthermore, urine sample or vaginal swab specimens are suitable for Chlamydia Rapid Test (Mahilum-Tapay et al., 2007), and FirstBurst is another novel urine collection device for men to detect C. trachomatis infection (Wisniewski et al., 2008). Simple and more comfortable specimen collection from patient is essential. This may lead to men and women with asymptomatic infections who are willing to be tested which could lead to more efficient screening and control of the spread of infection.
Cell culture method
Laboratory diagnosis of C. trachomatis can be done by a traditional cell culture method. Since chlamydiae are obligate intracellular bacterium, the specimen can be cultured in a monolayer of tissue culture (McCoy) cells with cycloheximide, which assists chlamydial replication. After two to three days, C. trachomatis forms a cytoplasmic inclusion containing glycogen which can be stained with iodine and visualized by microscopy (Levinson, 2004). In addition, the specific major outer membrane protein (MOMP) of C. trachomatis can be directly stained by fluorescein-conjugated monoclonal antibodies after cell culture (Fredlund et al., 2004). The disadvantages of using traditional cell culture method are that it needs a rapid cold transport system to preserve the small amount of live organisms, is a time consuming technique, can be easily contaminated, is expensive, and requires trained technicians (Table 2). In addition, due to its low sensitivity (40%-50%), it is seldom applied to diagnosis of Chlamydia infection even though it was regarded as the gold standard test in the 1980s due to its high specificity (near 100%). Because of its lower sensitivity, it is now preferably used for clinical legal cases such as child sex abuse and for research purposes such as testing antibiotic resistance to treatment (Robert E. Johnson et al., 2002, Domeika et al., 2009). Overall, the method shows a very high specific detection of C. trachomatis but is time consuming and uses complex technology (Johnson et al., 2002).
Antigen Detection Methods
In addition, C. trachomatis can be directly detected using microscopy via direct fluorescent antibody test (DFA). Specimens from a swab or endocervical brush are stained with fluorescent monoclonal antibodies which bind to C. trachomatis elementary bodies on a slide and can then be visualized via UV microscopy. DFA method detecting C. trachomatis antigens involves either the MOMP or LPS molecule and anti-MOMP antibodies and anti-LPS monoclonal which are specific for C. trachomatis organisms (Robert E. Johnson et al., 2002). This assay is highly specific like cell culture and has been approved to detect rectal, cervical, urethral, and conjunctival specimens (Table 2) (Domeika et al., 2009). However, it is time-consuming, requires an expensive fluorescent microscope, needs an experienced microscopist to identify the stained C. trachomatis elementary bodies and is limited to a certain number of specimens (Black, 1997, Robert E. Johnson et al., 2002).
Chlamydia antigens can also be detected via enzyme-linked immunosorbent assay (ELISA) (Mims et al., 2004). This method mainly detects chlamydial lipopoly saccharide (LPS) which are adhered with monoclonal or polyclonal antibody. Enzymes specific to the antibody bind to the complex resulting in the production of color produces. This method can detect a large number of specimens (Table 2). However, it is easy to create false-positive results due to cross-reaction with other LPS microorganism (Robert E. Johnson et al., 2002). Even though these two methods are quicker than traditional cell culture and specific, they fail to deliver the high sensitivity in required asymptomatic patients (Van Dyck et al., 2001).
Serological test are helpful in detecting C. pneumonia. and C. psittaci, but due to the high frequency of C. trachomatis infection, many people already have antibodies against these species (Levinson, 2004). Even though many studies have proposed that high titres of antichlamydial antibodies present in chlamydial diseases such as pelvic inflammation and tubal infertility, chlamydial antibodies could have a delayed presence or absence during infection (Domeika et al., 2009). Also, chlamydial antibodies can last long after the infection has been cleared from infection. Thus, serological tests are not regarded as a reliable method in detecting C. trachomatis infection.
Diagnosis of Chlamydia trachomatis infection using NAAT
Nucleic acid amplification technology (NAAT)
Nucleic acid amplification technology (NAAT) has been recognized as the most reliable and efficient laboratory method in diagnosis of Chlamydia infections (Ward, 2003). It is a nonculture test and different commercial methods are available which have their own specific target sequences and amplification methods (Johnson et al., 2002). Specimen collection for NAATs is obtained from endocervical swabs or urine sample from women and urethral swabs or urine sample from men (Johnson et al., 2002).
There are several commercial kits available, and in the UK, Roche COBAS AMPLICOR PCR (polymerase chain reaction), BD ProbeTec ET SDA (strand displacement amplification) and the GEN-PROBE APTIMA 2 Combo TMA (transcription mediated amplification), are the three most generally applied assays (Dean et al., 2008)
1. COBAS AMPLICOR PCR (polymerase chain reaction)
This assay uses primers, CP24 and CP27, which are specific for 207 bp nucleotides of plasmid DNA from Chlamydia trachomatis (Dean et al., 2008). The specimens are prepared for PCR amplification mixture in tubes. During the PCR reaction, the double-stranded DNA is denatured by raising the reaction temperature, and the sequence specific primers which target the Chlamydia trachomatis plasmid DNA are annealed by reducing the temperature. The primers are extended by Taq polymerase which synthesizes double stranded DNA molecules. Therefore, this process utilizes the Chlamydia trachomatis plasmid as a target which increases the sensitivity of the assay by amplification and quantitation of the target DNA (Sevestre et al., 2009).
The PCR amplification products are then analyzed by COBAS AMPLICOR. The amplified double-stranded DNA molecules are denatured into single-stranded DNA by adding chemical denaturation solution. The denatured DNA is then coated with magnetic particles. Biotin-labeled Chlamydia trachomatis amplicon is hydrolyzed into specific oligonucleotide probes which can bind to the magnetic particles. As a result, this specific probe can increase the specificity of the test results (Dean et al., 2008).
After the hybridisation reaction, unbound magnetic particles are washed away and the specific probes with magnetic particles bind with horseradish peroxidase conjugate. Substrate solution containing hydrogen peroxide and tetramethylbenzidine (TMB) is added. The oxidation of TMB is catalyzed by horseradish peroxidase and results in the production of color. The COBAS AMPLICOR analyser measures the absorbance at 660nm wavelength (Ward, 2003, Dean et al., 2008). This assay needs to be carried out under thermal cycles and the specimens can be provided from swab and urine specimens respectively.
2. BD ProbeTec ET SDA (strand displacement amplification)
This assay uses amplification primers and fluorescent labeled detector probes to simultaneously amplify and detect target DNA. Specimens are first lysed using heat and prepared into two microwell strips and incubated with SDA reagents (Battle et al., 2001). One microwell contains priming components consisting of amplification primers and fluorescent labeled detector probes, and the other is a control to confirm that the samples were inhibited by the SDA reaction (Dean et al., 2008) . After incubation, the reaction mixture is transferred into amplification plates, and the plates sealed prevent contamination. The reaction mixture containing DNA polymerase and a restriction endonuclease are incubated and fluoresce is detected during the incubation, as the fluorescent labeled probe binds to the amplicons (Ward, 2003, Dean et al., 2008). As a result, this system can efficiently detect the real-time amplification of target DNA in sterile environment decreasing contamination (Van Der Pol et al., 2001).
3. GEN-PROBE APTIMA 2 Combo TMA (transcription mediated amplification)
This assay involves transcription-mediated amplification (TMA) of the target and dual kinetic assay (DKA). rRNA molecules specific to Chlamydia trachomatis are amplified by capture oligomers which are complementary regions of the target molecules (Gaydos et al., 2003). The oligomers: target complexes are formed during hybridisation, and the complexes are captured by reducing the temperature to ~25?. In the target capture stage, the specific sequences are isolated by magnetic particles and the specimen matrix containing amplification reaction inhibitors are removal (Dean et al., 2008).
Following capture of the target, it is the amplified. The TMA uses the target of 23S rRNA from Chlamydia trachomatis. The rRNA amplification is detected by the DKA which applies single-stranded chemiluminescent DNA probes, labeled with different acridinium ester molecules which are complementary to the target amplicon forming RNA:DNA hybrids (Dean et al., 2008, Gaydos et al., 2003). The light produced from the RNA : DNA hybrids is measured by a luminometer, and are reported as Relative Light Units (RLU) (Dean et al., 2008). Therefore, this assay uses rRNA by target capture which increases the system specificity.
Overall, many different approaches have been developed to allow amplification of target Chlamydia trachomatis nucleic acids, providing specific and sensitive increasing methods with a much higher throughput than previous methods employed.
The Advantages and limitations of NAATs
NAATs have high specificity and sensitivity (90-95%) allowing for specimens to be collected using noninvasive methods such as first-catch urine samples and self-collected vaginal swabs (Herring et al., 2006, Hadgu and Sternberg, 2009). Furthermore, the amplification of target DNA or rRNA via NAATs produces robust copies for detection and is suitable approach to be used for asymptomatic individuals (Domeika et al., 2009).
However, there are some limitations of NAATs. It requires well-trained laboratory technicians to avoid false positive results due to sample contamination (Herring et al., 2006). Therefore, the method also needs specific facilities and laboratory based equipment which is not adequate in the clinic (Domeika et al., 2009). Thus, NAATs cannot be performed in every setting, and specimens need to be sent away so that the bacterial nucleic acids can be extracted and amplified. Furthermore, patients cannot get instant results and need to wait until the test results are available. These disadvantages delay the diagnosis and discourage patients to take the test.
New point of care Chlamydia Rapid Test
Chlamydia trachomatis infection is the most common reported STI, and the majority people are asymptomatic or have mild symptoms. Previously, invasive methods such as urethral swabs in men and endocervical swabs from women were employed to collect specimens. These screening methods could discourage people to get regularly tested, preventing the control and spread of Chlamydia trachomatis infection(Mahilum-Tapay et al., 2007). Currently, NAATs are recognized as the preferable method for the diagnosis of Chlamydia trachomatis infection due to its high sensitivity and specificity, which allows for invasive sample collection from urine or vaginal swabs. However, it requires expensive laboratory instrument and well trained technicians. Therefore, Chlamydia Rapid Test (CRT) has been developed by the Diagnostics Development Unit, University of Cambridge (Mahilum-Tapay et al., 2007, Herring et al., 2006).
CRT is a new immunoassay and noninvasive test. The specimens are collected from first-void urine samples (before giving urine test samples, testers wait for 2 hours), vaginal swabs, or self collection. The extracted samples are mixed with the lyophilised amplification and detection reagents. A clear pink color develops, and the test strip added, which is coated with a monoclonal antibody to recognize chlamydial lipopolysaccharide (Mahilum-Tapay et al., 2007). The test result can be obtained within 30 minutes, providing an efficient diagnosis and screening tool for Chlamydia trachomatis infection. Overall, CRT provides high specificity (98.3%) with good reproducible results but lower sensitivity (81.6%) than NAATs. The novel rapid test can provide instant results and comfortable self sample collection. Thus, the method can attract more people to be tested and provide immediate treatment, allowing more efficient control of the spread of Chlamydia trachomatis infection via person to person and extend the screening to every setting not only limited in laboratory setting (Herring et al., 2006).
Chlamydiae are obligate intercellular Gram-negative bacteria, which can only grow in host cells. Chlamydial infection can occur in different kinds of tissue, but it is mainly apparent in the genital tract epithelial in women. Also, it usually results in re-infection and over 80% of infected people are asymptomatic. Chlamydia trachomatis infection is the most prevalent reported STI and can cause blindness, infertility and ectopic pregnancy. Traditional methods of Chlamydia trachomatis detection employed painful and invasive sample collection. Therefore, infectious patients and asymptomatic men and women could be discouraged to be tested of Chlamydia trachomatis infection. This trend has increased in the rate of Chlamydia trachomatis infection transmission. More improved techniques have been developed proving high specificity and sensitivity of results, allowing for noninvasive sample collection and higher throughput screening.
NAATs employs rapid amplification and detection of Chlamydia trachomatis target DNA. However, the limitations to the approach includes the technology well trained technicians and time period needed for test results to be obtained. Therefore, currently the new point of new point of care Chlamydia Rapid Test is used. This new and novel method allows for instant results and self sample collection, making this method the clinical preferable screening test. Overall, these new methods developed may allow for increased screening and treatment of Chlamydia trachomatis infection, providing more efficient control and reducing the Chlamydia trachomatis transmission.