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Qualitative detection of Chlamydia trachomatis in both males and females



About Chlamydia

 

Introduction

 

The Chlamydiae are small, non-motile, gram negative bacteria that depend on the host cell for energy. In this way they resemble viruses, relying on the host cell for ATP synthesis and being unable to grow independently of the host cell. Their unique intracellular life cycle distinguishes the Chlamydiae from similar bacterial species. There are four known species of Chlamydia: Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae and Chlamydia pecorum. Chlamydia trachomatis has many different sub-groups called serovars which have been sub-divided into groups, dependant upon the disease condition caused1.

 

Epidemiology

 

Chlamydia trachomatis is the most common bacterial sexually transmitted disease. World-wide there are thought to be 50 million new cases of Chlamydia trachomatis infection annually1. In the UK, the prevalence of Chlamydia infection in women in high-risk populations, such as GUM (Genito-Urinary Medicine) clinic attendees,  has been reported to be 16.4%. In low risk populations such as those attending Family Planning Clinics, Obstetrics and Gynaecology clinics and GP surgeries, the mean prevalence is 4.5%-8.0%2. Estimated prevalence of Chlamydia infection amongst men attending a STD (Sexually Transmitted Disease) clinic is 15.4%3. The biggest challenge to the control of the disease is that as many as 80% of women and 50% of men have no symptoms. The most at risk groups are women under 20 years of age. This is thought to be due to anatomical differences in the cervix of younger women. Use of oral contraceptives and a high number of sexual partners also increases the risk of infection1.

 

Life Cycle

 

Chlamydia trachomatis exhibits an affinity for the epithelial cells of mucosal membranes such as those found on the surfaces of the cervix, urethra, rectum, nasopharynx and conjunctiva, and enter these cells by a phagocytic process4. Within infected cells, Chlamydiae occur in intracytoplasmic vesicles, or inclusion bodies. Within these inclusion bodies, morphological development takes place and  two distinct particles are observed: a small, dense infective particle, the elementary body which is transformed in the host cell into the larger less dense form, the reticulate body. These non-infective but metabolically active  reticulate bodies synthesise proteins and their own DNA and RNA, then replicate by binary fission to form microcolonies within the inclusion bodies. Between 18-24 hours post infection, the reticulate bodies divide and then ultimately some of the reticulate bodies reorganise into large numbers of elementary bodies. Between 48 and 72 hours post infection, the host cells ruptures releasing elementary bodies which can infect new host cells5. See below.

 

Life Cycle of Chlamydia

 

(Reproduced by permission from Gerald. J. Stine  (1992) The Biology of Sexually Transmitted Diseases. Wm. C. Brown, USA.)5

 

 

Chlamydia trachomatis Disease

 

Women:

 

Although women exhibit few symptoms, the disease manifests itself as cervicitis, urethritis and endometriosis, pelvic inflammatory disease and abscesses of the Bartholin glands1. The usual site of infection is the cylindrical epithelial cells of the endocervix. The cervix may appear congested and swollen. Bleeding may occur on contact and inter-menstrual bleeding and post coital bleeding occurring. Some women may have increased vaginal discharge. However, a large percentage of women may have a perfectly normal looking cervix. Asymptomatic infection of the urethra and rectum may accompany infection of the cervix6.

 

Pelvic inflammatory disease refers to infection of the endometrium, fallopian tubes and ovaries. Although it can be caused by a variety of infections, the leading cause is Chlamydia trachomatis. The disease may lead to infertility, increased risk of ectopic pregnancy and chronic pelvic pain. Early diagnosis and treatment of Chlamydia trachomatis is essential to avoid the infection spreading to the upper genital tract7.

 

Men:

 

In men, the main manifestation of Chlamydia trachomatis is urethritis, causing 40-50% of all non-gonococcal cases4. Urethritis may be characterised by a discharge and pain on micturition, although as many as 25% of men identified with urethral infections caused by Chlamydia trachomatis have no signs or symptoms of infection6. Infection of the urethra can progress to acute epididymitis4. As many as 60% of epididymitis infections are caused by Chlamydia trachomatis, which can have adverse effects on fertility8. Infections may also spread to the rectum, causing proctitis, and is more common in homosexual men. Left untreated, Chlamydia trachomatis may lead to arthritis or Reiters syndrome1.

 

Other Effects of Chlamydia trachomatis:

 

Chlamydia trachomatis during pregnancy increases the risk of still birth, neonatal death, early delivery and low birth weight. Chlamydia trachomatis can be passed from mother to child in the birth canal and is the leading cause of conjunctivitis and pneumonia in neonates. Infants with chlamydial pneumonia are at increased risk of pulmonary dysfunction in later life1.

 

Rarely, patients diagnosed with pelvic inflammatory disease or salpingitis may also develop Fitz-Hugh-Curtis syndrome (adhesions on the liver) or peri-appendicitis9.

 

Laboratory Diagnosis of Chlamydia trachomatis

 

Cell Culture:

 

The development of host cell lines revolutionised the detection of Chlamydiae and innoculation of cell lines still remains the gold standard method for the detection of Chlamydia trachomatis, i.e. the method to which all other methods of detection should be compared 9,10. However, the Chlamydiae are very fragile organisms. The swabs (female cervical or male urethral) must be transported to the laboratory in viral transport medium and inoculated into the cell culture as soon as possible. Keeping the specimen cold until arrival at the laboratory is critical for the viability of the organism. Chlamydiae isolated from urine samples are  not suitable for cell culture as urine is toxic to cell lines11. Once inoculated, the cell lines are incubated for 48-72 hours. In cell culture, Chlamydiae grow and produce intracytoplasmic inclusions. The preferred method for identifying inclusions is to stain the infected cells with a species-specific,  fluorescein-labelled monoclonal antibody for Chlamydia trachomatis. The stained inclusions can then be viewed microscopically10.

 

Compared with other diagnostic tests for Chlamydia trachomatis, the major advantage of cell culture isolation is excellent specificity (approaching 100%) and because of this, cell culture remains the standard for medico-legal cases of sexual abuse or assault. Cell culture technique is a highly skilled process and takes 2-3 days to obtain a result12. Also since only viable organisms are detected, the transportation and storage requirements are very stringent.  Finally specimens may also contain contaminating microorganisms or substances e.g. red blood cells, which are toxic to the cell lines used to isolate Chlamydiae10.

 

Polymerase Chain Reaction/Ligase Chain Reaction

 

Amplifying the DNA sequence of Chlamydia trachomatis provides a means of detection where the sensitivity is unparalleled by any other method. Even if only one elementary body is recovered this can be amplified to detectable levels. Positive results are possible from viable and non-viable Chlamydia trachomatis organisms, therefore specimen collection and handling is not as critical as with cell culture13,14. The method can also be used on male specimens4.

 

PCR/LCR has its disadvantages however, and in trials its specificity has not compared well with cell culture. This is because other organisms with similar sequences or contaminants may be amplified using the PCR/LCR approach. In addition to this, PCR technology is protected by patent and therefore commercial exploitation is costly10.

 

Rapid Immunoassay

 

Clearview Chlamydia MF is a rapid immunoassay for the detection of Chlamydia trachomatis antigen. These methods of detection of Chlamydia trachomatis have obvious advantages over cell culture. The tests detect an antigen present within the chlamydial cell, therefore a viable organism is not necessary in order to perform the test. Following on from this, it is not necessary to process swabs immediately as it is with cell culture. Clearview Chlamydia MF  can also be used together with culture methods as a means of confirmation of results.

 

The tests are more rapid than other methods of Chlamydia trachomatis detection. Tests can therefore be performed alongside the patient, avoiding the necessity to send the patient away to wait for results. Evaluation of near-patient testing has shown that a doctor is more likely to test a patient if the equipment is available in the surgery and likewise, patients are more likely to attend the surgery if they can be given results to tests during a visit13. The test can be used for male urine specimens rather than for urethral specimens, again reducing the trauma experienced by the patient. In the past, screening of males for Chlamydia trachomatis has been neglected because of the inconvenience and traumatic experience of collecting urethral swabs4. The test can also be performed using female endocervical swab specimens alongside the patient. The ease of use combined with rapid results through near patient testing may arguably have a positive impact on the numbers of patients screened for Chlamydia trachomatis. This may reduce the burden of the cost healthcare for people with Chlamydia trachomatis infections which have led to complications such as pelvic inflammatory disease, ectopic pregnancies and infertility.

 

Clearview Chlamydia MF: Assay Principle

 

The absorbent pad in the Sample Window contains a latex labelled monoclonal antibody directed against a genus-specific lipolysaccharide epitope of Chlamydia. When a specimen containing a Chlamydia trachomatis antigen is added to the sample pad, the chlamydial antigen binds to the latex labelled anti-chlamydial antibody. This moves along the test strip and binds to a region of immobilised anti-Chlamydia antibody in the Result Window.  If there is no Chlamydia trachomatis antigen present then the Result Window will remain clear.

 

At Start

 

Clearview CHlamydia MF: Assay Principle

 

 

A Positive Result

 

Clearview Chlamydia MF also provides an integral control feature. Unbound latex-labelled mouse anti-chalmydia antibody travels along the test strip to the Control Window which contains a region of immobilised rabbit anti-mouse antibody. Unbound mouse antibody binds to the immobilised anitbody in the Control Window forming a line and showing the test has worked correctly.

 

A Negative Result

 

A Negative Result

 

References

 

  1. Black C.M. (1997) Current methods of Laboratory Diagnosis of Chlamydia trachomatis Infections: Clinical Microbiology Reviews. 10(1), 160-184.
  2. Department of Health (1988). Main Report of the Chief Medical Officers Expert Advisory Group on Chlamydia trachomatis. Department of Health, London.
  3. Higgins SP, Klapper PE, Struthers JK, Bailey AS, Gough AP, Moore R, Corbitt G, Bhattacharyya MN (1998) Detection of male genital infection with Chlamydia trachomatis and Neisseria gonorrhoea using automated multiplex PCR system. Int. J. STD AIDS. 9(1), 21-4.
  4. Jaschek G., Gaydons C.A., Welsh L.E. & Quinn T.C. (1993) Direct Detection of Chlamydia trachomatis in Urine Specimens from Symptomatic and Asymptomatic Men by Using a Rapid Polymerase Chain Reaction: J. Clinical Microbiology. 31, 1209-1212.
  5. Stine G.J. (1992) The Biology of Sexually Transmitted Diseases: Wm C. Brown, USA.
  6. Cates W. & Wasserheit J.N. (1991) Genital chlamydial infections: Epidemiology and reproductive sequelae: Am J Obstet Gynecol. 164. 1771-81.
  7. Hillis SD & Wasserheit JN (1996) Screening for Chlamydia - A Key to the Prevention of Pelvic Inflamation Disease; The New England Journal of Medcine. 334(21), 1399-1401.
  8. Schachter J.; Sexually Transmitted Chlamydia trachomatis infection (1982) J. Postgraduate Medicine. 72, 60-69.
  9. Sam J.W., Jacobs J.E. & Birnbaum B.A. (2002) Spectrum of CT findings in Acute Pyogenic Pelvic Inflammatory Disease: Radiographics. 22(6), 1327-43.
  10. `Taylor-Robinson D. (1996) Test for infection with Chlamydia trachomatis: International Journal of TD & AIDS. 7, 19-26.
  11. Chan E. L. (2002) Laboratory testing for Chlamydia trachomatis urogenital infections. Journal of Family Planning and Reproductive Health Care. 28(3), 153-154.
  12. Jones R. B. et al (1986) Effect of blind passage and multiple sampling on recovery of Chlamydia trachomatis from urogenital specimens. Journal of Clinical microbiology. 24, 1029-1033.
  13. Kluytmans JAJW, Goessens WHF, Mouton JW, Van Rijsoort-Vos JH, Niesters HGM, Quint WGV, Habbema L, Stolz E & Wagenvoort JHT (1993) Evaluation of Clearview and magic Lite Tests, Polymerase Chain Reaction and Cell Culture for Detection of Chlamydia trachomatis in Urogenital Specimens; J Clin Micro. 31(12), 3204-3210.
  14. Dille B.J., Butzen C.C., & Birkenmeyer L.G. (1993) Amplification of Chlamydia trachomatis DNA by Ligase Chain Reaction: J. Clinical Microbiology. 31, 729-731.
  15. Rink E., Hilton S, Szeczepura J., Sibbald B., Davies C., Freeling P. & Stilwell J. (1993) Impact of Introducing Near Patient Testing for Standard Investigations in General Practice; BMJ. 307, 775-778.


 

 

 

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