< > Home : FAQ : Professional : Chlamydiales : Immunobiology : Infections : Diagnosis & Treatment : Links : Contact Us

Classic diagnostics: Micro IF and EIA

[There is a huge literature on chlamydial serology and seroepidemiology, much of it well known, quite a lot of it poor quality. Here I propose  to discuss the micro immunofluorescence test and its undoubted contribution to chlamydial research, plus newer attempts to improve chlamydial serology. The following section will briefly review whether the concept of the 'serodiagnosis' of chlamydial infections is helpful].

The micro immunofluorescence test (MIF). 

The MIF test as classically performed consists of microdots of dried whole chlamydiae arrayed with a Chinese calligraphy nib (or whatever) on a microscope slide, to which they are glued by yolk or other protein. Dilutions of the test serum are incubated on these microdots and any bound immunoglobulin due to the presence of antibody is detected by fluorescence microscopy using a fluorescein isothiocyanate-labelled antiglobulin. 

The MIF test has played a key role in chlamydial research from the mid sixties onwards. When used with differentially adsorbed antisera it led to the serological classification of C. trachomatis into 15 serotypes, a classification which has stood the test of time and, furthermore, has been vindicated by  elucidation of the primary structure and variability of the C. trachomatis major outer membrane protein [see: MOMP and serological classification]. In the early days when it was not easy to grow chlamydiae from clinical material, the MIF test  afforded valuable insights into chlamydial epidemiology generally and, more recently, into the possible role of C. pneumoniae in chronic diseases [see: C. pneumoniae: serological evidence]. It made the reputations of some of our foremost chlamydial researchers. It is therefore hardly surprising that this test has in many ways dominated chlamydial thinking. It is arguably the best method available for measuring antibody to chlamydiae, at least until we know how some of the newer tests perform. But, for all these achievements, it is a truly dreadful test! Reasons for this statement are as follows:

1. It cannot be regarded as chlamydial specific.  In its classic form, the MIF uses whole, undefined chlamydial antigens. Some of these, for example chlamydial lipopolysaccharide or heat shock protein , have the capability of cross reacting with antibody to other bacteria [e.g. Haralambieva et al., 2001] or to other proteins. This is seldom adequately controlled in published studies and the antigen preparations used are rarely checked for contamination by Mycoplasma spp. Various attempts have been made in recent years to overcome this by removing or inactivating lipopolysaccharide etc. It is, however, almost impossible to determine whether such methods are successful because of the absence of unambiguous standards. There is generally poor agreement between the presence of chlamydial antibody by MIF and objective criteria of current infection, such as PCR or other direct detection test [Black et al., 1994; Emre et al., 1994; see also Persson & Boman 2000]. This is hardly surprising. It is well known for many infections that antibody can persist in the absence of demonstrable current infection. Even paired acute and convalescent sera do not necessarily discriminate. Thus, only 3 of 8 patients with culture proven C. pneumoniae infection showed rising titres [Chirgwin et al., 1991]. 

2. It is only poorly chlamydial species specific [Bourke et al., 1989; Ozanne & Lefebvre, 1992]. Thus, high level C. trachomatis antibody often cross reacts in MIF with whole Chlamydophila antigens, albeit at lower titres. Marston et al., 2002 describe the experimental development of a phage display peptide antigen selected for its ability to react with the paratope [antibody binding site] of a C. pneumoniae specific monoclonal antibody. However it is not possible to judge whether this antigen would be clinically useful [MEW comment: it seems unlikely].

3. It is subjective and tiring. MIF requires considerable experience, e.g. to distinguish species specific or surface (ring) staining from other kinds of staining or from non specific reactions. It is tedious, painstaking and eye straining work, particularly for screening large numbers of patients. 

4. It is only semi-quantitative and difficult to automate for screening purposes.

5. It has poor reproducibility. While this has recently been particularly apparent for MIF of C. pneumoniae antibody [Peeling et al., 2000] it is true for MIF generally. Interchange of sera between reference laboratories often led to different conclusions. This reflects both the difficulty and subjectivity of the test itself and the lack of agreed methods, a problem which bedevils chlamydial research generally [Dowell et al., 2001]. 

6. It does not distinguish between acute, sub acute and persistent / chronic infection.  Despite attempts [for C. pneumoniae] to assert the contrary based on different immunoglobulin classes, there is no convincing evidence one can distinguish between these types of infection serologically . Moreover many patients with acute C. trachomatis urethritis do not develop a significant IgM antibody response.

Non classic MIF and other methods: 'improved' serology?

An obvious way of trying to make chlamydial serology more user friendly is to use enzyme immunoassay . Chlamydial antigen , bound to a polystyrene microwell in bicarbonate buffer, is incubated with dilutions of the test serum, washed, reacted with enzyme labelled anti-immunoglobulin, and the bound enzyme quantitated by colorimetric assay in a spectrophotometer. Automated, even robotic equipment is available for doing this kind of assay. With appropriate standards it can be made fully quantitative, and certainly it is possible to screen large number of sera. Unfortunately one also looses some of the significant advantages of MIF, such as the ability to discriminate specific 'ring' staining from non specific 'diffuse' staining. 

Commercial companies have produced all sorts of modified serological methodologies in recent years, some of them using synthetic defined antigens such as MOMP peptides or recombinant acylated chlamydial lipopolysaccharide .  While the move to more defined antigens seems sensible, the Achilles heel of this approach is that it has to be validated against the MIF 'gold standard'. As already indicated, no test can be better than its reference standard [see: specificity]. Why validate a supposed better test against an obviously poor test? The dilemma is illustrated by some of the results for the Labsystems EIA. Bas et al., 2001 compared a number of different methods for measuring chlamydial antibody, either in patients with C. trachomatis infections or in blood donors and concluded that, using the Labsystems EIA for C. pneumoniae antibodies, no cross reactions between C. trachomatis and C. pneumoniae were found. However Gnarpe et al., 2000 reported that high titre C. trachomatis antibodies cross-reacted with the Labsystems C. pneumoniae EIA, while Stralin et al., 2001 found that patients with serological evidence of C. psittaci infection all reacted with the Labsystems C. pneumoniae EIA. Furthermore, of seven patients positive by the MIF test for C. pneumoniae, only three / seven were reactive with the EIA. In this case, is it the EIA or the MIF which is performing most specifically? Without direct evidence of C. pneumoniae infection, it is impossible to say.

For hyperlinked papers on some of these new serological methods see Nikkari et al., 2001; Ossewarde et al., 2000; Persson & Boman, 2000 and Tuuminen et al., 2000 and the next section. 

[MEW] August 2003

NEXT: Classic diagnostics: Do we need serodiagnosis?

References

Bas, S., Muzzin, P., Ninet, B.,  Bornand, J. B., Scieux, C. and Vischer, T. L. (2001). Chlamydial serology: comparative diagnostic value of immunoblotting, microimmunofluorescence test, and immunoassays using different recombinant proteins as antigens. Journal of Clinical Microbiology 39, 1368 - 1377. Full article   

Black, C. M., Fields, P. I., Messmer, T. O., Berdal, B. P. (1994). Detection of Chlamydia pneumoniae in clinical specimens by polymerase chain reaction using nested primers. European Journal of Clinical Microbiology and Infectious Diseases 13, 752 - 756.

Boman, J. & Hammerschlag, M. R. (2002). Chlamydia pneumoniae and atherosclerosis: critical assessment of diagnostic methods and relevance to treatment studies. Clinical Microbiology Reviews 15, 1 - 20. [Review]

Bourke, S. J., Carrington, D., Frew, C. E., Stevenson, R. D. & Banham, S. W. (1989). Serological cross-reactivity among chlamydial strains in a family outbreak of psittacosis. Journal of Infection 19, 41 - 45.

Campbell, L. A., Roberts, S., Inoue, S., Kong, L. & Kuo, C. C. (2001). Evaluation of Chlamydia pneumoniae 43- and 53-kilodalton recombinant proteins for serodiagnosis by Western Blot. Clinical Diagnostic Laboratory Immunology 8, 1231 - 1233.

Chirgwin, K., Roblin, P. M., Gelling, M., Hammerschlag, M. R. & Schachter, J. (1991). Infection with Chlamydia pneumoniae in Brooklyn. Journal of Infectious Diseases 163, 757 - 761.

Dowell, S. F., et al., (2001). Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada). Clinical Infectious Diseases 33, 492 - 503.  [Review].

Emre, U., Roblin, P. M., Gelling, M., Dumornay, W., Rao, M., Hammerschlag, M. R. & Schachter, J. (1994). The association of Chlamydia pneumoniae infection and reactive airway disease in children. Archives of Pediatric and Adolescent Medicine 148, 727 - 732.

Gnarpe, J., Nääs, J. & Lundbäck, A. (2000). Comparison of a new commercial EIA kit and the microimmunofluorescence technique for the determination of IgG and IgA antibodies to Chlamydia pneumoniae. APMIS 108, 819 - 824

Haralambieva, I., Iankov, I., Petrov, D., Ivanova, R., Kamarinchev, B. & Mitov, I. (2001). Cross-reaction between the genus-specific lipopolysaccharide antigen of Chlamydia spp. and the lipopolysaccharides of Porphyromonas gingivalis, Escherichia coli O119 and Salmonella newington: implications for diagnosis. Diagnostic Microbiology and Infectious Disease 41, 99 - 106.

Marston EL, James AV, Parker JT, Hart JC, Brown TM, Messmer TO, Jue DL, Black CM, Carlone GM, Ades EW, Sampson J. (2002). Newly characterized species-specific immunogenic Chlamydophila pneumoniae peptide reactive with murine monoclonal and human serum antibodies. Clinical Diagnostic Laboratory Immunology 9, 446-52. Full article

Nikkari, S., Puolakkainen, M., Narvanen, A., Aakre, O., Toivanen, P. & Leirisalo-Repo, M. (2001). Use of a peptide based enzyme immunoassay in diagnosis of Chlamydia trachomatis triggered reactive arthritis. Journal of Rheumatology 28, 2487 - 2493.

Ossewaarde, J. M., Tuuminen, T., Boersma, W. G., Sandstrom, M., Palomaki, P. & Boman, J. (2000). A preliminary evaluation of a new enzyme immunoassay to detect Chlamydia pneumoniae-specific antibodies. Journal of Microbiological Methods 43, 117 - 125.

Ozanne, G. & Lefebvre, J. (1992). Specificity of the microimmunofluorescence assay for the serodiagnosis of Chlamydia pneumoniae infections. Canadian Journal of Microbiology 38, 1185 - 1189.

Peeling, R. W., Wang, S. P., Grayston, J. T. et al., (2000). Chlamydia pneumoniae serology: interlaboratory variation in microimmunofluorescence assay results. Journal of Infectious Diseases 181 Suppl 3, S426 - 429.

Persson, K., and Boman, J. (2000). Comparison of five serological tests for diagnosis of acute infections by Chlamydia pneumoniae. Clinical Diagnostic Laboratory Immunolology 7, 739 - 744 Full article   

Stralin, K., Fredlund, H. & Olcen, P. (2001).  Labsystems enzyme immunoassay for Chlamydia pneumoniae also detects Chlamydia psittaci infections. Journal of Clinical Microbiology 39, 3425 - 3426. Full letter [and authors reply]   

Tuuminen, T., Palomaki, P. & Paavonen, J. (2000). The use of serologic tests for the diagnosis of chlamydial infections. Journal of Microbiological Methods 42, 265 - 279. 

NEXT: Classic diagnostics: Do we need serodiagnosis?