1016 obtain under routine clinic conditions than serum specimens, and are frequently contaminated with blood which makes interpretation of findings difficult. In any individual patient it is always difficult to interpret the significance of a stable antibody level to a common infecting agent. Thus without evidence of either seroconversion or of a significant increase in antibody titre, the only definitive way to diagnose active C. trachomatis infection is by demonstration of the organism, which is at present most easily achieved by isolation.

HEAT INACTIVATION OF PLASMA o:-MANNOSIDASE AND ACID

PHOSPHATASE

——————————————————& mdash;—————’_ _ _ _ _ _ _ _ _!_ _ _ _ _ _ _ _ _ _

(mol

Public Health Laboratory, Bristol BS2 8EL

Specific activities methylumbelliferone/h/ml plasma x 10-’; mean±SD) of unheated plasma samples were: a-mannosidase, 1.7::tO.4 (normal) and 1.9+0.7 (CF); acid phosphatase, 3.9 ±1-9 (normal) and 6.2 ±1.9 (CF).

Kingsdown,

the inactivation temperature was increased to 47 °C for both enzymes. Residual oc-mannosidase activity ranged from 31 to 54% for normal plasma and from 34 to 65% for CF plasma. The range of residual acid phosphatase activity was 42-91% for normal plasma and 34-86% for CF plasma. Department of Chemical Pathology, and M.R.C. Clinical Genetics Unit, Institute of Child Health, London WC1N 1EH

A. D. PATRICK R. B. ELLIS

IMMUNOFLUORESCENCE TESTING FOR CHLAMYDIAL ANTIBODIES

SiR,-Dr Nisbet and colleagues (Oct. 20, p. 859) describe a modification of the micro-immunofluorescence (micro-IF) test for detecting chlamydial antibodies which, they suggest, may be useful in diagnostic laboratories who wish to undertake diagnosis of Chlamydia trachomatis infections but lack facilities for the culture of these organisms. Whilst technical simplifications of the micro-IF test are to be welcomed, I feel that, Nisbet eat al. may be too sanguine about the diagnostic value of such serological tests, for the following reasons: (1) Antibodies to these common sexually transmitted organisms can frequently be demonstrated in patients from whom chlamydiae cannot be recovered by cell culture tech-

niques. 1-1 (2) Most patients from whom chlamydix are recovered already have chlamydial antibodies in their sera at the time the organisms are isolated.’.24 Therefore seroconversion cannot usually be demonstrated and a significant rise in antibody titre in subsequent serum samples frequently does not occur. (3) The association referred to by Nisbet et al. between chlamydial antibodies in secretions from mucosal surfaces of the genital tract and isolation of the organism probably reflects the fact that chlamydial antibodies in secretions are found in people with high antibody levels in blood, a proportion of whom are culture-positive.4-7 There is no evidence that chlamydial antibodies in these secretions are more significantly associated with active chlamydial infections than are high antibody titres ,

in

sera.

Moreover, specimens of secretions

1. Richmond

are more

difficult

to

SJ, Caul EO. Single-antigen indirect immunofluorescence test for screening venereal disease clinic populations for chlamydial antibodies. In: Nongonococcal urethritis and related infections. Washington, D.C.: American Society for Microbiology, 1977: 259-65. 2. Saikku P, Paavonen J. Single-antigen immunofluorescence test for chlamydial antibodies. J Clin Microbiol 1978; 8: 119-22. 3. Wang SP, Grayston JT, Alexander ER, Holmes KK. Simplified microimmunofluorescence test with trachoma-lymphogranuloma venereum (Chlamydia trachomalis) antigens for use as a screening test for antibody. J Clin Microbiol 1975; 1: 250-55. 4. Treharne JD, Darougar S, Simmons PD, Thin RN. Rapid diagnosis of chlamydial infection of the cervix. Br J VenerDis 1978; 54: 403-8. 5. McComb DE, et al. Chlamydia trachomatis in women: antibody in cervical secretions as a possible indication of genital infection. J Infect Dis 1979; 139: 628-33. 6. Hammerschlag MR, Anderka M, Semine DZ, McComb D, McCormack WM. Prospective study of maternal and infantile infection with Chlamydia trachomatis. Pediatrics 1979; 64: 142-48. 7. Richmond SJ, Milne JD, Hilton AL, Caul EO. Antibodies to Chlamydia trachomatis in cervicovaginal secretions. Sexual Transm Dis (in press).

SHIRLEY J. RICHMOND

SIR,-We agree with Dr Nisbet and colleagues that the micro-immunofluorescence test provides a valuable tool for detecting type-specific antichlamydial antibodies. This technique has been used routinely in our laboratory to detect antichlamydial antibody in patients with a variety of clinical conditionsl-6 and for the rapid serodiagnosis of ocular7 and genital8 chlamydial infections. Nisbet et al. describe a system for preparing chlamydial antigen comprising growth of the agents to high titre in eggs, inoculation of flasks of hela 229 cells, centrifugation of inoculated cells, freeze-thawing of cells and centrifugation cycles to purify and concentrate the antigen. With this system one 30 ml flask of tissue culture yields at least 0.11 ml of antigen. The technique described by Wang et al.9 which we use for the mass production of antigens comprises the first step of this technique only-i.e., preparation of antigen direct from the infected egg. Ten embryonated eggs are inoculated with each strain and from these at least one heavily infected yolk sac is usually obtained. Antigen is purified and concentrated by centrifugation to achieve a 5-10% suspension and one yolk sac provides at least 5 ml of antigen. Thus the minimum yield from one egg is fifty times greater than that from one tissue culture flask. The need for tissue culture is obviated, and less time, effort, and materials are required to produce antigen. Egg grown antigens are very stable, both as purified preparations stored at -70°C (which have been kept for 5 years without loss of antigenicity) and as acetone fixed "dots" on slides (which have been transported abroad and used in field

trips). We find that the presence of yolk material in the antigen provides a useful background and aids location of antigen dots during microscopy. We have no difficulty in differentiating the specific fluorescence of chlamydial particles with the dull staining amorphous background and if rhodamine/bovine-albumin is used as a counterstain, the yolk material appears orange and contrasts well with the green staining chlamydial particles. Virus Laboratory, Institute of Ophthalmology London WC1H 9QS

T. FORSEY S. DAROUGAR

Treharne JD, Jones BR, Herring J. Results of microimmunofluorescence tests for the detection of type-specific antibody in certain chlamydial infections. Br J Vener Dis 1972, 48: 452-59. 2. Treharne JD, Dines RJ, Darougar S. Serological responses to chlamydial ocular and genital infections in the United Kingdom and Middle East. In: Nongonococcal urethritis and related infections Washington, D.C: American Society for Microbiology, 1977: 249-58. 3. Treharne JD et al. Antichlamydial antibody in tears and sera, and serotypes of Chlamydia trachomatis isolated from school children m Southern Tunisia. Br J Ophthalmol 1978; 62: 509-15. 4. Mardh P-A et al. Role of Chlamydia trachomatis in non-acute prostatitis. Br J Vener Dis 1978; 54: 330-34. 5. Treharne JD et al. Antibodies to Chlamydia trachomatis in acute salpingitis. Br J Vener Dis 1979; 55: 26-29. 6. Spalton DJ, Darougar S, Sanders MD, Forsey T. Juxta papillary choroiditis in association with rising antichlamydial antibody. Br J Ophthalmol (in 1.

Dwyer RStC,

press). Darougar S. et al. Rapid serological test for diagnosis of chlamydial ocular infections. Br J Ophthalmol 1978, 62: 503-08. 8. Treharne JD, Darougar S, Simmons PD, Thin RN. Rapid diagnosis of chlamydial infection of the cervix. Br J Vener Dis 1978; 54: 403-08. 9. Wang S-P, Grayston JT. Immunological relationship between genital TRIC, lymphogranuloma venereum and related organisms in a new microtitre indirect immunoflurorescence test. Am J Ophthalmol 1970, 70: 367-74.

7.

Immunofluorescence testing for chlamydial antibodies.

1016 obtain under routine clinic conditions than serum specimens, and are frequently contaminated with blood which makes interpretation of findings di...
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