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Molecular diagnostics : LCR, the ligase chain reaction

The Abbott LCx^®  test.

The Abbott LCx^® (Abbott Laboratories, Illinois, USA), based on a ligase chain reaction , was the second nucleic acid amplification-based method to be introduced for the diagnosis of chlamydial genital tract infections. It is based upon the cryptic chlamydial plasmid, which provides modest six to ten fold prior amplification compared to a single copy gene. The test is designed to be species-specific for C. trachomatis. 

Swabs are collected into a special transport medium while a different urine resuspension buffer is used for urine specimens. The transport medium on heating the sample, releases chlamydial DNA from cells and separates it into single strands. For amplification the sample is added to a reaction mix consisting of four oligonucleotide probes, thermostable ligase and polymerase, plus nucleotide triphosphates and buffer. Four oligonucleotide probes, working in pairs, hybridise to complementary single strand DNA. Polymerase fills in the gap of a few nucleotides between a given probe pair. Once the gap is filled, ligase joins the pair of probes so that an amplification product complementary  to the original target sequence has been created. This product then serves as a target for subsequent rounds of amplification in a thermal cycler.

When the temperature is raised above the melting point of the hybridised amplification product, it falls apart into single strands. Lowering the temperature permits more probes to hybridise to the newly available targets. Repeated thermal cycling generates exponential probe amplification, rather than the target amplification of PCR. 

For detection, the two pairs of nucleotide probes are labelled with an antigenic chemical grouping, a hapten  . Each probe has either a capture hapten, which enables it to be trapped by antibody to the hapten bound on a magnetic microparticle, or a detection hapten. The labelling of the probe pairs is such that each amplicon  ends up with a detection hapten at one end and a capture hapten at the other. On incubation in the automated LCx^® analyser, rabbit antibody-coated microparticles  capture the amplicon via the capture hapten. The particles are transferred to a glass fibre matrix and a wash step removes unligated probes having detection hapten only. The glass fibre bound microparticles with amplicons are then reacted with alkaline phosphatase-labelled rabbit antibody to the detection hapten. After washing the bound antibody is detected automatically by conventional enzyme immunoassay, using a fluorescent umbelliferyl phosphate as a substrate and detection in a fluorimeter [a photometer which measures fluorescent light emission]. The amount of fluorescence produced is proportional to the amount of captured amplicon. After detection, to reduce the risk that amplified product might subsequently contaminate the work area, the amplicons are chemically destroyed in the analyser with hydrogen peroxide plus a metal chelator . An Abbott analyser is necessary for the test. The kit includes negative controls (for contamination), positive controls (for calibration) but no internal amplification controls (for amplification inhibitors in specimens).

[MEW Comment: A weakness of this procedure would appear to be that it relies in part on polyclonal antibody, the specificity and avidity of which, from a manufacturing viewpoint is likely to be more difficult to control than either a hybridization reaction (many tests) or the avidin-biotin reaction used in the Roche Amplicor^®].

Clinical trials.

The Abbott LCx^® like the Roche Amplicor^® PCR has withstood the test of time. Both tests are broadly comparable in terms of sensitivity and specificity, but true estimates are difficult because of the problems of discrepant analysis. Published estimates of the sensitivity and specificity of PCR and LCR for detecting C. trachomatis are potentially biased because of study design limitations (confirmation of test results in many studies was  limited to subjects who were PCR or LCR positive but culture negative). Relative measures of test accuracy are less prone to bias in incomplete study designs. One study of urine and urethral samples from 1,138 asymptomatic men evaluated the probable levels of bias in sensitivity and specificity estimations, comparing PCR and LCR testing of urine versus culture of urethral specimens. Discordant results (PCR or LCR positive, but culture negative) were confirmed by using a sequence including the other DNA amplification test, direct fluorescent antibody testing, and a DNA amplification test to detect the gene encoding chlamydial major outer membrane protein. Very few false-positive results were found, indicating that the specificity levels of PCR, LCR, and culture are very high. The sensitivity of culture was estimated between 60 and 65%, giving a true sensitivity of PCR and LCR of between 90 and 97%.  [Cheng et al., 2001].  This is consistent with the manufacturer's figures for the purposes of calculating predictive values, of a sensitivity of 93.1% and a specificity of 97.1% [Table 2; LCx^® package insert dated April 2001].

Hjelm et al., 2001 evaluated cervical, vaginal and urine samples from 1,001 women in Uppsala, Sweden by LCx in comparison with cervical and urethral cell culture. A single urine specimen had a sensitivity of 80%, a single vaginal specimen a sensitivity of 96%, and a cervical specimen a sensitivity of 92%. However these differences were not statistically significant. Specificity was virtually 100% [Hjelm et al., 2001].

Puolakkainan et al., 1998 found the performance of the Roche Cobas Amplicor^® and Abbott LCx ^® ligase chain reaction to be broadly similar, with kappa coefficients of consistency of 0.93 and 0.95 for swab and urine specimens respectively. 

Another study evaluated the performance of the Abbott LCx^® LCR on a genitourinary medicine clinic population in the UK. The sensitivity on samples from 417 women was 100% for endocervical;  91% for vaginal specimens and 95% for female urine when compared with direct immunofluorescence on cervical samples. On  urethral and urine samples from 317 men it was 100% and 91%, respectively when compared with direct immunofluorescence on urethral smears. However, urethral swabs from a number of treated men remained LCR-positive when antigen was no longer detectable by immunofluorescence. An association between quantitative data from the LCR assay and the antigen load of the samples observed at immunofluorescence indicated a lack of significant inhibition of the LCx^® assay, probably because freezing had removed inhibitors prior to testing [Thomas et al., 2001]. 

As with other nucleic acid amplification based assays, significant reproducibility problems can occur during routine use of the LCx^® C. trachomatis assay. A study by Gronowski et al., 2001 outlines some of the problems which may be experienced that are missed by the recommended quality control procedures in the assay package insert. Usefully,  the methods used to resolve these problems in collaboration with Abbott are described. The full paper may be downloaded from the hyperlink provided below [Gronowski et al., 2000].  

The excellent sensitivity of the LCx^® assay makes it suitable for population screening studies in large populations, with urine the usual sample [see: non-invasive testing]. One monumental study, screened urine from 13,204 female US army recruits, with an overall prevalence of C. trachomatis infection of 9.2% and a peak prevalence for recruits from 5 southern states of some 15% [Gaydos et al., 1998].  Complete inhibition of LCR amplification by urinary inhibitors is rare; a reported 3.1% for urine from non pregnant women and 1% for pregnant women [Cherneskey et al., 1998]. For LCR, the main urinary inhibitor appears to be nitrite. Freezing the urines, or holding them in a refrigerator greatly reduces the amount of inhibition. It was anticipated that most urinary inhibition of the LCx^® would disappear in transit or during processing, meaning that a positive control for every urine is not required [Cherneskey et al., 1998]. The LCx^® was used for screening for chlamydial genital tract infections in a stratified probability sample survey of sexual attitudes and lifestyles on 11,161 men and women aged 16 - 44 years in Britain. Half of all the sexually experienced respondents aged 18 - 44 years were invited to provide a urine sample. C. trachomatis was found in 2.2% of men and 1.5% of women with age-specific prevalence highest among men aged 25 - 34 (3.1%) and women aged 16 - 24 years (3.0%). As would be expected, non-married status, age, and reporting partner concurrency or two or more sexual partners in the past year were independently associated with infection with C trachomatis [Fenton et al., 2001]. A population based survey of adults aged 18 - 35 years in Baltimore in the USA found using LCx^® that 3% of the population had untreated chlamydial infection [Turner et al., 2002].  Such studies are important for defining locally appropriate control strategies.

A number of new uses are being explored for the LCx^® assay. It has successfully been used to detect chlamydial DNA in the synovial fluid of patients with suspected reactive arthritis [Nikkari et al., 1997]. Two independent studies now indicate that the LCx^® assay, like PCR, can be successfully combined with cervical cytology screening using specimens collected in cytology preservative fluid [Augenot et al., 2001; Zhang et al., 2002]. In Denmark, Kjaer et al., 2000 conducted an interesting survey over 24 weeks on patient-collected and mailed urine or vaginal specimens of recurrent infection in women who had been treated for chlamydial infection. Two weeks after treatment 89% of patients tested negative, but there was a cumulative incidence of recurrent infection of 29% over the 24 weeks of follow up [Kjaer et al., 2000]. LCx^® may also be used to detect C. trachomatis in patient-administerd tampon specimens. For C. trachomatis the Roche Cobas Amplicor^® and the Abbott LCx^® gave similar results, though the Cobas Amplicor had the additional burden of needing prior DNA extraction to achieve comparable sensitivity. Detection of N. gonorrhoeae using the LCx^® was, however, lower than by Cobas Amplicor^® or by in-house PCR [Tabrizi et al., 2000].

Two rather different studies indicate the very considerable impact that the LCx^® has had. A survey of 70 clinical diagnostic laboratories in Washington state found that none of them were using nucleic acid amplification-based tests for C. trachomatis in 1995. However, by 1998, 23% of the responding laboratories were using the LCx^® assay, accounting for 36% of the 318,133 specimens tested. The majority of laboratories using nucleic acid amplification-based methods were the large labs. Cost and loss of revenue were the most frequently cited reasons for others not adopting such tests [Battle et al., 2001]. In Glasgow, Scotland, the main chlamydia laboratory introduced the LCx^® assay in 1997 for testing specimens from general practitioners and from genitourinary medicine clinics. Testing activity increased four and a half times between 1996-1997 and 1999-2000 and the detection rate rose from 4.8% to 7.8%; an additional 331 men and 844 women diagnosed at a cost per additional diagnosis of about £162 (~$227) for men and £263 (~$368) for women. It was considered that substantial public health gains were likely to be achieved, both at individual and public health level [Scoular et al., 2001].

For a review of the use of the ligase chain reaction see Benjamin et al., 2003.

[MEW] September, 2003.

[MEW comments: The Abbott LCx for Chlamydia trachomatis (and also for N. gonorrhoeae) was withdrawn early in 2003 due to continuing batch to batch problems with the performance of the test. Previously 37,957 LCx CT kits were withdrawn voluntarily by the company in 2001 because of high negative control rates resulting in invalid runs and non repeating positives [see FDA enforcement report recall nos Z-0859-1/Z-0860-1]. The global withdrawal of the LCx CT  will leave a significant gap before the deployment of Abbott's new molecular beacons diagnostic product .

The demise of this test is very much to be regretted since the Abbott LCx was one of the most important and widely used nucleic acid amplification based tests for the diagnosis of C. trachomatis infection. This web page will be retained for the time being as a record of the important studies performed with the test.

Details of the Abbott LCx for C. pneumoniae for research use only on C. pneumoniae are given in the report of the Antalya conference. This is potentially an important development as the C. pneumoniae field has been plagued by non-standard, home-brew, nucleic acid amplification tests. However, given the withdrawal of the C. trachomatis LCx, the current status of the C. pneumoniae research assay appears uncertain].

[MEW] September, 2003.

NEXT: Molecular beacons

References

Anguenot, J. L., de Marval, F., Vassilakos, P., Auckenthaler, R., Ibecheole, V. & Campana, A. (2001). Combined screening for Chlamydia trachomatis and squamous intra-epithelial lesions using a single liquid-based cervical sample. Human Reproduction 16, 2206 - 2210. [Cervical cytology plus LCR on a liquid preparation]

Battle, T. J., Golden, M. R., Suchland, K. L., Counts, J. M., Hughes, J. P., Stamm, W. E. & Holmes, K. K. (2001). Evaluation of laboratory testing methods for Chlamydia trachomatis infection in the era of nucleic acid amplification. Journal of Clinical Microbiology 39, 2924 -2927. Full article    [Interesting survey of the status of various diagnostic tests in Washington state].

Benjamin, W. H., Smith, K. R. & Waites, K. B. (2003). Ligase chain reaction. Methods in Molecular Biology 226, 135 - 150. [Useful review].

Cheng, H., Macaluso, M., Vermund, S. H. & Hook, E. W. 3rd. (2001). Relative accuracy of nucleic acid amplification tests and culture in detecting Chlamydia in asymptomatic men. Journal of Clinical Microbiology 39, 3927 - 3937. Full article  [Interesting and thoughtful study of an important issue]

Chernesky M, Chong S, Jang D, Luinstra K, Faught M, Mahony J. (1998). Inhibition of amplification of Chlamydia trachomatis plasmid DNA by the ligase chain reaction associated with female urines. Clinical Microbiology and Infection 4, 397 - 404. [Good study]

Fenton, K. A., Korovessis, C., Johnson, A. M., McCadden, A., McManus, S., Wellings, K., Mercer, C. H., Carder C., Copas, A. J., Nanchahal, K., Macdowall, W., Ridgway, G., Field, J. & Erens, B. (2001). Sexual behaviour in Britain: reported sexually transmitted infections and prevalent genital Chlamydia trachomatis infection. Lancet 358, 1851 - 1854. [Mega survey using the LCR]

Gaydos, C. A., Howell, M. R., Pare, B., Clark, K. L., Ellis, D. A., Hendrix, R. M., Gaydos, J. C., McKee, K. T. Jr, Quinn, T. C. (1998). Chlamydia trachomatis infections in female military recruits. New England Journal of Medicine 339, 739 - 744. [Huge survey of urine from 13,204 female military recruits in the US army using LCR] 

Gronowski, A.M., Copper, S., Raorto, D and Murray, P.R. (2000). Reproducibility Problems with the Abbott Laboratories LCx Assay for Chlamydia trachomatis and Neisseria gonorrhoeae. Journal of Clinical Microbiology 38, 2416 - 2418. Full article   [Full article well worth a read; abstract tells little]

Hjelm, E., Hallen, A. & Domeika, M. (2001). Cervical, urine and vaginal specimens for detection of Chlamydia trachomatis by ligase chain reaction in women: a comparison. Acta Dermatologica Venereologica 81, 285 - 288. 

Kjaer, H. O., Dimcevski, G., Hoff, G., Olesen, F. & Ostergaard, L. (2000). Recurrence of urogenital Chlamydia trachomatis infection evaluated by mailed samples obtained at home: 24 weeks' prospective follow up study. Sexually Transmitted Infections 76, 169 - 172. [Good study from a group that has done much to evaluate alternative approaches to screening and control]

Loeffelholz, M. J., Jirsa, S. J., Teske, R. K. & Woods, J. N. (2001). Effect of endocervical specimen adequacy on ligase chain reaction detection of Chlamydia trachomatis. Journal of Clinical Microbiology 39, 3838 - 3841. [Cervical samples containing endocervical cells give higher positivity rates in LCR].

Mahony, J., Chong, S., Jang, D., Luinstra, K., Faught, M., Dalby, D., Sellors, J. & Chernesky, M. (1998) Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chlamydia trachomatis nucleic acid by PCR, ligase chain reaction, and transcription-mediated amplification: identification of urinary substances associated with inhibition and removal of inhibitory activity. Journal of Clinical Microbiology 36, 3122 - 3126. Full article   [Excellent study]

Nikkari S, Puolakkainen M, Yli-Kerttula U, Luukkainen R, Lehtonen OP, Toivanen P. (1997). Ligase chain reaction in detection of Chlamydia DNA in synovial fluid cells. British Journal of Rheumatology 36, 763 - 765. Full article   

Scoular, A., McCartney, R., Kinn, S., Carr, S. & Walker, A. (2001). The 'real-world' impact of improved diagnostic techniques for Chlamydia trachomatis infection in Glasgow. Communicable Diseases and Public Health 4, 200 - 204.

Tabrizi, S. N., Fairley, C. K., Cehn, S., Giouzeppos, O., Paterson, B., Bowden, F. J. & Garland, S. M. (2000). Evaluation of patient-administered tampon specimens for Chlamydia trachomatis and Neisseria gonorrhoeae. Sexually Transmitted Diseases 27, 133 - 137.

Thomas, B. J., Pierpoint, T., Taylor-Robinson, D. & Renton, A. M. (2001). Qualitative and quantitative aspects of the ligase chain reaction assay for Chlamydia trachomatis in genital tract samples and urines. International Journal of STD and AIDS 12, 589 - 594. [Thoughtful]

Turner, C. F., Rogers, S. M., Miller, H. G., Miller, W. C., Gribble, J. N., Chromy, J. R., Leone, P. A., Cooley, P. C., Quinn, T. C. & Zenilman, J. M. (2002). Untreated gonococcal and chlamydial infection in a probability sample of adults. Journal of the American Medical Association 287, 726 - 733. Full text (html)  [Interesting population based study]

Zhang, W., Cohenford, M., Lentrichia, B., Isenberg, H. D., Simson, E., Li, H., Yi, J. & Zhang, D. Y. (2002). Detection of Chlamydia trachomatis by isothermal ramification amplification method: a feasibility study. Journal of Clinical Microbiology 40, 128 - 132. [LCx used as a control for new amplification methods. The future?]

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