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PubMedDate not supplied

Detection of Chlamydia trachomatis by a ligase chain reaction amplification method.

BACKGROUND AND OBJECTIVES: The ligase chain reaction is an in vitro DNA amplification technique that exponentially amplifies selected DNA sequences. GOAL: To evaluate a ligase chain reaction assay for the detection of Chlamydia trachomatis cryptic plasmid DNA (LCx Chlamydia) in patients routinely attending a sexually transmitted disease center in Italy. STUDY DESIGN: Urethral or cervical swabs were obtained from 501 consecutive patients (334 men and 167 women). The samples were assayed in parallel with LCx Chlamydia and conventional tissue culture; discordant results were further assayed by direct immunofluorescence and a ligase chain reaction with alternate primers. RESULTS: After resolution of discordant results, the LCx method showed a sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 99.3%, 96.7%, and 100% in men; 100%, 100%, 100%, and 100% in women; and 100%, 99.5%, 97.1%, and 100% overall, respectively. By comparison, the sensitivity of tissue culture was 81.4% in men, 50% in women, and 77.6% overall. CONCLUSIONS: The automated LCx method is sensitive, fast, and accurate and represents a useful diagnostic tool for C. trachomatis infection, even in low and medium prevalence populations.

Chlamydia Infections↗
PubMed2000

The ligase chain reaction as a primary screening tool for the detection of culture positive tuberculosis.

BACKGROUND: The ligase chain reaction Mycobacterium tuberculosis assay uses ligase chain reaction technology to detect tuberculous DNA sequences in clinical specimens. A study was undertaken to determine its sensitivity and specificity as a primary screening tool for the detection of culture positive tuberculosis. METHODS: The study was conducted on 2420 clinical specimens (sputum, bronchoalveolar lavage fluid, pleural fluid, urine) submitted for primary screening for Mycobacterium tuberculosis to a regional medical microbiology laboratory. Specimens were tested in parallel with smear, ligase chain reaction, and culture. RESULTS: Thirty nine patients had specimens testing positive by the ligase chain reaction assay. Thirty two patients had newly diagnosed tuberculosis, one had a tuberculosis relapse, three had tuberculosis (on antituberculous therapy when tested), and three had healed tuberculosis. In the newly diagnosed group specimens were smear positive in 21 cases (66%), ligase chain reaction positive in 30 cases (94%), and culture positive in 32 cases (100%). Using a positive culture to diagnose active tuberculosis, the ligase chain reaction assay had a sensitivity of 93.9%, a specificity of 99.8%, a positive predictive value of 83.8%, and a negative predictive value of 99.9%. CONCLUSIONS: This study is the largest clinical trial to date to report the efficacy of the ligase chain reaction as a primary screening tool to detect Mycobacterium tuberculosis infection. The authors conclude that ligase chain reaction is a useful primary screening test for tuberculosis, offering speed and discrimination in the early stages of diagnosis and complementing traditional smear and culture techniques.

Adolescent↗
PubMed1997

Detection of genitourinary tract Chlamydia trachomatis infection in pregnant women by ligase chain reaction assay.

OBJECTIVE: To compare the sensitivity and specificity of a ligase chain reaction assay of cervical swabs and voided urine with those of cervical swab tissue culture for the detection of genitourinary tract infection with Chlamydia trachomatis in pregnant women. METHODS: Infection with C trachomatis was assessed in cervical swabs by culture and in both cervical swabs and voided urine specimens by a ligase chain reaction assay specific for C trachomatis plasmid DNA. The matched cervical swab and voided urine specimens were collected from 462 women during routine visits to prenatal clinics. Standard criteria that defined infection included: 1) a positive cervical culture result or 2) a negative culture but a positive ligase chain reaction result in either the urine or cervical specimen that was confirmed by supplementary testing. Test performance was assessed by determination of sensitivity and specificity, and differences in paired results were determined using McNemar analysis. RESULTS: The prevalence of genitourinary C trachomatis infection was 6.1% (n = 28) by cervical culture (sensitivity 30.1%; specificity 100%), 18.2% (n = 84) by ligase chain reaction of cervical swabs (sensitivity 90.3%; specificity 100%), and 16.9% (n = 78) by ligase chain reaction of urine (sensitivity 83.9%; specificity 99.5%). Relative to the number of women with a positive culture or a confirmed ligase chain reaction-positive cervical swab, the sensitivity and specificity were 82.8% and 97.9%, respectively, for ligase chain reaction of urine and 96.6% and 100%, respectively, for ligase chain reaction of cervical swabs. Ligase chain reaction of cervical swabs and urine detected 89.3% and 82.1%, respectively, of women with a positive cervical culture. CONCLUSIONS: Ligase chain reaction assay of cervical or urine specimens detected considerably more pregnant women with C trachomatis infection of the genitourinary tract than did cervical culture. Ligase chain reaction testing of urine is a simple and effective means of screening pregnant women for genitourinary tract infection with C trachomatis.

Adult↗
PubMed1998

Early detection of Mycobacterium tuberculosis in BACTEC cultures by ligase chain reaction.

The LCx Mycobacterium tuberculosis ligase chain reaction system (Abbott Diagnostic Division, Abbott Park, Ill.) was used to detect M. tuberculosis in 150 consecutive BACTEC vials on the day on which a positive growth index (GI) was recorded. By LCx, M. tuberculosis DNA was detected in BACTEC vials on average 2.6 days before the presence of acid-fast bacilli could be confirmed by microscopic examination. A total of 106 of 108 M. tuberculosis isolates were detected without centrifugation from bottles presenting very low GIs (average, 70; median, 33). No false-positive result was obtained from nontuberculous mycobacteria or from isolates with contaminants.

Culture Media↗
PubMed1998

The inhibitory effect of phosphate on the ligase chain reaction used for detecting Chlamydia trachomatis.

AIMS: To examine the detection limit of the ligase chain reaction kit for Chlamydia trachomatis, to study the inhibitory effect of phosphate on the ligase chain reaction, and to clarify the mechanism of inhibition. METHODS: Three reference serovars of C trachomatis--D/UW-3/Cx, F/UW-6/Cx, and L2/434/Bu--were used to test the sensitivity of the chlamydia ligase chain reaction. Comparison was made of the inhibition by phosphate before and after DNA amplification. Phosphate in up to 2.4 mM concentration was added to specimens of C trachomatis serovar D (1 to 50 inclusion forming units (IFU)/reaction) before DNA amplification to examine the concentration dependency of phosphate inhibition of the ligase chain reaction. RESULTS: The detection limits were 0.6 IFU/reaction for serovar D/UW-3/Cx and F/UW-6/Cx, and 0.4 IFU/reaction for L2/434/Bu. Phosphate inhibited the ligase chain reaction only when it was added before the amplification stage. The specimens containing chlamydia at 1 to 50 IFU/reaction were negative when the concentration of phosphate added at the prethermocycle stage was more than 1.2 mM. CONCLUSIONS: Ligase chain reaction analysis is a reliable method of diagnosing C trachomatis infection because of its high sensitivity. It would be clearly superior to the currently used methods if the problem of inhibitors could be eliminated. The mechanism of inhibition of the ligase chain reaction by phosphate was thought to be blockade of the amplification of the target DNA. The efficacy of the ligase chain reaction could be inhibited by phosphate in the urine, so duplicate dilution analysis of some negative specimens should be useful.

Chlamydia Infections↗
PubMed1994

Identification of Erwinia stewartii by a ligase chain reaction assay.

A PCR-coupled ligase chain reaction (LCR) assay was developed to distinguish the plant pathogenic bacterium Erwinia stewartii from other erwiniae. This new technique allows discrimination to the species level on the basis of a single-base-pair difference in the 16S rRNA gene which is unique to E. stewartii. Portions of the 16S rRNA genes of E. stewartii and the closely related Erwinia herbicola were sequenced. From comparison of the two 16S rRNA gene regions, two primer pairs were constructed such that only E. stewartii DNA gave a product in the LCR assay. The ligated product was separated from the radioactively labelled primers by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Twenty-four different Erwinia species and strains were tested by PCR-coupled LCR to verify the specificity of the assay, and only E. stewartii strains gave a positive reaction. In addition, infected and healthy plant material was also assayed. E. stewartii was detected in infected plant material, even when large populations of epiphytic bacteria were present. No enrichment was necessary for detection of the pathogen in corn leaves. This assay has potential as a diagnostic technique for the detection of E. stewartii in infected plant and vector material.

Base Sequence↗
PubMed1994

Diagnosis of Chlamydia trachomatis urethral infection in symptomatic and asymptomatic men by testing first-void urine in a ligase chain reaction assay.

A multicenter study compared ligase chain reaction (LCR) of Chlamydia trachomatis plasmid DNA with culture of urethral swab specimens from 542 men (study A); a second study (B) compared LCR of first-void urine (FVU) with urethral swab cultures from 1043 men. Discordant results were resolved with direct fluorescent antibody staining of sediments from the FVU or urethral culture specimen and with a second LCR directed against a fragment of the major outer membrane protein gene. Test performance was calculated on the basis of an expanded reference standard. The LCR plasmid assay had a sensitivity of 98.0% in study A and 93.5% in study B; specificity was 99.8%-100%. The sensitivity of culturing urethral swabs from all study sites was 68.2% (range by sites, 40.0%-84.6%). The presence or absence of urethral symptoms did not alter the results. Use of this LCR test should allow more meaningful investigation and treatment of C. trachomatis infections in men.

Bacteriuria↗
PubMed1999

Detection of Chlamydia trachomatis endocervical infections by ligase chain reaction versus ACCESS Chlamydia antigen assay.

Ligase chain reaction (LCR) was compared with ACCESS immunoassay for detection of chlamydial infections in females. Despite efforts to improve ACCESS performance by evaluation of specimens that were in the test performance "grey zone," LCR remained more sensitive and was less expensive to perform. ACCESS had a sensitivity of 83.9%, a specificity of 99.7%, a positive predictive value of 96.3%, and a negative predictive value of 98.5%.

Antigens, Bacterial↗
PubMed2002

Quantification of Chlamydia trachomatis elementary bodies in urine by ligase chain reaction.

Modification of the standard Chlamydia trachomatis ligase chain reaction (LCR) detection assay resulted in a quantitative test. Sample rates from C. trachomatis standards ranging from 32 to 1,048,576 elementary bodies (EB)/ml of urine exhibited a linear correlation with concentration. Quantitative LCR (qLCR) was used to measure the number of EB per milliliter in 158 urine samples from women in Madagascar that tested positive for C. trachomatis by the standard LCR detection assay. C. trachomatis concentrations displayed an apparent bimodal distribution, with approximately one-third of samples (37%) in a peak ranging from 32 to 1,015 EB/ml (median = 297 EB/ml) and the remainder (63%) in a grouping with relatively higher concentrations, ranging from 1,086 to 218,670 EB/ml (median = 7,389 EB/ml). qLCR will be useful for studies of chlamydial infection aimed at understanding the associations of organism burden with clinical manifestations and transmission.

Chlamydia Infections↗
PubMed2003

Assessment of Chlamydia trachomatis infection of semen specimens by ligase chain reaction.

Diagnostic potential of the Chlamydia trachomatis ligase chain reaction system (LCx) to assess the presence of C. trachomatis in urine and semen specimens was evaluated. Paired urine and semen specimens from 153 asymptomatic male partners of subfertile couples attending our Center for Reproductive Medicine were examined by LCx. As controls, 19 semen samples from four donors who were participating in the programme for artificial insemination were used. Of these, 12 samples had previously been shown to be C. trachomatis-positive by an in-house PCR. C. trachomatis was detected by LCx in seven of 153 (5 %) urine samples. None of the 153 semen samples tested positive by LCx. Also, none of the 12 C. trachomatis-containing control semen samples were positive by LCx. By in-house PCR, seven urine specimens and two of 153 (1 %) semen samples tested positive. The corresponding urine samples of these male partners were also C. trachomatis-positive, as well as the 12 C. trachomatis-containing samples from donors. In conclusion, LCx is not sensitive enough to assess the presence of C. trachomatis in semen specimens; therefore, this method is not recommended to routinely screen semen specimens from donors who participate in programmes for artificial insemination or male partners of subfertile couples for C. trachomatis.

Chlamydia Infections↗
PubMed2002

Epidemiology and natural history of ligase chain reaction detected chlamydial and gonococcal infections.

OBJECTIVES: Ligase chain reaction (LCR) technology has dramatically increased the sensitivity of tests for sexually transmitted infections (STIs). It is unknown whether low copy infections (LCR positive, culture negative) have any clinical consequences. We assessed the clinical significance of untreated low copy Chlamydia trachomatis and Neisseria gonorrhoeae infections in a cohort of sexually active women. METHODS: We studied a cohort of sexually active women followed at 6 month intervals for up to 3 years. Frozen urine specimens from 181 women with negative cultures for C. trachomatis and N. gonorrhoeae who were 'high risk' (defined as being less than 40 years old at baseline, and having either Trichomonas vaginalis at baseline or a history of more than one sexual partner during the 12 months before baseline) were tested for C. trachomatis and N. gonorrhoeae by LCR (Abbott Laboratories, Abbott Park, IL, USA). The specimens from all visits for each person were pooled and LCR was performed on the pool. Laboratory results were linked to clinical information. We also tested all urine samples obtained from patients with a positive culture. RESULTS: 10 additional infections (nine C. trachomatis and one N. gonorrhoeae) were detected with LCR technique. None of the women with low copy infection had evidence of subsequent pelvic inflammatory disease or ectopic pregnancy. Pooling of urine samples resulted in a 47% decline in the number of tests performed. CONCLUSIONS: Additional STIs can be identified when using LCR. Pooling of urine specimens is a cost saving technique for C. trachomatis and N. gonorrhoeae testing.

Adult↗
PubMed2005

[Ligase chain reaction. Theory, practical use, prospects].

The authors present a review of papers on research into ligase chain reaction and its application in microbiology published in scientific periodicals by the end of 2003. The paper covers the theoretical ground of ligase chain reaction methodology, describes typical experimental procedures and gives examples of application of ligase chain reaction in modern diagnostic practice to detect infections, genetic diseases and for gene identification.

DNA Ligases↗
PubMed2001

Multisite pooling study using ligase chain reaction in screening for genital Chlamydia trachomatis infections.

BACKGROUND: Ligase chain reaction (LCR), a nucleic acid amplification assay, is a highly specific and sensitive test for detecting Chlamydia trachomatis in cervical and urethral swabs as well as first-void urine specimens. GOAL: To examine the suitability of using the LCR test to detect C trachomatis in pooled cervical specimens. STUDY DESIGN: The performance of LCR in pooled specimens was compared with individual specimen testing at six laboratories using 3,170 cervical swab specimens randomly selected from specimens received for routine testing in the participating laboratories. These samples then were combined consecutively into 634 pools of 5 specimens and 317 pools of 10 specimens. A reduced sample to cutoff ratio of 0.2 or more was used for the pooled specimens. RESULTS: Of the 188 positive specimens (98.9%), 186 were identified when single specimens were analyzed. When pools of 5 or 10 specimens were evaluated, 99.5% and 98.9% of the positive swabs, respectively, were identified correctly. Two positive specimens were detected only through pooling. CONCLUSIONS: Pooling samples for detection of C trachomatis by LCR is sensitive and specific. Depending on the prevalence of infection (positivity), LCR testing may result in cost savings, as compared with individual testing of specimens.

Cervix Uteri↗
PubMed2000

Retesting and follow-up of first-catch urines from men yield variable results with three Chlamydia trachomatis nucleic acid amplification tests.

First-catch urines from 276 asymptomatic male military recruits were screened by polymerase chain reaction for the detection of Chlamydia trachomatis. Eight initially positive specimens were retested by polymerase chain reaction, ligase chain reaction and transcription-mediated amplification. Urine specimens from six (2.2%) subjects were considered to contain C. trachomatis. However, retesting of serially collected urines from five of these six subjects using different nucleic acid amplification methods showed some discrepancy. This may have a major impact on the efficacy of screening programs for C. trachomatis in low prevalence populations.

Adolescent↗
PubMed1995

Detection of Chlamydia trachomatis in urine specimens from women by ligase chain reaction.

The performance of a plasmid-based ligase chain reaction (LCR) with urine specimens was compared with those of cell culture of cervical swabs and enzyme immunoassay with urine specimens for the detection of Chlamydia trachomatis infection in women who had attended a family planning clinic. The prevalence of chlamydial infection determined by LCR was 3.1%. Discrepant results among the three assays were resolved by testing urine by a second LCR assay based on the C. trachomatis chromosomal gene encoding the major outer membrane protein. Sensitivity, specificity, and positive and negative predictive values for the cell cultures were 56.3, 100, 100, and 98.4%, respectively, whereas those for the enzyme immunoassay were 18.8, 100, 100, and 97.1%, respectively, and those for LCR were 87.5, 100, 100, and 99.5%, respectively. LCR thus provides a highly sensitive and specific noninvasive screening method for detecting genital chlamydial infections in women.

Adult↗
PubMed1992

Evaluation of the ligase chain reaction (LCR) for the detection of point mutations.

The ligase chain reaction (LCR) was evaluated as an amplification method for an in vivo mutation assay. Specifically, the ligase was tested for its ability to selectively amplify a DNA sequence mutated at a single base, in the presence of an excess of wild-type DNA. As a model template a 370-bp DNA fragment of the mouse Ha-ras protooncogene containing an A to T mutation at the second position of codon 61 was used. With the commercially available ligase Ampligase (Epicenter), 250 molecules of mutant fragments could be detected by an enzyme-linked immunoassay with digoxigenin marker (giving a theoretical detection limit of 1 target gene per 10(4) copies of genome). In the analysis of mixtures with corresponding wild-type DNA fragments, a 1:1 mixture resulted in a clearly stronger signal than control samples lacking wild-type and mutant DNA. However, the signal obtained from a 100-fold dilution of the mutant DNA with wild-type DNA could not be distinguished from the background noise. In this particular form, LCR lacks sufficient selectivity to be applied to an in vivo situation, where the ratio of mutant to wild-type DNA sequences might be expected to lie around 1:10(6).

Animals↗
PubMed1993

Amplification of Chlamydia trachomatis DNA by ligase chain reaction.

Amplification of Chlamydia trachomatis DNA by polymerase chain reaction was compared with amplification by ligase chain reaction (LCR). Both amplification procedures were able to consistently amplify amounts of DNA equivalent to three C. trachomatis elementary bodies. All 15 C. trachomatis serovars were amplified to detectable levels by LCR, and no DNA from 16 organisms potentially found in clinical specimens or from Chlamydia psittaci and Chlamydia pneumoniae was amplified by LCR.

Base Sequence↗
PubMed2000

Opportunistic urine ligase chain reaction screening for sexually transmitted diseases in adolescents seeking care in an urban emergency department.

STUDY OBJECTIVE: Neisseria gonorrhoeae and Chlamydia trachomatis are the most common bacterial sexually transmitted diseases (STDs) in sexually active youth and many infections are asymptomatic or unrecognized. This study used ligase chain reaction assays for determination of prevalence of gonococcal and chlamydial infections in adolescents seeking care at an urban emergency department. METHODS: An unlinked prevalence study was performed with ligase chain reaction tests on voided urine specimens from a convenience sample of adolescents 14 years or older who sought care at the Children's Hospital of Alabama ED. Demographic data and data on care provided in the ED were determined from retrospective chart review of those patients whose urine specimens were tested. RESULTS: Of 282 urine specimens screened, 13.5% (38) yielded positive findings on ligase chain reaction testing for either or both pathogens (20 [7%] positive for N gonorrhoeae, 23 [8%] positive for C trachomatis). In the context of acute care, gonorrhea or chlamydial infection was diagnosed in 5 (1.8%). STD prevalence did not vary significantly by age. Only 39% (15/38) of patients with infections detected by ligase chain reaction testing received potentially effective antibiotics as a result of their urgent care evaluation. CONCLUSION: Many adolescents use the ED for nonurgent care and unsuspected STDs are often missed. Urine ligase chain reaction testing is a sensitive, noninvasive means of detecting STDs by which unsuspected adolescent STD cases can be detected in an ED setting.

Adolescent↗