Comparison of a ligase chain reaction-based assay and cell culture for detection of pharyngeal carriage of Chlamydia trachomatis.
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In 264 genitourinary medicine clinic attenders reporting recent fellatio, the prevalence of pharyngeal Chlamydia trachomatis determined by an expanded standard including cell culture and two in-house PCR tests was 1.5% in 194 women and zero in 70 men. The ligase chain reaction (Abbott LCx) had a specificity of 99.2% and a positive predictive value of 60%. (+info)
Efficient site-specific nonribozyme opening of hepatitis delta virus genomic RNA in infected livers.
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Examination of the 1,679-nucleotide (nt) unit-length hepatitis delta virus (HDV) RNAs in the livers of two HDV-infected woodchucks showed that 96% of the antigenomic RNA but only 50% of the genomic RNA was circular. We subsequently found that at least half of the linear unit-length genomic RNA was open at a unique location. Using a modified form of RNA ligation-mediated amplification of cDNA ends, we showed that the 5' end was located at nt 1212. Like the previously described ribozyme cleavage site at nt 686, the new site produced a 5'-OH. Nevertheless, we showed that this novel site was not produced by activity of the HDV genomic ribozyme. We speculate that the 5' end at nt 1212 reflects a preferred site of posttranscriptional endonucleolytic cleavage of genomic RNA. (+info)
The ligase chain reaction as a primary screening tool for the detection of culture positive tuberculosis.
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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. (+info)
Evaluation of nucleic acid amplification tests as reference tests for Chlamydia trachomatis infections in asymptomatic men.
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Urine ligase chain reaction (LCR) and PCR tests and urethral swab culture were compared for their abilities to detect Chlamydia trachomatis infection in 3,639 asymptomatic men by using one-, two-, and three-test reference standards. Frozen urine at four of five participating centers was also tested by a transcription-mediated amplification assay which was used as a reference test. LCR increased the yield of positive results by 27% and PCR increased the yield of positive results by 26% over the yield of positive results by culture (n = 295). LCR and PCR sensitivities were similar, ranging from 80.4 to 93.5%, depending on the reference standard. Culture sensitivity was substantially less. A multiple-test standard yielded LCR, PCR, and culture specificities of 99.6%, with or without discrepant analysis. Test performance varied among centers partly due to different interpretations of the testing protocols. The study confirms that urine LCR and PCR for the detection of C. trachomatis have substantially improved sensitivities over that of urethral swab culture for testing of asymptomatic men, enabling screening of this important target group. These tests, perhaps in combination, are also candidate reference tests for the conduct of test evaluation studies. (+info)
Optimised sample DNA preparation for detection of Chlamydia trachomatis in synovial tissue by polymerase chain reaction and ligase chain reaction.
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OBJECTIVE: Molecular biology techniques such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) are routinely used in research for detection of C trachomatis DNA in synovial samples, and these methods are now in use in some clinical laboratories. This study aimed at determining the method best suited to molecular diagnosis of C trachomatis by examining four standard DNA preparation methods using chlamydia spiked synovial tissue and chlamydia infected monocytes. METHODS: Synovial tissue from a chlamydia negative patient with rheumatoid arthritis was spiked with defined numbers of C trachomatis elementary bodies (EB). Purified human peripheral monocytes from normal donors were infected with the organism at a multiplicity of infection 1:1 in vitro and harvested after four days. DNA was prepared from all samples by four methods: (1) QIAmp tissue kit; (2) homogenisation in 65 degrees C phenol; (3) incubation at 97 degrees C; (4) proteinase K digestion at 97 degrees C. DNA from methods 1 and 2 was subjected to PCR using two different primer sets, each targeting the C trachomatis omp1 gene. LCR was done on DNA prepared by each method. RESULTS: In synovial tissue samples spiked with EB, and in monocytes persistently infected with the organism, preparation of template using the QIAmp tissue kit (method 1) and the hot phenol extraction technique (method 2) allowed sensitive detection of C trachomatis DNA. These methods also produced template from both sample types for LCR. DNA prepared by heat denaturation (method 3) allowed only low sensitivity chlamydia detection in LCR and did not work at all for PCR. Proteinase K digestion plus heat denaturation (method 4) gave template that did not allow amplification in either PCR or LCR assays. CONCLUSIONS: The sensitivity of detection for C trachomatis DNA in synovial tissue by PCR and LCR depends strongly on the method used for preparation of the amplification template. LCR targeting the multicopy chlamydial plasmid and two nested PCR assay systems targeting the single copy omp1 gene showed roughly equivalent sensitivity. Importantly, template preparation method and the specific PCR primer system used for screening must be optimised in relation to one another for highest sensitivity. (+info)
Clinical aspects of diagnosis of gonorrhea and Chlamydia infection in an acute care setting.
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We found a 10.4% prevalence of unrecognized genital gonorrhea and Chlamydia infection among young adults of an urban emergency department. Intensified detection and treatment policies are needed to prevent continued transmission and complications of sexually transmitted infections. (+info)
Emergency department screening for asymptomatic sexually transmitted infections.
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OBJECTIVES: This study assessed the prevalence and correlates of asymptomatic genital tract infection with Neisseria gonorrhoeae and Chlamydia trachomatis among emergency department patients. METHODS: Individuals seeking emergency department evaluation for nongenitourinary complaints provided urine samples for N gonorrhoeae and C trachomatis testing by ligase chain reaction and completed a sociodemographic and behavioral questionnaire. RESULTS: Asymptomatic N gonorrhoeae or C trachomatis was found in 9.7% of persons tested. Correlates of C trachomatis infection included younger age, residence in high-morbidity zip code areas, previous history of N gonorrhoeae or C trachomatis, and number of sex partners in the past year. CONCLUSIONS: Urine-based screening of asymptomatic emergency department patients detected significant numbers of N gonorrhoeae and C trachomatis infections. Targeted screening programs may contribute to community-level prevention and control of sexually transmitted infections. (+info)
Is the detection of Mycobacterium tuberculosis DNA by ligase chain reaction worth the cost: experiences from an inner London teaching hospital.
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AIMS: To evaluate the clinical usefulness and the costs of using a rapid, commercial ligase chain reaction test (LCx) to detect Mycobacterium tuberculosis directly from clinical samples. METHODS: A prospective study of 2120 routine clinical specimens from 1161 patients over a 13 month period. Investigations for mycobacterial disease by microscopy, culture, and the Abbott LCx assay were performed. Sequential LCx assays were monitored in a cohort of patients undergoing treatment. The costs of the assay were calculated using the WELCAN system. Sensitivity, specificity, and positive and negative predictive values of the LCx assay were compared with conventional tests. The performance of the assay in patients undergoing treatment and cost in terms of WELCAN units converted to pounds/annum was studied. RESULTS: The assay was 85%/88% sensitive and 98%/100% specific in culture confirmed/clinically confirmed cases of tuberculosis, respectively. The assay was not useful for the measurement of treatment outcomes. The test cost approximately 42,500 Pounds/annum. CONCLUSIONS: The assay is a rapid, sensitive, and specific adjunct to clinical diagnosis, especially in differentiating non-tuberculous mycobacteria. However, it does not differentiate old and treated tuberculosis from reactivated disease, it is not useful to monitor adherence to treatment, and it is expensive. (+info)