Bordetella pertussis diagnosed by polymerase chain reaction

The object of this work was to test the polymerase chain reaction (PCR) for demonstration of Bordetella pertussis (BP) in nasopharyngeal secretions. The method was applied to patients with recently diagnosed pertussis, as verified by BP culture. In order to test the sensitivity and specificity of PCR for the diagnosis of BP, we used known concentrations of BP, Bordetella parapertussis and Bordetella bronchiseptica in aqueous solutions. PCR was furthermore carried out on species of bacteria that might be isolated from the nasopharynx. The applicability of PCR to patient specimens was tested in 25 patients in whose nasopharyngeal secretions BP had been demonstrated after 4-7 days of culture. The detection limit of PCR in aqueous solution was 1-2 BP bacteria per reaction tube. PCR was 100% specific for BP, showing no response with other Bordetella species or other bacteria known to colonize the nasopharynx. Of 25 patient specimens, 16 were PCR-positive 4-7 days after the positive primary culture had been established; only 5 out of 13 patient specimens were positive by repeated conventional nasopharyngeal culture at that time. We conclude that PCR is a possible alternative to culture for the demonstration of BP, as PCR is considerably faster than culture and might be more sensitive.     

Comparison of performances of two commercially available tests, a PCR assay and a ligase chain reaction test, in detection of urogenital Chlamydia trachomatis infection

The diagnostic performance of a PCR test (Roche Cobas Amplicor CT/NG Test) and that of a ligase chain reaction (LCR) test (Abbott LCx Chlamydia trachomatis assay) were compared by using endocervical and urethral swab specimen culture as a reference test. First-void urine (FVU) and endocervical and urethral swab specimens were collected from 1,015 unselected patients attending a sexually transmitted disease clinic and a clinic for adolescents in Helsinki, Finland. Chlamydia trachomatis was cultured from samples from the endocervix or urethra. PCR was performed with fresh and frozen urine and the culture transport medium. LCR was performed with fresh and frozen urine and LCx swab transport medium. Diagnostic consistency and diagnostic accuracy were statistically tested. The test results were identical for 984 patients (97%). Discrepant results were observed for 31 patients. Overall, LCR and PCR showed excellent kappa coefficients of consistency for both swab and FVU specimens (0.93 and 0.95, respectively). Sixty-one patients (6%) were culture positive. Testing of FVU by LCR or PCR increased the overall positivity rates to 7.0 and 7.7%, respectively. While PCR of FVU detected the greatest number of C. trachomatis infections (sensitivity, 96.1%), for some PCR-positive FVU specimens the results could not be confirmed (specificity, 99.6%). PCR and LCR were more sensitive than culture (sensitivities, 92 and 93% versus 79% for culture) in the diagnosis of genital C. trachomatis infection. In conclusion, both tests can be recommended for use in the clinical laboratory and for the screening of asymptomatic C. trachomatis infections.     

Ligase chain reaction for detection of Neisseria gonorrhoeae in urogenital swabs

The ligase chain reaction (LCR) is an in vitro nucleic acid amplification technique that exponentially amplifies targeted DNA sequences. In a multicenter study, we evaluated the use of a 4-h LCR-based assay for the diagnosis of Neisseria gonorrhoeae infection of the cervix and male urethra. The LCR results were compared with those of culture for N. gonorrhoeae by using selective media. This assay amplifies target sequences within the N. gonorrhoeae opacity gene. Discordant LCR-positive and culture-negative specimens were further evaluated by testing by another LCR assay which used N. gonorrhoeae-specific pilin probe sets. A total of 1,539 female endocervical specimens and 808 male urethral swab specimens were evaluated in the study. An expanded "gold standard" was defined to include all culture-positive as well as culture-negative, confirmed LCR-positive specimens. After resolution of discrepant samples, the sensitivities of the N. gonorrhoeae LCR assays for the female and male specimens were 97.3 and 98.5%, respectively, with specificities of 99.6 and 99.8%, respectively. Resolved culture sensitivities were 83.9 and 96.5% for the female and male specimens, respectively. The LCR assay for gonorrhea is a rapid, highly sensitive nonculture method for detecting gonococcal infection of the cervix and male urethra.     

Evaluation of 2-SP transport medium for detection of Chlamydia trachomatis and Neisseria gonorrhoeae by two automated amplification systems and culture for chlamydia

AIMS: To assess the performance of 2-sucrose-phosphate based transport medium (2-SP) for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by an automated commercial polymerase chain reaction (PCR) and ligase chain reaction (LCR) compared to centrifugation culture on McCoy cells for C trachomatis. Second, to compare both amplification systems for initial diagnostic testing of a low prevalence population for sexually transmitted diseases. METHODS: Four hundred and eighty one consecutive urogenital and conjunctival specimens were examined. All tests were performed on the same specimen collected with a dacron swab and transported in 2-SP medium. Samples that were positive by culture or by both PCR and LCR were considered to be true positives. RESULTS: The prevalences of C trachomatis and of N gonorrhoeae were 2.7% and 0.4%, respectively. PCR had a resolved sensitivity and specificity of 100% and 99.8%, respectively, for C trachomatis, and 100% and 98.9%, respectively, for N gonorrhoeae. LCR was 100% sensitive and specific for both pathogens. The resolved sensitivity of the shell vial assay was 85%. No culture positive sample would have been missed by PCR or LCR. The inhibition rate for PCR was 4.8%. CONCLUSIONS: 2-SP medium proved to be suitable for both PCR and LCR. It is not limited to any one test manufacturer and allows the performance of amplification techniques and viral and chlamydia culture from the same specimen. The LCR was more reliable than PCR on initial testing. However, hands on time is longer, and no amplification control is available for LCR.     

Use of lambda phage DNA as a hybrid internal control in a PCR-enzyme immunoassay to detect Chlamydia pneumoniae

An inherent problem in the diagnostic PCR assay is the presence of ill-defined inhibitors of amplification which may cause false-negative results. Addition of an amplifiable fragment of foreign DNA in the PCR to serve as a hybrid internal control (HIC) would allow for a simple way to identify specimens containing inhibitors. Two oligonucleotide hybrid primers were synthesized to contain nucleic acid sequences of the Chlamydia pneumoniae 16S rRNA primers in a position flanking two primers that target the sequences of a 650-bp lambda phage DNA segment. By using the hybrid primers, hybrid DNA comprising a large sequence of lambda phage DNA flanked by short pieces of chlamydia DNA was subsequently generated by PCR, cloned into a plasmid vector, and purified. Plasmids containing the hybrid DNA were diluted and used as a HIC by adding them to each C. pneumoniae PCR test. Consequently, C. pneumoniae primers were able to amplify both chlamydia DNA and the HIC DNA. The production of a 689-bp HIC DNA band on an acrylamide gel indicated that the specimen contained no inhibitors and that internal conditions were compatible with PCR. Subsequently, a biotinylated RNA probe for the HIC was transcribed from a nested sequence of the HIC and was used for its hybridization. Detection of the HIC DNA-RNA hybrid was achieved by enzyme immunoassay (EIA). This PCR-EIA system with a HIC was initially tested with 12 previously PCR-positive and 14 previously PCR-negative specimens. Of the 12 PCR-positive specimens, 11 were reconfirmed as positive; 1 had a negative HIC value, indicating inhibition. Of the 14 previously PCR-negative specimens, 13 were confirmed as true negative; 1 had a negative HIC value, indicating inhibition. The assay was then used with 237 nasopharyngeal specimens from patients with pneumonia. Twenty-one of 237 (8.9%) were positive for C. pneumoniae, and 42 (17.7%) were found to inhibit the PCR. Specimens showing inhibitory activity were diluted 1:10 and were retested. Ten specimens were still inhibitory to the PCR and required further DNA purification. No additional positive samples were detected and 3 nasopharyngeal specimens remained inhibitory to PCR. Coamplification of a HIC DNA can help confirm true-negative PCR results by ruling out the presence of inhibitors of DNA amplification.     

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens from men and women by a coamplification PCR assay

A coamplification PCR test for the direct detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urethral and endocervical swabs and urine samples from men and women was compared to standard culture techniques. Processed specimens were amplified in single reaction tubes containing primers for both organisms, and PCR products were detected by a colorimetric microwell plate hybridization assay specific for each pathogen. Of 344 specimens from men, 45 (13.1%) urine specimens were PCR positive for C. trachomatis, 51 (14.8%) urethral swab specimens were PCR positive, and 29 urethral swab specimens (8.4%) were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis were 96.2 and 99.3%, respectively, in urethral swab specimens, compared to 88.2 and 98.6% for urine specimens. Of the 192 specimens from women, 28 (14.6%) urine specimens were PCR positive for C. trachomatis, 32 (16.7%) endocervical specimens were PCR positive, and 19 (9.9%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis for endocervical specimens were both 100% compared to 100 and 99.4%, respectively, for urine specimens from women. In men, 68 (19.8%) urine specimens were PCR positive for N. gonorrhoeae, 73 (21.2%) urethral swabs were PCR positive, and 59 (17.2%) urethral swabs were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 97.3 and 97.0%, respectively, for urethral specimens compared to 94.4 and 98.5% for urine specimens. In women, 18 (9.4%) urine specimens were PCR positive for N. gonorrhoeae, 23 (12.0%) were endocervical swab PCR positive, and 15 (7.8%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 100 and 99.4%, respectively, for endocervical specimens compared to 90.0 and 95.9% for female urine specimens. These results indicate that a multiplex PCR is highly sensitive for detecting both C. trachomatis and N. gonorrhoeae from a single urine or genital swab, providing a more cost-effective way of screening multiple pathogens.     

Evaluation of a novel PCR-based diagnostic assay for detection of Mycobacterium tuberculosis in sputum samples

We report on a PCR-based assay we have developed for the detection of Mycobacterium tuberculosis in sputum samples. One hundred sputum specimens, which included 34 culture-positive and 66 culture-negative specimens, were evaluated with this system. Of the 34 culture-positive specimens, 31 were PCR positive, and 60 of the culture-negative specimens were PCR negative. An internal standard has been included in the assay system to monitor PCR inhibition and to confirm the reliability of the PCR assay.     

Distribution of desmin and fibronectin in chick embryo gonad during testicular cord formation

In order to compare PCR with rapid virus culture for the early detection of CMV in bronchoalveolar lavage (BAL) after bone marrow transplantation, 26 asymptomatic patients were routinely evaluated for the presence of CMV on day 35 using these two techniques. Concurrent blood samples were also analyzed in all cases. CMV was detected synchronously by both culture and PCR in six of 26 (23%) BAL and in five of 26 (19%) blood specimens. Among these positive specimens, three BAL and blood samples were positive in the same patients. Five (19%) BAL and five (19%) blood samples were culture-negative but PCR-positive. No BAL or blood specimens were positive by culture alone. When considering matched BAL-blood samples, five were positive in only one fluid, BAL (n = 3) or blood (n = 2) using culture, while seven were positive in only one fluid, BAL (n = 4) or blood (in = 3) using PCR. Overall, six of 26 (23%) patients had culture-negative but PCR-positive results. Three of these six patients were positive only in BAL and two of them subsequently received antiviral therapy for development of symptoms suggestive of CMV infection. We suggest that asymptomatic patients with negative-culture but PCR-positive results on day 35 in BAL should be subsequently closely monitored for the presence of CMV.     

Polymerase chain reaction for the detection of Bordetella pertussis in clinical nasopharyngeal aspirates

A PCR procedure for the detection of Bordetella pertussis in nasopharyngeal aspirates (NPAs) was developed with primers derived from the pertussis toxin promoter region. The amplification resulted in a 191-bp PCR product specific for B. pertussis. A total of 681 NPAs collected from children with cough lasting >7 days was evaluated by PCR and culture; 104 aspirates were positive by PCR and 93 by culture. Sixteen cases were positive only by PCR and five culture positive aspirates were negative by PCR. An internal control was included in the assay to monitor the performance of the PCR and to identify possible inhibitory components in clinical samples. The PCR method was more efficient than culture in detecting B. pertussis in samples collected late in the disease, in antibiotic-treated children and in patients with mild disease.     

Quantification of Bordetella pertussis in clinical samples by colorimetric detection of competitive PCR products

Quantification of microorganisms is an important part of the normal diagnostic work of a clinical microbiology laboratory. Traditionally the diagnosis of pertussis is subject to a yes or no approach with no quantitative dimension. This can, however, be of interest as a factor when judging the risk of a patient spreading the bacterium and as a research tool. The aim of the present study was to develop a PCR-based quantitative assay for Bordetella pertussis DNA in clinical nasopharyngeal aspirates by combining a quantitative PCR with a colorimetric detection principle, DIANA (detection of immobilised amplified nucleic acid). A competitor to the PCR target sequence in IS-481, containing a lac-operator, was constructed and calibrated, and a test protocol prepared. A total of 46 clinical nasopharyngeal aspirates, previously diagnosed using a standard nested PCR assay and quantified by culture, were analysed by the quantitative PCR. The method showed acceptable precision and accuracy considering that it estimates the total number of bacterial genomes while culture detects viable bacteria. Recognised advantages were the simple colorimetric detection, the inborn indication of a working PCR assay, and the possibility of obtaining results even when partial inhibition of the PCR assay was seen. In addition, the quantitative PCR result can be obtained within one day compared to 3-10 days for culture. The present results and the qualities of the quantitative PCR suggest that this assay will be a useful complement in routine diagnostics and in research.     

Diagnosis of extrapulmonary tuberculosis by a commercial polymerase chain reaction kit

The Roche Amplicor Mycobacterium tuberculosis PCR test was compared with mycobacterial culture for direct detection of M. tuberculosis in extrapulmonary specimens. From January 1995 to October 1996, 124 clinical specimens from 112 patients were assessed, including 47 body fluids, 61 tissue specimens and 16 abscesses. The sensitivity and specificity of Amplicor PCR compared to culture were 63.6% and 93.1% respectively. Analysis of 7 PCR-positive, culture-negative specimens confirmed that all were from patients with recently diagnosed tuberculosis under treatment. Eight specimens were PCR negative-culture positive, including a pleural fluid containing inhibitory substances. On acid-fast bacilli (AFB) smear-negative specimens, sensitivity and specificity were 53 and 100% respectively. The best results for Amplicor PCR were obtained with abscesses and biopsies. It is concluded that this test, highly specific for the diagnosis of tuberculosis, is at least as sensitive on extrapulmonary specimens as on smear-negative respiratory specimens.     

Clinical evaluation of the automated COBAS AMPLICOR MTB assay for testing respiratory and nonrespiratory specimens

We evaluated the COBAS AMPLICOR PCR system (Roche Diagnostics) for the routine detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. Diagnostic culture, considered as the reference method, was performed with BACTEC, L?wenstein-Jensen, Stonebrink, and Kirchner media. Occasionally MB-Redox, ESP, or MGIT medium was also used. A total of 643 respiratory and 506 nonrespiratory specimens collected from 807 patients were investigated. Of the 95 culture-positive specimens, 80 were COBAS AMPLICOR MTB positive, and of the 1,054 culture-negative specimens, 1,044 were COBAS AMPLICOR MTB negative. After resolving discrepancies by review of the medical history, the overall sensitivity, specificity, and positive and negative predictive values for the COBAS AMPLICOR MTB assay, respectively, were 83.5, 98.8, 86.7, and 98.6% compared to those of diagnostic culture. In smear-positive specimens, the sensitivity of the COBAS AMPLICOR MTB assay was 96%, versus 48% for smear-negative specimens. No significant differences in the test performance between respiratory and nonrespiratory specimens were observed. The overall inhibition rate was less than 2%, excluding stool specimens. The clear advantages of the COBAS AMPLICOR PCR system are standardized procedures and reagents for specimen processing as well as an internal control for reliable monitoring of PCR inhibitors. By simplifying the work flow through a completely automated amplification and amplicon detection procedure, the COBAS AMPLICOR PCR system proved itself as a very useful component for routine diagnostic procedures.     

Improvement of cervical Chlamydia detection in asymptomatic family planning attendees by using Amplicor Chlamydia trachomatis assay

We evaluated the Amplicor C. trachomatis PCR assay (Roche Molecular Systems, N.J.) for the diagnosis of cervical infection in asymptomatic women attending a family planning clinic, aged between 18 and 25 years. Culture onto McCoy cells with fluorescent monoclonal staining was the reference system. Cervical specimens from 485 women were tested. The prevalence of C. trachomatis was 10.5% by culture and 11% by Amplicor. No specimen was positive by culture and negative by PCR. Three PCR-positive, culture-negative specimens were positive by MOMP-PCR and a second plasmid-based PCR. The resolved sensitivity of PCR and culture were 100% and 94.5%, respectively. Specificities for both were 100%, positive and negative predictive values for culture were 100% and 99.3%. Total test efficiency was 99.4%. The Amplicor C. trachomatis assay gave very clear results, quite above or below the cut-off value, and showed high sensitivity and specificity, improved ease of handling and represented a good alternative to culture for large scale diagnosis of asymptomatic C. trachomatis infection.     

Comparison of the Roche AMPLICOR MYCOBACTERIUM assay and Digene SHARP Signal System with in-house PCR and culture for detection of Mycobacterium tuberculosis in respiratory specimens

Two commercial primer kits and detection systems, the Roche AMPLICOR MYCOBACTERIUM test and the Digene primer-probe kit with the SHARP Signal System, were compared to in-house PCR as well as standard culture techniques. For the 27 culture-positive specimens, the Roche AMPLICOR MYCOBACTERIUM test detected 20 specimens, the Digene system detected 19, and in-house PCR detected 21. Of the 86 culture-negative specimens, 13 were positive by the Roche AMPLICOR MYCOBACTERIUM test, 16 were positive by the Digene system, and 21 were positive by in-house PCR. When clinical situations were evaluated, 11 of 13 culture-negative Roche AMPLICOR MYCOBACTERIUM test-positive specimens, 10 of 16 culture-negative Digene system-positive specimens, and 13 of 21 culture-negative-in-house PCR-positive specimens were diagnosed as true-positive specimens. The sensitivities of Roche AMPLICOR MYCOBACTERIUM test, the Digene system, and in-house PCR were 73.81, 69.05, and 80.95%, and the specificities were 97.18, 91.55 and 88.73%, respectively. The positive predictive values were 93.94, 82.86, and 80.95%, and the negative predictive values were 86.25, 83.33, and 88.73%, respectively. For the commercial kits, the Roche AMPLICOR MYCOBACTERIUM test seems to be more sensitive and specific than the Digene system. However, the Roche AMPLICOR MYCOBACTERIUM test cannot be used on nonrespiratory specimens.     

Direct genotypic detection of Mycobacterium tuberculosis rifampin resistance in clinical specimens by using single-tube heminested PCR

Recent analysis of the gene encoding the beta subunit of Mycobacterium tuberculosis RNA polymerase (rpoB) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. Earlier reports have described a high degree of sequence conservation of rpoB among mycobacteria other than M. tuberculosis and other GC-rich bacteria that can lead to false-positive amplification when applied directly to clinical specimens. We developed reagents for PCR amplification that are based on signature nucleotides discovered by comparative sequence analysis of the rpoB genes of organisms phylogenetically related to M. tuberculosis. The specificities of the reagents were challenged with 20 isolates of multiple-drug-resistant M. tuberculosis and more than 20 species of mycobacteria other than M. tuberculosis and other GC-rich organisms. A single-tube heminested PCR protocol was devised to obtain sensitivity equal to those of an IS6110-based PCR assay and culture in spiked sputum experiments. The assay correctly identified 21 of 24 (87.5%) culture-positive specimens, 13 of which were acid-fast smear-negative, in a panel of 51 clinical specimens. Three specimens that were false-positive initially were negative upon repeat testing when the assay was modified to eliminate the potential for aerosol carryover of the first-round amplification product during the open-tube addition of the second set of reaction reagents. This assay is the most sensitive and specific test to date for the direct detection of M. tuberculosis rpoB in clinical specimens. This rapid PCR-based assay can be used for the simultaneous identification of M. tuberculosis and its rifampin susceptibility genotype.     

Does Chlamydia trachomatis MoPn enter a microbiologically-inapparent state during experimental infection of the mouse genital tract?

Microbiologically-inapparent chlamydial infection may contribute towards the immunopathogenesis of these diseases. Although morphologically and physiologically aberrant non-cultivable chlamydiae can be induced reversibly in cell culture, evidence for these forms in infections of animals and humans is indirect. A mouse model of salpingitis caused by the mouse pneumonitis biovar of Chlamydia trachomatis (MoPn) was used to determine the existence of non-cultivable organisms in vivo. Following intravaginal inoculation, mice yielded high chlamydial counts for 7-14 dyas, with a decline in culture-positivity by 21-28 days. A significant elevation of IFN gamma production in infected tissues was measured for 21 days and, from 28-70 days, all mice were culture-negative and developed characteristic hydrosalpinges. MoPn was detected by PCR in vaginal swabs of 80% and 69% respectively of culture-negative animals at 21 and 28 days. In a second study, 100%, 63% and 50% of culture-negative genital tissue homogenates were PCR-positive at 21, 28 and 42 days. Immunosuppression with either cyclophosphamide or hydrocortisone failed to regenerate cultivable chlamydiae. Tissues were disrupted by homogenization and inoculated intranasally to MF1 mice which are extremely susceptible to MoPn, but all culture-negative specimens were non-infectious. The significance of the PCR-positive culture-negative specimens requires further investigation, since these may represent a non-cultivable state in the deeper tissues of the mouse genital tract which may be beyond the reach of reactivating triggers.     

Comparison of PCR- and hybrid capture-based human papillomavirus detection systems using multiple cervical specimen collection strategies

This study compared the performances of three human papillomavirus (HPV) detection tests with specimens collected by three alternative procedures. The HPV tests included the Hybrid Capture Tube test (HCT), the microplate-based Hybrid Capture II test (HC II), and the MY09-MY11 L1 consensus primer PCR-based assay. Initial cervical specimens were collected from study subjects with a broom device, and after Papanicolaou smears were made, residual specimens were placed into PreservCyt (PC), a liquid cytology medium. A second specimen was collected from each subject and placed into Digene Specimen Transport Medium (STM). The device for collection of the second specimen alternated with consecutive subjects between a conical cytology brush and a Dacron swab. At the 1.0-pg/ml cutoff, the results of the HC II agreed well with those of the PCR. Specifically, when PCR data were restricted to the types found by the HC II (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), there was greater than 90% agreement between the HC II and PCR results with both STM and PC. At a lower cutoff (0.2 pg/ml), HC II-positive results increased further, especially when the test was applied to the PC specimens. However, false-positive HC II results were more often observed at the 0.2-pg/ml cutoff. HC II yielded the highest HPV positivity with specimens placed into PC, followed by specimens collected with a conical brush and placed into STM and, last, by those collected with a Dacron swab and placed into STM. Our results demonstrate the utility of both the STM and PC specimen collection methods and show good agreement between the HC II and PCR.