Simple and rapid detection of Mycobacterium tuberculosis complex organisms in bovine tissue samples by PCR.

Mycobacterium bovis is a slowly growing microorganism, and confirmation of the diagnosis by conventional culture is a lengthy process. A simple, rapid method for the extraction of DNA from bovine tissue samples was developed and used in a PCR designed for the diagnosis of tuberculosis. Tissues from 81 cattle from tuberculosis-infected herds (group 1) and 19 cattle from tuberculosis-free herds (group 2) were tested in this PCR, and the results were compared with those of conventional culture. The PCR assay detected 71.4% of the culture-positive animals from group 1. Tissue from all animals in group 2 were negative in the PCR assay and by culture. The described method could be used as a rapid screening technique which would be complementary to culture of tissue specimens for the routine diagnosis of bovine tuberculosis. The PCR technique is much faster than culture and reduces the time for diagnosis from several months to 2 days. It also provides for the detection of M. bovis when rapidly growing Mycobacterium spp. are present in the sample and may be able to detect the presence of M. bovis in samples even when organisms have become nonviable.     

Polymorphic nucleotide within the promoter of nitrate reductase (NarGHJI) is specific for Mycobacterium tuberculosis

Mycobacterium tuberculosis rapidly reduces nitrate, leading to the accumulation of nitrite. This characteristic served for the past 40 years to differentiate M. tuberculosis from other members of the Mycobacterium tuberculosis complex (MTBC), such as Mycobacterium bovis (non-BCG [referred to here as simply "M. bovis"]), Mycobacterium bovis BCG, Mycobacterium africanum, or Mycobacterium microti. Here, a narG deletion in M. tuberculosis showed that rapid nitrite accumulation of M. tuberculosis is mediated by narGHJI. Analysis of narG mutants of M. bovis and M. bovis BCG showed that, as in M. tuberculosis, nitrite accumulation was mediated by narGHJI, and no other nitrate reductase was involved. However, in contrast to M. tuberculosis, accumulation was delayed for several days. Comparison of the narGHJI promoter revealed that, at nucleotide -215 prior to the start codon of narG, M. tuberculosis carried a thymine residue, whereas the bovine mycobacteria carried a cytosine residue. Using LightCycler technology we examined 62 strains of M. tuberculosis, M. bovis, M. bovis BCG, M. microti, and M. africanum and demonstrated that this single nucleotide polymorphism was specific for M. tuberculosis. For further differentiation within the MTBC, we included, by using LightCycler technology, the previously described analysis of oxyR polymorphism, which is specific for the bovine mycobacteria, and the RD1 polymorphism, which is specific for M. bovis BCG. Based on these results, we suggest a LightCycler format for rapid and unambiguous diagnosis of M. tuberculosis, M. bovis, and M. bovis BCG.     

Evaluation of DNA extraction techniques for detecting Mycobacterium tuberculosis complex organisms in Asian elephant trunk wash samples

Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens.     

Use of polymerase chain reaction for rapid diagnosis of tuberculosis.

A DNA amplification assay using the polymerase chain reaction technique designed for the rapid identification of Mycobacterium bovis organisms was used to test 211 human mycobacterial isolates and 177 clinical specimens previously submitted for routine mycobacterial culture. The procedures described could be used by routine or specialist laboratories for identification of M. tuberculosis complex organisms in 4 h and/or as a rapid screening method for the direct detection of M. tuberculosis complex organisms in specimens.     

Detection and identification of mycobacteria by amplification of a segment of the gene coding for the 32-kilodalton protein.

A polymerase chain reaction (PCR) assay for the rapid detection of mycobacterial DNA is described. Oligonucleotide primers, derived from the sequence of a gene coding for the 32-kDa antigen of Mycobacterium tuberculosis, amplified DNA from all 28 species of mycobacteria tested. All nonmycobacterial species tested were negative. An oligonucleotide probe hybridized to the PCR products of the strains belonging to the M. tuberculosis complex. This method could detect as little as 50 fg, as tested with purified M. tuberculosis DNA. By this amplification method, 127 sputum specimens were tested, with 7.9% of the specimens proving to be inhibitory in PCR. The sensitivity of detection by PCR compared with that by culture was 55.9%; when the inhibitory specimens were excluded, the sensitivity was 70.4%. The specificity of PCR combined with hybridization was 100%.     

Use of real-time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance-associated mutations in Mycobacterium tuberculosis

Very fast amplification of DNA in small volumes can be continuously monitored with a rapid cycler that incorporates fluorimetric detection. Primers were designed to amplify a 157-bp fragment of the rpoB gene spanning codons 526 and 531 and a 209-bp fragment of the katG gene spanning codon 315 of Mycobacterium tuberculosis. Most mutations associated with resistance to rifampin (RMP) and isoniazid (INH) in clinical isolates occur in these codons. Two pairs of hybridization probes were synthesized; one in each pair was 3' labeled with fluorescein and hybridized upstream of the codon with the mutation; the other two probes were 5' labeled with LightCycler-Red 640. Each pair of probes recognized adjacent sequences in the amplicon. After DNA amplification was finished by using a LightCycler, the temperature at which the Red 640 probe melted from the product was determined in a 3-min melt program. Twenty M. tuberculosis clinical isolates susceptible to streptomycin, INH, RMP, and ethambutol and 36 antibiotic-resistant clinical M. tuberculosis isolates (16 resistant to RMP, 16 to INH, and 4 to both antimicrobial agents) were amplified, and the presence of mutations was determined using single-strand conformation polymorphism analysis, the LiQor automated sequencer, and the LightCycler system. Concordant results were obtained in all cases. Within 30 min, the LightCycler method correctly genotyped all the strains without the need of any post-PCR sample manipulation. Overall, this pilot study demonstrated that real-time PCR coupled to fluorescence detection is the fastest available method for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.     

Rapid-cycle PCR and fluorimetry for detection of mycobacteria

In this study we used LightCycler PCR amplification and product detection by fluorescence resonance energy transfer probes to identify mycobacteria and differentiate between Mycobacterium tuberculosis complex, Mycobacterium avium, and other nontuberculous mycobacteria. Targeting the 16S rRNA gene, three different probes specific for mycobacteria, M. tuberculosis complex, and M. avium were constructed. As few as five genome copies of target nucleic acid were detected by the probes, illustrating the high sensitivity of the system. All 33 mycobacterial species tested but none of the closely related actinomycetes and other bacteria produced a specific fluorescence signal. A specificity of 100% was also demonstrated for the M. tuberculosis complex-specific probe and the M. avium-specific probe. Within 45 min, the LightCycler method correctly detected mycobacteria and specifically identified M. tuberculosis complex and M. avium without any post-PCR sample manipulation. In view of future clinical studies, we also constructed and tested an internal control which could be used to assure successful amplification and detection of mycobacteria. Monitoring of PCR inhibition will be essential for evaluation of this system for direct detection of mycobacteria in clinical specimens. Finally, we tested our system on sputum seeded with mycobacteria and were able to detect as few as 10 organisms. At present, this system is the fastest available method for identification and differentiation of mycobacteria from culture-positive specimens and offers an excellent alternative to previously established nucleic acid amplification-based techniques for the diagnostic mycobacterial laboratory.     

Diagnosis of varicella-zoster virus infections in the clinical laboratory by LightCycler PCR

Varicella-zoster virus (VZV) causes vesicular dermal lesions which are clinically evident as varicella (primary infection) or zoster (reactivated) diseases. The LightCycler system (Roche Molecular Biochemicals) is a newly developed commercially available system designed to rapidly perform PCR with real-time detection of PCR products using a fluorescence resonance energy transfer. We compared the detection of VZV from dermal specimens by shell vial cell culture (MRC-5) and by LightCycler PCR. Of 253 specimens, VZV was detected in 23 (9.1%) by shell vial cell cultures and 44 (17.4%) by LightCycler PCR directed to a nucleic acid target sequence in gene 28. Twenty-one of 44 (47.7%) specimens were exclusively positive by LightCycler PCR; the shell vial cell culture assay was never positive when DNA amplification was negative (specificity, 100%). VZV DNA was detected in 39 of 44 (88.6%) specimens positive during cycles 10 through 30 of the LightCycler PCR. These VZV DNA-positive specimens (cycles 10 to 30) and 5 of 11 other PCR positive specimens (cycles 31 to 36) were confirmed by another LightCycler PCR directed to another (gene 29) target of the viral genome. For routine laboratory practice, all specimens yielding amplified DNA to the VZV gene 28 target can be considered positive results. The increased sensitivity (91%) of the LightCycler PCR for detection of VZV, rapid turnaround time for reporting results, virtual elimination of amplicon carryover contamination, and equivalent costs compared to shell vial cell culture for detection of VZV indicate the need for implementation of this technology for routine laboratory diagnosis of this viral infection.     

Detection and identification of Mycobacterium tuberculosis by DNA amplification.

The polymerase chain reaction (PCR) was used to identify mycobacterial DNA sequences in uncultured clinical specimens. Two oligonucleotide primers derived from the sequence of a gene that codes for the 65-kilodalton antigen of Mycobacterium tuberculosis amplified DNA from all 11 species of mycobacteria tested. Amplified DNAs of nontuberculosis mycobacteria were found to be approximately 20 to 40 bases shorter than those from M. tuberculosis and Mycobacterium bovis BCG. DNA equivalent to that present in as few as 40 M. tuberculosis cells either alone or in the presence of DNA equivalent to that in 10(6) human cells could be detected. Results from analysis of cultured bacteria and clinical specimens showed PCR was sensitive and specific both in detecting mycobacteria and in differentiating M. tuberculosis and BCG from other species of mycobacteria. The PCR method with the primers reported here may become a useful tool in the early and rapid detection of mycobacterial infections in uncultured clinical specimens.     

Use of the real-time PCR assay in conjunction with MagNA Pure for the detection of mycobacterial DNA from fixed specimens.

Tuberculosis in immunocompromised patients is often caused by Mycobacterial species other than Mycobacterium tuberculosis. Thus, detection of and differentiation between M. tuberculosis and nontuberculosis species is necessary for diagnosis of disease in these patients. Furthermore, when tissue changes show granulomatous inflammation, quick confirmation testing for mycobacterial infection is needed for conclusive diagnosis. The aim of this study was to validate the utility of a real-time polymerase chain reaction (PCR) assay in conjunction with the MagNA Pure LC automated extraction system for the detection of mycobacterial DNA from formalin-fixed, paraffin-embedded specimens. A total of 46 archived, paraffin-embedded, fixed specimens showing granulomatous inflammation were studied for mycobacterial infection by real-time PCR. Bacterial DNA was extracted and isolated using the MagNA Pure extraction system. Real-time PCR was performed on the LightCycler using the Artus Real Art Mycob Diff ASR kit from Qiagen. Thirteen of the 46 patient specimens were positive for mycobacterial infection by acid-fast bacilli (AFB) stain. Of the13 reported positive by AFB stain, 12 where positive by real-time PCR. All 13 specimens reported positive by AFB were sent for culture confirmation. Eleven of 13 were returned positive by culture. Specimens reported as negative by culture and positive by real-time PCR were confirmed positive by a second PCR method from another reference laboratory. We believe that these studies are beneficial in the differential diagnosis of mycobacterial infection from fixed tissue specimens where tuberculosis might not have been clinically initially suspected and when specimens are not suitable for microbiologic examination.     

聚合酶链反应检测结核分枝杆菌DNA诊断关节结核的临床评价

目的评价聚合酶链反应(polymerase chain reaction,PCR)检测关节结核标本结核分枝杆菌脱氧核糖核酸(deoxyribonucleic acid,DNA)诊断关节结核的临床价值。方法对20份标准标本(5份结核分枝杆菌标准菌株、5份卡介苗和10份其它细菌标本)分别应用PCR盲法检测结核分枝杆菌DNA。对95例关节结核标本和98例非关节结核标本分别应用PCR检测结核分枝杆菌DNA。应用疾病诊断方法的临床评价原则对PCR诊断关节结核的敏感性、特异性和准确性进行评价。结果 (1)20份标准标本PCR检测中,结核分枝杆菌和卡介苗均为阳性,而其它细菌均为阴性;(2)95例关节结核标本中,PCR阳性78例、阴性17例;98例非关节结核标本中,PCR阳性9例、阴性89例;(3)PCR检测关节结核标本结核分枝杆菌DNA的敏感性为82.11%、特异性为90.81%、准确性为86.60%、阳性预测值为89.80%、阴性预测值为84.00%;(4)PCR整个检测过程自动化控制,可在3~6h内完成。结论 PCR是一种敏感、特异、快速、简便、无创、标本微量的关节结核标本结核分枝杆菌检测方法,对于关节结核的早期、快速诊断与鉴别诊断具有极其重要的临床价值。     

Evaluation of mtp40 genomic fragment amplification for specific detection of Mycobacterium tuberculosis in clinical specimens.

A PCR assay based on the species-specific mtp40 genomic fragment was developed for the specific detection and identification of Mycobacterium tuberculosis in different uncultured clinical specimens. The aim of the study was to evaluate the clinical applicability of this target DNA in comparison with those of conventional microbiological methods and to compare the results obtained with those obtained after amplification with the IS6110 repetitive element. Discrepant results were interpreted in conjunction with the patients' clinical data, medical histories, and response to therapy. A total of 172 specimens from 162 patients with respiratory symptoms were tested, 101 specimens were obtained from 92 patients clinically suspected of having tuberculosis, and 71 specimens were obtained from 70 patients without known mycobacterial infection. The results of our study suggest that PCR amplification with the mtp40 genomic fragment provides a highly sensitive and specific technique for the detection of M. tuberculosis strains in clinical samples. It allows for the differentiation between M. tuberculosis and other related mycobacteria, including M. bovis, and is more specific than the IS6110 target. For these and other reasons, we propose that the mtp40 assay is a possible alternative for the specific direct detection of M. tuberculosis in clinical laboratories.     

Rapid and specific detection of Mycobacterium tuberculosis from acid-fast bacillus smear-positive respiratory specimens and BacT/ALERT MP culture bottles by using fluorogenic probes and real-time PCR

A real-time PCR assay using the LightCycler (LC) instrument for the specific identification of Mycobacterium tuberculosis complex (MTB) was employed to detect organisms in 135 acid-fast bacillus (AFB) smear-positive respiratory specimens and in 232 BacT/ALERT MP (MP) culture bottles of respiratory specimens. The LC PCR assay was directed at the amplification of the internal transcribed spacer region of the Mycobacterium genome with real-time detection using fluorescence resonance energy transfer probes specific for MTB. The results from the respiratory specimens were compared to those from the Amplicor M. tuberculosis PCR test. Specimens from MP culture bottles were analyzed by Accuprobe and conventional identification methods. MTB was cultured from 105 (77.7%) respiratory AFB smear-positive specimens; 103 of these samples were positive by LC PCR and Amplicor PCR. Two samples negative in the LC assay contained rare numbers of organisms; both were positive in the Amplicor assay. Two separate samples negative by Amplicor PCR contained low and moderate numbers of AFB, respectively, and both of these were positive in the LC assay. There were 30 AFB smear-positive respiratory specimens that grew mycobacteria other than tuberculosis (MOTT), and all tested negative in both assays. Of the 231 MP culture bottles, 114 cultures were positive for MTB and all were positive by the LC assay. The remaining 117 culture bottles were negative in the LC assay and grew various MOTT. This real-time MTB assay is sensitive and specific; a result was available within 1 h of having a DNA sample available for testing.     

Polymerase chain reaction for detection of Mycobacterium tuberculosis.

A polymerase chain reaction for the specific detection of mycobacteria belonging to the Mycobacterium tuberculosis complex was developed. Using a single primer pair derived from the nucleotide sequence of protein antigen b of M. tuberculosis, we achieved specific amplification of a 419-base-pair DNA fragment in M. tuberculosis and M. bovis. After DNA was extracted from mycobacteria by using a simple, safe lysis procedure, we detected the 419-base-pair sequence in samples containing few mycobacteria. Preliminary data suggested that this technique could be applied to clinical specimens for early and specific diagnosis of tuberculosis.     

Detection of bacillus Galmette-Guérin (Mycobacterium bovis BCG) DNA in urine and blood specimens after intravesical immunotherapy for bladder carcinoma

A real-time PCR targeting IS6110 was employed for the detection of Mycobacterium tuberculosis DNA in specimens collected from 10 patients treated with intravesical M. bovis bacillus Galmette-Guérin (BCG) immunotherapy for bladder malignancy. BCG DNA was detected in all urine specimens taken 24 h after the instillations, as well as in 24% of the specimens collected 7 days after the instillations; it was also detected in a single specimen taken 6 weeks after the last instillation. BCG DNA was detected in 8.3% of the blood specimens taken 1 day after instillation, and its amplification was associated with cases of self-limiting fever. These findings give indications that this real-time PCR is helpful to recognize BCG bacteremic cases, which may lead to mycobacterial infection.     

Detection and identification of mycobacteria in formalin-fixed, paraffin-embedded tissues by nested PCR and restriction enzyme analysis.

A novel assay based on a nested PCR and restriction enzyme analysis of the PCR products was developed for the rapid detection and identification of Mycobacterium bovis and M. avium-M. intracellulare species in formalin-fixed, paraffin-embedded tissue (PET) specimens. On the basis of the nucleotide sequence data obtained in the present study, general nested primers were constructed to amplify a 424-bp segment of the gene encoding the 65-kDa surface antigen of mycobacteria. The nested PCR assay proved to be highly sensitive, since as little as 5 to 10 fg of extracted mycobacterial DNA was detected. The safety of the assay as a routine method for the diagnosis of M. bovis and M. avium-M. intracellulare in PET specimens was provided by taking various precautions. In order to prevent false positivity, specific tools and procedures were applied. To detect false-negative results and assess the efficiency of the PCR, an internal standard molecule of amplification was constructed. The digestion of the amplicons with the restriction endonuclease Sau96-I allowed the identification of M. bovis and M. avium-M. intracellulare in a large number of clinical specimens. The present results indicate that PCR combined with an internal control of amplification and restriction enzyme analysis of the amplicons provides a rapid, sensitive, and reliable method for routine diagnostic laboratories to detect and identify M. bovis and M. avium-M. intracellulare in PET specimens.     

Sensitive and specific method for rapid identification of Streptococcus pneumoniae using real-time fluorescence PCR

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the identification of organisms of clinical and epidemiological importance. As the leading cause of community-acquired pneumonia, Streptococcus pneumoniae was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. Seventy clinical isolates of S. pneumoniae, verified by latex agglutination, were screened against 26 negative control clinical isolates employing a TaqMan assay on a thermocycler (LightCycler). The probe, constructed from the lytA gene, correctly detected all S. pneumoniae genomes without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from patient specimens.     

Polymerase chain reaction using IS6110 primer to detect Mycobacterium tuberculosis in clinical samples

Nucleic acid amplification using IS6110 primers to detect Mycobacterium tuberculosis in clinical specimens has been extensively used as laboratory tool for the diagnosis for tuberculosis. Despite it's dramatic scientific value in practice, it is not as sensitive as expected for the detection of Mycobacterium tuberculosis. The results of the study suggest that PCR using 123 bp fragment of DNA belonging to IS6110 is specific (95.6%) but only has a sensitivity of 30% to detect M. tuberculosis in clinical specimens.     

Identification of the causative organism of tuberculous lymphadenitis in ethiopia by PCR

Tuberculous lymphadenitis (TBLN) is a common form of extrapulmonary tuberculosis with multiple differential diagnoses. Demonstration of the etiologic agent by smear microscopy or culture of fine needle aspirate (FNA) specimens is often unsuccessful. FNA specimens from 40 patients presenting at a rural health center in South Ethiopia and diagnosed as positive for TBLN on the basis of clinical and cytological criteria were analyzed for mycobacterial DNA by PCR. Thirty (75%) had cervical lymphadenitis and 11 (27.5%) were seropositive for human immunodeficiency virus (HIV). Three primer sets were initially used to identify the causative agent at the genus (antigen 85 complex), complex (IS6110 insertion sequence), and species (pncA gene and allelic variation) levels. Among the forty TBLN cases, 35 (87.5%) were positive by PCR at the genus and complex levels. Based on PCR for detection of allelic variation at position 169, 24 (68.6%) of the 35 were positive for Mycobacterium tuberculosis and 6 (17.1%) were positive for M. bovis. These six were positive in additional PCR assays using the JB21-JB22 primer set, which is highly specific for M. bovis. Five (14.1%) showed amplification for both M. tuberculosis and M. bovis with the allele-specific primer set. Cooccurrence of pyrazinamide (PZA)-sensitive and -resistant M. tuberculosis in those five cases was indicated, since all were negative in assays with the JB21-JB22 primer set. This feature was seen in 3 of 11 HIV-positive and 2 of 29 HIV-negative individuals (P < 0.001). Conclusion: among 35 PCR-positive cases of TBLN from southern Ethiopia, 29 (82.9%) were caused by M. tuberculosis and six (17.1%) were caused by M. bovis.