Chest ACCP Member Benefits
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Guest Access | Sign In via User Name/Password
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ruiz-Manzano, J.
Right arrow Articles by Ausina, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ruiz-Manzano, J.
Right arrow Articles by Ausina, V.
(Chest. 2000;118:648-655.)
© 2000 American College of Chest Physicians

Detection of Mycobacterium tuberculosis in Paraffin-Embedded Pleural Biopsy Specimens by Commercial Ribosomal RNA and DNA Amplification Kits*

Juan Ruiz-Manzano, MD; Joxe-Mari Manterola; Fredy Gamboa, PhD; Ana Calatrava, MD; Eduardo Monsó, MD; Carlos Martínez, MD and Vicente Ausina, MD

* From the Departments of Pneumology (Drs. Ruiz-Manzano, Monsó, and Martínez), Microbiology (Mr. Manterola, and Drs. Gamboa and Ausina), and Pathology (Dr. Calatrava), Hospital Universitari Germans Trias i Pujol, Badalona, Barcelona, Spain.

Correspondence to: Juan Ruiz-Manzano, MD, Pneumology Department, Hospital Universitari Germans Trias i Pujol, Carretera del Canyet s/n, 08916, Badalona, Barcelona, Spain; e-mail: jruiz{at}ns.hugtip.scs.es


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Study objectives: To evaluate the utility of two gene amplification systems in historical paraffin-embedded pleural biopsy (PEB) tissues from patients with pleural tuberculosis, and to compare the results to those obtained with conventional histologic and microbiological methods.

Design: A retrospective study.

Patients and methods: Seventy-four formalin-fixed PEB tissues collected and stored over 12 years (1984 through 1995) were retrieved. Gene amplifications were performed in 57 tissues from patients with diagnoses of pleural tuberculosis and in 17 from patients with carcinoma as controls, using the first version of the Amplified Mycobacterium tuberculosis Direct Test (AMTDT; Gen-Probe; San Diego, CA) and the LCx Mycobacterium tuberculosis Assay (LCxMTB; Abbott Laboratories; Abbott Park, IL).

Results: The sensitivities of the AMTDT and LCxMTB were 52.6% and 63.2%, respectively (p = not statistically significant). The specificity of both tests was 100%. Twenty tissue samples (35.1%) were positive by both systems, and 10 tissues (17.5%) were positive only by the AMTDT, while 16 tissues (28.1%) were positive only by the LCxMTB. Both tests gave negative results for 11 specimens (19.3%). When both tests were used, a positive diagnosis was achieved in 80.7% of the samples. Diagnosis of 73.7% of patient conditions had previously been made by smear examination of pleural biopsy and sputum, pleural liquid, or biopsy culture. The overall diagnostic yield with both culture and amplification techniques was 96.5% (55 of 57 patients) for pleural tuberculosis, with amplification techniques adding 22.8% of the diagnoses.

Conclusions: Amplification techniques are useful in archival PEB tissues, providing additional diagnoses beyond culturing, although the sensitivity should be improved, possibly by standardizing protocols.

Key Words: gene amplification • paraffin-embedded pleural tissue • pleural tuberculosis


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
The diagnosis of pleural tuberculosis—the second most frequent clinical presentation of human tuberculosis—is usually based on the observation of necrotizing granulomas in pleural biopsy tissue.1 Biochemical analysis for adenosine deaminase, lysozime, and interferon-{gamma} can contribute to diagnosis and, in some cases, render pleural biopsy unnecessary.1 2 3 4 Acid-fast staining of pleural fluid is a rapid, inexpensive method for diagnosing pleural tuberculosis, but sensitivity is low. The isolation of Mycobacterium tuberculosis in cultured pleural fluid or tissue biopsy specimens permits firm diagnosis, but, again, sensitivity is low and results can take as long as 3 to 6 weeks to arrive.

Various gene amplification techniques have demonstrated their utility in the diagnosis of pulmonary and extrapulmonary forms of tuberculosis.5 6 7 8 These techniques, which have usually involved homemade polymerase chain reaction (PCR) in pleural fluid samples,9 10 11 rather than commercially available kits, have performed poorly in the diagnosis of pleural tuberculosis. The aim of the present study was to evaluate the sensitivity and specificity of two commercial techniques of gene amplification for the diagnosis of pleural tuberculosis from formalin-fixed paraffin-embedded pleural biopsy (PEB) tissues, and to compare them with diagnostic results from pleural liquid and biopsy stains and cultures. We chose to study PEB tissues because few such "sterile" fluid specimens yield positive findings yearly. By retrieving stored samples, we were able to obtain a total of 57 tuberculous samples, our assumption being that the results obtained probably underestimate those achievable with fresh samples.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Patients and Clinical Specimens
From the archives of the Pathology Department of our hospital, we obtained sections of 74 blocks of 10% neutral buffered formalin-fixed PEB material corresponding to 74 patients. The tissue blocks were collected between 1984 and 1995, and were processed by standard procedures in the same laboratory. The sections were stained using the Ziehl-Neelsen technique for microscopic examination.

Fifty-seven patients, with 57 specimens, were given a diagnosis of pleural tuberculosis based on the following criteria: (1) isolation of M tuberculosis in sputum, pleural fluid, or biopsy specimen from patients with granulomas in pleural tissue (38 patients); (2) positive culture of M tuberculosis in the pleural biopsy specimen without granulomas (1 patient); and (3) patients whose diagnosis was based on the presence of necrotizing granulomas and the following criteria: lymphocytic liquid, positive purified protein derivative and adenosine deaminase activity > 40 IU/L (16 cases), or alternatively, on clinical and biochemical criteria exclusively; lymphocytic liquid, positive purified protein derivative and adenosine deaminase activity > 40 IU/L, in 2 young patients with recent contact with Ziehl-Neelsen-positive tuberculosis. Seventeen negative tissue controls were included, all of which were sections of pleural biopsy in patients with malignancy.

For 39 of the 57 patients with pleural tuberculosis, we also had records of the results of cultures of fresh pleural biopsy samples. They had been homogenized with 1 mL of sterile water and processed for microscopic examination using auramine-rhodamine and Ziehl-Neelsen stain techniques. Five hundred microliters of the homogenized sample was inoculated onto two tubes of Löwenstein-Jensen solid media and incubated at 37°C for up to 8 weeks. Pleural fluids from 41 patients were inoculated into the same culture media after centrifugation at 3,000g for 20 min. Other respiratory specimens, such as sputum from 18 patients, were processed for culture after decontamination by Tacquet-Tison technique on arrival in the laboratory.12 Mycobacteria were identified by routine biochemical methods and Accuprobe culture confirmation tests (Gen-Probe; San Diego, CA).13

RNA and DNA Extraction From PEB Tissues
Two 10-µm sections of each PEB sample were separately placed in two 1.5-mL Eppendorf tubes. The microtome blade was changed after each sample was sectioned to avoid cross-contamination. Each sample was deparaffinized by adding 1 mL of xylene, mixing on a rotary platform for 30 min at room temperature, and then discarding the supernatant after 8 min of centrifugation at 13,000g. This process was repeated twice for each sample. The remaining xylene was then extracted by adding 1 mL of absolute ethanol to each sample and vortexing for 30 s. The mixture was pelleted by centrifugation at 13,000g for 8 min and the ethanol removed. The xylene extraction procedure was repeated. Two drops of acetone were then added, and the tubes were incubated open in a water bath for 15 min at 60°C. The dried samples were resuspended in 100 µL of digestion buffer (2 mM Tris-HCl [pH 8.5], 0.06 mM ethylenediaminetetraacetate, and 0.005% sodium dodecyl-hydrogene sulfate) containing 500 µg/mL of proteinase K. Digestion took place at 55°C for 3 h. The preparations were vortexed for 30 s and then incubated at 95°C for 10 min to enhance mycobacterial lysis and inactivate the proteinase K. Then, the samples were spun for 2 min to pellet nonsoluble debris for removal. The supernatant was used without further purification. Further mycobacterial cell lysis was induced with sonication according to Amplified Mycobacterium tuberculosis Direct Test (AMTDT; Gen-Probe) and LCx Mycobacterium tuberculosis Assay (LCxMTB; Abbott Laboratories; Chicago, IL) protocols.14

AMTDT Protocol
The AMTDT kit uses an isothermal enzymatic amplification system of target ribosomal RNA, with the target amplicons detected using a chemiluminescent acridinium ester-labeled DNA probe. The test was performed in three steps according to the instructions on the package insert of the first version of the kit. Briefly, lysis was induced by adding 50 µL of digested sample to 200 µL of specimen dilution buffer in a lysing tube, and the mixture was sonicated in a Branson 1200 water bath sonicator (Branson Ultrasonics; Danbury, CT) for 15 min at room temperature. For amplification, 25 µL of reconstituted amplification reagent was placed in the bottom of a reaction tube and covered with 200 µL of mineral oil. Fifty microliters of lysate was transferred to the amplification tube, incubated at 95°C for 15 min, and then cooled at 42°C for 5 min. After addition of an enzyme reagent mix (25 µL), the mixture was incubated at 42°C for 2 h. Twenty microliters of the termination reagent was added to each amplification tube, and the mixtures were kept at 42°C for another 10 min. Detection was achieved by adding 100 µL of the reconstituted acridinium-labeled probe reagent to each tube and incubating the mixtures at 60°C for 15 min. The selection reagent (300 µL) was then added, and the mixture was reincubated at 60°C for 10 min. All temperature-controlled incubation steps were carried out in heating blocks, and all runs included AMTDT positive and negative amplification controls and positive and negative hybridization controls. The tubes were cooled at room temperature for 5 to 10 min prior to being read in a luminometer (Leader 50; Gen-Probe) with the cutoff value set at 30,000 relative light units (RLUs). Thus, samples showing > 30,000 RLUs were considered positive, and those showing < 30,000 RLUs were considered negative.

LCxMTB
One hundred microliters of each deparaffinized and digested specimen was placed in an LCxMTB kit respiratory specimen tube and centrifuged at 1,500g for 10 min. The supernatant was aspirated, and 1 mL of respiratory specimen resuspension buffer from the kit was added to the tube, which was centrifuged once again at 1,500g for 10 min. The supernatant was removed, and 0.5 mL of LCxMTB respiratory specimen resuspension buffer was pipetted into the specimen tube, after which the suspension was vortexed and placed in an LCxMTB covered dry bath for 20 min at 95°C. Finally, mycobacterial DNA was released by mechanical lysis in the LCxMTB lysor for 10 min. For amplification, 100 µL of the lysed specimen was added to the appropriately labeled LCxMTB tuberculosis amplification vial containing 100 µL of the reaction mixture of the ligase chain reaction, which contained thermostable DNA ligase and DNA polymerase, deoxyribonucleoside triphosphate, and four oligonucleotide probes labeled with haptens. Two calibrators and two negative controls were included in each test run. The specimens and controls were placed in the LCxMTB thermal cycler and amplified for 37 cycles of incubation for 1 s at 94°C, 1 s at 55°C, and 40 s at 69°C. For detection, the amplification vials were removed from the thermal cycler, transferred to the reaction cell in the carousel of the kit, and then locked into the LCxMTB analyzer. Each oligonucleotide probe has either a capture hapten or a detection hapten, each at a different end of the amplification product. In the analyzer, a sample of the amplification product is automatically transferred to an incubation well, where the microparticles coated with anticapture hapten bind the amplification product. The reaction mixture is then automatically transferred to a glass fiber matrix to which the microparticle complexes bind irreversibly. Washing removes the unligated probe, which has only the detection hapten. The bound microparticle complexes are then incubated with antidetection hapten alkaline phosphatase conjugate, which binds to the detection haptens. The antibody conjugate is then detected by addition of the substrate, 4-methylumbelliferyl phosphate, which is dephosphorylated by alkaline phosphatase, producing 4-methylumbelliferone, a fluorescent molecule that is measured by the optical assembly. A ratio of sample value to cutoff value > 1.0 indicates a positive result.

Statistical Analysis
Sensitivity values were compared using a McNemar test for comparison of percentages for paired data. A binomial test was used when the sample size was very small.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
The AMTDT was positive in 30 of 57 PEB blocks that were positive for tuberculous disease by microbiological and/or clinical and histopathologic criteria (Table 1 ). The AMTDT was negative in all 17 PEB blocks with clinical and histologic diagnoses of carcinoma. Thus, sensitivity and specificity were 52.6% and 100%, respectively, for the AMTDT (Table 2 ). The LCxMTB was positive in 36 of 57 PEB blocks that were positive for tuberculous disease (Table 1) . None of the 17 carcinoma controls had positive LCxMTB results. Therefore, sensitivity and specificity were 63.2% and 100%, respectively, for the LCxMTB (Table 2) . Table 2 also shows the values for sensitivity of both kits according to the means by which diagnosis was obtained. Differences were not statistically significant.


View this table:
[in this window]
[in a new window]

  		Table 1.. Comparison of Results Obtained in PEB Specimens by Culture, Histologic Study, and Two Genetic Amplification Techniques*

 

View this table:
[in this window]
[in a new window]

  		Table 2.. Sensitivity of AMTDT and LCxMTB M tuberculosis in PEB Samples*

 
Amplification was positive for a total of 20 samples (35.1%) in both systems, for 10 samples (17.5%) only in the AMTDT, and for 16 samples (28.1%) only in the LCxMTB; 11 samples (19.3%) were negative in both tests. At least one of the two amplification techniques was positive in 46 of the 57 tuberculous PEB tissues analyzed (80.7%).

Colonies of M tuberculosis had been isolated in the sputum cultures of 11 of 18 patients (61.1%), in 25 of 41 patients (61.0%) with pleural fluid samples, and in 28 of 39 patients (71.8%) whose pleural biopsy specimens had been cultured. Using either microscopic examination or culture of sputum, pleural fluid, or pleural biopsy, a pleural tuberculosis diagnosis was established in 42 patients (73.7%). By adding to these patients those confirmed by one or the other of the amplification techniques, a sure etiologic diagnosis was achieved in 55 of 57 patients (96.5%). Individually, AMTDT increased diagnosis by 14% (eight more cases), such that total diagnosis of AMTDT plus culture would reach 87.7%. LCxMTB increased diagnosis by 15.8% (nine more cases), such that diagnosis of LCxMTB plus culture would reach 89.5%. Amplification techniques, thus, contributed an additional 22.8% of the etiologically confirmed diagnoses.


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
The rapid detection of M tuberculosis by PCR or other gene amplification techniques may eventually become the standard laboratory method for diagnosing tuberculosis, as such techniques allow mycobacterial nucleic acid sequences to be multiplied sufficiently to permit earlier detection of the tuberculous bacillus. To date, several DNA or RNA amplification kits have been marketed and studied. The AMTDT and LCxMTB systems have been extensively evaluated in respiratory samples, in which they have been found highly sensitive and specific. Their utility in paucibacillary forms of tuberculosis or normally sterile fluids, such as pleural, articular, and cerebrospinal fluids, or in any PEB specimen, is still unknown. As few such specimens yield positive findings in any given year in our practice, we evaluated the AMTDT and LCxMTB amplification kits in PEB samples in storage at our laboratory in order to obtain a total of 57 tuberculous samples for study.

The AMTDT and LCxMTB kits gave sensitivity values of 52.6% and 62.3%, respectively, in PEB samples. These percentages rose to 61% and 69%, respectively, in PEB samples with positive Ziehl-Neelsen smears. The amplification of ribosomal RNA, as in the AMTDT, is theoretically more sensitive than DNA amplification, because approximately 2,000 copies are made per-cell rather than per-target sequence, but in our study, the LCxMTB gave slightly better results than did the AMTDT. The level of sensitivity in our PEB samples was lower than those reported by other authors for respiratory and extrarespiratory samples. The AMTDT has demonstrated excellent sensitivity (70 to 89%) and specificity (95 to 100%) in numerous assays with respiratory5 7 15 16 17 18 19 20 21 and extrarespiratory samples.8 21 22 23 Assessment of the LCxMTB in respiratory specimens has shown that sensitivity ranges from 96.7 to 100% in smear-positive specimens, from 36.8 to 85.7% in smear-negative specimens, and from 77 to 100% in specimens overall.24 25 26 27 In extrapulmonary tuberculosis, the sensitivities of the LCxMTB are 100% and 71% for smear-positive and smear-negative specimens, respectively, and between 73.3% and 78.5% for all specimens.26 28 The differences between our sensitivity data and those obtained by the cited authors are not surprising, however, given that we dealt with samples containing far fewer bacilli. The 100% specificity of AMTDT and LCxMTB in our PEB samples, in other hand, agrees with the high specificities reported in all cited publications.

Our results, like those of other authors who have worked with paucibacillary specimens, thus show that gene amplification techniques give relatively low yield in such specimens. In paraffin-embedded skin specimens from patients with a diagnosis of lupus vulgaris, Degitz et al29 found M tuberculosis DNA in 53.3% (8 of 15) by PCR, similar to our results in pleural tissues and to results in skin cultures. Moreover, they reported four PCR-positive findings (66.7%) in six papulonecrotic tuberculid samples.30 Other authors31 detected M tuberculosis DNA in seven of nine paraffin-embedded samples (77.8%) of papulonecrotic tuberculid. Marchetti et al,32 working with four nested PCR assays in 37 autopsy samples from HIV-positive patients, demonstrated that the sensitivity of PCR depends on the DNA concentration used for amplification, on target DNA size, or on the number of target sequences present in the M tuberculosis gene, with PCR sensitivity ranging from 47 to 87%. Similarly, Mycobacterium leprae was detectable by PCR amplification in 87.1% of biopsy specimens from multibacillary patients and in 36.4% of biopsy specimens from paucibacillary patients.33 Results in fresh pleural samples are similar. Vlaspolder et al,21 from 61 pleural exudate specimens, reported 20% sensitivity with the AMTDT (one transcriptase-mediated amplification positive out of five culture positives). Other authors, from 65 pleural fluids analyzed, obtained a level of sensitivity of PCR that was similar to that of mycobacterial culture.22 Other normally sterile fluids, such as pericardial or cerebrospinal fluids, are also paucibacillary, and the sensitivity of culture and amplification techniques in them is still low. In tuberculous pericarditis, Cegielski et al,34 using a homemade PCR process, reported a high sensitivity (80%) with 15 fresh pericardial tissues but very poor results (15%) with 13 pericardial fluid specimens; those results were similar to findings by Vlaspolder et al21 for pleural fluids. Bonington et al,35 on the other hand, managed an overall sensitivity of 25% (10 of 40 patients) in tuberculous meningitis with the Amplicor PCR system (Roche Molecular Systems; Branchburg, NJ).

We have found a few reports of high PCR sensitivities in paucibacillary samples, however. Palacios et al,36 working with 186 pleural exudates, found the LCxMTB system to be more sensitive (16 positive) than solid or liquid culture (12 positive). Querol et al,9 studying 21 patients with tuberculous pleurisy, found the sensitivity of a homemade PCR of pleural fluid to be 81%, making it more sensitive than microscopic examination, pleural fluid culture (52%), or biopsy culture (67%), those culture results being similar to ours.4 Folgueira et al,10 working with 20 patients with smear-negative tuberculous pleuritis, achieved a sensitivity of 85% with pleural fluid culture and 100% sensitivity with a homemade PCR process using undiluted and 10-fold diluted lysates in order to reduce the number of PCR inhibitors. In that study, lysis accomplished by nonionic detergent and proteinase K proved more effective than sonication for releasing DNA from mycobacteria. Other researchers, using a nested PCR and restriction enzyme analysis of the PCR amplicons, found the technique useful for the detection of Mycobacterium bovis and Mycobacterium avium–M avium-intracellulare complex in animal paraffin-embedded tissues.37

Less than optimal sensitivity in amplification techniques in pleural specimens, when taken in conjunction with numerous reports of false-positives due to contamination with DNA fragments (amplicons) from previous amplification techniques, leads us to consider whether results might be improved by standardizing protocols. In an international quality control study, only 5 of 30 laboratories correctly identified the presence or absence of mycobacterial DNA38 ; similarly, another comparison study reported false-positive PCR results ranging from 3 to 20%. Results from one laboratory in the study were false-positives in 77% of samples.39 To reduce the likelihood of false-positives in our study, the microtome blade was changed after sectioning each sample to avoid cross-contamination. AMTDT and LCxMTB, unlike the Roche Amplicor Mycobacterium tuberculosis PCR test and Cobas Amplicor M tuberculosis, which use uracil-DNA-glycosylase, have no system for neutralizing contamination by amplicons. We therefore used negative controls in all runs, finding no false-positives in the 17 carcinoma samples. As mentioned, the sensitivity of nucleic amplification techniques depends on the number of mycobacteria, their homogenous distribution in the sample, and the presence of amplification inhibitors in the analyzed sample.40 Our relatively low sensitivities of 52.6% and 63.2% in AMTDT and LCxMTB, respectively, could therefore be due to inhibitors in PEB tissues, to destruction of DNA and RNA in PEB blocks stored for many years in the laboratory,29 or to limited sensitivity of the commercial kits. When assessing the overall utility of these tests, however, it is important to remember that we were able to detect M tuberculosis in > 80% of the pleural PEB specimens with one technique or the other or both, although in 45.6%, one or the other technique was negative. The stated minimum number of bacilli in the samples for detection to occur is 10 cfu,5 although nine bacilli in a 5-µm section of mouse tissue were detected after up to 7 days of fixation in 10% neutral buffered formalin.41 Kirschner et al42 showed that inhibitors of amplification were found in 11.5% of biopsy specimens and in 25% of pleural fluids. Thus, strategies to improve the amplification sensitivity in PEB tissues include the following: (1) improving fixation procedures, among which it seems that 10% neutral buffered formalin is perhaps the best fixative29 41 ; (2) improving lysis and enzymatic method with proteinase K, which seems better than boiling or sonication9 37 ; (3) using 500 µL rather than 50 µL of decontaminated sediment to amplify in the AMTDT,43 or reducing the resuspension of buffer volume in the LCxMTB36 ; (4) inactivating polymerase inhibitors by the guanidinium isothiocyanate method,26 44 45 by phenol-chloroform and ice-cold ethanol,31 32 or by using capture resin46 ; (5) choosing the best primers, which are those that amplify short DNA sequences32 ; and (6) increasing the number of amplification cycles36 or doing nested PCR with a second amplification using internal primers.47 Finally, although sensitivity and specificity remain the main improvement targets for amplification techniques, other evident goals are full automation and the use of an internal control in each sample, as suggested by Ros Bascuñana and Belák37 and Rosenstraus et al.48 Specifically, Baselga et al31 and Marchetti et al32 have suggested that the ß-globin gene might serve that function.


   Conclusion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
Amplification techniques offer rapid results in the diagnosis of pleural tuberculosis, when they prove sensitive. The sensitivities of the AMTDT and LCxMTB were 52.6% and 63.2%, respectively, in PEB tissues, and the specificity was 100% in both tests. By using both, a sensitivity of 80.7% was achieved in tuberculous PEB samples. Amplification techniques added 22.8% to etiologic diagnoses provided by culture alone.

We have shown that gene amplification in PEB tissues can achieve retrospective diagnoses of pleural tuberculosis. Although amplification techniques proved somewhat useful in archival PEB tissues, and may be more useful in fresh samples, sensitivity must be improved, possibly by standardizing protocols. We believe these results in PEB samples justify the performance of larger studies to establish the real value of these methods in fresh pleural tissues, our hypothesis being that the results will be at least as good as those achievable in PEB tissues.


   Acknowledgements
 
The authors would like to thank Dr. Irma Casas for the statistical analysis and Ms. Mary Ellen Kerans for assistance with the English version of the article.


   Footnotes
 
Abbreviations: AMTDT = Amplified Mycobacterium tuberculosis Direct Test; LCxMTB = LCx Mycobacterium tuberculosis Assay; PCR = polymerase chain reaction; PEB = paraffin-embedded pleural biopsy; RLU = relative light unit

Received for publication September 28, 1999. Accepted for publication March 28, 2000.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

  1. Light, RW, Ferrer, J (1999) Diagnóstico de la tuberculosis pleural. Arch Bronconeumol 35,105-107[Medline]
  2. Valdés, L, San José, E, Álvarez, D, et al (1993) Diagnosis of tuberculous pleurisy using the biologic parameters adenosine deaminase, lysozyme, and interferon-{gamma}. Chest 103,458-465[Abstract/Free Full Text]
  3. Villena, V, López-Encuentra, A, Echave-Sustaeta, J, et al (1996) Interferon-gamma in 388 immunocompromised and immunocompetent patients for diagnosing pleural tuberculosis. Eur Respir J 9,2635-2639[Abstract]
  4. Haro, M, Ruiz-Manzano, J, Gallego, M, et al (1996) Tuberculosis pleural: análisis de 105 casos. Enferm Infecc Microbiol Clín 14,285-289
  5. Abe, C, Hirano, K, Wada, M, et al (1993) Detection of Mycobacterium tuberculosis in clinical specimens by polymerase chain reaction and Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test. J Clin Microbiol 31,3270-3274[Abstract/Free Full Text]
  6. Clarridge, JE, III, Shawar, RM, Shinninck, TM, et al (1993) Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory. J Clin Microbiol 31,2049-2056[Abstract/Free Full Text]
  7. Ichiyama, S, Iinuma, Y, Tawada, Y, et al (1996) Evaluation of Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and Roche PCR- microwell plate hybridization method (AMPLICOR MYCOBACTERIUM) for direct detection of mycobacteria. J Clin Microbiol 34,130-133[Abstract]
  8. Gamboa, F, Manterola, JM, Viñado, B, et al (1997) Direct detection of Mycobacterium tuberculosis complex in nonrespiratory specimens by Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test. J Clin Microbiol 35,307-310[Abstract]
  9. Querol, JM, Mínguez, J, García-Sánchez, E, et al (1995) Rapid diagnosis of pleural tuberculosis by polymerase chain reaction. Am J Respir Crit Care Med 142,1977-1981
  10. Folgueira, L, Delgado, R, Palenque, E, et al (1993) Detection of Mycobacterium tuberculosis DNA in clinical samples by using a simple lysis method and polymerase chain reaction. J Clin Microbiol 31,1019-1021[Abstract/Free Full Text]
  11. Stauffer, F, Mutschlechner, R, Hasenberger, P, et al (1995) Detection of Mycobacterium tuberculosis complex in clinical specimens by a commercial polymerase chain reaction kit. Eur J Clin Microbiol Infect Dis 14,1046-1051[CrossRef][ISI][Medline]
  12. Tacquet A, Tison F. Nouvelle technique d'isolement des mycobactéries par le lauryl-sulphate de sodium. Ann Inst Pasteur (Lille) 1961; 100:676–680
  13. Goto, M, Oka, S, Okuzumi, K, et al (1991) Evaluation of acridinium-ester-labeled DNA probes for identification of Mycobacterium tuberculosis and Mycobacterium avium-Mycobacterium intracellulare complex in culture. J Clin Microbiol 29,2473-2476[Abstract/Free Full Text]
  14. Wright, DK, Manos, MM (1990) Sample preparation from paraffin-embedded tissues. Innis, MA Gelfand, DH Sninsky, JJet al eds. PCR protocols: a guide to methods and applications ,153-158 Academic Press New York, NY.
  15. Bodmer, T, Gurtner, A, Schopfer, K, et al (1994) Screening of respiratory tract specimens for the presence of Mycobacterium tuberculosis by using the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test. J Clin Microbiol 32,1483-1487[Abstract/Free Full Text]
  16. Ehlers, S, Pirmann, M, Zaki, W, et al (1994) Evaluation of a commercial rRNA target amplification assay for detection of Mycobacterium tuberculosis complex in respiratory specimens. Eur J Clin Microbiol Infect Dis 13,827-829[CrossRef][ISI][Medline]
  17. Gamboa, F, Fernández, G, Padilla, E, et al (1998) Comparative evaluation of initial and new versions of the Gen-Probe Amplified Mycobacterium tuberculosis in respiratory and nonrespiratory specimens. J Clin Microbiol 36,684-689[Abstract/Free Full Text]
  18. Jonas, V, Alden, MJ, Curry, JI, et al (1993) Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA. J Clin Microbiol 31,2410-2416[Abstract/Free Full Text]
  19. La Rocco, MT, Wanger, A, Ocera, H, et al (1994) Evaluation of a commercial rRNA amplification assay for direct detection of Mycobacterium tuberculosis in processed sputum. Eur J Clin Microbiol Infect Dis 13,726-731[CrossRef][ISI][Medline]
  20. Pfyffer, GE, Kissling, P, Wirth, R, et al (1996) Direct detection of Mycobacterium tuberculosis complex in respiratory specimens by a target-amplified test system. J Clin Microbiol 32,918-923[Abstract/Free Full Text]
  21. Vlaspolder, F, Singer, P, Roggeveen, C (1995) Diagnostic value of an amplification method (Gen-Probe) compared with that of culture for diagnosis of tuberculosis. J Clin Microbiol 33,2699-2703[Abstract]
  22. Pfyffer, GE, Kissling, P, Jahn, EMI, et al (1996) Diagnostic performance of Amplified Mycobacterium tuberculosis Direct Test with cerebrospinal fluid, other nonrespiratory, and respiratory specimens. J Clin Microbiol 34,834-841[Abstract]
  23. Ehlers, S, Ignatius, R, Regnath, T, et al (1996) Diagnosis of extrapulmonary tuberculosis by Gen-Probe Amplified Mycobacterium tuberculosis Direct Test. J Clin Microbiol 34,2275-2279[Abstract]
  24. Alonso, P, Orduña, A, Bratos, MA, et al (1998) Clinical evaluation of a commercial ligase-based gene amplification method for detection of Mycobacterium tuberculosis. Eur J Clin Microbiol Infect Dis 17,371-376[ISI][Medline]
  25. Ausina, V, Gamboa, F, Gazapo, E, et al (1997) Evaluation of the semiautomated Abbott LCxMTB Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis in respiratory specimens. J Clin Microbiol 35,1996-2002[Abstract]
  26. Tortoli, E, Lavinia, F, Simonetti, MT (1997) Evaluation of a commercial ligase chain reaction kit (Abbott LCxMTB) for direct detection of Mycobacterium tuberculosis in pulmonary and extrapulmonary specimens. J Clin Microbiol 35,2424-2426[Abstract]
  27. Yuen, KY, Yam, WC, Wong, LP, et al (1997) Comparison of two automated DNA amplification systems with a manual one-tube nested PCR assay for diagnosis of pulmonary tuberculosis. J Clin Microbiol 35,1385-1389[Abstract]
  28. Gamboa, F, Domínguez, J, Padilla, E, et al (1998) Rapid diagnosis of extrapulmonary tuberculosis by ligase chain reaction amplification. J Clin Microbiol 36,1324-1329[Abstract/Free Full Text]
  29. Degitz, K, Steidl, M, Neubert, U, et al (1993) Detection of mycobacterial DNA in paraffin-embedded specimens of lupus vulgaris by polymerase-chain reaction. Arch Dermatol Res 285,168-170[CrossRef][ISI][Medline]
  30. Degitz, K, Steidl, M, Thomas, P, et al (1993) Etiology of tuberculids. Lancet 341,239-240[CrossRef][ISI][Medline]
  31. Baselga, E, Margall, N, Barnadas, MA, et al (1996) Mycobacterium tuberculosis DNA in papulonecrotic tuberculid [letter]. Arch Dermatol 132,92-94[CrossRef][ISI][Medline]
  32. Marchetti, G, Gori, A, Catozzi, L, et al (1998) Evaluation of PCR in detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues: comparison of four amplification assays. J Clin Microbiol 36,1512-1517[Abstract/Free Full Text]
  33. Wichitwechkarn, J, Karnjan, S, Shuntawuttisettee, S, et al (1995) Detection of Mycobacterium leprae infection by PCR. J Clin Microbiol 33,45-49[Abstract]
  34. Cegielski, JP, Devlin, BH, Morris, AJ, et al (1997) Comparison of PCR, culture, and histopathology for diagnosis of tuberculous pericarditis. J Clin Microbiol 35,3254-3257[Abstract]
  35. Bonington, A, Strang, JIG, Klapper, PE, et al (1998) Use of Roche AMPLICOR Mycobacterium tuberculosis PCR in early diagnosis of tuberculous meningitis. J Clin Microbiol 36,1251-1254[Abstract/Free Full Text]
  36. Palacios, JJ, Ferro, J, Ruiz Palma, N, et al (1998) Comparison of the ligase chain reaction with solid and liquid culture media for routine detection of Mycobacterium tuberculosis in nonrespiratory specimens. Eur J Clin Microbiol Infect Dis 17,767-772[CrossRef][ISI][Medline]
  37. Ros Bascuñana, C, Belák, K (1996) Detection and identification of mycobacteria in formalin-fixed, paraffin-embedded tissues by nested PCR and restriction enzyme analysis. J Clin Microbiol 34,2351-2355[Abstract]
  38. Noordhoek, GT, van Embden, JDA, Kolk, AHJ (1996) Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: an international collaborative quality control study among 30 laboratories. J Clin Microbiol 34,2522-2525[Abstract]
  39. Noordhoek, GT, Kolk, AHJ, Bjune, G, et al (1994) Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories. J Clin Microbiol 32,277-284[Abstract/Free Full Text]
  40. Roth, A, Schaberg, T, Mauch, H (1997) Molecular diagnosis of tuberculosis: current clinical validity and future perspectives. Eur Respir J 10,1877-1891[Abstract]
  41. Rish, JA, Eisenach, KD, Cave, MD, et al (1996) Polymerase chain reaction detection of Mycobacterium tuberculosis in formalin-fixed tissue. Am J Respir Crit Care Med 153,1419-1423[Abstract]
  42. Kirschner, P, Rosenau, J, Springer, B, et al (1996) Diagnosis of mycobacterial infections by nucleid acid amplification: 18 months prospective study. J Clin Microbiol 34,304-312[Abstract]
  43. Bodmer, T, Möckl, E, Mühlemann, K, et al (1996) Improved performance of Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test when 500 instead of 50 µL of decontaminated sediment is used. J Clin Microbiol 34,222-223[ISI][Medline]
  44. Kox, LFF, Rhienthong, D, Medo, A, et al (1994) A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol 32,672-678[Abstract/Free Full Text]
  45. Chomczynski, P, Sacchi, N (1987) Single-step method of RNA isolation by acid-guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162,156-159[ISI][Medline]
  46. Amicosante, M, Richeldi, L, Trenti, G, et al (1995) Inactivation of polymerase inhibitors for Mycobacterium tuberculosis DNA amplification in sputum by using capture resin. J Clin Microbiol 33,629-630[Abstract]
  47. Pierre, C, Lecossier, D, Boussougant, Y, et al (1991) Use of a reamplification protocol improves sensitivity of detection of Mycobacterium tuberculosis in clinical samples by amplification of DNA. J Clin Microbiol 29,712-717[Abstract/Free Full Text]
  48. Rosenstraus, M, Wang, Z, Chang, S-Y, et al (1998) An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance. J Clin Microbiol 36,191-197[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Mol. Diagn.Home page
I. S. Johansen, V. O. Thomsen, A. Forsgren, B. F. Hansen, and B. Lundgren
Detection of Mycobacterium tuberculosis Complex in Formalin-Fixed, Paraffin-Embedded Tissue Specimens with Necrotizing Granulomatous Inflammation by Strand Displacement Amplification
J. Mol. Diagn., August 1, 2004; 6(3): 231 - 236.
[Abstract] [Full Text]

Home page
 ChestHome page
N. A. Hasaneen, M. E. Zaki, H. M. Shalaby, and A. S. El-Morsi
Polymerase Chain Reaction of Pleural Biopsy Is a Rapid and Sensitive Method for the Diagnosis of Tuberculous Pleural Effusion
Chest, December 1, 2003; 124(6): 2105 - 2111.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ruiz-Manzano, J.
Right arrow Articles by Ausina, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ruiz-Manzano, J.
Right arrow Articles by Ausina, V.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS