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00019606-200806000-00008ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2008 Lippincott Williams & Wilkins, Inc.17June 2008 p 112-117Real-time Quantitative PCR in the Diagnosis of Tuberculosis in Formalin-fixed Paraffin-embedded Pleural Tissue in Patients From a High HIV Endemic AreaOriginal ArticlesBaba, Kamaldeen MD* † ‡; Pathak, Sharad MD§ ∥; Sviland, Lisbeth PhD† ¶; Langeland, Nina PhD* ♯; Hoosen, Anwar A. PhD‡; Asjo, Birgitta PhD§ ∥; Dyrhol-Riise, Anne M. PhD* ♯; Mustafa, Tehmina PhD† ***Institute of Medicine†Centre for International Health**Section of Microbiology and Immunology§Centre for Research in Virology, The Gade Institute, University of BergenDepartments of ∥Microbiology and Immunology¶Pathology♯Medicine, Haukeland University Hospital, Bergen, Norway‡Department of Microbiological Pathology, University of Limpopo (Medunsa Campus), Pretoria, South AfricaFunded by University of Bergen, Norway and the Regional Health Authorities of Western Norway.Competing interests: The authors have no financial or nonfinancial competing interests to declare.Kamaldeen Baba and Sharad Pathak contributed equally to this work.Reprints: Tehmina Mustafa, PhD, Section of Microbiology and Immunology, The Gades Institute, Armauer Hansens Building, Haukeland University Hospital, Bergen N-5021, Norway (e-mail: [email protected]).AbstractThe aim of the study was to improve the diagnosis of pleural tuberculosis (TB) based on formalin-fixed biopsies from patients living in high TB and human immunodeficiency virus (HIV) endemic areas. A real-time polymerase chain reaction (real-time PCR) assay targeting a segment of the gene for mycobacterial 65-kd heat shock protein was developed and evaluated on pleural biopsies from 25 patients clinically diagnosed as having TB, on the basis of the good response to treatment, and from 11 controls. A nested polymerase chain reaction (N-PCR) assay for the repetitive genetic sequence insert IS6110, common to Mycobacterium tuberculosis complex organisms, was performed for comparison. When compared with N-PCR, the real-time PCR assay gave a sensitivity and specificity of 83% and 72%, respectively. When compared with clinical diagnosis, the sensitivity and specificity of real-time PCR (68% and 73%, respectively) was comparable with the sensitivity and specificity of the N-PCR assay (64% and 82%, respectively). There were no major differences in the diagnostic validity for the confirmed TB/HIV coinfected patients compared with the results from the whole TB group. In conclusion, the overall accuracy of the real-time PCR assay was comparable with that of the N-PCR and both were equally useful as diagnostic tools in the setting of a HIV coinfection. The real-time PCR has the additional advantage of a short turn-around time, low risk of sample contamination, and offers the possibility to quantify bacterial load, making it a powerful tool for the rapid diagnosis of TB pleuritis.The worldwide control of tuberculosis (TB) is dependent on reliable and prompt diagnosis.1 However, diagnosis of pleural TB by means of acid-fast bacilli (AFB) smears and culture has low sensitivity and specificity. Histology of pleural biopsies might demonstrate atypical patterns, especially in patients infected with the human immunodeficiency virus (HIV),2 and it is not specific for mycobacterial species. Although Mycobacterium tuberculosis is the most common mycobacterial infection, there is an upsurge of nontuberculous mycobacterias (NTMs), especially Mycobacterium avium, in patients with HIV coinfection.3The nucleic acid amplification techniques offer better sensitivities and specificities in the diagnosis of pleural TB than the more traditional diagnostic approaches.4,5 The nested polymerase chain reaction (N-PCR) assay allows DNA amplification from formalin-fixed biopsies with a remarkably higher sensitivity and specificity compared with the single step PCR assay.6 However, the conventional single step PCR and N-PCR techniques have several limitations. There is a potential for the release of amplified nucleic acid products into the environment with the risk of contamination of subsequent analyses. The visualization of PCR products by gels is laborious and time-consuming and DNA is not quantified. In contrast, real-time PCR has the advantage of a short turn-around time (1 to 2 h), a closed detection system, and offers the possibility to quantify bacterial load.7 All these factors are important when considering whether a method can be implemented as a routine operation in the clinical microbiology laboratory. However, it is still not clear whether real-time PCR will offer the same sensitivity and specificity as conventional N-PCR when applied to formalin-fixed and paraffin-embedded pleural tissue.In this study, we have developed a real-time PCR method on the basis of the amplification of a segment of the gene for 65-kd heat shock protein (hsp65), which is shared by the M. tuberculosis complex organisms and other human NTMs, including M. avium. We have investigated the ability of this method to detect and quantify mycobacterial DNA in formalin-fixed paraffin-embedded pleural biopsies from TB patients. Real-time PCR results were compared with those from N-PCR and with TB diagnoses that were based on clinical criteria.MATERIALS AND METHODSStudy Participants and Pleural BiopsiesThirty-six formalin-fixed paraffin-embedded pleural biopsies, obtained between January 2004 and October 2005, were retrieved from the archives of the Department of Anatomical Pathology, Dr George Mukhari Hospital, Ga-Rankuwa, University of Limpopo (MEDUNSA campus), Pretoria, South Africa. The 36 biopsies were selected as follows: out of the total of 102 biopsies obtained by the hospital during the study period, 31 biopsies had been performed owing to clinically suspected TB. Clinical records were available for 26 of these patients. Laboratory investigations (histology, TB culture from biopsies and/or sputum, AFB-staining, HIV status, and CD4 cell count), clinical symptoms and findings, and response to therapy were reviewed from the patient files retrospectively. One patient turned out not to have TB and was thus excluded. The remaining 25 biopsies were included as clinically defined TB cases with a good response to anti-TB drugs. Eleven biopsies from patients with pleural malignancies or hydrothorax because of trauma were used as controls. Two sections from each of the 36 biopsies were stained with hematoxylin-eosin and Ziehl-Nielsen (ZN) for examination of AFB.The study was approved by the ethical committees at the hospitals and universities in both South Africa and Norway.DNA ExtractionFour to five 10-μm sections were cut from the larger biopsies, and 6 to 10-μm sections from the smaller ones. The microtome blade was cleaned with xylene or 100% ethanol after sectioning each biopsy to prevent carryover contamination. Proteinase K digestion of the tissue was performed by using the MagAttract DNA Mini M48 kit (Qiagen, West Sussex, UK) as described earlier.6 This kit provides high-quality genomic DNA suitable for direct use in downstream applications such as amplification and reduces the presence of potential inhibitors from the dewaxing procedure. Briefly, tissues were incubated with 190 μL of Buffer G2 for 5 minutes at 76°C with vigorous shaking on a thermomixer at 900 rpm. The samples were allowed to cool to 56°C, 10 μL of Proteinase K solution was added, and the samples were incubated for 48 hours with continuous mixing on a thermomixer at 550 rpm. Another 10 μL of Proteinase K solution was added after 24 hours. DNA was extracted with Biorobot M48 using the QIAsoft operating system according to the manufacturer's instructions (Qiagen, Hilden, Germany). Duplicate or triplicate samples were used for each biopsy. Extracted DNAs were used as templates for the 2 PCR methods. All PCR work was carried out according to the recommended guidelines for molecular diagnostic methods with unidirectional workflow and separate physical sites for reagent preparation, amplification, and postamplification procedures to avoid contamination. Defined tissue (either ZN positive or culture positive) and mycobacterial DNA positive controls went through the same extraction procedures as the patient samples with consistently positive results in the PCR assays.N-PCRA conventional single step PCR was performed for 45 thermal cycles with primers targeting a 123-bp long segment of the IS6110 element of M. tuberculosis complex using the Taqman master mix kit (Qiagen, Hilden, Germany) as previously described.6 The primer sequences were P1: 5′-CCTGCGAGCGTAGGCGTCGG-3′ and P2: 5′-CTCGTCCAGCGCCGCTTCGG-3′.N-PCR was performed with a second set of primers (S1: 5′-TTC GGACCACCAGCACCTAA-3′ and S2: 5′-TCGGTGACAAAGGCCACGTA-3′) for 35 thermal cycles, targeting an internal 92-bp long segment of the amplicon from the first PCR as previously described.6 A case was labeled positive if PCR products were detected in any of the 3 triplicates from each case.Real-time PCRA real-time Taq Man PCR was developed targeting a 103-bp long segment of the hsp65 gene. The Primer Express 2.0 software (Applied Biosystems, Foster City, CA) was used to evaluate and generate primer/probe pairs compatible with either the primer TB11 or TB12.8 The forward primer, reverse primer, and fluorescent-tagged Taq Man MGB probe were MycoFP1 (5′-CGAGGCGATGGACA-AGGT-3′), TB12 (5′-CTTGTCGAACCGCATACCCT-3′), and MycoPr1 (5′-VIC-AACGAGGGCGTCATCACCGTCG-MGB-3′), respectively. The assay was optimized according to protocol 4304449c from Applied Biosystems (data not shown), but the reaction mixture was scaled down to 20 μL. The final volume of 20 μL real-time PCR mixture contained 5 μL of extracted DNA from clinical samples, 900 nM of each primer, 250 nM of the probe, and 10 μL of 2× TaqMan Universal PCR MasterMix (Applied Biosystems). The real-time PCR was performed with a 7500 Fast-Real-Time System (Applied Biosystems) with the following thermal cycling conditions: 50°C for 2 minutes to activate AMPErase UNG, followed by AmpliTaq Gold activation at 95°C for 10 minutes, and 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Each sample (duplicate or triplicate from each biopsy) was run in 2 to 3 parallels. In addition, each run included no-template negative controls, containing 5 μL of sterile water instead of extracted DNA, and several positive standards in the form of a standard curve generated from known dilutions of Genomic DNA from Bacille Calmette-Guérin (BCG) (Mycobacterium spp. ATCC 19015D). Because the BCG genome only contains 1 copy of the hsp65 gene, the number of targets in each positive standard was calculated by dividing the amount of genomic DNA in each standard by the molecular weight of the BCG genome. The weight of the BCG genome was calculated to be 4.77 fg on the basis of a genome size of 4.35×106 bp.9 The real-time PCR results were analyzed using Sequence Detection System software v1.3 (Applied Biosystems). The threshold was set automatically to 10 SDs above the background level, and threshold cycle (CT) values below 40 cycles were considered a positive result, as recommended by the manufacturer (Applied Biosystems). The assay has the potential to detect down to 1 genome per reaction, with the lowest limit of quantification being in the range of 5 to 10 genomes per reaction. Samples with <5 to 10 detectable genomes displayed a wider variation in CT values between replicates, reflecting an increased uncertainty in the quantification of mycobacterial DNA.Statistical AnalysisStatistical analysis was done using cross-tabulation to calculate sensitivity and specificity and with 95% confidence interval for the proportions.RESULTSPatient CharacteristicsA total of 36 pleural biopsies from 25 TB and 11 non-TB patients were included in the study. There were equal numbers of women and men (18/18). The median age was 32 years (range: 19 to 73) and 55 years (range: 20 to 83) in the TB and non-TB groups, respectively. HIV serology was available for 18 (72%) of the TB cases and 16 (89%) of those were confirmed as being HIV-positive. Only 2 (18%) of the non-TB cases were HIV tested and both were negative.TB culture results were available for 14 (56%) of the TB cases and only 3 (21%) were positive (1 from a pleural biopsy and 2 from sputum). Culture was performed for 5 (45%) of the control cases and all were negative. Only 2 (14%) of the TB cases demonstrated positive ZN staining on histologic examination and both had TB empyema.Detection of Mycobacterial DNA by Single-step PCR, N-PCR, and Real-time PCRSingle-step PCR, N-PCR, and real-time PCR were performed on all 36 biopsies. Among the 25 cases defined as TB, single-step PCR, N-PCR, and real-time PCR were positive in 8%, 64%, and 68% of the cases, respectively (Table 1). The N-PCR and real-time PCR results were concordant in 28 (78%) patients, whereas for 8 samples there was disagreement between the tests (Table 2). Among the 11 clinically defined non-TB patients, 3 were positive by the real-time PCR, and 2 of these 3 were also positive by the N-PCR.JOURNAL/dimp/04.03/00019606-200806000-00008/table1-8/v/2021-02-17T195951Z/r/image-tiff Correlation of Mycobacterial DNA in Pleural Biopsies Detected by N-PCR and Real-time PCR With HIV Status and Histologic FeaturesJOURNAL/dimp/04.03/00019606-200806000-00008/table2-8/v/2021-02-17T195951Z/r/image-tiff Real-time PCR Compared With N-PCRThe 2 ZN-positive pleural biopsies showed high number of AFB and had the highest bacterial genomic load (160 and 1661 genomes/5 μL of DNA) as quantified by real-time PCR. However, for the majority of the TB cases, ZN staining was negative and real-time PCR revealed a very low genomic load, ranging from being barely detectable to an average of 4.4 genomes/5 μL of DNA extract, which is consistent with the paucibacillary nature of the clinical samples.PCR Assays Compared With Histologic DiagnosisBy histologic examination, well-formed granulomas (focal aggregates of epithelioid macrophages and multinucleated Langhans giant cells surrounded by a rim of lymphocytes) were seen in pleural biopsies from 14 (56%) of the TB cases, but only 3 of these cases had granulomas with classic central caseous necrosis (Table 1). N-PCR and real-time PCR were positive in 50% and 71% of these cases, respectively. In the 11 (44%) TB cases without well-formed granulomas, a histologic pattern of focal and diffuse chronic inflammation consisting of lymphocytes and macrophages (10 cases) or an acute inflammatory reaction consisting of predominantly neutrophils (1 cases) were seen. N-PCR and real-time PCR were positive in 82% and 64% of these cases, respectively.Out of the 16 confirmed HIV coinfected TB patients, 11 (69%) had well-formed granulomas, and N-PCR and real-time PCR were positive in 10 (63%) and 12 (75%) of the HIV/TB cases, respectively (Table 1). In the remaining 9 TB cases (2 HIV-negative and 7 not HIV tested), well-formed granulomas were seen in 3 (27%) of the cases. N-PCR and real-time PCR were positive in 6 (67%) and 5 (56%) cases, respectively.CD4 cell counts were available only for 5 of the HIV patients. We found no correlation between histologic pattern and CD4 cell count as the 2 cases with relatively high CD4 cell counts (453 and 460 cells/μL) did not demonstrate any granulomas, whereas 2 of the 3 cases with low CD4 cell counts (71, 77, and 64 cells/μL) did.Diagnostic Validity of the Real-time PCR AssayWhen compared with N-PCR, the sensitivity and specificity of the real-time PCR assay were 83% and 72%, respectively, for the whole TB group. As all the cases in this study had been clinically defined as suffering from TB on the basis of the clinical criteria including good response to antituberculous therapy, the diagnostic validities of the 2 PCR assays were also compared with clinical diagnosis as gold standard. This resulted in comparable sensitivities and specificities for the 2 assays: 68% and 73% for real-time PCR and 64% and 82% for N-PCR, respectively (Table 3). The diagnostic accuracy for both assays was calculated to be 69%. Diagnostic validity was also calculated for the confirmed HIV/TB coinfected patients separately and compared with the results obtained from the whole TB group to study the influence of HIV infection. Overall, there were no major differences between the 2 groups.JOURNAL/dimp/04.03/00019606-200806000-00008/table3-8/v/2021-02-17T195951Z/r/image-tiff Validity of N-PCR and Real-time-PCR as Diagnostic Tests Compared With Clinical Diagnosis* as Gold StandardDISCUSSIONThis study has evaluated and compared the performance of the real-time PCR with the N-PCR for the diagnosis of pleural TB in formalin-fixed archival pleural biopsies. Our results indicate that a limited amount of mycobacterial DNA is present in such biopsies below the detection limit of the conventional single-step PCR. Hence, a more sensitive diagnostic tool is required to allow for the accurate diagnosis of pleural TB. We report that our real-time PCR assay was as sensitive as the N-PCR (68% and 64%, respectively) and that both assays offered markedly better sensitivities than the conventional single-step PCR.To our knowledge, this is the first study evaluating the usefulness of the real-time PCR assay for the diagnosis of pleural TB in formalin-fixed paraffin-embedded biopsies from patients coming from a high TB-HIV endemic area. Our findings are in agreement with recently published studies regarding the performance of similar real-time assays for other paucibacillary mycobacterial specimens.10–12 In this cohort, HIV serology was available for only half of the patients and almost 90% of these were positive. However, we have performed a study of TB pleuritis in another cohort of patients coming from the same area, in whom we found a comparable HIV prevalence and relatively low CD4 cell counts (Baba et al, personal communication), indicating advanced HIV disease in patients with TB pleuritis from this area. Thus, our data seems to indicate that both N-PCR and real-time PCR are useful diagnostic tools for patients with HIV coinfection, a common clinical situation in TB endemic areas. The real-time PCR seemed to perform even better in the HIV group, but a firm conclusion cannot be made owing to the low number of cases.The interassay agreement between N-PCR and the real-time PCR assay was found to be 78%. The lack of absolute agreement is most probably attributable to the different genetic targets used for the 2 PCR methods. The N-PCR in this study employs the repetitive genetic insert IS6110 as a target. This target has the advantage of being specific for the M. tuberculosis complex, but the copy number of this genetic insert can range from 0 to 30 copies per genome, depending on the specific organism.13–17 Although it is technically possible to use the IS6110 as a target in a real-time PCR, any meaningful quantification of bacterial load for clinical purposes would be practically impossible as the exact number of IS6110 per genome will remain unknown. In contrast to IS6110, the hsp65 gene exists as a single copy. We therefore preferred this target for the quantitative real-time PCR assay to allow quantification of bacterial load. Consequently, the N-PCR assay might have a significantly greater number of targets available and this might result in a higher detection rate of mycobacterial DNA, particularly in paucibacillary specimens. We found 3 TB cases to be positive with N-PCR, whereas they were negative by the real-time PCR. Marchetti et al18 and Azov et al19 found a similar interassay disagreement between N-PCR using IS6110 and N-PCR using the hsp65 gene as targets. On the other hand, M. tuberculosis strains lacking the IS6110 element have also been described.13–16 Infection with such a strain will not be detectable with the N-PCR, whereas it might be detected by the real-time PCR. This can explain our finding of 4 TB cases that were negative with N-PCR, whereas they were positive by the real-time PCR.In addition to detecting M. tuberculosis complex, the real-time PCR assay might also detect NTMs. Hence, another potential explanation for the interassay disagreement can be the detection of NTMs by the real-time PCR in some of the patients with TB pleuritis. However, because pleuritis is not a common clinical manifestation for NTM infections, we believe that this is less likely. Also, all patients defined as being positive for TB responded adequately to conventional TB treatment. This outcome would have been less probable if NTMs had been the dominating pathogen.Other potential reasons for the observed interassay disagreement include DNA damage during sample processing, which could influence the number of available initial undamaged targets per genome, and differences in amplification efficiency between the 2 assays.Both the N-PCR and the real-time PCR assays were positive in biopsies from a few of the controls who did not have clinical TB pleuritis. Although contamination is a possibility, we believe this is less likely because all our negative controls worked adequately and all PCR work was done according to recommended guidelines, with unidirectional workflow and separate physical sites for reagent preparation, amplification, and postamplification procedures. However, it should be noted that all the biopsies were from patients living in a high TB endemic area. Hence, it is possible that these “false-positive cases” in patients with malignant diseases actually represent latent or subclinical TB infections that had not yet been clinically diagnosed.In this study, only 2 of the TB cases were ZN positive and only 3 had a typical histologic picture of central caseous granuloma, whereas a diffuse mixed inflammatory reaction with no granulomas was seen in about half of the TB cases. Our observations are in agreement with those of earlier studies, in which inflammation without granulomas was found in pleural and other tissue biopsies from the majority of HIV patients.2,20 Interestingly, the 2 HIV-TB coinfected cases with the relatively high CD4 cell count did not have granulomas, whereas 2 of the 3 patients with low CD4 cell count demonstrated granuloma formation. Although no firm statement can be made owing to the low number of cases, this finding indicates that immune dysfunction might have already taken place at an early stage of the HIV disease. Further, the number of CD4 cells in peripheral blood does not necessary correlate with immune responses in other compartments, as described earlier.21,22 However, the atypical histologic features of TB found in many HIV coinfected patients emphasizes the importance of developing better diagnostic tools for this group of patients.CONCLUSIONSIn conclusion, our real-time PCR assay offers an acceptable sensitivity and specificity for the diagnosis of TB pleuritis. However, it should be kept in mind that our real-time assay might amplify NTMs. Further, a negative result does not exclude the possibility of pleural involvement by M. tuberculosis. The technique offers a comparable diagnostic accuracy with that of the N-PCR assay, but has the additional advantage of a short turn-around time, low risk of sample contamination, and offers the possibility to quantify bacterial load, making it a powerful tool for the rapid diagnosis of TB pleuritis. Thus, the test could be a useful technique for the diagnosis of TB in pleuritis patients including those with HIV coinfection, but it needs to be fully evaluated in a larger cohort of patients.ACKNOWLEDGMENTSThe authors thank Prof Muthuphei, Dr Kekana, and Mr Moroka of Department of Anatomical Pathology, University of Limpopo (Medunsa Campus), Pretoria South Africa for assisting in retrieving biopsies from the archives. Special thanks to Silke Appel, Broegelmann Research Laboratory, The Gade Institute, University of Bergen, Norway for valuable advice in developing the real-time PCR assay.REFERENCES1. World Health Organization. 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AreaBaba Kamaldeen MD; Pathak, Sharad MD; Sviland, Lisbeth PhD; Langeland, Nina PhD; Hoosen, Anwar A. PhD; Asjo, Birgitta PhD; Dyrhol-Riise, Anne M. PhD; Mustafa, Tehmina PhDOriginal ArticlesOriginal Articles217p 112-117