Performance Characteristics of Nested Polymerase Chain Reaction vs ...

AJCP / Original Article

Performance Characteristics of Nested Polymerase Chain Reaction vs Real-Time Polymerase Chain Reaction Methods for Detecting Mycobacterium tuberculosis Complex in Paraffin-Embedded Human Tissues

CME/SAM

An Na Seo, MD, PhD,1,2 Hyo Jin Park, MD, PhD,1 Hye Seung Lee, MD, PhD,1 Jung Ok Park, MS,1 Ho Eun Chang, MS,3 Kyung Han Nam, MD,1 Gheeyoung Choe, MD, PhD,1,4 and Kyoung Un Park, MD, PhD3,5 From the 1Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Gyeonggido, Republic of Korea; 2Department of Pathology, Kyungpook National University School of Medicine, Jung-gu, Daegu, Republic of Korea; 3Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam, Gyeonggido, Republic of Korea; and Departments of 4Pathology and 5Laboratory Medicine, Seoul National University College of Medicine, Jongnogu, Seoul, Republic of Korea. Key Words: Mycobacterium tuberculosis; Nested PCR; Real-time PCR; Formalin-fixed, paraffin-embedded tissues Am J Clin Pathol September 2014;142:384-390 DOI: 10.1309/AJCP2QZRH4ZNPRDD

ABSTRACT Objectives: Nucleic acid amplification tests on formalinfixed, paraffin-embedded (FFPE) tissue specimens enable Mycobacterium tuberculosis complex (MTB) detection and rapid tuberculosis diagnosis in the absence of microbiologic culture tests. We aimed to evaluate the efficacy of different polymerase chain reaction (PCR) methods for detecting Mycobacterium species in FFPE tissues. Methods: We examined 110 FFPE specimens (56 nonmycobacterial cases, 32 MTB, and 22 nontuberculous mycobacteria [NTM] determined by acid-fast bacilli [AFB] culture) to assess five PCR methods: nested PCR (N-PCR) (Seeplex MTB Nested ACE Detection; Seegene, Seoul, South Korea), an in-house real-time PCR (RT-PCR) method, and three commercial RT-PCR methods (AccuPower MTB RT-PCR [Bioneer, Seoul, Korea], artus M tuberculosis TM PCR [Qiagen, Hilden, Germany], and AdvanSure tuberculosis/ NTM RT-PCR [LG Life Sciences, Seoul, Korea]). Results: The results of N-PCR, in-house RT-PCR, and AdvanSure RT-PCR correlated well with AFB culture results (concordance rates, 94.3%, 87.5%, and 89.5%, respectively). The sensitivity of N-PCR (87.5%) was higher than that of the RT-PCR methods, although these differences were not statistically significant between N-PCR and the in-house and AdvanSure RT-PCR methods (68.8% and 80.0%, respectively). All the PCR methods had high specificities, ranging from 98.2% to 100%. Only two NTM cases were detected by AdvanSure RT-PCR, implying a very low sensitivity. Conclusions: Well-designed RT-PCR and N-PCR can effectively identify MTB in FFPE specimens. 384 384

Am J Clin Pathol 2014;142:384-390 DOI: 10.1309/AJCP2QZRH4ZNPRDD

Upon completion of this activity you will be able to: • list the types of samples that can be tested for Mycobacterium tuberculosis by polymerase chain reaction (PCR) and discuss advantages and disadvantages of PCR over special stains and microbial culture. • compare limitations and advantages of nested PCR vs real-time PCR for detection of M tuberculosis. • outline a testing algorithm for nontuberculous mycobacteria, including specimen procurement and test strategy. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 422. Exam is located at www.ascp.org/ajcpcme.

Despite intense efforts to control tuberculosis (TB), it remains an important global health problem. Tuberculosis is the second leading cause of death due to an infectious disease worldwide, with emergence of the human immunodeficiency virus (HIV).1,2 Thus, the accurate and rapid diagnosis of Mycobacterium tuberculosis complex (MTB) is critical for disease control and patient management.3 Traditionally, MTB is detected by a sputum smear and microbiologic culture tests, considered the gold standard for identifying MTB.3,4 However, the disease can also present as extrapulmonary mass lesions, which are frequently biopsied without clinical suspicion for TB. In these cases, fresh tissues for culture are often unavailable; thus, histologic confirmation based on formalin-fixed, paraffin-embedded (FFPE) specimens is the only method for diagnosing TB.5-7 Histologically, biopsy specimens typically demonstrate granulomas with or without © American Society for Clinical Pathology

AJCP / Original Article

caseous necrosis; however, atypical patterns such as abscess formation or active inflammation without granulomas may be observed, especially in HIV-infected patients.8 Moreover, granulomas are found in other diseases such as fungal infection and sarcoidosis.7,9 Furthermore, acid-fast bacilli (AFB) staining has low sensitivity and specificity, although it is easy to perform on FFPE tissues.7,10 Therefore, more sensitive and specific tools that can be used in daily practice need to be developed for the diagnosis of TB using FFPE tissue. With the developments in molecular biology, nucleic acid amplification methods offer better sensitivity and specificity in the diagnosis of TB than do traditional diagnostic methods.4,11 Single-step polymerase chain reaction (PCR), nested PCR (N-PCR), and real-time PCR (RT-PCR) are the available molecular techniques for the detection of MTB. Among these, RT-PCR has been known to offer increased sensitivity and specificity compared with conventional single-step PCR or N-PCR.12,13 However, most studies demonstrate the sensitivity and specificity of RT-PCR using fresh tissues.14,15 The performance of RT-PCR is affected by the specimen type, sample storage time, DNA extraction procedure, primer concentration, and probe sequence.16-20 However, only a few studies have evaluated the usefulness of RT-PCR for the detection of MTB using FFPE specimens compared with culture results as a reference.7,21 In this study, we aimed to compare the diagnostic performances of N-PCR and four RT-PCR methods, including three commercial methods for detecting MTB. The sensitivities and specificities were analyzed using mycobacterial culture results as a reference.

Materials and Methods Sample Collection Samples from patients who were consecutively admitted to the Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Republic of Korea, between June 2009 and May 2011 were collected and analyzed by MTB N-PCR. We reviewed the corresponding medical records, pathologic reports, and histologic findings and selected cases with mycobacterial culture results from corresponding fresh tissue and sufficient FFPE tissue for PCR. Two pathologists (H.J.P. and A.N.S.) independently reviewed H&E-stained slides. Excluding cases with aspirated cytology or small biopsy specimens, which would have insufficient DNA to perform PCR, a total of 110 cases suitable for the study were selected. Biopsy specimens of 32 MTB cases, 22 nontuberculous mycobacteria (NTM) cases, and 56 nonmycobacterial cases were included. The lesions were present in various organs, including the lung, lymph node, © American Society for Clinical Pathology

pericardium, synovium, bone, and soft tissue. The study was conducted in accordance with the principles outlined in the World Medical Association’s Declaration of Helsinki. The study protocol was reviewed and approved by the institutional review board of the Seoul National University Bundang Hospital (IRB no. B-1212/183-302). AFB Culture As a reference method, mycobacterial culture tests were performed. Fresh tissues of 110 cases obtained from the same lesions or organs of the same patients with FFPE tissues were used for culture tests. Fresh tissue was cultured in 3% Ogawa media (Shin-yang Chemical, Seoul, Korea) for 8 weeks as described previously by Lee et al.7 Smears of the isolated mycobacterial colonies were identified using auraminerhodamine fluorescence and the Ziehl-Neelsen method. MTB and NTM isolated from culture were differentiated and identified by the AccuProbe test (Gen-Probe, San Diego, CA), according to the manufacturer’s protocols. DNA Extraction To extract DNA from FFPE specimens, we cut each paraffin block into 8-mm sections. Blades were cleaned or replaced after sectioning each paraffin block to prevent carryover contamination. Each FFPE tissue section was incubated in 1 mL of phosphate-buffered saline at 70°C for 5 minutes. This was repeated until deparaffinization was complete. DNA was extracted using the following methods: a Chelex 100 Resin boiling method (Bio-Rad Laboratories, Hercules, CA) for N-PCR, the in-house RT-PCR method, the artus M tuberculosis TM PCR method (Qiagen, Hilden, Germany), and the AdvanSure TB/NTM RT-PCR method (LG Life Sciences, Seoul, Korea), as well as an extraction method using the ExiPrep extraction kit for the AccuPower MTB RT-PCR method (Bioneer, Seoul, Korea). Briefly, for the Chelex boiling extraction method, each section was boiled in 100 mL of a 5% Chelex solution at 100°C for 20 minutes. For DNA extraction using the ExiPrep extraction kit, each section was incubated with 200 mL of tissue lysis buffer containing 10 mL of proteinase K (stock concentration, 40 mg/mL) at 60°C and then centrifuged at 13,000 rpm for 2 minutes. The supernatant was transferred to a sample loading well of buffer cartridge. MTB N-PCR In 110 FFPE tissue samples, IS6110 (GenBank accession no. AJ242908) and MPB64 (GenBank accession no. AL123456.3) were amplified using the N-PCR kit (Seeplex MTB Nested ACE Detection; Seegene, Seoul, South Korea) according to the manufacturer’s standard instructions. Firstrun PCRs were performed with a mixture comprising 3 mL of extracted DNA, 10 mL of 2× Multiple Master Mix, 4 mL

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Am J Clin Pathol 2014;142:384-390 DOI: 10.1309/AJCP2QZRH4ZNPRDD

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Seo et al / Detection of MTB in Formalin-Fixed, Paraffin-Embedded Human Tissues

of 5× MTB nested autocapillary electrophoresis (ACE) first primer mixture (PM), and 3 mL of 8-methoxypsoralen (MOP) solution (Seegene). N-PCR was performed with a mixture comprising 10 mL of 2× Multiple Master Mix, 4 mL of 5× MTB nested ACE first PM, and 5 mL of 8-MOP solution. The PCR cycling condition was as follows: one cycle of 94°C for 15 minutes; 39 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds; and one cycle of 72°C for 10 minutes. The PCR products (204 base pairs for MTB) were analyzed by 2% agarose gel electrophoresis. RT-PCR for MTB Detection For RT-PCR amplification, the in-house RT-PCR protocol and three commercial RT-PCR kits (the AccuPower MTB & NTM [Dual] RT-PCR kit [Bioneer], the artus M tuberculosis TM PCR kit [Qiagen], and the AdvanSure TB/NTM RT-PCR kit [LG Life Sciences]) were used. The primer sequences and detailed process of the in-house RT-PCR protocol have been published previously.7 The Exicycler 96 detection system (Bioneer) was used for the AccuPower MTB & NTM (Dual) RT-PCR kit, Rotor-Gene Q (Corbett Research, Sydney, Australia) was used for the artus M tuberculosis TM PCR kit, and the SLAN RT-PCR detection system (LG Life Sciences) was used for the AdvanSure TB/NTM RT-PCR kit. The amount of DNA used varied, as per the instructions for the kit/assay.

Statistical Analysis All statistical analyses were performed using SPSS software (version 18.0; SPSS, Chicago, IL). The concordance between N-PCR or the RT-PCR methods and mycobacterial culture results was analyzed using the k coefficient. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using cross-tabulation. The results of the MTB assessment using mycobacterial culture tests were used as the reference in this study. The one-way analysis of variance was performed to assess correlations among the PCR methods. All tests were two-sided, and statistical significance was considered when the P value was