1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

The Journal of Molecular Diagnostics, Vol. 17, No. 5, September 2015

jmd.amjpathol.org

Clinical Usefulness of PCR for Differential Diagnosis of Tuberculosis and Nontuberculous Mycobacterial Infection in Paraffin-Embedded Lung Tissues Q17

Yo Na Kim,* Kyoung Min Kim,* Ha Na Choi,* Ju Hyung Lee,y Ho Sung Park,* Kyu Yun Jang,* Woo Sung Moon,* Myoung Jae Kang,* Dong Geun Lee,* and Myoung Ja Chung* From the Departments of Pathology* and Preventive Medicine,y Chonbuk National University, Medical School, Research Institute of Clinical Medicine and Research Institute for Endocrine Sciences, Jeonju, Chonbuk, Republic of Korea Accepted for publication April 2, 2015. Address correspondence to Myoung Ja Chung, M.D., Ph.D., Department of Pathology, Chonbuk National University Medical School, San 2-20 Keumam-Dong, Dukjingu, Jeonju 561-180, Republic of Korea. E-mail: mjchung@jbnu. ac.kr.

Q3

The need for isolation of nontuberculous mycobacteria (NTM) from clinical specimens has increased in recent years. Our aim was to determine the clinical usefulness of PCR for differential diagnosis of tuberculosis and nontuberculous mycobacterial infection in lung tissue that show chronic granulomatous inflammation. A total of 199 formalin-fixed, paraffin-embedded specimens, including 137 Mycobacterium tuberculosis (MTB), 17 NTM cases, and 45 other than mycobacterial cases were collected. We performed acid-fast staining, MTB and NTM nested PCRs, and MTB and NTM real-time PCRs. No histologic difference between MTB and NTM infections was observed. Sensitivity and specificity for detecting MTB were 70.1% and 95.1% by nested PCR, respectively, and 70.8% and 100.0% by real-time PCR, respectively. Sensitivity and specificity for detecting NTM were 52.9% and 96.15% by nested PCR, respectively, and 35.3% and 100.0% by real-time PCR, respectively. Mycobacteria were identified by acid-fast staining in 50 of 154 cases (32.5%). All 50 acid-fast stainingpositive cases showed positive nested and real-time PCR results (n Z 47 MTB PCR positive; n Z 3 NTM PCR positive), and results agreed with final diagnosis. PCR will be useful for the rapid diagnosis of mycobacterial infection and differentiation of MTB from NTM in formalin-fixed, paraffin-embedded specimens, especially in acid-fast staining-positive specimens. (J Mol Diagn 2015, 17: 1e8; http:// dx.doi.org/10.1016/j.jmoldx.2015.04.005)

More than 140 species are found in the genus Mycobacterium,1 which are divided into three groups: M. tuberculosis complex (MTB), M. leprae, and nontuberculous mycobacteria (NTM). NTM is mycobacteria other than M. tuberculosis complex and M. leprae. The pathogenicity, virulence, and response to drugs of NTM are different from tuberculosis.2 Pulmonary disease is the most common clinical manifestation of NTM infection. In the endemic area of tuberculosis, pulmonary lesion that showed chronic granulomatous inflammation and caseation necrosis was regarded as pulmonary tuberculosis. The virulence of most NTM is low, and isolation of NTM from clinical specimens is considered contamination.3 However, they are proven human pathogens, and, although some NTM are as virulent as MTB, most NTM infections are resistant to antituberculosis

therapy.1,2,4 Attention to NTM infection is growing due to increasing frequency and improvement in clinical detection techniques. The recovery rates of NTM from acid-fast bacillus (AFB) smear-positive clinical specimens vary widely between studied populations, from 7.3% to 12.2% in the Republic of Korea to 21.1% in Spain and 24.8% to 48.5% in the United States.5 Recovery rate of NTM from patients with the AFB smearpositive sputum is approximately 10% in the Republic of Korea.5 Therefore, there is an increasing need to identify the Supported by the National Research Foundation of Korea (NRF) grant 2008-0062279, funded by the Korean Government (MSIP). Chonbuk National University Hospital Biobank was supported by the Ministry of Health and Welfare. Disclosures: None declared.

Copyright ª 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2015.04.005

FLA 5.2.0 DTD  JMDI420_proof  22 July 2015  9:09 pm  EO: JMD14_0161

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 Q1 Q2 122 123 124

125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186

Kim et al causative organism (MTB or NTM) when lung specimens show chronic granulomatous inflammation with caseation necrosis, because the correct diagnosis is vital for proper management. AFB culture is the gold standard for diagnosis of mycobacterial infection,2 whereas PCR is extensively evaluated as an alternative rapid diagnostic tool. Real-time PCR is a widely used molecular biology technique that is based on PCR amplification and simultaneous quantification of the targeted DNA molecule. Compared with the conventional nested PCR (N-PCR) method, real-time PCR does not require additional processing, such as agarose gel electrophoresis, which reduces the risk of contamination. It has the additional advantage of short turnaround time and increased specificity, thus making it a valuable diagnostic tool for the rapid diagnosis of mycobacterial infection.6 Studies have shown that the performance of MTB PCR on formalin-fixed, paraffin-embedded (FFPE) human tissues show relatively high sensitivity and specificity.6e8 However, studies on NTM PCR with FFPE specimens are limited,7 and its usefulness is not established. This study was conducted to evaluate the clinical usefulness of PCR on FFPE lung tissues that show chronic granulomatous inflammation for differential diagnosis of tuberculosis and nontuberculous mycobacterial infection and to compare the ability of N-PCR and real-time PCR to detect mycobacterial infection in FFPE specimens. Diagnostic yield of PCR was compared with acid-fast staining, AFB cultures, and histopathologic findings.

Materials and Methods

specimens but histopathologic findings suggestive of MTB (granuloma with caseation necrosis, granuloma, or caseation necrosis alone) and good clinical response to MTB medications.9 All NTM cases satisfied the 2007 guidelines from the American Thoracic Society/Infectious Diseases Society of America.2 Briefly, for the diagnosis of NTM, patients were required to satisfy both the clinical and microbiological requirements. Clinical requirements included patients with pulmonary symptoms and lung lesion on chest radiograph or a high-resolution computed tomography scan, including fibro- Q4 cavitary lesion or bronchiectasis with multiple nodules. Microbiological requirements included patients with positive culture for NTM (from at least two separate sputum samples or at least one bronchial wash) or with positive NTM culture on biopsy with chronic granulomatous inflammation. The final diagnoses of enrolled cases were classified as 137 MTB cases, 17 NTM cases, and 45 other than mycobacterial (OTM) infection. Of the 137 MTB cases, 91 were diagnosed as definite MTB and 46 as probable MTB. All 154 mycobacterial cases were negative for periodic acidSchiff and Grocott methenamine silver staining. This study Q5 enrolled 45 OTM cases as negative control, on which lung biopsy and AFB culture with clinical impression of mycobacterial infection were performed in the same period. Final diagnosis of the 45 OTM cases were 32 pneumonia, 5 lung cancer, 5 fungal infection, 2 sarcoidosis, and 1 parasite infection. The AFB cultures of all OTM cases were negative. The study was approved by the Institutional Review Board for Human Subjects Research of Chonbuk National University Hospital.

Specimens

Acid-Fast Staining and AFB Cultures

Specimens were collected from 269 patients admitted to the Chonbuk National University Hospital between January 2010 and April 2012 for suspected mycobacterial infection and who had a diagnostic lung biopsy. When tissue showed chronic granulomatous inflammation, AFB stain and MTB/ NTM N-PCR were performed with the tissue as a routine test for identification of the organism. AFB cultures were performed with bronchial washings that were taken within a few days (0 to 3 days) before the tissue collection date. The results of AFB stain, N-PCR, and AFB culture were obtained by reviewing medical records. In addition, we performed real-time PCR examination. For real-time PCR, DNA was extracted from the archived FFPE blocks that were previously used for N-PCR. The presence of granulomatous inflammation in the recut sections was confirmed by hematoxylin and eosin stain. The hematoxylin and eosinstained slides were reviewed by two pathologists (M.J.C. and Y.N.K.). Of these, FFPE tissue blocks from 70 cases were insufficient for further real-time PCR, and 199 cases were included in the present study. Diagnostic criteria for MTB were definite MTB, positive mycobacterial culture from respiratory specimens, and probable MTB, negative mycobacterial cultures from respiratory

Ziehl-Neelsen staining was performed on FFPE specimens to detect AFB according to standard protocol,10 and all acidfastestained slides were reviewed by two pathologists (M.J.C. and Y.N.K.). AFB cultures were established with 2% Ogawa media (Korean Institute of Tuberculosis, Cheongwon, Republic of Korea) for 8 weeks and BACTEC MGIT 960 cultures (Becton Dickinson, Sparks, MD) for 6 weeks. Isolated mycobacterial colonies from both solid culture media and liquid culture media were examined with the TB Ag MPT64 Rapid test (SD Bioline; Standard Diagnostics, Suwon, Republic of Korea) for detection of MTB.11 Commercial DNA probe, REBA Myco-ID (YD Diagnostics, Yongin, Republic of Korea) was used to detect and differentiate MTB and NTM species.12

2

jmd.amjpathol.org

DNA Extraction from FFPE Specimens Five to ten 5-mm sections were cut from FFPE tissue blocks. The knife was replaced after the tissue of each case was sectioned to prevent contamination. Tissue sections were deparaffinized with 100% xylene twice, each for 5 minutes. The samples were washed with 100% ethanol twice, each for 5 minutes, to remove the residual xylene. After

-

The Journal of Molecular Diagnostics

FLA 5.2.0 DTD  JMDI420_proof  22 July 2015  9:09 pm  EO: JMD14_0161

Q6

187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248

249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310

PCR for Diagnosis of TB and NTM in FFPE deparaffinization, DNA extraction was performed with a QIAamp DNA Mini Kit (catalog no. 51306; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The adequacy of the extracted DNA was evaluated by PCR analysis of the b-globin gene.

Conventional N-PCR for MTB and NTM N-PCR for MTB and NTM was performed with the Absolute NTM & MTB PCR kits (BioSewoom Inc., Seoul, Republic of Korea), according to the manufacturer’s instructions. It is a multiplex PCR assay. For the detection of the IS6110 gene in the MTB genome the specific primers provided were able to amplify a 181-bp product. Specific primers were able to amplify a 242-bp product for the detection of the 16s rRNA gene in the mycobacteria genome. Simultaneous amplification of 181- and 242-bp PCR products in a clinical sample or amplification of the 181 bp product alone, visualized on electrophoresis, were both interpreted as MTB positive. Visualization of a 242 bp on electrophoresis was determined as NTM positive. Briefly, a 4-mL volume of extracted DNA was used for the first PCR amplification (35 cycles of denaturing at 94 C for 30 seconds, annealing at 68 C for 30 seconds, and extension at 72 C for 30 seconds), and a 1.5-mL aliquot was used for the second PCR amplification (25 cycles of denaturing at 94 C for 30 seconds, annealing at 68 C for 30 seconds, and extension at 72 C for 30 seconds). The amplification was performed with an automated PCR Thermocycler (Mastercycler; Eppendorf, Hamburg, Germany). PCR products were electrophoresed and visualized on a 2% agarose gel. Adequacy of the extracted DNA was evaluated by PCR for the b-globin gene.

Real-Time PCR for MTB and NTM Real-time PCR for MTB and NTM was performed with the Real-Q MTB & NTM Kit (BioSewoom Inc.), according to the manufacturer’s instructions.13 The Real-Q MTB & NTM Kit (BioSewoom Inc.) was used to perform real-time PCR reactions. The Real-Q MTB & NTM Kit is a ready-to-use kit for the detection of the MTB and mycobacteria in a multiplex real-time PCR assay, based on the TaqMan probe system. The kit supplies three assays. The MTB assay is labeled with VIC; it contains a specific forward primer and probe for the detection of the IS6110 gene in the MTB genome. The mycobacteria assay is labeled with FAM. It contains a specific forward primer and probe located on 16s rRNA. The reverse primer is located on the ITS region between 16s and 23s rRNA in the mycobacteria genome. The mycobacteria assay can amplify all Mycobacterium species, but the MTB assay can detect MTB alone. The internal control cyanine 5-labeled assay is used to check for possible PCR inhibition. In clinical sample assays, the positive reaction was determined as an indication for the use of the Real-Q MTB

The Journal of Molecular Diagnostics

-

& NTM Kit. Positive VIC (MTB assay) and cyanine 5 signals (internal control assay) and positive FAM (mycobacteria assay), VIC (MTB assay), and cyanine 5 signals (internal control assay) simultaneously were interpreted as MTB positive. Positive FAM signal (mycobacteria assay) and cyanine 5 signal (internal control assay) were interpreted as NTM positive. Briefly, a final volume of 25 mL of real-time PCR mixture contained 2.5 mL of extracted DNA from FFPE tissue, 900 nmol/L of each primer, 250 nmol/L of the probe, and 10 mL of 2 TaqMan Universal PCR MasterMix. Real-time PCR was performed with a RotorGene Q (Qiagen) 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 40 cycles of 95 C for 15 seconds, and 67 C for 45 seconds. Each run included no-templateenegative controls that contained 2.5 mL of sterile water instead of DNA, and two positive standards. Threshold cycle (CT) values

Clinical Usefulness of PCR for Differential Diagnosis of Tuberculosis and Nontuberculous Mycobacterial Infection in Paraffin-Embedded Lung Tissues.

The need for isolation of nontuberculous mycobacteria (NTM) from clinical specimens has increased in recent years. Our aim was to determine the clinic...
594KB Sizes 1 Downloads 10 Views