Identification of Mycobacterium tuberculosis and Rifampin Resistance in Clinical Specimens Using the Xpert MTB/RIF Assay

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1 32 Available online at Identification of Mycobacterium tuberculosis and Rifampin Resistance in Clinical Specimens Using the Xpert MTB/RIF Assay Cheol-Hong Kim 1,2, In Gyu Hyun 1,2, Yong IL Hwang 1,2, Dong-Gyu Kim 1,2, Chang Youl Lee 1,2, Myung Goo Lee 1,2, Ki-Suck Jung 1,2, Heungjeong Woo 1, Jeongwon Hyun 3, Hyun Soo Kim 3, and Myung Jae Park 4 Departments of 1 Internal Medicine, 2 Lung Research Institute, and 3 Laboratory Medicine, Hallym University College of Medicine, Chuncheon, and 4 Department of Internal Medicine, Kyung Hee University School of Medicine, Seoul, Korea Abstract. Purpose. The Xpert MTB/RIF assay is a novel real-time polymerase chain reaction technique for the detection of the Mycobacterium tuberculosis (MTB) complex and rifampin (RIF) resistance. We evaluated the performance of this assay in identifying MTB and resistance to RIF in clinical specimens. Materials and Methods. We analyzed clinical specimens from 383 patients with suspected TB who were hospitalized at a secondary hospital in Korea. Specimens were processed using the Xpert MTB/RIF assay, acid-fast bacilli smear and culture, and drug susceptibility test (DST). Results. Among the 444 clinical samples analyzed, the Xpert MTB/RIF assay identified MTB in 56 (13.8%) of 405 respiratory specimens, but did not detect MTB in the remaining 39 non-respiratory specimens. Of the 65 pulmonary TB patients, 52 (80.0%) were confirmed by using mycobacterial culture as a reference standard. The sensitivity, specificity, PPV, and NPV of the Xpert MTB/RIF assay were 73.85%, 99.03%, 94.12%, and 94.72%, respectively. Among five patients with RIF resistance determined by the Xpert MTB/RIF assay, four (80%) were confirmed as suffering from multidrug-resistant (MDR) TB by DST. Conclusions. The Xpert MTB/RIF assay appears to be an accurate, simple, and useful technique for detecting MTB, especially in respiratory specimens. However, RIF resistance, if detected, should be verified with DST. Key words: pulmonary tuberculosis, real-time polymerase chain reaction, rifampin. Introduction Early detection of active tuberculosis (TB) and multi-drug-resistant (MDR) TB strains allows initiation of appropriate treatment and is essential for effective healthcare; diagnostic delay can lead to secondary resistance, increased mortality, and continued person-to-person transmission [1-3]. Acid-fast bacilli (AFB) smears have low sensitivity and negative predictive value (NPV), and AFB culture, which is the gold standard for TB diagnosis, is difficult and usually requires 2 8 weeks before the final results can be obtained [2,4]. In Korea, conventional drug susceptibility testing (DST) can take 7 13 weeks [5]. Address correspondence to Hyun Soo Kim, M.D., Department of Laboratory Medicine, Hallym University Dongtan Sacred Heart Hospital; 7 Keunjaebong-gil, Hwaseong-si, Gyeonggi-do, , Korea; Tel: ; Fax: ; hskim0901@ empal.com, or In-Gyu Hyun, M.D.; Department of Internal Medicine, Hallym University Dongtan Sacred Heart Hospital; 7 Keunjaebong-gil, Hwaseong-si, Gyeonggi-do, , Korea; Tel: ; Fax: ; ighyun@hallym.or.kr The Xpert MTB/RIF assay, which is based on realtime polymerase chain reaction (PCR) analysis of the TB-specific rpob gene, is an automated molecular diagnostic test for simultaneous detection of mycobacterium tuberculosis (MTB) and rifampin (RIF) resistance directly from clinical specimens [6]. The assay has shown high sensitivity and specificity [7-9]. The Xpert MTB/RIF assay was recently introduced in our new hospital, a secondary care hospital in Korea, for the simple and rapid diagnosis of TB and RIF resistance. The aim of this study was to evaluate the performance of the Xpert MTB/RIF assay in the detection of MTB and RIF resistance in clinical specimens during routine clinical practice. Korea has an annual TB incidence of 108 per 100,000, and 1,212 cases of MDR-TB were laboratory-confirmed in 2012 [10] /15/ by the Association of Clinical Scientists, Inc.

2 Xpert MTB/RIF assay on clinical specimens 33 Table 1. Characteristics of the 383 patients in whom the Xpert MTB/RIF assay was performed. Characteristics Age, years 56.31±17.88 Male 217(56.7) Number of clinical specimens 444 Respiratory 405(91.2) Non Respiratory 39(8.8) Co-morbidities Hypertension 85(22.2) Diabetes mellitus 53(13.8) Bronchiectasis 43(11.2) Malignancy 26(6.8) Cardiovascular disease 23(6.0) Stroke 15(3.9) Bronchial asthma 12(3.1) Dementia 13(3.4) Chronic liver disease 5(1.3) Chronic kidney disease 7(1.8) Taking immunosuppressive drugs 3(0.8) HIV/AIDS 1(0.3) Final diagnosis Pulmonary TB 65(17.0) MTB culture positive 52(13.6) MTB culture negative 13(3.4) Extrapulmonary TB 10(2.6) Bacterial pneumonia 139(36.3) Lung cancer 38(9.9) Hemoptysis 24(6.3) Influenza 14(3.7) Nontuberculous mycobacterial lung disease 12(3.1) Transudative pleural effusion 12(3.1) Lung abscess 10(2.6) Atelectasis 10(2.6) Empyema 9(2.3) Inactive TB sequelae 6(1.6) Malignant pleural effusion 5(1.3) Fungus ball 5(1.3) Pulmonary edema 4(1.0) Others* 20(5.2) Data are presented as mean±sd or n (%). *,Interstitial lung disease, 3; anthracofibrosis, 3; eosinophilic pneumonia, 2; bronchiectasis, 2; lung metastasis, 2; non specific lymphadenitis, 2; chronic respiratory failure, 2; sarcoidosis, 1; pyogenic pericarditis, 1; norcardiosis, 1; myofibrosis, 1; Abbreviations: MTB, Mycobacterium tuberculosis. Materials and Methods Study design. Between February and December 2013, we retrospectively reviewed the medical records of patients admitted to Hallym University Dongtan Sacred Heart Hospital for suspected TB. This institution attends to a population of approximately 500,000 inhabitants in southwestern Gyeonggi-do, Korea. The local institutional review board approved the study, with a waiver for obtaining consent from individual patients. Clinical samples. Respiratory and non-respiratory specimens were obtained from the patients as part of standard diagnostic methods for the diagnosis of TB. The respiratory specimens consisted of sputum, bronchial washes, and bronchoalveolar lavage fluid. Nonrespiratory specimens from normally sterile sites included pleural fluid, pericardial fluid, and tissue and lymph node samples. In total, 444 clinical samples were analyzed. Acid-fast bacillus smear and mycobacterial culture. Clinical specimens were pretreated with N-acetyl-Lcysteine-2% NaOH and centrifuged (3,000xg for 20 min at 4 C) for decontamination. AFB smears were performed by using auramine-rhodamine fluorescent staining and were confirmed by Ziehl-Neelsen staining. Decontaminated specimens were cultured on 3% Ogawa solid media (Asan Pharmaceutical, Seoul, Korea) and the BD BACTEC MGIT 960 system (Becton Dickinson, Sparks, MD, USA), an automated liquid culture system. MTB was identified using the BD MGIT TB identification test (Becton Dickinson), based on rapid immunochromatography. Drug susceptibility testing (DST). All positive cultures were tested for drug susceptibility to isoniazid (INH) and RIF using the absolute concentration method, which was defined as 1% bacterial growth in Löwenstein-Jensen medium at concentrations of 0.2 µg/ ml for INH and 40.0 µg/ml for RIF [11]. Xpert MTB/RIF assay. The Xpert MTB/RIF assay (Cepheid Inc., Sunnyvale, CA, USA) was performed according to the manufacturer s instructions. The primers in the Xpert MTB/RIF assay amplify a portion of the rpob gene containing the 81 base-pair core region. The probes are able to differentiate between the conserved wild-type sequence and mutations in the core region that are associated with RIF resistance [12,13]. The testing is conducted on GeneXpert, the test device platform,

3 34 which simplifies molecular testing by fully integrating and automating the three processes (sample preparation, amplification, and detection) required for real-time PCR-based molecular testing. Briefly, a decontaminated 0.5-mL sample was mixed with 1.5 ml of reagent that contained sodium hydroxide and isopropanol, and shaken vigorously times. The mixture was incubated for 15 min at room temperature and then shaken again. Next, 2 3 ml of each sample were transferred to a test cartridge and inserted into the GeneXpert, which processed the samples automatically. Statistical analysis. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Xpert MTB/RIF assay and nested PCR were calculated and 95% confidence intervals were estimated. Clinical data are presented as means ±SD and n (%). We compared the accuracy of the Xpert MTB/RIF assay and that of culture-based DST for determining RIF resistance. P<0.05 was considered significant. Statistical analyses were performed using dbstat software version 4.0 (dbstat Inc, Chuncheon, Korea). Results Patient characteristics. The Xpert MTB/RIF assay was performed using respiratory and non-respiratory clinical specimens from 383 patients with suspected TB. The age of these patients was 56.3± years, and 217 (56.7%) patients were male. Hypertension, diabetes mellitus, bronchiectasis, and malignancy were co-morbidities found in 85(22.2%), 53(13.8%), 43(11.2%), and 26(6.8%) of the patients, respectively. MTB culture-positive and MTB culture-negative pulmonary TB were finally diagnosed in 52(13.6%) and 13(3.4%) patients, respectively, based on their clinical symptoms and radiographic findings, even though their AFBsmearsshowedpositiveresults.Extrapulmonary TB was diagnosed in 10 patients (2.6%), TB pleurisy in 6, TB meningitis in 2, TB peritonitis in 1, and intestinal TB in 1 (Table 1). Detection of Mycobacterium tuberculosis (MTB). Among the 444 clinical samples, 405(91.2%) were respiratory specimens (sputum, 176; bronchial washes, 225; and bronchoalveolar lavage fluid, 4) and 39(8.8%) were non-respiratory specimens (pleural fluid, 36; tissue, 1; pericardial fluid, 1; and lymph node, 1). Xpert MTB/RIF assay and AFB culture were performed for all specimens. The MTB/RIF assay detected MTB in 56(12.6%) of the 444 specimens tested, all of which were respiratory specimens (sputum, 25; bronchial washes, 30; Table 2. Results of the Xpert MTB/RIF assay in 444 clinical specimens. Positive results by the Xpert MTB/ RIF assay Respiratory specimens (n=405) Sputum (n=176) 25(14.2) B. washing (n=225) 30(13.3) BAL fluid (n=4) 1(25.0) Non respiratory specimens* (n = 39) 0 *, Pleural fluid, 36; tissue, 1; pericardial fluid, 1; lymph node, 1. Abbreviations: MTB, Mycobacterium tuberculosis; RIF, rifampin; B. washing, bronchial washing; BAL, bronchoalveolar lavage. Table 3. Results of the Xpert MTB/RIF assay in 383 patients. Positive results with the Xpert MTB/RIF assay Pulmonary TB (n=65) 48(94.1) MTB culture positive (n=52) 46(90.2) MTB culture negative (n=13) 2(3.9) Extrapulmonary TB (n=10)* 0 Nontuberculous mycobacterial disease (n=12) 1(2.0) Bacterial pneumonia (n=139) 1(2.0) Lung cancer (n=38) 1(2.0) Others (n=119) 0 Total 51 (100) * TB pleurisy, 6; TB meningitis, 2; TB peritonitis and intestinal TB, 1. Abbreviations: MTB, Mycobacterium tuberculosis; RIF, rifampin. and bronchoalveolar lavage fluid, 1), and it did not detect MTB in any non-respiratory samples (Table 2). The results of the Xpert MTB/RIF assay and AFB culture are shown in Table 3. The Xpert MTB/RIF assay results detected TB infection in 51(13.3%) out of the 383 suspected TB patients, and identified 48(73.9%) out of the 65 patients as suffering from pulmonary TB. None of the cases of extrapulmonary TB were detected by the Xpert assay. Three cases showed false positive results in the Xpert assay; the actual diagnoses were non-tuberculous mycobacterial (NTM) disease, bacterial pneumonia, and lung cancer.

4 Xpert MTB/RIF assay on clinical specimens 35 Table 4. Accuracy of the Xpert MTB/RIF assay and mycobacterial culture in the diagnosis of pulmonary TB. Xpert MTB/RIF assay (n=373) Mycobacterial culture (n=373) P Sensitivity, % (95% CI) 73.85(48/65)(61.46, 83.96) 80.00(52/65)(68.23, 88.89) Specificity, % (95% CI) 99.03(305/308)(97.18, 99.79) 100(308/308)(98.79, 100) PPV, % (95% CI) 94.12(48/51)(83.74, 98.70) 100(52/52)(93.08, 100) NPV, % (95% CI) 94.72(305/322)(91.68, 96.89) 95.95(308/321)(93.11, 97.80) In this analysis, cases of extrapulmonary TB were excluded. Abbreviations: MTB, Mycobacterium tuberculosis; RIF, rifampin; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value. Diagnostic accuracy of the Xpert MTB/RIF assay and mycobacterial culture to detect MTB for diagnosis of pulmonary TB. The overall sensitivity, specificity, PPV, and NPV of the Xpert MTB/RIF assay and AFB culture to detect MTB were 73.85(48/65) (61.46, 83.96) vs. 80.0%(52/65) (68.23, 88.89), 99.03(305/308) (97.18, 99.79) vs. 100%(98.79, 100), 94.12%(48/51) (83.74, 98.70) vs. 100%(52/52) (93.08,100), and 94.72%(305/322) (91.68, 96.89) vs %(308/321) (93.11, 97.80), respectively. No differences were observed between the Xpert MTB/ RIF assay and mycobacterial culture with regard to accuracy (p>0.05). In this analysis, cases of extrapulmonary TB were excluded (Table 4). Resistance to rifampin determined by the Xpert MTB/RIF assay and conventional drug susceptibility test (DST). Among 52 pulmonary TB patients with positive mycobacterial cultures, the results of 36 patients (69.2%) were available from conventional DST. Specimens from three cases were contaminated, and specimens from 13 cases failed to grow on solid culture. Among the five cases with positive Xpert results, four (80%) were identified as MDR-TB by DST (new, 2; previously treated, 2). Although the Xpert assay detected MTB in the clinical specimens, one of the specimens was labeled as indeterminate for RIF resistance (Table 5). Discussion The Xpert MTB/RIF assay is an automated nucleic acid amplification test for clinical specimens that can identify MTB and resistance to RIF with minimal labor. When tested in areas with high TB incidence, the Xpert assay was highly accurate, with a sensitivity of 88% and specificity of 98% [9]. In Korea, the sensitivity and specificity of the Xpert assay for the diagnosis of pulmonary TB were reported to be 79.5% and 100%, respectively [7]. These data, however, were obtained from a tertiary referral hospital that required a consultation form from secondary hospitals or primary clinics. Our data were obtained at a 500-bed secondary hospital that freely treated local inhabitants without requiring a consultation form. The impact of the Xpert MTB/RIF assay has not been adequately studied in the community-based secondary-care setting of our country. It is important to generate additional evidence in settings with intermediate TB incidence because the Xpert assay has been recently approved by the US Federal Drug Administration [14]. In the present study, mycobacterial culture on solid media using clinical specimens was used as a reference standard. AFB smears have a low sensitivity for diagnosis of TB in Korea, as the proportion of NTM isolates from AFB smear-positive specimens significantly increased from 9% in 2001 to 64% in 2011 (p<0.001) [15]. Therefore, AFB smears alone are no longer an acceptable diagnostic method for detecting MTB in clinical specimens in Korea. Among the 383 individuals suspected of having TB, 52 patients were diagnosed with active pulmonary TB, which was confirmed by mycobacterial culture. The sensitivity, specificity, PPV, and NPV of the Xpert MTB/RIF assay for diagnosis of pulmonary TB were 73.85%, 99.03%, 94.12%, and 94.2%, respectively. These results were comparable with

5 36 those of mycobacterial culture (p>0.05). This sensitivity, however, was lower than the 79.5% 90.4% reported in recent analyses [7,9,16]. Mycobacterial culture-negative samples (n=13) account for the observed limitation of the sensitivity of the Xpert MTB/RIF assay, as the outcome of the Xpert assay is affected by a low bacillary load in the specimens [17]. The patients in this study likely presented early in their disease course as a result of routine screening in a secondary-care setting. Some patients presented scant sputum, which required us to obtain specimens bronchoscopically through bronchial washing and bronchoalveolar lavage. In our study, the most common specimens tested were bronchial washes (50.7%), followed by sputum (39.6%) and pleural fluid (8.1%). Positive Xpert results were obtained in 31 bronchoscopic samples (bronchial washing, 30; bronchoalveolar lavage fluid, 1), which suggests that the Xpert assay performed on bronchoscopy specimens accurately detects MTB, especially in patients with scarce sputum [18,19]. The diagnosis of extrapulmonary TB remains an important challenge in public health. The paucibacillary nature of the samples results in low sensitivity of AFB smear and culture techniques. Bunsow et al. reported sensitivity of 33.3% and specificity of 99.7% for the Xpert assay in non-respiratory specimens [20]. Tortoli et al. reported higher sensitivity and similar specificity of the Xpert assay in extrapulmonary specimens (79.0% and 97.3%) [21]. However, in the present study, the Xpert assay did not detect MTB in non-respiratory specimens, although the number of non-respiratory specimens was much lower than the number of respiratory specimens in our study (405 vs. 39). The Xpert assay started with the addition of a sample treatment reagent to each specimen, to limit bioaerosol infection risk to the testing personnel. The sample reagent was used to reduce the viability of M. tuberculosis in a sample, following international decontamination standards [22]. Therefore, one possible explanation for the discrepancy between our findings and those of the previous study is that the presence of sodium hydroxide and isopropanol alcohol for decontamination in the sample reagent may affect the quality of the specimens tested, thus reducing assay sensitivity [9,23]. It also should be Table. 5. Resistance to RIF determined by thexpert MTB/ RIF assay and conventional DST in 52 mycobacterial culture-positive patients. Conventional DST Resistance to RIF by Xpert MTB/ RIF assay Susceptible (n=31) 1 INH mono-resistant (n=1) 0 MDR (n=4)* 4 Contaminated (n=3) 0 Not evaluated (n=13) 0 *New, 2; previously treated, 2;, fail to grow on solid culture;, indeterminate, 1. Abbreviations: MTB, Mycobacterium tuberculosis; RIF, rifampin; INH, isoniazid; DST, drug susceptibility test; MDR, multi-drug resistant. noted that unprocessed specimens had a significantly higher sensitivity than processed specimens in smear-negative patients on the Xpert assay [9]. In our study, most of the specimens from extrapulmonary TB cases came from the pleural fluid (92.3%). The Xpert sensitivity on pleural fluid varied from 0% to 100% between studies [24]. Friedrich et al. reported that the sensitivity of the Xpert assay in pleural fluid was 25% for diagnosis of pleural TB [25]. Denkinger et al. showed that Xpert pooled sensitivity in pleural fluid was 46.4% against culture and 21.4% against a composite reference standard, and also noted that sample processing varied greatly among the studies [26]. The poor sensitivity of the Xpert assay in pleural fluid is probably due to the paucibacillary nature of the disease. Thus, a pleural biopsy is the preferred sample type for diagnosis of pleural TB, because pleural fluid is not a suitable specimen for microbiological diagnosis [27]. PCR inhibitors may exist in the pleural fluid itself or in blood contamination of the sample [28]. However, further studies need to be performed to increase the sensitivity of the Xpert assay on nonrespiratory specimens, including sterile body fluid, lymph node and tissue by optimizing sample collection and preparation. The incidence of MDR-TB is relatively low in Korea but has recently increased, from 516 cases of laboratory-confirmed MDR-TB in 2011 to 1212

6 Xpert MTB/RIF assay on clinical specimens 37 cases in 2012 [10]. Rapid detection of RIF resistance is important for treatment and prevention of person-to-person transmission of MDR-TB in areas with an intermediate TB burden, as RIF resistance is most commonly seen in MDR-TB strains and has a prevalence of >95% in such isolates [29,30]. In foreign studies, the sensitivity and specificity of the Xpert assay to detect RIF resistance has been reported to be 94.4% 100% or 98.3% 100%, respectively [8,31]. In a domestic study, however, the Xpert assay showed a sensitivity of 57.1% and a specificity of 100% for detecting RIF resistance [7]. In our study, five patients were identified as having RIF resistance by the Xpert assay, of which four (80%) had confirmed resistance on culture-based DST. All of these patients had MDR- TB (new, 2; previously treated, 2). A sample from one patient showed false positivity for RIF resistance, because susceptibility was observed in the DST results, while that from another showed indeterminate results. These findings suggest that selection of an adequate anti-tb drug is important for patients with potential MDR-TB as determined by the result of the Xpert assay alone. Concerns have been raised regarding the limited efficacy of the Xpert assay for determining RIF resistance [31-33], and the WHO recommends that RIF resistance determined by the Xpert assay should be confirmed with further tests and that treatment regimens should be based on DST [34]. We conclude that the Xpert MTB/RIF assay may be an important diagnostic tool for pulmonary TB disease and for the identification of RIF resistance in a secondary-care setting in an area with intermediate TB burden. However, a positive Xpert result for RIF resistance should be confirmed with further DST because of the potential for false-positive results. 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