- Email: [email protected]
RIF and GenoType MTBDRplus assays for the rapid diagnosis of bone and joint tuberculosis
International Journal of Infectious Diseases 36 (2015) e27–e30
Contents lists available at ScienceDirect
International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid
Xpert MTB/RIF and GenoType MTBDRplus assays for the rapid diagnosis of bone and joint tuberculosis Yunting Gu a, Guirong Wang a, Weijie Dong b, Yunxu Li a, Yifeng Ma a, Yuanyuan Shang a, Shibing Qin b,1,*, Hairong Huang a,1,* a
National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug-resistant Tuberculosis Research, Ma-Chang Rd 97, Beijing 101149, China Department of Orthopedics, Beijing Bone and Joint Tuberculosis Diagnosis and Treatment Center, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, China
b
A R T I C L E I N F O
Article history: Received 13 February 2015 Received in revised form 27 April 2015 Accepted 16 May 2015 Corresponding Editor: Eskild Petersen, Aarhus, Denmark Keywords: Bone and joint tuberculosis Xpert MTB/RIF GenoType MTBDRplus
S U M M A R Y
Background: Bone and joint tuberculosis (BJTB) constitutes about 10–20% of the extrapulmonary tuberculosis (EPTB) cases in China. The GenoType MTBDRplus assay (MTBDR) has been endorsed by the World Health Organization (WHO) for the diagnosis of pulmonary TB (PTB), while the Xpert MTB/RIF assay (Xpert) has also been endorsed by the WHO for the diagnosis of both PTB and EPTB. The diagnostic utility of these two techniques for BJTB was investigated prospectively. Methods: Sixty pus specimens were obtained from orthopedic patients. Smear, culture, Xpert, and MTBDR assays were performed for each specimen, and MGIT 960-based drug susceptibility testing (DST) was conducted for all of the isolates recovered. The diagnostic efficiency of Xpert and MTBDR was evaluated on the basis of bacteriological examination and the composite reference standard (CRS). Results: Fifty of the 60 patients were considered to have BJTB according to the CRS. The sensitivities of smear, culture, Xpert, and MTBDR were 26% (13/50), 48% (24/50), 82% (41/50), and 72% (36/50) respectively, while the specificities of all of the tests were 100% (10/10). Xpert was 100% concordant with MGIT 960-based DST for the detection of rifampicin resistance. MTBDR had a sensitivity of 83.3% and a specificity of 100% for the detection of rifampicin resistance and a sensitivity of 85.7% and specificity of 100% for the detection of isoniazid resistance. Conclusion: With their high sensitivities, short turnaround times, and ability to diagnose TB and detect drug resistance simultaneously, both Xpert and MTBDR are feasible as diagnostic tools for BJTB in clinical practice. ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
1. Introduction Tuberculosis (TB) remains one of the predominant infectious diseases affecting adults in developing countries.1 China ranks second among the 22 high-burden countries that together account for 80% of TB cases and 22% of multidrug-resistant TB (MDR-TB) cases worldwide.2 Extrapulmonary TB (EPTB) accounts for about 10–20% of all cases of active TB in China,3 and among cases of EPTB, 38.6% is lymph node TB, 19.9% is bone and joint TB (BJTB), 16.7% is urinary and genital TB, 9.1% is intestinal and peritoneal TB, and
* Corresponding author. Tel.: +86-10-89509159. E-mail addresses: [email protected] (S. Qin), [email protected] (H. Huang). 1 Yunting Gu and Guirong Wang contributed equally to this study.
6.4% is cerebral and nervous system TB.4 Therefore, rapid BJTB case detection and the early identification of patients with MDR-TB have become extremely important in high-burden countries such as China. The diagnosis of BJTB is challenging due to limitations in sampling from the affected sites and the paucibacillary nature of the specimens. Limitations in sensitivity, the amount of time required for assays, and the requirement for invasive procedures and expertise render the conventional diagnostics of BJTB less efficient. In the last decade, molecular tests have been developed to detect the presence of Mycobacterium tuberculosis bacilli and to identify mutations conferring resistance to the most important first-line TB antibiotics, isoniazid (INH) and/or rifampicin (RIF).5,6 In 2008 the GenoType MTBDRplus assay (MTBDR), which detects resistance to both INH and RIF within 48 h, was endorsed by the
http://dx.doi.org/10.1016/j.ijid.2015.05.014 1201-9712/ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
e28
Y. Gu et al. / International Journal of Infectious Diseases 36 (2015) e27–e30
World Health Organization (WHO) for the diagnosis of pulmonary TB (PTB).7 In 2010, another assay was endorsed by the WHO for the diagnosis of PTB, known as the Xpert MTB/RIF assay (Xpert).8 As a fully automated real-time PCR technique,9 Xpert can provide RIF susceptibility results within 2 h.10,11 Since Xpert also has comparable performance to the composite reference standard (CRS) for detecting EPTB,12 the WHO recommended Xpert for the diagnosis of EPTB in 2013.13 A series of meta-analyses have shown that nucleic acid amplification tests (NAATs) have high specificity, high positive predictive value, and highly variable sensitivity for the diagnosis of EPTB.14–16 However, both MTBDR and Xpert are yet to be rigorously evaluated for specimens collected from BJTB. Previous studies have involved the comparison of molecular tests with the outcomes of culture, which is known to be a very suboptimal reference standard for EPTB. Therefore in this study, the CRS17 was used in addition to culture in order to evaluate the realistic diagnostic efficiency of Xpert and MTBDR for BJTB. 2. Materials and methods 2.1. Ethics statement Ethical approval for this study was obtained from the Beijing Chest Hospital Ethics Committee. Written informed consent was acquired from each participant.
automated MGIT 960 apparatus (Becton Dickinson) for a maximum of 42 days, and monitored continuously. The MGIT 960 outcomes were recorded as per the manufacturer’s instructions. Standard drug susceptibility testing (DST) with INH and RIF was carried out for the positive cultures using the MGIT 960 IR kit (Becton Dickinson) following the manufacturer’s instructions. 2.5. GenoType MTBDRplus assay The DNA template for PCR was extracted from 1 ml of pus specimen using a GenoLyse kit (Hain Lifescience GmbH, Nehren, Germany). PCR amplification, hybridization, and the interpretation of outcomes were performed in accordance with the manufacturer’s instructions (Hain Lifescience).19 2.6. Xpert MTB/RIF assay The assay was performed as per the manufacturer’s instructions (Cepheid, Sunnyvale, CA, USA). Briefly, 1 ml of pus specimen was mixed with 2 ml Xpert sample reagent, vortexed for at least 10 s, and incubated at room temperature for 10 min. The mixture was again vortexed for another 10 s and incubated at room temperature for 5 min. A total of 2 ml of the mixture was transferred into the Xpert cartridge and loaded into the GeneXpert instrument. The automatic detection procedure was then run. 3. Results
2.2. Patient categories 3.1. Patients categorized by CRS Sixty patients with suspected BJTB were recruited between March and June 2014 from the Beijing Chest Hospital. These patients who were diagnosed with suspected BJTB had pus specimens collected during operations, and before operations, and all had been administered anti-TB treatment for at least 2 weeks. Pus specimens of at least 5 ml were collected under sterile conditions from each of the 60 orthopedic patients during surgical debridement. Based on the CRS, patients were categorized into four groups: (1) confirmed BJTB cases (culture-positive), (2) probable BJTB cases (culture-negative but biopsy or surgical specimens showed caseating granuloma and the patient responded well to anti-TB therapy), (3) possible BJTB cases (responded to anti-TB therapy and had radiographic findings consistent with osteoarticular TB, but lacked bacteriological and histopathological corroborating evidence), and (4) non-TB (culture and all other tests for TB were negative and the patient improved without receiving TB treatment).17 2.3. Smear microscopy Direct smears were prepared from the specimen and stained using the auramine staining technique. The specimens were then examined by light-emitting diode (LED) microscopy. The smears were read and interpreted in accordance with the WHO guidelines.18 2.4. Mycobacterial culture and susceptibility testing by MGIT 960 Two milliliters of pus specimen were decontaminated with Nacetyl-L-cysteine sodium hydroxide (BBL MycoPrep; Becton Dickinson, Sparks, MD, USA) for 20 min, neutralized with sterile phosphate-buffered saline (PBS, pH 6.8), added to a final volume of 45 ml, and centrifuged at 3000 g for 15 min at 4 8C. The supernatant was discarded, and the remaining pellet was resuspended in 1.5 ml of PBS. Each MGIT 960 tube was inoculated with 0.5 ml of the resulting specimen, incubated at 37 8C in an
According to the CRS, 50 patients were considered to have BJTB and 10 patients to have no evidence of TB. Of these 50 BJTB patients, 24 (40%) were culture-positive ‘confirmed BJTB’ cases, 22 (36.67%) were ‘probable BJTB’ cases, four (6.67%) were ‘possible BJTB’ cases, and 10 (16.67%) patients had no evidence of TB. Table 1 shows the demographic characteristics of patients in the different categories. According to the CRS, confirmed BJTB patients had a longer duration of illness (18 20 months) than those in the other three categories of patient (8 8 months, 8 8 months, and 7 10 months, respectively). Among the 24 confirmed BJTB patients, six (25%) were determined to have MDR-TB upon phenotypic DST. 3.2. Sensitivity and specificity of Xpert and MTBDR tests According to the CRS, the sensitivities of smears, culture, Xpert, and MTBDR tests were 26% (13/50), 48% (24/50), 82% (41/50), and 72% (36/50) respectively, while their specificities were 100% (10/ 10). The sensitivities of Xpert and MTBDR were 100% (24/24) and 87.5% (21/24) for confirmed BJTB, 72.73% (16/22) and 63.64% (14/ 22) for probable BJTB, and both were 25% (1/4) for possible BJTB. Both the Xpert and MTBDR test detected all smear-positive specimens, whereas Xpert detected all of the smear-negative/ culture-positive specimens but the MTBDR test detected only 75% (9/12) of these. It is worth mentioning that the Xpert and MTBDR tests produced positive outcomes for 64% (16/25) and 56% (14/25), respectively, of smear-negative/culture-negative/CRS-supposed BJTB patients. 3.3. Detection of drug resistance Standard MGIT 960 DST was performed for the 24 M. tuberculosis isolates recovered from pus samples. Six RIF-resistant isolates and seven INH-resistant isolates were identified by MGIT DST (six MDR strains and one INH mono-resistant strain) (Table 2). Xpert was 100% concordant with MGIT DST with regard to the detection of RIF resistance. Compared to phenotypic DST, MTBDR
Y. Gu et al. / International Journal of Infectious Diseases 36 (2015) e27–e30
e29
Table 1 Demographic characteristics of patients in the different categoriesa Characteristics
Confirmed BJTB (n = 24)
Probable BJTB (n = 22)
Possible BJTB (n = 4)
Non-TB (n = 10)
Age, years Male Spine lesion Joint lesion Duration of illness, months Responded well to anti-TB treatment Period of anti-TB treatment before sample collection, months Bone destruction 1 vertebral body 2 vertebral bodies 3 vertebral bodies 4 vertebral bodies
42 (18–82) 11 (45.83%) 18 (75%) 6 (25%) 18 20 (2–96) 23 (95.83%) 1.45 2.80 (0.5–12)
33 (21–77) 8 (36.36%) 16 (72.73%) 6 (27.27%) 8 8 (1–24) 22 (100%) 1.80 1.66 (0.5–16)
19 (16–28) 2 (50%) 3 (75%) 1 (25%) 8 8 (1–18) 4 (100%) 12.33 15.31 (3–30)
57 (21–70) 7 (70%) 5 (50%) 5 (50%) 7 10 (1–36) 0 1.25 1.03 (0.5–3)
1 (4.17%) 13 (54.17%) 0 3 (12.5%)
2 (9.09%) 13 (59.09%) 1 (4.55%) 0
1 (25%) 1 (25%) 0 0
0 3 (30%) 2 (20%) 0
BJTB, bone and joint tuberculosis; TB, tuberculosis. a Data are presented as the median (range), number (percentage), or mean standard deviation (range).
Table 2 Sensitivities and specificities of the Xpert MTB/RIF and GenoType MTBDRplus tests for the detection of rifampicin and/or isoniazid resistance in comparison with drug susceptibility testing Method
Sensitivity for RIF Resistance (%)
Specificity for RIF Resistance (%)
Sensitivity for INH Resistance (%)
Specificity for INH Resistance (%)
Xpert MTBDR
6/6 (100) 5/6 (83.33)
18/18 (100) 18/18 (100)
6/7 (85.71)
17/17 (100)
RIF, rifampicin, INH, isoniazid.
missed one case of RIF resistance and one case of INH resistance, resulting in a sensitivity of 83.33% and 85.71%, respectively. The MTBDR test had 100% specificity for the detection of resistance to both drugs. 4. Discussion A few studies have shown the effectiveness of Xpert in the diagnosis of EPTB,20,21 in which the reported sensitivity ranged from 87.3% to 95% and the specificity in contrast to CRS was consistently over 97%. In the present study, the performance of the Xpert and MTBDR assays in the diagnosis of BJTB using pus specimens was investigated. Among the sample recruited, the sensitivities of the Xpert and MTBDR tests were 82% (41/50) and 72% (36/50), respectively, while their specificities were 100% according to the CRS, a value similar to those reported from previous studies mainly focusing on other forms of EPTB. In the present study, according to the CRS, Xpert and MTBDR identified 75.68% (28/37) and 62.16% (23/37) of smear-negative specimens, as well as 65.38% (17/26) and 57.69% (15/26) of culture-negative specimens, respectively. The low sensitivity of culture (48%) in comparison with that of the molecular tests (72– 82%) against the CRS can be explained as follows:21 (1) all of the BJTB patients in this assay had undergone anti-TB treatment for various periods of time before sample collection, which is believed to affect the recovery rate of culture more than the outcome of molecular tests; (2) the paucibacillary nature of pus specimens with a tendency of M. tuberculosis to form clumps led to an uneven distribution of the bacilli; and (3) there was a loss of viable bacilli during decontamination. Due to the small number of patients in the possible BJTB and non-TB categories, further demographic characteristics could not be analyzed. However, the confirmed BJTB category of patients seemed to have a notably longer duration of illness than the other three categories of patients (Table 1). This phenomenon is consistent with the general observation that the longer the duration of illness, the more severe the disease is, and the more chance of obtaining bacteriological evidence to confirm the diagnosis of TB.
A clear advantage of molecular tests is their rapid turnaround time. For patients with a positive molecular test result, the time needed to obtain bacterial evidence of TB is reduced dramatically from a few weeks to a few days. BJTB cases used to be treated empirically on clinical and radiological grounds, and a full course of anti-TB chemotherapy was also required.22 Early determination of the identity and sensitivity of the mycobacteria is very important not just from a public health and epidemiological perspective, but also for treatment and management.23 The presence of MDR-TB has resulted in a less efficient and more prolonged and complicated course of treatment. Obtaining conventional DST results takes at least 2 months from sample collection to the outcome, while molecular tests make it possible for the appropriate management of MDR-TB to be started without delay.24 In this study, the sensitivity of Xpert for detecting RIF resistance was shown to be 100%, and the sensitivities of MTBDR in the detection of RIF and INH resistance were 83.3% and 85.7%, respectively. Xpert demonstrated a higher sensitivity characteristic than the MTBDR in this assay, and this merit may be the result of the sample processing method, inoculum size, and the full automation steps.24 MTBDR requires several manual steps to prepare the DNA template, which might cause the loss of DNA during processing; the manual hybridization step could also decrease the sensitivity of the assay. However, the Xpert assay only detects RIF resistance, which might lead to an over-estimation of MDR-TB in regions where RIF mono-resistance is frequently seen. Although in this scenario, MTBDR has an advantage over Xpert, the drawbacks such as redundancy of operation and subjective interpretation of the outcome, decrease the feasibility of this assay.25 In conclusion, both Xpert and MTBDR are feasible diagnostic tools for BJTB, with high sensitivity, a short turnaround time, and the ability to diagnose TB and detect drug resistance simultaneously. Conflict of interest The authors declare no conflicts of interest, real or perceived, financial or non-financial.
e30
Y. Gu et al. / International Journal of Infectious Diseases 36 (2015) e27–e30
Acknowledgement 12.
This work was supported by research funding from Beijing Natural Science Foundation Grants (7132049), Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201304), and the Key Project of Department of Science and Technology Beijing, China (D141107005214002). References 1. Lienhardt C, Glaziou P, Uplekar M, Lonnroth K, Getahun H, Raviglione M. Global tuberculosis control: lessons learnt and future prospects. Nat Rev Microbiol 2012;10:407–16. 2. World Health Organization. Global tuberculosis control: surveillance, planning, financing. Geneva, Switzerland: WHO; 2008. 3. Jianjun L, Hongyan Y. [Epidemiology and prevention and control countermeasure of tuberculosis in China] (in Chinese). Preventive Medicine Tribune 2006;12:638–40. 4. Jiansheng H, Mei S, Yaling S. [Epidemiological analysis of extrapulmonary tuberculosis in Shanghai] (in Chinese). Chinese Journal of Tuberculosis and Respiratory Diseases 2000;23:606–8. 5. Barnard M, Albert H, Coetzee G, O’Brien R, Bosman ME. Rapid molecular screening for multidrug-resistant tuberculosis in a high volume public health laboratory in South Africa. Am J Respir Crit Care Med 2008;177:787–92. 6. Bwanga F, Hoffner S, Haile M, Joloba ML. Direct susceptibility testing for multi drug resistant tuberculosis: a meta-analysis. BMC Infect Dis 2009;9:67. 7. World Health Organization. Molecular line probe assays for rapid screening of patients at risk of multi-drug resistant tuberculosis (MDRTB). Geneva, Switzerland: WHO; 2008. 8. World Health Organization. WHO endorses new rapid tuberculosis test. Geneva, Switzerland: WHO; 2010. 9. Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 2010;363:1005–15. 10. Blakemore R, Story E, Helb D, Kop J, Banada P, Owens MR, et al. Evaluation of the analytical performance of the Xpert MTB/RIF assay. J Clin Microbiol 2010;48:2495–501. 11. Boehme CC, Nicol MP, Nabeta P, Michael JS, Gotuzzo E, Tahirli R, et al. Feasibility, diagnostic accuracy, and effectiveness of decentralised use of the Xpert MTB/
13.
14.
15.
16. 17.
18.
19. 20.
21.
22. 23.
24.
25.
RIF test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study. Lancet 2011;377:1495–505. Walmsley S. ACP Journal Club: review: Xpert MTB/RIF assay detects extrapulmonary TB in lymph nodes and CSF, but not pleural fluid. Ann Intern Med 2015;162:JC11. World Health Organization. Automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF system for the diagnosis of pulmonary and extrapulmonary TB in adults and children. Policy update. Geneva, Switzerland: WHO; 2013. Daley P, Thomas S, Pai M. Nucleic acid amplification tests for the diagnosis of tuberculous lymphadenitis: a systematic review. Int J Tuberc Lung Dis 2007;11:1166–76. Pai M, Flores LL, Hubbard A, Riley LW, Colford JM. Nucleic acid amplification tests in the diagnosis of tuberculous pleuritis: a systematic review and metaanalysis. BMC Infect Dis 2004;4:6. Pai M, Ling DI. Rapid diagnosis of extrapulmonary tuberculosis using nucleic acid amplification tests: what is the evidence? Future Microbiol 2008;3:1–4. NICE guidelines for the diagnosis of TB. National Institute of Health and Care Excellence, UK; 2011. Available at: http://www.nice.org.uk/guidance/cg117/ chapter/guidance. Mohammed A, Gisela B, Fadila B, David D, Lanfranco F, Knut F, et al. Technical guide: sputum examination for tuberculosis by direct microscopy in low income countries, 5th ed., Paris, France: International Union against Tuberculosis and Lung Disease; 2000. Hain Lifescience. GenoType MTBDRplus version 1. 0 product manual. Nehren, Germany: Hain Lifescience; 2009. Maynard-Smith L, Larke N, Peters JA, Lawn SD. Diagnostic accuracy of the Xpert MTB/RIF assay for extrapulmonary and pulmonary tuberculosis when testing non-respiratory samples: a systematic review. BMC Infect Dis 2014;14:709. Vadwai V, Boehme C, Nabeta P, Shetty A, Alland D, Rodrigues C. Xpert MTB/RIF: a new pillar in diagnosis of extrapulmonary tuberculosis. J Clin Microbiol 2011;49:2540–5. Pigrau-Serrallach C, Rodriguez-Pardo D. Bone and joint tuberculosis. Eur Spine J 2013;22:556–66. World Health Organization. Checklist of prerequisites to country implementation of Xpert MTB/RIF and key action points at country level. WHO/HTM/TB/ 2011. 12. Geneva, Switzerland: WHO; 2011. Steingart KR, Schiller I, Horne DJ, Pai M, Boehme CC, Dendukuri N. Xpert1 MTB/ RIF assay for pulmonary tuberculosis and rifampicin resistance in adults. Cochrane Database Syst Rev 2014;1:CD009593. Nahid P, Kim PS, Evans CA, Alland D, Barer M, Diefenbach J, et al. Clinical research and development of tuberculosis diagnostics: moving from silos to synergy. J Infect Dis 2012;205(Suppl 2):S159–68.
Contents lists available at ScienceDirect
International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid
Xpert MTB/RIF and GenoType MTBDRplus assays for the rapid diagnosis of bone and joint tuberculosis Yunting Gu a, Guirong Wang a, Weijie Dong b, Yunxu Li a, Yifeng Ma a, Yuanyuan Shang a, Shibing Qin b,1,*, Hairong Huang a,1,* a
National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug-resistant Tuberculosis Research, Ma-Chang Rd 97, Beijing 101149, China Department of Orthopedics, Beijing Bone and Joint Tuberculosis Diagnosis and Treatment Center, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, China
b
A R T I C L E I N F O
Article history: Received 13 February 2015 Received in revised form 27 April 2015 Accepted 16 May 2015 Corresponding Editor: Eskild Petersen, Aarhus, Denmark Keywords: Bone and joint tuberculosis Xpert MTB/RIF GenoType MTBDRplus
S U M M A R Y
Background: Bone and joint tuberculosis (BJTB) constitutes about 10–20% of the extrapulmonary tuberculosis (EPTB) cases in China. The GenoType MTBDRplus assay (MTBDR) has been endorsed by the World Health Organization (WHO) for the diagnosis of pulmonary TB (PTB), while the Xpert MTB/RIF assay (Xpert) has also been endorsed by the WHO for the diagnosis of both PTB and EPTB. The diagnostic utility of these two techniques for BJTB was investigated prospectively. Methods: Sixty pus specimens were obtained from orthopedic patients. Smear, culture, Xpert, and MTBDR assays were performed for each specimen, and MGIT 960-based drug susceptibility testing (DST) was conducted for all of the isolates recovered. The diagnostic efficiency of Xpert and MTBDR was evaluated on the basis of bacteriological examination and the composite reference standard (CRS). Results: Fifty of the 60 patients were considered to have BJTB according to the CRS. The sensitivities of smear, culture, Xpert, and MTBDR were 26% (13/50), 48% (24/50), 82% (41/50), and 72% (36/50) respectively, while the specificities of all of the tests were 100% (10/10). Xpert was 100% concordant with MGIT 960-based DST for the detection of rifampicin resistance. MTBDR had a sensitivity of 83.3% and a specificity of 100% for the detection of rifampicin resistance and a sensitivity of 85.7% and specificity of 100% for the detection of isoniazid resistance. Conclusion: With their high sensitivities, short turnaround times, and ability to diagnose TB and detect drug resistance simultaneously, both Xpert and MTBDR are feasible as diagnostic tools for BJTB in clinical practice. ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
1. Introduction Tuberculosis (TB) remains one of the predominant infectious diseases affecting adults in developing countries.1 China ranks second among the 22 high-burden countries that together account for 80% of TB cases and 22% of multidrug-resistant TB (MDR-TB) cases worldwide.2 Extrapulmonary TB (EPTB) accounts for about 10–20% of all cases of active TB in China,3 and among cases of EPTB, 38.6% is lymph node TB, 19.9% is bone and joint TB (BJTB), 16.7% is urinary and genital TB, 9.1% is intestinal and peritoneal TB, and
* Corresponding author. Tel.: +86-10-89509159. E-mail addresses: [email protected] (S. Qin), [email protected] (H. Huang). 1 Yunting Gu and Guirong Wang contributed equally to this study.
6.4% is cerebral and nervous system TB.4 Therefore, rapid BJTB case detection and the early identification of patients with MDR-TB have become extremely important in high-burden countries such as China. The diagnosis of BJTB is challenging due to limitations in sampling from the affected sites and the paucibacillary nature of the specimens. Limitations in sensitivity, the amount of time required for assays, and the requirement for invasive procedures and expertise render the conventional diagnostics of BJTB less efficient. In the last decade, molecular tests have been developed to detect the presence of Mycobacterium tuberculosis bacilli and to identify mutations conferring resistance to the most important first-line TB antibiotics, isoniazid (INH) and/or rifampicin (RIF).5,6 In 2008 the GenoType MTBDRplus assay (MTBDR), which detects resistance to both INH and RIF within 48 h, was endorsed by the
http://dx.doi.org/10.1016/j.ijid.2015.05.014 1201-9712/ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
e28
Y. Gu et al. / International Journal of Infectious Diseases 36 (2015) e27–e30
World Health Organization (WHO) for the diagnosis of pulmonary TB (PTB).7 In 2010, another assay was endorsed by the WHO for the diagnosis of PTB, known as the Xpert MTB/RIF assay (Xpert).8 As a fully automated real-time PCR technique,9 Xpert can provide RIF susceptibility results within 2 h.10,11 Since Xpert also has comparable performance to the composite reference standard (CRS) for detecting EPTB,12 the WHO recommended Xpert for the diagnosis of EPTB in 2013.13 A series of meta-analyses have shown that nucleic acid amplification tests (NAATs) have high specificity, high positive predictive value, and highly variable sensitivity for the diagnosis of EPTB.14–16 However, both MTBDR and Xpert are yet to be rigorously evaluated for specimens collected from BJTB. Previous studies have involved the comparison of molecular tests with the outcomes of culture, which is known to be a very suboptimal reference standard for EPTB. Therefore in this study, the CRS17 was used in addition to culture in order to evaluate the realistic diagnostic efficiency of Xpert and MTBDR for BJTB. 2. Materials and methods 2.1. Ethics statement Ethical approval for this study was obtained from the Beijing Chest Hospital Ethics Committee. Written informed consent was acquired from each participant.
automated MGIT 960 apparatus (Becton Dickinson) for a maximum of 42 days, and monitored continuously. The MGIT 960 outcomes were recorded as per the manufacturer’s instructions. Standard drug susceptibility testing (DST) with INH and RIF was carried out for the positive cultures using the MGIT 960 IR kit (Becton Dickinson) following the manufacturer’s instructions. 2.5. GenoType MTBDRplus assay The DNA template for PCR was extracted from 1 ml of pus specimen using a GenoLyse kit (Hain Lifescience GmbH, Nehren, Germany). PCR amplification, hybridization, and the interpretation of outcomes were performed in accordance with the manufacturer’s instructions (Hain Lifescience).19 2.6. Xpert MTB/RIF assay The assay was performed as per the manufacturer’s instructions (Cepheid, Sunnyvale, CA, USA). Briefly, 1 ml of pus specimen was mixed with 2 ml Xpert sample reagent, vortexed for at least 10 s, and incubated at room temperature for 10 min. The mixture was again vortexed for another 10 s and incubated at room temperature for 5 min. A total of 2 ml of the mixture was transferred into the Xpert cartridge and loaded into the GeneXpert instrument. The automatic detection procedure was then run. 3. Results
2.2. Patient categories 3.1. Patients categorized by CRS Sixty patients with suspected BJTB were recruited between March and June 2014 from the Beijing Chest Hospital. These patients who were diagnosed with suspected BJTB had pus specimens collected during operations, and before operations, and all had been administered anti-TB treatment for at least 2 weeks. Pus specimens of at least 5 ml were collected under sterile conditions from each of the 60 orthopedic patients during surgical debridement. Based on the CRS, patients were categorized into four groups: (1) confirmed BJTB cases (culture-positive), (2) probable BJTB cases (culture-negative but biopsy or surgical specimens showed caseating granuloma and the patient responded well to anti-TB therapy), (3) possible BJTB cases (responded to anti-TB therapy and had radiographic findings consistent with osteoarticular TB, but lacked bacteriological and histopathological corroborating evidence), and (4) non-TB (culture and all other tests for TB were negative and the patient improved without receiving TB treatment).17 2.3. Smear microscopy Direct smears were prepared from the specimen and stained using the auramine staining technique. The specimens were then examined by light-emitting diode (LED) microscopy. The smears were read and interpreted in accordance with the WHO guidelines.18 2.4. Mycobacterial culture and susceptibility testing by MGIT 960 Two milliliters of pus specimen were decontaminated with Nacetyl-L-cysteine sodium hydroxide (BBL MycoPrep; Becton Dickinson, Sparks, MD, USA) for 20 min, neutralized with sterile phosphate-buffered saline (PBS, pH 6.8), added to a final volume of 45 ml, and centrifuged at 3000 g for 15 min at 4 8C. The supernatant was discarded, and the remaining pellet was resuspended in 1.5 ml of PBS. Each MGIT 960 tube was inoculated with 0.5 ml of the resulting specimen, incubated at 37 8C in an
According to the CRS, 50 patients were considered to have BJTB and 10 patients to have no evidence of TB. Of these 50 BJTB patients, 24 (40%) were culture-positive ‘confirmed BJTB’ cases, 22 (36.67%) were ‘probable BJTB’ cases, four (6.67%) were ‘possible BJTB’ cases, and 10 (16.67%) patients had no evidence of TB. Table 1 shows the demographic characteristics of patients in the different categories. According to the CRS, confirmed BJTB patients had a longer duration of illness (18 20 months) than those in the other three categories of patient (8 8 months, 8 8 months, and 7 10 months, respectively). Among the 24 confirmed BJTB patients, six (25%) were determined to have MDR-TB upon phenotypic DST. 3.2. Sensitivity and specificity of Xpert and MTBDR tests According to the CRS, the sensitivities of smears, culture, Xpert, and MTBDR tests were 26% (13/50), 48% (24/50), 82% (41/50), and 72% (36/50) respectively, while their specificities were 100% (10/ 10). The sensitivities of Xpert and MTBDR were 100% (24/24) and 87.5% (21/24) for confirmed BJTB, 72.73% (16/22) and 63.64% (14/ 22) for probable BJTB, and both were 25% (1/4) for possible BJTB. Both the Xpert and MTBDR test detected all smear-positive specimens, whereas Xpert detected all of the smear-negative/ culture-positive specimens but the MTBDR test detected only 75% (9/12) of these. It is worth mentioning that the Xpert and MTBDR tests produced positive outcomes for 64% (16/25) and 56% (14/25), respectively, of smear-negative/culture-negative/CRS-supposed BJTB patients. 3.3. Detection of drug resistance Standard MGIT 960 DST was performed for the 24 M. tuberculosis isolates recovered from pus samples. Six RIF-resistant isolates and seven INH-resistant isolates were identified by MGIT DST (six MDR strains and one INH mono-resistant strain) (Table 2). Xpert was 100% concordant with MGIT DST with regard to the detection of RIF resistance. Compared to phenotypic DST, MTBDR
Y. Gu et al. / International Journal of Infectious Diseases 36 (2015) e27–e30
e29
Table 1 Demographic characteristics of patients in the different categoriesa Characteristics
Confirmed BJTB (n = 24)
Probable BJTB (n = 22)
Possible BJTB (n = 4)
Non-TB (n = 10)
Age, years Male Spine lesion Joint lesion Duration of illness, months Responded well to anti-TB treatment Period of anti-TB treatment before sample collection, months Bone destruction 1 vertebral body 2 vertebral bodies 3 vertebral bodies 4 vertebral bodies
42 (18–82) 11 (45.83%) 18 (75%) 6 (25%) 18 20 (2–96) 23 (95.83%) 1.45 2.80 (0.5–12)
33 (21–77) 8 (36.36%) 16 (72.73%) 6 (27.27%) 8 8 (1–24) 22 (100%) 1.80 1.66 (0.5–16)
19 (16–28) 2 (50%) 3 (75%) 1 (25%) 8 8 (1–18) 4 (100%) 12.33 15.31 (3–30)
57 (21–70) 7 (70%) 5 (50%) 5 (50%) 7 10 (1–36) 0 1.25 1.03 (0.5–3)
1 (4.17%) 13 (54.17%) 0 3 (12.5%)
2 (9.09%) 13 (59.09%) 1 (4.55%) 0
1 (25%) 1 (25%) 0 0
0 3 (30%) 2 (20%) 0
BJTB, bone and joint tuberculosis; TB, tuberculosis. a Data are presented as the median (range), number (percentage), or mean standard deviation (range).
Table 2 Sensitivities and specificities of the Xpert MTB/RIF and GenoType MTBDRplus tests for the detection of rifampicin and/or isoniazid resistance in comparison with drug susceptibility testing Method
Sensitivity for RIF Resistance (%)
Specificity for RIF Resistance (%)
Sensitivity for INH Resistance (%)
Specificity for INH Resistance (%)
Xpert MTBDR
6/6 (100) 5/6 (83.33)
18/18 (100) 18/18 (100)
6/7 (85.71)
17/17 (100)
RIF, rifampicin, INH, isoniazid.
missed one case of RIF resistance and one case of INH resistance, resulting in a sensitivity of 83.33% and 85.71%, respectively. The MTBDR test had 100% specificity for the detection of resistance to both drugs. 4. Discussion A few studies have shown the effectiveness of Xpert in the diagnosis of EPTB,20,21 in which the reported sensitivity ranged from 87.3% to 95% and the specificity in contrast to CRS was consistently over 97%. In the present study, the performance of the Xpert and MTBDR assays in the diagnosis of BJTB using pus specimens was investigated. Among the sample recruited, the sensitivities of the Xpert and MTBDR tests were 82% (41/50) and 72% (36/50), respectively, while their specificities were 100% according to the CRS, a value similar to those reported from previous studies mainly focusing on other forms of EPTB. In the present study, according to the CRS, Xpert and MTBDR identified 75.68% (28/37) and 62.16% (23/37) of smear-negative specimens, as well as 65.38% (17/26) and 57.69% (15/26) of culture-negative specimens, respectively. The low sensitivity of culture (48%) in comparison with that of the molecular tests (72– 82%) against the CRS can be explained as follows:21 (1) all of the BJTB patients in this assay had undergone anti-TB treatment for various periods of time before sample collection, which is believed to affect the recovery rate of culture more than the outcome of molecular tests; (2) the paucibacillary nature of pus specimens with a tendency of M. tuberculosis to form clumps led to an uneven distribution of the bacilli; and (3) there was a loss of viable bacilli during decontamination. Due to the small number of patients in the possible BJTB and non-TB categories, further demographic characteristics could not be analyzed. However, the confirmed BJTB category of patients seemed to have a notably longer duration of illness than the other three categories of patients (Table 1). This phenomenon is consistent with the general observation that the longer the duration of illness, the more severe the disease is, and the more chance of obtaining bacteriological evidence to confirm the diagnosis of TB.
A clear advantage of molecular tests is their rapid turnaround time. For patients with a positive molecular test result, the time needed to obtain bacterial evidence of TB is reduced dramatically from a few weeks to a few days. BJTB cases used to be treated empirically on clinical and radiological grounds, and a full course of anti-TB chemotherapy was also required.22 Early determination of the identity and sensitivity of the mycobacteria is very important not just from a public health and epidemiological perspective, but also for treatment and management.23 The presence of MDR-TB has resulted in a less efficient and more prolonged and complicated course of treatment. Obtaining conventional DST results takes at least 2 months from sample collection to the outcome, while molecular tests make it possible for the appropriate management of MDR-TB to be started without delay.24 In this study, the sensitivity of Xpert for detecting RIF resistance was shown to be 100%, and the sensitivities of MTBDR in the detection of RIF and INH resistance were 83.3% and 85.7%, respectively. Xpert demonstrated a higher sensitivity characteristic than the MTBDR in this assay, and this merit may be the result of the sample processing method, inoculum size, and the full automation steps.24 MTBDR requires several manual steps to prepare the DNA template, which might cause the loss of DNA during processing; the manual hybridization step could also decrease the sensitivity of the assay. However, the Xpert assay only detects RIF resistance, which might lead to an over-estimation of MDR-TB in regions where RIF mono-resistance is frequently seen. Although in this scenario, MTBDR has an advantage over Xpert, the drawbacks such as redundancy of operation and subjective interpretation of the outcome, decrease the feasibility of this assay.25 In conclusion, both Xpert and MTBDR are feasible diagnostic tools for BJTB, with high sensitivity, a short turnaround time, and the ability to diagnose TB and detect drug resistance simultaneously. Conflict of interest The authors declare no conflicts of interest, real or perceived, financial or non-financial.
e30
Y. Gu et al. / International Journal of Infectious Diseases 36 (2015) e27–e30
Acknowledgement 12.
This work was supported by research funding from Beijing Natural Science Foundation Grants (7132049), Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201304), and the Key Project of Department of Science and Technology Beijing, China (D141107005214002). References 1. Lienhardt C, Glaziou P, Uplekar M, Lonnroth K, Getahun H, Raviglione M. Global tuberculosis control: lessons learnt and future prospects. Nat Rev Microbiol 2012;10:407–16. 2. World Health Organization. Global tuberculosis control: surveillance, planning, financing. Geneva, Switzerland: WHO; 2008. 3. Jianjun L, Hongyan Y. [Epidemiology and prevention and control countermeasure of tuberculosis in China] (in Chinese). Preventive Medicine Tribune 2006;12:638–40. 4. Jiansheng H, Mei S, Yaling S. [Epidemiological analysis of extrapulmonary tuberculosis in Shanghai] (in Chinese). Chinese Journal of Tuberculosis and Respiratory Diseases 2000;23:606–8. 5. Barnard M, Albert H, Coetzee G, O’Brien R, Bosman ME. Rapid molecular screening for multidrug-resistant tuberculosis in a high volume public health laboratory in South Africa. Am J Respir Crit Care Med 2008;177:787–92. 6. Bwanga F, Hoffner S, Haile M, Joloba ML. Direct susceptibility testing for multi drug resistant tuberculosis: a meta-analysis. BMC Infect Dis 2009;9:67. 7. World Health Organization. Molecular line probe assays for rapid screening of patients at risk of multi-drug resistant tuberculosis (MDRTB). Geneva, Switzerland: WHO; 2008. 8. World Health Organization. WHO endorses new rapid tuberculosis test. Geneva, Switzerland: WHO; 2010. 9. Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 2010;363:1005–15. 10. Blakemore R, Story E, Helb D, Kop J, Banada P, Owens MR, et al. Evaluation of the analytical performance of the Xpert MTB/RIF assay. J Clin Microbiol 2010;48:2495–501. 11. Boehme CC, Nicol MP, Nabeta P, Michael JS, Gotuzzo E, Tahirli R, et al. Feasibility, diagnostic accuracy, and effectiveness of decentralised use of the Xpert MTB/
13.
14.
15.
16. 17.
18.
19. 20.
21.
22. 23.
24.
25.
RIF test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study. Lancet 2011;377:1495–505. Walmsley S. ACP Journal Club: review: Xpert MTB/RIF assay detects extrapulmonary TB in lymph nodes and CSF, but not pleural fluid. Ann Intern Med 2015;162:JC11. World Health Organization. Automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF system for the diagnosis of pulmonary and extrapulmonary TB in adults and children. Policy update. Geneva, Switzerland: WHO; 2013. Daley P, Thomas S, Pai M. Nucleic acid amplification tests for the diagnosis of tuberculous lymphadenitis: a systematic review. Int J Tuberc Lung Dis 2007;11:1166–76. Pai M, Flores LL, Hubbard A, Riley LW, Colford JM. Nucleic acid amplification tests in the diagnosis of tuberculous pleuritis: a systematic review and metaanalysis. BMC Infect Dis 2004;4:6. Pai M, Ling DI. Rapid diagnosis of extrapulmonary tuberculosis using nucleic acid amplification tests: what is the evidence? Future Microbiol 2008;3:1–4. NICE guidelines for the diagnosis of TB. National Institute of Health and Care Excellence, UK; 2011. Available at: http://www.nice.org.uk/guidance/cg117/ chapter/guidance. Mohammed A, Gisela B, Fadila B, David D, Lanfranco F, Knut F, et al. Technical guide: sputum examination for tuberculosis by direct microscopy in low income countries, 5th ed., Paris, France: International Union against Tuberculosis and Lung Disease; 2000. Hain Lifescience. GenoType MTBDRplus version 1. 0 product manual. Nehren, Germany: Hain Lifescience; 2009. Maynard-Smith L, Larke N, Peters JA, Lawn SD. Diagnostic accuracy of the Xpert MTB/RIF assay for extrapulmonary and pulmonary tuberculosis when testing non-respiratory samples: a systematic review. BMC Infect Dis 2014;14:709. Vadwai V, Boehme C, Nabeta P, Shetty A, Alland D, Rodrigues C. Xpert MTB/RIF: a new pillar in diagnosis of extrapulmonary tuberculosis. J Clin Microbiol 2011;49:2540–5. Pigrau-Serrallach C, Rodriguez-Pardo D. Bone and joint tuberculosis. Eur Spine J 2013;22:556–66. World Health Organization. Checklist of prerequisites to country implementation of Xpert MTB/RIF and key action points at country level. WHO/HTM/TB/ 2011. 12. Geneva, Switzerland: WHO; 2011. Steingart KR, Schiller I, Horne DJ, Pai M, Boehme CC, Dendukuri N. Xpert1 MTB/ RIF assay for pulmonary tuberculosis and rifampicin resistance in adults. Cochrane Database Syst Rev 2014;1:CD009593. Nahid P, Kim PS, Evans CA, Alland D, Barer M, Diefenbach J, et al. Clinical research and development of tuberculosis diagnostics: moving from silos to synergy. J Infect Dis 2012;205(Suppl 2):S159–68.