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Differences in drug resistance profiles of Mycobacterium tuberculosis isolates causing pulmonary and extrapulmonary tuberculosis in a medical centre in Taiwan, 2000–2010
International Journal of Antimicrobial Agents 38 (2011) 125–129
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Differences in drug resistance profiles of Mycobacterium tuberculosis isolates causing pulmonary and extrapulmonary tuberculosis in a medical centre in Taiwan, 2000–2010 Chih-Cheng Lai a , Wei-Lun Liu a , Che-Kim Tan b , Yu-Chuang Huang c,d , Kuei-Pin Chung c,d , Meng-Rui Lee d , Po-Ren Hsueh c,∗ a
Department of Intensive Care Medicine, Chi-Mei Medical Center, Liouying, Tainan, Taiwan Department of Intensive Care Medicine, Chi-Mei Medical Center, Tainan, Taiwan c Department of Laboratory Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan d Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan b
a r t i c l e
i n f o
Article history: Received 23 March 2011 Accepted 24 March 2011 Keywords: Drug resistance rate Extrapulmonary tuberculosis Mycobacterium tuberculosis Taiwan
a b s t r a c t Few studies have investigated the drug resistance profiles of Mycobacterium tuberculosis (MTB) isolates recovered from different sites of infection. A total of 4521 non-duplicate MTB isolates, including 3723 (82.3%) from respiratory specimens and 798 (17.7%) from non-respiratory sources, were recovered from patients treated at a medical centre in Taiwan from 2000 to 2010. Trend analysis showed a significant decrease (P < 0.05) in the rates of resistance to isoniazid, rifampicin and ethambutol, a decrease in resistance to any one of four agents, namely isoniazid, rifampicin, ethambutol or streptomycin, and a decrease in resistance to both isoniazid and rifampicin (multidrug resistance) amongst pulmonary MTB isolates. A similar decrease in resistance to isoniazid and ethambutol was noted amongst non-pulmonary isolates. Rates of drug resistance were significantly higher amongst MTB isolates recovered from respiratory specimens than amongst those from non-respiratory specimens to 0.2 g/mL isoniazid (15.3% vs. 9.4%; P < 0.0001), 1 g/mL rifampicin (5.5% vs. 3.3%; P = 0.0108), 5 g/mL ethambutol (7.3% vs. 3.8%; P = 0.0004), and both isoniazid and rifampicin (4.8% vs. 2.5%; P = 0.0051). Resistance rates amongst isolates causing tuberculous lymphadenitis were significantly lower than amongst those causing genitourinary tuberculosis (TB) to isoniazid (3.5% vs. 19.4%, P = 0.0012) and to isoniazid, rifampicin, ethambutol or streptomycin (9.6% vs. 22.6%, P = 0.0003). In conclusion, the rates of resistance to first-line anti-TB agents and to multiple agents differed amongst MTB isolates obtained from different infectious sources. Continuous monitoring of resistance of MTB isolates from various sites is necessary in order to establish an effective TB surveillance programme. © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Tuberculosis (TB) remains the deadliest contagious infectious disease and continues to be a major public health burden worldwide. Globally, there were an estimated 9.4 million incident cases of TB in 2008 [1]. Although there were an estimated 140 new cases per 100 000 population in 2008, the incidence rate is declining in most parts of the world [1]. A recent epidemiological study [2] showed that despite the decrease in the number of cases of pulmonary and extrapulmonary TB in the USA, the portion of extrapulmonary TB
∗ Corresponding author. Present address: Departments of Laboratory Medicine and Internal Medicine, National Taiwan University Hospital, No. 7 Chung-Shan S. Road, Taipei 100, Taiwan. E-mail address: [email protected] (P.-R. Hsueh).
has increased as a proportion of total TB cases from 3963 (7.6%) of 52 255 cases in 1962 [3] to 3940 (15.7%) of 25 107 cases in 1993 [4], and to 2889 (21.0%) of 13 779 cases in 2006 [5]. In countries with comprehensive diagnostic and reporting systems, extrapulmonary TB accounts for 15–25% of reported cases [6]. Therefore, it is essential to better understand extrapulmonary TB in order to achieve the worldwide goal of TB elimination. Current anti-TB therapies are fraught with problems because of the increasing occurrence of drug resistance. Drug-resistant Mycobacterium tuberculosis (MTB) strains were detected shortly after the introduction of anti-TB drugs in the last century and now pose a critical threat to global TB control [7]. However, there is considerable variation in the incidence of anti-TB drug resistance worldwide [8]. In addition to geographical variation, two studies [2,9] reported that multidrug-resistant (MDR) TB occurs less often in cases of extrapulmonary TB than in cases of pulmonary TB. In
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order to optimise standard anti-TB drug therapy and to increase the success rate of control programmes, it is important to understand the drug resistance patterns in each region and each clinical category of TB, including pulmonary and extrapulmonary TB. Therefore, in this study we investigated the prevalence of drug resistance of MTB isolates in Taiwan as well as the difference in patterns of drug resistance amongst MTB isolates at various sites of TB infection. 2. Materials and methods 2.1. Setting and bacterial isolates This study was conducted at the National Taiwan University Hospital (NTUH), a 2500-bed tertiary care centre in Taipei, northern Taiwan. Isolates obtained from patients who had a positive culture for MTB at NTUH during the period January 2000 through December 2010 were included in this retrospective analysis. Non-duplicate isolates were defined as a single isolate collected for evaluation from a single patient who visited the hospital. If a patient had multiple isolates, only the first isolate was analysed. All specimens were processed and pre-treated as described elsewhere [10]. A fluorometric BACTEC technique (BACTECTM MGITTM 960 System; Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) was used for routine culture. 2.2. Drug susceptibility testing Testing of susceptibility to first-line anti-TB drugs, including isoniazid (0.2 g/mL and 1.0 g/mL), rifampicin (1 g/mL), ethambutol (5 g/mL and 10 g/mL) and streptomycin (2 g/mL and 10 g/mL) was performed in the Mycobacteriology Laboratory at NTUH. Testing of susceptibility to second-line anti-TB drugs, including rifabutin (0.5 g/mL), ofloxacin (1 g/mL), ethionamide (5 g/mL) and para-aminosalicylic acid (2 g/mL), began in NTUH on 1 January 2005. Drug susceptibility testing for all of the abovementioned anti-TB drugs was performed using the agar proportion method [10]. A MTB suspension was inoculated onto Middlebrook 7H10 agar (BBL Microbiology Systems, Cockeysville, MD) containing anti-TB drugs at their respective concentrations. The number of colony-forming units (CFU) growing on the drug-containing medium was compared with the CFU count on drug-free medium. Isolates for which growth on the drug-containing medium presented 1% of the number of colonies that developed on the drug-free medium were considered to be resistant to that agent. For quality control, the standard sensitive strain (M. tuberculosis H37Rv) and the resistant strain (Vertulo) were also tested for drug susceptibility using the same procedures. Drug resistance was defined as resistance to isoniazid (0.2 g/mL), rifampicin (1 g/mL), ethambutol (5 g/mL) or streptomycin (2 g/mL); any drug resistance (ADR) was defined as resistance to any one of the four agents. A MDR isolate was defined as being resistant to at least isoniazid (0.2 g/mL) and rifampicin (1 g/mL). 2.3. Statistical analysis Differences in drug susceptibility between pulmonary and extrapulmonary MTB isolates were analysed by 2 test. Drug resistance trends over time were evaluated by the Cochran–Armitage trend test. A P-value of <0.05 was considered statistically significant. 3. Results A total of 4521 non-duplicate isolates from 4521 patients were collected during the 11-year study period. These isolates were
Fig. 1. Trends in rates of resistance to (A) isoniazid, ethambutol, rifampicin and streptomycin and (B) any of the abovementioned four agents (resistant to any drug) as well as to multidrug resistance [resistant to at least isoniazid (0.2 g/mL) and rifampicin (1 g/mL)] amongst Mycobacterium tuberculosis isolates recovered from patients treated at National Taiwan University Hospital (Taipei, Taiwan) from 2000–2010, determined using the modified proportional method. P-values <0.05 were considered statistically significant.
recovered from various clinical specimens, including 3723 (82.3%) from respiratory specimens (sputum, bronchial washing and lung biopsy) and 798 (17.7%) from extrapulmonary specimens. Amongst the 798 extrapulmonary specimens, 317 (39.7%) were from pleural effusion specimens, 142 (17.8%) from skin, soft tissue or skeletal samples, 115 (14.4%) from lymph node specimens, 73 (9.1%) from ascitic fluid, 62 (7.8%) from genitourinary specimens, 36 (4.5%) from cerebral spinal fluid, 18 (2.3%) from pericardial fluid, 17 (2.1%) from blood, 11 (1.4%) from gastrointestinal tract samples and 7 (0.9%) from hepatobiliary tract samples. Trend analysis showed a significant decrease in the rates of resistance to isoniazid, ethambutol and rifampicin as well as a decrease in acquired resistance to any one of four agents, namely isoniazid, ethambutol, rifampicin or streptomycin, during the 11-year study period (Fig. 1; Table 1). In addition, the prevalence of MDR-TB also significantly declined. A similar decreasing trend in drug resistance was noted amongst MTB isolates causing pulmonary TB. With regard to extrapulmonary TB, the analysis showed a decreasing trend in resistance to isoniazid and ethambutol. Drug resistance patterns of the 4521 non-duplicate MTB isolates are summarised in Table 2. MTB isolates from respiratory specimens had a higher rate of resistance to isoniazid, ethambutol and rifampicin than isolates from non-respiratory specimens. In addition, MDR MTB isolates and isolates that were resistant
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Table 1 Trends in rates of resistance to isoniazid, ethambutol, rifampicin and streptomycin and to any one of these four drugs as well as multidrug resistance amongst Mycobacterium tuberculosis isolates recovered from patients with pulmonary and extrapulmonary tuberculosis treated at National Taiwan University Hospital (Taipei, Taiwan), 2000–2010. Agent
Pulmonary isolates (n) Isoniazid (0.2 g/mL) Ethambutol (5 g/mL) Rifampicin (1 g/mL) Streptomycin (2 g/mL) ADRb MDRc Extrapulmonary isolates (n) Isoniazid (0.2 g/mL) Ethambutol (5 g/mL) Rifampicin (1 g/mL) Streptomycin (2 g/mL) ADRb MDRc
% of isolates resistant to agent at indicated concentration
P-value of trend analysisa
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
106 18.9 29.2 3.8 6.6 35.8 2.8 27 22.2 18.5 3.7 14.8 40.7 3.7
170 18.2 12.4 7.1 12.9 25.9 6.5 18 16.7 5.6 5.6 5.6 16.7 5.6
221 14.5 18.6 5.0 11.3 33.9 4.1 52 9.6 1.9 0.0 3.8 11.5 0.0
368 20.4 9.0 9.8 13.3 25.3 9.8 80 11.3 5.0 6.3 13.8 17.5 6.3
567 14.5 8.1 7.1 10.2 21.3 6.7 87 11.5 6.9 6.9 10.3 18.4 4.6
467 16.3 8.8 7.5 11.6 23.8 5.8 107 11.2 3.7 4.7 3.7 15.0 1.9
420 12.6 2.6 3.8 9.0 18.6 2.6 94 6.4 1.1 2.1 6.4 11.7 1.1
380 20.8 5.3 6.6 12.1 27.6 5.3 71 7.0 2.8 2.8 2.8 8.5 2.8
362 10.8 3.9 3.9 8.3 16.9 3.3 82 6.1 1.2 2.4 11.0 14.6 2.4
336 13.4 2.7 1.2 12.5 18.8 1.2 86 2.3 0.0 0.0 8.1 8.1 0.0
326 12.0 1.5 2.8 7.1 15.3 2.8 94 13.8 6.4 2.1 13.8 23.4 2.1
0.005 <0.0001 <0.0001 0.17 <0.0001 <0.0001 0.043 0.022 0.067 0.628 0.172 0.116
ADR, resistant to any drug; MDR, multidrug-resistant. a P-values <0.05 marked in bold. b Resistance to any one of the four agents, namely isoniazid (0.2 g/mL), rifampicin (1 g/mL), ethambutol (5 g/mL) or streptomycin (2 g/mL). c Resistant to at least isoniazid (0.2 g/mL) and rifampicin (1 g/mL).
Table 2 Rates of resistance to anti-tuberculosis agents amongst Mycobacterium tuberculosis isolates recovered from patients with various sites of infections in a medical centre in Taiwan, 2000–2010. Agent
% of isolates resistant to agent at indicated concentration
All
First-line agent (no. of isolates tested) Isoniazid (0.2 g/mL) Isoniazid (1 g/mL) Ethambutol (5 g/mL) Ethambutol (10 g/mL) Rifampicin (1 g/mL) Streptomycin (2 g/mL) Streptomycin (10 g/mL) ADRb MDRc Second-line agent (no. of isolates tested) Ofloxacin (1 g/mL) PAS (2 g/mL) Ethionamide (5 g/mL) Rifabutin (0.5 g/mL)
Pulmonary
P-value (pulmonary vs. extrapulmonary)a
Extrapulmonary Pleural
Skin and soft tissue
Lymph nodes
Peritoneum
Genitourinary
All sites
142
115
73
62
798
3.5 2.6 1.7 0.0 1.7 4.4 2.6 9.6 0.9 80
5.5 2.8 1.4 0.0 1.4 9.6 5.5 12.3 1.4 49
19.4 9.7 4.8 0.0 4.8 9.7 1.6 22.6 4.8 39
9.4 5.6 3.8 0.8 3.3 8.4 4.0 15.4 2.5 506
<0.0001 0.0011 0.0004 0.2140 0.0108 0.0747 0.0028 <0.0001 0.0051
0.0 1.3 1.3 1.3
0.0 0.0 0.0 2.0
5.1 0.0 0.0 2.6
1.0 0.8 0.6 1.2
0.5866 0.1599 0.1117 0.0819
4521
3723
317
14.3 8. 7 6.7 1.3 5.1 10.2 6.1 21.3 4.4 2693
15.3 9.3 7.3 1.4 5.5 10.6 6.5 22.5 4.8 2187
10.1 6.6 4.1 1.3 3.5 9.5 5.4 15.1 2.8 195
1.3 1. 6 1. 5 2.3
1.4 1.8 1.7 2.6
1.0 1.0 0.5 0.5
9.9 4.9 2.8 0.0 1.4 7.0 2.8 14.1 1.4 87 0.0 0.00 0.00 0.00
ADR, resistant to any drug; MDR, multidrug-resistant; PAS, para-aminosalicylic acid. a P-values <0.05 marked in bold. b Resistance to any one of the four agents, namely isoniazid (0.2 g/mL), rifampicin (1 g/mL), ethambutol (5 g/mL) or streptomycin (2 g/mL). c Resistant to at least isoniazid (0.2 g/mL) and rifampicin (1 g/mL).
to at least one of the first-line anti-TB agents were more commonly found in respiratory isolates than in non-respiratory isolates. However, there were no significant differences in resistance to second-line anti-TB agents between MTB isolates from respiratory specimens and those isolated from extrapulmonary specimens. A comparison of drug resistance profiles of MTB isolates from various extrapulmonary sites revealed that isoniazid-resistant MTB was significantly more common in pleural fluid and genitourinary specimens than in peripheral lymph node specimens (10.1% and 19.4% vs. 3.5%, P = 0.046 and 0.0012), and that ADR MTB was significantly more common in genitourinary specimens than in peripheral lymph node specimens (22.6% vs. 9.6%, P = 0.0003).
4. Discussion Despite recent progress in global control efforts, TB remains one of the most serious challenges to public health, and Taiwan is no exception. For example, in 2007 the estimated prevalence was 111 per 100 000 population and in 2008 there were 14 265 new cases (62.0 per 100 000 population) of TB infection [11]. In addition, ca. 20% of all incident cases of TB were extrapulmonary [12]. Although drug resistance rates are regarded as one of the most important aspects of surveillance in the national TB control programme in Taiwan, few studies have compared the drug resistance profiles of MTB isolates that
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cause pulmonary TB with those that cause extrapulmonary TB [2,9]. In this study it was found that MDR-TB occurs more commonly in cases of pulmonary TB than in cases of extrapulmonary TB, a finding consistent with that reported by Peto et al. [2]. Similarly, rates of resistance to isoniazid, ethambutol or rifampicin as well as the rates of acquired resistance to any one of a number of anti-TB drugs were higher amongst isolates from pulmonary specimens than amongst isolates from extrapulmonary sites. That finding implies that extrapulmonary TB is negatively associated with anti-TB drug resistance in Taiwan, as it is in the USA and Russia [2,9]. One possible explanation is that most patients with extrapulmonary TB in this study received treatment after culture confirmation. In contrast, most patients with pulmonary TB received treatment based on clinical and radiological findings but before culture confirmation. Therefore, drug resistance may be acquired more commonly in cases of pulmonary TB than in cases of extrapulmonary TB. It is also possible that different strains of MTB with different drug resistance patterns may cause infection at different sites; however, studies using molecular typing methods would be needed to evaluate this hypothesis. In this study, the drug resistance profiles of MTB isolates in specimens from various infectious sites in patients with extrapulmonary TB were also investigated. Isoniazid-resistant MTB was more common in TB pleurisy and genitourinary TB than in peripheral TB lymphadenitis, and ADR MTB was more common in genitourinary TB than in peripheral TB lymphadenitis. However, the case number in each category of extrapulmonary TB was limited, therefore additional studies with larger case numbers are needed to elucidate the differences between each type of TB. In the USA, drug-resistant TB was detected in 14.2% of patients with TB in 1991 [13] and in 10% of TB patients in 1997 [14]. A surveillance study of 25 217 MTB isolates in the UK during the period 1993–1999 revealed that 6.1% (n = 1523) were resistant to one or more drugs, 5.6% (n = 1397) were resistant to isoniazid with or without resistance to other drugs, and 1.2% (n = 299) were MDR. Although the numbers of drug-resistant isolates increased over the study period, the proportions remained little changed [15]. In Germany, resistance rates rose slightly from 2001 to 2005 and then fell again somewhat in 2006 and 2007, perhaps because of a decline in immigration [16]. A similar decrease in the rate of resistance has been reported in Hong Kong and Saudi Arabia [17,18]. In this study of 4521 MTB isolated during the period 2000–2010 at a single medical centre in northern Taiwan, there was a significant decreasing trend in the rates of resistance to isoniazid, ethambutol and rifampicin, a decrease in the rate of acquired resistance to isoniazid, ethambutol, rifampicin or streptomycin, and a significant decrease in the rate of resistance to multiple agents. In fact, the drug susceptibility pattern has been shown to vary throughout Taiwan [19]. The reason for the variability may be due to the use of different testing methods between studies [20–27] or to differences in local epidemiology, or both. Therefore, clinicians need to be knowledgeable of the local epidemiology of drug-resistant TB, and laboratories should maintain up-to-date drug susceptibility data on local isolates of MTB. The drug resistance rate of MTB isolates causing extrapulmonary TB was also found to remain stable during the study period, with the exception of resistance to ethambutol and isoniazid, but the rate of drug resistance amongst isolates causing pulmonary TB declined. The difference in drug resistance may be explained by the limited number of cases of extrapulmonary TB in this study. None the less, these findings support the importance of continuous surveillance of MTB resistance at different infectious sites. This retrospective and laboratory-based surveillance study has two major limitations. First, we were unable to distinguish
precisely between newly diagnosed and previously treated cases and therefore were only able to report the combined resistance rate. Second, this study was conducted in a tertiary care centre and, as such, its findings might not reflect the overall situation in Taiwan. In conclusion, these findings indicate that the rates of single drug-resistant and MDR MTB isolates were significantly lower amongst non-respiratory specimens than amongst respiratory specimens. Continuous monitoring of antimicrobial resistance amongst MTB isolates from various sites of infections is necessary. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] World Health Organization. Global tuberculosis control: a short update to the 2009 report. http://whqlibdoc.who.int/publications/2009/ 9789241598866 eng.pdf [accessed 11 April 2011]. [2] Peto HM, Pratt RH, Harrington TA, LoBue PA, Armstrong LR. Epidemiology of extrapulmonary tuberculosis in the United States, 1993–2006. Clin Infect Dis 2009;49:1350–7. [3] Communicable Disease Center. Reported tuberculosis data, 1962. Atlanta, GA: US Department of Health, Education, and Welfare; 1962. No. 638. [4] US Centers for Disease Control and Prevention (CDC). Reported tuberculosis in the United States, 1993. Atlanta, GA: US Department of Health and Human Services; 1994. [5] US Centers for Disease Control and Prevention (CDC). Reported tuberculosis in the United States, 2006. Atlanta, GA: US Department of Health and Human Services; 2007. [6] TB Epidemiology and Surveillance Workshop. World Health Organization (WHO); 2006. http://www.who.int/tb/surveillanceworkshop/trend analysis/increasing decreasing over under diagnosis of extrapulmonary tb. htm [accessed 6 October 2006]. [7] Yew WW, Leung CC. Management of multidrug-resistant tuberculosis: update 2007. Respirology 2008;13:21–46. [8] World Health Organization. Global tuberculosis control—epidemiology, strategy, financing. WHO Report 2009. Geneva, Switzerland: WHO; 2009. WHO/HTM/TB/2009.411. [9] Vishnevski˘ı BI, Steklova LN. The rate and pattern of drug resistance in Mycobacterium tuberculosis at various sites of the disease. Probl Tuberk Bolezn Legk 2008;12:5–8 [in Russian]. [10] Lai CC, Tan CK, Huang YT, Chou CH, Hung CC, Yang PC, et al. Extensively-drug resistant Mycobacterium tuberculosis during a trend of declining drug resistance between 2000 and 2006 at a medical center in Taiwan. Clin Infect Dis 2008;47:e57–63. [11] Centers for Disease Control, Department of Health, R.O.C. (Taiwan). Taiwan Tuberculosis Control Report 2009. http://www.cdc.gov.tw/public/ Data/9123117221971.pdf [accessed 15 April 2011]. [12] Chen TM, Lee PY, Perng RP. Extrapulmonary tuberculosis: experience at Veterans General Hospital–Taipei, 1985 to 1987. J Formos Med Assoc 1991;90:1163–70. [13] Blocnited AB, Cauthen GM, Onorato IM, Dansbury KG, Kelly GD, Driver CR, et al. Nationwide survey of drug-resistant tuberculosis in the United States. JAMA 1994;271:665–71. [14] Espinal MA, Lazlo A, Simonsen L, Boulahbal F, Kim SJ, Reniero A, et al. Global trends in resistance to antituberculosis drugs: World Health Organization–International Union against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance. N Engl J Med 2001;344:1294–303. [15] Djuretic J, Herbert J, Drobniewski F, Yates M, Smith EG, Magee JG, et al. Antibiotic resistant tuberculosis in the United Kingdom: 1993–1999. Thorax 2002;57:477–82. [16] Robert Koch-Institut. Bericht zur Epidemiologie der Tuberkulose in Deutschland für 2007. Berlin, Germany: Robert Koch-Institut; 2009. http://www.rki.de [accessed 11 April 2011]. [17] Kam KM, Yip CW. Surveillance of Mycobacterium tuberculosis drug resistance in Hong Kong, 1986–1999, after the implementation of directly observed treatment. Int J Tuberc Lung Dis 2001;5:815–23. [18] Al-Tawfiq JA, Al-Muraikhy AA, Abed MS. Susceptibility pattern and epidemiology of Mycobacterium tuberculosis in a Saudi Arabian hospital: a 15-year study from 1989 to 2003. Chest 2005;128:3229–32. [19] Hsueh PR, Liu YC, So J, Liu CY, Yang PC, Luh KT. Mycobacterium tuberculosis in Taiwan. J Infect 2006;52:77–85. [20] Yu MC, Suo J, Chiang CY, Bai KJ, Lin TP, Luh KT. Initial drug resistance of Mycobacterium tuberculosis in Taiwan. J Formos Med Assoc 1997;96:890–4. [21] Chiang IH, Yu MC, Bai KJ, Wu MP, Hsu CJ, Lin TP, et al. Drug resistance patterns of tuberculosis in Taiwan. J Formos Med Assoc 1998;97:581–3. [22] Wang PD, Lin RS. Drug-resistant tuberculosis in Taipei, 1996–1999. Am J Infect Control 2001;29:41–7.
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Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Differences in drug resistance profiles of Mycobacterium tuberculosis isolates causing pulmonary and extrapulmonary tuberculosis in a medical centre in Taiwan, 2000–2010 Chih-Cheng Lai a , Wei-Lun Liu a , Che-Kim Tan b , Yu-Chuang Huang c,d , Kuei-Pin Chung c,d , Meng-Rui Lee d , Po-Ren Hsueh c,∗ a
Department of Intensive Care Medicine, Chi-Mei Medical Center, Liouying, Tainan, Taiwan Department of Intensive Care Medicine, Chi-Mei Medical Center, Tainan, Taiwan c Department of Laboratory Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan d Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan b
a r t i c l e
i n f o
Article history: Received 23 March 2011 Accepted 24 March 2011 Keywords: Drug resistance rate Extrapulmonary tuberculosis Mycobacterium tuberculosis Taiwan
a b s t r a c t Few studies have investigated the drug resistance profiles of Mycobacterium tuberculosis (MTB) isolates recovered from different sites of infection. A total of 4521 non-duplicate MTB isolates, including 3723 (82.3%) from respiratory specimens and 798 (17.7%) from non-respiratory sources, were recovered from patients treated at a medical centre in Taiwan from 2000 to 2010. Trend analysis showed a significant decrease (P < 0.05) in the rates of resistance to isoniazid, rifampicin and ethambutol, a decrease in resistance to any one of four agents, namely isoniazid, rifampicin, ethambutol or streptomycin, and a decrease in resistance to both isoniazid and rifampicin (multidrug resistance) amongst pulmonary MTB isolates. A similar decrease in resistance to isoniazid and ethambutol was noted amongst non-pulmonary isolates. Rates of drug resistance were significantly higher amongst MTB isolates recovered from respiratory specimens than amongst those from non-respiratory specimens to 0.2 g/mL isoniazid (15.3% vs. 9.4%; P < 0.0001), 1 g/mL rifampicin (5.5% vs. 3.3%; P = 0.0108), 5 g/mL ethambutol (7.3% vs. 3.8%; P = 0.0004), and both isoniazid and rifampicin (4.8% vs. 2.5%; P = 0.0051). Resistance rates amongst isolates causing tuberculous lymphadenitis were significantly lower than amongst those causing genitourinary tuberculosis (TB) to isoniazid (3.5% vs. 19.4%, P = 0.0012) and to isoniazid, rifampicin, ethambutol or streptomycin (9.6% vs. 22.6%, P = 0.0003). In conclusion, the rates of resistance to first-line anti-TB agents and to multiple agents differed amongst MTB isolates obtained from different infectious sources. Continuous monitoring of resistance of MTB isolates from various sites is necessary in order to establish an effective TB surveillance programme. © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Tuberculosis (TB) remains the deadliest contagious infectious disease and continues to be a major public health burden worldwide. Globally, there were an estimated 9.4 million incident cases of TB in 2008 [1]. Although there were an estimated 140 new cases per 100 000 population in 2008, the incidence rate is declining in most parts of the world [1]. A recent epidemiological study [2] showed that despite the decrease in the number of cases of pulmonary and extrapulmonary TB in the USA, the portion of extrapulmonary TB
∗ Corresponding author. Present address: Departments of Laboratory Medicine and Internal Medicine, National Taiwan University Hospital, No. 7 Chung-Shan S. Road, Taipei 100, Taiwan. E-mail address: [email protected] (P.-R. Hsueh).
has increased as a proportion of total TB cases from 3963 (7.6%) of 52 255 cases in 1962 [3] to 3940 (15.7%) of 25 107 cases in 1993 [4], and to 2889 (21.0%) of 13 779 cases in 2006 [5]. In countries with comprehensive diagnostic and reporting systems, extrapulmonary TB accounts for 15–25% of reported cases [6]. Therefore, it is essential to better understand extrapulmonary TB in order to achieve the worldwide goal of TB elimination. Current anti-TB therapies are fraught with problems because of the increasing occurrence of drug resistance. Drug-resistant Mycobacterium tuberculosis (MTB) strains were detected shortly after the introduction of anti-TB drugs in the last century and now pose a critical threat to global TB control [7]. However, there is considerable variation in the incidence of anti-TB drug resistance worldwide [8]. In addition to geographical variation, two studies [2,9] reported that multidrug-resistant (MDR) TB occurs less often in cases of extrapulmonary TB than in cases of pulmonary TB. In
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order to optimise standard anti-TB drug therapy and to increase the success rate of control programmes, it is important to understand the drug resistance patterns in each region and each clinical category of TB, including pulmonary and extrapulmonary TB. Therefore, in this study we investigated the prevalence of drug resistance of MTB isolates in Taiwan as well as the difference in patterns of drug resistance amongst MTB isolates at various sites of TB infection. 2. Materials and methods 2.1. Setting and bacterial isolates This study was conducted at the National Taiwan University Hospital (NTUH), a 2500-bed tertiary care centre in Taipei, northern Taiwan. Isolates obtained from patients who had a positive culture for MTB at NTUH during the period January 2000 through December 2010 were included in this retrospective analysis. Non-duplicate isolates were defined as a single isolate collected for evaluation from a single patient who visited the hospital. If a patient had multiple isolates, only the first isolate was analysed. All specimens were processed and pre-treated as described elsewhere [10]. A fluorometric BACTEC technique (BACTECTM MGITTM 960 System; Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) was used for routine culture. 2.2. Drug susceptibility testing Testing of susceptibility to first-line anti-TB drugs, including isoniazid (0.2 g/mL and 1.0 g/mL), rifampicin (1 g/mL), ethambutol (5 g/mL and 10 g/mL) and streptomycin (2 g/mL and 10 g/mL) was performed in the Mycobacteriology Laboratory at NTUH. Testing of susceptibility to second-line anti-TB drugs, including rifabutin (0.5 g/mL), ofloxacin (1 g/mL), ethionamide (5 g/mL) and para-aminosalicylic acid (2 g/mL), began in NTUH on 1 January 2005. Drug susceptibility testing for all of the abovementioned anti-TB drugs was performed using the agar proportion method [10]. A MTB suspension was inoculated onto Middlebrook 7H10 agar (BBL Microbiology Systems, Cockeysville, MD) containing anti-TB drugs at their respective concentrations. The number of colony-forming units (CFU) growing on the drug-containing medium was compared with the CFU count on drug-free medium. Isolates for which growth on the drug-containing medium presented 1% of the number of colonies that developed on the drug-free medium were considered to be resistant to that agent. For quality control, the standard sensitive strain (M. tuberculosis H37Rv) and the resistant strain (Vertulo) were also tested for drug susceptibility using the same procedures. Drug resistance was defined as resistance to isoniazid (0.2 g/mL), rifampicin (1 g/mL), ethambutol (5 g/mL) or streptomycin (2 g/mL); any drug resistance (ADR) was defined as resistance to any one of the four agents. A MDR isolate was defined as being resistant to at least isoniazid (0.2 g/mL) and rifampicin (1 g/mL). 2.3. Statistical analysis Differences in drug susceptibility between pulmonary and extrapulmonary MTB isolates were analysed by 2 test. Drug resistance trends over time were evaluated by the Cochran–Armitage trend test. A P-value of <0.05 was considered statistically significant. 3. Results A total of 4521 non-duplicate isolates from 4521 patients were collected during the 11-year study period. These isolates were
Fig. 1. Trends in rates of resistance to (A) isoniazid, ethambutol, rifampicin and streptomycin and (B) any of the abovementioned four agents (resistant to any drug) as well as to multidrug resistance [resistant to at least isoniazid (0.2 g/mL) and rifampicin (1 g/mL)] amongst Mycobacterium tuberculosis isolates recovered from patients treated at National Taiwan University Hospital (Taipei, Taiwan) from 2000–2010, determined using the modified proportional method. P-values <0.05 were considered statistically significant.
recovered from various clinical specimens, including 3723 (82.3%) from respiratory specimens (sputum, bronchial washing and lung biopsy) and 798 (17.7%) from extrapulmonary specimens. Amongst the 798 extrapulmonary specimens, 317 (39.7%) were from pleural effusion specimens, 142 (17.8%) from skin, soft tissue or skeletal samples, 115 (14.4%) from lymph node specimens, 73 (9.1%) from ascitic fluid, 62 (7.8%) from genitourinary specimens, 36 (4.5%) from cerebral spinal fluid, 18 (2.3%) from pericardial fluid, 17 (2.1%) from blood, 11 (1.4%) from gastrointestinal tract samples and 7 (0.9%) from hepatobiliary tract samples. Trend analysis showed a significant decrease in the rates of resistance to isoniazid, ethambutol and rifampicin as well as a decrease in acquired resistance to any one of four agents, namely isoniazid, ethambutol, rifampicin or streptomycin, during the 11-year study period (Fig. 1; Table 1). In addition, the prevalence of MDR-TB also significantly declined. A similar decreasing trend in drug resistance was noted amongst MTB isolates causing pulmonary TB. With regard to extrapulmonary TB, the analysis showed a decreasing trend in resistance to isoniazid and ethambutol. Drug resistance patterns of the 4521 non-duplicate MTB isolates are summarised in Table 2. MTB isolates from respiratory specimens had a higher rate of resistance to isoniazid, ethambutol and rifampicin than isolates from non-respiratory specimens. In addition, MDR MTB isolates and isolates that were resistant
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Table 1 Trends in rates of resistance to isoniazid, ethambutol, rifampicin and streptomycin and to any one of these four drugs as well as multidrug resistance amongst Mycobacterium tuberculosis isolates recovered from patients with pulmonary and extrapulmonary tuberculosis treated at National Taiwan University Hospital (Taipei, Taiwan), 2000–2010. Agent
Pulmonary isolates (n) Isoniazid (0.2 g/mL) Ethambutol (5 g/mL) Rifampicin (1 g/mL) Streptomycin (2 g/mL) ADRb MDRc Extrapulmonary isolates (n) Isoniazid (0.2 g/mL) Ethambutol (5 g/mL) Rifampicin (1 g/mL) Streptomycin (2 g/mL) ADRb MDRc
% of isolates resistant to agent at indicated concentration
P-value of trend analysisa
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
106 18.9 29.2 3.8 6.6 35.8 2.8 27 22.2 18.5 3.7 14.8 40.7 3.7
170 18.2 12.4 7.1 12.9 25.9 6.5 18 16.7 5.6 5.6 5.6 16.7 5.6
221 14.5 18.6 5.0 11.3 33.9 4.1 52 9.6 1.9 0.0 3.8 11.5 0.0
368 20.4 9.0 9.8 13.3 25.3 9.8 80 11.3 5.0 6.3 13.8 17.5 6.3
567 14.5 8.1 7.1 10.2 21.3 6.7 87 11.5 6.9 6.9 10.3 18.4 4.6
467 16.3 8.8 7.5 11.6 23.8 5.8 107 11.2 3.7 4.7 3.7 15.0 1.9
420 12.6 2.6 3.8 9.0 18.6 2.6 94 6.4 1.1 2.1 6.4 11.7 1.1
380 20.8 5.3 6.6 12.1 27.6 5.3 71 7.0 2.8 2.8 2.8 8.5 2.8
362 10.8 3.9 3.9 8.3 16.9 3.3 82 6.1 1.2 2.4 11.0 14.6 2.4
336 13.4 2.7 1.2 12.5 18.8 1.2 86 2.3 0.0 0.0 8.1 8.1 0.0
326 12.0 1.5 2.8 7.1 15.3 2.8 94 13.8 6.4 2.1 13.8 23.4 2.1
0.005 <0.0001 <0.0001 0.17 <0.0001 <0.0001 0.043 0.022 0.067 0.628 0.172 0.116
ADR, resistant to any drug; MDR, multidrug-resistant. a P-values <0.05 marked in bold. b Resistance to any one of the four agents, namely isoniazid (0.2 g/mL), rifampicin (1 g/mL), ethambutol (5 g/mL) or streptomycin (2 g/mL). c Resistant to at least isoniazid (0.2 g/mL) and rifampicin (1 g/mL).
Table 2 Rates of resistance to anti-tuberculosis agents amongst Mycobacterium tuberculosis isolates recovered from patients with various sites of infections in a medical centre in Taiwan, 2000–2010. Agent
% of isolates resistant to agent at indicated concentration
All
First-line agent (no. of isolates tested) Isoniazid (0.2 g/mL) Isoniazid (1 g/mL) Ethambutol (5 g/mL) Ethambutol (10 g/mL) Rifampicin (1 g/mL) Streptomycin (2 g/mL) Streptomycin (10 g/mL) ADRb MDRc Second-line agent (no. of isolates tested) Ofloxacin (1 g/mL) PAS (2 g/mL) Ethionamide (5 g/mL) Rifabutin (0.5 g/mL)
Pulmonary
P-value (pulmonary vs. extrapulmonary)a
Extrapulmonary Pleural
Skin and soft tissue
Lymph nodes
Peritoneum
Genitourinary
All sites
142
115
73
62
798
3.5 2.6 1.7 0.0 1.7 4.4 2.6 9.6 0.9 80
5.5 2.8 1.4 0.0 1.4 9.6 5.5 12.3 1.4 49
19.4 9.7 4.8 0.0 4.8 9.7 1.6 22.6 4.8 39
9.4 5.6 3.8 0.8 3.3 8.4 4.0 15.4 2.5 506
<0.0001 0.0011 0.0004 0.2140 0.0108 0.0747 0.0028 <0.0001 0.0051
0.0 1.3 1.3 1.3
0.0 0.0 0.0 2.0
5.1 0.0 0.0 2.6
1.0 0.8 0.6 1.2
0.5866 0.1599 0.1117 0.0819
4521
3723
317
14.3 8. 7 6.7 1.3 5.1 10.2 6.1 21.3 4.4 2693
15.3 9.3 7.3 1.4 5.5 10.6 6.5 22.5 4.8 2187
10.1 6.6 4.1 1.3 3.5 9.5 5.4 15.1 2.8 195
1.3 1. 6 1. 5 2.3
1.4 1.8 1.7 2.6
1.0 1.0 0.5 0.5
9.9 4.9 2.8 0.0 1.4 7.0 2.8 14.1 1.4 87 0.0 0.00 0.00 0.00
ADR, resistant to any drug; MDR, multidrug-resistant; PAS, para-aminosalicylic acid. a P-values <0.05 marked in bold. b Resistance to any one of the four agents, namely isoniazid (0.2 g/mL), rifampicin (1 g/mL), ethambutol (5 g/mL) or streptomycin (2 g/mL). c Resistant to at least isoniazid (0.2 g/mL) and rifampicin (1 g/mL).
to at least one of the first-line anti-TB agents were more commonly found in respiratory isolates than in non-respiratory isolates. However, there were no significant differences in resistance to second-line anti-TB agents between MTB isolates from respiratory specimens and those isolated from extrapulmonary specimens. A comparison of drug resistance profiles of MTB isolates from various extrapulmonary sites revealed that isoniazid-resistant MTB was significantly more common in pleural fluid and genitourinary specimens than in peripheral lymph node specimens (10.1% and 19.4% vs. 3.5%, P = 0.046 and 0.0012), and that ADR MTB was significantly more common in genitourinary specimens than in peripheral lymph node specimens (22.6% vs. 9.6%, P = 0.0003).
4. Discussion Despite recent progress in global control efforts, TB remains one of the most serious challenges to public health, and Taiwan is no exception. For example, in 2007 the estimated prevalence was 111 per 100 000 population and in 2008 there were 14 265 new cases (62.0 per 100 000 population) of TB infection [11]. In addition, ca. 20% of all incident cases of TB were extrapulmonary [12]. Although drug resistance rates are regarded as one of the most important aspects of surveillance in the national TB control programme in Taiwan, few studies have compared the drug resistance profiles of MTB isolates that
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cause pulmonary TB with those that cause extrapulmonary TB [2,9]. In this study it was found that MDR-TB occurs more commonly in cases of pulmonary TB than in cases of extrapulmonary TB, a finding consistent with that reported by Peto et al. [2]. Similarly, rates of resistance to isoniazid, ethambutol or rifampicin as well as the rates of acquired resistance to any one of a number of anti-TB drugs were higher amongst isolates from pulmonary specimens than amongst isolates from extrapulmonary sites. That finding implies that extrapulmonary TB is negatively associated with anti-TB drug resistance in Taiwan, as it is in the USA and Russia [2,9]. One possible explanation is that most patients with extrapulmonary TB in this study received treatment after culture confirmation. In contrast, most patients with pulmonary TB received treatment based on clinical and radiological findings but before culture confirmation. Therefore, drug resistance may be acquired more commonly in cases of pulmonary TB than in cases of extrapulmonary TB. It is also possible that different strains of MTB with different drug resistance patterns may cause infection at different sites; however, studies using molecular typing methods would be needed to evaluate this hypothesis. In this study, the drug resistance profiles of MTB isolates in specimens from various infectious sites in patients with extrapulmonary TB were also investigated. Isoniazid-resistant MTB was more common in TB pleurisy and genitourinary TB than in peripheral TB lymphadenitis, and ADR MTB was more common in genitourinary TB than in peripheral TB lymphadenitis. However, the case number in each category of extrapulmonary TB was limited, therefore additional studies with larger case numbers are needed to elucidate the differences between each type of TB. In the USA, drug-resistant TB was detected in 14.2% of patients with TB in 1991 [13] and in 10% of TB patients in 1997 [14]. A surveillance study of 25 217 MTB isolates in the UK during the period 1993–1999 revealed that 6.1% (n = 1523) were resistant to one or more drugs, 5.6% (n = 1397) were resistant to isoniazid with or without resistance to other drugs, and 1.2% (n = 299) were MDR. Although the numbers of drug-resistant isolates increased over the study period, the proportions remained little changed [15]. In Germany, resistance rates rose slightly from 2001 to 2005 and then fell again somewhat in 2006 and 2007, perhaps because of a decline in immigration [16]. A similar decrease in the rate of resistance has been reported in Hong Kong and Saudi Arabia [17,18]. In this study of 4521 MTB isolated during the period 2000–2010 at a single medical centre in northern Taiwan, there was a significant decreasing trend in the rates of resistance to isoniazid, ethambutol and rifampicin, a decrease in the rate of acquired resistance to isoniazid, ethambutol, rifampicin or streptomycin, and a significant decrease in the rate of resistance to multiple agents. In fact, the drug susceptibility pattern has been shown to vary throughout Taiwan [19]. The reason for the variability may be due to the use of different testing methods between studies [20–27] or to differences in local epidemiology, or both. Therefore, clinicians need to be knowledgeable of the local epidemiology of drug-resistant TB, and laboratories should maintain up-to-date drug susceptibility data on local isolates of MTB. The drug resistance rate of MTB isolates causing extrapulmonary TB was also found to remain stable during the study period, with the exception of resistance to ethambutol and isoniazid, but the rate of drug resistance amongst isolates causing pulmonary TB declined. The difference in drug resistance may be explained by the limited number of cases of extrapulmonary TB in this study. None the less, these findings support the importance of continuous surveillance of MTB resistance at different infectious sites. This retrospective and laboratory-based surveillance study has two major limitations. First, we were unable to distinguish
precisely between newly diagnosed and previously treated cases and therefore were only able to report the combined resistance rate. Second, this study was conducted in a tertiary care centre and, as such, its findings might not reflect the overall situation in Taiwan. In conclusion, these findings indicate that the rates of single drug-resistant and MDR MTB isolates were significantly lower amongst non-respiratory specimens than amongst respiratory specimens. Continuous monitoring of antimicrobial resistance amongst MTB isolates from various sites of infections is necessary. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] World Health Organization. Global tuberculosis control: a short update to the 2009 report. http://whqlibdoc.who.int/publications/2009/ 9789241598866 eng.pdf [accessed 11 April 2011]. [2] Peto HM, Pratt RH, Harrington TA, LoBue PA, Armstrong LR. Epidemiology of extrapulmonary tuberculosis in the United States, 1993–2006. Clin Infect Dis 2009;49:1350–7. [3] Communicable Disease Center. Reported tuberculosis data, 1962. Atlanta, GA: US Department of Health, Education, and Welfare; 1962. No. 638. [4] US Centers for Disease Control and Prevention (CDC). Reported tuberculosis in the United States, 1993. Atlanta, GA: US Department of Health and Human Services; 1994. [5] US Centers for Disease Control and Prevention (CDC). Reported tuberculosis in the United States, 2006. Atlanta, GA: US Department of Health and Human Services; 2007. [6] TB Epidemiology and Surveillance Workshop. World Health Organization (WHO); 2006. http://www.who.int/tb/surveillanceworkshop/trend analysis/increasing decreasing over under diagnosis of extrapulmonary tb. htm [accessed 6 October 2006]. [7] Yew WW, Leung CC. Management of multidrug-resistant tuberculosis: update 2007. Respirology 2008;13:21–46. [8] World Health Organization. Global tuberculosis control—epidemiology, strategy, financing. WHO Report 2009. Geneva, Switzerland: WHO; 2009. WHO/HTM/TB/2009.411. [9] Vishnevski˘ı BI, Steklova LN. The rate and pattern of drug resistance in Mycobacterium tuberculosis at various sites of the disease. Probl Tuberk Bolezn Legk 2008;12:5–8 [in Russian]. [10] Lai CC, Tan CK, Huang YT, Chou CH, Hung CC, Yang PC, et al. Extensively-drug resistant Mycobacterium tuberculosis during a trend of declining drug resistance between 2000 and 2006 at a medical center in Taiwan. Clin Infect Dis 2008;47:e57–63. [11] Centers for Disease Control, Department of Health, R.O.C. (Taiwan). Taiwan Tuberculosis Control Report 2009. http://www.cdc.gov.tw/public/ Data/9123117221971.pdf [accessed 15 April 2011]. [12] Chen TM, Lee PY, Perng RP. Extrapulmonary tuberculosis: experience at Veterans General Hospital–Taipei, 1985 to 1987. J Formos Med Assoc 1991;90:1163–70. [13] Blocnited AB, Cauthen GM, Onorato IM, Dansbury KG, Kelly GD, Driver CR, et al. Nationwide survey of drug-resistant tuberculosis in the United States. JAMA 1994;271:665–71. [14] Espinal MA, Lazlo A, Simonsen L, Boulahbal F, Kim SJ, Reniero A, et al. Global trends in resistance to antituberculosis drugs: World Health Organization–International Union against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance. N Engl J Med 2001;344:1294–303. [15] Djuretic J, Herbert J, Drobniewski F, Yates M, Smith EG, Magee JG, et al. Antibiotic resistant tuberculosis in the United Kingdom: 1993–1999. Thorax 2002;57:477–82. [16] Robert Koch-Institut. Bericht zur Epidemiologie der Tuberkulose in Deutschland für 2007. Berlin, Germany: Robert Koch-Institut; 2009. http://www.rki.de [accessed 11 April 2011]. [17] Kam KM, Yip CW. Surveillance of Mycobacterium tuberculosis drug resistance in Hong Kong, 1986–1999, after the implementation of directly observed treatment. Int J Tuberc Lung Dis 2001;5:815–23. [18] Al-Tawfiq JA, Al-Muraikhy AA, Abed MS. Susceptibility pattern and epidemiology of Mycobacterium tuberculosis in a Saudi Arabian hospital: a 15-year study from 1989 to 2003. Chest 2005;128:3229–32. [19] Hsueh PR, Liu YC, So J, Liu CY, Yang PC, Luh KT. Mycobacterium tuberculosis in Taiwan. J Infect 2006;52:77–85. [20] Yu MC, Suo J, Chiang CY, Bai KJ, Lin TP, Luh KT. Initial drug resistance of Mycobacterium tuberculosis in Taiwan. J Formos Med Assoc 1997;96:890–4. [21] Chiang IH, Yu MC, Bai KJ, Wu MP, Hsu CJ, Lin TP, et al. Drug resistance patterns of tuberculosis in Taiwan. J Formos Med Assoc 1998;97:581–3. [22] Wang PD, Lin RS. Drug-resistant tuberculosis in Taipei, 1996–1999. Am J Infect Control 2001;29:41–7.
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