INT J TUBERC LUNG DIS 19(2):157–162 Q 2015 The Union http://dx.doi.org/10.5588/ijtld.13.0905
Nationwide survey of anti-tuberculosis drug resistance in Japan Tuberculosis Research Committee (RYOKEN), Tokyo, Japan SUMMARY OBJECTIVE:
To determine the prevalence of antituberculosis drug resistance in Japan. D E S I G N : A nationwide drug resistance survey was conducted by convenience sampling based on a voluntary hospital consortium. Mycobacterium tuberculosis isolates (n ¼ 2292) were collected from August 2007 to July 2008. Drug susceptibilities were analysed according to the patients’ treatment history, age, sex, comorbidities and residential area. We determined susceptibility to the following drugs: isoniazid (INH), rifampicin (RMP), streptomycin (SM), ethambutol (EMB) and levofloxacin (LVX). R E S U L T S : The frequencies of drug-resistant isolates from new cases were as follows: INH, 3.1%; RMP, 0.7%; SM, 5.6%; EMB, 1.3%; and 8.5% to any drug. The frequencies of drug-resistant isolates from previ-
ously treated patients were as follows: INH, 12.3%; RMP, 6.7%; SM, 12.3%; and EMB, 2.6%. The frequencies of LVX-resistant isolates from new and previously treated patients were respectively 3.2% and 6.1% (n ¼ 852). The frequencies of multidrug-resistant isolates from new and previously treated patients were respectively 0.4% and 4.1%, with only one extensively drug-resistant case. C O N C L U S I O N : In general, the prevalence of drugresistant TB in Japan during this period was low. However, LVX resistance among new patients was relatively high. Japan should establish a nationwide surveillance system. K E Y W O R D S : tuberculosis; MDR-TB; drug susceptibility testing
TUBERCULOSIS (TB) is one of the main infectious diseases in Japan. The notification rate of TB all forms in 2010 was 18.2 per 100 000 population. TB prevalence in Japan has decreased continuously since the 1960s, except for a few years during the late 1990s. More than 60% of patients are aged .60 years, and these cases are likely the result of reactivation of latent tuberculous infection (LTBI) acquired decades ago. In contrast, although the prevalence is relatively low in the elderly population, there are over 4000 patients aged ,40 years, implying recent transmission.1 TB prevalence in Japan thus differs between these age groups. The prevalence of drug-resistant TB among TB cases in Asian countries is not low in general due to the extensive and possibly inappropriate use of antituberculosis drugs. In the Philippines, multidrugresistant TB strains (MDR-TB, defined as resistance to at least isoniazid [INH] and rifampicin [RMP]) were isolated from respectively 4.0% and 20.9% of new and treated cases in 2004; these frequencies were similar in Shanghai, China (3.9% and 12.4%) in 2004 and Viet Nam (2.7% and 19.3%) in 2006.2 The Ryoken Consortium has conducted nationwide drug resistance surveys 13 times since 1957. The
last survey, conducted in 2002, reported a significant reduction in drug-resistant isolates among previously treated cases from 1997 and revealed a 30.9% prevalence of extensively drug-resistant TB (XDRTB) among patients with MDR-TB.3 The Consortium conducted its most recent nationwide survey in 2007. In the present study, we analysed data from the latest survey to determine the prevalence of drug-resistant isolates of Mycobacterium tuberculosis among TB patients.
MATERIALS AND METHODS Sampling strategy and eligible subjects From August 2007 to July 2008, we acquired samples from patients with all types of TB, regardless of smear results, from the 67 voluntary participating hospitals of the Ryoken Consortium located throughout Japan. The hospitals provide tertiary TB care. All patients with M. tuberculosis-positive cultures and confirmed TB were eligible. Enrolment of patients We enrolled all newly diagnosed TB patients with positive cultures. Patients who were being treated for
Correspondence to: Satoshi Mitarai, Department of Mycobacterium Reference and Research, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama, Kiyose, Tokyo 204-8533, Japan. Tel: (þ81) 42 493 5762. Fax: (þ81) 42 492 4600. e-mail: [email protected] Article submitted 13 December 2013. Final version accepted 26 September 2014.
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TB at the time of survey were declared ineligible. A standardised questionnaire was used to acquire the treatment histories of patients, and a patient was classified as a new or previously treated case according to World Health Organization (WHO) criteria. Briefly, a new patient was defined as one who had never received anti-tuberculosis drug(s) for .1 month, while a previously treated patient had previously received anti-tuberculosis treatment for 71 month.4 We also acquired information regarding patient demographics (age, sex, area of residence, nationality), site/type of TB, and bacteriological/ radiological findings. The clinical information was provided directly by each patient; laboratory and radiological data were collected from patient records after obtaining informed consent. Laboratory methods One culture isolate from each enrolled patient was sent to the Research Institute of Tuberculosis (RIT), Tokyo, Japan, where it was subcultured and confirmed to be M. tuberculosis using the Capiliae TBNeo test (Tauns Laboratories, Inc, Numazu, Japan). If this test was negative, the species was identified by directly sequencing the bacterium’s 16S rRNA gene according to a published method.5 Confirmed M. tuberculosis isolates were subjected to drug susceptibility testing (DST) using the proportion method with 1% Ogawa medium. Resistance was expressed as the percentage of colonies growing in the presence of the following critical concentrations of the drugs: INH, 0.2 lg/ml; RMP, 40 lg/ml; streptomycin (SM), 10 lg/ml; ethambutol (EMB), 2.5 lg/ml; and levofloxacin (LVX), 1.0 lg/ml. The critical concentrations employed in 1% Ogawa medium were equivalent to those in Lowenstein-Jensen (LJ) medium. For MDR¨ TB isolates, further DST was performed using kanamycin (KM), capreomycin (CPM), amikacin (AMK), ethionamide (ETH), para aminosalicylic acid (PAS), cycloserine (CS) and pyrazinamide (PZA). The LJ proportion method was used for all drugs except PZA, according to the WHO standard.6 DST of PZA was performed using a MGIT BACTECe MGITe 960 PZA Kit (BD, Sparks, MD, USA). Due to limited capacity, 40% of the isolates were randomly selected for testing LVX resistance. Genotyping of MDR-TB isolates was performed by determining the variable number of tandem repeats (VNTR) of 15 loci using a published method.7 The VNTR profiles of MDR-TB isolates were analysed using the MIRU-VNTRplus application (http://www. miru-vntrplus.org). Statistical analysis Data were entered into a Microsoft Excel (Microsoft, Redmond, WA, USA) spreadsheet and then transferred to a JMP 10.0.1 (Statistical Analysis System, Cary, NC, USA) file. Analyses were conducted to
determine the prevalence of drug resistance among new or previously treated cases and their sum. Statistical analysis was performed using the v2 test for the comparison of proportions; P , 0.05 was considered statistically significant. We compared the subjects’ distribution according to sex, age and region (the six geographical regions of Japan) with those of all newly notified cases in 2007. These factors were adjusted by weighed analysis, if necessary, to calculate drug resistance rates. Fifteen patients were excluded from the analyses, as their ages were unavailable. Ethical considerations The Ryoken General Assembly approved the survey protocol, including ethics issues. The survey followed the Ethical Guidelines for Epidemiological Research of the Ministry of Education, Culture, Sports, Science and Technology; Ministry of Health, Labour, and Welfare of Japan. Clinical information on patients was collected after obtaining informed consent. All patients diagnosed with TB, with or without drugresistant strains, received appropriate treatment.
RESULTS Bacterial isolates and patient characteristics We registered 2292 TB patients: 1573 were males and 719 females; the mean age was 64.8 6 19.7 years (range 1–100). The age and sex distributions were similar to those of recorded TB cases across Japan in 2007.8 There were 2097 new and 195 previously treated cases. Of these, 1627 were smear-positive and 665 smear-negative. Patients were diagnosed with pulmonary TB (n ¼ 2012, 87.8%), extra-pulmonary TB (n ¼ 51, 2.2%) or both (n ¼ 229, 10.0%); 1292 patients had comorbidities; the most frequent of these were diabetes mellitus (DM) (n ¼ 387, 30.0%), malignant tumour (current and past, n ¼ 104, 8.0%) and steroid use (n ¼ 73, 5.7%). Six (0.26%) patients were human immunodeficiency virus (HIV) infected. Drug resistance We subjected 2292 M. tuberculosis isolates to DST. The prevalence of resistance to the major four drugs is summarised in Table 1. The frequency of isolates with resistance to any of the drugs was 9.6% (95% confidence interval [CI] 8.4–10.8), and the isolates represented respectively 8.5% (95%CI 7.4–9.8) and 20.5% (95%CI 15.4–26.7) of new and previously treated patients. We tested isolates for LVX resistance and found that 3.4% (95%CI 2.4–4.8) were resistant; this represented respectively 3.2% (95%CI 2.2–4.7) and 6.1% (95%CI 2.1–7.9) of patients without and with prior treatment. Of the LVX-resistant isolates from new patients, 88.0% (22/25) were monoresistant.
Anti-tuberculosis drug resistance survey
Table 1
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Summary of the prevalence of drug resistance in Japan by treatment history New
Previously treated
Combined
Characteristics
n
% (95%CI)
n
% (95%CI)
n
% (95%CI)
Total All susceptible
2097 1918
100 91.5 (90.3–92.7)
195 155
100 79.5 (73.3–84.6)
2292 2073
100 90.4 (89.2–91.6)
Any resistance INH RMP SM EMB LVX (n ¼ 852)† Monoresistance INH RMP SM EMB Multidrug resistance INH þ RMP INH þ RMP þ EMB INH þ RMP þ SM INH þ RMP þ EMB þ SM Polyresistance INH þ EMB INH þ SM EMB þ SM INH þ EMB þ SM RMP þ EMB RMP þ SM RMP þ EMB þ SM
179 64 15 118 27 25 147 36 5 95 11
8.5 3.1 0.7 5.6 1.3 3.2 7.0 1.7 0.2 4.5 0.5
(7.4–9.8) (2.4–3.9) (0.4–1.2) (4.7–6.7) (0.9–1.9) (2.2–4.7) (6.0–8.2) (1.2–2.3) (0.03–0.4) (3.6–5.4) (0.2–0.8)
40 24 13 24 5 4 21 7 3 11 0
20.5 12.3 6.7 12.3 2.6 6.1 10.8 3.6 1.5 5.6
9 2 3 1 3 23 3 13 3 3 1 0 0
0.4 0.1 0.1 0.05 0.1 1.1 0.1 0.6 0.1 0.1 0.05
(0.2–0.8) (0.03–0.4) (0.05–0.4) (0.001–0.3) (0.03–0.4) (0.7–1.6) (0.05–0.4) (0.4–1.1) (0.05–0.4) (0.05–0.4) (0.001–0.3) 0 0
8 3 1 2 2 11 2 7 0 0 0 2 0
4.1 1.5 0.5 1.0 1.0 5.6 1.0 3.6
(15.4–26.7) (8.4–17.7) (3.9–11.1) (8.4–17.7) (1.1–5.9) (2.4–14.6) (7.2–15.9) (1.0–6.2) (0.5–4.4) (2.4–8.9) 0
(1.8–7.9) (0.5–4.4) (0.09–2.8) (0.3–3.7) (0.3–3.7) (3.2–9.8) (0.3–3.7) (1.8–7.2) 0 0 0 1.0 (0.3–3.7) 0
219 88 28 142 32 29 168 43 8 106 11
9.6 3.8 1.2 6.2 1.4 3.4 7.3 1.9 0.3 4.6 0.5
(8.4–10.8) (3.1–4.7) (0.8–1.8) (5.3–7.3) (1.0–2.0) (2.4–4.8) (6.3–8.5) (1.3–2.4) (0.1–0.6) (3.8–5.5) (0.2–7.6)
17 5 4 3 5 34 5 20 3 3 1 2 0
0.7 0.2 0.2 0.1 0.2 1.5 0.2 0.9 0.1 0.1 0.04 0.1
(0.5–1.2) (0.09–0.5) (0.07–4.5) (0.05–0.4) (0.09–0.5) (1.1–2.1) (0.09–0.5) (0.6–1.3) (0.05–0.4) (0.05–0.4) (0.01–0.2) (0.02–0.3) 0
* LVX resistance among 852 randomly selected isolates. CI ¼ confidence interval; INH ¼ isoniazid; RMP ¼ rifampicin; SM ¼ streptomycin; EMB ¼ ethambutol; LVX ¼ levofloxacin.
MDR-TB isolates from 15 Japanese and 2 foreign nationals accounted for 0.7% (95%CI 0.5–1.2) of the total and represented respectively 0.4% (95%CI 0.2– 0.8) and 4.1% (95%CI 2.1–7.9) of patients without and with prior treatment. The resistance pattern of MDR-TB isolates to other drugs was as follows: 18.8% (95%CI 6.6–43.0) to KM, 12.5% (95%CI 3.5–36.0) to CPM, 18.8% (95%CI 6.6–43.0) to AMK, 31.3% (95%CI 14.2–55.6) to ETH, 37.5% (95%CI 18.5–61.4) to PAS, 12.5% (95%CI 3.5– 36.0) to CS and 50.0% (95%CI 28.0–72.0) to PZA. There was one (5.9%) XDR-TB isolate.9 VNTR analysis of the MDR-TB isolates detected 12 isolates (70.6%) of the Beijing lineage; the other lineages represented were East-African Indian (EAI) (n ¼ 2, 11.8%), NEW-1 (n ¼ 1, 5.9%), Haarlem (n ¼ 1, 5.9%) and S lineages (n ¼ 1, 5.9%). Table 2 shows the correlation between age and resistance as a function of prior treatment. There was no significant difference between INH and RMP resistance among isolates from new patients. Although differences were observed among INH- and RMP-resistant isolates from previously treated patients, none were statistically significant, probably due to the small number of specimens. With increasing age, there was a decreasing trend in the proportion of new cases with resistance to INH or SM (P ¼ 0.045 and P ¼ 0.008, respectively). There
were no significant sex-specific differences (data not shown), even among patients with MDR-TB. The data were categorised according to types of main comorbidities (Table 3). In previously treated DM patients, we observed significantly higher RMP resistance (P ¼ 0.038) than in those who did not have comorbidities. There were no significant differences between isolates from the other two patient groups compared with those from controls without comorbidities. Of the foreign nationals, 51 were new and 2 previously treated. Among the isolates from new patients, the frequencies of resistance to INH, RMP, SM and EMB were respectively 11.8% (95%CI 5.5– 23.4), 2.0% (95%CI 0.3–10.3), 5.9% (95%CI 2.0– 15.9) and 2.0% (95%CI 0.3–10.3). INH resistance was significantly higher in foreign nationals compared with Japanese nationals (P ¼ 0.004). The prevalence of resistance was generally similar among isolates from patients residing in the six geographical districts, with the exception of a significant difference in INH resistance between isolates from new patients from Kinki (Middle-southern) and Kyusyu (Southern) (P ¼ 0.002). Table 4 shows the adjusted proportion of drug resistance based on TB statistics for 2007 in Japan. There was no significant difference between raw and adjusted rates of prevalence. Table 5 shows changes in drug resistance from the Ryoken studies for 1997, 2002 and 2007. Ryoken
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Table 2
Association of age with drug resistance by treatment history
Age years
n
New cases (n ¼ 2082)* 619 18 20–29 131 30–39 192 40–49 166 50–59 242 60–69 288 70–79 459 780 586 P value, Cochran-Armitage test for trend Previously treated cases (n ¼ 195) 619 0 20–29 8 30–39 8 40–49 8 50–59 26 60–69 27 70–79 52 780 66 P value, Cochran-Armitage test for trend
INH (% (95%CI)
6.1 3.6 4.2 2.9 2.8 3.3 2.0
12.5 12.5 25.0 23.1 11.1 15.4 4.5
0 (2.0–10.3) (1.0–6.3) (1.1–7.3) (0.8–5.0) (0.9–4.7) (1.6–4.9) (0.9–3.2) 0.045
RMP % (95%CI)
0.8 0.5 1.2 1.7 0.7 0.7 0.3
0 (0.1–4.2) (0.1–2.9) (0.3–4.3) (0.04–3.3) (0.2–2.5) (0.2–1.9) (0.1–1.2) 0.351
SM % (95%CI)
6.9 8.3 8.4 6.2 5.9 5.9 3.2
0 (2.5–11.3) (4.4–12.3) (4.2–12.7) (3.1–9.3) (3.2–8.6) (3.7–8.0) (1.8–4.7)‡ 0.008
EMB % (95%CI)
4.6 1.0 1.2 0.8 1.0 1.5 0.7
0 (1.0–8.2)† (0.3–3.7) (0.3–4.3) (0.2–3.0) (0.4–3.0) (0.4–2.7) (0.3–1.7) 0.051
0 0 0 0 (2.2–47.1) 12.5 (2.2–47.1) 25.0 (7.1–59.1) 0 (2.2–47.1) 0 12.5 (2.2–47.1) 0 (7.1–59.1) 0 25.0 (7.1–59.1) 0 (11.0–42.1) 7.7 (2.1–24.1) 7.7 (2.1–24.1) 11.5 (4.0–29.0)† (3.9–28.1) 11.1 (3.9–28.1) 25.9 (13.1–44.7) 3.7 (0.7–18.3) (8.0–27.5) 5.8 (2.0–15.6) 9.6 (4.2–20.7) 1.9 (0.3–10.1) (1.6–12.5) 6.1 (2.4–14.6) 7.6 (3.3–16.5) 0 0.074 0.899 0.098 0.250
Any resistance % (95%CI)
11.5 13.0 12.1 8.7 8.7 9.2 5.0
0 (7.1–18.0) (9.0–18.5) (7.9–17.9) (5.7–12.9) (5.9–12.5) (6.8–12.1) (3.5–7.0) 0.001
25.0 12.5 25.0 26.9 37.0 21.2 10.6
0 (7.1–59.1) (2.2–47.1) (7.1–59.1) (13.7–46.1) (21.5–55.8) (12.2–34.0) (5.2–20.3) 0.117
* 15 patients, ages unknown. † Significantly higher than average (P , 0.05). ‡ Significantly lower than average (P , 0.05). INH ¼ isoniazid; CI ¼ confidence interval; RMP ¼ rifampicin; SM ¼ streptomycin; EMB ¼ ethambutol.
surveys employed the absolute concentration method before 1997. Direct comparison using the proportion method showed a clear reduction in drug resistance in isolates from previously treated patients (P , 0.001). Among new patients, SM resistance showed a decreasing trend, whereas there was a significant increase in EMB resistance over time (P ¼ 0.004). MDR-TB prevalence was significantly lower (P ¼ 0.015) than that reported by the 2002 Ryoken study: 0.7% (95%CI 0.4–1.1) and 9.8% (95%CI 7.3–13.1) in new and previously treated patients, respectively.3
DISCUSSION A nationwide survey of anti-tuberculosis drug resistance was conducted from 2007 to 2008 in Japan, and it is important to note its main limitations. As the Table 3
study sites were selected for convenience rather than random sampling, the national representativeness of the survey cannot be assured. However, as the distribution of patients by age, sex and region was similar to that of patients in the national TB register, it is likely that the study results provide a valid estimation of drug resistance patterns in the national TB patient population. In addition, the original target sample size of 2828 was based on the prevalence of drug resistance observed in 2002; however, sample size in the present study was 2292 (81.0%). The number of patients studied represents approximately 8.9% of all TB cases registered in Japan in 2007.8 The authors recognise that the participating hospitals and the number of patients do not guarantee an optimal representation of the standard of TB care in Japan. However, the survey included all of Japan. Among
Prevalence of drug resistance in patients with and those without comorbidities
Comorbidity
n
INH % (95%CI)
RMP % (95%CI)
Patients without comorbidity (n ¼ 1000; male, 645; female, 355) New 921 3.0 (1.9–4.2) 0.9 (0.3–1.5) Previously treated 79 12.7 (5.2–20.2) 6.3 (0.8–11.8) Diabetes mellitus (n ¼ 387: male, 301; female, 86) New 350 3.1 (1.8–5.5) 0.9 (0.3–2.5) Previously treated 37 21.6 (11.4–37.2) 18.9 (9.5–34.2)*
SM % (95%CI)
EMB % (95%CI)
MDR-TB % (95%CI)
Any resistance % (95%CI)
4.9 (3.5–6.3) 16.5 (8.1–24.8)
1.6 (0.8–2.4) 2.5 (0.7–8.8)
0.4 (0.0–0.9) 3.8 (1.3–10.6)
8.0 (6.3–9.8) 24.1 (14.4–33.7)
6.3 (4.2–9.3) 13.5 (5.9–28.0)
1.1 (0.4–2.9) 8.1 (2.8–21.3)
0.3 (0.1–1.6) 13.5 (5.9–28.0)
10.0 (7.3–13.6) 29.7 (17.5–45.8)
Malignant tumour (n ¼ 104: male, 80; female, 24) New 91 5.5 (2.4–12.2) Previously treated 13 0
0 0
5.5 (2.4–12.2) 0
1.1 (0.2–6.0) 0
0 0
9.9 (5.3–17.7) 0
Steroid use (n ¼ 73: male, 40; female, 33) New 67 3.0 (0.8–10.2) Previously treated 6 16.7 (3.0–56.4)
0 0
3.0 (0.8–10.2) 33.3 (9.7–70.0)
0 0
0 0
6.0 (2.3–14.4) 33.3 (9.7–70.0)
* Significant difference compared with patients without comorbidities (P ¼ 0.038). INH ¼ isoniazid; CI ¼ confidence interval; RMP ¼ rifampicin; SM ¼ streptomycin; EMB ¼ ethambutol; MDR-TB ¼ multidrug-resistant tuberculosis.
Anti-tuberculosis drug resistance survey
Table 4 Prevalence of anti-tuberculosis-drug resistance, raw and adjusted data for cases by age group, sex and district* Drug INH Raw Adjusted RMP Raw Adjusted SM Raw Adjusted EMB Raw Adjusted
New % (95%CI)
Previously treated % (95%CI)
3.1 (2.3–3.8) 3.2 (1.1–5.3)
12.3 (7.7–17.0) 11.3 (8.0–15.5)
0.7 (0.4–1.1) 0.7 (0.2–1.2)
6.7 (3.1–10.2) 5.3 (3.2–8.5)
5.6 (4.6–6.6) 5.6 (3.4–7.8)
12.3 (7.7–17.0) 11.8 (8.5–16.1)
1.3 (0.8–1.8) 1.3 (0.6–1.9)
2.6 (0.3–4.8) 2.1 (1.0–4.6)
* Raw data were adjusted on the basis of the prevalence of bacteriologically confirmed tuberculosis cases in six different districts in Japan (2007 official statistics of the Japanese Ministry of Health, Labour and Welfare). CI ¼ confidence interval; INH ¼ isoniazid; RMP ¼ rifampicin; SM ¼ streptomycin; EMB ¼ ethambutol.
behavioural risk factors, only alcoholism was considered, as other factors such as drug use and incarceration are not common. This survey revealed a relatively low level of resistance to the main antituberculosis drugs in new and previously treated patients. The proportions of new and previously treated patients in this survey with resistance to INH, RMP, SM and EMB were lower than the global averages of resistance to these drugs.2 Compared with data from studies conducted in 1997 and 2002, the SM resistance of isolates from new patients and resistance to all drugs of isolates from previously treated patients show a clear declining trend.3,10 SM is not commonly used in Japan for the initial treatment of TB due to the introduction of the standard 6-month short-course regimen; we therefore conclude that the reduction in SM resistance could be natural. The use of EMB is now common, and resistant strains have likely accumulated in the population, which is reflected by the increase in resistant isolates from new patients, although the level is still low. The prevalence of INH resistance among new TB cases appears to have increased slightly from 2002 to 2007. However, this difference was not statistically significant. The reason for the Table 5
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slight increase in INH resistance among isolates from new patients is unknown; however, the use of INH to treat LTBI, which increased after the introduction of the QuantiFERONw-TB (QIAGEN, Venlo, The Netherlands) test, should be carefully monitored.11 Similar to other bacterial infections,12,13 we show here that the prevalence of LVX resistance was relatively high. It is of great interest that the majority of LVX-resistant isolates identified here were not resistant to other anti-tuberculosis drugs. Although guidelines for the treatment of infectious diseases in Japan do not recommend the use of LVX in outpatient clinics, it is widely prescribed,14 and LVX is administered to many patients with TB before the disease is diagnosed. Of the MDR-TB isolates in the present study, the Beijing lineage represented 70.6%. The proportion was consistent with that of a previous report, where the majority of the isolates were non-MDR-TB.15 Several studies have indicated that MDR-TB is caused more frequently by the Beijing lineage; however, this was not clearly apparent from our present data.16,17 The association of resistance to any drug showed a relatively decreasing trend with increasing age among new as well as previously treated patients. This was true for INH- and SM-resistant isolates from new patients, and has been observed repeatedly in past surveys conducted in Japan. Moreover, this is a general trend for other drugs, although it is not statistically significant, primarily due to the limited number of patients. The age-dependence of drug resistance has been reported for isolates from patients residing in the United States during the 1970s,18 and was recently confirmed by the WHO,19 indicating that in older patients the disease is caused by LTBI reactivation that occurs when there are very few drugresistant bacteria. Comorbidities that induce immunocompromised status may result in poor treatment outcomes and development of drug resistance.20,21 However, the very low prevalence of HIV-infected patients in Japan and the small number included here did not allow additional analyses or the evaluation of statistical significance. In the present survey, RMP resistance was significantly high and was detected among
Sequential changes in drug resistance through 1997–2007
Category
Year
New
1997 2002 2007 1997 2002 2007
Previously treated*
INH % (95%CI) 4.4 2.8 3.1 33.0 18.9 12.3
(3.4–5.6) (2.2–3.5) (2.3–3.8) (27.6–38.8) (15.2–22.7) (7.7–17.0)
RMP % (95%CI) 1.4 1.0 0.7 21.6 11.0 6.7
(0.9–2.2) (0.7–1.4) (0.4–1.1) (17.1–26.9) (8.0–14.0) (3.1–10.2)
SM % (95%CI) 7.5 7.0 5.6 24.2 14.4 12.3
(6.2–9.0)* (6.0–7.9)* (4.6–6.6)* (19.5–29.8) (11.0–17.8) (7.7–17.0)
EMB % (95%CI) 0.4 0.9 1.3 15.2 8.4 2.6
(0.2–0.8)† (0.5–1.2)† (0.8–1.8)† (11.3–20.0) (5.7–11.1) (0.3–4.8)
MDR-TB % (95%CI) 0.8 0.7 0.4 19.7 9.8 4.1
* Significant decreasing trend (Cochran-Armitage test, P , 0.05) † Significant increasing trend (Cochran-Armitage test, P ¼ 0.004). INH ¼ isoniazid; CI ¼ confidence interval; RMP ¼ rifampicin; SM ¼ streptomycin; EMB ¼ ethambutol; MDR-TB ¼ multidrug-resistant tuberculosis.
(0.4–1.4) (0.4–1.1) (0.2–0.8) (15.3–24.9) (7.3–13.1) (2.1–7.9)
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isolates from previously treated DM patients. In the 2002 Ryoken survey, drug resistance was higher in defaulters than in simple relapses.3 In the present study, previously treated DM patients were more likely to have MDR-TB than previously treated patients without comorbidities (P ¼ 0.125). Although the detailed treatment history of patients with RMP resistance in the present survey is unknown, inappropriate treatment may possibly account for higher resistance. The decreasing trend in resistance among previously treated patients during the past decade suggests improvements in anti-tuberculosis treatment. The present survey also indicates that continuous monitoring of drug resistance will be required to establish an optimal TB control programme and that a drug resistance surveillance system should be established. Acknowledgement The authors extend their sincere appreciation to all the participants of this study. This study was supported by a Health Science Research Grant from the Ministry of Health, Labour and Welfare (H18-Shinko-Ippan-012 and H21-Shinko-Ippan-016), Tokyo, Japan. Conflicts of interest: none declared
References 1 Japan Anti-Tuberculosis Association. Statistics of TB 2010. Tokyo, Japan: JATA, 2011. 2 World Health Organization. Anti-tuberculosis drug resistance in the world. Report No. 4. WHO/HTM/TB/2008.394. Geneva, Switzerland: WHO, 2008. 3 Tuberculosis Research Committee (RYOKEN). Drug-resistant Mycobacterium tuberculosis in Japan: a nationwide survey, 2002. Int J Tuberc Lung Dis 2007; 11: 1129–1135. 4 World Health Organization. Guidelines for surveillance of drug resistance in tuberculosis. 4th ed. WHO/HTM/TB/2009.422. Geneva, Switzerland: WHO, 2009. 5 Springer B, Stockman L, Teschner K, Roberts G D, Bottger E C. Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods. J Clin Microbiol 1996; 34: 296–303. 6 World Health Organization. Policy guidance on drugsusceptibility testing (DST) of second-line anti-tuberculosis drugs. WHO/HTM/TB/2008.392. Geneva, Switzerland: WHO, 2008. 7 Supply P, Allix C, Lesjean S, et al. Proposal for standardization of optimized mycobacterial interspersed repetitive unitvariable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 2006; 44: 4498–4510.
8 Japan Anti-Tuberculosis Association. Statistics of TB 2007. Tokyo, Japan: JATA, 2008. 9 World Health Organization. XDR-TB: extensively drug resistant TB. Geneva, Switzerland: WHO, 2006. http://www. who.int/tb/challenges/mdr/xdr/en/ Accessed October 2014. 10 Abe C, Hirano K, Wada M, Aoyagi T. Resistance of Mycobacterium tuberculosis to four first-line anti-tuberculosis drugs in Japan, 1997. Int J Tuberc Lung Dis 2001; 5: 46–52. 11 Ministry of Health, Labour and Welfare. Statistics of tuberculosis in Japan. Tokyo, Japan: Ministry of Health, Labour and Welfare, 2011. http://www.mhlw.go.jp/bunya/ kenkou/kekkaku-kansenshou03/11.html Accessed October 2014. 12 Yokota S, Sato T, Okubo T, et al. Prevalence of fluoroquinolone-resistant Escherichia coli O25:H4-ST131 (CTX-M-15-nonproducing) strains isolated in Japan. Chemotherapy 2012; 58: 52–59. 13 Yokota S, Ohkoshi Y, Sato K, Fujii N. Emergence of fluoroquinolone-resistant Haemophilus influenzae strains among elderly patients but not among children. J Clin Microbiol 2008; 46: 361–365. 14 Sugawara T, Ohkusu Y, Gu Y, Kawanohara H, Taniguchi K, Okabe N. Estimation of amount of antibiotics used by pharmacy surveillance. Jpn J Infect Prev Cont 2012; 27: 195– 198. [Japanese] 15 Murase Y, Mitarai S, Sugawara I, Kato S, Maeda S. Promising loci of variable numbers of tandem repeats for typing Beijing family Mycobacterium tuberculosis. J Med Microbiol 2008; 57: 873–880. 16 de Steenwinkel J E, ten Kate M T, de Knegt G J, et al. Drug susceptibility of Mycobacterium tuberculosis Beijing genotype and association with MDR-TB. Emerg Infect Dis 2012; 18: 660–663. 17 Niemann S, Diel R, Khechinashvili G, Gegia M, Mdivani N, Tang Y W. Mycobacterium tuberculosis Beijing lineage favors the spread of multidrug-resistant tuberculosis in the Republic of Georgia. J Clin Microbiol 2010; 48: 3544–3550. 18 Kopanoff D, Kilburn J O, Glassroth J L, Snider D Jr, Farer L, Good R. A continuing survey of tuberculosis primary drug resistance in the United States: March 1975 to November 1977. A United States Public Health Service cooperative study. Am Rev Respir Dis 1978; 118: 835–842. 19 World Health Organization. Multidrug and extensively drugresistant TB (M/XDR-TB): 2010 global report on surveillance and response. WHO/HTM/TB/2010.3. Geneva, Switzerland: WHO, 2010. 20 Chang J T, Dou H Y, Yen C L, et al. Effect of type 2 diabetes mellitus on the clinical severity and treatment outcome in patients with pulmonary tuberculosis: a potential role in the emergence of multidrug-resistance. J Formos Med Assoc 2011; 110: 372–381. 21 Fisher-Hoch S P, Whitney E, McCormick J B, et al. Type 2 diabetes and multidrug-resistant tuberculosis. Scand J Infect Dis 2008; 40: 888–893.
Anti-tuberculosis drug resistance survey
i
RESUME
D´eterminer la pr´evalence de la r´esistance aux m´edicaments antituberculeux au Japon. S C H E´ M A : Une enquˆete nationale de pharmacor´esistance a e´ t´e r´ealis´ee grˆace a` un e´ chantillonnage de convenance bas´e sur la participation volontaire des hopitaux. ˆ Les isolats de Mycobacterium tuberculosis (n ¼ 2292) ont e´ t´e recueillis entre aout ˆ 2007 et juillet 2008. La sensibilit´e aux m´edicaments a e´ t´e analys´ee en fonction de l’historique du traitement des patients, de leur age, ˆ sexe, des comorbidit´es et du lieu de r´esidence. Nous avons d´etermin´e la sensibilit´e aux m´edicaments suivants : isoniazide (INH), rifampicine (RMP), streptomycine (SM), e´ thambutol (EMB) et l´evofloxacine (LVX). R E´ S U LT A T S : Les fr´equences d’isolats pharmacor´esistants e´ manant de nouveaux cas e´ taient les suivantes : INH OBJECTIF :
3,1% ; RMP 0,7% ; SM, 5,6% ; EMB, 1,3% ; et enfin 8,5% a` un m´edicament au moins. Les fr´equences de r´esistance e´ manant de patients d´ej`a trait´es e´ taient les suivantes : INH 12,3% ; RMP, 6,7% ; SM, 12,3% ; et EMB, 2,6%. Les fr´equences des isolats r´esistant a` la LVX e´ manant de patients nouveaux et pr´ealablement trait´es e´ taient respectivement de 3,2% et 6,1% (n ¼ 852). Les fr´equences de r´esistance multiple des isolats de patients nouveaux et d´ej`a trait´es e´ taient respectivement de 0,4% et 4,1%, avec un seul cas ultra-r´esistant. C O N C L U S I O N : D’une mani`ere g´en´erale, la pr´evalence de TB pharmacor´esistante au Japon e´ tait faible durant cette p´eriode. Cependant, la r´esistance a` la LVX chez de nombreux patients e´ tait assez e´ lev´ee. Le Japon devrait donc cr´eer un syst`eme de surveillance national. RESUMEN
O B J E T I V O: Determinar la prevalencia de resistencia a los medicamentos antituberculosos en el Japon. ´ M E´ T O D O: Se llevo ´ a cabo una encuesta nacional sobre farmacorresistencia mediante un muestreo de conveniencia de pacientes de los hospitales de un consorcio que voluntariamente participaron en el estudio. Se recogieron aislados cl´ınicos de Mycobacterium tuberculosis entre agosto del 2007 y julio del 2008 (n ¼ 2292). La sensibilidad a los medicamentos se analiz o´ en funci on ´ de los antecedentes de tratamiento, la edad, el sexo, las enfermedades concomitantes y la zona de residencia de los pacientes. Se examino´ la sensibilidad a los siguientes medicamentos antituberculosos: isoniazida (INH), rifampicina (RMP), estreptomicina (SM), etambutol (EMB) y levofloxacino (LVX). R E S U LT A D O S: La frecuencia de farmacorresistencia en los aislados provenientes de casos nuevos fue como
sigue: a INH 3,1%; RMP 0,7%; SM 5,6% y a EMB 1,3%; se observo´ resistencia a algun ´ medicamento en el 8,5% de los casos. La frecuencia de farmacorresistencia en los aislados provenientes de casos previamente tratados fue la siguiente: a INH 12,3%; RMP 6,7%; SM 12,3% y a EMB 2,6%. La frecuencia de resistencia a LVX en los casos nuevos fue 3,2% y en los casos con antecedente de tratamiento fue 6,1% (n ¼ 852). La frecuencia de multidrogorresistencia en los casos nuevos fue 0,4% y en los casos previamente tratados fue 4,1%; solo se observo´ un caso de tuberculosis extremadamente drogorresistente. ´ N: En general, se encontr o CONCLUSIO ´ una baja prevalencia de tuberculosis farmacorresistente en el Japon ´ durante el per´ıodo estudiado. Se observo, ´ no obstante, una resistencia relativamente alta a LVX en los casos nuevos. Es importante establecer un sistema de vigilancia de la resistencia a escala nacional.
Nationwide survey of anti-tuberculosis drug resistance in Japan Tuberculosis Research Committee (RYOKEN), Tokyo, Japan SUMMARY OBJECTIVE:
To determine the prevalence of antituberculosis drug resistance in Japan. D E S I G N : A nationwide drug resistance survey was conducted by convenience sampling based on a voluntary hospital consortium. Mycobacterium tuberculosis isolates (n ¼ 2292) were collected from August 2007 to July 2008. Drug susceptibilities were analysed according to the patients’ treatment history, age, sex, comorbidities and residential area. We determined susceptibility to the following drugs: isoniazid (INH), rifampicin (RMP), streptomycin (SM), ethambutol (EMB) and levofloxacin (LVX). R E S U L T S : The frequencies of drug-resistant isolates from new cases were as follows: INH, 3.1%; RMP, 0.7%; SM, 5.6%; EMB, 1.3%; and 8.5% to any drug. The frequencies of drug-resistant isolates from previ-
ously treated patients were as follows: INH, 12.3%; RMP, 6.7%; SM, 12.3%; and EMB, 2.6%. The frequencies of LVX-resistant isolates from new and previously treated patients were respectively 3.2% and 6.1% (n ¼ 852). The frequencies of multidrug-resistant isolates from new and previously treated patients were respectively 0.4% and 4.1%, with only one extensively drug-resistant case. C O N C L U S I O N : In general, the prevalence of drugresistant TB in Japan during this period was low. However, LVX resistance among new patients was relatively high. Japan should establish a nationwide surveillance system. K E Y W O R D S : tuberculosis; MDR-TB; drug susceptibility testing
TUBERCULOSIS (TB) is one of the main infectious diseases in Japan. The notification rate of TB all forms in 2010 was 18.2 per 100 000 population. TB prevalence in Japan has decreased continuously since the 1960s, except for a few years during the late 1990s. More than 60% of patients are aged .60 years, and these cases are likely the result of reactivation of latent tuberculous infection (LTBI) acquired decades ago. In contrast, although the prevalence is relatively low in the elderly population, there are over 4000 patients aged ,40 years, implying recent transmission.1 TB prevalence in Japan thus differs between these age groups. The prevalence of drug-resistant TB among TB cases in Asian countries is not low in general due to the extensive and possibly inappropriate use of antituberculosis drugs. In the Philippines, multidrugresistant TB strains (MDR-TB, defined as resistance to at least isoniazid [INH] and rifampicin [RMP]) were isolated from respectively 4.0% and 20.9% of new and treated cases in 2004; these frequencies were similar in Shanghai, China (3.9% and 12.4%) in 2004 and Viet Nam (2.7% and 19.3%) in 2006.2 The Ryoken Consortium has conducted nationwide drug resistance surveys 13 times since 1957. The
last survey, conducted in 2002, reported a significant reduction in drug-resistant isolates among previously treated cases from 1997 and revealed a 30.9% prevalence of extensively drug-resistant TB (XDRTB) among patients with MDR-TB.3 The Consortium conducted its most recent nationwide survey in 2007. In the present study, we analysed data from the latest survey to determine the prevalence of drug-resistant isolates of Mycobacterium tuberculosis among TB patients.
MATERIALS AND METHODS Sampling strategy and eligible subjects From August 2007 to July 2008, we acquired samples from patients with all types of TB, regardless of smear results, from the 67 voluntary participating hospitals of the Ryoken Consortium located throughout Japan. The hospitals provide tertiary TB care. All patients with M. tuberculosis-positive cultures and confirmed TB were eligible. Enrolment of patients We enrolled all newly diagnosed TB patients with positive cultures. Patients who were being treated for
Correspondence to: Satoshi Mitarai, Department of Mycobacterium Reference and Research, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama, Kiyose, Tokyo 204-8533, Japan. Tel: (þ81) 42 493 5762. Fax: (þ81) 42 492 4600. e-mail: [email protected] Article submitted 13 December 2013. Final version accepted 26 September 2014.
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TB at the time of survey were declared ineligible. A standardised questionnaire was used to acquire the treatment histories of patients, and a patient was classified as a new or previously treated case according to World Health Organization (WHO) criteria. Briefly, a new patient was defined as one who had never received anti-tuberculosis drug(s) for .1 month, while a previously treated patient had previously received anti-tuberculosis treatment for 71 month.4 We also acquired information regarding patient demographics (age, sex, area of residence, nationality), site/type of TB, and bacteriological/ radiological findings. The clinical information was provided directly by each patient; laboratory and radiological data were collected from patient records after obtaining informed consent. Laboratory methods One culture isolate from each enrolled patient was sent to the Research Institute of Tuberculosis (RIT), Tokyo, Japan, where it was subcultured and confirmed to be M. tuberculosis using the Capiliae TBNeo test (Tauns Laboratories, Inc, Numazu, Japan). If this test was negative, the species was identified by directly sequencing the bacterium’s 16S rRNA gene according to a published method.5 Confirmed M. tuberculosis isolates were subjected to drug susceptibility testing (DST) using the proportion method with 1% Ogawa medium. Resistance was expressed as the percentage of colonies growing in the presence of the following critical concentrations of the drugs: INH, 0.2 lg/ml; RMP, 40 lg/ml; streptomycin (SM), 10 lg/ml; ethambutol (EMB), 2.5 lg/ml; and levofloxacin (LVX), 1.0 lg/ml. The critical concentrations employed in 1% Ogawa medium were equivalent to those in Lowenstein-Jensen (LJ) medium. For MDR¨ TB isolates, further DST was performed using kanamycin (KM), capreomycin (CPM), amikacin (AMK), ethionamide (ETH), para aminosalicylic acid (PAS), cycloserine (CS) and pyrazinamide (PZA). The LJ proportion method was used for all drugs except PZA, according to the WHO standard.6 DST of PZA was performed using a MGIT BACTECe MGITe 960 PZA Kit (BD, Sparks, MD, USA). Due to limited capacity, 40% of the isolates were randomly selected for testing LVX resistance. Genotyping of MDR-TB isolates was performed by determining the variable number of tandem repeats (VNTR) of 15 loci using a published method.7 The VNTR profiles of MDR-TB isolates were analysed using the MIRU-VNTRplus application (http://www. miru-vntrplus.org). Statistical analysis Data were entered into a Microsoft Excel (Microsoft, Redmond, WA, USA) spreadsheet and then transferred to a JMP 10.0.1 (Statistical Analysis System, Cary, NC, USA) file. Analyses were conducted to
determine the prevalence of drug resistance among new or previously treated cases and their sum. Statistical analysis was performed using the v2 test for the comparison of proportions; P , 0.05 was considered statistically significant. We compared the subjects’ distribution according to sex, age and region (the six geographical regions of Japan) with those of all newly notified cases in 2007. These factors were adjusted by weighed analysis, if necessary, to calculate drug resistance rates. Fifteen patients were excluded from the analyses, as their ages were unavailable. Ethical considerations The Ryoken General Assembly approved the survey protocol, including ethics issues. The survey followed the Ethical Guidelines for Epidemiological Research of the Ministry of Education, Culture, Sports, Science and Technology; Ministry of Health, Labour, and Welfare of Japan. Clinical information on patients was collected after obtaining informed consent. All patients diagnosed with TB, with or without drugresistant strains, received appropriate treatment.
RESULTS Bacterial isolates and patient characteristics We registered 2292 TB patients: 1573 were males and 719 females; the mean age was 64.8 6 19.7 years (range 1–100). The age and sex distributions were similar to those of recorded TB cases across Japan in 2007.8 There were 2097 new and 195 previously treated cases. Of these, 1627 were smear-positive and 665 smear-negative. Patients were diagnosed with pulmonary TB (n ¼ 2012, 87.8%), extra-pulmonary TB (n ¼ 51, 2.2%) or both (n ¼ 229, 10.0%); 1292 patients had comorbidities; the most frequent of these were diabetes mellitus (DM) (n ¼ 387, 30.0%), malignant tumour (current and past, n ¼ 104, 8.0%) and steroid use (n ¼ 73, 5.7%). Six (0.26%) patients were human immunodeficiency virus (HIV) infected. Drug resistance We subjected 2292 M. tuberculosis isolates to DST. The prevalence of resistance to the major four drugs is summarised in Table 1. The frequency of isolates with resistance to any of the drugs was 9.6% (95% confidence interval [CI] 8.4–10.8), and the isolates represented respectively 8.5% (95%CI 7.4–9.8) and 20.5% (95%CI 15.4–26.7) of new and previously treated patients. We tested isolates for LVX resistance and found that 3.4% (95%CI 2.4–4.8) were resistant; this represented respectively 3.2% (95%CI 2.2–4.7) and 6.1% (95%CI 2.1–7.9) of patients without and with prior treatment. Of the LVX-resistant isolates from new patients, 88.0% (22/25) were monoresistant.
Anti-tuberculosis drug resistance survey
Table 1
159
Summary of the prevalence of drug resistance in Japan by treatment history New
Previously treated
Combined
Characteristics
n
% (95%CI)
n
% (95%CI)
n
% (95%CI)
Total All susceptible
2097 1918
100 91.5 (90.3–92.7)
195 155
100 79.5 (73.3–84.6)
2292 2073
100 90.4 (89.2–91.6)
Any resistance INH RMP SM EMB LVX (n ¼ 852)† Monoresistance INH RMP SM EMB Multidrug resistance INH þ RMP INH þ RMP þ EMB INH þ RMP þ SM INH þ RMP þ EMB þ SM Polyresistance INH þ EMB INH þ SM EMB þ SM INH þ EMB þ SM RMP þ EMB RMP þ SM RMP þ EMB þ SM
179 64 15 118 27 25 147 36 5 95 11
8.5 3.1 0.7 5.6 1.3 3.2 7.0 1.7 0.2 4.5 0.5
(7.4–9.8) (2.4–3.9) (0.4–1.2) (4.7–6.7) (0.9–1.9) (2.2–4.7) (6.0–8.2) (1.2–2.3) (0.03–0.4) (3.6–5.4) (0.2–0.8)
40 24 13 24 5 4 21 7 3 11 0
20.5 12.3 6.7 12.3 2.6 6.1 10.8 3.6 1.5 5.6
9 2 3 1 3 23 3 13 3 3 1 0 0
0.4 0.1 0.1 0.05 0.1 1.1 0.1 0.6 0.1 0.1 0.05
(0.2–0.8) (0.03–0.4) (0.05–0.4) (0.001–0.3) (0.03–0.4) (0.7–1.6) (0.05–0.4) (0.4–1.1) (0.05–0.4) (0.05–0.4) (0.001–0.3) 0 0
8 3 1 2 2 11 2 7 0 0 0 2 0
4.1 1.5 0.5 1.0 1.0 5.6 1.0 3.6
(15.4–26.7) (8.4–17.7) (3.9–11.1) (8.4–17.7) (1.1–5.9) (2.4–14.6) (7.2–15.9) (1.0–6.2) (0.5–4.4) (2.4–8.9) 0
(1.8–7.9) (0.5–4.4) (0.09–2.8) (0.3–3.7) (0.3–3.7) (3.2–9.8) (0.3–3.7) (1.8–7.2) 0 0 0 1.0 (0.3–3.7) 0
219 88 28 142 32 29 168 43 8 106 11
9.6 3.8 1.2 6.2 1.4 3.4 7.3 1.9 0.3 4.6 0.5
(8.4–10.8) (3.1–4.7) (0.8–1.8) (5.3–7.3) (1.0–2.0) (2.4–4.8) (6.3–8.5) (1.3–2.4) (0.1–0.6) (3.8–5.5) (0.2–7.6)
17 5 4 3 5 34 5 20 3 3 1 2 0
0.7 0.2 0.2 0.1 0.2 1.5 0.2 0.9 0.1 0.1 0.04 0.1
(0.5–1.2) (0.09–0.5) (0.07–4.5) (0.05–0.4) (0.09–0.5) (1.1–2.1) (0.09–0.5) (0.6–1.3) (0.05–0.4) (0.05–0.4) (0.01–0.2) (0.02–0.3) 0
* LVX resistance among 852 randomly selected isolates. CI ¼ confidence interval; INH ¼ isoniazid; RMP ¼ rifampicin; SM ¼ streptomycin; EMB ¼ ethambutol; LVX ¼ levofloxacin.
MDR-TB isolates from 15 Japanese and 2 foreign nationals accounted for 0.7% (95%CI 0.5–1.2) of the total and represented respectively 0.4% (95%CI 0.2– 0.8) and 4.1% (95%CI 2.1–7.9) of patients without and with prior treatment. The resistance pattern of MDR-TB isolates to other drugs was as follows: 18.8% (95%CI 6.6–43.0) to KM, 12.5% (95%CI 3.5–36.0) to CPM, 18.8% (95%CI 6.6–43.0) to AMK, 31.3% (95%CI 14.2–55.6) to ETH, 37.5% (95%CI 18.5–61.4) to PAS, 12.5% (95%CI 3.5– 36.0) to CS and 50.0% (95%CI 28.0–72.0) to PZA. There was one (5.9%) XDR-TB isolate.9 VNTR analysis of the MDR-TB isolates detected 12 isolates (70.6%) of the Beijing lineage; the other lineages represented were East-African Indian (EAI) (n ¼ 2, 11.8%), NEW-1 (n ¼ 1, 5.9%), Haarlem (n ¼ 1, 5.9%) and S lineages (n ¼ 1, 5.9%). Table 2 shows the correlation between age and resistance as a function of prior treatment. There was no significant difference between INH and RMP resistance among isolates from new patients. Although differences were observed among INH- and RMP-resistant isolates from previously treated patients, none were statistically significant, probably due to the small number of specimens. With increasing age, there was a decreasing trend in the proportion of new cases with resistance to INH or SM (P ¼ 0.045 and P ¼ 0.008, respectively). There
were no significant sex-specific differences (data not shown), even among patients with MDR-TB. The data were categorised according to types of main comorbidities (Table 3). In previously treated DM patients, we observed significantly higher RMP resistance (P ¼ 0.038) than in those who did not have comorbidities. There were no significant differences between isolates from the other two patient groups compared with those from controls without comorbidities. Of the foreign nationals, 51 were new and 2 previously treated. Among the isolates from new patients, the frequencies of resistance to INH, RMP, SM and EMB were respectively 11.8% (95%CI 5.5– 23.4), 2.0% (95%CI 0.3–10.3), 5.9% (95%CI 2.0– 15.9) and 2.0% (95%CI 0.3–10.3). INH resistance was significantly higher in foreign nationals compared with Japanese nationals (P ¼ 0.004). The prevalence of resistance was generally similar among isolates from patients residing in the six geographical districts, with the exception of a significant difference in INH resistance between isolates from new patients from Kinki (Middle-southern) and Kyusyu (Southern) (P ¼ 0.002). Table 4 shows the adjusted proportion of drug resistance based on TB statistics for 2007 in Japan. There was no significant difference between raw and adjusted rates of prevalence. Table 5 shows changes in drug resistance from the Ryoken studies for 1997, 2002 and 2007. Ryoken
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Table 2
Association of age with drug resistance by treatment history
Age years
n
New cases (n ¼ 2082)* 619 18 20–29 131 30–39 192 40–49 166 50–59 242 60–69 288 70–79 459 780 586 P value, Cochran-Armitage test for trend Previously treated cases (n ¼ 195) 619 0 20–29 8 30–39 8 40–49 8 50–59 26 60–69 27 70–79 52 780 66 P value, Cochran-Armitage test for trend
INH (% (95%CI)
6.1 3.6 4.2 2.9 2.8 3.3 2.0
12.5 12.5 25.0 23.1 11.1 15.4 4.5
0 (2.0–10.3) (1.0–6.3) (1.1–7.3) (0.8–5.0) (0.9–4.7) (1.6–4.9) (0.9–3.2) 0.045
RMP % (95%CI)
0.8 0.5 1.2 1.7 0.7 0.7 0.3
0 (0.1–4.2) (0.1–2.9) (0.3–4.3) (0.04–3.3) (0.2–2.5) (0.2–1.9) (0.1–1.2) 0.351
SM % (95%CI)
6.9 8.3 8.4 6.2 5.9 5.9 3.2
0 (2.5–11.3) (4.4–12.3) (4.2–12.7) (3.1–9.3) (3.2–8.6) (3.7–8.0) (1.8–4.7)‡ 0.008
EMB % (95%CI)
4.6 1.0 1.2 0.8 1.0 1.5 0.7
0 (1.0–8.2)† (0.3–3.7) (0.3–4.3) (0.2–3.0) (0.4–3.0) (0.4–2.7) (0.3–1.7) 0.051
0 0 0 0 (2.2–47.1) 12.5 (2.2–47.1) 25.0 (7.1–59.1) 0 (2.2–47.1) 0 12.5 (2.2–47.1) 0 (7.1–59.1) 0 25.0 (7.1–59.1) 0 (11.0–42.1) 7.7 (2.1–24.1) 7.7 (2.1–24.1) 11.5 (4.0–29.0)† (3.9–28.1) 11.1 (3.9–28.1) 25.9 (13.1–44.7) 3.7 (0.7–18.3) (8.0–27.5) 5.8 (2.0–15.6) 9.6 (4.2–20.7) 1.9 (0.3–10.1) (1.6–12.5) 6.1 (2.4–14.6) 7.6 (3.3–16.5) 0 0.074 0.899 0.098 0.250
Any resistance % (95%CI)
11.5 13.0 12.1 8.7 8.7 9.2 5.0
0 (7.1–18.0) (9.0–18.5) (7.9–17.9) (5.7–12.9) (5.9–12.5) (6.8–12.1) (3.5–7.0) 0.001
25.0 12.5 25.0 26.9 37.0 21.2 10.6
0 (7.1–59.1) (2.2–47.1) (7.1–59.1) (13.7–46.1) (21.5–55.8) (12.2–34.0) (5.2–20.3) 0.117
* 15 patients, ages unknown. † Significantly higher than average (P , 0.05). ‡ Significantly lower than average (P , 0.05). INH ¼ isoniazid; CI ¼ confidence interval; RMP ¼ rifampicin; SM ¼ streptomycin; EMB ¼ ethambutol.
surveys employed the absolute concentration method before 1997. Direct comparison using the proportion method showed a clear reduction in drug resistance in isolates from previously treated patients (P , 0.001). Among new patients, SM resistance showed a decreasing trend, whereas there was a significant increase in EMB resistance over time (P ¼ 0.004). MDR-TB prevalence was significantly lower (P ¼ 0.015) than that reported by the 2002 Ryoken study: 0.7% (95%CI 0.4–1.1) and 9.8% (95%CI 7.3–13.1) in new and previously treated patients, respectively.3
DISCUSSION A nationwide survey of anti-tuberculosis drug resistance was conducted from 2007 to 2008 in Japan, and it is important to note its main limitations. As the Table 3
study sites were selected for convenience rather than random sampling, the national representativeness of the survey cannot be assured. However, as the distribution of patients by age, sex and region was similar to that of patients in the national TB register, it is likely that the study results provide a valid estimation of drug resistance patterns in the national TB patient population. In addition, the original target sample size of 2828 was based on the prevalence of drug resistance observed in 2002; however, sample size in the present study was 2292 (81.0%). The number of patients studied represents approximately 8.9% of all TB cases registered in Japan in 2007.8 The authors recognise that the participating hospitals and the number of patients do not guarantee an optimal representation of the standard of TB care in Japan. However, the survey included all of Japan. Among
Prevalence of drug resistance in patients with and those without comorbidities
Comorbidity
n
INH % (95%CI)
RMP % (95%CI)
Patients without comorbidity (n ¼ 1000; male, 645; female, 355) New 921 3.0 (1.9–4.2) 0.9 (0.3–1.5) Previously treated 79 12.7 (5.2–20.2) 6.3 (0.8–11.8) Diabetes mellitus (n ¼ 387: male, 301; female, 86) New 350 3.1 (1.8–5.5) 0.9 (0.3–2.5) Previously treated 37 21.6 (11.4–37.2) 18.9 (9.5–34.2)*
SM % (95%CI)
EMB % (95%CI)
MDR-TB % (95%CI)
Any resistance % (95%CI)
4.9 (3.5–6.3) 16.5 (8.1–24.8)
1.6 (0.8–2.4) 2.5 (0.7–8.8)
0.4 (0.0–0.9) 3.8 (1.3–10.6)
8.0 (6.3–9.8) 24.1 (14.4–33.7)
6.3 (4.2–9.3) 13.5 (5.9–28.0)
1.1 (0.4–2.9) 8.1 (2.8–21.3)
0.3 (0.1–1.6) 13.5 (5.9–28.0)
10.0 (7.3–13.6) 29.7 (17.5–45.8)
Malignant tumour (n ¼ 104: male, 80; female, 24) New 91 5.5 (2.4–12.2) Previously treated 13 0
0 0
5.5 (2.4–12.2) 0
1.1 (0.2–6.0) 0
0 0
9.9 (5.3–17.7) 0
Steroid use (n ¼ 73: male, 40; female, 33) New 67 3.0 (0.8–10.2) Previously treated 6 16.7 (3.0–56.4)
0 0
3.0 (0.8–10.2) 33.3 (9.7–70.0)
0 0
0 0
6.0 (2.3–14.4) 33.3 (9.7–70.0)
* Significant difference compared with patients without comorbidities (P ¼ 0.038). INH ¼ isoniazid; CI ¼ confidence interval; RMP ¼ rifampicin; SM ¼ streptomycin; EMB ¼ ethambutol; MDR-TB ¼ multidrug-resistant tuberculosis.
Anti-tuberculosis drug resistance survey
Table 4 Prevalence of anti-tuberculosis-drug resistance, raw and adjusted data for cases by age group, sex and district* Drug INH Raw Adjusted RMP Raw Adjusted SM Raw Adjusted EMB Raw Adjusted
New % (95%CI)
Previously treated % (95%CI)
3.1 (2.3–3.8) 3.2 (1.1–5.3)
12.3 (7.7–17.0) 11.3 (8.0–15.5)
0.7 (0.4–1.1) 0.7 (0.2–1.2)
6.7 (3.1–10.2) 5.3 (3.2–8.5)
5.6 (4.6–6.6) 5.6 (3.4–7.8)
12.3 (7.7–17.0) 11.8 (8.5–16.1)
1.3 (0.8–1.8) 1.3 (0.6–1.9)
2.6 (0.3–4.8) 2.1 (1.0–4.6)
* Raw data were adjusted on the basis of the prevalence of bacteriologically confirmed tuberculosis cases in six different districts in Japan (2007 official statistics of the Japanese Ministry of Health, Labour and Welfare). CI ¼ confidence interval; INH ¼ isoniazid; RMP ¼ rifampicin; SM ¼ streptomycin; EMB ¼ ethambutol.
behavioural risk factors, only alcoholism was considered, as other factors such as drug use and incarceration are not common. This survey revealed a relatively low level of resistance to the main antituberculosis drugs in new and previously treated patients. The proportions of new and previously treated patients in this survey with resistance to INH, RMP, SM and EMB were lower than the global averages of resistance to these drugs.2 Compared with data from studies conducted in 1997 and 2002, the SM resistance of isolates from new patients and resistance to all drugs of isolates from previously treated patients show a clear declining trend.3,10 SM is not commonly used in Japan for the initial treatment of TB due to the introduction of the standard 6-month short-course regimen; we therefore conclude that the reduction in SM resistance could be natural. The use of EMB is now common, and resistant strains have likely accumulated in the population, which is reflected by the increase in resistant isolates from new patients, although the level is still low. The prevalence of INH resistance among new TB cases appears to have increased slightly from 2002 to 2007. However, this difference was not statistically significant. The reason for the Table 5
161
slight increase in INH resistance among isolates from new patients is unknown; however, the use of INH to treat LTBI, which increased after the introduction of the QuantiFERONw-TB (QIAGEN, Venlo, The Netherlands) test, should be carefully monitored.11 Similar to other bacterial infections,12,13 we show here that the prevalence of LVX resistance was relatively high. It is of great interest that the majority of LVX-resistant isolates identified here were not resistant to other anti-tuberculosis drugs. Although guidelines for the treatment of infectious diseases in Japan do not recommend the use of LVX in outpatient clinics, it is widely prescribed,14 and LVX is administered to many patients with TB before the disease is diagnosed. Of the MDR-TB isolates in the present study, the Beijing lineage represented 70.6%. The proportion was consistent with that of a previous report, where the majority of the isolates were non-MDR-TB.15 Several studies have indicated that MDR-TB is caused more frequently by the Beijing lineage; however, this was not clearly apparent from our present data.16,17 The association of resistance to any drug showed a relatively decreasing trend with increasing age among new as well as previously treated patients. This was true for INH- and SM-resistant isolates from new patients, and has been observed repeatedly in past surveys conducted in Japan. Moreover, this is a general trend for other drugs, although it is not statistically significant, primarily due to the limited number of patients. The age-dependence of drug resistance has been reported for isolates from patients residing in the United States during the 1970s,18 and was recently confirmed by the WHO,19 indicating that in older patients the disease is caused by LTBI reactivation that occurs when there are very few drugresistant bacteria. Comorbidities that induce immunocompromised status may result in poor treatment outcomes and development of drug resistance.20,21 However, the very low prevalence of HIV-infected patients in Japan and the small number included here did not allow additional analyses or the evaluation of statistical significance. In the present survey, RMP resistance was significantly high and was detected among
Sequential changes in drug resistance through 1997–2007
Category
Year
New
1997 2002 2007 1997 2002 2007
Previously treated*
INH % (95%CI) 4.4 2.8 3.1 33.0 18.9 12.3
(3.4–5.6) (2.2–3.5) (2.3–3.8) (27.6–38.8) (15.2–22.7) (7.7–17.0)
RMP % (95%CI) 1.4 1.0 0.7 21.6 11.0 6.7
(0.9–2.2) (0.7–1.4) (0.4–1.1) (17.1–26.9) (8.0–14.0) (3.1–10.2)
SM % (95%CI) 7.5 7.0 5.6 24.2 14.4 12.3
(6.2–9.0)* (6.0–7.9)* (4.6–6.6)* (19.5–29.8) (11.0–17.8) (7.7–17.0)
EMB % (95%CI) 0.4 0.9 1.3 15.2 8.4 2.6
(0.2–0.8)† (0.5–1.2)† (0.8–1.8)† (11.3–20.0) (5.7–11.1) (0.3–4.8)
MDR-TB % (95%CI) 0.8 0.7 0.4 19.7 9.8 4.1
* Significant decreasing trend (Cochran-Armitage test, P , 0.05) † Significant increasing trend (Cochran-Armitage test, P ¼ 0.004). INH ¼ isoniazid; CI ¼ confidence interval; RMP ¼ rifampicin; SM ¼ streptomycin; EMB ¼ ethambutol; MDR-TB ¼ multidrug-resistant tuberculosis.
(0.4–1.4) (0.4–1.1) (0.2–0.8) (15.3–24.9) (7.3–13.1) (2.1–7.9)
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The International Journal of Tuberculosis and Lung Disease
isolates from previously treated DM patients. In the 2002 Ryoken survey, drug resistance was higher in defaulters than in simple relapses.3 In the present study, previously treated DM patients were more likely to have MDR-TB than previously treated patients without comorbidities (P ¼ 0.125). Although the detailed treatment history of patients with RMP resistance in the present survey is unknown, inappropriate treatment may possibly account for higher resistance. The decreasing trend in resistance among previously treated patients during the past decade suggests improvements in anti-tuberculosis treatment. The present survey also indicates that continuous monitoring of drug resistance will be required to establish an optimal TB control programme and that a drug resistance surveillance system should be established. Acknowledgement The authors extend their sincere appreciation to all the participants of this study. This study was supported by a Health Science Research Grant from the Ministry of Health, Labour and Welfare (H18-Shinko-Ippan-012 and H21-Shinko-Ippan-016), Tokyo, Japan. Conflicts of interest: none declared
References 1 Japan Anti-Tuberculosis Association. Statistics of TB 2010. Tokyo, Japan: JATA, 2011. 2 World Health Organization. Anti-tuberculosis drug resistance in the world. Report No. 4. WHO/HTM/TB/2008.394. Geneva, Switzerland: WHO, 2008. 3 Tuberculosis Research Committee (RYOKEN). Drug-resistant Mycobacterium tuberculosis in Japan: a nationwide survey, 2002. Int J Tuberc Lung Dis 2007; 11: 1129–1135. 4 World Health Organization. Guidelines for surveillance of drug resistance in tuberculosis. 4th ed. WHO/HTM/TB/2009.422. Geneva, Switzerland: WHO, 2009. 5 Springer B, Stockman L, Teschner K, Roberts G D, Bottger E C. Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods. J Clin Microbiol 1996; 34: 296–303. 6 World Health Organization. Policy guidance on drugsusceptibility testing (DST) of second-line anti-tuberculosis drugs. WHO/HTM/TB/2008.392. Geneva, Switzerland: WHO, 2008. 7 Supply P, Allix C, Lesjean S, et al. Proposal for standardization of optimized mycobacterial interspersed repetitive unitvariable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 2006; 44: 4498–4510.
8 Japan Anti-Tuberculosis Association. Statistics of TB 2007. Tokyo, Japan: JATA, 2008. 9 World Health Organization. XDR-TB: extensively drug resistant TB. Geneva, Switzerland: WHO, 2006. http://www. who.int/tb/challenges/mdr/xdr/en/ Accessed October 2014. 10 Abe C, Hirano K, Wada M, Aoyagi T. Resistance of Mycobacterium tuberculosis to four first-line anti-tuberculosis drugs in Japan, 1997. Int J Tuberc Lung Dis 2001; 5: 46–52. 11 Ministry of Health, Labour and Welfare. Statistics of tuberculosis in Japan. Tokyo, Japan: Ministry of Health, Labour and Welfare, 2011. http://www.mhlw.go.jp/bunya/ kenkou/kekkaku-kansenshou03/11.html Accessed October 2014. 12 Yokota S, Sato T, Okubo T, et al. Prevalence of fluoroquinolone-resistant Escherichia coli O25:H4-ST131 (CTX-M-15-nonproducing) strains isolated in Japan. Chemotherapy 2012; 58: 52–59. 13 Yokota S, Ohkoshi Y, Sato K, Fujii N. Emergence of fluoroquinolone-resistant Haemophilus influenzae strains among elderly patients but not among children. J Clin Microbiol 2008; 46: 361–365. 14 Sugawara T, Ohkusu Y, Gu Y, Kawanohara H, Taniguchi K, Okabe N. Estimation of amount of antibiotics used by pharmacy surveillance. Jpn J Infect Prev Cont 2012; 27: 195– 198. [Japanese] 15 Murase Y, Mitarai S, Sugawara I, Kato S, Maeda S. Promising loci of variable numbers of tandem repeats for typing Beijing family Mycobacterium tuberculosis. J Med Microbiol 2008; 57: 873–880. 16 de Steenwinkel J E, ten Kate M T, de Knegt G J, et al. Drug susceptibility of Mycobacterium tuberculosis Beijing genotype and association with MDR-TB. Emerg Infect Dis 2012; 18: 660–663. 17 Niemann S, Diel R, Khechinashvili G, Gegia M, Mdivani N, Tang Y W. Mycobacterium tuberculosis Beijing lineage favors the spread of multidrug-resistant tuberculosis in the Republic of Georgia. J Clin Microbiol 2010; 48: 3544–3550. 18 Kopanoff D, Kilburn J O, Glassroth J L, Snider D Jr, Farer L, Good R. A continuing survey of tuberculosis primary drug resistance in the United States: March 1975 to November 1977. A United States Public Health Service cooperative study. Am Rev Respir Dis 1978; 118: 835–842. 19 World Health Organization. Multidrug and extensively drugresistant TB (M/XDR-TB): 2010 global report on surveillance and response. WHO/HTM/TB/2010.3. Geneva, Switzerland: WHO, 2010. 20 Chang J T, Dou H Y, Yen C L, et al. Effect of type 2 diabetes mellitus on the clinical severity and treatment outcome in patients with pulmonary tuberculosis: a potential role in the emergence of multidrug-resistance. J Formos Med Assoc 2011; 110: 372–381. 21 Fisher-Hoch S P, Whitney E, McCormick J B, et al. Type 2 diabetes and multidrug-resistant tuberculosis. Scand J Infect Dis 2008; 40: 888–893.
Anti-tuberculosis drug resistance survey
i
RESUME
D´eterminer la pr´evalence de la r´esistance aux m´edicaments antituberculeux au Japon. S C H E´ M A : Une enquˆete nationale de pharmacor´esistance a e´ t´e r´ealis´ee grˆace a` un e´ chantillonnage de convenance bas´e sur la participation volontaire des hopitaux. ˆ Les isolats de Mycobacterium tuberculosis (n ¼ 2292) ont e´ t´e recueillis entre aout ˆ 2007 et juillet 2008. La sensibilit´e aux m´edicaments a e´ t´e analys´ee en fonction de l’historique du traitement des patients, de leur age, ˆ sexe, des comorbidit´es et du lieu de r´esidence. Nous avons d´etermin´e la sensibilit´e aux m´edicaments suivants : isoniazide (INH), rifampicine (RMP), streptomycine (SM), e´ thambutol (EMB) et l´evofloxacine (LVX). R E´ S U LT A T S : Les fr´equences d’isolats pharmacor´esistants e´ manant de nouveaux cas e´ taient les suivantes : INH OBJECTIF :
3,1% ; RMP 0,7% ; SM, 5,6% ; EMB, 1,3% ; et enfin 8,5% a` un m´edicament au moins. Les fr´equences de r´esistance e´ manant de patients d´ej`a trait´es e´ taient les suivantes : INH 12,3% ; RMP, 6,7% ; SM, 12,3% ; et EMB, 2,6%. Les fr´equences des isolats r´esistant a` la LVX e´ manant de patients nouveaux et pr´ealablement trait´es e´ taient respectivement de 3,2% et 6,1% (n ¼ 852). Les fr´equences de r´esistance multiple des isolats de patients nouveaux et d´ej`a trait´es e´ taient respectivement de 0,4% et 4,1%, avec un seul cas ultra-r´esistant. C O N C L U S I O N : D’une mani`ere g´en´erale, la pr´evalence de TB pharmacor´esistante au Japon e´ tait faible durant cette p´eriode. Cependant, la r´esistance a` la LVX chez de nombreux patients e´ tait assez e´ lev´ee. Le Japon devrait donc cr´eer un syst`eme de surveillance national. RESUMEN
O B J E T I V O: Determinar la prevalencia de resistencia a los medicamentos antituberculosos en el Japon. ´ M E´ T O D O: Se llevo ´ a cabo una encuesta nacional sobre farmacorresistencia mediante un muestreo de conveniencia de pacientes de los hospitales de un consorcio que voluntariamente participaron en el estudio. Se recogieron aislados cl´ınicos de Mycobacterium tuberculosis entre agosto del 2007 y julio del 2008 (n ¼ 2292). La sensibilidad a los medicamentos se analiz o´ en funci on ´ de los antecedentes de tratamiento, la edad, el sexo, las enfermedades concomitantes y la zona de residencia de los pacientes. Se examino´ la sensibilidad a los siguientes medicamentos antituberculosos: isoniazida (INH), rifampicina (RMP), estreptomicina (SM), etambutol (EMB) y levofloxacino (LVX). R E S U LT A D O S: La frecuencia de farmacorresistencia en los aislados provenientes de casos nuevos fue como
sigue: a INH 3,1%; RMP 0,7%; SM 5,6% y a EMB 1,3%; se observo´ resistencia a algun ´ medicamento en el 8,5% de los casos. La frecuencia de farmacorresistencia en los aislados provenientes de casos previamente tratados fue la siguiente: a INH 12,3%; RMP 6,7%; SM 12,3% y a EMB 2,6%. La frecuencia de resistencia a LVX en los casos nuevos fue 3,2% y en los casos con antecedente de tratamiento fue 6,1% (n ¼ 852). La frecuencia de multidrogorresistencia en los casos nuevos fue 0,4% y en los casos previamente tratados fue 4,1%; solo se observo´ un caso de tuberculosis extremadamente drogorresistente. ´ N: En general, se encontr o CONCLUSIO ´ una baja prevalencia de tuberculosis farmacorresistente en el Japon ´ durante el per´ıodo estudiado. Se observo, ´ no obstante, una resistencia relativamente alta a LVX en los casos nuevos. Es importante establecer un sistema de vigilancia de la resistencia a escala nacional.
