Mycobacterium tuberculosis Detection of Multidrug ...

Multicenter Evaluation of Genechip for Detection of Multidrug-Resistant Mycobacterium tuberculosis Yu Pang, Hui Xia, Zhiying Zhang, Junchen Li, Yi Dong, Qiang Li, Xichao Ou, Yuanyuan Song, Yufeng Wang, Richard O'Brien, Kai Man Kam, Junying Chi, Shitong Huan, Daniel P. Chin and Yanlin Zhao J. Clin. Microbiol. 2013, 51(6):1707. DOI: 10.1128/JCM.03436-12. Published Ahead of Print 20 March 2013.

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Multicenter Evaluation of Genechip for Detection of MultidrugResistant Mycobacterium tuberculosis Yu Pang,a Hui Xia,a Zhiying Zhang,b Junchen Li,b Yi Dong,b Qiang Li,a Xichao Ou,a Yuanyuan Song,a Yufeng Wang,a Richard O’Brien,c Kai Man Kam,d Junying Chi,a Shitong Huan,e Daniel P. Chin,e Yanlin Zhaoa National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, Chinaa; Program for Appropriate Technology in Health, Beijing, Chinab; Foundation for Innovative New Diagnostics, Geneva, Switzerlandc; Microbiology Division, Public Health Laboratory Services Branch, Centre for Health Protection, Hong Kong, Chinad; Bill and Melinda Gates Foundation, China Office, Beijing, Chinae

T

uberculosis (TB) remains a serious threat to global public health (1). The World Health Organization (WHO) estimated that in 2011 there were 8.7 million new cases of TB (13% coinfections with HIV) and 1.4 million people died from TB (2). Multidrug-resistant TB (MDR-TB) (caused by resistance to both rifampin and isoniazid) and HIV/AIDS are considered the greatest obstacles to controlling TB, especially in developing countries with serious epidemics of MDR-TB (3, 4). Of the 440,000 cases of MDR-TB that emerge in the world each year, about 50% are from India and China (5, 6). Because of the slow grow of Mycobacterium tuberculosis, traditional drug sensitivity testing (DST) requires about 2 to 3 months to diagnose the illness (5, 7). Even a rapid liquid-medium culture technique requires about 1 to 2 months for diagnosis (8). This lengthy timeline delays the start of effective drug therapy and allows transmission of the disease to continue (9–11). The rapid diagnosis of TB, especially MDR-TB, is urgently needed to improve public health (9, 12, 13). Several methods have been developed for the rapid detection of MDR-TB, based on a test for encoding regions of the genes associated with drug resistance (9, 14, 15). For more than 95% of rifampin-resistant M. tuberculosis strains, mutation is observed in a “hot spot” region of 81 base pairs of the gene rpoB (16, 17). In addition, more than 85% of isoniazid-resistant strains are associated with mutations in the gene katG and the promoter region of the gene inhA (18, 19). The WHO has recommended the use of some of these rapid tests, including GenoType MTBDR (Hain, Germany) and GeneXpert (Cepheid Corp., USA), based on their good performance (20). These molecular tools provide clinical reports with shorter turnaround times than traditional DST and have fewer biosafety concerns than the latter. This means that they might be used in laboratories with limited resources (21). To diagnose MDR-TB rapidly, CapitalBio Corporation devel-

June 2013 Volume 51 Number 6

oped the Genechip MDR kit, based on multiple PCRs combined with reverse hybridization (14). Similar to GenoType MTBDR, the Genechip MDR kit is used to evaluate resistance to rifampin and isoniazid by detecting mutations in the rpoB, katG, and inhA genes of M. tuberculosis in the specimens. A previous study reported satisfactory performance of the kit; the rate of concordance between the biochip assay and DNA sequencing results was 100%, and the rates of concordance with conventional DST results were 91.8% for isolates and 94.6% for sputum samples for rifampin resistance and 70.2% for isolates and 78.1% for sputum samples for isoniazid resistance (14). According to the plan drawn up by the government of China, rapid detection of drug-resistant M. tuberculosis will be adopted overall in prefectural and municipal laboratories by 2015. However, data are still lacking on the performance of Genechip in prefectural and municipal laboratories. Therefore, this technique requires broader evaluation among prefectural and municipal TB laboratories, using more specimens than in previous evaluations, to provide a theoretical basis for possible expansion of the use of Genechip in China. The purpose of this study was to evaluate the efficacy of Genechip for diagnosing rifampin and isoniazid resistance and M. tuberculosis in pre-

Received 5 January 2013 Returned for modification 7 February 2013 Accepted 11 March 2013 Published ahead of print 20 March 2013 Address correspondence to Yanlin Zhao, [email protected]. Y.P. and H.X. contributed equally to this article. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.03436-12

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Drug-resistant tuberculosis (TB), especially multidrug-resistant TB (MDR-TB), is still one of the most serious threats to TB control worldwide. Early diagnosis of MDR-TB is important for effectively blocking transmission and establishing an effective protocol for chemotherapy. Genechip is a rapid diagnostic method based on molecular biology that overcomes the poor biosafety, time consumption, and other drawbacks of traditional drug sensitivity testing (DST) that can detect MDR-TB. However, the Genechip approach has not been effectively evaluated, especially in limited-resource laboratories. In this study, we evaluated the performance of Genechip for MDR-TB in 1,814 patients in four prefectural or municipal laboratories and compared its performance with that of traditional DST. The results showed that the sensitivity and specificity of Genechip were 87.56% and 97.95% for rifampin resistance and 80.34% and 95.82% for isoniazid resistance, respectively. In addition, we found that the positive grade of the sputum smears influenced the judgment of results by Genechip. The test judged only 75% of the specimens of “scanty” positive grade. However, the positive grade of the specimens showed no influence on the accuracy of Genechip. Overall, the study suggests that, in limited-resource laboratories, Genechip showed high sensitivity and specificity for rifampin and isoniazid resistance, making it a more effective, rapid, safe, and cost-beneficial method worthy of broader use in limited-resource laboratories in China.

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fectural and municipal laboratories with limited resources and to provide data for possible widespread use of Genechip. MATERIALS AND METHODS

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RESULTS

Patient enrollment. A total of 2,247 patients who tested positive in smear examinations were included in this evaluation. One specimen, at the highest positive grade, was collected from each patient and digested with NALC-NaOH for DST and Genechip testing. Of the specimens for DST, 208 (9.26%) were negative in culture, while 12 (0.53%) were contaminated. In addition, 42 (2.18%) specimens were identified as nontuberculous mycobacteria, and the DST results for 60 (2.67%) specimens were invalid. The results for 1,918 specimens were valid for evaluation of DST. Of the specimens for Genechip, however, 94 (4.18%) specimens were negative for M. tuberculosis, 83 (3.69%) specimens were identified as nontuberculous mycobacteria, and the results for 43 (1.91%) specimens were invalid. The results for 2,027 specimens were valid for evaluation of Genechip. Summarizing the results of two tests, the results for 1,814 specimens were usable for evaluation of the performance of Genechip (Fig. 1). Performance evaluation of Genechip for rifampin resistance. Genechip can be used for both rifampin resistance and isoniazid resistance. The sensitivity and specificity of Genechip for rifampin resistance are shown in Table 1. The diagnostic results for 1,756 (96.8%) patients by Genechip were consistent with those by DST. Of the 201 patients with rifampin-resistant TB diagnosed by DST, 176 cases were confirmed by Genechip, indicating a sensitivity of 87.6%. However, of the 1,613 patients with rifampin-sensitive TB diagnosed by DST, 1,580 cases were confirmed by Genechip, indicating a specificity of 98.0%. Among the four sites in this study, the sensitivity of the Genechip test to rifampin in Lianyungang (83.1%) was lowest, while that in Hohhot (94.74%) was highest. However, the specificities in all four sites were more than 97%. Performance evaluation of Genechip for isoniazid resistance. The rate of coincidence of the results on isoniazid resistance by DST and Genechip was 93.8% (1,702 cases/1,814 cases) (Table 2). Of the 234 patients with isoniazid-resistant TB diagnosed by DST, 188 cases were confirmed by Genechip, indicating a sensitivity of 80.3%. However, of the 1,580 patients with isoniazidsensitive TB diagnosed by DST, 1,514 cases were confirmed by Genechip, indicating a specificity of 95.8%. All of the sensitivities in Lianyungang, Kaifeng, and Yongchuan were approximately 80%. However, the sensitivity of the Genechip test in Hohhot was relatively low (71.4%), with the 95% confidence interval (CI) being 52.1% to 90.7%, which might be associated with small quantities of specimens. Specificities in all four sites were about 95%. Influence of positive grade of sputum specimens on Genechip performance. The smear positivity (positive grade) of sputum reflects the bacterial content in the specimen, which might influence the Genechip results. We calculated the proportions of results of “failure of interpretation” and “no mycobacterium” in the specimens at various positive grades. The results showed that the invalid result rate decreased with increasing positive grade, being 16.8% and 1.4% for specimens with positive grades of scanty and 4⫹, respectively. The positive grade of the specimens was significantly related to the invalid result rate (test of trend, chi-square ⫽ 53.93; P ⬍ 0.01) (Table 3). Meanwhile, we analyzed the influence of the positive grade of the specimens on the diagnostic results for rifampin resistance, as shown in Table 4. The sensitivity for the specimens at the scanty



Ethics. All patients gave written informed consent before they were included in this study. Ethics approval was granted by the Program for Appropriate Technology in Health (PATH) and the Ethics Committee of the Chinese Center for Disease Control and Prevention. Design of study. From January 2011 to February 2012, we evaluated Genechip in four prefectural or municipal cities in China, i.e., Hohhot City of Inner Mongolia Autonomous Region, Kaifeng City of Henan Province, Lianyungang City of Jiangsu Province, and Yongchuan District of Chongqing City. The cities belong to different administrative divisions in China. All smear-positive patients within the four cities were enrolled consecutively in the study, irrespective of comorbidities or HIV status. Sputum smear microscopy was performed for all patients and was the major screening tool used in the study. We collected two or three sputum specimens (2 ml each) from the patients. The specimens were collected at the same time and were transported to the corresponding prefectural or municipal laboratories within 3 days after collection, for concurrent DST and Genechip testing. Estimation of quantity of specimens. Sample size calculations assumed the sensitivity of Genechip for the detection of MDR-TB to be estimated as 75%, with an allowable error of 10%. When ␣ is 0.05, the case number needed (N) is calculated as 72 with the following formula: N ⫽ [(1.962 ⫻ 0.75 ⫻ 0.25)/0.12] ⫽ 72. However, on the basis of the estimated MDR rate (8.32%) among smear-positive patients published in a national prevalence survey, a total of 866 patients were included in this study (22). Given losses due to contamination, negative culture results, invalid DST results, or other causes, another 20% smear-positive patients were enrolled, that is, at least 1,040 patients were included in this study. Laboratory methods. Direct smears of each sputum specimen were examined using Ziehl-Neelsen staining for acid-fast bacilli. The grading of smears was performed according to the China Center for Disease Control and Prevention system, which starts with negative and proceeds to scanty and to 4⫹ (national guidelines for TB laboratories from the China Center for Disease Control and Prevention). One specimen, with the highest positive smear grade, was selected for each patient. The specimens were digested with N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) (4%) for 15 min and inoculated onto modified Lowenstein-Jensen (LJ) medium, according to a previous report (22). Bacterial colonies were collected 4 to 8 weeks later for traditional DST and identification by previously reported methods (22). A 1.2-ml portion of digested specimen was added to a 1.5-ml centrifuge tube, centrifuged at 13,000 rpm for 15 min at 4°C, washed with phosphate-buffered saline (0.01 M, pH 7.4), and further centrifuged at 13,000 rpm for 5 min at 4°C. The precipitates then were applied to the Genechip according to the manufacturer’s instructions (14). Staff members of all laboratories were trained and approved by the National Reference Laboratory of Tuberculosis. Each laboratory conducted a 2-month trial operation before the study, to ensure laboratory personnel skills. Sequencing. From the specimens with inconsistent results in DST and Genechip testing, we amplified the corresponding gene fragments and sequenced them by the methods reported previously (23). We entered the sequencing results into the Basic Local Alignment Search Tool (BLAST), an international data bank (www.ncbi.nlm.nih.gov/BLAST), for comparison with the corresponding genes of M. tuberculosis strain H37Rv. Analysis of data. In analysis of the data, DST with modified LJ medium was used as the reference standard, and calculation of sensitivity, specificity, positive predictive value, and negative predictive value was performed by using this method as the standard. All of the data were evaluated by the National Reference Laboratory of Tuberculosis, included in SPSS 15.0 as a data bank, and analyzed by the software. The chi-square

test was used for statistical analysis. If the P value was less than 0.05, then the difference was judged to be significant.

Multicenter Evaluation of Genechip for MDR-TB


FIG 1 Enrollment and outcomes.

positive grade was 75%, which was slightly lower than that for specimens at other positive grades (about 85%). However, no significant difference was observed in the test results for specimens at various positive grades. All of the specificities for specimens at various positive grades were more than 97% and showed no significant differences. Distribution of inconsistent results on rifampin resistance. Of the 1,814 cases we evaluated, 58 cases presented inconsistent rifampin resistance results between DST and Genechip. We sequenced the rifampin resistance-determining region (RRDR) of gene rpoB in the specimens. Of the 33 false-positive cases, 22 cases contained the same mutation type as that identified by Genechip, while no mutation was found in the other 11 cases. In addition, we observed no mutation in 18 of the 25


false-negative cases, and the other 7 cases showed the mutation located in the RRDR. Although the Genechip and DNA sequencing shared the same principle of determining rifampin susceptibility by scanning the RRDR, we also observed 18 cases with inconsistent results, including 11 cases of false-positive results and 7 cases of false-negative results, which we analyzed for their respective mutation types (Table 5). Of the false-positive results, 45.45% were due to substitution of C with T at codon 531. In addition, we observed mutations at codon 516 in two cases, codon 526 in two cases, codon 511 in one case, and codon 533 in one case. For false-negative results, however, four cases were due to mutations at codon 522, while three cases were due to mutations at codon 531. The mutations at codons 522 and 531 are beyond the detectable range of Genechip.

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TABLE 1 Performance of Genechip for detecting rifampin resistance

Genechip resulta

Site

No. of cases with DST result of: R

S

Total

Sensitivity (% [95% CI])

Specificity (% [95% CI])

Positive predictive value (%)

Negative predictive value (%)

R S Total

18 1 19

1 189 190

19 190 209

94.74 (84.70–100.00)

99.47 (98.44–100.00)

94.74

99.47

Lianyungang

R S Total

59 12 71

13 452 465

72 464 536

83.10 (74.38–91.82)

97.20 (95.71–98.70)

81.94

97.41

Kaifeng

R S Total

51 4 55

9 489 498

60 493 553

92.73 (85.86–99.59)

98.19 (97.02–99.36)

85.0

99.19

Yongchuan

R S Total

48 8 56

10 450 460

58 458 516

85.71 (76.55–94.88)

97.83 (96.49–99.16)

82.76

98.25

Total

R S Total

176 25 201

33 1,580 1,613

209 1,605 1,814

87.56 (83.00–92.12)

97.95 (97.26–98.64)

84.21

98.44

a

R, resistant; S, sensitive.

DISCUSSION

The Genechip kit may be used for detection of M. tuberculosis drug resistance, especially in clinical sputum specimens (14). However, so far there has not been any multicenter evaluation of the kit in comparison with modified LJ medium solid culture, especially in regions with large burdens of drug-resistant TB. Our study indicated that, in low-resource TB laboratories in China, the kit showed high sensitivity and specificity for drug-resistant M. tuberculosis. Both Genechip and the line probe assay are based on genotyp-

ing of the genes associated with drug resistance in M. tuberculosis by reverse hybridization. Several evaluations of line probe techniques have been reported previously. A meta-analysis showed that the total sensitivity and specificity of the line probe assay were 98.4% and 98.9% for rifampin resistance and 88.7% and 99.2% for isoniazid resistance, respectively, which were higher than the values in this study (15). The difference may be due to the following reasons. First, all of the evaluation results for the meta-analysis were from high-level reference laboratories, while the four laboratories in this study were limited-resource laboratories in which

TABLE 2 Performance of Genechip for detecting isoniazid resistance

Genechip resulta

No. of cases with DST result of:

Sensitivity (% [95% CI])

Specificity (% [95% CI])



R

S

Total

Hohhot

R S Total

15 6 21

4 184 188

19 190 209

71.43 (52.11–90.75)

97.87 (95.81–99.94)

78.95

96.84

Lianyungang

R S Total

68 18 86

12 438 450

80 456 536

79.07 (70.47–87.67)

97.33 (95.84–98.82)

85.0

96.05

Kaifeng

R S Total

54 12 66

32 455 487

86 467 553

81.82 (72.51–91.12)

93.43 (91.23–95.63)

62.79

97.43

Yongchuan

R S Total

51 10 61

18 437 455

69 447 516

83.61 (74.32–92.90)

96.04 (94.25–97.84)

73.91

97.76

Total

R S Total

188 46 234

66 1,514 1,580

254 1,560 1,814

80.34 (75.25–85.43)

95.82 (94.84–96.81)

74.02

97.05

Site

a


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Hohhot


TABLE 3 Genechip results for specimens with various acid-fast bacillus-positive smear grades

Failure of interpretation

No mycobacterium

Scanty 1⫹ 2⫹ 3⫹ 4⫹

4 9 10 10 5

15 44 22 10 3

Total

38

94

a

Total

Total no. of specimens for Genechip

Invalid result rate (%)a

19 53 32 20 8

113 594 561 460 519

16.81 8.92 5.70 4.35 1.54

132

2,247

5.89

No. of cases with invalid result of:

Positive smear grade

Chi-square ⫽ 53.93; P ⬍ 0.01.

TABLE 4 Performance of Genechip for detecting rifampin resistance among sputum specimens with various acid-fast bacillus-positive smear grades No. of cases with DST result of:

Sensitivity (%)

Specificity (%)



3 63 66

75.00

100.00

100.00

98.41

7 365 372

33 370 403

83.87

98.12

78.79

98.65

61 6 67

9 369 378

70 375 445

91.04

97.62

87.14

98.40

R S Total

41 7 48

6 343 349

47 350 397

85.42

98.28

87.23

98.00

R S Total

42 6 48

13 423 436

55 429 484

87.50

97.02

76.36

98.60

Positive smear grade

Genechip resulta

R

S

Total

Scanty

R S Total

3 1 4

0 62 62

1⫹

R S Total

26 5 31

2⫹

R S Total

3⫹

4⫹

a



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laboratory conditions and a lack of practical experience might have influenced the evaluation results. Second, inconsistent results between DST and Genechip were observed for some specimens in this study. One possible reason for this was that this study used a solid drug susceptibility test, which might not be as sensitive as a liquid drug sensitivity test. There also might be problems of contamination and possible overgrowth with nontuberculous mycobacteria. Genechip has more advantages than conventional DST. The entire Genechip test may be completed within 6 h, which saves time for drawing up an effective regimen for anti-TB chemotherapy. Also, Genechip is based on testing for nucleic acid of M. tuberculosis and not a large quantity of live M. tuberculosis, which is safer than conventional DST. Furthermore, the high throughput of Genechip reduces the time laboratory personnel must spend on testing. Our evaluation also revealed some disadvantages in the further use of Genechip. First, Genechip analysis required a confocal laser scanner, which was not readily available in some laboratories. Second, the design of the test did not include a probe to detect codon

522 in rpoB. Several inconsistent results in this study were due to a mutation at codon 522. If the mutation was included in the detection range of Genechip, then the sensitivity of the test likely would increase to 90%. In addition, the Genechip contained only two genes associated with isoniazid resistance, katG and inhA. As reported in previous studies, these genes covered only about 85% of isoniazid-resistant strains in China (24). However, the oxyR-ahpC region was associated with about 10% of isoniazid-resistant strains (25). We recommend that oxyR-ahpC be included in Genechip to increase the sensitivity of rapid detection. The positive grade of sputum specimens was an effective indicator of the M. tuberculosis concentration, which might influence the Genechip result based on the amplification of genes associated with the drug resistance of M. tuberculosis in specimens. The study showed that the proportion of invalid results decreased significantly as the positive grade of specimens increased. The percentage of invalid results reached 16.8% for the specimens with a scanty positive grade, indicating that Genechip should be used cautiously for specimens with a scanty positive grade. Invalid results were found for some specimens even at high positive grades; 1.4% of specimens had a positive grade of 4⫹. These specimens were bloody or caseous sputum samples with high foreign protein contents, which resulted in high protein contents in the DNA samples and inhibited Genechip amplification. However, the smear positivity grade of the specimens showed no significant influence on the sensitivity, specificity, or positive and negative predictive values of Genechip, indicating that the test results had a high grade of accuracy. According to previous literature, about 90% of cases of rifampin-resistant TB were also resistant to isoniazid (26). In other words, 90% of patients with rifampin-resistant TB had MDR-TB. For this reason, rifampin resistance serves as a marker for the diagnosis of MDR-TB by several techniques (26, 27). In our study, however, only 60% to 70% of cases of rifampin-resistant TB in the four sites in this study were resistant to isoniazid, which might be

Pang et al.

TABLE 5 Analysis of inconsistent results of Genechip and sequencing Mutated codon (mutation)a Type of cases

Patient no.

Genechip

Sequencing

False positive

L121 L147 L292 H41 L52 Y184 Y307 Y313 L154 L182 K110

511 (T-C) 516 (A-G) 516 (A-T) 526(C-G) 526 (C-G) 531 (C-T) 531 (C-T) 531 (C-T) 531 (C-T) 531 (C-T) 533 (T-C)

S S S S S S S S S S S

a b

Y263 Y513 K89 K92 Y214 L649 L659

S S S S S S S

511 (1/11 [9.09]) 516 (2/11 [18.18]) 526 (2/11 [18.18]) 531 (5/11 [45.45])

533 (1/11 [9.09]) b

522 (C-T) 522 (C-T) 522 (C-T) 522 (C-T) 531 (C-T) 531 (C-G) 531 (CG-AT)b

522 (4/7 [57.14])

531 (3/7 [42.86])

S, sensitive, indicating no mutation of the rpoB gene. This type of mutation is not included in the Genechip probe.

due to the relatively high rates and differentiation of M. tuberculosis in China, which might show a different drug resistance pattern than those observed in other countries. Because rifampin and isoniazid are the most important drugs for TB treatment, if patients with rifampin-resistant TB were treated with the same protocol as patients with MDR-TB, then the chance to treat these patients with isoniazid might be lost. The above-mentioned results indicated that, in China, rifampin monoresistance may not be used as a definitive marker for the diagnosis of MDR-TB. This study is the first large-scale evaluation of Genechip in basic-level TB laboratories, which showed that Genechip exhibited high sensitivity and specificity for rifampin resistance and isoniazid resistance of M. tuberculosis. Provided an adequate laboratory setup is available, we conclude that Genechip can be a more effective, rapid, safe, and cost-beneficial alternative to conventional DST for rifampicin and isoniazid and is a method worth further study for expanded use in basic-level laboratories in China. ACKNOWLEDGMENTS We thank the Bill and Melinda Gates Foundation for financial support. We thank the Program for Appropriate Technology in Health (PATH) for assistance in implementing the project, analyzing data, and writing this article. We also thank all staff members in the project laboratories who contributed to this work.

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False negative

Muted codon detected by Genechip or sequencing (no. of cases with mutated codon/total no. [%])


16. 17. 18.

19. 20.

22.


23.

24.

25.

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