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A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis
Biologicals xxx (2017) 1e6
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A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis Fatemeh Sakhaee a, 1, Morteza Ghazanfari a, 1, Nayereh Ebrahimzadeh a, Farzam Vaziri a, b, Fatemeh Rahimi Jamnani a, b, Mehdi Davari a, Safoora Gharibzadeh c, d, Fatemeh Hemati Mandjin a, Abolfazl Fateh a, b, *, Seyed Davar Siadat a, b a
Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran Research Centre for Emerging and Reemerging Infectious Diseases, Pasteur Institute of Iran, Tehran, Iran d Department of Epidemiology and Biostatistics, Pasteur Institute of Iran, Tehran, Iran b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 May 2017 Received in revised form 4 July 2017 Accepted 8 July 2017 Available online xxx
This study aimed to evaluate the frequency of resistance to first- and second-line drugs using phenotypic and genotypic methods and its correlation with resistance-linked mutations in Mycobacterium tuberculosis (M. tb) isolated in Iran. Three different methods, including the indirect proportion method(PM), direct and indirect nitrate reductase assay(NRA), and direct sequencing were used to assess drug resistance. In this study, sensitivity, specificity, agreement, costs, and turnaround time of these methods were compared in 395 smear positive isolates. Compared to the PM, the NRA and the direct sequencing methods demonstrated higher specificity, sensitivity, and agreement for detection of all anti-tuberculosis drugs. The NRA had a short turnaround time and was more cost-effective than the other methods. Mutations in codon 531 in rpoB, 315 in katG, 18 in rpsL, and 306 in embB were associated with high-level resistance to the first-line drugs, and mutations in codon 94 in gyrA, and A1401G in rrs were correlated with resistance to the second-line drugs. We found that the NRA is a highly sensitive, specific, inexpensive, and rapid test with strong potential to be a useful and interesting alternative tool, particularly in low-income countries. In addition, these molecular data will be helpful for developing new molecular methods for detecting first- and second-line drug-resistant M. tb. © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: First- and second-line drugs Mycobacterium tuberculosis Nitrate reductase assay Proportion method Direct sequencing
1. Introduction Tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis (M. tb) and is still a major global health problem in most parts of the world. Approximately 9.6 million new cases and 1.5 million deaths were reported in 2014. In 2015, about 480,000 new cases of multidrug-resistant TB (MDR-TB) were estimated, and 250,000 deaths were reported; majority of these cases were in Asia. About 9.7% of MDR-TB patients develop extensively drug-resistant TB (XDR-TB). Thus far, at least 117 countries in the world have reported at least one case of XDR-TB [1]. MDR-TB is defined as M. tb that develops resistance to both isoniazid (INH) and rifampin (RIF),
* Corresponding author. Departments of Mycobacteriology & Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran. E-mail address: [email protected] (A. Fateh). 1 These authors contributed equally to this work.
while XDR-TB is defined as MDR-TB that is resistant to any member of the fluoroquinolone family and at least one of the three secondline injectable drugs [2]. For the successful management of MDR-TB and XDR-TB, an inexpensive, easy-to-use, and rapid drug susceptibility test (DST) is required for fast initiation of the most effective antibiotic therapy. “Standard procedures for DST, such as the indirect or direct proportion procedure, are easy to perform; however, these methods have the disadvantages of being very time consuming (4e6 weeks) and requiring trained and experienced laboratory technicians. In addition to being expensive, molecular methods such as the Xpert MTB/RIF® and line probe assay need expert laboratory technicians. Further, these methods may not be able to distinguish the active infection causing organisms from the dead organisms by assessing their DNA. Although these methods have shorter turnaround times, it appears that low-income conventional laboratories are unable to perform these procedures for M. tb detection [3e5].
http://dx.doi.org/10.1016/j.biologicals.2017.07.003 1045-1056/© 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sakhaee F, et al., A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.07.003
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F. Sakhaee et al. / Biologicals xxx (2017) 1e6
In the past few years, several methods for rapid DST have been developed and evaluated, such as the radiometric method BACTEC 460-TB [6], colorimetric redox method [7], MGIT TBc Identification Test [8], E-Tests [9], and Genotype MTBDRplus [10] and INNO-LiPA [11] that need expensive substance and equipment. So, in most developing countries is not possible [12]. The microscopic observation DST, although inexpensive and fast, requires extensive training of the staff [13]. Several methods based on colorimetry such as the 3-(4), 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and the resazurin microtiter assay cause aerosol production and are dangerous [14,15]. The accuracy and reliability of the majority of these methods have been proven; however, they cannot be readily used in developing countries owing to limitations such as the high cost; on the other hand, colorimetric redox methods and nitrate reductase assay (NRA) require further evaluation in the field. Several studies have revealed that resistance to TB is usually correlated with specific genetic mutations. Moreover, the characteristics of the mutations indicated some variation in different countries [16]. Considering these facts, there is an urgent need to establish a drug sensitivity test that is inexpensive, safe, easy-to-use, and fast. The NRA (also called the Griess method) on solid medium is a suitable susceptibility test. The NRA is a noncommercial colorimetric test based on the principle that M. tb has the capability of reducing nitrate to nitrite and that the addition of the Griess reagent changes the color of the culture medium, making the test easy to interpret [17]. The aim of this study was to compare the turnaround time, cost, ease of use, specificity, and sensitivity of the three methods: the NRA (direct and indirect), indirect proportion method (PM), and direct sequencing for testing the drug susceptibility of first- and second-line anti-tubercular drugs. In addition, we investigated the prevalence of MDR-TB and XDR-TB in Iran. Therefore, we analyzed eight drug-resistant loci, including katG, rpoB, rpsL, embB, gyrA, gyrB, rrs, and tlyA to determine whether mutations in the target genes were related to the resistance to first- and second-line drugs. 2. Materials and methods 2.1. Patients and sample processing From April 2013 to September 2016, a total of 12725 suspected TB patients (whose sputum, urine, pus, pericardial fluid, synovial fluid, bronchial fluid, and cerebrospinal fluid samples as well as tissue biopsy were collected) were referred to the Departments of Mycobacteriology & Pulmonary Research of the Pasteur Institute of Iran (PII). The current study was approved by the Ethical Committee of the PII; in addition, written informed consent was obtained from each study participant. All samples were digested and decontaminated using the Nacetyl-L-cysteine-sodium hydroxide procedure, and cultures were conducted on two slopes of Lowenstein-Jensen (L.J) medium, according to the Centers for Disease Control and Prevention (CDC) manual. Ziehl-Neelsen (ZN) acid-fast staining was used to detect the existence of acid-fast bacilli [18]. The inclusion criteria were smear (positivity score of 1 þ or more) and culture positive samples and the exclusion criteria were Mycobacterium bovis positive that was determined using phenotypic test (susceptible to thiophene-2carboxylic acid hydrazide, negative for niacin accumulation and nitrate reduction), non-tuberculosis mycobacteria positive that was identified using phenotypic (catalase 68 C, arylsulfatase, iron uptake, etc.) and genotypic (gyrB and 16SrDNA PCR-sequencing) tests, BCGosis (extraction data from patient records), and smear negative samples.
2.2. Indirect proportion method (IPM) DST by IPM on L.J medium was carried out based on the CDC protocol as described in the section “Patients and sample processing”. First-line DST was carried out using the PM on L.J medium using the following drug concentrations: 0.2 mg/mL of isoniazid (INH), 40.0 mg/mL of rifampin (RIF), 4.0 mg/mL of streptomycin (STR), and 2.0 mg/mL of ethambutol (EMB). DST against ofloxacin (OFX), kanamycin (KM), and capreomycin (CAP) was performed by the PM at a concentration of 2, 30, and 40 mg/mL, respectively. All drugs were procured from the Sigma-Aldrich Company (St. Louis, USA) [19]. Slopes of L.J medium were incubated at 37 C for up to 60 days. 2.3. Direct and indirect nitrate reductase assay (NRA) € DST by the indirect NRA was conducted as described by Angeby et al. [20] on standard L.J medium (with or without antimicrobial agents). Critical concentrations for the drugs were the same as those for the indirect PM. In the direct NRA, after the specimen decontamination by Nacetyl-L-cysteine-sodium hydroxide procedure and re-suspension of the pellet with phosphate buffer, 1:10 dilution was prepared in sterile distilled water. Thereafter, for each specimen, 0.2 ml of the diluted preparation was inoculated into the L.J medium (with or without antimicrobial agents) according to the indirect method. The reference sensitive strain M. tb CRBIP7.126 was applied as a negative control for DST and Mycobacterium intracellulare CRBIP7.168 as a negative nitrate control for NRA. 2.4. DNA extraction and PCR-sequencing Genomic DNA was extracted from samples using the High Pure PCR Template Preparation kit (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) according to the manufacturer's instructions. The loci selected for first- and second-line drug resistance included gyrA and gyrB (OFX) and rrs and tlyA (KM and CAP), rpoB (RIF), katG and inhA (INH), rrs (STR), and embC (EMB). The following primers were designed using the Primer Premier Software (ver 6.23 Primer, PREMIER Biosoft International, USA) (Table 1). PCR was carried out in a 50-mL reaction mixture containing 50 ng of DNA, 10 pM of each primer, and 25 mL of 2 PCR Master Mix. The PCR conditions for first-line drug resistance were 95 C for 8 min followed by 35 cycles of 95 C for 35 s, 67 C for 40 s, 72 C for 40 s, and 72 C for 5 min. For second-line drug resistance, the most suitable PCR conditions were: 94 C for 6 min followed by 35 cycles of 94 C for 45 s, 58 C for 35 s, 72 C for 40 s and 72 C for 5 min. The
Table 1 The primers used of this study. Gene
Primer sequence
To
From
Product size (bp)
rpoB
F: 50 -GGTCGGCGAGCTGATCCAAA-30 R:50 -TTCTTGCCCAGCGCCATCTC -30 F: 50 -AAGACGTTCGGGTTCGGCTT -30 R: 50 -TCGCTACCACGGAACGACGA -30 F: 50 -ATGACAGACACGACGTTGCC-30 R: 50 -TTTGTAGGCATCAGCGGTGC-30 F: 50 -GTCAAATCGTTTGTGCAGAAG -30 R: 50 -GCATGAACCGGAACAACAAC-30 F: 50 -CTTGTCTCATGTTGCCAGCA-30 R: 50 -CGGCTACCTTGTTACGACTT-30 F: 50 -GCGGAGAAGGGTTGAGT-30 R: 50 -GGACGACCAGCAGAACA-30 F: 50 -GTCAAGACCGCGGCTCTG-30 R:50 -TTCTTGACACCCTGCGTATC-30
1122 2116 535 1441 1 716 1255 1755 1105 1485 28 873 101 353
1141 2135 554 1460 20 735 1275 1774 1124 1504 44 889 119 372
1014
katG gyrA gyrB rrs tlyA rpsL
926 735 520 400 862 272
Please cite this article in press as: Sakhaee F, et al., A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.07.003
F. Sakhaee et al. / Biologicals xxx (2017) 1e6
PCR products were assessed using 1.5% agarose gel electrophoresis. After purifying using the High Pure PCR Product Purification Kit (Roche Diagnostics Deutschland GmbH, Mannheim, Germany), the PCR products were sequenced using an ABI Automated Sequencer (Applied Biosystems, Foster City, CA, USA). The sequencing data were examined using BioEdit v7.2.5 software (Abbott Company, Calsbad, CA, USA). 2.5. Statistical analysis SPSS version 22 software (SPSS Inc., Chicago, IL, USA) was used to compute the statistical measures of agreement, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the direct and indirect NRA using the indirect PM as the gold standard method. 3. Results 3.1. Sample selection and patient characteristics As per the biochemical tests of the 12725 suspected TB patients, 395 (3.1%) tested positive for M. tb. Out of the 395 patients, 297 (75.2%) and 98 (24.8%) were men and women, respectively. An assessment of the association between the DST results and the microscopic grade revealed that out of the 395 subjects, 63 (15.9%) had grade (þ4), 74 (18.7%) had grade (þ3), 127 (32.2%) had grade (þ2), and 131 (33.2%) had grade (þ1). Out of the 395 M. tb positive subjects, 22 (5.7%) had MDR-TB, and these included four (1.0%) XDR-TB isolates. 3.2. Indirect PM Indirect PM remains the gold standard for culture-based DST. As per the results of the indirect PM, of the 395 strains, 310, 22, and 4 were found to be sensitive to all first- and second-line drugs, MDRTB, and XDR-TB, respectively. In addition, 63 strains were resistant to one or two drugs. Out of the 63 strains, 32, 18, and 2 strains were resistant to STR, EMB, and INH, respectively; two strains were resistant to both STR and RIF; and 9 strains were resistant to both STR and EMB. 3.3. Direct NRA and indirect NRA The results of both the methods were similar. Out of the 310
3
sensitive strains, 10 strains were different. Five strains were resistant to STR, and three isolates were resistant to EMB. Two strains sensitive to INH and KM as per the PM were falsely identified as being resistant by the NRA. As per the NRA, two and one of the MDR-TB strains were found to be sensitive to INH and RIF, respectively. For STR, there were 17 and 5 isolates resistant and sensitive as per the indirect PM, while in NRA, 15 and 7 strains were resistant and sensitive, respectively. Consequently, NRA showed two false negative results. Of the 22 MDR-TB isolates, 13 tested resistant and nine tested sensitive to EMB using the indirect PM. As per the results of the NRA for EMB, 17 and 5 isolates were resistant and sensitive, respectively. As a result, four isolates, recognized as being sensitive by the indirect PM, were falsely identified as resistant by the NRA. Out of the 22 isolates for second-line drugs, 12 tested resistant and 10 tested sensitive to OFX by the indirect PM. The results of the NRA for OFX were similar to those of the indirect PM in all the strains. Therefore, we determined a perfect agreement with the gold-standard indirect PM for OFX. For CAP, the indirect PM showed 12 resistant and 10 sensitive isolates, while the NRA showed 10 resistant and 12 sensitive isolates. For KM, seven strains were correctly identified as resistant by both methods, and 15 strains indicated discordant results. The sensitivity was 91.7% for INH, 95.8% for RIF, 73.3% for STR, 82.5% for EMB, 100.0% for OFX, 83.3% for CAP, and 100.0% for KAN. The specificity was more than 98.0% for all the drugs. The PPV was 100.0% for RIF, OFX, and KAN; 95.7% for INH; 89.8% for STR; 82.5% for EMB; and 70.0% for KAN. The NPV was higher than 95.4% for all first- and second-line drugs. The agreements were 99.2%, 99.8%, 94.7%, 89.4%, 100.0%, 99.5%, and 99.2% for INH, RIF, STR, EMB, OFX, CAP, and KAN, respectively (Table 2). 3.4. PCR-sequencing In all RIF-resistant strains, mutations were found in the 531, 526, 525, and 511 positions. In the INH-resistant strains, all strains had a mutation at the katG codon (141, 309, and 315 positions). Among the STR-resistant isolates, all strains had mutations in the rpsL codon (43 and 88 positions). In EMB-resistant strains, the majority of the mutations were at position 306 or 406 of embB. In all FQ-resistant isolates, mutations were observed in the quinolone resistance-determining regions (QRDRs) of gyrA at codon 94; in gyrB, three FQ-resistant isolates had a mutation at codon 495. The most common mutation was observed at position
Table 2 Sensitivity, Specificity, PPV, NPV and agreement of the NRA compared with those of the IPM for M. tuberculosis. Drug
INH RIF STR EMB OFX CAP KAN
IPM
S R S R S R S R S R S R S R
Nitrate Reductase Assay (NRA) S
R
Sensitivity (%) (95% CI)
Specificity (%) (95% CI)
PPV (%) (95% CI)
NPV (%) (95% CI)
Agreement (%)
370 2 371 1 330 16 348 7 383 0 383 2 385 0
1 22 0 23 5 44 7 33 0 12 0 10 3 7
91.7 (73.0e99.0)
99.7 (98.5e100.0)
95.7 (78.1e99.9)
99.5 (98.1e99.9)
99.2
95.8 (78.9e99.9)
100.0 (99e100.0)
100.0 (85.2e100.0)
99.7 (98.5e100.0)
99.8
73.3 (60.3e83.9)
98.5 (96.6e99.5)
89.8 (77.8e96.6)
95.4 (92.6e97.3)
94.7
82.5 (67.2e92.7)
98.0 (96.2e99.2)
82.5 (67.2e92.7)
98.0 (96.0e99.2)
89.4
100.0 (73.5e100.0)
100.0 (99.0e100.0)
100.0 (73.5e100.0)
100.0 (99.0e100.0)
100.0
83.3 (51.6e97.9)
100.0 (99.0e100.0)
100.0 (69.2e100.0)
99.5 (98.1e99.9)
99.5
100.0 (59e100.0)
99.2 (97.8e99.8)
70.0 (34.8e93.3)
100.0 (99.0e100.0)
99.2
R: Resistant; S: Susceptible; PPV: positive predictive value; NPV: negative predictive value; INH: isoniazid; RIF: rifampicin; STR: streptomycin; EMB: ethambutol; OFX: Ofloxacin; CAP: Capreomycin; KM: Kanamycin.
Please cite this article in press as: Sakhaee F, et al., A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.07.003
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F. Sakhaee et al. / Biologicals xxx (2017) 1e6
Table 3 Mutation in first- and second-line in resistant Mycobacterium tuberculosis. Gene Codon Number Amino acid changes rpoB
of incubation. Turnaround time, as per the AFB score, revealed that 95% of the samples with a positivity score of þ1 were positive on day 10. The median time for this development was 10 days (range 10e18 days) for the direct NRA and the indirect NRA. PCRsequencing results were obtained in up to 14 days.
531 526 525 516
10 4 3 4
TCG-TTG (Ser-Leu) CAC-TAC (His-Tyr) ACC-CCC (Thr-Pro) GAC-GGC (Asp-Val)
141 309 315
5 4 15
TTG-TTC (Leu-Phe) GGT-GTT (Gly-Val) AGC-(ACC, AGG, GCC, ACC) (Ser-Thr) (Ser-Arg) (Ser-Gly) (Ser-Asp)
The estimated costs of DST using the direct NRA, indirect NRA, indirect PM, and PCR-sequencing were US$3.85, US$4.10, US$4.55, and US$12.35, respectively. This estimation does not include the cost of equipment and manpower.
42 18
43 88
AAG-AGG (Lys-Arg) AAG-(AGG, CAG, ACG) (Lys-Arg) (Lys-Gln) (Lys-Thr)
4. Discussion
306
32
406
8
ATG-(ATA, GTG, CTG, ATT) (Met-Ile) (Met-Val) (Met-Leu) (Met-Ile) GGC-(GAC, GCC) (Gly-Asp) (Gly-Ala)
94
10
GAC-(GGC, AAC) (Asp-Gly) (Asp-Asn) (75.0% vs. 25.0
495
3
GAC-CAC (Asp-His)
1401
6
A1401G
118 160
6 3
CTG-CGG (Leu-Arg) TTG-TGG (Leu-Trp)
3.6. Estimate the cost of testing
katG
rpsL
embB
gyrA gyrB rrs tlyA
1401 (adenine to guanine) of the rrs gene in KM- and CAP-resistant isolates. All strains had mutations in tlyA, which correlated with resistance to capreomycin alone, at codons 118 and 160 (Table 3). Compared with the results of the indirect PM, the sensitivity of DNA sequencing for identifying RIF, INH, STR, EMB, KAN, CAP, and OFX resistance in the current study was 95.5%, 90.1%, 88.2%, 100.0%, 83.3%, 85.7%, and 75.0%, respectively. The specificity was more than 85.7% for all drugs (Table 4). 3.5. Turn-around time of results More results were obtained within the first 40 days of the study. Smear-positive samples with a positivity score of more than þ1 became positive in less than 40 days and those with grades of þ1 became positive after 40 days. For the indirect PM, the median time for this development was 38 days (range 28e64 days). Out of the 395 isolates that were subjected to the NRA during the study period, 328 (83.0%) strains showed positive results on the 10th day, 54 (13.7%) on the 14th day, and 13 (3.3%) on the 18th day
Table 4 Evaluation of DNA sequencing of seven drug resistance-associated loci and phenotypic DST. Number of isolates Drugs Gene Resistant
Sensitive/resistant Accuracy value to one or two drugs
With Without With Without Sensitivity Specificity mutation mutation mutation mutation (%) (%) RIF INH STR EMB OFX KM CAP
rpoB katG rpsL embB gyrA gyrB rrs tlyA
21 20 15 13 10 3 6 9
1 2 2 0 2 9 1 3
3 2 53 27 0 0 2 4
370 371 320 343 370 370 371 369
95.5 90.1 88.2 100.0 83.3 33.3 85.7 75.0
99.1 99.4 85.7 92.7 100.0 100.0 99.4 98.6
INH: isoniazid; RIF: rifampicin; STR: streptomycin; EMB: ethambutol; OFX: Ofloxacin; CAP: Capreomycin; KM: Kanamycin.
In recent years, the increase in the MDR-/XDR-TB strains has become a cause for concern. A rapid and reliable DST method is required to curb the global incidence of MDR-/XDR-TB. In the current study, DST with the NRA method that involves culture isolates or direct inoculation of the positive sample is reproducible and simple. There was excellent agreement between the results of the NRA and the indirect PM. RIF and INH, along with OFX and injectable drugs (KM and CAP) are the most important drugs for the treatment of MDR-/XDR-TB. NRA sensitivities for RIF and INH were 91.7% and 95.8%, respectively, while the specificity was >99.0% for both the drugs. In our study, there was complete agreement between the NRA and the indirect PM for OFX. As OFX is the most important second-line drug in the treatment of XDR-TB and is considered a selective marker for detecting XDR-TB, the results of this study further validate the utilization of the NRA as a routine procedure for screening XDR-TB. This result was consistent with those of many previous studies that suggested that NRA can be a powerful assay method for rapid detection of MDR-TB strains [21,22]. In this study, we evaluated drug resistance using PCRsequencing as a molecular method. Molecular procedures to determine mutations correlated with drug resistance can remarkably reduce diagnostic delay, and sometimes, have proven to be more specific than the PM. Before the large-scale implementation of molecular methods in determining first- and second-line drug resistance, it is necessary to identify the genes involved in drug resistance [23]. Mutations in the katG are considered the reason for most of the INH resistance in M. tb isolates. In our study, the sensitivity for katG was 90.1%. This sensitivity is consistent with that reported in one previous study [24], however, it differed from that in another past research [16]. Moreover, some isolates possessed no mutation in the katG gene, which suggests that there may be alternative mechanisms of INH resistance, such as mutation at the inhA and oxyReahpC promoter regions or an active drug efflux pump [16]. Mutations in the RIF-resistant determining region (RDRR) of rpoB are observed in more than 95% of RIF-resistant isolates. Within the rpoB RRDR, there are three specific mutation sites (codons 531, 526, and 516) wherein the majority of the mutations occur [16,24]. The current study confirmed these results; 85.7% of the MDR-TB isolates had a mutation in one of these three codons. In addition, our results indicated a sensitivity of 95.5% in MDR-TB for phenotypic RIF resistance; this result is in agreement with that of a previous study [16]. OFX, KAN, and CAP are important second-line drugs for the treatment of MDR-TB patients. DNA gyrase, encoded by gyrA and gyrB, is inhibited by fluoroquinolone drugs. Therefore, mutations in both genes lead to fluoroquinolone resistance. In the gyrA QRDR region, mutation in codons 94, 90, and 91 is the major cause of fluoroquinolone resistance [16,25]. In our study, 100.0% of OFX-
Please cite this article in press as: Sakhaee F, et al., A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.07.003
F. Sakhaee et al. / Biologicals xxx (2017) 1e6
resistant isolates had mutation in codon 94, suggesting that mutation in codon 94 of gyrA is significant for determining OFX resistance among XDR-TB isolates in Iran. Resistance to KAN is frequently due to mutations in the rrs coding for 16S ribosomal RNA. Codon A1401G of rrs has been recognized as the primary cause of KAN resistance that associates with high-level resistance [26]. In the current study, in contrast to the findings of other studies [16,25], 100.0% of the KAN resistance was associated with mutation at the A1401G position. Hence, crossresistance among KAM may be predicted on the basis of mutation in the A1401G codon. This difference can probably be due to the difference in the geographical areas. Several mechanisms, such as mutation in eis, play a role in KAN resistance. Four mutations were recognized in KAN-sensitive strains (A1449G, C1459G, G1473T and T1491A), which were not reported to create KAN resistance in previous studies [16,27]. Compared with the results of phenotypic tests, the specificity for identifying RIF, INH, STR, EMB, KAN, CAP, and OFX resistance by DNA-sequencing in the current study was 95.5%, 90.1%, 88.2%, 100.0%, 83.3%, 85.7%, and 75.0%, respectively, in concordance with the range of 84.8%e100.0% reported by earlier studies [24,28,29]. It is noteworthy that some portions of first- and second-line drug resistance cannot be explained. Therefore, whole-genome sequencing might be needed to identify the additional sites involved in drug resistance and to improve the performance of the sequencing-based methods [16,23]. In the current study, the mean turnaround time with the NRA, according to the AFB score was different; however, most results were obtained within 10 days. This is one of the most important advantages of this method. In the event that, indirect PM require on average of 4 or 5 weeks. However, the majority of the positive results (83.0%) were acquired within 10 days, and only 13.7% were obtained in 14 days. Further reductions in the turnaround time are practically possible if another reading is taken in 18 days, as proposed in the main protocol [30]. These results were concordant with those of several studies that reported a mean turnaround time of 6e12 days with indirect NRA and 14e21 days with direct NRA [3,31]. However, there is a gain of 2e4 weeks in DST. If drug resistance is rapidly detected, patients with MDR-/XDR-TB will get an efficient drug therapy regimen and thus will be less infectious to the community. The reduction in the turnaround time necessary to obtain susceptibility results is of fundamental importance [32]. Cost is a significant factor in low-income areas where this disease is most common. In the current study, the cost per sample was lower when the NRA and indirect NRA were used as compared to that when indirect PM and PCR-sequencing were used. In agreement with our study, several studies have shown that the NRA is cheaper than other methods [12,33]. Moreover, when compared to the indirect PM, these methods have advantages, including a lower turnaround time, lower cost, ease of performance, simplicity, highly trained personnel, no requirement for any proprietary equipment or costly reagents, and greater biosafety (because there is no need to perform serial dilutions), making it a widely used method for DST [31,34e36]. The results of a meta-analysis regarding NRA conducted by Coban et al. stated that this method is a reliable, cost-effective, and rapid DST colorimetric test; the study proposed the use of this method for the detection of MDR-/XDR-TB [32]. Also, it seems to be good options for TB diagnostic laboratories, and especially for laboratories lacking access to sophisticated equipment, rapid molecular methods, as is often the case in laboratories in resource-limited countries [36]. In addition, the results of DNA-sequencing indicated that it could detect high rates of first- and second-line drug mutations among MDR-/XDR-TB isolates in Iran. These results are useful for designing rapid diagnostic methods for fast detection of MDR-/
5
XDR-TB, such as gene chips, multiplex allele-specific PCR, and reverse dot blot hybridization methods as well as more suitable anti-TB therapy regimens, thereby aiding in reducing the transmission of MDR-/XDR-TB [37e39]. In conclusion, considering the inherent advantages of the direct and indirect NRA, including low cost, high reproducibility, quickness, ease of performance, and no requirement of expensive reagents and equipment, it has the potential to be a useful phenotypic tool for rapid DST of M. tb in developing countries with modest laboratory facilities. Moreover, since phenotypic DST is the gold standard for detecting drug resistance in MDR-/XDR-TB and no replacement molecular technique has yet been established, PCRsequencing, which provides rapid and accurate screening for most of the gene mutations linked to MDR-/XDR-TB, can currently be used to limit the emergence of MDR-/XDR-TB isolates for better management of TB drug resistance in Iran. Acknowledgements The authors would like to thank TB staff members from the Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran. References [1] World Health Organization. Global tuberculosis report. 2015. [2] Tasbiti AH, Yari S, Ghanei M, Shokrgozar MA, Fateh A, Bahrmand A. Low level of extensively drug-resistant tuberculosis among MDR-TB isolates and its relationship to risk factors: surveillance in Tehran-Iran, 2006e2014. Osong Public Health Res Perspect 2017;8:116e23. [3] Kammoun S, Smaoui S, Marouane C, Slim L, Messadi-Akrout F. Drug susceptibility testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on microscopy-positive sputum samples. Int J Mycobacteriol 2015;4:202e6. € [4] Musa HR, Ambroggi M, Souto A, Angeby KK. Drug susceptibility testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on microscopy-positive sputum samples. J Clin Microbiol 2005;43:3159e61. [5] Ani AE, Daylop Y, Agbaji O, Idoko J. Drug susceptibility test of Mycobacterium tuberculosis by nitrate reductase assay. J Infect Dev Ctries 2009;3:016e9. [6] Rodrigues C, Shenai S, Almeida D, Sadani M, Goyal N, Vadher C, et al. Use of bactec 460 TB system in the diagnosis of tuberculosis. Indian J Med Microbiol 2007;25:32e7. [7] Martin A, Portaels F, Palomino JC. Colorimetric redox-indicator methods for the rapid detection of multidrug resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis. J Antimicrob Chemother 2007;59: 175e83. [8] Brent AJ, Mugo D, Musyimi R, Mutiso A, Morpeth S, Levin M, et al. Performance of the MGIT TBc identification test and meta-analysis of MPT64 assays for identification of the Mycobacterium tuberculosis complex in liquid culture. J Clin Microbiol 2011;49:4343e6. [9] Freixo I, Caldas P, Martins F, Brito R, Ferreira R, Fonseca L, et al. Evaluation of Etest strips for rapid susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 2002;40:2282e4. [10] Bai Y, Wang Y, Shao C, Hao Y, Jin Y. GenoType MTBDR plus assay for rapid detection of multidrug resistance in Mycobacterium tuberculosis: a metaanalysis. PLoS one 2016;11:e0150321. [11] S¸kenders G, Holtz T, Riekstina V, Leimane V. Implementation of the INNO-LiPA Rif. TB® line-probe assay in rapid detection of multidrug-resistant tuberculosis in Latvia. Int J Tuberc Lung Dis 2011;15:1546e53. [12] Kohli A, Bashir G, Fatima A, Jan A, Ahmad J. Rapid drug-susceptibility testing of Mycobacterium tuberculosis clinical isolates to first-line antitubercular drugs by nitrate reductase assay: a comparison with proportion method. Int J Mycobacteriol 2016;5:469e74. [13] Minion J, Leung E, Menzies D, Pai M. Microscopic-observation drug susceptibility and thin layer agar assays for the detection of drug resistant tuberculosis: a systematic review and meta-analysis. Lancet Infect Dis 2010;10: 688e98. €rner H. Use of 3-(4, 5-Dimethylthiazol-2[14] Mshana RN, Tadesse G, Abate G, Mio yl)-2, 5-diphenyl tetrazolium bromide for rapid detection of RifampinResistantMycobacterium tuberculosis. J Clin Microbiol 1998;36:1214e9. [15] Affolabi D, Sanoussi ND, Odoun M, Martin A, Koukpemedji L, Palomino JC, et al. Rapid detection of multidrug-resistant Mycobacterium tuberculosis in Cotonou (Benin) using two low-cost colorimetric methods: resazurin and nitrate reductase assays. J Med Microbiol 2008;57:1024e7. [16] Chen Y, Zhao B, H-c Liu, Sun Q, Zhao X-q Liu Z-g, et al. Prevalence of mutations conferring resistance among multi-and extensively drug-resistant Mycobacterium tuberculosis isolates in China. J Antibiot (Tokyo) 2016;69:149e52.
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[17] Griess P. Bemerkungen zu der Abhandlung der HH. Weselsky und Benedikt “Ueber einige Azoverbindungen”. Berichte Dtsch Chem Ges 1879;12:426e8. [18] Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory: US Department of Health and Human Services. Centers for Disease Control: Public Health Service; 1985. [19] World Health Organization. Guidelines for drug susceptibility testing for second-line anti-tuberculosis drugs for DOTS-plus. 2001. € [20] Angeby KK, Klintz L, Hoffner SE. Rapid and inexpensive drug susceptibility testing of Mycobacterium tuberculosis with a nitrate reductase assay. J Clin Microbiol 2002;40:553e5. [21] Martin A, Palomino JC, Portaels F. Rapid detection of ofloxacin resistance in Mycobacterium tuberculosis by two low-cost colorimetric methods: resazurin and nitrate reductase assays. J Clin Microbiol 2005;43:1612e6. [22] Abilleira F, Brum C, von Groll A, da Silva PE. Evaluation of direct microplate nitrate reductase assay as a rapid method for the detection of multiple and dica 2015;35:285e91. extensively tuberculosis drug resistance. Biome [23] Hu Y, Hoffner S, Wu L, Zhao Q, Jiang W, Xu B. Prevalence and genetic characterization of second-line drug-resistant and extensively drug-resistant Mycobacterium tuberculosis in Rural China. Antimicrob Agents Chemother 2013;57:3857e63. [24] Campbell PJ, Morlock GP, Sikes RD, Dalton TL, Metchock B, Starks AM, et al. Molecular detection of mutations associated with first-and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis. Antimicrob Agents Chemother 2011;55:2032e41. [25] Zhang Z, Lu J, Wang Y, Pang Y, Zhao Y. Prevalence and molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis isolates in China. Antimicrob Agents Chemother 2014;58:364e9. [26] Du Q, Dai G, Long Q, Yu X, Dong L, Huang H, et al. Mycobacterium tuberculosis rrs A1401G mutation correlates with high-level resistance to kanamycin, amikacin, and capreomycin in clinical isolates from mainland China. Diagn Microbiol Infect Dis 2013;77:138e42. [27] Zaunbrecher MA, Sikes RD, Metchock B, Shinnick TM, Posey JE. Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci 2009;106:20004e9. € m A, Morcillo N, Imperiale B, Hoffner SE, Jure en P. Detection of first[28] Engstro and second-line drug resistance in Mycobacterium tuberculosis Clinical
Isolates using Pyrosequencing. J Clin Microbiol 2012. JCM. 06664e11. [29] Yuan X, Zhang T, Kawakami K, Zhu J, Li H, Lei J, et al. Molecular characterization of multidrug and extensively drug-resistant Mycobacterium tuberculosis strains in Jiangxi, China. J Clin Microbiol 2012;5. 06860e11. [30] Safonova S, Blinova L, Golyshevskaya V. Griess' method for M. tuberculosis drug susceptibility testing implementation, validation and programmatic value. Int J Tuberc Lung Dis 2001;5:S12. [31] Gupta A, Sen MR, Mohapatra TM, Anupurba S. Evaluation of the performance of nitrate reductase assay for rapid drug-susceptibility testing of Mycobacterium tuberculosis in north India. J Health Popul Nutr 2011;29:20. [32] Coban AY, Deveci A, Sunter AT, Martin A. Nitrate reductase assay for rapid detection of isoniazid, rifampin, ethambutol, and streptomycin resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis. J Clin Microbiol 2014;52:15e9. [33] Bwanga F, Haile M, Joloba ML, Ochom E, Hoffner S. Direct nitrate reductase assay versus microscopic observation drug susceptibility test for rapid detection of MDR-TB in Uganda. PLoS One 2011;6:e19565. [34] Shikama M, Ferro e Silva R, Villela G, Sato D, Martins M, Giampaglia C, et al. Multicentre study of nitrate reductase assay for rapid detection of rifampicinresistant M. tuberculosis. Int J Tuberc Lung Dis 2009;13:377e80. [35] Martin A, Imperiale B, Ravolonandriana P, Coban AY, Akgunes A, Ikram A, et al. Prospective multicentre evaluation of the direct nitrate reductase assay for the rapid detection of extensively drug-resistant tuberculosis. J Antimicrob Chemother 2014;69:441e4. [36] Palomino JC, Martin A, Portaels F. Rapid drug resistance detection in Mycobacterium tuberculosis: a review of colourimetric methods. Clin Microbiol Infect 2007;13:754e62. [37] Suresh N, Singh UB, Arora J, Pande JN, Seth P, Samantaray JC. Rapid detection of rifampicin-resistant Mycobacterium tuberculosis by in-house, reverse line blot assay. Diagn Microbiol Infect Dis 2006;56:133e40. [38] Guo Y, Zhou Y, Wang C, Zhu L, Wang S, Li Q, et al. Rapid, accurate determination of multidrug resistance in M. tuberculosis isolates and sputum using a biochip system. Int J Tuberc Lung Dis 2009;13:914e20. [39] Evans J, Segal H. Novel multiplex allele-specific PCR assays for the detection of resistance to second-line drugs in Mycobacterium tuberculosis. J Antimicrob Chemother 2010;65:897e900.
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A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis Fatemeh Sakhaee a, 1, Morteza Ghazanfari a, 1, Nayereh Ebrahimzadeh a, Farzam Vaziri a, b, Fatemeh Rahimi Jamnani a, b, Mehdi Davari a, Safoora Gharibzadeh c, d, Fatemeh Hemati Mandjin a, Abolfazl Fateh a, b, *, Seyed Davar Siadat a, b a
Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran Research Centre for Emerging and Reemerging Infectious Diseases, Pasteur Institute of Iran, Tehran, Iran d Department of Epidemiology and Biostatistics, Pasteur Institute of Iran, Tehran, Iran b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 May 2017 Received in revised form 4 July 2017 Accepted 8 July 2017 Available online xxx
This study aimed to evaluate the frequency of resistance to first- and second-line drugs using phenotypic and genotypic methods and its correlation with resistance-linked mutations in Mycobacterium tuberculosis (M. tb) isolated in Iran. Three different methods, including the indirect proportion method(PM), direct and indirect nitrate reductase assay(NRA), and direct sequencing were used to assess drug resistance. In this study, sensitivity, specificity, agreement, costs, and turnaround time of these methods were compared in 395 smear positive isolates. Compared to the PM, the NRA and the direct sequencing methods demonstrated higher specificity, sensitivity, and agreement for detection of all anti-tuberculosis drugs. The NRA had a short turnaround time and was more cost-effective than the other methods. Mutations in codon 531 in rpoB, 315 in katG, 18 in rpsL, and 306 in embB were associated with high-level resistance to the first-line drugs, and mutations in codon 94 in gyrA, and A1401G in rrs were correlated with resistance to the second-line drugs. We found that the NRA is a highly sensitive, specific, inexpensive, and rapid test with strong potential to be a useful and interesting alternative tool, particularly in low-income countries. In addition, these molecular data will be helpful for developing new molecular methods for detecting first- and second-line drug-resistant M. tb. © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: First- and second-line drugs Mycobacterium tuberculosis Nitrate reductase assay Proportion method Direct sequencing
1. Introduction Tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis (M. tb) and is still a major global health problem in most parts of the world. Approximately 9.6 million new cases and 1.5 million deaths were reported in 2014. In 2015, about 480,000 new cases of multidrug-resistant TB (MDR-TB) were estimated, and 250,000 deaths were reported; majority of these cases were in Asia. About 9.7% of MDR-TB patients develop extensively drug-resistant TB (XDR-TB). Thus far, at least 117 countries in the world have reported at least one case of XDR-TB [1]. MDR-TB is defined as M. tb that develops resistance to both isoniazid (INH) and rifampin (RIF),
* Corresponding author. Departments of Mycobacteriology & Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran. E-mail address: [email protected] (A. Fateh). 1 These authors contributed equally to this work.
while XDR-TB is defined as MDR-TB that is resistant to any member of the fluoroquinolone family and at least one of the three secondline injectable drugs [2]. For the successful management of MDR-TB and XDR-TB, an inexpensive, easy-to-use, and rapid drug susceptibility test (DST) is required for fast initiation of the most effective antibiotic therapy. “Standard procedures for DST, such as the indirect or direct proportion procedure, are easy to perform; however, these methods have the disadvantages of being very time consuming (4e6 weeks) and requiring trained and experienced laboratory technicians. In addition to being expensive, molecular methods such as the Xpert MTB/RIF® and line probe assay need expert laboratory technicians. Further, these methods may not be able to distinguish the active infection causing organisms from the dead organisms by assessing their DNA. Although these methods have shorter turnaround times, it appears that low-income conventional laboratories are unable to perform these procedures for M. tb detection [3e5].
http://dx.doi.org/10.1016/j.biologicals.2017.07.003 1045-1056/© 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sakhaee F, et al., A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.07.003
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In the past few years, several methods for rapid DST have been developed and evaluated, such as the radiometric method BACTEC 460-TB [6], colorimetric redox method [7], MGIT TBc Identification Test [8], E-Tests [9], and Genotype MTBDRplus [10] and INNO-LiPA [11] that need expensive substance and equipment. So, in most developing countries is not possible [12]. The microscopic observation DST, although inexpensive and fast, requires extensive training of the staff [13]. Several methods based on colorimetry such as the 3-(4), 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and the resazurin microtiter assay cause aerosol production and are dangerous [14,15]. The accuracy and reliability of the majority of these methods have been proven; however, they cannot be readily used in developing countries owing to limitations such as the high cost; on the other hand, colorimetric redox methods and nitrate reductase assay (NRA) require further evaluation in the field. Several studies have revealed that resistance to TB is usually correlated with specific genetic mutations. Moreover, the characteristics of the mutations indicated some variation in different countries [16]. Considering these facts, there is an urgent need to establish a drug sensitivity test that is inexpensive, safe, easy-to-use, and fast. The NRA (also called the Griess method) on solid medium is a suitable susceptibility test. The NRA is a noncommercial colorimetric test based on the principle that M. tb has the capability of reducing nitrate to nitrite and that the addition of the Griess reagent changes the color of the culture medium, making the test easy to interpret [17]. The aim of this study was to compare the turnaround time, cost, ease of use, specificity, and sensitivity of the three methods: the NRA (direct and indirect), indirect proportion method (PM), and direct sequencing for testing the drug susceptibility of first- and second-line anti-tubercular drugs. In addition, we investigated the prevalence of MDR-TB and XDR-TB in Iran. Therefore, we analyzed eight drug-resistant loci, including katG, rpoB, rpsL, embB, gyrA, gyrB, rrs, and tlyA to determine whether mutations in the target genes were related to the resistance to first- and second-line drugs. 2. Materials and methods 2.1. Patients and sample processing From April 2013 to September 2016, a total of 12725 suspected TB patients (whose sputum, urine, pus, pericardial fluid, synovial fluid, bronchial fluid, and cerebrospinal fluid samples as well as tissue biopsy were collected) were referred to the Departments of Mycobacteriology & Pulmonary Research of the Pasteur Institute of Iran (PII). The current study was approved by the Ethical Committee of the PII; in addition, written informed consent was obtained from each study participant. All samples were digested and decontaminated using the Nacetyl-L-cysteine-sodium hydroxide procedure, and cultures were conducted on two slopes of Lowenstein-Jensen (L.J) medium, according to the Centers for Disease Control and Prevention (CDC) manual. Ziehl-Neelsen (ZN) acid-fast staining was used to detect the existence of acid-fast bacilli [18]. The inclusion criteria were smear (positivity score of 1 þ or more) and culture positive samples and the exclusion criteria were Mycobacterium bovis positive that was determined using phenotypic test (susceptible to thiophene-2carboxylic acid hydrazide, negative for niacin accumulation and nitrate reduction), non-tuberculosis mycobacteria positive that was identified using phenotypic (catalase 68 C, arylsulfatase, iron uptake, etc.) and genotypic (gyrB and 16SrDNA PCR-sequencing) tests, BCGosis (extraction data from patient records), and smear negative samples.
2.2. Indirect proportion method (IPM) DST by IPM on L.J medium was carried out based on the CDC protocol as described in the section “Patients and sample processing”. First-line DST was carried out using the PM on L.J medium using the following drug concentrations: 0.2 mg/mL of isoniazid (INH), 40.0 mg/mL of rifampin (RIF), 4.0 mg/mL of streptomycin (STR), and 2.0 mg/mL of ethambutol (EMB). DST against ofloxacin (OFX), kanamycin (KM), and capreomycin (CAP) was performed by the PM at a concentration of 2, 30, and 40 mg/mL, respectively. All drugs were procured from the Sigma-Aldrich Company (St. Louis, USA) [19]. Slopes of L.J medium were incubated at 37 C for up to 60 days. 2.3. Direct and indirect nitrate reductase assay (NRA) € DST by the indirect NRA was conducted as described by Angeby et al. [20] on standard L.J medium (with or without antimicrobial agents). Critical concentrations for the drugs were the same as those for the indirect PM. In the direct NRA, after the specimen decontamination by Nacetyl-L-cysteine-sodium hydroxide procedure and re-suspension of the pellet with phosphate buffer, 1:10 dilution was prepared in sterile distilled water. Thereafter, for each specimen, 0.2 ml of the diluted preparation was inoculated into the L.J medium (with or without antimicrobial agents) according to the indirect method. The reference sensitive strain M. tb CRBIP7.126 was applied as a negative control for DST and Mycobacterium intracellulare CRBIP7.168 as a negative nitrate control for NRA. 2.4. DNA extraction and PCR-sequencing Genomic DNA was extracted from samples using the High Pure PCR Template Preparation kit (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) according to the manufacturer's instructions. The loci selected for first- and second-line drug resistance included gyrA and gyrB (OFX) and rrs and tlyA (KM and CAP), rpoB (RIF), katG and inhA (INH), rrs (STR), and embC (EMB). The following primers were designed using the Primer Premier Software (ver 6.23 Primer, PREMIER Biosoft International, USA) (Table 1). PCR was carried out in a 50-mL reaction mixture containing 50 ng of DNA, 10 pM of each primer, and 25 mL of 2 PCR Master Mix. The PCR conditions for first-line drug resistance were 95 C for 8 min followed by 35 cycles of 95 C for 35 s, 67 C for 40 s, 72 C for 40 s, and 72 C for 5 min. For second-line drug resistance, the most suitable PCR conditions were: 94 C for 6 min followed by 35 cycles of 94 C for 45 s, 58 C for 35 s, 72 C for 40 s and 72 C for 5 min. The
Table 1 The primers used of this study. Gene
Primer sequence
To
From
Product size (bp)
rpoB
F: 50 -GGTCGGCGAGCTGATCCAAA-30 R:50 -TTCTTGCCCAGCGCCATCTC -30 F: 50 -AAGACGTTCGGGTTCGGCTT -30 R: 50 -TCGCTACCACGGAACGACGA -30 F: 50 -ATGACAGACACGACGTTGCC-30 R: 50 -TTTGTAGGCATCAGCGGTGC-30 F: 50 -GTCAAATCGTTTGTGCAGAAG -30 R: 50 -GCATGAACCGGAACAACAAC-30 F: 50 -CTTGTCTCATGTTGCCAGCA-30 R: 50 -CGGCTACCTTGTTACGACTT-30 F: 50 -GCGGAGAAGGGTTGAGT-30 R: 50 -GGACGACCAGCAGAACA-30 F: 50 -GTCAAGACCGCGGCTCTG-30 R:50 -TTCTTGACACCCTGCGTATC-30
1122 2116 535 1441 1 716 1255 1755 1105 1485 28 873 101 353
1141 2135 554 1460 20 735 1275 1774 1124 1504 44 889 119 372
1014
katG gyrA gyrB rrs tlyA rpsL
926 735 520 400 862 272
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F. Sakhaee et al. / Biologicals xxx (2017) 1e6
PCR products were assessed using 1.5% agarose gel electrophoresis. After purifying using the High Pure PCR Product Purification Kit (Roche Diagnostics Deutschland GmbH, Mannheim, Germany), the PCR products were sequenced using an ABI Automated Sequencer (Applied Biosystems, Foster City, CA, USA). The sequencing data were examined using BioEdit v7.2.5 software (Abbott Company, Calsbad, CA, USA). 2.5. Statistical analysis SPSS version 22 software (SPSS Inc., Chicago, IL, USA) was used to compute the statistical measures of agreement, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the direct and indirect NRA using the indirect PM as the gold standard method. 3. Results 3.1. Sample selection and patient characteristics As per the biochemical tests of the 12725 suspected TB patients, 395 (3.1%) tested positive for M. tb. Out of the 395 patients, 297 (75.2%) and 98 (24.8%) were men and women, respectively. An assessment of the association between the DST results and the microscopic grade revealed that out of the 395 subjects, 63 (15.9%) had grade (þ4), 74 (18.7%) had grade (þ3), 127 (32.2%) had grade (þ2), and 131 (33.2%) had grade (þ1). Out of the 395 M. tb positive subjects, 22 (5.7%) had MDR-TB, and these included four (1.0%) XDR-TB isolates. 3.2. Indirect PM Indirect PM remains the gold standard for culture-based DST. As per the results of the indirect PM, of the 395 strains, 310, 22, and 4 were found to be sensitive to all first- and second-line drugs, MDRTB, and XDR-TB, respectively. In addition, 63 strains were resistant to one or two drugs. Out of the 63 strains, 32, 18, and 2 strains were resistant to STR, EMB, and INH, respectively; two strains were resistant to both STR and RIF; and 9 strains were resistant to both STR and EMB. 3.3. Direct NRA and indirect NRA The results of both the methods were similar. Out of the 310
3
sensitive strains, 10 strains were different. Five strains were resistant to STR, and three isolates were resistant to EMB. Two strains sensitive to INH and KM as per the PM were falsely identified as being resistant by the NRA. As per the NRA, two and one of the MDR-TB strains were found to be sensitive to INH and RIF, respectively. For STR, there were 17 and 5 isolates resistant and sensitive as per the indirect PM, while in NRA, 15 and 7 strains were resistant and sensitive, respectively. Consequently, NRA showed two false negative results. Of the 22 MDR-TB isolates, 13 tested resistant and nine tested sensitive to EMB using the indirect PM. As per the results of the NRA for EMB, 17 and 5 isolates were resistant and sensitive, respectively. As a result, four isolates, recognized as being sensitive by the indirect PM, were falsely identified as resistant by the NRA. Out of the 22 isolates for second-line drugs, 12 tested resistant and 10 tested sensitive to OFX by the indirect PM. The results of the NRA for OFX were similar to those of the indirect PM in all the strains. Therefore, we determined a perfect agreement with the gold-standard indirect PM for OFX. For CAP, the indirect PM showed 12 resistant and 10 sensitive isolates, while the NRA showed 10 resistant and 12 sensitive isolates. For KM, seven strains were correctly identified as resistant by both methods, and 15 strains indicated discordant results. The sensitivity was 91.7% for INH, 95.8% for RIF, 73.3% for STR, 82.5% for EMB, 100.0% for OFX, 83.3% for CAP, and 100.0% for KAN. The specificity was more than 98.0% for all the drugs. The PPV was 100.0% for RIF, OFX, and KAN; 95.7% for INH; 89.8% for STR; 82.5% for EMB; and 70.0% for KAN. The NPV was higher than 95.4% for all first- and second-line drugs. The agreements were 99.2%, 99.8%, 94.7%, 89.4%, 100.0%, 99.5%, and 99.2% for INH, RIF, STR, EMB, OFX, CAP, and KAN, respectively (Table 2). 3.4. PCR-sequencing In all RIF-resistant strains, mutations were found in the 531, 526, 525, and 511 positions. In the INH-resistant strains, all strains had a mutation at the katG codon (141, 309, and 315 positions). Among the STR-resistant isolates, all strains had mutations in the rpsL codon (43 and 88 positions). In EMB-resistant strains, the majority of the mutations were at position 306 or 406 of embB. In all FQ-resistant isolates, mutations were observed in the quinolone resistance-determining regions (QRDRs) of gyrA at codon 94; in gyrB, three FQ-resistant isolates had a mutation at codon 495. The most common mutation was observed at position
Table 2 Sensitivity, Specificity, PPV, NPV and agreement of the NRA compared with those of the IPM for M. tuberculosis. Drug
INH RIF STR EMB OFX CAP KAN
IPM
S R S R S R S R S R S R S R
Nitrate Reductase Assay (NRA) S
R
Sensitivity (%) (95% CI)
Specificity (%) (95% CI)
PPV (%) (95% CI)
NPV (%) (95% CI)
Agreement (%)
370 2 371 1 330 16 348 7 383 0 383 2 385 0
1 22 0 23 5 44 7 33 0 12 0 10 3 7
91.7 (73.0e99.0)
99.7 (98.5e100.0)
95.7 (78.1e99.9)
99.5 (98.1e99.9)
99.2
95.8 (78.9e99.9)
100.0 (99e100.0)
100.0 (85.2e100.0)
99.7 (98.5e100.0)
99.8
73.3 (60.3e83.9)
98.5 (96.6e99.5)
89.8 (77.8e96.6)
95.4 (92.6e97.3)
94.7
82.5 (67.2e92.7)
98.0 (96.2e99.2)
82.5 (67.2e92.7)
98.0 (96.0e99.2)
89.4
100.0 (73.5e100.0)
100.0 (99.0e100.0)
100.0 (73.5e100.0)
100.0 (99.0e100.0)
100.0
83.3 (51.6e97.9)
100.0 (99.0e100.0)
100.0 (69.2e100.0)
99.5 (98.1e99.9)
99.5
100.0 (59e100.0)
99.2 (97.8e99.8)
70.0 (34.8e93.3)
100.0 (99.0e100.0)
99.2
R: Resistant; S: Susceptible; PPV: positive predictive value; NPV: negative predictive value; INH: isoniazid; RIF: rifampicin; STR: streptomycin; EMB: ethambutol; OFX: Ofloxacin; CAP: Capreomycin; KM: Kanamycin.
Please cite this article in press as: Sakhaee F, et al., A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.07.003
4
F. Sakhaee et al. / Biologicals xxx (2017) 1e6
Table 3 Mutation in first- and second-line in resistant Mycobacterium tuberculosis. Gene Codon Number Amino acid changes rpoB
of incubation. Turnaround time, as per the AFB score, revealed that 95% of the samples with a positivity score of þ1 were positive on day 10. The median time for this development was 10 days (range 10e18 days) for the direct NRA and the indirect NRA. PCRsequencing results were obtained in up to 14 days.
531 526 525 516
10 4 3 4
TCG-TTG (Ser-Leu) CAC-TAC (His-Tyr) ACC-CCC (Thr-Pro) GAC-GGC (Asp-Val)
141 309 315
5 4 15
TTG-TTC (Leu-Phe) GGT-GTT (Gly-Val) AGC-(ACC, AGG, GCC, ACC) (Ser-Thr) (Ser-Arg) (Ser-Gly) (Ser-Asp)
The estimated costs of DST using the direct NRA, indirect NRA, indirect PM, and PCR-sequencing were US$3.85, US$4.10, US$4.55, and US$12.35, respectively. This estimation does not include the cost of equipment and manpower.
42 18
43 88
AAG-AGG (Lys-Arg) AAG-(AGG, CAG, ACG) (Lys-Arg) (Lys-Gln) (Lys-Thr)
4. Discussion
306
32
406
8
ATG-(ATA, GTG, CTG, ATT) (Met-Ile) (Met-Val) (Met-Leu) (Met-Ile) GGC-(GAC, GCC) (Gly-Asp) (Gly-Ala)
94
10
GAC-(GGC, AAC) (Asp-Gly) (Asp-Asn) (75.0% vs. 25.0
495
3
GAC-CAC (Asp-His)
1401
6
A1401G
118 160
6 3
CTG-CGG (Leu-Arg) TTG-TGG (Leu-Trp)
3.6. Estimate the cost of testing
katG
rpsL
embB
gyrA gyrB rrs tlyA
1401 (adenine to guanine) of the rrs gene in KM- and CAP-resistant isolates. All strains had mutations in tlyA, which correlated with resistance to capreomycin alone, at codons 118 and 160 (Table 3). Compared with the results of the indirect PM, the sensitivity of DNA sequencing for identifying RIF, INH, STR, EMB, KAN, CAP, and OFX resistance in the current study was 95.5%, 90.1%, 88.2%, 100.0%, 83.3%, 85.7%, and 75.0%, respectively. The specificity was more than 85.7% for all drugs (Table 4). 3.5. Turn-around time of results More results were obtained within the first 40 days of the study. Smear-positive samples with a positivity score of more than þ1 became positive in less than 40 days and those with grades of þ1 became positive after 40 days. For the indirect PM, the median time for this development was 38 days (range 28e64 days). Out of the 395 isolates that were subjected to the NRA during the study period, 328 (83.0%) strains showed positive results on the 10th day, 54 (13.7%) on the 14th day, and 13 (3.3%) on the 18th day
Table 4 Evaluation of DNA sequencing of seven drug resistance-associated loci and phenotypic DST. Number of isolates Drugs Gene Resistant
Sensitive/resistant Accuracy value to one or two drugs
With Without With Without Sensitivity Specificity mutation mutation mutation mutation (%) (%) RIF INH STR EMB OFX KM CAP
rpoB katG rpsL embB gyrA gyrB rrs tlyA
21 20 15 13 10 3 6 9
1 2 2 0 2 9 1 3
3 2 53 27 0 0 2 4
370 371 320 343 370 370 371 369
95.5 90.1 88.2 100.0 83.3 33.3 85.7 75.0
99.1 99.4 85.7 92.7 100.0 100.0 99.4 98.6
INH: isoniazid; RIF: rifampicin; STR: streptomycin; EMB: ethambutol; OFX: Ofloxacin; CAP: Capreomycin; KM: Kanamycin.
In recent years, the increase in the MDR-/XDR-TB strains has become a cause for concern. A rapid and reliable DST method is required to curb the global incidence of MDR-/XDR-TB. In the current study, DST with the NRA method that involves culture isolates or direct inoculation of the positive sample is reproducible and simple. There was excellent agreement between the results of the NRA and the indirect PM. RIF and INH, along with OFX and injectable drugs (KM and CAP) are the most important drugs for the treatment of MDR-/XDR-TB. NRA sensitivities for RIF and INH were 91.7% and 95.8%, respectively, while the specificity was >99.0% for both the drugs. In our study, there was complete agreement between the NRA and the indirect PM for OFX. As OFX is the most important second-line drug in the treatment of XDR-TB and is considered a selective marker for detecting XDR-TB, the results of this study further validate the utilization of the NRA as a routine procedure for screening XDR-TB. This result was consistent with those of many previous studies that suggested that NRA can be a powerful assay method for rapid detection of MDR-TB strains [21,22]. In this study, we evaluated drug resistance using PCRsequencing as a molecular method. Molecular procedures to determine mutations correlated with drug resistance can remarkably reduce diagnostic delay, and sometimes, have proven to be more specific than the PM. Before the large-scale implementation of molecular methods in determining first- and second-line drug resistance, it is necessary to identify the genes involved in drug resistance [23]. Mutations in the katG are considered the reason for most of the INH resistance in M. tb isolates. In our study, the sensitivity for katG was 90.1%. This sensitivity is consistent with that reported in one previous study [24], however, it differed from that in another past research [16]. Moreover, some isolates possessed no mutation in the katG gene, which suggests that there may be alternative mechanisms of INH resistance, such as mutation at the inhA and oxyReahpC promoter regions or an active drug efflux pump [16]. Mutations in the RIF-resistant determining region (RDRR) of rpoB are observed in more than 95% of RIF-resistant isolates. Within the rpoB RRDR, there are three specific mutation sites (codons 531, 526, and 516) wherein the majority of the mutations occur [16,24]. The current study confirmed these results; 85.7% of the MDR-TB isolates had a mutation in one of these three codons. In addition, our results indicated a sensitivity of 95.5% in MDR-TB for phenotypic RIF resistance; this result is in agreement with that of a previous study [16]. OFX, KAN, and CAP are important second-line drugs for the treatment of MDR-TB patients. DNA gyrase, encoded by gyrA and gyrB, is inhibited by fluoroquinolone drugs. Therefore, mutations in both genes lead to fluoroquinolone resistance. In the gyrA QRDR region, mutation in codons 94, 90, and 91 is the major cause of fluoroquinolone resistance [16,25]. In our study, 100.0% of OFX-
Please cite this article in press as: Sakhaee F, et al., A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.07.003
F. Sakhaee et al. / Biologicals xxx (2017) 1e6
resistant isolates had mutation in codon 94, suggesting that mutation in codon 94 of gyrA is significant for determining OFX resistance among XDR-TB isolates in Iran. Resistance to KAN is frequently due to mutations in the rrs coding for 16S ribosomal RNA. Codon A1401G of rrs has been recognized as the primary cause of KAN resistance that associates with high-level resistance [26]. In the current study, in contrast to the findings of other studies [16,25], 100.0% of the KAN resistance was associated with mutation at the A1401G position. Hence, crossresistance among KAM may be predicted on the basis of mutation in the A1401G codon. This difference can probably be due to the difference in the geographical areas. Several mechanisms, such as mutation in eis, play a role in KAN resistance. Four mutations were recognized in KAN-sensitive strains (A1449G, C1459G, G1473T and T1491A), which were not reported to create KAN resistance in previous studies [16,27]. Compared with the results of phenotypic tests, the specificity for identifying RIF, INH, STR, EMB, KAN, CAP, and OFX resistance by DNA-sequencing in the current study was 95.5%, 90.1%, 88.2%, 100.0%, 83.3%, 85.7%, and 75.0%, respectively, in concordance with the range of 84.8%e100.0% reported by earlier studies [24,28,29]. It is noteworthy that some portions of first- and second-line drug resistance cannot be explained. Therefore, whole-genome sequencing might be needed to identify the additional sites involved in drug resistance and to improve the performance of the sequencing-based methods [16,23]. In the current study, the mean turnaround time with the NRA, according to the AFB score was different; however, most results were obtained within 10 days. This is one of the most important advantages of this method. In the event that, indirect PM require on average of 4 or 5 weeks. However, the majority of the positive results (83.0%) were acquired within 10 days, and only 13.7% were obtained in 14 days. Further reductions in the turnaround time are practically possible if another reading is taken in 18 days, as proposed in the main protocol [30]. These results were concordant with those of several studies that reported a mean turnaround time of 6e12 days with indirect NRA and 14e21 days with direct NRA [3,31]. However, there is a gain of 2e4 weeks in DST. If drug resistance is rapidly detected, patients with MDR-/XDR-TB will get an efficient drug therapy regimen and thus will be less infectious to the community. The reduction in the turnaround time necessary to obtain susceptibility results is of fundamental importance [32]. Cost is a significant factor in low-income areas where this disease is most common. In the current study, the cost per sample was lower when the NRA and indirect NRA were used as compared to that when indirect PM and PCR-sequencing were used. In agreement with our study, several studies have shown that the NRA is cheaper than other methods [12,33]. Moreover, when compared to the indirect PM, these methods have advantages, including a lower turnaround time, lower cost, ease of performance, simplicity, highly trained personnel, no requirement for any proprietary equipment or costly reagents, and greater biosafety (because there is no need to perform serial dilutions), making it a widely used method for DST [31,34e36]. The results of a meta-analysis regarding NRA conducted by Coban et al. stated that this method is a reliable, cost-effective, and rapid DST colorimetric test; the study proposed the use of this method for the detection of MDR-/XDR-TB [32]. Also, it seems to be good options for TB diagnostic laboratories, and especially for laboratories lacking access to sophisticated equipment, rapid molecular methods, as is often the case in laboratories in resource-limited countries [36]. In addition, the results of DNA-sequencing indicated that it could detect high rates of first- and second-line drug mutations among MDR-/XDR-TB isolates in Iran. These results are useful for designing rapid diagnostic methods for fast detection of MDR-/
5
XDR-TB, such as gene chips, multiplex allele-specific PCR, and reverse dot blot hybridization methods as well as more suitable anti-TB therapy regimens, thereby aiding in reducing the transmission of MDR-/XDR-TB [37e39]. In conclusion, considering the inherent advantages of the direct and indirect NRA, including low cost, high reproducibility, quickness, ease of performance, and no requirement of expensive reagents and equipment, it has the potential to be a useful phenotypic tool for rapid DST of M. tb in developing countries with modest laboratory facilities. Moreover, since phenotypic DST is the gold standard for detecting drug resistance in MDR-/XDR-TB and no replacement molecular technique has yet been established, PCRsequencing, which provides rapid and accurate screening for most of the gene mutations linked to MDR-/XDR-TB, can currently be used to limit the emergence of MDR-/XDR-TB isolates for better management of TB drug resistance in Iran. Acknowledgements The authors would like to thank TB staff members from the Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran. References [1] World Health Organization. Global tuberculosis report. 2015. [2] Tasbiti AH, Yari S, Ghanei M, Shokrgozar MA, Fateh A, Bahrmand A. Low level of extensively drug-resistant tuberculosis among MDR-TB isolates and its relationship to risk factors: surveillance in Tehran-Iran, 2006e2014. Osong Public Health Res Perspect 2017;8:116e23. [3] Kammoun S, Smaoui S, Marouane C, Slim L, Messadi-Akrout F. Drug susceptibility testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on microscopy-positive sputum samples. Int J Mycobacteriol 2015;4:202e6. € [4] Musa HR, Ambroggi M, Souto A, Angeby KK. Drug susceptibility testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on microscopy-positive sputum samples. J Clin Microbiol 2005;43:3159e61. [5] Ani AE, Daylop Y, Agbaji O, Idoko J. Drug susceptibility test of Mycobacterium tuberculosis by nitrate reductase assay. J Infect Dev Ctries 2009;3:016e9. [6] Rodrigues C, Shenai S, Almeida D, Sadani M, Goyal N, Vadher C, et al. Use of bactec 460 TB system in the diagnosis of tuberculosis. Indian J Med Microbiol 2007;25:32e7. [7] Martin A, Portaels F, Palomino JC. Colorimetric redox-indicator methods for the rapid detection of multidrug resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis. J Antimicrob Chemother 2007;59: 175e83. [8] Brent AJ, Mugo D, Musyimi R, Mutiso A, Morpeth S, Levin M, et al. Performance of the MGIT TBc identification test and meta-analysis of MPT64 assays for identification of the Mycobacterium tuberculosis complex in liquid culture. J Clin Microbiol 2011;49:4343e6. [9] Freixo I, Caldas P, Martins F, Brito R, Ferreira R, Fonseca L, et al. Evaluation of Etest strips for rapid susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 2002;40:2282e4. [10] Bai Y, Wang Y, Shao C, Hao Y, Jin Y. GenoType MTBDR plus assay for rapid detection of multidrug resistance in Mycobacterium tuberculosis: a metaanalysis. PLoS one 2016;11:e0150321. [11] S¸kenders G, Holtz T, Riekstina V, Leimane V. Implementation of the INNO-LiPA Rif. TB® line-probe assay in rapid detection of multidrug-resistant tuberculosis in Latvia. Int J Tuberc Lung Dis 2011;15:1546e53. [12] Kohli A, Bashir G, Fatima A, Jan A, Ahmad J. Rapid drug-susceptibility testing of Mycobacterium tuberculosis clinical isolates to first-line antitubercular drugs by nitrate reductase assay: a comparison with proportion method. Int J Mycobacteriol 2016;5:469e74. [13] Minion J, Leung E, Menzies D, Pai M. Microscopic-observation drug susceptibility and thin layer agar assays for the detection of drug resistant tuberculosis: a systematic review and meta-analysis. Lancet Infect Dis 2010;10: 688e98. €rner H. Use of 3-(4, 5-Dimethylthiazol-2[14] Mshana RN, Tadesse G, Abate G, Mio yl)-2, 5-diphenyl tetrazolium bromide for rapid detection of RifampinResistantMycobacterium tuberculosis. J Clin Microbiol 1998;36:1214e9. [15] Affolabi D, Sanoussi ND, Odoun M, Martin A, Koukpemedji L, Palomino JC, et al. Rapid detection of multidrug-resistant Mycobacterium tuberculosis in Cotonou (Benin) using two low-cost colorimetric methods: resazurin and nitrate reductase assays. J Med Microbiol 2008;57:1024e7. [16] Chen Y, Zhao B, H-c Liu, Sun Q, Zhao X-q Liu Z-g, et al. Prevalence of mutations conferring resistance among multi-and extensively drug-resistant Mycobacterium tuberculosis isolates in China. J Antibiot (Tokyo) 2016;69:149e52.
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