Multiplex PCR for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Isolated from Bandung, Indonesia

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1 MICOBIOLOGY INDONESIA, December 007, p 4 Volume, Number 3 ISSN Multiplex PC for apid Detection of ifampin and Isoniazid esistance in Mycobacterium tuberculosis Isolated from Bandung, Indonesia HEA NOVIANA,3*, ZEILY NUACHMAN, MAELIA AMDANI, AND AS NOE Biochemistry Division, School of Biological Science, Institut eknologi Bandung, Jalan Ganesha 0, Bandung 403, Indonesia 3 Microbiology Department, Medical Faculty, Universitas Katolik Atma Jaya, Jalan Pluit aya No., Jakarta 4440, Indonesia Mycobacterium tuberculosis resistant to rifampin and isoniazid, known as multidrugresistant M. tuberculosis (MDB) strains, is an emerging problem of great importance to public health, with higher mortality rates than for drugsensitive strains. ifampin resistance is due to mutations on the hot spot region of the rpob gene, especially at positions 56 and 53, and isoniazid resistance is due to mutation on katg at position 35. Mechanisms of resistance are an appropriate target for molecular genotyping diagnostic methods. Here we examined the multiplex PC assays for the rapid detection targeting rpob56, rpob53, and katg mutations. Sixtyone M. tuberculosis strains were studied based on rpob56, rpob53, and katg35 assays employing multiplex PC. Of the 6 strains, the susceptibility tests determined 4 isolates were MDB strains, 0, 4, and 5 isolates were resistant to rifampin, isoniazid, and at least to six drugs which prescibed for B, respectively. he mutation profiles of the 4 MD strains assayed by multiplex PC were and 3.% on rpob and katg, respectively. Six rifampinresistant isolates (60%) had a mutation on rpob, 5% isoniazidresistant isolates had mutation on katg, and 0% of the isolates that were sensitive to all drugs tested had a mutation on rpob. Sequencing analysis revealed sensitivity of the multiplex PC assay for rpob was 9.4% and was 00% for katg. here was a 9% difference between phenotype and genotype properties of all isolates detected. In conclusion, the sensitivity of multiplex PC method was sufficient for preliminary detection of rpob and katg mutations, but resistance M. tuberculosis to rifampin and isoniazid were not always conferred by mutated alleles on rpob and, especially, on katg. Key words: multiplex PC, rpob, katg, sequencing Mycobacterium tuberculosis, the causative agent of tuberculosis (B), is still the single most deadly infectious disease in developing countries. he spread of MDB, defined as M. tuberculosis resistant to at least isoniazid (INH) and rifampin (IF), is worsening and emerging as a global emergency. It has increased worldwide and reached epidemic proportions in many countries (Fang et al. 999; ie et al. 00; ei et al. 003). Indonesia is third in the rank of the highly burdened countries, after China and India. In 00, the HO estimates there are B patients per people. he mortality rate is at least people each year in Indonesia (HO 006). IF in combination with INH is a frontline antituberculosis agent that is prescribed daily in millions of doses worldwide. esistance to IF is conferred by mutations in the rpob hot spot region, encoding the subunitb of NA polymerase. he two most frequent mutations in the rpob are mutations at codon rpob56 and rpob53 (Gillespie 00; Mokrousov et al. 003; Van der Zanden et al. 003; erngren and Hoffner 003). INH is a prodrug, which is peroxidatively activated by the M. tuberculosis catalaseperoxidase katg to produce damaging cell wall of the bacteria (hitney and ainberg 00). Mutations at codon S35 (AGCaACC) were found in 6070% of INH resistant strains (Van Doorn et al. 00; amaswamy et al. 003). his mutation was reported to be associated with intermediate or high levels of resistance to INH (.0 to 0 mg l ) (Mokrousov et al. 00a; Caws et al. 006). he inha and kasa genes may participate in INH resistance. * Corresponding author, Phone: ext. 54, Fax: , hnoviana@yahoo.com ecent advances in the development of rapid and reliable diagnostic methods have allowed for detection of resistance to antituberculosis drugs without the need for a viable culture. In this work we examined multiplex PC assay as a means for rapid detection of rpob56, rpob53, and katg35 mutation related to IF and INH resistance, respectively. he aim is to confirm results from molecular assays of multiplex PC by nucleotides sequencing. MAEIALS AND MEHODS Strains Collection. M. tuberculosis clinical isolates were recovered from different patients originating from Bandung, est Java with new or previous diagnosed pulmonary B. Clinical specimens were collected from sputum, pleural exudate, and cerebrospinal fluid of patients between January and November 005 at Balai Pengembangan dan Latihan Kesehatan (BPLK) laboratory for Mycobacteria, Department of Health and otinsulu Lung Diseases Hospital, Bandung, est Java. Susceptibility esting. Drug susceptibility was determined using the proportion method on Lowenstein Jensen medium with the following critical concentration of the drugs: INH (0. mg ), IF (40 mg l ), streptomycin (0 mg l ), kanamycin (40 mg l ), ethambutol (0 mg l ), and pyrazinamide (40 mg l ). he control was a tube of the medium without drugs inoculated by the strains of M. tuberculosis being tested. All the tubes were incubated at 37 C, growth was monitored after 6 weeks of incubation and susceptibility results were then read. An isolate was considered resistant if the bacterial growth in the culture

2 NOVIANA E AL. Microbiol Indones 5 tube was % compared with bacterial growth in the respective control tube (Kumari and Ichhpujani 000). Preparation DNA Samples of M. tuberculosis. M. tuberculosis colonies were lysed by 0 µl lysis buffer (50 mm rishcl ph.5; mm EDA ph.5; 0.5% ween 0); and 4 ml ProteinaseK, using a 5 C incubation temperature for an hour. he lysate was heated to 95 C to disrupt Proteinase K, and the supernatant was then collected for further analysis. For both multiplex PC and PC for nucleotides sequencing, we used 5 ml DNA samples from this preparation. Multiplex PC. he multiplex PC assay (modification of Mokrousov et al. 00b; Mokrousov et al. 003) was employed for mutational assay on rpob56, rpob53, and katg35. Multiplex PC, either for rpob or katg, consisted of three primers targeting codons rpob56, rpob53, and katg35. A negative control was amplified using a standard strain of M. tuberculosis, H37v. he primer sequences that used are shown in able. he inner forward primers are positioned so that their 3 OH ends pair with the second bases of the respective codons in the wildtype allele. If a mutation occurs, this results in a mismatch at the 3 end of the inner primer sequence, in the absence of the variable PC product ( bp for rpob56, 67 bp for rpob5, and 9 bp for katg35). he invariable fragments of 49 and 435 bp are amplified by two outer primers, forward and reverse, flanked the rpob hot spot and katg region, respectively. he amplification a pair of forward and reverse primer is used to control the quality of the multiplex PC. Meanwhile, the amplification employing similar forward and reverse primer without inner primer is used for nucleotide sequencing analysis. he multiplex PC reactions for katg35 were carried out in a thermal controller (PerkinElmer PC system 400, Inc.) under the following conditions: initial denaturation at 96 C for 3 min; 30 cycles at 94 C for min, 5 C for 40 s, and 7 C for 30 s with the final elongation at 7 C for 3 min. he PC reactions for rpob56 and rpob53 were carried out under the following conditions: initial denaturation at 96 C for 3 min; 5 cycles of 95 C for 45 s, 60 C for min, and 7 C for 30 s. hen 5 cycles at 95 C for 40 s, 59 C for 50 s, and 7 C for 30 s. hen 5 cycles at 94 C for 50 s, 55 C for 40 s, and 70 C for 30 s with the final elongation at 7 C for 3 min. he PC product was electrophoresed on.5% w/v agarose gel incorporating 0.5 g ml ethidium bromide for visualization using a UV transilluminator. Primer Forward rpob everse rpob Inner rpob53 Inner rpob56 Forward katg everse katg Inner katg35 ESULS Sixtyone strains of M. tuberculosis were collected from two laboratories and analyzed for their phenotype and able Nucleotide sequences of primers used in the multiplex PC assay and PC for nucleotide sequencing Nucleotide sequences 5 GCGCC GCGACAAGGA3 5 GACCCGCGCG ACA C3 5 ACAAGCGCCGAC G C3 5 GCGGGGGACCCA3 5 GCAGAGGGGCGACACG3 5 AACGGGCCGGGAGGG3 5 AACGACC CGAGCCGC3 genotype properties. he susceptibility tests showed 4 strains were resistant to at least IF and INH (MDB), 0 strains were sensitive to INH but resistant at least to IF, 4 strains were sensitive to IF but resistant at least to INH, and 5 strains were considering sensitive to all of the drugs tested. he results of susceptibility test are shown in able. Molecular analysis of M. tuberculosis is a promising alternative to bacteriological assays. he genetic basis for IF and INH resistance are mutations in the rpob and katg genes, respectively. Using the method PCbased, therefore the time to identify IF and INH resistant strains were further is shortened to h. In addition, it does not require any special probe preparation and can be applied to simultaneous analysis of several variable segments of the bacterial genome. esults of the multiplex PC assays are shown in Fig and. Based on multiplex PC results, this study determined the existance of various genotype combinations of M. tuberculosis strains as shown in able 3. DISCUSSION here are many tools developed to simplify methods on B diagnostics. he susceptibility test remains the gold standard method for phenotyping of M. tuberculosis. In total, drugsusceptibility testing of 6 M. tuberculosis isolates showed that resistance to IF was the most frequent (5.%) in this study, followed by INH resistance (75.4%). his study identified 4 strains of MDB with nine of them (.4%) resistant to six drugs tested. esistance to ethambuthol, streptomycin, kanamycin, and pyrazinamide was observed for 60.7, 4.0, 6., and.3%, respectively. aniguchi et al. (996) suggest that there was a strong correlation of specific amino acid substitutions of rpob and katg, and its minimum inhibitory concentration to IF and INH. his study revealed a correlation between the level of resistance and specific mutation on rpob56, rpob53, and katg35. wenty one strains of MDB had the capability to grow on media containing IF 300 µg ml and 9 strains able esistance pattern of MD M. tuberculosis strains iso No. of strains a IF INH EM S KN PY otal a n = 6, IF: rifampin, INH: isoniazid, EM: ethambutol, S: streptomycin, KN: kanamycin, PY: pyrazinamide.

3 6 Volume, 007 Microbiol Indones of MDB has the capability to grow on media containing INH 0 µg ml (data not shown). Mycobacterium tuberculosis strains carrying a mutated allele on rpob53 49 bp 67 bp rpob53 M rpob56 rpob53 M rpob56 nonspecific bands 49 bp bp Fig Profiles generated by multiplex PC targeting rpob56 and rpob3 (note: samples which amplified a product of 67 and bp were considered to be wildtype rpob53 and rpob56, the strains with no amplified product of 67 and bp were considered to be mutant, M: 00 bp marker lane). nonspecific bands 435 bp 9 bp Fig Profiles generated by multiplex PC targeting katg35 (note: the strains with amplified product of 9bp were considered to be wildtype, the strains with no amplified product of 9 bp were considered to be mutant, M: 00 bp marker lane). able 3 Genotype combinations of MD M. tuberculosis strains isolated from Bandung employing multiplex PC No. of strains Genotype (n=6) katg35 rpob56 rpob : wildtype allele; : mutant allele. had a high level resistance to IF (MICe 00 mg l ) compared with strains carrying a mutated allele on rpob56. Likewise, M. tuberculosis strains carrying a mutated allele on katg35 had a high level resistance to INH (MIC between mg l and e 00 mg l ) than strains that had no carrying mutated allele on katg35 (MIC 0.0 mg l ). Multiplex PC assays for IF and INH resistance tests rely on detection of mutations on rpob56, rpob53, and katg35. Of the 6 M. tuberculosis strains, approximately 6.9% were carrying mutated alleles of rpob56 and rpob53, whereas 7.9% carrying mutated allele of katg35. Mutation on rpob56 and rpob53 in this study were lower than reported earlier, with more than 95% of the rifampinresistant M. tuberculosis strains caused by these mutations (Van der Zanden et al. 003). Fortytwo MDB strains consisted of 6 (3.%), 5 (35.7%), and 3 (7.%) strains carrying the mutated allele of rpob56, rpob53, and both rpob56 and rpob53, respectively. Eight strains of MDB were not detected carrying mutated alleles at either codon 56 or 53. Meanwhile, of the 4 MDB strains analyzed by multiplex PC for katg35, only 6 strains (3.%) were detected carrying mutated allele of katg35. he results of genotype analysis on katg35 also were lower than reported in previous studies, which showed a 6070% frequency for katg35 mutation. Mokrousov et al. (00a) found a 93.6% prevalence of the katg35 mutation in strains from patients with both new and previous diagnosed cases of B. Of the ten strains sensitive to INH, but resistant to IF when subjected to assay, seven were not demonstrated as having variable amplified product of or 67 bp. his means that only three strains were detected carrying mutated alleles on rpob56 and/or rpob53. Interestingly, one strain sensitive to IF but resistant to INH, and one strain sensitive to all six drugs, demonstrated had no amplified bp product. Both strains that sensitive to IF were considered to carrying mutated alleles on rpob56. hose two strains might develop resistance to IF at a low level (<40 mg l ) so that in the susceptible test to IF (40 mg l ) they show as sensitive. Meanwhile, only one of four strains sensitive to IF, but resistant to INH, was detected carrying a mutated allele on katg35. here was a 500 and 000 bp of nonspecific band in a few products of multiplex PC for both rpob56 and rpob53, and katg35, respectively. he unspecific binding of primers or the abundant of DNA templates might cause this nonspecific band (measurement of DNA samples were not done so that the concentration of DNA in every sample was different). All the isolates were sequenced in order to avoid misdiagnosis through false positive and negative results of multiplex PC, especially M. tuberculosis strains which demonstrated that no mutation occurred. However, sequencing analysis revealed approximately 9% correlation between multiplex PC of rpob56, rpob53, katg35, and sequencing. he sequencing showed one M. tuberculosis MD strain had a mutated allele carrying CAC (his) to CGC (arg) at codon rpob56, but this was not detected by multiplex PC although it was reconfirmed a second time. In the absence of the allelespecific PC product, this can be

4 NOVIANA E AL. Microbiol Indones 7 caused by inappropriate positioning of the PC primers, which resulted in an underestimation of the prevalence of this mutation. Although Mokrousov et al. (003) had been stably adjusted to assess reproducibility of multiplex PC, it might be that the inner primer sequence and the effectiveness of the thermal controller was the crucial factor which affected the process of the multiplex PC. he remaining isolates showed similarity between multiplex PC and sequencing. his finding demonstrates that although the level of sensitivity of multiplex PC to identify rpob56, rpob53, and katg35 mutations was not 00% because it was based on the specificity of multiplex PC. However, this method can be used as preliminary assay for the detection of mutation on rpob56, rpob53, and katg35. It is logical that a mutation would not be selected for in a population if it did not confer some form of selective advantage. he sequencing analysis recognized new mutated alleles in this study. Four of the eight MDB strains carrying wildtype alleles on rpob56, rpob53, and katg35 had a mutation at a position both outside and within the hot spot of rpob, either rpob56 and rpob53. It is remain unclear and undetermined what is the resistance mechanism of the remaining four MDB strains. Interestingly, one strain sensitive to at least six antib drugs demonstrated a new mutated allele carrying CCC (Pro) to CAC (His) at codon rpob535, but the clinical significance of this mutation is not clear. he high percentage of isolates lacking mutations suggests that phenotyping methods remain an important complement to genotyping methods for drug susceptibility testing. he genotype analysis determined 5 of the 5 (.%) strains which have phenotype resistant to IF had no mutation on both rpob56 and rpob53. here were also 9 of the 46 (63%) strains phenotype resistant to INH which had no mutation on katg35. herefore, this study revealed a 9% difference between phenotype and genotype properties of all isolates detected by multiplex PC of rpob56, rpob53, and katg35. Our results suggest that this molecular method only determines the expected mutations, especially on rpob56 and rpob53. Susceptibility tests for the detection of INH resistance remain the most crucial assay because of the frequency of resistance corresponding to katg35 mutation being low. herefore, although the molecular methods may aid in the rapid detection of mutations associated with drug resistance, the results always need to be confirmed by phenotyping methods. In conclusion, the molecular analysis of M. tuberculosis is a promising alternative to bacteriological assays. he study demonstrates multiplex PC is rapid, easy to perform, and sufficiently accurate for preliminary assay. It was essential that the test always be verified by standard culturebased methods because there were resistant strains of M. tuberculosis that were carrying no mutated alleles, whether on rpob or katg. It is hope that the development of novel molecular methods using a rapid and reliable genetic approach will facilitate the appropriate and timely delivery of antituberculosis therapy. ACKNOLEDGEMEN e thank the technicians of the Balai Pengembangan dan Latihan Kesehatan, Public Health Province Laboratory for Mycobacteria which contributed to the research. e thank Francisca, intin, and Isak Solikin for assistance from their facilities and their advice and discussion. his study was supported by BPPS and Universitas Katolik Indonesia Atma Jaya. EFEENCES Caws M, Duy PM, ho DQ, Lan NN Mutations prevalent among rifampin and isoniazidresistant Mycobacterium tuberculosis isolates from a hospital in Vietnam. J Clin Microbiol 44: Fang Z, Doig C, ayner A, Kenna, att B, Forbes KJ Molecular evidence for heterogeneity of the multipledrugresistant Mycobacterium tuberculosis population in Scotland (990997). J Clin Microbiol 37: Gillespie SH. 00. Evolution of drug resistance in Mycobacterium tuberculosis: clinical and molecular perspective. Antimicrob Agents Chemother 46:6774. Kumari S, Ichhpujani L Guidelines on Standard Operating Procedures for Microbiology. 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5 Volume, 007 Microbiol Indones erngren J, Hoffner SE Drugsusceptible Mycobacterium tuberculosis Beijing genotype does not develop mutation conffered resistance to rifampin at an elevated rate. J Clin Microbiol 4:5054. hitney JB, ainberg MA. 00. Isoniazid the frontline of resistance in Mycobacterium tuberculosis. J Med 6:43. [HO] orld Health Organization eport Global tuberculosis control: surveillance, planning, financing. Geneva: HO.