PCR-RFLP for rapid, low-cost identification of isoniazid-resistant Mycobacterium ACCEPTED

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1 JCM Accepts, published online ahead of print on April 00 J. Clin. Microbiol. doi:./jcm Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved PCR-RFLP for rapid, low-cost identification of isoniazid-resistant Mycobacterium tuberculosis. Maxine Caws 1, *, Dau Quang Tho 1, Phan Minh Duy 1, Nguyen Thi Ngoc Lan, Dai Viet Hoa, Mili Estee Torok 1,, Tran Thi Hong Chau, Nguyen van Vinh Chau, Nguyen Tran Chinh, Jeremy Farrar 1,. 1 Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ben Ham Tu, District, Ho Chi Minh City, Vietnam Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases, Hung Vuong, District, Ho Chi Minh City, Vietnam. The Hospital for Tropical Diseases, Ben Ham Tu, District, Ho Chi Minh City Viet Nam Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Old Road, Headington, Oxford, United Kingdom. * corresponding author: Dr Maxine Caws, Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ben Ham Tu, Quan, Ho Chi Minh City, Vietnam. Tel: ( ) 01 Fax: ( ) 0 mcaws@hotmail.com Downloaded from on May, 01 by guest 1

2 1 1 1 Abstract PCR Restriction Fragment Length Poymorphism (PCR-RFLP) is a simple, robust technique for the rapid identification of isoniazid-resistant Mycobacterium tuberculosis (M. tuberculosis). One hundred consecutive isolates from a Vietnamese TB hospital were tested by mspa1 I PCR-RFLP for the detection of isoniazid-resistant katg_1 mutants. The test had a sensitivity of 0% and a specificity of 0% against conventional phenotypic drug susceptibility testing. Positive and negative predictive values were 1 and 0., respectively. None of the discrepant isolates had mutant katg_1 codons by sequencing. This test is cheap (<1.USD per test), specific and suitable for the rapid identification of isoniazid resistance in regions with a high prevalence of katg_1 mutants among isoniazid resistant M.tuberculosis. Downloaded from on May, 01 by guest

3 Introduction Tuberculosis (TB) control is threatened by increasing resistance to first-line antituberculous agents. The WHO estimates that in the year 00.% of new TB cases globally were multi-drug resistant TB (MDR-TB) (), defined as resistance to at least isoniazid and rifampicin. Treatment regimens for MDR-TB are more complex, less potent, more toxic and more expensive than first-line regimens, resulting in poorer tolerability and adherence, and higher morbidity and mortality rates. Early identification of drug resistant and particularly MDR strains is routine in developed low-prevalence countries but the methods are currently unavailable and are too costly for resource-poor nations where the burden of disease is greatest. Early identification of MDR-TB is crucial in order to permit the timely administration of appropriate drug regimens and could potentially improve the efficacy of MDR treatment schemes such as DOTS-PLUS. Isoniazid is a pro-drug, which is activated through cleavage by catalase peroxidase, the product of the katg gene. The active drug has a complex mode of action which remains to be fully elucidated but principally targets mycolic acid biosynthesis pathways. Upregulation of the inha gene product, an enoyl-acp reductase, can overwhelm the impact of isoniazid. Resistance to isoniazid is achieved through mutations in the katg gene, predominantly at codon 1, and in the inha promoter for the majority of isolates. Mutations in other genes such as ahpc and ndh have been implicated in isoniazid resistance, but their role is not yet proven. A role for kasa gene mutation in isoniazid resistance has recently been Downloaded from on May, 01 by guest

4 discredited (). Many isolates are isoniazid-resistant without any mutations in known target genes and the mechanism of resistance for these isolates remains obscure. Phenotypic sensitivity testing of M. tuberculosis is time consuming requiring - weeks from primary specimen in settings with rapid liquid culture systems such as BACTEC MGIT 0, and - weeks in settings with solid media culture (1). Several approaches to the rapid identification of isoniazid resistance have been reported in the literature, including real-time PCR (), single-strand-conformation polymorphism analysis (SSCP) (0), dot-blots (), chips (1, 1) and multiplex-allele-specific PCR (MAS- PCR) (1). The PCR-RFLP approach has several advantages; it is cheap, robust and simple to both perform and interpret, requiring only basic PCR equipment. MspA1 I digestion for the identification of katg_1 mutation, as described here, has previously been described (,, ). The mspa1 I restriction enzyme step also allows the detection of mutations other than katg_s1t at the 1 codon site, increasing the sensitivity of the test over MAS-PCR and still giving a positive identification of both mutant and wild type genes. It does not detect the very rare katg_s1r (AGC CGC) mutation (). Commercial molecular hybridization tests, such as INNO-LiPA RIF TB (Innogenetics, Zwijnaarde, Belgium) and MDRTBI (Hain lifesciences, Nehren, Germany), while sensitive and specific (), are too costly for routine use where the burden of disease is greatest. Both these tests are based on reverse hybridization of PCR product to membrane-bound probes. The INNO-LiPA RIF TB test detects mutations in the rpob gene and consequently will detect only rifampicin resistance. However, rifampicin Downloaded from on May, 01 by guest

5 resistance can be used as a surrogate marker of MDR-TB in many settings (1, ). The MDRTBi test has probes targeted to both rpob and katg mutations for the positive identification of MDR strains. The identification of isoniazid resistance in the absence of rifampicin resistance is important in settings where primary resistance to both isoniazid and streptomycin is high. Dual resistance to isoniazid and streptomycin, without MDR, has been strongly associated with an increased risk of treatment failure (OR=., % CI 1.1-) or relapse (OR.,% CI 1.-.) and with acquired MDR (OR=., % CI 1.-) on a standard WHO regimen [ months streptomycin, rifampicin, isoniazid and pyrazinamide, months isoniazid and ethambutol (SRHZ/HE )] (1). Around % of primary isolates are isoniazid resistant in Ho Chi Minh City and 1% are isoniazid and streptomycin dual resistant (1). The aim of the current study was to develop a rapid, simple PCR-RFLP screening test for the identification of isoniazid resistance from positive M. tuberculosis cultures. Previous sequencing data on isoniazid resistant isolates from Vietnam indicated that around 0% of isoniazid resistant isolates could be detected through katg_1 analysis, with katg_s1t being by far the most prevalent mutation (1%) (). A restriction enzyme test specific for digestion of the CMGCKG site, found at wild type katg_1, was therefore developed. The test was expected to detect all the mutations at codon 1 previously identified in Ho Chi Minh City: GC (S1T), GA (S1N), GT (S1I), AG (S1G) and GC/CG (S1T) (). Rare isolates (<1% isoniazid resistant isolates) which lack the katg gene would not produce an amplicon in the initial PCR (). Downloaded from on May, 01 by guest

6 To assess specificity the PCR-RFLP was tested on DNA extracts from M. tuberculosis isolates with previouly determined katg sequences (). The test was 0% specific and was therefore tested prospectively on 0 positive M. tuberculosis cultures collected from patients at the Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases (PNT TB hospital), Ho Chi Minh City. Costs of the test in routine use were also calculated, based on minimum equipment requirements and reagent costs available locally, and a batch size of isolates. Methods Sample collection Initial testing was done on cultures with known katg gene sequences: 0 with S1T mutations; 0 isoniazid sensitive isolates (wild type katg), x S1N, x S1I, 1 x S1G, one double mutant S1T/T0T and one isolate with katg whole gene deletion. The test was then prospectively evaluated on 0 M. tuberculosis cultures from pulmonary TB patients at PNT TB Hospital during April 00. An aliquot of the first 0 isolates processed for phenotypic drug susceptibility testing each Wednesday was also tested by PCR-RFLP. Drug susceptibility testing (DST) and the PCR-RFLP test were performed blind by two different operators. The sensitivity and specificity of the PCR- RFLP were determined at the end of the study. Any isolate that produced discrepant results with the two tests was sequenced in the katg gene to determine the katg_ 1 sequence. DNA extraction A single colony of culture was dispersed in 0µl distilled water and an equal volume of chloroform was added. The tube was vortexed, heated at 0 C for 0 minutes, vortexed Downloaded from on May, 01 by guest

7 again and centrifuged at 1,000g for minutes. Five µl of the upper aqueous layer was taken for PCR. Three control isolates were included with each run; one wild-type positive control, one katg_s1t mutant positive control and one negative control (water). PCR. PCR of a 1bp region of the katg gene was carried out with primers '- GGTCGACATTCGCGAGACGTT-' and '-CGGTGGATCAGCTTGTACCAG-' from Martilla et al. (). The reaction mix had a final concentration of 0.µM each primer, 1x bioline buffer, 0.1mM each dntp, mm MgCl with 0.U taq per reaction (Bioline, UK). µl DNA extract was added to 1µl reaction mix and the PCR carried out in Tetrad PCR machine (MJ research, Hercules, Biorad,UK) with the following program: cycles of 1 C, 1 C, 1 C followed by a further cycles of 1 C, 1 C, 1 secs at C. Restriction enzyme digest Ten µl of restriction enzyme mix was added to each PCR reaction to give a final volume of 0µl containing final 0.U mspa1 I (New England Biolabs, USA), 0.x NEB buffer, 0µg/ml BSA, according to manufacturer s instructions. The mix was incubated at C for hours in the PCR machine. Products were electrophoresed on 1.% agarose gel. A 0 bp band indicates a wild-type katg_1 codon. A bp band indicates mutant type katg_ 1 codon and therefore izoniazid resistance. (figure 1). Phenotypic sensitivity testing Downloaded from on May, 01 by guest

8 Phenotypic sensitivity testing for isoniazid resistance was carried out at 0.µg/ml by 1% proportion method at PNT TB hospital, according to WHO protocols. Sequencing PCR products were amplified using primers as for the PCR-RFLP test with a program using 0 cycles of C for 1 sec, C for 1 seconds and C for 1 secs. PCR products were purified with QIAgen PCR purification kits (Qiagen, UK) and then served as templates for cycle sequencing reactions. Both strands of each product were sequenced with CEQ Dye Terminator Cycle Sequencing Quick Start kits (Beckman Coulter, Singapore) in half volume reaction using the same primers. The thermal cycling program was: ºC for 0 sec, appropriate Ta for 0 sec and 0 ºC for min, for 0 cycles followed by holding at ºC. The cycle sequencing products were subjected to ethanol precipitation steps according to manufacturer s instructions and sequenced on the CEQ000 system (Beckman Coulter, Singapore). MAS-PCR Multiple-allele specific PCR (MAS-PCR) was used to screen for inha -1C T mutation in discrepant isolates. Primers were TB '-CCTCGCTGCCCAGAAAGGGA-' (0.µm) and TB '- ATCCCCCGGTTTCCTCCGGT-' (0.0µM) from Telenti et al., with an internal primer inha Rmut '-AGTCACCCCGACAACCTATTA-' (0.µM). The PCR reaction mix used 0.mM dntp's,.mm MgCl, 0.U Bioline Taq (Bioline, UK) and 1x taq buffer, supplied by the manufacturer. Reactions were amplified in a Downloaded from on May, 01 by guest Tetrad PCR machine (MJ research, Hercules, Biorad, UK) with an initial min

9 denaturation at C, followed by 0 cycles of 0 secs at C, 1 secs at C, and 0 secs at C, with a final minute elongation at C. The presence of bands at 1 bp and bp indicates a mutant allele (inha -1C T). Wild-type isolates generate only a single band at bp. Costs Cost calculations were based on a batch size of ten isolates, with all reagents purchased through local distributors and an exchange rate of 1,00 VND to the US dollar. Equipment cost estimates were based upon locally available catalogue prices. The cheapest appropriate models were selected for calculations, rather than the relatively expensive multiplate gradient models used in our laboratory, as only a basic thermocycler is required to perform the test. Costs were averaged and rounded up to the nearest 0 USD. Final costs were estimated in US dollars. Results. Retrospective culture analysis: The test was 0% specific for the detection of katg_1 mutant codon on retrospective cultures with known katg_1 sequences. The twenty isoniazid sensitive isolates produced a wild-type bp band and resistant isolates with miscellaneous mutations at katg_1 generated a bp band (figure 1). The katg deleted isolate failed to generate an amplicon, as predicted, and the result was therefore 'uninterpretable'. This DNA extract generated product with other PCR based tests, including spoligotyping, rpob and inha sequnecing (data not shown). Downloaded from on May, 01 by guest

10 Prospective culture analysis: Of 0 M. tuberculosis cultures tested at PNT TB Hospital by PCR-RFLP, were resistant, wild-type and one isolate generated both wild-type and resistant bands and was therefore deemed to be a mixed culture. By phenotypic DST, isolates were resistant to isoniazid (one of which was identified as Mycobacterium fortuitum (M. fortuitum), were sensitive, and isolates failed to return a result due to failed growth or contamination- (both of which were sensitive by PCR-RFLP). Excluding the isolates which failed to return a result by DST and the M. fortuitum isolate, the sensitivity of the PCR-RFLP test was 0%, the specificity was 0%. The positive predictive value (PPV) and negative predictive value (NPV) were 1 and 0., respectively. Nine discrepant isolates were sequenced in the katg gene, including isolates returning a PCR-RFLP sensitive/dst resistant result, 1 isolate (RE1) returning a mixed resistant/sensitive PCR-RFLP result with a resistant DST result and the isolate returning a resistant PCR-RFLP result but identified as M.fortuitum by DST (RE10). These isolates were also screened for inha -1C T mutation by MAS PCR. A summary of the discrepant isolates is presented in table 1. Sequencing results confirmed isolate RE10 contained M.tuberculosis DNA, and carried a katg_s1t mutation. Isolate RE1 produced a hetero-peak for the relevant mutation at codon 1 (GC/WT), confirming that it contained a mixed population of resistant and sensitive organisms. The remaining isolates carried a wild-type katg_1 codon, with one isolate (RE) carrying a novel mutation in katg, CA (LM), confirming that in all these isolates resistance was attributable to mutations at sites other than katg_1. Downloaded from on May, 01 by guest

11 The median time to positive culture was days (range 1-, data available for only cultures). Phenotypic DST testing required a further 1 days to return a result, with PCR- RFLP returning a result within 1 day. Costs Capital equipment required for the test would be a basic PCR machine (approximately 000USD), a gel tank (0USD), power pack (100USD), tranilluminator (00USD), a camera (00USD) and four pipettes (00USD). The total capital costs would be approximately 0USD. However, this equipment can also be applied for other tests once purchased and is already available in many reference level laboratories. Processing isolates per batch, with controls (positive mutant control, positive wild type control, negative control), the cost of each test is 1.USD per isolate. Labour costs were not included in this calculation. Discussion The mspa1 I PCR-RFLP rapid test described showed 0% specificitiy and 0% sensitivity in the routine identification of isoniazid-resistant M.tuberculosis. It is affordable and simple to perform. It was also possible to obtain a screening result for isolates by PCR-RFLP which failed to return a result by phenotypic DST, due to failed regrowth or contamination. Isolate RE10 was originally identified as M.fortuitum at PNT laboratory and the presence of M.tuberculosis DNA, shown by katg sequencing, in the original culture extract of isolate RE10 suggests a laboratory contamination event at the phenotypic DST subculture stage led to the growth of M.fortuitum. However, it is impossible to rule out DNA contamination of a M.fortuitum sample with M.tuberculosis Downloaded from on May, 01 by guest

12 DNA during PCR-RFLP sample processing for DNA extraction. Unfortunately, clinical data was not available from this patient to determine if later samples isolated M.tuberculosis. The early detection of isoniazid resistance is an important step in reducing the use of drug resistance amplifying regimens, particularly with the increasing availability of second line drugs through schemes such as DOTS-PLUS. Mutations in KatG_1 are associated with higher minimum inhibitory concentrations (MICs) for isoniazid than mutations in the inha promoter and therefore are probably of the greatest clinical significance (1,, ). Variations in outcome on standard treatment regimens with a primary isoniazid resistant organism are multi-factorial but mean inhibitory concentration (MIC) of the isolate may be a contributing factor. Vietnam has an estimated primary isoniazid resistance rate of % in Ho Chi Minh City (1). Despite having a well-functioning National Tuberculosis Program (NTP) administering DOTS to WHO guidelines (), drug-resistance continues to rise. It has been speculated that this is due to high rates of primary isoniazid resistance in conjunction with the use of isonizid/ethambutol continuation phase regimens (1). Of greatest concern is the evidence that this may be fuelling the development of MDR-TB, which has seen an increase from.% in 1 to an estimated.% in 00 (1). There is therefore an urgent need for a cheap, rapid, simple test to diagnose isoniazid resistance prior to continuation phase, when currently around % of patients in Vietnam effectively receive monotherapy due to polyresistance. Primary isoniazid resistance has been associated with the development of MDR TB on treatment (1). The katg_1 mutation is associated with the development and transmission of MDR-TB, whereas Downloaded from on May, 01 by guest 1

13 other isoniazid-resistance conferring mutations, such as inha -1C T are not (,, ). The test described here is economical (1.USD per isolate) and simple to perform. In routine laboratories the cost per isolate is likely to be lower as we used a batch size of isolates for calculations, whereas larger batch sizes of 0-0 could easily be processed, with careful precautions against contamination, including the use of 'clean' and 'dirty' containment areas, dutp's with enzyme digestion, and increasing the number of negative controls in larger batch sizes. Currently, in the Vietnamese NTP, TB patients are only tested for drug resistance after microbiological failure of first-line therapy. This is primarily because of cost and workload considerations. Although the PRA test is not 0% sensitive for the detection of isoniazid resistance, it is 0% specific and can therefore be used as a rapid screening test to identify the majority of patients with isoniazid resistant organisms. At 1.USD per test, it is also affordable. The test can be completed in one day and requires around - hours hands-on time, if pre-prepared PCR mastermixes are used. We are currently evaluating the test for routine use directly with smear-positive sputum. Strategies for the rapid identification of drug resistance should be region specific. A test screening for katg_1 mutations is likely to be of most use in regions with a high prevalence of this mutation, such as China, Russia, the former Soviet States, Vietnam and Peru (, ). In Europe a higher prevalence of inha gene mutations indicates a multiplex PCR or hybridisation approach is warranted (,, 1). The reasons for variations in mutation prevalence are unclear and may be related to TB control program differences or to variations in the predominant M. tuberculosis strains within a region. Assessment of Downloaded from on May, 01 by guest 1

14 strategies for the rapid identification of drug resistance should be made in the context of knowledge of the predominant resistance-conferring mutations in a region. In Vietnam several factors should be considered: high underlying isoniazid resistance; the rarity of rifampicin resistance without isoniazid resistance, diverse rifampicin resistance mutation patterns complicating the identification of rifampicin resistance (). These factors suggest an approach based upon rapid isoniazid resistance screening, with subsequent rifampicin resistance testing in isoniazid resistant isolates. The implementation of rapid resistance screening tests in high incidence areas must be carefully considered. In Vietnam, as in many developing countries, patients who fail primary therapy are treated with a standard first line re-treatment regimen. Even after identification of drug resistance by DST, TB therapy is rarely modified even though treatment success rates with standard first line drug re-treatment regimens are known to be low. The advent of DOTS-PLUS schemes supported by the Green Light Committee for the economical procurement of drugs make the implementation of rapid drug resistance screening and the use of second-line treatment regimes feasible within a wellestablished NTP such as is found in Vietnam. The use of the PRA test to rapidly identify isoniazid resistance and guide timely modification of therapy could potentially reduce both the generation and transmission of MDR-TB. Acknowledgements This work was funded by the Wellcome Trust Grant 00/Z/0/Z. We would like to thank the staff of Pham Ngoc Thach Microbiology laboratory for assistance with this study. Downloaded from on May, 01 by guest 1

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18 Table 1. KatG gene sequences of isolates showing discrepant results with PCR- RFLP and phenotypic DST tests. Isolate number PCR-RFLP result DST result katg sequencing inha -1C T MAS PCR RE10 RESISTANT M.fortuitum S1T WT RE1 SENSITIVE/RESITANT RESISTANT S1T/WT* WT RE1 SENSITIVE RESISTANT WT -1C T RE0 SENSITIVE RESISTANT WT WT RE0 SENSITIVE RESISTANT WT WT RE SENSITIVE RESISTANT WT WT RE SENSITIVE RESISTANT LM -1C T RE0 SENSITIVE RESISTANT WT -1C T RE SENSITIVE RESISTANT WT WT * Isolate RE1 showed a heteropeak (G/C) at katg_1, nucleotide. Downloaded from on May, 01 by guest 1

19 1 Figure 1. PCR-RFLP for katg_1 mutation conferring isoniazid resistance in M.tuberculosis. 1 Figure 1 legend: Lanes 1, + sensitive clinical isolates. Lanes: +: resistant clinical isolates. Lane : sensitive control (0bp band). Lane : Resistant control ( bp band). Lane : 0bp ladder. Lane : negative control. 1 Downloaded from on May, 01 by guest 1