JAC Advance Access originally published online on December 18, 2008
Journal of Antimicrobial Chemotherapy 2009 63(2):295-301; doi:10.1093/jac/dkn506
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Original research |
Application of denaturing HPLC to rapidly identify rifampicin-resistant Mycobacterium tuberculosis in low- and high-prevalence areas
1 HPA West Midlands Laboratory, Heart of England NHS Foundation Trust, Bordesley Green East, Birmingham B9 5SS, UK 2 Department of Microbiology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, People’s Republic of China 3 Division of Immunity and Infection, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
* Corresponding author. Tel: +44-121-424-0250; Fax: +44-121-772-6229; E-mail: jason.evans{at}heartofengland.nhs.uk
Received 4 August 2008; returned 7 September 2008; revised 4 November 2008; accepted 20 November 2008
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Objectives: Since the emergence of multidrug-resistant and extensively drug-resistant tuberculosis, there has been a call for a rapid assay to detect rifampicin-resistant strains that can be implemented into a routine service to analyse all strains in a specific geographical location. Denaturing HPLC (dHPLC) is a rapid screening test that can detect mutations in PCR amplicons. The aim of this study was to evaluate the dHPLC analysis of rifampicin-resistant Mycobacterium tuberculosis isolates using an extensive strain collection from Hong Kong and the UK and a collection of 84 consecutive clinical isolates.
Methods: DNA from 51 rifampicin-resistant M. tuberculosis strains from the UK and Hong Kong identified from 1996 to 2005 was extracted and each mutation was defined by capillary electrophoresis. A 400 bp PCR product was amplified from each strain, heteroduplexed with a known susceptible control (H37Rv) and analysed by dHPLC at 67.0°C.
Results: Forty-five out of 51 (88.2%) rifampicin-resistant strains with known DNA mutations were detected by dHPLC. Two out of 84 clinical isolates were phenotypically rifampicin-resistant and dHPLC detected a mutation in the rpoB amplicon for both these isolates. dHPLC detected a mutation in 1 out of 82 phenotypically rifampicin-susceptible isolates (M482T, a non-cluster I/II mutation). In a combined analysis of all strains and isolates, mutation detection by dHPLC analysis exhibited 88.2% sensitivity and 98.8% specificity.
Conclusions: This study shows that dHPLC analysis is sensitive and specific and could be implemented in a routine clinical service.
Keywords: antitubercular agents , microbial drug resistance , polymerase chain reaction
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Introduction |
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Multidrug-resistant tuberculosis (MDR-TB) and the recent emergence of extensively drug-resistant tuberculosis (XDR-TB) have highlighted the need for rapid detection of drug resistance, especially for first-line drugs as MDR-TB or XDR-TB isolates need to be identified as quickly as possible to initiate laboratory testing for second or third-line drugs and public health interventions.1–3 Rifampicin-resistant strains arise by spontaneous mutation in the genome often due to lack of compliance with treatment. The global burden of Mycobacterium tuberculosis is increased by local transmission or global migration of patients with drug-resistant strains.4
Unlike many Western European countries, the numbers of TB cases and rates in the UK have been slowly increasing each year since the late 1980s. The fewest number of notified TB cases since records began was 5086 notified cases of TB in 1987 with 8497 clinical cases reported in 2006, representing a rate of 14.0 per 100 000 population. Rifampicin resistance rates in the UK are relatively constant with a rate of 1.3% of all isolates from 2000 to 2006 resistant to rifampicin, including 1.5% of all new isolates in 2006.5 In Hong Kong in 2002, there was a total of 6607 TB cases, at a rate of 97.3/100 000 of the population with 0.92% of all isolates resistant to rifampicin and isoniazid.6,7
The introduction of phenotypic drug susceptibility testing using sensitive liquid culture systems has reduced the time taken to detect resistant isolates, although it can still take up to a month from initial sample collection to the provision of a final drug susceptibility result.8 Rapid detection of drug-resistant isolates is essential as it ensures that an effective treatment regimen can be implemented and alerts public health teams to enhance contact tracing efforts.
In the Midlands region of the UK, 93 out of 109 (85.3%) phenotypically rifampicin-resistant isolates identified from 1992 to 2007 are also phenotypically isoniazid-resistant, making detection of mutations in the rpoB gene a useful indicator of MDR-TB with more than 95% of mutations conferring resistance to rifampicin in M. tuberculosis isolates located in the 81 bp cluster II region of the RNA polymerase subunit B (rpoB) gene.4,5,9 Current methods for genotypic detection of resistance routinely used in laboratories include: DNA sequence analysis, which is regarded as the gold standard for mutation detection,4 reverse line hybridization,10 single-strand conformation polymorphism,11 DNA macroarrays12,13 and real-time PCR that utilizes a variety of molecular probes.14–16 Denaturing HPLC (dHPLC) offers an attractive alternative to these methods as it is relatively inexpensive, same-day results can be obtained, potentially any mutation in the amplified fragment can be detected and it can be applied on a universal basis to analyse all isolates in a region.
dHPLC is a rapid technique that can analyse large fragments, is relatively inexpensive and can potentially detect any nucleotide substitution within the 81 bp hotspot region of the rpoB gene.4 dHPLC is widely used in human genetics and has begun to be used in the identification of bacterial species.17,18 It is a cost-effective method that detects mutations in DNA sequences via the differential retention times of homoduplex wild-type DNA and heteroduplex DNA containing mutations19–21 and has been used to detect mutations conferring antibiotic resistance,22–25 and in DNA fingerprinting of bacterial strains,26,27 including the M. tuberculosis complex.28,29
A preliminary study of dHPLC analysis of the rpoB gene for mutations conferring resistance to rifampicin demonstrated that this method could detect mutations with a recent study extending this to a larger collection of strains.22,25 While the recent study demonstrated the utility of dHPLC in detecting mutations, the sensitivity was 83.3% and specificity was 91.0% when compared with DNA sequencing. The reduction in specificity was particularly related to the test not detecting the L511P mutation. In combination, these previous studies have detected >20 distinct mutations in the rpoB gene by dHPLC.22,24,25
In the study presented here, we describe and evaluate a dHPLC analysis method for the rpoB gene that is a highly sensitive and specific screening method for detecting drug resistance-conferring mutations. We created a strain collection with a wide range of known rpoB mutations by combining collections of rifampicin-resistant strains from a low-incidence area (Midlands, UK) with strains from a high-incidence area (Hong Kong). dHPLC analysis of the rpoB gene was developed using rifampicin-resistant strains with known rpoB mutations defined by capillary electrophoresis and then applied to 84 consecutive clinical DNA extracts to evaluate the clinical utility as an integral part of a dHPLC-based MIRU-VNTR DNA fingerprinting scheme for a population of 10 million in the Midlands, UK.28
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Bacterial strains, identification and drug susceptibility testing
Thirty-five non-duplicate archived rifampicin-resistant M. tuberculosis strains identified from 1996 to 2005 stored at –20°C in the Health Protection Agency Midlands Regional Centre for Mycobacteriology and 16 rifampicin-resistant strains from the Department of Microbiology, The Chinese University of Hong Kong, were selected for analysis in this study. Before this study was started, phenotypic resistance to rifampicin was confirmed by the detection of rpoB mutations in rifampicin-resistant strains by capillary electrophoresis until an internationally comparable collection (including the strains from Hong Kong) of mutations was achieved.
M. tuberculosis H37Rv (ATCC 27294) was used as a rifampicin-susceptible control. In order to evaluate the clinical utility as part of a routine strain typing service, 84 consecutive clinical isolates originating from patients in the Midlands and identified as M. tuberculosis by reverse line hybridization (Hain Lifescience, Nehren, Germany) were selected for analysis by dHPLC.30 All isolates were cultured in liquid culture (Becton, Dickinson, Oxford, UK) at 37°C until a positive growth index was detected.8 Phenotypic drug susceptibility testing was undertaken using the BD MGIT 960 liquid culture system.31 Strains from Hong Kong were initially identified using phenotypic methods and the absolute concentration method for phenotypic drug susceptibility testing.6
One millilitre of a positive liquid culture was centrifuged for 15 min at 10 000 g. Supernatant was discarded, and cells were resuspended in 300 µL of sterile distilled water and incubated for 20 min at 95°C. Samples were then incubated for 15 min in an ultrasonic water bath, centrifuged for 5 min at 10 000 g and the resulting supernatant was transferred to a clean 1.5 mL microcentrifuge tube for immediate use or storage at –70°C.30
Primer selection and rpoB PCR conditions
Two sets of oligonucleotides that amplified fragments of the M. tuberculosis rpoB gene (accession no. L27989 [GenBank] ) were designed using primer 3 (Table 1).32 An in silico analysis of the designed primers was undertaken using insilico.ehu.es and NCBI BLAST to check that only amplicons from the M. tuberculosis complex would be amplified.33,34 Primer sets rpoB-1F and rpoB-1R amplified a 500 bp fragment that was used to define mutations by capillary electrophoresis. This flanking set of primers ensured that the entire sequence of the 400 bp fragment amplified by the internal primers and analysed by dHPLC (rpoB-2F and rpoB-2R) was defined. Each PCR reaction contained: 30.6 µL of sterile distilled water, 0.5 µM of each primer, 5 µL of 10x PCR buffer, 200 µM of each dNTP, 1 mM MgCl2, 1 U of Optimase DNA Polymerase (Transgenomic, Glasgow, UK) and 1 µL of template DNA, giving a total reaction volume of 50 µL. Amplification was performed on an MBS thermal cycler (ThermoFisher, Loughborough, UK) using the following programme: an initial denaturation at 95°C for 2 min; 35 cycles of denaturation at 95°C for 30 s, primer annealing at 57.0°C for 30 s, extension at 72°C for 40 s; and a terminal extension step of 5 min at 72°C.
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Identification of rifampicin resistance-conferring mutations by DNA sequencing
Rifampicin resistance-conferring mutations in the rpoB gene were confirmed by DNA sequence analysis of all 51 phenotypically rifampicin-resistant isolates and in any of the 84 consecutive clinical isolates identified as possessing a mutation by dHPLC analysis. Amplicons of 500 bp, generated using the flanking primers rpoB-1F and rpoB-1R, were purified using the QIAquick PCR Purification Kit (Qiagen, Crawley, UK). Purified amplicon (10 ng) was labelled using the BigDye Terminator (version 3.0) cycle sequencing kit (Applied Biosystems, Warrington, UK), with both forward and reverse DNA strands analysed on a PRISM 3700 DNA Analyzer (Applied Biosystems).
To ensure equivocal concentrations for dHPLC analysis, 5 µL of each amplicon was injected onto a WAVE System (Transgenomic, Glasgow, UK) and analysed using the non-denaturing sizing programme.28 M. tuberculosis H37Rv was also quantified and used as the control wild-type DNA. The peak height (mV) of each amplicon was used to calculate the proportions required in the heteroduplex formation stage to achieve equivocal concentrations of wild-type and sample DNA.
DNA extracted from M. tuberculosis H37Rv and the analysed sample were combined in a 50 µL mix, heated to 95°C for 5 min and cooled to 25°C at a rate of 1.8°C/s on a ThermoFisher MBS Thermal Cycler.
dHPLC analysis of amplified hybridized DNA
dHPLC detects mutations in DNA sequences via the differential retention times of homoduplex wild-type DNA and heteroduplex DNA containing mutations on a polyvinyldibenzene column at a partially denaturing analysis temperature. Ten microlitres of each heteroduplex sample was injected onto a WAVE 4500 System, bound to the column via triethylammonium acetate and then eluted by the addition of increasing volumes of acetonitrile at an increasing gradient of 5%/min using the Rapid DNA programme (3.3 min, at a flow rate of 1.5 mL/min). For both collections of 51 phenotypically rifampicin-resistant isolates and 84 consecutive clinical isolates, dHPLC chromatograms were visually assessed and the presence of heteroduplex DNA was determined by the presence of two or more peaks when compared with the single peak homoduplex of M. tuberculosis H37Rv with the homoduplex peak being >4 mV.
Sensitivity, specificity, positive predictive values (PPVs) and negative predictive values (NPVs) of detecting true rifampicin resistance were calculated for the collection of known mutations and 84 consecutive clinical extracts and both collections combined.
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Identification of rifampicin resistance-conferring mutations by DNA sequence analysis
DNA sequence analysis using capillary electrophoresis of a 400 bp fragment of the rpoB gene defined mutations in 51 rifampicin-resistant strains, 35 from the UK and 16 from Hong Kong, creating 16 distinct mutations in 10 different codons forming 18 different combinations (Table 2). Seventeen out of 18 mutations were in the 81 bp cluster II ‘hotspot’ region with one mutation outside this region (I572F). An example dHPLC chromatogram of each mutation is shown in Figure 1.
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dHPLC analysis of rifampicin-resistant strains with defined rpoB mutations
A total of 45 (88.2%) out of 51 rifampicin-resistant strains with confirmed DNA mutations were detected by dHPLC at 67.0°C (Table 2).
dHPLC analysis of consecutive clinical isolates
Eighty-four consecutive isolates with unknown rifampicin susceptibility results were analysed by dHPLC. Of these 84 DNA extracts, 83 (98.8%) were amplified at the first attempt by the rpoB PCR. The lone amplification failure was re-amplified using a new DNA extract and was phenotypically rifampicin-susceptible. There were two isolates that were phenotypically rifampicin-resistant and dHPLC detected a mutation in the rpoB amplicon for both of these isolates. The mutations detected in these two rifampicin-resistant isolates were S531L and S531W. Of the 82 phenotypically rifampicin-susceptible isolates, dHPLC detected a mutation in 1 amplicon with no mutations detected in the other 81 DNA extracts. The mutation in this amplicon was confirmed by DNA sequence analysis and was found to be M482T.
dHPLC analysis exhibited sensitivity of 88.2% for the collection of rifampicin-resistant strains with defined rpoB mutations. For the 84 consecutive clinical isolates, dHPLC analysis exhibited sensitivity of 100.0%, specificity of 98.9%, PPV of 66.7% and NPV of 100.0%. For a combined analysis of both rifampicin-resistant strains with defined rpoB mutations and 84 consecutive clinical samples, detection of mutations in the rpoB gene by dHPLC analysis exhibited sensitivity of 88.2%, specificity of 98.8%, PPV of 97.8% and NPV of 93.1%.
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This study shows that dHPLC analysis of mutations in the rpoB gene is highly sensitive and specific and that this assay can be used in a routine service with confidence.
The assay described in this study can be carried out on the same day as the molecular identification of positive liquid cultures and could therefore provide data on rifampicin resistance up to 2 weeks before phenotypic drug susceptibility testing results would be known.8 Universal application of this dHPLC-based assay to all positive M. tuberculosis isolates would be able to detect epidemiologically significant unsuspected MDR-TB cases earlier. These results would direct the initiation of effective drug regimens and prophylaxis for contacts. This should enhance TB control as it would optimize treatment and prophylaxis earlier than would normally be the case.35,36
We would envisage the rpoB dHPLC assay being used as a screening test for every positive culture with capillary electrophoresis and phenotypic susceptibility testing carried out to confirm positive results. The low consumable costs and lower capital cost compared with a DNA sequencer make dHPLC very attractive.25 The dHPLC assay costs under $4.00 per isolate in terms of consumables. The utility of this assay is enhanced as it has been added directly onto an established routine clinical MIRU-VNTR DNA fingerprinting service undertaken for all M. tuberculosis strains using the same platform and methods.28 Perhaps the greatest utility in adding this assay to a routine DNA fingerprinting service that would analyse all isolates would be the detection of unsuspected drug resistance. Although there are defined risk factors for MDR-TB in the UK,37 an increase in unsuspected cases with drug-resistant TB may only be rapidly detected by a prospective service analysing all isolates. Theoretically, the utility of this assay could be enhanced even further by developing it for direct detection of rifampicin resistance in specimens as PCR amplicons are quantified before analysis so that PCR-positive specimens could be analysed. However, there are well-documented deficiencies when detecting M. tuberculosis by PCR directly from specimens.38
The strain collection used here contained 16 distinct mutations in 10 different codons forming 18 different combinations defined by capillary electrophoresis. The dHPLC assay can detect prevalent mutations both in the UK and in Hong Kong, suggesting that it should have global applicability. dHPLC detected 45 (88.2%) out of 51 rpoB mutations defined by capillary electrophoresis. Our methodology successfully detected the L511P mutation that had not been detected previously.25 However, a D516Y mutation was not detected in a single strain in this collection when tested the first time; on repeat testing, it was detected.
The most prevalent mutation in this study was S531L. In the collection of previously defined rpoB mutations, 20 (83%) out of 24 strains with this mutation were detected by dHPLC. The apparent reduction in sensitivity for the S531L mutation may be an effect of the sample collection and it may be necessary to analyse a larger collection of S531L mutations to assess if the detection rate would increase or remain the same. The sensitivity of mutation detection by dHPLC can be improved by either the addition of GC clamps or the reduction of the length of the PCR amplicon fragment analysed. GC clamps increase the melting temperature of the amplicon to stabilize variations in melting temperature across the length of the fragment. However, M. tuberculosis DNA is already high in GC content, so GC clamps would not have much effect. We believe that the length of the PCR amplicon in this study is optimal as it covers the hotspot and known non-hotspot mutations (I572F). Reducing the amplicon fragment length may actually miss some non-hotspot mutations and reduce the sensitivity.
This study amplified and analysed a larger PCR fragment (400 bp) than has been used in three previous studies (
250 bp).22,24,25 The 400 bp fragment analysed in this study covers the whole of the 81 bp cluster II ‘hotspot’ region as well as DNA regions distinct from the 81 bp region, with one such mutation (I572F) being identified in this study.
For the routine analysis of this dHPLC assay, various levels of quality control were implemented: proprietary oven calibration standards were applied on a regular basis throughout this study; the three most prevalent, geographically relevant known rpoB mutations were included on every run; and 1 in every 100 PCR amplicons would be amplified blindly from a known rifampicin-resistant isolate as an internal quality control.
When the clinical utility of this assay was assessed by applying it to 84 consecutive clinical isolates, high values for sensitivity (100.0%), specificity (98.9%) and NPV (100.0) were observed. The dHPLC assay detected an rpoB mutation in one rifampicin-susceptible isolate, which was confirmed as a non-cluster I/II non-resistance-conferring mutation (M482T) by capillary electrophoresis.39 Mutations in two rifampicin-resistant isolates were detected by dHPLC and were confirmed as S531L and S531W.
Two previous smaller studies demonstrated that dHPLC could detect mutations in the rpoB gene in a small collection of drug-resistant strains (nine mutations and eight drug-resistant strains),22,24 with a larger study using a strain collection with a wider set of mutations that also evaluated the clinical utility when applied to the routine analysis of 3000 isolates in Hong Kong. Phenotypic susceptibility testing was performed in parallel and 84 (2.7%) out of 3107 isolates were judged to be rifampicin-resistant, with 70 (83.3%) of them being detected using dHPLC.25
The overall high values for sensitivity, specificity, PPV and NPV for our methodology compared with a previous study show that dHPLC could be reliably implemented in a routine service.25
For our study, we employed a combination of a retrospective analysis of strains with defined mutations and a prospective analysis of consecutive clinical isolates. These two collections were utilized as we purposefully obtained an internationally comparable collection of known rpoB mutations for the retrospective analysis. Prospective studies of consecutive clinical isolates are the most appropriate methodology to obtain reliable sensitivity, specificity and predictive values. However, rates of rifampicin resistance are still relatively low in the populations studied so that identifying a substantial number of rifampicin-resistant isolates requires lengthy study periods and high numbers of isolates.
TB is primarily a disease of developing countries. Previous studies have demonstrated the application of the rpoB dHPLC assay in high-incidence regions such as South-East Asia.24,25 Like many other molecular assays for rpoB mutation detection, regional and structured implementation of this assay in the developing world still needs to be achieved. Increases in global TB rates have seen a concomitant rise in TB rates in the UK that intensifies the need for a drug-resistance detection assay that can be readily universally applied to all samples as a recent study has shown that conventionally recognized risk factors for MDR-TB were absent in almost half (43%) of all MDR-TB cases.40
The PCR amplification stage of the dHPLC assay is a relatively basic PCR and so extensive training is not required. Result analysis is automated but this is not always necessary as positive results are readily identifiable visually. The costs of confirmation will be minimal as only 1% to 2% of all M. tuberculosis isolates in the UK are rifampicin-resistant.
In the study presented here, DNA was extracted from liquid cultures that are more rapid in isolating M. tuberculosis than solid cultures, thus increasing the speed of the dHPLC analysis even further, whereas in the previous study DNA was extracted from cells cultivated on solid LJ cultures.8,25
This study expands the number of rpoB mutations detected by dHPLC, including those mutations not detected in previous studies and shows the ease with which dHPLC analysis of rpoB mutations can be integrated into routine genotyping of M. tuberculosis. It is likely that MDR- and XDR-TB will be encountered with greater frequency in the future and having an economical and rapid method for their detection will be of considerable use.
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Part of this study was supported by the UK Department of Health.
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None to declare.
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DNA sequence analysis was undertaken at the Functional Genomics and Proteomics Laboratories, Division of Biosciences, University of Birmingham. This facility is funded by grant 6/JIF13209 from the BBSRC. We wish to thank all microbiology laboratories in the Midlands that refer specimens to the Health Protection Agency Midlands Regional Centre for Mycobacteriology, public health teams, health protection units and the staff at the Midlands Regional Centre for Mycobacteriology for isolation and identification.
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