Hairong Huang34 Chuanyou Li34 Shaofa Xu34 and Li Li12
1 Department of Tuberculosis Tianjin Haihe Hospital Tianjin 300350 China2 Tianjin Respiratory Disease Research Institute Tianjin 300350 China3National Tuberculosis Clinical Laboratory Beijing Chest Hospital Capital Medical University Beijing 101149 China4 Beijing Key Laboratory in Drug Resistant Tuberculosis Research Beijing Tuberculosis ampThoratic Tumor Research InstituteBeijing 101149 China
Correspondence should be addressed to Shaofa Xu xushaofa263net and Li Li hhyyjhkll126com
Received 5 June 2014 Revised 29 October 2014 Accepted 3 November 2014
Academic Editor Madhab K Chattopadhyay
Copyright copy 2015 Zaoxian Mei et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
To find the potential reasons for the discrepancies in the drug susceptibility test (DST) of M tuberculosis isolates twenty pairedisolates with disputed drug susceptibilities to isoniazid (INH) were selected according to the MGIT960 testing and Lowenstein-Jensen (L-J) proportionmethodsTheirMICs were confirmed again by brothmicrodilutionmethod and by L-J proportionmethodThe spoligotyping results showed that of all the 20 paired strains 11 paired isolates belonged to the Beijing genotype and 6 pairedisolates belonged to SIT1634 and that each of the remaining 3 paired isolates had two genotypes namely SIT1 and SIT1634Those 3paired isolates with different intrapair spoligotypes were further confirmed asmixed infection by the results that those three pairs ofisolates with different 12 locusMIRU intrapair types and one pair carried different base pair at codon 315 (AGC versus AAC) Totallymutations in the katG gene were identified in 13 paired isolates No mutations were found in the regulatory sequences and openreading frames (ORF) of the inhA and ahpC genes in any of the tested isolates Those results showed that the different test systemsand the mixed infection with particular genotypes ofM tuberculosis strains contributed to the drug susceptibility discrepancies
1 Introduction
Performance of drug susceptibility testing (DST) to measuredrug resistance is important not only before treatment butalso in the course of therapy to identify acquired resistanceespecially in the areas with a high incidence of MDR-TB [1]Conventional DST methods rely on egg-based (Lowenstein-Jensen L-J) or agar-based (Middlebrook) media but theseare laborious and time-consuming procedures requiring 3to 8 weeks to obtain results [2] A number of new methodsfor DST including the mycobacterial growth indicator tube(MGIT) [3] 119864 test [4] and Alamar blue [5] methods havebeen introduced over the last decade to detect mycobacteriarapidly and to improve their growth rates [6 7]
The BACTEC MGIT960 method has been assessed inmany countries and its degree of agreement with conven-tional DST methods inM tuberculosis has been assessed [8ndash10]Meta-analysis of published results revealed high accuracyand high predictive value associated with the use of BACTECMGIT960 [11] However there are still discrepancies in theDST results obtained for different anti-TB drugs betweenBACTEC MGIT960 and other DST methods The discrep-ancies in INH susceptibility between the MGIT960 and L-J proportion methods for example varied from 0 to 1[9] however few investigations have been reported thataddressed the possible mechanisms underlying the discrep-ancies between the MGIT960 system and L-J proportionmethods
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 651980 7 pageshttpdxdoiorg1011552015651980
2 BioMed Research International
Total 20 positive byMGIT960 but negative by agar proportion method
Polluted
Total 330 DST positive to INH both by MGIT960 and by agar proportion methods
Total 17 were NTM
Total 1412 culture positive isolates
Total 1216 culture positive isolates by L-J
Total 1303 culture positive isolates by MGIT960
Total 339 DST positive to Total 357 DST positive to INH by MGIT960INH by agar proportion
method
Figure 1 Strains selected in this experiment A total of 1014 culturepositive isolates were included in this study which were isolated in2006 We focus on the INH as it is a very important antibiotic incuring tuberculosis In this study of all the total 1412 culture positiveisolates 1216 were positive on the L-J medium of which 339 wereresistant by L-J method to INH and 1303 isolates were positive bythe MGIT960 of which 357 were resistant to INH by the MGIT960system Total 330 were DST positive to INH by both MGIT960 andagar proportion methods of which 20 isolates with positive bothby MGIT960 system but negative by agar proportion method wereexamined in this study
Discrepancies can arise from many reasons for exampledifferent DST systems used mixed infection with differentM tuberculosis strains and last but not least contaminationIn this study 20 paired isolates with disputed drug suscep-tibilities to INH were selected according to the MGIT960testing and L-J proportion methods The name of the ldquopairedisolatesrdquo referred to the two isolates obtained separately fromthe cultures after the DST by MGIT960 and L-J proportionmethods from the same sputum of the patient The reasonsfor the DST discrepancies were analyzed by the spoligotypingand VNTR genotyping methods and drug resistance-relatedmutations tested in INH resistance-related genes
2 Materials and Methods
21 Strains and Antibiotics A total of 20 pairedM tuberculo-sis isolates with DST discrepancies were collected in TianjinHaihe Hospital in the year of 2006 from total 1412 isolates(Figure 1) ldquopaired isolatesrdquo were from the the culture of theMGIT960 and L-J proportion method respectively whichwas mentioned above Meanwhile 96 randomly selectedisolates whose MGIT960 and agar proportion DST resultswere in agreement were also collected from the same hospi-tal The 20 paired M tuberculosis isolates were determinedto be sensitive to INH using the conventional L-J propor-tion method (1 120583gmL) [12] but resistant to INH using the
BACTEC MGIT960 method (01 120583gmL Becton DickinsonMicrobiology Systems MD USA) [9]M tuberculosisH37Rv(ATCC27294) obtained from theChineseNational ReferenceLaboratory was used as a control
22 Determination of the MIC of INH by Middlebrook 7H9Broth Microdilution and L-J Agar Dilution Resazurin wasused as an indicator to test the MIC of INH in the Mid-dlebrook 7H9 broth microdilution method [13] Briefly a100 120583L volume of Middlebrook 7H9 broth containing 005Tween 80 and 10 OADC (Sigma USA) was dispensed intothe wells of a 96-well cell culture plate (Corning Coast)INH concentrations in Middlebrook 7H9 medium were asfollows 01 02 04 08 10 12 16 and 18mgL Recoveredisolates were collected from L-J slants and homogenizedTurbidity was adjusted to the number 1 McFarland standard(approximately 1 times 107 CFUmL) and the suspension isdiluted 1 10 and 100 120583L of the dilution is added in each wellthat contains 100 120583L of the appropriate INH dilution Thefinal inoculum concentrationwas 5times 104 CFUmLThe plateswere sealed and incubated at 37∘C for one week Twenty-five microliter of 002 resazurin (Sigma Chem Co USA)solution was then added to each well and the plates wereincubated for an additional 2 days A change in color fromblue to pink indicated the growth of bacteria and the MICwas read as the minimum INH concentration that preventedthe color change in the presence of resazurin
Determination of the MIC of INH using the L-J pro-portion method followed the protocol of the Chinese Anti-Tuberculosis Association [12] INH concentrations used inthe L-J medium were 20 18 16 12 10 08 06 04and 02mgL About 105 CFU were inoculated on the INH-containingmedium slants and results were recorded after 5-6weeks
23 Genomic DNA Isolation Polymerase Chain Reaction(PCR) and Sequence Analysis Colonies were first removedfrom the recovering slants by scraping resuspended in 500120583Lof TE (10mM Tris 1mM EDTA (pH 80)) and killed byheating at 80∘C for 30min The DNA extraction methodprimers (from CyberSyn Co Beijing China) and PCRconditions were as described previously [14] The primerswere designed to amplify the katG gene including the regionaround codon 315 the inhA regulatory region the inhAORF and oxyR-ahpC regions (Table 1) [15 16] Both strandswere sequenced for confirmation Mutations were identifiedby BLAST comparisons with M tuberculosis H37Rv as thereference (GenBank number NC 0009623)
24Molecular Typing by Spoligotyping and the 12-LocusMIRUMethod Spoligotyping was performed with a commercialkit (Isogen Bioscience BV Maarssen The Netherlands)according to the manufacturerrsquos instructions Amplificationof the direct variant regions for spoligotyping was performedessentially as described previously [17] Interpretation ofspoligotype patterns and assignment of octal codes werebased on SITVIT2 database (Pasteur Institute of GuadeloupeParris France) which is an updated version of the
BioMed Research International 3
Table 1 Primers used for PCR amplification in this study
Gene Forward primer 51015840-31015840 Reverse primer 51015840-31015840
katG GCT GCT GTG GCC GGT CAA GA CGT CCT TGG CGG TGT ATT GCinhA reg CCT CGC TGC CCA GAA AGG GA ATC CCC CGG TTT CCT CCG GTinhA ORF GAA CTC GAC GTG CAA AAC CAT CGA AGC ATA CGA ATAoxyR-ahpC CTG CGA CGG TGC TGG CACG CAC GCT GCT GCG GGT GAT TGA T
previously released SpolDB4 database (httpwwwpasteur-guadeloupefr8081SITVITDemotsSpoligojsp) as previ-ously described [18]
The numbers of tandem repeats (TRs) at each locus inthe isolates were determined on the basis of the number ofwhole repeats in a PCR product of the size estimated from thegel [19] Polymerase chain reaction assays for the 12 chosenloci were repeated and compared within and between gelsto ensure consistent estimation of size and TR copy number[20]
3 Results
31 Genotyping Analysis Genotyping analysis can determinenot only whether an infection results from transmission ofthe given tuberculosis isolate but also whether the infectioninvolves more than one strain of M tuberculosis Resultsfrom our genotyping analysis showed that 10 paired isolatesbelong to the Spoligotype International Type SIT1 (Beijinggenotype 000000000003771) and 6 paired isolates belongto the Spoligotype International Type SIT1634 (MANU2777777777723771) (Table 2) a spoligotype that was notfound in the 96 randomly selected clinical isolates (Table 3)Three paired isolates were mixtures of the SIT1 and SIT1634spoligotypes and one pair was a mixture of SIT1 and theSIT269 (Beijing genotype 000000000000771) spoligotypesCompared with our set of 96 randomly selected isolates fromTianjin only the Beijing and MANU genotypes were presentand the percentage of the MANU genotype was extremelyhigh (20 paired isolates 1540 375 96 random clinicalisolates 396 3125)
Results obtained by using the 12-locusMIRUmethod [19]showed that 20 pairs of isolates had 14 MIRU patterns Boththe spoligotyping and the MIRU patterns were different in
the isolates named as 6 12 and 18 pairs individually Theisolates named as 7 pairs had different spoligotypes but thesame MIRU type (Table 2)
32 MICs of the Tested Strains To identify the differencesbetween the liquid Middlebrook 7H9 and L-J proportionmethods in DST we tested the MICs of each of the 16 pairedINH-resistant isolates and 4 pairs of isolates which consistedof different genotypes using both Middlebrook 7H9 brothmicrodilution and L-J proportion methods The MICs of allthe 24 tested isolates were determined to be greater than01 120583gmL (01 to 06 120583gmL) using the Middlebrook 7H9broth microdilution method and greater than 03120583gmL (04to 18 120583gmL) using the L-J proportionmethod (Table 4)TheMICs of 5 pairs of the tested isolates using the L-J proportionmethod were higher than 1 120583gmL the cutoff concentrationfor determining drug susceptibility in the L-J agar proportionmethod in this study (Table 4)
33 Sequence Analysis of the Putative INH-Target GenesMutations in the katG gene were identified in 13 pairedisolates of which each of 12 paired isolates carried the samemutations and one pair which showed a DST discrepancyby MGIT960 and L-J proportion methods carried differentbase pair at codon 315 (AGC versus AAC) The AGC315AACmutation was found in 4 paired isolates while 9 pairedisolates carried themutation AGC315ACCTheAGC315AACand AGC 315ACC mutations were not associated with speci-ficity to the Beijing or MANU2 genotypes among the testedisolates Seven paired isolates did not contain mutations inthe katG gene and nomutations were found in the regulatorysequences and open reading frames (ORF) of the inhA andahpC genes in any of the tested isolates (Table 4)
4 BioMed Research International
Table 2 Genotypes of the 20 isolates with discrepancies in theirINH DST as determined by the Middlebrook 7H9 broth microdi-lution and L-J agar dilution methods
Note order of 12 MIRU loci is 2 4 10 16 20 23 24 26 27 31 39 and 40
4 Discussion
Different DST methods have been developed and are used inroutine clinical practice such as the conventional L-Jmethodsand the automated MBBacT (Organon Teknika TurnhoutBelgium) ESPII (Difco Laboratories Detroit Michigan)BACTEC 9000MB (Becton DickensonMicrobiology System
Table 3 Spoligotyping patterns of the 96 randomly selected Mtuberculosis isolates
Number ofisolates Shared types Spoligotyping pattern
Sparks MD) and BACTECMGIT 960 (BBL Becton Dickin-sonMicrobiology Systems CockeysvilleMD) systems [5 21ndash23]TheDST results would be influenced bymany steps of theprotocol including the culture and the DST methods In thisstudy we analyzed the discrepancy of the drug susceptibilitytest by the MGIT and L-J methods for the isolates collectedfrom the culture by MGIT and L-J respectively
Except for the median time to report the DST resultstheM tuberculosis complex culture positivity rates were alsogreatly different in MGIT and L-J [24] which indicated thepossible culture preference to somewhat And the detectiontime accuracy and performance capacity are also variableby different DST methods Studies reported that the reasonsfor the different performance capacity among these methodsmainly resulted from the different DST systems [23 25]The most obvious difference is the drug concentrations usedfor the DST In MGIT system the sensitive strains weresusceptible to the INH less than the 01 120583gmL while theconcentration of the INH was 1120583gmL in L-J system in thisstudy [12 26 27] Of all the 20 paired cases 15 cases hadMIC in borderlines between theMGIT and theDSTmethodswhich was a usual reason for the discordant
Many reports showed that there was a good concordancebetween DST on L-J and MGIT for INH in DST [25ndash27] Inthis study we still found that 20 paired isolates with the samegenotypes individually showed the discrepancy in the drugsusceptibilities to INH according to the MGIT960 testingand L-J proportionmethods Lawson et al demonstrated thatthere was a substantial degree of agreement between the twomethods with similar INH and rifampicin DST patterns butmore frequent detection of streptomycin resistance and lessfrequent detection of ethambutol with L-J than MGIT-960However the differenceswere not statistically significant [25]Amultiple center evaluation showed that the discrepancies inINH susceptibility between theMGIT960 and L-J proportionmethods varied from 0 to 1 [9]
Mixed infection with the different genotypes of Mtuberculosis in the same patient also affected the DST resultseven by the same testing systems [28 29] In this studyheterogeneous genotypeswere found in the isolates fromeachof the 4 patientsThree patients were infected by the different
BioMed Research International 5
Table 4 MIC of INH and the katG inhA and oxyR-ahpCmutations of the 20 pairs ofM tuberculosis isolates with DST discrepancies
Note katG315 is the predominant mutation The wild type is AGClowast16 isolates with consistent genotype in pair and 4 pairs of isolates (bold) with different genotypes in pair
stains with Spoligotype International Type SIT1634 (Manu2)andBeijing genotypes and 1 patientwas infected by the strainswith two different Beijing genotypes And also our test on themutations of the putative INH-target genes katG inhA andahpC further confirmed one patient (number 18) with mixedinfection by the heterogeneous genotypes (Table 4)
Some mycobacterial characteristics might be associatedwith particular genotypes A well-known but controversial
example is that the Beijing family strains of M tuberculosisare often associated with relapse [30] drug resistance [31]and an increased ability to cause disease to be transmittedwithin certain geographic settings [32 33] The isolateswith particular genotypes such as Spoligotype InternationalType SIT1634 (Manu2) in this study showed higher rate ofresistance in MGIT960 system than in L-J system In thisstudy we found that the percentage of ldquoMANUrdquo genotype
6 BioMed Research International
strains was markedly increased in paired isolates whoseDST results showed discrepancies (375) compared to therandomly selected clinical isolates (3125) An unusuallyhigh proportion of strains belonging to the ldquoManurdquo clade(2715) were also reported by Helal et al [18] InterestinglyManu2 strains (SIT1634) have rarely been reported in Tianjinor even in China as a whole [34 35] or in the SPOLDB4database (excluding this study 119899 = 3 1 from India and 2 fromthe USA)
In this study all the 40 isolates were determined asresistant by MGIT and sensitive by L-J of which twenty-seven isolates were found with mutations in katG315 and 13isolates were found with no mutations in katG315 (Table 4)Those results of the mutations found in the INH-targetedgenes supported that the DST result by the MGIT was moreaccurate than that by the L-J andwe also found that theMICsof some isolates by L-J agar method were very higher thanthose in the first execution in clinic which indicated to someextent that the operation needs to be improved in proportionmethod on L-J agar
5 Conclusion
Our study confirmed that the discrepancies of the DSTin M tuberculosis clinical isolates did exist for INH Oneof the reasons for the discrepancy is the different testsystems between the BACTEC MGIT960 system and thetraditional L-J proportion method Mixed infection by thestrains with MANU2 and Beijing genotype patterns couldalso contributed to drug discrepancies
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Zaoxian Mei and Zhaogang Sun contributed equally to thiswork
Acknowledgments
The authors thank the Beijing Bio-Bank of Clinical Resourceson Tuberculosis and the Outpatient Department of Tian-jin Haihe Hospital for supplying M tuberculosis H37Rvand clinical isolates They also thank the National Spe-cial Key Project of China on Major Infectious Diseases(2013ZX10003009) Beijing Municipal Administration ofHospitals ClinicalMedicine Development of Special FundingSupport (ZYLX201304) and the Beijing High-level TechnicalPersonnel Training Project in Health (2011-3-069) for finan-cial support
References
[1] J G Pasipanodya S Srivastava and T Gumbo ldquoMeta-analysisof clinical studies supports the pharmacokinetic variability
hypothesis for acquired drug resistance and failure of antituber-culosis therapyrdquo Clinical Infectious Diseases vol 55 no 2 pp169ndash177 2012
[2] P T Kent and G P Kubica Public Health Mycobacteriology AGuide for Level III Laboratory Centers for Diseases ControlAtlanta Ga USA 1985
[3] S B Walters and B A Hanna ldquoTesting of susceptibilityof Mycobacterium tuberculosis to isoniazid and rifampin bymycobacterium growth indicator tube methodrdquo Journal ofClinical Microbiology vol 34 no 6 pp 1565ndash1567 1996
[4] AWanger and KMills ldquoTesting ofMycobacterium tuberculosissusceptibility to ethambutol isoniazid rifampin and strepto-mycin by using Etestrdquo Journal of Clinical Microbiology vol 34no 7 pp 1672ndash1676 1996
[5] L A Collins and S G Franzblau ldquoMicroplate Alamar blue assayversus BACTEC 460 system for high-throughput screening ofcompounds against Mycobacterium tuberculosis and Mycobac-terium aviumrdquo Antimicrobial Agents and Chemotherapy vol 41no 5 pp 1004ndash1009 1997
[6] C Piersimoni ldquoTB control strategies present and future indiagnostic methods what the clinician can ask to the labrdquoMonaldi Archives for Chest Disease vol 57 no 5-6 pp 306ndash3102002
[7] G L Woods G Fish M Plaunt and T Murphy ldquoClinicalevaluation of Difco ESP culture system II for growth anddetection ofmycobacteriardquo Journal of ClinicalMicrobiology vol35 no 1 pp 121ndash124 1997
[8] R A Adegbola P Hill I Baldeh et al ldquoSurveillance of drug-resistant Mycobacterium tuberculosis in The Gambiardquo Interna-tional Journal of Tuberculosis and Lung Disease vol 7 no 4 pp390ndash393 2003
[9] C M S Giampaglia M C Martins G B De Oliveira Vieiraet al ldquoMulticentre evaluation of an automated BACTEC 960system for susceptibility testing ofMycobacterium tuberculosisrdquoInternational Journal of Tuberculosis and Lung Disease vol 11no 9 pp 986ndash991 2007
[10] L Heifets and G A Cangelosi ldquoDrug susceptibility testing ofMycobacterium tuberculosis a neglected problem at the turnof the centuryrdquo International Journal of Tuberculosis and LungDisease vol 3 no 7 pp 564ndash581 1999
[11] C Piersimoni A Olivieri L Benacchio and C ScarparoldquoCurrent perspectives on drug susceptibility testing ofMycobac-terium tuberculosis complex the automated nonradiometricsystemsrdquo Journal of Clinical Microbiology vol 44 no 1 pp 20ndash28 2006
[12] Chinese Anti-Tuberculosis Association Protocols for Tubercu-losis Diagnosis in Laboratory Chinese Educational and CulturalPublisher Beijing China 1st edition 2006
[13] J-C Palomino A Martin M Camacho H Guerra JSwings and F Portaels ldquoResazurin microtiter assay platesimple and inexpensive method for detection of drug resis-tance inMycobacterium tuberculosisrdquo Antimicrobial Agents andChemotherapy vol 46 no 8 pp 2720ndash2722 2002
[14] N Siddiqi M Shamim S Hussain et al ldquoMolecular char-acterization of multidrug-resistant isolates of Mycobacteriumtuberculosis from patients in North Indiardquo Antimicrobial Agentsand Chemotherapy vol 46 no 2 pp 443ndash450 2002
[15] M Y Lipin V N Stepanshina I G Shemyakin and T MShinnick ldquoAssociation of specific mutations in katG rpoB rpsLand rrs geneswith spoligotypes ofmultidrug-resistantMycobac-terium tuberculosis isolates in RussiardquoClinical Microbiology andInfection vol 13 no 6 pp 620ndash626 2007
BioMed Research International 7
[16] Z Sun J Zhang X Zhang S Wang Y Zhang and C LildquoComparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strainsand clinical isolatesrdquo International Journal of AntimicrobialAgents vol 31 no 2 pp 115ndash121 2008
[17] J Kamerbeek L Schouls AKolk et al ldquoSimultaneous detectionand strain differentiation of Mycobacterium tuberculosis fordiagnosis and epidemiologyrdquo Journal of Clinical Microbiologyvol 35 no 4 pp 907ndash914 1997
[18] Z H Helal M S E-D Ashour S A Eissa et al ldquoUnexpectedlyhigh proportion of ancestral manu genotype Mycobacteriumtuberculosis strains cultured from tuberculosis patients inEgyptrdquo Journal of Clinical Microbiology vol 47 no 9 pp 2794ndash2801 2009
[19] P Supply E Mazars S Lesjean V Vincent B Gicquel andC Locht ldquoVariable human minisatellite-like regions in theMycobacterium tuberculosis genomerdquo Molecular Microbiologyvol 36 no 3 pp 762ndash771 2000
[20] M B Boniotti M Goria D Loda et al ldquoMolecular typingof Mycobacterium bovis strains isolated in Italy from 2000to 2006 and evaluation of variable-number tandem repeatsfor geographically optimized genotypingrdquo Journal of ClinicalMicrobiology vol 47 no 3 pp 636ndash644 2009
[21] W H Benjamin Jr K B Waites A Beverly et al ldquoComparisonof the MBBacT system with a revised antibiotic supplementkit to the BACTEC 460 system for detection of mycobacteriain clinical specimensrdquo Journal of Clinical Microbiology vol 36no 11 pp 3234ndash3238 1998
[22] World Health Organisation The Use of Liquid Medium forCulture and DST in Middle- and Low-Income Countries 2007httpwwwwhointtbdotslaboratorypolicyenindex3htm
[23] W W Yew S C W Tong K S Lui S K F Leung CH Chau and E P Wang ldquoComparison of MBBacT systemand agar proportion method in drug susceptibility testingof Mycobacterium tuberculosisrdquo Diagnostic Microbiology andInfectious Disease vol 39 no 4 pp 229ndash232 2001
[24] Y Balabanova F Drobniewski V Nikolayevskyy et al ldquoAnintegrated approach to rapid diagnosis of tuberculosis andmul-tidrug resistance using liquid culture andmolecular methods inRussiardquo PLoS ONE vol 4 no 9 Article ID e7129 2009
[25] L Lawson N Emenyonu S T Abdurrahman et al ldquoCom-parison ofMycobacterium tuberculosis drug susceptibility usingsolid and liquid culture in Nigeriardquo BMC Research Notes vol 6no 1 article 215 2013
[26] S J Kim ldquoDrug-susceptibility testing in tuberculosis methodsand reliability of resultsrdquo European Respiratory Journal vol 25no 3 pp 564ndash569 2005
[27] World Health Organization ldquoPolicy guidance on drug-sus-ceptibility testing (DST) of second-line antituberculosisdrugsrdquo 2008 httpwwwwhointtbfeatures archivexdr mdrpolicy guidance
[28] I C Shamputa L Jugheli N Sadradze et al ldquoMixed infectionand clonal representativeness of a single sputum sample intuberculosis patients from a penitentiary hospital in GeorgiardquoRespiratory Research vol 7 article 99 2006
[29] I C Shamputa L Rigouts L A Eyongeta et al ldquoGenotypic andphenotypic heterogeneity among Mycobacterium tuberculosisisolates from pulmonary tuberculosis patientsrdquo Journal of Clin-ical Microbiology vol 42 no 12 pp 5528ndash5536 2004
[30] M N T Huyen T N Buu E Tiemersma et al ldquoTuberculosisrelapse in vietnam is significantly associated with Mycobac-terium tuberculosis Beijing genotype infectionsrdquo The Journal ofInfectious Diseases vol 207 no 10 pp 1516ndash1524 2013
[31] EuropeanConcertedAction onNewGenerationGeneticMark-ers and Techniques for the Epidemiology and Control ofTuberculosis ldquoBeijingW genotypeMycobacterium tuberculosisand drug resistancerdquo Emerging Infectious Diseases vol 12 no 5pp 736ndash743 2006
[32] B C De Jong P C Hill A Aiken et al ldquoProgression to activetuberculosis but not transmission varies by Mycobacteriumtuberculosis lineage in the Gambiardquo Journal of Infectious Dis-eases vol 198 no 7 pp 1037ndash1043 2008
[33] M Hanekom G D van der Spuy E Streicher et al ldquoA recentlyevolved sublineage of the Mycobacterium tuberculosis Beijingstrain family is associated with an increased ability to spreadand cause diseaserdquo Journal of Clinical Microbiology vol 45 no5 pp 1483ndash1490 2007
[34] Y Kong M D Cave L Zhang et al ldquoAssociation betweenMycobacterium tuberculosis BeijingW lineage strain infectionand extrathoracic tuberculosis Insights from epidemiologicand clinical characterization of the three principal geneticgroups of M tuberculosis clinical isolatesrdquo Journal of ClinicalMicrobiology vol 45 no 2 pp 409ndash414 2007
[35] Y Pang Y Zhou B Zhao et al ldquoSpoligotyping and drugresistance analysis of Mycobacterium Tuberculosis strains fromnational survey in Chinardquo PLoS ONE vol 7 no 3 Article IDe32976 2012
Total 20 positive byMGIT960 but negative by agar proportion method
Polluted
Total 330 DST positive to INH both by MGIT960 and by agar proportion methods
Total 17 were NTM
Total 1412 culture positive isolates
Total 1216 culture positive isolates by L-J
Total 1303 culture positive isolates by MGIT960
Total 339 DST positive to Total 357 DST positive to INH by MGIT960INH by agar proportion
method
Figure 1 Strains selected in this experiment A total of 1014 culturepositive isolates were included in this study which were isolated in2006 We focus on the INH as it is a very important antibiotic incuring tuberculosis In this study of all the total 1412 culture positiveisolates 1216 were positive on the L-J medium of which 339 wereresistant by L-J method to INH and 1303 isolates were positive bythe MGIT960 of which 357 were resistant to INH by the MGIT960system Total 330 were DST positive to INH by both MGIT960 andagar proportion methods of which 20 isolates with positive bothby MGIT960 system but negative by agar proportion method wereexamined in this study
Discrepancies can arise from many reasons for exampledifferent DST systems used mixed infection with differentM tuberculosis strains and last but not least contaminationIn this study 20 paired isolates with disputed drug suscep-tibilities to INH were selected according to the MGIT960testing and L-J proportion methods The name of the ldquopairedisolatesrdquo referred to the two isolates obtained separately fromthe cultures after the DST by MGIT960 and L-J proportionmethods from the same sputum of the patient The reasonsfor the DST discrepancies were analyzed by the spoligotypingand VNTR genotyping methods and drug resistance-relatedmutations tested in INH resistance-related genes
2 Materials and Methods
21 Strains and Antibiotics A total of 20 pairedM tuberculo-sis isolates with DST discrepancies were collected in TianjinHaihe Hospital in the year of 2006 from total 1412 isolates(Figure 1) ldquopaired isolatesrdquo were from the the culture of theMGIT960 and L-J proportion method respectively whichwas mentioned above Meanwhile 96 randomly selectedisolates whose MGIT960 and agar proportion DST resultswere in agreement were also collected from the same hospi-tal The 20 paired M tuberculosis isolates were determinedto be sensitive to INH using the conventional L-J propor-tion method (1 120583gmL) [12] but resistant to INH using the
BACTEC MGIT960 method (01 120583gmL Becton DickinsonMicrobiology Systems MD USA) [9]M tuberculosisH37Rv(ATCC27294) obtained from theChineseNational ReferenceLaboratory was used as a control
22 Determination of the MIC of INH by Middlebrook 7H9Broth Microdilution and L-J Agar Dilution Resazurin wasused as an indicator to test the MIC of INH in the Mid-dlebrook 7H9 broth microdilution method [13] Briefly a100 120583L volume of Middlebrook 7H9 broth containing 005Tween 80 and 10 OADC (Sigma USA) was dispensed intothe wells of a 96-well cell culture plate (Corning Coast)INH concentrations in Middlebrook 7H9 medium were asfollows 01 02 04 08 10 12 16 and 18mgL Recoveredisolates were collected from L-J slants and homogenizedTurbidity was adjusted to the number 1 McFarland standard(approximately 1 times 107 CFUmL) and the suspension isdiluted 1 10 and 100 120583L of the dilution is added in each wellthat contains 100 120583L of the appropriate INH dilution Thefinal inoculum concentrationwas 5times 104 CFUmLThe plateswere sealed and incubated at 37∘C for one week Twenty-five microliter of 002 resazurin (Sigma Chem Co USA)solution was then added to each well and the plates wereincubated for an additional 2 days A change in color fromblue to pink indicated the growth of bacteria and the MICwas read as the minimum INH concentration that preventedthe color change in the presence of resazurin
Determination of the MIC of INH using the L-J pro-portion method followed the protocol of the Chinese Anti-Tuberculosis Association [12] INH concentrations used inthe L-J medium were 20 18 16 12 10 08 06 04and 02mgL About 105 CFU were inoculated on the INH-containingmedium slants and results were recorded after 5-6weeks
23 Genomic DNA Isolation Polymerase Chain Reaction(PCR) and Sequence Analysis Colonies were first removedfrom the recovering slants by scraping resuspended in 500120583Lof TE (10mM Tris 1mM EDTA (pH 80)) and killed byheating at 80∘C for 30min The DNA extraction methodprimers (from CyberSyn Co Beijing China) and PCRconditions were as described previously [14] The primerswere designed to amplify the katG gene including the regionaround codon 315 the inhA regulatory region the inhAORF and oxyR-ahpC regions (Table 1) [15 16] Both strandswere sequenced for confirmation Mutations were identifiedby BLAST comparisons with M tuberculosis H37Rv as thereference (GenBank number NC 0009623)
24Molecular Typing by Spoligotyping and the 12-LocusMIRUMethod Spoligotyping was performed with a commercialkit (Isogen Bioscience BV Maarssen The Netherlands)according to the manufacturerrsquos instructions Amplificationof the direct variant regions for spoligotyping was performedessentially as described previously [17] Interpretation ofspoligotype patterns and assignment of octal codes werebased on SITVIT2 database (Pasteur Institute of GuadeloupeParris France) which is an updated version of the
BioMed Research International 3
Table 1 Primers used for PCR amplification in this study
Gene Forward primer 51015840-31015840 Reverse primer 51015840-31015840
katG GCT GCT GTG GCC GGT CAA GA CGT CCT TGG CGG TGT ATT GCinhA reg CCT CGC TGC CCA GAA AGG GA ATC CCC CGG TTT CCT CCG GTinhA ORF GAA CTC GAC GTG CAA AAC CAT CGA AGC ATA CGA ATAoxyR-ahpC CTG CGA CGG TGC TGG CACG CAC GCT GCT GCG GGT GAT TGA T
previously released SpolDB4 database (httpwwwpasteur-guadeloupefr8081SITVITDemotsSpoligojsp) as previ-ously described [18]
The numbers of tandem repeats (TRs) at each locus inthe isolates were determined on the basis of the number ofwhole repeats in a PCR product of the size estimated from thegel [19] Polymerase chain reaction assays for the 12 chosenloci were repeated and compared within and between gelsto ensure consistent estimation of size and TR copy number[20]
3 Results
31 Genotyping Analysis Genotyping analysis can determinenot only whether an infection results from transmission ofthe given tuberculosis isolate but also whether the infectioninvolves more than one strain of M tuberculosis Resultsfrom our genotyping analysis showed that 10 paired isolatesbelong to the Spoligotype International Type SIT1 (Beijinggenotype 000000000003771) and 6 paired isolates belongto the Spoligotype International Type SIT1634 (MANU2777777777723771) (Table 2) a spoligotype that was notfound in the 96 randomly selected clinical isolates (Table 3)Three paired isolates were mixtures of the SIT1 and SIT1634spoligotypes and one pair was a mixture of SIT1 and theSIT269 (Beijing genotype 000000000000771) spoligotypesCompared with our set of 96 randomly selected isolates fromTianjin only the Beijing and MANU genotypes were presentand the percentage of the MANU genotype was extremelyhigh (20 paired isolates 1540 375 96 random clinicalisolates 396 3125)
Results obtained by using the 12-locusMIRUmethod [19]showed that 20 pairs of isolates had 14 MIRU patterns Boththe spoligotyping and the MIRU patterns were different in
the isolates named as 6 12 and 18 pairs individually Theisolates named as 7 pairs had different spoligotypes but thesame MIRU type (Table 2)
32 MICs of the Tested Strains To identify the differencesbetween the liquid Middlebrook 7H9 and L-J proportionmethods in DST we tested the MICs of each of the 16 pairedINH-resistant isolates and 4 pairs of isolates which consistedof different genotypes using both Middlebrook 7H9 brothmicrodilution and L-J proportion methods The MICs of allthe 24 tested isolates were determined to be greater than01 120583gmL (01 to 06 120583gmL) using the Middlebrook 7H9broth microdilution method and greater than 03120583gmL (04to 18 120583gmL) using the L-J proportionmethod (Table 4)TheMICs of 5 pairs of the tested isolates using the L-J proportionmethod were higher than 1 120583gmL the cutoff concentrationfor determining drug susceptibility in the L-J agar proportionmethod in this study (Table 4)
33 Sequence Analysis of the Putative INH-Target GenesMutations in the katG gene were identified in 13 pairedisolates of which each of 12 paired isolates carried the samemutations and one pair which showed a DST discrepancyby MGIT960 and L-J proportion methods carried differentbase pair at codon 315 (AGC versus AAC) The AGC315AACmutation was found in 4 paired isolates while 9 pairedisolates carried themutation AGC315ACCTheAGC315AACand AGC 315ACC mutations were not associated with speci-ficity to the Beijing or MANU2 genotypes among the testedisolates Seven paired isolates did not contain mutations inthe katG gene and nomutations were found in the regulatorysequences and open reading frames (ORF) of the inhA andahpC genes in any of the tested isolates (Table 4)
4 BioMed Research International
Table 2 Genotypes of the 20 isolates with discrepancies in theirINH DST as determined by the Middlebrook 7H9 broth microdi-lution and L-J agar dilution methods
Note order of 12 MIRU loci is 2 4 10 16 20 23 24 26 27 31 39 and 40
4 Discussion
Different DST methods have been developed and are used inroutine clinical practice such as the conventional L-Jmethodsand the automated MBBacT (Organon Teknika TurnhoutBelgium) ESPII (Difco Laboratories Detroit Michigan)BACTEC 9000MB (Becton DickensonMicrobiology System
Table 3 Spoligotyping patterns of the 96 randomly selected Mtuberculosis isolates
Number ofisolates Shared types Spoligotyping pattern
Sparks MD) and BACTECMGIT 960 (BBL Becton Dickin-sonMicrobiology Systems CockeysvilleMD) systems [5 21ndash23]TheDST results would be influenced bymany steps of theprotocol including the culture and the DST methods In thisstudy we analyzed the discrepancy of the drug susceptibilitytest by the MGIT and L-J methods for the isolates collectedfrom the culture by MGIT and L-J respectively
Except for the median time to report the DST resultstheM tuberculosis complex culture positivity rates were alsogreatly different in MGIT and L-J [24] which indicated thepossible culture preference to somewhat And the detectiontime accuracy and performance capacity are also variableby different DST methods Studies reported that the reasonsfor the different performance capacity among these methodsmainly resulted from the different DST systems [23 25]The most obvious difference is the drug concentrations usedfor the DST In MGIT system the sensitive strains weresusceptible to the INH less than the 01 120583gmL while theconcentration of the INH was 1120583gmL in L-J system in thisstudy [12 26 27] Of all the 20 paired cases 15 cases hadMIC in borderlines between theMGIT and theDSTmethodswhich was a usual reason for the discordant
Many reports showed that there was a good concordancebetween DST on L-J and MGIT for INH in DST [25ndash27] Inthis study we still found that 20 paired isolates with the samegenotypes individually showed the discrepancy in the drugsusceptibilities to INH according to the MGIT960 testingand L-J proportionmethods Lawson et al demonstrated thatthere was a substantial degree of agreement between the twomethods with similar INH and rifampicin DST patterns butmore frequent detection of streptomycin resistance and lessfrequent detection of ethambutol with L-J than MGIT-960However the differenceswere not statistically significant [25]Amultiple center evaluation showed that the discrepancies inINH susceptibility between theMGIT960 and L-J proportionmethods varied from 0 to 1 [9]
Mixed infection with the different genotypes of Mtuberculosis in the same patient also affected the DST resultseven by the same testing systems [28 29] In this studyheterogeneous genotypeswere found in the isolates fromeachof the 4 patientsThree patients were infected by the different
BioMed Research International 5
Table 4 MIC of INH and the katG inhA and oxyR-ahpCmutations of the 20 pairs ofM tuberculosis isolates with DST discrepancies
Note katG315 is the predominant mutation The wild type is AGClowast16 isolates with consistent genotype in pair and 4 pairs of isolates (bold) with different genotypes in pair
stains with Spoligotype International Type SIT1634 (Manu2)andBeijing genotypes and 1 patientwas infected by the strainswith two different Beijing genotypes And also our test on themutations of the putative INH-target genes katG inhA andahpC further confirmed one patient (number 18) with mixedinfection by the heterogeneous genotypes (Table 4)
Some mycobacterial characteristics might be associatedwith particular genotypes A well-known but controversial
example is that the Beijing family strains of M tuberculosisare often associated with relapse [30] drug resistance [31]and an increased ability to cause disease to be transmittedwithin certain geographic settings [32 33] The isolateswith particular genotypes such as Spoligotype InternationalType SIT1634 (Manu2) in this study showed higher rate ofresistance in MGIT960 system than in L-J system In thisstudy we found that the percentage of ldquoMANUrdquo genotype
6 BioMed Research International
strains was markedly increased in paired isolates whoseDST results showed discrepancies (375) compared to therandomly selected clinical isolates (3125) An unusuallyhigh proportion of strains belonging to the ldquoManurdquo clade(2715) were also reported by Helal et al [18] InterestinglyManu2 strains (SIT1634) have rarely been reported in Tianjinor even in China as a whole [34 35] or in the SPOLDB4database (excluding this study 119899 = 3 1 from India and 2 fromthe USA)
In this study all the 40 isolates were determined asresistant by MGIT and sensitive by L-J of which twenty-seven isolates were found with mutations in katG315 and 13isolates were found with no mutations in katG315 (Table 4)Those results of the mutations found in the INH-targetedgenes supported that the DST result by the MGIT was moreaccurate than that by the L-J andwe also found that theMICsof some isolates by L-J agar method were very higher thanthose in the first execution in clinic which indicated to someextent that the operation needs to be improved in proportionmethod on L-J agar
5 Conclusion
Our study confirmed that the discrepancies of the DSTin M tuberculosis clinical isolates did exist for INH Oneof the reasons for the discrepancy is the different testsystems between the BACTEC MGIT960 system and thetraditional L-J proportion method Mixed infection by thestrains with MANU2 and Beijing genotype patterns couldalso contributed to drug discrepancies
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Zaoxian Mei and Zhaogang Sun contributed equally to thiswork
Acknowledgments
The authors thank the Beijing Bio-Bank of Clinical Resourceson Tuberculosis and the Outpatient Department of Tian-jin Haihe Hospital for supplying M tuberculosis H37Rvand clinical isolates They also thank the National Spe-cial Key Project of China on Major Infectious Diseases(2013ZX10003009) Beijing Municipal Administration ofHospitals ClinicalMedicine Development of Special FundingSupport (ZYLX201304) and the Beijing High-level TechnicalPersonnel Training Project in Health (2011-3-069) for finan-cial support
References
[1] J G Pasipanodya S Srivastava and T Gumbo ldquoMeta-analysisof clinical studies supports the pharmacokinetic variability
hypothesis for acquired drug resistance and failure of antituber-culosis therapyrdquo Clinical Infectious Diseases vol 55 no 2 pp169ndash177 2012
[2] P T Kent and G P Kubica Public Health Mycobacteriology AGuide for Level III Laboratory Centers for Diseases ControlAtlanta Ga USA 1985
[3] S B Walters and B A Hanna ldquoTesting of susceptibilityof Mycobacterium tuberculosis to isoniazid and rifampin bymycobacterium growth indicator tube methodrdquo Journal ofClinical Microbiology vol 34 no 6 pp 1565ndash1567 1996
[4] AWanger and KMills ldquoTesting ofMycobacterium tuberculosissusceptibility to ethambutol isoniazid rifampin and strepto-mycin by using Etestrdquo Journal of Clinical Microbiology vol 34no 7 pp 1672ndash1676 1996
[5] L A Collins and S G Franzblau ldquoMicroplate Alamar blue assayversus BACTEC 460 system for high-throughput screening ofcompounds against Mycobacterium tuberculosis and Mycobac-terium aviumrdquo Antimicrobial Agents and Chemotherapy vol 41no 5 pp 1004ndash1009 1997
[6] C Piersimoni ldquoTB control strategies present and future indiagnostic methods what the clinician can ask to the labrdquoMonaldi Archives for Chest Disease vol 57 no 5-6 pp 306ndash3102002
[7] G L Woods G Fish M Plaunt and T Murphy ldquoClinicalevaluation of Difco ESP culture system II for growth anddetection ofmycobacteriardquo Journal of ClinicalMicrobiology vol35 no 1 pp 121ndash124 1997
[8] R A Adegbola P Hill I Baldeh et al ldquoSurveillance of drug-resistant Mycobacterium tuberculosis in The Gambiardquo Interna-tional Journal of Tuberculosis and Lung Disease vol 7 no 4 pp390ndash393 2003
[9] C M S Giampaglia M C Martins G B De Oliveira Vieiraet al ldquoMulticentre evaluation of an automated BACTEC 960system for susceptibility testing ofMycobacterium tuberculosisrdquoInternational Journal of Tuberculosis and Lung Disease vol 11no 9 pp 986ndash991 2007
[10] L Heifets and G A Cangelosi ldquoDrug susceptibility testing ofMycobacterium tuberculosis a neglected problem at the turnof the centuryrdquo International Journal of Tuberculosis and LungDisease vol 3 no 7 pp 564ndash581 1999
[11] C Piersimoni A Olivieri L Benacchio and C ScarparoldquoCurrent perspectives on drug susceptibility testing ofMycobac-terium tuberculosis complex the automated nonradiometricsystemsrdquo Journal of Clinical Microbiology vol 44 no 1 pp 20ndash28 2006
[12] Chinese Anti-Tuberculosis Association Protocols for Tubercu-losis Diagnosis in Laboratory Chinese Educational and CulturalPublisher Beijing China 1st edition 2006
[13] J-C Palomino A Martin M Camacho H Guerra JSwings and F Portaels ldquoResazurin microtiter assay platesimple and inexpensive method for detection of drug resis-tance inMycobacterium tuberculosisrdquo Antimicrobial Agents andChemotherapy vol 46 no 8 pp 2720ndash2722 2002
[14] N Siddiqi M Shamim S Hussain et al ldquoMolecular char-acterization of multidrug-resistant isolates of Mycobacteriumtuberculosis from patients in North Indiardquo Antimicrobial Agentsand Chemotherapy vol 46 no 2 pp 443ndash450 2002
[15] M Y Lipin V N Stepanshina I G Shemyakin and T MShinnick ldquoAssociation of specific mutations in katG rpoB rpsLand rrs geneswith spoligotypes ofmultidrug-resistantMycobac-terium tuberculosis isolates in RussiardquoClinical Microbiology andInfection vol 13 no 6 pp 620ndash626 2007
BioMed Research International 7
[16] Z Sun J Zhang X Zhang S Wang Y Zhang and C LildquoComparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strainsand clinical isolatesrdquo International Journal of AntimicrobialAgents vol 31 no 2 pp 115ndash121 2008
[17] J Kamerbeek L Schouls AKolk et al ldquoSimultaneous detectionand strain differentiation of Mycobacterium tuberculosis fordiagnosis and epidemiologyrdquo Journal of Clinical Microbiologyvol 35 no 4 pp 907ndash914 1997
[18] Z H Helal M S E-D Ashour S A Eissa et al ldquoUnexpectedlyhigh proportion of ancestral manu genotype Mycobacteriumtuberculosis strains cultured from tuberculosis patients inEgyptrdquo Journal of Clinical Microbiology vol 47 no 9 pp 2794ndash2801 2009
[19] P Supply E Mazars S Lesjean V Vincent B Gicquel andC Locht ldquoVariable human minisatellite-like regions in theMycobacterium tuberculosis genomerdquo Molecular Microbiologyvol 36 no 3 pp 762ndash771 2000
[20] M B Boniotti M Goria D Loda et al ldquoMolecular typingof Mycobacterium bovis strains isolated in Italy from 2000to 2006 and evaluation of variable-number tandem repeatsfor geographically optimized genotypingrdquo Journal of ClinicalMicrobiology vol 47 no 3 pp 636ndash644 2009
[21] W H Benjamin Jr K B Waites A Beverly et al ldquoComparisonof the MBBacT system with a revised antibiotic supplementkit to the BACTEC 460 system for detection of mycobacteriain clinical specimensrdquo Journal of Clinical Microbiology vol 36no 11 pp 3234ndash3238 1998
[22] World Health Organisation The Use of Liquid Medium forCulture and DST in Middle- and Low-Income Countries 2007httpwwwwhointtbdotslaboratorypolicyenindex3htm
[23] W W Yew S C W Tong K S Lui S K F Leung CH Chau and E P Wang ldquoComparison of MBBacT systemand agar proportion method in drug susceptibility testingof Mycobacterium tuberculosisrdquo Diagnostic Microbiology andInfectious Disease vol 39 no 4 pp 229ndash232 2001
[24] Y Balabanova F Drobniewski V Nikolayevskyy et al ldquoAnintegrated approach to rapid diagnosis of tuberculosis andmul-tidrug resistance using liquid culture andmolecular methods inRussiardquo PLoS ONE vol 4 no 9 Article ID e7129 2009
[25] L Lawson N Emenyonu S T Abdurrahman et al ldquoCom-parison ofMycobacterium tuberculosis drug susceptibility usingsolid and liquid culture in Nigeriardquo BMC Research Notes vol 6no 1 article 215 2013
[26] S J Kim ldquoDrug-susceptibility testing in tuberculosis methodsand reliability of resultsrdquo European Respiratory Journal vol 25no 3 pp 564ndash569 2005
[27] World Health Organization ldquoPolicy guidance on drug-sus-ceptibility testing (DST) of second-line antituberculosisdrugsrdquo 2008 httpwwwwhointtbfeatures archivexdr mdrpolicy guidance
[28] I C Shamputa L Jugheli N Sadradze et al ldquoMixed infectionand clonal representativeness of a single sputum sample intuberculosis patients from a penitentiary hospital in GeorgiardquoRespiratory Research vol 7 article 99 2006
[29] I C Shamputa L Rigouts L A Eyongeta et al ldquoGenotypic andphenotypic heterogeneity among Mycobacterium tuberculosisisolates from pulmonary tuberculosis patientsrdquo Journal of Clin-ical Microbiology vol 42 no 12 pp 5528ndash5536 2004
[30] M N T Huyen T N Buu E Tiemersma et al ldquoTuberculosisrelapse in vietnam is significantly associated with Mycobac-terium tuberculosis Beijing genotype infectionsrdquo The Journal ofInfectious Diseases vol 207 no 10 pp 1516ndash1524 2013
[31] EuropeanConcertedAction onNewGenerationGeneticMark-ers and Techniques for the Epidemiology and Control ofTuberculosis ldquoBeijingW genotypeMycobacterium tuberculosisand drug resistancerdquo Emerging Infectious Diseases vol 12 no 5pp 736ndash743 2006
[32] B C De Jong P C Hill A Aiken et al ldquoProgression to activetuberculosis but not transmission varies by Mycobacteriumtuberculosis lineage in the Gambiardquo Journal of Infectious Dis-eases vol 198 no 7 pp 1037ndash1043 2008
[33] M Hanekom G D van der Spuy E Streicher et al ldquoA recentlyevolved sublineage of the Mycobacterium tuberculosis Beijingstrain family is associated with an increased ability to spreadand cause diseaserdquo Journal of Clinical Microbiology vol 45 no5 pp 1483ndash1490 2007
[34] Y Kong M D Cave L Zhang et al ldquoAssociation betweenMycobacterium tuberculosis BeijingW lineage strain infectionand extrathoracic tuberculosis Insights from epidemiologicand clinical characterization of the three principal geneticgroups of M tuberculosis clinical isolatesrdquo Journal of ClinicalMicrobiology vol 45 no 2 pp 409ndash414 2007
[35] Y Pang Y Zhou B Zhao et al ldquoSpoligotyping and drugresistance analysis of Mycobacterium Tuberculosis strains fromnational survey in Chinardquo PLoS ONE vol 7 no 3 Article IDe32976 2012
previously released SpolDB4 database (httpwwwpasteur-guadeloupefr8081SITVITDemotsSpoligojsp) as previ-ously described [18]
The numbers of tandem repeats (TRs) at each locus inthe isolates were determined on the basis of the number ofwhole repeats in a PCR product of the size estimated from thegel [19] Polymerase chain reaction assays for the 12 chosenloci were repeated and compared within and between gelsto ensure consistent estimation of size and TR copy number[20]
3 Results
31 Genotyping Analysis Genotyping analysis can determinenot only whether an infection results from transmission ofthe given tuberculosis isolate but also whether the infectioninvolves more than one strain of M tuberculosis Resultsfrom our genotyping analysis showed that 10 paired isolatesbelong to the Spoligotype International Type SIT1 (Beijinggenotype 000000000003771) and 6 paired isolates belongto the Spoligotype International Type SIT1634 (MANU2777777777723771) (Table 2) a spoligotype that was notfound in the 96 randomly selected clinical isolates (Table 3)Three paired isolates were mixtures of the SIT1 and SIT1634spoligotypes and one pair was a mixture of SIT1 and theSIT269 (Beijing genotype 000000000000771) spoligotypesCompared with our set of 96 randomly selected isolates fromTianjin only the Beijing and MANU genotypes were presentand the percentage of the MANU genotype was extremelyhigh (20 paired isolates 1540 375 96 random clinicalisolates 396 3125)
Results obtained by using the 12-locusMIRUmethod [19]showed that 20 pairs of isolates had 14 MIRU patterns Boththe spoligotyping and the MIRU patterns were different in
the isolates named as 6 12 and 18 pairs individually Theisolates named as 7 pairs had different spoligotypes but thesame MIRU type (Table 2)
32 MICs of the Tested Strains To identify the differencesbetween the liquid Middlebrook 7H9 and L-J proportionmethods in DST we tested the MICs of each of the 16 pairedINH-resistant isolates and 4 pairs of isolates which consistedof different genotypes using both Middlebrook 7H9 brothmicrodilution and L-J proportion methods The MICs of allthe 24 tested isolates were determined to be greater than01 120583gmL (01 to 06 120583gmL) using the Middlebrook 7H9broth microdilution method and greater than 03120583gmL (04to 18 120583gmL) using the L-J proportionmethod (Table 4)TheMICs of 5 pairs of the tested isolates using the L-J proportionmethod were higher than 1 120583gmL the cutoff concentrationfor determining drug susceptibility in the L-J agar proportionmethod in this study (Table 4)
33 Sequence Analysis of the Putative INH-Target GenesMutations in the katG gene were identified in 13 pairedisolates of which each of 12 paired isolates carried the samemutations and one pair which showed a DST discrepancyby MGIT960 and L-J proportion methods carried differentbase pair at codon 315 (AGC versus AAC) The AGC315AACmutation was found in 4 paired isolates while 9 pairedisolates carried themutation AGC315ACCTheAGC315AACand AGC 315ACC mutations were not associated with speci-ficity to the Beijing or MANU2 genotypes among the testedisolates Seven paired isolates did not contain mutations inthe katG gene and nomutations were found in the regulatorysequences and open reading frames (ORF) of the inhA andahpC genes in any of the tested isolates (Table 4)
4 BioMed Research International
Table 2 Genotypes of the 20 isolates with discrepancies in theirINH DST as determined by the Middlebrook 7H9 broth microdi-lution and L-J agar dilution methods
Note order of 12 MIRU loci is 2 4 10 16 20 23 24 26 27 31 39 and 40
4 Discussion
Different DST methods have been developed and are used inroutine clinical practice such as the conventional L-Jmethodsand the automated MBBacT (Organon Teknika TurnhoutBelgium) ESPII (Difco Laboratories Detroit Michigan)BACTEC 9000MB (Becton DickensonMicrobiology System
Table 3 Spoligotyping patterns of the 96 randomly selected Mtuberculosis isolates
Number ofisolates Shared types Spoligotyping pattern
Sparks MD) and BACTECMGIT 960 (BBL Becton Dickin-sonMicrobiology Systems CockeysvilleMD) systems [5 21ndash23]TheDST results would be influenced bymany steps of theprotocol including the culture and the DST methods In thisstudy we analyzed the discrepancy of the drug susceptibilitytest by the MGIT and L-J methods for the isolates collectedfrom the culture by MGIT and L-J respectively
Except for the median time to report the DST resultstheM tuberculosis complex culture positivity rates were alsogreatly different in MGIT and L-J [24] which indicated thepossible culture preference to somewhat And the detectiontime accuracy and performance capacity are also variableby different DST methods Studies reported that the reasonsfor the different performance capacity among these methodsmainly resulted from the different DST systems [23 25]The most obvious difference is the drug concentrations usedfor the DST In MGIT system the sensitive strains weresusceptible to the INH less than the 01 120583gmL while theconcentration of the INH was 1120583gmL in L-J system in thisstudy [12 26 27] Of all the 20 paired cases 15 cases hadMIC in borderlines between theMGIT and theDSTmethodswhich was a usual reason for the discordant
Many reports showed that there was a good concordancebetween DST on L-J and MGIT for INH in DST [25ndash27] Inthis study we still found that 20 paired isolates with the samegenotypes individually showed the discrepancy in the drugsusceptibilities to INH according to the MGIT960 testingand L-J proportionmethods Lawson et al demonstrated thatthere was a substantial degree of agreement between the twomethods with similar INH and rifampicin DST patterns butmore frequent detection of streptomycin resistance and lessfrequent detection of ethambutol with L-J than MGIT-960However the differenceswere not statistically significant [25]Amultiple center evaluation showed that the discrepancies inINH susceptibility between theMGIT960 and L-J proportionmethods varied from 0 to 1 [9]
Mixed infection with the different genotypes of Mtuberculosis in the same patient also affected the DST resultseven by the same testing systems [28 29] In this studyheterogeneous genotypeswere found in the isolates fromeachof the 4 patientsThree patients were infected by the different
BioMed Research International 5
Table 4 MIC of INH and the katG inhA and oxyR-ahpCmutations of the 20 pairs ofM tuberculosis isolates with DST discrepancies
Note katG315 is the predominant mutation The wild type is AGClowast16 isolates with consistent genotype in pair and 4 pairs of isolates (bold) with different genotypes in pair
stains with Spoligotype International Type SIT1634 (Manu2)andBeijing genotypes and 1 patientwas infected by the strainswith two different Beijing genotypes And also our test on themutations of the putative INH-target genes katG inhA andahpC further confirmed one patient (number 18) with mixedinfection by the heterogeneous genotypes (Table 4)
Some mycobacterial characteristics might be associatedwith particular genotypes A well-known but controversial
example is that the Beijing family strains of M tuberculosisare often associated with relapse [30] drug resistance [31]and an increased ability to cause disease to be transmittedwithin certain geographic settings [32 33] The isolateswith particular genotypes such as Spoligotype InternationalType SIT1634 (Manu2) in this study showed higher rate ofresistance in MGIT960 system than in L-J system In thisstudy we found that the percentage of ldquoMANUrdquo genotype
6 BioMed Research International
strains was markedly increased in paired isolates whoseDST results showed discrepancies (375) compared to therandomly selected clinical isolates (3125) An unusuallyhigh proportion of strains belonging to the ldquoManurdquo clade(2715) were also reported by Helal et al [18] InterestinglyManu2 strains (SIT1634) have rarely been reported in Tianjinor even in China as a whole [34 35] or in the SPOLDB4database (excluding this study 119899 = 3 1 from India and 2 fromthe USA)
In this study all the 40 isolates were determined asresistant by MGIT and sensitive by L-J of which twenty-seven isolates were found with mutations in katG315 and 13isolates were found with no mutations in katG315 (Table 4)Those results of the mutations found in the INH-targetedgenes supported that the DST result by the MGIT was moreaccurate than that by the L-J andwe also found that theMICsof some isolates by L-J agar method were very higher thanthose in the first execution in clinic which indicated to someextent that the operation needs to be improved in proportionmethod on L-J agar
5 Conclusion
Our study confirmed that the discrepancies of the DSTin M tuberculosis clinical isolates did exist for INH Oneof the reasons for the discrepancy is the different testsystems between the BACTEC MGIT960 system and thetraditional L-J proportion method Mixed infection by thestrains with MANU2 and Beijing genotype patterns couldalso contributed to drug discrepancies
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Zaoxian Mei and Zhaogang Sun contributed equally to thiswork
Acknowledgments
The authors thank the Beijing Bio-Bank of Clinical Resourceson Tuberculosis and the Outpatient Department of Tian-jin Haihe Hospital for supplying M tuberculosis H37Rvand clinical isolates They also thank the National Spe-cial Key Project of China on Major Infectious Diseases(2013ZX10003009) Beijing Municipal Administration ofHospitals ClinicalMedicine Development of Special FundingSupport (ZYLX201304) and the Beijing High-level TechnicalPersonnel Training Project in Health (2011-3-069) for finan-cial support
References
[1] J G Pasipanodya S Srivastava and T Gumbo ldquoMeta-analysisof clinical studies supports the pharmacokinetic variability
hypothesis for acquired drug resistance and failure of antituber-culosis therapyrdquo Clinical Infectious Diseases vol 55 no 2 pp169ndash177 2012
[2] P T Kent and G P Kubica Public Health Mycobacteriology AGuide for Level III Laboratory Centers for Diseases ControlAtlanta Ga USA 1985
[3] S B Walters and B A Hanna ldquoTesting of susceptibilityof Mycobacterium tuberculosis to isoniazid and rifampin bymycobacterium growth indicator tube methodrdquo Journal ofClinical Microbiology vol 34 no 6 pp 1565ndash1567 1996
[4] AWanger and KMills ldquoTesting ofMycobacterium tuberculosissusceptibility to ethambutol isoniazid rifampin and strepto-mycin by using Etestrdquo Journal of Clinical Microbiology vol 34no 7 pp 1672ndash1676 1996
[5] L A Collins and S G Franzblau ldquoMicroplate Alamar blue assayversus BACTEC 460 system for high-throughput screening ofcompounds against Mycobacterium tuberculosis and Mycobac-terium aviumrdquo Antimicrobial Agents and Chemotherapy vol 41no 5 pp 1004ndash1009 1997
[6] C Piersimoni ldquoTB control strategies present and future indiagnostic methods what the clinician can ask to the labrdquoMonaldi Archives for Chest Disease vol 57 no 5-6 pp 306ndash3102002
[7] G L Woods G Fish M Plaunt and T Murphy ldquoClinicalevaluation of Difco ESP culture system II for growth anddetection ofmycobacteriardquo Journal of ClinicalMicrobiology vol35 no 1 pp 121ndash124 1997
[8] R A Adegbola P Hill I Baldeh et al ldquoSurveillance of drug-resistant Mycobacterium tuberculosis in The Gambiardquo Interna-tional Journal of Tuberculosis and Lung Disease vol 7 no 4 pp390ndash393 2003
[9] C M S Giampaglia M C Martins G B De Oliveira Vieiraet al ldquoMulticentre evaluation of an automated BACTEC 960system for susceptibility testing ofMycobacterium tuberculosisrdquoInternational Journal of Tuberculosis and Lung Disease vol 11no 9 pp 986ndash991 2007
[10] L Heifets and G A Cangelosi ldquoDrug susceptibility testing ofMycobacterium tuberculosis a neglected problem at the turnof the centuryrdquo International Journal of Tuberculosis and LungDisease vol 3 no 7 pp 564ndash581 1999
[11] C Piersimoni A Olivieri L Benacchio and C ScarparoldquoCurrent perspectives on drug susceptibility testing ofMycobac-terium tuberculosis complex the automated nonradiometricsystemsrdquo Journal of Clinical Microbiology vol 44 no 1 pp 20ndash28 2006
[12] Chinese Anti-Tuberculosis Association Protocols for Tubercu-losis Diagnosis in Laboratory Chinese Educational and CulturalPublisher Beijing China 1st edition 2006
[13] J-C Palomino A Martin M Camacho H Guerra JSwings and F Portaels ldquoResazurin microtiter assay platesimple and inexpensive method for detection of drug resis-tance inMycobacterium tuberculosisrdquo Antimicrobial Agents andChemotherapy vol 46 no 8 pp 2720ndash2722 2002
[14] N Siddiqi M Shamim S Hussain et al ldquoMolecular char-acterization of multidrug-resistant isolates of Mycobacteriumtuberculosis from patients in North Indiardquo Antimicrobial Agentsand Chemotherapy vol 46 no 2 pp 443ndash450 2002
[15] M Y Lipin V N Stepanshina I G Shemyakin and T MShinnick ldquoAssociation of specific mutations in katG rpoB rpsLand rrs geneswith spoligotypes ofmultidrug-resistantMycobac-terium tuberculosis isolates in RussiardquoClinical Microbiology andInfection vol 13 no 6 pp 620ndash626 2007
BioMed Research International 7
[16] Z Sun J Zhang X Zhang S Wang Y Zhang and C LildquoComparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strainsand clinical isolatesrdquo International Journal of AntimicrobialAgents vol 31 no 2 pp 115ndash121 2008
[17] J Kamerbeek L Schouls AKolk et al ldquoSimultaneous detectionand strain differentiation of Mycobacterium tuberculosis fordiagnosis and epidemiologyrdquo Journal of Clinical Microbiologyvol 35 no 4 pp 907ndash914 1997
[18] Z H Helal M S E-D Ashour S A Eissa et al ldquoUnexpectedlyhigh proportion of ancestral manu genotype Mycobacteriumtuberculosis strains cultured from tuberculosis patients inEgyptrdquo Journal of Clinical Microbiology vol 47 no 9 pp 2794ndash2801 2009
[19] P Supply E Mazars S Lesjean V Vincent B Gicquel andC Locht ldquoVariable human minisatellite-like regions in theMycobacterium tuberculosis genomerdquo Molecular Microbiologyvol 36 no 3 pp 762ndash771 2000
[20] M B Boniotti M Goria D Loda et al ldquoMolecular typingof Mycobacterium bovis strains isolated in Italy from 2000to 2006 and evaluation of variable-number tandem repeatsfor geographically optimized genotypingrdquo Journal of ClinicalMicrobiology vol 47 no 3 pp 636ndash644 2009
[21] W H Benjamin Jr K B Waites A Beverly et al ldquoComparisonof the MBBacT system with a revised antibiotic supplementkit to the BACTEC 460 system for detection of mycobacteriain clinical specimensrdquo Journal of Clinical Microbiology vol 36no 11 pp 3234ndash3238 1998
[22] World Health Organisation The Use of Liquid Medium forCulture and DST in Middle- and Low-Income Countries 2007httpwwwwhointtbdotslaboratorypolicyenindex3htm
[23] W W Yew S C W Tong K S Lui S K F Leung CH Chau and E P Wang ldquoComparison of MBBacT systemand agar proportion method in drug susceptibility testingof Mycobacterium tuberculosisrdquo Diagnostic Microbiology andInfectious Disease vol 39 no 4 pp 229ndash232 2001
[24] Y Balabanova F Drobniewski V Nikolayevskyy et al ldquoAnintegrated approach to rapid diagnosis of tuberculosis andmul-tidrug resistance using liquid culture andmolecular methods inRussiardquo PLoS ONE vol 4 no 9 Article ID e7129 2009
[25] L Lawson N Emenyonu S T Abdurrahman et al ldquoCom-parison ofMycobacterium tuberculosis drug susceptibility usingsolid and liquid culture in Nigeriardquo BMC Research Notes vol 6no 1 article 215 2013
[26] S J Kim ldquoDrug-susceptibility testing in tuberculosis methodsand reliability of resultsrdquo European Respiratory Journal vol 25no 3 pp 564ndash569 2005
[27] World Health Organization ldquoPolicy guidance on drug-sus-ceptibility testing (DST) of second-line antituberculosisdrugsrdquo 2008 httpwwwwhointtbfeatures archivexdr mdrpolicy guidance
[28] I C Shamputa L Jugheli N Sadradze et al ldquoMixed infectionand clonal representativeness of a single sputum sample intuberculosis patients from a penitentiary hospital in GeorgiardquoRespiratory Research vol 7 article 99 2006
[29] I C Shamputa L Rigouts L A Eyongeta et al ldquoGenotypic andphenotypic heterogeneity among Mycobacterium tuberculosisisolates from pulmonary tuberculosis patientsrdquo Journal of Clin-ical Microbiology vol 42 no 12 pp 5528ndash5536 2004
[30] M N T Huyen T N Buu E Tiemersma et al ldquoTuberculosisrelapse in vietnam is significantly associated with Mycobac-terium tuberculosis Beijing genotype infectionsrdquo The Journal ofInfectious Diseases vol 207 no 10 pp 1516ndash1524 2013
[31] EuropeanConcertedAction onNewGenerationGeneticMark-ers and Techniques for the Epidemiology and Control ofTuberculosis ldquoBeijingW genotypeMycobacterium tuberculosisand drug resistancerdquo Emerging Infectious Diseases vol 12 no 5pp 736ndash743 2006
[32] B C De Jong P C Hill A Aiken et al ldquoProgression to activetuberculosis but not transmission varies by Mycobacteriumtuberculosis lineage in the Gambiardquo Journal of Infectious Dis-eases vol 198 no 7 pp 1037ndash1043 2008
[33] M Hanekom G D van der Spuy E Streicher et al ldquoA recentlyevolved sublineage of the Mycobacterium tuberculosis Beijingstrain family is associated with an increased ability to spreadand cause diseaserdquo Journal of Clinical Microbiology vol 45 no5 pp 1483ndash1490 2007
[34] Y Kong M D Cave L Zhang et al ldquoAssociation betweenMycobacterium tuberculosis BeijingW lineage strain infectionand extrathoracic tuberculosis Insights from epidemiologicand clinical characterization of the three principal geneticgroups of M tuberculosis clinical isolatesrdquo Journal of ClinicalMicrobiology vol 45 no 2 pp 409ndash414 2007
[35] Y Pang Y Zhou B Zhao et al ldquoSpoligotyping and drugresistance analysis of Mycobacterium Tuberculosis strains fromnational survey in Chinardquo PLoS ONE vol 7 no 3 Article IDe32976 2012
Table 2 Genotypes of the 20 isolates with discrepancies in theirINH DST as determined by the Middlebrook 7H9 broth microdi-lution and L-J agar dilution methods
Note order of 12 MIRU loci is 2 4 10 16 20 23 24 26 27 31 39 and 40
4 Discussion
Different DST methods have been developed and are used inroutine clinical practice such as the conventional L-Jmethodsand the automated MBBacT (Organon Teknika TurnhoutBelgium) ESPII (Difco Laboratories Detroit Michigan)BACTEC 9000MB (Becton DickensonMicrobiology System
Table 3 Spoligotyping patterns of the 96 randomly selected Mtuberculosis isolates
Number ofisolates Shared types Spoligotyping pattern
Sparks MD) and BACTECMGIT 960 (BBL Becton Dickin-sonMicrobiology Systems CockeysvilleMD) systems [5 21ndash23]TheDST results would be influenced bymany steps of theprotocol including the culture and the DST methods In thisstudy we analyzed the discrepancy of the drug susceptibilitytest by the MGIT and L-J methods for the isolates collectedfrom the culture by MGIT and L-J respectively
Except for the median time to report the DST resultstheM tuberculosis complex culture positivity rates were alsogreatly different in MGIT and L-J [24] which indicated thepossible culture preference to somewhat And the detectiontime accuracy and performance capacity are also variableby different DST methods Studies reported that the reasonsfor the different performance capacity among these methodsmainly resulted from the different DST systems [23 25]The most obvious difference is the drug concentrations usedfor the DST In MGIT system the sensitive strains weresusceptible to the INH less than the 01 120583gmL while theconcentration of the INH was 1120583gmL in L-J system in thisstudy [12 26 27] Of all the 20 paired cases 15 cases hadMIC in borderlines between theMGIT and theDSTmethodswhich was a usual reason for the discordant
Many reports showed that there was a good concordancebetween DST on L-J and MGIT for INH in DST [25ndash27] Inthis study we still found that 20 paired isolates with the samegenotypes individually showed the discrepancy in the drugsusceptibilities to INH according to the MGIT960 testingand L-J proportionmethods Lawson et al demonstrated thatthere was a substantial degree of agreement between the twomethods with similar INH and rifampicin DST patterns butmore frequent detection of streptomycin resistance and lessfrequent detection of ethambutol with L-J than MGIT-960However the differenceswere not statistically significant [25]Amultiple center evaluation showed that the discrepancies inINH susceptibility between theMGIT960 and L-J proportionmethods varied from 0 to 1 [9]
Mixed infection with the different genotypes of Mtuberculosis in the same patient also affected the DST resultseven by the same testing systems [28 29] In this studyheterogeneous genotypeswere found in the isolates fromeachof the 4 patientsThree patients were infected by the different
BioMed Research International 5
Table 4 MIC of INH and the katG inhA and oxyR-ahpCmutations of the 20 pairs ofM tuberculosis isolates with DST discrepancies
Note katG315 is the predominant mutation The wild type is AGClowast16 isolates with consistent genotype in pair and 4 pairs of isolates (bold) with different genotypes in pair
stains with Spoligotype International Type SIT1634 (Manu2)andBeijing genotypes and 1 patientwas infected by the strainswith two different Beijing genotypes And also our test on themutations of the putative INH-target genes katG inhA andahpC further confirmed one patient (number 18) with mixedinfection by the heterogeneous genotypes (Table 4)
Some mycobacterial characteristics might be associatedwith particular genotypes A well-known but controversial
example is that the Beijing family strains of M tuberculosisare often associated with relapse [30] drug resistance [31]and an increased ability to cause disease to be transmittedwithin certain geographic settings [32 33] The isolateswith particular genotypes such as Spoligotype InternationalType SIT1634 (Manu2) in this study showed higher rate ofresistance in MGIT960 system than in L-J system In thisstudy we found that the percentage of ldquoMANUrdquo genotype
6 BioMed Research International
strains was markedly increased in paired isolates whoseDST results showed discrepancies (375) compared to therandomly selected clinical isolates (3125) An unusuallyhigh proportion of strains belonging to the ldquoManurdquo clade(2715) were also reported by Helal et al [18] InterestinglyManu2 strains (SIT1634) have rarely been reported in Tianjinor even in China as a whole [34 35] or in the SPOLDB4database (excluding this study 119899 = 3 1 from India and 2 fromthe USA)
In this study all the 40 isolates were determined asresistant by MGIT and sensitive by L-J of which twenty-seven isolates were found with mutations in katG315 and 13isolates were found with no mutations in katG315 (Table 4)Those results of the mutations found in the INH-targetedgenes supported that the DST result by the MGIT was moreaccurate than that by the L-J andwe also found that theMICsof some isolates by L-J agar method were very higher thanthose in the first execution in clinic which indicated to someextent that the operation needs to be improved in proportionmethod on L-J agar
5 Conclusion
Our study confirmed that the discrepancies of the DSTin M tuberculosis clinical isolates did exist for INH Oneof the reasons for the discrepancy is the different testsystems between the BACTEC MGIT960 system and thetraditional L-J proportion method Mixed infection by thestrains with MANU2 and Beijing genotype patterns couldalso contributed to drug discrepancies
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Zaoxian Mei and Zhaogang Sun contributed equally to thiswork
Acknowledgments
The authors thank the Beijing Bio-Bank of Clinical Resourceson Tuberculosis and the Outpatient Department of Tian-jin Haihe Hospital for supplying M tuberculosis H37Rvand clinical isolates They also thank the National Spe-cial Key Project of China on Major Infectious Diseases(2013ZX10003009) Beijing Municipal Administration ofHospitals ClinicalMedicine Development of Special FundingSupport (ZYLX201304) and the Beijing High-level TechnicalPersonnel Training Project in Health (2011-3-069) for finan-cial support
References
[1] J G Pasipanodya S Srivastava and T Gumbo ldquoMeta-analysisof clinical studies supports the pharmacokinetic variability
hypothesis for acquired drug resistance and failure of antituber-culosis therapyrdquo Clinical Infectious Diseases vol 55 no 2 pp169ndash177 2012
[2] P T Kent and G P Kubica Public Health Mycobacteriology AGuide for Level III Laboratory Centers for Diseases ControlAtlanta Ga USA 1985
[3] S B Walters and B A Hanna ldquoTesting of susceptibilityof Mycobacterium tuberculosis to isoniazid and rifampin bymycobacterium growth indicator tube methodrdquo Journal ofClinical Microbiology vol 34 no 6 pp 1565ndash1567 1996
[4] AWanger and KMills ldquoTesting ofMycobacterium tuberculosissusceptibility to ethambutol isoniazid rifampin and strepto-mycin by using Etestrdquo Journal of Clinical Microbiology vol 34no 7 pp 1672ndash1676 1996
[5] L A Collins and S G Franzblau ldquoMicroplate Alamar blue assayversus BACTEC 460 system for high-throughput screening ofcompounds against Mycobacterium tuberculosis and Mycobac-terium aviumrdquo Antimicrobial Agents and Chemotherapy vol 41no 5 pp 1004ndash1009 1997
[6] C Piersimoni ldquoTB control strategies present and future indiagnostic methods what the clinician can ask to the labrdquoMonaldi Archives for Chest Disease vol 57 no 5-6 pp 306ndash3102002
[7] G L Woods G Fish M Plaunt and T Murphy ldquoClinicalevaluation of Difco ESP culture system II for growth anddetection ofmycobacteriardquo Journal of ClinicalMicrobiology vol35 no 1 pp 121ndash124 1997
[8] R A Adegbola P Hill I Baldeh et al ldquoSurveillance of drug-resistant Mycobacterium tuberculosis in The Gambiardquo Interna-tional Journal of Tuberculosis and Lung Disease vol 7 no 4 pp390ndash393 2003
[9] C M S Giampaglia M C Martins G B De Oliveira Vieiraet al ldquoMulticentre evaluation of an automated BACTEC 960system for susceptibility testing ofMycobacterium tuberculosisrdquoInternational Journal of Tuberculosis and Lung Disease vol 11no 9 pp 986ndash991 2007
[10] L Heifets and G A Cangelosi ldquoDrug susceptibility testing ofMycobacterium tuberculosis a neglected problem at the turnof the centuryrdquo International Journal of Tuberculosis and LungDisease vol 3 no 7 pp 564ndash581 1999
[11] C Piersimoni A Olivieri L Benacchio and C ScarparoldquoCurrent perspectives on drug susceptibility testing ofMycobac-terium tuberculosis complex the automated nonradiometricsystemsrdquo Journal of Clinical Microbiology vol 44 no 1 pp 20ndash28 2006
[12] Chinese Anti-Tuberculosis Association Protocols for Tubercu-losis Diagnosis in Laboratory Chinese Educational and CulturalPublisher Beijing China 1st edition 2006
[13] J-C Palomino A Martin M Camacho H Guerra JSwings and F Portaels ldquoResazurin microtiter assay platesimple and inexpensive method for detection of drug resis-tance inMycobacterium tuberculosisrdquo Antimicrobial Agents andChemotherapy vol 46 no 8 pp 2720ndash2722 2002
[14] N Siddiqi M Shamim S Hussain et al ldquoMolecular char-acterization of multidrug-resistant isolates of Mycobacteriumtuberculosis from patients in North Indiardquo Antimicrobial Agentsand Chemotherapy vol 46 no 2 pp 443ndash450 2002
[15] M Y Lipin V N Stepanshina I G Shemyakin and T MShinnick ldquoAssociation of specific mutations in katG rpoB rpsLand rrs geneswith spoligotypes ofmultidrug-resistantMycobac-terium tuberculosis isolates in RussiardquoClinical Microbiology andInfection vol 13 no 6 pp 620ndash626 2007
BioMed Research International 7
[16] Z Sun J Zhang X Zhang S Wang Y Zhang and C LildquoComparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strainsand clinical isolatesrdquo International Journal of AntimicrobialAgents vol 31 no 2 pp 115ndash121 2008
[17] J Kamerbeek L Schouls AKolk et al ldquoSimultaneous detectionand strain differentiation of Mycobacterium tuberculosis fordiagnosis and epidemiologyrdquo Journal of Clinical Microbiologyvol 35 no 4 pp 907ndash914 1997
[18] Z H Helal M S E-D Ashour S A Eissa et al ldquoUnexpectedlyhigh proportion of ancestral manu genotype Mycobacteriumtuberculosis strains cultured from tuberculosis patients inEgyptrdquo Journal of Clinical Microbiology vol 47 no 9 pp 2794ndash2801 2009
[19] P Supply E Mazars S Lesjean V Vincent B Gicquel andC Locht ldquoVariable human minisatellite-like regions in theMycobacterium tuberculosis genomerdquo Molecular Microbiologyvol 36 no 3 pp 762ndash771 2000
[20] M B Boniotti M Goria D Loda et al ldquoMolecular typingof Mycobacterium bovis strains isolated in Italy from 2000to 2006 and evaluation of variable-number tandem repeatsfor geographically optimized genotypingrdquo Journal of ClinicalMicrobiology vol 47 no 3 pp 636ndash644 2009
[21] W H Benjamin Jr K B Waites A Beverly et al ldquoComparisonof the MBBacT system with a revised antibiotic supplementkit to the BACTEC 460 system for detection of mycobacteriain clinical specimensrdquo Journal of Clinical Microbiology vol 36no 11 pp 3234ndash3238 1998
[22] World Health Organisation The Use of Liquid Medium forCulture and DST in Middle- and Low-Income Countries 2007httpwwwwhointtbdotslaboratorypolicyenindex3htm
[23] W W Yew S C W Tong K S Lui S K F Leung CH Chau and E P Wang ldquoComparison of MBBacT systemand agar proportion method in drug susceptibility testingof Mycobacterium tuberculosisrdquo Diagnostic Microbiology andInfectious Disease vol 39 no 4 pp 229ndash232 2001
[24] Y Balabanova F Drobniewski V Nikolayevskyy et al ldquoAnintegrated approach to rapid diagnosis of tuberculosis andmul-tidrug resistance using liquid culture andmolecular methods inRussiardquo PLoS ONE vol 4 no 9 Article ID e7129 2009
[25] L Lawson N Emenyonu S T Abdurrahman et al ldquoCom-parison ofMycobacterium tuberculosis drug susceptibility usingsolid and liquid culture in Nigeriardquo BMC Research Notes vol 6no 1 article 215 2013
[26] S J Kim ldquoDrug-susceptibility testing in tuberculosis methodsand reliability of resultsrdquo European Respiratory Journal vol 25no 3 pp 564ndash569 2005
[27] World Health Organization ldquoPolicy guidance on drug-sus-ceptibility testing (DST) of second-line antituberculosisdrugsrdquo 2008 httpwwwwhointtbfeatures archivexdr mdrpolicy guidance
[28] I C Shamputa L Jugheli N Sadradze et al ldquoMixed infectionand clonal representativeness of a single sputum sample intuberculosis patients from a penitentiary hospital in GeorgiardquoRespiratory Research vol 7 article 99 2006
[29] I C Shamputa L Rigouts L A Eyongeta et al ldquoGenotypic andphenotypic heterogeneity among Mycobacterium tuberculosisisolates from pulmonary tuberculosis patientsrdquo Journal of Clin-ical Microbiology vol 42 no 12 pp 5528ndash5536 2004
[30] M N T Huyen T N Buu E Tiemersma et al ldquoTuberculosisrelapse in vietnam is significantly associated with Mycobac-terium tuberculosis Beijing genotype infectionsrdquo The Journal ofInfectious Diseases vol 207 no 10 pp 1516ndash1524 2013
[31] EuropeanConcertedAction onNewGenerationGeneticMark-ers and Techniques for the Epidemiology and Control ofTuberculosis ldquoBeijingW genotypeMycobacterium tuberculosisand drug resistancerdquo Emerging Infectious Diseases vol 12 no 5pp 736ndash743 2006
[32] B C De Jong P C Hill A Aiken et al ldquoProgression to activetuberculosis but not transmission varies by Mycobacteriumtuberculosis lineage in the Gambiardquo Journal of Infectious Dis-eases vol 198 no 7 pp 1037ndash1043 2008
[33] M Hanekom G D van der Spuy E Streicher et al ldquoA recentlyevolved sublineage of the Mycobacterium tuberculosis Beijingstrain family is associated with an increased ability to spreadand cause diseaserdquo Journal of Clinical Microbiology vol 45 no5 pp 1483ndash1490 2007
[34] Y Kong M D Cave L Zhang et al ldquoAssociation betweenMycobacterium tuberculosis BeijingW lineage strain infectionand extrathoracic tuberculosis Insights from epidemiologicand clinical characterization of the three principal geneticgroups of M tuberculosis clinical isolatesrdquo Journal of ClinicalMicrobiology vol 45 no 2 pp 409ndash414 2007
[35] Y Pang Y Zhou B Zhao et al ldquoSpoligotyping and drugresistance analysis of Mycobacterium Tuberculosis strains fromnational survey in Chinardquo PLoS ONE vol 7 no 3 Article IDe32976 2012
Note katG315 is the predominant mutation The wild type is AGClowast16 isolates with consistent genotype in pair and 4 pairs of isolates (bold) with different genotypes in pair
stains with Spoligotype International Type SIT1634 (Manu2)andBeijing genotypes and 1 patientwas infected by the strainswith two different Beijing genotypes And also our test on themutations of the putative INH-target genes katG inhA andahpC further confirmed one patient (number 18) with mixedinfection by the heterogeneous genotypes (Table 4)
Some mycobacterial characteristics might be associatedwith particular genotypes A well-known but controversial
example is that the Beijing family strains of M tuberculosisare often associated with relapse [30] drug resistance [31]and an increased ability to cause disease to be transmittedwithin certain geographic settings [32 33] The isolateswith particular genotypes such as Spoligotype InternationalType SIT1634 (Manu2) in this study showed higher rate ofresistance in MGIT960 system than in L-J system In thisstudy we found that the percentage of ldquoMANUrdquo genotype
6 BioMed Research International
strains was markedly increased in paired isolates whoseDST results showed discrepancies (375) compared to therandomly selected clinical isolates (3125) An unusuallyhigh proportion of strains belonging to the ldquoManurdquo clade(2715) were also reported by Helal et al [18] InterestinglyManu2 strains (SIT1634) have rarely been reported in Tianjinor even in China as a whole [34 35] or in the SPOLDB4database (excluding this study 119899 = 3 1 from India and 2 fromthe USA)
In this study all the 40 isolates were determined asresistant by MGIT and sensitive by L-J of which twenty-seven isolates were found with mutations in katG315 and 13isolates were found with no mutations in katG315 (Table 4)Those results of the mutations found in the INH-targetedgenes supported that the DST result by the MGIT was moreaccurate than that by the L-J andwe also found that theMICsof some isolates by L-J agar method were very higher thanthose in the first execution in clinic which indicated to someextent that the operation needs to be improved in proportionmethod on L-J agar
5 Conclusion
Our study confirmed that the discrepancies of the DSTin M tuberculosis clinical isolates did exist for INH Oneof the reasons for the discrepancy is the different testsystems between the BACTEC MGIT960 system and thetraditional L-J proportion method Mixed infection by thestrains with MANU2 and Beijing genotype patterns couldalso contributed to drug discrepancies
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Zaoxian Mei and Zhaogang Sun contributed equally to thiswork
Acknowledgments
The authors thank the Beijing Bio-Bank of Clinical Resourceson Tuberculosis and the Outpatient Department of Tian-jin Haihe Hospital for supplying M tuberculosis H37Rvand clinical isolates They also thank the National Spe-cial Key Project of China on Major Infectious Diseases(2013ZX10003009) Beijing Municipal Administration ofHospitals ClinicalMedicine Development of Special FundingSupport (ZYLX201304) and the Beijing High-level TechnicalPersonnel Training Project in Health (2011-3-069) for finan-cial support
References
[1] J G Pasipanodya S Srivastava and T Gumbo ldquoMeta-analysisof clinical studies supports the pharmacokinetic variability
hypothesis for acquired drug resistance and failure of antituber-culosis therapyrdquo Clinical Infectious Diseases vol 55 no 2 pp169ndash177 2012
[2] P T Kent and G P Kubica Public Health Mycobacteriology AGuide for Level III Laboratory Centers for Diseases ControlAtlanta Ga USA 1985
[3] S B Walters and B A Hanna ldquoTesting of susceptibilityof Mycobacterium tuberculosis to isoniazid and rifampin bymycobacterium growth indicator tube methodrdquo Journal ofClinical Microbiology vol 34 no 6 pp 1565ndash1567 1996
[4] AWanger and KMills ldquoTesting ofMycobacterium tuberculosissusceptibility to ethambutol isoniazid rifampin and strepto-mycin by using Etestrdquo Journal of Clinical Microbiology vol 34no 7 pp 1672ndash1676 1996
[5] L A Collins and S G Franzblau ldquoMicroplate Alamar blue assayversus BACTEC 460 system for high-throughput screening ofcompounds against Mycobacterium tuberculosis and Mycobac-terium aviumrdquo Antimicrobial Agents and Chemotherapy vol 41no 5 pp 1004ndash1009 1997
[6] C Piersimoni ldquoTB control strategies present and future indiagnostic methods what the clinician can ask to the labrdquoMonaldi Archives for Chest Disease vol 57 no 5-6 pp 306ndash3102002
[7] G L Woods G Fish M Plaunt and T Murphy ldquoClinicalevaluation of Difco ESP culture system II for growth anddetection ofmycobacteriardquo Journal of ClinicalMicrobiology vol35 no 1 pp 121ndash124 1997
[8] R A Adegbola P Hill I Baldeh et al ldquoSurveillance of drug-resistant Mycobacterium tuberculosis in The Gambiardquo Interna-tional Journal of Tuberculosis and Lung Disease vol 7 no 4 pp390ndash393 2003
[9] C M S Giampaglia M C Martins G B De Oliveira Vieiraet al ldquoMulticentre evaluation of an automated BACTEC 960system for susceptibility testing ofMycobacterium tuberculosisrdquoInternational Journal of Tuberculosis and Lung Disease vol 11no 9 pp 986ndash991 2007
[10] L Heifets and G A Cangelosi ldquoDrug susceptibility testing ofMycobacterium tuberculosis a neglected problem at the turnof the centuryrdquo International Journal of Tuberculosis and LungDisease vol 3 no 7 pp 564ndash581 1999
[11] C Piersimoni A Olivieri L Benacchio and C ScarparoldquoCurrent perspectives on drug susceptibility testing ofMycobac-terium tuberculosis complex the automated nonradiometricsystemsrdquo Journal of Clinical Microbiology vol 44 no 1 pp 20ndash28 2006
[12] Chinese Anti-Tuberculosis Association Protocols for Tubercu-losis Diagnosis in Laboratory Chinese Educational and CulturalPublisher Beijing China 1st edition 2006
[13] J-C Palomino A Martin M Camacho H Guerra JSwings and F Portaels ldquoResazurin microtiter assay platesimple and inexpensive method for detection of drug resis-tance inMycobacterium tuberculosisrdquo Antimicrobial Agents andChemotherapy vol 46 no 8 pp 2720ndash2722 2002
[14] N Siddiqi M Shamim S Hussain et al ldquoMolecular char-acterization of multidrug-resistant isolates of Mycobacteriumtuberculosis from patients in North Indiardquo Antimicrobial Agentsand Chemotherapy vol 46 no 2 pp 443ndash450 2002
[15] M Y Lipin V N Stepanshina I G Shemyakin and T MShinnick ldquoAssociation of specific mutations in katG rpoB rpsLand rrs geneswith spoligotypes ofmultidrug-resistantMycobac-terium tuberculosis isolates in RussiardquoClinical Microbiology andInfection vol 13 no 6 pp 620ndash626 2007
BioMed Research International 7
[16] Z Sun J Zhang X Zhang S Wang Y Zhang and C LildquoComparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strainsand clinical isolatesrdquo International Journal of AntimicrobialAgents vol 31 no 2 pp 115ndash121 2008
[17] J Kamerbeek L Schouls AKolk et al ldquoSimultaneous detectionand strain differentiation of Mycobacterium tuberculosis fordiagnosis and epidemiologyrdquo Journal of Clinical Microbiologyvol 35 no 4 pp 907ndash914 1997
[18] Z H Helal M S E-D Ashour S A Eissa et al ldquoUnexpectedlyhigh proportion of ancestral manu genotype Mycobacteriumtuberculosis strains cultured from tuberculosis patients inEgyptrdquo Journal of Clinical Microbiology vol 47 no 9 pp 2794ndash2801 2009
[19] P Supply E Mazars S Lesjean V Vincent B Gicquel andC Locht ldquoVariable human minisatellite-like regions in theMycobacterium tuberculosis genomerdquo Molecular Microbiologyvol 36 no 3 pp 762ndash771 2000
[20] M B Boniotti M Goria D Loda et al ldquoMolecular typingof Mycobacterium bovis strains isolated in Italy from 2000to 2006 and evaluation of variable-number tandem repeatsfor geographically optimized genotypingrdquo Journal of ClinicalMicrobiology vol 47 no 3 pp 636ndash644 2009
[21] W H Benjamin Jr K B Waites A Beverly et al ldquoComparisonof the MBBacT system with a revised antibiotic supplementkit to the BACTEC 460 system for detection of mycobacteriain clinical specimensrdquo Journal of Clinical Microbiology vol 36no 11 pp 3234ndash3238 1998
[22] World Health Organisation The Use of Liquid Medium forCulture and DST in Middle- and Low-Income Countries 2007httpwwwwhointtbdotslaboratorypolicyenindex3htm
[23] W W Yew S C W Tong K S Lui S K F Leung CH Chau and E P Wang ldquoComparison of MBBacT systemand agar proportion method in drug susceptibility testingof Mycobacterium tuberculosisrdquo Diagnostic Microbiology andInfectious Disease vol 39 no 4 pp 229ndash232 2001
[24] Y Balabanova F Drobniewski V Nikolayevskyy et al ldquoAnintegrated approach to rapid diagnosis of tuberculosis andmul-tidrug resistance using liquid culture andmolecular methods inRussiardquo PLoS ONE vol 4 no 9 Article ID e7129 2009
[25] L Lawson N Emenyonu S T Abdurrahman et al ldquoCom-parison ofMycobacterium tuberculosis drug susceptibility usingsolid and liquid culture in Nigeriardquo BMC Research Notes vol 6no 1 article 215 2013
[26] S J Kim ldquoDrug-susceptibility testing in tuberculosis methodsand reliability of resultsrdquo European Respiratory Journal vol 25no 3 pp 564ndash569 2005
[27] World Health Organization ldquoPolicy guidance on drug-sus-ceptibility testing (DST) of second-line antituberculosisdrugsrdquo 2008 httpwwwwhointtbfeatures archivexdr mdrpolicy guidance
[28] I C Shamputa L Jugheli N Sadradze et al ldquoMixed infectionand clonal representativeness of a single sputum sample intuberculosis patients from a penitentiary hospital in GeorgiardquoRespiratory Research vol 7 article 99 2006
[29] I C Shamputa L Rigouts L A Eyongeta et al ldquoGenotypic andphenotypic heterogeneity among Mycobacterium tuberculosisisolates from pulmonary tuberculosis patientsrdquo Journal of Clin-ical Microbiology vol 42 no 12 pp 5528ndash5536 2004
[30] M N T Huyen T N Buu E Tiemersma et al ldquoTuberculosisrelapse in vietnam is significantly associated with Mycobac-terium tuberculosis Beijing genotype infectionsrdquo The Journal ofInfectious Diseases vol 207 no 10 pp 1516ndash1524 2013
[31] EuropeanConcertedAction onNewGenerationGeneticMark-ers and Techniques for the Epidemiology and Control ofTuberculosis ldquoBeijingW genotypeMycobacterium tuberculosisand drug resistancerdquo Emerging Infectious Diseases vol 12 no 5pp 736ndash743 2006
[32] B C De Jong P C Hill A Aiken et al ldquoProgression to activetuberculosis but not transmission varies by Mycobacteriumtuberculosis lineage in the Gambiardquo Journal of Infectious Dis-eases vol 198 no 7 pp 1037ndash1043 2008
[33] M Hanekom G D van der Spuy E Streicher et al ldquoA recentlyevolved sublineage of the Mycobacterium tuberculosis Beijingstrain family is associated with an increased ability to spreadand cause diseaserdquo Journal of Clinical Microbiology vol 45 no5 pp 1483ndash1490 2007
[34] Y Kong M D Cave L Zhang et al ldquoAssociation betweenMycobacterium tuberculosis BeijingW lineage strain infectionand extrathoracic tuberculosis Insights from epidemiologicand clinical characterization of the three principal geneticgroups of M tuberculosis clinical isolatesrdquo Journal of ClinicalMicrobiology vol 45 no 2 pp 409ndash414 2007
[35] Y Pang Y Zhou B Zhao et al ldquoSpoligotyping and drugresistance analysis of Mycobacterium Tuberculosis strains fromnational survey in Chinardquo PLoS ONE vol 7 no 3 Article IDe32976 2012
strains was markedly increased in paired isolates whoseDST results showed discrepancies (375) compared to therandomly selected clinical isolates (3125) An unusuallyhigh proportion of strains belonging to the ldquoManurdquo clade(2715) were also reported by Helal et al [18] InterestinglyManu2 strains (SIT1634) have rarely been reported in Tianjinor even in China as a whole [34 35] or in the SPOLDB4database (excluding this study 119899 = 3 1 from India and 2 fromthe USA)
In this study all the 40 isolates were determined asresistant by MGIT and sensitive by L-J of which twenty-seven isolates were found with mutations in katG315 and 13isolates were found with no mutations in katG315 (Table 4)Those results of the mutations found in the INH-targetedgenes supported that the DST result by the MGIT was moreaccurate than that by the L-J andwe also found that theMICsof some isolates by L-J agar method were very higher thanthose in the first execution in clinic which indicated to someextent that the operation needs to be improved in proportionmethod on L-J agar
5 Conclusion
Our study confirmed that the discrepancies of the DSTin M tuberculosis clinical isolates did exist for INH Oneof the reasons for the discrepancy is the different testsystems between the BACTEC MGIT960 system and thetraditional L-J proportion method Mixed infection by thestrains with MANU2 and Beijing genotype patterns couldalso contributed to drug discrepancies
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Zaoxian Mei and Zhaogang Sun contributed equally to thiswork
Acknowledgments
The authors thank the Beijing Bio-Bank of Clinical Resourceson Tuberculosis and the Outpatient Department of Tian-jin Haihe Hospital for supplying M tuberculosis H37Rvand clinical isolates They also thank the National Spe-cial Key Project of China on Major Infectious Diseases(2013ZX10003009) Beijing Municipal Administration ofHospitals ClinicalMedicine Development of Special FundingSupport (ZYLX201304) and the Beijing High-level TechnicalPersonnel Training Project in Health (2011-3-069) for finan-cial support
References
[1] J G Pasipanodya S Srivastava and T Gumbo ldquoMeta-analysisof clinical studies supports the pharmacokinetic variability
hypothesis for acquired drug resistance and failure of antituber-culosis therapyrdquo Clinical Infectious Diseases vol 55 no 2 pp169ndash177 2012
[2] P T Kent and G P Kubica Public Health Mycobacteriology AGuide for Level III Laboratory Centers for Diseases ControlAtlanta Ga USA 1985
[3] S B Walters and B A Hanna ldquoTesting of susceptibilityof Mycobacterium tuberculosis to isoniazid and rifampin bymycobacterium growth indicator tube methodrdquo Journal ofClinical Microbiology vol 34 no 6 pp 1565ndash1567 1996
[4] AWanger and KMills ldquoTesting ofMycobacterium tuberculosissusceptibility to ethambutol isoniazid rifampin and strepto-mycin by using Etestrdquo Journal of Clinical Microbiology vol 34no 7 pp 1672ndash1676 1996
[5] L A Collins and S G Franzblau ldquoMicroplate Alamar blue assayversus BACTEC 460 system for high-throughput screening ofcompounds against Mycobacterium tuberculosis and Mycobac-terium aviumrdquo Antimicrobial Agents and Chemotherapy vol 41no 5 pp 1004ndash1009 1997
[6] C Piersimoni ldquoTB control strategies present and future indiagnostic methods what the clinician can ask to the labrdquoMonaldi Archives for Chest Disease vol 57 no 5-6 pp 306ndash3102002
[7] G L Woods G Fish M Plaunt and T Murphy ldquoClinicalevaluation of Difco ESP culture system II for growth anddetection ofmycobacteriardquo Journal of ClinicalMicrobiology vol35 no 1 pp 121ndash124 1997
[8] R A Adegbola P Hill I Baldeh et al ldquoSurveillance of drug-resistant Mycobacterium tuberculosis in The Gambiardquo Interna-tional Journal of Tuberculosis and Lung Disease vol 7 no 4 pp390ndash393 2003
[9] C M S Giampaglia M C Martins G B De Oliveira Vieiraet al ldquoMulticentre evaluation of an automated BACTEC 960system for susceptibility testing ofMycobacterium tuberculosisrdquoInternational Journal of Tuberculosis and Lung Disease vol 11no 9 pp 986ndash991 2007
[10] L Heifets and G A Cangelosi ldquoDrug susceptibility testing ofMycobacterium tuberculosis a neglected problem at the turnof the centuryrdquo International Journal of Tuberculosis and LungDisease vol 3 no 7 pp 564ndash581 1999
[11] C Piersimoni A Olivieri L Benacchio and C ScarparoldquoCurrent perspectives on drug susceptibility testing ofMycobac-terium tuberculosis complex the automated nonradiometricsystemsrdquo Journal of Clinical Microbiology vol 44 no 1 pp 20ndash28 2006
[12] Chinese Anti-Tuberculosis Association Protocols for Tubercu-losis Diagnosis in Laboratory Chinese Educational and CulturalPublisher Beijing China 1st edition 2006
[13] J-C Palomino A Martin M Camacho H Guerra JSwings and F Portaels ldquoResazurin microtiter assay platesimple and inexpensive method for detection of drug resis-tance inMycobacterium tuberculosisrdquo Antimicrobial Agents andChemotherapy vol 46 no 8 pp 2720ndash2722 2002
[14] N Siddiqi M Shamim S Hussain et al ldquoMolecular char-acterization of multidrug-resistant isolates of Mycobacteriumtuberculosis from patients in North Indiardquo Antimicrobial Agentsand Chemotherapy vol 46 no 2 pp 443ndash450 2002
[15] M Y Lipin V N Stepanshina I G Shemyakin and T MShinnick ldquoAssociation of specific mutations in katG rpoB rpsLand rrs geneswith spoligotypes ofmultidrug-resistantMycobac-terium tuberculosis isolates in RussiardquoClinical Microbiology andInfection vol 13 no 6 pp 620ndash626 2007
BioMed Research International 7
[16] Z Sun J Zhang X Zhang S Wang Y Zhang and C LildquoComparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strainsand clinical isolatesrdquo International Journal of AntimicrobialAgents vol 31 no 2 pp 115ndash121 2008
[17] J Kamerbeek L Schouls AKolk et al ldquoSimultaneous detectionand strain differentiation of Mycobacterium tuberculosis fordiagnosis and epidemiologyrdquo Journal of Clinical Microbiologyvol 35 no 4 pp 907ndash914 1997
[18] Z H Helal M S E-D Ashour S A Eissa et al ldquoUnexpectedlyhigh proportion of ancestral manu genotype Mycobacteriumtuberculosis strains cultured from tuberculosis patients inEgyptrdquo Journal of Clinical Microbiology vol 47 no 9 pp 2794ndash2801 2009
[19] P Supply E Mazars S Lesjean V Vincent B Gicquel andC Locht ldquoVariable human minisatellite-like regions in theMycobacterium tuberculosis genomerdquo Molecular Microbiologyvol 36 no 3 pp 762ndash771 2000
[20] M B Boniotti M Goria D Loda et al ldquoMolecular typingof Mycobacterium bovis strains isolated in Italy from 2000to 2006 and evaluation of variable-number tandem repeatsfor geographically optimized genotypingrdquo Journal of ClinicalMicrobiology vol 47 no 3 pp 636ndash644 2009
[21] W H Benjamin Jr K B Waites A Beverly et al ldquoComparisonof the MBBacT system with a revised antibiotic supplementkit to the BACTEC 460 system for detection of mycobacteriain clinical specimensrdquo Journal of Clinical Microbiology vol 36no 11 pp 3234ndash3238 1998
[22] World Health Organisation The Use of Liquid Medium forCulture and DST in Middle- and Low-Income Countries 2007httpwwwwhointtbdotslaboratorypolicyenindex3htm
[23] W W Yew S C W Tong K S Lui S K F Leung CH Chau and E P Wang ldquoComparison of MBBacT systemand agar proportion method in drug susceptibility testingof Mycobacterium tuberculosisrdquo Diagnostic Microbiology andInfectious Disease vol 39 no 4 pp 229ndash232 2001
[24] Y Balabanova F Drobniewski V Nikolayevskyy et al ldquoAnintegrated approach to rapid diagnosis of tuberculosis andmul-tidrug resistance using liquid culture andmolecular methods inRussiardquo PLoS ONE vol 4 no 9 Article ID e7129 2009
[25] L Lawson N Emenyonu S T Abdurrahman et al ldquoCom-parison ofMycobacterium tuberculosis drug susceptibility usingsolid and liquid culture in Nigeriardquo BMC Research Notes vol 6no 1 article 215 2013
[26] S J Kim ldquoDrug-susceptibility testing in tuberculosis methodsand reliability of resultsrdquo European Respiratory Journal vol 25no 3 pp 564ndash569 2005
[27] World Health Organization ldquoPolicy guidance on drug-sus-ceptibility testing (DST) of second-line antituberculosisdrugsrdquo 2008 httpwwwwhointtbfeatures archivexdr mdrpolicy guidance
[28] I C Shamputa L Jugheli N Sadradze et al ldquoMixed infectionand clonal representativeness of a single sputum sample intuberculosis patients from a penitentiary hospital in GeorgiardquoRespiratory Research vol 7 article 99 2006
[29] I C Shamputa L Rigouts L A Eyongeta et al ldquoGenotypic andphenotypic heterogeneity among Mycobacterium tuberculosisisolates from pulmonary tuberculosis patientsrdquo Journal of Clin-ical Microbiology vol 42 no 12 pp 5528ndash5536 2004
[30] M N T Huyen T N Buu E Tiemersma et al ldquoTuberculosisrelapse in vietnam is significantly associated with Mycobac-terium tuberculosis Beijing genotype infectionsrdquo The Journal ofInfectious Diseases vol 207 no 10 pp 1516ndash1524 2013
[31] EuropeanConcertedAction onNewGenerationGeneticMark-ers and Techniques for the Epidemiology and Control ofTuberculosis ldquoBeijingW genotypeMycobacterium tuberculosisand drug resistancerdquo Emerging Infectious Diseases vol 12 no 5pp 736ndash743 2006
[32] B C De Jong P C Hill A Aiken et al ldquoProgression to activetuberculosis but not transmission varies by Mycobacteriumtuberculosis lineage in the Gambiardquo Journal of Infectious Dis-eases vol 198 no 7 pp 1037ndash1043 2008
[33] M Hanekom G D van der Spuy E Streicher et al ldquoA recentlyevolved sublineage of the Mycobacterium tuberculosis Beijingstrain family is associated with an increased ability to spreadand cause diseaserdquo Journal of Clinical Microbiology vol 45 no5 pp 1483ndash1490 2007
[34] Y Kong M D Cave L Zhang et al ldquoAssociation betweenMycobacterium tuberculosis BeijingW lineage strain infectionand extrathoracic tuberculosis Insights from epidemiologicand clinical characterization of the three principal geneticgroups of M tuberculosis clinical isolatesrdquo Journal of ClinicalMicrobiology vol 45 no 2 pp 409ndash414 2007
[35] Y Pang Y Zhou B Zhao et al ldquoSpoligotyping and drugresistance analysis of Mycobacterium Tuberculosis strains fromnational survey in Chinardquo PLoS ONE vol 7 no 3 Article IDe32976 2012
[16] Z Sun J Zhang X Zhang S Wang Y Zhang and C LildquoComparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strainsand clinical isolatesrdquo International Journal of AntimicrobialAgents vol 31 no 2 pp 115ndash121 2008
[17] J Kamerbeek L Schouls AKolk et al ldquoSimultaneous detectionand strain differentiation of Mycobacterium tuberculosis fordiagnosis and epidemiologyrdquo Journal of Clinical Microbiologyvol 35 no 4 pp 907ndash914 1997
[18] Z H Helal M S E-D Ashour S A Eissa et al ldquoUnexpectedlyhigh proportion of ancestral manu genotype Mycobacteriumtuberculosis strains cultured from tuberculosis patients inEgyptrdquo Journal of Clinical Microbiology vol 47 no 9 pp 2794ndash2801 2009
[19] P Supply E Mazars S Lesjean V Vincent B Gicquel andC Locht ldquoVariable human minisatellite-like regions in theMycobacterium tuberculosis genomerdquo Molecular Microbiologyvol 36 no 3 pp 762ndash771 2000
[20] M B Boniotti M Goria D Loda et al ldquoMolecular typingof Mycobacterium bovis strains isolated in Italy from 2000to 2006 and evaluation of variable-number tandem repeatsfor geographically optimized genotypingrdquo Journal of ClinicalMicrobiology vol 47 no 3 pp 636ndash644 2009
[21] W H Benjamin Jr K B Waites A Beverly et al ldquoComparisonof the MBBacT system with a revised antibiotic supplementkit to the BACTEC 460 system for detection of mycobacteriain clinical specimensrdquo Journal of Clinical Microbiology vol 36no 11 pp 3234ndash3238 1998
[22] World Health Organisation The Use of Liquid Medium forCulture and DST in Middle- and Low-Income Countries 2007httpwwwwhointtbdotslaboratorypolicyenindex3htm
[23] W W Yew S C W Tong K S Lui S K F Leung CH Chau and E P Wang ldquoComparison of MBBacT systemand agar proportion method in drug susceptibility testingof Mycobacterium tuberculosisrdquo Diagnostic Microbiology andInfectious Disease vol 39 no 4 pp 229ndash232 2001
[24] Y Balabanova F Drobniewski V Nikolayevskyy et al ldquoAnintegrated approach to rapid diagnosis of tuberculosis andmul-tidrug resistance using liquid culture andmolecular methods inRussiardquo PLoS ONE vol 4 no 9 Article ID e7129 2009
[25] L Lawson N Emenyonu S T Abdurrahman et al ldquoCom-parison ofMycobacterium tuberculosis drug susceptibility usingsolid and liquid culture in Nigeriardquo BMC Research Notes vol 6no 1 article 215 2013
[26] S J Kim ldquoDrug-susceptibility testing in tuberculosis methodsand reliability of resultsrdquo European Respiratory Journal vol 25no 3 pp 564ndash569 2005
[27] World Health Organization ldquoPolicy guidance on drug-sus-ceptibility testing (DST) of second-line antituberculosisdrugsrdquo 2008 httpwwwwhointtbfeatures archivexdr mdrpolicy guidance
[28] I C Shamputa L Jugheli N Sadradze et al ldquoMixed infectionand clonal representativeness of a single sputum sample intuberculosis patients from a penitentiary hospital in GeorgiardquoRespiratory Research vol 7 article 99 2006
[29] I C Shamputa L Rigouts L A Eyongeta et al ldquoGenotypic andphenotypic heterogeneity among Mycobacterium tuberculosisisolates from pulmonary tuberculosis patientsrdquo Journal of Clin-ical Microbiology vol 42 no 12 pp 5528ndash5536 2004
[30] M N T Huyen T N Buu E Tiemersma et al ldquoTuberculosisrelapse in vietnam is significantly associated with Mycobac-terium tuberculosis Beijing genotype infectionsrdquo The Journal ofInfectious Diseases vol 207 no 10 pp 1516ndash1524 2013
[31] EuropeanConcertedAction onNewGenerationGeneticMark-ers and Techniques for the Epidemiology and Control ofTuberculosis ldquoBeijingW genotypeMycobacterium tuberculosisand drug resistancerdquo Emerging Infectious Diseases vol 12 no 5pp 736ndash743 2006
[32] B C De Jong P C Hill A Aiken et al ldquoProgression to activetuberculosis but not transmission varies by Mycobacteriumtuberculosis lineage in the Gambiardquo Journal of Infectious Dis-eases vol 198 no 7 pp 1037ndash1043 2008
[33] M Hanekom G D van der Spuy E Streicher et al ldquoA recentlyevolved sublineage of the Mycobacterium tuberculosis Beijingstrain family is associated with an increased ability to spreadand cause diseaserdquo Journal of Clinical Microbiology vol 45 no5 pp 1483ndash1490 2007
[34] Y Kong M D Cave L Zhang et al ldquoAssociation betweenMycobacterium tuberculosis BeijingW lineage strain infectionand extrathoracic tuberculosis Insights from epidemiologicand clinical characterization of the three principal geneticgroups of M tuberculosis clinical isolatesrdquo Journal of ClinicalMicrobiology vol 45 no 2 pp 409ndash414 2007
[35] Y Pang Y Zhou B Zhao et al ldquoSpoligotyping and drugresistance analysis of Mycobacterium Tuberculosis strains fromnational survey in Chinardquo PLoS ONE vol 7 no 3 Article IDe32976 2012
