Currenttuberculosisdiagnostictools&roleofureasebreathtest

MamoudouMaiga,AhmedAbaza&WilliamR.Bishai

Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University, Maryland, USA

ReceivedMarch1,2011

Tuberculosis (TB) remains a significant public health issue worldwide especially in developing countries, where the disease is endemic, and effective TB diagnostic as well as treatment-monitoring tools are serious barriers to defeating the disease. Detection of pathogen-specific metabolic pathways offers a potential alternative to current methods, which focus on bacterial growth, bacterial nucleic acid amplification, or detection of host immune response to the pathogen. Metabolic pathway detection may provide rapid and effective new tools for TB that can improve TB diagnostics for children and HIV infected patients. Metabolic breath tests are attractive because these are safe, and provide an opportunity for rapid point of care diagnostics and tool for drug efficacy evaluation during clinical trials. Our group has developed a rabbit urease breath test model to evaluate the sensitivity and the specificity of urease based detection of Mycobacterium tuberculosis. TB infected rabbits were given stable isotopically labelled urea as the substrate. The urea tracer was metabolized to 13C-CO2 and detected in exhaled breaths using portable infrared spectrometers. The signal correlated with bacterial load both for primary diagnostics and treatment monitoring. Clinical trials are currently ongoing to evaluate the value of the test in clinical management settings. Urea breath testing may provide a useful diagnostic and biomarker assay for tuberculosis and treatment response.

Key words Biomarker-TBdiagnosticandtreatmentmonitoring-UBT

Review Article

Introduction

Abouttwobillionpeople(one-thirdoftheworld’spopulation) carry latent tuberculosis (TB) infectionandmorethanninemillionofthembecomesickeachyearwith activeTB,which can be spread to others.It disproportionately affects people in resource-poorsettings,particularlythoseinAsiaandAfrica1,2.Morethan90percentofnewTBcasesanddeathsoccurinthosepartsoftheworld,posingsignificantchallengesto the livelihood of individuals and developingeconomies,asTBprimarilyaffectspeopleduringtheirmostproductiveyears1,2.Poorhealthsystems,limitedlaboratorycapacityforcasedetection,treatmentbarriers

and complications (unreliable drug supply, patientsnot completing treatment, or prescribing errors), TBand HIV co-infection, and the emergence of drug-resistance3,4makeTBamajorchallengefacingpublichealth programmes, particularly in the 22 countrieswiththehighestTBburden(Fig.1).Inaddition,TBisdifficulttodiagnoseanditsdiagnosisismoredifficultin individualswithTB/HIVco-infection.ThecurrentdiagnostictechniquesareeitherinadequatetodetectTBcaseswithprecision,oraretimeconsuming,expensive,andrequirehighlyequippedlaboratorieswhicharenotavailable in developing countries, where the diseaseis endemic. In this paper, we reviewed the currentavailablediagnostictoolsforTBandthepotentialrole

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ofureasebreathtest(UBT)tobothdiagnoseTBandmonitortreatmentresponse.

Current TB diagnostic tools

Sputum smear is used to detect acid fast bacilli(AFB) in clinical specimens by Ziehl-Neelsen(Z-N) or fluorescent staining. It is a cost-effectivetool for diagnosing patientswithTB and tomonitorthe progress of treatment especially in developingcountries.However,therearemanydrawbackssuchasthedifficultyofobtainingthesputumsampleespeciallyinchildrenandthelowsensitivityespeciallyinHIV-infectedindividualswithAFBsmearpositivityrangingfrom31to90percent5.

Sputum culture is the gold standard for TBdiagnosiswithanexcellentsensitivityandspecificity.The traditional method of inoculating solid mediumsuchasLowerstein-Jensen(L-J)or7H10/7H11mediais slow and takes 6-8 wk of incubation to diagnosethe infection and furthermore time to determine thesusceptibilitypatterns.Thatresultsindelayininitiationofappropriatetherapy.

TheintroductionoftheBACTECTB460radiometricsystem (Becton Dickinson, Sparks, MD, USA) inthe 1980s allowed the detection of Mycobacterium tuberculosis in a few days as compared with theconventionalculturemedia.Theintroductionoftheserapid and automated systems have increased thesensitivityofisolationofMycobacteriumfromclinicalsamples andhasbrought down the time required forpositiveculturesubstantially(9-10days).Fastercultureresults inM. tuberculosis infectedpatients can resultin evidence-based therapy and faster implementation

oftheplannedtreatment6.Althoughacombinationoftraditionalsolidandliquidmediaisrecommendedandhelpful for the primary isolation ofMycobacterium,the process still requires several days and expensiveinfrastructure.

The microscopic observation drug susceptibility(MODS) assay relies on light microscopy tovisualizethecharacteristiccordingmorphologyofM. tuberculosisinliquidculture.MODShasshortertimeto culture positivity (average 8 days) comparedwithLJmedium(average~26days).Thecost-effectivenessofMODS (~US$2/`102/- per sample) is favourablecompared with L-J culture (~ US $6/`307/- persample)7. Hence,MODS appears to be a promising,novelandinexpensivetoolthatcanrapidlydetectTBand drug resistance directly from sputum samples.However,theeffectivenessofMODSinHIV/TBcasedetection, which is a very common co-infection indevelopingcountries,isnotyetclearlyestablished.Alsothetest isonlyvalidatedfor isoniazidandrifampicinsusceptibilitytesting,excludingtheotherfirstlinesandsecondlinesTBtherapy.

TheGen-ProbeassayspecificforM. tuberculosis complexisarapiddetection,nucleicacidamplification(NAA)test,whereresultscanbeobtainedasfastasintwohours(providedifapositivecultureispresent);italsohasahighsensitivityof99percentandspecificityof99.2percent8.Itholdsthedisadvantageofneedingofpositiveculturethatcantakeseveraldays.

Spoligotyping is a technique for concurrentdetection and typing of M. tuberculosis complexbacteria9. This method relies upon amplification ofa highly polymorphic direct repeat locus in the M. tuberculosisgenomeandresultscanbeobtainedwithinoneday.Thus,theclinicalusefulnessofspoligotypingisdeterminedbyitsrapidity,bothinidentifyingcausativebacteria and in providing molecular epidemiologicinformationonstrains.Spoligotypingholdsdrawbacksof being expensive, and requiring well-equippedlaboratoryandskilledlaboratorypersonnel,whicharenotalwaysavailableinendemicareas.

Line-probe assays (INNO-LiPA Rif. TB andHAINGenotypeMTBDRplus) and themicrofluidicPCR device (GenExpert/Cepheid) allow both rapiddetectionofTBandalsodrugsresistancepatterns10,11.The INNO-LiPARif.TB can simultaneously detectM. tuberculosisandthepresenceofamutationintherpoBgene,whichconfersresistancetorifampicinwithasensitivityof95percentandspecificitynearly100

Fig. 1. The 22 high burden TB countries in the word (Source:KaiserFamilyFoundation,www.GlobalHealthFacts.org,basedonWHO,Global tuberculosis control 2010, Slide Date:March 04,2011;reprintedwithpermissionfromtheHenryJ.KaiserFamilyFoundation,California,USA).

per cent when isolates from culture were used12,13.However,INNO-LiPARif.TBislessaccuratewhendirectly applied on clinical sputum specimens12. Thenew Hain “GenoType MTBDR plus” test can beused both on culture-based isolates and directly onsmear positive sputum samples from patients withpulmonaryTB.TheMTBDRassayhastheadvantageofidentifyingmutationsintherpoBgene(rifampicinresistance)aswellasmutationsinthekatGgene(high-levelofisoniazidresistance)andinhAgene(low-levelofisoniazidresistance).TheMTBDRassaycandetectatleast90percentofMDR-TBcasesinafewhours13.GeneXpert MTB/ RIF uses heminested real-timepolymerasechainreaction(PCR)assaytoamplifyM. tuberculosisspecificsequenceoftherpoBgene,whichisprobedwithmolecularbeaconsformutationswithinthe rifampin-resistance determining region14,15. ThesensitivityofasingledirectMTB/RIFtestforculture-confirmed tuberculosiswas92.2percentandrose to96.0percentwiththeadditionaltestingofapelletedsample16. The MTB/RIF test provided sensitivedetection of tuberculosis and rifampicin resistancedirectly fromuntreated sputum in less than2hwithminimalhands-ontime16.

Suchnovel technologieshavebeen criticized forbeing expensive and excluding many drugs for theresistancepatternsprofile.

Urease activity of M. tuberculosis and applications

The M. tuberculosis urease enzyme encoded byureA, ureB and ureC (Rv1848,Rv1849 andRv1850)hydrolyzes urea into carbon dioxide and ammonia17.The M. tuberculosisureaseisalikelybacterialvirulencefactor,whichmaysubvertthehostacidificationofthephagosome microenvironment. Urease activity hasbeenusedinthepastasakeytestintheidentificationofmicroorganisms18.Sohngenreportedureaseactivityinmycobacteriain1913(asnotedbyUrabeandSaito)19.Withtheclassificationofmycobacteriainthe1950sbyTimpe20andRunyon21,medicalandtaxonomicinterestinmycobacteriology increased.During the followingdecade, biochemical and morphological findingsestablished mycobacteriology as a distinct area fordevelopment22-24 and tests based on urease activityhavebeenconsistentlyutilizedfortheidentificationofmycobacterial species25-28. Despite the role of ureasetesting for mycobacterial species identification, littlehasbeendonetoemployurease-basedtestsaspoint-of-careprimaryTBdiagnosticsuntil2009,whenourgroup studied the usefulness of rabbit urease breathtest forTBdiagnosis and treatmentmonitoring29.An

attractive aspect of urease-based diagnostics is thathumanslackureaseenzymes.Hence,thedetectionofureaseenzymeactivityinhumanssignifiesthepresenceofmicrobialpathogenorcommensalwhich isureasepositive.

Metabolomic application of Helicobacter pylori urease activity

as M. tuberculosis, H. pylori also possessesureaseactivitytopromoteitssurvival.Gastricureasedetectionhasbeenclassicallyutilizedforrapidandnon-invasivediagnosticsofH. pyloriinfection30.Thisassayintroducedduring1980s,usesanoraldoseoflabelled13C-ureawithsamplingoftheratioof13CO2 to 12CO2 in exhaledairaftertheureaishydrolyzedinthestomachby theH. pylori urease (Fig.2).This ratio is readilymeasuredusingsmallportableinfraredspectrometers,and theentireprocedurecanbecompletedwithin30min31.ThetestcalledH. pyloriureasebreathtest(UBT-HP) received United States FDA approval in 1993and is currently popular for point-of-care diagnosisof H. pylori active infection and its post-treatmenteradication.ThissuccessoftheH. pyloriureasebreathtestcomprisesthebasisofdevelopingasimilarbreathtestforTB.

Rabbit model of TB-urease breath test (UBT-TB)

OurapproachhasbeentodevelopabreathtestforuseinTBsimilartotherapiddiagnosticforH. pylori infectionandasabiomarkerofitseradicationbydrugtreatment30.Wedelivered13C-ureaintolungsofrabbitsinfectedwithM. bovis or M. tuberculosis(bothofwhichareureaseproducingmycobacteria)viabronchoscopicinfection, and estimated lungburdensof disease and

Fig. 2. Labelled urea is degraded by H. pylori in the stomachwhereby the labelledCO2produced is thenexhaledanddetectedin the lungs.Thedetectionof the labelledCO2 implies infectionwithH. pylori.

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pulmonary tuberculosis associated with a variety ofpathologicpresentations29.

Isotopic gas ratio analysis can be conductedwith compact infrared spectrophotometers such asPOConeTMorUBiT(OtsukaPharmaceuticalCo.,Ltd.,Tokyo, Japan). The exhaled breath sample is easilyobtainedthroughone-wayvalvebagsandanalyzedbysuchdevices.ThesedevicescanbemodifiedandfiltersincorporatedforuseforTBinfectedpatients(toavoidcontaminations).

The UBT-TB test may also provide a novelbiomarker for TB drug therapy trials to improvetherapeuticstrategiesandvalidatenovelTBdrugandvaccinecandidates.Ifvalidated,itmayprovidevaluableearly information on whether bacterial burdens areresponding to antimicrobial therapy. As opposed tocurrentlyutilizedsputummicrobiologyanalysesbeingusedfordrugsusceptibility,ureabreathtestingforM. tuberculosismayallowarapidassessmentoftreatmentefficacyand theneed for treatmentmodifications fordrugresistantTB33,34.

UBT,asametabolomicbasedtest,worksonlivebacteria. A large cell population of M. tuberculosis duringthediseaseandevenduringTBtreatmentisnon-replicatingandnonculturableinnormalconditions35.However,UBThasthepotentialtodetectthispopulationandmonitoritsresponsetochemotherapy,althoughtherelativeexpressionofureaseactivitybydormantcellsrequiresinvestigations.

Specificity of UBT-TB

UBT-TB specificity may be limited by otherurease-producingmicrobialspecies,suchasH. pylori,

response to therapy by comparing the UBT resultsovertime29.Theserabbitsconverted13C-labelledureainto 13CO2inamatterofminutes(Fig.3).The13CO2 in exhaledbreathwasrapidlydetectablewithanisotopicratiomassspectrometerorasmallinfraredabsorbancespectrometer. The UBT signal in rabbits declines astherabbitsaretreatedwithantimicrobialandincreasesagainaftertherapyishalted29.IncontrasttooralUBT-HP, our experiments with rabbit UBT implementeddirect instillationof 13C-urea substrate into the lungsorby intravenous injection to increase thespecificityforM. tuberculosis.The rabbitUBTmodelhasbeenlimitedbythefactthatexhaledairisdifficulttoobtainandanimalsneedtobeanesthetized(Fig.4).Potential utilities of UBT-TB for patient management

UBT provides the possibility of using exhaledbreathsamplestodiagnoseTB.Itisattractivebecauseitoffersapathtowardsanovelpoint-of-carediagnosticfor TB and TB treatment response. Point-of-carediagnostics are needed for TB especially in high-burdensettings inorder to reduce thedisease impactby providing the possibility to quickly prescribe theappropriate treatment.Thus theUBTmaybeable toserveasarapidindicatoroftreatmentfailureasoccursinpatientswithMDR-TB.Resistance toTBdrugs isgenotypic and new nucleic acid amplification tests(such asGeneXpert) offer significant steps forwardinrapidlydetectingcertainformsofresistantclinicalisolates.However,aproportionofresistantinfectionswillnotbeidentifiedmolecularlyandsomemolecularresistant genotypes may not follow the phenotypeexpected32.UBTwillhavetheadvantagetofollowthein vivo real timeimpactof the treatmentonbacterialdeath. Furthermore, the conversion rate per minuteshowed that the testmay rapidly and reliably detect

Fig. 3.UBTsignalincreasedinrabbitsinfectedbyM. tuberculosis anddeclinedsignificantlyinresponsetopartialTBtherapy.Source:Ref29(Reprintedwithpermission).

Fig. 4. Materials to get exhaled air breath from anaesthetizedrabbitsandtransferintoBreathekTMairbag.

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whichiscommoninhighTBincidenceareasreaching70to90percentofthepopulation36.Interestingly,thespecificitymaybeamelioratedbyoralnon-absorbableurease inhibitors such as bismuth subsalicylate(commonly used to treat dyspepsia) or proton pumpinhibitors (PPIs) by allowing selective suppressionof gastrointestinalH. pylori37. TB therapy known tohavethepotentialtosuppressH. pylori inthefirsttwoweeksalso increases thespecificity forTB treatmentmonitoring38.

In addition, specificity may be gained byintroducingthe13C-ureatracerviatherespiratorytractor intravenously, thereby avoid gastricHelicobacter species.

Anadditionalapproachwouldbetoco-administeran unlabelled urea drink not enriched with 13C, butrather has this at normal abundance. This approachwouldsaturategutureaseproducerswith“cold”tracerand, therefore, any CO2 produced by gut organismswouldnotbeenrichedin13CO2andthusnotconfoundthesignalfromthelungs.

Ongoing clinical trials on UBT-TB

In collaboration with partners, our group isinvestigating the sensitivity and the specificity oforal UBT both for TB diagnostic and treatmentresponse in Mali and South Africa39. Pranactin®-Citric (BreathtekTM), an oral granulated powder of13C-urea approved by United States Food and Drugadministration (FDA) forUBT-HPwill beusedwithdifferenttimepointstoassessthebestmomenttotestforpulmonaryTB.Whilethetestmaybeconfoundedby co-existing gastric H. pylori infection, bismuthsubsalicylatewillbealsoevaluatedincertainarmsofthestudies tospecificallyreducefalse-positivesignalfromH. pylori.ThestudieswillcompareUBT-TBtoTBstandardtestsandwillincludebothHIVco-infectedandnon-HIVTBpatients.Tolerabilitystudiesuponthenebulizeddeliveryof13C-ureasolutiondirectlytothelungsarealsoinplanningstages.

Conclusion

DespiteprogressinthelastdecadeinTBdiagnostictool discovery, effort is still needed to have impactonthe timeandtheaccuracyof theresults.Theureabreath test may fulfill the criteria for service as aneffective point-of-care diagnostic which requires aneasily instituted, cost-effective and rapidly appliedtechnology. UBTmay also be valuable for responseto therapyandtherebyofferearlyinformationonthepossibility of drugs resistant infections monitoring.

Further clinical studies are needed to evaluate thebenefits of metabolic breath tests compared to thecurrentlyavailableones.

Acknowledgment AuthorsacknowledgethefinancialsupportofHowardHughesMedical Institute grant (80026555) andUSNational Institute ofAllergy and Infectious Diseases grants (AI30036,AI37856 andAI36973).

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