In2010,theCDCreportedupdatedguidelines(250)fortheuseofIGRAsasaidsin diagnosing bothLTBIandactive TBdisease, andin2014, theAmericanAcademyof Pediatrics released a technical report entitled Interferon-Gamma Release Assays for DiagnosisofTuberculosisInfectionandDiseaseinChildren(251).Theprimarypurposeof IGRAsistoidentifythosewhowillbenefitfromLTBItherapy.Priorto2001,theTSTwas theonlyimmunologicaltestforTBinfectionapprovedintheUnitedStates.In2001,the Quantifier-TB (QFT) test (Cellestis Limited,Carnegie, Victoria, Australia) was the first IGRAapprovedbytheFDA(250).In2005,theQuantiFERON-TBGold(QFT-G)testwas FDAapproved(252).Previousguidelineswerereportedin2003fortheuseofQFT(253) andin2005fortheuseof QFT-G(252). Since2005, twonew IGRAshavebeenFDA approved:theQuantiFERON-TBGoldIn-Tube(QFT-GIT)test(Qiagen,Germantown,MD [formerlyCellestisLimited])andtheT-Spot.TB(T-Spot)test(OxfordImmunotecLimited, Abingdon,UnitedKingdom)(250).Thesetestsmayimprovespecificityanddifferfrom previousIGRAsintheantigensused,methods,andinterpretivecriteria.CDCguidelines reportedin2010addresstheuseofthesenewerIGRAs(250).
The ATS/IDSA/CDCclinical practice guidelines reportedin2017 (98)recommend performinganIGRAinsteadofaTSTforindividuals5yearsofageorolderwhomeet thefollowingcriteria:theindividualsarelikelytobeinfectedwiththeM.tuberculosis
complex,theindividualshavealoworintermediateriskofdiseaseprogression,ithas been decided that testing for LTBI is warranted, and theindividuals either have a history of BCG vaccination or areunlikely to return to have their TST results read. Additionally,theguidelinessuggestperforminganIGRAratherthanaTSTforallother individuals5yearsofageorolderwhoarelikelytobeinfectedwiththeM.tuberculosis
complex,whohavealoworintermediateriskofdiseaseprogression,andforwhomit hasbeendecidedthattestingforLTBIiswarranted.Thereareinsufficientdatato
recommendapreferenceforeitheraTSToranIGRAasthefirst-linediagnostictestfor individuals5yearsofageorolderwhoarelikelytobeinfectedwiththeM.tuberculosis
complex, whohave ahighriskof progressiontodisease, andforwhomithasbeen determinedthatdiagnostictestingforLTBIiswarranted.
CommerciallyavailableIGRAsmeasuretheTcellimmuneresponsetoantigensthat arepresentinM.tuberculosiscomplexbutabsentinM.bovisBCGvaccinestrainsand mostNTM.ItshouldbenotedthatalthoughtheQFT-GITandT-Spottestsdonotdetect theM.bovisBCGvaccinestrains,bothassaysdetecttheM.tuberculosiscomplex,which includesM.bovis.TheIGRAsmeasureIFN-␥releasedbysensitizedTcellsincubatedin thepresenceofM.tuberculosiscomplex-specificantigens.Untilthedevelopmentofthe IGRAs,theTSTwastheonlymethodtodetermineLTBI.TheTSThasbeenusedformore than100years andhasmodestsensitivityandspecificity.TheIGRAshavespecificity, especially inBCG-vaccinatedpopulations, andarepredictedtobe betterdiagnostic toolsforLTBIthantheTST(250,254–256).
FortheQFT-GITtest,bloodisdrawndirectlyintoaheparinizedtubecoatedwiththe
M. tuberculosis complex-specificantigens ESAT-6, CFP-10, andTB7.7. A second tube containing phytohemagglutinin (PHA) acts as a mitogen control that indicates cell functionality.Athirdtube,whichcontainsnoantigenorPHA,servesasanilcontrol. Approximately 1ml ofbloodisdrawnintoeachtube. Thetubesmustbe incubated within16hofcollection.Afterincubationfor16to24hat37°C,theplasmaiscollected, andtheconcentrationofIFN-␥isdeterminedbyusinganenzyme-linkedimmunosor- bentassay(ELISA).Themanufacturerprovidescalculationsandcriteriafordetermining apositive result. The test detects infections causedby theM. tuberculosiscomplex, which includesM. tuberculosis,M. bovis,andM. africanum.Specimens frompatients infectedwithM.kansasii,M.szulgai,andM.marinummayrespondintheassay,asthese organismshavethegenesencodingESAT-6andCFP-10(257).
In 2015, the fourth-generation QuantiFERON-TB Gold Plus (QFT-Plus) ELISA was releasedinmarketsoutsidetheUnitedStates.Thisassayalsomeasurestheresponseto theESAT-6andCFP-10peptideantigens,butitisdesignedtoseparatelymeasurethe responseofCD8⫹cytotoxicTlymphocytesandCD4⫹ThelperlymphocytestotheTB
antigens.The testhas twoTB antigentubes: onetube contains ESAT-6andCFP-10 peptides that are designed to stimulateresponses from CD4⫹ cells, anda second
tube contains aset of peptides targeted to induce responsesfrom CD8⫹ cells. M.
tuberculosis-responsive CD8⫹cells have been foundin patients with LTBIand with
active TB disease and aremore frequently found in active TB disease than inLTBI (258–260).StudieshavefoundthatCD8⫹cellresponsesmaybeassociatedwithrecent
M.tuberculosiscomplexexposure(261–263).TheQFT-PlusCD8⫹Tcellresponsemay
help distinguish active from latent TB and discriminate between recent and old infections; however, this awaits further studies. The sensitivity of QFT-Plus using culture-confirmed M. tuberculosis complex cases as a surrogate for LTBI is 95.3% according tothepackage insert(264).Thespecificityinpersons withnoknownrisk factorsforTBis97.6%(264).
TheT-SpotassaywasFDAapprovedinJuly2008.Dependingonthepatient’sage, 2to8mlofbloodisdrawnintoalithium-heparintubeandprocessedwithin30hof collection if T-Cell Xtend is used or within 8 h if T-Cell Xtend is not used (265). Alternatively, bloodmay becollected intoamononuclearcell preparationtubeand processedwithin 8 h of collection.Densitygradientcentrifugationisusedtoseparate the mononuclear cells.T-Cell Xtendcontains antibodiesthatcross-link granulocytes tored bloodcellsandseparate thegranulocytes,whichmay reducetheviability of mononuclear cells and reduce their ability to release interferon gamma, from the mononuclear cells during centrifugation (265). The cells are counted, and approxi- mately250,000 cellsareaddedto microtiterwellsthat arecoatedwith monoclonal antibodiestoIFN-␥.ESAT-6andCFP-10antigensarethenaddedtotwowells,witha third well being used as a nil control and a fourth well containing PHA as a cell functionalitycontrol.Themicrotiterplatesareincubatedfor16to20hat37°Candthen washedtoremovethecells.Aconjugatedsecondaryantibodyisthenadded,which
TABLE 16Persons at increased risk forM. tuberculosiscomplex infection or at increased risk for progression from latent tuberculosis infection to active tuberculosisa
Patient population Description
Persons at increased risk for TB infection Those with close contact with known or suspected active TB cases Foreign-born persons from areas that have a high incidence of active TB
Residents and employees in congregate settings whose clients are at an increased risk for active TB, especially if visits are frequent or prolonged
Persons who visit areas with a high prevalence of active TB disease
Health care workers who serve persons who are at an increased risk for active TB
Populations defined locally as having an increased incidence of TB infection or active disease (e.g., low-income populations and persons who abuse drugs or alcohol)
Infants, children, and adolescents exposed to adults who are at an increased risk for LTBI or active TB Persons at increased risk for progression
to active TB
Persons living with HIV
Those who are receiving immunosuppressive therapy (e.g., TNF-␣bantagonists, systemic
corticosteroids, or immunosuppressive drug therapy following organ transplantation) Those who were infected withMycobacterium tuberculosiswithin the last 2 yr
Those with a history of untreated or inadequately treated active TB, including those with fibrotic changes on chest radiograph consistent with prior TB
Those with silicosis; diabetes mellitus; chronic renal failure; leukemia; lymphoma; or cancer of the head, neck, or lung
Those who have had a gastrectomy or jejunoileal bypass Those who weigh⬍90% of their ideal body wt
Cigarette smokers and those who abuse drugs or alcohol
Those defined locally as having an increased incidence of active TB, including medically underserved or low-income populations
Infants and children aged⬍5 yr who are at an increased risk for a poor outcome (e.g., meningitis, disseminated disease, or death) if active TB occurs
aAdaptedfromreference250. bTNF-␣,tumornecrosisfactoralpha.
bindstotheIFN-␥secretedbytheTcells(andcapturedbytheprimaryantibody).A substrateisthenadded,whichproducesspotswhereIFN-␥ wassecretedbytheTcells. ThespotsthatrepresentsensitizedeffectorTcellsarecounted.FortheT-Spot.TBassay, apositivetestresultisbasedonthenumberofspot-formingunits.IntheUnitedStates, theFDA-approvedcriterionforapositivetestresultis⬎8spots,⬍4spotsareconsid- eredanegativetestresult,and5to7spotsareconsidereda“borderline”result.Asfor alltests, theresultsshould beinterpretedinconjunctionwithresults ofotherdiag- nostic tests and epidemiological informationto help determine theM. tuberculosis
complexinfectionstatusofthepatient.
The2010CDCguidelinesstatethatFDA-approvedIGRAs,includingQFT-G,QFT-GIT, andT-Spot,canbeusedsimilarlytotheTSTforthediagnosisofM.tuberculosiscomplex infectioninchildrenandadults(250);thisincludespersonsatanincreasedriskforM. tuberculosiscomplexinfectionandthoseatanincreasedriskforprogressiontoactive TB, aslistedinTable 16.Individuals inthesetwo categories will likelybenefit from treatment for LTBI. IGRAs may also be used for surveillance purposes, in contact investigations,toscreenhealthcareworkersandothergroupsatriskforLTBI,andto identifypersonswhoarelikelytobenefitfromtreatment.Theguidelinesissuecaution in using IGRAsfor children ⬍5 years of age.Specificity isexpected to be high for children,butadditionalstudiesareneededtoevaluatetheperformanceofIGRAsfor thispopulation.
TheCDCguidelines alsostatethatlaboratoriesshouldreportboththequalitative testresultinterpretationandthequantitativeassayvaluesalongwiththecriteriaused forthetestresultinterpretation(250).Reportingthequantitativevalue isimportant, especiallywheninterpretingresultsfromserialtesting(e.g.,healthcareworkers),when IFN-␥ levels are close to the cutoff value separating positive and negative results. SeveralstudieshaveshownthatinindividualsseriallytestedwithQFT-IT, thosewith quantitativevaluesnearthecutoffof0.35IU/mlaremorelikelytohavevariabilityin theirqualitativeresultinterpretation,positiveornegative,thanindividualswithquan-
titativeresultsfartherfromthecutoffvalue(212–214).Providingthisinformationwill allowthehealthcareprovidertomakeabetterassessmentoftheIGRAresults.
There are both advantages and disadvantages to consider when implementing IGRAs.UnliketheTST,IGRAresultscanbeavailablewithin24h,ifperformedon-site, withouttheneedforasecondpatientvisit.InTBcontactinvestigations,asignificant numberofindividualsdonotreturntohavetheirskintestsread,andresultsarenot available to help public health professionals prioritize follow-up (266). In addition, errorsintheplacementandreadingofskintestscanresultinmisinterpretationofthe results andinappropriate patientmanagement. Disadvantagesof IGRAs includethe need todraw blood, stringent blood collection andmixing requirements,andtime limitationsforinitialspecimenprocessingorincubation.TheT-SpotXtendreagentcan beusedtoextendthetimefromcollectiontosampleprocessinginthelaboratoryto 30 h,as notedabove.However, this still maynot be enough timetotransport the specimentoadistantreferenceorpublichealthlaboratory.IGRAsarecomplexassays. ComparedtotheTST,whichrequires5stepstocomplete,QFT-GITrequiresatleast126 separatestepsforonetestresult(267).ThecostofIGRAsishigherthanthatoftheTST, butstudieshaveshownthatthelowerratesofpositivityofIGRAsandtheadvantage of a single patient encounter, which provides results for a higher percentage of patients,mayleadtooverallsavingstothehealthcaresystem(268).
IGRAshavethepotentialtoimprovethediagnosisandmanagementofTB;however, there are still outstanding issues concerning theperformance characteristics of the assays, interpretation of the results, and limitations of IGRAs that require further research. For example, although quantitative results are useful for evaluating the likelihoodofconversionsorreversionswhenvaluesareclosetotheassaycutoffvalue, thereisnoconsensusontheinterpretationandsignificanceofIGRAconversionsand reversions at this time. Quantitative IGRA results have not been shown to have prognosticvalue,anditisstillnotknownifIGRAsarebetterthantheTSTinpredicting progressiontoactiveTB.
SeveralfactorshavebeenshowntoimpacttheresultsofIGRAs(206–211,269–274). Preanalyticalfactorssuchastheamountoftimefrombloodcollectiontoincubation cansignificantlyaffectIGRAresults(269).Otherstudieshaveshownthatthetimeofday whenbloodisdrawncanalsoinfluencetheIGRAresult(270).AstudybyWhitworthet al.showedthatdifferentincubationtimeswithintheparametersoftheassayprotocols canaffectthequantitativeresultsandtheinterpretationoftheresultsof theassays (271). Another study showedvariability of results related to howthe QuantiFERON tubes areshaken and thequantity of blood ineach tube (275). It isimportant for laboratories to standardize and control the parameters of the assays to optimize reproducibility.
HealthcareworkersareatanincreasedriskofexposuretoM.tuberculosiscomplex bacteriaandhavetraditionallybeenscreenedforLTBIbyusingtheTST.Twoexcellent systematicreviewshavesummarizeddatafromthestudiesthathaveevaluatedtheuse ofIGRAsonhealthcareworkers(276,277).SomestudieshaveshownhighratesofIGRA conversionsthatarenotconsistentwiththelowratesofTBseenintheUnitedStates and the exposure risks of health care workers (278). In 2016, Gamsky et al. (279) reportedastudyontheuseofserialQuantiFERON-TBIGRAsonemergencyresponders in a low-TB-prevalence county in California from 2001 to 2013. Those researchers concludedthattheQuantiFERON-TBIGRAshouldnotbeusedforthediagnosisofLTBI inlow-riskpopulationsbecauseof frequentandirreproducible positiveresults.Most studies have also shownhighrates of reversions, which aremore likelyto be seen amongthosewithIFN-␥valuesorspotcountsnearthediagnosticcutoff(278).Some healthcarefacilitieshaveestablishedtheirowncutoffsorhaveimplementedretesting strategiestoeliminatefalse-positiveconversions(280,281).A2015studybyKingetal. thatincludedover19,000healthcareworkersfrom19U.S.hospitalsshowedpositivity andconversionratesconsistentwithknownTBriskfactorsusingtheT-Spot.TBassay (282).Suggestionsforthestandardizationofoccupationaltestingprogramshavebeen proposedtolimitthevariabilityintestresultsandinterpretations(283).
BecauseofthecomplexityofIGRAsandthemanyfactorsthatcanimpacttheresults oftheassays,itisespeciallyimportanttohaveastrongqualityassuranceprogramin place.Keyqualityassurancemonitorsshouldbeidentified,suchasthepercentageof positiveresults;thepercentageofindeterminateorinvalidresults;thedistributionof IGRAvalues,especiallythosenearthecutoffvalue;andcorrelationofpositiveresults withthepatients’riskfactorsforTBinfection.Gauretal.foundthatthebloodvolume andtubeshakingimpactIGRAresults(275).Inmanylaboratories,IGRAsareperformed intheserologydepartmentandnotintheTBlaboratory.Thepersonnelperformingthe testingmustestablishacloserelationshipwiththestateTBprogram,localpublichealth programs, infection control programs, employee health programs, and health care providerstodeterminethecorrelationofIGRAresultswithclinicalpresentation,chest radiographyresults,resultsofotherdiagnostictests,andepidemiologicalriskfactors forTBofthepatients.Itisimportantforthelaboratorytoeducatehealthcareproviders aboutthevariability andlimitationsof IGRAssothat theresultscan be interpreted appropriately.
USINGAREFERENCELABORATORY
Theneedfor,andavailabilityof,diagnosticmycobacteriologyserviceshasunder- gonesubstantialchangesintherecentpast.Thesechangesaredueto(i)theoverall decreasedincidenceandprevalenceofTBoverthedecades,withaconcurrentincrease intheisolationofNTM;(ii)thefactthatmycobacterialinfectionsvarygeographically andbypatientpopulations;(iii)thefactthatmanycommunityhospitalsandclinicssee fewornopatientswithNTMinfections;(iv)thecentralizationofclinicalservicesthat providecareforpatientswithmycobacterialinfections(e.g.,publichealthclinics);(v) theincreasingcomplexityandsophisticationoftestingrequiredtoprovidediagnostic mycobacteriologyservices;(vi)theneedtocloselylinkclinicalandlaboratoryservices aspartofintegratedsystemsforcaringforpatientswithmycobacterialinfections;and (vii)theongoingemphasisoncontrollingcostsinhealthcare.Notsurprisingly,many clinical laboratories have opted to discontinue offering a full range of diagnostic mycobacteriologyservices,insteadreferringpartsoralloftheseservicestoreference laboratories.
WhenToReferSpecimens/IsolatestoaReferenceLaboratory
Clinicalmicrobiologylaboratoriesarefacedwithdecisions astowhich laboratory services tooffer in-houseversuswhichonestosendtoareferencelaboratory (103). Factorsthatmustbeconsideredincludethescopeofclinicalservicesprovidedbythe hospitalorclinicsservedbyeachlaboratory,testvolumes,thetechnicalcapacityofthe laboratory,whetherthelaboratoryispartofalargerhealthcaresystemornetwork,and theavailabilityofreferencelaboratoryservices.Formosthospital-basedclinicalmicro- biologylaboratories,providingafullscopeofmycobacterialculturesandsusceptibility testingisneithernecessarynorappropriate.However,almostallhospital-basedlabo- ratoriesshouldofferAFBsmears.
Amorecontemporarydecisioniswhethertoofferrapiddiagnostictestingbymethods suchastheXpertassayoralineprobeassaysuchastheGenoTypeMTBDRplusassay.This isadecisionnotonlyforlaboratoriesthatperformbothAFBsmearsandcultures,where rapidassaysmustbeintegratedintoatraditionaltestingapproach,butalsoforlaboratories that mayneedaccess toarapid diagnostictest in urgentsituations. The Xpertassay recentlyreceivedanexpandedproductclaimthatallowsclinicianstoremovepatientswith suspectedTBfromAIIbasedonnegativetestresults(147).Becauseallrapidassaysaremore sensitivewhentestingAFBsmear-positivespecimens,theresultsofrapidmolecularassays mustbeinterpretedinconjunctionwiththeresultsofAFBsmears(evenwhenAFBsmears