Questions Characteristics

General Tuberculosis ® Information

T-SPOT.TB Test Description and Frequently Asked Performance

T-SPOT.TB Test Performance Questions Characteristics

T-SPOT.TB Advantages over Tuberculin Skin Test

T-SPOT.TB Testing through Oxford Diagnostics Laboratories®

T-SPOT.TB Test Results

T-SPOT.TB Methodology

Screening Control Programs

Contact Investigations

ReferencesReferences Table of Contents

General Tuberculosis Information

Tuberculosis: Definition, Infection and Disease 1. What is the scale of the TB problem? 2. How is TB spread? 3. What is TB infection (Latent Tuberculosis Infection “LTBI” or “latent TB”)? 4. What is TB disease (“active TB”)? 5. Are certain groups of individuals at an increased risk of exposure to Mycobacterium tuberculosis? 6. Are certain individuals at an increased risk of progressing from latent TB infection to TB disease? 7. How important is treatment for TB disease? 8. Why is the treatment period for TB disease so long? 9. How important is treatment for TB infection (“LTBI” or “latent TB”)?

TB Detection 10. Is there a test for the detection of TB infection (“LTBI” or “latent TB”)? • Tuberculin Skin Test (TST) • Interferon-Gamma Release Assays (IGRA) 11. Is there a test for the detection of TB disease (“active TB”)? 12. What are the limitations of the TST? Bacille Calmette-Guérin (BCG) Vaccination 13. What is the BCG vaccination?

T-SPOT.TB Test Description and Performance 14. What is the intended use of the T-SPOT.TB test? 15. Why does the T-SPOT.TB test measure interferon-gamma? 16. Does the T-SPOT.TB test differentiate between latent TB infection and TB disease? 17. What data is there to support the T-SPOT.TB test in clinical use? 18. How soon after exposure to Mycobacterium tuberculosis can an infection be detected with the T-SPOT.TB test?

T-SPOT.TB Test Performance Characteristics 19. What is the sensitivity and the specificity of the T-SPOT.TB test? 20. Why is sensitivity important in a test for Mycobacterium tuberculosis infection? 21. Why is specificity important in a test for Mycobacterium tuberculosis infection? 22. Can the T-SPOT.TB test be used in testing samples from patients with weakened immune systems? 23. Are any patient groups excluded from testing with the T-SPOT.TB test? 24. Is a positive T-SPOT.TB test result expected in patients with a previous history of tuberculosis? 25. Are infections with mycobacteria other than Mycobacterium tuberculosis expected to produce positive T-SPOT.TB test results? 26. Can the T-SPOT.TB test detect infections of drug-resistant tuberculosis strains such as MDR-TB, XDR-TB or TDR-TB? 27. What regulatory approvals does the T-SPOT.TB test have? 28. Is the T-SPOT.TB test affected by previous BCG vaccination?

T-SPOT.TB Advantages over Tuberculin Skin Test 29. What are the advantages of the T-SPOT.TB test? 30. What are the advantages of the T-SPOT.TB test over TST? 31. Can the T-SPOT.TB test be used in place of a TST?

T-SPOT.TB Testing through Oxford Diagnostic Laboratories Ordering and cost 32. Which requisition form should be used? 33. How much does the test cost? Blood Collection 34. How are T-SPOT.TB test samples collected? 35. How much blood is needed for the T-SPOT.TB test? 36. Which blood collection tubes can be used? 37. Can blood collection tubes containing EDTA (purple top tubes) be used? 38. Do you accept other types of specimens other than blood? 39. What information is required on the blood collection tube? 40. How should blood samples be stored prior to sending to the laboratory? 41. After collecting a blood sample, how long do I have to send it to the laboratory? 42. In which circumstances would the laboratory reject a blood sample? Specimen Packaging and Shipping 43. How should samples be packaged? 44. How can additional supplies be ordered (e.g. requisition forms)? 45. Where can samples be sent for processing using the T-SPOT.TB test? 46. How are samples shipped to the laboratory? 47. Can I ship samples using my own carrier? 48. How can I obtain the dangerous goods training needed to conform to IATA Packaging Instructions 650? 49. When can samples be sent to Oxford Diagnostic Laboratories? Laboratory Certification 50. Is Oxford Diagnostic Laboratories CLIA-certified? 51. What does College of American Pathologists (CAP) accreditation mean? 52. Is Oxford Diagnostic Laboratories CAP-accredited? 53. Is the T-SPOT.TB test covered by insurance?

T-SPOT.TB Test Results 54. How will I receive T-SPOT.TB test results? 55. How are T-SPOT.TB test results interpreted? 56. How are T-SPOT.TB test results reported? 57. What action should be taken if the T-SPOT.TB test is positive? 58. What is a T-SPOT.TB borderline result? 59. Are borderline T-SPOT.TB test results the same as invalid results? 60. What should I do with a borderline T-SPOT.TB test result? 61. What should I do with an invalid T-SPOT.TB test result? 62. What is the expected frequency of invalid results with the T-SPOT.TB test? 63. Why does the T-SPOT.TB test include a Positive Control with each patient sample? 64. Why does the T-SPOT.TB test include a Nil (Negative) Control with each patient sample? 65. How quickly are T-SPOT.TB test results available?

T-SPOT.TB Methodology 66. How do I prepare blood samples for the T-SPOT.TB test? 67. How is the T-SPOT.TB test performed? 68. Are all the materials required for the test provided in the kit? 69. How many tests (patient samples) can be processed with the T-SPOT.TB 8 kit? 70. What is the T-Cell Xtend® reagent and how is it used? 71. How long can blood samples be stored prior to processing with the T-Cell Xtend reagent? 72. Does the T-Cell Xtend reagent impact T-SPOT.TB test performance? 73. Is a standard curve required each time the T-SPOT.TB test is performed? 74. Which buffer should be used for washing the 96-well plate for the T-SPOT.TB test? 75. What is the purpose of the cell washing and counting steps in the T-SPOT.TB test? 76. Can finished assay plates be stored for future reading? 77. Is there a dedicated T-SPOT.TB product support service for laboratories?

Screening Control Programs 78. What is the impact of TB testing on healthcare resources?

Contact Investigations 79. In contact investigations, should a baseline T-SPOT.TB test be performed?

References General Information Tuberculosis Tuberculosis

Tuberculosis: Definition, Infection and Disease immune system. immune system. strains, HIV, andotherconditionsthatweaken the immigration, theemergenceofdrug-resistantTB to beasignificantdiseaseduefactorssuchas with than one-thirdoftheworld’s populationisinfected Health Organization(WHO)estimatesthatmore from aninfectiousdiseaseworldwide.TheWorld for over70years,TBistheleadingcauseofdeath Although effectivetreatmenthasbeenavailable problem the Tuberculosis (TB) What isthescaleof REFERENCE: 1 . TB continues Mycobacterium tuberculosis.TBcontinues ?

1 1

General Tuberculosis Information General Tuberculosis Information (TB) spread How isTuberculosis REFERENCE: 2 frequency anddurationofexposure. infectiousness oftheTBcontactandproximity, the immunestatusofexposedindividual, theprobabilityofinfectioninclude that determine inhale theseaerosolscanbecomeinfected.Factors sneeze, sing,speakorspit.Personswhothen complex organismsintotheairwhentheycough, aerosols containingMycobacteriumtuberculosis (lung)TBcanpropel Individuals withpulmonary TB ispassedfrompersontothroughtheair. 2 ?

2 “latent TB”) Infection “LTBI” or (Latent Tuberculosis What isTBinfection immune system. immune system. disease isincreasedinthosewithaweakened The riskofprogressingfromTBinfection toTB developing TBdiseaseatsomestagein theirlife. after infection,whiletheotherhalfareat riskof will developTBdiseasewithinthefirsttwo years lives. Approximatelyhalfoftheseindividuals will developTBdiseaseduringthecourseoftheir immunocompetent personswithlatentTBinfection diagnostic findings.Itisestimatedthat10%of of epidemiological,historical,medicaland the probabilityofinfectionrequiresacombination positive T-SPOT.TB testresult;however, assessing disease. TBinfectedindividualsusuallyhavea infectious anddonothavesymptomsofTB complex organismsintheirbodiesbutarenot Mycobacteriumtuberculosis TB”) harbordormant Individuals withTBinfection(“LTBI” or“latent 3 ?

General Tuberculosis Information General Tuberculosis Information diagnostic findings. diagnostic findings. consideration ofepidemiological,medicaland degree ofriskprogressionwhichisderivedfrom antibiotics. Urgencyoftreatmentisdictatedby diagnosed, latentTBinfectioncanbetreatedwith immunosuppressive therapy. Whenappropriately undergone anorgantransplant,orarereceiving to TBdisease,asarepatientswhohave are atagreatlyincreasedriskofprogressing For example,patientslivingwithHIV/AIDS REFERENCE: 2 3 4 General Information 4 Tuberculosis

What is TB disease (“active TB”)?

TB disease, or active TB, develops when the immune system cannot prevent Mycobacterium tuberculosis complex organisms from multiplying in the body. After exposure, persons can develop latent TB infection or TB disease. TB disease most commonly occurs in the lungs (pulmonary TB) but may occur in other body organs or spaces, particularly in immunocompromised patients or children, and may be localized or disseminated as occurs in miliary TB. Symptoms of pulmonary TB disease may include fever, cough, night sweats, weight loss, and fatigue. Without treatment, TB mortality rates are high. Individuals with TB disease usually have a positive T-SPOT.TB test result; however, assessing the probability of disease requires a combination of epidemiological, historical, medical and diagnostic findings. The definitive diagnosis of TB disease is made on the basis of isolation and identification of the TB mycobacterium in culture. Identification of the mycobacterium by other means, such as genetic tests of its presence, are increasingly being accepted as proof of infection. Persons with latent

5 General Tuberculosis Information epidemiological factors. factors. epidemiological age, concomitantillness,medicationandother TB disease,withthatriskbeingmodulatedby TB infectionarealsoatriskforprogressionto REFERENCE: 2, 3 6 General Information 5 Tuberculosis

Are certain groups of individuals at an increased risk of exposure to Mycobacterium tuberculosis?

Yes, certain groups are more likely to be exposed to Mycobacterium tuberculosis, which may lead to TB infection and/or disease. These include:

• known close contacts of a person with infectious TB disease, primarily pulmonary TB

• persons living in, immigrating from, or traveling to TB-endemic regions of the world

• persons who work or reside in facilities or settings with individuals who are at high risk for TB (e.g. hospitals, homeless shelters, correctional facilities, nursing homes, boarding schools, or residential facilities for persons living with HIV/AIDS)

REFERENCE: 2, 3 7 General Tuberculosis Information disease latent TBinfectionto of progressingfrom at anincreasedrisk individuals Are certain • • • • • • • • individuals,including: certain The riskofprogressiontoTBdiseaseishigherin Cigarette smokers head orneck Persons withleukemia orcancerofthelung, following transplantation TNF-α corticosteroids, immunosuppressive agentssuchassystemic Those undergoingmedicaltreatmentswith healed TB Persons withradiographicevidenceofprior Organ orhematologictransplantrecipients Persons livingwithHIV/AIDS Children undertheageoffive Persons withweakenedimmunesystems ? antagonists and therapies antagonistsandtherapies 6 8 • • • • • REFERENCE: 2, 3 Persons infectedwithTBwithinprior2years jejunoileal bypass gastrectomyor Persons whounderwent chronic renalfailure/hemodialysis Persons withsilicosis,diabetesmellitusor Persons withalowbodyweight Drug oralcoholabusers

General Tuberculosis Information General Tuberculosis Information disease treatment forTB is How important REFERENCE: 2, 3 resistant todrugs,makingitmoredifficulttreat. is notadequatelytreatedcanreactivateorbecome of treatmenteveniftheirsymptomsimprove.TBthat that individualswithTBcompletetheirentirecourse longer inthosewithdrug-resistantTB.Itiscrucial duration oftreatmentis6–9months,butmuch reducing transmissiontoothers.Ingeneral,the treatment includenotonlycuringthepatient,but Treatment forTBdiseaseisvital.Thegoalsof ? 7 10 disease toreactivateordevelopdrug-resistance. potentially allowingtuberculosis(TB) may survive, somebacteria treatment isinconsistentortooshort, ensure thatallofthebacteriaaredestroyed.If long; therefore,lengthytreatmentisrequiredto tuberculosis complexorganismsisrelatively replicating. ThereplicationcycleofMycobacterium can onlydosowhilethebacteriaareactively Most antibioticscapableofdestroyingbacteria so long period forTBdisease Why isthetreatment ? 8 11

General Tuberculosis Information General Tuberculosis Information “latent TB”) infection (“LTBI” or treatment forTB is How important REFERENCE: 4, 5 tuberculosis-infection/en/ Visit: www.who.int/tb/publications/latent- managing latentTBinfection. itsfirstguidelineson Organization (WHO)setforth eliminating TBdisease,theWorld Health and treatmentascriticalcomponentsofultimately In 2015,recognizinglatentTBinfectiontesting epidemiological, medicalanddiagnosticfindings. progression whichisderivedfromconsiderationof of treatmentisdictatedbydegreerisk the riskofTBdiseasebyupto90%.Urgency Completedtreatmentregimensreduce efforts. preventing TBdiseaseandoverallelimination Treatment forlatentTBinfectionisfundamentalin 9 ? 12 “latent TB”) infection (“LTBI” or the detectionofTB Is thereatestfor TB Detection • tests: broadly dividedintotuberculinskintestsandblood There areseveralmethodstodetectTBinfection, • • resultant indurationismeasured. derivative (PPD)intotheskin.In48-72hours, the injection ofasmallamountpurified protein for over100years,requiresanintradermal A TST, whichhasbeenusedtodetectTBinfection

Tuberculin SkinTest (TST) Release Assays(IGRAs) Interferon-Gamma 10 ? 13

General Tuberculosis Information • More recently, blood-based tests, referred to as General Information Tuberculosis Tuberculosis interferon-gamma release assays (IGRAs), have been introduced. The technology of the T-SPOT.TB test, an IGRA, is based on the release of interferon-gamma secreted by individual effector T cells (both CD4+ and CD8+) after being stimulated by TB-specific antigens.

REFERENCE: 6, 7 14 and latent TB infection. and latentTBinfection. does notdifferentiatebetweenactiveTB disease ELISA-based TBbloodtest,detectsinfection but evaluations. TheT-SPOT. TB test,likeaTSTandthe radiography andothermedicaldiagnostic disease patientswhenusedinconjunctionwith may beusedasadiagnosticaidinsuspectedTB antibiotics usedfortreatment.TheT-SPOT. TB test susceptibilityofthestraintoarange determine thediagnosisofTBandto categorically confirm sputum orothersitesarecollectedandculturedto culture, smearmicroscopy, PCR).Samplesfromthe thediagnosis(e.g.chestx-ray,confirm sputum TB diseasemayundergoanumberofteststo is criticaltoTBcontrol.Thosesuspectedofhaving Identification ofindividualswithactiveTBdisease (“active TB”) detection ofTBdisease Is thereatestforthe REFERENCE: 2 11 ? 15

General Tuberculosis Information General Information Tuberculosis Tuberculosis 12

What are the limitations of a tuberculin skin test (TST)?

Limitations of a TST, as well as factors which can influence test performance and accuracy, include:

• Noncompliance

o Requires a minimum of 2 visits

o Failure to return for TST interpretation

• Subjective Results

o Misreading of induration

o Poor inter-observer reproducibility

o Cutoff dependent on individual risk factors

• False-Positive Results

o Cross-reactivity with BCG vaccine

o Cross-reactivity with non-tuberculous mycobacteria (NTM)

o Errors in TST placement or reading

16 • • REFERENCE: 6, 7 Costly False-Negative Results o o o o o o o interpretation Staff mustbetrainedinTSTplacementand Solution waste Inaccurate results Errors inTSTplacementorreading Immunosuppression Immunocompromised status response) Anergy (inabilitytoproduceanimmune • • Biologics, corticosteroids HIV, cancer 17

General Tuberculosis Information General Information Tuberculosis Tuberculosis 13 BCG Vaccination

What is Bacille Calmette-Guérin (BCG) vaccination?

The BCG vaccine is used in many countries with a high prevalence of TB to prevent childhood tuberculous meningitis and miliary disease, but confers limited protective value in adults. The BCG vaccine is also used as an immunotherapeutic agent for individuals with bladder cancer. BCG- vaccinated individuals may produce a positive TST, even if they are not infected with Mycobacterium tuberculosis complex organisms. This is a common cause of TST inaccuracy.

REFERENCE: 8, 20 18 Description Test TB Test T-SPOT. and Performance

T-SPOT.TB Test Description and Performance T-SPOT.TB Test Description and Performance test use oftheT-SPOT.TB What istheintended REFERENCE: 8 evaluations. diagnostic assessment, radiographyandothermedical and isintendedforuseinconjunctionwithrisk M. tuberculosisinfection(includingdisease) The T-SPOT. TB testisanindirectfor infection. diagnosis ofM.tuberculosis heparin. Itisintendedforuseasanaidinthe collected insodiumcitrateorlithium in thevicinityofTcellshumanwholeblood ESAT-6 andCFP10bycapturinginterferon-gamma stimulation byMycobacteriumtuberculosisantigens for thedetectionofeffectorTcellsthatrespondto The T-SPOT. TB testisaninvitrodiagnostic ?

20 gamma? measure interferon- T-SPOT. TB test Why doesthe REFERENCE: 8, 10 individual TB-specificeffectorTcells. secretedby test directlycapturesinterferon-gamma antigens usedintheT-SPOT.TB test.TheT-SPOT.TB invitrowhenstimulatedbythe interferon-gamma circulating intheirperipheralblood,whichsecrete TB-infected patientshaveTB-specificeffectorTcells macrophages toingestanddestroymycobacteria. toactivate infection andsecreteinterferon-gamma CD4+ andCD8+),whichthenmigratetothesiteof and developintoTB-specificeffectorTcells(both T cellsbecomesensitizedtoTB-specificantigens Mycobacterium tuberculosis.Postinfection,naïve against intracellularpathogenssuchas iscrucialtoimmunedefense Interferon-gamma 15

T-SPOT.TB Test Description and Performance T-SPOT.TB Test Description and Performance disease infection andTB between latentTB test differentiate Does theT-SPOT.TB REFERENCE: 8, 10 not differentiatelatentTBinfectionfromdisease. ELISA-based TBbloodtest,theT-SPOT. TB testdoes No, likeatuberculinskintest(TST)andthe ? 16

22 • • • • • • • • including: wide rangeofclinicalandepidemiologicalsettings meta-analyses, havebeenpublishedcoveringa reviewed publications,includinganumberof populations continuestogrow. Hundredsofpeer- of TBinfection(latentandactive)invariouspatient oftheT-SPOT.performance TB testforthedetection The bodyofscientificevidencedemonstratingthe test inclinicaluse theT-SPOT.support TB What dataisthereto Renal patientsundergoing dialysis Malnourished individuals Children, adultsandtheelderly Healthcare workerscreening anti-TNF-α therapycandidates HIV infectedpatients TB) extra- pulmonary and TB disease(includingpulmonary Latent TBinfection(“LTBI”) 17 ?

T-SPOT.TB Test Description and Performance T-SPOT.TB Test Description and Performance • • • • • Transplant patients TB contacttracing High prevalencecountries Low prevalencecountries Patients withoncologicaldisorders 24 test with theT-SPOT.TB infection bedetected tuberculosis canan Mycobacterium exposure to How soonafter REFERENCE: 2, 6,9,11 weeks afterlastexposure. TST within8weeksofexposure,beretested 8-10 release assay(IGRA)or initial interferon-gamma of apersonwithTBdisease,whohave a negative Prevention (CDC)itisrecommendedthat contacts bytheCentersforDiseaseControland set forth According tothecorecurriculumontuberculosis (typically 2-8weeks). no laterthantuberculinskintest(TST)conversion is notyetwelldefined,butexpectedtooccur forconversionfollowingexposure The timeinterval ? 18

T-SPOT.TB Test Description and Performance Performance Test TB Test T-SPOT. Characteristics

T-SPOT.TB Test Performance Characteristics 19

What is the sensitivity and the specificity of the T-SPOT.TB test ? Performance Test TB Test T-SPOT. Characteristics

The sensitivity of the T-SPOT.TB test is 95.6% and the specificity of the T-SPOT.TB test is 97.1%.

REFERENCE: 8 27 20

Why is sensitivity important in a test for Mycobacterium Performance Test TB Test T-SPOT. Characteristics tuberculosis infection?

Sensitivity (the ability to detect TB-infected individuals) is one of the key measures of diagnostic performance. A test with high sensitivity, such as the T-SPOT.TB test, will have few false- negative results. This is especially important in patients that carry a greater risk of progression from latent TB infection to TB disease, such as those with weakened immune systems.

REFERENCE: 8 28 21

Why is specificity important in a test for Mycobacterium Performance Test TB Test T-SPOT. Characteristics tuberculosis infection?

In addition to sensitivity, specificity (the ability to identify non-infected individuals) is another key measure of diagnostic performance. A test with high specificity, such as the T-SPOT.TB test, will have few false-positive results, minimizing unnecessary follow-up diagnostic or therapeutic procedures.

REFERENCE: 8 29 22

Can the T-SPOT.TB test be used in testing samples from patients Performance Test TB Test T-SPOT. Characteristics with weakened immune systems?

Yes, the T-SPOT.TB test is well-suited for use in patients with weakened immune systems. Each sample undergoes a cell count which is used to create a normalized (standardized and known number) suspension of cells that are subsequently incubated with TB-specific antigens. Immunocompromised patients may have a reduced number of peripheral blood mononuclear cells (PBMCs) - the types of white blood cells used in the T-SPOT.TB test. In these patients, multiple blood tubes can be pooled to obtain the required number of cells to perform the test.

30 Pivotal clinical study data submitted to the US Food and Drug Administration (FDA) for pre-market approval extensively evaluated the T-SPOT.TB test in immunocompromised individuals including, but not limited to, subjects with HIV, silicosis, diabetes, end-stage renal disease and organ transplant. A negative tuberculin skin test was associated with being immunocompromised; in contrast, no Performance Test TB Test T-SPOT. association was observed between T-SPOT.TB test Characteristics result and immunocompromised status.

REFERENCE: 8 31 23

Are any patient groups excluded from testing with the Performance Test TB Test T-SPOT. Characteristics T-SPOT.TB test?

According to the ATS/IDSA/CDC 2016 guidelines, the T-SPOT.TB test is acceptable for all patient groups and preferred in situations where a patient is likely to be infected with a low to intermediate risk of progression. For those patients greater than 5 years of age who have a history of BCG vaccination or are unlikely to return to have their TST read, the IGRA is also preferred. A TST is preferable in healthy children under the age of 5, for whom testing is warranted, due to the limited data available in this age group. The American Academy of Pediatrics (AAP) also prefers a TST be used in patients under 5 years of age but acknowledge that some experts will use an IGRA in children 2 to 4 years of age, especially if they have received a BCG vaccine.

REFERENCE: 8, 9, 21, 35 32 of tuberculosis with aprevioushistory expected inpatients T-SPOT. TB testresult Is apositive REFERENCE: 12, 13,14,34 the patientremains positive aftertreatment. and medicalexaminationscouldbeconsidered if Depending onrequirements,otherdiagnostic tests studyisrequired. bacteriabutfurther of dormant in theT-SPOT. TB test,maysuggestthepresence T cells,thecellsstimulatedbyTB-specific antigens or bloodtestpositive.Thepersistenceof effector ofindividualsremainskin that alargeproportion phase (latentTBinfection).Somedatahaveshown tothequiescent sterilizing cureandthereturn clinicalcurereferstobotha changes. Theterm culture, improvementofsymptomsandchestx-ray Clinical cureisdescribedbynegativesputum that individualstestnegativeafterTBtreatment. question. Studiesdonotconsistentlydemonstrate Unfortunately, thereisnoclearanswertothis 24

? 33

T-SPOT.TB Test Performance Characteristics 25

Are infections with mycobacteria other than Mycobacterium Performance Test TB Test T-SPOT. Characteristics tuberculosis expected to produce positive T-SPOT.TB test results?

Mycobacterium tuberculosis is the causative agent of most cases of tuberculosis and thus, T-SPOT.TB test sensitivity was determined from subjects with active, culture-confirmed Mycobacterium tuberculosis infection. Individuals infected with other Mycobacterium tuberculosis complex organisms (such as M. bovis, M. africanum, M. microti, M. canetti) usually have T cells in their blood which recognize the antigens, ESAT-6 and CFP10, used in the T-SPOT.TB test and are also anticipated to produce positive T-SPOT.TB test results. ESAT-6 and CFP10 antigens are absent from most non-tuberculous mycobacteria (NTM) with the exception of M. marinum, M. szulgai

34 and M. kansasii. While it is unclear if ESAT-6 and CFP10 are present in the genome of all subspecies of M. gordonae, it is possible that this NTM may also produce a positive result. All other non- tuberculous mycobacteria, including M. avium, are not expected to cross-react with the antigens used in the T-SPOT.TB test. Performance Test TB Test T-SPOT. Characteristics

REFERENCE: 8 35 26

Can the T-SPOT.TB test detect infections of drug-resistant Performance Test TB Test T-SPOT. Characteristics tuberculosis strains such as MDR-TB, XDR-TB or TDR-TB?

Yes, the T-SPOT.TB test can detect all strains of Mycobacterium tuberculosis complex organisms, including multi-drug-resistant tuberculosis (MDR-TB), extensively-drug-resistant tuberculosis (XDR-TB) and totally-drug-resistant tuberculosis (TDR-TB). The antigens in the T-SPOT.TB test are common to all Mycobacterium tuberculosis strains. However, the test does not predict whether the organism is sensitive to usual drug treatments. Drug susceptibility testing occurs after isolation and identification of the organism by other methods.

36 27

What regulatory approvals does the T-SPOT.TB test have ? Performance Test TB Test T-SPOT. Characteristics

The T-SPOT.TB test has carried the CE mark, which allows it to be marketed in the EU, since 2004. Additionally, the T-SPOT.TB test received US Food and Drug Administration (FDA) premarket approval in 2008, was approved in China in 2010 and in Japan in 2012. Approvals have also been obtained in many other countries, including, but not limited to, Canada, Taiwan, Russia, Singapore, Thailand, Peru, Nigeria and Mexico.

37 28

Is the T-SPOT.TB test affected by previous BCG vaccination Performance ? Test TB Test T-SPOT. Characteristics

Unlike a tuberculin skin test, there is no association between BCG vaccination and T-SPOT.TB test results. The BCG vaccine is an attenuated derivative of virulent Mycobacterium bovis, the bovine or animal form of the TB mycobacterium. The T-SPOT.TB test utilizes antigens (ESAT-6 and CFP10) that are located on a genomic region designated as RD1, region of differentiation 1. The RD1 region is present in all virulent M. bovis strains but is deleted from all BCG strains. Because the antigens used in the T-SPOT.TB test are not present in the BCG vaccine, the T-SPOT.TB test does not produce a false-positive result due to BCG vaccination. It should be noted, however, that patients infected with virulent M. bovis are likely to produce a positive T-SPOT.TB result.

REFERENCE: 8, 22 38 TB T-SPOT.

T-SPOT.TB Advantages over Tuberculin Skin Test Tuberculin Advantages Over Tuberculin Skin Test (TST) 29

What are the advantages of the T-SPOT.TB test?

The T-SPOT.TB test is the only TB test with sensitivity

TB T-SPOT. and specificity exceeding 95% in pivotal clinical Advantages over Tuberculin Skin Test Tuberculin studies. The T-SPOT.TB test is reliable even in challenging testing populations, including BCG- vaccinated and immunocompromised persons, and relies on routine phlebotomy procedures.

REFERENCE: 8 40 30 What are the advantages of the T-SPOT.TB test over a tuberculin skin test

(TST)? TB T-SPOT. Advantages over Tuberculin Skin Test Tuberculin

The T-SPOT.TB test has a number of advantages over a TST, including:

• Single visit to complete test (versus 2 - 4 with TST)

• Improved sensitivity (reliable in patients with weakened immune systems)

• Improved specificity (not affected by BCG vaccine and most non-tuberculous mycobacteria)

41 31 Can the T-SPOT.TB test be used in place of a tuberculin skin test (TST)? TB T-SPOT. Advantages over

Tuberculin Skin Test Tuberculin According to the ATS/CDC/IDSA 2016 guidelines, an interferon-gamma release assay (IGRA), such as the T-SPOT.TB test, is acceptable in all LTBI testing situations. An IGRA is preferred in situations where the patient is likely to be infected and has a low to intermediate risk of progression to disease, as well as in situations where the patient is unlikely to be infected but LTBI testing is required. IGRAs are also preferred in patients that are at least 5 years of age that are BCG-vaccinated or unlikely to return for their TST reading. Please refer to the full guidelines for complete information.

REFERENCE: 8, 9, 35 42 T-SPOT.TB Testing through TB T-SPOT. Laboratories Testing through Testing

Oxford Diagnostic Oxford Diagnostics Laboratories T-SPOT.TB Testing through Oxford Diagnostics Laboratories form should beused? form Which requisition Ordering andcost Form shouldbeused. Form The OxfordDiagnosticLaboratoriesTest Requisition 32 44 information. TeamClient Support at1-877-598-2522forpricing Please contacttheOxfordDiagnosticLaboratories test cost? How muchdoesthe 33 45

T-SPOT.TB Testing through Oxford Diagnostics Laboratories T-SPOT.TB Testing through Oxford Diagnostics Laboratories REFERENCE: 2, 8 samples collected? T-SPOT. TB test How are Blood Collection blood collectiontubesusingroutinephlebotomy. T-SPOT. TB testsamplescanbedrawnintostandard

34 46 35 How much blood is needed for the T-SPOT.TB test?

Typically, in immunocompetent patients, sufficient peripheral blood mononuclear cells (PBMCs) to perform the T-SPOT.TB test can be obtained with the following age-dependent guidelines:

• Adults and children ≥ 10 years of age: 6 mL TB T-SPOT. Laboratories Testing through Testing • Children ≥2 to <10 years of age: 4 mL Oxford Diagnostics

• Children <2 years of age: 2 mL

REFERENCE: 8 47 T-SPOT.TB Testing through Oxford Diagnostics Laboratories REFERENCE: 8 can beused? collection tubes Which blood and lithiumheparin). accepts standardgreentoptubes(sodiumheparin Oxford DiagnosticLaboratoriesinMemphis,TN 36

48 37 Can blood collection tubes containing EDTA (purple top tubes) be used?

No, EDTA (ethylenediaminetetraacetic acid) affects cells’ secretion of interferon-gamma due to its chelating (calcium binding) properties. Blood TB T-SPOT. collection tubes that contain this anti-coagulant Laboratories Testing through Testing therefore cannot be used. Oxford Diagnostics

REFERENCE: 8 49 T-SPOT.TB Testing through Oxford Diagnostics Laboratories other thanblood? types ofspecimens Do youacceptother or sodiumheparintubesareaccepted. No, onlywholebloodsamplescollectedinlithium 38 50 39 What information is required on the blood collection tube?

All blood collection tubes must be labeled with the following information:

• Patient’s first and last name

• Date of birth or unique patient identifier TB T-SPOT. Laboratories • Date and time of sample collection through Testing Oxford Diagnostics

51 T-SPOT.TB Testing through Oxford Diagnostics Laboratories ? the laboratory prior tosending samples bestored How shouldblood transport andshouldnotbecentrifuged. transport between 64-77°F(18-25°C),untilpackagedfor Blood samplesmustbestoredatroomtemperature, 40 52 41 After collecting a blood sample, how long do I have to send it to the laboratory?

Blood samples must be shipped the same day they

are collected, unless prior arrangements have been TB T-SPOT. Laboratories Testing through Testing made. Oxford Diagnostics

53 T-SPOT.TB Testing through Oxford Diagnostics Laboratories reject abloodsample would thelaboratory In whichcircumstances • • • • • • • • below: why abloodsamplewouldberejectedarelisted Examplesofreasons outlined onthetestreport. If abloodsampleisrejected,thereasonwillbe Diagnostic Laboratories shippingcontainer Blood specimensare notreceivedinanOxford Blood collectiontubeisunder-filled Blood collectiontubeisleakingorbroken heparin orsodium Specimen iscollectedintubesotherthan lithium frozen Specimen isreceivedclotted,refrigeratedor to shipping Blood collectiontubehasbeencentrifugedprior Blood collectiontubeisnotproperlylabeled sample collection Specimens arenotshippedonthesamedayas 42 ? 54 43 Specimen Packaging and Shipping

How should samples be packaged?

Please visit www.oxforddiagnosticlabs.com/client- TB T-SPOT. Laboratories Testing through Testing support/send-specimens/how-to-videos for an Oxford Diagnostics instructional video on packaging samples.

55 T-SPOT.TB Testing through Oxford Diagnostics Laboratories forms) (e.g. requisition supplies beordered How canadditional support/order-supplies Visit: www.oxforddiagnosticlabs.com/client- tabontheCompanywebsite. the ClientSupport found inthe“OrderSupplies”sectionlocated at 1-877-598-2522orcompletethereorderform TeamDiagnostic LaboratoriesClientSupport To ordersupplies,contacttheOxford ? 44

56 45

Where can samples be sent for processing using the T-SPOT.TB test?

Please contact the Oxford Diagnostic Laboratories Client Support Team at 1-877-598-2522. TB T-SPOT. Laboratories Testing through Testing Oxford Diagnostics

57 T-SPOT.TB Testing through Oxford Diagnostics Laboratories laboratory shipped tothe How aresamples samples(s) duringtransit. provides temperaturestabilityfortheblood phasechangematerial cost. State-of-the-art containers andpackingmaterialsatnoadditional Oxford DiagnosticLaboratoriesprovidesshipping 46 ? 58 47

Can I ship samples using my own carrier?

Oxford Diagnostic Laboratories has established an agreement with FedEx® services. If you have questions concerning this carrier, please contact the Client Support team at 1-877-598-2522.

FedEx is a registered trademark of FedEx Corporation. TB T-SPOT. Laboratories Testing through Testing Oxford Diagnostics

59 T-SPOT.TB Testing through Oxford Diagnostics Laboratories 650 Packaging Instructions toIATAconform training neededto dangerous goods How canIobtain dangerous good training. dangerous goodtraining. education/online/dangerousgoods tocomplete Visit www.mayomedicallaboratories.com/ ? 48 60 49

When can samples be sent to Oxford Diagnostics Laboratories?

Excluding certain holidays, samples are accepted Tuesday through Saturday, meaning samples can be drawn Monday through Friday and shipped TB T-SPOT. overnight to our laboratory for next-day processing. Laboratories Testing through Testing Oxford Diagnostics

61 T-SPOT.TB Testing through Oxford Diagnostics Laboratories certified Laboratories CLIA- Is OxfordDiagnostic Certification Laboratory credentials Visit www.oxforddiagnosticlabs.com/the-lab/ (CLIAIDNumber:44D2035207). CLIA-certified Yes, OxfordDiagnosticLaboratoriesis ? 50

62 51

What does College of American Pathologists (CAP) accreditation mean?

CAP’s Laboratory Accreditation Program (LAP) includes the most comprehensive and scientifically endorsed laboratory standards. CAP accreditation TB T-SPOT. means that a laboratory has met or exceeded these Laboratories Testing through Testing standards through a peer-based inspection model. Oxford Diagnostics

63 T-SPOT.TB Testing through Oxford Diagnostics Laboratories accredited Laboratories CAP- Is OxfordDiagnostic credentials Visit www.oxforddiagnosticlabs.com/the-lab/ CAP-accredited (LAPNumber:8044548). Yes, OxfordDiagnosticLaboratoriesis 52 ?

64 53

Is the T-SPOT.TB test covered by insurance?

Yes, the T-SPOT.TB test is covered by the vast majority of commercial and government insurers and is described by a unique CPT® code: 86481, as “Tuberculosis test, cell mediated immunity antigen response measurement; enumeration of gamma interferon-producing T-cells in cell suspension.” TB T-SPOT. Laboratories Testing through Testing

Oxford Immunotec does not make any Oxford Diagnostics recommendations or representation of use for any particular reimbursement code for any given test/procedure.

CPT is a registered trademark of the American Medical Association.

65 T-SPOT.TB Test Results TB T-SPOT. Test Results Test soon asresultsarereleased. haveaccesstotestresultsas SNAP™ ClientPortal Customer Agreement.Authorizedusersofthe as directedintheOxfordDiagnosticLaboratories Test totheorderingprovideror resultsarereported T-SPOT.TB testresults receive I will How 54 ? 66

T-SPOT.TB Test Results 55

How are T-SPOT.TB test results interpreted?

T-SPOT.TB test results are qualitative and are reported as positive, borderline (equivocal) or negative, given that the test controls perform as expected. Test results are determined by firstly enumerating the spots (captured interferon-gamma from individual T cells) in each of the patient's four test wells (Positive Control, Nil Control, Panel A, Panel B). Spots can be counted from the test wells using a magnifying glass, stereomicroscope, or a digital image captured from a microscope. TB T-SPOT. Test Results Test Qualitative results are interpreted by subtracting the spot count in the Nil (Negative) Control from the spot count in Panels A and B. Detailed information regarding test result interpretation can be found in the T-SPOT.TB package insert.

REFERENCE: 8, 33 67 56

How are T-SPOT.TB test results reported?

T-SPOT.TB test results are reported as positive, negative, borderline, or invalid. TB T-SPOT. Test Results Test

REFERENCE: 8 68 57

What action should be taken if the T-SPOT.TB test is positive?

Patients testing positive with the T-SPOT.TB test likely have TB infection and should be clinically evaluated for active TB disease. Risk of one or the other can be assessed on the basis of a combination of epidemiological, historical, medical and diagnostic findings. A definitive diagnosis of active TB disease is made on isolation and identification of the mycobacterium from the patient. TB T-SPOT. Test Results Test

REFERENCE: 8 69 58

What is a T-SPOT.TB borderline result?

The vast majority of T-SPOT.TB test results are either positive or negative. A small percentage of test results can be borderline (equivocal), where the higher of (Panel A minus Nil Control) and (Panel B minus Nil Control) is 5, 6 or 7 spots. The borderline category is intended to reduce the likelihood of false-positive or false-negative results around the cutoff point of the T-SPOT.TB test. As opposed to an indeterminate or invalid result, a borderline result is clinically interpretable and should be followed by retesting TB T-SPOT. as a substantial proportion of individuals may test Results Test positive upon retesting. In a study of US healthcare workers, 23% of subjects with a borderline test result retested as positive, suggesting the borderline category is useful in maintaining test sensitivity. According to the CDC's Updated Guidelines for Using Interferon Gamma Release Assays to Detect Mycobacterium tuberculosis Infection published in 2010, "incorporation of a borderline category for the [T-SPOT.TB test] as approved by FDA increases test accuracy by classifying results near the cut point (at which small variations might affect the interpretation) as neither positive or negative.”

REFERENCE: 8, 9, 15, 36 70 59

Are borderline T-SPOT.TB test results the same as invalid results?

No, borderline results are clinically interpretable whereas invalid results cannot be interpreted due to the failure of the test’s Positive and/or Nil (Negative) Control. In both cases, however, retesting by collecting another blood sample is recommended. TB T-SPOT. Test Results Test

REFERENCE: 8, 9, 15 71 60

What should I do with a borderline T-SPOT.TB test result?

Borderline results are clinically interpretable and should be followed. Retesting by collecting another sample is recommended. In a study of US healthcare workers, 23% of subjects with a borderline test result retested as positive, suggesting the borderline category is useful in maintaining test sensitivity. Upon retesting, if the test result remains borderline, other diagnostic tests and/or epidemiologic information should be used to help TB T-SPOT. Test Results Test determine the TB infection status of the patient.

REFERENCE: 8, 15 72 61

What should I do with an invalid T-SPOT.TB test result?

Invalid results are not clinically interpretable and may occur if the Positive and/or Nil (Negative) Control does not perform as expected. Retesting by collecting another sample is recommended. Upon retesting, if the test result remains invalid, other diagnostic tests and/or epidemiologic information should be used to help determine the TB infection status of the patient. Invalid results are uncommon and may be related to factors such as TB T-SPOT. Test Results Test inappropriate blood storage conditions, delay in sample transport, patient specific conditions, or laboratory error.

REFERENCE: 8, 15 73 62

What is the expected frequency of invalid results with the T-SPOT.TB test?

Invalid results are infrequent with the T-SPOT.TB test. Oxford Diagnostic Laboratories maintains a low overall invalid rate and does not charge for invalid T-SPOT.TB test results. Contact technical support for additional information (1-877-598-2522). TB T-SPOT. Test Results Test

74 63

Why does the T-SPOT.TB test include a Positive Control with each patient sample?

The Positive Control serves as an indicator of patient cell functionality. Patient cells are incubated with a non-specific stimulator, phytohemagglutinin, in the Positive Control sample well. The Positive Control spot count is ≥ 20 in the vast majority of patient samples. A failed Positive Control is uncommon but may be related to factors such as TB T-SPOT. Test Results Test inappropriate blood storage conditions, delay in sample transport, technical factors or patient specific conditions. A small proportion of patients may have T cells which show only a limited response to phytohemagglutinin.

REFERENCE: 8, 16 75 64

Why does the T-SPOT.TB test include a Nil (Negative) Control with each patient sample?

The Nil (Negative) Control is designed to control for non-specific T cell reactivity. Patient cells are incubated with sterile media in the Nil Control well. For the test to be considered valid, there must be ≤ 10 spots in the Nil Control well. A failed TB T-SPOT. Nil Control is uncommon but may be related to Results Test technical factors or a patient specific condition.

REFERENCE: 8, 16 76 65

How quickly are T-SPOT.TB test results available?

Laboratory processing of the T-SPOT.TB test can be completed in approximately 24 hours. Oxford Diagnostic Laboratories in Memphis, TN reports T-SPOT.TB test results within 36 - 48 hours of receiving the blood sample. TB T-SPOT. Test Results Test

REFERENCE: 8, 17 77 T-SPOT.TB Methodology TB T-SPOT. Methodology T-SPOT.TB Methodology REFERENCE: 8 T-SPOT.TB test blood samplesforthe How doIprepare (1-877-598-2522). orbycontactingtechnicalsupport package insert sample preparationcanbefoundintheT-SPOT.TB on the bloodcellfractions.Detailedinformation techniques andequipmenttoseparate laboratory Blood samplesareprocessedusingstandard 66 ? 75

enumerated to determine thepatient’senumerated todetermine result. the plateiswashedandnumbersof spotsare washed andasolublesubstrateisadded. Finally, a conjugatedantibodyisadded.Theplatethen (ESAT-6 incubation, andCFP10).Afterovernight two to aPositiveControl,Nil(Negative)Controland to fourmicrotiterwells,wheretheyareexposed them. AstandardnumberofPBMCsareadded mononuclear cells(PBMCs),washesandcounts separatestheperipheralblood The laboratory from thepatientandsenttolaboratory. method. Awholebloodsampleiscollected ELISA (enzyme-linkedimmunosorbentassay) which hassomesimilaritiestoaconventional ELISPOT (enzyme-linkedimmunospot)method, The T-SPOT. TB testisasimplifiedandvalidated test performed How istheT-SPOT.TB REFERENCE: 8 specific antigens Mycobacterium tuberculosisspecificantigens 67 ?

T-SPOT.TB Methodology T-SPOT.TB Methodology REFERENCE: 8 provided inthekit required forthetest Are allthematerials the T-SPOT.TB packageinsert. list ofadditionalrequiredmaterialscanbefoundin for incubationwiththe2suppliedTBantigens.A and Nil(Negative)Controlwell2otherwells designed toprovide4wellsperpatient,aPositive required tosuccessfullyruntheassay. Thekitis The T-SPOT. TB testkitcontainsthecorematerials 68 ? 77 T-SPOT.TB 8kit be processedwiththe (patient samples)can How manytests REFERENCE: 8 per batch,withoutwastingstrips. processing between2and24patientsamples (4 wellseach),providingtheflexibilityof wells. Eachstripprocesses2patientsamples The T-SPOT. TB 8kitcontainstwelvestripsofeight Each kitprocesses24patientsamples. 69 ?

T-SPOT.TB Methodology T-SPOT.TB Methodology REFERENCE: 8, 29,30 how isitused Xtend reagentand What istheT-Cell contained withintheT-SPOT. TB packageinsert. theapprovalis FDA in2010.Datasupporting The T-Cell Xtendreagentwasapprovedbythe collection. extends bloodstabilityto32hoursaftersample activated granulocytes,theT-Cell Xtendreagent production.Byremoving vitro interferon-gamma due toactivatedgranulocyteswhich,suppressin show reducedresponsestoantigenstimulation from wholebloodstoredover8hoursappearto mononuclear cells(PBMCs).Tseparated density gradientseparationofperipheralblood the T-Cell Xtendreagenttowholebloodprior removed duringcentrifugation.Labpersonneladd with redbloodcells,creatingacomplexwhichis monoclonal antibodythatcross-linksgranulocytes The T-Cell Xtendreagentcontainsabispecific 70 ? 79 reagent with theT-Cell Xtend prior toprocessing samples bestored How longcanblood REFERENCE: 8 regarding samplestability. T-SPOT. fordetailedinformation TB packageinsert hours ofsamplecollection.Pleaserefertothe Blood samplesshouldbeprocessedwithin32 ? 71

T-SPOT.TB Methodology T-SPOT.TB Methodology REFERENCE: 18, 29,30,32 performance T-SPOT. TB test reagent impact Does theT-Cell Xtend and foundhighconcordancesensitivity. with andwithouttheuseofT-Cell Xtend reagent studies haveevaluatedT-SPOT. TB testperformance validationprotocolsandpeer-reviewedInternal 72 ?

81 performed the T-SPOT. TB testis required eachtime Is astandardcurve REFERENCE: 33 standard curve. inplasma,relativetoa such asinterferon-gamma thetotalconcentrationofcytokines, determine immunosorbent assays,orELISA-basedtests, the single-celllevel.Incontrast,enzyme-linked secreted bycellsastheyarebeingreleased,at capture cytokines,suchasinterferon-gamma, the T-SPOT.TB test.ELISPOT-based testsdirectly linked immunospot(ELISPOT)-basedtests,suchas arenotapplicabletoenzyme- No. Standardcurves 73 ? 82

T-SPOT.TB Methodology T-SPOT.TB Methodology REFERENCE: 8 the T-SPOT.TB test the 96-wellplatefor be usedforwashing should Which buffer be used. Sterile phosphatebufferedsaline(PBS)should 74 ?

83 T-SPOT.TB test counting stepsinthe the cellwashingand What isthepurposeof REFERENCE: 23, 24 values. substance orhaveaPBMCcountoutside ofnormal and maybeprescribedapotentiallyinterfering progressing fromlatentTBinfectionto disease, immunocompromised patientshaveahigher riskof immune systems.Immunosuppressedand inpatientswithweakened importance particular test. Thewashingandcountingstepsmaybeof ensure astandardnumberofcellsareusedinthe correct forvariationsinpatientPBMCcountsand allow foranadjustmentincellconcentrationto blood mononuclearcells(PBMCs)arecountedto drugs. Afterwashing,theperipheral inflammatory tricyclic antidepressants,andnonsteroidalanti- such asendogenousinterferon-gamma, substances, plasma andpotentiallyinterfering The cellwashingstepenablesremovalof 75 ? 84

T-SPOT.TB Methodology T-SPOT.TB Methodology REFERENCE: 8 future reading plates bestoredfor Can finishedassay temperature environment. they areproperlystoredinadry, dark,room re-examination, forupto12months,aslong be archivedforretrospectivequalitycontrolor read immediately. Completedtestplatesmay test platesremainstableanddonotneedtobe Yes. Oncedeveloped,thespotsincompleted 76 ? 85 laboratories for service support T-SPOT. TB product dedicated a there Is REFERENCE: 8 the T-SPOT.TB test. customers andlaboratoriesinterestedinoffering toexistingcustomersaswellnew offers support Team,Support availableat1-877-598-2522, and troubleshootingassistance.OurProduct customized training,productevaluationsupport tocustomers,whichincludes technical support Oxford Immunotecprovidescomprehensive 77 ? 86

T-SPOT.TB Methodology Screening Control Programs Programs Screening Control transmission). higher costsoftreating disease(includingonward toTBdiseaseleading tomorbidityand convert stock. False-negativeTSTs may andnon-returners time periodleadingtopotentialwasteof unused a TST, onceopened,hastobeused withinashort compounded bythefactthatsolution usedin and readingaTSTarerelativelyhigh.These are and laborcostsassociatedwithadministering when containinganoutbreak.Indirectresource wasted resourcesandpotentiallydangerousgaps tohavetheirtestread.Thismayresultin not return two officevisits.Uptoone-thirdofindividualsdo TST requiresthepersonbeingtestedhaveatleast care systemsandpatients. with useofatuberculinskintest(TST)tobothhealth elimination ofsignificantindirectcostsassociated overall costofTBcontrol.Thisislargelyduetothe T-SPOT. TB testincontrolprogramswillreducethe Studies haveshownthattheincorporationof healthcare resources of TBtestingon What istheimpact 78 ? 88

Screening Control Programs False-positive TST results, often due to cross- reactivity with BCG vaccine or environmental non- tuberculous mycobacteria can lead to unnecessary anti-TB treatment and associated toxicity testing and clinical follow-up.

REFERENCE: 23, 28 89 Contact Investigations Contact Investigations 79

In contact investigations, should a baseline T-SPOT.TB test be performed?

Testing too soon after exposure may lead to a false-negative test result, as the individual's cellular immune response to Mycobacterium tuberculosis may not yet be detectable. However, a baseline test may be useful in determining whether a person had a pre-existing infection prior to exposure. Such a baseline test should be performed as soon as possible following exposure since conversion typically occurs 2 – 8 weeks post exposure.

According to the CDC's Core Curriculum on Tuberculosis: What the Clinician Should Know, contacts of a person with TB disease who have a negative initial interferon-gamma release assay (IGRA) or TST within 8 weeks of exposure be retested 8 – 10 weeks after last exposure. Contact Investigations

REFERENCE: 2, 6, 9, 11 91 References References References Wrighton-Smith 7. Huebner 6. 5. 4. 3. 2. Dye 1. References 12. 11. Cavalcanti 10. Mazurek 9. Oxford 8. 1993; 17:968-975. (DC); 2006. Disease ControlPrioritiesinDevelopingCountries,2nded.Washington Alleyne G,ClaesonM,EvansDB,JhaP, MillsA,MusgroveP, editors. Vaccine Immunol 2010;17:1985-1990. tuberculosis inMycobacteriumbovis BCG-vaccinatedpopulations.Clin Mycobacterium tuberculosisantigensfordiagnosisoflatent andactive Weng X,ZhangW. releaseassaysusing Evaluationofgamma interferon Zhang S,ShaoL,MoChenJ,Wang F, MengC,ZhongM,QiuL,Wu M, 1241. releaseassayresults.ClinVaccineinterferon Immunol 2007;14:1239- with negativetuberculinskintestresultsinassociationpositive gamma customers Arend SM.Follow-upstudyoftuberculosis-exposedsupermarket Franken WP, KosterBF, BossinkAW, ThijsenSF, BouwmanJJ,vanDisselJT, Tuberculosis. PulmMed2012;2012:745483. TNF-Alpha, IFN-Gamma,andIL-10intheDevelopmentofPulmonary infection -UnitedStates,2010.MMWRRecommRep2010;59:1-25. ReleaseAssaystodetectMycobacteriumtuberculosis Interferon-Gamma IE, CentersforDiseaseC,Prevention.Updatedguidelinesusing UK. 2013. J OccupEnvironMed2012;54:806-815. skin test:impactoncostsandadherencetotesting(theSWITCHstudy). releaseassayversustuberculin health careworkerswithinterferon-gamma infection. NEnglJMed2011;365:2155-2166. TBTCPTS. Threemonthsofrifapentineandisoniazidforlatenttuberculosis Wing D,CondeMB,BozemanL,HorsburghCR,Jr., ChaissonRE,Team E, HackmanJ,HamiltonCD,MenziesD,KerriganA,Weis SE,Weiner M, Sterling 2015. Guidelines ontheManagementofLatentTuberculosis Infection.Geneva; WHO. WHO. 2013. ofHealthandHumanServices 6th Edition.Department CDC. C, FloydK.Tuberculosis. In:JamisonDT, Breman JG, MeashamAR, Core CurriculumonTuberculosis: WhattheClinicianShouldKnow. Immunotec Ltd.T-SPOT. PI-TB-US-V5Abingdon, TB PackageInsert Guidelines ontheManagementofLatentTuberculosis Infection. Global Tuberculosis 2016. Report TR, Villarino ME, BorisovAS,ShangN,GordinF, Bliven-Sizemore RE, ScheinMF, BassJB,Jr. Thetuberculinskintest.ClinInfectDis GH, JerebJ,Vernon A,LoBueP, GoldbergS,CastroK,Committee YV, BrelazMC,NevesJK,FerrazJC,PereiraVR. Roleof P, Sneed L,HumphreyF, Tao E.Screening X,Bernacki 93 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. Med 2010;10: 7. analysis.BMCPulm of suspectedlatenttuberculosis: acosteffectiveness screening strategies(singleordual)forthediagnosis A, DhedaK.Different Pooran A,BoothH,MillerRF, ScottG,BadriM,HuggettJF, Rook G,Zumla inhibitors. JImmunol1999;163: 111-119. activated Tlymphocytes:regulationofcellactivationbycyclooxygenase-2 Iniguez MA,PunzonC,FresnoM.Inductionofcyclooxygenase-2 on 1996;34:27-37. inTcells.Immunopharmacology interferon-gamma IL-1 betaandTNF-alphareleaseinhumanbloodmonocytes IL-2 and Xia Z,DePierreJW, NassbergerL.Tricyclic antidepressants inhibitIL-6, Guerin attenuation.JInfectDis2003;187:117-123. Deletion ofRD1fromMycobacteriumtuberculosismimicsBacilleCalmette- DR. Lewis KN,LiaoR,GuinnKM,HickeyMJ,SmithS,BehrMA,Sherman 134:e1763-1773. diagnosis oftuberculosisinfectionanddiseaseinchildren.Pediatrics 2014; releaseassaysfor Starke JR,CommitteeOnInfectiousD.Interferon-gamma BCG Vaccines 2009. ConsultationonStandardizationandEvaluationof WHO. WHOInformal 345290. of BloodSamples,DaresSalaam,Tanzania. Tuberc ResTreat 2012;2012: Maintenance ofSensitivitytheT-SPOT. Storage TB AssayafterOvernight Talbot EA,MaroI, FergusonK,AdamsLV, MteiL,MateeM,vonReynCF. 2012; 50:2469-2471. tuberculosisriskfactors.JClinMicrobiol reagent inindividualswithdifferent of increasedbloodstoragetimeswiththeT-SPOT.TB assaywithT-Cell Xtend Wang SH,StewSS,CyktorJ,CarruthersB,Turner J,RestrepoBI.Validation Tuberc LungDis2009; 13:1416-1421. T-SPOT.TB, inapopulationwithlowprevalenceoftuberculosis.Int J releaseassay,Bienek DR,ChangCK.Evaluationofaninterferon-gamma Immunol 2003;132:225-231. unresponsiveness: roleofselectiveinterleukin-2deficiency. ClinExp Y, SmolenJS.FunctionalandmolecularaspectsoftransientTcell Koller MD,KienerHP, AringerM,GraningerWB,MeuerS,Samstag Medicine, 2015,Vol.192: 367-373. andCriticalCare ofRespiratory at 19USHospitals.AmericanJournal Gamma ReleaseTest inHealthcareWorker (IGRA)Performance Screening Matsubara J,O'DeaL,BalserWrighton-Smith P. T-SPOT JG, Humphrey-CarothersF, RiellyAF, P, Drewry MurrayK,DeWittM, King TC,UpfalM,GottliebA,AdamoP, E,KadlecekCP, Bernacki Jones biomarkers oftuberculosis.NatRevImmunol2011;11:343-354. Walzl G,RonacherK,HanekomW, ScribaTJ,ZumlaA.Immunological change aftertreatmentoftuberculosis Janssens JP. DoresultsoftheT-SPOT. releaseassay TB interferon-gamma Bosshard V, Roux-LombardP, T, Perneger MetzgerM,Vivien R,RochatT, ? RespirMed2009;103:30-34. ® .TB Interferon- 94

References References 36. 35. 34. 33. 32. 31. 30. 29. 28. 27. 26. infection -UnitedStates,2010.MMWRRecommRep.2010;59(RR-5):1-25. GammaReleaseAssaystodetectMycobacterium tuberculosis Interferon Mazurek GH,JerebJ,Vernon A,etal.Updatedguidelines forusing org/content/early/2016/12/08/cid.ciw694.long. Diagnosis ofTuberculosis inAdultsandChildren.http://cid.oxfordjournals. Centers forDiseaseControlandPreventionClinicalPracticeGuidelines: Official AmericanThoracicSociety/InfectiousDiseasesSocietyof America/ antigens. EurRespirJ.2010Aug;36(2):355-61. T-cell responsestoMycobacteriumtuberculosis-specific interferon-gamma Chee CB,KhinMarKW, GanSH,etal.Tuberculosis on treatmenteffect cell levelusingtheELISPOTassay. Methods2006;38:274-282. Cox JH,FerrariG,JanetzkiS.Measurementofcytokinereleaseatthesingle enables delayedprocessing.ScandJInfectDis2010;42:845-850. of latenttuberculosisinfectioninadultandpediatricpopulationsthat releaseassayforthediagnosis Evaluation ofamodifiedinterferon-gamma Wang SH,PowellDA,NagarajaHN,MorrisJD,SchlesingerLS, Turner J. J Infect2012;64:197-203. blood collection and interferon-gamma releaseassayusingT-Cellblood collectionandinterferon-gamma Xtend Bouwman JJ,ThijsenSF, BossinkAW. Improvingthetimeframebetween which inhibitTcellfunction.JImmunolMethods2009;341:68-75. of bloodincreasesthefrequencyactivatedCD11b+CD15+granulocytes McKenna KC,BeattyKM,Vicetti MiguelR,BilonickRA.Delayedprocessing Abingdon, UK.2013. Oxford ImmunotecLtd.T-Cell PI-TT.610-US-V4 XtendPackageInsert Saf 2014;62:460-467. releaseassays.Workplacetuberculin skintestandinterferon-gamma Health hospital employees:compliance,clearancetoworktime,andcostusing Foster-Chang SA,ManningML,ChandlerL.Tuberculosis screeningofnew Respir J2007;30:321-332. gamma releaseassaytestingforthetreatmentoflatenttuberculosis. Eur Diel R,Wrighton-Smith P, ZellwegerJP. ofinterferon- Cost-effectiveness screening oflatenttuberculosisinfection.EurRespirJ2006;28:45-50. Wrighton-Smith P, ZellwegerJP. Directcostsofthreemodelsforthe ® . 95 ®

T-SPOT, T-Cell Xtend and Oxford Diagnostic Laboratories are registered trademarks of Oxford Immunotec Ltd. SNAP is a trademark of Oxford Immunotec, Inc. The Oxford Diagnostic Laboratories and the Oxford Immunotec logos are registered trademarks of Oxford Immunotec Ltd. © 2017 Oxford Immunotec. All rights reserved. TB-US-TG-0081-V1