Clinical and bacteriological correlates of whole blood interferon gamma (IFN- 毭 ) in newly detected cases of pulmonary TB

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1 224 Asia Pacific Joural of Tropical Medicie (2010) Cotets lists available at ScieceDirect Asia Pacific Joural of Tropical Medicie joural homepage: Documet headig Cliical ad bacteriological correlates of whole blood iterfero gamma (IFN- 毭 ) i ewly detected cases of pulmoary TB Badyopadhyay M 1, Bhakta A 2, Chakrabarty S 3, Pal M 4, Bharati P 4* 1 Departmet of Microbiology, RG Kar Medical College, Kolkata, West Begal, Idia 2 Departmet of Aatomy, NRS Medical College, Kolkata, West Begal, Idia 3 School of Studies i Athropology, Pt. Ravishakar Shukla Uiversity, Raipur, Chhattisgarh, Idia 4 Idia Statistical Istitute, 203 BT Road, Kolkata ARTICLE INFO Article history: Received 16 November 2009 Received i revised form 16 December 2009 Accepted 15 February 2010 Available olie 20 March 2010 Keywords: Iterfero gamma(ifn- 毭 ) Tuberculosis(TB) patiets Quatifero-tuberculosis gold(qft) positivity Maharashtra Idia ABSTRACT Objective: To determie the relatioship of the capacity to produce iterfero gamma (IFN- 毭 ) i whole blood, bacteriological, hematological, radiographic ad cliical presetatios i ew, HIV seroegative cases of pulmoary tuberculosis (TB). Methods: 80 cases ad 50 cotrol subjects aged 15 years owards, represetative of Kasturba Hospital ad Nursig schools of Wardha district of Maharashtra state i Idia were examied for their health coditio with stadard methodology. Results: Amog these TB patiets, 73.8% were Quatifero-TB gold (QFT) positive with IFN- 毭 cocetratio as 0.35 IU or more ad there was oe i healthy cotrols. The mea IFN- 毭 cocetratios varied betwee 9.58 IU (50-59 yrs) ad 2.58 IU ( 曒 60 yrs), showig o tred. The differeces i positivity ad mea IFN- 毭 cocetratios were statistically isigificat. Both the QFT positivity ad IFN- 毭 cocetratios were higher i ormal lymphocyte percet as compared to below ad above ormal, but differeces were ot statistically sigificat. Coclusios: The IFN- 毭 cocetratios are ot correlated with ay of the predictors of disease severity studied, the levels are sigificatly higher i observatio group as compared to healthy group. 1. Itroductio Worldwide, there were 8.8 millio ew cases of active tuberculosis (TB) ad a estimated 1.7 millio deaths from TB i 2003[1]. Idia occupies oly 2% of the lad area ad 15% of the total populatio but shares 30% of the TB burde. Oe perso dies from TB i Idia every miute, more tha 1 000, every day ad every year[2]. Sputum smear for acid fast bacilli (AFB) is the primary microbiologic method for diagosis of TB. Culture for AFB requires more time, traied persoal ad laboratory facilities. Huma immuodeficiecy virus (HIV) coifectio ca further complicate TB diagosis with atypical cliical symptoms of pulmoary tuberculosis (PTB) ad a higher likelihood of egative sputum smears for AFB[3]. TB is a importat opportuistic disease amog HIV ifected *Correspodig author: Dr. Premaada Bharati Professor, Biological Athropology Uit, Idia Statistical Istitute, 203 B.T. Road, Kolkata , West Begal, Idia Tel: (091) (033) , Fax: (91) (033) bharati@isical.ac.i, pbharati@gmail.com persos world wide. I developig coutries of Africa, Southeast Asia & Lati America a estimated 10 millios persos are co ifected. The tuberculi ski test (TST) which is the stadard for diagosis of latet tuberculosis ifectio (LTBI), is the oly test available for that purpose i resource-limited settigs, ad sometimes is used to aid i the diagosis of active TB; however, its specificity is limited by cross reactivity with o tuberculous mycobacteria ad bacille calmette-gueri (BCG) vaccie strais of M. bovis, ad its sesitivity ca be affected by malutritio ad immuo-suppressio[4]. Mycobacterium tuberculosis (M. tuberculosis) ifectio is chiefly cotrolled by activatio of macrophages through type 1 cytokie productio by T cells (Th1), ad iterfero gamma (IFN- 毭 ) is cetral to this process[5]. Patiets with defective IFN- 毭 receptor develop severe mycobacterial disease[6]. Several ex vivo studies have focused o the role of IFN- 毭 ad its regulatory cytokies i active pulmoary TB (PTB) patiets ad they suggested that cytokie aalysis

2 Badyopadhyay M et al./asia Pacific Joural of Tropical Medicie (2010) i whole blood cultures may be able to discrimiate betwee active TB ifectio ad healthy cotrols[7]. Vakayalapati[8] shows that Th1 (helper T-cell 1) cytokies i sputum, serum ad brochoalveolar lavage fluid (BALF) correlate with disease activity durig active TB of the lug. Advaces i geomics ad immuology have led to promisig alterative- i vitro iterfero gamma release assay (IGRA), based o the pricipal that T cells of idividuals ifected with M. tuberculosis release IFN- 毭 whe they re-ecouter TB specific atiges[9,10]. Newly discovered atiges that are specific to the M. tuberculosis complex are ot produced by M. bovis. BCG vaccie strais offer the opportuity to develop IGRA with high sesitivity for TB ad LTBI, ad specificity for LTBI ad potetially for TB as well[11].three promisig atiges for use i such assays are the 6-kDa early secreted atigeic target (ESAT-6), 10-kDa culture filtrate protei (CFP-10), ad the Rv2654 atige (TB 7.7)[12]. These atiges, ecoded by gees located withi the regio of differece 1 (RD1) segmet of the M. tuberculosis geome, are more specific because they do ot share with ay of the BCG vaccie strais or certai species of o tuberculosis mycobacterium (NTM)[12]. ESAT-6 ad CFP-10 ca elicit strog IFN- 毭 resposes from T-cells of persos ifected with MTB but ot from T-cells of those vacciated with BCG or at low risk of ifectio[11]. Although IGRAs are promisig ad offer logistic advatage, uresolved issues remai. Oe such is whether they ca be used for diagosis of active TB ad for determiig the disease severity. Effector respose may be drive by atige load, ad there is evidece that reductio of the atige load by treatmet decreases T cell resposes. The sesitivity of IFN- 毭 i active PTB cases has bee reported to be 75%-89% usig TB specific atiges (ESAT-6, CFA-10 & TB7.7) ad 96% usig purified protei derivative (PPD)[13]. Several studies have reported sigificatly higher IFN- 毭 cocetratios i serum[14] ad brocho alveolar lavage fluid (BALF)[15] of active TB patiets as compared to healthy cotrols. Sice IFN- 毭 productio correlates strogly with ieffective immuity to TB, Sodhi et al[16] determied, if productio of this cytokie varied i patiets with differet cliical maifestatios of TB ad observed that the radiographic extet of disease ad the site of disease were the oly idepedet predictors of IFN- 毭 productio i HIV-egative ad ifected patiets (P=0.001). It is cocluded that reduced IFN- 毭 productio by peripheral blood moouclear cells (PBMCs) is a marker of severe TB i both HIV-egative ad ifected patiets with TB. Iokuchi et al[17] i their attempt to determie if whole blood IFN- 毭 productio IS correlated with the radiographic extet of PTB before treatmet by usig PPD ad phytohemagglutii (PHA) for stimulatio, observed that PHA stimulated IFN- 毭 productio was lower i patiets tha healthy voluteers (P<0.05) ad iversely correlated with disease extet (P<0.01). Tuberculous PPD stimulated IFN- 毭 productio, however, it did ot correlate with disease extet. It is cocluded that PPD-IFN- 毭 / PHA- IFN- 毭 may be useful for diagosis of TB but ot for evaluatig the disease severity, ad it is suggested that PHA-IFN- 毭 could be cosidered as a marker of disease severity. Tsiouris et al[18] suggested that TST combied with IGRA or with a sigle sputum smear may play a role i excludig the diagosis of TB i some settigs. IGRA with Quatifero TB Gold (QFT-G) i tube kit (Cellestis Ltd Australia), which has bee performed previously i estimatig prevalece of LTBI amog health care workers[19], study of IFN- 毭 resposes amog health care workers before ad after LTBI prevetive therapy ad diagosis of LTBI i childre[20]. There has bee practically o study from Idia that has studied the correlatio of IFN- 毭 respose followig stimulatio of PBMCs i whole blood usig I-tube IGRA maufactured by Cellestis Ltd, Australia, with differet parameters suggestig disease severity i PTB patiets. Keepig i view the above, the preset study had bee udertake 镡 to determie if the capacity to produce IFN- 毭 i whole blood is related to bacteriological, hematological, radiographic ad cliical presetatios i ew, HIV seroegative cases of PTB; 镢 to cofirm ew cases of PTB by Zeihl Neelse staiig, culture o Lowestei Jese medium ad X- ray chest; 镤 to determie HIV status of recruited patiets usig 4th geeratio ezyme-liked immuosorbet assay (ELISA) kit; 镥 to determie the disease severity i recruited patiets through smear/culture gradig ad extet of disease by chest radiography; 镦 to determie total leukocyte cout, absolute lymphocyte cout ad lymphocyte percet i recruited patiets usig automated coulter couter; 镧 to determie IFN- 毭 cocetratios i whole blood amog patiets ad cotrols usig Quatifero TB Gold (QFT) I tube commercial test kit; 镨 to correlate IFN- 毭 results ad cocetratios with smear, culture, symptoms suggestive of PTB, radiographic ad hematological fidigs. 2. Materials ad methods The preset work was performed i the Departmet of Microbiology, Mahatma Gadhi Istitute of Medical Scieces, Sevagram, which is located i a rural Istitute i Wardha district of Maharashtra state i Cetral Idia. The study was coducted from November 2004 to October Study populatio A total of 80 subjects attedig the Kasturba Hospital attached to the Istitute ad fulfillig the followig iclusio criteria: Adult above 15 years of age; ewly diagosed case of active PTB ( smear ad/or culture positive ad/or evidece of TB by chest X ray); ew case havig received treatmet for o more tha 4 weeks at the time of iclusio; HIV seroegative. The exclusio criteria were as followig: Refusal to participate; ustable cardio-respiratory coditio; past H/O active TB or those who have received treatmet for more tha oe moth; pregat wome; HIV seropositive. The cotrol group icluded 50 healthy studets of MGIMS & Kasturba Nursig School. All patiets ad cotrols were

3 226 Badyopadhyay M et al./asia Pacific Joural of Tropical Medicie (2010) erolled after obtaiig writte iformed coset. Where ever ecessary this was doe i local laguage. The study was started after obtaiig the approval of the Istitutes ethical committee. All patiets were subjected to followig studies: 2.2. Sputum smear examiatio by ZN method Three sputum specimes were examied [spot/overight/ spot as per revised atioal tuberculosis cotrol programme (RNTCP) criteria]. Gradig of smears was doe as TRC maual[21] Sputum culture All sputum samples were ioculated o two LJ slopes ad icubated i a CO 2 icubator at 37 曟 for a maximum of 8 weeks before declarig them egative. All slopes were examied weekly ad the growth graded as TRC maual ad the isolate idetified by stadard methods[21] X-ray chest This was doe for all patiets ad examied by two idepedet cosultats blided to laboratory results. The presece or absece of cavitatios was oted ad the extet of disease was assessed as follows: Miimal disease The total extet of disease did ot ivolve more tha a area of lug equivalet to that above the secod chodrosteral juctio ad the spie of fourth thoracic vertebra Moderately advaced disease Dese ad cofluet lesios did ot exceed a area of oe third of the volume of oe lug; o cofluet lesios of slight or moderate desity did ot exceed the area of oe lug Far advaced disease More extesive tha moderately advaced disease HIV status This was determied by 4th geeratio ELISA kit, Viroostika (BioMerieux), which is capable of detectig HIV-1, HIV-2 atibodies ad p24 atige. The test was performed as the directios of the maufacturer Haemotological examiatio Total lymphocyte cout (TCL) ad lymphocyte percet were determied for all patiets usig coulter couter IFN- 毭 estimatio The mai kit was QuatiFERON -TB Gold KIT (Cellestis Limited, Australia). The test was performed i followig stages Collectio of blood, icubatio & harvest of plasma Two ml blood was collected ad 1 ml each was added to the two tubes up to the 1 ml mark: Nil Cotrol (adjusts for backgroud) ad TB Atige tube provided with the kit. Both tubes were shake vigorously ad icubated immediately upright at 37 曟 for 24 hrs. Followig the icubatio, the tubes were cetrifuged at rpm for 5-10 miutes to harvest plasma. Movemet of gel plugs to separate plasma from cells idicated that cetrifugatio was successful. Plasma ( 毺 L) was pipetted out ad trasferred to separate 1mL sterile micro tube racks i a 96 well format (two for each patiet). All plasma samples were stored at 2-4 曟 till evaluated (4 weeks maximum) Preparatio of reagets All reagets were prepared as kit istructios All plasma samples ad reagets, except for Cojugate 100 伊 Cocetrate, were brought to room temperature (22 暲 5) 曟 before use. At least 60 miutes were allowed for equilibratio. Oe ELISA plate for 44 samples was used at oe time. The stadards (S1 to S4) ad cojugate were recostituted as kit istructios. The lay out of samples ad cotrols was as Table 1. Prior to assay plasma samples were mixed. 50 毺 L of freshly prepared diluted cojugate was added to all wells usig a multichael pipette. Followig this 50 毺 L of plasma was added to test 50 毺 L of the four stadards i duplicate i respective wells as per layout (Table 1). The cojugate ad plasma samples/stadards were thoroughly mixed usig microplate shaker (Waveform=20, amplitude=6, time=1 miute). Plate was covered with a lid ad icubated at room temperature (22 暲 5) 曟 for (120 暲 5) miutes. Wells were washed with 毺 L of workig stregth wash buffer forsicycles at room temperature with a fully automated ELISA washer. After the last washig plates were tapped face dow o a absorbet wipe to remove residual wash buffer. 100 毺 L of ezyme substrate solutio was added to each well ad mixed thoroughly o the shaker. Plate was covered with a lid ad icubated at room temperature for 30 miutes, startig from the time substrate was added to the first well. 50 毺 L of the ezyme stop solutio was added to each well i the same sequece ad same speed as was the substrate added, followig completio of icubatio. The stop solutio was getly mixed. Optical desity was read withi 5 miutes of termiatig the reactio usig 450 m filter, with a referece filter betwee 620 m ad 650 m. Optical desity (OD) values were used to calculate results. OD values were directly fed from the ELISA reader to the computer ad the QFT-G i tube aalysis software was used to aalyze raw data ad to calculate result. Iterpretatio of results show i Table 2. All relevat patiet related iformatio was recorded Statistical aalysis This was doe usig SPSS 10.0 versio for Widows ad Smart viewers software. Meas were compared usig the

4 Badyopadhyay M et al./asia Pacific Joural of Tropical Medicie (2010) Studets't' test. Proportios, after covertig ito umbers, were compared usig the Chi-square test ad the Fisher's exact test, as appropriate. P<0.05 was cosidered as sigificat. 3. Results Amog the TB patiets, 73.7% were QFT positive with IFN- 毭 cocetratio as 0.35 IU or more, ad there was oe i healthy cotrols. The IFN- 毭 cocetratios were sigificatly higher i patiets as compared to the cotrol group (P=0.015). The age of the patiets icluded i the study raged betwee 18 to 80 years, with the mea age beig years. As per the kit criteria, both positive ad egative test results were see i all the age groups studied. The positive results varied betwee 92.3% (< 20 yrs) to 60.0% (30-39 yrs) ad egative betwee 7.7% (<20 yrs) to 40.0% (30-39 yrs). The mea IFN- 毭 cocetratios varied betwee 9.58 IU (50-59 yrs) ad 2.58 IU ( 曒 60 yrs), showig o tred. The positivity (81.0%) ad mea IFN- 毭 cocetratios (7.11 IU) were higher i the females with isigificat differece (Table 3). Table 4 showed the correlatio with cliical features. More patiets with fever showed positive results (74.7%) ad had higher mea IFN- 毭 cocetratios (6.82 IU) with isigificat differece. Patiets without cough showed higher positivity (100.0%) but lower mea IFN- 毭 cocetratios (5.64 IU). However, those without cough were small i umber. Haemoptysis is a importat presetatio of advaced disease. However, both positivity (75.0%) ad mea IFN- 毭 cocetratios (7.24 IU) were higher i those without haemoptysis with isigificat differece. Loss of weight was see i 66 subjects. Those loss of weight patiets showed higher positivity (74.2%) ad lower mea IFN- 毭 cocetratios (6.49 IU). Table 5 showed the correlatio with bacteriological status, icludig smear ad culture. The higher the smear ad/or culture gradig the higher the severity of the disease, hece higher positivity ad IFN- 毭 cocetratios are expected i them. O correlatio of smear with IFN- 毭, it was observed that maximum positivity was see i those with 2+ smear (92.9%) ad there was o upward tred i positivity with icrease i umber of bacilli/ml of sputum, as those with 1-9 bacilli i their sputum showed higher positivity (76.9%) tha those with 3+ smears (69.2%). It was iterestig to ote that the mea IFN- 毭 cocetratio was the maximum i smear egative cases (15.16 IU). Noe of the differeces were sigificat. As see i the smear correlatio, maximum positivity was see i patiets with 2+ culture report (81.8%) ad there was o upward tred as expected, either i the positivity or mea IFN- 毭 cocetratios with icrease i culture gradig. The lowest mea IFN- 毭 cocetratios were recorded i patiets with 3+ culture report. The degree of damage to the lugs is determied through chest radiography. The greater the damage, the higher the extet of disease. Though, the mea IFN- 毭 cocetratios were higher i those with cavity, more patiets without cavity showed positive QFT results. Noe of these differeces were statistically sigificat. O correlatig the various grades of disease severity, though positivity was higher i those with moderate disease, mea IFN- 毭 cocetratios were foud to be higher i those with miimal disease. However, oe of these differeces were sigificat (Table 6). Table 7 showed the correlatio of hematological fidigs with IFN- 毭. O the basis of TLC couts patiets were divided ito three groups: ormal couts ( cells/ 毺 L of blood), below ormal (<4 000 cells/ 毺 L) ad above ormal (> cells/ 毺 L). Though the positivity was the maximum i those havig TLC>11 000, the mea IFN- 毭 cocetratios were the highest i those with TLC< Noe of the observatios were sigificat. Both the percet positivity (68.4%) ad mea IFN- 毭 cocetratios (5.25 IU) were lower i those showig higher tha ormal absolute lymphocyte cout (>4 500) tha those with lower cout (78.6% & 8.03 IU, respectively), but the differeces were ot sigificat. Both the QFT positivity ad IFN- 毭 cocetratios were foud to be higher i those havig ormal lymphocyte percet as compared to those below ad above ormal, but the these differeces were ot statistically sigificat. 4. Discussio PTB is oe of the most importat ifectious diseases i the world. Both, TB occurrig i immuo-deficiet patiets ad multi-drug TB have emerged as cliical problems. T cells play a cetral role i the huma immue respose to mycobacterial pathoges ad the balace of T-cell cytokies produced i respose to ifectio is believed to have a profoud effect o cliical outcome. Although the specific cytokies that mediate immuologic resistace to mycobacteria i humas remais uidetified, IFN- 毭 appears to be the key cytokie ivolved, as icreased susceptibility to mycobacterial ifectios has bee reported i idividuals with IFN- 毭 receptor deficiecy or mutatios i IFN- 毭 receptor 1[6]. The primary fuctio of this cytokie is to activate the macrophage ad eable this cell to carry out its mycobacterial effecter fuctios. To date, the immue respose to M. tuberculosis has bee assessed by ski testig usig the purified protei derivative from M. tuberculosis; however, TST measures both effecter ad memory T-cell resposes as agaist ew geeratio IFN- 毭 assays which measures mostly effecter T-cell resposes[22]. Both TST ad IFN- 毭 assays have bee used for diagosig MTB ifectio, however, whether the results of whole blood IFN- 毭 assays is correlated with disease severity is ot clear at preset. I the preset study the sesitivity of the QFT TB Gold test i HIV sero-egative active PTB cases was 73.7%, takig 曒 0.35 IU/mL IFN- 毭 cocetratio i whole blood as positive, as per the kit, followig stimulatio with TB specific atiges. Tsiouris et al[18] have reported a overall sesitivity of 75% i all ad 82% i ew PTB patiets. Cliical studies have estimated the sesitivity of QFT TB Gold to be 89% approximately, for active TB disease. I compariso to QFT TB Gold, which utilizes TB specific atiges for stimulatio,

5 228 Badyopadhyay M et al./asia Pacific Joural of Tropical Medicie (2010) Table 1 Sample layout. Row A 1N 5N 9N 13N 17N S1 S1 25N 29N 33N 37N 41N B 1A 5A 9A 13A 17A S2 S2 25A 29A 33A 37A 41A C 2N 6N 10N 14N 18N S3 S3 26N 30N 34N 38N 42N D 2A 6A 10A 14A 18A S4 S4 26A 30A 34A 38A 42A E 3N 7N 11N 15N 19N 21N 23N 27N 31N 35N 39N 43N F 3A 7A 11A 15A 19A 21A 23A 27A 31A 35A 39A 43A G 4N 8N 12N 16N 20N 22N 24N 28N 32N 36N 40N 44N H 4A 8A 12A 16A 20A 22A 24A 28A 32A 36A 40A 44A Note: 1N: Sample 1 Nil cotrol plasma; 1A: Sample 1 TB specific atige plasma; S1, S2, S3, S4: Stadard cotrols with IFN- 毭 cocetratio: 4IU, 1 IU, 0.25 IU ad 0.1 IU, respectively. Table 2 Iterpretatio of results. NIL(IU/mL) TB atige mius NIL (IU/mL) Result Iterpretatio < 8.0 < 0.35 > 0.35 & < 25 % of Nil value Negative M.tuberculosis ifectio NOT likely > 0.35 & > 25 % of Nil value Positive 1 M.tuberculosis ifectio likely > Ay Idetermiate 3 Results are idetermiate for TB atige resposiveess 1 Where M. tuberculosis ifectio is ot suspected, iitially positive results ca be cofirmed by retestig the origial plasma samples i duplicate i the QuatiFERON -TB Gold ELISA. If repeat testig of oe or both replicates is positive, the idividual should be cosidered test positive. 2 I cliical studies, less tha 0.25% of subjects had IFN- 毭 levels of > 8.0 IU/mL for the Nil cotrol. 3 Refer to Trouble shootig sectio of the packig isert for possible causes. Table 3 QFT result ad IFN- 毭 cocetratio i health, age ad sex. Group Result[(%)] Mea cocetratio Positive Negative Health * Healthy cotrols 50 Nil 50(100.0) Patiets 80 59(73.7) 21(26.3) 6.71 Age(yrs) < (92.3) 1(7.7) (77.3) 5(22.7) (60.0) 4(40.0) (66.7) 3(33.3) (80.0) 1(20.0) 9.58 曒 (66.7) 7(33.3) 2.58 Sex Male 59 42(71.2) 17(28.8) 6.57 Female 21 17(81.0) 4(19.0) 7.12 *: Sigificat at 1% level. Table 4 QFT result ad IFN- 毭 cocetratio by cliical features. Parameters Result[(%)] Mea cocetratio Positive Negative Fever Yes 75 56(74.7) 19(25.3) 6.82 No 5 3(60.0) 2(40.0) 5.05 Cough Yes 77 56(72.7) 21(27.3) 6.75 No 3 3(100.0) Haemoptysis Yes 16 11(68.8) 5(31.3) 4.58 No 64 48(75.0) 16(25.0) 7.24 Weight loss Yes 66 49(74.2) 17(25.8) 6.49 No 14 10(71.4) 4(28.6) 7.73

6 Table 5 QFT result ad IFN- 毭 cocetratio by sputum smear ad culture. Group Badyopadhyay M et al./asia Pacific Joural of Tropical Medicie (2010) Result[(%)] Positive Negative Mea cocetratio Sputum smear Negative 2 1(50.0) 1(50.0) bacilli 13 10(76.9) 3(23.1) (68.4) 12(31.6) (92.9) 1(7.1) (69.2) 4(30.8) 3.21 Culture Negative 7 4(57.1) 3(42.9) 4.94 < 20 coloies 7 4(57.1) 3(42.9) (72.4) 8(27.6) (81.8) 4(18.2) (80.0) 3(20.0) 3.69 Total 80 59(73.7) 21(26.2) 6.71 Table 6 QFT result ad IFN- 毭 cocetratio by Chest X ray positivity ad gradig. Result[(%)] Mea cocetratio Group Positive Negative X-ray positivity Not suggestive of TB 4 2(50.0) 2(50.0) 6.13 Suggestive of TB 39 26(66.7) 13(33.3) 5.87 Suggestive of TB + cavity 37 21(56.8) 16(43.2) 7.82 Total 80 49(73.7) 31(26.2) 6.71 X-ray gradig Negative 4 2(50.0) 2(50.0) 9.71 M: Miimal, Md: Moderate, Ad:Advace, D:Disease, C: Cavity. MD 8 5(62.5) 3(37.5) Md. D 23 18(78.3) 5(21.7) 7.13 Ad. D 45 34(75.6) 11(24.4) 5.67 Table 7 QFT result ad IFN- 毭 cocetratio by TLC, absolute lymphocyte cout ad lymphocyte percetage. Group Result[(%)] Positive Negative Mea cocetratio TLC groups(cells/ 毺 L) < (63.6) 4(36.4) (67.6) 11(32.4) 4.32 > (82.9) 6(17.1) 7.90 Sigificat level P = P = Absolute lymphocyte cout ( 毺 L) (78.6) 9(21.4) 8.03 > (68.4) 12(31.6) 5.25 Sigificat level P =0.303 P =0.186 Lymphocyte( %) < (67.7) 10(32.3) (82.8) 5(17.2) 7.71 > (70.0) 6(30.0) 7.54 Sigificat level P = P = 0.054

7 230 Badyopadhyay M et al./asia Pacific Joural of Tropical Medicie (2010) sesitivity & specificity of whole blood gamma iterfero released followig stimulatio with PPD has bee reported to be 96% ad 98% for the diagosis of TB[13]. I the preset study IFN- 毭 cocetratios were sigificatly higher i patiets with disease tha healthy cotrols, as report of Vakayalapati et al[8]. Tsao et al[15] demostrated high levels i BALF as compared to serum of active PMTB ad they advocate IFN- 毭 estimatio i BALF rather tha serum i such cases, as PTB is a local disease ad IFN- 毭 is secreted by local immue cells. Moreover, they observed that T-lymphocytes from BALF spotaeously secreted sigificatly greater quatities of IFN- 毭 as compared to those i peripheral blood. Some workers have determied a defect i peripheral blood lymphocytes from tubercular samples for IFN- 毭 productio after stimulatio with mitoges or atiges[21]. Together with reasos for depressed PPD iduced IFN- 毭 productio i PBMC of ew TB patiets (both HIV ifected ad o ifected) i compariso to healthy cotrols viz: geetic defect i IFN- 毭 productio / respose, compartmetalizatio of atige resposive cells to sites of active disease, active ad selective depletio of circulatig MTB resposive T cells durig TB or icrease i spotaeous ad MTB iduced programmed cell death amog PBMC, it could also possibly explai the reaso for low IFN- 毭 productio or egative QFT result i our study. Iokuchi et al[17] observed phyto-hemaggluti (PHA) iduced IFN- 毭 productio to be sigificatly low i patiets but o such differece with PPD iduced IFN- 毭. All our patiets were HIV sero-egative, however, i absece of TB HIV ifectio aloe reduces IFN- 毭 productio i respose to atiges or mitoges. Tsiouris et al[18] observed sigificatly lower IFN- 毭 cocetratios i HIV ifected patiets tha i HIV egative patiets with culture positive TB (P=0.033). Though the patiets i age group <20 yrs i the preset study showed highest positivity (92.3%), o sigificat chage i sesitivity was observed with advace i age. Quatifero TB Gold appears to fuctio well i persos of all ages i report of Mori et al [23] o 110 culture cofirmed TB patiets stratified by age. A sigificat declie i test sesitivity was observed by them for the Matoux test i cotrast to QFT TB Gold, suggestig measuremet of i vitro IFN- 毭 resposes appropriate i the elderly. IFN- 毭 cocetratios were the lowest i age group 曒 60 i our study, however these cocetratios were ot sigificat compared to other age groups, as reported by Sodhi et al [16] ad Tsiouris et al[18]. MTB iduced IFN- 毭 productio was correlated with extet of disease, which was assessed either by the sigs ad symptoms of PTB, bacteriological gradig (both smear ad culture), radiographic extet of disease ad hematological parameters viz. TLC, absolute lymphocyte cout ad lymphocyte percet. With respect to sigs & symptoms, QFT positivity ad IFN- 毭 cocetratios were more i patiets with fever ad weight loss while it was low i those with cough ad haemoptysis, however, the differeces were ot sigificat whe compared to those without them, suggestig that there was o correlatio of IFN- 毭 respose with sigs ad symptoms. Fever, weight loss, cough ad haemoptysis are usually associated with more itesive disease, hece a higher IFN- 毭 respose is expected i patiets presetig with them. Tsao et al[15] reported sigificatly higher IFN- 毭 levels i active TB patiets with fever or loss of body weight, but they were observed i BALF ad ot serum. Studies have reported that the severity of TB is associated with depressio i lymphocytes ad CD 4 + cell couts i HIV egative patiets, a feature that could be due to either compartmetalizatio of atige-resposive cells to sites of active MTB ifectio or active ad selective depletio of circulatig MTB resposive T cells durig TB. This could result i uresposiveess or poor respose of the peripheral blood cells to produce IFN- 毭, as both the umber ad fuctio of lymphocytes ca ifluece the amout of IFN- 毭 productio[17]. I the preset study, ot oly ormal ad above ormal lymphocyte cout, lymphocyte percet ad leukocyte cout were observed i the active TB patiets, but also the IFN- 毭 cocetratios did ot correlate with the couts, rather the IFN- 毭 cocetratios were more i those with low leukocyte ad absolute lymphocyte couts as compared to those havig ormal or above ormal couts. Sodhi et al[16] also did ot observe ay correlatio betwee IFN- 毭 cocetratios with either absolute lymphocyte cout or lymphocyte percet. Iokuchi et al[17] observed sigificatly lower total lymphocytes i active TB patiets as compared to cotrols ad reported that though the PHA iduced IFN- 毭 per lymphocyte remaied sigificatly lower (P<0.05) i patiets tha healthy voluteers, tuberculous PPD iduced IFN- 毭 was ot differet betwee the two groups. Uderstadig correlatio of IFN- 毭 respose with TB would be perfect, provided it is studied i active TB cases cofirmed by a defiite marker. There could be o less a perfect marker as a positive smear ad/or culture for this ad i additio the severity of PTB ca be assessed through bacterial burde (smear ad/or culture gradig). Keepig this i mid we tried to fid if the severity of disease, as assessed through smear or culture had ay correlatio with either QFT results or IFN- 毭 cocetratios. This study did't show ay such correlatio. We could ot come across ay studies i the literature that have looked ito this type of correlatio. However, a study o estimatio of sesitivity of IFN- 毭 i culture cofirmed cases of TB has bee doe[23], which have show the sesitivity of IFN- 毭 to be 89% agaist 66% by TST i such cases. Tsao et al[15] demostrated highest IFN- 毭 levels i BALF of patiets with higher bacterial load as assessed by smear ad culture ad have suggested that IFN- 毭 actually might be a marker of local iflammatio ad/or bacterial load i the lugs. Tsiouris et al[18] compared the sesitivity of QFT TB Gold kit, TST ad sputum smear i diagosis of culture cofirmed cases of TB ad foud it to be 82%, 92% ad 53% respectively i ew PTB cases. Sice they observed that the sesitivity of the TST combied with i tube IFN- 毭 estimatio or sputum smear for AFB was high i PTB suspects (TST+QFT=96%, TST+ oe sputum smear=93%), they are of the opiio that such combied testig strategy could be useful, i some settigs, to exclude a diagosis of PTB, cosiderig the poor sesitivity of smear microscopy ad specificity of TST. However, Iokuchi et al[17] did ot fid ay correlatio betwee PPD iduced IFN- 毭 ad sputum smear. Sodhiratmadja et al[21] observed strog correlatio of IFN- 毭

8 Badyopadhyay M et al./asia Pacific Joural of Tropical Medicie (2010) (PBMCs stimulated by M. tuberculosis) with radiographic extet of disease, both by uivariate (P=0.0004) ad multivariate (P=0.001) aalysis i PTB HIV sero egatives patiets oly. O compariso with differet degrees of disease extet, estimated by X ray, IFN- 毭 levels were sigificatly low (P=0.0001) i far advaced tha moderately advaced i report of Dlugovitzky et al[14]. PPD iduced IFN- 毭 i whole blood was foud to be sigificatly low i far advaced disease (P<0.01) by Iokuchi et al [17]. Our study showed the same result, though the differeces were ot statistically sigificat. I the preset study mea IFN- 毭 levels were observed to be higher i patiets presetig with cavity as compared to those without it. This is i cotrast to that reported by Sodhi et al[16]. However, the differeces i both were ot sigificat. Moreover, the later measured IFN- 毭 released by PBMC's followig stimulatio with killed MTB, while our study measured it i whole blood followig stimulatio with TB specific atiges. Sodhi et al [16] suggested that reduced MTB iduced IFN- 毭 productio by PBMCs reflects disease severity. Tsao et al[15] regarded it as ot oly a marker of local iflammatio but also bacterial load whe measured i BALF. I o k u c h i et al[17] foud tuberculous PPD-IFN- 毭 / PHA-IFN- 毭 a useful marker for TB diagosis ad reduced PHA- IFN- 毭 a marker of disease severity, while Tsiouris fid IGRA plus TST useful i excludig the diagosis of TB. Though the IFN- 毭 cocetratios is ot correlated with ay of the predictors of disease severity studied, the levels were foud to be sigificatly higher i diseased as compared to healthy subjects. Cosiderig the exorbitat cost of the kit (Rs / per kit-44 tests), the study could be performed o limited patiets oly. To evaluate the correlatio betwee disease severity ad IFN- 毭 released by cells, further studies i larger populatio ot oly i whole blood but also i those from local site of ifectio eed to be doe. Coflict of iterest statemet We declare that we have o coflict of iterest. Refereces [1]World Health Orgaizatio. Global tuberculosis cotrol: Surveillace, plaig, fiacig. WHO/ HTM/ TB/ Report, 349. Geeva: World Health Orgaizatio; [2]RNTCP status report TB Idia; [3]Satos Mde L, Poce MA, Vedramii SH, Villa TC, Satos NS, Wysocki AD, et al. The epidemiological dimesio of TB/HIV coifectio. Rev Lat Am Efermagem 2009; 17: [4]Kiwauka JP. Iterpretatio of tuberculi ski-test results i the diagosis of tuberculosis i childre. Afr Health Sci 2005; 5: [5]Al-Attiyah R, Mustafa AS, Abal AT, El-Shamy AS, Dalemas W, Skeiky YA. I vitro cellular immue resposes to complex ad ewly defied recombiat atiges of Mycobacterium tuberculosis. Cli Exp Immuol 2004; 138: [6]Newport MJ, Huxley CM, Husto S, Hawrylowicz CM, Oostra BA, Williamso R, et al. A mutatio i the iterfero-gamma-receptor gee ad susceptibility to mycobacteria ifectio. N Egl J Med 1996; 335: [7]Eu-Kyeog Jo, Park JK, Dockrell HM. Dyamics of cytokie geeratio i patiets with active pulmoary tuberculosis. Curr Opi Ifect Dis 2003; 16: [8]Vakayalapati R, Wizel B, Weis SE, Klucar P, Shams H, Samte B, et al. Serum cytokie cocetratios do ot parallel Mycobacterium tuberculosis-iduced cytokie productio i patiets with tuberculosis. Cli Ifect Dis 2003; 36: [9]Hewiso RG, Vordermeier HM, Smith NH, Gordo SV. Recet advaces i our kowledge of Mycobacterium bovis: a feelig for the orgaism. Vet Microbiol 2006; 112: [10]Okada M. Novel vaccies agaist M. tuberculosis. Kekkaku 2006; 81: [11]Doherty TM, Demissie A, Olobo J, Wolday D, Britto S, Eguale T, et al. Immue resposes to the Mycobacterium tuberculosis-specific atige ESAT-6 sigal subcliical ifectio amog cotacts of tuberculosis patiets. J Cli Microbiol 2002; 40: [12]Yotsumoto H. Specific immue-based diagosis of tuberculosis ifectio. Risho Byori 2008; 56: [13]Rav P, Rose MV, Søborg B, Aderse AB. New diagostic test for tuberculosis. Ugeskr Laeger 2009; 171: [14]Dlugovitzky D, Bay ML, Ratei L, Fioreza G, Vietti L, Farroi MA, et al. Ifluece of disease severity o itrite ad cytokie productio by peripheral blood moouclear cells (PBMC) from patiets with pulmoary tuberculosis (TB). Cli Exp Immuol 2000; 122: [15]Tsao TCY, Huag CC, Chiou WK, Yag PY, Hsieh MJ, Tsao KC. Levels of iterfero-g ad iterleuki-2 receptor- 毩 for brochoalveolar lavage fluid ad serum were correlated with cliical grade ad treatmet of pulmoary tuberculosis. It J Tuberc Lug Dis 2002; 8: [16]Sodhi A, Jia-Hua G, Claudia S, Qia D, Bares PF. Cliical correlates of iterfero-g productio i patiets with tuberculosis. Cli Ifect Dis 1997; 25: [17]Iokuchi N, Kazuyuki S, Hiroshi S, Usui T, Hirakata Y, Fukushima K, et al. Relatioship betwee whole-blood iterferogamma productio ad extet of radiographic disease i patiets with pulmoary tuberculosis. Diag Microbiol Ifect Dis 2003; 46: [18]Tsiouris SJ, David C, Toro PL, Austi J, Stei Z, El-Sadr W. Sesitivity aalysis ad potetial uses of a ovel gamma iterfero release assay for diagosis of tuberculosis. J Cli Microbiol 2006; 44: [19]Madhukar P, Kaustubh G, Rajish J, Dogra S, Kalatri S, Mediratta DK, et al. Mycobacterium tuberculosis ifectio i health care workers i rural Idia: Compariso of a whole-blood iterfero - 毭 assay with tuberculi ski testig. JAMA 2005; 293: [20]Dogra S, Narag P, Mediratta DK, Chaturvedi P, Reigold AL, Colford JM Jr, et al. Compariso of a whole blood iterfero- 毭 assay with tuberculi ski testig for the detectio of tuberculosis ifectio i hospitalized childre i rural Idia. J Ifect 2007; 54: [21]Sahiratmadja E, Alisjahbaa B, de Boer T, Ada I, Maya A, Dausatoso H, et al. Dyamic chages i pro- ad ati-iflammatory cytokie profiles ad gamma iterfero receptor sigalig itegrity correlate with tuberculosis disease activity ad respose to curative treatmet. Ifect Immu 2007; 75: [22]Dheda K, Udwadia ZF, Huggett JF, Johso MA, Rook GA. Utility of the atige-specific iterfero- 毭 assay for the maagemet of tuberculosis. Curr Opi Pulm Med 2005; 11: [23]Mori T, Sakatai M, Yamagishi F, Takashima T, Kawabe Y, Nagao K, et al. Specific detectio of tuberculosis ifectio: a iterfero-gammabased assay usig ew atiges. Am J Respir Crit Care Med 2004; 170: