Volume. 35, No. 2 july. 2011 - The Chest and Heart Association of ...

Chest & Heart Journal Vol. 35, No.-2, July 2011with category IV regimens which are mainly secondline drug 5 .Sputum smears microscopy by Ziehl-Neelsenstaining (Z-N) for acid fast bacilli (AFB) remainsthe most important and widely available diagnosticmethod for tuberculosis in high prevalencecountries. Its specificity is very high on diagnosisof new TB cases, but not for the case detectionduring treatment 6 . ZN stained sputum smearmicroscopy cannot distinguish between live anddead AFB 7 . Smear may often found positive dueto prolonged excretion of dead bacilli. Atcontinuous treatment phase of Cat I a Z-N positivesmear is already more indicative of resistance andin 50% cases during Cat II it even indicates MultiDrug Resistant Tuberculosis (MDR-TB). Later,during the treatment and at the end of thetreatment, it usually indicates failure. However,one third of sputum specimens of these failurepatients did not grow TB bacilli on culture, whichmay have been due to dead AFB, resulting in falsedeclaration of failure and unnecessary re-treatment.Besides, rapid determination of drug susceptibilityremains problematic all over the world andespecially in TB endemic countries. A reliable testto distinguish live from dead AFB might thus bevery useful as a criterion to change treatment early,i.e. to MDR treatment or prolong intensive phaseand as a true indicator of failure. FluoresceinDiacetate (FDA), a salt of the green fluorophorefluorescein, is detectable by fluorescence microscopyonly after its cleavage by cell esterase. These arepresent in viable cell only 8 . .Materials and Methods:Patients diagnosed as Category II TreatmentFailure by physician referred to the NationalTuberculosis Referral Laboratory (NTRL) wasenrolled for sputum examination. Those patientswho showed AFB positive by Z-N method wereincluded for FDA staining and conventionalculture. A total 100 patients of different age andsex were enrolled in this study. Sample collectionand laboratory work were done in the NTRL,NIDCH, during the period of January 2010 toDecember 2010.Staining procedures:Ziehl-Neelsen (ZN) staining was performed as perstandard procedure 9. . The FDA staining procedurewas adapted from the publication by Tsukiyama etal. 8 . After drying, unfixed smears were coveredfor 30 min with the FDA reagent, which was madeup weekly by diluting 100µl FDA stock (5% w/vfluorescein diacetate in acetone, stored in aliquotsat 20° C for up to 2 years) in 10 ml phosphatebuffer solution.. After rinsing with water and destainingwith 1% acid alcohol for 1–2 min, bacilliwere killed using 5% watery phenol for 10 min,followed by a final water rinse and air-drying inthe dark. One length of the smear was read undera fluorescence microscope at 1000 magnification.Conventional culture:Culture were done on Lowenstein-Jensen media(LJ) media as was described by Kantor et al., 10 .ResultAmong 100 patients, sputum Z-N staining gradingshowed maximum positivity, i.e., 3 + in 48 (48%)cases, followed by 1 + in 29 (29%) cases, 2 + in 17(17%) cases and scanty in 6 (6%) cases. Amongthese cases 90 (90%) were positive and 10 (10%)cases were negative by FDA staining. Of these 90cases, FDA staining grading showed, 8 (8.8%) caseswere scanty, 30 (33.3%) cases were 1 + , 25 (27.7%)cases were 2 + , and 27 (30%) cases were 3 + .The results of FDA staining were compared withconventional culture, among 100 Z-N positive TBcases 86 cases were positive and 9 were negativeby both the methods. Out of 10 FDA negative, 1(10%) case was found culture positive and 4 (4.5%)cases of FDA positive were culture negative.Sensitivity of FDA staining was 98.5% andspecificity was 70.0%Table-IDistribution of the study patients according toFDA grading (n=100).FDA grading Number of patients Percentage(n=90) 100S 8 8.81 + 30 33.32 + 25 27.73 + 27 30.0Total 90 100Note:FDA: Fluorescein Diacetate104