LEAVES FROM HISTORY - 6

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1 LEAVES FROM HISTORY - 6 Edward Livingston Trudeau ( ) Father of the Sanatorium Movement in America E d w a rd L i v i n g s t o n T r u d e a u was among t h e most famous pioneers of anti- tuberculosis movement in the USA. Having been diagnosed to be suffereing from the disease at the young age of 25 years, he was advised to retire to the hills and live among pine trees. He was then a medical student at the C, College, New York. He chose the picturesque Adirondack mountains in New York state, where, by the side of the Saranac lake, he built his little red cottage to regain health in Other tuberculosis patients followed his example and soon the area became dotted with cottages and came to be known as the Adirondack Cottage Sanatorium, to later become the world famous Trudeau Sanatorium. This sanatorium closed clown in Dr Trudeau also founded the Saranac Laboratory within the sanatorium in This was the first laboratory of its k i n d in the USA. Research at this laboratory made rapid strides and the world famous strains of tubercle bacilli H 37 R v and H 37 R a were identified there by Dr. Stgeinken. Having lost his brother and daughter to tuberculosis, and writing about his own despondency during the dreary stay in Little Red Cottage : All my dreams of achievement now shattered, and in their place only exile and the inevitable end, he survived to earn the encomium of Father of the Sanatorium Movement in America. He was elected as the first President of the National Tuberculosis Association of America.

2 Original Article Ind J Tub, 2007, 48, 9 A COMPARISON OF LIQUID AND SOLID CULTURE MEDIA WITH RAD1OMETRIC SYSTEM FOR DETECTION OF MYCOBACTERIA IN CLINICAL SPECIMENS A. Bhargava 1, A Jain 2 and S.K. Agrawal 3 (Received on , Accepted on ) Summary : The present study was done on 100 consecutive specimens, received in the tuberculosis laboratory for culture of mycobactcri.i. to determine the sensitivity of and time taken by Lowenstcin Jensen (IJ) medium, Middlcbrook 7H9(MB7H9) broth and BACTEC 460 TB radiomelric system. Out of 100, a total of 59 specimens tested positive for acid fast bacilli (AFB) by microscopy and 65 were culture positive by one or another culture technique. In all, 59 were positive on LJ medium with sensitivity of 90.8% and average detection time of five weeks, 60 were positive by BACTHC radiomctric system with sensitivity of 92.3% and average detection time of three weeks and MB7H9 broth showed positive growth in 45 specimens with average detection time of six weeks and sensitivity of 62.9%. Two cases were detected by LJ medium. 3 by BACTHC and one by MB7F19 broth exclusively. I he results suggest that although LJ and BACTEC arc more sensitive culture methods than MB7H9 broth and BACTEC is more rapid and sensitive than the other two, yet none arc 100% sensitive. Simultaneous use of all the three is suggested for more efficient isolation of mycobacterium species. Key words : M\cohactcria. Culture medium, DACTkC, Kadiomctric method, IJ medium. MB7II9 medium INTRODUCTION Tuberculosis is a major public health problem, particularly in the developing world where the prevalence of infection is reported to be as high as 40% and (he annual risk of infection is 2-4% 1. The number of cases of tuberculosis world-wide is estimated to be more than 30 million with an annual incidence of 10 million cases of which about 95% are in developing countries 2 Many cases remain undiagnoscd since conventional methods currently used for diagnosis are not so sensitive and are time consuming. Culture method is still considered as the gold standard because of being 100% specific. Culture is essential in situations like drug sensitivity testing, change of drug regimen in resistant cases, epidemiological surveys, mass case-finding programmes, identification of mycobacterial species and assessment of tuberculosis programmes. There is a great need for evolving rapid, less cumbersome and more sensitive culture methods for detection of mycobacteria. Hence, the present study was done lo evaluate Lowenstcin Jensen medium, Middlcbrook 7H9 broth and BACTCC 12B medium for isolation of mycobacterial species in terms of sensitivity and time taken in various clinical specimens. MATERIAL AND METHODS The study material comprised 100 consecutive clinical specimens obtained from suspected cases of tuberculosis as received in the tuberculosis laboratory of the Post-Graduate Department of Microbiology, King George s Medical College, Lucknow from April to June Specimens included 73 sputa. 12 CSF, 9 fine needle aspirates, 3 pus and 3 other body fluids. All the specimens were subjected to decontamination and concentration by Petroff s method using 4%NaOH Holding time was 30 minutes for sputum and pus and 15 minutes for other specimens. A smear was prepared from each treated specimen and stained by Ziehl Neelsen method. A 0.5 ml of the treated specimen was inoculated simultaneously on LJ medium slope 3, in MB7H9 broth (Himedia, Mumbai)and HACTI C 12B vials(becton- Dickinson, USA). The inoculated bottles/vials were incubated at 37 C in the presence of 5-10% CO 2 and read every alternate day (L J & MB7H9 vials). Reading of Bactec 12B vials was done according to 1. Senior Resident. 2. Professor 3. Professor and Head of Department Department of Microbiology King George s Medical College, Lucknow. Correspondence: Dr. Amita Jain, Post-Graduate Department of Microbiology. King George s Medical College, 1 ucknow

3 10 A BHARGAVA E T AL the manufacturer s instructions. Presence of growth was confirmed by making a smear from the colonies/ culture broths, stained by Ziehl-Neelsen stain and examined for the presence of acid fast bacilli Culture media showing presence of AFB were recorded as positive. The average time taken for culture to become positive was also recorded for each medium. A culture was regarded negative if it showed no AFB even after 12 weeks of incubation. RESULTS Out of the 100 specimens, 59 were AFB positive on smear examination and 65 on culture (Table 1). The table shows that 40 positive specimens were detected by all the three culture media, 15 were positive both on EJ medium and in BACTEC 12B vials, 2 were positive by LJ medium and MB7H9 broth and 2 were positive by MB7H9 broth and BACTEC 460 TB system; 2 specimens showed growth only on EJ medium, 3 grew exclusively in BACTEC 12B vials and one case was detected by MB7H9 broth alone. Six pulmonary and 2 extrapulmonary specimens which were smear negative were positive on culture, and 2 smear positive pulmonary specimens did not grow on any of the culture media used (patients were already on antituberculosis drugs). On comparing one culture medium with combination of the other two media, it was observed that BACTEC 12B medium missed 5 positive specimens detected by the other two media and detected 3 tuberculosis cases that were missed by the other two media. LJ medium gave a nearly similar performance, missing 6 positives but detecting 2 cases exclusively. Although MB7H9 broth could not detect 20 positive cases detected by the other two media, yet it was able to detect one case exclusively. In terms of average time taken for detection, BACTEC radiometric system proved to be fastest with average of 20 days, MB7H9 broth was slowest taking 6 weeks and EJ culture medium took an average of five weeks (Graph 1) DISCUSSION Considering the significance of culture in diagnosing mycobacterial diseases, various culture media and techniques are being evolved and evaluated world-wide to improve the

4 COMPARISON OF CULTURE MEDIA FOR MYCOBACTERIA 11 Graph 1: Average isolation time from each culture medium diagnostic standards. The introduction of different systems based on radiometric detection of growth, in various liquid culture media, represents an important step in providing a more rapid and efficient method than the conventional egg-based LJ solid culture medium 4-9. BACTEC system has been reported to be more sensitive and rapid, but certain inconveniences like need for syringe inoculation, use of radiolabeled products and cost have also been noted 10. The use of radiometric culture methods for confirmation of diagnosis of tuberculosis in childhood has been well documented 11. In our study, we observed maximum sensitivity, negative predictive value and diagnostic value for BACTEC 460 TB system and minimum for Middlebrook 7H9 broth culture. Values obtained by LJ medium were only marginally lower compared to those achieved by BACTEC method (Table 2). Table 2 : Evaluation of three culture media for isolation of mycobacteria Parameter LJ Medium BACTEC 12B medium MB7H9 broth Total positives Sensitivity(%) NPV(%) DA(%) Total specimens Total culture positive = 65 NPV = Negative predictive value DA = Diagnostic accuracy Among the earlier studies, isolation on BACTEC was 91% and with LJ 73%, while in another study it was 12.7% and 10.8% respectively; both reported a high contamination rate of 5% 12,13. We, however, found no contamination because all our specimens were treated by Petroff s method. Some authors also have used Petroff s method, but only for specimens like sputum and pus; body fluids like CSF & pleural fluids were not so treated, perhaps, causing contamination 6. A few other studies showing better positivity, sensitivity and faster recovery of mycobacterium by BACTEC as compared to egg based media have also been reported 10, Mean isolation time of mycobacteria by BACTEC method was reported to be 15 days compared to 28 days by the conventional method 13. We found that BACTEC system detected mycobacteria, on an average, days earlier than the LJ medium. BACTEC should be used along with LJ medium, because of its higher sensitivity and faster detection, to get best results, if the cost and maintenance of the system can be met. But BACTEC, as a substitute for conventional LJ medium is not recommended as the latter has nearly equal sensitivity. Some researchers have concluded that liquid media and Lowenstein Jensen medium must both be used. According to most, liquid media allow both faster detection of certain atypical mycobacteria and increased accuracy but are not suitable for growing tubercle bacilli 18. Middlebrook 7H9 broth.if used alone was found to be unsatisfactory for primary isolation of mycobacteria, although one case was isolated exclusively by it. Another study revealed isolation of

5 12 A. BHARGAVA ET AL three cases of mycobacterial origin exclusively by MB7H9 broth and this suggested the use of liquid medium in addition to an egg based medium 19 Therefore, in the case of non-availability of BACTEC 460TB system, MB7H9 broth is an economical and less cumbersome alternative method to be used simultaneously with LJ medium to increase the overall mycobactcrium detection by 3% from that if LJ is used alone (Table 3). Hence, simultaneous use of all the above three, or at least two of the three media (one liquid and one egg based solid medium) is recommended. Table3: Culture positivity rate with various combinations of three media Media Combinations Positivity All three combined (n 65) 100% HACTEC + LJ medium 98.5% HACTEC + MB 7H9 96.9% LJ medium + MB7H9 95.4% BACTEC alone 92.3% LJ medium alone 90.8% MD7H9 broth alone 69.2% REFERNCES 1 Baily GVI. I uhcrculosis control in India: Current problems and possible solutions; Ind J Tub, 1983, 30,45 2 Lahoiatory Practices for diagnosis of tuberculosis; Morbidity & Mortality : fk eavy /^o/v^/aw/^, 1995, 44(31) Procedures Tor the Isolation and Identification of Mycohnctcria, CDC. Publication, No edition. 4. Middlebrook. 0. Reggiardo. X. Tigret, WD. Automatable Radiometric Detection of Growth of Mycobacterium /;;/wcv(/mv.s in selective media,.-f»? M?v /?M /);.? Ginesu. I Pirina. I. Seehi, LA, llolicotli. P.. Santoru. L. Poeru. 1,.et al Microbiological diagnosis of tuberculosis - A comparison of old & new methods, J Chem;1998, 10(4) Giangc. JM. Diagnostic tests for tuberculosis & their evaluation, Seminars in Respiratory Infections, 1994,9(2), 71 7 Abe. ( I loso ima. S. Kukasawa. Y., Kaxsumi, Y., Takahasi, M Ilirano. K... c t a l. Comparison ofmb-check, HACII C and egg based media Cor recovery of m)cohactcria../(ym W/cvo Isenherg. III).. D Amalo. Rl\, lleifets. I,, Murray, PR.. Scandamaglia. M. Jacobs. ct al. CollaborativcTcasibility study of a biphase syslcni(rochc Scpti-Chck AFB) for rapid detection and isolation of mycobacleria; JC/in Micro Morgan. MA., Horslineier, CD.. DC Young Roberts, (if).. Comparison of a radiomelrie method (MACTKC) and conventional culture media Cor recovery of mycobaeteria from smear negative specimens,. J Clin Micro, , Carbonnellc, B., Carpcntier, E., Baunaud, R., Castets, M, Chippaux, (., Danjoux, MF., ct al. Use of BACTKC 460 TB method for the bacteriological diagnosis of luberculosis : Results of a multiccnlrc study: Pathologic Kiologie. 1995, 43(5) Schaaf, US., Nel, I D.. Beyers, N., Gie. RP.. Scott. I.. Donald. PR.. A decade of experience with Mycohaciernuu tuberculosis culture from children ^seasonal influence on incidence of childhood tuberculosis; Tubercle & l.ung Disease, (1), Anargyros, P.. Astill. OS., Lin, IS., Comparison of unproved BACTliC & Lowcnstcin Jcnscn medium for culture of mycobactcria from clinical specimens: J Clin Micro, 1990,28(6), Prcsslich, J.., Lahounik, E.. Krans, G., The BAC lir system in the diagnosis of tuberculosis: Comparison of a conventional & the radiometric method for culluring differentiation & susceptibility testing of mycobactcria: Zentralhlatl 1 Tir Bakleriologte, Mikrobiologie and Hygiene. Series A Medical Microbiologv, Infectious Diseases, Virology, Parasilology, (4) 14. Luquin, M., Gamboa, F., Barcelo. MG., Manterola, JM.. Matas. L.. (limene/, m., ct al: Comparison of a hiphasic non-radiomelric system with Lowcnstcin Jensen & BACTliC 460 system for recovery of mycobactcria from clinical specimens; Tubercle & l.ung Disease, (5), Palacios, J.L Oarcia. JM., I crro, J..Rodriguc/, J.. Sanchc/, A., Villar. H.. et al Comparison of MB-chcck and L.I medium for recovery of mycobactcria: Microscopy Research A Technique, 1997, 38(5) Rohncr, P..Ninct, B. Metral. C., Kmlcr. S., Auckcnthaler. R.. Evaluation of MB/Bac l system and comparison to the BACTKC 460 system and solid media for isolation of mycobactcria from clinical specimens:./ C Iin Micro, (2) Casal. N. Gutierre/..1.. Vaqucro, M.. Comparative evaluation of the mycobacterial growth indicator tube with the BACTKC 460 TB system and IJ medium for isolation of mycobactcria from clinical specimens. //;/./ Tub and imiigdis, 1997, 1(1) Allcrhcrger. I.. Fille, M.. Streif. \V., I.uef, (J., Schmut/hard. K., Dierich, MP.. Microbiological diagnosis of Tuberculosis; Wiener Medizinesche Woclienschnft, 1994, 144(8-9) Martin, T.. Chekc, D., Natyshak. I., Broth culture, the modem guinea pig for isolation of mycobactcria: Inbercle (1); 53