MANUSCRIPT JCM Revised. sputa subjected to long-term storage ACCEPTED

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1 JCM Accepts, published online ahead of print on 22 November 2006 J. Clin. Microbiol. doi: /jcm Copyright 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. MANUSCRIPT JCM Revised Usefulness of the BACTEC MGIT 960 system for isolation of Mycobacterium tuberculosis from sputa subjected to long-term storage Manuela Pardini 1, Francis Varaine 2, Maryline Bonnet 3, Graziella Orefici 1, Marco Rinaldo Oggioni 4, the LONG-DRUG Study Group 5 and Lanfranco Fattorini 1* Running title: MGIT 960 culture for M. tuberculosis recovery 1 Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Rome, Italy; 2 Médecins Sans Frontières, Paris, France; 3 Epicentre, Paris, France ; 4 Dipartimento di Biologia Molecolare, Università di Siena, Italy ; 5 The LONG-DRUG study group is composed by Marco Rinaldo Oggioni, Francesca Meacci and Viviana D Amato, Dipartimento di Biologia Molecolare, Università di Siena, Italy; Graziella Orefici, Lanfranco Fattorini and Manuela Pardini, Istituto Superiore di Sanità, Rome, Italy; Maryline Bonnet, Epicentre, Paris, France; Peter W. Andrew, Mike Barer, Hasan Yesilkaya, University of Leicester, United Kingdom; Heinz Rinder, LGL, Oberschleiβheim, Germany; Sabine Rüsch-Gerdes and Stefan Niemann, Research Centre Borstel, Germany; Germano Orrù, University of Cagliari, Italy; Francis Varaine, Médecins Sans Frontières, Paris, France; Thierry Jarosz, 3Es, Paris, France. * Corresponding author: Dr Lanfranco Fattorini, Dipartimento di Malattie Infettive, Parassitarie e Immunomediate Istituto Superiore di Sanità Viale Regina Elena Rome Italy Tel: Fax: lanfranco.fattorini@iss.it

2 Summary Recovery of Mycobacterium tuberculosis from sputa positive or negative for acid-fast bacilli stored for 17 ± 7 days and inoculated in BACTEC MGIT 960 system (MGIT) was higher than that from sputa inoculated in Lowenstein-Jensen medium. MGIT is useful for isolation of M. tuberculosis from sputa subjected to long-term storage. 2

3 The transportation of sputa for isolation of Mycobacterium tuberculosis (Mtb) from remote settings to laboratories located abroad usually takes more than one week and results in increased contamination rate and a loss of positive cultures (3, 6). Growth detection of Mtb from samples inoculated in solid media such Lowenstein-Jensen (LJ) may take several weeks, with a delay in patient treatment. The BACTEC MGIT 960 system (MGIT) is a fully automated, nonradiometric, faster technique in liquid medium largely used for Mtb recovery, but few investigations have been reported on its usefulness for the culture of sputa subjected to long-term storage (1, 3). Sample preservatives such as cetyl-pyridium chloride (CPC) are shown to be useful for Mtb isolation on LJ from sputa subjected to long-term storage (1, 6). Using MGIT we found that the addition of CPC to the sputa did not significantly increase Mtb yield (data not shown). In the present study, the comparison of Mtb recovery and contamination rate from long-term stored sputa without CPC inoculated in LJ and MGIT was performed. Sputa were collected by the humanitarian medical aid agency Médecins Sans Frontières (MSF) at Guliripchi Tuberculosis Hospital, Sukhumi, Abkhazia, a region of the former Soviet Union, from December 2003 to December Smear microscopy by the Ziehl-Neelsen method was performed on all unconcentrated specimens and reported as smear negative (S-) or smear positive (S+) (range, 1+ to 4+) (6). Sputa were kept refrigerated at +4 C at the hospital of Sukhumi, sent by car in groups of 20 to 30 per parcel to the office of MSF at Tbilisi and finally mailed by an express courier to the Istituto Superiore di Sanità, Rome, for culture; overall, the mean time ± standard deviation between collection and sample processing was 17 ± 7 days (range, 7 to 39 days). Sputa were processed blinded by the N-acetyl-L-cysteine-NaOH (NALC) method using a commercial kit (MycoPrep, Becton Dickinson, Cockeysville, Md); the sediment was suspended in PBS and inoculated in LJ (100 µl) (Biomérieux, Marcy l Etoile, France) and BACTEC MGIT 960 tubes (Becton-Dickinson) (500 µl) according to the manufacturer s instructions. The LJ slants were incubated at 37 C in 5% CO 2 and examined weekly for 8 weeks. Mtb was identified in positive cultures by DNA probe (Gene Probe, San Diego, Calif.) (5, 6). A total of 1101 sputa including 344 baseline samples from 261 patients and 757 follow-up samples from 555 patients on anti-tb treatment were examined (Table 1). Inoculation of (S+)-sputa in MGIT significantly increased Mtb recovery from 50 to 66% and decreased the contamination rate from 33 to 13%, in comparison with LJ. In particular, positivity increased from 62 to 82% for baseline samples and from 39 to 50% for follow-up samples; the contamination rate decreased from 35 to 14% for baseline samples and from 30 to 13% for follow-up samples, in comparison with LJ. When the combination of LJ and MGIT media was considered, Mtb recovery increased from 2 to 3%, in comparison with MGIT alone. 3

4 A similar trend was observed for S(-) sputa, with Mtb recovery increasing from 19 to 47% and the contamination rate decreasing from 41 to 13%, in baseline samples, in comparison with LJ. In S(-) samples collected during the follow-up, culture positivity was higher in MGIT than in LJ, but the difference did not reach statistically significant values. The contamination rate of S(-) sputa was significantly lower for MGIT than for LJ and decreased by 2% or more when the combination of LJ and MGIT media was considered. Correct collection and transportation of sputa is crucial to ensure accurate Mtb recovery from remote survey settings (3). Overall, our results indicated that the rate of Mtb recovery by MGIT, or LJ plus MGIT, of S+ or S- long-term stored sputa was clearly higher than in LJ. Positivity rate by MGIT of our S+ samples was lower than that reported for routinely processed S+ samples cultured in the same medium (7). This can be explained by the knowledge that increasing storage time at room temperature reduces Mtb viability and increases contamination (4). After collection samples were kept refrigerated, but ground transportation and mailing from Sukhumi to Rome occurred at room temperature and took several days; this period may have affected Mtb viability and contamination. Contamination rates similar to those found by us were also reported by other investigators when late arrival of the specimens delayed sample processing (7). The use of one liquid and one solid medium is recommended by the Centers for Disease Control and prevention (2). Indeed, when the combination of LJ and MGIT media was considered, overall positivity increased (68%) and contamination rate decreased (11%). We are aware that comparative studies between long-term stored and routinely examined samples are difficult because they are affected by several variables. When data were stratified for baseline and follow-up samples, positivity rate of S+ baseline samples processed by LJ plus MGIT was 85%, indicating that this method is helpful to increase Mtb isolation from long-term stored sputa. MGIT was very useful for Mtb recovery also for S- sputa, either at baseline or follow-up. In the baseline samples, Mtb isolation by MGIT (or LJ plus MGIT) increased from 19 to 47% compared to LJ, and in the follow-up was similar to that reported by other investigators (8). From the clinical point of view this observation is very important since S- patients, either at the baseline or during follow-up, have a higher chance to be treated with anti-tb therapy if culture is performed in MGIT plus LJ, in comparison with LJ alone. Overall, our results indicate that MGIT is a suitable liquid method to culture Mtb from samples collected in remote areas of the world. 4

5 Acknowledgements We thank Marco Pataracchia for technical assistance. This research was supported in part by European Community grant QLK-CT (LONG-DRUG study). 5

6 References 1. Bobadilla-del-Valle, M., A. Ponce-de-Leon, M. Kato-Maeda, A. Hernandez-Cruz, J.J. Calva-Mercado, B. Chavez-Mazari, B.A. Caballero-Rivera, J.C. Nolasco-Garcia, and J. Sifuentes-Osornio Comparison of sodium carbonate, cetyl-pyridinium chloride, and sodium borate for preservation of sputa for culture of Mycobacterium tuberculosis. J Clin Microbiol. 41: Centers for Disease Control and Prevention Essential components of a tuberculosis prevention and control program. Morbid. Mortal. Weekly Rep. 44-RR: Lumb, R., M. Ardian, G. Waramori, H. Syahrial, E. Tjitra, G. P. Maguire, N. M. Anstey, and P. M. Kelly An alternative method for sputum storage and transport for Mycobacterium tuberculosis drug resistance surveys. Int. J. Tuberc. Lung. Dis. 10: Paramavisan, C. N., A. S. L. Narayana, R. Prabhakar, M. S. Rajagopal, P. R. Somasundaram, and S. P. Tripathy Effect of storage of sputum specimens at room temperatureon smear and culture results. Tubercle. 64: Pardini, M., E. Iona, F. Varaine, H. Karakozian, H. Arzumanian, L. Brunori, G. Orefici, the LONG-DRUG Study Group, and L. Fattorini Mycobacterium tuberculosis drug resistance, Abkhazia. Emerg. Infect. Dis. 11: Pardini, M., F. Varaine, E. Iona, E. Arzumanian, F. Checchi, M. R. Oggioni, G. Orefici, and L. Fattorini Cetyl-pyridinium chloride is useful for isolation of Mycobacterium tuberculosis from sputa subjected to long-term storage. J. Clin. Microbiol. 43: Pfyffer, G. E., H. M. Welscher, P. Kissling, C. Cieslak, M. J. Casal, J. Gutierrez, and S. Rüsch-Gerdes Comparison of the Mycobacteria Growth Indicator Tube (MGIT) with radiometric and solid culture for recovery of acid-fast bacilli. J. Clin. Microbiol. 35: Ramarokoto, H., H. Randriamiharisoa, A. Rakotoarisaonina, T. Rasolovavalona, V. Rasolofo, S. Chanteau, M. Ralamboson, B. Cauchoix, and D. Rakotondramarina Bacteriological follow-up of tuberculosis treatment: a comparative study of smear microscopy and culture results at the second month of treatment. Int. J. Tuberc. Lung. Dis. 6:

7 Table 1: Comparison of smears and overall culture results Group (No. of samples) All samples (1101) Baseline (344) Follow-up (757) Smear result No. of samples LJ No. (%) of positive cultures MGIT No. (%) of contaminated cultures Combined LJ/MGIT a LJ MGIT Combined LJ/MGIT b S (9) 60 (13) * 61 (13) * 138 (30) 54 (12) ** 43 (9) ** S (50) 422 (66) ** 440 (68) ** 211 (33) 86 (13) ** 71 (11) ** S (19) 15 (47) * 15 (47) * 13 (41) 4 (13) ** 2 (6) ** S (62) 257 (82) ** 264 (85) ** 110 (35) 43 (14) ** 39 (13) ** S (8) 45 (11) 47 (11) 125 (29) 50 (12) ** 41 (10) ** S (39) 165 (50) ** 176 (53) ** 101 (30) 43 (13) ** 32 (10) ** ( a ), LJ and/or MGIT cultures were positive for Mtb; ( b ), both LJ and MGIT cultures were contaminated. (*), P<0.05; (**), P<0.01, versus LJ ( Fisher s exact text) 7

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