Background Mycobacterial culture and identification provide a definitive diagnosis of TB. Culture on L？wenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients. Methods and Findings The biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001). Conclusions The biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.
References
[1]
World Health Organization (2010) Global tuberculosis control - surveillance, planning, financing. World Health Organization. WHO/HTM/TB/2008.393.
[2]
World Health Organization (2010) Multidrug and extensively drug-resistant TB (M/XDR-TB): 2010 global report on surveillance and response. World Health Organization. WHO/HTM/TB/2010.3.
[3]
World Health Organization (2010) Anti-tuberculosis drug resistance in the world (Fourth Global Report). World Health Organization. WHO/HTM/TB/2008.394.
[4]
Keshavjee S, Gelmanova IY, Farmer PE, Mishustin SP, Strelis AK, et al. (2008) Treatment of extensively drug-resistant tuberculosis in Tomsk, Russia: a retrospective cohort study. The Lancet 372: 1403–1409.
[5]
Gandhi NR, Moll A, Sturm AW, Pawinski R, Govender T, et al. (2006) Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. The Lancet 368: 1575–1580.
[6]
Liberato IR, de Albuquerque MF, Campelo AR, de Melo HR (2004) Characteristics of pulmonary tuberculosis in HIV seropositive and seronegative patients in a Northeastern region of Brazil. Rev Soc Bras Med Trop 37: 46–50.
[7]
Caws M, Thwaites G, Stepniewska K, Nguyen TN, Nguyen TH, et al. (2006) Beijing genotype of Mycobacterium tuberculosis is significantly associated with human immunodeficiency virus infection and multidrug resistance in cases of tuberculous meningitis. J Clin Microbiol 44: 3934–3939.
[8]
World Health Organization (2009) A guide to monitoring and evaluation for collaborative TB/HIV activities. World Health Organization. WHO/HTM/TB/2009.414.
[9]
Raviglione MC, Narain JP, Kochi A (1992) HIV-associated tuberculosis in developing countries: clinical features, diagnosis, and treatment. Bull WHO 70: 515–526.
[10]
World Health Organization (1998) Laboratory services in TB control. Part III: Culture. World Health Organization. WHO/TB/98.258.
[11]
Bemer P, Palicova F, Rusch-Gerdes S, Drugeon HB, Pfyffer GE (2002) Multicenter evaluation of fully automated BACTEC Mycobacteria Growth Indicator Tube 960 system for susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 40: 150–154.
[12]
World Health Organization (2009) Treatment of tuberculosis: guidelines – 4th ed. World Health Organization. WHO/HTM/TB/2009.420.
[13]
Greco S, Rulli M, Girardi E, Piersimoni C, Saltini C (2009) Diagnostic accuracy of in-house PCR for pulmonary tuberculosis in smear-positive patients: meta-analysis and metaregression. J Clin Microbiol 47: 569–576.
[14]
International Union Against Tuberculosis and Lung Disease (2000) Technical guide. Sputum examination for tuberculosis by direct microscopy in low-income countries. Paris: International Union Against Tuberculosis and Lung Disease.
[15]
Pfyffer GE, Brown-Elliot BA, Wallace RJ (2003) General characteristics, isolation, and staining procedures. Manual of Clinical Microbiology. 8th edition. Washington, DC: ASM Press. pp. 532–559.
[16]
Rieder HL, Van Deun A, Kam KM, Kim SJ, Chonde TM, et al. (2007) Priorities for Tuberculosis Bacteriology Services in Low-Income Countries. International Union Against Tuberculosis and Lung Disease. ISBN: 2-914365-29-2.
[17]
Tengerdy RP, Nagy JG, Martin B (1967) Quantitative measurement of bacterial growth by the reduction of tetrazolium salts. Appl Microbiol 15: 954–955.
[18]
Hanna BA, Ebrahimzadeh A, Elliott LB, Morgan MA, Novak SM, et al. (1999) Multicenter evaluation of the BACTEC MGIT 960 system for recovery of mycobacteria. J Clin Microbiol 37: 748–752.
[19]
Giampaglia CM, Martins MC, Inumaru VT, Butuem IV, Telles MA (2005) Evaluation of a rapid differentiation test for the Mycobacterium tuberculosis complex by selective inhibition with rho-nitrobenzoic acid and thiophene-2-carboxylic acid hydrazide. Int J Tuberc Lung Dis 9: 206–209.
[20]
Angeby KA, Klintz L, Hoffner SE (2002) Rapid and inexpensive drug susceptibility testing of Mycobacterium tuberculosis with a nitrate reductase assay. J Clin Microbiol 40: 553–555.
[21]
Kumar M, Khan IA, Verma V, Kalyan N, Qazi GN (2005) Rapid, inexpensive MIC determination of Mycobacterium tuberculosis isolates by using microplate nitrate reductase assay. Diagn Microbiol Infect Dis 53: 121–124.
[22]
Affolabi D, Odoun M, Sanoussi N, Martin A, Palomino JC, et al. (2008) Rapid and inexpensive detection of multidrug-resistant Mycobacterium tuberculosis with the nitrate reductase assay using liquid medium and direct application to sputum samples. J Clin Microbiol 46: 3243–3245.
[23]
Collins L, Franzblau SG (1997) Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother 41: 1004–1009.
[24]
Jadaun GP, Agarwal C, Sharma H, Ahmed Z, Upadhyay P, et al. (2007) Determination of ethambutol MICs for Mycobacterium tuberculosis and Mycobacterium avium isolates by resazurin microtitre assay. J Antimicrob Chemother 60: 152–155.
[25]
Mirovic V, Lepsanovic Z (2002) Evaluation of the MB/BacT system for recovery of mycobacteria from clinical specimens in comparison to Lowenstein-Jensen medium. Clin Microbiol Infect 8: 709–714.
