Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR

Background Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess...
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Autor*in:	Pathak, Divya [verfasserIn] Chakravorty, Soumitesh Hanif, Mahmud Tyagi, Jaya Sivaswami

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2007

Schlagwörter:	Bacterial Load Supernatant Fraction Bacterial Lysis Smear Microscopy Sputum Specimen

Anmerkung:	© Pathak et al; licensee BioMed Central Ltd. 2007. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (

Übergeordnetes Werk:	Enthalten in: BMC microbiology - London : BioMed Central, 2001, 7(2007), 1 vom: 04. Sept.
Übergeordnetes Werk:	volume:7 ; year:2007 ; number:1 ; day:04 ; month:09

Links:	Volltext

DOI / URN:	10.1186/1471-2180-7-83

Katalog-ID:	SPR027177696

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520			\|a Background Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage. Results Eighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20°C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples. Conclusion We conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment.
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700	1		\|a Tyagi, Jaya Sivaswami \|4 aut
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allfields	10.1186/1471-2180-7-83 doi (DE-627)SPR027177696 (SPR)1471-2180-7-83-e DE-627 ger DE-627 rakwb eng Pathak, Divya verfasserin aut Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR 2007 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Pathak et al; licensee BioMed Central Ltd. 2007. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage. Results Eighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20°C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples. Conclusion We conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment. Bacterial Load (dpeaa)DE-He213 Supernatant Fraction (dpeaa)DE-He213 Bacterial Lysis (dpeaa)DE-He213 Smear Microscopy (dpeaa)DE-He213 Sputum Specimen (dpeaa)DE-He213 Chakravorty, Soumitesh aut Hanif, Mahmud aut Tyagi, Jaya Sivaswami aut Enthalten in BMC microbiology London : BioMed Central, 2001 7(2007), 1 vom: 04. Sept. (DE-627)326644997 (DE-600)2041505-9 1471-2180 nnns volume:7 year:2007 number:1 day:04 month:09 https://dx.doi.org/10.1186/1471-2180-7-83 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2007 1 04 09
spelling	10.1186/1471-2180-7-83 doi (DE-627)SPR027177696 (SPR)1471-2180-7-83-e DE-627 ger DE-627 rakwb eng Pathak, Divya verfasserin aut Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR 2007 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Pathak et al; licensee BioMed Central Ltd. 2007. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage. Results Eighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20°C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples. Conclusion We conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment. Bacterial Load (dpeaa)DE-He213 Supernatant Fraction (dpeaa)DE-He213 Bacterial Lysis (dpeaa)DE-He213 Smear Microscopy (dpeaa)DE-He213 Sputum Specimen (dpeaa)DE-He213 Chakravorty, Soumitesh aut Hanif, Mahmud aut Tyagi, Jaya Sivaswami aut Enthalten in BMC microbiology London : BioMed Central, 2001 7(2007), 1 vom: 04. Sept. (DE-627)326644997 (DE-600)2041505-9 1471-2180 nnns volume:7 year:2007 number:1 day:04 month:09 https://dx.doi.org/10.1186/1471-2180-7-83 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2007 1 04 09
allfields_unstemmed	10.1186/1471-2180-7-83 doi (DE-627)SPR027177696 (SPR)1471-2180-7-83-e DE-627 ger DE-627 rakwb eng Pathak, Divya verfasserin aut Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR 2007 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Pathak et al; licensee BioMed Central Ltd. 2007. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage. Results Eighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20°C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples. Conclusion We conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment. Bacterial Load (dpeaa)DE-He213 Supernatant Fraction (dpeaa)DE-He213 Bacterial Lysis (dpeaa)DE-He213 Smear Microscopy (dpeaa)DE-He213 Sputum Specimen (dpeaa)DE-He213 Chakravorty, Soumitesh aut Hanif, Mahmud aut Tyagi, Jaya Sivaswami aut Enthalten in BMC microbiology London : BioMed Central, 2001 7(2007), 1 vom: 04. Sept. (DE-627)326644997 (DE-600)2041505-9 1471-2180 nnns volume:7 year:2007 number:1 day:04 month:09 https://dx.doi.org/10.1186/1471-2180-7-83 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2007 1 04 09
allfieldsGer	10.1186/1471-2180-7-83 doi (DE-627)SPR027177696 (SPR)1471-2180-7-83-e DE-627 ger DE-627 rakwb eng Pathak, Divya verfasserin aut Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR 2007 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Pathak et al; licensee BioMed Central Ltd. 2007. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage. Results Eighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20°C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples. Conclusion We conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment. Bacterial Load (dpeaa)DE-He213 Supernatant Fraction (dpeaa)DE-He213 Bacterial Lysis (dpeaa)DE-He213 Smear Microscopy (dpeaa)DE-He213 Sputum Specimen (dpeaa)DE-He213 Chakravorty, Soumitesh aut Hanif, Mahmud aut Tyagi, Jaya Sivaswami aut Enthalten in BMC microbiology London : BioMed Central, 2001 7(2007), 1 vom: 04. Sept. (DE-627)326644997 (DE-600)2041505-9 1471-2180 nnns volume:7 year:2007 number:1 day:04 month:09 https://dx.doi.org/10.1186/1471-2180-7-83 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2007 1 04 09
allfieldsSound	10.1186/1471-2180-7-83 doi (DE-627)SPR027177696 (SPR)1471-2180-7-83-e DE-627 ger DE-627 rakwb eng Pathak, Divya verfasserin aut Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR 2007 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Pathak et al; licensee BioMed Central Ltd. 2007. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage. Results Eighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20°C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples. Conclusion We conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment. Bacterial Load (dpeaa)DE-He213 Supernatant Fraction (dpeaa)DE-He213 Bacterial Lysis (dpeaa)DE-He213 Smear Microscopy (dpeaa)DE-He213 Sputum Specimen (dpeaa)DE-He213 Chakravorty, Soumitesh aut Hanif, Mahmud aut Tyagi, Jaya Sivaswami aut Enthalten in BMC microbiology London : BioMed Central, 2001 7(2007), 1 vom: 04. Sept. (DE-627)326644997 (DE-600)2041505-9 1471-2180 nnns volume:7 year:2007 number:1 day:04 month:09 https://dx.doi.org/10.1186/1471-2180-7-83 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2007 1 04 09
language	English
source	Enthalten in BMC microbiology 7(2007), 1 vom: 04. Sept. volume:7 year:2007 number:1 day:04 month:09
sourceStr	Enthalten in BMC microbiology 7(2007), 1 vom: 04. Sept. volume:7 year:2007 number:1 day:04 month:09
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	Bacterial Load Supernatant Fraction Bacterial Lysis Smear Microscopy Sputum Specimen
isfreeaccess_bool	false
container_title	BMC microbiology
authorswithroles_txt_mv	Pathak, Divya @@aut@@ Chakravorty, Soumitesh @@aut@@ Hanif, Mahmud @@aut@@ Tyagi, Jaya Sivaswami @@aut@@
publishDateDaySort_date	2007-09-04T00:00:00Z
hierarchy_top_id	326644997
id	SPR027177696
language_de	englisch
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author	Pathak, Divya
spellingShingle	Pathak, Divya misc Bacterial Load misc Supernatant Fraction misc Bacterial Lysis misc Smear Microscopy misc Sputum Specimen Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR
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topic_title	Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR Bacterial Load (dpeaa)DE-He213 Supernatant Fraction (dpeaa)DE-He213 Bacterial Lysis (dpeaa)DE-He213 Smear Microscopy (dpeaa)DE-He213 Sputum Specimen (dpeaa)DE-He213
topic	misc Bacterial Load misc Supernatant Fraction misc Bacterial Lysis misc Smear Microscopy misc Sputum Specimen
topic_unstemmed	misc Bacterial Load misc Supernatant Fraction misc Bacterial Lysis misc Smear Microscopy misc Sputum Specimen
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title	Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR
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title_full	Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR
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author_browse	Pathak, Divya Chakravorty, Soumitesh Hanif, Mahmud Tyagi, Jaya Sivaswami
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title_sort	lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by pcr
title_auth	Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR
abstract	Background Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage. Results Eighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20°C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples. Conclusion We conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment. © Pathak et al; licensee BioMed Central Ltd. 2007. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
abstractGer	Background Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage. Results Eighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20°C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples. Conclusion We conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment. © Pathak et al; licensee BioMed Central Ltd. 2007. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
abstract_unstemmed	Background Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage. Results Eighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20°C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples. Conclusion We conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment. © Pathak et al; licensee BioMed Central Ltd. 2007. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
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title_short	Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR
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Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR

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