Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays

Background Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. Methods and results Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × $ 10^{−4} $–1 × $ 10^{−5} $ (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × $ 10^{5} $) patient sample. Conclusions Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h. Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Wayengera, Misaki [verfasserIn] Mwebaza, Ivan Welishe, Johnson Bayiyana, Alice Kateete, David P. Wampande, Eddie Kirimunda, Samuel Kigozi, Edgar Katabazi, Fred Musubika, Carol Kyobe, Samuel Babirye, Peace Asiimwe, Benon Joloba, Moses L.

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2017

Schlagwörter:	Tuberculosis Immunodiagnosis Thymidylate Kinase Sputum Cultures

Anmerkung:	© The Author(s) 2017

Übergeordnetes Werk:	Enthalten in: BMC Research Notes - London, 2008, 10(2017), 1 vom: 08. Aug.
Übergeordnetes Werk:	volume:10 ; year:2017 ; number:1 ; day:08 ; month:08

Links:	Volltext

DOI / URN:	10.1186/s13104-017-2649-y

Katalog-ID:	SPR030322863

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100	1		\|a Wayengera, Misaki \|e verfasserin \|0 (orcid)0000-0001-7239-8079 \|4 aut
245	1	0	\|a Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays
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520			\|a Background Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. Methods and results Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × $ 10^{−4} $–1 × $ 10^{−5} $ (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × $ 10^{5} $) patient sample. Conclusions Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h.
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700	1		\|a Asiimwe, Benon \|4 aut
700	1		\|a Joloba, Moses L. \|4 aut
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allfields	10.1186/s13104-017-2649-y doi (DE-627)SPR030322863 (SPR)s13104-017-2649-y-e DE-627 ger DE-627 rakwb eng Wayengera, Misaki verfasserin (orcid)0000-0001-7239-8079 aut Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2017 Background Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. Methods and results Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × $ 10^{−4} $–1 × $ 10^{−5} $ (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × $ 10^{5} $) patient sample. Conclusions Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h. Tuberculosis (dpeaa)DE-He213 Immunodiagnosis (dpeaa)DE-He213 Thymidylate Kinase (dpeaa)DE-He213 Sputum (dpeaa)DE-He213 Cultures (dpeaa)DE-He213 Mwebaza, Ivan aut Welishe, Johnson aut Bayiyana, Alice aut Kateete, David P. aut Wampande, Eddie aut Kirimunda, Samuel aut Kigozi, Edgar aut Katabazi, Fred aut Musubika, Carol aut Kyobe, Samuel aut Babirye, Peace aut Asiimwe, Benon aut Joloba, Moses L. aut Enthalten in BMC Research Notes London, 2008 10(2017), 1 vom: 08. Aug. (DE-627)559431805 (DE-600)2413336-X 1756-0500 nnns volume:10 year:2017 number:1 day:08 month:08 https://dx.doi.org/10.1186/s13104-017-2649-y kostenfrei 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_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_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 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 10 2017 1 08 08
spelling	10.1186/s13104-017-2649-y doi (DE-627)SPR030322863 (SPR)s13104-017-2649-y-e DE-627 ger DE-627 rakwb eng Wayengera, Misaki verfasserin (orcid)0000-0001-7239-8079 aut Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2017 Background Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. Methods and results Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × $ 10^{−4} $–1 × $ 10^{−5} $ (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × $ 10^{5} $) patient sample. Conclusions Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h. Tuberculosis (dpeaa)DE-He213 Immunodiagnosis (dpeaa)DE-He213 Thymidylate Kinase (dpeaa)DE-He213 Sputum (dpeaa)DE-He213 Cultures (dpeaa)DE-He213 Mwebaza, Ivan aut Welishe, Johnson aut Bayiyana, Alice aut Kateete, David P. aut Wampande, Eddie aut Kirimunda, Samuel aut Kigozi, Edgar aut Katabazi, Fred aut Musubika, Carol aut Kyobe, Samuel aut Babirye, Peace aut Asiimwe, Benon aut Joloba, Moses L. aut Enthalten in BMC Research Notes London, 2008 10(2017), 1 vom: 08. Aug. (DE-627)559431805 (DE-600)2413336-X 1756-0500 nnns volume:10 year:2017 number:1 day:08 month:08 https://dx.doi.org/10.1186/s13104-017-2649-y kostenfrei 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_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_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 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 10 2017 1 08 08
allfields_unstemmed	10.1186/s13104-017-2649-y doi (DE-627)SPR030322863 (SPR)s13104-017-2649-y-e DE-627 ger DE-627 rakwb eng Wayengera, Misaki verfasserin (orcid)0000-0001-7239-8079 aut Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2017 Background Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. Methods and results Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × $ 10^{−4} $–1 × $ 10^{−5} $ (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × $ 10^{5} $) patient sample. Conclusions Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h. Tuberculosis (dpeaa)DE-He213 Immunodiagnosis (dpeaa)DE-He213 Thymidylate Kinase (dpeaa)DE-He213 Sputum (dpeaa)DE-He213 Cultures (dpeaa)DE-He213 Mwebaza, Ivan aut Welishe, Johnson aut Bayiyana, Alice aut Kateete, David P. aut Wampande, Eddie aut Kirimunda, Samuel aut Kigozi, Edgar aut Katabazi, Fred aut Musubika, Carol aut Kyobe, Samuel aut Babirye, Peace aut Asiimwe, Benon aut Joloba, Moses L. aut Enthalten in BMC Research Notes London, 2008 10(2017), 1 vom: 08. Aug. (DE-627)559431805 (DE-600)2413336-X 1756-0500 nnns volume:10 year:2017 number:1 day:08 month:08 https://dx.doi.org/10.1186/s13104-017-2649-y kostenfrei 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_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_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 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 10 2017 1 08 08
allfieldsGer	10.1186/s13104-017-2649-y doi (DE-627)SPR030322863 (SPR)s13104-017-2649-y-e DE-627 ger DE-627 rakwb eng Wayengera, Misaki verfasserin (orcid)0000-0001-7239-8079 aut Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2017 Background Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. Methods and results Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × $ 10^{−4} $–1 × $ 10^{−5} $ (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × $ 10^{5} $) patient sample. Conclusions Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h. Tuberculosis (dpeaa)DE-He213 Immunodiagnosis (dpeaa)DE-He213 Thymidylate Kinase (dpeaa)DE-He213 Sputum (dpeaa)DE-He213 Cultures (dpeaa)DE-He213 Mwebaza, Ivan aut Welishe, Johnson aut Bayiyana, Alice aut Kateete, David P. aut Wampande, Eddie aut Kirimunda, Samuel aut Kigozi, Edgar aut Katabazi, Fred aut Musubika, Carol aut Kyobe, Samuel aut Babirye, Peace aut Asiimwe, Benon aut Joloba, Moses L. aut Enthalten in BMC Research Notes London, 2008 10(2017), 1 vom: 08. Aug. (DE-627)559431805 (DE-600)2413336-X 1756-0500 nnns volume:10 year:2017 number:1 day:08 month:08 https://dx.doi.org/10.1186/s13104-017-2649-y kostenfrei 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_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_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 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 10 2017 1 08 08
allfieldsSound	10.1186/s13104-017-2649-y doi (DE-627)SPR030322863 (SPR)s13104-017-2649-y-e DE-627 ger DE-627 rakwb eng Wayengera, Misaki verfasserin (orcid)0000-0001-7239-8079 aut Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2017 Background Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. Methods and results Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × $ 10^{−4} $–1 × $ 10^{−5} $ (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × $ 10^{5} $) patient sample. Conclusions Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h. Tuberculosis (dpeaa)DE-He213 Immunodiagnosis (dpeaa)DE-He213 Thymidylate Kinase (dpeaa)DE-He213 Sputum (dpeaa)DE-He213 Cultures (dpeaa)DE-He213 Mwebaza, Ivan aut Welishe, Johnson aut Bayiyana, Alice aut Kateete, David P. aut Wampande, Eddie aut Kirimunda, Samuel aut Kigozi, Edgar aut Katabazi, Fred aut Musubika, Carol aut Kyobe, Samuel aut Babirye, Peace aut Asiimwe, Benon aut Joloba, Moses L. aut Enthalten in BMC Research Notes London, 2008 10(2017), 1 vom: 08. Aug. (DE-627)559431805 (DE-600)2413336-X 1756-0500 nnns volume:10 year:2017 number:1 day:08 month:08 https://dx.doi.org/10.1186/s13104-017-2649-y kostenfrei 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_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_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 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 10 2017 1 08 08
language	English
source	Enthalten in BMC Research Notes 10(2017), 1 vom: 08. Aug. volume:10 year:2017 number:1 day:08 month:08
sourceStr	Enthalten in BMC Research Notes 10(2017), 1 vom: 08. Aug. volume:10 year:2017 number:1 day:08 month:08
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	Tuberculosis Immunodiagnosis Thymidylate Kinase Sputum Cultures
isfreeaccess_bool	true
container_title	BMC Research Notes
authorswithroles_txt_mv	Wayengera, Misaki @@aut@@ Mwebaza, Ivan @@aut@@ Welishe, Johnson @@aut@@ Bayiyana, Alice @@aut@@ Kateete, David P. @@aut@@ Wampande, Eddie @@aut@@ Kirimunda, Samuel @@aut@@ Kigozi, Edgar @@aut@@ Katabazi, Fred @@aut@@ Musubika, Carol @@aut@@ Kyobe, Samuel @@aut@@ Babirye, Peace @@aut@@ Asiimwe, Benon @@aut@@ Joloba, Moses L. @@aut@@
publishDateDaySort_date	2017-08-08T00:00:00Z
hierarchy_top_id	559431805
id	SPR030322863
language_de	englisch
fullrecord	<?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01000caa a22002652 4500</leader><controlfield tag="001">SPR030322863</controlfield><controlfield tag="003">DE-627</controlfield><controlfield tag="005">20230519085444.0</controlfield><controlfield tag="007">cr uuu---uuuuu</controlfield><controlfield tag="008">201007s2017 xx \|\|\|\|\|o 00\| \|\|eng c</controlfield><datafield tag="024" ind1="7" ind2=" "><subfield code="a">10.1186/s13104-017-2649-y</subfield><subfield code="2">doi</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-627)SPR030322863</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(SPR)s13104-017-2649-y-e</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-627</subfield><subfield code="b">ger</subfield><subfield code="c">DE-627</subfield><subfield code="e">rakwb</subfield></datafield><datafield tag="041" ind1=" " ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="100" ind1="1" ind2=" "><subfield code="a">Wayengera, Misaki</subfield><subfield code="e">verfasserin</subfield><subfield code="0">(orcid)0000-0001-7239-8079</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2017</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">Text</subfield><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">Computermedien</subfield><subfield code="b">c</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">Online-Ressource</subfield><subfield code="b">cr</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="500" ind1=" " ind2=" "><subfield code="a">© The Author(s) 2017</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Background Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. Methods and results Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. 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author	Wayengera, Misaki
spellingShingle	Wayengera, Misaki misc Tuberculosis misc Immunodiagnosis misc Thymidylate Kinase misc Sputum misc Cultures Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays
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topic_title	Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays Tuberculosis (dpeaa)DE-He213 Immunodiagnosis (dpeaa)DE-He213 Thymidylate Kinase (dpeaa)DE-He213 Sputum (dpeaa)DE-He213 Cultures (dpeaa)DE-He213
topic	misc Tuberculosis misc Immunodiagnosis misc Thymidylate Kinase misc Sputum misc Cultures
topic_unstemmed	misc Tuberculosis misc Immunodiagnosis misc Thymidylate Kinase misc Sputum misc Cultures
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title	Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays
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title_full	Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays
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author_browse	Wayengera, Misaki Mwebaza, Ivan Welishe, Johnson Bayiyana, Alice Kateete, David P. Wampande, Eddie Kirimunda, Samuel Kigozi, Edgar Katabazi, Fred Musubika, Carol Kyobe, Samuel Babirye, Peace Asiimwe, Benon Joloba, Moses L.
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title_sort	immuno-diagnosis of mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays
title_auth	Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays
abstract	Background Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. Methods and results Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × $ 10^{−4} $–1 × $ 10^{−5} $ (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × $ 10^{5} $) patient sample. Conclusions Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h. © The Author(s) 2017
abstractGer	Background Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. Methods and results Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × $ 10^{−4} $–1 × $ 10^{−5} $ (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × $ 10^{5} $) patient sample. Conclusions Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h. © The Author(s) 2017
abstract_unstemmed	Background Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. Methods and results Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × $ 10^{−4} $–1 × $ 10^{−5} $ (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × $ 10^{5} $) patient sample. Conclusions Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h. © The Author(s) 2017
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title_short	Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays
url	https://dx.doi.org/10.1186/s13104-017-2649-y
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author2	Mwebaza, Ivan Welishe, Johnson Bayiyana, Alice Kateete, David P. Wampande, Eddie Kirimunda, Samuel Kigozi, Edgar Katabazi, Fred Musubika, Carol Kyobe, Samuel Babirye, Peace Asiimwe, Benon Joloba, Moses L.
author2Str	Mwebaza, Ivan Welishe, Johnson Bayiyana, Alice Kateete, David P. Wampande, Eddie Kirimunda, Samuel Kigozi, Edgar Katabazi, Fred Musubika, Carol Kyobe, Samuel Babirye, Peace Asiimwe, Benon Joloba, Moses L.
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up_date	2024-07-03T15:26:26.531Z
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Methods and results Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × $ 10^{−4} $–1 × $ 10^{−5} $ (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × $ 10^{5} $) patient sample. 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Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays

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