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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.

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 × 105) 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.

SUBMITTER: Wayengera M

PROVIDER: S-EPMC5549350 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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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.

Wayengera Misaki M Mwebaza Ivan I Welishe Johnson J Bayiyana Alice A Kateete David P DP Wampande Eddie E Kirimunda Samuel S Kigozi Edgar E Katabazi Fred F Musubika Carol C Kyobe Samuel S Babirye Peace P Asiimwe Benon B Joloba Moses L ML

BMC research notes 20170808 1

<h4>Background</h4>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 th ...[more]

PMID: 28789704

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Project description:Clinical and laboratory diagnosis of Active Tuberculosis (ATB) and latent Mycobacterium Tuberculosis (M. tuberculosis) infections (LTBI) among people living with HIV/AIDS (PLWHA) presents formidable challenges. In the past, WHO issued an advisory against the use of existing TB sero-diagnostics. Emerging evidence, however, points to a precision of TB sero-diagnostics based on secretory rather than structural M. tuberculosis antigens. We hypothesized that secretory levels of M. tuberculosis thymidylate kinase (TMKmt) can Designate ATBI from LTBI and no TB (NTB). Here, we report in-house validation studies of levels of TMKmt antigen (Ag) and host specific TMKmt antibody (Ab) amongst HIV +ve and HIV -ve participants. Direct TMKmt Ag and host specific IgG Ab detection EIAs were conducted on broadly consented, stored serum (N=281[Ag] vs. 214 [Ab] respective) samples stratified as either HIV +ve or HIV-ve ATB relative to LTBI and No TB. On one hand, UG-peptide 1 and its PAb-based EIAs accurately diagnosed ATB relative to LTBI and NTB among HIV +ve subjects {irrespectively: (a) Ag detection ATB=OD>0.490; 95% CI: 0.7446 to 0.8715 vs. LTBI=OD<0.490; 95% CI 0.4325 to 0.4829 vs. NTB=OD<0.26; 95% CI 0.1675 to 0.2567 and (b) TMKmt specific IgG detection ATB=OD>1.00; 95% CI 1.170 to 1.528 [HIV +ve] and 2.044 to 2.978 [HIV -ve] respectively vs. LTBI=OD<1.00; 95% CI 0.2690 to 0.6396 vs. NTB=OD<; 95% CI 0.1527 to 0.8751}. HIV -ve ATB presented with Ag levels greater than NTB and less than LTBI (i.e. ATB -ve=<0.490 ODs>0.26), but displayed better ant-TMKmt IgG responses (OD>2.00; 95% CI 2.044 to 2.978) relative to HIV +ve ATB (OD<1.600; 95% CI 1.170 to 1.528); suggesting a better control of M. tuberculosis-septicemia. On the other hand, UG-peptide 2 and its PAb-based EIAs did not demonstrate ATB diagnostic potential regardless of HIV sero-status, except towards designating NTB. TMKmt Ab and Ag detecting EIAs based on UG-peptide 1 and its derivative PAb can accurately demarcate ATB from LTBI and NTB among HIV +ve subjects.

Project description:Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.

Project description:BackgroundThe Abbott RealTime MTB is an assay for the detection of Mycobacterium tuberculosis (MTB) complex DNA from respiratory specimens in combination with the Abbott RealTime RIF/INH assay for the detection of genetic resistance markers for isoniazid (INH) and rifampicin (RIF) from MTB positive isolates. Hence, this study aimed to evaluate the performance of the Abbott RealTime MTB and RIF/INH assays.MethodsA cross-sectional study was conducted on 289 study subjects presumptive to have pulmonary tuberculosis at Nigist Eleni Mohammed Memorial Hospital, South Ethiopia from April 2017 to June 2018. Two morning expectorated sputum specimens were collected from each study participant. One sample was tested directly by Xpert MTB/RIF assay at Nigist Eleni Mohammed Memorial Hospital and the other sample was used for smear microscopy, TB culture, Abbott RealTime MTB, and Abbott RealTime INH/RIF assays at International Clinical Laboratories, Addis Ababa, Ethiopia. The diagnostic performance of the Abbott RealTime MTB and INH/RIF assays were calculated against MGIT liquid culture and phenotypic drug susceptibility testing (DST) as the gold standard.ResultsFor the detection of MTB the Abbott RealTime MTB assay exhibited sensitivity 92.4% (95% CI 83.6-96.9), specificity 95.4% (95% CI 91.1-97.7), PPV 89.0% (95% CI 79.7-94.5) and NPV 96.9% (95% CI 93.0-98.7). For the detection of RIF resistance MTB, Abbott RealTime MTB RIF/INH concurred with phenotypic DST and Xpert MTB/RIF, while for the detection of INH resistance MTB, the sensitivity, specificity, PPV and NPV of the Abbott MTB RIF/INH assay was 84.2% (95% CI 60.4-96.6), 100% (95% CI 89.7-100), 100% and 91.9% (95% CI 80.0-96.9), respectively.ConclusionsThe Abbott RTMTB and RIF/INH assays revealed high sensitivity and specificity in MTB diagnosis and provided reliable INH and RIF resistance profiles. This assay has a similar diagnostic performance to the Xpert MTB/RIF assay with the advantages of high-throughput.

Dataset Information

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.

Publications

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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