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[Identification of mycobacteria by the acridinium-ester labeled DNA probes for M. tuberculosis and M. avium-intracellulare complex in culture and its clinical application].

The detectability of mycobacterium in culture by non-isotopic, chemiluminescent DNA probes for Mycobacterium tuberculosis (M. tuberculosis) and M. avium-intracellulare complex (MAC) was evaluated and compared with that by 125I-labeled DNA probe for the same mycobacteria. The sensitivity and specificity of the AE-DNA probes for MAC were 97.2% and 100%, respectively, for the conventional method and were both 100% for the 125I-labeled DNA probes. The detection limits of the AE-DNA probes tests for both M. tuberculosis and MAC were 10(5)-10(6) CFU/tube which were almost the same as those of the 125I-labeled DNA probes tests. Because the procedure is simple, rapid (it can be completed within 90 min), and safe (it does not use radioisotopes), it can be easily performed in any clinical laboratory.

Acridines

[Differentiation of M. tuberculosis and M. avium complex using various monoclonal antibodies].

M. avium-complex (MAC) is the cause of the most bacterial infections in AIDS patients. Because of the high resistance of MAC, a rapid differentiation between M. tuberculosis and MAC is of great interest. In an enzyme-linked immunosorbent assay (ELISA) we tested three monoclonal antibodies BS 103, BS 104, BS 113 and the combination of BS 103/BS 113, which bind selectively to the cell wall of M. tuberculosis. 98 MAC isolates from AIDS patients and 233 M. tuberculosis isolates from patients with lung disease were tested after primary culture of the bacteria on Lowenstein-Jensen media. ELISA could clearly discriminate MAC from M. tuberculosis with a specificity of 100% and a sensitivity of 98-100%. With these monoclonal antibodies the time for differentiation can be reduced by several weeks.

Antibodies, Monoclonal

Potency of PPD preparations derived from M. tuberculosis and M. bovis.

No significant difference was found between the biological activity of PPD-preparations derived from M. tuberculosis and M. bovis, although the latter was more potent. In potency tests performed in guinea-pigs, the use of difference mycobacteria as sensitizing agents had no influence on the result of the assay. We could not demonstrate a significantly greater potency of homologous versus heterologous PPD. Evaluation of the 95 p.c. fiducial limits established in the course of variance analysis suggested a greater reliability of reading at 48 than at 24 hrs after allergen application. It also seems possible that the results of the potency assays of PPDs derived from different mycobacterium strains are greatly influenced by the level of the working dilutions employed.

Animals

Detection and identification of Mycobacterium tuberculosis, M. bovis/BCG, and M. avium by two-step polymerase chain reaction. Comparison with ELISA using A60 antigen.

We propose a rapid two-step PCR to amplify a 767-bp sequence present in the gene coding for the 65-kD antigen of mycobacteria. The high G+C content (80%) permitted annealing to occur at 70 degrees C, enhancing the specificity. The amplified fragment contains a restriction site for differentiation between M. tuberculosis, M. bovis/BCG, and M. avium. Complete diagnosis can be achieved in less than four hours without labelled probe or nucleic acid transfer.

Antigens, Bacterial

[Genetic engineering production of H37Rv M. tuberculosis proteins].

A complete library of M. tuberculosis H37Rv genes was produced by incorporating the DNA fragments of M. tuberculosis H37Rv into the lambda pSI phasmid. For this, DNA isolated from the mycobacteria was treated by EcoRI restrictases and the fragments of 8-17 thousand nucleotide pairs were crosslinked with the phasmid DNA. Hybrid DNA molecules were packed into the caspids from the proteins of E. coli BHB2688 and BHB2690 strains. By estimates, this library contained 98% of M. tuberculosis H37Rv genome so that any required gene can be found at 0.99 probability. The needed genes were sought by monoclonal antibodies against a protein with a molecular mass of 17-19 kDa (IT-12, IT-51, IT-54) obtained from the WHO. The protein gene was also produced by the method for raising the end sequences using synthesis of two oligonucleotides SP30 complementary to segments that limit this gene in the presence of Taq-DNA-polymerase and DNA of M. tuberculosis H37Rv. Copies of a gene (MT-1) were produced by denaturation, firing and raising. This gene was incorporated in the puC119 plasmid and expressed in E. coli cells, the direction of reading being checked up. A protein with a molecular mass of 19 kDa was detected in E. coli extracts with the expressed pMT-2 plasmid using monoclonal antibodies IT-12 and IT-54 in enzyme-linked immunoassay and immunoblotting.

Antibodies, Monoclonal

[Isolation of M. tuberculosis in abdominal specimens].

The isolation of mycobacteria in abdominal specimens during a 10 years period is presented. Twenty-three clinical cases have been reviewed; patients were divided in three groups: 1) Peritoneal and intestinal tuberculosis. 2) Pulmonary tuberculosis with isolation of M. tuberculosis in feces, and 3) Miliary tuberculosis. We emphasize the low yielding of bacilloscopy, the low number of colonies in cultures and the importance of the microbiological study of abdominal specimens in the confirmatory diagnosis. The predominant symptoms of peritoneal tuberculosis were abdominal pain and distention and fever. The study of the ascitic fluid showed in most of the cases lymphocytic exudate and the pathological study of biopsies showed granulomas with caseous necrosis. Three patients had another associated abdominal disease. Isolation of M. tuberculosis in feces does not invariably mean the presence of intestinal tuberculosis. We confirm the frequent association of disseminated tuberculosis and HIV1 infection.

Abdomen

[Production and characteristics of monoclonal antibodies reacting with human-type M. tuberculosis].

Monoclonal antibodies to M. tuberculosis were produced by hybrid technology. They were described via enzyme immunoassay and immunoblotting. It was shown that these antibodies should be included into IgG class. Besides, they are oriented towards and antigen with a molecular weight of 20 kDa, react with H37Rv at a concentration of 12 ng/ml and with BCG at a concentration of 50 micrograms/ml and fail to react with M. Intracellulare, M. Scrofulaceum and E. coli.

Animals

Thiacetazone: in vitro activity against Mycobacterium avium and M. tuberculosis.

Bacteriostatic and bactericidal activity of thiacetazone was determined for 68 M. avium clinical isolates and 14 wild drug-susceptible M. tuberculosis strains. The drug had equally low bactericidal activity against both mycobacterial species. The inhibitory activity against most of the M. avium strains was greater than it was against M. tuberculosis. The broth determined MICs for 65 of 68 M. avium strains were between 0.02 and 0.15 micrograms/ml, while the MICs for M. tuberculosis ranged from 0.08 to 1.2 micrograms/ml.

Dose-Response Relationship, Drug

[Resistance testing of M. avium-intracellulare and M. tuberculosis of AIDS patients with new drugs and drug combinations].

The minimal inhibitory concentration (MIC) of rifabutin for M. tuberculosis was 0.006 to 0.06 micrograms/ml, and 0.12 to 0.25 micrograms/l for clofazimine. Accordingly, M. tuberculosis is inhibited by concentrations of these two medications that are far lower than the levels normally found in the serum. In the case of M. avium, the MIC of the new drugs such as rifabutin and clofazimine are, in contrast to the MICs for M. tuberculosis, merely of the order of the achievable serum concentrations. The minimum bactericidal concentrations of these two substances are much higher than the bacteriostatic concentrations, which probably explains the frequent therapeutic failures, while in the case of ciprofloxacin, the prevailing situation is much more favourable. The growth of all M. avium strains is inhibited (= sensitive) when elevated concentrations (double "breakpoint" concentrations) of a triple-drug combination comprising rifampicin, ethambutol and ciprofloxacin, or a combination of ethambutol, rifampicin, ciprofloxacin and prothionamid are tested at "normal breakpoint" concentrations.

Acquired Immunodeficiency Syndrome

A tiled amplicon protocol for culture-free whole-genome sequencing of M. tuberculosis from clinical specimens.

Whole-genome sequencing of Mycobacterium tuberculosis can be a valuable tool for TB surveillance and treatment, providing insights into transmission patterns and comprehensive drug susceptibility testing. However, the slow growth of M. tuberculosis means traditional culture-based sequencing methods can take weeks to return results, which has limited the widespread adoption of these techniques and limited their use in clinical decision-making. Tiled amplicon sequencing is a fast, reliable, and cost-effective method of whole-genome sequencing that can be done directly on clinical specimens and has been implemented at scale in academic and public health laboratories across the world; it was the cornerstone of SARS-CoV-2 sequencing and has been adapted for a wide range of viral pathogens. However, similar methods are not yet available for far larger bacterial genomes. Extending this approach to M. tuberculosis would significantly reduce the cost, labor, and turnaround time for whole-genome sequencing. We designed a tiled amplicon panel consisting of 5,128 primers that covers the entire M. tuberculosis genome, the largest tiled amplicon sequencing panel we are aware of to date. Applying our amplicon panels to clinical samples of sputum, we show the ability to recover whole-genome bacterial sequences without the need for culture. The resulting sequence data can be used to determine M. tuberculosis lineage and reliably identify markers of drug resistance. Using this approach in clinical settings could reduce the time needed for comprehensive drug susceptibility testing from weeks to days and enable genomic epidemiology to be performed at scale, even in resource-limited settings.IMPORTANCEWe have developed and tested an amplicon panel, TB-seq, for the priority pathogen Mycobacterium tuberculosis, demonstrating recovery of near-full genomes directly from patient sputum, including mixed and low-concentration samples. This approach significantly reduces the turnaround time for this slow-growing bacterium while maintaining high accuracy in detecting clinically relevant mutations, including those associated with drug resistance. Given the global burden of tuberculosis and the critical need for faster diagnostic solutions, we believe our method has the potential to improve clinical decision-making and public health strategies.

Mycobacterium tuberculosis

Protein splicing in the maturation of M. tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence.

The M. tuberculosis recA locus comprises an 85 kd open reading frame but produced 38 kd RecA and 47 kd products in E. coli. No RNA processing was detected; rather, an 85 kd precursor protein was spliced, releasing a 47 kd spacer protein, and joining its terminal fragments to form mature RecA protein. "Spacer" protein was also produced in M. tuberculosis and from a hybrid spacer-LacZ alpha fusion molecule. Mutagenesis at codon wobble positions at one splice junction showed that protein rather than nucleotide sequence determined splicing activity. Other mutants defined additional regions needed for splicing and allowed processing to be followed. Splicing was essential for RecA activity in E. coli. The possibility that splicing is a manifestation of a novel class of genetic element is discussed.

Amino Acid Sequence

[Isolation of a strain of M. tuberculosis which is considered to be rifampicin-dependent, from a patient with long-lasted smear positive and culture difficult (SPCD) mycobacteria].

During the course of clinical examination of drug sensitivity tests for M. tuberculosis, a strain of M. tuberculosis which is considered to be rifampicin-dependent was isolated from a patient with persisting smear positive, culture negative (SPCN) or culture difficult (SPCD) mycobacteria status. The strain isolated produced a few tiny colonies on the control Ogawa-egg yolk medium, whereas it showed abundant growth like a bacteria plaque on the medium containing rifampicin 50 micrograms/ml. Furthermore, the growth of the strain on Ogawa medium containing rifampicin 50 micrograms/ml is much better than that on the medium containing rifampicin 10 micrograms/ml.

Colony Count, Microbial

Observations on the cultivation of M. leprae and M. tuberculosis in medium 'V' and 'V 1'.

Skin scrapings from five different active sites were collected from 14 leprosy patients and inoculated into medium V. Skin scrapings from three leprosy patients were inoculated into medium V 1. All the cultures were incubated at 8-10 degrees C. M. tuberculosis H37Rv, pretreatment isolates and streptomycin resistant strains were inoculated into medium V, with and without antibiotics, and incubated at 8-10 degrees C as well as 37 degrees C. Smears were made from the M. leprae and M. tuberculosis cultures at 0 hours and at different time points. The number of bacilli in the smears were counted. There was no increase in the number of M. leprae or M. tuberculosis in any of the cultures.

Culture Media

The genes coding for the antigen 85 complexes of Mycobacterium tuberculosis and Mycobacterium bovis BCG are members of a gene family: cloning, sequence determination, and genomic organization of the gene coding for antigen 85-C of M. tuberculosis.

A gene encoding the 33-kDa secreted protein of Mycobacterium tuberculosis (antigen 85-C) was isolated and sequenced. The corresponding DNA sequence contains a 1,020-bp coding region. The deduced amino acid sequence corresponds to a 340-residue protein consisting of a 46-amino-acid signal peptide and a 294-amino-acid mature protein. Comparison with previously described genes for the 30-kDa antigen (the alpha antigen of M. bovis BCG, also called antigen 85-B) and the 32-kDa antigens from M. bovis BCG and M. tuberculosis (antigens 85-A) indicates that the three genes share considerable sequence homology (70.8 to 77.5%) but may also code for distinctive epitopes. Strong differences among the three sequences are clearly visible upstream and downstream from the region coding for the mature proteins. The three genes have been detected in the genome of M. bovis BCG by Southern blot hybridization with three type-specific probes. Furthermore, hybridization of large DNA fragments (100 to 1,000 kbp) from M. tuberculosis separated by pulsed-field gel electrophoresis showed that the three genes coding for the antigen 85 complex are not clustered within the bacterial genome.

Amino Acid Sequence