8.4. Culture-negative Pulmonary Tuberculosis CONTENTS in Adults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 650 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604 8.5. Radiographic Evidence of Prior Tuberculosis: Inactive Tuberculosis . . . . . . . . . . . . . . . . . . . . . 650

American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America: treatment of tuberculosis.

Isoniazid (isonicotinic acid hydrazide, INH) is one of the most widely used antituberculosis drugs, yet its precise target of action on Mycobacterium tuberculosis is unknown. A missense mutation within the mycobacterial inhA gene was shown to confer resistance to both INH and ethionamide (ETH) in M. smegmatis and in M. bovis. The wild-type inhA gene also conferred INH and ETH resistance when transferred on a multicopy plasmid vector to M. smegmatis and M. bovis BCG. The InhA protein shows significant sequence conservation with the Escherichia coli enzyme EnvM, and cell-free assays indicate that it may be involved in mycolic acid biosynthesis. These results suggest that InhA is likely a primary target of action for INH and ETH.

https://science.sciencemag.org/content/sci/263/5144/227.full.pdf

inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis

In 1994, CDC published the Guidelines for Preventing the Transmission of Mycobacterium tuberculosis in HealthCare Facilities, 1994. The guidelines were issued in response to 1) a resurgence of tuberculosis (TB) disease that occurred in the United States in the mid-1980s and early 1990s, 2) the documentation of several high-profile health-care--associated (previously termed "nosocomial") outbreaks related to an increase in the prevalence of TB disease and human immunodeficiency virus (HIV) coinfection, 3) lapses in infection control practices, 4) delays in the diagnosis and treatment of persons with infectious TB disease, and 5) the appearance and transmission of multidrug-resistant (MDR) TB strains. The 1994 guidelines, which followed statements issued in 1982 and 1990, presented recommendations for TB infection control based on a risk assessment process that classified health-care facilities according to categories of TB risk, with a corresponding series of administrative, environmental, and respiratory protection control measures. The TB infection control measures recommended by CDC in 1994 were implemented widely in health-care facilities in the United States. The result has been a decrease in the number of TB outbreaks in health-care settings reported to CDC and a reduction in health-care-associated transmission of Mycobacterium tuberculosis to patients and health-care workers (HCWs). Concurrent with this success, mobilization of the nation's TB control programs succeeded in reversing the upsurge in reported cases of TB disease, and case rates have declined in the subsequent 10 years. Findings indicate that although the 2004 TB rate was the lowest recorded in the United States since national reporting began in 1953, the declines in rates for 2003 (2.3%) and 2004 (3.2%) were the smallest since 1993. In addition, TB infection rates greater than the U.S. average continue to be reported in certain racial/ethnic populations. The threat of MDR TB is decreasing, and the transmission of M. tuberculosis in health-care settings continues to decrease because of implementation of infection-control measures and reductions in community rates of TB. Given the changes in epidemiology and a request by the Advisory Council for the Elimination of Tuberculosis (ACET) for review and update of the 1994 TB infection control document, CDC has reassessed the TB infection control guidelines for health-care settings. This report updates TB control recommendations reflecting shifts in the epidemiology of TB, advances in scientific understanding, and changes in health-care practice that have occurred in the United States during the preceding decade. In the context of diminished risk for health-care-associated transmission of M. tuberculosis, this document places emphasis on actions to maintain momentum and expertise needed to avert another TB resurgence and to eliminate the lingering threat to HCWs, which is mainly from patients or others with unsuspected and undiagnosed infectious TB disease. CDC prepared the current guidelines in consultation with experts in TB, infection control, environmental control, respiratory protection, and occupational health. The new guidelines have been expanded to address a broader concept; health-care--associated settings go beyond the previously defined facilities. The term "health-care setting" includes many types, such as inpatient settings, outpatient settings, TB clinics, settings in correctional facilities in which health care is delivered, settings in which home-based health-care and emergency medical services are provided, and laboratories handling clinical specimens that might contain M. tuberculosis. The term "setting" has been chosen over the term "facility," used in the previous guidelines, to broaden the potential places for which these guidelines apply.

/pdf/guidelines-for-preventing-the-transmission-of-mycobacterium-4h27m134c1.pdf

Guidelines for preventing the transmission of Mycobacterium tuberculosis in health-care settings, 2005

Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update.

The live attenuated bacillus Calmette-Guerin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.

Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.

The fluctuation test shows that Mycobacterium tuberculosis mutates to resistance to isoniazid, streptomycin, ethambutol and rifampin spontaneously and at random. The average mutation rates for the drugs, in the same order, were calculated to be 2.56 × 10−8, 2.95 × 10−8, 10−7, and 2.25 × 10−10 mutation per bacterium per generation. The relatively high mutation rate to ethambutol resistance and the low mutation rate to rifampin resistance were confirmed by analyzing the increase in the proportion of mutants with time in a growing population of the tubercle bacilli. The highest proportions of mutants to be expected in unselected populations of the tubercle bacilli were calculated from the results of fluctuation tests.

https://aem.asm.org/content/aem/20/5/810.full.pdf

Probability distribution of drug-resistant mutants in unselected populations of Mycobacterium tuberculosis.

When an actively growing culture of the H37Ra strain of Mycobacterium tuberculosis was exposed to isoniazid at a concentration of 0.5 μg/ml, the cells began to lose their ability to synthesize mycolic acids immediately. After 60 min, the cells had completely lost this ability. The synthesis of the three mycolate components—α-mycolate, methoxymycolate, and β-mycolate—was inhibited. The viability of the isoniazid-treated cells was unaffected up to about 60 min of exposure, after which time there was a gradual decline in the viability to about 18% after 180 min. Correspondingly, growth of the drug-treated cells slowed down and stopped after 24 hr. The inhibition of the synthesis of mycolic acids was reversible if the drug was removed before the loss of viability set in. Incubation of the viable cells in the absence of the drug for 24 hr restored the mycolate synthesis. These results strongly suggest that the inhibition of the synthesis of the mycolic acids is closely associated with the primary mechanism of action of isoniazid on the tubercle bacilli. The sequence of events which leads to the loss of viability of cells exposed to isoniazid is described.

Effect of Isoniazid on the In Vivo Mycolic Acid Synthesis, Cell Growth, and Viability of Mycobacterium tuberculosis

The results of drug susceptibility tests were found to be affected by changes that occur spontaneously in populations of Mycobacterium avium maintained in the laboratory. Because the transparent colony type variant was resistant to antituberculosis chemotherapeutic agents and the opaque colony type variant was usually susceptible to these agents, the transition of transparent to opaque colony type was investigated. The rate of the transition was found to be temperature dependent and, in agreement with a previous report, was found to be about 10 −4 to 10 −5 per generation at 37 C. Reversion was found to occur at a rate of 10 −6 to 10 −7 at 37 C. The mutation rate from susceptibility to resistance to rifampin, kanamycin, and erythromycin was about 10 −8 to 10 −9 mutations per bacterium per generation. Judged from our data, the high rate of the transparent to opaque variation was not caused either by mutator effects or by the occurrence of extrachromosomal genes in these bacteria, but could have been due to selective mechanisms still incompletely understood.

Effect of temperature on the rate of the transparent to opaque colony type transition in Mycobacterium avium.

The kinetics of inactivation of mycobacteria by ultraviolet light were investigated. Mycobacterium tuberculosis and M. marinum were shown to be capable of photo-reactivation.

Ultraviolet light inactivation and photoreactivation in the mycobacteria.

A single transport system was found to accumulate l- and d-alanine, glycine and d-serine in Mycobacterium tuberculosis. The results of inhibition experiments suggested that the antibiotics d-cycloserine and O-carbamyl-d-serine were also transported by the alanine-glycine-d-serine system. A d-cycloserine-resistant permease-competent (d-CS(r)/perm(+)) mutant and a d-cycloserine-resistant permease-defective (d-CS(r)/perm(-)) mutant were isolated. The d-CS(r)/perm(-) mutant was not found to be more resistant to the drug than was the d-CS(r)/perm(+) mutant. The data were consistent with the conclusion that resistance to d-cycloserine in the tubercle bacilli is primarily due to mutations in the gene(s) controlling the enzyme d-alanyl-d-alanine synthetase. The mutation rate was calculated to be about 10(-10) mutations per bacterium per generation.

Hugo L. David

Papers

Probability distribution of drug-resistant mutants in unselected populations of Mycobacterium tuberculosis.

Effect of Isoniazid on the In Vivo Mycolic Acid Synthesis, Cell Growth, and Viability of Mycobacterium tuberculosis

Effect of temperature on the rate of the transparent to opaque colony type transition in Mycobacterium avium.

Ultraviolet light inactivation and photoreactivation in the mycobacteria.

Resistance to D-cycloserine in the tubercle bacilli: mutation rate and transport of alanine in parental cells and drug-resistant mutants