Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction.

A polymerase chain reaction (PCR) assay for the rapid detection of Mycobacterium tuberculosis in sputum samples is described. The target DNA is a 123-base pair (bp) segment of IS6110, which is repeated in the M. tuberculosis chromosome and is specific for the M. tuberculosis complex. Methodology used to lyse the mycobacteria, extract the DNA, and amplify the 123-bp target DNA is presented. The amplified PCR product is detected by examination of ethidium-bromide-stained acrylamide gels. An internal control using the same primers as the target DNA has been constructed to assess the efficacy of each individual reaction. Of 162 sputum samples tested, 82 were smear-positive for acid-fast bacilli. Of the 94 specimens from patients in whom pulmonary tuberculosis was diagnosed, 51 were culture-positive, smear-positive, or both. Fifty of these were PCR positive. Of the 42 specimens from patients with nontuberculous mycobacterial pulmonary disease, 41 were PCR negative. All 26 specimens from patients without mycobacterial infection were PCR negative. This assay provides a sensitive and specific means for the laboratory diagnosis of tuberculosis within 48 h that is relatively simple to perform.

Host determinants of susceptibility to Mycobacterium tuberculosis.

Susceptibility to tuberculosis is determined by a number of host factors and may vary according to characteristics of the infecting strain. Native resistance is probably mediated via the macrophage and is under genetic control. Other factors that contribute to native resistance are nutrition, associated disease states, age, and sex. Acquired resistance is mediated via macrophages and controlled by T-helper cells. The ability of an activated macrophage to phagocytize and kill virulent tubercle bacilli is influenced by the function and number of T-helper cells. The macrophages are affected by concurrent disease states and immunosuppressive drugs. Macrophages provide local immunity, once activated, and T-helper lymphocytes provide the long-term systemic immunity.

Glucocorticoid induction of beta-adrenergic receptors in the DDT1 MF-2 smooth muscle cell line involves synthesis of new receptor.

We have shown that glucocorticoids induce the appearance of beta 2-adrenergic receptors in membranes of the ductus deferens smooth muscle cell line (DDT1 MF-2). A concomitant increase in isoproterenol stimulated adenylate cyclase activity in the absence of exogenously applied GTP was observed as was a significantly increased (p less than 0.05) sensitivity of the adenylate cyclase system to exogenously applied GTP. However, no significant difference in the maximal velocity of adenylate cyclase between control and steroid treatment was measurable in the presence of sodium fluoride. Induction of beta 2-adrenergic receptors in DDT1 MF-2 cells is correlated with the presence of steroid receptors (androgen and glucocorticoid) in the cells since estrogens and progesterones had no effect on receptor levels. Finally, utilizing dense amino acid labeling of cells to measure old versus newly synthesized receptor sites by a density shift method, we have documented that glucocorticoid induction of beta 2-adrenergic receptors involves synthesis of new receptor protein.