Widespread use of DNA restriction fragment length polymorphism (RFLP) to differentiate strains of Mycobacterium tuberculosis to monitor the transmission of tuberculosis has been hampered by the need to culture this slow-growing organism and by the level of technical sophistication needed for RFLP typing. We have developed a simple method which allows simultaneous detection and typing of M. tuberculosis in clinical specimens and reduces the time between suspicion of the disease and typing from 1 or several months to 1 or 3 days. The method is based on polymorphism of the chromosomal DR locus, which contains a variable number of short direct repeats interspersed with nonrepetitive spacers. The method is referred to as spacer oligotyping or "spoligotyping" because it is based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides. Most of the clinical isolates tested showed unique hybridization patterns, whereas outbreak strains shared the same spoligotype. The types obtained from direct examination of clinical samples were identical to those obtained by using DNA from cultured M. tuberculosis. This novel preliminary study shows that the novel method may be a useful tool for rapid disclosure of linked outbreak cases in a community, in hospitals, or in other institutions and for monitoring of transmission of multidrug-resistant M. tuberculosis. Unexpectedly, spoligotyping was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.

Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.

(MRSA) with reduced suscept-ibility to vancomycin (MIC 8 mg/L). The strain was isolated from a surgical wound infection which was refrac -tory to vancomycin therapy.In May 1996, a 4 month-old male infant underwent heartsurgery for pulmonary atresia. Two weeks followingsurgery, the infant became febrile and developed a purulent discharge from the sternal surgical incision site;culture of the purulent material yielded MRSA. The patientwas treated with vancomycin (45 mg/kg daily) for 29 days,but fever and discharge of pus continued, and the C-reactive protein (CRP) remained elevated (40 mg/L). Thetreatment was changed to a combination of vancomycin andarbekacin (an aminoglycoside approved for MRSA infec-tion in Japan). After 12 days of this regimen, the purulentdischarge subsided, the wound began to heal, and the CRPdeclined from 40 to 9 mg/L. The antimicrobial therapy wasdiscontinued. However, 12 days later the surgical siteappeared inﬂamed with the development of a subcutaneousabscess accompanied by a sudden onset of fever and a raised CRP level of 35 mg/L. Therapy was resumed with the com -bination of arbekacin and ampicillin/sulbactam which hasbeen shown to have synergic activity against MRSA.

Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility.

The emergence of multidrug-resistant bacteria is a phenomenon of concern to the clinician and the pharmaceutical industry, as it is the major cause of failure in the treatment of infectious diseases. The most common mechanism of resistance in pathogenic bacteria to antibiotics of the aminoglycoside, beta-lactam (penicillins and cephalosporins), and chloramphenicol types involves the enzymic inactivation of the antibiotic by hydrolysis or by formation of inactive derivatives. Such resistance determinants most probably were acquired by pathogenic bacteria from a pool of resistance genes in other microbial genera, including antibiotic-producing organisms. The resistance gene sequences were subsequently integrated by site-specific recombination into several classes of naturally occurring gene expression cassettes (typically "integrons") and disseminated within the microbial population by a variety of gene transfer mechanisms. Although bacterial conjugation once was believed to be restricted in host range, it now appears that this mechanism of transfer permits genetic exchange between many different bacterial genera in nature.

https://science.sciencemag.org/content/sci/264/5157/375.full.pdf

Inactivation of antibiotics and the dissemination of resistance genes.

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

Statistical Methods in Medical Research.

Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis

Molecular approach to identifying route of transmission of tuberculosis in the community.

A prospective 2-month trial involving 617 respiratory tract specimens was conducted to compare sensitivity, specificity, and predictive values of the newly developed Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test kit (AMTDT; Gen-Probe, Inc., San Diego, Calif.) for the rapid detection of Mycobacterium tuberculosis and of fluorescent acid-fast staining versus combined BACTEC 12B and solid-medium cultures as the "gold standard." A total of 590 specimens were culture and AMTDT negative. Twenty-one (3.4%) cultures yielded M. tuberculosis. Of these, 15 (71.4%) were detected by AMTDT, whereas 6 (28.6%) were missed. M. tuberculosis did not grow in six (28.6%) of AMTDT-positive specimens derived from three patients under treatment for tuberculosis. AMTDT exhibited a sensitivity, a specificity, a negative predictive value, and a positive predictive value of 71.4, 99, 99, and 71.4%, respectively. In comparison, the same values for fluorescent microscopy were 66.7, 98.3, 98.8, and 58.3%, respectively. AMTDT was easy to perform and highly specific. However, a screening test would require an improved sensitivity and, when feasible, the implementation of an internal amplification control.

Screening of respiratory tract specimens for the presence of Mycobacterium tuberculosis by using the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test.

Screening ofRespiratory Tract Specimens forthePresence of Mycobacterium tuberculosis byUsingtheGen-Probe Amplified Mycobacterium Tuberculosis Direct Test

Coxiella burnetii was isolated from the valve material of two patients who underwent valvectomy because of progressive congestive heart failure due to endocarditis. In each case antibiotic therapy was administered for several months prior to valvectomy. Classical histopathological examination of the valves did not reveal an etiology. However, coxiella-like organisms were demonstrated in valvular material with Koster, Stamp, and Giemsa stains, and the organisms were grown in cell culture. Antibody titers were consistent with the diagnosis of chronic C. burnetii infection. This report illustrates the advantage of simple and fast staining techniques and cell culture for the demonstration and isolation of C. burnetii in the heart valve tissue of patients with Q fever endocarditis.

K Schopfer

Papers

Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis

Molecular approach to identifying route of transmission of tuberculosis in the community.

Screening of respiratory tract specimens for the presence of Mycobacterium tuberculosis by using the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test.

Screening ofRespiratory Tract Specimens forthePresence of Mycobacterium tuberculosis byUsingtheGen-Probe Amplified Mycobacterium Tuberculosis Direct Test

Isolation of Coxiella burnetii from heart valves of patients treated for Q fever endocarditis.