Amalio Telenti

Genotypic analysis of Mycobacterium tuberculosis in two distinct populations using molecular beacons : Implications for rapid susceptibility testing

ABSTRACT Past genotypic studies of Mycobacterium tuberculosismay have incorrectly estimated the importance of specific drug resistance mutations due to a number of sampling biases including an overrepresentation of multidrug-resistant (MDR) isolates. An accurate assessment of resistance mutations is crucial for understanding basic resistance mechanisms and designing genotypic drug resistance assays. We developed a rapid closed-tube PCR assay using fluorogenic reporter molecules called molecular beacons to detect reportedly common M. tuberculosis mutations associated with resistance to isoniazid and rifampin. The assay was used in a comparative genotypic investigation of two different study populations to determine whether these known mutations account for most cases of clinical drug resistance. We analyzed samples from a reference laboratory in Madrid, Spain, which receives an overrepresentation of MDR isolates similar to prior studies and from a community medical center in New York where almost all of the resistant isolates and an equal number of susceptible controls were available. The ability of the molecular beacon assay to predict resistance to isoniazid and rifampin was also assessed. The overall sensitivity and specificity of the assay for isoniazid resistance were 85 and 100%, respectively, and those for rifampin resistance were 98 and 100%, respectively. Rifampin resistance mutations were detected equally well in isolates from both study populations; however, isoniazid resistance mutations were detected in 94% of the isolates from Madrid but in only 76% of the isolates from New York (P = 0.02). In New York, isoniazid resistance mutations were significantly more common in the MDR isolates (94%) than in single-drug-resistant isolates (44%; P < 0.001). No association between previously described mutations in thekasA gene and isoniazid resistance was found. The first mutations that cause isoniazid resistance may often occur in sequences that have not been commonly associated with isoniazid resistance, possibly in other as yet uncharacterized genes. The molecular beacon assay was simple, rapid, and highly sensitive for the detection of rifampin-resistant M. tuberculosis isolates and for the detection of isoniazid resistance in MDR isolates.

Inteins in mycobacterial GyrA are a taxonomic character

The A subunit of DNA gyrase in mycobacteria is frequently subjected to splicing events as its gene, gyrA, harbours an insertion encoding an intein. Investigation of a number of different isolates of Mycobacterium kansasii, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium ulcerans and Mycobacterium xenopi demonstrated that the presence of GyrA inteins is not random but a taxonomic character specific for a given taxon at a species or subspecies level.

Time Frame for Infectious Complications in the Surgical-Trauma Intensive-Care Unit

Fever is a frequent diagnostic dilemma in the intensive-care unit (ICU), and a definitive diagnosis of infection may be established in less than 10-15 % of patients. This uncertainty results in excessive use of empirical antibiotics and unnecessary diagnostic investigations. The diagnostic approach can benefit from the careful description of various ICU time periods characterized by different infection rates and a restricted number of ICU-related infectious complications. Incorporation of the concept of ‘infection time frame’ may allow a better targeting of populations at risk.

Nucleic acid amplification methods in diagnostic virology

The speed, sensitivity and specificity of nucleic acid amplification techniques make them very attractive tools for diagnostic virology. Priorities should be defined for the stepwise implementation of these methods. Their application is not limited to the purpose of direct diagnosis, but includes quantitation of viral load, evaluation of genetic determinants of resistance to antiviral drugs, typing of strains, and the analysis of new viral pathogens. However, a widespread use of these techniques by clinical laboratories will require fully standardized, carefully controlled systems. Very few commercial systems are as yet available.

Appendicitis: Spectrum of Bacteria and the Role of Antimicrobial Therapy

The use of perioperative antibiotics has been shown to decrease infectious complications after appendectomy. Controversy remains as to the optimal antibiotic combination and duration of therapy. Most