Novel rapid PCR for the detection of Ile491Phe rpoB mutation of Mycobacterium tuberculosis, a rifampicin-resistance-conferring mutation undetected by commercial assays.

OBJECTIVES: Neither the liquid medium-based Bactec MGIT, nor commercial molecular assays such as the Xpert MTB/RIF and the MTBDRplus V2.0 assays are capable of detecting up to 30% of rifampicin-resistant Mycobacterium tuberculosis strains in Swaziland because of the large proportion of the rpoB Ile491Phe mutations. In other countries, the frequency of this mutation is thought to be low. METHODS: We designed a real-time multiplex allele-specific PCR assay to identify the rpoB Ile491Phe mutation responsible for these undetected resistant M. tuberculosis strains. RESULTS: The technique showed 100% similarity with rpoB sequencing on a panel of 78 strains from Swaziland. CONCLUSIONS: We propose that the detection of the rpoB Ile491Phe rpoB mutation should complement commercial assays for the diagnosis of rifampicin-resistant M. tuberculosis in routine conditions, particularly in countries where this specific mutation is frequent. The technique proposed in this paper is adapted for most reference laboratories.

Consensus numbering system for the rifampicin resistance-associated rpoB gene mutations in pathogenic mycobacteria.

The rpoB gene codes for the RNA polymerase ß subunit, which is the target of rifampicin, an essential drug in the treatment of tuberculosis and other mycobacterial infections. This gene is present in all bacteria, but its length and nucleotide sequence vary between bacterial species, including mycobacteria. Mutations in the rpoB gene alter the structure of this protein and cause drug resistance. To describe the resistance-associated mutations, the scientific and medical communities have been using, since 1993, a numbering system based on the Escherichia coli sequence annotation. Using E. coli reference for describing mutations in mycobacteria leads to misunderstandings, particularly with the increasing use of whole genome sequencing, which brought an alternative numbering system based on the Mycobacterium tuberculosis rpoB sequence. We propose using a consensus numbering system for the reporting of resistance mutations based on the reference genomes from the species interrogated (such as strain H37Rv for M. tuberculosis). This manuscript provides the necessary figures and tables allowing researchers, microbiologists and clinicians to easily convert other annotation systems into one common language.

[Quantitative evaluation of neurovegetative systemic changes based on the degree of minute-rhythmic coupling and control quality in acute and chronic diazépam therapy of neuroses]. / Quantitative Bewertung von neurovegetativen Systemveränderungen anhand des minutenrhythmischen Kopplungsgrades und der Regelgüte während akuter und chronischer Diazepamtherapie bei neurotischen Patienten

In 10 patients with definite forms of neurosis, controlphysiologic and biorhythmometric investigations were carried out to establish the effectiveness of diazepam therapy. After acute application of 10 mg diazepam, an increase in the degree of minute-rhythmic coupling correlated positively with a decrease of the control area, of the time adjustment of the heart rate after load-related deflection, and with an increase in a derived complex parameter of control quality. Chronic therapy with diazepam reversed the positive tendency of the biorhythmometric and control parameters. The results permit the conclusion that the minute-rhythmic coupling degree lends itself to diagnostic evaluation of the actual synchronization state or the related neurovegetative reaction state.