An improved simple method for the identification of Mycobacteria by MALDI-TOF MS (Matrix-Assisted Laser Desorption- Ionization mass spectrometry).

The aim of this study was to establish a simple method for the rapid identification of Mycobacteria species by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass spectrometry) using the Bruker MALDI-TOF Biotyper system (Bruker Daltonik, Bremen, Germany). A multicentre, prospective, and single blind study was performed in three European Hospitals, two Spanish and one UK hospital from May to August 2018. The BD BACTEC MGIT (Becton Dickinson, Berks, UK) liquid culture system was used in all three centres for the growth of Mycobacteria. When signal positive, tubes were removed from the analyser and in addition to standard laboratory procedures were subcultured on blood agar plates for MALDI-TOF analysis. Plates were incubated aerobically for 1 to 7 days at 37 °C and inspected every day. Once any growth was visible, it was transferred to the steel target plate, overlaid with 1 µl of neat formic acid and 1 µl HCCA matrix (alpha hydroxyl 4 cinnamic acid), and analysed in a Bruker Biotyper MALDI-TOF. Results given by MALDI-TOF were compared with the reference methods used for identification in the different centres. At two Spanish hospitals, identification by MALDI-TOF was only attempted on presumptive non-tuberculosis mycobacteria (NTM) and the results were initially compared with the results obtained by a commercial reverse hybridisation assay, GenoType CM/AS (Hain Lifescience, Tübingen, Germany). At the UK Hospital, identification of any presumptive mycobacteria was attempted and compared with the results obtained by whole genome sequencing (WGS). Overall in 142/167 (85%) of cases the identifications obtained were concordant; all Mycobacterium tuberculosis (MTB) isolates 43/43 (100%), 57/76 (75%) of the rapid growing nontuberculous mycobacteria (NTM), and 42/48 (85%) slow growing NTM tested were identified correctly. We report a new, easy, cheap and quick method for isolation and identification of Mycobacterium spp. without the need for additional steps or equipment and this method is in routine used in all three centres.

UV-fixed-thick-blotch preparation improves sensitivity of auramine staining.

We describe here a very simple modification of the auramine staining procedure based on preparation of a UV-fixed thick blotch which allowed us to reach an overall sensitivity of 0.82 (592 acid-fast bacillus [AFB]-positive specimens/722 initial respiratory specimens with positive mycobacterial culture) and sensitivities of 0.93 (526 AFB-positive specimens/564 culture-positive specimens) for Mycobacterium tuberculosis complex and 0.42 (66 AFB-positive specimens/158 culture-positive specimens) for nontuberculous mycobacteria.

Antibiotic susceptibility and molecular epidemiology of nasopharyngeal pneumococci from Spanish children.

Nasopharyngeal pneumococci were collected from 635 Spanish children aged 6 months to 6 years attending four primary healthcare centres (n = 276) or two hospital emergency rooms (n = 359); 36% of the children had received >/=1 dose of pneumococcal conjugate vaccine (PCV7). Overall, the carriage rate of Streptococcus pneumoniae was 31%, with no significant differences in carriage rates according to setting. Colonization with vaccine serotypes was significantly associated with the absence of PCV7 immunization (29.4% vs. 5.9%, p <0.001). Forty-seven per cent of all isolates were penicillin- and/or erythromycin-non-susceptible; 13 international antibiotic-resistant clones were represented among non-susceptible pneumococci and were similarly distributed among vaccine and non-vaccine serotypes.

New contributions to the ethnopharmacology of Spain.

In the course of an ethnobotanical research project carried out in the Province of Granada (Southern Spain), 267 taxa have been catalogued as being used in folk medicine. Based on the literature consulted, phytotherapeutic use for 34 of these was recorded for the first time.