Bacterial analysis by MALDI-TOF mass spectrometry: An inter-laboratory comparison

Bacterial analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been demonstrated in numerous laboratories, and a few attempts have been made to compare results from different laboratories on the same organism. It has been difficult to understand the causes behind the observed differences between laboratories when different instruments, matrices, solvents, etc. are used. In order to establish this technique as a useful tool for bacterial identification, additional efforts in standardizing the methods by which MALDI mass spectra are obtained and comparisons of spectra from different instruments with different operators are needed. Presented here is an extension of our previous single-laboratory reproducibility study with three different laboratories in a controlled experiment with aliquots of the same bacterial culture, matrix stock solution, and calibrant standards. Using automated spectral collection of whole-cell bacteria and automated data processing and analysis algorithms, fingerprints from three different laboratories were constructed and compared. Nine of the ions appeared reproducibly within all three laboratories, with additional unique ions observed within each of the laboratories. An initial evaluation of the ability to use a fingerprint generated within one laboratory for bacterial identification of a sample from another laboratory is presented, and strategies for improving identification rates between laboratories is discussed

What Qualities Are Most Important to Making a Point of Care Test Desirable for Clinicians and Others Offering Sexually Transmitted Infection Testing?

To investigate the possible effects of different levels of attributes of a point-of-care test (POCT) on sexually transmitted infection (STI) professionals' decisions regarding an ideal POCT for STI(s).An online survey was designed based on a large-scale in-depth focus discussion study among STI experts and professionals. The last section of the survey "build your own POCT" was designed by employing the discrete choice experiment approach. Practicing clinicians from two venues, STI-related international conference attendees and U.S. STD clinic clinicians were invited to participate in the survey. Conditional logistical regression modeling was used for data analysis.Overall, 256 subjects took the online survey with 218 (85%) completing it. Most of the participants were STD clinic clinicians who already used some POCTs in their practice. "The time frame required" was identified as a major barrier that currently made it difficult to use STI POCTs. Chlamydia trachomatis was the organism chosen as the top priority for a new POCT, followed by a test that would diagnose early seroconversion for HIV, and a syphilis POCT. Without regard to organism type selected, sensitivity of 90-99% was always the most important attribute to be considered, followed by a cost of $20. However, when the test platform was prioritized for early HIV seroconversion or syphilis, sensitivity was still ranked as most important, but specificity was rated second most important.STI professionals preferred C. trachomatis as the top priority for a new POCT with sensitivity over 90%, low cost, and a very short completion time

Use of 16S rRNA, 23S rRNA, and gyrB Gene Sequence Analysis To Determine Phylogenetic Relationships of Bacillus cereus Group Microorganisms

In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group

Rapid Identification of Biothreat and Other Clinically Relevant Bacterial Species by Use of Universal PCR Coupled with High-Resolution Melting Analysis▿

A rapid assay for eubacterial species identification is described using high-resolution melt analysis to characterize PCR products. Unique melt profiles generated from multiple hypervariable regions of the 16S rRNA gene for 100 clinically relevant bacterial pathogens, including category A and B biothreat agents and their surrogates, allowed highly specific species identification