ELISA Test for the Serological Detection of Scedosporium/Lomentospora in Cystic Fibrosis Patients

The detection and diagnosis of the opportunistic fungi Scedosporium spp. and Lomentospora prolificans still relies mainly on low-sensitive culture-based methods. This fact is especially worrying in Cystic Fibrosis (CF) patients in whom these fungal species are frequently isolated and may increase the risk of suffering from an infection or other health problems. Therefore, with the purpose of developing a serologic detection method for Scedosporium/Lomentospora, four different Scedosporium boydii protein extracts (whole cell protein extract, secretome, total cell surface and conidial surface associated proteins) were studied by ELISA to select the most useful for IgG detection in sera from CF patients. The four extracts were able to discriminate the Scedosporium/Lomentospora-infected from Aspergillus-infected and non-infected patients. However, the whole cell protein extract was the one selected, as it was the one with the highest output in terms of protein concentration per ml of fungal culture used, and its discriminatory capacity was the best. The ELISA test developed was then assayed with 212 sera from CF patients and it showed to be able to detect Scedosporium spp. and Lomentospora prolificans with very high sensitivity and specificity, 86%–100% and 93%–99%, respectively, depending on the cut-off value chosen (four values were proposed A450nm= 0.5837, A450nm= 0.6042, A450nm= 0.6404, and A450nm= 0.7099). Thus, although more research is needed to reach a standardized method, this ELISA platform offers a rapid, low-cost and easy solution to detect these elusive fungi through minimally invasive sampling, allowing the monitoring of the humoral response to fungal presence.

Bacterial Whole Cell Protein Profiling: Methodology, Applications and Constraints

Background:In the era of modern microbiology, several methods are available for identification and typing of bacteria, including whole genome sequencing. However, in microbiological laboratories or hospitals where genomic based molecular typing methods and/or trained manpower are unavailable, whole cell protein profiling using sodium dodecyl sulfate polyacrylamide gel electrophoresis might be a useful alternative/supplementary method for bacterial identification, strain typing and epidemiology. Whole cell protein profiling by SDS-PAGE is based on the principle that under standard growth conditions, a bacterial strain expresses the same set of proteins, the pattern of which can be used for bacterial identification.Objective:The objective of this review is to assess the current status of whole cell protein profiling by SDS-PAGE and its advantages and constraints for bacterial identification and typing.Results and Conclusions:Several earlier and recent studies prove the potential and utility of this technique as an adjunct or supplementary method for bacterial identification, strain typing and epidemiology. There is no denying the fact that utility of this technique as an adjunct or supplementary method for bacterial identification and typing has already been demonstrated and its practical applications need to be evaluated further.

Partial Proteome Map of Campylobacter Jejuni Strain Nctc11168 by Gel-Free Proteomics Analysis

A proteome map of the foodborne pathogen Campylobacter jejuni NCTC11168 was analyzed using a state-of-the-art gel-free proteomic approach for the first time. A whole cell protein extract was prepared from the C. jejuni strain NCTC11168 grown in brain heart infusion (BHI) broth at 42°C under microaerobic conditions. A gel-free technique using isobaric tags for relative and absolute quantitation (iTRAQ) was employed to create a protein expression profile of the strain. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to identify the proteins. Protein functionalities were searched to classify them. A total of 235 proteins were identified in the whole cell protein fraction of C. jejuni NCTC11168 cells using iTRAQ analysis. Functional grouping of the identified proteins showed that forty percent of these proteins were associated with energy metabolism, protein synthesis and genetic information processing. iTRAQ was faster, easier and proved more sensitive than two-dimensional gel-based proteomics approaches previously applied to C. jejuni, making it an attractive tool for further studies of cellular physiological response. DOI: http://dx.doi.org/10.3126/ijasbt.v2i4.11253 Int J Appl Sci Biotechnol, Vol. 2(4): 464-477