Currently microorganisms are best identified using 16S rRNA and 18S rRNA gene sequencing. However, in recent years matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. The process is rapid, sensitive, and economical in terms of both labor and costs involved. The technology has been readily imbibed by microbiologists who have reported usage of MALDI-TOF MS for a number of purposes like, microbial identification and strain typing, epidemiological studies, detection of biological warfare agents, detection of water- and food-borne pathogens, detection of antibiotic resistance and detection of blood and urinary tract pathogens etc. The limitation of the technology is that identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains. This review provides an overview of the status and recent applications of mass spectrometry for microbial identification. It also explores the usefulness of this exciting new technology for diagnosis of diseases caused by bacteria, viruses, and fungi.

MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis.

Interleukin-6 and its receptors: A highly regulated and dynamic system

MALDI-TOF-mass spectrometry (MS) has been successfully adapted for the routine identification of microorganisms in clinical microbiology laboratories in the past 10 years. This revolutionary technique allows for easier and faster diagnosis of human pathogens than conventional phenotypic and molecular identification methods, with unquestionable reliability and cost–effectiveness. This article will review the application of MALDI-TOF-MS tools in routine clinical diagnosis, including the identification of bacteria at the species, subspecies, strain and lineage levels, and the identification of bacterial toxins and antibiotic-resistance type. We will also discuss the application of MALDI-TOF-MS tools in the identification of Archaea, eukaryotes and viruses. Pathogenic identification from colony-cultured, blood-cultured, urine and environmental samples is also reviewed.

MALDI-TOF-mass spectrometry applications in clinical microbiology.

Recent threats posed by pathogenic microorganisms in food, recreational waters, and as agents of bioterror have underscored the need for the development of more rapid, accurate, and cost-effective methods of microbial characterization and identification. This chapter focuses on the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to rapidly characterize and identify microorganisms through generation of characteristic fingerprints of intact cells. While most efforts have focused on bacteria, this technology has also been applied to fungi and viruses. Results of most studies suggest that MALDI-TOF MS can be used to rapidly and accurately characterize microorganisms. A variety of quantitative approaches have been employed in the analysis of MALDI-TOF MS fingerprints of microorganisms. The reproducibility of fingerprints of intact cells remains a primary concern and limitation associated with this approach. Protocols and instrumentation used have varied considerably and likely account for much of the variability in reproducibility reported. Key first steps to overcoming this limitation will be the development of standard approaches to quantifying reproducibility and the development of standard protocols for sample preparation and analysis.

Microbial Fingerprinting using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS): Applications and Challenges

Cirrhosis is a common final pathway for most chronic liver diseases; representing an increasing burden worldwide and is associated with increased morbidity and mortality. Current evidence has shown that, after an initial injury, the immune response has a significant participation in the ongoing damage, and progression from chronic viral hepatitis (CVH) to cirrhosis, driving the activation and maintenance of main fibrogenic pathways. Among immune deregulations, those related to the subtype 17 of T helper lymphocytes (Th17)/interleukin-17 (IL-17) axis have been recognized as key immunopathological and prognostic elements in patients with CVH. The Th17/IL-17 axis has been found involved in several points of fibrogenesis chain from the activation of stellate cells, increased expression of profibrotic factors as TGF-β, promotion of the myofibroblastic or epithelial-mesenchymal transition, stimulation of the synthesis of collagen, and induction of imbalance between matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). It also promotes the recruitment of inflammatory cells and increases the expression of proinflammatory cytokines such as IL-6 and IL-23. So, the Th17/IL-17 axis is simultaneously the fuel and the flame of a sustained proinflammatory and profibrotic environment. This work aims to present the immunopathologic and prognostic role of the Th17/IL-17 axis and related pathways in fibrogenesis and progression to cirrhosis in patients with liver disease due to hepatitis B virus (HBV) and hepatitis C virus (HCV).

Immunity and Fibrogenesis: The Role of Th17/IL-17 Axis in HBV and HCV-induced Chronic Hepatitis and Progression to Cirrhosis.

Background
Increased numbers of Interleukin-17-producing CD4+ T cells (Th17) have been found in association with hepatitis B virus (HBV)-induced liver injury. However, the mechanism underlying the increase of Th17 responses in patients with HBV infection remains unclear. In this study, we investigate the possible regulatory mechanisms of increased Th17 responses in patients with chronic hepatitis B(CHB).

/pdf/elevated-il-6-receptor-expression-on-cd4-t-cells-contributes-4614davthv.pdf

Elevated IL-6 Receptor Expression on CD4 + T Cells contributes to the increased Th17 Responses in patients with Chronic Hepatitis B

Background: Variations in the hepatitis B virus (HBV) genome may develop spontaneously or under selective pressure from antiviral therapy. Such variations may confer drug resistance or affect virus replication capacity, resulting in failure of antiviral therapy.

Methods: A duplex PCR was used to amplify the region of the reverse transcriptase gene, the precore promoter, and the basal core promoter of the HBV genome. Four multiplex primer-extension reactions were used to interrogate 60 frequently observed HBV variants during antiviral therapy. Automated MALDI-TOF mass spectrometry (MS) was used for mutation detection. Capillary sequencing was used to confirm the MS results.

Results: The limit of quantification was 1000 HBV copies/mL for multiplex detection of HBV variants. Fifty-three variants (88.3%) were analyzed successfully in at least 90% of the sera from 88 treatment-naive patients and 80 patients with virologic breakthrough. MS was able to detect twice as many minor variants as direct sequencing while achieving close to full automation. MS and direct sequencing showed only 0.1% discordance in variant calls.

Conclusions: This platform based on multiplex primer extension and MALDI-TOF MS was able to detect 60 HBV variants in 4 multiplex reactions with accuracy and low detection limits.

/pdf/multiplex-detection-of-60-hepatitis-b-virus-variants-by-srre0933h6.pdf

Multiplex Detection of 60 Hepatitis B Virus Variants by MALDI-TOF Mass Spectrometry

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Elevated IL-6 Receptor Expression on CD4 + T Cells contributes to the increased Th17 Responses in patients with Chronic Hepatitis B

Multiplex Detection of 60 Hepatitis B Virus Variants by MALDI-TOF Mass Spectrometry