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.

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

Viral infections are the most common among diseases that globally require around 60 percent of medical care. However, in the heat of the pandemic, there was a lack of medical equipment and inpatient facilities to provide all patients with viral infections. The detection of viral infections is possible in three general ways such as (i) direct virus detection, which is performed immediately 1-3 days after the infection, (ii) determination of antibodies against some virus proteins mainly observed during/after virus incubation period, (iii) detection of virus-induced disease when specific tissue changes in the organism. This review surveys some global pandemics from 1889 to 2020, virus types, which induced these pandemics, and symptoms of some viral diseases. Non-analytical methods such as radiology and microscopy also are overviewed. This review overlooks molecular analysis methods such as nucleic acid amplification, antibody-antigen complex determination, CRISPR-Cas system-based viral genome determination methods. Methods widely used in the certificated diagnostic laboratory for SARS-CoV-2, Influenza A, B, C, HIV, and other viruses during a viral pandemic are outlined. A comprehensive overview of molecular analytical methods has shown that the assay's sensitivity, accuracy, and suitability for virus detection depends on the choice of the number of regions in the viral open reading frame (ORF) genome sequence and the validity of the selected analytical method.

/pdf/advances-and-insights-in-the-diagnosis-of-viral-infections-31v9czly8h.pdf

Advances and insights in the diagnosis of viral infections.

MALDI-TOF mass spectrometry is a diagnostic tool of microbial identification and characterization based on the detection of the mass of molecules. In the majority of clinical laboratories, this technology is currently being used mainly for bacterial diagnosis, but several approaches in the field of virology have been investigated. The introduction of this technology in clinical virology will improve the diagnosis of infections produced by viruses but also the discovery of mutations and variants of these microorganisms as well as the detection of antiviral resistance. This review is focused on the main current applications of MALDI-TOF MS techniques in clinical virology showing the state of the art with respect to this exciting new technology.

/pdf/application-of-maldi-tof-mass-spectrometry-in-clinical-3ken9liyl8.pdf

Application of MALDI-TOF Mass Spectrometry in Clinical Virology: A Review

OBJECTIVE To discuss the clinical features, treatment and prognosis of patients with AIDS complicated by tuberculosis(TB). METHODS The clinical features of 23 patients with AIDS complicated by tuberculosis admitted from 1997 to July 2004 were retrospectively analyzed. RESULTS Of the 23 patients, most (94.3%) were young or middle-aged, and 11 (47.8%) died within half a year. The main HIV transmission was via sexual contact in 15 (65.2%) patients. Loss of body weight by 5 - 15 kg was present in all patients, cough for over 1 month in 15 (65.2%), and multiple opportunistic infections were complicated in most cases. Out of the 23 cases, 14 (60.9%) showed only pulmonary TB, and 8 (34.8%) showed lymph node TB. In 12 cases with infiltrated pulmonary TB, X-ray showed bilateral infiltration and no cavity formation was found. Slightly positive PPD test was found in 2 (8.7%) cases, and positive acid-fast bacilli was detected in sputum in 1 case (4.4%). The pre-treatment CD(4)(+) cell number in 23 patients was much lower than that in AIDS patients without complicated TB (P < 0.05). The pre-treatment CD(4)(+) cell number in patients died shortly after diagnosis was much lower than that in survived patients (P < 0.05). The HIV RNA level in the 23 patients was much higher than that in patients without complication of TB (P < 0.05). The mortality in patients treated with therapy against both TB and HIV was much lower than that in patients untreated or treated only with anti-TB therapy (P < 0.05). CONCLUSIONS For patients with AIDS complicated by TB, the high negative rate in PPD test and the atypical chest X-ray manifestations are common. However, lymph node TB is quite common with high mortality. The pre-treatment CD(4)(+) level decreases significantly, and is associated with mortality. The TB bacilli may accelerate HIV virus duplication. It is suggested that the patients be treated with a combination of anti-TB and anti-HIV therapies.

Clinical features of 23 cases of AIDS complicated by tuberculosis

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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Clinical features of 23 cases of AIDS complicated by tuberculosis

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