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.

Cryptosporidiosis-an overview.

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

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a rapidly spreading illness, coronavirus disease 2019 (COVID-19), affecting more than seventeen million people around the world. Diagnosis and treatment guidelines for clinicians caring for patients are needed. In the early stage, we have issued “A rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-nCoV) infected pneumonia (standard version)”; now there are many direct evidences emerged and may change some of previous recommendations and it is ripe for develop an evidence-based guideline. We formed a working group of clinical experts and methodologists. The steering group members proposed 29 questions that are relevant to the management of COVID-19 covering the following areas: chemoprophylaxis, diagnosis, treatments, and discharge management. We searched the literature for direct evidence on the management of COVID-19, and assessed its certainty generated recommendations using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. Recommendations were either strong or weak, or in the form of ungraded consensus-based statement. Finally, we issued 34 statements. Among them, 6 were strong recommendations for, 14 were weak recommendations for, 3 were weak recommendations against and 11 were ungraded consensus-based statement. They covered topics of chemoprophylaxis (including agents and Traditional Chinese Medicine (TCM) agents), diagnosis (including clinical manifestations, reverse transcription-polymerase chain reaction (RT-PCR), respiratory tract specimens, IgM and IgG antibody tests, chest computed tomography, chest x-ray, and CT features of asymptomatic infections), treatments (including lopinavir-ritonavir, umifenovir, favipiravir, interferon, remdesivir, combination of antiviral drugs, hydroxychloroquine/chloroquine, interleukin-6 inhibitors, interleukin-1 inhibitors, glucocorticoid, qingfei paidu decoction, lianhua qingwen granules/capsules, convalescent plasma, lung transplantation, invasive or noninvasive ventilation, and extracorporeal membrane oxygenation (ECMO)), and discharge management (including discharge criteria and management plan in patients whose RT-PCR retesting shows SARS-CoV-2 positive after discharge). We also created two figures of these recommendations for the implementation purpose. We hope these recommendations can help support healthcare workers caring for COVID-19 patients.

https://link.springer.com/content/pdf/10.1186/s40779-020-00270-8.pdf

Chemoprophylaxis, diagnosis, treatments, and discharge management of COVID-19: An evidence-based clinical practice guideline (updated version)

Background: The urgency of the 2019 novel coronavirus outbreak has led to demand of effective antiviral agents Preliminary experiment indicated that Arbidol co

Clinical Efficacy of Arbidol in Patients with 2019 Novel Coronavirus-Infected Pneumonia: A Retrospective Cohort Study

Objective To investigate the infection of Cryptosporidium and its epidemiologicul characteristics in AIDS patients of Southern China. Methods Stool samples eolleted from AIDS cinfirmed patients. The samples were detected for ooeyst of Cryptesporidium by acid fast bacteria stain and indirect fluorescent antibody stain respectively, CD4 eount was detected by Flow Cytometry. Results 212 samples of fresh stool obtained from the AIDS patients who live in Guangdong and Yunnan province. The total infection rate of Cryptesporidium in AIDS patients was 4.25% (9/212), the infectious rate of ooeyst in the group of 50-59 yeats old was signifieantly higher than those in 30-39 (P ＜ 0.01); the infectious rate of oocyst in patients with antiretrovind therapy(ART) was also significantly lower (P = 0.0000);we found the patients coinfected with Cryptosporidium with CD4 count all below 100 cells/μl. However, there were no any difference between the infectious rate to the patient's gender, areas and stool shape. Conclusion AIDS patients infected by Cryptesporidium are not rare in southern China, and the infectious rate was lower than western country. Patients reeeived ART could decrease the infectious rate of Cryptesporidium, Cryptosporidium always happen in patient whose CD4 count was very low(＜ 100 cells/μl).

Key words: 
Cryptosporidiosis; Acquired immunodeficiency syndrome

Detection of cryptosporidium infection among AIDS patients in Guangdong and Yunnan

Objective To develop a high-throughput clinical method on drag-resistance gene mutations of HBV using MALDI-TOF-MS. Method Using MassArray Assay Design software designed the iPLEX primers and followed the iPLEX instruction for amplification, SAP reaction, primer extenction, desalination, dispensing, MALDI-TOF-MS screening and data analysis of the gene mutation locus. 138 serum samples of chronic HBV patients with single drug-resistance or multiple drug-resistance on Lamivudin, adefovi, Entecavir were detected. Result The HBV gene mutation platform was successfully developed and applied on the high-throughput dectection of clinical serum samples. It was also a high throughput assay which could be used to detect for more than 138 samples once. The MALDI-TOF-MS technology and the DNA sequencing simultaneously examine 33 samples, in which result of 10 sample is inconsistent, the including 2 samples by MALDI-TOF-MS technology has not tested, 1 sample has 2 inconsistent mutations. Conclusion Detction of HBV gene mutations using MALDI-TOF-MS is highly-sensitive, highly-accuate, high-thoughput,fast achieved and suithle to use in the diagnosis and monitoring of HBV.

Key words: 
Hepatitis B Virus; MALDI-TOF-MS; DNA; Seqences analysis

[High throughput detection of drug-resistance gene mutations in HBV using MALDI-TOF mass spectrometry].

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 Efficacy of Arbidol in Patients with 2019 Novel Coronavirus-Infected Pneumonia: A Retrospective Cohort Study

Detection of cryptosporidium infection among AIDS patients in Guangdong and Yunnan

[High throughput detection of drug-resistance gene mutations in HBV using MALDI-TOF mass spectrometry].

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