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Detection of pathogenic microbes in cerebrospinal fluid using metagenomic next-generation sequencing

Stewart, Laura (2022) Detection of pathogenic microbes in cerebrospinal fluid using metagenomic next-generation sequencing. Master Essay, University of Pittsburgh.

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HIV-associated neurocognitive disorder (HAND) is a significant public health concern as many of the people living with human immunodeficiency virus (HIV) may suffer from it despite effective antiretroviral therapy (ART), and the specific cause is often unclear. Current screening methods of HAND typically use quantitative PCR (qPCR) to identify pathogens of interest in human cerebrospinal fluid (CSF). However, qPCR has a limitation of missing unexpected pathogens or coinfections. In addition, qPCR targeting multiple pathogens requires a large volume (several mL) of CSF, which is difficult to obtain. Recently, a next-generation sequencing (NGS) - based workflow called Galileo ONE was developed by Arc Bio. Galileo ONE enables comprehensive detection of microbes including viruses, bacteria, and fungi simultaneously using a small volume (400 to 800 µL) of plasma. In this study, we investigated the suitability of Galileo ONE for microbial detection in CSF with the ultimate goal of detecting pathogens causing HAND. Following Galileo ONE instructions, DNA and RNA were extracted from the CSF samples spiked with known amounts of cytomegalovirus (CMV) or HIV, and from clinical CSF samples containing known pathogens previously detected by qPCR. Following the library preparation and NGS, the sequences were generated and analyzed by Cloud-based Galileo ONE Analytics for identification and quantitation of microbes in the CSF samples. Both CMV and HIV were successfully detected in the spiked CSF samples by Galileo ONE. For the clinical CSF samples, some pathogens were detected by both qPCR and Galileo ONE. However, some pathogens which were detected by qPCR were not detected by Galileo ONE. In summary, the Galileo ONE platform shows great potential for clinical diagnosis but requires further improvement to be a suitable tool to unravel the cause and pathogenesis of HAND.


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Item Type: Other Thesis, Dissertation, or Long Paper (Master Essay)
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Stewart, Lauraljs102@pitt.eduljs102
ContributionContributors NameEmailPitt UsernameORCID
Committee ChairChen, YueCHENY@pitt.eduCHENYUNSPECIFIED
Committee MemberRinaldo, CharlesRINALDO@pitt.eduRINALDOUNSPECIFIED
Committee MemberPhan, Tungphantg@upmc.eduTUP5UNSPECIFIED
Date: 12 May 2022
Date Type: Completion
Number of Pages: 29
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Infectious Diseases and Microbiology
Degree: MPH - Master of Public Health
Thesis Type: Master Essay
Refereed: Yes
Date Deposited: 12 May 2022 17:15
Last Modified: 12 May 2022 17:15


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