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Application of next generation sequencing for detection of protozoan pathogens in shellfish.

ABSTRACT: Food and waterborne protozoan pathogens can cause serious disease in people. Three common species Cryptosporidium parvum, Giardia enterica and Toxoplasma gondii can contaminate diverse shellfish species, including commercial oysters. Current methods of protozoan detection in shellfish are not standardized, and few are able to simultaneously identify multiple species. Here, we present a novel metabarcoding assay targeting the 18S rRNA gene followed by next generation sequencing (NGS) for simultaneous detection of Cryptosporidium spp., Giardia spp. and T. gondii spiked into oyster samples. We further developed a bioinformatic pipeline to process and analyze 18S rRNA data for protozoa classification. The ability of the NGS assay to detect protozoa was later compared with conventional PCR. Results demonstrated that background amplification of oyster and other eukaryotic DNA competed with that of protozoa for obtained sequence reads. Sequences of target protozoans were obtained across all spiking levels; however, low numbers of target sequences in negative controls imply that a threshold for true positives must be defined for assay interpretation. While this study focused on three target parasites, the ability of this approach to detect numerous known and potentially unknown protozoan pathogens make it a promising screening tool for monitoring protozoan contamination in food and water.

SUBMITTER: DeMone C

PROVIDER: S-EPMC7708651 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Application of next generation sequencing for detection of protozoan pathogens in shellfish.

DeMone Catherine C Hwang Mei-Hua MH Feng Zeny Z McClure J Trenton JT Greenwood Spencer J SJ Fung Rebecca R Kim Minji M Weese J Scott JS Shapiro Karen K

Food and waterborne parasitology 20201118

Food and waterborne protozoan pathogens can cause serious disease in people. Three common species Cryptosporidium parvum, Giardia enterica and Toxoplasma gondii can contaminate diverse shellfish species, including commercial oysters. Current methods of protozoan detection in shellfish are not standardized, and few are able to simultaneously identify multiple species. Here, we present a novel metabarcoding assay targeting the 18S rRNA gene followed by next generation sequenci ...[more]

PMID: 33299933

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Project description:BackgroundDifferential diagnosis of patients with suspected infections is particularly difficult, but necessary for prompt diagnosis and rational use of antibiotics. A substantial proportion of these patients have non-infectious diseases that include malignant tumors. This study aimed to explore the clinical value of metagenomic next-generation sequencing (mNGS) for tumor detection in patients with suspected infections.MethodsA multicenter, prospective case study involving patients diagnosed with suspected infections was conducted in four hospitals in Shanghai, China between July 2019 and January 2020. Based upon mNGS technologies and chromosomal copy number variation (CNV) analysis on abundant human genome, a new procedure named Onco-mNGS was established to simultaneously detect pathogens and malignant tumors in all of the collected samples from patients.ResultsOf 140 patients screened by Onco-mNGS testing, 115 patients were diagnosed with infections; 17 had obvious abnormal CNV signals indicating malignant tumors that were confirmed clinically. The positive percent agreement and negative percent agreement of mNGS testing compared to clinical diagnosis was 53.0% (61/115) and 60% (15/25), vs. 20.9% (24/115) and 96.0% (24/25), respectively, for conventional microbiological testing (both P <0.01). Klebsiella pneumoniae (14.8%, 9/61) was the most common pathogen detected by mNGS, followed by Escherichia coli (11.5%, 7/61) and viruses (11.5%, 7/61). The chromosomal abnormalities of the 17 cases included genome-wide variations and local variations of a certain chromosome. Five of 17 patients had a final confirmed with malignant tumors, including three lung adenocarcinomas and two hematological tumors; one patient was highly suspected to have lymphoma; and 11 patients had a prior history of malignant tumor.ConclusionThis preliminary study demonstrates the feasibility and clinical value of using Onco-mNGS to simultaneously search for potential pathogens and malignant tumors in patients with suspected infections.

Dataset Information

Application of next generation sequencing for detection of protozoan pathogens in shellfish.

Publications

Application of next generation sequencing for detection of protozoan pathogens in shellfish.

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