Internal Transcribed Spacer Amplicon Sequencing In Cerebrospinal Fluid And Genotyping Of Cryptococcal Meningitis

Objective: Cryptococcal meningitis is a severe infectious disease associated with high morbidity and mortality.Rapidity and accuracy of diagnosis contribute to better prognosis,but readily available tools,such as microscopy,culture,and antigens do not perform well all the time.Dramatic advances in next-generation sequencing has allowed for a relatively unbiased,quick and accurate profiling of microbial communities in many types of body fluid samples.Our study attempted to diagnose and genotype cryptococci in the cerebrospinal fluid(CSF)samples from patients with cryptococcal meningitis using sequencing of internal transcribed spacer(ITS)amplicons approach.Methods: The CSF samples were collected from 11 clinically diagnosed cryptococcal meningitis patients and 4 non-infectious controls.ITS1 ribosomal deoxyribonucleic acid(rDNA)gene of 15 whole samples were amplified by universal forward primer ITS1 and reverse primer ITS2 and sequenced by Illumina MiSeq Benchtop Sequencer.The qualified data were analyzed for biological information such as operational taxonomic units(OTU)clustering and species annotation,and the dominant species were determined according to the proportion of OTU,and the OTU annotated as Cryptococcus neoformans was classified into ITS types.The diagnostic results of ITS amplicon sequencing were compared with the results of cerebrospinal fluid India ink microscopy and fungal culture.The genomic DNA of fungal colony cultured by blood plate culture was extracted.The transcriptional spacer region ITS1 of ITS ribosomal rDNA and some fragments of fungus-specific housekeeping gene CAP59 were amplified by PCR,and Sanger sequencing was performed in ABI PRISM 3730 XL gene analyzer to further verify the diagnosis results.Results: 1.The number of OTU sequences belonging to Cryptococcus neoformans accounted for 95.9% to 99.97% of the total number of OTU sequences in Sample No.1-11.Cryptococcus neoformans was recognized as the dominant fungal species in the microflora of the samples.Combined with the clinical manifestations of the patients,it was considered that Patients No.1-11 had cryptococcal meningitis.The ITS genotype in 11 CSF samples was defined as ITS type 1,consistent with previous studies in China.2.The sequencing of ITS amplicon of Sample No.12-15 yielded 8-60 optimized sequences,which were too few to detect reliable pathogens.3.The diagnostic results of ITS amplicon sequencing in 15 cases of cerebrospinal fluid were consistent with the diagnostic results of CSF ink staining and fungal culture,which were also confirmed by Sanger sequencing of ITS-1 region and the partial fragment of fungus-specific housekeeping gene CAP59 of the fungi extracted from blood plate.In addition,the typing results were confirmed by Sanger sequencing of the previous regions.Conclusions: Cerebrospinal fluid ITS amplicon sequencing can be used as a complementary method for etiological diagnosis of Cryptococcal meningitis.It can detect the pathogen cryptococcus neoformans in cerebrospinal fluid as a whole and quickly.At the same time,it is also a genotyping approach without culture,which is beneficial to the diagnosis and treatment of cryptococcal meningitis.