Department of Neurology and Institute of Neurology, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian 350005, China.
School of Postgraduate Education, Fujian Medical University, Fuzhou, Fujian 350122, China.
Chin Med J (Engl). 2019 Dec 5;132(23):2827-2834. doi: 10.1097/CM9.0000000000000541.
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. Our study attempted to diagnose and genotype cryptococcus in the cerebrospinal fluid (CSF) samples from patients with cryptococcal meningitis using the approach of metataxonomics of Internal Transcribed Spacer (ITS) amplicons.
The CSF samples were collected from 11 clinically suspected cryptococcal meningitis patients and four non-infectious controls. Samples were recruited from the First Affiliated Hospital of Fujian Medical University Hospital, Fuzhou Fourth Hospital and the 476th Hospital of Chinese People's Liberation Army from December 2017 to December 2018. ITS1 ribosomal deoxyribonucleic acid (rDNA) genes of 15 whole samples were amplified by universal forward primer ITS1 (CTTGGTCATTTAGAGGAAGTAA) and reverse primer ITS2 (GCTGCGTTCTTCATCGATGC), sequenced by Illumina MiSeq Benchtop Sequencer. The results were confirmed by sanger sequencing of ITS1 region and partial CAP59 gene of microbial isolates from 11 meningitic samples. Pair-wise comparison between infectious group and control group was conducted through permutational multivariate analysis (PERMANOVA) in R software.
The 30,000 to 340,000 high-quality clean reads were obtained from each of the positively stained or cultured CSF samples and 8 to 60 reads from each control. The samples from 11 infected patients yielded detectable cryptococcal-specific ITS1 DNA with top abundance (from 95.90% to 99.97%), followed by many other fungal groups (each <1.41%). ITS genotype was defined in 11 CSF samples, corresponding to ITS type 1, and confirmed by Sanger sequencing. A statistically significant difference (r = 0.65869, P = 0.0014) between infectious group and control group was observed.
The metataxonomics of ITS amplicons facilitates the diagnosis and genotype of cryptococcus in CSF samples, which may provide a better diagnostic approach of cryptococcal infection.
隐球菌性脑膜炎是一种严重的传染性疾病，发病率和死亡率均较高。快速准确的诊断有助于改善预后，但现有的检测工具，如显微镜检查、培养和抗原检测，并非始终能发挥良好的作用。本研究尝试采用内转录间隔区（ITS）扩增子的分类组学方法，对隐球菌性脑膜炎患者的脑脊液（CSF）样本进行隐球菌的诊断和基因分型。
11 例疑似隐球菌性脑膜炎的临床患者和 4 例非感染性对照的 CSF 样本被收集。样本来自 2017 年 12 月至 2018 年 12 月期间福建医科大学附属第一医院、福州第四医院和中国人民解放军第 476 医院。使用通用正向引物 ITS1（CTTGGTCATTTAGAGGAAGTAA）和反向引物 ITS2（GCTGCGTTCTTCATCGATGC）对 15 个全样本的 ITS1 核糖体脱氧核糖核酸（rDNA）基因进行扩增，使用 Illumina MiSeq Benchtop Sequencer 进行测序。通过对 11 例脑膜炎样本中微生物分离物的 ITS1 区和部分 CAP59 基因进行 Sanger 测序，对结果进行确认。通过 R 软件中的置换多元方差分析（PERMANOVA）对感染组和对照组进行两两比较。
每个经染色或培养的 CSF 样本均获得了 30,000 至 340,000 个高质量的清洁读段，每个对照样本获得了 8 至 60 个读段。从 11 例感染患者的样本中均检测到了可检测的隐球菌特异性 ITS1 DNA，其丰度最高（95.90%至 99.97%），其次是许多其他真菌群（每个均<1.41%）。在 11 例 CSF 样本中确定了 ITS 基因型，对应于 ITS 1 型，并通过 Sanger 测序进行了确认。感染组和对照组之间存在显著差异（r=0.65869，P=0.0014）。
ITS 扩增子的分类组学有助于 CSF 样本中隐球菌的诊断和基因分型，可能为隐球菌感染提供更好的诊断方法。
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