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. 2024 Feb 29;23(1):22.
doi: 10.1186/s12941-024-00682-7.

Nanopore targeted sequencing-based diagnosis of central nervous system infections in HIV-infected patients

Xihong Yang #  1 , Shuilian Zhou #  2   3 , Ziwei Chang  1 , Xiaotong Xi  2   3 , Jiahui Li  1 , Mengjiao Miao  4 , Yaling Chen  1 , Wei Chen  5 , Hongying Zhang  6 , Ran Ding  7   8 , Zhiliang Hu  9   10   11
Affiliations

Nanopore targeted sequencing-based diagnosis of central nervous system infections in HIV-infected patients

Xihong Yang et al. Ann Clin Microbiol Antimicrob. .

Abstract

Background: Early and accurate etiological diagnosis is very important for improving the prognosis of central nervous system (CNS) infections in human immunodeficiency virus (HIV)-infected patients. The goal is not easily achieved by conventional microbiological tests. We developed a nanopore targeted sequencing (NTS) platform and evaluated the diagnostic performance for CNS infections in HIV-infected patients, with special focus on cryptococcal meningitis (CM). We compared the CM diagnostic performance of NTS with conventional methods and cryptococcal polymerase chain reaction (PCR).

Methods: This study included 57 hospitalized HIV-infected patients with suspected CNS infections from September 2018 to March 2022. The diagnosis established during hospitalization includes 27 cases of CM, 13 CNS tuberculosis, 5 toxoplasma encephalitis, 2 cytomegalovirus (CMV) encephalitis and 1 Varicella-zoster virus (VZV) encephalitis. The 2 cases of CMV encephalitis also have co-existing CM. Target-specific PCR amplification was used to enrich pathogen sequences before nanopore sequencing. NTS was performed on stored cerebrospinal fluid (CSF) samples and the results were compared with the diagnosis during hospitalization.

Results: 53 (93.0%) of the patients were male. The median CD4 cell count was 25.0 (IQR: 14.0-63.0) cells/uL. The sensitivities of CSF culture, India ink staining, cryptococcal PCR and NTS for CM were 70.4% (95%CI: 51.5 - 84.1%), 76.0% (95%CI: 56.6 - 88.5%), 77.8% (59.2 - 89.4%) and 85.2% (95%CI: 67.5 - 94.1%), respectively. All those methods had 100% specificity for CM. Our NTS platform could identify Cryptococcus at species level. Moreover, NTS was also able to identify all the 5 cases of toxoplasma encephalitis, 2 cases of CMV encephalitis and 1 VZV encephalitis. However, only 1 of 13 CNS tuberculosis cases was diagnosed by NTS, and so did Xpert MTB/RIF assay.

Conclusions: NTS has a good diagnostic performance for CM in HIV-infected patients and may have the ability of simultaneously detecting other pathogens, including mixed infections. With continuing improving of the NTS platform, it may be a promising alterative microbiological test for assisting with the diagnosis of CNS infections.

Keywords: Central nervous system (CNS) infections; Cryptococcal meningitis; HIV; Nanopore targeted sequencing.

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Conflict of interest statement

All authors have no conflict of interest to declare.

Figures

Fig. 1
Fig. 1
Procedure of nanopore targeted sequencing. The clinical application of the nanopore targeted sequencing (NTS) workflow, which consists of five parts. (1) Sample collection: cerebrospinal fluid of suspected meningitis patients was collected. (2) Nucleic acid extraction: cerebrospinal fluid sample DNA and RNA nucleic acid extraction. (3) Targeted pathogen enrichment: specific polymerase chain reaction (PCR) targeted enrichment of target pathogenic microorganisms. (4) Amplicon library preparation and mixed library: libraries were constructed for all samples using an SQK-PBK004 kit and library mixing was performed. (5) Sequencing and data analysis: library pools were loaded onto Gridion×5 sequencer for 1 h, and original data was analyzed
Fig. 2
Fig. 2
Venn diagrams of positive results of different methods. Positive results of different methods in 27 cryptococcal meningitis cases, including cryptococcal antigen (CrAg), culture, India ink staining, nanopore targeted sequencing (NTS), polymerase chain reaction (PCR). (A) CrAg, culture, India ink staining. (B) CrAg, NTS, PCR. (C) culture, India ink staining, NTS, PCR.
Fig. 3
Fig. 3
Associations of cryptococcal sequencing reads with CrAg and PCR. (A) The comparation of nanopore targeted sequencing (NTS) sequencing reads between groups of cerebrospinal fluid (CSF) cryptococcal antigen (CrAg) titer ≤1:640 and CSF CrAg titer > 1:640 of 27 patients with cryptococcal meningitis, and NTS sequencing reads of each case. (B) The relationship between NTS sequencing reads and cryptococcal PCR cycle threshold (Ct) value of 28 patients. Of 7 cases with negative PCR results, the Ct value was considered as maximum thermal cycles of 40. In both A and B, when NTS sequencing reads was 0, it was adjusted to 0.2 for the purpose of analysis
Fig. 4
Fig. 4
Other pathogens reported by nanopore targeted sequencing. (A) Pathogens except cryptococcus that detected by nanopore targeted sequencing (NTS), and the NTS sequencing reads of each case. Grouped by whether the detection was clinically important. (B) Comparations of clinically composite diagnosed cases, positive NTS and polymerase chain reaction (PCR) cases of each pathogen
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