Diagnosis and analysis of unexplained cases of childhood encephalitis in Australia using metagenomic next-generation sequencing

Abstract

Encephalitis is most often caused by a variety of infectious agents, the identity of which is commonly determined through diagnostic tests utilising cerebrospinal fluid (CSF). Immune-mediated disorders are also a differential in encephalitis cases. We investigated the clinical characteristics and potential aetiological agents of unexplained encephalitis through metagenomic next-generation sequencing of residual clinical samples of multiple tissue types and independent clinical review. A total of 43 specimens, from both sterile and non-sterile sites, were collected from 18 encephalitis cases with no cause identified by the Australian Childhood Encephalitis study. Samples were subjected to total RNA sequencing to determine the presence and abundance of potential pathogens, to reveal mixed infections, pathogen genotypes, and epidemiological origins, and to describe the possible aetiologies of unexplained encephalitis. From this, we identified five RNA and two DNA viruses associated with human infection from both non-sterile (nasopharyngeal aspirates, nose/throat swabs, urine, stool rectal swab) and sterile (cerebrospinal fluid, blood) sites. These comprised two human rhinoviruses, two human seasonal coronaviruses, two polyomaviruses and one picobirnavirus. With the exception of picobirnavirus all have been previously associated with respiratory disease. Human rhinovirus and seasonal coronaviruses may be responsible for five of the encephalitis cases reported here. Immune-mediated encephalitis was considered clinically likely in six cases and RNA sequencing did not identify a possible pathogen in these cases. The aetiology remained unknown in nine cases. Our study emphasises the importance of respiratory viruses in the aetiology of unexplained child encephalitis and suggests that the routine inclusion of non-CNS sampling in encephalitis clinical guidelines/protocols could improve the diagnostic yield.

Direct Metagenomic Diagnosis of Community-Acquired Meningitis: State of the Art

“…Further, there was concordant documentation of 56 different pathogens, including 30 bacterial pathogens, 19 viral pathogens, six fungi and one Balamuthia mandrillaris ( B. mandrillaris ) as the only amoeba detected in a CSF sample ( Table 1 and Figure 4 ). Of the 217 CSF samples in which mNGS investigation yielded results discordant with the routine method, mNGS conclusively found a pathogen in 38/103 CSF samples in which routine investigations found no pathogen, as reported in 25/51 studies: mNGS over-detected RNA viruses, including Human rhinovirus and Human coronavirus ( Li et al, 2021 ), Coxsackievirus 9 and Mumps virus ( Kawada et al, 2016 ), Saint-Louis Encephalitis virus ( Chiu et al, 2017 ), Powassan Virus ( Piantadosi et al, 2018 ), Toscana virus ( Tschumi et al, 2019 ), Jamestown Canyon virus ( Solomon et al, 2021 ), Enterovirus A71 ( Leon et al, 2020 ), and Hepatitis E virus ( Carbo et al, 2020 ), while only HSV-1, HHV-6, and EBV were identified as DNA viruses ( Zhang Y. et al, 2019 ; Carbo et al, 2020 ). Bacterial pathogens documented in 11% of these cases included Listeria monocytogenes in four cases ( Yao et al, 2016 ; Lan et al, 2020 ), Ureaplasma parvum in three cases ( Wang et al, 2020 ; Xing X.-W. et al, 2021 ; Zhan et al, 2021 ) and one case each was detected of Klebsiella pneumoniae ( Zeng et al, 2021 ), Pasteurella multocida ( Morsli et al, 2022 ), Enterococcus faecalis ( Zhang et al, 2021a ), Nocardia farcinica ( Zhang et al, 2021b ), Streptococcus suis ( Zhang et al, 2020 ), and Psychrobacter sp.…”

“…As for the geographic origin of published studies, 28 (56%) were published in Asia, including 25 (49%) from Chinese laboratories ( Table 1 ), one each from Saudi Arabia ( Guan et al, 2021 ), Bangladesh ( Saha et al, 2019 ), and Japan ( Kawada et al, 2016 ) (6.1%); nine in the United States ( Table 1 ); one in Mexico ( Joanna María et al, 2016 ); five in France (10%) (Tabel 1); two in Netherlands ( Edridge et al, 2019 ; Carbo et al, 2020 ); and one each in Switzerland, the United Kingdom, and Spain ( Tschumi et al, 2019 ; Leon et al, 2020 ; Manso et al, 2020 ). One study was also published in Guinea and another in Australia ( Eibach et al, 2019 ; Li et al, 2021 ; Figure 2 ).…”

“…The sample pre-treatment reported in 18/51 (35%) studies included centrifugation (∼15,000 g ), filtration and vortexing in 12 studies, DNase treatment in four studies ( Piantadosi et al, 2018 ; Edridge et al, 2019 ; Manso et al, 2020 ; Morsli et al, 2021d ) and proteinase K treatment in five studies ( Table 1 ; Guan et al, 2015 ; Miller et al, 2019 ; Carbo et al, 2020 ; Manso et al, 2020 ; Morsli et al, 2021a , c ; Piantadosi et al, 2021 ), while no pre-treatment was performed in 15 studies and no such information was provided in 16 studies ( Table 1 ). Extracted nucleic acids (retro-transcribed RNA and DNA) were always quantified using either a Qubit fluorometer and a Qubit DNA and RNA High Sensitivity Assay Kit (Life Technology, United States) ( Table 1 ; Guan et al, 2015 ; Miller et al, 2019 ; Carbo et al, 2020 ; Manso et al, 2020 ; Morsli et al, 2021a , c ; Piantadosi et al, 2021 ), or NanoDrop spectrophotometer (Thermo Fisher Scientific, United States) ( Li et al, 2021 ). Optimisation in the human-to-microbial ratio of DNA was achieved by intermediate microbial genome enrichment or human genome depletion ( Table 1 ).…”

“…Standard BGISEQ pair-end protocols were used in 6/51 studies. Two studies used the PACEseq mNGS test (Hugobiotech, Beijing, China) ( Mao et al, 2021 ; Zeng et al, 2021 ), two studies used VIDISCA and Ion Torrent end-repair library ( Edridge et al, 2019 ; Gao et al, 2021 ), and one study each used the NEBNext Ultra II Directional RNA Library prep kit (New England Biolabs, Ipswich, MA, United States) ( Carbo et al, 2020 ; Leon et al, 2020 ), the KAPA Hyper Prep Kit (Kapa Biosystem, Potters Bar, United Kingdom) ( Yin et al, 2021 ), the NuGEN Ovation Ultralow Library System V2 kit (NuGEN Technologies, United States) ( Guan et al, 2015 ) the Trio RNA-Seq kit (NuGEN Technologies) ( Li et al, 2021 ), and the Roche 454 GS FLX single-end library (Roche Diagnostics, Branford, CT, United States) ( Joanna María et al, 2016 ). In three studies, the mNGS library was constructed following the single-end Oxford Nanopore library preparation protocol ( Morsli et al, 2021a , b , 2022 ).…”

“…no pre-treatment was performed in 15 studies and no such information was provided in 16 studies (Table 1). Extracted nucleic acids (retro-transcribed RNA and DNA) were always quantified using either a Qubit fluorometer and a Qubit DNA and RNA High Sensitivity Assay Kit (Life Technology, United States) (Table 1; Guan et al, 2015;Miller et al, 2019;Carbo et al, 2020;Manso et al, 2020;Morsli et al, 2021a,c;Piantadosi et al, 2021), or NanoDrop spectrophotometer (Thermo Fisher Scientific, United States) (Li et al, 2021). Optimisation in the human-to-microbial ratio of DNA was achieved by intermediate microbial genome enrichment or human genome depletion (Table 1).…”