Materials and Methods

RNA and DNA were extracted from a CSF sample (200ml) by using ELITe InGenius instrument (Elitech, France), which integrates extraction, amplification for viruses and bacteria. Epstein-Barr virus, Herpes simplex virus type 1, Herpes simplex virus type 2, Herpesvirus-6, Cytomegalovirus, Varicella Zoster virus, Adenovirus, Measles virus, Picornavirus, JC Polyomavirus, West Nile Virus, Toscana Virus, Mycoplasma pneumoniae, Borrelia and Mycobacterium tuberculosis were tested. HTLV-1 DNA was extracted from CSF by use of an EZ1&2 DNA Blood kit (Qiagen GmbH, Germany) and amplified as described (Moens B et al., 2009).

Next Generation Sequencing of Cerebrospinal fluid

DNA was extracted with RNeasy plus mini kit (Qiagen GmbH, Germany) where a selective depletion of the genomic DNA and the Ribosomal RNA (rRNA) is carried out using specific filtering columns. Quantification of the obtained RNA was carried out with the Qubit RNA high sensitivity assay kit and the Qubit 3.0 fluorometer (Thermo Fisher Scientific, Rodano, (MI), Italy). The retro transcription and library preparation were assessed using the NEBNext Single Cell/Low Input RNA Library Prep kit for Illumina (New England Biolabs, Milan, Italy) according to the manufacturer’s protocol. The obtained library was then quantified with Qubit DNA high sensitivity assay kit and the Qubit 3.0 fluorometer (Thermo Fisher Scientific). The median size was assessed with a 2100 biological analyzer (Agilent Technologies, Milan, Italy) using a high sensitivity DNA Chip. Libraries were then pooled and denatured according to Illumina’s instructions and charged on a V2 micro MiSeq 300 cycles flow cell (Illumina S.r.l. Milan, Italy). FastQ analysis was carried with the CZ ID and Genome detective online platform (https://czid.org) (https://www. genomedetective.com)

Case Presentation

On January 5th, 2022, a 25-year-old man was admitted to the Emergency Department of the ‘S. Maria delle Scotte’ Hospital in Siena, Italy, with fever, cough, pneumonia, hypoasthenia in limbs (left>right), pain in the left leg and speech disorder. Laboratory results, including complete blood count, metabolic panel and blood cultures, were unremarkable; only an increase of leukocytes (11,96 x 103/mm³) was evidenced. The oropharyngeal swab revealed the presence of RSV by RT-PCR, while influenza virus type A/B and SARS-CoV-2 were negative. Thorax RX did not show parenchymal alterations at foci, nor signs of pleural effusion. A few days later, the patient developed weakness in the lower limbs, more pronounced on the left side, along with pain and language impairment and on January 17th he was admitted to the Neurology Unit. The neurological examination revealed difficulty walking on tiptoes. Romberg test showed slight multidirectional swaying. Bilateral facial diplegia with the remaining cranial nerves was within normal limits. No strength deficits against gravity or resistance in all four limbs were present. Deep tendon reflexes were normal and symmetrical in the upper limbs, absent in the lower limbs except for the patellar reflexes. Cerebellar tests were correctly performed. The patient reported numbness in the lower limbs, especially on the left side. Laboratory tests (complete blood count, negative procalcitonin, C-reactive protein, lactate dehydrogenase, creatine kinase, serum electrolytes, liver and kidney function) were normal. Chest X-ray did not show any abnormalities. A CT scan and later an MRI scan of the cranial and lumbar spine were normal. Cerebrospinal fluid (CSF) analysis was normal; In particular, there was no albuminocytologic dissociation. Isoelectric focusing showed the presence of some oligoclonal IgG at the same isoelectric point in serum and cerebrospinal fluid. CSF polymerase chain reaction assay (PCR) for Epstein-Barr virus, Herpes simplex virus type 1, Herpes simplex virus type 2, Herpesvirus 6, Cytomegalovirus, Varicella Zoster virus, Adenovirus, HTLV-1, Measles virus, Picornavirus, JC Polyomavirus, West Nile Virus, Toscana Virus, Mycoplasma pneumoniae, Borrelia and Mycobacterium tuberculosis was negative. Furthermore, the search for anti-ganglioside and sulfatide antibodies and anti-onconeural antigen antibodies were negative in both blood and cerebrospinal fluid. A neurophysiological study documented an acute motor axonal neuropathy (AMAN). The patient was diagnosed as a case of AMAN (a GBS subtype) based on history, clinical findings and EMG examination. He was treated with plasma-exchange (5 sessions) with good tolerability and complete clinical recovery. During the ten-day stay in hospital, the patient improved, in particular the orbicularis oculi bilateral deficit decreased, although less improvement for the orbicularis oris muscle deficit was observed. Due to the negative results in molecular diagnosis, a second-generation sequencing technology (NGS), not limited by targeted primers, was performed on the CSF. NGS has a fast detection speed and is capable of diagnosing infectious diseases of the central nervous system with speed and efficiency.

Discussion and Conclusions

Direct information on pathogenic microorganisms from brain tissue and CSF can be obtained via NGS with high throughput, thus speeding up the detection process and eliminating the drawbacks of targeted diagnostic methods. Current diagnostic procedures are complicated by the extensive and growing list of viral pathogens that cause encephalitis, and in up to 50% of cases the etiology remains unknown. The need to specify targets in advance is eliminated by sequence-agnostic metagenomic nextgeneration sequencing (mNGS), which is thus highly promising for the diagnosis of encephalitis and other forms of nervous diseases caused by infectious agents. However, the low relative abundance of virus-derived material in clinical specimens compared to hostderived material makes viral mNGS a difficult approach. To ensure reliable detection and accurate identification of viruses in a sample, it is essential to improve this ratio and achieve a sufficient amount of viral reads [15-19]. We set a procedure to enhance the sensitivity of the assay, trying to detect viral agents that were not revealed by classical molecular tests. In this case, the patient was first hospitalized, due to pneumonia, and RSV was detected as the etiologic agent. After a few days, the disease worsened and the man began to show neurological problems. Analyses were then focused on pathogens infecting the central nervous system, without success. Therefore, NGS was performed on the patient’s CSF in order to assess the presence of a virus. The blood-brain barrier (BBB), the essential homeostatic bridge to neural function, plays a vital role in neural protection and molecular passage of microorganisms. Hematogenous spread of viral pathogens is the common mechanism allowing cellular migration of the virus, therefore neurotropism is necessary for viral pathogens to invade the CNS. Before the onset of GB syndrome, most patients showed an antecedent illness, most commonly an upper respiratory tract infection [12]. Extrapulmonary manifestations of RSV involving neurologic, cardiovascular, and hepatic organ systems are well reported [20]. However, neurologic abnormalities, such as convulsions, following cardiopulmonary arrest, apnea, and nuchal rigidity have been mainly reported in children infected with RSV [8]. Saravanos et al. [21] undertook a systematic review that included articles concerning patients <15 years old with RSVassociated severe acute neurologic complications. However, due to the high incidence of RSV viral infection and low prevalence of complications, host factors must also play a role [21]. By contrast, scant evidence exists in the literature implicating RSV as a neuropathogen in the adult population. RSV was detected in the CSF of some patients, as shown in this report, making direct viral invasion of the CNS plausible. A case of Respiratory Syncytial Virus-associated encephalopathy, in which the virus was detected in cerebrospinal fluid and intratracheal aspiration despite negative rapid test results, has been reported [22]. The exact mechanism of CNS penetration and pathologic response of RSV are unknown; however, it is fundamental for diagnosis to verify whether the subject presenting neurologic complications has or had previous respiratory infection, and to enlarge the panel of viruses to look for, including respiratory viruses. While in children the virus antigens seem to trigger an inflammatory response producing a ‘cytokine storm’, which mediates immune cell over activation and cellular dysfunction within the CNS, in adults the virus seems a direct cause of the disease, as shown in this case. The detection of RSV in CSF suggests that it could have potential neurotropism and play a role in the pathogenesis of the nervous system. Therefore, the pathogenesis of respiratory virus-associated encephalopathy is likely diverse and may include both direct and indirect pathways. To prevent and treat infections that affect the nervous system, the responsible pathogens must be identified, and NGS could be a valid tool to aid the diagnosis of these infectious diseases. This case underscores the importance of physicians being mindful of the clinical presentation of GBS, and the importance to also investigate on the presence of respiratory viruses, such as RSV, as etiologic agents.

Table 1: Literature cases of Guillan-Barrè syndrome associated with RSV infection.

A summary of the literature cases of Guillan-Barrè syndrome associated with RSV infection. For each article we have registered the following data: first author, clinical manifestation, molecular diagnosis, therapy and clinical course.

Acknowledgements: This study was supported by the Italian Ministry of Health- CUP B93C2200121000, CCM 2022 ‘SURVEID’.

Ethical approval and consent to participate: The study has been performed in accordance with local ethics committee requirements and has therefore been performed in accordance with the ethical standards laid down in the Declaration of Helsinki in 1964 and in its later amendments. Written informed consent was obtained from the patient.

Conflict of interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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