Metagenomic next-generation sequencing for etiological diagnosis of an unexpected rabies case with unclear exposure history

Metagenomic next-generation sequencing for etiological diagnosis of an unexpected rabies case with unclear exposure history | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Metagenomic next-generation sequencing for etiological diagnosis of an unexpected rabies case with unclear exposure history jing Wu, Yingjie QI, Wenyan Zhang, Lixue Liu, Jiangrong Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4380249/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Feb, 2025 Read the published version in BMC Infectious Diseases → Version 1 posted 4 You are reading this latest preprint version Abstract Background Rabies is an acute and lethal zoonotic disease caused by the rabies virus (RABV). After onset, there are no effective drugs or treatment methods. We reported a fatal rabies case with no clear history of animal bites and atypical early clinical symptoms. And unexpectedly, we discovered that unique RABV sequences were detected in the patient's saliva by metagenomic next-generation sequencing (mNGS). The patient was diagnosed with rabies based on the results of saliva mNGS, real-time reverse transcriptase polymerase chain reaction (RT-PCR) conducted by local Centers for Disease Control and Prevention (CDC), the detection of human rabies virus IgG antibodies performed in our laboratory, as well as the clinical symptoms of pruritus, agitation, paralysis, and excessive salivation at disease onset. Case presentation A 49-year-old female from Hefei, Anhui Province, China, presented to a local hospital with fever, pruritus, chest distress, and shortness of breath. During the consultation, the patient exhibited agitation and was later admitted to the intensive care unit (ICU) for endotracheal intubation and mechanical ventilation due to worsened agitation and dyspnea. Cerebrospinal fluid (CSF) and blood samples were collected and pathogenic microorganism identification was performed by culture and mNGS. However, all results were negative. In addition, the patient did not display typical rabies-specific symptoms such as aerophobia, hydrophobia or photophobia from onset to admission. Subsequently, saliva samples were collected for mNGS detection following consultation with experts at our hospital. Nucleic acid sequences uniquely aligned to the rabies virus (RABV) were identified in these samples. The result was further confirmed by local CDC through RT-PCR which detected part of the N gene of RABV in the saliva sample. Then the patient was transferred to our hospital's ICU for isolation. Unfortunately, she died on the 10th day of admission due to multiple organ failure. The patient remained in a comatose state during symptomatic supportive medical care in ICU, and the progression of the disease was accompanied by intermittent elevated body temperature, excessive salivation, and limb weakness. The detection of human rabies virus IgG antibodies reported positive during the advanced stage of the disease. We consistently verified with the patient's family member that there was no clear history of animal bites and no history of RABV vaccination. Furthermore, we performed phylogenetic analysis of partial L and G gene sequences of RABV obtained by mNGS (designated HFG23-L and HFG23-G, respectively), the results showed that both HFG23-L and HFG23-G belonged to the China I lineage, and shared 99.7% similarity with the Fengtai strain isolated from dogs in Beijing. The results suggested that the origin of RABV in this case may be a dog from the northern China. Conclusions The patient's non-specific prodromal symptom, along with negative culture and mNGS results of blood and CSF, impose challenges on promptly and definitively diagnosing rabies in this case. The identification of unique RABV sequence through mNGS in the patient's saliva sample suggested that mNGS could serve as a valuable screening tool for the etiological diagnosis of rabies, especially when timely laboratory testing was unavailable or when patients lacked a clear exposure history. rabies rabies virus saliva metagenomic next-generation sequencing phylogenetic analysis unclear exposure history Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Human rabies is a deadly disease caused by RABV, which is a negative strand (antisense) RNA virus belonging to the family Rhabdoviridae and genus Lyssavirus [ 1 ]. In China, dog-associated RABV is the main cause of human rabies [ 2 ]. And RABV is present in the saliva of wild and domestic infected-dogs and in the tissues at the injury site [ 1 ]. RABV infection in humans manifests as a furious or dumb forms, characterized by a progression through four clinical stages: incubation, prodrome, acute neurological signs of encephalomyelitis, and ultimately death [ 3 ]. The primary clinical symptoms of rabies include aerophobia, hydrophobia, photophobia, dysphagia, mania, acute fatal encephalomyelitis and paralysis. Once rabies symptoms occur, the mortality rate of human is 100% [ 4 ]. A total of 3,302 rabies cases were reported from 2015 to 2021 in China [ 5 ]. Anhui province is divided into medium epidemic regions of rabies. In 2017, there were 28,243 persons obtaining postexposure prophylaxis treatment from clinics, and 39 rabies cases were reported in Anhui province. Furthermore, 12 rabies cases were reported in Anhui province in 2021[ 5 ]. Though the number of rabies cases is declining in China from 2015, RABV is still a fatal factor for human life. To surveil and control rabies, early and accurate diagnosis is essential for clinicians. Clinical diagnosis and lab testing are always used to diagnose rabies [ 6 ]. While it is difficult for clinicians to diagnose rabies without recommended clinical symptoms (including hydrophobia and aerophobia) and a clear exposure history (wild/domestic animal bites or scratch) [ 7 ]. Lab testing methods will not be specifically applied for rabies detection due to the negative clinical diagnosis, resulting in RABV missing detection and spreading [ 8 ]. Metagenomic next-generation sequencing (mNGS) is able to theoretically detect all pathogens in clinical samples within 24 hours [ 9 ]. mNGS is still capable of identifying infectious agents when lab testing results are negative. More positive rabies cases were detected with saliva samples (about 32%), followed by CSF (about 21%) and blood samples (about 10%) [ 10 ]. Therefore, for rabies virus detection, saliva samples are preferred, followed by CSF (cerebrospinal fluid) samples and blood samples. In this study, the case was lack of recommended clinical symptoms and animal contact history. Furthermore, lab testing results of CSF and blood samples from the case were all negative. These clinical and lab testing results were tricky for clinicians to diagnose rabies. Finally, mNGS characterized the RABV in the saliva samples of patient. And probable origin of rabies virus was also investigated in this study. Case presentation A 49-year-old woman presented with unexplained fever and pruritus, which did not improve after self-administration of antipyretic medication. Despite receiving antiviral treatment at a local clinic the following day, her symptoms were not relieved. Subsequently, she was admitted to a reputable local hospital due to chest distress and shortness of breath. Based on blood test results, potassium supplementation was initiated for the treatment of hypokalemia. Throughout the course of treatment, the patient exhibited agitation. Consultations with neurology and psychiatry specialists failed to alleviate or ameliorate her manic symptoms which continued to worsen. Due to concurrent complaints of chest congestion, dyspnea and respiratory distress, she was transferred to the intensive care unit (ICU) for endotracheal intubation and mechanical ventilation. Five days later, the patient underwent bedside temporary cardiac pacemaker treatment due to an unstable heart rate. During hospitalization, based on the aforementioned symptoms, the physician suspected infection. Cerebrospinal fluid (CSF) and blood samples were collected from the patient for culture identification. Additionally, CSF was subjected to metagenomic next-generation sequencing (mNGS). However, both culture and sequencing results were negative. Then, several specialists from our hospital were invited for consultation. According to the patient's clinical symptoms and manifestations, doctors recommended further investigation into potential pathogenic agents. Therefore, saliva samples were collected from the patient and sent to our hospital's laboratory for mNGS detection. mNGS results showed that the patient was infected by RABV, with 1566 unique viral reads and 89.06% coverage rate of the RABV genome (Fig. 1 A). Consequently, on December 7, 2023, the patient was transferred to ICU in our hospital for isolation treatment. Upon admission, the patient presented with unconsciousness and exhibited temperature of 37.3°C, pulse rate of 102/min, respiratory rate of 16/min, and blood pressure of 92/57 mmHg. The patient exhibited non-cooperation during the physical examination. Unequal pupil size (anisocoria) was observed, along with sensitivity to light and blood stains in the oral cavity. The neck displayed supple and lack of resistance. Intubation was performed, and ventilation was supported by a ventilator, accompanied by coarse breath sounds bilaterally in both lungs. No evident sputum rales were present, and bilateral Babinski sign as well as meningeal irritation sign were negative. The abdomen appeared flat, soft, and non-tender without any signs of edema in both lower limbs or jaundice on the skin or mucosa throughout the body. Additionally, no deformity of the head was noted. The patient has not exhibited any rabies-specific symptoms, such as aerophobia, hydrophobia, or photophobia, since the onset of fever and pruritus. After the detection of the RABV sequence through mNGS, a saliva sample was sent to the local CDC for thorough examination, and presence of the RABV was confirmed by RT-PCR (Fig. 1 B, Table 1 ). The patient had been in a comatose state, and the progression of the disease was accompanied by elevated white blood cell (WBC) count, increased levels of C-reactive protein (CRP), serum amyloid A (SAA), liver and kidney function indicators, electrolyte imbalance, continuous decline in red blood cells count and hemoglobin levels, intermittent temperature elevation, unstable pulse (Fig. 2 , Table 2 ), sialorrhea, and quadriplegia. During the initial four days of hospitalization, the patient had a Glasgow Coma Scale (GCS) score of 4T. On the fifth day of admission, there was a progressive decrease in blood pressure observed. At the same time, the detection of human rabies virus IgG antibodies (The Human Rabies Virus IgG Rapid Test Kit (Colloidal Gold), Beijing Beier Bioengineering Co., Ltd, Beijing, China) was conducted which yielded a positive result. The patient had not received prior rabies vaccination. Based on the presence of pruritus, accompanied by dysphoria, respiratory failure, and arrhythmia, the diagnosis of rabies was confirmed for this patient. The patient was managed with comprehensive supportive care in ICU, including respiratory support, fluid resuscitation, antimicrobial therapy, nutrition supplementation, and other appropriate symptomatic interventions. However, the therapeutic response was not prominently observed. Unfortunately, the patient died to multiple organ failure ten days after admission. Table 1 The primers of RT-qPCR Name Sequence Position in N gene Size RV-F1 AGTCTCCYTAYTCATCRAATGC 889–993 105 RV-R1 GCAATAACTGTYGCATTRAGAG RV-R2 GCAATRACTGTCGCATTAAGAG RV-PF (FAM,BQ1) CATATAGCATCCRACAAAGTGAATGAG The RT-qPCR was performed targeting at the conserved regions of the nucleoprotein gene N Table 2 Partial laboratory testing results of patients in our hospital Laboratory Test 2023.12.08 12.09 12.10 12.11 12.14 12.15 12.16 White blood cell (×109/L) 10.89 10.65 22.74 6.58 C-reactive protein(mg/L) 23.89 14.61 124.21 210.28 Red blood cell(×109/L) 3.78 3.32 3.32 2.62 Hemoglobin(g/L) 79 70 72 57 Prothrombin(s) 14 12.1 D-Dimer(µg/ml) 4.94 2.06 Alanine aminotransferase(U/L) 907 572 356 223 100 Aspartate aminotransferase(U/L) 684 172 60 173 101 Creatinine(µmol/L) 74 61 37 283 352 250 Glucose(mmol/L) 7.35 10.11 4.43 K+(mmol/L) 4.72 4.65 4.6 4.49 7.85 6.45 3.62 Serum amyloid A(mg/L) 255 246.6 253.4 Procalcitonin(ng/ml) 0.17 38.16 creatine kinase-MB(ng/ml) 7 5 18 lactic dehydrogenase(U/L) 787 348 209 266 Myoglobin(ng/ml) 82 80 62 1203 B-type natriuretic peptide(pg/ml) 3127 2330 > 35000 > 35000 Sputum culture negative negative Before the patient's saliva mNGS results indicated a RABV infection, the patient had been in a comatose state and unable to communicate directly or effectively. Therefore, we corroborated the history of animal bites with the patient's daughter, and confirmed that incident of small animal bites, particularly from dogs, did not happened to the patient. To uncover possible infectious source of RABV in this case, phylogenetic analysis was implemented. Partial sequence fragment of L or G gene was obtained (namely HFG23-L and HFG23-G, respectively), and grouped into China I lineage of rabies virus (Fig. 3 , Fig. 4 ). HFG23-L and HFG23-G both showed about 99.7% homology with the corresponding sequences of strain Fengtai RABV (KC660078). Fengtai strain was isolated from a dog in Beijing, China. The results indicated that rabies virus in this study, infecting 49-year-old female, may originate from a dog. Discussion Timely diagnosis of rabies is indispensable for public health administration to control and surveil rabies [ 6 ]. In this study, the patient only felt itching and irritable mood in the early period, and then became coma in a few days. The patient did not exhibit any rabies-specific symptoms of hydrophobia or aerophobia during the hospital visit. In addition, culture results of the patient's CSF and blood samples, as well as mNGS results of CSF, were negative, confusing clinicians to get an accurate diagnosis. Animal contact details were clear for previously reported rabies cases, and therefore, clinicians were able to get hints for rabies diagnosis on time [ 11 – 13 ]. Nevertheless, animal contact history was denied by the patient’s daughter. Therefore, the patient was a great challenge for clinicians to make rabies diagnosis due to her quickly worsening situations and unclear causative reason. However, upon admission of the patient to our hospital's ICU for isolation treatment, RABV was identified in saliva samples from patient by mNGS. Previous researches showed that positive rate of rabies in saliva samples (about 32%) is higher than that of CSF and blood samples [ 10 ]. It is recommended that saliva, rather than CSF or blood (serum), is considered as the primary choice for RABV detection in the stage of rabies development, particularly when prodromal symptoms are atypical and exposure history is uncertain. mNGS may be a useful tool for clinical diagnosis of similar cases in the future [ 14 – 17 ]. In addition, more experience should be accumulated for accurate detection of rabies associated with non-specific clinical symptoms. Rabies virus sequences were assembled, and analyzed by Neighbor-Joining (NJ) method. Phylogenetic tree of partial sequence of L (1966 bp/6575 bp, 30.36%) and G (1476 bp/1675 bp, 88.12%) gene showed that the virus belonged to China I lineage of RABV. China I lineage is a dog-associated prevalent lineage in China [ 18 , 19 ]. The homology of L and G gene partial sequences were about 99.7% with strain Fengtai rabies virus. G gene of rabies virus is conserved, and equivalent with N gene at phylogenetic position [ 20 ]. The similarity of G gene to virus strains isolated in Anhui was about 97%, indicating the virus was phylogenetically closer to Fengtai strain. It means that the virus may not belong to local rabies virus strains. For this patient, we did not get complete virus genome, because additional samples can’t be collected after her death. All the results demonstrated that the virus may originated from a dog in this study. Although there was no certain dog bite or scratch history for the patient, current evidence inferred that the patient may have contacted with a dog. Additionally, local CDC verified the RABV detected by mNGS in saliva samples. Two diagnostic kits based on RT-PCR method were employed to detect RABV in saliva sample, but failed. Successful verification of RARV was achieved by a third diagnostic kit. Therefore, RT-PCR did not always detect rabies virus even though 1566 viral reads were present in saliva samples. L and G partial sequences were obtained from saliva sample, however, L and G gene are not used by commercial reagent kits to confirm rabies diagnosis with RT-PCR method. Although two small N gene fragments (< 500 bp) in the saliva sample were assembled, only one N gene fragment was targeted by RT-PCR primers. Therefore, RT-PCR method should be optimized to be much more sensitive and accurate, and extend the target regions of primers. The results indicated that mNGS was much more sensitive than routine lab testing methods such as RT-PCR, and may be a potential tool for CDC to validate rabies virus or other infectious diseases. According to the published data, the number of rabies cases declines significantly in China year by year. But the regional distribution of rabies did not shrink at district and county level [ 5 ]. In China, dog population was about 136,109 per thousand people in 2022 [ 21 ]. Dog issues are present in China such as free roaming dogs and lack of dog vaccination awareness [ 22 ]. In 2020, rabies infection mainly affected the age group of 40–70 years, corresponding reported cases accounted for 69% of all cases [ 22 ]. Rabies is preventable with timely wound treatment, vaccine and immunoglobulin reagent. However, post-exposure prophylaxis vaccination rates vary greatly in different regions of China. In this study, the patient, 49-year-old, lived in a county in Anhui province. She may contact with a dog, but didn’t have post-exposure prophylaxis following official recommendation. Anhui province belongs to medium risk groups, and 12 cases were reported in 2021. To avoid occurrence of similar tragedy in Anhui, people need to pay attention on animal contacts. There is no effective therapy for rabies except vaccines. However, the first recovery case was reported in 2005 [ 23 ]. Willoughby and colleagues cured the patient by suppressing her brain function and activate her immune response. Afterwards, there is no rabies case rescued successfully by clinicians. The whole process needs high experienced multidiscipline clinicians, these essentials may help human conquer rabies in the future. Therefore, to eliminate rabies till 2030, education and public advocacy should be promoted among all age groups in China [ 22 ]. Conclusion The manifestations of rabies exhibit significant variation during the onset period, and unspecific early symptoms, rapid disease progression, and an unclear exposure history impose challenges on the diagnosis, intervention, and nursing of rabies cases. Utilizing appropriate molecular testing tools (such as mNGS) is imperative for confirming cases of rabies. It is crucial to enhance knowledge and awareness regarding rabies while providing suitable post-exposure prophylaxis in order to mitigate the incidence of human rabies and associated fatalities. Abbreviations rabies virus (RABV) metagenomic next-generation sequencing (mNGS) Center for Disease Control and Prevention (CDC) intensive care unit (ICU) central nervous system (CNS) C-reactive protein (CRP) cerebrospinal fluid (CSF) Declarations Acknowledgements Not applicable. Author contributions Epidemiological investigation and medical information: Y. Yang, X. Zhang. Sequencing analysis: L. Liu, J. Chen, W. Zhang. Writing and original draft preparation: J. Wu, Y. Qi, Y. Shi. X. Liu Supervision: J. Wu, Y. Shi. Commenting and editing: all authors. Funding This work was supported by the seventh cycle clinical key (cultivation) specialty in Hefei Department Construction Project (Clinical Laboratory) (Z033-1). Availability of data and materials The data supporting this study’s the findings are available from the corresponding author upon reasonable request. Ethics approval and consent to participate This study was approved and performed according to the guidelines of the Medical Research Ethics Committee of the First Affiliated Hospital of University of Science and Technology of China (Anhui Provincial Hospital) (No.2024-RE-63). Consent for publication Not applicable. Competing interests The authors of this study declare no commercial relationships and no competing interest. References Jackson AC. Human Rabies: a 2016 Update. Curr Infect Dis Rep. 2016;18:38. Miao F, Li N, Yang J, Chen T, Liu Y, Zhang S, et al. Neglected challenges in the control of animal rabies in China. One Health. 2021;12:100212. Hemachudha T, Ugolini G, Wacharapluesadee S, Sungkarat W, Shuangshoti S, Laothamatas J. Human rabies: neuropathogenesis, diagnosis, and management. Lancet Neurol. 2013;12:498–513. 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Cite Share Download PDF Status: Published Journal Publication published 24 Feb, 2025 Read the published version in BMC Infectious Diseases → Version 1 posted Editorial decision: Revision requested 09 May, 2024 Submission checks completed at journal 08 May, 2024 Editor assigned by journal 08 May, 2024 First submitted to journal 07 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4380249","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":300456814,"identity":"588b4ff0-ed14-44b8-a915-d05b07241f46","order_by":0,"name":"jing Wu","email":"","orcid":"","institution":"Infection Hospital Area of the First Affiliated Hospital of University of Science and Technology of China (Hefei Infectious Disease Hospital)","correspondingAuthor":false,"prefix":"","firstName":"jing","middleName":"","lastName":"Wu","suffix":""},{"id":300456815,"identity":"a2ab3c25-89bb-46b6-8fc1-85b3e923ab5d","order_by":1,"name":"Yingjie QI","email":"","orcid":"","institution":"Infection Hospital Area of the First Affiliated Hospital of University of Science and Technology of China (Hefei Infectious Disease Hospital)","correspondingAuthor":false,"prefix":"","firstName":"Yingjie","middleName":"","lastName":"QI","suffix":""},{"id":300456816,"identity":"a1e432b5-c7c9-4c1d-a37d-c32aaffc3638","order_by":2,"name":"Wenyan Zhang","email":"","orcid":"","institution":"The Center of Disease Control and Prevention in Hefei","correspondingAuthor":false,"prefix":"","firstName":"Wenyan","middleName":"","lastName":"Zhang","suffix":""},{"id":300456817,"identity":"8509fee0-513d-44e6-9fb3-195b67e21d10","order_by":3,"name":"Lixue Liu","email":"","orcid":"","institution":"BGI Genomics","correspondingAuthor":false,"prefix":"","firstName":"Lixue","middleName":"","lastName":"Liu","suffix":""},{"id":300456818,"identity":"f99acaa0-21fe-4174-9045-aa5f4237a147","order_by":4,"name":"Jiangrong Chen","email":"","orcid":"","institution":"BGI Genomics","correspondingAuthor":false,"prefix":"","firstName":"Jiangrong","middleName":"","lastName":"Chen","suffix":""},{"id":300456819,"identity":"0e1f7e23-ff7a-4b11-9365-177021895f49","order_by":5,"name":"Yun Yang","email":"","orcid":"","institution":"Infection Hospital Area of the First Affiliated Hospital of University of Science and Technology of China (Hefei Infectious Disease Hospital)","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Yang","suffix":""},{"id":300456821,"identity":"f99c8c54-ccff-4e88-b981-8c0b522f63e8","order_by":6,"name":"Xuanshun Zhang","email":"","orcid":"","institution":"Infection Hospital Area of the First Affiliated Hospital of University of Science and Technology of China (Hefei Infectious Disease Hospital)","correspondingAuthor":false,"prefix":"","firstName":"Xuanshun","middleName":"","lastName":"Zhang","suffix":""},{"id":300456824,"identity":"1ba56da3-5e43-48f5-970d-da626b6b5d52","order_by":7,"name":"Xinru Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of USTC, University of Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Xinru","middleName":"","lastName":"Liu","suffix":""},{"id":300456826,"identity":"1ed3f883-d972-4e1c-bd96-9c549864be48","order_by":8,"name":"Yuru Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACfvbG9h8f/9XUN7Y3EKlFsufwAckZbMcYm3sOEKnF4EZagjQPGzNj+4wEYl12I8fAmAeoh3fm4403GGpsognqYOx5Y5A4R0KGTXJ2WrEFw7G03AZCWpjZcwwOvDFg4zGcnWMmwdhwmLAWNoYcwwaeBGYJ+5tniNTCw5GWzMhzgNmAcQYPkVokeA4fY5zZcCyBsQfolwRi/GJ/vLGN4WNDTQJj++GNNz7U2BDWggwMJBJIUQ7RQqqOUTAKRsEoGBkAAKF8QDciewo5AAAAAElFTkSuQmCC","orcid":"","institution":"Infection Hospital Area of the First Affiliated Hospital of University of Science and Technology of China (Hefei Infectious Disease Hospital)","correspondingAuthor":true,"prefix":"","firstName":"Yuru","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2024-05-07 04:54:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4380249/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4380249/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12879-025-10687-y","type":"published","date":"2025-02-24T15:58:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56621206,"identity":"12f81e04-42d5-42ab-824b-67605a206e5e","added_by":"auto","created_at":"2024-05-16 18:06:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":96983,"visible":true,"origin":"","legend":"\u003cp\u003eThe metagenomic next-generation sequencing and polymerase chain reaction (PCR) results of RABV. Distribution map of rabies virus genome reads detected in the laboratory(A), Detection of rabies virus amplification curve in specimens by RT-qPCR (B), from left to right are positive control, sample, compound well sample, and negative control, with CT values of 25.717, 33.661, 34.105, and 0.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380249/v1/a2dd89028cca6b5c7b88a28d.jpg"},{"id":56621208,"identity":"6a7aa49d-4b1f-4bc5-8c8e-455430bf72c6","added_by":"auto","created_at":"2024-05-16 18:06:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113538,"visible":true,"origin":"","legend":"\u003cp\u003ePatient temperature and pulse rate chart\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380249/v1/5b314e575694e9ed678feb04.jpg"},{"id":56621212,"identity":"ac53c5eb-f3ab-4ef1-b11a-56751fc5b665","added_by":"auto","created_at":"2024-05-16 18:06:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":255715,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of G genes. NJ (Neighbor Joining) tree of 107 partial G gene sequences. China I-VI lineages are listed, and HGF23-G gene is marked with red frame.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380249/v1/dd8c9e5604aa4dd6b9768afd.jpg"},{"id":56621210,"identity":"5f91ab1c-faad-47d0-93fb-910d9d4c8446","added_by":"auto","created_at":"2024-05-16 18:06:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":173775,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of L genes. NJ (Neighbor Joining) tree of 28 partial L gene sequences. China I-V lineages are listed, and HGF23-L gene is marked with red frame.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380249/v1/ff5e1cf5c2d5069b8ecfcab2.jpg"},{"id":77622851,"identity":"f1e076c9-5b09-446a-9d49-12f2ad9bc8ea","added_by":"auto","created_at":"2025-03-03 16:10:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1205155,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4380249/v1/afb7ace2-8292-4820-9d60-986986569ffb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metagenomic next-generation sequencing for etiological diagnosis of an unexpected rabies case with unclear exposure history","fulltext":[{"header":"Background","content":"\u003cp\u003eHuman rabies is a deadly disease caused by RABV, which is a negative strand (antisense) RNA virus belonging to the family \u003cem\u003eRhabdoviridae\u003c/em\u003e and genus \u003cem\u003eLyssavirus\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In China, dog-associated RABV is the main cause of human rabies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. And RABV is present in the saliva of wild and domestic infected-dogs and in the tissues at the injury site [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. RABV infection in humans manifests as a furious or dumb forms, characterized by a progression through four clinical stages: incubation, prodrome, acute neurological signs of encephalomyelitis, and ultimately death [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The primary clinical symptoms of rabies include aerophobia, hydrophobia, photophobia, dysphagia, mania, acute fatal encephalomyelitis and paralysis. Once rabies symptoms occur, the mortality rate of human is 100% [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA total of 3,302 rabies cases were reported from 2015 to 2021 in China [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Anhui province is divided into medium epidemic regions of rabies. In 2017, there were 28,243 persons obtaining postexposure prophylaxis treatment from clinics, and 39 rabies cases were reported in Anhui province. Furthermore, 12 rabies cases were reported in Anhui province in 2021[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Though the number of rabies cases is declining in China from 2015, RABV is still a fatal factor for human life.\u003c/p\u003e \u003cp\u003eTo surveil and control rabies, early and accurate diagnosis is essential for clinicians. Clinical diagnosis and lab testing are always used to diagnose rabies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. While it is difficult for clinicians to diagnose rabies without recommended clinical symptoms (including hydrophobia and aerophobia) and a clear exposure history (wild/domestic animal bites or scratch) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Lab testing methods will not be specifically applied for rabies detection due to the negative clinical diagnosis, resulting in RABV missing detection and spreading [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMetagenomic next-generation sequencing (mNGS) is able to theoretically detect all pathogens in clinical samples within 24 hours [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. mNGS is still capable of identifying infectious agents when lab testing results are negative. More positive rabies cases were detected with saliva samples (about 32%), followed by CSF (about 21%) and blood samples (about 10%) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, for rabies virus detection, saliva samples are preferred, followed by CSF (cerebrospinal fluid) samples and blood samples. In this study, the case was lack of recommended clinical symptoms and animal contact history. Furthermore, lab testing results of CSF and blood samples from the case were all negative. These clinical and lab testing results were tricky for clinicians to diagnose rabies. Finally, mNGS characterized the RABV in the saliva samples of patient. And probable origin of rabies virus was also investigated in this study.\u003c/p\u003e"},{"header":"Case presentation","content":"\u003cp\u003eA 49-year-old woman presented with unexplained fever and pruritus, which did not improve after self-administration of antipyretic medication. Despite receiving antiviral treatment at a local clinic the following day, her symptoms were not relieved. Subsequently, she was admitted to a reputable local hospital due to chest distress and shortness of breath. Based on blood test results, potassium supplementation was initiated for the treatment of hypokalemia. Throughout the course of treatment, the patient exhibited agitation. Consultations with neurology and psychiatry specialists failed to alleviate or ameliorate her manic symptoms which continued to worsen. Due to concurrent complaints of chest congestion, dyspnea and respiratory distress, she was transferred to the intensive care unit (ICU) for endotracheal intubation and mechanical ventilation. Five days later, the patient underwent bedside temporary cardiac pacemaker treatment due to an unstable heart rate. During hospitalization, based on the aforementioned symptoms, the physician suspected infection. Cerebrospinal fluid (CSF) and blood samples were collected from the patient for culture identification. Additionally, CSF was subjected to metagenomic next-generation sequencing (mNGS). However, both culture and sequencing results were negative. Then, several specialists from our hospital were invited for consultation. According to the patient's clinical symptoms and manifestations, doctors recommended further investigation into potential pathogenic agents. Therefore, saliva samples were collected from the patient and sent to our hospital's laboratory for mNGS detection. mNGS results showed that the patient was infected by RABV, with 1566 unique viral reads and 89.06% coverage rate of the RABV genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Consequently, on December 7, 2023, the patient was transferred to ICU in our hospital for isolation treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon admission, the patient presented with unconsciousness and exhibited temperature of 37.3\u0026deg;C, pulse rate of 102/min, respiratory rate of 16/min, and blood pressure of 92/57 mmHg. The patient exhibited non-cooperation during the physical examination. Unequal pupil size (anisocoria) was observed, along with sensitivity to light and blood stains in the oral cavity. The neck displayed supple and lack of resistance. Intubation was performed, and ventilation was supported by a ventilator, accompanied by coarse breath sounds bilaterally in both lungs. No evident sputum rales were present, and bilateral Babinski sign as well as meningeal irritation sign were negative. The abdomen appeared flat, soft, and non-tender without any signs of edema in both lower limbs or jaundice on the skin or mucosa throughout the body. Additionally, no deformity of the head was noted. The patient has not exhibited any rabies-specific symptoms, such as aerophobia, hydrophobia, or photophobia, since the onset of fever and pruritus. After the detection of the RABV sequence through mNGS, a saliva sample was sent to the local CDC for thorough examination, and presence of the RABV was confirmed by RT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The patient had been in a comatose state, and the progression of the disease was accompanied by elevated white blood cell (WBC) count, increased levels of C-reactive protein (CRP), serum amyloid A (SAA), liver and kidney function indicators, electrolyte imbalance, continuous decline in red blood cells count and hemoglobin levels, intermittent temperature elevation, unstable pulse (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), sialorrhea, and quadriplegia. During the initial four days of hospitalization, the patient had a Glasgow Coma Scale (GCS) score of 4T. On the fifth day of admission, there was a progressive decrease in blood pressure observed. At the same time, the detection of human rabies virus IgG antibodies (The Human Rabies Virus IgG Rapid Test Kit (Colloidal Gold), Beijing Beier Bioengineering Co., Ltd, Beijing, China) was conducted which yielded a positive result. The patient had not received prior rabies vaccination. Based on the presence of pruritus, accompanied by dysphoria, respiratory failure, and arrhythmia, the diagnosis of rabies was confirmed for this patient. The patient was managed with comprehensive supportive care in ICU, including respiratory support, fluid resuscitation, antimicrobial therapy, nutrition supplementation, and other appropriate symptomatic interventions. However, the therapeutic response was not prominently observed. Unfortunately, the patient died to multiple organ failure ten days after admission.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe primers of RT-qPCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePosition in N gene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSize\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRV-F1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGTCTCCYTAYTCATCRAATGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e889\u0026ndash;993\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRV-R1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCAATAACTGTYGCATTRAGAG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRV-R2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCAATRACTGTCGCATTAAGAG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRV-PF\u003c/p\u003e \u003cp\u003e(FAM,BQ1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCATATAGCATCCRACAAAGTGAATGAG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eThe RT-qPCR was performed targeting at the conserved regions of the nucleoprotein gene N\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePartial laboratory testing results of patients in our hospital\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLaboratory Test\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2023.12.08\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.09\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.11\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12.16\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWhite blood cell (\u0026times;109/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC-reactive protein(mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e124.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e210.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRed blood cell(\u0026times;109/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemoglobin(g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProthrombin(s)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-Dimer(\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlanine aminotransferase(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e907\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAspartate aminotransferase(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e684\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e173\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCreatinine(\u0026micro;mol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e352\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose(mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK+(mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum amyloid A(mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e246.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e253.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProcalcitonin(ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecreatine kinase-MB(ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elactic\u0026nbsp;dehydrogenase(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e787\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e348\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e266\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyoglobin(ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1203\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB-type natriuretic peptide(pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;35000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;35000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSputum culture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBefore the patient's saliva mNGS results indicated a RABV infection, the patient had been in a comatose state and unable to communicate directly or effectively. Therefore, we corroborated the history of animal bites with the patient's daughter, and confirmed that incident of small animal bites, particularly from dogs, did not happened to the patient. To uncover possible infectious source of RABV in this case, phylogenetic analysis was implemented. Partial sequence fragment of L or G gene was obtained (namely HFG23-L and HFG23-G, respectively), and grouped into China I lineage of rabies virus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). HFG23-L and HFG23-G both showed about 99.7% homology with the corresponding sequences of strain Fengtai RABV (KC660078). Fengtai strain was isolated from a dog in Beijing, China. The results indicated that rabies virus in this study, infecting 49-year-old female, may originate from a dog.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTimely diagnosis of rabies is indispensable for public health administration to control and surveil rabies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In this study, the patient only felt itching and irritable mood in the early period, and then became coma in a few days. The patient did not exhibit any rabies-specific symptoms of hydrophobia or aerophobia during the hospital visit. In addition, culture results of the patient's CSF and blood samples, as well as mNGS results of CSF, were negative, confusing clinicians to get an accurate diagnosis. Animal contact details were clear for previously reported rabies cases, and therefore, clinicians were able to get hints for rabies diagnosis on time [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Nevertheless, animal contact history was denied by the patient\u0026rsquo;s daughter. Therefore, the patient was a great challenge for clinicians to make rabies diagnosis due to her quickly worsening situations and unclear causative reason. However, upon admission of the patient to our hospital's ICU for isolation treatment, RABV was identified in saliva samples from patient by mNGS. Previous researches showed that positive rate of rabies in saliva samples (about 32%) is higher than that of CSF and blood samples [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It is recommended that saliva, rather than CSF or blood (serum), is considered as the primary choice for RABV detection in the stage of rabies development, particularly when prodromal symptoms are atypical and exposure history is uncertain. mNGS may be a useful tool for clinical diagnosis of similar cases in the future [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition, more experience should be accumulated for accurate detection of rabies associated with non-specific clinical symptoms.\u003c/p\u003e \u003cp\u003eRabies virus sequences were assembled, and analyzed by Neighbor-Joining (NJ) method. Phylogenetic tree of partial sequence of L (1966 bp/6575 bp, 30.36%) and G (1476 bp/1675 bp, 88.12%) gene showed that the virus belonged to China I lineage of RABV. China I lineage is a dog-associated prevalent lineage in China [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The homology of L and G gene partial sequences were about 99.7% with strain Fengtai rabies virus. G gene of rabies virus is conserved, and equivalent with N gene at phylogenetic position [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The similarity of G gene to virus strains isolated in Anhui was about 97%, indicating the virus was phylogenetically closer to Fengtai strain. It means that the virus may not belong to local rabies virus strains. For this patient, we did not get complete virus genome, because additional samples can\u0026rsquo;t be collected after her death. All the results demonstrated that the virus may originated from a dog in this study. Although there was no certain dog bite or scratch history for the patient, current evidence inferred that the patient may have contacted with a dog.\u003c/p\u003e \u003cp\u003eAdditionally, local CDC verified the RABV detected by mNGS in saliva samples. Two diagnostic kits based on RT-PCR method were employed to detect RABV in saliva sample, but failed. Successful verification of RARV was achieved by a third diagnostic kit. Therefore, RT-PCR did not always detect rabies virus even though 1566 viral reads were present in saliva samples. L and G partial sequences were obtained from saliva sample, however, L and G gene are not used by commercial reagent kits to confirm rabies diagnosis with RT-PCR method. Although two small N gene fragments (\u0026lt;\u0026thinsp;500 bp) in the saliva sample were assembled, only one N gene fragment was targeted by RT-PCR primers. Therefore, RT-PCR method should be optimized to be much more sensitive and accurate, and extend the target regions of primers. The results indicated that mNGS was much more sensitive than routine lab testing methods such as RT-PCR, and may be a potential tool for CDC to validate rabies virus or other infectious diseases.\u003c/p\u003e \u003cp\u003eAccording to the published data, the number of rabies cases declines significantly in China year by year. But the regional distribution of rabies did not shrink at district and county level [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In China, dog population was about 136,109 per thousand people in 2022 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Dog issues are present in China such as free roaming dogs and lack of dog vaccination awareness [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In 2020, rabies infection mainly affected the age group of 40\u0026ndash;70 years, corresponding reported cases accounted for 69% of all cases [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Rabies is preventable with timely wound treatment, vaccine and immunoglobulin reagent. However, post-exposure prophylaxis vaccination rates vary greatly in different regions of China. In this study, the patient, 49-year-old, lived in a county in Anhui province. She may contact with a dog, but didn\u0026rsquo;t have post-exposure prophylaxis following official recommendation. Anhui province belongs to medium risk groups, and 12 cases were reported in 2021. To avoid occurrence of similar tragedy in Anhui, people need to pay attention on animal contacts.\u003c/p\u003e \u003cp\u003eThere is no effective therapy for rabies except vaccines. However, the first recovery case was reported in 2005 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Willoughby and colleagues cured the patient by suppressing her brain function and activate her immune response. Afterwards, there is no rabies case rescued successfully by clinicians. The whole process needs high experienced multidiscipline clinicians, these essentials may help human conquer rabies in the future. Therefore, to eliminate rabies till 2030, education and public advocacy should be promoted among all age groups in China [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe manifestations of rabies exhibit significant variation during the onset period, and unspecific early symptoms, rapid disease progression, and an unclear exposure history impose challenges on the diagnosis, intervention, and nursing of rabies cases. Utilizing appropriate molecular testing tools (such as mNGS) is imperative for confirming cases of rabies. It is crucial to enhance knowledge and awareness regarding rabies while providing suitable post-exposure prophylaxis in order to mitigate the incidence of human rabies and associated fatalities.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003erabies virus (RABV)\u003c/p\u003e\n\u003cp\u003emetagenomic next-generation sequencing (mNGS)\u003c/p\u003e\n\u003cp\u003eCenter for Disease Control and Prevention (CDC)\u003c/p\u003e\n\u003cp\u003eintensive care unit (ICU)\u003c/p\u003e\n\u003cp\u003ecentral nervous system (CNS)\u003c/p\u003e\n\u003cp\u003eC-reactive protein (CRP)\u003c/p\u003e\n\u003cp\u003ecerebrospinal fluid (CSF)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEpidemiological investigation and medical information: Y. Yang, X. Zhang. Sequencing analysis: L. Liu, J. Chen, W. Zhang. Writing and original draft preparation: J. Wu, Y. Qi, Y. Shi. X. Liu Supervision: J. Wu, Y. Shi. Commenting and editing: all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the seventh cycle clinical key (cultivation) specialty in Hefei Department Construction Project (Clinical Laboratory) (Z033-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting this study\u0026rsquo;s the findings are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved and performed according to the guidelines of the Medical Research Ethics Committee of the First Affiliated Hospital of University of Science and Technology of China (Anhui Provincial Hospital) (No.2024-RE-63).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors of this study declare no commercial relationships and no competing interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJackson AC. Human Rabies: a 2016 Update. Curr Infect Dis Rep. 2016;18:38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao F, Li N, Yang J, Chen T, Liu Y, Zhang S, et al. Neglected challenges in the control of animal rabies in China. One Health. 2021;12:100212.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHemachudha T, Ugolini G, Wacharapluesadee S, Sungkarat W, Shuangshoti S, Laothamatas J. Human rabies: neuropathogenesis, diagnosis, and management. Lancet Neurol. 2013;12:498\u0026ndash;513.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Jin Z, Sun G-Q, Zhou T, Ruan S. Analysis of Rabies in China: Transmission Dynamics and Control. PLoS ONE. 2011;6:e20891.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang N, Song C, Tao X, Zhu W. Epidemiologic Features of Human Rabies in China from 2015\u0026ndash;2021. Zoonoses. 2023;3:29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMani RS, Madhusudana SN. 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Infect Dis Ther. 2023;12:1175\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Liu J-J, Ding S-J, Cai L, Feng Y, Yu P-C, et al. Human rabies in China: evidence-based suggestions for improved case detection and data gathering. Infect Dis Poverty. 2020;9:60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmoako YA, El-Duah P, Sylverken AA, Owusu M, Yeboah R, Gorman R, et al. Rabies is still a fatal but neglected disease: a case report. J Med Case Rep. 2021;15:575.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTolentino J\u0026uacute;nior DS, Marques MSV, Krummenauer A, Duarte MMS, Rocha SM, de Brito MG, et al. Rabies outbreak in Brazil: first case series in children from an indigenous village. Infect Dis Poverty. 2023;12:78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDorji T, Lamichaney J, Gyaltshen C, Lungten L, Dhakal GP, Dorjee S, et al. Human rabies encephalomyelitis in the background of rabies outbreak in animals in Gelephu, Bhutan, 2023: a case report. Infect Dis Poverty. 2023;12:94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan D, Li Z, Li R, Tan P, Zhang R, Li J. mNGS in clinical microbiology laboratories: on the road to maturity. \u003cem\u003eCrit Rev Microbiol\u003c/em\u003e. 2019. Taylor and Francis Ltd., 45: 668\u0026ndash;685.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo F, Kang L, Zhang L. mNGS for identifying pathogens in febrile neutropenic children with hematological diseases. Int J Infect Dis. 2022;116:85\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Q, Wu B, Yang D, Yang C, Jin Z, Cao J, et al. Optimal specimen type for accurate diagnosis of infectious peripheral pulmonary lesions by mNGS. BMC Pulm Med. 2020;20:268.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu M, Liu Y, Xu D, Wan L, Zhao R. mNGS helped diagnose scrub typhus presenting as a urinary tract infection with high D-dimer levels: a case report. BMC Infect Dis. 2021;21:1219.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTao X, Liu S, Zhu W, Rayner S. Rabies surveillance and control in China over the last twenty years. Biosaf Health 2021 Elsevier B V, 3: 142\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao FM, Chen T, Liu Y, Zhang SF, Zhang F, Li N, et al. Emerging New Phylogenetic Groups of Rabies Virus in Chinese Ferret Badgers. Biomed Environ Sci. 2018;31:479\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa J, Li S, Yang Y, Wang Q, Huo Y. Epidemiological and phylogenetic analysis of rabies virus isolated from humans in Henan province, China. Arch Virol. 2019;164:2811\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChina Pet population and ownership. 2022 update | China Pet Market. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chinapetmarket.com/industry-news/china-pet-population-and-ownership-2022-update/\u003c/span\u003e\u003cspan address=\"https://www.chinapetmarket.com/industry-news/china-pet-population-and-ownership-2022-update/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 5 May 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen T, Welburn SC, Sun L, Yang G-J. Progress towards dog-mediated rabies elimination in PR China: a scoping review. Infect Dis Poverty. 2023;12:30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilloughby RE, Tieves KS, Hoffman GM, Ghanayem NS, Amlie-Lefond CM, Schwabe MJ, et al. Survival after Treatment of Rabies with Induction of Coma. N Engl J Med. 2005;352:2508\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"rabies, rabies virus, saliva, metagenomic next-generation sequencing, phylogenetic analysis, unclear exposure history","lastPublishedDoi":"10.21203/rs.3.rs-4380249/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4380249/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRabies is an acute and lethal zoonotic disease caused by the rabies virus (RABV). After onset, there are no effective drugs or treatment methods. We reported a fatal rabies case with no clear history of animal bites and atypical early clinical symptoms. And unexpectedly, we discovered that unique RABV sequences were detected in the patient's saliva by metagenomic next-generation sequencing (mNGS). The patient was diagnosed with rabies based on the results of saliva mNGS, real-time reverse transcriptase polymerase chain reaction (RT-PCR) conducted by local Centers for Disease Control and Prevention (CDC), the detection of human rabies virus IgG antibodies performed in our laboratory, as well as the clinical symptoms of pruritus, agitation, paralysis, and excessive salivation at disease onset.\u003c/p\u003e\u003ch2\u003eCase presentation\u003c/h2\u003e \u003cp\u003eA 49-year-old female from Hefei, Anhui Province, China, presented to a local hospital with fever, pruritus, chest distress, and shortness of breath. During the consultation, the patient exhibited agitation and was later admitted to the intensive care unit (ICU) for endotracheal intubation and mechanical ventilation due to worsened agitation and dyspnea. Cerebrospinal fluid (CSF) and blood samples were collected and pathogenic microorganism identification was performed by culture and mNGS. However, all results were negative. In addition, the patient did not display typical rabies-specific symptoms such as aerophobia, hydrophobia or photophobia from onset to admission. Subsequently, saliva samples were collected for mNGS detection following consultation with experts at our hospital. Nucleic acid sequences uniquely aligned to the rabies virus (RABV) were identified in these samples. The result was further confirmed by local CDC through RT-PCR which detected part of the N gene of RABV in the saliva sample. Then the patient was transferred to our hospital's ICU for isolation. Unfortunately, she died on the 10th day of admission due to multiple organ failure. The patient remained in a comatose state during symptomatic supportive medical care in ICU, and the progression of the disease was accompanied by intermittent elevated body temperature, excessive salivation, and limb weakness. The detection of human rabies virus IgG antibodies reported positive during the advanced stage of the disease. We consistently verified with the patient's family member that there was no clear history of animal bites and no history of RABV vaccination. Furthermore, we performed phylogenetic analysis of partial L and G gene sequences of RABV obtained by mNGS (designated HFG23-L and HFG23-G, respectively), the results showed that both HFG23-L and HFG23-G belonged to the China I lineage, and shared 99.7% similarity with the Fengtai strain isolated from dogs in Beijing. The results suggested that the origin of RABV in this case may be a dog from the northern China.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe patient's non-specific prodromal symptom, along with negative culture and mNGS results of blood and CSF, impose challenges on promptly and definitively diagnosing rabies in this case. The identification of unique RABV sequence through mNGS in the patient's saliva sample suggested that mNGS could serve as a valuable screening tool for the etiological diagnosis of rabies, especially when timely laboratory testing was unavailable or when patients lacked a clear exposure history.\u003c/p\u003e","manuscriptTitle":"Metagenomic next-generation sequencing for etiological diagnosis of an unexpected rabies case with unclear exposure history","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-16 18:06:19","doi":"10.21203/rs.3.rs-4380249/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-09T05:33:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-08T13:25:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-08T13:25:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2024-05-07T04:52:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"732342c6-ee3e-486f-a5c7-b0d59cc8c6fc","owner":[],"postedDate":"May 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-03T16:06:29+00:00","versionOfRecord":{"articleIdentity":"rs-4380249","link":"https://doi.org/10.1186/s12879-025-10687-y","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2025-02-24 15:58:13","publishedOnDateReadable":"February 24th, 2025"},"versionCreatedAt":"2024-05-16 18:06:19","video":"","vorDoi":"10.1186/s12879-025-10687-y","vorDoiUrl":"https://doi.org/10.1186/s12879-025-10687-y","workflowStages":[]},"version":"v1","identity":"rs-4380249","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4380249","identity":"rs-4380249","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}