- Email: [email protected]
Next-generation sequencing of the cerebrospinal fluid in the diagnosis of neurobrucellosis
Accepted Manuscript Title: Next-generation sequencing of the cerebrospinal fluid in the diagnosis of neurobrucellosis Authors: Siyuan Fan, Haitao Ren, Yanping Wei, Chenhui Mao, Zhenzi Ma, Lu Zhang, Li Wang, Ying Ge, Taisheng Li, Liying Cui, Honglong Wu, Hongzhi Guan PII: DOI: Reference:
S1201-9712(17)30310-7 https://doi.org/10.1016/j.ijid.2017.11.028 IJID 3107
To appear in:
International Journal of Infectious Diseases
Received date: Revised date: Accepted date:
16-9-2017 19-11-2017 23-11-2017
Please cite this article as: Fan Siyuan, Ren Haitao, Wei Yanping, Mao Chenhui, Ma Zhenzi, Zhang Lu, Wang Li, Ge Ying, Li Taisheng, Cui Liying, Wu Honglong, Guan Hongzhi.Next-generation sequencing of the cerebrospinal fluid in the diagnosis of neurobrucellosis.International Journal of Infectious Diseases https://doi.org/10.1016/j.ijid.2017.11.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Next-generation sequencing of the cerebrospinal fluid in the diagnosis of neurobrucellosis
Siyuan Fana, M.D., Haitao Rena, B.S., Yanping Weia, M.D., Chenhui Maoa, M.D.,
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Zhenzi Mab, M.S., Lu Zhangb, M.S., Li Wangb, B.S., Ying Gec, M.D., Taisheng Lic,
a
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M.D., Ph.D., Liying Cuia, M.D., Ph.D., Honglong Wub, M.S., Hongzhi Guana, M.D.
Department of Neurology, Peking Union Medical College Hospital, Chinese Academy
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of Medical Sciences and Peking Union Medical College, Beijing 100730, China The Beijing Genomics Institute (Tianjin), Tianjin 200308, China
c
Department of Infectious Diseases, Peking Union Medical College Hospital, Chinese
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b
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Academy of Medical Sciences and Peking Union Medical College, Beijing 100730,
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China
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Corresponding author: Hongzhi Guan.
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Department of Neurology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China Telephone Number: (0086) 10-69156371
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Fax Number: (0086) 10-69156371 Email: [email protected]
Co-corresponding author: Honglong Wu. 1
The Beijing Genomics Institute (Tianjin), Tianjin 200308, China Telephone Number: (0086) 22-59096478 Fax Number: (0086) 22-59096420
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Email: [email protected]
Highlights
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• Patients with dramatic different symptoms were diagnosed as neurobrucellosis (NB). • The patients were diagnosed using next-generation sequencing (NGS) of the CSF.
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• This is the first case series of NB diagnosed with NGS of the CSF.
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Abstract
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Backgrounds
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Brucellosis is the most common zoonotic infection in the world. Brucellosis with nervous system involvement is known as ‘neurobrucellosis’ (NB). The diagnosis of
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NB is difficult because its clinical manifestations are nonspecific and the sensitivity of
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routine culture tests is low. Methods
Next-generation sequencing (NGS) of cerebrospinal fluid (CSF) was used to detect
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pathogens in patients with clinically suspected central nervous system (CNS) infections in a tertiary referral center in China between June 1, 2016 and June 1, 2017. The clinical characteristics and NGS results of patients with the diagnosis of NB are reviewed in the study. 2
Results Four patients were quickly diagnosed as NB using NGS of the CSF in patients with clinically suspected CNS infections, although the clinical manifestations varied dramatically among these patients. The NGS of the CSF revealed that the identified
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sequence reads corresponding to Brucella species range from 11 to 104, with genomic coverage ranging from 0.043% to 0.4%. The quick diagnosis leading to prompt
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treatment with the appropriate antibiotics. Conclusions
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This study demonstrates the power of NGS of the CSF coupled with a bioinformatic
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pipeline in the diagnosis of NB.
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Keywords
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Introduction
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Neurobrucellosis; cerebrospinal fluid; next-generation sequencing; quick diagnosis
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Brucellosis is the most common zoonotic infection in the world. More than 500,000 new cases are reported annually worldwide 1. It is a multisystem infection, with variable clinical presentations. Brucellosis with nervous system involvement is known as
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‘neurobrucellosis’ (NB). The clinical features and cerebrospinal fluid (CSF) findings of NB are usually nonspecific, and the sensitivity and specificity of routine culture and serological tests vary, making the diagnosis of NB difficult. In endemic areas, NB should be considered in the differential diagnosis of patients presenting with 3
neurological symptoms and concomitant fever 2. Next-generation sequencing (NGS) is a new technique that is increasingly used for the clinical diagnosis of infectious diseases of the central nervous system (CNS) 3-7. Here, we present a case study of neurobrucellosis, which were diagnosed quickly with NGS
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of the CSF, allowing prompt treatment with the appropriate antibiotics.
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Materials and methods Case series
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The case series included four consecutive patients admitted to the neurology department
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of Peking Union Medical College Hospital (PUMCH), a tertiary referral center in China
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between June 1, 2016 and June 1, 2017. This study is part of a research project which
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aims to detect pathogens in patients with clinically suspected CNS infections using
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NGS of CSF. The demographic, clinical, radiological, and pathogenic findings, and treatment and outcome data were extracted from the Hospital Information System of
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PUMCH, a medical information tool that permits access to the electronic clinical data
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for all patients treated at PUMCH since 2013. The project was approved by the ethics committee of PUMCH. Written informed consent was obtained for each patient before
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his/her participation in the study.
DNA Extraction Following the manufacturer’s manual, a 300 μL CSF sample from each patient or a negative ‘no-template’ control (NTC) was transferred to a new sterile tube, and the 4
DNA was extracted directly with the TIANamp Micro DNA Kit (DP316, Tiangen Biotech, Beijing, China). After adding 10 μL proteinase K and 300 μL buffer GB (with carrier RNA), the sample was incubated at 56℃ for 10 minutes. Then it was added with 300 μL cold absolute ethyl alcohol and incubated at room temperature for 5 minutes.
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The liquid was transferred to a new adsorption column and washed with buffer GD and buffer PW. DNA was then dissolved in 40 μL of TE buffer. The extracted DNA was
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used for the construction of DNA libraries.
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Library construction and sequencing
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The DNA extracted in the previous step was sonicated with Bioruptor® Pico, according
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to the manufacturer’s protocol, to generate 200–300-bp fragments. The DNA libraries
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were then constructed according to the standard protocol of the BGISEQ-100
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sequencing platform (BGI-Tianjin, Tianjin, China). DNA was end-repaired and an endrepaired adapter was added overnight. After ligation, the DNA was amplified by PCR
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and purified with AMPure XP beads (Beckman Coulter, Pasadena, CA, USA). To
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measure the adapters before sequencing, quality control was performed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) combined with quantitative PCR. The quantified libraries were subjected to emulsion PCR with the
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OneTouch system. The DNA was then sequenced with the BGISEQ-100 platform.
Data processing and analysis High-quality sequencing data were generated by filtering out short (< 35 bp), low5
quality and low-complexity reads, and with the subsequent elimination of the human host sequences, which were mapped to the human reference genomes (hg19 and YH sequences) with Burrows–Wheeler alignment, a vigoroso alignment tool. Finally, the remaining sequencing data were aligned to the Microbial Genome Database, which
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contains viral, bacterial, fungal, and parasite genomic sequences to identify the pathogenic sequences. An advanced data analysis was then performed, as for the
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mapped data.
The reference genomes were downloaded from the National Center for Biotechnology
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Information (ftp://ftp.ncbi.nlm.nih.gov/genomes/). The database used in our study
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included 1494 bacteria, 2700 viruses, 73 fungi, and 48 parasites, all related to human
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disease. We calculated the sequence depth and genomic coverage for each species using
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SoapCoverage from the SOAP website (http://soap.genomics.org.cn/).
PCR and Sanger validation
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To verify the results of NGS, we used sequence-specific PCR to amplify the target
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fragment with specific primers: BLA-2F-F: TGGCTCGGTTGCCAATATC and BLA2F-R: CGCTTGCCTTTCAGGTCTG. Agarose gel electrophoresis was used to analyze the PCR products, and Sanger sequencing with an ABI PRISM 3730 DNA Analyzer
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(Applied Biosystems, Foster City, CA, USA) was used to validate the sequencing results. Finally, the sequences were aligned to the NTdatabase with the online software NCBI
Blast
(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSear 6
ch&LINK_LOC=blasthome).
Results Clinical Findings
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Four patients with NB were identified in a group of patients with clinically suspected infectious diseases of CNS. Two were women and two were men, ranging in age from
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38 to 49 years. The clinical manifestations in these patients are summarized in Table 1.
The major complications are summarized in Table 2. All four patients were previously
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healthy and none of them had a definite history of epidemiological contact, although
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they were all from endemic areas. The clinical manifestations varied among these
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patients. Case 1 presented with transient left-sided numbness and weakness,
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progressive numbness and weakness of bilateral lower limbs, and bilateral hearing loss.
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Case 2 presented with recurrent right-sided weakness and slurred speech. Case 3 presented with headache and blurred vision. Case 4 presented with recurrent seizures.
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Fever was present in only two patients. Peripheral blood Human immunodeficiency
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virus antibodies, rapid plasma reagin (RPR), Lyme disease antibodies, Mycobacterium PCR, and Cytomegalovirus (CMV) PCR were negative in all four patients. Gram staining, acid-fast bacilli, Mycobacterium PCR, India-ink staining, Cryptococcus
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antigen, RPR, TORCH, T-SPOT.TB, and CMV PCR analyses of the CSF were also negative in these patients. Brain and spinal magnetic resonance imaging (MRI) with contrast was performed in all patients. Case 1 showed abnormal enhancement of the cranial nerve (CN) Ⅴ (Fig 1A), Ⅶ and Ⅷ, and diffuse enhancement of the spinal 7
meninges (Fig 1B), together with a T2-weighted hyperintense lesion on the right temporal lobe. Case 2 showed abnormal enhancement of the CN Ⅲ(Fig 1C), Ⅴand Ⅵ, and diffuse meningeal enhancement, which improved (Fig 1D) after treatment for 2 months. Case 3 showed meningeal abscesses with ring enhancement and surrounding
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edema near the right frontal lobe (Fig 1E–F) and parietal lobe. Case 4 showed white matter lesions with fluid-attenuated inversion recovery (FLAIR) hyperintensity (Fig
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1H). The CSF analyses and peripheral blood results are summarized in Table 3. Lumbar punctures revealed increased opening pressures, elevated white blood cell counts with
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prominent mononuclear cells, elevated protein levels, and reduced glucose levels in all
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cases. It took 7 days for CSF culture to be positive for brucellosis in Case 1. Peripheral
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blood Rose Bengal tests were positive for cases 2, 3, and 4, CSF serological tests for
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brucellosis were not performed in these patients. The symptoms of these patients
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improved gradually after the initiation of empiric antibiotic treatment with doxycycline, rifampin, and ceftriaxone. Case 1 experienced cerebral hemorrhagic transformation in
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the infarction area of the right temporal lobe before discharge.
NGS results
DNA library were constructed from 300 μL of CSF sample in each patient. Brucella
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species-specific DNA was detected in all four patients. The number of raw reads range from 19,083,456 to 30,738,055. The identified sequence reads corresponding to Brucella species range from 11 to 104, with genomic coverage ranging from 0.043% to 0.4%, reads per million (RPM) range from 0.58 to 3.38. The details of the CSF NGS 8
results are given in Table 4, Fig 2 and Supplementary Table 1–5. There were several contaminating sequences in the results (Fig 2 and Supplementary Table 2–5). Propionibacterium acnes, a common contaminating microorganism, was present in all four patients with NB. Methylobacterium radiotolerans, M. populi, and Truepera
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radiovictrix were detected in case 1; Porphyromonas gingivalis, Thermus thermophilus, and Rhizobium etli were detected in case 2; Bradyrhizobium S23321 and Ralstonia
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solanacearum were detected in case 3; and Micrococcus luteus, M. radiotolerans, and Asticcacaulis excentricus were detected in case 4. The contaminating sequences were
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either present in the NTC samples (Supplementary Table 2–5) or common
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contaminating microorganisms. However, there were no Brucella reads in the negative
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NTC samples. Mycobacterium tuberculosis, Treponema pallidum, and Cryptococcus
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were not detected by NGS of CSF for all four patients.
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The presence of Brucella DNA in the CSF was confirmed with a PCR analysis of cases
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sample.
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1, 2, and 4. Case 3 didn’t undergo Sanger sequencing because there was no extra CSF
Discussion
Four patients from a group of patients with clinically suspected infectious diseases of
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CNS, with dramatically different presenting symptoms, were diagnosed with NB using NGS of the CSF. To our knowledge, this is the first case series in which NGS of the CSF was used for the diagnosis of NB. Human brucellosis is the most common zoonotic disease in the world, with more than 9
500,000 new cases annually 8. However, it is estimated that the real number is 26 times higher than the number of reported cases 9. About 5% of brucellosis cases have NB10. Precise and prompt diagnosis and the initiation of appropriate antibiotic treatment are crucial for a favorable prognosis.
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Unfortunately, the diagnosis of NB is a considerable clinical challenge. Firstly, the clinical features and CSF findings of NB are usually nonspecific, and it shares strong
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similarities with other CNS infections, including tuberculosis, syphilis, Lyme disease,
and Cryptococcus infection. The clinical manifestations and neuroimages of our four
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patients varied dramatically, and they had no history of epidemiological contact. The
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symptoms of our patients were unusual for brucellosis, although these symptoms had
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been reported in the previous studies11-13. Brain MRI of case 4 showed diffuse white
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matter changes. This neuroimage change was reported in the previous study11, 14. Given
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the rarity of the symptoms and neuroimages, neurobrucellosis would be a forgotten diagnosis for these patients, and may not even be considered in the differential
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diagnoses. Case 4 was diagnosed as NB when he was admitted for the second time
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because of nonspecific symptoms. Secondly, although positive culture is the gold standard for diagnosis, the sensitivity of routine culture tests is low. About 28% of patients with CNS brucellosis had positive blood cultures and 15% had positive CSF
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cultures
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. Only one patient in our study was culture positive. Thirdly, as
demonstrated in case 1, CSF culture for NB is sometimes time-consuming, which makes prompt diagnosis a difficult mission. Fourthly, although serology is more sensitive and faster than routine culture test; brucellosis seropositivity is high in 10
endemic area (2.6-14.4% among Turkish people13); it may not always distinguish active and previous infections. NGS is a new technique that is increasingly used for the clinical diagnosis of infectious diseases of CNS. Theoretically, with sufficiently long reads, multiple hits in the
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microbial genome, and a complete reference database, almost all microorganisms can be uniquely identified based on specific nucleic acid sequences. Several case reports
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and clinical studies have demonstrated the use of NGS as a diagnostic tool for infectious
diseases 5-7. Mongkolrattanothai K, et al recently reported a pediatric patient who was
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unexpectedly diagnosed as neurobrucellosis using NGS17. These four patients were also
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“unexpectedly” diagnosed as neurobrucellosis in our multicenter study which aims to
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detect pathogens in patients with clinically suspected CNS infections using NGS of
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CSF. Simultaneously, Mycobacterium tuberculosis, Treponema pallidum, and
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Cryptococcus were not detected by NGS of CSF for all four patients. The diagnoses were finally confirmed by CSF culture or Sanger sequencing in our study. In our
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multicenter study, we found no NB patients with negative NGS results, implying the
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high sensitivity of NGS in the diagnosis of NB. Contamination is a common problem in NGS. There were several contaminating microorganisms in our study, of which Propionibacterium acnes was most common. The contamination could come from
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cross-contamination between samples during the NGS library preparation, skin or body flora during the process of lumbar puncture, or an incorrect process of sample demultiplexing or formation of artificial products17,18. It is essential to differentiate the clinical relevant pathogens from contaminating microorganisms in NGS. We can 11
remove plenty of background microorganisms according to the NGS results of “NTC” control. Besides, we found that some background microorganisms appear frequently in the result of NGS. For example, Propionibacterium acnes, Burkholderia, Bradyrhizobium appear with high species-specific reads and RPM in the NGS result of
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most patients at our center (unpublished data). Therefore, before explaining the results, researchers should be cautious with the common background microorganisms at their
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center. NGS of the CSF is a fast method, taking less than 48 hours at our center. With the results of CSF NGS, treatment can be initiated promptly, meanwhile, standard
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diagnostic methods for the target pathogen can be used to confirm the diagnosis.
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Finally, NGS of CSF is a new technique in the diagnosis of CNS infections, which is
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frequently unavailable in endemic and resource-limited areas. Therefore, we emphasize
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that NGS of CSF is especially suitable for these puzzling cases.
Conclusion
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This study demonstrates the power of NGS of the CSF coupled with a bioinformatic
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pipeline in the diagnosis of NB.
Funding source
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None.
Conflicts of Interest We declare that we have no conflicts of interest. 12
Acknowledgments
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The authors thank the patients for participating in this study.
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Fig 1. Brain and spinal magnetic resonance imaging (MRI) of the patients. Case 1 showed abnormal enhancement of cranial nerve (CN) Ⅴ (Fig 1A), and diffuse enhancement of spinal meningeal (Fig 1B). Case 2 showed abnormal enhancement of CN Ⅲ (Fig 1C), which were improved (Fig 1D) after treatment of 2 months. Case 3
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showed meningeal abscesses with ring enhancement and surrounding edema near right frontal lobe (Fig 1E-F). Case 4 showed white matter lesions of fluid attenuated
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inversion recovery (FLAIR) hyperintensity (Fig 1H), comparing to multiple small
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scattered white matter lesions (Fig 1G) in the beginning of the disease.
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Fig 2. Next generation sequencing (NGS) of cerebrospinal fluid (CSF) in the patients. The identified sequence reads corresponding to Brucella species were 30 (Fig 2A), 11
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(Fig 2C), 24 (Fig 2E), and 104 (Fig 2G); with a genomic coverage of 0.11% (Fig 2B), 0.043% (Fig 2D), 0.093% (Fig 2F), and 0.4% (Fig 2H), for case 1, 2, 3, and 4, respectively. Propionibacterium acnes, as a common contamination microorganism, presented in all four patients (Fig 2A, C, E, G). Methylobacterium radiotolerans, 17
Methylobacterium populi, and Truepera radiovictrix presented in case 1 (Fig 2A); Porphyromonas gingivalis, Thermus thermophilus, and Rhizobium etli presented in case 2 (Fig 2C); Bradyrhizobium S23321 and Ralstonia solanacearum presented in case 3 (Fig 2E); Micrococcus luteus, Methylobacterium radiotolerans, and Asticcacaulis
A
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excentricus presented in case 4 (Fig 2G).
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Table 1. Clinical features of the four patients with neurobrucellosis.
No.
e
Gen
Fev
Headach
Sweati
Weight
Back
Hearing
Joint
Seiz
Meningeal
(yr.
der
er
e
ng
loss
pain
loss
pain
ure
signs
Mal
＋
＋
－
＋
＋
＋
＋
－
－
－
－
＋
＋
－
＋
－
－
－
－
＋
＋
＋
＋
＋
－
＋
－
－
－
＋
－
－
)
1
38
2
46
3
38
4
49
e Fem ale Fem ale Mal e
A
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M
A
N
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＋ = positive; － = negative; No. = number; yr. = year.
19
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e
Ag
－
＋
＋
－
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Cas
Table 2. The major complications of the four patients with neurobrucellosis Case
CN
Polyneuropathy
No.
involvement
/radiculopathy
1
＋
2
Encephalitis
Stroke
Brian abscess
＋
＋
－
＋
－
＋
－
＋
－
＋
－
3
－
－
＋
－
－
＋
4
－
－
＋
＋
－
－
A
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M
A
N
U
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＋ = positive; － = negative; CN = cranial nerves; No. = number;
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Meningitis
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Table 3. CSF and Peripheral blood tests of the four patients with neurobrucellosis. Case
CSF
Peripheral Blood
Pressure
WBC
MN
Protein
Glucose
(mmH2O)
(cells/µL)
(%)
(g/L)
(mmol/L)
1
210
607
99
9.3
1.2
Brucella
－
－
2
>330
361
91
3.14
0.7
－
＋
－
3
>330
178
99
1.09
0.8
－
＋
－
4
220
86
93
1.28
1.7
－
＋
－
Rose Bengal Test
Culture
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Culture
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No.
＋ = positive; － = negative; CSF = cerebrospinal fluid; No. = number; WBC = white
A
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M
A
N
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blood cell; MN = mononuclear cell.
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Table 4. The NGS of CSF of the four patients with neurobrucellosis. From Onset to CSF
Sample
Collection Time
Volume
(months)
(μL)
1
24
300
2
12
3 4
Case
Genomic Coverage
Raw Reads
RPM
30
22865302
1.31
0.11
300
11
19083456
0.58
0.043
6
300
24
24691420
0.97
0.093
4
300
104
30738055
3.38
0.4
Specific Reads
(%)
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No.
Brucella Species-
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M
A
N
U
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NGS = next-generation sequencing; CSF = cerebrospinal fluid; No. = number; RPM = reads per million
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S1201-9712(17)30310-7 https://doi.org/10.1016/j.ijid.2017.11.028 IJID 3107
To appear in:
International Journal of Infectious Diseases
Received date: Revised date: Accepted date:
16-9-2017 19-11-2017 23-11-2017
Please cite this article as: Fan Siyuan, Ren Haitao, Wei Yanping, Mao Chenhui, Ma Zhenzi, Zhang Lu, Wang Li, Ge Ying, Li Taisheng, Cui Liying, Wu Honglong, Guan Hongzhi.Next-generation sequencing of the cerebrospinal fluid in the diagnosis of neurobrucellosis.International Journal of Infectious Diseases https://doi.org/10.1016/j.ijid.2017.11.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Next-generation sequencing of the cerebrospinal fluid in the diagnosis of neurobrucellosis
Siyuan Fana, M.D., Haitao Rena, B.S., Yanping Weia, M.D., Chenhui Maoa, M.D.,
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Zhenzi Mab, M.S., Lu Zhangb, M.S., Li Wangb, B.S., Ying Gec, M.D., Taisheng Lic,
a
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M.D., Ph.D., Liying Cuia, M.D., Ph.D., Honglong Wub, M.S., Hongzhi Guana, M.D.
Department of Neurology, Peking Union Medical College Hospital, Chinese Academy
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of Medical Sciences and Peking Union Medical College, Beijing 100730, China The Beijing Genomics Institute (Tianjin), Tianjin 200308, China
c
Department of Infectious Diseases, Peking Union Medical College Hospital, Chinese
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b
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Academy of Medical Sciences and Peking Union Medical College, Beijing 100730,
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China
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Corresponding author: Hongzhi Guan.
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Department of Neurology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China Telephone Number: (0086) 10-69156371
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Fax Number: (0086) 10-69156371 Email: [email protected]
Co-corresponding author: Honglong Wu. 1
The Beijing Genomics Institute (Tianjin), Tianjin 200308, China Telephone Number: (0086) 22-59096478 Fax Number: (0086) 22-59096420
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Email: [email protected]
Highlights
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• Patients with dramatic different symptoms were diagnosed as neurobrucellosis (NB). • The patients were diagnosed using next-generation sequencing (NGS) of the CSF.
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• This is the first case series of NB diagnosed with NGS of the CSF.
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Abstract
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Backgrounds
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Brucellosis is the most common zoonotic infection in the world. Brucellosis with nervous system involvement is known as ‘neurobrucellosis’ (NB). The diagnosis of
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NB is difficult because its clinical manifestations are nonspecific and the sensitivity of
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routine culture tests is low. Methods
Next-generation sequencing (NGS) of cerebrospinal fluid (CSF) was used to detect
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pathogens in patients with clinically suspected central nervous system (CNS) infections in a tertiary referral center in China between June 1, 2016 and June 1, 2017. The clinical characteristics and NGS results of patients with the diagnosis of NB are reviewed in the study. 2
Results Four patients were quickly diagnosed as NB using NGS of the CSF in patients with clinically suspected CNS infections, although the clinical manifestations varied dramatically among these patients. The NGS of the CSF revealed that the identified
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sequence reads corresponding to Brucella species range from 11 to 104, with genomic coverage ranging from 0.043% to 0.4%. The quick diagnosis leading to prompt
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treatment with the appropriate antibiotics. Conclusions
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This study demonstrates the power of NGS of the CSF coupled with a bioinformatic
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pipeline in the diagnosis of NB.
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Keywords
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Introduction
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Neurobrucellosis; cerebrospinal fluid; next-generation sequencing; quick diagnosis
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Brucellosis is the most common zoonotic infection in the world. More than 500,000 new cases are reported annually worldwide 1. It is a multisystem infection, with variable clinical presentations. Brucellosis with nervous system involvement is known as
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‘neurobrucellosis’ (NB). The clinical features and cerebrospinal fluid (CSF) findings of NB are usually nonspecific, and the sensitivity and specificity of routine culture and serological tests vary, making the diagnosis of NB difficult. In endemic areas, NB should be considered in the differential diagnosis of patients presenting with 3
neurological symptoms and concomitant fever 2. Next-generation sequencing (NGS) is a new technique that is increasingly used for the clinical diagnosis of infectious diseases of the central nervous system (CNS) 3-7. Here, we present a case study of neurobrucellosis, which were diagnosed quickly with NGS
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of the CSF, allowing prompt treatment with the appropriate antibiotics.
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Materials and methods Case series
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The case series included four consecutive patients admitted to the neurology department
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of Peking Union Medical College Hospital (PUMCH), a tertiary referral center in China
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between June 1, 2016 and June 1, 2017. This study is part of a research project which
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aims to detect pathogens in patients with clinically suspected CNS infections using
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NGS of CSF. The demographic, clinical, radiological, and pathogenic findings, and treatment and outcome data were extracted from the Hospital Information System of
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PUMCH, a medical information tool that permits access to the electronic clinical data
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for all patients treated at PUMCH since 2013. The project was approved by the ethics committee of PUMCH. Written informed consent was obtained for each patient before
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his/her participation in the study.
DNA Extraction Following the manufacturer’s manual, a 300 μL CSF sample from each patient or a negative ‘no-template’ control (NTC) was transferred to a new sterile tube, and the 4
DNA was extracted directly with the TIANamp Micro DNA Kit (DP316, Tiangen Biotech, Beijing, China). After adding 10 μL proteinase K and 300 μL buffer GB (with carrier RNA), the sample was incubated at 56℃ for 10 minutes. Then it was added with 300 μL cold absolute ethyl alcohol and incubated at room temperature for 5 minutes.
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The liquid was transferred to a new adsorption column and washed with buffer GD and buffer PW. DNA was then dissolved in 40 μL of TE buffer. The extracted DNA was
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used for the construction of DNA libraries.
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Library construction and sequencing
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The DNA extracted in the previous step was sonicated with Bioruptor® Pico, according
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to the manufacturer’s protocol, to generate 200–300-bp fragments. The DNA libraries
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were then constructed according to the standard protocol of the BGISEQ-100
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sequencing platform (BGI-Tianjin, Tianjin, China). DNA was end-repaired and an endrepaired adapter was added overnight. After ligation, the DNA was amplified by PCR
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and purified with AMPure XP beads (Beckman Coulter, Pasadena, CA, USA). To
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measure the adapters before sequencing, quality control was performed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) combined with quantitative PCR. The quantified libraries were subjected to emulsion PCR with the
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OneTouch system. The DNA was then sequenced with the BGISEQ-100 platform.
Data processing and analysis High-quality sequencing data were generated by filtering out short (< 35 bp), low5
quality and low-complexity reads, and with the subsequent elimination of the human host sequences, which were mapped to the human reference genomes (hg19 and YH sequences) with Burrows–Wheeler alignment, a vigoroso alignment tool. Finally, the remaining sequencing data were aligned to the Microbial Genome Database, which
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contains viral, bacterial, fungal, and parasite genomic sequences to identify the pathogenic sequences. An advanced data analysis was then performed, as for the
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mapped data.
The reference genomes were downloaded from the National Center for Biotechnology
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Information (ftp://ftp.ncbi.nlm.nih.gov/genomes/). The database used in our study
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included 1494 bacteria, 2700 viruses, 73 fungi, and 48 parasites, all related to human
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disease. We calculated the sequence depth and genomic coverage for each species using
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SoapCoverage from the SOAP website (http://soap.genomics.org.cn/).
PCR and Sanger validation
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To verify the results of NGS, we used sequence-specific PCR to amplify the target
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fragment with specific primers: BLA-2F-F: TGGCTCGGTTGCCAATATC and BLA2F-R: CGCTTGCCTTTCAGGTCTG. Agarose gel electrophoresis was used to analyze the PCR products, and Sanger sequencing with an ABI PRISM 3730 DNA Analyzer
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(Applied Biosystems, Foster City, CA, USA) was used to validate the sequencing results. Finally, the sequences were aligned to the NTdatabase with the online software NCBI
Blast
(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSear 6
ch&LINK_LOC=blasthome).
Results Clinical Findings
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Four patients with NB were identified in a group of patients with clinically suspected infectious diseases of CNS. Two were women and two were men, ranging in age from
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38 to 49 years. The clinical manifestations in these patients are summarized in Table 1.
The major complications are summarized in Table 2. All four patients were previously
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healthy and none of them had a definite history of epidemiological contact, although
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they were all from endemic areas. The clinical manifestations varied among these
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patients. Case 1 presented with transient left-sided numbness and weakness,
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progressive numbness and weakness of bilateral lower limbs, and bilateral hearing loss.
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Case 2 presented with recurrent right-sided weakness and slurred speech. Case 3 presented with headache and blurred vision. Case 4 presented with recurrent seizures.
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Fever was present in only two patients. Peripheral blood Human immunodeficiency
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virus antibodies, rapid plasma reagin (RPR), Lyme disease antibodies, Mycobacterium PCR, and Cytomegalovirus (CMV) PCR were negative in all four patients. Gram staining, acid-fast bacilli, Mycobacterium PCR, India-ink staining, Cryptococcus
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antigen, RPR, TORCH, T-SPOT.TB, and CMV PCR analyses of the CSF were also negative in these patients. Brain and spinal magnetic resonance imaging (MRI) with contrast was performed in all patients. Case 1 showed abnormal enhancement of the cranial nerve (CN) Ⅴ (Fig 1A), Ⅶ and Ⅷ, and diffuse enhancement of the spinal 7
meninges (Fig 1B), together with a T2-weighted hyperintense lesion on the right temporal lobe. Case 2 showed abnormal enhancement of the CN Ⅲ(Fig 1C), Ⅴand Ⅵ, and diffuse meningeal enhancement, which improved (Fig 1D) after treatment for 2 months. Case 3 showed meningeal abscesses with ring enhancement and surrounding
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edema near the right frontal lobe (Fig 1E–F) and parietal lobe. Case 4 showed white matter lesions with fluid-attenuated inversion recovery (FLAIR) hyperintensity (Fig
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1H). The CSF analyses and peripheral blood results are summarized in Table 3. Lumbar punctures revealed increased opening pressures, elevated white blood cell counts with
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prominent mononuclear cells, elevated protein levels, and reduced glucose levels in all
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cases. It took 7 days for CSF culture to be positive for brucellosis in Case 1. Peripheral
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blood Rose Bengal tests were positive for cases 2, 3, and 4, CSF serological tests for
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brucellosis were not performed in these patients. The symptoms of these patients
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improved gradually after the initiation of empiric antibiotic treatment with doxycycline, rifampin, and ceftriaxone. Case 1 experienced cerebral hemorrhagic transformation in
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the infarction area of the right temporal lobe before discharge.
NGS results
DNA library were constructed from 300 μL of CSF sample in each patient. Brucella
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species-specific DNA was detected in all four patients. The number of raw reads range from 19,083,456 to 30,738,055. The identified sequence reads corresponding to Brucella species range from 11 to 104, with genomic coverage ranging from 0.043% to 0.4%, reads per million (RPM) range from 0.58 to 3.38. The details of the CSF NGS 8
results are given in Table 4, Fig 2 and Supplementary Table 1–5. There were several contaminating sequences in the results (Fig 2 and Supplementary Table 2–5). Propionibacterium acnes, a common contaminating microorganism, was present in all four patients with NB. Methylobacterium radiotolerans, M. populi, and Truepera
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radiovictrix were detected in case 1; Porphyromonas gingivalis, Thermus thermophilus, and Rhizobium etli were detected in case 2; Bradyrhizobium S23321 and Ralstonia
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solanacearum were detected in case 3; and Micrococcus luteus, M. radiotolerans, and Asticcacaulis excentricus were detected in case 4. The contaminating sequences were
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either present in the NTC samples (Supplementary Table 2–5) or common
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contaminating microorganisms. However, there were no Brucella reads in the negative
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NTC samples. Mycobacterium tuberculosis, Treponema pallidum, and Cryptococcus
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were not detected by NGS of CSF for all four patients.
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The presence of Brucella DNA in the CSF was confirmed with a PCR analysis of cases
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sample.
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1, 2, and 4. Case 3 didn’t undergo Sanger sequencing because there was no extra CSF
Discussion
Four patients from a group of patients with clinically suspected infectious diseases of
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CNS, with dramatically different presenting symptoms, were diagnosed with NB using NGS of the CSF. To our knowledge, this is the first case series in which NGS of the CSF was used for the diagnosis of NB. Human brucellosis is the most common zoonotic disease in the world, with more than 9
500,000 new cases annually 8. However, it is estimated that the real number is 26 times higher than the number of reported cases 9. About 5% of brucellosis cases have NB10. Precise and prompt diagnosis and the initiation of appropriate antibiotic treatment are crucial for a favorable prognosis.
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Unfortunately, the diagnosis of NB is a considerable clinical challenge. Firstly, the clinical features and CSF findings of NB are usually nonspecific, and it shares strong
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similarities with other CNS infections, including tuberculosis, syphilis, Lyme disease,
and Cryptococcus infection. The clinical manifestations and neuroimages of our four
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patients varied dramatically, and they had no history of epidemiological contact. The
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symptoms of our patients were unusual for brucellosis, although these symptoms had
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been reported in the previous studies11-13. Brain MRI of case 4 showed diffuse white
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matter changes. This neuroimage change was reported in the previous study11, 14. Given
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the rarity of the symptoms and neuroimages, neurobrucellosis would be a forgotten diagnosis for these patients, and may not even be considered in the differential
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diagnoses. Case 4 was diagnosed as NB when he was admitted for the second time
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because of nonspecific symptoms. Secondly, although positive culture is the gold standard for diagnosis, the sensitivity of routine culture tests is low. About 28% of patients with CNS brucellosis had positive blood cultures and 15% had positive CSF
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cultures
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. Only one patient in our study was culture positive. Thirdly, as
demonstrated in case 1, CSF culture for NB is sometimes time-consuming, which makes prompt diagnosis a difficult mission. Fourthly, although serology is more sensitive and faster than routine culture test; brucellosis seropositivity is high in 10
endemic area (2.6-14.4% among Turkish people13); it may not always distinguish active and previous infections. NGS is a new technique that is increasingly used for the clinical diagnosis of infectious diseases of CNS. Theoretically, with sufficiently long reads, multiple hits in the
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microbial genome, and a complete reference database, almost all microorganisms can be uniquely identified based on specific nucleic acid sequences. Several case reports
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and clinical studies have demonstrated the use of NGS as a diagnostic tool for infectious
diseases 5-7. Mongkolrattanothai K, et al recently reported a pediatric patient who was
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unexpectedly diagnosed as neurobrucellosis using NGS17. These four patients were also
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“unexpectedly” diagnosed as neurobrucellosis in our multicenter study which aims to
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detect pathogens in patients with clinically suspected CNS infections using NGS of
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CSF. Simultaneously, Mycobacterium tuberculosis, Treponema pallidum, and
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Cryptococcus were not detected by NGS of CSF for all four patients. The diagnoses were finally confirmed by CSF culture or Sanger sequencing in our study. In our
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multicenter study, we found no NB patients with negative NGS results, implying the
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high sensitivity of NGS in the diagnosis of NB. Contamination is a common problem in NGS. There were several contaminating microorganisms in our study, of which Propionibacterium acnes was most common. The contamination could come from
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cross-contamination between samples during the NGS library preparation, skin or body flora during the process of lumbar puncture, or an incorrect process of sample demultiplexing or formation of artificial products17,18. It is essential to differentiate the clinical relevant pathogens from contaminating microorganisms in NGS. We can 11
remove plenty of background microorganisms according to the NGS results of “NTC” control. Besides, we found that some background microorganisms appear frequently in the result of NGS. For example, Propionibacterium acnes, Burkholderia, Bradyrhizobium appear with high species-specific reads and RPM in the NGS result of
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most patients at our center (unpublished data). Therefore, before explaining the results, researchers should be cautious with the common background microorganisms at their
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center. NGS of the CSF is a fast method, taking less than 48 hours at our center. With the results of CSF NGS, treatment can be initiated promptly, meanwhile, standard
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diagnostic methods for the target pathogen can be used to confirm the diagnosis.
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Finally, NGS of CSF is a new technique in the diagnosis of CNS infections, which is
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frequently unavailable in endemic and resource-limited areas. Therefore, we emphasize
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that NGS of CSF is especially suitable for these puzzling cases.
Conclusion
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This study demonstrates the power of NGS of the CSF coupled with a bioinformatic
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pipeline in the diagnosis of NB.
Funding source
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None.
Conflicts of Interest We declare that we have no conflicts of interest. 12
Acknowledgments
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The authors thank the patients for participating in this study.
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Fig 1. Brain and spinal magnetic resonance imaging (MRI) of the patients. Case 1 showed abnormal enhancement of cranial nerve (CN) Ⅴ (Fig 1A), and diffuse enhancement of spinal meningeal (Fig 1B). Case 2 showed abnormal enhancement of CN Ⅲ (Fig 1C), which were improved (Fig 1D) after treatment of 2 months. Case 3
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showed meningeal abscesses with ring enhancement and surrounding edema near right frontal lobe (Fig 1E-F). Case 4 showed white matter lesions of fluid attenuated
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inversion recovery (FLAIR) hyperintensity (Fig 1H), comparing to multiple small
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scattered white matter lesions (Fig 1G) in the beginning of the disease.
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Fig 2. Next generation sequencing (NGS) of cerebrospinal fluid (CSF) in the patients. The identified sequence reads corresponding to Brucella species were 30 (Fig 2A), 11
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(Fig 2C), 24 (Fig 2E), and 104 (Fig 2G); with a genomic coverage of 0.11% (Fig 2B), 0.043% (Fig 2D), 0.093% (Fig 2F), and 0.4% (Fig 2H), for case 1, 2, 3, and 4, respectively. Propionibacterium acnes, as a common contamination microorganism, presented in all four patients (Fig 2A, C, E, G). Methylobacterium radiotolerans, 17
Methylobacterium populi, and Truepera radiovictrix presented in case 1 (Fig 2A); Porphyromonas gingivalis, Thermus thermophilus, and Rhizobium etli presented in case 2 (Fig 2C); Bradyrhizobium S23321 and Ralstonia solanacearum presented in case 3 (Fig 2E); Micrococcus luteus, Methylobacterium radiotolerans, and Asticcacaulis
A
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excentricus presented in case 4 (Fig 2G).
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Table 1. Clinical features of the four patients with neurobrucellosis.
No.
e
Gen
Fev
Headach
Sweati
Weight
Back
Hearing
Joint
Seiz
Meningeal
(yr.
der
er
e
ng
loss
pain
loss
pain
ure
signs
Mal
＋
＋
－
＋
＋
＋
＋
－
－
－
－
＋
＋
－
＋
－
－
－
－
＋
＋
＋
＋
＋
－
＋
－
－
－
＋
－
－
)
1
38
2
46
3
38
4
49
e Fem ale Fem ale Mal e
A
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A
N
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＋ = positive; － = negative; No. = number; yr. = year.
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e
Ag
－
＋
＋
－
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Cas
Table 2. The major complications of the four patients with neurobrucellosis Case
CN
Polyneuropathy
No.
involvement
/radiculopathy
1
＋
2
Encephalitis
Stroke
Brian abscess
＋
＋
－
＋
－
＋
－
＋
－
＋
－
3
－
－
＋
－
－
＋
4
－
－
＋
＋
－
－
A
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M
A
N
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＋ = positive; － = negative; CN = cranial nerves; No. = number;
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Meningitis
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Table 3. CSF and Peripheral blood tests of the four patients with neurobrucellosis. Case
CSF
Peripheral Blood
Pressure
WBC
MN
Protein
Glucose
(mmH2O)
(cells/µL)
(%)
(g/L)
(mmol/L)
1
210
607
99
9.3
1.2
Brucella
－
－
2
>330
361
91
3.14
0.7
－
＋
－
3
>330
178
99
1.09
0.8
－
＋
－
4
220
86
93
1.28
1.7
－
＋
－
Rose Bengal Test
Culture
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Culture
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No.
＋ = positive; － = negative; CSF = cerebrospinal fluid; No. = number; WBC = white
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M
A
N
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blood cell; MN = mononuclear cell.
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Table 4. The NGS of CSF of the four patients with neurobrucellosis. From Onset to CSF
Sample
Collection Time
Volume
(months)
(μL)
1
24
300
2
12
3 4
Case
Genomic Coverage
Raw Reads
RPM
30
22865302
1.31
0.11
300
11
19083456
0.58
0.043
6
300
24
24691420
0.97
0.093
4
300
104
30738055
3.38
0.4
Specific Reads
(%)
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No.
Brucella Species-
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A
N
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NGS = next-generation sequencing; CSF = cerebrospinal fluid; No. = number; RPM = reads per million
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