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The value of next-generation sequencing for the diagnosis of Streptococcus suis meningitis

SUMMARY

OBJECTIVE:

The aim of this study was to investigate the value of next-generation sequencing for the diagnosis of Streptococcus suis meningitis.

METHODS:

Patients with meningitis in the Department of Neurology of the Hainan General Hospital were recruited and divided into a next-generation sequencing group and a control group. In the next-generation sequencing group, we used the next-generation sequencing method to detect the specific pathogenic bacteria in the patients. In the control group, we used the cerebrospinal fluid bacterial culture method to detect the specific pathogenic bacteria in the patients.

RESULTS:

A total of 28 participants were recruited for this study, with 14 participants in each group. The results showed similarities in both the average age and average course of the disease between the two groups (p>0.05). The white blood cell count, percentage of neutrophils, and level of C-reactive protein in the next-generation sequencing group were significantly higher than those in the control group (p<0.05). There were similarities in both the temperature and intracranial pressure between the two groups (p>0.05). In the next-generation sequencing group, all patients (100%) were detected as having had the S. suis meningitis infection by next-generation sequencing, while only 6 (43%) patients in the control group had been detected as having the S. suis meningitis infection by cerebrospinal fluid bacterial culture.

CONCLUSIONS:

The positive detection rate of S. suis by the next-generation sequencing method was significantly higher compared with using a cerebrospinal fluid bacterial culture. Therefore, the next-generation sequencing method is valuable for the diagnosis of S. suis meningitis and is worthy of clinical application.

Keywords
Streptococcus suis; Cerebrospinal fluid; Next-generation sequencing; Meningitis; Bacterial culture

INTRODUCTION

Streptococcus suis (Ss) meningitis is an acute infectious disease caused by Ss, invading the central nervous system. The main form of transmission is through close contact between people and pigs; the pathogen invades the body through damaged skin or mucous membranes and infects the central nervous system via the blood. It is the primary type of Ss infection among humans, and its main clinical symptoms include fever, headache, and hearing loss, and in severe cases, consciousness disturbances may occur11. Hlebowicz M, Jakubowski P, Smiatacz T. Streptococcus suis meningitis: epidemiology, clinical presentation and treatment. Vector Borne Zoonotic Dis. 2019;19(8):557-62. https://doi.org/10.1089/vbz.2018.2399
https://doi.org/10.1089/vbz.2018.2399...
. Most of the cerebrospinal fluid of patients with Ss meningitis showed purulent meningitis22. Su ZW, Xu J, Ye SJ, Xiao KJ. Characteristics clinical manifestations and cerebrospinal fluid change in patients infected with Streptococcus suis meningitis. Chinese Journal of Infection Control. 2006;4:315-316,319., and some patients reflected similar viral meningitis33. Liu LX, Li HF, Chen Y, Zhang AM. A case of septic shock secondary to Streptococcus suis infection with suspected viral meningitis. Chinese Journal of Neurology. 2017;50:681-3. or tuberculous meningitis44. Li L. A case report of human infection with Streptococcus suis meningitis misdiagnosed as tuberculous meningitis. J Youjiang Medicine. 2014;42(1):113-4. https://doi.org/10.3969/j.issn.1003-1383.2014.01.037
https://doi.org/10.3969/j.issn.1003-1383...
.

Next-generation sequencing (NGS) is a large-scale parallel sequencing technology that allows for thousands to billions of deoxyribonucleic acid (DNA) fragments to be simultaneously and independently sequenced55. Gu W, Miller S, Chiu CY. Clinical metagenomic next-generation sequencing for pathogen detection. Annu Rev Pathol. 2019;14:319-38. https://doi.org/10.1146/annurev-pathmechdis-012418-012751
https://doi.org/10.1146/annurev-pathmech...
. Since most infectious agents include DNA or RNA genomes, existing research findings on NGS technology have been widely applied to infectious diseases. Studies have shown that the application of NGS to infectious disease outbreaks of unknown causes and patients with suspected infections has positive results66. Gardy JL, Loman NJ. Towards a genomics-informed, real-time, global pathogen surveillance system. Nat Rev Genet. 2018;19(1):9-20. https://doi.org/10.1038/nrg.2017.88
https://doi.org/10.1038/nrg.2017.88...
. The applications of NGS included whole-genome sequencing of microbial isolates, microbiome studies, drug resistance testing of viruses or culture isolates, and lineage tracing77. Rossen JWA, Friedrich AW, Moran-Gilad J, ESCMID Study Group for Genomic and Molecular Diagnostics (ESGMD). Practical issues in implementing whole-genome-sequencing in routine diagnostic microbiology. Clin Microbiol Infect. 2018;24(4):355-60. https://doi.org/10.1016/j.cmi.2017.11.001
https://doi.org/10.1016/j.cmi.2017.11.00...
. Additionally, NGS technology exhibited efficient potential through unbiased pathogen detection. It has been demonstrated in the field of microbiology and in clinical contexts, and its application is rapidly becoming routine88. Schlaberg R, Chiu CY, Miller S, Procop GW, Weinstock G, Professional Practice Committee and Committee on Laboratory Practices of the American Society for Microbiology, et al. Validation of metagenomic next-generation sequencing tests for universal pathogen detection. Arch Pathol Lab Med. 2017;141(6):776-86. https://doi.org/10.5858/arpa.2016-0539-RA
https://doi.org/10.5858/arpa.2016-0539-R...
.

Traditional blood culture and cerebrospinal fluid culture detection of pathogens have the disadvantages of long detection times and low positive rates. This can make it easy to misdiagnose and affect patients’ prognoses. Cerebrospinal fluid pathogen NGS technology is an emerging molecular diagnostic method that can quickly detect intracranial pathogens. The literature evidenced its use in the diagnosis of encephalitis; however, its applicability for routine diagnosis has not been evaluated99. Brown JR, Bharucha T, Breuer J. Encephalitis diagnosis using metagenomics: application of next generation sequencing for undiagnosed cases. J Infect. 2018;76(3):225-40. https://doi.org/10.1016/j.jinf.2017.12.014
https://doi.org/10.1016/j.jinf.2017.12.0...
. Accordingly, this study investigated the value of NGS for the diagnosis of Ss meningitis.

METHODS

Participants

From August 2018 to January 2021, patients with meningitis in the Department of Neurology of the Hainan General Hospital were recruited and divided into an NGS group and a control group. This study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of our hospital. All participants signed informed consent forms for inclusion in the study.

The diagnostic criteria of Ss meningitis are as follows:
  1. Coma may occur in patients with acute onset, fever, chills, general discomfort, fatigue, headache, dizziness, nausea, and vomiting (possibly jet vomiting). There are no bleeding spots, ecchymosis spots, and ecchymosis spots on the skin. No shock.

  2. The meningeal stimulation sign was positive, and the cerebrospinal fluid showed suppurative changes.

  3. The NGS or cerebrospinal fluid bacterial culture method was used to detect specific pathogens in patients.

Inclusion and exclusion criteria

The inclusion criteria were as follows:
  1. Patients who had been diagnosed with meningitis;

  2. Those older than 18 years;

  3. Patients suspected of suffering from Ss meningitis:
    1. recent history of close contact with sick pigs and their products, especially those with damaged skin and mucosa;

    2. acute onset, chills, fever, and other acute infectious poisoning symptoms; and

  4. Patients who provided signed informed consent for inclusion in the study.

The exclusion criteria were as follows:
  1. Patients who had an advanced malignant tumor and

  2. Patients whose data were incomplete.

METHODS

In the NGS group, we used the NGS method to detect specific pathogenic bacteria in the patients. In the control group, we used the cerebrospinal fluid bacterial culture method to detect the specific pathogenic bacteria in the patients.

Cerebrospinal fluid detection

For the lumbar puncture, approximately 2–3 mL of cerebrospinal fluid was collected, sterilely sealed, and stored at -20°C or transported on dry ice to Yuguo Biotechnology (Beijing) Co., Ltd., for metagenomics NGS detection. Following the TIANGEN DNA Mini kit DP316 (TIANGEN Biotechnology Company, Beijing, TIANGEN DNA) instructions, 200 μL of cerebrospinal fluid was collected to extract DNA and purify it. Nucleic acid concentration and quality were detected and confirmed using the Qubit dsDNA HS kit (Thermo Fisher Scientific Co., Ltd., Shanghai) and agarose gel electrophoresis.

The Qiagen library construction kit (QIAseq™ Ultralow Input Library Kit) operation manual was followed to complete the DNA library construction. The Qubit dsDNA HS kit (Thermo Fisher Scientific Co., Ltd.) and agarose gel electrophoresis were used to detect DNA concentration and quality. Qualified DNA libraries with different barcode tags were collected and sequenced by the Illumina NextSeq sequencing platform.

After obtaining the sequencing data, high-quality data were generated by filtering out joints, low-quality, low-complexity, and short sequences. Then, the human sequence that matched the human reference genome was removed using the SNAP software. Burrow-Wheeler alignment was used to compare the remaining data with the microbial genome database. The database collects a large number of microbial genomes from NCBI, including more than 20,000 microorganisms, 11,910 bacteria species, 7,103 virus species, 1,046 fungi species, and 305 parasite species. Finally, the microbiological composition of the sample is obtained. It took approximately 2–3 days to obtain the NGS microbial identification results from the cerebrospinal fluid.

Main observation indexes

In this study, the main observation indexes included sex, age, the course of the disease, temperature, white blood cell count, the percentage of neutrophils, the level of C-reactive protein, intracranial pressure, and cerebrospinal fluid detection.

Statistical analysis

This study used the SPSS Statistics version 20.0 (IBM, Chicago, IL, USA) software to conduct the statistical analysis. The continuous variables of normal distribution were expressed as mean±standard deviation, the continuous variables of non-normal distribution were expressed as a median (interquartile range), and the categorical variables were expressed as a frequency (percentage [%]). For the two comparisons, each value was compared using a t-test when each datum conformed to a normal distribution, while non-normally distributed continuous data were compared using nonparametric tests. The counting data were tested by the chi-square test; p<0.05 was considered statistically significant.

RESULTS

General characteristics

A total of 28 participants were recruited for this study, including 4 females and 24 males. There were 14 patients in the NGS group; the average age was 54.79±10.34 years and the average course of the disease was 5.93±3.52 days. There were 14 patients in the control group; the average age was 46.71±18.96 years and the average course of the disease was 8.07±7.49 days. The results showed similarities in both the average age (p=0.151) and the average course of the disease (p=0.350) between the two groups.

Comparison of routine and biochemical indexes between the two groups

In the NGS group, the temperature was 39.10±0.44°C, the white blood cell count was 12.45±4.21×109, the percentage of neutrophils was 82.48±8.50%, and the level of C-reactive protein was 134.07±88.86 mg/L. In the control group, the temperature was 38.46±0.98°C, the white blood cell count was 7.95±2.91×109, the percentage of neutrophils was 71.99±9.23%, and the level of C-reactive protein was 4.70±6.03 mg/L. The results showed that the white blood cell count (p=0.005), the percentage of neutrophils (p=0.004), and the level of C-reactive protein (p<0.001) in the NGS group were significantly higher than in the control group. There were also similarities in temperature (p=0.082) between the two groups. The details are shown in Figure 1.

Figure 1.
(A) The comparison of white blood cell between the two groups. (B) The comparison of the percentage of neutrophils between the two groups. (C) The comparison of the level of C-reactive protein between the two groups. **p<0.01. ***p<0.001.

Cerebrospinal fluid test results between the two groups

In the NGS group, the intracranial pressure was 214.29±67.14 mmH2O. In the control group, the intracranial pressure was 190.86±55.99 mmH2O. The results showed similar intracranial pressure between the two groups (p=0.350).

In the NGS group, all patients (100%) had been detected as having the Ss meningitis infection by NGS, while only 6 (43%) patients in the control group had been detected as having the Ss meningitis infection by cerebrospinal fluid bacterial culture.

DISCUSSION

S. suis is a Gram-positive and anaerobic zoonotic pathogen that can be divided into 35 serotypes, the most common of which is serotype II. Anyone is susceptible to the Ss pathogen and, following infection, it can cause meningitis (the most common manifestation), sepsis, endocarditis, and arthritis. Most of these conditions can lead to serious cochlear nerve and vestibular nerve damage sequelae1010. Lun ZR, Wang QP, Chen XG, Li AX, Zhu XQ. Streptococcus suis: an emerging zoonotic pathogen. Lancet Infect Dis. 2007;7(3):201-9. https://doi.org/10.1016/S1473-3099(07)70001-4
https://doi.org/10.1016/S1473-3099(07)70...
. The pathogen enters the central nervous system through the brain microvascular epithelial cells or choroidal epithelial cells via the blood-brain barrier or blood-cerebrospinal fluid barrier and causes inflammation of the meninges and brain parenchyma1111. Dutkiewicz J, Zając V, Sroka J, Wasiński B, Cisak E, Sawczyn A, et al. Streptococcus suis: a re-emerging pathogen associated with occupational exposure to pigs or pork products. Part II – Pathogenesis. Ann Agric Environ Med. 2018;25(1):186-203. https://doi.org/10.26444/aaem/85651
https://doi.org/10.26444/aaem/85651...
. The main pathogenic risk factors include occupational exposure to pigs and raw pork or eating raw pork products1212. Takeuchi D, Kerdsin A, Akeda Y, Chiranairadul P, Loetthong P, Tanburawong N, et al. Impact of a food safety campaign on streptococcus suis infection in humans in Thailand. Am J Trop Med Hyg. 2017;96(6):1370-7. https://doi.org/10.4269/ajtmh.16-0456
https://doi.org/10.4269/ajtmh.16-0456...
. The diagnosis of this disease depends on the positive culture of Ss in the blood-cerebrospinal fluid1313. Jiang Y, Yao KH. Talking about human infection with Streptococcus suis. J Chinese Journal of Infectious Diseases. 2008;26(9):573-6. https://doi.org/10.3321/j.issn:1000-6680.2008.09.019
https://doi.org/10.3321/j.issn:1000-6680...
. However, due to factors such as low blood-cerebrospinal fluid, insufficient sampling, or early antibiotic treatment, the positive rate of blood-cerebrospinal fluid cultures is low, and the positive rate of bacterial blood cultures is approximately 32%. The positive rate of cerebrospinal fluid bacterial cultures is 18.5%, leading to a higher incidence of poor prognosis1414. Yang XX, Jiang N, Wu JY, Tang RZ, Wang SJ, Wang T, et al. Clinical analysis of 75 cases of human infection with Streptococcus suis. J Chinese Journal of Internal Medicine. 2007;46(9):764-5. https://doi.org/10.3760/j.issn:0578-1426.2007.09.020
https://doi.org/10.3760/j.issn:0578-1426...
. Early diagnosis and treatment can help improve the clinical outcome of patients. Therefore, a rapid and accurate detection method is needed to assist with diagnosis in clinical practice. The NGS of cerebrospinal fluid is used as a molecular diagnosis of infectious diseases of unknown pathogens. This new technology has attracted significant attention55. Gu W, Miller S, Chiu CY. Clinical metagenomic next-generation sequencing for pathogen detection. Annu Rev Pathol. 2019;14:319-38. https://doi.org/10.1146/annurev-pathmechdis-012418-012751
https://doi.org/10.1146/annurev-pathmech...
.

The common symptoms and signs of Ss meningitis include fever, headache, and neck stiffness1515. Susilawathi NM, Tarini NMA, Fatmawati NND, Mayura PIB, Suryapraba AAA, Subrata M, et al. Streptococcus suis-Associated Meningitis, Bali, Indonesia, 2014–2017. Emerg Infect Dis. 2019,25(12):2235-42. https://doi.org/10.3201/eid2512.181709
https://doi.org/10.3201/eid2512.181709...
. Bilateral hearing loss can occur during early onset with an incidence as high as 66.4%1616. Feng YJ, Zhang HM, Wu ZW, Wang SH, Cao M, Hu D, et al. Streptococcus suis infection: an emerging/reemerging challenge of bacterial infectious diseases? Virulence. 2014;5(4):477-97. https://doi.org/10.4161/viru.28595
https://doi.org/10.4161/viru.28595...
. All five patients had a fever, headache, and bilateral deafness. The incidence was 100%, which was higher than noted in existing literature reports1515. Susilawathi NM, Tarini NMA, Fatmawati NND, Mayura PIB, Suryapraba AAA, Subrata M, et al. Streptococcus suis-Associated Meningitis, Bali, Indonesia, 2014–2017. Emerg Infect Dis. 2019,25(12):2235-42. https://doi.org/10.3201/eid2512.181709
https://doi.org/10.3201/eid2512.181709...
,1616. Feng YJ, Zhang HM, Wu ZW, Wang SH, Cao M, Hu D, et al. Streptococcus suis infection: an emerging/reemerging challenge of bacterial infectious diseases? Virulence. 2014;5(4):477-97. https://doi.org/10.4161/viru.28595
https://doi.org/10.4161/viru.28595...
. Reports in the literature showed that the classification of cerebrospinal fluid white blood cells in this disease was dominated by multiple nuclear cells, which were transformed into mononuclear cells or lymphocytes quickly after antibiotic treatment22. Su ZW, Xu J, Ye SJ, Xiao KJ. Characteristics clinical manifestations and cerebrospinal fluid change in patients infected with Streptococcus suis meningitis. Chinese Journal of Infection Control. 2006;4:315-316,319.. If the epidemiological data of patients are not clear, the condition can be easily confused with tuberculous meningitis, resulting in a misdiagnosis.

The NGS of cerebrospinal fluid pathogens as a molecular technology to assist in the diagnosis of central nervous system infections can quickly and sensitively identify pathogens1717. Dai YY, Chen L, Chang WJ, Lu HW, Cui P, Ma XL. Culture-negative streptococcus suis infection diagnosed by metagenomic next-generation sequencing. Front Public Health. 2019;7:379. https://doi.org/10.3389/fpubh.2019.00379
https://doi.org/10.3389/fpubh.2019.00379...
. This can enable clinicians to make reasonable interpretations of test results, based on a patient’s clinical manifestations, and select an appropriate antibiotic treatment plan1818. Simner PJ, Miller HB, Breitwieser FP, Monsalve GP, Pardo CA, Salzberg SL, et al. Development and optimization of metagenomic next-generation sequencing methods for cerebrospinal fluid diagnostics. J Clin Microbiol. 2018;56(9):e00472-18. https://doi.org/10.1128/JCM.00472-18
https://doi.org/10.1128/JCM.00472-18...
.

The results showed that all patients (100%) had been detected as having the Ss meningitis infection by NGS in the NGS group, while only 6 (43%) patients in the control group had been detected as having the Ss meningitis infection by cerebrospinal fluid bacterial culture. Therefore, the positive detection rate of Ss using the NGS method was significantly higher compared with detection via cerebrospinal fluid bacterial culture.

This study had multiple limitations. First, it was not a randomized controlled trial. Second, the sample size was limited and, as such, a larger trial with more participants is necessary for further study.

CONCLUSIONS

The positive detection rate of Ss by the NGS method was significantly higher compared with detection via cerebrospinal fluid bacterial culture. Therefore, the NGS method represents value for the diagnosis of Ss meningitis and is worthy of clinical application.

REFERENCES

  • 1.
    Hlebowicz M, Jakubowski P, Smiatacz T. Streptococcus suis meningitis: epidemiology, clinical presentation and treatment. Vector Borne Zoonotic Dis. 2019;19(8):557-62. https://doi.org/10.1089/vbz.2018.2399
    » https://doi.org/10.1089/vbz.2018.2399
  • 2.
    Su ZW, Xu J, Ye SJ, Xiao KJ. Characteristics clinical manifestations and cerebrospinal fluid change in patients infected with Streptococcus suis meningitis. Chinese Journal of Infection Control. 2006;4:315-316,319.
  • 3.
    Liu LX, Li HF, Chen Y, Zhang AM. A case of septic shock secondary to Streptococcus suis infection with suspected viral meningitis. Chinese Journal of Neurology. 2017;50:681-3.
  • 4.
    Li L. A case report of human infection with Streptococcus suis meningitis misdiagnosed as tuberculous meningitis. J Youjiang Medicine. 2014;42(1):113-4. https://doi.org/10.3969/j.issn.1003-1383.2014.01.037
    » https://doi.org/10.3969/j.issn.1003-1383.2014.01.037
  • 5.
    Gu W, Miller S, Chiu CY. Clinical metagenomic next-generation sequencing for pathogen detection. Annu Rev Pathol. 2019;14:319-38. https://doi.org/10.1146/annurev-pathmechdis-012418-012751
    » https://doi.org/10.1146/annurev-pathmechdis-012418-012751
  • 6.
    Gardy JL, Loman NJ. Towards a genomics-informed, real-time, global pathogen surveillance system. Nat Rev Genet. 2018;19(1):9-20. https://doi.org/10.1038/nrg.2017.88
    » https://doi.org/10.1038/nrg.2017.88
  • 7.
    Rossen JWA, Friedrich AW, Moran-Gilad J, ESCMID Study Group for Genomic and Molecular Diagnostics (ESGMD). Practical issues in implementing whole-genome-sequencing in routine diagnostic microbiology. Clin Microbiol Infect. 2018;24(4):355-60. https://doi.org/10.1016/j.cmi.2017.11.001
    » https://doi.org/10.1016/j.cmi.2017.11.001
  • 8.
    Schlaberg R, Chiu CY, Miller S, Procop GW, Weinstock G, Professional Practice Committee and Committee on Laboratory Practices of the American Society for Microbiology, et al. Validation of metagenomic next-generation sequencing tests for universal pathogen detection. Arch Pathol Lab Med. 2017;141(6):776-86. https://doi.org/10.5858/arpa.2016-0539-RA
    » https://doi.org/10.5858/arpa.2016-0539-RA
  • 9.
    Brown JR, Bharucha T, Breuer J. Encephalitis diagnosis using metagenomics: application of next generation sequencing for undiagnosed cases. J Infect. 2018;76(3):225-40. https://doi.org/10.1016/j.jinf.2017.12.014
    » https://doi.org/10.1016/j.jinf.2017.12.014
  • 10.
    Lun ZR, Wang QP, Chen XG, Li AX, Zhu XQ. Streptococcus suis: an emerging zoonotic pathogen. Lancet Infect Dis. 2007;7(3):201-9. https://doi.org/10.1016/S1473-3099(07)70001-4
    » https://doi.org/10.1016/S1473-3099(07)70001-4
  • 11.
    Dutkiewicz J, Zając V, Sroka J, Wasiński B, Cisak E, Sawczyn A, et al. Streptococcus suis: a re-emerging pathogen associated with occupational exposure to pigs or pork products. Part II – Pathogenesis. Ann Agric Environ Med. 2018;25(1):186-203. https://doi.org/10.26444/aaem/85651
    » https://doi.org/10.26444/aaem/85651
  • 12.
    Takeuchi D, Kerdsin A, Akeda Y, Chiranairadul P, Loetthong P, Tanburawong N, et al. Impact of a food safety campaign on streptococcus suis infection in humans in Thailand. Am J Trop Med Hyg. 2017;96(6):1370-7. https://doi.org/10.4269/ajtmh.16-0456
    » https://doi.org/10.4269/ajtmh.16-0456
  • 13.
    Jiang Y, Yao KH. Talking about human infection with Streptococcus suis. J Chinese Journal of Infectious Diseases. 2008;26(9):573-6. https://doi.org/10.3321/j.issn:1000-6680.2008.09.019
    » https://doi.org/10.3321/j.issn:1000-6680.2008.09.019
  • 14.
    Yang XX, Jiang N, Wu JY, Tang RZ, Wang SJ, Wang T, et al. Clinical analysis of 75 cases of human infection with Streptococcus suis. J Chinese Journal of Internal Medicine. 2007;46(9):764-5. https://doi.org/10.3760/j.issn:0578-1426.2007.09.020
    » https://doi.org/10.3760/j.issn:0578-1426.2007.09.020
  • 15.
    Susilawathi NM, Tarini NMA, Fatmawati NND, Mayura PIB, Suryapraba AAA, Subrata M, et al. Streptococcus suis-Associated Meningitis, Bali, Indonesia, 2014–2017. Emerg Infect Dis. 2019,25(12):2235-42. https://doi.org/10.3201/eid2512.181709
    » https://doi.org/10.3201/eid2512.181709
  • 16.
    Feng YJ, Zhang HM, Wu ZW, Wang SH, Cao M, Hu D, et al. Streptococcus suis infection: an emerging/reemerging challenge of bacterial infectious diseases? Virulence. 2014;5(4):477-97. https://doi.org/10.4161/viru.28595
    » https://doi.org/10.4161/viru.28595
  • 17.
    Dai YY, Chen L, Chang WJ, Lu HW, Cui P, Ma XL. Culture-negative streptococcus suis infection diagnosed by metagenomic next-generation sequencing. Front Public Health. 2019;7:379. https://doi.org/10.3389/fpubh.2019.00379
    » https://doi.org/10.3389/fpubh.2019.00379
  • 18.
    Simner PJ, Miller HB, Breitwieser FP, Monsalve GP, Pardo CA, Salzberg SL, et al. Development and optimization of metagenomic next-generation sequencing methods for cerebrospinal fluid diagnostics. J Clin Microbiol. 2018;56(9):e00472-18. https://doi.org/10.1128/JCM.00472-18
    » https://doi.org/10.1128/JCM.00472-18
  • Funding: this study was supported by the Hainan Province Science and Technology Special Fund (No. ZDYF2021SHFZ112) and the Scientific research projects of health industry in Hainan Province (no. 20A200223).

Publication Dates

  • Publication in this collection
    25 Nov 2022
  • Date of issue
    2022

History

  • Received
    25 Apr 2022
  • Accepted
    06 July 2022
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