This article is excerpted from "China Medical Device Industry Development Report" B29, author Wang Jiawei, director of the Department of Neurology and director of the Hospital Center of Beijing Tongren Hospital Affiliated to Capital Medical University, postdoctoral fellow, doctor

[Editor's Note] This article is excerpted from "China Medical Device Industry Development Report (2021)" B29, author Wang Jiawei, director of the Department of Neurology and director of the Hospital Center of Beijing Tongren Hospital Affiliated to Capital Medical University, postdoctoral fellow, doctoral doctor, professor, chief physician, doctoral supervisor, member of the American Society of Neuroscience; Xia Han, Yuguo Biotechnology (Beijing) Co., Ltd., general manager of the legal representative; Ma Zili, Yuguo Biotechnology (Beijing) Co., Ltd., deputy general manager.

[Abstract] Pathogen microbial detection can detect and analyze pathogens or metabolites of infectious diseases. It is one of the subdivisions of in vitro diagnostic reagent (In Vitro Diagnosis, IVD). At present, the diagnosis technology of pathogenic microorganisms is constantly developing. Traditional pathogenic microorganism culture technologies mainly include isolation and culture, smear microscopy, biochemical identification, antigen-antibody immunity, etc., which face the low positive rate and long diagnosis cycle, and cannot meet the requirements of detecting the pathogen . Molecular biological detection methods are developing rapidly, and there are many methods to amplify a small number of nucleic acid molecules to an easy-to-detection level. These detection methods include constant temperature amplification technology (LAMP), real-time fluorescence quantitative PCR, etc. With the emergence of new technologies such as second-generation sequencing, it provides new solutions for the diagnosis of clinical pathogenic microorganisms , especially the difficulty in culturing in ordinary laboratories, slow growth, unknown pathogens, rare pathogens, etc. However, the second-generation sequencing method still faces several challenges, such as high detection costs, strict requirements on the experimental environment, and some test results need to be interpreted manually. New approaches and new technologies for molecular diagnosis are changing the way we practice clinical microbiology, which affects the pathogenic microbiology-related testing industry.

[Keywords] Pathogen Nucleic Acid Second-generation Sequencing

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(hereinafter the main text)

Pathogen microbial detection can detect and analyze pathogens or metabolites of infectious diseases and is an in vitro diagnostic reagent (In Vitro Diagnosis, IVD) one of the sub-sectors. Microbial testing is mainly the detection and analysis of metabolites of pathogens or pathogens of infectious diseases in humans, including bacterial culture, identification and drug sensitivity analysis, etc. The purpose is to provide a basis for diagnosis and treatment for clinical treatment, so as to choose the most suitable drugs and treatment methods for patients. Nucleic acid amplification-based techniques can provide sensitive and specific results with shorter detection times than before.

In December 2019, the new crown epidemic broke out in Wuhan. With the development and spread of the new crown epidemic, the new coronavirus in 2020 has become a well-known word for the public. In 2020, the State Food and Drug Administration of approved 1,026 three-category registration certificates, and as many as 111 products for pathogenic microbial testing, of which 54 products related to novel coronavirus (2019-nCoV) have been approved.

National Health Commission issued the National Health Commission as of February 1, 2021, the capacity of single-tube nucleic acid testing has been improved to 16 million copies per day, which has increased by more than 11 times compared with 1.26 million copies per day in March 2020. This is enough to show that the field of molecular diagnosis is in a golden period of rapid development.

- Nucleic acid detection method Application in the field of pathogenic microorganisms

The basic goal of the clinical microbiology laboratory is to identify and identify pathogens such as viruses, bacteria, fungi or parasites in clinical samples, and provide other information that helps guide clinical management and even prognosis where possible, such as antibiotic sensitivity or the presence or absence of virulence factors.

So far, clinical microbiology laboratories have achieved these goals mainly through growth-based detection and biochemical testing.For example, bacteria determine antibiotic sensitivity based on their unique micromorphology, nutrients required for growth and ability to catalyze certain reactions, the cytopathic effect of viruses in tissue culture , and the micromorphology of fungi and parasites; the growth of microbial in the presence of antibiotic is determined. These technologies are reliable but time-consuming. The use of nucleic acid detection has become increasingly the standard method for clinical microbial laboratory for detection, quantification and/or identification, gradually replacing the methods of phenotypic feature identification and microscopic observation. The detection and quantification of

clinical sample-specific DNA and RNA base sequences have become a powerful tool for clinical diagnosis of bacteria, viruses, parasites and fungal infections. Nucleic acid testing has four main uses. First, it is used to qualitatively/quantitatively detect pathogens in clinical samples. Second, it is used to identify pathogens that are difficult to identify by traditional methods. Third, it is used to determine whether two or more isolates of the same pathogen are related (ie whether they belong to the same "clone" or "strain"). Fourth, it is used to predict the sensitivity of pathogens to drugs. Some of these have been approved by the National Drug Administration (NMPA) for clinical diagnosis [[2]].

There are many ways to amplify a small amount of nucleic acid molecules to easily detectable levels. These detection methods include constant temperature amplification technology (LAMP), real-time fluorescence quantitative PCR (qPCR) and sequencing methods. In any case, exponential amplification of pathogen-specific DNA or RNA sequences depends on primers annealing to the target sequence. The amplified nucleic acid can be detected after the reaction is completed, or during the amplification process (real-time detection). The sensitivity of nucleic acid amplification detection is much higher than traditional detection methods such as culture method and immunologic detection antibodies, and immunologic detection antibodies are mainly used for retrospective diagnosis. Virus culture is a more time-consuming approach compared to other methods.

With the emergence of new treatment options for HIV/AIDS -related diseases, cytomegalovirus infection, and hepatitis B and C virus infection, the response to treatment needs to be monitored by determining the genotype and viral load at different times after the onset of treatment. Quantitative nucleic acid amplification methods can be used to detect HIV (PCR), cytomegalovirus (PCR), hepatitis B virus (PCR), and hepatitis C virus (PCR and TMA). Many laboratories have validated and quantitatively analyzed these pathogens and other pathogens (such as EB virus) through analyte-specific reagents for nucleic acid amplification methods [[3]].

At present, the State Administration of Medicine has approved the detection of nucleic acid test kits of a variety of pathogens, including the detection of Mycobacterium tuberculosis , Neisseria gonorrhea, Chlamydia trachoma, Streptococcus group B and methicillin-resistant Staphylococcus aureus . At the same time, the multiple nucleic acid testing method approved by the State Food and Drug Administration can also be used to detect some respiratory or reproductive tract pathogens simultaneously (see Table 1). Likewise, many laboratories have used commercially available reagents and analyte-specific reagents for diagnostic tests.

Table 1 Summary of multi-respiratory nucleic acid detection methods approved by the State Drug Administration

product name

respiratory virus nucleic acid detection kit (constant temperature amplification chip method)

411htm RNA

3 respiratory pathogen multiplex detection kit (PCR capillary electrophoresis fragment analysis method)

Methodology	Manufacturing Enterprise	Sample type	Expected use	Expected use	Expected use ml3
constant temperature amplification chip method	Chengdu Boao Jingxin Biotechnology Co., Ltd. l3	pharyngeal swab	Novel coronavirus (2019-nCoV) S and N target genes as well as Influenza A virus , the novel H1N1 influenza A virus (2009), H3N2 influenza A virus, influenza B virus, respiratory syncytial virus nucleic acid.
influenza A/B and respiratory syncytial virus nucleic acid joint detection kit (real-time fluorescence PCR method) Xpert Xpress Flu/RSV Assay	real-time fluorescence PCR	American Saipai Company Cepheid
PCRcapillary electrophoresis fragment analysis method	sputum or throat swab	Influenza A virus (H7N9, H1N1, H3N2, H5N2), influenza A virus H1N1 (2009), seasonal H3N2 virus, influenza B virus (Victoria strain and Yamagata strain), adenovirus ( Group B, Group C and Group E), Boca virus , rhinovirus , parainfluenza virus (type 1, type 2, type 3 and type 4), coronavirus (229E, OC43, NL63 and HKU1), respiratory syncytial virus (group A and group B), metapneumonia virus, Mycoplasma pneumonia and Chlamydia (Chlamydia trachoma and Chlamydia pneumonia). Among them, the test results of adenovirus , parainfluenza virus, coronavirus, respiratory syncytial virus and chlamydia are not classified.

respiratory pathogen nucleic acid detection kit (double amplification method)

double amplification method

double amplification method

Wuhan Zhongzhi Biotechnology Co., Ltd.

pharyngeal swab

H1N1/H3N2 type of influenza A virus, influenza B virus, respiratory syncytial virus, human parainfluenza virus type 1/2/3 type B, adenovirus B/E genus, Mycoplasma pneumoniae, and Chlamydia pneumoniae nucleic acid. This product can distinguish different pathogens in the sample, but cannot distinguish different types of the same pathogen.

double amplification method

Wuhan Zhongzhi Biotechnology Co., Ltd.

pharyngeal swab

respiratory syncytial virus, human parainfluenza virus type 1/2/3, and adenovirus genus B/E nucleic acid.

Respiratory Virus Nucleic Acid Six-Related Test Kit (PCR Fluorescence Probe Method)

PCR Fluorescence Probe Method

PCR Fluorescence Probe Method

nose swab

influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, parainfluenza virus type I and parainfluenza virus type III nucleic acid.

Respiratory Pathogen Nucleic Acid Detection Kit (PCR- Fluorescent Probe Method)

PCR-Fluorescent Probe Linux Method

Shengxiang Biotechnology Co., Ltd.

nucleic acid test helps detect and identify pathogenic bacteria that are difficult to grow or cannot be cultivated, such as Legionella , Ericite, Rickettsia, Babeworm, and Borreti. In addition, in order to respond to major public health emergencies, the State Food and Drug Administration has issued the "Medical Device Emergency Approval Procedure" to implement rapid approval of pathogen testing products involving public health issues, such as H1N1, Ebola , and new coronavirus. Taking the new coronavirus detection reagent as an example, the State Food and Drug Administration issued the "2020 Medical Device Registration Work Report" showing that in 2020, the State Food and Drug Administration approved a total of 54 new coronavirus detection reagents (25 nucleic acid detection reagents, 26 antibody detection reagents, and 3 antigen detection reagents), of which nucleic acid rapid detection products include 8, forming a comprehensive new coronavirus detection product coverage system, with a production capacity of 24.018 million people per day, helping epidemic prevention and control.

Application of the new generation of metagenomic sequencing technology

metagenomic sequencing technology (metagenomics next-generation sequencing, mNGS) high-throughput sequencing nucleic acids in human clinical samples, and then compare them one by one with the pathogen database and analyze the sequence information. The types of pathogenic microorganisms contained in the sample are determined based on the sequence information obtained by the big data analysis and comparison. This technical method can detect a variety of pathogenic microorganisms (including viruses, bacteria, fungi, parasites, etc.) [[4]]. It can promote the development of diagnostic methods and discovery of new pathogens, promote the epidemiological research of infected diseases, and provide materials for infection control measures, public health response to the epidemic and vaccine development.

(I) The detection cycle is relatively short

Traditional clinical testing methods are still the main means of clinical front-line testing, but the detection cycles of various methods vary. Some can quickly obtain results, such as PCR and its derivative technology; but there are also many periods that are longer, such as traditional culture averages 3-5 days, and Mycobacterium tuberculosis/non-Myanobacterium tuberculosis requires 42 days of culture. Based on the technological innovation of detection methods, mNGS pathogen identification is a new method for clinical laboratory testing in recent years. In the clinical testing and diagnosis of severely infected patients, patients have gained treatment time, reduced the use of other tried drugs, and the comprehensive treatment cost may be lower. Compared with mNGS sequencing, millions or even hundreds of millions of DNA or RNA sequences can be measured at one time, greatly improving the efficiency of whole genome sequencing and compressing the detection cycle.

(II) Positive detection rate high

Traditional clinical detection methods, for pathogens covering bacteria, fungi, viruses, etc., the gold standard for pathogen diagnosis is still that the culture result is positive, but most pathogens in actual testing cannot be cultured and culture results cannot be obtained.Research on the application of mNGS in the diagnosis and treatment of infectious diseases has found that mNGS is more sensitive than traditional culture, and has more obvious advantages in the diagnosis of tuberculosis /fungal/virus and anaerobic bacteria . For example, some pathogenic microorganisms that are not easy to detect when used by common methods, such as Nukaci, virus, atypical pathogens that cannot be cultivated and bacteria that cannot grow after use. There are literature reports that NGS-based metagenomic sequencing has a prominent role in the detection of pathogens of abscessive lesions and has a high positive rate [[5]]. The PCR detection of , which also has high sensitivity and specificity, cannot complete the screening of multiple pathogens at one time, so the detection efficiency is not high [[6]].

(III) One-time detection coverage is wide

Conventional clinical etiology diagnosis often can only be used for detection of several target pathogens, and cannot effectively detect all pathogen microorganisms in clinical samples. For example, the mass spectrometry method has a limited number of bacterial detection; the immunology method is simple to operate, but due to the wide variety of pathogens in clinical samples, it is impossible to detect multiple antigens, and antibody detection . The new generation of gene chip technology can only target the screening of known pathogen genomes and cannot detect new unknown pathogens [[7]]. More than 2/3 of infectious diseases clinically cannot identify the infected pathogens, resulting in the inability to target medication, and the clinician's judgment of target pathogens is uneven, and trial and error situations occur from time to time [[8]].

mNGS can complete untargeted detection of samples in a short time, and thousands of pathogens can be detected in a single time [[9]].

(IV) Can identify unexpected and rare pathogens. The accuracy of unknown or mutant pathogens is difficult to identify. The current identification of unknown pathogens mainly revolves around three detections: viral antigen detection, nucleic acid detection and virus isolation and culture. Virus isolation and culture technology has defects, resulting in certain shortcomings in the accuracy of the analysis of unknown pathogens; and PCR detection methods require that the sequence of the detection of pathogens must be known.

New pathogen infections have emerged in recent years. The infection of new pathogens in SARS virus , Ebola virus , Zika virus , etc. have been emerging continuously. Traditional detection methods cannot identify unknown pathogens. mNGS can detect pathogens when there is no prior information or clinical tendency at all, and it has absolute advantages in the identification and detection of rare, rare or new infectious pathogens.

In 2017, Professor Zhang Wenhong 's team of , Huashan Hospital, Fudan University, used NGS technology to detect pseudorabies virus (PRV) in the vitreous fluid of a patient with endophthalmitis, which was confirmed for the first time that PRV can infect humans and cause endophthalmitis [[10]]. In 2016, the world's first case of mellophilus-related meningitis was discovered using NGS [[11]].

In 2015, a new Bolna virus was detected using mNGS in three patients with encephalitis . The new Bolna virus originated from mottled squirrels has been confirmed to be a pathogen of zoonotic diseases that can lead to severe lethal neurological infections in humans. For pathogens such as cysticus , Brucella , spirochete , spirochete , and , which are not common in clinical practice and are not routinely tested in most hospitals, or pathogens such as Listeria , and nukaci, which are not clinically common and have not been routinely tested, mNGS testing can avoid the shortcomings of routine examinations [[12]]. All of the above reflect the advantages of mNGS in identifying unanticipated and rare pathogenic microorganisms.

(V) Drug resistance, virulence, virus molecular typing

Popular infectious diseases such as: HIV, multiple or broad-spectrum drug-resistant Mycobacterium tuberculosis, Mycoplasma trachoma, etc., generally have strong drug resistance. Through research on drug resistance, more targeted drugs can be selected to treat patients. High-coverage mNGS can obtain drug resistance mutation information, evaluate the drug sensitivity of pathogens, and help accurately guide clinical medication.

Virulence analysis occupies an extremely important position in the prevention of infectious diseases. The severity and outcome of the infectious disease can be evaluated based on the virulence. In mNGS sequencing, currently the main characterization of highly virulent bacteria [[13]].

(VI) Elimination of infection

Negative results help clinically enhance central nervous system non-infectious etiology. For a clinical sample, when both mNGS and mtNGS are negative, and the internal reference detection value indicates that the minimum virus known to be less than 10 molecules within a unit volume (usually 1 mL) in the sample, the negative value can be considered, that is, the patient does not have infectious diseases, and may have immune diseases and tumors [[14]].

016 "Research on the etiology of infectious cerebral (membrane)itis" in the field of neurology was the first batch of national key projects for precision medicine under the leadership of National Ministry of Science and Technology and of the Health and Family Planning Commission. Professor Zhao Gang's team of Xijing Hospital of the Air Force Military Medical University, Professor Wang Jiawei's team of Beijing Tongren Hospital Affiliated to Capital Medical University, Professor Bu Hui's team of Hebei Medical University Second Hospital, and Yuguo Biology, once again demonstrated the advantages of mNGS: without prior hypothesis, change the traditional guess first and then measure, overcome the limitations of traditional targeted diagnosis; the sensitivity and specificity are higher than traditional methods; rare pathogens and special pathogens can be detected; one-time test, comprehensive coverage, wider detection range, shorter time, and higher efficiency [[15]].

(VII) Challenges faced by mNGS

mNGS method has taken the lead in clinical application of pathogen infections. However, this method still faces several challenges, which are reflected in the high testing costs, strict requirements on the experimental environment, and some test results need to be interpreted manually.

Currently, the mNGS detection service provided by most sequencing manufacturers has a sequencing data volume of up to tens of megabytes, making the sequencing cost high. How to reduce the amount of mNGS data, reduce sequencing costs without affecting accuracy, and meet the standards of mature NGS products such as NIPT ("non-invasive prenatal chromosomal aneuploidy") is a challenge to mNGS technology and application, but it will also be an industry development trend.

Since there are various large amounts of microorganisms in the environment, nucleic acid fragments of microorganisms can be detected from the laboratory air, biological reagents , consumables, etc., so unlike NGS application fields such as tumors and non-invasive prenatality, pathogenic microorganism sequencing requires a higher cleanliness laboratory environment to avoid interference with the results of background and contaminated pathogens.

Human samples, especially open cavity samples, such as alveolar lavage fluid, sputum, etc., are inevitably detected during NGS sequencing. Therefore, how to interpret and detect various microorganisms and determine which pathogens are pathogens is still a challenge for clinical practice.

Overall, mNGS has many advantages in pathogenic screening for infectious diseases. For some infected diseases that are fast-moving and onset, pathogenic microorganisms can be identified in a short time, and quickly provide a basis for clinical diagnosis. Compared with some traditional methods such as culture, the positive rate is high, and of course the negative results also have the effect of eliminating infection. It can also identify unexpected and rare pathogenic microorganisms. With the increase in data volume and the development of technology, it can even detect drug resistance and analyze virulence and virus molecular typing.

The current mNGS has a high economic cost and is unlikely to become a clinical front-line detection method in the short term, but it is still an effective broad-spectrum pathogen screening method for special populations such as fever due to unknown reasons, ineffective primary antibacterial treatment, and immunodeficiency. In addition, the use of mNGS with traditional molecular, serological detection and other methods in the detection of infection diagnosis can play a key role [[16]].

Future development trends and prospects

Just as microscopes make microorganisms visible and open the door to the world of microbiology, the technological advances in today's genomics provide microbiologists with powerful new methods that can characterize the genetic map behind all microorganisms at unprecedented resolution, thereby elucidating the complex interactions between them, their environment and human health.

genomics of infectious diseases contains a broad and active cutting-edge field, which may change clinical practice related to infectious diseases. Although genetics has always played a key role in elucidating the infection process and managing clinical infectious diseases, genomics extends our thinking and approaches from single gene research to the entire genome sequence, structure and function, and is discovering new possibilities for research and opportunities to change clinical practice.

both have unprecedented sensitivity, specificity and speed research and development diagnostic methods, and design novel public health interventions. Technical and statistical innovations in genomics are reshaping our understanding of the impact of the microbial world on human health.

References

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