mNGS Leads the Future of Microbial Testing

Infectious diseases have brought serious disease burden to human beings. Traditional pathogen laboratory diagnosis technology has the problems of poor sensitivity and specificity and long detection cycle. Infections caused by viruses and atypical pathogens place higher demands on clinical infection diagnosis technology. Limited by the limitations of microbial culture methods and the slow progress of new culture technologies, it is currently believed that less than 1% of all microorganisms have been studied through isolation and culture. The development of sequencing technology has improved the efficiency of microbiome research and accelerated the in-depth study of microorganisms.

01 Metagenomic Next-Generation Sequencing (mNGS)

Metagenomic next-generation sequencing (mNGS) is a diagnostic technology that performs high-throughput sequencing and comprehensive analysis of the genetic material (DNA and RNA) of microorganisms and hosts in samples. mNGS is changing the landscape of clinical microbiology testing and the traditional mode of diagnosis and treatment of infectious diseases.

The mNGS pathogen detection method can be divided into several stages, including pre-test, in-test, and post-test. The pre-test stage includes sample collection, transportation, storage, and processing. The test stage needs to ensure that nucleic acid is extracted from the sample without introducing contamination. The extracted nucleic acid will include host nucleic acid, pathogen DNA or RNA. For the large amount of host nucleic acid, the host nucleic acid removal operation is carried out, and then the library construction and machine sequencing steps are carried out. Finally, the sequencing results are automatically analyzed and the results are interpreted through manual interpretation.

02 Applications of mNGS

A large number of published case reports and clinical studies have shown that mNGS has been successfully applied to dozens of sample types, including cerebrospinal fluid (CSF), respiratory secretions, feces, urine, blood, and tissues. It has shown great advantages in diagnosing complex and serious infections, such as those associated with bloodstream infections, respiratory infections, bone and joint infections, and encephalitis.

When the following clinical indications occur:

(1) Fever of unknown cause or fever syndrome;

(2) Infection is not excluded in critical and severe cases;

(3) Immunocompromise with suspected secondary infection;

(4) Suspected local infection, if the etiology is not clearly diagnosed and not treated in time, will have serious consequences;

(5) Highly suspected infectious diseases, for which conventional anti-infective treatment is ineffective;

(6) Chronic infection or chronic disease does not exclude infection;

(7) Food poisoning, drowning, travel, and special environments (not excluding infectious diseases).

It is recommended to conduct mNGS at the same time as conventional testing. However, mNGS is not suitable for evaluating the effectiveness of anti-infection treatment, and conventional microbiological examinations can easily identify pathogen infections.

When the following microbiological indications occur:

(1) The emergence of suspected emerging pathogens or special pathogens;

(2) Traditional detection methods cannot detect microorganisms but there are indeed signs of microbial infection;

(3) Considering multi-pathogen infection, traditional technical means can only identify part of it.

It is recommended to conduct mNGS at the same time as conventional testing. When the pathogen of an infectious disease has been identified or a pathogen is under consideration and information about virulence factors is needed, mNGS species identification combined with mNGS detection of virulence genes can be considered.

03 Specimen Collection for mNGS

Basic requirements for specimen collection: (1) Specimens should be collected in a strictly sterile manner to avoid contamination; (2) Nucleic acid stabilizers should be added to the specimens; (3) Specimens must be transported to prevent contamination and use a cold chain for rapid transportation; (4) If specimens cannot be tested in time, they should be stored in a refrigerator below -80°C or in liquid nitrogen.

04 The Promise of mNGS

Numerous studies have shown that mNGS appears promising for strain-level typing, antimicrobial resistance (AMR) detection, virulence analysis, and phylogenetic relatedness of detected pathogens. More and more physicians recognize it as a last resort to solve infection problems.

Although mNGS testing still faces challenges, including high costs, long reporting time, low sequencing depth, the current testing process has not been standardized (threshold, background bacteria, etc.), the quality assurance system is not perfect, the report is not standardized, the interpretation of the report is often out of touch with the clinic, and the combination with traditional methods needs to be strengthened, etc., mNGS technology is a very promising technology. In terms of clinical research and scientific research, it still needs to continue research and evaluation so that it can provide more valuable services for the clinic.

References:

[1] Wang Zhenqi, Bai Jingwei, Zhao Xiuying. Research progress of metagenomic next-generation sequencing technology in clinical applications[J]. Beijing Medical Journal, 2023, 45(3): 243-248.

[2] Li Wei, Zhuo Chao, Xu Jiancheng, et al. Application of mNGS in laboratory and clinical practice[J]. International Journal of Laboratory Medicine, 2023, 44(1): 1-7.

[3] Chinese Medical Association Laboratory Medicine Branch. Expert consensus on standardized management of bioinformatics analysis of metagenomic sequencing pathogenic microorganism detection[J]. Chinese Journal of Laboratory Medicine, 2021, 44(9): 799-807.

[4]Han D, Li Z, Li R, Tan P, Zhang R, Li J. mNGS in clinical microbiology laboratories: on the road to maturity. Crit Rev Microbiol. 2019 Sep-Nov;45(5-6):668-685.