How is mNGS used in infectious diseases? This is the best interpretation I've ever seen!

Infectious diseases refer to local or systemic inflammation or organ dysfunction caused by bacteria, viruses, fungi, atypical pathogens, parasites, etc.
and their products, which have great harm and high case fatality rate, and are common diseases
that seriously threaten human health in today's world.


Pathogenic microbial detection is an important method for diagnosing infectious diseases, and at the same time has very important guiding significance
for treatment options.
In recent years, the emerging metagenomics second-generation sequencing (mNGS) detection method has effectively improved the detection rate and made up for the shortcomings of traditional detection methods, but at the same time, mNGS in practical applications also has been exaggerated and deified, which stems from too many people's half-knowledge of mNGS, I hope this article can help you improve your understanding of mNGS and correct application
.

Author: Wang Chengsheng Danzhou Municipal People's Hospital Affiliated to Hainan Medical College Department of Infectious DiseasesThis
article is authorized by the author to publish the medical pulse pass, please do not reprint without authorization!

1

The advantages of mNGS over traditional pathogenic detection methods

The traditional pathogenic microbial identification methods mainly include smear microscopy, isolation and culture, biochemical reaction and mass spectrometry, but its shortcomings such as long cycle, complex process and low sensitivity, etc.
, have limitations in sensitivity, specificity, timeliness, information volume, etc.
, such as mycobacterial identification takes up to 30 to 40 days, while some caustic bacteria and viruses require extremely harsh culture conditions, and even can not be cultured, and for unknown or rare pathogenic microorganisms can not be quickly identified
。 According to statistics, about 70% of patients with infectious diseases cannot determine the pathogen information due to traditional detection methods, and cannot be treated in a timely and effective manner, thus worsening
their condition.
Metagenomics second-generation sequencing (mNGS) does not require isolation and culture of pathogens, nor does it rely on known nucleic acid sequences, and can directly and quickly and objectively identify more pathogenic microorganisms (including viruses, bacteria, fungi, parasites) in clinical samples, without specific amplification, which greatly saves detection time, improves diagnostic efficiency, and has an incomparable advantage
in the identification of unknown species and pathogens that are difficult to cultivate 。 Traditional isolation culture methods usually only produce one microorganism, when there is a mixed bacteria (including mixed different types of microorganisms) infection usually miss detection, when the colonization bacteria exceed the load of pathogenic bacteria, usually only the colonization bacteria are cultured and cause missed detection and misleading clinical, while mNGS can detect a variety of microorganisms (including mixed bacteria and different types of microorganisms
).
。 Therefore, the rapid, specific and high-throughput pathogen detection method (mNGS) is of great significance for the effective diagnosis and timely prevention and treatment of infectious diseases, especially under the current global incidence of infectious diseases continues to rise, and the development trend of pathogens showing diversification and complexity, mNGS has been greatly promoted and popularized
.

2

The importance of mNGS is insufficient
.

From the above content, mNGS is a very good technology and detection method, and has obvious advantages over traditional detection methods, how does the author think it is exaggerated and deified? First of all, we must understand the main reason and ultimate purpose of the pathogenic test.
It is to find the causative agent, and to understand which drugs it is sensitive to and resistant to, so as to help us select drugs more accurately and assess the condition and prognosis
。 Many people mistakenly think that mNGS can do everything, thinking that with mNGS you can find pathogens and know what anti-infective drugs to use, this deification is actually a "lack of awareness" that is blindly produced by mNGS - in fact, unfortunately, mNGS can only detect a large number of microorganisms including parasitic bacteria, colonization bacteria, background bacteria and pathogenic bacteria, but can not directly tell you which is the pathogen, there is no drug susceptibility results, can not tell you which drug is sensitive, which drug resistance, Therefore, you can not blindly and rely too much on it, and it is expensive (at least thousands of yuan), especially the test project in many areas need to pay for themselves, medical insurance is not reimbursed, for ordinary people, this amount is a large amount and burden
.

3

How to apply mNGS correctly?

mNGS can not directly tell us which is the pathogen, nor can it tell us what drugs it is sensitive to, what drugs are resistant, out of the clinic, costing tens of thousands of yuan of mNGS test results are a waste of paper - but mNGS is by no means useless, at critical times it may play a very important key role, or even save the "last straw", so we must learn to apply
correctly 。 MNGS has many advantages over traditional pathogenic detection methods, but there are also some important (and even critical) deficiencies, so it is necessary to pay attention to the following matters and relevant expert consensus recommendations: (1) mNGS can not completely replace the conventional pathogenic detection program, and it needs to be used in conjunction with traditional methods to improve the sensitivity and specificity
of pathogen diagnosis.
(2) Because of the high cost of mNGS testing and its own shortcomings, it cannot be used as a first-line detection method for mild infectious diseases, let alone as the first choice
.
(3) For patients with focal infection, routine infection indicators and traditional pathogenic detection should be completed first, if the pathogenic diagnosis results have not been obtained, and the clinical treatment effect is poor, mNGS can be used as the second-line preferred detection method
.
(4) For patients with respiratory infection, if there is still no clear etiological basis through traditional laboratory tests within 3 days and empiric anti-infection therapy is ineffective, it is recommended to retain respiratory specimens for mNGS
.
(5) For patients with sepsis, it is recommended to use a combination of traditional laboratory culture and mNGS to improve the detection rate
of pathogens.
(6) For patients with high suspicion of viral pneumonia and continuous progression, multiple PCR detection of respiratory virus can be improved first, and if it is negative, mNGS can be performed, and nucleic acid RNA reverse transcription
should be carried out at the same time.
(7) For patients with new, rare and difficult infectious diseases and immunodeficiency, mNGS can significantly improve the detection rate of pathogens, so mNGS can be used as a first-line detection method
for the above diseases.
(8) For patients with severe illness, critical illness or immunosuppression and immunodeficiency who are clinically suspected (and supportive) of infection, because it is extremely important to clarify the pathogenic results early, it is recommended to collect specimens of suspected (and supportive) infection sites for mNGS
while improving traditional laboratory and molecular biology testing.

4

What should I pay attention to when interpreting mNGS results?

Correct interpretation of test results is an important premise for correctly guiding the clinic, mNGS is not omnipotent, it has many limitations and influencing factors, so in the face of mNGS results, it should be closely combined with the patient's clinical manifestations and laboratory tests under the premise of interpretation and verification, especially to pay attention to the following knowledge points: (1) mNGS detected pathogens can only represent the presence of these detected microorganisms in the specimen, but it cannot be determined whether it is colonizing bacteria, reagent engineering bacteria, background bacteria or pathogenic bacteria, Therefore, it is necessary to understand the normal microecological flora of the collection site of the specimen, as well as possible conditional pathogenic bacteria and colonization bacteria, and also pay attention to the exclusion of reagent engineering bacteria and background bacteria
.
(2) mNGS alone can not directly determine whether there is infection and its pathogen, it is necessary to combine clinical data, such as current symptoms and signs, commonly used infection indicators (such as blood routine, C-reactive protein, procalcitonin, erythrocytes, etc.
) and imaging whether there is an infectious basis
.
(3) For patients with focal abscess or tissue infection, the overall sensitivity and specificity of mNGS are better, and the sensitivity of bacterial detection is better than that of fungi
.
(4) For respiratory infections, mNGS shows good detection efficacy in the detection of viruses and rare pathogens; However, in the detection of pathogens such as bacteria and fungi, mNGS cannot accurately determine the colonization or infection status of the microflora, and it still needs to rely on clinicians to conduct further analysis
in combination with the patient's condition.
(5) mNGS detection capacity is not omnipotent, intracellular infectious bacteria (tuberculosis bacillus, legionella, brucella, etc.
) due to the release of less content into the body fluid and resulting in low
detection sensitivity.
In addition, pathogens with thicker cell walls, such as fungi, have lower nucleic acid extraction efficiency, resulting in lower
detection rates and sensitivity.
(6) mNGS has a low detection rate for intracellular bacteria and thick-walled microorganisms, so even if the detection sequence number of some / some intracellular bacteria / thick-walled bacteria is not high, it is necessary to consider the possibility
of pathogenic pathogens.
(7) The mNGS test report indicates that a certain microorganism has a high number of detected sequences and high genomic coverage, indicating that the pathogenic microorganism has been detected; In the case of exclusion of background, contaminating and colonizing bacteria, the microorganism may be considered to be a pathogenic pathogen
.
(8) RNA transcription itself has higher abundance and complexity, and is easy to degrade, and has higher requirements for transportation and preservation, so there are still certain difficulties
in the clinical detection of RNA viruses by mNGS.
Common RNA viruses are HIV, hepatitis C virus, Japanese encephalitis virus, influenza virus, rhinovirus, poliovirus, coxsackievirus, dengue virus, rotavirus, SARS virus, Ebola virus, novel coronavirus, etc
.
When clinically infected with this type of virus, the role of mNGS is likely to be unexpected
.
(9) For patients whose mNGS results are negative but are highly suggestive of infection based on other auxiliary test results (such as culture results) and infection cannot be ruled out, it is recommended to repeat mNGS testing
again if necessary.
(10) mNGS information is large, it is impossible to list all detected pathogens in the report, for rare pathogens, intracellular bacteria, etc.
, may be due to the small number of detected sequences, low microbial abundance, failed to list in the report, if there is a suspected infection of special pathogens in the clinic, you can trace the original database for query
.
(11) The current mNGS can not fully guide the selection of anti-infective drugs of drug-resistant bacteria, and the information obtained by microorganisms needs to be combined with the clinical characteristics of patients, the epidemiological data of local bacterial resistance, and the susceptibility results of traditional pathogenic culture to help guide the selection of
antibacterial drugs.
References:[1] Expert consensus group on the application of metagenomic analysis and diagnostic techniques in acute critical infections
.
Expert consensus on the application of metagenomic analysis and diagnostic techniques in acute and critical infections[J] .
Chinese Journal of Emergency Medicine,2019,28( 2 ): 151-155.

[2] Editorial Board of Chinese Journal of Infectious Diseases.
Expert consensus on the clinical application of Chinese metagenomics second-generation sequencing technology for the detection of infectious pathogens[J] .
Chinese Journal of Infectious Diseases,2020,38(11): 681-689.
DOI: 10.
3760/cma.
j.
cn311365-20200731-00732.

[3] Laboratory Medicine Branch of Chinese Medical Association.
Expert consensus on the standardization of clinical application of high-throughput metagenome sequencing technology for the detection of pathogenic microorganisms.
Chinese Journal of Laboratory Medicine, 2020,43(12): 1181-1195.