Today, it mainly introduces the current status and development direction of rapid respiratory virus detection technology. It is difficult to define "fast detection", which means getting the results within "two hours", or does it take "one day" to get the results? Because for some rare pathogens, rare pathogens or new pathogens, it is difficult to make a clear diagnosis in a few hours, and it requires more complex technology and longer time to confirm. The "fast detection" mentioned below refers to the technology that produces faster results than traditional methods. I may have also been involved in
related content on different occasions, and I am very honored to share it with you again.
respiratory infectious diseases
respiratory tract is connected to the outside world. Viruses, bacteria, fungi, parasites, etc. in the environment can cause respiratory infection through inhalation, mainly upper and lower respiratory tract infection. In recent years, we have paid more attention to the virus, especially since the COVID-19 epidemic that has continued to the present day at the end of 2019. People are particularly concerned about infectious diseases in respiratory tract or infectious diseases in other parts of the virus, such as the influenza virus , adenovirus , which causes respiratory tract infection, and the digestive tract virus norovirus that has caused hospital infection. In addition, human colonized viruses, such as certain coronaviruses, rhinoviruses, , cytomegaloviruses, etc., may also cause respiratory infections. It is precisely because the types of respiratory pathogens are relatively complex that the clinical symptoms caused are clinically indistinguishable from some bacterial and fungal infections. This requires some laboratory technology to detect pathogens and clarify the type of pathogens in order to clearly determine the diagnosis and determine the next step of clinical treatment.
Guidelines for diagnosis and treatment of adult community-acquired pneumonia in China (2016 edition)
2016 edition CAP Guidelines mentioned that The virus detection rate in adult CAP patients in my country is 15%-34.9%, and influenza virus ranks first. Other viruses include parainfluenza virus, rhinovirus, adenovirus, human metapneumonia virus and respiratory syncytial virus. 5.7%-65.7% of patients with positive virus tests may be associated with bacterial or atypical pathogen infection. For children, respiratory syncytial virus accounts for a large proportion of the virus, and Mycoplasma pneumoniae is the main one among the atypical pathogens. Therefore, as the laboratory department or microbial laboratory of pediatric hospitals, the detection of these special viruses or atypical pathogens is also very critical.
In addition, I would like to remind everyone that with the improvement of detection technology, we now find that for Chlamydia pneumoniae, the probability of infection with Chlamydia pneumonia is not very high, and the proportion of Chlamydia psittaphorus infection in is increasing, which is often related to the feeding history of birds. For Chlamydia, Chlamydia trachoma should not be simply detected in Chlamydia pneumonia, neonatal, and Chlamydia trachoma are very important pathogens; for patients who are frequently exposed to respiratory infections in avian birds, Chlamydia psittisca has become a very important pathogen. It is recommended that manufacturers design reagents based on different epidemiological spectrums and the main pathogen types of infection, especially multiple detection reagents. Clinical laboratories should also use this as a basis to select relevant reagents.
clinical microbial pathogen detection methodology comparison
The figure above shows it from a review of CRITICAL REVIEWS IN MICROBIOLOGY (DOI: 10.1080/1040841X.2019.1681933), telling us what are the main clinical microbial detection methods currently included. Molecular biological methods and antigen detection methods are the most commonly used methods to detect viruses, and are recommended in foreign and domestic guidelines. Many guidelines do not recommend using the rapid antibody detection method to diagnose respiratory pathogenic infections, such as adenovirus infection or influenza virus infection. Detection of antibodies, whether it is IgM or IgG, a single test is not very valuable. The main feature of antigen detection is good specificity, but the sensitivity is slightly poor. Under the premise of suitable sampling, the sensitivity will be between 40% and 70% depending on the patient's viral load, and different literature reports have certain differences.In addition, after many bacteria invade the human body, the degradation products enter the blood and eventually appear in our urine. Then, the urine can detect related pathogenic infections. For example, when Streptococcus pneumonia is infected, the polysaccharide component of the bacterial wall of Streptococcus pneumonia is detected in urine, or when Legionella (serum type 1) is infected, Legionella urinary antigen is detected. The sensitivity of the urinary antigen detection of Streptococcus pneumoniae is 50-80%, and the specificity is above 90%. The sensitivity will be higher during severe infection, but if the pathogen load is small, the sensitivity will be low if the infection is low or the infection stages. At present, there is no suitable urinary antigen detection target for respiratory virus infection. The main nasopharyngeal swabs are collected to detect antigens, including influenza virus, adenovirus, or coronavirus. Antigen detection is suitable for use in situations where pathogen load is relatively high.
and molecular biological method . According to the number of targets, PCR method can be divided into multiple PCR or single PCR. The future development direction is mainly multiple PCR, at least two PCR. At present, there are not many problems with conventional molecular reagent companies in designing products for two or three targets. However, we hope to target more pathogens in the future. That is, after obtaining a sample, it can conduct rapid testing of multiple pathogen targets in a short period of time, and realize the "rapid detection" mode of "sample in, report out" , and complete 10, 20 or even more pathogen testing within two hours. I think this is a very important development direction. Multiple detection can reduce detection costs. Of course, for some rare pathogens or extremely rare pathogens, it is impossible for the laboratory to design reagents specifically for this pathogen, which is not cost-effective in terms of economic benefits. So in these cases, doing metagenome sequencing will be more valuable. At present, dozens or even hundreds of primers can be added to a system, amplified first, and then sequenced. This is called tNGS. In theory, this method is also very sensitive and may be reduced in the future, but it has not been widely used in clinical testing.
When it comes to molecular biology methods, we always think of PCR method, metagenomic sequencing and other methods. In fact, there are many methods of molecular biology, such as constant temperature amplification technology, gene chip , etc., but these applications are relatively less than fluorescence quantitative PCR and metagenomic sequencing. In recent years, constant temperature amplification technology has gradually increased. PCR must have a process of repeated heating and cooling, which has an impact on the reaction speed and also has high requirements for instruments and equipment. The constant temperature amplification removes the repeated rise and fall process, which increases the speed, but the sensitivity and specificity of constant temperature amplification will generally be lower than that of PCR. There is also the CRISPR technology discovered by Professor Zhang Feng from the United States, which is now slowly being applied to the field of molecular biological detection. It has high sensitivity and may become a very important direction for molecular biological detection in the future.
Pathogen detection technology development - nucleic acid detection
For the traditional microbiological method , virus culture methods are now less and less used, and will be used when some specialized scientific research institutions and CDC departments need to carry out live virus research. Because virus culture requires higher hardware conditions and personnel protection in laboratories, it is difficult to maintain conventional front-line clinical laboratories in terms of labor costs and economic benefits. Later, it slowly transitioned to immunology method , which were divided into antigen detection and antibody detection. As mentioned earlier, antibody detection is not suitable for routine respiratory virus detection and clinical diagnosis, and is more suitable for retrospective analysis. Antigen detection is simple and easy to use, and has other advantages. Molecular Biology Method , special design of different primers for related amplification according to DNA virus or RNA virus.
Novel coronavirus antigen detection
This is a recent article published in China. That is, antigenic methods are used to detect novel coronavirus infection. Some countries in Europe and the United States welcome antigen detection methods. They are very fast and simple to operate. The disadvantage is that their sensitivity is low. We have never used such a test reagent because there are fewer positives in China.In some high-incidence areas, antigen detection has certain value and can be used for the initial screening of suspicious people.
Colloidal gold method cannot be used as the diagnosis standard for negative virus infection
Why we have always said that antigen detection cannot be used as the diagnosis standard for negative virus infection? It is because the sensitivity is insufficient. For example, the patient is indeed infected with influenza virus, adenovirus infection or respiratory syncytial virus infection, but if the pathogen load is low, or the sampling is a pharyngeal swab is not taken, or the corresponding part is not taken during sampling, it should have entered from the nasal cavity, and the degree of entry should be deeper. When it reaches the position of nasopharyngeal , the swab is actually lighter, and the nasal swab is taken. Or the course of the disease is long. All of the above situations will greatly reduce the positivity rate. Therefore, if the antigen test result is negative and the patient's symptoms are similar, it cannot be ruled out. It is recommended to use more sensitive molecular biological detection methods. There are still certain differences in the sensitivity of different molecular biological detection methods, and there are quite a difference between different manufacturers, so we recommend that laboratories should make corresponding evaluations before using a certain reagent.
influenza virus
Before there is no new coronavirus, we pay special attention to the flu virus every year. Every autumn and winter, our laboratory detects a considerable number of influenza A and B viruses every day. Due to the COVID-19 pandemic, people have begun to wear masks and take personal protection measures. The flu has basically become "extinct" from 2020 to early 2021. It can be seen that this method of controlling the new coronavirus is very effective in controlling influenza and controlling the epidemic of respiratory syncytial virus or adenovirus.
respiratory syncytial virus (RSV)
respiratory syncytial virus (RSV) is mainly seen in some elderly people with relatively low immunity and preschool children. RSV accounts for 42-45% of the causes of lower respiratory tract infection in children under 2 years old, especially newborns and babies within 6 months. Generally speaking, the symptoms are relatively mild, mainly respiratory symptoms such as cough and wheezing, which can cause interstitial pneumonia, bronchiolitis, etc. If the condition is serious or recurring, it can develop into bronchial asthma, leading to respiratory failure, etc. When RSV is infected with other respiratory viruses, it can cause serious consequences.
lancet : The virus dominated by RSV has become the main cause of pneumonia in severe childhood in low- and middle-income countries
This is a multi-center study spanning seven countries in two continents of Asia and Africa. It has been the largest and most comprehensive study of its kind since 1980. It was published in the 2019 journal The Lancet. Among the children: pneumonia caused by respiratory syncytial virus accounts for 31.1% of all cases, while bacteria accounts for only 27.3%, Mycobacterium tuberculosis accounts for 5.9%, and the rest are fungi and other pathogens. This document tells us that RSV has become a very important class of infected viruses, especially in children. Therefore, as a children's hospital, it is recommended that at least one method of detecting respiratory syncytial virus, which can use antigen quick detection reagents, or molecular biological detection methods. We should not focus all our energy on the new coronavirus. In fact, other viruses will still have a certain impact on us.
Antigen detection of respiratory syncytial virus is not necessarily very sensitive, and some people cannot detect it, especially when the pathogen load is relatively low. It is recommended to use nucleic acid testing methods at this time.
As shown in the figure above, if some companies prepare their own reagents, they can consider influenza A/B and respiratory syncytial virus detection together.
As shown in the figure above, in addition to common viruses, pertussis, chlamydia, legionella, etc. can also be included for multiple rapid pathogenic testing. The test results are related to the pathogenic load. If the pathogen load is large, conventional methods will get positive results; but if the pathogen load is very small, such as the CT value of PCR detection is more than 30 cycles, the detection result is not necessarily very accurate at this time, and the sensitivity may decrease, so you need to pay attention.
Traditional pathogenic detection methods still have clinical insecurity needs
mNGS, WGS and tNGS applications
Metagenome sequencing has been widely used in recent years. During the discovery of the new coronavirus, it depends on the use of metagenomic sequencing. In other words, I have always emphasized that for rare pathogens, new pathogens, or travel-related infections that are infected by traveling abroad, it is difficult for laboratories to design detection methods for this type of pathogen, and it does depend on metagenomic sequencing.
In fact, genome sequencing does not necessarily require metagenomic sequencing. There are other methods. As shown in the figure above, WGS is mainly used to sequence a certain pure colony to detect its virulence characteristics, drug resistance characteristics, plasmid distribution, evolution, homology analysis, etc. For example, for mycobacterium, it takes a long time to do drug sensitivity experiments and requires special conditions. However, WGS genome sequencing can be performed to detect drug-resistant genes. Most of their drug resistance is caused by point mutations, which matches the results of the real drug sensitivity experiments well.
and tNGS, which refers to targeted sequencing, first amplifying the fragments that need to be detected, and then sequencing them. For example, if I only care about the bacteria in the sample, I can amplify the 16s fragment first and then sequence it. If you are concerned about fungi, you can amplify the ITS fragments and then sequence them. You can also add dozens or hundreds of pathogen primers that I care about to this system, and then sequence them after amplification. This advantage is that it does not require testing the human genome, reduces costs and improves sensitivity. When we do mNGS, a considerable portion of the sequencing is actually consumed on the human genome.
The above figure shows that a domestic scientific researchers published an article in JCM some time ago. He is doing the classification of non-tuberculous mycobacterium. He first amplifies many sequences before sequencing them. Using tNGS is also a very important development direction.
There is one thing I want to remind everyone that we do virus testing not all have clinical value. As shown in the above figure, some viruses are prone to appear in asymptomatic infected people, including rhinovirus, some coronaviruses, etc., but influenza viruses rarely appear in asymptomatic patients. Interpretation of the results means that we need to look at the type of pathogen and also pay attention to the clinical situation.
The above figure shows, this is an article summarized by the China-Japan Respiratory Center some time ago. It was found that if respiratory syncytial virus is infected with influenza A or influenza B, the mortality rate of patients increases. This tells us that during clinical testing, we cannot only focus on one pathogen. One virus can cause another bacterial infection after infection, such as Streptococcus pneumoniae or Staphylococcus aureus, or two viruses are infected at the same time. This situation may occur. If another pathogen infection is ignored, the treatment may be biased and may cause serious clinical consequences.
summary
Viral infections in children and adults cannot be ignored. Many microorganisms have neglected this aspect of viral infection. Viral pneumonia in people with low immunity should attract our attention. With the application of genome sequencing technology, viral infection has gradually become known to everyone. Rapid multiple PCR detection is an important direction for future development, and I hope that reagent companies can understand clinical needs. In addition, although metagenomic sequencing is developing, it has unique advantages in diagnosing new viruses or rare respiratory virus infections and multiple infections, and we should recognize its advantages and disadvantages. Different NGS sequencing will gradually be applied to more scenarios, providing clinicians with more convenience.
Online Q&A discussion
Host Wang Yimin: For the rapid detection of bedside pathogenic etiology (M-ROSE) technology, the M-ROSE technology I understand is the technology of putting a microscope on the bedside and quickly detecting specimens on the bedside. Whether it is through traditional Gram staining or adding some Reynolds staining for cytology, to see the relationship between cells and bacteria, to judge whether the pathogen is pathogenic, contamination, or colonization, in addition to the detection technology, have there been any improvement in these aspects?
Lu Binghuai: M-ROSE technology is a relatively popular technology in recent years. After obtaining the samples in clinical practice, the samples are stained quickly and quickly. Recently, I know that some companies are doing automatic scanning and identifying them through artificial intelligence to roughly distinguish corresponding cells and bacteria. I found that most of the clinical observations are under high power microscope (400 times). In fact, if you look under high power microscope, you can only see a rough impression, just a simple speculation. Clinical microbiology laboratories are used to use oil mirrors (1000 times) microscopy. If the sample has a large number of bacteria or fungi, it is OK to use a high-power microscope; but when the number of pathogens is small, it can only be inferred under a high-power microscope.
The main development direction in the future, fast multiple PCR detection should be a very important direction. For example, the patient is a very serious infection caused by streptococci . Sometimes Streptococcus pneumoniae cannot be seen under the microscope. After using the drug, Streptococcus has disappeared, but its infection is very serious. M-ROSE and laboratory microscopy are fast detection methods with low sensitivity. For example, for Mycobacterium tuberculosis, the positive rate detected by microscopy will not exceed 30%. Currently, Xpert is more sensitive to mycobacterium tuberculosis. I think moving the rapid diagnosis forward to the bedside, the patient immediately obtains the sample and goes to the test immediately, which is a very good development direction. First, if such experiments can be carried out, it shows that clinical attention is given to pathogenic testing; second, it is conducive to the rapid development of artificial intelligence morphology, and this development may only occur when there is demand, and this combination of technology is very promising.
Host Wang Yimin: Some colleagues ask you about nucleic acid testing issues. Now the new coronavirus is because we may consider the risks of environmental exposure and infection, and more about collecting oropharyngeal swabs or nasopharyngeal swabs. However, for other pathogens, sputum may be collected or alveolar lavage fluid may be performed. So, can other pathogens be tested just like the nucleic acid test for the new crown? Can it be simpler and only oropharyngeal swabs or nasopharyngeal swabs be taken?
Lu Binghuai: For many respiratory viruses, taking nasopharyngeal swabs is a good way. Because relatively few colonization of these viruses (such as influenza virus and adenovirus) in normal people, it can be roughly speculated that the patient has related infections. However, this is not the case for bacteria and fungi. For example, there may be a large number of colonization of Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae in the nasopharynx. The above samples collected in the nasopharynx of the respiratory tract contain bacteria and fungi to reflect infection in the lower respiratory tract. No matter how good the technology is, sampling is the most important prerequisite. The best sampling method is to collect samples at the closest location to the infected site. Therefore, for pneumonia, even if it is a viral infection, I believe that the detection sensitivity of alveolar lavage fluid will be higher than that of nasopharyngeal swab. There is another reason. Most of the new coronavirus is done now for screening, rather than the patients who have really developed symptoms of lung infection. For most respiratory virus infections, if there are lung symptoms, the nasopharyngeal swab may also be negative, and the best choice is to collect alveolar lavage fluid. For example, a respiratory doctor previously suspected that a patient had tuberculosis infection. After coming to the hospital in the morning, he collected sputum samples for sputum test, and the result was negative. In the afternoon, he happened to have an alveolar lavage fluid, which was positive. Therefore, the closer the samples collected at the infection site, they must reflect the real pathogen of the infection. It is absolutely impossible to say that the special situation of collecting nucleic acids for the new coronavirus for large-scale screening of the population is extended.
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This article was edited by "Breathing Realm" by Da Ben. Thanks to Mr. Lu Binghuai for review and modification!
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