1. Changes in the pathogenic spectrum of community-acquired pneumonia (CAP) before and after the epidemic
1, respiratory tract infection symptoms and causes
pathogenic microorganism invades the respiratory tract and reproduces is called respiratory tract infection. According to its location, it is divided into upper respiratory tract infection and lower respiratory tract infection .
literature reports that 70-80% of pathogens of upper respiratory tract infection are viral; bacterial pathogens account for 20-25%.
lower respiratory tract infection symptoms include pneumonia , bronchitis and bronchitis , etc., which are mainly caused by infections such as bacteria, Mycoplasma , Chlamydia , Legionella, viruses, etc. The bacteria of CAP mainly include Haemophilus influenzae , Streptococcus pneumoniae , and Moraxella cataracta . In recent years, the proportion of infection of Klebsiella pneumoniae has gradually increased.
2, common pathogens of respiratory infection
Common pathogens of respiratory infection are divided into viruses, bacteria, fungi, mycoplasma, chlamydia, etc. (Figure 1).
viral pathogens are divided into RNA virus and DNA virus. Unlike DNA virus detection, RNA viruses undergo PCR detection. is first reverse transcribed into cDNA, and then PCR detection is performed.
RNA viruses include influenza virus (A/B), parainfluenza virus, respiratory syncytial virus , coronavirus, etc. Influenza virus has always been the focus of our attention, and respiratory syncytial virus is more likely to cause infections in infants, children, and the elderly. Coronaviruses like SARS and SARS-CoV-2 (COVID-19) have had serious impacts on global politics and economy, and should not be underestimated. Common coronaviruses include 229E, NL63, OC43 and HKU1, which are the main pathogenic microorganisms of common cold . The symptoms of infection caused by mild symptoms, healthy people have a certain degree of resistance to it and often do not receive special attention. Of course, serious infection with the common coronavirus can also cause death. In 2022, I have seen 2 patients, including obesity and diabetes (over 65 years old), who died of common coronavirus infection. The application of mNGS technology and multiple PCR technology has made our understanding of coronavirus more profound.
DNA viruses include adenovirus , cytomegalovirus , EB virus, etc. Adenovirus can cause severe infections, and there are also cases where adenovirus is detected in healthy people. Cytomegalovirus and EB viruses are infected in infants and young children. As patients get older and unexpected situations occur, such as transplantation, hematologic diseases, infection with HIV and tumors, the latent/recessively infected cytomegalovirus and EB virus may "rekind", causing very serious infections. The lung transplant department is very concerned about infections caused by cytomegalovirus.
bacterial pathogens are divided into morphologically: cocci and bacillus , and are divided into Gram-negative bacteria and positive bacteria according to the characteristics of Gram staining. Streptococcus pneumoniae in
gram-positive bacteria is an important pathogen that causes CAP. This bacteria is difficult to cultivate and is prone to death due to unreasonable delivery or storage. The test is negative. Streptococcus pneumoniae can cause severe infections, and it is difficult for patients to detect it after using antibiotic . Therefore, the proportion of CAP caused by Streptococcus pneumoniae tends to be underestimated. Using molecular biology to detect Streptococcus pneumoniae is a good choice. Klebsiella pneumoniae and Haemophilus influenzae have a higher chance of causing CAP. Outpatients with Pseudomonas aeruginosa infection may be more likely to be seen in patients with bronchodilation and chronic obstructive pulmonary disease (COPD). CAP caused by Acinetobacter baumannii is very rare. The above bacteria may also be colonized in normal populations, and detection may not necessarily mean infection, especially if the sample is not qualified to be retained. Respiratory tract sample smears often help distinguish infection from colonization.
Legionella can cause very serious infections. It is recommended that Legionella use molecular biological detection schemes, such as PCR or multiple PCR technology, etc. It is not recommended to use antibody to detect methods. It is also difficult to cultivate, takes time and requires special culture media.
fungal pathogens mainly include yeast, Aspergillus , mucoriasis, etc.
Other atypical pathogens include Mycoplasma pneumoniae, Chlamydia pneumoniae , Chlamydia psilocytica and Rickettsia. It is worth noting that Chlamydia psittaci. In fact, Chlamydia psittaci has been mainly diagnosed through sequencing in recent years. Some molecular biological reagents in clinical practice can also detect Chlamydia psittaci. With the widespread application of molecular biological detection technology and metagenomic sequencing (mNGS) technology, we found that the infection rate and detection rate of Chlamydia psittaci in our laboratory have exceeded Chlamydia pneumoniae and have become a very important type of pathogen. Psittaci disease can cause serious clinical infections, and the proportion of patients entering ICU is also quite high.
In addition, new pathogens are constantly emerging: such as SARS virus , highly pathogenic avian influenza, human infection with H7N9 avian influenza, novel coronavirus (SARS-CoV-2), etc.
Figure 1 Common pathogens classification
3, research on viral infection in patients with CAP
3 Chinese immune function is normal for adults to obtain viral pneumonia the main reason for influenza virus infection , followed by non-influenza virus infection.
influenza and non-influenza virus infection account for 63.5% and 26.2% of adult CAP patients with normal immune function in my country. Analysis of patients with single viral infection also confirmed that influenza virus infection is the main cause of viral pneumonia, followed by non-influenza virus infections such as adenovirus and respiratory syncytial virus (Figure 2) [1].
Figure 2 Research on viral infection in adult CAP patients with normal immune function in my country
Respiratory virus is an important cause of CAP disease
In the past 20 years, the outbreak of various infectious diseases has highlighted the importance of viruses in the onset of CAP. Such as SARS virus, MERS virus and avian influenza virus have appeared one after another, and new viruses will continue to emerge in the future (Figure 3).
Figure 3 Outbreaks of various infectious diseases in 2002-2019 [2-6]
4. Before and after the epidemic, the pathogen spectrum of CAP was changed
Before the epidemic, the pathogen species were relatively stable
2021 The National Center for Disease Control and Prevention (CDC) published an epidemiological survey study, investigating the number of pathogenic species in four sources of samples: oropharyngeal swab (TV), nasopharyngeal swab (PV), sputum sample (SP) and bronchial alveolar lavage fluid (BALF) from September 2015 to August 2018 (Figure 4) [7] , and analyzed the etiology trends of community-acquired pneumonia (CAP). The pathogen spectrum detected covers almost all CAP pathogens, including bacteria, viruses, fungi, and atypical pathogens.
Figure 4 From 2015 to 2018, the pathogen distribution of oropharyngeal swabs, nasopharyngeal swabs, sputum samples and bronchial alveolar lavage fluid samples in 19 hospitals in Beijing from 2015 to 2018
From the perspective of the pathogen distribution of oropharyngeal swab samples, the probability of infection of adenovirus, coronavirus (excluding typing), rhinovirus , influenza A, influenza B, Mycoplasma pneumoniae, etc. is relatively high (Figure 5). Before the epidemic, the distribution of pathogenic species was relatively stable.
Figure 5 Pathogen distribution in oropharyngeal swab samples
CAP pathogen spectrum changes after the epidemic
In 2020, the influenza weekly report - North and South Epidemic Investigation Data released by the Chinese Center for Disease Control and Prevention showed that there were very few infections in influenza A and abortion B after the epidemic, and basically disappeared (Figure 6) [8].
Figure 6 Southern and Northern Epidemic Investigation Data
The curve of positive test for influenza viruses in different years shows that from 2020 to 2021, influenza viruses were basically at the baseline level (Figure 7) [8].The reason is related to the prevention and control measures taken by my country for the new crown pneumonia epidemic, such as wearing masks, washing hands frequently, reducing contact, etc., which are also effective in preventing and controlling the influenza virus. However, starting from November 2021, the number of infections of abortion B has been continuously detected and increased, resulting in a new growth trend. At the same time, the detection rate of respiratory syncytial virus has also increased significantly compared with the same period last year.
Figure 7 Influenza positive test reported by North and South China Laboratory in 2010-2021
Distribution of influenza pathogens from April 2019 to October 2020 shows that there are fewer viruses in type A H1N1, and more viruses in type A H3N2 virus and type B Victoria viruses (Figure 8) [8].
Figure 8 From April 2019 to October 2020, the distribution of influenza pathogens in mainland China
The trend of international and domestic influenza changes are basically the same. In early 2020, a epidemic investigation study of Swiss showed that the co-infection rate of novel coronavirus (SARS-CoV-2) and community-acquired respiratory virus (CARV) was 1.8% [9]. This reminds us to pay attention to the coronavirus, and not to ignore infections from other viruses.
Early epidemiological investigation studies from Jiangsu Province show that among the infected people detected by the new coronavirus, rhinovirus, adenovirus, influenza virus, Mycoplasma pneumoniae, whooping cough , and metapneumonia virus can also be detected. These viruses have a certain co-infection rate with the new coronavirus.
Figure 9 Jiangsu Province SARS-CoV-2 merged with respiratory virus pathogens
In the context of the new crown epidemic, although SARS-CoV-2 is the focus of everyone's attention, China's CDC also pays special attention to influenza viruses.
In May 2021, small-scale cases of avian influenza virus (H5N6) appeared in Sichuan Province, with a total of 5 patients (49-65 years old) diagnosed with H5N6, which generally did not cause serious consequences (Figure 10). However, it is alarming that three of the patients died.
During the flu season every year, even if the new pneumonia epidemic is effectively prevented and controlled, we should also be vigilant with other epidemic viruses.
Figure 10 (A) Geographical distribution of H5N6 virus in human infection, (B) Timeline and basic demographic information of human infection
In order to show that influenza A virus has different pairings of HA and NA, a document published by my country's CDC experts yellows the subtype of influenza A virus that has been detected in humans or animals. The ones that have not been detected are represented by "X" (Figure 11). In the future, a certain "X" virus may also become a large-scale epidemic.
Figure 11 (Left) Influenza A virus subtype and its earliest detection year, (right) Natural host and mammalian host of influenza A virus
2. Introduction to laboratory methods for detecting respiratory viruses
1. Clarify the target pathogen
31. Clarify the target pathogen
Only as long as the target pathogen is clarified, clinical anti-infection treatment can be targeted, and can be treated in a timely and effective manner, and can also prevent the abuse of clinical antibiotics. Different pathogen types adopt different treatment regimens (Figure 12).
Case: China-Japan Friendship Hospital once received a positive patient with Chlamydia psittiscima. The patient is a pregnant woman with severe lung infection and is of average treatment effect through ECMO, antibiotics and antiviral drugs. After clarifying the pathogen is Chlamydia psittisca, symptomatic treatment is given, and moxifloxacin , tigecycline, or azithromycin is used, which has better results.
Figure 12 Clinical anti-infection treatment plan [10-18]
2, respiratory pathogen epidemiological situation
Although there are many types of pathogens for respiratory infection, epidemiological investigation shows that the common pathogens of respiratory infection mainly include influenza A and B viruses, respiratory syncytial virus, adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella cataractia, etc., basically covering most CAP cases and can be a key target for nucleic acid detection .
Metagenomic sequencing (mNGS) is used more frequently in the diagnosis of pathogenic diseases in China, because there is a lack of fast multimolecular biological targeted detection in the development of microbial detection in China. Foreign countries, especially European and American countries, have more applications for multiple targeted detection.
In fact, patients may be infected with one or more pathogens, and pathogens may have co-infection, so multiple detection will be a trend in the future. If we have a fast and accurate detection method for multiple (such as more than 20) respiratory pathogens, we can obtain results within 2 hours and test them at any time, which will provide more help to clinical treatment.
In clinical work, you can quickly discover the goals and strive to win. In the diagnosis and treatment of respiratory infections, timely detection of pathogens and intervention can improve the patient's outcome.
3, Pathogen detection technology
With the rapid development of technology, pathogen detection has gone from traditional pathogen isolation and culture, to immunology technology, and then to serological examination, and finally entered the era of molecular detection.
① Pathogen isolation and culture
In the past few decades, pathogen microbial recognition has been dependent on smears and in vitro isolation and culture. But this method takes a long time. Some pathogens, such as viruses and atypical bacterial infections, have problems such as difficult to operate and high false negative rates. They are currently mainly used in scientific research and have been replaced by molecular biology and antigen detection methods in clinical practice [19]. However, antibiotic treatments such as Staphylococcus aureus or Klebsiella pneumoniae and Pseudomonas aeruginosa still require culture, and culture can provide drug sensitivity results.
②Immunology technology
Pathogen antigen detection is short, easy to operate, and does not require complex operating instruments. It is the mainstream method for respiratory virus detection in many laboratories now. This method is not sensitive and has good specificity, and is often used to quickly screen [20]. For example, the influenza A virus that broke out in Beijing at the end of 2017 showed the advantages of high efficiency and low cost.
③Serological examination
serology mainly detects antibodies, inflammatory markers reflect the status of the body's infection in the early stage, and can also be used for monitoring disease progression and efficacy, but its disadvantage is that it cannot be used for the diagnosis of infected pathogens [21]. In most cases, a single test antibody is not very valuable for clinical diagnosis. Even if IgM antibody is positive, its value for clinically ill infection diagnosis is very limited.
④Molecular detection
Molecular detection technology has gradually been used in pathogen diagnosis since its inception in the 1980s. This method is efficient and convenient, with high detection sensitivity and specificity, and is increasingly valued in pathogen detection [22].
4, domestic and foreign guidelines recommend
Viral diagnostic methods recommended by the US CDC: antigen detection----fast; molecular diagnosis---fast, early, routine; virus isolation----difficulty in culture; serological detection antibodies-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Overall, the basic concepts of domestic and foreign pathogen detection guidelines are the same. The virus's antigen and nucleic acid test can be used for early diagnosis; antibody test can be used for retrospective investigation and research, but it has little significance for early diagnosis of cases.
During the epidemic season, it is necessary to use molecular biological detection methods with high sensitivity and specificity to perform rapid detection of respiratory viruses such as influenza viruses in patients infected with CAP. Accurate diagnosis can reduce additional tests and the use of antibiotics [24]. Real-time quantitative PCR has high sensitivity and specificity, and is the first choice for rapid diagnosis of respiratory virus infections such as influenza viruses and avian influenza viruses. Nucleic acid testing can be used for the early diagnosis of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella infections [25]. When screening for
quickly, antigen testing can be used.
3. The value of multiple rapid molecular biological detection
In most cases, it is recommended to use molecular detection methods, especially multiple PCR virus detection.
Multiple PCR Virus Detection
Foreign relies heavily on FilmArray fast multiplex PCR system (Figure 13), which can detect various pathogen targets and antibiotic resistance genes that can cause respiratory tract infections, blood flow infections and gastrointestinal infections, as well as antibiotic resistance genes, which are very helpful for clinical diagnosis.
Figure 13 FilmArray Fast Multiple PCR System
Of course, Saipe's joint nucleic acid detection kit (Xpert Xpress Flu/RSV Assay) is also very helpful for clinical pathogenic diagnosis of respiratory infection (Figure 14).
Figure 14 Xpert Xpress Flu/RSV Assay
Multiple PCR is a representative of molecular detection technology, with broad spectrum coverage of pathogens, truly realizing high-throughput detection.
There are also products in China for 13 target detection. Currently, the commercial multiple PCR kits can cover common viruses and atypical pathogens that cause respiratory infection, including influenza A/B virus, respiratory syncytial virus, adenovirus, rhinovirus, Mycoplasma pneumoniae and Chlamydia (can cover Chlamydia psittisca and Chlamydia pneumoniae).
In recent years, the field of rapid and fully automatic molecular detection in China has been very active. I believe that soon we will have high-quality and low-cost domestic products for clinical use.
Metagenome sequencing (mNGS)
Traditional pathogenic detection methods still have clinically unmet needs. For new influenza viruses, traditional methods cannot identify their pathogens, which may cause a trend of outbreaks like the new coronavirus, and will be applied to metagenomic sequencing.
is different from the domestic literature that strongly advocates metagenomic sequencing technology. Some foreign literatures are cautious about the use of metagenomic sequencing for clinical use. A document published by CHEST in 2020 shows that compared with traditional methods, metagenomic sequencing has no obvious advantage in the detection of fungi [26]. In clinical practice, metagenomic sequencing has limited advantages for tuberculosis detection. However, if microbiology laboratory cannot provide more effective services for the clinical practice, it is also an inevitable trend for clinicians to choose metagenomic sequencing.
Nanopore sequencing technology
Nanopore sequencing technology (third generation technology) refers to single molecule real-time sequencing technology. Main advantages: direct sequencing, fast, low equipment cost. Compared with the previous two generations of sequencing technologies, its biggest feature is that the sequencing process does not require PCR to amplify , and the sequencing fragments are long.
Targeted sequencing tNGS
t refers to targeted Current technology can add hundreds of pathogen primers to a reaction well, and post-sequencing 100 to 200 targets, or add a capture probe to capture the target pathogen nucleic acid and sequencing it. In this way, interference from human and background bacteria is eliminated, and targeted amplification and sequencing are only carried out (Figure 15).
Figure 15 Targeted sequencing of multiple respiratory pathogens 153 types of
Summary
Before and after the epidemic, the CAP pathogen spectrum has changed.
Respiratory infectious pathogenesis Multiple nucleic acid detection has important clinical significance: identification and early warning of severe diseases; precise treatment reduces medical costs.
Multiple broad-spectrum fast molecular diagnostic solutions provide a solid backing for the precise management of respiratory infectious diseases.
mNGS technology has become an important means of pathogenic means. Correct interpretation of results is the key to making good use of this tool. 1. Changes in the pathogenic spectrum of community-acquired pneumonia (CAP) before and after the epidemic
1, respiratory tract infection symptoms and causes
pathogenic microorganism invades the respiratory tract and reproduces is called respiratory tract infection. According to its location, it is divided into upper respiratory tract infection and lower respiratory tract infection .
literature reports that 70-80% of pathogens of upper respiratory tract infection are viral; bacterial pathogens account for 20-25%.
lower respiratory tract infection symptoms include pneumonia , bronchitis and bronchitis , etc., which are mainly caused by infections such as bacteria, Mycoplasma , Chlamydia , Legionella, viruses, etc. The bacteria of CAP mainly include Haemophilus influenzae , Streptococcus pneumoniae , and Moraxella cataracta . In recent years, the proportion of infection of Klebsiella pneumoniae has gradually increased.
2, common pathogens of respiratory infection
Common pathogens of respiratory infection are divided into viruses, bacteria, fungi, mycoplasma, chlamydia, etc. (Figure 1).
viral pathogens are divided into RNA virus and DNA virus. Unlike DNA virus detection, RNA viruses undergo PCR detection. is first reverse transcribed into cDNA, and then PCR detection is performed.
RNA viruses include influenza virus (A/B), parainfluenza virus, respiratory syncytial virus , coronavirus, etc. Influenza virus has always been the focus of our attention, and respiratory syncytial virus is more likely to cause infections in infants, children, and the elderly. Coronaviruses like SARS and SARS-CoV-2 (COVID-19) have had serious impacts on global politics and economy, and should not be underestimated. Common coronaviruses include 229E, NL63, OC43 and HKU1, which are the main pathogenic microorganisms of common cold . The symptoms of infection caused by mild symptoms, healthy people have a certain degree of resistance to it and often do not receive special attention. Of course, serious infection with the common coronavirus can also cause death. In 2022, I have seen 2 patients, including obesity and diabetes (over 65 years old), who died of common coronavirus infection. The application of mNGS technology and multiple PCR technology has made our understanding of coronavirus more profound.
DNA viruses include adenovirus , cytomegalovirus , EB virus, etc. Adenovirus can cause severe infections, and there are also cases where adenovirus is detected in healthy people. Cytomegalovirus and EB viruses are infected in infants and young children. As patients get older and unexpected situations occur, such as transplantation, hematologic diseases, infection with HIV and tumors, the latent/recessively infected cytomegalovirus and EB virus may "rekind", causing very serious infections. The lung transplant department is very concerned about infections caused by cytomegalovirus.
bacterial pathogens are divided into morphologically: cocci and bacillus , and are divided into Gram-negative bacteria and positive bacteria according to the characteristics of Gram staining. Streptococcus pneumoniae in
gram-positive bacteria is an important pathogen that causes CAP. This bacteria is difficult to cultivate and is prone to death due to unreasonable delivery or storage. The test is negative. Streptococcus pneumoniae can cause severe infections, and it is difficult for patients to detect it after using antibiotic . Therefore, the proportion of CAP caused by Streptococcus pneumoniae tends to be underestimated. Using molecular biology to detect Streptococcus pneumoniae is a good choice. Klebsiella pneumoniae and Haemophilus influenzae have a higher chance of causing CAP. Outpatients with Pseudomonas aeruginosa infection may be more likely to be seen in patients with bronchodilation and chronic obstructive pulmonary disease (COPD). CAP caused by Acinetobacter baumannii is very rare. The above bacteria may also be colonized in normal populations, and detection may not necessarily mean infection, especially if the sample is not qualified to be retained. Respiratory tract sample smears often help distinguish infection from colonization.
Legionella can cause very serious infections. It is recommended that Legionella use molecular biological detection schemes, such as PCR or multiple PCR technology, etc. It is not recommended to use antibody to detect methods. It is also difficult to cultivate, takes time and requires special culture media.
fungal pathogens mainly include yeast, Aspergillus , mucoriasis, etc.
Other atypical pathogens include Mycoplasma pneumoniae, Chlamydia pneumoniae , Chlamydia psilocytica and Rickettsia. It is worth noting that Chlamydia psittaci. In fact, Chlamydia psittaci has been mainly diagnosed through sequencing in recent years. Some molecular biological reagents in clinical practice can also detect Chlamydia psittaci. With the widespread application of molecular biological detection technology and metagenomic sequencing (mNGS) technology, we found that the infection rate and detection rate of Chlamydia psittaci in our laboratory have exceeded Chlamydia pneumoniae and have become a very important type of pathogen. Psittaci disease can cause serious clinical infections, and the proportion of patients entering ICU is also quite high.
In addition, new pathogens are constantly emerging: such as SARS virus , highly pathogenic avian influenza, human infection with H7N9 avian influenza, novel coronavirus (SARS-CoV-2), etc.
Figure 1 Common pathogens classification
3, research on viral infection in patients with CAP
3 Chinese immune function is normal for adults to obtain viral pneumonia the main reason for influenza virus infection , followed by non-influenza virus infection.
influenza and non-influenza virus infection account for 63.5% and 26.2% of adult CAP patients with normal immune function in my country. Analysis of patients with single viral infection also confirmed that influenza virus infection is the main cause of viral pneumonia, followed by non-influenza virus infections such as adenovirus and respiratory syncytial virus (Figure 2) [1].
Figure 2 Research on viral infection in adult CAP patients with normal immune function in my country
Respiratory virus is an important cause of CAP disease
In the past 20 years, the outbreak of various infectious diseases has highlighted the importance of viruses in the onset of CAP. Such as SARS virus, MERS virus and avian influenza virus have appeared one after another, and new viruses will continue to emerge in the future (Figure 3).
Figure 3 Outbreaks of various infectious diseases in 2002-2019 [2-6]
4. Before and after the epidemic, the pathogen spectrum of CAP was changed
Before the epidemic, the pathogen species were relatively stable
2021 The National Center for Disease Control and Prevention (CDC) published an epidemiological survey study, investigating the number of pathogenic species in four sources of samples: oropharyngeal swab (TV), nasopharyngeal swab (PV), sputum sample (SP) and bronchial alveolar lavage fluid (BALF) from September 2015 to August 2018 (Figure 4) [7] , and analyzed the etiology trends of community-acquired pneumonia (CAP). The pathogen spectrum detected covers almost all CAP pathogens, including bacteria, viruses, fungi, and atypical pathogens.
Figure 4 From 2015 to 2018, the pathogen distribution of oropharyngeal swabs, nasopharyngeal swabs, sputum samples and bronchial alveolar lavage fluid samples in 19 hospitals in Beijing from 2015 to 2018
From the perspective of the pathogen distribution of oropharyngeal swab samples, the probability of infection of adenovirus, coronavirus (excluding typing), rhinovirus , influenza A, influenza B, Mycoplasma pneumoniae, etc. is relatively high (Figure 5). Before the epidemic, the distribution of pathogenic species was relatively stable.
Figure 5 Pathogen distribution in oropharyngeal swab samples
CAP pathogen spectrum changes after the epidemic
In 2020, the influenza weekly report - North and South Epidemic Investigation Data released by the Chinese Center for Disease Control and Prevention showed that there were very few infections in influenza A and abortion B after the epidemic, and basically disappeared (Figure 6) [8].
Figure 6 Southern and Northern Epidemic Investigation Data
The curve of positive test for influenza viruses in different years shows that from 2020 to 2021, influenza viruses were basically at the baseline level (Figure 7) [8].The reason is related to the prevention and control measures taken by my country for the new crown pneumonia epidemic, such as wearing masks, washing hands frequently, reducing contact, etc., which are also effective in preventing and controlling the influenza virus. However, starting from November 2021, the number of infections of abortion B has been continuously detected and increased, resulting in a new growth trend. At the same time, the detection rate of respiratory syncytial virus has also increased significantly compared with the same period last year.
Figure 7 Influenza positive test reported by North and South China Laboratory in 2010-2021
Distribution of influenza pathogens from April 2019 to October 2020 shows that there are fewer viruses in type A H1N1, and more viruses in type A H3N2 virus and type B Victoria viruses (Figure 8) [8].
Figure 8 From April 2019 to October 2020, the distribution of influenza pathogens in mainland China
The trend of international and domestic influenza changes are basically the same. In early 2020, a epidemic investigation study of Swiss showed that the co-infection rate of novel coronavirus (SARS-CoV-2) and community-acquired respiratory virus (CARV) was 1.8% [9]. This reminds us to pay attention to the coronavirus, and not to ignore infections from other viruses.
Early epidemiological investigation studies from Jiangsu Province show that among the infected people detected by the new coronavirus, rhinovirus, adenovirus, influenza virus, Mycoplasma pneumoniae, whooping cough , and metapneumonia virus can also be detected. These viruses have a certain co-infection rate with the new coronavirus.
Figure 9 Jiangsu Province SARS-CoV-2 merged with respiratory virus pathogens
In the context of the new crown epidemic, although SARS-CoV-2 is the focus of everyone's attention, China's CDC also pays special attention to influenza viruses.
In May 2021, small-scale cases of avian influenza virus (H5N6) appeared in Sichuan Province, with a total of 5 patients (49-65 years old) diagnosed with H5N6, which generally did not cause serious consequences (Figure 10). However, it is alarming that three of the patients died.
During the flu season every year, even if the new pneumonia epidemic is effectively prevented and controlled, we should also be vigilant with other epidemic viruses.
Figure 10 (A) Geographical distribution of H5N6 virus in human infection, (B) Timeline and basic demographic information of human infection
In order to show that influenza A virus has different pairings of HA and NA, a document published by my country's CDC experts yellows the subtype of influenza A virus that has been detected in humans or animals. The ones that have not been detected are represented by "X" (Figure 11). In the future, a certain "X" virus may also become a large-scale epidemic.
Figure 11 (Left) Influenza A virus subtype and its earliest detection year, (right) Natural host and mammalian host of influenza A virus
2. Introduction to laboratory methods for detecting respiratory viruses
1. Clarify the target pathogen
31. Clarify the target pathogen
Only as long as the target pathogen is clarified, clinical anti-infection treatment can be targeted, and can be treated in a timely and effective manner, and can also prevent the abuse of clinical antibiotics. Different pathogen types adopt different treatment regimens (Figure 12).
Case: China-Japan Friendship Hospital once received a positive patient with Chlamydia psittiscima. The patient is a pregnant woman with severe lung infection and is of average treatment effect through ECMO, antibiotics and antiviral drugs. After clarifying the pathogen is Chlamydia psittisca, symptomatic treatment is given, and moxifloxacin , tigecycline, or azithromycin is used, which has better results.
Figure 12 Clinical anti-infection treatment plan [10-18]
2, respiratory pathogen epidemiological situation
Although there are many types of pathogens for respiratory infection, epidemiological investigation shows that the common pathogens of respiratory infection mainly include influenza A and B viruses, respiratory syncytial virus, adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella cataractia, etc., basically covering most CAP cases and can be a key target for nucleic acid detection .
Metagenomic sequencing (mNGS) is used more frequently in the diagnosis of pathogenic diseases in China, because there is a lack of fast multimolecular biological targeted detection in the development of microbial detection in China. Foreign countries, especially European and American countries, have more applications for multiple targeted detection.
In fact, patients may be infected with one or more pathogens, and pathogens may have co-infection, so multiple detection will be a trend in the future. If we have a fast and accurate detection method for multiple (such as more than 20) respiratory pathogens, we can obtain results within 2 hours and test them at any time, which will provide more help to clinical treatment.
In clinical work, you can quickly discover the goals and strive to win. In the diagnosis and treatment of respiratory infections, timely detection of pathogens and intervention can improve the patient's outcome.
3, Pathogen detection technology
With the rapid development of technology, pathogen detection has gone from traditional pathogen isolation and culture, to immunology technology, and then to serological examination, and finally entered the era of molecular detection.
① Pathogen isolation and culture
In the past few decades, pathogen microbial recognition has been dependent on smears and in vitro isolation and culture. But this method takes a long time. Some pathogens, such as viruses and atypical bacterial infections, have problems such as difficult to operate and high false negative rates. They are currently mainly used in scientific research and have been replaced by molecular biology and antigen detection methods in clinical practice [19]. However, antibiotic treatments such as Staphylococcus aureus or Klebsiella pneumoniae and Pseudomonas aeruginosa still require culture, and culture can provide drug sensitivity results.
②Immunology technology
Pathogen antigen detection is short, easy to operate, and does not require complex operating instruments. It is the mainstream method for respiratory virus detection in many laboratories now. This method is not sensitive and has good specificity, and is often used to quickly screen [20]. For example, the influenza A virus that broke out in Beijing at the end of 2017 showed the advantages of high efficiency and low cost.
③Serological examination
serology mainly detects antibodies, inflammatory markers reflect the status of the body's infection in the early stage, and can also be used for monitoring disease progression and efficacy, but its disadvantage is that it cannot be used for the diagnosis of infected pathogens [21]. In most cases, a single test antibody is not very valuable for clinical diagnosis. Even if IgM antibody is positive, its value for clinically ill infection diagnosis is very limited.
④Molecular detection
Molecular detection technology has gradually been used in pathogen diagnosis since its inception in the 1980s. This method is efficient and convenient, with high detection sensitivity and specificity, and is increasingly valued in pathogen detection [22].
4, domestic and foreign guidelines recommend
Viral diagnostic methods recommended by the US CDC: antigen detection----fast; molecular diagnosis---fast, early, routine; virus isolation----difficulty in culture; serological detection antibodies-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Overall, the basic concepts of domestic and foreign pathogen detection guidelines are the same. The virus's antigen and nucleic acid test can be used for early diagnosis; antibody test can be used for retrospective investigation and research, but it has little significance for early diagnosis of cases.
During the epidemic season, it is necessary to use molecular biological detection methods with high sensitivity and specificity to perform rapid detection of respiratory viruses such as influenza viruses in patients infected with CAP. Accurate diagnosis can reduce additional tests and the use of antibiotics [24]. Real-time quantitative PCR has high sensitivity and specificity, and is the first choice for rapid diagnosis of respiratory virus infections such as influenza viruses and avian influenza viruses. Nucleic acid testing can be used for the early diagnosis of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella infections [25]. When screening for
quickly, antigen testing can be used.
3. The value of multiple rapid molecular biological detection
In most cases, it is recommended to use molecular detection methods, especially multiple PCR virus detection.
Multiple PCR Virus Detection
Foreign relies heavily on FilmArray fast multiplex PCR system (Figure 13), which can detect various pathogen targets and antibiotic resistance genes that can cause respiratory tract infections, blood flow infections and gastrointestinal infections, as well as antibiotic resistance genes, which are very helpful for clinical diagnosis.
Figure 13 FilmArray Fast Multiple PCR System
Of course, Saipe's joint nucleic acid detection kit (Xpert Xpress Flu/RSV Assay) is also very helpful for clinical pathogenic diagnosis of respiratory infection (Figure 14).
Figure 14 Xpert Xpress Flu/RSV Assay
Multiple PCR is a representative of molecular detection technology, with broad spectrum coverage of pathogens, truly realizing high-throughput detection.
There are also products in China for 13 target detection. Currently, the commercial multiple PCR kits can cover common viruses and atypical pathogens that cause respiratory infection, including influenza A/B virus, respiratory syncytial virus, adenovirus, rhinovirus, Mycoplasma pneumoniae and Chlamydia (can cover Chlamydia psittisca and Chlamydia pneumoniae).
In recent years, the field of rapid and fully automatic molecular detection in China has been very active. I believe that soon we will have high-quality and low-cost domestic products for clinical use.
Metagenome sequencing (mNGS)
Traditional pathogenic detection methods still have clinically unmet needs. For new influenza viruses, traditional methods cannot identify their pathogens, which may cause a trend of outbreaks like the new coronavirus, and will be applied to metagenomic sequencing.
is different from the domestic literature that strongly advocates metagenomic sequencing technology. Some foreign literatures are cautious about the use of metagenomic sequencing for clinical use. A document published by CHEST in 2020 shows that compared with traditional methods, metagenomic sequencing has no obvious advantage in the detection of fungi [26]. In clinical practice, metagenomic sequencing has limited advantages for tuberculosis detection. However, if microbiology laboratory cannot provide more effective services for the clinical practice, it is also an inevitable trend for clinicians to choose metagenomic sequencing.
Nanopore sequencing technology
Nanopore sequencing technology (third generation technology) refers to single molecule real-time sequencing technology. Main advantages: direct sequencing, fast, low equipment cost. Compared with the previous two generations of sequencing technologies, its biggest feature is that the sequencing process does not require PCR to amplify , and the sequencing fragments are long.
Targeted sequencing tNGS
t refers to targeted Current technology can add hundreds of pathogen primers to a reaction well, and post-sequencing 100 to 200 targets, or add a capture probe to capture the target pathogen nucleic acid and sequencing it. In this way, interference from human and background bacteria is eliminated, and targeted amplification and sequencing are only carried out (Figure 15).
Figure 15 Targeted sequencing of multiple respiratory pathogens 153 types of
Summary
Before and after the epidemic, the CAP pathogen spectrum has changed.
Respiratory infectious pathogenesis Multiple nucleic acid detection has important clinical significance: identification and early warning of severe diseases; precise treatment reduces medical costs.
Multiple broad-spectrum fast molecular diagnostic solutions provide a solid backing for the precise management of respiratory infectious diseases.
mNGS technology has become an important means of pathogenic means. Correct interpretation of results is the key to making good use of this tool.
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Live guest of this issue
Lu Binghuai
Chinese and Japanese Hospital, Clinical Microbiology and Infection Laboratory of Respiratory and Critical Care Medicine.Chief physician, MD, professor and master's supervisor at Peking University School of Medicine. Part-time job in society: Member of the Microbiology Group of the Testing Branch of the Chinese Medical Association, and Member of the Standing Committee of the Clinical Microbiology and Infection Branch of the Chinese Medical Promotion Association. Research direction: clinical microbial and pathogenic diagnosis, drug resistance mechanism. He has presided over a number of national and provincial and ministerial scientific research projects. Published nearly 30 professional articles included in SCI.
This article was compiled by Asiya, editor of "Breathing Realm". Thanks to Professor Lu Binghuai for review and revision!
* Thanks to Saipe (Shanghai) Trading Co., Ltd. for its strong support!
Complete this article
Live director: Liu Di; Typesetting: Jerry
Chief physician, MD, professor and master's supervisor at Peking University School of Medicine. Part-time job in society: Member of the Microbiology Group of the Testing Branch of the Chinese Medical Association, and Member of the Standing Committee of the Clinical Microbiology and Infection Branch of the Chinese Medical Promotion Association. Research direction: clinical microbial and pathogenic diagnosis, drug resistance mechanism. He has presided over a number of national and provincial and ministerial scientific research projects. Published nearly 30 professional articles included in SCI.This article was compiled by Asiya, editor of "Breathing Realm". Thanks to Professor Lu Binghuai for review and revision!
* Thanks to Saipe (Shanghai) Trading Co., Ltd. for its strong support!
Complete this article
Live director: Liu Di; Typesetting: Jerry
