In the 1990s. The whole genome sequence of Haemophilus influenzae was determined by the first generation sequencing technology for the first time. This is the first time in human history to use sequencing technology, and it seems that this technology has been sealed for more than ten years.
It is said that before being beheaded, a person is not afraid, and will say something like "/kloc-he will still be a hero after 0/8 years". Sure enough, 18 years later, in 2008, three recipients of different organs from the same donor in Australia died of encephalopathy-related diseases. The pathogen was not detected by traditional methods of etiology, immunology and molecular biology. Some tissue samples of donors and recipients were analyzed by NGS, which indicated that there was arenavirus infection, which was later confirmed by other methods. Therefore, NGS was first applied to the pathogen detection of infectious diseases. The next generation sequencing technology-metagenome sequencing technology mNGS is carried forward.
MNGS is a large-scale parallel sequencing of all nucleic acid components in the sample to be tested, and then complete nucleic acid sequence information in the sample is obtained through bioinformatics analysis of sequencing data. In addition, the composition information such as the species and quantity of microorganisms, and the characteristics or mechanism information such as drug resistance and virulence are expounded. MNGS does not require targeted amplification. You don't need to know the sequence information of pathogen genome, so you can detect all pathogens including bacteria, viruses, fungi and parasites, which is like carpet bombing in war. Carpet bombing wastes ammunition and carpet sequencing wastes money. mNGS charges 35,000 yuan at a time, and it is not covered by medical insurance.
Three or five thousand dollars, just the name of a pathogenic microorganism, three short words, and a long word not exceeding 10, can't be described with a gold word. Since the patient spent so much money and the doctor earned some money from it (accounting fee is about 500 yuan), it is necessary to study some sequencing theories to better interpret this report.
The concept is boring, and it is not suitable for writing many books on popular science. Once a book is written, readers are reluctant to read it. If readers don't want to read popular science, you will be very lonely in popular science. Here, we will focus on a relative abundance, which is an important indicator to judge what an infected pathogen is, and is significantly affected by the following factors: (1) The difference in genome size of pathogens. (2) Cell wall components have great influence on the efficiency of nucleic acid extraction, and the detection process has great interference on the treatment process and quality of refractory pathogens (such as fungi and tuberculosis). ) and the relative abundance of different pathogenic nucleic acids. (3) The quality of the database also affects the relative abundance of each pathogenic nucleic acid in the sample, which leads to the inconsistency between the detection results and the actual total amount.
The effect of carpet bombing varies greatly with different terrain. If the African savannah is bombed, basically, none of those lions can escape from zebra giraffes. If the complex barren hills are bombed, pangolins, weasels and the like can survive. Similarly, the detection efficiency of mNGS for infectious pathogens in different parts shows great differences: for respiratory tract samples, mNGS can detect 865,438+0.7%-94.8% of culture positive samples. For the infection of central nervous system, MNGS can only detect 22.4% pathogens that can't be identified by routine detection methods (including smear, culture and serum immunological detection), but 44.8% pathogens that can be identified by routine detection methods are missed. Therefore, there may not be a great advantage in the detection sensitivity of common bacterial pathogens (especially culturable pathogens).
The main reasons are the limited sequencing depth caused by controlling the reporting time and detection cost, and the interference of host nucleic acids and contaminated nucleic acids in the detection and result analysis process, which will make NGS even less sensitive to common pathogens than conventional culture methods. Compared with efforts to improve the efficacy of routine detection methods (such as high-quality direct smear staining microscopy, immunological detection, etc.), it is probably more difficult for most clinical microbiology laboratories to improve the role of NGS in a short time.
Another more heartfelt news is that the so-called pathogen in this report, which we bought for 1000 yuan, may not be the real pathogen, but misleading the treatment. This is like the information that heroes exchanged with their lives and blood, which turned out to be false information. On the contrary, our troops were trapped in the encirclement of the enemy. One method is to use the biological information obtained by sequencing to analyze the genetic characteristics related to pathogenic ability, such as adhesion, invasion, immune escape and toxin production of pathogenic bacteria. Using bioinformatics technology to analyze the complete sequence data of clinical specimens. Based on the theory of medical microbiology, immunology and infectious diseases, it is speculated that different pathogenic bacteria play a role in the occurrence and development of infection, which is helpful to distinguish normal flora, colonization and infection, which requires doctors to be microbiologists, immunologists and biologists. As far as I know, biologists are absolutely disdainful of being doctors, and they are all busy developing various vaccines to make a lot of money.
This is what I want to say about the significance of mNGS in the diagnosis of infectious diseases. Apart from the above shortcomings, others are advantages.