Expert consensus

Multiple pathogen detection is an advanced diagnostic technology that can simultaneously detect and identify multiple pathogens, including bacteria, viruses, fungi and parasites. This detection method plays a vital role in the diagnosis of infectious diseases, especially when the pathogens are diverse and complex, and can quickly and accurately identify the pathogens, thereby formulating effective treatment plans.

As a new type of gene-level detection technology, pathogen metagenomic high-throughput sequencing technology (mNGS) does not require pre-setting, cultivation, or preference, and can comprehensively detect all pathogen genetic materials (including DNA and RNA) in the specimen and perform high-throughput sequencing. Based on bioinformatics algorithms, this technology can be compared and analyzed with the pathogen database to identify the types and functional information of pathogenic microorganisms in the sample, providing accurate etiological evidence for clinical anti-infection.

In order to standardize the application of pathogen metagenomic high-throughput sequencing technology in clinical localized testing, authoritative organizations such as the Chinese Pharmacists Association, the Chinese Medical Association's Bacterial Infection and Drug Resistance Prevention Branch, and the National Health Commission's Clinical Antimicrobial Susceptibility Breakpoint Research and Standard Development Expert Committee jointly published the "Expert Consensus on Clinical Localized Testing Specifications for Pathogen Metagenomic High-throughput Sequencing" in the "Chinese Journal of Preventive Medicine" in January 2024. This consensus provides specific guidance on how to conduct mNGS testing locally, provided that the laboratory complies with relevant national policies and regulations and ensures the reliability of the detection method performance.

Performance confirmation of dry experiment

After the bioinformatics analysis process is established, its performance needs to be confirmed. It can be done through three types of data sets: (1) Clinical specimen sequencing data set: clinical specimen sequencing data with sufficient annotations and clear characteristics (FASTQ or other formats); (2) Computer simulation sequencing data: sequence simulation software is used to simulate and generate sequencing data sets, which requires that the data sets have the same/similar technical parameters as the real sequencing data quality (library fragment length distribution, sequencing mode, sequencing error type and frequency, sequencing quality value, sequencing depth, etc.), and the background component information of clinical specimens such as host nucleic acid, microorganisms and reagent background nucleic acid should be simulated as much as possible; (3) Combined data set: sequence simulation software is used to quantitatively simulate the sequencing data of one or more target pathogens, and then the simulated sequences are added to the real sequencing data of target pathogen-negative specimens to form a simulated data set close to clinical specimens. The above data are used to carry out performance confirmation and determine the main performance indicators of the dry experiment process, including accuracy, precision, recall and F1-Score (i.e., the harmonic mean of precision and recall). If false positive results of parasites occur during the comparison process (parasites are eukaryotic organisms with high similarity to host sequences, and some parasite genome data are contaminated by human sequences), it is necessary to consider setting a higher positive reporting threshold or cleaning the genome data of the corresponding parasites. The common parasite reporting threshold is that the RPM is more than 10 times that of the negative control. The genome data cleaning often uses the target parasite genome to be compared with the human genome alone and remove highly homologous sequences.

Full-process performance confirmation

Confirm whether the detection ability of the full process of mNGS wet and dry experiments for pathogens can achieve the expected clinical use. In the full-process performance confirmation, at least Gram-positive bacteria, Gram-negative bacteria, mycobacteria, filamentous fungi, yeast, DNA viruses, RNA viruses, and parasites should be included, and attention should be paid to intracellular bacteria and pathogenic microorganisms that are difficult to break through the cell wall.

The performance confirmation parameters can refer to the "Guidelines for Performance Validation of Molecular Diagnostic Test Procedures", "Performance Validation of Clinical Microbiological Culture, Identification and Drug Sensitivity Testing Systems", "Performance Validation Scheme for Pathogen Metagenomic High-Throughput Sequencing", etc. It is recommended that the laboratory mNGS detection system should confirm the performance parameters such as method compliance rate, cross-reaction, precision, accuracy, detection limit, anti-interference ability (such as for high human specimens), stability, etc.

Preparation of sample trays

The sample trays should contain simulated references and real clinical specimens with known pathogen detection results. The setting of the sample tray should strictly follow the specimen types involved in the intended clinical use, and the microorganisms contained should be representative and cover the pathogens in the intended use as much as possible. Standard microbial strains can be selected from ATCC or CMCC strains, etc. The human cell concentration in the simulated reference should be reasonably set according to different specimen types (such as the median human cell concentration of bronchoalveolar lavage fluid is 105cells/ml). The simulated reference product should contain at least 8 types of pathogenic microorganisms, including Gram-positive bacteria, Gram-negative bacteria, filamentous fungi, yeast, mycobacteria, DNA viruses, RNA viruses, and parasites. Before preparing the simulated reference product, the minimum detection limit (LoD) of all microorganisms to be put into the product should be studied to confirm whether it can meet the requirements of the detection performance in the intended use.

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Product data

We have newly launched a multi-pathogen quality control product. The representative pathogen composition is shown in the table below. The above pathogens can be traced, and the genome data of each pathogen can be provided by Kebai.

Contains 4 Gram-positive bacteria, 4 Gram-negative bacteria, and 2 fungi.

鉴于细菌破壁提取难易程度对检测结果的影响，我们分别使用了市面上不同厂家的试剂盒对如上质控品做了提取，并使用相应数字PCR探针检测对应的拷贝数，如图所示，不同的提取试剂检测得到的结果会有较大的差异，与该试剂盒提取的偏好性相关。

同时，我们也对此产品进行了mNGS的检测，在无差别检测比对得到的数据中，10个菌都可以检出，但是比例与数字PCR也有同样的差异。

然而作为一款质控品，无论是数字PCR还是mNGS的方法，不同提取方法，均可以做到阳性的检出。

We can customize more different combinations of pathogen quality control products and reference products, and select representative bacteria, fungi, DNA viruses, RNA viruses, etc. according to customer requirements. You are welcome to inquire.