NGS Makes Pathogen Detection More Accurate

Status of Infectious Diseases

Approximately 15 million people worldwide die from infections every year. At present, infectious diseases are still a major threat to global public health. According to the statistics of WHO, about 15 million people worldwide die of infection every year, accounting for 25.5% of the global annual total mortality rate. Although in the past decades, the treatment and vaccination against pathogens have greatly reduced the impact of infectious diseases, with the emergence of new pathogenic microorganisms, the increase of drug-resistant pathogenic microorganisms, and the increase of immunosuppressed hosts, the incidence rate and mortality of infection remain high.

Status of Diagnosis and Treatment

Pathogenetic diagnosis has always been the most important segment in the diagnosis of infectious diseases. Traditional pathogenic diagnosis involves the clinician making a series of differential diagnoses based on the patient's clinical presentation, and then testing for these, with one test usually corresponding to only one pathogen. Usually, bacterial/fungal infections are mainly cultured, while viral infections are mainly detected by PCR and serology (with a window period). Traditional pathogenicity testing has a lower positive rate and throughput, is more time-consuming, and can only be used for one to a dozen types of pathogens, and is therefore generally only suitable for clinical diagnosis of mild infections.

Traditional microbial detection methods have many deficiencies in sensitivity, specificity, timeliness, and information content, and they are unable to rapidly identify unknown or rare pathogenic microorganisms, making it difficult to meet the high demands of clinical diagnosis. According to the literature, >50% of central nervous system infections, >40% of sepsis, and >30% of severe pneumonia fail to detect pathogens and lead to treatment failure due to the limitations of traditional diagnostic techniques and methods.

NGS Technology Shines in Clinical Pathogen Detection

In recent years, with the rapid development of high-throughput sequencing technology, also known as next-generation sequencing (NGS), the operation process has been simplified and the detection throughput has been growing, while the detection cost has been greatly reduced, so that it presents certain technical advantages in the detection of infectious pathogens, and has been more and more widely used. Currently, there are two main ways to identify microorganisms based on NGS: metagenomics next generation sequencing (mNGS) and targeted next generation sequencing (tNGS).

01 Metagenomics Next Generation Sequencing (mNGS)

In 2014, mNGS was used for the first time in the pathogenic diagnosis of clinical infection patients, successfully saving the life of a 14-year-old boy. mNGS has been increasingly researched and applied in the clinic since then. mNGS, compared with the traditional pathogenicity testing, requires no pre-setting and has no preference. mNGS can complete the detection of bacteria, fungi, viruses and parasites in one go by directly extracting the DNA/RNA from the clinical samples, performing high-throughput sequencing, and comparing with specialized pathogen databases and conducting bioinformatics analysis. With the advantages of short time-consumption, no bias and wide coverage, it plays an increasingly important role in the diagnosis of infectious disease pathogens, and is especially suitable for the diagnosis of pathogens in patients with difficult and critical conditions, immunodeficiency, or suspected infections of rare and emerging pathogens.

◆ mNGS detection improves diagnosis of central nervous system infections

In 2019, Charles Chiu's team from the University of California, USA, published a "Clinical Metagenomic Sequencing for Diagnosis of Meningitis and Encephalitis" study in the New England Journal of Medicine. They tested cerebrospinal fluid (CSF) samples from patients with meningitis and encephalitis. By comparing the results of the mNGS test with conventional tests, the PPA (positive coincidence rate) of mNGS for infectious diagnosis through direct CSF testing was 80.0%, The NPA (negative coincidence rate) was 98.2%, while the routine examination was 67.5% and 99.4%. mNGS testing has improved the diagnosis of central nervous system infections and demonstrated the potential use of mNGS in clinical patient testing.

◆mNGS improves diagnostic accuracy and survival rate in ICU patients with severe pneumonia

A study conducted by Prof. Ruilan Wang's team at Shanghai First People's Hospital included 178 patients with severe pneumonia, and for the first time explored the relationship between the early provision of clinical pathogenetic evidence by mNGS and the survival of ICU patients with severe pneumonia. Compared with traditional methods, mNGS can provide faster and more accurate pathogenetic results, especially the diagnostic value of caustic Streptococcus pneumoniae, Haemophilus influenzae, and Pseudomonas aeruginosa is obvious, as well as the obvious advantage in the diagnosis of mixed infections. After early definitive diagnosis by mNGS, clinical adjustment of patients' treatment plan according to the results resulted in an increase in patients' 28-day and 90-day survival rates, with the 90-day survival rate increasing from 57.7% to 83.3%.

◆mNGS can quickly and accurately identify rare/emerging pathogens

Yersinia pneumoniae lacks a gold standard for pathogen diagnosis in clinical diagnosis. Although there is no significant specificity in the G test and elevated serum lactate dehydrogenase, usually when these two tests are negative, they can serve as a basis for excluding pulmonary spore bacteria. Recent studies have found that the sensitivity (94.59%) and specificity (100%) of blood mNGS pulmonary spore bacteria are higher than those of G test combined with serum lactate dehydrogenase (sensitivity 89.19%, specificity 56.0%), especially in specificity. Wu et al. detected 29 patients with Yersinia pneumoniae infection through mNGS, of which only 8 were detected through Wright Giemesa staining smear. Gu et al. and Chen et al. reported that in cases of Chlamydia psittaci pneumonia, conventional pathogen detection methods are difficult to detect. However, using BALF mNGS to detect Chlamydia psittaci can reduce diagnostic delays and adopt precise anti infection treatment as soon as possible. Prior to mNGS detection, no significant pathogens were detected using traditional etiological methods, providing a strong basis for infection using mNGS.

◆mNGS can also improve the success rate of treatment in immunosuppressed patients

Professor Zhang Wenhong's team from Huashan Hospital affiliated with Fudan University included 108 suspected immunosuppressive patients who received hormone therapy. All patients underwent routine microbiological testing (CMT) and mNGS testing at the same time. The results showed that the treatment success rate (81.8%) of patients who adjusted the antibiotic regimen based on the mNGS test results was significantly higher than those who used empirical antibiotic therapy (52.6%). For the diagnosis of multiple infectious pathogens, compared to CMT, mNGS reduces the detection time by nearly half; When the cumulative dosage of steroid hormones exceeds 1000 mg, the positive rate of CMT significantly decreases, and mNGS has no effect.

Although mNGS has advantages such as short detection time, unbiased, and wide coverage, it also has certain limitations. Firstly, the cost of mNGS detection is relatively high. Secondly, due to the high proportion of human derived nucleic acids in clinical samples, the sensitivity of pathogen detection is reduced, and there is also a risk of missed detection of drug resistance and virulence genes. Furthermore, due to the fact that mNGS sequencing is a random fragment sequencing, it may not necessarily cover the typing section during sequencing, which may result in inaccurate typing of nearby pathogens. In response to the many issues of sensitivity and cost in clinical detection of mNGS methods, tNGS is gradually entering the clinical field of vision.

02 Targeted Next Generation Sequencing (tNGS)

tNGS combines the advantages of "PCR" and "NGS" and uses ultra multiple PCR to enrich target pathogens in a positive manner. This can greatly reduce the amount of sequencing data while increasing the information of pathogenic microorganisms in the sample and improving sensitivity, thus achieving dual optimization of performance and cost. Generally used for hospital infection screening, it can cover 95% of the most common pathogens in clinical practice.

03 How to Choose Between mNGS/tNGS

Macrogenomic sequencing (mNGS) and targeted sequencing (tNGS) are both pathogen detection strategies based on high-throughput sequencing technologies and other ancillary molecular detection technologies. The difference between the two is the preparation or provision of different detection targets for NGS. At the application level, the biggest difference is the coverage of different pathogen species and the cost of detection. 

There are few comprehensive and systematic evaluations of detection performance indicators such as sensitivity, specificity and accuracy of the two, and the results of related studies are shared below:

1）Prof. Peng Hu's team performed clinical characteristics, auxiliary examination collection and sample collection, including blood, sputum, bronchoalveolar lavage fluid (BALF), pleural effusion and lymph node tissues, as well as mNGS and tNGS tests, on 102 inpatients who were hospitalized at the Second Affiliated Hospital of Chongqing Medical University during the period of 2021.2-2021.7.

The study suggested that overall there was no significant difference in microbial detection rates between tNGS and mNGS across samples (82.17% vs. 86.51%, P=0.41); in particular, sputum (97.14% vs. 90.91%, P=0.28), BALF (80.95% vs. 90.48%, P=0.08), and pleural effusion (both 33.30%). mNGS additionally detected 20 microorganisms not included in the tNGS panel, whereas tNGS detected 20 cases of rhinovirus A/B/C (RNA viruses), but not mNGS (DNA processes).

Based on this, it can be inferred that the current efficacy of tNGS in detecting respiratory pathogenic microorganisms is equivalent to that of mNGS. The detection spectrum of mNGS is wider and the coverage of pathogens is more comprehensive, and tNGS has higher applicability in specific pathogen detection.

2）The study conducted tNGS and mNGS detection on 201 BALF specimens, and compared them with comprehensive clinical standards (conventional pathogenic examination, retrospective analysis, and orthogonal experiments) to evaluate the performance of the two methods in pathogen detection.

The mNGS process detected 2995 potential pathogens (2860 bacteria, 4 mycobacteria, 98 viruses, 18 fungi, and 15 parasites) from 173 specimens, with 28 specimens tested negative. The tNGS process identified 294 potential pathogens (247 bacteria, 1 mycobacterium, 43 viruses, and 3 fungi) from 123 BALF body fluid samples; 78 specimens tested negative. The overall accuracy of tNGS is 65.6%, while the overall accuracy of mNGS is 67.1%. Pathogens that were not detected by conventional detection methods were detected through both NGS methods. Overall, mNGS and tNGS have similar detection ranges for bacteria, fungi, and viruses. The reproducibility of the two detection processes is 100%.

 Summary

Whether it is traditional pathogen detection or NGS detection, various detection strategies have their own strengths. In the short term, mNGS/tNGS is unlikely to replace traditional diagnostic methods, but in some clinical situations, it may be a supplementary and necessary detection method. Therefore, if mNGS/tNGS is used in combination with traditional culture, other molecular biology, serological detection and other methods, through comprehensive analysis, it may truly achieve rapid and accurate diagnosis of infectious diseases, thus benefiting more doctors and patients.

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