Metagenomic sequencing used in infectious diseases

Metagenomics research is widely used in the analysis of soil, water, and other environmental samples, as well as the biodiversity analysis of microbial communities associated with human diseases. Since metagenomics does not require separate isolation and cultivation of pathogens, clinical samples can be directly analyzed by nucleic acid extraction and purification, which provides new technical means and ideas for the detection of infectious disease pathogens, especially unknown pathogens.

Metagenomics-based next-generation sequencing (mNGS) can obtain the genomes of all species in the sample, which can identify all pathogenic pathogens at the same time, greatly reducing the time and manpower required to eliminate suspected pathogens one by one, and can detect unknown pathogens. Identification of known pathogens with large mutations brings new ideas for the prevention and control of infectious diseases. At present, mNGS is widely used in public health monitoring, pathogen detection, and traceability of infectious diseases.

In this article, several applications of metagenomics sequencing in prevention and control of infections diseases were introduced.

1. Pathogen monitoring

Collecting clinical samples, food, and vector samples of a certain area or population for metagenomic sequencing, monitoring potential pathogens, and predicting and warning of possible epidemics can effectively prevent public health emergencies. Metagenomic sequencing can overcome the limitations of traditional methods, achieve high-precision identification of pathogens, and provide guidance for daily pathogen monitoring.

In one study, researchers collected samples of bronchoalveolar lavage fluid, sputum, and throat swabs from 24 seasonal influenza patients. Real-time quantitative PCR results showed that H3N2 and H1N1 were positive, but could not be further typed. Sequencing and comparing it with the reported genome can pinpoint its specific type, and mixed infections with other viruses or bacteria can be found in some samples.

With the development of global trade, the risk of spreading pathogens by means of food has increased, and food monitoring has become increasingly important. However, due to the scope of detection, the possibility of food carrying pathogens cannot be completely ruled out. By sequencing the imported genomes of imported animal meat products, researchers have detected viruses that may infect humans, including coronaviruses, flaviviruses, poxviruses, and hantaviruses.

The same strategy for metagenomic research can also monitor vector organisms, assess the possibility of pathogens infecting humans, and prevent zoonotic diseases. To assess the risk of humans carrying the virus in wild mice, researchers collected and sequenced intestinal extracts from 20 wild mice in Berlin, Germany, and detected boca virus, sandpo virus, norovirus, and rotavirus. Rotavirus strains are closely related to human pathogenicity.

2. Pathogen detection

Unexplained diseases and unknown pathogens increase the difficulty of clinical diagnosis, carrying out effective treatment, and lead to worsening of the patient's condition and even death. Metagenomics-based sequencing is playing an increasingly important role in pathogen detection.

A few years ago, an acute intestinal hemorrhagic epidemic broke out in Germany. There was no specific means to detect pathogenic infections at the beginning of the epidemic, and isolation and culture took a long time and were difficult. In a retrospective study of the outbreak, researchers used metagenomic testing to take 40 Shiga toxin-producing E. coli (STEC) -positive stool samples during the outbreak, and used 5 STEC-negative diarrhea samples as controls DNA was directly extracted for sequencing without isolation and culture. The disease-causing strain STEC O104: H4 was detected in samples from STEC-positive patients, and Shiga toxin-producing gene fragments were further detected. Clostridium difficile, Campylobacter jejuni, and Salmonella were also detected in 5 control groups. This demonstrates the potential of the metagenomics to use non-cultured samples to detect pathogens and analyze their virulence.

A large-scale yellow fever epidemic broke out in Angola in April 2016. More than 1,700 infections have been reported, with an additional 238 deaths.

Yellow fever has never before been reported in East Asia or China. In March 2016, the first case of imported yellow fever occurred in China. The patient was a Chinese labor importer who worked in Angola and returned to China.

With the help of metagenomic sequencing technology, the blood sample of this case came through a whole genome sequencing within 24 hours. The related sequencing reads were assembled and spliced, and the consistent sequences obtained were compared by BLAST. The results were most closely related to the Angola71 strain. Subsequent quantitative PCR backtracked the sequencing results.

3. Pathogen traceability

When investigating the epidemic of infectious diseases, after sequencing the samples, metagenomics can also trace the pathogens, find potential transmission routes, and identify and cut off the source of infection in a timely manner.

Zika virus is transmitted through mosquito bites, and pregnant women can cause microcephaly in newborns. The outbreak of the Zika virus in 2016 caused widespread concern.

Researchers performed high-throughput sequencing of amniotic fluid samples from two pregnant women directly, and detected the entire genome of Zika virus, indicating that Zika virus can cross the placental barrier to infect fetuses, and then use PCR and ELISA to verify the test results, further Phylogenetic analysis of the genome revealed that it has a 97-100% similarity with Zika virus in French Polynesia, which provides an important reference for establishing a link between Zika virus and microcephaly and determining the source of the virus.

In addition, researchers have developed a method for directly sequencing clinical samples. Enriching the virus genome by multiplex PCR and performing sequencing analysis can obtain the entire virus sequence. Based on the above method, another group of research teams obtained the Zika virus sequence of infected patients. After comparison and traceability analysis, it was found that the sequence is similar to the virus sequence in Aedes aegypti in North America. It is speculated that the virus was introduced from Guatemala and may continue the spread to northward. It provided guidance for preventing further transmission of the Zika virus. According to the World Health Organization, the incidence of Zika virus dropped significantly in 2017 and 2018.

Using the metagenomic method can also study some historical epidemics or cases, so as to trace the origin of infectious diseases. A study collected 22 bone and tooth samples from the United Kingdom, Denmark, and Sweden between 1010-1383 and the use of metagenomic sequencing to detect leprosy, and to analyze the differences of leprosy in different regions. It is speculated that the leprosy of the Americas may originate from Europe, and the genotype of leprosy in the Middle East is related to the genotype of medieval Europe.

In addition, after extracting nucleic acid from a 1797 mummy lung residual tissue sample and sequencing it, it was found that it was infected by a mixed infection of two different genotypes of Mycobacterium tuberculosis distributed in Europe and North America. The spread of Mycobacterium in the world is of great significance.

4. Efficient control of emerging infectious diseases

Zhang Wenhong's team used metagenomics to identify and cure for the first time human infections caused by the transmission of porcine herpes virus across species. This result was derived from a case in the Department of Infectious Diseases of Huashan Hospital.

In June 2017, the Department of Infectious Diseases of Huashan Hospital admitted a patient with fever to be checked. Following up on his medical history, the patient worked in a pig farm on weekdays and was splashed into the eyes by sewage from the pigsty the day before the onset of illness. Considering the possibility of central nervous system infection and endophthalmitis caused by human-pig comorbidity, the vitreous fluid and cerebrospinal fluid of Huashan Hospital's Infectious Diseases Department were examined for next-generation sequencing based on metagenomics (mNGS) to confirm the etiology diagnosis.

The report showed that no pathogen was detected in the patient's cerebrospinal fluid, and 4,832 swine herpes virus sequences were detected in the vitreous fluid of the eye, with a coverage rate of 84%. However, suspicious herpes virus, a virus that uses pigs as its natural host, has been reported in humans with suspected cases, but no confirmed cases have been reported.

Combining the patient's history of exposure to pigsty pollutants, clinical manifestations of endophthalmitis, and sequencing results of intraocular surgical specimens, the clinical team highly suspected swine herpes virus infection and immediately gave patients antiviral treatment.

The Huashan infection team quickly divided into three teams to work together. The genetic and immunological levels were subsequently used to identify the presence of swine herpes virus cross-species human cases. Multiple evidences assist the clinical diagnosis of swine herpes virus infection in patients, and patients can be relieved by intensive and precise treatment.

Based on the continuous development of mNGS, the use of metagenomics technology for pathogen monitoring, detection and traceability can accurately respond to new outbreaks of infectious diseases, opening up a new field for public health control.

For more information on new developments in gene testing, molecular diagnostics, and precision medicine, please pay attention to CD Genomics.

CD Genomics was established in 2004, aiming at providing the research community with high quality Next Generation Sequencing, PacBio SMRT sequencing, and microarray services. Due to the demand of increased services, CD Genomics has already updated its technology platform to mainstream NGS and microarray instruments. At present, the company’s senior bioinformaticians have ever viewed more than ten thousands of trace files and accumulated abundant experience with our Illumina HiSeq 2500, HiSeq 4000, Miseq Benchtop Sequencer, PacBio Sequel, PacBio RS II, Ion Torrent PGM, and ABI 3730/3730XL analyzer, etc.