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Using metagenomics to investigate infections

Article on applications of metagenomics to investigate infections
© COG-Train

The gold standard for diagnosis or detection of viral infection is targeted PCR (polymerase chain reaction) to amplify and detect small regions of the DNA or RNA of specific viruses. Real-time PCR is fast, with results in as little as 1–2 hours and inexpensive, starting at only £2 per reaction for laboratory-developed tests (LDTs). PCR assays are designed to target the organism of interest so are highly specific and can detect as little as one target copy per reaction so are very sensitive. In syndromes that are well characterised with infections largely caused by known and expected viruses, PCR is unrivalled.

However, some syndromes such as neurological infections in immunocompromised patients can be caused by hundreds of different pathogens including viruses, bacteria and parasites. In these instances, there is often insufficient specimen available to test for every possible organism, especially since PCR reactions are limited to just a handful of targets per reaction. Consequently, in the majority of cases of encephalitis, the causative agent is not known. In these instances, metagenomics is increasingly being applied to aid in the diagnosis of infection.

Metagenomics is the deep sequencing of all of the genetic material in a specimen. Following depletion of host DNA and RNA, sequencing libraries are prepared from the remaining DNA and RNA. This will include residual human DNA and RNA but also the DNA and/or RNA of any organisms present in the specimen. The sequences generated are compared to a database of known sequences to identify any organisms present. The main advantage of metagenomics for the diagnosis of infection is it requires no prior assumptions about the organism causing infection. With PCR we have to think of the likely aetiologies and test for them, which may or may not yield an answer.

Conversely, since metagenomics is untargeted, any organism will be identified so long as there is some similarity to sequences in the analysis database. This means rare, unexpected or novel organisms can be identified. In addition, when an organism is identified, assuming the viral load is high enough, sequence data for that organism will be generated which can inform typing, molecular epidemiology and/or genotypic resistance analysis. This was the approach used to initially determine the SARS-CoV-2 genome sequence.

The primary advantage of using metagenomics to diagnose infection is that it is untargeted. However, it is the untargeted nature of the technique that also leads to its main disadvantage. Despite the depletion of human DNA and RNA prior to sequencing library preparation, the vast majority of data sequenced is of human origin, with as little as 0.000002% of sequences belonging to the causative organism. Identifying this ‘needle in a haystack’ requires a depth of sequencing which comes at a substantial cost compared to PCR and can result in only a small amount of sequences generated for the organism of interest.

Illustrative image comparing pathogen-specific real-time PCR versus metagenomics for pathogen detection. Target-specific PCR amplification (real-time PCR) pros: fast, inexpensive, sensitive; cons: unexpected or novel pathogens not detected, multiple reactions required for multiple pathogens, limited by specimen volume. Sequencing of total DNA or cDNA (metagenomics) pros: pan-pathogen detection in a single reaction; cons: relatively expensive with slower time-to-result than PCR

Click to enlarge

Figure 1 – Illustration of pathogen-specific real-time PCR versus metagenomics for pathogen detection. Source: Journal of infection

References

Astrovirus VA1/HMO-C: an increasingly recognised neurotropic pathogen in immunocompromised patients

Norovirus whole genome sequencing by SureSelect target enrichment: a robust and sensitive method

Encephalitis diagnosis using metagenomics: application of next generation sequencing for undiagnosed cases

Beyond viruses: clinical profiles and etiologies associated with encephalitis

The epidemiology of acute encephalitis

Metagenomics for neurological infections – expanding our imagination

Case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis consortium

A Novel Coronavirus from Patients with Pneumonia in China, 2019

A new coronavirus associated with human respiratory disease in China

© COG-Train
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