Revolutionising the diagnosis of respiratory infections using clinical metagenomics

A new rapid clinical metagenomics test for respiratory infections has been developed by researchers based at the Quadram Institute, (QI) and the University of East Anglia (UEA) that diagnoses bacterial Lower Respiratory Tract Infections (LRTIs). The test is more informative, cheaper and faster than current culture-based methods and is used to provide early targeted antimicrobial treatment and improve patient outcomes. The method is already in use in UK and Chinese hospitals and creating commercial impact.

Although there are alternative rapid technologies currently available on the market, such as multiplex polymerase chain reaction (PCR) kits, they can only detect the top causes of infection and resistance and are not a suitable replacement for the depth of information provided by current culture methods.

In 2013 Prof. O’Grady and his team began studying the practical applications of a portable sequencing device, MinION, developed by Oxford Nanopore Technologies (ONT). In the following years they became pioneers of the technology and published the first paper on practical application of MinION in 2015[i]. The team then focussed on using the DNA sequencing technology for the diagnosis of infectious diseases, a research area known as clinical metagenomics. They soon recognised that the large amounts of human DNA present in clinical samples compared with the tiny amounts of DNA from target microbes was a major barrier in obtaining clear and reliable results. This was the incentive to develop novel human cell/DNA depletion technologies, funded by an MRC New Investigator Award[ii], to remove human DNA and make clinical metagenomics feasible in terms of cost and turnaround time. A saponin-mediated differential lysis method was developed as a result of this work, which incorporates a step that rapidly and efficiently removes human genetic material from the patient’s sample, leaving only pathogen DNA for sequencing.

“Clinical metagenomics has the promise to revolutionise the diagnosis of infectious diseases. Our study describes the first rapid, affordable and accurate clinical metagenomic test that could readily be used on a routine basis in a clinical setting.” Professor Justin O’Grady, QI/UEA.

Dr Themoula Charalampous loading a MiniON at QI

As part of the INHALE study[iii] (https://www.ucl.ac.uk/inhale-project) UEA/QI PhD student, Themoula Charalampous, optimised the saponin depletion method and used it in conjunction with MinION sequencing to identify pathogens in sputum samples taken from patients with suspected lower respiratory tract infections (LRTIs)[iv]. These samples were provided through collaboration with the Norfolk and Norwich University Hospital Clinical Microbiology Laboratory.

The optimised method had high clinical specificity and sensitivity for pathogen identification, so even pathogens in low numbers could be successfully identified. The test took only 6 hours from sample-to-result thereby demonstrated that LRTIs were a tractable application for clinical metagenomics.

“Respiratory samples are difficult to work with because they are mainly comprised of human genetic material. Removing this makes detecting the pathogens easier and reduces the sequencing cost and time.” Dr Themoula Charalampous, QI.

A relationship was developed with leading Chinese pharmaceutical company, Simcere Pharmaceutical Group, which led to a UK-China Innovate UK AMR grant[v] that enabled further development. The saponin method now forms the foundation of a Simcere respiratory metagenomics test used in Chinese hospitals for detection of hospital-acquired and ventilator-acquired pneumonia, and severe community-acquired pneumonia. Samples are tested centrally in Nanjing in conjunction with culture to guide antimicrobial therapy, or on site in some hospitals as a rapid diagnostic.

Clinical metagenomics for respiratory infections

Since May 2019, the Simcere test has been used to test for respiratory infection in 12,000 samples collected in Chinese hospitals, generating greater than £2 million in revenue.

The test can be performed in 6 hours, compared to 2-3 days using the current culture methods. It identifies the infecting pathogen/s and provides antimicrobial resistance information to inform treatment decisions, resulting in improved patient outcomes whilst reducing the use of broad-spectrum antibiotics and helping in the fight against antimicrobial resistance. The portability of the MinION sequencing device means that it can be used closer to the patient, reducing the time spent sending samples to a central laboratory as the test can be conducted easily in a hospital lab. The method is also now being used at St Thomas’ Hospital in London to rapidly diagnose pneumonia and inform effective treatment and will be rolled out further over the coming months and years. Implementation of this test will improve patient outcomes and reduce the financial burden on healthcare providers in the UK, China and beyond while generating significant income for the collaborative partners.

“The pipeline that we’ve developed in this study produces data that can be used not only for clinical diagnostics but for public health applications such as outbreak detection and hospital infection control” Dr Gemma Kay, QI.

 

'MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance' infographic

workflow

The clinical metagenomics for respiratory infections workflow

According to the World Health Organisation (WHO), acute LRTIs, such as pneumonia and viral bronchitis caused of the deaths of 808,694 children under the age of five globally in 2017, more than any other single determinant[vi]. They account for around 3 million deaths worldwide each year and are the top cause of death in high- and low-income countries. These infections are caused by numerous bacteria and identifying the one responsible can be a challenge. Traditional diagnosis through culture of clinical samples, has poor sensitivity (may fail to detect the pathogen) and is too slow (>2 day’s turnaround) to guide early, targeted antimicrobial therapy. In the absence of a microbiological diagnosis, patients are treated with broad-spectrum or best guess antibiotics. Such an approach can result in poor patient outcomes and susceptibility to other infections (e.g. Clostridium difficile). Unnecessary prescription of antibiotics[vii] has also been linked to an increase in occurrence of antimicrobial resistance[viii] and associated patient recovery times.

In the US, the direct annual cost of community-acquired pneumonia has been estimated to be at least $17 billion[ix], and in Europe, overall annual costs are estimated to be € 10.1 billion[x]. Each year in the UK alone 220,000-484,000 people suffer from community-acquired pneumonia, 175,000 are admitted to hospital and at least 7,000 other hospital patients contract hospital-acquired pneumonia. The NICE pneumonia costing statement[xi] confirms that improved speed of diagnosis and targeted treatment are likely to improve outcomes for the patient, including time required for recovery. Cost savings achieved by more appropriate use of antibiotics would include reduced loss of workforce productivity, repeat appointments in primary care and length of stay in secondary care[xii]. Respiratory illnesses can also be fatal in some cases (5-14% of cases requiring hospital admission) and as such, faster, more targeted interventions could very feasibly save lives and reduce suffering. This picture is comparable worldwide.

It has been stated in academic journals that a new diagnostic test for pneumonia with at least 95% sensitivity, 85% specificity and minimal infrastructure requirements could save 405,000 children’s lives every year[xiii]. This clinical metagenomics method has been proven to have 96.6% agreement with cultured samples so it could potentially save numerous lives if implemented globally.

The research described here supports the national post Millennium Development Goal agenda to reduce the broad spectrum use of antibiotics, and international guidelines to reduce the escalation of antimicrobial resistance and childhood mortality rates including the WHO/ UNICEF Global Action Plan for Pneumonia and Diarrhoea.

In the battle to combat antimicrobial resistance, better and more rapid diagnostic tests are urgently needed to detect potential pathogens so that the most appropriate treatment can be prescribed.

Metagenomic sequencing had the potential to change the way we diagnose infection, combining rapidity with comprehensiveness beyond that of current methods. However, the use of metagenomic sequencing was not a viable alternative to culture until a method was developed by Prof. O’Grady to bypass the large amount of human DNA present in clinical samples.

There is still a lot of work to be done. Accurately predicting antimicrobial susceptibility of pathogens in lung samples that contain commensal flora is challenging and more research needs to be done. Another issue is viral detection, as the current test excludes viruses.

Funding for this work came from the Biotechnology and Biological Sciences Research Council, the Medical Research Council, Innovate UK, Rosetrees Trust and the National Institute for Health Research. Grant numbers: MR/N013956/1; RP-PG-0514-20018; Application Number:104596-610264; M468

References

[i] Ashton, P. M., Nair, S., Dallman, T., Rubino, S., Rabsch, W., Mwaigwisya, S., Wain, J., & O’Grady, J. (2015). MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island. Nature biotechnology33(3), 296–300. https://doi.org/10.1038/nbt.3103.

[ii] MRC New Investigator Research Grant (MR/N013956/1): “Improving the management of sepsis through rapid pathogen and antibiotic resistance detection in blood.” Total value GBP205,000. Aug 2016-Mar 2018; O’Grady PI.

[iii] NIHR PGfAR grant: “INHALE: Potential of Molecular Diagnostics for Hospital-Acquired and Ventilator-Associated Pneumonia in UK Critical Care.” Total value GBP2,500,000. Jan 2016-Dec 2020. O’Grady Co-I and workpackage 1 lead.

[iv] Charalampous, T., Kay, G. L., Richardson, H., Aydin, A., Baldan, R., Jeanes, C., Rae, D., Grundy, S., Turner, D. J., Wain, J., Leggett, R. M., Livermore, D. M., & O’Grady, J. (2019). Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection. Nature biotechnology37(7), 783–792. https://doi.org/10.1038/s41587-019-0156-5.

[v] Innovate UK UK-China AMR grant: Development of Key Technologies for Real-Time Diagnosis, Surveillance and Intervention of Resistant-Bacterial Infections Based on Nanopore Sequencing”. Total value GBP730,065. 2019-2022; O’Grady academic PI.

[vi] WHO fact sheet https://www.who.int/news-room/fact-sheets/detail/pneumonia.

[vii] Rennie M. D’Souza, Care-Seeking Behaviour, Clinical Infectious Diseases, Volume 28, Issue 2, February 1999, Page 234, https://doi.org/10.1086/515120.

[viii] Okeke, I. N., Laxminarayan, R., Bhutta, Z. A., Duse, A. G., Jenkins, P., O’Brien, T. F., Pablos-Mendez, A., & Klugman, K. P. (2005). Antimicrobial resistance in developing countries. Part I: recent trends and current status. The Lancet. Infectious diseases5(8), 481–493. https://doi.org/10.1016/S1473-3099(05)70189-4.

[ix] Thomas M. File Jr and Thomas J. Marrie. Burden of Community-Acquired Pneumonia in North American Adults. Pages 130-141. Postgraduate Medicine, 13 Mar 2015. https://doi.org/10.3810/pgm.2010.03.2130.

[x] Welte, T., Torres, A., and Nathwani, D. Clinical and economic burden of community-acquired pneumonia among adults in Europe, BMJ journals Vol 67, Issue 1. http://dx.doi.org/10.1136/thx.2009.129502

[xi] NICE pneumonia costing statement. https://www.nice.org.uk/guidance/cg191/resources/costing-statement-193352797.

[xii] For example, the NICE costings state that an average 7-day course of oral antibiotics for pneumonia is approximately £5.00 and treatment in an NHS hospital in the UK costs between £1,650 (without complications) and £4,107 (with complications), per person for an average of an eight day stay (a single day in a hospital bed costs £192).

[xiii] Lim, Y., Steinhoff, M., Girosi, F., Holtzman, D., Campbvell, H., Boer, R., Black, R and Mulholland, K. Reducing the global burden of acute lower respiratory infections in children, the contribution of a new diagnostics. Nature diagnostics, 2006. https://globalhealthdiagnostics.tghn.org/site_media/media/articles/Reducing_the_global_burden_of_acute_lower_respiratory_infections_in_children.pdf

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