Gliddon, H

Hayward, A

Shorten, R

Story, A

UCL Discovery

A sputum sample processing method forcommunity and mobile tuberculosisdiagnosis using the Xpert MTB/RIF assayTo the Editor:Tuberculosis (TB) remains a global public health emergency and claims the lives of more people annuallythan any other infectious disease. TB disproportionately affects people living in poverty, with 95% of casesand 98% of deaths occurring in developing countries [1]. In high income countries, TB is nowconcentrated in populations who originate from high burden countries and in socially vulnerable andexcluded groups, including homeless people, drug and alcohol users and prisoners. These populationsoften experience poor access to health services and are at increased risk of delayed diagnosis, onwardtransmission, drug resistance, severe disease and preventable death.Despite a decline in reported TB cases since 2011, London accounted for 2210 of the 5664 cases reportedin England in 2016; this amounts to 40% of all cases reported nationally and equates to more TB casesthan the Netherlands (889 cases), Greece (443), Norway (299) and Denmark (330) combined [2]. Therecent decline in cases has not been seen among homeless and vulnerable populations who now accountfor one in six of all TB cases reported in London, where the numbers of people sleeping rough has morethan doubled since 2010 [3]. TB patients in London with social risk factors are more likely to beinfectious, have drug-resistant TB and not complete treatment, and homelessness is an independent riskfactor for multidrug-resistant TB [4].Active case finding using targeted mobile digital radiology is widely recommended and used in highincome countries to reduce diagnostic delays and prevent onward transmission. Find&Treat are a specialistoutreach team dedicated to the early detection and management of TB and blood-borne viral diseaseamong homeless and vulnerable people. The team operate a Mobile Health Unit (MHU) routinelytargeting homeless hostels, day centres, street kitchens and drug treatment services in London, andthroughout the UK, in response to TB incidents and outbreaks. The MHU is equipped to provide digitalchest radiographs, which are interpreted on the spot by experienced reporting radiographers, to identifyabnormalities consistent with active pulmonary TB. Radiography alone is not diagnostic for pulmonary TBand patients with abnormal chest radiographs are required to provide sputum samples or attend localhealthcare facilities for further investigations. This delay in diagnosis confirmation increases the risk ofvulnerable patients disengaging from services and becoming lost to follow-up care. The Xpert MTB/RIFassay (Cepheid, Sunnyvale, CA, USA) is a nucleic acid amplification test that simultaneously detectsMycobacterium tuberculosis complex and markers of rifampicin resistance in less than 2 h. This test hasthe potential to allow patients with abnormal chest radiographs to be investigated in the community.However, roll-out beyond laboratory settings in high income countries has been limited by health andsafety considerations regarding sputum sample handling. To overcome this, the Find&Treat servicesourced and utilised a “septum sample pot”, which allows samples to be treated and processed safely onthe MHU. Ethical approval for this study was granted by the NRES Committee East of England – Essex,Research Ethics Committee (On March 20, 2014; reference: 10/H0302/51).@ERSpublicationsThe Xpert MTB/RIF assay can rapidly diagnose tuberculosis, but sputum samples cannot besafely processed unless in a lab. The septum sample pot allows safe handling of sputum and hasallowed a mobile TB unit to run the assay in community settings. http://ow.ly/HOA130mS6LGCite this article as: Gliddon HD, Shorten RJ, Hayward AC, et al. A sputum sample processingmethod for community and mobile tuberculosis diagnosis using the Xpert MTB/RIF assay. ERJOpen Res 2019; 5: 00165-2018 [https://doi.org/10.1183/23120541.00165-2018].Copyright ©ERS 2019. This article is open access and distributed under the terms of the Creative Commons AttributionNon-Commercial Licence 4.0.https://doi.org/10.1183/23120541.00165-2018 ERJ Open Res 2019; 5: 00165-2018ORIGINAL RESEARCH LETTERThe MHU is equipped with a Gene Xpert IV system (Cepheid) to run the Xpert MTB/RIF assay as apoint-of-care (POC) test. The high sensitivity and specificity of the assay has been extensively studied andit is now recommended by the World Health Organization for TB diagnosis [5, 6]. Following addition ofthe sample to a cartridge, the assay is entirely automated. One of the major barriers to the roll-out of theXpert MTB/RIF assay in the community is the biosafety requirements of adding the sample reagent to thesputum sample, vortexing or shaking vigorously (and so potentially producing aerosolised droplet nucleiof M. tuberculosis) and then adding the processed sample to the cartridge. This is typically performed in amicrobiological safety cabinet, which is not available to the staff of the MHU. To overcome the risk ofproducing aerosolised droplet nuclei, patients with abnormal chest radiographs are given a “septumsample pot” (figure 1), which comprises a container with a rubber VACUETTE valve stopper (productnumber 724300, Urine Beaker with Safety Stopper 100 mL, white lid, single-packed, sterile; GreinerBio-One GmbH, Kremsmünster, Austria). Patients step off the MHU to produce a sputum sample in theMobile radiographyunitPatients from a vulnerable populationXpertMTB/RIFassayPositive, ornegative and: – Weight loss  – Night sweats – HaemoptysisReferral to TB clinic for assessment and treatmentReferral to community TB serviceand repeat chest radiographafter 3 monthsSuccessful completion of treatmentNegative,no symptomsAbnormalDigital chestradiographClearSputum collected and processed safely for Xpert MTB/RIF assayRubbervalveSamplereagentaddedFIGURE 1 Patient pathway for the diagnosis of pulmonary tuberculosis (TB) on the mobile health unit. Patientsfrom a vulnerable population are first offered a digital chest radiograph. If indicative of a potential pulmonaryTB infection, a sputum sample is collected in a secure septum sample pot for processing before performingthe Xpert MTB/RIF assay. The rubber valve stopper in the lid of the septum sample pot allows access with apipette, without needing to open the lid and manipulate the sample. Patients are referred to TB clinics if theXpert MTB/RIF assay result is positive, or if they display symptoms typical of TB.https://doi.org/10.1183/23120541.00165-2018 2ORIGINAL RESEARCH LETTER | H.D. GLIDDON ET AL.open air and away from other people to avoid possible transmission of M. tuberculosis or other airbornepathogens. The lid is fastened by the patient once the sputum sample has been added and can then be safelytransferred to staff on the MHU. The Xpert MTB/RIF sample reagent is then added with a pipette thatenters through the valve (septum) on the lid, the pipette is then removed, disposed of appropriately, and theseptum self-seals allowing the sample and reagent to be mixed through vigorous shaking. This samplereagent lyses M. tuberculosis and therefore makes the sample safe to work with. Studies have shown thatwhen live M. tuberculosis cultures were added to fresh sputum samples and treated with the sample reagentin a 1:2 ratio, an 8-log decrease in viability was observed following an incubation time of 15 min [7]. Theprocessed sample can then be retrieved using a separate pipette, again through the septum valve, and addedto the Xpert cartridge. Training of MHU personnel covers the need for an adequate sputum sample volume(minimum 2 mL). Staff can estimate the volume of sputum visually and are trained to add 2–3 times thisvolume of sample reagent using a plastic pipette via the rubber septum. In addition, staff assess the qualityof sputum and reject salivary samples that may provide false negative results.Prior to the implementation of the Xpert MTB/RIF assay, a TB nurse specialist or outreach workeraccompanied patients with suspected pulmonary TB to local hospitals for assessment, conventionalmicrobiological investigation and possible admission. The MHU screens between 8000–10000 patientsannually. Approximately 1% of patients screened have abnormal chest radiology suggestive of active TB;however, approximately 75% of referred patients are subsequently diagnosed as negative for active TB. Theresource implications of referrals, including staff time during the accompanied visit, administration,clinician time, and urgent pathology samples sent to the laboratories is significant. In addition, theinconvenience to patients is considerable and many are reluctant to go to hospital. The aims ofimplementing Xpert MTB/RIF on the MHU are to: 1) rapidly identify cases of active TB, withaccompanied referral to link them to care and commence treatment the same day; 2) reduce the risk ofpatients disengaging from services and becoming lost to follow-up; and 3) prevent unnecessary referrals tosecondary care. The new pathway using the Xpert MTB/RIF assay for patients with radiologicalabnormalities (figure 1) has been well received by staff and patients. In addition to enabling simple andsafe POC diagnosis, this pathway exploits the high negative predictive value of Xpert MTB/RIF [8].Patients able to produce a voluntary sputum sample who have a negative Xpert MTB/RIF result can beassessed as presenting an extremely low transmission risk and be further investigated for TB or otherrespiratory disease in the community, including patients with a false negative Xpert MTB/RIF result whoare more likely to be smear negative and therefore less likely to be infectious [9].This novel approach enables safe processing of sputum specimens in the community, without the need forcontainment facilities or a microbiological safety cabinet. We believe this is the first example of amolecular diagnostic test for TB being used in the community at the POC for socially vulnerable andexcluded TB patients in Europe. This affordable technology has the potential to improve biosafetymeasures in high TB burden countries where GeneXpert is often used outside laboratory settings in areaswhere containment measures are not adopted for direct Xpert testing and achieving adequate ventilationfor safe handling of specimens may be challenging [10]. New smaller, portable, battery-powered devicesthat run one GeneXpert cartridge (i.e. the GeneXpert Omni [11]) will further enable POC TB moleculardiagnosis to be performed in other settings where rapid confirmation of pulmonary disease is highlyadvantageous and barriers to further diagnostic testing are operationally and financially challenging, suchas in prisons and custodial settings.Harriet D. Gliddon1, Robert J. Shorten2,3, Andrew C. Hayward4 and Alistair Story51London Centre for Nanotechnology, University College London, London, UK. 2Public Health England,Public Health Laboratory Manchester, Manchester Royal Infirmary, Manchester, UK. 3Dept of Infection,Centre for Clinical Microbiology, University College London, London, UK. 4University College London,Institute of Epidemiology and Health Care, London, UK. 5Find&Treat, University College LondonHospitals, London, UK.Correspondence: Alistair Story, Find&Treat, University College London Hospitals NHS Foundation Trust,4th Floor East, 250 Euston Rd, London, NW1 2PG, UK. E-mail: al.story@nhs.netReceived: Sept 19 2018 | Accepted after revision: Nov 07 2018Acknowledgements: The authors would like to thank UCLH for funding the MHU and Sushma Patel (Cepheid) forsourcing the sample pots.Conflict of interest: H.D. Gliddon has nothing to disclose. R.J. Shorten reports personal fees from Cepheid (sponsoredattendance at an international conference in 2016), outside the submitted work. A.C. Hayward has nothing to disclose.A. Story has nothing to disclose.https://doi.org/10.1183/23120541.00165-2018 3ORIGINAL RESEARCH LETTER | H.D. GLIDDON ET AL.Support statement: This service development research was funded through NIHR Programme GrantRP-PG-0407-10340. H.D. Gliddon acknowledges support from the i-sense Engineering and Physical Sciences ResearchCouncil (EPSRC) IRC in Early Warning Sensing Systems for Infectious Diseases (EP/K031953/1; www.i-sense.org.uk).Funding information for this article has been deposited with the Crossref Funder Registry.References1 Benatar SR, Upshur R. Tuberculosis and poverty: what could (and should) be done? Int J Tuberc Lung Dis 2010;14: 1215–1221.2 European Centre for Disease Control and Prevention/WHO Regional Office for Europe. Tuberculosis surveillanceand monitoring in Europe 2018, 2016 data. Stockholm, European Centre for Disease Prevention and Control,2018. https://ecdc.europa.eu/sites/portal/files/documents/ecdc-tuberculosis-surveillance-monitoring-Europe-2018-19mar2018.pdf Date last accessed: July 30, 2018.3 Ministry of Housing, Communities and Local Government. Rough Sleeping Statistics Autumn 2017, England.London, Ministry of Housing, Communities and Local Government, 2017. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/682001/Rough_Sleeping_Autumn_2017_Statistical_Release_-_revised.pdf Date last accessed: July 29, 2018. Date last updated: February 16, 2018.4 Public Health England. Tackling tuberculosis in under-served populations: a resource for TB control boards andtheir partners. London, PHE 2017. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/592274/Tackling_TB_in_Under-Served_Populations_-_A_Resource_for_TBCBs_and_partners.pdf Date last accessed: June 29, 2018.5 Steingart KR, Schiller I, Horne DJ, et al. Xpert® MTB/RIF assay for pulmonary tuberculosis and rifampicinresistance in adults. Cochrane Database Syst Rev 2014: 1: CD009593.6 Boehme CC, Nicol MP, Nabeta P, et al. Feasibility, diagnostic accuracy, and effectiveness of decentralised use ofthe Xpert MTB/RIF test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementationstudy. Lancet 2011; 377: 1495–1505.7 Helb D, Jones M, Story E, et al. Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use ofon-demand, near-patient technology. J Clin Microbiol 2010; 48: 229–237.8 Zeka AN, Tasbakan S, Cavusoglu C. Evaluation of the GeneXpert MTB/RIF assay for rapid diagnosis oftuberculosis and detection of rifampin resistance in pulmonary and extrapulmonary specimens. J Clin Microbiol2011; 49: 4138–4141.9 Opota O, Senn L, Prod’hom G, et al. Added value of molecular assay Xpert MTB/RIF compared to sputum smearmicroscopy to assess the risk of tuberculosis transmission in a low-prevalence country. Clin Microbiol Infect 2016;22: 613–619.10 World Health Organization. Tuberculosis Laboratory Safety Manual. Geneva, World Health Organization, 2012.http://apps.who.int/iris/bitstream/handle/10665/77949/9789241504638_eng.pdf;jsessionid=A248722DEDD0D0C0B6ABC546AA389ADF?sequence=1 Date last accessed: September 11, 2018.11 Drain PK, Garrett NJ. The arrival of a true point-of-care molecular assay-ready for global implementation? LancetGlob Health 2015; 3: e663–e664.https://doi.org/10.1183/23120541.00165-2018 4ORIGINAL RESEARCH LETTER | H.D. GLIDDON ET AL.

A sputum sample processing method for community and mobile tuberculosis diagnosis using the Xpert MTB/RIF assay

http://discovery.ucl.ac.uk/10066815/1/A%20sputum%20sample%20processing%20method%20for%20community%20and%20mobile%20tuberculosis%20diagnosis%20using%20the%20Xpert%20MTB%3ARIF%20assay.pdf

A sputum sample processing method for community and mobile tuberculosis diagnosis using the Xpert MTB/RIF assay

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