WO2022101161A2 - Point-of-care test for a group of individuals - Google Patents

Point-of-care test for a group of individuals Download PDF

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Publication number
WO2022101161A2
WO2022101161A2 PCT/EP2021/081026 EP2021081026W WO2022101161A2 WO 2022101161 A2 WO2022101161 A2 WO 2022101161A2 EP 2021081026 W EP2021081026 W EP 2021081026W WO 2022101161 A2 WO2022101161 A2 WO 2022101161A2
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WO
WIPO (PCT)
Prior art keywords
sample
individuals
swabs
disease
nucleic acid
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PCT/EP2021/081026
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French (fr)
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WO2022101161A3 (en
Inventor
Christofer Toumazou
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Dnanudge Limited
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Publication date
Priority claimed from GB2017742.4A external-priority patent/GB2600774A/en
Priority claimed from US17/094,190 external-priority patent/US11384403B2/en
Application filed by Dnanudge Limited filed Critical Dnanudge Limited
Priority to CN202180089635.1A priority Critical patent/CN116724123A/en
Publication of WO2022101161A2 publication Critical patent/WO2022101161A2/en
Publication of WO2022101161A3 publication Critical patent/WO2022101161A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to a point-of-care test for a group of individuals. It is applicable in particular, though not necessarily, to tests for SARS-CoV-2.
  • RT-PCR Reverse Transcriptase PCR
  • the present teaching is based on studies conducted by the applicant into testing more than one sample at the same time.
  • a method of testing a group of individuals for the presence of a disease or condition comprises: providing a sputum sample from each individual of the group of individuals; collecting and pooling a portion of each sample (i.e. each individual’s sample), using a device, the device comprising an absorbent material for absorbing at least some of the portion of each sample (in turn, i.e.
  • a negative result from the analysis indicates that said one or more nucleic acid sequence(s) is not present in the pooled sample and each individual of the group of individuals does not have the disease or condition and a positive result indicates that said one or more nucleic acid sequence(s) is present in the pooled sample and one or more of each individual of the group of individuals may have the disease or condition.
  • a method of testing a group of two or more individuals for the presence of a disease or condition comprises transferring a biological sample from each of said individuals onto one of a plurality of swabs so that each of the swabs comprises a biological sample from at least one of said individuals.
  • the swabs are inserted into a receiving chamber of an analyser so that all of the swabs are present together in the receiving chamber and a pooled sample is formed in the receiving chamber.
  • the analyser is capable of analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition.
  • a negative result from the analysis indicates that said one or more nucleic acid sequence(s) is not present in the pooled sample and each individual of the group of individuals does not have the disease or condition.
  • a positive result indicates that said one or more nucleic acid sequence(s) is present in the pooled sample and one or more of each individual of the group of individuals has the disease or condition.
  • Analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition may comprise amplifying, if present in the pooled sample, the or each of the nucleic acid sequence(s), wherein the or each nucleic acid sequence may be DNA sequences.
  • analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition may comprise deriving from the or each nucleic acid sequence, if present in the pooled sample, one or more other nucleic acid sequence(s) and amplifying the or each of the derived other nucleic acid sequence(s), wherein the or each first mentioned nucleic acid sequence may be RNA sequences and said one or more other nucleic acid sequences may be DNA sequences.
  • the method may comprise, when all of the swabs are present together in the receiving chamber, introducing a lysis buffer into the receiving chamber to release nucleic acids into the lysis buffer to form the pooled sample.
  • the lysis buffer may be forced into the receiving chamber under pressure generated using a pneumatic pressure unit and the method may comprise measuring the pressure generated by the pneumatic pressure unit as the lysis buffer is introduced and stopping introduction of the lysis buffer when a predetermined pressure is reached.
  • the method may further comprise measuring a value indicative of the volume of lysis buffer introduced into the receiving chamber and determining from said value, one or more control parameters for controlling the pneumatic pressure unit to generate a pressure to force the lysis buffer from the receiving chamber through a frit or filter, whereby the released nucleic acids bind to said frit or filter.
  • the one or more control parameters may be used to control one or more of a distance moved by a piston of the pneumatic pressure unit, an amount of pressure generated by the pneumatic pressure unit, and an amount of time for which to maintain the pressure.
  • the analyser may comprise a removable single-use cartridge in which the receiving chamber is provided.
  • the cartridge may comprise a further chamber containing a lysis buffer used to form the pooled sample.
  • the swabs may be inserted into the receiving chamber before the cartridge is inserted into or attached to the analyser.
  • the swabs may be inserted into the receiving chamber through a port that is sealed before the cartridge is inserted into or attached to the analyser.
  • the cartridge may comprise a plurality of circumferentially spaced chambers, including the receiving chamber, arranged about a central chamber.
  • the central chamber is rotatable relative to the other chambers to bring one or more openings of the central chamber into selective alignment with an opening of each of the other chambers to allow transfer of fluid between the central chamber and a selected one of the other chambers.
  • the frit or filter may be provided in an opening of the central chamber.
  • the biological sample of each of said individuals may be collected using a different one of said swabs. At least one of the swabs may comprise biological samples from more than one of said individuals.
  • the step of transferring a biological sample from each of said individuals onto one of a plurality of swabs may comprise collecting sputum samples from the individuals.
  • the number of swabs present together in the receiving chamber may be from 2 to 10.
  • the method according to the first or the second aspect may comprise retaining a biological sample of each of said individuals or collecting a further biological sample from each of said individuals. If a positive result is obtained, the retained portions or the further biological samples are subjected to analysis, using the or further analyser(s) to detect which of the retained portions or further biological samples contain said one or more sequences associated with the disease or condition and thereby identify which individuals of the group of individuals has the disease or condition.
  • the disease or condition may be a disease or condition that affects the respiratory tract.
  • the disease or condition, which affects the respiratory tract may be an infection, such as a bacterial, viral or fungal infection.
  • the infection may be a viral infection and the virus may be a common cold, influenza, respiratory syncytial, adeno, or corona virus.
  • the virus may be a corona virus including SARS, MERS and COVID-19 (SARS-CoV-2) virus.
  • the one or more nucleic acids to be detected may comprise at least 2, 3, 4, 5, 6, 7, or 8 specific nucleic acid sequences which are specific to the disease or condition.
  • the one or more nucleic acids to be detected may comprise at most 4, 6, 8, 10, or 12 specific nucleic acid sequences which are specific to the disease or condition.
  • the one or more nucleic acids may encode a native or mutant protein associated with the disease or condition.
  • the one or more nucleic acids may be, or may include the rdrp-l P2, rdrp-IP4, e-gene, n1 , n2, and/or n3 gene(s), or specific fragments thereof.
  • the group of individuals may be asymptomatic.
  • the group of individuals may be asymptomatic emergency hospital admissions, staff and/or residents in care homes, family and/or support groups, workplace and/or conference groups, film and/or television production teams, theatre and/or concert production teams, sports teams, and staff and/or students in pre-school, school, college or university.
  • the groups may comprise at least 2 individuals and up to 20 individuals, such as 2 - 20, 2 - 15, 2 — 12, 2 — 10, 2 — 8, 2 - 6, or 2 - 4, as well as any integers and ranges in between.
  • the swabs may be buccal, nasopharyngeal, or oropharyngeal swabs.
  • some or all of the swabs may be nasopharyngeal or oropharyngeal swabs.
  • the method according to the first or the second aspect may preferably be a point-of-care testing method, in particular a testing method that can be performed outside a dedicated laboratory setting, such that the sample can tested at the same site (geographic location, e.g. hospital or care home) at which it was obtained.
  • a method of managing a disease outbreak comprises using the method as described above in connection with the first aspect to determine whether one or more individuals in a group of two or more individuals has said disease. If such a determination is made, the result (e.g. an electronic communication of the result) is sent to the individuals of the group identified as having the disease via an electronic communication device (e.g. a mobile phone or personal computer device) together with an instruction to isolate.
  • an electronic communication device e.g. a mobile phone or personal computer device
  • kits for use in a method according to any preceding claim comprises two or more swabs, each swab being for collecting a biological sample from a respective individual in a group of two or more individuals.
  • Each swab has a corresponding sealable enclosure device comprising indicia, or a label, specifically designed to identify the biological sample with the individual from whom it was collected.
  • the kit also comprises instructions explaining how to take a biological sample.
  • the kit may comprise two or more of said swabs and two or more of said sealable enclosure devices comprising the indicia, or the label specifically designed to identify the biological sample with the individual from whom it was collected.
  • the kit may further comprise a further sealable enclosure device for receiving the two or more swabs and comprising indicia, or one or more labels, specifically designed to identify the further sealable enclosure with the individuals in said group and instructions to place the two or more swabs in the further sealable enclosure.
  • an analyser for testing a group of two or more individuals for the presence of a disease or condition.
  • the analyser is configured or configurable to analyse a pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition.
  • the analyser comprises: a receiving chamber into which two or more swabs can be inserted; a fluid transport system for delivering a lysis buffer to the receiving chamber in order to form the pooled sample by releasing nucleic acid sequences into the lysis buffer and comprising a pneumatic pressure unit for generating pressure to force the lysis buffer into the receiving chamber and a pressure sensor for measuring the pressure generated by the pneumatic pressure unit during delivery of the lysis buffer; and a control unit configured or configurable to control the fluid transport system to stop delivery of the lysis buffer once the pressure measured by the pressure sensor exceeds a predetermined value.
  • the receiving chamber and at least a portion of the fluid transport system may comprise a single use cartridge.
  • the analyser may comprise a frit or filter to which to reversibly bind nucleic acid sequences, the fluid transport system being configurable to force the lysis buffer containing the pooled sample through the frit or filter under pressure generated by the pneumatic pressure unit.
  • the control unit is further configured or configurable to: measure a value indicative of the volume of lysis buffer delivered to the receiving chamber; determine from said value one or more control parameters for controlling the pneumatic pressure unit; and control the generation of pressure by the pneumatic pressure unit according to said one or more control parameters.
  • the one or more control parameters may be used to control one or more of a distance moved by a piston of the pneumatic pressure unit, an amount of pressure generated by the pneumatic pressure unit, and an amount of time for which to maintain the pressure.
  • a method a testing a group of individuals for the presence of a disease or condition comprising: providing a sputum sample from each individual of the group of individuals; collecting and pooling a portion of each sample, using a device, the device comprising an absorbent material for absorbing at least some of the portion of each sample in order to form a pooled sample absorbed to the absorbent material of the device; transferring at least a portion of the pooled sample from the absorbent material of the device to an analyser capable of analysing the transferred pooled sample or portion of the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition; and wherein a negative result from the analysis indicates that said one or more nucleic acid sequence(s) is not present in the pooled sample and each individual of the group of individuals does not have the disease or condition and a positive result indicates that said one or more nucleic acid sequence(s) is present in the pooled sample and one or more of each individual of the group of individuals may
  • analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition comprises amplifying, if present in the pooled sample, the or each of the nucleic acid sequence(s), wherein the or each nucleic acid sequence may be DNA sequences.
  • analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition comprises deriving from the or each nucleic acid sequence, if present in the pooled sample, one or more other nucleic acid sequence(s) and amplifying the or each of the derived other nucleic acid sequence(s), wherein the or each first mentioned nucleic acid sequence may be RNA sequences and said one or more other nucleic acid sequences may be DNA sequences.
  • virus is a corona virus including SARS, MERS and COVID-19 (SARS-CoV-2) virus.
  • the corona virus is COVID-19 (SARS-CoV-2).
  • the one or more nucleic acids to be detected comprises at least 2, 3, 4, 5, 6, 7, or 8 specific nucleic acid sequences which are specific to the disease or condition.
  • nucleic acids to be detected comprises at most 4, 6, 8, 10, or 12 specific nucleic acid sequences which are specific to the disease or condition.
  • nucleic acids encodes a native or mutant protein associated with the disease or condition.
  • nucleic acids is, or includes the rdrp-l P2, rdrp-IP4, e-gene, n1 , n2, and/or n3 gene(s), or specific fragments thereof.
  • nucleic acid which acts as the positive control is the ribonuclease P gene or specific fragment thereof.
  • the groups comprises at least 2 individuals and up to 40 individuals, such as 2 - 40, 2 -30, 2 - 20, 2 - 15, 2 - 12, 2 - 10, 2 - 8, 2 - 6, or 2 - 4, as well as any integers and ranges in between.
  • the device is in the form of a swab, such as a buccal, nasopharyngeal, or oropharyngeal swab.
  • a method of managing a disease outbreak comprising: using the method of any preceding clause to determine whether one or more individuals of a group of individuals has said disease or condition; if such a determination is made, sending the result to the individuals of the group identified as having the disease or condition via an electronic communication device together with an instruction to isolate.
  • kits for use in a method according to any preceding clause comprising: two or more sample receptacles for collecting a respective sputum sample from each of two or more individuals in a group of individuals, each receptacle comprising indicia, or a label specifically designed to identify the sputum sample, with the individual from which is was obtained; and instructions explaining how to take a sputum sample.
  • kit according to clauses 24 or 25 further comprising a single pool collection swab comprising an absorbent material for absorbing and pooling a portion of each sample from all individuals in the group of individuals.
  • 27 The kit according to either of clauses 25 - 26, further comprising instructions explaining how to take a sputum sample and/or how obtain a pooled sample.
  • Figure 1 NudgeBox (28 x 15-5 x 13-5 cm; 5 kg) and DnaCartridge (25 x 78 x 85 mm; 40 g);
  • Figure 2 Cartridge with IsohelixTM swab (with stopper and bung) inserted into the sample chamber;
  • Figure 3 Number of replicates amplifying on the DnaNudgeTM platform for nasopharyngeal and sputum samples.
  • the data does not suggest that nasopharyngeal or sputum samples present a consistently higher viral load, with significant patient-to- patient variation.
  • Figures 4A-C PCR amplification curves from the DnaNudge platform for a pool of 1 positive and 39 negative samples. For test one ( Figures 4A and 4B), one e-gene replicate and one n2-gene replicate amplified. In the second test ( Figure 4C), two n1 - gene replicates amplified. Curves with no replicates amplifying are not shown. The positive sample was in position 2 in the pool for both tests;
  • Figure 5 Number of replicates amplifying for individual and 10-pooled positive samples. Pooled samples in all cases had lower number of replicates amplifying than the original individual samples;
  • FIG. 6 Relative efficiency (in terms of average number of tests performed) for various pooling strategies, as compared to individual testing only. Pools of up to 12 were analysed as the maximum recommended by UK NHS [4], For the scenarios simulated, all pooling strategies have an efficiency greater than 1 for prevalence up to 10%. In all cases simulated, the nested pooling scenario results in a higher efficiency than the simple pooling case; however the trade-off is a longer wait for the final confirmatory result in case of a positive result in one or more of the pooled samples.
  • Figure 7 For prevalence below 2%, pooling of up to 12 samples has a high probability (>75%) of returning a negative result. This would result in a significant increase of testing throughput in an asymptomatic patient cohort. As prevalence increases, to maintain high efficiency of pooling, the pool size should decrease. At 5% prevalence, to maintain a 75% probability of a negative first test, the pool size should decrease to 5.
  • Figure 8 Photograph of a DnaCartridge into which four swabs have been inserted.
  • Figure 9 Schematic cross section view of a DnaCartridgeTM.
  • Figure 10 System diagram of a NudgeBoxTM and DnaCartridgeTM.
  • Figure 1 1 Flow diagram of the steps used to prepare the pooled sample prior to PCR.
  • Figure 12 Flow diagram of the steps used for releasing the DNA/RNA by lysis of virus collected on the swabs.
  • the DnaNudgeTM CovidNudgeTM test is a point-of-care, real-time RT-PCR test that provides a sample-to-answer diagnosis of SARS-CoV-2 without the need for any laboratory facilities or sample preparation. This is described in detail in more detail and with reference to WO2108055407 and Gilbani and Toumazou [2] to which the skilled reader is directed.
  • the platform comprises a single-use DnaCartridgeTM and a processing unit (the NudgeBoxTM) illustrated in Figure 1.
  • the DnaCartridge is a disposable, sealed, and integrated lab-on-chip device that enables sample-to-result PCR.
  • the DnaCartridge consists of two main parts: an amplification unit (AU) and a sample preparation unit (SPU).
  • AU amplification unit
  • SPU sample preparation unit
  • a nasopharyngeal swab sample is inserted directly into the swab chamber of the sample preparation unit immediately after collection. The swab is broken, leaving the swab tip and the sample within the chamber, which is then sealed.
  • Cartridges are placed in the NudgeBox processing unit, which provides the pneumatic, thermal, imaging, and mechanics required to run a real-time RT-PCR reaction outside of a laboratory setting.
  • the sample preparation unit consists of chambers containing buffers to extract and purify RNA from the swab sample, as well as a lyophilised RT-PCR mastermix to mix with the extracted RNA.
  • the DnaCartridgeTM fits on top of a motor-driven spigot in the NudgeBoxTM, which rotates the SPU through each stage of sample processing before filling the wells of the AU, inside which the RT-PCR reaction takes place.
  • SARS-CoV-2 SARS-CoV-2
  • diseases or condition which can be detected by way of one or more nucleic acid sequences which are associated with the disease or condition to be detected.
  • examples include respiratory conditions, especially infections caused by bacterial, viral or fungal agents.
  • the AU comprises dried primers and probes uniquely spotted into 72 reaction wells enabling multiplex analysis.
  • the array comprises six viral targets (rdrp-l P2, rdrp-IP4, e- gene, n1 , n2, and n3) and one host gene as a sample adequacy control (Ribonuclease P, RNaseP). Each gene target has nine or ten technical replicates within the array, while the human control has six replicates.
  • results from the DnaNudge test are transmitted to the Cloud where an algorithm is run to determine the results [2], These results are transmitted back to an Operator app run on an iPad, as well as being sent onwards to hospital IT systems, and are reported as positive, negative, indeterminate, invalid or aborted according to Table 1.
  • the single-use cartridge is disposed of following standard laboratory disposal procedures.
  • the diagnostic accuracy of the CovidNudge test was assessed in April and May 2020 by comparing nasopharyngeal swab samples from individuals at three hospitals in London and Oxford against nasal and throat swabs tested on laboratory RT-PCR platforms [2], The sensitivity of the DnaNudge point-of-care test compared with laboratory-based testing was 94% (95% Cl 86-98) with a specificity of 100% (95% Cl 99-100). Following this clinical validation, the CovidNudge test achieved the CE Mark in July 2020 enabling the technology to be used as standard of care in UK healthcare settings. Since July, over 20,000 patient samples have been tested on the CovidNudge platform across 8 separate hospital sites in London.
  • CovidNudge as a point-of-care diagnostic has enabled effective triage and timely therapeutic and infection control interventions for emergency admission patients in clinical areas including adult and paediatric A&E, maternity, mental health and renal transplantation, and the technology has been fully embedded as an integral part of the emergency admission pathway at the deployment sites.
  • test results available within 90 minutes of sample collection patients can be admitted into the appropriate care pathway bypassing the need for isolation if test results are negative, while enabling sites to meet operational targets to admit, transfer or discharge patients from A&E within 4 hours.
  • the first method is to pool aliquots of transport media each containing a single patient sample (sample pooling), while the second method is to combine swabs from multiple patients into a single volume of transport media (swab pooling).
  • sample pooling the individual patient samples are separately preserved, so that if a pooled test reports a positive result, the patient samples needed for the individual follow-up tests already exist.
  • the disadvantage is that, since the volume of each sample is reduced, the analytical sensitivity is decreased (i.e. limit of detection (LOD) increases) because individual samples are further diluted.
  • LOD limit of detection
  • Sputum samples were collected into a sample tube (Oragene500, DNAgenotek); the stabilising solution released by the Oragene collection tube has been shown to inactivate the SARS-CoV-2 virus due to the presence of an ionic detergent which renders ineffective enveloped viruses such as SARS-Cov-2 [5].
  • RNA/DNA buccal swab (SK-2, Isohelix) was used. The cap from the swab was removed while retaining the bung with stopper, and the swab was mixed in the sputum in stabilising solution by rubbing gently for 5 seconds to get a good sputum sample on the swab.
  • any excess sputum residue hanging from the swab was removed by wiping the swab gently against the inside edge of the tube.
  • the Isohelix swab was then inserted into the cartridge pressing the stopper in place, the swab tail was removed leaving the swab in the chamber, and the cartridge was sealed using the Isohelix bung ( Figure 2).
  • the cartridge was then inserted into the NudgeBox and a test run following standard procedure [2], 292 paired samples were obtained, and results are shown in Table 2.
  • the pooled samples were dipped in turn with a single Isohelix swab following the method outlined in Appendix 2, then inserted into a DnaCartridge and tested following standard procedure [2], The position of the positive sample within the pool was varied to ensure that all possible permutations were tested (i.e. positive sample in first position, second position, ninth position, tenth position), with at least two tests per ‘position’ of the positive sample.
  • 200 negative samples were also divided into pools of 10 and tested, and results are shown in Table 3. while the positive and indeterminate results were tested as one positive sample with nine negative samples. An indeterminate result is when only one or two replicates amplify, i.e. the signal is at the limits of detection.
  • a negative sputum sample was spiked with viral genetic material (Microbiologies HE0062S process control pellet) dissolved in molecular water.
  • the viral solution was serially diluted and aliquots of 25uL were added to a 25uL sputum sample and absorbed onto an Isohelix swab for testing.
  • the LOD was measured as 250 copies per swab; this rose to 1250 copies per swab for 10 pool testing.
  • the 5-fold increase in LOD for a 10-pool test demonstrates that dipping a swab into successive sputum samples provides less sample dilution than would be expected through traditional sample pooling, where an n-fold reduction in viral concentration would be expected for an n-pool test.
  • Nested pooled test in this scenario if the pooled test is positive, smaller patient pools are repeated to narrow down the search for the positive result(s).
  • a nested pooled test can be denoted as ( n1
  • nx ), where n1 > n2 > n3 ... > nx, and where n1 denotes the size of the first pool, n2, n3 etc. are the sizes of subsequent sub-pools, and nx 1 . So a pooling strategy of ( 12
  • 1 ) would start with an initial pool size of 12, followed by 4 pooled tests of 3 samples each, followed by individual testing of any of the n 3 sub-pools that had tested positive. Using this nomenclature, the single pooled test can be considered as a nested pool of ( n
  • Figure 7 shows the probability of the first pooled test returning a negative result, as a function of prevalence.
  • an alternative or complementary approach to sample pooling is to create a pooled sample by introducing multiple swabs into a single volume of transport media. This can be achieved using the DnaNudge platform by introducing the multiple swabs into the sample (or “receiving”) chamber of the DnaCartridge.
  • nasopharyngeal or oropharyngeal swabs can be used to collect samples from individuals in the usual way and the swab tips inserted through the swab port (inlet) of the sample chamber one after another, as shown in Figure 8. The tails of each of the swabs are then removed, e.g.
  • pooled sample is then formed by flooding the sample chamber with a lysis buffer and the pooled sample tested as described above. Surprisingly, this form of pooled testing has been found to be effective without requiring extensive modifications to the DnaNudge platform.
  • Figure 9 shows a cross sectional view of a DnaCartridge 900 comprising the swab port 901 opening into the sample chamber 903, a rotatable central chamber 905 and a lysis buffer chamber 907.
  • the swab tips introduced into the sample chamber through the swab port 901 are not shown for clarity.
  • a pooled sample is created by introducing lysis buffer from the lysis buffer chamber 907 into the sample chamber 903 to release DNA and/or RNA present in virus on each of the swab tips into solution by breaking down membranes of the virus.
  • the lysis buffer is first drawn into the central chamber 905 from the lysis buffer chamber 907 using a pneumatic pressure unit (as described below in connection with Figure 10).
  • the lysis buffer is then introduced into the sample chamber 903 by using the pneumatic pressure unit to apply a positive relative pressure between the central chamber 905 and the sample chamber 903.
  • the pneumatic pressure unit comprises a pressure sensor that is used to monitor the pressure opposing the flow of lysis buffer into the sample chamber 903. When the pressure exceeds a predetermined threshold, the flow of lysis buffer is stopped to ensure that the sample chamber 903 is not overfilled with lysis buffer. Any lysis buffer remaining in the central chamber 905 is then expelled to a waste chamber or returned to the lysis buffer chamber 907.
  • the NudgeBox system can be used for different numbers of swab tips and/or different sizes of swab tip without needing to be reconfigured.
  • the system is able to automatically adjust the amount of lysis buffer delivered to the sample chamber 903 to account for the volume of the sample chamber 903 that has been taken up by the swab tip(s).
  • the amount of lysis buffer transferred to the sample chamber 903 is determined, e.g. by monitoring the length of time for which the pressure is applied by the pneumatic pressure unit or, the distance moved by a piston (e.g. syringe plunger) within a pneumatic pressure unit.
  • the lysis buffer is permitted to remain in the sample chamber 903 until the DNA and/or RNA has been released from the swab tips.
  • the lysis buffer containing the extracted DNA and/or RNA is then returned to the central chamber 905 by using the pneumatic pressure unit to applying a negative relative pressure between the central chamber 905 and the sample chamber 903.
  • the lysis buffer flows through a silica frit (not shown in Figure 9, but visible in Figure 8) as it re-enters the central chamber 905, causing the DNA and/or RNA to adhere to the frit.
  • the pneumatic pressure unit is operated such that a large majority (e.g. greater than 90%) or all of the lysis buffer is transferred back to the central chamber 905, but without exposing the frit to conditions that would damage it.
  • the negative pressure may be applied for only a limited period, which is determined from the amount of lysis buffer transferred to the sample chamber 903, to avoid maintaining the negative relative pressure across the frit for too long after lysis buffer has been removed from the sample chamber 903.
  • the magnitude of the negative pressure may be varied according to the amount of lysis buffer that needs to be transferred.
  • swabs with smaller tip sizes allows more of them to be accommodated within the sample chamber 903 and therefore greater pooling of the samples. It may therefore be preferable to use nasopharyngeal or oropharyngeal swabs rather than larger buccal swabs, although combinations of different types of swab can also be used, e.g. depending on swab availability or factors that favour the use of one swab over another for different individuals. It is also possible to use one or more pooled samples created by the sputum pooling method described above.
  • a swab used to create a pooled sample from one or more sputum samples can be combined with one or more nasopharyngeal or oropharyngeal swab, which may allow individuals who were not able to provide a sputum sample to be included in the pooled test.
  • multiple swabs with pooled samples created for different groups of individuals by sputum pooling can be combined, which may facilitate sample collection, such as in circumstances where the group of individuals needing to be tested are in different locations, e.g. hospital wards or college dormitories.
  • FIG 10 shows a system diagram of the analyser 1000 (a NudgeBox in this case), which receives the DnaCartridge 1001 in order to test the pooled sample.
  • the analyser comprises a mechanical unit 1003, a pressure unit 1005, thermal unit 1007 temperature control of the PCR, an imaging unit 1009 for measuring fluorescence produced by the test and a control unit 101 1 that controls each of the other units when the test is being performed.
  • the pressure unit 1005 in this example comprises a syringe 1013, a pressure sensor 1015 and a valve 1017 that is used to isolate the pressure unit from the DnaCartridge when the pressure unit is not being used.
  • the mechanical unit rotates the central chamber 905, to place it in fluid communication with both the syringe and one of the other chambers of the DnaCartridge 1001 (e.g. the sample chamber 903 or the lysis buffer chamber 907).
  • a piston or plunger
  • the pressure sensor 1015 is used to monitor the pressure within the syringe 1013, e.g. by measuring the force required to move the piston.
  • Figure 11 shows the steps for preparing the pooled sample prior to amplification of DNA from the pooled sample by PCR, the DNA being present initially in the pooled sample or derived from RNA present in the pooled sample by reverse transcription.
  • Step 1 101 Deliver lysis buffer to the swab chamber to release DNA and/or RNA contained in virus collected on the swabs from each of the individuals.
  • Step 1 102 Pass the lysed sample through a frit to capture the DNA/RNA on the frit.
  • Step 103 Pass a wash buffer through the frit to clean non-DNA and non-RNA debris from the frit.
  • Step 1 104 Pass an elution buffer through the frit to release the DNA/RNA from the frit.
  • Step 1 105: Reconstitute lyophilized mastermix with the elution buffer containing the DNA/RNA.
  • Step 1106 Fill an analysis unit with the DNA/RNA, mastermix, and elution buffer.
  • Step 1107 (optional): Perform reverse transcription of the RNA to form DNA corresponding to the RNA. • Step 1108. Start the PCR amplification of the DNA.
  • Figure 12 shows the steps used for releasing the DNA/RNA by lysis of the virus collected on the swabs.
  • Step 1201 Rotate the mixing chamber to align its port with the port of the lysis buffer chamber.
  • Step 1202. De-pressurise the mixing chamber to create a vacuum and let the lysis buffer into the mixing chamber.
  • Step 1203. Rotate the mixing chamber to align its port with the port of the swab chamber.
  • Step 1204 Pressurise the mixing chamber to force the lysis buffer out and to the swab chamber; measure the pressure and the time to calculate volume and pressure in the swab chamber; record the time and/or volume reaching a set threshold pressure.
  • Step 1205. Rotate back the mixing chamber to align its port with the port of the lysis buffer chamber.
  • Step 1206 Dispense the retained lysis buffer back to the lysis chamber.
  • Step 1207 Wait for a certain time (e.g. 1 min) to let the lysis buffer extract DNA/RNA from the swabs.
  • Step 1208 Rotate the mixing chamber to align its port with the port of the swab chamber.
  • Step 1209 De-pressurise the mixing chamber to create a vacuum and let the lysis buffer from the swab chamber enter the mixing chamber through the frit and so that the lysed DNA/RNA material binds to the frit. Set the negative pressure to a value based on the recorded time/volume during the lysis dispense step (step 1204 above).
  • Step 1210 Rotate the mixing chamber to ward a waste chamber to dispense the lysis while the DNA/RNA material is bound to the frit.
  • Isohelix swab is dipped into each of the pooled samples in turn, and the dipped samples are then discarded as contaminated. If a positive result is returned from the pool test, the Isohelix swabs for the individual samples within the pool are available for retesting.
  • Oragene 500 sample collection tune (DNAgenotek) i.
  • the healthcare professional should prepare the patient for the procedure by asking them to sit upright, rinse their mouth with water and spit out prior to sputum collection.
  • ii. The patient should be asked to take a few deep breaths to help loosen secretions; please note, if patient is on a nebuliser, give nebuliser first and wait 10 minutes before taking a sample.
  • iii The patient should cover their mouth before forcing out a deep cough to release the sputum. Sputum should be collected in the sample tube provided. Ideally the sputum sample should be no less than the size of a small fingernail. iv.
  • Method i Obtain a number (up to 10) of individual sputum samples following steps (i) - (vi) in Appendix 1 . Samples should be labelled as known positive or negative.
  • ii. Position all sputum sample tubes in a line using a test tube rack or similar receptacle holder and unscrew the lids of each of the tubes.
  • iii. Remove cap from Isohelix swab retaining the bung with stopper, and gently dip the swab in the first sputum sample for 5 seconds, performing 2 gentle rubs for each side of the swab against the sputum and the inside of the tube.

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Abstract

The present invention relates to the use of pooled samples to optimise the efficiency and utility of a rapid, lab-free point-of-care test. It is applicable in particular, though not necessarily, to tests for SARS-CoV-2.

Description

POINT-OF-CARE TEST FOR A GROUP OF INDIVIDUALS
Technical Field
The present invention relates to a point-of-care test for a group of individuals. It is applicable in particular, though not necessarily, to tests for SARS-CoV-2.
Background
Despite the emergence of new rapid serological tests for COVID-19, molecular testing such as Reverse Transcriptase PCR (RT-PCR) remains the standard of care for detection of SARS-CoV-2 due to its higher sensitivity and specificity [1]. However, standard laboratory RT-PCR can be time consuming and requires samples to be processed in centralised laboratory facilities. Factoring in sample transportation time and the requirement to process samples in batches means that the turnaround time for laboratory RT-PCR testing can often exceed 24 hours. Point-of-care diagnostic tests which can be run outside of traditional laboratory settings have the potential to accelerate clinical decision making and enable effective triage and infection control measures in frontline clinical and community settings.
Summary
The present teaching is based on studies conducted by the applicant into testing more than one sample at the same time.
According to a first aspect of the present invention there is provided a method of testing a group of individuals for the presence of a disease or condition. The method comprises: providing a sputum sample from each individual of the group of individuals; collecting and pooling a portion of each sample (i.e. each individual’s sample), using a device, the device comprising an absorbent material for absorbing at least some of the portion of each sample (in turn, i.e. one after another) in order to form a pooled sample absorbed to the absorbent material of the device; transferring at least a portion of the pooled sample from the absorbent material of the device to an analyser and using the analyser to analyse the transferred pooled sample or portion of the pooled sample for the presence of said one or more nucleic acid sequences associated with the disease or condition. A negative result from the analysis indicates that said one or more nucleic acid sequence(s) is not present in the pooled sample and each individual of the group of individuals does not have the disease or condition and a positive result indicates that said one or more nucleic acid sequence(s) is present in the pooled sample and one or more of each individual of the group of individuals may have the disease or condition.
According to a second aspect of the present invention, there is provided a method of testing a group of two or more individuals for the presence of a disease or condition. The method comprises transferring a biological sample from each of said individuals onto one of a plurality of swabs so that each of the swabs comprises a biological sample from at least one of said individuals. The swabs are inserted into a receiving chamber of an analyser so that all of the swabs are present together in the receiving chamber and a pooled sample is formed in the receiving chamber. The analyser is capable of analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition. A negative result from the analysis indicates that said one or more nucleic acid sequence(s) is not present in the pooled sample and each individual of the group of individuals does not have the disease or condition. A positive result indicates that said one or more nucleic acid sequence(s) is present in the pooled sample and one or more of each individual of the group of individuals has the disease or condition.
Analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition may comprise amplifying, if present in the pooled sample, the or each of the nucleic acid sequence(s), wherein the or each nucleic acid sequence may be DNA sequences. Alternatively, analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition may comprise deriving from the or each nucleic acid sequence, if present in the pooled sample, one or more other nucleic acid sequence(s) and amplifying the or each of the derived other nucleic acid sequence(s), wherein the or each first mentioned nucleic acid sequence may be RNA sequences and said one or more other nucleic acid sequences may be DNA sequences.
The method may comprise, when all of the swabs are present together in the receiving chamber, introducing a lysis buffer into the receiving chamber to release nucleic acids into the lysis buffer to form the pooled sample.
The lysis buffer may be forced into the receiving chamber under pressure generated using a pneumatic pressure unit and the method may comprise measuring the pressure generated by the pneumatic pressure unit as the lysis buffer is introduced and stopping introduction of the lysis buffer when a predetermined pressure is reached.
The method may further comprise measuring a value indicative of the volume of lysis buffer introduced into the receiving chamber and determining from said value, one or more control parameters for controlling the pneumatic pressure unit to generate a pressure to force the lysis buffer from the receiving chamber through a frit or filter, whereby the released nucleic acids bind to said frit or filter. The one or more control parameters may be used to control one or more of a distance moved by a piston of the pneumatic pressure unit, an amount of pressure generated by the pneumatic pressure unit, and an amount of time for which to maintain the pressure.
The analyser may comprise a removable single-use cartridge in which the receiving chamber is provided. Optionally, the cartridge may comprise a further chamber containing a lysis buffer used to form the pooled sample. The swabs may be inserted into the receiving chamber before the cartridge is inserted into or attached to the analyser. The swabs may be inserted into the receiving chamber through a port that is sealed before the cartridge is inserted into or attached to the analyser.
The cartridge may comprise a plurality of circumferentially spaced chambers, including the receiving chamber, arranged about a central chamber. The central chamber is rotatable relative to the other chambers to bring one or more openings of the central chamber into selective alignment with an opening of each of the other chambers to allow transfer of fluid between the central chamber and a selected one of the other chambers. The frit or filter may be provided in an opening of the central chamber.
The biological sample of each of said individuals may be collected using a different one of said swabs. At least one of the swabs may comprise biological samples from more than one of said individuals. The step of transferring a biological sample from each of said individuals onto one of a plurality of swabs may comprise collecting sputum samples from the individuals.
The number of swabs present together in the receiving chamber may be from 2 to 10.
The method according to the first or the second aspect may comprise retaining a biological sample of each of said individuals or collecting a further biological sample from each of said individuals. If a positive result is obtained, the retained portions or the further biological samples are subjected to analysis, using the or further analyser(s) to detect which of the retained portions or further biological samples contain said one or more sequences associated with the disease or condition and thereby identify which individuals of the group of individuals has the disease or condition.
The disease or condition may be a disease or condition that affects the respiratory tract. The disease or condition, which affects the respiratory tract may be an infection, such as a bacterial, viral or fungal infection. The infection may be a viral infection and the virus may be a common cold, influenza, respiratory syncytial, adeno, or corona virus. The virus may be a corona virus including SARS, MERS and COVID-19 (SARS-CoV-2) virus.
The one or more nucleic acids to be detected may comprise at least 2, 3, 4, 5, 6, 7, or 8 specific nucleic acid sequences which are specific to the disease or condition. The one or more nucleic acids to be detected may comprise at most 4, 6, 8, 10, or 12 specific nucleic acid sequences which are specific to the disease or condition. The one or more nucleic acids may encode a native or mutant protein associated with the disease or condition. The one or more nucleic acids may be, or may include the rdrp-l P2, rdrp-IP4, e-gene, n1 , n2, and/or n3 gene(s), or specific fragments thereof.
The group of individuals may be asymptomatic. The group of individuals may be asymptomatic emergency hospital admissions, staff and/or residents in care homes, family and/or support groups, workplace and/or conference groups, film and/or television production teams, theatre and/or concert production teams, sports teams, and staff and/or students in pre-school, school, college or university. The groups may comprise at least 2 individuals and up to 20 individuals, such as 2 - 20, 2 - 15, 2 — 12, 2 — 10, 2 — 8, 2 - 6, or 2 - 4, as well as any integers and ranges in between.
The swabs may be buccal, nasopharyngeal, or oropharyngeal swabs. For example, some or all of the swabs may be nasopharyngeal or oropharyngeal swabs.
The method according to the first or the second aspect may preferably be a point-of-care testing method, in particular a testing method that can be performed outside a dedicated laboratory setting, such that the sample can tested at the same site (geographic location, e.g. hospital or care home) at which it was obtained.
According to a third aspect of the present invention, there is provided a method of managing a disease outbreak. The method comprises using the method as described above in connection with the first aspect to determine whether one or more individuals in a group of two or more individuals has said disease. If such a determination is made, the result (e.g. an electronic communication of the result) is sent to the individuals of the group identified as having the disease via an electronic communication device (e.g. a mobile phone or personal computer device) together with an instruction to isolate.
According to a fourth aspect of the present invention, there is provided a kit for use in a method according to any preceding claim. The kit comprises two or more swabs, each swab being for collecting a biological sample from a respective individual in a group of two or more individuals. Each swab has a corresponding sealable enclosure device comprising indicia, or a label, specifically designed to identify the biological sample with the individual from whom it was collected. The kit also comprises instructions explaining how to take a biological sample.
For each of the individuals in the group, the kit may comprise two or more of said swabs and two or more of said sealable enclosure devices comprising the indicia, or the label specifically designed to identify the biological sample with the individual from whom it was collected. The kit may further comprise a further sealable enclosure device for receiving the two or more swabs and comprising indicia, or one or more labels, specifically designed to identify the further sealable enclosure with the individuals in said group and instructions to place the two or more swabs in the further sealable enclosure.
According to a fifth aspect of the present invention, there is provided an analyser for testing a group of two or more individuals for the presence of a disease or condition. The analyser is configured or configurable to analyse a pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition. The analyser comprises: a receiving chamber into which two or more swabs can be inserted; a fluid transport system for delivering a lysis buffer to the receiving chamber in order to form the pooled sample by releasing nucleic acid sequences into the lysis buffer and comprising a pneumatic pressure unit for generating pressure to force the lysis buffer into the receiving chamber and a pressure sensor for measuring the pressure generated by the pneumatic pressure unit during delivery of the lysis buffer; and a control unit configured or configurable to control the fluid transport system to stop delivery of the lysis buffer once the pressure measured by the pressure sensor exceeds a predetermined value. The receiving chamber and at least a portion of the fluid transport system may comprise a single use cartridge.
The analyser may comprise a frit or filter to which to reversibly bind nucleic acid sequences, the fluid transport system being configurable to force the lysis buffer containing the pooled sample through the frit or filter under pressure generated by the pneumatic pressure unit. The control unit is further configured or configurable to: measure a value indicative of the volume of lysis buffer delivered to the receiving chamber; determine from said value one or more control parameters for controlling the pneumatic pressure unit; and control the generation of pressure by the pneumatic pressure unit according to said one or more control parameters.
The one or more control parameters may be used to control one or more of a distance moved by a piston of the pneumatic pressure unit, an amount of pressure generated by the pneumatic pressure unit, and an amount of time for which to maintain the pressure.
Further examples of the present teaching are defined by the following numbered clauses.
1 . A method a testing a group of individuals for the presence of a disease or condition, the method comprising: providing a sputum sample from each individual of the group of individuals; collecting and pooling a portion of each sample, using a device, the device comprising an absorbent material for absorbing at least some of the portion of each sample in order to form a pooled sample absorbed to the absorbent material of the device; transferring at least a portion of the pooled sample from the absorbent material of the device to an analyser capable of analysing the transferred pooled sample or portion of the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition; and wherein a negative result from the analysis indicates that said one or more nucleic acid sequence(s) is not present in the pooled sample and each individual of the group of individuals does not have the disease or condition and a positive result indicates that said one or more nucleic acid sequence(s) is present in the pooled sample and one or more of each individual of the group of individuals may have the disease or condition. 2. The method according to clause 1 , wherein analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition comprises amplifying, if present in the pooled sample, the or each of the nucleic acid sequence(s), wherein the or each nucleic acid sequence may be DNA sequences.
3. The method according to clause 1 , wherein analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition comprises deriving from the or each nucleic acid sequence, if present in the pooled sample, one or more other nucleic acid sequence(s) and amplifying the or each of the derived other nucleic acid sequence(s), wherein the or each first mentioned nucleic acid sequence may be RNA sequences and said one or more other nucleic acid sequences may be DNA sequences.
4. The method according to clause 1 and comprising, prior to said step of collecting and retaining a portion of each sample, collecting and retaining a first portion of each sample using a separate device for each sample, each separate device comprising an absorbent material for absorbing the first portion of each said sample, wherein, when a positive result is obtained, subjecting the retained first portion of each sample to analysis, using the or further analyser(s) in order to detect which retained samples contain said one or more sequences associated with the disease or condition and thereby identify which individuals of the group of individuals has the disease or condition.
5. The method according to any preceding clause, wherein the disease or condition is a disease or condition, which affects the respiratory tract.
6. The method according to clause 5, wherein the disease or condition, which affects the respiratory tract is an infection, such as a bacterial, viral or fungal infection.
7. The method according to clause 6 wherein the infection is a viral infection and the virus is a common cold, influenza, respiratory syncytial, adeno, or corona virus.
8. The method according to clause 7 wherein the virus is a corona virus including SARS, MERS and COVID-19 (SARS-CoV-2) virus.
9. The method according to clause 8 wherein the corona virus is COVID-19 (SARS-CoV- 2). 10. The method according to any preceding clause, wherein the one or more nucleic acids to be detected comprises at least 2, 3, 4, 5, 6, 7, or 8 specific nucleic acid sequences which are specific to the disease or condition.
1 1 . The method according to any preceding clause, wherein the one or more nucleic acids to be detected comprises at most 4, 6, 8, 10, or 12 specific nucleic acid sequences which are specific to the disease or condition.
12. The method according to any preceding clause, wherein the one or more nucleic acids encodes a native or mutant protein associated with the disease or condition.
13. The method according to any of clauses 9 - 12, wherein the one or more nucleic acids is, or includes the rdrp-l P2, rdrp-IP4, e-gene, n1 , n2, and/or n3 gene(s), or specific fragments thereof.
14. The method according to any preceding clause wherein the method includes a positive control, to confirm that the sample or samples contains nucleic acid from the individual or individuals, not associated with the disease or condition and that the sample has been obtained correctly.
15. The method according to clause 14, wherein the nucleic acid, which acts as the positive control is the ribonuclease P gene or specific fragment thereof.
16. The method according to any preceding clause wherein the group of individuals are asymptomatic.
17. The method according to any preceding clause wherein the group of individuals are asymptomatic emergency hospital admissions, staff and/or residents in care homes, family and/or support groups, workplace and/or conference groups, film and/or television production teams, theatre and/or concert production teams, sports teams, and staff and/or students in pre-school, school, college or university.
18. The method according to any preceding clause, wherein the groups comprises at least 2 individuals and up to 40 individuals, such as 2 - 40, 2 -30, 2 - 20, 2 - 15, 2 - 12, 2 - 10, 2 - 8, 2 - 6, or 2 - 4, as well as any integers and ranges in between. 19. The method according to any preceding clause, wherein the device is in the form of a swab, such as a buccal, nasopharyngeal, or oropharyngeal swab.
20. The method according to clause 19, wherein the swab is an Isohelix™ swab.
21 . The method according to any preceding clause, wherein the and/or first portions of the sample are contacted with or inserted a cartridge which is to be introduced, or has been introduced into the analyser.
22. The method according to any preceding clause wherein the analyser is a NudgeBox™ analyser.
23. A method of managing a disease outbreak comprising: using the method of any preceding clause to determine whether one or more individuals of a group of individuals has said disease or condition; if such a determination is made, sending the result to the individuals of the group identified as having the disease or condition via an electronic communication device together with an instruction to isolate.
24. A kit for use in a method according to any preceding clause, the kit comprising: two or more sample receptacles for collecting a respective sputum sample from each of two or more individuals in a group of individuals, each receptacle comprising indicia, or a label specifically designed to identify the sputum sample, with the individual from which is was obtained; and instructions explaining how to take a sputum sample.
25. A kit for use in a method according to any of clauses 1 - 23, the kit comprising: two or more sample receptacles for collecting a respective sputum sample from each of two or more individuals in a group of individuals; and two or more swabs, each of said two or more swabs comprising an absorbent material for absorbing a first portion of said sample from each of said two or more individuals and two or more respective sealable enclosure devices for receiving said two or more swabs, wherein each receptacle and/or enclosure device comprises indicia, or a label specifically designed to identify the sputum sample, with the individual from which is was obtained.
26. The kit according to clauses 24 or 25 further comprising a single pool collection swab comprising an absorbent material for absorbing and pooling a portion of each sample from all individuals in the group of individuals. 27. The kit according to either of clauses 25 - 26, further comprising instructions explaining how to take a sputum sample and/or how obtain a pooled sample.
All embodiments described herein should not be construed as limiting and various alternatives will be evident to the skilled addressee. All papers and patents referred to herein and their entire contents are hereby incorporated by way of reference.
Brief Description of the drawings
Figure 1 : NudgeBox (28 x 15-5 x 13-5 cm; 5 kg) and DnaCartridge (25 x 78 x 85 mm; 40 g);
Figure 2: Cartridge with Isohelix™ swab (with stopper and bung) inserted into the sample chamber;
Figure 3: Number of replicates amplifying on the DnaNudge™ platform for nasopharyngeal and sputum samples. The data does not suggest that nasopharyngeal or sputum samples present a consistently higher viral load, with significant patient-to- patient variation. The average number of replicates across all samples was similar for both sputum (mean replicates = 24.7) and nasopharyngeal (mean replicates = 27.1 ), with the number of replicates amplifying ranging from (5 - 54) for sputum samples and (6 - 56) for nasopharyngeal samples;
Figures 4A-C: PCR amplification curves from the DnaNudge platform for a pool of 1 positive and 39 negative samples. For test one (Figures 4A and 4B), one e-gene replicate and one n2-gene replicate amplified. In the second test (Figure 4C), two n1 - gene replicates amplified. Curves with no replicates amplifying are not shown. The positive sample was in position 2 in the pool for both tests;
Figure 5: Number of replicates amplifying for individual and 10-pooled positive samples. Pooled samples in all cases had lower number of replicates amplifying than the original individual samples;
Figure 6: Relative efficiency (in terms of average number of tests performed) for various pooling strategies, as compared to individual testing only. Pools of up to 12 were analysed as the maximum recommended by UK NHS [4], For the scenarios simulated, all pooling strategies have an efficiency greater than 1 for prevalence up to 10%. In all cases simulated, the nested pooling scenario results in a higher efficiency than the simple pooling case; however the trade-off is a longer wait for the final confirmatory result in case of a positive result in one or more of the pooled samples. Figure 7: For prevalence below 2%, pooling of up to 12 samples has a high probability (>75%) of returning a negative result. This would result in a significant increase of testing throughput in an asymptomatic patient cohort. As prevalence increases, to maintain high efficiency of pooling, the pool size should decrease. At 5% prevalence, to maintain a 75% probability of a negative first test, the pool size should decrease to 5.
Figure 8: Photograph of a DnaCartridge into which four swabs have been inserted.
Figure 9: Schematic cross section view of a DnaCartridge™.
Figure 10: System diagram of a NudgeBox™ and DnaCartridge™.
Figure 1 1 : Flow diagram of the steps used to prepare the pooled sample prior to PCR. Figure 12: Flow diagram of the steps used for releasing the DNA/RNA by lysis of virus collected on the swabs.
Detailed Description
The DnaNudge™ CovidNudge™ test is a point-of-care, real-time RT-PCR test that provides a sample-to-answer diagnosis of SARS-CoV-2 without the need for any laboratory facilities or sample preparation. This is described in detail in more detail and with reference to WO2108055407 and Gilbani and Toumazou [2] to which the skilled reader is directed. The platform comprises a single-use DnaCartridge™ and a processing unit (the NudgeBox™) illustrated in Figure 1. The DnaCartridge is a disposable, sealed, and integrated lab-on-chip device that enables sample-to-result PCR. The DnaCartridge consists of two main parts: an amplification unit (AU) and a sample preparation unit (SPU). A nasopharyngeal swab sample is inserted directly into the swab chamber of the sample preparation unit immediately after collection. The swab is broken, leaving the swab tip and the sample within the chamber, which is then sealed. Cartridges are placed in the NudgeBox processing unit, which provides the pneumatic, thermal, imaging, and mechanics required to run a real-time RT-PCR reaction outside of a laboratory setting. The sample preparation unit consists of chambers containing buffers to extract and purify RNA from the swab sample, as well as a lyophilised RT-PCR mastermix to mix with the extracted RNA. The DnaCartridge™ fits on top of a motor-driven spigot in the NudgeBox™, which rotates the SPU through each stage of sample processing before filling the wells of the AU, inside which the RT-PCR reaction takes place.
The following description is directed to the detection of SARS-CoV-2, but this should not be construed as limiting and the teaching can easily be extended to the detection of any disease or condition, which can be detected by way of one or more nucleic acid sequences which are associated with the disease or condition to be detected. Examples include respiratory conditions, especially infections caused by bacterial, viral or fungal agents.
The AU comprises dried primers and probes uniquely spotted into 72 reaction wells enabling multiplex analysis. The array comprises six viral targets (rdrp-l P2, rdrp-IP4, e- gene, n1 , n2, and n3) and one host gene as a sample adequacy control (Ribonuclease P, RNaseP). Each gene target has nine or ten technical replicates within the array, while the human control has six replicates. Following completion of the PCR reaction, the results from the DnaNudge test are transmitted to the Cloud where an algorithm is run to determine the results [2], These results are transmitted back to an Operator app run on an iPad, as well as being sent onwards to hospital IT systems, and are reported as positive, negative, indeterminate, invalid or aborted according to Table 1. Following the test, the single-use cartridge is disposed of following standard laboratory disposal procedures.
Figure imgf000013_0001
Table 1 : DnaNudge test result reporting
The diagnostic accuracy of the CovidNudge test was assessed in April and May 2020 by comparing nasopharyngeal swab samples from individuals at three hospitals in London and Oxford against nasal and throat swabs tested on laboratory RT-PCR platforms [2], The sensitivity of the DnaNudge point-of-care test compared with laboratory-based testing was 94% (95% Cl 86-98) with a specificity of 100% (95% Cl 99-100). Following this clinical validation, the CovidNudge test achieved the CE Mark in July 2020 enabling the technology to be used as standard of care in UK healthcare settings. Since July, over 20,000 patient samples have been tested on the CovidNudge platform across 8 separate hospital sites in London. Current NHS guidelines state that all patients admitted to UK hospitals must have a test for COVID-19; if a rapid test is not available, then patients presenting as emergency admissions must be isolated in side rooms until their laboratory test is returned, allowing the appropriate care pathway to be determined. These side rooms must then be fully cleaned regardless of the COVID-19 test result before they can be occupied by a subsequent patient, placing additional burden on an already stretched nursing resource.
The deployment of CovidNudge as a point-of-care diagnostic has enabled effective triage and timely therapeutic and infection control interventions for emergency admission patients in clinical areas including adult and paediatric A&E, maternity, mental health and renal transplantation, and the technology has been fully embedded as an integral part of the emergency admission pathway at the deployment sites. With test results available within 90 minutes of sample collection, patients can be admitted into the appropriate care pathway bypassing the need for isolation if test results are negative, while enabling sites to meet operational targets to admit, transfer or discharge patients from A&E within 4 hours.
Sample Pooling
Due to the user-friendly nature of the DnaNudge test with no sample preparation required, in clinical setting the tests are performed by frontline healthcare workers rather than central laboratory staff. The simplicity of use means that each NudgeBox processes one sample at a time, thus each clinical area deploys multiple NudgeBoxes to manage the timely testing of emergency admissions at peak throughput times. Although an increase in testing throughput can be accommodated by deploying additional NudgeBoxes, the use of pooled patient samples has been proposed as a method to increase throughput of molecular testing for SARS-CoV-2 [3, 4], The US Food and Drug Administration (FDA) currently recommends two approaches to patient specimen pooling [3]. The first method is to pool aliquots of transport media each containing a single patient sample (sample pooling), while the second method is to combine swabs from multiple patients into a single volume of transport media (swab pooling). Both methods have advantages and disadvantages. For sample pooling, the individual patient samples are separately preserved, so that if a pooled test reports a positive result, the patient samples needed for the individual follow-up tests already exist. However, the disadvantage is that, since the volume of each sample is reduced, the analytical sensitivity is decreased (i.e. limit of detection (LOD) increases) because individual samples are further diluted. This limits the practical size of the sample pool; the FDA recommends that the analytical sensitivity of the test with n-sample pooling should be evaluated, starting with a maximum n=5. In contrast, swab pooling does not lead to a reduction in analytical sensitivity since the entire sample from each patient is pooled into a single volume of transport media. However, if the test is reported as positive, there is no way to deconvolve which of the individual samples are positive without taking another sample from each patient in the pool. For this reason, the UK NHS currently only recommends sample pooling for asymptomatic patients [4], The present teaching may overcome one or more of the above identified disadvantages.
Despite the efficiency gains in testing throughput that can potentially be achieved through pooling, in practice the technique has not been widely used in clinical settings. For standard laboratory-based platforms there may be several contributing factors; many test suppliers have not validated the use of pooling; pooling can potentially delay the reporting of results since if a pooled test reports positive, the individual samples need to be retested; pooling is most efficient when prevalence is low, however low prevalence may reduce testing demand, enabling the volume of tests required to be adequately handled by parallel processing of single patient samples.
For point-of-care testing however, the potential benefits of testing pooled samples may be more easily realised. Near-patient tests such as CovidNudge produce rapid results thus a positive pooled test may be quickly followed up with individual testing, while a negative pooled test allows the elimination from isolation in COVID wards of multiple patients in parallel rather than sequentially. However, neither of the pooling methods described earlier are compatible with the DnaNudge CovidNudge platform. This is because the CovidNudge test eliminates the requirement for swab samples to be diluted in liquid transport media, therefore no simple method exists for combining multiple nasopharyngeal swabs from individual patients. This in part led to a consideration of the use of alternative fluid samples and sputum, in particular, as an alternative sample media.
Sputum Sampling
To investigate whether sputum samples are compatible with the DnaNudge platform, a validation study comparing nasopharyngeal swab samples with sputum was undertaken. Assessment took place using samples from two separate groups: patients admitted to hospital via the emergency department at Chelsea & Westminster NHS Foundation Trust, and members of the London Symphony Orchestra. Testing of emergency admissions at Chelsea & Westminster NHS Foundation Trust was done as a service evaluation approved by the point-of-care committee. Patients over 18 testing positive (n = 71 ) and negative (n = 103) on the DnaNudge platform via nasopharyngeal sampling agreed to provide a sputum sample. Members of the London Symphony Orchestra (n = 1 18) were undergoing regular COVID-19 screening on the DnaNudge platform. All participants consented to supplying a sputum sample following the procedure in Appendix 1 in addition to providing a nasopharyngeal swab. Sputum samples were collected into a sample tube (Oragene500, DNAgenotek); the stabilising solution released by the Oragene collection tube has been shown to inactivate the SARS-CoV-2 virus due to the presence of an ionic detergent which renders ineffective enveloped viruses such as SARS-Cov-2 [5].
Following sputum and nasopharyngeal sample collection, the samples were tested on the DnaNudge platform. To test the sputum samples, an RNA/DNA buccal swab (SK-2, Isohelix) was used. The cap from the swab was removed while retaining the bung with stopper, and the swab was mixed in the sputum in stabilising solution by rubbing gently for 5 seconds to get a good sputum sample on the swab. When extracting the swab from the sample tube, any excess sputum residue hanging from the swab was removed by wiping the swab gently against the inside edge of the tube. The Isohelix swab was then inserted into the cartridge pressing the stopper in place, the swab tail was removed leaving the swab in the chamber, and the cartridge was sealed using the Isohelix bung (Figure 2). The cartridge was then inserted into the NudgeBox and a test run following standard procedure [2], 292 paired samples were obtained, and results are shown in Table 2.
Figure imgf000016_0001
Table 2: Nasopharyngeal and sputum paired samples tested on the DnaNudge platform. Sputum samples demonstrated 98.6% sensitivity (95% Cl = 92.4 - 99.96%) and 100% specificity (95% Cl = 96.9 - 100%) against nasopharyngeal samples.
We quantitively assessed the relative concentration of viral load in nasal and sputum samples by plotting the number of SARS-CoV-2 gene target replicates that amplified in each sample as shown in Figure 3.
Sputum Pooling Following the confirmation that sputum is a suitable sample type for the DnaNudge Covid-19 test, we investigated the pooling of multiple sputum samples to increase testing throughput. Pools were tested with one positive sample and the rest as negative samples following the method outlined in Appendix 2. An initial exploratory analysis was performed starting at a pool of two and then incrementing the pool by adding further negative samples. This exploratory analysis was still able to report a positive result from a single positive sputum sample in a pool of 30. A pool of 40 samples was also run twice and both times showed two replicates amplifying, as shown in Figure 4C.
In practice a pool size of 30 or 40 samples is unlikely to be practical unless prevalence is very low, therefore we performed further experiments by choosing a pool size n = 10 (NHS guidelines currently recommend a pool size between 6 and 12 [4]). FDA guidelines regarding the validation of n-pooled tests recommend that samples from at least 20 positive patients and (20 x n) negative patients should be collected and tested with one positive and (n - 1 ) negative samples per pool [3]. Therefore 20 samples from the positive data set and 180 samples from the negative data set were divided into pools of 10, with one positive and nine negative samples per pool [3]. The samples were selected from the original dataset to cover a range of viral loads (i.e. number of replicates that amplified in the original sample test). The pooled samples were dipped in turn with a single Isohelix swab following the method outlined in Appendix 2, then inserted into a DnaCartridge and tested following standard procedure [2], The position of the positive sample within the pool was varied to ensure that all possible permutations were tested (i.e. positive sample in first position, second position, ninth position, tenth position), with at least two tests per ‘position’ of the positive sample. 200 negative samples were also divided into pools of 10 and tested, and results are shown in Table 3.
Figure imgf000017_0001
while the positive and indeterminate results were tested as one positive sample with nine negative samples. An indeterminate result is when only one or two replicates amplify, i.e. the signal is at the limits of detection. We further examined the number of replicates that amplified (i) when testing the original positive sputum sample, and (ii) when testing the positive sputum sample in a pool of 10, and the results are shown in Figure 5.
In Figure 5, the ‘position’ of the positive sample in the pool is illustrated by the sample colour; no significant correlation between sample position and number of replicates could be seen. This was surprising as it may be expected that the samples would effectively be diluted as more samples are added to the pool and hence reduce the effectiveness of the method. Without being bound by theory, we hypothesise that this may due to the nature of the Isohelix™ buccal swab which is patterned with a matrix designed to efficiently collect and ‘trap’ buccal cell samples for DNA testing. This patterned matrix has the effect of securely capturing and holding the sputum samples, and so each additional pooled sample simply adds more sputum material onto the swab matrix.
Limits of Detection for Pooling
Following the qualitative investigation above, a quantitative investigation of the reduction in sensitivity through pooling was performed by assessing the LOD. A negative sputum sample was spiked with viral genetic material (Microbiologies HE0062S process control pellet) dissolved in molecular water. The viral solution was serially diluted and aliquots of 25uL were added to a 25uL sputum sample and absorbed onto an Isohelix swab for testing. For individual sample testing the LOD was measured as 250 copies per swab; this rose to 1250 copies per swab for 10 pool testing. The 5-fold increase in LOD for a 10-pool test demonstrates that dipping a swab into successive sputum samples provides less sample dilution than would be expected through traditional sample pooling, where an n-fold reduction in viral concentration would be expected for an n-pool test.
Putting Pooling into Practice
The efficiency gained through pooling of samples is highly dependent on the prevalence of positive patients in the cohort to be tested. If prevalence is very high, pooling can lead to a decrease in testing efficiency due to the need to repeat individual follow-up tests when the pooled sample returns a positive result, and pooling is not recommended when prevalence rises above 10% [4, 5]. We investigated the average efficiency of pooling as a function of prevalence, where the probability P(neg) of returning a negative result from a pooled test is calculated as:
P(neg) = (1 — p~)n where p = prevalence and n = pool size. We considered two scenarios: • Single pooled test: in this scenario, if the pooled test is reported as positive, the patient samples are all individually tested to determine which of the samples is positive
• Nested pooled test: in this scenario if the pooled test is positive, smaller patient pools are repeated to narrow down the search for the positive result(s).
A nested pooled test can be denoted as ( n1 | n2 | n3 .... | nx ), where n1 > n2 > n3 ... > nx, and where n1 denotes the size of the first pool, n2, n3 etc. are the sizes of subsequent sub-pools, and nx = 1 . So a pooling strategy of ( 12 | 3 | 1 ) would start with an initial pool size of 12, followed by 4 pooled tests of 3 samples each, followed by individual testing of any of the n = 3 sub-pools that had tested positive. Using this nomenclature, the single pooled test can be considered as a nested pool of ( n | 1 ), i.e. if the initial n-pool tests positive, the next round of testing would test all n samples individually.
To explore the relative efficiency of single and nested pooling, we simulated the result of different pooling scenarios. We selected a range of single and nested pooling scenarios, with a maximum initial pool size of 12 as per Public Health England (PHE) guidelines [3]. We also limited the total number of nested testing cycles to three (i.e. one or two rounds of pooling with a final individual testing round). This is because three rounds of testing has already tripled the time required to receive a final result for a positive patient; further rounds of pool testing would delay the result further and may detract from the benefit of having a rapid test at the point of care.
For each initial pool size, all possible input sample permutations were evaluated. For a pool size of n, this results in 2n input sample vectors. Each input vector was evaluated in turn to determine the total number of tests that would need to be run in the given pooling scenario. Finally, the probability of the specific input vector being realised was calculated as a function of prevalence. This enabled the calculation of the relative efficiency for different pooling strategies as a function of prevalence, and results are summarised in Figure 6.
Figure 7 shows the probability of the first pooled test returning a negative result, as a function of prevalence.
These results support the proposal that nested pool testing may be used in accordance with the teaching herein as an alternative to single confirmatory tests. Swab Pooling
As discussed above, an alternative or complementary approach to sample pooling is to create a pooled sample by introducing multiple swabs into a single volume of transport media. This can be achieved using the DnaNudge platform by introducing the multiple swabs into the sample (or “receiving”) chamber of the DnaCartridge. For example, nasopharyngeal or oropharyngeal swabs can be used to collect samples from individuals in the usual way and the swab tips inserted through the swab port (inlet) of the sample chamber one after another, as shown in Figure 8. The tails of each of the swabs are then removed, e.g. using disposable scissors, and a stopper or bung inserted into the swab port to seal the swab tips within the sample chamber. A pooled sample is then formed by flooding the sample chamber with a lysis buffer and the pooled sample tested as described above. Surprisingly, this form of pooled testing has been found to be effective without requiring extensive modifications to the DnaNudge platform.
Figure 9 shows a cross sectional view of a DnaCartridge 900 comprising the swab port 901 opening into the sample chamber 903, a rotatable central chamber 905 and a lysis buffer chamber 907. The swab tips introduced into the sample chamber through the swab port 901 are not shown for clarity. During testing of the swab tips, a pooled sample is created by introducing lysis buffer from the lysis buffer chamber 907 into the sample chamber 903 to release DNA and/or RNA present in virus on each of the swab tips into solution by breaking down membranes of the virus. The lysis buffer is first drawn into the central chamber 905 from the lysis buffer chamber 907 using a pneumatic pressure unit (as described below in connection with Figure 10). The lysis buffer is then introduced into the sample chamber 903 by using the pneumatic pressure unit to apply a positive relative pressure between the central chamber 905 and the sample chamber 903. The pneumatic pressure unit comprises a pressure sensor that is used to monitor the pressure opposing the flow of lysis buffer into the sample chamber 903. When the pressure exceeds a predetermined threshold, the flow of lysis buffer is stopped to ensure that the sample chamber 903 is not overfilled with lysis buffer. Any lysis buffer remaining in the central chamber 905 is then expelled to a waste chamber or returned to the lysis buffer chamber 907.
By dynamically controlling the filling of the sample chamber 903 with lysis buffer, the NudgeBox system can be used for different numbers of swab tips and/or different sizes of swab tip without needing to be reconfigured. In other words, the system is able to automatically adjust the amount of lysis buffer delivered to the sample chamber 903 to account for the volume of the sample chamber 903 that has been taken up by the swab tip(s).
After or during introduction of the lysis buffer into the sample chamber 903, the amount of lysis buffer transferred to the sample chamber 903 is determined, e.g. by monitoring the length of time for which the pressure is applied by the pneumatic pressure unit or, the distance moved by a piston (e.g. syringe plunger) within a pneumatic pressure unit. The lysis buffer is permitted to remain in the sample chamber 903 until the DNA and/or RNA has been released from the swab tips. The lysis buffer containing the extracted DNA and/or RNA is then returned to the central chamber 905 by using the pneumatic pressure unit to applying a negative relative pressure between the central chamber 905 and the sample chamber 903. The lysis buffer flows through a silica frit (not shown in Figure 9, but visible in Figure 8) as it re-enters the central chamber 905, causing the DNA and/or RNA to adhere to the frit. The pneumatic pressure unit is operated such that a large majority (e.g. greater than 90%) or all of the lysis buffer is transferred back to the central chamber 905, but without exposing the frit to conditions that would damage it. For example, the negative pressure may be applied for only a limited period, which is determined from the amount of lysis buffer transferred to the sample chamber 903, to avoid maintaining the negative relative pressure across the frit for too long after lysis buffer has been removed from the sample chamber 903. Alternatively or additionally, the magnitude of the negative pressure may be varied according to the amount of lysis buffer that needs to be transferred.
Using swabs with smaller tip sizes allows more of them to be accommodated within the sample chamber 903 and therefore greater pooling of the samples. It may therefore be preferable to use nasopharyngeal or oropharyngeal swabs rather than larger buccal swabs, although combinations of different types of swab can also be used, e.g. depending on swab availability or factors that favour the use of one swab over another for different individuals. It is also possible to use one or more pooled samples created by the sputum pooling method described above. For example, a swab used to create a pooled sample from one or more sputum samples can be combined with one or more nasopharyngeal or oropharyngeal swab, which may allow individuals who were not able to provide a sputum sample to be included in the pooled test. As another example, multiple swabs with pooled samples created for different groups of individuals by sputum pooling can be combined, which may facilitate sample collection, such as in circumstances where the group of individuals needing to be tested are in different locations, e.g. hospital wards or college dormitories. Figure 10 shows a system diagram of the analyser 1000 (a NudgeBox in this case), which receives the DnaCartridge 1001 in order to test the pooled sample. The analyser comprises a mechanical unit 1003, a pressure unit 1005, thermal unit 1007 temperature control of the PCR, an imaging unit 1009 for measuring fluorescence produced by the test and a control unit 101 1 that controls each of the other units when the test is being performed. The pressure unit 1005 in this example comprises a syringe 1013, a pressure sensor 1015 and a valve 1017 that is used to isolate the pressure unit from the DnaCartridge when the pressure unit is not being used. In use, the mechanical unit rotates the central chamber 905, to place it in fluid communication with both the syringe and one of the other chambers of the DnaCartridge 1001 (e.g. the sample chamber 903 or the lysis buffer chamber 907). A piston (or plunger) is then moved backwards or forwards within the syringe to generate positive or negative pressure within the central chamber 905 to either force fluid from the central chamber 905 into the other chamber 903, 907 or else draw fluid from the other chamber 903, 907 into the central chamber 905. The pressure sensor 1015 is used to monitor the pressure within the syringe 1013, e.g. by measuring the force required to move the piston.
Figure 11 shows the steps for preparing the pooled sample prior to amplification of DNA from the pooled sample by PCR, the DNA being present initially in the pooled sample or derived from RNA present in the pooled sample by reverse transcription.
• Step 1 101 : Deliver lysis buffer to the swab chamber to release DNA and/or RNA contained in virus collected on the swabs from each of the individuals.
• Step 1 102: Pass the lysed sample through a frit to capture the DNA/RNA on the frit.
• Step 103: Pass a wash buffer through the frit to clean non-DNA and non-RNA debris from the frit.
• Step 1 104: Pass an elution buffer through the frit to release the DNA/RNA from the frit.
• Step 1 105: Reconstitute lyophilized mastermix with the elution buffer containing the DNA/RNA.
Step 1106: Fill an analysis unit with the DNA/RNA, mastermix, and elution buffer. Step 1107 (optional): Perform reverse transcription of the RNA to form DNA corresponding to the RNA. • Step 1108. Start the PCR amplification of the DNA.
Figure 12 shows the steps used for releasing the DNA/RNA by lysis of the virus collected on the swabs.
• Step 1201 : Rotate the mixing chamber to align its port with the port of the lysis buffer chamber.
• Step 1202. De-pressurise the mixing chamber to create a vacuum and let the lysis buffer into the mixing chamber.
• Step 1203. Rotate the mixing chamber to align its port with the port of the swab chamber.
• Step 1204: Pressurise the mixing chamber to force the lysis buffer out and to the swab chamber; measure the pressure and the time to calculate volume and pressure in the swab chamber; record the time and/or volume reaching a set threshold pressure.
• Step 1205. Rotate back the mixing chamber to align its port with the port of the lysis buffer chamber.
• Step 1206. Dispense the retained lysis buffer back to the lysis chamber.
• Step 1207. Wait for a certain time (e.g. 1 min) to let the lysis buffer extract DNA/RNA from the swabs.
• Step 1208. Rotate the mixing chamber to align its port with the port of the swab chamber.
• Step 1209: De-pressurise the mixing chamber to create a vacuum and let the lysis buffer from the swab chamber enter the mixing chamber through the frit and so that the lysed DNA/RNA material binds to the frit. Set the negative pressure to a value based on the recorded time/volume during the lysis dispense step (step 1204 above).
• Step 1210. Rotate the mixing chamber to ward a waste chamber to dispense the lysis while the DNA/RNA material is bound to the frit.
Practical Use Cases
While pooling of samples has the potential to offer significant efficiency gains, in clinical practice at the point of care it may be of imperative importance to know the definitive result for a patient as soon as possible (e.g. when planning for an emergency surgery). In such settings, the requirement for immediacy of results may rule out the use of pooling to eliminate the risk that a second round of testing may be required. However, there are specific use cases where pooling is not only feasible, but also desirable, particularly when testing asymptomatic population groups. Example use cases cover:
• Asymptomatic emergency hospital admissions
• Care Homes
• Family and Support “Bubbles”
• Workplace “Bubbles”: Film and television production, theatres and concerts, sports teams
• School classrooms
Our sputum sample validation has demonstrated that a single sputum sample can be dipped by a (clean) swab multiple times and stored to enable repeat testing. This provides a method by which sputum pooling can be simply used in practice without the requirement for pipetting small volumes into a pool to avoid sample cross contamination, which is impractical at the point of care. Once collected, each individual sputum sample is dipped with a separate Isohelix swab(s) following the method in Appendix 1 , and the cap of each Isohelix swab is then replaced to store the labelled samples for later processing if necessary. Following, a single Isohelix swab is dipped into each of the pooled samples in turn, and the dipped samples are then discarded as contaminated. If a positive result is returned from the pool test, the Isohelix swabs for the individual samples within the pool are available for retesting.
References
[1 ] Lisboa Bastos M. et al, Diagnostic accuracy of serological tests for covid-19: systematic review and meta-analysis. BMJ 2020;370:m2516 | doi: 10.1136/bmj.m2516
[2] Gilbani M., Toumazou C. et al. Assessing a novel, lab-free, point-of-care test for SARS-CoV-2 (CovidNudge): a diagnostic accuracy study. Lancet Microbe. https://doi.Org/10.1016/32666-5247(20)30121 -X
[3] Molecular Diagnostic Template for Commercial Manufacturers https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency- use-authorizations-medical-devices/vitro-diagnostics-euas
[4] https://www.england.nhs.uk/coronavirus/publication/pooling-of-asymptomatic-sars- cov-2-covid-19-samples-for-pcr-or-other-testing/
[5] https://www.dnagenotek.com/US/pdf/MK-01430.pdf Appendix 1: Taking a Sputum Sample
Equipment Required
• SK-2 buccal swab with tube (Isohelix)
• Oragene 500 sample collection tune (DNAgenotek) i. The healthcare professional should prepare the patient for the procedure by asking them to sit upright, rinse their mouth with water and spit out prior to sputum collection. ii. The patient should be asked to take a few deep breaths to help loosen secretions; please note, if patient is on a nebuliser, give nebuliser first and wait 10 minutes before taking a sample. iii. The patient should cover their mouth before forcing out a deep cough to release the sputum. Sputum should be collected in the sample tube provided. Ideally the sputum sample should be no less than the size of a small fingernail. iv. It is important that the healthcare professional checks the quality of the sputum to ensure it is not simply saliva, but rather sputum (mixture of phlegm and mucous). If the patient is unable to provide any sputum, advise to keep hydrated where possible, and encourage deep breathing to try again in an hour. v. The sample tube should be held upright in one hand and the funnel lid closed with the other hand by pressing firmly until a loud click is heard. The liquid in the lid will be released into the tube to mix with the sputum. vi. Holding the tube upright, unscrew the funnel from the tube and discard the funnel as clinical waste. Use the small screw cap to close the sample tube tightly. Shake the capped tube for 5 seconds. vii. Remove the cap from the Isohelix swab while retaining the bung with stopper, and mix the swab in the sputum, rubbing gently for 10 seconds to get a good sputum sample on the swab. When extracting the swab from the sample tube, remove any excess sputum residue hanging from the swab by wiping the swab gently against the inside edge of the tube. viii. Remove the cap from the DnaCartridge and insert the swab end at a vertical angle into the Cartridge (the DnaCartridge cap can be discarded) ix. Press the Isohelix cap with stopper into the DnaCartridge and gently remove tail of the swab, this will leave swab tip and sample in the swab chamber. x. Discard the swab tail in a “sharps” bin. xi. Lock the DnaCartridge closed with the Isohelix bung and run the test as per standard procedure. Appendix 2: Pooled tests using sputum samples
Pooling of samples should only be performed by an experienced nurse or healthcare professional who has been adequately trained in the technique.
Equipment required:
• SK-2 buccal swab with tube (Isohelix)
• Oragene 500 sample collection tubes (DNAgenotek)
Method i. Obtain a number (up to 10) of individual sputum samples following steps (i) - (vi) in Appendix 1 . Samples should be labelled as known positive or negative. ii. Position all sputum sample tubes in a line using a test tube rack or similar receptacle holder and unscrew the lids of each of the tubes. iii. Remove cap from Isohelix swab retaining the bung with stopper, and gently dip the swab in the first sputum sample for 5 seconds, performing 2 gentle rubs for each side of the swab against the sputum and the inside of the tube. Remove any excess sputum hanging off the swab by rubbing the swab gently against the inside of the tube. iv. Once this is done for the first sample, insert the same swab into the next and repeat the process for all 10 samples. v. Load the Isohelix swab into the DnaCartridge following steps (viii) - (xii) in Appendix 1 vi. Any negative samples that have been dipped with the Isohelix swab after the swab has been dipped into a positive sample must be discarded as contaminated.

Claims

1 . A method of testing a group of individuals for the presence of a disease or condition, the method comprising: providing a sputum sample from each individual of the group of individuals; collecting and pooling a portion of each sample, using a device, the device comprising an absorbent material for absorbing at least some of the portion of each sample in order to form a pooled sample absorbed to the absorbent material of the device; transferring at least a portion of the pooled sample from the absorbent material of the device to an analyser and using the analyser to analyse the transferred pooled sample or portion of the pooled sample for the presence of said one or more nucleic acid sequences associated with the disease or condition; and wherein a negative result from the analysis indicates that said one or more nucleic acid sequence(s) is not present in the pooled sample and each individual of the group of individuals does not have the disease or condition and a positive result indicates that said one or more nucleic acid sequence(s) is present in the pooled sample and one or more of each individual of the group of individuals may have the disease or condition.
2. A method of testing a group of two or more individuals for the presence of a disease or condition, the method comprising: transferring a biological sample from each of said individuals onto one of a plurality of swabs so that each of the swabs comprises a biological sample from at least one of said individuals; inserting the swabs into a receiving chamber of an analyser so that all of the swabs are present together in the receiving chamber and a pooled sample is formed in the receiving chamber, the analyser being capable of analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition; and wherein a negative result from the analysis indicates that said one or more nucleic acid sequence(s) is not present in the pooled sample and each individual of the group of individuals does not have the disease or condition and a positive result indicates that said one or more nucleic acid sequence(s) is present in the pooled sample and one or more of each individual of the group of individuals has the disease or condition.
3. The method according to claim 2, wherein analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition comprises amplifying, if present in the pooled sample, the or each of the nucleic acid sequence(s), wherein the or each nucleic acid sequence may be DNA sequences.
4. The method according to claim 2, wherein analysing the pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition comprises deriving from the or each nucleic acid sequence, if present in the pooled sample, one or more other nucleic acid sequence(s) and amplifying the or each of the derived other nucleic acid sequence(s), wherein the or each first mentioned nucleic acid sequence may be RNA sequences and said one or more other nucleic acid sequences may be DNA sequences.
5. The method according to any one of claims 2 to 4 and comprising, when all of the swabs are present together in the receiving chamber, introducing a lysis buffer into the receiving chamber to release nucleic acids into the lysis buffer to form the pooled sample.
6. The method according to any one of claims 2 to 5, wherein the lysis buffer is forced into the receiving chamber under pressure generated using a pneumatic pressure unit, the method comprising measuring the pressure generated by the pneumatic pressure unit as the lysis buffer is introduced and stopping introduction of the lysis buffer when a predetermined pressure is reached.
7. The method according to claim 6 and comprising: measuring a value indicative of the volume of lysis buffer introduced into the receiving chamber; determining from said value, one or more control parameters for controlling the pneumatic pressure unit to generate a pressure to force the lysis buffer from the receiving chamber through a frit or filter, whereby the released nucleic acids bind to said frit or filter.
8. The method according to claim 7, wherein the one or more control parameters are used to control one or more of a distance moved by a piston of the pneumatic pressure unit, an amount of pressure generated by the pneumatic pressure unit, and an amount of time for which to maintain the pressure.
9. The method according to any one claims 2 to 8, wherein the analyser comprises a removable single-use cartridge in which the receiving chamber is provided, and optionally the cartridge comprises a further chamber containing a lysis buffer used to form the pooled sample.
10. The method according to claim 9, wherein the swabs are inserted into the receiving chamber before the cartridge is inserted into or attached to the analyser.
1 1 . The method according to claim 10, wherein the swabs are inserted into the receiving chamber through a port that is sealed before the cartridge is inserted into or attached to the analyser.
12. The method according to any one of claims 9 to 1 1 , wherein the cartridge comprises a plurality of circumferentially spaced chambers, including the receiving chamber, arranged about a central chamber, the central chamber being rotatable relative to the other chambers to bring one or more openings of the central chamber into selective alignment with an opening of each of the other chambers to allow transfer of fluid between the central chamber and a selected one of the other chambers.
13. The method according to claim 12, when dependent on claim 7 or 8, wherein the frit or filter is provided in an opening of the central chamber.
14. The method according to any one of claims 2 to 13, wherein the biological sample of each of said individuals is collected using a different one of said swabs.
15. The method according to any one of claims 2 to 13, wherein at least one of the swabs comprises biological samples from more than one of said individuals.
16. The method according to any one of claims 2 to 15, wherein said step of transferring a biological sample from each of said individuals onto one of a plurality of swabs comprises collecting sputum samples from the individuals.
17. The method according to any one of claims 2 to 16, wherein the number of swabs present together in the receiving chamber is from 2 to 10.
18. The method according to any preceding claim and comprising: retaining a biological sample of each of said individuals or collecting a further biological sample from each of said individuals; and if a positive result is obtained, subjecting the retained portions or the further biological samples to analysis, using the or further analyser(s) to detect which of the retained portions or further biological samples contain said one or more sequences associated with the disease or condition and thereby identify which individuals of the group of individuals has the disease or condition.
19. The method according to any one of the preceding claims, wherein the disease or condition is a disease or condition that affects the respiratory tract, such as a bacterial, viral or fungal infection, the infection optionally being a viral infection and the virus being a common cold, influenza, respiratory syncytial, adeno, or corona virus, such as SARS, MERS and COVID-19 (SARS-CoV-2) virus.
20. The method according to any preceding claim, wherein the one or more nucleic acids to be detected comprises at least 2, 3, 4, 5, 6, 7, or 8 specific nucleic acid sequences which are specific to the disease or condition, and/or wherein the one or more nucleic acids to be detected comprises at most 4, 6, 8, 10, or 12 specific nucleic acid sequences which are specific to the disease or condition.
21 . The method according to any preceding claim, wherein the one or more nucleic acids encodes a native or mutant protein associated with the disease or condition.
22. The method according to claim 21 or 22, wherein the one or more nucleic acids is, or includes the rdrp-IP2, rdrp-IP4, e-gene, n1 , n2, and/or n3 gene(s), or specific fragments thereof.
23. The method according to any preceding claim, wherein the group of individuals are asymptomatic.
24. The method according to any preceding claim, wherein the group of individuals are asymptomatic emergency hospital admissions, staff and/or residents in care homes, family and/or support groups, workplace and/or conference groups, film and/or television production teams, theatre and/or concert production teams, sports teams, and staff and/or students in pre-school, school, college or university.
25. The method according to any preceding claim, wherein the groups comprises at least 2 individuals and up to 20 individuals, such as 2 - 20, 2 - 15, 2 - 12, 2 - 10, 2 - 8, 2 - 6, or 2 - 4, as well as any integers and ranges in between.
29
26. The method according to any preceding claim, wherein the device or some or all of the swabs are buccal, nasopharyngeal, or oropharyngeal swabs, preferably Isohelix™ swabs.
27. The method according to any one of the preceding claims, wherein the and/or first portions of the sample are contacted with or inserted into a cartridge which is to be introduced, or has been introduced into the analyser.
28. The method according to any one of the preceding claim, wherein the and/or further analyser(s) is or are a NudgeBox™ analyser.
29. A method of managing a disease outbreak comprising: using the method of any preceding claim to determine whether one or more individuals in a group of two or more individuals has said disease; if such a determination is made, sending the result to the individuals of the group identified as having the disease via an electronic communication device together with an instruction to isolate.
30. A kit for use in a method according to any preceding claim, the kit comprising: two or more sample receptacles for collecting a respective sputum sample from each of two or more individuals in a group of individuals, each receptacle comprising indicia, or a label specifically designed to identify the sputum sample, with the individual from which is was obtained; and instructions explaining how to take a sputum sample.
31 . A kit for use in a method according to any one of claims 1 to 29, the kit comprising: two or more sample receptacles for collecting a respective sputum sample from each of two or more individuals in a group of individuals; and two or more swabs, each of said two or more swabs comprising an absorbent material for absorbing a first portion of said sample from each of said two or more individuals and two or more respective sealable enclosure devices for receiving said two or more swabs, wherein each receptacle and/or enclosure device comprises indicia, or a label specifically designed to identify the sputum sample, with the individual from which is was obtained.
32. The kit according to claim 31 , further comprising
30 a single pool collection swab comprising an absorbent material for absorbing and pooling a portion of each sample from all individuals in the group of individuals.
33. The kit according to claim 31 or 32, further comprising instructions explaining how to take a sputum sample and/or how obtain a pooled sample.
34. A kit for use in a method according to any one of claims 2 to 17, the kit comprising: two or more swabs, each swab being for collecting a biological sample from a respective individual in a group of two or more individuals, each swab having a corresponding sealable enclosure device comprising indicia, or a label, specifically designed to identify the biological sample with the individual from whom it was collected; and instructions explaining how to take a biological sample.
35. The kit according to claim 34, wherein, for each of the individuals in the group, the kit comprises two or more of said swabs and two or more of said sealable enclosure devices comprising the indicia, or the label specifically designed to identify the biological sample with the individual from whom it was collected.
36. The kit according to claim 34 and comprising a further sealable enclosure device for receiving the two or more swabs and comprising indicia, or one or more labels, specifically designed to identify the further sealable enclosure with the individuals in said group and instructions to place the two or more swabs in the further sealable enclosure.
37. An analyser for testing a group of two or more individuals for the presence of a disease or condition, the analyser being configured or configurable to analyse a pooled sample for the presence of one or more nucleic acid sequence(s) associated with the disease or condition, the analyser comprising: a receiving chamber into which two or more swabs can be inserted; a fluid transport system for delivering a lysis buffer to the receiving chamber in order to form the pooled sample by releasing nucleic acid sequences into the lysis buffer and comprising a pneumatic pressure unit for generating pressure to force the lysis buffer into the receiving chamber and a pressure sensor for measuring the pressure generated by the pneumatic pressure unit during delivery of the lysis buffer; and
31 a control unit configured or configurable to control the fluid transport system to stop delivery of the lysis buffer once the pressure measured by the pressure sensor exceeds a predetermined value.
38. The analyser according to claim 37, said receiving chamber and at least a portion of the fluid transport system comprising a single use cartridge.
39. The analyser according to claim 37 or 38 and comprising a frit or filter to which to reversibly bind nucleic acid sequences, the fluid transport system being configurable to force the lysis buffer containing the pooled sample through the frit or filter under pressure generated by the pneumatic pressure unit, and wherein the control unit is further configured or configurable to: measure a value indicative of the volume of lysis buffer delivered to the receiving chamber; determine from said value one or more control parameters for controlling the pneumatic pressure unit; and control the generation of pressure by the pneumatic pressure unit according to said one or more control parameters.
40. The analyser according to claim 39, wherein the one or more control parameters are used to control one or more of a distance moved by a piston of the pneumatic pressure unit, an amount of pressure generated by the pneumatic pressure unit, and an amount of time for which to maintain the pressure.
32
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