A Novel Microfluidic Dielectrophoresis Technology to Enable Rapid Diagnosis of Mycobacteria tuberculosis in Clinical Samples

To achieve the global efforts to end tuberculosis, affordable diagnostics suitable for true point-of-care implementation are required to reach the missing millions. In addition, diagnostics with increased sensitivity and expanded drug susceptibility testing are needed to address drug resistance and to diagnose low-bacterial burden cases. The laboratory-on-a-chip technology described herein used dielectrophoresis to selectively isolate Mycobacterium tuberculosis from sputum samples, purifying the bacterial population ahead of molecular confirmation by multiplex real-time quantitative PCR. After optimization using a panel of 50 characterized sputum samples, the performance of the prototype was assessed against the current gold standards, screening 100 blinded sputum samples using characterized and biobanked sputum provided by Foundation for Innovative New Diagnostics. Concordance with culture diagnosis was 100% for smear-negative samples and 87% for smear-positive samples. Of the smear-positive samples, the high burden sample concordance was 100%. Samples were diagnosed on the basis of visual assessment of the dielectrophoresis array and by multiplex real-time quantitative PCR assay. The results described herein demonstrate the potential of the CAPTURE-XT technology to provide a powerful sample preparation tool that could function as a front-end platform for molecular detection. This versatile tool could equally be applied as a visual detection diagnostic, potentially associated with bacterial identification for low-cost screening or coupled with an expanded PCR assay for genotypic drug susceptibility testing. To achieve the global efforts to end tuberculosis, affordable diagnostics suitable for true point-of-care implementation are required to reach the missing millions. In addition, diagnostics with increased sensitivity and expanded drug susceptibility testing are needed to address drug resistance and to diagnose low-bacterial burden cases. The laboratory-on-a-chip technology described herein used dielectrophoresis to selectively isolate Mycobacterium tuberculosis from sputum samples, purifying the bacterial population ahead of molecular confirmation by multiplex real-time quantitative PCR. After optimization using a panel of 50 characterized sputum samples, the performance of the prototype was assessed against the current gold standards, screening 100 blinded sputum samples using characterized and biobanked sputum provided by Foundation for Innovative New Diagnostics. Concordance with culture diagnosis was 100% for smear-negative samples and 87% for smear-positive samples. Of the smear-positive samples, the high burden sample concordance was 100%. Samples were diagnosed on the basis of visual assessment of the dielectrophoresis array and by multiplex real-time quantitative PCR assay. The results described herein demonstrate the potential of the CAPTURE-XT technology to provide a powerful sample preparation tool that could function as a front-end platform for molecular detection. This versatile tool could equally be applied as a visual detection diagnostic, potentially associated with bacterial identification for low-cost screening or coupled with an expanded PCR assay for genotypic drug susceptibility testing. Tuberculosis (TB) presents a multifaceted challenge to diagnostics, which impedes treatment access and sustains global transmission.1Walzl G. McNerney R. du Plessis N. Bates M. McHugh T.D. Chegou N.N. Zumla A. Tuberculosis: advances and challenges in development of new diagnostics and biomarkers.Lancet Infect Dis. 2018; 18: e199-e210Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar Pulmonary tuberculosis, the most common presentation of the Mycobacterium tuberculosis (MTB) infection in humans, is primarily diagnosed from analysis of sputum samples.2World Health Organization: Global Tuberculosis Report 2019. Geneva, Switzerland: World Health Organization, 2019. Available at: https://www.who.int/publications/i/item/9789241565714 (accessed January 26, 2021)Google Scholar Traditionally, smear microscopy is used with Ziehl-Neelsen staining to specifically highlight acid-fast bacterium.3Dzodanu E.G. Afrifa J. Acheampong D.O. Dadzie I. Diagnostic yield of fluorescence and Ziehl-Neelsen staining techniques in the diagnosis of pulmonary tuberculosis: a comparative study in a district health facility.Tuberc Res Treat. 2019; 2019: 1-6Google Scholar Mycobacterial burden is measured as grades of smear positivity (3+ to scanty), which is used to evaluate disease severity and associated infectiousness of the patient. Although this method is low cost and requires minimal laboratory facilities, sensitivity is poor, with a detectable limit of 104 bacilli/mL of sputum, and the quality of the diagnostic can vary between site and operators.4Lombardi G. Di Gregori V. Girometti N. Tadolini M. Bisognin F. Dal Monte P. Diagnosis of smear-negative tuberculosis is greatly improved by Xpert MTB/RIF.PLoS One. 2017; 12e0176186Crossref Scopus (47) Google Scholar,5Singhal R. Myneedu V.P. Microscopy as a diagnostic tool in pulmonary tuberculosis.Int J Mycobacteriol. 2015; 4: 1-6Crossref PubMed Google Scholar More recent improvements to smear microscopy have been implemented, including fluorescent cell labeling with the auramine stain, but lower burden infections still remain undiagnosed.6Steingart K.R. Henry M. Ng V. Hopewell P.C. Ramsay A. Cunningham J. Urbanczik R. Perkins M. Aziz M.A. Pai M. Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review.Lancet Infect Dis. 2006; 6: 570-581Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar For higher sensitivity interrogation of so-called smear-negative samples, culture has remained the World Health Organization–recommended method.7World Health Organization: Improving the diagnosis and treatment of smear-negative pulmonary and extrapulmonary tuberculosis among adults and adolescents: Recommendations for HIV-prevalent and resource-constrained settings. Geneva, Switzerland: World Health Organization, 2007. Available at: https://apps.who.int/iris/handle/10665/69463 (accessed May 11, 2023)Google Scholar The time to culture positivity in liquid culture system has long been used to predict patient outcome. Sputum samples are first decontaminated to eliminate competing, faster growing commensal bacteria and then incubated in growth medium to determine the presence of MTB.8Grandjean L. Martin L. Gilman R.H. Valencia T. Herrera B. Quino W. Ramos E. Rivero M. Montoya R. Escombe A.R. Coleman D. Mitchison D. Evans C.A. Tuberculosis diagnosis and multidrug resistance testing by direct sputum culture in selective broth without decontamination or centrifugation.J Clin Microbiol. 2008; 46: 2339-2344Crossref PubMed Scopus (26) Google Scholar Although undisputedly the most sensitive diagnostic, with limits of detection from 1 to 10 bacilli/mL of sputum, the slow growth rate of MTB and requirement of biosafety level 3 facilities to effectively perform this method of diagnosis remain limitations, particularly in resource-restricted settings.9Balcha T.T. Sturegård E. Winqvist N. Skogmar S. Reepalu A. Jemal Z.H. Tibesso G. Schön T. Björkman P. Intensified tuberculosis case-finding in HIV-positive adults managed at Ethiopian health centers: diagnostic yield of xpert MTB/RIF compared with smear microscopy and liquid culture.PLoS One. 2014; 9e85478Crossref PubMed Scopus (53) Google Scholar Meeting the Sustainable Development Goals for Tuberculosis requires the design and implementation of new technologies to address the key bottlenecks in TB control and elimination.10Falzon D. Migliori G.B. Jaramillo E. Weyer K. Joos G. Raviglione M. Digital health to end tuberculosis in the sustainable development goals era: achievements, evidence and future perspectives.Eur Respir J. 2017; 501701632Crossref Scopus (6) Google Scholar Novel diagnostic technologies are required to improve case detection, thereby reaching the missing millions, estimated in 2019 to be 2.9 million individuals globally.11Mechal Y. Benaissa E. El Mrimar N. Benlahlou Y. Bssaibis F. Zegmout A. Chadli M. Malik Y.S. Touil N. Abid A. Maleb A. Elouennass M. Evaluation of GeneXpert MTB/RIF system performances in the diagnosis of extrapulmonary tuberculosis.BMC Infect Dis. 2019; 19: 1069Crossref PubMed Scopus (32) Google Scholar,12World Health Organization: Global tuberculosis report 2020. Geneva, Switzerland: World Health Organization, 2020. Available at: https://www.who.int/publications/i/item/9789240013131 (accessed January 26, 2021)Google Scholar Cepheid's GeneXpert MTB/RIF assay and the GeneXpert MTB/RIF Ultra (Cepheid, Sunnyvale, CA) is the World Health Organization's main recommended rapid diagnostic test for detection of TB and rifampicin resistance as other more portable technologies are currently not available to provide accurate and rapid diagnosis.13World Health OrganizationWHO meeting report of a technical expert consultation: non-inferiority analysis of Xpert MTF/RIF Ultracompared to Xpert MTB/RIF. World Health Organization, Geneva, Switzerland2017Google Scholar There is still a need for rapid, accurate, and robust TB diagnostic tests suitable for use at the point of care,14World Health Organization: Global tuberculosis report 2018. Geneva, Switzerland: World Health Organization, 2018. Available at: https://apps.who.int/iris/handle/10665/274453 (accessed January 26, 2021)Google Scholar with high sensitivity for children and patients with HIV co-infection. The imperative for these much-needed advances will not just be based on increased sensitivity of detection in new assay formats for pathogen-specific DNA assays, but equally on developing in vitro diagnostic platforms applicable to high-endemic, underresourced areas of the world.15Seki M. Kim C.K. Hayakawa S. Mitarai S. Recent advances in tuberculosis diagnostics in resource-limited settings.Eur J Clin Microbiol Infect Dis. 2018; 37: 1405-1410Crossref PubMed Scopus (21) Google Scholar This requires devices that are portable and not dependent on infrastructure, yet suitable for use with difficult to manipulate sputum samples and capable of providing purified samples to test, as well as enhanced sensitivity of detection.16Van Rie A. Page-Shipp L. Scott L. Sanne I. Stevens W. Xpert MTB/RIF for point-of-care diagnosis of TB in high-HIV burden, resource-limited countries: hype or hope?.Expert Rev Mol Diagn. 2010; 10: 937-946Crossref PubMed Scopus (0) Google Scholar Herein we describe a prototype microfluidic chip-based system that can process solubilized sputum from patients with suspected TB, MTB for visual analysis a for smear and provide a purified sample for molecular confirmation by real-time quantitative PCR and for CAPTURE-XT technology dielectrophoresis of MTB from the patient and of the from other and that can detection or with molecular N. in dielectrophoresis PubMed Google Scholar the of a and the medium in which to manipulate the The is by of the of and the medium in which or that a can be by a to an array of with the in which the are the system was first has been the of and coupled with for as and and in the development of has been in A. V. analysis in microfluidic 2017; PubMed Scopus Google of cell Scopus Google L. of of dielectrophoresis in and for bacteria Scopus Google Scholar a and as yet to TB diagnosis that could provide a test with improved performance for low bacterial burden sputum samples, as smear or smear-negative of current diagnostic and low prototype microfluidic was used ahead of a assay to performance against gold TB diagnostics on a blinded of 100 sputum samples provided by the Foundation for Innovative New to of sputum were provided by from a with from 100 Samples were and patient was diagnostics were used for of the disease including at culture and smear microscopy TB Available at: (accessed May 11, 2023)Google Scholar Sputum samples from and and were provided blinded with a Sputum samples were at required for 50 samples were provided with smear and culture for use in The panel of samples culture samples culture and samples between to culture The blinded was of and samples by as these are the most samples to as for MTB for TB sputum sample was at for the of and in Samples were for and incubated at for of 3 cell Samples were then incubated at for a the of Samples were for and an and then to at for in samples were in a biosafety level 3 The of are based on a described by and I. of and by PubMed Google Scholar and and of J. Full Text PDF PubMed Google Scholar 1 a of to the of the the were with in were that the of the from a and to a for were from the microfluidic using and were with to sample was were by and to a function and was 1 from to on the 1 and of the control platform were used with The first with laboratory a with and J. I. E. V. M. T. S. S. B. V. K. P. A. an platform for PubMed Scopus Google Scholar control was a The of control a and were a in M.A. N. N. of control using 2014; PubMed Google Scholar In with of were used to the presence or of to the of the and were using an in the sputum samples were with stain, are to areas of high by of by an To the of the test were to For and the sample was which the are and the sample has to the end of the sample the are the bacteria the for and a presence can be by fluorescence visual analysis is the is the bacteria the for from the microfluidic The purified sample can then be used for molecular The process is in sputum was the and the to a of to the The was with a current and remained the sample to 1 was the from the the was After visual analysis of the with fluorescent the presence or of was and with was the of were using a in to 1 For optimization of the system sputum samples with were Mycobacterium tuberculosis, and Mycobacterium Health Organization: Global Tuberculosis Report 2019. Geneva, Switzerland: World Health Organization, 2019. Available at: https://www.who.int/publications/i/item/9789241565714 (accessed January 26, 2021)Google Scholar were as and in liquid were and at of culture to in with and The were incubated at The were for a culture was quantitative PCR assay using was for MTB and and which was and were using the A. T. R. an enhanced to PubMed Scopus Google Scholar with on minimal for rapid was as an the and were to the sensitivity of MTB detection. analysis of the and by was to and for are in of was on the basis of the GeneXpert which sample with of as and in in Mycobacterium tuberculosis (MTB) of MTB have of from to real-time quantitative in Mycobacterium tuberculosis (MTB) of MTB have of from to in a new real-time quantitative testing panel of and a of and bacteria was from The of and from of was against the panel to determine the of the PCR was using PCR the with 1 DNA and of and PCR were for by of at at and at and a at DNA sample was and with TB DNA as an The PCR were using the CA) For molecular analysis of the CAPTURE-XT using the was the and a was from the were as as well as a which was sputum and sputum samples were to in a for to samples to be of the level 3 Samples were then for cell by the of for for for for 1 for 1 and for of of was used as in as described with and samples that been were and were for testing using a of devices were to to the PCR was with and an The was to culture to and the presence of low of MTB in the this was using selective medium with and 100 10 and 10 or and growth and 100 10 and 10 at for to 3 which the liquid culture was and 100 10 and 10 incubated at for 3 to and assessed for in between sputum samples and were estimated using and real-time of MTB by the is in This the of solubilized sputum the microfluidic the and of the of the and on the of from to as more sample is The the are After of the the is and the bacteria are with a on the to the bacterial cell were with M. in as a to To determine the required of were applied to the and to the was The of the on the was with and are in N. J. by 2006; Scopus (32) Google J. R. J. analysis of in a PubMed Google Scholar with and the of for the of The was to be at 10 and at 10 were for development for To CAPTURE-XT technology as a TB diagnostic sputum samples be with the without or that the of the bacterial to the the used as and be applied of the these optimization of time of and were using sputum with MTB or M. fluorescent microscopy and quantitative the of and was Of the a at the of sputum that was enhanced by at and by the of Sputum required the of and in a with and at the improvements in and with this The method in a of bacteria and not of the The bacteria in a in and was of the of the sample is required as the of the to the is This was by with The of by was in samples but was from a and at with the without Although 100% be the the at was to the microfluidic at of to of was testing with sputum from TB and by conventional gold a visual diagnostic, by the presence of bacteria on the molecular analysis was used to the presence of MTB as to other in the For this a assay was for for and a well as or a assay with limits of detection of bacteria as from were for against a panel of including most of the MTB a of and or bacterial was in the design for MTB on purified DNA in PCR in PCR with the panel of control was assessed with for Mycobacterium which with and and Mycobacterium which with The results of testing are in For the of with a sample the was for optimization and of the CAPTURE-XT technology and were using biobanked sputum samples. Samples been characterized using gold TB diagnostics, including culture and smear of these characterized samples using the method was with a visual in the of a microscopy of the and a molecular from of the The 50 samples were for optimization of the to be used on the 100 blinded sample as well as to demonstrate To perform quantitative on the optimization a with was using the samples be on the samples be with the samples with the burden was with a or with a samples without the of from or After these sputum samples were used with the method to performance ahead of the blinded were smear and smear and culture and and that smear and culture as the of is smear For these samples, the are smear for Sputum Samples of samples smear and culture real-time quantitative smear and culture in a new Evaluation of of smear with gold that were and were and which are by the Foundation for Innovative New smear and culture that were and were and which are by the in a new of samples smear and culture real-time quantitative smear and culture Foundation for Innovative New smear and culture of the using sample in to the smear microscopy and the visual bacterial on the There was a between and estimated by smear The for these samples for a of samples are in This between bacteria and measured by the potential of this bacterial and method for use ahead of molecular diagnostic concordance between gold diagnostic and the visual for these samples is in concordance was for the smear-positive samples, from smear to of the samples were in the and were as on by been Concordance with for the smear-negative samples was to the results and and not reach the these demonstrate a of that and and by concordance and a for the smear-positive sputum samples. MTB a between the sputum and the samples. The in from sputum sample to for and was and This to a of and that the bacterial as by and then are MTB is with MTB in the compared with sputum samples. the results from the samples with the of the samples, the method described was used for the analysis of a provided without a assessment of the technology against gold The 100 blinded sputum samples were using the from visual analysis were at the end of sample and used to the diagnostic in a as MTB or MTB were for molecular analysis with sample with a of was samples a between and were In this the were and a of positivity was and by laboratory of and of samples were between the visual and PCR samples were as Of the 100 samples were as were as and were as were to for and were with concordance concordance of was samples, with and for and smear Concordance was lower for smear-negative samples, with between gold and the CAPTURE-XT To the samples, from the samples were to analysis was on a of the CAPTURE-XT platform and in with technology To sensitivity was the sputum sample was to visual To improve the PCR was with and the in a multiplex of the analysis improved results with an concordance of for smear-positive samples with smear samples The concordance of samples remained at The PCR improved of from PCR and from to on this sample that was as smear and culture culture in the the the method a concordance of for samples, with an concordance for culture of The sensitivity and were 87% and 100% The results of the study are in of samples with by Foundation for Innovative New samples by smear and Concordance as of samples for which the the diagnostic or MTB as the gold Mycobacterium in a new of samples with by Foundation for Innovative New samples by smear and Concordance as of samples for which the the diagnostic or MTB as the gold Mycobacterium The of TB, as of the Sustainable Development Goals by the requires not tests with improved sensitivity but with the potential to be used in the of and health of individuals D. Migliori G.B. Jaramillo E. Weyer K. Joos G. Raviglione M. Digital health to end tuberculosis in the sustainable development goals era: achievements, evidence and future perspectives.Eur Respir J. 2017; 501701632Crossref Scopus (6) Google Scholar,12World Health Organization: Global tuberculosis report 2020. Geneva, Switzerland: World Health Organization, 2020. Available at: https://www.who.int/publications/i/item/9789240013131 (accessed January 26, 2021)Google Scholar The technology in this study the potential of CAPTURE-XT for the and detection by of MTB from sputum samples. the prototype CAPTURE-XT platform a of 100% and a sensitivity of In GeneXpert has been to have a of and a sensitivity of and Ultra has a of and a sensitivity of Y. Benaissa E. El Mrimar N. Benlahlou Y. Bssaibis F. Zegmout A. Chadli M. Malik Y.S. Touil N. Abid A. Maleb A. Elouennass M. Evaluation of GeneXpert MTB/RIF system performances in the diagnosis of extrapulmonary tuberculosis.BMC Infect Dis. 2019; 19: 1069Crossref PubMed Scopus (32) Google S. M. Y. J. S. S. A. J. B. P. N. A. E. A. D. R. M. D. The new Xpert MTB/RIF detection of Mycobacterium tuberculosis and resistance to in an assay suitable for point-of-care 2017; Scopus Google Scholar Concordance between gold diagnostics and with were 100% for and smear-positive samples. bacterial burden samples culture which are more to results in diagnostic a concordance of in the N. A. D. B. in sputum smear microscopy results for acid-fast by and in J Dis. Google Scholar of was improved were This could be to the of which can be in 1 to or on and are in of and a more S. I. in Mycobacterium tuberculosis in the PubMed Scopus (0) Google Scholar The study described used as a assay to of and of MTB by from solubilized sputum the used in this study was This and the as to positivity for of the low bacterial burden samples with to is a of this which samples to determine a more quantitative Although is on the be the for GeneXpert is which was that was for this In addition, samples that were in the system on by and been as This was to MTB bacteria that In the for samples from sputum were but were of the of which samples with low burden are to be as this system have an increased In this was in a biosafety with which in a the and of the assay and the potential sample the system a portable front-end these The sample study was to of For improved sensitivity and an time to devices with are to microfluidic can the bacteria in a sample in with a visual for an diagnosis analysis for samples and for drug resistance to of the and design are to in improved For this biobanked samples were at have in a of cell or the bacterial cell required to a to the of the samples in not A. D. of tuberculosis Clin Microbiol. PubMed Scopus Google Scholar the of be with to the with which can be for microfluidic To the performance analysis of this samples are required to a for point-of-care The fluorescent in this study was not for MTB and could have in a an improve the of the visual analysis of this M. P. B. N. R. detection of Mycobacterium tuberculosis in sputum with a 2018; PubMed Scopus Google Scholar This assay be used in the future for of In we have as a of using biobanked TB sputum and an that CAPTURE-XT is a sensitive and platform for TB This has potential as a TB diagnostic or as a front-end sample preparation technology for visual and molecular detection In addition, the assay could detection of for drug this technology capable of the Foundation for Innovative New for of biobanked sputum samples. with with of and from the The is the are and then at to the