| Objective:(1)To design and screen a microfluidic paper-based device(μPAD)with high genomic DNA extraction efficiency of mycobacteria,and given enough thought to the tough cell wall of mycobacteria.(2)To establish an optimized genomic DNA extraction protocol based on the self-designed μPAD.(3)To establish the multiplex loop-mediated isothermal amplification(LAMP)detection system for Mycobacterium tuberculosis complex(MTBC)and the nontuberculous mycobacteria(NTMs)which are common causes of nontuberculous mycobacterial pulmonary diseases(NTM-PD).(4)To evaluate the parameters of μPAD and multiple LAMP systems by using standard bacterial strains and quantitative genomic DNA of standard bacterial strains,and build multiple visualization strategies for different application settings,and finally obtain the optimized Point-of-Care nucleic acid detection device(3-D μPAD POCT)for multiple mycobacteria;(5)To evaluate the diagnostic performance of 3-D μPAD POCT device in multicenter study by taking the reference method and recommended method for detection of mycobacterial infection as a control,in order to comprehensively evaluate the clinical diagnostic performance of the POCT device developed in this study.Methods:Part Ⅰ(1)Paper-based microfluidic design and fabrication: Chosen Whatman No.1 filter paper as the medium of paper-based microfluidic.The Xerox Color Qube 8570 printer and matched Wax-based Ink was selected to print the wax on filter paper according to the designed pattern,then the patterned papers were heated to form the hydrophobic barrier,and which was measured to verify whether the vertical and lateral diffusion of melting wax in the paper are consistent with Capillary Flow formula that established by Washburn EW.and coworkers.And then,the resolution of patterned hydrophobic barrier was further determined according to Washburn Capillary Flow formula.Once the above parameters were confirmed,the vector design software Adobe illustrator CS6 will be employed to draw the plan view of millimeter-level 3-D paper microfluid(3-D μPAD)with different fluid channels.Following,the 3-D μPAD with different designs will be fabricated by heating on the hotplate,and the optimized one was obtained through the comprehensive screening of multiple indicators,including cost,operability,simplicity,and loss of target DNA.Finally,the surface topography of the optimized 3-D μPAD was examined using a field emission scanning electron microscope(FESEM),and the bulk water absorption amount and flow velocity were measured by using the pure water added with the pigment.(2)Establishment and optimization of the rapid nucleic acid extraction protocol:Through domestic and foreign literature investigations to establish a suitable protocol based on the optimized 3-D μPAD to achieve rapid extraction of mycobacterial genomic DNA in the field of rapid detection application settings.And the standard strains and clinical samples were employed to evaluate the DNA extraction efficiency of the rapid nucleic acid extraction protocol by comparison with the Nucli SENS easy MAG automatic extraction system.(3)Construction of multiple mycobacterial LAMP systems: Based on previously published literature and the results of genome sequence alignment,the target genes and/or sequences which can specifically identify Mycobacterium tuberculosis complex(MTBC),M.avium,M.intracellulare,M.abscessus,M.kansasii,and M.fortuitum were pooled for LAMP primer sets design.And then,the online primer design software Primer Explorer V5 was used to design the LAMP primer sets.The primer sets were screened through the system comprised of commercial Q-LAMP kit and quantitative genomic DNA of target mycobacteria as templates.The primer sets obtained by screening were furtherly used to construct the in-house LAMP amplification systems,aiming to reduce the cost and replace the commercial system.Finally,the parameters including optimal primers ratio,optimal reaction temperature,sensitivity,and specificity of in-house LAMP amplification systems were verified.(4)Visualization strategy construction: The non-DNA binding fluorescent dye-Calcein,and Chelation-Manganese chloride were used to visualize the amplicons of established LAMP systems.In addition,a closed lateral flow chromatography device that can detect amplicons was designed and fabricated by integrating PMMA and disposable nucleic acid detection test strips.Part Ⅱ(1)Participant’s enrollment: The participants were recruited by a two phases strategy in 3 centers.The first phase is from October 2018 to September 2020,in total of 1,213 inpatients or outpatients suspected with PTB or NTM-PD were prospectively recruited from the Tuberculosis Unit of the Department of Respiratory Medicine,West China Hospital of Sichuan University.The second phase was from April 2020 to October 2020,462 and 316 highly suspected PTB and/or NTM-PD patients were retrospectively enrolled from the First Affiliated Hospital of Zhengzhou University and Lanzhou Pulmonary Hospital,respectively,based on their clinical manifestation,results of imaging and AFB smear.(2)Clinical application evaluation: the 3-D μPAD POCT device developed in this study,the well-reputed Cepheid Xpert MTB/FIF detection method,and the mycobacterial culture(gold standard)were used to carry out the parallel comparison test.All enrolled clinical samples were tested in a double-blind method,and the results were analyzed to evaluate the performance of 3-D μPAD POCT device.(3)Statistical analysis: The continuous variables and categorical variables were analyzed through SPSS 24.0.The Cochran-Mantel-Haenszel test was used to compare the diagnostic performance of the three detection methods compared with the final clinical diagnosis.P value <0.05 represents a significant statistical difference.Graph Pad Prism v8.0 and R version 3.5.3 were employed to rebuild the graphs.Results:Part Ⅰ(1)Paper-based microfluidics design and fabrication: We finally selected Whatman No.1 qualitative filter paper(cat# 1001-917)as the production medium for paper-based microfluidics,and the wax was directly printed on the surface of the properly cut filter paper by Color Qube 8570 wax printer.When heated on the hotplate,the patterned waxy layer on the surface of paper melts and penetrates the paper forming the patterned hydrophobic barrier.Through the specific measurement of the width of the finally formed hydrophobic barrier and hydrophilic channel,we confirmed that the dynamics of selected wax material during the heat treatment was completely consistent with Washburn EW.Capillary Flow formula.By making a series of patterned hydrophobic barriers,we confirmed that wax printing can form the smallest functional hydrophobic barrier with a printing line width of 300 μm,and the average width of the barrier can be 850 ± 50 μm.Based on the above parameters,we have designed and manufactured three generation of 3-D μPAD.After screening multiple indicators for operability,simplicity,and depletion of target DNA,we finally selected the third generation 3-D μPAD as the ultimate tool for extraction and purification of mycobacterial genomic DNA in this study.The surface topography results of field emission scanning electron microscope and stereoscopic microscope showed that the 3-DμPAD printed by Xerox Color Qube 8570 wax printer can form the functional hydrophobic barrier in Whatman No.1 filter paper(cat# 1001-917).And the flow characterization test results showed that after the third-generation 3-D μPAD was folded to form a three-dimensional fluid channel,the bulk water absorption amount in each waste area is 200 μL,and the velocity of the flow through the three-dimensional fluid channel is about 3 μL/s.(2)Establishment of the rapid nucleic acid extraction protocol: We have established a nucleic acid extraction strategy of "physical fragmentation + boiling lysis +3-D μPAD",which can achieve rapid and efficient extraction of mycobacteria genomic DNA in point-of-care settings independent of large instruments and complete laboratory conditions.The DNA extraction efficiency of 3-D μPAD was evaluated with mimic samples and clinical samples by comparison with easy MAG automated extraction system.The results showed that the DNA recovery rate of 3-D μPAD was about 85% of that of Nucli SENS easy MAG automated extraction system.(3)Construction of multiple mycobacterial LAMP systems: A total of 312 sets of LAMP primer were designed to target 14 genes and sequences which can specifically identify Mycobacterium tuberculosis complex(MTBC),M.avium,M.intracellulare,M.abscessus,M.kansasii,and M.fortuitum.And 217 primer sets were eliminated through online screening.The other 95 primer sets were screened through the system comprised of commercial Q-LAMP kit,Hongshi SLAN Fluorescence quantitative PCR analyzer and quantitative genomic DNA of target mycobacteria as templates.After testing for specificity,optimal primer concentration and sensitivity,12 primer sets that can be used to construct the in-house LAMP amplification systems were finally pooled.They were 5 primer sets in the MTBC primer group(IS6110-28,IS6110-106,IS 6110-125,IS1081-13 and IS1081-22),the primer set ITS-4 in the MIN primer group,the primer set tuf-92 in the MAB primer group,the primer set MKAN＿rs12360-43 in the MKAN primer group,the primer set ku-8 and ku-24 in the MFOR primer group,and the primer sets of IS1245-49 and IS1245-18 in the MAC primer group.One of primer set was selected from each primer group according to the performance of specificity,sensitivity to construct the in-house LAMP amplification systems,aiming to reduce the cost and replace the commercial system.Finally,the sensitivity of six in-house LAMP amplification systems were verified through 1.5%agarose gel,and the results showed that the in-house systems have the same sensitivity as the commercial Q-LAMP kit.(4)Visualization strategy construction: Adding Calcein and manganese chloride in a series of concentration ratios to the already constructed in-house LAMP reaction system,the Fluorescence quantitative PCR analyzer was employed to capture the fluorescence signal in a real-time way,and the colorimetry changes of the reaction system under ultraviolet light and visible light were recorded.After a comprehensive evaluation of the results,we selected 1:26 as the optimal concentration ratio of Calcein and manganese chloride.The sensitivity of in-house LAMP amplification systems using the optimal concentration ratio of Calcein and manganese chloride as an indicator were verified,and the result was compared with that of 1.5% agarose gel electrophoresis and commercial Q-LAMP kit.The comparison results showed that the in-house systems using the optimal concentration ratio of Calcein and manganese chloride as an indicator have the same sensitivity as the commercial Q-LAMP kit.This visualization strategy can be applied to the visual presentation of multiple mycobacterial nucleic acid amplification results.We fabricated a closed lateral flow device by integrating polymethyl methacrylate(PMMA)and disposable nucleic acid test strip forming a new visualization strategy.The amplicons in the amplification system can be released and detected without forming aerosols by using this device.The sensitivity of in-house LAMP amplification systems using the closed lateral flow device as a visualization strategy were verified,and the result was compared with that of 1.5% agarose gel electrophoresis,commercial Q-LAMP kit,and Calcein.The comparison results showed that the in-house systems using the closed lateral flow device as a visualization strategy have the same sensitivity as the commercial Q-LAMP kit.This visualization strategy can also be applied to the visual presentation of multiple mycobacterial nucleic acid amplification results.Part Ⅱ(1)Participants: A total of 815 suspected PTB or NTM-PD patients were recruited from West China Hospital of Sichuan University from October 2018 to September 2020,292 of them were finally diagnosed with PTB,27 of them were finally diagnosed with NTM-PD,2 patients were diagnosed with mixed infection of MTBC and NTM,and 496 patients were confirmed with pulmonary infection and tumor.A total of 396 suspected PTB or NTM-PD patients were recruited from the First Affiliated Hospital of Zhengzhou University from April 2020 to October 2020,277 of them were finally diagnosed with PTB,24 of them were finally diagnosed with NTM-PD,and 95 patients were confirmed with pulmonary infection and tumor.A total of 172 suspected PTB or NTM-PD patients were recruited from Lanzhou Pulmonary Hospital from April 2020 to October 2020,111 of them were finally diagnosed with PTB,22 of them were finally diagnosed with NTM-PD,and 39 patients were confirmed with pulmonary infection.(2)Clinical application evaluation results: Without stratifying the sample type and the type of diagnosis,the clinical sensitivity of the 3-D μPAD POCT device for PTB in cohort I was 77.74%(95% CI: 72.63%-82.13%),and the specificity was 97.90%(95 % CI: 96.28%-98.82%),the positive predictive value(PPV)is 95.38%(95% CI:91.91%-97.40%),the negative predictive value(NPV)is 88.75%(95% CI:85.92%-91.08%),and the diagnostic accuracy is 90.69%(95% CI: 88.50%-92.49%);clinical sensitivity to NTM-PD is 96.55%(95% CI: 82.82%-99.39%),specificity is98.99%(95% CI: 97.66%-99.57%),the PPV is 84.85%(95% CI: 69.08%-93.35%),the NPV is 99.80%(95% CI: 98.86%-99.96%),and the diagnostic accuracy is 98.86%(95% CI: 88.50%-92.49%).Without stratifying the sample type and the type of diagnosis,the clinical diagnosis sensitivity of the 3-D μPAD POCT device for PTB in cohort II is 90.25%(95%CI: 86.19%-93.21%),and the specificity was 96.64%(95 % CI: 91.68%-98.69%),the PPV is 98.43%(95% CI: 96.02%-99.39%),the NPV is 80.99%(95% CI:73.75%-86.59%),the diagnostic accuracy is 92.17%(95% CI: 89.10%-94.43%);the clinical sensitivity of NTM-PD is 91.67%(95% CI: 74.15%-97.68%),and the specificity is 98.66%(95% CI: 96.89%-99.42%),the PPV is 81.48%(95% CI:63.30%-81.82%),the NPV is 99.46%(95% CI: 98.05%-99.85%),and the diagnostic accuracy is 98.23%(95% CI: 96.4%-99.14%).Without stratifying the sample type and the type of diagnosis,the clinical sensitivity of the 3-D μPAD POCT device in cohort III for PTB is 88.29%(95% CI:80.99%-93.03%),and the specificity is 96.72%(95 % CI: 88.81%-99.1%),the PPV is98.00%(95% CI: 93%-99.45%),the NPV is 81.94%(95% CI: 71.52%-89.13%),and the diagnostic accuracy is 91.28%(95% CI: 86.11%-94.64%);the clinical sensitivity for NTM-PD is 86.36%(95% CI: 66.66%-95.25%),and the specificity is 89.74%(95%CI: 76.42%-95.94%),the PPV is 82.61%(95% CI: 62.86%-93.022%),the NPV is92.11%(95% CI: 79.2%-97.28%),and the diagnostic accuracy is 88.52%(95% CI:78.16%-94.33%).In the bacteriological diagnosed PTB subgroup of cohort I,the clinical sensitivity of the 3-D μPAD POCT device is 90.20%(95% CI: 85.84%-93.33%),and the specificity is 97.78%(95% CI: 96.07%-98.76%),PPV was 95.26%(95% CI:91.71%-97.33%),NPV was 95.28%(95% CI: 93.08%-96.81%),and the diagnostic accuracy is 95.28%(95% CI: 93.50%-96.58%).In the clinical diagnosed PTB subgroup of cohort I,the clinical sensitivity of the 3-D μPAD POCT device is only 12.77%(95% CI: 5.985%-25.17%),and the PPV is 35.29%(95% CI: 17.31%-58.70%),the specificity and NPV are consistent with the subgroup of bacteriological diagnosed,and the diagnostic accuracy is 90.42%(95% CI: 87.66%-92.62%).In the bacteriological diagnosed PTB subgroups of Cohort II and Cohort III,the diagnostic performance of the three methods has been improved compared with that without stratification.Such as,the sensitivity of the 3-D μPAD POCT device in cohort II improved from90.25%(95%CI: 86.19%-93.21%)to 93.28%(95%CI: 89.63%-95.71%),and the diagnostic accuracy improved from 92.17%(95% %CI: 89.1%-94.43%)to 93.94%(95%CI: 90.99%-95.96%);the sensitivity of 3-D μPAD POCT device in cohort Ⅲimproved from 88.29%(95%CI: 80.99%-93.03%)to 93.33%(95%CI: 86.87%-96.73%),the diagnostic accuracy improved from 91.28%(95%CI: 86.11%-94.64%)to 93.75%(95%CI: 88.55%-96.68%).In the sputum subgroup of Cohort I,the clinical sensitivity of the 3-D μPAD POCT device is 92.17%(95% CI: 85.79%-95.83%),and the specificity is 99.44%(95%CI: 96.9%-99.9%),the PPV is 99.07%(95% CI: 94.89%-99.83%),the NPV is 95.19%(95% CI: 91.11%-97.45%),and the diagnostic accuracy is 96.6%(95% CI:93.85%-98.14%).In the BALF subgroup of cohort I,the clinical sensitivity of the 3-DμPAD POCT device is 88.46%(95% CI: 81.83%-92.88%),and the specificity is96.85%(95% CI: 94.29%-98.28%),the PPV is 92.00%(95% CI: 85.9%-95.6%),the NPV is 95.34%(95% CI: 92.46%-97.16%),and the diagnostic accuracy is 94.41%(95% CI: 91.87%-96.18%).In the sputum subgroup of cohort II,the clinical sensitivity of the 3-D μPAD POCT device is 92.80%(95% CI: 86.88%-96.17%),and the specificity is 95.24%(95%CI: 86.91%-98.37%),the PPV is 97.48%(95% CI: 92.85%-99.14%),the NPV is86.96%(95% CI: 77.03%-92.98%),and the diagnostic accuracy is 93.62%(95% CI:89.18%-96.31%).In the BALF subgroup of cohort II,the clinical sensitivity of the3-D μPAD POCT device is 90.97%(95% CI: 85.17%-94.65%),and the specificity is96.88%(95% CI: 84.26%-99.45%),the PPV is 99.24%(95% CI: 95.83%-99.87%),the NPV is 70.45%(95% CI: 55.78%-81.84%),and the diagnostic accuracy is 92.05%(95% CI: 87.09%-95.20%).In the sputum subgroup of cohort III,the clinical sensitivity of the 3-D μPAD POCT device is 98.31%(95% CI: 91.00%-99.70%),and the specificity is 100%(95%CI: 84.54%-100%),the PPV is 100%(95% CI: 93.79%-100%),the NPV is 95.45%(95% CI: 78.20%-99.19%),and the diagnostic accuracy is 98.75%(95% CI:93.25%-99.78%).In the BALF subgroup of cohort III,the clinical sensitivity of the3-D μPAD POCT device is 86.96%(95% CI: 74.33%-93.88%),and the specificity is88.89%(95% CI: 67.20%-96.90%),the PPV is 95.24%(95% CI: 84.21%-98.68%),the NPV is 72.73%(95% CI: 51.85%-86.85%),and the diagnostic accuracy is 87.50%(95% CI: 77.23%-93.53%).Conclusion:In this study,we successfully developed a point-of-care testing device named3-D μPAD POCT device by integration of paper-based microfluidic,loop-mediated isothermal amplification and lateral flow chromatography technology for the first time,which can simultaneously and rapidly detect the genomic DNA of MTBC,M.avium,M.intracellulare,M.abscessus,M.kansasii,and M.fortuitum.The 3-D μPAD POCT device integrates all the necessary functions for rapid detection of mycobacterial nucleic acid,including rapid extraction of mycobacterial genomic DNA,isothermal amplification of target sequences,and visual interpretation of the amplicons.And the device can complete rapid screening and identification of PTB and 5 common NTM-PD diseases in the point-of-care setting without precision instrument and equipped medical laboratory conditions.Multi-center clinical application evaluation shows that the3-D μPAD POCT device has a good performance in the rapid detection of MTBC and NTM infections,and it is expected to become a new tool to assist the rapid differential diagnosis of PTB and NTM-PD. |
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