A novel triple lines lateral-flow assay （LFA） with enhanced sensitivity for the detection of Leishmania infantum DNA in dog blood samples was designed and successfully applied. The enhanced LFA methodology takes adv...A novel triple lines lateral-flow assay （LFA） with enhanced sensitivity for the detection of Leishmania infantum DNA in dog blood samples was designed and successfully applied. The enhanced LFA methodology takes advantage of the gold nanoparticle tags （AuNPs） conjugated to polyclonal secondary antibodies, which recognize anti-FITC antibodies. The polyclonal nature of the secondary antibodies allows for multiple binding to primary antibodies, leading to enhanced AuNP plasmonics signal. Furthermore, endogenous control consisting of the amplified dog 18S rRNA gene was introduced to avoid false negatives. Using this strategy, 0.038 spiked Leishmania parasites per DNA amplification reaction （1 parasite/100 μL of DNA sample） were detected. Detection limit of LFA was found to be lower than that of the conventional techniques. In summary, our novel LFA design is a universal and simple sensing altemative that can be extended to several other biosensing scenarios.展开更多
Background: Although existing mycological tests (bronchoalveolar lavage [BAL] galactomannan [GM], serum GM, serum (1,3)-β-D-glucan [BDG], and fungal culture) are widely used for diagnosing invasive pulmonary aspergil...Background: Although existing mycological tests (bronchoalveolar lavage [BAL] galactomannan [GM], serum GM, serum (1,3)-β-D-glucan [BDG], and fungal culture) are widely used for diagnosing invasive pulmonary aspergillosis (IPA) in non-hematological patients with respiratory diseases, their clinical utility in this large population is actually unclear. We aimed to resolve this clinical uncertainty by evaluating the diagnostic accuracy and utility of existing tests and explore the efficacy of novel sputum-basedAspergillus assays.Methods: Existing tests were assessed in a prospective and consecutive cohort of patients with respiratory diseases in West China Hospital between 2016 and 2019 while novel sputum assays (especially sputum GM andAspergillus-specific lateral-flow device [LFD]) in a case-controlled subcohort. IPA was defined according to the modified European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Sensitivity and specificity were computed for each test and receiver operating characteristic (ROC) curve analysis was performed.Results: The entire cohort included 3530 admissions (proven/probable IPA=66, no IPA=3464) and the subcohort included 127 admissions (proven/probable IPA=38, no IPA=89). Sensitivity of BAL GM (≥1.0 optical density index [ODI]: 86% [24/28]) was substantially higher than that of serum GM (≥0.5 ODI: 38% [39/102]) (χ^(2)=19.83,P<0.001), serum BDG (≥70 pg/mL: 33% [31/95]) (χ^(2)=24.65,P<0.001), and fungal culture (33% [84/253]) (χ^(2)=29.38,P<0.001). Specificity varied between BAL GM (≥1.0 ODI: 94% [377/402]), serum GM (≥0.5 ODI: 95% [2130/2248]), BDG (89% [1878/2106]), and culture (98% [4936/5055]). Sputum GM (≥2.0 ODI) had similar sensitivity (84% [32/38]) (Fisher’s exactP=1.000) to and slightly lower specificity (87% [77/89]) (χ^(2)=5.52,P=0.019) than BAL GM (≥1.0 ODI). Area under the ROC curve values were comparable between sputum GM (0.883 [0.812-0.953]) and BAL GM (0.901 [0.824-0.977]) (P=0.734). Sputum LFD had similar specificity (91% [81/89]) (χ^(2)=0.89,P=0.345) to and lower sensitivity (63% [24/38]) (χ^(2)=4.14,P=0.042) than BAL GM (≥1.0 ODI), but significantly higher sensitivity than serum GM (≥0.5 ODI) (χ^(2)=6.95,P=0.008), BDG (χ^(2)=10.43,P=0.001), and fungal culture (χ^(2)=12.70,P<0.001).Conclusions: Serum GM, serum BDG, and fungal culture lack sufficient sensitivity for diagnosing IPA in respiratory patients. Sputum GM and LFD assays hold promise as rapid, sensitive, and non-invasive alternatives to the BAL GM test.展开更多
文摘A novel triple lines lateral-flow assay （LFA） with enhanced sensitivity for the detection of Leishmania infantum DNA in dog blood samples was designed and successfully applied. The enhanced LFA methodology takes advantage of the gold nanoparticle tags （AuNPs） conjugated to polyclonal secondary antibodies, which recognize anti-FITC antibodies. The polyclonal nature of the secondary antibodies allows for multiple binding to primary antibodies, leading to enhanced AuNP plasmonics signal. Furthermore, endogenous control consisting of the amplified dog 18S rRNA gene was introduced to avoid false negatives. Using this strategy, 0.038 spiked Leishmania parasites per DNA amplification reaction （1 parasite/100 μL of DNA sample） were detected. Detection limit of LFA was found to be lower than that of the conventional techniques. In summary, our novel LFA design is a universal and simple sensing altemative that can be extended to several other biosensing scenarios.
基金supported by grants from Sichuan Science and Technology Program(No.2019YFS0231)1,3,5 project for disciplines of excellence,West China Hospital,Sichuan University(No.2018-119)the National Natural Science Foundation of China(No.81870014).
文摘Background: Although existing mycological tests (bronchoalveolar lavage [BAL] galactomannan [GM], serum GM, serum (1,3)-β-D-glucan [BDG], and fungal culture) are widely used for diagnosing invasive pulmonary aspergillosis (IPA) in non-hematological patients with respiratory diseases, their clinical utility in this large population is actually unclear. We aimed to resolve this clinical uncertainty by evaluating the diagnostic accuracy and utility of existing tests and explore the efficacy of novel sputum-basedAspergillus assays.Methods: Existing tests were assessed in a prospective and consecutive cohort of patients with respiratory diseases in West China Hospital between 2016 and 2019 while novel sputum assays (especially sputum GM andAspergillus-specific lateral-flow device [LFD]) in a case-controlled subcohort. IPA was defined according to the modified European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Sensitivity and specificity were computed for each test and receiver operating characteristic (ROC) curve analysis was performed.Results: The entire cohort included 3530 admissions (proven/probable IPA=66, no IPA=3464) and the subcohort included 127 admissions (proven/probable IPA=38, no IPA=89). Sensitivity of BAL GM (≥1.0 optical density index [ODI]: 86% [24/28]) was substantially higher than that of serum GM (≥0.5 ODI: 38% [39/102]) (χ^(2)=19.83,P<0.001), serum BDG (≥70 pg/mL: 33% [31/95]) (χ^(2)=24.65,P<0.001), and fungal culture (33% [84/253]) (χ^(2)=29.38,P<0.001). Specificity varied between BAL GM (≥1.0 ODI: 94% [377/402]), serum GM (≥0.5 ODI: 95% [2130/2248]), BDG (89% [1878/2106]), and culture (98% [4936/5055]). Sputum GM (≥2.0 ODI) had similar sensitivity (84% [32/38]) (Fisher’s exactP=1.000) to and slightly lower specificity (87% [77/89]) (χ^(2)=5.52,P=0.019) than BAL GM (≥1.0 ODI). Area under the ROC curve values were comparable between sputum GM (0.883 [0.812-0.953]) and BAL GM (0.901 [0.824-0.977]) (P=0.734). Sputum LFD had similar specificity (91% [81/89]) (χ^(2)=0.89,P=0.345) to and lower sensitivity (63% [24/38]) (χ^(2)=4.14,P=0.042) than BAL GM (≥1.0 ODI), but significantly higher sensitivity than serum GM (≥0.5 ODI) (χ^(2)=6.95,P=0.008), BDG (χ^(2)=10.43,P=0.001), and fungal culture (χ^(2)=12.70,P<0.001).Conclusions: Serum GM, serum BDG, and fungal culture lack sufficient sensitivity for diagnosing IPA in respiratory patients. Sputum GM and LFD assays hold promise as rapid, sensitive, and non-invasive alternatives to the BAL GM test.
