Competitive ELISA for a serologic test to detect dengue serotype-specific anti-NS1 IgGs using high-affinity UB-DNA aptamers.


Journal

Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288

Informations de publication

Date de publication:
09 09 2021
Historique:
received: 16 04 2021
accepted: 24 08 2021
entrez: 10 9 2021
pubmed: 11 9 2021
medline: 18 11 2021
Statut: epublish

Résumé

Serologic tests to detect specific IgGs to antigens related to viral infections are urgently needed for diagnostics and therapeutics. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers that recognize the four categorized serotypes. Using UB-DNA aptamers specific to each serotype of dengue NS1 proteins (DEN-NS1), we developed our aptamer-antibody sandwich ELISA for dengue diagnostics. Furthermore, IgGs highly specific to DEN-NS1 inhibited the serotype-specific NS1 detection, inspiring us to develop the competitive ELISA format for dengue serotype-specific IgG detection. Blood samples from Singaporean patients with primary or secondary dengue infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than the recent infection. Using this dengue competitive ELISA format, cross-reactivity tests of 21 plasma samples from Singaporean Zika virus-infected patients revealed two distinct patterns: 8 lacked cross-reactivity, and 13 were positive with unique dengue serotype specificities, indicating previous dengue infection. This antigen-detection ELISA and antibody-detection competitive ELISA combination using the UB-DNA aptamers identifies both past and current viral infections and will facilitate specific medical care and vaccine development for infectious diseases.

Identifiants

pubmed: 34504185
doi: 10.1038/s41598-021-97339-8
pii: 10.1038/s41598-021-97339-8
pmc: PMC8429655
doi:

Substances chimiques

Antibodies, Viral 0
Antigens, Viral 0
Aptamers, Nucleotide 0
Immunoglobulin G 0
Viral Nonstructural Proteins 0

Types de publication

Evaluation Study Journal Article Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

18000

Informations de copyright

© 2021. The Author(s).

Références

Maple, P. A. C. & Sikora, K. How useful is COVID-19 antibody testing—A current assessment for oncologists. Clin. Oncol. 33, e73–e81 (2021).
doi: 10.1016/j.clon.2020.10.008
Balestri, R., Magnano, M., Rizzoli, L. & Rech, G. Do we have serological evidences that chilblain-like lesions are related to SARS-CoV-2? A review of the literature. Dermatol Ther 33, e14229 (2020).
pubmed: 32844512 doi: 10.1111/dth.14229
Deeks, J. J. et al. Antibody tests for identification of current and past infection with SARS-CoV-2. Cochrane Database Syst. Rev. 6, CD013652 (2020).
pubmed: 32584464
Espejo, A. P. et al. Review of current advances in serologic testing for COVID-19. Am. J. Clin. Pathol. 154, 293–304 (2020).
pubmed: 32583852 doi: 10.1093/ajcp/aqaa112
Motley, M. P., Bennett-Guerrero, E., Fries, B. C. & Spitzer, E. D. Review of viral testing (polymerase chain reaction) and antibody/serology testing for severe acute respiratory syndrome-coronavirus-2 for the intensivist. Crit. Care Explor. 2, e0154 (2020).
pubmed: 32696013 pmcid: 7314351 doi: 10.1097/CCE.0000000000000154
Goldberg, M. E. & Djavadi-Ohaniance, L. Methods for measurement of antibody/antigen affinity based on ELISA and RIA. Curr. Opin. Immunol. 5, 278–281 (1993).
pubmed: 8507406 doi: 10.1016/0952-7915(93)90018-N
Kozel, T. R. & Burnham-Marusich, A. R. Point-of-care testing for infectious diseases: Past, present, and future. J. Clin. Microbiol. 55, 2313–2320 (2017).
pubmed: 28539345 pmcid: 5527409 doi: 10.1128/JCM.00476-17
Pang, J., Chia, P. Y., Lye, D. C. & Leo, Y. S. Progress and challenges towards point-of-care diagnostic development for dengue. J. Clin. Microbiol. 55, 3339–3349 (2017).
pubmed: 28904181 pmcid: 5703800 doi: 10.1128/JCM.00707-17
Masyeni, S. et al. Serological cross-reaction and coinfection of dengue and COVID-19 in Asia: Experience from Indonesia. Int. J. Infect. Dis. 102, 152–154 (2021).
pubmed: 33115680 doi: 10.1016/j.ijid.2020.10.043
Yan, G. et al. Covert COVID-19 and false-positive dengue serology in Singapore. Lancet Infect. Dis. 20, 536 (2020).
pubmed: 32145189 pmcid: 7128937 doi: 10.1016/S1473-3099(20)30158-4
Bhatt, S. et al. The global distribution and burden of dengue. Nature 496, 504–507 (2013).
pubmed: 23563266 pmcid: 3651993 doi: 10.1038/nature12060
Ang, L. W. et al. A 15-year review of dengue hospitalizations in Singapore: Reducing admissions without adverse consequences, 2003 to 2017. PLoS Negl. Trop. Dis. 13, e0007389 (2019).
pubmed: 31091272 pmcid: 6519799 doi: 10.1371/journal.pntd.0007389
Halstead, S. B. & O’Rourke, E. J. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J. Exp. Med. 146, 201–217 (1977).
pubmed: 406347 pmcid: 2180729 doi: 10.1084/jem.146.1.201
Halstead, S. B. & O’Rourke, E. J. Antibody-enhanced dengue virus infection in primate leukocytes. Nature 265, 739–741 (1977).
pubmed: 404559 doi: 10.1038/265739a0
Halstead, S. B. In vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody. J. Infect. Dis. 140, 527–533 (1979).
pubmed: 117061 doi: 10.1093/infdis/140.4.527
Halstead, S. B. Dengue. Lancet 370, 1644–1652 (2007).
pubmed: 17993365 doi: 10.1016/S0140-6736(07)61687-0
Guzman, M. G. & Harris, E. Dengue. Lancet 385, 453–465 (2015).
pubmed: 25230594 doi: 10.1016/S0140-6736(14)60572-9
Guzman, M. G., Gubler, D. J., Izquierdo, A., Martinez, E. & Halstead, S. B. Dengue infection. Nat. Rev. Dis. Primers 2, 16055 (2016).
pubmed: 27534439 doi: 10.1038/nrdp.2016.55
Wilder-Smith, A., Ooi, E. E., Horstick, O. & Wills, B. Dengue. Lancet 393, 350–363 (2019).
pubmed: 30696575 doi: 10.1016/S0140-6736(18)32560-1
Priyamvada, L. et al. Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus. Proc. Natl. Acad. Sci. U S A 113, 7852–7857 (2016).
pubmed: 27354515 pmcid: 4948328 doi: 10.1073/pnas.1607931113
Mathew, A. et al. B-cell responses during primary and secondary dengue virus infections in humans. J. Infect. Dis. 204, 1514–1522 (2011).
pubmed: 21930609 pmcid: 3222107 doi: 10.1093/infdis/jir607
Corbett, K. S. et al. Preexisting neutralizing antibody responses distinguish clinically inapparent and apparent dengue virus infections in a Sri Lankan pediatric cohort. J. Infect. Dis. 211, 590–599 (2015).
pubmed: 25336728 doi: 10.1093/infdis/jiu481
Priyamvada, L. et al. B cell responses during secondary dengue virus infection are dominated by highly cross-reactive, memory-derived plasmablasts. J. Virol. 90, 5574–5585 (2016).
pubmed: 27030262 pmcid: 4886779 doi: 10.1128/JVI.03203-15
Katzelnick, L. C. et al. Antibody-dependent enhancement of severe dengue disease in humans. Science 358, 929–932 (2017).
pubmed: 29097492 pmcid: 5858873 doi: 10.1126/science.aan6836
St. John, A. L. & Rathore, A. P. S. Adaptive immune responses to primary and secondary dengue virus infections. Nat. Rev. Immunol. 19, 218–230 (2019).
pubmed: 30679808 doi: 10.1038/s41577-019-0123-x
Patel, B. et al. Dissecting the human serum antibody response to secondary dengue virus infections. PLoS Negl. Trop. Dis. 11, 0005554 (2017).
doi: 10.1371/journal.pntd.0005554
Reich, N. G. et al. Interactions between serotypes of dengue highlight epidemiological impact of cross-immunity. J. R. Soc. Interface 10, 20130414 (2013).
pubmed: 23825116 pmcid: 3730691 doi: 10.1098/rsif.2013.0414
Villar, L. et al. Efficacy of a tetravalent dengue vaccine in children in Latin America. N. Engl. J. Med. 372, 113–123 (2015).
pubmed: 25365753 doi: 10.1056/NEJMoa1411037
Capeding, M. R. et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 384, 1358–1365 (2014).
pubmed: 25018116 doi: 10.1016/S0140-6736(14)61060-6
Ferguson, N. M. et al. Benefits and risks of the Sanofi-Pasteur dengue vaccine: Modeling optimal deployment. Science 353, 1033–1036 (2016).
pubmed: 27701113 pmcid: 5268127 doi: 10.1126/science.aaf9590
Sridhar, S. et al. Effect of dengue serostatus on dengue vaccine safety and efficacy. N. Engl. J. Med. 379, 327–340 (2018).
pubmed: 29897841 doi: 10.1056/NEJMoa1800820
Aguiar, M., Halstead, S. B. & Stollenwerk, N. Consider stopping dengvaxia administration without immunological screening. Exp. Rev. Vaccines 16, 301–302 (2017).
doi: 10.1080/14760584.2017.1276831
Halstead, S. B. Dengvaxia sensitizes seronegatives to vaccine enhanced disease regardless of age. Vaccine 35, 6355–6358 (2017).
pubmed: 29029938 doi: 10.1016/j.vaccine.2017.09.089
Luo, R. et al. Rapid diagnostic tests for determining dengue serostatus: A systematic review and key informant interviews. Clin. Microbiol. Infect. 25, 659–666 (2019).
pubmed: 30664935 pmcid: 6543064 doi: 10.1016/j.cmi.2019.01.002
Muller, D. A., Depelsenaire, A. C. & Young, P. R. Clinical and laboratory diagnosis of dengue virus infection. J. Infect. Dis. 215, S89–S95 (2017).
pubmed: 28403441 doi: 10.1093/infdis/jiw649
Peeling, R. W. et al. Evaluation of diagnostic tests: Dengue. Nat. Rev. Microbiol. 8, S30–S38 (2010).
pubmed: 21548185 doi: 10.1038/nrmicro2459
Lebani, K. et al. Isolation of serotype-specific antibodies against dengue virus non-structural protein 1 using phage display and application in a multiplexed serotyping assay. PLoS ONE 12, e0180669 (2017).
pubmed: 28683141 pmcid: 5500353 doi: 10.1371/journal.pone.0180669
Roltgen, K. et al. Development of dengue virus serotype-specific NS1 capture assays for the rapid and highly sensitive identification of the infecting serotype in human sera. J. Immunol. 200, 3857–3866 (2018).
pubmed: 29661824 doi: 10.4049/jimmunol.1701790
Bosch, I. et al. Serotype-specific detection of dengue viruses in a nonstructural protein 1-based enzyme-linked immunosorbent assay validated with a multi-national cohort. PLoS Negl. Trop. Dis. 14, e0008203 (2020).
pubmed: 32579555 pmcid: 7351204 doi: 10.1371/journal.pntd.0008203
Bosch, I. et al. Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum. Sci. Transl. Med. 9, eaan1589 (2017).
pubmed: 28954927 pmcid: 6612058 doi: 10.1126/scitranslmed.aan1589
Ng, D. H. L. et al. Fever patterns, cytokine profiles, and outcomes in COVID-19. Open Forum Infect. Dis. 7, ofaa375 (2020).
pubmed: 32999893 pmcid: 7499767 doi: 10.1093/ofid/ofaa375
Stringari, L. L. et al. Covert cases of severe acute respiratory syndrome coronavirus 2: An obscure but present danger in regions endemic for dengue and chikungunya viruses. PLoS ONE 16, e0244937 (2021).
pubmed: 33406122 pmcid: 7787539 doi: 10.1371/journal.pone.0244937
Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).
pubmed: 1697402 doi: 10.1038/346818a0
Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).
pubmed: 2200121 doi: 10.1126/science.2200121
Kimoto, M., Yamashige, R., Matsunaga, K., Yokoyama, S. & Hirao, I. Generation of high-affinity DNA aptamers using an expanded genetic alphabet. Nat. Biotechnol. 31, 453–457 (2013).
pubmed: 23563318 doi: 10.1038/nbt.2556
Matsunaga, K., Kimoto, M. & Hirao, I. High-affinity DNA aptamer generation targeting von Willebrand factor A1-domain by genetic alphabet expansion for systematic evolution of ligands by exponential enrichment using two types of libraries composed of five different bases. J. Am. Chem. Soc. 139, 324–334 (2017).
pubmed: 27966933 doi: 10.1021/jacs.6b10767
Hirao, I., Kimoto, M. & Lee, K. H. DNA aptamer generation by ExSELEX using genetic alphabet expansion with a mini-hairpin DNA stabilization method. Biochimie 145, 15–21 (2018).
pubmed: 28916151 doi: 10.1016/j.biochi.2017.09.007
Matsunaga, K. et al. Architecture of high-affinity unnatural-base DNA aptamers toward pharmaceutical applications. Sci. Rep. 5, 18478 (2015).
pubmed: 26690672 pmcid: 4686876 doi: 10.1038/srep18478
Matsunaga, K. et al. High-affinity five/six-letter DNA aptamers with superior specificity enabling the detection of dengue NS1 protein variants beyond the serotype identification. Nucleic Acids Res. (in press) https://doi.org/10.1093/nar/gkab515 (2021).
doi: 10.1093/nar/gkab515
Sharma, M. et al. Magnitude and functionality of the NS1-specific antibody response elicited by a live-attenuated tetravalent dengue vaccine candidate. J. Infect. Dis. 221, 867–877 (2019).
pmcid: 7325620 doi: 10.1093/infdis/jiz081
Halstead, S. B., Russell, P. K. & Brandt, W. E. NS1, dengue’s dagger. J. Infect. Dis. 221, 857–860 (2019).
Hirao, I. et al. An unnatural hydrophobic base pair system: Site-specific incorporation of nucleotide analogs into DNA and RNA. Nat. Methods 3, 729–735 (2006).
pubmed: 16929319 doi: 10.1038/nmeth915
Kimoto, M., Soh, S. H. G., Tan, H. P., Okamoto, I. & Hirao, I. Cognate base-pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase. Biopolymers 112, e23407 (2021).
pubmed: 33156531 doi: 10.1002/bip.23407
Hirao, I. et al. Extraordinary stable structure of short single-stranded DNA fragments containing a specific base sequence: d(GCGAAAGC). Nucleic Acids Res. 17, 2223–2231 (1989).
pubmed: 2704619 pmcid: 317592 doi: 10.1093/nar/17.6.2223
Hirao, I. et al. Most compact hairpin-turn structure exerted by a short DNA fragment, d(GCGAAGC) in solution: An extraordinarily stable structure resistant to nucleases and heat. Nucleic Acids Res. 22, 576–582 (1994).
pubmed: 8127706 pmcid: 307846 doi: 10.1093/nar/22.4.576
Yoshizawa, S., Kawai, G., Watanabe, K., Miura, K. & Hirao, I. GNA trinucleotide loop sequences producing extraordinarily stable DNA minihairpins. Biochemistry 36, 4761–4767 (1997).
pubmed: 9125496 doi: 10.1021/bi961738p
Kimoto, M., Nakamura, M. & Hirao, I. Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications. Nucleic Acids Res. 44, 7487–7494 (2016).
pubmed: 27387284 pmcid: 5009754
Hamashima, K., Kimoto, M. & Hirao, I. Creation of unnatural base pairs for genetic alphabet expansion toward synthetic xenobiology. Curr. Opin. Chem. Biol. 46, 108–114 (2018).
pubmed: 30059833 doi: 10.1016/j.cbpa.2018.07.017
Kimoto, M., Shermane Lim, Y. W. & Hirao, I. Molecular affinity rulers: Systematic evaluation of DNA aptamers for their applicabilities in ELISA. Nucleic Acids Res. 47, 8362–8374 (2019).
pubmed: 31392985 pmcid: 6895277 doi: 10.1093/nar/gkz688
Chao, D. Y., Galula, J. U., Shen, W. F., Davis, B. S. & Chang, G. J. Nonstructural protein 1-specific immunoglobulin M and G antibody capture enzyme-linked immunosorbent assays in diagnosis of flaviviral infections in humans. J. Clin. Microbiol. 53, 557–566 (2015).
pubmed: 25502522 pmcid: 4298564 doi: 10.1128/JCM.02735-14
Shu, P. Y. et al. Comparison of capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) and nonstructural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections. Clin. Diagn. Lab. Immunol. 10, 622–630 (2003).
pubmed: 12853395 pmcid: 164246
Tyson, J. et al. Combination of nonstructural protein 1-based enzyme-linked immunosorbent assays can detect and distinguish various dengue virus and zika virus infections. J. Clin. Microbiol. 57, e01464-e11418 (2019).
pubmed: 30429254 pmcid: 6355536 doi: 10.1128/JCM.01464-18
Raafat, N., Blacksell, S. D. & Maude, R. J. A review of dengue diagnostics and implications for surveillance and control. Trans. R. Soc. Trop. Med. Hyg. 113, 653–660 (2019).
pubmed: 31365115 pmcid: 6836713 doi: 10.1093/trstmh/trz068
Blacksell, S. D. et al. Comparison of seven commercial antigen and antibody enzyme-linked immunosorbent assays for detection of acute dengue infection. Clin. Vaccine Immunol. 19, 804–810 (2012).
pubmed: 22441389 pmcid: 3346317 doi: 10.1128/CVI.05717-11
Azimzadeh, A., Weiss, E. & Van Regenmortel, M. H. Measurement of affinity of viral monoclonal antibodies using Fab’-peroxidase conjugate. Influence of antibody concentration on apparent affinity. Mol Immunol 29, 601–608 (1992).
pubmed: 1584228 doi: 10.1016/0161-5890(92)90196-5
Zhang, L., Li, Z., Jin, H., Hu, X. & Su, J. Development and application of a monoclonal antibody-based blocking ELISA for detection of antibodies to Tembusu virus in multiple poultry species. BMC Vet. Res. 14, 201 (2018).
pubmed: 29940964 pmcid: 6019803 doi: 10.1186/s12917-018-1537-6
Chang, S. F. et al. Retrospective serological study on sequential dengue virus serotypes 1 to 4 epidemics in Tainan City, Taiwan, 1994 to 2000. J. Microbiol. Immunol. Infect. 41, 377–385 (2008).
pubmed: 19122918
Chia, P. Y. et al. Clinical features of patients with Zika and dengue virus co-infection in Singapore. J. Infect. 74, 611–615 (2017).
pubmed: 28344113 doi: 10.1016/j.jinf.2017.03.007
Tsai, W. Y. et al. Distinguishing secondary dengue virus infection from Zika virus infection with previous dengue by a combination of 3 simple serological tests. Clin. Infect. Dis. 65, 1829–1836 (2017).
pubmed: 29020159 pmcid: 5850648 doi: 10.1093/cid/cix672
Gao, X. et al. Delayed and highly specific antibody response to nonstructural protein 1 (NS1) revealed during natural human ZIKV infection by NS1-based capture ELISA. BMC Infect. Dis. 18, 275 (2018).
pubmed: 29898684 pmcid: 6000977 doi: 10.1186/s12879-018-3173-y
Felix, A. C. et al. Cross reactivity of commercial anti-dengue immunoassays in patients with acute Zika virus infection. J. Med. Virol. 89, 1477–1479 (2017).
pubmed: 28229481 doi: 10.1002/jmv.24789
Dejnirattisai, W. et al. Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nat. Immunol. 17, 1102–1108 (2016).
pubmed: 27339099 pmcid: 4994874 doi: 10.1038/ni.3515
Fernandez, E. et al. Human antibodies to the dengue virus E-dimer epitope have therapeutic activity against Zika virus infection. Nat. Immunol. 18, 1261–1269 (2017).
pubmed: 28945244 pmcid: 5679314 doi: 10.1038/ni.3849
Yap, T. L. et al. Engineered NS1 for sensitive, specific zika virus diagnosis from patient serology. Emerg. Infect. Dis. 27, 1427–1437 (2021).
pubmed: 33900180 pmcid: 8084482 doi: 10.3201/eid2705.190121
Chao, D. Y. et al. Comprehensive evaluation of differential serodiagnosis between zika and dengue viral infections. J. Clin. Microbiol. 57, 1056 (2019).
doi: 10.1128/JCM.01506-18
Zaidi, M. B. et al. Serological tests reveal significant cross-reactive human antibody responses to Zika and Dengue viruses in the Mexican population. Acta Trop 201, 105201 (2020).
pubmed: 31562846 doi: 10.1016/j.actatropica.2019.105201
Fu, P. et al. Enzyme linked aptamer assay: Based on a competition format for sensitive detection of antibodies to Mycoplasma bovis in serum. Anal Chem 86, 1701–1709 (2014).
pubmed: 24417693 doi: 10.1021/ac4042203
Zumrut, H. E. et al. Ligand-guided selection of aptamers against T-cell receptor-cluster of differentiation 3 (TCR-CD3) expressed on Jurkat.E6 cells. Anal. Biochem. 512, 1–7 (2016).
pubmed: 27519622 pmcid: 5593316 doi: 10.1016/j.ab.2016.08.007
Zumrut, H. E. & Mallikaratchy, P. R. Ligand guided selection (LIGS) of artificial nucleic acid ligands against cell surface targets. ACS Appl. Bio Mater. 3, 2545–2552 (2020).
pubmed: 34013167 doi: 10.1021/acsabm.9b00938
Tan, C. W. et al. A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction. Nat. Biotechnol. 38, 1073–1078 (2020).
pubmed: 32704169 doi: 10.1038/s41587-020-0631-z
Byrnes, J.R. et al. A SARS-CoV-2 serological assay to determine the presence of blocking antibodies that compete for human ACE2 binding. medRxiv (2020) (in press).

Auteurs

Ken-Ichiro Matsunaga (KI)

Institute of Bioengineering and Bioimaging, 31 Biopolis Way, The Nanos, #07-01, Singapore, 138669, Singapore.

Michiko Kimoto (M)

Institute of Bioengineering and Bioimaging, 31 Biopolis Way, The Nanos, #07-01, Singapore, 138669, Singapore.

Vanessa Weixun Lim (VW)

National Centre for Infectious Diseases, 16 Jalan Tan Tock Seng, Singapore, 308442, Singapore.

Tun-Linn Thein (TL)

National Centre for Infectious Diseases, 16 Jalan Tan Tock Seng, Singapore, 308442, Singapore.

Shawn Vasoo (S)

National Centre for Infectious Diseases, 16 Jalan Tan Tock Seng, Singapore, 308442, Singapore.
Department of Infectious Diseases, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore, 308433, Singapore.
Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Dr., Experimental Medicine Building, Singapore, 636921, Singapore.

Yee-Sin Leo (YS)

National Centre for Infectious Diseases, 16 Jalan Tan Tock Seng, Singapore, 308442, Singapore.
Department of Infectious Diseases, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore, 308433, Singapore.
Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Dr., Experimental Medicine Building, Singapore, 636921, Singapore.
Saw Swee Hock School of Public Health, National University of Singapore, 12 Science Drive 2, #10-01, Singapore, 117549, Singapore.

William Sun (W)

Institute of Bioengineering and Bioimaging, 31 Biopolis Way, The Nanos, #07-01, Singapore, 138669, Singapore.
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117593, Singapore.

Ichiro Hirao (I)

Institute of Bioengineering and Bioimaging, 31 Biopolis Way, The Nanos, #07-01, Singapore, 138669, Singapore. ichiro@ibb.a-star.edu.sg.

Articles similaires

[Redispensing of expensive oral anticancer medicines: a practical application].

Lisanne N van Merendonk, Kübra Akgöl, Bastiaan Nuijen
1.00
Humans Antineoplastic Agents Administration, Oral Drug Costs Counterfeit Drugs

Smoking Cessation and Incident Cardiovascular Disease.

Jun Hwan Cho, Seung Yong Shin, Hoseob Kim et al.
1.00
Humans Male Smoking Cessation Cardiovascular Diseases Female
Humans United States Aged Cross-Sectional Studies Medicare Part C
1.00
Humans Yoga Low Back Pain Female Male

Classifications MeSH