Introduction. Timely detection of invasive aspergillosis (IA) caused by fungal pathogens, i.e. Aspergillus fumigatus and Aspergillus flavus, in immunocompromised patients is crucial in preventing high mortality.
Aim. To develop a simple immunoassay for the detection of galactomannan (GM), an IA biomarker.
Methodology. GM from A. fumigatus and A. flavus clinical strains was purified and characterized by X-ray diffraction, IR spectroscopy and 13C/1H nuclear magnetic resonance (NMR) for polyclonal antibody (pAb) production in rabbits. An enzyme-linked immunosorbent assay (ELISA) was standardized using concanavalin A to capture Aspergillus GM and pAbs to detect it. Gold nanoparticles (AuNPs) were synthesized and conjugated to pAbs for the development of a dot-blot immunoassay. The developed dot-blot was evaluated with 109 clinical serum and bronchoalveolar lavage samples.
Results. Spectroscopy studies characterized the d-galactofuranosyl groups of GM responsible for the immune response and generation of pAbs. The ELISA employing pAbs showed a sensitivity of 1 ng ml−1 for Aspergillus GM. Furthermore, a sensitive, visual, rapid dot-blot assay developed by the conjugation of pAbs to AuNPs (~24±5 nm size, −36±2 mV zeta potential) had a detection limit of 1 pg ml−1 in serum. The pAbs interacted with Aspergillus spp. but did not cross-react with other fungal pathogen genera such as Penicillium and Candida. Evaluation of the dot-blot with 109 clinical samples showed high sensitivity (80 %) and specificity (93.2 %), with an overall assay accuracy of 89%.
Conclusion. The developed nano-gold immunodiagnostic assay has immense potential for practical use in rapid, specific and sensitive on-site diagnosis of IA, even under resource-limited settings.
BaoZ,
ChenH,
ZhouM,
ShiG,
LiQ et al. Invasive pulmonary aspergillosis in patients with chronic obstructive pulmonary disease: a case report and review of the literature. Oncotarget2017; 8:38069–38074 [View Article]
PasqualottoAC.
Differences in pathogenicity and clinical syndromes due to Aspergillus fumigatus and Aspergillus flavus
. Med Mycol2009; 47:S261–S270 [View Article]
AtchadeE,
Jean-BaptisteS,
HouzéS,
ChabutC,
MassiasL et al. Fatal invasive aspergillosis caused by Aspergillus niger after bilateral lung transplantation. Med Mycol Case Rep2017; 17:4–7 [View Article]
De PauwB,
WalshTJ,
DonnellyJP,
StevensDA,
EdwardsJE et al. Revised definitions of invasive fungal disease from the European organization for research and treatment of Cancer/Invasive fungal infections cooperative group and the National Institute of allergy and infectious diseases Mycoses Study Group (EORTC/MSG) consensus group. Clin Infect Dis2008; 46:1813–1821 [View Article]
KlontRR,
MeisJF,
VerweijPE.
Critical assessment of issues in the diagnosis of invasive aspergillosis. Clin Microbiol Infect2001; 7:32–37 [View Article]
Cuenca-EstrellaM,
Bernal-MartinezL,
BuitragoMJ,
CastelliMV,
Gomez-LopezA et al. Update on the epidemiology and diagnosis of invasive fungal infection. Int J Antimicrob Agents2008; 32:S143–S147 [View Article]
Mennink-KerstenMASH,
DonnellyJP,
VerweijPE.
Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis. Lancet Infect Dis2004; 4:349–357 [View Article]
ThorntonCR.
Development of an immunochromatographic lateral-flow device for rapid serodiagnosis of invasive aspergillosis. Clin Vaccine Immunol2008; 15:1095–1105 [View Article]
HurstSF,
ReyesGH,
McLaughlinDW,
ReissE,
MorrisonCJ.
Comparison of commercial latex agglutination and sandwich enzyme immunoassay with a competitive binding inhibition enzyme immunoassay for detection of antigenemia and antigenuria in a rabbit model of invasive aspergillosis. Clin and Diagn Lab Immunol2000; 7:477–485
FrensG.
Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature Physical Science1973; 241:20–22 [View Article]
ThorntonCR,
JohnsonG,
AgrawalS.
Detection of invasive pulmonary aspergillosis in haematological malignancy patients by using lateral-flow technology. J Vis Exp2012; 61:1–5
JaedickeKM,
TaylorJJ,
PreshawPM.
Validation and quality control of ELISAs for the use with human saliva samples. J Immunol Methods2012; 377:62–65 [View Article]
LatgéJP,
DebeaupuisJP,
MoutaouakilM,
DiaquinM,
SarfatiJ et al. Galactomannan and the circulating antigens of Aspergillus fumigatus
. In
LatgéJP,
BouciasD.
(editors) Fungal Cell Wall and Immune Response53, NATO ASI Series (Series H: Cell Biology). Berlin, Heidelberg: Springer; 1991 pp 143–145
MartinsJT,
CerqueiraMA,
BourbonAI,
PinheiroAC,
SouzaBWS et al. Synergistic effects between κ-carrageenan and locust bean gum on physicochemical properties of edible films made thereof. Food Hydrocoll2012; 29:280–289 [View Article]
FigueiroS,
GOESJ,
MOREIRAR,
SombraA.
On the physico-chemical and dielectric properties of glutaraldehyde crosslinked galactomannan–collagen films. Carbohydr Polym2004; 56:313–320 [View Article]
LatgéJP,
KobayashiH,
DebeaupuisJP,
DiaquinM,
SarfatiJ et al. Chemical and immunological characterization of the extracellular galactomannan of Aspergillus fumigatus
. Infect Immun1994; 62:5424–5433
M. Barreto-BerterE,
R. TravassosL,
GorinPAJ.
Chemical structure of the d-galacto-d-mannan component from hyphae of Aspergillus niger and other Aspergillus spp
. Carbohydr Res1980; 86:273–285 [View Article]
UnkeferCJ,
GanderJE.
The 5-O-beta-D-galactofuranosyl-containing peptidophosphogalactomannan of Penicillium charlesii. Characterization of the mannan by 13C NMR spectroscopy. J Biol Chem1990; 265:685–689
KobayashiH,
ShibataN,
MitobeH,
OhkuboY,
SuzukiS.
Structural study of phosphomannan of yeast-form cells of Candida albicans J-1012 strain with special reference to application of mild acetolysis. Arch Biochem Biophys1989; 272:364–375 [View Article]
HaidoRM,
SilvaMH,
EjzembergR,
LeitãoEA,
HearnVM et al. Analysis of peptidogalactomannans from the mycelial surface of Aspergillus fumigatus
. Med Mycol1998; 36:313–321 [View Article]
KrylovVB,
ArgunovDA,
SolovevAS,
PetrukMI,
GerbstAG et al. Synthesis of oligosaccharides related to galactomannans from Aspergillus fumigatus and their NMR spectral data. Org Biomol Chem2018; 16:1188–1199 [View Article]
GuoS,
MaoW,
YanM,
ZhaoC,
LiN et al. Galactomannan with novel structure produced by the coral endophytic fungus Aspergillus ochraceus
. Carbohydr Polym2014; 105:325–333 [View Article]
GoldsteinIJ,
HollermanCE,
SmithEE.
Protein-Carbohydrate interaction. II. inhibition studies on the interaction of concanavalin A with polysaccharides. Biochemistry1965; 4:876–883 [View Article]
ChaiLYA,
KullbergB-J,
JohnsonEM,
TeerenstraS,
KhinLW et al. Early serum galactomannan trend as a predictor of outcome of invasive aspergillosis. J Clin Microbiol2012; 50:2330–2336 [View Article]
SulahianA,
BoutboulF,
RibaudP,
LeblancT,
LacroixC et al. Value of antigen detection using an enzyme immunoassay in the diagnosis and prediction of invasive aspergillosis in two adult and pediatric hematology units during a 4-year prospective study. Cancer2001; 91:311–318 [View Article]
CaillotD,
CasasnovasO,
BernardA,
CouaillierJF,
DurandC et al. Improved management of invasive pulmonary aspergillosis in neutropenic patients using early thoracic computed tomographic scan and surgery. JCO1997; 15:139–147 [View Article]
BeckerMJ,
de MarieS,
WillemseD,
VerbrughHA,
Bakker-WoudenbergIA.
Quantitative galactomannan detection is superior to PCR in diagnosing and monitoring invasive pulmonary aspergillosis in an experimental rat model. J Clin Microbiol2000; 38:1434–1438
ChindampornA,
ChakrabartiA,
LiR,
SunPL,
TanBH et al. Survey of laboratory practices for diagnosis of fungal infection in seven Asian countries: an Asia fungal Working Group (AFWG) initiative. Med Mycol2017; 0:1–10
RamachandranS,
SinghalM,
McKenzieKG,
OsbornJL,
ArjyalA et al. A rapid, multiplexed, high-throughput flow-through membrane immunoassay: a convenient alternative to ELISA. Diagnostics2013; 3:244–260 [View Article]
NotermansS,
VeenemanGH,
van ZuylenCW,
HoogerhoutP,
van BoomJH.
(1→5)-linked beta-D-galactofuranosides are immunodominant in extracellular polysaccharides of Penicillium and Aspergillus species. Mol Immunol1988; 25:975–979 [View Article]
FengX,
WenH,
ZhangZ,
ChenX,
MaX et al. Dot immunogold filtration assay (DIGFA) with multiple native antigens for rapid serodiagnosis of human cystic and alveolar echinococcosis. Acta Trop2010; 113:114–120 [View Article]
KalitaP,
DasguptaA,
SritharanV,
GuptaS.
Nanoparticle-Drug bioconjugate as dual functional affinity ligand for rapid point-of-care detection of endotoxin in water and serum. Anal Chem2015; 87:11007–11012 [View Article]
DelaneyKP,
BransonBM,
UniyalA,
PhillipsS,
CandalD et al. Evaluation of the performance characteristics of 6 rapid HIV antibody tests. Clin Infect Dis2011; 52:257–263 [View Article]
HuangJY,
LinHT,
ChenTH,
ChenCA,
ChangHT et al. Signal amplified gold nanoparticles for cancer diagnosis on paper-based analytical devices. ACS Sens2018; 3:174–182 [View Article]
EamsobhanaP,
GanXX,
MaA,
WangY,
WanachiwanawinD et al. Dot immunogold filtration assay (DIGFA) for the rapid detection of specific antibodies against the rat lungworm Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) using purified 31-kDa antigen. J Helminthol2014; 88:396–401 [View Article]
PandeySK,
SuriCR,
ChaudhryM,
TiwariRP,
RishiP.
A gold nanoparticles based immuno-bioprobe for detection of Vi capsular polysaccharide of Salmonella enterica serovar Typhi. Mol Biosyst2012; 8:1853–1860 [View Article]
ZhouY,
TianXL,
LiYS,
PanFG,
ZhangYY et al. An enhanced ELISA based on modified colloidal gold nanoparticles for the detection of Pb(II). Biosens Bioelectron2011; 26:3700–3704 [View Article]