[en] The detection of low virus concentrations in biological matrices, especially stool samples, is facing significant limitations as far as common
diagnostic methods (enzyme-linked-immunosorbent assay (ELISA) or quantitative real-time PCR (qPCR)) are considered. Here the
development of a new immuno real-time PCR (iPCR) is described and its performance in the detection of human adenoviruses (HAdVs)
in spiked stools is compared with those of ELISA and qPCR assays. For the iPCR, detection of the sandwich formed by the complexation
of capture antibody-antigen-detection antibody was performed by qPCR thanks to the substitution of peroxydase by a chimeric DNA.
This modification increased the detection sensitivity 200-fold compared to ELISA. The direct qPCR results revealed that only 0.3–9.5% of
the spiked HAdV were detectable, resulting from important losses of DNA occurring at the extraction step. This step was not necessary
in the iPCR workflow, avoiding this drawback. The losses of viral particles occurred at the elution step from the stool only. The
recovery rate of the iPCR was thus better and ranged between 21 and 54%. As a result, iPCR enabled the detection of lower virus
concentrations in stool samples compared to those detected by ELISA and qPCR. The iPCR could be considered as a ‘hyper sensitive
ELISA’ for early detection of HAdV infections, especially in the case of immunocompromised patients after haematopoietic stem cell
transplant.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Bonot, Sebastian
Ogorzaly, Leslie
Elmoualij, Benaïssa ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Histologie
Zorzi, Willy ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Histologie
Cauchie, Henry-Michel ; Université de Liège - ULiège > DER Sc. et gest. de l'environnement (Arlon Campus Environ.) > DER Sc. et gest. de l'environnement (Arlon Campus Environ.)
Language :
English
Title :
Detection of small amounts of human adenoviruses in stools: comparison of a new immuno real-time PCR assay with classical tools.
Publication date :
12 December 2014
Journal title :
Clinical Microbiology and Infection
ISSN :
1198-743X
eISSN :
1469-0691
Publisher :
Blackwell Science, Oxford, United Kingdom
Special issue title :
Detection of small amounts of human adenoviruses in stools: comparison of a new immuno real-time PCR assay with classical tools.
Harrach B, Benkö M, Both G et al. Family - Adenoviridae. In: King A, Adams M, Carstens E, Lefkowitz E, eds, Virus taxonomy: ninth report of the international committee on taxonomy of viruses. San Diego: Elsevier, 2012; 125-141.
Mena KD, Gerba CP. Waterborne adenovirus. Rev Environ Contam Toxicol 2009; 198: 133-167.
Yates MV. Adenoviruses and ultraviolet light: an introduction. Ozone Sci Eng 2008; 30: 70-72.
Garnett CT, Talekar G, Mahr JA et al. Latent species C adenoviruses in human tonsil tissues. J Virol 2009; 83: 2417-2428.
Fox JP, Hall CE, Cooney MK. The Seattle virus watch: VII. Observations of adenovirus infections. Am J Epidemiol 1977; 105: 362-386.
D'Ambrosio E, Del Grosso N, Chicca A, Midulla M. Neutralizing antibodies against 33 human adenoviruses in normal children in Rome. J Hyg 1982; 89: 155-161.
Chakrabarti S, Mautner V, Osman H et al. Adenovirus infections following allogeneic stem cell transplantation: incidence and outcome in relation to graft manipulation, immunosuppression, and immune recovery. Blood 2002; 100: 1619-1627.
Lindemans CA, Leen AM, Boelens JJ. How I treat adenovirus in hematopoietic stem cell transplant recipients. Blood 2010; 116: 5476-5485.
Feuchtinger T, Lang P, Handgretinger R. Adenovirus infection after allogeneic stem cell transplantation. Leuk Lymphoma 2007; 48: 244-255.
Leen AM, Rooney CM. Adenovirus as an emerging pathogen in immunocompromised patients. Brit J Haematol 2005; 128: 135-144.
Matthes-Martin S, Feuchtinger T, Shaw PJ et al. European guidelines for diagnosis and treatment of adenovirus infection in leukemia and stem cell transplantation: summary of ECIL-4 (2011). Transpl Infect Dis 2012; 14: 555-563.
Heim A. Advances in the management of disseminated adenovirus disease in stem cell transplant recipients: impact of adenovirus load (DNAemia) testing. Expert Rev Anti Infect Ther 2011; 9: 943-945.
Lion T, Kosulin K, Landlinger C et al. Monitoring of adenovirus load in stool by real-time PCR permits early detection of impending invasive infection in patients after allogeneic stem cell transplantation. Leukemia 2010; 24: 706-714.
Jeulin H, Salmon A, Bordigoni P, Venard V. Diagnostic value of quantitative PCR for adenovirus detection in stool samples as compared with antigen detection and cell culture in haematopoietic stem cell transplant recipients. Clin Microbiol Infec 2011; 17: 1674-1680.
Iker BC, Bright KR, Pepper IL, Gerba CP, Kitajima M. Evaluation of commercial kits for the extraction and purification of viral nucleic acids from environmental and fecal samples. J Virol Methods 2013; 191: 24-30.
Oikarinen S, Tauriainen S, Viskari H et al. PCR inhibition in stool samples in relation to age of infants. J Clin Virol 2009; 44: 211-214.
Sano S, Smith CL, Cantor CR. Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 1992; 258: 120-122.
Malou N, Raoult D. Immuno-PCR: a promising ultrasensitive diagnostic method to detect antigens and antibodies. Trends Microbiol 2011; 19: 295-302.
Ogorzaly L, Bonot S, El Moualij B, Zorzi W, Cauchie HM. Development of a quantitative immunocapture real-time PCR assay for detecting structurally intact adenoviral particles in water. J Virol Methods 2013; 194: 235-241.
Gofflot S, El Moualij B, Zorzi D et al. Immuno-quantitative polymerase chain reaction for detection and quantitation of prion protein. J Immunoassay Immunochem 2004; 25: 241-258.
Hernroth BE, Conden-Hansson AC, Rehnstam-Holm AS, Girones R, Allard AK. Environmental factors influencing human viral pathogens and their potential indicator organisms in the blue mussel, Mytilus edulis: the first Scandinavian report. Appl Environ Microbiol 2002; 68: 4523-4533.
Tian T, Mandrell R. Detection of norovirus capsid proteins in faecal and food samples by a real time immuno-PCR method. J Appl Microbiol 2006; 100: 564-574.
Adler M, Schulz S, Fischer R, Niemeyer CM. Detection of rotavirus from stool samples using a standardized immuno-PCR ("Imperacer") method with end-point and real-time detection. Biochem Biophys Res Commun 2005; 333: 1289-1294.
Tate J, Ward G. Interferences in immunoassay. Clin Biochem Rev 2004; 25: 105-120.
Davies C. Clinical Concepts. In: David W, ed., The immunoassay handbook, 4th edn. Oxford: Elsevier, 2013; 661-672.
Lind K, Kubista M. Development and evaluation of three real-time immuno-PCR assemblages for quantification of PSA. J Immunol Methods 2005; 304: 107-116.
Niemeyer CM, Adler M, Wacker R. Detecting antigens by quantitative immuno-PCR. Nat Protoc 2007; 2: 1918-1930.
Shulman LM, Hindiyeh M, Muhsen K et al. Evaluation of four different systems for extraction of RNA from stool suspensions using MS-2 coliphage as an exogenous control for RT-PCR inhibition. PLoS ONE 2012; 7: e39455.
Petrich A, Mahony J, Chong S et al. Multicenter comparison of nucleic acid extraction methods for detection of severe acute respiratory syndrome coronavirus RNA in stool specimens. J Clin Microbiol 2006; 44: 2681-2688.
