Limits...
Use of sensitive, broad-spectrum molecular assays and human airway epithelium cultures for detection of respiratory pathogens.

Pyrc K, Stożek K, Wojcik K, Gawron K, Zeglen S, Karolak W, Wojarski J, Ochman M, Hubalewska-Mazgaj M, Bochenek G, Sanak M, Zembala M, Szczeklik A, Potempa J - PLoS ONE (2012)

Bottom Line: Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ~76% of cases.This additional step resulted in the detection of pathogens in all samples tested.Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Department, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. k.a.pyrc@uj.edu.pl

ABSTRACT
Rapid and accurate detection and identification of viruses causing respiratory tract infections is important for patient care and disease control. Despite the fact that several assays are available, identification of an etiological agent is not possible in ~30% of patients suffering from respiratory tract diseases. Therefore, the aim of the current study was to develop a diagnostic set for the detection of respiratory viruses with sensitivity as low as 1-10 copies per reaction. Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ~76% of cases. To improve assay performance and facilitate the identification of novel species or emerging strains, cultures of fully differentiated human airway epithelium were used to pre-amplify infectious viruses. This additional step resulted in the detection of pathogens in all samples tested. Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used.

Show MeSH

Related in: MedlinePlus

Detection of viruses in clinical material. Detection of respiratory viral pathogens in clinical specimens (nasal lavages).M: size marker (GeneRuler 50–1000 bp DNA; Fermentas); PC: positive control; Clinical samples are denoted with consecutive numbers. Samples considered to be positive are marked with “+” sign. CoV1: alphacoronavirus, CoV2: betacoronavirus; RSV: respiratory syncytial virus; IAV: influenza A virus; PIV: parainfluenza virus type 1, 2 or 3; BoV: bocavirus; AdV: adenovirus; IBV: influenza B virus; hMPV: human metapneumovirus; EV: enterovirus. Analysis was performed on 1.5% agarose gel.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3293820&req=5

pone-0032582-g005: Detection of viruses in clinical material. Detection of respiratory viral pathogens in clinical specimens (nasal lavages).M: size marker (GeneRuler 50–1000 bp DNA; Fermentas); PC: positive control; Clinical samples are denoted with consecutive numbers. Samples considered to be positive are marked with “+” sign. CoV1: alphacoronavirus, CoV2: betacoronavirus; RSV: respiratory syncytial virus; IAV: influenza A virus; PIV: parainfluenza virus type 1, 2 or 3; BoV: bocavirus; AdV: adenovirus; IBV: influenza B virus; hMPV: human metapneumovirus; EV: enterovirus. Analysis was performed on 1.5% agarose gel.

Mentions: A total of 34 samples were subjected to analysis. Briefly, RNA or DNA was isolated and samples processed as described in “Materials and Methods”. Further, samples were used as an input for the nested PCR developed within the current study. The results are presented in Table 2 and show that an etiological agent was identified in ∼76% of cases, and the frequency of occurrence of certain species was consistent with the available literature data [40]. Furthermore, two or more pathogens were identified in the same sample in ∼65% of cases (Table 3, Figure 5).


Use of sensitive, broad-spectrum molecular assays and human airway epithelium cultures for detection of respiratory pathogens.

Pyrc K, Stożek K, Wojcik K, Gawron K, Zeglen S, Karolak W, Wojarski J, Ochman M, Hubalewska-Mazgaj M, Bochenek G, Sanak M, Zembala M, Szczeklik A, Potempa J - PLoS ONE (2012)

Detection of viruses in clinical material. Detection of respiratory viral pathogens in clinical specimens (nasal lavages).M: size marker (GeneRuler 50–1000 bp DNA; Fermentas); PC: positive control; Clinical samples are denoted with consecutive numbers. Samples considered to be positive are marked with “+” sign. CoV1: alphacoronavirus, CoV2: betacoronavirus; RSV: respiratory syncytial virus; IAV: influenza A virus; PIV: parainfluenza virus type 1, 2 or 3; BoV: bocavirus; AdV: adenovirus; IBV: influenza B virus; hMPV: human metapneumovirus; EV: enterovirus. Analysis was performed on 1.5% agarose gel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293820&req=5

pone-0032582-g005: Detection of viruses in clinical material. Detection of respiratory viral pathogens in clinical specimens (nasal lavages).M: size marker (GeneRuler 50–1000 bp DNA; Fermentas); PC: positive control; Clinical samples are denoted with consecutive numbers. Samples considered to be positive are marked with “+” sign. CoV1: alphacoronavirus, CoV2: betacoronavirus; RSV: respiratory syncytial virus; IAV: influenza A virus; PIV: parainfluenza virus type 1, 2 or 3; BoV: bocavirus; AdV: adenovirus; IBV: influenza B virus; hMPV: human metapneumovirus; EV: enterovirus. Analysis was performed on 1.5% agarose gel.
Mentions: A total of 34 samples were subjected to analysis. Briefly, RNA or DNA was isolated and samples processed as described in “Materials and Methods”. Further, samples were used as an input for the nested PCR developed within the current study. The results are presented in Table 2 and show that an etiological agent was identified in ∼76% of cases, and the frequency of occurrence of certain species was consistent with the available literature data [40]. Furthermore, two or more pathogens were identified in the same sample in ∼65% of cases (Table 3, Figure 5).

Bottom Line: Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ~76% of cases.This additional step resulted in the detection of pathogens in all samples tested.Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Department, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. k.a.pyrc@uj.edu.pl

ABSTRACT
Rapid and accurate detection and identification of viruses causing respiratory tract infections is important for patient care and disease control. Despite the fact that several assays are available, identification of an etiological agent is not possible in ~30% of patients suffering from respiratory tract diseases. Therefore, the aim of the current study was to develop a diagnostic set for the detection of respiratory viruses with sensitivity as low as 1-10 copies per reaction. Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ~76% of cases. To improve assay performance and facilitate the identification of novel species or emerging strains, cultures of fully differentiated human airway epithelium were used to pre-amplify infectious viruses. This additional step resulted in the detection of pathogens in all samples tested. Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used.

Show MeSH
Related in: MedlinePlus