Determining the analytical specificity of PCR-based assays for the diagnosis of IA

What is Aspergillus?

C. Oliver Morton, P. Lewis White, Rosemary A. Barnes, Lena Klingspor, Manuel Cuenca-Estrella, Katrien Lagrou, Stéphane Bretagne, Willem Melchers, Carlo Mengoli, Angela M. Caliendo, Massimo Cogliati, Yvette Debets-Ossenkopp, Rebecca Gorton, Ferry Hagen, Catriona Halliday, Petr Hamal, Kathleen Harvey-Wood, Katia Jaton, Gemma Johnson, Sarah Kidd & 12 others Martina Lengerova, Cornelia Lass-Florl, Chris Linton, Laurence Millon, C. Orla Morrissey, Melinda Paholcsek, Alida Fe Talento, Markus Ruhnke, Birgit Willinger, J. Peter Donnelly, Juergen Loeffler, on behalf of the EAPCRI

Research output: Contribution to journalArticleResearchpeer-review

7 Citations (Scopus)

Abstract

A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

Original languageEnglish
Pages (from-to)402-413
Number of pages12
JournalMedical Mycology
Volume55
Issue number4
DOIs
Publication statusPublished - 1 Jun 2017

Keywords

  • analytical specificity
  • Aspergillus PCR
  • cross reactivity
  • detection range

Cite this

Morton, C. O., White, P. L., Barnes, R. A., Klingspor, L., Cuenca-Estrella, M., Lagrou, K., ... on behalf of the EAPCRI (2017). Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus? Medical Mycology, 55(4), 402-413. https://doi.org/10.1093/mmy/myw093
Morton, C. Oliver ; White, P. Lewis ; Barnes, Rosemary A. ; Klingspor, Lena ; Cuenca-Estrella, Manuel ; Lagrou, Katrien ; Bretagne, Stéphane ; Melchers, Willem ; Mengoli, Carlo ; Caliendo, Angela M. ; Cogliati, Massimo ; Debets-Ossenkopp, Yvette ; Gorton, Rebecca ; Hagen, Ferry ; Halliday, Catriona ; Hamal, Petr ; Harvey-Wood, Kathleen ; Jaton, Katia ; Johnson, Gemma ; Kidd, Sarah ; Lengerova, Martina ; Lass-Florl, Cornelia ; Linton, Chris ; Millon, Laurence ; Morrissey, C. Orla ; Paholcsek, Melinda ; Talento, Alida Fe ; Ruhnke, Markus ; Willinger, Birgit ; Donnelly, J. Peter ; Loeffler, Juergen ; on behalf of the EAPCRI. / Determining the analytical specificity of PCR-based assays for the diagnosis of IA : What is Aspergillus?. In: Medical Mycology. 2017 ; Vol. 55, No. 4. pp. 402-413.
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abstract = "A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8{\%}, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2{\%}). For genus specific assays the most often missed species were A. lentulus (25.0{\%}), A. versicolor (24.1{\%}), A. terreus (16.1{\%}), A. flavus (15.2{\%}), A. niger (13.4{\%}), and A. fumigatus (6.2{\%}). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.",
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Morton, CO, White, PL, Barnes, RA, Klingspor, L, Cuenca-Estrella, M, Lagrou, K, Bretagne, S, Melchers, W, Mengoli, C, Caliendo, AM, Cogliati, M, Debets-Ossenkopp, Y, Gorton, R, Hagen, F, Halliday, C, Hamal, P, Harvey-Wood, K, Jaton, K, Johnson, G, Kidd, S, Lengerova, M, Lass-Florl, C, Linton, C, Millon, L, Morrissey, CO, Paholcsek, M, Talento, AF, Ruhnke, M, Willinger, B, Donnelly, JP, Loeffler, J & on behalf of the EAPCRI 2017, 'Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?', Medical Mycology, vol. 55, no. 4, pp. 402-413. https://doi.org/10.1093/mmy/myw093

Determining the analytical specificity of PCR-based assays for the diagnosis of IA : What is Aspergillus? / Morton, C. Oliver; White, P. Lewis; Barnes, Rosemary A.; Klingspor, Lena; Cuenca-Estrella, Manuel; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem; Mengoli, Carlo; Caliendo, Angela M.; Cogliati, Massimo; Debets-Ossenkopp, Yvette; Gorton, Rebecca; Hagen, Ferry; Halliday, Catriona; Hamal, Petr; Harvey-Wood, Kathleen; Jaton, Katia; Johnson, Gemma; Kidd, Sarah; Lengerova, Martina; Lass-Florl, Cornelia; Linton, Chris; Millon, Laurence; Morrissey, C. Orla; Paholcsek, Melinda; Talento, Alida Fe; Ruhnke, Markus; Willinger, Birgit; Donnelly, J. Peter; Loeffler, Juergen; on behalf of the EAPCRI.

In: Medical Mycology, Vol. 55, No. 4, 01.06.2017, p. 402-413.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Determining the analytical specificity of PCR-based assays for the diagnosis of IA

T2 - What is Aspergillus?

AU - Morton, C. Oliver

AU - White, P. Lewis

AU - Barnes, Rosemary A.

AU - Klingspor, Lena

AU - Cuenca-Estrella, Manuel

AU - Lagrou, Katrien

AU - Bretagne, Stéphane

AU - Melchers, Willem

AU - Mengoli, Carlo

AU - Caliendo, Angela M.

AU - Cogliati, Massimo

AU - Debets-Ossenkopp, Yvette

AU - Gorton, Rebecca

AU - Hagen, Ferry

AU - Halliday, Catriona

AU - Hamal, Petr

AU - Harvey-Wood, Kathleen

AU - Jaton, Katia

AU - Johnson, Gemma

AU - Kidd, Sarah

AU - Lengerova, Martina

AU - Lass-Florl, Cornelia

AU - Linton, Chris

AU - Millon, Laurence

AU - Morrissey, C. Orla

AU - Paholcsek, Melinda

AU - Talento, Alida Fe

AU - Ruhnke, Markus

AU - Willinger, Birgit

AU - Donnelly, J. Peter

AU - Loeffler, Juergen

AU - on behalf of the EAPCRI

PY - 2017/6/1

Y1 - 2017/6/1

N2 - A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

AB - A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

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Morton CO, White PL, Barnes RA, Klingspor L, Cuenca-Estrella M, Lagrou K et al. Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus? Medical Mycology. 2017 Jun 1;55(4):402-413. https://doi.org/10.1093/mmy/myw093