TY - JOUR
T1 - Quantitative multiplexed detection of common pulmonary fungal pathogens by labeled primer polymerase chain reaction
AU - Gu, Zhengming
AU - Buelow, Daelynn R.
AU - Petraitiene, Ruta
AU - Petraitis, Vidmantas
AU - Walsh, Thomas J.
AU - Hayden, Randall T.
N1 - Publisher Copyright:
© 2014, College of American Pathologists. All rights reserved.
PY - 2014/11/1
Y1 - 2014/11/1
N2 - Context. - Invasive fungal infections are an important cause of morbidity and mortality among immunocompromised patients. Objective. - To design and evaluate a multiplexed assay aimed at quantitative detection and differentiation of the 5 molds that are most commonly responsible for pulmonary infections. Design. - Using labeled primer polymerase chain reaction chemistry, an assay was designed to target the 5.8S and 28S ribosomal RNA genes of Aspergillus spp, Fusarium spp, Scedosporium spp, and members of the order Mucorales (Rhizopus oryzae, Rhizopus microsporus, Cunninghamella bertholletiae, Mucor circinelloides, Lichtheimia corymbifera, and Rhizomucor pusillus). This assay was split into 2 multiplexed reactions and was evaluated using both samples seeded with purified nucleic acid from 42 well-characterized clinical fungal isolates and 105 archived samples (47 blood [45%], 42 bronchoalveolar lavage fluid [40%], and 16 tissue [15%]) collected from rabbit models of invasive pulmonary fungal infections. Results. - Assay detection sensitivity was less than 25 copies of the target sequence per reaction for Aspergillus spp, 5 copies for Fusarium spp and Scedosporium spp, and 10 copies for the Mucorales. The assay showed quantitative linearity from 5 × 101 to 5 × 105 copies of target sequence per reaction. Sensitivities and specificities for bronchoalveolar lavage fluid, tissue, and blood samples were 0.86 and 0.99, 0.60 and 1.00, and 0.46 and 1.00, respectively. Conclusions. - Labeled primer polymerase chain reaction permits rapid, quantitative detection and differentiation of common agents of invasive fungal infection. The assay described herein shows promise for clinical implementation that may have a significant effect on the rapid diagnosis and treatment of patients' severe infections caused by these pulmonary fungal pathogens.
AB - Context. - Invasive fungal infections are an important cause of morbidity and mortality among immunocompromised patients. Objective. - To design and evaluate a multiplexed assay aimed at quantitative detection and differentiation of the 5 molds that are most commonly responsible for pulmonary infections. Design. - Using labeled primer polymerase chain reaction chemistry, an assay was designed to target the 5.8S and 28S ribosomal RNA genes of Aspergillus spp, Fusarium spp, Scedosporium spp, and members of the order Mucorales (Rhizopus oryzae, Rhizopus microsporus, Cunninghamella bertholletiae, Mucor circinelloides, Lichtheimia corymbifera, and Rhizomucor pusillus). This assay was split into 2 multiplexed reactions and was evaluated using both samples seeded with purified nucleic acid from 42 well-characterized clinical fungal isolates and 105 archived samples (47 blood [45%], 42 bronchoalveolar lavage fluid [40%], and 16 tissue [15%]) collected from rabbit models of invasive pulmonary fungal infections. Results. - Assay detection sensitivity was less than 25 copies of the target sequence per reaction for Aspergillus spp, 5 copies for Fusarium spp and Scedosporium spp, and 10 copies for the Mucorales. The assay showed quantitative linearity from 5 × 101 to 5 × 105 copies of target sequence per reaction. Sensitivities and specificities for bronchoalveolar lavage fluid, tissue, and blood samples were 0.86 and 0.99, 0.60 and 1.00, and 0.46 and 1.00, respectively. Conclusions. - Labeled primer polymerase chain reaction permits rapid, quantitative detection and differentiation of common agents of invasive fungal infection. The assay described herein shows promise for clinical implementation that may have a significant effect on the rapid diagnosis and treatment of patients' severe infections caused by these pulmonary fungal pathogens.
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U2 - 10.5858/arpa.2013-0592-OA
DO - 10.5858/arpa.2013-0592-OA
M3 - Article
C2 - 25357108
AN - SCOPUS:84923884691
SN - 0003-9985
VL - 138
SP - 1474
EP - 1480
JO - Archives of Pathology and Laboratory Medicine
JF - Archives of Pathology and Laboratory Medicine
IS - 11
ER -