Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens.

Coccidioidomycosis (Valley fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing...

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Autores principales:	Sudha Chaturvedi, Tanya R Victor, Anuradha Marathe, Ketevan Sidamonidze, Kelly L Crucillo, Vishnu Chaturvedi
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270
Acceso en línea:	https://doaj.org/article/3b948a5b227c462281ecb814a8589caa
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spelling	oai:doaj.org-article:3b948a5b227c462281ecb814a8589caa2021-12-02T20:24:06ZReal-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens.1935-27271935-273510.1371/journal.pntd.0009765https://doaj.org/article/3b948a5b227c462281ecb814a8589caa2021-09-01T00:00:00Zhttps://doi.org/10.1371/journal.pntd.0009765https://doaj.org/toc/1935-2727https://doaj.org/toc/1935-2735Coccidioidomycosis (Valley fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 168 isolates and four primary specimens as C. posadasii and 30 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals (rhesus monkey and rhinoceros) were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the treatment and surveillance of coccidioidomycosis.Sudha ChaturvediTanya R VictorAnuradha MaratheKetevan SidamonidzeKelly L CrucilloVishnu ChaturvediPublic Library of Science (PLoS)articleArctic medicine. Tropical medicineRC955-962Public aspects of medicineRA1-1270ENPLoS Neglected Tropical Diseases, Vol 15, Iss 9, p e0009765 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270
spellingShingle	Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Sudha Chaturvedi Tanya R Victor Anuradha Marathe Ketevan Sidamonidze Kelly L Crucillo Vishnu Chaturvedi Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens.
description	Coccidioidomycosis (Valley fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 168 isolates and four primary specimens as C. posadasii and 30 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals (rhesus monkey and rhinoceros) were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the treatment and surveillance of coccidioidomycosis.
format	article
author	Sudha Chaturvedi Tanya R Victor Anuradha Marathe Ketevan Sidamonidze Kelly L Crucillo Vishnu Chaturvedi
author_facet	Sudha Chaturvedi Tanya R Victor Anuradha Marathe Ketevan Sidamonidze Kelly L Crucillo Vishnu Chaturvedi
author_sort	Sudha Chaturvedi
title	Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens.
title_short	Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens.
title_full	Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens.
title_fullStr	Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens.
title_full_unstemmed	Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens.
title_sort	real-time pcr assay for detection and differentiation of coccidioides immitis and coccidioides posadasii from culture and clinical specimens.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/3b948a5b227c462281ecb814a8589caa
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Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens.

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