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VALIDATION OF A NEW DUPLEX REAL-TIME POLYMERASE CHAIN REACTION FOR CHLAMYDIA TRACHOMATIS DNA DETECTION IN OCULAR SWAB SAMPLES
Author
Favacho, Joana da Felicidade Ribeiro
Leite, Keren Kariene
Jacomasso, Thiago
Farias, Aline Burda
Franco Filho, Luciano Chaves
Gomes, Samara Tatielle Monteiro
Reis, Herald Souza
Mota, Gardene Dourado
Schluga, Pedro Henrique de Caires
Tassi, Walleyd Sami
Rampazzo, Rita de Cássia Pontello
West, Sheila Kay
Gaydos, Charlotte Ann
Cunha, Antonio José Ledo Alves
Costa, Alexandre Dias Tavares
Leite, Keren Kariene
Jacomasso, Thiago
Farias, Aline Burda
Franco Filho, Luciano Chaves
Gomes, Samara Tatielle Monteiro
Reis, Herald Souza
Mota, Gardene Dourado
Schluga, Pedro Henrique de Caires
Tassi, Walleyd Sami
Rampazzo, Rita de Cássia Pontello
West, Sheila Kay
Gaydos, Charlotte Ann
Cunha, Antonio José Ledo Alves
Costa, Alexandre Dias Tavares
Affilliation
Ministry of Health. Secretariat of Health and Environment Surveillance. Evandro Chagas Institute. Ananindeua, PA, Brasil.
Institute of Molecular Biology of Paraná. Curitiba, PR, Brasil.
Institute of Molecular Biology of Paraná. Curitiba, PR, Brasil.
Institute of Molecular Biology of Paraná. Curitiba, PR, Brasil.
Ministry of Health. Secretariat of Health and Environment Surveillance. Evandro Chagas Institute. Ananindeua, PA, Brasil.
Ministry of Health. Secretariat of Health and Environment Surveillance. Evandro Chagas Institute. Ananindeua, PA, Brasil.
Ministry of Health. Secretariat of Health and Environment Surveillance. Evandro Chagas Institute. Ananindeua, PA, Brasil.
Ministry of Health. Secretariat of Health and Environment Surveillance. Evandro Chagas Institute. Ananindeua, PA, Brasil.
Institute of Molecular Biology of Paraná. Curitiba, PR, Brasil.
Institute of Molecular Biology of Paraná. Curitiba, PR, Brasil.
Institute of Molecular Biology of Paraná. Curitiba, PR, Brasil.
Johns Hopkins University. Dana Center for Preventative Ophthalmology. Baltimore, USA.
Johns Hopkins University. Division of Infectious Diseases. International Sexually Transmitted Disease Research Laboratory. Baltimore, USA.
Federal University of Rio de Janeiro. Institute of Studies in Public Health. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Institute of Molecular Biology of Paraná. Curitiba, PR, Brasil.
Institute of Molecular Biology of Paraná. Curitiba, PR, Brasil.
Institute of Molecular Biology of Paraná. Curitiba, PR, Brasil.
Ministry of Health. Secretariat of Health and Environment Surveillance. Evandro Chagas Institute. Ananindeua, PA, Brasil.
Ministry of Health. Secretariat of Health and Environment Surveillance. Evandro Chagas Institute. Ananindeua, PA, Brasil.
Ministry of Health. Secretariat of Health and Environment Surveillance. Evandro Chagas Institute. Ananindeua, PA, Brasil.
Ministry of Health. Secretariat of Health and Environment Surveillance. Evandro Chagas Institute. Ananindeua, PA, Brasil.
Institute of Molecular Biology of Paraná. Curitiba, PR, Brasil.
Institute of Molecular Biology of Paraná. Curitiba, PR, Brasil.
Institute of Molecular Biology of Paraná. Curitiba, PR, Brasil.
Johns Hopkins University. Dana Center for Preventative Ophthalmology. Baltimore, USA.
Johns Hopkins University. Division of Infectious Diseases. International Sexually Transmitted Disease Research Laboratory. Baltimore, USA.
Federal University of Rio de Janeiro. Institute of Studies in Public Health. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Abstract
Trachoma is the world-leading infectious cause of preventable blindness and is caused by the bacteria Chlamydia trachomatis. In developing countries, diagnosis is usually based on clinical evaluation. Serological-based tests are cheaper than molecular-based ones, but the latter are more sensitive and specific. The present study developed a new duplex qPCR which concomitantly detects the C. trachomatis cryptic plasmid and the human 18S rRNA gene, with an LOD95% for C. trachomatis DNA of 13.04 genome equivalents per reaction. The new qPCR was tested using 50 samples from an endemic area and 12 from a non-endemic area that were previously characterized using direct immunofluorescence assay (DFA) and clinical evaluation. Among the 50 endemic samples, 3 werefound to be positive by clinical evaluation (6%), 18 were found to be positive by DFA (36%), and 48 were found to be positive by qPCR (96%). Next, the new duplex qPCR was validated using 50 samples previously characterized by qPCR. Validation was carried out on a benchtop instrument (ABI7500) or on a portable point-of-care instrument (Q3-Plus), showing 95% specificity and 100% sensitivity. The ubiquitous presence of C. trachomatis DNA in samples from the endemic region confirms that constant monitoring is of paramount importance for the effective measurement of the elimination of trachoma. The newly developed duplex qPCR presented in this study, along with its validation in a portable qPCR system, constitutes important tools toward achieving this goal.
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