Validation of a New Duplex Real-Time Polymerase Chain Reaction for Chlamydia trachomatis DNA Detection in Ocular Swab Samples

Author:

Favacho Joana da Felicidade Ribeiro1ORCID,Leite Keren Kariene2,Jacomasso Thiago2ORCID,Farias Aline Burda2,Franco Filho Luciano Chaves1ORCID,Gomes Samara Tatielle Monteiro1ORCID,dos Reis Herald Souza1ORCID,Mota Gardene Dourado1,Schluga Pedro Henrique de Caires2,Tassi Walleyd Sami2,Rampazzo Rita de Cássia Pontello2,West Sheila Kay3,Gaydos Charlotte Ann4,da Cunha Antonio José Ledo Alves5,Costa Alexandre Dias Tavares6ORCID

Affiliation:

1. Evandro Chagas Institute, Secretariat of Health and Environment Surveillance, Ministry of Health (IEC/SVSA/MS), Ananindeua 67030-000, PA, Brazil

2. Institute of Molecular Biology of Paraná (IBMP), Curitiba 81350-010, PR, Brazil

3. Dana Center for Preventative Ophthalmology, Johns Hopkins University, Baltimore, MD 21287, USA

4. International Sexually Transmitted Disease Research Laboratory, Division of Infectious Diseases, Johns Hopkins University, Baltimore, MD 21218, USA

5. Institute of Studies in Public Health, Federal University of Rio de Janeiro, (IESC/UFRJ), Rio de Janeiro 21941-592, RJ, Brazil

6. Carlos Chagas Institute, Fundação Oswaldo Cruz (FIOCRUZ PR), Curitiba 81350-010, PR, Brazil

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 were found 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.

Funder

Banco Nacional de Desenvolvimento Econômico e Social

National Health Fund

Fundação de Desenvolvimento Científico e Tecnológico em Saúde

Publisher

MDPI AG

Reference82 articles.

1. Brazil, Ministry of Health (2016). Guia Prático para Operacionalização da Campanha Nacional de Hanseníase, Verminoses, Tracoma e Esquistossomose, Ministério da Saúde.

2. Trachoma;Wright;Lancet,2008

3. Chlamydia Conjunctivitis and Central Retinal Vein Occlusion;Stewart;Am. J. Ophthalmol.,2005

4. A survey of trachoma: The histopathology and the mechanism of progressive cicatrization of eyelid tissues;Guzey;Ophthalmologica,2000

5. Upper eyelid entropion and dry eye in cicatricial trachoma without trichiasis;Lucena;Arq. Bras. Oftalmol.,2012

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