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Research Article

Multiplex Real-Time Polymerase Chain Reaction Assay To Detect Acanthamoeba spp., Vermamoeba vermiformis, Naegleria fowleri, and Balamuthia mandrillaris in Different Water Sources

Elizabeth Córdoba-Lanús Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna, La Laguna, Tenerife, Islas Canarias, Spain;
Consorcio Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain;

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María Reyes-Batlle Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna, La Laguna, Tenerife, Islas Canarias, Spain;
Consorcio Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain;
Departamento de Obstetricia y Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología, Medicina Legal y Forense y Parasitología, Facultad de Ciencias de la Salud, Universidad de La Laguna, Tenerife, Spain

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Angélica Domínguez-de-Barros Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna, La Laguna, Tenerife, Islas Canarias, Spain;

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Patricia Pérez-Pérez Departamento de Obstetricia y Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología, Medicina Legal y Forense y Parasitología, Facultad de Ciencias de la Salud, Universidad de La Laguna, Tenerife, Spain

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Rubén L. Rodríguez-Expósito Consorcio Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain;
Departamento de Obstetricia y Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología, Medicina Legal y Forense y Parasitología, Facultad de Ciencias de la Salud, Universidad de La Laguna, Tenerife, Spain

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Alma García-Ramos Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna, La Laguna, Tenerife, Islas Canarias, Spain;

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Inés Sifaoui Consorcio Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain;
Departamento de Obstetricia y Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología, Medicina Legal y Forense y Parasitología, Facultad de Ciencias de la Salud, Universidad de La Laguna, Tenerife, Spain

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Omar García-Pérez Consorcio Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain;
Departamento de Obstetricia y Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología, Medicina Legal y Forense y Parasitología, Facultad de Ciencias de la Salud, Universidad de La Laguna, Tenerife, Spain

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Germán Aneiros-Giraldez Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna, La Laguna, Tenerife, Islas Canarias, Spain;

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José E. Piñero Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna, La Laguna, Tenerife, Islas Canarias, Spain;
Consorcio Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain;
Departamento de Obstetricia y Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología, Medicina Legal y Forense y Parasitología, Facultad de Ciencias de la Salud, Universidad de La Laguna, Tenerife, Spain

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Jacob Lorenzo-Morales Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna, La Laguna, Tenerife, Islas Canarias, Spain;
Consorcio Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain;
Departamento de Obstetricia y Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología, Medicina Legal y Forense y Parasitología, Facultad de Ciencias de la Salud, Universidad de La Laguna, Tenerife, Spain

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DOI:
https://doi.org/10.4269/ajtmh.24-0028
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Ahead of Print
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Free-living amoebae (FLA) are widely distributed in the environment. Among these, Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Vermamoeba vermiformis have been reported as human pathogens with health effects ranging from lethal encephalitis to different epithelial disorders. Despite this, FLA still present many diagnostic challenges. The aim of this study was to develop a rapid and efficient multiplex real-time quantitative polymerase chain reaction (qPCR) to simultaneously detect Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis in different water sources. For the validation of the qPCR assay, 38 samples (19 tap water and 19 stagnant water sources) were analyzed. The qPCR assay accurately identified the four types of FLA with no cross-reactivity. Considering water samples with results subsequently confirmed by conventional PCR, the multiplex qPCR assay detected 18/38 (47.4%) positive samples (Acanthamoeba spp. in 44.7% and V. vermiformis in 31.6%) and growth in nonnutritive agar (NNA) cultures identified 7/38 (18.4%) positive samples. Of the tap water samples analyzed, 26.3% of samples positive for FLA were detected by growth in NNA culture whereas 31.6% were identified by qPCR. In addition, FLA were detected in 2/19 stagnant water samples (10.5%) by growth in NNA culture and in 12/19 stagnant water samples (63.2%) by qPCR. Neither N. fowleri nor B. mandrillaris was detected in the water samples analyzed. In conclusion, the qPCR developed showed its potential as a rapid tool for detection of Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis. Moreover, FLA species were detected in half of the water sources evaluated, suggesting the importance of the surveillance of these potential infectious agents.

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Author Notes

Financial support: This work was funded by the Consorcio Centro de Investigación Biomédica (CIBER) de Enfermedades Infecciosas (CIBERINFEC) (CB21/13/00100), Instituto de Salud Carlos III, Madrid, Spain; by Cabildo Insular de Tenerife 2023–2028; by Ministerio de Sanidad, Spain; and by Project 2022CLISA26 “PROTCAN” from Fundación Cajacanarias/Fundación Bancaria la Caixa. R. L. Rodríguez-Expósito, I. Sifaoui and O. García-Pérez were funded by Cabildo Insular de Tenerife 2023–2028 PROYECTO (CC20230222, CABILDO.23) and P. Pérez-Pérez (TESIS2021010070) was funded by a grant from the Agencia Canaria de Investigación, Innovación y Sociedad de la Información, with 85% cofunded by Fondo Social Europeo (FSE).

Authors’ contributions: E. Córdoba-Lanús, J. E. Piñero, and J. Lorenzo-Morales participated in the conception, design, and funding acquisition. E. Córdoba-Lanús, M. Reyes-Batlle, A. Domínguez-de-Barros, P. Pérez-Pérez, R. L. Rodríguez-Expósito, A. García-Ramos, I. Sifaoui, O. García-Pérez, G. Aneiros-Giraldez, J. E. Piñero, and J. Lorenzo-Morales participated in experimentation and data acquisition. E. Córdoba-Lanús, M. Reyes-Batlle, A. Domínguez-de-Barros, P. Pérez-Pérez, A. García-Ramos, J. E. Pinero, and J. Lorenzo-Morales participated in analysis and interpretation and in drafting of the manuscript for important intellectual content and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final manuscript.

Data availability: Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as additional information.

Current contact information: E. Córdoba-Lanus, M. Reyes-Batlle, A. Domínguez-de-Barros, P. Pérez-Pérez, R. L. Rodrıguez-Exposito, A. García-Ramos, I. Sifaoui, O. García-Pérez, G. Aneiros-Giraldez, J. E. Piñero, and J. Lorenzo-Morales. Instituto Universitario de Enfermedades Tropicales y Salud Publica de Canarias, Universidad de La Laguna, La Laguna, Tenerife, Islas Canarias, Spain, and Avda. Astrofísico Francisco Sánchez S/N, ZC:38206, San Cristóbal de La Laguna, Tenerife, Spain, E-mails: acordoba@ull.edu.es, mreyesba@ull.edu.es, angelica4arealejos@gmail.com, pperezp@ull.edu.es, rrodrige@ull.edu.es, almagarx00@gmail.com, isifaoui@ull.edu.es, omargp6@gmail.com, germanzo_12@msn.com, jpinero@ull.edu.es, and jmlorenz@ull.edu.es.

Address correspondence to Elizabeth Córdoba-Lanús or José E. Piñero, Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez S/N, 38206 La Laguna, Tenerife, Islas Canarias, Spain. E-mails: acordoba@ull.edu.es or jpinero@ull.edu.es
Copyright:
© American Society of Tropical Medicine and Hygiene 2024
Received:
15 Jan 2024
|
Accepted:
23 Mar 2024
|
Published Online:
06 Aug 2024
Cover The American Journal of Tropical Medicine and Hygiene
The American Journal of Tropical Medicine and Hygiene
Print ISSN:
0002-9637
Online ISSN:
1476-1645
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