Development of a New Genus-Specific Quantitative Real-Time PCR Assay for the Diagnosis of Scrub Typhus in South America

Ju Jiang; Constanza Martínez-Valdebenito; Thomas Weitzel; Christina M Farris; Gerardo Acosta-Jamett; Katia Abarca; Allen L Richards

doi:10.3389/fmed.2022.831045

Development of a New Genus-Specific Quantitative Real-Time PCR Assay for the Diagnosis of Scrub Typhus in South America

Front Med (Lausanne). 2022 Apr 27:9:831045. doi: 10.3389/fmed.2022.831045. eCollection 2022.

Authors

Ju Jiang^{1

2}, Constanza Martínez-Valdebenito³, Thomas Weitzel^{4

5}, Christina M Farris¹, Gerardo Acosta-Jamett^{6

7}, Katia Abarca³, Allen L Richards^{2

8}

Affiliations

¹ Naval Medical Research Center, Silver Spring, MD, United States.
² The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, MD, United States.
³ Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile.
⁴ Laboratorio Clínico, Clínica Alemana de Santiago, Facultad de Medicina Clínica Alemana, Universidad del Desarrollo, Santiago, Chile.
⁵ Instituto de Ciencias e Innovación en Medicina (ICIM), Facultad de Medicina Clínica Alemana, Universidad del Desarrollo, Santiago, Chile.
⁶ Instituto de Medicina Preventiva Veterinaria, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile.
⁷ Programa de Investigación Aplicada en Fauna Silvestre, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile.
⁸ Department of Preventive Medicine and Biostatistics, Uniformed Services University of the Health Sciences, Bethesda, MD, United States.

Abstract

Scrub typhus is a potentially severe rickettsiosis, caused by Orientia tsutsugamushi in the Asia-Pacific region. Recently, however, two distinct pathogens, "Candidatus Orientia chuto" and "Candidatus Orientia chiloensis", have been discovered in the Middle East and South America, respectively. Since the novel pathogens differ significantly from O. tsutsugamushi, many established diagnostic methods are unreliable. This work describes the development and validation of a new quantitative real-time PCR (qPCR) assay (Orien16S) for the detection of all known Orientia species. Based on a 94 bp sequence of the 16S rRNA gene (rrs), Orien16S recognized DNA samples from O. tsutsugamushi (n = 41), Ca. O. chiloensis (n = 5), and Ca. O. chuto (n = 1), but was negative for DNA preparations from closely related rickettsiae and other members of the order Rickettsiales (n = 22) as well as unrelated bacterial species (n = 11). After its implementation in Chile, the assay was verified, correctly identifying all tested eschar and buffy coat samples (n = 28) of clinical suspected cases. Furthermore, Orien16S detected Orientia DNA in trombiculid mites collected in endemic regions in southern Chile. The presented novel qPCR assay provides a useful tool for detecting Orientia and diagnosing scrub typhus from all geographical regions.

Keywords: Candidatus Orientia chiloensis; Chile; Orien16S; Orientia; South America; molecular diagnostic techniques; quantitative real-time PCR (qPCR); scrub typhus.