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
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.