Genotyping of Chlamydophila psittaci by Real-Time PCR and High-Resolution Melt Analysis

Abstract

Human infection with Chlamydophila (Chlamydia) psittaci can lead to psittacosis, a disease that occasionally results in severe pneumonia and other medical complications. C. psittaci is currently grouped into seven avian genotypes: A through F and E/B. Serological testing, outer membrane protein A (ompA) gene sequencing, and restriction fragment length polymorphism analysis are currently used for distinguishing these genotypes. Although accurate, these methods are time-consuming and require multiple confirmatory tests. By targeting the ompA gene, a real-time PCR assay has been developed to rapidly detect and genotype C. psittaci by light-upon-extension chemistry and high-resolution melt analysis. Using this assay, we screened 169 animal specimens; 98 were positive for C. psittaci (71.4% genotype A, 3.1% genotype B, 4.1% genotype E, and 21.4% unable to be typed). This test may provide insight into the distribution of each genotype among specific hosts and provide epidemiological and epizootiological data in human and mammalian/avian cases. This diagnostic assay may also have veterinary applications during chlamydial outbreaks, particularly with respect to identifying the sources and tracking the movements of a particular genotype when multiple animal facilities are affected.

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

“…HRM analysis has been used in numerous studies since the mid-2000s on a wide range of human pathogens, including those within the genera Campylobacter, Brucella, Leishmania, Bordetella, Clostridium, Mycobacterium, Mycoplasma, Chlamydophila, Cryptosporidium, and Staphylococcus, among others (445)(446)(447)(448)(449)(450)(451)(452). Recent studies have shown HRM analysis to be a powerful technique for characterizing antibiotic resistance and typing respiratory disease agents, including M. pneumoniae, C. pneumoniae, and Chlamydophila psittaci (453)(454)(455)(456)(457)(458)(459). To our knowledge, HRM analysis for intraspecies or interspecies Legionella discrimination has been included in only a few studies (460)(461)(462)(463); however, this approach seems promising for future Legionella diagnostics or typing.…”

Psittacosis Outbreak among Workers at Chicken Slaughter Plants, Virginia and Georgia, USA, 2018

“…Staff collected samples from areas where workers were close to or directly handled live chickens or carcasses. Environmental samples were tested for chlamydial species by using real-time PCR, followed by high-resolution melt analysis ( 8 ), at the University of Georgia Infectious Disease Laboratory (Athens, GA, USA).…”

Development of a Novel Real-Time PCR Assay with High-Resolution Melt Analysis To Detect and Differentiate OXA-48-Like β-Lactamases in Carbapenem-Resistant Enterobacteriaceae

“…HRMA has been successfully used for genotyping and for determination of single-nucleotide polymorphisms (SNPs) in both research and clinical settings (40)(41)(42), with exceptional sensitivity and specificity (43). However, only a few studies have utilized HRMA in bacterial genotyping to distinguish rapidly among sequences with high levels of homology (44)(45)(46)(47)(48), and only one other study used HRMA to detect single-nucleotide mutations (A to G) in bacteria (44). For our study, in order to determine confidently the presence of bla OXA-232 -carrying CRE in our institution, it was crucial that the assay be able to discriminate between bla OXA-232 and other bla OXA-48 variants.…”