Detection of Microsporum canis and Trichophyton mentagrophytes by loop-mediated isothermal amplification (LAMP) and real-time quantitative PCR (qPCR) methods.

BACKGROUND: Dermatophytes are infectious zoonotic fungal agents that are common in animals worldwide. A new loop-mediated isothermal amplification (LAMP) method and quantitative (q)PCR can be used for identifying these agents. Both methods have high specificity and sensitivity, and are simple and quick to use. HYPOTHESIS/OBJECTIVES: To develop a LAMP and a rapid multiplex qPCR method for detecting Microsporum canis and Trichophyton mentagrophytes, which are the most common fungal species isolated from cats and dogs. MATERIAL AND METHODS: Both methods targeted the CHS-1 gene. Their specificity and sensitivity were tested using 64 M. canis and 44 T. mentagrophytes field strains. The validation of the methods was performed using 250 clinical fungal-positive hair samples. RESULTS: The specificity value was 100% for both methods. For LAMP, the sensitivity value was 96.9% for M. canis and 93.2% for T. mentagrophytes. For qPCR, the sensitivity values were 98.4% for M. canis and 97.7% for T. mentagrophytes. Similar specificity and sensitivity results were obtained from the validation study using 250 clinical hair samples. LAMP and multiplex qPCR took 30 and 45 min (respectively) for both targets. The limit of detection (LOD) assays for both targets were 10 and 1 spore/mL for LAMP and multiplex qPCR, respectively. CONCLUSION: These findings demonstrate that the LAMP and multiplex qPCR methods targeting CHS-1 gene developed in this study can be used both for point-of-care testing and in the laboratory for detecting M. canis and T. mentagrophytes with high specificity and sensitivity with an internal control.

Molecular Epidemiology of Fowl Aviadenoviruses in Broiler Chickens from Vaccinated and Nonvaccinated Breeders.

Fowl aviadenoviruses (FAdVs) are widely distributed among poultry populations, leading to various diseases, immunosuppression, and economic losses. Molecular characterization and phylogenetic analysis of circulating FAdV isolates play a critical role in epidemiologic studies, contributing to the control, monitoring, and prevention of related outbreaks. This study aimed to determine the serotypes of FAdV and reveal the molecular epidemiology in broiler chicken flocks. Samples were taken based on epidemiologically important parameters, such as vaccination status, age, and transmission route. A total of 20 vaccinated flocks (VF, flocks originated from vaccinated breeder lines) and 20 nonvaccinated flocks (NVF, flocks originated from nonvaccinated breeder lines) were randomly selected from flocks reporting suspected FAdV clinical symptoms and deaths. Vaccination was administered by intramuscular injection into the pectoral muscle with a commercial inactivated vaccine at 12 and 18 wk. Liver and cloacal swab samples were collected from each flock over two different production cycles and for three different age groups (1-day-old, 14-day-old, and 28-day-old chickens). The liver and cloacal swap samples were analyzed for FAdV using PCR targeting the hexon loop-1 gene. Molecular detection revealed that 30.0% (24/80) of all flocks were FAdV positive, with 50.0% (20/40) positivity in NVF and 10.0% (4/40) in VF. Sequence analysis of the hexon loop-1 gene revealed that all samples were FAdV-8b serotype (OR670689-OR670712), with 100.0% similarity. One randomly selected FAdV-8b sample was analyzed by whole-genome sequence analysis. This is the first study in Turkey to deposit an FAdV whole-genome sequence (44,139 bp) into the GenBank database (PP236873). Given the significantly lower FAdV positivity rates in VF compared to NVF, the findings indicate that vaccination is an effective tool for protecting against FAdV-related infections.

Epidemiología molecular de los aviadenovirus del pollo en pollos de engorde de reproductoras vacunadas y no vacunadas. Los aviadenovirus del pollo (FAdV) están ampliamente distribuidos entre las poblaciones avícolas, lo que provoca diversas enfermedades, inmunosupresión y pérdidas económicas. La caracterización molecular y el análisis filogenético de los aislados circulantes de aviadenovirus del pollo desempeñan un papel fundamental en los estudios epidemiológicos, contribuyendo al control, seguimiento y prevención de brotes relacionados. Este estudio tuvo como objetivo determinar los serotipos de aviadenovirus del pollo y revelar la epidemiología molecular en parvadas de pollos de engorde. Las muestras se tomaron en función de parámetros epidemiológicamente importantes, como el estado de vacunación, la edad y la vía de transmisión. Se seleccionaron aleatoriamente un total de 20 parvadas vacunadas (VF, parvadas originadas a partir de líneas de reproductoras vacunadas) y 20 parvadas no vacunadas (NVF, parvadas originadas a partir de líneas reproductoras no vacunadas) de parvadas que reportaron signos clínicos y mortalidad sugestiva de aviadenovirus del pollo. La vacunación se administró mediante inyección intramuscular en el músculo de la pechuga con una vacuna inactivada comercial a las 12 y 18 semanas. Se recolectaron muestras de hisopos de hígado y cloacales de cada parvada durante dos ciclos de producción diferentes y para tres grupos de edad diferentes (pollos de 1 día, 14 días y 28 días). Las muestras de hígado y de hisopos cloacales se analizaron para detectar aviadenovirus del pollo mediante un método de PCR dirigido a la asa-1 del gene del hexon. La detección molecular reveló que el 30.0% (24/80) de todas las parvadas eran positivas para aviadenovirus del pollo, con un 50% (20/40) de positividad en las parvadas no vacunadas y un 10.0% (4/40) en las parvadas vacunadas. El análisis de secuencia de la asa-1 del gene del hexon reveló que todas las muestras eran del serotipo FAdV-8b (OR670689-OR670712), con un 100.0% de similitud. Se analizó una muestra del FAdV-8b seleccionada aleatoriamente mediante análisis de la secuencia del genoma completo. Este es el primer estudio realizado en Turquía que deposita una secuencia del genoma completo de aviadenovirus del pollo (44,139 pb) dentro de la base de datos GenBank (PP236873). Dadas las tasas de positividad significativamente más bajas de aviadenovirus del pollo en las parvadas vacunadas en comparación con las parvadas no vacunadas, los hallazgos indican que la vacunación es una herramienta eficaz para proteger contra las infecciones relacionadas con aviadenovirus del pollo.

Genomic surveillance during the first two years of the COVID-19 pandemic - country experience and lessons learned from Türkiye.

Background: Türkiye confirmed its first case of SARS-CoV-2 on March 11, 2020, coinciding with the declaration of the global COVID-19 pandemic. Subsequently, Türkiye swiftly increased testing capacity and implemented genomic sequencing in 2020. This paper describes Türkiye's journey of establishing genomic surveillance as a middle-income country with limited prior sequencing capacity and analyses sequencing data from the first two years of the pandemic. We highlight the achievements and challenges experienced and distill globally relevant lessons. Methods: We tracked the evolution of the COVID-19 pandemic in Türkiye from December 2020 to February 2022 through a timeline and analysed epidemiological, vaccination, and testing data. To investigate the phylodynamic and phylogeographic aspects of SARS-CoV-2, we used Nextstrain to analyze 31,629 high-quality genomes sampled from seven regions nationwide. Results: Türkiye's epidemiological curve, mirroring global trends, featured four distinct waves, each coinciding with the emergence and spread of variants of concern (VOCs). Utilizing locally manufactured kits to expand testing capacity and introducing variant-specific quantitative reverse transcription polymerase chain reaction (RT-qPCR) tests developed in partnership with a private company was a strategic advantage in Türkiye, given the scarcity and fragmented global supply chain early in the pandemic. Türkiye contributed more than 86,000 genomic sequences to global databases by February 2022, ensuring that Turkish data was reflected globally. The synergy of variant-specific RT-qPCR kits and genomic sequencing enabled cost-effective monitoring of VOCs. However, data analysis was constrained by a weak sequencing sampling strategy and fragmented data management systems, limiting the application of sequencing data to guide the public health response. Phylodynamic analysis indicated that Türkiye's geographical position as an international travel hub influenced both national and global transmission of each VOC despite travel restrictions. Conclusion: This paper provides valuable insights into the testing and genomic surveillance systems adopted by Türkiye during the COVID-19 pandemic, proposing important lessons for countries developing national systems. The findings underscore the need for robust testing and sampling strategies, streamlined sample referral, and integrated data management with metadata linkage and data quality crucial for impactful epidemiological analysis. We recommend developing national genomic surveillance strategies to guide sustainable and integrated expansion of capacities built for COVID-19 and to optimize the effective utilization of sequencing data for public health action.