Development of diagnostic tests for pathogen identification and detection of antimicrobial resistance on WHO global priority pathogens using modular real-time nucleic acid amplification test / Desarrollo de pruebas de diagnóstico para la identificación de patógenos y la detección de resistencia a los antimicrobianos en patógenos prioritarios a nivel mundial de la OMS utilizando pruebas modulares de amplificación de ácidos nucleicos en tiempo real

Background: Concerns regarding antimicrobial resistance (AMR) have resulted in the World Health Organization (WHO) designating so-called global priority pathogens (GPPs). However, little discussion has focused on the diagnosis of GPPs. To enable the simultaneous identification of pathogens and AMR, we developed a modular real-time nucleic acid amplification test (MRT-NAAT). Methods: Sequence-specific primers for each modular unit for MRT-NAAT pathogen identification and AMR sets were designed. The composition of the reaction mixture and the real-time PCR program were unified irrespective of primer type so to give MRT-NAAT modularity. Standard strains and clinical isolates were used to evaluate the performance of MRT-NAAT by real-time PCR and melting curve analysis. Probit analysis for the MRT-NAAT pathogen identification set was used to assess the limit of detection (LoD). Results: The MRT-NAAT pathogen identification set was made up of 15 modular units 109–199 bp in product size and with a Tms of 75.5–87.5 °C. The LoD was < 15.548 fg/μL, and nine modular units successfully detected the target pathogens. The MRT-NAAT AMR set included 24 modular units 65–785 bp in product size with a Tms of 75.5–87.5 °C; it showed high performance for detecting GPP target genes and variants. Conclusions; MRT-NAAT enables pathogen identification and AMR gene detection and is time-effective. By unifying the reaction settings of each modular unit, the modularity where combinations of primers can be used according to need could be achieved. This would greatly help in reflecting the researcher’s need and the AMR status of a certain region while successfully detecting pathogens and AMR genes.(AU)

Factors influencing oral microbiome analysis: from saliva sampling methods to next-generation sequencing platforms.

The exploration of oral microbiome has been increasing due to its relatedness with various systemic diseases, but standardization of saliva sampling for microbiome analysis has not been established, contributing to the lack of data comparability. Here, we evaluated the factors that influence the microbiome data. Saliva samples were collected by the two collection methods (passive drooling and mouthwash) using three saliva-preservation methods (OMNIgene, DNA/RNA shield, and simple collection). A total of 18 samples were sequenced by both Illumina short-read and Nanopore long-read next-generation sequencing (NGS). The component of the oral microbiome in each sample was compared with alpha and beta diversity and the taxonomic abundances, to find out the effects of factors on oral microbiome data. The alpha diversity indices of the mouthwash sample were significantly higher than that of the drooling group with both short-read and long-read NGS, while no significant differences in microbial diversities were found between the three saliva-preservation methods. Our study shows mouthwash and simple collection are not inferior to other sample collection and saliva-preservation methods, respectively. This result is promising since the convenience and cost-effectiveness of mouthwash and simple collection can simplify the saliva sample preparation, which would greatly help clinical operators and lab workers.

Development of diagnostic tests for pathogen identification and detection of antimicrobial resistance on WHO global priority pathogens using modular real-time nucleic acid amplification test.

BACKGROUND: Concerns regarding antimicrobial resistance (AMR) have resulted in the World Health Organization (WHO) designating so-called global priority pathogens (GPPs). However, little discussion has focused on the diagnosis of GPPs. To enable the simultaneous identification of pathogens and AMR, we developed a modular real-time nucleic acid amplification test (MRT-NAAT). METHODS: Sequence-specific primers for each modular unit for MRT-NAAT pathogen identification and AMR sets were designed. The composition of the reaction mixture and the real-time PCR program were unified irrespective of primer type so to give MRT-NAAT modularity. Standard strains and clinical isolates were used to evaluate the performance of MRT-NAAT by real-time PCR and melting curve analysis. Probit analysis for the MRT-NAAT pathogen identification set was used to assess the limit of detection (LoD). RESULTS: The MRT-NAAT pathogen identification set was made up of 15 modular units 109-199 bp in product size and with a Tms of 75.5-87.5 °C. The LoD was < 15.548 fg/µL, and nine modular units successfully detected the target pathogens. The MRT-NAAT AMR set included 24 modular units 65-785 bp in product size with a Tms of 75.5-87.5 °C; it showed high performance for detecting GPP target genes and variants. CONCLUSIONS: MRT-NAAT enables pathogen identification and AMR gene detection and is time-effective. By unifying the reaction settings of each modular unit, the modularity where combinations of primers can be used according to need could be achieved. This would greatly help in reflecting the researcher's need and the AMR status of a certain region while successfully detecting pathogens and AMR genes.

Zika Virus Infection During Research Vaccine Development: Investigation of the Laboratory-Acquired Infection via Nanopore Whole-Genome Sequencing.

Zika virus (ZIKV) emerged as a serious public health problem since the first major outbreak in 2007. Current ZIKV diagnostic methods can successfully identify known ZIKV but are impossible to track the origin of viruses and pathogens other than known ZIKV strains. We planned to determine the ability of Whole Genome Sequencing (WGS) in clinical epidemiology by evaluating whether it can successfully detect the origin of ZIKV in a suspected case of laboratory-acquired infection (LAI). ZIKV found in the patient sample was sequenced with nanopore sequencing technology, followed by the production of the phylogenetic tree, based on the alignment of 38 known ZIKV strains with the consensus sequence. The closest viral strain with the consensus sequence was the strain used in the laboratory, with a percent identity of 99.27%. We think WGS showed its time-effectiveness and ability to detect the difference between strains to the level of a single base. Additionally, to determine the global number of LAIs, a literature review of articles published in the last 10 years was performed, and 53 reports of 338 LAIs were found. The lack of a universal reporting system was worrisome, as in the majority of cases (81.1%), the exposure route was unknown.