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Rapid Phenotypic and Genotypic Antimicrobial Susceptibility Testing Approaches for Use in the Clinical Laboratory.

ABSTRACT: The rapid rise in increasingly resistant bacteria has become a major threat to public health. Antimicrobial susceptibility testing (AST) is crucial in guiding appropriate therapeutic decisions and infection prevention practices for patient care. However, conventional culture-based AST methods are time-consuming and labor-intensive. Therefore, rapid AST approaches exist to address the delayed gap in time to actionable results. There are two main types of rapid AST technologies- phenotypic and genotypic approaches. In this review, we provide a summary of all commercially available rapid AST platforms for use in clinical microbiology laboratories. We describe the technologies utilized, performance characteristics, acceptable specimen types, types of resistance detected, turnaround times, limitations, and clinical outcomes driven by these rapid tests. We also discuss crucial factors to consider for the implementation of rapid AST technologies in a clinical laboratory and what the future of rapid AST holds.

SUBMITTER: Hattab S

PROVIDER: S-EPMC11351821 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Rapid Phenotypic and Genotypic Antimicrobial Susceptibility Testing Approaches for Use in the Clinical Laboratory.

Hattab Siham S Ma Adrienne H AH Tariq Zoon Z Vega Prado Ilianne I Drobish Ian I Lee Rachel R Yee Rebecca R

Antibiotics (Basel, Switzerland) 20240822 8

The rapid rise in increasingly resistant bacteria has become a major threat to public health. Antimicrobial susceptibility testing (AST) is crucial in guiding appropriate therapeutic decisions and infection prevention practices for patient care. However, conventional culture-based AST methods are time-consuming and labor-intensive. Therefore, rapid AST approaches exist to address the delayed gap in time to actionable results. There are two main types of rapid AST technologies- phenotypic and gen ...[more]

PMID: 39200086

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Project description:Antimicrobial susceptibility testing (AST) should be fast and accurate, leading to proper interventions and therapeutic success. Clinical microbiology laboratories rely on phenotypic methods, but the continuous improvement and decrease in the cost of whole-genome sequencing (WGS) technologies make them an attractive alternative. Studies evaluating the performance of WGS-based prediction of antimicrobial resistance (AMR) for selected bacterial species have shown promising results. There are, however, significant gaps in the literature evaluating the applicability of WGS as a diagnostics method in real-life clinical settings against the range of bacterial pathogens experienced there. Thus, we compared standard phenotypic AST results with WGS-based predictions of AMR profiles in bacterial isolates without preselection of defined species, to evaluate the applicability of WGS as a diagnostics method in clinical settings. We collected all bacterial isolates processed by all Danish Clinical Microbiology Laboratories in 1 day. We randomly selected 500 isolates without any preselection of species. We performed AST through standard broth microdilution (BMD) for 488 isolates (n = 6,487 phenotypic AST results) and compared results with in silico antibiograms obtained through WGS (Illumina NextSeq) followed by bioinformatics analyses using ResFinder 4.0 (n = 5,229 comparisons). A higher proportion of AMR was observed for Gram-negative bacteria (10.9%) than for Gram-positive bacteria (6.1%). Comparison of BMD with WGS data yielded a concordance of 91.7%, with discordant results mainly due to phenotypically susceptible isolates harboring genetic AMR determinants. These cases correspond to 6.2% of all isolate-antimicrobial combinations analyzed and to 6.8% of all phenotypically susceptible combinations. We detected fewer cases of phenotypically resistant isolates without any known genetic resistance mechanism, particularly 2.1% of all combinations analyzed, which corresponded to 26.4% of all detected phenotypic resistances. Most discordances were observed for specific combinations of species-antimicrobial: macrolides and tetracycline in streptococci, ciprofloxacin and β-lactams in combination with β-lactamase inhibitors in Enterobacterales, and most antimicrobials in Pseudomonas aeruginosa. WGS has the potential to be used for surveillance and routine clinical microbiology. However, in clinical microbiology settings and especially for certain species and antimicrobial agent combinations, further developments in AMR gene databases are needed to ensure higher concordance between in silico predictions and expected phenotypic AMR profiles.

Project description:BackgroundGenomic analysis has revealed extensive contamination among laboratory-maintained microbes including malaria parasites, Mycobacterium tuberculosis and Salmonella spp. Here, we provide direct evidence for recent contamination of a laboratory schistosome parasite population, and we investigate its genomic consequences. The Brazilian Schistosoma mansoni population SmBRE has several distinctive phenotypes, showing poor infectivity, reduced sporocysts number, low levels of cercarial shedding and low virulence in the intermediate snail host, and low worm burden and low fecundity in the vertebrate rodent host. In 2021 we observed a rapid change in SmBRE parasite phenotypes, with a ~10x increase in cercarial production and ~4x increase in worm burden.MethodsTo determine the underlying genomic cause of these changes, we sequenced pools of SmBRE adults collected during parasite maintenance between 2015 and 2023. We also sequenced another parasite population (SmLE) maintained alongside SmBRE without phenotypic changes.ResultsWhile SmLE allele frequencies remained stable over the eight-year period, we observed sudden changes in allele frequency across the genome in SmBRE between July 2021 and February 2023, consistent with expectations of laboratory contamination. (i) SmLE-specific alleles rose in the SmBRE population from 0 to 41-46% across the genome between September and October 2021, documenting the timing and magnitude of the contamination event. (ii) After contamination, strong selection (s = ~0.23) drove replacement of low fitness SmBRE with high fitness SmLE alleles. (iii) Allele frequency changed rapidly across the whole genome, except for a region on chromosome 4 where SmBRE alleles remained at high frequency.ConclusionsWe were able to detect contamination in this case because SmBRE shows distinctive phenotypes. However, this would likely have been missed with phenotypically similar parasites. These results provide a cautionary tale about the importance of tracking the identity of parasite populations, but also showcase a simple approach to monitor changes within populations using molecular profiling of pooled population samples to characterize fixed single nucleotide polymorphisms. We also show that genetic drift results in continuous change even in the absence of contamination, causing parasites maintained in different labs (or sampled from the same lab at different times) to diverge.

Project description:Standard antimicrobial susceptibility testing (AST) approaches lead to delays in the selection of optimal antimicrobial therapy. Here, we sought to determine the accuracy of antimicrobial resistance (AMR) determinants identified by Nanopore whole-genome sequencing in predicting AST results. Using a cohort of 40 clinical isolates (21 carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae, 10 non-carbapenemase-producing carbapenem-resistant K. pneumoniae, and 9 carbapenem-susceptible K. pneumoniae isolates), three separate sequencing and analysis pipelines were performed, as follows: (i) a real-time Nanopore analysis approach identifying acquired AMR genes, (ii) an assembly-based Nanopore approach identifying acquired AMR genes and chromosomal mutations, and (iii) an approach using short-read correction of Nanopore assemblies. The short-read correction of Nanopore assemblies served as the reference standard to determine the accuracy of Nanopore sequencing results. With the real-time analysis approach, full annotation of acquired AMR genes occurred within 8 h from subcultured isolates. Assemblies sufficient for full resistance gene and single-nucleotide polymorphism annotation were available within 14 h from subcultured isolates. The overall agreement of genotypic results and anticipated AST results for the 40 K. pneumoniae isolates was 77% (range, 30% to 100%) and 92% (range, 80% to 100%) for the real-time approach and the assembly approach, respectively. Evaluating the patients contributing the 40 isolates, the real-time approach and assembly approach could shorten the median time to effective antibiotic therapy by 20 h and 26 h, respectively, compared to standard AST. Nanopore sequencing offers a rapid approach to both accurately identify resistance mechanisms and to predict AST results for K. pneumoniae isolates. Bioinformatics improvements enabling real-time alignment, coupled with rapid extraction and library preparation, will further enhance the accuracy and workflow of the Nanopore real-time approach.

Project description:There is a growing global concern regarding the rise of antimicrobial resistance among Ureaplasma spp. isolates. However, studies on the antimicrobial susceptibility profiles, resistance mechanisms, and clonality of Ureaplasma spp. clinical isolates are still limited and cover only some geographic regions. Firstly, Ureaplasma species from the urogenital tracts of patients in Shanghai, China, were isolated by using the culture medium (A8 and 10B broth), and identified the genotype by polymerase chain reaction (PCR). Secondly, the antimicrobial susceptibility tests were determined by using broth microdilution assay. Then, the resistance genetic determinants to fluoroquinolones (FQs), macrolides, and tetracyclines were investigated through PCR/DNA sequencing. Finally, the molecular epidemiology of Ureaplasma species was studied by multilocus sequence typing (MLST). Among 258 isolates, Ureaplasma parvum (UPA) and Ureaplasma urealyticum (UUR) were found in 226 (87.60%) and 32 (12.40%) isolates, respectively. The minimum inhibitory concentrations (MICs) of 258 Ureaplasma spp. strains ranged from 0.015 to 64μg/ml for all 11 kinds of antimicrobials. Regardless of species, the isolates were most sensitive to AZI (1.94%), JOS (3.49%), and CLA (4.23%). Among them, there were 39 (15.12%) multidrug-resistant (MDR) strains, including 32 UPA isolates. The resistance rates of UPA to CIP (91.59%), and ROX (36.28%) were significantly higher than those of UUR. Twenty six FQ-resistant isolates had amino acid substitutions in gyrA and in parC (Ser83Leu). Mutations were detected in genes encoding ribosomal proteins L4 (Thr84Ile) and L22 (Ser81Pro) in macrolide-resistant isolates. Tet(M) was found in four UPA isolates. These mutations were mainly found in UPA isolates. Sequence type 1 (ST1) was the predominant ST, which contained 18 isolates. In conclusion, this study showed a higher resistance rate (especially to ROX and CIP), higher substitution rate, and higher MDR rate among UPA strains. The most active antimicrobial agents were AZI, JOS, and CLA. Identifying UPA or UUR in clinical isolates could help clinicians to choose appropriate drugs for treatment. The main resistance mechanisms may involve gene substitution of Ser83Leu in parC and Ser81Pro in L22. ST1 was the predominant ST of Ureaplasma isolates with MDR to FQs and macrolides in Shanghai, China.

Dataset Information

Rapid Phenotypic and Genotypic Antimicrobial Susceptibility Testing Approaches for Use in the Clinical Laboratory.

Publications

Rapid Phenotypic and Genotypic Antimicrobial Susceptibility Testing Approaches for Use in the Clinical Laboratory.

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