Genomic reconstruction of an azole-resistantCandida parapsilosisoutbreak and the creation of a multilocus sequence typing scheme: a retrospective observational and genomic epidemiology study
Authors: Brassington Phillip , Klefisch Frank-Rainer , Graf Barbara , Pfüller Roland , Kurzai Oliver , Walther Grit , Barber Amelia E. 
Published: July 2024

SummaryBackgroundFluconazole-resistantCandida parapsilosishas emerged as a significant healthcare-associated pathogen with a propensity to spread patient-to-patient and cause nosocomial outbreaks, similar toCandida auris. This study investigates a prolonged outbreak of fluconazole-resistantC. parapsilosisacross multiple years and healthcare centers in Berlin, Germany.MethodsIn this retrospective observational study, we used whole-genome sequencing of isolates from the outbreak in Berlin and other regions within Germany and compared them with isolates from a global distribution to understand the molecular epidemiology of this outbreak. Additionally, we used the genomic dataset of global samples to identify loci with high discriminatory power to establish a multi-locus sequence typing (MLST) strategy forC. parapsilosis.FindingsA clonal, azole-resistant strain ofC. parapsilosiswas observed causing 33 cases of invasive infection from 2018-2022 in multiple hospitals within the outbreak city. Whole genome sequencing revealed that outbreak strains were separated by an average of 36 single nucleotide variants, while outbreak strains differed from outgroup samples from Berlin and other regions of Germany by an average of 2,112 variants. Temporal and genomic reconstruction of the outbreak cases indicated that transfer of patients between healthcare facilities was likely responsible for the persistent reimportation of the drug-resistant clone and subsequent person-to-person transmission. German outbreak strains were closely related to strains responsible for an outbreak in Canada and to others isolated in the Middle East and East Asia. Including the outbreak clone, we identified three distinctERG11Y132F azole-resistant lineages in Germany, marking the first description of this azole-resistance in the country and its endemic status. Using the novel MLST strategy, a global collection of 386 isolates was categorized into 62 sequence types, with the outbreak strains all belonging to the same sequence type.InterpretationThis study underscores the emergence of drug resistant fungal pathogens that can spread patient-to-patient within a healthcare system, but also around the globe. This highlights the importance of monitoringC. parapsilosisepidemiology globally and of continuous surveillance and rigorous infection control measures at the local scale. Through large-scale genomic epidemiology, our study offers a high-resolution view of how a drug-resistant clone behaved in a local healthcare system and how this clone fits into the global epidemiology of this pathogen. We also demonstrate the utility of the novel typing scheme for genetic epidemiology and outbreak investigations as a faster and less expensive alternative to whole genome sequencing.FundingGerman Federal Ministry for Education and Research, German Research Foundation, German Ministry of HealthResearch in contextEvidence before this studyWe searched PubMed and Google Scholar from database inception to Apr 25, 2024, using the search terms “Candida parapsilosis”, “outbreak”, “azole resistance”, and/or “fluconazole” in PubMed and Google Scholar. We applied no language or study type restrictions. The epidemiology of candidemia has undergone dramatic changes in recent years. New pathogenic species, such asCandida auris, have emerged, and existing species likeCandida parapsilosishave increased in prominence. There has also been a worrying increase in drug resistance amongCandidaspecies. Moreover, numerous drug-resistant outbreaks ofC. parapsilosishave been reported worldwide and are challenging to control due to their prolonged and intermittent nature. The overwhelming majority of previous work has used microsatellite markers to infer genetic relationships among outbreaks strains, obscuring whether they are really clonal in nature, our understanding of the temporal and transmission dynamics of these outbreaks, and the genetic relationship between outbreak clones.Added value of this studyThis study adds to the existing evidence by utilizing whole genome sequencing in conjunction with hospital records to analyze a prolonged outbreak of clonal, azole-resistantC. parapsilosisthat occurred across multiple years and medical centers. This study demonstrates that patient transfers can result in the reimportation of outbreak clones, posting a significant challenge for infection control. We also reveal that the outbreak clone is closely related to drug-resistant isolates from other continents, highlighting the global spread of drug-resistantC. parapsilosis. Furthermore, the study addresses the need for rapid strain differentiation in outbreak settings by establishing and validating a set of four loci for Sanger sequence-based typing, which provide a highly discriminatory tool for epidemiologic investigations.Implications of all the available evidenceThis study underscores the global challenge of azole-resistantC. parapsilosisand its importance as the causative agent of nosocomial outbreaks. Clinicians should be aware of the evolving epidemiology ofC. parapsilosisand the prevalence of drug-resistant strains, emphasizing the importance of appropriate antifungal stewardship and infection control measures. The study emphasizes the challenges caused by inter-hospital transmission and their role in persistent outbreaks, highlighting the need for robust surveillance and coordination among healthcare facilities. While whole genome sequencing (WGS) is becoming more widely available, it is still not available in many settings due to cost, limitations in bioinformatic expertise, and the absence of standardized methodology and data interpretation. The establishment of a sequence-based typing scheme is a valuable tool for rapid assessment of samples, which can aid in outbreak tracking and containment efforts, and provide results more rapidly even in settings where WGS is available.