Genomic reconstruction of an azole-resistant Candida parapsilosis outbreak and the creation of a multi-locus sequence typing scheme: a retrospective observational and genomic epidemiology study.

Fluconazole-resistant Candida parapsilosis has emerged as a significant health-care-associated pathogen with a propensity to spread patient to patient and cause nosocomial outbreaks, similar to Candida auris. This study investigates a long-lasting outbreak of fluconazole-resistant C parapsilosis that was initially detected in December, 2018, and January, 2019, and officially declared in November, 2019; lasted multiple years; and involved several health-care centres in Berlin, Germany. In this retrospective, observational, and genomic epidemiology study, we used whole-genome sequencing (WGS) of isolates sent by German health-care facilities and laboratories to the National Reference Center for Invasive Fungal Infections (Jena, Germany) for antifungal susceptibility testing between Jan 1, 2016, and Dec 31, 2022. We included all potential outbreak samples (ie, isolates originating from Berlin that were resistant to fluconazole and voriconazole but susceptible to posaconazole) and all non-outbreak isolates that originated from outside of Berlin and were resistant to at least one azole. We also included a number of non-outbreak isolates from outside Berlin that were susceptible or resistant to azoles so that the total study dataset included a matching amount of outbreak and non-outbreak samples from Germany. We used admission and discharge records for patients involved in the outbreak and constructed a network of patient transfers in time and space. We used WGS data for included samples, complemented with WGS data for global samples obtained from the National Center for Biotechnology Information Sequence Read Archive, to construct single-nucleotide variant (SNV)-based phylogeny and perform SNV distance-based analyses. Additionally, we used the whole genomic dataset to identify loci with high discriminatory power to establish a multi-locus sequence typing (MLST) strategy for C parapsilosis. We identified 38 clonal, azole-resistant isolates of C parapsilosis causing 33 cases of invasive infection during a 2018-22 outbreak in multiple hospitals in Berlin. We also sequenced the genomes of 37 non-outbreak isolates. WGS revealed that outbreak strains were separated by a mean of 36 SNVs (SD 20), whereas outbreak strains differed from outgroup samples from Berlin and other regions of Germany by a mean of 2112 SNVs (828). Temporal and genomic reconstruction of the outbreak cases indicated that transfer of patients between health-care facilities was probably 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 isolates from Kuwait, Türkiye, and South Korea. Including the outbreak clone, we identified three distinct azole-resistant lineages carrying ERG11 Y132F in Germany. We identified four 750 bp loci in CPAR2_101400, CPAR2_101470, CPAR2_108720, and CPAR2_808110 for inclusion in our MLST strategy. Application of the MLST method to a global collection of 386 isolates identified 62 sequence types, with the outbreak strains all belonging to the same sequence type. This study underscores the emergence of drug-resistant C parapsilosis that can spread patient to patient within a health-care system, but also, possibly, internationally. Our findings highlight the importance of monitoring C parapsilosis epidemiology globally and of continuous surveillance and rigorous infection control measures at the local scale. We also developed a novel MLST scheme for genetic epidemiology and outbreak investigations, which could represent a faster and less expensive alternative to WGS. German Federal Ministry for Education and Research, German Research Foundation, and German Ministry of Health.

Brassington PJT ，Klefisch FR ，Graf B ，Pfüller R ，Kurzai O ，Walther G ，Barber AE ... -  《Lancet Microbe》

Can a Liquid Biopsy Detect Circulating Tumor DNA With Low-passage Whole-genome Sequencing in Patients With a Sarcoma? A Pilot Evaluation.

A liquid biopsy is a test that evaluates the status of a disease by analyzing a sample of bodily fluid, most commonly blood. In recent years, there has been progress in the development and clinical application of liquid biopsy methods to identify blood-based, tumor-specific biomarkers for many cancer types. However, the implementation of these technologies to aid in the treatment of patients who have a sarcoma remains behind other fields of cancer medicine. For this study, we chose to evaluate a sarcoma liquid biopsy based on circulating tumor DNA (ctDNA). All human beings have normal cell-free DNA (cfDNA) circulating in the blood. In contrast with cfDNA, ctDNA is genetic material present in the blood stream that is derived from a tumor. ctDNA carries the unique genomic fingerprint of the tumor with changes that are not present in normal circulating cfDNA. A successful ctDNA liquid biopsy must be able to target these tumor-specific genetic alterations. For instance, epidermal growth factor receptor (EGFR) mutations are common in lung cancers, and ctDNA liquid biopsies are currently in clinical use to evaluate the status of disease in patients who have a lung cancer by detecting EGFR mutations in the blood. As opposed to many carcinomas, sarcomas do not have common recurrent mutations that could serve as the foundation to a ctDNA liquid biopsy. However, many sarcomas have structural changes to their chromosomes, including gains and losses of portions or entire chromosomes, known as copy number alterations (CNAs), that could serve as a target for a ctDNA liquid biopsy. Murine double minute 2 (MDM2) amplification in select lipomatous tumors or parosteal osteosarcoma is an example of a CNA due to the presence of extra copies of a segment of the long arm of chromosome 12. Since a majority of sarcomas demonstrate a complex karyotype with numerous CNAs, a blood-based liquid biopsy strategy that searches for these CNAs may be able to detect the presence of sarcoma ctDNA. Whole-genome sequencing (WGS) is a next-generation sequencing technique that evaluates the entire genome. The depth of coverage of WGS refers to how detailed the sequencing is, like higher versus lower power on a microscope. WGS can be performed with high-depth sequencing (that is, > 60×), which can detect individual point mutations, or low-depth sequencing (that is, 0.1× to 5×), referred to as low-passage whole-genome sequencing (LP-WGS), which may not detect individual mutations but can detect structural chromosomal changes including gains and losses (that is, CNAs). While similar strategies have shown favorable early results for specific sarcoma subtypes, LP-WGS has not been evaluated for applicability to the broader population of patients who have a sarcoma. Does an LP-WGS liquid biopsy evaluating for CNAs detect ctDNA in plasma samples from patients who have sarcomas representing a variety of histologic subtypes? This was a retrospective study conducted at a community-based, tertiary referral center. Nine paired (plasma and formalin-fixed paraffin-embedded [FFPE] tissue) and four unpaired (plasma) specimens from patients who had a sarcoma were obtained from a commercial biospecimen bank. Three control specimens from individuals who did not have cancer were also obtained. The paired and unpaired specimens from patients who had a sarcoma represented a variety of sarcoma histologic subtypes. cfDNA was extracted, amplified, and quantified. Libraries were prepared, and LP-WGS was performed using a NextSeq 500 next-generation sequencing machine at a low depth of sequencing coverage (∼1×). The ichorCNA bioinformatics algorithm, which was designed to detect CNAs from low-depth genomic sequencing data, was used to analyze the data. In contrast with the gold standard for diagnosis in the form of histopathologic analysis of a tissue sample, this test does not discriminate between sarcoma subtypes but detects the presence of tumor-derived CNAs within the ctDNA in the blood that should not be present in a patient who does not have cancer. The liquid biopsy was positive for the detection of cancer if the ichorCNA algorithm detected the presence of ctDNA. The algorithm was also used to quantitatively estimate the percent ctDNA within the cfDNA. The concentration of ctDNA was then calculated from the percent ctDNA relative to the total concentration of cfDNA. The CNAs of the paired FFPE tissue and plasma samples were graphically visualized using aCNViewer software. This LP-WGS liquid biopsy detected ctDNA in 9 of 13 of the plasma specimens from patients with a sarcoma. The other four samples from patients with a sarcoma and all serum specimens from patients without cancer had no detectable ctDNA. Of those 9 patients with positive liquid biopsy results, the percent ctDNA ranged from 6% to 11%, and calculated ctDNA quantities were 0.04 to 5.6 ng/mL, which are levels to be expected when ctDNA is detectable. In this small pilot study, we were able to detect sarcoma ctDNA with an LP-WGS liquid biopsy searching for CNAs in the plasma of most patients who had a sarcoma representing a variety of histologic subtypes. These results suggest that an LP-WGS liquid biopsy evaluating for CNAs to identify ctDNA may be more broadly applicable to the population of patients who have a sarcoma than previously reported in studies focusing on specific subtypes. Large prospective clinical trials that gather samples at multiple time points during the process of diagnosis, treatment, and surveillance will be needed to further assess whether this technique can be clinically useful. At our institution, we are in the process of developing a large prospective clinical trial for this purpose.

Anderson CJ ，Yang H ，Parsons J ，Ahrens WA ，Jagosky MH ，Hsu JH ，Patt JC ，Kneisl JS ，Steuerwald NM ... -  《-》

Multicentre Study of Candida parapsilosis Blood Isolates in Türkiye Highlights an Increasing Rate of Fluconazole Resistance and Emergence of Echinocandin and Multidrug Resistance.

Worldwide emergence of clonal outbreaks caused by fluconazole-resistant (FLCR) and the recent emergence of echinocandin- and multidrug-resistant (ECR and MDR) Candida parapsilosis isolates pose serious threats to modern clinics. Conducting large-scale epidemiological studies aimed at determining the genetic composition and antifungal resistance rates is necessary to devise antifungal stewardship and infection control strategies at international, national and local levels. Despite being severely hit by outbreaks due to FLCR C. parapsilosis isolates, such knowledge at the national level is lacking in Türkiye. Herein, we conducted a prospective multicentre study involving five major clinical centres in Türkiye to determine antifungal resistance rates, underlying mechanisms and genetic composition of all isolates. In total, 341 isolates were collected from 265 patients including clinical information. Antifungal susceptibility testing against common antifungals was performed in addition to sequencing of ERG11 and FKS1. Last, isolates were genotyped with short tandem repeat (STR) genotyping to investigate potential nosocomial transmission. The FLCR rate was 26.7% (91/341), out of which 75.8% (69/91) harboured the ERG11Y132F mutation. Patients infected with FLCR isolates had a higher mortality rate compared to their susceptible counterparts (49% for FLCR vs. 42% for susceptible). ECR rate was 2.1% (7/341) and isolates carried FKS1F652L/R658G/W1370R mutations. Concerningly, four ECR isolates were MDR. FLCR isolates grouped in distinct clusters without evidence of inter-hospital transmission, whereas large clusters containing susceptible isolates from all centres were noted. Overall, the increasing prevalence of FLCR C. parapsilosis at national level and the emergence of ECR and MDR isolates pose serious clinical challenges in Türkiye. Therefore, conducting large-scale epidemiological studies are critical to determine the trend of antifungal resistance and to tailor pertinent antifungal stewardship and infection control strategies to effectively curb the spread of drug-resistant C. parapsilosis.

Ünal N ，Spruijtenburg B ，Arastehfar A ，Gümral R ，de Groot T ，Meijer EFJ ，Türk-Dağı H ，Birinci A ，Hilmioğlu-Polat S ，Meis JF ，Lass-Flörl C ，Ilkit M ... -  《-》

Precise genome editing underlines the distinct contributions of mutations in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in Candida parapsilosis.

Hartuis S ，Ourliac-Garnier I ，Robert E ，Albassier M ，Duchesne L ，Beaufils C ，Kuhn J ，Le Pape P ，Morio F ... -  《-》

Inter-Institutional Dynamics and Impact of Fluconazole-Resistant Candida parapsilosis.

Infections with fluconazole-resistant Candida parapsilosis have been increasing in Israeli hospitals with unclear implications for patient outcomes. To determine the frequency, mechanisms, molecular epidemiology, and outcomes of azole-resistant C. parapsilosis bloodstream infections in four hospitals in Israel. C. parapsilosis bloodstream isolates were collected at four hospitals in central Israel during varying periods from 2005 to 2022. Antifungal susceptibility testing was done using CLSI broth microdilution. Risk factors for fluconazole resistance were investigated using logistic regression. ERG11 gene sequencing was performed on all isolates. Genetic relatedness was determined using multilocus microsatellite genotyping. Clinical cure, microbiological eradication, and mortality rates were compared between fluconazole-susceptible and resistant isolates. A total of 192 patient-specific C. parapsilosis isolates were analysed. Resistance to fluconazole and voriconazole was detected in 80 (41%) and 14 (7.2%) isolates, respectively. The ERG11 Y132F substitution was found in 91% of fluconazole-resistant and 1% of fluconazole-susceptible isolates. Increasing age, intensive care hospitalisation, haemodialysis, and recent exposure to antibiotics were risk factors for fluconazole-resistant C. parapsilosis. Distinct but related genotypes predominated at each centre, indicating extensive dissemination within hospitals and limited transmission among them. Fluconazole resistance was associated with increased likelihood of microbiological failure but no significant difference in clinical cure and mortality. We found high rates of fluconazole resistance in C. parapsilosis, attributable to nosocomial spread of hospital-specific clones bearing the Y132F substitution. Fluconazole resistance was associated with a higher risk of microbiological but not clinical failure. Strategies to limit nosocomial transmission of C. parapsilosis are needed.

Korem M ，Reich S ，Rahav G ，Yahav D ，Weinberger M ，Novikov A ，Mizrahi N ，Ben-Ami R ... -  《-》