Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

IMPORTANCE: Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite." This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omics technologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and, therefore, are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, which can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

Small colony variants (SCVs) are relatively common among some bacterial species and are associated with poor prognosis and recalcitrant infections. Similarly, Candida glabrata - a major intracellular fungal pathogen - produces small and slow-growing respiratory-deficient colonies, termed "petite." Despite reports of clinical petite C . glabrata strains, our understanding of petite behavior in the host remains obscure. Moreover, controversies exist regarding in-host petite fitness and its clinical relevance. Herein, we employed whole-genome sequencing (WGS), dual-RNAseq, and extensive ex vivo and in vivo studies to fill this knowledge gap. WGS identified multiple petite-specific mutations in nuclear and mitochondrially-encoded genes. Consistent with dual-RNAseq data, petite C . glabrata cells did not replicate inside host macrophages and were outcompeted by their non-petite parents in macrophages and in gut colonization and systemic infection mouse models. The intracellular petites showed hallmarks of drug tolerance and were relatively insensitive to the fungicidal activity of echinocandin drugs. Petite-infected macrophages exhibited a pro-inflammatory and type I IFN-skewed transcriptional program. Interrogation of international C . glabrata blood isolates ( n =1000) showed that petite prevalence varies by country, albeit at an overall low prevalence (0-3.5%). Collectively, our study sheds new light on the genetic basis, drug susceptibility, clinical prevalence, and host-pathogen responses of a clinically overlooked phenotype in a major fungal pathogen. Importance: Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite". This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omicstechnologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and therefore are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex-vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Analysis of fks1 and fks2 gene mutations in invasive Candida glabrata strains from Pakistan.

INTRODUCTION: The gradual increase in caspofungin usage in Pakistan raises a concern of emergence of echinocandin resistance in local Candida glabrata strains. We sequenced and determined mutations in fks1 and fks2 genes in invasive Candida glabrata strains from Pakistan. MATERIAL AND METHODS: Thirty-six invasive C. glabrata strains were selected with median (min-max) minimum inhibitory concentrations (MICs) of 0.06 (0.015-0.25) mg/L for caspofungin, 0.015 (0.008-0.06) mg/L for micafungin and 0.06 (0.015-0.12) mg/L for anidulafungin. fks1 and fks2 gene fragments were sequenced using Sanger methodology. Sequences were analysed with MEGA-6 software to identify specific single-nucleotide polymorphisms (SNP) against wild-type sequences of C. glabrata. RESULTS: In fks1 gene, non-synonymous mutation D632H was observed in one isolate with caspofungin MIC of 0.25 mg/L. Synonymous mutation at position A742 was observed in 26/36 (72%) of the isolates. 34/36 (94.5%) isolates analysed for fks2 gene were observed as wild type. A novel non-synonymous mutation at I661T was observed in fks2 gene in one isolate with caspofungin MIC of 0.12 mg/L and anidulafungin and micafungin MIC of 0.06 and 0.015 mg/L, respectively. Novel fks2 synonymous mutations at position T647, K652 and I706 were observed in 16/36 (44%), 25/36 (69%) and 23/36 (63%) isolates, respectively. CONCLUSION: Low frequencies of both non-synonymous and synonymous polymorphisms were observed in invasive C. glabrata strains. Since S663P in fks2 gene is associated with caspofungin resistance, a novel mutation at 661 codon identified in our study needs correlation with treatment outcome data and mandates periodic genomic surveillance.

C. auris and non-C. auris candidemia in hospitalized adult and pediatric COVID-19 patients; single center data from Pakistan.

We compared candidemia due to Candida auris and other non-C.auris cases in hospitalized COVID-19 patients over a period of 9 months at our institution. Candidemia cases in all admitted patients (with or without COVID-19) from April to December 2020 were identified. Electronic records were accessed to record clinical data of COVID-19 patients with candidemia. For statistical analysis, independent samples Mann-Whitney U test was used for continuous and Fisher's exact test was used for categorical variables.A total of 26 candidemia cases (four C.auris, 22 non-C.auris) in 2438 admitted COVID-19 (10.7 per 1000 admissions) and 59 candidemia cases (six C.auris, 53 non-C.auris) in admitted non-COVID patients (8.2 per 1000 admission) were identified. The proportion of C.auris candidemia in COVID-19 and non-COVID-19 patients was 15.4 and 10%, respectively. 4/26 of COVID-19 candidemia patients were aged ≤ 15 years (10 months--15 years). Comparison of C.auris and non-C. auris candidemia cases reveal significant difference in prior antifungal exposure, present in 100% C. auris candidemia versus 27% non-C. auris candidemia patients (P-value 0.014). Although not statistically significant, C. auris candidemia patients had a longer stay in hospital before candidemia (20 vs. 9 days), higher isolation rate of multidrug resistant bacteria (100 vs. 50%), increased rate of prior colonization of Candida species (50 vs. 14%) and lower mean beta-d-glucan levels (48.73 pg/ml vs. 138.146 pg/ml). Both C. auris and non-C. auris COVID-19 patients had similar mortality rate (67 vs. 65%). A significant number of critically ill COVID-19 patients developed candidemia in our study highlighting the need for prompt diagnosis and management. LAY SUMMARY: 26 candidemia cases (4 Candida auris;22 non-C. auris) in COVID-19 patients (April-December 2020) are reported from Pakistan. Compared to non-C. auris, C. auris candidemia patients had higher prior antifungal exposure, longer hospital stay, higher rates of MDR bacteria and Candida colonization.