Detection and characterisation of a sixth Candida auris clade in Singapore: a genomic and phenotypic study.

BACKGROUND: The emerging fungal pathogen Candida auris poses a serious threat to global public health due to its worldwide distribution, multidrug resistance, high transmissibility, propensity to cause outbreaks, and high mortality. We aimed to characterise three unusual C auris isolates detected in Singapore, and to determine whether they constitute a novel clade distinct from all previously known C auris clades (I-V). METHODS: In this genotypic and phenotypic study, we characterised three C auris clinical isolates, which were cultured from epidemiologically unlinked inpatients at a large tertiary hospital in Singapore. The index isolate was detected in April, 2023. We performed whole-genome sequencing (WGS) and obtained hybrid assemblies of these C auris isolates. The complete genomes were compared with representative genomes of all known C auris clades. To provide a global context, 3651 international WGS data from the National Center for Biotechnology Information (NCBI) database were included in a high-resolution single nucleotide polymorphism (SNP) analysis. Antifungal susceptibility testing was done and antifungal resistance genes, mating-type locus, and chromosomal rearrangements were characterised from the WGS data of the three investigated isolates. We further implemented Bayesian logistic regression models to classify isolates into known clades and simulate the automatic detection of isolates belonging to novel clades as their WGS data became available. FINDINGS: The three investigated isolates were separated by at least 37 000 SNPs (range 37 000-236 900) from all existing C auris clades. These isolates had opposite mating-type allele and different chromosomal rearrangements when compared with their closest clade IV relatives. The isolates were susceptible to all tested antifungals. Therefore, we propose that these isolates represent a new clade of C auris, clade VI. Furthermore, an independent WGS dataset from Bangladesh, accessed via the NCBI Sequence Read Archive, was found to belong to this new clade. As a proof-of-concept, our Bayesian logistic regression model was able to flag these outlier genomes as a potential new clade. INTERPRETATION: The discovery of a new C auris clade in Singapore and Bangladesh in the Indomalayan zone, showing a close relationship to clade IV members most commonly found in South America, highlights the unknown genetic diversity and origin of C auris, particularly in under-resourced regions. Active surveillance in clinical settings, along with effective sequencing strategies and downstream analysis, will be essential in the identification of novel strains, tracking of transmission, and containment of adverse clinical effects of C auris infections. FUNDING: Duke-NUS Academic Medical Center Nurturing Clinician Researcher Scheme, and the Genedant-GIS Innovation Program.

Emergence of SARS-CoV-2 Spike Mutations during Prolonged Infection in Immunocompromised Hosts.

Immunocompromised hosts with prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been implicated in the emergence of highly mutated SARS-CoV-2 variants. Spike mutations are of particular concern because the spike protein is a key target for vaccines and therapeutics for SARS-CoV-2. Here, we report the emergence of spike mutations in two immunocompromised patients with persistent SARS-CoV-2 reverse transcription (RT)-PCR positivity (>90 days). Whole-genome sequence analysis of samples obtained before and after coronavirus disease 2019 (COVID-19) treatment demonstrated the development of partial therapeutic escape mutations and increased intrahost SARS-CoV-2 genome diversity over time. This case series thus adds to the accumulating evidence that immunocompromised hosts with persistent infections are important sources of SARS-CoV-2 genome diversity and, in particular, clinically important spike protein diversity. IMPORTANCE The emergence of clinically important mutations described in this report highlights the need for sustained vigilance and containment measures when managing immunocompromised patients with persistent COVID-19. Even as jurisdictions across the globe start lifting pandemic control measures, immunocompromised patients with persistent COVID-19 constitute a unique group that requires close genomic monitoring and enhanced infection control measures, to ensure early detection and containment of mutations and variants of therapeutic and public health importance.

SARS-CoV-2 N Gene G29195T Point Mutation May Affect Diagnostic Reverse Transcription-PCR Detection.

Rapid onsite whole-genome sequencing of two suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene diagnostic escape samples revealed a previously unreported N gene point mutation at genome position 29195. Because the G29195T mutation occurs within a region probed by a commonly referenced U.S. CDC N gene reverse transcription (RT)-PCR assay, we hypothesize that the G29195T mutation rendered the N gene target of a proprietary commercial assay undetectable. The putative diagnostic escape G29195T mutation demonstrates the need for nearly real-time surveillance, as emergence of a novel SARS-CoV-2 variant with the potential to escape diagnostic tests continues to be a threat. IMPORTANCE Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples. Potential diagnostic escapes, such as those of clinical samples harboring the G29195T mutation, may result in false-negative SARS-CoV-2 RT-PCR results. The rapid detection and sharing of potential diagnostic escapes are essential for diagnostic laboratories and manufacturers around the world, to optimize their assays as SARS-CoV-2 continues to evolve.

Effects of genetic immunization of Swiss outbred mice with human thyroid stimulating hormone receptor cDNA plasmids harboring gain-of-function mutations.

Animal models of Graves' disease have been generated in recent years with various vaccination protocols using wild-type TSH receptor. In this study, we report the findings of genetic immunization of Swiss outbred mice with three different mutated human TSH receptor plasmids, each containing one constitutive activating mutation located at the ectodomain (S281N), exoloop (I486F), and transmembrane segment (D633H) respectively. Although the overall rate of thyrotoxicosis in the mice was < 10%, anti-TSH receptor antibodies could be detected in many animals by flow cytometry, radioreceptor assay, and functional bioassays using recombinant human TSH receptor. Mice injected with plasmids harboring activated mutants (S281N and D633H) showed production of predominantly stimulating antibodies, whilst those treated with wild-type receptor plasmids generated mainly blocking sera. Most of these antibodies displaced radiolabeled bovine TSH, and their epitopes, independent of functional characteristics, were mapped to the first 271 amino acids of the TSH receptor. This supports recent findings that binding of stimulatory or blocking antibodies lie in close proximity within the leucine-rich repeat region.

Detection of thyroid peroxidase mRNA and protein in orbital tissue.

OBJECTIVE: We have previously reported that the absence of thyroid peroxidase antibodies (TPOAb) in Graves' disease (GD) was associated with an increased risk of Graves' ophthalmopathy (GO). This observation raised the possibility that TPOAb could act as a protective factor. However, the presence of thyroid peroxidase (TPO) in the orbit has not been previously reported. The aim of this study was to confirm or exclude the presence of orbital TPO. METHODS AND DESIGN: Relative TPO mRNA expression from GO (n=6) and normal (n=5) orbital fat tissue was determined using real-time PCR technique. Orbital fat in the normal group from blepharoplasty represents extraconal (anterior) fat. mRNA expression in fibroblasts grown from these tissues before and after adipocyte differentiation was also documented. Finally, Western blotting was carried out to verify translation of TPO mRNA transcripts. RESULTS AND DISCUSSION: TPO transcripts were detected in the orbital fat tissue obtained from normal and GO subjects using the real-time PCR technique. TPO expression was increased in GO compared to normal (N) tissues. However, TPO expression in cultured fibroblasts was similar in both groups and adipogenesis did not appear to alter TPO expression. Protein was detected by Western blot analysis using the TPO MAB 47 (mAb 47). The predicted 110-kDa band was detected in orbital fat as well as in orbital fibroblasts. Our results suggest the presence of TPO in GO and N orbital tissues. We hypothesise that immune responses directed against orbital TPO might play a role in modulating the clinical expression of GO.

Cysteine 390 mutation of the TSH receptor modulates its ectodomain as an inverse agonist on the serpentine domain with decrease in basal constitutive activity.

Mutations of individual cysteine residues at codon 301, 390, 398 and 408 of the thyrotropin receptor (TSHr) to serine resulted in cell surface expression of only C301S and C390S mutants. C390S mutation was a silencing mutation with decreased basal constitutive activity. Although the C301S and C390S mutants did not show any significant TSH binding, they generated cyclic AMP upon TSH stimulation. These mutants were also able to interact with stimulating and blocking anti-TSHr antibodies. In fact, C390S receptor is a more sensitive tool for blocking antibody detection than wild type receptor. Introduction of C390S to activating mutations in the ectodomain (S281N), exloop (I486F) and transmembrane (D633H) segments could not mute/nullify receptor activation. These data indicate that the C390S ectodomain behaves as a more effective inverse agonist on the noisy transmembrane segment and suggest that the basal and activated states of the receptor operate through two independent pathways.

Association of Graves' disease with intragenic polymorphism of the thyrotropin receptor gene in a cohort of Singapore patients of multi-ethnic origins.

Thyrotropin (TSH) receptor (TSHr) mutations have been investigated in relation to Graves' disease (GD) genetic susceptibility under the hypothesis that a modified antigen may have novel immunogenic properties. The prevalence of three germline polymorphisms--D36H, P52T, and D727E--were studied in a cohort of multiracial GD patients together with their associations with disease state, Graves' ophthalmopathy, and thyroid autoantibodies titers. Polymerase chain reaction products of exon 1 and 10e of the TSHr were generated from 164 GD patients (109 Chinese, 34 Malays, and 21 Indians) and 240 individuals with no thyroid illnesses (74 Chinese, 84 Malays, and 82 Indians). Mutations were detected by single-strand conformational polymorphism and confirmed with direct sequencing. The D36H mutation was absent, while significant ethnic differences in the distribution of the P52T and D727E mutations were found. The levels of thyroid autoantibodies also differed significantly amongst the three ethnic groups, with the Indian cohort having the lowest titer. Both the P52T and D727E mutations were not associated with GD. An intron mutation, C/G+63IVS1, was detected and showed significant association with GD. Overall, it conferred a twofold increase risk of GD, while subgroup analysis showed increased odds ratios of 2.4 for Chinese (p = 0.008) and 2.8 for Indian (p = 0.049) but not for the Malay ethnic group. Together with recent identification of disease susceptibility markers in the region of the TSHr gene, these results are supportive of genetic factors existing in this region that may be in linkage disequilibrium with the inheritance of various TSHr polymorphisms.

Effects of mutations involving cysteine residues distal to the S281HCC motif at the C-terminus on the functional characteristics of a truncated ectodomain-only thyrotropin receptor anchored on glycosylphosphatidyl-inositol.

BACKGROUND: Cysteine (Cys) residues pair to form disulfide bonds that are important in maintaining structure and function of the thyrotropin receptor (TSHR). There are 11 Cys residues in the ectodomain (ECD). Cys 41 at the N-terminus and Cys 283 at the SHCC motif have been identified as important for ligand binding. The present study evaluated the effects of mutating Cys distal to the S281HCC motif at the C-terminus of the ECD on the functional characteristics of TSHR. METHODS: We introduced (i) individual Cys and (ii) consecutive cumulative Cys mutations into the starting template SHCS-TSHR, a truncated TSHR-ECD moiety previously shown to behave like the wild-type TSHR. Each mutant receptor was evaluated for relative specific binding (RSB), calculated as a measure of TSH-binding ability after normalization with receptor surface expression. RESULTS: In the first approach, RSB was severely affected when Cys 390 and Cys 398 were individually switched to serine. Failed receptor trafficking occurred with Cys 408 mutation. These findings were likely results of altered receptor conformation due to illegitimate disulfide bridge formation. Only SHCS-301 TSHR bound TSH in a specific manner, and it formed the base for sequential Cys mutations. Through this second approach, both Cys 301 and 390 could be removed simultaneously without hindering TSH binding significantly. Cys 398, however, was shown to be critical. Its absence resulted in huge loss of TSH binding. Leaving Cys 283 and 398 as the only Cys pair in the C-terminus alone could support 40% of the total ligand-binding capacity. CONCLUSIONS: From these data, we proposed Cys 398 as a stable disulfide bond partner of Cys 283, corroborating with a model based on evolutionary history of TSHR across species. This pairing of Cys 283 and Cys 398 also provides an objective alternative to conventional hypotheses on Cys coupling based on other predictive models.