Low toxicity contributes to Sporothrix globosa invade the skin of patients in low-epidemic areas of China.

OBJECTIVE: This study aims to assess the clinical characteristics of sporotrichosis in low-endemic areas of China, including the prevalence geography, genotypic traits of patients, clinical manifestations, and strain virulence and drug sensitivities. The objective is to improve the currently used clinical management strategies for sporotrichosis. METHODS: Retrospective data were collected from patients diagnosed with sporotrichosis through fungal culture identification. The isolates from purified cultures underwent identification using CAL (Calmodulin) gene sequencing. Virulence of each strain was assessed using a Galleria mellonella (G. mellonella) larvae infection model. In vitro susceptibility testing against commonly used clinical antifungal agents for sporotrichosis was conducted following CLSI criteria. RESULTS: In our low-endemic region for sporotrichosis, the majority of cases (23) were observed in middle-aged and elderly women with a history of trauma, with a higher incidence during winter and spring. All clinical isolates were identified as Sporothrix globosa (S. globosa). The G. mellonella larvae infection model indicated independent and dose-dependent virulence among strains, with varying toxicity levels demonstrated by the degree of melanization of the G. mellonella. Surprisingly, lymphocutaneous types caused by S. globosa exhibited lower in vitro virulence but were more common in affected skin. In addition, all S.globosa strains displayed high resistances to fluconazole, while remaining highly susceptible to terbinafine, itraconazole and amphotericin B. CONCLUSION: Given the predominance of elderly women engaged in agricultural labour in our region, which is a low-epidemic areas, they should be considered as crucial targets for sporotrichosis monitoring. S. globosa appears to be the sole causative agent locally. However, varying degrees of melanization in larvae were observed among these isolates, indicating a divergence in their virulence. Itraconazole, terbinafine and amphotericin B remain viable first-line antifungal options for treating S.globosa infection.

Deletion of Non-histidine Domains of Histidine Kinase CHK1 Diminishes the Infectivity of Candida albicans in an Oral Mucosal Model.

Objectives: The histidine kinase (HK) CHK1 and other protein kinases in Candida albicans are key players in the development of hyphae. This study is designed to determine the functional roles of the S_Tkc domain (protein kinase) and the GAF domain of C. albicans CHK1 in hyphal formation and mucosal invasion. Methods: The domain mutants CHK25 (ΔS_Tkc CHK1/Δchk1) and CHK26 (ΔS_TkcΔgaf CHK1/Δchk1) were first constructed by the his1-URA3-his1 method and confirmed by sequencing and Southern blots. A mouse tongue infection model was used to evaluate the hyphal invasion and fungal loads in each domain mutant, full-gene deletion mutant CHK21 (chk1Δ/chk1Δ), re-constituted strain CHK23 (chk1Δ/CHK1), and wild type (WT) from day 1 to day 5. The degree of invasion and damage to the oral mucosa of mice in each strain-infected group was evaluated in vivo and compared with germ tube rate and hyphal formation in vitro. Result: When compared with severe mucosal damage and massive hyphal formation in WT- or CHK23-infected mouse tongues, the deletion of S_Tkc domain (CHK25) caused mild mucosal damage, and fungal invasion was eliminated as we observed in full-gene mutant CHK21. However, the deletion of S_Tkc and GAF (CHK26) partially restored the hyphal invasion and mucosal tissue damage that were exhibited in WT and CHK23. Regardless of the in vivo results, the decreased hyphal formation and germ tube in vitro were less apparent and quite similar between CHK25 and CHK26, especially at the late stage of the log phase where CHK26 was closer to WT and CHK23. However, growth defect and hyphal impairment of both domain mutants were similar to CHK21 in the early stages. Conclusion: Our data suggest that both protein kinase (S_Tkc) and GAF domains in C. albicans CHK1 are required for hyphal invasiveness in mucosal tissue. The appropriate initiation of cell growth and hyphal formation at the lag phase is likely mediated by these two functional domains of CHK1 to maintain in vivo infectivity of C. albicans.

Evaluation of Droplet Digital PCR Assay for the Diagnosis of Candidemia in Blood Samples.

Numerous studies have shown that droplet digital PCR (ddPCR) is a promising tool for the diagnosis of pathogens, especially in samples with low concentrations of pathogenic DNA. An early diagnosis of candidemia is critical for the effective treatment of patients. In this study, we evaluated the sensitivity and specificity of ddPCR assay for Candida DNA detection both in vitro by mixing fungal cells with human blood and in vivo by analyzing blood samples from infected mice and patients with suspected candidemia. The results showed that ddPCR assay could detect a minimum of 4.5 DNA copies per reaction in blood samples. ddPCR showed higher sensitivity and specificity for Candida DNA detection than traditional culture and quantitative PCR (qPCR) methods and also exhibited significantly better positive and negative predictive values than the culture and qPCR methods that were commonly used in clinical practice. Hence, our study demonstrates that ddPCR assay is a promising method for the timely diagnosis of candidemia and could be useful for monitoring the treatment of candidemia.

Rapid detection of fungi from blood samples of patients with candidemia using modified calcofluor white stain.

We reported a modified CFW assay for rapid detection of fungi in blood samples and evaluated its efficacy in vivo and in vitro. The positive rate, sensitivity, and negative predictive values of the modified CFW method were all significantly higher than those of traditional fungal culture and KOH methods.