In Vitro Antifungal Activity of Azoles and Other Antifungal Agents Against Pathogenic Yeasts from Vulvovaginal Candidiasis in China.

BACKGROUND: Vulvovaginal candidiasis (VVC) is a public health issue worldwide. Little is known of the optimal treatment of recurrent VVC (RVVC) has not been established. OBJECTIVE: Through the in vitro antifungal susceptibility profiling of VVC isolates, we hope to foster significant improvements in the control and treatment of this disease. METHODS: Candida isolates from VVC patients were collected from 12 hospitals in 10 cities across China. Species were identified by phenotype analysis and DNA sequencing. Species were identified by phenotype analysis and DNA sequencing. Susceptibilities to 11 drugs were determined by Clinical and Laboratory Standards Institute broth microdilution. RESULTS: 543 strains were isolated from those VVC patients enrolled in this study, of which, 15.7% were from RVVC. The most commonly identified species was C. albicans (460, 84.71%), and the most commonly non-albicans Candida spp. (NAC) was C. glabrata (47, 8.66%). NAC also included C. Krusei, Meyerozyma Guillermondii, Meyerozyma Caribbica, C. Tropicalis, C. Parapsilosis, and C. Nivariensis. Most C. albicans isolates were susceptible to caspofungin (99.8%), followed by fluconazole (92%) and voriconazole (82.6%). The proportion of C. albicans strains with wild type (WT) MICs that were susceptible to amphotericin B and caspofungin were 98%, followed by posaconazole at 95%, itraconazole at 86%, fluconazole at 74% and voriconazole at 54%. The fluconazole MICs for C. albicans were lower than those for NAC (P < 0.05), while the itraconazole MICs showing no significant difference (P > 0.05). The susceptible rate of uncomplicated VVC to fluconazole was 92%. The proportion of WT strains to fluconazole in RVVC was much lower than that in other types of VVC (67 vs. 77%, P < 0.05). However, the proportions of WT strains to itraconazole in RVVC was over 85%, which was much higher than that to fluconazole (87 vs. 67%, P < 0.05). CONCLUSIONS: C. albicans was still the predominant pathogen for VVC in China, while C. glabrata was the main species in NAC. Fluconazole could still be used as an empirical treatment for uncomplicated VVC. However, fluconazole may not be the first choice for the therapy of RVVC. In such cases, itraconazole appears to be the more appropriate treatment. As for VVC caused by NAC, nonfluconazole drugs, such as itraconazole, may be a good choice.

Phaeohyphomycosis in China.

Background: Due to more attentions paid to melanized fungi over the past few decades and under the background of the global coronavirus disease 2019 pandemic (COVID-19) the fact that the virus itself and the immunosuppressive agents such as glucocorticoids can further increase the risk of infections of deep mycoses, the number of patients with phaeohyphomycosis (PHM) has a substantial increase. Their spectrum is broad and the early diagnosis and treatments are extremely sticky. This study aims to more comprehensively understand the clinical features of phaeohyphomycosis in China over 35 years and to establish a more applicable systematical classification and severity grades of lesions to guide treatments and prognosis. Methods: We reviewed 174 cases of proven phaeohyphomycosis reported in Chinese and English language literature from 1987 to 2021 and we also made the accurate classification definitions and detailed information about the epidemiology, species of clinical dematiaceous fungi, minimum inhibitory concentration values, clinical features, treatments, and prognosis. Results: The mortality of cerebral, disseminated and pulmonary phaeohyphomycosis are 55%, 36%, and 25%. Nearly 19% of patients had poor quality of life caused by the complications such as disability, disfigurements, and blindness. The overall misdiagnosis rate of phaeohyphomycosis was 74%. Moderate to severe rashes are accounting for 82% of subcutaneous phaeohyphomycosis. The areas of the head and face are mostly affected accounting for 16% of severe rashes. Nearly 30% of invasive infections of phaeohyphomycosis are triggered by recurrent lesions. Voriconazole, itraconazole, amphotericin B deoxycholate (AmB-DOC), and terbinafine were most commonly used but diagnosis and treatments of phaeohyphomycosis remain challenging in reality. Conclusions: Our classifications are likely to be more practical and easier to popularize, and there are still also plenty of characteristics in these non-specific lesions. There're no significant variations in cure rates, or death rates between three grades of lesions. But patients with severe rashes have longer courses and lower effective rates.

Successful Treatment of Eczema-Like Mucormycosis in a Child by Combination of Intravenous Drip and Percutaneous Injection Amphotericin B.

We report a case of eczema-like cutaneous mucormycosis caused by Rhizopus arrhizus. A 4-year-old child was presented to our hospital with a history of gradually enlarging papule and plaque in the periumbilical area for nearly 4 years since 2 weeks after his birth, and it has been misdiagnosed as eczema for nearly 3 years. Based on histopathology examination, the fungus culture test and DNA sequencing, it was revealed that R. arrhizus should be the responsible fungus for skin infection. The patient was successfully cured by combination of intravenous drip and percutaneous injection amphotericin B for nearly 3 months, and no recrudescence was seen during a follow-up of 6-month observation.

In vitro susceptibility of dematiaceous fungi to nine antifungal agents determined by two different methods.

BACKGROUND: The infection rate for dematiaceous fungi has increased rapidly over the most recent decades. However, the treatment for such infections has been lacking in empirical support with oral antifungal agents. OBJECTIVES: To provide a better regimen for dematiaceous infections in future, the Sensititre YeastOne® colorimetric antifungal panels were used and compared with Laboratory Standards Institute (CLSI) M38-A2 reference broth microdilution method. METHODS: Two methods were used for nine antifungals against 67 dematiaceous fungi. RESULTS: Via two methods, we found that the MICs of itraconazole, voriconazole, posaconazole and amphotericin B were lower than fluconazole. The values obtained with CLSI method for four triazoles, 5-flucytosine and amphotericin B were in high essential agreements with those observed by YeastOne® method. The results of echinocandins across the two methods showed some divergence, which might be attributed to the methodology itself, particularly when sensitivity was determined in the lowest concentration of the drug. In YeastOne® method, the results were defined as MICs, but as MECs in CLSI method. CONCLUSIONS: The YeastOne® method appeared to be both easier and more efficient for dematiaceous fungi when compared with the CLSI method, and the agreement between the two methods was good for most common antifungals.

Subcutaneous Phaeohyphomycosis Caused by Exophiala oligosperma in an Immunocompetent Host: Case Report and Literature Review.

We report a case of subcutaneous infection caused by Exophiala oligosperma. Erythematous ulcerated plaque with exudate was major clinical features. Histopathological examination showed yeast-like cells and fungal hyphae. Mycological and molecular identification revealed E. oligosperma as etiologic agent. Local debridement and oral itraconazole were effective. To the best of our knowledge, this is the first report of phaeohyphomycosis caused by E. oligosperma in mainland China. This report highlights the potential role of E. oligosperma as an emerging cause of infection in immunocompetent patients.

Coccidioidomycosis: Imported and possible domestic cases in China: A case report and review, 1958-2017.

We report a case of imported pulmonary coccidioidomycosis caused by Coccidioides posadasii in a patient who was misdiagnosed as tuberculosis and mistreated with antituberculosis medications for 18 months. The symptoms were not relieved until antifungal treatment was started. An extensive review of the coccidioidomycosis cases occurring in China reveals 38 cases, 16 of which had no associated history of travel to any traditional endemic areas. We speculate that some factors may drive Coccidioides spp. transference to China, which then causes those domestic infections. Moreover, we indicate the first, to the best of our knowledge, possible endemic areas in China.

Successful Treatment by Surgery of a Primary Cutaneous Mucormycosis Caused by Mucor irregularis.

We report a case of primary cutaneous mucormycosis caused by Mucor irregularis. A 66-year-old man was presented to our hospital with a history of gradually enlarging plaque on the right leg for about a year. The identification of pathogen based on the fungus morphology and DNA sequencing revealed M. irregularis as the responsible fungus for skin lesion. The lesion was removed incidentally by a surgery procedure, and no recrudescence was seen during a follow-up of 24-month observation.

The influence of the mating type on virulence of Mucor irregularis.

Mucor irregularis is an emerging fungal pathogen that cause cutaneous infection and could cause death. However, little is known about its mechanism of pathogenesis. There is evidence suggesting virulence vary with mating types in fungi, including the Mucorales. Here, we characterized the mating type locus of M. irregularis and the mating type ratio of 17 clinical isolates in China. Genomic data indicated M. irregularis is heterothallic having two mating types - bearing either SexP or SexM allele. Also, we employed a mice model to study the inflammation and pathological effects of different mating types. The comparison of the inflammatory response, cytokine profiles and Th-1, Th-2 and Th-17 cells numbers in each mating type treated mice showed that the severity and disease progress were enhanced in (+) mating type treated mice. One (+/0) mutant strain, with multiple mutations at the mating locus, had defects in sexual mating ability but appeared to be more virulent than the (-) mating type. Although (+) mating type appeared to be more virulent, most of our clinical isolates presented belonged to (-) mating type. Our findings support the involvement of MAT genes in sexual fertility, and the influence of mating type on the severity of cutaneous infection.

Systematic Analysis of the Lysine Succinylome in Candida albicans.

Candida albicans is the most common human fungal pathogen for both immunocompetent and immunocompromised individuals. Lysine succinylation is a frequently occurring post-translational modification that is found in many organisms; however, the role of succinylation is still under investigation. Here, we initiated a first screening of lysine succinylation in C. albicans. We identified 1550 succinylation sites from 389 proteins in C. albicans, demonstrating that succinylation is conservative in this organism. However, the lysine succinylation sites showed some difference in C. albicans, with the overlapping rates between C. albicans and other species ranging from 55% for Saccharomyces cerevisiae, 40% for human, 35% for mouse, and to only 16% for Escherichia coli. The further bioinformatics analysis indicated that the succinylated proteins were involved in a wide range of cellular functions with diverse subcellular localizations. Furthermore, we discovered that lysine succinylation could coexist with phosphorylation and/or acetylation in C. albicans. The KEGG enrichment pathway analysis of these succinylated proteins suggested that succinylation may play an indispensable role in the regulation of the tricarboxylic acid cycle. The bioinformatic data obtained from this study therefore enable the depth-resolved physiological roles of lysine succinylation in C. albicans.

Microsatellite analysis of genotype distribution patterns of Candida albicans vulvovaginal candidiasis in Nanjing, China and its association with pregnancy, age and clinical presentation.

PURPOSE: To characterize the genotype distribution pattern of Candida albicans associated with vulvovaginal candidiasis (VVC) in Nanjing, China by microsatellite genotyping. METHODS: A questionnaire was completed by each patient diagnosed with VVC. A total of 208 independent C. albicans was isolated from 208 patients. Microsatellite genotyping characterized the genotype distribution by analysis of the CAI locus marker. RESULTS: PCR of CAI fragments showed the three major genotypes contained 30:45, 21:21 and 32:46 alleles among the 51 genotypes detected, accounting for 29.3, 13.0 and 12.0 % of 208 clinical isolates. Genotype distributions had a similar pattern among different clinical presentations (P = 0.219). In both groups of the (21-30) and (31-40) years, 30:45 was the most frequent genotype allele detected. In the (21-30) year females, 16.5 % of the isolated strains had the genotype 21:21, while the same genotype in the group of (31-40) years was 6.9 %. Genotype distributions were significant difference between the pregnant and non-pregnant women (P < 0.001). 30:45 was detected only one in the 23 pregnant women. CONCLUSIONS: The results indicated a unique genotype distribution of C. albicans associated with VVC in Nanjing, eastern China and a different distribution pattern was also detected in pregnant women compared to non-pregnant women.

Combination of Estrogen and Immunosuppressive Agents to Establish a Mouse Model of Candidiasis with Concurrent Oral and Vaginal Mucosal Infection.

Mouse model is an appropriate tool for pathogenic determination and study of host defenses during the fungal infection. Here, we established a mouse model of candidiasis with concurrent oral and vaginal mucosal infection. Two C. albicans strains sourced from clinical candidemia (SC5314) and mucosal infection (ATCC62342) were tested in ICR mice. The different combinational panels covering estrogen and immunosuppressive agents, cortisone, prednisolone and cyclophosphamide were used for concurrent oral and vaginal candidiasis establishment. Prednisolone in combination with estrogen proved an optimal mode for concurrent mucosal infection establishment. The model maintained for 1 week with fungal burden reached at least 10(5) cfu/g of tissue. This mouse model was evaluated by in vivo pharmacodynamics of fluconazole and host mucosal immunity of IL-17 and IL-23. Mice infected by SC5314 were cured by fluconazole. An increase in IL-23 in both oral and vaginal homogenates was observed after infection, while IL-17 only had a prominent elevation in oral tissue. This model could properly mimic complicated clinical conditions and provides a valuable means for antifungal assay in vivo and may also provide a useful method for the evaluation of host-fungal interactions.

Multiple cyp51A-based mechanisms identified in azole-resistant isolates of Aspergillus fumigatus from China.

Seventy-two A. fumigatus clinical isolates from China were investigated for azole resistance based on mutations of cyp51A. We identified four azole-resistant strains, among which we found three strains highly resistant to itraconazole, two of which exhibit the TR34/L98H/S297T/F495I mutation, while one carries only the TR34/L98H mutation. To our knowledge, the latter has not been found previously in China. The fourth multiazole-resistant isolate (with only moderate itraconazole resistance) carries a new G432A mutation.

Phaeohyphomycosis caused by Exophiala spinifera: an increasing disease in young females in mainland China? Two case reports and review of five cases reported from mainland China.

No more than 30 cases of phaeohyphomycosis caused by Exophiala spinifera have been reported up to now in English and Chinese literature over the past half century. Here, we reported two cases of phaeohyphomycosis caused by E. spinifera and reviewed all the five cases reported from mainland China. These two involved were both young female, one patient experiencing recurrence during pregnancy and the other developing multiple-site infections without apparent immunodeficiency. The aetiological agents were both identified as E. spinifera by molecular analysis. Oral itraconazole was proved effective enough for the first patient, while the combination of itraconazole and terbinafine was needed for the second patient. It seems that infections due to E. spiniferais increasing in China mainland nowadays, usually involving young female.

The application of laser microdissection in molecular detection and identification of aspergillus fumigatus from murine model of acute invasive pulmonary aspergillosis.

Invasive aspergillosis (IA) is a major concern in patients with severe immune deficiency. As antifungal susceptibility varies in different fungal pathogens, accurate and timely identification of species is becoming imperative for guidance of therapy and reducing high mortality rates in patients with IA. But, in fact, the diagnosis is challenging and new validated techniques are required for the detection and identification of clinically relevant isolates. The laser capture microdissection (LCM) system enables analysis of cytologically and/or phenotypically defined cell types from heterogeneous tissue and has been used in diagnosis and fungal species identification in pulmonary aspergillosis of white storks. To establish the experimental foundation for clinical application of the system, we microdissected and collected Blankophor-stained single hyphal strands from tissue cryosections of murine model of invasive pulmonary aspergillosis (IPA) with A. fumigatus by LCM, subsequently processed for DNA extraction, PCR sequencing, and species molecular identification. The sensitivity of LCM-PCR sequencing was 89 % (89/100), and the specificity was 100 %. Moreover, the positive predictive value and negative predictive value were 100 and 78.43 %, respectively. The result approved that the LCM-based methods had the potential for accurately diagnosis and rapidly identification fungal pathogens of IPA.

In vitro analyses of mild heat stress in combination with antifungal agents against Aspergillus fumigatus biofilm.

Aspergillus fumigatus biofilms still present a challenge for effective treatment in clinical settings. While mild heat stress has been introduced as a treatment for infectious diseases, the effectiveness of mild heat stress on A. fumigatus biofilm formation and antifungal susceptibility is still unknown. In the present study, confocal laser scanning microscopy (CLSM) was used to image and quantify Aspergillus fumigatus biofilm formation under three different regimens of continuous mild heat stress: at 37, 39, and 41°C. Furthermore, fungal growth has been investigated under the above conditions in combination with antifungal drugs (amphotericin B [AMB], micafungin [MCF], and voriconazole [VOC]) at early and late stages. CLSM analysis showed that higher temperatures induce earlier germination and greater hyphal elongation but poorer polar growth and reduced biofilm thickness. In the early stage of biofilm formation, the combination of treatment at 39 or 41°C with MCF or VOC produced no visible difference in biomass formation from similar treatments at 37°C with the same drug. Interestingly, AMB treatment at 37°C inhibited early stage biofilm formation to a much greater extent than at 39 and 41°C. At the late stage of biofilm formation, the mild heat treatments at 39 and 41°C with AMB, MCF, and VOC inhibited biomass formation compared to that at 37°C. The present data show that mild heat stress has a negative regulatory effect on biofilm formation in vitro, and antifungal drug improvement with mild heat treatment at late-stage biofilm formation provides useful indications of possible effective strategies for clinical management of aspergillosis.

The first case of phaeohyphomycosis caused by Rhinocladiella basitona in an immunocompetent child in China.

We present the first case of phaeohyphomycosis caused by Rhinocladiella basitona (R. basitona) in China and describe the mycological characteristics of this pathogen. A 11-year-old girl was presented with plaque on her face for 3 years. Diagnosis was based on histopathology, mycology, and molecular identification. The patient was treated with terbinafine and itraconazole. This case is the second of phaeohyphomycosis caused by R. basitona in the world (previously belonging to Geniculosporium).

Cytokine profile of a self-healing Fonsecaea pedrosoi infection in murine model.

Chromoblastomycosis is a chronic infectious disease of the skin and subcutaneous tissue. However, the host-defence response to this fungal infection has not been investigated thoroughly. This study was carried out to analyse the sequential events and the change of local cytokine release in a murine model infected with Fonsecaea pedrosoi in footpad. The anti-inflammatory Th2 cytokine IL-10 demonstrated an upward trend up to 7 days post infection followed by a steady decline. The titers of TNF-α (a pro-inflammatory Th1 cytokine) increased up to 7 days post infection followed by a relatively steady-state until full recovery. The anti-inflammatory cytokine IL-4 showed a similar pattern as TNF-α. The pro-inflammatory cytokine IFN-γ did not increased until 7 days post infection, while demonstrated an upward trend up to 30 days when the mice reached a full recovery from infection.

Cutaneous and mucosal phaeohyphomycosis caused by Exophiala spinifera in a pregnant patient: case report and literature review.

We report a case of mucocutaneous phaeohyphomycosis caused by Exophiala spinifera. Crusty plaques and nodules were major clinical features. Histological examination revealed brown yeast-like cells and hyphae. Mycological and molecular data identified E. spinifera as etiologic agent. Oral itraconazole was effective, which was in accordance with the results of in vitro susceptibility testing. We speculated that her pregnancy may play a role of risk factor in the infection by E. spinifera.

[Effect of phospholipomannan of Candida albicans on the production of interleukin 6 and interleukin 8 in monocytes].

OBJECTIVE: To investigate whether Candida albicans-native phospholipomannan (PLM) induce an inflammation response through Toll-like receptor(TLRé2 in human acute monocytic leukemia cell line (THP-1) cells. METHODS: Human THP-1 monocytes were challenged with PLM in vitro. The mRNA expressions of TLR2, TLR4, proinflammatory cytokine [interleukin(IL)-6], and chemokine (IL-8) were assayed by real time reverse transcription polymerase chain reaction. The secretions of IL-6 and IL-8 were measured by enzyme-linked immunosorbent assay. The expression of TLR2 was analyzed with Western blot. RESULTS: PLM increased the mRNA expressions and secretions of proinflammatory cytokines (IL-6) and chemokines (IL-8) in THP-1 cells (all P=0.0000). PLM up-regulated the mRNA and protein levels of TLR2 (P=0.0000), whereas the mRNA level of TLR4 was not altered. PLM hydrolyzed with ß-D-mannoside manno hydrolase failed to induce gene and protein expressions of TLR2, IL-6, and IL-8. Anti-TLRS-neutralizing antibody blocked the PLM-induced secretions of IL-6 and IL-8 in THP-1 cells (P = 0.0003, P = 0.0010). CONCLUSION: Canidada albicans-native PLM may contribute to the inflammatory responses during Candida infection in a TLR2-dependent manner.