Table of Content

28 October 2021, Volume 16 Issue 5

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Original articles

Multicenter retrospective study on antifungal susceptibility and molecular epidemiology of Candida parapsilosis isolated from bloodstream infections in China

ZHANG Li, YU Shuying, NING Yating, XIAO Meng, XIAO Yuling, CUI Lanying, CHEN Zhongju, YAN Yan, YE Liyan, XU Yingchun

2021, 16(5):  289-295. 

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Objectives The present study was to conduct molecular epidemiology and antifungal susceptibilities of C. parapsilosis retrospectively based on multicenter isolates nationwide in China. Methods Atotal of 377 non-duplicated C. parapsilosis isolates from bloodstream infections were collected from unique patients from 42 hospitals across China. Antifungal susceptibility to fluconazole and voriconazole was determined for all isolates using the CLSI M44-A2 disc diffusion method. C. parapsilosis isolates were genotyped using four highly polymorphic microsatellite markers. Results Of the 377 patients, 46.2% were over 60 years old. 26.5%, 23.9%, 21.8% and 12.5% patients were located in surgical ward, medicine ward, general ICU and surgical ICU respectively. Among 377 strains, 24 (6.4%) were resistant to fluconazole and 10 (2.7%) to voriconazole. Microsatellite typing showed that all strains were divided into 172 microsatellite types, 125 MT types only appeared in a single strain; in 28 hospitals, there were 65 cases of which different patients in the same hospital were infected with the same MT type of strains, among which 6 cases indicated different patients were infected with the same MT type of fluconazole resistant strains in the same hospital. Conclusions Here we presented the nationwide molecular epidemiology study of C. parapsilosis causing bloodstream infections in China, and identified several cases of which different patients in the same hospital were infected with the same MT type of strains including antifungal resistant isolates, indicating probable horizontal transmission of C. parapsilosis in hospitals.

The effect of cinnamaldehyde on the growth and biofilm formation of Trichosporon asahii

ZHANG Weizheng, XIAO Yinglun, PENG Xiangming, LI Song, ZHANG Xuan, CHEN Cha

2021, 16(5):  296-302. 

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Objective To find the effect of cinnamaldehyde on the growth and biofilm formation of Trichosporon asahii(T. asahii). Methods Clinical isolates were selected for gene sequencing and VITEK MS identification, and the biofilm model was established in vitro. The inhibitory effects of cinnamaldehyde at 1050 mg/L, 525 mg/L, 262.5 mg/L and 131.25 mg/L on the growth of T. asahii were determined by multiple dilution method.The effects of different concentrations of cinnamaldehyde on the biofilm formation of T. asahii. were observed by silver staining method. Results Each concentration of cinnamaldehyde could affect the growth of T. asahii and prolong the formation time of biofilm. The higher the concentration of cinnamaldehyde, the more obvious the effect was. When the concentration of cinnamaldehyde reached 1050 mg/L, the inhibition effect of cinnamaldehyde on the growth of T. asahii was the most significant, the growth of T. asahii was not observed in the medium wells at the concentration. Silver staining showed that no biofilm was formed in the medium at this concentration, indicating that the concentration of cinnamaldehyde at 1050mg/L had a significant inhibitory effect on the formation of biofilm of T. asahii. Conclusion Cinnamaldehyde can affect the growth and biofilm formation of T. asahii.

Construction of CRISPR-Cas9 knockout vector of Fonsecaea monophora polyketide synthase gene

LI Minying, HUANG Huan, LI Qian, LUO Mingfen, WANG Xiaoyue, LIU Hongfang, ZENG Weiying, XI Liyan

2021, 16(5):  303-307,313. 

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Objective To construct a Fonsecaea monophora polyketide synthase 1 (PKS1) gene knock-out vector by CRISPR/Cas9 system with pFC332 plasmid. Methods Primers of the gRNA skeleton fragment from pFC334 plasmid were designed, and then the gRNA skeleton fragment inserted with the AarⅠ restriction site was amplified by PCR. After that, the pFC334 plasmid was linearized with PstⅠ and MerI enzymes and a pFC334-AarⅠ vector was constructed. Subsequently, both pFC334-AarⅠ vector and pFC332 plasmid were digested with MreI and BglII. Digested products were ligated together in order to produce a novel recombinant vector called pFC332-gRNA-AarⅠ, with gRNA skeleton fragment. Finally, multiple gRNAs targeting PKS1 were designed and integrated into the pFC332-gRNA-AarⅠ vector, respectively. Results The obtained pFC332-gRNA-AarⅠ plasmid was 16533bp long, identified by sequencing. PCR, enzyme digestion and sequencing were applied to indicate that the PKS1 gene knock-out vector by CRISPR/Cas9 system was successfully constructed. Conclusion The Fonsecaea monophora polyketide synthase 1 (PKS1) gene knock-out vector by CRISPR/Cas9 system was successfully constructed, which laid a good foundation for the research of PKS1 gene and the biological functions of DHN-melanin in Fonsecaea monophora.

The efficacy and safety of domestic caspofungin for empirical therapy in patients with agranulocytosis and fever

YANG Xiaozhu, LIANG Aibin, ZHENG Xiaoyun, FU Jianfei, HOU Ming, SUN Aining, LU Hua, JIN Jie, HU Jianda

2021, 16(5):  308-313. 

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Objective In the hospitalized patients with agranulocytosis and persistent fever, the efficacy and safety of domestic caspofungin were further evaluated on the basis of the previous study which comparing the pharmacokinetic characteristics of domestic and original caspofungin for empirical antifungal therapy. Methods This study was a prospective, multi-center, open clinical trial (ClinicalTrials.gov Identifier:NCT03857399). Hospitalized patients with agranulocytosis and persistent fever were enrolled. After the empirical treatment of caspofungin, the efficacy and safety of domestic caspofungin in all patients were evaluated. Results Of the 32 patients enrolled, there were 32 patients in full analysis set (FAS) and 30 patients in Per-protocol Set (PPS). In the FAS and PPS, the clinical efficacy rate of domestic caspofungin were 71.88%(23/32) and 76.67%(23/30), the treatment courses were (13.22±5.80) days and (13.70±5.62) days, respectively. Within 0 to 2 days after treatment, the pyretolysis rate were 78.13%(25/32) and 80%(24/30), and the proportion of patients whose absolute neutrophil count over 0.5×10⁹/L were 75%(24/32) and 76.67%(23/30) in the two set. The 28-day survival rate after treatment were 81.25%(26/32) and 83.33%(25/30), respectively. Domestic caspofungin was well tolerated. Most of the adverse events were grade 1 to grade 2 and drug-unrelated, no adverse events led to dosage decreased or withdrew. The incidence of drug-related adverse events was 3.1%(1/32), including 1 case of hepatic dysfunction. No drug-related serious adverse events occurred. Conclusions Domestic caspofungin had achieved great clinical efficacy and safety as empirical antifungal therapy in patients with agranulocytosis and persistent fever.

The levels of HMGBl,IL-17 and IL-23 in patients with vulvovaginal candidiasis and its clinical significance

GAO Aili, DAI Xinyue, LI Huaping, WANG Huanli, MA Shaoyin, CHENG Jiaoquan, ZHU Huilan

2021, 16(5):  314-318,325. 

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Objective To investigate the expression and clinical significance of IL-17,IL-23,HMGB1 in vulvovaginal candidiasis. Method Various vulvovaginal candidiasis patients in our hospital were selected as the observation groups,and healthy volunteers were selected as the control group. ELISA was used to detect the expression of HMGB1,IL-17 and IL-23 in serum and vaginal discharge secretion of all subjects,and the relationship between the expression of these indexes and the clinical parameters of VVC was analyzed.Result In this study, 105 VVC patients were divided into mild and moderate VVC group (51 cases), severe VVC group (21 cases) and recurrent vulvovaginal candidiasis(RVVC) group (33 cases). 39 cases with normal physical examination results were selected as control group. In peripheral blood, the levels of HMGB1 in severe VVC group and RVVC group were higher than that in normal group (t=2.971, P=0.008; t=2.365, P=0.005). The levels of IL-17 in mild to moderate and severe VVC group were higher than that in control group (t=2.370, P=0.024; t=2.987, P=0.008), and the levels of IL-23 in mild, moderate and severe VVC group and RVVC group were higher than that in control group (t=2.948, P=0.008; t=2.955, P=0.008; t=2.940, P=0.009); In the vaginal discharge, the levels of IL-17, mild, moderate and severe VVC group and RVVC group were higher than those in control group (t=2.273, P=0.029; t=2.097, P=0.049; t=2.075, P=0.046), the levels of IL-23 in mild to severe VVC group were significantly higher than that in control group (r=2.624, P=0.017). From a correlation analysis between HMGB1, IL-17, IL-23 and clinical parameters of VVC, in peripheral blood, the level of HMGB1 was positively correlated with the expression of IL-17 (r=0.328, P<0.001; r=0.172, P<0.004), and the level of IL-23 was positively correlated with the expression of IL-17 (r=0.689, P<0.001), There was a positive correlation between IL-23 level and IL-17 expression in the vaginal discharge (r=0.268, P<0.001). in peripheral blood, the level of HMGB1 was positively correlated with severity of disease (r=0.286, P=0.001). In the vaginal discharge, the level of IL-17 was positively correlated with the severity of the disease (r=0.273, P=0.001). Conclusion Levels of HMGB1, IL-17 and IL-23 were significantly higher in VVC patients,and they were closely related to disease severity. These indexes may play an important role in the pathogenesis of VVC.

The epidemiology and antifungal susceptibility of invasive Candida isolates in Southern Sichuan

XU Lingling, ZENG Zhangrui, DING Yinhuan, YANG Kui

2021, 16(5):  319-325. 

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Objective To analyze species distribution, sample types and antifungal susceptibility of invasive Candida infection, to provide basis for clinical diagnosis and treatment. Methods The clinical data of patients with invasive Candida infection were retrospectively analyzed in the Affiliated Hospital of Southwest Medical University from January 2014 to December 2019. Results A total of 646 patients with non-repetitive invasive Candida infection were collected, including 355 males (54.95%) and patients over 60 years old (55.11%). The main isolates of Candida species were Candida albicans (314, 48.61%), followed by Candida glabrata (226, 34.98%) and Candida tropicalis (71, 10.99%). The number of infection patients was the highest in 2017 (141 cases, 21.83%), and then showed a downward trend. The main specimen types were deep wound secretion (229 cases, 35.45%), followed by clean urine (174 cases, 26.93%) and blood (117 cases, 18.11%). The top three departments of invasive Candida infection were internal medicine (255 cases, 39.47%), surgery (214 cases, 33.13%) and pediatrics (78 cases, 12.07%). The total resistance rates of all Candida species to fluconazole, itraconazole and voriconazole were 18.82%, 36.15% and 19.38%, respectively, and the susceptibilities of amphotericin B and 5-flucytosine was 100.00%. Conclusions Candida albicans was the main invasive infection species in this area and elderly patients were the main infected group.Non-Candida albicans was the main Candida species. Candida species were highly resistant to azole antifungal drugs, but highly sensitive to amphotericin B and 5-flucytosine.

In vitro antifungal activity of domestic posaconazole against pathogenic fungi isolated from genital candidiasis

TAN Jingwen, GAO Zhiqin, YANG Hong, YANG Lianjuan

2021, 16(5):  326-329,334. 

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Objective To determine the in vitro antifungal activity of domestic posaconazole against the clinical isolates isolated from genital candidiasis. Methods We used the CLSI (Clinical and Laboratory Standards Institute) broth microdilution method M27-A4 to evaluate the minimal inhibitory concentration (MIC) of posaconazole and other common antifungal agents against 60 clinical isolates of Candida spp. Results The MIC range, geometric mean (GM), MIC₅₀ and MIC₉₀ of posaconazole against fluconazole-resistant C. albicans strain is 0.25-0.50 μg/mL, 0.40 μg/mL, 0.5 μg/mL and 0.5 μg/mL respectively. The data changed to 0.03-0.125 μg/mL, 0.10 μg/mL, 0.125 μg/mL and 0.125 μg/mL when posaconazole against C. parapsilosis. To C. tropicalis, the MIC range, GM, MIC₅₀ and MIC₉₀ is 0.125-8.0 μg/mL, 0.57 μg/mL, 0.5 μg/mL and 2 μg/mL respectively. To C. glabrata, the MIC range, GM, MIC₅₀ and MIC₉₀ is 0.06-2.0 μg/mL, 0.25 μg/mL, 0.125 μg/mL and 1 μg/mL respectively. And the MIC range and GM of posaconazole against C. krusei is 0.125-0.50 μg/mL and 0.29 μg/mL. The MIC₅₀ and MIC₉₀ of posaconazole against C. krusei is 0.25 μg/mL and 0.5 μg/mL, respectively. Conclusions Posaconazole is active against the fluconazole-resistant strains and non-albicans Candida strains associate with genital candidiasis.

Effects of Candida in different membrane production capacity on Toll-like receptor pathway and pregnancy outcome in patients with vaginitis during pregnancy

LI Caiyun, KE Tingting, LI Gang, ZHAO Mei, WANG Wen

2021, 16(5):  330-334. 

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Objective To analyze the effect of Candida in different states on Toll-like receptor signaling pathway in peripheral blood of patients with vaginitis during pregnancy and its relationship with pregnancy outcome. Methods Candida isolated from patients with vaginitis during pregnancy was selected to construct a biofilm model in vitro. Crystal violet and fluorescence staining were used to legally observe the growth and morphology of the biofilm to find strong membrane-producing group and non-film production group. Then 178 healthy parturients were selected as the control group. RT-PCR method was used to detect the expression of TLR2, TLR4 and MyD88 genes in peripheral blood mononuclear cells of each group of patients; ELISA was used to determine the concentration of cytokines in each group, and statistical analysis of maternal premature delivery, abnormal amniotic fluid and premature rupture of membranes And other bad endings. Results A total of 178 Candida strains were isolated, including 64 strains with strong membrane production. The peripheral blood TLR2, TLR4 and MyD88 gene expression of this group of patients was lower than that of the control group, while the non-film production group was higher than the control group (P<0.05). Compared with the control group, the plasma levels of IL-1β, TNF, and IFN-γ in the strong membrane-producing group were significantly lower than those in the control group (P<0.05). The incidence of adverse pregnancy outcomes in the control group in the non-membrane group and in the strong membrane group were 2.8%, 4.3%, and 21.8%. The difference was statistically significant. Conclusion Pregnant women infected with Candida of strong membrane-producing group could down-regulate the expression of TLR2 and TLR4, resulting in a weakened cellular immune response and an imbalanced Th1/Th2 ratio, which might cause changes in the expression of cytokines and could also increase the risk of adverse pregnancy outcomes.

Deletion and functional studies of SER5 gene in Cryptococcus neoformans

Tong Shuangli, WU Jie, LI Shasha, SANG Junjun

2021, 16(5):  335-340. 

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Objective To construct SER5 gene deletion and reconstruction mutants for Cryptococcus neoformans and study its functions. Methods Knocking-out and reconstruction the SER5 gene were performed in strain H99, and the function of Ser5 were analyzed by in vitro stress response experiment, melanin induction experiment, capsule induction experiment, urease determination, growth curve determination, and yeast two-hybrid experiment. Results Gene deletion and reconstruction mutants of SER5 were successfully constructed. ser5Δ had no significant effect on the in vitro stress, capsule, urea decomposition and growth of C.neoformans, and it had a significant reduction in the melanin synthesis of C. neoformans.19 genes were screened by yeast two-hybrid experiment that the coding proteins may interact with Ser5. Conclusion Ser5 affected the melanin production of C. neoformans, and it might interact with endoplasmic reticulum protein, protein YOP1, vesicle-associated membrane protein 7, inorganic phosphate transporter pho88, GDP-mannose transporter 1, vesicle transport through interaction with t-SNAREs 1, suggesting that Ser5 might be involved in the synthesis or transport of melanin in C. neoformans.

Case reports

Cutaneous Exserohilum rostratum infection secondary to marine biological stings

GUO Yanyang, ZHANG Zhe, HAI Luming, GAO Jixin, YAN Dong, ZHU Guannan, WANG Gang, FU Meng

2021, 16(5):  346-350. 

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To report one case of cutaneous Exserohilum rostratum infection secondary to marine biological stings. A 47-year-old female presented with dark red plaques covered with ulcers on the flexor side of her right leg over 4 weeks, after being stabbed by unidentified marine organisms while traveling in Thailand. Skin tissues from the patient were obtained by skin biopsy for histopathological examination, then fungal culture and molecular biological identification of the strains were performed. Histopathological examination revealed fungal structure within focal necrotic dermis and lobular adipose necrosis. Fungal culture showed that horizontal expanding dark brown, woolly colonies. Catenulate conidia and macroconidia with branched hyphae were found by PAS and methenamine sliver staining. Under microscope, brown conidia with 3-4 septates and dark bands were observed. Exserohilum rostratum was identified as the pathogen by sequence analysis of ITS region. According to clinical features and laboratory results, the patient was diagnosed as cutaneous Exserohilum rostratum infection. Treatment:Lesions lapsed after treatment with itraconazole 400 mg twice daily for one month, and tempering to 200 mg once daily for 3 months. Skin infections caused by Exserohilum rostratum were unusual, and impairment of skin barrier function caused by marine organisms stings and exposured to fungi in the environment were the main risk factors.