Chinese Journal of Mycology

Correlation analysis of TFP1 gene and calcineurin pathway of Candida albicans

MENG Lingning, LIU Jinyan, XIANG Mingjie, SHEN Han

2021, 16(4): 217-221.

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Objective To construct the TFP1 gene knockout strains of Candida albicans and analyze the relationship between TFP1 gene and genes related to calcineurin pathway. Methods The upstream and downstream flanking sequences of TFP1 gene and the screened markers were connected to form the gene knockout fragments through fusion PCR, and then transferred into Candida albicans SN152 by lithium acetate transfection method. TFP1^+/- strains were then screened out. The second allele was knocked out by the same method. Quantitative real-time PCR was used to detect the expression levels of RTA2 and CRZ1 genes of TFP1^-/- strain, TFP1^+/- strain and SN152 strain. Results The Candida albicans SN152 TFP1^-/- double allelic deletion strains were successfully constructed, and the expression levels of RTA2 and CRZ1 genes in the SN152 TFP1^-/- strains were both decreased. Conclusion TFP1 gene of Candida albicans is related to calcineurin pathway.

Species identification and in vitro antifungal susceptibility profile of 39 isolates of Aspergillus section Flavi isolated from nails in Iran

XU Xue, DENG Shuwen, WANG Xiaodong, ZHAO Rongfen, ZHANG Hong, YU Nong, Mohammad Javad Najafzadeh, SHI Dongmei, LU Min

2021, 16(4): 222-228.

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Objective In this study, species identification and in vitro antifungal susceptibility profile of 39 isolates of Aspergillus section Flavi isolated from onychomycosis in Mashhad, Iran were investigated. Methods β-tubulin/calmodulin gene sequencing combined with MALDI-TOF MS were used for identification of 39 isolates of Aspergillus section Flavi; Antifungal susceptibility to 10 antifungal drugs including terbinafine, new triazole and echinocandins were determined on all the isolates according to CLSI M38-A3. Results Among the 39 Aspergillus section Flavi based on morphological identification, 38 isolates were identified as A. flavus or A. oryzae by molecular method, and identified as A. flavus by MALDI-TOF MS, one isolate was A. minisclerotigenes by molecular method. However, MALDI-TOF MS misidentified one isolate of A.minisclerogenes as A.flavus. The antifungal susceptibility results indicated that terbinafine, posaconazole and echinocandins had good activity against the most Aspergillus isolates tested in this study. Conclusions Molecular identification combined with MALDI-TOF MS could correctly identify Aspergillus section Flavi to species level, particularly for A. flavus/A. oryzae which molecular identification could not differentiated, MALDI-TOF MS could identify correctly. Expanding reference strain database is necessary for precise identification of Aspergillus section Flavito species level. Terbinafine, posaconazole and echinocandins could be potentially effective drugs for the treatment of onycomycosis due to Aspergillus section Flavi.

Application of MADLI-TOF MS in rapid identification of Talaromyces marneffei

GUO Penghao, WU Zhongwen, QIU Danping, XIE Meiying, LIU Min, LIAO Kang

2021, 16(4): 229-233.

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Objective Evaluation of the application of MADLI-TOF MS in rapid identification of T. marneffei. Methods T. marneffei isolates were collected from Guangzhou, Yulin and Fangchenggang. All the isolates identified by ITS region amplification and sequencing. The strains were pretreated by glass bead grinding and formic acid/acetonitrile extraction. The spectra of the mold and yeast phase colonies of the establishment strains on different plates and at different time points were obtained. The collected spectra were used for the establishment of the super-spectrum. The remaining isolates and other common clinical fungi isolates were used to evaluate the self-established database. Results The super-spectrum of T. marneffei with 39 characteristic peaks was established. The RUO SARAMIS database was expanded. All the validation strains were correctly identified by the expanded database, with an accuracy of 100%. Conclusion MADLI-TOF MS can quickly and accurately identify T. marneffei by the expanded database.

Distribution characteristics of invasive yeast infections in China Hospital Invasive Fungal Surveillance Net(CHIF-NET) in 2012

DUAN Simeng, XIAO Meng, HUANG Jingjing, WANG He, ZHANG Ge, ZHANG Jingjia, KANG Wei, WANG Tong, XIAO Yuling, NI Fang, GAO Lanmei, YE Liyan, GUO Penghao, WAN Zhe, CHEN Zhongju, YANG Qing, LIN Jie, HU Qingfeng, LI Hongling, YUE Na, CUI LanYing, LI Gang, FAN Hui, SU Danhong, LIU Liwen, TIAN Sufei, ZHAO Shengyuan, DU Xuefei, ZHOU Hongwei, XU Yingchun

2021, 16(4): 234-242.

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Objective Incidence rate of invasive yeasts increased significantly in China. This study reported the datas of the China Hospital Invasive Fungal Surveillance Net (CHIF-NET) in 2012. Methods A total of 1619 Candida strains were collected from 22 hospitals in China. The sensitivity of fluconazole and voriconazole were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry and ITS sequencing. The drug sensitivities of fluconazole and voriconazole were determined by kirby Bauer, K-B, recommended by the American institute of clinical laboratory standardization (CLSI). Results In this study, 626 strains from blood samples accounted for 38.67%.There were 993 strains from other parts (including ascitic fluid, pus, venous catheter, cerebrospinal fluid, bronchoalveolar lavage fluid, bile, pleural fluid, tissue, hydrarthrosis and gastric juice), accounting for 61.33%. Among them, 354 strains isolated from ascites accounted for 21.8%, 159 strains isolated from pus accounted for 9.8%, and fewer strains isolated from other sample types (<8%). Among 1619 strains of Candida isolated Other parts except blood, 491 strains were mainly Candida albicans, accounting for 49.45%,followed by 164 strains of Candida tropicalis, accounting for 16.52%, and the number of other species was small (<10%). Among the patients with invasive yeast detected, there were 1,006 male patients accounting for 62.1% and 612 female patients accounting for 37.8%. The age range was 0-94 years old, among which 498 patients ≥ 65 years accounted for 30.76%. In invasive candida infection, most Candida albicans were sensitive to fluconazole and voriconazole (R<1%), followed by Candida parapsilosis (R<6%) and Candida tropicalis (R<7%). In addition, different Candida species have different geographical distribution. Conclusion Studies howed that the distribution of invasive yeast infection in China was related to many factors including infection site, region and age. Therefore, continuous monitoring is needed.

Diagnostic value of DNA microarray chip for cutaneous mycobacterial infections

YU Qian, WEN Yiyang, GAO Zhiqin, YANG Lianjuan

2021, 16(4): 243-246,256.

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Objective To study the diagnostic value of DNA microarray chip for cutaneous mycobacterial infections. Methods Six skin tissues from clinical cutaneous mycobacterial infections suspected cases were underwent pathogenic detection by traditional bacteria culture method and DNA microarray chip technology. Results All patients had history of aquatic exposure or trauma, the clinical manifestation patterns were single lesion or sporotrichoid infection type. Among 6 suspected cases, there were 5 cases with positive culture of mycobacteria strains, which include 4 strains of Mycobacterium marinum, 1 strain of M. chelonae. The chip examinations were positive in 6 cases, included 5 M. marinum cases and 1 M. chelonae case. Compared with traditional bacteria culture method, DNA microarray chip technology had a higher sensitivity (6/6 vs 5/6) and was less time-consuming. Conclusion The DNA microarray ship technology provided a simple, rapid, and highly sensitive clinical assay, it could be used for mycobacteria species identification of skin tissue samples from cutaneous mycobacterial infections, allowing earlier aetiological diagnosis and pathogen-adapted antimicrobial therapy.

Rapid identification technology of invasive filamentous fungi by MALDI-TOF MS

ZHANG Jing, JIANG Bin, PENG Na, OUYANG Pengwen, Xu Wen, Ning Xingwang, LIU Qiong, XIE Liangyi

2021, 16(4): 247-251.

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Objective To evaluate the application value of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology in the identification of clinical common filamentous fungi in clinical practice. Methods From April 2017 to July 2019, 65 filamentous fungi were isolated and cultured from various types of clinical specimens of ourhospital and 26 common filamentous fungi donated by a large third-class hospital. After the 91 filamentous fungi were identified by traditional morphological identification, DNA sequencing and MALDI-TOF MS, the results of DNA sequencing were compared with those of the other two methods to evaluate the application of MALDI-TOF MS in the identification of clinical filamentous fungi. Results The filamentous fungi cultured at 28℃ and 35℃ were identified by mass spectrometer, the Chi-square test(χ² test,P=0.05) showed that P>0.05, suggesting that there was no significant difference in culture temperature between the two groups.Using DNA sequencing results as gold standard, the correct identification rate of traditional morphological methods was 81.3% (74/91), while that of MALDI-TOF MS technology was 97.8% (89/91). In this study, the correct identification rate of filamentous fungi by mass spectrometry was 90.1% (82/91) after extracting protein by acetonitrile formate extraction method, while that of protein extracted by magnetic bead grinding method was 97.8%(89/91). Conclusion In clinical routine work, when using mass spectrometer to identify common filamentous fungi, the culture temperature will not affect the mass spectrometry identification results; the correctness of mass spectrometry after improved magnetic bead grinding method to extract protein is better than that recommended by mass spectrometer to extract acetonitrile formate; compared with traditional morphological identification, MALDI-TOF MS technology is faster, more objective, more efficient and more accurate in identification of common filamentous fungi.

Analysis of the clinical characteristics of 113 children with vulvovaginal candidiasis

ZHENG Bingjie, WANG Yirong, CHEN Xilan, YAO Chenlong, WANG chun, QIAN Qiufang, LIN Xiao

2021, 16(4): 252-256.

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Objective To analyze the clinical characteristics of vulvovaginal candidiasis (VVC)in 113 children. Methods The data of 113 children with VVC were analyzed retrospectively for the last 5 years. The clinical characteristics, mycological examination results and in vitro sensitivity of strains to amphotericin B, flucytosine, fluconazole, itraconazole and voriconazole were analyzed. Results The age distribution of 113 children ranged from 3 months to 16 years old, with an average age of 10.96 years old, and 76.1% of them were 10-12 years old. The main clinical symptoms were pruritus vulvae (47.8%) and increased secretion of vulva (36.3%). A total of 108 Candida strains were isolated and identified, with the highest isolation rate of Candida albicans, (91.7%), followed by Candida glabrata (5.6%), C. parapsilosis (1%), C. krusei (1%) and C. tropicalis (1%). All Candida albicans were sensitive to amphotericin B. The resistance rates to itraconazole, fluconazole and voriconazole were 11.1%, 10.1% and 8.1% respectively, and the resistance rate to flucytosine was 1%. The external antifungal drug was mainly used to treatment. Conclusion The 113 cases of VVC in children were mainly caused by Candida albicans. The drug resistance of different Candida strains was different, and the standard treatment needs to be further discussed.

Adult kerion caused by Trichophyton tonsurans: a case report

ZHOU Chenglong, ZHANG Xiaoli

2021, 16(4): 266-268,283.

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The patient was a 22-year-old female with erythema at the top of the head with hair loss for 3 weeks. Dermatological examination:large erythema on the top of the head with hair loss, visible follicular pustules and subcutaneous abscess. The patient was diagnosed as kerion caused by Trichophyton tonsurans after direct microscopic examination, fungal culture and molecular identification. Oral itraconazole (0.2 g/d) and 2% ketoconazole shampoo were given. After twelve weeks of treatment, the skin lesions completely faded, and hair grows in the alopecia area.

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