Table of Content

28 February 2020, Volume 15 Issue 1

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Special topic on combating COVID-19 infection

Experts opinion on the laboratory diagnosis and treatment of invasive fungal infection secondary to severe novel coronavirus pneumonia

YU Jin, LIU Wei, CHEN Wei, ZHANG Xiao-mei, WAN Zhe, WANG Ai-ping, LI Ruo-yu

2020, 15(1):  1-5. 

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The morbidity rate and transmissibility of the novel coronavirus of zoonotic origin (COVID-19) infected pneumonia are high, and the fatality rate among severe cases is high. Severe cases have risk factors of invasive fungal infections (IFIs), with some of them being reported to have developed combined or secondary fungal infections. To minimize the fatality rate among severe cases, IFIs-related microbiological, serologic and molecular biology examinations should be aggressively applied for the early discovery of invasive fungal infections and the early usage of target antifungal agents.

Biosafety proposal for fungal examination in combating COVID-19 infection

CHEN Wei, ZHANG Xiao-mei, WAN Zhe, LI Ruo-yu, YU Jin, WANG Ai-ping, LIU Wei

2020, 15(1):  6-9. 

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With the outbreak and spread of the COVID-19 infection, biosafety in clinical laboratory has become increasingly important. Based on Chinese National Recommendations and relevant guidelines, we propose this document about biosafety when fungal examination. This proposal consists of applicability, safety principles, personal precaution, specimen collection, specimen transport, specimen handling, analysis, post-analysis and occupational exposure, and gives recommendations on biosafety precaution and biosafety protection. We hope that it can be practically implemented and continuously improved to ensure laboratory safety.

Original articles

Epidemiology and antifungal susceptibilities of yeast isolates causing invasive infections

FAN Xin, GUO Li-na, YANG Yang, ZHANG Ge, DUAN Si-meng, ZHANG Jing-jia, XU Ying-chun, YANG Chun-xia

2020, 15(1):  10-14. 

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Objective To investigate the species distribution and antifungal susceptibility profiles of yeast isolates causing invasive infections in Beijing Chao-Yang Hospital. Methods All the duplicated yeast isolates causing invasive infection were collected from January 2012 to December 2013 and January 2016 to December 2017. All the isolates correctly identified by MALDI-TOF MS supplemented by ITS region amplification and sequencing. Antifungal susceptibility testing to seven common agents was performed according to the CLSI broth microdilution method. Results A total of 398 yeast meet the enrollment criteria were collected and the majority was Candida spp. (n=383,96.2%). Among the Candida spp., C. albicans (n=202) was still the most common species. C. tropicalis and C. glabrata was ranked second and third position, respectively. Although the C. albicans was predominant species, all isolates were 100% susceptible to seven common antifungal agents. Six azole resistant isolates detected in this study were 3 C. tropicalis isolates, 1 C. glabrata isolate and 1 C. parapsilosis isolate. For micafungin, only 1 C. glabrata intermediate and 1 resistant isolate were detected. For amphotericin B, the MIC of all the isolates in this study were ≤ 2 μ g/mL, but for 5-flucytosine, two C. albicans isolates which MIC>64 μ g/mL were detected. Conclusion The azole resistance of common Candida spp. isolated in our hospital was lower than the level of Beijing and national surveillance data. But further detection of FKS gene mutation was needed to verify the echinocandins resistance in C. glabrata detected in this study.

Clinical distribution of yeast infection in aseptic parts of patients with liver disease and sensitivity analysis of antifungal drugs

ZHANG Jing-xia, CUI En-bo, ZHANG Ju-ling, GUO Li-na, XU Ying-chun, QU Fen

2020, 15(1):  15-21. 

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Objective Invasive yeast infection is closely related to the infection rate of patients with low immunity such as intensive care unit and organ transplantation. This study analyzed the clinical distribution characteristics of yeast infection in the sterile site, and analyzed the in vitro antifungal activity of seven drugs. Methods The minimum inhibitory concentration (MIC) of 188 yeast strains collected from the 302th hospital of PLA from 2012 to 2017 was determined. The clinical distribution characteristics of 188 yeasts were analyzed. Results Candida albicans (45.7%) is the main yeast causing infection in the aseptic site of patients with liver disease, followed by Candida tropicalis (18.1%) and Candida glabrata (16.0%), and the infected site is mainly ascites fluid (39.9%); Mainly between 50 and 65 years old (43.6%), and mainly patients were male (70.7%). Candida glabrata is the mainly non-Candida albican that causes infection in the age of 50-65 years; drug sensitivity test demonstrated that the MIC₅₀ of itraconazole against Candida albicans was 0.25 μ g/mL and MIC₉₀ was 0.5 μ g/mL, while the MIC₅₀ of caspofungin to Candida krusei was 0.25 μ g/mL while MIC₉₀ reached at 0.5 μ g/mL except for Candida albicans on itraconazole the sensitivity is low (19.8%) and the sensitivity of Candida krusei to the caspofungin is low (33.3%), triazole drugs, echinocanins and 5-fluorocytosine have high in vitro antifungal activity against yeast. Conclusion Candida albicans was still the main strain of invasive yeast infection. The infected patients are mainly male. The antifungal drugs have different antifungal activities against Candida albicans and non-Candida albicans. The antifungal drugs should be selected reasonably.

Optimization of rapid detection method for sporotrichosis

WU Yong-zhuo, LIU Xiao-ming

2020, 15(1):  22-25. 

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Objectives A modified method for extracting DNA from Sporothrix. To study a rapid method to detect and identify sporothrix schenckii by using Species-specific oligonucleotide primer PCR techniques,and lay the foundation of molecular diagnosis for sporotrichosis. Methods Viscozyme L enzyme was used to replace of the traditional method. The species-specific oligonucleotide primer pair was used in this study. It was designed from nucleotide sequences of calmodulin gene in Sporothrix schenckii. 52 strains of Sporothrix schenckii and 6 strains of common fungus were amplified by PCR. Results Genomic DNA of Sporothrix schenckii were extracted with the amended (CTAB)method. All strains of Sporothrix schenckii showed a specific fragment of about 430bp with the species-specific primer pair. Using the same primer pair,the other species had no specific amplification. Conclusions Compared with the traditional method,higher yield and purity of genomic DNA were obtained with less contamination. The result indicted that this was a simple and highly efficient method. This method was specific,sensitive,reliable,rapid and simple for identifying Sporothrix schenckii and could be used for clinical molecular diagnosis.

Clinical analysis of 9 cases of Pneumocystis jirovecii pneumonia in adult haematology patients

GUO Kai, YUE Wen-qin, WANG Tie-gong, YANG Jian-min

2020, 15(1):  26-30. 

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Objective To discuss the clinical characteristics,risk factors, treatment methods,prognosis and prevention measures of the Pneumocystis jirovecii pneumonia (PCP) in adult hematologic disorders. Methods The clinical manifestations, laboratory tests, imaging features, treatment and outcomes of adult patients with hematology diagnosed as PCP from January 2014 to July 2019 were collected. Results In all 9 patients with PCP, 8 patients were hematologic malignancies and 7 patients were with allogeneic hematopoietic stem cell transplantation. The median time of diagnosed as PCP after transplantation was 8 months. 8 patients were associated with cytomegalovirus, 4 of which were multiple infections in all 9 cases. All cases had fever during the infection, and 7 cases were accompanied by cough and sputum production. The serum 1-3-β-D glucan was ranging from 104.3 to 1377.1 pg/mL, the median was 293.3pg/mL, higher than the reference value. Diffuse plaques or ground-glass opacity were the primary manifestation in imaging findings. Nodules or pleural effusion were occurred in severe cases. All patients were treated with TMP/SMZ combined with voriconazole or caspofungin or with caspofungin alone, 8 patients were cured and one patient died. Conclusion The PCP in adult hematologic disease patients progresses rapidly,and which is usually with multiple infections. Fever and cough were the main clinical manifestation of adult hematologic disease complicated with PCP. Bilateral patchy areas in CT findings and serum 1-3-β-D glucan concentration increases both contribute to the diagnosis of PCP. Finding Pneumocystis in the bronchoalveolar lavage fluid (BALF) is the gold standard for diagnosis. Anti-PCP prophylaxis should be given for high-risk patients. Early treatment of PCP is the key to reduce death and improve the prognosis of PCP in adult hematologic disease patients.

Study on the effect of flunixin meglumine on the biofilm formation of Candida krusei and on the combination with azoles

MA Sheng-yan, ZHOU Xue-zhang

2020, 15(1):  31-36. 

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Objective This study was designed to evaluate the antifungal activity of flunixin meglumine (FM) against Candida krusei biofilm and the antifungal effects of azole combined with flunixin meglumine (FM) in vitro. Method Candida krusei clinically isolated from cows with fungal mastitis was used as a test strain. The antifungal activity of FM against Candida krusei was preliminarily investigated by measuring the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) by microdilution method. The effects of FM on the early 1-3h adhesion phase of Candida krusei biofilm formation and the mature biofilm after 24h were determined by XTT method and effects of cell surface hydrophobicity (CSH) were determined by Water-hydrocarbon two-phase. The static antifungal activity of FM combined with antifungal azoles against Candida krusei in vitro was detected by checkerboard method using the partial FICI as the detection index. Enzyme-labeled turbidimetry was used to plot the time-kill curves of different drugs after treatment. Results The MIC of FM single drug against Candida krusei was 0.625 mg/mL; FM could reduce cell surface hydrophobicity,FM had significant inhibitory effect on Candida krusei biofilm at different growth stages (early adhesion and maturation), and its inhibitory effect was time-and concentration-dependent. The FICI of FM combined with itraconazole against Candida krusei ranged from 0.5 to 1.5, showing synergistic/additive effects. The FICI index of FM combined with ketoconazole was ranged from 0.375 to 1.25, showing a synergistic/additive effect. In the time-kill curves, the combination of FM and azoles could enhance the inhibitory effect of azoles on Candida krusei and maintain a longer action time. Conclusion FM is an effective antifungal synergist and can promote the antifungal activity of itraconazole and ketoconazole.

Epidemiology, antifungal drugs susceptibilities and prognosis of catheter-related candidemia in cardiovascular disease patients

LIU Yu-lei, LIU Mei-qing, WANG Li, YUAN Hui, GUO Li-na

2020, 15(1):  37-42. 

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Objective To analyze the pathogenic epidemiological data of intravascular catheter-associated candidemia and the prognosis of patients. Methods Laboratory and clinical data of patients with catheter-related candidemia in AnZhen hospital from 2012 to 2017 were collected, and statistical analysis was conducted on the distribution of pathogenic bacteria, sensitivity of antifungal drugs, antifungal treatment and prognosis data. Results 71 subjects were included. A total of 27 patients with catheter-related candidemia were diagnosed, 51.9%(14) of which were elderly patients. Candida albicans accounted for 48.1%(13)of all the isolates. The susceptibility rate of all isolates to antifungal drugs that commonly used on clinic was above 90%. Compared with patients that undergo no bloodstream infection,patients with catheter-related candidemia have much more decline on Barthel index(-27.8 vs -13.5, P=0.00),longer length of hospitalization stay(40.0 days vs 12.8, P=0.00),longer length of ICU stay(14.4 days vs 2.3, P=0.00),much more hours of respirator support(318.4 vs 25.9, P=0.00), and less proportion of recovery or improvement(P=0.02,Fisher's Exact Test). Conclusion Catheter-associated candidemia is mainly occur in elderly patients. Candida albicans take the most place of the pathogenic isolates. Yeast colonize on the skin or in vivo may be the main source of infection. Catheter-related candidemia can significantly affect the prognosis of patients and aggravate the burden of medical care. Reasonable preventive measures should be taken to prevent the occurrence of catheter-related candidemia.

The construction of Aspergillus fumigatus mutant library and screening of itraconazole drug-resistant mutant

LIU Jing, XU Wen, TAN Zhi-wei, LIU Yao-yao, ZHOU Ling-ling, FU Yu, LONG Nan-biao

2020, 15(1):  47-51. 

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Objective This study aim to construct a A. fumigatus mutant library and to screen the drug resistance mutant for preliminarily investigation of the mechanism of A. fumigatus drug resistance. Methods The A. fumigatus mutant library was constructed by Agrobacterium tumefaciens T-DNA insertion. For drug resistance mutant of A. fumigatus, plates that containing anti-fungal drug itraconazole were used. Results The present study obtained 1805 A. fumigatus mutants. Diagnostic PCR result showed that each tested mutant had a hygromycin marker, indicating that T-DNA were inserted into the host genome. Further, 16 strains of A. fumigatus mutants were successfully obtained by screening the mutant library using itraconazole, among which 2 exhibited obvious Amphotericin B resistance. Conclusion Sixteen A. fumigatus drug-resistant strains were obtained by construct and screening the A. fumigatus mutant library, which laid a foundation for further study on the mechanism of drug resistance of A. fumigatus.

Observation of the efficacy of luliconazole cream combined willow acid solution sprays in malassezia folliculitis

ZHANG Huan-mei

2020, 15(1):  52-54. 

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Objective To observe the therapeutic effect and safety of luliconazole cream combined willow acid solution sprays in malassezia folliculitis. Methods 102 patients with malassezia folliculitis were randomly divided into three groups. The patients in the experimental group were treated with luliconazole cream combined willow acid solution sprays, once alternately in turn each morning and evening for 4 weeks; the patients in the first control group were treated with luliconazole cream, once a day for 4 weeks; and the patients in the second control group were treated with willow acid solution sprays, twice a day for 4 weeks. The therapeutic effect and the fungus clearance rate were observed after the treatment for 4 weeks. Results The therapeutic rate of the experimental group was 88.24%,which was higher than that of the first control group, there was a significant difference between the two groups(P=0.03).There was no significant difference between the experimental group and the second control group(P=0.08).The fungus clearance rate in the experimental group(91.18%) was higher than the first control group(75%) and the second control group(80.65%),there was no significant difference among the three groups(P=0.21). Conclusion Luliconazole cream combined willow acid solution sprays is effective and safe in the treatment of malassezia folliculitis.