| Objectives1.The recombinase polymerase amplification assays was developed for rapid,specific and sensitive detection of C.auris,and also applied to the differential diagnosis of common clinical candida.2.To investigate the strain characteristics of C.auris and an epidemiological investigation was conducted on the infection of C.auris among inpatients in a top third-class hospital in Guangzhou.3.To investigate the different efficacies of common disinfection methods used in medical institutions against C.auris and other Candida species.Method1.The fragment of 5.8S,all of ITS2 rRNA genes of Candida species were used as the target sequence to design the C.auris specific primers and the ITS region for Candida universal primer.The analytical sensitivities of the RPA assay and the PCR assay were determined by testing 10-fold serial dilutions of standard C.auris DNA(CBS12766)ranging from 100 to 106 copies/reaction.A total of 65 isolates including three C.auris,sixty-one clinical common yeast isolates and four bacterial isolates were tested to evaluate the specificity of the RPA assay.Blood spiking experiments were used to simulate candidemia to evaluate the value of RPA in clinical rapid diagnosis.C.albicans,C.tropicalis,C.glabrata,C.parapsilosis,C.krusei,and C.auris were evaluated for the RPA detection method for universal identification of Candida.2.Taking C.albicans as a reference,the growth of C.auris and C.albicans under conventional culture conditions,different pH values and salt concentrations were measured and compared.The fermentation performance of two Candida species were determined by microbiochemical reaction tube.Candida specimens were collected from a top third-class hospital in Guangzhou.The internal transcribed spacer(ITS)and D1/D2 regions of the C.auris were amplified by PCR and the amplified products were sequenced to identify the species.3.The minimum inhibitory concentration of three disinfectant products("84"disinfectant,IodineTincture disinfectant,and quaternary ammonium)and 75%ethanol against C.auris and other Candida species were measured.A pig skin model was used to evaluate the efficacy of three hand hygiene products in killing pathogens.The killing effect of ultraviolet-C(253.7 nm)and the LK/CXD bed unit ozone disinfection machine on C.auris was also evaluated.Results1.The length of C.auris-specific primers designed in this study was 30 bp,and amplified a 163-bp-long RPA product.Our RPA approach was proved to reliably identify all of the tested C.auris strains,distinguishing these isolates from other strains with a specificity of 100%.Our assay was able to detect a template provided atconcentrations 103 copies/reaction,which was the same as that of PCR.Moreover,the results were obtained within a short time,20 minutes,which was much less than the 2 hours needed for PCR detection.The results of direct detected by RPA and PCR in blood sample,showed that the detection limits of RPA and PCR were 1.02 x 103 CFU/ml and 1.02 × 104CFU/ml,respectively.2.The length of Candida universal primer designed in this study is 30bp,which can realize the preliminary differential diagnosis between five common clinical Candida species and C.auris,by amplifying different sizes of products.In the mixed infection experiment,two kinds of easily distinguishable bands can be produced in the case of the ratio of C.albicans to C.auris(5:1,1:1 and 1:5).3.The logarithmic growth period of C.auris was about 8-24 hours,and entered the stable growth period after 24 hours.The optimum pH range of growth was 5-7,and the growth mode was the same as that of C.albicans.C.auris grew better than C.albicans in NaCl medium with 5%and 10%concentration,and could ferment glucose,sucrose,trehalose and sorbitol.4.A total of 1637 strains of Candida were detected in this study.No C.auris was isolated from hospitalized patients.5.Thirty seconds of pig skin washing with bacteriostatic hand sanitizer followed by drying and 15 s of ethanol-based gel can completely eradicate the colonization of C.auris(3.00 log10 CFU).The antifungal activity of ultraviolet-C to C.auris inoculated on bed sheets was significantly reduced(P<0.01)at a distance of 1 meter.C.glabrata and C.auris showed greater resistance to ozone than other Candida species.The ozone could completely eradicate C.auris(3.60 log10 CFU)on bed sheets at dosage of 300 mg/m3 for 40 minutes of exposure.Conclusion1.We have developed an simple,rapid and reliable RPA detection method,which can be used for molecular diagnosis and differential diagnosis of C.auris,and can directly detect C.auris in blood samples.Its advantages are more suitable for clinical use in remote areas and laboratories with poor economic conditions.2.At present,the infection rate of C.auris in China is low,and C.auris is the most suitable for growth under weak acid or neutral conditions and has high salt tolerance.3.We recommend extending the disinfection times of ultraviolet-C and oz one and emphasizing the effectiveness of washing skin with soap,drying skin,and then applying an ethanol-based gel to remove C.auris from skin. |
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