Value Evaluation Of Real-time Fluorescence PCR For The Rapid Diagnosis Of Sporotrichosis In Clinical Settings

Background:Sporotrichosis is a chronic or subacute deep fungal disease caused by the Sporothrix complex affecting the skin,subcutaneous tissue,mucous membranes and nearby lymphatic system.The mycosis is found all over the world,with an overall prevalence of up to 2.5 percent in the subtropics and tropics,and a particularly high incidence in our country’s northeast.Sporotrichosis is a zoonotic disease and the species that could infecting humans are these followings: Sporothrix globosa,Sporothrix brasiliensis,Sporothrix Schenckii,Sporothrix Mexicanica and Sporothrix rueri.Sporothrix globosa is the most prevalent species in China,according to the current research;Sporothrix Mexicanica and Sporothrix brasiliensis are found predominantly in Mexico and Brazil;while Sporothrix Schenckii is widely distributed worldwide.Because of the low infectivity and lethality,it has not attracted much attention worldwide for the rapid diagnosis of sporotrichosis.Culture is still the gold standard for diagnosing sporomycosis,however because of the long culture period,it takes 2 weeks to receive results,and the results are easily influenced by other bacterias and false negatives,it is not appropriate for rapid clinical diagnosis and epidemiological inquiry.The focus of this research is to use clinical specimens to investigate the feasibility of using animal model experiments in the clinical and to assess the technology value in the clinical for the fast diagnosis of patients with S.globosa.As a result,a highly sensitive and specific approach for the quick detection of sporotrichosis will be developed.Objective:This study will provide a novel technology for the fast diagnosis of Sporotrichosis by establishing a real-time PCR technology according to the internal transcribed spacer（ITS）sequence for the genome of S.globosa,and to evaluate the sensitivity and specificity of this technology in the clinical.Methods:From December 2020 to December 2021,formalin-fixed and paraffin-embedded（FFPE）tissues and pathological tissues from patients with sporotrichosis and other patients other than sporotrichosis who were diagnosed by both histopathological examination and fungal culture were collected in the Dermatology department of the Second Hospital of Jilin University.The samples were separated into two parts,each with 32 cases.The first group（group A）consisted of FFPE tissues and the second group（group B）consisted of freshly drilled pathological tissues.Besides,each group contained 32 specimens other than sporotrichosis as negative controls.Extracted nucleic acids from the samples,and conducted q PCR amplification reactions with particular primers and probes.The number of effective amplification cases of the experiments in groups A and B were recorded,and the specificity sensitivity of q PCR reactions were computed to evaluate the efficacy of this method in the rapid diagnosis of S.globosa in clinical practice.Results:Group A: 23 cases of effective amplification in the positive control group and 2 case of effective amplification in the negative control group,with a sensitivity of 71.88% and specificity of 93.75%.The statistics of g PCR test and gold standard diagnosis were generally consistent（kappa=0.839,P < 0.001）.Group B: 29 cases of effective amplification in the positive control group and 1 case of effective amplification in the negative control group,with a sensitivity of 90.63% and specificity of 96.88%.The statistics of q PCR test and gold standard diagnosis were highly consistent（kappa=0.945,P <0.001）The diagnostic performance of Group B is better than Group A in both consistency tests and ROC curves.Conclusions:1.The sensitivity of the q PCR method in fresh pathological tissues was 90.63% and the specificity was 96.88%;however,the sensitivity and specificity in FFPE were 71.88% and 93.75%,which were significantly lower than those in the fresh pathological tissue group.The application of this technique in fresh tissues has more diagnostic value.2.Using q PCR,it takes only about 4 hours from the taking of a patient sample to a diagnostic result,compared to 2 weeks or more for the traditional gold standard.Therefore,the application of q PCR method from fresh tissue has the advantages of short time consumption,high sensitivity and high specificity,providing a basis for rapid diagnosis,and is expected to be promoted in clinical practice.