Development Of Droplet Digital PCR-bases Assay For Detection Of Fungi In Respiratory Tract And Its Clinical Practice

Fungi are common and diverse heterotrophic eukaryotes in the nature world,there are more than 300 species are able to affect human health.The main infection caused by fungi is devided into superficial infection(e.g.fungal dermatosis)and deep infection(or invasive fungal infection).There are about 1.7 billion people worldwide affected by superficial fungal infections without life-threatening.On the contrary.Invasive fungal infection has high mortality,For example,the delay in diagnosis and treatment of invasive aspergillosis lead to a mortality rate of nearly 100%in some group.Invasive fungal disease cause 1.5 million people death annually,the increase of immunosuppressive population(such as HIV,hematopathy,organ transplant patients,etc)and invasive medical intervention are the main reasons for the rising trend of invasive fungal infection.Lung is the most common affected organ of deep fungal infection.Therefore,it has high clinical application value to rapid and early detection of common pathogenic fungi in respiratory tract.Candida and Aspergillus are the most common opportunistic infection fungal in respiratory tract,it is the first time that the sputum samples were used as the test materials to development of detection method for multi-fungal target(Candida and Aspergillus)in respiratory tract based on Droplet digital PCR in this study,then the clinical application of this method in diagnosis of pulmonary aspergillosis was studied.The results showed that this method has high sensitivity,good specificity and robust clinical applicability,it is a promising method for rapid and accurate detection of common clinical respiratory fungi.To date,the new methods for the detection of pulmonary fungal infection mainly include G test,GM test,mass spectrometry,sequencing and PCR,the sample are used in test were blood,bronchoalveolar lavage fluid,brush biopsy and lung tissue,however,all of this samples requires invasive procedure to obtain and may injure patients.Sputum is a non-sterile sample that can obtained without invasive procedure,at present,there are few reports on the detection of fungal infection on sputum samples.one of the reason is the complexity.Eight common respiratory fungi(Candida albicans,Candida tropicalis,Candida glabrata,Candida parapsilosis,Aspergillus fumigatus,Aspergillus flavus,Aspergillus niger and Aspergillus terreus)were detected and validated based on sputum samples and ddPCR.The results show that the method has a high analytical sensitivity and all of fungi target in this assay was linear from 2 to 20000 copies with R2>0.99,Primer and probe set show a good specificity that did not cross-react with other Bacteria,Fungi and Homo sapiens.And all of the clinical Candida or Aspergillus isolates were detected by this method.The mock sputum sample which only contain 5 CFU aspergillus spore(A.fumigatus or A.flavus)also can be detected effectively by this method.Further,Among 29 sputum samples were tested by ddPCR,the consistency of this method and culture was 96.5%.one of culture false negative sputum sample was detected by ddPCR and multi-positive results were found in 7 samples.Finally,we used Next-generation sequencing to verify these results,ddPCR were proved to be accurate and reliable.Subsequently,ddPCR was used for the first time in the diagnosis of pulmonary aspergillosis.Pulmonary aspergillosis is a serious disease caused by aspergillosis infection,many studies have shown that the aspergillus load in sample is related to infection status,but there is no standard for quantification of aspergillus load with few reports.ddPCR is absolute quantification method that can analyze complex sample,it can accrate quantification of the aspergillus load in sputum samples.Four target aspergillus species(A.fumigatus.A.flavus,A.niger and A.terreus)which can cover more than 90%of clinical isolates were selected for detection in this study.First the 18 pulmonary aspergillosis(PA)group patients and 99 non-pulmonary aspergillosis(non-PA)group were analyzed with clinical characteristics and risk factors for pulmonary aspergillosis.The ddPCR results of 160 sputum samples presented that the detection rate of aspergillus was 84.2%and 8.2%in PA group and No-group respectively,the main detection of aspergillus is A.fumigatus.Then the PA group were further divided into included or excluded allergic bronchopulmonary aspergillosis(ABPA)patients to analyzed by receiver-operating Characteristic curve(ROC).The results show that the sensitivity and specificity were 81.6%and 95.1%respectively in ABPA-included PA group when the detection threshold is 20.2 copies,and in ABPA-excluded PA group,the sensitivity and specificity were 94.7%and 99.2%respectively when the threshold is 39.9 copies,Both of the Area Under Curve(AUC)were great than 0.9.it means that the method has high diagnostic value for PA.Finally,ddPCR results of 17 sputum samples which were collected at different time points from 4 PA patients were used for monitoring of PA,the results suggest that the change of ddPCR detection results was consistent with the progress or improvement trend of patients,condition.In this study,a non-invasive,accurate,rapid and reliable detection method for fungi in respiratory tract was development based on ddPCR platform and sputum sample.This method can not only accurately identify trace amounts of common fungi in respiratory tract,but also effectively detect each fungal target in compound infection samples with high sensitivity,specificity and detection rate,which can be used for the early diagnosis of pulmonary fungal infection.This dectection method can be used as a robust tool for the determination of the accurate fungal load in each samples.The results suggest that ddPCR can be used for the diagnosis of pulmonary aspergillosis,and it is expected to provide a reliable solution for monitoring pulmonary aspergillosis patients condition in the future.