The Correlation Of Pneumocystis Jirovecii With Respiratory Diseases And Its Molecular Epidemiology

Objective:Pneumocystis is an opportunistic pathogenic fungi with multiple and widespread hosts,which is commonly found in the lung tissue from human and many other kinds of animals.It is widely distributed around the world and the culture of Pneumocystis is very difficult.However,Pneumocystis has high host specificity.Pneumocystis infected can cause severe Pneumocystis pneumonia（Pneumocystis jirovecii Pneumonia,PJP or Pneumocystis pneumonia,PCP）,PJP/PCP primarily occur among immunocompromised individuals,congenital or acquired,including paitents infected by HIV（Human immunodeficiency virus,HIV）,malignant tumor patients who received chemotherapy or radiotherapy,organ transplant recipient and other patients with immunocompromised diseases.In recent years,the incidence of PJP/PCP is increasing year by year annually among patients infected without HIV,such as IBD（Inflammatory bowel disease）,IPA（Invasive pulmonary aspergillosis）,AID（Autoimmune disease）.In addition,the phenomenon of P.jirovecii colonization commonly occurs in immunocompetent patients and individuals,especially in those with pulmonary diseases such as COPD（chronic obstructive pulmonary disease）.Some scholars conclude that P.jirovecii colonization might be associated with the occurrence and development of respiratory system diseases especially in COPD.However,the correlation between them needs further confirmation and investigation.The further study is of great significance for exploring the pathogenesis,prevention and controll strategies and therapeutic measures of respiratory diseases such as COPD.Once PJP/PCP has occurs,the development of the disease is rapid and the mortality is extremely high.Although the status of P.jirovecii colonization is lacking the specific clinical manifestations,P.jirovecii colonization is the more important source of infection with highly infectious,which is also the most important risk factors for nosocomial infections.Therefore,promptly diagnosis of the patients with PJP and carriers with P.jirovecii,and the molecular epidemiological study are of great significance for tracing the infection sources,preventing the transmission of PJP and controlling the nosocomial infections.We established a new method to detecting the presence of P.jirovecii gene and detected the presence of P.jirovecii gene in respiratory secretions from patients with COPD and other respiratory diseases.Then the positive presence of the gene were analysed by sequencing and typing.The positive rate and the molecular epidemiologic characteristics were investigated in this study.PCP animal model were established and the clinical data were collected.Histopathological characteristics,counts of the inflammatory cells and the concentration variations of inflammatory factors in lung tissues which were caused by infected colonized Pneumocystis were monitored.On that basis,we analyzed and studied the pathogenicity of Pneumocystis and the correlation between Pneumocystis and respiratory diseases such as COPD.The study results provided the more advanced methods for clinically detecting the colonization of P.jirovecii and diagnosing PJP/PCP,the reliable data for molecular epidemiology of P.jirovecii,the new train of thoughts to the study in pathogenesy of P.jirovecii,and the new approaches for the pathogenesis preventive and curative strategies for COPD.Methods 1.Establishment of Nested PCR system and its specific and sensitive detections Nested PCR system for detecting the P.jirovecii mt LSU rRNA gene was established.The specific detection was compared with other common respiratory pathogens including bacteria and fungi respectively.The sensitive detections were performed by using gradient dilution of plasmids（mt LSU-pMD18T）constructed with mt LSU target genes.2.Establishment of real-time fluorescence LAMP and its specific and sensitive detections LAMP（Loop-Mediated Isothermal Amplification）for detecting the presence of P.jirovecii 18s rRNA gene was established and optimized.And on this base,real-time fluorescence LAMP was further developed and then the system was optimized.The specific detections were compared with other common respiratory pathogens including bacteria and fungi respectively.The sensitive detections were performed by using gradient dilution of plasmids（18S rRNA-pMD18T）constructed with 18S rRNA target genes.3.Clinical studies of the correlations between P.jirovecii and respiratory diseases The respiratory secretions from patients with respiratory diseases were collected.P.jirovecii cysts were detected with Giemsa staining method and GMS（Gomori’s methenamine silver nitrate）staining method.The colonization of P.jirovecii genes in respiratory secretions from patients with respiratory diseases were tested by PCR,Nested PCR and real-time fluorescence LAMP mentioned above.According to the positive rates,the molecular epidemiological characteristics of the role of P.jirovecii in the patients with respiratory diseases were analysed.All clinical data of the patients were collected and analysed to study the correlations between P.jirovecii and respiratory diseases.4.Genotyping of P.jirovecii ITS and related analysis of molecular epidemiological The respiratory secretions,which were from the positive presence of P.jirovecii genes tested by the methods described above,were amplified,cloned,sequenced and analyzed.ITS（ITS1-5.8S rRNA-ITS2）was used as the target gene.Then sequences we acquired were blasted and compared with the genotypes which had been reported or published in the GenBank database.The genotype diversity were analysed in the isolated P.jirovecii from different regions in China.Our study can expect to provide reliable information for characteristics of genotypes distribution and provide important data for molecular epidemiology of P.jirovecii,establish the theoretical foundation for deducing the infection reservoirs and transmission mechanism of P.jirovecii,and furthermore,provide significant reference values for the prophylaxis and treatment of PJP/PCP.5.The experimental studies of the Pneumocystis pathogenicity and the correlations with COPD To study the pathogenicity of Pneumocystis and its role in the pathogenesis and development of COPD,PCP rat model was established.The histopathologically sections of lung tissue from PCP rats were made,which were observed to diagnose whether there were similar pathological characteristics to the COPD.Counts of different inflammatory cells in BALF（bronchoalveolar lavage fluid）and splenic tissues were detected by FCM（flow cytometry）.The expressions of mRNA of MMPs（matrix metalloproteinases）（including MMP-2,MMP-8,MMP-9 and MMP-12）and HSP-27（Heat Shock Proteins 27）,which were intimately related to COPD,were detected by RT-qPCR.The expressions of MMPs and HSP-27 proteins were tested by IHC（immunohistochemical method）,ELISA（enzyme-linked immuno sorbent assay）and Western Blot.The activities of MMP-2 and MMP-9 were detected by gelatin zymography.Results:1.Results of Nested PCR Nested PCR system for detecting the colonization of P.jirovecii mt LSU rRNA gene was successfully established.Specific identifications were detected and compared between Pneumocystis and other common respiratory fungi and bacteria.We found that other common respiratory fungi and bacteria were all negative except Pneumocystis,which indicated that Nested PCR assay we established had high specificity.The constructed plasmids with mt LSU-pMD18T were diluted gradiently for the sensitivity test.We found that the lowest detection limits of PCR and Nested PCR were 10⁴ copies/μl and 10² copies/μl respectively,which means that the detection sensitivity of Nested PCR was 100 times higher than conventional PCR.Our study suggested that Nested PCR system we established had higher sensitivity.2.Results of real-time fluorescence LAMP LAMP and real-time fluorescence LAMP for detecting the presence of P.jirovecii 18S rRNA gene were successfully established.Specific identifications of LAMP and real-time fluorescence LAMP were respectively detected and compared between Pneumocystis and other common respiratory fungi and bacteria.We found that other common respiratory fungi and bacteria were all negative except Pneumocystis,which indicated that both LAMP and real-time fluorescence LAMP we established had high specificity.The constructed plasmids with 18S rRNA-pMD18T were diluted gradiently for the sensitivity test.We found that the lowest detection limits of PCR,LAMP and real-time fluorescence LAMP were 10⁴ copies/μl,50 copies/μl and10 copies/μl respectively.Our study suggested that the detection sensitivities of LAMP and real-time fluorescence LAMP we established had higher sensitivities.3.Results of Clinical studies on the correlations between P.jirovecii and respiratory diseasesRespiratory secretions from 403 cases of HIV negative patients with pulmonary diseases were collected,which were tested respectively by PCR,Nested PCR and real-time fluorescence LAMP.The gene positive rates of them were 40.45%（163/403）,58.56%（236/403）and 69.73%（281/403）,respectively.P.jirovecii cysts in only one case was detected by Giemsa staining and GMS（Gomori’s methenamine silver nitrate）staining.The consistency of the gene positive rate between conventional PCR and real-time fluorescence LAMP was 100%.The consistency of the gene positive rate between Nested PCR and real-time fluorescence LAMP was 87.59%.The clinical data from P.jirovecii gene positive cases of HIV negative patients with pulmonary diseases were collected.The results of statistical analysis from clinical parameters showed that P.jirovecii colonizations or infections in HIV negative patients with pulmonary diseases were closely correlated with 1,3-β-D-Glucose（BDG）,CRP（C-Reactive Protein）,counts of CD₄⁺T lymphocytes,HRCT（high-resolution computed tomography）and FEV1/FVC of lung function tests,especially HRCT and FEV1/FVC,which indicated certain correlation existed between P.jirovecii and the pathogenesis and development of pulmonary diseases such as COPD.4.Results of P.jirovecii ITS genotyping and related analysis of molecular epidemiological Respiratory secretions from 403 cases with the positive presence of P.jirovecii gene mentioned above were amplified by Nested PCR and the target gene was ITS1-5.8S rRNA-ITS2 gene.There were 25 cases whose sequences were in conformity with the analysis of genotypes of P.jirovecii ITS gene.These specimens were cloned,screened and sequenced.139 sequences were acquired.According to the characteristics of ITS1-5.8S rRNA-ITS2 gene structure and its standard for nomenclature and classification,the sequences we acquired were analyzed by homology comparison.19genotypes of P.jirovecii ITS gene were acquired incuding 5 genotypes previous reported and 14 new genotypes found in this study.Bi type was the the most frequent genotype.The Bi type and Eb type respectively were the most common genotypes in northeast China region and north China region.Eb type appeared both in cases from northeast China region and north China region.5.Results of experimental studies of the Pneumocystis pathogenicity and the correlations with COPD（1）Histopathological changes of lung tissue from PCP rats:The degree of histopathological changes varied somewhat in lungs of the different PCP rats.The fusion even destruction of alveolus cavity and ruptures of alveolar walls were observed.The thickened alveoli septum and diffuse pulmonary interstitial tissue hyperplasia were observed.The lesions were also embodied in red foamy alveolar exudates.Interstitial edema and inflammatory cell infiltrated primarily including lymphocytes and macrophages were observed in pulmonary interstitial tissue.The lung tissue from the most serious rat was markedly atelectatic and consolidation.All the histopathological changes were exactly similar to the reported COPD rats induced by cigarette smoking.（2）Results of flow cytometry:Compared with the control group,counts of CD₈⁺T lymphocytes（P<0.01）,M1 macrophages（P<0.01）and granulocytes（P<0.01）in BALF and splenic tissue from PCP group were significantly increased respectively,counts of CD₄⁺T lymphocytes were drastically reduced（P<0.01）,with statistically significant differences.（3）Results of the COPD-correlated cytokines in rats PCP model:Compared with the normal control group,the mRNA levels and protein levels of MMPs and HSP-27 levels in serum,BALF and lung tissues respectively were significantly higher in the PCP model group（P<0.01）by RT-qPCR,IHC,ELISA and Western Blot.The activites of MMP-2 and MMP-9 were higher in the PCP model group（P<0.01）by gelatin zymography.Conclusion:1.Nested PCR for P.jirovecii mt LSU rRNA gene with higher specificity and sensibility was established.Respiratory secretions from HIV negative patients with pulmonary diseases were detected by Nested PCR with higher positive rate of P.jirovecii mt LSU rRNA gene presence（58.56%）.2.Real-time fluorescence LAMP with higher specificity and sensibility for detecting the presence of P.jirovecii 18S rRNA gene was established,which was applied to determine the presence of P.jirovecii gene in respiratory secretions from clinical patients.The positive rate of P.jirovecii 18S rRNA gene presence in HIV negative patients with pulmonary diseases was higher（69.73%）.3.P.jirovecii colonizations or infections in HIV negative patients with pulmonary diseases were closely correlated with the elevated levels of BDG and CRP,the reduced levels of CD₄⁺T lymphocytes counts,GGO imaging manifestations of HRCT and the reduced levels of FEV1/FVC of lung function tests,especially GGO imaging manifestations of HRCT and the reduced levels of FEV1/FVC.4.The genotypes of P.jirovecii ITS gene were different from different geographical areas in China.There was also similarity of P.jirovecii ITS gene in northeast China region and north China region.Genetic diversity of P.jirovecii ITS gene existed in China.5.Pneumocystis colonization or infection was positively associated with the expressions of cytokines associated with the development of COPD.The correlation might exist between them.