| Part Ⅰ Clinical analysis of pulmonary cryptococcosisObjective:To improve clinical diagnosis and treatment of pulmonary cryptococcosis(PC),clinical symptoms,laboratory examination,imaging findings,diagnosis,treatment and prognosis were analyzed.This study aimed to determine the association between clinical features,immune status,computed tomography(CT)findings,pathological findings,prognosis and serum cryptococcal antigen(CrAg)of PC.Methods:Clinical characteristics of 768 PC patients hospitalized in Shanghai Pulmonary Hospital from October 2014 to September 2020 were investigated.According to the immune status,the patients were divided into an immunocompetent group with 462 cases and an immunocompromised group with 306 cases.The information from patients’ relevant follow-up was obtained on regular clinic visits and by telephone follow-up.Of 768 cases,553 were tested for serum CrAg by Lateral flow immunoassay(LFA).Furthermore,the clinical data of those 553 cases were examined,dividing into serum LFA positive group(n=386)and serum LFA negative group(n=167).Results:(1)Among 768 cases,402 were pathologically confirmed and 366 were clinically diagnosed.The patients consisted of 485 males and 283 females,with a mean age of(50.6± 12.04)years(range,18 to 88 years),40 to 65 years old was the high incidence age.(2)49.09%PC patients were asymptomatic,all of which were found on chest CT in routine physical examination.The main symptoms were cough,expectoration,chest pain,dyspnea and fever.In the immunocompetent group,asymptomatic patients were more common,while in the immunocompromised group,symptoms and abnormal physical examination were more common.The incidence of fever,asthenia,hemoptysis,rales and positive signs of nervous system in the immuno-compromised group were significantly higher than those in the immunocompetent group(P<0.05).(3)The incidence of leukopenia and neutropenia in the immuno-compromised group was higher than that in the immunocompetent group,and the CD3+and CD4+T lymphocyte counts were lower than those in the immunocompetent group,the differences were statistically significant(P<0.05).(4)Imaging findings showed that the lesions of the 76.69%patients were located in 1/3 of the lung field,close to the pleura;the lesions were more common in the right or lower lungs,accounting for 45.83%and 46.22%respectively.Imaging findings showed solitary nodule/mass in 284,multiple nodule/mass in 140,patch consolidation in 150,mixed lesions in 176,interstitial pneumonia in 11,and diffuse miliary in 4.In the immunocompetent group,63.64%of patients showed nodular/mass shadows,which was significantly higher than that in the immunocompromised group(P<0.05),while in the immunocompromised group,53.27%of patients showed patch consolidation and mixed lesions,which was significantly higher than that in the immunocompetent group(P<0.05).The incidence of lesions with cavitation,halo sign and pleural effusion in immunocompromised group was higher than that in immunocompetent group(P<0.05).PET-CT examination showed that the SUVmax of patch consolidation and mixed lesion pattern was higher than that of solitary nodule/mass pattern(P<0.05).(5)Among the 402 cases with PC confirmed by pathology,354 cases were non-caseous granuloma and 48 cases were inflammatory consolidation.The incidence of inflammatory consolidation was higher in the immunocompromised group than in the immunocompetent group(P<0.05).(6)Of the 768 PC patients,379 were treated with antifungal drugs alone,144 received surgical resection alone,237 received surgery combined with postoperative antifungal drug therapy,and 8 received immunomodulatory therapy.Up to October 31,2021,except for 15 patients who were still treated with antifungal drugs,366 were cured,298 were improved,61 were stable,and 28 were deteriorated.Fluconazole resistance rate was 6.35%.(7)Of 768 cases,the positive rates of LFA in serum,BALF,lung puncture fluid,were 69.8%,90.91%,and 93.33%,respectively.Of 11 PC patients with cryptococcal meningitis,the positive rates of LFA in cerebrospinal fluid were 100%.The positive rate in the immunocompromised group was higher than that in the immunocompetent group(P<0.05).(8)The proportion of patients with underlying diseases or immune impairments in LFA-positive group was respectively 55.44%and 57.25%,which were significantly higher than those in LFA-negative group(P<0.05).Compared with LFA-negative group,the median CD4+T cell count and CD4+/CD8+ratio in LFApositive group were lower(P<0.05).The positive rate of serum LFA and geometric mean of titer in the immunocompromised group were higher than those in the immunocompetent group(P<0.05).(9)66.47%of LFA-negative patients were asymptomatic,while 70.47%of LFA-positive patients had cough,expectoration,chest pain,fever and other symptoms.(10)Imaging findings of 65.80%serum positive LFA patients showed patch consolidation or mixed lesions,while those of 77.84%cases with serum negative LFA showed nodular mass.The incidence of pleural effusion,halo sign,cavitation and bronchial inflation was greater in the LFApositive group than in the LFA-negative group.The geometric mean values of CrAg titers in PC patients presenting with solitary nodule/mass shadow,multiple nodule/mass shadow,patch consolidation shadow,mixed lesion,interstitial pneumonia,and diffuse miliary shadow were 1:2.48,1:10.7,1:33.04,1:14.26,1:2032.36,1:1279.38,respectively.Serum CrAg titers in patients with nodular masses were lower than that in the other types of lesions(P<0.05).(11)The effective rate and deterioration rate of serum LFA negative group and serum LFA positive group were 89.31%,3.14%and 87.06%,4.85%,respectively.The difference between the two groups was statistically significant(P<0.05).(12)Serum LFA were re-detected in 375 PC patients during treatment.Serum CrAg titer of 89 patients were negative before treatment.Serum CrAg titers of 286 patients ranged from 1:5 to 1:2560 before treatment,with a geometric mean of 1:42.85.Up to the time of treatment cessation,the serum CrAg titer became negative in 240 cases and still positive in 46 cases with the titer ranged from 1:1 to 1:40.The time of turning negative in immunocompromised cases was longer than that in immunocompetent cases,and the rate of turning negative was less than that in immunocompetent cases(P<0.05).Conclusions:(1)PC is mostly found in middle-aged and elderly men without immunosuppression or underlying diseases.Clinical manifestations and imaging features of PC were diverse,and it was easy to be missed and misdiagnosed.(2)The detection of serum CrAg by LFA can be used for the diagnosis of PC.Once PC is suspected,the clinician should detect CrAg by LFA in serum or other specimens as soon as possible.If LFA test is negative,TBLB or PNLB should be selected for diagnosis according to the imaging characteristics.(3)PC patients with serum LFA negative who are difficultly diagnosed by TBLB or PNLB,are mostly treated with surgery,surgical resection and adequate course of antifungal therapy can achieve good results,and triazole drugs are highly effective in the treatment.If fluconazole resistance occurs,voriconazole can be replaced.(4)Serum CrAg test results were found to be associated with the clinical manifestations,immune status,CT findings,pathological features,curative effect and prognosis of PC patients.(5)After antifungal therapy,the serum LFA test results of some PC patients could still be positive with low titer for a long time.So,drug withdrawal should be comprehensively evaluated according to the completion of the antifungal treatment,Clinical symptoms,immune status and the results of chest CT.Part Ⅱ Expression of PD-1/PD-L1 in pulmonary cryptococcosisObjective:To investigate the role of PD-1/PD-L1 signaling pathway in the pathogenesis of PC by comparing the expression of programmed death molecule1(PD-1)and its ligand(PD-L1)in serum,bronchoalveolar lavage fluid and tissues of patients with PC.Methods:Peripheral venous blood was collected from 45 PC patients(experimental group)and 10 healthy controls(control group)in Shanghai Pulmonary Hospital from January 2019 to September 2020.Serum concentrations of PD-1 and PD-L1 in the two groups were detected by ELISA.BALF of 20 PC patients(experimental group)and 5 chronic cough patients(control group)hospitalized in Shanghai Pulmonary Hospital from January 2019 to September 2020 were collected,and the concentrations of PD-1 and PD-L1 in BALF of the two groups were detected by ELISA.The pathological tissues of 25 PC patients undergoing thoracic surgery(experimental group)and the normal lung tissues of 5 patients(control group)were collected.After immunohistochemical preparation,digital tissue section scanner was used to collect images.The expression of PD-1 and PD-L1 in lung tissues of the two groups was analyzed by using the Servicebio image analysis system.Results:(1)The median serum concentrations of PD-1 and PD-L1 in PC patients and healthy controls were 97.49pg/ml,0.024pg/ml and 62.78pg/ml,0.016pg/ml,respectively,which were higher in PC patients than in controls(P<0.05).(2)The median BALF concentrations of PD-1 and PD-L1 in PC patients and chronic cough controls were 132.97pg/ml,0.049pg/ml and 74.77pg/ml,0.046pg/ml,respectively.The PD-1 concentrations of BALF in PC patients were higher than those in controls(P<0.05).(3)The positive cell ratio,H-Score and positive score of PD-1 in the lesion tissue of PC patients were higher than those of normal lung tissue(P<0.05).The positive cell ratio,positive cell density,H-Score and positive score of PD-L1 in the lesion tissue of PC patients were higher than those of normal lung tissue(P<0.05).Conclusions:The expression of PD-1/PD-L1 was up-regulated in serum,BALF and lesion tissue of PC patients,which indicated that PD-1/PD-L1 signaling pathway may be involved in the immune process of infection.In the future,drugs that intervene in PD-1/PD-L1 signaling pathway can be developed for the clinical treatment of refractory PC. |
