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Pneumocystis , an important opportunistic fungal pathogen that parasitizes in multiple mammalian lungs, may cause life-threatening Pneumocystis pneumonia (PCP) and even death among immunocompromised individuals. With the rapid development of high-throughput sequencing and multi-omics technologies, systematic comparative analyses of genome, transcriptome, and whole-genome sequencing results demonstrate that Pneumocystis is a type of obligate biotrophic fungi, and requires obtaining nutrition from hosts. In addition, sexual reproduction is an essential process for Pneumocystis survival, production and transmission, and asexual reproduction facilitates Pneumocystis survival, which provides new insights into understanding of the whole developmental process of Pneumocystis in the host lung and inter-host transmission of Pneumocystis . This review summarizes the advances in the reproduction mode of Pneumocystis and underlying mechanisms, which provides insights into prevention and treatment of PCP, notably for the prophylaxis against nosocomial transmission of PCP.

Objective: To investigate the pathogenicity of Pneumocystis and its association with the development of chronic obstructive pulmonary disease (COPD)., Methods: The rat model of Pneumocystis pneumonia (PCP) was induced by intraperitoneal injection with dexamethasone, which was confirmed by pathogenic detection. The pathologic changes of rat lung specimens were examined using conventional HE staining, and the expression of inflammatory cells were detected by flow cytometry in bron-choalveolar lavage fluid (BALF) and splenic tissues of the rat model of PCP. In addition, the serum levels of matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured using enzyme-linked immunosorbent assay (ELISA)., Results: Fusion and atrophy of alveolar spaces and hyperplasia of lung tissue were seen in the lung specimens of the rat model of PCP, and foam-like alveolar exudates and infiltration of inflammation cells were observed in the alveolar space, while severe infections exhibited consolidation of lung, which was similar to pathological features of COPD. The counts of CD8 + T lymphocytes ( t = -7.920 and -12.514, P < 0.01), macrophages ( t = -7.651 and -14.590, P < 0.01) and granulocytes ( t = -10.310 and -16.578, P < 0.01) significantly increased and the counts of CD4 + T lymphocytes ( t = 6.427 and 18.579, P < 0.01) significantly reduced in the BALF and splenic specimens of the rats with PCP relative to those without PCP. In addition, higher serum MMP-8 ( t = -8.689, P < 0.01) and MMP-9 levels ( t = -7.041, P < 0.01) were measured in rats with PCP than in those without PCP., Conclusions: Pneumocystis infection may be associated with the development and progression of COPD.

Objective: To investigate the clinical manifestations and prognostic factors of hospital death in connective tissue disease patients with acute respiratory failure caused by pneumocystis pneumonia (PCP) admitted to two medical intensive care units(MICU). Methods: A retrospective review was conducted for all connective tissue disease (CTD) patients with acute respiratory failure from PCP in MICU of 2 academic medical centers between 2010 and 2015. The patients were divided into survivors and non-survivors. Demographic and clinical data, including laboratory, radiological and microbiological findings, as well as therapy, clinical course, mortality and prognostic factors of hospital mortality were included in the analysis. Logistic regression models were used to determine the effect of prognostic factors on hospital death after adjusting for covariates of which the p values were less than 0.1. Results: A total of 41 patients with connective tissue disease were identified. The PaO(2)/FiO(2) ratio (PFR) on ICU admission was 120 mmHg(55-180 mmHg, 1 mmHg=0.133 kPa). Common clinical features included dyspnea (90.2%, 37/41), fever (87.8%, 36/41) and dry cough(65.9%, 30/41). 58.5%(24/41) and 17.1%(7/41) patients were co-infected by CMV and aspergillus, respectively. The overall mortality rate was 75.6%(31/41). Compared with survivors, the age, APACHEⅡ score and incidence of barotrauma in non-survivors were higher (39±17 vs 58±15, t =3.018, P =0.002), (15±6 vs 19±5, t =2.528, P =0.019), (0/10 vs 12/31, χ(2)=5.473, P =0.021), while PFR on ICU admission was lower in non-survivors (172±68 vs 116±49, t =-1.893, P =0.007). Logistic analysis showed that PFR on ICU admission was the independent risk factor for hospital death ( OR =1.004, 95% CI: 1.002-1.006, P =0.048). Conclusions: Mortality rate among patients with acute respiratory failure secondary to CTD related PCP is still high, and the poor prognostic factors of hospital mortality included PFR on ICU admission and barotrauma.

Pneumocystis jirovecii pneumonia is an opportunistic infection among immunocompromised people. Studies have found that the increased resistance to sulfa drugs of Pneumocystis jirovecii may be associated with the mutation of dihydropteroate synthase ( DHPS ) gene and dihydrofolate reductase ( DHFR ) genes, but the mechanism is still unclear. The mutation of DHPS and DHFR genes may be the result of sulfa drugs selection or spontaneous genetic polymorphism, and it can be acquired from person-to-person transmission. This article reviews the cause, molecular epidemiology of DHPS and DHFR gene mutation, and the relationship between DHPS and DHFR gene mutation and clinical outcomes.

Objective: To establish an animal model for Pneumocystis pneumonia (PCP) and to study the etiological and molecular biological technology for PCP detection., Methods: SD and Wistar rats were divided into experimental and control groups randomly. The animals in the experimental group were immunosuppressed by subcutaneous injection with dexamethasone 2 mg per time per rat, twice a week, while those in the control group underwent the same way of injection with physiological saline simultaneously. After the induction for 8 weeks, all the rats were killed and their bronchoalveolar lavage fluid (BALF) and lung tissues were collected for smear making and microscopic detection. Meanwhile, the BALF samples were detected by PCR, and the products were sequenced and compared with rat source PCP in GenBank., Results: A total of 34 samples of lung tissue and BALF were observed. The etiological detection showed that the infection rates of the rats in the experimental and control groups were 29.2% (7/24) and 0, respectively. In the experimental group, the infection rates of SD and Wistar rats were 25.0% (3/12) and 33.3% (4/12), respectively, and the difference between them was not statistically significant (P = 0.31). The positive detection rates of the lung smears and BALF from SD rats in the experimental group were 25.0% (3/12) and 16.7% (2/12), respectively, while those in Wistar rats in the experimental group were 33.3% (4/12) and 16.7% (2/12), respectively, and there were no statistically significant difference between them (P = 0.34, 0.24). A total of 28 samples of BALF were detected by PCR, and the positive detection rates of rats in the experimental group and control group were 91.7% (26/28) and 0, respectively. The sequence analysis of the PCR products showed that it shared 100% homology with the genes of rat source PCP in Gen Bank (JX499145, GU133622 and EF646865)., Conclusions: The animal model of PCP can be established by subcutaneous injection with dexamethasone. As animal models, there are no significant difference between SD rats and Wistar rats. PCR method is suitable for PCP detection at the early stage of infection, while etiological detection with high missing rate is not a right option.

Objective: To evaluate the diagnostic utility of bronchoscopy in solid organ transplant recipients with pulmonary opportunistic infections., Methods: From December 2006 to September 2011, 117 cases of bronchoscopy with protected specimen brush and bronchoalveolar lavage were performed in 114 solid organ transplant recipients at Tianjin First Central Hospital. The indication for bronchoscopy was suspected pulmonary infections. The bronchoscopic manifestations were described and the specimens analyzed with regards to bacteriology, cytomegalovirus, P carinii, mycobacterium tuberculosis and other fungal cultures., Results: A definite infectious etiology was confirmed in 63 patients (53.8%). And opportunistic infections were the most frequent etiology (56/63, 88.9% including 7 cases with two mixed opportunistic infections). Among 63 pathogens, P carinii was demonstrated in 36 episodes, cytomegalovirus in 24 episodes and mycobacterium tuberculosis in 3 episodes. Bacterial infections (mainly Gram-negative) accounted for 16 of 63 episodes (25.9% including 9 cases with mixed opportunistic/bacterial infection). In accordance with the diagnostic results, antibiotic treatment was changed in 45 cases., Conclusions: As an extremely useful tool, bronchoscopy is of great value for pathogenic detection in transplant recipients with suspected pulmonary infections, especially for opportunistic infections. And the bronchoscopic findings may guide targeted antimicrobial therapy.

In order to evaluate the diagnostic value of fibrotic bronchoscopy (FB) in the pulmonary infiltration following bone marrow transplantation (BMT), 18 patients with pulmonary complications after BMT from November 2003 to March 2006 were performed with FB. Bronchoalveolar lavage (BAL) and brushing were performed in patients who had received short-term empirical therapy without good response, and transbronchial lung biopsy (TBLB) was carried out in 3 cases. The results showed that 9 out of 10 cases with pulmonary infection, including bacterial pneumonia (n = 3), aspergillosis (n = 2), pneumocystis carinii pneumonia (n = 3) and viral infection (n = 1) were diagnosed by using FB. One case was diagnosed as tuberculosis after open lung biopsy following negative results from twice BAL. 2 out of 8 cases were diagnosed by TBLB as noninfectious pulmonary complications. In conclusion, FB, especially with BAL, is a safe and useful procedure for the evaluation of pulmonary complications, which is particularly suitable for diagnosis of pulmonary infection after BMT. Furthermore, TBLB should be recommended in order to avoid open lung biopsy, if the patients tolerate the operation.

Objective: To construct prokaryotic recombinant expression plasmid carrying Pneumocystis carinii Mr 55 000 antigen (p55) gene fragment and express the recombinant protein., Methods: P. carinii pneumonia (PcP) rat models were established by subcutaneous injection of dexamethasone for 14 weeks. Total RNA was extracted from lung of P. carinii rat and p55 antigen gene fragment was cloned by RT-PCR, which was identified by sequencing. The 690 bp fragment was cloned to pGEX-4T-1, the recombinant plasmid was screened and identified by restriction analysis and PCR. The recombinant plasmid was finally induced with IPTG to express a new fusion protein, and the products were analyzed by SDS-PAGE and Western blot., Results: A fragment of 690 bp was obtained by RT-PCR. The recombinant pGEX-4T-1/690 was constructed. SDS-PAGE revealed that the molecular weight of the recombinant protein was approximately Mr 62,000, the maximum amount of the fusion protein produced was 11.6% of the total protein. The recombinant protein can be recognized by GST antibody and by the sera from P. carinii infected rats using Western blotting., Conclusion: Prokaryotic expression plasmid pGEX-4T-1/690 has been constructed and the recombinant fusion protein shows antigenicity.

Objective: To establish axenic cultivation of Pneumocystis carinii (P.c)., Methods: The organisms of P.c were isolated from the bronchoalveolar lavage fluid (BALF) of the rats with Pneumocystis carinii pneumonia (PCP) and cultured in a medium which was based on IMDM(GIBCO) supplemented with S-adenosyl-L-methionine, putrescine, N-acetyl glucosamine, putrescine, L-cysteine and L-glutamine, and newborn calf serum. The organisms cultured in the system were identified by observing the morphology of cysts in smears stained with Gomori's methenamine silver nitrate stain (GMS). Ultrastructure of the cysts/trophozoites was examined by transmission electron microscopy. The sequences of mitochondrial large ribosomal DNA subunit of the cultured organisms were compared with the Pneumocystis carinii f.sp. ratti variant isolate (GenBank No U20173) and Pneumocystis carinii f.sp.hominis (GenBank No M58605)., Results: Five isolates of P. carinii received from BALF of 8 rats with PCP were cultured axenically and continuously in the system. The cultured organisms could be stored in frozen condition and used to reinitiate culture, and were amplified by 19-22 times within 72 h. The morphology, ultrastructure and gene sequencing of the cultured organisms confirmed that the isolated organisms were P. carinii., Conclusion: Five continuously and axenicly cultured isolates of P. carinii have been received.

Objectives: To examine sICAM-1 in Pneumocystis carinii pneumonia (PCP) and the effect of TMP-SMZ therapy on its level and on pathological and immunological changes in rats., Methods: 50 female Wistar rats were randomly divided into normal group (N group), PCP model group (PCP group) and TMP-SMZ therapy group (SMZ group). 1 mg of dexamethasone was injected intramuscularly twice a week for rats in PCP and SMZ groups to induce PCP. Normal saline was injected for N group in the same way. When the infection was confirmed, TMP-SMZ was given to rats in SMZ group by 25 mg/(kg.d) for 5 days for 3 courses with an interval of 2 weeks. sICAM-1 in serum was detected by ELISA, and the pathological changes in lungs and liver and the Pc in alveoli of lungs were observed., Results: The level of sICAM-1 in PCP and SMZ groups at the 3rd week [(1.847+/-0.50) ng/ml, (1.787+/-0.59) ng/ml] was lower notably than that at 0 week [(2.407+/-0.81) ng/ml, [(2.478+/-0.59) ng/ml respectively] (P<0.05), and then increased gradually. It was significantly higher in PCP group at 9th week [(3.233+/-0.83) ng/ml] and at 12th week [(3.984+/-0.87) ng/ml] than that of 0 week (P<0.05). Its level in SMZ group at 12th week [(3.621+/-l.62) ng/ml] was also higher than that in 0 week [(2.478+/-0.59) ng/ml] (P<0.05). sICAM-1 level in both PCP and SMZ groups at 9th week and 12th week was higher than that of N group (P<0.05, P<0.01). There was no significant difference between SMZ and PCP groups at 9th week and 12th week (P>0.05)., Conclusion: The sICAM-1 level in rats was low but significantly increases after the induction of PCP.

Objective: To study the alveolar ultrastructural changes and the interaction between Pneumocystis carinii (PC) and alveoli in Pneumocystis carinii pneumonia (PCP) patients after renal transplantation., Methods: Twenty-seven patients with suspected PCP after renal transplantation were examinated by bronchoscopy with bronchoalveolar lavage (BAL) and transbronchial lung biopsy (TBLB). The BAL fluid was centrifuged and the sediments were stained for PC. Cases for which electron microscope showed alveolar tissue in TBLB specimen were included. Then the clinical features, PC, alveolar epithelial damage, exudate in alveolar space, inflammatory cell infiltration, and fibrous tissue in the interstitial space were analyzed and evaluated., Results: Twenty-three cases were studied. The mean time from renal transplantation to onset of illness was 5.6 months, and that from onset of illness to hospitalization was 5.5 days. Clinical features included fever, dyspnea, unproductive cough, and scanty chest signs, to hypoxemic respiratory failure. Chest CT showed diffuse lung interstitial changes in 22 of the 23 cases, 9 with consolidation. After treatment with SMZco, the fever resolved in 1 - 5 days, and the general state of the patients became better, and 19 patients were dischaged within 1 month. PC in BAL fluid was found by special staining in 18 patients, while PC was found by electron microscope in 14 patients. In most cases PC was few in the lung tissue, but in 3 cases abundant PC filled the alveolar space. PC was seen in two forms, the cyst and the trophozoite. Electron and light microscopes showed alveolar exudate, inflammation in interstitium and alveolar space, interstitial fibrosis, and alveolar epithelial damage in all patients., Conclusion: In PCP patients after renal transplantation there was marked alveolar damage, which was the major pathological change in the lung.

Objective: To explore the susceptibility of different strains of mice to P. carinii infection and the host immune response to the organism., Methods: C57BL/6 and BALB/c strains of mice (15 each) were exposed to SCID mice infected with P. carinii by co-housing. Observations were made on the number of parasites in the lungs, level of CD4+ T cell and CD8+ T cell in BALF, and serum IgG at 4, 5 and 6 wk after contagion., Results: The number of P. carinii grew in the lungs of BALB/c mice was found much greater than those in C57BL/6 mice. A few number of P. carinii cysts were detected in the lungs of both strains of mice by 4 wk after co-housing, the number of cysts increased at 5 wk in the lungs of BALB/c mice but not in that of C57BL/6 mice. P. carinii-specific IgG in the sera and high level of CD62low CD4- and CD8-positive T cells in the lungs were found at 5 wk. The parasites were cleared from the lungs at 6 wk in all infected mice, shortly after acquired immune response was initiated., Conclusion: BALB/c mice are more susceptible to P. carinii than the C57BL/6 mice by natural transmission, and all the immunocompetent mice can clear the infection of P. carinii by cellular and humoral immune responses.

Twenty Wistar rats were injected with cortisone acetate twice a week subcutaneously for 6-12 wks. By 7 weeks Pneumocystis carinii pneumonia was induced in rats successfully. The cysts of Pneumocystis carinii from infected lungs of cortisonized rats were identified by phase-contrast microscopy, Giemsa's stain, Chalvardjian's stain and Gomori's methenamine silver nitrate stain. The process of isolation and purification of Pneumocystis carinii from infected rat lungs included the following three test steps. First, the tissue was cut into small pieces and homogenized and filtered through #60, #100, #200-gauge wire mesh respectively. Secondly, the homogenate was digested with collagenase, the optimal concentration of collagenase being 0.2%. Thirdly, the discontinuous percoll density gradient centrifugation was used to separate P. carinii cysts. The majority of P carinii cysts were present in a density zone of approximately 1.033g/ml and were essentially free from host lung debris, the latter being removed due to their higher density, 1.040g/ml.

A biotin labelled human cytomegalovirus (HCMV) DNA probe of EcoRI D fragment (17kb) from HCMV AD 169 strain was prepared. In situ hybridization of HCMV was performed in paraffin section of tissues collected from an autopsy case of congenital and combined immunodeficiency syndrome associated with systemic HCMV and pneumocystis carinii infections. The pathological lesions of HCMV infection showed three different patterns: 1, necrotic granuloma type, 2, diffuse type, and 3, scattered type. HCMV DNA was identified in all the inclusion bodies and piled or granular positive material was found in the nuclei or cytoplasms of some giant cells. In addition, some morphologically intact cells might be observed positive for CMV DNA either in nuclei or in cytoplasms.