Next-generation sequencing technology for the diagnosis of Pneumocystis pneumonia in an immunocompetent female: A case report.

BACKGROUND: Pneumocystis pneumonia (PCP) is a serious fungal infection usually seen in patients with human immunodeficiency virus, and it is more frequently found and has a high fatality rate in immunocompromised people. Surprisingly, it rarely occurs in immunocompetent patients. However, the clinical diagnosis of this pathogen is made more difficult by the difficulty of obtaining accurate microbiological evidence with routine tests. This case reports a PCP patient with normal immune function who was diagnosed through next-generation sequencing (NGS). CASE SUMMARY: A 23-year-old female who had no special disease in the past was admitted to the hospital with a persistent fever and cough. Based on the initial examination results, the patient was diagnosed with bipulmonary pneumonia, and empirical broad-spectrum antibiotic therapy was administered. However, due to the undetermined etiology, the patient's condition continued to worsen. She was transferred to the intensive care unit because of acute respiratory failure. After the diagnosis of Pneumocystis jirovecii infection through NGS in bronchoalveolar lavage fluid and treatment with trimethoprim/sulfamethoxazole and caspofungin, the patient gradually recovered and had a good prognosis. CONCLUSION: This case emphasizes that, for patients with normal immune function the possibility of PCP infection, although rare, cannot be ignored. NGS plays an important role in the diagnosis of refractory interstitial pneumonia and acute respiratory failure.

Bioaerosols emission characteristics from wastewater treatment aeration tanks and associated health risk exposure assessment during autumn and winter.

Aeration tanks from activated sludge wastewater treatment plants (WWTPs) can release a large amount of bioaerosols that can pose health risks. However, risk characterization of bioaerosols emissions form wastewater treatment plants is currently not systematically carried out and still in its infancy. Therefore, this study investigated emission characteristic of two indicator model bioaerosols Staphylococcus aureus and Escherichia coli, emitted from aeration tanks of a municipal WWTP. Monte Carlo simulation was then used to quantitatively assess microbial risk posed by different aeration modes under optimistic and conservative estimates. Further to this, two different exposure scenarios were considered during 3 days sampling campaign in autumn and winter. Results showed that the bioaerosol concentration from microporous aeration tank (20-262 CFU m-3) was one order of magnitude lower than rotating disc aeration tank. Average aerosolization rate was 7.5 times higher with mechanical aeration mode. Health risks of exposed populations were 0.4 and 9.6 times higher in winter than in autumn for E. coli and S. aureus bioaerosol, respectively. Health risks of staff members were 10 times higher than academic visitors. Interesting results were observed for academic visitors without personal protective equipment (PPE) respectively exposed to S. aureus and E. coli bioaerosol in autumn and winter: while the derived infection risk met the United States Environmental Protection Agency (U.S. EPA) benchmark under optimistic estimation, the disease risk burden was over the World Health Organization (WHO) benchmark under conservative estimation. These revealed that only satisfying one of the two benchmarks didn't mean absolute acceptable health risk. This study could facilitate the development of better understanding of bioaerosol quantitative assessment of risk characterizations and corresponding appropriate risk control strategies for wastewater utilities.

Seasonal variation of quantitative microbial risk assessment for three airborne enteric bacteria from wastewater treatment plant emissions.

Airborne E. coli, fecal coliform, and Enterococcus are all related to sewage worker's syndrome and therefore used as target enteric bioaerosols about researches in wastewater treatment plants (WWTPs). However, most of the studies are often inadequately carried out because they lack systematic studies reports bioaerosols emission characteristics and health risk assessments for these three enteric bacteria during seasonal variation. Therefore, quantitative microbial risk assessment based on Monte Carlo simulation was utilized in this research to assess the seasonal variations of health risks of the three enteric bioaerosols among exposure populations (academic visitors, field engineers, and office staffs) in a WWTP equipped with rotating-disc and microporous aeration modes. The results show that the concentrations of the three airborne bacteria from the rotating-disc aeration mode were 2-7 times higher than the microporous aeration mode. Field engineers had health risks 1.5 times higher than academic visitors due to higher exposure frequency. Health risks of airborne Enterococcus in summer were up to 3 times higher than those in spring and winter. Similarly, health risks associated to E. coli aerosol exposure were 0.3 times higher in summer compared to spring. In contrast, health risks associated with fecal coliform aerosol were between 2 and 19 times lower in summer compared to spring and winter seasons. Data further suggest that wearing of N95 mask could minimize health risks by 1-2 orders of magnitude. This research shed light on seasonal variation of health risks associated with bioaerosol emission from wastewater utilities.

The relationship between activity level and cognitive function in Chinese community-dwelling elderly.

To study the relationship between daily activity level and cognitive function in community-dwelling elderly. We collected demographic features, cognitive function, activity level and self-rating depression scale scores in 53 community-dwelling olderly aged 60 years or above. The activity level and moderate-to-vigorous physical activity (MVPA) time were assessed by using the accelerometer for 7 consecutive days. We compared activity level, MVPA time and depression scores between cognitive impaired and normal groups. Cognitive functions were compared in groups with different MVPA level, and the correlation between cognitive function and MVPA time was analysed. Of the 53 subjects, 27 had varying degrees of cognitive impairment. Individuals with cognitive impairment shown significantly shorter MVPA time and higher depression score compared to the cognitive normal group (P < 0.05). After controlling for confounding factors (age, BMI), MVPA time was associated with cognitive function (r = 0.358, P = 0.009). The memory factor score correlated with MVPA time (r = 0.357, P = 0.012) and mean activity level (r = 0.287, P = 0.046). Moderate-to-vigorous physical activity in the elderly was positively related to their cognitive function. Strengthening daily activities may beneficial for the elderly to maintain better cognitive function.

Short term effects of small incision lenticule extraction surgery on corneal endothelium.

AIM: To assess the effects of small incision lenticule extraction (SMILE) surgery on the corneal endothelium at 1d to 1mo postoperatively. METHODS: A retrospective, observational study was conducted on 47 patients (47 eyes) who received SMILE surgery. Patients were grouped according to contact lens wear condition. The corneal endothelium was examined preoperatively and at 1d, 1wk and 1mo postoperatively. The corneal endothelium was analyzed for endothelial cell density (ECD), percentage of hexagonal cells, and coefficient of variation (CV) of cell size. RESULTS: There were no significant decrease in the ECD, percentage of hexagonal cells or increase in CV at 1d, 1wk and 1mo postoperatively (P>0.05). However, there was a small increase of ECD by 2.88% in contact lens wearers (78.26±113.62 cell/mm(2), P<0.05). CONCLUSION: SMILE has no significant adverse effects on the corneal ECD and morphology during 1mo follow-up time.

[Protective effect of Icariin against TGF-ß1-induced injury of rat Leydig cells].

OBJECTIVE: To study the effect of Icariin on rat Leydig cells with TGF-ß1-induced injury. METHODS: We determined the optimal concentration of Icariin for protecting primarily cultured Leydig cells against TGF-ß1-induced injury by methyl thiazolyl tetrazolium assay. We detected the effects of Icariin on the secretion of estradiol (E2) and activity of aromatase in the injured Leydig cells by radioimmunoassay and Tritium water release experiment and its effect on the gap junctional intercellular communication (GJIC) between the Leydig cells by fluorescence distribution after photobleaching. RESULTS: Different concentrations of Icariin showed different degrees of protective effect on the TGF-ß1-treated Leydig cells, the effect observed at 20 µg/ml and at its optimum at 160 µg/ml. After treatment of the injured Leydig cells with Icariin at 160 µg/ml, significant improvement was observed in the E2 secretion and aromatase activity (P<0.01) as well as in the GJIC between the Leydig cells (P<0.01). CONCLUSIONS: Icariin can effectively protect rat Leydig cells against TGF-ß1-induced injury, which is largely attributed to its effects of increasing E2 synthesis, enhancing aromatase activity, and improving GJIC between Leydig cells.

[Inhibitory effects of doxycycline on argyrophilic nucleolar organizing regions and a-smooth muscle actin expression in proliferative bovine corneal myofibroblasts in vitro].

OBJECTIVE: To investigate the effect of doxycycline on the nucleolar organizing regions and a-smooth muscle actin expression in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repair mechanisms. METHODS: Cell culture and identification: bovine corneal fibroblasts were cultured after the stroma was incubated in 1.0 and 2.0 g/L type I collagenase in two stages.Isolated cells were plated at mantaryay culture flask in 10% of BSA RPMI-1640. Vimentin and alpha-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry. The cells staining positive for Vimentin and α-SMA indicated the presence of corneal myofibroblasts. Bovine corneal myofibroblasts were treated with different concentrations of doxycycline (10, 20, 40, 60, 80 mg/L) , a bland control group and the dexamethasone group (120 mg/L) were set up, each group had 30 cases. The argyrophilic nucleolar organizing regions (AgNOR) staining and the immunohistochemistry for α-SMA were performed when the cells were treated for 24 hours and 48 hours. The AgNOR count (Ag-c), AgNOR area (Ag-a) and the expression of α-SMA in the bovine corneal myofibroblasts among each experiment group and control group were compared using one-way ANOVA, further pairwise comparisons using Independent-Samples t test. RESULTS: Cell culture techniques were successfully used to establish a method for the isolation and culture of bovine corneal myofibroblasts. Microscopic examination and immunohistochemical staining confirmed that the cells cultured were bovine corneal myofibroblasts. The Ag-c and Ag-a of bovine corneal myofibroblasts progressively decreased as the concentrations of doxycycline was increase. 24 h:bland control group Ag-c was 6.40 ± 0.6, 60 mg/L doxycycline group Ag-c was 2.23 ± 0.43;bland control group Ag-a was (34.80 ± 2.36) µm(2), 60 mg/L doxycycline hormone group Ag-a was (19.91 ± 2.15) µm(2). 48 h: bland control group Ag-c was 7.27 ± 0.6,60 mg/L doxycycline hormone group Ag-c was 2.80 ± 0.76, bland control group Ag-a was (36.27 ± 1.99) µm(2), 60 mg/L doxycycline group Ag-a was (13.75 ± 2.09) µm(2). The differences were statistically significant: in the same time intervention (FAg-c 24 h = 252.55, FAg-a 24 h = 202.16, P < 0.05, FAg-c 48 h = 169.38, FAg-a 48 h = 853.23, P < 0.05), in the same concentrations intervention (tAg-c = 6.98, tAg-a = 11.62, P < 0.05). And 60 mg/L of doxycycline had an obviously inhibitory action as 120 mg/L dexamethasone in the same treated hours (dexamethasone group Ag-a 24 h = 30.56 ± 3.66, dexamethasone group Ag-a 48 h = 28.35 ± 1.23 ),the differences were not statistically significant (tAg-a 24h = 1.182, P = 0.242,tAg-a 48 h = 0.21, P = 0.832). As the concentrations investigated, doxycycline can inhibit the expression of α-SMA in the bovine corneal myofibroblasts (189.90 ± 7.48, 140.20 ± 7.79, 113.20 ± 8.98, 98.00 ± 3.50, 85.50 ± 4.99), the difference was statistically significant (F = 761.79, P = 0.00). While dexamethasone had no significant role in the expression of α-SMA (bland control group was 225.10 ± 6.74, the dexamethasone group was 228.50 ± 7.12), and the statistically difference was not obvious (t = 1.096, P = 0.287). CONCLUSIONS: As the concentrations of doxycycline was increased from 10 mg/L to 80 mg/L, the AgNOR count and AgNOR area of bovine corneal myofibroblasts can be significantly reduced in vitro. Compared with dexamethasone, doxycycline significantly suppressed the expression of α-SMA in bovine corneal myofibroblasts in a dose-dependent positive trend.

TGF-ß1 regulation of estrogen production in mature rat Leydig cells.

BACKGROUND: Besides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-ß1 (TGF-ß1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-ß1 regulates estradiol (E2) synthesis in adult rat Leydig cells and then to assess the impact of TGF-ß1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells. METHODOLOGY/PRINCIPAL FINDINGS: Primary cultured Leydig cells were incubated in the presence of recombinant TGF-ß1 and the production of E2 as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-ß1 down-regulates the level of E2 secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E2 and TGF-ß1, and E2 treatment in turn restored the inhibition of TGF-ß1 on GJIC. CONCLUSIONS: Our results indicate, for the first time in adult rat Leydig cells, that TGF-ß1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E2 secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.

[TGF-beta1 reduces connexin43-mediated gap junctional intercellular communication in rat Leydig cells].

OBJECTIVE: To observe the effects of TGF-beta on the expression of connexin43 (Cx43) and Cx43-mediated gap junctional intercellular communication (GJIC) in rat Leydig cells, and investigate the association of its effects on Leydig cells with its ability of changing GJIC. METHODS: Primarily cultured purified Leydig cells were divided into a blank control group, a positive control group (treated with the GJIC inhibitor Carbenoxolone), and four TGF-beta1 groups (treated with TGF-beta1 at the concentration of 1, 2, 5 and 10 ng/ml, respectively, for 20 hours). The localization and expression of Cx43 were detected by immunofluorescence and Western blot, and the changes in GJIC analyzed by FRAP assay. RESULTS: Cx43 was expressed as scattered bright spots in the cytoplasm and membrane of Leydig cells. TGF-beta1 significantly elevated the expression of Cx43 in the cytoplasm, but caused no evident change in the membrane. Western blot showed an evident increase in the phosphorylation of Cx43 with the increased concentration of TGF-beta1 as compared with that of the blank control group (P < 0.05). After 20 hours of treatment with TGF-beta1 at 5 ng/ml, the fluorescence intensity of Leydig cells was markedly reduced (P < 0.01), with a mean fluorescence recovery rate of merely (43.58 +/- 1.87)%. CONCLUSION: TGF-beta1 could significantly down-regulate GJIC between adjacent Leydig cells, and this inhibitory effect may be achieved by promoting the expression of Cx43 in the cytoplasm and elevating the phosphorylation of Cx43.

[Effects of Wenyang Shengjing Decoction containing serum on the estradiol secretion of Leydig cells of sterile rats of shen-yang deficiency].

OBJECTIVE: To study the effects of Wenyang Shengjing Decoction (WSD) containing serum on the estradiol (E2) secretion, the synthesized cytochrome P450 aromatase (P450arom) activities, as well as the expression of its encode gene CYP19 in Leydig cells of male sterile rats of adenine induced Shen-yang deficiency (SYD). METHODS: Experimental rats were randomly divided into 4 groups, i.e., the normal control group, the high, middle, and low dose WSD groups, 5 in each group. The normal saline, low, middle, and high dose WSD were respectively given to rats of all groups for 10 successive days. Blood was drawn from rats' heart 2 h after the last gastrogavage. The serum was separated after centrifuge. Leydig cells isolated and purified from SYD rats were primary cultured in vitro and divided into 5 groups in random, i. e., the blank control group, the model group, the high, middle, and low dose WSD groups (1.2, 1.0, and 0.8 g/mL, respectively). The content of E2 released in the culture supernate was determined by radioimmunoassay. The P450arom activity was detected by tritium release assay. Meanwhile, the mRNA and protein expressions of CYP19 were analyzed using fluorescent quantitative PCR and Western blot respectively. RESULTS: Compared with the blank control group, the E2 secretion of the supernate of Leydig cells obviously decreased in the model groups, accompanied with the inhibition of P450arom activities, significant decreased protein and mRNA expressions of CYP19 (P < 0.01, P < 0.05). Compared with the model group, after intervened by WSD containing serum, the E2 secretion in the Leydig cells could be significantly increased, the P450arom activities up-regulated, the CYP19 expressions up-regulated at the protein and mRNA levels partially in a dose-dependent manner (P < 0.01, P < 0.05). CONCLUSIONS: WSD containing serum could effectively elevate the E2 secretion in Leydig cells, which might be partially achieved through up-regulating P450arom activities and enhancing the gene expression of CyP19. This might be one of its mechanisms of action for treating male infertility of SYD.