Leishmania. Hepatomegaly is one of the most frequent clinical manifestations of VL, whereas immunopathology of the symptom has not been well investigated. Using our chronic model of experimental VL, we examined the influence of Leishmania donovani infection on the liver by clinical, histological, and biochemical analyses. The infected mice showed increased liver weight 24 weeks post-infection. Although an increase in serum ALT and inflammatory cell accumulation were observed in the livers of infected mice, no apparent parenchymal necrosis or fibrosis was observed. Tissue water content analyses demonstrated that increased liver weight was predominantly due to an increase in water weight. Together with the finding of hepatic sinusoidal dilation, these results suggested that edema associated with sinusoidal dilation causes hepatomegaly in L. donovani infection. Immunostaining of platelets and erythrocytes showed no thrombus formation or damage to the sinusoidal endothelium in the liver of infected mice. Taken together, these results suggest that hepatomegaly during experimental VL is caused by non-obstructive sinusoidal dilation.Visceral leishmaniasis (VL) is the most severe form of leishmaniasis caused by protozoan parasites of the genus  Leishmania species, Leishmania donovani and Leishmania infantum are well known to infect internal organs and cause VL.Leishmaniasis is one of the neglected tropical diseases and is estimated to affect 0.7–1 million people annually [Enlargement of the liver  is a common symptom in VL patients; 30–100% of VL patients with parasitological confirmation present with liver enlargement ,9,10,11.Leishmania infection, they have received less attention as a tool for studying the immunopathology of VL. Despite the fact that hepatic abnormalities are characteristics of chronic disease in human VL patients [Leishmania amastigotes as infecting pathogens, and result in self-limiting liver infection with a peak parasite burden at approximately four weeks post-infection [Although experimental models of VL are useful to study protective responses against patients , much repatients ,17,18. Tnfection ,20. Hamsnfection . In contnfection . Our mouIn general, hepatomegaly is caused by various mechanisms, including congestion, portal hypertension, and hepatocyte hypertrophy . These aL. donovani in BALB/cA mice induced a significant increase in the weight of the spleen and liver at 24 weeks post-infection, whereas no apparent change in body weight was induced by the infection . Increased water weight (0.40 g), rather than dry weight (0.12 g), predominantly accounted for the increased whole liver weight (0.52 g) induced by L. donovani infection . In addition to nitric oxide, carbon monoxide is also involved in vasodilation in the liver [hmox1 gene (data not shown), which encodes the enzyme involved in production of carbon monoxide. Future studies on nitric oxide and carbon monoxide, as well as other soluble factors, may help to elucidate the detailed mechanisms of sinusoidal dilation leading to hepatomegaly during experimental VL.There are several models of hepatic sinusoidal dilation. Marzano et al. proposed possible causes as obstruction of the sinusoids or central veins, including thrombosis due to damage to the sinusoidal endothelium, and non-obstructive mechanisms including hepatocyte shrinkage, increased inflow of blood, and an increase in soluble factors, which induce sinusoidal dilation . One of he liver ,36. The L. donovani infection, serum biochemical analyses as well as histological analyses were performed. One finding was the reduction of serum albumin in the infected mice  mice were purchased from Japan Clea, Tokyo, Japan. All mice were maintained under specific pathogen-free conditions. The mice were used for experiments at the age of 6–8 weeks. iversity ) were cuL. donovani promastigotes (passage number = 2) in late log or stationary phase were washed with phosphate-buffered saline  by centrifugation at 1600× g for 10 min and were resuspended with PBS at the concentration of 1 × 108 cells/mL. Mice were infected with 1× 107 L. donovani promastigotes by intravenous injection into the tail vein. After 12 or 24 weeks, cardiac puncture was performed on those mice under isoflurane anesthesia to collect the whole blood at the time of euthanasia, and the spleen and liver were then harvested. Impression smears of the spleen and liver were fixed for 5 min in methanol and stained for 25 min with 5% Giemsa solution . Amastigotes were counted by microscopic observation of the stained smear at 1000× magnification, and Leishman-Donovan Units (LDU) were enumerated as the number of amastigotes per 1000 host nuclei times the tissue weight in grams as performed in a previous study [us study .The tissues collected at the time of sacrifice were fixed with Carnoy’s solution composed of 60% methanol, 30% chloroform, and 10% acetic acid for 48 h and embedded in paraffin. Four-micrometer-thick sections were prepared from the paraffin-embedded tissues. The sections were dewaxed and treated with periodic acid for 5 min. Next, the sections were covered with Schiff’s reagent  for 20 min and with sulfite water for 6 min for color development. Then, they were stained with Mayer’s hematoxylin solution (WAKO) for 90 s and rinsed in running tap water for 5 min. For the quantification of glycogen, images were split into three colors using a Fiji plugin “Colour Deconvolution” (set vector at “H PAS”), purple signals were inverted to monochrome, and average color intensity was quantified.The tissues collected at the time of sacrifice were fixed with 10% buffered formalin . The fixed tissues were sent to Biopathology Institute Co., Ltd.  to make frozen sections and stain neutral fat with Oil red-O. For the quantification of triglyceride (TG) images were split to three colors using a plugin “Colour Deconvolution” (set vector at “RGB”), red signals were inverted to monochrome, and average color intensity was quantified.The tissues collected at the time of sacrifice were fixed with 10% buffered formalin and then embedded in paraffin. Four-micrometer-thick liver sections were dewaxed and stained with hematoxylin and eosin (H&E) for histopathological and stereological analyses. Digital images of hepatic tissue were obtained using a light microscope  connected to a digital camera . Eight photomicrographs from H&E-stained sections  were randomly obtained for each mouse. For the stereological analysis, a test system of 192 or 768 points was used in a standard test area ,42. In s2O2 in methanol for 30 min. After blocking with Block Ace , 2.5 μg/mL solution of anti-mouse KI-67 antibody (Thermo Fisher #14-5698-80), 6 μg/mL solution of anti-mouse CD42b antibody (Abcam #ab183345) and 0.5 μg/mL solution of anti-mouse Ter119 antibody (BioLegend #116211) was applied to the sections of liver, and the sections were incubated for 1 hour at room temperature and washed with PBS. Next, Histofine® Simple Stain Mouse MAX PO (Rat) , or Histofine® SAB PO(R) kit second antibody  followed by HRP-conjugated streptavidin  was applied, and the sections were incubated for 1 h at room temperature and washed with PBS. After incubating with 3,3’-diaminobenzidine (DAB) substrate (Nichirei) to yield an enzymatic color change, the sections were counterstained with Mayer’s hematoxylin solution for 60 s and rinsed with tap water for 40 min. The pictures of each section were split to blue, blown, and green using a Fiji plugin “Colour Deconvolution” (set vector at “HDAB”), and the number of KI-67 positive and negative nuclei was counted.Immunohistochemical staining was performed to characterize the nuclei of proliferating cells. The tissues collected at the time of sacrifice were fixed with Carnoy’s solution for 48 h and embedded in paraffin. Paraffin-embedded tissues were dewaxed and boiled in 10 mM sodium citrate buffer (pH 6.0) for 20 min, followed by washing with tap water. Endogenous peroxidase was inactivated with 0.3% HThe cut tissue pieces (~100 mg of liver for each mouse) were placed at the bottom of a 1.5 mL centrifuge tube (BIOBIK #CF-0150) and dried at 90 °C for 24 h using a CTU-mini . Of note, the dry weight did not change by further drying after 24 h. The dry weight of the whole liver was calculated as the product of whole liver wet weight and the dry: wet weight ratio of the cut piece. The water content of each sample was calculated as below.Serum was separated from the collected blood and analyzed for biochemical tests by Oriental Yeast Co., Ltd. . The following methods were used for the individual tests; total protein by biuret method, albumin by BCG method, globulin calculated as , AST by JSCC transferable method, ALT by JSCC transferable method, and TG by the GPO-HMMPS method.t test or two-way ANOVA coupled with Sidak’s multiple comparisons test was used for comparison of two independent groups, i.e. naive and L. donovani-infected mice. p values less than 0.05 were considered statistically significant.Statistical analysis was performed using GraphPad Prism 9 software package . Results are presented as mean ± standard deviation (SD). Either unpaired


Sepsis is a potentially lethal complication for both flexible ureteroscopy (fURS) and percutaneous nephrolithotomy (PCNL). This study is aimed at comparing the sepsis rate after fURS and PCNL and the risk factors for sepsis in patients with solitary proximal ureteral stone.  We reviewed the data of patients with calculi between 10 mm to 20 mm who underwent fURS or PCNL surgery from Tongji Hospital's database. A total of 910 patients were eligible with 412 fURS cases and 498 PCNL cases. We used univariate analysis and multivariate logistic regression analysis to identify the risk factors for sepsis. Subgroup analysis was performed using logistic regression analysis. P = 0.004), serum WBC ≥ 10,000 cells/μL , albumin − globulin ratio < 1.2 , positive urine culture , and prolonged operation time . Subgroup analysis was conducted using potential risk factors: stone size, serum WBC, urine culture, and albumin-globulin ratio (AGR). In subgroup of positive urine culture, patients were more likely to develop sepsis after fURS than PCNL.  In the cohort, 27 (6.6%) and 19 (3.8%) patients developed sepsis after fURS and PCNL, respectively. Multivariate analysis shows that the risk factors for sepsis are fURS (OR = 3.160,  PCNL may be a better choice than fURS to reduce postoperative sepsis, especially for patients with positive urine culture. Urolithiasis is one of the most common diseases in the urinary system. It affects patients globally because of its high incidence rate that is 7-13% in North America, 5-9% in Europe, and 1-5% in Asia .Sepsis is one of the most intractable surgery complications, which leads to a longer length of stay and even lethal sepsis shock in some cases . AccordiBesides, PCNL is usually utilized to treat larger stone than fURS , 11. ConThe research was approved by the Ethics Committee of Tongji Medical College (2019S1035). The retrospective study included patients from January 2012 to December 2018. The inclusion criteria were (1) unilateral, solitary, and proximal ureteral stones; (2) PCNL or fURS to treat urolithiasis; (3) stone size ranging from 10 mm to 20 mm; (4) patient age ≥ 18 years. The exclusion criteria were anatomical abnormality: solitary kidney, horseshoe kidney, transplant kidney, and kidney duplication.μL or >12,000 cells/μL.The primary outcome was postoperative sepsis. According to the 2001 International Sepsis Definitions Conference, postoperative sepsis was defined as the concurrence of infection and at least two of the following criteria with 48 hours of surgery: (1) heart rate > 90/minute, (2) respiratory rate > 20/minute, (3) body temperature > 38°C, and leukocyte count < 4,000 cells/The patient data was retrospectively collected from the hospital's database. Preoperative factors were recorded such as patient age, sex, body mass index (BMI), comorbidities , stone size and laterality, presence of hydronephrosis and indwelling stent, hematological tests , biochemical tests , urine tests (urine WBC and urine nitrite), urine culture, and American Society of Anesthesiologists (ASA) score. Size of ureteral access sheath and flexible ureteroscope for fURS and size of sheath and nephroscope for PCNL were also recorded. Operation time was documented from the commencement of operation to the end of anesthesia. The laboratory tests were routinely performed and obtained for all patients. Patients who have infectious indicators  received at least a full antibiotic regimen for seven days until the tests turned negative. Otherwise, one dose of antibiotics was applied for prophylactic purpose.t-test was used to compare continuous variables (expressed by mean ± standard deviation) with a normal distribution. Continuous variables with a skewed distribution were showed as median (interquartile range [IQR]) and compared by the Mann-Whitney U test. The chi-square test or Fisher's exact test was utilized to detect the difference between groups with categorical variables (expressed by proportions). The logistic regression method was used to identify the risk factors of sepsis. The difference was considered statistically significant when P value <0.05.Statistical analysis was conducted using Statistical Product and Service Solutions (SPSS) version 24.0. The Student's n = 103), bilateral stone (n = 677), renal stone > 4 mm (n = 1205), and ureteral stone below the fourth lumbar (n = 375). Finally, a total of 910 patients with 10-20 mm solitary proximal ureteral stone were eligible for analysis . The multivariate analysis indicates that five variables are independent risk factors of sepsis (P = 0.004), serum WBC ≥ 10,000 cells/μL , albumin − globulin ratio < 1.2 , positive urine culture , and prolonged operation time . Sex (P = 0.354) and stone size (P = 0.716) are not considered as independent risk factors.The detailed basic information of the eligible patients is shown in f sepsis : fURS . In the positive urine culture group, patients who receive fURS have 5.71 times the risk of developing sepsis than patients who receive PCNL (P < 0.001).Urologists are more likely to choose PCNL to treat larger stones. We divided patients into a larger stone size group (15-20 mm) and a smaller stone size group (10-15 mm) to evaluate the sepsis rate between fURS and PCNL . Both grSepsis is one of the most severe complications in patients who underwent lithotomy, which may both occur after PCNL or fURS , 13. It We previously report that positive urine culture is an independent predictor for post-fURS sepsis and assemble a nomogram to predict the occurrence of post-fURS sepsis . UchindaKreydin and Eisner have systemically summarized the risk factors for sepsis after PCNL , and thePositive urine culture is a predictor of postoperative sepsis for both fURS and PCNL. The evidence reveals that urine culture can be an essential reference factor for surgical choice to reduce the incidence of sepsis. In patients with 10-20 mm ureteral stone, we find that PCNL is better than fURS when patients have a positive urine culture. Our works can optimize the surgery strategy for patients with a high risk of infection .The main limitation of the study is the single-center retrospective nature, which may cause selection bias. We include a relatively large number of patients to stabilize the results. Subgroup analysis is also performed to compare sepsis incidence in patients with different conditions. Further prospective and multicenter studies are needed.In summary, we find that PCNL might be a better choice than fURS to reduce postoperative sepsis, especially when patients have a positive urine culture.


Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of excessive inflammation. We aimed to describe the clinical and laboratory findings of HLH patients secondary to Visceral leishmaniasis (VL) and their treatment outcome during a 4-year follow-up period compared to primary HLH.Forty children with primary HLH confirmed by genetic study and 20 children with HLH secondary to VL confirmed by a blood or bone marrow polymerase chain reaction from 2014 to 2018 in Shiraz, Fars province, Southern Iran, were enrolled.r = 0.371, P = 0.004). Patients with primary HLH experienced more thrombocytopenia (P = 0.012) and higher alanine transaminase (P = 0.016), while patients with VL-associated HLH had higher ferritin (P = 0.034) and erythrocyte sedimentation rate (P = 0.011). Central nervous system (CNS) involvement occurred in 38.3% of patients. The mortality rate was higher in patients with CNS disease . The 3-yr overall survival rate was 35.9%. . In Cox regression analysis, platelet count < 100,000/ μ l  correlated with increased mortality in patients with primary HLH.The median age at diagnosis was 11.5 months (range 1–170), and 56.7% were male. Fever and splenomegaly were the most frequent clinical presentations. 93.3% of the subjects had an HScore > 169, which had a good correlation with HLH-2004 criteria (VL is a potential source of secondary HLH in regions with high endemicity. Treatment of the underlying disease in VL-associated HLH is sufficient in most cases, with no need to start etoposide-based chemotherapy. Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of excessive inflammation and tissue destruction due to abnormal immune activation. The absence of normal downregulation by activated macrophages and lymphocytes is possibly the cause of hyper inflammation and dysregulated immune state . InfantsHLH presents as a febrile illness associated with multiple organ involvement. Thus, initial signs and symptoms of HLH can mimic common infections, fever of unknown origin, hepatitis, or encephalitis . TherefoMycobacterium tuberculosis, among many others. Moreover, autoimmune and rheumatologic disorders, malignancies, and metabolic disorders may trigger the cytokine storm [On the other hand, secondary  HLH is generally used to describe patients without a known familial mutation who typically have a clear trigger for developing HLH. It usually follows an infectious disease caused by organisms, including Epstein-Barr virus (EBV), cytomegalovirus (CMV), hepatitis viruses, varicella infection, leishmania, ne storm .A few small case series of primary or secondary HLH have been reported in Iran –10. AddiThis prospective observational study was conducted in a tertiary oncology referral center in Shiraz, Fars province in Southern Iran, 2014–2018. During the study period, patients with a confirmed diagnosis of HLH were included. HLH was diagnosed based on the diagnostic criteria proposed by the Histiocyte Society in 2004 [Patients with a positive family history or with disease-causing mutations in the genes encoding perforin (FHL2), Munc 13–4 (FHL3), Syntaxin 11 (FHL4), Munc 18–2 (FHL5), Lyst (Chediak-Higashi syndrome), and Rab 27A (Griscelli syndrome type 2), signaling lymphocyte activation molecule associated protein (XLP1) and X-linked inhibitor of apoptosis (XLP2) were defined as primary HLH. Patients with a confirmed VL diagnosis with a blood or bone marrow polymerase chain reaction (PCR) or indirect fluorescent antibody (IFA) test who fulfilled HLH-2004 criteria were defined as VL-associated HLH. In addition, information on demographic characteristics, clinical, laboratory, and radiological findings at presentation, treatment response, and survival outcomes were collected. HScore was calculated in each patient to estimate the probability of HLH in our patients and compare it with HLH-2004 diagnostic criteria. Participants in this study were assented to come in the survey according to the local Ethics Committee of the Shiraz University of Medical Sciences (IR.sums.med.rec. 1396.s195). A written informed consent was signed by the parents or the legal guardians of the participants.Based on our HLH-management protocol, the initial diagnostic workup included serological tests for EBV, CMV, human immunodeficiency virus, Brucella, Salmonella, and VL. In any suspected case of viral-associated HLH or leishmania-associated HLH, an additional molecular test with real-time polymerase chain reaction (RT-PCR) was requested to quantify viral or parasite load. RT-PCR provides valuable information about baseline virologic or parasitic status and is very useful for monitoring and response to treatment. The molecular diagnosis of disease was assessed with whole-exome sequencing with the next-generation sequencing (NGS) method in most study patients. The mutations found by NGS were confirmed by Sanger method.sCD25 was measured using a commercial ELISA kit  (sensitivity: 4 pg/ml) based on the manufacturer’s instructions. We determined the levels of sCD25 within the sample by setting up a standard curve of known target protein concentrations provided in the kit. Normal ranges for sCD25 in this study were 1.9–13.1 pg/ml.CNS involvement was defined if the patients had neurologic signs and symptoms including seizure, altered level of consciousness, or focal neurologic signs in addition to one of these two conditions: 1. pleocytosis and/or proteinosis in cerebrospinal fluid or 2. radiologic abnormalities on magnetic resonance imaging (MRI) such as high signal intensity lesions, hemorrhage, atrophy, and leptomeningeal enhancement.2/day twice a week for 2 weeks and then once weekly for the next 6 weeks. Besides, dexamethasone 10 mg/m2/day for 2 weeks was started. The dose was halved every 2 weeks and tapered and discontinued on the last week of induction therapy. Patients with evidence of CNS disease were additionally treated with intrathecal methotrexate if the symptoms did not improve with systemic treatment. The continuation therapy included etoposide 150 mg/m2/day every other week with pulses of dexamethasone 10 mg/m2/day for 3 days and oral cyclosporine 6 mg/kg/day aiming at blood trough level around 200 mcg/L. The treatment continued for up to 40 weeks unless hematopoietic stem cell transplantation from a matched donor was available. Most of the patients with leishmaniasis-associated HLH (94.7%) were only treated with liposomal amphotericin B  and 95% confidence interval (CI). A P-value of less than 0.05 was considered statistically significant.Data were analyzed by the Statistical Package for the Social Sciences . Descriptive data were presented as median, interquartile range (IQR), and percentages. The comparison of qualitative variables among different groups was made by the Chi-square test or the Fisher exact test. Quantitative variables with non-normal distribution were compared with the non-parametric Mann-Whitney U test between two groups. The Pearson correlation tested the correlation between the 2004-HLH diagnosis criteria and HScore. Univariate analysis was used to test possible covariates associated with outcome measures, including CNS involvement, relapse, and death. Those variables with a Sixty patients with a confirmed diagnosis of HLH were enrolled. The clinical features and laboratory findings of patients at their initial presentation are shown in Table Fever and splenomegaly comprised the most frequent initial clinical findings, with 100 and 96.7% prevalence, respectively. Similarly, high serum ferritin (> 500 ng/ml) and high serum lactic dehydrogenase (LDH) (> 450 U/dL) were found exclusively in all patients. The study population’s median serum ferritin was 4825 ng/ml . Extreme hyperferritinemia  was detected in 16 (26.7%) of individuals. sCD25 was measured in one-third of the study population. It was increased in 14 (70%) out of 20 individuals.P = 0.012) and increased alanine transaminase (ALT) (P = 0.016) compared to secondary HLH. Moreover, the risk of death was significantly higher in primary HLH (P < 0.001). On the other hand, patients with VL-associated HLH had higher serum ferritin (P = 0.034). Moreover, they included a higher proportion of patients with high ESR (> 20 mm/hr) than primary HLH (P = 0.011). None of the patients in this group showed skin eruptions, bleeding tendency, or bone marrow hemophagocytosis. Nobody experienced disease relapse, and all of them survived.The clinical and laboratory characteristics of patients with primary and VL-associated secondary HLH were compared in Table P > 0.99). HScore significantly correlated with HLH-2004 criteria .Fifty-six (93.3%) patients achieved a score ≥ of 169, with no difference in the two groups of primary and secondary HLH (P = 0.133). In univariate analysis, age < 2 years (P = 0.024), bone marrow hemophagocytosis (P = 0.027), and bicytopenia (P = 0.02) were associated with CNS involvement (Table P = 0.016). However, no risk factor was found in patients with VL-associated HLH. Binary logistic regression revealed that only age < 2 years (Odds ratio (OR) 6.446, 95% CI 1.538–27.012, P = 0.011) increased the odds of CNS involvement. On the other hand, patients with bicytopenia had less chance of neurologic disease . Patients with CNS disease had a worse outcome than those without CNS involvement (death rate 61% vs. 35%) with marginal statistical significance (P = 0.051).CNS involvement occurred in 23 (38.3%) patients. The proportion of patients with CNS involvement was not significantly different between primary and secondary HLH groups  showed a treatment response. Twenty-nine patients (87%) finished their treatment successfully. They were all alive and off-treatment at the end of the study. Treatment was ongoing in 4 more patients with an excellent initial response to treatment. Twenty-six (43.3%) patients died during treatment that all of them had primary HLH.P < 0.001).Fig. P = 0.001), total bilirubin > 1 mg/dL (P = 0.008), and erythrocyte sedimentation rate (ESR) ≤ 20 mm/hr. (P = 0.02) were associated with mortality. In Cox regression analysis, platelet count < 100,000/ μ l  remained an independent risk factor of mortality.Table During the 40 months follow-up period, six patients (10%) experienced disease relapse. All of them occurred in boys with primary HLH. In multivariable analysis, no independent risk factor was found.The current study represents the first study on HLH patients in a large referral oncology center in Southern Iran. It is also one of the most extensive case series of secondary HLH associated with VL. It was reported that VL is associated with about 2% of all cases of HLH . HoweverThe clinical sign and symptoms and laboratory parameters in HLH associated with VL were very similar to primary HLH. Fever and splenomegaly were the most consistent presenting signs and symptoms detected in nearly all patients. Serum ferritin at diagnosis was higher than 900 ng/ml in all patients, and about 30% of them had extreme hyperferritinemia . However, it did not correlate with disease severity or adverse outcomes such as death or relapse. Allen et al. reported that ferritin level over 10,000 ng/ml was 90% sensitive and 96% specific for HLH .On the other hand, a recent review concluded that HLH is a rare cause of extreme hyperferritinemia, and more common causes such as liver disease and malignancies should be ruled out first . Since tPatients with VL-associated HLH experienced higher serum ferritin and ESR. Though the underlying infection may be a simple explanation, enhanced ferroportin-mediated iron efflux due to overexpression of growth differentiation factor 15 may play a role . On the Nearly 40% of our patients had evidence of CNS involvement. The rate of CNS involvement in HLH varies in different studies ranging from 18 to 73% . Some reMany reports are indicating that CNS involvement might be associated with worse outcomes (19). Although the overall mortality rate was higher in those with CNS events, this association was not shown in multivariable analysis. It is generally agreed that CNS involvement poses a higher risk of relapse unless hematopoietic stem cell transplantation (HSCT) is undergone early after starting systemic therapy .HScore had a good correlation with HLH-2004 criteria for the diagnosis of HLH in our study. It was reported that HScore carries a diagnostic sensitivity of 90% and specificity of 79% for HLH . A cutofThe treatment response was 55% in our study cohort with a 3-yr OS rate of 36%. It was much lower in patients with primary HLH than secondary HLH (24% vs. 100%). All patients with VL-associated HLH were successfully treated with liposomal amphotericin. Nobody needed to be treated with etoposide or cyclosporine. Dexamethasone was initially started in a few patients with severe disease but was tapered and discontinued after a few weeks. The outcome was excellent, with no report of death or HLH relapse during the 40 months follow-up. Targeted treatment of the etiologic pathogen usually is the only treatment strategy recommended in secondary infection-associated HLH such as brucella and leishmania. Specific antimicrobial therapy usually is associated with complete recovery in most cases –31.High-dose intravenous gamma-globulin (IVIG) has been used successfully in viral-associated hemophagocytic syndrome ; howeverPlatelet count less than 100,000/μl was independently associated with more than four times the increased risk of death in our patients with primary HLH. The outcome of patients with HLH and associated prognostic factors differ among different studies. A recent study in Japan reported a much better outcome in their patients with a 3-yr OS rate of 74% . Koh et Our study faced some limitations. Firstly, some specific laboratory tests, such as NK cell function, were unavailable in our center. Besides, sCD25 was measured in a portion of our patients since it was available in our center in 2017. These tests’ unavailability might lead to a delayed diagnosis and poor outcome, evident as a lower 3-yr OS rate in our study cohort than many other reports.VL is a potential source of secondary HLH in regions with high endemicity. The main clinical findings and complications such as CNS involvement are the same as primary HLH. It is associated with higher serum ferritin and ESR but a lower incidence of thrombocytopenia and transaminitis than primary HLH. Treatment of the underlying disease in VL-associated HLH is sufficient in most cases, with no need to start etoposide-based chemotherapy. The prognosis is excellent, with no reported death or recurrence of disease in our case series.


China is one of 30 countries with a high tuberculosis (TB) burden, and poor adherence to TB treatment is one of the biggest challenges for TB control. We aimed to explore the barriers and facilitators of treatment adherence among drug-sensitive tuberculosis (DS-TB) patients under the “Integrated model” in Western China, to provide evidence-based treatment and control regimens for DS-TB patients to improve adherence behaviours.Both qualitative and quantitative research methods were used to explore the factors associated with self-reported adherence (SRA) behaviours. Questionnaire surveys with DS-TB patients and in-depth interviews with leaders from the Centers for Disease Control and Prevention (CDC) and community health sectors (CHCs), healthcare workers (HCWs) from CHCs, and DS-TB patients were conducted.A total of 459 eligible patients were included in the quantitative survey, and two patients and 13 healthcare providers were included in the in-depth interviews. The percentage of patients who experienced a missed dose, lack of follow-up sputum examination, and interrupted treatment were 19.0%, 11.3%, and 9.2%, respectively. Patients aged 20–39 had a higher risk of missed dose [OR (95% CI): 2.302 (1.001–5.305)] and a lower risk of interrupted treatment [OR (95% CI): 0.278 (0.077–0.982)] than patients more than 60 years. Patients who were of Han ethnicity (OR [95% CI]: 0.524 [0.301–0.912]) received psychological support (OR [95% CI]: 0.379 [0.144–0.998]) from their family and had a lower risk of missed doses. Patients who had drug side effects had a higher risk of interrupted treatment (OR [95% CI]: 2.587 [1.237–5.412]). Patients who possessed higher knowledge had a lower risk of lack of follow-up sputum examination [OR (95% CI): 0.817 (0.673–0.991)]. The results of the qualitative study also reported that patients’ poor TB knowledge was the main reason for their non-SRA behaviours.Patient-centred strategies should be implemented to improve health literacy and strengthen psychological support. More effective case management should be designed and implemented based on different patient characteristics to improve adherence behaviours in further studies. Tuberculosis (TB) is a communicable disease with a long history , 2, and In response of non-adherence to treatment for TB patients, the WHO proposed direct observation of treatment and short course (DOTs), which needs to be monitored on a regular basis to maintain treatment adherence for six to eight months for DS-TB patients and 18–24 months for MD-TB/ RR-TB , 10. DOTThere is an average of 695 per 100,000 infection rate of TB in the western part of China and an average of 463 per 100,000 and 291 per 100,000 infection rate in the central and eastern part of China that shows the western part of China suffers the higher TB prevalence than in the eastern and central part of China . ChongqiThis study aimed to evaluate the self-reported adherence (SRA) behaviours and the association between the above factors and SRA behaviours under the “integrated model’ among DS-TB patients in Southwest China, and to explore the reasons for poor adherence behaviours from the perspectives of both healthcare demanders (patients) and healthcare providers, to further provide evidence-based treatment and control regimens for DS-TB patients.A cross-sectional study was conducted in Guizhou and Chongqing using both qualitative and quantitative research methods to explore the factors predicting SRA behaviours among patients with DS-TB.Consecutive sampling was used to recruit patients with DS-TB as participants for the quantitative study. Participants who enrolled in our study met the inclusion and exclusion criteria. The inclusion criteria were as follows: (1) TB patients who were registered in a TB dispensary; (2) patients aged > 15 years; (3) received at least four months of TB treatment and no more than four months after complete treatment. Exclusion criteria: (1) patients with intellectual disability and difficulty in speech and hearing; (2) patients with severe complications  or who underwent hospitalisation; and (3) patients who refused to participate in this survey.This questionnaire was designed by our research team, who reviewed the existing literature reports and then consulted related experts before the pilot study. Then, a pilot test was conducted with 100 participants, and the Cronbach’s alpha value of the pilot study was 0.753. Trained investigators executed all questionnaires from our research group, and the completed questionnaires were checked and examined by trained investigators for quality control.A self-designed structured questionnaire was administered to collect data, including socio-demographic information, knowledge about TB , and cuThe reliability and validity analysis of the scoring system was conducted to test the validity and consistency of the questionnaire, and the Kaiser–Meyer–Olkin (KMO) and Cronbach’s alpha values were used to evaluate the internal consistency and construct validity of the scoring system . The resPurposive sampling was used to select DS-TB patients for in-depth interviews and healthcare providers , and health care workers (HCWs) from CHCs for key informant interviews. The inclusion and exclusion criteria for patients with TB were consistent with those of the quantitative study. Healthcare providers who are engaged in the management of TB patients were included in this study. The sample size was determined by the number of participants required to reach data saturation.Semi-structured topic guides were used in all interviews. The topic guide for participants included adherence behaviours and associated barriers and facilitators from both patients’ and healthcare providers’ perspectives. Three senior researchers conducted interviews in Chinese. Each interview lasted approximately 40–60 min. All interviews were audio-recorded and transcribed for the analysis.2) test, T test, and logistic regression analysis were conducted to explore the factors associated with SRA behaviours. A two-tailed probability level of p < 0.05 was selected as the statistically significant level.Quantitative data were compiled in Epi Data 3.1, and analysed using Statistical Package for Social Science (SPSS 22.0) . The test for normality revealed that the numerical data were normal, so numerical variables were reported as mean ± SD and categorical variables as percentages. Missing data were excluded from the analysis. Chi-square  patients were included in this study after quality check of questionnaire. The sociodemographic characteristics are shown in Table Totally 15 participants were included in qualitative study. Two DS-TB patients were included in the in-depth interviews. One male patient was married, a local resident who lived in an urban area, covered by BMI, and had no work or education level of primary school. Another female patient was unmarried; migrants lived in a rural area, covered by BMI, and worked as a clerk in a private enterprise. Both were newly diagnosed treatment patients. A total of 13 healthcare providers, including eight leaders from the CDC, two leaders, and three HCWs from CHCs, were purposively selected for key informant interviews. Leaders from the CDC were in charge of TB prevention and control, and leaders and HCWs from CHCs were in charge of TB patient management.The SRA behaviours are depicted in Fig. As Table As for TB knowledge score, patients who reported interrupted treatment and lack of follow-up sputum exam were scored 2.81 ± 1.50 and 2.87 ± 1.51 respectively, significantly lower than those who adhered to treatment (3.41 ± 1.59) and sputum exam (3.42 ± 1.59) (P < 0.05)  received family support, including psychological , economic support , and nutritional support . As Table Of the patients with DS-TB, 54.2% reported drug side-effects, and most (88.5%) patients had symptoms Table . CompareIn total, 92.4%, 90.8%, and 52.3% of DS-TB patients were satisfied with the management of HCWs in CDC/TB dispensaries, CHCs, and village clinics, respectively. Furthermore, 0.7%, 7.4%, and 46.8% of DS-TB patients self-reported that they did not receive management from HCWs in CDC/TB dispensaries, CHCs, and village clinics, respectively. Patients who were satisfied with the management of HCWs in the CDC/TB dispensary had a lower risk of interrupted treatment when compared with patients who self-reported not being satisfied (7.5% versus 28.1%) and did not receive the management (7.5% versus 33.3%) from HCWs in the CDC/TB dispensary (P < 0.05). Patients who were satisfied with the management of HCWs in village clinics had a lower risk of missed dose when compared with patients who self-reported not being satisfied (14.2% versus 75.0%) and not receiving HCWs in village clinics (14.2% versus. 23.3%) (P < 0.05) (Table Results of binary logistic regression analysis Table  demonstrThe results of the qualitative study are shown in Table Standard treatment regimens in DOTs require six months of treatment for initial TB patients and eight months for retreatment TB patients . AdherenIn this study, SRA behaviours included missed dose, interrupted treatment, and lack of follow-up sputum examination. We found that almost 20% of DS-TB patients had missed doses, which occurred most frequently, followed by a lack of follow-up sputum examination and interrupted treatment. The incidence of poor adherence was much higher in a previous study on MDR-TB patients in Chongqing  but loweUnlike the previous study, patients with MDR-TB were mainly young and middle-aged . Most DSInadequate knowledge about TB was a determinant factor for adherence behaviours, which has been reported in many previous studies –48. Our Previous studies indicated that patients who received social support from friends and families were more likely to take their medication regularly; besides, financial and food support were the main factors associated with adherence , 49, 50,Previous studies reported that drug side-effects were the major reasons for poor adherence behaviours , 52, 54,Many studies have indicated that the health system is a major factor influencing adherence behaviours for TB patients, including case management from CHCs and satisfaction with healthcare services , 56, 57.This study had several limitations. First, only two of the 88 counties in Guizhou province were selected as representative counties of Guizhou, and the sample counties would increase in future studies to ensure good representation. Second, information bias existed in this study for some patients with low education levels, and they had a poor understanding of the contents of the questionnaire.Poor adherence to treatment is one of the biggest challenges for TB control. Strategies to improve health literacy and strengthen psychological support are urgently needed for TB patients. More attention should be paid to patients from minorities with drug side-effects, aged 20–39 and over 60 years old. Patient-centred intervention measures should be designed and implemented to improve adherence behaviours.


The aim of this multicenter cross-sectional study was to analyze a cohort of breast (BC) and gynecological cancer (GC) patients regarding their interest in, perception of and demand for integrative therapeutic health approaches.BC and GC patients were surveyed at their first integrative clinic visit using validated standardized questionnaires. Treatment goals and potential differences between the two groups were evaluated.p = 0.006), obstipation (< 0.005)].340 patients  participated in the study. The overall interest in IM was 95.3% and correlated with older age, recent chemotherapy, and higher education. A total of 89.4% were using integrative methods at the time of enrolment, primarily exercise therapy (57.5%), and vitamin supplementation (51.4%). The major short-term goal of the BC patients was a side-effects reduction of conventional therapy (70.4%); the major long-term goal was the delay of a potential tumor progression (69.3%). In the GC group, major short-term and long-term goals were slowing tumor progression (73.1% and 79.1%) and prolonging survival (70.1% and 80.6%). GC patients were significantly more impaired by the side-effects of conventional treatment than BC patients [pain (Our data demonstrate a high overall interest in and use of IM in BC and GC patients. This supports the need for specialized IM counseling and the implementation of integrative treatments into conventional oncological treatment regimes in both patient groups. Primary tumor site, cancer diagnosis, treatment phase, and side effects had a relevant impact on the demand for IM in our study population. Over the past decade “Complementary and Alternative Medicine” (CAM) as well as “Integrative Oncology” have become increasingly popular as part of a holistic treatment approach in gynecologic oncology. This is partly due to the fact that advances in conventional cancer treatments have led to a prolonged life expectancy and a more pronounced interest in short-term and long-term quality of life (QoL).According to the National Center for Complementary and Integrative Health (NCCIH), CAM is the popular name for non-mainstream health care practices that are not part of the standard medical treatment and are used to support or replace conventional medical therapies  –3. IntegThere has been a marked increase in the demand for IM in the oncological patient population over the past years . IM is bA significant correlation between an interest in IM and a younger age (< 60 years) and the absence of metastases at the time of diagnosis has been described in BC patients , 22−27. Although there is data on the prevalence of IM use for oncological patients, there is little data regarding IM for those patients treated in highly specialized academic oncological centers (comprehensive cancer centers), and information is still limited on the potential impact of counseling for different cancer patient subgroups .The aim of this multicenter cross-sectional study was to evaluate the interest in, use of and demand for IM in patients with BC and GC in the setting of a comprehensive cancer center. In addition, we wanted to identify individual treatment goals that patients sought to achieve using IM, as well as the frequency of the applied IM methods. Lastly, we wanted to elucidate the association between patient and therapy-related characteristics and adverse effects and to gather information on two different specialized integrative consultancy services in the south and north of Germany.n = 229, UKE n = 111) aged 23−84 who sought counseling in a specialized IM service. Both comprehensive cancer centers had implemented IM as part of clinical routine in a standardized form years ago. The physicians who counsel with regard to IM are specialists in gynecological oncology and members of the “Arbeitsgemeinschaft Gynäkologische Onkologie Kommission Integrative Medicine” (AG IMed) who are certified in naturopathic and anthroposophic medicine as well as nutrition sciences.Over a period of 20 months, the multicenter nonsponsored cross-sectional study of the Department of Gynecology and Obstetrics, Technical University Munich (TUM), Klinikum rechts der Isar and the Department of Gynecology and Gynecologic Oncology at the University Medical Center Hamburg-Eppendorf (UKE) prospectively enrolled 340 patients with gynecologic malignancies .p < 0.05. The data management and statistical analyses were performed using the statistical software SPSS, Version 20 .Descriptive statistics, such as absolute and relative frequencies as well as means and standard deviations were generated to determine the prevalence and patterns of CAM use, the demand for IM approaches, the differences in the types of cancer, the frequency of metastases, the current treatment, symptomatic complaints and the treatment goals the patients hoped to achieve. An one-way ANOVA and chi-squared test were conducted for hypothesis testing on potential associations between the interest in IM and the application of CAM, as well as sociodemographic characteristics. A potential association between the type of cancer and the two comprehensive cancer centers were analyzed using chi-square analysis for the comparison of absolute and relative frequencies and Fisher´s exact test . Patients with missing values were excluded from the analysis of the corresponding variables. The level of significance was set at n = 229, UKE n = 111) with a median age of 53 (range 23−84) were included in the study. The distribution of the different types of carcinomas is displayed in Table n = 94/339) had metastasized disease  . There was a significant association between a higher degree of education and a higher interest in IM (p = 0.021)  > 91.8% (56/61), current CTX: 60.5% (124/205) > 86.8% (178/205), no-CTX: 65.2% (43/66) > 95.5% (63/66), metastasis: 67.4% (62/92) > 89.1% (82/92)). Patients who had recently undergone a CTX applied more methods of IM at the time of enrolment than patients without a prior CTX or currently under CTX ). The amount of different IM methods increased with higher educational levels.A total of 63.8% of patients had previously (before study enrolment) applied some IM methods and 89.2% patients were using some IM method at the time of the study enrolment  undergone a CTX while 19.8% had never received CTX). The The difference between the use of different IM methods at present and in the past is depicted in Fig. Both in the BC group (BCG) and the GCG, the reduction of potential side effects of the conventional therapies, the stabilization of body, soul, and spirit, as well as active participation in the treatment of their cancer were the most important short-term goals. The delay of a potential progression of disease, an attempt to prolong survival as well as the stabilization of body, soul, and spirit were major long-term goals Table . A possin = 49/67 in the GCG vs. 58.1%, n = 157/270 in the BCG; p = 0.024). In addition, significantly more patients with GC reported the prolongation of survival time ; long-term: 80.6%, n = 54/67 in the GCG vs. 63.7%, n = 172/270 in the BCG (p = 0.008)) and a reduction of cancer-associated symptoms ; long-term: 46.3%, n = 31/67 in the GCG vs. 30.7%, n = 83/270 in the BCG (p = 0.016)) as a short-term or long-term goal.At the time of enrolment 249 patients (96.1%) with BC and 57 GC patients (91.9%) indicated an interest in IM and a total of 238 (89.1%) BC patients and 60 (89.6%) GC patients were using some IM method. The methods used in both groups are shown in Figs. n = 127/207 (61.4%) vs. recent CTX: n = 35/61 (57.4%) vs. no CTX: n = 27/66 (40.9%), p = 0.014) and obstipation (current CTX: n = 47/207 (22.7%) vs. recent CTX: n = 4/61 (6.6%) vs. no CTX: n = 6/66 (9.1%), p = 0.002) than patients after recent CTX or without CTX. Patients with the recent CTX had more pain (recent CTX: n = 37/61 (60.7%) vs. current CTX: n = 78/207 (37.7%) vs. no CTX: n = 20/66 (30.3%), p = 0.001), climacteric symptoms (recent CTX: n = 14/61 (23.0%) vs. current CTX: n = 22/207 (10.6%) vs. no CTX: n = 13/66 (19.7%), p = 0.025) and impaired sexual activity (recent CTX: n = 17/61 (27.9%) vs. current CTX: n = 32/207 (15.5%) vs. no CTX: n = 6/66 (9.1%), p = 0.014) than patients with current CTX or without CTX. Patients with palliative therapy were more affected by fatigue  vs. adjuvant: n = 55/126 (43.7%) vs. neoadjuvant: n = 57/90 (63.3%), p = 0.001) and pain  vs. adjuvant: n = 52/126 (41.3%) vs. neoadjuvant: n = 23/90 , p = 0.001) than patients treated in a curative setting.Chemotherapy-associated side effects, such as reduced cognition, fatigue, pain, menopausal symptoms, diarrhea, obstipation, depression, and reduced sexual activity were also analyzed in BC and GC patients Table . More paIn line with previous research, our data reflect the recent trend towards an increased interest in IM (> 90%) among BC- and GC patients. Likewise, there was comparably high use of IM at the time of study enrolment (89.1% in the BCG and 89.6% in the GCG), and a mean number of four IM methods were being applied (BCG 4.45 SD ± 3.98 and GCG 3.54 SD ± 2.82).When compared with the existing evidence, this demonstrates an even higher use of IM; a number of studies may have underestimated the high prevalence of IM among women with BC and GC , 31–33. With an estimated incidence of almost two million BC cases and nearly 300,000 new cases of OC in 2018 around the globe, the interest in and frequent use of IM applies to a large number of patients . Many stThere is immense variation in preferred IM practices between studies from different countries; practices are often chosen based on a specific cultural background , 42–44. The current medical field of exercise interventions in BC is enormous. Large observational studies on BC patients show an inverse relationship between physical activity (before and after diagnosis) and overall mortality, BC-specific mortality, and BC events  , 46. Exep = 0.006) and obstipation (p = < 0.005). Symptom-related research in patients with a malignancy has shown that physical activity is associated with improvement concerning physiological complaints, such as pain, peripheral neuropathy, and fatigue as well as in the psychological symptoms of anxiety and depression [In OC patients, physical activity seems just as important, although evidence is scarce, particularly on long-term outcomes. OC continues to have a poor prognosis, with a 5-year survival rate of approximately 43% across all stages (RKI 2016). Faced with a poor prognosis, stressful treatments and a high likelihood of recurrence, women with OC confront significant physical and psychological morbidities that are likely to have negative influence on their health-related QoL. In our cohort, GC patients are more impaired in their QoL than patients with BC, reaching statistical significance in the category pain .Reducing the side effects of conventional oncological therapies, stabilizing body, soul, and spirit, and active treatment participation were the most important short-term goals of BC patients Table . The majThe data regarding the goals of GC patients in integrative oncology are scarce. Within our cohort, prolonging survival, slowing the progression of disease, and improving disease-related QoL were major short-term and long-term goals in GC patients. Interestingly, neither a delay of disease progression nor an improvement in survival was a short-term or a long-term goal in 30−40% of all patients  apply IM methods, starting at the time of diagnosis, and that most are satisfied with them. Only 6.5% of these women reported no benefit from IM . A recenThis again underlines the importance of specialized integrative services at comprehensive cancer centers where patients can be counseled on safe and effective IM methods to help alleviate treatment-based and disease-related side-effects while keeping potential interactions with chemotherapies and other conventional treatments in mind and creating a larger body of evidence.Trained health-care professionals, a standardized setting, good communications skills, time, and an open discussion on IM issues are key to protect patients from an inappropriate and potentially dangerous use of IM. Oncologists should proactively communicate information on IM and explain possible supportive treatment options at the same time they make patients aware of the potential harm of some of these therapies.The results of the current study have some limitations. First, our survey data are prone to recall bias since it is based on the self-reporting. Patients presenting to our associated integrative medicine service were generally interested in IM; thereby, introducing an element of selection bias. Secondly, regardless of their stage of disease, BC and GC patients must be considered a rather heterogeneous group. Understandably, different stages and phases of disease may result in different demands for integrative medicine support services. Thirdly, some of the studies quoted include mind/body therapies, others solely evaluated "biological therapies". Overall, the wide range of any generous definition of IM and CAM may result in a study overestimating the number of IM users .To our knowledge, this is the first bicentric observational study analyzing patients with both BC and GC who seek counseling in IM at two large comprehensive cancer centers in Germany. Patients of all ages and tumor stages are strongly interested in IM as a supplement to their conventional oncological treatment. In contrast to other studies, this survey underlines that patients older than 60 years are also interested in IM. The results show that nutritional counseling as well as physiotherapeutic approaches such as physical activity, massage, and lymph drainage should be routinely offered to patients and financially supported as part of their cancer treatment. Further psychological counseling as well as mind–body approaches should be offered within a comprehensive oncological treatment approach.The alleviation of physical symptoms and the desire to improve health behaviors is of paramount importance to BC and GC patients in regards to their supportive care needs. BC and GC patients place a special focus on IM methods to achieve these goals. The primary tumor site, BC versus ovarian cancer, treatment phase and side effects had a relevant impact on the patients` demand for IM in the current study. Therefore, it is crucial to incorporate IM counseling into the conventional oncological therapy. Since comprehensive cancer centers spearhead oncological research and innovations, these centers need to ensure a financially sustainable integration of IM into the oncological treatment and follow-up. In addition, evidence-informed IM needs to expand beyond the walls of academic medical centers in order to ensure access to IM for patients from diverse socioeconomic backgrounds in an oncological setting.


We conducted population pharmacokinetic (PPK) analysis and Monte Carlo simulations to determine the appropriate prophylactic dose of fluconazole to prevent invasive candidiasis in patients with hematological malignancies. Patients receiving chemotherapy or hematopoietic stem cell transplantation at Yokohama City University Hospital between November 2018 and March 2020 were included. Additionally, patients receiving oral fluconazole for prophylaxis were recruited. We set the free area under the curve/minimum inhibitory concentration (MIC) = 50 as the target and determined the largest MIC (breakpoint MIC) that could achieve more than 90% probability of target attainment. The blood fluconazole concentration of 54 patients (119 points) was used for PPK analysis. The optimal model was the one-compartment model with first-order administration and first-order elimination incorporating creatinine clearance (CLcr) as a covariate of clearance and body weight as a covariate of distribution volume. We conducted Monte Carlo simulation with fluconazole at 200 mg/day or 400 mg/day dosing schedules and patient body weight and CLcr ranging from 40 to 70 kg and 40–140 mL/min, respectively. The breakpoint MICs on the first dosing day and at steady state were 0.5–1.0 μg/mL and 1.0–2.0 μg/mL for 200 mg/day and 1.0–2.0 μg/mL and 2.0–4.0 μg/mL for 400 mg/day, respectively. The recommended dose was 400–700 mg/day for the loading dose and 200–400 mg/day for the maintenance dose. Our findings suggest that the optimal prophylactic dose of fluconazole in hematological malignancy patients depends on CLcr and body weight, and a sufficient loading and maintenance dose may be needed to completely prevent invasive candidiasis. Invasive candidiasis is one of the most common fungal infections and includes candidemia and deep-seated candidiasis . HematolCandida infection in the pre-engraftment of allogeneic transplantation, autologous transplantation, and chemotherapy-induced neutropenia [In the Japanese Domestic Guidelines for Management of Deep-seated Mycosis 2014, fluconazole, itraconazole, and micafungin are recommended for the prophylaxis of invasive tropenia . Gastroitropenia . Althougtropenia , it is ntropenia . TherefoTherapeutic efficacy has been reported to be correlated with fluconazole dose , and henPopulation pharmacokinetic (PPK) analysis can be used to obtain the pharmacokinetic parameters required for drug dosage optimization. Several PPK analyses of fluconazole have been reported ,18,19,20In this study, we built a PPK model of fluconazole in patients with hematological malignancies and investigated the optimal dosage using Monte Carlo simulation.This study was approved by the Yokohama City University Ethics Committee  and the Keio University Faculty of Pharmacy . Written informed consent was obtained from all participants who were administered fluconazole at the Yokohama City University Hospital.Among the patients admitted to the Yokohama City University Hospital in Japan between November 2018 and March 2020 who received chemotherapy or hematopoietic stem cell transplantation for hematological malignancy, only those who met the following criteria were enrolled in this study: age ≥ 16 years and prophylactic fluconazole administration. Patients aged < 16 years who could not provide consent or critically ill patients were excluded.Fluconazole was administered orally at a dose of 200 mg once daily every 24 h. Blood samples were collected at 1−4 points at indicated time (1 h before (trough levels), 2, 4, or 12 h) after fluconazole administration. Plasma was immediately extracted and stored at −20 °C at the hospital. Plasma was then transported to the Keio University Faculty of Pharmacy and stored at −80 °C until measurement. Clinical and patient background data on the first day of fluconazole administration were collected. Body surface area (BSA), creatinine clearance (CLcr), and estimated GFRcre (eGFRcre) were calculated using the Dubois formula , Cockcro® RP-18 GP 250–4.6 (5 μm) analytical column was used, the measurement temperature was 40 °C, the mobile phase was 10 mM phosphate buffer (pH 5.70): methanol = 65:35, and the wavelength of the ultraviolet absorption detector (SPD-20A) was set to 266 nm.Fluconazole Standard solutions ) or samples were mixed with dichloromethane, and the organic phase was evaporated and then redissolved in the mobile phase where the resulting solution was used as a sample. A calibration curve was prepared using the standard solution, and the concentration of the sample was measured. Fluconazole concentrations were measured using high-performance liquid chromatography . The HPLC conditions were as follows: MightysilPPK analysis was performed using the Phoenix NLME™ . First-order conditional estimation-extended least squares (FOCE-ELS) methods that are equivalent to the NONMEM FOCE methodology with interaction (FOCE-I) were used throughout the modeling process. First, one-, two-, and three-compartment models with first-order administration and first-order elimination were tested to determine fluconazole distribution. The PK models were assessed using statistical methods. Additive, multiplicative, additive and multiplicative models were tested to describe the residual variability model. The optimal base compartment model was evaluated for statistical significance using the objective function value  and Akaike information criterion. Subsequently, the final model was evaluated, including covariates that could affect fluconazole PK parameters. The evaluated covariates were age, body weight, height, BSA, body mass index, CLcr estimated using the Cockcroft-Gault formula, and eGFRcre. The covariates were tested using a stepwise forward inclusion and backward elimination model building process.The suitability of the final model was verified using the visual predictive check (VPC) and bootstrap methods (1000 bootstrap runs).® NLME™  and Phoenix WinNonlin™  were used. Following previous reports, the f AUC/minimum inhibitory concentration (MIC) and the protein binding rate were set at 50 [In the PTA analysis, we randomly generated data for 10,000 simulated patients by CLcr and body weight using Monte Carlo simulations and calculated the area under the curve (AUC). Phoenix et at 50 ,26 and 1Twenty-four patient backgrounds were assumed: four weights  and six CLcr . Fluconazole  was simulated.Candida albicans in CLSI M60 1st Edition [f AUC/MIC = 50, was determined by performing Monte Carlo simulations.To determine the prophylactic dose, the target MIC was set at 2.0 μg/mL with reference to the sensitive norm of  Edition  and prev Edition ,29. The 2, 87.1 (31.1–193.6) mL/min, and 71.2 (25.9–148.5) mL/min, respectively. Patients with many types of hematological malignancies were included, and post-transplantation cases were included in this analysis. The fluconazole dose was 200 mg/day in all patients.The characteristics of the 54 patients are listed in The correlation coefficient of the calibration curve was 1.0. The accuracy of the assay ranged from 86.9% to 100.7%. The %CVs of the intraday and inter-day assays using the four test samples were <5.3% and <7.5%, respectively.p < 0.05). The median pharmacokinetic estimates of the final multivariate model were CL/F = 1.2 L/h and V/F = 62.3 L.A total of 125 blood samples were collected from patients, and 119 were used for analysis, excluding six points for which renal function changed substantially during administration. The results of the final model and bootstrap method performed using PPK model of fluconazole are shown in The diagnostic plots and VPC confirmed the appropriateness of the model, as shown in Monte Carlo simulations assuming a fixed dose of 200 mg or 400 mg/day were performed to indicate the breakpoint MIC in each scenario . On the f AUC/MIC = 50 for a target MIC = 2.0 μg/mL, additional Monte Carlo simulations were performed at 300, 400, 500, and 600 mg/day in each scenario. On Day 1, the required doses were proportional to body weight when CLcr was the same  parameter of fluconazole has been reported to be AUC/MIC [f AUC/MIC = 50, which Cojutti et al. [Candida albicans in CLSI M60 1st Edition [f AUC/MIC = 50 and MIC = 2 μg/mL). Fluconazole 400 mg/day prophylaxis was considered sufficiently feasible to reduce fungal infections in patients with hematological malignancies. Goodman et al. reported that fluconazole 400 mg/day was adequate to reduce fungal infections in bone marrow transplantation recipients [We showed that fluconazole 400 mg/day achieved  AUC/MIC , we adopi et al.  and Pai i et al.  used, as Edition  and prev Edition ,29. We scipients ; our anacipients ,14; howeWe determined the recommended dose based on body weight and CLcr and showed a loading dose of 400–700 mg/day and maintenance dose of 200–400 mg/day. The breakpoint MIC of fluconazole on the first day of administration was lower than the breakpoint MIC on the steady state. This may be because it takes time for blood concentrations to reach a steady state because the half-life of fluconazole is 31 h . As the f AUC/MIC was 50 in this study. Although f AUC/MIC = 50 is widely used, there is little information regarding the f AUC/MIC of fluconazole. More research on the f AUC/MIC of fluconazole is needed. Fourth, the epidemiology of Candida infections varies across countries. A high target MIC is required to completely prevent breakthrough infection, which is unrealistic. Therefore, the target MIC was set with reference to the sensitive norm of Candida albicans in the CLSI M60 1st Edition. Even though there are several limitations, this is the first informative study to show the optimal dosing regimen based on Monte Carlo simulation for hematological malignancy patients. Robust evidence through prospective studies using these regimens must be obtained.This study has several limitations. First, this was a single-center study. It only included patients with hematological malignancies. We believe this is the first report to provide fluconazole pharmacokinetics information in patients with hematological malignancies whose ARC is a problem. Because the sample size and measurement points of this study were equal to or greater than previously published PPK analysis of fluconazole ,18,19,20This PPK analysis included only patients with hematological malignancies, and the PPK model of hematological malignancy was built for the first time. Model-based Monte Carlo simulations suggested that fixed doses of fluconazole 200 mg/day may be ineffective with CLcr > 60 mL/min. Here, the optimal dosing regimens based on body weight and CLcr render fluconazole prophylaxis more reliable.


Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with an unmet need of biomarkers that can aid in the diagnostic and prognostic assessment of the disease and response to treatment. In this two-part explorative proteomic study, we demonstrate how proteins associated with tissue remodeling, inflammation and chemotaxis such as MMP7, CXCL13 and CCL19 are released in response to aberrant extracellular matrix (ECM) in IPF lung. We used a novel ex vivo model where decellularized lung tissue from IPF patients and healthy donors were repopulated with healthy fibroblasts to monitor locally released mediators. Results were validated in longitudinally collected serum samples from 38 IPF patients and from 77 healthy controls. We demonstrate how proteins elevated in the ex vivo model , and other serum proteins found elevated in IPF patients such as HGF, VEGFA, MCP-3, IL-6 and TNFRSF12A, are associated with disease severity and progression and their response to antifibrotic treatment. Our study supports the model’s applicability in studying mechanisms involved in IPF and provides additional evidence for both established and potentially new biomarkers in IPF.  Idiopathic pulmonary fibrosis (IPF) is an irreversible interstitial pneumonia of progressive nature. Patients with IPF usually present exertional dyspnea and cough that over time ultimately result in restricted ventilation, hypoxemia, respiratory failure and death within a few years from diagnosis . DiagnosSeveral growth factors, cytokines and chemokines involved in regulating coagulation, angiogenesis, inflammation and repair responses have shown to be implicated in the pathogenesis of IPF in a number of preclinical and clinical studies ,7,8,9,10Decellularized lung tissue with retained material properties offers an opportunity to study interactions between cells and ECM of the lung in health and disease. We have created a novel ex vivo model that mimics the structural and biomechanical properties of the lung-ECM by using decellularized tissue, devoid of cellular content, from healthy donors and IPF patients . By repoThis translational study serves two purposes—to study potential biomarkers associated with tissue remodeling, inflammation and chemotaxis by using an ex vivo model, and to analyze these candidate biomarkers in serum from IPF patients and investigate their possible associations to disease severity and progression. We hypothesize that the microenvironment of the lung in IPF governs cellular activity including release of mediators that is reflected in the blood circulation and is associated to disease activity and progression. The repopulated scaffolds derived from end-stage IPF patients and healthy individuals exhibited preserved tissue morphology and viable cell cultures as previously described by Elowsson Rendin et al. . The fibFour   of the 1Analysis of demographic and clinical data of the enrolled patients showed that IPF patients were older, a majority men with a history of smoking, with a restrictive lung function and low DLCO . Classification according to Gender-Age-Physiology stage (GAP), a staging system providing the average risk of mortality in IPF-patients , and CPIp-value < 0.05, ANOVA adjusted for age) (We identified 44 proteins with elevated levels in the baseline samples (corrected for age) . Many of the proteins (30 out of 44) regulated inflammation and chemotaxis. Five of these  had a NPX-difference > 1, i.e., a doubling of protein expression between groups, and 14 proteins had a 1 < NPX-difference > 0.5, meaning an increased protein level of more than 50% . Among tBioinformatic analysis of the elevated proteins observed in IPF patients revealed a network of associated protein-protein interactions , with noPathway analysis of all elevated proteins in IPF revealed several significant associations with signaling pathways such as chemokine/cytokine signaling, NF-kB signaling and TNF receptor binding .A complete list of all proteins and their age-unadjusted protein expressions are available in Thirty proteins demonstrated a significantly altered protein expression in IPF follow up samples compared to healthy controls, out of which 28 proteins were elevated in IPF. Eighteen  were assA complete list of all proteins analyzed and their protein amount at follow up is available in To investigate the proteins’ potential in reflecting the severity of IPF, proteins were correlated with common clinical measures of disease severity at both baseline and follow up . SeveralOther proteins that rendered associations to several measures were, e.g., ARG-1, which was abundantly expressed in IPF patients compared to controls. While no associations with lung function or CPI was observed at baseline, ARG-1 demonstrated an inverse correlation with all lung function measures; FVC %, TLC % and DLCO %, and a positive correlation with CPI at follow up. An opposite but similar pattern was observed for PDGF-subunit B, which was not differentially expressed compared to controls and only associated to FVC % at baseline. At follow up however, PDGF-subunit B correlated with FVC %, TLC %, DLCO % and CPI.We further investigated associations between longitudinal changes in protein levels and lung function . In agrep = 0.04; 0.51 (95% CI: 0.02–1.01), p = 0.03; 0.14 (95% CI: 0.02–0.25), p = 0.03 and 0.36 (95% CI: −0.01–0.73), p = 0.03, respectively). Despite not reaching statistical significance when comparing the two groups, levels of ARG1 at follow up were numerically increased in progressive patients (mean difference 0.47 (95% CI: −0.08–1.02), p = 0.05). CXCL13 levels in progressive patients at follow up were also numerically increased compared to stable patients but not statistically significant (0.43 (95% CI: −0.07–0.93), p = 0.17). IL12 demonstrated an opposite pattern, where progressive patients had numerically lower, but not statistically significant levels at follow up compared to stable patients (−0.60 (95% CI: −1.13–0.07), p = 0.09). Next, patients were divided into stable and progressive disease. Twenty patients had progressed while 17 patients remained stable at follow up. Patients with progressive disease had higher levels of IL6, NOS3, MMP7 and CASP-8 at follow up (mean difference 0.45 (95% CI of difference: 0.03–0.88), p = 0.01)), HGF (0.14 (95% CI: −0.04–0.33); stable: −0.1(95% CI: −0.25–0.05), p = 0.04)), VEGFA (0.08 (95%CI: −0.07–0.23); stable: −0.12 (95% CI: −0.24- (−0.0001)), p = 0.03)), CASP-8 (−0.035 (95% CI: −0.31–0.24); stable: −0.49 (−0.79- (−0.19)), p = 0.02) and MMP7 (0.04 (95% CI: −0.03–0.12); stable: −0.06 (−0.13–0.005), p = 0.03)). Furthermore, levels of TNFRSF12A (0.15 (95% CI: 0.007–0.30); stable: −0.19 (−0.35- (−0.04)), p = 0.0008)) increased during the observation period in progressive patients while it decreased in patients who were stable.We further investigated changes in protein levels between baseline and follow up in patients with progressive and stable disease . PatientBased on the results obtained above , we procp = 0.047). Following adjustments for age, gender and baseline FVC % and DLCO %, baseline values of both TNFRSF12A  and its ligand TWEAK  were predictive of non-progression. In contrast, multivariate Cox regression analyses of the change in each respective biomarker revealed that patients with increasing levels of MMP7 , TNFRSF12A , TWEAK , VEGFA , NOS3 , CD40  and CXCL13  progressed faster than patients with decreased levels.In the univariate Cox proportional hazards regression analyses  of baselWe also analyzed the protein levels in serum from IPF patients with regards to treatment with antifibrotics. Patients who had initiated treatment between baseline and follow up (n = 13) had a change in level of 30 proteins as opposed to 8 proteins in patients treated throughout the observation period (n = 12). In patients not treated with antifibrotics (n = 11), only TRAIL was changed during the period . PatientExperimentally induced models of lung fibrosis such as the bleomycin model are commonly used to study fibrotic development and therapeutic interventions in vivo ,24 howevTen out of the 12 proteins identified as significantly elevated in repopulated IPF scaffolds were also significantly elevated in serum from IPF patients, demonstrating strong cell-ECM crosstalk and translatability of the ex vivo as a pre-clinical tool. The two proteins from the ex vivo model that were not found to be elevated at serum baseline, TNFRSF21 and DCN, were however significantly elevated in serum samples that were unadjusted for age . InteresIPF is thought to be a result of repeated micro-injuries in the lung epithelium creating an abnormal and ongoing wound repair that initiates an inflammatory response. The massive recruitment and infiltration of inflammatory cells such as monocytes ,26, and Several proteins engaged in inflammation and chemotaxis were elevated by fibroblasts cultured in IPF scaffolds, of which CXCL13 and CCL19 share a close connection in the recruitment of B cells and T cells ,50. IntrWhen examining the protein interaction of all elevated mediators in IPF, we found a distinct cluster of proteins regulating inflammation and chemotaxis such as CXCL12, which is involved in fibrocyte homing to the lung . The cluWith our longitudinal collection of samples, we are able to study if associations to common measures of disease severity are maintained over time, providing further evidence for the role of both established proteins and candidates in IPF. Taken together, our data demonstrate the significance of several proteins involved in remodeling, of which MMP7 was indicative of being the most prominent across our analyses. Other reoccurring remodeling proteins with regards to associations to severity and progression were HGF, VEGFA, MMP12, NOS3 and TNFRSF12A. The elevated levels of HGF and its correlations to disease severity and progression observed in this study correspond with other studies of fibrotic lung diseases ,59,60,61As the fibrotic destruction of the alveoli in IPF ultimately leads to a reduced gas diffusion capacity over time, the strong association observed between changes in TNFRSF12A and DLCO % is interesting. TNFRS12A is a member of the TNF receptor superfamily, which has been described to act as a downstream target of TGF-β and to contribute to pathological angiogenesis through the binding of the multifunctional cytokine TWEAK ,68. In fOf relevance, MCP-3 has been found in several studies in blood from patients hospitalized with COVID-19 infection ,81,82. IThe results on how protein levels are altered with regard to progression/non-progression should only be seen as hypothesis generating given the low numbers of patients. However, our findings show that progressive disease has higher levels of IL6, NOS3, MMP7 and CASP-8 compared to stable patients at follow-up. Albeit not reaching statistical significance, levels of ARG1 and CXCL13 were numerically higher in patients who had progressed at follow up, while levels of IL-12 were numerically lower in progressors. In parallel, progressive patients also demonstrated increasing levels of among others, NOS3, VEGFA, HGF, MMP7 and TNFRSF12A, further emphasizing several ongoing vascular and tissue remodeling processes linked to progression of the disease. Adding results from the Kaplan–Meier and cox-regression analyses demonstrate that the progression of disease involves multiple pathways. The analysis of the predictive value of baseline TWEAK and TNFRSF12 and their “protective” effects against progression comes off as paradoxical due to their associations to disease severity and the subsequent analyses of how patients with elevations in these proteins show a faster rate to progression. Prospective studies are certainly needed to confirm these results and evaluate the role of TWEAK and TNFRSF12A in fibrosis and disease development.We provide explorative data on the potential effects of antifibrotic treatments on protein levels. In patients who were untreated at baseline but treated at follow up and thus the group that perhaps best can convey the direct outcome of treatment, a reduced effect was demonstrated both on remodeling associated proteins , and inflammatory proteins . From a pharmacodynamic perspective, the decrease in tyrosine kinase associated proteins are in line with the mechanisms of which the approved antifibrotic treatments exert their effects ,83,84. MAs pointed out in several biomarker-related studies over the years, a “one-protein” approach to establish diagnosis or prognosis is likely not feasible given the large number of active pathophysiological processes in IPF. Rather, an evaluation of several markers reflecting different pathophysiological events may be more relevant. A main limitation of our study is the relatively small cohort sizes, which limits the power in our analyses and likely overestimates the risks reflected by the hazard ratio and confidence intervals in the cox-regression analyses. The absence of a replication cohort to validate our results and the potential influence of antifibrotic treatments, immunomodulatory agents such as corticosteroids, comorbidities or other unmeasured variables on the expression of analyzed biomarkers are also factors that ought to be weighted in when interpreting our results. Despite this, using our human ex vivo model, we have identified markers of remodeling, inflammation and chemotaxis that are differentially expressed compared to controls, and are associated with disease severity. We also demonstrate how changes in protein levels may track with changes in lung function. These types of longitudinal measures are relatively unique among studies of biomarkers in clinical studies of IPF patients. By combining clinical data with biological markers evaluated in experimental human models we believe that more accurate information will be achieved which, in turn, may increase our understanding of the pathobiology of IPF. Our data suggest that remodeling events linked to inflammatory processes governed by the cell-ECM interplay are the main drivers of the disease.This study was designed in two parts; firstly, to examine local fibroblast activities in IPF using a novel ex vivo model, and secondly to compare and establish the systemic proteomic profile in serum collected at baseline and follow up from IPF patients. By linking the proteomic data to clinical data such as lung function, we sought to find markers indicative of disease severity and progression. 3 blocks and directly frozen in 2-methylbutan chilled with liquid nitrogen. Lung tissue originated from four IPF patients; two females (57 years and 62 years old) and two males (57 years and 68 years old) with prior smoking history with forced vital capacity (FVC) ranging between 1.1–3.1 L. Healthy donor tissue was retrieved from two females (41 years and 55 years old) and two males (62 years and 86 years old) of which three were non-smokers and one a current smoker. Lung tissue was cryosectioned into 350 µm slices and treated for cellular removal according to previously established decellularization procedure [Human distal lung tissue was dissected from healthy donor lungs , or explanted lungs from IPF patients that were diagnosed consistent with ERS and ATS criteria . Native lung samples were cut into 1 cmrocedure ,86. In s2 at 37 °C. After 24 h, lung scaffolds derived from healthy donors or IPF patients were mounted on custom-made holders (8 mm inner diameter) to maintain lung tissue in a stretch formation, as fibroblast overtime contract surrounding tissue. Repopulated scaffolds were cultured up to 9 days in complete SILAC DMEM Flex Media  supplemented with 10% dialyzed serum , glucose (4500 µg/mL), amphotericin B (2.5 µg/mL), penicillin-streptomycin (1%), gentamicin (50 µg/mL), 1% Glutamax, with medium changed at day 1, 3 and 6. For detailed methodology, please see Healthy distal lung fibroblasts, derived from one donor, were used to repopulate scaffolds placed in 24-well suspension plates with mild agitation at 10% COWe used serum from patients included in the Swedish IPF-registry ,88 that Two serum samples from each patient were obtained: at baseline and upon follow-up. The baseline samples (n = 38) were taken at a median time of 2 months (IQR: 11.8 months) from diagnosis. Follow-up samples were obtained at time points ranging from 6–30 months (median: 16 months (IQR: 9.5 months)) from baseline. Thirty-seven patients were included in the follow-up analyses as one sample did not meet the quality control guidelines set for protein analysis. Associated clinical data included forced vital capacity, % predicted (FVC %), diffusing capacity for carbon monoxide, % predicted (DLco %) and total lung capacity, % predicted (TLC %). Four measures of disease severity were considered: FVC %, DLco %, TLC %, and the composite physiologic index (CPI), which correlates with the fibrotic extent seen in radiology . DiseaseSerum from the COpd and Smoking from an oMIC perspective (COSMIC) cohort conducted at Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden (NCT02627872) was used as a healthy control cohort ,94,95. OA panel of 92 proteins were analyzed both in cell culture media from the ex vivo model A and in Bioinformatic analysis of protein interactions was analyzed and illustrated with STRING database v 11, showing full network with line thickness denoting confidence strength and textmining, experiments, databases and co-expression as active interaction sources, illustrated with Cytoscape v.3.8.2. Note that the following proteins are shown with corresponding gene identification in STRING; ADGRG1 = GPR56, MCP-3 = CCL7, MCP-4 = CCL13, Gal-9 = LGALS9, Gal-1 = LGALS1 and PD-L1 = CD274. Pathway analysis was performed with g:Profiler with ranked gene list according to mean NPX difference using KEGG and Reactome databases for biological function annotations. p-value was <0.05. Even though the statistical tests have been adjusted for covariates and post hoc comparisons to avoid erroneous significance levels the study employs multiple hypothesis testing, where each hypothesis was analyzed separately and the existence of patterns in and the consistency of the results were considered in the analysis. Statistical analyses were performed in SAS , GraphPad Prism 9 and R . Continuous variables are reported as means with SD. Categorical variables are presented as counts and percentages. Bioinformatical analysis with q:Profiler was performed on all known genes as statistical domain scope with over-representation analysis with Bonferroni correction with significant threshold of 0.01.Two-way ANOVA with Sidak’s adjustment for multiple comparison was used for the identification of differentially expressed proteins between repopulated healthy and IPF scaffolds in the ex vivo model. Serum samples were analyzed with one way ANOVA for comparisons of IPF patients vs. controls. As the mean age of the IPF cohort is 18.2 years older than the healthy cohort, the ANOVA was adjusted for the age discrepancy only to avoid overfitting. Protein selections were corrected for multiple testing using the Benjamini–Hochberg method, controlling for a false discovery rate (FDR) at 5%. Differences between groups were assessed using Mann–Whitney test, while paired data was analyzed with Wilcoxon signed rank test. Correlation analyses between protein expression and clinical variables were done using Spearman’s rank correlation. Analyses of time to progression were performed using Kaplan–Meier curves to illustrate the associations of changes in protein level (stratified by increase or decrease in levels observed at follow up) with progression. Progression-free survival was defined as the time from baseline until the date of first disease progression. Comparisons were done using the log-rank test. Patients were censored at the time of the last spirometry performed during the observation period of maximum 36 months from baseline. Univariate and multivariate Cox regression analyses were used to investigate the associations between continuous levels of proteins deemed important in progression and drop of either >10% in FVC% or >15% in DLCO% over a 36-month period. The covariates considered in the multivariate analysis included age, gender, FVC% and DLCO% at baseline. Results were considered significant if the In conclusion, we have explored proteins related to remodeling of the ECM, inflammation and chemotaxis both ex vivo and in serum from patients with IPF. We emphasized the crosstalk between fibroblasts and ECM, revealed in our preclinical experiments, as an important interplay in IPF as their local interaction in part was reflected in the systemic profile of IPF patients including correlation to disease severity. The constant release of recruitment factors by fibroblasts in a fibrotic environment creates a persistent homing of inflammatory cells such as monocytes, T cells and B cells to the site of injury where lack of intact barriers in epithelial and vascular BM, created by the activity of, e.g., MMPs, further enables cellular infiltration and chronic tissue injury . By corr