Epidemiology of Clostridium difficile infection: results of a hospital-based study in Krakow, Poland.

Over the past two decades Clostridium difficile infection (CDI) has appeared as a major public health threat. We performed a retrospective study based on the records of patients hospitalized for CDI at the University Hospital in Krakow, Poland, between 2008 and 2014. In the study period, CDI occurred in 1009 individuals. There were 790 (78%) individuals who developed infection only once, whereas 219 (22%) developed infection more than once. The percentage of deaths within 14 days of CDI confirmation was 2·4%, with a mean age of 74·2 ± 15·9 years. Crude mortality was 12·9% in medical wards, 5·6% for surgical wards and 27·7% in the ICU setting. The time span between diagnosis and death was 5·1 days on average. Between 2008 and 2012 a 6·5-fold increase of CDI frequency with a posterior stabilization and even reduction in 2013 and 2014 was observed. According to the data analysed, 2/3 patients in our population developed CDI during their hospitalization even though they were admitted for different reasons. Medical wards pose a significantly higher risk of CDI than the surgical ones. Age is a risk factor for CDI recurrence. In the case of patients who died, death occurred shortly after diagnosis. The first CDI episode poses much higher risk of mortality than the consecutive ones.

Clostridium difficile the hospital plague.

Clostridium difficile infection (CDI) has become one of the major public health threats in the last two decades. An increase has been observed not only in the rate of CDI, but also in its severity and mortality. Symptoms caused by this pathogen are accompanied by intense local and systemic inflammation. We confirmed that Raman microspectroscopy can help us in understanding CDI pathogenesis. A single erythrocyte of patients with CDI shows a difference, approximately 10 times, in the intensity of the Raman spectra at the beginning of hospitalization and after one week of treatment. The intensity level is an indicator of the spread of the inflammation within the cell, confirmed by standard laboratory tests. Many of the observed bands with enormously enhanced intensity, e.g. 1587, 1344, 1253, 1118 and 664 cm(-1), come from the symmetric vibration of the pyrrole ring. Heme variation of recovered cells in the acute CDI state between the first and the seventh day of treatment seems to show increased levels of oxygenated hemoglobin. Intense inflammation alters the conformation of the protein which is reflected in the significant changes in the amide I, II and III bands. There is an observed shift and a significant intensity increase of 1253 and 970 cm(-1) amide III and skeletal protein backbone CC stretching vibration bands, respectively. Principal Component Analysis (PCA) was used to find the variance in the data collected on the first and seventh day. PC2 loading in the 1645-1500 cm(-1) range shows an increase of heme, Tyr, Trp, or Phe vibrations because of changes in the protein microenvironment due to their exposure. Positive maxima at 1621, 1563 and 1550 in the PC2 loading originated from the ring vibrations. These observations indicate that Clostridium difficile toxins induce cytopathogenicity by altering cellular proteins.

The role of local and systemic cytokines in patients infected with Clostridium difficile.

It is widely accepted that the pathogenesis of Clostridium difficile infection (CDI) is multifactorial, dependent on pathogen virulence factors produced by the organism as well as disorders of the gastrointestinal tract, the alteration in intestinal flora and the immune response of the host. In particular, the immune response in the course of CDI and the involvement of cytokines in the pathogenesis of CDI is not fully understood. The aim of our study was to evaluate the relationship between proinflammatory and anti-inflammatory cytokines and the course of CDI in vivo. We prospectively studied 80 patients. Our study group included 40 patients aged 30-87 years (mean age 66.9 years) with CDI hospitalized at Infectious Diseases Department and Gastroenterology and Hepatology Clinic, University Hospital in Cracow, and 40 healthy volunteers aged 24-62 years (mean age 51.1 years). The serum concentrations of cytokines IL-1ß, IL-6, IL-8, IL-10, tumor necrosis factor (TNF-α), myeloperoxidase (MPO), and prostaglandin E2 (PGE2) were measured using ELISA assays. Additionally, the routine biochemical parameters were assessed including the following: white blood cells with differential leukocyte count, platelets counts, and blood plasma levels of creatinine, alanine transaminase, and C-reactive protein were determined. We noted a significant increase in the concentration of the following cytokines in the CDI group when compared to the control group: IL-1b (4.7 vs. 3.6 pg/ml), IL-6 (21.0 vs. 0.04 pg/ml), IL-10 (8.5 vs. 0.5 pg/ml), TNF-α (7.1 vs. 0.09 pg/ml). In addition the serum concentration of MPO (1056.0 vs. 498.0 pg/ml), and PGE2 (2036.7 vs. 1492.0 pg/ml) showed a significant increase in CDI patients as compared with control subjects. Most CDI patients did not show any increase in the concentration of IL-8. We did observe a direct relationship between TNF-α and creatinine. The course of CDI is characterized by an initial local inflammatory process followed by a systemic inflammatory response, which manifests clinically as fever, and includes leukocytosis, an increase in the level of neutrophils in the blood, and an increase in the serum concentrations of TNF-α, IL-1ß, IL-6, IL-10, MPO and PGE2. Despite the leading role of IL-8 in the local inflammatory process, we postulate TNF-α and IL-6 play a key role in the systemic inflammatory response in CDI, and the plasma TNF-α level seems to act as a major factor of poor prognosis in patients with CDI.

Raman micro-spectroscopy tracing human lymphocyte activation.

The activation of lymphocytes occurs when they are exposed to viruses or other foreign antigens. The aim of this work was to verify if Raman spectroscopy can be used to screen the activation of lymphocytes during viral infection. There are distinct peaks that reveal differences between activated and intact cells. The most important marker of the lymphocyte activation process is the prominent 521 cm(-1) disulfide band which marks the immunoglobulin formation. The up shift of the S-S mode from the broad band centered at 510 cm(-1) of human normal immunoglobulin to a single band at 521 cm(-1) of human B cells indicates a selection of the optimal geometry of the disulphide bridges to bind to a foreign antigen. Polarization data is used to detect the structural alteration between domain fragments. Differences in other band intensities may be due to different protein compositions in both the investigated forms. B cell activation causes the change of the intracellular cytoplasm composition due to the secretion of immunoglobulins during the fighting of the infection.

Expression and release of leptin and proinflammatory cytokines in patients with ulcerative colitis and infectious diarrhea.

Leptin plays not only an important role in regulation of food intake, but also in the mechanism of inflammation. The universal presence of leptin in the cells of immune system and its secretion by these cells caused increasing interest in the role of this hormone in ulcerative colitis (UC). We determined the role of leptin in 80 patients, aged from 18 to 69 years, including 50 patients with active UC and 30 patients with infectious diarrhea. The tests were performed within 48 hours of the first symptoms, in the period of remission of UC and 8 weeks after resolution of infectious diarrhea. Endoscopy was performed in each patient, and the biopsy samples were taken for the assessments of expression of mRNA for leptin, IL-1ß, IL-6 and TNF-α by RT-PCR and Western blot. Blood tests included concentrations of leptin, IL-1ß, IL-6 and TNF-α. In addition, the plasma levels of leptin, IL-1ß, IL-6 and TNF-α were assessed by ELISA. Serum concentrations of leptin was significantly increased in patients with exacerbation of UC over that in patients with UC in remission. The serum leptin concentration was significantly higher in patients with infectious diarrhea, than the patients that recovered from infectious diarrhea. The leptin protein was overexpressed in the biopsy samples of the mucosa of large intestine compared to those with exacerbation of UC, and in patients after successful recovery from infectious diarrhea. The leptin mRNA was overexpressed in patients with infectious diarrhea compared with that in the group of patients after successful recovery from this condition. Serum concentrations of leptin failed to correlate with severity of exacerbation of UC and with extent of intestinal inflammatory lesions in patients with UC. However, the correlation was observed between serum concentrations of leptin in patients with exacerbation of UC and serum concentrations of proinflammatory cytokines IL-1ß and TNF-α. We conclude that 1) the increased leptin in exacerbated UC is related to the increased serum proinflammatory cytokines IL-1ß, TNF-α and IL-6 levels; 2) In patients with infectious diarrhea, the concentrations of leptin in intestinal mucosa correlates with serum concentrations of cytokines IL-1ß, IL-6 and TNF-α and with an increased expression of leptin mRNA in intestinal mucosa but not with alterations in serum levels of this hormone; 3) leptin may serve as useful predictive marker of inflammation in inflammatory bowel disease (IBD).