LPS Guides Distinct Patterns of Training and Tolerance in Mast Cells.

Mast cells (MCs) are tissue-resident, long lived innate immune cells with important effector and immunomodulatory functions. They are equipped with an eclectic variety of receptors that enable them to sense multiple stimuli and to generate specific responses according on the type, strength and duration of the stimulation. Several studies demonstrated that myeloid cells can retain immunological memory of their encounters - a process termed 'trained immunity' or 'innate immune memory'. As MCs are among the one of first cells to come into contact with the external environment, it is possible that such mechanisms of innate immune memory might help shaping their phenotype and effector functions; however, studies on this aspect of MC biology are still scarce. In this manuscript, we investigated the ability of MCs primed with different stimuli to respond to a second stimulation with the same or different ligands, and determined the molecular and epigenetic drivers of these responses. Our results showed that, while the stimulation with IgE and ß-glucan failed to induce either tolerant or trained phenotypes, LPS conditioning was able to induce a profound and long-lasting remodeling of the signaling pathways involved in the response against LPS or fungal pathogens. On one side, LPS induced a strong state of unresponsiveness to secondary LPS stimulation due to the impairment of the PI3K-AKT signaling pathway, which resulted in the reduced activation of NF-κB and the decreased release of TNF-α and IL-6, compared to naïve MCs. On the other side, LPS primed MCs showed an increased release of TNF-α upon fungal infection with live Candida albicans, thus suggesting a dual role of LPS in inducing both tolerance and training phenotypes depending on the secondary challenge. Interestingly, the inhibition of HDAC during LPS stimulation partially restored the response of LPS-primed MCs to a secondary challenge with LPS, but failed to revert the increased cytokine production of these cells in response to C. albicans. These data indicate that MCs, as other innate immune cells, can develop innate immune memory, and that different stimulatory environments can shape and direct MC specific responses towards the dampening or the propagation of the local inflammatory response.

Risk Factors and Outcome of C. difficile Infection after Hematopoietic Stem Cell Transplantation.

Patients who undergo hematopoietic stem cell transplants (HSCT) are at major risk of C. difficile (CD) infection (CDI), the most common cause of nosocomial diarrhea. We conducted a retrospective study, which enrolled 481 patients who underwent autologous (220) or allogeneic HSCT (261) in a 5-year period, with the aim of identifying the incidence, risk factors and outcome of CDI between the start of conditioning and 100 days after HSCT. The overall cumulative incidence of CDI based upon clinical evidence was 5.4% (95% CI, 3.7% to 7.8%), without any significant difference between the two types of procedures. The median time between HSCT and CDI diagnosis was 12 days. Out of 26 patients, 19 (73%) with clinical and symptomatic evidence of CDI were positive also for enzymatic or molecular detection of toxigenic CD; in particular, in 5 out of 26 patients (19%) CD binary toxin was also detected. CDI diagnoses significantly increased in the period 2018-2019, since the introduction in the microbiology lab unit of the two-step diagnostic test based on GDH immunoenzymatic detection and toxin B/binary toxin/027 ribotype detection by real-time PCR. Via multivariate analysis, abdominal surgery within 10 years before HSCT (p = 0.002), antibiotic therapy within two months before HSCT (p = 0.000), HCV infection (p = 0.023) and occurrence of bacterial or fungal infections up to 100 days after HSCT (p = 0.003) were significantly associated with a higher risk of CDI development. The 26 patients were treated with first-line vancomycin (24) or fidaxomicine (2) and only 2 patients needed a second-line treatment, due to the persistence of stool positivity. No significant relationship was identified between CDI and the development of acute graft versus host disease (GVHD) after allogeneic HSCT. At a median follow-up of 25 months (range 1-65), the cumulative incidence of transplant related mortality (TRM) was 16.6% (95% CI 11.7% to 22.4%) and the 3-year overall survival (OS) was 67.0% (95% CI 61.9% to 71.6%). The development of CDI had no significant impact on TRM and OS, which were significantly impaired in the multivariate analysis by gastrointestinal and urogenital comorbidities, severe GVHD, previous infections or hospitalization within two months before HSCT, active disease at transplant and occurrence of infections after HSCT. We conclude that 20% of all episodes of diarrhea occurring up to 100 days after HSCT were related to toxigenic CD infection. Patients with a history of previous abdominal surgery or HCV infection, or those who had received broad spectrum parenteral antibacterial therapy were at major risk for CDI development. CDIs were successfully treated with vancomycin or fidaxomicin after auto-HSCT as well as after allo-HSCT.

Orthopoxvirus Seroprevalence in Cats and Veterinary Personnel in North-Eastern Italy in 2011.

Orthopoxviruses (OPV) are emerging zoonotic pathogens, and an increasing number of human infections is currently reported in Europe and in other continents, warranting heightened attention on this topic. Following two OPV infections reported in veterinarians scratched by sick cats in 2005 and 2007 in North-Eastern-Italy, involving a previously undescribed OPV, a similar strain was isolated by a sick cat from the same territory in 2011, i.e., 6 years later, raising attention on OPV circulation in this region. A surveillance program was launched to assess the OPV seroprevalence among the veterinarians working in local veterinary clinics and in the local wild and domestic cat population; seroprevalence was 33.3% in veterinarians and 19.5% in cats. Seroprevalence in cats was unevenly distributed, peaking at 40% in the area where OPV-infected cats had been observed.

Mast Cells Respond to Candida albicans Infections and Modulate Macrophages Phagocytosis of the Fungus.

Mast cells (MCs) are long-lived immune cells widely distributed at mucosal surfaces and are among the first immune cell type that can get in contact with the external environment. This study aims to unravel the mechanisms of reciprocal influence between mucosal MCs and Candida albicans as commensal/opportunistic pathogen species in humans. Stimulation of bone marrow-derived mast cells (BMMCs) with live forms of C. albicans induced the release of TNF-α, IL-6, IL-13, and IL-4. Quite interestingly, BMMCs were able to engulf C. albicans hyphae, rearranging their α-tubulin cytoskeleton and accumulating LAMP1+ vesicles at the phagocytic synapse with the fungus. Candida-infected MCs increased macrophage crawling ability and promoted their chemotaxis against the infection. On the other side, resting MCs inhibited macrophage phagocytosis of C. albicans in a contact-dependent manner. Taken together, these results indicate that MCs play a key role in the maintenance of the equilibrium between the host and the commensal fungus C. albicans, limiting pathological fungal growth and modulating the response of resident macrophages during infections.

Epidemiological and clinical features of imported malaria at the three main hospitals of the Friuli-Venezia Giulia Region, Italy.

BACKGROUND: Imported malaria cases continue to occur in non-endemic regions among travellers returning from tropical and subtropical countries. At particular risk of acquiring malaria is the group of travellers identified as immigrants who return to their home country with the specific intent of visiting friends or relatives (VFRs) and who commonly believe they are immune to malaria and fail to seek pre-travel advice. Our aim was to review the current trends of imported malaria in the three main hospitals of the Friuli-Venezia Giulia region (FVG), North Eastern Italy, focusing in particular on patient characteristics and laboratory findings. METHODS: In this retrospective study, we examined all malaria cases among patients admitted from January 2010 through December 2014 to the emergency department of the three main hospitals located in FVG. RESULTS: During the 5-year study period from 2010 to 2014, there were a total of 140 patients with a diagnosis of suspected malaria and who received microscopic confirmation of malaria. The most common species identified was P. falciparum, in 96 of 140 cases (69%), followed by P. vivax (13%), P. ovale (4%), P. malariae (4%), and mixed infection (4%). The most common reason for travel was VFRs (54%), followed by work (17%), and recent immigration (15%). Moreover, 78% of all patients took no chemoprophylaxis, 80 (79%) of whom were foreigners. Notably, the percentage of Italian travellers who took chemoprophylaxis was only 20% (8 of 39 Italian cases), and the regimen was appropriate in only four cases. Parasitaemia greater than 5% was observed in 11 cases (10%), all due to P. falciparum infection. CONCLUSIONS: We highlight that VFRs have the highest proportion of malaria morbidity and the importance of improving patient management in this category. These data are useful for establishing appropriate malaria prevention measures and recommendations for international travellers.

Determination of PCT on admission is a useful tool for the assessment of disease severity in travelers with imported Plasmodium falciparum malaria.

Procalcitonin (PCT) and C-reactive protein (CRP) may be useful to predict complicated forms of malaria. A total of 30 consecutive travelers diagnosed with Plasmodium falciparum malaria over a two-year period were included in the study. Patients with complicated Plasmodium falciparum malaria showed higher levels of parasitemia (P = 0.0001), PCT (P = 0.0018), CRP (P = 0.0005), bilirubinemia (P = 0.004), and a lower platelet count (P<0.0001) compared with patients with uncomplicated forms. PCT levels above 5 ng/mL showed the highest value of specificity (0.86) and positive predictive factor (0.67) among other parameters, and equal sensitivity (0.67) was displayed by CRP levels above 150 mg/dl. None of the patients with complicated malaria showed PCT levels within normal limits (<0.5 ng/ml). Both PCT and CRP correlated with parasitemia (P<0.001) and showed areas under ROC curve of 0.83. At multivariate analysis, only PCT was associated with an increased risk of complicated malaria (OR 8.2, IC 95% 1.2-57.2, P = 0.03). The determination of PCT on admission showed better results compared to CRP, platelet count, and bilirubinemia and can be useful in non-endemic areas for the initial clinical assessment of disease severity in travelers with Plasmodium falciparum malaria.

Antifungal activity of cathelicidin peptides against planktonic and biofilm cultures of Candida species isolated from vaginal infections.

Vulvovaginal candidiasis (VVC) is a frequent gynecological condition caused by Candida albicans and a few non-albicans Candida spp. It has a significant impact on the quality of life of the affected women also due to a considerable incidence of recurrent infections that are difficult to treat. The formation of fungal biofilm may contribute to the problematic management of recurrent VVC due to the intrinsic resistance of sessile cells to the currently available antifungals. Thus, alternative approaches for the prevention and control of biofilm-related infections are urgently needed. In this regard, the cationic antimicrobial peptides (AMPs) of the innate immunity are potential candidates for the development of novel antimicrobials as many of them display activity against biofilm formed by various microbial species. In the present study, we investigated the in vitro antifungal activities of the cathelicidin peptides LL-37 and BMAP-28 against pathogenic Candida spp. also including C. albicans, isolated from vaginal infections, and against C. albicans SC5314 as a reference strain. The antimicrobial activity was evaluated against planktonic and biofilm-grown Candida cells by using microdilution susceptibility and XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide] reduction assays and, in the case of established biofilms, also by CFU enumeration and fluorescence microscopy. BMAP-28 was effective against planktonically grown yeasts in standard medium (MIC range, 2-32µM), and against isolates of C. albicans and Candida krusei in synthetic vaginal simulated fluid (MIC range 8-32µM, depending on the pH of the medium). Established 48-h old biofilms formed by C. albicans SC5314 and C. albicans and C. krusei isolates were 70-90% inhibited within 24h incubation with 16µM BMAP-28. As shown by propidium dye uptake and CFU enumeration, BMAP-28 at 32µM killed sessile C. albicans SC5314 by membrane permeabilization with a faster killing kinetics compared to 32µM miconazole (80-85% reduced biofilm viability in 90min vs 48h). In addition, BMAP-28 at 16µM prevented Candida biofilm formation on polystyrene and medical grade silicone surfaces by causing a >90% reduction in the viability of planktonic cells in 30min. LL-37 was overall less effective than BMAP-28 against planktonic Candida spp. (MIC range 4-≥64µM), and was ineffective against established Candida biofilms. However, LL-37 at 64µM prevented Candida biofilm development by inhibiting cell adhesion to polystyrene and silicone surfaces. Finally, Candida adhesion was strongly inhibited when silicone was pre-coated with a layer of BMAP-28 or LL-37, encouraging further studies for the development of peptide-based antimicrobial coatings.

Epidemiology, diagnosis and treatment of Clostridium difficile infection.

Clostridium difficile infection (CDI) is considered to be the main cause of bacterial infectious diarrhea in nosocomial settings. Since the beginning of the new century a continuous rise in the incidence of severe CDI has been observed worldwide. Even though some CDI cases are not associated with previous antibiotic exposure, this remains as the principal risk factor for the development of CDI. The rate of recurrences represents perhaps one the most challenging aspect on the management of CDI. There are several microbiological tests available, but glutamate dehydrogenase antigen test can be selected as the first screening step in a diagnostic algorithm, with positive samples then confirmed using a toxin(s) test, to distinguish toxinogenic from nontoxinogenic CDI. Although metronidazole and vancomycin are and have been the mainstay treatment options for CDI, there are some unmet medical and therapeutical needs. Usually oral metronidazole is recommended for initial treatment of nonsevere CDI and vancomycin for treatment of severe disease. Fidaxomicin may be considered in patients who cannot tolerate vancomycin, although more data are needed. For treatment of a nonsevere initial recurrence of CDI, oral metronidazole should be used, but for treatment of subsequent recurrences or more severe cases fidaxomicin may be helpful.

Acute renal failure due to visceral leishmaniasis by Leishmania infantum successfully treated with a single high dose of liposomal amphotericin B.

We report a visceral leishmaniasis case in an immunocompetent immigrant with acute renal failure. Parasites were demonstrated in bone marrow, peripheral blood, and kidney samples. A collapsing focal segmental glomerulosclerosis was documented, which was successfully treated with a single infusion of 10 mg/kg liposomal amphotericin B.

Trends in antibiotic sensitivity pattern and molecular detection of tet(O)-mediated tetracycline resistance in campylobacter jejuni isolates from human and poultry sources.

This study was conducted to determine the trends in Campylobacter antibiotic resistance occurring in our setting and to assess the differences in the isolates using patterns of plasmid profiles. One hundred Campylobacter jejuni strains of human and poultry origin isolated in 2002-2003 (phase A) and 2005-2006 (phase B) in the Kingdom of Bahrain were evaluated. Susceptibility to erythromycin, ciprofloxacin and tetracycline was determined, and plasmid extraction and polymerase chain reaction detection of the tet(O) gene was carried out. A single erythromycin-resistant isolate was identified, in sharp contrast to the high ciprofloxacin resistance which also showed an increment in phase B. Tetracycline resistance was higher in chicken (80.9%) compared to human (41.3%) isolates (P<0.01). Most isolates harbored two plasmids (23 kb and 35 kb) with significant correlation between tetracycline resistance and plasmid carriage in chicken isolates. The findings show continued effectiveness of erythromycin for campylobacteriosis but an increasing trend of high ciprofloxacin and tetracycline resistance. Tetracycline resistance is most likely due to the transfer of plasmids carrying the tet(O) gene between isolates.

Human granulocytic anaplasmosis in northeastern Italy.

Sporadic cases of human granulocytic anaplasmosis (HGA) have been reported in areas with a high prevalence of tick-borne diseases (TBDs) in Europe. We aimed at estimating the sero-prevalence of A. phagocytophilum and other TBDs in northeastern Italy in outpatients with a history of recent tick bite or suspected TBD. In the 1-year study, 79 patients were enrolled and 30 (38%) received a diagnosis of TBD: 24 (30%) with Lyme disease and 5 (6%) with HGE. Our findings indicate the presence of HGA in northeastern Italy; so, since co-infection with Lyme disease appeared to be frequent, physicians assessing patients after a tick bite should consider HGA in the diagnosis.

Human papillomavirus infection among women attending health facilities in the Kingdom of Bahrain.

OBJECTIVE: To investigate the occurrence of human papillomavirus (HPV) infection and the associated risk factors in Bahrain's female population. METHODS: This study was carried out between March to December 2004, which includes cervical scrapings for Pap smear and HPV-DNA testing using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis, obtained from 100 women attending the Gynecology Clinic at Salmaniya Medical Center and Sheikh Sabah Health Center in the Kingdom of Bahrain. We distributed questionnaires that include the sociodemographic data as well as information on risk factors such as smoking, parity, and the contraceptive used. RESULTS: Eleven women (11%) with normal cytology were HPV-positive. The RFLP analysis detected HPV-types 16, 18, 45, 62 and 53. Positive women were significantly older (43.3 +/- 10.1 years) than negatives (36.5 +/- 9.9 years; p=0.04), however, there was no difference in age of first sexual contact (positive: 18.1 +/- 5.7 years versus negative: 20.6 +/- 4.4 years). Polygamy, smoking and hormonal contraception was not identified as risk factors, but positive women showed higher parity. CONCLUSION: In this study on HPV infection in Bahrain, the 11% positivity with high risk HPV types, in the presence of normal cytology suggests that in addition to the cervical cancer screening program, offer of HPV testing deserves consideration.

African trypanosomiasis gambiense, Italy.

African trypanosomiasis caused by Trypanosoma brucei gambiense has not been reported in Italy. We report 2 cases diagnosed in the summer of 2004. Theses cases suggest an increased risk for expatriates working in trypanosomiasis-endemic countries. Travel medicine clinics should be increasingly aware of this potentially fatal disease.

Antimicrobial activity of SMAP-29 against the Bacteroides fragilis group and clostridia.

OBJECTIVES: The cathelicidin-derived peptide SMAP-29 exerts rapid and broad-spectrum antimicrobial activity against aerobic bacteria and fungi. In this study, the effects of the peptide against the Bacteroides fragilis group, including antibiotic-resistant isolates, Clostridium perfringens and Clostridium difficile reference and clinical isolates, were investigated. METHODS: The microbicidal activity of SMAP-29 against eight reference and 100 clinical anaerobic strains from a national collection was assessed using a microdilution susceptibility assay, and by determining the killing kinetics on selected strains. The killing mechanism was investigated further by means of a two-colour fluorescent permeabilization assay, and by scanning electron microscopy (SEM). RESULTS: The Bacteroides fragilis group, Clostridium reference strains and most clinical isolates were inhibited in vitro by 1-2 microM (3.2-6.4 mg/L) SMAP-29, and killed by 1.5- to 2-fold higher peptide concentrations. The anaerobic bacterial cells were 90%-100% permeabilized within 2 h of exposure to bactericidal concentrations of the peptide. The SEM images of bacteria exposed to SMAP-29 provide morphological evidence that the envelope is an important target of the bactericidal activity of this peptide. These results are consistent with earlier studies indicating that SMAP-29 kills aerobic bacteria with a membranolytic mechanism, and suggest that both aerobic and anaerobic bacteria share surface features that are targeted by this peptide. CONCLUSIONS: These studies demonstrate that the spectrum of antibacterial activity of SMAP-29 includes the B. fragilis group and Clostridium species, and encourage further investigations of the therapeutic potential of this peptide.