Postmortem histological freeze-thaw artifacts: a case report of a frozen infant and literature review.

Freezing and thawing have the potential to alter the gross and histologic appearance of tissues, causing damage to individual cells and disrupting the overall architecture. In forensic investigations, freezing and thawing can play a crucial role in cases of unknown cause of death. Perpetrators may use freezing preservation to conceal the body or obscure the time of death. Freezing can also occur naturally when a body is exposed to the elements, sometimes even leading to death itself. We present a case report involving an autopsy performed on an infant, who died of natural causes, after undergoing freezing and thawing. The objective of this study was to identify and discuss the histological artifacts observed in different tissues as a result of the freeze-thaw process. Histologically, the infant's tissues exhibited the most common features described in the literature. Ice crystal artifacts, characterized by expansion of the extracellular space and tissue clefts, were found in the heart, brain, liver, lungs, and kidneys. On the contrary, adipose tissue was not affected, likely due to the scarcity of water. Freeze-thaw artifacts should be taken into account whether a body is known to have been frozen or to add further data if found already defrosted.

Co-infections in critically ill adults with severe acute respiratory syndrome coronavirus 2 infection: an Italian multi-center prospective study.

BACKGROUND: To date, few studies have described Hospital-acquired infections (HAIs) during COVID-19 outbreak. To examine the incidence of HAIs in critically ill adult patients with SARS-CoV-2 infection and to observe risk factors, and the impact on outcome of HAI. METHODS: A prospective multicenter study was conducted that included adult patients with SARS-CoV-2 infection admitted to 18 Italian Intensive Care Units from September 2020 to November 2021. RESULTS: A total of 589 patients were included. A total of 233 patients were diagnosed with at least one HAI (39.6%). The co-infection/co-colonization rate >48 hours after admission was 31.0 per 1000 person-days (95% CI 18.8-34.8). Age, length of ICU stay >7 days, obesity, type 2 diabetes mellitus, cardiovascular disease, inserted central venous catheter, intubation, APACHE II score >25, mechanical ventilation (MV) >48 hours, obesity and inserted urinary catheter are associated outcomes for infection acquisition. The overall mortality rate of patients was found to be significantly higher in patients who had acquired a HAI (RR=4.37; 95% CI 3.30-5.78; P<0.001). CONCLUSIONS: Associated factors for HAI acquisition and mortality in ICU patients were identified and cause for revision of existing infection control policies.

Active screening of COVID-19-associated pulmonary aspergillosis with serum beta-glucan and endotracheal aspirates galactomannan and fungal culture.

BACKGROUND: Since February 2021 active screening of COVID-19-associated pulmonary aspergillosis (CAPA) has been implemented in our institution. OBJECTIVES: To evaluate CAPA incidence in our centre and evaluate performance of our screening protocol. METHODS: We screened once per week, collecting endotracheal aspirates for fungal culture and galactomannan (GM) and serum for 1,3-ß-D-glucan (BG). In case of positivity (GM more than 4.5, platelia assay, and/or BG >7 pg/ml, wako and/or positive fungal culture), second-level investigations were performed to pursue CAPA diagnosis according to ECMM/ISHAM criteria: bronchoalveolar lavage (BAL) fungal culture and GM, chest computed tomography (CT), serum GM. RESULTS: A total of 102 patients were screened (median age 64 years, range 39-79; 28 (27.4%) females). Twenty-two patients were diagnosed with CAPA (21%). 12 patients were positive for serum BG, 17 patients were positive for endotracheal aspirates GM and 27 patients were positive for endotracheal aspirates fungal culture. Thirty-two BALs were performed, and 26 patients underwent CT chest. Following the second level investigations 61% of the patients with positive screening tests were diagnosed with CAPA. Serum BG above 20 pg/ml or positive serum GM were always associated with typical CT chest signs of aspergillosis. Compared with 1 single positive test, having 2 positive screening test was significantly more associated with CAPA diagnosis (p = .0004). CONCLUSIONS: Active CAPA screening with serum 1,3-ß-D-glucan and endotracheal aspirates galactomannan and fungal cultures and consequent second level investigations led to high number of CAPA diagnosis. Combining more positive fungal biomarkers was more predictive of CAPA diagnosis.

Fulminant Sepsis and Perinatal Death at 23 Weeks Due to Fusobacterium nucleatum.

Introduction: Fusobacterium nucleatum is a gram-negative anaerobe, a constituent of the oral microflora, responsible for chronic periodontal diseases. Case Report: We describe a preterm infant with premature rupture of membranes at 23 weeks of gestational age due to F. nucleatum. The newborn died soon after birth. Placental histopathology showed severe necrotizing chorioamnionitis and funisitis with gram-negative bacilli. After autopsy, F. nucleatum was microbiologically isolated from the lung. The mother had dental hygiene 1 day before delivery, presenting mild and diffuse gingivitis. At admission, she had leukocytosis, foul-smelling vaginal discharge, but no fever. Conclusion: This case highlights the possibility of F. nucleatum spreading from oral cavity after a dental procedure to the placenta with chorioamnionitis and fetal infection. This raises the question of whether dental procedures during pregnancy should be accompanied by prophylactic antibiotics.

Mycobacterium chimaera Identification Using MALDI-TOF MS Technology: A Practical Approach for the Clinical Microbiology Laboratories.

Mycobacterium chimaera (MC) is an environmental, slowly growing, non-tuberculous mycobacterium (NTM) belonging to Mycobacterium avium complex (MAC), which recently has been linked to severe cardiovascular infections following open heart and vascular surgery. The majority of the diagnostic laboratory tests used in routine are not able to distinguish MC from M. intracellulare (MI), because of the great genetic similarity existing between these two species. The Genotype Mycobacterium NTM-DR™ represents a valid method to differentiate between these species, but it is expensive, requiring also specialized personnel. Recently, MALDI-TOF MS has been proposed to identify relevant NTM. However, a software implementation is required to distinguish between MC and MI, presenting the two microorganisms' overlapping spectra. The present study evaluates the feasibility of applying a MALDI-TOF logarithmic-based analysis in the routine of a clinical microbiology laboratory, and proposes an easy-to-use template spreadsheet to make the results quickly interpretable. The protocol was previously validated through the identification of 87 strains of MC/MI collected from clinical and environmental samples, and it was identified using the GenoType Mycobacterium NTM-DR™ and/or WGS. The proposed protocol provides accurate identification for the isolates tested; moreover, it is less expensive and more rapid than sequencing methods and can be implemented with minimum effort.

Second Trimester Fetal Loss Due to Citrobacter koseri Infection: A Rare Cause of Preterm Premature Rupture of Membranes (PPROM).

Citrobacter koseri is a facultative anaerobic, motile, non-spore-forming Gram-negative bacillus, which belongs to the family of Enterobacteriaceae. Severe infections due to Citrobacter spp. have been reported in the urinary tract, respiratory airways, intra-abdominal organs, skin and soft tissue, eye, bone, bloodstream, and central nervous system. In newborns, C. koseri is a well-known cause of meningitis, cerebral abscesses, brain adhesions, encephalitis, and pneumocephalus. Infection can be acquired through vertical maternal transmission or horizontal hospital settings; however, in many cases, the source is unknown. Preterm premature rupture of membranes (PPROM), caused by C. koseri, has rarely been described. Herein, we describe a case of PPROM at 16 weeks and 3 days of gestation, leading to anhydramnios. The parents opted for legal termination of the pregnancy, as the prognosis was very poor. C. koseri was isolated postmortem from a placental subamniotic swab and parenchymal sample, as well as fetal blood and lung. To the best of our knowledge, this is the first case of early second-trimester PPROM in which C. koseri infection was demonstrated.

Impact of COVID-19 outbreak on ICU nurses' mental health. An Italian multicenter study. / Impatto dell'epidemia di COVID-19 sulla salute mentale degli infermieri di Terapia Intensiva. Uno studio multicentrico Italiano.

AIM: AIM: The aim of this study was to evaluate variations in ICU nurses ' mental health status over the COVID-19 outbreak by quantifying the extent of symptoms of depression, anxiety and PTSD over time. METHODS: METHODS: This study was an Italian multicenter prospective cohort study assessing caseness of anxiety, depression and PTSD at 6 and 12 months from the beginning of the COVID-19 outbreak in Italy. RESULTS: RESULTS: A total of 359 nurses, 233 (64.9%) were males and 126 (35.1%) were females were enrolled. At 6 months the caseness prevalence for anxiety, depression and PTSD were 31.3%, 32.1% and 18.7% respectively. At 12 months the caseness prevalence for anxiety, depression and PTSD were 34.8%, 36.4% and 24.1 % respectively. No statistically significant increase between 6 and 12 months was recorded for the caseness prevalence anxiety (p= .29) and depression (p= .19). However, an increase for the caseness prevalence PTSD at 12 months was observed (p= .049). The significant risk factors for the 221 patients with at least one disorders were age 31-40 (RR= 1.44, IC= 1.25-1.89; p < .001), female gender (RR= 1.31, IC= 1.02-1.51; p=. 042) and had 0-5 years of professional experience (RR= 1.36, IC= 1.02-1.63; p = .031). CONCLUSION: The results of our study may provide support for the implementation of some interventions for well-being in COVID-19 outbreak condition.

Diagnostic approach and epidemiology of Microbial Keratitis: findings from an Italian Tertiary Care center.

Rapid identification of causative microorganisms of microbial keratitis (MK) and knowledge of the most common local pathogens are prerequisites for rational antimicrobial therapy. We retrospectively reviewed the characteristics of MK diagnosed at the IRCCS Arcispedale Santa Maria Nuova of Reggio Emilia (Italy) in a 5-years period, where the Ophthalmologist Unit is a reference center for corneal infections. During the study period, 183 MK were evaluated through corneal scrapings cultures. The positivity rate was 54,1%. A total of 107 microorganisms have been isolated: Acanthamoeba species was the etiologic agent in 19 cases. Pseudomonas aeruginosa and Staphylococcus aureus were more frequently isolated in bacterial keratitis, while Fusarium spp., Candida albicans, and Alternaria alternata were predominant among the fungal isolates. Strict cooperation between ophthalmologists and clinical microbiologists is advisable to allow the best diagnostic approach for MK.

Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease.

Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment.

Klebsiella pneumoniae Chorioamnionitis: An Underrecognized Cause of Preterm Premature Rupture of Membranes in the Second Trimester.

Klebsiella pneumoniae is a Gram-negative, rod-shaped bacterium, responsible for hospital and community acquired pneumonia, urinary tract and wound infections, and bloodstream dissemination. K. pneumoniae infection in pregnancy, leading to acute chorioamnionitis (AC), preterm premature rupture of membranes (PPROM) and early pregnancy loss in the second trimester, has been rarely reported. Herein, we present a case of K. pneumoniae AC that caused intrauterine fetal demise (IUFD) at 19 weeks + 5 days. The 36-year-old mother was admitted at 18 weeks + 1 day of gestation for threatened abortion. IUFD occurred 11 days after. Fetal postmortem showed severe AC and funisitis, neutrophils within alveoli and intestinal lumen, associated with rod-like bacteria. Fetal blood and lung cultures grew K. pneumoniae, ß-lactamase-non-producing strain. Antibiogram revealed sensitivity for piperacillin/tazobactam. Three days after IUFD, the mother presented with fever (37.8 °C) which persisted for one week. Maternal blood and urine cultures were negative. According to fetal microbiological results, available 6 days after IUFD, initial treatment with amoxicillin/clavulanic acid was replaced with piperacillin/tazobactam with full patient recovery. Therefore, in the event of PPROM and IUFD, fetal microbiological investigations should always be performed to isolate the proper etiologic agent and start the correct medical treatment.

Excisional penetrating keratoplasty for fungal interface keratitis after endothelial keratoplasty: surgical timing and visual outcome.

PURPOSE: In this paper, we describe two cases of fungal interface infectious keratitis (IIK) developed after endothelial keratoplasty (EK) who underwent delayed therapeutic penetrating keratoplasty (TPK) with a poor visual outcome. Furthermore, we conducted a review of the literature and analyzed the visual outcomes of TPK in relation to the time from IIK diagnosis. METHODS: We searched the literature for fungal IIK cases occurred after EK and treated by TPK. We identified 17 cases of fungal IIK, mostly caused by Candida spp. (88%). RESULTS: Infection was diagnosed at a median time of 21 (range 1-90 days) days after EK. The median lag time between infection diagnosis and TPK was 30 (range 7-393) days. The median distance corrected visual acuity (DCVA) measured 4-12 months after surgery was 20/40 (range 20/200-20/20). When TPK was performed within one month from diagnosis, the final median DCVA was 20/30 (range 20/100-20/20), with 83% of patients achieving ≥ 20/40 vision. When TPK was carried out later, the final median DCVA was 20/50 (range 20/200-20/22) with 44% of patients achieving ≥ 20/40 vision. One patient in the early surgery and four patients in the late surgery group showed postoperative DCVA ≤ 20/100 despite clear grafts. CONCLUSION: TPK with removal of the sequestered infection is advocated as a safe and effective measure to treat a post-EK infection. Early surgery allows a reduced exposure time to infection and therefore may result in better visual outcomes and lower risk of complications caused by prolonged inflammation.

[Burnout syndrome among Italian nursing staff during the COVID 19 emergency. Multicentric survey study]. / Sindrome di Burnout tra il personale infermieristico italiano durante l'emergenza COVID 19. Indagine conoscitiva multicentrica.

AIM: To analyze the prevalence of Burnout defined by the MBI-GS among Italian nursing staff during the COVID-19 emergency. INTRODUCTION: The recent health emergency which in the first half of 2020 has seen Italy involved in the management of patients with COVID-19 (COronaVIrus Disease 19), has led to further stress for hospitals both in terms of beds and in terms of workload for healthcare workers. METHOD: A multicenter study was conducted. A convenience sample of 208 nurses participated in the study. Between March 20 and July 20, 2020, the Maslach Burnout Inventory-General Survey (MBI-GS) questionnaire was made available online by using the Google Forms platform in order to detect and measure the severity of Burnout Syndrome (BOS). RESULTS: BOS-related symptoms (medium or high score) for individual MBI-GS domains were identified in at least 68% of nurses. One hundred and sixty-one nurses (77.4%) had a risk of emotional exhaustion, 143 (68.7%) of depersonalization, and 162 (77.9%) of reduced professional accomplishment. High risk was observed among nurses who managed COVID patients at their own operating unit (RR = 1.27, p = 0.016). Severe BOS risk was observed in 146 nurses analyzed (70.2%) with an increased risk among Intensive Care nurses. CONCLUSIONS: Our results suggest that the nurses during the coronavirus pandemic, had high levels of work-related suffering and were at risk of physical and emotional exhaustion. Further research should be undertaken to establish causal relationships between BOS and personal and environmental risk factors among healthcare professionals in relation to the COVID-19.

Interface infectious keratitis after anterior and posterior lamellar keratoplasty. Clinical features and treatment strategies. A review.

Interface infectious keratitis (IIK) is a novel corneal infection that may develop after any type of lamellar keratoplasty. Onset of infection occurs in the virtual space between the graft and the host where it may remain localised until spreading with possible risk of endophthalmitis. A literature review identified 42 cases of IIK. Thirty-one of them occurred after endothelial keratoplasty and 12 after deep anterior lamellar keratoplasty. Fungi in the form of Candida species were the most common microorganisms involved, with donor to host transmission of infection documented in the majority of cases. Donor rim cultures were useful to address the infectious microorganisms within few days after surgery. Due to the sequestered site of infection, medical treatment, using both topical and systemic antimicrobials drugs, was ineffective on halting the progression of the infection. Injection of antifungals, right at the graft-host interface, was reported successful in some cases. Spreading of the infection with development of endophthalmitis occurred in five cases after Descemet stripping automated endothelial keratoplasty with severe sight loss in three cases. Early excisional penetrating keratoplasty showed to be the treatment with the highest therapeutic efficacy, lowest rate of complications and greater visual outcomes.

Clinical Validation of SensiTest Colistin, a Broth Microdilution-Based Method To Evaluate Colistin MICs.

The global spread of multidrug-resistant Gram-negative bacteria has led to the return of colistin for treating severe infections. Recently, different plasmid-mediated genes conferring resistance to this drug were described and reported worldwide. International committees (EUCAST/CLSI) reevaluated inconsistencies surrounding colistin antimicrobial susceptibility testing (AST), concluding that broth microdilution (BMD) should serve as the reference method for AST. The development of an accurate, reproducible commercial test based on BMD is therefore highly desirable. SensiTest Colistin (STC), a BMD-based compact 4-test panel containing the lyophilized antibiotic in 7 2-fold dilutions (0.25 to 16 µg/ml) was here compared with the EUCAST-CLSI standard reference method (BMD) and, for some isolates, with the automated Phoenix 100 system (PHX). A total of 353 bacterial strains were evaluated by two different laboratories; 137 isolates were resistant to colistin (19 were intrinsically resistant, 83 harbored the mcr-1 gene). Essential agreement (EA) between STC and BMD was obtained for 339 out of the 353 strains tested (96.0%). Overall categorical agreement was obtained for 349 out of the 353 strains analyzed (98.9%). Two major errors (MEs; 0.93%) and two very major errors (VMEs; 1.46%) were documented. STC appeared to be a simple but highly reliable test with good reproducibility even with panels stored at room temperature or at 35°C. Moreover, STC showed a good performance with strains carrying the mcr-1 gene, with a 98.8% EA. As the secondary endpoint of our study, VMEs for PHX were documented for 6 isolates (10%).

Detection of mcr-4 positive Salmonella enterica serovar Typhimurium in clinical isolates of human origin, Italy, October to November 2016.

In this study we report the detection of the recently described mcr-4 gene in two human isolates of Salmonella enterica serovar Typhimurium. The strains were isolated from faecal samples of two Italian patients with gastroenteritis, collected in 2016. The identified mcr-4 genes (variant mcr-4.2) differed from the mcr-4 gene originally described in a Salmonella strain of swine origin from Italy. Salmonella species could represent a hidden reservoir for mcr genes.

[Treating Clostridium difficile infection with faecal transplantation: donor microbiological testing]. / Terapia delle infezioni da Clostridium difficile con trapianto di feci: controlli microbiologici sul donatore.

Clostridium difficile associated diseases (CDADs) or C. difficile infections (CDIs) are increasing in incidence, severity and mortality. Among patients with CDIs, those with recurrent disease are less responsive to traditional therapies with commonly used drugs, such as metronidazole and vancomycin. Faecal microbiota transplantation is an old therapeutic procedure that has been recently proposed as a safe and effective treatment for CDI patients non-responsive to antibiotic therapy. In this paper we discuss the microbiological procedures that should be performed on faecal microbiota donors.

Laboratory diagnosis of Clostridium difficile associated diarrhoea and molecular characterization of clinical isolates.

We evaluated a three-step algorithm for laboratory diagnosis of Clostridium difficile-associated diarrhoea (CDAD). First, stool specimens were screened using an EIA test for glutamate dehydrogenase detection. Screen-positive specimens were tested by a rapid cytotoxintoxin A/B assay and subjected to stool culture. All cultures positive for C. difficile underwent toxigenic culture. The results showed that toxigenic culture allowed us to recover 37/156 (24.4%) stool samples harbouring toxigenic C. difficile that would have been missed by using faecal cytotoxin assay alone. This determined an increase in infection prevalence of 4.2% (from 11.4% to 15.6 %). Furthermore, to characterize the clinical Clostridium difficile isolates and the distribution of PCR ribotypes circulating in the San Carlo Borromeo hospital, molecular typing using semi-automated repetitive-sequence-based PCR (rep- PCR) and PCR ribotyping, and an evaluation of the antibiotic resistance were also performed. Among them, 71 indistinguishable strains were detected by rep-PCR and 83 by PCR-ribotyping revealing C. difficile outbreaks in our hospital. A total of 6 different ribotypes were obtained by PCR ribotyping. The most frequent ribotype was 018 (88.2%) that also showed resistance to moxifloxacin. In one case, uncommon PCR ribotype 186 was also identified.

Microarray technology for yeast identification directly from positive blood cultures. A multicenter Italian experience.

The authors evaluated the performance of the MycArray™ Yeast ID (Myconostica Ltd, UK) assay in the identification of a total of 88 yeast isolates recovered in culture as compared to that obtained through routine methods. The turn-around time for species identification directly from cultures by the MycArray was 6 hours, much quicker than classical methods and all yeasts were correctly identified. In two cases a double identification including Saccharomyces cerevisiae was noted, but it was not confirmed by culture. The results show that MycArray Yeast ID can be a potential tool for rapid detection and identification of Candida species.

In vitro activity of Amphotericin B against zygomycetes isolated from deep mycoses: a comparative study between incubation in aerobic and hyperbaric atmosphere.

Many case reports suggest that the use of hyperbaric oxygen therapy associated with surgical and medical approaches may contribute to restricting the growth of zygomycetes in patient tissue. The primary aim of this study was to obtain data concerning the in vitro susceptibility of 22 zygomycetes to antifungals such as amphotericin B and posaconazole, and to compare the in vitro development of these fungi in aerobic normobaric versus hyperoxic normobaric and hyperbaric atmosphere. None of the zygomycetes grew after 24-hour and 72-hour incubation in a hyperoxic hyperbaric (2 or 3 ATA) atmosphere. However, when plates were maintained at room temperature in aerobic conditions, colonies were observed from 36-96 h after inoculation, while minimum inhibitory concentration (MIC) values remained the same. This preliminary in vitro study focuses on the in vitro examination of combination therapies to potentiate antifungal activity. Both hyperoxic hyperbaric conditions and a single antifungal agent, as well as combinations of different antifungal drugs were used. Results suggest an impressive in vitro fungistatic activity of the hyperoxic hyperbaric atmosphere, even if the antifungal effect is strictly time-dependent using these incubation conditions.

Interlaboratory evaluation of VITEK2 system and Sensititre YeastOne® for antifungal susceptibility testing of yeasts isolated from blood cultures against four antifungal agents.

An interlaboratory evaluation (seven centers) of VITEK2 System and Sensititre YeastOne® was conducted to test the antifungal susceptibilities of yeasts. The MICs of amphotericin B, fluconazole, flucytosine, and voriconazole were determined for 70 isolates of Candida spp. Our results demonstrated a higher interlaboratory agreement of VITEK 2 System than Sensititre YeastOne©. A good concordance between the two methods was observed for amphotericin B, fluconazole, voriconazole and 5-fluorocytosine (from 81.4% to 88.6%). The study suggests the potential value of the VITEK2 System as a convenient alternative method for testing the susceptibility of yeasts. It also indicates the need for further optimization of MIC endpoint criteria to improve interlaboratory agreement.