Major cluster of paediatric 'true' primary chilblains during the COVID-19 pandemic: a consequence of lifestyle changes due to lockdown.

BACKGROUND: Over the last months, during the COVID-19 pandemic, a growing number of chilblain-like lesions were reported mainly in children and rarely in young adults. The relationship with SARS-CoV-2 infection was postulated, often without any laboratory, instrumental or clinical confirmation. The disclosure of information about chilblain-like lesions as a COVID-19 manifestation in social media has created concern in children's families and paediatricians. OBJECTIVES: To verify whether the chilblain-like lesions were caused by SARS-CoV-2 infection. METHODS: Prospective study on a case series including children who presented with acral lesions at the Pediatric Dermatology Outpatient and Pediatric Emergency Unit of the University of Bologna, from 1 April to 30 April 2020. We reported demographical, laboratory and clinical features, history of close contact with COVID-19 patients, presence of similar skin lesions in other family members, precipitating and risk factors for chilblain onset. RESULTS: We evaluated eight patients (five females, three males) aged between 11 and 15 years. We excluded acute or previous SARS-CoV-2 infection with RT-PCR nasopharyngeal swab, serum antibody levels using chemiluminescent immunoassays. Other acute infections causing purpuric lesions at the extremities were negative in all patients. Skin lesion biopsy for histological and immunohistochemical evaluation was made in two cases and was consistent with chilblain. PCR assay on skin lesion biopsy for parvovirus B19, Mycoplasma pneumoniae and SARS-CoV-2 was performed in a patient and resulted negative. We identified common precipitating and risk factors: physical (cold and wet extremities, low BMI), cold and wet indoor and outdoor environment, behaviours, habits and lifestyle. We therefore reached a diagnosis of primary chilblains. CONCLUSIONS: During the COVID-19 pandemic, a 'cluster' of primary chilblains developed in predisposed subjects, mainly teenagers, due to cold exposure in the lockdown period. Laboratory findings support our hypothesis, although it is also possible that an unknown infectious trigger may have contributed to the pathogenesis.

Prospective Epstein-Barr virus-related post-transplant lymphoproliferative disorder prevention program in pediatric allogeneic hematopoietic stem cell transplant: virological monitoring and first-line treatment.

BACKGROUND: In 28 pediatric allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients, we aimed to evaluate: (i) the impact of routine Epstein-Barr virus (EBV) DNA monitoring on the development of EBV-related post-transplant lymphoproliferative disorder (EBV-PTLD); (ii) the incidence of EBV infection and the potential risk factors; and (iii) the suitability of whole blood (WB) as clinical specimen to monitor the risk of patients to develop EBV-PTLD. METHODS: Quantitative real-time polymerase chain reaction assay was performed on WB samples for all patients. EBV DNA quantification also in peripheral blood mononuclear cells (PBMCs) samples was adopted for the patients at higher risk of developing EBV-PTLD (≥ 10,000 copies/mL WB). RESULTS: High EBV DNAemia levels were observed in 37.5% of the actively infected recipients (57.1%). Severe aplastic anemia, matched-unrelated donor transplant, the reduced-intensity conditioning regimen and, to a lesser extent, the in vivo T-cell depletion with anti-thymocyte immunoglobulin were associated with high viral load. A significant correlation between EBV DNA levels in WB and PBMC samples was obtained (r = 0.755, P < 0.001). A similar kinetics of EBV DNA in the 2 blood compartments was observed. Clinically, both specimen types appeared to be equally informative to assess the risk of patients to develop PTLD. On the basis of EBV DNAemia levels, in 3 patients (10.7%) immunosuppressive therapy was reduced and 1 patient (3.5%) received early treatment for probable EBV disease. No patients developed EBV-PTLD. CONCLUSION: WB proved to be a suitable clinical specimen to monitor EBV DNA load after allo-HSCT for the management of EBV infection and PTLD prevention.

Clinical evaluation of the Roche Elecsys CMV IgG Avidity assay.

Congenital cytomegalovirus (CMV) infection has potentially severe consequences in newborns. The testing of pregnant women for CMV-specific antibodies may be useful for the identification of women at risk of transmitting the infection to the fetus. The determination of CMV IgG avidity helps to establish the timing of infection as IgG avidity matures during the course of infection. This study examines the performance of the Elecsys CMV IgG Avidity assay using preselected samples from patients at different phases of CMV infection. The Elecsys CMV IgG Avidity assay was tested at three sites using sequential samples from patients with recent primary CMV infection, as well as single samples from patients with recent primary or past CMV infection. The Elecsys assay discriminated well between early (low avidity) and late (high avidity) phases of infection in sequential serum samples. Overall, 98.8% of low-avidity samples corresponded to infection onset <180 days before sampling and 77.8% of all high-avidity results corresponded to infection onset >90 days before sampling. The assay's sensitivity was 90-97%, with specificity ranging from 89 to 100%, depending on the consideration of gray-zone avidity values. Single samples from recent primary or past infection showed similar distributions of avidity results. The Elecsys CMV IgG Avidity assay results are in agreement with preselected samples from patients with primary or past CMV infection, showing that the test is an adequate predictor of the phase of infection.

Clinical evaluation of new automated cytomegalovirus IgM and IgG assays for the Elecsys(®) analyser platform.

Cytomegalovirus (CMV) is a leading cause of physical and neurological abnormalities in newborns. Hence, the diagnosis of CMV infection in pregnant women is necessary in order to allow appropriate management of their pregnancy. New assays have been developed for the Roche Elecsys® immunoassay platform that detect CMV-specific immunoglobulin (Ig)M and IgG, with the IgM assay designed to target IgM produced at the start of infection rather than IgM persisting later in infection. This study aimed to evaluate the performance of the new assays compared with other commercial kits widely distributed in laboratories. The performance of the Elecsys and comparator CMV IgM and IgG assays was assessed using 967 preselected patient samples characterised by CMV infection status, as well as being compared using 1,668 unselected clinical samples. The Elecsys CMV IgM and IgG assays performed consistently with comparator assays using the preselected samples. The Elecsys CMV IgM assay showed improved sensitivity compared with the Enzygnost® assay in primary infection (91.2 % vs. 79.4 %) and improved specificity over the Architect® assay in potentially cross-reacting samples (94.1 % vs. 82.4 %). The Elecsys IgM assay reported fewer positive results in the later stages of CMV infection compared with ETI-CYTOK-M ELISA, while the Elecsys IgG assay reported slightly fewer negative results in the early stages of infection compared with ETI-CYTOK-G ELISA. There was good agreement between Elecsys and comparator assays using unselected clinical samples (range 90.4-99.4 %). The Elecsys CMV IgM and IgG assays compare well with routinely used assays and are suitable for clinical use.

Antibiotic Resistance and Therapy Outcome in H. pylori Eradication Failure Patients.

Helicobacter pylori (H. pylori) eradication fails in a definite amount of patients despite one or more therapeutic attempts. Curing these patients is progressively more difficult, due to development of antibiotic resistance. Current guidelines suggest testing antibiotic susceptibility in H. pylori isolates following two therapeutic attempts. AIM: to evaluate the development of antibiotic resistance, MIC values trends and therapeutic outcomes in patients who failed at least one H. pylori eradication therapy. METHODS: consecutive patients, referred to perform upper gastrointestinal endoscopy (UGIE) to our Unit from January 2009 to January 2019 following at least one therapeutic attempt were considered. Bacterial resistance towards clarithromycin, metronidazole and levofloxacin was tested. Patients received either a susceptibility-guided therapy or Pylera®. RESULTS: a total of 1223 patients were H. pylori positive, and antibiotic susceptibility was available for 1037. The rate of antibiotic resistance and MIC values significantly increased paralleling the number of previous therapeutic attempts. Eradication rates of antibiogram-tailored therapies remained stable, except for the sequential therapy if used as a third line. As a rescue treatment, the Pylera® therapy achieved cure rates comparable to those of the other culture-guided therapies. CONCLUSIONS: A significant increase in the secondary resistance towards the three tested antibiotics was observed, both as rate and MIC values, in correlation with the number of therapy failures. These findings should be considered when administering an empirical second-line therapy. Pylera® therapy eradication rates are comparable to culture-tailored therapies.

Management of cytomegalovirus infection in pregnancy: is it time for valacyclovir?

BACKGROUND: Congenital cytomegalovirus (CMV) infection is the leading infectious cause of neurological impairment for which, currently, there are no approved antenatal treatment options. OBJECTIVES: The aim of this article was to summarize the available evidence on the use of valacyclovir during pregnancy to prevent and treat congenital CMV infection and disease. SOURCES: Two databases (PubMed and ClinicalTrial.gov) were reviewed. CONTENT: Six relevant documents were identified, namely one observational study, three clinical trials, two case reports. Most relevant findings were those from two clinical trials. A phase 2/3 placebo-controlled study showed a decrease of 71% (5 of 45 vs 14 of 47) in rate of CMV vertical transmission in women treated with 8 g/day valacyclovir following primary CMV infection in pregnancy. A phase 2, single-arm clinical trial, showed that 8 g/day valacyclovir administered to mothers of symptomatic infected foetuses increased the portion of asymptomatic neonates to 82% (34 of 41), compared with 43% (20 of 47) in untreated pregnancies from a historical cohort. IMPLICATIONS: Studies in favour of using valacyclovir during pregnancy for prevention and treatment of congenital CMV infection are emerging but are still few. Randomized clinical trials on large cohorts of patients investigating the efficacy on prevention and treatment of congenital CMV are required. Unfortunately, this will be probably not be feasible at least in the short period. In the meantime, data on the 'off label' use of valacyclovir for CMV in pregnancy could be collected within a multicentre observational study.

Automatic environmental disinfection with hydrogen peroxide and silver ions versus manual environmental disinfection with sodium hypochlorite: a multicentre randomized before-and-after trial.

BACKGROUND: New technologies for automated disinfection have been developed, including the use of hydrogen peroxide atomized by specific equipment, with associated silver compounds. AIMS: To compare the effectiveness of an automated disinfection system with hydrogen peroxide <8% and silver ion versus a manual method with 0.5% sodium hypochlorite solution when evaluating the reduction of microbial mesophilic contamination and Clostridium difficile presence; and to evaluate the time required for both of these processes. METHODS: This was a randomized multicentre trial performed in different hospital wards that had been occupied previously by patients with Clostridium difficile infection. When patients were discharged their rooms were randomized to one of two decontamination arms. The surfaces where sampled using swabs, before and after disinfection. Swab samples were cultured for quantitative detection of microbial mesophilic contamination and qualitative detection of C. difficile. FINDINGS: Before disinfection, 13% of surfaces decontaminated with hydrogen peroxide and silver ions and 20% of surfaces decontaminated with sodium hypochlorite showed presence of C. difficile spores. After disinfection, the samples containing C. difficile were 0% (P < 0.001) in the group decontaminated with hydrogen peroxide and silver ions, and were 3% (P < 0.001) in the group decontaminated with sodium hypochlorite. This difference was not statistically significant; nor was the difference in the reduction of the microbial mesophilic contamination. CONCLUSION: The differences between the groups were not statistically significant; however, the disinfection with hydrogen peroxide and silver ions is preferable due to less dependence on operators.

Results of intestinal and multivisceral transplantation in adult patients: Italian experience.

PURPOSE: We report our experience with intestinal and multivisceral transplantation in Italy. METHODS: We performed 23 adult isolated intestinal transplants and seven multivisceral ones, three with liver, between December 2000 and June 2005. Indications for transplantation were loss of venous access (n = 14), recurrent sepsis (n = 10), and electrolyte-fluid imbalance (n = 6), 14 of whom also presented with total parenteral nutrition (TPN)-related liver dysfunction. Immunosuppression was based on induction agents like daclizumab (followed by tacrolimus and steroids) in the first period; alemtuzumab or thymoglobulin (with tacrolimus) in a second period after 2002. RESULTS: The mean follow-up was 742 +/- 550 days. Three-year patient actuarial survival rate was 88% for intestinal transplants and 42% for multivisceral (P = .015). Three-year graft actuarial survival rate was 73% for intestinal patients and 42.8% for multivisceral (P = .1). Graft loss was mainly due to rejection (57%). Complications were mainly represented by bacterial infections (92% of patients), relaparotomies (82%), and rejections (72%). Full bowel function without any parenteral nutrition or intravenous fluid support was achieved in 60% of recipients with functioning bowel including 95% on a regular diet. One patient underwent abdominal wall transplantation as well. DISCUSSION AND CONCLUSION: Intestinal transplantation has achieved high rates of patient and graft survival with even longer follow-up. Early referral of patients, especially in cases of TPN-liver disease, is mandatory to obtain good outcomes and avoid high mortality rates on the transplant waiting list. Immunosuppressive management remains the key factor to increase the success rate.

Abstension from treatment of low-level pp65 cytomegalovirus antigenemia after liver transplantation: a prospective study.

BACKGROUND: Ganciclovir is a highly effective and relatively safe drug to treat cytomegalovirus (CMV) infection in liver transplant patients; CMV resistance to ganciclovir is progressively emerging due to the extensive use of the drug in transplant and AIDS patients; CMV pp65 antigenemia allows early diagnosis of CMV infection and quantitation of the viral load; preemptive antigenemia-guided therapy of CMV infection can prevent CMV disease but the threshold of antigenemia value above which treatment has to be instituted is unclear. METHODS: To demonstrate the safety of abstention from preemptive treatment in the presence of low levels of antigenemia 77 consecutive liver transplant recipients were prospectively evaluated. Antigenemia was tested twice a week from transplantation until discharge, then once a week until the third postoperative month. In absence of risk factors for CMV disease, namely donor positive/recipient negative CMV serology, treatment with antibodies to lymphocytes and retransplantation, only patients with antigenemia of more than 50 or symptoms possibly related to CMV infection had preemptive treatment. RESULTS: A total of 32 patients had at least one positive antigenemia test with a value less than 50; 22 (68.7%) spontaneously cleared the virus, 3 were treated with i.v. ganciclovir for the presence of fever, and the other 7 (21,8%) progressed to values of antigenemia of more than 50 and were treated even if asymptomatic. No CMV disease was observed in these patients. CONCLUSION: CMV antigenemia less than 50 in liver transplant recipients with low and intermediate risk for CMV disease does not mandate preemptive ganciclovir treatment. Close surveillance with repeated determination of antigenemia until its negativization and careful clinical and laboratory monitoring is advisable.

Clinical evaluation of a new recombinant antigen-based cytomegalovirus immunoglobulin M immunoassay in liver transplant recipients.

BACKGROUND: Human cytomegalovirus (CMV) is a significant cause of morbidity and mortality among transplant recipients. Monitoring transplant recipients by CMV IgM serology has been questioned by several studies due to the reported insensitivity of serologic tests relative to antigen detection methods. METHODS: In this retrospective study, we have evaluated the performance of the new recombinant antigen-based Abbott AxSYM CMV IgM assay and compared it with CMV culture technique in a cohort of 40 liver transplant recipients who did not receive antiviral prophylaxis. RESULTS: The sensitivity, specificity, and positive and negative predictive values for detection of CMV disease by the AxSYM CMV IgM assay were 90.0%, 60.0%, 69.2%, and 85.7%, respectively, and by culture the values were 100%, 55.0%, 69.0%, and 100%, respectively. Detection of CMV IgM occurred before or at the time of CMV disease in only R+ recipients. CONCLUSION: Although this assay is a sensitive test for CMV-specific IgM, detection of CMV IgM preceded detection of virus by culture in patients only when the liver transplant recipient was CMV immune before transplantation (R+).

Human cytomegalovirus structural components: intracellular and intraviral localization of p38.

Rabbit antisera raised against the product of ORF UL 80 of human cytomegalovirus (CMV) genome (HindIII L fragment of AD169 strain) as well as IgM from acutely infected patients recognize an antigen of Mr 38 kDa. In the viral particle this antigen is bound via S-S bridge to a lower Mr compound to form a final complex of 62 kDa that is also recognized by rabbit antisera as well as patients' IgM. P38 is present both in the nucleus and in the cytoplasm of CMV-infected cells starting from 24 h p.i. and increasingly thereafter. Immunoelectron microscopy revealed that this antigen is mainly associated with the internal portion of viral capsids both in the nucleus and in the cytoplasm.

Maternal IgG avidity and IgM detected by blot as diagnostic tools to identify pregnant women at risk of transmitting cytomegalovirus.

In this study, we determined the avidity index (AI) of anticytomegalovirus (CMV) immunoglobulin G (IgG) and the anti-CMV immunoglobulin M (IgM) profile in 124 pregnant women, 87 of whom were considered at risk of transmitting CMV infection to their offspring and 37 of whom were at no risk. IgG avidity and blot for IgM were performed on two serum samples from each woman, at 6-18 weeks' gestation and at 20-23 weeks' gestation. Pregnancy outcomes were monitored. The results obtained showed that the determination of anti-CMV IgG avidity at 6-18 weeks' gestation can identify all women who would have an infected fetus/newborn (100% sensitivity), whereas IgM detected by blot had poorer results (69% sensitivity). Interestingly, at 20-23 weeks' gestation, the sensitivity of IgM detection by blot was higher than that obtained by avidity (75 % and 63%, respectively) and the combination of IgG avidity and IgM by blot yielded the best results (81% sensitivity).

Evaluation of two novel enzyme immunoassays using recombinant antigens to detect cytomegalovirus-specific immunoglobulin M in sera from pregnant women.

BACKGROUND: Two novel enzyme linked immunosorbent assays (ELISA) (Abbott IMx CMV IgM 2.0, and Cobas Core CMV IgM EIA recomb, research version) which use recombinant antigens to detect cytomegalovirus (CMV)-specific IgM antibodies were evaluated. OBJECTIVES: A new ELISA is normally evaluated against a gold standard commercial kit, which in this case does not exist. We therefore evaluated the two novel recombinant ELISA against four conventional ELISAs and a recently developed CMV IgM immunoblot containing four purified viral and four recombinant proteins. STUDY DESIGN: A total of 280 sera from pregnant women and 42 potentially cross-reactive sera were investigated using the six ELISAs, including 101 sera which were also tested using the new IgM immunoblot. RESULTS: Relative sensitivity, relative specificity and overall agreement differed according to the reference assay. The Cobas Core CMV EIA recomb showed much higher agreement with the ELISA consensus, and the IMx CMV IgM 2.0 with the immunoblot. CONCLUSION: The evaluation of these new IgM assays in terms of their agreement with either commercial ELISA kits or the IgM immunoblot demonstrates that the question 'which reference method?' is still open. However the recombinant IgM assays may improve the diagnosis of CMV infection in pregnancy since the recombinant technology offers helpful tools for identifying diagnostically relevant proteins and allows the use of standardized pure preparations of antigens. For serological diagnosis of CMV infection in pregnancy two IgM assays that can be relied upon should be performed. IgM positive sera should be tested with supplementary assays to differentiate primary from non-primary infection.

New developments in the diagnosis of maternal and congenital CMV infection.

Recent advances in the screening of pregnant women with Cytomegalovirus (CMV) IgM, CMV IgG and CMV IgG avidity serologic tests, has led to a more accurate diagnosis of CMV infection. When serologic screening is performed early in gestation, it is possible to identify those women at risk of intrauterine transmission of the virus, i.e., those women with a primary CMV infection, who should be enrolled in prenatal diagnosis. The use of quantitative PCR on amniotic fluid from pregnant women at 21-22 weeks of gestation in prenatal diagnosis is an effective diagnostic tool to distinguish between CMV infection and CMV disease in the fetus and newborn. Quantitative PCR on peripheral blood leukocytes from CMV infected newborns can be used to monitor viral load, especially during treatment with ganciclovir. These advances in serology and quantitative virology should lead to more accurate diagnosis of maternal and congenital CMV infection.

Prokaryotic expression of a large fragment of the most antigenic cytomegalovirus DNA-binding protein (ppUL44) and its reactivity with human antibodies.

We isolated and characterized from a lambda gt11 expression library clones expressing portions of human cytomegalovirus (HCMV)-p52. This nonstructural viral protein is encoded by UL44 and is known to be one of the best IgM reactive antigens. The reactivity of these clones was studied with human antibody and the gene fragment coding for the most immune-reactive portion of p52 (aa 202-434) was cloned in a prokaryotic expression vector, pROS, which overexpresses the antigen as a fusion protein to a truncated molecule of beta-galactosidase.