Virological surveillance of SARS-CoV-2 in an Italian Northern area: differences in gender, age and Real Time RT PCR cycle threshold (Ct) values in three epidemic periods.

BACKGROUND AND AIM OF THE WORK: Coronavirus Disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is a global public health emergency. The aim of this study was to investigate cases characteristics and Real Time RT PCR cycle threshold (Ct) values distribution of COVID-19 in an Italian Northern area during three periods: first period, February-May 2020; second period, June-August 2020; third period, September 2020-February 2021. METHODS: Real Time RT PCR was used to detect SARS-CoV-2 in respiratory samples (oro/nasopharyngeal swabs). RESULTS: A total of 254,744 samples were tested during the study period. Out of 20,188 positive samples (7.92%), 10,303 were females (51.04%) and 9,885 were males (48.96%). The percentage of positivity varied during the three different periods: 14.1% in the first period, 1.4% in the second and 9.2% in the third. The lowest Ct values were observed in the first phase of pandemic, with an overall average of 25.64. Overall average of the Ct values was lower in males than in females, 26.29 ± 6.04 and 26.84 ± 5.99 respectively. The oldest patients recorded lower Ct values. CONCLUSIONS: The findings of our study represent further evidence in support of the fact that male sex and older age showed lower Ct values, which means higher viral loads and higher infectious potential. These knowledges are useful to better understand the epidemiological aspects of COVID-19 and to perform effective Public Health Policies.

Virological surveillance of SARS-CoV-2 in an Italian northern area: comparison of Real Time RT PCR cycle threshold (Ct) values in three epidemic periods.

Aim of the study was to investigate the differences in Ct values in nasopharingeal swabs collected in three SARS-CoV-2 epidemic periods: first one from February 23 to March 25 (14 days from lockdown started on March 11); the second one from  March  26 to May 18 (14 days  from the end of strict lockdown on May 4) and the third one from May 19 until June 15. Viral RNA was detected in nasopharyngeal swabs obtained both from inpatients and outpatients. COVID-19 infection was confirmed according to the Ct values for N1 and N2 genes ascertained by Real-Time RT-PCR assay as described by the CDC. We calculated the prevalence of nasopharyngeal swabs tested positive for SARS-CoV-2, the mean and median of the Cts and the percentage of samples equal or below the Ct value of 25 in the 3 periods considered. The average value of Ct increased, going from 24.80 in the first epidemic period to 26.64 in the second period to 28.50 in the third period (p <0.001). The percentage of samples with Ct lower than or equal to 25 also decreased sharply from 54.7% to 20.0%. These findings need to be integrated with epidemiological and clinical data.

Immunity status against tetanus in young migrants: a seroprevalence study.

BACKGROUND AND AIM OF THE WORK: Thanks to the highly effective vaccine, tetanus became sporadic in high-income countries with well-established primary childhood immunization programs, but it is common in low-income countries. The migrants, leaving countries with poor immunization programs or where vaccinations have been interrupted, may represent a new risk group for tetanus in host countries. A seroprevalence study was conducted to estimate the immunological status against tetanus in young migrants without vaccination documentation. METHODS: After a careful assessment by vaccination services of the Local Health Authority, all migrants recently arrived in Italy were included in the serosurvey. Titers of anti-tetanus toxoid were measured using a commercial ELISA kit. Subjects were stratified by age and by WHO region. Antibody titers <0.10 IU/ml were considered to be seronegative, between 0.10 and 1.00 IU/ml as intermediate protection, and >1.00 IU/ml high protection. RESULTS: From January 2004 to December 2019, 2,326 blood samples were collected. Mean age was 13.9 years with no differences between WHO regions. The percentage of the subjects without protective antibodies was 22.3%, with an intermediate level was 45.2%, with high titer was 32.5%. Among migrant coming from African and Eastern Mediterranean WHO regions, the highest percentages of seronegative titers and, at the same time, the low percentages of high protective levels were found. Titers decreased with age. CONCLUSIONS: The significant proportion of seronegative migrants and the decrease of protective titers increasing age, confirm the importance of the evaluation of the immunological status to employ the appropriate vaccination strategy.

Passive air sampling: the use of the index of microbial air contamination.

BACKGROUND: Bioaerosol plays an important role in human life with potentially infectious, allergic and toxic effects. Active and passive methods can be used to assess microbial air contamination, but so far there is not a unanimous consensus regarding the indications about methods to be used and how to interpret the results. The passive method has been standardized by the Index of Microbial Air contamination (IMA). Classes of contamination and maximum acceptable levels of IMA have been proposed, related to different infection or contamination risks. The aim of this study was to provide information about the use of the passive sampling method, with reference to the IMA standard. METHODS: We searched PubMed and Scopus for articles published until January 2020 reporting the citation of the article by Pasquarella et al. "The index of microbial air contamination. J Hosp Infect 2000". Only studies in English language where the IMA standard was applied were considered. Studies regarding healthcare settings were excluded. RESULTS: 27 studies were analyzed; 12 were performed in Europe, 8 in Asia, 5 in Africa, 2 in America. Cultural heritage sites, educational buildings and food industries were the most common indoor monitored environments; in 8 studies outdoor air was monitored. CONCLUSIONS: This review has provided a picture of the application of standard IMA in different geographic areas and different environments at risk of airborne infection/contamination. The analysis of the results obtained, together with a wider collection of data, will provide a useful contribution towards the definition of reference limits for the various types of environments to implement targeted preventive measures.

[Fungal contamination in a University building].

BACKGROUND: It is recognized that airborne fungi can cause illnesses in humans but data on environmental exposure are still poor. The aim of this study was to evaluate the fungal airborne contamination in a university building. METHODS: The study was performed in February and May 2018; air samples were collected, before activity (on Monday) and during activity, (on Friday), both through active (CFU/m3) and passive (Index of microbial air contamination, IMA) method. Fungi were identified by using the scotch test. RESULTS: In February the median fungal contamination value decreased from 14 CFU/m3 before activity to 7 CFU/m3 during activity, while IMA median remains 0. Instead in May both increased during activity (from 87 to 140 CFU/m3; from 5.5 to 7.5 IMA). Overall values increased in May compared to February. Aspergillus spp., Penicillium spp.,  Cladosporium spp. Absidia spp. were the genera most frequently isolated in both months, while in May Chaetomium spp. e Ulocladium spp. were recovered too. CONCLUSIONS: Seasonal trend in the levels of fungal contamination of the air was observed, with a statistically significant increase in May. This study represents the first step of a wider study aimed at enhancing knowledge about air fungal contamination.

A novel mutation in the NR0B1 gene in a family with monozygotic twin sisters and congenital adrenal hypoplasia affected children.

OBJECTIVE: Congenital adrenal hypoplasia (CAH) is a rare disorder that can be inherited in an X-linked or autosomal recessive pattern. CAH is frequently associated with hypogonadotropic hypogonadism (HHG) with absent or arrested puberty and impaired fertility caused by abnormalities in spermatogenesis. It is estimated that more than 50% of boys with idiopathic adrenal insufficiency have mutations in the NR0B1 gene product, DAX1. CASE REPORT: The proband is a young boy born after an uneventful pregnancy and delivery to non-consanguineous parents. At age 4 years and 4 months he came to our attention because of severe vomiting, abdominal pain, dehydration, and asthenia. The proband underwent a detailed clinical investigation including genetic testing. Sequencing analysis of the NR0B1 gene coding region from the affected child revealed a novel hemizygous deletion [c.385delC; p.(Leu129Cysfs*135)]. This mutation was also present in the heterozygous healthy mother and in her twin sister and in the first cousin of the proband. Monozygosity of the twin sisters was demonstrated. This suggests a de novo mutation and gonadal mosaicism for the deletion. CONCLUSIONS: Adrenal hypoplasia typically presents as adrenal insufficiency during the first few months of life, however, not necessarily as shown by our index case. HHG is thought to affect all NR0B1 mutated patients who reach puberty and, as understanding of the disease has improved, more of these patients survive while presenting different features of the disease, this emphasizing the value of genetic testing in boys with primary adrenal insufficiency and suspected X-linked CAH.

Markers of insulin sensitivity in placentas and cord serum of intrauterine growth-restricted newborns.

OBJECTIVE: Intrauterine growth restriction (IUGR) has been related to a higher incidence of insulin resistance in adult life, which is associated with low adiponectin and high resistin, insulin and interleukin (IL)-6 serum concentrations. This study assessed cortisol, insulin, total insulin receptor, resistin, adiponectin and IL-6 concentrations, as markers of insulin sensitivity, in placental lysates and cord serum of IUGR and appropriate for gestational age (AGA) newborns, to establish relationships among peptides and with growth parameters at birth. DESIGN AND PATIENTS: Whole villous tissue and cord serum at birth were collected from 24 AGA and 18 IUGR newborns of comparable gestational age. MEASUREMENTS: Hormonal and peptide concentrations were assayed in placental lysates and cord serum using specific commercial kits. Concentrations in lysates were adjusted per mg of total protein content. RESULTS: Cortisol, insulin and resistin concentrations and the total amount of insulin receptor were similar in both groups. IL-6 concentration in lysates was significantly higher in IUGR compared with AGA newborns. Adiponectin was significantly lower in lysates from IUGR compared with AGA newborns. Placental insulin and resistin concentrations were positively correlated. Placental adiponectin concentration was positively correlated with the weight of the placenta, birthweight and head circumference. IL-6 concentration in lysates was negatively correlated with birth length, birthweight and head circumference. CONCLUSIONS: This study evaluated the markers of insulin sensitivity in the placentas of subjects born IUGR, showing new potential roles for adiponectin and IL-6 in particular, and suggesting a role for the placenta in the programming of these hormones.

The IGF system and cytokine interactions and relationships with longitudinal growth in prepubertal patients with cystic fibrosis.

OBJECTIVE: Growth delay is a feature of patients with cystic fibrosis (CF). CF is a condition characterized by chronic inflammation that has been shown to modify the IGF system, which is essential for normal growth, and is related to pulmonary function in CF patients. We aimed to verify whether circulating levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, insulin and the IGF system were related and/or had relationships with linear growth in children with CF. DESIGN AND PATIENTS: Seventeen prepubertal CF patients (nine males and eight females) in a stable clinical condition were enrolled. Auxological parameters, pulmonary function and the Shwachman-Kulczycki (S-K) score were assessed, and serum samples were drawn at baseline and after 12 months. MEASUREMENTS: TNF-alpha, IL-6, IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3 and insulin were assayed using specific commercial kits. RESULTS: At baseline, TNF-alpha serum concentration was related to serum IGF-I concentration (R = 0.53), IGF-II bioactivity (IGF-II/IGFBP-3 molar ratio, R = +0.52) and insulin concentration (R = +0.63). Changes in serum IL-6 and IGFBP-2 concentrations during the 12-month observation were positively correlated (R = +0.63). Changes in height standard deviation score (Ht SDS) were correlated with IGF-I serum concentrations at baseline (R =+0.67) and after 12 months (R = +0.70), with IGF-I bioavailability and with IGFBP-1 serum concentrations (R = -0.88). Body mass index (BMI) SDS correlated with IGF bioavailability. CONCLUSIONS: This study showed a relationship between inflammatory status and the IGF system, and an effect of these interactions on longitudinal growth. Moreover, a role for insulin in growth was identified. Better control of inflammation and preservation of insulin secretion could benefit these patients.

Ghrelin inhibits steroid biosynthesis by cultured granulosa-lutein cells.

CONTEXT: Growing evidence indicates that ghrelin may participate in the regulation of different aspects of reproductive function. The genes encoding for this peptide and its receptor are expressed in the human ovary, but their functional role is still unknown. OBJECTIVE: The aim of our study was to assess whether ghrelin has any effect on steroid synthesis by human granulosa-lutein cells and to identify the receptor isoform through which this potential effect is exerted. DESIGN, PATIENTS, AND METHODS: Thirty-five women with spontaneous ovulatory cycles undergoing in vitro fertilization for infertility due to uni- or bilateral tubal impatency or male factor were studied. Granulosa-lutein cells obtained from follicular fluid were incubated with increasing amounts of human acylated ghrelin (10(-11) to 10(-7) mol/liter) either alone or together with a 1:500 concentration of a specific anti-ghrelin receptor antibody [GH secretagogue receptor 1a (GHS-R1a)]. Culture media were tested for estradiol (E(2)) and progesterone (P(4)). The expression of GHS-R1a and GHS-R1b in human granulosa-lutein cells was also studied by real-time quantitative PCR. RESULTS: E(2) and P(4) concentrations in the culture media were significantly reduced by ghrelin in a dose-dependent fashion. The maximal decrease in E(2) (25%) and P(4) (20%) media concentrations was obtained with the 10(-7) and 10(-8) mol/liter ghrelin concentrations, respectively. The inhibitory effect of all ghrelin concentrations used was antagonized by the specific anti-ghrelin receptor-1a antibody added to the culture media and not by the specific anti-ghrelin receptor-1b antibody. Both 1a and 1b isoforms of the GHS-R were expressed in human granulosa-lutein cells, with the latter exceeding the former's expression (GHS-R1b/GHS-R1a ratio, 143.23 +/- 28.15). CONCLUSIONS: Ghrelin exerts an inhibitory effect on granulosa-lutein cells steroidogenesis by acting through its functional GHS-R1a. This suggests that ghrelin may serve an autocrine-paracrine role in the control of gonadal function and be part of a network of molecular signals responsible for the coordinated control of energy homeostasis and reproduction.

Changes and relationships of IGFS and IGFBPS and cytokines in coeliac disease at diagnosis and on gluten-free diet.

OBJECTIVE: To evaluate changes and relationships of IGFs and IGFBPs, serum interleukin 6 (IL-6) and tumour necrosis factor (TNF)-alpha, and auxological parameters at diagnosis of coeliac disease (CD) and at 6 months and 12 months after starting a gluten-free diet (GFD), compared with a control population. PATIENTS: Twenty patients were enrolled at diagnosis (9 male, 11 female; age 9.6 +/- 0.8 years). A healthy population of 18 subjects (5 male, 13 female; age 11.3 +/- 0.6 years) comparable for age, sex and pubertal status served as controls at baseline. MEASUREMENTS: Blood samples were taken at diagnosis, and at 6 months and 12 months after starting the GFD. Serum IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, IL-6 and TNF-alpha were measured using commercial kits. Height (Ht) standard deviation score (SDS), body mass index (BMI) SDS and Ht velocity SDS were evaluated at diagnosis and at 6 months and 12 months after starting GFD. RESULTS: In CD patients, both Ht SDS and BMI SDS increased during the first year of treatment, and Ht velocity SDS increased during the second 6 months of follow-up (P < 0.05). At diagnosis, IGF-I, IGF-II and IGFBP-3 were lower compared with controls, IGFBP-1 was similar, IGFBP-2, IL-6 and TNF-alpha were higher (P < 0.05). When on GFD, all peptides normalized and IGFBP-1 decreased. The IGF-I/IGFBP-2 and IGF-I/IGFBP-3 molar ratios were significantly reduced at diagnosis compared with those of controls, but were increased for both groups when on GFD. Although there was no apparent abnormality at diagnosis, the IGF-II/IGFBP-2 molar ratio increased significantly on GFD. Ht velocity SDS was positively correlated with IGFBP-3 (P < 0.05) and with the IGF-I/IGFBP-2 molar ratio (P < 0.05). Serum IL-6 was negatively correlated with IGF-I and positively with IGFBP-1 (P < 0.05). CONCLUSIONS: The data obtained from this study confirm changes in the IGF and cytokine systems at diagnosis of CD which tend to normalize on the gluten-free diet. The two systems show relationships with each other and with linear growth.

Changes in interleukin-6 and IGF system and their relationships in placenta and cord blood in newborns with fetal growth restriction compared with controls.

OBJECTIVES: The IGF system is central to fetal growth. Recently, the relationships between cytokines and the IGF system have been shown in specific tissues. It is unknown whether these occur in the placenta. The aim of this study was to assess whether interleukin-6 (IL-6) modulated the IGF system. METHODS: Whole villous tissue and cord serum were collected from fetal growth restriction (FGR) neonates diagnosed before birth with altered Doppler velocimetry and controls. Sixteen FGR and 20 controls, born after week 32 of gestation from elective Caesarean sections, were compared. Total RNA was extracted from the placenta samples, reverse transcribed, and real-time quantitative reverse transcriptase (RT)-PCR was performed to quantify cDNA for IGF-I, IGF-II, IGF binding protein (IGFBP)-1, IGFBP-2, and IL-6. The same proteins were assayed in placenta lysates and cord serum using specific commercial kits and western immunoblotting. RESULTS: FGR subjects had significantly more IGFBPs-1 and -2, and IL-6 mRNA and corresponding proteins in the placenta. In particular, the less phosphorylated isoforms of IGFBP-1 were highly increased. IL-6 and IGFBPs-2 mRNA, and IL-6 and IGFBP-1 peptides were positively and significantly correlated in the placenta. The IGF-II peptide was also significantly increased in FGR placentas. In cord serum, IGFBPs-1 and -2 were significantly more elevated in the FGR neonates. Serum IL-6 was significantly and positively correlated with both IGFBP-1 and IGFBP-2. CONCLUSIONS: The placenta of FGR neonates has higher IGF-II, IGFBP-1, IGFBP-2, and IL-6 contents compared with controls. At birth, IGFBPs-1 and -2 are increased in the cord blood of FGR neonates. IL-6 and IGFBP-2 gene expressions are closely related in the placenta. We suggest that the increase in IL-6 and IGFBP-2 could be subsequent to hypoxia and nutrient deficiency. As IGFBP-2 has a strong affinity for IGF-II, which is crucial for fetal growth, it could be an important bioregulator of IGF-II in the placenta.

Inflammation is a modulator of the insulin-like growth factor (IGF)/IGF-binding protein system inducing reduced bioactivity of IGFs in cystic fibrosis.

OBJECTIVE: In inflammatory bowel diseases, increased serum interleukin (IL)-6 levels are associated with high serum insulin-like growth factor-binding protein 2 (IGFBP-2) levels, and cytokines modify the insulin-like growth factor (IGF)/IGFBP system in models in vitro. In cystic fibrosis (CF) the IGF/IGFBP system has not been extensively studied, and relationships with proinflammatory cytokines have not been explored. The aim of this study was to investigate the IGF/IGFBP system and verify changes dependent on IL-1beta, IL-6, tumour necrosis factor alpha (TNFalpha), and insulin. METHODS: Eighteen subjects with CF (mean age 26.6 +/- 1.1 years) and 18 controls, comparable for age, sex, and body mass index, were enrolled. Serum IGF-I, IGF-II, IGFBP-2, IGFBP-3, IL-1beta, IL-6, TNFalpha, insulin and C-peptide were measured. Different molecular forms of IGFBP-2 and IGFBP-3 were investigated by Western immunoblotting. The patients were analysed as a whole and as two subgroups depending on established clinical criteria (Swachman-Kulczycki score). RESULTS: Patients had higher serum concentrations of IL-1beta, IL-6, TNFalpha and IGFBP-2 than controls. Serum concentrations of IGF-I and IGF-II were significantly lower and insulin and C-peptide levels significantly increased in CF compared with healthy controls whereas IGFBP-3 serum concentrations were similar, with comparable IGF-I/IGFBP-3 and decreased IGF-I/IGFBP-2 and IGF-II/IGFBP-2 molar ratios. From correlation analysis we detected a significant positive correlation between IGFBP-2 and IL-6 and a negative correlation between IGFBP-2 and IGFBP-3. CONCLUSIONS: Our findings suggest that inflammation is an important modulator of the IGF/IGFBP system with an overall reduction in IGF bioactivity in CF.

Differentiation of leptospires of the serogroup Pomona by monoclonal antibodies, pulsed-field gel electrophoresis and arbitrarily primed polymerase chain reaction.

All reference strains described as representing separate serovars belonging to the serogroup Pomona and a clinical leptospiral isolate (LP2) from this serogroup were analyzed using a battery of 9 monoclonal antibodies, pulsed-field gel electrophoresis (PFGE) and arbitrarily primed polymerase chain reaction (AP-PCR). Monoclonal antibody analysis provided taxonomic results which were in agreement with the current classification of the serogroup Pomona into six serovars and allowed the classification of the isolate LP2 in the serovar pomona. PFGE and AP-PCR, although in general agreement with monoclonal antibody analysis, also were able to demonstrate some differences in the restriction patterns of strains Pomona, Monjakov and CB. These results indicate that these strains, grouped within serovar pomona after the introduction of bacterial restriction endonuclease analysis as the typing method, but formerly described as representing separate serovars (pomona, monjakov and cornelli, respectively), are similar but not identical to one another. This was also the case with strains 5621, the serovar mozdok reference strain, and K1, formerly described as serovar dania reference strain, but currently recognized to be a mozdok-like strain. These findings suggest that the deletion of some serovars within the serogroup Pomona, namely mozdok, cornelli, and dania, should be reconsidered. Thus, PFGE appears to be a useful tool for the serovar identification of leptospires belonging to the serogroup Pomona and for shedding light on the problem of their classification.