Validation of a Loop-Mediated Isothermal Amplification-Based Kit for the Detection of Legionella pneumophila in Environmental Samples According to ISO/TS 12869:2012.

Legionella pneumophila is a freshwater opportunistic pathogen and the leading cause of severe pneumonia known as Legionnaires' disease. It can be found in all water systems and survives in biofilms, free-living amoebae, and a wide variety of facilities, such as air conditioning and showers in hospitals, hotels and spas. The reference cultural method allows for the isolation and identification in many days, and in addition, it does not detect viable but rather non-culturable bacteria, increasing the risk of infection. In this context, a new LAMP-based (loop-mediated isothermal amplification) kit was developed, allowing for the rapid, sensitive, and labor-saving detection of L. pneumophila. The kit, "Legionella pneumophila Glow", was validated according to ISO/TS 12869:2012, testing sensitivity, inclusivity and exclusivity, and kit robustness. Sensitivity showed that the "Legionella pneumophila Glow" kit can detect up to 28 plasmid copies/µL. Robustness tests showed consistent results, with both contamination levels and the matrices used giving reproducible results. Furthermore, real samples were evaluated to compare the performance of the two methods. The LAMP kit "Legionella pneumophila Glow" proved a useful option for the rapid, efficient, and labor-saving screening of different typologies of water samples, offering significant advantages over the traditional method, as it is characterized by a high sensitivity, ease of use for laboratory testing, and a large reduction in analysis time, making it an asset to official controls.

What Is the Role of Psychological Factors in Long COVID Syndrome? Latent Class Analysis in a Sample of Patients Recovered from COVID-19.

Background: This study aimed to identify clusters of long COVID-19 symptoms using latent class analysis and investigate the psychological factors involved in the onset of this syndrome. Method: Five hundred and six subjects recovering from COVID-19 completed a series of standardized questionnaires to evaluate the personality traits, alexithymia, and post-traumatic stress. Results: Five classes were identified: Brain fog (31.82%), No symptoms (20.95%), Sensory disorders (18.77%), Breath impairment (17.59%), and Multiple disorders (10.87%). Women reported post-COVID-19 respiratory symptoms and multiple disorders to a greater extent than men. Hospitalized subjects were more likely to report persistent symptoms after COVID-19 than asymptomatic or home-treated subjects. Antagonism, hyperarousal, and difficulty identifying emotions significantly predicted post COVID-19 symptoms. Conclusions: These findings open new questions for research on long COVID-19 and how states of emotional dysregulation can alter the physiological processes of the body and contribute to the onset of organic pathologies.

Risk of post-traumatic stress symptoms in hospitalized and non-hospitalized COVID-19 recovered patients. A cross-sectional study.

High rates of post-traumatic stress disorder (PTSD) symptoms have been found among patients with more severe COVID-19-related symptoms, and hospitalization is generally recognized as a risk factor for developing PTSD. Furthermore, other personality characteristics may increase the risk of developing post-traumatic stress symptoms following a COVID-19 infection. This study aimed to assess personality traits, alexithymia, dissociation, anxiety, and depression in patients who have recovered from COVID-19 and the impact of these variables on the presence of post-traumatic stress symptoms. Five hundred and six participants completed a battery of standardized questionnaires. All the scales used in this study are valid and reliable measures of their respective constructs. Results showed that high levels of alexithymia, dissociation, anxiety, and depression statistically significantly predicted the three main clusters of PTSD symptoms (avoidance, intrusion, and hyperarousal) in individuals who have recovered from COVID-19. Furthermore, negative affectivity and psychoticism significantly predicted PTSD symptoms in our sample. Finally, individuals hospitalized by COVID-19 are more at risk of developing intrusion and hyperarousal symptoms than those who never needed hospital care. Our findings are a valuable contribution in identifying the main risk factors of psychological distress related to COVID-19 to address the long-term mental health needs of people who have experienced the disease.

COVID-19 and non-COVID-19 pneumonia: a comparison.

BACKGROUND: The COVID-19 pandemic has caused the relocation of huge financial resources to departments dedicated to infected patients, at the expense of those suffering from other pathologies. AIM: To compare clinical features and outcomes in COVID-19 pneumonia and non-COVID-19 pneumonia patients. PATIENTS AND METHODS: 53 patients (35 males, mean age 61.5 years) with COVID-19 pneumonia and 50 patients (32 males, mean age 72.7 years) with non-COVID-19 pneumonia, consecutively admitted between March and May 2020 were included. Clinical, laboratory and radiological data at admission were analyzed. Duration of hospitalization and mortality rates were evaluated. RESULTS: Among the non-COVID patients, mean age, presence of comorbidities (neurological diseases, chronic kidney disease and chronic obstructive pulmonary disease), Charlson Comorbidity Index and risk factors (tobacco use and protracted length of stay in geriatric healthcare facilities) were higher than in COVID patients. The non-COVID-19 pneumonia group showed a higher (24% vs. 17%), although not statistically significant in-hospital mortality rate; the average duration of hospitalization was longer for COVID patients (30 vs. 9 days, p = .0001). CONCLUSIONS: In the early stages of the COVID pandemic, our centre noted no statistical difference in unadjusted in-hospital mortality between COVID and non-COVID patients. Non-COVID patients had higher Charlson Comorbidity Scores, reflecting a greater disease burden in this population.Key MessagesIn March 2020, the COVID-19 disease was declared a pandemic, with enormous consequences for the organization of health systems and in terms of human lives; this has caused the relocation of huge financial resources to departments dedicated to infected patients, at the expense of those suffering from other pathologies.Few published reports have compared COVID-19 and non-COVID-19 pneumonia. In our study, performed in a geographic area with a low prevalence of SARS-CoV-2 infection, we found few statistically significant differences in terms of clinical characteristics between the two groups analyzed.In the early stages of the COVID pandemic, our centre noted no statistical difference in unadjusted in-hospital mortality between COVID and non-COVID patients. Non-COVID patients had higher Charlson Comorbidity Scores, reflecting a greater disease burden in this population.

Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants.

Contagious agalactia (CA), an infectious disease of small ruminants, caused by Mycoplasma agalactiae, is responsible for severe losses to dairy sheep production with substantial socioeconomic impacts on small-scale farmers. The diagnosis of CA is still problematic, time-consuming and requires well-equipped labs for confirmation of outbreaks. Therefore, rapid, accurate and cost-effective diagnostic tests are urgently needed. This work aims to validate a novel Loop-Mediated Isothermal Amplification (LAMP) test, based on the p40 target gene, for the detection of M. agalactiae in dairy sheep in order to confirm its potential practical use as a rapid and cheap field test. The LAMP system proposed in this study consists of a portable device composed of real-time fluorometer with the automatic interpretation of results displayed in a tablet. A total of 110 milk samples (90 positives and 20 negatives) were analysed to optimise the analysis procedure and to investigate the efficacy and robustness of the LAMP method. All samples were analysed using LAMP and conventional real-time PCR to compare the diagnostic sensitivity of the methods. The sensitivity of the LAMP was 10-fold higher than that of real-time PCR, with a detection limit up to 103 CFU/ml. The LAMP assay was able to detect M. agalactiae in 81 of 90 (90%, 95%CI 0.84-0.96) positive milk samples compared to 69 (77%, 95%CI 0.59-0.95) positive samples detected by real-time PCR; no positive signal occurred for any of the negative milk samples in either test. Therefore, the LAMP assay was found to be more sensitive than real-time PCR, low-cost, easy to perform, fast and not affected by contamination, indicating its potential as an effective diagnostic tool in the field level for the diagnosis of CA.

Validation of a Commercial Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Anisakis spp. DNA in Processed Fish Products.

Parasites belonging to the Anisakis genera are organisms of interest for human health because they are responsible for the Anisakiasis zoonosis, caused by the ingestion of raw or undercooked fish. Furthermore, several authors have reported this parasite to be a relevant inducer of acute or chronic allergic diseases. In this work, a rapid commercial system based on Loop-Mediated Isothermal Amplification (LAMP) was optimised and validated for the sensitive and rapid detection of Anisakis spp. DNA in processed fish products. The specificity and sensitivity of the LAMP assay for processed fish samples experimentally infected with Anisakis spp. larvae and DNA were determined. The LAMP system proposed in this study was able to give positive amplification for all the processed fish samples artificially contaminated with Anisakis spp., giving sensitivity values equal to 100%. Specificity tests provided no amplification for the Contracaecum, Pseudoterranova, or Hysterothylacium genera and uninfected samples. The limit of detection (LOD) of the LAMP assay proposed was 102 times lower than the real-time PCR method compared. To the best of our knowledge, this is the first report regarding the application of the LAMP assay for the detection of Anisakis spp. in processed fish products. The results obtained indicate that the LAMP assay validated in this work could be a reliable, easy-to-use, and convenient tool for the rapid detection of Anisakis DNA in fish product inspection.

Cigarette smoke affects the onco-suppressor DAB2IP expression in bronchial epithelial cells of COPD patients.

Cigarette smoke is a risk factor for COPD and lung cancer. In cancer, epigenetic modifications affect the expression of Enhancer of Zester Homolog 2 (EZH2), and silenced disabled homolog 2 interacting protein gene (DAB2IP) (onco-suppressor gene) by Histone H3 tri-methylation in lysine 27 (H3K27me3). In"ex vivo"studies, we assessed EZH2, H3K27me3 and DAB2IP immunoreactivity in bronchial epithelial cells from COPD patients (smokers, ex-smokers), Smoker and control subjects. In"in vitro" experiments we studied the effect of cigarette smoke extract (CSE) on EZH2/H3K27me3/DAB2IP expression, apoptosis, invasiveness, and vimentin expression in 16HBE, primary cells, and lung cancer cell lines (A549) long-term exposed to CSE. Finally, in "in vitro"studies, we tested the effect of GSK343 (selective inhibitor of EZH2). EZH2 and H3K27me3 expression was higher, while DAB2IP was lower levels, in bronchial epithelium from COPD and Smokers than in Controls. CSE increased EZH2, H3K27me3 expression and decreased DAB2IP, cell apoptosis and invasiveness in epithelial cells. GSK343 restored the effects of CSE. Cigarette smoke affects EZH2 expression, and reduced DAB2IP via H3K27me3 in COPD patients. The molecular mechanisms associated with EZH2 expression, generate a dysregulation of cell apoptosis, mesenchymal transition, and cell invasiveness in bronchial epithelial cells, encouraging the progression of airway inflammation toward lung cancer in COPD patients.

Effects of aging on sensation of dyspnea and health-related quality of life in elderly asthmatics.

BACKGROUND AND AIMS: As the awareness of sensory stimuli is often impaired in older subjects, it has been hypothesized that the aging process may influence the perception of dyspnea. This study aimed at evaluating the aging-related difference in perception of spontaneously occurring dyspnea in adult asthmatics and at whether any such aging-related differences have an effect on the health-related quality of life (HRQOL). METHODS: 18 elderly asthmatics (EA) aged >65 years and 20 young asthmatics (YA) (age range 16-44 years) were recruited. In all subjects, 12-month asthma symptom score and respiratory function were recorded. Dyspnea was measured at rest by the Visual Analog Scale (VAS) and HRQOL by the St. George's Respiratory Questionnaire (SGRQ). RESULTS: Although the groups did not differ for FEV1% predicted, the EA showed lower VAS scores (9.9+/-19.8 mm vs 19.5+/-17.0, p<0.05). As regards HRQOL, only the "Symptom" section of SGRQ showed lesser impairment in EA. CONCLUSIONS: Aging is associated with blunted sensation of dyspnea. This only partly attenuates the disease-related impairment in quality of life, and other factors are presumed to counterbalance this effect.