Culture and Real-time Polymerase Chain reaction sensitivity in the diagnosis of invasive meningococcal disease: Does culture miss less severe cases?

BackgroundInvasive meningococcal disease (IMD) is a highly lethal disease. Diagnosis is commonly performed by culture or Realtime-PCR (qPCR).AimsOur aim was to evaluate, retrospectively, whether culture positivity correlates with higher bacterial load and fatal outcome. Our secondary aim was to compare culture and qPCR sensitivity.MethodsThe National Register for Molecular Surveillance was used as data source. Cycle threshold (CT), known to be inversely correlated with bacterial load, was used to compare bacterial load in different samples.ResultsThree-hundred-thirteen patients were found positive for Neisseria meningitidis by qPCR, or culture, or both; 41 died (case fatality rate 13.1%); 128/143 (89.5%) blood samples and 138/144 (95.8%) CSF were positive by qPCR, 37/143 (25.9%) blood samples and 45/144 (31.2%) CSF were also positive in culture. qPCR was 3.5 times (blood) or 3.1 times (CSF) more sensitive than culture in achieving a laboratory diagnosis of IMD (OR 24.4; 95% CI 12.2-49.8; p ConclusionsIn conclusion our study demonstrated that qPCR is significantly (at least 3 times) more sensitive than culture in the laboratory confirmation of IMD. The study also demonstrated that culture negativity is not associated with lower bacterial loads and with less severe cases. On the other side, in patients with sepsis, qPCR can predict fatal outcome since higher bacterial load, evaluated by qPCR, appears strictly associated with most severe cases and fatal outcome. The study also showed that molecular techniques such as qPCR can provide a valuable addition to the proportion of diagnosed and serotyped cases of IMD

Significant impact of pneumococcal conjugate vaccination on pediatric parapneumonic effusion: Italy 2006-2018

Abstract Etiology and serotyping of parapneumonic effusion (PPE) and the impact of vaccination was evaluated over a 12-year period, before and after the PCV13 introduction (2011) for Italian children From 0 to 16 years of age. Five hundred and two children were evaluated; 226 blood and 356 pleural fluid samples were obtained and tested using Realtime-PCR and culture. In the pre-PCV13 era S. pneumoniae was the most frequent pathogen identified (64/90; 71.1%) with a large predominance of serotypes 1 (42.4%), 3 (23.7%), 7F (5.1%) and 19A (11.9%). The impact of vaccination, calculated on children 0–8 years of age, demonstrated a significant reduction of PPE: with an incidence rate of 2.82 (95%CL 2.32–3.41) in the pre-PCV13 era and an age-standardized rate (ASR) of 0.66 (95% CL 0.37–1.99) in the post-PCV13 era, p  In conclusion, our findings indicate that routine immunization with PCV13 has significantly reduced the burden of childhood PPE in vaccinated children, without increasing PPE due to other bacteria and without serotype shift. Moreover, the impact of PCV13 may be underestimated due to the increase in pneumococcal surveillance in Italy. Data has also shown that Real-time PCR is an essential tool to better define the etiology of PPE and to monitor vaccination plans. Longer studies will be necessary to evaluate the role of herd protection in PPE prevention

Mutazioni del gene SMC1A e tumore del colon retto a stadi precoci di sviluppo tumorale

Il gene SMC1A codifica per una proteina del complesso denominato coesina, responsabile della coesione dei cromatidi fratelli e della corretta segregazione cromosomica durante la meiosi e la mitosi. Il core della coesina è costituito da 4 subunità differenti: SMC1A , SMC3, RAD21 e STAG1/2. Negli ultimi anni, mutazioni nel complesso proteico coesina e nei suoi fattori regolatori, NIPBL, ESCO2, sono state associate a malattie, come la sindrome di Cornelia de Lange e la sindrome di Roberts, complessivamente note con il nome di “coesinopatie”. Evidenze sperimentali suggeriscono che la coesina è implicata anche nella regolazione dell’espressione genica e nel mantenimento della stabilità genomica. Infatti la coesina regola l'espressione genica e in particolare di geni legati allo sviluppo e alla proliferazione cellulare modulando l’interazione a distanza tra il promotore e l’enhancer. Inoltre i fattori della coesina sono coinvolti nel controllo del ciclo cellulare a livello del checkpoint G2/M, dopo il danno al DNA. Per di più, l'espressione dei geni della coesina e dei suoi regolatori è down-regolata in numerosi tumori umani (tumore della cute, carcinoma della vescica e del seno). La recente analisi di 132 carcinomi del colon-retto, caratterizzati da instabilità cromosomica (CIN), ha permesso di identificare 11 mutazioni somatiche in cinque geni, di cui quattro coinvolti nella coesione dei cromatidi fratelli: SMC1A, NIPBL, SMC3, STAG3. In particolare sono state identificate 4 mutazioni nel gene SMC1A. Il tumore colon-rettale è una neoplasia maligna che colpisce l'intestino crasso e si manifesta con maggiore frequenza a livello del retto, del sigma e dell'ano; meno interessati sono il colon ascendente, il trasverso e il cieco. Il tumore è dovuto alla proliferazione incontrollata delle cellule della mucosa che rivestono il lume con formazione di lesioni precancerose (adenomi), che hanno un alto rischio di evolvere in una forma tumorale. Lo sviluppo del tumore del colon-retto è ben descritto e ad ogni stadio istologico si associa una specifica mutazione nei geni soppressori o protooncogeni. Tuttavia il tumore colon-rettale è anche caratterizzato da una elevata instabilità cromosomica di cui non è nota l'origine. Oltre alla forma CIN, che rappresenta circa il 90-95% dei tumori colon rettali, vi è una forma caratterizzata da instabilità dei microsatelliti (MIN) che costituisce circa il 5-10%. Le forme MIN non mostrano instabilità cromosomica. Tenendo in considerazione il ruolo della coesina nella corretta segregazione cromosomica, nell’ambito di questa tesi ho eseguito lo screening mutazionale del gene SMC1A in tumori del colon retto a stadi precoci di sviluppo tumorale. Inoltre ho eseguito la stessa analisi in un numero ristretto di tumori colon rettali MIN. Il DNA è stato estratto da sezioni bioptiche in paraffina ed il gene SMC1A è stato amplificato utilizzando primers specifici per le regioni introne/esone. Successivamente il prodotto di PCR è stato sequenziato e la sequenza nucleotidica ottenuta è stata confrontata con la sequenza wild-type di riferimento. Questo ci ha permesso di identificare due mutazioni a carico del gene SMC1A che non sono state descritte precedentemente. Queste mutazioni non sono state identificate nella popolazione generale, suggerendo che non sono varianti rare. Infine, è interessante notare come, i tumori MIN non mostrano mutazioni a carico del gene SMC1A. Complessivamente, i dati prodotti durante la mia tesi, suggeriscono che le mutazioni del gene SMC1A siano specifiche per forme CIN del tumore colon rettale e che potrebbero svolgere un ruolo importante nella insorgenza dell'instabilità cromosomica

X-rays Activate Telomeric Homologous Recombination Mediated Repair in Primary Cells

Cancer cells need to acquire telomere maintenance mechanisms in order to counteract progressive telomere shortening due to multiple rounds of replication. Most human tumors maintain their telomeres expressing telomerase whereas the remaining 15%-20% utilize the alternative lengthening of telomeres (ALT) pathway. Previous studies have demonstrated that ionizing radiations (IR) are able to modulate telomere lengths and to transiently induce some of the ALT-pathway hallmarks in normal primary fibroblasts. In the present study, we investigated the telomere length modulation kinetics, telomeric DNA damage induction, and the principal hallmarks of ALT over a period of 13 days in X-ray-exposed primary cells. Our results show that X-ray-treated cells primarily display telomere shortening and telomeric damage caused by persistent IR-induced oxidative stress. After initial telomere erosion, we observed a telomere elongation that was associated to the transient activation of a homologous recombination (HR) based mechanism, sharing several features with the ALT pathway observed in cancer cells. Data indicate that telomeric damage activates telomeric HR-mediated repair in primary cells. The characterization of HR-mediated telomere repair in normal cells may contribute to the understanding of the ALT pathway and to the identification of novel strategies in the treatment of ALT-positive cancers

Intrinsic positive end-expiratory pressure during ventilation through small endotracheal tubes during general anesthesia: Incidence, mechanism, and predictive factors

Study Objective To assess the safety of mechanical ventilation and effectiveness of extrinsic positive end-expiratory pressure (PEEP) (PEEPe) in improving peripheral oxygen saturation (SpO2) during direct microlaryngeal laser surgery; to assess the incidence, amount, and nature (dynamic hyperinflation or airflow obstruction) of ensuing intrinsic PEEP (PEEPi); and to find a surrogate PEEPiindicator. Design Quasiexperimental. Setting S. Raffaele Hospital (Milano), November 2009 to December 2010. Patients Fifty-two adults scheduled for direct microlaryngeal laser surgery. Exclusion criterion is pregnancy. Interventions Twenty-one percent O2mechanical ventilation through 4.5- to 5.5-mm internal diameter endotracheal tubes; in 29 patients, after measurement of PEEPi, an identical amount of PEEPewas added; and PEEPi. Measurements SpO2, peak (Pawpeak) and plateau (Pawplateau) airway pressure, and end-expiratory carbon dioxide were measured every 5 minutes. Respiratory compliance (Crs) was computed. PEEPiwas measured (end-expiratory occlusion method). Main Results PEEPi\ue2\u89\ua5 5 cm H2O occurred in 14 patients (27%) after intubation, in 16 (30%) at the beginning, and in 14 (27.3%) at the end of surgery. Thirty-one patients (59.4%) exhibited PEEPi\ue2\u89\ua5 5 cm H2O on at least 1 time point. PEEPiat the beginning of surgery was positively correlated with Pawplateau, Crs, tidal volume, and body mass index. Body mass index was the only predictor for the occurrence of PEEPi\ue2\u89\ua5 5 cm H2O. At the beginning of surgery, the Pawplateaureceiver operating characteristic curve predicting PEEPi\ue2\u89\ua5 5 cm H2O had area under the receiver operating characteristic curve of 0.85; best cutoff value of 15.5 cm H2O (sensitivity, 88.9%; specificity, 75%; correctly classified cases, 86.1%). When PEEPewas applied, in 23 cases (82.1%), total PEEP equaled PEEPe+ PEEPi; in 3 (10.7%), it was lower; and in 2 (7.1%), it was higher. Application of PEEPeincreased SpO2(P<.05) and Crs(P<.05). Conclusions During ventilation through small endotracheal tubes, PEEPi(mostly due to dynamic hyperinflation) is common. Hemodynamic complications, barotrauma, and O2desaturation (reversible with PEEPe) are rare. Pawplateauprovided by ventilators is useful in suspecting and monitoring the occurrence of PEEPiand allows detection of lung overdistension as PEEPeis applied

The Italian Version of the Michigan Hand Outcomes Questionnaire (MHQ): Translation, Cross-Cultural Adaptation and Validation

Background: Patient-reported outcome measures are largely used in clinical practice and scientific community. Michigan hand questionnaire (MHQ) is widely recognized as a standardized reliable instrument for the assessment of any kind of hand disorders.Aim of the study: translation, cross-cultural adaptation and validation of the Italian version of MHQ.Methods: The study was composed by two phases. Phase 1 consisted in translation and cross-cultural adaptation of MHQ, from original language version (English) into Italian, according to the standard procedure of translation and back translation. The final Italian version of MHQ was tested on 136 Italian patients with hand disorders (Phase 2), in addition to Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaire, Visual analogue scale (VAS) for pain assessment and grip strength, to psychometric properties of the new version, 55 patients repeated the questionnaire after 7 days for test-retest to assess the reproducibility of the questionnaire. All data were subsequently analyzed (descriptive statistics, multitrait analysis, reliability and construct validity assessment).Results: Phase 1 was performed without major problems, thus the final Italian version was approved for Phase 2. The questionnaire was clear and easily understood (missing data 0-2.9%). Multitrait analysis brought very good results for each outcome measure. High reliability (Cronbach's alpha: 0.91-0.99) and very good reproducibility (Intraclass correlation coefficients: 0.83-0.98) were revealed. High to moderate correlation was found between MHQ and DASH, grip strength and VAS.Conclusions: The Italian version of MHQ has demonstrated to be reliable and valid

Bioelectrical Impedance Analysis (BIA) to measure alterations on hydroelectrolytic equilibrium of peripheral cells in COVID-19 patients with nonthyroidal illness syndrome (NTIS)

In COVID-19 patients the occurrence of low T3 serum values is associated with disease severity and death, but hormonal substitutive replacement is still debated. Clinical trials reported so far failed to demonstrate clear beneficial effects of T3, T4 or both treatments. With the aim to analyze the peripheral effects of the acute deficiency of T3 in blood, we analyzed body fluid composition in 74 COVID-19 patients, admitted to our University Hospital during the last pandemic wave. COVID-19 patients were sub divided into those that presented low FT3 serum levels, i.e., 1.7 pg/ml (n. 42), 13 of which showed very low FT3 serum values, i.e., 1.1 pg/ml, and those that showed normal FT3 serum levels, i.e., > 1.7 pg/ml (n. 32). Body fluid composition was analyzed by Bioelectrical Impedance Analysis (BIA). We demonstrated that COVID-19 patients with low FT3 serum values exhibited increased values of the Total Body Water/Free Fat Mass (TBW/FFM) ratio. Patients with the lowest FT3 serum values had also the highest level of TBW/FFM ratio, an indicator of the fraction of FFM as water and one of the best-known body-composition constants in mammals. We found an inverse correlation between FT3 serum values and this constant. Reduced FT3 serum values in COVID-19 patients were correlated with the increase in the total body water (TBW), the extracellular water (ECW) and the sodium/potassium exchangeable ratio (Nae:Ke), and with the reduction of the intracellular water (ICW). Since the Na+/K+ pump is a well-known T3 target, we measured the mRNA expression levels of the two genes coding for the two major isoforms of this pump. We demonstrated that COVID-19 patients with NTIS had lower levels of mRNA of both genes in the peripheral blood mononuclear cells (PBMC)s obtained from our patients during the acute phase of the disease. In conclusion, we demonstrated that the acute T3 deficit in our COVID-19 patients has marked effects on the hydroelectrolytic equilibrium of their peripheral blood mononuclear cells. The Na+/K+ pump is a possible target of T3 action, involved in the pathogenesis of the anasarcatic condition observed in these patients. Measurement of BIA parameters is a useful method to analyze water and salt retention in COVID-19 patients hospitalized in ICU that develop NTIS and may represent a novel reliable outcome to evaluate the benefit of T3 treatment in future clinical interventional trials

Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy

Abstract Background Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial. Methods Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays. Results Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm–1, which is enriched in human induced pluripotent stem cells. Conclusions Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts