Comparison of different NAT assays for the detection of microorganisms belonging to the class Mollicutes

Abstract Background Mollicutes detection can be cumbersome due to their slow growth in vitro. For this reason, the use of DNA based on generic molecular tests represents an alternative for rapid, sensitive and specific detection of these microorganism. For this reason, six previously described nucleic acid testing assays were compared to evaluate their ability to detect microorganisms belonging to the class Mollicutes. Methods A panel of 61 mollicutes, including representatives from the Mycoplasma, Acholeplasma, Mesoplasma, Spiroplasma and Ureaplasma genus, were selected to evaluate the sensitivity and specificity of these assays. A total of 21 non-mollicutes, including closely related non-mollicutes species, were used to evaluate specificity. Limits of detection were calculated to determine the analytical sensitivity of the assays. The two best performing assays were subsequently adapted into real-time PCR format, followed by melting curve analysis. Results Both assays performed satisfactorily, with a 100% specificity described for both assays. The detection limits were found to be between 10−4 and 10−5 dilutions, equivalent to 15 to 150 genome copies approximately. Based on our work, both van Kuppeveld and Botes real-time PCR assays were found to be the best performing tests in terms of sensitivity and specificity. Furthermore, Botes real-time PCR assay could detect phytoplasmas as well. Conclusions These assays can be very useful for the rapid, specific and sensitive screening cell line contaminants, clinical samples as well as detecting non-culturable, unknown species of mollicutes or mollicutes whose growth is slow or difficult

Inhibition of Bruton's TK regulates macrophage NF-kappa B and NLRP3 inflammasome activation in metabolic inflammation

Background and Purpose: There are no medications currently available to treat metabolic inflammation. Bruton's tyrosine kinase (BTK) is highly expressed in monocytes and macrophages and regulates NF-\u3baB and NLRP3 inflammasome activity; both propagate metabolic inflammation in diet-induced obesity. Experimental Approach: Using an in vivo model of chronic inflammation, high-fat diet (HFD) feeding, in male C57BL/6J mice and in vitro assays in primary murine and human macrophages, we investigated if ibrutinib, an FDA approved BTK inhibitor, may represent a novel anti-inflammatory medication to treat metabolic inflammation. Key Results: HFD-feeding was associated with increased BTK expression and activation, which was significantly correlated with monocyte/macrophage accumulation in the liver, adipose tissue, and kidney. Ibrutinib treatment to HFD-fed mice inhibited the activation of BTK and reduced monocyte/macrophage recruitment to the liver, adipose tissue, and kidney. Ibrutinib treatment to HFD-fed mice decreased the activation of NF-\u3baB and the NLRP3 inflammasome. As a result, ibrutinib treated mice fed HFD had improved glycaemic control through restored signalling by the IRS-1/Akt/GSK-3\u3b2 pathway, protecting mice against the development of hepatosteatosis and proteinuria. We show that BTK regulates NF-\u3baB and the NLRP3 inflammasome specifically in primary murine and human macrophages, the in vivo cellular target of ibrutinib. Conclusion and Implications: We provide \u201cproof of concept\u201d evidence that BTK is a novel therapeutic target for the treatment of diet-induced metabolic inflammation and ibrutinib may be a candidate for drug repurposing as an anti-inflammatory agent for the treatment of metabolic inflammation in T2D and microvascular disease

Outbreak da batteri saprofiti gram negativi in emodialisi: esperienza monocentrica

Ralstonia insidiosa (Ri), Ralstonia pickettii (Rp) e Burkholderia cepacia (B) sono batteri Gram negativi saprofiti di ambienti umidi responsabili di batteriemia in pazienti emodializzati portatori di catetere venoso centrale (CVCp). Nella dialisi di Ancona ad aprile 2021 7 su 39 pz con CVCp di età media di 67,8±11,4 aa hanno avuto batteriemia da Ri (n.4), Rp (n.2) e B (n.1). 4/7 sepsi, 3 batteriemie asintomatiche individuate con screening. Tutti i pazienti sono stati trattati con antibiotico mirato e lock-therapy del CVCp sostituito in tutti i pz. Analisi microbiologiche eseguite su farmaci iniettabili, disinfettanti, soluzioni dialitiche, impianto di trattamento dell’acqua, anello di distribuzione e su spezzoni di tubi di carico e scarico dei monitor sottoposti a sonicazione.. Tutti i pazienti hanno risposto alla terapia antibiotica. Le analisi microbiologiche dell’acqua di dialisi previste dalle linee guida SIN pre e post oubreak erano conformi. Ri e B sono stati isolati nel biofilm dei tubi di carico di 10 monitor e in un rubinetto dell’anello. I tubi di carico sono stati sostituiti e eseguiti cicli di disinfezione chimica integrata anello-monitors. Ad oggi nessun caso di batteriemia da Ri, Rp, B.La nostra esperienza conferma quanto riportato in letteratura circa la possibilità di batteriema da batteri saprofiti nei pazienti in emodialisi con CVCp, sottolinea l’importanza di identificare la sorgente dell’infezione e suggerisce una possibile soluzione

Inhibition of Bruton's tyrosine kinase regulates macrophage NF-κB and NLRP3 inflammasome activation in metabolic inflammation

Background and Purpose Currently there are limited medicines available for the treatment of metabolic inflammation in diseases such as obesity and type 2 diabetes (T2D). Although initially associated with B‐ells, Bruton's tyrosine kinase (BTK) is present in a wide variety of cells including monocytes and macrophages, and has been implicated in the regulation of the NF‐κB and NLRP3 inflammasome activity. Experimental Approach Using in vivo models of chronic inflammation [high‐fat‐diet (HFD) feeding] and in vitro assays in primary murine and human macrophages we investigated if ibrutinib, an FDA approved medicine that targets BTK, may represent a novel anti‐inflammatory drug for the use in treating metabolic inflammation. Key results HFD feeding was associated with increased BTK expression and activation, which was significantly correlated with monocyte/macrophage accumulation in the liver, adipose tissue and kidney. Treatment of mice fed HFD with ibrutinib inhibited the activation of BTK and reduced monocyte/macrophage recruitment to the liver, adipose tissue and kidney. Reduced inflammatory gene expression associated with decreased activation of NF‐κB and the NLRP3 inflammasome in vivo. As a result, ibrutinib treated mice fed HFD had improved glycaemic control through restored signalling by the IRS‐1/Akt/GSK‐3β pathway; protecting mice against the development of hepatosteatosis and proteinuria. We show that inhibition of BTK reduces activation of NF‐κB and the NLRP3 inflammasome specifically in primary murine and human macrophages; which are the primary target of ibrutinib in vivo in the setting of metabolic inflammation. Conclusions and Implications In the present study we provide ‘proof of concept' evidence that BTK is a novel therapeutic target for the treatment of diet ‐metabolic inflammation. Ibrutinib may be a candidate for drug repurposing as an anti‐inflammatory for the treatment of metabolic inflammation in T2D and microvascular disease