Cultivo de Acanthamoeba spp. en agua apirógena / Acanthamoeba spp. culture in pyrogen-free water / Cultura de Acanthamoeba spp. em água apirogênica

Resumen El objetivo de este estudio fue evaluar el cultivo de cepas de Acanthamoeba spp. en agua destilada estéril apirógena de uso farmacéutico. Se utilizaron dos cepas de genotipo T4 [un aislamiento de encefalitis granulomatosa amebiana (EGA) y una ambiental] y cepas correspondientes a los genotipos T5 y T15. Los quistes de cada una de las cepas se sembraron en placas de Petri con agar no nutritivo con diferentes soluciones (agua destilada estéril apirógena uso médico para preparaciones inyectables, agua destilada filtrada, medio Page) y combinados con suspensiones de Escherichia coli. Las placas se incubaron a 37 °C y se monitorearon diariamente durante 15 días para la detección de trofozoítos. El crecimiento se evaluó mediante examen microscópico directo. Cada cultivo contó con cuatro repeticiones para cada una de las cepas (n=96). En conclusión, se hallaron diferencias estadísticamente significativas en el crecimiento de las cepas por día. Las cepas T5 y T4 (encefalitis amebiana granulomatosa) desarrollaron mayor cantidad de trofozoítos en el primer día respecto de la cepa T15 (H=16,42; p=0,001). En el agua apirógena con E. coli se obtuvo un crecimiento igual a la solución de Page con E. coli (H=24,64; p=0,0001). No se hallaron diferencias estadísticamente significativas en la cantidad de trofozoítos obtenidos en agua apirógena con E. coli y solución de Page con E. coli en la cepa T4 (EGA) (U=4; p<0,05) pero sí en la cepa T4 ambiental (U=0; p<0,05).

Abstract The objective of this study was to evaluate the culture of strains of Acanthamoeba spp. in sterile apyrogenic distilled water for pharmaceutical use. Two T4 genotype strains [one isolate of granulomatous amebic encephalitis (GAE) and one environmental], a T5 and T15 genotype strains were used. The cysts of each of the strains were seeded in Petri dishes with non-nutritive agar with different solutions (pyrogenic sterile distilled water for medical use for injectable preparations, filtered distilled water, Page medium) and combined with Escherichia coli suspensions. Plates were incubated at 37 °C and monitored daily for 15 days for the detection of trophozoites. Growth was assessed by direct microscopic examination. Each medium culture counted four replicates for each of the strains (n=96). Concluding, statistically significant differences were found in the growth of the strains per day. Strains T5 and T4 (granulomatous amebic encephalitis) developed a greater number of trophozoites on the first day compared to strain T15 (H=16.42; p=0.001). In apyrogenic water with E. coli, a growth equal to Page's solution with E. coli was obtained (H=24.64; p=0.0001). No statistically significant differences were found in the amount of trophozoites obtained in apyrogenic water with E. coli and Page's solution with E. coli in strain T4 (GAE) (U=4; p<0.05), but significant differences were found in the environmental T4 strain (U=0; p<0.05).

Resumo O objetivo deste trabalho foi avaliar o cultivo de cepas Acanthamoeba spp. em água destilada apirogênica estéril para uso farmacêutico. Foram utilizadas duas cepas de genótipo T4 [um isolamento de encefalite granulomatosa amebiana (EGA) e uma ambiental], e cepas correspondentes aos genótipos T5 e T15. Os cistos de cada uma das cepas foram semeados em placas de Petri com ágar não nutritivo com diferentes soluções (água destilada estéril apirogênica para uso médico para preparações injetáveis, água destilada filtrada, meio Page) e combinados com suspensões de Escherichia coli. As placas foram incubadas a 37 °C e monitoradas diariamente durante 15 dias para detecção de trofozoítos. O crescimento foi avaliado através de exame microscópico direto. Cada cultura contou com quatro réplicas para cada uma das cepas (n=96). Em conclusão, diferenças estatisticamente significativas foram encontradas no crescimento das cepas por dia. As cepas T5 e T4 (encefalite amebiana granulomatosa) desenvolveram maior número de trofozoítos no primeiro dia em relação à cepa T15 (H=16,42; p=0,001). Em água apirogênica com E. coli, foi obtido crescimento igual ao da solução de Page com E. coli (H=24,64, p=0,0001). Não foram encontradas diferenças estatisticamente significativas na quantidade de trofozoítos obtidos em água apirogênica com E. coli e solução de Page com E. coli na cepa T4 (EGA) (U=4; p%<0,05), mas sim na cepa T4 ambiental (U=0; p<0,05).

La meningoencefalitis bacteriana que era en realidad una apoplejía hipofisaria

Introducción: La apoplejía hipofisaria es un síndrome que se produce como consecuencia de una lesión isquémica o hemorrágica en la glándula pituitaria dando lugar a un déficit de hormonas hipofisarias. Se manifiesta en forma de deterioro neurológico con cefalea en trueno como síntoma prínceps, siendo la irritación meníngea una manifestación infrecuente. Métodos: Presentamos el caso de una mujer de 53 años con antecedente de madroadenoma productor de prolactina que comienza con cefalea, náuseas y deterioro de nivel de consciencia. Se detecta un hipopituitarismo incompleto con nivel de cortisol normal. El líquido cefalorraquídeo (LCR) es consistente con una pleocitosis aséptica sin respuesta a terapias antibióticas. Asocia paresia oculomotora y una RM craneal revela sangrado en el adenoma hipofisario con compromiso de seno cavernoso. Resultados: la sospecha inicial es una meningoencefalitis bacteriana por la fiebre, estupor y LCR con pleocitosis, si bien no se identifica microorganismo y no hay respuesta a antibióticos. El LCR de la apoplejía muestra una pleocitosis aséptica por irritación meníngea del espacio subaracnoideo por el sangrado y la necrosis de la glándula. El hipopituitarismo puede ser parcial o completo, siendo más frecuente el déficit selectivo. Especial atención merece el déficit de ACTH por la morbimortalidad que conlleva el fallo adrenal. La oftalmoparesia traduce implicación de seno cavernoso por incremento en la presión selar. Conclusiones: Destacamos la importancia de tener una sospecha diagnóstica de apoplejía ante un cuadro neurológico agudo para dirigir las investigaciones pertinentes con determinación hormonal y así iniciar una terapia sustitutiva temprana y una actitud neuroquirúrgica en caso de ser necesaria; precisando un manejo multidisciplinar.

Introduction: Pituitary apoplexy is a syndrome that occurs as a result of an ischemic or hemorrhagic lesion in the pituitary gland, leading to a deficiency of pituitary hormones. It manifests in the form of neurological deterioration with thunderclap headache as the main symptom, with meningeal irritation being an infrequent manifestation. Methods: We present the case of a 53-year-old woman with a history of prolactin-producing madroadenoma that began with headache, nausea and impaired level of consciousness. Incomplete hypopituitarism with normal cortisol level is detected. Cerebrospinal fluid (CSF) is consistent with an aseptic pleocytosis unresponsive to antibiotic therapy. It is associated with oculomotor paresis and a cranial MRI reveals bleeding in the pituitary adenoma with involvement of the cavernous sinus. Results: the initial suspicion is bacterial meningoencephalitis due to fever, stupor and CSF with pleocytosis, although no microorganism is identified and there is no response to antibiotics. CSF from stroke shows aseptic pleocytosis due to meningeal irritation of the subarachnoid space from bleeding and necrosis of the gland. Hypopituitarism can be partial or complete, selective deficiency being more frequent. ACTH deficiency deserves special attention due to the morbidity and mortality that adrenal failure entails. Ophthalmoparesis translates involvement of the cavernous sinus due to an increase in sellar pressure. Conclusions: We emphasize the importance of having a suspected diagnosis of apoplexy in case of an acute neurological condition, to direct the pertinent investigations with hormonal determination and thus initiate early replacement therapy and a neurosurgical approach if necessary; requiring a multidisciplinary management.

Molecular pathogenesis of acetaminophen-induced liver injury and its treatment options / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Acetaminophen, also known as N-acetyl-p-aminophenol (APAP), is commonly used as an antipyretic and analgesic agent. APAP overdose can induce hepatic toxicity, known as acetaminophen-induced liver injury (AILI). However, therapeutic doses of APAP can also induce AILI in patients with excessive alcohol intake or who are fasting. Hence, there is a need to understand the potential pathological mechanisms underlying AILI. In this review, we summarize three main mechanisms involved in the pathogenesis of AILI: hepatocyte necrosis, sterile inflammation, and hepatocyte regeneration. The relevant factors are elucidated and discussed. For instance, N-acetyl-p-benzoquinone imine (NAPQI) protein adducts trigger mitochondrial oxidative/nitrosative stress during hepatocyte necrosis, danger-associated molecular patterns (DAMPs) are released to elicit sterile inflammation, and certain growth factors contribute to liver regeneration. Finally, we describe the current potential treatment options for AILI patients and promising novel strategies available to researchers and pharmacists. This review provides a clearer understanding of AILI-related mechanisms to guide drug screening and selection for the clinical treatment of AILI patients in the future.

Análisis de riesgo de un microorganismo objetable en un suplemento dietario para la liberación de lotes

RESUMEN La contaminación con microorganismos objetables en los productos farmacéuticos no estériles puede tener el potencial de disminuir o inactivar la actividad farmacológica y terapéutica del principio activo y por ende representar un peligro de gran riesgo para la salud de la persona que está en tratamiento con fármacos; adicionalmente la presencia de estos microorganismos dependiendo de su capacidad para producir enfermedad en huéspedes susceptibles pueden ocasionar infecciones no esperadas o enfermedades que afecten al paciente o consumidor de estos medicamentos no estériles. El objetivo del trabajo fue desarrollar un análisis de riesgo microbiológico de microorganismos objetables en un suplemento dietario de una industria farmacéutica como herramienta en la gestión de seguridad. La metodología fue observacional-descriptivo de corte transverso. En la industria farmacéutica (en el suplemento dietario) se desarrolló un análisis de riesgo estudiando todos los parámetros, teniendo como resultado, que las cápsulas blandas con aceite de Krill, no presentan microorganismo objetable del tipo patógeno, como producto de bajo riesgo, por lo que es aprobado para la liberación del lote siguiendo estos parámetros. En Paraguay no existen normativas acerca del análisis de microorganismos objetables, en comparación con otros países como Argentina, y desde hace tiempo en EE. UU y países europeos (por reportes de la FDA) por lo cual, este trabajo es relevante y de vital importancia para establecer legislaciones nacionales con miras a la fabricación de medicamentos seguros, confiables y eficaces.

ABSTRACT Contamination with objectionable microorganisms in non sterile pharmaceutical products may have the potential to decrease or inactivate the pharmacological and therapeutic activity of the active principle and therefore represent a danger of great risk to the health of the person who is being treated with drugs, additionally, the presence of these microorganisms, depending on their ability to produce disease in susceptible hosts, can cause unexpected infections or diseases that affect the patient or consumer of these non sterile drugs. The objective of the work was to develop a microbiological risk analysis of objectionable microorganisms in a dietary supplement of a pharmaceutical industry as a tool in safety management. This was an observational-descriptive cross-sectional study. In the pharmaceutical industry (in the dietary supplement) a risk analysis was developed studying all the parameters, having as a result that the soft capsules with Krill oil do not present objectionable microorganisms of the pathogenic type, as a low risk product, therefore it is approved for batch release following these parameters. In Paraguay there are no regulations about the analysis of objectionable microorganisms, in comparison with other countries such as Argentina, and for a long time in the US and European countries (due to FDA reports). Therefore, this work is relevant and very vital important to establish national legislation with a view of the manufacture of safe, reliable and effective drugs.

Microbial lipids production from wastes by Metschnikowia pulcherrima: a review / 生物工程学报

Biodiesel is an alternative fuel to addressing the energy shortage problem. Microbial lipids have attracted widespread attention as one of the potential feed-stocks for cost-effective and efficient biodiesel production. However, the large-scale production of microbial lipids is hampered by the complexity and the high cost of aseptic culturing approach. Metschnikowia pulcherrima is an oleaginous yeast with strong environmental adaptability. It is capable of utilizing a wide spectrum of substrates, and can be cultured under non-sterile conditions. Therefore, this yeast has great potential to replace the traditional oleaginous microorganisms, particularly in the area of recycling wastewater and solid waste for the production of biodiesel. Based on the analysis of lipid production and application conditions of M. pulcherrima, this review summarized the unique advantages of M. pulcherrima and the key factors affecting lipids production. We further discussed the feasibility of cultivating M. pulcherrima on various organic wastes under non-sterile conditions for lipids production. Moreover, we analyzed the challenges associated with M. pulcherrima's in the yield and mechanism for lipids production, and proposed perspectives for how to achieve efficient biodiesel production using this yeast.

Application of coding navigation index and specialized placement scheme for surgical instruments / 中华医院管理杂志

As the operation volume in hospitals is increasing year by year, surgical instruments are widely used, which pose a great challenge to the daily management of surgical instruments. The authors investigated the existing problems which occurred in the management of surgical instruments, and came out with instruments coding navigation index and specialized placement scheme for the operating room as solutions. The specific measures included dividing sterile items into specialized categories, setting cabinets respectively for specialized and general subjects, building equipment coding and identification, establishing surgical instruments navigation index and carrying out training program, to serve as reference for efficient and fine management of surgical instruments.

Management of corneal perforations: An update

Corneal perforation is a potentially devastating complication that can result from numerous conditions that precipitate corneal melting. It is associated with significant morbidity and prompt intervention is necessary to prevent further complications. Causes include microbial keratitis, ocular surface disease, and autoimmune disorders and trauma. Various management options have been described in the literature to facilitate visual rehabilitation. This rview discusses the treatment options that range from temporising measures such as corneal gluing through to corneal transplantation, with decision making guided by the location, size, and underlying aetiology of the perforation.

Comparison of 16S Ribosomal RNA Targeted Sequencing and Culture for Bacterial Identification in Normally Sterile Body Fluid Samples: Report of a 10-Year Clinical Laboratory Review

As 16S ribosomal RNA (rRNA)-targeted sequencing can detect DNA from non-viable bacteria, it can be used to identify pathogens from clinical samples even in patients pretreated with antibiotics. We compared the results of 16S rRNA-targeted sequencing and culture for identifying bacterial species in normally sterile body fluid (NSBF): cerebrospinal, pericardial, peritoneal and pleural fluids. Over a 10-year period, a total of 312 NSBF samples were evaluated simultaneously using 16S rRNA-targeted sequencing and culture. Results were concordant in 287/312 (92.0%) samples, including 277 (88.8%) negative and 10 (3.2%) positive samples. Of the 16 sequencing-positive, culture-negative samples, eight showed clinically relevant isolates that included Fusobacterium nucleatum subsp. nucleatum, Streptococcus pneumoniae, and Staphylococcus spp. All these samples were obtained from the patients pretreated with antibiotics. The diagnostic yield of 16S rRNA-targeted sequencing combined with culture was 11.2%, while that of culture alone was 6.1%. 16S rRNA-targeted sequencing in conjunction with culture could be useful for identifying bacteria in NSBF samples, especially when patients have been pretreated with antibiotics and when anaerobic infection is suspected.

Prevalence and Clinical Characteristics of Asymptomatic Pyuria in Chronic Kidney Disease

BACKGROUND: Pyuria seems to be common in chronic kidney disease (CKD), irrespective of urinary tract infection (UTI). It has been hypothesized that sterile pyuria occurs in CKD because of chronic renal parenchymal inflammation. However, there are limited data on whether CKD increases the rate of pyuria or how pyuria in CKD should be interpreted. We investigated the prevalence and characteristics of asymptomatic pyuria (ASP) in CKD via urinary white blood cell (WBC) analysis.METHODS: Urine examination was performed for all stable hemodialysis (HD) and non-dialysis CKD patients of the outpatient clinic (total N=298). Patients with infection symptoms or recent history of antibiotic use were excluded. Urine culture and WBC analysis were performed when urinalysis revealed pyuria.RESULTS: The prevalence of ASP was 30.5% (24.1% in non-dialysis CKD and 51.4% in HD patients). Over 70% of the pyuria cases were sterile. The majority of urinary WBCs were neutrophils, even in sterile pyuria. However, the percentage of neutrophils was significantly lower in sterile pyuria. In multivariate logistic regression analysis, the degree of pyuria, percentage of neutrophils, and presence of urinary nitrites remained independently associated with sterile pyuria.CONCLUSIONS: The prevalence of ASP was higher in CKD patients and increased according to CKD stage. Most ASP in CKD was sterile. Ascertaining the number and distribution of urinary WBCs may be helpful for interpreting ASP in CKD.

Role and related mechanism of Mst-1 on regulating hypoxic reoxygenation induced autophagy and apoptosis in cardiomyocytes of mouse / 中华心血管病杂志

Objective: To explore the role and related mechanism of mammalian sterile 20-like kinase 1(Mst-1)in regulating hypoxia reoxygenation (HR) induced myocardial cell autophagy and apoptosis. Methods: Enzyme digestion method combined with differential adherent method was used to culture neonatal mouse myocardial cells. HR model was established by hypoxia for 24 hours and reoxygenation for 6 hours. The experimental groups including control group (normal cultured cardiomyocytes), Mst-1 empty virus group (cardiomyocytes transfected with recombinant lentiviral empty vector for 48 hours), Mst-1 knockdown group (recombinant lentivirus carrying Mst-1small interfering RNA (siRNA) was transfected into cardiomyocytes for 48 hours), Mst-1 overexpression group (cardiomyocytes were transfected with recombinant lentivirus carrying Mst-1 gene for 48 hours), HR group (cardiomyocytes exposed to HR), Mst-1 knockdown+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1siRNA) and Mst-1 overexpression+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1 gene). Real-time fluorescence quantitative RCR (qPCR) and Western blot were used to detect the relative expression of Mst-1 mRNA and protein in the cells, immunofluorescence staining was used to detect cardiomyocyte troponin T (cTnT), and autophagosomes and autophagy enzyme changes. TUNEL method was used to detect myocardial cell apoptosis, Western blot was adopted to detect autophagy-related protein microtubule-related protein 1 light chain 3 (LC3) Ⅱ/LC3 Ⅰ, P62 and apoptosis-related protein cleaved-caspase 9, pro-caspase 9, cleaved-caspase-3, pro-caspase-3, and myeloid leukemia 1 (MCL-1) expression. MCL-1 inhibitor A1210477 was used to validate the signaling pathway of Mst-1 on regulating cardiomyocyte apoptosis and autophagy. Results: Immunofluorescence detection revealed that the cultured cells expressed cardiomyocyte-specific marker cTnT. The expression of Mst-1 in cardiomyocytes increased in HR model. Lentiviral transfection could effectively inhibit or overexpress Mst-1 in treated cells. The levels of autophagosomes and autophagolysosomes in cardiomyocytes undergoing HR and in Mst-1 overexpression+HR group were lower than those of control group, while autophagosomes and autophagolysosomes in cardiomyocytes of Mst-1 knockdown+HR group was significantly higher than in the HR group (all P<0.05). The TUNEL results showed that the proportion of TUNEL positive cells was significantly increased in the HR group and Mst-1 overexpression+HR group than in the control group, while the proportion of TUNEL positive cells was significantly decreased in the Mst-1 knockdown group+HR group as compared to the HR group (all P<0.05). Western blot results showed that the LC3 Ⅱ/LC3 Ⅰ levels were significantly lower, while the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly higher in the HR group and Mst-1 overexpression+HR group than in control group (all P<0.05). The LC3 Ⅱ/LC3 Ⅰ value was significantly higher, and the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly lower in the Mst-1 knockdown+HR group than in the HR group (P both<0.05). The expression level of P-MCL-1 protein was significantly lower in cardiomyocytes of HR and Mst-1 overexpression+HR group than in control group, and the expression level of P-MCL-1 protein was higher in Mst-1 knockdown+HR group than in HR group (P both<0.05). The recovery experiment showed that inhibiting MCL-1 in cells can block the regulatory effect of Mst-1 siRNA on cell autophagy and apoptosis. Conclusion: Inhibiting Mst-1 expression in cardiomyocytes can promote the autophagy of cardiomyocytes induced by hypoxic reoxygenation and reduce the apoptosis of cardiomyocytes via activating McL-1.

A study of sterile pyuria in dengue fever

Background: Dengue fever is an important tropical disease which is endemic in around 110 countries. It infects 50-100 million people worldwide per year. In India case fatality rate is 1-5% for severe Dengue. Between 2015-2017, 790 deaths have been recorded according to NVBDCP data. Global burden of Dengue has increased at least fourfold over last three decades and now 2.5 billion people at risk of disease. This study aims at determining sterile pyuria as a manifestation in patients presenting with Dengue fever, as patients may present with similar symptoms as that of urinary tract infection, thereby preventing unnecessary use of antibiotics.Methods: It is a Cross sectional observational study conducted on 100 consecutive patients with serologically proven Dengue fever. Patients satisfying inclusion and exclusion criteria underwent relevant investigations and in patients with urine routine showing pyuria, urine culture and sensitivity was done to rule out urinary tract infection and look for sterile pyuria.Results: Among 100 patients of dengue studied, age distribution being 18years to 70years, mean age was 33.27±13.2 years of them 78 were male and 22 were female. 41% patients showed pyuria in urine. 25 % patients were culture positive most common being E. coli and 16% patients had sterile pyuria.Conclusions: Sterile pyuria is not a well-recognized entity in Dengue fever and is often missed. This study shows that sterile pyuria is quite common manifestation in dengue fever which resembles urinary tract infection and therefore does not require any empirical antibiotic treatment.

Analysis on biological functions of restriction factor SAMHD1 of primates / 吉林大学学报(医学版)

Objective: To discuss the antiviral effect, the inhibitory effect on LINE-1 retrotransposon activity and the redection of interferon production signal pathway of restriction factor SAMHD1 of the primates, and to provide the basis for further study of the SAMHD1 of the primates. Methods: The U937 cells stably expressing the SAMHD1 of primates were established; the U937-control cells established with pLVX -puro were used as negative control group, and the U937-SAMHD1 cells stably expressing the SAMHD1 protein of the different primates were used as experimental groups; the cells were treated with PMA to induce cell differentiation. The virus infection rates of HIV-1 in the cells in various groups were determined by flow cytometry. The HEK293T cells transfected with the expression plasmid of SAMDH1 were used as control group, and the cells co-transfected with the SAMHD1 and HIV-2/SIV Vpx expression plasmids were used as experimental groups. The cells were obtained 48 h after transfection, and the expression levels of SAMHD1 protein were determined by Western blotting method. The intracellular location of SAMHD1 protein was determined by immunofluorescence. The HEK293T cells transfected with LINE-1-GFP report plasmid were used as control group, and the cells co-transfected with LINE-1-GFP and SAMHD1 expression plasmids were used as experimental groups. The rates of GFP positive cells (activity of SAMHD1 to LINE-1 transposon) were determined by flow cytometry. The HEK293T cells transfected with IFN- Luc report plasmid were used as control group, and the cells co-transfected with IFN-Luc and pSAMHDl expression plasmids were used as experimental groups. The expression levels of luciferase in HEK293T cells were determined by chemiluminescence instrument. Results: Compared with negative control group, the virus infection rates of HIV-1 in experimental groups with stable expression of SAMHD1 in the primates were significantly decreased (P<0. 01). Compared with control group, the expression levels of SAMHD1 protein of the primates in experimental groups were decreased (P<0. 05 or P<0. 01). The immunofluorescence results showed that the SAMHD1 protein of the primates was localized in the nucleus. Compared with control group, the rates of GFP positive cells (activity of SAMHD1 to LINE-1 transposon) in experimental groups were significantly decreased (P< 0.05 or P<0.01). Compared with control group, the expression levels of luciferase in the HEK293T cells in experimental groups were significantly decreased (P<0. 05). Conclusion: The SAMHD1 protein of the different primates can resist the HIV-1 infection, inhibit the LINE-1 retrotransposon and antagonize the IFN production by natural immune system.

Abordagem microbiológica e estatística aplicada ao monitoramento ambiental em áreas produtivas farmacêuticas / Microbiological and statistical approach applied to environmental monitoring of pharmaceutical manufacturing areas

O controle microbiológico durante a produção de preparações farmacêuticas é de grande importância para garantir a qualidade do produto final, quanto às propriedades terapêuticas e de segurança ao paciente. O monitoramento ambiental é uma valiosa ferramenta como forma de mensurar a efetividade das medidas que integram a estratégia de controle de contaminação microbiana. Neste contexto, pouco destaque tem sido dado à manufatura de produtos farmacêuticos não-estéreis, por representarem as classes cujos riscos de contaminação microbiana são menores, quando comparados aos produtos parenterais. Dessa maneira, este estudo teve como objetivo caracterizar os isolados microbianos de amostras de ar ativo e passivo e de superfícies de áreas produtivas não-estéreis. Ainda, visou-se avaliar estatisticamente os dados de monitoramento ambiental, como base para o desenvolvimento de uma abordagem para determinação de limites de alerta e ação. Os resultados obtidos revelaram que a maioria dos microrganismos encontrados são de origem humana, seguidos por bactérias e fungos provenientes do solo. As diferenças sazonais foram observadas, principalmente, para a ocorrência de fungos, mais prevalentes no período seco. Foi desenvolvida uma abordagem estatística baseada em (1) determinação de subgrupos racionais, (2) avaliação da distribuição estatística e (3) determinação de limites, utilizando, como critério, o índice de capacidade do processo (Cpk). Um melhor entendimento do perfil microbiano das áreas produtivas e a determinação de limites de acordo com a distribuição real dos dados levará à destinação dos recursos necessários a ações que visem a qualidade do produto e a segurança do paciente

The microbiological control during the production of pharmaceutical preparations is of great importance for quality assurance of the final product regarding to therapeutic properties and patient safety. Environmental monitoring is a valuable tool to measure the effectiveness of the actions that integrate the microbial contamination control strategy. In this context, little attention has been given to the manufacture of non-sterile pharmaceutical products, because they represent classes whose microbial contamination risks are lower when compared to parenteral products. Considering this scenario, this study aimed to characterize microbial isolates from surfaces, active and passive air sampling of non-sterile manufacturing areas. Furthermore, it was expected to statistically evaluate the environmental monitoring data, as a basis for the development of an approach for determining alert and action limits. The results showed that most of the microorganisms found are from human source, followed by bacteria and fungi typically found in the soil. The seasonal differences were mainly observed for fungi recovery, which were more prevalent in the dry period. A statistical approach was developed based on (1) the determination of rational subgroups, (2) evaluation of the statistical distribution and (3) limit determination, using the process capacity index (Cpk) as criteria. A better understanding of the typical manufacturing areas microbial profile and the determination of limits according to the actual data distribution will lead to the allocation of the necessary resources to actions focusing on product quality and patient safety

Avaliação da segurança da esterilização por peróxido de hidrogênio vaporizado nas práticas atuais do centro de material e esterilização / Safety Assessment of Vaporized Hydrogen Peroxide Sterilization in the Current Practices of Central Sterile Service Departments

Introdução: O recente avanço das técnicas cirúrgicas demandou a utilização de métodos de esterilização a baixa temperatura, tal como o peróxido de hidrogênio vaporizado (VH2O2), para possibilitar a esterilização de produtos para saúde (PPS) termossensíveis. Embora amplamente utilizado nos Centros de Material e Esterilização (CME), os problemas relacionados à esta tecnologia são muitos, tais como: baixa difusibilidade do VH2O2, ausência de normas construtivas e de validação, limitação das tecnologias para medir a concentração do agente esterilizante, validação e monitoramento dos ciclos, além de práticas inadequadas de carregamento e esterilização de PPS. Objetivo: Avaliar, por meio de indicadores físicos (IF) e biológicos (IB), se as práticas atuais do CME, na escolha das cargas e do carregamento, interferem na segurança dos esterilizadores de VH2O2: Sterrad® 100S ciclo curto, Sterrad® 100NX ciclo padrão e Vpro® Max ciclo sem lúmen. Método: Pesquisa de campo, exploratória e descritiva, realizada em hospitais que utilizam os equipamentos supracitados na rotina. Os dados foram coletados após aprovação do Comitê de Ética e Pesquisa e autorização dos hospitais participantes. Foram avaliados três esterilizadores de cada modelo de CMEs distintos, sendo que a escolha dos ciclos para coleta dos dados deu-se pela maior criticidade e frequência de utilização nos locais de estudo. Os IB foram posicionados, unitariamente, em envelopes duplos de Tyvek® (considerados pacote-teste), com fechamento por selagem térmica. Em cada esterilizador, foi realizado um ensaio vazio em meio ciclo, com três pacotes-testes nas seguintes posições: superior/anterior; superior/centro e inferior/posterior. Sequencialmente, foram realizados ensaios com a mesma carga em triplicata, compostos pelos PPS que representam um desafio à rotina, também, em meio ciclo. Internamente, adicionaram-se IB, além de dois pacotes-testes posicionados na parte superior/anterior e inferior /posterior, sendo o primeiro colocado no ponto mais próximo da entrada do agente esterilizante, e o segundo colocado no ponto mais distante. Resultados: do total de 9 ensaios com carga para cada modelo de esterilizador, obtemos IB positivos em 22% dos ensaios do modelo 100S, 33% no modelo 100NX e 100% no modelo V-pro. Os IB positivos foram relacionados à sobreposição de pacotes, à presença de tapete imantado, às bandejas de acondicionamento sem instrução de uso do fabricante (IUF) para o método e, principalmente, à difusibilidade do VH2O2 prejudicada em pontos mais distantes da entrada do VH2O2 na câmara de esterilização. Outras hipóteses que podem ter contribuído para o resultado foram a falta de IUF dos PPSs para o método e o uso de massa adicional que consome o VH2O2. Não foi possível correlacionar os resultados dos IB positivos aos parâmetros físicos do esterilizador, bem como ao total de peso da carga. Conclusão: O presente estudo pode afirmar que as cargas e o carregamento interferem na segurança dos esterilizadores por VH2O2. As IUF validadas dos esterilizadores, dos PPSs, dos sistemas de barreira estéril e das bandejas de acondicionamento devem ser respeitadas, como também a qualificação dos equipamentos com carga que representem um desafio da rotina utilizando-se IB em meio ciclo. Este procedimento pode detectar a insuficiência de exposição dos PPSs ao agente esterilizante para atender o Nível de Segurança de Esterilidade (SAL - Sterility Assurance Level) de 10-6 . Atualmente, o monitoramento de rotina de todos os ciclos com IB é o procedimento mais seguro para este método de esterilização.

Introduction: The recent advances in surgical techniques have required the use of low- temperature sterilization methods, such as vaporized hydrogen peroxide (VH2O2), to enable the sterilization of thermo-sensitive health care products. Although widely applied in Central Sterile Service Departments (CSSD), problems related to this technology are many such as: low diffusivity, lack of constructive and validation standards, technology limitation to measure the concentration of the sterilizing agent, cycle validation and monitoring, as well as inadequate loading and sterilization practices for health care products. Objective: Evaluating, through physical (PI) and biological (BI) indicators, whether the current practices of Central Sterile Service Departments, when choosing the load and the loading processes, interfere with the safety of VH2O2 sterilizers: Sterrad® 100S short cycle, Sterrad® 100NX standard cycle and Vpro® Max lumenless cycle. Method: Field, exploratory and descriptive research study performed in hospitals that use the aforementioned equipment in the working routine. Data were collected after approval by the Research Ethics Committee and authorization of the participating hospitals. Three sterilizers from each model and from different CSSD were evaluated, and the choice of cycles for data collection followed the criticality and frequency of use in the study sites. The BIs were unitarily positioned in double-Tyvek® (test-package) envelopes, with heat-sealing closure. In each sterilizer, a half-cycle no-loading test was performed, with three testing-packages in the following positions: upper/anterior; upper/center and lower/posterior. Subsequently, triplicate assays were performed with the same load, containing health care products that represent a routine challenge, also, in half cycle. BIs were added internally, in addition to two testing-packages, positioned in the upper/anterior and inferior/posterior area, the first placed at the point closest to the entrance of the sterilizing agent in the chamber, and the second at the farthest point. Results: For total of 9 assays with load from each sterilizers model, positive BIs 22% on assays in the 100S model test, 33% in the 100NX model and 100% in the V-pro model. The positive BIs were related to package overlapping; presence of magnetic mat; packaging trays, without the recommendations of the equipment manufacturers; and impaired diffusion of the VH2O2 sterilizing agent in the sterilization chamber, especially at points distant from the agent entrance. Other hypotheses that may have contributed to the result were the lack of IFU of the health care products for the method, as well as the use of additional mass consuming VH2O2. It was not possible to correlate the positive BI results to the physical parameters of the sterilizer, as well as to the total weight of the load. Conclusion: The current research study can conclude that the loads and the loading interfere in the safety of the sterilizers by VH2O2. The validated instructions for use of sterilizers manufacturers, health care products, sterile barrier systems and packaging trays must be respected, as well as the qualification of loading equipment, representing a challenge to the routine using BI in half cycle. This procedure can detect the insufficient exposure of health care products to the sterilizing agent to meet the Sterility Assurance Level (SAL) of 10-6 . Currently, routine monitoring of all cycles with BIs is the safest procedure for this sterilization method.

Avaliação da limpeza e esterilização de cânulas de lipoaspiração após contaminação com gordura, Mycobacteroides abscessus subespécie bolletii e Geobacillus stearothermophilus: estudo experimental / Evaluation of liposuction cannulas cleaning and sterilization after contamination with fat, Mycobacteroides abscessus subspecies bolletii and Geobacillus stearothermophilus: an experimental study

Introdução: A lipoaspiração ou lipossucção é a intervenção cirúrgica destinada a remover depósitos superficiais e profundos de gordura subcutânea do tecido adiposo localizado. Em alguns casos, para obter o resultado estético desejado, é realizada a lipoenxertia. Neste processo, faz-se um transplante autólogo de tecido gorduroso para preencher, aumentar ou modelar as estruturas flácidas, depressões ou áreas com pouco tecido adiposo. As cânulas utilizadas para realizar a lipoaspiração apresentam um design desafiador para os processos de limpeza, favorecendo o acúmulo de resíduos de gordura em seu interior. Há registros de surtos infecciosos causados por microrganismos que sobreviveram ao processo de esterilização, relacionados à falha na limpeza dos instrumentais cirúrgicos, reforçando a premente necessidade de investigar se os resíduos de gordura no lúmen das cânulas são passíveis de remoção, garantindo assim, a eficácia da esterilização e a segurança em seu reuso. Objetivos: Fase I - avaliar a eficácia da remoção da gordura humana do lúmen das cânulas submetidas a seis diferentes Procedimentos Operacionais Padrão (POP) de limpeza, comparando-os com os grupos controle positivos e negativos. Fase II - Avaliar o alcance do nível de segurança de esterilidade de 10-6 , quando submetida à esterilização por vapor saturado sob pressão as cânulas de lipoaspiração intencionalmente contaminadas com 6 L(residual mínimo após limpeza) e 50 L (residual máximo após limpeza) de gordura ----------------|a humana, ao serem desafiados frente à cepa de Mycobacteroides abscessus subespécie massiliense (INCQS no 00594) e a cepa do esporo Geobacillus stearothermophilus (ATCC no 7.953). Método: a pesquisa caracterizou-se como pesquisa experimental laboratorial. A Fase I dos experimentos consistiu em submeter as cânulas de lipoaspiração de 3mm e 5mm de diâmetro com lúmen intencionalmente contaminado com gordura humana, a seis distintos POP de limpeza com variações na inclusão/exclusão/sequência dos passos básicos de limpeza, atualmente, adotados pela Enfermagem em Centros de Material e Esterilização (CME), quais sejam: 1. Flush inicial com água por meio de seringa de 10mL com detergente no lúmen das cânulas; 2. Flush automatizado a alta pressão e alta temperatura por meio do sistema de vapor fluente; 3. Imersão em solução de detergente enzimático com lipase e alternativamente no detergente alcalino; 4. Limpeza manual como o método que antecedeu a limpeza automatizada; 5. Limpeza automatizada em lavadora ultrassônica com retrofluxo intermitente com conectores para canulados. A gordura contaminante nos corpos de prova permaneceu por 120 minutos de contato, e após a drenagem do contaminante as cânulas ficaram expostas ao ar ambiente por 60 minutos. Após a aplicação dos seis distintos POP de limpeza, procedeu-se a extração e quantificação dos resíduos de gordura humana pela técnica de extração com solvente éter de petróleo a quente. A partir dos resultados obtidos nesta fase, realizou-se a Fase II caracterizada como microbiológica - utilizando a maior (50 L) e a menor (6,0 L ) média dos valores obtidos do resíduo de gordura para avaliar se esses quantitativos constituir-se-iam como fator protetor para os microrganismos no processo de esterilização por vapor saturado sob pressão alcançando o nível de segurança de esterilidade de 10-6 . Resultados: a Fase I da pesquisa demonstrou que mesmo utilizando todos os recursos atualmente, disponíveis no CME, não foi possível remover totalmente os resíduos de gordura inoculada nas cânulas de lipoaspiração restando valores residuais mínimos e máximos de gordura de 6,00 mg e 52 mg respectivamente. O POP que apresentou melhor desempenho na remoção de resíduo de gordura foi o método que empregou os seguintes recursos e sequência: 1. Flush inicial com água por meio de seringa de 10mL com detergente enzimático com lipase no lúmen das cânulas; 2. Imersão em solução de detergente enzimático com lipase; 3. Limpeza manual como o método que antecedeu a limpeza automatizada; 4. Limpeza automatizada em lavadora ultrassônica com retrofluxo intermitente com conectores para canulados; 5. Flush automatizado a alta pressão e alta temperatura por meio do sistema de vapor fluente. Os resultados microbiológicos da Fase II comprovaram a premissa de que a sujidade pode proteger microrganismos, constatando-se a sobrevivência, tanto da Mycobacteroides abscessus subespécie massiliense como do Geobacillus stearothermophilus, em ciclos de esterilização por vapor saturado sob pressão a 134o C, nos tempos de 1,30 minuto (meio ciclo) e 3 minutos (ciclo completo). Conclusões: As cânulas de lipoaspiração não são passíveis de limpeza pelos recursos atuais disponíveis pelos CME e houve recuperação dos microrganismos testados Mycobacteroides abscessus subespécie massiliense e Geobacillus stearothermophilus, demonstrando o risco de infecção relacionada ao reuso deste produto para saúde (PPS). Ressalta-se que dentre resíduos de matéria orgânica a serem removidos dos PPS, a gordura merece uma atenção especial porquanto há evidências de que os microrganismos em presença de óleos e gorduras necessitam de um tempo de exposição ao agente esterilizante até oito vezes maior que se estivesse na presença de água.

Introduction: Liposuction is the surgical intervention intended to remove superficial and deep deposits of subcutaneous fat from localized adipose tissue. In some cases, fat grafting is used to achieve the desired aesthetic result. In this process, an autologous fat tissue transplant is performed to fill, augment, or model flaccid structures, depressions, or areas with little adipose tissue. The cannulas used to perform liposuction have a challenging design for the cleaning processes, favoring the accumulation of fat residues inside. There are records of infectious outbreaks from microorganisms that survived the sterilization process, related to failure in cleaning surgical instruments, reinforcing the urgent need to investigate whether fat residues in the cannula lumen can be removed, thus ensuring the efficacy of sterilization and safety in its reuse. Objectives: Phase I to evaluate the efficacy of removing human fat from the cannula lumen undergoing six different cleaning standard operating procedures (SOPs), comparing them with the positive and negative control groups. Phase II - to evaluate the safety level of sterility reached of 10-6 , when liposuction cannula intentionally contaminated with 6 L (minimum residual after cleaning) and 50 L (maximum residual after cleaning) of human fat undergo sterilization with saturated steam, and are challenged with a strain of Mycobacteroides abscessus subspecies massiliense (INCQS no. 00594) and a strain of Geobacillus stearothermophilus spore (ATCC no. 7953). Method: the research was characterized as laboratorial and experimental. Phase I of the experiments consisted of submitting the 3mm- and 5mm- diameter liposuction cannula - with lumen intentionally contaminated with human fat, to six different cleaning SOPs with variations in the inclusion/exclusion/sequence of basic cleaning steps currently adopted by the nursing staff in sterile processing department, namely: 1. Initial flush with water using a 10mL syringe with detergent in the cannula lumen; 2. High-pressure, high-temperature automated flush through a flowing steam system; 3. Immersion in an enzymatic detergent solution with lipase, and alternatively in an alkaline detergent; 4. Manual cleaning as the method that preceded the automated cleaning; 5. Automated cleaning in ultrasonic washer with intermittent backflow with connectors for cannula. The contaminant fat in the specimens remained for 120 minutes of contact, and after draining the contaminant the cannulas were exposed to ambient air for 60 minutes. After the application of the six different cleaning SOPs, extraction and quantification of human fat residues were carried out using the hot petroleum ether extraction technique. Based on the results obtained in this phase, Phase II - characterized as microbiological - was performed using the largest (52L) and the lowest (6.0 L) average of values obtained from the fat residue to evaluate whether these quantitative values were a protective factor for microorganisms in the saturated steam sterilization process, reaching the sterile assurance level of 10-6 . Results: Phase I of the research demonstrated that even using all the currently available technologies in sterile processing department, it was not possible to completely remove fat residues inoculated in the liposuction cannula, with remaining minimum and maximum fat residual values of 6.0 mg and 52 mg, respectively. The SOP presenting better performance in the removal of fat residues was the method that used the following features and sequence: 1. Initial flush with water using a 10mL syringe with enzymatic detergent with lipase in the lumen of the cannula; 2. Immersion in an enzymatic detergent solution with lipase; 3. Manual cleaning using the method that preceded the automated cleaning; 4. Automated cleaning in ultrasonic washer with intermittent backflow with cannula connectors; 5. High-pressure and high-temperature automated flush using fluent steam system. The minimum and maximum residual fat values extracted were 6.0 mg and 52 mg. The microbiological results of Phase II have confirmed the premise that soil can protect microorganisms, with survival of both Mycobacteroides abscessus subspecies massiliense and Geobacillus stearothermophilus being observed after steam sterilization cycles under pressure at 134o C for 1,30 minute (half cycle) and 3 minutes (complete cycle). Conclusions: Liposuction cannula cannot be cleaned with the current resources available in Sterile Processing Departments, and the microorganisms tested, and Mycobacteroides abscessus subspecies massiliense and Geobacillus stearothermophilus, were recovered, demonstrating risk of infection related to the reuse of this health product. It should be emphasized that among the residues of organic matter to be removed from health products, fat deserves special attention because there is evidence that microorganisms in the presence of oils and fats need a time of exposure to the sterilizing agent up to eight times greater than if they were in the presence of water.

Courtship activity, copulation & insemination success in a mosquito vector fed a herbal aphrodisiac: Implications for sterile insect technology

Background & objectives: In sterile insect technology (SIT), mating competitiveness is a pre-condition for the reduction of target pest populations and a crucial parameter for judging efficacy. Still, current SIT trials are being hindered by decreased effectiveness due to reduced sexual performance of released males. Here, we explored the possible role of a herbal aphrodisiac in boosting the mating activity of Aedes aegypti. Methods: Males were fed one of two diets in this study: experimental extract of Eurycoma longifolia (MSAs) and sugar only (MSOs). Differences in life span, courtship latency, copulation activity and mating success were examined between the two groups. Results: No deaths occurred among MSA and MSO males. Life span of MSOs was similar to that of MSAs. The courtship latency of MSAs was shorter than that of MSOs (P<0.01). MSAs had greater copulation success than MSOs (P<0.001). In all female treatments, MSAs mated more than MSOs, but the differences in rate were significant only in the highest female density (P<0.05). In MSAs, mating success varied significantly with female density (P<0.01), with the 20-female group (P<0.01) having the lowest rate. Single MSA had better mating success at the two lowest female densities. In MSOs, there were no significant differences in mating success rate between the different female densities. Interpretation & conclusions: Our results suggested that the herbal aphrodisiac, E. longifolia, stimulated the sexual activity of Ae. aegypti and may be useful for improving the mating competitiveness of sterile males, thus improving SIT programmes.

Delayed formation of sterile abscess after zygomaticomaxillary complex fracture treatment with bioabsorbable plates

We present a patient who showed a sterile abscess after facial bone fixation with bioabsorbable plates and screws. He had zygomaticomaxillary complex and periorbital fracture due to falling down. The displaced bones were treated by open reduction and internal fixation successfully using bioabsorbable plate system. However, at postoperative 11 months, abrupt painless swelling was noted on the previous operation sites, left lateral eyebrow and lower eyelid. By surgical exploration, pus-like discharge and degraded materials were observed and debrided. The pathologic analysis revealed foreign body reaction with sterile abscess. This complication followed by bioabsorbable device implantation on maxillofacial bone surgery has been rarely reported in which we call attention to the maxillofacial plastic surgeons.

Comparative Analysis of In-use Period after First Opening or Reconstitution of European Union Imported Sterile Products and Domestic Sterile Products in Our Hospital / 中国药师

Objective: To provide reference and suggestions for improving the in-use period information of sterile products after first opening or reconstitution in China. Methods: The package inserts of sterile products in our hospital were investigated. The sterile products needed to be reconstituted before use and with multiple-dose package were selected. The in-use period information of European imported sterile products and domestic sterile products were comparatively analyzed. Results: The presentation rate of in-use period in the package inserts of domestic sterile products was 32. 25% , which was less than 55. 56% of the other imported sterile products, and significantly less than 93. 55% of European imported sterile products. Conclusion: The relevant content of in-use period of sterile products after first opening or reconstitution is supplemented relatively late in China's stability testing guidance for drug substances and products, moreover, the specific technical proposals are absent. It is suggested that the drug administrative department and its techni-cal supportive agencies should improve the relevant guidance and guide pharmaceutical research enterprises and manufacturing enterpri-ses to carry out in-use stability studies, furthermore, gradually improve the relevant in-use period information in package inserts.

Study on the Application of Flow Cytometry in Drug Sterility Test / 中国药师

Objective: To establish a method for the rapid detection of sterile preparations. Methods: Microbes in the sterile preparations were labeled by fluorescent dye SYTO9,and detected by flow cytometry. Meanwhile,the results from the routine examina-tion method recorded in Chinese Pharmacopoeia and those from the flow cytometry were compared. Results:The detection limit of in-jection and injection of sterile powder using flow cytometry was less than 10 cfu. It could be directly detected when the microbial con-tamination of the test samples was greater than 105cfu. When the amount of pollution was <10 cfu-104cfu, the detection time was shortened by 40%-70% when compared with that of the routine method. Conclusion:Flow cytometry used for the detection of microbi-ological contamination can shorten the time for positive result,which can be an effective supplement to the routine method.

Improvement and application of the opening method of mechanical ventilator humidification tank liquid / 中国实用护理杂志

Objective@#To improve the opening method of humidifier, enhance the efficiency and success rate of opening the humidifiers.@*Methods@#Use all the recycled humidifier from respirators in the wards from 1st of May to 31st of October 2015 as control group. Use the recycled humidifiers from 1st Feb to 31st July 2016 as the test group. Use metal tools to open humidifier bottles in the control group while use the newly developed and improved opening device to open the bottles in the test group to test and compare the damage rate.@*Results@#The testing group that used the practical opening device for humidifier has a decrease in damage rate from 5.97% (16/268) to 1.22% (3/245), and this difference before and after was statistically significant (χ2=8.082, P<0.01).@*Conclusions@#The use of the opening device for humidifier can enhance the intact rate, greatly improve working efficiency and save the cost.