Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves a reporter probe. In this study, we demonstrated that using 2-3 gRNAs in parallel can create a synergistic effect, resulting in a 4.5 × faster cleaving rate of the probe and increased sensitivity compared to using individual gRNAs. The synergy is due to the simultaneous activation of CRISPR-Cas12a and the improved performance of each gRNA. This approach was able to detect as few as 10 viral copies of the N-gene of SARS-CoV-2 RNA after a preamplification step using reverse transcription loop-mediated isothermal amplification. The method was able to accurately detect 100% of positive and negative clinical samples in ∼25 min using a fluorescence plate reader and ∼45 min with lateral flow strips.

Diagnostics of COVID-19 Based on CRISPR-Cas Coupled to Isothermal Amplification: A Comparative Analysis and Update.

The emergence of the COVID-19 pandemic prompted fast development of novel diagnostic methods of the etiologic virus SARS-CoV-2. Methods based on CRISPR-Cas systems have been particularly promising because they can achieve a similar sensitivity and specificity to the benchmark RT-qPCR, especially when coupled to an isothermal pre-amplification step. Furthermore, they have also solved inherent limitations of RT-qPCR that impede its decentralized use and deployment in the field, such as the need for expensive equipment, high cost per reaction, and delivery of results in hours, among others. In this review, we evaluate publicly available methods to detect SARS-CoV-2 that are based on CRISPR-Cas and isothermal amplification. We critically analyze the steps required to obtain a successful result from clinical samples and pinpoint key experimental conditions and parameters that could be optimized or modified to improve clinical and analytical outputs. The COVID outbreak has propelled intensive research in a short time, which is paving the way to develop effective and very promising CRISPR-Cas systems for the precise detection of SARS-CoV-2. This review could also serve as an introductory guide to new labs delving into this technology.

[Biliary stents for reconstruction in bile duct injuries. The role of bile acids]. / Férulas después de la reconstrucción de la vía biliar. El papel de los ácidos biliares.

UNLABELLED: Biliary stents, placed either surgically, endoscopically or radiologically are a frequent palliative and/or curative option for treatment of obstructive jaundice. Stones or sludge formation above or inside the stent are of major concern in that they cause dysfunction of the stent that needs prompt replacement. To evaluate the efficacy of ursodeoxycholic acid (UDCA) for long-term management of surgically placed biliary stents after iatrogenic injury, a prospective, controlled, randomized trial was conducted. METHOD: Patients with biliary tract reconstruction caused by iatrogenic injury that required a transanastomotic stent were randomized into a control group (N: 29) and into a UDCA (15 mg/kg day)-treated group (N: 30). Patients were followed on an external basis and a cholangiogram was obtained to verify stent patency. Liver function tests were also analyzed. RESULTS: The two groups were compared and no differences were observed. UDCA treated patients had significant elevation of transaminases and alkaline phosphatase. Two patients in the control group and four in the treated group developed lithiasis and/or sludge. CONCLUSION: No advantages were shown with administration of UDCA to maintain the biliary stents patent and to prevent neoformation of lithiasis and/or sludge. We conclude that no benefit is obtained with administration of UDCA to patients with biliary reconstruction and a transanastomotic stent.

To stent or not to stent bilioenteric anastomosis after iatrogenic injury: a dilemma not answered?

BACKGROUND: Bile duct injury is a complex and serious complication whose frequency has not diminished. A bilidigestive anastomosis (Roux-en-Y hepaticojejunostomy) is usually needed after complex injuries. Placement of an anastomotic stent is a matter of debate and to our knowledge there is no study that compares the results between stenting and not stenting the anastomosis. DESIGN: A retrospective review of medical records of patients operated on for biliary reconstruction after iatrogenic injury. SETTING: Tertiary care academic university hospital. PATIENTS: A comparative study was performed of patients operated on between 1995 and 1999, who were referred to our hospital for acute or elective reconstruction of the biliary tract following iatrogenic injury. All patients underwent Roux-en-Y hepaticojejunostomy. The patients were divided into 2 groups: those who underwent Roux-en-Y hepaticojejunostomy with a transanastomotic stent and those who underwent Roux-en-Y hepaticojejunostomy without a transanastomotic stent. MAIN OUTCOME MEASURES: Operative mortality, anastomotic dysfunction, biliary fistula, reoperations, postoperative complications, postoperative liver function tests. RESULTS: Sixty-three patients with high and complex biliary injuries (Bismuth type III, IV; Strasberg D, E). Thirty-seven cases had reconstruction with the placement of a transanastomotic stent and 26 did not have a stent placed. No operative mortality was observed. The postoperative outcomes of both groups were compared and no differences found. Good results were observed in more than 80% of the patients. Reoperations were more frequent in the nonstented group (15% vs. 5%) and complications were more frequent in the stented group (16% vs. 7%). CONCLUSIONS: Good results are obtained with a Roux-en-Y hepaticojejunostomy after complex injuries. The use of transanastomotic stents has to be selective according to the individual characteristics of each patient and the experience of each surgeon. We recommend their use when unhealthy (ie, ischemic, scarred) and small ducts (<4 mm) are found.

Diagnóstico serológico de leptospirosis / Serologic diagnosis of leptospirosis

Leptospirosis debe considerarse un diagnóstico diferencial de ictericia infecciosa en especial en países del trópico. En el estudio se realizó una comparación entre el campo obscuro y la serología. Para obtener nuestra población se evaluaron los pacientes que consultaron por cuadro de ictericia al servicio de Infectología del Hospital General de Enfermedades delIGSS. Se evaluaron un total de 102 pacientes de los cuales 90 eran masculinos y 12 femeninos.La edad en que más se presentaron casos fue de 15-45 años. Del total de 102 pacientes, en 48 se reportó positivo el campo obscuro y en 54 pacientes negativo; en 29 pacientes se reportó una serología positiva para leptospira (IgM-IgG) y 73 pacientes una serología negativa para leptospira. De los pacientes en el que la serología fue positiva en el campo obscuro, fue reportado negativo en 19 y de los 73 pacientes en los que la serología fue reportada negativa, el campo obscuro fue positivo en 38. Los datos anteriores nos dan un porcentaje de falsos positivos del 35 por ciento para el campo obscuro y un porcentaje del 47 por ciento de falsos negativos. Presentando el campo obscuro una sensibilidad del 65 por ciento y una especificidad del 53 por ciento en relación a la serología. Con los datos anteriores concluimos que el campo obscuro no es un método estándar para el diagnóstico de Leptospirosis