Implementation of an open-source robotic platform for SARS-CoV-2 testing by real-time RT-PCR.

The current global pandemic due to the SARS-CoV-2 has pushed the limits of global health systems across all aspects of clinical care, including laboratory diagnostics. Supply chain disruptions and rapidly-shifting markets have resulted in flash-scarcity of commercial laboratory reagents; this has motivated health care providers to search for alternative workflows to cope with the international increase in demand for SARS-CoV-2 testing. The aim of this study is to present a reproducible workflow for real time RT-PCR SARS-CoV-2 testing using OT-2 open-source liquid-handling robots (Opentrons, NY). We have developed a framework that includes a code template which is helpful for building different stand-alone robotic stations, capable of performing specific protocols. Such stations can be combined together to create a complex multi-stage workflow, from sample setup to real time RT-PCR. Using our open-source code, it is easy to create new stations or workflows from scratch, adapt existing templates to update the experimental protocols, or to fine-tune the code to fit specific needs. Using this framework, we developed the code for two different workflows and evaluated them using external quality assessment (EQA) samples from the European Molecular Genetics Quality Network (EMQN). The affordability of this platform makes automated SARS-CoV-2 PCR testing accessible for most laboratories and hospitals with qualified bioinformatics personnel. This platform also allows for flexibility, as it is not dependent on any specific commercial kit, and thus it can be quickly adapted to protocol changes, reagent, consumable shortages, or any other temporary material constraints.

rUTI Resolution After FMT for Clostridioides difficile Infection: A Case Report.

Clostridioides difficile infection (CDI) is the leading cause of nosocomial infectious diarrhea. Fecal microbiota transplantation (FMT) is a successful treatment for recurrent CDI (rCDI), and in some patients FMT has been associated with the resolution of recurrent urinary tract infections (rUTI). Recent evidence suggests that the origin of most bacterial infections in the urinary tract is the gut. Thus, the possibility of using FMT to displace pathogens commonly involved in rUTIs has major therapeutic implications. We report the case of a 93-year-old female patient with a rCDI and rUTI that underwent FMT and reported a complete clinical resolution of CDI; unexpectedly, no new symptomatic UTI episodes were diagnosed post-FMT. We characterized the gut microbiota of the stool donor and of the patient before and after the procedure. Our patient presented a dysbiosis with clear predominance of Enterobacteriaceae (74%) before FMT, which was significantly reduced to 0.07% after FMT. These findings were maintained for almost a year. We also observed an increase in microbial diversity indices compared with the pre-FMT sample reaching diversity values comparable to the donor stool samples. We reasoned that the disappearance of UTIs in our patient resulted from the reduction of Enterobacteriaceae in the gut microbiota. Our findings support previous evidence suggesting the potential of FMT for rUTI, particularly in cases due to multi-drug resistant pathogens where conventional antibiotic treatment is not an option.

Decontamination Strategies and Bloodstream Infections With Antibiotic-Resistant Microorganisms in Ventilated Patients: A Randomized Clinical Trial.

Importance: The effects of chlorhexidine (CHX) mouthwash, selective oropharyngeal decontamination (SOD), and selective digestive tract decontamination (SDD) on patient outcomes in ICUs with moderate to high levels of antibiotic resistance are unknown. Objective: To determine associations between CHX 2%, SOD, and SDD and the occurrence of ICU-acquired bloodstream infections with multidrug-resistant gram-negative bacteria (MDRGNB) and 28-day mortality in ICUs with moderate to high levels of antibiotic resistance. Design, Setting, and Participants: Randomized trial conducted from December 1, 2013, to May 31, 2017, in 13 European ICUs where at least 5% of bloodstream infections are caused by extended-spectrum ß-lactamase-producing Enterobacteriaceae. Patients with anticipated mechanical ventilation of more than 24 hours were eligible. The final date of follow-up was September 20, 2017. Interventions: Standard care was daily CHX 2% body washings and a hand hygiene improvement program. Following a baseline period from 6 to 14 months, each ICU was assigned in random order to 3 separate 6-month intervention periods with either CHX 2% mouthwash, SOD (mouthpaste with colistin, tobramycin, and nystatin), or SDD (the same mouthpaste and gastrointestinal suspension with the same antibiotics), all applied 4 times daily. Main Outcomes and Measures: The occurrence of ICU-acquired bloodstream infection with MDRGNB (primary outcome) and 28-day mortality (secondary outcome) during each intervention period compared with the baseline period. Results: A total of 8665 patients (median age, 64.1 years; 5561 men [64.2%]) were included in the study (2251, 2108, 2224, and 2082 in the baseline, CHX, SOD, and SDD periods, respectively). ICU-acquired bloodstream infection with MDRGNB occurred among 144 patients (154 episodes) in 2.1%, 1.8%, 1.5%, and 1.2% of included patients during the baseline, CHX, SOD, and SDD periods, respectively. Absolute risk reductions were 0.3% (95% CI, -0.6% to 1.1%), 0.6% (95% CI, -0.2% to 1.4%), and 0.8% (95% CI, 0.1% to 1.6%) for CHX, SOD, and SDD, respectively, compared with baseline. Adjusted hazard ratios were 1.13 (95% CI, 0.68-1.88), 0.89 (95% CI, 0.55-1.45), and 0.70 (95% CI, 0.43-1.14) during the CHX, SOD, and SDD periods, respectively, vs baseline. Crude mortality risks on day 28 were 31.9%, 32.9%, 32.4%, and 34.1% during the baseline, CHX, SOD, and SDD periods, respectively. Adjusted odds ratios for 28-day mortality were 1.07 (95% CI, 0.86-1.32), 1.05 (95% CI, 0.85-1.29), and 1.03 (95% CI, 0.80-1.32) for CHX, SOD, and SDD, respectively, vs baseline. Conclusions and Relevance: Among patients receiving mechanical ventilation in ICUs with moderate to high antibiotic resistance prevalence, use of CHX mouthwash, SOD, or SDD was not associated with reductions in ICU-acquired bloodstream infections caused by MDRGNB compared with standard care. Trial Registration: ClinicalTrials.gov Identifier: NCT02208154.

Risk factors for antimicrobial-resistant Neisseria gonorrhoeaeand characteristics of patients infected with gonorrhea / Factores de riesgo para infección por Neisseria gonorrhoeae resistente a antimicrobianos y características de los pacientes con infección gonocócica

Background: There are very few data available regarding risk factors associated with antibiotic resistant-Neisseria gonorrhoeae. Methods: A study was conducted on 110 samples from 101 patients with gonococcal infection, in order to describe their characteristics and compare them with the antimicrobial susceptibility profile of their samples. Results: An association was observed between resistant infections and heterosexual men, older age, concurrent sexually transmitted infection, and unsafe sexual behaviors. Conclusion: There is a need for improved data on the risk factors associated with antibiotic resistant gonococcal infection in order to identify risk groups, and to propose public health strategies to control this infection (AU)

Introducción: Disponemos de poca información sobre los factores de riesgo asociados a la infección por gonococo resistente a antibióticos. Métodos: Estudiamos 110 muestras de 101 pacientes, describimos sus característica y las comparamos según el perfil de susceptibilidad antimicrobiana de sus muestras. Resultados: observamos una asociación significativa entre las infecciones por cepas resistentes y varones heterosexuales, edad avanzada, infección de transmisión sexual concurrente y comportamientos sexuales de riesgo. Conclusiones: es necesario ampliar el estudio sobre los factores de riesgo asociados a infecciones por gonococo resistente con el objetivo de implementar medidas estratégicas para controlar la infección (AU)

Risk factors for antimicrobial-resistant Neisseria gonorrhoeae and characteristics of patients infected with gonorrhea.

BACKGROUND: There are very few data available regarding risk factors associated with antibiotic resistant-Neisseria gonorrhoeae. METHODS: A study was conducted on 110 samples from 101 patients with gonococcal infection, in order to describe their characteristics and compare them with the antimicrobial susceptibility profile of their samples. RESULTS: An association was observed between resistant infections and heterosexual men, older age, concurrent sexually transmitted infection, and unsafe sexual behaviors. CONCLUSION: There is a need for improved data on the risk factors associated with antibiotic resistant gonococcal infection in order to identify risk groups, and to propose public health strategies to control this infection.

Xpert(R) MTB/RIF: utilidad en el diagnóstico de la tuberculosis y de la resistencia a la rifampicina / Xpert(R) MTB/RIF: Usefulness for the diagnosis of tuberculosis and resistance to rifampicin

La aparición de la técnica Xpert(R) MTB/RIF supuso una revolución en el diagnóstico de la tuberculosis, especialmente en zonas con alta incidencia y pocos recursos. Permite la detección de Mycobacterium tuberculosis complex y, simultáneamente, de las mutaciones más comunes de resistencia a rifampicina en menos de 2 h. Su sensibilidad en muestras respiratorias es muy alta, pero disminuye en muestras extrapulmonares y en niños. Aunque más rápida y simple que los métodos convencionales, presenta ciertas limitaciones y aún son necesarias nuevas y mejores herramientas diagnósticas que contribuyan a reducir los casos y las muertes por tuberculosis. Esta revisión pretende compendiar la evidencia científica en torno al rendimiento diagnóstico de Xpert(R) MTB/RIF en diferentes tipos de muestras y poblaciones, así como analizar sus ventajas y limitaciones para el diagnóstico de tuberculosis (AU)

The advent of the Xpert(R) MTB/RIF technique was a revolution in the diagnosis of tuberculosis, especially in areas with high incidence and low resources. It allows the detection of Mycobacterium tuberculosis complex and simultaneously the most common resistance mutations to rifampicin in less than 2h. For respiratory samples the sensitivity is very high, but it decreases for extrapulmonary samples and children. Although it is faster and simpler than conventional methods, it presents some limitations and new and better techniques are needed to reduce the number of cases and deaths caused by tuberculosis. This review aims to assess the scientific evidence around the diagnostic performance of Xpert(R) MTB/RIF in different types of samples and populations, as well as analyse its strengths and limitations for TB diagnosis (AU)

Xpert® MTB/RIF: Usefulness for the diagnosis of tuberculosis and resistance to rifampicin. / Xpert® MTB/RIF: utilidad en el diagnóstico de la tuberculosis y de la resistencia a la rifampicina.

The advent of the Xpert® MTB/RIF technique was a revolution in the diagnosis of tuberculosis, especially in areas with high incidence and low resources. It allows the detection of Mycobacterium tuberculosis complex and simultaneously the most common resistance mutations to rifampicin in less than 2h. For respiratory samples the sensitivity is very high, but it decreases for extrapulmonary samples and children. Although it is faster and simpler than conventional methods, it presents some limitations and new and better techniques are needed to reduce the number of cases and deaths caused by tuberculosis. This review aims to assess the scientific evidence around the diagnostic performance of Xpert® MTB/RIF in different types of samples and populations, as well as analyse its strengths and limitations for TB diagnosis.

[Molecular diagnostic methods of respiratory infections. Has the scheme diagnosis changed?]. / Métodos moleculares de diagnóstico de infecciones respiratorias. ¿Ha cambiado el esquema diagnóstico?

Lower respiratory tract infections remain one of the most common causes of mortality worldwide, which is why early diagnosis is crucial. Traditionally the microbiological diagnosis of these infections has been based on conventional methods including culture on artificial media for isolation of bacteria and fungi and cell cultures for virus and antibody or antigen detection using antigen-antibody reactions. The main drawback of the above mentioned methods is the time needed for an etiological diagnosis of the infection. The techniques based on molecular biology have drawn much attention in recent decades as tools for rapid diagnosis of infections. Some techniques are very expensive, especially those that can detect various microorganisms in the same reaction, therefore the question that arises is whether the cost of such testing is justified by the information obtained and by the clinical impact that its implementation will determine. In this article we make a review of the various techniques of molecular biology applied to the diagnosis of pneumonia and focus primarily on analysing the impact they may have on the management of patients with acute respiratory tract infections.

Métodos moleculares de diagnóstico de infecciones respiratorias. ¿Ha cambiado el esquema diagnóstico? / Molecular diagnostic methods of respiratory infections. Has the scheme diagnosis changed?

Las infecciones respiratorias bajas siguen siendo una de las causas más frecuentes de mortalidad en todo el mundo, de ahí que el diagnóstico precoz sea fundamental. Tradicionalmente, el diagnóstico microbiológico de este tipo de infecciones se ha basado en métodos convencionales que incluyen cultivos en medios artificiales para aislamiento de bacterias y hongos y cultivos celulares para virus, así como en la detección antigénica o de anticuerpos mediante reacciones antígeno-anticuerpo. El principal inconveniente de las metodologías anteriormente citadas es el tiempo necesario para obtener un diagnóstico etiológico de la infección. Las técnicas basadas en la biología molecular han irrumpido con fuerza en las últimas décadas como herramientas de diagnóstico rápido de las infecciones. Algunas de estas técnicas -sobre todo aquellas que pueden detectar diversos microorganismos en la misma reacción- acostumbran a ser caras, por lo que la cuestión que se plantea es si el gasto de tales ensayos se ve justificado por la información obtenida y por el impacto clínico que su implementación determina. En este artículo se pretende hacer una revisión de las diversas técnicas de biología molecular aplicadas al diagnóstico de las infecciones respiratorias, centrándose fundamentalmente en la neumonía, y analizar el impacto que pueden tener en el manejo del paciente con infección respiratoria aguda

Lower respiratory tract infections remain one of the most common causes of mortality worldwide, which is why early diagnosis is crucial. Traditionally the microbiological diagnosis of these infections has been based on conventional methods including culture on artificial media for isolation of bacteria and fungi and cell cultures for virus and antibody or antigen detection using antigen-antibody reactions. The main drawback of the above mentioned methods is the time needed for an etiological diagnosis of the infection. The techniques based on molecular biology have drawn much attention in recent decades as tools for rapid diagnosis of infections. Some techniques are very expensive, especially those that can detect various microorganisms in the same reaction, therefore the question that arises is whether the cost of such testing is justified by the information obtained and by the clinical impact that its implementation will determine. In this article we make a review of the various techniques of molecular biology applied to the diagnosis of pneumonia and focus primarily on analysing the impact they may have on the management of patients with acute respiratory tract infections