Rapid molecular diagnostics of COVID-19 by RT-LAMP in a centrifugal polystyrene-toner based microdevice with end-point visual detection.

Infection caused by the new coronavirus (SARS-CoV-2) has become a serious worldwide public health problem, and one of the most important strategies for its control is mass testing. Loop-mediated isothermal amplification (LAMP) has emerged as an important alternative to simplify the diagnostics of infectious diseases. In addition, an advantage of LAMP is that it allows for easy reading of the final result through visual detection. However, this step must be performed with caution to avoid contamination and false-positive results. LAMP performed on microfluidic platforms can minimize false-positive results, in addition to having potential for point-of-care applications. Here, we describe a polystyrene-toner (PS-T) centrifugal microfluidic device manually controlled by a fidget spinner for molecular diagnosis of COVID-19 by RT-LAMP, with integrated and automated colorimetric detection. The amplification was carried out in a microchamber with 5 µL capacity, and the reaction was thermally controlled with a thermoblock at 72 °C for 10 min. At the end of the incubation time, the detection of amplified RT-LAMP fragments was performed directly on the chip by automated visual detection. Our results demonstrate that it is possible to detect COVID-19 in reactions initiated with approximately 10-3 copies of SARS-CoV-2 RNA. Clinical samples were tested using our RT-LAMP protocol as well as by conventional RT-qPCR, demonstrating comparable performance to the CDC SARS-CoV-2 RT-qPCR assay. The methodology described in this study represents a simple, rapid, and accurate method for rapid molecular diagnostics of COVID-19 in a disposable microdevice, ideal for point-of-care testing (POCT) systems.

Loop-mediated isothermal amplification in disposable polyester-toner microdevices.

In recent years, isothermal DNA amplification methods have emerged as an alternative in molecular diagnostics due to its ease of operation. The purpose of using isothermal amplification is to simplify the diagnostics work by i) eliminating heating cycles, ii) reducing equipment costs, and iii) providing rapid and accurate results in laboratories with limited resources. Here we show a simple and fast method for E. coli detection in disposable polyester-toner (PeT) microdevice. The amplification by LAMP of the malB gene from E. coli was carried out in a microchamber with 5-µL capacity and the reaction was thermally controlled with a thermoblock at 66 °C for 60 min. The passivation of the surface of PeT channels with BSA improved the efficiency of the LAMP reaction. The detection of amplified LAMP fragments was performed directly on-chip by visual detection and validated with off-chip detection to compare results. Visualization of amplicons directly in the microchip yielded positive reactions as low as 10 double-stranded DNA copies. Separation by gel electrophoresis was able to detect amplicons in reactions that initiated only with one copy of double-stranded DNA. We demonstrate that LAMP in PeT microchip is an important tool for molecular diagnostics at the point-of-care.

[Meaning of the death/dying process for entering nursing students]. / Significado do processo morte/morrer para os acadêmicos ingressantes no curso de enfermagem.

This is a qualitative study, based on Heidegger's existential phenomenology, with the purpose of revealing the meaning of death/dying for freshmen students of the nursing program. The study was developed in a public university in the northwest of Paraná, Brazil between August and October 2010, and 33 entering students from a Nursing program were interviewed. The following existential themes emerged from the phenomenological analysis: "Understanding death as a difficult process to be understood"; "Understanding death as a natural process"; and "Perceiving death as a passage to another life". The present study allowed to understand that knowledge and death are entangled in the temporality and the historicity of every being, which requires a scientific, ethical and philosophical understanding of the death/dying phenomenon so that the scholar may prepare for the humanized care of the patient and his/her family.

Detecting lineage-defining mutations in SARS-CoV-2 using colorimetric RT-LAMP without probes or additional primers.

Despite the advance of vaccination worldwide, epidemic waves caused by more transmissible and immune evasive genetic variants of SARS-CoV-2 have sustained the ongoing pandemic of COVID-19. Monitoring such variants is expensive, as it usually relies on whole-genome sequencing methods. Therefore, it is necessary to develop alternatives that could help identify samples from specific variants. Reverse transcription loop-mediated isothermal amplification is a method that has been increasingly used for nucleic acid amplification, as it is cheaper and easier to perform when compared to other molecular techniques. As a proof of concept that can help distinguish variants, we present an RT-LAMP assay capable of detecting samples carrying a group of mutations that can be related to specific SARS-CoV-2 lineages, here demonstrated for the Variant of Concern Gamma. We tested 60 SARS-CoV-2 RNA samples extracted from swab samples and the reaction showed a sensitivity of 93.33%, a specificity of 88.89% and a kappa value of 0.822 for samples with a Ct ≤ 22.93. The RT-LAMP assay demonstrated to be useful to distinguish VOC Gamma and may be of particular interest as a screening approach for variants in countries with poor sequencing coverage.

Ten-minute direct detection of Zika virus in serum samples by RT-LAMP.

Zika virus (ZIKV) is a current threat to global health. In most of cases, ZIKV infection has no symptoms; however in some cases, ZIKV can cause paralysis (Guillain-Barré syndrome), and in pregnant women, it can cause birth defects in infants. Rapid and accurate diagnosis can help improve disease control as well as being vital to prenatal care for women living in endemic areas. Molecular diagnostics based on isothermal amplification techniques are an excellent alternative to conventional methods of DNA amplification, such as PCR. Here, we develop and optimized a rapid and sensitive method for direct detection of ZIKV in Serum samples based on RT-LAMP and visual detection. The reaction was thermally controlled with a thermoblock for 10 min at 72 °C. The results show that the use of the Bst 3.0 enzyme and an adequate optimization can further reduce the time needed for the RT-LAMP reaction to detect ZIKV. Our results demonstrate that it is possible to detect ZIKV through RT-LAMP directly from a Serum sample, without prior RNA extraction. As little as 10-3 copies of RNA in a 10 µL reaction (20 zepto-molar) was detected by RT-LAMP from a panel of 51 Serum samples (16 samples from pregnant women and 35 samples from newborns infected with ZIKV during pregnancy). The RT-LAMP has proven to be a valuable tool for molecular diagnosis of Zika, presenting a great potential for point-of-care applications, especially in developing countries.

[Route taken by families of children hospitalized in a teaching hospital]. / Itinerario percorrido pelas familias de criancas internadas em um hospital escola.

Exploratory and descriptive study, of qualitative nature, developed with the purpose of knowing the route of the child's family admitted to the hospital. The data were collected at the pediatric unit of the Regional University Hospital of Maringá (HUM), through semi-structured interviews with the accompanying family members. After analysis of content, two categories emerged: the role of the family in the identification of deviations in the child's health, and the (un)readiness of the health services: impacts on the child health attendance. The study evidenced an expectation of the family in face of the child's illness, searching for alternative strategies and late seeking for health services. These, in turn, show gaps along the attendance process, from diagnosis to treatment, thus raising familiar stress and increasing the possibilities of aggravation of the clinical condition of the child.