Mejoramiento de un método no comercial para la extracción de ADN de moscas de interés médico-legal

La obtención de ADN de moscas de interés médico-legal es de relevancia para una variedad de aplicaciones. Aunque existen métodos de extracción comerciales de ADN, su uso rutinario es limitado, en algunos escenarios. En este contexto, el uso de métodos no comerciales constituye una alternativa; sin embargo, su optimización es clave para mejorar el flujo de trabajo y los resultados. Este trabajo evaluó el impacto de variaciones a un método de precipitación salina sobre la concentración y la pureza del ADN recuperado. No se encontraron diferencias significativas en la concentración de ADN extraído entre los diferentes tiempos de incubación, probados durante la fase de extracción, mientras que el incremento en el volumen de etanol absoluto, en la fase de precipitación de ADN, mejoró significativamente la concentración de ADN obtenido. Las modificaciones propuestas reducen el tiempo de ejecución y la concentración de ADN obtenido comparado con el protocolo original.

Obtaining DNA from flies of medico-legal interest is relevant for a variety of applications. Although commercial extraction methods offer optimal DNA, their routine use is limited in some settings. In this context, the use of non-commercial methods constitutes an alternative in laboratories with limited resources however, its optimization is key to improving the workflow and the results. This work evaluated the impact of variations to a saline precipitation method on the concentration and purity of the recovered DNA. No significant differences were found in the concentration of extracted DNA between the different incubation times tested during the extraction phase. In contrast, the increased volume of absolute ethanol in the DNA precipitation phase significantly improved the concentration of DNA obtained. The proposed modifications reduce the runtime and DNA concentration obtained compared with the original protocol.

The generation and detection of 2019-nCoV subgenomic RNA / 中华检验医学杂志

The 2019 novel coronavirus (2019-nCoV) is a newly discovered pathogen in 2019. The coronavirus disease (COVID-19) has spread around the world and has greatly affected global health and the world economy. It is a positive-sense single-stranded RNA virus, which generates subgenomic RNA from discontinuous transcription of the replication-transcription complex (RTC). This discontinuous transcription is regulated by transcriptional regulatory sequences/elements, produced by switching templates on genomic RNA. At present, the detection methods of subgenomic RNA include the next generation sequencing, nanopore sequencing, reverse transcription dropletdigital polymerase chain reaction, reverse transcription real-time quantitative polymerase chain reaction, etc. Subgenomic RNA is produced only when the virus infects cell, so it may be a novel marker for viral replication.

Application and consideration of metagenomic next-generation sequencing technology in the diagnosis of insect-borne diseases / 中华检验医学杂志

Insect-borne diseases are serious life-threatening infectious diseases. Rapid and accurate etiological diagnosis are the premise of timely and effective clinical treatments, reducing mortality and sequelae. Laboratory diagnoses of insect-borne diseases mainly focus on targeted serological detection and polymerase chain reaction, which is difficult to detect rare insect borne pathogens. At present, the metagenomic next-generation sequencing (mNGS) technology has moved from scientific research into clinical application. The detection of nucleic acid sequences of all organisms in infected samples by mNGS exhibited significant advantages in the diagnosis and traceability of rare pathogens. But at the same time, mNGS is also suffered with challenges such as background microbial interference, false results caused by database restrictions, pathogen resistance and host immune status information that are urgently needed for clinical treatments. This article systematically summarized applications of mNGS in the diagnosis of insect-borne pathogens and the challenges and difficulties it faces. With the continuous optimization of mNGS in the detection, it will bring new development and innovation to the etiology diagnosis of clinical infectious diseases.

Fetal RHD screening from circulating cell-free DNA in RhD-negative pregnant women / 中华围产医学杂志

Objective:To investigate the RHD genotypes of RhD-negative pregnant women and explore the optimum strategy for fetal RHD screening among this population in the region. Methods:This prospective study recruited 33 cases of RhD-negative singleton pregnancies at ≥12 weeks of gestation in Nanjing Drum Tower Hospital from March to November 2021. On the basis of RHD genotyping, quantitative real-time polymerase chain reaction (PCR) was used to amplify the exons 5 and 10 of RHD gene in the circulating cell-free DNA of RhD-negative pregnant women harboring whole RHD gene deletion and RHD-CE(2-9)- D. High-throughput sequencing was performed to detect chr1:25648453 locus from circulating cell-free DNA in plasma of RhD-negative pregnant women harboring RHD 1227A mutation to screen the fetal RhD blood group. Neonatal umbilical cord blood samples were collected for verifying fetal RHD genotyping. Descriptive statistical analysis was used. Results:Whole RHD gene deletion homozygous genotype ( n=20, 60.6%), RHD-CE(2-9) -D/whole RHD gene deletion heterozygous genotype ( n=5, 21.2%), RHD 1227A/whole RHD gene deletion heterozygous genotype ( n=7, 15.2%) and RHD 711delC/whole RHD gene deletion heterozygous genotype ( n=1) were identified in the 33 RhD-negative pregnant women. In the 25 cases with whole RHD gene deletion homozygous genotype or RHD-CE(2-9)- D/whole RHD gene deletion heterozygous genotype, 22 fetuses were RhD-positive and three were RhD-negative based on prenatal screening, which were confirmed by the neonatal serological test results after birth. In the seven cases carrying RHD 1227A/whole RHD gene deletion heterozygous genotype, all fetuses were RhD-positive, which were consistent with the results of serological detection after delivery. The case harboring RHD 711delC/whole RHD gene deletion heterozygous genotype did not receive fetal RHD screening. Conclusions:This study suggests that whole RHD gene deletion homozygous genotype is the most common allele in RhD-negative population in this area, followed by RHD 1227A/whole RHD gene deletion heterozygous genotype and RHD- CE(2-9)- D/whole RHD gene deletion heterozygous genotype. For women with whole RHD gene deletion homozygous genotype, RHD- CE(2-9)- D, or RHD 1227A mutation, fetal RHD screening with quantitative real-time PCR and high-throughput sequencing are important for the management of RhD-negative pregnant women.

Application value of modified magnetic bead screening for enrichment of cell-free fetal DNA from maternal peripheral blood in non-invasive prenatal testing / 中华围产医学杂志

Objective:To evaluate the value of modified magnetic bead screening for enrichment of cell-free fetal DNA (cffDNA) in non-invasive prenatal testing (NIPT).Methods:This study retrospectively recruited 31 cases with low concentration of cffDNA (<6.00%), Z value in the gray zone (3.00-4.00) at the first detection, or false-positive (confirmed by invasive prenatal diagnosis) or false-negative (confirmed by postnatal chromosome test) results among 11 000 pregnant women who underwent routine NIPT in Beijing Haidian District Maternal and Child Health Care Hospital from October 2017 to December 2019. Plasma samples collected for the first-time routine NIPT were used to enrich cffDNA using modified magnetic beads for NIPT (modified NIPT). Wilcoxon rank sum test was used to compare the modified NIPT with the routine NIPT in detecting the cffDNA concentrations of male fetuses.Results:Among the 31 pregnant women, there were 13 cases with low cffDNA concentration in routine NIPT, 11 having false-positive results in the routine NIPT (three for trisomy 13, four for trisomy 18 and four for trisomy 21, all were confirmed by invasive prenatal diagnosis), six with gray-zone Z values in the first-time NIPT (retesting indicating low risk) and one having false negative result for trisomy 21 (confirmed by postnatal chromosome test). Cell-free DNA (cfDNA) fragments less than 150 bp were effectively enriched using the modified magnetic bead screening and the concentration of cffDNA was increased from 4.43% (2.45%-17.61%) in routine NIPT to 13.46% (7.75%-36.64%) in the modified NIPT ( Z=-14.22, P<0.01). Results of the modified NIPT indicated that 13 cases with low cffDNA concentration of routine NIPT were successfully detected as low risk, as well as the risks in the six cases with gray-zone Z value and six of the 11 false-positive cases in the routine NIPT were low, which were consistent with the retest results of the routine NIPT, while high risk was found in one false-negative case. Conclusions:The modified NIPT could reduce the false positive rate by lowering the failure rate caused by low concentration of cffDNA and is able to identify false-negative cases. Compared with the routine NIPT, it shows a higher success rate and a lower false positive rate.

Storage and purification adaptations for the isolation of total RNA from the dura mater / Adaptações no armazenamento e purificação para o isolamento de RNA total da dura-máter

Abstract Background RNA extraction is a step that precedes several molecular techniques. The fibrous tissue, more specifically the dura mater, has several limitations in routine protocols, and lacks optimization protocols to overcome these problems. Objective To test stock reagents and purification kits, optimizing commercial kit protocols for RNA extraction from the dura mater. Methods Dura mater samples were obtained from eight Wistar rats and maintained in two different stabilizers. The samples were purified using four different protocols, and the RNA was evaluated for the yield and purity in NanoDrop 2000 (Thermo Scientific, Wilmington, DE, United States). Beta-actin gene was used for analyzing gene expression, since is one of the most used reference genes. Results The RNA preservation was similar in both stabilizers. The addition of an incubation step prior the purification protocols allowed better tissue digestion and RNA recovery. The RNA purified using the protocols membrane-based showed higher quality than liquid-liquid purification. This impact was observed in the 3-week evaluation using RT-qPCR. Conclusion Stabilizers are efficient for RNA preservation and membrane-based purification protocols are more suitable for RNA recovery from dura mater tissue, allowing the evaluation of gene expression in this type of tissue. Adaptations in the dura mater RNA extraction protocol differ from the pre-established protocols because it takes into account the peculiarity of fibrous tissue and low cellularity. In addition to providing a low-cost mechanism, based on techniques that are part of the laboratory routine, it is possible to improve the quality of the extracted material, ensuring greater efficiency in the use of subsequent techniques.

Resumo Antecedentes A extração de RNA é uma etapa que antecede várias técnicas moleculares. O tecido fibroso, mais especificamente a dura-máter, apresenta várias limitações nos protocolos de rotina e carece de protocolos de otimização para superar estes problemas. Objetivo Testar reagentes de estoque e kits de purificação, otimizando protocolos de kits comerciais para extração de RNA da dura-máter. Métodos Amostras de dura-máter foram obtidas de oito ratos Wistar e mantidas em dois estabilizadores diferentes. As amostras foram purificadas em quatro protocolos diferentes e o RNA foi avaliado quanto ao rendimento e pureza no NanoDrop 2000 (Thermo Scientific, Wilmington, DE, United States). O gene da beta-actina foi utilizado para analisar a expressão gênica, uma vez que é um dos genes de referência mais utilizados. Resultados A preservação do RNA foi semelhante em ambos os estabilizadores. A adição de uma etapa de incubação antes dos protocolos de purificação permitiu uma melhor digestão do tecido e recuperação de RNA. O RNA purificado pelos protocolos baseados em membrana apresentou qualidade superior ao da purificação líquido-líquido. Este impacto foi observado na avaliação de três semanas usando RT-qPCR. Conclusão Os estabilizadores são eficientes para preservação do RNA e os protocolos de purificação baseados em membrana são mais adequados para recuperação de RNA do tecido da dura-máter, permitindo a avaliação da expressão gênica neste tipo de tecido. As adaptações no protocolo de extração de RNA da dura-máter diferem dos protocolos preestabelecidos porque leva em consideração a peculiaridade do tecido fibroso e com baixa celularidade. Além de fornecer um mecanismo de baixo custo, baseado em técnicas que fazem parte da rotina laboratorial, é possível melhorar a qualidade do material extraído, garantindo maior eficácia no uso de técnicas subsequentes.

Clinical value of noninvasive prenatal testing in vanishing twin pregnancies / 中华围产医学杂志

Objective:To analyze the clinical value of noninvasive prenatal testing (NIPT) in vanishing twin (VT) pregnancies.Methods:A total of 164 VT pregnancies that underwent NIPT in Peking University Third Hospital from January 2017 to December 2020 were enrolled. Gestational age at onset of vanishing, results of NIPT and invasive prenatal diagnosis, blood sampling time points, and pregnancy outcomes were retrospectively analyzed using two independent samples t test and Chi-square test. Results:(1) Of the 164 cases, six had positive results for NIPT, but negative results for karyotype analysis or single nucleotide polymorphism genotyping, with a false positive rate of 3.7% (6/164) for NIPT and all of them were delivered at term. Four pregnancies terminated in the second trimester, including two fetal malformation cases and one unexplained intrauterine death whose single nucleotide polymorphisms results are all normal and one inevitable abortion case due to premature rupture of membrane who refused amniocentesis. The other 154 women all gave birth to normal phenotype babies including 12 preterm ones. (2) The false-positive rate of NIPT was lower in VT pregnancies diagnosed at less than eight gestational weeks than those diagnosed after [1.5% (2/134) vs 13.3% (4/30), χ2=6.68, P=0.010]. The false-positive rate was 6.9% (4/58) in women diagnosed at or below eight weeks between the occurrence of VT and blood sampling and was 1.9% (2/106) in those with interval more than eight weeks, but without significant difference ( χ2=1.44, P=0.231). Conclusions:Although VT pregnancies exist false-positive results in NIPT, screening is still recommended based on fully informed consent to reduce unnecessary invasive prenatal diagnosis. The earlier the onset of VT, the lower the NIPT false positive rate, but whether extending the sampling interval would reduce the risk of false-positive needs further study.

Pharmaceutical applications of framework nucleic acids

DNA is a biological polymer that encodes and stores genetic information in all living organism. Particularly, the precise nucleobase pairing inside DNA is exploited for the self-assembling of nanostructures with defined size, shape and functionality. These DNA nanostructures are known as framework nucleic acids (FNAs) for their skeleton-like features. Recently, FNAs have been explored in various fields ranging from physics, chemistry to biology. In this review, we mainly focus on the recent progress of FNAs in a pharmaceutical perspective. We summarize the advantages and applications of FNAs for drug discovery, drug delivery and drug analysis. We further discuss the drawbacks of FNAs and provide an outlook on the pharmaceutical research direction of FNAs in the future.

Efficacy of entecavir versus tenofovir disoproxil fumarate in treatment of chronic hepatitis B patients with high viral load / 临床肝胆病杂志

Objective To investigate the efficacy of entecavir (ETV) versus tenofovir disoproxil fumarate (TDF) and the treatment measures for poor response in previously untreated chronic hepatitis B (CHB) patients with high viral load. Methods A total of 165 CHB patients who received antiviral therapy and met the inclusion criteria in Department of Infectious Diseases, The First Affiliated Hospital of Guangxi Medical University, from June 2016 to July 2021 were enrolled. The patients enrolled had a baseline HBV DNA level of > 6lg copies/ml and were previously untreated CHB patients who had used ETV or TDF for 48 weeks, and quantitative real-time PCR was used to measure HBV DNA. Virologic response rate was calculated after 48 weeks of treatment; a logistic regression analysis was used to investigate the influencing factors for the response of HBV DNA 40 U/L) at baseline, 89.2% (107/120) achieved an HBV DNA load of 30 years (77.8% vs 47.2%, 85.2% vs 66.7%). For the patients who did not achieve complete virologic response (HBV DNA ≥100 copies/mL) after 48 weeks of treatment, 87.9% (29/33) achieved complete virologic response after the original treatment regimen was prolonged for 48 weeks, and 100% (9/9) of the patients achieved complete virologic response after switching to or adding the first-line nucleos(t)ide analogues (NUCs) without cross-resistance sites with the original regimen for another 48 weeks. Conclusion The patients aged > 30 years should receive antiviral therapy as early as possible, regardless of viral load and ALT level, especially those with a family history of liver cirrhosis or hepatocellular carcinoma; the patients aged ≤30 years who have a normal ALT level and a high viral load should consider initiating antiviral therapy after providing informed consent. For the patients with poor response after 48 weeks of treatment, first-line NUCs without cross-resistance sites with the original regimen should be switched to or added in time.

Challenges in the use of nanostructures as carriers of nucleic acids in clinical practice

ABSTRACT The delivery of nucleic acids to cells is considered a crucial step for the success of genetic modifications aimed at therapeutic purposes or production of genetically modified animals. In this context, nanotechnology is one of the most promising fields of science, with the potential to solve several existing problems. Nanostructures have desirable characteristics to be used as carriers, such as nanometric size, large surface area, cell internalization capacity, prolonged and controlled release, among others. Genetically modified animals can contribute to the production of biopharmaceuticals, through the expression of high-associated-value molecules. The production of these animals, also known as biofactories, further enhances Brazilian agribusiness, since it allows adding value to the final product, and favors the integration between the agricultural market and the pharmaceutical sector. However, there is a growing concern about the safety and possible harmful effects of nanostructures, since data on the safe use of these materials are still insufficient. The objective of this review was to address aspects of the use of nanostructures, mainly carbon nanotubes as nucleic acid carriers, aiming at the production of genetically modified animals, with the certainty that progress in this field of knowledge depends on more information on the mechanisms of interaction between nanostructures, cells and embryos, as well as on its toxicity.

Application of circulating cell-free nucleic acid in clinical diagnosis and treatment of colorectal cancer / 国际肿瘤学杂志

The incidence of colorectal cancer (CRC) is increasing year by year, and early diagnosis is of great significance to improve the prognosis of patients. Circulating cell-free nucleic acid (cfNA) has the advantages of non-invasive, real-time monitoring, and overcoming tumor heterogeneity. The characteristics of cfNA content, mutation, methylation and fragmentation patterns provid important reference value for early diagnosis, curative effect monitoring, prognosis judgment and medication guidance of CRC. However, the practical application of cfNA in the clinical diagnosis and treatment of CRC still needs to solve the problems of unstandardized detection technology, high detection cost, screening of markers with high diagnostic efficacy, and construction of multi-combination models. These challenges will provide new directions for future cfNA research.

Influence of three different nucleic acid extraction methods on 2019-nCoV / 中华检验医学杂志

Objective:To study the difference in the extraction efficiency of the novel coronavirus (2019-nCoV) nucleic acid by using magnetic beads method, centrifugal column method and one-step method.Methods:On March 5, 2021, 10 throat swabs were collected from the staff working in the nucleic acid sampling room in Department of Clinical Laboratory, Affiliated Taikang Xianlin Drum Tower Hospital, Medical School of Nanjing University. The positive quality control samples were mixed into the swabs and used as mock positive samples. The RNA was extracted from simulated positive samples and their diluted samples by using magnetic beads method, centrifugation column method and one-step method. The purity ( A260/ A280 ratio) and concentration of the nucleic acid obtained were measured by micro-uv photometry, and fluorescence quantitative PCR was performed to compare the CT value and extraction efficiency. The three methods were used to extract the simulated weak positive specimens and to compare the difference of CT values after amplification. The measurement data that followed normal distribution were expressed by xˉ±s, the t test was used for comparing in the same group, and single factor analysis of variance was used for comparing among multiple groups. A P value smaller than 0.05 indicated a significant difference. Results:2019-nCoV nucleic acid extracted by magnetic bead method, centrifugal column method and one-step method could amplify positive results. There was no significant difference between the CT value of RNA amplification extracted by magnetic bead method and one-step method ( t=? 0.995 , P=0.376). The CT values of orf1ab gene amplified by centrifugal column method, magnetic bead method and one-step method were 29.28±0.06, 30.82±0.14 and 29.79±0.01 respectively ( F=11.196 , P=0.041). The CT values of E gene were 28.52±0.40, 27.33±0.78 and 27.38±0.13 respectively ( F=3.407, P=0.169). The CT values of N gene were 28.61±1.02, 27.24±0.20 and 27.25±0.47, respectively ( F=2.880 , P=0.020). The CT values of human genes extracted by centrifugal column method, magnetic bead method and one-step method were 19.68±0.36, 20.14±0.06 and 20.58±0.49 respectively, which was statistically significant ( F=4.904, P=0.048). The CT value of amplified human gene was affected by the dilution of human samples twice. The CT value of undiluted samples was smaller than that of diluted samples twice, with a difference of 2.95±0.22, which was statistically significant ( t=?3.025, P=0.039). The extraction time of one-step method, magnetic bead method and centrifugal column method were (15.00±1.50), (20.00±1.50) and (40.00±5.5) min respectively, and the difference was statistically significant ( F=688 , P=0.027). Conclusions:Magnetic bead method, centrifugal column method and one-step method can be used to extract 2019-nCoV nucleic acid, for the centrifugal column method has a higher extraction efficiency than the magnetic bead method and the one-step method. The one-step method is the fastest, followed by the magnetic bead method and the centrifugal column method. A large number of clinical samples can be processed using the magnetic bead method and one-step method. One-step rapid nucleic acid test can also be performed on samples from emergency and fever clinics. It is not recommended to dilute specimens for testing. In order to improve the detection rate, extracting RNA from highly suspected samples with negative initial nucleic acid test by centrifugal column method is suggested.

Current status and research progress of POCT-based detection technology for 2019-nCoV / 中华检验医学杂志

In view of the significant changes in the prevention and control of COVID-19 at home and abroad, "external prevention of importation and internal prevention of rebound" has become the focus of prevention and control of the epidemic in China. Due to the limitation of testing sites, equipment and reaction time, traditional detection methods cannot meet the needs of real-time and rapid detection of 2019-nCoV. Point-of-care testing (POCT) is rapid, portable and flexible. It plays an increasingly important role in the rapid detection and screening of 2019-nCoV. Here, we review the current status and research progress of POCT for 2019-nCoV in terms of antigens, specific antibodies and nucleic acids, in order to provide reference for epidemic prevention and control and clinical management.

Acid-base and lipophilic properties of peptide nucleic acid derivatives / 药物分析学报

The first combined experimental and theoretical study on the ionization and lipophilic properties of peptide nucleic acid(PNA)derivatives,including eleven PNA monomers and two PNA decamers,is described.The acidity constants(pKa)of individual acidic and basic centers of PNA monomers were measured by automated potentiometric pH titrations in water/methanol solution,and these values were found to be in agreement with those obtained by MoKa software.These results indicate that single nucleobases do not change their pKa values when included in PNA monomers and oligomers.In addition,immobilized artificial membrane chromatography was employed to evaluate the lipophilic properties of PNA monomers and oligomers,which showed the PNA derivatives had poor affinity towards membrane phospholipids,and confirmed their scarce cell penetrating ability.Overall,our study not only is of po-tential relevance to evaluate the pharmacokinetic properties of PNA,but also constitutes a reliable basis to properly modify PNA to obtain mimics with enhanced cell penetration properties.

La biopsia líquida en el diagnóstico y monitoreo de pacientes oncológicos: Oportunidades y retos en Latinoamérica / Liquid biopsy in the diagnosis and monitoring of cancer patients: opportunities and challenges in Latin America

Resumen En los últimos años el estudio de los ácidos nucleicos circulantes ha tenido grandes avances en el campo de la oncología, lo que ha permitido avanzar de forma importante en las aplicaciones clínicas de la biopsia líquida en diferentes áreas como el pronóstico, la estadificación, la predicción de recurrencia, la selección y monitorización de tratamientos, entre otros. Lo anterior se debe en gran parte al desarrollo de nuevas y mejores tecnologías, algunas de las cuales incluso han sido autorizadas para el diagnóstico y seguimiento clínico de ciertos tipos de cáncer. No obstante, la utilización de la biopsia líquida como herramienta de apoyo clínico sigue siendo objeto de estudio. Debido a la importancia que ha cobrado este avance tecnológico a nivel mundial, se realizó una revisión de literatura con el fin de establecer el estado actual del uso de biopsia líquida en oncología, así como sus aplicaciones clínicas actuales, con un énfasis en Latinoamérica.

Abstract In recent years, the study of circulating nucleic acids has made great progress in the field of oncology, allowing for significant advances in clinical applications of liquid biopsy in diverse areas such as prognosis, staging, recurrence prediction, selection and monitoring of treatments, among others. This advance is largely due to the development of new and better technologies, some of which have even been validated for the diagnosis and clinical follow-up of certain types of cancer. However, the use of liquid biopsy as an additional tool in clinical oncology remains under study. Given the worldwide importance of this technological advance, a literature review was conducted to establish the current status of the use of liquid biopsy in oncology, as well as its current clinical applications, with a particular focus on Latin America.

Vacunas para SARS-CoV-2: diferentes estrategias de los desarrollos en curso / Vaccines for SARS-CoV-2: different strategies of ongoing developments

El objetivo de este artículo es proporcionar una guía que sirva para la interpretación y seguimiento de los esfuerzos que se están desarrollando en todo el mundo con el objetivo de obtener una vacuna que pueda generar inmunidad contra el nuevo coronavirus SARS-CoV-2 de 2019, el agente causante de la enfermedad por coronavirus denominada COVID-19. Cinco meses después de haber sido detectada la enfermedad, ya hay 102 vacunas en distintos estadios de desarrollo, registradas por la Organización Mundial de la Salud (OMS), correspondientes a 8 plataformas vacunales con diferentes estrategias, y todos los días aparecen nuevas. Esto representará un enorme desafío de organismos internacionales, para la evaluación, comparación y selección de aquellas que cumplan con los criterios regulatorios indispensables de seguridad y eficacia y que, por otro lado, puedan ser producidas en cantidades suficientes para abastecer la demanda mundial. (AU)

The objective of this article is to provide a guide to help the interpretation and monitoring the efforts that are being carried out worldwide to obtain a vaccine that will be able to generate immunity against the new 2019 SARS-CoV-2 coronavirus, the viral agent causes the disease named COVID-19. Five months after the disease was detected, there are already 102 vaccines at different stages of development, registered by World Health Organization (WHO), corresponding to 8 vaccination platforms base on different strategies, and every day new ones appear. This will represent a huge challenge for international organizations, to evaluate, compare and selects those that will meet the essential regulatory criteria of safety and efficacy and that, would be able to be produced in enough quantities to supply the worldwide demand. Key words: SARS-Cov-2 vaccine, vaccine platform, COVID-19 strategy, attenuated virus, viral vector, viral proteins, viral DNA, viral RNA, nucleic acids, viral like particles, WHO. (AU)

Analysis of follow-up information and pregnancy outcomes of cell free DNA prenatal screening / 中华妇产科杂志

Objective@#To evaluate the efficacy of cell free DNA (cf-DNA) screening in prenatal care by analyzing the follow-up information and pregnancy outcomes.@*Methods@#All cf-DNA cases conducted in Women′s Hospital of Nanjing Medical University from August 2011 to December 2017 were enrolled. The general information of the pregnancies, cf-DNA results, confirmatory testing results, and the follow-up results were collected. The pregnancy outcomes were analyzed in cases with low risk cf-DNA results as well as with high risk results for common trisomies, which were trisomy 21 (T21), trisomy 18 (T18), and trisomy 13 (T13). The sensitivity, specificity, positive predictive value and negative predictive value of cf-DNA screening were calculated.@*Results@#(1) A total of 43 615 cf-DNA cases were involved, with 44 cases (0.10%, 44/43 615) test failure results, 314 cases (0.72%, 314/43 571) high risk results for common trisomies and 43 257 cases (99.27%, 43 257/43 571) low risk results. (2) Among 277 cases (88.21%, 277/314) high risk cases were successfully followed up, and 228 cases (82.31%, 228/277) underwent invasive confirmatory prenatal diagnosis. In the low risk results, 36 826 cases (85.13%, 36 826/43 257) were successfully followed up, and 572 (1.55%, 572/36 826) cases were found to have adverse pregnancy outcomes, among which 4 false negative cf-DNA results were confirmed. (3) In the 37 103 successfully followed up cf-DNA cases, the sensitivity for T21, T18, T13 were calculated as 97.96%, 96.67% and 100.00%, respectively; the specificity for T21, T18, T13 were calculated as 99.96%, 99.95% and 99.95%, respectively. The positive predictive value for T21, T18, T13 were calculated as 90.57%, 63.04% and 17.39%, respectively. The negative predictive value for T21, T18, T13 were calculated as 99.99%, 99.98% and 100.00%.@*Conclusions@#Cf-DNA is effective in detecting common trisomies, with a high sensitivity and specificity. However, the follow-up information revealed several potential limitations in current clinical practice, such as a number of cases with high risk results rejected invasive confirmatory testing, as well as the genetic diagnostic results for most low risk cases with an adverse pregnancy outcome aren′t obtained. Genetic counseling and the follow-up for all the cf-DNA cases should be emphasized in the future.

Impact of maternal X chromosome aneuploidies on cell free DNA prenatal screening / 中华妇产科杂志

Objective@#To investigate the impact of maternal X chromosome aneuploidies on cell free DNA (cf-DNA) prenatal screening.@*Methods@#After genetic counseling, invasive prenatal diagnosis was provided for the 124 cases with high risk of sex chromosome aneuploidie (SCA) indicated by cf-DNA prenatal screening. For cases with discordant results of fetal prenatal diagnosis and cf-DNA prenatal screening, maternal leukocyte was collected for copy number variation sequencing (CNV-seq) to detect whether the maternal X chromosome was carrying variations.@*Results@#Totally, 124 cases with high risks of SCA indicated by cf-DNA prenatal screening, 9 cases refused to take invasive prenatal diagnosis, while the remaining 115 cases received. Among the 115 cases, 41 cases received accordant results with cf-DNA prenatal screening while 74 cases discordant. Among the 74 cases with discordant results, 19 cases were indicated with maternal X chromosome variations by maternal leukocyte CNV-seq, which accounting for 25.7% (19/74) of the SCA false positive cases, and 15.3% (19/124) of all SCA cases.@*Conclusions@#Pregnant women with X chromosome variations may affect the results of cf-DNA prenatal screening, resulting in false positive or false negative outcomes, it should be emphasized that the cf-DNA results may be affected by maternal X chromosome variations. In cases with discordant results of prenatal diagnosis and cf-DNA prenatal screening, maternal leukocyte CNV-seq is recommended to find the reasons of false positive or negative results. And cf-DNA prenatal screening is not recommended for pregnant women who are already known with X chromosome variations.

Circulating nucleic acids in the plasma and serum as potential biomarkers in neurological disorders

Neurological diseases are responsible for approximately 6.8 million deaths every year. They affect up to 1 billion people worldwide and cause significant disability and reduced quality of life. In most neurological disorders, the diagnosis can be challenging; it frequently requires long-term investigation. Thus, the discovery of better diagnostic methods to help in the accurate and fast diagnosis of neurological disorders is crucial. Circulating nucleic acids (CNAs) are defined as any type of DNA or RNA that is present in body biofluids. They can be found within extracellular vesicles or as cell-free DNA and RNA. Currently, CNAs are being explored as potential biomarkers for diseases because they can be obtained using non-invasive methods and may reflect unique characteristics of the biological processes involved in several diseases. CNAs can be especially useful as biomarkers for conditions that involve organs or structures that are difficult to assess, such as the central nervous system. This review presents a critical assessment of the most current literature about the use of plasma and serum CNAs as biomarkers for several aspects of neurological disorders: defining a diagnosis, establishing a prognosis, and monitoring the disease progression and response to therapy. We explored the biological origin, types, and general mechanisms involved in the generation of CNAs in physiological and pathological processes, with specific attention to neurological disorders. In addition, we present some of the future applications of CNAs as non-invasive biomarkers for these diseases.