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1.
Braz. j. biol ; 83: e249125, 2023. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1339338

ABSTRACT

Abstract COVID-19 is reported as an extremely contagious disease with common symptoms of fever, dry cough, sore throat, and tiredness. The published literature on incidence and gender-wise prevalence of COVID-19 is scarce in Pakistan. Therefore, the present study was designed to compare the distribution, incubation period and mortality rate of COVID-19 among the male and female population of district Attock. The data were collected between 01 April 2020 and 07 December 2020 from the population of district Attock, Pakistan. A total of 22,962 individuals were screened and 843 were found positive for RT-qPCR for SARS-CoV-2. The confirmed positive cases were monitored carefully. Among the positive cases, the incidence of COVID-19 was 61.7% among males and 38.2% among females. The average recovery period of males was 18.89±7.75 days and females were 19±8.40 days from SARS-CoV-2. The overall mortality rate was 8.06%. The death rate of male patients was significantly higher (P<0.05) compared to female patients. Also, the mortality rate was higher (P<0.05) in male patients of 40-60 years of age compared to female patients of the same age group. Moreover, the mortality rate significantly increased (P<0.05) with the increase of age irrespective of gender. In conclusion, the incidence and mortality rate of COVID-19 is higher in males compared to the female population. Moreover, irrespective of gender the mortality rate was significantly lower among patients aged <40 years.


Resumo Covid-19 é relatada como uma doença extremamente contagiosa com sintomas comuns de febre, tosse seca, dor de garganta e cansaço. A literatura publicada sobre incidência e prevalência de Covid-19 com base no gênero é escassa no Paquistão. Portanto, o presente estudo teve como objetivo comparar a distribuição, o período de incubação e a taxa de mortalidade de Covid-19 entre a população masculina e feminina do distrito de Attock. Os dados foram coletados entre 1 de abril de 2020 e 7 de dezembro de 2020 da população do distrito de Attock, Paquistão. Um total de 22.962 indivíduos foi selecionado, e 843 foram considerados positivos para RT-qPCR para SARS-CoV-2. Os casos positivos confirmados foram monitorados cuidadosamente. Entre os casos positivos, a incidência de Covid-19 foi de 61,7% no sexo masculino e 38,2% no feminino. O período médio de recuperação dos homens foi de 18,89 ± 7,75 dias e das mulheres 19 ± 8,40 dias do SARS-CoV-2. A mortalidade geral foi de 8,06%. A taxa de mortalidade de pacientes do sexo masculino foi significativamente maior (P < 0,05) em comparação com pacientes do sexo feminino. Além disso, a taxa de mortalidade foi maior (P < 0,05) em pacientes do sexo masculino com 40-60 anos de idade em comparação com pacientes do sexo feminino da mesma faixa etária. Além disso, a taxa de mortalidade aumentou significativamente (P < 0,05) com o aumento da idade, independentemente do sexo. Em conclusão, a incidência e a taxa de mortalidade de Covid-19 são maiores no sexo masculino em comparação com a população feminina. E também, independentemente do sexo, a taxa de mortalidade foi significativamente menor entre os pacientes com idade < 40 anos.


Subject(s)
Humans , Male , Female , COVID-19 , Pakistan/epidemiology , Incidence , Real-Time Polymerase Chain Reaction , SARS-CoV-2
2.
Arch. argent. pediatr ; 120(5): 336-339, oct. 2022. tab
Article in English, Spanish | LILACS, BINACIS | ID: biblio-1391180

ABSTRACT

Frenar la propagación de la enfermedad por el coronavirus 2019 (COVID-19, por su sigla en inglés) es fundamental, y se puede realizar mediante técnicas de detección rápidas y efectivas. El objetivo fue comparar la precisión diagnóstica de un test rápido de antígeno (TRAg,) con la reacción en cadena de polimerasa con retrotranscripción (RT-qPCR, por su sigla en inglés) y describir los umbrales de amplificación (Ct, por su sigla en inglés). Participaron niños de 1 mes a 11 años que tuvieran menos de 7 días de síntomas, sin resultado detectable en los últimos 90 días, e inmunocompetentes. Se incluyeron 1855 pacientes con una prevalencia de COVID-19 del 4,7 %. La sensibilidad global del TRAg fue del 60,2 % y su especificidad, del 99,8 %; en niños mayores de 5 años los valores fueron de 69,8 % y 99,8 %, respectivamente. Los valores de Ct de las muestras discordantes fueron más altos. En conclusión, la precisión diagnóstica muestra que TRAg tiene una especificidad similar a la RT-qPCR, pero una sensibilidad considerablemente menor, sobre todo en niños de menos de 5 años.


Stopping the spread of coronavirus disease 2019 (COVID-19) is critical and can be achieved through rapid and effective detection techniques. Our objective was to compare the diagnostic accuracy of rapid antigen tests (RAgT) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) and to describe amplification cycle thresholds (Cts). Participants were children aged 1 month to 11 years with symptoms for less than 7 days, who did not have a detectable result in the past 90 days, and were immunocompetent. A total of 1855 patients were included; the prevalence of COVID-19 was 4.7%. For the RAgT, overall sensitivity was 60.2% and specificity, 99.8%; in children older than 5 years, values were 69.8% and 99.8%, respectively. Ct values for discordant samples were higher. To conclude, the diagnostic accuracy indicated that the specificity of RAgT is similar to that of RT-qPCR, but its sensitivity is notably lower,especially in children younger than 5 years.


Subject(s)
Humans , Infant , Child, Preschool , Child , SARS-CoV-2 , COVID-19/diagnosis , Cross-Sectional Studies , Sensitivity and Specificity , Clinical Laboratory Techniques/methods , Real-Time Polymerase Chain Reaction , COVID-19 Testing
3.
Int. j. morphol ; 40(3): 728-734, jun. 2022. ilus, tab
Article in English | LILACS-Express | LILACS | ID: biblio-1385653

ABSTRACT

SUMMARY: The main objective of this study was to analyze by real-time quantitative polymerase chain reaction (RT-qPCR) the expression patterns of the myosin heavy chain (MHC) isoforms (MHC-I, MHC-IIa, MHC-IIx) in the sphenomandibularis portion of the temporalis muscle. We expected to find differences between the sphenomandibularis and the other portions of the temporalis that could be related to the functional characteristics of the sphenomandibularis identified by electromyography. We dissected the right temporalis muscle of ten adult human individuals (five men and five women). Samples of the anterior and posterior temporalis and of the sphenomandibularis portion were obtained from each dissected muscle. These samples were analyzed by RT-qPCR to determine the percentages of expression of the MHC-I, MHC-IIa and MHC-IIx isoforms. No significant differences were identified between the anterior and the posterior temporalis in the expression patterns of the MHC-I, MHC-IIa and MHC-IIx isoforms. However, there were significant differences between the sphenomandibularis and the anterior temporalis. Specifically, the sphenomandibularis portion had a higher percentage of expression of the MHC-I isoform (P=0.04) and a lower percentage of expression of the MHC-IIx isoform (P=0.003). The pattern of expression that we observed in the sphenomandibularis reflects a greater resistance to fatigue, a lower contraction speed, and a lower capacity of force generation in the sphenomandibularis compared to the anterior temporalis. These characteristics are consistent with electromyographic findings on the functional differences between these two portions.


RESUMEN: El principal objetivo de este estudio fue analizar mediante real-time quantitative polymerase chain reaction (RT-qPCR) los patrones de expresión de las isoformas de la cadena pesada de la miosina (MHC-I, MHC-IIa y MHC-IIx) en la porción esfenomandibular del músculo temporal. Se esperó encontrar diferencias entre el esfenomandibular y las otras porciones del músculo temporal que se pudieran relacionar con las características funcionales del esfenomandibular, identificadas mediante electromiografía. Para obtener estos resultados, se diseccionó el músculo temporal derecho en diez humanos adultos (cinco hombres y cinco mujeres) y se obtuvieron muestras de la porción anterior y posterior del músculo temporal y de su porción esfenomandibular. Estas muestras fueron analizadas mediante RT-qPCR para determinar los porcentajes de expresión de las isoformas MHC-I, MHC- IIa y MHC-IIx. No se identificaron diferencias significativas de los patrones de expresión entre la porción anterior y la porción posterior del músculo temporal, pero sí que se observaron diferencias significativas entre la porción anterior del músculo temporal y su porción esfenomandibular. Concretamente, la porción esfenomandibular presentó un mayor porcentaje de expresión de la isoforma MHC-I (P=0.04) y un menor porcentaje de expresión de la isoforma MHC-IIx (P=0.003). El patrón de expresión que hemos observado en la porción esfenomandibular del músculo temporal refleja una mayor resistencia a la fatiga, una velocidad de contracción más lenta y una menor capacidad de generar fuerza si se compara esta porción con la porción anterior del músclo temporal. Estas características son consistentes con las diferencias funcionales que presentan estas dos porciones, que han sido descritas mediante electromiografía.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Temporal Muscle/metabolism , Myosin Heavy Chains/metabolism , Sphenoid Bone , RNA, Messenger/metabolism , Immunohistochemistry , Protein Isoforms , Electromyography , Real-Time Polymerase Chain Reaction
4.
Rev. Inst. Adolfo Lutz ; 81(Único): e37345, mar.1, 2022. tab, graf
Article in English | LILACS, ColecionaSUS, SES-SP, CONASS, SESSP-ACVSES, SESSP-IALPROD, SES-SP, SESSP-IALACERVO | ID: biblio-1391112

ABSTRACT

The present study aims to correlate the sample-to-cutoff ratios (S/CO) distributions of reactive results for HTLV-1/2 antibodies with the detection of proviral DNA in a population of blood donor candidates. It was carried out a retrospective data search of 632 HTLV-1/2 reactive samples, submitted to confirmatory testing from January 2015 to December 2019. Serological screening was performed by chemiluminescent microparticle immunoassay Architect rHTLV-I/II, whereas confirmatory testing was performed by in-house real-time polymerase chain reaction method. 496 out of 632 samples (78%) had undetectable HTLV-1/2 proviral DNA and 136 (22%) had detectable proviral DNA. HTLV infection was not confirmed in any individual for whom the S/CO ratio value was <4, and proviral DNA detection rates gradually escalated as S/CO ratio values increased. The sensitivity and predictive positive value found for the Architect rHTLV-I/II was 100% and 22%, respectively. The receiver operating characteristic (ROC) curve analysis showed that the optimal S/CO ratio value for predicting the presence of HTLV-1/2 was 18.11. High S/CO ratios were more associated with the detection of proviral DNA. The S/CO ratio value <4 suggests excluding true HTLV infection and the risk of blood transmission (AU).


O estudo tem como objetivo correlacionar às distribuições das razões sample-to-cutoff (S/CO) de resultados reagentes para anticorpos HTLV-1/2 com a detecção de DNA proviral em uma população de candidatos à doação de sangue. Realizou-se uma busca retrospectiva de dados de 632 amostras reagentes para HTLV-1/2 submetidas à testagem confirmatória entre janeiro de 2015 a dezembro de 2019. A triagem sorológica foi realizada pelo imunoensaio quimioluminescente de micropartículas Architect rHTLV-I/II, enquanto o teste confirmatório foi realizado pelo método de PCR em tempo real in-house. 496 de 632 amostras (78%) apresentaram DNA proviral indetectável e 136 (22%) apresentaram DNA proviral detectável. A infecção por HTLV não foi confirmada em nenhum indivíduo com valor de S/CO <4 e as taxas de detecção de DNA proviral escalonaram gradualmente à medida que as razões S/CO aumentaram. A sensibilidade e valor preditivo positivo encontrados para o Architect rHTLV-I/II foram 100% e 22%, respectivamente. Utilizando análise de curva ROC, o valor de razão S/CO ideal para predizer a presença de DNA proviral foi de 18,11. Razões S/CO elevadas foram mais associadas à detecção de DNA proviral. Em suma, o valor de S/CO <4 sugere a exclusão de infecção por HTLV e o risco de transmissão pelo sangue (AU).


Subject(s)
Blood Donors , Immunoassay , Human T-lymphotropic virus 1 , Human T-lymphotropic virus 2 , Real-Time Polymerase Chain Reaction , Infections
5.
Rev. chil. infectol ; 39(1): 35-44, feb. 2022. ilus, tab
Article in Spanish | LILACS-Express | LILACS | ID: biblio-1388330

ABSTRACT

INTRODUCCIÓN: El umbral de ciclo (en inglés cycle threshold-Ct) de la reacción de polimerasa en cadena en tiempo real con transcripción reversa (RT-qPCR) indica la concentración relativa de una secuencia de ARN; este valor se ha relacionado con la expresión de cuadros clínicos en infecciones virales. OBJETIVO: Determinar la correlación entre el valor Ct y la clasificación clínica de la COVID-19. MÉTODO: Se realizó un estudio transeccional correlacional; los valores Ct se obtuvieron mediante RT-qPCR dirigida al gen N del SARS-CoV-2 agrupándolos mediante un estimador robusto central y relacionándose con la clasificación clínica de la COVID-19. RESULTADOS: De los 718 casos incluidos en el estudio; 77,7% (558) fueron leves; 21,3% (153) moderados y 1% (7) graves. El valor Ct se agrupó en niveles: Ct bajo 18,83 - 30,10 y Ct alto > 30,10. Existió correlación significativa inversa débil (p = 0,002; rho de Spearman = -0,117) entre el valor Ct y la clasificación clínica. Las características: sexo, edad menor a 65 años, fiebre, escalofrío, diarrea, anosmia y sobrepeso-obesidad estuvieron asociadas al valor de Ct. CONCLUSIÓN: A menor valor Ct se espera una clasificación de mayor gravedad de la COVID-19; no obstante, debido a que la correlación es débil, su utilidad como predictor de gravedad es limitada.


BACKGROUND: The cycle threshold (Ct) of real-time reverse transcription PCR (RT-qPCR) indicates the relative concentration of an RNA sequence, this value has been related to clinical profile in viral infections. AIM: To determine the correlation between the Ct value and the clinical classification of COVID-19. METHOD: A correlational cross-sectional study was carried out, the Ct values were obtained by RT-qPCR directed to the N gene of SARS-CoV-2, grouping them by means of a central robust estimator and related to the clinical classification of COVID-19. RESULTS: Of the 718 cases included in the study; 77.7% (558) were mild; 21.3% (153) moderate and 1% (7) severe. The Ct value was grouped into levels: low Ct 18.83-30.10 and high Ct> 30.10. There was a weak inverse significant correlation (p = 0.002; Spearman's rho = -0.117) between the Ct value and the clinical classification. The characteristics: sex, age under 65 years, fever, chills, diarrhea, anosmia, and overweightobesity were associated with the Ct value. CONCLUSION: The lower the Ct value, a classification of greater severity of COVID-19 is expected, however, because the correlation is weak, its usefulness as a severity predictor is limited.


Subject(s)
Humans , Male , Female , COVID-19/diagnosis , Cross-Sectional Studies , Real-Time Polymerase Chain Reaction , COVID-19 Testing , SARS-CoV-2/genetics
6.
Article in Chinese | WPRIM | ID: wpr-927948

ABSTRACT

Artemisia Argyi Folium, a traditional Chinese medicine of important medicinal and economic value, sees increasing demand in medicinal and moxibustion product market. Screening stable and reliable reference genes for quantitative real-time PCR(qRT-PCR) is a prerequisite for the analysis of gene expression in Artemisia argyi. In this study, eight commonly used reference genes, Actin, 18s, EF-1α, GAPDH, SAND, PAL, TUA, and TUB, from the transcriptome of A. argyi, were selected as candidate genes. The expression of each gene in different tissues(roots, stems, and leaves) of A. argyi and in leaves of A. argyi after treatment with methyl jasmonate(MeJA) for different time(0, 4, 8, 12 h) was detected by qRT-PCR. Then, geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder were employed to evaluate their expression stability. The results demonstrated that Actin was the most stable reference gene in different tissues and in leaves treated with MeJA, and coming in the second was SAND. Furthermore, the expression of DXS and MCT which are involved in terpenoid backbone biosynthesis was detected in different tissues and after MeJA treatment. The results showed that the expression patterns of DXS and MCT in different tissues and under MeJA treatment calculated with Actin and SAND as internal reference genes were consistent, which validated the screening results. In conclusion, Actin is the most suitable reference gene for the analysis of gene expression in different tissues of A. argyi and after MeJA treatment. This study provides valuable information for gene expression analysis in A. argyi and lays a foundation for further research on molecular mechanism of quality formation of Artemisia Argyi Folium.


Subject(s)
Artemisia/genetics , Gene Expression Profiling , Genes, Plant/genetics , Plant Leaves/genetics , Real-Time Polymerase Chain Reaction , Reference Standards , Transcriptome
7.
Article in English | WPRIM | ID: wpr-927630

ABSTRACT

OBJECTIVE@#To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation.@*METHODS@#Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 -/-, arhgef10 +/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively.@*RESULTS@#WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 -/- zebrafish had more severe symptoms than arhgef10 +/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.@*CONCLUSION@#Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.


Subject(s)
Animals , Annexin A5 , Apoptosis , Blotting, Western , CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Cell Line , Cell Proliferation , Cells, Cultured , Flow Cytometry , Genotype , Humans , In Situ Hybridization , Larva/physiology , Phenotype , RNA/isolation & purification , Real-Time Polymerase Chain Reaction/standards , Rho Guanine Nucleotide Exchange Factors/metabolism , Sincalide/analysis , Spectrophotometry/methods , Zebrafish/physiology
8.
Frontiers of Medicine ; (4): 240-250, 2022.
Article in English | WPRIM | ID: wpr-929208

ABSTRACT

The continuing discoveries of novel classes of RNA modifications in various organisms have raised the need for improving sensitive, convenient, and reliable methods for quantifying RNA modifications. In particular, a subset of small RNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), are modified at their 3'-terminal nucleotides via 2'-O-methylation. However, quantifying the levels of these small RNAs is difficult because 2'-O-methylation at the RNA 3'-terminus inhibits the activity of polyadenylate polymerase and T4 RNA ligase. These two enzymes are indispensable for RNA labeling or ligation in conventional miRNA quantification assays. In this study, we profiled 3'-terminal 2'-O-methyl plant miRNAs in the livers of rice-fed mice by oxidative deep sequencing and detected increasing amounts of plant miRNAs with prolonged oxidation treatment. We further compared the efficiency of stem-loop and poly(A)-tailed RT-qPCR in quantifying plant miRNAs in animal tissues and identified stem-loop RT-qPCR as the only suitable approach. Likewise, stem-loop RT-qPCR was superior to poly(A)-tailed RT-qPCR in quantifying 3'-terminal 2'-O-methyl piRNAs in human seminal plasma. In summary, this study established a standard procedure for quantifying the levels of 3'-terminal 2'-O-methyl miRNAs in plants and piRNAs. Accurate measurement of the 3'-terminal 2'-O-methylation of small RNAs has profound implications for understanding their pathophysiologic roles in biological systems.


Subject(s)
Animals , High-Throughput Nucleotide Sequencing , Humans , Methylation , Mice , MicroRNAs/genetics , Oxidative Stress , RNA, Small Interfering/metabolism , Real-Time Polymerase Chain Reaction
9.
Article in Chinese | WPRIM | ID: wpr-935355

ABSTRACT

Objective: To evaluate the application of real-time RT-PCR and semi-nested RT-PCR in the detection of norovirus in oysters and analyzing the genetic characteristics of the isolates. Methods: Real-time fluorescent RT-PCR and semi-nested RT-PCR were used to detect norovirus GⅠ/GⅡ in fresh oysters collected from the markets in Beijing from November 2014 to October 2015. The detection rate of the parallel test was also analyzed. In addition, the reliability of semi-nested RT-PCR was evaluated by agreement rate and consistency test (Kappa value). The positive products of norovirus GⅠ/GⅡ capsid protein region gene by semi-nested RT-PCR were sequenced. Software BioEdit 7.0.9.0 was used for sequence alignment, and software Mega 6.0 was used to construct the evolutionary tree. Results: In 72 samples, the detection rate of norovirus was 31.94% (23/72) by real-time RT-PCR, 38.89% (28/72) by semi-nested RT-PCR and 48.61% (35/72) by parallel test. The coincidence rate of the two methods was 73.61%, a moderate degree (Kappa value =0.43). A total of 13 norovirus strains were successfully sequenced, and 11 strains (7 GⅡ.17 strains, 2 GⅡ. 4 Sydney_ 2012 strains, 1 GⅡ. 1 strain and 1 GⅡ. 21 strain) were obtained from norovirus positive samples by two RT-PCR methods, two strains (1 GⅡ. 17 strain and 1 GⅡ. 3 strain) were obtained from real-time RT-PCR negative samples which were positive for norovirus by semi-nested RT-PCR. The similarity between these strains and reference strains from diarrhea patients, environmental sewage, and shellfish products were 84.4% - 100.0%. Conclusions: The parallel test of norovirus in oysters by two RT-PCR methods can improve the detection rate and detect more genotypes. Norovirus strains in oysters were highly homologous with reference strains from diarrheal patients, environmental sewage, and shellfish products. Therefore, surveillance, prevention and control for norovirus should be carried out in people who have frequent contacts with oysters and related environments.


Subject(s)
Animals , Beijing , Humans , Norovirus/genetics , Ostreidae , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction
10.
J. bras. nefrol ; 43(4): 530-538, Dec. 2021. tab, graf
Article in English, Portuguese | LILACS | ID: biblio-1350900

ABSTRACT

Abstract Introduction: Cytomegalovirus (CMV) is one of the most common agents of infection in solid organ transplant patients, with significant morbidity and mortality. Objective: This study aimed to establish a threshold for initiation of preemptive treatment. In addition, the study compared the performance of antigenemia with qPCR results. Study design: This was a prospective cohort study conducted in 2017 in a single kidney transplant center in Brazil. Clinical validation was performed by comparing in-house qPCR results, against standard of care at that time (Pp65 CMV Antigenemia). ROC curve analysis was performed to determine the ideal threshold for initiation of preemptive therapy based on the qPCR test results. Results: Two hundred and thirty two samples from 30 patients were tested with both antigenemia and qPCR, from which 163 (70.26%) were concordant (Kappa coefficient: 0.435, p<0.001; Spearman correlation: 0.663). PCR allowed for early diagnoses. The median number of days for the first positive result was 50 (range, 24-105) for antigenemia and 42 (range, 24-74) for qPCR (p<0.001). ROC curve analysis revealed that at a threshold of 3,430 IU/mL (Log 3.54), qPCR had a sensitivity of 97.06% and a specificity of 74.24% (AUC 0.92617 ± 0.0185, p<0.001), in the prediction of 10 cells/105 leukocytes by antigenemia and physician's decision to treat. Conclusions: CMV Pp65 antigenemia and CMV qPCR showed fair agreement and a moderate correlation in this study. The in-house qPCR was revealed to be an accurate method to determine CMV DNAemia in kidney transplant patients, resulting in positive results weeks before antigenemia.


Resumo Introdução: Citomegalovírus (CMV) é um dos agentes infecciosos mais comuns em pacientes com transplante de órgãos sólidos, com morbidade e mortalidade significativas. Objetivo: Este estudo visou estabelecer um limite para o início do tratamento preemptivo. Além disso, comparou o desempenho da antigenemia com os resultados da qPCR in house. Desenho do estudo: Este foi um estudo de coorte prospectivo realizado em 2017 em um centro único de transplante renal no Brasil. A validação clínica foi realizada comparando resultados de qPCR in house, com o padrão de atendimento na época (Antigenemia para CMV Pp65). A análise da curva ROC foi realizada para determinar o limite ideal para o início da terapia preemptiva baseado nos resultados do teste qPCR in house. Resultados: 232 amostras de 30 pacientes foram testadas com antigenemia e qPCR, das quais 163 (70,26%) foram concordantes (Coeficiente Kappa: 0,435, p<0,001; Correlação Spearman: 0,663). PCR permitiu diagnósticos precoces. O número médio de dias para o primeiro resultado positivo foi 50 (intervalo, 24-105) para antigenemia e 42 (intervalo, 24-74) para qPCR (p<0,001). A análise da curva ROC revelou que em um limite de 3.430 UI/mL (Log 3,54), qPCR teve sensibilidade de 97,06% e especificidade de 74,24% (AUC 0,92617 ± 0,0185, p<0,001), na previsão de 10 células/10(5) leucócitos por antigenemia e na decisão do médico de tratar. Conclusões: Antigenemia para CMV Pp65 e qPCR para CMV mostraram uma concordância aceitável e uma correlação moderada neste estudo. qPCR in house revelou-se um método preciso para determinar DNAemia do CMV em pacientes transplantados renais, obtendo resultados positivos semanas antes da antigenemia.


Subject(s)
Humans , Kidney Transplantation , Cytomegalovirus Infections/diagnosis , World Health Organization , DNA, Viral , Prospective Studies , Viral Load , Real-Time Polymerase Chain Reaction , Antigens, Viral
11.
Braz. j. biol ; 81(3): 692-700, July-Sept. 2021. graf
Article in English | LILACS | ID: biblio-1153403

ABSTRACT

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.


Resumo A contaminação bacteriana dos componentes sanguíneos é um grande desafio na medicina transfusional, principalmente nos concentrados de plaquetas (PCs) devido às condições de armazenamento que favorecem a proliferação bacteriana. Neste estudo, desenvolvemos um protocolo de PCR em tempo real rápido, sensível e específico para a triagem bacteriana de PCs. Um método baseado em PCR em tempo real, controlado internamente, foi otimizado e validado com um Master Mix Universal PCR 16S (IBMP / Fiocruz), que detecta uma região conservada do gene 16S rRNA bacteriano. O background de DNA não específico foi completamente eliminado tratando a PCR Master Mix com monoazida de etídio (EMA). O limite de detecção inferior observado foi de 10 cópias equivalentes do genoma com um valor de Ct 34 ± 1,07, a curva de calibração foi gerada com diluições seriada de 10 vezes do DNA de E. coli. O tempo de processamento, incluindo a purificação microbiana do DNA, foi de aproximadamente 4 horas. O método desenvolvido mostrou alta sensibilidade sem amplificação inespecífica e menor tempo de detecção do que os métodos microbiológicos tradicionais, demonstrando ser um meio eficiente de triagem de PCs pré-transfusionais.


Subject(s)
Blood Platelets , Escherichia coli , Bacteria/genetics , DNA, Bacterial/genetics , RNA, Ribosomal, 16S , Real-Time Polymerase Chain Reaction
12.
Rev. cuba. med. mil ; 50(3): e1381, 2021. tab, graf
Article in Spanish | LILACS, CUMED | ID: biblio-1357313

ABSTRACT

Introducción: Desde el surgimiento de los primeros casos en la pandemia de la COVID-19, se ha desarrollado una carrera vertiginosa en crear un espacio de investigación para el diagnóstico, tratamiento y control de la enfermedad. Objetivo: Describir las características clínicas y radiológicas de los pacientes con la COVID-19. Métodos: Se realizó un estudio descriptivo, en el período comprendido de marzo a octubre del año 2020, se estudiaron 404 pacientes de todas las edades, ingresados, con diagnóstico confirmado con PCR en tiempo real. Las variables utilizadas fueron: edad, sexo, síntomas y radiografía del tórax. Resultados: El 54,5 por ciento de los pacientes fueron del sexo femenino y entre ellos asintomáticos el 55,9 por ciento; el 36,9 por ciento tenía entre 40 a 59 años de edad, en los menores de 20 años, el 64,9 por ciento no presentó síntomas de la enfermedad al ingreso. Estuvieron asintomáticos el 53,5 por ciento; el 76,6 por ciento de las radiografías positivas correspondieron a los sintomáticos, la tos fue el síntoma más frecuente. La mayor positividad en la radiografía del tórax se encontró en los pacientes mayores de 60 años, se observó como patrón más frecuente, la opacidad en velo, de distribución periférica. Conclusiones: Predominan los pacientes asintomáticos, la positividad de las radiografías es mayor en los ancianos(AU)


Introduction: Since the emergence of the first cases of COVID-19 pandemic, a dizzying race has developed in creating a research space for the diagnosis, treatment and control of the disease. Objective: To describe the clinical and radiological characteristics of patients with COVID-19. Methods: A descriptive study was carried out, in the period from March to October 2020, 404 patients of all ages, admitted, with confirmed diagnosis with real-time PCR, were studied. The variables used were: age, sex, symptoms and chest X-ray. Results: 54.5 percent of the patients were female and 55,9 percent of them were asymptomatic, 36,9 percent were between 40 and 59 years old, in those under 20 years 64,9 percent were not. They presented symptoms of the disease upon admission 53,5 percent were asymptomatic, 76,6 percent of the positive radiographs corresponded to the symptomatic ones, coughing was the most frequent symptom. The greatest positivity in the chest X-ray was found in patients older than 60 years, the most frequent pattern was the opacity in the peripheral distribution veil. Conclusions: Asymptomatic patients predominate, the positivity of radiographs is higher in the elderly(AU)


Subject(s)
Humans , Polymerase Chain Reaction , Racial Groups , Real-Time Polymerase Chain Reaction , COVID-19 , Radiography, Thoracic/methods , Epidemiology, Descriptive
13.
Mem. Inst. Invest. Cienc. Salud (Impr.) ; 19(2)ago. 2021. tab, ilus
Article in Spanish | LILACS, BDNPAR | ID: biblio-1337801

ABSTRACT

Los Flavivirus constituyen virus transmitidos por artrópodos, principalmente mosquitos. Pueden producir enfermedades en humanos y animales, también incluyen virus específicos de insectos que solo infectan y se replican en los insectos, no así en vertebrados. En Paraguay los virus dengue, fiebre amarilla y Zika fueron detectados en infecciones humanas, pero los estudios de flavivirus en mosquitos son aún escasos. Por ello, el objetivo del presente estudio fue implementar un sistema de detección de flavivirus en mosquitos en el IICS-UNA. Primero, se organizaron capacitaciones en colecta, preparación de pools y procesamiento por técnicas de RT-PCRs convencionales realizadas por expertos internacionales a profesionales locales (bioquímicos y biólogos). Además, se implementaron planillas de registro de datos y de control de transporte de muestras de los lugares de colectas hasta el IICS-UNA. Se prepararon en total 201 pools de 1 a 35 mosquitos cada uno agrupados por especie, localidad, entre otros criterios. Para asegurar la integridad del RNA extraído se realizó la detección de un control interno (Actina-1), siendo todos los pools positivos para el mismo, 91/201 pools fueron positivos para flavivirus. Se realizó la secuenciación de 19/91 pools positivos para flavivirus identificándose flavivirus de insectos (detectándose principalmente Culex Flavivirus, cell fusing agents Flavivirus y Kamiti river virus), evidenciando la elevada distribución de estos virus. Estos resultados demuestran que fue factible implementar el sistema de detección de flavivirus en mosquitos, lo cual podría contribuir a fortalecer la vigilancia y control de estas virosis, así como el conocimiento sobre la importancia ecológica de flavivirus de insectos


Flaviviruses are viruses transmitted by arthropods, mainly mosquitoes. They can cause diseases in humans and animals, they also include specific insect viruses that only infect and replicate in insects, not in vertebrates. In Paraguay, dengue, yellow fever, and Zika viruses were detected in human infections, but studies of flaviviruses in mosquitoes are still scarce. Therefore, the objective of the present study was the implementation of a flavivirus detection system in mosquitoes at IICS-UNA. First, trainings on collection, pool preparation and processing by conventional RT-PCR techniques were organized by international experts for local professionals (biochemists and biologists). In addition, data log sheets and sample transport control forms from the collection sites to the IICS were implemented. A total of 201 pools of 1 to 35 mosquitoes were prepared, each grouped by species, locality, among others. To ensure the integrity of the extracted RNA, an internal control (Actin-1) detection was performed, all pools being positive for it; 91/201 pools were positive for flaviviruses. The sequencing of 19/91 pools positive for flavivirus was carried out, identifying flavivirus in all cases of insects (mainly detecting Culex Flavivirus, cell fusing agents Flavivirus and Kamiti river virus), evidencing the high distribution of these viruses. These results demonstrate that it was feasible to implement the flavivirus detection system in mosquitoes, which could contribute to strengthen the detection, surveillance and control of these viruses, as well as, the knowledge about the ecological importance of insect flaviviruses


Subject(s)
Animals , Real-Time Polymerase Chain Reaction , Flavivirus , Culicidae/virology , Paraguay
14.
Electron. j. biotechnol ; 52: 59-66, July. 2021. ilus, tab
Article in English | LILACS | ID: biblio-1283592

ABSTRACT

BACKGROUND: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). RESULTS: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. CONCLUSIONS: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.


Subject(s)
Animals , Mice , Adenosine Deaminase , Cytosine , CRISPR-Cas Systems , Gene Editing/methods , Base Sequence , Blotting, Western , Models, Animal , Real-Time Polymerase Chain Reaction , Mutation
15.
Electron. j. biotechnol ; 52: 67-75, July. 2021. tab, graf, ilus
Article in English | LILACS | ID: biblio-1283594

ABSTRACT

BACKGROUND: Adipogenesis and fibrogenesis can be considered as a competitive process in muscle, which may affect the intramuscular fat deposition. The CCAAT/enhancer-binding protein beta (C/EBPb) plays an important role in adipogenesis, which is well-characterized in mice, but little known in bovine so far. RESULTS: In this study, real-time qPCR revealed that the level of C/EBPb was increased during the developmental stages of bovine and adipogenesis process of preadipocytes. Overexpression of C/EBPb promoted bovine fibroblast proliferation through mitotic clonal expansion (MCE), a necessary process for initiating adipogenesis, by significantly downregulating levels of p21 and p27 (p < 0.01). Also, the PPARc expression was inhibited during the MCE stage (p < 0.01). 31.28% of transfected fibroblasts adopted lipid-laden adipocyte morphology after 8 d. Real-time qPCR showed that C/EBPb activated the transcription of early stage adipogenesis markers C/EBPa and PPARc. Expression of ACCa, FASN, FABP4 and LPL was also significantly upregulated, while the expression of LEPR was weakened. CONCLUSIONS: It was concluded C/EBPb can convert bovine fibroblasts into adipocytes without hormone induction by initiating the MCE process and promoting adipogenic genes expression, which may provide new insights into the potential functions of C/EBPb in regulating intramuscular fat deposition in beef cattle.


Subject(s)
Cattle/metabolism , Adipocytes/metabolism , CCAAT-Enhancer-Binding Protein-beta/metabolism , Fibroblasts/metabolism , Adipose Tissue/metabolism , Clone Cells , Cell Proliferation , Adipogenesis , Real-Time Polymerase Chain Reaction , Mitosis , Muscles
16.
Hematol., Transfus. Cell Ther. (Impr.) ; 43(2): 141-146, Apr.-June 2021. tab, graf
Article in English | LILACS | ID: biblio-1286688

ABSTRACT

ABSTRACT Background Acute lymphoblastic leukemia (ALL) is the most common malignancy in children characterized by the overproduction and accumulation of immature lymphoid cells in the bone marrow and peripheral blood. The BMI-1 is an important component of the Polycomb Repressive Complex-1 (PRC1). It is an important molecule for the self-renewal of hematopoietic stem cells (HSCs). The BMI-1 expression is generally high in HSCs and decreases after cell differentiation. The BMI-1 is required for the maintenance of normal and cancer stem cells and has been reported as an oncogene in various tumors. The NANOG is a homeodomain transcription factor responsible for maintaining the stem cell compartment at the blastocyst stage of developing embryos. The NANOG gene has been proven to be transcribed in CD34+ cells and different leukemic cells. Methods The ribonucleic acid (RNA) was extracted from the peripheral blood mononuclear cells (PBMNCs) of 30 pediatric ALL patients (16 B-ALL and 14 T-ALL) and 14 healthy controls. The Bmi-1 and NANOG expression levels were determined using the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Results Compared to normal controls, patients with ALL exhibited upregulated levels of Bmi-1 (p = 0.03). Patients who overexpressed Bmi-1 and NANOG displayed a significantly worse survival than low-expressing patients (hazard ratio (HR) 5.74, 95% confidence interval (CI):1.48-22, p = 0.012 and HR 3.8, 95% CI:1.009-14.3, p = 0.048, respectively). Conclusions Taken together, these data suggest that the Bmi-1 and NANOG might serve as a novel survival predictor in ALL patients. Our observation also suggests that the Bmi-1 and NANOG could serve as new therapeutic targets for treatment of pediatric ALL.


Subject(s)
Humans , Male , Female , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Real-Time Polymerase Chain Reaction , Polycomb-Group Proteins , Polycomb Repressive Complex 1 , Nanog Homeobox Protein
17.
Electron. j. biotechnol ; 51: 8-16, May. 2021. tab, graf, ilus
Article in English | LILACS | ID: biblio-1343314

ABSTRACT

BACKGROUND: Myogenic regulatory factors (MRFs) such as MyoD, Myf6 and Myf5 play a vital role in the growth and development of muscles. Jeju Native Pig (JNP) is the top ranker in Korea amongst the indigenous livestock reared for meat purpose. Few studies covering transcript abundance of the MRFs and related to their co-expression with Pax7 in JNP have been conducted. Despite having better quality pork, JNP does not have a comparative growth rate with respect to western breeds. Therefore, the present study was designed with the objective to study the relative transcript levels of MRFs in the postnatal myogenesis of longissimus dorsi muscles in JNP and Berkshire breeds. RESULTS: Relative transcript levels were analyzed by qRT-PCR and blot expression analysis through Western blotting. Immunocytochemistry was performed to analyze their expressions at cellular levels. ToppCluster aided in the analysis of gene ontology of biological processes. The quantitative transcript levels of MyoD and Pax7 were significantly (P < 0.05) higher in Berkshire than in JNP. Myotube formation was observed under the co-expression of MyoD and Pax7. ToppCluster helped in the understanding of the linking of biological processes of the MRFs with the different signaling pathways. MyBPH had significantly (P < 0.05) high transcript levels during the chosen age groups in JNP than Berkshire. CONCLUSIONS: The current study can be helpful in understanding the genetic basis for myogenesis in postnatal stage. Moreover, it can act as stepping stone for the identification of marker genes related to body growth and meat quality in JNP.


Subject(s)
Animals , Swine , Myogenic Regulatory Factors/metabolism , Muscle Development/genetics , Immunohistochemistry , Genetic Markers , Blotting, Western , Myogenic Regulatory Factors/genetics , PAX7 Transcription Factor/metabolism , Real-Time Polymerase Chain Reaction , Gene Ontology , Pork Meat
18.
Electron. j. biotechnol ; 51: 40-49, May. 2021. tab, ilus, graf
Article in English | LILACS | ID: biblio-1343322

ABSTRACT

BACKGROUND: Scavenger receptor class B (SRB) is a multifunctional protein in animals that participates in physiological processes, including recognition of a wide range of ligands. Astaxanthin is a major carotenoid found in shrimp. However, the molecular mechanism of astaxanthin and SRB protein binding has not been reported. RESULTS: In the present study, a member of the SRB subfamily, named PmSRB, was identified from the transcriptome of black tiger shrimp (Penaeus monodon). The open reading frame of PmSRB was 1557 bp in length and encoded 518 amino acids. The structure of PmSRB included a putative transmembrane structure at the N-terminal region and a CD36 domain. Multiple sequence alignment indicated that the CD36 domain were conserved. Phylogenetic analysis showed four separate branches (SRA, SRB, SRC, and croquemort) in the phylogenetic tree and that PmSRB was clustered with SRB of Eriocheir sinensis. Quantitative real-time polymerase chain reaction showed that the PmSRB gene was widely expressed in all tissues tested, with the highest expression level observed in the lymphoid organ and brain. Subcellular localization analysis revealed that PmSRB-GFP (green fluorescent protein) fusion proteins were predominantly localized in the cell membrane. The recombinant proteins of PmSRB showed binding activities against astaxanthin in vitro. CONCLUSIONS: PmSRB was identified and characterized in this study. It is firstly reported that PmSRB may take as an important mediator of astaxanthin uptake in shrimp.


Subject(s)
Animals , Penaeidae , Receptors, Scavenger/metabolism , In Vitro Techniques , Blotting, Western , Chromatography, High Pressure Liquid , Sequence Alignment , Xanthophylls , Receptors, Scavenger/isolation & purification , Receptors, Scavenger/genetics , Real-Time Polymerase Chain Reaction/methods , Transcriptome
19.
Arq. Ciênc. Vet. Zool. UNIPAR (Online) ; 24(1, cont.): e2403, jan-jun. 2021. tab, graf
Article in Portuguese | LILACS, VETINDEX | ID: biblio-1252764

ABSTRACT

O Whatman FTA-Card® é um papel-filtro quimicamente tratado, destinado à coleta, transporte, armazenamento de amostras para posterior extração de ácidos nucléicos. A tecnologia FTA-Card® é utilizada para manter estável DNA e RNA em temperatura ambiente, podendo ser utilizados para fixação de uma ampla variedade de material orgânico ou tecidos. Foram realizados testes para certificar sua eficiência na conservação do material a ser analisado com o intuito de eliminar a cadeia fria de conservação, agilizando o processo e diminuindo os custos da execução de exames moleculares associados ao diagnóstico de patologias. Foram testadas oito amostras de felinos na forma de sangue total e soro, para a extração utilizou-se o kit Magazorb RNA Total mini-prep kit (Promega®, EUA), para o diagnóstico foi utilizada a técnica de PCR em tempo real para amplificar o gene CI2 de mamíferos, a fim de visualizar a eficácia na conservação de ácidos nucleicos. A utilização desse método torna possível que o material biológico seja enviado por serviços de transporte postais, reduzindo os custos e viabilizando diagnósticos provenientes de áreas mais remotas.(AU)


Whatman FTA-Card® is a chemically treated filter paper intended for the collection, transport, and storage of samples for later extraction of nucleic acids. FTA-Card® technology is used to keep DNA and RNA stable at room temperature and can be used to fix a wide variety of organic material or tissues. Tests were carried out to certify its efficiency in the conservation of the material to be analyzed in order to eliminate the cold conservation chain, speeding up the process and decreasing the costs of performing molecular tests associated with the diagnosis of pathologies. By using this method, biological material can be sent by postal transport services, reducing costs and making diagnoses from more remote areas feasible. Samples of feline specimens were tested in the form of whole blood and serum, using the Magazorb RNA Total mini-prep kit (Promega®, USA) for the extraction. Diagnosis was performed using real-time PCR technique to amplify the mammalian CI2 gene in order to visualize the effectiveness in conserving nucleic acids.(AU)


Whatman FTA-Card® es un papel de filtro tratado químicamente, destinado a la recogida, transporte, almacenamiento de muestras para su posterior extracción de ácidos nucleicos. La tecnología FTA-Card® se usa para mantener el ADN y el ARN estables a la temperatura ambiente y se puede usar para la fijación de una amplia variedad de materiales o tejidos orgánicos. Se realizaron pruebas para certificar su eficiencia en la conservación del material a analizar con el fin de eliminar la cadena de frío de conservación, agilizando el proceso y reduciendo los costos de realización de pruebas moleculares asociadas al diagnóstico de patologías. Se analizaron ocho muestras felinas en forma de sangre total y suero, para la extracción se utilizó el mini-prep kit Magazorb RNA Total (Promega®, USA), para el diagnóstico se utilizó la técnica de PCR en tiempo real para amplificar el CI2 de mamífero gen, con el fin de visualizar la efectividad en la conservación de ácidos nucleicos. El uso de ese método permite el envío de material biológico por los servicios de transporte postal, lo que reduce los costes y permite realizar diagnósticos desde zonas más remotas.(AU)


Subject(s)
Biocompatible Materials , DNA , Real-Time Polymerase Chain Reaction
20.
Rev. invest. clín ; 73(2): 120-126, Mar.-Apr. 2021. graf
Article in English | LILACS | ID: biblio-1251872

ABSTRACT

ABSTRACT Background: Underestimation of the number of cases during the coronavirus disease 2019 (COVID-19) pandemic has been a constant concern worldwide. Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA using real-time reverse-transcription polymerase chain reaction (RT-PCR) is the most common method to confirm a case. However, these tests have suboptimal sensitivity. Objective: The objective of the study was to estimate the number of COVID-19 confirmed cases, intensive care unit (ICU) admissions and deaths in Mexico, accounting for the probabilities of false-negative tests. Methods: We used publicly available, national databases of all SARS-CoV-2 tests performed at public laboratories in Mexico between February 27 and October 31, 2020. We used the estimated probabilities of false-negative tests based on the day of clinical sample collection after symptom initiation calculated previously. With the resulting model, we estimated the corrected daily number of cases, ICU admissions, and deaths. Results: Among 2,024,822 people tested in Mexico between February 27 and October 31 with an available result, we estimated 1,248,583 (95% confidence interval 1,094,850-1,572,818) cases, compared to 902,343 cases reported with positive tests. ICU admissions and deaths were 15% and 8% higher than reported, respectively. Conclusion: Accounting for SARS-CoV-2 RT-PCR-based diagnostic testsҠprecision is a simple way to improve estimations for the true number of COVID-19 cases among tested persons.


Subject(s)
Humans , COVID-19 Testing/methods , COVID-19/diagnosis , Databases, Factual , Sensitivity and Specificity , Reverse Transcriptase Polymerase Chain Reaction , False Negative Reactions , Real-Time Polymerase Chain Reaction/methods , COVID-19/mortality , COVID-19/epidemiology , Hospitalization/statistics & numerical data , Intensive Care Units/statistics & numerical data , Mexico/epidemiology
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