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Background/Objectives: Runaway inflammation is a key feature of COVID-19. NR3C1 gene encodes for glucocorticoid receptor which plays an important role in inflammation reaction. The variant rs41423247 cause increased glucocorticoid receptors sensitivity. This study aimed to investigate the impact of variants of NR3C1 gene on the course of COVID-19 pneumonia in patients with necessarily artificial lung ventilation. Method(s): The study group included 20 patients (9 women and 11 men) with diagnosis viral COVID-19 pneumonia on artificial lung ventilation at the intensive care unit. All patients underwent daily standard examinations according clinical protocols. Determination of NR3C1 gene variants was carried out by using PCRRFLP. Result(s): There were found the significant negative correlations between NR3C1 gene variants and level of SpO2 (rS = -0.601, p = 0.008), Glasgow Coma Scale score (rS = -0.523, p = 0.026). Also, it was defined a protective effect of genotype CC at risk of development acute respiratory distress syndrome in this patients (chi2 = 4.38, p = 0.037, OR = 0.05 (CI:0.01-0.66)). Conclusion(s): The investigated variant rs41423247 of the NR3C1 gene may be the genetic predictor of complicated course of COVID-19 pneumonia. .
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Background: From the time when the first outbreak of coronavirus disease (COVID-19), only a small proportion of infected people developed a severe infection, which is usually a sequel of cytokine overproduction. Genetic variations in the genes of some cytokines can influence the transcription rate of these cytokines. Objective: The going research article tried to evaluate the link between tumor necrosis factor (TNF)-α-308 gene polymorphism and COVID-19 severity. Materials and Methods: Blood samples were obtained from 60 patients with COVID-19 and verified by reverse transcriptase polymerase chain reaction (PCR) in nasopharyngeal swabs. Patients were categorized into two categories based on the severity of the disease: severe COVID-19 included 30 patients and mild/moderate COVID-19 with 30 patients. The nucleic DNA was obtained from the whole blood, and TNF-α-308G>A polymorphism was genotyped utilizing PCR-restriction fragment length polymorphism. Results: Homozygous (GG) and heterozygous (GA) genotypes were more frequent among severe than among mild cases, although the differences were not significant. At the allelic level, the frequency of a mutant allele (A) was higher in severe than in mild cases with a noticeable distinction (odds ratio = 2.49, 95% confidence interval = 1.1-5.64, P = 0.029). Conclusion: Allele A of TNF-α-308G>A may be deemed a threat for the severity of COVID-19. © 2023 Medical Journal of Babylon.
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Mycobacterium tuberculosis complex (MTBC), the causative organisms of tuberculosis (TB), has afflicted man for millennia. TB was declared a global health emergency by the World Health Organization in 1993. Before the 2020 Covid pandemic, it was responsible for 10 million new cases annually and was the leading infectious disease killer worldwide. MTBC strain typing represents an important complementary tool to guide TB control measures. TB programmes can use genotyping results in combination with epidemiological information to determine if recent transmission has likely occurred, and hence identify outbreaks that require targeted public health action. MTBC genotyping also can differentiate between relapse or re-infection, detect false-positive cases, and identify and monitor the circulating TB strains in the population over time. Restriction Fragment Length Phenotyping (RFLP), introduced in the 1990s, was labour intensive, required large amounts of DNA and was not easily comparable between laboratories. These disadvantages are overcome by the PCR-based MIRU-VNTR and spoligotyping methods. More recently, whole genome sequencing (WGS) of MTBC has been shown to provide high resolution identification of recent transmission chains and their direction, as well as drug resistance prediction. Its increasing reliability and affordability has enabled this technology to transition from the research arena to clinical care and public health functions. Its application in high TB burden countries will hopefully revitalize global TB control efforts which have set back by the Covid pandemic.Copyright © 2023
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The occurrence of Cryptosporidium species infection and its risk factors in neonatal goats is less explored. Also it is due to the fact that diseases like colibacillosis and neonatal viral enteritis complex caused by Group A rotaviruses and Bovine corona viruses can co-exist with Cryptosporidium and can lead to mixed infections and the latter is often overlooked. Therefore, in the current research we explored the cryptosporidial occurrence in neonatal goats of Mathura district of Uttar Pradesh, India. In this study, a total of 644 faecal samples were collected from neonatal goats at different villages and certain organized farms of Mathura district age-wise, season-wise and breed-wise, and were examined for Cryptosporidium based on modified Ziehl-Neelsen technique, conventional 18SSU rRNA nested PCR assay. The overall prevalence of Cryptosporidium infection in goats based on microscopy was 36.80% (237/644;p value <0.0001) and 18SSU rRNA nested PCR 52.95% (341/644;p value <0.0001) respectively. Cryptosporidium species typing was also done using 18SSU rRNA nested PCR-RFLP product using enzymes Mbo-II, Ssp-I and Vsp-I, which revealed species including C. parvum C. bovis, C. ryanae, C. hominis and C. andersoni. Also the infection was clinically associated based on age, gender and seasons to identify the causal relationships that precipitate the cryptosporidial infection in goat kids. Since mZN microscopy based screening requires expertise and may sometimes be confuse with other weak acid fast bodies and also due to low sensitivity, combination of diagnostic tests are used in this study to identify the best test combination that yields best statistical fit in terms of kappa-agreement and McNemar's test. Cryptosporidiosis is caused by an enteric protozoan parasite and the first report in sheep and goat was observed in early 1980s, with other important etiological agents for neonatal diarrhoea, mortality and morbidity in neonatal kids and lambs, responsible for economic losses.Copyright © 2023 Indian Veterinary Assocaition. All rights reserved.
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Objectives: Toll-like receptors (TLRs) are an important family of receptors that recognize infectious agents and play an important role in the innate immune system. TLRs are a potential candidate to control infection in the early stages of the disease and to produce vaccines against SARS-CoV-2. In addition, studies have suggested that TLR polymorphisms are also associated with antiviral responses against SARS-CoV-2. Therefore, we aimed to investigate the relationship of TLR7 and TLR8 polymorphisms with COVID-19 disease prognosis. Materials-Methods: A total of 120 COVID-19 patients, including 40 outpatients, 40 patients with mild and moderate clinical status, hospitalized and severe pneumonia, and 40 patients followed in the intensive care unit (ICU), were included in the study. The classification of disease severity was made according to WHO criteria. TLR7 (rs179009), TLR8 -129 C/G (rs3764879) and TLR8 Met1Val (rs3764880) polymorphisms were genotyped using the PCR-RFLP method. Result(s): Since TLR7 and TLR8 are located on the X chromosome, men and women were analyzed separately. There was no significant difference between the groups in terms of 3 polymorphisms in males. On the other hand, in women, individuals carrying AG genotype and G allele for TLR8 Met1Val polymorphism were found at a higher rate in patients hospitalized in ICU than in patients followed in the service (p <0.05). In terms of the other two polymorphisms, no significant difference was found between the groups in women. Conclusion(s): We suggest that the AG genotype and G allele of TLR8 Met1Val polymorphism can be considered as an important risk factor that increases the severity of the disease in women.
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Introduction: It is known that the development of COVID-19 in the human body consists of complex system of biological mechanisms underlying the complex interplay between infectious agents and the human host. This raised the question about hosts' genetic variants as predictors of clinical phenotype. The aim of our study was to analyze the effect of the NOS3 gene (VNTR intron 4 a/b), NR3C1 gene (C647G, rs41423247) and the SFTPB gene (C1580T, rs11130866) variants on the course of severe COVID-19 pneumonia in patients. Material(s) and Method(s): The study group included 20 patients (13 men and 7 women) with diagnosis "viral COVID19 pneumonia" treated at the intensive care unit. Investigation of the NOS3, NR3C1 and SFTPB genes variants was carried out by a molecular method using PCR-RFLP and allele-specific PCR, respectively. Result(s): The correlation analysis showed a significant association of the NOS3 gene variants and level of SpO2 (rS=-0.488, p=0.029;SpO2=93.1+/-2.4% for b/b and SpO2=82.0+/-1.1% for a/a genotypes). Also a significant positive correlation was between NR3C1 gene variants and duration of nasal intermittent positive pressure ventilation (nIPP) therapy (rS=0.454, p=0.044;for 647CC - 1.5+/-1.0 days and for 674GG - 3.9+/-2.5 days), presence of fever (need for antipyretics) (rS=0.525, p=0.017;647C vs 647G alleles - chi2=5.8, p=0.016). No significant correlations were found for the variants of SFTPB gene. The obtained results support a hypothesis about the combined influence of different pathways genes variants (NOS3 and NR3C1) on severity of COVID-19. However, in order to draw definite conclusions, further multifaceted research in this area are need.
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BACKGROUND: It has been reported that the SARS-CoV-2 pandemic originated in Wuhan, China in December 2019 and spread rapidly worldwide. The virus gets entry into target cells via angiotensin-converting enzyme 2 (ACE2) receptors and its gene is highly polymorphic. INTRODUCTION: the variations in SARS-CoV-2 susceptibility and severity can be explained on a genetic level by studying the polymorphism in ACE2 receptor polymorphism. OBJECTIVE: A prospective case-control study was designed to compare the ACE2 levels in SARS-CoV-2 patients with the healthy controls in the local population, for which a total of 100 EDTA-containing blood samples were included (50 SARS-CoV-2 IgM positive case and 50 healthy controls). METHODS: PCR-RFLP was performed to investigate the polymorphism of ACE2 in genomic DNA and the ACE2 plasma levels were determined through ELISA. RESULTS: No significant difference in allelic and genotype frequencies (GG, GA, AA) were observed while the ACE2 plasma levels were found to be decreased in positive samples. CONCLUSION: No significant association of the ACE2 gene polymorphism (G8790A) was found with the SARS-CoV-2 susceptibility in the Pakistani population which intimates the search for other genetic factors within the local population.
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The infection generated by SARS-CoV-2 has caused more than 200 million cases and 4.5 million deaths worldwide. SARS-CoV-2 has accumulated mutations that allow it to be classified into different lineages. Some of these lineages have been designated variants by the WHO: under monitoring (VUM), of interest (VOI), or of concern (VOC). Methodology. Different strategies for genomic surveillance of these SARS-CoV-2 variants have been described in each country. In Venezuela, the strategies include the amplification of a fragment of the spike, PCR-RFLP, and sequencing of the complete viral genome, which has allowed us to monitor the introduction of VOCs and VOIs to the country. Results. By October 2021, in Venezuela, the circulation of three VOCs, Alpha, Gamma, and Delta, and the two VOIs (Lambda and Mu) have been described. Globally, the Delta variant predominates in practically all continents except some Latin American countries, although it is estimated that it will soon prevail in the region as well. Discussion. The circulation of variants in the countries is a very dynamic process and Venezuela does not escape from this reality;therefore, it is important to continue genomic surveillance of this virus. Copyright © 2021, Revista Salus. All rights reserved.
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Objetivo: Relatar um caso imunohematologico complexo, descrevendo as estrategias sorologicas e moleculares realizadas para a caracterizacao fenotipica de um paciente com ausencia de dois antigenos de alta frequencia U (MNS:-5) e Yta (YT:-1), assim como, a determinacao do(s) anticorpo(s) de alta frequencia presente no soro. Materiais e metodos: Trata-se de um trabalho descritivo em que foram utilizados dados clinicos de prontuario disponiveis no laboratorio de referencia de imunohematologia (LRI) onde foram testadas as amostras. Resultados: Paciente de 84 anos, com anemia sintomatica, COVID-19 e abdomen agudo obstrutivo, teve a amostra encaminhada para o LRI para confirmacao de anticorpo anti-U no soro e genotipagem eritrocitaria. O soro do paciente foi testado com painel de hemacias comerciais e raras sendo reativo com todas as hemacias do painel em Gel-Liss/Enzima, exceto com hemacias S-s-U-/Yt(a+) demonstrando apenas a especificidade de anti-U. O teste de adsorcao alogenica com hemacias de doador tratadas com ZZAP excluiu a presenca de anticorpo anti-Yta no soro aloadsorvido. Para avaliar a importancia clinica do anticorpo, foi realizada a tecnica de MMA (Monocyte Monolayer Assay), sendo que o teste apresentou menos que 5% de atividade fagocitica dos Monocitos, alem disso, o fato da classe da Imunoglobulina nao pertencer a IgG1 ou IgG3 (cartao ID-Card DAT IgG1/IgG3, BioRad), indicaram que esse anticorpo nao possui importancia clinica. O DNA do paciente foi extraido do sangue total utilizando o Kit MagNa Pure, Roche. A genotipagem realizada pelas tecnicas do BloodChip Kit ID CoreXT - Grifol, PCR-SSP e PCR-RFLP demonstrou o seguinte resultado: GYPB*delecao;YT*2/2. O fenotipo foi confirmado como U- e Yt(a-) com antissoros provenientes do SCARF. Discussao: Antigenos de alta frequencia quando ausentes, resultam na dificuldade da busca de sangue compativel para a transfusao, assim como na disponibilidade de insumos e tecnicas bem padronizadas para a investigacao imunohematologica. Determinados antigenos de alta frequencia quando ausentes na membrana eritrocitaria podem afetar a expressao de antigenos de outros sistemas. Atraves de uma tecnica semi-automatizada de genotipagem identificamos que a amostra de um paciente tinha a ausencia de dois antigenos de alta frequencia U (MNS:-5) e Yta(YT:-1). Por se tratar de um caso complexo, estrategias sorologicas e moleculares foram realizadas para a caracterizacao fenotipica do paciente, determinacao do anticorpo de alta frequencia presente no soro e sua importancia clinica. Conclusao: A tecnica do BloodChip permitiu uma analise molecular mais completa, sendo possivel verificar a ausencia de dois antigenos de alta frequencia: U e Yta, o que tornou mais dificil a busca por doador fenotipo compativel para o paciente. Paineis de hemacias e soros raros foram capazes de definir a especificidade do anti-U e excluir a presenca do anti-Yta e de outros anticorpos que poderiam estar mascarados no soro. Apesar do anti-U nao aparentar importancia clinica, recomendamos a transfusao de concentrado de hemacias U- (MNS:-5), uma vez que esse anticorpo esta implicado em reacoes transfusionais hemoliticas. Copyright © 2022
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Uncovering risk factors playing roles in the severity of Coronavirus disease 2019 (Covid‐19) are important for understanding pathoimmunology of the disease caused by severe acute respiratory syndrome Coronavirus 2 (SARS CoV‐2). Genetic variations in innate immune genes have been found to be associated with Covid‐19 infections. A single‐nucleotide polymorphism (SNP) in a promoter region of tumor necrosis factor alpha (TNF‐α) gene, TNF‐α −308G>A, increases expression of TNF‐α protein against infectious diseases leading to immune dysregulations and organ damage. This study aims to discover associations between TNF‐α −308G>A SNP and Covid‐19 infection. Polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) was used for genotyping a general Kurdish population and Covid‐19 patients. The homozygous mutant (AA) genotype was found to be rare in the current studied population. Interestingly, the heterozygous (GA) genotype was significantly (p value = 0.0342) higher in the Covid‐19 patients than the general population. This suggests that TNF‐α −308G>A SNP might be associated with Covid‐19 infections. Further studies with larger sample sizes focusing on different ethnic populations are recommended.
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Aim: The Sars Corona Virus (SARS CoV) belongs to the Nidovirales order, the Coronaviridae family, and the genus Coronavirus. The SARS CoV has enveloped, linear, positive sense and single-stranded RNA. The disease caused by SARS-CoV 2 as named as COVID-19. Toll-like receptors (TLRs) initiate signaling cascades leading to the activation of the innate immune system. TLR3 activates antiviral immune responses through the production of inflammatory cytokines and type I interferons. In this study we aimed to investigate TLR 3 c.1377C/T and -7C/A polymorphisms in COVID-19 infection. Methods: In this study, we investigated the frequencies of TLR3 (c.1377C/T and -7 C/A) polymorphisms in 150 COVID-19 patients and 171 healthy adults as controls in Sivas Cumhuriyet University. Firstly, DNA was isolated using phenol-chloroform methods. Then we performed polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP). We also investigated whether these polymorphisms were related to the severity of COVID-19 disease. Results: We found that both TLR3 polymorphisms were associated with COVID-19 disease. TLR3 c.1377C/T TT genotype frequencies were found statistically significant between patients and controls (p=0.019). In TLR3 -7C/A polymorphism we found statistically significant difference in A allele frequencies (p=0.03). There is an also statistically significant difference in distribution of TLR3 -7C/A CT genotype frequency between patients and controls (p=0.04). However, there is no statistically significant association between severe/non-severe and two TLR3 polymorphisms. Conclusion: Our findings suggest that TLR3 c.1377C/T and -7C/A polymorphisms may be important on susceptibility or clinical course of COVID-19.
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Vitamin D [25 (OH)D] plays a role in many of biological processes, such as bone metabolism, immunomodulation, cell proliferation, differentiation, and regulation. Also, it has anti-inflammatory, antifibrotic, and antioxidant effects. Due to the immunomodulatory effects of 25 (OH)D, its deficiency is blamed for a higher risk for COVID-19 infection. Serum concentrations of 25 (OH)D were inversely associated with proinflammatory cytokines such as increased IL-6, CRP levels, and increased risk of pneumonia or ARDS. Lower 25 (OH)D concentrations are associated with a higher risk for infections, especially from the respiratory tract [1]. Chronic vitamin D deficiency can induce the renin-angiotensin system activation and leads to fibrotic changes that can cause lung injury by inducing proinflammatory cytokine production in human monocytes/macrophages (2). Increased frequency of COVID-19 infection at high latitudes and worse prognosis of these cases made clinicians to think that 25 (OH)D levels may affect the risk and prognosis of COVID-19 infection [3]. In previous reports, in the early pandemic, a higher prevalence of vitamin D deficiency has been reported to be related to high rates of COVID-19 infection, higher risk of invasive mechanical ventilation (IMV), and mortality [6]. Whilst, it is reported that 25 (OH)D may not protect against COVID-19 infection in recent studies. Moreover, it was not associated with disease severity or lethality [4-6]. The active form of vitamin D binds to its receptor (VDR) and modulates its responses. VDR is located on chromosome 12q13, consisting of 9 exons. Vitamin D-VDR signaling regulates the expression of a wide range of physiological functions. Herein, VDR polymorphisms cause a dysfunctional receptor that affects VDR activity. Both innate and adaptive immune responses can vary according to different polymorphisms of VDR. Also VDR polymorphisms have been previously found to be associated with bacterial infections such as tuberculosis [7] and severe Respiratory Syncytial Virus (RSV) bronchiolitis in respect to vitamin D deficiency [8]. Moreover, it was demonstrated that different VDR polymorphisms such as FokI, BsmI, ApaI, and TaqI could change the course of RSV infection in several studies, respectively [8-10]. This study aimed to evaluate if there is any association between the VDR gene polymorphism at FokI, TaqI, BsmI, and ApaI alleles and the prognosis of COVID-19 in respect to vitamin D deficiency. Two-hundred ninety-seven (n=297) patients with reverse-transcription polymerase chain reaction (RT-PCR)-confirmed COVID-19 who were admitted to Marmara University Education and Research Hospital between April and October 2020 were enrolled. The severity of COVID-19 patients was classified into 1-10 according to WHO criteria. The patients' requirement for noninvasive mechanical ventilation (NIMV) or reservoir mask, their requirement for admission to intensive care unit (ICU), mortality, and WHO clinical progression scales were reviewed. Four variant regions of vitamin D receptor (VDR);FokI, BsmI, ApaI, and TaqI were determined using the Restriction Fragment Length Polymorphism (RFLP) technique. To conclude;The effect of VDR polymorphisms on the receptor function causes intensive care unit treatment, disease severity and mortality differences among patients with covid-19 infection in the clinical set-up. VDR Ff genotype was related with disease severity, TT with disease severity and aa with mortality respectively. As a result we have detected that 25 (OH)D levels were not related to COVID-19 infection severity and mortality. Additionally, it indicated that VDR polymorphisms are independently associated with the severity of COVID-19 and the survival of patients. More extensive studies are needed to determine the impact of polymorphisms on COVID-19 and explain the underlying cause.
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Background: Candida infection is on the rise with an increasing number of nonalbicans species. Therefore, the need to speciate Candida rapidly and accurately is of the utmost importance. The purpose of this study was to speciate Candida using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), to analyze the correlation of the isolates with the clinical condition, and to study the outcome of the patient. Materials and Methods: PCR-RFLP using universal primers ITS1 and ITS4 was done to speciate all isolates of Candida;patient details were collected to analyze the clinical condition and the outcome of the patient. Results: The most common species of Candida isolated was Candida tropicalis 14 (56%) followed by Candida albicans 5 (20%), Candida auris 3 (14%), Candida parapsilosis 1 (4%), Candida orthopsilosis 1 (4%), and Candida kefyr 1 (4%). Majority of the samples that were collected were urine samples 15 (60%). The average duration of hospital stay was found to be 13.8 days. A number of underlying risk factors were present such as patients with diabetes, sepsis, malignancy, covid19 infection, surgical patients, preterm patients, elderly patients, and patients on long-term steroids. Conclusion: Candidemia is on the rise nowadays with nonalbicans species responsible for the majority of the infections. Since the outcome of the patient depends on rapid diagnosis and prompt initiation of antifungal agents PCR-RFLP proves to be a rapid and reliable test to identify most of the prevailing species of Candida. © 2021 Journal of Medical Society ;Published by Wolters Kluwer-Medknow.
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A common mutation has occurred in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), known as D614G (A23403G). There are discrepancies in the impact of this mutation on the virus's infectivity. Also, the whole genome sequencings are expensive and time-consuming. This study aims to develop three fast economical assays for prompt identifications of the D614G mutation including Taqman probe-based real-time reverse transcriptase polymerase chain reaction (rRT PCR), an amplification refractory mutation system (ARMS) RT and restriction fragment length polymorphism (RFLP), in nasopharyngeal swab samples. Both rRT and ARMS data showed G614 mutants indicated by the presence of HEX probe and 176 bp, respectively. Additionally, the results of the RFLP data and DNA sequencings confirmed the prevalence of the G614 mutants. These methods will be important, in epidemiological, reinfections and zoonotic aspects, through detecting the G614 mutant in retro-perspective samples to track its origins and future re-emergence of D614 wild type.
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In late 2019, an outbreak of respiratory disease named COVID-19 started in the world. To date, thousands of cases of infection are reported worldwide. Most researchers focused on epidemiology and clinical features of COVID-19, and a small part of studies was performed to evaluate the genetic characteristics of this virus. Regarding the high price and low availability of sequencing techniques in developing countries, here we describe a rapid and inexpensive method for the detection of D614G mutation in SARS-CoV-2. Using bioinformatics databases and software, we designed the PCR-RFLP method for D614G mutation detection. We evaluated 144 SARS-CoV-2 positive samples isolated in six months in Northeastern Iran. Our results showed that the prevalent type is S-D in our isolates, and a small number of isolated belongs to the S-G type. Of 144 samples, 127 (88.2%) samples have belonged to type S-D, and 13 (9%) samples typed S-G. The first S-G type was detected on 2020 June 10. We have little information about the prevalence of D614G mutation, and it seems that the reason is the lack of cheap and fast methods. We hope that this method will provide more information on the prevalence and epidemiology of D614G mutations worldwide.