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BACKGROUND: Polymerase Chain Reaction (PCR) has become an important diagnostic and research tool of modern molecular biology globally. Real-time PCR allows for rapid and reliable quantification of mRNA transcription. Reference genes are used as internal reaction control to normalise mRNA levels between different samples in order to allow for an exact comparison of mRNA transcription level. METHODS: In this study, twelve commonly used human reference genes were investigated in Human Embryonic Kidney Cell Lines (HEK293) using real-time qPCR with SYBR green. The genes included beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes was investigated using the geNorm application. RESULTS: The range of expression stability in the genes analysed was (from the most stable to the least stable): UBC, TOP1, ATP5B, CYC1, GAPDH, SDHA, YWHAZ, CTB, 18S, EIFA-2, B2M and RPL13A. The optimal number of reference targets in the experiment was calculated to be 2 (geNorm V<0.15) when comparing a normalization factor based on the 2 or 3 most stable targets). CONCLUSION: The expression stability varied greatly between the 12 candidate reference genes. UBC, TOP1, ATP5B, CYC1 and GAPDH respectively showed the highest stability in HEK293 cells based on both expression stability and expression level. Overall, our data suggest that UBC and TOP1show the least variation and the highest expression stability. This report validates the need for rational selection of reference genes for data normalization to ensure accuracy of quantitative PCR assays.
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Expressão Gênica , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Antígenos de Neoplasias/genética , DNA Topoisomerases Tipo I/genética , Células HEK293 , Humanos , RNA Mensageiro/metabolismo , Valores de ReferênciaRESUMO
To determine the prevalence of asymptomatic bacteriuria, bacteriology and sensitivity pattern in Ilorin using the gold standard of urine culture. A prospective study was carried out from 1st July to 31st October 2007, at the University of Ilorin Teaching Hospital [UITH] on 125 consenting asymptomatic pregnant women, A structured proforma was used to collect information from the women and a midstream urine specimen collected for bacteriological culture, Of the 125 pregnant women, 50 had bacteriuria on urine culture giving a prevalence of 40%. The mean age of the women was 28.5 years with a standard deviation of 4.95. The age ranged between 14 and 40 years. Staphylococcus aureus was the commonest pathogen isolated [72%], followed by Proteus spp [14%]. Most of the organisms showed good sensitivity to Nitrofurantoin and gentamicin. The prevalence of asymptomatic bacteriuria in Ilorin is high and routine urine culture is advocated for all pregnant women at booking
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Humanos , Masculino , Feminino , Estudos Prospectivos , Gravidez , Prevalência , Staphylococcus aureus , Proteus , Nitrofurantoína , Gentamicinas , Cuidado Pré-NatalRESUMO
BackgroundReports on the evaluation of immune responses to different COVID-19 vaccines are limited. Similarly, effects of age and gender have not been well explored as variables that could impact on the vaccine-induced antibody response. Therefore, seroprevalence of anti-SARS-CoV-2 specific antibodies in vaccinated and vaccine naive adult Nigerians was determined in this study. MethodologyA total of 141 adults were enrolled into this study. Presence or absence of SARS-CoV-2 infection was confirmed by real-time reverse-transcriptase polymerase-chain reaction (RT-PCR) assay on nasopharyngeal and oropharyngeal swab specimens. Anti-SARS-CoV-2 Specific IgG and IgM antibodies were qualitatively detected using a Rapid Diagnostic Test kit. ResultsPre-vaccination, 77% of the study participants had never had PCR-confirmed COVID-19 test yet 66.7% of them were seropositive for SARS-CoV-2 antibodies. Of 111 COVID-19 vaccinated participants, 69.2% and 73.8% of them had SARS-CoV-2 specific IgG post-first and second doses of COVID-19 vaccine respectively. However, 23.1% and 21.4% of the participants who have had first and second doses respectively had no detectable anti-SARS-CoV-2 antibodies. The proportion of participants with SARS-CoV-2 specific IgG was insignificantly higher in those between the ages of 18 - 40 years and 41 - 59 years compared with individuals aged [≥]60 years. No significant association was observed between gender and seropositivity for SARS-CoV-2 antibodies. ConclusionThere is high SARS-CoV-2 antibody seroprevalence among Nigerian adults who never had PCR-confirmed COVID-19. Also, there is the need for anti-SARS-CoV-2 antibodies screening post vaccination as this could be essential in achieving herd immunity. Age and gender do not seem to have significant association with seropositivity.
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BackgroundFinding effective therapeutics for COVID-19 continues to be an urgent need, especially considering use context limitations and high cost of currently approved agents. The NACOVID trial investigated the efficacy and safety of repurposed antiprotozoal and antiretroviral drugs, nitazoxanide and atazanavir/ritonavir, used in combination for COVID-19. MethodsIn this pilot, randomized, open-label trial conducted in Nigeria, patients diagnosed with mild to moderate COVID-19 were randomly assigned to receive standard of care (SoC) or SoC plus a 14-day course of nitazoxanide (1000 mg b.i.d.) and atazanavir/ritonavir (300/100 mg od) and followed through day 28. Study endpoints included time to clinical improvement, SARS-CoV-2 viral load change, and time to complete symptom resolution. Safety and pharmacokinetics of nitazoxanide active metabolite, tizoxanide, were also evaluated. This trial was registered with ClinicalTrials.gov (NCT04459286). FindingsThere was no difference in time to clinical improvement between the SoC (n = 26) and SoC plus intervention arms (n = 31; Cox proportional hazards regression analysis adjusted hazard ratio, aHR = 0.898, 95% CI: 0.492-1.638, p = 0.725). No difference was observed in the pattern of saliva SARS-CoV-2 viral load changes from days 2 to 28 in the 35% of patients with detectable virus at baseline (20/57) between the two arms (aHR = 0.948, 95% CI: 0.341-2.636, p = 0.919). There was no significant difference in time from enrolment to complete symptom resolution (aHR = 0.535, 95% CI: 0.251 - 1.140, p = 0.105). Atazanavir/ritonavir increased tizoxanide plasma exposure by 68% and median trough plasma concentration was 1546 ng/ml (95% CI: 797-2557), above its putative EC90 in 54% of patients. Tizoxanide was not detectable in saliva. InterpretationThese findings should be interpreted in the context of incomplete enrolment (64%) and the limited number of patients with detectable SARS-CoV-2 in saliva at baseline in this trial. FundingThe University of Liverpool. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThe potential efficacy of nitazoxanide as a repurposed drug for COVID-19 is being investigated in a number of studies due to confirmed in vitro activity against SARS-CoV-2. Available data from completed randomised controlled trials in which clinical improvement, effect on viral load, and symptom resolution were evaluated as outcomes do not offer conclusive evidence. Added value of this studyIn the NACOVID trial, we sought to take advantage of a model-informed strategy and known interaction between nitazoxanide and atazanavir/ritonavir to achieve optimal concentration of tizoxanide in plasma, and possibly in respiratory tracts of patients with mild to moderate COVID-19. While this strategy significantly enhanced tizoxanide exposure in the plasma of patients, our data indicated poor penetration into the respiratory tracts. Specifically, there were no differences in time to clinical improvement, viral load changes, and symptom resolutions between patients who were given standard of care alone and those who combined it with study intervention. Implications of all the available evidenceThe clinical benefit of nitazoxanide remains uncertain. The present study highlights the need for early insight into target site biodistribution of potential COVID-19 therapeutics to better inform candidate selection for clinical trials.
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Identifying the dissemination patterns and impacts of a virus of economic or health importance during a pandemic is crucial, as it informs the public on policies for containment in order to reduce the spread of the virus. In this study, we integrated genomic and travel data to investigate the emergence and spread of the B.1.1.318 and B.1.525 variants of interest in Nigeria and the wider Africa region. By integrating travel data and phylogeographic reconstructions, we find that these two variants that arose during the second wave emerged from within Africa, with the B.1.525 from Nigeria, and then spread to other parts of the world. Our results show how regional connectivity in downsampled regions like Africa can often influence virus transmissions between neighbouring countries. Our findings demonstrate the power of genomic analysis when combined with mobility and epidemiological data to identify the drivers of transmission in the region, generating actionable information for public health decision makers in the region.
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Investment in Africa over the past year with regards to SARS-CoV-2 genotyping has led to a massive increase in the number of sequences, exceeding 100,000 genomes generated to track the pandemic on the continent. Our results show an increase in the number of African countries able to sequence within their own borders, coupled with a decrease in sequencing turnaround time. Findings from this genomic surveillance underscores the heterogeneous nature of the pandemic but we observe repeated dissemination of SARS-CoV-2 variants within the continent. Sustained investment for genomic surveillance in Africa is needed as the virus continues to evolve, particularly in the low vaccination landscape. These investments are very crucial for preparedness and response for future pathogen outbreaks. One-Sentence SummaryExpanding Africa SARS-CoV-2 sequencing capacity in a fast evolving pandemic.