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Introduction/Objective In both the past and for the foreseeable future, SARS-CoV-2 (the coronavirus that causes COVID-19 disease) will continue to evolve. This evolution has already and will lead to new variants that will then cause surges of infection. These outbreaks in the past with the variant responsible have previously been reported individually. However, a timeline perspective on the changing SARS-CoV-2 variant landscape is sparse in the literature, particularly for testing performed at a Veteran Affairs Medical Center (VAMC). The Veteran population has increased comorbidities compared to the general population leading to susceptibility to infection including SARS-CoV-2. Hence, it is of utmost importance to explore the trending variants of SARS-CoV-2 in the veteran population as this epidemiological information may help in preventing transmission, which remains key in the management of COVID-19. Methods/Case Report Samples from selected patients from March 2021 to June 2022 who tested positive for SARS- CoV-2 by reverse transcriptase polymerase chain reaction with a cycle threshold or number <30 (required for sequencing) were sent for SARS-CoV-2 sequencing analysis. Results (if a Case Study enter NA) There were a total of 19 VAMC patients who were sequenced during the entire study period (March 2021 to June 2022). From March to May 2021, there were 8 patients, from which 6 demonstrated Pango Lineage B.1.1.7, 1 demonstrated Pango Lineage B.1.526.1, and 1 demonstrated Pango Lineage B.1. Later in 2021 (August to October 2021), there were 4 patients all of which demonstrates the Delta variant;2 of these 4 demonstrated the Delta subvariant Pango Lineage AY.25 and the other 2 demonstrated Pango Lineage AY.44. By May to June 2022, there were 7 patients, all of whom demonstrated infection by the Omicron variant. Interestingly, 6 of these 7 patients demonstrated the newly emerging subvariant BA.2.12.1 and the remaining 1 demonstrated BA.2.9. Conclusion SARS-CoV-2 has continued to evolve throughout the course of the pandemic, which has led to variants and subvariants that have predominated for a time to cause an outbreak only to be replaced later by a different strain. This timeline epidemiological perspective demonstrates that the Veteran population has also been affected by the variants that have led to outbreaks in the past within the general population.
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Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed an RTqPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase/DNA polymerase (RTX) (Ellefson et al., 2016). Here we show that RTX performs comparably to the other assays sanctioned by the CDC and validated in kit format. We demonstrate two modes of RTX use - (i) dye-based RT-qPCR assays that require only RTX polymerase, and (ii) TaqMan RT-qPCR assays that use a combination of RTX and Taq DNA polymerases (as the RTX exonuclease does not degrade a TaqMan probe). We also provide straightforward recipes for the purification of this alternative reagent RTX. We anticipate that in low resource or point-of-need settings researchers could obtain the available constructs and begin to develop their own assays, within whatever regulatory framework exists for them.Copyright © 2021 Bio-protocol LLC. All Rights Reserved.
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Background: With NHS PrEP now available for those at risk, we aimed to identify missed opportunities for people newly diagnosed with HIV who attended sexual and reproductive health (SRH) services, and to determine the HIV outcomes associated with people acquiring HIV with previous or recent PrEP use. Method(s): A retrospective observational study reviewed all new HIV diagnoses from the last 2 years to see if they were eligible for PrEP and offered in SRH services. Data was collected using electronic medical records on HIV outcomes - virological suppression, resistance and antiretroviral choice. Result(s): There were 74 new HIV diagnoses. 41 people were eligible but only 10 were known to have accessed PrEP at our services. 21% were heterosexual and of black ethnicity - it was not possible to ascertain whether they were eligible for PrEP from the notes. Of the 10 people with recent PrEP use, 2 stopped due to side effects;headaches, vomiting, fatigue and renal toxicity concerns. For the remaining adherence concerns were reported - taking event based dosing (EBD) incorrectly and difficulty accessing services. 80% of people achieved virological suppression. 90% were put on a second generation integrase or protease inhibitor. No one developed nucleoside reverse transcriptase inhibitor (NRTI) resistance. 6 people eligible for PrEP had attended SRH services but not given PrEP. 2 attended during the IMPACT trial being full and referred to IwantPrEPnow. 2 attended during COVID where baseline bloods were done with follow up but subsequently tested positive. 2 people refused PrEP with 1 deeming themselves to be low risk. Conclusion(s): Our data highlights several missed opportunities for starting same-day PrEP which potentially may have prevented HIV acquisition. If PrEP is not issued on the day, adequate follow up must be ensured. Reassuringly those who acquired HIV with recent PrEP use have achieved good virological control without NRTI mutations. Counselling on potential side effects, EBD dosing and ongoing HIV risk are essential. Despite NHS PrEP available over 2 years, our data shows we are still failing to meet the demand of PrEP not only in men who have sex with men but also in other key at risk groups.
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Background: In 2018 we reported the emergence of the new HIV-1 recombinant CRF94-02BF2 involved in a large transmission cluster of 49 French MSM mostly infected in 2016-2017. This CRF94 raised concerns of enhanced virulence. Prevention actions were undertaken in the area and population affected. This study reported the molecular and epidemiological evolution of this CRF94 until June 2022. Method(s): In 2021-2022, French sequence databases were screened for patients infected with HIV-1 subtype CRF94 or similar strain. HIV subtyping was confirmed by phylogenetic analysis of genes encoding both protease and reverse transcriptase (1070bps), and integrase (696bps) using IQ-Tree. Five whole genomes, related but distinct from CRF94, were obtained with the DeepChek assay Whole Genome kits. Recombination breakpoints were estimated using RDP4 and SimPlot. Mann-Whitney and LogRank tests were used for statistical analyses to compare patients' characteristics. Result(s): In June 2022, 49 new HIV-1 sequences were collected: 14 clustered with the 49 previous CRF94, 32 formed a new cluster next to but distinct from CRF94, and 3 strains could not be classified. Analysis of 5 whole genomes from the new cluster revealed a new recombinant, the CRF132-94B, mainly consisting of CRF94 which recombined with subtype B in the POL and accessory genes. Vif gene changed from the F2 to the B subtype. Both CRF94 and 132 clusters involved >95% of MSM, mostly infected < 1 year before diagnosis. However, there were differences: 97% were diagnosed in 2013-2019 for CRF94 vs 90% in 2020-2022 for CRF132. At time of diagnosis, 33% of patients infected with CRF94 knew the Prep vs 95% for CRF132. In the cluster CRF94, patients were older (34 vs 30 years, p=0.02), had higher viral loads (5.42 vs 4.42 log10 copies/Ml;p< 0.001), a lower CD4 cell counts (358 vs 508 /mm3, p=0.002). On treatment, the patients with the CRF94 reached viremia < 50 copies/Ml significantly later than those infected with CRF132 (p=0.0002). The prevention activities targeting the CRF94 cluster could explained the few patients infected with this strain after 2018. The CRF132 is mainly located in another Paris region area, but no specific transmission place has been identified. Conclusion(s): After 2019, the CRF94 spread seems greatly slowed down but the very close CRF132-94B has given birth to a new highly active cluster in 2020- 2022, despite the COVID social-distancing and a strong knowledge of the Prep. CRF132 appears to be less virulent perhaps due to the Vif gene change. Identified breakpoints positions of the new HIV-1 CRF132-94B. GenBank accession numbers of the five references : ON901787 to ON901791.
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The COVID-19 outbreak has fashioned to severe threat to each and every individual in social and economic aspects in the country. This required improved wisdom to know how it is different and dominant, to diagnose and determine effective vaccines to avoid the transmission of these deadly causative agents. From this review, the probable property of these deadly transmissible viruses is related to that of SARS-CoV-2 as a fright zone of viruses. It also provides some sparks about effective and accurate diagnosis and treatment strategies. The effective management and control of panic zone of virus (PZV) and SARS-CoV-2 are more important to reduce the pandemic situation.Copyright © 2023 Inderscience Enterprises Ltd.
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IDWeek is the joint annual meeting of the Infectious Diseases Society of America (IDSA), Society for Healthcare Epidemiology of America (SHEA), the HIV Medicine Association (HIVMA), the Pediatric Infectious Diseases Society (PIDS) and the Society of Infectious Diseases Pharmacists (SIDP). For the first time since the COVID-19 public health emergency began, IDWeek 2022 returned to in-person attendance. It was held in Washington, D.C., and the meeting comprised 5 days of live sessions and on-demand content that included posters and oral presentations.Copyright © 2023 Clarivate.
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During the coronavirus pandemic, increasing evidence has confirmed that the SARS-CoV-2 virus is susceptible to increased risk of stroke. On the other hand, the relationship between the SARS-CoV-2 virus and CADASIL was among the topics discussed in the literature with a small number of cases. In this case report, we present multiple cerebral infarcts in an asymptomatic CADASIL patient and we aim to shed light on the complex nature of cerebrovascular manifestations of the SARS-CoV-2 virus. A 50-year-old man with an unremarkable past medical history was admitted to our department with fever and neurologic manifestations on the 6th day of self-isolation due to positive reverse-transcriptase-polymerasechain-reaction assay in a nasopharyngeal sample for SARS-CoV-2. Neurological deficits were related to the acute vascular lesions located in the border-zone areas of both hemispheres, corpus callosum, and cerebellar peduncles on brain MRI. Lesions in chronic nature in the bilateral subcortical white matter predominantly involving the external capsule and temporal poles were also challenging. As a result of a comprehensive study that could explain the neurological status and imaging findings, the CADASIL diagnosis is reached by genetic testing for NOTCH-3. The experience, in this case, suggests considering patients with suspicious MRI findings for CADASIL diagnosis during the coronavirus pandemic. Further studies are needed to explain the underlying pathophysiological mechanisms related to cerebrovascular manifestations of SARS-CoV-2.Copyright © 2022 by Turkish Cerebrovascular Diseases Society.
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The approval of mRNA vaccine technique against COVID-19 opens a door to research and the creation of new drugs against different infectious pathologies or even cancer, since for several diseases the therapeutic options are limited, and different viral diseases are treated only symptomatically. For these reasons, this study proposed a hypothesis supported by biological studies, that it provides a theoretical basis for the possible development of a drug that used the mRNA technique and the ribonucleolytic action of a ribonuclease for a possible antiviral therapy, and analyzed a future perspective of this technique in order to provide a bibliographic basis on this hypothesis and motivate researchers to carry out biological studies on this topic.Copyright © 2022, Health Biotechnology and Biopharma. All rights reserved.
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The emergence of new variants of SARS-CoV-2 associated with varying infectivity, pathogenicity, diagnosis, and effectiveness against treatments challenged the overall management of the COVID-19 pandemic. Wastewater surveillance (WWS), i.e., monitoring COVID-19 infections in communities through detecting viruses in wastewater, was applied to track the emergence and spread of SARS-CoV-2 variants globally. However, there is a lack of comprehensive understanding of the use and effectiveness of WWS for new SARS-CoV-2 variants. Here we systematically reviewed published articles reporting monitoring of different SARS-CoV-2 variants in wastewater by following the PRISMA guidelines and provided the current state of the art of this study area. A total of 80 WWS studies were found that reported different monitoring variants of SARS-CoV-2 until November 2022. Most of these studies (66 out of the total 80, 82.5%) were conducted in Europe and North America, i.e., resource-rich countries. There was a high variation in WWS sampling strategy around the world, with composite sampling (50/66 total studies, 76%) as the primary method in resource-rich countries. In contrast, grab sampling was more common (8/14 total studies, 57%) in resource-limited countries. Among detection methods, the reverse transcriptase polymerase chain reaction (RT-PCR)-based sequencing method and quantitative RT-PCR method were commonly used for monitoring SARS-CoV-2 variants in wastewater. Among different variants, the B1.1.7 (Alpha) variant that appeared earlier in the pandemic was the most reported (48/80 total studies), followed by B.1.617.2 (Delta), B.1.351 (Beta), P.1 (Gamma), and others in wastewater. All variants reported in WWS studies followed the same pattern as the clinical reporting within the same timeline, demonstrating that WWS tracked all variants in a timely way when the variants emerged. Thus, wastewater monitoring may be utilized to identify the presence or absence of SARS-CoV-2 and follow the development and transmission of existing and emerging variants. Routine wastewater monitoring is a powerful infectious disease surveillance tool when implemented globally.
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The development of the polymerase chain reaction (PCR), for which Kary Mullis received the 1992 Novel Prize in Chemistry, revolutionized molecular biology. At around the time that prize was awarded, research was being carried out by Russel Higuchi which led to the discovery that PCR can be monitored using fluorescent probes, facilitating quantitative real-time PCR (qPCR). In addition, the earlier discovery of reverse transcriptase (in 1970) laid the groundwork for the development of RT-PCR (used in molecular cloning). The latter can be coupled to qPCR, termed RT-qPCR, allowing analysis of gene expression through messenger RNA (mRNA) quantitation. These techniques and their applications have transformed life science research and clinical diagnosis.Copyright © The Authors.
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The secondary metabolites produced by the plant are used for growth, development and protection of plants against animals. The metabolites produced by plants are used to treat many diseases. In this research performed the docking study of secondary metabolites of plant products against infectious diseases like Covid-19, HIV and Tuberculosis. All the plant constituents inhibiting the selected target. The following plant constituents inhibiting the target by binding with the target at low energy. The binding energy of Nimbin found to be - 169.44 Varicella zoster virus protease, the binding energy of curcumin found to be -135.3 Covid-19 main protease and the binding energy of Thalassiolin A found to be -131.17 against Reverse transcriptase. The present work focused on the bioactive phytochemicals against infectious diseases with their molecular docking study.
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912 Table 1Association of demographic and clinical features with serology status of SARS-CoV-2Results88 pediatric patients up to the age of 18 years attending the pediatric department at AIIMS Patna were enrolled for the study. Only two patients had history of positive RT-PCR test for COVID-19 infection in the past. 63.6% (56 out of 88) had seropositive status against SARS-Cov-2. Various demographic and clinical variables described in table 1 were analysed and none of the demographic features had statistically significant association with serology status of SARS-CoV-2. Out of 88 children, 57 (64.8%) were males and 31(35.2%) were females. 58% of the children were from urban areas and 42% were from rural areas. The majority of the patients i.e 58 (65.9%) belonged to lower socioeconomic class and 30 (34.0%) belonged to upper class according to modified Kuppuswamy scale 2021. The corticosteroid therapy was received by 13 patients for various clinical indications among which 5 (38%) had seropositive status and 8(61.5%) had seronegative status against SARS-CoV-2 and the association was statistically significant with p-value of 0.041and Odd’s ratio ( 95% CI) of 0.29 (0.087-0.994) suggesting that patients who received corticosteroid therapy had 29% lesser chances of getting seropositive status compared to those who did not receive the therapy.ConclusionAmong the participants, 63.6% were seropositive against SARS-CoV-2 while only 2.2% had history of COVID 19 RTPCR positivity in past. The patients who received corticosteroids had lesser chances of getting positive antibody status against SARS-CoV-2 infection compared to those who did not receive the same.
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The authors correctly stated that the Centers for Disease Control and Prevention (CDC) performed testing for SARS-CoV-2 and found no evidence of SARS-CoV-2 infection in autopsy tissues from the decedents. Molecular analysis included polymerase chain reaction (PCR) assays on nucleic acid extracted from FFPE heart tissue, including SARS-CoV-2 and enterovirus reverse transcriptase PCR (RT-PCR) assays2,3 and conventional PCR for parvovirus B19. Clostridium septicum produces multiple toxins that cause necrosis of striated muscle cells9,11 and inhibit influx of neutrophils to infected tissues;indeed, paucity of neutrophilic infiltrates in tissues infected with C septicum is considered a hallmark of this disease.9,12 Clostridium septicum is not considered normal flora of the human intestinal tract,13,14 but rather an opportunistic invader of immunologically compromised hosts, particularly persons with colonic adenocarcinoma, leukemia, diabetes, bowel ischemia, or cyclic, congenital, or acquired neutropenia.7,8 Spontaneous infections have been described for a few pediatric patients with no recognized risk factor and for whom microscopic breaches in the mucosa of the large intestine were considered the likely portal of entry.8,15 No representative samples of the small or large intestine were provided to the IDPB for evaluation;however, histologic evidence of bacterial invasion of the external surfaces of the adrenals, kidneys, liver, and spleen support an intraabdominal source of infection. The findings and conclusions in this letter are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. doi: 10.5858/arpa.2022-0084-LE In Reply.-We thank the Centers for Disease Control and Prevention's (CDC's) Infectious Diseases Pathology Branch laboratory for performing these tests and for sharing the full extent of its workup.
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In January 2020, Guangdong Province, China imported several suspected cases with SARS-CoV-2 from Wuhan City, Hubei Province. China, which were detected as SARS-CoV-2 positive in laboratory. To further understand the SARS-CoV-2 virulence, as well as drug development and epidemic prevention and control needs, we established a SARS-CoV-2 isolation procedure. Vero-E6 cells were infected with the positive bronchoalveolar-lavage sample. The cells were monitored daily for cytopathic effects using light microscopy. The presence of viral nucleic acid in the supernatant was detected by RT-PCR. RNA extracted from culture supernatants were used as a template to clone and sequence the genome. We used Illumina sequencing to characterize the virus genome and results showed that the isolated virus was SARS-CoV-2.
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Introduction: Rapid cost effective, Point-of-Care (PoC) Truenat assay for the diagnosis of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) have been developed to shorten the Turn Around Time (TAT) of reporting with a wireless data transfer system. Aim: To explore the SARS-CoV-2 positivity using closed system Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Materials and Methods: An observational cross-sectional study was carried out in Molecular Laboratory of, Department of Microbiology, Jawaharlal Nehru Institute of Medical Sciences, Imphal, Manipur, India, using Truenat RT-PCR (Molbio diagnostics) and data was entered from May 2020 to April 2021. Manufacturer's literature was followed while performing the test. Screening of sample was done with Envelope (E) gene test and confirmed with RNA-dependent RNA polymerase gene (RdRP) gene test. Statistical analysis was done using Microsoft Excel sheet by calculating the percentage, proportions. Results: A total of 1,528 individuals were tested for SARS-Cov-2 and 73 tests were reported positive. The positivity rate by age was highest among 21-30 years. The positivity rate was higher among males than females. Among 1,105 asymptomatic individual, 27 (2.4%) were positive and among 423 symptomatic, 46 (10.9%) were positive. Conclusion: Using Truenat, positivity rate among symptomatic Coronavirus Disease 2019 (COVID-19) suspected persons was about four times more than positivity rate among exposed contact persons who are asymptomatic.
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Background-aim: World Health Organization (WHO) announced that diagnostic testing for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV2) should be performed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Most of these methods use different gene props and therefore the sensitivity and specificity of each method may different. In this study, we have compared two RT-PCR methods using two different genes for detection SARS-COV2. Methods: A total of random 40 nasopharyngeal swab samples were collected, transported and received in iced box shipment. All samples were performed on two separate semi-automated PCR systems (Qiagen and Abbott m2000). For Qiagen method, 200uL from each sample were added in 96-well QiAcube plate which loaded in QiAcube HT (SN:019658;Qiagen, Germany) to extract RNA. Following extraction, master mix prepared using SARS-COV2-RT-PCR kit 1.0 (REF: 821005;Altona, Germany) for 44 samples including 40 patient samples, two negative controls using nuclease-free water (with and without internal control), and two positive controls (with and without internal control). Extraction elute of each sample (20uL) added to master mix (10uL) to have a total volume of 30uL which uploaded into Rotor-Gene Q (SN:R0219307;Qiagen, Germany). The primer pair used to amplify S gene and E gene in SARS-COV2. Amplifications were done as follow: reverse transcriptase (20 minutes at 55oC);initial denaturation (2 minutes at 95oC);45 cycles of denaturation (15 seconds at 95oC), annealing for (45 seconds at 55oC), and extension (15 seconds at 72oC). Results reported as valid for internal control less than 35 cycle threshold (CT). The Abbott m2000 System uses SARS-CoV-2 assay was a dual target assay for the RdRp and N genes. All 40 samples were extracted using m2000sp (Abbott, United States) as recommend by manufacture using 100uL. An RNA sequence that was unrelated to the SARS-CoV-2 target sequence was introduced into each specimen at the beginning of sample preparation. This unrelated RNA sequence was simultaneously amplified by RT-PCR and serves as an internal control (IC) to demonstrate that the process has proceeded correctly for each sample. Following extraction, master mix (20uL) added to extraction elute of each sample (30uL) to have a total volume of 50uL which uploaded into m2000rp (Abbott, United States). Amplification were done as follow: reverse transcriptase (25 minutes at 55oC);initial denaturation (5 minutes at 94oC);40 cycles of denaturation (20 seconds at 94oC), annealing for (55 seconds at 55oC), and extension (15 seconds at 72oC). Result: All samples had valid extraction process with a CT value of internal control between 26.97 to 28.89. A total of 30 samples displayed positive results and 10 samples exhibited negative results with 100% agreement for both methods. This has resulted with a 100% accuracy between both methods. Conclusions: Both semi-automated methods from Qiagen and Abbott are comparable and accurate despite different technology and different primer genes.
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Background: Knowing the true incidence of HIV-1 infections (recent infections) among people newly diagnosed is pivotal to monitoring the course of the epidemic. We have developed a Primer ID Next Gen Sequencing (PID-NGS) assay to identify recent infection by measuring within-host viral diversity over multiple regions of the HIV-1 genome. We implemented a state-wide project to identify recent infections and transmitted drug resistance mutations (DRMs) in diagnostic samples in near real time. Methods: Serum samples from individuals with newly HIV-1 diagnoses (diagnostic sample collected within 30 days of diagnosis) were sequenced. PID-NGS libraries were constructed covering the coding regions for protease, a portion of reverse transcriptase, integrase, and the env gene. The use of the PID-NGS strategy allows for significant error correction and also a definition of the sampling depth of the viral population. Recent infection was defined as within 9-month of infection. DRMs were summarized at detection sensitivities of 30%, 10% and 1% based on viral population sampling depth. Results: From Jan 2018 to Jun 2021, we successfully sequenced partial genomes from 743 individuals with new diagnoses. Year 2020 had the lowest number of new diagnoses (Fig 1a, red bar). Overall, 39.2% of samples were inferred to have represented infection within the previous 9 months. Percent of recent infection varied significantly over the years, increasing from 29.6% in late 2018 to 50.9% in early 2020, but decreasing significantly to 32.7% in 2021 (Fig 1a, blue lines). Individuals younger than 30 y/o were more likely to be identified with recent infection (p<0.01). NNRTI DRMs, especially K103N, were the most abundant DRMs. Fig 1b shows the trend of DRMs over the four years. We observed a trend of decrease in the overall NNRTI DRMs and an increase in the NRTI DRMs in the population. Further analysis suggests that the increase in NRTI DRMs were from TAMs and their revertants, while clinically important NRTI DRMs (K65R and M184) were low (<1%). Conclusion: We have demonstrated a state-wide, all-in-one platform to monitor HIV-1 recency and DRMs in new diagnoses. The number of new diagnoses decreased significantly in 2020 in concert with the COVID-19 pandemic which suggests a decrease in overall HIV testing. The decline in the percentage of recent infections in early 2021 signals a return to broader HIV-1 testing and diagnosis. The increase of other NRTI DRMs suggests ongoing evolution at these sites within the viral population.
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BackgroundThis study aimed to compare the testing strategies for COVID‐19 (i.e., individual, simple pooling, and matrix pooling) in terms of cost.MethodsWe simulated the total expenditures of each testing strategy for running 10,000 tests. Three parameters were used: positive rate (PR), pool size, and test cost. We compared the total testing costs under two hypothetical scenarios in South Korea. We also simulated country‐specific circumstances in India, South Africa, South Korea, the UK, and the USA.ResultsAt extreme PRs of 0.01% and 10%, simple pooling was the most economic option and resulted in cost reductions of 98.0% (pool size ≥80) and 36.7% (pool size = 3), respectively. At moderate PRs of 0.1%, 1%, 2%, and 5%, the matrix pooling strategy was the most economic option and resulted in cost reductions of 97.0% (pool size ≥88), 86.1% (pool size = 22), 77.9% (pool size = 14), and 59.2% (pool size = 7), respectively. In both hypothetical scenarios of South Korea, simple pooling costs less than matrix pooling. However, the preferable options for achieving cost savings differed depending on each country's cost per test and PRs.ConclusionsBoth pooling strategies resulted in notable cost reductions compared with individual testing in most scenarios pertinent to real‐life situations. The appropriate type of testing strategy should be chosen by considering the PR of COVID‐19 in the community and the test cost while using an appropriate pooling size such as five specimens.
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In the light of the recent events in the world concerning COVID-19 virus, it is important to review the challenges faced by the world by another pandemic, AIDS. The painstaking research by the scientists, the pharmaceutical companies, the medical professionals have led to this day when AIDS patients are living their whole life span. Though we do not have any vaccine for AIDS but by intelligent use of medication, we have been able to combat the disease to a large extent. HIV is a RNA virus, whose treatment is mainly done by finding the structure and function of the proteins that are vital to its life cycle. Designing a drug/inhibitor to make those proteins ineffective constitutes the next step. WHO has recognized AIDS as a pandemic almost 40 years back but the world is yet to find a cure or a vaccine. The current treatment method is called HAART, Highly Active Anti Retroviral Therapy, where different types of inhibitors,eg. Reverse Transcriptase inhibitors, Protease inhibitors;each arresting a different important protein are given in combination. The virus replicates very fast and forms mutations which render it ineffective to the inhibitors thus resistance to the inhibitors develop. Hence development of new types of inhibitors is crucial to the problem. There are certain similarities between AIDS and COVID-19, both in terms of the attacking virus and effective medication, which make it more important than ever that the research on HIV is revisited and knowledge we gain from it is used to battle the new pandemic.
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The present invention describes a method for Tusing a bacterial CRISPR Cas Ribonucleoprotein complex for detecting single nucleotide variants in RNA or DNA or more broadly, any DNA or RNA fragment, without the need for sequencing. The principle ofdiscrimination is derived from the natural property of the enzyme being used for the invention, Francisellanovicida Cas9 (FnCas9) which shows very low binding affinity to mismatched substrates. DNA is isolated either from blood, saliva, or any other biological sources like bacteria and amplified if required. For virus infected patients, samples are collected as a nasal swab and inactivated. Total RNA isolated from the sample is converted to cDNA using the reverse transcriptase enzyme. The DNA (when test material is DNA) or cDNA (when test material is RNA, like for COVID-19) is subjected to Polymerase Chain reaction, amplifying using specific primers and tagging the amplified DNA products with a ligand of choice. The detection mix consists of labelled PCR products, sgRNA-fnCAS9 complex. The detection complex can be visualized using a wide array of technologies like lateral flow, gel based cleavage assay, fluorescence based detection, in both low, medium or plate based high-throughput format. Science behind this technology will be discussed in the presentation.