Felecia Garrett – Page 2 – International Epidemiologic Databases to Evaluate AIDS (IeDEA)

How To Stabilize Oral Microbiome Samples

July 15, 2020 by Felecia Garrett

The study of the human microbiome is having an increasing presence and impact on biomedical research projects. Although one of the best described microbiota to date is the intestinal microbiome , which has captured the attention of the scientific community due, among other things, to its high cell density, we must not lose sight of the interest that the study of other microbiota of clinical interest such as nasal, oral, urogenital and / or cutaneous.

In this post we will focus on analyzing the options to stabilize samples of the oral microbiome , which represents approximately 26% of the total human microbiome, and which is attracting the interest of a growing number of researchers in the field of cell biology, microbiology and immunology due to the increasingly evident contribution of this microbiome to states of health and disease not only at the level of the oral cavity (gingivitis, periodontitis …), but also at the systemic level in brain, liver and lung diseases.

The Importance Of Stabilizing Oral Microbiome Samples

Once the microbiome sample is collected, it is essential to proceed to its immediate stabilization to avoid overgrowth and / or degradation of certain populations, and thus guarantee that the result of the subsequent analysis is a faithful reflection of the state and real composition in vivo at the time of collection, ensuring that the obtained microbiological profile represents the phenotype of interest.

Given that the cell cycle of many microorganisms can be around 40 minutes, unless there is a reliable method to stabilize samples of the oral microbiome at the time of collection, the reproducibility and comparability of the microbial profiles between different collection sites or between different studies, it would be practically impossible.

Traditionally, these samples are usually refrigerated or even frozen in order to avoid bacterial growth and degradation, but the truth is that this practice hinders the logistics of the studies and makes the processes more expensive, having to pay maximum attention to maintaining the cold chain. from the collection, transportation, storage and processing of samples.

In order to avoid the need to be subject to the maintenance of this cold chain, and to optimize the quality of the samples, the Canadian DNA Genotek has developed a new battery of kits that allow the collection and stabilization of oral microbiome samples at room temperature during extended periods of time.

Features And Benefits Of Kits To Stabilize Oral Microbiome Samples

By obtaining reliable profiles of the microbiota at the time of collection, they minimize noise in your data analysis.
They improve patient / donor adherence through an intuitive and friendly collection method.
Immediate stabilization of the sample minimizes microbial growth and DNA degradation.
Eliminates the costs associated with the cold chain in shipping and warehousing.
Optimize sample handling.
Maintains the integrity of nucleic acids despite fluctuations in ambient temperature.

Below we summarize the different formats of the kits to stabilize oral microbiome samples and their respective characteristics:

All of these kits eliminate the need for cold chain shipping and warehousing, are tailored for high-throughput processes, and are suitable for NGS applications.

In short, the study of the oral microbiome requires the correct collection and stabilization of the samples to avoid bacterial overgrowth and DNA degradation, guaranteeing the subsequent obtaining of reliable results. Maintaining a cold chain during sample transport and storage can result in additional costs and difficulties in handling the samples, making kits that allow sample collection and stabilization at room temperature a great alternative. booming among researchers.

Isolation and Immunological Detection of Mycobacterium Tuberculosis from HIV and Non-HIV Patients in Benue State, Nigeria.

May 22, 2020 by Felecia Garrett

Immunological methods are essential instruments for tuberculosis epidemiology; though its use is underutilized in Nigeria. In this research, we report the epidemiological outlook of Mycobacterium tuberculosis amongst HIV sufferers in Benue State, Nigeria.

Sputum samples had been collected from 425 suspected TB sufferers from July 2016 to February 2018 and subjected to acid-fast microscopy, GeneXpert MTB/RIF, processed utilizing NALC-NaOH and cultured on Lowenstein-Jensen media. The isolates obtained had been recognized by SD-Bioline® assay.

ResultsThe prevalence of TB by acid-fast microscopy was 35(15.9%). The prevalence of TB by acid-fast bacilli was considerably (χ² = 8.458; P = 0.003) highest among the many 15-34 years age group (22.0%) in contrast with different age teams. TB prevalence was considerably (χ² = 4.751; P = 0.029) greater amongst sufferers from rural areas than these from city middle (23.8% vs 14.1%).

GeneXpert assay detected 64(15.1%) TB circumstances of which sufferers from rural areas had considerably (χ² = 8.104; P = 0.017) greater prevalence of TB than sufferers from city areas (23.8% vs 12.9%). The total rifampicin resistance TB was 3.1%. Also, sufferers from rural areas had considerably (χ² = 10.625; P = 0.005) greater rifampicin resistance in contrast with affected person from city areas (8.3% vs 1.3%). Of the 126(29.7%) mycobacterial isolates, 42(33.33%) had been recognized as MTBC and 84 (66.67%) as NTM by SD-Bioline® assay.

The research revealed that Mycobacterium tuberculosis an infection remains to be a significant public well being drawback, with comparatively excessive prevalence charge of rifampicin resistance amongst HIV optimistic sufferers. Further research are wanted for early detection and remedy intervention needed for an infection management.

Fulminant central nervous system varicella-zoster virus an infection unexpectedly recognized by metagenomic next-generation sequencing in an HIV-infected affected person: a case report.

Varicella-zoster virus (VZV) an infection might be recognized clinically as soon as classical rash happens however the prognosis is difficult when typical rash is absent. We reported a case of fulminant central nervous system (CNS) VZV an infection in a human immunodeficiency virus (HIV)-infected affected person with out typical VZV-related rash. CNS VZV an infection was surprising recognized by metagenomic next-generation sequencing (mNGS).A 28-year-old HIV-infected affected person introduced with neurological signs for Three days.

The affected person, who was not suspected of VZV an infection at admission, rapidly progressed to deep coma throughout the first 24 h of hospitalization. An unbiased mNGS was carried out on DNA extract from 300 μL cerebrospinal fluid (CSF) with the BGISEQ-50 platform.

The sequencing detection recognized 97,248 (out of 38,561,967) sequence reads uniquely aligned to the VZV genome, and these reads coated a excessive share (99.91%) of the VZV. Presence of VZV DNA in CSF was additional verified by VZV-specific polymerase chain response and Sanger sequencing.

Altogether, these outcomes confirmed CNS VZV an infection.This research means that mNGS could also be a helpful diagnostic device for CNS VZV an infection. As mNGS might establish all pathogens instantly from CSF pattern in a single run, it has the promise of strengthening our capacity to diagnose CNS infections in HIV-infected sufferers.

A Pilot Study of the Humoral Response Against the AntiSense Protein (ASP) in HIV-1-Infected Patients.

May 22, 2020 by Felecia Garrett

The existence of an antisense Open Reading Frame (ORF) that encodes a putative AntiSense Protein (ASP) on the proviral genome of Human Immunodeficiency Virus type 1 (HIV-1) was a source of debate for 30 years.

During the last years, some progresses have been made to characterize the cellular immune response against ASP in HIV-1 seropositive patients. However, no tools were available for the detection of antibodies to ASP in the plasma of HIV-1-infected patients during the natural course of the infection.

The aim of our study was to develop a Luciferase Immuno-Precipitation System (LIPS) to monitor the quantitative detection of ASP-specific antibodies in the plasma of HIV-1-infected patients [antiretroviral therapy (ART) naive-patients, patients under ART and HIV-1 controllers], patients who discontinued antiretroviral drugs (ARV). We further used this approach to delineate the epitopes of ASP targeted by antibodies.

Antibodies directed against ASP were detected in 3 out of 19 patients who discontinued ARV (15%) and in 1 out of 10 ART-naive patients (10%), but were neither detected in HIV-1 infected patients under ART nor in HIV-1 controllers. Individual variations in levels of ASP-specific antibodies were detected overtime.

Both the conserved prolin-rich motif and the core 60-189 region of ASP were found to be essential for antibody recognition in the four patients tested positive for anti-ASP antibodies, who were all untreated at the time of sampling.

Moreover, for two of these patients, increased levels of ASP-specific antibodies were observed concomitantly to viremia declines. Overall, our method may represent a useful tool to detect a humoral response to ASP in HIV-1-infected patients, which allowed us to confirm the expression of ASP during the course of HIV-1 infection. Further studies will be needed to fully characterize the humoral response to ASP in HIV-1-infected patients.

A Pilot Study of the Humoral Response Against the AntiSense Protein (ASP) in HIV-1-Infected Patients.

Comparison of two Immunoassays for Concurrent Detection of HCV Antigen and Antibodies among HIV/HCV Co-infected Patients in Dried Serum/Plasma Spots.

Hepatitis C virus (HCV) antigen/antibody (Ag/Ab) assays offer the benefit of reducing the window period compared to assays that detect only HCV-Ab. In this study the performance of the Murex Ag/Ab (Murex, Abbott) and Monolisa Ag/Ab Ultra (Monolisa, Bio-Rad) ELISAs was compared for the use of filter dried serum/plasma spots (DS/PS) with a focus on the sensitivity and the percentage of correct positive test results.

Correct positive ELISA results were assumed for samples that subsequently tested positive for HCV RNA by RT-qPCR, or RNA negative samples that tested positive in a Western blot (confirmed ELISA results).

Sensitivity was evaluated from DS/PS eluates using HCV seroconversion panels [plasma samples of subtypes-(St) 1a, 2b)] and longitudinal HCV antibody-positive serum panels (St 1b, 2b, 3a, and 4d). The proportion of correct positive test results was evaluated using 1102 newly-diagnosed HIV-positive clinical dried serum spots DSS eluates for screening of potential HCV co-infection.

For the plasma HCV seroconversion samples, which were used as a reference for DSS eluates, the Murex became reactive earlier for antigen-positive bleeds. However, for the HCV antibody-positive eluates and dilutions thereof, the Monolisa demonstrated a superior sensitivity.

Of the clinical DSS 22.8% (28/123) of samples reactive in the Murex were negative in a subsequent RT-qPCR and Western blot, while only 1.9% (2/105) of the samples reactive in the Monolisa were negative in these confirmatory assays. Our results indicate that the Monolisa provides fewer false positive results for HCV detection in DSS, whereas for undiluted plasma or serum samples, the Murex can serve as an additional diagnostic tool to narrow the window period.

Burden of tuberculosis and challenges related to screening and diagnosis in Ethiopia.

May 22, 2020 by Felecia Garrett

One-third of tuberculosis (TB) circumstances in Ethiopia are lacking from look after causes that aren’t properly studied. The goal of this examine was to assess TB burden and establish challenges related to TB screening and diagnosis in Ethiopia.

A facility-based cross-sectional examine was carried out in seven well being services chosen from two areas and 2 metropolis administrations of Ethiopia utilizing stratified random sampling procedures.

The information of 1,059,065 sufferers have been included from outpatient division, HIV clinic, diabetic, and maternal-child well being clinics. Data have been collected from October to December 2018 utilizing a retrospective assessment of three years’ facility information (2015 to 2017) supplemented by a semi-structured interview with purposively chosen well being care employees and heads of the well being services.

ResultsA complete of 1,059,065 sufferers visited the well being services in three years, of these, 978,480 (92.4%) have been outpatients. Of the entire, 20,284 (2%) have been presumptive TB circumstances (with 14 days or extra cough), 12.2% (2483/20,284) of which had TB. For the kind of TB, 604 (24.3%) have been smear-positive pulmonary TB (PTB), 789 (31.8%) have been smear-negative PTB, 719 (29%) have been extra-pulmonary TB, and information have been lacking for the remaining.

TB screening was built-in into HIV clinic, outpatient division, diabetic clinic however not with the maternal and little one clinics. High affected person load, weak TB laboratory specimen referral system, and scarcity of TB diagnostic instruments together with Xpert MTB/RIF assay and chest X-ray, have been the most important challenges in the screening and diagnosis of TB.

The burden of TB was excessive in the examine setting, and frequent interruption of laboratory reagents and provides hampered TB screening and diagnostic companies. Realizing the END-TB technique in such resource-limited settings requires sustainable TB diagnostic capability and improved case detection mechanisms, with nationwide TB applications strongly built-in into the final well being care system.

Remodeling of the core leads HIV-1 pre-integration complicated in the nucleus of human lymphocytes.

Retroviral replication proceeds by obligate integration of the viral DNA into the host genome. In explicit, HIV-1 genome to enter the nucleus, should be led by the nuclear pore complicated (NPC). During HIV-1 cytoplasmic journey, the viral core acts like a shell to defend the viral genetic materials from antiviral sensors and guarantee an ample setting for the reverse transcription.

However, the comparatively slim measurement of the nuclear pore channel requires that the HIV-1 core reshapes right into a construction that matches the pore. On the opposite hand, the group of the viral CA proteins that stay related to the pre-integration complicated (PIC) throughout and after nuclear translocation remains to be enigmatic.

In this examine, we analysed the progressive organizational modifications of viral CA proteins inside the cytoplasm and the nucleus by immuno-gold labelling. Furthermore, we arrange a novel know-how, HIV-1 ANCHOR, which allows the precise detection of the retrotranscribed DNA by fluorescence microscopy, thereby providing the chance to uncover the structure of the potential HIV-1 PIC.

Thus, we mixed the immunoelectron microscopy and ANCHOR applied sciences to reveal the presence of DNA- and CA-positive complexes by correlated light- and electron microscopy (CLEM).

During and after nuclear translocation, HIV-1 seems as a posh of viral DNA adorned by a number of viral CA proteins remodelled in a “pearl necklace” form. Thus, we might describe how CA proteins reshape across the viral DNA to allow the doorway of the HIV-1 in the nucleus.

This explicit CA protein complicated composed by the integrase and the retrotranscribed DNA leads HIV-1 genome contained in the host nucleus.Our findings contribute to the understanding of the early steps of HIV-1 an infection and present new insights into the group of HIV-1 CA proteins throughout and after viral nuclear entry. Of notice, we at the moment are ready to visualize the viral DNA in viral complexes, opening up new views for future research on viral nuclear destiny.

CRISPR/Cas systems for editing genes in HIV treatment Research

March 13, 2020 by Felecia Garrett

What are the riscks in using gene therapy are we close to approval of such treatments?

The main concern is that gene therapy might create a Frankenstein cell, they would hit different genes rather than the intended goal, so it is great that this did not occur.China appears to be moving fast on such research and may get remedies approved earlier than the United States, June explained. He’s financial ties to some gene therapy companies and is leading another study testing CRISPR to fight cancer in the united states. Three patients have been treated up to now and some results are expected at the end of this season.

What is CRISPR/Cas9 system ?

CRISPR -clustered regularly interspaced short palindromic repeats is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences have been derived from DNA fragments of bacteriophages that had formerly infected the prokaryote.CRISPR works in coordination with CRISPR associated (CAS) genes, enabling prokaryotic adaptive immune systems to comprehend, react to, and remove foreign DNA. CRISPR/Cas chemical editing is called a molecular scissors since it essentially cuts the foreign DNA (plasmid and bacteriophage) from the prokaryotic genome. Analogies of CRISPR/Cas gene editing in prokaryotes are created to RNA interference or receptor found in eukaryotic organisms

Could CRISPR/Cas system be used for inactivation of HIV infection?

Scientists have made RNA guides to guide Cas9 to cleave unique areas of HIV viral DNA comprising essential genes along with the long terminal repeat.3 CRISPR/Cas9 gene editing led to significant reduction of HIV viral generation and disease in various research mobile versions, such as human pluripotent stem cells, primary CD4+ T cells, and CD4+ T cell lines. Theoretically, the removal of HIV viral DNA or enzymes necessary for viral expression in infected cells need to have the ability to inactivate or eliminate HIV disease.

Regrettably, researchers also revealed cells developed CRISPR/Cas9 resistance mutations in certain experiments. All these mutations clustered in the viral website once the Cas9 was led . Cas9 wasn’t able to cleave the targeted website and thus the CRISPR/CAS9 strikes were unsuccessful.HIV, like other viruses, was known to possess a well-developed capability to evolve immunity to other assaulting mechanisms like the human immune system and anti inflammatory drugs.

CRISPR/CAS9 HIV mechanism — **CRISPR/CAS9 mode of action in HIV infected cells**

What is the most successful strategy for gene therapy of HIV, so far?

Combating HIV-1 disease, CRISPR/Cas9 was used to ablate chemokine receptor type 5 (CCR5) expression. CCR5 is a co-receptor and necessary for specific HIV types to get entrance into T-cells and lead to HIV disease. The mechanics of CCR5-ZFN relies on genetically engineered alterations of CCR5 that make CCR5 non-functional and tissues resistant to HIV infection. What is yet to be found is whether CCR5-ZFN/SB-728-T could replicate itself to further resistant cells and may restore immune cell functioning in infected hosts by preventing HIV entry into cells.