Point-of-care platform integrated with deep-learning, convolutional neural network algorithms effectively evaluates canine and feline peripheral blood smears.

Objective: To perform a diagnostic assessment of a point-of-care veterinary multiuse platform integrated with a model comprised of deep-learning, convolutional neural network algorithms for evaluating canine/feline peripheral blood smears compared to board-certified clinical pathologists (CPs).

Methods: This study had a blinded, randomized, incomplete block design, and results were compared between CPs and algorithms. Blood smears from convenience samples from veterinary diagnostic reference laboratories from October to December 2021 were used.

Multicenter evaluation of the Vetscan Imagyst system using Ocus 40 and EasyScan One scanners to detect gastrointestinal parasites in feces of dogs and cats.

The Vetscan Imagyst system (Zoetis) is a novel, artificial intelligence-driven detection tool that can assist veterinarians in the identification of enteric parasites in dogs and cats. This system consists of a sample preparation device, an automated digital microscope scanner, and a deep-learning algorithm. The EasyScan One scanner (Motic) has had good diagnostic performance compared with manual examinations by experts; however, there are drawbacks when used in veterinary practices in which space for equipment is often limited.

Further evaluation and validation of the VETSCAN IMAGYST: in-clinic feline and canine fecal parasite detection system integrated with a deep learning algorithm.

Background: Fecal examinations in pet cats and dogs are key components of routine veterinary practice; however, their accuracy is influenced by diagnostic methodologies and the experience level of personnel performing the tests. The VETSCAN IMAGYST system was developed to provide simpler and easier fecal examinations which are less influenced by examiners' skills. This system consists of three components: a sample preparation device, an automated microscope scanner, and analysis software.

Evaluation of the VETSCAN IMAGYST: an in-clinic canine and feline fecal parasite detection system integrated with a deep learning algorithm.

Background: Fecal examination is an important component of routine companion animal wellness exams. Sensitivity and specificity of fecal examinations, however, are influenced by sample preparation methodologies and the level of training and experience of personnel who read fecal slides. The VETSCAN IMAGYST system consists of three components: a sample preparation device, a commercially available scanner, and an analysis software.

RNA Sequencing of the Human Herpesvirus 6B Transcriptome To Identify Targets for Clinical Assays Distinguishing between Latent and Active Infections.

Human herpesvirus 6B (HHV-6B) DNA is frequently detected in human samples. Diagnostic assays distinguishing HHV-6B reactivation from latency are limited. This has impaired strategies to diagnose and treat HHV-6B-associated diseases.

Comparative genomic, transcriptomic, and proteomic reannotation of human herpesvirus 6.

Background: Human herpesvirus-6A and -6B (HHV-6) are betaherpesviruses that reach > 90% seroprevalence in the adult population. Unique among human herpesviruses, HHV-6 can integrate into the subtelomeric regions of human chromosomes; when this occurs in germ line cells it causes a condition called inherited chromosomally integrated HHV-6 (iciHHV-6). Only two complete genomes are available for replicating HHV-6B, leading to numerous conflicting annotations and little known about the global genomic diversity of this ubiquitous virus.

Outcomes of hematopoietic cell transplantation using donors or recipients with inherited chromosomally integrated HHV-6.

Human herpesvirus 6 (HHV-6) species have a unique ability to integrate into chromosomal telomeres. Mendelian inheritance via gametocyte integration results in HHV-6 in every nucleated cell. The epidemiology and clinical effect of inherited chromosomally integrated HHV-6 (iciHHV-6) in hematopoietic cell transplant (HCT) recipients is unclear.

Cell Culture Systems To Study Human Herpesvirus 6A/B Chromosomal Integration.

Human herpesviruses 6A/B (HHV-6A/B) can integrate their viral genomes in the telomeres of human chromosomes. The viral and cellular factors contributing to HHV-6A/B integration remain largely unknown, mostly due to the lack of efficient and reproducible cell culture models to study HHV-6A/B integration. In this study, we characterized the HHV-6A/B integration efficiencies in several human cell lines using two different approaches.

Superiority of Digital Reverse Transcription-PCR (RT-PCR) over Real-Time RT-PCR for Quantitation of Highly Divergent Human Rhinoviruses.

Human rhinoviruses (HRV) comprise 3 species representing more than 150 genotypes. As an important human respiratory pathogen, molecular detection is an indispensable tool for diagnosis and surveillance. However, the sequence diversity of HRV genotypes poses challenges for developing robust molecular methods that detect all genotypes with equal efficiencies.

Efficient identification of inherited chromosomally integrated human herpesvirus 6 using specimen pooling.

Background: Human herpesvirus 6 (HHV-6) has a unique ability to integrate into chromosomal telomeres. Vertical transmission via germ cell integration results in offspring with inherited chromosomally integrated (ci)HHV-6 in all nucleated cells, affecting ∼1% of the population.

Objectives: Inherited ciHHV-6 may be a direct or indirect mediator of human disease, but efficient identification of affected individuals is a fundamental roadblock to larger studies exploring the clinical importance of this condition.

Detection of Human Herpesvirus 6B (HHV-6B) Reactivation in Hematopoietic Cell Transplant Recipients with Inherited Chromosomally Integrated HHV-6A by Droplet Digital PCR.

The presence of inherited chromosomally integrated human herpesvirus 6 (ciHHV-6) in hematopoietic cell transplant (HCT) donors or recipients confounds molecular testing for HHV-6 reactivation, which occurs in 30 to 50% of transplants. Here we describe a multiplex droplet digital PCR clinical diagnostic assay that concurrently distinguishes between HHV-6 species (A or B) and identifies inherited ciHHV-6. By applying this assay to recipient post-HCT plasma and serum samples, we demonstrated reactivation of HHV-6B in 25% (4/16 recipients) of HCT recipients with donor- or recipient-derived inherited ciHHV-6A, underscoring the need for diagnostic testing for HHV-6 infection even in the presence of ciHHV-6.

Digital detection of endonuclease mediated gene disruption in the HIV provirus.

Genome editing by designer nucleases is a rapidly evolving technology utilized in a highly diverse set of research fields. Among all fields, the T7 endonuclease mismatch cleavage assay, or Surveyor assay, is the most commonly used tool to assess genomic editing by designer nucleases. This assay, while relatively easy to perform, provides only a semi-quantitative measure of mutation efficiency that lacks sensitivity and accuracy.

Past, present, and future perspectives on the diagnosis of Roseolovirus infections.

Diagnosis of Roseolovirus infections mandates careful selection of patients, samples, and testing methods. We review advances in the field and highlight research priorities. Quantitative (q)PCR can accurately identify and distinguish between human herpesvirus 6 (HHV-6) species A and B.

A multiplexed droplet digital PCR assay performs better than qPCR on inhibition prone samples.

We demonstrate the development of a multiplex droplet digital PCR assay for human cytomegalovirus (CMV), human adenovirus species F, and an internal plasmid control that may be useful for PCR inhibition-prone clinical samples. This assay performs better on inhibition-prone stool samples than a quantitative PCR assay for CMV and is the first published clinical virology droplet digital PCR assay to incorporate an internal control.

Prevalence of chromosomally integrated human herpesvirus 6 in patients with human herpesvirus 6-central nervous system dysfunction.

We identified 37 hematopoietic cell transplantation recipients with human herpesvirus 6 (HHV-6) central nervous system dysfunction and tested donor-recipient pairs for chromosomally integrated HHV-6 (ciHHV-6). One patient had ciHHV-6A with possible HHV-6A reactivation and encephalitis. There was no ciHHV-6 enrichment in this group, but larger studies are needed to determine if patients with ciHHV-6 are at increased risk for HHV-6-associated diseases or other complications.

Human herpesvirus 6 can be detected in cerebrospinal fluid without associated symptoms after allogeneic hematopoietic cell transplantation.

Background: Human herpesvirus 6 (HHV-6) is an opportunistic pathogen after hematopoietic cell transplantation (HCT) that is associated with central nervous system (CNS) dysfunction.

Objectives: The aim of this study was to determine the frequency and significance of HHV-6 DNA detection in cerebrospinal fluid (CSF) after HCT.

Study Design: We identified patients with HHV-6 DNA in CSF using quantitative PCR.

Clinical utility of droplet digital PCR for human cytomegalovirus.

Human cytomegalovirus (CMV) has historically been the major infectious cause of morbidity and mortality among patients receiving hematopoietic cell or organ transplant. Standard care in a transplant setting involves frequent monitoring of CMV viral load over weeks to months to determine when antiviral treatment may be required. Quantitative PCR (qPCR) is the standard molecular diagnostic method for monitoring.

AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped.

The potential advantages of digital PCR for clinical virology diagnostics.

Digital PCR (dPCR), a new nucleic acid amplification technology, offers several potential advantages over real-time or quantitative PCR (qPCR), the current workhorse of clinical molecular virology diagnostics. Several studies have demonstrated dPCR assays for human cytomegalovirus or HIV, which give more precise and reproducible results than qPCR assays without sacrificing sensitivity. Here we review the literature comparing dPCR and qPCR performance in viral molecular diagnostic assays and offer perspective on the future of dPCR in clinical virology diagnostics.

Identification of chromosomally integrated human herpesvirus 6 by droplet digital PCR.

Background: Human herpesvirus 6 (HHV-6) latently infects a majority of adults. In about 1% of the population HHV-6 exists in a chromosomally integrated form (ciHHV-6) that resides in every somatic and germ cell and can be transmitted through the germ line. Patients with ciHHV-6 have been misdiagnosed and unnecessarily treated for active HHV-6 infection, sometimes with important side effects, based on results from quantitative molecular HHV-6 tests.

Rapid detection of human cytomegalovirus UL97 and UL54 mutations directly from patient samples.

Human cytomegalovirus (CMV) is a significant contributor to morbidity and mortality in immunocompromised patients, particularly in the transplant setting. The availability of anti-CMV drugs has improved treatment, but drug resistance is an emerging problem. Here, we describe an improved, rapid, sequencing-based assay for the two genes in CMV where drug resistance occurs, the UL97 and UL54 genes.

Viral diagnostics in the era of digital polymerase chain reaction.

Unlike quantitative polymerase chain reaction (qPCR), digital PCR (dPCR) achieves sensitive and accurate absolute quantitation of a DNA sample without the need for a standard curve. A single PCR reaction is divided into many separate reactions that each have a positive or negative signal. By applying Poisson statistics, the number of DNA molecules in the original sample is directly calculated from the number of positive and negative reactions.

Engineered Escherichia coli silver-binding periplasmic protein that promotes silver tolerance.

Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications.