Clinical Characterization of Arrhythmia-Induced Cardiomyopathy in Patients With Tachyarrhythmia and Idiopathic Heart Failure.

BACKGROUND: Arrhythmia-induced cardiomyopathy (AIC) is a known entity, but prospective evidence for its characterization is limited. OBJECTIVES: This study aimed to: 1) determine the relative frequency of the pure form of AIC in the clinically relevant cohort of patients with newly diagnosed, otherwise unexplained left ventricular systolic dysfunction (LVSD) and tachyarrhythmia; 2) assess the time to recovery from LVSD; and 3) identify parameters for an early diagnosis of AIC. METHODS: Patients were prospectively included, underwent effective rhythm restoration, and were followed-up at 2, 4, and 6 months to evaluate clinical characteristics, biomarkers, and cardiac imaging including cardiac magnetic resonance imaging. Patients with recurred arrhythmia were excluded from analysis. RESULTS: 41 of 50 patients were diagnosed with AIC 6 months after rhythm restoration. Left ventricular (LV) ejection fraction increased 2 months after rhythm restoration from 35.4% ± 8.2% to 52.7% ± 8.0% in AIC patients vs 37.0% ± 9.5% to 43.3% ± 7.0% in non-AIC patients. From month 2 to 6, LV ejection fraction continued to increase in AIC patients (57.2% ± 6.1%; P < 0.001) but remained stable in non-AIC patients (44.0% ± 7.8%; P = 0.628). Multivariable logistic regression analysis revealed that lower LV end-diastolic diameter at baseline could be used for early diagnosis of AIC, whereas biomarkers and other morphological or functional parameters, including late LV gadolinium enhancement, did not show suitability for early diagnosis. CONCLUSIONS: We observed a high prevalence of AIC in patients with otherwise unexplained LVSD and concomitant tachyarrhythmia, suggesting that this condition may be underdiagnosed in clinical practice. Most patients recovered fast, within months, from LVSD. A low initial LV end-diastolic diameter may constitute an early marker for diagnosis of AIC.

Cardiac function in relation to functional status and fatigue in patients with post-COVID syndrome.

Patients with Post-COVID syndrome (PCS) are frequently referred for cardiologic evaluation. We assessed cardiac function and biomarkers in relation to functional status and fatigue in patients with PCS. This prospective single-center cohort study included 227 patients with persisting symptoms after COVID-19 infection. Most frequent complaints were fatigue (70%), dyspnea (56%), neurocognitive symptoms (34%) and chest pain (28%). Standardized questionnaires were used to assess Post-COVID-Functional-Scale (PCFS) and fatigue (MFI-20). The fatigue severity was inversely related to age and did not correlate with cardiovascular diseases, echocardiographic findings, or biomarkers. Similarly, mild to moderate functional impairment (PCFS 1-3) did not correlate with cardiovascular alterations. However, the subgroup of patients with significant functional impairment (PCFS = 4) had more frequent cardiovascular comorbidities, biomarkers and impaired global longitudinal strain (GLS). Patients with elevated troponin T showed abnormal GLS, reduced left ventricular ejection fraction and impaired tricuspid annular plane systolic excursion. The majority of patients with PCS shows a normal cardiac function. Only the small subgroup of patients with severe functional impairment and patients with elevated troponin T is at risk for impaired cardiac function and likely to benefit from specialized care by a cardiologist.

Neuropsychiatric phenotype of post COVID-19 syndrome in non-hospitalized patients.

The post COVID-19 syndrome (PCS) is an emerging phenomenon worldwide with enormous socioeconomic impact. While many patients describe neuropsychiatric deficits, the symptoms are yet to be assessed and defined systematically. In this prospective cohort study, we report on the results of a neuropsychiatric consultation implemented in May 2021. A cohort of 105 consecutive patients with merely mild acute course of disease was identified by its high symptom load 6 months post infection using a standardized neurocognitive and psychiatric-psychosomatic assessment. In this cohort, we found a strong correlation between higher scores in questionnaires for fatigue (MFI-20), somatization (PHQ15) and depression (PHQ9) and worse functional outcome as measured by the post COVID functional scale (PCFS). In contrast, neurocognitive scales correlated with age, but not with PCFS. Standard laboratory and cardiopulmonary biomarkers did not differ between the group of patients with predominant neuropsychiatric symptoms and a control group of neuropsychiatrically unaffected PCS patients. Our study delineates a phenotype of PCS dominated by symptoms of fatigue, somatisation and depression. The strong association of psychiatric and psychosomatic symptoms with the PCFS warrants a systematic evaluation of psychosocial side effects of the pandemic itself and psychiatric comorbidities on the long-term outcome of patients with SARS-CoV-2 infection.

Consensus Definitions of BK Polyomavirus Nephropathy in Renal Transplant Recipients for Clinical Trials.

BACKGROUND: BK polyomavirus (BKPyV) infection and BK polyomavirus nephropathy (BKPyVAN) are important causes of allograft dysfunction and premature allograft loss in renal transplant recipients. RESULTS AND DISCUSSION: Controlled clinical trials to evaluate new agents for prevention and treatment are needed but are hampered by the lack of outcome measures that accurately assess the effect of the intervention, are clinically relevant, and are acceptable from a regulatory perspective. METHODS: To facilitate consistent end points in clinical trials and to support clinical research and drug development, definitions of BKPyV infection and disease have been developed by the BK Disease Definitions Working Group of the Transplantation Associated Virus Infection Forum with the Forum for Collaborative Research, which consists of scientists, clinicians, regulators, and industry representatives. CONCLUSIONS: These definitions refine established principles of "proven" BKPyV disease and introduce a "probable" disease category that could be used in clinical trials to prevent or treat BKPyVAN in renal transplant recipients.

An HIV Diagnostic Testing Algorithm Using the cobas HIV-1/HIV-2 Qualitative Assay for HIV Type Differentiation and Confirmation.

Human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) diagnostic testing algorithms recommended by the Centers for Disease Control involve up to three tests and rely mostly on detection of viral antigen and host antibody responses. HIV-1 p24 antigen/HIV-1/HIV-2 antibody-reactive specimens are confirmed with an immunochromatographic HIV-1/HIV-2 antibody differentiation assay, and negative or indeterminate results from the differentiation assay are resolved by an HIV-1-specific nucleic acid amplification test (NAT). The performance of a proposed alternative algorithm using the cobas HIV-1/HIV-2 qualitative NAT as the differentiation assay was evaluated in subjects known to be infected with HIV-1 (n = 876) or HIV-2 (n = 139), at low (n = 6,017) or high (n = 1,020) risk of HIV-1 infection, or at high-risk for HIV-2 infection (n = 498) (study A). The performance of the cobas HIV-1/HIV-2 qualitative test was also evaluated by comparison to an HIV-1 or HIV-2 alternative NAT (study B). The HIV-1 and HIV-2 overall percent agreements (OPA) in study A ranged from 95% to 100% in all groups. The positive percent agreements (PPA) for HIV-1 and HIV-2 were 100% (876/876) and 99.4% (167/168), respectively, for known positive groups. The negative percent agreement in the HIV low-risk group was 100% for both HIV-1 and HIV-2. In study B, the HIV-1 and HIV-2 OPA ranged from 99% to 100% in all groups evaluated (n = 183 to 1,030), and the PPA for HIV-1 and HIV-2 were 100% and 99.5%, respectively, for known positive groups. The cobas HIV-1/HIV-2 qualitative assay can discriminate between HIV-1 and HIV-2 based on HIV RNA and can be included in an alternative diagnostic algorithm for HIV.

Case report: isolated pulmonary valve endocarditis in a 39-year-old patient with intravenous drug abuse.

BACKGROUND: Isolated pulmonary valve endocarditis is a very rare form of right-sided infective endocarditis. Due to the anatomy, in most cases, just the tricuspid valve is involved. Diagnosis can be challenging because of non-specific symptoms (fever, dyspnoea, haemoptysis, and pleuritic chest pain) and difficulty of detection by echocardiography. Risk factors include intravenous drug abuse, congenital heart disorders, alcohol abuse, male sex and central venous catheters, or pacemaker leads. CASE SUMMARY: A 39-year-old homeless male patient, who was a current intravenous drug user, presented with fever, dyspnoea, and haemoptysis. The chest X-ray showed bilateral infiltrates. Empiric antibiotic treatment was initiated. Blood cultures showed the presence of Streptococcus dysgalactiae. Atypical causes of pneumonia were excluded. Systemic embolism was suspected, and a computed tomography scan of brain, thorax, and abdomen was performed. Multiple septic embolic lesions were detected in both lungs. Echocardiography revealed an isolated pulmonary valve endocarditis. Penicillin G and gentamycin were administered intravenously for a duration of 6 and 2 weeks, respectively. The patient was discharged in stable condition but did not return for outpatient clinical appointments. DISCUSSION: To detect rare causes of right-sided infective endocarditis, repeated echocardiograms with special focus on the pulmonary valve may be required. Usually, antibiotic treatment alone leads to recovery. In special situations (heart failure, septic shock, or large vegetation size) surgery is required. Due to the high risk of postoperative complications, surgery in intravenous drug users should be avoided if possible.

Improved Sensitivity of a Dual-Target HIV-1 Qualitative Test for Plasma and Dried Blood Spots.

The use of nucleic acid detection for HIV type 1 (HIV-1) detection is strongly recommended in infants <18 months of age, in whom serology is unreliable. This study evaluated the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qualitative Test v2.0 (TaqMan HIV-1 Qual Test, v2.0), a dual-target total nucleic acid real-time PCR assay. The limit of detection (LOD) of the new test in plasma and dried blood spots (DBS) was determined with the 2nd International HIV-1 RNA WHO standard. The specificity of the assay was tested with EDTA plasma (n = 1,301) and DBS from HIV-negative adults (n = 1,000). The sensitivity was determined using HIV-1-positive samples (n = 169 adult EDTA plasma, n = 172 adult DBS, and n = 100 infant DBS) that included group M, subtypes A to H, CRF01_AE, CRF02_AG, and groups O and N. All positive specimens and a subset of the negative specimens were also tested with the Abbott RealTime HIV-1 Qual assay (RealTime). The LOD of the TaqMan assay was 20 copies/ml in plasma and 300 copies/ml in DBS, with specificities of 99.8% in plasma and 99.9% in DBS. The TaqMan assay results were 100% concordant with RealTime results in EDTA plasma samples and in 100 HIV-1-negative adult DBS. Among 172 HIV-1-positive DBS from adults, the TaqMan assay showed positive results for all DBS while RealTime missed five DBS with low target concentrations. Infant DBS results were 100% concordant. The improved sensitivity of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qualitative Test, v2.0, compared to current commercially available assays may enable earlier diagnosis and treatment in adults and infants. The dual-target test may ensure HIV-1 detection even if a mutation is present in one of the two target regions. The DBS sample matrix facilitates virological testing in remote areas.

A simple method to elute cell-free HIV from dried blood spots improves their usefulness for monitoring therapy.

BACKGROUND: Dried blood spots (DBS) improve access to HIV viral load (VL) testing, but yield increased VL measurements compared to the plasma reference method because of cell-associated viral nucleic acid. In clinical settings, DBS methods may falsely categorize many patients as failing therapy. OBJECTIVES: Description of a simple method, free virus elution (FVE), to preferentially elute plasma-associated virus from DBS samples with phosphate-buffered saline, and an initial HIV VL performance comparison with standard DBS elution methods. STUDY DESIGN: The mechanism of action of FVE was studied with model DBS samples containing purified virus or washed HIV-containing cells, and with a DNA-specific HIV PCR. Using clinical samples, VL results from the new FVE method were compared to results from a dried fluid spot procedure (DFSP) protocol, which uses a guanidinium-based elution method, using plasma VL as the reference method. RESULTS: Model system experiments suggest that the method efficiently separates virus from cell-associated HIV, with a wide tolerance for incubation time and temperature. In 196 clinical samples, FVE reduced VL over-quantification from DBS, and improved DBS clinical concordance with plasma from 67% to 95%. CONCLUSIONS: A simple elution in PBS significantly reduced the over-quantification of HIV VL in DBS. Additional studies are needed to validate the method in fingerstick-collected specimens and to further understand the compartmentalization of HIV DNA and RNA in DBS specimens.

Wrestling with the repertoire: the promise and perils of next generation sequencing for antigen receptors.

Next generation sequencing technologies are revolutionizing the study of immune repertoires. These methods provide a previously unimaginable amount of sequence data, unfortunately accompanied by numerous challenges associated with error correction and interpretation that remain to be solved. For antigen receptors, these challenges will require dedicated solutions beyond those developed for genome sequencing, which may differ depending on the sequencing technology used and the purpose of the experiment. Many investigators are developing such methods, based on different sequencing platforms, but critical details of protocol and performance are proprietary. The field will move forward when these methods are shared and standardized, and when the accuracy, sensitivity and reproducibility of various sequencing, and analytic methods are evaluated using standardized samples in comparative experiments.

Blood T-cell receptor diversity decreases during the course of HIV infection, but the potential for a diverse repertoire persists.

HIV infection results in a decrease in circulating CD4(+) T-cell and naive T-cell numbers. If such losses were associated with an erosion of T-cell receptor (TCR) repertoire diversity in the peripheral T-cell pool, this might exacerbate the state of persistent immunodeficiency. Existing methods for the analysis of the TCR repertoire have demonstrated skewed distributions of TCR genes in HIV-infected subjects but cannot directly measure TCR diversity. Here we used AmpliCot, a quantitative assay based on DNA hybridization kinetics, to measure TCR diversity in a cross-sectional comparison of 19 HIV-infected persons to 18 HIV-uninfected controls. HIV-infected persons had a 10-fold decrease in total TCR repertoire diversity in 1.5 mL of blood compared with uninfected controls, with decreased diversity correlating most closely with a lower CD4(+) T-cell percentage. Nonetheless, the TCR repertoire diversity of sort-purified T-cell subpopulations in HIV-infected and HIV-uninfected subjects was comparable. These observations suggest that the TCR repertoire diversity changes in whole blood during HIV disease progression are primarily the result of changes in the number and proportion of T-cell subpopulations and that most HIV-infected persons may retain a sufficiently diverse TCR repertoire to permit immune reconstitution with antiretroviral therapy alone, without thymopoiesis.

Abacavir increases platelet reactivity via competitive inhibition of soluble guanylyl cyclase.

OBJECTIVE: To provide a molecular mechanism that explains the association of the antiretroviral guanosine analogue, abacavir, with an increased risk of myocardial infarction. DESIGN: Drug effects were studied with biochemical and cellular assays. METHODS: Human platelets were incubated with nucleoside analogue drugs ex vivo. Platelet activation stimulated by ADP was studied by measuring surface P-selectin with flow cytometry. Inhibition of purified soluble guanylyl cyclase was quantified using an ELISA to measure cGMP production. RESULTS: Pre-incubation of platelets in abacavir significantly increased activation in response to ADP in a time and dose-dependent manner. The active anabolite of abacavir, carbovir triphosphate, competitively inhibited soluble guanylyl cyclase activity with a K(i) of 55 µmol/l. CONCLUSION: Abacavir competitively inhibits guanylyl cyclase, leading to platelet hyperreactivity. This may explain the observed increased risk of myocardial infarction in HIV patients taking abacavir.

Measurement of absolute T cell receptor rearrangement diversity.

T cell receptor (TCR) diversity is critical for adaptive immunity. Existing methods for measuring such diversity are qualitative, expensive, and/or of uncertain accuracy. Here, we describe a method and associated reagents for estimating the absolute number of unique TCR Vß rearrangements present in a given number of cells or volume of blood. Compared to next generation sequencing, this method is rapid, reproducible, and affordable. Diversity of a sample is calculated based on three independent measurements of one Vß-Jß family of TCR rearrangements at a time. The percentage of receptors using the given Vß gene is determined by flow cytometric analysis of T cells stained with anti-Vß family antibodies. The percentage of receptors using the Vß gene in combination with the chosen Jß gene is determined by quantitative PCR. Finally, the absolute clonal diversity of the Vß-Jß family is determined with the AmpliCot method of DNA hybridization kinetics, by interpolation relative to PCR standards of known sequence diversity. These three component measurements are reproducible and linear. Using titrations of known numbers of input cells, we show that the TCR diversity estimates obtained by this approach approximate expected values within a two-fold error, have a coefficient of variation of 20%, and yield similar results when different Vß-Jß pairs are chosen. The ability to obtain accurate measurements of the total number of different TCR gene rearrangements in a cell sample should be useful for basic studies of the adaptive immune system as well as in clinical studies of conditions such as HIV disease, transplantation, aging, and congenital immunodeficiencies.

Design, construction, and validation of a modular library of sequence diversity standards for polymerase chain reaction.

Methods to measure the sequence diversity of polymerase chain reaction (PCR)-amplified DNA lack standards for use as assay calibrators and controls. Here we present a general and economical method for developing customizable DNA standards of known sequence diversity. Standards ranging from 1 to 25,000 sequences were generated by directional ligation of oligonucleotide "words" of standard length and GC content and then amplified by PCR. The sequence accuracy and diversity of the library were validated using AmpliCot analysis (DNA hybridization kinetics) and Illumina sequencing. The library has the following features: (i) pools containing tens of thousands of sequences can be generated from the ligation of relatively few commercially synthesized short oligonucleotides; (ii) each sequence differs from all others in the library at a minimum of three nucleotide positions, permitting discrimination between different sequences by either sequencing or hybridization; (iii) all sequences have identical length, GC content, and melting temperature; (iv) the identity of each standard can be verified by restriction digestion; and (v) once made, the ends of the library may be cleaved and replaced with sequences to match any PCR primer pair. These standards should greatly improve the accuracy and reproducibility of sequence diversity measurements.

Mu2 adaptin facilitates but is not essential for synaptic vesicle recycling in Caenorhabditis elegans.

Synaptic vesicles must be recycled to sustain neurotransmission, in large part via clathrin-mediated endocytosis. Clathrin is recruited to endocytic sites on the plasma membrane by the AP2 adaptor complex. The medium subunit (micro2) of AP2 binds to cargo proteins and phosphatidylinositol-4 ,5-bisphosphate on the cell surface. Here, we characterize the apm-2 gene (also called dpy-23), which encodes the only micro2 subunit in the nematode Caenorhabditis elegans. APM-2 is highly expressed in the nervous system and is localized to synapses; yet specific loss of APM-2 in neurons does not affect locomotion. In apm-2 mutants, clathrin is mislocalized at synapses, and synaptic vesicle numbers and evoked responses are reduced to 60 and 65%, respectively. Collectively, these data suggest AP2 micro2 facilitates but is not essential for synaptic vesicle recycling.

Growth hormone enhances thymic function in HIV-1-infected adults.

Growth hormone (GH) is an underappreciated but important regulator of T cell development that can reverse age-related declines in thymopoiesis in rodents. Here, we report findings of a prospective randomized study examining the effects of GH on the immune system of HIV-1-infected adults. GH treatment was associated with increased thymic mass. In addition, GH treatment enhanced thymic output, as measured by both the frequency of T cell receptor rearrangement excision circles in circulating T cells and the numbers of circulating naive and total CD4(+) T cells. These findings provide compelling evidence that GH induces de novo T cell production and may, accordingly, facilitate CD4(+) T cell recovery in HIV-1-infected adults. Further, these randomized, prospective data have shown that thymic involution can be pharmacologically reversed in humans, suggesting that immune-based therapies could be used to enhance thymopoiesis in immunodeficient individuals.

C. elegans AP-2 and retromer control Wnt signaling by regulating mig-14/Wntless.

While endocytosis can regulate morphogen distribution, its precise role in shaping these gradients is unclear. Even more enigmatic is the role of retromer, a complex that shuttles proteins between endosomes and the Golgi apparatus, in Wnt gradient formation. Here we report that DPY-23, the C. elegans mu subunit of the clathrin adaptor AP-2 that mediates the endocytosis of membrane proteins, regulates Wnt function. dpy-23 mutants display Wnt phenotypes, including defects in neuronal migration, neuronal polarity, and asymmetric cell division. DPY-23 acts in Wnt-expressing cells to promote these processes. MIG-14, the C. elegans homolog of the Wnt-secretion factor Wntless, also acts in these cells to control Wnt function. In dpy-23 mutants, MIG-14 accumulates at or near the plasma membrane. By contrast, MIG-14 accumulates in intracellular compartments in retromer mutants. Based on our observations, we propose that intracellular trafficking of MIG-14 by AP-2 and retromer plays an important role in Wnt secretion.

Direct measurement of T-cell receptor repertoire diversity with AmpliCot.

Many studies require the measurement of nucleic acid sequence diversity. Here we describe a method, called AmpliCot, that measures the sequence diversity of PCR products on the basis of DNA hybridization kinetics, thereby avoiding the time, expense and biases associated with cloning and sequencing. SYBR Green dye is used to measure DNA hybridization kinetics in a homogeneous, automated fashion. PCR products are prepared in wholly double-stranded homoduplex form for a baseline measurement of DNA concentration. The DNA is melted and then reannealed under stringent conditions that allow only homoduplexes to form. The sequence diversity of a sample is proportional to the product of its concentration and the time required for it to anneal. After validating AmpliCot with a library of diverse sequences, we use it to measure the diversity of expressed rearrangements of the gene encoding the T-cell antigen receptor (TCR) beta chain. AmpliCot measurements are in good agreement with previous estimates of murine TCR repertoire diversity that required extensive cloning and sequencing.

HLH-14 is a C. elegans achaete-scute protein that promotes neurogenesis through asymmetric cell division.

Achaete-Scute basic helix-loop-helix (bHLH) proteins promote neurogenesis during metazoan development. In this study, we characterize a C. elegans Achaete-Scute homolog, HLH-14. We find that a number of neuroblasts express HLH-14 in the C. elegans embryo, including the PVQ/HSN/PHB neuroblast, a cell that generates the PVQ interneuron, the HSN motoneuron and the PHB sensory neuron. hlh-14 mutants lack all three of these neurons. The fact that HLH-14 promotes all three classes of neuron indicates that C. elegans proneural bHLH factors may act less specifically than their fly and mammalian homologs. Furthermore, neural loss in hlh-14 mutants results from a defect in an asymmetric cell division: the PVQ/HSN/PHB neuroblast inappropriately assumes characteristics of its sister cell, the hyp7/T blast cell. We argue that bHLH proteins, which control various aspects of metazoan development, can control cell fate choices in C. elegans by regulating asymmetric cell divisions. Finally, a reduction in the function of hlh-2, which encodes the C. elegans E/Daughterless bHLH homolog, results in similar neuron loss as hlh-14 mutants and enhances the effects of partially reducing hlh-14 function. We propose that HLH-14 and HLH-2 act together to specify neuroblast lineages and promote neuronal fate.