Effective intravitreal gene delivery to retinal pigment epithelium with hyaluronic acid nanospheres.

Inherited retinal degeneration (IRD) can cause a wide range of different forms of vision loss and blindness, and in spite of extensive advancements in gene therapy research, therapeutic approaches for targeting IRDs are still lacking. We have recently developed an approach for the intravitreal co-delivery of hyaluronic-acid nanospheres (HA-NSs) with sulfotyrosine (ST), effectively reaching the outer retina from the vitreal cavity. Here, our goal was to understand whether DNA-filled HA-NSs could generate gene expression in the outer retina. TxRed-labeled HA-NSs were compacted with plasmid DNA carrying a GFP reporter gene and intravitreally injected into the mouse retina. Follow-up at 4 weeks showed widespread gene expression in the outer retina and reduced, albeit present, expression at 8 weeks post-injection. Further analysis revealed this expression to be largely localized to the retinal pigment epithelium (RPE). These data show that intravitreal delivery of HA-NSs is a promising non-viral platform for the delivery of therapeutic genes and can generate pan-tissue, persistent gene expression in the RPE.

Downregulation of rhodopsin is an effective therapeutic strategy in ameliorating peripherin-2-associated inherited retinal disorders.

Given the absence of approved treatments for pathogenic variants in Peripherin-2 (PRPH2), it is imperative to identify a universally effective therapeutic target for PRPH2 pathogenic variants. To test the hypothesis that formation of the elongated discs in presence of PRPH2 pathogenic variants is due to the presence of the full complement of rhodopsin in absence of the required amounts of functional PRPH2. Here we demonstrate the therapeutic potential of reducing rhodopsin levels in ameliorating disease phenotype in knockin models for p.Lys154del (c.458-460del) and p.Tyr141Cys (c.422 A > G) in PRPH2. Reducing rhodopsin levels improves physiological function, mitigates the severity of disc abnormalities, and decreases retinal gliosis. Additionally, intravitreal injections of a rhodopsin-specific antisense oligonucleotide successfully enhance the physiological function of photoreceptors and improves the ultrastructure of discs in mutant mice. Presented findings shows that reducing rhodopsin levels is an effective therapeutic strategy for the treatment of inherited retinal degeneration associated with PRPH2 pathogenic variants.

Practicing Serious Illness Conversations in a Pulmonary Medicine Teaching Clinic.

BACKGROUND: Physician trainees in pulmonary medicine are not provided with supervised practice opportunities to gain confidence and skill in having serious illness conversations in the ambulatory setting. OBJECTIVE: We incorporated a palliative medicine attending into an ambulatory pulmonology teaching clinic to provide supervised opportunities for serious illness conversations. METHODS: Trainees in a pulmonary medicine teaching clinic requested supervision from a palliative medicine attending based on a set of evidence-based pulmonary-specific triggers that indicate advanced disease. Semi-structured interviews were conducted to determine the trainee's perceptions of the educational intervention. RESULTS: The palliative medicine attending supervised 8 trainees in 58 patient encounters. The most common trigger for palliative medicine supervision was answering "no" to the "surprise question." At baseline, all trainees cited lack of time as the primary barrier to having serious illness conversations. Themes emerging from post-intervention semi-structured interviews included trainees learning that (1) patients are grateful to have conversations about the severity of their illness, (2) patients do not have a good sense of their prognosis, and (3) with improved skills, these conversations can be conducted efficiently. CONCLUSIONS: Pulmonary medicine trainees were provided practice opportunities for having serious illness conversations under the supervision of the palliative medicine attending. These practice opportunities effected trainee perception on important barriers to further practice.

Potassium Leak Channels and Mitochondrial Complex I Interact in Glutamatergic Interneurons of the Mouse Spinal Cord.

BACKGROUND: Volatile anesthetics induce hyperpolarizing potassium currents in spinal cord neurons that may contribute to their mechanism of action. They are induced at lower concentrations of isoflurane in noncholinergic neurons from mice carrying a loss-of-function mutation of the Ndufs4 gene, required for mitochondrial complex I function. The yeast NADH dehydrogenase enzyme, NDi1, can restore mitochondrial function in the absence of normal complex I activity, and gain-of-function Ndi1 transgenic mice are resistant to volatile anesthetics. The authors tested whether NDi1 would reduce the hyperpolarization caused by isoflurane in neurons from Ndufs4 and wild-type mice. Since volatile anesthetic behavioral hypersensitivity in Ndufs4 is transduced uniquely by glutamatergic neurons, it was also tested whether these currents were also unique to glutamatergic neurons in the Ndufs4 spinal cord. METHODS: Spinal cord neurons from wild-type, NDi1, and Ndufs4 mice were patch clamped to characterize isoflurane sensitive currents. Neuron types were marked using fluorescent markers for cholinergic, glutamatergic, and γ-aminobutyric acid-mediated (GABAergic) neurons. Norfluoxetine was used to identify potassium channel type. Neuron type-specific Ndufs4 knockout animals were generated using type-specific Cre-recombinase with floxed Ndufs4. RESULTS: Resting membrane potentials (RMPs) of neurons from NDi1;Ndufs4, unlike those from Ndufs4, were not hyperpolarized by 0.6% isoflurane (Ndufs4, ΔRMP -8.2 mV [-10 to -6.6]; P = 1.3e-07; Ndi1;Ndufs4, ΔRMP -2.1 mV [-7.6 to +1.4]; P = 1). Neurons from NDi1 animals in a wild-type background were not hyperpolarized by 1.8% isoflurane (wild-type, ΔRMP, -5.2 mV [-7.3 to -3.2]; P = 0.00057; Ndi1, ΔRMP, 0.6 mV [-1.7 to 3.2]; P = 0.68). In spinal cord slices from global Ndufs4 animals, holding currents (HC) were induced by 0.6% isoflurane in both GABAergic (ΔHC, 81.3 pA [61.7 to 101.4]; P = 2.6e-05) and glutamatergic (ΔHC, 101.2 pA [63.0 to 146.2]; P = 0.0076) neurons. In neuron type-specific Ndufs4 knockouts, HCs were increased in cholinergic (ΔHC, 119.5 pA [82.3 to 156.7]; P = 0.00019) and trended toward increase in glutamatergic (ΔHC, 85.5 pA [49 to 126.9]; P = 0.064) neurons but not in GABAergic neurons. CONCLUSIONS: Bypassing complex I by overexpression of NDi1 eliminates increases in potassium currents induced by isoflurane in the spinal cord. The isoflurane-induced potassium currents in glutamatergic neurons represent a potential downstream mechanism of complex I inhibition in determining minimum alveolar concentration.

FAAH inhibition ameliorates breast cancer in a murine model.

Breast cancer is the leading cancer among females worldwide. Disease outcome depends on the hormonal status of the cancer and whether or not it is metastatic, but there is a need for more efficacious therapeutic strategies where first line treatment fails. In this study, Fatty Acid Amide Hydrolase (FAAH) inhibition and endocannabinoids were examined as therapeutic alternatives. FAAH is an integral membrane enzyme that hydrolyzes endocannabinoids, rendering them inactive, and FAAH inhibition is predicted to increase cancer cell death. To test this, breast cancer cells were probed for FAAH expression using Western blot analysis, treated with FAAH inhibitors, exogenous endocannabinoids, and combinations of the two treatments, and assessed for viability. High levels of FAAH were observed in different breast cancer cell lines. FAAH inhibition was more effective than exogenous endocannabinoid treatment, and the combination of FAAH inhibitors and endocannabinoids was the most effective in inducing apoptosis of breast cancer cells in vitro. In addition, in vivo FAAH inhibition reduced breast cancer growth in immunodeficient mice. FAAH inhibition is a promising approach, and tremendous progress has been made in the field to validate this mechanism as an alternative to chemotherapy. Further research exploring the therapeutic potential and impact of FAAH expression on cancer cells is warranted.

TREK-1 and TREK-2 Knockout Mice Are Not Resistant to Halothane or Isoflurane.

BACKGROUND: A variety of molecular targets for volatile anesthetics have been suggested, including the anesthetic-sensitive potassium leak channel, TREK-1. Knockout of TREK-1 is reported to render mice resistant to volatile anesthetics, making TREK-1 channels compelling targets for anesthetic action. Spinal cord slices from mice, either wild type or an anesthetic- hypersensitive mutant, Ndufs4, display an isoflurane-induced outward potassium leak that correlates with their minimum alveolar concentrations and is blocked by norfluoxetine. The hypothesis was that TREK-1 channels conveyed this current and contribute to the anesthetic hypersensitivity of Ndufs4. The results led to evaluation of a second TREK channel, TREK-2, in control of anesthetic sensitivity. METHODS: The anesthetic sensitivities of mice carrying knockout alleles of Trek-1 and Trek-2, the double knockout Trek-1;Trek-2, and Ndufs4;Trek-1 were measured. Neurons from spinal cord slices from each mutant were patch clamped to characterize isoflurane-sensitive currents. Norfluoxetine was used to identify TREK-dependent currents. RESULTS: The mean values for minimum alveolar concentrations (± SD) between wild type and two Trek-1 knockout alleles in mice (P values, Trek-1 compared to wild type) were compared. For wild type, minimum alveolar concentration of halothane was 1.30% (0.10), and minimum alveolar concentration of isoflurane was 1.40% (0.11); for Trek-1tm1Lex, minimum alveolar concentration of halothane was 1.27% (0.11; P = 0.387), and minimum alveolar concentration of isoflurane was 1.38% (0.09; P = 0.268); and for Trek-1tm1Lzd, minimum alveolar concentration of halothane was 1.27% (0.11; P = 0.482), and minimum alveolar concentration of isoflurane was 1.41% (0.12; P = 0.188). Neither allele was resistant for loss of righting reflex. The EC50 values of Ndufs4;Trek-1tm1Lex did not differ from Ndufs4 (for Ndufs4, EC50 of halothane, 0.65% [0.05]; EC50 of isoflurane, 0.63% [0.05]; and for Ndufs4;Trek-1tm1Lex, EC50 of halothane, 0.58% [0.07; P = 0.004]; and EC50 of isoflurane, 0.61% [0.06; P = 0.442]). Loss of TREK-2 did not alter anesthetic sensitivity in a wild-type or Trek-1 genetic background. Loss of TREK-1, TREK-2, or both did not alter the isoflurane-induced currents in wild-type cells but did cause them to be norfluoxetine insensitive. CONCLUSIONS: Loss of TREK channels did not alter anesthetic sensitivity in mice, nor did it eliminate isoflurane-induced transmembrane currents. However, the isoflurane-induced currents are norfluoxetine-resistant in Trek mutants, indicating that other channels may function in this role when TREK channels are deleted.

Isoflurane inhibition of endocytosis is an anesthetic mechanism of action.

The mechanisms of volatile anesthetic action remain among the most perplexing mysteries of medicine. Across phylogeny, volatile anesthetics selectively inhibit mitochondrial complex I, and they also depress presynaptic excitatory signaling. To explore how these effects are linked, we studied isoflurane effects on presynaptic vesicle cycling and ATP levels in hippocampal cultured neurons from wild-type and complex I mutant (Ndufs4(KO)) mice. To bypass complex I, we measured isoflurane effects on anesthetic sensitivity in mice expressing NADH dehydrogenase (NDi1). Endocytosis in physiologic concentrations of glucose was delayed by effective behavioral concentrations of isoflurane in both wild-type (τ [unexposed] 44.8 ± 24.2 s; τ [exposed] 116.1 ± 28.1 s; p < 0.01) and Ndufs4(KO) cultures (τ [unexposed] 67.6 ± 16.0 s; τ [exposed] 128.4 ± 42.9 s; p = 0.028). Increasing glucose, to enhance glycolysis and increase ATP production, led to maintenance of both ATP levels and endocytosis (τ [unexposed] 28.0 ± 14.4; τ [exposed] 38.2 ± 5.7; reducing glucose worsened ATP levels and depressed endocytosis (τ [unexposed] 85.4 ± 69.3; τ [exposed] > 1,000; p < 0.001). The block in recycling occurred at the level of reuptake of synaptic vesicles into the presynaptic cell. Expression of NDi1 in wild-type mice caused behavioral resistance to isoflurane for tail clamp response (EC50 Ndi1(-) 1.27% ± 0.14%; Ndi1(+) 1.55% ± 0.13%) and halothane (EC50 Ndi1(-) 1.20% ± 0.11%; Ndi1(+) 1.46% ± 0.10%); expression of NDi1 in neurons improved hippocampal function, alleviated inhibition of presynaptic recycling, and increased ATP levels during isoflurane exposure. The clear alignment of cell culture data to in vivo phenotypes of both isoflurane-sensitive and -resistant mice indicates that inhibition of mitochondrial complex I is a primary mechanism of action of volatile anesthetics.

Mitochondrial Function and Anesthetic Sensitivity in the Mouse Spinal Cord.

BACKGROUND: Ndufs4 knockout (KO) mice are defective in mitochondrial complex I function and hypersensitive to inhibition of spinal cord-mediated response to noxious stimuli by volatile anesthetics. It was hypothesized that, compared to wild-type, synaptic or intrinsic neuronal function is hypersensitive to isoflurane in spinal cord slices from knockout mice. METHODS: Neurons from slices of the vestibular nucleus, central medial thalamus, and spinal cord from wild-type and the global Ndufs4 knockout were patch clamped. Unstimulated synaptic and intrinsic neuronal characteristics were measured in response to isoflurane. Norfluoxetine was used to block TREK channel conductance. Cholinergic cells were labeled with tdTomato. RESULTS: All values are reported as means and 95% CIs. Spontaneous synaptic activities were not different between the mutant and control. Isoflurane (0.6%; 0.25 mM; Ndufs4[KO] EC95) increased the holding current in knockout (ΔHolding current, 126 pA [95% CI, 99 to 152 pA]; ΔHolding current P < 0.001; n = 21) but not wild-type (ΔHolding current, 2 7 pA [95% CI, 9 to 47 pA]; ΔHolding current, P = 0.030; n = 25) spinal cord slices. Knockout and wild-type ΔHolding currents were significantly different (P < 0.001). Changes comparable to those in the knockout were seen in the wild type only in 1.8% (0.74 mM) isoflurane (ΔHolding current, 72 pA [95% CI, 43 to 97 pA]; ΔHolding current, P < 0.001; n = 13), the control EC95. Blockade of action potentials indicated that the increased holding current in the knockout was not dependent on synaptic input (ΔHolding current, 154 pA [95% CI, 99 to 232 pA]; ΔHolding current, P = 0.506 compared to knockout without blockade; n = 6). Noncholinergic neurons mediated the increase in holding current sensitivity in Ndufs4 knockout. The increased currents were blocked by norfluoxetine. CONCLUSIONS: Isoflurane increased an outwardly rectifying potassium current in ventral horn neurons of the Ndufs4(KO) mouse at a concentration much lower than in controls. Noncholinergic neurons in the spinal cord ventral horn mediated the effect. Presynaptic functions in Ndufs4(KO) slices were not hypersensitive to isoflurane. These data link anesthetic sensitivity, mitochondrial function, and postsynaptic channel activity.

Dysbiosis characteristics of gut microbiota in cerebral infarction patients.

OBJECTIVE: The aim of this study is to investigate the dysbiosis characteristics of gut microbiota in patients with cerebral infarction (CI) and its clinical implications. METHODS: Stool samples were collected from 79 CI patients and 98 healthy controls and subjected to 16S rRNA sequencing to identify stool microbes. Altered compositions and functions of gut microbiota in CI and its correlation with clinical features were investigated. Random forest and receiver operating characteristic analysis were used to develop a diagnostic model. RESULTS: Microbiota diversity and structure between CI patients and healthy controls were overall similar. However, butyrate-producing bacteria (BPB) were significantly reduced in CI patients, while lactic acid bacteria (LAB) were increased. Genetically, BPB-related functional genes were reduced in CI patients, whereas LAB-related genes were enhanced. The interbacterial correlations among BPB in CI patients were less prominent than those in healthy controls. Clinically, BPB was negatively associated with the National Institutes of Health Stroke Scale (NIHSS), while LAB was positively correlated with NIHSS. Both BPB and LAB played leading roles in the diagnostic model based on 47 bacteria. CONCLUSIONS: The abundance and functions of BPB in CI patients were significantly decreased, while LAB were increased. Both BPB and LAB displayed promising potential in the assessment and diagnosis of CI.

Impact of Novel Multiinstitutional Curriculum on Critical Care Fellow Ventilator Knowledge.

Background: Management of mechanical ventilation (MV) is a curricular milestone for trainees in pulmonary critical care medicine (PCCM) and critical care medicine (CCM) fellowships. Though recognition of ventilator waveform abnormalities that could result in patient complications is an important part of management, it is unclear how well fellows recognize these abnormalities.Objective: To study proficiency of ventilator waveform analysis among first-year fellows enrolled in a MV course compared with that of traditionally trained fellows.Methods: The study took place from July 2016 to January 2019, with 93 fellows from 10 fellowship programs completing the waveform examination. Seventy-three fellows participated in a course during their first year of fellowship, with part I occurring at the beginning of fellowship in July and part II occurring after 6 months of clinical work. These fellows were given a five-question ventilator waveform examination at multiple time points throughout the two-part course. Twenty fellows from three other fellowship programs who were in their first, second, or third year of fellowship and who did not participate in this course served as the control group. These fellows took the waveform examination a single time, at a median of 23 months into their training.Results: Before the course, scores were low but improved after 3 days of education at the beginning of the fellowship (18.0 ± 1.6 vs. 45.6 ± 3.0; P < 0.0001). Scores decreased after 6 months of clinical rotations but increased to their highest levels after part II of the course (33.7 ± 3.1 for part II pretest vs. 77.4 ± 2.4 for part II posttest; P < 0.0001). After completing part I at the beginning of fellowship, fellows participating in the course outperformed control fellows, who received a median of 23 months of traditional fellowship training at the time of testing (45.6 ± 3.0 vs. 25.3 ± 2.7; P < 0.0001). There was no difference in scores between PCCM and CCM fellows. In anonymous surveys, the fellows also rated the mechanical ventilator lectures highly.Conclusion: PCCM and CCM fellows do not recognize common waveform abnormalities at the beginning of fellowship but can be trained to do so. Traditional fellowship training may be insufficient to master ventilator waveform analysis, and a more intentional, structured course for MV may help fellowship programs meet the curricular milestones for MV.

Isolated cortical venous thrombosis after fetal demise.

Isolated cortical venous thrombosis (ICVT) occurring in the absence of dural venous thrombosis, constitutes about 2%-5% of all cerebral venous thrombosis. Its vague, non-specific presentation makes it a difficult and challenging diagnosis that needs an extensive workup especially in young patients. Outcome and prognosis depend mainly on early diagnosis and treatment. Here we discuss the clinical presentation, diagnosis and the treatment of a young woman diagnosed with ICVT with acute ischaemic venous stroke, in the setting of eclampsia and family history of coagulation disease.

Non-infectious pulmonary disorders in HIV.

INTRODUCTION: Since the advent of antiretroviral therapy (ART), non-infectious pulmonary disorders have become common comorbidities in the human immunodeficiency virus (HIV) positive population. Clinicians caring for those with HIV disease should be aware of the prevalence of non-infectious pulmonary disorders. A comprehensive understanding is required to diagnosis and manage these syndromes appropriately. Areas covered: This review focuses on the epidemiology, risk factors, pathogenesis, clinical feature and diagnosis, and treatment of HIV-related chronic obstructive pulmonary disease (COPD), lung cancer, pulmonary hypertension. Expert Commentary: The prevalence of COPD in the HIV population is frequent and requires appropriate diagnosis and treatment. HIV-positive individuals with lung cancer carry a poorer prognosis and require early diagnosis and treatment. A complex condition exists with pulmonary hypertension in the HIV population and requires a high degree of clinical suspicion for early diagnosis.

Practical Considerations in Sepsis Resuscitation.

BACKGROUND: Sepsis is a common condition managed in the emergency department, and the majority of patients respond to resuscitation measures, including antibiotics and i.v. fluids. However, a proportion of patients will fail to respond to standard treatment. OBJECTIVE: This review elucidates practical considerations for management of sepsis in patients who fail to respond to standard treatment. DISCUSSION: Early goal-directed therapy revolutionized sepsis management. However, there is a paucity of literature that provides a well-defined treatment algorithm for patients who fail to improve with therapy. Refractory shock can be defined as continued patient hemodynamic instability (mean arterial pressure, ≤ 65 mm Hg, lactate ≥ 4 mmol/L, altered mental status) after adequate fluid loading (at least 30 mL/kg i.v.), the use of two vasopressors (with one as norepinephrine), and provision of antibiotics. When a lack of improvement is evident in the early stages of resuscitation, systematically considering source control, appropriate volume resuscitation, adequate antimicrobial coverage, vasopressor selection, presence of metabolic pathology, and complications of resuscitation, such as abdominal compartment syndrome and respiratory failure, allow emergency physicians to address the entire clinical scenario. CONCLUSIONS: The care of sepsis has experienced many changes in recent years. Care of the patient with sepsis who is not responding appropriately to initial resuscitation is troublesome for emergency physicians. This review provides practical considerations for resuscitation of the patient with septic shock. When a septic patient is refractory to standard therapy, systematically evaluating the patient and clinical course may lead to improved outcomes.

Glutamatergic Neurotransmission Links Sensitivity to Volatile Anesthetics with Mitochondrial Function.

An enigma of modern medicine has persisted for over 150 years. The mechanisms by which volatile anesthetics (VAs) produce their effects (loss of consciousness, analgesia, amnesia, and immobility) remain an unsolved mystery. Many attractive putative molecular targets have failed to produce a significant effect when genetically tested in whole-animal models [1-3]. However, mitochondrial defects increase VA sensitivity in diverse organisms from nematodes to humans [4-6]. Ndufs4 knockout (KO) mice lack a subunit of mitochondrial complex I and are strikingly hypersensitive to VAs yet resistant to the intravenous anesthetic ketamine [7]. The change in VA sensitivity is the largest reported for a mammal. Limiting NDUFS4 loss to a subset of glutamatergic neurons recapitulates the VA hypersensitivity of Ndufs4(KO) mice, while loss in GABAergic or cholinergic neurons does not. Baseline electrophysiologic function of CA1 pyramidal neurons does not differ between Ndufs4(KO) and control mice. Isoflurane concentrations that anesthetize only Ndufs4(KO) mice (0.6%) decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) only in Ndufs4(KO) CA1 neurons, while concentrations effective in control mice (1.2%) decreased sEPSC frequencies in both control and Ndufs4(KO) CA1 pyramidal cells. Spontaneous inhibitory postsynaptic currents (sIPSCs) were not differentially affected between genotypes. The effects of isoflurane were similar on evoked field excitatory postsynaptic potentials (fEPSPs) and paired pulse facilitation (PPF) in KO and control hippocampal slices. We propose that CA1 presynaptic excitatory neurotransmission is hypersensitive to isoflurane in Ndufs4(KO) mice due to the inhibition of pre-existing reduced complex I function, reaching a critical reduction that can no longer meet metabolic demands.

A Longitudinal Regional Educational Model for Pulmonary and Critical Care Fellows Emphasizing Small Group- and Simulation-based Learning.

Recent trends have necessitated a renewed focus on how we deliver formal didactic and simulation experiences to pulmonary and critical care medicine (PCCM) fellows. To address the changing demands of training PCCM fellows, as well as the variability in the clinical training, fund of knowledge, and procedural competence of incoming fellows, we designed a PCCM curriculum that is delivered regionally in the Baltimore/Washington, DC area in the summer and winter. The educational curriculum began in 2008 as a collaboration between the Critical Care Medicine Department at the National Institutes of Health and the Pulmonary and Critical Care Section of the Department of Medicine at MedStar Washington Hospital Center and now includes 13 individual training programs in PCCM, critical care medicine, and pulmonary diseases in Baltimore and Washington, DC. Informal and formal feedback from the fellows who participated led to substantial changes to the course curriculum, allowing for continuous improvement. The educational consortium has helped build a local community of educators to share ideas, support each other's career development, and collaborate on other endeavors. In this article, we describe how we developed and deliver this curriculum and report on lessons learned.

Case series of Bartonella quintana blood culture-negative endocarditis in Washington, DC.

INTRODUCTION: Prior studies (predominantly from Europe) have demonstrated blood culture-negative endocarditis due to Bartonella. Our objective was to describe three cases of Bartonella quintana endocarditis identified within one year at a large hospital in Washington, DC, USA. CASE PRESENTATION: We constructed a descriptive case series from a retrospective review of medical records from April to December 2013 at an 800-bed urban hospital. All three patients (ages: 52, 55 and 57 years) were undomiciled/homeless men with a history of alcoholism. Although they had negative blood cultures, echocardiography demonstrated aortic/mitral valve perforation and regurgitation in one patient, aortic/mitral valve vegetation with mitral regurgitation in the second patient, and aortic valve vegetation with regurgitation in the third patient. The patients had positive Bartonella quintana serum immunoglobulin G (IgG) with negative immunoglobulin M (IgM). PCR on DNA extracted from cardiac valves was positive for Bartonella, and DNA sequencing of PCR amplicons identified Bartonella quintana. Patients received treatment with doxycycline/rifampin or doxycycline/gentamicin. CONCLUSION: Clinicians should consider Bartonella endocarditis as a differential diagnosis in patients who fit elements of the Duke Criteria, as well as having a history of homelessness and alcoholism.

The Role for Optical Density in Heparin-Induced Thrombocytopenia: A Cohort Study.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin utilization. An enzyme-linked immunosorbent assay (ELISA) is usually performed to assist in the diagnosis of HIT. ELISAs tend to be sensitive but lack specificity. We sought to use a new cutoff to define a positive HIT ELISA. METHODS: We conducted a prospective observational study of hospitalized patients undergoing ELISA testing. All patients who underwent ELISA testing were eligible for inclusion (n = 496). Irrespective of the results, all subjects had confirmatory testing with a serotonin release assay (SRA). We compared a threshold optical density (OD) > 1.00 to the current definition of a positive ELISA (OD > 0.40) as a screening test for a positive SRA. We used sensitivity, specificity, and area under the receiver operating curve to determine whether an OD > 1.00 would improve diagnostic accuracy for HIT. RESULTS: The SRA was positive in 10 patients (prevalence, 2.0%). Adjusting the definition of a positive HIT ELISA to > 1.00 maintained the sensitivity and negative predictive value at 100% in the cohort. The positive predictive value of the higher cutoff OD was more than triple the positive predictive value of an OD > 0.40 (41.7% vs 13.3%). No patient with a positive SRA had an OD measurement ≤ 1.00. CONCLUSIONS: Increasing the OD threshold enhances specificity without noticeably compromising sensitivity. Altering the definition of the HIT ELISA could prevent unnecessary testing and/or treatment with non-heparin-based anticoagulants in patients with possible HIT. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT00946400; URL: www.clinicaltrials.gov.