Increased Heparanase Levels in Urine during Acute Puumala Orthohantavirus Infection Are Associated with Disease Severity

Old–world orthohantaviruses cause hemorrhagic fever with renal syndrome (HFRS), characterized by acute kidney injury (AKI) with transient proteinuria. It seems plausible that proteinuria during acute HFRS is mediated by the disruption of the glomerular filtration barrier (GFB) due to vascular leakage, a hallmark of orthohantavirus–caused diseases. However, direct infection of endothelial cells by orthohantaviruses does not result in increased endothelial permeability, and alternative explanations for vascular leakage and diminished GFB function are necessary. Vascular integrity is partly dependent on an intact endothelial glycocalyx, which is susceptible to cleavage by heparanase (HPSE). To understand the role of glycocalyx degradation in HFRS–associated proteinuria, we investigated the levels of HPSE in urine and plasma during acute, convalescent and recovery stages of HFRS caused by Puumala orthohantavirus. HPSE levels in urine during acute HFRS were significantly increased and strongly associated with the severity of AKI and other markers of disease severity. Furthermore, increased expression of HPSE was detected in vitro in orthohantavirus–infected podocytes, which line the outer surfaces of glomerular capillaries. Taken together, these findings suggest the local activation of HPSE in the kidneys of orthohantavirus–infected patients with the potential to disrupt the endothelial glycocalyx, leading to increased protein leakage through the GFB, resulting in high amounts of proteinuria

Increased Heparanase Levels in Urine during Acute Puumala Orthohantavirus Infection Are Associated with Disease Severity

Old–world orthohantaviruses cause hemorrhagic fever with renal syndrome (HFRS), characterized by acute kidney injury (AKI) with transient proteinuria. It seems plausible that proteinuria during acute HFRS is mediated by the disruption of the glomerular filtration barrier (GFB) due to vascular leakage, a hallmark of orthohantavirus–caused diseases. However, direct infection of endothelial cells by orthohantaviruses does not result in increased endothelial permeability, and alternative explanations for vascular leakage and diminished GFB function are necessary. Vascular integrity is partly dependent on an intact endothelial glycocalyx, which is susceptible to cleavage by heparanase (HPSE). To understand the role of glycocalyx degradation in HFRS–associated proteinuria, we investigated the levels of HPSE in urine and plasma during acute, convalescent and recovery stages of HFRS caused by Puumala orthohantavirus. HPSE levels in urine during acute HFRS were significantly increased and strongly associated with the severity of AKI and other markers of disease severity. Furthermore, increased expression of HPSE was detected in vitro in orthohantavirus–infected podocytes, which line the outer surfaces of glomerular capillaries. Taken together, these findings suggest the local activation of HPSE in the kidneys of orthohantavirus–infected patients with the potential to disrupt the endothelial glycocalyx, leading to increased protein leakage through the GFB, resulting in high amounts of proteinuria

A comprehensive assessment of four whole blood stabilizers for flow-cytometric analysis of leukocyte populations

Though cryopreservation of cell fractions is widely used in flow cytometry studies, whole blood cryopreservation is more challenging due to the presence of erythrocytes and effects of fixatives commonly used for preservation. Here, we evaluated and compared head-to-head the performance of four commercial whole blood cryopreservation kits; (1) Cytodelics, (2) Stable-Lyse V2 and Stable-Store V2 (SLSS-V2), (3) Proteomic stabilizer (PROT-1), and (4) Transfix. We found that PROT-1, Transfix, and Cytodelics maintained the distribution of major leukocyte subsets-granulocytes, T cells, natural killer cells, and B cells, on a comparable level to unpreserved samples, despite the attenuation of fluorescence intensities in flow cytometric assays. Moreover, these three stabilizers also maintained the activated phenotypes of neutrophils upon stimulation with N-formylmethionyl-leucyl-phenylalanine and lipopolysaccharides. The upregulation of adhesion molecules (CD11b), Fc receptors (CD16), and granule proteins (CD66b), as well as the shedding of surface L-selectin (CD62L), was conserved most efficiently in PROT-1 and Cytodelics when compared to samples only treated with erythrocyte lysing. However, none of the stabilizers provided a reliable detection of CCR7 for accurate quantification of T cell maturation stages. We also evaluated the performance of Cytodelics in longitudinal clinical samples obtained from acute COVID-19 patients, where it allowed reliable detection of lymphopenia and granulocyte expansion. These results support the feasibility of whole blood cryopreservation for immunophenotyping by flow cytometry, particularly in longitudinal studies. In conclusion, the performance of different stabilizers is variable and therefore the choice of stabilizers should depend on cell type of interest, as well as antibody clones and experimental design of each study.Peer reviewe

Detecting Mutations in the Mycobacterium tuberculosis Pyrazinamidase Gene pncA to Improve Infection Control and Decrease Drug Resistance Rates in Human Immunodeficiency Virus Coinfection.

Hospital infection control measures are crucial to tuberculosis (TB) control strategies within settings caring for human immunodeficiency virus (HIV)-positive patients, as these patients are at heightened risk of developing TB. Pyrazinamide (PZA) is a potent drug that effectively sterilizes persistent Mycobacterium tuberculosis bacilli. However, PZA resistance associated with mutations in the nicotinamidase/pyrazinamidase coding gene, pncA, is increasing. A total of 794 patient isolates obtained from four sites in Lima, Peru, underwent spoligotyping and drug resistance testing. In one of these sites, the HIV unit of Hospital Dos de Mayo (HDM), an isolation ward for HIV/TB coinfected patients opened during the study as an infection control intervention: circulating genotypes and drug resistance pre- and postintervention were compared. All other sites cared for HIV-negative outpatients: genotypes and drug resistance rates from these sites were compared with those from HDM. HDM patients showed high concordance between multidrug resistance, PZA resistance according to the Wayne method, the two most common genotypes (spoligotype international type [SIT] 42 of the Latino American-Mediterranean (LAM)-9 clade and SIT 53 of the T1 clade), and the two most common pncA mutations (G145A and A403C). These associations were absent among community isolates. The infection control intervention was associated with 58-92% reductions in TB caused by SIT 42 or SIT 53 genotypes (odds ratio [OR] = 0.420, P = 0.003); multidrug-resistant TB (OR = 0.349, P < 0.001); and PZA-resistant TB (OR = 0.076, P < 0.001). In conclusion, pncA mutation typing, with resistance testing and spoligotyping, was useful in identifying a nosocomial TB outbreak and demonstrating its resolution after implementation of infection control measures

Hantavirus infection-induced B cell activation elevates free light chains levels in circulation

In humans, orthohantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). An earlier study reported that acute Andes virus HPS caused a massive and transient elevation in the number of circulating plasmablasts with specificity towards both viral and host antigens suggestive of polyclonal B cell activation. Immunoglobulins (Igs), produced by different B cell populations, comprise heavy and light chains; however, a certain amount of free light chains (FLCs) is constantly present in serum. Upregulation of FLCs, especially clonal species, associates with renal pathogenesis by fibril or deposit formations affecting the glomeruli, induction of epithelial cell disorders, or cast formation in the tubular network. We report that acute orthohantavirus infection increases the level of Ig FLCs in serum of both HFRS and HPS patients, and that the increase correlates with the severity of acute kidney injury in HFRS. The fact that the kappa to lambda FLC ratio in the sera of HFRS and HPS patients remained within the normal range suggests polyclonal B cell activation rather than proliferation of a single B cell clone. HFRS patients demonstrated increased urinary excretion of FLCs, and we found plasma cell infiltration in archival patient kidney biopsies that we speculate to contribute to the observed FLC excreta. Analysis of hospitalized HFRS patients' peripheral blood mononuclear cells showed elevated plasmablast levels, a fraction of which stained positive for Puumala virus antigen. Furthermore, B cells isolated from healthy donors were susceptible to Puumala virus in vitro, and the virus infection induced increased production of Igs and FLCs. The findings propose that hantaviruses directly activate B cells, and that the ensuing intense production of polyclonal Igs and FLCs may contribute to acute hantavirus infection-associated pathological findings. Author summary Orthohantaviruses are globally spread zoonotic pathogens, which can cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) with significant burden to human health. The pathogenesis mechanisms of orthohantavirus-caused diseases are not known in detail; however, excessive immune response towards the virus with concomitant pathological effects against host tissues appears to be a contributing factor. Here we report an increase of free immunoglobulin (Ig) light chains (FLCs), components required to make complete Ig molecules, in blood of acute HFRS and HPS. Samples collected during acute HFRS demonstrated increased FLCs levels in the urine and blood of patients hospitalized due the disease. Furthermore, the FLC levels positively correlated with markers of acute kidney injury. In addition, our results show that orthohantaviruses can infect and activate B cells to produce FLCs as well as whole Igs, which provides a mechanistic explanation of the increased FLC levels in patients. Taken together, our results suggest that aberrant antibody responses might play a role in the pathogenesis of orthantavirus infections.Peer reviewe

Characterization of low-density granulocytes in COVID-19

Author summary The emergence of SARS-COV-2 and the ensuing COVID-19 disease has revealed an unprecedented need to understand the pathological mechanisms of acute respiratory infections in more detail. Granulocytes are highly abundant cells of the innate immunity, and thus first responders towards acute infections. However, their excessive activation can cause unwanted tissue damage and detrimental effects in humans. This study identifies a population of low-density granulocytes (LDGs) in COVID-19 patient samples, which has been poorly described in the context of acute infections so far. These cells were subclassified and found to be mainly of immature phenotypes. Further characterization revealed COVID-19 LDGs as a phenotypically diverse population with immunosuppressive characteristics, which seemed to be in line with an elevated recruitment and activation of granulocytes. Altogether, these findings suggest LDG may play a role in COVID-19 disease progression. Severe COVID-19 is characterized by extensive pulmonary complications, to which host immune responses are believed to play a role. As the major arm of innate immunity, neutrophils are one of the first cells recruited to the site of infection where their excessive activation can contribute to lung pathology. Low-density granulocytes (LDGs) are circulating neutrophils, whose numbers increase in some autoimmune diseases and cancer, but are poorly characterized in acute viral infections. Using flow cytometry, we detected a significant increase of LDGs in the blood of acute COVID-19 patients, compared to healthy controls. Based on their surface marker expression, COVID-19-related LDGs exhibit four different populations, which display distinctive stages of granulocytic development and most likely reflect emergency myelopoiesis. Moreover, COVID-19 LDGs show a link with an elevated recruitment and activation of neutrophils. Functional assays demonstrated the immunosuppressive capacities of these cells, which might contribute to impaired lymphocyte responses during acute disease. Taken together, our data confirms a significant granulocyte activation during COVID-19 and suggests that granulocytes of lower density play a role in disease progression.Peer reviewe

Dynamics of Cough Frequency in Adults Undergoing Treatment for Pulmonary Tuberculosis.

Background: Cough is the major determinant of tuberculosis transmission. Despite this, there is a paucity of information regarding characteristics of cough frequency throughout the day and in response to tuberculosis therapy. Here we evaluate the circadian cycle of cough, cough frequency risk factors, and the impact of appropriate treatment on cough and bacillary load. Methods: We prospectively evaluated human immunodeficiency virus-negative adults (n = 64) with a new diagnosis of culture-proven, drug-susceptible pulmonary tuberculosis immediately prior to treatment and repeatedly until treatment day 62. At each time point, participant cough was recorded (n = 670) and analyzed using the Cayetano Cough Monitor. Consecutive coughs at least 2 seconds apart were counted as separate cough episodes. Sputum samples (n = 426) were tested with microscopic-observation drug susceptibility broth culture, and in culture-positive samples (n = 252), the time to culture positivity was used to estimate bacillary load. Results: The highest cough frequency occurred from 1 pm to 2 pm, and the lowest from 1 am to 2 am (2.4 vs 1.1 cough episodes/hour, respectively). Cough frequency was higher among participants who had higher sputum bacillary load (P < .01). Pretreatment median cough episodes/hour was 2.3 (interquartile range [IQR], 1.2-4.1), which at 14 treatment days decreased to 0.48 (IQR, 0.0-1.4) and at the end of the study decreased to 0.18 (IQR, 0.0-0.59) (both reductions P < .001). By 14 treatment days, the probability of culture conversion was 29% (95% confidence interval, 19%-41%). Conclusions: Coughs were most frequent during daytime. Two weeks of appropriate treatment significantly reduced cough frequency and resulted in one-third of participants achieving culture conversion. Thus, treatment by 2 weeks considerably diminishes, but does not eliminate, the potential for airborne tuberculosis transmission

Manejo de heridas por trauma en el servicio de urgencias

Todas las heridas, con independencia de su gravedad, pueden ser la consecuencia de un trastorno subyacente serio o la manifestación de una lesión con amenaza vital o para una extremidad. En dos publicaciones de la organización mundial de la salud se destaca que, a nivel mundial cada año mueren por traumatismos más de cinco millones de personas, lo cual representa una de cada 10 defunciones. El presente estudio es de tipo cuantitativo, descriptivo y de corte longitudinal – prospectivo, que nace de la necesidad de conocer el manejo, cuidado específico y tratamiento que se está llevando en la herida, para protocolizar el manejo de las heridas en urgencias de adultos de la Empresa Social del Estado Hospital Universitario Erasmo Meoz (ESE HUEM), y que sirva como base para crear la clínica de la herida en este servicio. La muestra se obtuvo por un muestreo no probabilístico por conveniencia y la recolección de información fue por observación y registro en lista de chequeo; se analizaron 49 pacientes que ingresaron al servicio de urgencias durante el mes de noviembre de 2007, con heridas por trauma; de esta muestra el 65% eran de sexo masculino; la mayor proporción de pacientes se encontraba en edades de los 15 – 25 años con un 37%. Sus profesiones en su mayoría eran comerciantes y albañiles con un 20% cada una; 25 pacientes consumen alcohol y/o tabaco. En cuanto a la técnica de limpieza de la herida la realiza la auxiliar de enfermería con un 42% y la técnica como tal es inadecuada en el 47% de los casos; en general se realiza una adecuada exploración de la herida y una buena elección de la sutura y la aguja a utilizar. Palabras claves: Valoración de Heridas, Técnica aséptica, Sutura.SUMMARYAll the wounds, independently of their gravity, can be the consequence of a serious underlying upheaval or the manifestation of an injury with vital threat or for an extremity. In two  publications of theWHOone stands out that, at world-wide level every year die by traumatismos more than five million people, which represents one of each 10 deaths. The present study is of type Quantitative, Descriptive and of longitudinal section - Prospective, that it is born of the necessity to know the handling, specific care and treatment that is taking in the wound, to formalize the handling of the wounds in Urgencies of Adults of that University Hospital Erasmo Meoz, that serves as it bases to create the Clinic of the  Wound for this service. The sample it was obtained by Sampling non Probabilístico by Convenience and the information harvesting was by observation and registry in control list,  49 patients who entered to the service of urgencies during the month of November of 2007, with hurt by trauma analyzed themselves; of this sample 65% were of masculine sex; the greater proportion of patients was in ages of the 15 - 25 years with a 37%. Their professions in their majority were retailers and bricklayers with a 20% each one; 25 patients consume Alcohol and/or Tobacco; as far as the technique of cleaning of the wound it makes the Aid of Infirmary with a 42% and the technique as so she is Inadequate in 47% of the cases; in general it is made a suitable exploration of the wound and a good election of the suture. Key Words: Valuation of Wounds, aseptic Technique, and Suture

Maturing neutrophils of lower density associate with thrombocytopenia in Puumala orthohantavirus-caused hemorrhagic fever with renal syndrome

Puumala orthohantavirus-caused hemorrhagic fever with renal syndrome (PUUV-HFRS) is characterized by strong neutrophil activation. Neutrophils are the most abundant immune cell type in the circulation and are specially equipped to rapidly respond to infections. They are more heterogenous than previously appreciated, with specific neutrophil subsets recently implicated in inflammation and immunosuppression. Furthermore, neutrophils can be divided based on their density to either low-density granulocytes (LDGs) or “normal density” polymorphonuclear cell (PMN) fractions. In the current study we aimed to identify and characterize the different neutrophil subsets in the circulation of PUUV-HFRS patients. PMNs exhibited an activation of antiviral pathways, while circulating LDGs were increased in frequency following acute PUUV-HFRS. Furthermore, cell surface marker expression analysis revealed that PUUV-associated LDGs are primarily immature and most likely reflect an increased neutrophil production from the bone marrow. Interestingly, both the frequency of LDGs and the presence of a “left shift” in blood associated with the extent of thrombocytopenia, one of the hallmarks of severe HFRS, suggesting that maturing neutrophils could play a role in disease pathogenesis. These results imply that elevated circulating LDGs might be a general finding in acute viral infections. However, in contrast to the COVID-19 associated LDGs described previously, the secretome of PUUV LDGs did not show significant immunosuppressive ability, which suggests inherent biological differences in the LDG responses that can be dependent on the causative virus or differing infection kinetics