Fetal cord plasma herpesviruses and preeclampsia: an observational cohort study.

A previous study suggested that fetal inheritance of chromosomally integrated human herpesvirus 6 (ici-HHV6) is associated with the hypertensive pregnancy disorder preeclampsia (PE). We aimed to study this question utilizing cord plasma samples (n = 1276) of the Finnish Genetics of Preeclampsia Consortium (FINNPEC) cohort: 539 from a pregnancy with PE and 737 without. We studied these samples and 30 placentas from PE pregnancies by a multiplex qPCR for the DNAs of all nine human herpesviruses. To assess the population prevalence of iciHHV-6, we studied whole-genome sequencing data from blood-derived DNA of 3421 biobank subjects. Any herpes viral DNA was detected in only two (0.37%) PE and one (0.14%) control sample (OR 2.74, 95% CI 0.25-30.4). One PE sample contained iciHHV-6B and another HHV-7 DNA. The control's DNA was of iciHHV-6B; the fetus having growth restriction and preterm birth without PE diagnosis. Placentas showed no herpesviruses. In the biobank data, 3 of 3421 subjects (0.08%) had low level HHV-6B but no iciHHV-6. While iciHHV-6 proved extremely rare, both fetuses with iciHHV-6B were growth-restricted, preterm, and from a pregnancy with maternal hypertension. Our findings suggest that human herpesviruses are not a significant cause of PE, whereas iciHHV-6 may pose some fetal risk.

Reactivation of a transplant recipient's inherited human herpesvirus 6 and implications to the graft.

BACKGROUND: The implications of inherited chromosomally integrated human herpesvirus 6 (iciHHV-6) in solid organ transplantation remain uncertain. Although this trait has been linked to unfavorable clinical outcomes, an association between viral reactivation and complications has only been conclusively established in a few cases. In contrast to these studies, which followed donor-derived transmission, our investigation is the first to examine the pathogenicity of a recipient´s iciHHV-6B and its impact on the graft. METHODS: We used hybrid capture sequencing for in-depth analysis of the viral sequences reconstructed from sequential liver biopsies. Moreover, we investigated viral replication through in situ hybridization (U38-U94 genes), real-time PCR (U89/U90 genes), immunohistochemistry, and immunofluorescence (against viral lysate). We also performed whole transcriptome sequencing of the liver biopsies to profile the host immune response. RESULTS: We report a case of reactivation of a recipient´s iciHHV-6B and subsequent infection of the graft. Using a novel approach integrating the analysis of viral and mitochondrial DNAs, we located the iciHHV-6B intra-graft. We demonstrated active replication via the emergence of viral minor variants across time points, in addition to positive viral mRNAs and antigen stainings in tissue sections. Furthermore, we detected significant upregulation of cell surface molecules, transcription factors, and cytokines associated with antiviral immune responses, arguing against immunotolerance. CONCLUSIONS: Our analysis underscores the potential pathological impact of iciHHV-6B, emphasizing the need for close monitoring of reactivation in transplant recipients. Most crucially, it highlights the critical role that the host's virome can play in shaping the outcome of transplantation, urging further investigations.

Presence of herpesviruses, parvoviruses, and polyomaviruses in sinonasal lymphoma.

PURPOSE: Sinonasal lymphoma (SL) is a rare lymphatic neoplasm of the nasal cavities, paranasal sinuses and nasopharynx. Whereas some risk factors for SL subtypes have been identified, their aetiology is unknown. Along with other predisposing factors, the viral association of lymphomas, such as Epstein-Barr virus (EBV) and Burkitt and Hodgkin lymphomas, is well-established. Modern molecular biology techniques have enabled the discovery of novel human viruses, exemplified by the protoparvovirus cutavirus (CuV), associated with cutaneous T-cell lymphoma. These findings, and the anatomical location of the sinonasal tract with its rich microbiome and infectious agents, justify in-depth studies among SL. METHODS: We analysed the presence of 20 viruses of Orthoherpesviridae, Parvoviridae, and Polyomaviridae by qPCR in 24 SL tumours. We performed RNAscope in situ hybridisation (RISH) to localize the viruses. Parvovirus-specific IgG was analysed by enzyme immunoassay and targeted next-generation sequencing (NGS) was applied to detect CuV in plasma. RESULTS: We detected viral DNA in 15/24 (63%) tumours; nine of EBV, six of human herpesvirus (HHV) -7, four each of HHV-6B and parvovirus B19, two of cytomegalovirus, and one each of CuV and Merkel-cell polyomavirus. We found tumours with up to four viruses per tumour, and localized CuV and EBV DNAs by RISH. Two of the ten plasma samples exhibited CuV IgG, and one plasma sample demonstrated CuV viremia by NGS. CONCLUSION: Viruses were frequent findings in SL. The EBV detection rate was high in diffuse large B-cell lymphoma, and co-detections with other viruses were prevalent.

The Cardiovascular Event Risk Associated with Tyrosine Kinase Inhibitors and the Lipid Profile in Patients with Chronic Myeloid Leukemia.

BACKGROUND: Second- and third-generation tyrosine kinase inhibitors (TKIs) are now available to treat chronic-phase chronic myeloid leukemia (CP-CML) in the first and second line. However, vascular adverse events (VAEs) have been reported for patients with CML treated with some TKIs. METHODS: We retrospectively evaluated the cumulative incidence (CI) and cardiovascular risk for 210 patients included in the Canarian Registry of CML. RESULT: With a mean follow up of 6 years, 19/210 (9.1%) patients developed VAEs, all of whom presented at least one cardiovascular risk factor at diagnosis. The mean time to VAE presentation was 54 months from the start of TKI treatment. We found a statistically significant difference between the CI for nilotinib-naïve vs. nilotinib-treated patients (p = 0.005), between dasatinib-naïve and dasatinib-treated patients (p = 0.039), and for patients who received three lines of treatment with first-line imatinib vs. first-line imatinib (p < 0.001). From the multivariable logistic regression analyses, the Framingham risk score (FRS) and patients with three lines of TKI with first-line imatinib were the only variables with statistically significant hazard ratios for VAE development. Significant increases in HDL-C and total cholesterol may also be predictive for VAE. CONCLUSIONS: In conclusion, it is important to estimate the cardiovascular risk at the diagnosis of CML as it can help determine whether a patient is likely to develop a VAE during TKI treatment.

Presence of Myeloid Mutations in Patients with Chronic Myeloid Leukemia Increases Risk of Cardiovascular Event on Tyrosine Kinase Inhibitor Treatment.

For chronic myeloid leukemia (CML) patients with a known risk of cardiovascular events (CVE), imatinib is often recommended for first-line tyrosine kinase inhibitor (TKI) treatment rather than a second-generation TKI (2G-TKI) such as nilotinib or dasatinib. To date, very few studies have evaluated the genetic predisposition associated with CVE development on TKI treatment. In this retrospective study of 102 CML patients, 26 CVEs were reported during an average follow-up of over 10 years. Next-generation sequencing identified pathogenic/likely pathogenic mutations in genes associated with myeloid malignancies in 24.5% of the diagnostic samples analyzed. Patients with a recorded CVE had more myeloid mutations (0.48 vs. 0.14, p = 0.019) and were older (65.1 vs. 55.7 years, p = 0.016). Age ≥ 60 years and receiving a 2G-TKI in first-line were CVE risk factors. The presence of a pathogenic somatic myeloid mutation was an independent risk factor for CVE on any TKI (HR 2.79, p = 0.01), and significantly shortened the CV event-free survival of patients who received first-line imatinib (by 70 months, p = 0.011). Indeed, 62% of patients on imatinib with mutations had a CVE vs. the 19% on imatinib with a mutation and no CVE. In conclusion, myeloid mutations detectable at diagnosis increase CVE risk, particularly for patients on imatinib, and might be considered for first-line TKI choice.

Electroencefalografía básica para monitores de profundidad anestésica / Basic electroencephalography for anesthesia depth monitors

Resumen: Los monitores de profundidad anestésica permiten guiar el estado hipnótico del paciente durante la anestesia general. Debido a su sencillez, tradicionalmente se han empleado índices de profundidad anestésica, obtenidos a través del procesamiento del electroencefalograma mediante algoritmos matemáticos, para orientar la monitorización del nivel de consciencia. Sus beneficios han sido ampliamente recogidos en la literatura científica; sin embargo, no están exentos de importantes limitaciones. No todos los anestésicos actúan en las mismas dianas moleculares ni dichos índices tienen en cuenta las características propias del paciente (comorbilidades, edades extremas, etcétera). Estas limitaciones podrían reducirse si interpretamos directamente toda la información que nos ofrecen los monitores. Presentamos una revisión que describe los conceptos básicos necesarios para su valoración directa, así como su correlación con los estados de profundidad anestésica del paciente.

Abstract: Anesthesia depth monitors allow to guide the patient's hypnotic state during general anesthesia. Traditionally, anesthetic depth indices have been used due to their simplicity to guide the monitoring of the level of consciousness. They have been obtained by processing the electroencephalogram using mathematical algorithms and their benefits have been widely reported in the scientific literature. However, they are not exempt from important limitations. Neither all anesthetics act on the same molecular targets, nor these mentioned indices take into account the patient's own characteristics (comorbidities, extreme ages, etc.). These limitations could be far reduced if we are able to understand all the information provided by the monitors. We present a review describing the basic concepts necessary for its direct assessment, as well as their correlation with the patient's anesthetic depth states.

Herpesviruses, polyomaviruses, parvoviruses, papillomaviruses, and anelloviruses in vestibular schwannoma.

Etiology of vestibular schwannoma (VS) is unknown. Viruses can infect and reside in neural tissues for decades, and new viruses with unknown tumorigenic potential have been discovered. The presence of herpesvirus, polyomavirus, parvovirus, and anellovirus DNA was analyzed by quantitative PCR in 46 formalin-fixed paraffin-embedded VS samples. Five samples were analyzed by targeted next-generation sequencing. Viral DNA was detected altogether in 24/46 (52%) tumor samples, mostly representing anelloviruses (46%). Our findings show frequent persistence of anelloviruses, considered normal virome, in VS. None of the other viruses showed an extensive presence, thereby suggesting insignificant role in VS.

Unmasking the tissue-resident eukaryotic DNA virome in humans.

Little is known on the landscape of viruses that reside within our cells, nor on the interplay with the host imperative for their persistence. Yet, a lifetime of interactions conceivably have an imprint on our physiology and immune phenotype. In this work, we revealed the genetic make-up and unique composition of the known eukaryotic human DNA virome in nine organs (colon, liver, lung, heart, brain, kidney, skin, blood, hair) of 31 Finnish individuals. By integration of quantitative (qPCR) and qualitative (hybrid-capture sequencing) analysis, we identified the DNAs of 17 species, primarily herpes-, parvo-, papilloma- and anello-viruses (>80% prevalence), typically persisting in low copies (mean 540 copies/ million cells). We assembled in total 70 viral genomes (>90% breadth coverage), distinct in each of the individuals, and identified high sequence homology across the organs. Moreover, we detected variations in virome composition in two individuals with underlying malignant conditions. Our findings reveal unprecedented prevalences of viral DNAs in human organs and provide a fundamental ground for the investigation of disease correlates. Our results from post-mortem tissues call for investigation of the crosstalk between human DNA viruses, the host, and other microbes, as it predictably has a significant impact on our health.

Uso y resultados del astegolimab en el manejo del asma severa: ¿qué se conoce?

El asma es una enfermedad inflamatoria crónica de las vías respiratorias que acarrea elevados costos en salud, afecta sustancialmente la calidad de vida y, dependiendo de ciertos factores de riesgo asociados, disminuye la capacidad funcional de quien lo padece. Para el 2019, el asma afectó a 262 millones de personas (4,3 % de la población mundial) y causó 461 000 muertes. Se estima que habrá 100 millones de personas adicionales con asma para el año 2025. El asma severa es un fenotipo resistente a corticoides que ocasiona mayor número de exacerbaciones, afecta sustancialmente la calidad de vida y capacidad funcional del afectado. Su manejo inicialmente se encamina a suprimir los síntomas, y este ha ido evolucionando hasta la comprensión, aún no completa, de los sistemas intrínsecos de su generación, con lo cual se han estudiado nuevas formas de incidir en su manejo, mediante la modulación de la respuesta inmune y la cascada inflamatoria, con la generación de medicamentos biológicos. A raíz del estudio e identificación de endotipos y fenotipos variados, se han diseñado este tipo de medicamentos, con distintos mecanismos de acción, que han demostrado una utilidad sólida en los últimos años. No obstante, existe evidencia de que se ha encontrado resistencia incluso a estos medicamentos, por lo que ha sido necesario seguir investigando nuevas dianas terapéuticas. El astegolimab es un novedoso anticuerpo monoclonal Ig G2 humano que bloquea la señalización de IL-33 al dirigirse a ST2, su receptor, por consiguiente, controla la respuesta inflamatoria en el asma severa. Actualmente, se encuentra en realización de ensayo clínico fase 2b, aunque experimentaciones previas han encontrado resultados positivos y significativos respecto a la inmunomodulación, función pulmonar, sintomatología y calidad de vida. En la actualidad, casi no existe literatura que haya analizado el potencial del astegolimab en el asma grave, y están disponibles prácticamente solo los ensayos que lo han evaluado y algunas revisiones que han compartido su farmacocinética y farmacodinamia. Sobre la base de lo anterior, el objetivo de esta revisión consiste en sintetizar evidencia relacionada con los resultados del uso del astegolimab en asma severa, discutiendo aspectos epidemiológicos y fisiopatológicos que resalten la necesidad del desarrollo de un fármaco seguro, eficaz y eficiente.

Asthma is a chronic inflammatory disease of the respiratory tract which causes high health costs, substantially affects the quality of life and, depending on certain associated risk factors, reduces the functional capacity of the sufferer. By 2019, asthma affected 262 million people (4.3 % of the world's population) and caused 461,000 deaths. It is estimated that there will be an additional 100 million people with asthma by 2025. Severe asthma is a phenotype resistant to corticosteroids which causes a greater number of exacerbations and substantially affects the quality of life and functional capacity of the affected person. Its management was initially aimed at suppressing the symptoms and then evolved to understand, although not completely, the intrinsic systems of its generation. Thus, new ways of influencing its management have been studied by modulating the immune response and the inflammatory cascade with the generation of biological drugs. As a result of the study and identification of various endotypes and phenotypes, drugs with different mechanisms of action have been designed and have demonstrated to be considerably useful in recent years. However, there is evidence that resistance even to these drugs has occurred, being necessary to continue researching new therapeutic targets. Astegolimab is a novel human IgG2 monoclonal antibody that blocks IL-33 signaling by targeting ST2, its receptor, thus controlling the inflammatory response in severe asthma. A phase 2b clinical trial is currently undergoing, although previous results have found positive and significant results regarding immunomodulation, pulmonary function, symptomatology and quality of life. At present, there is almost no literature that has analyzed the potential of astegolimab in severe asthma, and practically only trials that have evaluated it and some reviews that have shared its pharmacokinetics and pharmacodynamics are available. Based on the above, the aim of this review is to synthesize evidence related to the results of the use of astegolimab in severe asthma and discuss epidemiological and pathophysiological aspects that highlight the need for the development of a safe, effective and efficient drug.

Detection of Low-Copy Human Virus DNA upon Prolonged Formalin Fixation.

Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (±paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63-159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (<250 copies/million cells) and longer PCR amplicons (>150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials.

Survey of Viral Reactivations in Elite Athletes: A Case-Control Study.

Exercise-induced immune perturbations have been proposed to increase susceptibility to viral infections. We investigated the replication of persisting viruses as indicators of immune function in elite cross-country skiers after ten months of sustained high-performance exercise. The viruses evaluated, nine human herpesviruses (HHVs) and torque teno virus (TTV), are typically restrained in health but replicate actively in immunosuppressed individuals. We collected sera from 27 Finnish elite cross-country skiers at the end of the competition's season and 27 matched controls who perform moderate exercise. We quantified all the HHVs and-TTV via highly sensitive qPCRs. To verify equal past exposures between the groups, we assessed the IgG antibody prevalences toward HHV-4 (Epstein-Barr virus, EBV) and HHV-5 (human cytomegalovirus, HCMV). We found equal TTV DNA prevalences in athletes (63%) and controls (63%) and loads with respective geometric means of 1.7 × 103 and 1.2 × 103 copies/mL of serum. Overall, the copy numbers were low and consistent with those of healthy individuals. Neither of the groups presented with herpesvirus viremia despite similar past exposures to HHVs (seroprevalences of EBV 70% vs. 78% and HCMV 52% vs. 44% in athletes and controls, respectively). We found no evidence of increased replication of persistent viruses in elite athletes, arguing against impaired viral immunity due to high-performance exercise.

Comparison of approaches for IgG avidity calculation and a new highly sensitive and specific method with broad dynamic range.

BACKGROUND: Antimicrobial IgG avidity is measured in the diagnosis of infectious disease, for dating of primary infection or immunization. It is generally determined by either of two approaches, termed here the avidity index (AI) or end-point ratio (EPR), which differ in complexity and workload. While several variants of these approaches have been introduced, little comparative information exists on their clinical utility. METHODS: This study was performed to systematically compare the performances of these approaches and to design a new sensitive and specific calculation method, for easy implementation in the laboratory. The avidities obtained by AI, EPR, and the newly developed approach were compared, across parvovirus B19, cytomegalovirus, Toxoplasma gondii, rubella virus, and Epstein-Barr virus panels comprising 460 sera from individuals with a recent primary infection or long-term immunity. RESULTS: With optimal IgG concentrations, all approaches performed equally, appropriately discriminating primary infections from past immunity (area under the receiver operating characteristic curve (AUC) 0.93-0.94). However, at lower IgG concentrations, the avidity status (low, borderline, high) changed in 17% of samples using AI (AUC 0.88), as opposed to 4% using EPR (AUC 0.91) and 6% using the new method (AUC 0.93). CONCLUSIONS: The new method measures IgG avidity accurately, in a broad range of IgG levels, while the popular AI approach calls for a sufficiently high antibody concentration.

The Human Bone Marrow Is Host to the DNAs of Several Viruses.

The long-term impact of viruses residing in the human bone marrow (BM) remains unexplored. However, chronic inflammatory processes driven by single or multiple viruses could significantly alter hematopoiesis and immune function. We performed a systematic analysis of the DNAs of 38 viruses in the BM. We detected, by quantitative PCRs and next-generation sequencing, viral DNA in 88.9% of the samples, up to five viruses in one individual. Included were, among others, several herpesviruses, hepatitis B virus, Merkel cell polyomavirus and, unprecedentedly, human papillomavirus 31. Given the reactivation and/or oncogenic potential of these viruses, their repercussion on hematopoietic and malignant disorders calls for careful examination. Furthermore, the implications of persistent infections on the engraftment, regenerative capacity, and outcomes of bone marrow transplantation deserve in-depth evaluation.

Persistence of Human Bocavirus 1 in Tonsillar Germinal Centers and Antibody-Dependent Enhancement of Infection.

Human bocavirus 1 (HBoV1), a nonenveloped single-stranded DNA parvovirus, causes mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children. HBoV1 often persists in nasopharyngeal secretions for months, hampering diagnosis. It has also been shown to persist in pediatric palatine and adenoid tonsils, which suggests that lymphoid organs are reservoirs for virus spread; however, the tissue site and host cells remain unknown. Our aim was to determine, in healthy nonviremic children with preexisting HBoV1 immunity, the adenotonsillar persistence site(s), host cell types, and virus activity. We discovered that HBoV1 DNA persists in lymphoid germinal centers (GCs), but not in the corresponding tonsillar epithelium, and that the cell types harboring the virus are mainly naive, activated, and memory B cells and monocytes. Both viral DNA strands and both sides of the genome were detected, as well as infrequent mRNA. Moreover, we showed, in B-cell and monocyte cultures and ex vivo tonsillar B cells, that the cellular uptake of HBoV1 occurs via the Fc receptor (FcγRII) through antibody-dependent enhancement (ADE). This resulted in viral mRNA transcription, known to occur exclusively from double-stranded DNA in the nucleus, however, with no detectable productive replication. Confocal imaging with fluorescent virus-like particles moreover disclosed endocytosis. To which extent the active HBoV1 GC persistence has a role in chronic inflammation or B-cell maturation disturbances, and whether the virus can be reactivated, will be interesting topics for forthcoming studies.IMPORTANCE Human bocavirus 1 (HBoV1), a common pediatric respiratory pathogen, can persist in airway secretions for months hampering diagnosis. It also persists in tonsils, providing potential reservoirs for airway shedding, with the exact location, host cell types, and virus activity unknown. Our study provides new insights into tonsillar HBoV1 persistence. We observed HBoV1 persistence exclusively in germinal centers where immune maturation occurs, and the main host cells were B cells and monocytes. In cultured cell lines and primary tonsillar B cells, we showed the virus uptake to be significantly enhanced by HBoV1-specific antibodies, mediated by the cellular IgG receptor, leading to viral mRNA synthesis, but without detectable productive replication. Possible implications of such active viral persistence could be tonsillar inflammation, disturbances in immune maturation, reactivation, or cell death with release of virus DNA, explaining the long-lasting HBoV1 airway shedding.

A hybrid pipeline for reconstruction and analysis of viral genomes at multi-organ level.

BACKGROUND: Advances in sequencing technologies have enabled the characterization of multiple microbial and host genomes, opening new frontiers of knowledge while kindling novel applications and research perspectives. Among these is the investigation of the viral communities residing in the human body and their impact on health and disease. To this end, the study of samples from multiple tissues is critical, yet, the complexity of such analysis calls for a dedicated pipeline. We provide an automatic and efficient pipeline for identification, assembly, and analysis of viral genomes that combines the DNA sequence data from multiple organs. TRACESPipe relies on cooperation among 3 modalities: compression-based prediction, sequence alignment, and de novo assembly. The pipeline is ultra-fast and provides, additionally, secure transmission and storage of sensitive data. FINDINGS: TRACESPipe performed outstandingly when tested on synthetic and ex vivo datasets, identifying and reconstructing all the viral genomes, including those with high levels of single-nucleotide polymorphisms. It also detected minimal levels of genomic variation between different organs. CONCLUSIONS: TRACESPipe's unique ability to simultaneously process and analyze samples from different sources enables the evaluation of within-host variability. This opens up the possibility to investigate viral tissue tropism, evolution, fitness, and disease associations. Moreover, additional features such as DNA damage estimation and mitochondrial DNA reconstruction and analysis, as well as exogenous-source controls, expand the utility of this pipeline to other fields such as forensics and ancient DNA studies. TRACESPipe is released under GPLv3 and is available for free download at https://github.com/viromelab/tracespipe.

The landscape of persistent human DNA viruses in femoral bone.

The imprints left by persistent DNA viruses in the tissues can testify to the changes driving virus evolution as well as provide clues on the provenance of modern and ancient humans. However, the history hidden in skeletal remains is practically unknown, as only parvovirus B19 and hepatitis B virus DNA have been detected in hard tissues so far. Here, we investigated the DNA prevalences of 38 viruses in femoral bone of recently deceased individuals. To this end, we used quantitative PCRs and a custom viral targeted enrichment followed by next-generation sequencing. The data was analyzed with a tailor-made bioinformatics pipeline. Our findings revealed bone to be a much richer source of persistent DNA viruses than earlier perceived, discovering ten additional ones, including several members of the herpes- and polyomavirus families, as well as human papillomavirus 31 and torque teno virus. Remarkably, many of the viruses found have oncogenic potential and/or may reactivate in the elderly and immunosuppressed individuals. Thus, their persistence warrants careful evaluation of their clinical significance and impact on bone biology. Our findings open new frontiers for the study of virus evolution from ancient relics as well as provide new tools for the investigation of human skeletal remains in forensic and archaeological contexts.

HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses.

Infections with the nine human herpesviruses (HHVs) are globally prevalent and characterized by lifelong persistence. Reactivations can potentially manifest as life-threatening conditions for which the demonstration of viral DNA is essential. In the present study, we developed HERQ-9, a pan-HHV quantitative PCR designed in triplex reactions to differentiate and quantify each of the HHV-DNAs: (i) herpes simplex viruses 1 and 2 and varicella-zoster virus; (ii) Epstein-Barr virus, human cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus; and (iii) HHV-6A, -6B, and -7. The method was validated with prequantified reference standards as well as with mucocutaneous swabs and cerebrospinal fluid, plasma, and tonsillar tissue samples. Our findings highlight the value of multiplexing in the diagnosis of many unsuspected, yet clinically relevant, herpesviruses. In addition, we report here frequent HHV-DNA co-occurrences in clinical samples, including some previously unknown. HERQ-9 exhibited high specificity and sensitivity (LOD95s of ∼10 to ∼17 copies/reaction), with a dynamic range of 101 to 106 copies/µl. Moreover, it performed accurately in the coamplification of both high- and low-abundance targets in the same reaction. In conclusion, we demonstrated that HERQ-9 is suitable for the diagnosis of a plethora of herpesvirus-related diseases. Besides its significance to clinical management, the method is valuable for the assessment of hitherto-unexplored synergistic effects of herpesvirus coinfections. Furthermore, its high sensitivity enables studies on the human virome, often dealing with minute quantities of persisting HHVs.IMPORTANCE By adulthood, almost all humans become infected by at least one herpesvirus (HHV). The maladies inflicted by these microbes extend beyond the initial infection, as they remain inside our cells for life and can reactivate, causing severe diseases. The diagnosis of active infection by these ubiquitous pathogens includes the detection of DNA with sensitive and specific assays. We developed the first quantitative PCR assay (HERQ-9) designed to identify and quantify each of the nine human herpesviruses. The simultaneous detection of HHVs in the same sample is important since they may act together to induce life-threatening conditions. Moreover, the high sensitivity of our method is of extreme value for assessment of the effects of these viruses persisting in our body and their long-term consequences on our health.

Thrombotic Risk Detection in Patients with Polycythemia Vera: The Predictive Role of DNMT3A/TET2/ASXL1 Mutations.

The development of thrombotic events is common among patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). We studied the influence of pathogenic mutations frequently associated with myeloid malignancies on thrombotic events using next-generation sequencing (NGS) in an initial cohort of 68 patients with myeloproliferative neoplasms (MPN). As expected, the presence of mutations in DNMT3A, TET2, and ASXL1 (DTA genes) was positively associated with age for the whole cohort (p = 0.025, OR: 1.047, 95% CI: 1.006-1.090). Also, while not related with events in the whole cohort, DTA mutations were strongly associated with the development of vascular events in PV patients (p = 0.028). To confirm the possible association between the presence of DTA mutation and thrombotic events, we performed a case-control study on 55 age-matched patients with PV (including 12 PV patients from the initial cohort, 25 with event vs. 30 no event). In the age-matched case-control PV cohort, the presence of ≥1 DTA mutation significantly increased the risk of a thrombotic event (OR: 6.333, p = 0.0024). Specifically, mutations in TET2 were associated with thrombotic events in the PV case-control cohort (OR: 3.56, 95% CI: 1.15-11.83, p = 0.031). Our results suggest that pathogenic DTA mutations, and particularly TET2 mutations, may be an independent risk factor for thrombosis in patients with PV. However, the predictive value of TET2 and DTA mutations in ET and PMF was inconclusive and should be determined in a larger cohort.

Características Epidemiológicas en la Unidad de Cuidados IntensivosPediátricos, Hospital General Docente de Calderón, Quito / Epidemiological Characteristics in the Pediatric Intensive Care Unit, Calderón General Teaching Hospital, Quito

Introducción: La valoración de la gravedad, inestabilidad clínica y pronóstico es un componente vital a enfrentar en las Unidades de Cuidados Intensivos Pediátricos (UCIP). Por tal razón, la evaluación sistemática de los indicadores epidemiológicos en dichos servicios desempeña un papel fundamental en los servicios públicos de salud. El objetivo del presente estudio fue identificar las características epidemiológicas en la Unidad de Cuidados Intensivos del Hospital General Docente de Calderón, Quito, en el período 2018-2019. Métodos: Estudio descriptivo, retrospectivo y transversal basado en la revisión de historias clínicas de todos los pacientes pediátricos ingresados en el período 2018-2019, considerando las variables edad, género, causa de ingreso, apoyo ventilatorio, infecciones asociadas a cuidados de la salud (IACS), estancia en la UCIP y causa de egreso. Resultados: 217 casos fueron incluidos. Hubo predominio del sexo masculino 130/217 (60%) y en el grupo etario predominaron los lactantes 116/217(53%). Las infecciones fueron la principal causa de ingreso 137/217(63%). Un sector importante de pacientes necesitó ventilación mecánica invasiva 84/217(38.7%); las tasas de IACS fueron mínimas (neumonía 3.2%; infección de catéter de vía central 2.3%); el promedio de estancia hospitalaria en la UCIP fue 4.8±0.8 días y la mortalidad fue de 5.99% (IC95 5.78-6.21%). Conclusión: Los resultados concuerdan con los reportes internacionales en mortalidad, prevalencia y epidemiología

Introduction: The assessment of severity, clinical instability and prognosis is a vital component to face in the Pediatric Intensive Care Units (PICU). For this reason, the systematic evaluation of epidemiological indicators in these services plays a fundamental role in public health services. The objective of this study was to identify the epidemiological characteristics in the Intensive Care Unit of the General Teaching Hospital of Calderón, Quito, in the period 2018-2019. Methods: Descriptive, retrospective and cross-sectional study based on the review of medical records of all pediatric patients admitted in the period 2018-2019, considering the variables age, gender, cause of admission, ventilatory support, infections associated with health care ( IACS), stay in the PICU and cause of discharge. Results: 217 cases were included. There was a predominance of males 130/217 (60%) and in the age group, infants 116/217 (53%) predominated. Infections were the main cause of admission 137/217 (63%). An important sector of patients required invasive mechanical ventilation 84/217 (38.7%); IACS rates were minimal (pneumonia 3.2%; central line catheter infection 2.3%); the mean hospital stay in the PICU was 4.8 ± 0.8 days and mortality was 5.99% (CI95 5.78-6.21%). Conclusion: The results agree with international reports on mortality, prevalence and epidemiology

Experiencia clínica con la implantación de un protocolo multidisciplinar de cefalea postpunción dural / Clinical experience with the implementation of a postdural puncture headache multidisciplinary protocol

INTRODUCCIÓN: La cefalea postpunción dural (CPPD) es una complicación de la punción lumbar, práctica utilizada por múltiples especialistas con diferentes fines, y de la punción dural accidental (PDA) en una técnica epidural. El objetivo de disponer de un protocolo de manejo de la CPPD, con implicación multidisciplinar, es diagnosticar y tratar precozmente a cualquier paciente con CPPD, independientemente de qué especialidad observe la complicación. MATERIAL Y MÉTODOS: Estudio descriptivo, observacional y retrospectivo en el que se analiza la utilidad clínica de un protocolo de manejo de CPPD entre septiembre de 2014 y diciembre de 2017. RESULTADOS: Se analizaron un total de 60 interconsultas. Se observó una incidencia de PDA del 0,61 %, el 80 % de las PDA desarrollaron CPPD, y de ellas, el 72 % precisó un parche hemático. Con el seguimiento telefónico, 8 pacientes que inicialmente fueron dados de alta con CPPD leve precisaron reingreso por empeoramiento clínico, y se diagnosticó un paciente con parálisis del VI par craneal. Los especialistas que incluyeron pacientes en el protocolo fueron: anestesiólogos, obstetras, médicos de urgencias y neurólogos. CONCLUSIONES: El protocolo permitió un diagnóstico y tratamiento precoz de la CPPD, además de un seguimiento tras el alta hospitalaria del paciente

INTRODUCTION: Postdural puncture headache (PDPH) is a lumbar puncture complication, this practice is used by several medical specialists for different purposes as well as the accidental dural puncture (ADP) in an epidural technique. The objective of a PDPH multidisciplinary management protocol, is to diagnose and early treat any patient with PDPH, no matter which speciality observes this complication. MATERIAL AND METHODS: A descriptive, observational and retrospective study in which the clinical helpfulness of a PDPH management protocol is analyzed between September 2014 and December 2017. RESULTS: A total of 60 consult visits were analyzed. An incidence of ADP of 0.61 % was observed, 80 % of them developed PDPH, and out of these, 72 % required an epidural blood patching. Re-entry due to clinical worsening was required by 8 patients on telephone follow-up, who were initially discharged with mild PDPH, and one of them was diagnosed with abducens nerve palsy. Medical specialities with patients in this protocol were: anesthesiologists, obstetricians, emergency physicians and neurologists. CONCLUSIONS: The protocol allowed a diagnosis and early treatment of the PDPH as well as supplied the patient's follow-up after his hospital discharge