Low gH/gL (Sub)Species-Specific Antibody Levels Indicate Elephants at Risk of Fatal Elephant Endotheliotropic Herpesvirus Hemorrhagic Disease.

Elephant endotheliotropic herpesviruses (EEHVs), of which eleven (sub)species are currently distinguished, infect either Asian (Elephas maximus) or African elephants (Loxodonta species). While all adult elephants are latently infected with at least one EEHV (sub)species, young elephants, specifically those with low to non-detectable EEHV-specific antibody levels, may develop fatal hemorrhagic disease (EEHV-HD) upon infection. However, animals with high antibody levels against EEHV(1A) gB, an immunodominant antigen recognized by antibodies elicited against multiple (sub)species, may also occasionally succumb to EEHV-HD. To better define which animals are at risk of EEHV-HD, gB and gH/gL ELISAs were developed for each of the Asian elephant EEHV subspecies and assessed using 396 sera from 164 Asian elephants from European zoos. Antibody levels measured against gB of different (sub)species correlated strongly with one another, suggesting high cross-reactivity. Antibody levels against gH/gL of different subspecies were far less correlated and allowed differentiation between these (sub)species. Importantly, while high gB-specific antibody levels were detected in the sera of several EEHV-HD fatalities, all fatalities (n = 23) had low antibody levels against gH/gL of the subspecies causing disease. Overall, our data indicate that (sub)species-specific gH/gL ELISAs can be used to identify animals at risk of EEHV-HD when infected with a particular EEHV (sub)species.

Assessment of rare bleeding disorders in adolescents with heavy menstrual bleeding.

INTRODUCTION: There are a significant number of patients with mucocutaneous bleeding, specifically heavy menstrual bleeding (HMB), who do not have a diagnosed bleeding disorder. These patients receive nontargeted interventions and may have suboptimal treatments. Functional assays, particularly for fibrinolytic and rare platelet function defects, are not robust and not readily available. AIM: We aimed to prospectively evaluate the prevalence of genetic defects associated with rare bleeding disorders and describe alterations of coagulation and fibrinolysis in a cohort of adolescents with HMB. METHODS: We performed a prospective observational cohort study of patients with HMB and unexplained bleeding. The study utilized a next generation sequencing panel and investigational global assays of coagulation and fibrinolysis. Additionally, specific functional assays were performed to help characterize novel variants that were identified. RESULTS: In 10 of the 17 patients (∼59%), genetic variants were identified on molecular testing. Thrombin generation by calibrated thromboelastography was not significantly altered in this patient population. The clot formation and lysis assay showed a trend towards increased fibrinolysis with rapid phase of decline in 23% of the patients. Further corresponding functional assays and study population are described. CONCLUSION: Our study describes a unique correlative model in a homogenous cohort of patients with HMB and unexplained bleeding which may inform future diagnostic algorithms, genotype-phenotype correlations as well as aid in specific targeted treatment approaches. Larger future studies may inform risk stratification of patients and improve health related outcomes in patients with HMB.

Domestic pigs are susceptible to experimental infection with non-human primate-derived Reston virus without the need for adaptation.

Domestic pigs are a critical component of the food supply and one of the most commonly raised production animals. Pork consumption has driven the intensification of pig production expanding into environments conducive to increased emergence and spread of infectious diseases, including the spillover of pathogens into human populations. One of these emerging viruses, Reston virus (RESTV), is an enigma among the Orthoebolavirus genus in that its lack of human pathogenicity is in stark contrast to the high virulence associated with most other ebolaviruses. RESTV is, however, associated with outbreaks of highly lethal hemorrhagic disease in non-human primates (NHP), as well as poorly understood clinical manifestations of mixed virulence and lethality in naturally and experimentally infected domestic pigs. Our results show it is possible for RESTV derived from an NHP to infect domestic pigs resulting in a spectrum of disease, from asymptomatic to severe respiratory distress. Further, we report on the first experimental transmission of RESTV between infected pigs and a co-housed, naïve animal, as well as the first report of the successful use of group oral fluids for the detection of RESTV RNA and virus-specific IgA antibodies.

Apoptotic Cell Death in an Animal Model of Virus-Induced Acute Liver Failure-Observations during Lagovirus europaeus/GI.2 Infection.

Lagovirus europaeus/GI.2 causes severe and highly fatal Rabbit Hemorrhagic Disease (RHD). Because of its characteristics, this infection is used as an animal model for acute liver failure (ALF). Apoptosis is one of the key processes underlying ALF and has been described as one of the mechanisms of RHD pathogenesis. Apoptotic cell death has been quite well characterized in infection with different variants of GI.1 strains, but so far, the GI.2 genotype has not been widely studied. In this study, we performed an evaluation of apoptotic cell death in hepatocytes of rabbits infected with Lagovirus europaeus/GI.2. We analyzed the expression of genes involved in apoptotic cell death by real-time PCR and performed immunohistochemical (IHC) assays. We showed a significant increase in the expression of caspase-3 and the proapoptotic Bax and anti-apoptotic Bcl-2 in infected animals. In addition, we recorded increased Bax/Bcl-2 ratios. IHC analyses showed the presence of morphological signs of apoptosis in the hepatocytes of infected rabbits. Our results indicate that caspase-3 and proteins from the Bcl-2 families play a key role in apoptosis induced by Lagovirus europaeus/GI.2 infection.

Bleeding phenotype and hemostatic evaluation by thrombin generation in children with Noonan syndrome: A prospective study.

BACKGROUND: This study aimed to evaluate the bleeding phenotype and to conduct a comprehensive hemostatic evaluation in individuals with Noonan syndrome (NS), a dominantly inherited disorder caused by pathogenic variants in genes associated with the Ras/MAPK signaling pathway. METHODS: Children with a genetically confirmed diagnosis of NS underwent clinical evaluation, routine laboratory tests, platelet function testing, and thrombin generation (TG) assessment. RESULTS: The study included 24 children. The most frequently reported bleeding symptoms were easy bruising and epistaxis, while bleeding complications were observed in 15% of surgical procedures. Various hemostatic abnormalities were identified, including platelet dysfunction, von Willebrand disease, and clotting factor deficiencies. Abnormal platelet function was observed in 50% of the patients, and significantly lower TG parameters were found compared to controls. However, no significant correlation was observed between bleeding symptoms and TG results. CONCLUSIONS: The study suggests that the bleeding diathesis in NS is multifactorial, involving both platelet dysfunction and deficiencies of plasma coagulation factors. The potential role of TG assay as an ancillary tool for predicting bleeding tendencies in individuals with NS undergoing surgery warrants further investigation.

Long-term nintedanib treatment for progressive pulmonary fibrosis associated with Hermansky-Pudlak syndrome type 1 followed by lung transplantation.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disease that often causes progressive pulmonary fibrosis (HPS-PPF) in some genetic types with high mortality rates. No effective treatment for HPS-PPF other than lung transplantation has been established. Herein, we report a case of HPS type 1 with progressive pulmonary fibrosis treated with long-term nintedanib administration followed by lung transplantation. The resected lungs revealed diffuse interstitial lung lesions, including fibroblastic foci, suggesting the potential beneficial effects of anti-fibrotic drugs in HPS-PPF. Together with previous reports, the present case suggests that nintedanib might be a safe and effective drug for HPS-PPF.

Environmental impact on pediatric epistaxis and the utility of diagnostic studies: A single-institutional review.

OBJECTIVES: Pediatric epistaxis is a multifactorial disease entity. The objective of this study is to determine the socioeconomic and air-quality contributions to pediatric epistaxis. The study also evaluates the utility of diagnostic lab work as a predictor of bleeding rates and need for operative intervention. METHODS: A case series of pediatric patients treated in an outpatient Otolaryngology clinic at a tertiary care children's hospital in 2021 for epistaxis was performed. Patients with nasal bone trauma (n = 8), consult while inpatient (n = 7), and those with nasal masses (n = 2) were excluded; 181 patients met inclusion criteria. Demographic, clinical, socioeconomic, and air quality (tropospheric ozone, particulate matter) data were recorded. Associations with persistent bleeding and operative interventions were evaluated using logistic regression, Wilcoxon rank-sum, and Spearman rank correlation. RESULTS: Of the 181 patients, 75 (41.4%) were female. Forty-six of 181 (25.4%) had associated allergic symptoms. Twenty-six patients had allergy testing; 14/26 (53.8%) of these had positive results. Re-bleeding was more common in those with allergic symptoms (OR: 2.42, 95% CI: 1.22-4.78, p = 0.01). Patients with re-bleeding lived in counties with more days with ozone over the US standard (median 5 days, range 0-32 days) compared with those with no re-bleeding (median 3 days, range 0-32 days, p = 0.007). There was also an association between the number of visits for re-bleed and percent below poverty level (ρ = 0.259, p = 0.03) as well as the number of days with particulate matter levels over the US standard (ρ = 0.343, p = 0.01). Coagulopathy was present in 9/54 (16.7%) patients, with the majority being Von Willebrand disease (5/54, 9.3%). Easy bruising was not significantly associated with positive lab results. CONCLUSIONS: Environmental pollution, living in a zip code with more residents below the poverty level, and allergic rhinitis were positively associated with recurrent epistaxis. Understanding the geographic background of presenting patients may help direct workup and treatment options.

[Acute promyelocytic leukemia with asymptomatic cerebral hemorrhage].

A 43-year-old man presenting with oral bleeding was diagnosed with acute promyelocytic leukemia (APL). Induction chemotherapy consisting of all-trans retinoic acid and idarubicin was initiated, and disseminated intravascular coagulation (DIC) was treated with fresh frozen plasma and recombinant thrombomodulin infusions. The patient was free from neurological symptoms throughout the clinical course. However, cerebral hemorrhagic lesions were detected incidentally on magnetic resonance imaging performed to screen for leukemic central nervous system invasion at 2 weeks after treatment initiation. Imaging findings suggested subacute or later-phase cerebral hemorrhage. Platelet transfusions and other supportive care was provided. Serial imaging evaluations confirmed reduction of the hemorrhagic lesions. Hematological remission was achieved after induction chemotherapy, and no symptoms due to cerebral hemorrhage developed during the subsequent consolidation therapy. As patients with APL characteristically experience hemorrhagic events due to bleeding tendency caused by DIC, physicians should be aware of the possibility of asymptomatic cerebral hemorrhage in these patients.

Association between subconjunctival hemorrhage and hemorrhagic disorders: a nationwide population-based study.

Subconjunctival hemorrhage (SCH) is a benign eye condition that is often noticeable and leads to medical attention. Despite previous studies investigating the relationship between SCH and cardiovascular diseases, the relationship between SCH and bleeding disorders remains controversial. In order to gain further insight into this association, a nationwide cohort study was conducted using data from the National Health Insurance Service-National Sample Cohort version 2.0 from 2006 to 2015. The study defined SCH using a diagnostic code and compared the incidence and risk factors of intracerebral hemorrhage (ICH) and gastrointestinal (GI) bleeding in 36,772 SCH individuals and 147,088 propensity score (PS)-matched controls without SCH. The results showed that SCH was associated with a lower risk of ICH (HR = 0.76, 95% CI = 0.622-0.894, p = 0.002) and GI bleeding (HR = 0.816, 95% CI = 0.690-0.965, p = 0.018) when compared to the PS-matched control group. This reduced risk was more pronounced in females and in the older age group (≥ 50 years), but not observed in males or younger age groups. In conclusion, SCH dose not increase the risk of ICH and major GI bleeding and is associated with a decreased incidence in females and individuals aged ≥ 50 years.

Coagulation parameters in very preterm infants.

The aim of this study was to define normal percentile values of coagulation parameters in preterm infants below 32 weeks of gestational age. This retrospective cohort study was conducted at Istanbul Medical Faculty. Preterm infants who were born prior to 32 weeks of gestation, between 2011 and 2021 were included and evaluated for coagulation parameters. Blood samples obtained through umbilical catheters prior to administration of heparinized flushes/fluids, vitamin K or fresh frozen plasma (FFP). Infants with a major bleeding disorder, intrapartum asphyxia or a history of familial bleeding disorders were excluded. Infants were grouped according to their gestational ages and birth weights: less than 24, 25-26, 27-28, 29-30, 31-32 weeks and <500, 500-749, 750-999, 1000-1249, 1250-1499, more than 1500 g. Third to 97th percentile values of both prothrombin time (PT) and activated partial thromboplastin time (aPTT) were defined. A total of 420 preterm infants were included. The median value and range of gestational age and birth weight of the infants were 29 (22.3-32.9) weeks and 1150 (395-2790) g, respectively. PT values were similar between subgroups according to gestational age but longer in infants with a birth weight less than 1000 g. aPTT values in infants born less than 24 weeks of gestation were found significantly longer. As maturation of the coagulation system increases by gestational age, very preterm infants (<32 gestational week (GW)) are under increased risk of bleeding. Determination of normal percentile distribution of coagulation parameters for preterm infants will shed light on the interpretation of coagulation parameters of these infants and minimize unnecessary FFP administrations.

Generation and Characterisation of Monoclonal Antibodies against Nairobi Sheep Disease Virus Nucleoprotein.

Nairobi sheep disease (NSD), caused by the viral agent NSD virus (NSDV), is a haemorrhagic fever disease affecting and inducing high mortality in sheep and goat populations. NSDV belongs to the genus Orthonairovirus of the Nairoviridae family from the order Bunyavirales. Other viruses circulating in livestock such as Crimean-Congo haemorrhagic fever virus (CCHFV) and Dugbe virus (DUGV) are members of the same genus and are reported to share antigenic features. There are very few available materials to study NSDV infection both in vitro and in vivo. In the present work, we characterised two monoclonal antibodies generated in mice that recognise NSDV specifically but not CCHFV or DUGV, along with a potential use to define virus-infected cells, using flow cytometry. We believe this tool can be useful for research, but also NSDV diagnostics, especially through immunological staining.

High prevalence of heavy menstrual bleeding in women with rare bleeding disorders in the Netherlands: retrospective data from the RBiN study.

BACKGROUND: Heavy menstrual bleeding (HMB) is associated with a reduced quality of life and limitations in social and physical functioning. Data on HMB in women with rare bleeding disorders (RBDs), including coagulation factor deficiencies and fibrinolytic disorders, are scarce. OBJECTIVES: To analyze the prevalence, severity, and treatment of HMB in Dutch women with an RBD. METHODS: The Rare Bleeding Disorders in the Netherlands (RBiN) study included 263 patients with an RBD from all 6 hemophilia treatment centers (October 2017-November 2019). In this analysis, data of 111 women aged ≥16 years were studied. According to the International Society on Thrombosis and Haemostasis bleeding assessment tool, HMB symptoms were scored from 0 (no/trivial) to 4 (severe symptoms requiring medical intervention). HMB was defined as a score ≥1. Age at RBD diagnosis was extracted from patient files. RESULTS: HMB was reported by 80% of women (89/111) and was more prevalent in women with a fibrinolytic disorder (33/35; 94%) than in women with a coagulation factor deficiency (56/76; 74%) (P = .011). Of the 89 women with HMB, 82% (n = 73) ever required treatment. Multiple treatment modalities were frequently used, both in severe and mild deficiencies. Hormonal treatment was mostly used (n = 64; 88%), while antifibrinolytics were prescribed less frequently (n = 18; 25%). In women with HMB since menarche (n = 61; 69%), median age at RBD diagnosis was 28 years (IQR, 14-41). CONCLUSION: HMB is common in women with RBDs. Women with mild deficiencies also frequently reported HMB. Only a minority of women were treated with hemostatic agents. A significant diagnostic delay was observed after the onset of HMB symptoms.

Point-of-care platelet function testing results in a dog with Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS), also known as hemorrhagiparous thrombocytic dystrophy (OMIA 002207-9615), is a rare defect in platelet function recognized in both dogs and humans. It is caused by a deficiency in glycoprotein 1b-IX-V, the platelet surface protein which acts as a receptor for the von Willebrand factor. The characteristic features of BSS in humans and dogs include macrothrombocytes and mild-to-moderate thrombocytopenia with a bleeding tendency. This condition has previously been reported in European Cocker Spaniel dogs; however, the results of platelet function tests in these animals have not been reported. This case report describes a European Cocker Spaniel dog with spontaneously occurring Bernard-Soulier syndrome and the results of point-of-care platelet function tests, including a prolonged buccal mucosal bleeding time (>8 min), prolongation (>300 s) of PFA-200 COL/ADP, COL/EPI, and P2Y closure times, and reduced aggregation (15%-48%) with Plateletworks ADP, but with normal aggregation (92%) with Plateletworks AA. This is the first description of the results of platelet function tests in canine Bernard-Soulier syndrome.

Genetics of the rabbit haemorrhagic disease virus strains responsible for rabbit haemorrhagic disease outbreaks in Japan between 2000 and 2020.

Rabbit haemorrhagic disease (RHD) is a highly contagious and fatal disease in rabbits caused by the rabbit haemorrhagic disease virus (RHDV), which includes two genotypes, RHDV-GI.1 and RHDV2-GI.2. RHDVs tend to recombine among different strains, resulting in significant genetic evolution. This study evaluated the genetics of Japanese RHDV strains associated with six outbreaks between 2000 and 2020 using whole-genome sequencing, genomic recombination and phylogenetic analyses. Genomic recombination analysis using near-complete genomic sequences revealed that two Japanese strains detected in 2000 and 2002 were non-recombinant GI.1 (variant RHDVa-GI.1a) strains of different origins, most closely related to strains identified in PR China in 1997 and the USA in 2001, respectively. In contrast, four recent Japanese GI.2 strains detected between 2019 and 2020 were recombinant viruses harbouring structural protein (SP) genes from GI.2 strains and non-SP (NSP) genes from a benign rabbit calicivirus (RCV) strain of genotype RCV-E1-GI.3 (GI.3P-GI.2) or an RHDV G1-GI.1b variant (GI.1bP-GI.2). Phylogenetic analysis based on SP and NSP regions revealed that the GI.1bP-GI.2 recombinant virus detected in Ehime prefecture and the GI.3P-GI.2 recombinant viruses detected in Ibaraki, Tochigi and Chiba prefectures were most closely related to recombinant viruses identified in Australia in 2017 and Germany in 2017, respectively. These results suggested that past RHD outbreaks in Japan did not result from the evolution of domestic RHDVs but rather represented incursions of foreign RHDV strains, implying that Japan is constantly at risk of RHDV incursion from other countries.

Characterization of bleeding symptoms in Ehlers-Danlos syndrome.

BACKGROUND: Easy bruising is included as a major or minor criterion for the classification of multiple types of Ehlers-Danlos syndrome (EDS). Despite a longstanding recognition of the association between EDS and bleeding, we still lack a definitive understanding of the frequency, severity, and types of bleeding complications in patients with EDS. OBJECTIVES: To evaluate hemorrhagic symptoms using the International Society of Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) in a cohort of patients with defined types of EDS. METHODS: We utilized the ISTH-BAT to characterize hemorrhagic symptoms and their severity in a cohort of 52 patients with classical, classical-like, hypermobile, or vascular EDS and a matched group of 52 healthy control subjects. RESULTS: The mean ISTH-BAT score was 0.1 for healthy subjects and 9.1 for patients with EDS (p < .0001). An abnormal ISTH-BAT score was observed in 32 of 52 (62%) patients with EDS and 0 of 52 healthy controls (p < .0001). The most frequent bleeding symptoms were bruising, muscle hematomas, menorrhagia, epistaxis, bleeding from the oral cavity, and bleeding after tooth extraction. Menorrhagia that was life-threatening or required surgery was reported in 7 of 52 (14%) patients with EDS. CONCLUSION: Patients with multiple types of EDS exhibit a wide range of bleeding symptoms ranging from mild to life-threatening episodes.