Screening for SARS-CoV-2 and Other Coronaviruses in Urban Pigeons (Columbiformes) from the North of Spain under a 'One Health' Perspective.

Coronaviruses have a major impact on human and animal health. The SARS-CoV-2, a beta coronavirus responsible for the COVID-19 pandemic, is a clear example. It continues circulating and causes human deaths, and its high replication rate results in numerous variants. Coronaviruses adapt to birds and mammals and constitute a serious threat, and new viruses are likely to emerge. Urban pigeons (Columbiformes) are synanthropic birds of great interest from a 'One Health' perspective, due to their interaction with humans and other animals. Aware that they may act as viral reservoirs and contribute to their spread, we aimed to investigate the possible presence of SARS-CoV-2 and other coronaviruses in Columbiformes in the city of Logroño, Spain. Oropharyngeal and cloacal swabs were tested using real-time (N1 and E genes from SARS-CoV-2) and conventional PCR assays (RdRp gene from all coronaviruses). SARS-CoV-2 was not detected. A total of 13.3% of pigeons harbored coronaviruses closely related to Gamma coronavirus (Igacovirus) from Columbiformes in Finland, Poland and China. Monitoring the emergence of a new variant of SARS-CoV-2 capable of infecting Columbiformes should continue. SARS-CoV-2 is still circulating, the viral RNA of this virus has been detected in avian species (Phasianidae and Anatidae), and other coronaviruses are associated with animals that are in close contact with humans. The presence of Gamma coronavirus in urban pigeons must be considered for the risk of surveillance of human infections.

Surveillance and screening of Stomoxyinae flies from Mallorca Island (Spain) reveal the absence of selected pathogens but confirm the presence of the endosymbiotic bacterium Wolbachia pipientis.

Adult brachycera biting flies can significantly impact livestock through both direct effects (reduction of food intake, disturbance, painful bites, and blood loss) and indirect effects (pathogen transmission), leading to substantial economic losses and production damage. This study aimed to assess the presence of blood-sucking flies in six mixed-animal farm environments on the island of Mallorca (Balearic Islands, Spain) by employing multiple trapping methods. Additionally, distribution maps of brachycera biting fly species recorded in Spain were created, based on data extracted thorough review of scientific literature and citizen digital databases. Investigation of several pathogens, including equine infectious anemia virus (EIAV), Anaplasmataceae bacteria, and piroplasm protozoa, was carried out using different PCR targets (18S rRNA, 16S rRNA, groESL, and tat genes). Citizen science databases and literature review corroborated the consistent distribution trend for two Stomoxyinae species, underscoring the importance of citizen collaboration as a complement to traditional entomological surveillance. Our study confirmed the presence of two biting Stomoxyinae species: the prevalent stable fly Stomoxys calcitrans across all sampled farms, and the horn fly Haematobia irritans, which turned out to be less abundant. DNA barcoding techniques validated the identification of the two species. Neither EIAV nor bacterial/protozoan pathogens were detected using the selected PCR targets in either fly species. However, Wolbachia pipientis (clustered in the supergroup A together with the only sequence of W. pipientis from the USA) was identified through PCR targeting 16S rRNA, groESL and wsp genes in all pools of H. irritans (n = 13) collected from two of the examined farms. This study represents the first attempt to investigate pathogens in Stomoxyinae biting flies in Spain. The discovery of the endosymbiotic Wolbachia organism in H. irritans represents the first record in Spain and the second from Europe. This finding holds significant implications for future research on the applications of this bacterium in biocontrol programs.

Candidatus Neoehrlichia mikurensis Infection in Patient with Antecedent Hematologic Neoplasm, Spain1.

We report a confirmed case of Candidatus Neoehrlichia mikurensis infection in a woman in Spain who had a previous hematologic malignancy. Candidatus N. mikurensis infections should be especially suspected in immunocompromised patients who exhibit persistent fever and venous thrombosis, particularly if they live in environments where ticks are prevalent.

Epidemiological, Clinical, and Microbiological Characteristics in a Large Series of Patients Affected by Dermacentor-Borne-Necrosis-Erythema-Lymphadenopathy from a Unique Centre from Spain.

During recent decades, a tick-borne rickettsial syndrome, characterized by eschar and painful lymphadenopathy after Dermacentor marginatus-bite, has been described as an emerging rickettsiosis in Europe. Our group named it DEBONEL (Dermacentor-borne-necrosis-erythema-lymphadenopathy), regarding the vector and the main infection signs. Other groups called it TIBOLA (tick-borne-lymphadenophathy) and, later, SENLAT (scalp-eschar-and-neck-lymphadenopathy-after-tick-bite), expanding, in the latter, the etiological spectrum to other pathogens. Objective: To investigate the etiology of DEBONEL agents in our area, and to compare their epidemiological/clinical/microbiological characteristics. During 2001-2020, 216 patients clinically diagnosed of DEBONEL (the largest series from one center) in La Rioja (northern Spain) were examined. Rickettsia spp. were amplified in 14/104 (13.46%) blood samples, 69/142 (48.59%) eschar swabs, 7/7 (100%) biopsies, and 71/71 (100%) D. marginatus from patients. For samples in which Rickettsia was undetected, no other microorganisms were found. 'Candidatus Rickettsia rioja', Rickettsia slovaca, Rickettsia raoultii, and Rickettsia DmS1 genotype were detected in 91, 66, 4, and 3 patients, respectively. DEBONEL should be considered in patients with clinical manifestations herein described in areas associated to Dermacentor. The most frequently involved agent in our environment is 'Ca. R. rioja'. The finding of Rickettsia sp. DmS1 in ticks attached to DEBONEL patients suggests the implication of other rickettsia genotypes.

First case report of inherited Rubinstein-Taybi syndrome associated with a novel EP300 variant.

BACKGROUND: Rubinstein-Taybi syndrome (RSTS; OMIM #180849, #613684) is a rare autosomal dominant genetic condition characterized by broad thumbs and halluces, facial dysmorphism, short stature and variable degree of intellectual disability. RSTS is associated with mutations in CREBBP and EP300 genes in 50-60% and 5-8% of cases, respectively. The majority of cases are de novo heterozygous mutations. CASE PRESENTATION: Here we describe a familial RSTS case, associated with a novel EP300 mutation. The proband was a 9 years old female, with mild learning difficulties. Her mother, who also had learning difficulties, was found to have short and broad thumbs. MLPA and panel-based NGS of CREBBP and EP300 were performed. A novel heterozygous frameshift mutation in exon 31 of the EP300 gene (c.7222_7223del; p.(Gln2408Glufs*39)) was found in both. CONCLUSIONS: This case represents the first case of inherited EP300-RSTS. The location of the frameshift deletion not affecting HAT domain and PHD finger, could explain the mild phenotype and the well-preserved intelligence. These patients are mildly affected, and this case highlights the possible missed diagnosis. We would recommend molecular testing of apparently healthy parents, and in the case of inherited mutations, of all adult first degree relatives at risk.

A novel mutation in the CDH1 gene in a Spanish family with hereditary diffuse gastric cancer.

Hereditary diffuse gastric cancer (HDGC) is an inherited form of diffuse type gastric cancer. Germline CDH1 mutations have been identified in approximately 15-50 % of affected kindred that meet the clinical criteria for HDGC. If any of the criteria is met the individual is referred to genetic counseling and CDH1 testing is offered. In this report we present the case of a Spanish family with HDGC harboring a novel CDH1 mutation. A 47 year-old female with a diagnostic of gastric adenocarcinoma and some of her relatives were tested. Study of the entire CDH1 gene, including intron-exon boundaries, by PCR and sequencing and immunohistochemical determination of the expression of E-cadherin were performed. A novel heterozygous deletion in exon 9 of CDH1 gene (c.1220_1220delC, p.P407Qfs10), was found in the proband, one sister and a nephew. It generates a premature stop codon giving rise to a truncated protein that leads to a pathogenic variant. Expression of E-cadherin was absent or frankly reduced in the proband's tumor but normal in tumor cells of great-uncle. After these results, the sister underwent prophylactic total gastrectomy, and the nephew is under annual endoscopic surveillance. Personal or familial history of diffuse gastric cancer, above all at young age, should encourage CDH1 genetic testing. In this sense, the review of the criteria and the addition in the last guideline of the recommendation: "other families in which genetic testing may also be considered" broadens the number of individuals at risk detected. Since there are not reliable methods for early detection, DGC is usually diagnosed at an advanced stage and consequently associated with a poorer outcome. Thus, CDH1 mutations detection contributes to an improvement in diagnosis and therapeutic intervention.

Digital droplet PCR on disk.

Existing systems for digital droplet PCR (ddPCR) either suffer from low integration or are difficult to introduce to mass fabrication. Here we present an integrated system that is compatible to mass fabrication and combines emulsification, PCR, and fluorescence readout in a single chamber within a disposable cartridge (disk). Droplets are generated by injecting the sample into fluorinated oil via centrifugal step emulsification. The resulting emulsion is aligned in the PCR and readout zone by capillary action. During thermocycling, gas bubbles generated by degassing are removed by capillary driven transport through tapered regions in the PCR chamber. Thereby, the positioning of the emulsion within the readout zone of the PCR chamber is ensured at any time and no bubbles are present during readout. Manual handling of the disk solely requires pipetting of oil and PCR mix into the inlet structures, placing the disk into the thermocycler and subsequently into a microarray scanner. The functionality of the ddPCR process chain is demonstrated by quantitative detection of the cystic fibrosis causing mutation p.Phe508del, which is of interest for non-invasive prenatal testing (NIPT). The mutation was detected in a concentration range spanning four orders of magnitude. We envision that this work will lay the base for the development of highly integrated sample-to-digital-answer PCR systems that can be employed in routine clinical diagnosis.

A novel SLC6A8 mutation associated with motor dysfunction in a child exhibiting creatine transporter deficiency.

Creatine transporter (CT) deficiency is an X-linked disorder caused by mutations in the SLC6A8 gene. We describe a clinical, biochemical and molecular examination of a child with X-linked cerebral creatine deficiency. Increased urinary creatine/creatinine ratio, abnormal brain proton magnetic resonance spectroscopy and reduced creatine transport confirmed the clinical diagnosis. SLC6A8 analysis revealed a novel mutation that was hemizygous in the child and not detected in his mother. CT deficiency should be considered in children, especially males, with mental retardation.