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En 2016 se estableció un sistema anual de vigilancia entomológica para la detección de Aedes albopictus en la comunidad autónoma de La Rioja. En el verano de 2020, se detectó por primera vez la presencia de huevos en una zona urbana, lo que constituye el primer registro de esta especie en la región. La identificación de este mosquito invasor supone un cambio en el riesgo de aparición de casos de arbovirosis en La Rioja. La vigilancia entomológica es crucial para el diseño de estrategias de control que, junto con la vigilancia epidemiológica, puedan evitar la aparición de brotes autóctonos de arbovirosis. En este trabajo se detalla el proceso de la primera detección de mosquito tigre en La Rioja, así como las intervenciones llevadas a cabo y las perspectivas de futuro. In 2016, an annual entomological surveillance program for the detection of Aedes albopictus in the autonomous community of La Rioja was established. In the summer of 2020, the presence of eggs was detected for the first time in an urban area, which constitutes the first record of this species in the region. The identification of this invasive mosquito implies a change in the risk of occurrence of arbovirus cases in La Rioja. Entomological surveillance is crucial for the design of control strategies that, together with epidemiological surveillance, can prevent the appearance of autochthonous arbovirus outbreaks. This work is a detailed description of the first detection of tiger mosquito in La Rioja, as well as the completed interventions, and the perspectives for the future.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an arthropod-borne virus (arbovirus), mainly transmitted by ticks, belonging to the genus Orthonairovirus (family Nairoviridae, order Bunyavirales). CCHFV causes a potentially severe, or even fatal, human disease, and it is widely distributed in Africa, Asia, eastern Europe and, more recently, in South-western Europe. Until a few years ago, no cases of Crimean-Congo hemorrhagic fever (CCHF) had been reported in western Europe, with the exception of several travel-associated cases. In 2010, the CCHFV was reported for the first time in South-western Europe when viral RNA was obtained from Hyalomma lusitanicum ticks collected from deer in Cáceres (Spain). Migratory birds from Africa harboring CCHFV-infected ticks and flying to Spain appear to have contributed to the establishment of the virus (genotype III, Africa-3) in this country. In addition, the recent findings in a patient and in ticks from deer and wild boar of viral sequences similar to those from eastern Europe (genotype V, Europe-1), raise the possibility of the introduction of CCHFV into Spain through the animal trade, although the arrival by bird routes cannot be ruled out (Africa-4 has been also recently detected). The seropositive rates of animals detected in regions of South-western Spain suggest an established cycle of tick-host-tick in certain areas, and the segment reassortment detected in the sequenced virus from one patient evidences a high ability to adaptation of the virus. Different ixodid tick genera can be vectors and reservoirs of the virus, although Hyalomma spp. are particularly relevant for its maintenance. This tick genus is common in Mediterranean region but it is currently spreading to new areas, partly due to the climate change and movement of livestock or wild animals. Although to a lesser extent, travels with our pets (and their ticks) may be also a factor to be considered. As a consequence, the virus is expanding from the Balkan region to Central Europe and, more recently, to Western Europe where different genotypes are circulating. Thus, seven human cases confirmed by molecular methods have been reported in Spain from 2016 to August 2020, three of them with a fatal outcome. A One Health approach is essential for the surveillance of fauna and vector populations to assess the risk for humans and animals. We discuss the risk of CCHFV causing epidemic outbreaks in Western Europe.

The knowledge of the distribution, richness and epidemiological importance of soft ticks of the genus Argas is incomplete. In Spain, five Argas species have been recorded, including three ornitophilic nidicolous ticks, but their associated microorganisms remain unknown. This study aimed to investigate ticks from bird nests and their microorganisms. Ticks were collected extensively from natural cavities and nest-boxes used by European rollers (Coracias garrulus) and little owls (Athene noctua) in Southeastern and Central Spain. Ticks were morphologically and genetically identified and corresponding DNA/RNA tick extracts were analyzed [individually (n = 150) or pooled (n = 43)] using specific PCR assays for bacteria (Anaplasmataceae, Bartonella, Borrelia, Coxiella/Rickettsiella, and Rickettsia spp.), viruses (Flaviviruses, Orthonairoviruses, and Phenuiviruses), and protozoa (Babesia/Theileria spp.). Six Argas genotypes were identified, of which only those of Argas reflexus (n = 8) were identified to the species level. Two other genotypes were closely related to each other and to Argas vulgaris (n = 83) and Argas polonicus (n = 33), respectively. These two species have not been previously reported from Western Europe. Two additional genotypes (n = 4) clustered with Argas persicus, previously reported in Spain. The remaining genotype (n = 22) showed low sequence identity with any Argas species, being most similar to the African Argas africolumbae. The microbiological screening revealed infection with a rickettsial strain belonging to Rickettsia fournieri and Candidatus Rickettsia vini group in 74.7% of ticks, mainly comprising ticks genetically related to A. vulgaris and A. polonicus. Other tick endosymbionts belonging to Coxiella, Francisella and Rickettsiella species were detected in ten, one and one tick pools, respectively. In addition, one Babesia genotype, closely related to avian Babesia species, was found in one tick pool. Lastly, Anaplasmataceae, Bartonella, Borrelia, and viruses were not detected. In conclusion, five novel Argas genotypes and their associated microorganisms with unproven pathogenicity are reported for Spain. The re-use of nests between and within years by different bird species appears to be ideal for the transmission of tick-borne microorganisms in cavity-nesting birds of semiarid areas. Further work should be performed to clarify the taxonomy and the potential role of soft Argas ticks and their microorganisms in the epidemiology of zoonoses.

Infective endocarditis (IE) is a severe and life-threatening disease. Identification of infectious etiology is essential for establishing the appropriate antimicrobial treatment and decreasing mortality. The aim of this study was to explore potential utility of metagenomics for improving microbiological diagnosis of IE. In this work, next-generation sequencing (NGS) of V3-V4 region of the 16S rRNA gene was performed in 27 heart-valve tissues (18 natives, 5 intravascular devices, and 4 prosthetics) of patients diagnosed by IE. Initial microbiological diagnosis, blood culture (BC) and/or PCR, was compared with NGS-based diagnosis. Metagenomics matched with conventional techniques diagnosis in 24/27 cases (88.9%). The same bacterial family was assigned to 24 cases, the same genus to 23 cases, and the same specie for 13 cases. In 22 of them, the etiological agent was represented by percentages >99% of the reads and in two by ∼70%. Staphylococcus aureus was detected in a previously undiagnosed patient, making the microbiological diagnosis possible in one more sample than with previously used techniques. The remaining two patients showed no coincidence between traditional and NGS microbiological diagnoses. Minority records verified mixed infections in four cases and suggested confections in two cases, supported by clinical data. In conclusion: 16S rRNA gene-targeted NGS allowed to diagnose one case of IE without microbiological entity based on traditional techniques. However, the application of metagenomics to the study of IE in resected heart valves provides no benefits in comparison with BC and/or PCR. More studies are needed before implementation of NGS for the diagnosis of IE.

REIPI-SEIMC COVID-19 group and COVID@HULP group., [Background] The clinical presentation of COVID-19 in patients admitted to hospital is heterogeneous. We aimed to determine whether clinical phenotypes of patients with COVID-19 can be derived from clinical data, to assess the reproducibility of these phenotypes and correlation with prognosis, and to derive and validate a simplified probabilistic model for phenotype assignment. Phenotype identification was not primarily intended as a predictive tool for mortality., [Methods] In this study, we used data from two cohorts: the COVID-19@Spain cohort, a retrospective cohort including 4035 consecutive adult patients admitted to 127 hospitals in Spain with COVID-19 between Feb 2 and March 17, 2020, and the COVID-19@HULP cohort, including 2226 consecutive adult patients admitted to a teaching hospital in Madrid between Feb 25 and April 19, 2020. The COVID-19@Spain cohort was divided into a derivation cohort, comprising 2667 randomly selected patients, and an internal validation cohort, comprising the remaining 1368 patients. The COVID-19@HULP cohort was used as an external validation cohort. A probabilistic model for phenotype assignment was derived in the derivation cohort using multinomial logistic regression and validated in the internal validation cohort. The model was also applied to the external validation cohort. 30-day mortality and other prognostic variables were assessed in the derived phenotypes and in the phenotypes assigned by the probabilistic model., [Findings] Three distinct phenotypes were derived in the derivation cohort (n=2667)—phenotype A (516 [19%] patients), phenotype B (1955 [73%]) and phenotype C (196 [7%])—and reproduced in the internal validation cohort (n=1368)—phenotype A (233 [17%] patients), phenotype B (1019 [74%]), and phenotype C (116 [8%]). Patients with phenotype A were younger, were less frequently male, had mild viral symptoms, and had normal inflammatory parameters. Patients with phenotype B included more patients with obesity, lymphocytopenia, and moderately elevated inflammatory parameters. Patients with phenotype C included older patients with more comorbidities and even higher inflammatory parameters than phenotype B. We developed a simplified probabilistic model (validated in the internal validation cohort) for phenotype assignment, including 16 variables. In the derivation cohort, 30-day mortality rates were 2·5% (95% CI 1·4–4·3) for patients with phenotype A, 30·5% (28·5–32·6) for patients with phenotype B, and 60·7% (53·7–67·2) for patients with phenotype C (log-rank test p, [Interpretation] Patients admitted to hospital with COVID-19 can be classified into three phenotypes that correlate with mortality. We developed and validated a simplified tool for the probabilistic assignment of patients into phenotypes. These results might help to better classify patients for clinical management, but the pathophysiological mechanisms of the phenotypes must be investigated., [Funding] Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation, and Fundación SEIMC/GeSIDA., Funding: Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation, and Fundación SEIMC/GeSIDA.

The knowledge of the distribution, richness and epidemiological importance of soft ticks of the genus Argas is incomplete. In Spain, five Argas species have been recorded, including three ornitophilic nidicolous ticks, but their associated microorganisms remain unknown. This study aimed to investigate ticks from bird nests and their microorganisms. Ticks were collected extensively from natural cavities and nest-boxes used by European rollers (Coracias garrulus) and little owls (Athene noctua) in Southeastern and Central Spain. Ticks were morphologically and genetically identified and corresponding DNA/RNA tick extracts were analyzed [individually (n = 150) or pooled (n = 43)] using specific PCR assays for bacteria (Anaplasmataceae, Bartonella, Borrelia, Coxiella/Rickettsiella, and Rickettsia spp.), viruses (Flaviviruses, Orthonairoviruses, and Phenuiviruses), and protozoa (Babesia/Theileria spp.). Six Argas genotypes were identified, of which only those of Argas reflexus (n = 8) were identified to the species level. Two other genotypes were closely related to each other and to Argas vulgaris (n = 83) and Argas polonicus (n = 33), respectively. These two species have not been previously reported from Western Europe. Two additional genotypes (n = 4) clustered with Argas persicus, previously reported in Spain. The remaining genotype (n = 22) showed low sequence identity with any Argas species, being most similar to the African Argas africolumbae. The microbiological screening revealed infection with a rickettsial strain belonging to Rickettsia fournieri and Candidatus Rickettsia vini group in 74.7% of ticks, mainly comprising ticks genetically related to A. vulgaris and A. polonicus. Other tick endosymbionts belonging to Coxiella, Francisella and Rickettsiella species were detected in ten, one and one tick pools, respectively. In addition, one Babesia genotype, closely related to avian Babesia species, was found in one tick pool. Lastly, Anaplasmataceae, Bartonella, Borrelia, and viruses were not detected. In conclusion, five novel Argas genotypes and their associated microorganisms with unproven pathogenicity are reported for Spain. The re-use of nests between and within years by different bird species appears to be ideal for the transmission of tick-borne microorganisms in cavity-nesting birds of semiarid areas. Further work should be performed to clarify the taxonomy and the potential role of soft Argas ticks and their microorganisms in the epidemiology of zoonoses.

Pets from COVID‐19 owners were screened for SARS‐CoV‐2 (April‐May 2020). From 23 pets, an asymptomatic cat showed positive RT‐qPCRs results from oropharyngeal swab (negative rectal swab). Remaining pets were negative. This suggests that cats can contract the virus from their infected owners and may act as potential hosts for SARS‐CoV‐2. Their role in carrying live or infectious viruses and disseminating them needs more investigation.

During April-May 2020, the presence of respiratory syndrome coronavirus 2 (SARS-CoV-2) in pets living with coronavirus disease 2019 (COVID-19) owners was analyzed. From 23 pets, a cat without clinical symptoms showed positive results for SARS-CoV-2 in oropharyngeal swab using three RT-qPCR assays (negative rectal swab). SARS-CoV-2 was not detected in the remaining pets. Our finding suggests that cats may act as asymptomatic dispersers of SARS-CoV-2, although viral transmission from animals to humans seems unlikely.

Mosquitoes are important to public and animal health due to their capacity to transmit diseases. Since the Zika virus was declared a pandemic by the WHO in 2016, and it has been recorded in different regions of Mediterranean Area (included Spain), the Government of La Rioja (Northern Spain) through the Center of Rickettsiosis and Arthropod-Borne Diseases, implemented an entomological surveillance programme of mosquitoes in La Rioja and in a close area of Navarra. This surveillance extended to some of the pathogens that they can transmit. Here we describe the framework of the initial surveillance programme for the detection of mosquitoes and associated human pathogens. We outline the benefits and the limitation of the programme to date, and explore how greater benefits can be achieved, for example using a One Health approach. Entomological surveillance has been carried out with BG-Sentinel traps, human bait technique and other methods such as collecting adults in resting places or immature stages by dipping in several wetlands. Since Aedes albopictus, vector of arbovirus such as Dengue, Chikungunya, and Zika, has not been detected yet in the region, the entomological programme included the surveillance of this exotic species using ovitraps in the most important cities. Morphological identification was supported using the mitochondrial cytochrome C oxidase subunit I and the internal transcribed spacer 2 genes analysis. In 2016 and 2017, more than 6,000 mosquitoes were collected. The mosquito's community included 21 species associated with six genera: Anopheles (n = 4), Aedes (n = 5), Culex (n = 6), Culiseta (n = 4), Uranotaenia (n = 1) and Coquillettidia (n = 1). Eleven species represent new records for La Rioja and Navarra regions. Several species were collected biting humans and a great proportion of the sampled mosquito population are competent vectors of several pathogens, such as West Nile virus. Sequences closely related to mosquito-only flavivirus have been detected in 0.34% of analysed pools. At the same time, the epidemiological surveillance emphasis is placed in the early detection of mosquito-borne diseases in primary health and emergency services. The surveillance programme represents a relevant and necessary assessment of the risk of pathogen transmission in a region, and it allows for the establishment of the appropriate preventive measures.

ObjectiveThe aim of this study was to characterize the bacterial microbiome of hard ticks with affinity to bite humans in La Rioja (North of Spain).MethodsA total of 88 adult ticks (22 Rhipicephalus sanguineus sensu lato, 27 Haemaphysalis punctata, 30 Dermacentor marginatus and 9 Ixodes ricinus) and 120 I. ricinus nymphs (CRETAV collection, La Rioja, Spain), representing the main anthropophilic species in our environment, were subjected to a metagenomic analysis of the V3-V4 region of the 16S rRNA gene using an Illumina MiSeq platform. Data obtained with Greengenes database were refined with BLAST. Four groups of samples were defined, according to the four tick species.ResultsProteobacteria was the predominant phylum observed in all groups. Gammaproteobacteria was the most abundant class, followed by Alphaproteobacteria for R. sanguineus, H. punctata and D. marginatus but the relative abundance of reads for these classes was reversed for I. ricinus. This tick species showed more than 46% reads corresponding to 'not assigned' OTUs (Greengenes), and >97% of them corresponded to 'Candidatus Midichloriaceae' using BLAST. Within Rickettsiales, 'Candidatus Midichloria', Rickettsia, Ehrlichia, 'Candidatus Neoehrlichia' and Wolbachia were detected. I. ricinus was the most alpha-diverse species. Regarding beta-diversity, I. ricinus and H. punctata samples grouped according to their tick species but microbial communities of some R. sanguineus and D. marginatus specimens clustered together.ConclusionsThe metagenomics approach seems useful to discover the spectrum of tick-related bacteria. More studies are needed to identify and differentiate bacterial species, and to improve the knowledge of tick-borne diseases in Spain.

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes species of medical and veterinary importance. The presence of Anaplasma spp. in ticks from birds, as well as in Haemaphysalis punctata (Ixodida: Ixodidae) specimens collected from cattle and vegetation in northern Spain was investigated. A total of 336 ticks from birds [174 Ixodes frontalis (Ixodida: Ixodidae), 108 H. punctata, 34 Hyalomma marginatum (Ixodida: Ixodidae), 17 Ixodes ricinus (Ixodida: Ixodidae) and three Ixodes spp.], and 181 H. punctata specimens collected from cattle (n = 71) and vegetation (n = 110) were analysed. Anaplasma bovis was detected in five H. punctata, including two from birds (1.9%) and three from vegetation (2.7%). Four I. frontalis (2.3%) (one co-infected with 'Candidatus Midichloria mitochondrii') and one I. ricinus (5.9%) removed from birds, as well as four H. punctata (5.6%) collected from cattle showed Anaplasma phagocytophilum infection. In addition, Anaplasma centrale was found in two H. punctata, one from a cow (1.4%) and the other from vegetation (0.9%). This study represents the first evidence of the presence of A. bovis in European ticks, and reports the first detection of A. bovis and A. centrale in H. punctata, and the first finding of A. phagocytophilum and 'Ca. Midichloria mitochondrii' in I. frontalis.

Introduction Borrelia miyamotoi is a tick-borne pathogen belonging to the relapsing fever group. It had not been reported from Spain, but its wide distribution and the presence of the tick-vector (Ixodes ricinus) made us suspect its circulation. The aim of this study was to investigate the presence of Borrelia spp. in I. ricinus in Spain. Methods A total of 652 I. ricinus nymphs collected in northern Spain were processed. The DNA was extracted using incubations with ammonium hydroxide. Borrelia spp. DNA was amplified using Borrelia-specific PCR assays (glpQ, 16S rRNA and flagellin genes). Results B. miyamotoi was amplified in 4 specimens, and Borrelia burgdorferi sensu lato in 27 (8 Borrelia afzelii, 7 Borrelia garinii, 8 Borrelia lusitaniae, 3 Borrelia valaisiana and 1 B. burgdorferi sensu stricto). Conclusion B. miyamotoi should be considered in the differential diagnoses of patients with confirmed or suspected tick-bite in Spanish endemic areas for Lyme disease.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12881-016-0361-8) contains supplementary material, which is available to authorized users.

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.

The genus Borrelia includes species responsible for severe human diseases such as Lyme disease. Birds are involved in their epidemiology as dispersers of infected ticks (Ixodida: Ixodidae) and as reservoirs or amplifiers of the bacterium. Herein, the presence of Borrelia burgdorferi sensu lato (s.l.) Johnson, Schmid, Hyde, Steigerwalt & Brenner in 336 ticks collected from birds in the north of Spain from 2009 to 2011 was investigated. Nucleic acid extracts from 174 Ixodes frontalis (Panzer), 108 Haemaphysalis punctata Canestrini & Fanzango, 34 Hyalomma marginatum Koch, 17 Ixodes ricinus (L.), and 3 Ixodes spp. were screened for the presence of B. burgdorferi s.l. by PCR. Borrelia turdi was detected in 22 I. frontalis, 2 H. punctata, and 2 I. ricinus Additionally, 1 I. frontalis and 1 H. punctata were found to be infected with the human pathogen Borrelia valaisiana Moreover, 3 I. frontalis showed coinfection with both Borrelia species. This study corroborates the presence of B. turdi and B. valaisiana in ticks from birds in the north of Spain. The presence of these bacteria in larval specimens could suggest the role of birds as their reservoirs, or the occurrence of the cofeeding phenomenon. In addition, the detection of B. turdi and B. valaisiana in H. punctata and I. frontalis ticks, respectively, is reported for the first time.

Trombiculid mites (Acari: Trombiculidae) are distributed worldwide ectoparasites of a wide range of vertebrates. More than 50 species are known to bite humans, and about 20 have medical importance. The larval stages (chiggers) of the genus Leptotrombidium are vectors of Orientia tsutsugamushi, causative agent of scrub typhus. This life-threatening disease is widely endemic in Asian Pacific regions where more than one billion people are at risk of acquiring the infection and around one million new cases are estimated to occur annually. In addition, although underreported and often misdiagnosed, trombicu‐ liasis, defined as a dermatitis caused by the salivary secretion of biting chiggers, is present in America and Europe.

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.

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.

We amplifi ed gene sequences from Anaplasma phagocytophilum, Borrelia garinii, B. valaisiana, B. turdi, Rickettsia monacensis, R. helvetica, R. sibirica sibirica, and Rickettsia spp. (including Candidatus Rickettsia vini) in ticks removed from birds in Spain. The fi ndings support the role of passerine birds as possible dispersers of these tick-borne pathogens.

Background The effectiveness of PCR methods to amplify rickettsiae from clinical samples has still not been evaluated. Our aim was to determine the sensitivity and usefulness for Rickettsia species identification by PCR methods, targeting 16S rDNA, htrA, gltA, ompA, and ompB genes for molecular diagnosis of rickettsioses. Methods A total of 72 clinical samples (EDTA-blood, skin biopsies and ticks) taken from 52 patients in the early phase of the illness with PCR-confirmed rickettsioses were included. Single [16S rDNA, gltA (5′ end), and htrA genes] and sequential (nested or semi-nested) PCR assays [ompB, gltA (central region) and ompA genes] were performed. Results For single-stage PCR assays, the greatest sensitivity (33.3%) was obtained using the gltA (5′ end), while for sequential assays, the most sensitive results were obtained using the ompB assay (83.3%). The highest sensitivity (100%) was achieved using the three sequential PCRs. The ompA PCR method was the most reliable for identifying Rickettsia species, according to clinical features. Conclusions PCR-based amplification methods are useful rickettsial diagnostic tools in the early phase of the illness. The three sequential PCR assays here investigated (ompB, gltA and ompA) appear to be useful tools for molecular diagnosis of rickettsioses. ompB PCR assay is effective for primary screening, since it detects a high percentage of positive samples. ompA assay is the most useful method to identify a Rickettsia species in human pathology. Nevertheless, epidemiology, clinical symptoms and the vector involved in the infection have to be taken into account for the diagnosis of rickettsioses.

To the Editor: Crimean-Congo hemorrhagic fever virus (CCHFV; family Bunyaviridae, genus Nairovirus) causes outbreaks of severe hemorrhagic fever in humans, with case-fatality rates

In an attempt to know the potential risk of human disease after exposure to ticks in La Rioja (North of Spain), the objective of our study was to investigate the presence of Rickettsia species in Haemaphysalis ticks collected in our area. A total of 177 Haemaphysalis spp. belonging to three species (H. punctata, H. sulcata, and H. inermis) were subjected to DNA extraction and tested by polymerase chain reaction (PCR) targeting three rickettsial genes: gltA, ompB, and ompA. Six (3 H. inermis, 2 H. punctata, and 1 H. sulcata) of the 177 specimens were found to be infected (3.4%). The rickettsiae in H. inermis ticks (n = 3) were identified as Rickettsia aeschlimannii by sequencing of the genes coding for gltA, ompB, and ompA. Nucleotide sequences from H. punctata and H. sulcata samples that yielded PCR products (n = 3), showed99% similarity with sequences of Rickettsia endosymbiont of H. sulcata and 'Candidatus Rickettsia hoogstraalii' for gltA and ompB genes, respectively. Attempts to amplify ompA from these two H. punctata and one H. sulcata failed. This study suggests that H. inermis could be a vector for tick-borne spotted fever caused by R. aeschlimannii in the north of Spain. Further studies on characterization and culture of rickettsial endosymbionts found in Haemaphysalis spp. should be performed.