Predictive factors for nephrectomy in renal trauma; assessment of a 6-point score.

PURPOSE: To evaluate predictive and associated risk factors for nephrectomy in renal trauma and assess a 6-point score for surgical decision-making. PATIENTS AND METHODS: This multicenter, retrospective, and observational study assessed 247 subjects with blunt or penetrating kidney trauma. Kidney injuries were classified according to the American Association for the Surgery of Trauma (AAST) Injury Scoring Scale. Renal trauma was classified as "low-grade" (Grades I-III), Grade IV, and Grade V. Subjects were compared according to conservative treatment (CTrt.) or nephrectomy. Predictive factors were evaluated with a multiple regression model. A 6-point score was evaluated with a ROC analysis. RESULTS: Patients requiring nephrectomy had a lower mean arterial pressure MAP compared to CTrt, 64.71 mmHg (SD ± 10.26) and 73.86 (SD ± 12.42), respectively (p = < 0.001). A response to IV solutions was observed in 90.2% of patients undergoing CTrt. (p = < 0.001, OR = 0.211, 95%CI = 0.101-0.442). Blood lactate ≥ 4 mmol/L was associated with nephrectomy (p = < 0.001). A hematoma ≥ 25 mm was observed in 41.5% of patients undergoing nephrectomy compared to 20.1% of CTrt. (p = 0.004, OR = 9.29, 95% CI = 1.37-5.58). A logistic regression analysis (p = < 0.001) showed that blood lactate ≥ 4 mmol/L (p = 0.043), an inadequate response to IV solutions (p = 0.041) and renal trauma grade IV-V (p = < 0.001), predicted nephrectomy. A 6-point score with a cut-off value ≥ 3 points showed 83% sensitivity and 87% specificity for nephrectomy with an AUC of 89.9% (p = < 0.001). CONCLUSIONS: An inadequate response to IV solutions, a lactate level ≥ 4 mmol/L, and grade IV-V renal trauma predict nephrectomy. A score ≥ 3 points showed a good performance in this population.

Risk factors associated with urethral stricture recurrence after end-to-end urethroplasty and buccal mucosal graft urethroplasty.

OBJECTIVES: (1) Assess risk factors associated with urethral stricture recurrence (USR). (2) Assess urethral stricture recurrence after end-to-end urethroplasty (EE) and buccal mucosal graft urethroplasty (BMG). SUBJECTS AND METHODS: A total of 29 males with urethral stricture who underwent either an end-to-end urethroplasty or a buccal mucosal graft urethroplasty were included in this study and followed for 18 months. The association between risk factors and stricture recurrence was assessed. RESULTS: Overall mean patient age was 51.69 ± 14.22 years, time to recurrence was 3 months (IQR: 1-6.25), and stricture length was 2.57 ± 1.30 cm. Important risk factors for USR were stricture length ⩾ 2 cm (p = 0.024), older age (p = 0.042), BMI > 25 kg/m2 (p = 0.021), Qmax after catheter removal <15 ml/s (χ2 = 14.87 p ⩽ <0.001) and previous urethral procedures adjusted for re-do BMG urethroplasty (χ2 = 6.10, p = 0.021). End-to-end urethroplasty showed less USR than BMG, however, these differences were not statistically significant (41.6% vs 22.2%, respectively, p ⩾ 0.05). CONCLUSIONS: Stricture length, age, BMI, and previous urethral procedures predict USR, furthermore, an initial Qmax after catheter removal is an objective measure predictive of USR. There's no difference in USR rate between BMG and EE urethroplasties.

Descripción de la epidemia estacional por Gripe B en Cantabria durante los inicios de la pandemia por SARS-CoV-2 / Description of influenza B in seasonal epidemic in Cantabria during the beginning of the pandemia due to SARS-CoV-2

INTRODUCCIÓN: La cocirculación de los dos linajes de gripe B dificulta la predicción de la cepa a incluir en la vacuna trivalente. Comunidades autónomas (CCAA) como Cantabria continúan sin suministrar la vacuna tetravalente. El objetivo de este estudio fue analizar las características epidemiológicas de la gripe B en Cantabria (temporada 2019-2020), y determinar el linaje predominante y su relación con la vacuna recomendada. MÉTODOS: Estudio retrospectivo en el que el diagnóstico de gripe y los linajes de gripe B se determinaron mediante RTPCR. RESULTADOS: Todas las muestras pertenecieron al linaje Victoria. La coinfección vírica más frecuente fue por SARSCoV-2. La población afectada por gripe B fue fundamentalmente pediátrica y los pacientes no vacunados requirieron más frecuentemente ingreso hospitalario. CONCLUSIÓN: La gripe B presenta una mayor incidencia sobre población pediátrica, y la gripe A afecta más a población adulta. Sólo el 28,8% de los pacientes con gripe B que presentaban algún factor de riesgo estaban vacunados, existiendo la necesidad de aumentar la cobertura con vacunas tetravalentes para reducir la carga de enfermedad asociada al virus gripal B

INTRODUCTION: Co-circulation of the two Influenza B lineages hinders forecast of strain to include in trivalent vaccine. Autonomous Communities such as Cantabria continue without supplying tetravalent vaccine. The aim of this study was to analyse epidemiological characteristics of influenza type B in Cantabria (2019-2020 season) as well as to establish the predominant lineage and its relation to the recommended vaccine. METHODS: Retrospective study whereby flu diagnosis and lineage analysis were determined by RT-PCR. RESULTS: All samples belonged to the Victoria lineage. Most prevalent viral co-infection was due to SARS-CoV-2. The population affected by influenza B was mainly paediatric and non-vaccinated patients more frequently required hospital admittance. CONCLUSIONS: Influenza type B has a higher incidence in the paediatric population and type A affects more the adult population. Only 28.8% of patients with Influenza B that presented with some underlying condition or risk factor were vaccinated. This shows the need to increase coverage with tetravalent vaccines in order to reduce the burden of disease associated with the Influenza B virus

Primer caso descrito en España de infección por Wohlfahrtiimonas chitiniclastica / First case of infection by Wohlfahrtiimonas chitiniclastica described in Spain

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Post-traumatic endophthalmitis caused by Nocardia nova.

INTRODUCTION: Nocardia nova complex has been associated with infections in both immunocompetent and immunocompromised patients. Infection can be localized or disseminated, affecting skin and soft tissues, the respiratory system, bones and joints, the circulatory system and especially the central nervous system. Ocular infections such as keratitis, scleritis, conjunctivitis, dacryocystitis, orbital cellulitis and endophthalmitis due to Nocardia spp. are infrequently reported, and usually described after penetrating corneal trauma or ocular contact with plants and soils. CASE PRESENTATION: An immunocompetent male presented with a history of penetrating ocular trauma that had evolved to infectious endophthalmitis, which was refractory to different antibiotic treatments. No micro-organisms were isolated from repeated conjunctival smear and corneal scraping cultures between the ocular trauma (August 2014) and the endophthalmitis diagnosis (November 2015). After this period, N. nova sensu stricto was isolated in aqueous humour aspirate. Treatment was adjusted and clinical improvement was obtained after an adequate microbiological procedure, including an optimal sampling and an antimicrobial-susceptibility testing report. CONCLUSION: Nocardia identification to the species level and performance of antimicrobial-susceptibility tests are both essential tools for treatment adjustment and clinical improvement.

Stable and Broad Spectrum Cross-Protection Against Pepino Mosaic Virus Attained by Mixed Infection.

While recent pepino mosaic virus (PepMV; species Pepino mosaic virus, genus Potexvirus, family Alphaflexiviridae) epidemics seem to be predominantly caused by isolates of the CH2 strain, PepMV epidemics in intensive tomato crops in Spain are caused by both CH2 and EU isolates that co-circulate, representing a challenge in terms of control, including cross-protection. In this work, we hypothesized that mixed infections with two mild isolates of the EU and CH2 strains (PepMV-Sp13 and -PS5, respectively) may be useful in PepMV cross-protection in Spanish epidemics, providing protection against a broad range of aggressive isolates. Thus, we performed a range of field trials and an experimental evolution assay to determine the phenotypic and genetic stability of PepMV-Sp13 and -PS5 mixed infections, as well as their cross-protective efficiency. Our results showed that: (i) the phenotype of PepMV-Sp13 and -PS5 mixed infections was mild and did not change significantly when infecting different tomato cultivars or under different environmental conditions in Spain, (ii) PepMV-Sp13 and -PS5 mixed infections provided more efficient protection against two aggressive EU and CH2 isolates than single infections, and (iii) PepMV-Sp13 and -PS5, either in single or in mixed infections, were less variable than other two PepMV isolates occurring naturally in PepMV epidemics in Spain.

Characterization of Begomoviruses Sampled during Severe Epidemics in Tomato Cultivars Carrying the Ty-1 Gene.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) is a major species that causes a tomato disease for which resistant tomato hybrids (mainly carriers of the Ty-1/Ty-3 gene) are being used widely. We have characterized begomoviruses severely affecting resistant tomato crops in Southeast Spain. Circular DNA was prepared from samples by rolling circle amplification, and sequenced by massive sequencing (2015) or cloning and Sanger sequencing (2016). Thus, 23 complete sequences were determined, all belonging to the TYLCV Israel strain (TYLCV-IL). Massive sequencing also revealed the absence of other geminiviral and beta-satellite sequences. A phylogenetic analysis showed that the Spanish isolates belonged to two groups, one related to early TYLCV-IL isolates in the area (Group 1), and another (Group 2) closely related to El Jadida (Morocco) isolates, suggesting a recent introduction. The most parsimonious evolutionary scenario suggested that the TYLCV isolates of Group 2 are back recombinant isolates derived from TYLCV-IS76, a recombinant virus currently predominating in Moroccan epidemics. Thus, an infectious Group 2 clone (TYLCV-Mu15) was constructed and used in in planta competition assays against TYLCV-IS76. TYLCV-Mu15 predominated in single infections, whereas TYLCV-IS76 did so in mixed infections, providing credibility to a scenario of co-occurrence of both types of isolates.

Applied diagnostics in liver cancer. Efficient combinations of sorafenib with targeted inhibitors blocking AKT/mTOR.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. There is increasing interest in developing specific markers to serve as predictors of response to sorafenib and to guide targeted therapy. Using a sequencing platform designed to study somatic mutations in a selection of 112 genes (HepatoExome), we aimed to characterize lesions from HCC patients and cell lines, and to use the data to study the biological and mechanistic effects of case-specific targeted therapies used alone or in combination with sorafenib. We characterized 331 HCC cases in silico and 32 paired samples obtained prospectively from primary tumors of HCC patients. Each case was analyzed in a time compatible with the requirements of the clinic (within 15 days). In 53% of the discovery cohort cases, we detected unique mutational signatures, with up to 34% of them carrying mutated genes with the potential to guide therapy. In a panel of HCC cell lines, each characterized by a specific mutational signature, sorafenib elicited heterogeneous mechanistic and biological responses, whereas targeted therapy provoked the robust inhibition of cell proliferation and DNA synthesis along with the blockage of AKT/mTOR signaling. The combination of sorafenib with targeted therapies exhibited synergistic anti-HCC biological activity concomitantly with highly effective inhibition of MAPK and AKT/mTOR signaling. Thus, somatic mutations may lead to identify case-specific mechanisms of disease in HCC lesions arising from multiple etiologies. Moreover, targeted therapies guided by molecular characterization, used alone or in combination with sorafenib, can effectively block important HCC disease mechanisms.

Microenvironment Eradication of Hepatitis C: A Novel Treatment Paradigm.

OBJECTIVES: Prisons are major reservoirs of hepatitis C virus (HCV) in which a therapeutic approach has been particularly difficult so far. Our aim was to create a permanent program of HCV elimination in a prison based on a "test and treat" strategy. METHODS: This open-label clinical trial was conducted in the Spanish prison "El Dueso" between May 2016 and July 2017. Viremic patients were treated with a ledipasvir-sofosbuvir regimen (8-12 weeks) according to the 2015 Spanish Guidelines. A teleconsultation program was established to follow-up patients from the hospital. Non-responders were submitted for a phylogenetic analysis and offered retreatment. An evaluation of new cases of HCV infection was performed every 6 months and upon release in all inmates. RESULTS: 847 (99.5%) inmates accepted to participate. HCV antibodies were present in 110 (13.0%) and 86 (10.2%) had detectable viremia. Most of them were genotype 1 or 3 (82.6%) and had

Prevalencia en España de mecanismos de resistencia a quinolonas en enterobacterias productoras de betalactamasas de clase C adquiridas y/o carbapenemasas / Prevalence of quinolone resistance mechanisms in Enterobacteriaceae producing acquired AmpC Beta-lactamases and/or carbapenemases in Spain

Introducción: En los últimos años se ha observado un incremento de la resistencia a fluoroquinolonas en enterobacterias, estando asociado significativamente a la resistencia a betalactámicos. Nuestro objetivo fue conocer la prevalencia de mecanismos cromosómicos y plasmídicos de resistencia a quinolonas en aislados productores de betalactamasas de claseC adquiridas y/o carbapenemasas. Métodos: Se evaluó la presencia de mecanismos cromosómicos y plasmídicos de resistencia a quinolonas [mutaciones en la región determinante de resistencia a quinolonas de gyrA y parCy genes qnr, aac(6')-Ib-cr y qepA] en 289 aislados de enterobacterias productoras de betalactamasas de claseC adquiridas y/o carbapenemasas recogidos entre febrero y julio de 2009 en 35 hospitales españoles. Resultados: Se detectaron determinantes plasmídicos en 92 aislados (31,8%); en 83 aislados (28,7%) se detectó algún gen qnr, y en 20 (7%), la variante aac(6')-Ib-cr. El gen qnr más prevalente fue qnrB4 (20%), asociado en la mayoría de los casos a DHA-1. El 14,6% de los aislados con una CMI de ciprofloxacino superior a 0,25mg/l no presentaban mutaciones en gyrA ni parC, detectándose en el 90% de los mismos algún determinante plasmídico de resistencia a quinolonas. Conclusión: qnrB4 fue el determinante plasmídico más prevalente, claramente asociado a DHA-1. Los mecanismos plasmídicos en asociación con mecanismos cromosómicos diferentes a las mutaciones en los genes de las topoisomerasas (sobreexpresión de bombas de expulsión, alteración del lipopolisacárido o disminución de porinas) pueden dar lugar a valores de CMI de ciprofloxacino que superan los puntos de corte establecidos por los principales comités internacionales de definición de puntos de corte para interpretación de datos de sensibilidad (AU)

Background: Quinolone resistance in Enterobacteriaceae species has increased over the past few years, and is significantly associated to beta-lactam resistance. The aim of this study was to evaluate the prevalence of chromosomal- and plasmid-mediated quinolone resistance in acquired AmpC Beta-lactamase and/or carbapenemase-producing Enterobacteriaceae isolates. Methods: The presence of chromosomal- and plasmid-mediated quinolone resistance mechanisms [mutations in the quinolone resistance determining region (QRDR) of gyrA and parC and qnr, aac(6')-Ib-cr and qepA genes] was evaluated in 289 isolates of acquired AmpC Beta -lactamase- and/or carbapenemase-producing Enterobacteriaceae collected between February and July 2009 in 35 Spanish hospitals. Results: Plasmid mediated quinolone resistance (PMQR) genes were detected in 92 isolates (31.8%), qnr genes were detected in 83 isolates (28.7%), and the aac(6')-Ib-cr gene was detected in 20 isolates (7%). qnrB4 gene was the most prevalent qnr gene detected (20%), associated, in most cases, with DHA-1. Only 14.6% of isolates showed no mutations in gyrA or parC with a ciprofloxacin MIC of 0.5mg/L or higher, whereas PMQR genes were detected in 90% of such isolates. Conclusion: qnrB4 gene was the most prevalent PMQR gene detected, and was significantly associated with acquired AmpC Beta -lactamase DHA-1. PMQR determinants in association with other chromosomal-mediated quinolone resistance mechanisms, different to mutations in gyrA and parC (increased energy-dependent efflux, altered lipopolysaccharide or porin loss), could lead to ciprofloxacin MIC values that exceed breakpoints established by the main international committees to define clinical antimicrobial susceptibility breakpoints (AU)

Prevalence of quinolone resistance mechanisms in Enterobacteriaceae producing acquired AmpC ß-lactamases and/or carbapenemases in Spain. / Prevalencia en España de mecanismos de resistencia a quinolonas en enterobacterias productoras de betalactamasas de clase C adquiridas y/o carbapenemasas.

BACKGROUND: Quinolone resistance in Enterobacteriaceae species has increased over the past few years, and is significantly associated to beta-lactam resistance. The aim of this study was to evaluate the prevalence of chromosomal- and plasmid-mediated quinolone resistance in acquired AmpC ß-lactamase and/or carbapenemase-producing Enterobacteriaceae isolates. METHODS: The presence of chromosomal- and plasmid-mediated quinolone resistance mechanisms [mutations in the quinolone resistance determining region (QRDR) of gyrA and parC and qnr, aac(6')-Ib-cr and qepA genes] was evaluated in 289 isolates of acquired AmpC ß-lactamase- and/or carbapenemase-producing Enterobacteriaceae collected between February and July 2009 in 35 Spanish hospitals. RESULTS: Plasmid mediated quinolone resistance (PMQR) genes were detected in 92 isolates (31.8%), qnr genes were detected in 83 isolates (28.7%), and the aac(6')-Ib-cr gene was detected in 20 isolates (7%). qnrB4 gene was the most prevalent qnr gene detected (20%), associated, in most cases, with DHA-1. Only 14.6% of isolates showed no mutations in gyrA or parC with a ciprofloxacin MIC of 0.5mg/L or higher, whereas PMQR genes were detected in 90% of such isolates. CONCLUSION: qnrB4 gene was the most prevalent PMQR gene detected, and was significantly associated with acquired AmpC ß-lactamase DHA-1. PMQR determinants in association with other chromosomal-mediated quinolone resistance mechanisms, different to mutations in gyrA and parC (increased energy-dependent efflux, altered lipopolysaccharide or porin loss), could lead to ciprofloxacin MIC values that exceed breakpoints established by the main international committees to define clinical antimicrobial susceptibility breakpoints.

Genomic Diversity of Mycobacterium tuberculosis Complex Strains in Cantabria (Spain), a Moderate TB Incidence Setting.

BACKGROUND: Tuberculosis (TB) control strategies are focused mainly on prevention, early diagnosis, compliance to treatment and contact tracing. The objectives of this study were to explore the frequency and risk factors of recent transmission of clinical isolates of Mycobacterium tuberculosis complex (MTBC) in Cantabria in Northern Spain from 2012 through 2013 and to analyze their clonal complexity for better understanding of the transmission dynamics in a moderate TB incidence setting. METHODS: DNA from 85 out of 87 isolates from bacteriologically confirmed cases of MTBC infection were extracted directly from frozen stocks and genotyped using the mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) method. The MIRU-VNTRplus database tool was used to identify clusters and lineages and to build a neighbor joining (NJ) phylogenetic tree. In addition, data were compared to the SITVIT2 database at the Pasteur Institute of Guadeloupe. RESULTS: The rate of recent transmission was calculated to 24%. Clustering was associated with being Spanish-born. A high prevalence of isolates of the Euro-American lineage was found. In addition, MIRU-VNTR profiles of the studied isolates corresponded to previously found MIRU-VNTR types in other countries, including Spain, Belgium, Great Britain, USA, Croatia, South Africa and The Netherlands. Six of the strains analyzed represented clonal variants. CONCLUSION: Transmission of MTBC is well controlled in Cantabria. The majority of TB patients were born in Spain. The population structure of MTBC in Cantabria has a low diversity of major clonal lineages with the Euro-American lineage predominating.

Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding.

The long juvenile period of citrus trees (often more than 6 years) has hindered genetic improvement by traditional breeding methods and genetic studies. In this work, we have developed a biotechnology tool to promote transition from the vegetative to the reproductive phase in juvenile citrus plants by expression of the Arabidopsis thaliana or citrus FLOWERING LOCUS T (FT) genes using a Citrus leaf blotch virus-based vector (clbvINpr-AtFT and clbvINpr-CiFT, respectively). Citrus plants of different genotypes graft inoculated with either of these vectors started flowering within 4-6 months, with no alteration of the plant architecture, leaf, flower or fruit morphology in comparison with noninoculated adult plants. The vector did not integrate in or recombine with the plant genome nor was it pollen or vector transmissible, albeit seed transmission at low rate was detected. The clbvINpr-AtFT is very stable, and flowering was observed over a period of at least 5 years. Precocious flowering of juvenile citrus plants after vector infection provides a helpful and safe tool to dramatically speed up genetic studies and breeding programmes.

Sternal wound infection caused by Gordonia bronchialis: identification by MALDI-TOF MS.

INTRODUCTION: Gordonia spp. infections are uncommon. However, a few clinical cases have been reported in the literature, particularly those involving immunocompromised hosts. Advanced microbiology diagnosis techniques, such as matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS), have been recently introduced in clinical microbiology laboratories in order to improve microbial identification, resulting in better patient management. CASE PRESENTATION: Here, we present a new clinical case of persistent wound infection caused by Gordonia bronchialis in a 64-year-old woman after a mitral valve replacement, using two MALDI-TOF-based systems for identifying this micro-organism. CONCLUSION: Both MALDI-TOF systems were able to identify Gordonia spp.; thus, providing a useful tool that overcomes the current limitations of phenotypic identification associated with this micro-organism. Although the technique validation deserves additional verification, our study provides guidance about MALDI-TOF as a fast and easy method for Gordonia spp. identification.

Microbiological and clinical aspects of respiratory infections associated with Bordetella bronchiseptica.

Bordetella bronchiseptica is a well-known veterinary pathogen, but its implication in human disease is probably not fully recognized. The purpose of this study was to determine the clinical significance of 36 B. bronchiseptica isolates from respiratory samples of 22 patients. Therefore, we describe microbiological characteristics, including phenotypic and genotypic identification as well as antimicrobial susceptibilities of the isolates. Clonal relatedness was evaluated using pulsed-field gel electrophoresis (PFGE). Most of the patients had some underlying immunosuppressive condition. Eighteen out of 22 (82%) patients had respiratory symptoms, and the death of 2 patients was associated with respiratory infection.All strains were correctly identified at species level by the simultaneous use of phenotypic methods and were confirmed by specific amplification of the upstream region of the fla gene. Tigecycline, minocycline, doxycycline, colistin, and meropenem were the most active agents tested. PFGE analysis revealed that repeated infections involving each patient had been caused by the same strain.

Tsukamurella pulmonis bloodstream infection identified by secA1 gene sequencing.

Recurrent bloodstream infections caused by a Gram-positive bacterium affected an immunocompromised child. Tsukamurella pulmonis was the microorganism identified by secA1 gene sequencing. Antibiotic treatment in combination with removal of the subcutaneous port healed the patient.

Effectiveness of gene silencing induced by viral vectors based on Citrus leaf blotch virus is different in Nicotiana benthamiana and citrus plants.

Virus induced gene silencing (VIGS) is an effective technology for gene function analysis in plants. We assessed the VIGS effectiveness in Nicotiana benthamiana and citrus plants of different Citrus leaf blotch virus (CLBV)-based vectors, using insets of the phytoene desaturase (pds) gene. While in N. benthamiana the silencing phenotype was induced only by the construct carrying a 58-nt pds hairpin, in citrus plants all the constructs induced the silencing phenotype. Differences in the generation of secondary small interfering RNAs in both species are believed to be responsible for differential host-species effects. The ability of CLBV-based vectors to silence different endogenous citrus genes was further confirmed. Since CLBV-based vectors are known to be stable and induce VIGS in successive flushes for several months, these vectors provide an important genomic tool and it is expected that they will be useful to analyze gene function by reverse genetics in the long-lived citrus plants.

Citrus leaf blotch virus invades meristematic regions in Nicotiana benthamiana and citrus.

To invade systemically host plants, viruses need to replicate in the infected cells, spread to neighbouring cells through plasmodesmata and move to distal parts of the plant via sieve tubes to start new infection foci. To monitor the infection of Nicotiana benthamiana plants by Citrus leaf blotch virus (CLBV), leaves were agroinoculated with an infectious cDNA clone of the CLBV genomic RNA expressing green fluorescent protein (GFP) under the transcriptional control of a duplicate promoter of the coat protein subgenomic RNA. Fluorescent spots first appeared in agroinfiltrated leaves 11-12 days after infiltration, indicating CLBV replication. Then, after entering the phloem vascular system, CLBV was unloaded in the upper parts of the plant and invaded all tissues, including flower organs and meristems. GFP fluorescence was not visible in citrus plants infected with CLBV-GFP. Therefore, to detect CLBV in meristematic regions, Mexican lime (Citrus aurantifolia) plants were graft inoculated with CLBV, with Citrus tristeza virus (CTV), a virus readily eliminated by shoot-tip grafting in vitro, or with both simultaneously. Although CLBV was detected by hybridization and real-time reverse transcription-polymerase chain reaction (RT-PCR) in 0.2-mm shoot tips in all CLBV-inoculated plants, CTV was not detected. These results explain the difficulty in eliminating CLBV by shoot-tip grafting in vitro.

Development of viral vectors based on Citrus leaf blotch virus to express foreign proteins or analyze gene function in citrus plants.

Viral vectors have been used to express foreign proteins in plants or to silence endogenous genes. This methodology could be appropriate for citrus plants that have long juvenile periods and adult plants that are difficult to transform. We developed viral vectors based on Citrus leaf blotch virus (CLBV) by duplicating a minimum promoter (92 bp) either at the 3' untranslated region (clbv3'pr vector) or at the intergenic region between the movement and coat protein (CP) genes (clbvINpr vector). The duplicated fragment (-42/+50) around the transcription start site of the CP subgenomic RNA (sgRNA) had the full promoter activity and induced synthesis of a new sgRNA in infected plants. Agroinoculation with these vectors resulted in systemic infection of Nicotiana benthamiana and the resulting virions systemically infected citrus plants. A clbvINpr vector carrying the green fluorescent protein (GFP) gene expressed GFP in citrus plants and triggered gfp silencing in gfp-transgenic citrus plants, and vectors carrying fragments of the phytoene desaturase or the magnesium chelatase genes incited a silencing phenotype in citrus plants. These silenced phenotypes persisted in successive flushes. Because CLBV infections are symptomless in most citrus species, the effective silencing induced by CLBV-derived vectors will be helpful to analyze citrus gene function.

Recurrent respiratory infection caused by Bordetella bronchiseptica in an immunocompetent infant.

Bordetella bronchiseptica rarely infects immunocompetent humans. We report an unusual case of recurrent pertussis-like syndrome caused by B. bronchiseptica in a 7-month-old immunocompetent boy. Molecular analysis demonstrated that the isolates from the child and mother were identical.