Epidemiología de la enfermedad fúngica invasora por hongos filamentosos en el período 2005 a 2015, en un hospital universitario en Santiago, Chile / Epidemiology of invasive fungal disease by filamentous fungi in the period 2005 to 2015, in a university hospital in Santiago, Chile

Resumen Introducción: La enfermedad fúngica invasora (EFI) por hongos filamentosos es cada vez más frecuente. Objetivo: Estudiar la epidemiología de la EFI en adultos hospitalizados en nuestro centro. Metodología: Estudio retrospectivo de pacientes adultos de un hospital universitario en Santiago, Chile, con EFI por hongos filamentosos entre enero de 2005 y diciembre de 2015. Resultados: Se identificaron 125 episodios, siendo 48% categoria probada, 40% probable y 11% posible según criterios EORTC/MSG, incidencia global 0,47 x 1.000 egresos, 57% pacientes masculinos y edad de 50 ± 16 años. El 66,4% tenía patología hematológica, 11,2% trasplante de órgano sólido, 11,2% enfermedad reumatológica, 11,2% otra condición. Los factores de riesgo fueron neutropenia 44%, corticoterapia 21%, inmunosupresores 13%. Los hongos más frecuentemente identificados fueron Aspergillus spp (53,6%), Mucorales (16%), Fusarium spp (8,8%), Alternaria spp (5,6%), otros filamentosos (3,2%). Todos recibieron antifúngicos, 82% monoterapia, 18% terapia combinada, hubo defocación quirúrgica en 90% de mucormicosis. La mortalidad global fue 42%. Al comparar 2005-2009 vs 2010-2015, hubo un aumento significativo de la incidencia y una tendencia a menor mortalidad en el segundo período. Conclusiones: Durante un período de 10 años, observamos un aumento de la incidencia de EFI por filamentosos, aspergilosis fue la etiología más frecuente y la mortalidad global fue 42%.

Background: Invasive fungal disease (IFD) due to filamentous fungi is increasingly common. Aim: To study the epidemiology of EFI in hospitalized adults in our center. Methods: Retrospective study of adult patients of a university hospital in Santiago, Chile, with EFI due to filamentous fungi between January 2005 and December 2015. Results: 125 episodes were identified, being 48% proven, 40% probable and 11% possible according to EORTC/MSG criteria, overall incidence was 0.47/1,000 admissions, 57% male patients and age 50 ± 16 years. 66.4% had hematological pathology, 11.2% solid organ transplant, 11.2% rheumatology diseases, 11.2% other conditions. The risk factors were neutropenia 44%, corticosteroid therapy 21%, immunosuppressants 13%. The most frequent mould identified were Aspergillus spp (53.6%), Mucorales (16%), Fusarium spp (8.8%), Alternaria spp (5.6%) and other filamentous (3.2%). All received antifungals, 82% monotherapy, 18% combined therapy, there was surgical defocation in 90% of mucormycosis. The overall mortality was 42%. When comparing 2005-2009 vs 2010-2015, there was a significant increase in incidence and a tendency to lower mortality in the second period. Conclusions: Over a period of 10 years, we observed an increase in the incidence of EFI by filamentous, aspergillosis was the most frequent etiology and the overall mortality was 42%.

Generation and analysis of an Eucalyptus globulus cDNA library constructed from seedlings subjected to low temperature conditions

Eucalyptus globulus is the most important commercial temperate hardwood in the world because of its wood properties and due to its characteristics for biofuel production. However, only a very low number of expressed sequence tags (ESTs) are publicly available for this tree species. We constructed a cDNA from E. globulus seedlings subjected to low temperature and sequenced 9,913 randomly selected clones, generating 8,737 curated ESTs. The assembly produced 1,062 contigs and 3,879 singletons forming a Eucalyptus unigene set. Based on BLASTX analysis, 89.3 percent of the contigs and 88.5 percent of the singletons had significant similarity to known genes in the non-redundant database of GenBank. The Eucalyptus unigene set generated is a valuable public resource that provides an initial model for genes and regulatory pathways involved in cell wall biosynthesis at low temperature.

Generation and analysis of expressed sequence tags from Botrytis cinerea

Botrytis cinerea is a filamentous plant pathogen of a wide range of plant species, and its infection may cause enormous damage both during plant growth and in the post-harvest phase. We have constructed a cDNA library from an isolate of B. cinerea and have sequenced 11,482 expressed sequence tags that were assembled into 1,003 contigs sequences and 3,032 singletons. Approximately 81% of the unigenes showed significant similarity to genes coding for proteins with known functions: more than 50% of the sequences code for genes involved in cellular metabolism, 12% for transport of metabolites, and approximately 10% for cellular organization. Other functional categories include responses to biotic and abiotic stimuli, cell communication, cell homeostasis, and cell development. We carried out pair-wise comparisons with fungal databases to determine the B. cinerea unisequence set with relevant similarity to genes in other fungal pathogenic counterparts. Among the 4,035 non-redundant B. cinerea unigenes, 1,338 (23%) have significant homology with Fusarium verticillioides unigenes. Similar values were obtained for Saccharomyces cerevisiae and Aspergillus nidulans (22% and 24%, respectively). The lower percentages of homology were with Magnaporthe grisae and Neurospora crassa (13% and 19%, respectively). Several genes involved in putative and known fungal virulence and general pathogenicity were identified. The results provide important information for future research on this fungal pathogen.

Isolation, sequencing and phylogenetic analysis of the hemagglutinin, neuraminidase and nucleoprotein genes of the Chilean equine influenza virus subtypes H7N7 and H3N8

We report here on the isolation and sequencing of the hemagglutinin, neuraminidase and nucleoprotein genes of the Chilean equine influenza virus subtypes H7N7 (A⁄equi-1⁄Santiago⁄77, Sa77) and H3N8 (A⁄equi-2⁄Santiago⁄85, Sa85) . The sequences obtained allowed a variability analysis, which indicated significant differences when compared with other isolates. We found that Chilean isolates are more similar to the North American variety than to European isolates. Isolate Sa77 is a good candidate for inclusion in a vaccine as it is the latest isolate of the subtype H7N7 and is probably better-adapted to the equine host. Isolate Sa85, of subtype H3N8, also appears to be a good candidate since it has no significant differences in the main antigenic sites with recent isolates.

Production and immune response of recombinant Hsp60 and Hsp70 from the salmon pathogen Piscirickettsia salmonis

We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60) and 70 (Hsp70) of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80 percent homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.

Strategies to capture biotechnology opportunities in Chile

Two complementary strategies are proposed to help develop the biotechnology industry in Chile. The objectives of such propositions are based on identifying business opportunities, which can be transformed into biotechnology projects that complement the competitive advantages of the most active areas of the Chilean economy. As a result, the establishment of these initiatives may create the proper business environment where good information, investors' safeguards and economic incentives would be provided to encourage investors to support new biotechnology ventures focused on mining, aquaculture, forestry, as well as wine and fruit production. In addition, a complete description of the Chilean biotechnology industry is provided. Amongst other characteristics, this report shows that the industry lacks financial support from venture capital and foreign investors, has a relatively modest proportion of highly qualified employees that work in Research and Development, presents insufficient patent productivity and is mostly regulated in terms of the use of GMOs.

Isolation and expression of the genes coding for the membrane bound transglycosylase B (MltB) and the transferrin binding protein B (TbpB) of the salmon pathogen Piscirickettsia salmonis

We have isolated and sequenced the genes encoding the membrane bound transglycosylase B (MltB) and the transferring binding protein B (TbpB) of the salmon pathogen Piscirickettsia salmonis. The results of the sequence revealed two open reading frames that encode proteins with calculated molecular weights of 38,830 and 85,140. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from phylogenetically related microorganisms. Partial sequences coding the amino and carboxyl regions of MltB and a sequence of 761 base pairs encoding the amino region of TbpB have been expressed in E. coli. The strong humoral response elicited by these proteins in mouse confirmed the immunogenic properties of the recombinant proteins. A similar response was elicited by both proteins when injected intraperitoneally in Atlantic salmon. The present data indicates that these proteins are good candidates to be used in formulations to study the protective immunity of salmon to infection by P. salmonis.

Complete sequence of the genome of the human isolate of Andes virus CHI-7913: comparative sequence and protein structure analysis

We report here the complete genomic sequence of the Chilean human isolate of Andes virus CHI-7913. The S, M, and L genome segment sequences of this isolate are 1,802, 3,641 and 6,466 bases in length, with an overall GC content of 38.7 percent. These genome segments code for a nucleocapsid protein of 428 amino acids, a glycoprotein precursor protein of 1,138 amino acids and a RNA-dependent RNA polymerase of 2,152 amino acids. In addition, the genome also has other ORFs coding for putative proteins of 34 to 103 amino acids. The encoded proteins have greater than 98 percent overall similarity with the proteins of Andes virus isolates AH-1 and Chile R123. Among other sequenced Hantavirus, CHI-7913 is more closely related to Sin Nombre virus, with an overall protein similarity of 92 percent. The characteristics of the encoded proteins of this isolate, such as hydrophobic domains, glycosylation sites, and conserved amino acid motifs shared with other Hantavirus and other members of the Bunyaviridae family, are identified and discussed

The complete sequence of the mitochondrial genome of the Chinook salmon, Oncorhynchus tshawytscha

The complete sequence of the mitochondrial genome of Chinook salmon, Oncorhynchus tshawytscha, has been determined. The circular genome consisting of 16,644 base pairs encodes thirteen proteins, the 12S and 16S ribosomal RNAs, and 22 transfer RNAs. These genes are ordered in the same way as most other vertebrates. The nucleotide and amino acid sequences of the ribosomal RNAs and the thirteen protein-coding genes were compared with those of other salmonids such as Oncorhynchus mykiss, Salmo salar, Salvelinus fontinalis, Salvelinus alpinus and Coregonus lavaretus. The sequence features of the control region (D-loop), the origin of L-strand replication and a putative peptide codified by the 16S mitochondrial RNA are described and discussed

Cloning and expression of the coding regions of the heat shock proteins HSP10 and HSP16 from Piscirickettsia salmonis

The genes encoding the heat shock proteins HSP10 and HSP16 of the salmon pathogen Piscirickettsia salmonis have been isolated and sequenced. The HSP10 coding sequence is located in an open reading frame of 291 base pairs encoding 96 aminoacids. The HSP16 coding region was isolated as a 471 base pair fragment encoding a protein of 156 aminoacids. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from other prokaryotic organisms. Both proteins were expressed in E. coli as fusion proteins with thioredoxin and purified by chromatography on Nicolumn. A rabbit serum against P. salmonis total proteins reacts with the recombinant HSP10 and HSP16 proteins. Similar reactivity was determined by ELISA using serum from salmon infected with P. salmonis. The possibility of formulating a vaccine containing these two proteins is discussed.

Immunoresponse of Coho salmon immunized with a gene expression library from Piscirickettsia salmonis

We have used the expression library immunization technology to study the protection of Coho salmon Oncorhynchus kisutch to the infection with Piscirickettsia salmonis. Purified DNA from this bacterium was sonicated and the fragments were cloned in the expression vector pCMV-Bios. Two libraries were obtained containing 22,000 and 28,000 colonies and corresponding to approximately 8 and 10 times the genome of the pathogen, respectively. On average, the size of the inserts ranged between 300 and 1,000 bp. The plasmid DNA isolated from one of these libraries was purified and 20 micrograms were injected intramuscularly into 60 fish followed by a second dose of 10 micrograms applied 40 days later. As control, fish were injected with the same amount of DNA of the vector pCMV-Bios without insert. The titer of IgM anti-P. salmonis of vaccinated fish, evaluated 60 days post-injection, was significantly higher than that of the control group injected with the vector alone. Moreover, this response was specific against P. salmonis antigens, since no cross reaction was detected with Renibacterium salmoninarum and Yersinia ruckeri. The vaccinated and control fish were challenged 60 days after the second dose of DNA with 2.5 x 10(7) P. salmonis corresponding to 7.5 times the LD50. At 30 days post-challenge, 100 per cent mortality was obtained with the control fish while 20 per cent of the vaccinated animals survived. All surviving fish exhibited a lower bacterial load in the kidney than control fish. The expression library was also tested in Balb/c mice and it was found that the humoral immune response was specific to P. salmonis and it was dependent on the amount of DNA injected.

Cloning and comparison of ten gene sequences of a Chilean H. pylori strain with other H. Pylori strains revealed higher variability for VacA and CagA virulence factors

We have cloned and sequenced ten Helicobacter pylori genes from a Chilean strain (CH-CTX1) including: a cytotoxin VacA fragment, a CagA fragment (A17), a species-specific protein (TsaA), urease subunits (UreA, UreB), a flagellin subunit (FlaB), heat shock proteins (HspA and HspB), adhesin (HpaA) and a lipoprotein (Lpp20). We compared their deduced amino acid sequences with the corresponding sequences from three unrelated H. pylori strains, including fully sequenced strains 26695(UK) and J99(USA), and found that eight of them (UreA, UreB, FlaB, HspA, HspB, Lpp20, TsaA and HpaA) presented more than 97.3% identity. In contrast, VacA partial sequence showed lower identity values (93.2-94.9%). Moreover, we found major differences in the A17 region respect to the number and arrangement of the internal repeated elements when sequences from different strains were aligned. The A17 regions from strains CH-CTX1 and 26695 are very similar (91.8% identity) but lacked 6 repeated elements when compared to the Australian strains ATCC 43526 and NCTC 11637. The CCUG 17874 A17 region showed the largest deletion involving 9 repeats. A17 size differences between strains CCUG 17874 and CH-CTX1 were verified by PCR and polypeptide size. Such differences may explain variations in virulence among H. pylori strains as well as diversity in serum immunoreactivity.

The bacteriophage lambda DNA packaging enzyme: identification of four structural domains of the gpNu1 subunit using limited proteolysis

Lambda DNA terminase, the enzyme that cleaves virion-length chromosomes from multigenomic concatemers and packages them into the bacteriophage head, is composed of two subunits, gpNu1 and gpA. Direct determination of the structure of gpNu1, the smaller subunit, has not been possible because of its insolubility in aqueous solutions. Therefore, to identify smaller and potentially water-soluble domains of gpNu1, we analyzed the nature of the products obtained by limited digestion of the protein with several proteases. The gpNu1 subunit was obtained from E. coli cells transfected with the plasmid pH6-Nu1 that overproduces the protein. Incubation of gpNu1 solubized in 2.5 M guanidinium chloride with chymotrypsin resulted in the formation of at least eight discrete protein bands, while treatment with endoproteinase glu-C and bromelain yielded three and one major bands, respectively. The peptides generated by digestion with the various proteases were separated by two-dimensional gel electrophoresis and transferred to Immobilon membranes. Amino acid sequencing of the peptides allowed for the precise assignment of their N-terminal amino acid, while their estimated molecular weights permitted the identification of their C-terminal ends. The results reveal that in the presence of 2.5 M guanidinium chloride, gpNu1 is partially folded in at least four distinct structural domains that correspond to functional domains as determined by previously reported genetic experiments. This information is key to design new plasmids to overproduce these domains for further structural analysis.

Mortalidad en el Servicio de Neonatología del Hospital Clínico de la Universidad de Chile entre 1996 y 1999 / Mortality at neonatology service, Hospital Clínico, Universidad de Chile

Las características de los Servicios de Neonatología pueden ser tan disímiles que las comparaciones entre sí pueden ser inútiles, sin embargo es necesario tener referencias locales para medir el avance en los métodos en uso y al mismo tiempo trabajar en lograr consensos para obtener oportuna información a nivel local y nacional. Se muestra la mortalidad en el Servicio de Neonatología del Hospital Clínico de la Universidad de Chile entre los años 1996 y 1999. La información se extrajo desde una base de datos previamente consensuada entre los neonatólogos del Servicio y ordenada de acuerdo a los criterios de Wigglesworth modificados. Se describe la mortalidad por peso de nacimiento, edad gestacional y por grupos de diagnósticos

Cuerpo extraño en la vía aérea / Foreign body in the airway

La presencia de substancias extrañas, dentro de la vía aérea, condiciona una serie de cuadros clínicos que por su importancia y frecuencia es indispensable estudiar. Aún cuando la denominación de cuerpo extraño sugiere algo sólido, es necesario considerar dentro de esta denominación, a substancias líquidas y gaseosas, que por sus características, actuarán como cuerpos extraños. Por otra parte, la denominación de extraño, parece llevar implícita la reacción local o a distancia, que genera la presencia. Esta respuesta puede ser inespecífica o generar cuadros clínicos especiales. De este modo, deberíamos definir como cuerpos extraños, a aquellas substancias u objetos que, colocados dentro de la vía aérea, impiden su normal funcionamiento y generan una respuesta. Así, no actuarían como cuerpos extraños, las prótesis, pero sí, los dispositivos de una vía aérea artificial. Dentro de esta definición también cabrían los gases, ya que algunos de ellos, por sus especiales características, actúan sobre la vía aérea con mayor fuerza que los líquidos, especialmente aquéllos con alta temperatura o los que tienen efecto cáustico. Podemos, entonces, clasificar las substancias extrañas dentro de la vía aérea, según la tabla 1