Jorge Levican

Comparison of virological profiles of respiratory syncytial virus and rhinovirus in acute lower tract respiratory infections in very young Chilean infants, according to their clinical outcome

Abstract Background Respiratory syncytial virus (RSV) and rhinovirus (HRV) are the main cause of acute lower respiratory tract infections (ALRTIs) in infants. Viral and host-related risk factors for severe disease have also not been clearly established. Objective To assess whether certain viral features of RSV and, or HRV are associated with severe ALRTI. Study design RSV and HRV were studied in nasopharyngeal samples of infants by immunofluorescence, Luminex® and/or real-time RT-PCR assays. Quantitation and genotyping of RSV and HRV by PCR were done. Results Of 124 virus positive specimens, 74 (59.7%) had RSV; 22 (17.7%) HRV and 28 (22.6%) RSV-HRV co-infection. Hospitalization was required in 57/74 RSV infants (77.0%); in 10/22 HRV cases (45.5%) (p =0.006) and in 15/28 co-infected by both viruses (53.6%) (p =0.003). Severe cases were 33/74 (44.6%) RSV infections, 2/22 HRV cases (9.1%), (p <0.002) and 6/28 (21.4%) patients co-infected by RSV–HRV (p <0.026). Three genotypes (NA1, B7, B9) of RSV circulated during the study. In 33 severe infants, NA1 was detected in 19 cases (57.6%); B7 in 13 (39.4%) and B9 in 1 (3.0%) (p <0.01; OR=10.0). RSV loads were similar between outpatients and hospitalized infants (p =0.7) and among different severities (p =0.7). NA1 loads were higher than other strains (p =0.049). Three geno-groups of HRV circulated homogeneously. Conclusion In very young infants, RSV cause more severe disease than HRV. Co-infection does not increase the severity of illness. NA1 RSV genotype was associated with major frequency of hospitalization, severe respiratory disease and higher viral load.

Merkel cell polyomavirus in non-small cell lung carcinomas from Chile

Lung cancer is a leading pathology strongly associated with the smoking habit. However, a viral etiology for a subset of patients developing lung cancer has been suggested. Polyomaviruses (PyVs) are small double stranded DNA viruses associated with the development of some human diseases. However, a causal role of these viruses in human cancer has been difficult to demonstrate. In this study, eighty-six non-small cell lung carcinomas (NSCLCs), including adenocarcinomas (AdCs) and squamous cell lung carcinomas (SQCs) from Chile were analyzed for the presence of PyVs using polymerase chain reaction (PCR). All of the specimens were positive for a fragment of the betaglobin gene. We found that 4/86 (4.7%) of lung carcinomas were positive for PyVs. After sequencing and BlastN alignment, all four cases were identified as Merkel cell polyomaviruses (MCV) that corresponded to two AdCs and two SQCs. A non-significant statistical association was found between the presence of MCV and clinic-pathological features of the patients and tumors. In addition, 1/4 (25%) of the carcinomas were actively expressing large T antigen (LT) transcripts, as demonstrated by reverse-transcriptase PCR (RT-PCR). Thus a possible role of MCV in a very small subset of patients with lung cancer cannot be ruled out and warrants more investigation.

Human bocavirus in children with acute gastroenteritis, Chile, 1985-2010.

We detected human bocavirus in 89 (19.3%) of 462 fecal samples collected during 3 periods from 1985 through 2010 from children <5 years of age in Chile who were hospitalized with acute gastroenteritis. Our findings confirm the long-term circulation of human bocavirus in Chile.

Atrofia muscular espinal: Caracterización clínica, electrofisiológica y molecular de 26 pacientes

Background: Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder affecting the anterior horn cells of the spinal cord resulting in muscle weakness and atrophy, linked to the homozygous disruption of the survival motor neuron 1 (SMN1) gene. It is the leading genetic cause of infant death. It has been classified into three types based on the severity of symptoms. Type I SMA is the most severe form with death within the first 2 years of life. Type II and III SMA patients show intermediate and mild forms of the disorder. Aim: To describe the clinical and electrophysiological findings of 26 Chilean patients with SMA with molecular confirmation. Patients and Methods: Retrospective multicenter analysis of patients with SMA assessed between 2003 and 2010. The diagnosis was suspected on clinical and electrophysiological criteria. Since 2006 molecular genetics confirmation was implemented in one of our centers. Results: Twenty-six patients between 2 months and 18 years of age at presentation were analyzed; 15 (58%) were males. SMA I, II and III clinical criteria were observed in 4 (15.4 %), 11 (42.3%) and 11 (42.3%)patients, respectively. All had proximal muscle weakness and atrophy. Electromyography showed features of acute denervation or re-innervation with normal motor and sensory nerve conduction. Nine patients required a muscle biopsy. The genetic confirmation of the disease by PCR technique followed by restriction fragment length polymorphism method disclosed the SMN1 gene deletion in all 26 cases. All patients died secondary to respiratory failure, between eight and 14 months of life. Conclusions: An adequate clinical and molecular diagnosis of spinal muscular atrophy will help for a better management of these patients.

Detección de ADN de parvovirus B19 en donantes de sangre de tres hospitales en Santiago, Chile

BACKGROUND: Parvovirus B19 (B19) is associated with a wide range of diseases in humans, whose severity depends on the immunological and hematological status of the host. It is transmitted mainly through the airway but also by transfusions. AIM: To determine the B19 DNA carrier frequency in a population of volunteer blood donors from three hospitals blood banks in Santiago, Chile, and to determine the viral load in DNA positive cases. MATERIAL AND METHODS: A total of 477 serum samples were analyzed. The screening of B19 DNA was carried out by nested polymerase chain reaction (PCR) directed to the non-structural region of the virus (NS1). The viral load in positives cases was quantified by NS1 Real Time PCR. RESULTS: Parvovirus B19 was detected in four samples, rendering a frequency of 1:119. The viral loads ranged from less than 2000 to 5626 x 10(5) genome equivalents/ml. CONCLUSIONS: Parvovirus B19 was present in four of 477 blood bank blood donors from three hospitals in Santiago.

Role of BK human polyomavirus in cancer

Human polyomaviruses (HPyV), which are small DNA viruses classified into the polyomaviridae family, are widely distributed in human populations. Thirteen distinct HPyVs have been described to date. Some of these viruses have been found in human tumors, suggesting an etiological relationship with cancer. In particular, convincing evidence of an oncogenic role has emerged for a specific HPyV, the Merkel cell polyomavirus (MCPyV). This HPyV has been linked to rare skin cancer, Merkel cell carcinoma (MCC). This finding may be just the tip of the iceberg, as HPyV infections are ubiquitous in humans. Many authors have conjectured that additional associations between HPyV infections and neoplastic diseases will likely be discovered. In 2012, the International Agency for Research on Cancer (IARC) evaluated the carcinogenicity of the BK virus (BKPyV), reporting that BKPyV is “possibly carcinogenic to humans.” This review explores the BKPyV infection from a historical point of view, including biological aspects related to viral entry, tropism, epidemiology and mechanisms potentially involved in BKPyV-mediated human carcinogenesis. In order to clarify the role of this virus in human cancer, more epidemiological and basic research is strongly warranted.

Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis

Infectious pancreatic necrosis virus (IPNV) is a non-enveloped virus belonging to the Birnaviridae family. IPNV produces an acute disease in salmon fingerlings, with high mortality rates and persistent infection in survivors. Although there are reports of IPNV binding to various cells, the viral receptor and entry pathways remain unknown. The aim of this study was to determine the endocytic pathway that allows for IPNV entry. We observed that IPNV stimulated fluid uptake and virus particles co-localysed with the uptake marker dextran in intracellular compartments, suggesting a role for macropinocytosis in viral entry. Consistent with this idea, viral infection was significantly reduced when the Na+/H+ exchanger NHE1 was inhibited with 5-(N-Ethyl-N-isopropyl) amiloride (EIPA). Neither chlorpromazine nor filipin complex I affected IPNV infection. To examine the role of macropinocytosis regulators, additional inhibitors were tested. Inhibitors of the EGFR pathway and the effectors Pak1, Rac1 and PKC reduced viral infection. Together, our results indicate that IPNV is mainly internalized into CHSE-214 cells by macropinocytosis.

[BK and JC polyomavirus detection in leukocyte extracts of peripheral blood samples of HIV+ patients from the north area of Santiago].

INTRODUCTION Polyomavirus BK (BKPyV) and JC (JCPyV) are persistent pathogens able to reactivate in im-munocompromised patients, involving mostly urinary and central nervous system. There are no Chilean studies in HIV positive patients. OBJECTIVE To detect BKPyV and JCPyV in blood of Chilean HIV positive adult patients and to correlate these results with clinical-related variables. MATERIALS AND METHODS 96 stored blood samples from HIV patients belonging to the north area of Santiago were analyzed. Viral genomes of both viruses were detected by real-time PCR. For statistical analysis, chi-square (Pearson) and Mann-Whitney tests were used and p-values < 0.05 were considered significant. RESULTS 33% of the samples were positive for BKPyV and a significant correlation was found between the presence of BKPyV genome and the absence of detectable HIV viral load. We demonstrated the need to consider more than one amplification target to detect the BKPyV genome. All the samples were negative for JCPyV genome. DISCUSSION BKPyV prevalence in Chilean HIV patients is higher than most of international studies. New studies regarding the interaction between both viruses are required. These patients should undergo periodic evaluations by urologist and nephrologist.

High genetic diversity of species A rotaviruses detected in swine farms in Chile.

Rotavirus A is one of the main causative agents of diarrhoea in lactating and weaned pigs worldwide. Its impact in the swine industry is well documented. However, in Chile, the current epidemiological status of rotavirus on porcine farms is unknown. This study evaluated the current epidemiologic status of rotavirus A infection in Chile using on-farm detection techniques, electrophoretic confirmation, genotyping and phylogenetic clustering by analysis of partial sequences of VP4 and VP7 genes. Rotavirus A was detected in four out of five farms with an overall prevalence of 17.7 % in diarrhoeic pigs. The average age of diarrhoea onset was at 32±6.2 days, corresponding to weaning pigs, and rotavirus was not detected in lactating piglets. Molecular characterization indicated that genotypes G5, G3, P[7] and P[13] are currently the most widely represented on these pigs farms. The phylogenetic analysis showed that farms shared similar G types (VP7), which might denote a common origin. Meanwhile, [P] types (VP4) showed considerable genetic diversity, and this might represent a high rate of reassortment of this genetic segment in rotavirus circulating in the researched area. These findings demonstrate the importance of considering both the geographical and production factors to accurately determine rotavirus prevalence status at the national level, and have relevant implications in determining effective strategies for rotavirus infection control on porcine farms.

Abstract 5551: Somatic mutations in KRAS, BRAF and PIK3CA genes in Chilean patients with sporadic colorectal cancer: Correlation with clinical and histological features

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: KRAS, BRAF and PIK3CA proto-oncogenes encode for proteins involved in epidermal growth factor receptor (EGFR) signaling pathways. Activating mutations in these genes may contribute to tumor development and induce resistance to biological therapies in patients with metastatic colorectal cancer (CRC). Furthermore, microsatellite instability (MSI) has been proposed as a prognostic and predictive marker in sporadic CRC. Patients and methods: In tumors of patients undergoing surgery for sporadic CRC at our institution, somatic mutations in KRAS, BRAF and PIK3CA genes were assessed by single-strand conformation polymorphism(SSCP) and direct sequencing. MSI analysis was performed using the 5 markers of the standard NIH panel. Clinical and histopathological data of the patients were obtained from our prospectively maintained database. Results: A total of 58 patients with a nearly equal gender distribution and a mean age of 62 years were included. A total of 31 mutations in 26 patients were identified. The frequency of mutations was 28% for KRAS, 9% for BRAF and 17% for PIK3CA. No difference was found in the mutation frecuency between the different tumor locations (42,1% in right colon vs. 46,2% in left colon/rectum, P= NS) or lymphnode status (37,5% in node positive vs. 50% in node negative tumors, P=0,346). Tumors with deeper invasion showed a higher frecuency of mutations, but this tendency did not reach statistical significance (32% in pT1-2 vs. 51% in pT3-4, P=0,157). In 26% of the patients, MSI-high was observed, more frecuently in tumors of the right colon (57,9% vs 10,3%, P<0,001). Patients with BRAF mutations, a higher proportion of MSI-high was found compared to BRAF wildtype (27% vs. 2,3%, P=0,013). There was no correlation between the lymphnode positivity and MSI status. Conclusion: Our results show that the frequency of mutations in patients with sporadic CRC in Chile is similar to that described in other countries. The presence of MSI-high is correlated with tumors in the right colon and with mutations in the BRAF gene. The tendency of finding more mutations in locally advanced and node-negative tumors needs to be confirmed in future studies with a larger number of patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5551. doi:1538-7445.AM2012-5551