Prevalence and genotypes of the adenovirus infection as well detection of co-infection with bocavirus in Mexican immunosuppressed and non-immunosuppressed children with pneumonia.

BACKGROUND: Adenovirus (AdV) causes respiratory infection; recent observations suggest that some subtypes have more ability to develop fatal disease. AdV infection has been associated with co-infection with human bocavirus (HBoV). We analysed the frequency of AdV infection, its subtypes and the presence of co-infection with HBoV, as well the clinical characteristics of such co-infection in Mexican paediatric immunosuppressed (IP) and non-immunosuppressed patients (non-IP) diagnosed with pneumonia. METHODS: A total of 5185 nasopharyngeal swabs from two groups of children with pneumonia, one IP and the other non-IP, were analysed for the detection of AdV by immunofluorescence and confirmed by PCR and culture. HBoV was identified by PCR. Positive samples for AdV and AdV/HBoV were typed using PCR sequencing, the clinical characteristics of the AdV/HBoV co-infection were analysed. RESULTS: Thirty-seven of the 5185 (0.71%) samples were positive for AdV, of those 27/37 (73%) were detected in non-IP and 10/37 (27%) in the IP group. Twelve were typed as follows: 9/12 (75%) as Species B1 subtype 3, of those 8/9 (88.9%) in non-IP and 1/9 in the IP group. One of twelve AdV2 subtype B11a was identified in one non-IP and the remaining two out of 12 successfully typed, were identified as Species C subtypes 2 and 6 in the group of non-IP. The presence of both AdV and HBoV1 in co-infection was observed in 2/37 (5.4%) non-IP with a syndrome like influenza. CONCLUSIONS: In this 5 year analysis of samples from non-IP and IP hospitalized paediatric patients with a diagnosis of pneumonia, a low incidence of AdV was found. B1 was the most frequent subtype and frequently found in non-IP, and two cases of co-infection AdV/HBoV1 were detected in two non-IP with a influenza-like syndromes. This is the first report of HBoV and AdV co-infection in Mexico. The frequency of AdV and HBoV co-infection was lower than that reported in other populations.

Parainfluenza virus type 1 induces epithelial IL-8 production via p38-MAPK signalling.

Human parainfluenza virus type 1 (HPIV-1) is the most common cause of croup in infants. The aim of this study was to describe molecular mechanisms associated with IL-8 production during HPIV-1 infection and the role of viral replication in MAPK synthesis and activation. An in vitro model of HPIV-1 infection in the HEp-2 and A549 cell lines was used; a kinetic-based ELISA for IL-8 detection was also used, phosphorylation of the mitogen-activated protein kinases (MAPKs) was identified by Western blot analysis, and specific inhibitors for each kinase were used to identify which MAPK was involved. Inactivated viruses were used to assess whether viral replication is required for IL-8 production. Results revealed a gradual increase in IL-8 production at different selected times, when phosphorylation of MAPK was detected. The secretion of IL-8 in the two cell lines infected with the HPIV-1 is related to the phosphorylation of the MAPK as well as viral replication. Inhibition of p38 suppressed the secretion of IL-8 in the HEp-2 cells. No kinase activation was observed when viruses were inactivated.

In situ molecular identification of the influenza A (H1N1) 2009 Neuraminidase in patients with severe and fatal infections during a pandemic in Mexico City.

BACKGROUND: In April 2009, public health surveillance detected an increased number of influenza-like illnesses in Mexico City's hospitals. The etiological agent was subsequently determined to be a spread of a worldwide novel influenza A (H1N1) triple reassortant. The purpose of the present study was to demonstrate that molecular detection of pandemic influenza A (H1N1) 2009 strains is possible in archival material such as paraffin-embedded lung samples. METHODS: In order to detect A (H1N1) virus sequences in archived biological samples, eight paraffin-embedded lung samples from patients who died of pneumonia and respiratory failure were tested for influenza A (H1N1) Neuraminidase (NA) RNA using in situ RT-PCR. RESULTS: We detected NA transcripts in 100% of the previously diagnosed A (H1N1)-positive samples as a cytoplasmic signal. No expression was detected by in situ RT-PCR in two Influenza-like Illness A (H1N1)-negative patients using standard protocols nor in a non-related cervical cell line. In situ relative transcription levels correlated with those obtained when in vitro RT-PCR assays were performed. Partial sequences of the NA gene from A (H1N1)-positive patients were obtained by the in situ RT-PCR-sequencing method. Sequence analysis showed 98% similarity with influenza viruses reported previously in other places. CONCLUSIONS: We have successfully amplified specific influenza A (H1N1) NA sequences using stored clinical material; results suggest that this strategy could be useful when clinical RNA samples are quantity limited, or when poor quality is obtained. Here, we provide a very sensitive method that specifically detects the neuraminidase viral RNA in lung samples from patients who died from pneumonia caused by Influenza A (H1N1) outbreak in Mexico City.

Reducing occurrence and severity of pneumonia due to pandemic H1N1 2009 by early oseltamivir administration: a retrospective study in Mexico.

BACKGROUND: Anti-viral treatment has been used to treat severe or progressive illness due to pandemic H1N1 2009. A main cause of severe illness in pandemic H1N1 2009 is viral pneumonia; however, it is unclear how effective antiviral treatment is against pneumonia when administered >48 hours after symptom onset. Therefore, we aimed to determine how time from symptom onset to antiviral administration affected the effectiveness of antiviral treatment against pneumonia due to pandemic (H1N1) 2009. METHODS/PRINCIPAL FINDINGS: A retrospective medical chart review of 442 patients was conducted in a hospital in Mexico. Subjects had tested positive for pandemic H1N1 2009 virus by real-time reverse-transcriptase-polymerase-chain-reaction and were administered oseltamivir. Median time from symptom onset to oseltamivir administration was 5.0 days (range, 0-43). 442 subjects, 71 (16.1%) had severe pneumonia which required mechanical ventilation, 191 (43.2%) had mild to moderate pneumonia, and 180 (40%) did not have pneumonia. Subjects were divided into four groups based on time to oseltamivir administration: ≤2, 3-7, 8-14, and >14 days. Severity of respiratory features was associated with time to treatment, and multivariate analysis indicated that time to oseltamivir administration was associated with severity of respiratory features. A proportional odds model indicated that 50% probability for occurrence of pneumonia of any severity and that of severe pneumonia in patients who would develop pneumonia reached at approximately 3.4 and 21 days, respectively, after symptom onset. Patients with a shorter time to oseltamivir administration were discharged earlier from the hospital. CONCLUSIONS: Earlier initiation of oseltamivir administration after symptom onset significantly reduced occurrence and severity of pneumonia and shortened hospitalization due to pandemic H1N1 2009. Even when administered >48 hours after symptom onset, oseltamivir showed considerable potential for reducing pneumonia. Application of these results would benefit patients affected by future influenza pandemics.

Clinical comparison of two Mexican coccidioidins.

Coccidioidin, an extract from the saprophytic mycelial form of Coccidioides spp., has been a very useful antigen preparation both for skin and serological tests for coccidioidomycosis. Unfortunately, coccidioidin is not currently available for skin testing in the United States. Coccidioidin has been produced commercially in Mexico by a vaccine and reagents laboratory of the Mexican Federal Government. It also has been produced at the Microbiology Laboratory of the Faculty of Medicine, Universidad Nacional Autónoma de México exclusively as an antigen for research projects. The objective of the study was to compare both coccidioidins in their reactivity and safety when applied in humans. One hundred and eighty-four volunteers were tested; median age was 33 (range 14-82). When the cutoff point is set in 5 mm, 88 subjects (47.8%) had a positive test for the commercial coccidioidin and 76 (41.3%; CI(95%) 0.50, 1.15; P = 0.20) were positive with the research antigen. Seventy-five subjects were positive for both antigens and 96 were negative for both. Fifty-nine subjects (31.3%) reported an adverse reaction after the application of the antigen; they were mostly very mild local reactions. Mexican research coccidioidin is a safe and reliable antigen that can be used for the detection of coccidioidomycosis infection in mammals.

Modification of the cytoprotective protein C pathway during Dengue virus infection of human endothelial vascular cells.

Dengue fever (DF) is the most prevalent arthropod-borne viral disease of humans. No safe vaccine is available, there is no experimental animal model and no specific treatment (antiviral) for Dengue virus (DV) infection exists. The pathogenic mechanisms of the severe forms of the disease, such as Dengue shock syndrome (DSS) and Dengue haemorrhagic fever (DHF), in which endothelial damage is the pathognomonic sign, are not fully understood. Clinical observations have revealed significant abnormalities in the coagulation and inflammation systems, with increased levels of soluble thrombomodulin (sTM) in the plasma of patients with DHF/DSS (grade III or IV). Blood sTM was proposed as an early predictor of DSS during the febrile stage. However, the role of the DV in endothelial injury during DSS is unclear. Here, we present novel insights into the participation of DV in the downregulation of the thrombomodulin-thrombin-protein C complex formation at the endothelial surface, with a reduction in activated protein C (APC). APC is the most important vasoprotective protein because it downregulates thrombin generation (by the inactivation of procoagulant factors Va and VIIIa) and has anti-inflammatory, antiapoptotic, and barrier protection properties. These biological functions of APC are associated with the endothelial protein C receptor (EPCR) and protease-activated receptor 1 (PAR-1) signalling pathways, which link the coagulation-inflammation responses. We found alterations in the antithrombotic and cytoprotective protein C pathways during DV infection of human endothelial vascular cells, which may explain the vasculopathy observed during DHF/DSS. Clarification of the basic principles that underlie these processes has important implications for the design of new therapeutic strategies for DHF/DSS.

Crosstalk between coagulation and inflammation during Dengue virus infection.

Dengue fever is the most prevalent viral disease transmitted by vectors (Aedes aegypti, Aedes albopictus) in worldwide. More than 100 million cases occur annually with a mortality rate of 5% and no safe vaccine is available. The pathogenesis of Dengue, where host and viral factors participate in the establishment of Dengue haemorrhagic fever (DHF) and Dengue shock syndrome (DSS) remains unresolved. Clinical observations have revealed significant abnormalities in coagulation and inflammation systems, with increased levels of tissue factor (TF) and the chemokine IL-8, correlating with the severity of the disease and implicating damage to endothelial vascular cells (EVC). Here we present novel insights concerning the crosstalk between the regulatory signaling pathways of the coagulation-inflammation processes, during Dengue virus (DV) infection of EVC. We found that DV up-regulates Protease Activated receptor type-1 (inflammation) and TF (coagulation) receptors, via the phosphorylation of p38 and ERK1/2 MAPKs, which favor the activation of NF-kappaB transcription factor. This induces pro-inflammatory (IL-8) or pro-adhesive (VCAM-1) gene expression which may lead to EVC activation. The elucidation of the basic principles that signal these processes has important implications for the design of new therapeutic strategies for DHF/DSS.

incorporación de la técnica de RT-PCR para la detección del virus sincitial respiratorio / Incorporation of the RT-PCR technique to the detection of respiratory syncitial virus

Objetivo: Estandarizar e incorporar técnicas de biología molecular, RT-PCR para la detección del virus sincitial respiratorio que complementen y enriquezcan el diagnóstico.Material y métodos: se utilizaron cepas de referencia del virus sincitial respiratorio de los grupos A y B (virus stock ATCC), las cuales se propagaron y titularon en células HEp-2. Se realizaron pruebas de inmunofluorescencia indirecta y de la prueba RT-PCR. Para ello se extrajo el ARN del virus con trizol, se realizó una transcripción reversa para obtener ADNc y, para la PCR se utilizaron oligonucleótidos que amplifican un fragmento del gen de la proteína G del virus sincitial respiratorio. Para comprobar la especificidad de la prueba se utilizaron virus de la misma familia (sarampión y parainfluenza) y virus de diferentes familias (influenza y adenovirus). Al evaluar la sensibilidad se utilizaron diferentes diluciones del ADNc viral.Resultados: en el cultivo, el efecto citopático se puede observar claramente del cuarto al octavo día. La prueba de inmunofluorescencia como se sabe es una técnica sensible y específica para el virus. La RT-PCR que se desarrollo, fue efectiva para la amplificación del ADNc; además, mostró ser específica para el virus sincitial respiratorio, ya que no amplifica material genético de otros virus incluyendo a aquellos que pertenecen a la misma familia. La sensibilidad de la prueba es alta, alcanzando a amplificar hasta picogramos de ADNc.Conclusiones: la rapidez de la inmunofluorescencia, permite dar un diagnóstico presuntivo que después puede ser comprobado por el aislamiento y propagación del virus en cultivo celular. La técnica de RT-PCR con los oligonucleótidos que utilizamos fue sensible, específica y rápida, por tanto debe ser tomada en cuenta para el apoyo al diagnóstico clínico. Con lo anterior, no se trata de sustituir las técnicas tradicionales, sino contar con mayores opciones y además reforzarlas; de esta manera, se podrá fundamentar mejor el diagnóstico de las infecciones virales.

Virus influenza: enigma del pasado y del presente / Influenza virus: past and present riddle

Antecedentes. El virus de la influenza es biológica y bioquímicamente único. En el pasado causó pandemias con mortalidad elevada, y en la actualidad continúan originando epidemias con alto impacto en la salud y en la economía. Son tres los tipos inmunológicos del virus A, B y C que infectan al humano; además, los del tipo A también pueden infectar a un amplio rango de animales, en particular diversas especies de aves, cerdos y caballos. Características. Los virus presentan un genoma de ARN de polaridad negativo, segmentado, esta característica facilita el elevado grado de variabilidad, particularmente en los virus de tipo A, cuya variación es originada principalmente por mutación o por recombinación genética, este fenómeno se incrementa si el virus pasa de una especie animal a otra, lo que genera nuevos subtipos virales, los cambios más importantes se dan en las glucoproteínas, hemaglutinación y neuraminidasa. Inmunidad. Se ha descrito que la resistencia depende de la inmunidad hacia las proteínas de superficie, especialmente la hemaglutinina por tal motivo, cuando aparece un nuevo subtipo viral la población humana es sensible a la infección. Actualmente se cuenta con algunas vacunas, sin embargo, el éxito que se tiene con ellas es limitado en humanos. Las campañas de información sobre control y detección de casos por ahora son de gran importancia. Así, a pesar de los avances que se han logrado en la ciencia, el virus de la influenza continúa siendo un enigma

Infección en pacientes con inflamación y enfermedad pulmonar obstructiva crónica o asma / Infection in patients with chronic obstructive pulmonary disease and inflammation of asthma

Objetivo: Determinar la frecuencia de infección viral y bacteriana en inflamación aguda de pacientes con enfermedad pulmonar obstructiva crónica o asma. Material y Métodos: Se incluyeron 140 pacientes; 46 con enfermedad pulmonar obstructiva crónica y 94 con asma, todos con inflamación aguda del aparato respiratorio y edades entre 20 a 70 años y como grupo de control 80 pacientes con inflamación aguda por infección respiratoria aguda sin antecedentes de enfermedad pulmonar crónica o asma. Se tomo muestra de exudado nasofaringeo para el aislamiento viral y bacteriano y se dio seguimiento para ver la evolución. Resultados: El 49 por ciento de los pacientes del grupo de trabajo presentaron infección viral y 13 por ciento infección bacteriana, el virus más frecuente fue parainfluenza, los casos más severos se observaron en este grupo. En el grupo control, 55 por ciento presentó infección viral, el más frecuente fue parainfluenza. Conclusiones: Los virus son agentes importantes que pueden incrementar la inflamación aguda en pacientes con enfermedad pulmonar obstructiva crónica o asma, generalmente son los mismos que infectan a personas sin daño previo, pero las infecciones pueden ser más severas cuando hay un daño del aparato respiratorio

Efectos biológicos en células HEp-2, en la multiplicación del adenovirus tipo 6 y en la concentración de interferón en plasma humano en presencia de un RNA de transferencia fúngico / Biological effects in HEp-2 cells, adenovirus type 6 and interferon concentration in human plasma in the presence of a fungal transfer RNA

Introducción: Trabajos previos mostraron la capacidad antiviral in vitro, así como la utilidad como inductor de interferón natural (IFN-n) in vivo de un RNA de transferencia (tRNA) de origen fúngico, por este motivo se sigue estudiando esta molécula como una alternativa para el tratamiento antiviral. Objetivos: Estudiar el efecto del tRNA fúngico en la viabilidad, síntesis de DNA y en la multiplicación del adenovirus tipo 6 (AV-6) en células HEp-2, comparado su efecto con el producido por el polyl:polyC o por IFN-Ó. Valorar su capacidad como unductor a largo plazo de la síntesis de IFN-n in vivo. Material y métodos: Células HEp-2 se incubaron con diferentes concentraciones de las moléculas mencionadas durante 24 horas, y se determinó la viabilidad y la síntesis de DNA celular; adicionalmente cultivos tratados en las mismas condiciones se infectaron con 200 unidades formadoras de placas (ufp) del AV-6, se incubaron cinco días adicionales, y se valoró el grado de protección. In vivo a siete voluntarios clínicamente sanos se les administró intramuscularmente una dosis única de 100 mg del tRNA, y se determinó mediante la técnica de inhibición del efecto citopático (ECP) el nivel sérico de IFN-n, cinco días después de la inoculación. Resultados. El tRNA protegió a las células HEp-2 contra la infección por AV-6, mejor que el polyl:polyC y de forma similar al IFN-Ó. Ninguna de las tres sustancias afectó significativamente la viabilidad celular. En cambio, la síntesis de DNA sí disminuyó de manera directa en relación con la concentración de los inductores, no así con el IFN-Ó. En los plasmas de los sujetos tratados con el tRNA fúngico se encontró un aumento en la concentración de IFN-n (de 84.2 ñ 107.6 a 171.42 ñ 129.5 UI/mL) a los cinco días, aunque la diferencia no fue estadísticamente significativa. Conclusión. El tRNA fúngico mostró actividad antiviral contra el AV-6, no afectó la viabilidad pero sí la síntesis celular de DNA, y con una capacidad de mantener elevada hasta por cinco días la concentración plasmática de IFN-n en humanos

Fijación y tinción de las placas de los virus herpes simple tipo 1 y sincitial respiratorio / Fixing and staining of tipe 1 herpes simplex and respiratory syncytial viruses plaques

El presente trabajo, se realizó en el laboratorio de Virología del Instituto Nacional de Enfermedades Respiratorias, México, D.F. Los objetivos fueron: encontrar las condiciones óptimas para la fijación y tinción de las placas virales producidas por los virus herpes simple tipo 1 y sincitial respiratorio: Ambos virus, fueron propagados en monocapas de células Vero y se observaron a diferentes tiempos. Para la fijación de las monocapas infectadas se probaron cinco sustancias: formaldehído, etanol, metanol, acetona y éter-metanol y se tiñeron con tres colorantes: Giemsa, Wrigth y cristal violeta. Los mejores resultados se obtuvieron al usar éter al 5 por ciento en metanol como fijador y Giemsa como colorante. El tiempo más corto en que se observó el efecto citopático fue: 18 h para el virus sincitial respiratorio y 15 h para el virus herpes simple. por lo que el uso de estas sustancias, facilitó la visualización del efecto citopático en tiempos breves