title: In situ molecular identification of the influenza A (H1N1) 2009 Neuraminidase in patients with severe and fatal infections during a pandemic in Mexico City.
abstract: 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.
authors: Department of Genetics and Molecular Biology, CINVESTAV-IPN, Mexico City, Mexico.
authors: Rodolfo
authors: R
authors: Ocadiz-Delgado
authors: Martha Estela
authors: ME
authors: Albino-Sanchez
authors: Enrique
authors: E
authors: Garcia-Villa
authors: Maria Guadalupe
authors: MG
authors: Aguilar-Gonzalez
authors: Carlos
authors: C
authors: Cabello
authors: Dora
authors: D
authors: Rosete
authors: Fidencio
authors: F
authors: Mejia
authors: Maria Eugenia
authors: ME
authors: Manjarrez-Zavala
authors: Carmen
authors: C
authors: Ondarza-Aguilera
authors: Rosa Ma
authors: RM
authors: Rivera-Rosales
authors: Patricio
authors: P
authors: Gariglio
mesh_terms: D001344:Autopsy; D005260:Female; D015870:Gene Expression; D049674:History, 21st Century; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics*; D007251:Influenza, Human / Q000175:diagnosis* / Q000453:epidemiology* / Q000266:history; D008168:Lung / Q000473:pathology / Q000821:virology; D008297:Male; D008800:Mexico / Q000453:epidemiology; D009439:Neuraminidase / Q000235:genetics*; D058873:Pandemics; D012367:RNA, Viral; D017422:Sequence Analysis, DNA
chemical_list: D012367:RNA, Viral; D009439:Neuraminidase