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Open Access Highly Accessed Research article

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

Rodolfo Ocadiz-Delgado1, Martha Estela Albino-Sanchez1, Enrique Garcia-Villa1, Maria Guadalupe Aguilar-Gonzalez1, Carlos Cabello2, Dora Rosete2, Fidencio Mejia2, Maria Eugenia Manjarrez-Zavala2*, Carmen Ondarza-Aguilera34, Rosa Ma Rivera-Rosales3 and Patricio Gariglio1

Author Affiliations

1 Department of Genetics and Molecular Biology, CINVESTAV-IPN, Mexico City, Mexico

2 Department of Virology and Micology Research, Instituto Nacional de Enfermedades Respiratorias Ismael Cosio Villegas, Calz. De Tlalpan 4502, Colonia Sección XVI, CP 14080, Mexico, DF, Mexico

3 Department of Pathoplogy, Instituto Nacional de Enfermedades Respiratorias Ismael Cosio Villegas, Mexico City, Mexico

4 Clínic 32, Instituto Mexicano del Seguro Social, Mexico City, Mexico

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BMC Infectious Diseases 2013, 13:20  doi:10.1186/1471-2334-13-20

Published: 18 January 2013

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.

Keywords:
Influenza virus; In situ RT-PCR; Influenza diagnosis; Influenza pneumonia; Influenza pandemic in Mexico City