In situ molecular identification of the Influenza A (H1N1) 2009 Neuraminidase in patients with severe and fatal infections during a pandemic in Mexico City
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
BMC Infectious Diseases 2013, 13:20 doi:10.1186/1471-2334-13-20Published: 18 January 2013
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.
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.
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.
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.