During April 2009, the number of atypical pneumonia cases increased in Mexico City’s hospitals and spread to almost all boroughs in the city; these cases were related to a new influenza A (H1N1) virus strain that was identified as the etiological agent [1–4]. In less than a month the virus spread worldwide and on June 11, 2009 the World Health Organization (WHO) declared the start of the first 21st century influenza pandemic.
Influenza A viruses belong to the Orthomyxoviridae family; they are characterized by a unique genome structure with a single-negative RNA strand, which codifies, among others, for two transmembrane proteins: hemagglutinin (HA) and neuraminidase (NA) [5–7]. HA plays an important role during the cell entry of influenza viruses. This protein is essential during the initial steps of infection because it is responsible for the attachment of the virus to sialic acid (SA) cellular receptor. This interaction explains, at least in part, the host range and tissue tropism of influenza viruses [5, 8]. The NA of influenza viruses is a homotetrameric glycoprotein anchored by a fibrous stalk in the viral membrane. The protein possesses a globular head comprised of four monomers that constitute the active site composed of nine conserved residues. Its primary role in the infectious cycle is to liberate the viral progeny from infected cells. Its enzymatic activity catalyzes SA removal from its linkage to galactose, thereby destroying the receptor and allowing the virus to disseminate and infect other cells [8, 9]. Furthermore, NA is also the main target of the antiviral drugs zanamivir and oseltamivir. These drugs closely resemble the structure of the natural substrate of the NA and thus prevent the removal of the SA residue from the glycopeptide receptor by the viral neuraminidase . In addition to the increased transmissibility and the low or lack of immunity of the human population, the fact that new reassortment events may alter the pathogenicity of circulating strains, makes it crucial to monitor the progress of the pandemics at the molecular level [2, 10, 11].
Molecular methods are becoming more widely used for the detection of respiratory pathogens, in part because of their superior sensitivity, relatively rapid turnaround time, and ability to identify pathogens that are slow growing or difficult to culture. The recent novel H1N1 influenza A pandemic has been useful to underscore how quickly new molecular tests can become available for clinical use . Previously, several groups have been used immunohistochemical or immunofluorescence detection for determine the localization of influenza virus antigens, as well as in situ hybridization for detection of viral sequences and ultrastructural examination to detect viral particles, in cases of fatal H1N1 influenza A virus infection during the period 2009–2010 [13–19]. These analyses were performed on sections from different tissues such as respiratory tissues (trachea, lung), heart, liver, and placenta [13–19]. Over the years, Reverse-transcriptase PCR is the recommended test for diagnosis and confirmation of infections due to pandemic 2009 influenza A(H1N1) virus . Recently, modifications of this technology have emerged, some of which allow the rapid detection of multiple pathogens in a single test such as multiplex molecular technologies, reverse transcriptase-PCR, real-time PCR, microarrays and nucleic acid sequencing-based amplifications [12, 21, 22]. Other studies have also shown the usefulness of rapid immunoassays for seasonal influenza virus . These methods have greatly enhanced the capability for surveillance and characterization of influenza viruses and their clinical utility for the detection of respiratory pathogens. However, these methods can not be easily applied for the analysis of paraffin-embedded tissues. In situ RT-PCR has some major strengths for the detection of specific nucleic acid sequences. First and foremost, one can detect particular sequences on archival material; second, this technique combines the extreme sensitivity of PCR with the cell localization ability similar to in situ hybridization . A third strength of in situ RT-PCR relates to the issue of sample contamination in solution-phase PCR. Sample contamination, which can lead to false-positive results in PCR, limits its value as a diagnostic test for viral infections; this limitation is not encountered in in situ RT-PCR. Fourth, this technique is the only amplification technique that allows direct target-specific incorporation of a reporter nucleotide (such as DIG-digoxigenin-dUTP-labeled nucleotide) [24, 25], thus eliminating the need for a hybridization step. Clearly, in situ RT-PCR has been useful for any target that is low copy and, thus, difficult to detect with standard in situ hybridization, which has a detection threshold of 10 to 20 copies per cell .
The purpose of the present study was to demonstrate that sensitive and specific molecular detection of pandemic influenza A (H1N1) 2009 strains is possible in archival material such as paraffin-embedded lung samples. This strategy would be useful to perform current and retrospective studies in a specific and reproducible manner.