case_images: PMC3266250_01
case_images: Detection of the 2009 pdm influenza A(H1N1) virus by RT-SmartAmp assay in the fatal case.. 
  A: Nasopharyngeal swab samples were collected at 11, 28, and 52 hours after the onset of fever from the patient who was transferred by ambulance to the National Center for Global Health and Medicine. The 2009 pdm influenza A(H1N1) virus was immediately detected by the RT-SmartAmp assay as described in Materials and Methods. B: Chest radiography of the patient was taken at 11 and 28 hours after the onset of fever. C: Partial sequence of the HA segment of the 2009 pdm influenza A(H1N1) virus was analyzed after extraction of viral genome RNA from the swab samples. An arrow indicates the mutation that caused an amino acid substitution at 185 from aspartate to asparagine (N) in the HA protein.
case_images: pone.0030236.g004.jpg
case_images: PMC3266250_01_pone.0030236.g004.jpg
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case_images: At that time, the result of the rapid influenza diagnostic test was negative, whereas the RT-SmartAmp assay gave a positive result from her nasopharyngeal swab sample (Figure 4A). Figure 4B depicts the chest radiography of the patient, demonstrating a drastic progression of severe viral pneumonia, which is characterized by pathological lesions of diffuse alveolar damage (DAD). However, the RT-SmartAmp assay clearly detected the 2009 pdm virus in both samples (Figure 4A). It appeared that the 2009 pdm influenza A(H1N1) virus level was gradually decreasing over time, as shown in Figure 4A. HA protein of the 2009 pdm influenza A(H1N1) virus was asparagine (N), instead of aspartic acid (D) (Figure 4C), in both swab and tracheal fluid samples collected from the patient. HA-Asn185 is located in the Ca antigenic site, being found in the HA protein of the reported sequences of the 1918 influenza viruses.
case_images: PMC3266250_02
case_images: Detection of the 2009 pdm influenza A(H1N1) virus by the RT-SmartAmp assay in the oseltamivir-resistance case.. 
  A: Images of the head CT scanning taken on hospital day 1 (left) and day 13 (right). B: Chest radiography of the patient taken on hospital day 1 (left) and day 5 (right). C: The RT-SmartAmp assay with tracheal fluid ( ) and nasopharyngeal swab ( ) samples collected on hospital day 9. This figure depicts the time courses of the RT-SmartAmp assay reactions with those samples as well as with positive ( ) and negative ( ) controls. D: Partial sequence of the NA segment of the 2009 pdm influenza A(H1N1) virus was analyzed after extraction of viral genome RNA from the swab samples. An arrow indicates the mutation that caused an amino acid substitution at 275 from histidine (N) to tyrosine (Y) in the NA protein.
case_images: pone.0030236.g005.jpg
case_images: PMC3266250_02_pone.0030236.g005.jpg
case_images: pone-0030236-g005
case_images: A. A brain CT scan revealed diffuse and severe edema of the whole brain (Figure 5A). Moreover, chest radiography of the patient demonstrated slight infiltrates in both middle and lower lung fields and mild atelectasis of the right upper lung lobe (Figure 5B). The rapid influenza diagnostic kit gave negative results, whereas the RT-SmartAmp assay still detected the 2009 pdm influenza A(H1N1) virus in the tracheal fluid sample (Figure 5C). A repeat brain CT scan showed alleviated edema in her whole brain (Figure 5A). Sequence analyses with both nasopharyngeal swabs and tracheal fluid samples from this patient revealed that the amino acid residue 275 in the NA protein of the 2009 pdm influenza A(H1N1) virus was tyrosine (Y), instead of histidine (H) (Figure 5D), providing evidence that this patient was infected with oseltamivir-resistant viruses.