One-Step Detection of the 2009 Pandemic Influenza A(H1N1) Virus by the RT-SmartAmp Assay and Its Clinical Validation

<div><h3>Background</h3><p>In 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society.</p> <h3>Methodology</h3><p>To address the clinical need for rapid diagnosis, we have developed a new method, the “RT-SmartAmp assay”, to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses.</p> <h3>Results and Conclusions</h3><p>We evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.</p> </div

Efficacy and Safety of Synbiotics in Patients Undergoing Autologous Hematopoietic Stem Cell Transplantation: A Randomized, Double-blinded, Placebo-controlled Pilot Study

Objective: High-dose chemotherapy with autologous hematopoietic stem cell transplantation (auto-HSCT) is an effective treatment option for relapsed and refractory aggressive malignant lymphoma. However, patients frequently experience treatment-induced gastrointestinal symptoms. Synbiotics, including live microorganisms and nondigestible food ingredients, reportedly ameliorate chemotherapy-induced mucosal damage. In this study, we assessed the efficacy and safety of synbiotics in patients undergoing auto-HSCT. Methods: This randomized, double-blinded study included patients with malignant lymphoma eligible for auto-HSCT. The patients were randomly assigned to either a synbiotic group receiving Bifidobacterium longum (BB536) and guar gum or a placebo group receiving a placebo containing dextrin. The supplements were administered twice daily from the start of conditioning chemotherapy up to 28 days after auto-HSCT. The primary endpoint was the duration of total parenteral nutrition (TPN). Results: In total, 12 patients were included and randomized. The median duration of TPN was 15 (range, 12-33) days in the synbiotic group and 17.5 (range, 0-32) days in the placebo group. The median duration of grade ≥3 diarrhea was shorter in the synbiotic group than in then placebo group (2.5 vs. 6.5 days), as was the duration of hospital stay (31.5 vs. 43 days). The oral intake and quality of life regarding diarrhea and anorexia improved in the synbiotic group after engraftment. Synbiotic infections, including bacteremia, were not observed. Conclusion: Synbiotics may reduce gastrointestinal toxicity, thereby reducing nutritional problems and improving the quality of life of patients undergoing auto-HSCT, without severe adverse events

Time after the onset of fever and the number of patients who were diagnosed by the RT-SmartAmp assay as positive for infection with the 2009 pdm influenza A(H1N1) virus.

<p>Time after the onset of fever and the number of patients who were diagnosed by the RT-SmartAmp assay as positive for infection with the 2009 pdm influenza A(H1N1) virus.</p

Comparison of results detected by the rapid diagnosis kits and the RT-SmartAmp assay.

<p>As depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030236#pone.0030236.s003" target="_blank">Figure S3</a>, two swab samples were collected as one pair from each patient. Swab samples were separately used for the rapid influenza diagnostic tests and RT-SmartAmp assay. All the samples were transferred to RIKEN Yokohama Institute for sequence analysis and RT-Q-PCR detection.</p

Detection of the 2009 pdm influenza A(H1N1) virus by RT-SmartAmp assay in the fatal case.

<p><b>A:</b> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030236#s4" target="_blank">Materials and Methods</a>. <b>B:</b> Chest radiography of the patient was taken at 11 and 28 hours after the onset of fever. <b>C:</b> 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.</p