Diagnosis of Novel Pandemic Influenza Virus 2009 H1N1 in Hospitalized Patients

Abstract

A real-time RT-PCR assay was standardized and evaluated for the detection of the recent pandemic 2009 H1N1 strain that circulated around the world causing colossal loss of human life. We amplified the conserved regions of the hemagglutinin (HA) gene of 438 clinical specimens using real-time RT-PCR assay for rapid identification of pandemic influenza virus. The real-time RT-PCR was optimized and the primers and probes were tested against a panel of known negative and positive controls. RNA isolated from the HeLa cell line served as quality control. The conventional RT-PCR which is an established method of influenza virus diagnosis was compared to realtime RT-PCR. Of 438 clinical specimens tested, 212 specimens were found positive for influenza A virus (SD 46.669) in which 139 specimens were diagnosed positive for the pandemic 2009 H1N1 while 73 were the seasonal influenza viruses. We report that the real-time RT-PCR assay offers both, a high sensitivity and specificity when compared with the traditional identification method. The real-time RT-PCR assay allows rapid identification of the pandemic swine 2009-H1N1 at very low viral loads that are negative by the traditional RT-PCR. This optimized assay can be a very useful tool to assist both epidemiologists and the clinicians.

The emerging influenza virus threat: status and new prospects for its therapy and control

“…Nasopharyngeal specimens are always preferred over throat swabs or other specimens [61]. The best time to collect the clinical specimen is on the second or third day of symptoms (when viral shedding is at its peak), as the results obtained will be more reliable than when samples are obtained earlier or later in the course of disease [28,51,55,61,62]. There are a number of methods available for influenza diagnosis including rapid antigen tests, viral culture, serology, conventional reverse transcription polymerase chain reaction (PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), real-time reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assay.…”

“…While virus culture is believed to be one of the most accurate methods for identifying viral strains and subtypes, it can sometimes be an impractical choice for physicians who usually need to initiate antiviral drug therapy within 48 hours of the onset of symptoms [59]. The virus culture method also becomes a secondary choice during pandemic situations when a large number of infected people rush to hospitals for diagnosis and treatment [51,62]. The most sensitive diagnostic tool available to date is the real-time reverse transcription polymerase chain reaction (RRT-PCR) test [62,65].…”

Evaluation of Respiratory Virus Pathogen from Patients Presenting with Influenza like Illness at Various Hospitals in Delhi, India

“…Chest Institute, Delhi from September, 2012 to August, 2013. The patients with influenza-like-illness and respiratory complications presenting to the Out-Patient Department (OPD) during the study period underwent diagnostic testing for evidence of a panel of respiratory virus [36].Appropriate clinical specimens (throat swabs and nasal swabs) were collected in viral transport medium, as described previously following Centers for Disease Control and Prevention (CDC), Atlanta and WHO guidelines prior to informed consent from the patients or guardian [37][38][39]. All the collected clinical specimens were subjected to one-step Reverse Transcriptase-PCR (RT-PCR) for a panel of respiratory viruses, from Seeplex RV15 one-step Ace detection kit (Seegene, Korea) as per manufacturer's instructions.…”

Advancements in Nucleic Acid Based Therapeutics against Respiratory Viral Infections

“…Seasonal influenza viruses are one of the foremost public health concerns, especially influenza type A viruses that have been associated with the annual epidemics worldwide [33]. The recent 2009 H1N1 pandemic caused high morbidity [34,35] and mortality [36,37,38], and was a reminder that influenza viruses are highly unpredictable [3,39,40,41] and continuous efforts are needed to manage pandemic scenarios in short time. Antiviral drugs that are currently approved to treat influenza include the Adamantanes (M2 ion-channel protein inhibitors) and the neuraminidase inhibitors such as zanamivir and oseltamivir [42].…”