Search

Background and Aim: The pandemic (H1N1) 2009 influenza (H1N1pdm09) virus has affected both human and animal populations worldwide. The transmission of the H1N1pdm09 virus from humans to animals is increasingly more evident. Captive animals, particularly zoo animals, are at risk of H1N1pdm09 virus infection through close contact with humans. Evidence of exposure to the H1N1pdm09 virus has been reported in several species of animals in captivity. However, there is limited information on the H1N1pdm09 virus infection and circulation in captive animals. To extend the body of knowledge on exposure to the H1N1pdm09 virus among captive animals in Thailand, our study investigated the presence of antibodies against the H1N1pdm09 virus in two captive animals: Camelids and Eld's deer. Materials and Methods: We investigated H1N1pdm09 virus infection among four domestic camelid species and wild Eld's deer that were kept in different zoos in Thailand. In total, 72 archival serum samples from camelid species and Eld's deer collected between 2013 and 2014 in seven provinces in Thailand were analyzed for influenza antibodies using hemagglutination inhibition (HI), microneutralization, and western blotting (WB) assays. Results: The presence of antibodies against the H1N1pdm09 virus was detected in 2.4% (1/42) of dromedary camel serum samples and 15.4% (2/13) of Eld's deer serum samples. No antibodies were detected in the rest of the serum samples derived from other investigated camelids, including Bactrian camels (0/3), alpacas (0/5), and llamas (0/9). The three positive serum samples showed HI antibody titers of 80, whereas the neutralization titers were in the range of 320-640. Antibodies specific to HA and NP proteins in the H1N1pdm09 virus were detected in positive camel serum samples using WB. Conversely, the presence of the specific antibodies in the positive Eld's deer serum samples could not be determined using WB due to the lack of commercially labeled secondary antibodies. Conclusion: The present study provided evidence of H1N1pdm09 virus infection in the captive dromedary camel and Eld's deer in Thailand. Our findings highlight the need for continuous surveillance for influenza A virus in the population of dromedary camels and Eld's deer. The susceptible animal populations in close contact with humans should be closely monitored. Further study is warranted to determine whether Eld's deer are indeed a competent reservoir for human influenza virus.

Receipt of seasonal inactivated trivalent vaccine neither increased nor decreased the risk for pandemic influenza virus infection., We conducted a population-based study in Manitoba, Canada, to investigate whether use of inactivated trivalent influenza vaccine (TIV) during the 2008–09 influenza season was associated with subsequent infection with influenza A(H1N1)pdm09 virus during the first wave of the 2009 pandemic. Data were obtained from a provincewide population-based immunization registry and laboratory-based influenza surveillance system. The test-negative case–control study included 831 case-patients with confirmed influenza A(H1N1)pdm09 virus infection and 2,479 controls, participants with test results negative for influenza A and B viruses. For the association of TIV receipt with influenza A(H1N1)pdm09 virus infection, the fully adjusted odds ratio was 1.0 (95% CI 0.7–1.4). Among case-patients, receipt of 2008–09 TIV was associated with a statistically nonsignificant 49% reduction in risk for hospitalization. In agreement with study findings outside Canada, our study in Manitoba indicates that the 2008–09 TIV neither increased nor decreased the risk for infection with influenza A(H1N1)pdm09 virus.

Monitoring of antiviral resistance is strongly recommended for immunocompromised patients., Oseltamivir has been widely used for pandemic (H1N1) 2009 virus infection, and by April 30, 2010, a total of 285 resistant cases were reported worldwide, including 45 in the United Kingdom. To determine risk factors for emergence of oseltamivir resistance and severe infection, a case–control study was conducted in the United Kingdom. Study participants were hospitalized in England or Scotland during January 4, 2009–April 30, 2010. Controls had confirmed oseltamivir-sensitive pandemic (H1N1) 2009 virus infections, and case-patients had confirmed oseltamivir-resistant infections. Of 28 case-patients with available information, 21 (75%) were immunocompromised; 31 of 33 case-patients (94%) received antiviral drugs before a sample was obtained. After adjusting for confounders, case-patients remained significantly more likely than controls to be immunocompromised and at higher risk for showing development of respiratory complications. Selective drug pressure likely explains the development of oseltamivir resistance, especially among immunocompromised patients. Monitoring of antiviral resistance is strongly recommended in this group.

To describe clinical aspects of pandemic (H1N1) 2009 virus–associated pneumonia in children, we studied 80 such children, including 17 (21%) with complications, who were admitted to 5 hospitals in Japan during August–November 2009 after a mean of 2.9 symptomatic days. All enrolled patients recovered (median hospitalization 6 days). Timely access to hospitals may have contributed to favorable outcomes.

A swine influenza outbreak occurred on a commercial pig farm in Thailand. Outbreak investigation indicated that pigs were co-infected with pandemic (H1N1) 2009 virus and seasonal influenza (H1N1) viruses. No evidence of gene reassortment or pig-to-human transmission of pandemic (H1N1) 2009 virus was found during the outbreak.

Younger children may require a longer isolation period and more aggressive treatment., Pandemic (H1N1) 2009 virus causes severe illness, including pneumonia, which leads to hospitalization and even death. To characterize the kinetic changes in viral load and identify factors of influence, we analyzed variables that could potentially influence the viral shedding time in a hospital-based cohort of 1,052 patients. Viral load was inversely correlated with number of days after the onset of fever and was maintained at a high level over the first 3 days. Patients with pneumonia had higher viral loads than those with bronchitis or upper respiratory tract infection. Median viral shedding time after the onset of symptoms was 9 days. Patients 13 years of age (11 days vs. 7 days). These results suggest that younger children may require a longer isolation period and that patients with pneumonia may require treatment that is more aggressive than standard therapy for pandemic (H1N1) 2009 virus.

To the Editor: In March 2009, a novel influenza virus A (H1N1) emerged in Mexico, and, because of widespread human-to-human transmission, a global pandemic was declared in June 2009 (1). Although most cases have involved humans, pandemic (H1N1) 2009 has sporadically infected swine and turkeys and has also been reported in a small number of pet ferrets, cats, and captive cheetahs, and in a dog (2). Many of these animals were cared for by persons who experienced influenza-like illness and the owner of 1 cat who died had confirmed pandemic (H1N1) 2009 respiratory disease before the cat became ill, which suggests probable human-to animal-transmission of the virus (2). During mid-December 2009–mid-January 2010, eight striped skunks (Mephitis mephitis) died on a mink farm near Vancouver, British Columbia, Canada. On January 12, 2010, two of the skunks were brought to the Animal Health Centre in Abbotsford, British Columbia, for postmortem examination. One skunk exhibited purulent nasal exudates. In both skunks, investigators observed splenomegaly and severe pneumonia, characterized by heavy, dark red to purple, lung lobes involving >70% of the lung field. Microscopic examination showed moderate rhinitis and severe bronchopneumonia with intralesional bacteria, areas of interstitial pneumonia, and occasional nematode larvae. Also observed were splenic extramedullary hematopoiesis, plasmacytosis of both lymph nodes and spleen, and mild plasmacytic glomerulonephritis with proteinuria. Routine bacteriologic culture of lung showed heavy growth of Streptococcus dysgaslactiae subsp. equisimilis, Staphylococcus aureus, and Hafnia alvei. That death was caused by uncomplicated mixed bacterial bronchopneumonia in 2 (and possibly up to 8) adult skunks over a 6-week period was considered unlikely. The presence of lungworm was considered incidental. However, the areas of interstitial pneumonia suggested that a primary viral pathogen was likely. Molecular testing was conducted initially on fresh lung, liver, kidney, and spleen for canine distemper virus and, subsequently, for influenza A virus. The splenic and nodal plasmacytosis and plasmacytic glomerulonephritis also prompted testing for Aleutian disease virus (ADV). Organ samples were negative for canine distemper virus and positive for ADV. Detection of influenza A virus nucleoprotein and matrix genes and hemagglutinin and neuraminidase typing was performed with real-time reverse transcription–PCR. Organ samples were positive for pandemic (H1N1) 2009, which was confirmed by sequence analysis of DNA fragments obtained in the hemagglutinin, neuraminidase, and matrix gene testing. Primary viral interstitial pneumonia is frequently complicated by opportunistic bacterial bronchopneumonia and influenza virus A infection has been shown to predispose to pulmonary bacterial toxicity (3). Thus, we concluded that primary pandemic (H1N1) 2009 interstitial pneumonia had predisposed the 2 skunks to mixed bacterial bronchopneumonia and death. The skunks were also infected with ADV, presumably as a result of viral shedding by the minks, which are known to be ADV carriers. Striped skunks can be experimentally infected with ADV, and antibodies to ADV have been detected in wild skunks (4). Although ADV does not cause pneumonia (4), co-infection with ADV and influenza A virus is associated with higher mortality rates in minks with respiratory disease (5). Thus, ADV co-infection may have contributed to the severity of the pneumonia and the death of the skunks. The source of the pandemic (H1N1) 2009 virus is unclear. Nasal discharge was also observed in many of the minks, which suggests that they had a respiratory viral infection. However, no diagnostic workup was undertaken. Although severe outbreaks of interstitial pneumonia on mink farms can occur (6), most natural influenza A virus infections in minks are either mild or asymptomatic (5). Thus, the minks may also have been infected with pandemic (H1N1) 2009. Many of the pandemic (H1N1) 2009 infections reported in animals are believed to have been the result of exposure to infected humans (2). Workers on the mink farm did not experience influenza-like illness. However, humans with asymptomatic pandemic (H1N1) 2009 infection may have transmitted it to the mink. Because the skunks visited the mink farm daily, transmission of pandemic (H1N1) 2009 from humans to minks to skunks is a possibility. In view of the detection of pandemic (H1N1) 2009 virus in 2 striped skunks with fatal pneumonia, this species should now be regarded as a potential source of influenza A virus. Wild animals participate in the transmission of influenza A viruses between species, and the presence of wildlife on farms is known to be a risk factor for infection of poultry (7). Similar to raccoons, skunks express both α2,3 and α2,6 sialic acid receptors for avian and human influenza viruses in the respiratory tract (M. Shrenzel, San Diego Zoo, pers. comm.), which is believed to create the opportunity for mixed influenza infections with potential for genetic reassortment (8). Skunks, like raccoons, are highly mobile animals with large home ranges in rural and urban areas, which provides numerous opportunities for influenza A virus exposure and transmission to poultry, livestock, pets, and, ultimately, humans. The inclusion of striped skunks in wildlife influenza surveillance programs may be warranted.

To the Editor: Dengue is a mosquito-borne viral infection caused by 4 related dengue viruses. Each of these viruses is capable of causing classic dengue fever or dengue hemorrhagic fever (DHF), but may also cause nonspecific febrile illnesses. As a result, dengue is often difficult to diagnose clinically, especially because peak dengue season often coincides with that of other common febrile illnesses in tropical regions (1). Concurrent outbreaks of influenza and dengue have been reported (2,3); this circumstance often leads to delayed recognition of the presence of one or other disease in the community. In April 2009, a new strain of influenza A virus known as pandemic (H1N1) 2009 virus was first detected in the United States (4). Pandemic (H1N1) 2009 infections were first detected in Puerto Rico in June 2009, and 59 deaths caused by the virus have been confirmed to date. This influenza outbreak coincided with the typical dengue season in Puerto Rico, which led to diagnostic difficulties; both infections disproportionately affected young persons, who often had similar, nonspecific symptoms. We describe a case of laboratory-confirmed co-infection of dengue virus and pandemic (H1N1) 2009, and discuss the difficulties in distinguishing the 2 illnesses clinically. A 33-year-old woman (healthcare worker) in Ponce, Puerto Rico, sought treatment at an emergency department of a hospital in the southern part of the island with a 3-day history of febrile illness. Her symptoms began with throat irritation and earache; subsequently, cough, fever, and headache developed. She reported palpitations and generalized malaise but no other symptoms. The patient had no notable medical history and denied taking any medicines apart from over-the-counter antipyretics. She reported recent exposure to influenza at work and multiple recent mosquito bites. On physical examination, she had a temperature of 37°C, a heart rate of 91 bpm, and blood pressure of 125/82 mm Hg. A tourniquet test result was positive. Her pharynx was erythematous without exudate, and she had rhinorrhea. She had no lymphadenopathy, rash, petechiae, or purpura. Several small, red papules, which the patient described as recent mosquito bites, were on her legs. The remainder of her examination showed no unusual findings. Laboratory studies showed a leukocyte count of 5,300 cells/mm3 with a normal differential count, hematocrit 35.2%, and thrombocyte count of 239,000 cells/dL. Results of a chest radiograph was unremarkable. A nasopharyngeal swab was positive for influenza A virus by rapid test. Nasopharyngeal and serum samples were sent to the Centers for Disease Control and Prevention (Dengue Branch) for influenza and dengue testing. The patient was diagnosed with suspected pandemic (H1N1) 2009 infection and prescribed oseltamivir for 5 days. She returned for a follow-up visit 12 days after the onset of symptoms. She reported having 2 more days of fever after her initial visit, but had no rash, petechiae, bleeding, or progression of respiratory symptoms. A second serum specimen was obtained during this visit. The initial serum specimen was positive for dengue virus by serotype-specific, singleplex, real-time reverse transcription–PCR (5). Her nasopharyngeal specimen was positive by PCR for pandemic (H1N1) 2009 influenza. The second, convalescent-phase, serum specimen was negative for dengue immunoglobulin (Ig) M by IgM antibody–capture ELISA. The acute-phase and convalescent-phase samples were positive for IgG against dengue by ELISA (6), which indicated a secondary dengue infection. IgG titers exceeded the limits of the test for acute-phase and convalescent-phase samples, showing unusually elevated levels of IgG against dengue. Distinguishing dengue and influenza by clinical features alone can be difficult. In an investigation of simultaneous dengue and influenza A outbreaks in Puerto Rico in 1977, similar percentages of persons with confirmed dengue and confirmed influenza had classic dengue symptoms (2). Hemorrhagic manifestations, like those typically seen in DHF, have been reported with influenza A in prior outbreaks (7,8) and with pandemic (H1N1) 2009 (Centers for Disease Control and Prevention, unpub. data). Previous influenza A outbreaks were initially believed to be outbreaks of DHF until careful laboratory investigation proved otherwise (8). Our patient did not have the typical signs and symptoms of dengue (rash, eye pain, thrombocytopenia, arthralgia, petechiae, or bleeding) that would differentiate her condition from that of patients with other febrile illnesses. She did have a positive tourniquet test result and fever, which have been advocated as screening criteria for dengue infection in children, at the time of initial examination (9). Data for the specificity and sensitivity of these criteria in adults are sparse, however, and some studies have shown a high incidence of positive tourniquet test results in patients with laboratory-confirmed influenza (7,8). Our report demonstrates that co-infection with dengue virus and pandemic (H1N1) 2009 can occur. Previous studies also have shown cases of probable co-infection with seasonal influenza and dengue (1,10), including 1 fatal case (1). Because many dengue-endemic countries are experiencing pandemic (H1N1) 2009 outbreaks, providers should consider the possibility of viral co-infection, especially in severe cases, and should consider testing for both viruses.

Please cite this paper as: Pabbaraju et al. (2011) Comparison of a singleplex real‐time RT‐PCR assay and multiplex respiratory viral panel assay for detection of influenza “A” in respiratory specimens. Influenza and Other Respiratory Viruses 5(2), 99–103. Background Evaluation of different molecular tests for the detection of pandemic (H1N1) 2009 virus is important before the next wave of the pandemic. Objectives To compare a hydrolysis probe‐based real‐time RT‐PCR assay recommended by the CDC to the xTAG® respiratory viral panel (RVP) (Luminex® Molecular Diagnostics) for the detection of influenza A. Methods Eleven thousand eight hundred and ninety‐eight respiratory specimens were tested by the real‐time RT‐PCR and RVP assays for the detection of influenza A. The distribution of seasonal H1, H3 and pandemic H1N1 subtypes in these specimens was compared. Results The RVP assay was generally unable to identify influenza A–positive samples with a low viral load, whereas the real‐time RT‐PCR assay detected most of these samples resulting in a subset of specimens that could not be confirmed as either seasonal or pandemic influenza A subtypes. Conclusions When the prevalence of influenza A is high, the CDC recommended real‐time RT‐PCR has significant advantages as a frontline assay, namely higher sensitivity and shorter time to reporting a result. Anticipated scenarios would be during the peaks of the pandemic and episodes of seasonal influenza. Furthermore, the better sensitivity of the RT‐PCR makes it the preferred assay to detect influenza in patients with severe respiratory disease tested late in their clinical course. If pandemic (H1N1) 2009 virus is not the dominant virus and there is a high proportion of other respiratory viruses circulating, laboratories will be faced with the decision to use the RVP assay for the detection of pandemic (H1N1) 2009 virus.

Background Influenza A virus is a leading causative pathogen of human acute respiratory infection. Recently, the co-circulation of pandemic (H1N1) 2009 and seasonal H3N2 viruses was reported, and sporadic cases with reassortant avian H7N9 virus are continually reported in China. We aimed to establish a multiplex one-step real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay to simultaneously detect and discriminate FluA subtypes, including human seasonal H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus, in one reaction tube. Methods Clinical samples, including throat swabs and sputum, were collected from the patients with influenza-like illness (ILIs). Total viral RNA from each sample or viral culture was extracted, and the specific detection of FluA virus and its subtypes was performed using a multiplex rRT-PCR assay. Results The limitation of detection (LOD) of the multiplex assay was 5.4 × 10−2 50% tissue culture infective dose (TCID50) per reaction or 4.8 × 101 copies per reaction for each virus of the three viruses. For simultaneously detecting the three viruses, the LOD was 1.8 × 10−2 TCID50 per reaction or 1.6 × 10 copies per reaction for testing the total FluA virus RNA and 5.6 × 10−2 TCID50 per reaction or 5.1 × 10 copies per reaction for the H3, H1, and H7 genes in one reaction tube. The multiplex assay specifically detected these viruses, and no cross-reaction with other pathogens was found. Moreover, the assay had reliable clinical sensitivity (100%) and valuable clinical specificity (>95%). The detection of FluA with the matrix (M) gene contributed to the further determination of these subtypes, and the Rnase P gene (RP) was considered an internal control to favourably evaluate the quality of the clinical samples. Conclusions These findings indicate that the multiplex assay can simultaneously detect and discriminate FluA subtypes with reliable sensitivity and specificity, which is required for the early clinical diagnosis and viral surveillance of patients with FluA infection.