Evidence of M cells as portals of entry for antigens in the nasopharyngeal lymphoid tissue of humans

The nasopharyngeal tonsils (adenoids) are prominent components of human nasal-associated lymphoid tissues (NALT). However, the role of the nasopharyngeal tonsils in antigen uptake for initiation of the mucosal immune response is unknown. The aims of this study were to describe the ultrastructure and function of the M cells of the human nasopharyngeal tonsils and to clarify their capacity for antigen uptake. Tissues obtained from eight patients undergoing adenectomy were examined by light and electron microscopy. Lymphoepithelium covers the nasopharyngeal lymphoid tissue and consists of ciliary epithelium, non-ciliary epithelial cells, M cells, goblet cells, and many intraepithelial lymphoid cells. M cells have irregular and broad cytoplasm-containing microvilli on their surface and small vesicles in their cytoplasm. Many lymphoid cells were enfolded by M cells. The uptake of horseradish peroxidase (HRP) in the tissue in organ culture was studied using histochemical techniques. Excised adenoid tissue was incubated in RPMI 1640 culture media with HRP for 10, 30, and 60 min. HRP which had adhered to the surface was taken up in vesicles and then transported in vesicles and tubules by M cells. The M cells of nasopharyngeal lymphoid tissue were ultrastructurally and functionally similar to those in human Peyer’s patches and colonic lymphoid follicles. These findings indicate that NALT bears similarities to the gut-associated lymphoid tissue, and its antigen uptake capacity may be important for initiation of immunity in the upper aerodigestive tract. Received: 8 July 1999 / Accepted: 17 December 1999     

Influenza Virus Receptor Specificity : Disease and Transmission

This Commentary discusses the role of influenza virus specificity in viral transmission.To be successful, respiratory viruses must efficiently infect their hosts through the respiratory mucosa, replicate, and be shed in the appropriate areas of the respiratory tract in the form of highly infectious transmissible material that infects a new host. The interaction between the viral attachment protein and its cellular receptor are among the critical molecular determinants that regulate respiratory virus infection, replication, and shedding, and therefore transmission. Thus, the presence or absence of cellular receptors in specific hosts and tissues is one of the factors that determines whether the host is susceptible or not to viral infection, the tissues and cell types where the virus replicates, and the route of viral transmission.For influenza viruses, it has been known for a long time that the viral attachment protein, hemagglutinin (HA), binds to and uses sialic acid-containing molecules as receptors. The use of such abundant and ubiquitous molecules as receptors, while providing the apparent advantage to the virus of allowing infection of multiple cell types and species, also results in binding to nonproductive receptors present in respiratory secretions, surfaces of dead cells, and even other virions. It is because of this capability that influenza virus has evolved a second viral surface protein, neuraminidase, as a receptor-destroying enzyme that cleaves sialic acid, allowing the virus to be released after binding to sialic acid–containing molecules that do not lead to viral infection.This picture of multiple interactions of the influenza virus with its receptor is further complicated by early findings indicating that not all sialic acid–containing molecules bind with equal efficiency to HA and that different viral strains show different receptor specificities according to their host tropism. Most influenza viruses circulate in waterfowl, and these avian influenza virus strains contain an HA with preference for binding to sialic acids linked to the rest of the sugar by an α2-3 linkage. In contrast, HAs from human influenza virus strains show enhanced binding to α2-6–linked sialic acids.¹ This correlates with an abundance of α2-6-linked sialic acids in the upper respiratory tract of humans, and of α2-3–linked sialic acids in the intestinal mucosa of birds, where replication of human and avian strains of influenza viruses takes place, respectively.² More detailed analysis of the abundance of these types of linkages in the whole human respiratory tract and in the context of severe infection in humans with highly pathogenic avian H5N1 influenza viruses led to the conclusion that α2-3–linked sialic acids are more abundant in the human lower respiratory tract, which correlates with an enhanced tropism of H5N1 viruses for deep areas in the human lung. This may contribute to both severe disease, as viral replication in the lower respiratory tract is more likely to induce pneumonia, as well as to the lack of efficient transmission of H5N1 viruses from human to human, because the virus is less likely to infect the upper respiratory tract, where α2-3–linked sialic acids are sparse.^3,4 Consistent with a role of sialic acid binding preference in transmission, it was found that changes in receptor specificity in the HA of the 1918 human H1N1 pandemic influenza virus from α2-6 to α2-3 linkages dramatically decreased its aerosol transmission in the ferret model, although surprisingly this was not accompanied with decreased viral shedding.⁵ Thus, whether more efficient HA binding to α2-6–linked sialic acids results in an increased tropism for the human upper respiratory tract and in increased viral transmission in humans is an attractive hypothesis that still requires more research to be proven.In this issue of The American Journal of Pathology, van Riel et al⁶ have provided new evidence that supports this hypothesis. Using three representative human influenza A virus strains, corresponding to seasonal H1N1, H3N2, and the new pandemic H1N1 viruses, and three avian influenza viruses of the H5 and H7 subtypes, including a highly pathogenic H5N1 strain, the authors have analyzed the pattern of HA-mediated binding of virions to different human tissues of the upper respiratory tract. For this purpose, they have used a previously developed technique by the same group, named virus histochemistry. In this technique, influenza virions are labeled with FITC, and on incubation with fixed human tissue sections, virions that remain bound are visualized using a peroxidase-labeled anti-FITC secondary antibody.⁴ All human influenza virus strains readily attached and decorated ciliated epithelial and goblet cells from human upper respiratory tract tissues, such as nasal septum, concha, and nasopharynx. In contrast, avian influenza viruses were poor binders to the same tissues. Interestingly, the same group has previously shown that avian influenza viruses are nevertheless able to bind well to cells in tissue sections from the human lower respiratory tract, especially to type II pneumocytes and to alveolar macrophages, whereas human influenza viruses have preference for type I pneumocytes and rarely bind to type II pneumocytes and macrophages.⁷ Taking both observations together, a pattern emerges where binding to macrophages and type II pneumocytes in the lower respiratory tract may promote infection of these cells and destruction of alveolar structures and induction of high levels of cytokines, leading to severe disease, but at the expense of loosing binding to and viral replication in cells of the upper respiratory tract, which is likely to be essential for transmission. If this is the case, the most pathogenic influenza viruses for humans are also the less transmissible.An important advance of the study by van Riel et al⁶ is the comprehensive analysis that has been conducted using multiple human tissue samples derived from the respiratory tract. This provides a fingerprint pattern for influenza viruses that efficiently transmit in humans or that are more likely to induce lower respiratory disease. Although it is clear that host tropism and virulence is dependent of multiple virus and host factors, and not only of HA receptor specificity, virus histochemisty might give the first indication whether a particular viral strain is more likely to transmit in humans or to cause severe disease in humans. For example, some severe cases of influenza virus infection with the new pandemic H1N1 virus have been correlated with the presence of specific mutations in the HA, and it will be interesting to use virus histochemistry to compare the pattern of binding of these mutant viruses to human respiratory tissue. Importantly, as the hallmark of pandemic influenza consists in the introduction in humans of a novel virus strain expressing an HA derived from an animal strain for which there is little pre-existing immunity in humans, efficient transmission in humans of the new pandemic virus requires that its HA binds to sialic acid–containing receptors present in cells of the human upper respiratory tract.However, there are still many unknowns with respect to the relationship between HA receptor specificity and influenza virus host and tissue tropism. It is, for example, quite clear now that the determinants of influenza tropism are more complex than the simplistic early view of classifying HAs by preferential binding to α2-6 and α2-3 sialic acids. The development of glycan arrays that allow to determine more precisely the ability of influenza viruses to bind to different sugar molecules have demonstrated a wide variety of complex binding patterns according to the specific viral strain.⁸ It is now clear that not only the linkage between the sialic acid and the next sugar influences binding of a specific viral and/or HA strain, but also the type of sialic acid as well as the rest of the carbohydrate. Because both the distribution of all possible different sialic acid–containing sugars in respiratory tissues and the types of molecules that can be used as receptors for productive infection by influenza viruses remain unclear, it remains to be determined what the different patterns of binding to specific carbohydrates by different influenza virus strains mean. It is also not known whether high affinity binding necessarily correlates with high infectivity, as this might inhibit viral spreading by facilitating virus retention in noninfectable surfaces of the respiratory tract. Finally, whether infection of specific cells in the respiratory tract facilitates virus mobilization into infectious aerosols and respiratory droplets, and therefore also facilitates transmission, is again unclear. Thus, more research is required to understand how the complexity of interactions of influenza viruses with their receptor determines the outcome of viral infection and transmission. A better understanding of these processes might facilitate the design of specific antivirals that stop influenza virus transmission and infection of the lower respiratory tract.     

Uptake of microparticles into the epithelium of human nasopharyngeal lymphoid tissue

The M cells of nasopharyngeal lymphoid tissue (NALT) have been considered to play an important role for vaccine delivery systems in humans. A number of investigations have reported particle uptake data in NALT of rodents. However, there have been no reports indicating any involvement of the nasopharyngeal lymphoid tissue in human vaccination. In the present study, we investigated whether the epithelium of human adenoid tissues might incorporate fluorescent microparticles using electron and fluorescent microscopy. The dissected adenoid tissues were incubated with various sizes and concentrations of fluorescent microparticles for 120 min at 37°C. Furthermore, the effect of surface coatings of microparticles with cations on the uptake into the epithelium of adenoid tissues was investigated. Transmission electron microscopy revealed that microparticles were taken up by the M cells of human nasopharyngeal lymphoid tissues. The NALT-M cells showed greater uptake of the smallest particles, 0.2 μm in diameter, than those of 0.5, 1.0, or 2.0 μm diameter. It was also revealed that surface coatings with poly-l-lysin or chitosan resulted in efficient uptake into the NALT. These results indicate that nasal administration of antigenic microparticles, which were coated with cationic materials, probably leads to a useful method of transnasal vaccination against respiratory and intestinal infections in humans.     

Enhanced detection of respiratory viruses using cryopreserved R-Mix ReadyCells.

BACKGROUND: R-Mix, which contains a fresh mixture of two cell lines, Mv1Lu (mink lung cells) and A549 cells, has shown good sensitivity and specificity for respiratory virus culture. However, it has until recently only been available in North America, in part due to the shipping constraints associated with cell aging and the difficulty in providing these cells to hard to reach regions. Recently, cryopreserved R-Mix ReadyCells for longer storage were developed. These cells, which are shipped on dry ice and have a shelf life as long as 6 months from date of manufacture, can be thawed and used as needed with minimal addition of refeeding media. OBJECTIVE: Assess the potential for cryopreserved R-Mix ReadyCells to replace conventional culture. STUDY DESIGN: Two hundred and twenty-three nasopharyngeal aspirates confirmed as respiratory virus-positive by conventional culture were inoculated into cryopreserved R-Mix ReadyCells and re-inoculated into conventional culture cells simultaneously. After 1 and 3 days of incubation cryopreserved R-Mix ReadyCells and conventional culture cells were screened using a respiratory virus fluorescent antibody pool for the detection of seven major respiratory viruses (influenza A and B viruses, parainfluenza 1, 2 and 3 viruses, respiratory syncytial virus and adenovirus). Positive pool results were further differentiated with specific monoclonal antibodies against the individual viruses. RESULTS: After 1 day of incubation detection rates for conventional culture were 25%, 39%, 39%, 49%, and 10% for influenza A virus, influenza B virus, parainfluenza viruses, respiratory syncytial virus, and adenovirus, respectively. Corresponding detection rates for cryopreserved R-Mix ReadyCells were 78%, 91%, 72%, 81%, and 65%. Average detection rates of cryopreserved R-Mix ReadyCells for all respiratory viruses were 80% after 1 day incubation and 95% after 3 days incubation, compared to 35% and 70% by conventional culture. CONCLUSION: The cryopreserved R-Mix ReadyCells system offers a highly sensitive and rapid method for detection of respiratory viruses that may allow it to replace conventional cell culture systems.     

Seasonal and Pandemic Human Influenza Viruses Attach Better to Human Upper Respiratory Tract Epithelium than Avian Influenza Viruses

Influenza viruses vary markedly in their efficiency of human-to-human transmission. This variation has been speculated to be determined in part by the tropism of influenza virus for the human upper respiratory tract. To study this tropism, we determined the pattern of virus attachment by virus histochemistry of three human and three avian influenza viruses in human nasal septum, conchae, nasopharynx, paranasal sinuses, and larynx. We found that the human influenza viruses—two seasonal influenza viruses and pandemic H1N1 virus—attached abundantly to ciliated epithelial cells and goblet cells throughout the upper respiratory tract. In contrast, the avian influenza viruses, including the highly pathogenic H5N1 virus, attached only rarely to epithelial cells or goblet cells. Both human and avian viruses attached occasionally to cells of the submucosal glands. The pattern of virus attachment was similar among the different sites of the human upper respiratory tract for each virus tested. We conclude that influenza viruses that are transmitted efficiently among humans attach abundantly to human upper respiratory tract, whereas inefficiently transmitted influenza viruses attach rarely. These results suggest that the ability of an influenza virus to attach to human upper respiratory tract is a critical factor for efficient transmission in the human population.Influenza is an important cause of morbidity and mortality in humans during seasonal, pandemic, and zoonotic outbreaks. Seasonal influenza is estimated to cause 250,000 to 500,000 deaths per year worldwide. Pandemic influenza viruses of the previous century resulted in an estimated 1 to 4 million deaths for the 1957 H2N2 (Asian flu) and the 1968 H3N2 (Hong Kong flu) influenza pandemics, and 20 to 50 million deaths for the 1918 H1N1 (Spanish flu) influenza pandemic.^1,2 The first influenza pandemic of the 21st century, the currently ongoing new H1N1 virus outbreak (Mexican flu), has caused at least 3486 deaths as of September 13, 2009 (http://www.who.int/csr/don/2009_09_18/en/index.html). The zoonotic highly pathogenic avian influenza virus (HPAIV) H5N1, which is causing an ongoing outbreak in poultry, only occasionally infects humans, but has a high mortality rate, with 262 deaths out of 400+ confirmed infections as of August 2009 (http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_08_11/en/index.html).The pandemic potential of an influenza virus depends largely on its efficiency of human-to-human transmission. Human influenza viruses, including seasonal H1N1 and H3N2 viruses, and the pandemic H1N1 virus, are transmitted efficiently.³ In contrast, the zoonotic HPAIV H5N1 is only rarely transmitted from human to human.⁴ However, the factors determining efficient virus transmission among humans are poorly understood.Tropism of influenza virus for the human upper respiratory tract (URT) has been speculated to be an important determinant for the efficiency of virus transmission, based both on receptor distribution and virus replication studies.^5,6 Based on lectin histochemistry, the human URT has abundant receptors for human influenza viruses, which are efficiently transmitted.^5,7 This fits with the ability for human influenza viruses to replicate in human URT tissues based on in vivo,⁸ ex vivo,⁷ and in vitro studies.^9,10,11 In contrast, the human URT has only limited receptors for avian influenza viruses.^5,7 This fits with the absence or rarity of HPAIV H5N1 transmission among humans.⁴ However, it is discordant with a study of Nicholls and others, who showed that HPAIV H5N1 can replicate in URT tissues. They explained this discordance by suggesting that HPAIV H5N1 attached to receptors not detected by the lectins used. Therefore, there is currently no consensus on the tropism of HPAIV H5N1 for the human URT. In addition, the studies to date have not studied the human URT systematically, and it is not known what the tropism of the new H1N1 virus is for the human URT.To address the question whether URT tropism of influenza viruses is linked to efficient transmission, we determined the pattern of attachment of selected influenza viruses in the human URT: human influenza viruses, including seasonal H1N1 and H3N2 viruses and pandemic H1N1 virus, which are transmitted efficiently, and avian influenza viruses, including a HPAIV H5N1, isolated from a fatal human case, which is not transmitted efficiently among humans. We measured the pattern of virus attachment by use of virus histochemistry instead of lectin histochemistry.⁶ Virus histochemistry measures the attachment of influenza virus to its host cell directly. Therefore, any receptors other than SA-α-2,3-Gal terminated saccharides and SA-α-2,6-Gal terminated saccharides also would be detected by virus histochemistry. We have used this technique previously to show that the pattern of attachment in the human lower respiratory tract is different for human and avian influenza viruses, which correlates with differences in primary disease.¹²     

Spectrum of respiratory viruses circulating in eastern India: Prospective surveillance among patients with influenza‐like illness during 2010–2011

In developing countries, viruses causing respiratory disease are a major concern of public health. During January 2010–December 2011, 2,737 patients with acute respiratory infection from the outpatient departments as well as patients admitted to hospitals were screened for different respiratory viruses. Nasal and or throat swabs were collected and transported to the laboratory where initial screening of influenza A and influenza B viruses was performed. The samples were tested further for influenza C virus, parainfluenza viruses 1–4, human rhinovirus, metapneumovirus and respiratory syncytial virus by conventional RT‐ PCR. The study revealed that the majority of the patients were under 5 years of age; both due to their higher susceptibility to respiratory infections and presentation to hospitals. Out of 2,737 patients enrolled in this study, 59% were found positive for one or more respiratory viruses. Influenza B infection was detected in 12% of patients followed by influenza A (11.7%), respiratory syncytial virus (7.1%), parainfluenza virus‐2 (6%), metapneumovirus (3%), parainfluenza virus‐3 (1%), parainfluenza virus‐4 (0.6%), parainfluenza virus‐1 (0.3%), influenza C (0.2%) and human rhinovirus (0.2%). Distinct seasonal infection was observed only for influenza A and influenza B viruses. J. Med. Virol. 85:1459–1465, 2013 . © 2013 Wiley Periodicals, Inc.

     

Tropism and Innate Host Responses of the 2009 Pandemic H1N1 Influenza Virus in ex Vivo and in Vitro Cultures of Human Conjunctiva and Respiratory Tract

The novel pandemic influenza H1N1 (H1N1pdm) virus of swine origin causes mild disease but occasionally leads to acute respiratory distress syndrome and death. It is important to understand the pathogenesis of this new disease in humans. We compared the virus tropism and host-responses elicited by pandemic H1N1pdm and seasonal H1N1 influenza viruses in ex vivo cultures of human conjunctiva, nasopharynx, bronchus, and lung, as well as in vitro cultures of human nasopharyngeal, bronchial, and alveolar epithelial cells. We found comparable replication and host-responses in seasonal and pandemic H1N1 viruses. However, pandemic H1N1pdm virus differs from seasonal H1N1 influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in its receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection with H1N1pdm. A greater viral replication competence in bronchial epithelium at 33°C may also contribute to the slight increase in virulence of the pandemic influenza virus. In contrast with highly pathogenic influenza H5N1 virus, pandemic H1N1pdm does not differ from seasonal influenza virus in its intrinsic capacity for cytokine dysregulation. Collectively, these results suggest that pandemic H1N1pdm virus differs in modest but subtle ways from seasonal H1N1 virus in its intrinsic virulence for humans, which is in accord with the epidemiology of the pandemic to date. These findings are therefore relevant for understanding transmission and therapy.The recent pandemic caused by a novel H1N1 virus (H1N1pdm) arose from the reassortment of three or more viruses of swine origin, including the North American triple reassortant H3N2 and H1N2 viruses, classical swine H1N1, and European swine H1N1/H3N2 viruses.^1,2 Most patients with pandemic H1N1pdm have mild influenza-like illness, but a minority of patients develop a primary viral pneumonia, sometimes leading to acute respiratory distress syndrome and death.^3,4 Many, but not all, patients with severe disease have pregnancy, obesity, or underlying disease states such as asthma, obstructive airways disease, diabetes, and chronic cardiovascular or renal disease. The disease associated with H1N1pdm so far appears to be comparable with that of seasonal influenza and less severe than that seen in the 1918 pandemic or in zoonotic disease caused by highly pathogenic avian influenza (HPAI) H5N1. However, unlike seasonal influenza where morbidity and mortality are mainly seen in the elderly, pandemic H1N1pdm appears to spare this age-group, possibly because of the presence of cross-neutralizing antibody generated by prior repeated seasonal H1N1 infection.⁵ In California, the median age of all cases was 17 years, of hospitalized cases 26 years, and for fatal cases was 45 years.It is therefore important to understand how the pathogenesis and tissue tropism of H1N1pdm virus in humans differs from seasonal influenza viruses. However, there is so far limited information in this regard. The H1N1pdm virus does not possess the genetic motifs of virulence associated with either the HPAI H5N1 or 1918 H1N1 viruses.² In experimentally infected ferrets, macaques, and mice, H1N1pdm causes moderately more severe illness compared with seasonal influenza although being much less virulent than HPAI H5N1 or the 1918 pandemic Spanish flu virus.^6,7,8 In these animal models, H1N1pdm virus was able to infect the alveolar epithelium more readily than seasonal H1N1 virus, but whether this holds true for humans is not known.⁷ Though H1N1pdm was initially reported to have a predominantly α2-6 sialic acid (Sia) receptor binding preference⁸ similar to human seasonal influenza viruses, recent glycan array data indicates that there is binding to both “human” Sia α2-6 and “avian” Sia α2-3.⁹ H1N1pdm virus differs from seasonal influenza viruses in their ability to infect and cause illness in mice without prior adaptation. As the mouse respiratory tract has a predominance of Sia α2-3, rather than Sia α2-6 receptors, these findings support the contention that H1N1pdm viruses have a broader Sia receptor binding profile.⁸ Taken together, these observations suggest that H1N1pdm virus differs in subtle but important ways from seasonal influenza viruses in receptor usage and tissue tropism, and this may be important in its pathogenesis and transmission.Cytokine dysregulation is believed to contribute to the pathogenesis of human disease caused by HPAI H5N1 as well as the 1918 pandemic H1N1 viruses.^{10,11,12,13,14} It is not known whether the H1N1pdm virus differs from seasonal influenza in the induction of proinflammatory host responses in human tissues. The lungs of H1N1pdm-infected mice had a markedly different cytokine profile when compared with seasonal influenza infected animals with elevated levels of interleukin (IL)-4, IL-10, and interferon (IFN)-γ. The lungs of H1N1pdm-infected macaques also had higher levels of chemokines MCP-1, MIP-1α, IL-6, and IL-18.⁶ However, it is not known whether these host responses simply reflect the greater or more extensive replication of the H1N1pdm virus in the lung when compared with seasonal influenza viruses or are attributable to intrinsic differences in the virus itself being able to induce a more potent innate host response as occurs with the highly pathogenic avian influenza H5N1 virus. When primary human cells (macrophages and type I-like pneumocytes) are infected with seasonal and HPAI H5N1 influenza viruses of comparable infectious titers, the HPAI H5N1 viruses differentially hyperinduce a range of proinflammatory responses over a single virus replication cycle.^10,11,14 Thus it is clear that the H5N1 virus has inherent properties that lead to an exaggerated innate immune response. It is relevant to use a similar approach to investigate the host innate immune responses induced by pandemic H1N1pdm compared with that of seasonal influenza H1N1 virus in primary human respiratory epithelium.We have previously used ex vivo cultures of nasopharynx, tonsillar tissue, and lung for investigating virus tropism.¹⁵ We have also established in vitro cultures of polarized primary human respiratory epithelial cells, including type I–like alveolar epithelial cells, nasopharyngeal epithelial cells, and differentiated bronchial epithelial cells for investigating tissue tropism and innate immune host responses elicited by influenza viruses.^10,14,15 These in vitro cultures of bronchial epithelium differentiated at an air–liquid interface (ALI) provide a good representation of the human bronchial epithelium and have a ciliated epithelium as well as mucus producing goblet cells. We have also recently established ex vivo tissue culture models of human conjunctival epithelium. We now use these ex vivo human tissue cultures as well as the primary human respiratory epithelial cell cultures to compare the virus replication competence, cell tropism, and host innate immune responses of the pandemic H1N1pdm virus with that of seasonal influenza H1N1 viruses and, where relevant, avian HPAI H5N1 and H7N7 viruses.We demonstrate that whereas seasonal H1N1 and pandemic H1N1pdm viruses replicate comparably in ex vivo cultures of human nasopharynx and lung tissues, the human conjunctiva is preferentially infected by H1N1pdm rather than seasonal influenza H1N1 or H3N2 viruses. Pandemic H1N1pdm replicates more efficiently than seasonal H1N1 virus in differentiated bronchial epithelial cells in vitro at 33°C, but the two viruses replicate comparably at 37°C. We also demonstrate that the pandemic H1N1pdm virus does not differ from the human seasonal influenza viruses in their ability to induce proinflammatory cytokines and therefore does not appear to have the same potential to induce cytokine dysregulation as that manifested by HPAI H5N1 or the 1918 H1N1 virus.     

Comparison of Midturbinate Flocked-Swab Specimens with Nasopharyngeal Aspirates for Detection of Respiratory Viruses in Children by the Direct Fluorescent Antibody Technique

Paired nasopharyngeal aspirate (NPA) and midturbinate flocked-swab specimens from 153 children with respiratory symptoms were examined by the direct fluorescent antibody (DFA) technique. Seventy-four infants (49%) had a viral infection documented by DFA. The flocked-swab specimens had 93% sensitivity and 96.7% agreement with the NPA specimens, with a kappa coefficient of 93.4% (95% confidence interval, 0.877, 0.991).The direct fluorescent antibody (DFA) technique revolutionized the rapid detection of respiratory viruses. Since its inception in 1968, it has been one of the mainstays in clinical virology laboratories throughout the world (4). The ability of DFA to detect respiratory viruses depends on many things, but it all begins with good specimen collection. The nasopharyngeal aspirate (NPA) has been considered the best specimen to detect respiratory viruses in infants (4). However, it is difficult to collect because it requires special equipment, such as a catheter, trap, and vacuum source, and specialized training. A traditional nasopharyngeal swab is the next best specimen, especially in older children or adults, because it utilizes common supplies; however, the collection end of the swab, comprised of wound Dacron fibers, has limited absorbent capacity to trap virus-infected exfoliated epithelial cells. A nylon nasopharyngeal flocked swab with enhanced absorptive properties introduced in 2006 compared favorably to the NPA for the detection of respiratory viruses by DFA (2). Recently, a midturbinate flocked swab developed by Smieja, et al. (7), and marketed by Copan, Inc., has offered a more intuitive approach for the collection of nasopharyngeal specimens (1). It has compared favorably to the NPA and the flocked nasopharyngeal swab in the diagnosis of respiratory viruses by culture, antigen detection, and PCR, none of which require intact exfoliated epithelial cells for visualization; there is no published experience of midturbinate flocked swabs with DFA in children (1, 5, 6). The midturbinate flocked swab differs from the nasopharyngeal swab. It has a sampling depth indication gauge and also has a larger absorptive capacity than the smaller nasopharyngeal swab.The present study was designed to compare the efficacy of the midturbinate flocked swab with the NPA in the detection of respiratory viruses by DFA.The study was conducted from 5 January 2010 through 11 March 2010. All children 2 years of age or less admitted to the infant''s floor of the hospital with respiratory symptoms were enrolled. The study was reviewed by the Children and Youth Institutional Review Board, who waived the need for a formal review because the study was deemed an evaluation comparing a new specimen collection device to the standard nasopharyngeal aspirate; parents were allowed to opt out of the use of the new specimen device. A nasopharyngeal aspirate specimen was collected through one nostril. A second specimen was collected through the other nostril with a midturbinate FLOQ swab (Copan Diagnostics, Inc., Murrieta, CA) designed for children 2 years of age or less; the swab was inserted up to the collar on the shaft. Both specimens were placed in 3 ml of Copan UTM transport medium, transported to the virus laboratory, and processed within 6 h. The suspension was centrifuged, and the cellular pellet washed. The cells were then spotted to glass slides. The cells were stained for DFA using a D3 Ultra respiratory screening identification kit (Diagnostic Hybrids, Inc. [DHI], Athens, OH). The kit screened for respiratory syncytial virus (RSV), influenza viruses (IFV) A and B, parainfluenza viruses (PFV) 1, 2, and 3, and adenovirus (AdV). An additional stain for human metapneumovirus (hMPV) (DHI) was included. The DFA readers were not blinded to the specimen source. The degree of DFA agreement between specimens collected by NPA and midturbinate flocked swabs was calculated with Cohen''s kappa coefficient of agreement.One hundred fifty-three infants entered the study. Paired specimens were collected from every infant. Respiratory viruses were identified in 74 (48.6%). Respiratory syncytial virus was most frequent, found in 47 patients (30%), with hMPV in 25 (16.3%), PFV in 1 (0.7%), AdV in 1 (0.7%), and IFV in none (0.0%). The 2009 H1N1 influenza A virus had last been identified in the laboratory in November 2009, more than 1 month before the start of the study. DFA of NPA specimens identified all the viruses. DFA of the flocked-swab specimens failed to detect 4 RSV and 1 hMPV isolate that had been detected in the NPA specimens. The negative DFA test results on flocked-swab specimens agreed with the negative DFA test results on NPA specimens. Overall, the positive and negative DFA test results on flocked-swab specimens had 96.7% agreement with the DFA test results on NPA specimens, with a Kappa coefficient of 93.4% (95% confidence interval [CI], 0.877, 0.991; P < 0.00001). The sensitivity of the flocked swab was 93.2% (95% CI, 0.849, 0.978).The midturbinate flocked swab proved to be comparable to the NPA for the detection of common respiratory viruses, such as RSV and hMPV, in a DFA test in the present study. The absence of IFV and the low numbers of AdV and PFV isolates in specimens prevented an assessment of the swab''s utility in detecting these viruses; however, earlier studies with nasopharyngeal flocked swabs suggested that the midturbinate swab would give similar results (3). In an earlier study, the sensitivity of the NPA in detecting either IFV or RSV was greater than the sensitivity of flocked nasopharyngeal swabs, although the difference was not statistically significant; the differences may be attributed to the greater number of respiratory epithelial cells available for examination in NPA specimens (2). The advantage of the midturbinate collection over nasopharyngeal collection resides in the relative ease of collection and the resultant patient cooperation, especially among the very young; however, the observations made in the present study may not extend beyond the pediatric population.     

Time-resolved fluoroimmunoassay with monoclonal antibodies for rapid diagnosis of influenza infections

Monoclonal antibodies that are broadly reactive with either influenza A or influenza B viruses were used to develop a 2- to 3-h antigen capture time-resolved fluoroimmunoassay (TR FIA) for detecting influenza viral antigens in both original nasopharyngeal aspirate specimens and in tissue cultures inoculated with nose or throat swab specimens. The lower limit of sensitivity of the assay was about 10 pg of protein as determined with purified influenza A nucleoprotein expressed by recombinant DNA. When the TR FIA was performed with 96 nasopharyngeal aspirate specimens collected during outbreaks of influenza A (H3N2) virus and the results were compared with serodiagnosis results with paired sera, the specificity and sensitivity of TR FIA for the demonstration of influenza A infections were 95 and 85%, respectively. In culture confirmation assays, more than 80% of the swab specimens that grew influenza A or B virus within 7 days could be identified by the TR FIA within 48 h of the inoculation of cells. The results are consistent with those previously reported for respiratory syncytial virus and extend the applicability of monoclonal antibody-based TR FIA for the rapid diagnosis of acute respiratory viral infections.     

Human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals

Viral attachment to the host cell is critical for tissue and species specificity of virus infections. Recently, pattern of viral attachment (PVA) in human respiratory tract was determined for highly pathogenic avian influenza virus of subtype H5N1. However, PVA of human influenza viruses and other avian influenza viruses in either humans or experimental animals is unknown. Therefore, we compared PVA of two human influenza viruses (H1N1 and H3N2) and two low pathogenic avian influenza viruses (H5N9 and H6N1) with that of H5N1 virus in respiratory tract tissues of humans, mice, ferrets, cynomolgus macaques, cats, and pigs by virus histochemistry. We found that human influenza viruses attached more strongly to human trachea and bronchi than H5N1 virus and attached to different cell types than H5N1 virus. These differences correspond to primary diagnoses of tracheobronchitis for human influenza viruses and diffuse alveolar damage for H5N1 virus. The PVA of low pathogenic avian influenza viruses in human respiratory tract resembled that of H5N1 virus, demonstrating that other properties determine its pathogenicity for humans. The PVA in human respiratory tract most closely mirrored that in ferrets and pigs for human influenza viruses and that in ferrets, pigs, and cats for avian influenza viruses.     

Detection of influenza virus by centrifugal inoculation of MDCK cells and staining with monoclonal antibodies.

Two methods for detection of influenza virus in 451 clinical respiratory specimens were compared: (i) 24-well-plate centrifugation with Madin-Darby canine kidney (MDCK) cells and staining with monoclonal antibody pools to influenza viruses A and B (Centers for Disease Control, Atlanta, Ga.) in an indirect immunofluorescence assay after incubation for 40 h, and (ii) conventional tissue cell culture with primary monkey cells and hemadsorption. For 100 of these specimens, direct examination of smears by the direct fluorescence assay with monoclonal antibodies (Boots Cell Tech/API Analytab Products, Plainview, N.Y.) was also performed. Influenza A virus was recovered from 28 specimens by tissue cell culture after incubation for an average of 4.75 days (range, 2 to 14 days). Influenza B virus was recovered from 35 specimens by tissue culture after incubation for an average of 5.4 days (range, 3 to 14 days). By the centrifugation assay, 23 specimens were positive for influenza A virus and 30 were positive for influenza B virus. All specimens positive by the centrifugation assay were also positive by conventional tissue cell culture. The sensitivities of the centrifugation assay were 82% for detection of influenza A virus and 86% for influenza B virus (84% overall); the specificity of the assay was 100%. Of the 100 specimens studied by direct examination, 15 were positive for influenza virus by both conventional culture and centrifugation assay; however, the direct-smear results for these 15 specimens were negative in 13 cases and inconclusive in 2. The centrifugation assay is a rapid and specific method for detection of influenza A and B viruses in clinical specimens, and it can serve as a valuable and cost-efficient adjunct to conventional culture methods.     

Respiratory viral infections are underdiagnosed in patients with suspected sepsis

The study aim was to investigate the prevalence and clinical relevance of viral findings by multiplex PCR from the nasopharynx of clinically septic patients during a winter season. During 11 weeks of the influenza epidemic period in January–March 2012, consecutive adult patients suspected to be septic (n = 432) were analyzed with cultures from blood and nasopharynx plus multiplex PCR for respiratory viruses on the nasopharyngeal specimen. The results were compared with those from microbiology analyses ordered as part of standard care. During the winter season, viral respiratory pathogens, mainly influenza A virus, human metapneumovirus, coronavirus, and respiratory syncytial virus were clinically underdiagnosed in 70% of patients positive by the multiplex PCR assay. During the first four weeks of the influenza epidemic, few tests for influenza were ordered by clinicians, indicating low awareness that the epidemic had started. Nasopharyngeal findings of Streptococcus pneumoniae and Haemophilus influenzae by culture correlated to pneumonia diagnosis, and in those patients laboratory signs of viral co-infections were common but rarely suspected by clinicians. The role of respiratory viral infections in patients presenting with a clinical picture of sepsis is underestimated. Specific antiviral treatment might be beneficial in some cases and may reduce spread in a hospital setting. Diagnosing viral infections may promote reduction of unnecessary antibiotic use. It can also be a tool for decisions concerning patient logistics, in order to minimize exposure of susceptible patients and personnel.