Effects of serial swabs on the nasopharyngeal mucosa: our experience in SARS-CoV2 screening.

INTRODUCTION: The purpose of the study was to analyze the effect of swabs on nasal mucosa. METHODOLOGY: Since May 2020, our department was responsible for screening coronavirus disease 2019 (COVID-19) among the employees of a company that continued its activity during the pandemic. The screening protocol consisted of two swabs per week. The samples were analyzed through objective endoscopic and subjective clinical evaluations with sino-nasal outcome test (SNOT Test) at three time points (T0, T1 - three months, T2 - six months). RESULTS: 23.76% of patients showed an increase in the SNOT score at T1, and the score decreased at T2. This could be due to the phenomenon of "adaptation" of the nasal mucosa. Endoscopic control showed that at T1, secretion, hyperemia, and edema are the most common signs. At T2, however, the crusts accounted for 52.94% of all damage. It is evident that at T1 the endoscopically detected signs of "acute" damage were more represented than at T2, while the signs of "chronic" damage increased as the number of swabs increased. CONCLUSIONS: We demonstrated that mucosal damage and perceived symptoms were absolutely acceptable compared to the diagnostic advantage obtained with serial screening.

High burden of viruses and bacterial pathobionts drives heightened nasal innate immunity in children.

Studies during the COVID-19 pandemic showed that children had heightened nasal innate immune responses compared with adults. To evaluate the role of nasal viruses and bacteria in driving these responses, we performed cytokine profiling and comprehensive, symptom-agnostic testing for respiratory viruses and bacterial pathobionts in nasopharyngeal samples from children tested for SARS-CoV-2 in 2021-22 (n = 467). Respiratory viruses and/or pathobionts were highly prevalent (82% of symptomatic and 30% asymptomatic children; 90 and 49% for children <5 years). Virus detection and load correlated with the nasal interferon response biomarker CXCL10, and the previously reported discrepancy between SARS-CoV-2 viral load and nasal interferon response was explained by viral coinfections. Bacterial pathobionts correlated with a distinct proinflammatory response with elevated IL-1ß and TNF but not CXCL10. Furthermore, paired samples from healthy 1-year-olds collected 1-2 wk apart revealed frequent respiratory virus acquisition or clearance, with mucosal immunophenotype changing in parallel. These findings reveal that frequent, dynamic host-pathogen interactions drive nasal innate immune activation in children.

Intranasal adenovirus-vectored Omicron vaccine induced nasal immunoglobulin A has superior neutralizing potency than serum antibodies.

The upper respiratory tract is the initial site of SARS-CoV-2 infection. Nasal spike-specific secretory immunoglobulin A (sIgA) correlates with protection against Omicron breakthrough infection. We report that intranasal vaccination using human adenovirus serotype 5 (Ad5) vectored Omicron spike in people who previously vaccinated with ancestral vaccine could induce robust neutralizing sIgA in the nasal passage. Nasal sIgA was predominantly present in dimeric and multimeric forms and accounted for nearly 40% of total proteins in nasal mucosal lining fluids (NMLFs). A low-level IgG could also be detected in NMLFs but not IgM, IgD, and IgE. After a complete nasal wash, sIgA in the nasal passage could be replenished rapidly within a few hours. A comparison of purified paired serum IgA, serum IgG, and nasal sIgA from the same individuals showed that sIgA was up to 3-logs more potent than serum antibodies in binding to spikes and in neutralizing Omicron subvariants. Serum IgG and IgA failed to neutralize XBB and BA.2.86, while nasal sIgA retained potent neutralization against these newly emerged variants. Further analysis showed that sIgA was more effective than IgG or IgA in blocking spike-mediated cell-to-cell transmission and protecting hACE2 mice from XBB challenge. Using a sIgA monoclonal antibody as a reference, we estimated that the total nasal sIgA contains about 2.6-3.9% spike-specific sIgA in NMLFs collected approximately one month after intranasal vaccination. Our study provided insights for developing intranasal vaccines that can induce sIgA to build an effective and mutation-resistant first-line immune barrier against constantly emerging variants.

Delayed Mucosal Antiviral Responses Despite Robust Peripheral Inflammation in Fatal COVID-19.

BACKGROUND: While inflammatory and immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in peripheral blood are extensively described, responses at the upper respiratory mucosal site of initial infection are relatively poorly defined. We sought to identify mucosal cytokine/chemokine signatures that distinguished coronavirus disease 2019 (COVID-19) severity categories, and relate these to disease progression and peripheral inflammation. METHODS: We measured 35 cytokines and chemokines in nasal samples from 274 patients hospitalized with COVID-19. Analysis considered the timing of sampling during disease, as either the early (0-5 days after symptom onset) or late (6-20 days after symptom onset) phase. RESULTS: Patients that survived severe COVID-19 showed interferon (IFN)-dominated mucosal immune responses (IFN-γ, CXCL10, and CXCL13) early in infection. These early mucosal responses were absent in patients who would progress to fatal disease despite equivalent SARS-CoV-2 viral load. Mucosal inflammation in later disease was dominated by interleukin 2 (IL-2), IL-10, IFN-γ, and IL-12p70, which scaled with severity but did not differentiate patients who would survive or succumb to disease. Cytokines and chemokines in the mucosa showed distinctions from responses evident in the peripheral blood, particularly during fatal disease. CONCLUSIONS: Defective early mucosal antiviral responses anticipate fatal COVID-19 but are not associated with viral load. Early mucosal immune responses may define the trajectory of severe COVID-19.

Kids' noses resist COVID-19.

Children resist COVID-19, and previous studies reported increased innate immunity in their upper airways. A new paper by Watkins et al. (https://doi.org/10.1084/jem.20230911) shows that the nasal mucosa of children is characterized by often asymptomatic viral and/or bacterial infections that dynamically regulate distinct innate immune programs.

Detection of SARS-CoV-2-Specific Secretory IgA and Neutralizing Antibodies in the Nasal Secretions of Exposed Seronegative Individuals.

Mucosal immunity may contribute to clearing SARS-CoV-2 infection prior to systemic infection, thereby allowing hosts to remain seronegative. We describe the meaningful detection of SARS-CoV-2-specific nasal mucosal antibodies in a group of exposed-household individuals that evaded systemic infection. Between June 2020 and February 2023, nasopharyngeal swab (NPS) and acute and convalescent blood were collected from individuals exposed to a SARS-CoV-2-confirmed household member. Nasal secretory IgA (SIgA) antibodies targeting the SARS-CoV-2 spike protein were measured using a modified ELISA. Of the 36 exposed individuals without SARS-CoV-2 detected by the RT-PCR of NPS specimens and seronegative for SARS-CoV-2-specific IgG at enrollment and convalescence, 13 (36.1%) had positive SARS-CoV-2-specific SIgA levels detected in the nasal mucosa at enrollment. These individuals had significantly higher nasal SIgA (median 0.52 AU/mL) compared with never-exposed, never-infected controls (0.001 AU/mL) and infected-family participants (0.0002 AU/mL) during the acute visit, respectively (both p < 0.001). The nasal SARS-CoV-2-specific SIgA decreased rapidly over two weeks in the exposed seronegative individuals compared to a rise in SIgA in infected-family members. The nasal SARS-CoV-2-specific SIgA may have a protective role in preventing systemic infection.

Nasal cathelicidin is expressed in early life and is increased during mild, but not severe respiratory syncytial virus infection.

Respiratory syncytial virus is the major cause of acute lower respiratory tract infections in young children, causing extensive mortality and morbidity globally, with limited therapeutic or preventative options. Cathelicidins are innate immune antimicrobial host defence peptides and have antiviral activity against RSV. However, upper respiratory tract cathelicidin expression and the relationship with host and environment factors in early life, are unknown. Infant cohorts were analysed to characterise early life nasal cathelicidin levels, revealing low expression levels in the first week of life, with increased levels at 9 months which are comparable to 2-year-olds and healthy adults. No impact of prematurity on nasal cathelicidin expression was observed, nor were there effects of sex or birth mode, however, nasal cathelicidin expression was lower in the first week-of-life in winter births. Nasal cathelicidin levels were positively associated with specific inflammatory markers and demonstrated to be associated with microbial community composition. Importantly, levels of nasal cathelicidin expression were elevated in infants with mild RSV infection, but, in contrast, were not upregulated in infants hospitalised with severe RSV infection. These data suggest important relationships between nasal cathelicidin, upper airway microbiota, inflammation, and immunity against RSV infection, with interventional potential.

Interferon signaling in the nasal epithelium distinguishes among lethal and common cold coronaviruses and mediates viral clearance.

All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at an air-liquid interface (ALI). HCoV-229E, HCoV-NL63, and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33 °C) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally directed IFNs as potential therapeutics.

Age-specific nasal epithelial responses to SARS-CoV-2 infection.

Children infected with SARS-CoV-2 rarely progress to respiratory failure. However, the risk of mortality in infected people over 85 years of age remains high. Here we investigate differences in the cellular landscape and function of paediatric (<12 years), adult (30-50 years) and older adult (>70 years) ex vivo cultured nasal epithelial cells in response to infection with SARS-CoV-2. We show that cell tropism of SARS-CoV-2, and expression of ACE2 and TMPRSS2 in nasal epithelial cell subtypes, differ between age groups. While ciliated cells are viral replication centres across all age groups, a distinct goblet inflammatory subtype emerges in infected paediatric cultures and shows high expression of interferon-stimulated genes and incomplete viral replication. In contrast, older adult cultures infected with SARS-CoV-2 show a proportional increase in basaloid-like cells, which facilitate viral spread and are associated with altered epithelial repair pathways. We confirm age-specific induction of these cell types by integrating data from in vivo COVID-19 studies and validate that our in vitro model recapitulates early epithelial responses to SARS-CoV-2 infection.

Histochemical Evidence for Reduced Immune Response in Nasal Mucosa of Patients with COVID-19.

The primary entry point of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nasal mucosa, where viral-induced inflammation occurs. When the immune response fails against SARS-CoV-2, understanding the altered response becomes crucial. This study aimed to compare SARS-CoV-2 immunological responses in the olfactory and respiratory mucosa by focusing on epithelia and nerves. Between 2020 and 2022, we obtained post mortem tissues from the olfactory cleft from 10 patients with histologically intact olfactory epithelia (OE) who died with or from COVID-19, along with four age-matched controls. These tissues were subjected to immunohistochemical reactions using antibodies against T cell antigens CD3, CD8, CD68, and SARS spike protein for viral evidence. Deceased patients with COVID-19 exhibited peripheral lymphopenia accompanied by a local decrease in CD3+ cells in the OE. However, SARS-CoV-2 spike protein was sparsely detectable in the OE. With regard to the involvement of nerve fibers, the present analysis suggested that SARS-CoV-2 did not significantly alter the immune response in olfactory or trigeminal fibers. On the other hand, SARS spike protein was detectable in both nerves. In summary, the post mortem investigation demonstrated a decreased T cell response in patients with COVID-19 and signs of SARS-CoV-2 presence in olfactory and trigeminal fibers.

Intranasal neomycin evokes broad-spectrum antiviral immunity in the upper respiratory tract.

Respiratory virus infections in humans cause a broad-spectrum of diseases that result in substantial morbidity and mortality annually worldwide. To reduce the global burden of respiratory viral diseases, preventative and therapeutic interventions that are accessible and effective are urgently needed, especially in countries that are disproportionately affected. Repurposing generic medicine has the potential to bring new treatments for infectious diseases to patients efficiently and equitably. In this study, we found that intranasal delivery of neomycin, a generic aminoglycoside antibiotic, induces the expression of interferon-stimulated genes (ISGs) in the nasal mucosa that is independent of the commensal microbiota. Prophylactic or therapeutic administration of neomycin provided significant protection against upper respiratory infection and lethal disease in a mouse model of COVID-19. Furthermore, neomycin treatment protected Mx1 congenic mice from upper and lower respiratory infections with a highly virulent strain of influenza A virus. In Syrian hamsters, neomycin treatment potently mitigated contact transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In healthy humans, intranasal application of neomycin-containing Neosporin ointment was well tolerated and effective at inducing ISG expression in the nose in a subset of participants. These findings suggest that neomycin has the potential to be harnessed as a host-directed antiviral strategy for the prevention and treatment of respiratory viral infections.

Curcumin and quercetin co-encapsulated in nanoemulsions for nasal administration: A promising therapeutic and prophylactic treatment for viral respiratory infections.

One of the most frequent causes of respiratory infections are viruses. Viruses reaching the airways can be absorbed by the human body through the respiratory mucosa and mainly infect lung cells. Several viral infections are not yet curable, such as coronavirus-2 (SARS-CoV-2). Furthermore, the side effect of synthetic antiviral drugs and reduced efficacy against resistant variants have reinforced the search for alternative and effective treatment options, such as plant-derived antiviral molecules. Curcumin (CUR) and quercetin (QUE) are two natural compounds that have been widely studied for their health benefits, such as antiviral and anti-inflammatory activity. However, poor oral bioavailability limits the clinical applications of these natural compounds. In this work, nanoemulsions (NE) co-encapsulating CUR and QUE designed for nasal administration were developed as promising prophylactic and therapeutic treatments for viral respiratory infections. The NEs were prepared by high-pressure homogenization combined with the phase inversion temperature technique and evaluated for their physical and chemical characteristics. In vitro assays were performed to evaluate the nanoemulsion retention into the porcine nasal mucosa. In addition, the CUR and QUE-loaded NE antiviral activity was tested against a murine ß-COV, namely MHV-3. The results evidenced that CUR and QUE loaded NE had a particle size of 400 nm and retention in the porcine nasal mucosa. The antiviral activity of the NEs showed a percentage of inhibition of around 99 %, indicating that the developed NEs has interesting properties as a therapeutic and prophylactic treatment against viral respiratory infections.

Protective mucosal SARS-CoV-2 antibodies in the majority of the general population in the Netherlands.

Antibodies to SARS-CoV-2 on the mucosal surfaces of the respiratory tract are understood to contribute to protection against SARS-CoV-2 infection. We aimed to describe the prevalence, levels, and functionality of mucosal antibodies in the general Dutch population. Nasal samples were collected from 778 randomly selected participants, 1-90 years of age, nested within the nationwide prospective SARS-CoV-2 PIENTER corona serosurvey in the Netherlands. Spike-specific immunoglobulin (Ig)G was detected in the nasal samples of 94.6% (in case of the wild-type S1 variant) and 94.9% (Omicron BA.1) of the individuals, whereas 44.2% and 62.7% of the individuals were positive for wild-type and Omicron BA.1 S1 IgA, respectively. The lowest prevalence of mucosal antibodies was observed in children under 12 years of age. The prevalence and levels of IgA and IgG were higher in individuals with a history of SARS-CoV-2 infection. Mucosal antibodies inhibited the binding of Wuhan, Delta, and Omicron BA.1 receptor binding domain to human angiotensin-converting enzyme 2 in 94.4%, 95.4%, and 92.6% of the participants, respectively. Higher levels of mucosal antibodies were associated with a lower risk of future infection.

Apoptosis and pyroptosis in the nasal mucosa of Syrian hamster during SARS-CoV-2 infection and reinfection.

In SARS-CoV-2 infection, it has been observed that viral replication lasts longer in the nasal mucosa than in the lungs, despite the presence of a high viral load at both sites. In hamsters, we found that the nasal mucosa exhibited a mild inflammatory response and minimal pathological injuries, whereas the lungs displayed a significant inflammatory response and severe injuries. The underlying cellular events may be induced by viral infection in three types of cell death: apoptosis, pyroptosis, and necroptosis. Our findings indicate that apoptosis was consistently activated during infection in the nasal mucosa, and the levels of apoptosis were consistent with the viral load. On the other hand, pyroptosis and a few instances of necroptosis were observed only on 7 dpi in the nasal mucosa. In the lungs, however, both pyroptosis and apoptosis were prominently activated on 3 dpi, with lower levels of apoptosis compared to the nasal mucosa. Interestingly, in reinfection, obvious viral load and apoptosis in the nasal mucosa were detected on 3 dpi, while no other forms of cell death were detected. We noted that the inflammatory reactions and pathological injuries in the nasal mucosa were milder, indicating that apoptosis may play a role in promoting lower inflammatory reactions and milder pathological injuries and contribute to the generation of long-term viral replication in the nasal mucosa. Our study provides valuable insights into the differences in cellular mechanisms during SARS-CoV-2 infection and highlights the potential significance of apoptosis regulation in the respiratory mucosa for controlling viral replication.

Differential interferon responses to influenza A and B viruses in primary ferret respiratory epithelial cells.

Influenza B viruses (IBV) cocirculate with influenza A viruses (IAV) and cause periodic epidemics of disease, yet antibody and cellular responses following IBV infection are less well understood. Using the ferret model for antisera generation for influenza surveillance purposes, IAV resulted in robust antibody responses following infection, whereas IBV required an additional booster dose, over 85% of the time, to generate equivalent antibody titers. In this study, we utilized primary differentiated ferret nasal epithelial cells (FNECs) which were inoculated with IAV and IBV to study differences in innate immune responses which may result in differences in adaptive immune responses in the host. FNECs were inoculated with IAV (H1N1pdm09 and H3N2 subtypes) or IBV (B/Victoria and B/Yamagata lineages) and assessed for 72 h. Cells were analyzed for gene expression by quantitative real-time PCR, and apical and basolateral supernatants were assessed for virus kinetics and interferon (IFN), respectively. Similar virus kinetics were observed with IAV and IBV in FNECs. A comparison of gene expression and protein secretion profiles demonstrated that IBV-inoculated FNEC expressed delayed type-I/II IFN responses and reduced type-III IFN secretion compared to IAV-inoculated cells. Concurrently, gene expression of Thymic Stromal Lymphopoietin (TSLP), a type-III IFN-induced gene that enhances adaptive immune responses, was significantly downregulated in IBV-inoculated FNECs. Significant differences in other proinflammatory and adaptive genes were suppressed and delayed following IBV inoculation. Following IBV infection, ex vivo cell cultures derived from the ferret upper respiratory tract exhibited reduced and delayed innate responses which may contribute to reduced antibody responses in vivo.IMPORTANCEInfluenza B viruses (IBV) represent nearly one-quarter of all human influenza cases and are responsible for significant clinical and socioeconomic impacts but do not pose the same pandemic risks as influenza A viruses (IAV) and have thus received much less attention. IBV accounts for greater severity and deaths in children, and vaccine efficacy remains low. The ferret can be readily infected with human clinical isolates and demonstrates a similar course of disease and immune responses. IBV, however, generates lower antibodies in ferrets than IAV following the challenge. To determine whether differences in initial innate responses following infection may affect the development of robust adaptive immune responses, ferret respiratory tract cells were isolated, infected with IAV/IBV, and compared. Understanding the differences in the initial innate immune responses to IAV and IBV may be important in the development of more effective vaccines and interventions to generate more robust protective immune responses.

Human Nasal Organoids Model SARS-CoV-2 Upper Respiratory Infection and Recapitulate the Differential Infectivity of Emerging Variants.

The human upper respiratory tract, specifically the nasopharyngeal epithelium, is the entry portal and primary infection site of respiratory viruses. Productive infection of SARS-CoV-2 in the nasal epithelium constitutes the cellular basis of viral pathogenesis and transmissibility. Yet a robust and well-characterized in vitro model of the nasal epithelium remained elusive. Here we report an organoid culture system of the nasal epithelium. We derived nasal organoids from easily accessible nasal epithelial cells with a perfect establishment rate. The derived nasal organoids were consecutively passaged for over 6 months. We then established differentiation protocols to generate 3-dimensional differentiated nasal organoids and organoid monolayers of 2-dimensional format that faithfully simulate the nasal epithelium. Moreover, when differentiated under a slightly acidic pH, the nasal organoid monolayers represented the optimal correlate of the native nasal epithelium for modeling the high infectivity of SARS-CoV-2, superior to all existing organoid models. Notably, the differentiated nasal organoid monolayers accurately recapitulated higher infectivity and replicative fitness of the Omicron variant than the prior variants. SARS-CoV-2, especially the more transmissible Delta and Omicron variants, destroyed ciliated cells and disassembled tight junctions, thereby facilitating virus spread and transmission. In conclusion, we establish a robust organoid culture system of the human nasal epithelium for modeling upper respiratory infections and provide a physiologically-relevant model for assessing the infectivity of SARS-CoV-2 emerging variants. IMPORTANCE An in vitro model of the nasal epithelium is imperative for understanding cell biology and virus-host interaction in the human upper respiratory tract. Here we report an organoid culture system of the nasal epithelium. Nasal organoids were derived from readily accessible nasal epithelial cells with perfect efficiency and stably expanded for more than 6 months. The long-term expandable nasal organoids were induced maturation into differentiated nasal organoids that morphologically and functionally simulate the nasal epithelium. The differentiated nasal organoids adequately recapitulated the higher infectivity and replicative fitness of SARS-CoV-2 emerging variants than the ancestral strain and revealed viral pathogenesis such as ciliary damage and tight junction disruption. Overall, we established a human nasal organoid culture system that enables a highly efficient reconstruction and stable expansion of the human nasal epithelium in culture plates, thus providing a facile and robust tool in the toolbox of microbiologists.

Nasal symbiont Staphylococcus epidermidis restricts the cellular entry of influenza virus into the nasal epithelium.

Our recent study presented that human nasal commensal Staphylococcus epidermidis could potentiate antiviral immunity in the nasal mucosa through interferon-related innate responses. Here, we found that human nasal commensal S. epidermidis promoted protease-protease inhibitor balance in favor of the host and prevented influenza A virus (IAV) replication in the nasal mucosa and lungs. A relatively higher induction of Serpine1 exhibited in S. epidermidis-inoculated nasal epithelium and S. epidermidis-induced Serpine1 significantly decreased the expression of serine proteases. Furthermore, the transcription of urokinase plasminogen activator (uPA) and Serpine1 was biologically relevant in S. epidermidis-inoculated nasal epithelium, and the induction of uPA might be related to the sequential increase of Serpine1 in human nasal epithelium. Our findings reveal that human nasal commensal S. epidermidis manipulates the cellular environment lacking serine proteases in the nasal epithelium through Serpine1 induction and disturbs IAV spread to the lungs at the level of the nasal mucosa.

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity.

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.