Group Movement in Entomopathogenic Nematodes: Aggregation Levels Vary Based on Context.

Maintenance of an aggregated population structure implies within-species communication. In mixed-species environments, species-specific aggregations may reduce interspecific competition and promote coexistence. We studied whether movement and aggregation behavior of three entomopathogenic nematode species changed when isolated, as compared to mixed-species arenas. Movement and aggregation of Steinernema carpocapsae, S. feltiae and S. glaseri were assessed in sand. Each species demonstrated significant aggregation when alone. Mixed-species trials involved adding two species of nematodes, either combined in the center of the arena or at separate corners. While individual species became less aggregated than in single-species conditions when co-applied in the same location, they became more aggregated when applied in separate corners. This increased aggregation in separate-corner trials occurred even though the nematodes moved just as far when mixed together as they did when alone. These findings suggest that maintenance of multiple species within the same habitat is driven, at least in part, by species-specific signals that promote conspecific aggregation, and when the species are mixed (as occurs in some commercial formulations involving multiple EPN species), these signaling mechanisms are muddled.

Enhancing entomopathogenic nematode efficacy with Pheromones: A field study targeting the pecan weevil.

Consistent efficacy is required for entomopathogenic nematodes to gain wider adoption as biocontrol agents. Recently, we demonstrated that when exposed to nematode pheromone blends, entomopathogenic nematodes showed increased dispersal, infectivity, and efficacy under laboratory and greenhouse conditions. Prior to this study, the impact of entomopathogenic nematode-pheromone combinations on field efficacy had yet to be studied. Steinernema feltiae is a commercially available entomopathogenic nematode that has been shown to increase mortality in insect pests such as the pecan weevil Curculio caryae. In this study, the pecan weevil was used as a model system to evaluate changes in S. feltiae efficacy when treated with a partially purified ascaroside pheromone blend. Following exposure to the pheromone blend, the efficacy of S. feltiae significantly increased as measured with decreased C. caryae survival despite unfavorable environmental conditions. The results of this study highlight a potential new avenue for using entomopathogenic nematodes in field conditions. With increased efficacy, using entomopathogenic nematodes will reduce reliance on conventional management methods in pecan production, translating into more environmentally acceptable practices.

Movers and shakers: Do nematodes that move more invade more?

Entomopathogenic nematodes (EPNs) are roundworms that parasitize insects with the aid of symbiotic bacteria. These nematodes have been used both as model organisms and for biological control of pests. The specialized third stage of an EPN, known as an infective juvenile (IJ) must forage to find a host with strategies varying from species to species (cruising, ambushing, and intermediate). Some IJs move more than others to find a host, despite an increased risk of predation and desiccation. This hints at potential underlying benefits (e.g., increased invasion) for EPNs that move more. We assessed whether EPNs that moved farther down a soil column also exhibit higher levels of invasion when compared to nematodes that remained at or near their point of origin. We found that movers in the cruisier and intermediate species: Steinernema riobrave, Heterorhabditis bacteriophora, and H. indica had higher invasion rates compared to their counterparts that did not move. S. carpocapsae, an ambusher, did not exhibit invasion differences between EPNs that moved versus those that did not. For the three cruiser/intermediate EPNs we tested, our results support our hypothesis that EPNs that tend to move more enjoy related benefits such as increased invasion potential. Further studies are required to explore other parameters that may interact with movement. The results of this study can potentially be used to develop EPN strains that move more and invade more, and thus can potentially be more effective biological control agents.

Effectiveness of the live zoster vaccine during the 10 years following vaccination: real world cohort study using electronic health records.

OBJECTIVES: To assess the effectiveness of live zoster vaccine during more than 10 years after vaccination; and to describe methods for ascertaining vaccine effectiveness in the context of waning. DESIGN: Real world cohort study using electronic health records. SETTING: Kaiser Permanente Northern California, an integrated healthcare delivery system in the US, 1 January 2007 to 31 December 2018. POPULATION: More than 1.5 million people aged 50 years and older followed for almost 9.4 million person years. MAIN OUTCOME MEASURE: Vaccine effectiveness in preventing herpes zoster, postherpetic neuralgia, herpes zoster ophthalmicus, and admission to hospital for herpes zoster was assessed. Change in vaccine effectiveness by time since vaccination was examined using Cox regression with a calendar timeline. Time varying indicators were specified for each interval of time since vaccination (30 days to less than one year, one to less than two years, etc) and adjusted for covariates. RESULTS: Of 1 505 647 people, 507 444 (34%) were vaccinated with live zoster vaccine. Among 75 135 incident herpes zoster cases, 4982 (7%) developed postherpetic neuralgia, 4439 (6%) had herpes zoster ophthalmicus, and 556 (0.7%) were admitted to hospital for herpes zoster. For each outcome, vaccine effectiveness was highest in the first year after vaccination and decreased substantially over time. Against herpes zoster, vaccine effectiveness waned from 67% (95% confidence interval 65% to 69%) in the first year to 15% (5% to 24%) after 10 years. Against postherpetic neuralgia, vaccine effectiveness waned from 83% (78% to 87%) to 41% (17% to 59%) after 10 years. Against herpes zoster ophthalmicus, vaccine effectiveness waned from 71% (63% to 76%) to 29% (18% to 39%) during five to less than eight years. Against admission to hospital for herpes zoster, vaccine effectiveness waned from 90% (67% to 97%) to 53% (25% to 70%) during five to less than eight years. Across all follow-up time, overall vaccine effectiveness was 46% (45% to 47%) against herpes zoster, 62% (59% to 65%) against postherpetic neuralgia, 45% (40% to 49%) against herpes zoster ophthalmicus, and 66% (55% to 74%) against admission to hospital for herpes zoster. CONCLUSIONS: Live zoster vaccine was effective initially. Vaccine effectiveness waned substantially yet some protection remained 10 years after vaccination. After 10 years, protection was low against herpes zoster but higher against postherpetic neuralgia. TRIAL REGISTRATION: ClinicalTrials.gov number NCT01600079; EU PAS register number EUPAS17502.

Ingested histamine and serotonin interact to alter Anopheles stephensi feeding and flight behavior and infection with Plasmodium parasites.

Blood levels of histamine and serotonin (5-HT) are altered in human malaria, and, at these levels, we have shown they have broad, independent effects on Anopheles stephensi following ingestion by this invasive mosquito. Given that histamine and 5-HT are ingested together under natural conditions and that histaminergic and serotonergic signaling are networked in other organisms, we examined effects of combinations of these biogenic amines provisioned to A. stephensi at healthy human levels (high 5-HT, low histamine) or levels associated with severe malaria (low 5-HT, high histamine). Treatments were delivered in water (priming) before feeding A. stephensi on Plasmodium yoelii-infected mice or via artificial blood meal. Relative to effects of histamine and 5-HT alone, effects of biogenic amine combinations were complex. Biogenic amine treatments had the greatest impact on the first oviposition cycle, with high histamine moderating low 5-HT effects in combination. In contrast, clutch sizes were similar across combination and individual treatments. While high histamine alone increased uninfected A. stephensi weekly lifetime blood feeding, neither combination altered this tendency relative to controls. The tendency to re-feed 2 weeks after the first blood meal was altered by combination treatments, but this depended on mode of delivery. For blood delivery, malaria-associated treatments yielded higher percentages of fed females relative to healthy-associated treatments, but the converse was true for priming. Female mosquitoes treated with the malaria-associated combination exhibited enhanced flight behavior and object inspection relative to controls and healthy combination treatment. Mosquitoes primed with the malaria-associated combination exhibited higher mean oocysts and sporozoite infection prevalence relative to the healthy combination, with high histamine having a dominant effect on these patterns. Compared with uninfected A. stephensi, the tendency of infected mosquitoes to take a second blood meal revealed an interaction of biogenic amines with infection. We used a mathematical model to project the impacts of different levels of biogenic amines and associated changes on outbreaks in human populations. While not all outbreak parameters were impacted the same, the sum of effects suggests that histamine and 5-HT alter the likelihood of transmission by mosquitoes that feed on hosts with symptomatic malaria versus a healthy host.

Maternal SARS-CoV-2 vaccination and infant protection against SARS-CoV-2 during the first six months of life.

We examined the effectiveness of maternal vaccination against SARS-CoV-2 infection in 30,311 infants born at Kaiser Permanente Northern California from December 15, 2020, to May 31, 2022. Using Cox regression, the effectiveness of ≥2 doses of COVID-19 vaccine received during pregnancy was 84% (95% confidence interval [CI]: 66, 93), 62% (CI: 39, 77) and 56% (CI: 34,71) during months 0-2, 0-4 and 0- 6 of a child's life, respectively, in the Delta variant period. In the Omicron variant period, the effectiveness of maternal vaccination in these three age intervals was 21% (CI: -21,48), 14% (CI: -9,32) and 13% (CI: -3,26), respectively. Over the entire study period, the incidence of hospitalization for COVID-19 was lower during the first 6 months of life among infants of vaccinated mothers compared with infants of unvaccinated mothers (21/100,000 person-years vs. 100/100,000 person-years). Maternal vaccination was protective, but protection was lower during Omicron than during Delta. Protection during both periods decreased as infants aged.

Extended surveillance to assess safety of 9-valent human papillomavirus vaccine.

The safety of 9-valent HPV vaccine (9vHPV) has been established with regard to common and uncommon adverse events. However, investigation of rare and severe adverse events requires extended study periods to capture rare outcomes. This observational cohort study investigated the occurrence of three rare and serious adverse events following 9-valent human papillomavirus (9vHPV) vaccination compared to other vaccinations, in US individuals 9-26 years old, using electronic health record data from the Vaccine Safety Datalink (VSD). We searched for occurrences of Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), and stroke following 9vHPV vaccination from October 4, 2015, through January 2, 2021. We compared the risks of GBS, CIDP, and stroke following 9vHPV vaccination to risks of those outcomes following comparator vaccines commonly given to this age group (Td, Tdap, MenACWY, hepatitis A, and varicella vaccines) from January 1, 2007, through January 2, 2021. We observed 1.2 cases of stroke, 0.3 cases of GBS, and 0.1 cases of CIDP per 100,000 doses of 9vHPV vaccine. After observing more than 1.8 million doses of 9vHPV, we identified no statistically significant increase in risks associated with 9vHPV vaccination for any of these adverse events, either combined or stratified by age (9-17 years of age vs. 18-26 years of age) and sex (males vs. females). Our findings provide additional evidence supporting 9vHPV vaccine safety, over longer time frames and for more serious and rare adverse events.

Maternal SARS-CoV-2 Vaccination and Infant Protection Against SARS-CoV-2 During the First 6 Months of Life.

We examined the effectiveness of maternal vaccination against SARS-CoV-2 infection in 30,288 infants born at Kaiser Permanente Northern California from December 15, 2020, to May 31, 2022. Using Cox regression, the effectiveness of maternal vaccination was 85% (95% confidence interval [CI]: 67, 93), 64% (CI: 43, 78) and 57% (CI: 36,71) during the first 2, 4 and 6 months of life, respectively, in the Delta variant period. In the Omicron variant period, the effectiveness of maternal vaccination in these three age intervals was 22% (CI: -18,48), 14% (CI: -10,32) and 12% (CI: -4,26), respectively. Over the entire study period, the incidence of hospitalization for COVID-19 was lower during the first 6 months of life among infants of vaccinated mothers compared with infants of unvaccinated mothers (21/100,000 person-years vs. 100/100,000 person-years). Maternal vaccination was protective, but protection was lower during Omicron than during Delta. Protection during both periods decreased as infants aged.

Anopheles stephensi Feeding, Flight Behavior, and Infection With Malaria Parasites are Altered by Ingestion of Serotonin.

Approximately 3.4 billion people are at risk of malaria, a disease caused by infection with Plasmodium spp. parasites, which are transmitted by Anopheles mosquitoes. Individuals with severe falciparum malaria often exhibit changes in circulating blood levels of biogenic amines, including reduced serotonin or 5-hydroxytryptamine (5-HT), and these changes are associated with disease pathology. In insects, 5-HT functions as an important neurotransmitter for many behaviors and biological functions. In Anopheles stephensi, we show that 5-HT is localized to innervation in the head, thorax, and midgut, suggesting a gut-to-brain signaling axis that could support the effects of ingested 5-HT on mosquito biology and behavioral responses. Given the changes in blood levels of 5-HT associated with severe malaria and the key roles that 5-HT plays in insect neurophysiology, we investigated the impact of ingesting blood with healthy levels of 5-HT (1.5 µM) or malaria-associated levels of 5-HT (0.15 µM) on various aspects of A. stephensi biology. In these studies, we provisioned 5-HT and monitored fecundity, lifespan, flight behavior, and blood feeding of A. stephensi. We also assessed the impact of 5-HT ingestion on infection of A. stephensi with the mouse malaria parasite Plasmodium yoelii yoelii 17XNL and the human malaria parasite Plasmodium falciparum. Our data show that ingestion of 5-HT associated with severe malaria increased mosquito flight velocity and investigation of visual objects in response to host odor (CO2). 5-HT ingestion in blood at levels associated with severe malaria also increased the tendency to take a second blood meal 4 days later in uninfected A. stephensi. In mosquitoes infected with P. y. yoelii 17XNL, feeding tendency was decreased when midgut oocysts were present but increased when sporozoites were present. In addition to these effects, treatment of A. stephensi with 5-HT associated with severe malaria increased infection success with P. y. yoelii 17XNL compared to control, while treatment with healthy levels of 5-HT decreased infection success with P. falciparum. These changes in mosquito behavior and infection success could be used as a basis to manipulate 5-HT signaling in vector mosquitoes for improved control of malaria parasite transmission.

Safety of measles and pertussis-containing vaccines in children with autism spectrum disorders.

OBJECTIVES: To determine whether children aged 4-7 years with a diagnosis of autism spectrum disorders (ASD) were at increased risk of fever, febrile seizures, or emergency department (ED) visits following measles- or pertussis-containing vaccines compared with children without ASD. METHODS: The study included children born between 1995-2012, aged 4-7 years at vaccination, and members of six healthcare delivery systems within Vaccine Safety Datalink. We conducted self-controlled risk interval analyses comparing rates of outcomes in risk and control intervals within each group defined by ASD status, and then compared outcome rates between children with and without ASD, in risk and control intervals, by estimating difference-in-differences using logistic regressions. RESULTS: The study included 14,947 children with ASD and 1,650,041 children without ASD. After measles- or pertussis-containing vaccination, there were no differences in association between children with and without ASD for fever (ratio of rate ratio for measles-containing vaccine = 1.07, 95% CI 0.58-1.96; for pertussis-containing vaccine = 1.16, 95% CI 0.63-2.15) or ED visits (ratio of rate ratio for measles-containing vaccine = 1.11, 95% CI 0.80-1.54; for pertussis-containing vaccine = 0.87, 95% CI 0.59-1.28). Febrile seizures were rare. Pertussis-containing vaccines were associated with small increased risk of febrile seizures in children without ASD. CONCLUSION: Children with ASD were not at increased risk for fever or ED visits compared with children without ASD following measles- or pertussis-containing vaccines. These results may provide further reassurance that these vaccines are safe for all children, including those with ASD.

Infected host responses across entomopathogenic nematode phylogeny.

We used a phylogenetic framework to examine the relationship between entomopathogenic nematode (EPN) vertical dispersal and infectivity when EPNs are exposed to a mixture of compounds found in late-stage EPN-infected insect cadavers. EPNs from five phylogenetically close and distant species (Heterorhabditis bacteriophora, H. georgiana, H. megidis, H. indica and Steinernema feltiae) were exposed to cadaver macerate produced by their own species' infection and by H. bacteriophora infected hosts. We found that only three of the five species (H. bacteriophora, H. indica and S. feltiae) responded to exposure to their own macerate by increasing rates of dispersal. When we exposed all five species to a H. bacteriophora infected host macerate, we found that only H. bacteriophora responded by increasing dispersal, and that the most distantly related species (S. feltiae) essentially halted dispersal. These findings suggest that (1) responses to cadaver macerate vary, and (2) there may be a relationship between inherent dispersal rates and sensitivity to macerate exposure, as the most rapidly dispersing species (H. megidis) showed no response to macerate exposure.

Hypotonic-hyporesponsive Episodes After Diphtheria, Tetanus and Acellular Pertussis Vaccination.

BACKGROUND: Hypotonic-hyporesponsive episode (HHE) after whole cell pertussis vaccination is a known adverse event. Less is known about the risk of HHE after administration of acellular pertussis vaccines. METHODS: Using parental interviews, this study actively surveyed for HHE among infants after doses 1 and 2 of acellular pertussis vaccine. RESULTS: We interviewed the parents of 52,531 infants. HHE was reported at a rate of 22.8 per 100,000 doses (95% CI: 11.8-39.9) of acellular pertussis vaccine, approximately 45 episodes per 100,000 children. CONCLUSIONS: These rates are lower than HHE rates reported after whole cell pertussis vaccines and within the range of HHE rates reported in other studies of acellular pertussis vaccines.

Efficacy of Naturally Occurring and Commercial Entomopathogenic Nematodes Against Sugar Beet Wireworm (Coleoptera: Elateridae).

Wireworms are the larval stage of click beetles (Coleoptera: Elateridae), and some of their species are serious pests of many crops. In the present study, we evaluated the efficacy of naturally occurring and commercial entomopathogenic nematode species against the sugar beet wireworm, Limonius californicus (Mannerheim), in the laboratory. First, efficacies of Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) collected from an irrigated (S. feltiae-SSK) and a dryland (S. feltiae-SSC) field and the two commercial entomopathogenic nematode species, S. carpocapsae (Weiser) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), were examined. Efficacies of the two field-collected S. feltiae isolates were also compared against a commercial S. feltiae strain. In the first bioassay, S. feltiae-SSK caused 63.3% wireworm mortality, followed by 30% caused by S. carpocapsae, 23.3% by S. feltiae-SSC, and 6.7% by H.bacteriophora. In the second assay, S. feltiae-SSK killed 56.7% of the wireworms, ≈2.1- and ≈5.7-fold higher than S. feltiae-SSC and the commercial isolate, respectively.

Histamine Ingestion by Anopheles stephensi Alters Important Vector Transmission Behaviors and Infection Success with Diverse Plasmodium Species.

An estimated 229 million people worldwide were impacted by malaria in 2019. The vectors of malaria parasites (Plasmodium spp.) are Anopheles mosquitoes, making their behavior, infection success, and ultimately transmission of great importance. Individuals with severe malaria can exhibit significantly increased blood concentrations of histamine, an allergic mediator in humans and an important insect neuromodulator, potentially delivered to mosquitoes during blood-feeding. To determine whether ingested histamine could alter Anopheles stephensi biology, we provisioned histamine at normal blood levels and at levels consistent with severe malaria and monitored blood-feeding behavior, flight activity, antennal and retinal responses to host stimuli and lifespan of adult female Anopheles stephensi. To determine the effects of ingested histamine on parasite infection success, we quantified midgut oocysts and salivary gland sporozoites in mosquitoes infected with Plasmodium yoelii and Plasmodium falciparum. Our data show that provisioning An. stephensi with histamine at levels consistent with severe malaria can enhance mosquito behaviors and parasite infection success in a manner that would be expected to amplify parasite transmission to and from human hosts. Such knowledge could be used to connect clinical interventions by reducing elevated histamine to mitigate human disease pathology with the delivery of novel lures for improved malaria control.

A Risk Score to Predict Clostridioides difficile Infection.

BACKGROUND: Clostridioides difficile infection (CDI) is a major cause of severe diarrhea. In this retrospective study, we identified CDI risk factors by comparing demographic and clinical characteristics for Kaiser Permanente Northern California members ≥18 years old with and without laboratory-confirmed incident CDI. METHODS: We included these risk factors in logistic regression models to develop 2 risk scores that predict future CDI after an Index Date for Risk Score Assessment (IDRSA), marking the beginning of a period for which we estimated CDI risk. RESULTS: During May 2011 to July 2014, we included 9986 CDI cases and 2 230 354 members without CDI. The CDI cases tended to be older, female, white race, and have more hospitalizations, emergency department and office visits, skilled nursing facility stays, antibiotic and proton pump inhibitor use, and specific comorbidities. Using hospital discharge as the IDRSA, our risk score model yielded excellent performance in predicting the likelihood of developing CDI in the subsequent 31-365 days (C-statistic of 0.848). Using a random date as the IDRSA, our model also predicted CDI risk in the subsequent 31-365 days reasonably well (C-statistic 0.722). CONCLUSIONS: These results can be used to identify high-risk populations for enrollment in C difficile vaccine trials and facilitate study feasibility regarding sample size and time to completion.

Determining Which of Several Simultaneously Administered Vaccines Increase Risk of an Adverse Event.

INTRODUCTION: Childhood immunization schedules often involve multiple vaccinations per visit. When increased risk of an adverse event is observed after simultaneous (same-day) vaccinations, it can be difficult to ascertain which triggered the adverse event. This methods paper discusses a systematic process to determine which of the simultaneously administered vaccine(s) are most likely to have caused an observed increase in risk of an adverse event. METHODS: We use an example from the literature where excess risk of seizure was observed 1 day after vaccination, but same-day vaccination patterns made it difficult to discern which vaccine(s) may trigger the adverse event. We illustrate the systematic identification process using a simulation that retained the observed pattern of simultaneous vaccination in an empirical cohort of vaccinated children. We simulated "true" effects for diphtheria-tetanus-acellular pertussis (DTaP) and pneumococcal conjugate (PCV) on risk of seizure the day after vaccination. We varied the independent and interactive effects of vaccines (on the multiplicative scale). After applying the process to simulated data, we evaluated risk of seizure 1 day after vaccination in the empirical cohort. RESULTS: In all simulations, we were able to determine which vaccines contributed to excess risk. In the empirical data, we narrowed the association with seizure from all vaccines in the schedule to three likely candidates, DTaP, PCV, and/or Haemophilus influenzae type B (HiB) (p < 0.01, attributable risk when all three were administered together: five per 100,000). Disentangling their associations with seizure would require a larger sample or more variation in the combinations administered. When none of these three were administered, no excess risk was observed. CONCLUSION: The process outlined could provide valuable information on the magnitude of potential risk from individual and simultaneousvaccinations. Associations should be further investigated with independent data as well as biologically based, statistically independent hypotheses.

Conspecific and heterospecific pheromones stimulate dispersal of entomopathogenic nematodes during quiescence.

Ascaroside pheromones stimulate dispersal, a key nematode behavior to find a new food source. Ascarosides produced by entomopathogenic nematodes (EPNs) drive infective juvenile (IJ) emergence from consumed cadavers and dispersal in soil. Without ascarosides from host cadavers, Steinernema feltiae (EPN) reduce dispersal substantially. To determine whether other Steinernema spp. exhibit the same behavior, we compared S. feltiae and S. carpocapsae IJs without host cadaver pheromones. Unlike S. feltiae, S. carpocapsae IJs continued to disperse. However, S. carpocapsae IJs exhibited a temperature-dependent quiescent period. The IJ quiescent period increased at ≤20 °C but did not appear at ≥25 °C. Consistent with this, S. carpocapsae IJ quiescence increased from 30 min to 24 h at ≤20 °C over 60 days. The quiescent period was overcome by dispersal pheromone extracts of their own, other Steinernema spp. and Heterorhabditis spp. Furthermore, S. carpocapsae IJ ambush foraging associated behaviors (tail standing, waving, and jumping) were unaffected by the absence or presence of host cadaver pheromones. For S. feltiae, IJ dispersal declined at all temperatures tested. Understanding the interaction between foraging strategies and pheromone signals will help uncover molecular mechanisms of host seeking, pathogenicity and practical applications to improve the EPN's efficacy as biocontrol agents.

Retrospective study of the use of an influenza disease two-tiered classification system to characterize clinical severity in US children.

In children <5 years, influenza is associated with higher risk of serious disease and hospitalization when compared with other age groups. Influenza vaccination reduces the risk of influenza and vaccination may attenuate the severity of disease. Recent studies in Europe suggest that classifying influenza disease as mild versus moderate-to-severe (M-S) using a novel definition may be clinically significant. We retrospectively evaluated whether this M-S definition also characterized influenza severity in a cohort of US children. We included children <18 years at Kaiser Permanente Northern California with PCR-confirmed influenza during the 2013-2014 influenza season. We classified children as M-S if they had ≥1 symptom: fever >39°C, acute otitis media, lower respiratory tract infection (LRTI), or extra-pulmonary complications; otherwise, they were classified as mild. We used multivariable log-binomial models to assess whether M-S influenza disease was associated with increased healthcare utilization. Nearly half of the 1,105 influenza positive children were classified as M-S. Children 6-35 months had the highest proportion of M-S disease (35.1%), mostly due to LRTI (63.2%) and fever (44.6%). Children ≥6 months who had M-S disease were associated with a 1.6 to 2.8 times increased likelihood of having had an emergency department or any follow-up outpatient visits. Those who had M-S disease were associated with an increased likelihood of receiving antibiotics, with the highest likelihood in children 6-35 months (RR 9.0, 95% CI 4.1, 19.8). While more studies are needed, an influenza classification system may distinguish children with more clinically significant disease.