Advertisement

Influenza-like illness is associated with high pneumococcal carriage density in Malawian children

  • Author Footnotes
    1 TKN and AL contributed equally to this work.
    Tinashe K. Nyazika
    Correspondence
    Corresponding authors at: Malawi-Liverpool-Wellcome Trust Clinical Research Programme, University of Malawi College of Medicine, Blantyre, Malawi.
    Footnotes
    1 TKN and AL contributed equally to this work.
    Affiliations
    Malawi-Liverpool-Wellcome Trust Clinical Research Programme, University of Malawi College of Medicine, Blantyre, Malawi

    Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom

    Department of Pathology, College of Health Sciences, University of Malawi, Blantyre, Malawi
    Search for articles by this author
  • Author Footnotes
    1 TKN and AL contributed equally to this work.
    Alice Law
    Footnotes
    1 TKN and AL contributed equally to this work.
    Affiliations
    Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom
    Search for articles by this author
  • Todd D. Swarthout
    Affiliations
    Malawi-Liverpool-Wellcome Trust Clinical Research Programme, University of Malawi College of Medicine, Blantyre, Malawi

    Division of Infection and Immunity, NIHR Global Health Research Unit on Mucosal Pathogens, University College London, London, United Kingdom
    Search for articles by this author
  • Lusako Sibale
    Affiliations
    Malawi-Liverpool-Wellcome Trust Clinical Research Programme, University of Malawi College of Medicine, Blantyre, Malawi
    Search for articles by this author
  • Danielle ter Braake
    Affiliations
    Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom

    Department of Biomolecular Health Sciences, Division of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht, the Netherlands
    Search for articles by this author
  • Neil French
    Affiliations
    Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom
    Search for articles by this author
  • Robert S. Heyderman
    Affiliations
    Division of Infection and Immunity, NIHR Global Health Research Unit on Mucosal Pathogens, University College London, London, United Kingdom
    Search for articles by this author
  • Dean Everett
    Affiliations
    The Queens Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom
    Search for articles by this author
  • Author Footnotes
    2 AK, KCJ and DRN contributed equally to this work.
    Aras Kadioglu
    Footnotes
    2 AK, KCJ and DRN contributed equally to this work.
    Affiliations
    Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom
    Search for articles by this author
  • Author Footnotes
    2 AK, KCJ and DRN contributed equally to this work.
    Kondwani C. Jambo
    Correspondence
    Corresponding authors at: Malawi-Liverpool-Wellcome Trust Clinical Research Programme, University of Malawi College of Medicine, Blantyre, Malawi.
    Footnotes
    2 AK, KCJ and DRN contributed equally to this work.
    Affiliations
    Malawi-Liverpool-Wellcome Trust Clinical Research Programme, University of Malawi College of Medicine, Blantyre, Malawi

    Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
    Search for articles by this author
  • Author Footnotes
    2 AK, KCJ and DRN contributed equally to this work.
    Daniel R. Neill
    Footnotes
    2 AK, KCJ and DRN contributed equally to this work.
    Affiliations
    Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom
    Search for articles by this author
  • Author Footnotes
    1 TKN and AL contributed equally to this work.
    2 AK, KCJ and DRN contributed equally to this work.
Open AccessPublished:July 22, 2020DOI:https://doi.org/10.1016/j.jinf.2020.06.079

      Highlights

      • Influenza-like illness (ILI) in children is associated with high pneumococcal carriage density.
      • Children with ILI harboured more viral organisms than asymptomatic healthy children.
      • Children with ILI patients had higher IL-8 levels in nasal aspirates than asymptomatic healthy children.

      Summary

      Background

      High pneumococcal carriage density is a risk factor for invasive pneumococcal disease (IPD) and transmission, but factors that increase pneumococcal carriage density are still unclear.

      Methods

      We undertook a cross-sectional study to evaluate the microbial composition, cytokine levels and pneumococcal carriage densities in samples from children presenting with an influenza-like illness (ILI) and asymptomatic healthy controls (HC).

      Results

      The proportion of children harbouring viral organisms (Relative risk (RR) 1.4, p = 0.0222) or ≥ 4 microbes at a time (RR 1.9, p < 0.0001), was higher in ILI patients than HC. ILI patients had higher IL-8 levels in nasal aspirates than HC (median [IQR], 265.7 [0 – 452.3] vs. 0 [0 – 127.3] pg/ml; p = 0.0154). Having an ILI was associated with higher pneumococcal carriage densities compared to HC (RR 4.2, p < 0.0001).

      Conclusion

      These findings suggest that children with an ILI have an increased propensity for high pneumococcal carriage density. This could in part contribute to increased susceptibility to IPD and transmission in the community.

      Keywords

      Introduction

      Pneumococcal carriage is a prerequisite for disease and an important step for transmission. In Malawi, pneumococcus is known to be the major cause of meningitis and second cause of bacteraemia and community acquired pneumonia.
      • Everett D.B.
      • Mukaka M.
      • Denis B.
      • Gordon S.B.
      • Carrol E.D.
      • van Oosterhout J.J.
      • et al.
      Ten years of surveillance for invasive streptococcus pneumoniae during the era of antiretroviral scale-up and cotrimoxazole prophylaxis in Malawi.
      • Wall E.C.
      • Cartwright K.
      • Scarborough M.
      • Ajdukiewicz K.M.
      • Goodson P.
      • Mwambene J.
      • et al.
      High mortality amongst adolescents and adults with bacterial meningitis in sub-Saharan Africa: an analysis of 715 cases from Malawi.
      • Wall E.C.
      • B.Everett D.
      • Mukaka M.
      • Bar-Zeev N.
      • Feasey N.
      • Jahn A.
      et al. Bacterial meningitis in Malawian adults, adolescents, and children during the era of antiretroviral scale-up and Haemophilus influenzae type B vaccination, 2000-2012.
      • Aston S.J.
      • Ho A.
      • Jary H.
      • Huwa J.
      • Mitchell T.
      • Ibitoye S.
      • et al.
      Etiology and risk factors for mortality in an adult community-acquired pneumonia cohort in Malawi.
      • Carrol E.D.
      • Mankhambo L.A.
      • Jeffers G.
      • Parker D.
      • Guiver M.
      • Newland P.
      • et al.
      The diagnostic and prognostic accuracy of five markers of serious bacterial infection in Malawian children with signs of severe infection.
      The majority of invasive pneumococcal disease (IPD) cases are seen in children under the ages of 5 and carriage rates are known to increase with age between 0 and 5yrs.
      • Meiring S.
      • Cohen C.
      • Quan V.
      • de Gouveia L.
      • Feldman C.
      • Karstaedt A.
      • et al.
      HIV infection and the epidemiology of invasive pneumococcal disease (IPD) in South African adults and older children prior to the introduction of a pneumococcal conjugate vaccine (PCV).
      • Kalata N.L.
      • Nyazika T.K.
      • Swarthout T.D.
      • Everett D.
      • French N.
      • Heyderman R.S.
      • et al.
      Pneumococcal pneumonia and carriage in Africa before and after introduction of pneumococcal conjugate vaccines, 2000–2019: protocol for systematic review.
      • Richards L.
      • Ferreira D.M.
      • Miyaji E.N.
      • Andrew P.W.
      • Kadioglu A
      The immunising effect of pneumococcal nasopharyngeal colonisation; protection against future colonisation and fatal invasive disease.
      IPD cases have been shown to be on the decrease in Africa, including Malawi with uptake of the PCV.
      • Iroh Tam P.-.Y.
      • Thielen B.K.
      • Obaro S.K.
      • Brearley A.M.
      • Kaizer A.M.
      • Chu H.
      • et al.
      Childhood pneumococcal disease in Africa – a systematic review and meta-analysis of incidence, serotype distribution, and antimicrobial susceptibility.
      ,
      • Ngocho J.S.
      • Magoma B.
      • Olomi G.A.
      • Mahande M.J.
      • Msuya S.E.
      • de Jonge M.I.
      • et al.
      Effectiveness of pneumococcal conjugate vaccines against invasive pneumococcal disease among children under five years of age in Africa: a systematic review.
      Pneumococcal meningitis frequently seen in those between the ages of 6 –18 months, while bacteraemia is commonly seen amongst those aged 6 – 36 months and pneumococcal pneumonia occurring in children between 3 – 60 months of age.
      • Iroh Tam P.-.Y.
      • Thielen B.K.
      • Obaro S.K.
      • Brearley A.M.
      • Kaizer A.M.
      • Chu H.
      • et al.
      Childhood pneumococcal disease in Africa – a systematic review and meta-analysis of incidence, serotype distribution, and antimicrobial susceptibility.
      ,
      • Tan T.Q.
      Pediatric invasive pneumococcal disease in the United States in the era of pneumococcal conjugate vaccines.
      In Malawi, pneumococcal disease peaks during the colder and drier months, with serotypes 1, 6A/B, 14 and 23F being major causes of IPD in children.
      • Everett D.B.
      • Mukaka M.
      • Denis B.
      • Gordon S.B.
      • Carrol E.D.
      • van Oosterhout J.J.
      • et al.
      Ten years of surveillance for invasive streptococcus pneumoniae during the era of antiretroviral scale-up and cotrimoxazole prophylaxis in Malawi.
      ,
      • Carrol E.D.
      • Mankhambo L.A.
      • Jeffers G.
      • Parker D.
      • Guiver M.
      • Newland P.
      • et al.
      The diagnostic and prognostic accuracy of five markers of serious bacterial infection in Malawian children with signs of severe infection.
      ,
      • Carrol E.D.
      • Guiver M.
      • Nkhoma S.
      • Mankhambo L.A.
      • Marsh J.
      • Balmer P.
      • et al.
      High pneumococcal DNA loads are associated with mortality in Malawian children with invasive pneumococcal disease.
      • Cornick J.E.
      • Everett D.B.
      • Broughton C.
      • Denis B.B.
      • Banda D.L.
      • Carrol E.D.
      • et al.
      Invasive streptococcus pneumoniae in children, Malawi, 2004–2006.
      • Wall E.C.
      • Everett D.B.
      • Mukaka M.
      • Bar-Zeev N.
      • Feasey N.
      • Jahn A.
      • et al.
      Bacterial meningitis in Malawian adults, adolescents, and children during the era of antiretroviral scale-up and Haemophilus influenzae type b vaccination, 2000-2012.
      Pneumococcal carriage is common in children, with approximately 80% of under-fives in Malawi carrying at a given time,
      • Swarthout T.D.
      • Fronterre C.
      • Lourenço J.
      • Obolski U.
      • Gori A.
      • Bar-Zeev N.
      • et al.
      High residual carriage of vaccine-serotype Streptococcus pneumoniae after introduction of pneumococcal conjugate vaccine in Malawi.
      and is influenced by the microbial composition of the upper respiratory tract (URT), including viral co-infections.
      • Ding T.
      • Song T.
      • Zhou B.
      • Geber A.
      • Ma Y.
      • Zhang L.
      • et al.
      Microbial composition of the human nasopharynx varies according to influenza virus type and vaccination status.
      • Kumpitsch C.
      • Koskinen K.
      • Schöpf V.
      • Moissl-Eichinger C
      The microbiome of the upper respiratory tract in health and disease.
      • Schenck L.P.
      • Surette M.G.
      • Bowdish D.M.E
      Composition and immunological significance of the upper respiratory tract microbiota.
      • de Steenhuijsen Piters W.A.A.
      • Sanders E.A.M.
      • Bogaert D
      The role of the local microbial ecosystem in respiratory health and disease.
      • Thevaranjan N.
      • Whelan F.J.
      • Puchta A.
      • Ashu E.
      • Rossi L.
      • Surette M.G.
      • et al.
      Streptococcus pneumoniae colonization disrupts the microbial community within the upper respiratory tract of aging mice.
      • Heinsbroek E.
      • Tafatatha T.
      • Phiri A.
      • Swarthout T.D.
      • Alaerts M.
      • Crampin A.C.
      • et al.
      Pneumococcal carriage in households in Karonga District, Malawi, before and after introduction of 13-valent pneumococcal conjugate vaccination.
      Colonisation studies have demonstrated that microbe–microbe competition and synergy occur in the URT.
      • Schenck L.P.
      • Surette M.G.
      • Bowdish D.M.E
      Composition and immunological significance of the upper respiratory tract microbiota.
      ,
      • Hibbing M.E.
      • Fuqua C.
      • Parsek M.R.
      • Peterson S.B
      Bacterial competition: surviving and thriving in the microbial jungle.
      ,
      • Shak J.R.
      • Vidal J.E.
      • Klugman K.P
      Influence of bacterial interactions on pneumococcal colonization of the nasopharynx.
      For example, strong positive associations of pneumococcal nasopharyngeal carriage with rhinovirus, influenza virus, respiratory syncytial virus (RSV) and parainfluenza virus have been reported.
      • Grijalva C.G.
      • Griffin M.R.
      • Edwards K.M.
      • Williams J.V.
      • Gil A.I.
      • Verastegui H.
      • et al.
      The role of influenza and parainfluenza infections in nasopharyngeal pneumococcal acquisition among young children.
      • Howard L.M.
      • Zhu Y.
      • Griffin M.R.
      • Edwards K.M.
      • Williams J.V.
      • Gil A.I.
      • et al.
      Nasopharyngeal pneumococcal density during asymptomatic respiratory virus infection and risk for subsequent acute respiratory illness.
      • Morpeth S.C.
      • Munywoki P.
      • Hammitt L.L.
      • Bett A.
      • Bottomley C.
      • Onyango C.O.
      • et al.
      Impact of viral upper respiratory tract infection on the concentration of nasopharyngeal pneumococcal carriage among Kenyan children.
      Viral-induced local inflammation in the nasal mucosa has been implicated as an important factor that promotes pneumococcal carriage and transmission.
      • Vissers M.
      • Ahout I.M.
      • van den Kieboom C.H.
      • van der Gaast de Jongh C.E.
      • Groh L.
      • Cremers A.J.
      • et al.
      High pneumococcal density correlates with more mucosal inflammation and reduced respiratory syncytial virus disease severity in infants.
      • Short K.R.
      • Reading P.C.
      • Wang N.
      • Diavatopoulos D.A.
      • Wijburg O.L
      Increased nasopharyngeal bacterial titers and local inflammation facilitate transmission of streptococcus pneumoniae.
      • Jochems S.P.
      • Marcon F.
      • Carniel B.F.
      • Holloway M.
      • Mitsi E.
      • Smith E.
      • et al.
      Inflammation induced by influenza virus impairs human innate immune control of pneumococcus.
      Respiratory viral infections are associated with increased pneumococcal carriage density.
      • Howard L.M.
      • Zhu Y.
      • Griffin M.R.
      • Edwards K.M.
      • Williams J.V.
      • Gil A.I.
      • et al.
      Nasopharyngeal pneumococcal density during asymptomatic respiratory virus infection and risk for subsequent acute respiratory illness.
      ,
      • Vissers M.
      • Ahout I.M.
      • van den Kieboom C.H.
      • van der Gaast de Jongh C.E.
      • Groh L.
      • Cremers A.J.
      • et al.
      High pneumococcal density correlates with more mucosal inflammation and reduced respiratory syncytial virus disease severity in infants.
      ,
      • Murad C.
      • Dunne E.M.
      • Sudigdoadi S.
      • Fadlyana E.
      • Tarigan R.
      • Pell C.L.
      • et al.
      Pneumococcal carriage, density, and co-colonization dynamics: a longitudinal study in Indonesian infants.
      • Wolter N.
      • Tempia S.
      • Cohen C.
      • Madhi S.A.
      • Venter M.
      • Moyes J.
      • et al.
      High Nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia.
      • Vu H.T.T.
      • Yoshida L.M.
      • Suzuki M.
      • Nguyen H.A.T.
      • Nguyen C.D.L.
      • Nguyen A.T.T.
      • et al.
      Association between nasopharyngeal load of streptococcus pneumoniae, viral coinfection, and radiologically confirmed pneumonia in Vietnamese children.
      Specifically, a direct correlation between heightened mucosal inflammation and high pneumococcal carriage density has been reported in children with RSV infection.
      • Vissers M.
      • Ahout I.M.
      • van den Kieboom C.H.
      • van der Gaast de Jongh C.E.
      • Groh L.
      • Cremers A.J.
      • et al.
      High pneumococcal density correlates with more mucosal inflammation and reduced respiratory syncytial virus disease severity in infants.
      Inflammation in the nasal mucosa drives epithelial denudation, up-regulation of platelet-activating factor receptor (PAFr) and polymeric immunoglobulin receptor (pIgR) on epithelial cells, resulting in increased adherence of pneumococci and evasion of the nasal mucosal surface.
      • Jochems S.P.
      • Weiser J.N.
      • Malley R.
      • Ferreira D.M
      The immunological mechanisms that control pneumococcal carriage.
      • Moreno A.T.
      • Oliveira M.L.S.
      • Ho P.L.
      • Vadesilho C.F.M.
      • Palma G.M.P.
      • Ferreira J.M.C.
      • et al.
      Cross-reactivity of antipneumococcal surface protein C (PspC) antibodies with different strains and evaluation of inhibition of human complement factor H and secretory IgA binding via PspC.
      • Li Y.
      • Jin L.
      • Chen T
      The effects of secretory IgA in the mucosal immune system.
      • Maestro B.
      • Sanz J.
      Choline binding proteins from streptococcus pneumoniae: a dual role as enzybiotics and targets for the design of new antimicrobials.
      These observations highlight the important association, clinical and epidemiological, between virally induced inflammation in the URT and the pneumococcus.
      Influenza-like illness (ILI) is prevalent amongst children,
      • Rudge J.W.
      • Inthalaphone N.
      • Pavlicek R.
      • Paboriboune P.
      • Flaissier B.
      • Monidarin C.
      • et al.
      "Epidemiology and aetiology of influenza-like illness among households in metropolitan Vientiane, Lao PDR’: a prospective, community-based cohort study.
      • Muscatello D.
      • Barr M.
      • Thackway S.V.
      • MacIntyre C.R
      Epidemiology of influenza-like Illness during pandemic (H1N1) 2009, New South Wales, Australia.
      • Azziz-Baumgartner E.
      • Alamgir A.
      • Rahman M.
      • Homaira N.
      • Sohel B.M.
      • Sharker M.Y.
      • et al.
      Incidence of influenza-like illness and severe acute respiratory infection during three influenza seasons in Bangladesh, 2008–2010.
      with respiratory viral and bacterial pathogens reported as the key aetiological agents.
      • Hui D.S.
      • Woo J.
      • Hui E.
      • Foo A.
      • Ip M.
      • To K.-.W.
      • et al.
      Influenza-like illness in residential care homes: a study of the incidence, aetiological agents, natural history and health resource utilisation.
      ,
      • Taylor S.
      • Lopez P.
      • Weckx L.
      • Borja-Tabora C.
      • Ulloa-Gutierrez R.
      • Lazcano-Ponce E.
      • et al.
      Respiratory viruses and influenza-like illness: epidemiology and outcomes in children aged 6 months to 10 years in a multi-country population sample.
      Recognising that ILI is common in Malawi and that the aetiology of ILI induces inflammation which increases both risk of carriage and carriage density, we hypothesised that children with an influenza-like illness (ILI) residing in Malawi, are more likely to harbour higher pneumococcal carriage densities than asymptomatic healthy controls. We conducted a cross-sectional study in children, recruiting those with and without ILI symptoms. Here, we show that children with an ILI harboured more respiratory microbes per individual, exhibited higher levels of IL-8 in the nasal mucosa, and had increased likelihood of high pneumococcal carriage densities, than asymptomatic healthy controls. These findings have potential broader implications in the development of interventions to curb pneumococcal disease and transmission.

      Methods

      Study design and participants

      HIV-uninfected children were recruited into a comparative cross-sectional study from the Gateway clinic, a government primary healthcare facility and surrounding communities, between June and September 2017. All participants were from within Blantyre, a commercial city in the southern part of Malawi. Children between the ages of 1 and 10 years were conveniently sampled into two groups; children fulfilling the WHO influenza-like-illness (ILI) case definitions and community asymptomatic healthy controls. ILI was defined as having an acute respiratory illness (ARI) of recent onset (within 10 days of screening) manifested by fever (≥38.0°C), cough but not requiring hospitalisation.
      • Fitzner J.
      • Qasmieh S.
      • Mounts A.W.
      • Alexander B.
      • Besselaar T.
      • Briand S.
      • et al.
      Revision of clinical case definitions: influenza-like illness and severe acute respiratory infection.
      We excluded participants that had received antibiotics at least 14-days prior to recruitment into the study. HIV status was confirmed using a single rapid test kit, STAT-VIEW HIV 1/2 assay (ChemBio Diagnostic Systems Inc, USA), and willingness to test was part of the inclusion criteria. Written informed consent were obtained from parents/guardians for children under the age of 8 and assents for children aged 8 and above. Ethical approval was obtained from the local ethics committee COMREC (P.07/16/1990) and University of Liverpool (Ref:0783).

      Nasopharyngeal swab

      A nylon nasal swab (FLOQSwabs™, Copan Diagnostics, USA) was inserted into the nostril to the nasopharynx, a depth equal to the outer ear and left in place for 2–3 s before slow removal with a rotating movement. Two different swabs were used to sample both nares, using the same method. The swabs were placed in 1 ml skim milk-tryptone-glucose-glycerol (STGG) media and transported to the Malawi-Liverpool-Wellcome Trust laboratories for processing.

      Nasal aspirates

      Nasopharyngeal secretions were aspirated through a disposable sterile catheter connected to a mucus trap and vacuum source. The catheter was inserted into a nostril, directed posteriorly and toward the opening of the external ear to allow extraction from the nasopharynx. Suction (100–120 mmHg for children; 120–150 mmHg for adults) was applied whilst the catheter was slowly withdrawn using a rotating motion. Mucus from the other nostril was collected with the same catheter, using the same method. After mucus collection from both nostrils, the catheter was flushed with 2 ml sterile phosphate buffered saline. Mucus aspirates were vortexed, aliquoted and frozen at −80 °C within 1-h of collection.

      Streptococcus pneumoniae culture and lytA PCR

      Nasopharyngeal (NP) swabs, after collection and prior to initial freezing, were vortexed and 100 µl of the suspension cultured on sheep blood agar with gentamicin (SBG; 7% sheep blood agar, 5µL gentamicin/mL) overnight at 37°C and 5% CO2. Plates showing no S. pneumoniae growth were incubated for a further 24-h before being reported as negative. S. pneumoniae was identified by colony morphology and optochin disc (Oxoid, Basingstoke, UK) susceptibility and bile solubility test was done on isolates with zone diameter <14 mm. Nasopharyngeal pneumococcal carriage was detected via qPCR, targeting S. pneumoniae virulence gene lytA and semi-quantitative microbiological culture (Miles and Misra), which was positively correlated with regards to detection and density determination and has been shown previously.
      • Gritzfeld J.F.
      • Cremers A.J.H.
      • Ferwerda G.
      • Ferreira D.M.
      • Kadioglu A.
      • Hermans P.W.M.
      • et al.
      Density and duration of experimental human pneumococcal carriage.
      Samples were classified as positive for pneumococci by the presence of growth by culture and/or if lytA qPCR signals were >10 DNA copies, <40 cycles.

      Multiplex real-time PCR detection of 33 respiratory microbes

      Total nucleic acids were extracted from 300 µl aliquots of the nasal aspirate by manual extraction using the QIAamp DNA Mini Kit (Qiagen, Manchester, UK), according to manufacturer's instructions. 10 µl of total nucleic acid extracted was used for the Fast-track Diagnostics (FTDⓇ; Luxembourg) multiplex. The multiplex real-time PCR uses the principle of the TaqMan technology to detect pathogen genes. The kit detects 33 respiratory microbes namely, influenza A virus; influenza B virus; influenza C virus; influenza A(H1N1) virus (swine-lineage); human parainfluenza viruses 1, 2, 3 and 4; human coronaviruses NL63, 229E, OC43 and HKU1; human metapneumoviruses A/B; human rhinovirus; human respiratory syncytial viruses A/B; human adenovirus; enterovirus; human parechovirus; human bocavirus; Pneumocystis jirovecii; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Streptococcus pneumoniae; Haemophilus influenzae B; Staphylococcus aureus; Moraxella catarrhalis; Bordetella spp.; Klebsiella pneumoniae; Legionella pneumophila/longbeachae; Salmonella spp.; Haemophilus influenzae. All targeted microorganisms in a sample with a cycle threshold value of >10 and ≤37 were considered positive for that pathogen.

      Cytokine measurements

      IL-8, IL-10 and interferon (IFN)-γ levels in nasal aspirates were quantified using Quantikine ELISA kits (R&D systems, Minneapolis, USA), according to manufacturer instructions. Levels of active TGF-β within nasal aspirates was determined using luciferase-reporting transformed mink lung epithelial cells (MLEC) stably transfected with the expression construct p800neoLUC, containing a plasminogen activator inhibitor-1 (PAI-1) promoter fused to the firefly luciferase reporter gene. MLEC cells were cultured, and TGF-β quantified from aspirates, as previously described.
      • Abe M.
      • Harpel J.G.
      • Metz C.N.
      • Nunes I.
      • Loskutoff D.J.
      • Rifkin D.B
      An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.
      ,
      • Neill D.R.
      • Coward W.R.
      • Gritzfeld J.F.
      • Richards L.
      • Garcia-Garcia F.J.
      • Dotor J.
      • et al.
      Density and duration of pneumococcal carriage is maintained by transforming growth factor β1 and T regulatory cells.

      Statistical analyses

      We performed statistical analyses and graphical presentation using GraphPad Prism 8 (GraphPad Software, USA). Statistically significant differences between groups were determined using the Mann-Whitney U test, and the data is summarised as median with interquartile ranges (IQR). Categorical data were summarised as proportions and compared using the Fisher's exact test, with effect size reported as Relative Risk.

      Results

      Demographic characteristics of study population

      A total of 47 HIV-uninfected children were recruited, comprising of 21 asymptomatic healthy children and 26 influenza-like illness outpatients (Table 1). The asymptomatic healthy controls were predominantly female (71.4%), while those with an influenza-like illness were predominantly male (75.0%). Furthermore, the ILI group was relatively younger (1 – 4yrs, 80.8 vs. 47.6%, p = 0.029), and the age eligible children were more likely to have received the 13-valent pneumococcal conjugate vaccine (PCV13) (84.6 vs. 52.4%, p = 0.025). PCV13 was rolled out in Malawi in 2011.
      Table 1Demographic characteristics of study population in relation to ILI status.
      VariableAll participants
      All participants were HIV negative.


      (n=47)
      Healthy controls

      (n=21)
      ILI
      ILI was defined as Influenza-like-illness according to WHO syndromic case-definitions.


      (n=26)
      p value
      Fishers Exact test of categorical data; t-test for continuous data.
      Age, years, median (IQR)

      3 (2 – 6)


      5 (2 – 6)


      2 (1 – 4)


      0.078
      1–<53110 (47.6)21 (80.8)0.029
      ≥5–101611 (52.4)5 (19.2)
      Sex0.245
      Female29 (61.7)15 (71.4)14 (53.8)
      PCV-vaccinated
      PCV pneumococcal conjugate vaccine – all 3 routine scheduled doses.
      0.025
      Yes33 (70.2)11 (52.4)22 (84.6)
      a All participants were HIV negative.
      b ILI was defined as Influenza-like-illness according to WHO syndromic case-definitions.
      c Fishers Exact test of categorical data; t-test for continuous data.
      d PCV pneumococcal conjugate vaccine – all 3 routine scheduled doses.

      ILI patients exhibit increased propensity for harbouring a viral organism and/or multiple microbes in nasal aspirates

      Using an FTD multiplex real-time PCR, we detected viruses, bacteria and fungi in the nasal aspirates among the ILIs and asymptomatic healthy controls (Fig. 1A). ILI patients were more likely than healthy controls to have a virus in their nasal aspirate (Relative risk (RR), 1.4 [95% CI 1.069–1.953], p = 0.0222). The prevalence of ILI-associated pathogens including influenza virus, human rhinovirus and enterovirus was 11.5%, 11.5% and 23% in ILI patients, and 4.7%, 4.7% and 14.3% in healthy controls, respectively. Furthermore, ILI patients were more likely to have greater than four microbes per individual compared to the asymptomatic healthy controls (RR, 1.9 [95% CI 1.427–2.395], p < 0.0001) (Fig. 1B). These findings suggest that children with ILI are not only more likely to harbour a viral organism but also multiple respiratory microbes within the nasal mucosa.
      Fig. 1
      Fig. 1Prevalence of respiratory pathogens in nasal aspirates of ILI patients and healthy controls. A multiplex real-time PCR was used to detect 33 respiratory pathogens in nasal aspirates of children with an influenza-like illness and healthy controls. A) Prevalence of detectable viral, bacterial and fungal pathogens in nasal aspirates. B) Prevalence of multiple pathogens in nasal aspirates of children with an influenza-like illness compared to healthy controls. Chi-square tests was used to compare the two groups. ILI= Influenza-like illness (cases) (n = 26); HC= Healthy child (control) (n = 21).

      ILI patients harbour higher levels of pro-inflammatory IL-8 in the URT than asymptomatic healthy controls

      Having established that an ILI is associated with increased likelihood of detecting a viral organism and/or multiple microbes, we investigated the cytokine microenvironment in the upper respiratory tract (URT) by measuring the levels of pro- and anti-inflammatory cytokines in nasal aspirates of ILI patients and healthy controls. The levels of pro-inflammatory cytokine IL-8 were higher in nasal aspirates from children presenting with ILI, compared to healthy controls (median [IQR], 265.7 [0 – 452.3] vs. 0 [0 – 127.3] pg/ml; p = 0.0154) (Fig. 2A). Furthermore, children with ILI were more likely to have detectable IL-8 than asymptomatic healthy controls (RR, 1.9 [95% CI 1.46 – 2.72], p < 0.0001). In contrast, the levels of IFN-γ, IL-10 and active TGF-β in nasal aspirates were similar between children presenting with ILI and healthy controls (all p > 0.05; Fig. 2B-D). Collectively, these findings suggest that children presenting with ILI likely have ongoing URT inflammation.
      Fig. 2
      Fig. 2Cytokine levels in nasal aspirates of ILI patients and healthy controls. Levels of IL-8, FN-γ and IL-10 were measured by ELISA in nasal aspirates from ILI patients and healthy controls. Active TGF-β was also measured using a luciferase-reporting transformed mink lung epithelial cell assay. A) Concentration of IL-8 in nasal aspirates in healthy children and those with an ILI. B) Concentration of IFN-γ in nasal aspirates in healthy children and those with an ILI. C) Concentration of IL-10 in nasal aspirates in healthy children and those with an ILI. D) Concentration of active TGF-β in nasal aspirates in healthy children and those with an ILI. Data were analysed using Mann Whitney test. The bars represent median. ILI was defined as Influenza-like-illness according to WHO syndromic case-definitions. ILI= Influenza-like illness (cases) (n = 26); HC= Healthy child (control) (n = 21).

      ILI patients exhibit higher likelihood of greater pneumococcal carriage densities than asymptomatic healthy controls

      Following observations that ILI patients were likely to have an inflamed URT mucosa, we determined whether this could impact pneumococcal carriage dynamics. Combining the pneumococcal carriage detection data from culture and lytA PCR on NP swabs the prevalence of carriage was higher in ILI patients than asymptomatic healthy controls (84.6 vs. 57.1%, p = 0.037) (Fig. 3A). There was a strong concordance between culture and lytA PCR (Sensitivity 0.9310, Specificity 0.7647) (Table 2). The difference in prevalence between the two groups is likely due to age differences,
      • Roca A.
      • Bottomley C.
      • Hill P.C.
      • Bojang A.
      • Egere U.
      • Antonio M.
      • et al.
      Effect of age and vaccination with a pneumococcal conjugate vaccine on the density of pneumococcal nasopharyngeal carriage.
      however the ages of carriage positives were similar in both groups (median, range; 2 (1–9) vs. 3 (1 −9), p = 0.4476). There was also no difference in median bacterial density between ILI patients and asymptomatic healthy controls (3.24 [2.23 – 3.50] vs. 3.89 [3.06 – 4.37]; p = 0.1115) (Fig. 3B). There was a strong correlation between pneumococcal density as measured by lytA PCR and culture (r = 0.6800, p < 0.0001) (Supplementary Fig. 1). However, further analysis revealed that children with ILI were more likely to harbour bacterial densities of ≥ 104 cfu/ml than asymptomatic healthy controls (RR, 4.2 [95% CI 2.396 – 7.919], p < 0.0001) (Fig. 3C). Taken together, these findings suggest that having an ILI is associated with increased propensity for high pneumococcal carriage density.
      Fig. 3
      Fig. 3Point prevalence of Streptococcus pneumoniae nasopharyngeal carriage and density in ILI patients and healthy controls. Quantitative PCR targeting lytA gene and culture were used to determine pneumococcal carriage rates and carriage densities in nasal swabs. FTD multiplex PCR was used to detect 33 respiratory organisms in nasal aspirates. A) S. pneumoniae carriage prevalence among healthy controls and ILI children based on aggregation of culture and lytA PCR. Data were analysed using a Chi-square test. HC (n = 21), ILI (n = 26). B) S. pneumoniae carriage densities between ILI patients and healthy controls. Data were analysed using Mann Whitney test. The bars represent median. HC (n = 11), ILI (n = 16) C). S. pneumoniae carriage densities [(medium/low carriage (< 104 copies) vs. high carriage ≥104)] between ILI patients and healthy controls. Data was analysed using a Fischer's exact test. HC (n = 11), ILI (n = 16). ILI was defined as Influenza-like-illness according to WHO syndromic case-definitions. ILI= Influenza-like illness (cases); HC= Healthy child (control).
      Table 2Relationship between culture and LytA PCR.
      Culture PositiveCulture NegativeTotalDiagnostic accuracy


      LytA Positive


      27 (93.1%)


      4 (23.5%)


      31 (67.4%)
      Sensitivity

      0.9310 (95% CI 0.7804 – 0.9877)

      Positive predictive value

      0.8710 (95% CI 0.7115 – 0.9487)


      LytA Negative


      2 (6.9%)


      13 (76.5%)


      15 (22.6%)
      Specificity

      0.7647 (95% CI 0.5274 – 0.9044)

      Negative predictive value

      0.8667 (95% CI 0.6212 – 0.9763)
      Total29 (63.0%)17 (37.0%)46 (100%)

      Discussion

      This study describes the relationship between having an influenza-like illness (ILI) in children and pneumococcal carriage. Pneumococcus is the leading cause of pneumonia and invasive bacterial infections in all ages, with the greatest incidence being in children under the age of 5.
      • Azzari C.
      • Cortimiglia M.
      • Nieddu F.
      • Moriondo M.
      • Indolfi G.
      • Mattei R.
      • et al.
      Pneumococcal serotype distribution in adults with invasive disease and in carrier children in Italy: should we expect herd protection of adults through infants’ vaccination?.
      • Troeger C.
      • Forouzanfar M.
      • Rao P.C.
      • Khalil I.
      • Brown A.
      • Swartz S.
      • et al.
      Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the Global Burden of Disease Study 2015.
      • Wahl B.
      • O'Brien K.L.
      • Greenbaum A.
      • Majumder A.
      • Liu L.
      • Chu Y.
      • et al.
      Burden of streptococcus pneumoniae and Haemophilus influenzae type b disease in children in the era of conjugate vaccines: global, regional, and national estimates for 2000–15.
      In this study, ILI patients had higher likelihood of greater pneumococcal carriage densities than asymptomatic healthy controls. This is consistent with studies that have shown that viral infection driven local inflammation in the nasal mucosa is associated with increased pneumococcal carriage load.
      • Howard L.M.
      • Zhu Y.
      • Griffin M.R.
      • Edwards K.M.
      • Williams J.V.
      • Gil A.I.
      • et al.
      Nasopharyngeal pneumococcal density during asymptomatic respiratory virus infection and risk for subsequent acute respiratory illness.
      • Morpeth S.C.
      • Munywoki P.
      • Hammitt L.L.
      • Bett A.
      • Bottomley C.
      • Onyango C.O.
      • et al.
      Impact of viral upper respiratory tract infection on the concentration of nasopharyngeal pneumococcal carriage among Kenyan children.
      • Vissers M.
      • Ahout I.M.
      • van den Kieboom C.H.
      • van der Gaast de Jongh C.E.
      • Groh L.
      • Cremers A.J.
      • et al.
      High pneumococcal density correlates with more mucosal inflammation and reduced respiratory syncytial virus disease severity in infants.
      ,
      • Jochems S.P.
      • Marcon F.
      • Carniel B.F.
      • Holloway M.
      • Mitsi E.
      • Smith E.
      • et al.
      Inflammation induced by influenza virus impairs human innate immune control of pneumococcus.
      ,
      • Wolter N.
      • Tempia S.
      • Cohen C.
      • Madhi S.A.
      • Venter M.
      • Moyes J.
      • et al.
      High Nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia.
      In line with the role of IL-8 in maintaining an inflammatory microenvironment at the site of infection,
      • Weight C.M.
      • Venturini C.
      • Sherin P.
      • Jochems S.P.
      • Reiné J.
      • Nikolaou E.
      • et al.
      Microinvasion by streptococcus pneumoniae induces epithelial innate immunity during colonisation at the human mucosal surface.
      having an ILI was associated with high levels of IL-8. Inflammation leads to increased adherence of pneumococci to the nasal mucosal surface,
      • Jochems S.P.
      • Weiser J.N.
      • Malley R.
      • Ferreira D.M
      The immunological mechanisms that control pneumococcal carriage.
      ,
      • Adamou J.E.
      • Wizemann T.M.
      • Barren P.
      • Langermann S
      Adherence of streptococcus pneumoniae to human bronchial epithelial cells (BEAS-2B).
      but also clearance of pneumococci from the URT.
      • van Rossum A.M.C.
      • Lysenko E.S.
      • Weiser J.N
      Host and bacterial factors contributing to the clearance of colonization by streptococcus pneumoniae in a murine model.
      Recent work from the experimental human challenge model has demonstrated that prior nasal infection with live attenuated influenza virus (LAIV) induces inflammation and impairs innate immune function, leading to increased pneumococcal carriage densities.
      • Jochems S.P.
      • Marcon F.
      • Carniel B.F.
      • Holloway M.
      • Mitsi E.
      • Smith E.
      • et al.
      Inflammation induced by influenza virus impairs human innate immune control of pneumococcus.
      It is therefore plausible that local inflammation in the URT during an ILI episode promotes a conducive environment for pneumococcal survival and replication.
      On the other hand, the high pneumococcal carriage density could be one of the aetiological factors for the development of an influenza-like illness. ILI in this study was defined by clinical presentation, following the WHO guidelines.
      • Fitzner J.
      • Qasmieh S.
      • Mounts A.W.
      • Alexander B.
      • Besselaar T.
      • Briand S.
      • et al.
      Revision of clinical case definitions: influenza-like illness and severe acute respiratory infection.
      It is well known that ILI may be caused by both viral and bacterial infections, of which S. pneumoniae is a potential aetiological agent.
      • Hui D.S.
      • Woo J.
      • Hui E.
      • Foo A.
      • Ip M.
      • To K.-.W.
      • et al.
      Influenza-like illness in residential care homes: a study of the incidence, aetiological agents, natural history and health resource utilisation.
      In our cohort, we observed a high propensity for respiratory viral organisms in the ILI patients compared to healthy controls. One of the common pathogens associated with ILI is influenza virus.
      • Rudge J.W.
      • Inthalaphone N.
      • Pavlicek R.
      • Paboriboune P.
      • Flaissier B.
      • Monidarin C.
      • et al.
      "Epidemiology and aetiology of influenza-like illness among households in metropolitan Vientiane, Lao PDR’: a prospective, community-based cohort study.
      ,
      • Renois F.
      • Talmud D.
      • Huguenin A.
      • Moutte L.
      • Strady C.
      • Cousson J.
      • et al.
      Rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-PCR DNA microarray systems.
      The prevalence of influenza virus in the patients with ILI was 11.5%, and this is consistent with previous studies in Malawi that reported influenza prevalence between 8.3% and 13.7% among patients with severe acute respiratory illness (SARI) and community cases of ILI.
      • Peterson I.
      • Bar-Zeev N.
      • Kennedy N.
      • Ho A.
      • Newberry L.
      • SanJoaquin M.A.
      • et al.
      Respiratory virus–associated severe acute respiratory illness and viral clustering in Malawian children in a setting with a high prevalence of HIV infection, malaria, and malnutrition.
      ,
      • Ho A.
      • Aston S.J.
      • Jary H.
      • Mitchell T.
      • Alaerts M.
      • Menyere M.
      • et al.
      Impact of human immunodeficiency virus on the burden and severity of influenza illness in Malawian adults: a prospective cohort and parallel case-control study.
      The ILI patients were more likely to have harbour more than four potentially pathogenic respiratory organisms per individual in their nasal aspirate compared to healthy controls. This highlights the complexity of identifying the underlying infective aetiology of ILI in children in high transmission and disease-burdened settings.
      Nevertheless, the lack of data on the definitive aetiological agents in our ILI patients constitutes a study limitation. It is clear from our study and others
      • Hui D.S.
      • Woo J.
      • Hui E.
      • Foo A.
      • Ip M.
      • To K.-.W.
      • et al.
      Influenza-like illness in residential care homes: a study of the incidence, aetiological agents, natural history and health resource utilisation.
      ,
      • Assane D.
      • Makhtar C.
      • Abdoulaye D.
      • Amary F.
      • Djibril B.
      • Amadou D.
      • et al.
      Viral and bacterial etiologies of acute respiratory infections among children under 5 years in Senegal.
      ,
      • Setiawaty V.
      • Puspa K.D.
      • Adam K.
      • Pangesti K.N
      Upper respiratory tract bacteria in influenza-like illness cases in Indonesia using multiplex PCR method.
      that the majority of ILI cases likely not caused by influenza but by other viruses or bacteria. Defining the aetiology of ILI in children in high transmission and disease-burdened settings like Malawi should be a research priority, as it could help in development of potential interventions to curb transmission of potentially pathogenic respiratory organisms in the community. The other limitation of study is the imbalance in our two study groups in terms of age and gender, which potentially skewed our pneumococcal carriage prevalence data. However, pneumococcal carriage density was unlikely impacted by age in our study, since the median age of the carriage positive children was similar between the two groups. Furthermore, we found similar carriage rates among the PCV-13 age eligible children, but we were not able to serotype the pneumococcus in order to elucidate its impact on vaccine serotypes.
      Unexpectedly, we found relatively high prevalence of other pathogens, including notably Salmonella and pneumocystis jirovecii. For the bacterial genus Salmonella, we were not able to identify the organisms to species level in order to differentiate between pathogenic and commensal organisms. In Malawi, at least 10.3% of bloodstream infections have been reported to be caused by S. typhi.
      • Musicha P.
      • Cornick J.E.
      • Bar-Zeev N.
      • French N.
      • Masesa C.
      • Denis B.
      • et al.
      Trends in antimicrobial resistance in bloodstream infection isolates at a large urban hospital in Malawi (1998–2016): a surveillance study.
      Whilst, Pneumocystis jirovecii is estimated between 6.8 –51%, to be the causative agent of children presenting with acute lower respiratory infection in Africa.
      • Lanaspa M.
      • O'Callaghan-Gordo C.
      • Machevo S.
      • Madrid L.
      • Nhampossa T.
      • Acácio S.
      • et al.
      High prevalence of pneumocystis jirovecii pneumonia among Mozambican children <5 years of age admitted to hospital with clinical severe pneumonia.
      • Graham S.M.
      • Mtitimila E.I.
      • Kamanga H.S.
      • Walsh A.L.
      • Hart C.A.
      • Molyneux M.E
      Clinical presentation and outcome of pneumocystis carinii pneumonia in Malawian children.
      • Morrow B.M.
      • Samuel C.M.
      • Zampoli M.
      • Whitelaw A.
      • Zar H.J
      Pneumocystis pneumonia in South African children diagnosed by molecular methods.
      • Morris A.
      • Lundgren J.D.
      • Masur H.
      • Walzer P.D.
      • Hanson D.L.
      • Frederick T.
      • et al.
      Current epidemiology of pneumocystis pneumonia.
      In conclusion, we have demonstrated that having an ILI is associated with increased propensity for high pneumococcal carriage density in children. These findings have potential implications in the development of interventions to curb pneumococcal disease and transmission, since high-density pneumococcal carriage is an important risk factor for pneumonia and transmission.

      Declaration of Competing Interest

      We declare no competing interests.

      Author contributions

      AL, AK, KCJ and DRN conceived the study. TKN, AL, TDS, NF, RH, DE, AK, KCJ, DRN contributed reagents. TKN, AL, LSK, DtB, KCJ and DRN performed the experiments. TKN, AL, KCJ, DRN performed the data analysis. TKN and AL drafted the manuscript. TKN, AL, TDS, LSK, NF, RH, DE, AK, KCJ and DRN critically revised successive versions of the paper and approved the final version.

      Acknowledgements

      The authors thank all study participants, the clinical team (Mrs Mercy Juma, Mr Andrew Sigoloti, Ms Chifundo Kondoni) and supporting staff of Malawi-Liverpool-Wellcome Trust Clinical Research Programme and Queen Elizabeth Central Hospital for their support and cooperation during the study.

      Funding

      This work was supported by the CDC through a cooperative agreement (grant 5U01CK000146–04 ). TKN is supported by training grant awarded as part of the Wellcome Strategic award number 101113/Z/13/Z084 to The Malawi-Liverpool-Wellcome Trust Clinical Research Programme . KCJ was supported by the Wellcome (UK) through an Intermediate Fellowship number 105831/Z/14/Z. DRN is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant number 204457/Z/16/Z ). AL and AK were supported by a UK Medical Research Council Programme Grant ( MR/P011284/1 ). RSH, NF and TS are supported by the National Institute for Health Research (NIHR) Global Health Research Unit on Mucosal Pathogens using UK aid from the UK Government. The views expressed in this publication are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care”.

      Appendix. Supplementary materials

      References

        • Everett D.B.
        • Mukaka M.
        • Denis B.
        • Gordon S.B.
        • Carrol E.D.
        • van Oosterhout J.J.
        • et al.
        Ten years of surveillance for invasive streptococcus pneumoniae during the era of antiretroviral scale-up and cotrimoxazole prophylaxis in Malawi.
        PLoS ONE. 2011; 6: e17765
        • Wall E.C.
        • Cartwright K.
        • Scarborough M.
        • Ajdukiewicz K.M.
        • Goodson P.
        • Mwambene J.
        • et al.
        High mortality amongst adolescents and adults with bacterial meningitis in sub-Saharan Africa: an analysis of 715 cases from Malawi.
        PLoS ONE. 2013; 8: e69783
        • Wall E.C.
        • B.Everett D.
        • Mukaka M.
        • Bar-Zeev N.
        • Feasey N.
        • Jahn A.
        et al. Bacterial meningitis in Malawian adults, adolescents, and children during the era of antiretroviral scale-up and Haemophilus influenzae type B vaccination, 2000-2012.
        Clin Infect Dis. 2014; 58: e137-e145
        • Aston S.J.
        • Ho A.
        • Jary H.
        • Huwa J.
        • Mitchell T.
        • Ibitoye S.
        • et al.
        Etiology and risk factors for mortality in an adult community-acquired pneumonia cohort in Malawi.
        Am J Respir Crit Care Med. 2019; 200: 359-369
        • Carrol E.D.
        • Mankhambo L.A.
        • Jeffers G.
        • Parker D.
        • Guiver M.
        • Newland P.
        • et al.
        The diagnostic and prognostic accuracy of five markers of serious bacterial infection in Malawian children with signs of severe infection.
        PLoS ONE. 2009; 4: e6621
        • Meiring S.
        • Cohen C.
        • Quan V.
        • de Gouveia L.
        • Feldman C.
        • Karstaedt A.
        • et al.
        HIV infection and the epidemiology of invasive pneumococcal disease (IPD) in South African adults and older children prior to the introduction of a pneumococcal conjugate vaccine (PCV).
        PLoS ONE. 2016; 11e0149104
        • Kalata N.L.
        • Nyazika T.K.
        • Swarthout T.D.
        • Everett D.
        • French N.
        • Heyderman R.S.
        • et al.
        Pneumococcal pneumonia and carriage in Africa before and after introduction of pneumococcal conjugate vaccines, 2000–2019: protocol for systematic review.
        BMJ Open. 2019; 9e030981
        • Richards L.
        • Ferreira D.M.
        • Miyaji E.N.
        • Andrew P.W.
        • Kadioglu A
        The immunising effect of pneumococcal nasopharyngeal colonisation; protection against future colonisation and fatal invasive disease.
        Immunobiology. 2010; 215: 251-263
        • Iroh Tam P.-.Y.
        • Thielen B.K.
        • Obaro S.K.
        • Brearley A.M.
        • Kaizer A.M.
        • Chu H.
        • et al.
        Childhood pneumococcal disease in Africa – a systematic review and meta-analysis of incidence, serotype distribution, and antimicrobial susceptibility.
        Vaccine. 2017; 35: 1817-1827
        • Ngocho J.S.
        • Magoma B.
        • Olomi G.A.
        • Mahande M.J.
        • Msuya S.E.
        • de Jonge M.I.
        • et al.
        Effectiveness of pneumococcal conjugate vaccines against invasive pneumococcal disease among children under five years of age in Africa: a systematic review.
        PLoS ONE. 2019; 14e0212295
        • Tan T.Q.
        Pediatric invasive pneumococcal disease in the United States in the era of pneumococcal conjugate vaccines.
        Clin Microbiol Rev. 2012; 25: 409-419
        • Carrol E.D.
        • Guiver M.
        • Nkhoma S.
        • Mankhambo L.A.
        • Marsh J.
        • Balmer P.
        • et al.
        High pneumococcal DNA loads are associated with mortality in Malawian children with invasive pneumococcal disease.
        Pediatr Infect Dis J. 2007; 26: 416-422
        • Cornick J.E.
        • Everett D.B.
        • Broughton C.
        • Denis B.B.
        • Banda D.L.
        • Carrol E.D.
        • et al.
        Invasive streptococcus pneumoniae in children, Malawi, 2004–2006.
        Emerg Infect Dis. 2011; 17: 1107-1109
        • Wall E.C.
        • Everett D.B.
        • Mukaka M.
        • Bar-Zeev N.
        • Feasey N.
        • Jahn A.
        • et al.
        Bacterial meningitis in Malawian adults, adolescents, and children during the era of antiretroviral scale-up and Haemophilus influenzae type b vaccination, 2000-2012.
        Clin Infect Dis Off Publ Infect Dis Soc Am. 2014; 58: e137-e145
        • Swarthout T.D.
        • Fronterre C.
        • Lourenço J.
        • Obolski U.
        • Gori A.
        • Bar-Zeev N.
        • et al.
        High residual carriage of vaccine-serotype Streptococcus pneumoniae after introduction of pneumococcal conjugate vaccine in Malawi.
        Nat Commun. 2020; 11: 2222
        • Ding T.
        • Song T.
        • Zhou B.
        • Geber A.
        • Ma Y.
        • Zhang L.
        • et al.
        Microbial composition of the human nasopharynx varies according to influenza virus type and vaccination status.
        MBio. 2019; 10 (e01296-19)
        • Kumpitsch C.
        • Koskinen K.
        • Schöpf V.
        • Moissl-Eichinger C
        The microbiome of the upper respiratory tract in health and disease.
        BMC Biol. 2019; 17: 87
        • Schenck L.P.
        • Surette M.G.
        • Bowdish D.M.E
        Composition and immunological significance of the upper respiratory tract microbiota.
        FEBS Lett. 2016; 590: 3705-3720
        • de Steenhuijsen Piters W.A.A.
        • Sanders E.A.M.
        • Bogaert D
        The role of the local microbial ecosystem in respiratory health and disease.
        Philos Trans R Soc B Biol Sci. 2015; 37020140294
        • Thevaranjan N.
        • Whelan F.J.
        • Puchta A.
        • Ashu E.
        • Rossi L.
        • Surette M.G.
        • et al.
        Streptococcus pneumoniae colonization disrupts the microbial community within the upper respiratory tract of aging mice.
        Infect Immun. 2016; 84: 906-916
        • Heinsbroek E.
        • Tafatatha T.
        • Phiri A.
        • Swarthout T.D.
        • Alaerts M.
        • Crampin A.C.
        • et al.
        Pneumococcal carriage in households in Karonga District, Malawi, before and after introduction of 13-valent pneumococcal conjugate vaccination.
        Vaccine. 2018; 36: 7369-7376
        • Hibbing M.E.
        • Fuqua C.
        • Parsek M.R.
        • Peterson S.B
        Bacterial competition: surviving and thriving in the microbial jungle.
        Nat Rev Microbiol. 2010; 8: 15-25
        • Shak J.R.
        • Vidal J.E.
        • Klugman K.P
        Influence of bacterial interactions on pneumococcal colonization of the nasopharynx.
        Trends Microbiol. 2013; 21: 129-135
        • Grijalva C.G.
        • Griffin M.R.
        • Edwards K.M.
        • Williams J.V.
        • Gil A.I.
        • Verastegui H.
        • et al.
        The role of influenza and parainfluenza infections in nasopharyngeal pneumococcal acquisition among young children.
        Clin Infect Dis. 2014; 58: 1369-1376
        • Howard L.M.
        • Zhu Y.
        • Griffin M.R.
        • Edwards K.M.
        • Williams J.V.
        • Gil A.I.
        • et al.
        Nasopharyngeal pneumococcal density during asymptomatic respiratory virus infection and risk for subsequent acute respiratory illness.
        Emerg Infect Dis. 2019; 25: 2040-2047
        • Morpeth S.C.
        • Munywoki P.
        • Hammitt L.L.
        • Bett A.
        • Bottomley C.
        • Onyango C.O.
        • et al.
        Impact of viral upper respiratory tract infection on the concentration of nasopharyngeal pneumococcal carriage among Kenyan children.
        Sci Rep. 2018; 8: 1-8
        • Vissers M.
        • Ahout I.M.
        • van den Kieboom C.H.
        • van der Gaast de Jongh C.E.
        • Groh L.
        • Cremers A.J.
        • et al.
        High pneumococcal density correlates with more mucosal inflammation and reduced respiratory syncytial virus disease severity in infants.
        BMC Infect Dis. 2016; 16: 129
        • Short K.R.
        • Reading P.C.
        • Wang N.
        • Diavatopoulos D.A.
        • Wijburg O.L
        Increased nasopharyngeal bacterial titers and local inflammation facilitate transmission of streptococcus pneumoniae.
        mBio. 2012; 3 (e00255-12)
        • Jochems S.P.
        • Marcon F.
        • Carniel B.F.
        • Holloway M.
        • Mitsi E.
        • Smith E.
        • et al.
        Inflammation induced by influenza virus impairs human innate immune control of pneumococcus.
        Nat Immunol. 2018; 19: 1299-1308
        • Murad C.
        • Dunne E.M.
        • Sudigdoadi S.
        • Fadlyana E.
        • Tarigan R.
        • Pell C.L.
        • et al.
        Pneumococcal carriage, density, and co-colonization dynamics: a longitudinal study in Indonesian infants.
        Int J Infect Dis. 2019; 86: 73-81
        • Wolter N.
        • Tempia S.
        • Cohen C.
        • Madhi S.A.
        • Venter M.
        • Moyes J.
        • et al.
        High Nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia.
        J Infect Dis. 2014; 210: 1649-1657
        • Vu H.T.T.
        • Yoshida L.M.
        • Suzuki M.
        • Nguyen H.A.T.
        • Nguyen C.D.L.
        • Nguyen A.T.T.
        • et al.
        Association between nasopharyngeal load of streptococcus pneumoniae, viral coinfection, and radiologically confirmed pneumonia in Vietnamese children.
        Pediatr Infect Dis J. 2011; 30: 11-18
        • Jochems S.P.
        • Weiser J.N.
        • Malley R.
        • Ferreira D.M
        The immunological mechanisms that control pneumococcal carriage.
        PLoS Pathog. 2017; 13e1006665
        • Moreno A.T.
        • Oliveira M.L.S.
        • Ho P.L.
        • Vadesilho C.F.M.
        • Palma G.M.P.
        • Ferreira J.M.C.
        • et al.
        Cross-reactivity of antipneumococcal surface protein C (PspC) antibodies with different strains and evaluation of inhibition of human complement factor H and secretory IgA binding via PspC.
        Clin Vaccine Immunol. 2012; 19: 499-507
        • Li Y.
        • Jin L.
        • Chen T
        The effects of secretory IgA in the mucosal immune system.
        BioMed Res Int. 2020; 2020: 1-6
        • Maestro B.
        • Sanz J.
        Choline binding proteins from streptococcus pneumoniae: a dual role as enzybiotics and targets for the design of new antimicrobials.
        Antibiotics. 2016; 5: 21
        • Rudge J.W.
        • Inthalaphone N.
        • Pavlicek R.
        • Paboriboune P.
        • Flaissier B.
        • Monidarin C.
        • et al.
        "Epidemiology and aetiology of influenza-like illness among households in metropolitan Vientiane, Lao PDR’: a prospective, community-based cohort study.
        PLoS ONE. 2019; 14e0214207
        • Muscatello D.
        • Barr M.
        • Thackway S.V.
        • MacIntyre C.R
        Epidemiology of influenza-like Illness during pandemic (H1N1) 2009, New South Wales, Australia.
        Emerg Infect Dis. 2011; 17: 1240-1247
        • Azziz-Baumgartner E.
        • Alamgir A.
        • Rahman M.
        • Homaira N.
        • Sohel B.M.
        • Sharker M.Y.
        • et al.
        Incidence of influenza-like illness and severe acute respiratory infection during three influenza seasons in Bangladesh, 2008–2010.
        Bull World Health Organ. 2012; 90: 12-19
        • Hui D.S.
        • Woo J.
        • Hui E.
        • Foo A.
        • Ip M.
        • To K.-.W.
        • et al.
        Influenza-like illness in residential care homes: a study of the incidence, aetiological agents, natural history and health resource utilisation.
        Thorax. 2008; 63: 690-697
        • Taylor S.
        • Lopez P.
        • Weckx L.
        • Borja-Tabora C.
        • Ulloa-Gutierrez R.
        • Lazcano-Ponce E.
        • et al.
        Respiratory viruses and influenza-like illness: epidemiology and outcomes in children aged 6 months to 10 years in a multi-country population sample.
        J Infect. 2017; 74: 29-41
        • Fitzner J.
        • Qasmieh S.
        • Mounts A.W.
        • Alexander B.
        • Besselaar T.
        • Briand S.
        • et al.
        Revision of clinical case definitions: influenza-like illness and severe acute respiratory infection.
        Bull World Health Organ. 2018; 96: 122-128
        • Gritzfeld J.F.
        • Cremers A.J.H.
        • Ferwerda G.
        • Ferreira D.M.
        • Kadioglu A.
        • Hermans P.W.M.
        • et al.
        Density and duration of experimental human pneumococcal carriage.
        Clin Microbiol Infect. 2014; 20: O1145-O1151
        • Abe M.
        • Harpel J.G.
        • Metz C.N.
        • Nunes I.
        • Loskutoff D.J.
        • Rifkin D.B
        An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.
        Anal Biochem. 1994; 216: 276-284
        • Neill D.R.
        • Coward W.R.
        • Gritzfeld J.F.
        • Richards L.
        • Garcia-Garcia F.J.
        • Dotor J.
        • et al.
        Density and duration of pneumococcal carriage is maintained by transforming growth factor β1 and T regulatory cells.
        Am J Respir Crit Care Med. 2014; 189: 1250-1259
        • Roca A.
        • Bottomley C.
        • Hill P.C.
        • Bojang A.
        • Egere U.
        • Antonio M.
        • et al.
        Effect of age and vaccination with a pneumococcal conjugate vaccine on the density of pneumococcal nasopharyngeal carriage.
        Clin Infect Dis. 2012; 55: 816-824
        • Azzari C.
        • Cortimiglia M.
        • Nieddu F.
        • Moriondo M.
        • Indolfi G.
        • Mattei R.
        • et al.
        Pneumococcal serotype distribution in adults with invasive disease and in carrier children in Italy: should we expect herd protection of adults through infants’ vaccination?.
        Hum Vaccines Immunother. 2016; 12: 344-350
        • Troeger C.
        • Forouzanfar M.
        • Rao P.C.
        • Khalil I.
        • Brown A.
        • Swartz S.
        • et al.
        Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the Global Burden of Disease Study 2015.
        Lancet Infect Dis. 2017; 17: 1133-1161
        • Wahl B.
        • O'Brien K.L.
        • Greenbaum A.
        • Majumder A.
        • Liu L.
        • Chu Y.
        • et al.
        Burden of streptococcus pneumoniae and Haemophilus influenzae type b disease in children in the era of conjugate vaccines: global, regional, and national estimates for 2000–15.
        Lancet Glob Health. 2018; 6: e744-e757
        • Weight C.M.
        • Venturini C.
        • Sherin P.
        • Jochems S.P.
        • Reiné J.
        • Nikolaou E.
        • et al.
        Microinvasion by streptococcus pneumoniae induces epithelial innate immunity during colonisation at the human mucosal surface.
        Nat Commun. 2019; 10: 1-15
        • Adamou J.E.
        • Wizemann T.M.
        • Barren P.
        • Langermann S
        Adherence of streptococcus pneumoniae to human bronchial epithelial cells (BEAS-2B).
        Infect Immun. 1998; 66: 820-822
        • van Rossum A.M.C.
        • Lysenko E.S.
        • Weiser J.N
        Host and bacterial factors contributing to the clearance of colonization by streptococcus pneumoniae in a murine model.
        Infect Immun. 2005; 73: 7718-7726
        • Renois F.
        • Talmud D.
        • Huguenin A.
        • Moutte L.
        • Strady C.
        • Cousson J.
        • et al.
        Rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-PCR DNA microarray systems.
        J Clin Microbiol. 2010; 48: 3836-3842
        • Peterson I.
        • Bar-Zeev N.
        • Kennedy N.
        • Ho A.
        • Newberry L.
        • SanJoaquin M.A.
        • et al.
        Respiratory virus–associated severe acute respiratory illness and viral clustering in Malawian children in a setting with a high prevalence of HIV infection, malaria, and malnutrition.
        J Infect Dis. 2016; 214: 1700-1711
        • Ho A.
        • Aston S.J.
        • Jary H.
        • Mitchell T.
        • Alaerts M.
        • Menyere M.
        • et al.
        Impact of human immunodeficiency virus on the burden and severity of influenza illness in Malawian adults: a prospective cohort and parallel case-control study.
        Clin Infect Dis. 2018; 66: 865-876
        • Assane D.
        • Makhtar C.
        • Abdoulaye D.
        • Amary F.
        • Djibril B.
        • Amadou D.
        • et al.
        Viral and bacterial etiologies of acute respiratory infections among children under 5 years in Senegal.
        Microbiol Insights. 2018; 11: 1-5
        • Setiawaty V.
        • Puspa K.D.
        • Adam K.
        • Pangesti K.N
        Upper respiratory tract bacteria in influenza-like illness cases in Indonesia using multiplex PCR method.
        HSJI. 2013; 4: 83-86
        • Musicha P.
        • Cornick J.E.
        • Bar-Zeev N.
        • French N.
        • Masesa C.
        • Denis B.
        • et al.
        Trends in antimicrobial resistance in bloodstream infection isolates at a large urban hospital in Malawi (1998–2016): a surveillance study.
        Lancet Infect Dis. 2017; 17: 1042-1052
        • Lanaspa M.
        • O'Callaghan-Gordo C.
        • Machevo S.
        • Madrid L.
        • Nhampossa T.
        • Acácio S.
        • et al.
        High prevalence of pneumocystis jirovecii pneumonia among Mozambican children <5 years of age admitted to hospital with clinical severe pneumonia.
        Clin Microbiol Infect. 2015; 21 (e9-1018.e15): 1018
        • Graham S.M.
        • Mtitimila E.I.
        • Kamanga H.S.
        • Walsh A.L.
        • Hart C.A.
        • Molyneux M.E
        Clinical presentation and outcome of pneumocystis carinii pneumonia in Malawian children.
        Lancet. 2000; 355: 369-373
        • Morrow B.M.
        • Samuel C.M.
        • Zampoli M.
        • Whitelaw A.
        • Zar H.J
        Pneumocystis pneumonia in South African children diagnosed by molecular methods.
        BMC Res Notes. 2014; 7: 26
        • Morris A.
        • Lundgren J.D.
        • Masur H.
        • Walzer P.D.
        • Hanson D.L.
        • Frederick T.
        • et al.
        Current epidemiology of pneumocystis pneumonia.
        Emerg Infect Dis. 2004; 10: 1713-1720