The importance of considering BACTERIAL LOAD in OTITIS MEDIA research

Transcription

1 The importance of considering BACTERIAL LOAD in OTITIS MEDIA research Heidi Smith-Vaughan, Robyn Marsh, Michael Binks, Mirjam Kaestli, Peter S Morris, Amanda J Leach

2 OM in Indigenous children OM is endemic in children in remote Indigenous communities High NP carriage rates of OM pathogens (~80%) Prevalence of perforation ~20% (<2.5yo) Many will progress to CSOM AOM CSOM

3 Microbes in the nasopharynx and ear discharge In Northern Territory Indigenous children Ear discharge 3 Nasopharyngeal microbes Pseudomonas aeruginosa Staphylococcus aureus Alloiococcus otitidis 4 Proteus Fungi (Aspergillus) Yeast Nasopharynx 1 Haemophilus influenzae Streptococcus pneumoniae Moraxella catarrhalis Staphylococcus aureus Respiratory viruses 2 1. Leach et al PIDJ: 13(11). 2. Binks et al BMC Infect Dis Leach et al PIDJ; 27(8). 4. Marsh et al Submitted.

4 Bacterial load why it s important

5 Probability of suppurative OM NP Bacterial load is associated with OM severity As bacterial load in the NP increases, so does the probability of acute otitis media 70% 60% 50% 40% 30% 20% 10% S. pneumoniae H. influenzae M. catarrhalis Sp + Hi + Mc ӿ Total bacteria 0% 10^0 1 10^1 10^2 2 10^3 3 10^4 4 10^5 5 10^6 6 10^7 7 Num Number of of bacteria Aboriginal and non-aboriginal children (18-36mo) Smith-Vaughan et al. BMC Ear Nose Throat Disord. 2006;6:10

6 Geometric mean load (cells/ml) Bacterial load at onset of OM For Indigenous children, interventions need to be early in life NASOPHARYNGEAL SWABS 1.E+07 1.E E+05 1.E+04 1.E+03 1.E+02 1.E+01 1.E+00 9/10 with OM All OM * * * S. pneumoniae H. influenzae M. catarrhalis S. aureus Total bacterial load 0-3 weeks 3-6 weeks 6-13 weeks 10 Children with no OM 0-3 weeks of age S. pneumoniae H. influenzae M. catarrhalis S. aureus Total bacterial lo Smith-Vaughan et al. Int J Pediatr Otorhinolaryngol :57-61

7 Geometric mean load (cells/ml) Bacterial load at onset of OM 1.E+07 1.E E+05 1.E+04 1.E+03 1.E+02 1.E+01 1.E+00 NASOPHARYNGEAL SWABS * * * * * ** * 9/10 with OM All OM S. pneumoniae H. influenzae M. catarrhalis S. aureus Total bacterial load 0-3 weeks 3-6 weeks 6-13 weeks No OM 9/10 OM S. pneumoniae H. influenzae M. catarrhalis S. aureus Total bacterial load Smith-Vaughan et al. Int J Pediatr Otorhinolaryngol :57-61

8 Related examples from literature 1. S. pneumoniae load in NP associated with pneumonia 1 2. S. pneumoniae load in serum associated with pneumonia severity 2 3. Bacterial load positively associated with inflammation 3 1. Vu et al. Pediatr Infect Dis (1): Werno et al. J Med Microbiol Epub ahead of print. 3. Hill et al Am J Med 109:

9 An ear discharge case study To explore bacterial load in children with acute otitis media with perforation (AOMwiP)

10 Methods 55 NT Indigenous children aged 4 55 months AOMwip <2% pars tensa and <6 weeks duration no ABs in previous week paired NP and ED swabs Culture optimised for Spn and Hi qpcr to estimate bacterial load

11 % of nasopharyngeal swabs Prevalence of bacteria in the nasopharynx 100 NASOPHARYNX Culture PCR Hi Spn Mc None 1 only 2 only all 3 0 Hi Spn Mc None 1 onl y 2 onl y

12 % of nasopharyngeal swabs Prevalence of bacteria in the nasopharynx 100 NASOPHARYNX Culture 40 PCR 20 0 Hi Spn Mc None 1 only 2 only all 3

13 % of nasopharyngeal swabs Prevalence of bacteria in ear discharge 100 EAR DISCHARGE Culture PCR 20 0 Hi Spn Mc None 1 only 2 only all 3

14 Geometric mean load (cells/ml) BACTERIAL LOAD in the nasopharynx PCR ON NASOPHARYNGEAL SWABS 1x10 8 1x10 7 9,000,000 1x ,000 1x10 5 ** ** 1x10 4 1x10 3 1x Total Bacterial Load

15 Geometric mean load (cells/ml) Bacterial load in ear discharge 1x10 8 1x10 7 PCR ON EAR DISCHARGE SWABS ** 1x10 6 1x10 5 1x10 4 1x10 3 1x ** ** Total Bacterial Load

16 Geometric mean load (cells/ml) Comparing paired NP and ear discharge swabs (pos swabs only) 1x10 8 1x10 7 ** 1x10 6 1x10 5 * NS * 1x10 4 1x10 3 NP ED 1x Hi Spn Mc TBL Total Bacterial Load

17 Proportion of the total bacterial load

18 Main findings OM has a complex pathogenesis...and bacterial loads can help us understand the potential importance of various pathogens in OM H. Influenzae is a more dominant pathogen in ear discharge from children with AOMwip than Spn or Mc...but there are more than the usual suspects (Robyn Marsh presentation)

19 Using qpcr

20 Acknowledgments We would like to thank the families who participated in these studies and for their continued support of our research. We are grateful to the NHMRC (Australia) and Channel 7 Children s Research Foundation for research support. Menzies Ear Health Research Team and Child Health Laboratory Team for clinical specimens, clinical data, and laboratory support. The OMOZ2012 Organisers!

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22 Using qpcr...1 DNA extraction consistency decide on a method (incl. enzymatic vs bead- beating pretreatments) and stick to it. Store and prepare standards in TE (stability) Apply strict criteria for accepting results R 2 must be > or = 0.99 Efficiency must be > or = 0.80 Replicates are <or =0.5 Cq different Negative control. If probe-free PCR, check the melt curve Internal control LOD - lowest standard concentration at which specific amplification was detected in at least 95% of replicates LOQ lowest standard concentration at which replicates consistently within 0.5Cq POSBLOQ median value between LOQ and LOQ

23 Background viruses in the NP In Northern Territory Indigenous children (to 2 years of age) Nasopharyngeal viruses Human rhinovirus (38%) Human polyomavirus (14%) Human adenovirus (13%)* Human bocavirus (8%) Human coronavirus (4%) Binks et al BMC Infect Dis. 11:161

24 Background virology of OM -2 Human Adenovirus and AOM

25 The critical step and critical people The clinical staff!!! Critical for meaningful results Good specimens No contamination (eg. of tubes or swabs) Frozen (LN2 shipper or dry ice) soon after collection and not thawed

26 Plotting the individual specimens