Andrew LawrenceMicrobiology and Infectious Diseases Dept.SA Pathology at Women’s and Children’s HospitalAdelaide, SANeisse...
Neisseria and related spp. ofhuman origin• Neissseria meningitidis• Neisseria gonorrhoeae• N. lactamica, N. cinerea, N.pol...
Neisseria spp.• Characteristic diplococcus (bean shaped)• Inhabit mucous membranes of warmblooded hosts• Aerobic, non-moti...
Gram’s Stain of CSF ShowingGN Diplococci
Neisseria spp:Clinical Significance• NG always considered a pathogenirrespective of site of isolation• Other Neiss. Inhabi...
Specimens and culture mediaMCMeningitis/bacteraemiaCSF, skinlesions, blood,nasopharynxSelective/non-selectiveMcatPneumonia...
Neisseria spp: ID• Sugar fermentation (GC = glu pos onlywhile MC = glu, malt pos)• Growth on selective agar• Latex/ co-agg...
N.meningitidis cell wallProtects against host defences13 Serogroups (ctrA diagnostic test)Porins (porA and porB) - serosub...
Meningococcal Disease. Editor Cartwright K 1995Neisseria meningitidis showing blebs50% lipooligosaccharide50% OMPs, phosph...
Meningococcal purpuric rash
IMD and the Military• 1812 US Civil War - meningitis outbreaks• Turn of century - bacteriologic confirmation• Increased ra...
IMD and the MilitaryMeningococcal meningitis in the US Army, 1910-1946Evolution of the Meningococcus, Vedros editor
IMD and the Military• WW1 (US)- IMD at all training campsMost crowded had most disease• During WW1 5839 cases of IMD and 2...
IMD and the Military• Throughout WW2 13,922 cases of IMD• 559 deaths• Cases fatality rate 4% - sulphadiazineprophylaxsis• ...
IMD and the Military• Large epidemics in winters 1917-18, 1942-43• Smaller secondary peaks approx 9 months later• New recr...
IMD and the Military• Post WW2 sulphadiazine used forprophylaxis but in 1963 (Vietnam era) sdzresistance developed• Serogr...
Typical meningococcal petechial rash
Meningococcal sepsis - digital gangrene
Meningococcal Disease inAustralia• July 1900 - Feb 1901 large outbreakSydney 69% mortality• Post WW1 increased rates• 1915...
The annual number of deaths from meningococcal disease wasobtained from the compilation of mortality data collected by the...
Typing Methods• Phenotyping• Genotyping• Why type N.meningitidis?Tracking infections - short term or long termEpidemiologi...
MeningococciPhenotypic Typing Methods• Sero-grouping• Sero-typing• Sero-subtyping• Multilocus Enzyme Electrophoresis (14-2...
Meningococci Phenotyping• Meningococcal Pheno-types– Serogroups (Capsular polysaccharide)• A, B, C, D, E, H, I, K, L, X, Y...
Serogrouping• Murex antisera (gpB is a monoclonal)• Slide agglutination done on fresh organisms(overnight incubation)• Lig...
2D Model of Nm PorAMeningococcal Disease. Editor Cartwright K 1995
Serotyping/sub-typing
Genotypic Typing Methods• Pulse Field Gel Electrophresis - wholechromosome• porA PCR with RFLP analysis• Random Amplified ...
Genotyping• Identification of capsule serotype (serogroup) byPCR• Identification of a clone or lineage by MLST andPFGE• Id...
Genotyping• Not dependent on gene expression• Confirmation of IMD from culture negativespecimens• Capsule and subtype dete...
Genotyping• MLST is more robust than MLEE• porA/fetA/penA sequencing almost routine– www. database• Recombination events m...
Ponte Sant’Angelo
Strain Characterisation and Typing• http://neisseria.org/nm/• Martin Maiden– Probably 20,000 MN sequence typed now– ST-11 ...
Strain Characterisation and TypingAndrew Fox – EMGM Questionnaire• Working group on strain characterisation• Movement is t...
The diversity and dynamics of Neisseria meningitidis populations generate arequirement for high resolution, comprehensive,...
Antigenic Formula• Based on -Serogroup (SiaD gene detection)Serotype (porB gene sequencing)Serosubtype (porA gene sequenci...
Fet A (ferric enterobactin bindingprotein)• Protein expressed in response to iron limitation• Expressed during invasive in...
FetA Sequence Typing• Work on FetA done in the context ofvaccine development for SG B MC• Thompson etal 2003 – 107 MC isol...
Urwin etal 2004• This combination of PorA and FetAvariants would potentially protect against95 (89%) of the 107 diversemen...
Claus et al 2007(Deletion of the Meningococcal fetA Gene Used for Antigen SequenceTyping of Invasive and Commensal Isolate...
Claus et al 2007• Mutation in fetA is non-lethal• Iron uptake not affected• Serum sensitivity mildly enhanced• The fact th...
fetA typing at WCH 2008F1-5 8 BF1-7 1 W135F3-1 1 BF3-3 1 BF5-1 2 BF5-5 1 B• 14 isolates tested andall could be typed• 5 cl...
# FetA VR SG ST SST Year SiteNM 638 F1-5 B NT P1.4 2008 CSFNM 641 F1-5 B NT P1.4 2008 BloodNM 646 F1-5 B NT P1.4 2008 Bloo...
Conclusions• fetA typing works well for isolates but werequire some modification to the protocolfor clinical samples• fetA...
AntimicrobialSusceptibility/Resistance
Mechanisms of resistance to β-Lactams in Nm• Production of β-Lactamases -rare 5isolates• Alteration of the Structure of Pe...
Mechanisms of resistance to β-Lactams in Nm• Lability of β-lactamase-β-Lactaminteraction determines resistance• Stability ...
Antibiotic Resistance• Move towards molecular testing for penicillinsusceptibility• Paper recently published outlining a E...
Penicillin Binding Proteins• Alterations to PBP’s structure (mostly PBP2) lead to increases in MIC ranging from0.125-1 mg/...
Figure 1Phenotype distribution among the tested meningococcalisolates with known minimal inhibitory concentration (MIC mg/...
Antibiotic Resistance- penA sequencing Project• Reduced susceptibility = alterations in PBP2 binding due tomutations in 3’...
Antibiotic Resistance- penA sequencing Project• No clonal expansion observed• Sequence of 5 amino acid residues werealways...
PBP 2• Alteration of the gene encoding PBP2(penA) leads to this structural change• Occurs by horizontal exchange of geneti...
penA translation← Increasing MIC
Aims• To determine the correlation of penApolymorphisms and penicillin MIC• To determine if the polymorphisms are aresult ...
Background:Definitions of Penicillin Susceptibility:CSLI Guidelines:Susceptible: MIC ≤ 0.06ug/mLIntermediate MIC: 0.12-0.2...
Penicillin Susceptibility in SouthAustralian Isolates
Method:• Using protocols described by Taha et al2007.• All isolates with MIC>0.094 selected forsequencing.• 10 isolates wi...
• 57 isolates retrieved for penA sequecing• Of these, 23 did not yield the expectedproduct on initial PCR.• 34 yielded the...
Conclusions:• A subpopulation of penicillin less-susceptibleisolates have alterations to the transpeptidaseregion of the p...
Antimicrobial Susceptibility• E Test (CLSI guidelines) or agar dilution
ETest vs Agar Dilution
Australian MeningococcalSurveillance Programme (AMSP1994- continuing)(National Neisseria Network)
AMSP Requirements• Serogrouping within 48 hours• Phenotyping• Antibiotic susceptibility data• Genotyping• Methodology harm...
Number of laboratory confirmed cases of invasive meningococcal disease,Australia, 2007, by State or Territory and serogrou...
Australian MeningococcalSurveillance Program : 2007• SeasonalityJan 1- Mar 31 16.5 %April 1 - June 30 18.1%July 1 - Sept 3...
AMSP Data :Antibiotic Susceptibility• penicillin 76/177 (21%) fully sensitive101/177 (79%) less sensitive(MIC 0.06 - 0.5 u...
010203040506070800-4y 5-14y 15-19 20-24 25-44 45+BCFigure 1 Number of serogroup B and C cases of IMDconfirmed by all metho...
Anatomical source of samples positive for a laboratory confirmed case of IMD Australia 2007Specimen type Isolate of MC PCR...
Lab Diagnosis IMD (CSF/Blood): Overview• Cell count• Gram’s Stain• Culture• Antigen Detection• Serology• Nucleic Acid Ampl...
Diagnostic PCR• Manchester PHLS Ref Lab method• ctrA gene (WCH 20 cfu/mL)• siaDB and siaDC gene (WCH 200cfu/mL)• (porA gen...
Diagnostic PCR• Manchester PHLS Ref Lab method (in 1998additional 56% cases confirmed by using NAA)• ctrA gene (capsular b...
Diagnostic meningococcal PCRctrA ------ IS1106siaD B/C ------ siaD WY
Corbett Rotor-Gene 6000
Materials & Methods• Samples - clinical samples (blood/CSF)- cultures of N.meningitidis- cultures of other bacteria• WCH P...
Organisms other thanN.meningitidisStaph.aureus Mycoplasma hominisStrep. agalactiae Moraxella catarrhalisStrep. pneumoniae ...
DNA from Bacterial CulturesTested by RotorGeneRotorgenePosRotorgeneNegTotalN.meningo 81 2* 83Other#0 18 18•# Organisms oth...
Clinical Sample (blood/csf) Tested byWCH Current Method and RotorgeneRotorGenePosRotorGeneNegWCH PCRPos32 1#WCH PCRNeg2* 6...
Conclusions• rapid test time (approx. 2 hours cf > 4 hours)• sensitive & specific• early active management of patient and ...
Recent CaseLimb NecrosisIntracellular diplococci
Positive patient
Avantages of MolecularTechniques• Can perform assays despite treatmentand/or reluctance to lumbar puncture• No requirement...
Disadvantages• Unable to perform antimicrobialsusceptibilities• Specialised equipment and laboratories• Cost• Interpretati...
Bibliography-Diagnostics• Chakrabarti P Indian J Med Res. 2009 Feb;129(2):182-8.Application of 16S rDNA based seminested P...
Loop-mediated isothermal amplification (LAMP)of gene sequences and simple visual detection ofproductsNorihiro Tomita, Yasu...
Boving J Clin Microbiol. 2009 Apr;47(4):908-13.Eight-plex PCR and liquid-array detection of bacterial and viral pathogens ...
Sampling methods to detect carriage of Neisseria meningitidis;literature review.J Infect. 2009 Feb;58(2):103-7 Roberts J, ...
Application of atmospheric pressure matrix-assisted laserdesorption/ionization mass spectrometry for rapid identification ...
Simultaneous detection of Haemophilus influenzae type bpolysaccharide-specific antibodies and Neisseria meningitidis serog...
Trop Med Int Health. 2009 Jan;14(1):111-7. Epub 2008 Nov 12.Field evaluation of rapid diagnostic tests for meningococcalme...
FEMS Immunol Med Microbiol. 2008 Jul;53(2):178-82.Simultaneous single-tube PCR-based assay for the direct identification o...
Arch Dermatol. 2008 Jun;144(6):770-3..Value of a novel Neisseria meningitidis--specific polymerase chainreaction assay in ...
Acknowledments• Prof John Tapsall and his laboratory atPOW Hospital• NNN Laboratories• Kathryn Whetter• Stuart McKessar an...
Neisseria meningitidis _clinical_laboratory_diagnosis_-plus (1)
Neisseria meningitidis _clinical_laboratory_diagnosis_-plus (1)
Neisseria meningitidis _clinical_laboratory_diagnosis_-plus (1)
Neisseria meningitidis _clinical_laboratory_diagnosis_-plus (1)
Neisseria meningitidis _clinical_laboratory_diagnosis_-plus (1)
Neisseria meningitidis _clinical_laboratory_diagnosis_-plus (1)
Neisseria meningitidis _clinical_laboratory_diagnosis_-plus (1)
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Neisseria meningitidis _clinical_laboratory_diagnosis_-plus (1)

  1. 1. Andrew LawrenceMicrobiology and Infectious Diseases Dept.SA Pathology at Women’s and Children’s HospitalAdelaide, SANeisseria meningitidis:Clinical LaboratoryDiagnosis - Plus
  2. 2. Neisseria and related spp. ofhuman origin• Neissseria meningitidis• Neisseria gonorrhoeae• N. lactamica, N. cinerea, N.polysacharea, N. subflava, N. sicca,N.mucosa, N.flavescens, N.elongata• Moraxella (Branhamella) catarrhalis –coccoid form
  3. 3. Neisseria spp.• Characteristic diplococcus (bean shaped)• Inhabit mucous membranes of warmblooded hosts• Aerobic, non-motile, no spores, growoptimally at 37°C, growth stimulated byCO2 and NG has obligate requirement,oxidase positive, produce acid fromcarbohydrates oxidatively, most catalasepositive (cf Kingella –ox + but cat -)
  4. 4. Gram’s Stain of CSF ShowingGN Diplococci
  5. 5. Neisseria spp:Clinical Significance• NG always considered a pathogenirrespective of site of isolation• Other Neiss. Inhabitants of upperrespiratory tract and most not pathogenic• Only a few strains of meningococci arepathogenic ie hypervirulent strains
  6. 6. Specimens and culture mediaMCMeningitis/bacteraemiaCSF, skinlesions, blood,nasopharynxSelective/non-selectiveMcatPneumonia Sputum or BALetcNon-selectiveOtitis media Tympanocentesis(not routine)Non-selectiveSinus Sinus biopsy oraspirateSelective/Non-selective
  7. 7. Neisseria spp: ID• Sugar fermentation (GC = glu pos onlywhile MC = glu, malt pos)• Growth on selective agar• Latex/ co-agglutination tests/slideagglutination for serogrouping MC(Pastorex/Murex)• Vitek card• NATs – serogroup specific
  8. 8. N.meningitidis cell wallProtects against host defences13 Serogroups (ctrA diagnostic test)Porins (porA and porB) - serosubtype, serotypeOpacity proteins - adherence to leukocytes / host cells,Fet A (Fe binding protein formally FrpB)Potent endotoxin13 ImmunotypesAdherence to epith andRB cells
  9. 9. Meningococcal Disease. Editor Cartwright K 1995Neisseria meningitidis showing blebs50% lipooligosaccharide50% OMPs, phospholipids and capsularpolysaccharide
  10. 10. Meningococcal purpuric rash
  11. 11. IMD and the Military• 1812 US Civil War - meningitis outbreaks• Turn of century - bacteriologic confirmation• Increased rates in 1907 - Cubanoccupation and 1913 Mexican bordermobilisation• New recruits = higher levels of disease cfseasoned troops
  12. 12. IMD and the MilitaryMeningococcal meningitis in the US Army, 1910-1946Evolution of the Meningococcus, Vedros editor
  13. 13. IMD and the Military• WW1 (US)- IMD at all training campsMost crowded had most disease• During WW1 5839 cases of IMD and 2279deaths (army) with fatality rate of 39% -pre antibiotic era• Between WW1 and WW2 - 2 epidemicscoincidental with epidemics as a whole inUS civilian population
  14. 14. IMD and the Military• Throughout WW2 13,922 cases of IMD• 559 deaths• Cases fatality rate 4% - sulphadiazineprophylaxsis• 67% cases in troops who had been in thearmy for less than 3 months
  15. 15. IMD and the Military• Large epidemics in winters 1917-18, 1942-43• Smaller secondary peaks approx 9 months later• New recruits at greater risk than “regular” troops• Both epidemics coincident with civilian populationepidemics• Serogroup A accounted for approx. 90% cases• Post demobilisation rates of disease returned to“normal”Summary
  16. 16. IMD and the Military• Post WW2 sulphadiazine used forprophylaxis but in 1963 (Vietnam era) sdzresistance developed• Serogroup B and C (1967) disease reportedand increased - sdz prophylaxis stopped• 1969-1970 trials of C polysaccharidevaccines - efficacy of 90%• 1971 routine use of serogroup C ps vaccine• 1982 all trainees given A/C/Y/W135 psvaccine
  17. 17. Typical meningococcal petechial rash
  18. 18. Meningococcal sepsis - digital gangrene
  19. 19. Meningococcal Disease inAustralia• July 1900 - Feb 1901 large outbreakSydney 69% mortality• Post WW1 increased rates• 1915 Large outbreak in Victoria beginningat Seymour military camp - 644 casesmortality 52%• 1939 – 1946 conflict saw increases butmortality 23% - sulphonamide therapy
  20. 20. The annual number of deaths from meningococcal disease wasobtained from the compilation of mortality data collected by theAustralian Bureau of Statistics.Annual notification rates and deaths from meningococcal disease for allstates/territories in Australia, 1915–2003Australia’s century of meningococcal disease:development and the changing ecology of anaccidental pathogenMahomed S Patel MJA 2007; 186 (3): 136-141
  21. 21. Typing Methods• Phenotyping• Genotyping• Why type N.meningitidis?Tracking infections - short term or long termEpidemiological spread of NM acrosshouseholds/countries/the worldVaccine developmentEvolution of the organism
  22. 22. MeningococciPhenotypic Typing Methods• Sero-grouping• Sero-typing• Sero-subtyping• Multilocus Enzyme Electrophoresis (14-20 enzyme mobilities) - ET Type• Immunotyping
  23. 23. Meningococci Phenotyping• Meningococcal Pheno-types– Serogroups (Capsular polysaccharide)• A, B, C, D, E, H, I, K, L, X, Y, Z, W135, 29E,non-encapsulated• Only B and C common currently– Serotypes• based on outer membrane proteinepitopesSerotypes - Por BSerosubtypes - Por A• eg C:2a,15
  24. 24. Serogrouping• Murex antisera (gpB is a monoclonal)• Slide agglutination done on fresh organisms(overnight incubation)• Light inoculum• Often need light box to read slide• Must be done in a bio-safety cabinet• group B strains sometimes stringy andautoagglutinate• non-encapsulate
  25. 25. 2D Model of Nm PorAMeningococcal Disease. Editor Cartwright K 1995
  26. 26. Serotyping/sub-typing
  27. 27. Genotypic Typing Methods• Pulse Field Gel Electrophresis - wholechromosome• porA PCR with RFLP analysis• Random Amplified Polymorphic DNA PCR• *porA/porB Sequencing• *fetA sequencing• *penA sequencing• *Multi Locus Gene Sequencing- house keeping genes• Ribotyping• VNTR
  28. 28. Genotyping• Identification of capsule serotype (serogroup) byPCR• Identification of a clone or lineage by MLST andPFGE• Identification of serosubtype by porA sequencing• Identification of serotype by porB genesequencing• Penicillin binding protein 2 variable regions bypenA• Iron binding protein variable regions by fetA
  29. 29. Genotyping• Not dependent on gene expression• Confirmation of IMD from culture negativespecimens• Capsule and subtype determinationdirectly from clinical samples• Electronic data portable and reproducible• MLST aid to longer term epidemiologicalstudies. PFGE short term studies.
  30. 30. Genotyping• MLST is more robust than MLEE• porA/fetA/penA sequencing almost routine– www. database• Recombination events more easily pickedup by sequencing
  31. 31. Ponte Sant’Angelo
  32. 32. Strain Characterisation and Typing• http://neisseria.org/nm/• Martin Maiden– Probably 20,000 MN sequence typed now– ST-11 28x more likely to cause disease compared with“ordinary NM”– Different clonal complexes are associated with differentserogroups/particular antigens and reflect human immunity– Capsule null locus carried in 16% NM– NM are continually producing variants but these arecontinuously cleaved = not much change– Once vaccines have been introduced there is a majorchange in population structure– Future lies in using/interpreting sequence data now indatabases rather than new ST techniquesEMGM 2007
  33. 33. Strain Characterisation and TypingAndrew Fox – EMGM Questionnaire• Working group on strain characterisation• Movement is toward genotyping but some GT data notbeing submitted• Phenotyping reagents still available and are supportedby NIBSC• Want a hub system for smaller labs not doingsequencing (Oxford can do sequence for 75p!)• Recommendation from last EMGM was to do porAsequencing• MLST also needs to be maintained to monitor populationbiology• fetA probably helpful when combined with otherAgs• http://neisseria.org/nm/ EMGM 2007
  34. 34. The diversity and dynamics of Neisseria meningitidis populations generate arequirement for high resolution, comprehensive, and portable typing schemes formeningococcal disease surveillance. Molecular approaches, specifically DNAamplification and sequencing, are the methods of choice for various reasons,including: their generic nature and portability, comprehensive coverage, and readyimplementation to culture negative clinical specimens. The following target genesare recommended: (1) the variable regions of the antigen-encoding genes porA andfetA and, if additional resolution is required, the porB gene for rapid investigationof disease outbreaks and investigating the distribution of antigenic variants; (2) theseven multilocus sequence typing loci–these data are essential for the mosteffectivenational, and international management of meningococcal disease, as wellas being invaluable in studies of meningococcal population biology and evolution.These targets have been employed extensively in reference laboratoriesthroughout the world and validated protocols have been published. It is furtherrecommended that a modified nomenclature be adopted of the form:serogroup: PorA type:FetA type: sequence type (clonal complex)Molecular typing of meningococci: recommendations for targetchoice and nomenclatureKeith A. Jolley, Carina Brehony & Martin C.J. MaidenDepartment of Zoology, University of Oxford, Oxford, UK
  35. 35. Antigenic Formula• Based on -Serogroup (SiaD gene detection)Serotype (porB gene sequencing)Serosubtype (porA gene sequencing)C:2a:P1.7-2,4fetA (gene sequencing) eg F1-5Sequence Type• B: P1.19,15: F5-1: ST-33 (cc32).Angelo Zaia NNN 2008
  36. 36. Fet A (ferric enterobactin bindingprotein)• Protein expressed in response to iron limitation• Expressed during invasive infection but clear role inthe bloodstream not defined• Receptor function in iron acquisition pathways =attractive vaccine candidate as expression essentialfor growth in vivo• FetA antibodies found in convalescent antisera• mAbs raised in mice are bactericidal and specific forthe parent isolate
  37. 37. FetA Sequence Typing• Work on FetA done in the context ofvaccine development for SG B MC• Thompson etal 2003 – 107 MC isolates -60 fetA alleles encoding 56 proteinsequences• Variability due to point mutations as wellas horizontal genetic exchange• Nomenclature established with 6 FetAvariant families identified• Concluded that FetA unlikely to be asingular vaccine candidate
  38. 38. Urwin etal 2004• This combination of PorA and FetAvariants would potentially protect against95 (89%) of the 107 diversemeningococcal isolates used to developMLST
  39. 39. Claus et al 2007(Deletion of the Meningococcal fetA Gene Used for Antigen SequenceTyping of Invasive and Commensal Isolates from Germany:Frequencies and Mechanisms)• fetA negative isolates in Germany identified from2201 invasive isolates during 2001-2007• 11 (0.5%) found• Randomly distributed geographically and noassociation with a particular ST observed• Due to deletions in fetA gene (similar to thesituation with porA)• 12/821 carrier strains also contained thesedeletions
  40. 40. Claus et al 2007• Mutation in fetA is non-lethal• Iron uptake not affected• Serum sensitivity mildly enhanced• The fact that fetA negative strains arepresent has implications for vaccinedevelopment• There is a trend to carrier strains havingfetA deletions at a higher rate
  41. 41. fetA typing at WCH 2008F1-5 8 BF1-7 1 W135F3-1 1 BF3-3 1 BF5-1 2 BF5-5 1 B• 14 isolates tested andall could be typed• 5 clinical samplestested with 2 givingweak PCR productonly and 3 no product
  42. 42. # FetA VR SG ST SST Year SiteNM 638 F1-5 B NT P1.4 2008 CSFNM 641 F1-5 B NT P1.4 2008 BloodNM 646 F1-5 B NT P1.4 2008 BloodNM 648 F1-5 B NT P1.4 2008 BloodNM 649 F1-5 B NT P1.4 2008 BloodNM 651 F1-5 B NT P1.4, 1.1 2008 CSFNM 653 F1-5 B NT NST 2008 BloodNM 656 F1-5 B NT P1.4 2008 BloodNM 645 F1-7 W135 NT P1.4 2008 BloodNM 640 F3-1 B NT NST 2008 BloodNM 637 F3-3 B 15 NST 2008 BloodNM 639 F5-1 B 15 NST 2008 BloodNM 655 F5-1 B NT P1.4 2008 BloodNM 652 F5-5 B NT P1.4 2008 BloodfetA summary 2008
  43. 43. Conclusions• fetA typing works well for isolates but werequire some modification to the protocolfor clinical samples• fetA can help with strain differentiation• F1-5 most common type in SA• Await report from EMGM 2009 forrecommendations on standardnomenclature for Australian isolates
  44. 44. AntimicrobialSusceptibility/Resistance
  45. 45. Mechanisms of resistance to β-Lactams in Nm• Production of β-Lactamases -rare 5isolates• Alteration of the Structure of PenicillinBinding Proteins (PBP2 particularly) due toacquisition of DNA from other organisms -major mechanism• Alteration in the permeability of cellularmembranes possibly by decreasedexpression of class 3 porin
  46. 46. Mechanisms of resistance to β-Lactams in Nm• Lability of β-lactamase-β-Lactaminteraction determines resistance• Stability of the high molecular weightPBP-β-lactam interaction determinessusceptibility• There is however a range of interactionsand rates of enzyme turnover whichleads to variations of susceptibility
  47. 47. Antibiotic Resistance• Move towards molecular testing for penicillinsusceptibility• Paper recently published outlining a Europeanstudy and methods for testing penA genesequences (result of initiative from EMGM2005 Dublin)• Standard methods for penA sequencing• penA sequencing can also be used todifferentiate strains which have the same ST
  48. 48. Penicillin Binding Proteins• Alterations to PBP’s structure (mostly PBP2) lead to increases in MIC ranging from0.125-1 mg/L• Alterations to PBP 1 can lead to higherlevel MIC increases• Accumulations of these plus other PBPsmay lead to further increases in MIC andtreatment failure as in S.pneumoniae andgonococci
  49. 49. Figure 1Phenotype distribution among the tested meningococcalisolates with known minimal inhibitory concentration (MIC mg/L)(n=1644, 98% of total isolates).Target gene sequencing to characterize penicillin G susceptibility of N.meningitidis TAHA M et al personal communication 2007Reduced Pen susc
  50. 50. Antibiotic Resistance- penA sequencing Project• Reduced susceptibility = alterations in PBP2 binding due tomutations in 3’ half of the penA gene• 402 bp fragment of penA sequenced• 1670 strains from 60 years and 22 countries tested• 808 csf; 585 blood (ie 83% were invasive isolates-55%B,29%C)• Phenotyping, genotyping and penicillin G MIC testsperformed• 65% had reduced susc to penicillin• 139 different penA alleles found (penA1-penA139)• 38 of these were highly related and clustered to correspondto susceptible strains• Remaining 101 were diverse and accounted for 38% of totalisolates
  51. 51. Antibiotic Resistance- penA sequencing Project• No clonal expansion observed• Sequence of 5 amino acid residues werealways altered in these strains• Correlation not 100% which suggests othermechanisms may be involved• Evidence of mosaic structures throughinterspecies recombination were detected• Data argues for use of penA sequencing toidentify isolates with reduced susc. Topenicillin• Database now available on web
  52. 52. PBP 2• Alteration of the gene encoding PBP2(penA) leads to this structural change• Occurs by horizontal exchange of geneticmaterial from other organisms egN.flavescens via transformation
  53. 53. penA translation← Increasing MIC
  54. 54. Aims• To determine the correlation of penApolymorphisms and penicillin MIC• To determine if the polymorphisms are aresult of the interspecies transfer of geneticmaterial• To discover if any unique penA allelesexist within our isolates.
  55. 55. Background:Definitions of Penicillin Susceptibility:CSLI Guidelines:Susceptible: MIC ≤ 0.06ug/mLIntermediate MIC: 0.12-0.25ug/mLResistant: ≥0.5ug/mLProposed EMGM Guidelines:Intermediate: MIC 0.06-1ug/mLResistant ≥1ug/mL
  56. 56. Penicillin Susceptibility in SouthAustralian Isolates
  57. 57. Method:• Using protocols described by Taha et al2007.• All isolates with MIC>0.094 selected forsequencing.• 10 isolates with MIC≤0.094 randomlyselected.
  58. 58. • 57 isolates retrieved for penA sequecing• Of these, 23 did not yield the expectedproduct on initial PCR.• 34 yielded the expected product and werethen sequenced.• 23 different alleles were found amongstthese isolates• 11 of these alleles were unique.• Excluding allele 1, the most commonalleles were two unique alleles, each foundin 3 isolates.
  59. 59. Conclusions:• A subpopulation of penicillin less-susceptibleisolates have alterations to the transpeptidaseregion of the penA gene• In these isolates, substitutions F504L , A510Vand I515V are always present.• Additional mutations in amino acid positions 485-552 often correlate with a higher MIC(>0.125ug/mL).• A subpopulation of penicillin less-susceptibleisolates have a more significantly altered gene,and require further study.
  60. 60. Antimicrobial Susceptibility• E Test (CLSI guidelines) or agar dilution
  61. 61. ETest vs Agar Dilution
  62. 62. Australian MeningococcalSurveillance Programme (AMSP1994- continuing)(National Neisseria Network)
  63. 63. AMSP Requirements• Serogrouping within 48 hours• Phenotyping• Antibiotic susceptibility data• Genotyping• Methodology harmonisation
  64. 64. Number of laboratory confirmed cases of invasive meningococcal disease,Australia, 2007, by State or Territory and serogroup.State/TerritorySerogroup TotalB C A Y W135 NG*ACT 4 0 1 5NSW 78 7 5 1 10 101NT 1 1 2Qld 30 3 1 1 1 36SA 11 1 1 1 14Tas 3 0 1 1 5Vic 46 2 4 3 4 59WA 19 0 1 20Australia 192 14 0 12 8 16 242* not serogrouped
  65. 65. Australian MeningococcalSurveillance Program : 2007• SeasonalityJan 1- Mar 31 16.5 %April 1 - June 30 18.1%July 1 - Sept 30 31.9%Oct 1 - December 31 34.5 %• M/F Ratio (all) 1.08:1Mortality 4.2%(higher for gp C strains)Tapsall JW CDI 2008
  66. 66. AMSP Data :Antibiotic Susceptibility• penicillin 76/177 (21%) fully sensitive101/177 (79%) less sensitive(MIC 0.06 - 0.5 ug/mL)• ciprofloxacin (1 isolate 0.6 –slightly raised) Sensitive (0.002ug/mL)• ceftriaxone Sensitive (<0.002 ug/mL)• Rifampicin (1 isolate slightly raised) Sensitive (0.004 - 0.38 ug/mL)• chloramphenicol Sensitive (038 - 1.0 ug/mL)
  67. 67. 010203040506070800-4y 5-14y 15-19 20-24 25-44 45+BCFigure 1 Number of serogroup B and C cases of IMDconfirmed by all methods, by age, Australia, 2007AMSP Report CDI 2007
  68. 68. Anatomical source of samples positive for a laboratory confirmed case of IMD Australia 2007Specimen type Isolate of MC PCR positive* TotalBlood 93 63 156CSF +/- Blood 29 45 74Other+ 5 2 7Serology alone** 5Total 127 110 242*PCR positive in the absence of a positive culture;**serology positive in the absence of positive culture or PCR.+ Joint and fluid samples (4 isolates from joints and 2 by PCR of joint fluid;1 culture from peritoneal fluid)
  69. 69. Lab Diagnosis IMD (CSF/Blood): Overview• Cell count• Gram’s Stain• Culture• Antigen Detection• Serology• Nucleic Acid Amplification Assays• RDTs
  70. 70. Diagnostic PCR• Manchester PHLS Ref Lab method• ctrA gene (WCH 20 cfu/mL)• siaDB and siaDC gene (WCH 200cfu/mL)• (porA gene)• SamplesCSF, whole blood, serum, tissue• Sensitivity (Poritt 2001) 94% (csf)• Specificity (Poritt 2001) 100% (csf)
  71. 71. Diagnostic PCR• Manchester PHLS Ref Lab method (in 1998additional 56% cases confirmed by using NAA)• ctrA gene (capsular biosynthesis locus – transportof capsular polysaccharide)• siaDB and siaDC gene (polymerisation of sialicacid to polysialic acid chain and regions of thegene are serogroup specific)• porA gene (porB gene for typing)
  72. 72. Diagnostic meningococcal PCRctrA ------ IS1106siaD B/C ------ siaD WY
  73. 73. Corbett Rotor-Gene 6000
  74. 74. Materials & Methods• Samples - clinical samples (blood/CSF)- cultures of N.meningitidis- cultures of other bacteria• WCH PCR protocol using ctrA primers developed for ELISAPCR (Manchester UK) with gel detection of PCR product• Real-time PCR using CtrA primers developed for Taqmanassays- FAM/ BHQ-1 dual-labelled 20mer probe with ctrA specificsequence for product detection
  75. 75. Organisms other thanN.meningitidisStaph.aureus Mycoplasma hominisStrep. agalactiae Moraxella catarrhalisStrep. pneumoniae Ur. Urealyticum B.Strep. Pyogenes Burk.cepaciaB.pertussis Legionella pneumophilaPs.Aeruginosa Haemophilus influenzaeProteus mirabilis Chlamydia trachomatisM.pneumoniae Chl. pneumoniaeN.gonorrhea
  76. 76. DNA from Bacterial CulturesTested by RotorGeneRotorgenePosRotorgeneNegTotalN.meningo 81 2* 83Other#0 18 18•# Organisms other than N.meningitidis•*These subsequently identified as N.lactamica
  77. 77. Clinical Sample (blood/csf) Tested byWCH Current Method and RotorgeneRotorGenePosRotorGeneNegWCH PCRPos32 1#WCH PCRNeg2* 68*36 cycles before positive (total 39 cycles)# non repeatable ctrA, siaD neg and pos gp B isolatefrom pharynxTotal3370
  78. 78. Conclusions• rapid test time (approx. 2 hours cf > 4 hours)• sensitive & specific• early active management of patient and patient’scontacts
  79. 79. Recent CaseLimb NecrosisIntracellular diplococci
  80. 80. Positive patient
  81. 81. Avantages of MolecularTechniques• Can perform assays despite treatmentand/or reluctance to lumbar puncture• No requirement for culturegenotyping possible (porA/B, MLST)Some resistance genes detectable• Rapid (diagnostics, typing)• Sensitive and specific• Automation
  82. 82. Disadvantages• Unable to perform antimicrobialsusceptibilities• Specialised equipment and laboratories• Cost• Interpretation difficulties• Trained staff
  83. 83. Bibliography-Diagnostics• Chakrabarti P Indian J Med Res. 2009 Feb;129(2):182-8.Application of 16S rDNA based seminested PCR for diagnosis of acute bacterial meningitis• The overall sensitivity, specificity, positivepredictive value and negative predictive value of16S rDNA PCR were 79.24, 97.6, 89.36 and94.88 per cent respectively when culture wasconsidered as gold standard. The detection limitof 16S rDNA PCR was determined to be 1000cfu/ml of E. coli and 4000 cfu/ml of S.pneumoniae
  84. 84. Loop-mediated isothermal amplification (LAMP)of gene sequences and simple visual detection ofproductsNorihiro Tomita, Yasuyoshi Mori, Hidetoshi Kanda & Tsugunori Notomi• Loop-mediated isothermal amplification(LAMP) is a simple, rapid, specific andcost-effective nucleic acid amplificationmethod when compared to PCR, nucleicacid sequence-based amplification, self-sustained sequence replication and stranddisplacement amplification. This protocoldetails an improved simple visualdetection system for the results of theLAMP reaction.
  85. 85. Boving J Clin Microbiol. 2009 Apr;47(4):908-13.Eight-plex PCR and liquid-array detection of bacterial and viral pathogens incerebrospinal fluid from patients with suspected meningitis• Eight-plex PCR and liquid-array detectionof bacterial and viral pathogens incerebrospinal fluid from patients withsuspected meningitis.• Luminex 100 suspension array system• N. meningitidis (sens 100% and spec99.7%)
  86. 86. Sampling methods to detect carriage of Neisseria meningitidis;literature review.J Infect. 2009 Feb;58(2):103-7 Roberts J, Greenwood B, Stuart J• The evidence to date suggests thatmeningococcal carriage should beassessed by swabbing the posteriorpharyngeal wall through the mouth, andthat swabs should be plated directly onsite or placed in transport medium for <5h.
  87. 87. Application of atmospheric pressure matrix-assisted laserdesorption/ionization mass spectrometry for rapid identification ofNeisseria species.J Biomol Tech. 2008 Jul;19(3):200-4 Gudlavalleti SK, et al• five serogroups (A, B, C, W135, and Y) ofNeisseria meningitidis subjected to onprobe/peptide extraction and tryptic digestionfollowed by AP-MALDI-Tandem MS• Amino acid sequences derived from threeprotonated peptides were used to probe adatabase yielding 3 of neisserial proteins whichare potential biomarkers for neisserial speciesidentification
  88. 88. Simultaneous detection of Haemophilus influenzae type bpolysaccharide-specific antibodies and Neisseria meningitidis serogroupA, C, Y, and W-135 polysaccharide-specific antibodies in a fluorescent-bead-based multiplex immunoassay.Clin Vaccine Immunol. 2009 Mar;16(3):433-6de Voer RM, Schepp RM, Versteegh FG, van der Klis FR, Berbers GA.• meningococcal serogroup A, C, Y, and W-135 multiplex immunoassay (MIA)• Used for serological analysis for childhoodvaccine studies
  89. 89. Trop Med Int Health. 2009 Jan;14(1):111-7. Epub 2008 Nov 12.Field evaluation of rapid diagnostic tests for meningococcalmeningitis in Niger.Boisier P, Mahamane AE, Hamidou AA, Sidikou F, Djibo S, Nato F,Chanteau S.• Rapid Diagnostic Tests (RDTs) used for detecting antigen to NM inCSF in Niger, Africa• Using RDTs, health facilities reported 382 negative results (73.9%),114 NmA (22.1%), 12 NmW135 (2.3%)• CONCLUSION: We confirmed that dipstick RDTs to identify N.meningitidis serogroups A, C, W135 and Y can be reliably operatedby non-specialized staff in basic health facilities. RDTs proved veryuseful to recommend vaccination in NmA epidemics, and also toavoid vaccination in epidemics due to serogroups not included invaccines (NmX).
  90. 90. FEMS Immunol Med Microbiol. 2008 Jul;53(2):178-82.Simultaneous single-tube PCR-based assay for the direct identification ofthe five most common meningococcal serogroups from clinical samples.Drakopoulou Z et al• Neisseria meningitidis serogroups (A, B,C, W-135 and Y) in 530 clinical samplesobtained from 428 patients (271 blood and259 cerebrospinal fluid). The sensitivityand the specificity was calculated to 100%[positive predictive value 100% (95%, CI99.0-100%) and negative predictive value100% (95% CI 99.0-100%)].
  91. 91. Arch Dermatol. 2008 Jun;144(6):770-3..Value of a novel Neisseria meningitidis--specific polymerase chainreaction assay in skin biopsy specimens as a diagnostic tool in chronicmeningococcemia.Parmentier L, et al• In 2 patients with CM, we established thediagnosis by a newly developed PCR-based approach performed on skin biopsyspecimens
  92. 92. Acknowledments• Prof John Tapsall and his laboratory atPOW Hospital• NNN Laboratories• Kathryn Whetter• Stuart McKessar and WCH Laboratory• SA Pathology RAH Site MolecularPathology Unit

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