Laboratory Diagnosis of Pertussis Past, Present and Future by goingbackto85

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									Laboratory Diagnosis of Pertussis:
Past, Present and Future

           Andrew Whitelaw

                  NHLS
        University of Cape Town
Whooping cough
 First clinical
 description 1578
 Causative organism
 first isolated 1906
   Octave Gengou and
   Jules Bordet
   Initially Haemophilus
   pertussis, later
   reclassified as
   Bordetella pertussis
Introduction
 Classic presentation usually
 older children
 In very young, presentation
 can be atypical eg apnoea.
 Higher mortality in            CDC

 neonates (3% vs 0,1%)
 Concerns about increasing
 numbers cases
Incidence reduced
by vaccination
Still occurs in very
young
Possibly increasing
in adults /
adolescents
Reservoir to infect
unvaccinated
children
           Data from HPA, UK
  South Africa




Local epidemiology of pertussis unknown
Effect of HIV co-infection on pertussis
unknown
The bacterium
 Gram negative cocco-bacillus
 Strict aerobe
 Fastidious
   Sensitive to toxic substances / metabolites
   B. pertussis most fastidious
   Other species less so
 Slow growing
   Inversely proportional to fastidiousness
Microscopy
 Gram stain of specimens not useful
 Can be detected by DFA on nasopharyngeal
 secretions
   Variable sensitivity and specificity (30-70% sens,
   up to 85% false positives)
   Depends on comparator
   Depends on experience
   DFA not accepted as proof of pertussis in USA
   (and some other countries)
Specimen for culture
Nasopharyngeal swabs (calcium
alginate or dacron fibres, NOT cotton
or rayon)
Nasal washes
Nasal / nasopharyngeal aspirate

Aspirates / washes preferred
  Better sample volume
  Better yield than swab (11%)
Cough plates no longer recommended
Transport

            Casamino acids (casein
            hydrosylate) if <2 hr delay
            Amies transport medium plus
            charcoal (<24 hr delay)
            Regan Lowe transport (>24 hrs
            delay)
Culture
Regan-Lowe medium                With peptones,
  Charcoal based                 glycerol, blood,
                                 antibiotic
Bordet-Gengou medium             (cephalexin)
  Potato infusion based
RL preferred
  Better yield
  Longer shelf life
Incubate ambient, avoid drying out, 35C (37
too high!), up to 12 days
Inoculate BA in parallel
Identification from culture
 RL – round, silver colonies
 BG – haemolysis visible
 Gram, oxidase, catalase
 Urea
 Growth on BA, MAC
 PCR
Sensitivity of culture
 Poor
 Transport, fastidiousness etc
 Decreases with time
   After about 3 weeks not recommended
   Often problem in adolescents / adults
Serology
 Various methods– ELISA probably commonest
 Antibodies to PT, FHA, pertactin, fimbriae
 IgG and IgA to PT and FHA most reliable
   90% infected get IgG to PT and FHA
   PT – specific for B. pertussis
   FHA expressed by B. parapertussis, cross
   reactivity with other bacteria (eg H. influenzae)
Serology
 Ideally need paired sera
   Often impractical
 Use single specimen with high titre
   Cutoff of 100 or 125 EU/ml (anti-PT IgG)
 Interpretation post vaccination difficult
 Failure to mount antibody response described
 in <3 month olds
 In older age groups – possibly best option
Molecular diagnosis
 Regarded as current gold standard by
 many
 Numerous in-house assays described
 Limited commercial assays
Specimen for PCR
 Nasal washes
 Nasal / nasopharyngeal aspirate
 Dacron or Rayon swabs



 Alginate can inhibit PCR
 Aluminium in shaft may also inhibit PCR
Specimen handling for PCR
 Less of an issue!

 Can be transported at ambient temp
 Transport medium not critical
   Doesn’t seem to interfere with PCR

 DNA extraction – commercial methods
 preferable to in-house
PCR targets
 IS481
   Insertion sequence, multiple copies
   Can be found in B. holmesii, parapertussis,
   bronchiseptica, avium

 ptxA-Pr (pertussis toxin promoter)
   Toxin operon present in other species
   Promoter specific for B. pertussis
   Often only single copy
Which PCR?
 Real time probably similar to block based
 Sequence specific detection (FRET, beacons
 etc)
   Expense
 IS481 vs ptx-Pr
   More sensitive (by up to 30% but varies widely)
   Less specific (by up to 10% less – still >80%)

 One option – IS481, confirm positive with
 ptx-Pr
   Also adds to expense and time
Sensitivity of PCR
 Affected by
   Age of patients
   Age of sample (also decreases with time –
   useful up to 4 weeks)
   Type of sample
   Definition of “gold standard”
 Up to 100%, low as 15%
Other PCR Issues
 Lack of QC programmes
 False positives causing scares
 Lack of standardisation across various
 centres
   DNA extraction
   Primers
   Platforms
   Detection
GSH experience
 45 patients (19 – 294 days old)
 All with resp illness
 Culture NPA 7 days
 Nested block based PCR for IS481
 Real-time for ptx-Pr (Lightcycler, Sybr
 Green)
GSH experience
 5/45 cult pos                 (11,1%)
 29/45 IS481 pos               (64,4%)
 16/45 ptx-Pr pos              (35,5%)
   9/13 discordant results – only inner nested
   primers pos
   ?lab contam
   ?not B. pertussis
   ?B. pertussis missed by ptx-Pr assay
 Serology not done (not available, and young
 cohort)
And the answer is…
 Ideally:

 Cough / symptoms <3 weeks: PCR plus
 culture
 Cough 3-4 weeks: PCR plus serology
 Cough > 4 weeks: serology alone

 If serology not available - ?
Thanks
 Craig Corcoran
 Iva Shankland     Lab work
 Charmaine Barthus
 Heather Zar
                  Clinical work
 Ralph Diedericks

 NHLS research trust
References
  Muller et al. Laboratory Diagnosis of Pertussis: State of the Art in 1997. J Clin
  Micro 1997; 35: 2435
  Singh et al. Whooping Cough: The Current Scene. Chest 2006; 130: 1547
  Andre et al. Comparison of Serological and Real-Time PCR Assays To
  Diagnose Bordetella pertussis Infection in 2007. J Clin Micro 2008; 46: 1672
  Centers for Disease Control and Prevention. Outbreaks of Respiratory Illness
  Mistakenly Attributed to Pertussis — New Hampshire, Massachusetts, and
  Tennessee, 2004–2006. MMWR 2007;56:837
  Wirsing von König. Pertussis of Adults and Infants. Lancet Infect Dis 2002; 2:
  744
  Mattoo & Cherry. Molecular pathogenesis, epidemiology and clinical
  manifestations of respiratory infections due to Bordetella pertussis and other
  Bordetella species. Clin Micro Rev 2005; 18: 326
  Tozzi AE et al. Diagnosis and Management of pertussis. CMAJ 2005; 172: 509

								
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