Lesson 3 WT
neisseria infections
• Diagnosis of neisseria infections
• Diagnostical model:gonorrhoe – swab
from vagina, uretral discharge
• Microscopy, cultivation, biochemical
tests, detection of enzym,ATB
susceptibility tests
Diagnosis of neisseria infections - gonococcal
• Gram stain - very sensitive (90%) and specific (98%) –
in purulent uretritis in men and purulent artritis.In other
cases (cervicitis, anorectal infections,pharyngitis) –
sensitivity and specificity is very low. Staining of
cultures.
• Cultivation - Selective media Thayer Martin´s,
chocolate agar + ATB (vancomycin), 5% CO2 !drying
and cold! – collected material cannot be placed in the
refrigerator
• Biochemical identification – differenciation from
Neisseria meningitis a nonpatogenic neisseria –
theoretical meaning
• Genetic probes- detection from clinical material
Cultivation of Neisseria gonorrhoe
• Swab from vagina or discharge – on blood
agar, modified blood agar, chocolate agar
+ ATB – inhibition of contaminating flora
• Grey cololnies after application of
cytocromoxidase – become black – slide
sc. Gram, - biochemical tests for diff.dg.
From other Neisseria
Detection of catalasa of
Neisseria gonorrhoe – summer
term
ATB susceptibility testing
• Disc diffusion method
• 6-8 ATB discs in one plate
• Zone of inhibitionof the growth in mms –
comparison with standards
Without zone of inhibition – resistence to tested ATB
Zone of inhibition of growth sufficiently large
ATB disc
Growth of tested bacteria
Insufficient zone of inhibition
ATB susceptibility
• Neisseria gonorrhoe - PNC – penicilinase production
- chromosome type of
resistence - changes in cell surface,
- ceftriaxon
- TTC, chinolons, makrolides - azitromycin – therapy
of chlamydia infection
Microscopy
• Vagina swab in susp. gonorrhoe: - Gram
staining: G - diplococci, coffee beans,
epitelial cells., leukocytes
• From culture colonies: G- diplococci
Haemophilus infections
• Noninvasive – upper respiratory tract infection or
superinfection (overinfection) or normal flora of
URT mucous membrane – non encapsulated
strains of H.influenzae, H.parainfluenzae
• Invasive – infections connected with
bacteraemia – encapsulated strains of
H.influenzae – in 98% od type b H.i.b –
meningitis, sepsis, prim. pneumonia, artritis,
cellulitis – age distribution from 6th mnths – 3rd
year
Haemophilus infections
• Microscopy – Gram staining Haemophilus influenzae
• Cultivation -Haemophilus influenzae on chocolate
agar- satelite growth, identification withgrow factors
X,V,XV
• Biochemical activity – not common
• Antigen detection of H. influenzae typ b – diagnosis of
bacterial infection of CNS
• antigennic structure H. influenzae typ b – capsule
detection (a,b,c,d,e,f type)
Microscopy
• - Gram staining- Haemophilus influenzae - G - rods ( coccoid
to filamentous
• Capsule detection in H.influenzae – Burri method,
- quellung reaction- aplication of specific antiserum (anti a, b, c,
d, e or f) to the testing culture and staining sc Burri or in native
smear growing – magnification of capsule
- aglutination with specific antiserum - agglutination and
clearing of suspension after application of specific antiserum to
the colony on slide
Cultivation
• -Haemophilus influenzae on chocolate agar and on blood agara
– satelite growing,identification with grow factors discs X,V,XV
• Chocolate agar – heat destruction of ery let haemin to be available from
cells for bacteria and NAD present in ery - factor X a V
• Blood agar - Stafylococcus aureus spreads NAD (V factor) – in his
environment Haemophilus can grow
• Requiremnt of X or V or both factors serve for idnetificatio of heamophilus
sp - H.i. – need both XV, H. parainfluenzae only V
• Antigen detection of Hib – rapid test for detection of bacterial
infection of CNS
• Presence of H.i.b in CSM, blood,
urine can be detected by use of rapid test
of latex agglutination
Antibody against Hib is bound
on latex particules
If Hib is present in the sample,
it will make a strong bound
to latex and eye visible agglutination
is detected. Very rapid dg - 30 minutes. Reaction of nonvital
bacteria- even after previous therapy with ATB
• Need of rather a big quantitiy of bacteria – correlating with
visible bacteria in microscopic smear and clear clinical signs.