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.