3-month-old child has temperature 37,8-38o C and signs of common cold.

Approximately after week begins cough, it becomes more intensive day by day, with vomiting and apnoe. Blood test shows leucocytosis with predominance of lymphocytes. Cough will slowly disappear in some weeks, but lasts altogether 6 weeks.

Corynebacteriae, Bordetella, Mycobacteriae

Irja Lutsar 2007

What is the agent of the disease? What cause such clinical symptoms (pathogenesis)
cough predominance of lymphocytes

How the disease spread (epidemiology) Diagnostics of the disease Treatment and prophylactics

Bordetella: morphology
Very small Gramnegative coccobacilli Obligatory aerobes Nonfermentative; oxidase-positive, ureasenegative (B. parapertussis oxydase(-), urease(+); B. bronchiseptica oxydase(+), urease(+)) Very fastidious for media: Bordet-Gengou blood agar, charcoal-caoline agar Sensitive to environmental conditions B.pertussis (Gram staining)

Human pathogenic Bordetellae

B.pertussis
Pertussis a disease with paroxysmal cough

B.parapertussis
Mild form of pertussis

B.bronchiseptica
Respiratory disease in animals, but also bronchitis in humans

Epidemiology of pertussis

Epidemiology of pertussis: USA

Human disease only

N Engl J Med 2005;352:1215-1222

Pertussis in some European countries in 1945-2006

700 600 500 400 300

Pertussis in Estonia in different age groups in 1991- 2005

300,00 250,00 200,00 150,00 100,00

immunisation

50 40 30 20 10 0

86 88 90 92 94 96 98 00 02 04 06 19 19 19 19 19 19 19 20 20 20 20

200 100

Schoolentry imm stopped

50,00 0,00

91

92

93

94

95

96

97

98

99

00

01

02

03

04

45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96 99 02 05 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 20 20

19

19

19

19

19

19

19

19

19

20

20

20

20

20

Estonia

Poland

Lithuania

Romania

Czech

Turkey

<1

1_4

5_9

10_14

>15

B.pertussis: virulence factors

Adhesins
Filamentous hemagglutinin (FHA) Pertactin (P69 protein) Pertussis toxin (PT) Fimbriae

B.pertussis: adhesins
Filamentous hemagglutinin binds to glycoproteins of ciliary epithelium; binds to CR3 of PMNL; initiates phagocytosis Pertactin (p69 protein) as with FHA Pertussis toxin classic A-B exotoxin; S2 subunit binds to glycolipid of ciliary epithelial cell and S3 subunit binds to ganglioside of phagocytes Fimbriae binds to mammalian cells and stimulates humoral immunity; exact role in pathogenesis of the disease unknown

Toxins
Pertussis toxin Adenylate cyclase Dermonecrotic toxin Tracheal cytotoxin Lipopolysaccharide

B.pertussis uses adhesines for binding to ciliary epithelium

B.pertussis: toxins
Pertussis toxin S1 subunit inactivates membrane surface protein that controls adenylate-cyclate activity ; cAMP augmentation of slime production (characteristic to paroxyxsmal phase) Adenylate-cyclase/hemolysin - intracellular cAMP; protects from phagocytosis Dermonecrotic toxin necrose of skin in experimental animal model; role in pathogenesis of pertussis not known Tracheal cytotoxin a fragment of peptidoglycan that kills ciliary epithelium and induces release of IL-1 LPS two distinct LPS (lipid A and lipid X); activates alternative complement pathway, their role if the disease unknown

20

05

Pertussis toxin (PT)

Exsaturation of cAMP is important in pathogenesis

Formation of Adenylate cyclasis does not stop cAMP exsaturation H2O production slime

B.pertussis: toxins
Pertussis toxin S1 subunit inactivates membrane surface protein that controls adenylate-cyclate activity ; cAMP augmentation of slime production (characteristic to paroxyxsmal phase) Adenylate-cyclase/hemolysin - intracellular cAMP; protects from phagocytosis Dermonecrotic toxin necrose of skin in experimental animal model; role in pathogenesis of pertussis not known Tracheal cytotoxin a fragment of peptidoglycan that kills ciliary epithelium and induces release of IL-1 LPS two distinct LPS (lipid A and lipid X); activates alternative complement pathway, their role if the disease unknown

Pertussis: clinical findings

Catarrhal period similar to common cold Paroxysmal cough with slime production, ending up with vomiting In vaccinated children clinical findings are milder

Clinical findings and diagnosis of pertussis

1. Incubation 2. Catarrhal period 3. Paroxysmal period 4. Convalescent
PCR + serology serology PCR + culture

Pertussis: diagnosis
B.pertussis extremely sensitive to drying Nasopharyngeal aspirate (epithelial cells are required) culture on site or use of transport media (Regan-Lowe transport media) Bacterial culture charcoal-horse blood agar (ReganLowe medium),
Freshly made medium, prolonged incubation (7 days), in humidated air, at 35o C results depend on quality of sampling and transport technique, stages of the disease, history of AB therapy Appr. 50% have positive results

PCR sensitivity 80-100% Serological tests?

Antibodies against IgA, IgM, IgG by ELISA method against FHA, PT In convalescent period significant rise of antibody titre

Downloaded from: StudentConsult (on 26 February 2006 01:47 PM) 2005 Elsevier

B. pertussis on charcoal agar

B. pertussis Bordet-Gengou agar

Pertussis: treatment and prophylaxis

Treatment
Supportive care Macrolides (erythromycin, clarithromycin, azitromycin)

Prophylactics
Whole cell vaccine inactivated bacteria
Side effects

Acellular vaccines (PT, pertactin, FHA, fimbriae; monovalent and polyvalent) In combinations with diphtheria and tetanus toxoid Immunity lasts for 5-7 years; booster doses required Isolation of patients Erythromycin for contact persons
Identifcation: small colonies on charcoal agar; oxydase.positive

Aerobic, non-spore, G-pos. bacilli

Regular shape
Listeria and Erysipelothrix

L.monocytogenes

Corynebacteriae
G+ rods, irregular, facultatively anaerobes Virulence factors:
Diphtheria exotoxin (C.ulcerans, C.pseudotuberculosis) UTI pathogens produce urease Adhesion to foreign bodies Some multiresistant to antibiotics (C.amycolatum, C.jeikeikum, C.urealyticum)

Coryneforms or diphtheroids
Corynebacterium

C.diphtheriae

Acid-fast bacilli (longchained mycolic acid in cell wall)

Nocardia, Rhodococcus, Mycobacterium

M.tuberculosis

Normally colonise skin, upper respiratory tract, GI tract, urogenital tract Usually endogenous infections (normal microflora) Clinical findings: septicaemia, endocarditis, foreign body infection, wound infections, UTI, RTI

Corynebacterium diphtheriae
Gram-positive irregular rod (V and Y shape) Very variable in size (0,3-0,8 m X 1,0-8,0 m) oxydase- , catalase+ Aerobes or facultative anaerobes Lipids in cell wall: mesodiaminopimelinic acid, arabinogalactane and mycolic acid Resistant in environment because of previous factors Different biotypes depending on morphology and biochemical properties of colonies belfanti, gravis, intermedium, mitis Toxigenic strains

Clinical finding in diphtheria

Depending on: localisation, immune status of organism, virulence of microbe Disease caused only by toxigenic strains Colonisation Respiratory diphtheria (acute beginning, pseudomembranous plaque on tonsills, toxic status, cardiac damage Cutaneous diphtheria
Spreading by direct contacts, nonhealing ulcer following skin lesion

Diphtheria toxin
Typical A-B exotoxin Tox gene transported into cell by -phage of lysogenic bacteriophage Three functional regions
Catalytic region (A unit) Receptor binding region Translocation region

Effect of diphtheria toxin

Tox-gene Inhibition of protein synthesis

Receptor heparin-binding epidermal growth factor

NS and cardiac muscule cells

Effect on protein synthesis by inhibition of elongation factor 2 (EF-2)

Diphtheria: epidemiology
From human to human Historically mostly in children Most important cause of death in 2-14 y old children prior immunisation After immunisation single cases in Brazil, India, Indonesia, Filipines Last outbreak in former SU in middle 1990ties
150 000 cases Mortality 3-23% 60% of patients were adults Caused by low immunity Prior the outbreak increased carriage of toxigenous strains
600 500

Diphtheria in Estonia, 19452005

Difteeria haigestumus , 1991-2005
1,4

Nitaja 100 000 el. kohta

400

N i t a ja 1 0 0 0 0 0 e l . k o h t a

1,2 1

0,8 0,6 0,4 0,2 0

91 92 9 3 94 95 96 97 9 8 99 00 01 02 0 3 04 05 19 19 19 1 9 19 19 19 19 1 9 20 20 20 20 2 0 20

300

immunisation

200

100

Aasta

0
4 19 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 4 4 5 5 5 5 5 6 6 6 6 6 7 7 7 7 7 8 8 8 8 8 9 9 9 9 9 0 0 0 1 9 1 9 19 1 9 1 9 1 9 19 1 9 1 9 1 9 19 1 9 1 9 19 1 9 1 9 19 1 9 1 9 19 19 1 9 1 9 19 1 9 1 9 19 2 0 2 0 2 0

Aastad

Vaccination against diphtheria in Estonia

Diagnosis of diphtheria
Microscopy is less informative Culture nasopharynx and throat
Nonselective, enriched blood agar cysteine-tellurite agar tellurite inihibits growth of most bacteria in nasopharynx Lfflers medium (classical medium for diphtheria) not recommended as first choice anymore

Toxigenicity testing (production of exotoxin)

Elek test (a tissue culture neutralisation assay) In vivo neutralisation assay (guinea pigs) NAT test
False positive results Jgiste et al. 2000

C.diphtheriae colonies in enriched blood agar plate

Elek precipitation test

C.diphtheria strains Tox-negative

Precipitate Tox-positive

Filter paper with antitoxin (500 IU)

Diphtheria: treatment and prophylaxis

Prophylaxis
Immunisation with formaline inactivated diphtheria toxoid
Effective against acute forms of disease, but does not eradicate circulation of C.diphtheriae Effective antibody titre 0,1 0,001 IU/ml Immunity is vaning, booster doses required every 10 years Combined vaccine DTP or dT Vaccines for children and adults (different amount of toxoid)

Mycobacterium
Non-motile, non-sporeforming, aerobic G+ rods Cell wall rich in lipids, hydrophobic, resistant to disinfectants & laboratory stains Acid-fast (once stained cannot be decolorised with acids) Very slow growth (3-8 weeks of incubation) Appr. 100 species identified; not all are human pathogens M.tuberculosis spreads from human to human with aerosol Other mycobacteria from environment

Treatment
Antitoxic serum (antitoxin)!!! Penicillin or erythromycin Symptomatic treatment No effective antibodies after natural infection Immunisation after infection needed

Components of cell wall of mycobacteria

Most common mycobacteria in human

Microorganism
M.tuberculosis group

Pathogenicity Pathogen sometimes pathogen

Clinical findings Tuberculosis

Lymphadenitis, TBC-like in immunocompromised

M.tuberculosis M.bovis (BCG strain) M. leprae

Leprosy (Hansens disease)

Non-tuberculous mycobacteria

M.kansasii *M.avium complex M.abscessus

The components include the (A) plasma membrane, (B) peptidoglycans, (C) arabinogalactan, (D) mannose-capped lipoarabinomannan, (E) plasma-associated and cell wall-associated proteins (PPD), (F) mycolic acids, and (G) glycolipid surface molecules associated with the mycolic acids. (Redrawn from Karakousis et al: Cell Downloaded from: StudentConsult (on 26 February 2006 09:39 PM) Microbiol 6:105-116, 2004.)
2005 Elsevier

Usually pathogen

TBC-like in immunocompromised
Pulmonary disease in COPD patients or elderly people; disseminated disease in AIDS patients

M.chelonae M.fortuitum

Opportunistic pathogen

Catheter infections, foreign body infections, wound infections

*M.avium and M.intracellulare

Mycobacterium avium-complex, Ziehl-Neelsen acid fast stain

M.tuberculosis: pathogenesis
Intracellular pathogen, that is able to establish lifelong infection Spreads with respiratory droplets Microbes enter alveolar macrophages, but are not phagocyted; microbes are able to multiply in phagosome Clinical findings (and histology) primarily components of immune response rather than toxic effects of microorganisms Intracellular replication of microorganisms activation of T-helper cells (CD4+) & cytotoxic T cells (CD8+), release of interferon activation of macrophages Collection of macrophages (granulomas) killing of microbes or forming necrotic/caseousus granulomas with dormant bacteriae reactivation and late infection

Tuberculosis: from infection to immune response

TBC pathogenesis

TBC: clinical findings

Pulmonary TBC

Figure 29-4 Pulmonary tuberculosis.

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Patients with tuberculosis (Dec. 31, 2003)

2001 2002 2003 Pulmonary tuberculosis Extrapulmonary Altogether MDR Adolescents Recoveries deaths by tuberculosis 597 54 651 236 4 610 72 376 65 441 226 4 579 82 446 56 502 187 3 502 64

Extrapulmonary tuberculosis in Estonia in 2003

Extrapulmonary TBC Bacterially proved MDR from proved Thoracal lymphous TBC Tuberculous pleuritis Bone-joint system TBC Urinary-genital TBC Peripherial l/s TBC Intestinal and peritoneal
No CNS TBC
TB register 2004 TB register 2004

Primary recidives Altogether 55 11 66 30 6 36 4 1 5 3 0 3 19 19 16 6 22 11 3 14 3 2 5 3 3

TBC clinical findings

TBC diagnostics
Cell mediated immunity (skin tests)
PPD (purified protein derivates)
Reactivity to other mycobacteria, and BCG

INF- (Quatiferon-gamma test)

Microscopy detection acid-fast bacteria

Induced sputum; gastric aspirate Ziehl-Neelsen method (staining with carbol fuxine)
Detects 30-50% of culture positives

KNS TBC

Bone TBC

Tubercules (granulomas) in an asymptomatic patient

Fluorescent auramine-rhodamine dyes Specificity 95%, + test corresponds with higher infectivity

M. tuberculosis in lung, Ziehl-Neelsen acid fast stain

TBC diagnostics
Bacterial culture
Induced sputum in morning, gastric aspirate in children Culture from other sites often negative Slow growth (3-4 weeks) Lwenstein-Jensen agar; new enriched broth media 10-14 days Preliminary identification by morphology of colonies Final indentification by biochemical tests

PCR, ligase chain reaction, transcription mediated amplification

Low sensitivity

Mycobacterium tuberculosis on Lowenstein-Jensen agar

Mycobacteria on the LwensteinJensen agar

Characteristic non-pigmented buffy colonies M. tuberculosis

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M.kansasii

MAC

TBC: epidemiology
Spread by close human to human contacts With the improvement of living condition prevalence decreased Low prevalence in Western countries but not in Africa and Asia With HIV epidemic TBC is in rise again

TBC higher incidence if Africa

per 100 000 population

< 10 10 to 24 25 to 49 50 to 99 100 to 299 300 or more No Estimate

The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement.
WHO 2002

Tuberculosis in Estonia primary cases 1962 2005

200 150 100 50 0
62 19 64 19 66 19 68 19 70 19 72 19 74 19 76 19 78 19 80 19 82 19 84 19 86 19 88 19 90 19 92 19 94 19 96 19 98 19 00 20 02 20 04 20 06 20

Tuberculosis in Estonia primary cases 1962 2005

200 150 100 50 0
62 19 64 19 66 19 68 19 70 19 72 19 74 19 76 19 78 19 80 19 82 19 84 19 86 19 88 19 90 19 92 19 94 19 96 19 98 19 00 20 02 20 04 20

TB register 2004; Tervisekaitseserver

Arenenud Lne Euroopa riigid

TB register 2004; Tervisekaitsesrver

Tuberculosis in Baltic sea region countries in 2001

90 80 87,9 70 71,8 60 50 40 30 20 10 10,9 6,7 0
140 120

Tuberculosis cases by gender and age groups in 2003

Tuberkuloosihaigestumine
60,3 41,7 14,1 7,7 8,1 1

MDR TB %

100 80 60

Kokku Mehed Naised

4,4 1,1

5,9

40

Venemaa

Lti

Leedu

Eesti

Soome

Rootsi

Norra

20 0 0-14 15-24 25-34 35-44 45-54 55-64 65<

TB register 2004

TB register 2004

TBC prophylaxis
Immunisation
BCG attenuated M.bovis strain Used in endemic countries Efficacy 0 80% Prevention of disseminated disease (CNS TBC, disseminated)

TBC: treatment
M.tuberculosis
M.tuberculosis is resistant to most antibiotics Long-lasting combination therapy
INH, etambuthol, rifampicin, pyrasinamide; MDR Second line - fluoroquinolones

Chemoprophylaxis
People exposed to M.tuberculosis
INH or Rifampin (+ pyrazinamide)

HIV+ and other immune compromised

DOT MDR, XDR

MAC
macrolides + etambuthol or rifabutin

Mechanisms of action of some antibiotics targeting M. tuberculosis

Rifampin and rifabutin

Rifamycin B synthetic derivates, originate from Streptomyces mediterranei Binds to DNA-depending RNA polymerase synthesis and inhibits the beginning of RNA synthesis Very active against M.tuberculosis and many G+ bacteria G- bacteria are resistant (decreased uptake of hydrophobic agent) Fast development of resistance (mutation of chromosomal gene coding -subunit of RNA polymerase)

Nature Biotechnology 23, 187 - 188 (2005)

10

XDR-TB (pinc) in 2007

Nature Medicine 13, 295 - 298 (2007)

Mycobacterium leprae
Causes leprosy (Hansens disease) Two clinical forms
Tuberculoid leprosy (strong cellular immune response, many lyphocytes and granulomas in damaged tissues, but few bacteria) Lepromatous leprosy (no cellular immunity; abundance bacteria in dermal macrophages)

Leprosy clinical picture

Epidemiology
From human to human (skin contact, inhalation) Incidence remarkably decreased WHO 286 000 cases in the world by the end of 2004 Reason for decrease - effective treatment (dapsone + rifampicin + clofasimin)

Tuberculoid leprosy

Lepromatous leprosy

MAC
M.avium and M.intracellulare Weakly G+, high resistance to acids; cell wall rich in lipids Intracellular growth; clinical finding characterise organisms immune response Epidemiology
Occurs world-wide; most ofter in the areas with low prevalence of TBC Sources: contaminated water and food; inhalation? Immunocompromised patients (AIDS, COPD) Clinical findings

MAC
Lady Windermeres syndrome Asymptomatic colonisation Chronic pulmonary disease Infiltrates of middle lobe and nodules in elderly women Pulmonary nodules Disseminated disease in HIV patients

Treatment
Macrolides + ethambutol + rifabutin Macrolide-prophylaxis in AIDS patients

11