Transcript Objectives

Objectives
• By the end of this lecture the student must be:
 A) Identify the genus Legionellae, Yersinia,
Francisella and Brucella
 B) describe the chemical tests for this genus
• C) Differentiate between different sps.
• D) List and match the symptoms, diagnosis
and treatment for different sps.
Legionellae
•
•
•
•
•
•
•
•
•
•
•
Gram negative rod belonging to Legionellaceae
Stains poorly with gram stain
Facultative intracellular pathogen
> 57 species, ½ of species implicated in human disease
L. pneumophila is the most important human pathogen
Recently became opportunistic human pathogen
~ 90% of all cases of legionellosis (Legionnaires' disease)
15 serogroups, 88.6% of cases are caused by serogroup 1
Serogroup 1 seems to be more virulent
Not grow on Sheep Blood Agar
Requires specialized media to grow
• Cysteine and iron are essential for growth
2
L. pneumophila
• Reservoir principally aquatic
• L. pneumophila is not a free-living organism but an intracellular
parasite of amoebae
• Natural environment
– Lakes and rivers
• Artificial environment
– Showers, taps, air conditioning system
– Normally found in hot water (up to 500C) tanks
– Multiply in pipes in sediment & live many years
– Sediment provides shelter and nutrition for other bacteria that
can supply cysteine
• Important to detect L. pneumophila serogroup1 in water
3
Legionnaires disease
• The portal of entry is the respiratory tract via inhalation
• Legionellae are associated with water-based aerosols
1. Air conditioning cooling towers
2. Sauna
•
Person to person spread does not occur
• Nosocomial infections
– Due to presence of bacteria in water taps, sinks & showers
4
Legionnaires disease
• In general, healthy people rarely get disease
• People under Risk
• People with defect in cell mediated immunity
1. Typically occurred in older man who smokes and
consumes substantial amount of alcohol
2. Patients with AIDS, cancers, Renal transplants, patient
treated with corticosteriods or elderly
5
L. pneumophila
• Initial host response: acute inflammatory response of
aveoli & then bronchioles (similar to pneumococcal infection)
• Neutrophils accumulate followed by macrophage
• Different from pneumococcal infection
1. Organisms located inside of macrophage
2. Inhibit lysosomal fusion & acidification of phagocyte
6
Clinical Presentations
• Pontiac fever
– Pontiac, Michigan in 1968
– Incubation period 1-2 days
– Flu-like, Milder (no mortality) and self-limiting
– Persists for 2-5 days, then spontaneously resolves
• Legionnaire's disease
– Incubation period 2-10 days
– Atypical pneumonia
– Most cases resolve spontaneously in 7-10 days but
– In older or immunocomprmised may be fatal
– 15-75% mortality
7
Laboratory Diagnosis of Legionella
• Specimen: sputum and blood
• Culture: on Buffered Charcoal Yeast Extract Agar (BCYE)
– Grow after 3-5 days
– Appear as small colonies with ground glass appearance
• Microscopy -Difficult because of
• Lack of staining
• Intracellular nature
• Require large number of organisms to detect
• Detection of antigen in respiratory secretion or urine
• Seroconversion: ≥4 fold rise in specific serum antibody titer
• Direct fluorescent antibody (DFA) staining
8
Yersinia, Francisella and Brucella
• Small Gram-negative rods
• Zoontic diseases
• True pathogens: isolation always associated with disease; i.e.,
always clinically significant
• NOTE: Previously studied nonfermenters were all opportunistic
pathogens
• Very virulent and able to penetrate any body area they touch
– Skin through insect bite, animal bite or direct contact with animals
– Lung after inhalation of infected aerosolized matter
• Non-fermentative
• Facultative intracellular pathogens
• Common treatment: Aminoglycosides and/or Doxycycline which
must be given for prolonged period so as to reach the hidden
intracellular bacteria
9
1. Brucella
• Brucella are small gram-negative coccobacilli, strict aerobic, nonfermentative lacking a capsule, flagella, endospores
• Oxidase and catalase are positive
• Some species require 5-10% CO2 (B. abortus ) for primary isolation
in Lab.
• Requires specialized media and prolonged incubation for growth in
culture
• Facultative Intracellular pathogen
• Brucella spp. is the causative agent of brucellosis
– Brucellosis is a zoonotic infection transmitted to humans
– The disease is rarely, if ever, transmitted between humans
– Brucellosis also called Undulant fever, Malta fever,
Mediterranean fever
10
Human Brucellosis & Associated Species
Severe
11
Epidemiology
Brucellosis occurs worldwide; major endemic areas include
countries of the Mediterranean basin, Arabian Gulf, Indian
subcontinent and Mexico, Central and South America
Major in Saudi Arabia especially Aseer
B. melitensis is the species that infects humans most frequently
The incubation period ranges from a few days to a few months
The disease is manifested as fever accompanied by a wide array of
other symptoms
12
• Human brucellosis is mostly found among farmers, Vet and others
who work with infected animals
• The organism enters the body through
1. Ingestion of contaminated food such as raw milk, cheese made
from unpasteurized milk or raw meat
2. Direct inoculation through skin abrasions from handling animal
carcass, placenta or contact with animal vaginal secretions
3. Inhalation of infectious aerosols
• Localize on reticule-endothelia system (Lymph nodes, liver,
spleen, bone marrow)
• Many organisms are killed by macrophage but some survive
where they are protected from antibodies
13
Clinical Signs of Human Brucellosis
Multi-systemic disease with a broad spectrum of symptoms
Human brucellosis is manifested by intermittent fever
Patient has 3-4 weeks fever and then 3-4 weeks without fever
Incubation Period:1-6 weeks
Asymptomatic infections are common
The disease is extremely variable and the clinical signs may appear
abruptly in symptomatic cases
 After incubation period, an acute febrile illness with non-specific flulike signs occur
 Fever, malaise, chills, night sweats, fatigue, weakness,
myalgias, weight loss, arthralgias and nonproductive cough
 Splenomegaly, hepatomegaly and chest pain
 Anorexia, nausea, vomiting, diarrhea and constipation
14






Clinical Signs of Human Brucellosis
 In many patients, the symptoms last for 2-4 weeks and are
followed by spontaneous recovery
 Others develop an intermittent fever
 Most with this undulant (Rising and falling) form recover
completely in 3-12 Months
 A few become chronically ill
 Relapses (10%) can occur months after the initial symptoms
 Chronic disease and recurrence are common. Why?
 Because it can survive in phagocytes & multiply to high
concentrations
 Complications are seen occasionally, particularly in the undulant
and chronic forms
 The most common complications are arthritis, spondylitis,
epididymo-orchitis and chronic fatigue
15
Brucellosis in Animals
 Brucella cause abortion in animals
 Brucella infects organs rich in erythritol
 Breast, uterus, placenta and epididymis
 Does Brucella cause abortion in humans?
 Brucella does not cause abortion in Human. Why?
 Because of absence of erythritol in human placenta and fetus
 Asymptomatic carriage, sterility or abortions
 Transmitted between animals in aborted tissues
16
Laboratory Diagnosis
A. Culture
• When brucellosis is suspected, Lab. should be informed to
maintain cultures for minimum of 4 weeks
• Blood culture is done into liver infusion broth during febrile attack
• Inoculate 2 tubes, one of them in 10-20% CO2 and the other in the
normal atmospheric conditions
• Rate of isolation from blood ranges from 15-70% depending on
methods used and incubation period
• Bone marrow or lymph node cultures can be done if the blood
culture is negative
• Bone marrow cultures have higher yield than blood
• Most Labs. use rapid isolation techniques, (e.g. BACTEC)
• PCR used for rapid diagnosis of Brucella in blood specimens
17
Laboratory Diagnosis
Serological Tests
• Most serological studies for diagnosis of Brucellosis are
based on antibody detection
• Antibodies appear in the patient’s serum 7-10 days after
the onset of clinical features
• These include:
1. Serum agglutination (Standard tube agglutination)
2. ELISA
3. Complement fixation
4. Indirect Coombs
18
Serum agglutination
• This test is insensitive and should not be relied on for diagnosis
• A titer of >1:160 supports the diagnosis of brucellosis
• A fourfold increase in agglutinating antibodies over 4 to 12 weeks provides even
stronger evidence for the diagnosis
• A titer of 1:100 may be met in normal people
– Due to previous subclinical infection
• False-negative reactions
– Result from prozone phenomenon (High serum concentration)
• False-positive reactions
– Result from cross-reaction with antibodies to Yersinia, Cholera and Tularemia
• These can be avoidable by routinely diluting serum beyond 1:320
• Must be done using wide range of dilutions of patient’s serum
• In certain conditions antibodies are blocked giving no visible agglutination, so
Coomb’s test must be done
19
Prevention and Control
• Brucellosis can be eradicated from animals by testing them
and slaughtering the positive
• Immunization of animals with a live attenuated vaccine
• Pasteurization of milk and dairy products
• Use of proper safety techniques in clinical laboratory
working with Brucella
20
Treatment
• Intracellular localization of brucellae, believed to offer some
protection against antimicrobials, thus drugs with good intracellular penetration are necessary for cure
• Tetracyclines among most active drugs for treating brucellosis
• Relapse rate unacceptably high with single-drug therapy
• Combination therapy is recommended
• There are two major regimens:
• Regimen A: Doxycycline 100 mg orally twice daily for 6 weeks
+ Streptomycin 1 gm IM once daily for the first 14 - 21 days
• Regimen B: Doxycycline 100 mg orally twice daily plus
rifampin 600-900 mg (15 mg/kg) orally once daily for 6 weeks
21
Yersinia Species
Yersinia formerly classified in the Pasteurellaceae family
So Yersinia formerly called Pasteurella
Yersinia reclassified in the Enterobacteriaceae family
Three species of Yersinia cause disease in humans
Y. pseudotuberculosis
Y. enterocolitica
– Both are animal pathogens
– Y. pseudotuberculosis causes pseudotuberculosis in animals
– Y. enterocolitica sometimes infects man causing Yersiniosis
– Fever, diarrhea, abdominal pain and arthritis
– Yersiniosis is usually self-limiting and does not require treatment
– Both are enteric food and water borne pathogens
– Acquired by ingestion of contaminated food
3. Y. pestis
22
•
•
•
•
1.
2.
Y. pestis (formerly Pasteurella pestis)
•
•
•
•
•
•
•
•
•
•
•
•
•
Discovered in 1894 in Hong Kong by Alexandre Yersin
Gram negative short bacilli (coccobacilli)
Exhibit bipolar staining (special stain e.g. Giemsa stain)
Dark blue bioplar and the remaining appears light blue
Facultative anaerobes, Fermentative, non-motile, non-spore forming
Oxidase negative (Pasteuralla and Brucella are oxidase positive)
Have capsule when freshly isolates
Loss of their capsule when passed in laboratory
Found in low frequency in wild rodent populations (rat)
Rat flea is prime transmitter of disease
Disease:
Plague (Black death)
Disease has an extremely important place in human history
23
Virulence Factors
• Pathogenic Y. pestis produce two antiphagocytic components;
• Fraction 1 (F1) capsular antigen and the VW antigens
• Both are required for virulence
• They are only produced when the organism grows at 37°C
• They not expressed at temperatures < 37°C
• Yersinia is not virulent in fleas Why?
– Because their body temperature normally levels around 25°C
• The bacteria are capable of surviving and multiplying within
monocytes, but not PMNs, and upon emerging from the monocytic
host, the bacteria possess their F1 and VW antigens
24
Transmission Cycles
25
Transmission of Y. pestis
• Bubonic and Septicemic plague
– Disease endemic to rat species
– Flea bites rat and then Y. pestis replicates in flea’s digestive tract
– A solid mass forms, Obstructs the fleas gut, Transfers to the flea’s
mouth and then Introduced to human by flea bits
– Bubonic and Septicemic can occur directly when rat bites human
• Bubonic and Septicemic can not be transferred human to human
• Pneumonic – primary:
– Contracted when infected droplets are directly inhaled
– This can be passed from person to person
• Secondary: develops when bubonic or septicemic goes untreated 
moves to lungs and then can be spread to someone else
26
Types of Plague
• Incubation period 2-6 days
1. Bubonic Plague
– Most common
– Infection of the lymph system (attacks immune system)
– Fever, headache, chills, weakness, swollen and tender lymph
glands
2. Pneumonic Plague
– Most serious type of plague
– Infection of the lungs leading to pneumonia
– Fever, headache, weakness, rapid onset of pneumonia
(accompanied by: shortness of breath, chest pain, cough, bloody
or watery sputum)
3. Septicemic Plague
– Bacteria reproduces in the blood
– Can be contracted like bubonic plague but is most often seen as
a complication of untreated bubonic or pneumonic plague
– Fever, chills, weakness, abdominal pain, shock, bleeding
underneath skin or other organs
27
Stages of Disease
• Bacteria travel through the blood to the nearest lymph nodes
• In lymph nodes, Y. pestis is ingested by fixed macrophages
– Y. pestis grows in macrophages but not PMNs and replicates
– Elicits an inflammatory response (the bubo)
– Bubo is swelling of the lymph nodes
• Bacteria from the bubo leak into blood stream (septicemic plague)
• Lysis of the bacteria releases LPS, which causes septic shock
• Eventually bacteria reach the lung, where they parasitize the lung
macrophages (pneumonic plague)
• At the pneumonic stage, the bacteria can be spread to others via
aerosols (respiratory droplets)
• Direct inhalation at this point of the disease, induces more rapid
development (than flea)
– At this stage the bacteria have well developed virulence factors
28
needed to colonize the human body
Lab diagnosis
• Diagnosis is based primarily on clinical suspicion
• Specimen
– Blood (Septicemic)
– Sputum or (Pneumonic)
– Pus from the bubo (Bubonic)
• Direct smear
– By Giemsa stain shows the characteristic morphology of Y.
pestis
• Smear is the best diagnostic procedure
• Culture
• Blood agar: non-hemolytic
• or enteric media (MacConkey agar): non-lactose ferment
– After 24-48 hrs the colonies were pinpoint grey to greyish white, and
slightly mucloid
29
Treatment
• Without treatment, fatality rates: up to 90% for bubonic
plague, 100% for Septicemic or pneumonic plague
• Treatment, fatality rate= (5-20%)
• Rapid treatment is critical to improved survival
• No major antibiotic resistances have developed
• Plague is usually treated with Streptomycin (Drug of choice)
• Other antibiotics
fluoroquinolones
:
tetracyclines,
chloramphenicol,
– Course of treatment: 10-14 days
30
Prevention and Control
•
•
•
•
•
Keep rodent population down by proper disposal of garbage
Anti-rats, anti fleas measures
Eliminate crowded living conditions of substandard housing
Strict Isolation of cases (Quarantine)
Vaccines
– Effective but protects for less than a year
– Heat killed vaccine consists of whole killed Y. pestis cells
– Requires a series of injections over a 6 month period
– Live attenuated vaccines: Injection of non-pathogenic mutant,
derived from a fully virulent strain
• Chemoprophylaxis
– Sulfonamides and tetracyclines during epidemics
31
Francisella tularensis
• Small gram-negative coocobacilli (0.2 by 0.2-0.7 µm)
• Nonmotile, non-spore forming, strict aerobic, and non-fermentative
• Fastidious and slow-growing
 Requires cysteine-supplemented specialized media
 Blood-glucose-cysteine agar
 Requires prolonged growth
• Facultative intracellular bacterium
• Survive up to weeks at low temperatures in water, soil, and animal
carcasses
• Major target organs are lymph nodes, lungs, pleura, spleen, liver
and kidney
• One of the most infectious pathogenic bacteria known
• Substantial capacity to cause illness and death
32
Francisella tularensis
• Disease:
– Tularemia Also Known As…
– Rabbit fever, Deer-fly fever, Glandular tick fever
– It resembles bubonic plague
• Virulence Factors
– Antiphagocytic capsule
– Thin lipid capsule present in pathogenic strains
– Facultative intracellular parasite that can survive in
macrophages of the reticuloendothelial system
33
Clinical Presentation of Tularemia
NOTE: Also Gastrointestinal & Pneumonic forms of disease
34
Two subspecies (biovars)
Characteristics
Jellison Type A
Jellison Type B
Geographic distribution North America
Europe, Asia
Source of infection
Rabbit and ticks
Contaminated water or
rodents
Severity
Highly virulent (Sever)
Relatively virulent (Mild)
Mortality
5-6% in untreated <0.5% in untreated
cutaneous disease
cutaneous disease
Biochemical properties
•Glycerol fermentation Positive
•Citrulline ureidase
Positive
Negative
Negative
35
Infection
• Incubation period
– 1-21 days (average=3-5 days)
• Infective dose
– 10-50 organisms
• Duration of illness
– 2 weeks
• Mortality
– treated: low
– untreated: moderate
– Ulceroglandular and glandular tularemia are rarely fatal
(mortality rate < 3%)
– Pulmonic or Septicemic tularemia is more acute form of
disease (mortality rate 30-60 %)
• Vaccine efficacy
– good, ~80%
36
Route of Transmission
Skin or conjunctiva
Skin
GI tract
Respiratory tract
Mode of Transmission
Handling of infected animals
Bite of arthropod (vector) which has fed on an
infected animal (Reservoir)
Ingestion of improperly cooked meat or
contaminated water
Aerosol inhalation
• Very small number of organisms to become infected (10-50)
• Humans cannot transmit infection to others
• Reservoirs
– Rabbits, Aquatic Rodents, Rats, Squirrels, Lemmings, Mice
• Vectors
– Ticks, Mosquitoes, Biting Flies
• Ulceroglandular Tularemia (Most common)
– Transmitted through a bite from an arthropod vector which
has fed on an infected animal (Reservoir)
37
Diagnosis of F. tularensis
 Because this bacterium so virulent, most labs will not
culture it from pus or blood
 It is not advisable to drain infected lymph nodes
 Fastidious and slow-growing
 Requires cysteine-supplemented specialized media
 Requires prolonged growth
 Diagnosis rests on
• Clinical picture
• Skin test similar to tuberculin and brucellin
• Measurement of antibodies titer
38
Prevention
• Avoidance of reservoirs and vectors
• Protective clothing and gloves
• Laboratory personnel should be made aware of potential for Fransicella in
clinical specimens
• Best Immunity (Permanent)
– Previous infection with avirulent strain
• Live Vaccine Strain (LVS)
– Best prophylactic
• Only effective vaccine against tularemia
• Doesn’t provide 100% immunity
• Possibility of varying immunogenicity between different batches
• Possibility of a spontaneous return to virulence
• Foshay’s Vaccine (killed bacteria)
– Provides lesser immunity towards systemic and fatal aspects of disease
than LVS
39