A(nother) Mysterious Soft Tissue
Infection from Tasmania
Alistair McGregor –
Royal Hobart Hospital
Mitchell Brown –
Westmead Hospital
1
Ms CC
2
Aged 44
Operating theatre technician in
Hobart
Previously well
No significant past history
No meds
Driving on the West Coast of
Tasmania after a bush walk
Bitten whilst removing juvenile
Ringtail Possum from the road
Ms CC - Progress
Day 1
–
Day 4
–
Superficial bite to dorsum of right index finger
Purulent discharge from site, swollen tender
epitrochlear nodes, systemically unwell with
documented fever and rigors
Day 5
–
Attended GP
3
No swabs but bloods taken, commenced oral
Cephalexin
Progress –
contd.
4
Day 16
– Returned to GP - “no better”
Finger lesion healing, nodes larger,
remains unwell
Finger lesion swabbed ,further bloods,
ultrasound = two enlarged nodes with
surrounding oedema
Commenced Flucloxacillin
Progress –
contd.
5
Day 29
– Skin breakdown over epitrochlear nodes with
purulent discharge, axillary lymphadenopathy
– Swab repeated, call to local ID physician
Discuss with micro lab ? AAFB
Oral Amoxycillin / Clavulanate and Ciprofloxacin
Refer if no improvement
Progress – contd.
6
Day 33
– Improvement in systemic symptoms but persisting
epitrochlear mass with discharging sinus and axillary
swelling
– Incision and drainage, further micro specimens sent
Day 36
– Increased axillary swelling. Ultrasound = hetrogeneous
mass in axilla, “adenopathy with central necrosis”
– Referred to ID clinic at RHH
Progress – contd.
Day 46 - ID Clinic
–
–
–
Scar on dorsum of right index finger
Two ulcers proximal to right medial epicondyle
with surrounding erythema and induration
Axillary lymphadenopathy
Provisional Diagnosis
Ulcerating Lymphadenitis secondary to a
possible Mycobacterial infection
7
Progress – contd.
Admitted to hospital
Excisional biopsy of epitrocheal mass and
aspiration of axillary mass
–
–
–
–
Commenced on empiric antimicrobial regime
–
8
Histology = Necrotising granulomatous inflammation
Microscopy (Gram and acid fast stains) = Negative
Culture ( Bacterial, mycobacterial, fungal) = No
growth
PCR – 16sRNA (Westmead Hospital)
Isoniazid, Ethambutol, Rifabutin, Moxifloxacin
Discharged with outpatient follow - up
Specimens
arrive in Sydney
9
R axillary Lymph node biopsy received, tissue and in
broth
16s rRNA PCR and sequencing
Culture performed in parallel, extended incubation
Culture results
Culture = NG
@ 7 days, also
@ 2 weeks
BA (O2, CO2
AN) BHV AN
BHI broth
10
16S rRNA sequencing
•3 primers targeting conserved area of the U1, U3 and U5 regions
of the 16S rRNA gene, amplifying the intervening variable regions
•Sequences are merged where possible to create a construct
spanning most of the ~1500bp
•Assay prone to contamination
•In this instance only a small clean amplicon of the U3 region was
achieved, severely limiting the strength of BLASTN result.
HOWEVER….
11
Something unexpected….
12
97% sequence
homology
Francisella
tularensis
not conclusive
Investigate
further
Gram Stain - axilla aspirate
13
GNCB small, most
easily seen with
hindsight! From
centrifuged aspirate in
Thioglycolate broth
DFA – CDC LRN protocols
Francisella tularensis
detected by DFA (CDC LRN
Protocol)
FITC labelled rabbit
polyclonal anti- F. tularensis
antibodies bind to membrane
proteins and
lipopolysaccharides
14
Francisella tularensis RT PCR
Francisella tularensis detected by RT PCR on
the Lightcycler (CDC LRN Protocol)
3 undisclosed targets – 2/3 presumptive ID
(3/3)
15
Preliminary Result Summary
Specimen
Elbow tissue
F. tularensis
PCR
F. tularensis
DFA
F. tularensis
Culture
Negative
Positive
NG
Inhibited
Positive
NG
Positive
Positive
NG
Positive
Positive
NG
18/4/11
Fungal BC
24/4/11
Axilla aspirate
1
27/4/11
Axilla aspirate
2
27/4/11
16
Further testing
17
Preliminary results now suggestive of
Tularaemia
Discussion with RHH clinically possible
Confirmatory testing desirable
Renewed effort to culture organism
Virapid® TULARAEMIA kit
Control
Patient
serum
Test Line
18
Immunochromatographic qualitative
detection method
total anti Francisella
tularensis
antibodies
recA gene sequencing
19
Recombinase A gene ubiquitous in bacteria,
vital in homologous recombination and DNA
repair.
Useful for speciation of some genera
RESULT: 939/965 bp amplicon showed 99%
homology with several tularensis ssp
including holarctica, tularensis, mediasiatica
and novicida
PCR for speciation
Published primers – Birgit Huber et al IJSEM 2010
7 primer pairs
2 of these were positive
RD1 – Francisella tularensis
RD6 – Size of amplicon used to subspeciate
629bp F. tularensis ssp holarctica
840bp F. tularensis ssp holarctica bv japonica
1325bp F. tularensis ssp tularensis
CC isolate = 835bp by capillary fragment separation (close
match to F. tularensis ssp holarctica bv. japonica)
20
Rpt Rpt Culture
21
10% chocolatised blood GC base + Isovitalex
(Cysteine)
BCYE (legionella)
Shell vials (cell culture)
Possum serum in A8 plates (Mycoplasma)
Brucella broth
Blood culture bottles
Tulare County, California
22
Francisella
23
Identified in 1911 following an outbreak of
‘bubonic plague’ (suppuration of
inflammed lymph nodes mimics bubonic
plague)
Tularaemia - Rabbit Fever, Pahvant
Valley fever, Deer fly fever, Lemming
fever, Ohara’s disease
At least 6 clinical presentations;
ulceroglandular >75%
glandular
oropharyngeal
pneumonic
oculoglandular
typhoidal
Endemic in North America, Europe and
parts of Asia
Edward Francis
Francisella tularensis ssp. tularensis
Francisella tularensis ssp. holarctica
Francisella tularensis ssp. mediasiatica
24
Francisella tularensis ssp. tularensis
Classic type A tularaemia
Most severe disease in humans and
in animals
Francisella tularensis zoonotic,
found in >300 species of mammals,
invertebrates, birds and amphibians.
Arthropod vectors include ticks,
biting flies
and mosquitos
Francisella
novicida
ssp. tularensis most often from
terrestrial environments with rabbits
primary hosts and ticks the main
athropod vector
USA 1940-1942 6% mortality
Pneumonic/typhoidal up to 80%
Biochemical differences - Ferments
glycerol, Citrulline ureidase pos
Francisella tularensis ssp. tularensis
Francisella tularensis ssp. holarctica
€€
Francisella tularensis ssp. mediasiatica
25
Francisella novicida
Francisella tularensis ssp. holarctica
type B Tularaemia
less severe disease in humans and in animals
Greater geographic distribution / ?more heterogeneity
ssp. holarctica possibly more associated with aquatic environments
Russia, Sweden, Austria, Czechoslovakia and Turkey 1940-1942 <1% mortality
Biochemical differences – glycerol fermentation neg, Citrulline ureidase neg
Francisella tularensis ssp. tularensis
Francisella tularensis ssp. holarctica
Francisella tularensis ssp. mediasiatica
Francisella novicida
Francisella novicida
Reported 2003 from a foot wound of an
immunocompromised patient in the NT1
less severe disease in humans and in
most animals
Limited distribution
26
Margaret Whipp et al J Med Microbiol 2003 vol 52 no. 9 839-842
Francisella tularensis ssp. tularensis
Francisella tularensis ssp. holarctica
Francisella tularensis ssp. mediasiatica
Francisella novicida
27
Francisella tularensis ssp. mediasiatica
less severe disease in humans and in
most animals
Limited distribution – Central Asia
Francisella
28
Can survive 3-4 months in mud, water and dead animals
GNCB, intracellular, fastidious and slow growing aerobic
bacteria, requires cysteine containing media
Beta-lactamase producer
Risk of laboratory acquired infections
SSBA Tier 2 agent – PC3 facility (approved premises) is required
for handling
Day 75
A result was received
29
Progress – contd.
Diagnosis : “Ulcero – glandular” Francisella infection –
species?
Patient recalled, antimicrobials modified:
–
Francisella serology sent and repeated ( CDC Atlanta)
–
30
Moxifloxacin, Doxycyline
Stable low positive titre 1:128
Next two weeks – remained unwell.
Readmitted for IV Gentamicin, rapid clinical response.
Home on oral antibiotics (Moxi / Doxy)
Reviewed at three months – well
Ms CC - Implications
Is this F. tularensis and if so what subspecies?
–
Environmental reservoir?
Animal vectors?
Risk to humans?
–
31
Two prior Australian reports:
F. novicida, F. philomiragia
Laboratory workers, veterinarians, others?
Public and animal health implications?
Acknowledgements
Westmead
Jimmy Ng, Trang Nguyen
Dr Vitali Sintchenko
Dr Neisha Jeoffreys
Greg James, Marion Yuen
CIDM Media Production Unit
RHH
32
Dr Justin Jackson
Dr Chong Ong
Hello……… Possums!!!
33
Questions?