Tissue Triage and Special
Studies for Tracking
Mycobacterial Disease
Dan Milner, MD
June 6, 2009
Washington, D.C.
MAPA/PANE
Mycobacterial Disease
• Tuberculosis
– Immunocompromised and competent
• Atypical Mycobacteria
– Immunocompromised (TB-like, non-TB-like)
• Atypical Mycobacteria
– Immunocompetent (rare, syndromes)
• Other Mycobacteria
2 Billion Worldwide
Countries of Birth for Foreign-born
Persons Reported with TB
United States, 2003
Mexico
(26%)
Other
Countries
(36%)
S. Korea
(2%)
Haiti
(3%) China
India
(5%) (8%)
Philippines
(12%)
Viet Nam
(8%)
Tuberculosis
Tuberculosis
MYCOBACTERIA
•
Mycobacteria tuberculosis
–
–
–
•
–
–
–
–
Mycobacteria gordonae
–
•
High incidence in AIDS patients and
elderly women
Sepsis and positive blood cultures
common
AIDS: fever of unknown origin and weight
loss common; pulmonary disease
uncommon
Immunocompetent hosts or elderly:
pulmonary disease common
Most common AFB involving bone marrow
Rare human infections
Mycobacteria szulgi
– Rare human infections
Mycobacteria kansasii
–
Ghon complex in primary TB (middle lobe
or apical lower lobe)
Secondary TB in apical portion of upper
lobes
Tuberculous effusions - mature
lymphocytes
–
–
Mycobacteria avium-intracellular
–
•
•
•
Mycobacteria marinum
–
–
•
Superficial granulomatous skin
infection (“swimming pool” or “fish tank”
granulomas) involving traumatized skin
of the extremities in contact with poorly
chlorinated fresh water
Sporotrichosis-like lesions (chain of
ulcers up the arm along the lymphatics)
Mycobacteria simiae
–
•
Chronic pulmonary disease similar to
classic TB except it is noninfectious
and has less extrapulmonary or
disseminated diseases
15% of patients have disseminated
disease
Disseminated disease in the
immunocompromised and patients with
late stage AIDS
Pulmonary disease in humans
Mycobacteria scrofulaceum
–
Scrofula---unilateral painless
lymphadenitis, involving lymph nodes
high in the neck in healthy children
MYCOBACTERIA
•
Mycobacteria fortuitum/chelonae
complex
–
–
–
–
•
Mycobacteria ulcerans
–
•
Skin infections with draining abscesses
May have involvement of lungs, bone,
CNS, and prosthetic heart valves, and
disseminated disease
Associated with post-surgical wound,
needle injections, renal transplant
recipients
Sporotrichosis-like lesions (chain of ulcers
up the arm along the lymphatics) in
immunocompromised hosts
“Bairnsdale (Buruli) ulcer”---a painless
“boil” or lump in skin of extremities at the
site of previous trauma, developing into a
shallow non-healing ulcer with a necrotic
base (Tropical disease)
Mycobacterium leprae
–
–
Most usual presentation: numbness in the
earlobes or nose
Several varieties:
•
•
Lepromatous leprosy
Tuberculoid leprosy
•
Mycobacteria haemophilum
–
–
•
Mycobacteria xenopi
–
•
TB-like pulmonary disease; Rare
infection in AIDS patients and
immunocompetent hosts
Mycobacteria paratuberculosis
–
•
Painful subcutaneous nodules,
swellings and ulcers progressing into
abscesses and draining fistulas
Disseminated disease in AIDS patients
Associated with Crohn’s disease
Mycobacteria bovis
–
–
–
Typical produces TB in cattle, but may
infect humans
Human disease similar to that caused
by M. tuberculosis
Urinary bladder infections with BCG
chemo of bladder cancer
Pre-biopsy
• Clinical history
–
–
–
–
BCG vaccination
Positive PPD, Quantiferon Test
Chest x-ray
History of exposure
• Close family member
• Endemic area
– Risk factors for atypical mycobacteria
•
•
•
•
•
Elderly/poor lung health
Exposure to salt water
Travel to endemic areas
Rate of infection
Involvement of lymph nodes
QuantiFERON-TB Gold Test
• Blood samples are mixed with antigens (substances that
can produce an immune response) and controls.
• After incubation of the blood with antigens for 16 to 24
hours, the amount of interferon-gamma (IFN-gamma) is
measured.
• If the patient is infected with M. tuberculosis, their white
blood cells will release IFN-gamma in response to
contact with the TB antigens.
• Clinical evaluation and additional tests (such as a chest
radiograph, sputum smear, and culture) are needed to
confirm the diagnosis of LTBI or TB disease.
Safety
• Personal Protective Equipment
– Gloves
– Gowns
– Face Shield
– Respirator Mask
– Booties
– Coveralls
– Hair net
• Low risk Specimens
– >24 Formalin Fixation*
• Very High Risk Specimens
– Anything else
*Prions require NaOH fixation & many bacterial toxins may be heat and formalin stable
Post-biopsy
• Morphology
– Gross
• Caseation
– Tumor vs. necrotic granulomas
– Cytology
• Necrosis and giant cells with histiocytes
– Frozen section
• Necrotizing granulomas
– Permanents
• Necrotizing granulomas
• Additional Initial Testing
– Were cultures sent of diagnostic material?
– Were direct molecular probes sent of diagnostic material?
• BWH = TB only, State lab = expanded panel
Culture Methods
•
•
Standard mycobacterial cultures requires 6-8 weeks for isolation from
conventional media
Automated radiometric culture methods (eg, BACTEC) are increasingly
being used for the rapid growth of mycobacteria.
– Employs a liquid Middlebrook 7H12 medium containing radiometric palmitic acid
labeled with radioactive carbon 14 (14C)
– Several antimicrobial agents are added to this medium to prevent the growth of
nonmycobacterial contaminants.
– Production of 14CO2 by the metabolizing organisms provides a growth index for
the mycobacteria.
– Growth generally is detected within 9-16 days.
•
Another rapid method for isolation of mycobacteria is SEPTICHEK.
– nonradiometric approach has a biphasic broth-based system that decreases the
mean recovery time versus conventional methods.
•
Mycobacterial growth indicator tubes (MGITs) have round-bottom tubes with
oxygen-sensitive sensors at the bottom.
– MGITs indicate microbial growth and provide a quantitative index of M
tuberculosis growth.
Transcription
Mediated
Amplification
Ancillary Testing
• Histology
– AFB staining
• Ziehl Neelsen (AFB)
– Carbol fuchsin (with heat), acid alcohol (hydrochloric), methylene blue
• Kinyoun (Atypical mycobacteria, Nocardia)
– Carbol fuchsin (cold with phenol), acid alcohol (sulfuric), methylene
blue
• Fite-Faraco (M. leprae)
– De-paraffinize in xylene/vegetable oil, carbol fuchsin (cold), sulfuric
acid (no alcohol), methylene blue
– Gram, Silver, PAS
• Molecular
– PCR at State Lab (for M.tb, any tissue)
– PCR with sequencing at Genetic Assays, Inc. (TN)
Tissue Biopsy
Issue Report
With RCC
Necrotizing?
AFB
Stains?
Culture
Sent?
At BWH sputum smears
AFB
are(as
read
in house described)
(x 3) on target
Acidfor
fast
stains
previously
Necrotizing granulomas
include
a
patients suspected ofa having
TB.
range of
organisms from
mycobacteria
(TB,
FFPE
PCR with Sequencing/
differential of mycobacterial
disease
Granulomas?
atypicals,
Issue rhodococcus,
consistent report
(both
TB and
atypical),
fungalleprae, etc), nocardia,
At this point, you Samples
have
a biopsy
showing
necrotizing
can also bediptheroids,
directly inoculated
into a BACTEC
cryptosporidium
(stool), etc.
disease,
Bartonella
spp,
granulomas with
positive
AFB
stains
for
which
system which is hands
free.
If the
clinical orFor
histological
suspicion
of TB
cases where
organisms
are is
clearly
Campylobacter
(mesenteric
cultures
have
been
sent.
Granulomatous disease has a broad
AFB stain
butare
no culture was
very(with
high,minimal
levelsseen
andonrepeat
AFBs
The clinicians
will
often
push
for
more
information
and
biopsies),
sarcoidosis
differential
depending
onofhistory,
site, are sent,
The vast
majority
specimens
sent send
to the6 State
unstained slides to Genetic
acceptable.
that can be provided
in three
basic
flavors
focal
necrosis,
rheumatoid
nodules,
milieu, is
and
type offor
granulomas
present. Assays, Inc, and they will perform PCR
processing.
(though moreLaboratory
possible).
Thorough
of an AFB stain requires
and a few other rare entities. evaluation
and
sequencing
to determine species.
1. Rare organisms seen in a patient with
20hxminutes under oil (think about
scanning a
Pathology
lab
pays
for
FBGC
only
/
Non-immune
Granulomas
vs.
consistent with
TB, youcan
canbe
favor
Culture
as TB.
short
as
7 days (rapid growers) or as charge in
Pap
smear).
Mycobacterial
disease
hasinthe
patients without insurance.
Immune
Granulomas
2. Many organisms
seen
(especially
clustered
long
as 60
days.(T-cells,
Inimplication
general, mycobacterial
and nocardia are
public health
and
macs),histiocytes+/you canlargest
favor atypicals.
necrosis)
found with
but this
thistest
is not
In rare cases,
mayalways
be sent
should,
therefore,
always
be inflammation
3. Skin biopsy with
neuropathy
from
endemic
Following positive culture,
additional
tests
may
be
true
(e.g.,
atypicals
withing of
Macs,
leprae
even cluster
in the presence
cultures
carefully
country,
call itgranulomas
leprosy
(willexcluded
not
grow):
EM
may be
When
are
seen,
infection
necessary to speciate
and nerve
determine
susceptibilities.
pending
but should be discussed with
within
bundles).
helpful.should be ruled out
your pathology team including
4. Skin biopsy with exposure to salt water, favor M.
marinum
microbiology if possible.
Tissue Biopsy
Issue Report
With RCC
Necrotizing?
Culture
Sent?
FFPE PCR with Sequencing/
Issue consistent report
Granulomas?
Host?
Consider
Other
diagnoses
AFB
Stains?
Prior to biopsy (via discussion with clinicians or from the history)
you may have a high suspicion of mycobacterial disease (or simply
infection NOS).
This is usually because of some particular host factor (e.g., history
of TB, iatrogenic immunosuppression, AIDs, malignancy, classic
associations, mononeuropathy not from US, clinical Buruli ulcer,
etc).
In these situations, despite the fact that granulomas are missing (or
lack necrosis if present), it is prudent to order AFB stains to rule out
these organisms.
If a patient is a perfectly normal host with no clinical reason to
suspect a mycobacterial infection, other diagnosis (including other
infections) should be considered (this branch, of course, takes you
back into the rest of surgical pathology).
For cases of necrotizing granulomas with negative AFB stains (even after repeat if necessary),
Tissue
Biopsy pathogens should be excluded included correlation with culture, serological
fungal
and bacterial
tests (beta-1,3-glucan and galactomannan; bartonella serology), and discussion with the clinicians.
Issue Report
With
RCC of
In some cases, biopsies are limited by patient conditions (transbronch vs. VATS),
so review
radiology prior to sign out is important forAFB
understanding the question,
Culture “Is this a biopsy of the lesion
Necrotizing?
or something peripheral?” Some samples
can be “non-diagnostic”
Stains?
Sent?by virtue of their geography
rather than their quality.
When cultures are not sent and you suspect a fungal pathogen, the CDC can perform IHC for a
wide range of organisms (including fungus, bacterial, viruses, and protozoa)
as well
as Sequencing/
have access
FFPE PCR
with
to
IFA which are not routinely available (most require fresh tissue).
Granulomas?
Issue consistent report
Host?
Silver/PAS
Stains?
Consider
Other
diagnoses
Consider
Non-infectious
diagnoses
Culture
Sent?
Issue Report
With RCC
IHC at CDC,
Limited PCR/Sequencing
WS/Steiner?
Confirm Serology Sent/
Issue consistent report
Tissue Biopsy
Issue Report
With RCC
Necrotizing?
AFB
Stains?
Culture
Sent?
FFPE PCR with Sequencing/
Issue consistent report
Granulomas?
Host?
Silver/PAS
Stains?
Consider
Other
diagnoses
Consider
Non-infectious
diagnoses
Culture
Sent?
Issue Report
With RCC
IHC at CDC,
Limited PCR/Sequencing
WS/Steiner?
Confirm Serology Sent/
Issue consistent report