The Tell-Tale Stain

111 th General Meeting ASM
New Orleans, LA
May 24
Session 242 3:00 – 5:30 PM
Certify Your Worth: ABMM Credentialing

EVEN MORE

1. I am extremely knowledgeable. (I should be
up there talking.)
2. Better than average. (But compared to this
crowd… way, way above average.)
3. I am here with a ―friend‖.
(He/she promised to buy the drinks later.)
4. I think Donald Trump Gram-stained the nation

Edgar Allen Poe
Author of the ―Tell-Tale Heart ―
and ―The Raven‖, etc.
Jill E. Clarridge, III, Ph.D., D(ABMM), F(AAM)
Professor, University of Washington
Chief, Microbiology, Serology and Molecular
Diagnostics , VA Med Center
Seattle WA

Modified GMS Stain
Once upon a silver staining,
Oh, be sure I’m not complaining,
Saw I numerous rods and cocci
as I had never seen before.

The Modified Gomori Methenamine-Silver Nitrate
Stain (GMS Stain) will aid in the histological
visualization of which of following?
1. Fungi including Pneumocystis carinii
2. Nocardia asteroides
3. Mycobacteria
4. Corynebacteria
5. Treponema pallidum
6. All of above and others

Case A 28 year old female with chronic hepatitis B
with 2-3 year history of rash and lesions was referred to
dermatology for biopsy.
HISTORY OF PRESENT ILLNESS:
• the rash started on her extremities as
tender, dark nodules which then
progress to become flat, darker lesions
which finally develop into scars.
• the rash (most) eventually resolved
spontaneously.

She reports history of tick bites in Missouri, which she
thought caused several of the nodules on her right leg.
A biopsy was performed
Cultures were negative.
Rare AFB seen by
tissue AF stain
Granulomas seen
Special stains for bacteria, fungi and
AFB were reported negative except
for GMS.

FURTHER HISTORY
Patient has associated symptoms of
peripheral neuropathy, swelling in hands and feet,
"bone pain", joint pain,
hair loss on head and body.
She reports the hand and foot pain and swelling are worsened
by cold weather. The joint pain is localized to knees, wrists and
hands.
She reports chemical exposure to hydraulic fluid but this has
only been in last 1 month.
Hypopigmented area with loss of
sensation on abdomen
She reports travel to her birthplace
of Palau, most recently in 2007.

What is the diagnosis for this patient?
1. Rocky Mountain Spotted Fever
2. Erythema Induratum of Bazin
3. Leprosy (Hansen’s Disease)
4. Syphilis

Erythema Induratum of Bazin :
It is an inflammation in the subcutaneous fatty tissue and that is
caused by a cellular immune response to M. tuberculosis .
It usually involves the lower extremities.
The diagnosis is made on the basis of histopathology and tests
showing a positive cellular immune response to TB.
Tiny case –somewhat related to main case
• A woman with a two-year history of non-healing leg ulcers
Her PPD was positive

She was started on anti-tuberculous chemotherapy.
Four months later lesions were healing.
The biopsy showed vasculitis and tuberculoid granulomas
Special stains for bacteria, fungi and AFB were negative.
Cultures were negative
M. tuberculosis PCR can support the diagnosis, but a negative
PCR result does not exclude EIB.

What do you think is the etiological agent of leprosy?
1. This is like asking who is buried in Grant’s tomb!
2. Mycobacteria leprae
3. Mycobacteria lepromatosis

For this case it is Mycobacteria leprae.
16S rRNA gene sequencing was positive for M. leprae
Dendrogram showing
the relatedness of the
16S rRNA gene
sequences of selected
Mycobacteria
But what is Mycobacteria lepromatosis

New Mycobacterium species from 2 patients who died of diffuse
lepromatous leprosy (DLL).
DNA extraction from heavily infected freshly frozen liver tissue or
paraffin-embedded skin tissue
Six genes of the organism were sequenced.
Significant genetic differences with M. leprae were found, including a
2.1% divergence of the 16S ribosomal RNA (rRNA) gene,
The unique clinicopathologic features of DLL led them to propose
Mycobacterium lepromatosis sp nov.

Historically the varying clinical presentations has been thought to
be primarily due to host response
Borderline Indeterminate Lepromatous
New species might explain varying severity and clinical features of
HD.
This has been challenged –M. lepromatosis could be just another
related Mycobacteria-not causing leprosy.
However, the sequence has also been detected in another leprosy
patient

Hansen’s Disease Factoids
Worldwide— about 250,000 new cases/year
United States – 150 new cases/year
The largest numbers of cases in the United States are in California,
Texas, Hawaii, Louisiana, Florida; New York and Puerto Rico.
Most are in immigrants from countries where the disease is endemic.
What is the source of the about 50 cases acquired in the US?

Hansen’s Disease Factoids
Mycobacterium leprae multiplies very slowly (generation time 12.5
days) and is an obligate intracellular parasite.
It grows best at 27ºC to 33ºC, which correlates with its affecting
cooler areas of the body like the skin nerve segments close to the
skin, and the mucous membranes of the upper respiratory tract.
M. leprae has never been cultured in artificial media but grows in the
tissues of the armadillo, which has a body temperature of 34ºC.
Leprosy is found in wild armadillos in the south central United States
and possibly in some primates
Most patients today are treated with dapsone, rifampin and
clofazimine. Also levofloxacin, minocycline and clarithromycin are
used in selectively
Chemotherapy rapidly renders the patient noninfectious.

Which is not true?
1. Mycobacterium leprae can be transmitted transplacentally.
2. Leprosy is known as a sexually transmitted disease
3. Kissing or eating an armadillo can be harmful to your health
4. Leprosy which was brought to the New World by Europeans
and Africans infected the native armadillo
5. Armadillos when dead in the road are called hill-billy speed
bumps.

Transmission:
The exact mechanism of transmission is unclear.
Leprosy is probably transmitted by droplet infection and direct
contact with discharges from respiratory mucous membranes
of infected persons.
Household and prolonged, close contact appear to be important
in transmission.
In children

Handling or eating armadillos could expose
humans to the bacterium that causes leprosy.
Probable Zoonotic Leprosy in the Southern United States
Truman, Singh, Sharma, Busso, ougemont, Paniz-Mondolfi,
Kapopoulou, Brisse, Scollard, Gillis, Cole. NEJM April 28, 2011
1. Genotyped about 150 strains using 51 singlenucleotide
polymorphisms , an 11-bp insertion–
deletion with 10 variable-number tandem repeats.
2. Found local infected humans share the same
leprosy bacterium strain as local armadillos.
3. Humans contract the disease by handling
armadillos or consuming their meat.
4. More than 20 percent of armadillos around Carville
infected with M. leprae
5. The animals are most common in Texas, but they
are expanding their range in Florida, and
northward into the Midwest.

Now back to Staining M. leprae
Skin smears (touch preps) may be taken from lesions. A biopsy
should be taken from entirely within a lesion or for example, the ear
lobe
The effects of therapy are sometimes followed by counting the
numbers of bacilli in a biopsy or smear from the patient.
But what is the best stain to demonstrate Mycobacteria leprae in
tissue and how should it be performed
1. Routine tissue AFB stain
2. Fite’s stain
3. Plain old Kinyoun stain as
used in the microbiology lab
4. Silver stain

KINYOUN’S STAIN
We obtained a slice of the
tissue from the block,
deparafinned it and
performed our routine stain
in microbiology
FITE'S ACID FAST STAIN Combines
peanut oil with the deparaffinizing
solvent (xylene), minimizing the
exposure of the bacteria's cell wall
to organic solvents, thus protecting
the precarious acid-fastness of the
organism.

Routine tissue AFB stain Fewest AFB
Fite’s stain More AFB
Kinyoun stain as used micro lab About same # but darker
Siver stain More organisms, not specific
Thus stain method is important for
quantitating to follow course of
disease
The exact procedure should be
known as many labs tended to
perform the stains in their own
unique way. This so confusing

1831 - Edgar’s older brother, William, dies in
Baltimore, probably of tuberculosis
1836 - Edgar (aged 27) and Virginia (aged 13) marry in
Richmond, Virginia.
1847 - Virginia Poe dies of tuberculosis
1849 – Edgar Allen Poe dies
Final Question: Of what did Edgar Allen Poe die?
1. Died subsequent to being mugged and falling in a puddle
while in an alcoholic stupor (Competitor wrote this after he
died)
2. Some AFB disease (Tuberculosis or Leprosy would make
sense with this talk)
3. Unknown (Wikipedia)
4. He did not die (Celebrity Ghost Stories)

Special thanks to colleagues
From the National Hansen's Disease Programs in
Baton Rouge, Louisiana, which functions as a
referral and consulting center with related
research and training activities:
Dr. David Scollard.
Dr. Richard W. Truman
Dr. Thomas P. Gillis
From the University of Washington and Seattle VAMC
Dr. Greg Raugi
Dr. Rich Miller
Dr. Jeff Virgin
Susan Izak
Dr. Angela Talley for the
erythema induratum of Bazin
case