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Journal of Clinical Microbiology, September 1999, p. 3059-3061, Vol. 37, No. 9
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Recurrent, Disseminated Mycobacterium
marinum Infection Caused by the Same Genotypically Defined
Strain in an Immunocompromised Patient
G. Frank
Holmes,
Susan M.
Harrington,
Mark J.
Romagnoli, and
William G.
Merz*
Department of Pathology, The Johns Hopkins
Medical Institutions, Baltimore, Maryland 21287-7093
Received 15 January 1999/Returned for modification 11 March
1999/Accepted 27 May 1999
 |
ABSTRACT |
An 81-year-old male with myasthenia gravis developed a cutaneous
infection with Mycobacterium marinum, which apparently
resolved following local heat therapy. Five months later, the patient
developed new skin lesions and pancytopenia. M. marinum was
isolated from his bone marrow. Pulsed-field gel electrophoresis was
performed to determine if the skin and bone marrow isolates were
clonally related. Digestion of the genomic DNA with the restriction
enzymes SpeI and AseI yielded indistinguishable
banding patterns. An epidemiologically unrelated control strain showed
significant banding differences. The results suggest that the
patient's recurrent, disseminated infection was due to recrudescence
of his initial infection rather than reinfection by another strain.
| |
TEXT |
Mycobacterium marinum is
a photochromogenic mycobacterium that is ubiquitous in the aquatic
environment (7). First isolated in 1926 by Aronson
(1) from saltwater fish that had died in the Philadelphia
Aquarium, it was not recognized as a cause of human disease until 1951, when Norden and Linnel (11) isolated the organism from
granulomatous skin lesions in swimmers. Infection typically arises when
traumatized skin comes into contact with infected water in swimming
pools, aquariums, oceans, or lakes. Because of the organism's optimal
growth at 30 to 32°C, infection is usually limited to the skin
in the peripheral, cooler parts of the body, particularly the hands.
The typical lesion is a single nodule that occasionally
progresses along the lymphatics in a sporotrichoid fashion
(7). Less commonly, patients develop deeper infections,
involving tendon sheaths or periarticular tissues (13), and,
rarely, septic arthritis or osteomyelitis can ensue (2, 4).
Disseminated cutaneous lesions have also been described to occur in
both immunocompetent (8, 17) and immunocompromised patients
(5, 6). Because of the organism's poor growth at 37°C,
however, systemic dissemination is extraordinarily rare and has only
been reported to occur in immunocompromised patients (9, 12,
14).
We present a case of systemically disseminated M. marinum
infection in an immunocompromised patient, who had been diagnosed 5 months earlier with cutaneous disease that had apparently resolved following local heat therapy. The second isolate was recovered from the
patient's bone marrow, which was cultured after the patient became
pancytopenic. Pulsed-field gel electrophoresis (PFGE) was performed to
determine if the skin and bone marrow isolates were clonally related.
Case report.
An 81-year-old white male presented with a
painful, erythematous plaque on the dorsum of his left forearm. He had
a 10-year history of myasthenia gravis, for which he was taking
prednisone (40 mg every other day) and azathioprine (275 mg/day). He
was seen by a dermatologist, who felt the lesion probably represented chronic erysipelas, and was treated with ciprofloxacin for 1 month. Although there was some initial improvement, the lesion persisted and
began to ulcerate. A separate ulcer developed over the
metacarpophalangeal joint of his left index finger. A biopsy was
performed, and this revealed a lymphohistiocytic infiltrate in the
dermis with necrosis and acute inflammation. The findings were most
suggestive of an infectious process, but special stains for fungi,
bacteria, and acid-fast organisms were negative. The skin lesion was
cultured for mycobacteria on Lowenstein-Jensen and 12B Bactec media at 30 and 37°C. Growth occurred in Bactec media after 8 days at 30°C. Initial testing by using commercial DNA-RNA hybridization probes (Gen-Probe, San Diego, Calif.) revealed negative results for M. tuberculosis complex, M. avium complex, M. gordonae, and M. kansasii. By Runyon typing, the
organism was found to be a photochromogen that preferred growth at
30°C compared to 37°C. Biochemical testing (16) showed
positive reactions for urease, Tween hydrolysis (both 5- and 10-day
readings), and pyrazinamidase activity (both 4- and 7-day readings).
Negative tests included nitrate reduction (tube test), arylsulfatase
(3-day test), and heat-stable catalase (68°C), and the organism did
not grow on MacConkey agar without crystal violet (5- and 11-day
readings). The results of the biochemical tests were indicative of
M. marinum and were confirmed by gas-liquid chromatographic
analysis of cellular fatty acids (18).
Following the diagnosis, it was learned that the patient maintained a
small aquarium at home. Because of gastrointestinal complaints while
taking ciprofloxacin, the patient refused antibiotic therapy, but he
agreed to wrap his arm daily with a heating pad. Three months later,
when the patient was admitted to the Johns Hopkins Hospital with a
myasthenic crisis, the skin lesions had almost completely resolved
except for some residual postinflammatory hyperpigmentation. At the
time of hospitalization, he did note some arthritic pain in his left
hand, but the pain was easily relieved by taking ibuprofen. After 5 courses of plasma exchange therapy, the patient's myasthenia gravis
stabilized and he was sent home.
Three weeks after being discharged, the patient was readmitted to the
hospital because of marked, painful swelling of his right arm.
Examination revealed a 30- by 8-cm area of erythematous induration with
focal ulceration and blister formation along the dorsal and radial
sides of the forearm and the medial side of the upper arm. The patient
was treated with clindamycin and gentamicin but was switched to
intravenous vancomycin after bacterial cultures from the lesion grew
methicillin-resistant Staphylococcus aureus. Antibiotics did
not cause the lesion to regress, however, and a similar 5- by 5-cm area
developed around the antecubital fossa of the left arm. Although 5 months had elapsed since the initial skin biopsy-based diagnosis, the
patient was also placed on intravenous doxycycline because of concerns
of M. marinum infection. Mycobacterial cultures from the
right arm, however, were negative.
The patient gradually became pancytopenic and developed rising levels
of creatinine and elevated liver function tests. Because of his
pancytopenia, azathioprine was discontinued, and bone marrow biopsy and
cultures were performed. The biopsy showed mild hypocellularity with a
slight lymphocytosis. Mycobacterial cultures of bone marrow specimens
incubated at 37°C were positive at 3 weeks. Before the isolate was
identified, however, the patient became encephalopathic and died. The
results of biochemical tests performed on the bone marrow isolate were
identical to the results from the skin isolate, confirming that the
organism grown from the bone marrow was in fact M. marinum.
Gas-liquid chromatographic analysis of cellular fatty acids also gave
an identification as M. marinum.
To determine whether the skin and bone marrow isolates were clonally
related, PFGE was performed. The two isolates of M. marinum from the patient were compared to M. marinum ATCC 927 in
order to demonstrate the ability of PFGE to discriminate a potentially related organism from an organism known to be unrelated
epidemiologically. PFGE was performed with some modification of
established protocols (15, 20). In a biosafety level 3 facility, the M. marinum isolates were grown for 2 weeks at
30°C in 10 ml of Middlebrook 7H9 broth (BBL, Cockeysville, Md.)
supplemented with 0.1% Tween 80. Before DNA extraction, ethambutol, a
cell wall-active agent, was added to 1 µg/ml, a concentration
expected to be subinhibitory. Cultures were further incubated about
16 h. Cells were harvested by centrifugation and resuspended in 1 ml of suspension reagent (1 M NaCl, 10 mM Tris-HCl [pH 7.6]). Cell
suspensions were mixed 1:1 with molten 1.6% InCert Agarose (FMC,
Rockland, Maine) and pipetted onto glass slides. Several plugs,
approximately 1 by 5 by 10 mm, were cut from the slide by using a
microscope coverglass and placed into about 5 ml of lysis solution (1 M
NaCl, 100 mM Na2EDTA [pH 7.5], 0.5% Brij 58, 0.2%
deoxycholate, 0.5% sodium lauroyl sarcosine, and 5 mg of lysozyme per
ml [Sigma, St. Louis, Mo.]). Plugs were incubated overnight at
37°C. Lysis solution was replaced with ESP reagent (0.5 M EDTA [pH
9], 1% sodium laroyl sarcosine, 500 mg of proteinase K per ml
[Sigma]), and the plugs were incubated overnight at 50°C. Plugs
were washed three times at 37°C for 1 h each time with TE (10 mM
Tris-HCl [pH 7.4] and 0.1 M Na2EDTA [pH 8]).
For DNA macrorestriction, the agarose inserts were placed in a solution
containing 200 µl of deionized water, 25 µl of 10× buffer, and 20 U of SpeI or AseI (New England Biolabs, Beverly, Mass.), supplemented with bovine serum albumin as indicated by the
manufacturer, and incubated for 4 h at 37°C. After a brief wash
with TE, inserts were loaded into 1.0% agarose gels (Bio-Rad, Hercules, Calif.). PFGE was performed with a Bio-Rad CHEF DRII apparatus with an increasing pulse time of 1 to 20 s over a total run time of 22 h in 0.5× TBE (1 M Tris-HCl, 0.9 M boric acid, 10 mM Na2EDTA [pH 8]) at 200 V. A well-characterized strain
of Enterococcus faecalis, OGIRF, was digested with
NotI to serve as the molecular weight marker
(10). The gel was stained with ethidium bromide for UV
transillumination and Polaroid photography.
Restriction profiles of the isolates' genomic DNA obtained by
SpeI digestion yielded 20 to 25 fragments ranging in size
from less than 20 to approximately 280 kbp (Fig.
1). The skin isolate (lane 2) and bone
marrow isolate (lane 3) demonstrated indistinguishable banding
patterns, whereas an epidemiologically unrelated, control isolate of
M. marinum (lane 4) showed significant (>5) multiband differences. A second restriction digestion with AseI was
also performed on the same gel. Once again, the skin isolate (lane 5)
and bone marrow isolate (lane 6) displayed indistinguishable banding
patterns. The control isolate (lane 7) showed at least four band
differences.
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FIG. 1.
PFGE of M. marinum. Lanes 2 and 5, patient's
skin isolate; lanes 3 and 6, patient's bone marrow isolate; lanes 4 and 7, M. marinum ATCC 927. Lanes 2 to 4 are digested with
SpeI, and lanes 5 to 7 are digested with AseI.
Lane 1, molecular size marker E. faecalis OGIRF digested
with NotI. Molecular sizes shown are given in kilobase
pairs.
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|
Discussion.
M. marinum infection in otherwise
healthy hosts can be self-limiting and disappear after several months,
or it can be treated with a variety of antimicrobial drugs, including
trimethoprim-sulfamethoxazole, rifampin and ethambutol, or doxycycline
(7). Alternatively, local heat therapy has also been used
with good results. Topical or local injection of steroids, however, is
contraindicated because it frequently causes exacerbation of the
infection and leads to greater difficulty in curing the patient
(3, 19).
In contrast to the situation with immunocompetent hosts, treating
M. marinum infection in immunocompromised patients can be very difficult (12, 14). One reason for the difficulty could in part be related to delays in accurate diagnosis. There is frequently a substantial lag time between the appearance of the skin lesion and
the correct diagnosis, ranging from a few weeks to several years
(7). Clinical misdiagnosis is common, and organisms are rarely identified on acid-fast stains of biopsy material. Such delays
in diagnosis can lead to local spread of the infection, especially in
immunocompromised hosts. Once the correct diagnosis is made and
appropriate antimicrobial therapy is started, however, M. marinum infections are still difficult to control in
immunosuppressed patients, necessitating many months of treatment with
antibiotics, and even then, the results can be disappointing.
The present case confirms the difficulty in treating M. marinum infections in immunocompromised patients. Local heat
therapy apparently cleared the cutaneous infection, but it might not
have prevented spread to deeper tissues. The patient's joint pain in the left hand could have represented involvement of the joint space. A
few weeks later, he had disseminated cutaneous lesions involving the
right and left arms, and the systemic dissemination to the bone marrow.
Despite intravenous doxycycline, the patient died in multiorgan system
failure, presumably from his M. marinum infection.
The skin and bone marrow isolates were the same genotypically defined
strain based on the identical patterns generated by PFGE after
macrodigestion. Significant multiband differences were seen with an
epidemiologically unrelated control strain. From a clinical standpoint,
the findings suggest that the second isolate probably represented
recrudescence of the patient's initial infection rather than
reinfection by another strain of M. marinum. The most likely
source of infection was the patient's aquarium, but unfortunately, cultures from the aquarium itself were not performed. It is unknown how
often the patient had contact with the aquarium in the intervening 5 months between the two positive cultures. The possibility that the
patient was reinfected by the same strain of M. marinum
after further contact with the aquarium therefore cannot be entirely excluded.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Microbiology,
Meyer B1-193, Department of Pathology, The Johns Hopkins Hospital,
Baltimore, MD 21287-7093. Phone: (410) 955-5077. Fax: (410) 614-8087. E-mail: wmerz{at}pathlan.path.jhu.edu.
| |
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Journal of Clinical Microbiology, September 1999, p. 3059-3061, Vol. 37, No. 9
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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