British Joumal of Dermatology (1976) 94» 435-
Mycobacterium marinum skin infections mimicking cutaneous leishmaniasis Z.EVEN-PAZ, H.HAAS, T.SACKS AND E.ROSENMANN Departments of Dermatology, Clinical Microbiology and Pathology, Hadassah-University Hospital and Hebrew University-Hadassah Medical School, Jerusalem, Israel Accepted for publication 11 July 1975
SUMMARY
Ten cases of skin infection due to Mycobacterium marinum are reported from Israel. Most of the infections were contracted in natural bathing pools at Ein Feshka on the shores of the Dead Sea, south of Jericho. The lesions closely resembled those of cutaneous leishmaniasis prevalent in this region. The condition does not seem to have been detected previously in the Middle East.
Skin granulomas due to Mycobacterium marinum (balnei) have been recognized since the early 1950s following reports by Linell & Norden (1954) and Herlitz (1953) on 80 cases infected at one swimming pool. Most earlier instances of 'swimming-pool granuloma' (Hellerstrom, 1952) were probably due to the same organism. Four epidemics account for 523 ofthe more than 600 cases so far published; these occurred in Sweden, the U.S.A. and Britain (Linell & Norden, 1954; Zettergren & Zetterberg, 1956; Philpott et al, 1963; Waddington, 1967). Sporadic cases or small outbreaks have been reported from Canada, Holland, Belgium, Czechoslovakia, Australia, New Zealand and Japan. The deteaion of M. marinum infection in the Middle East is of interest because the lesions mimic those of cutaneous leishmaniasis endemic in the same area. CASE REPORTS
Three types of lesion were seen (Figs i, 2). A general description follows and additional clinical data for each case are presented in Table i. The first type of lesion, present in all cases, consisted of chronic inflammatory nodules or plaques 1-4 cm in diameter, with scales, crusts or small ulcers on their surface. Beneath the crusts was a slight sero-sanguinous or purulent discharge. The surface ofthe plaques was uneven, occasionally with a smooth atrophic area. Small satellite papules were present in most cases and constituted the second type of lesion; they usually had a central scale, crust or ulcer. The third type of lesion was seen in four ofthe ten patients and consisted of one or more subcutaneous or intradermal cyst-like swellings. These were I-5-2-O cm in diameter, some distance proximal to the original lesions, and if multiple were arranged along an axis in sporotrichoid fashion. The condition began as one or more small papules which rapidly became swollen and discharged pus. The discharge became scanty or dried and the lesion enlarged. Satellite papules and sporotrichoid Correspondence: Dr Zvi Even-Paz, Chief Physician, Department of Dermatology and Venereology, HadassahUniversity Hospital, Jerusalem, P.O.B. 499, Israel.
435
436
Z.Even-Paz et al.
CD FIGURE I. Case 2. The nodular, ulcerated lesion on the hand appeared 2 weeks after a period of bathing at Ein Feshka. Four months later a smooth, cystic, sporotrichoid swelling developed 13 cm proximally; when incised this released a gelatinous material and subsequently ulcerated. M. marinum was isolated from both lesions. FIGURE 2. Case 3. The larger, irregular, nodular plaque with scales, small crusted ulcers and partial central atrophy developed on an unhealed graze of the right knee which occurred while bathing at Ein Feshka. The smaller lesion appeared 8 months later without further bathing. M. marinum was isolated from both lesions.
lesions appeared later, at intervals which varied up to 18 months. The lesions were usually asymptomatic, the regional lymph nodes were not enlarged and there was no disturbance of the general health. The clinical picture was similar to that of cutaneous leishmaniasis, locally called 'Jericho boil', and all but two ofthe patients had visited the Jericho area. When smears and cultures for Leishmania tropica and the intradermal leishmanin tests proved negative routine, bacteriological studies were done and a search made for mycobaeteria. Biopsies were done in eight ofthe cases. Bacteriological studies
Tissue scraped from incisions, biopsy material, exudates, crusts and scales, was examined. The specimens were ground aseptically, washed once in normal saline and suspended in phosphate buffer, pH 72. Specimens showing no growth on blood agar plates were cultured on Loewenstein-Jensen (L-J) medium without further treatment. Specimens showing growth were treated for 15 min with 6% sulphuric acid, washed with saline, resuspended in phosphate buffer and inoculated on L-J medium. The cultures were incubated at 32 C and 37 C. On microscopic examination of smears a few, scattered, acid-fast bacilli were seen in specimens from five out ofthe ten cases. In cultures from all ten cases, small creamy-white colonies which became yellow-orange after exposure to light appeared after 7-12 days at 32 C. There was no growth at 37 C. The cultures were identified as M. marinum by their photochromogenecity, rate of growth and need for lower temperature. Identification was confirmed by hydrolysis of Tween 80, negative nitrate reductase and positive arylsulphatase activity.
Mycobacterium marinum skin infections
437
TABLE I. Clinical data on ten cases of M. marinum skin infection Patient
Sex
Age
Sites affected
Trauma
I
M
40
Elbow, upper arm
—
Ein Feshka
Yes
2 wk2 mth.
2
M
9
Hand, forearm
—
Yes
2 wk2 mth.
3
F
9
Knee
Ein Feshka Galilee stream Medit'n Ein Feshka
—
Brief
4
M
39
Elbow, Graze (while upper arm bathing)
Ein Feshka Medit'n
Yes
Brief
5
M
2t
Knee, thigh
Sea of Galilee
Yes
?
6
F
47
—
—
Brief
7
F
Ein Feshka
—
6 mth.
Ein Feshka
—
Brief
Ein Feshka
—
1-2 mth.
Ein Feshka
—
2yr.
M
9
10
M
F
Forearm, hand 7 Knee
Graze (while bathing)
—
Graze (from gas range) Kick (before bathing)
Upper arm Insect bite (before bathing) 12 Buttock Insect bite (while bathing) — 38 Forearm
6i
Water exposure
Sporotrichoid lesions
Incub. period
Treatment and course None: complete resolution Grenz rays: partial resolution Grenz rays: partial resolution Grenz rays: complete resolution Griseofulvin: complete resolution Grenz rays: course not known Grenz rays: partial resolution None: complete resolution Grenz rays: partial resolution None: course not known
Drug sensitivity tests were done on L-J medium containing various dilutions of isoniazid, PAS, streptomycin, ethambutol and rifampicin. Disc susceptibility tests were performed on Dubos oleic agar base, using cotrimoxazole (25 |jg) and vancomycin (5 //g) discs (Baltimore Biological Laboratories, U.S.A.). The results are given in Table 2. Purified protein derivative (PPD) tests The results of intradermal tests with PPD from 6 various sources are given in Table 3. Histopathology Marked changes were observed in seven of the eight patients from whom biopsy specimens were taken (Fig. 3). The epidermis showed hyperkeratosis with focal parakeratosis, irregular acanthosis with pseudoepitheliomatous hyperplasia or extreme elongation of the rete pegs, and liquefaction degeneration ofthe basal layer. In the upper- and mid-dermis there was a mixed infiammatory infiltrate which sometimes extended into the epidermis. The cells were mainly lymphocytes and histiocytes but in two patients plasma cells predominated. There was a marked neutrophilic and eosinophilic exudate in two cases and in six cases microabscesses were present, usually located near
Z.Even-Paz et al.
438
TABLE 2. Sensitivity of nine M. marinum strains to antibacterial agents (compared with a standard strain of M. tuberculosis)
Strain Minimum inhibitory concentration (/'g/ml) from — patient Isoniazid PAS Streptomycin Ethambutol Rifampicin
Disc sensitivity test Cotrimoxazole Vancomycin
no. I 2
3 4 5 6 7 8 9
M. tuberculosis (H37RV)
>5 >5 5 5 >5 5 >5 5 5
(25 fig)
(5 f'g)
R* R R R R R R R R
> IOO > IOO > IOO
10
10
10
2
10
> roo
10
2
10
> > > > >
IOO IOO IOO IOO IOO
10
2
10
10
I
10
10
2
10
10
2
10
R* R R R R R R R
10
2
10
R
I
I
2
5
02
* Resistant (no inhibitory zone around discs).
the tip of an elongated rete peg or in a dermal papilla. Non-caseating epithelioid granulomas were seen in all seven cases and in one instance the epithelioid granulomas were invaded by neutrophils. In most cases multinucleated giant cells of Langhans' or foreign body type were present (Fig. 4a, b). In the eighth patient there was a mixed histiocytic and lymphocytic infiltrate, with a few neutrophils, around the deep dermal vessels: no granulomas, giant cells or microabscesses were seen. Acid-fast bacilli were found in one case. Leishman-Donovan bodies and fungi could not be demonstrated. Treatment and course
Five patients were treated twice with 500-700 rad Grenz rays 3-26 months after the onset of their condition; the interval between the radiations in the different cases varied from 3 weeks to 4 months (Table i: patients nos. 2, 3, 4, 7, 9). Complete resolution occurred within 2 months in patient no. 4 and moderate to marked resolution in the others, who are still under observation. Patient no. 6 received 700 rad Grenz rays once to the lesions but did not report for further examination. Complete resolution occurred in three patients without specifically directed treatment. In patient no. 8 this occurred 4 months after the lesions appeared, bacitracin ointment only being applied. The elbow T A BL E 3. Results of intradermal tests withfiveunits PPD from various sources (mm induration after 72 h) Patiem
M. marinum M. tuberculosis M. kansasii M. foriuitum Scotochromogens Battey
6
7
8
9
10
12
21
6 5
0
20
19 16
0
10
6
0
2
0
0
7
0
0
5
5
4
5 9
I
2
3
4
5
12
7
7
13
0
5
8 8
17
12
6
0
15
24
0
5
0
0
13
15
16
12
10
12
16 18
0
5
9 13
7
14 0
6 7
0
Mycobacterium marinum skin infections
439
FIGURE 3. Typical lesion with hyper- and parakeratosis, pseudoepitheliomatous hyperplasia and a dense inflammatory infiltrate in the dermis (H & E, x 42},
lesion in patiem no. i had been diagnosed elsewhere as psoriasis and had been treated by 4 intralesional injections of triamcinolone acetonide. After initial marked improvement the lesion became swollen, discharged pus and 2 months later a sporotrichoid swelling appeared on the upper arm. After a further 8 months complete regression occurred without other treatment. Patient no. 5 had been given fineparticle griseofulvin by another doctor shortly before the diagnosis was made. This treatment was continued and 3 months later there was resolution of the lesions. The last patient (no. 10) did not report after the initial visit at which material was taken for examination. DISCUSSION
Mycobacterium marinum is capable of living saprophytically, especially in an aquatic environment, and parasitically in cold-blooded animals and man. The fact that in man the lesions are nearly always
a F1G u R E 4. (a) Multinucleated giant cell of the Langhans type (H & E, x 420). (b) Multinucleated giant cell of the foreign body type (H & E, x 260).
44°
Z.Even-Paz et al.
limited to the skin has been attributed to the preference of the parasite for relatively cool growth conditions. Only occasionally has involvement of deeper structures been demonstrated or suspected (Winter & Runyon, 1965; Gould, McMeekin & Bright, 1968; Jolly & Seabury, 1972; Cortez & Pankey, 1973). The lesions are usually on one limb and few in number but widespread skin infection has occurred in patients with suspected immunological impairment (Gould et al., 1968; Sage & Derringtcn, 1973)Human infection is usually acquired whilst bathing in fresh, brackish or salt water. However, 'swimming-pool granuloma' is not a suitable synonym, for lakes, wells, rivers, inlets, bays, the seashore, home aquaria and a coal mine have been incriminated. Some patients have denied contact with any extra-domiciliary source of water. Previous trauma of the affected site, although common, is not always recalled. Eight of the ten patients in this series were apparently infected at Ein Feshka, a bathing area south of Jericho where fresh-water pools fed by springs empty into the Dead Sea via short canals. By coincidence, this region is highly endemic for cutaneous leishmaniasis, the lesions of which may be clinically indistinguishable from those due to M. marinum. This coincidence has probably delayed recognition of the latter condition. The incubation period was very variable. Although most commonly a few weeks to a few months, in one case it may have been as long as 2 years whilst in others it seemed virtually absent, the lesions rapidly developing on a traumatized site which did not heal. Jt was not possible to assess the incubation period in those cases without a history of trauma and with no limited exposure period to a suspected water-source. The examination of multiple samples is important for the detection of M. marinum in the lesions. Acid-fast bacilli are seen only rarely in histological sections and infrequently in smears, whilst cultures may not always be positive. In the present series, three to eight samples were taken from each patient. Of the total of forty-six samples, acid-fast bacilli were seen microscopically in six and there were only twenty-six positive cultures. M. marinum is classified in Runyon's Group I together with M. kansasii, since both are photochromogenie. M. kansasii grows at 37 C whilst M. marinum grows at 30-32'C. However, primary isolation of M marinum at 37 C has been reported (Feldman, Long & David, 1974): confirmation of identity may be made by the nitrate reduction test, usually negative for M. marinum and positive for M. kansasii (Silcox & David, 1971)- ^- ulcerans also requires the lower incubation temperature but its growth takes several weeks. Skin lesions due to M. ulcerans possess an abundance of acidfast bacilli and on culture the colonies are non-pigmcnted, tend to form cords and give a positive neutral red test. The histopathological picture is usually suggestive but not pathognomonic. The diagnosis is supported by the presence of a mixed inflammatory reaction with a dense mononuclear infiltrate, a few non-caseating granulomas with or without giant cells, and microabscesses. Central fibrinoid necrosis in the granulomas has been reported occasionally and cascating necrosis rarely (Linell & Norden, 1954; Schaefer & Davis, 1961). Acid-fast bacilli are not usually seen and their detection requires a prolonged search of serial sections. Clinical and histological differential diagnosis includes all other chronic infective granulomas, foreign body granuloma and iodine and bromine granulomas. Clinically, in addition, psoriasis, verrucous lichen planus and even verruca vulgaris may have to be considered. In the Middle East confusion is most likely with cutaneous leishmaniasis and some features differentiating the two conditions are given in Table 4. A positive reaction of 10 mm or more induration to the intradermal PPD (marinum) test supports the diagnosis but the result must be interpreted with caution. There is a high incidence of cross-reactivity with PPD from other sources and in the patients in this series the reaction to PPD (marinum) was not usually the strongest (Table 3).
Mycobacterium marinum skin infections
441
TABLE 4. Differential diagnosis of M. marinum and L. tropica skin infection Mycobacterium marinum infection
Leishmania tropica infection
Patient's history Geographic factors Seasonal factors Diurnal factors
Exposure to water source Minor trauma Incubation period
No[ known: reported so far from only a few widely dispersed areas Probably not relevant, but if infected whilst bathing outdoors usually during warmer season Probably not relevant, but if infected whilst bathing outdoors usually during daylight hotirs Commonly reported Commonly reported Very brief to prolonged
Sub-tropical and tropical endemic areas During warmer season Acquired at or after sunset Not relevant None, except insect bite Usually not less than 4 weeks. May be prolonged
Clinical appearance Number and sice Few, usually on limb at site liable to of lesions trauma: rarely bilateral Sporotrichoid Relatively common lesions Special examinations
Few or numerous. Common on face as well as limbs: often bilateral
Intradermal tests * Smears
PPD tests positive Leishmanin test negative Acid-fast bacilli found with difficulty
Culture
From tissues and discharge: M. marinum may not grow from all the specimens Acute and chronic inflammatory infiltrate. May be giant cells, epithelioid granulomas and microabscesses. A-F bacilli rare
PPD tests negative Leishmanin test positive Leishman-Donovan (LD) bodies found easily in early lesions From tissue at edge of lesion: L. tropica usually grows from all early lesions Chronic infiammatory infiltrate sometimes granulomatous. Numerous LD bodies in histiocytes in early lesions
Histologic picture
Rare
* In areas in which both leishmania] and mycobacterial infections occur the intradermal tests must be interpreted with care for positive reactions persist after cure. Spontaneous healing probably occurs within a few months to a few years in the majority of cases. Small, rapidly healing lesions may never be brought to the attention of a doctor: such cases, if frequent, may be responsible for an increased percentage of tuberculin-positive reactors in some areas (Judson & Feldman, 1974). The many treatments recommended include excision with skin grafting, curettage, electrodesiccation or fulguration, cryotherapy, local heat, ultraviolet irradiation, Grenz-ray therapy. X-ray therapy, intralesional injection of corticosteroids, potassium iodide, vaccine therapy, chemotherapy and the use of antibiotic and antituberculous drugs. In refractory cases a combination of treatment is indicated, possibly combined with oral corticosteroids. Drug therapy should be preceded by sensitivity studies, for the response of different strains may vary. All the nine strains examined in the present scries were sensitive to ethambutol and partially sensitive to streptomycin and rifampicin: they were resistant to isoniazid, PAS, cotrimoxazole and H
442
Z.Even-Paz et al.
vancomycin (Table 2). Strains sensitive to cotrimoxazole and vancomycin have been reported by others (Flowers, 1970; Adams et al, 1970). Resistance to isoniazid and PAS, uniformly reported for M. marinum^ is highly characteristic of all atypical tnycobacteria. Epidemiological studies are under way at Ein Feshka. Samples of water from the pools, canals and shower-room facilities, earth and stones from the bottom of the pools, and scrapings from rocks at the pool edges have been examined. Most of the specimens were highly contaminated with other microorganisms. Acid-fast bacilli were seen microscopically in three out of sixteen specimens and M. marinum was isolated from two out of fifty-four cultures. The studies will be reported on in detail at a later date and it is hoped that on their completion means may be devised to eradicate the source of infection. ACKNOWLEDGMENTS We wish to thank Dr Hugo L.David (Chief, Mycobacteriology Branch, Center for Disease Control, U.S.A. Public Health Service, Atlanta, Georgia) for supplying the various types of PPD, and Miss Rachel Albo (Department of Clinical Microbiology, Hadassah-University Hospital, Jerusalem) for her technical assistance. REFERENCES ADAMS, R.A., REMINGTON, J.S., STEINBERG, J. & SEIBERT, J.S. (1970) Tropical fish aquariums: a source of
Mycobacterium marinum infection resembling sporotrichosis. Journal of the American Medical Association, 211, 457. CORTEZ, L . M . & PANKEY, G.A. (1973) Mycobacterium marinum infections of the hand: Report of three cases and review of the literature. Journal of Bone and Joint Surgery, 55, 363. FELDMAN, R.A., LONG, M.W. & DAVID, H.L. (1974) Mycobacierium marinum: a leisure-time pathogen. Jouma/ of Infectious Diseases, 129, 618. FLOWERS, D.J. (1970) Human infection due to Mycobacterium marinum after a dolphin bite. Journal of Clinical Pathology, 23, 475. GOULD, W.M., MCMEEKIN, D.R. & BRIGHT, R.D. (1968) Mycobacterium marinum (balnei) infection: report of a case with cutaneous and laryngeal lesions. Archives of Dermatology, 97, 159. HELLERSTROM, S. (1952) Water-borne tuberculous and similar infections of the skin in swimming pools. Acta dermato-venereologica, 32, 449. HEBLITZ, S. (1953) Cutaneous infection contracted in a swimming pool and resembling tuberculosis. Acta dermato-venereologica, 33, 156. JOLLY, H.W. JR. & SEABURY, J.H. C1972) lnieciions-withMycobacteriummarinum. Archives of Dermatology, 106,32. JUDSDN, F.N. & FELDMAN, R.A. (1974) Mycobacterial skin tests in humans 12 years after infection with Mycobacterium marinum. American Review of Respiratory Diseases, 109, 544. LINELL, F . & NOBDEN, A. (1954) Mycobacterium balnei: a new acid-fast bacillus occurring in swimming pools and capable of producing skin lesions in humans. Acta Tuberculosea et Pneumologica Scandinavica, Supplement 33. PHILPOTT, J.A. JB., WOODBUBNE, A.R., PHILPOTT, O.S., ScHAEFEB, W.B. & MoLLOHAN, C S . (1963) Swimming poo! granuloma: astudy of 290 cases. Archives of Dermatology, 88, 158. SAGE, R.E. & DERBINGTON, A.W. (1973) Opportunistic Mycobacterium marinum infection mimicking Mycobacterium ulcerans in lymphosarcoma. Medical Journal of Australia, 2, 437. SCHAEFER, W.B. & DAVIS, C.L. (1961) A bacteriotogic and histologic study of skin granuloma due to Mycobacterium balnei. American Review of Respiratory Diseases, 84, 837. SILCOX, V.A. & DAVID, H.L. C1971) Differential identification of Mycobacterium kansasii and Mycobacterium marinum. Applied Microbiology, 21, 327. WADDINGTON, E. (1967) An outbreak of swimming pool granuloma. Transactions and Report of the St John's Hospital Dermatological Society, 53, 122. WINTER, F.E. & RUNYON, E.H. (1965) Prepatellar bursitis caused by Mycobacterium marinum {halnti). Journal of Bone and Joint Surgery, 47, 375. ZETTERGREN, L . & ZETTERBERG, B. (1956) The swimming pool disease: Mycobacteriosis balnearea. Acta Sodetatis Medicorum Upsaliensis, 61, 47.
Mycobacterium marinum skin infections mimicking cutaneous leishmaniasis Z.EVEN-PAZ, H.HAAS, T.SACKS AND E.ROSENMANN Departments of Dermatology, Clinical Microbiology and Pathology, Hadassah-University Hospital and Hebrew University-Hadassah Medical School, Jerusalem, Israel Accepted for publication 11 July 1975
SUMMARY
Ten cases of skin infection due to Mycobacterium marinum are reported from Israel. Most of the infections were contracted in natural bathing pools at Ein Feshka on the shores of the Dead Sea, south of Jericho. The lesions closely resembled those of cutaneous leishmaniasis prevalent in this region. The condition does not seem to have been detected previously in the Middle East.
Skin granulomas due to Mycobacterium marinum (balnei) have been recognized since the early 1950s following reports by Linell & Norden (1954) and Herlitz (1953) on 80 cases infected at one swimming pool. Most earlier instances of 'swimming-pool granuloma' (Hellerstrom, 1952) were probably due to the same organism. Four epidemics account for 523 ofthe more than 600 cases so far published; these occurred in Sweden, the U.S.A. and Britain (Linell & Norden, 1954; Zettergren & Zetterberg, 1956; Philpott et al, 1963; Waddington, 1967). Sporadic cases or small outbreaks have been reported from Canada, Holland, Belgium, Czechoslovakia, Australia, New Zealand and Japan. The deteaion of M. marinum infection in the Middle East is of interest because the lesions mimic those of cutaneous leishmaniasis endemic in the same area. CASE REPORTS
Three types of lesion were seen (Figs i, 2). A general description follows and additional clinical data for each case are presented in Table i. The first type of lesion, present in all cases, consisted of chronic inflammatory nodules or plaques 1-4 cm in diameter, with scales, crusts or small ulcers on their surface. Beneath the crusts was a slight sero-sanguinous or purulent discharge. The surface ofthe plaques was uneven, occasionally with a smooth atrophic area. Small satellite papules were present in most cases and constituted the second type of lesion; they usually had a central scale, crust or ulcer. The third type of lesion was seen in four ofthe ten patients and consisted of one or more subcutaneous or intradermal cyst-like swellings. These were I-5-2-O cm in diameter, some distance proximal to the original lesions, and if multiple were arranged along an axis in sporotrichoid fashion. The condition began as one or more small papules which rapidly became swollen and discharged pus. The discharge became scanty or dried and the lesion enlarged. Satellite papules and sporotrichoid Correspondence: Dr Zvi Even-Paz, Chief Physician, Department of Dermatology and Venereology, HadassahUniversity Hospital, Jerusalem, P.O.B. 499, Israel.
435
436
Z.Even-Paz et al.
CD FIGURE I. Case 2. The nodular, ulcerated lesion on the hand appeared 2 weeks after a period of bathing at Ein Feshka. Four months later a smooth, cystic, sporotrichoid swelling developed 13 cm proximally; when incised this released a gelatinous material and subsequently ulcerated. M. marinum was isolated from both lesions. FIGURE 2. Case 3. The larger, irregular, nodular plaque with scales, small crusted ulcers and partial central atrophy developed on an unhealed graze of the right knee which occurred while bathing at Ein Feshka. The smaller lesion appeared 8 months later without further bathing. M. marinum was isolated from both lesions.
lesions appeared later, at intervals which varied up to 18 months. The lesions were usually asymptomatic, the regional lymph nodes were not enlarged and there was no disturbance of the general health. The clinical picture was similar to that of cutaneous leishmaniasis, locally called 'Jericho boil', and all but two ofthe patients had visited the Jericho area. When smears and cultures for Leishmania tropica and the intradermal leishmanin tests proved negative routine, bacteriological studies were done and a search made for mycobaeteria. Biopsies were done in eight ofthe cases. Bacteriological studies
Tissue scraped from incisions, biopsy material, exudates, crusts and scales, was examined. The specimens were ground aseptically, washed once in normal saline and suspended in phosphate buffer, pH 72. Specimens showing no growth on blood agar plates were cultured on Loewenstein-Jensen (L-J) medium without further treatment. Specimens showing growth were treated for 15 min with 6% sulphuric acid, washed with saline, resuspended in phosphate buffer and inoculated on L-J medium. The cultures were incubated at 32 C and 37 C. On microscopic examination of smears a few, scattered, acid-fast bacilli were seen in specimens from five out ofthe ten cases. In cultures from all ten cases, small creamy-white colonies which became yellow-orange after exposure to light appeared after 7-12 days at 32 C. There was no growth at 37 C. The cultures were identified as M. marinum by their photochromogenecity, rate of growth and need for lower temperature. Identification was confirmed by hydrolysis of Tween 80, negative nitrate reductase and positive arylsulphatase activity.
Mycobacterium marinum skin infections
437
TABLE I. Clinical data on ten cases of M. marinum skin infection Patient
Sex
Age
Sites affected
Trauma
I
M
40
Elbow, upper arm
—
Ein Feshka
Yes
2 wk2 mth.
2
M
9
Hand, forearm
—
Yes
2 wk2 mth.
3
F
9
Knee
Ein Feshka Galilee stream Medit'n Ein Feshka
—
Brief
4
M
39
Elbow, Graze (while upper arm bathing)
Ein Feshka Medit'n
Yes
Brief
5
M
2t
Knee, thigh
Sea of Galilee
Yes
?
6
F
47
—
—
Brief
7
F
Ein Feshka
—
6 mth.
Ein Feshka
—
Brief
Ein Feshka
—
1-2 mth.
Ein Feshka
—
2yr.
M
9
10
M
F
Forearm, hand 7 Knee
Graze (while bathing)
—
Graze (from gas range) Kick (before bathing)
Upper arm Insect bite (before bathing) 12 Buttock Insect bite (while bathing) — 38 Forearm
6i
Water exposure
Sporotrichoid lesions
Incub. period
Treatment and course None: complete resolution Grenz rays: partial resolution Grenz rays: partial resolution Grenz rays: complete resolution Griseofulvin: complete resolution Grenz rays: course not known Grenz rays: partial resolution None: complete resolution Grenz rays: partial resolution None: course not known
Drug sensitivity tests were done on L-J medium containing various dilutions of isoniazid, PAS, streptomycin, ethambutol and rifampicin. Disc susceptibility tests were performed on Dubos oleic agar base, using cotrimoxazole (25 |jg) and vancomycin (5 //g) discs (Baltimore Biological Laboratories, U.S.A.). The results are given in Table 2. Purified protein derivative (PPD) tests The results of intradermal tests with PPD from 6 various sources are given in Table 3. Histopathology Marked changes were observed in seven of the eight patients from whom biopsy specimens were taken (Fig. 3). The epidermis showed hyperkeratosis with focal parakeratosis, irregular acanthosis with pseudoepitheliomatous hyperplasia or extreme elongation of the rete pegs, and liquefaction degeneration ofthe basal layer. In the upper- and mid-dermis there was a mixed infiammatory infiltrate which sometimes extended into the epidermis. The cells were mainly lymphocytes and histiocytes but in two patients plasma cells predominated. There was a marked neutrophilic and eosinophilic exudate in two cases and in six cases microabscesses were present, usually located near
Z.Even-Paz et al.
438
TABLE 2. Sensitivity of nine M. marinum strains to antibacterial agents (compared with a standard strain of M. tuberculosis)
Strain Minimum inhibitory concentration (/'g/ml) from — patient Isoniazid PAS Streptomycin Ethambutol Rifampicin
Disc sensitivity test Cotrimoxazole Vancomycin
no. I 2
3 4 5 6 7 8 9
M. tuberculosis (H37RV)
>5 >5 5 5 >5 5 >5 5 5
(25 fig)
(5 f'g)
R* R R R R R R R R
> IOO > IOO > IOO
10
10
10
2
10
> roo
10
2
10
> > > > >
IOO IOO IOO IOO IOO
10
2
10
10
I
10
10
2
10
10
2
10
R* R R R R R R R
10
2
10
R
I
I
2
5
02
* Resistant (no inhibitory zone around discs).
the tip of an elongated rete peg or in a dermal papilla. Non-caseating epithelioid granulomas were seen in all seven cases and in one instance the epithelioid granulomas were invaded by neutrophils. In most cases multinucleated giant cells of Langhans' or foreign body type were present (Fig. 4a, b). In the eighth patient there was a mixed histiocytic and lymphocytic infiltrate, with a few neutrophils, around the deep dermal vessels: no granulomas, giant cells or microabscesses were seen. Acid-fast bacilli were found in one case. Leishman-Donovan bodies and fungi could not be demonstrated. Treatment and course
Five patients were treated twice with 500-700 rad Grenz rays 3-26 months after the onset of their condition; the interval between the radiations in the different cases varied from 3 weeks to 4 months (Table i: patients nos. 2, 3, 4, 7, 9). Complete resolution occurred within 2 months in patient no. 4 and moderate to marked resolution in the others, who are still under observation. Patient no. 6 received 700 rad Grenz rays once to the lesions but did not report for further examination. Complete resolution occurred in three patients without specifically directed treatment. In patient no. 8 this occurred 4 months after the lesions appeared, bacitracin ointment only being applied. The elbow T A BL E 3. Results of intradermal tests withfiveunits PPD from various sources (mm induration after 72 h) Patiem
M. marinum M. tuberculosis M. kansasii M. foriuitum Scotochromogens Battey
6
7
8
9
10
12
21
6 5
0
20
19 16
0
10
6
0
2
0
0
7
0
0
5
5
4
5 9
I
2
3
4
5
12
7
7
13
0
5
8 8
17
12
6
0
15
24
0
5
0
0
13
15
16
12
10
12
16 18
0
5
9 13
7
14 0
6 7
0
Mycobacterium marinum skin infections
439
FIGURE 3. Typical lesion with hyper- and parakeratosis, pseudoepitheliomatous hyperplasia and a dense inflammatory infiltrate in the dermis (H & E, x 42},
lesion in patiem no. i had been diagnosed elsewhere as psoriasis and had been treated by 4 intralesional injections of triamcinolone acetonide. After initial marked improvement the lesion became swollen, discharged pus and 2 months later a sporotrichoid swelling appeared on the upper arm. After a further 8 months complete regression occurred without other treatment. Patient no. 5 had been given fineparticle griseofulvin by another doctor shortly before the diagnosis was made. This treatment was continued and 3 months later there was resolution of the lesions. The last patient (no. 10) did not report after the initial visit at which material was taken for examination. DISCUSSION
Mycobacterium marinum is capable of living saprophytically, especially in an aquatic environment, and parasitically in cold-blooded animals and man. The fact that in man the lesions are nearly always
a F1G u R E 4. (a) Multinucleated giant cell of the Langhans type (H & E, x 420). (b) Multinucleated giant cell of the foreign body type (H & E, x 260).
44°
Z.Even-Paz et al.
limited to the skin has been attributed to the preference of the parasite for relatively cool growth conditions. Only occasionally has involvement of deeper structures been demonstrated or suspected (Winter & Runyon, 1965; Gould, McMeekin & Bright, 1968; Jolly & Seabury, 1972; Cortez & Pankey, 1973). The lesions are usually on one limb and few in number but widespread skin infection has occurred in patients with suspected immunological impairment (Gould et al., 1968; Sage & Derringtcn, 1973)Human infection is usually acquired whilst bathing in fresh, brackish or salt water. However, 'swimming-pool granuloma' is not a suitable synonym, for lakes, wells, rivers, inlets, bays, the seashore, home aquaria and a coal mine have been incriminated. Some patients have denied contact with any extra-domiciliary source of water. Previous trauma of the affected site, although common, is not always recalled. Eight of the ten patients in this series were apparently infected at Ein Feshka, a bathing area south of Jericho where fresh-water pools fed by springs empty into the Dead Sea via short canals. By coincidence, this region is highly endemic for cutaneous leishmaniasis, the lesions of which may be clinically indistinguishable from those due to M. marinum. This coincidence has probably delayed recognition of the latter condition. The incubation period was very variable. Although most commonly a few weeks to a few months, in one case it may have been as long as 2 years whilst in others it seemed virtually absent, the lesions rapidly developing on a traumatized site which did not heal. Jt was not possible to assess the incubation period in those cases without a history of trauma and with no limited exposure period to a suspected water-source. The examination of multiple samples is important for the detection of M. marinum in the lesions. Acid-fast bacilli are seen only rarely in histological sections and infrequently in smears, whilst cultures may not always be positive. In the present series, three to eight samples were taken from each patient. Of the total of forty-six samples, acid-fast bacilli were seen microscopically in six and there were only twenty-six positive cultures. M. marinum is classified in Runyon's Group I together with M. kansasii, since both are photochromogenie. M. kansasii grows at 37 C whilst M. marinum grows at 30-32'C. However, primary isolation of M marinum at 37 C has been reported (Feldman, Long & David, 1974): confirmation of identity may be made by the nitrate reduction test, usually negative for M. marinum and positive for M. kansasii (Silcox & David, 1971)- ^- ulcerans also requires the lower incubation temperature but its growth takes several weeks. Skin lesions due to M. ulcerans possess an abundance of acidfast bacilli and on culture the colonies are non-pigmcnted, tend to form cords and give a positive neutral red test. The histopathological picture is usually suggestive but not pathognomonic. The diagnosis is supported by the presence of a mixed inflammatory reaction with a dense mononuclear infiltrate, a few non-caseating granulomas with or without giant cells, and microabscesses. Central fibrinoid necrosis in the granulomas has been reported occasionally and cascating necrosis rarely (Linell & Norden, 1954; Schaefer & Davis, 1961). Acid-fast bacilli are not usually seen and their detection requires a prolonged search of serial sections. Clinical and histological differential diagnosis includes all other chronic infective granulomas, foreign body granuloma and iodine and bromine granulomas. Clinically, in addition, psoriasis, verrucous lichen planus and even verruca vulgaris may have to be considered. In the Middle East confusion is most likely with cutaneous leishmaniasis and some features differentiating the two conditions are given in Table 4. A positive reaction of 10 mm or more induration to the intradermal PPD (marinum) test supports the diagnosis but the result must be interpreted with caution. There is a high incidence of cross-reactivity with PPD from other sources and in the patients in this series the reaction to PPD (marinum) was not usually the strongest (Table 3).
Mycobacterium marinum skin infections
441
TABLE 4. Differential diagnosis of M. marinum and L. tropica skin infection Mycobacterium marinum infection
Leishmania tropica infection
Patient's history Geographic factors Seasonal factors Diurnal factors
Exposure to water source Minor trauma Incubation period
No[ known: reported so far from only a few widely dispersed areas Probably not relevant, but if infected whilst bathing outdoors usually during warmer season Probably not relevant, but if infected whilst bathing outdoors usually during daylight hotirs Commonly reported Commonly reported Very brief to prolonged
Sub-tropical and tropical endemic areas During warmer season Acquired at or after sunset Not relevant None, except insect bite Usually not less than 4 weeks. May be prolonged
Clinical appearance Number and sice Few, usually on limb at site liable to of lesions trauma: rarely bilateral Sporotrichoid Relatively common lesions Special examinations
Few or numerous. Common on face as well as limbs: often bilateral
Intradermal tests * Smears
PPD tests positive Leishmanin test negative Acid-fast bacilli found with difficulty
Culture
From tissues and discharge: M. marinum may not grow from all the specimens Acute and chronic inflammatory infiltrate. May be giant cells, epithelioid granulomas and microabscesses. A-F bacilli rare
PPD tests negative Leishmanin test positive Leishman-Donovan (LD) bodies found easily in early lesions From tissue at edge of lesion: L. tropica usually grows from all early lesions Chronic infiammatory infiltrate sometimes granulomatous. Numerous LD bodies in histiocytes in early lesions
Histologic picture
Rare
* In areas in which both leishmania] and mycobacterial infections occur the intradermal tests must be interpreted with care for positive reactions persist after cure. Spontaneous healing probably occurs within a few months to a few years in the majority of cases. Small, rapidly healing lesions may never be brought to the attention of a doctor: such cases, if frequent, may be responsible for an increased percentage of tuberculin-positive reactors in some areas (Judson & Feldman, 1974). The many treatments recommended include excision with skin grafting, curettage, electrodesiccation or fulguration, cryotherapy, local heat, ultraviolet irradiation, Grenz-ray therapy. X-ray therapy, intralesional injection of corticosteroids, potassium iodide, vaccine therapy, chemotherapy and the use of antibiotic and antituberculous drugs. In refractory cases a combination of treatment is indicated, possibly combined with oral corticosteroids. Drug therapy should be preceded by sensitivity studies, for the response of different strains may vary. All the nine strains examined in the present scries were sensitive to ethambutol and partially sensitive to streptomycin and rifampicin: they were resistant to isoniazid, PAS, cotrimoxazole and H
442
Z.Even-Paz et al.
vancomycin (Table 2). Strains sensitive to cotrimoxazole and vancomycin have been reported by others (Flowers, 1970; Adams et al, 1970). Resistance to isoniazid and PAS, uniformly reported for M. marinum^ is highly characteristic of all atypical tnycobacteria. Epidemiological studies are under way at Ein Feshka. Samples of water from the pools, canals and shower-room facilities, earth and stones from the bottom of the pools, and scrapings from rocks at the pool edges have been examined. Most of the specimens were highly contaminated with other microorganisms. Acid-fast bacilli were seen microscopically in three out of sixteen specimens and M. marinum was isolated from two out of fifty-four cultures. The studies will be reported on in detail at a later date and it is hoped that on their completion means may be devised to eradicate the source of infection. ACKNOWLEDGMENTS We wish to thank Dr Hugo L.David (Chief, Mycobacteriology Branch, Center for Disease Control, U.S.A. Public Health Service, Atlanta, Georgia) for supplying the various types of PPD, and Miss Rachel Albo (Department of Clinical Microbiology, Hadassah-University Hospital, Jerusalem) for her technical assistance. REFERENCES ADAMS, R.A., REMINGTON, J.S., STEINBERG, J. & SEIBERT, J.S. (1970) Tropical fish aquariums: a source of
Mycobacterium marinum infection resembling sporotrichosis. Journal of the American Medical Association, 211, 457. CORTEZ, L . M . & PANKEY, G.A. (1973) Mycobacterium marinum infections of the hand: Report of three cases and review of the literature. Journal of Bone and Joint Surgery, 55, 363. FELDMAN, R.A., LONG, M.W. & DAVID, H.L. (1974) Mycobacierium marinum: a leisure-time pathogen. Jouma/ of Infectious Diseases, 129, 618. FLOWERS, D.J. (1970) Human infection due to Mycobacterium marinum after a dolphin bite. Journal of Clinical Pathology, 23, 475. GOULD, W.M., MCMEEKIN, D.R. & BRIGHT, R.D. (1968) Mycobacterium marinum (balnei) infection: report of a case with cutaneous and laryngeal lesions. Archives of Dermatology, 97, 159. HELLERSTROM, S. (1952) Water-borne tuberculous and similar infections of the skin in swimming pools. Acta dermato-venereologica, 32, 449. HEBLITZ, S. (1953) Cutaneous infection contracted in a swimming pool and resembling tuberculosis. Acta dermato-venereologica, 33, 156. JOLLY, H.W. JR. & SEABURY, J.H. C1972) lnieciions-withMycobacteriummarinum. Archives of Dermatology, 106,32. JUDSDN, F.N. & FELDMAN, R.A. (1974) Mycobacterial skin tests in humans 12 years after infection with Mycobacterium marinum. American Review of Respiratory Diseases, 109, 544. LINELL, F . & NOBDEN, A. (1954) Mycobacterium balnei: a new acid-fast bacillus occurring in swimming pools and capable of producing skin lesions in humans. Acta Tuberculosea et Pneumologica Scandinavica, Supplement 33. PHILPOTT, J.A. JB., WOODBUBNE, A.R., PHILPOTT, O.S., ScHAEFEB, W.B. & MoLLOHAN, C S . (1963) Swimming poo! granuloma: astudy of 290 cases. Archives of Dermatology, 88, 158. SAGE, R.E. & DERBINGTON, A.W. (1973) Opportunistic Mycobacterium marinum infection mimicking Mycobacterium ulcerans in lymphosarcoma. Medical Journal of Australia, 2, 437. SCHAEFER, W.B. & DAVIS, C.L. (1961) A bacteriotogic and histologic study of skin granuloma due to Mycobacterium balnei. American Review of Respiratory Diseases, 84, 837. SILCOX, V.A. & DAVID, H.L. C1971) Differential identification of Mycobacterium kansasii and Mycobacterium marinum. Applied Microbiology, 21, 327. WADDINGTON, E. (1967) An outbreak of swimming pool granuloma. Transactions and Report of the St John's Hospital Dermatological Society, 53, 122. WINTER, F.E. & RUNYON, E.H. (1965) Prepatellar bursitis caused by Mycobacterium marinum {halnti). Journal of Bone and Joint Surgery, 47, 375. ZETTERGREN, L . & ZETTERBERG, B. (1956) The swimming pool disease: Mycobacteriosis balnearea. Acta Sodetatis Medicorum Upsaliensis, 61, 47.
