First Etiologically Confirmed Cases of Mycobacterium Marinum Infection in Bulgaria

This study aimed to describe the first two microbiologically confirmed cases of cutaneous and soft tissue Mycobacterium marinum infection in Bulgaria. The isolation of the Nontuberculous Mycobacteria (NTM) strains and their species identification was performed at NRL TB, NCIPD using specific media and cultivation conditions, and PCR based Line Probe Assay (LPA) from the positive cultures. The two patients had closely related jobs to fishes and water reservoirs and both of them had a similar clinical manifestation of M. marinum infection known as “swimming pool” or “fish tank” granuloma. The prolonged specific treatment with at least two-drug combination of rifampicin plus ethambutol and some complications were a big challenge for clinicians as well as the patients.


INTRODUCTION
Mycobacterium marinum is a slow-growing NTM also known as atypical mycobacterium or environmental mycobacterium or MOTT (Mycobacterium Other Than Tuberculosis), and belongs to the same genus as the pathogens causing tuberculosis and leprosy. 1,2 M. marinum is found around the world in nondisinfected salt-and freshwater reservoirs such as swimming pools and fish tanks, hence the name of the condition: "swimming pool" or "fish tank" granuloma. 3 The mycobacteriosis involves usually fingers of the right hand -most people are right-handed and they fish, clean fish tanks or do some other aquatic activities using the right hand. Infection occurs most commonly at sites of small skin wounds or abrasions where bacteria enter. Sign of disease involve localized painless skin lesions, single or multiple nodules or ulcers. Less commonly M. marinum can spread in the body and cause joint or bone infections. 4 Lethal outcome occurs on very rare occasions, most often in immunocompromised persons. 5

MATERIALS AND METHODS
In both clinically described cases herein reported we examined skin samples and pus from the wound site. A smear microscopy for AFB (Ziel-Neelsen) was performed and cultivating on specific media -solid medium (Löwenstein -Jensen) and liquid media (MGIT) at lower temperature -room temperature, i.e. 25-28°C. For species identification of M. marinum we used Geno Type Mycobacterium Common Mycobacteria (CM) (HAIN Lifescience, Nehren, Germany), based on DNA strip technology for LPA: DNA amplification of 23S rRNA gene, reverse hybridization and immobilization on a membrane strip. PCR diagnosis was supplemented with phenotypic analysis, including examination of grow characteristics, temperature tolerance, and pigmentation of colonies.

CASE REPORT
The first microbiologically confirmed case of cutaneous M. marinum disease was in 2014. A 66-year-old woman presented with 12 small red-violet papules on the dorsum of the right hand and forearm, some of them having progressed to pustules and ulcers, more or less in straight line proximally (Fig. 1A). The patient was immunocompetent and clinically healthy. She was a pet shop owner. A few days after feed stem pricking under the middle finger nail of her right hand she cleaned an ornamental tropical fishes aquarium without using protective gloves. The fishes were bought from Romanian pet shop the previous month.
Culture at room temperature was positive only on MGIT, 25 days after inoculation (Fig. 2).
The first skin lesions on the right hand occurred approximately 3 weeks after a local hand injury. The progression from erythematous plaques through papules, pustules and ulcers was painless in the next month (Fig. 1A). The lesion was excised and sent for histological examination. Histology revealed chronic inflammatory granulation tissue and giant cell of the Langhans type. In NRL TB, NCIPD was taken wound secretion with skin sample for microbiological examination. Smear microscopy for AFB was negative.
There was no growth on liquid and solid media at 37°C. The cord-factor phenomenon was revealed using Ziel-Neelsen staining from positive MGIT culture. Lowenstein-Jensen was negative the end of 56 days cultivating. Immunochromatographic test (BD MGIT TBc Identification Test) did not show presence of MPT64 antigen in the strain, i.e. it was negative for M. tuberculosis complex in a differential diagnosis plan. The subsequent subculture from positive MGIT on Lowenstein-Jensen allowed us to trace the colony morphology, photo reactivity, pigmentation and growth rate. Twenty-eight days after the primary inoculation on MGIT, we identified M. marinum by Geno Type Mycobacterium CM using extracted DNA. A specific two-drug therapy with a combination of rifampicin (600 mg/day) and ethambutol (1000 mg/day) for at least 6 months was started after microbiological confirmation and was discontinued at 2 months because of the side effects as joint pain, candidosis, diarrhea and intestinal pain. The treatment continued with symptomatic therapy and the lesions slowly improved after 8 months of therapy (Fig. 1B). (Fig. 3). Then the carpal tunnel syndrome occurred and the patient underwent surgery.  Tissue biopsy sample was sent for culture and histology. Histology was the same as in Case 1. The smear microscopy for AFB was negative. The cultures at room temperature were positive on both liquid and solid media -on MGIT after 14 days from the inoculation and respectively on Lowenstein-Jensen after 54 days (Fig. 4). There was no growth on liquid and solid media at 37°C. The "cord-factor" was observed on Ziel-Neelsen staining from positive MGIT culture (Fig. 5). We extracted DNA from MGIT and identified M. marinum by Geno Type Mycobacterium CM. A treatment regimen of rifampicin 600 mg/day and ethambutol 1000 mg/day was begun following the etiological confirmation of the diagnosis. The patient rapidly responded to the treatment. But a month later he felt strong pain in the area of the operative wound and had again the symptoms of carpal tunnel syndrome. Redo surgery followed by revision, debridement, desinscertio, excision, decompression and reinsertion. The intra operationem taken samples were microbiologically negative. The two-regimen therapy continued plus clarithromycin 500 mg/day for the next 6 months and the patient's condition slowly improved.

DISCUSSION
The delay of diagnosis of M. marinum infection is common, probably due to the rarity of the infection. 4,8,9 In both cases reported herein, the patients have been consulted by dermatologists, rheumatologists, infectious disease specialists, orthopedists, surgeons, until some of them decided to send biopsies -more often for histology and rarely for microbiology. Usually histology is identical to mycobacteriosis and tuberculosis and then comes into consideration the history with swimming, fishing and other activities related to water tanks or marine life. 9 Of course, the history of M. marinum infection is very pivotal, but in our practice we had cases with fishing injuries history but subsequently we identified M. tuberculosis. With respect to the immunological tests, except for tuberculosis, they are also positive in M. marinum, M. kansasii, M. szulgai, and M. riyadhense mycobacteriosis. 10 In the second case the T-Spot.TB test was positive. The differential diagnosis between tuberculosis and mycobacteriosis should always be taken into consideration given that the treatment for the two diseases is different. For example, two potent drugs used in the intensive phase of tuberculosis such as isoniazide and pyrazinamide are not applicable to the M. marinum infection due to innate resistance. 2,11,12 Culture and species identification of M. marinum play a crucial role in the further development of the infection. M. marinum infections have slow, spontaneous resolution over period of 1 to 6 years. 5,12 Antimicrobial therapy (rifampicin, ethambutol, claritromycin, trimethoprim/sulfamethoxasole, tetracycline, and some of the quinolones) may be warranted to prevent progression to deep infection. Corticosteroids should be avoided, because of exacerbation of the condition. 5,11 Depending on the extent and severity of M. marinum infection, the therapy may continue from several weeks to 18 months. 2,5,11 Deep M. marinum infection typically requires both antimicrobial and surgical treatment. Debridement of the necrotic tissues may be necessary. In order to control the infection despite appropriate therapy and related debridement, amputation may be required. M. marinum systemic disease is rare and most commonly affects immunocompromised patients. 5,9,11