Rhinosinusitis and disseminated cutaneous infection caused by Mycobacterium chelonae in an immunocompromised patient.

Mycobacterium chelonae frequently involves the skin, and the disseminated form can be observed in immunocompromised patients. In contrast, rhinosinusitis caused by the bacterium is a rare manifestation, which occurs independently of immune status. We report here a rare case of M. chelonae infection presenting as both disseminated cutaneous infection and rhinosinusitis in an immunocompromised patient. He had received systemic corticosteroids for 11 months due to cryptogenic organizing pneumonia. Before admission, he sustained injuries to his left arm and hand; those injuries succumbed to an infection that would subsequently spread to his other limbs, face, and even nasal cavities. This valuable case suggests that disseminated cutaneous infection by M. chelonae could spread to other organs.

Discrimination of Mycobacterium abscessus subsp. massiliense from Mycobacterium abscessus subsp. abscessus in clinical isolates by multiplex PCR.

The rapidly growing mycobacterium M. abscessus sensu lato is the causative agent of emerging pulmonary and skin diseases and of infections following cosmetic surgery and postsurgical procedures. M. abscessus sensu lato can be divided into at least three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. Clinical isolates of rapidly growing mycobacteria were previously identified as M. abscessus by DNA-DNA hybridization. More than 30% of these 117 clinical isolates were differentiated as M. abscessus subsp. massiliense using combinations of multilocus genotyping analyses. A much more cost-effective technique to distinguish M. abscessus subsp. massiliense from M. abscessus subsp. abscessus, a multiplex PCR assay, was developed using the whole-genome sequence of M. abscessus subsp. massiliense JCM15300 as a reference. Several primer sets were designed for single PCR to discriminate between the strains based on amplicons of different sizes. Two of these single-PCR target sites were chosen for development of the multiplex PCR assay. Multiplex PCR was successful in distinguishing clinical isolates of M. abscessus subsp. massiliense from samples previously identified as M. abscessus. This approach, which spans whole-genome sequencing and clinical diagnosis, will facilitate the acquisition of more-precise information about bacterial genomes, aid in the choice of more relevant therapies, and promote the advancement of novel discrimination and differential diagnostic assays.

Therapeutic efficacy of rifalazil (KRM-1648) in a M. ulcerans-induced Buruli ulcer mouse model.

Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans infection that requires long-term antibiotic treatment and/or surgical excision. In this study, we investigated the therapeutic efficacy of the rifamycin derivative, rifalazil (RLZ) (also known as KRM-1648), in an advanced M. ulcerans infection model. Six-week-old female BALB/c mice were infected with 3.25 x 104 colony-forming units (CFU) of M. ulcerans subcutaneously into the bilateral hind footpads. At 33 days post-infection, when the footpads exhibited significant redness and swelling, mice were treated orally with 5 or 10 mg/kg of RLZ for up to 15 weeks. Mice were followed for an additional 15 weeks following treatment cessation. Untreated mice exhibited a progressive increase in footpad redness, swelling, and erosion over time, and all untreated mice reached to endpoint within 5-8 weeks post-bacterial injection. In the RLZ-treated mice, footpad redness and swelling and general condition improved or completely healed, and no recurrence occurred following treatment cessation. After 3 weeks of treatment, the CFU counts from the footpads of recovered RLZ-treated mice showed a 104 decrease compared with those of untreated mice. We observed a further reduction in CFU counts to the detection limit following 6 to 15 weeks of treatment, which did not increase 15 weeks after discontinuing the treatment. Histopathologically, bacteria in the treated mice became fragmented one week after RLZ-treatment. At the final point of the experiment, all the treated mice (5mg/kg/day; n = 6, 10mg/kg/day; n = 7) survived and had no signs of M. ulcerans infection. These results indicate that the rifamycin analogue, RLZ, is efficacious in the treatment of an advanced M. ulcerans infection mouse model.

Nineteen cases of Buruli ulcer diagnosed in Japan from 1980 to 2010.

The etiology, clinical manifestations, and treatment of 19 sporadic cases of Buruli ulcer (BU) in Japan are described. The cases originated in different regions of Honshu Island, with no evidence of patient contact with an aquatic environment. The majority (73.7%) of cases occurred in females, with an average age of 39.1 years for females and 56.8 years for males. All patients developed ulcers on exposed areas of the skin (e.g., face, extremities). Most ulcers were <5 cm in diameter (category I), except in one severe progressive case (category II). Pain was absent in 10 of the 19 cases. Fourteen ulcers were surgically excised, and nine patients needed skin grafting. All cases were treated with various antibiotic regimens, with no reported recurrences as of March 2011. Mycobacterium ulcerans-specific IS2404 was detected in all cases. Ten isolates had identical 16S rRNA gene sequences, which were similar to those of M. ulcerans. However, the rpoB gene showed a closer resemblance to Mycobacterium marinum or Mycobacterium pseudoshottsii. PCR identified pMUM001 in all isolates but failed to detect one marker. DNA-DNA hybridization misidentified all isolates as M. marinum. The drug susceptibility profile of the isolates also differed from that of M. ulcerans. Sequence analysis revealed "Mycobacterium ulcerans subsp. shinshuense" as the etiologic agent of BU in Japan. Clinical manifestations were comparable to those of M. ulcerans but differed as follows: (i) cases were not concentrated in a particular area; (ii) there was no suspected connection to an aquatic environment; (iii) drug susceptibility was different; and (iv) bacteriological features were different.

Multiple cases of cutaneous Mycobacterium massiliense infection in a "hot spa" in Japan.

Seven body polishers working in the same "hot spa" presented with multiple red nodules and papules on their hands and forearms. A causative agent was successfully isolated from two of the subjects and from a swab sample collected from the underside of a bed cover in the body-polishing facility. The two cutaneous isolates and the environmental isolate were rapidly growing mycobacteria that formed nonphotochromogenic smooth or smooth/rough colonies on Ogawa egg slants. They were identified as Mycobacterium massiliense by multigenotypic analysis using the 16S rRNA, hsp65, and rpoB genes and the 16S-23S rRNA internal transcribed spacer (ITS) region. However, the use of the 16S rRNA gene sequence and/or DNA-DNA hybridization (DDH Mycobacteria Kit) alone would not distinguish M. massiliense from mycobacteria in the M. chelonae-M. abscessus group. The three isolates were significantly more susceptible to clarithromycin, doxycycline, and minocycline than the M. abscessus and M. bolletii reference strains. One cutaneous isolate and the environmental isolate were in a related cluster by randomly amplified polymorphic DNA PCR (RAPD-PCR). Of the several mycobacterial species found in the day spa, only M. massiliense was isolated from biopsy specimens of the skin lesions, suggesting that this bacterium is a human skin pathogen. This is the first known report of cutaneous M. massiliense infections that could not be attributed to a prior invasive procedure. This is also the first report of M. massiliense infection in Japan.

[Non-tuberculous mycobacterium strains that show positive test for identification kits of M. tuberculosis complex].

OBJECTIVES: Saito et al. isolated novel mycobacterium strains from the sputum of 12 patients with pulmonary disease. They reported, that the strains were clearly different from Mycobacterium tuberculosis (TB) in cultural, biochemical and immunological properties, despite the high homology (99.1%) of the 16S rRNA gene sequence between the two. Recently, we isolated four strains having similar properties as the above strains among mycobacterial strains that were sent to the Research Institute of Tuberculosis for identification. We examined these isolates using commercial systems for identification of mycobacteria. MATERIALS: Four strains of the unidentified mycobacteria were used in this study. METHODS: Tests used in the study included cultural on solid media, biochemical characteristics, DNA sequence analyses of 16S rRNA and rpoB genes, TRC, COBAS AMPLICOR, COBAS TaqMan, MTD, DDH, Accu-Probe, Capilia TB, and a drug susceptibility tests. RESULTS: After three weeks of culture, smooth and non-photochromogenic colonies were formed. The niacin accumulation test was negative. The homologies of DNA sequence between the new strains and M. tuberculosis for 16S rRNA and rpoB genes were 97.8% and 90.2%, respectively. The tests with TRC and MTD kits were positive, whereas the tests with AMPLICOR, TaqMan, Accu-Probe and Capilia TB kits were negative. The organism was not identified with the DDH system.

Mycolactone is responsible for the painlessness of Mycobacterium ulcerans infection (buruli ulcer) in a murine study.

Buruli ulcer is a chronic skin disease caused by Mycobacterium ulcerans, which produces a toxic lipid mycolactone. Despite the extensive necrosis and tissue damage, the lesions are painless. This absence of pain prevents patients from seeking early treatment and, as a result, many patients experience severe sequelae, including limb amputation. We have reported that mice inoculated with M. ulcerans show loss of pain sensation and nerve degeneration. However, the molecules responsible for the nerve damage have not been identified. In order to clarify whether mycolactone alone can induce nerve damage, mycolactone A/B was injected to footpads of BALB/c mice. A total of 100 microg of mycolactone induced footpad swelling, redness, and erosion. The von Frey sensory test showed hyperesthesia on day 7, recovery on day 21, and hypoesthesia on day 28. Histologically, the footpads showed epidermal erosion, moderate stromal edema, and moderate neutrophilic infiltration up to day 14, which gradually resolved. Nerve bundles showed intraneural hemorrhage, neutrophilic infiltration, and loss of Schwann cell nuclei on days 7 and 14. Ultrastructurally, vacuolar change of myelin started on day 14 and gradually subsided by day 42, but the density of myelinated fibers remained low. This study demonstrated that initial hyperesthesia is followed by sensory recovery and final hypoesthesia. Our present study suggests that mycolactone directly damages nerves and is responsible for the absence of pain characteristic of Buruli ulcer. Furthermore, mice injected with 200 microg of mycolactone showed pulmonary hemorrhage. This is the first study to demonstrate the systemic effects of mycolactone.

[Intraspecies divergence of 16S rDNA, ITS, rpoB gene and hsp65 gene sequence for Mycobacterium lentiflavum].

OBJECTIVE: To clarify the genetic microheterogeneity of Mycobacterium lentiflavum and identify the predominant genotype. MATERIALS AND METHODS: Clinical isolates of M. lentiflavum used in this study were obtained from sixteen patients of lung diseases. In order to assess their intraspecies variability, four gene fragments, from the 16S rDNA (1471 bp), 16S-23S ITS (282 bp), rpoB (306 bp), and hsp65 (401 bp), were sequenced. RESULTS: Intraspecies variabilities were found in all of the four targeting fragments. As multilocus sequence typing with these four targets, 16 clinical isolates were divided into 3 genotypes, i.e., MLST2, MLST3, and MLST4. Among them, MLST2 to which 12 clinical isolates belonged, was a predominant genotype. Three strains belonged to MLST3 and the remaining one strain belonged to MLST4. Drug susceptibility study indicated that there was no clear relation between sequence types and drug susceptibility. CONCLUSION: Multilocus sequence typing could aid in characterization and in better understanding of the epidemiology of M. lentiflavum.

Case of Mycobacterium haemophilum misdiagnosed as Mycobacterium intracellulare due to one base insertion in the bacterial genome.

Mycobacterium haemophilum is a slow-growing, non-tuberculous mycobacteria that causes cutaneous infection. We describe a case of cutaneous infection in a 68-year-old Japanese man with polymyositis. This was caused by M. haemophilum harboring one base insertion in gene sequence. At first, the causal microorganism was misidentified as M. intracellulare by COBAS® TaqMan® MAI test. However, poor growth on Ogawa media and growth enhancement on 7H11C agar around a hemin-containing disk prompted us to reinvestigate the causal microorganisms, which were revealed to be M. haemophilum. Amplified polymerase chain reaction products were sequenced, and the 16S rRNA gene, rpoB, hsp65 and internal transcribed spacer region sequences showed a 100%, 100%, 99.66% and 99.7% match, respectively, with the corresponding regions of M. haemophilum, but it harbored a novel gene sequence in hsp65. The sequences determined by gene analysis of the M. haemophilum strain were deposited into the International Nucleotide Sequence Database. Although numerous cases of M. haemophilum infection have been reported in other countries, only six cases have been reported in Japan to date. It could be possible that this novel mutation lead to misdiagnosis. As M. haemophilum prefers a lower growth temperature (30-32°C) and it requires iron in the culture medium, M. haemophilum could be misidentified or overlooked. Accordingly, a M. haemophilum infection should be considered in cases of cutaneous infection of the body sites, of which surface temperature is low.

Naturally occurring a loss of a giant plasmid from Mycobacterium ulcerans subsp. shinshuense makes it non-pathogenic.

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a WHO-defined neglected tropical disease. All Japanese BU causative isolates have shown distinct differences from the prototype and are categorized as M. ulcerans subspecies shinshuense. During repeated sub-culture, we found that some M. shinshuense colonies were non-pigmented whereas others were pigmented. Whole genome sequence analysis revealed that non-pigmented colonies did not harbor a giant plasmid, which encodes elements needed for mycolactone toxin biosynthesis. Moreover, mycolactone was not detected in sterile filtrates of non-pigmented colonies. Mice inoculated with suspensions of pigmented colonies died within 5 weeks whereas those infected with suspensions of non-pigmented colonies had significantly prolonged survival (>8 weeks). This study suggests that mycolactone is a critical M. shinshuense virulence factor and that the lack of a mycolactone-producing giant plasmid makes the strain non-pathogenic. We made an avirulent mycolactone-deletion mutant strain directly from the virulent original.

[Two cases of lung infection due to Mycobacterium shimoidei, with special reference to bacteriological investigation].

Mycobacterium shimoidei (Tsukamura 1982) is an uncommon but widely distributed pathogen usually isolated from respiratory specimens. We report two cases of lung disease due to M. shimoidei and the associated bacteriological results. A 45-year-old man (Case 1) admitted to National Hospital Organization (NHO) Miyagi Hospital, a 75-year-old man (Case 2) admitted to NHO Higashi-Hiroshima Medical Center were found in initial chest X-ray and thoracic computed tomography (CT) to have a tuberculosis-like cavity in the left apex (Case 1) and the right apex (Case 2). In Case 1, the patient was treated with isoniazid and rifampicin for one month and lesions showed a partial response. In Case 2, the patient responded favorably with rifampicin, ethambutol, streptomycin, and clarithromycin therapy. Mycobacteria were repeatedly detected in smear and culture from sputum specimens in both patients. Isolates were nonphotochromogenic and rough. Isolated colonies developed after two to three weeks on 2% Ogawa egg medium. Organisms grew on 2% Ogawa egg medium at 30, 37, 42, and 45 degrees C, but not 25 degrees C. Both organisms were susceptible to 500 microg of p-nitrobenzoate per mL and 5mg of sodium chloride per mL. Isolates were negative for niacin accumulation, nitrate reduction, semiquantitative catalase, 68 degrees C catalase, 3-day aryl-sulfatase, iron uptake, and MPB64 antigen production, but positive for Tween 80 hydrolysis (5 and 10 days), acid phosphatase, and pyrazinamidase. Isolates had typical uv-HPLC chromatograms similar to M. shimoidei, demonstrating triple-peak clusters with peaks in the early cluster. 16S rRNA gene sequencing showed isolates to be consistent with Mycobacterium shimoidei. Based on composite characterization, isolates were identified as M. shimoidei. This is, to our knowledge, the third case of M. shimoidei infection reported in Japan.

[Mycobacterium shinshuense and Mycobacterium leprae infections: usefulness of genetical examinations].

Although Mycobacterium shinshuense and M. leprae infections are relatively rare in the fields of dermatology, an early diagnosis is one of the important prognostic factors of these infections. Applications of the genetical examinations such as PCR and 16S rDNA sequencing are helpful in early diagnosis with culture nagative cases. Short target PCR tests are available to detect DNA of M. shinshuense or M. leprae from clinical specimens including formalin fixed-paraffin embedded samples. A partial 16s rDNA sequencing is functional with enough intact bacterial DNA. A similarity search based on the partial 16S rDNA sequences using RIDOM database is an easy and powerful tool to estimate the species of mycobacteria, however, it is not enough for the identification in some cases. For instance, a clinical isolate of M. shinshuense is clearly discriminated from M. leprae (92.75% sequence identity), however, difficult to be identified from M. marinum and M. ulcerans (99.77% sequence identity). The phylogenetic tree based on 16S rDNA sequences is illustrating that both M. leprae (closely related to M. haemophilum) and M. shinshuense (closely related to M. marinum and M. ulcerans, and also M. tuberculosis) are relatively related species and distantly related to rapidly growing species among 30 species of pathogenic mycobacteria which have been isolated in Japan.

[Current practice of genetic diagnosis for Mycobacterium leprae].

Laboratory tests necessary for the diagnosis of leprosy have not been well introduced in general hospitals and clinical laboratories. Therefore, several tests have been performed in Leprosy Research Center, National Institute of Infectious Diseases since July, 1997, as a part of administrative examinations (tests done by request of Ministry of Health, Labour and Welfare). These examinations include histopathology, serum antibody titers (anti-PGL-I antibody), PCR test and bioactivity of anti-bacterial agents.

Complete Genome Sequence of Mycobacterium ulcerans subsp. shinshuense.

Mycobacterium ulcerans subsp. shinshuense produces mycolactone and causes Buruli ulcer. Here, we report the complete sequence of its genome, which comprises a 5.9-Mb chromosome and a 166-kb plasmid (pShT-P). The sequence will represent the essential data for future phylogenetic and comparative genome studies of mycolactone-producing mycobacteria.

Revisiting Buruli ulcer.

Buruli ulcer (BU), or Mycobacterium ulcerans infection, is a new emerging infectious disease which has been reported in over 33 countries worldwide. It has been noted not only in tropical areas, such as West Africa where it is most endemic, but also in moderate non-tropical climate areas, including Australia and Japan. Clinical presentation starts with a papule, nodule, plaque or edematous form which eventually leads to extensive skin ulceration. It can affect all age groups, but especially children aged between 5 and 15 years in West Africa. Multiple-antibiotic treatment has proven effective, and with surgical intervention at times of severity, it is curable. However, if diagnosis and treatment is delayed, those affected may be left with life-long disabilities. The disease is not yet fully understood, including its route of transmission and pathogenesis. However, due to recent research, several important features of the disease are now being elucidated. Notably, there may be undiagnosed cases in other parts of the world where BU has not yet been reported. Japan exemplifies the finding that awareness among dermatologists plays a key role in BU case detection. So, what about in other countries where a case of BU has never been diagnosed and there is no awareness of the disease among the population or, more importantly, among health professionals? This article will revisit BU, reviewing clinical features as well as the most recent epidemiological and scientific findings of the disease, to raise awareness of BU among dermatologists worldwide.

Mycobacterium haemophilum infection with prominent facial manifestation mimicking leprosy.

Mycobacterium haemophilum is a slow-growing non-tuberculous mycobacterium that is rarely known to cause human skin infection, particularly in immunocompromised patients. We recently experienced a 69-year-old Japanese woman with this infection who had been under immunosuppressive treatment for recalcitrant rheumatoid arthritis. The patient showed disseminated erythematous plaques and subcutaneous nodules on the face and extremities, and interestingly, the face manifested with a striking "facies leontina" appearance. Biopsy revealed abscess and granulomatous dermatitis with the involvement of peripheral nerve bundles and the presence of innumerable acid-fast bacilli, thus necessitating differentiation from lepromatous leprosy. M. haemophilum was identified by molecular characterization as well as by successful culture with iron supplements. Although drug susceptibility testing indicated responsiveness to multiple antibiotics administrated simultaneously for the treatment, it took over 6 months to achieve significant improvement, and we also employed concurrent oral potassium iodide administration and repeated surgical excision. This case highlights the importance of continuous combination therapy for successful outcome in this rare infection. Furthermore, application of potassium iodide for mycobacterial infection warrants further evaluation by accumulating more cases.

Exploration of a standard treatment for Buruli ulcer through a comprehensive analysis of all cases diagnosed in Japan.

Buruli ulcer (BU) is a refractory skin ulcer caused by Mycobacterium ulcerans or M. ulcerans ssp. shinshuense, a subspecies thought to have originated in Japan or elsewhere in Asia. Although BU occurs most frequently in tropical and subtropical areas such as Africa and Australia, the occurrence in Japan has gradually increased in recent years. The World Health Organization recommends multidrug therapy consisting of a combination of oral rifampicin (RFP) and i.m. streptomycin (SM) for the treatment of BU. However, surgical interventions are often required when chemotherapy alone is ineffective. As a first step in developing a standardized regimen for BU treatment in Japan, we analyzed detailed records of treatments and prognoses in 40 of the 44 BU cases that have been diagnosed in Japan. We found that a combination of RFP (450 mg/day), levofloxacin (LVFX; 500 mg/day) and clarithromycin (CAM; at a dose of 800 mg/day instead of 400 mg/day) was superior to other chemotherapies performed in Japan. This simple treatment with oral medication increases the probability of patient adherence, and may often eliminate the need for surgery.