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DERMATOLOGICA SINICA 31 (2013) 149e153

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Dermatologica Sinica

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CASE REPORT

Actinomycetoma caused by Nocardia otitidiscaviarum: Reportof a case in Taiwan with long-term follow-up

Min-Hui Chi 1, Rosaline Chung-Yee Hui 1,*, Chin-Fang Lu 2, Li-Cheng Yang 1,Shu-Ying Li 3

1Department of Dermatology, Chang Gung Memorial Hospital & Chang Gung University, College of Medicine, Taipei, Taiwan2Department of Dermatology, Chung Shan Hospital, Taipei, Taiwan3Mycotic Diseases Laboratory, Research and Diagnostic Center, Centers for Disease Control, Taipei, Taiwan

a r t i c l e i n f o

Article history:Received: Jun 18, 2012Revised: Nov 22, 2012Accepted: Nov 29, 2012

Keywords:16S ribosomal RNA (16S rRNA)actinomycetomaNocardia otitidiscaviarumnocardiosis

* Corresponding author. Department of DermatolHospital, 199, Tun-Hwa North Road, Taipei 10527135211x3397; fax: þ886 2 27191623.

E-mail address: rosaline.hui@gmail.com (R.C.-Y. Hu

1027-8117/$ e see front matter Copyright � 2012, Tahttp://dx.doi.org/10.1016/j.dsi.2012.11.005

a b s t r a c t

Actinomycetoma is a chronic granulomatous infection, with Nocardia species being one of the infectingpathogens. Infections caused by N. otitidiscaviarum are relatively rare compared with those caused byother Nocardia species. Conventional methods for the diagnosis of nocardiosis based on phenotypiccharacterization of the strains (e.g., morphology, histopathology) are relatively time consuming andnonspecific. Molecular techniques have become the better choice for prompt and accurate identificationof Nocardia isolates. We report a case of actinomycetoma due to N. otitidiscaviarum characterized by 16Sribosomal RNA (16S rRNA) gene sequence analysis and the long-term follow-up.

Copyright � 2012, Taiwanese Dermatological Association.Published by Elsevier Taiwan LLC. All rights reserved.

Introduction

Actinomycetoma is a chronic granulomatous infection of the skin,subcutaneous tissue, fascia, and bone caused by traumatic inoc-ulation of actinomycetes, with the feet being the predominantsite.1 Nocardia species are one of the infecting pathogens thatcause actinomycetoma. Primary cutaneous nocardiosis has threemain clinical manifestations: superficial skin infections, myce-toma, and sporotrichoid lymphocutaneous disease. More than 50species in the genus Nocardia have currently been characterizedby phenotypic and molecular methods2; however, nocardiosiscaused by N. otitidiscaviarum is relatively rare compared withinfections caused by other species. Herein, we report the case ofa 71-year-old manwith lymphocutaneous actinomycetoma on theleft calf that started as mycetoma at the ankle and 30 years offollow-up. The developments of different diagnostic methods arealso discussed.

Case report

A 71-year-old male coal miner had multiple erythematous indu-rated nodules, abscesses, and granulation tissue on the left lower

ogy, Chang Gung Memorial08, Taiwan. Tel.: þ886 2

i).

iwanese Dermatological Associatio

leg for nearly 30 years. He was initially seen in October 1982 witha 2-year history of multiple erythematous nodules and pusformation over the left ankle with no obvious history of trauma(Figure 1A). Mycetoma was diagnosed based on the clinical mani-festations and histopathologic finding of a typical sulfur granule ona specimen taken from the left ankle (Figure 1B). Because nomicroorganisms were isolated from numerous bacterial and fungalcultures, various antibiotic and antifungal agents, including peni-cillin, oxacillin, doxycycline, sulfamethoxazoleetrimethoprim(cotrimoxazole), gentamicin, griseofulvin, and ketoconazole, wereprescribed sequentially. However, the lesions persisted.

The patient was lost to follow-up for 14 years; however, hereturned in 2007 because of worsening skin lesions. The lesionscontinued to increase in number and spread upward from the leftankle to the left popliteal fossa in a sporotrichoid pattern over thenext 3 years, resulting in multiple erythematous protuberantnodules of granulation tissue and abscesses with purulentdischarge. The previous ankle lesion healed, leaving a whitish scar(Figure 2A). Moreover, he had fixed drug eruptions after repeateduse of cotrimoxazole. Subacute cutaneous lupus erythematosuspresented as erythematous annular macules over sun-exposedareas for 3 months starting in September 2008. This wasconfirmed by pathology and positive antinuclear antibody (1:320,speckled) and anti-SSA (>240 U/mL)/SSB (232 U/mL) antibodies,although antidouble-stranded DNA and anti-Smith antibodies werenegative. The lupus was controlled using low dose oral predniso-lone and hydroxychloroquine.

n. Published by Elsevier Taiwan LLC. All rights reserved.

Figure 1 (A) Initial presentation of multiple erythematous nodules with pus formation over the left ankle in October 1982; (B) histopathology of a section of the nodule showed onesulfur granule (arrow) surrounded by inflammatory cells present in the deep dermis (hematoxylin-eosin stain, �100).

M.-H. Chi et al. / Dermatologica Sinica 31 (2013) 149e153150

Repeat laboratory studies revealed normocytic anemia(hemoglobin, 10.5 g/dL; mean corpuscular volume, 85.8 fL) anda slightly elevated erythrocyte sedimentation rate (54 mm/h;normal, 0e20 mm/h) with normal flow cytometry analysis ofT lymphocytes, implying a chronic inflammatory process.Magnetic resonance imaging (MRI) of the left lower extremity

Figure 2 (A) Skin lesions on the left lower leg in March 2010 that indicated multiple nodules(B) histopathology revealed ulcers with necrotic dermis, diffuse fibrosis with inflammationfound in one microabscess (hematoxylin-eosin stain, �400); (C) after 9 days of culture, chalblood/MacConkey agar biplate, suggestive of Nocardia species.

disclosed extensive subcutaneous swelling in the left lower legand pyomyositis without bony destruction. Histopathology ofa specimen from one protuberant nodule showed ulcers coveredwith necrotic dermis and diffuse dermal and subcutaneousfibrosis with acute and chronic inflammation and scatteredmicroabscesses. One sulfur granule was found in a microabscess

with proximal purulent discharge and previously healed skin lesions on the left ankle;, and scattered microabscesses (hematoxylin-eosin stain, �20). One sulfur granule wasky-white colonies with rough and verrucous surface grew on the blood agar side of the

M.-H. Chi et al. / Dermatologica Sinica 31 (2013) 149e153 151

under the ulcer (Figure 2B). Filamentous structures in the sulfurgranule were partially acid-fast on Fite stain and negative onKinyoun acid-fast stain. Periodic acid-Schiff staining was alsonegative.

Nine-day cultures of native lesion tissue grew chalky-whitecolonies with a rough and verrucous-like surface, indicative ofNocardia species on the blood agar of a blood/MacConkey agarbiplate (Figure 2C). Identification of the species done at theMycotic Diseases Laboratory of the Centers for Disease Control inTaiwan revealed that the colonies were able to hydrolyze bothhypoxanthine and xanthine. To further identify the species, the16S ribosomal RNA gene of the clinical isolate 04-901-553856 wassequenced as well as the Institute of Food Microbiology (IFM)reference strains (now the Research Center of Pathogenic Fungiand Microbial Toxicoses, Chiba University). The sequence ofNocardia brasiliensis DSM43758 was obtained from the NationalCenter for Biotechnology Information (NCBI) database. Sequenceswere aligned using Clustal X (version 2.0.12)3 and a phylogenetictree was generated using the neighbor-joining method by MEGA(version 5.05).4 Bootstrap analysis with 1000 replications wasperformed. The 16S ribosomal RNA sequence of the isolate 04-901-553856 was found to cluster with that of a Nocardia otiti-discaviarum reference strain (IFM0239), with a bootstrap value of82% (Figure 3).

An antimicrobial susceptibility test revealed that the isolate wassusceptible to amikacin, gentamicin, tetracycline, meropenem,imipenem, and cotrimoxazole. After treatment failed with oraltetracycline and doxycycline, intravenous amikacin (400 mg twicedaily) and imipenemecilastatin (500 mg four times per day) werestarted together with topical tetracycline in May 2010. Imipeneme

cilastatin was associated with nausea and was subsequentlyreplaced with meropenem (1000 mg three times daily). In terms oflong-term treatment after discharge, desensitization to cotrimox-azole was attempted according to a previous protocol.5 The skinlesions diminished in size and had less discharge after more than 3weeks of treatment. Unfortunately, the patient died of acuterespiratory failure secondary to pneumonia a few months afterdischarge.

Figure 3 Phylogenetic tree based on 16S rRNA gene sequencing showed that the Nocardia isof Nocardia otitidiscaviarum (strain number Institute of Food Microbiology (IFM) 0239) (nowscale bar indicates 0.1 substitutions per nucleotide position.

Discussion

N. otitidiscaviarum was first recognized in 1924 from a Sumatrancavy or guinea pig with ear disease. In the 1930s, Erikson renamedthe organism Actinomyces caviae and in the 1960s, Gordon andMihm proposed the name Nocardia caviae. However, Nocardia oti-tidiscaviarumwas used in Bergey’s Manual of Systematic Bacteriologyand the name has since been routinely used in clinical laboratoriesto identify members of this complex.2 It can be isolated from soiland is known to cause primary cutaneous, lymphocutaneous,pulmonary, and even disseminated central nervous system infec-tions.2,6,7 Cases of N. otitidiscaviarum infections are relatively rareglobally and the disease spectrum in the English literature includesmycetoma (Table 1), primary lymphocutaneous disease,8 primarysubcutaneous abscess,9 keratitis,10 septic arthritis,11 pneumonia,12

meningitis,13 brain abscess,6 and disseminated infection.14 InTaiwan, nocardiosis involving the lungs and skin as well asdisseminated infections have been reported, with 2.7% (3/113) dueto N. otitidiscaviarum although only one patient has had a cuta-neous infection.7 In the United States2 and Germany,15 the preva-lence of Nocardia infections is 2.9% and 6.1%, respectively. Our casehad unusual primary cutaneous nocardiosis with initial actino-mycetoma followed by lymphocutaneous nocardiosis with sporo-trichoid spreading. This may be explained by the incompletetreatment of the primary lesion leading to the residual pathogens indeep tissue spreading along the lymphatic drainage.

Nocardia species grow readily on most nonselective media butmay take 2e14 days to appear. Therefore, the microbiologicallaboratory should be instructed to maintain the plates for at least 2weeks in a humidified environment to avoid dehydration ifNocardia infection is suspected.2 The colonial morphology varies,although most have a chalky-white appearance with a “cottoncandy”-like colony surface.2,15 The biochemical ability to hydrolyzeboth hypoxanthine and xanthine is suggestive ofN. otitidiscaviarum.2 Unfortunately, the traditional identification ofNocardia infections based on phenotypic features, such asmorphology and histopathology, is relatively time-consuming andnonspecific compared with genetic methods.

olates (number 04-901-553856) from the patient were identical to the reference strainthe Research Center of Pathogenic Fungi and Microbial Toxicoses, Chiba University). The

Table 1 Review of the clinical features, therapy, and outcome of actinomycetoma caused by Nocardia otitidiscaviarum.

Author Age/sex Location Diseaseduration (yr)

History oftrauma

Underlyingdisease

Treatment/duration Outcome

Sandhu et al19 20/M Right knee 2 No NA NA NAAlteras et al20 41/M Left foot 2 NA NA Isoniazid þ TMP-SMZ/>1 yr ImprovementAlteras et al21 39/F Right foot �1 No NA Isoniazid/>1 yr Tetracycline/>2 mo ImprovementSaúl et al22 54/M Left leg NA Yes NA Dapsone þ TMP-SMZ, po/1 yr Fistulas healed and no

further “grains” were observed46/M Right supra-

clavicular region3 No NA Dapsone þ TMP-SMZ, po/NA Flattening of the lesions

and no “grains” found bydirect examination

Saarinen et al23 50/M Right hand,right armpit,chest wall

10 NA NA Amikacin (IV)/ 3 weeks � 3 Rifampicin,po þ TMP-SMZ, po/52 mos

Cured

Bonifaz et al1 52/M Lumbar back 6 NA NA Amoxicillin-clavulanate, po/6 mos CuredMagalhães et al24 37/M Right hand 4 Yes NA TMP-SMZ, po/10 mos Improvement without relapseChen et al25 51/M Right lower leg 25 Yes None TMP-SMZ, po/1 yr Healed without relapsePresent case 71/M Left lower leg w30 No SCLE Amikacin þ imipenem-cilastatin; then

Amikacin þ meropenem (IV)/total3 wks TMP-SMZ, po/10 d

Skin lesions diminishedin size with less discharge

d ¼ day; IV ¼ intravenous; M ¼ male; mo ¼ month; NA ¼ not available; PO ¼ per os (by way of the mouth); SCLE ¼ sub-acute cutaneous lupus erythematosus; yr ¼ year.

M.-H. Chi et al. / Dermatologica Sinica 31 (2013) 149e153152

Many serologic tests including immunoassays and enzyme-linked immunosorbent assays that detect circulating antibodieshave been developed for the early diagnosis of nocardial disease.However, using a single serologic method for the detection ofnocardial infections may be limited due to the numerous speciescausing infections and the potential lack of sensitivity in immu-nocompromised patients.2 Molecular techniques such as multi-locus sequence analysis of gyrase B of the b subunit of DNAtopoisomerase (gyrB), 16S rRNA (16S), subunit A of SecA preproteintranslocase (secA1), the 65-kDa heat shock protein (hsp65), andRNA polymerase (rpoB) have been developed to identify Nocardiaspecies.16 Among these, 16S rRNA gene sequence analysis, whichwas developed in the 1980s for bacterial identification, is anexcellent and extensively used technique to identify Nocardiaisolates at the species level.2,6 In the present case report, the 16SrRNA phylogenetic tree revealed thatNocardia isolates (number 04-901-553856) from the patient were clustered with the referencestrain of N. otitidiscaviarum (strain number IFM0239).

Before the availability of antibiotic susceptibility of Nocardiaspecies, empirical antibiotics with cotrimoxazole or minocyclinewere the most frequently used treatment for localized or mildnocardiosis, while combination therapy with a carbapenem ora third-generation cephalosporin with or without amikacin wasrecommended in severe cases or for those with central nervoussystem involvement.6 For those who fail to respond to standardantibiotic therapy, successful treatment with linezolid17 andamoxicillin-clavulanate1 has been reported. The mean treatmentduration with amoxicillineclavulanate has been reported to be 9.6months, with a minimum of 4 months and a maximum of 22months.1 However, geographic variations and species distributionhave a significant impact on differences in terms of antibioticsusceptibility.

In Taiwan, Lai et al18 found that eight N. otitidiscaviarum isolateswere susceptible to linezolid, sulfamethoxazole, and amikacin, butnot to amoxicillineclavulanate, ceftriaxone, imipenem, or cipro-floxacin.18 Co-trimoxazole (sulfamethoxazole-trimethoprim) is themost common choice for treating actinomycetoma caused byNocardia otitidiscaviarum. The total treatment duration regardlessof antibiotic regimen is at least 10months and as long as 55months(Table 1). In the present case report, the drug of choice was basedon the antimicrobial susceptibility test and the patient’s pasthistory of fixed drug eruptions from co-trimoxazole.

In our case, the prolonged clinical course and unsatisfactorytherapeutic efficacy may be due to several factors, including the

accuracy of the initial diagnosis, an immunocompromised statusdue to old age, subacute cutaneous lupus erythematosus, intoler-able adverse events of antimicrobials leading to poor drugcompliance, and frequent switching. Prompt diagnosis with geno-typic methods and accurate antimicrobial susceptibility tests areuseful and important in clinical practice.

In conclusion, hereinwe report a case that highlights the clinicalcourse of actinomycetoma under various treatments for almost 30years,with a partial response. Identification of themicrobial isolateswithmolecular techniques, the selectionof antimicrobials accordingto antimicrobial susceptibility tests, and compliance for completetreatment course are crucial for a good therapeutic response. Todate, this patient is the first reported case of actinomycetomainduced by N. otitidiscaviarumwith long-term follow-up in Taiwan.

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