Nocardia elegans infection involving purulent arthritis in humans

J Infect Chemother (2012) 18:386–389 DOI 10.1007/s10156-011-0311-5

CASE REPORT

Nocardia elegans infection involving purulent arthritis in humans Takayuki Masaki • Kiyofumi Ohkusu Takayuki Ezaki • Hiroshi Miyamoto

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Received: 6 June 2011 / Accepted: 13 September 2011 / Published online: 4 October 2011 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2011

Abstract Nocardia elegans infection in humans is rare and is predominantly associated with pulmonary infections. We describe the first case of N. elegans infection associated with purulent arthritis in humans. The patient was a 66-year-old woman without underlying disease. She had swelling in her left ankle that was increasing in size, but it did not cause the patient substantial pain. Punctual discharge was collected for Gram staining and Kinyoun’s acid-fast staining. The results of microscopic findings were suggestive of the genus Nocardia. The 16S rRNA sequence of the isolate was completely identical (100%) with that of N. elegans, indicating that the isolate was N. elegans. All the previously reported 4 cases of N. elegans infection in humans were associated with respiratory infections; we present the first case of the infection involving purulent arthritis. Keywords Nocardia elegans  Nocardial infection  Purulent arthritis

aerobic, Gram-positive, and form extensively branched substrate hyphae that fragment into rod-shaped or coccoid nonmotile forms. Most Nocardia species are associated with human and animal infections. Although nocardiosis has been considered rare, the incidence of infection with Nocardia species has recently been reported to be increasing [1, 2]. Most commonly, nocardiosis presents as a pulmonary disease. The members of the N. asteroides complex primarily cause pulmonary disease and, except for N. nova, all other organisms are prone to extrapulmonary dissemination. Dissemination is especially prevalent with N. farcinica [3–6]. N. brasiliensis and N. transvalensis typically produce localized infection induced by an abrasion, although the latter species is uncommon in Japan. The first case of N. elegans pulmonary infection was reported by Yassin et al. [7], and 3 cases have since been reported. N. elegans infection is rare. Herein, we report for the first time that in addition to the development of pulmonary lesions and abscess, N. elegans can cause purulent arthritis.

Introduction The genus Nocardia belongs to the family Nocardiaceae within the order Actinomycetales. The organisms are

T. Masaki (&) The Chemo-Sero-Therapeutic Research Institute, 1-6-1 Ohkubo, Kumamoto 860-8568, Japan e-mail: [email protected] T. Masaki  H. Miyamoto Faculty of Medicine, Saga University, Saga, Japan K. Ohkusu  T. Ezaki Gifu University Graduate School of Medicine, Gifu, Japan

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Case report A 66-year-old woman without underlying disease was admitted to Nanbu Central Hospital in July 2006 because of persistent fever for 1 week. Swelling in her left ankle was increasing in size but did not cause her substantial pain, and there was no visible wound on her swollen ankle. The following results were obtained from laboratory tests: hemoglobin concentration, 12.3 g/dl; leukocyte count, 3,600/ll; platelet count, 371,000/ll; C-reactive protein concentration, 0.1 mg/dl; total protein concentration, 7.0 g/dl; albumin concentration, 3.7 g/dl; blood

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glucose concentration, 126 mg/dl. Blood glucose concentration was measured 2 h after a meal. Chest radiography showed no obvious abnormalities. Therefore, bursectomy was performed 1 week after admission, which subsequently relieved her symptoms without antibiotics. The punctual discharge (Fig. 1) was collected for microbiological examination. The collected specimen was smeared and Gram staining and Kinyoun’s acid-fast staining were performed (Fig. 2). Gram staining revealed that the organisms were Gram-positive, coccid, and had thin branching filaments. In Kinyoun’s acid-fast staining, the organisms appeared as thin, acid-fast, branching filaments. Because all direct microscopic examinations were suggestive of Nocardia morphology, the specimen was inoculated on 5% sheep blood agar, chocolate agar, and semisolid brucella medium. The sample was inoculated in sheep blood agar, incubated at 35°C, and examined daily for up to 3 days. The colony formed by the clinical isolate was white and had a wrinkled surface. The clinical isolate was an aerobic, Gram-positive, rod-shaped bacterium approximately 1 9 5 lm in size. The vegetative hyphae changed their color from white to orange and developed irregular branches penetrating into the agar with white aerial hyphae. The organism was further identified by standard bacteriological methods [8].

Fig. 1 The punctual discharge was collected for microbiological examination. The discharge is milky and does not smell bad

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Fig. 2 Photomicrograph of Kinyoun’s acid-fast stain of the pus showing acid-fast, branching filaments

The isolate failed to hydrolyze casein, xanthin, and tyrosine; however, it did hydrolyze aesculin and urea (Christensen urea agar; Nissui seiyaku, Japan). Identification of N. elegans was proved by the equivalence in growth at 35°C and the production of acid from glucose. These features were consistent with the previously reported phenotypic features of N. elegans [7]. Antibacterial susceptibility of the isolated N. elegans was determined by the microdilution method [9]. The isolated organism was resistant to ciprofloxacin (MIC, [4 lg/ml) and susceptible to clarithromycin (MIC, \0.5 lg/ml), imipenem (MIC, \0.25 lg/ml), sulfamethoxazole-trimethoprim (MIC, \19/1 lg/ml); however, no further antibacterial susceptibility testing for species identification was performed because a clinical isolate of N. elegans was considered extremely rare. Molecular identification was attempted by polymerase chain reaction (PCR) amplification and sequencing analyses. A 1,433-base pair (bp) sequence of the 16S rRNA gene was amplified with universal primers 8UA (50 -AGAGTTTGATCMTGGCTCAG-30 ) and 1485B (50 -A CGGGCGGTGTGTRC-30 ), as described elsewhere [10]. The purified PCR products were sequenced with an ABI Prism Big Dye sequencing kit (v1.1). phylogenetic trees were constructed by the neighbor-joining method [11]. Tree topology was evaluated by bootstrap analysis using the Clustal W software [12]. Visual comparison and manual calculation were used to determine sequence similarity values. On the basis of subsequent sequence determination of a 1,433-bp fragment amplified from the 30 end of the 16S rRNA gene, we identified the isolate as N. elegans. The diagnosis was confirmed by the DNA Data Bank of Japan (DDBJ), and the phylogenetic tree indicated that the isolate was located within a subset of the genus Nocardia as shown in Fig. 3. The sequence of the 16S rRNA gene was 100% identical with that of N. elegans type strain

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0.01

49

100 No.138 (This case) N.elegansIMMIBN-402T(AJ854057) N.africanaDSM44491T(AF430054)

78 34

65

89

N.veteranaDSM44445T(AF430055)

48 46

N.kruczakiaeDSM44877T(GQ376176) N.cerradoensisY9T(AF060790) N.aobensisIFM0372T(AB126876) N.vacciniiDSM43285T(AF430045)

N. vermiculataIFM0391T(AB126873) N.novaJCM6044T(AF430028)

Fig. 3 Phylogenetic tree of the 16S rRNA gene sequences of the isolate prepared by the neighbor-joining method. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node. The scale bar represents a 1% difference in nucleotide sequences

IMMIBN-402T (AJ854057). The sequence similarity was high enough to confirm the identification of N. elegans.

Discussion Nocardial organisms are associated with environmental specimens. They are ubiquitous saprophytes, including soil saprophytes [13, 14], and occasionally cause infection in humans [1, 15]. Members of the N. asteroides complex cause most infections in humans, especially immunocompromised patients. Further, the general manifestations of nocardial infections are mycetoma and pulmonary changes with generalized metastasis [13]. Extrapulmonary nocardiosis involving skin and brain is common, but nocardial infection involving purulent arthritis, similar to that observed in this case, is very rare. We report the first case of purulent arthritis caused by N. elegans.

In our case, nocardial infection was suspected because of the presence of thin branched filaments observed on Gram staining and Kinyoun’s acid-fast staining of the joint fluid from the patient, which could contribute to giving immediate information about the organism to clinicians. The organism could not be identified by conventional methods based on identification of the colony that appeared on the isolation plate at 2 days post inoculation. Therefore, we used genetic analysis and identified the isolate as N. elegans. The drug-susceptibility pattern of the isolate was identical with that of common Nocardia species. The traditional method for identification of Nocardia species relies on the phenotypic characteristics of biochemical testing, pigment production, growth characteristics, and colonial morphology [8, 16]. The extent of biochemical testing depends on the ease of characterization and biochemical activity of the Nocardia under investigation. Phenotypic methods are still used in some laboratories to identify Nocardia despite their acknowledged difficulty. However, the 16S rRNA gene is regarded as the definitive standard for identification of all bacteria and is widely recognized as a rapid and accurate method of identifying known and novel Nocardia [17]. The first case of N. elegans pulmonary infection was reported by Yassin and Brenner [7] and the second was described by Watanabe et al. [18]. Nocardial infection caused by N. elegans is rare. A review of the literature revealed 5 reported cases, including 1 case of infection in a cat in which the organism was isolated from an abscess on the foot pad of the cat (Table 1) [7, 19–21]. Previously reported N. elegans infections in humans were associated with respiratory infections. Thus, N. elegans causes infection not only at the traumatic wound but can also become the main causal agent of pulmonary infection.

Table 1 Summary of previously reported cases of Nocardia elegans infection References

Underling disease

Disease

Specimens

Identification

Yassin and Brenner [7]

Unknown

Pulmonary infection

Sputum

16S rRNA

Watanabe et al. [18]

Unknown

Bronchitis

Sputum

16S rRNA

Park et al. [19]

Transplantation

Lung abscess

Sputum

16S rRNA

Barrio et al. [20]

Cystic fibrosis

Pulmonary infection

Respiratory secretions

16S rRNA

Harada et al. [21]a

None

Suppurative pyogranulomatous

Pus

16S rRNA

This case

None

Purulent arthritis

Punctual discharge

16S rRNA

a

Infectious disease of cat

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Herein, we are the first to report that in addition to the development of pulmonary lesions and abscess, N. elegans can cause purulent arthritis.

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