Nocardia nova causing empyema necessitatis after lung re-transplantation : a case report

We report herein a case of thoracic infection due to Nocardia nova following lung re-transplantation performed for emphysema related to alpha-1-antitrypsin defi ciency. The infection extended from the lung into the pleural space, thoracic wall, and mediastinum, presenting as pericarditis and empyema necessitatis. Nocardia nova was identifi ed by 16S ribosomal deoxyribonucleic acid (rDNA) sequencing and phylogenetic analysis. According to a literature search of PubMed, LILACS and MEDLINE databases, we describe herein the fi rst case of empyema necessitatis caused by N. nova species in a transplanted patient.


INTRODUCTION
Empyema necessitatis refers to infl ammatory tissue that extends directly from the pleural cavity into the thoracic wall, forming a mass (a cold abscess requiring drainage) in the extra-pleural soft tissues.It is a very rare complication of different infectious etiologies; tuberculosis and actinomycosis are the most common causative organisms (1) .Recently, cases of nocardiosis with chest wall extension (2) and N. asteroides empyema necessitatis have been observed (3) .Herein, we report the fi rst case of empyema necessitatis caused by N. nova.

CASE REPORT
A 50-year-old woman underwent lung re-transplantation in July 2011, after developing chronic lung allograft dysfunction.The patient had a long history of emphysema secondary to alpha-1-antitrypsin defi ciency.She was subjected to doublelung volume-reduction surgery in 2003 and single-lung transplantation in 2006.The lung re-transplantation in 2011 was bilateral.Her immunosuppressive regimen consisted of prednisone, mycophenolate, and tacrolimus.Five months after re-transplantation, the patient presented to the transplant clinic with dyspnea.Bronchoscopy identifi ed stenosis of the left bronchial anastomosis.Endoscopic attempts to manage the stenosis failed and a lower left lobectomy was performed.
One month later, she presented to the clinic with dyspnea and a painful draining mass located on her left lateral chest wall.On examination, she was afebrile and an external fi stula was identifi ed in the left inframammary region.The patient received intravenous ceftriaxone as empiric antimicrobial therapy for subcutaneous infection, with consequent clinical improvement.However, when the therapy was stopped, the problem worsened and the chest radiograph revealed a pleural effusion.
In the investigation, the pleural fl uid consisted of a purulent exudate (Figure 1A).Gram-positive filamentous bacilli (Figure 1B) were detected in the exudate.These bacilli were decolorized by Ziehl-Neelsen staining.A contrast-enhanced axial computed tomography (CT) scan with a mediastinal window demonstrated atelectasis in the left lower lobe, associated with bilateral pleural and pericardial effusions.In addition, pleural and pericardial thickening was revealed, suggesting a complicated effusion.The clinical and cytological fi ndings were suggestive of actinomycosis.The patient was thus treated with penicillin, but failed to show any clinical improvement.Kinyoun staining was performed and revealed branching, weakly acid-fast, fi laments (Figure 1C).Aerobic culture of the pleural fl uid suggested Nocardia.

A B C
In the diagnosis, deoxyribonucleic acid (DNA) from isolated colonies (SBP72a/12: sampled from pleural fl uid; and SBP72b/12 sampled from the pleural cavity secretion) was isolated and molecular diagnosis was performed by analyzing the partial sequence (approximately 1,300 base pairs) of 16S ribosomal ribonucleic acid (rRNA) gene amplifi ed with universal primers (4) .Polymerase chain reaction (PCR) products were sequenced by ACTGene Análises Moleculares Ltda [Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil] using the automatic sequencer ABI-PRISM ® 3100 Genetic Analyzer (Applied Biosystems).Consensus sequences were assembled using Staden Package 4. Figure 2 shows the strong homology of the samples with others N. nova strains and the relationship with others species available in GenBank dataset.The partial 16S rRNA gene sequences of SBP72a/12 and SBP72b/12 were deposited in the GenBank database.
In the follow-up, N. nova was identifi ed, the patient received treatment with intravenous trimethoprim/sulfamethoxazole (TMP/SMX) 320/1,600mg every six hours for one month, with success.Central nervous system disease was ruled out.Finally, the patient was discharged on oral TMP/SMX (160/800 BID).At discharge, she was asymptomatic, and the lesions had improved both clinically and radiologically.At the time of her last followup visit, fi ve months after discontinuation of antimicrobial therapy, she remained well.

DISCUSSION
Traditional identifi cation of Nocardia to species level by phenotyping is cumbersome and the results are sometimes diffi cult to interpret.The recent introduction of molecular methods has had an enormous impact on the taxonomy of Nocardia and has resulted in the recognition of numerous new species.Worldwide, respiratory and disseminated infections are most often due to members of the previously broadly defi ned N. asteroides complex, which contains three strains: Nocardia asteroides sensu stricto, Nocardia farcinica, and N. nova complex.The latter includes N. nova species (5) .
In a search of PubMed/MEDLINE and LILACS databases, we found no previous report of empyema necessitatis caused by N. nova.The key words used for the search were Nocardia nova, nocardiosis, lung transplant, and empyema necessitatis, and it was performed until September 2015.We describe here the fi rst case of empyema necessitatis caused by N. nova species in a transplanted patient.
A contemporary case-control study found that the incidence of N. nova isolated from solid organ transplant recipients was higher than N. farcinica (49% vs. 28%, respectively) (6) .In a review of 17 patients with an underlying malignancy and Nocardia bacteremia, N. nova complex was demonstrated to be responsible for 35% of the cases.Bacterial adhesion to intravenous catheters may play a role in the incidence of intravascular N. nova infection (7) (8) .
Infection of the lung parenchyma and pleural space by N. nova has previously been observed (9) (10) (11) .However, direct extension from the lung into the pleural cavity, progressing to penetration of the mediastinum, with consequent pericarditis and empyema necessitates-specifi cally in a lung-transplant recipient -has not previously been reported.Physicians should be aware of this possibility in order to obtain and submit appropriate thoracic specimens to the laboratory to optimize the recovery of this microorganism.
In conclusion, we advocate maintaining a high index of suspicion for nocardiosis in a patient with underlying immunosuppression, and instituting an active approach to obtaining specimens for diagnostic studies.Opportunistic pathogens such as observed in this case, may be weakly acidfast and will be decolorized by the Ziehl-Neelsen procedure.Previously diagnosed as "Nocardia asteroides complex", routine microbiology laboratories should attempt to identify the organism to species level.Finally, clinicians should be aware that N. nova could be associated with empyema necessitatis.

FIGURE 1 .
FIGURE 1. Nocardia nova pictures.(A) Purulent pleural fl uid obtained via aspiration of the pleural cavity.(B) Staining performed directly from the pleural effusion showing Gram-positive rod-shaped bacteria.(C) Partially acid-fast branching fi laments showed by Kinyoun Staining.Note: Figures B and C were observed by microscopy at 1,000-times magnifi cation.

FIGURE 2 .
FIGURE 2. Nucleotide sequence analysis.Phylogenetic relationship derived from 16S rDNA sequences showing the homology between Nocardia nova (samples SBP 72a/12 and SBP 72b/12) and 16S rDNA sequences of other Nocardia species.The phylogenetic tree was built in the MEGA 5.2 software program, using the Neighbor-Joining algorithm and the Tamura 3-parameter substitution model.Rhodococcus opacus B4 (AP011115.1)was used as the out group reference.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to each branch.SBP72a/12 was isolated from pleural fl uid and SBP72b/12 was isolated from the pleural cavity.N: Nocardia; rDNA: ribosomal deoxyribonucleic acid; SBP: Setor de Bacteriologia e Pesquisa; MEGA: Molecular Evolutionary Genetics Analysis.