Molecular diagnosis of culture-negative cerebral nocardiosis due to Nocardia abscessus

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Diagnostic Microbiology and Infectious Disease 55 (2006) 237 – 240


Molecular diagnosis of culture-negative cerebral nocardiosis due to Nocardia abscessus He´le`ne Marchandina,4, Aure´lia Edenb, He´le`ne Jean-Pierrea, Jacques Reynesb, Estelle Jumas-Bilakc, Patrick Boirond, Fre´de´ric Laurentd a

Laboratoire de Bacte´riologie, Hoˆpital Arnaud de Villeneuve, 34295 Montpellier Cedex 5, France Service des Maladies Infectieuses et Tropicales, Hoˆpital Gui de Chauliac, 34295 Montpellier Cedex 5, France c Laboratoire de Bacte´riologie-Virologie, EA 3755, Faculte´ de Pharmacie, 34060 Montpellier Cedex 5, France d UMR CNRS 5557, Center for Microbial Ecology, Opportunistic Pathogens and Environment Research Group, Observatoire Franc¸ais des Nocardioses, Laboratoire de Mycologie Fondamentale et Applique´e aux Biotechnologies Industrielles, Faculte´ de Pharmacie, Universite´ Claude Bernard Lyon I, 69373 Lyon Cedex 8, France Received 9 October 2005; accepted 16 January 2006 b

Abstract We describe a case of culture-negative cerebral nocardiosis in a 34-year-old immunocompetent man who presented multiple cerebral abscesses. All bacteriologic cultures were negative. Nocardiosis was diagnosed by using a direct genus-specific 16S rDNA amplification method, and Nocardia abscessus was identified by hsp65 sequence analysis. The patient is alive and well on imipenem and doxycycline therapy, 14 months after onset. D 2006 Elsevier Inc. All rights reserved. Keywords: Nocardia abscessus; Cerebral infection; 16S rDNA; hsp65; PCR; Molecular diagnosis

Nocardiosis is a localized or disseminated infection occurring in both immunocompetent and immunocompromised subjects. It is caused by soil-borne aerobic actinomycetes. The infection is generally acquired by inhalation from the environment, and pulmonary disease is therefore the most common clinical form. However, hematogenous dissemination can lead to nervous system and skeletal softtissue involvement (Eisenblatter et al., 2002; Lerner, 1996). Nocardia asteroides, Nocardia farcinica, and Nocardia otitidiscaviarum are the main species causing cerebral nocardiosis. Two recently reported cases also identified Nocardia transvalensis in brain abscesses (Montoya et al.,

Part of this work was presented at the Third International Conference of Biology of Nocardia, Nocardiae’ 2005, 5–7 July 2005, Lyon, France. Boutruche S, Laurent F, Jean-Pierre H, Jumas-Bilak E, Godreuil S, Couble A, Boiron P, and Marchandin H. Nocardiosis over a five-year period in a French hospital: clinical, bacteriological and molecular findings of twenty cases. 4 Corresponding author. Tel.: +33-4-67-33-59-00; fax: +33-4-67-3358-93. E-mail address: [email protected] (H. Marchandin). 0732-8893/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2006.01.023

2003; Yorke and Rouah, 2003). We report a case of primary cerebral infection caused by Nocardia abscessus, a species incriminated in only a few previous cases of human infection (Yassin et al., 2000). This case illustrates the utility of direct molecular diagnosis of nocardiosis when culture is negative (Couble et al., 2005). In August 2004, a 34-year-old shepherd presented to a local hospital with headache and right-sided hemiparesis. Computed tomography (CT) of the brain showed cerebral lesions, which were first suspected to be of metastatic origin, and mannitol and corticosteroids were prescribed to reduce intracranial pressure. Further investigations failed to identify a primary neoplasm. Magnetic resonance imaging (MRI) of the brain, done 1 week after CT, revealed 8 nodular, bilateral, and ring-enhancing lesions. Craniotomic biopsy of the largest lesion (5 cm), located in the left cerebellum, yielded purulent fluid, ruling out a neoplastic origin. Direct examination showed altered polymorphonuclear cells, but bacteriologic cultures were negative. In October 2004, the patient was transferred to the Infectious Diseases Department of Montpellier University Hospital for further etiologic investigations. His medical history included


H. Marchandin et al. / Diagnostic Microbiology and Infectious Disease 55 (2006) 237 – 240

arterial hypertension, constitutional nystagmus, and a leg fracture requiring osteosynthesis in 2001. On admission, his treatment consisted of 0.6 mg kg!1 day!1 prednisone, which had been prescribed 2 months previously. Physical examination showed right-sided hemiplegia and a cerebellar syndrome. The temperature was normal. He had difficulties speaking and experienced dysarthria and diminishing visual acuity. The C-reactive protein level was below the detection limit, and the white blood cell count was 10 600/mm3 (742 lymphocytes/mm3). The CD4 T-cell count was 108/mm3 and the CD4/CD8 T-cell ratio was also low (0.5). Immunoglobulin (Ig) M and IgA were normal, but levels of total IgG (5 g/l) and IgG subclasses 1, 2, 3, and 4 were low. Cefotaxime was given for 1 week, followed by ceftriaxone, plus metronidazole and steroids for 15 days. After an initial clinical improvement, his condition again deteriorated. Serologic tests for Borrelia burgdorferi, Treponema pallidum, human immunodeficiency virus types 1 and 2, and cysticercosis were negative. He was Toxoplasma gondii IgG-positive but IgM-negative, and serum cryptococcal antigen assay was negative. Pyrimethamine, clindamycin, and prednisone were prescribed for suspected toxoplasmic encephalitis, but generalized seizures occurred after 3 weeks of this treatment. CT showed that the lesions were stable, but also revealed moderate deviation of midline structures. A second brain abscess was biopsied on November 10. A capsulated lesion located in the right frontal lobe was excised after craniotomy. Direct examination showed rare polymorphonuclear leukocytes. Gram staining was negative, but auramine staining was positive. Ziehl–Neelsen staining revealed partially acid-fast branched rods resembling actinomycetes. The specimen was seeded on trypticase soy agar, trypticase soy broth, Schaedler broth with vitamin K3 and 0.2% agar, and blood-chocolate agar (bioMe´rieux, Marcy l’Etoile, France), incubated at 378C with 5% CO2 for 10 days. Anaerobic cultures were

performed on Columbia sheep blood agar (bioMe´rieux) incubated for 21 days in an anaerobic jar. Samples were also tested with the BACTEC 460 TB system (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) and seeded on Lfwenstein–Jensen medium (bioMe´rieux) for mycobacteria. All these cultures remained negative. Blood cultures were also negative. Direct molecular diagnosis was attempted with a recently published method of Nocardia genusspecific 16S rDNA amplification (Couble et al., 2005). A 590-bp amplified fragment was obtained, confirming the diagnosis of nocardiosis while direct polymerase chain reaction (PCR) amplification of T. gondii DNA was negative. Species identification was performed by direct amplification and sequencing of hsp65 (Conville et al., 2000) and 16S rRNA genes (Rodrı´guez-Nava et al., 2005). Corresponding nucleotide sequences have been deposited in the GenBank database under accession numbers DQ351152 and DQ351151, respectively. For phylogenetic analysis, the hsp65 and 16S rRNA genes sequences were aligned with the corresponding sequences of representative Nocardia species contained in the GenBank and Bibi databases (Devulder et al., 2003) by using the multiple sequence alignment program CLUSTAL W (Thompson et al., 1994). Phylo_win software (Galtier et al., 1996) was used to infer evolutionary trees according to neighbor-joining methods (Saitou and Nei, 1987) using the Kimura 2-parameter model (Kimura, 1980). Tree robustness was assessed by bootstrap resampling (1000 replicates each). After sequences and phylogenetic analysis of 16S rRNA gene, it was not clear if the uncultured pathogen belonged to N. abscessus (100% of similarity) or to Nocardia asiatica (99.8% of similarity) (Fig. 1). Conversely, the pathogen was clearly identified as N. abscessus with 100% sequence similarity according to hsp65 gene sequence, whereas only 98.5% of sequence similarity were observed with N. asiatica. This was confirmed by hsp65-based phylogenetic analysis showing

Fig. 1. Neighbor-joining tree based on partial 16S rDNA sequences (569 nt) showing relationships between the sequence of the uncultured pathogen of the present study and those of the 11 closest Nocardia species. Nocardia crassostreae ATCC 700418T was the out-group organism. Bootstrap values are expressed as a percentage of 1000 replications and are shown at nodes with relevance to this study. The scale bar represents the number of substitutions per nucleotide position.

H. Marchandin et al. / Diagnostic Microbiology and Infectious Disease 55 (2006) 237 – 240

that N. asiatica is clearly more remote from N. abscessus and the uncultured pathogen sequenced in this study (Fig. 2). Treatment combining ceftriaxone (4 g/day), doxycycline (300 mg/day), and sulfamethoxazole (800 mg twice daily) plus trimethoprim (160 mg twice daily) was initiated after the biopsy. Fifteen days later, CT showed that the brain lesions had shrunk and that the deviation of midline structures had diminished. His neurologic status gradually improved, with motor recuperation, but the cerebellar syndrome and visual disorders were unchanged. Two months later, he developed a diffuse eruption with mouth ulceration, and Stevens–Johnson syndrome was diagnosed. Cotrimoxazole and ceftriaxone were permanently withdrawn and were replaced by intravenous imipenem (1 g 3 times a day) and doxycycline (300 mg/day) on January 19. He gradually recovered the ability to walk. Three months later, cerebral MRI showed the same number of lesions, but their size had diminished and the surrounding edema had disappeared. The same antibiotic treatment was maintained. In November 2005, the patient came again for a follow-up CT scan. He remained well and CT scan did not reveal active disease. Imipenem treatment was stopped and doxycycline (300 mg/day) was maintained and is to be continued for a further 3 months. N. abscessus was characterized in 2000 after polyphasic taxonomic analysis of four clinical isolates originating from various abscesses (Yassin et al., 2000). Few cases of nocardiosis due to N. abscessus have since been reported; they include a case of pericarditis (Wellinghausen et al., 2002) and 4 cases of N. abscessus isolation from wound fluid or pus (Horre et al., 2002, Wauters et al., 2005). Five additional strains were isolated from 5 Japanese patients between 1992 and 2000 (lung biopsy material, brain abscess, bronchial lavage fluid, and septum). Four of these patients had underlying diseases (Kageyama et al., 2004), but no clinical information was available on the 62-year-old man with brain abscess. Diego et al. (2005) recently


reported the first case of disseminated nocardiosis due to N. abscessus in an HIV-infected patient with central nervous system lesions. Thus, the present report represents the third case of N. abscessus brain abscess and the only documented case in an immunocompetent patient. N. abscessus infection may be more frequent than suggested by the literature. Indeed, N. abscessus was the main species isolated from patients with nocardiosis in our hospital between 1999 and 2004, accounting for 5 of 20 cases (Presented at the Third International Conference of Biology of Nocardia, 5–7 July 2005, Lyon, France) and represented 5.8% of 86 Nocardia isolates studied by Wauters et al. (2005). Cerebral nocardial abscesses are rare, accounting for approximately 1–2 % of all cerebral abscesses (Yorke and Rouah, 2003), but the morbidity and mortality rates are higher than those of other cerebral abscesses. Until recently, bacteriologic diagnosis of nocardiosis was based on culture followed by phenotypic and chemotaxonomic analyses. However, a recently published molecular method, which was retrospectively validated on culture-positive samples, permits direct diagnosis in clinical samples (Couble et al., 2005). The case described here represents the first clinical application of this method to a culturenegative specimen. As previously demonstrated for nocardial brain infection in mice (Loeffler et al., 2004), our findings suggest that PCR is more sensitive than culture for the detection of Nocardia in the human brain, particularly when viable bacterial counts have been reduced by antibiotic treatment. In our patient, 3 months of standard investigations, including bacteriologic analysis of pus sampled from a cerebellar lesion, failed to reach a diagnosis. This case also demonstrates the value of hsp65 sequence analysis for discriminating among closely related species, such as N. abscessus and N. asiatica, that cannot be distinguished by analyzing the partial 5V 16S rDNA sequence (500bp) (Cloud et al., 2004; Roth et al., 2003; Rodrı´guez-Nava et al., 2006; this study).

Fig. 2. Neighbor-joining tree based on partial hsp65 sequences (401 nt) showing relationships between the sequence of the present study and those of the 11 closest Nocardia species. N. crassostreae ATCC 700418T was the out-group organism. Bootstrap values are expressed as a percentage of 1000 replications and are shown at nodes with relevance to this study. The scale bar represents the number of substitutions per nucleotide position.


H. Marchandin et al. / Diagnostic Microbiology and Infectious Disease 55 (2006) 237 – 240

Although brain abscesses caused by Nocardia species occur predominantly in immunocompromised patients, no known risk factors are found in about 50% of cases (Lerner, 1996). Our patient was initially immunocompetent, apart from IgG and CD4 T-cell defects attributable to prior corticosteroid therapy. No portal of entry was identified; in particular, the patient’s pulmonary status was normal. Surgical drainage was not feasible because of the location and number of the lesions, and antimicrobial chemotherapy could not be based on susceptibility tests, as culture was systematically negative. Molecular identification to the species level did not help guide the choice of antimicrobial treatment either, as data on N. abscessus are scarce (Roth et al., 2003; Wellinghausen et al., 2002). Our patient responded well to imipenem and doxycycline. In conclusion, we describe the third reported case of N. abscessus brain abscess. Diagnosis was particularly difficult, as Nocardia was only visualized when stains specific for mycobacteria were performed, and all cultures were negative, probably because of ongoing antimicrobial chemotherapy. Direct molecular diagnosis was decisive in this case. Acknowledgments The authors are very grateful to Andre´e Couble for excellent technical assistance. References Cloud JL, Conville PS, Croft A, Harmsen D, Witebsky FG, Carroll KC (2004) Evaluation of partial 16S ribosomal DNA sequencing for identification of Nocardia species by using the MicroSeq 500 system with an expanded database. J Clin Microbiol 42:578 – 584. Conville PS, Fischer SH, Cartwright CP, Witebsky FG (2000) Identification of Nocardia species by restriction endonuclease analysis of an amplified portion of the 16S rRNA gene. J Clin Microbiol 38:158 – 164. Couble A, Rodriguez-Nava V, de Montclos MP, Boiron P, Laurent F (2005) Direct detection of Nocardia spp. in clinical samples by a rapid molecular method. J Clin Microbiol 43:1921 – 1924. Devulder G, Perriere G, Baty F, Flandrois J-P (2003) BIBI, a bioinformatics bacterial identification tool. J Clin Microbiol 41:1785 – 1787. Diego C, Ambrosioni JC, Abel G, Fernando B, Tomas O, Ricardo N, Jorge B (2005) Disseminated nocardiosis caused by Nocardia abscessus in an HIV-infected patient: first reported case. AIDS 19:1330 – 1331. Eisenblatter M, Disko U, Stoltenburg-Didinger G, Scherubl H, Schaal KP, Roth A, Ignatius R, Zeitz M, Hahn H, Wagner J (2002) Isolation of

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