An immunosuppressed patient with systemic vasculitis suffering from cerebral abscesses due to Nocardia farcinica identified by 16S rRNA gene universal PCR

Andreas Sonesson1, Björn Öqvist2, Per Hagstam3, Isabella M. Björkman-Burtscher4, Håkan Miörner1 and Ann Cathrine Petersson1

1 Department of Clinical Microbiology and Immunology, 2 Department of Nephrology, 3 Department of Infectious Diseases and 4 Department of Diagnostic Radiology, Lund University Hospital, Lund, Sweden

Correspondence and offprint requests to: Ann Cathrine Petersson, PhD, Department of Clinical Microbiology and Immunology, Lund University Hospital, S221 85 Lund, Sweden. Email: Ann-Cathrine.Petersson{at}skane.se

Keywords: amikacin; cerebral abscess; immunosuppression; Nocardia farcinica; PCR; 16S rDNA



   Introduction
 Top
 Introduction
 Case
 Discussion
 References
 
Nocardia spp. are Gram-positive, partially or variably acid-fast filamentous, branching rods of aerobic actinomycetes [1]. The most common primary site for infection in man is the respiratory tract [1,2]. Brain abscess is the most common clinical manifestation of central nervous system (CNS) infection [1]. The majority of the patients affected have underlying chronic diseases or suffer from endogenous or drug-induced immunosuppression [2,3], but the pathogen can also infect persons without any risk factors [2]. However, the course of the infection tends to be more severe and prolonged in immunocompromised hosts than in patients with normal cell-mediated immunity [4]. The symptoms of brain abscesses are often non-specific and include the classic triad of fever, headache and focal neurological deficiency [2,3]. It is important to establish the appropriate microbial diagnosis, as the list of potential microbial invaders among immunosuppressed patients, especially in transplant recipients, is large and antibiotic therapy differs [3]. The diagnostic work-up should include active diagnostic investigations for uncommon pathogens, such as radiological imaging, measurement of arabinitol in the urine, bronchoscopy in combination with bronchoalveolar lavage, biopsy and histological examinations, sputum examinations, culturing for bacteria, fungi and viral agents in body fluids, serological tests and microscopy, including acid-fast staining for opportunistic pathogens. Crypyococcus neoformans, Listeria monocytogenes, Aspergillus fumigatus, conventional bacteria, viral agents, Nocardia, Mycobacterium tuberculosis, Mucoraceae and Toxoplasma gondii have been reported to cause CNS infections in immunocompromised patients [3]. Nocardial cultures often demonstrate growth too late to be clinically useful or are discarded too early to allow growth of Nocardia [2].

It is thus important to prolong the incubation period for up to 3 weeks or more, and to use specially designed growth media to allow the growth of uncommon pathogens [1]. A new technique for fast and reliable diagnoses of CNS infections and uncommon pathogens such as Nocardia spp. has been needed for a long time [2].



   Case
 Top
 Introduction
 Case
 Discussion
 References
 
In December 2001, a 60-year-old man was referred to Lund University Hospital (LUH) for plasmapheresis. His past history revealed type II diabetes, which for the last year was insulin dependent. He was treated with warfarin due to relapsing deep vein thrombosis and recurrent ischaemic cardiovascular episodes (Table 1). In 1998, a renal biopsy had shown a myeloperoxidase (MPO)-positive systemic vasculitis with a severe pauci-immune crescentic glomerulonephritis. Despite immunosuppression, he rapidly progressed to end-stage renal failure and became dependent on haemodialysis. Because of persistent positive anti-neutrophil cytoplasmic antibody (ANCA), he was maintained on immunosuppressive medication (Figure 1A).


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Table 1. The most important investigations performed since 1998 and findings/outcome

 



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Fig. 1. (a) The MPO-ANCA titre during 2001. Reference interval <21 arbitrary units, marginal value 25 (Wieslab, Lund, Sweden). (b) The CRP values during 2001. Reference interval <5 mg/l (Department of Clinical Chemistry, Lund University Hospital, Lund, Sweden).

 
During the summer and autumn of 2001, the patient had been repeatedly admitted to a local hospital due to recurrent pulmonary infections, fever, elevated C-reactive protein (CRP) (Figure 1B) and cerebral confusion. Investigative interventions and treatment regimes are summarized in Table 1. Cranial computed tomography (CT) was done in late November; however, this was of low quality due to motion artefacts, and no abnormal lesions were detected. His mental status did not improve and a lumbar puncture was performed; microbiological investigations were all negative. A brain CT 2 weeks later revealed multiple, up to 12 mm, well-defined lesions located in the supraventricular white matter and a few in the basal ganglia. Magnetic resonance imaging (MRI) showed a total of 40 supra- and infratentorial lesions of the size of 2–12 mm (Figure 2). Apart from multiple brain abscesses with possible accompanying meningitis, CNS manifestation of the underlying vasculitis was also considered. The history of recurring pulmonary changes, a persistent elevated MPO-ANCA titre (Figure 1A) and the present cerebral involvement presumably were the reasons why the consultant advocated increased immunosuppression with cyclophosphamide and methylprednisolone (Table 1).



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Fig. 2. T1-weighted axial MRIs after contrast administration. MRI showed multiple brain lesions with a diameter between 2 and 12 mm, homogeneous or ring-shaped contrast enhancement, distributed to the supra- and infratentorial white and grey matter including the brainstem, and without any marked oedema or mass effect. The lesions showed homogeneous or ring-shaped slight hyperintensity on T1-weighted images prior to contrast enhancement (not shown).

 
The patient was referred to LUH for plasmapheresis on 28 December 2001, due to the persistent high MPO titre and the presumed diagnosis; cerebral vasculitis or brain abscesses. After four plasma exchanges, his condition was unchanged and the history, laboratory and radiological findings were re-evaluated. A follow-up MRI and another CT showed a slight decrease of the contrast enhancement, with no substantial changes in size or numbers of brain lesions. In spite of a slight improvement over the following days, the patient was still severely lethargic and unable to speak. A lumbar puncture performed just 1 week before death on 10 January 2002 showed a high polymorphonuclear count, heavily elevated spinal protein and more pronounced glucose consumption than in a previous sample. However, microscopy and cultures for bacteria and fungi were again negative, as were cerebrospinal fluid (CSF) investigations for tuberculosis, herpes simplex and cryptococcus. Blood cultures were still negative. Because of the negative findings, CSF analysis using 16S rRNA gene universal polymerase chain reaction (PCR) was performed. Ampicillin was added to the meropenem medication, for the possibility of Listeria meningitis. On 9 January 2002, contact with the neurosurgical department was initiated; the consultant did not advocate performing biopsy of the brain lesions in the patient's current state. The patient again deteriorated and died on 10 January 2002 subsequent to a respiratory arrest. Autopsy confirmed the cerebral lesions seen on radiological images as abscesses and also an acute myocardial infarction. PCR analysis of the CSF after 4 days revealed the presence of N. farcinica DNA, which subsequently was confirmed at autopsy in cultures from the brain lesions.

16S gene universal PCR
Aliquots of CSF, undiluted and diluted 1/20 in UV-treated distilled water, were heated at 95°C for 15 min and used as template without further purification. The amplification was carried out in 50 µl of reaction mixture of 1 x PCR buffer containing Tris–HCl, KCl and (NH4)2SO4, 2 mM MgCl2, 200 µM each of dATP, dCTP and dGTP, 300 µM dUTP, 1.25 U of HotStar Taq DNA polymerase (Qiagen, Hilden, Germany), 10 pmol of each primer and 5 µl of template. BAK11w (5'-AGA GTT TGA TC(A/C) TGG CTC AG-3') and p91E-Revers (5'-CCC GTC AAT TC(A/T/C) TTT GAG T-3') complementary to p91E [5,6] were used as PCR and sequencing primers.

A pre-PCR step of 15 min at 95°C was followed by 38 cycles at 93°C for 40 s, 55°C for 40 s and 72°C for 40 s. A final step of 5 min at 72°C terminated the amplification. Tubes with no target DNA and with Escherichia coli DNA were included as negative and positive controls, respectively.

Both strands of the ~900 bp PCR product were sequenced using the BigDye Terminator Cycle Sequencing Kit, and analysed on an ABI PRISM 3100 Genetic Analyser. The sequence showed homology over 853/854 bp (99.9%) to the 16S rRNA gene of N. farcinica available at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

Post-mortem culture
Samples collected at autopsy from brain abscesses and from CSF were inoculated onto aerobic and anaerobic blood agar plates, defibrinated horse blood agar (CO2), and into an anaerobic broth that was subcultured after 4 days. On day 3 after subculture of the enrichment broth, orange colonies grew on blood agar plates. Gram staining showed Gram-positive rods with a branched appearance and with a tendency to be partially acid fast in modified acid-fast staining. Staining results, the colony morphology, growth in basal glycerol broth with 50 mg/l lysozyme and production of urea associated the isolate with the genus Nocardia [1,7,8]. Additional tests showed opacification of Middlebrooke 7H10 agar, growth at both 45 and 35°C, and production of acid from rhamnose. The 16S rRNA gene sequence of the isolate was identical to the amplicon obtained from CSF. Minimal inhibitory concentrations (MICs) were determined by E-tests on IsoSensitest agar with 5% horse erythrocytes incubated at 37°C for 72 h. The isolate was susceptible to amikacin, intermediate to ciprofloxacin and resistant to tobramycin, ampicillin, cefotaxime, imipenem, meropenem and trimethoprim-sulfamethoxazole according to MIC breakpoints (www.srga.org).



   Discussion
 Top
 Introduction
 Case
 Discussion
 References
 
Nocardia spp. are well recognized but seldom encountered human pathogens that have a tropism for cerebral tissue [9]. This predilection stresses the importance of awareness of this pathogen as a cause of CNS infections, in particular among patients with impaired cell-mediated immunity presenting signs of brain abscesses following a history of recent pulmonary infections [1,2,4]. The X-ray examinations and clinical findings in this case indicated pulmonary infections prior to the development of brain abscesses. However, when a total of 27 chest X-ray examinations of the patient were reviewed, none showed cavitations or solid masses suspicious for nocardial infection. None of them were in accordance with granuloma and hence a nocardial pulmonary infection was never suspected. Without biopsy specimens from lung lesions, it is therefore not possible to verify that the patient in fact suffered from pulmonary nocardial infection. However, further diagnostic efforts should have been pursued in order to obtain a microbiological diagnosis, especially in an immunocompromised patient.

Culture is still the ‘gold standard’ when bacterial CNS infections are investigated. Unfortunately, the technique is hampered by several factors such as small sample volumes, antibiotic treatment, suboptimal duration of incubation and suboptimal growth medium [1,2]. The negative CSF cultures in the present case may also have been due to the highly encapsulated abscesses leaving no or very few viable cells in the spinal fluid. The post-mortem cultures grew bacteria only after broth enrichment in combination with prolonged incubation. The use of CT- or MRI-guided stereotactic aspiration has made it possible to obtain tissue from brain abscesses for microbiological analysis, as demonstrated in a recent publication [10]. This, however, necessitates an invasive operation only advisable under certain conditions [11,12]. Neurosurgeons advised not to perform biopsy of the brain lesions at this stage due to the bad condition of the patient. Hence, neurosurgical intervention was not performed in this case, in contrast to what has been recommended by other authors [12].

The universal 16S rRNA gene PCR and sequencing of the amplicon has been used for identification of fast and slow growing and uncultured bacteria in different types of clinical samples including CSF [5,6,13]. To our knowledge, no case has been reported previously using this technique as a diagnostic tool to identify N. farcinica in CSF. Nocardia infection may go undiagnosed for a long time, for several months or even years, and only 20% of cases with meningitis or brain abscesses may be diagnosed by CSF culture [1]. There is no information available in the literature as to at what stage in the disease Nocardia may be identified by PCR from CSF of infected patients. Further studies have to be performed. While culture often needs prolonged incubation for growth, for species identification and for antibiotic susceptibility testing [2], PCR and sequencing can be completed in a few days. Species identification by 16S rRNA gene sequence analysis may give an indication of how to design the drug treatment strategy. Nocardia farcinica show unique resistant patterns that include resistance to most antibiotics available except ciprofloxacin, imipenem, co-trimoxazole and amikacin [8]. However, susceptibility to ciprofloxacin, imipenem and co-trimoxazole varies, leaving amikacin as the only drug to which N. farcinica has been reported to be fully susceptible [1,2,8,12]. The strain isolated post-mortem showed increased MICs to all antibiotic drugs used in the course of treatment since the first sign of pulmonary infection. The strain was also resistant to trimethoprim-sulfamethoxazole and imipenem, leaving amikacin as the only drug of choice.

The technique employed in the described case fulfils the need for a fast technique for the diagnosis of uncommon slow growing or uncultivable pathogens in CNS infections. Species identification using 16S rRNA gene sequencing may also contribute to improved antibiotic treatment and survival of the immunocompromised patient.



   Acknowledgments
 
The skilful technical assistance of Maria Kelemen at the Department of Clinical Microbiology and Immunology, Lund University Hospital, is gratefully acknowledged.

Conflict of interest statement. None declared.



   References
 Top
 Introduction
 Case
 Discussion
 References
 

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Received for publication: 2.12.03
Accepted in revised form: 17. 6.04





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