first case of disseminated human infection with...
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First case of disseminated human infection with Nocardia cerradoensis 1
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Caroline Piaua,b,c#, Mallorie Kerjouana,d, Marc Le Mouela, Solene Patrat-Delona,e, Pierre-Louis 4
Henauxa,f, Vanessa Bruna,g, Marie-Pascale Morina,h, Philippe Gautiera, Veronica Rodriguez-5
Navai, Samer Kayala,b,c#. 6
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University Rennes-1, Medical School, Rennes, Francea; IGDR-UMR 6290-CNRS, Université 8
Rennes 1, Rennes, Franceb; CHU Rennes-Hôpital Pontchaillou, departments of Microbiologyc, 9
Pulmonologyd, Infectious Diseasese, Neusurgeryf, Radiologyg, Nephrologyh, Rennes France; 10
Observatoire Français des Nocardioses UMR 5557-CNRS, University Lyon 1, Lyon, Francei. 11
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Running Head : Disseminated Nocardia infection 13
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# Address correspondence to Samer KAYAL, [email protected] or Caroline PIAU, 16
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JCM Accepted Manuscript Posted Online 7 January 2015J. Clin. Microbiol. doi:10.1128/JCM.02979-14Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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ABSTRACT 21
Herein we report the first human disseminated infection with Nocardia cerradoensis in a 22
renal transplant patient, and isolated after a brain biopsy. Species identification was based 23
on 16S rRNA, gyrB and hsp65 gene analyses. Antibiotic treatment was successful by 24
combining carbapenems and aminoglycosides, before switching to oral trimethoprim-25
sulfamethoxazole. 26
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CASE REPORT 29
A 59 year-old woman, with a history of end-stage renal disease, secondary to 30
autosomal–dominant polycystic kidney disease, received a renal transplant in 2010. 31
Immunosupressive regimen included tacrolimus 12 mg/day and prednisolone 7.5 mg/day. 32
Prophylaxis with trimethoprim-sulfametoxazole (SXT) was given for six month after the 33
transplantation and then stopped due to intolerance symptoms. She had no history of recent 34
travel. Two weeks before admission she had had a mild headache with fever, and chills and 35
she was empirically treated with amoxicillin and prednisone for seven days. After a brief 36
improvement, she developed a non-productive cough with dyspnea. Based on the 37
hypothesis of pneumonia, antibiotic treatment was switched to ceftriaxone combined with 38
aerosol bronchodilators. Pulmonary symptoms rapidly worsened, and on September 2013 39
she was admitted to hospital for further investigations. 40
On examination, the patient was awake and complained of inspiratory dyspnea. 41
Diarrhea was noted a few days after the initiation of antibiotic treatment and resolved 42
spontaneously. Temperature was measured at 37.8 °C, blood pressure was 150/90 mmHg, 43
the pulse 97 beats/min, the respiratory rate 36/min, and oxygen saturation at 97% while she 44
was breathing ambient air. The auscultation evidenced bilateral basal crepitus and no other 45
abnormal sounds. Heart sounds were normal, abdomen was soft, without tenderness, 46
distention, or organomegaly, and neurologic examination was normal. No peripheral lymph 47
nodes were detected. Subcutaneous nodules of lower extremities appeared few days after 48
she was admitted (Fig 1.A). The white-cell count was 18.6 x 109/L, with 95% neutrophils and 49
the blood level of C-reactive protein was 159 mg/L. Chest X-ray and computed tomography 50
(CT) scan evidenced wall thickening of the right main bronchus, moderate ground glass 51
opacities of the right upper lobe, a right hilar lymph node, a small right parenchymal nodule 52
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(<10 mm), and a moderate homolateral pleural effusion (Fig1.B). Abdominal CT scan 53
revealed diffuse peritoneal and right retroperitoneal nodules (Fig1.C), and a thickening of 54
the cæcal wall. Bronchoalveolar lavage (BAL), bronchial and subcutaneous biopsies were 55
performed and the examination of stained smears (Gram, Ziehl–Neelsen, May Grunwald 56
Giemsa, Papanicolaou, Perls and hematoxilin safranin) showed no bacteria or abnormal cells. 57
Cultures for bacteria, fungi, and mycobacteria were all negative. Histological examination of 58
the subcutaneous nodule concluded in an erythema nodosum. The amplification of bacterial 59
16S rRNA gene on the cutaneous biopsy was also negative. The diagnosis of primary lung 60
carcinoma was then suggested and transbronchial right hilar lymph node puncture was 61
performed and did not show any malignant cell; bacterial cultures were not done due to 62
insufficient sampling. 63
On 20th day of hospitalization, the patient presented neurological worsening with 64
generalized weakness, dysarthria, lateral seizures and pyramidal syndrome on examination. 65
Brain CT-scan, without the administration of contrast material, followed by magnetic 66
resonance imaging (MRI) showed no hemorrhage of brain parenchyma and small non-67
specific lesions suggesting a differential diagnosis between multiple abscesses and 68
cystic/necrotic brain tumors (Fig 1.D). Stereotactic biopsy of brain parenchymal lesion was 69
done, and Gram staining evidenced filamentous Gram-positive bacilli (Fig 1.E). Bacterial 70
growth was obtained on chocolate and Buffered charcoal yeast extract plates (oriented by 71
Gram staining) incubated at 37°C for 48 hrs in an aerobic atmosphere; the colonies of 2 mm 72
diameter were white and rough. Forehead skin swab performed during neurological surgery 73
and two positive aerobic blood culture (Bactec®), both plated on chocolate agar, allowed 74
after 48 h of incubation the isolation of a bacteria with the same morphological features 75
than above. Cerebrospinal fluid remained sterile after 10 days incubation. 76
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Bacterial identification by Matrix Assisted Laser Desorption Ionization Time-of-Flight 77
Mass Spectrometry (MALDI-TOF; Brucker®-France), even after a full extraction, yielded an 78
identification of Nocardia species with a score value <1,7 (in 2014, 5627 species were 79
included in the Brucker’s spectrometry database). To better identify all the isolated bacteria, 80
we carried out the sequencing of 16S rRNA, gyrB, and hsp65 genes (1), which were queried 81
against GenBank database. The best nucleotide identities (%) are respectively given for each 82
gene: 1-16S rRNA gene: 99.84% with N. cerradoensis (2 different nucleotides [nt]/1325 nt 83
fragment), 99.62% with N. mikamii (5/1325), 99.47% with N. africana and N. aobensis 84
(7/1325) and 99.39% with N. veterana (8/1325); 2-gyrB gene: 98.98% with N. cerradoensis 85
(10/990) and 98.28% with N. mikamii (17/990), 3-hsp65 gene: 99.76% with N. cerradoensis 86
(1/434) and 98.84% with N. nova N. africana and N. aobensis (5/434). Finally, the retained 87
identification of isolated strain from brain abscess hereafter named OFN13.186 88
(Observatoire Français des Nocardioses; Lyon, France), is N. cerradoensis. 89
The antimicrobial susceptibility of the strain OFN13.186, was tested by using a broth 90
micro-dilution method according to the CLSI standard M24-A2 guidelines (2) .The MICs were 91
studied as previously described (1), by using Rapid Growing Mycobacteria Plate Format 92
(RAPMYCO) sensititre plates, incubated at 37°C for 72 h. Based on the previously established 93
breakpoints for the Nocardia genus (1), the strain was resistant to amoxicillin-clavulanic 94
acid, piperacillin-tazobactam, tobramycin, minocycilin and fluoroquinolones (Table 1). Taking 95
into account the intolerance of the patient to SXT, the empirical intravenous treatment was 96
started by associating meropenem (4 weeks) and amikacin (2 weeks), and allowed a rapid 97
clinical improvement and a regression of all the neurological disorders. The patient was 98
discharged, and after desensitization (3), antimicrobial treatment was switched to oral SXT 99
for 6 weeks. Two months after the initiation of antimicrobial therapy, intra-abdominal 100
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nodules and thoracic lymph nodes had completely regressed, but a lung parenchymal nodule 101
(5 mm) persisted. A follow-up of the pulmonary nodule, and a long-term prophylaxis with 102
SXT (800/160) was then maintained. 103
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The genus Nocardia is a ubiquitous group of environmental bacteria found in soil, 106
water or decaying organic matter and more than 50 species have been already described (4, 107
5). Opportunistic infections to Nocardia in humans mostly occur in patients with cell-108
mediated immunodeficiency like transplant recipients. Herein, and as far we know, we 109
report the first human case of diffuse nocardiosis due to the species Nocardia cerradoensis 110
which has been recently described as a new species from a strain isolated in Cerrado soil in 111
Brazil in 1985 as YT9 strain (6). 112
Immunosuppressive drugs required after kidney transplant induce immune cell 113
deficiency formally recognized as a risk factor for diffuse nocardiosis (7). However 114
nocardiosis remain a diagnostic challenge, because it is a rare etiology beside other 115
opportunistic pathogens, clinical presentation is non-specific and the bacterial cultures are 116
often difficult. The most common route of entry of Nocardia spp is inhalation or aspiration of 117
the organism as they may become airborne, particularly on dust particles (4, 5, 8). In this 118
case, the initial foremost respiratory symptoms and the main involvement of the right upper 119
lobe and hilar lymph node, suggested that the respiratory tract is the primary site of 120
infection. Although, primary cutaneous infection, or more occasionally spreading from oral 121
cavity or gastrointestinal tract may occur, we believe the cutaneous nodules, that appeared 122
after the onset of symptoms (Fig 1), as well as ceacal thickening and abdominal nodules, are 123
associated with disseminated disease. Additionally, cutaneous nodules mimicking erythema 124
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nodusum or cellulitis have been previously described in disseminated infections (9). Unlike 125
other bacterial brain abscesses, neurological symptoms in nocardiosis are highly variable, 126
generally insidious, without fever or signs of septicemia. The most common signs are focal 127
neurological deficits, non-focal findings, and seizures (10). In our patient, the acute CNS 128
symptoms associated to multiple small cerebral abscesses were concomitant to a rapid 129
progression of the dissemination since all the bacterial cultures became positive. However, 130
the most likely is that the brain metastases were initially present despite a normal 131
neurological examination. 132
At the onset of the infection, short antibacterial treatment (amoxicillin followed by 133
ceftriaxone for two weeks) may explain the brief improvement of clinical status, and 134
negative bacterial cultures despite the efforts to search a nocardiosis. Nocardia species 135
exhibit a species-predictable antimicrobial susceptibility pattern and early species 136
identification may be a crucial step to start an adapted antibiotherapy (11). When bacterial 137
culture were positive, the isolates were correctly identified with MALDI-TOF at the genus 138
level and the lack of accuracy to identify at species level is easily explained because N. 139
cerradoensis was not included in the database. Thus the identification of N. cerradoensis was 140
obtained by the sequencing of three housekeeping genes (16SrRNA, gyrB and hsp65) as 141
previously described (12, 13). The used criteria was actually, the one described by the 142
MM18-A document i.e. for identification at species level whatever the used gene, and 143
identity higher than 99,6% was required (14). 144
The probabilistic treatments in addition to their activity against Nocardia species 145
should also exhibit good diffusion to all sites of infection. In our case, we decided to initiate 146
the treatment with large spectrum antibiotics that also crosses blood-brain barrier. Actually, 147
Linezolid, SXT and amikacin show the less percentage of resistance (11, 15). Carbapenem 148
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display most of the time low MIC and meropenem exhibit the best cerebral diffusion among 149
this class. Time-kill studies showed that imipenem/amikacin and imipenem/moxifloxacin 150
combinations were bactericidal for most isolates whereas linezolid and SXT exhibited mainly 151
bacteriostatic activity (16). The noticed intolerance to SXT, and the initial identification 152
limited to the genus level, did not allow us to make a decision for antibiotic treatment based 153
on the in vitro predictable susceptibility. Therefore, the probabilistic treatment combined 154
meropenem plus amikacin, for which the bacteria have subsequently been shown to be 155
susceptible (Table 1). AST results, and patient improvement, lead us to reduce antibiotic 156
spectra and start a treatment with SXT 800/160 after desensitization protocol (3). 157
Prophylactic treatment was then instituted with SXT. 158
This is the first case of human disseminated infection with N. cerradoensis occurring 159
in an immunosuppressed host. Nevertheless, even if a nocardiosis was initially strongly 160
suggested among other opportunistic pathogens, it is essential to make the precise diagnosis 161
at species level to institute the adapted antimicrobial regimen. Finally, because the Nocardia 162
species can invade the CNS silently (17), we would like to emphasize, as recommended by 163
others (18), that MRI of the brain should be considered systematically for the diagnostic 164
evaluation even in the absence of neurological symptoms. 165
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NUCLEOTIDE SEQUENCE ACCESSION NUMBERS 167
The GenBank accession numbers for the indicated genes nucleotide sequences of Nocardia 168
cerradoensis (strain OFN 13.186) : gyrB gene (KP013615); 16S rRNA gene (KP013616) ; 169
hsp65 gene (KP013617). 170
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ACKNOWLEDGMENTS 172
This work was supported by the University Rennes1- Medical School, and CHU de 173
Rennes-France. We would like to thank E. Bergeron and D. Mouniée for their technical 174
support in this study. 175
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FIGURES 230
Figure 1 231
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LEGENDS FOR FIGURE 1 237
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Figure 1 : Disseminated nocardiosis. A- Subcutaneous nodules of lower 239
extremities; B- Right hilar lymph node (*); C-Retroperitoneal nodules 240
(arrows); D- Multiple Brain abscess (MRI); E- Gram stain (x100) of cerebral 241
biopsy. 242
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TABLE 244
Table 1 : 245
246
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Legend for Table 1: Antimicrobial susceptibility testing results for the strain 249
OFN13.186. MICs were determinate by broth microdilution method [ see 250
reference (1) for breakpoints and methods]. SXT= Trimethoprim-sulfamethoxazole. 251
Antibiotics Observed MICs Interpretation
Amikacin ≤4 S
Amoxicillin 4 S*
Amoxicillin-clavulanic acid >32/16 R
Cefotaxime ≤4 S
Ceftriaxone ≤4 S
Cefepime ≤4 S
Ciprofloxacin >4 R
Clarithromycin ≤2 S
Imipenem ≤2 S
Linezolid ≤4 S
Minocycline 2 I
Moxifloxacin 2 I
Tobramycin 16 R
SXT ≤1/19 S
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*Interpretative criteria used for amoxicillin was based on amoxicillin-clavulanic acid 252
break points (CLSI M24) 253
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