Meningoencephalitis with subdural empyema caused by Clostridium perfringens type A 1*Michael

Hugelshofer and Achermann et al.

1

Meningoencephalitis with subdural empyema caused by Clostridium 1

perfringens type A 2

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1*Michael Hugelshofer, 2*Yvonne Achermann, 2Helen Kovari, 1Wolfgang Dent, 4

3Michael Hombach, 3Guido Bloemberg 5

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1Neurointensive Care Unit, Department of Neurosurgery, University Hospital Zurich, 7

Switzerland 8

2Division of Infectious Diseases and Hospital Epidemiology, University and University 9

Hospital Zurich, Switzerland 10

3Institute of Medical Microbiology, University of Zurich, Switzerland 11

12

*Note: Michael Hugelshofer and Yvonne Achermann contributed equally to this 13

manuscript 14

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Keywords: Meningoencephalitis, Clostridium perfringens, 16s rRNA PCR, Alpha 16

Toxin, Molecular diagnostics 17

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Running title: Meningoencephalitis with Clostridium perfringens 19

20

Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00802-12 JCM Accepts, published online ahead of print on 15 August 2012

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Corresponding author: 21

Michael Hugelshofer 22

University Hospital Zurich 23

Neurointensive Care Unit, Department of Neurosurgery 24

Frauenklinikstrasse 10 25

CH-8091 Zurich, Switzerland 26

Phone: ++41 44 255 56 24 27

Fax: ++41 44 255 46 72 28

E-mail: [email protected] 29

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Abstract (49 words) 31

We report a clinical case of meningoencephalitis with subdural empyema in an 32

immunocompromised farmer caused by toxigenic C. perfringens type A, which was 33

identified by 16S RNA gene analysis of cerebrospinal fluid and subdural empyema. In 34

immunocompromised patients C. perfringens should be considered as a potential 35

pathogen of sepsis. 36

37

Case Report 38

In June 2011, a seventy-four year old farmer developed headache, fever, nausea 39

with emesis, and fatigue within twenty-four hours prior to admission to a community 40

hospital. Due to a systemic inflammatory response syndrome (SIRS), he was referred 41

to the intensive care unit (ICU). Empirical intravenous antibiotic treatment with 42

piperacillin-tazobactam and clarithromycin was started based on the clinical 43

presentation of pneumonia with a suspicious infiltrate in the basal lobe. The patient 44

was immunosuppressed secondary to unclassified myelodysplastic syndrome (MDS) 45

and was on continuous steroid treatment for bronchiolitis obliterans organizing 46

pneumonia. Three weeks prior to hospital admission, the farmer had been struck by a 47

cow, but did not seek medical attention. Two hours after admission, the patient 48

developed a generalized seizure with postictal reduced consciousness and left-sided 49

Todd’s paresis. A cerebral computed tomography (CT) showed a thin hypodense 50

subdural lesion parieto-occipital on the right side with adjacent brain edema, 51

suggestive for a chronic subdural hematoma (see Figure 1a). Therefore, the patient 52

was referred to a tertiary care hospital for neurosurgical intervention. There, in 53

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addition to fever and tachycardia, reduced consciousness, left sided hemiparesis and 54

slight neck stiffness was present. Laboratory findings revealed the following: white 55

blood cell count, 1.02 x 106/ml (34% neutrophils, 63% lymphocytes, 1% eosinophils, 56

2% monocytes); thrombocyte count, 23 x 106/ml; prothrombin ratio 58%, international 57

normalized ratio (INR) 1.3; serum C-reactive protein, 13.3 mg/dl; HIV- and Hepatitis B 58

negative. Based on clinical presentation with headache, fever, epileptic seizure and 59

slight neck stiffness, a central nervous system (CNS) infection was postulated. 60

Antibiotic treatment was changed to intravenous ceftriaxone, amoxicillin, 61

metronidazole, vancomycin and acyclovir in order to cover microorganism potentially 62

causing central nervous system (CNS) infection (including Listeria spp., anaerobic 63

bacteria, penicillin resistant Streptococcus pneumoniae and Herpes simplex virus). 64

Due to impaired coagulation with increased risk of spinal bleeding, a lumbar puncture 65

was initially declined. A cerebral CT scan showed a progression of the subdural 66

lesion to the right frontal lobe, suggestive for an active subdural process such as 67

empyema (see Figure 1a and 1c). Two days after reconstitution of coagulation, the 68

right frontal part of the lesion was evacuated through a burr hole for microbiological 69

workup. An intraventricular drain (IVD) was inserted on the contra lateral side. The 70

intraoperative macroscopic findings supported the radiological diagnosis of subdural 71

empyema. Samples from cerebrospinal fluid (CSF) and empyema were processed 72

for CSF cell count, cytospin and Gram-staining, bacterial broad range 16sRNA gene 73

PCR, fungal broad range PCR targeting the ITS region, and microbiological cultures. 74

Unfortunately, initial analysis of CSF cell count failed and CSF drainage suspended 75

immediately after surgery due to clotting of the catheter. The direct staining of both 76

specimens showed plump, Gram-positive rods with blunt ends suggestive for 77

Clostridium spp. C. perfringens was finally identified in both samples by bacterial 78

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broad range 16S rRNA gene PCR followed by sequence analysis of the amplicon (4). 79

A nested PCR as second amplification step was not required, for which the time to 80

result was substantially reduced. PCR amplicons were sequenced (±500 bp) and 81

homology analysis was performed using IDNS software (SmartGene, Zug, 82

Switzerland). A homology of 99%, with a minimal of 0.5% difference with the second 83

homologous species, allows for identification at the species level. The sequences of 84

the PCR products (465 and 468 bp, respectively) showed 100% identity to the 16S 85

rRNA gene of C. perfringens. The second homologous species was Clostridium 86

saccharobutylicum with an identity of 94.7 %, enabling an accurate assignment of C. 87

perfringens present in both specimens. The 16S sequence was submitted to 88

Genbank (Accesion number JQ782390). The sequence electropherograms gave no 89

indication for a possible polymicrobial infection. A broad range fungal PCR targeting 90

the ITS region (15) was performed using the DNA extracts of the cerebrospinal fluid 91

and the subdural empyema, which was negative for both samples. Subsequently, C. 92

perfringens toxin typing was performed by multiplex PCR (34) followed by sequence 93

analysis for confirmation, which showed the presence of the cpa gene (encoding α-94

toxin) in both CSF and subdural empyema samples. PCR for cpb (β-toxin), cpb2 (β2-95

toxin), etx (ε-toxin), iap (ι -toxin) and cpe (enterotoxin) genes were negative. 96

Anaerobic cultures on brucella agar remained negative even after 10 days of 97

incubation. Therefore, biochemical tests and susceptibility testing were not possible. 98

According to MIC data for C. perfringens and clinical breakpoints of European 99

Committee for Antimicrobial Susceptibility Testing (EUCAST) for Gram-positive 100

anaerobes (20, 32) antibiotic treatment was adapted to intravenous penicillin (4 Mio 101

IU qid) and metronidazole (500mg tid). The disease course was complicated by 102

severe septic shock with multi-organ failure consisting of disseminated intravascular 103

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coagulation (DIC) with IVD associated intracerebral haemorrhage in the left frontal 104

lobe (see Figure 1d), acute respiratory and renal failure, hepatic dysfunction, and 105

aggravated pancytopenia. Intensive care management with mechanical ventilation, 106

hemofiltration, extensive substitution of blood products and high dose catecholamine 107

treatment was needed. After stabilisation of septic shock, tracheal aspergillosis was 108

microbiologically and histologically diagnosed by biopsy of the tracheal wall and was 109

treated with voriconazole. The patient could be discharged from the ICU after one 110

month of intensive care treatment. He remained in a stable condition and 111

neurologically, continuous improvement of alertness and communication was 112

observed. Spontaneous directed movement of the left hemi-body with only slight 113

impairment of force further proved the functional recovery of the right hemisphere. 114

Unfortunately, right-sided hemiplegia caused by the IVD associated intracerebral 115

haemorrhage in the left frontal lobe (see Figure 1b), did not improve. Four months 116

after the diagnosis of C. perfringens type A infection, the patient again developed a 117

SIRS. Due to the poor prognosis associated with unclassified MDS, no further clinical 118

workup was initiated and he died a few days later. Autopsy revealed subacute 119

myocardial infarction and pneumonia as the direct cause of death. Neuropathological 120

findings confirmed healing subdural empyema with a hemorrhagic component and 121

yellow stained leptomeninges on the right side consistent with prior CNS infection 122

with C. perfringens type A. 123

The most frequent clinical manifestation of C. perfringens infection is myonecrosis or 124

gas gangrene. If only minor amounts of toxins are produced infections may present 125

as a mild, self-limiting disease. If local infections spread via hematogenous 126

dissemination, every organ might be involved, including the brain. Central nervous 127

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system manifestation of C. perfringens infection in humans is rare (9). Most 128

commonly the meninges are affected (1, 3, 6, 7, 10-14, 16-19, 21, 22, 24-26, 29, 30) 129

after clinical manifestation of sepsis (31). Rarely, focal or diffuse encephalitis with or 130

without pneumocephalus (2, 13, 22, 27, 29) and only one single case of subdural 131

empyema has been reported in current literature (24). In our patient due to the 132

clinical findings of generalized epileptic seizure, radiological evidence of progressive 133

brain edema, and detection of C. perfringens type A by molecular analysis of CSF 134

and subdural empyema we diagnosed a meningoencephalitis and subdural 135

empyema with C. perfringens. We were unable to cultivate C. perfringens probably 136

due to the antibiotic treatment started 3 days before sampling. However, the 137

diagnosis was unambiguous according to: i) Direct microscopic detection of plump 138

Gram-positive, rod-shaped bacteria in large quantity in two independent samples of 139

the subdural lesion and ii) 100% identification of the 16S rRNA gene PCR amplicon 140

with C. perfringens in both clinical samples (cerebrospinal fluid and subdural 141

empyema). In addition, the cpa gene, which codes for the virulence factor alpha toxin 142

of C. perfringens type A, was amplified and confirmed by sequence analysis. 143

Usually, infections with C. perfringens start from a recent surgical wound, trauma or 144

from intra-abdominal disease, such as infections of the biliary tract or other 145

gastrointestinal infections (23). However, in most of the cases of C. perfringens 146

meningitis the site of infectious origin remains unidentified (6, 11, 13, 14, 18, 21, 24, 147

25, 28) as in our patient. C. perfringens is a common part of the intestinal flora of 148

domestic animals, such as cattle, and are spread into the environment by feces (33). 149

In our case, gastrointestinal disease caused by a foodborne infection was ruled out. 150

There was a history of minor trauma caused by a strike of a cow’s hoof three weeks 151

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before onset of symptoms. Possibly C. perfringens type A was carried on the cow’s 152

hoof and transmitted into the wound. Due to the pre-existing coagulopathy and the 153

history of trauma, we assume a chronic subdural hematoma as the site of onset for 154

the CNS manifestation. Although repeated normal aerobic and anaerobic blood 155

cultures remained negative, a transient bacteraemia with C. perfringens type A is 156

likely to have happened in a chronically exposed and immunocompromised farmer. 157

Subdural empyema and diffuse encephalitis with C. perfringens are reported to be 158

fulminant and with a fatal outcome in 30-100% of untreated patients (9). The 159

functional recovery in our patient might be explained by the early targeted therapy 160

with penicillin and metronidazole. There are expert recommendations but no in vivo 161

data regarding the treatment of C. perfringens CNS manifestations (21). In vitro, 162

antibiotic drugs with activity against Gram-positive anaerobic bacteria (penicillin, 163

clindamycin, metronidazole) reportedly show low minimal inhibitory concentrations 164

(MICs) (32). To date, no C. perfringens isolates resistant to penicillin, clindamycin, or 165

metronidazole according to EUCAST clinical breakpoints for anaerobic Gram-positive 166

rods (20) have been described. In the treatment of a brain abscess with C. 167

perfringens, a favourable outcome after immediate surgical debridement or drainage 168

was reported (5, 8). 169

In conclusion, we report the first case of cerebral infection due to C. perfringens type 170

A manifested with subdural empyema and diffuse meningoencephalitis which was 171

diagnosed by 16S rRNA sequencing and a multiplex PCR approach. The patient was 172

successfully treated with targeted antibiotic therapy and surgical removal of subdural 173

empyema. Even though rarely reported, C. perfringens type A should be considered 174

as a differential pathogen in a patient with immunosuppression. If no pathogen can 175

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be cultivated due to on-going antibiotic treatment, molecular analysis such as broad-176

range 16sRNA PCR is a valuable and rapid tool leading to an accurate diagnosis.177

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271 Legends 272 273

Figure 1 274

Figure 1a 275

Native CT scan of patients head at admission to our institution. Arrows marking 276

hypodense lesion parieto-occipital. Extensive brain swelling of the right hemisphere 277

with elapsed sulci. 278

279

Figure 1b 280

Native CT scan of patients head before transfer to communal hospital. Arrows 281

marking residual hypodense lesion parieto-occipital. Relaxed right hemisphere with 282

pronounced sulci. Arrow heads marking residual blood of left frontal hemorrhage. 283

284

Figure 1c 285

Coronary native CT scan of patients head before surgical intervention. Arrows 286

marking hypodense right frontal lesion. Extensive brain swelling of the right 287

hemisphere with elapsed sulci. 288

289

Figure 1d 290

Coronary native CT scan of patients head after surgical intervention. Virtually 291

complete evacuation of right frontal subdural lesion. Arrow head marking 292

intraventricular catheter. Arrows marking small IVD associated hemorrhage. 293

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Meningoencephalitis with subdural empyema caused by Clostridium perfringens type A 1*Michael