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Clinical and Vaccine Immunology, November 2006, p. 1287-1290, Vol. 13, No. 11
1071-412X/06/$08.00+0     doi:10.1128/CVI.00269-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Compartmentalization of Interleukin-6 Response in a Patient with Septic Meningococcal Peritonitis

Alexandre Leite de Souza,^1* Jaques Sztajnbok,¹ Maristela Marques Salgado,² Carla C. Romano,³ Maria das Graças Adelino Alkmin,² Alberto J. S. Duarte,³ and Antonio Carlos Seguro^1,4

Intensive Care Unit, Emílio Ribas Institute of Infectology, São Paulo, Brazil,¹ Department of Immunology, Immunology and Microbiology Service, Adolfo Lutz Institute, São Paulo, Brazil,² Department of Dermatology/LIM56, University of São Paulo School of Medicine, São Paulo, Brazil,³ Department of Nephrology, Laboratory of Basic Research, University of São Paulo School of Medicine, São Paulo, Brazil⁴

Received 20 July 2006/ Returned for modification 21 August 2006/ Accepted 30 August 2006

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We report the first case of Neisseria meningitidis-induced septic peritonitis diagnosed by PCR assay of peritoneal fluid. Concentrations of interleukin-6 were notably higher in the peritoneal fluid than in the blood. PCR diagnosis of septic meningococcal peritonitis and the pathogenesis of the disease are discussed.

Case report. A previously healthy 6-year-old girl presented with a 24-h history of fever, headache, and intense myalgia. In the 2 h preceding her examination, she had developed petechiae on her abdomen, arms, face, and legs, accompanied by a deteriorating level of consciousness. Upon examination, petechial lesions were noted on both palpebral conjunctivae, and a dramatic progression from petechiae to confluent ecchymoses was observed on her skin, apparently fitting the clinical profile of disseminated intravascular coagulation. Her vital signs were as follows: axillary temperature, 38°C; pulse, 160 beats/min; respiration, 40 breaths/min; and blood pressure, 80/60 mm Hg. She was positive for Kernig's sign and Brudzinski's sign, and her Glasgow coma scale score was 13. The rest of the examination was unremarkable. The white blood cell (WBC) count was 28,000 WBCs/mm³ (93% polymorphonuclear neutrophils, 3% lymphocytes, 4% monocytes). The hemoglobin concentration was 9.3 g/dl, and the platelet count was 58,000 cells/mm³. Coagulation studies demonstrated an international normalized ratio of 2.12 and an activated partial-thromboplastin time of 55 s. Abnormal laboratory values included the following serum levels: creatine kinase, 784 U/liter; albumin, 2.7 g/dl; aspartate aminotransferase, 106 U/liter; and alanine aminotransferase, 63 U/liter. The acid/base response to this clinical profile was consistent with acidemia, metabolic acidosis, and respiratory alkalosis (arterial pH, 7.28; partial O₂ pressure, 96 mm Hg; bicarbonate, 15 mmol/liter; base deficit, –3 mmol/liter; partial CO₂ pressure, 26 mmol/liter). A computed tomography scan of the brain was normal, and a lumbar puncture was performed. The cerebrospinal fluid (CSF) was cloudy and contained 1,680 WBCs/mm³ (80% polymorphonuclear neutrophils, 15% lymphocytes, 5% monocytes), 104 mg/dl of protein, and 27 mg/dl of glucose. Gram staining of the CSF showed gram-negative diplococci, and Neisseria meningitidis serogroup C was detected by using a latex agglutination test. Blood samples and CSF samples were collected for culture. Counterimmunoelectrophoresis results for Neisseria meningitidis serogroup C in the serum were positive. The preliminary diagnosis was meningitis accompanied by meningococcemia. Therefore, antibiotic treatment was initiated with ceftriaxone (100 mg/kg of body weight/day) and dexamethasone (0.6 mg/kg/day for 4 days). In addition, supportive measures to maintain homeostasis, including electrolyte replacement, fluid reposition, vitamin K administration, and transfusion of fresh-frozen plasma, were implemented. The patient's close contacts received chemoprophylaxis.

Initially, the general status of the patient improved. However, 48 h after admission, the patient's headache and myalgia subsided and were replaced by severe abdominal pain. Upon examination, the patient's bowel sounds were found to be diminished, and the abdomen was tender and distended. A conventional X ray of the abdomen showed abnormal gas distribution in the bowel and generalized dilation of the bowel loops, although there was no fluid level or free air under the diaphragm. Appendicitis was suspected, and a midline laparotomy, including complete exploration of the abdominal cavity, was performed. The peritoneal cavity contained a moderate amount of yellowish exudate. The peritoneum appeared markedly inflamed, although the appendix was normal. An appendectomy was performed in the usual fashion. The clinical suspicion of appendicitis was later confirmed by histopathological findings, including alterations to the serous membrane that were indicative of peritonitis, and many gram-negative diplococci were present within the peritoneal specimen (Fig. 1). The peritoneal fluid contained 1,320 WBCs/mm³ (79% polymorphonuclear neutrophils, 15% lymphocytes, 6% monocytes), 2,100 mg/dl of protein, and 128 mg/dl of glucose. Gram and Ziehl-Neelsen staining revealed no microorganisms. The sample of peritoneal fluid obtained during a laparotomy performed after the patient had received five doses of ceftriaxone was negative, as were the cultures of CSF and blood. However, PCR results with the peritoneal fluid were positive for N. meningitidis serogroup C. The antibiotic therapy was therefore continued for an additional 10 days.

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FIG. 1. Specimen obtained from a biopsy of the patient's peritoneum (hematoxylin and eosin stain; original magnification, x1,000). The arrows indicate gram-negative diplococci.

PCR amplification of N. meningitidis serogroup C genes in peritoneal fluid was performed for three specific regions (5a): the capsular transport gene (ctrA), the polysialyltransferase gene (siaD) for serogroup B (siaD_B), and siaD for serogroup C (siaD_C). The following pairs of primers were used: ctrA1, 5' ATG CGG TGG CTG CGG TAG GT 3', and ctrA2, 5' CCG GCG AGA ACA CAA ACG ACA AG 3'; B1, 5' GGA TCA TTT CAG TGT TTT CCA CCA 3', and B2, 5' GCA TGC TGG AGG AAT AAG CAT TAA 3' (for siaD_B); and C1, 5' TCA AAT GAG TTT GCG AAT AGA AGG T 3', and C2, 5' CAA TCA CGA TTT GCC CAA TTG AC 3' (for siaD_C) (8). PCR conditions were based on those published by Taha (17). The amplification was performed with an Eppendorf thermocycler (Mastercycler Personal model; Hamburg, Germany). Two whole-cell suspensions of the N. meningitidis clinical strains Nm573/03 (for siaD_B PCR) and Nm576/03 (for siaD_C PCR), with optical densities at 620 nm of 0.2, in 0.02% phosphate-buffered saline (PBS) sodium azide (inactivated at 56°C for 30 min and stored at 4°C), were utilized as positive controls. The PCR products were electrophoresed on an agarose gel containing 22 µg ethidium bromide/50 ml of gel (15 min at 40 V/cm and 30 min at 80 V/cm) and sized with a 100-bp DNA ladder (Invitrogen, Carlsbad, CA). A single product of 523 bp for ctrA, 450 bp for siaD_B, or 250 bp for siaD_C was interpreted as a positive result. Sample-positive PCR results for ctrA and siaD_C can be seen in Fig. 2.

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FIG. 2. PCR amplification of the capsular transport gene (ctrA) for N. meningitidis (523-bp amplicon) is shown in lanes 1, 2, and 3, corresponding to the negative and positive controls (strain Nm573/03) and the peritoneal fluid, respectively. PCR amplification of the polysialyltransferase gene (siaD) for N. meningitidis serogroup B (450-bp amplicon) is shown in lanes 4, 5, and 6, representing the negative and positive controls (strain Nm573/03) and the peritoneal fluid, respectively. PCR amplification of the siaD gene for N. meningitidis serogroup C (250-bp amplicon) is shown in lanes 7, 8, and 9, pertaining to the negative and positive controls (strain Nm576/03) and peritoneal fluid, respectively. Lanes MW, 100-bp DNA ladder (0.8 µl/well).

In addition, commercially available enzyme-linked immunosorbent assay kits were used to measure cytokine levels in the CSF and blood collected upon the patient's admission, as well as in peritoneal fluid and blood recovered during laparotomy. Antibody-matched pairs and respective standards were purchased from Endogen and used according to the manufacturer's recommendations. The detection limit was 10 pg/ml. Briefly, the microplate was coated with anticytokine monoclonal antibody (4 mg/ml; R&D Systems) and incubated overnight at 4°C. The plate was washed in PBS-0.02% Tween 20 (Sigma) and blocked with PBS-4% bovine serum albumin for 2 h at room temperature. Then, the plate was washed five times with PBS-0.02% Tween 20, the standard recombinant cytokine (at concentrations of 1,000 to 10 pg/ml) and the supernatants were added in duplicate, plus biotinylated anticytokine monoclonal antibody (0.25 mg/ml; R&D Systems), and the plate was incubated for 2 h at room temperature. The microplate was washed again, and the reaction was revealed by the addition of streptavidin-peroxidase (diluted 1:200) for 30 min at 37°C. After the plate was washed, tetramethylbenzidine was added, and the plate was incubated for 30 min in the dark. The reaction was stopped with 1 M sulfuric acid, and the development of color was read at 450 nm in a microplate reader (Bio-Rad reader). The results are shown in Table 1.

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TABLE 1. Concentration of cytokines determined by enzyme-linked immunosorbent assay in a patient with septic meningococcal peritonitis

On day 7, the patient developed painful arthritis in both knees and ankles. Examination of the joints revealed signs of inflammatory activity but no clinical evidence of effusion. A presumptive diagnosis of polyarthritis (nonseptic) was made, and aspirin (50 mg/kg/day) was added to the antibiotic therapy for 5 days.

Based on all of the available clinical and laboratory evidence, the patient was diagnosed with severe sepsis and coagulopathy caused by Neisseria meningitidis, complicated by purulent peritonitis and probable nonseptic polyarthritis. The evolution was favorable in the postoperative period as well as over the long term.

Meningococcal disease is an infection caused by Neisseria meningitidis and has an epidemic and overwhelming nature. It is found the world over and presents a constellation of clinical profiles: meningitis, sepsis, severe sepsis, and septic shock (7). In patients with meningococcal infection, atypical presentations such as complications involving abdominal organs, which may converge to an acute abdomen, have also been well documented (1, 2). The case presented herein illustrates an atypical phenomenon in a child who developed purulent peritonitis caused by Neisseria meningitidis serogroup C. We have also discussed some aspects of the pathogenesis of meningococcal peritonitis and recommend the use of PCR techniques for the diagnostic evaluation of septic meningococcal peritonitis.

Discussion. Neisseria meningitidis has been implicated in various conditions that result in an acute abdomen: peritonitis (1), splenic rupture (2), cholecystitis (4), pelvic inflammatory disease (3), mesenteric adenitis (11), ileitis (11), and pericarditis (6). Peritonitis caused by Neisseria meningitidis is rare but affects adults (1) and children alike (11, 12) and has been linked to serogroups B (5), C (11), W135 (1), and Z (12). Few cases of meningococcal peritonitis have been reported (9).

Our patient presented with septic peritonitis, which was confirmed by PCR results positive for Neisseria meningitidis serogroup C in the peritoneal fluid. How penetration into the peritoneal compartment occurs is not clearly understood. We propose the following sequence of events. After invasion of the bloodstream, N. meningitidis initiates hematogenous seeding and migrates independently into the meningeal and peritoneal compartments, where it triggers the host inflammatory response that leads to an acute abdomen. Our findings support the existence of this pathophysiological pathway, as evidenced by the meningococci found in the blood, the CSF, and the peritoneal fluid by using counterimmunoelectrophoresis, a latex agglutination test, and PCR techniques, respectively. In addition, the inflammatory response was confirmed by measuring cytokine concentrations in peritoneal fluid, CSF, and blood (Table 1), as well as by performing inflammatory cell counts. The concentration of interleukin-6 (IL-6) was higher in the peritoneal fluid than in the serum. This cytokine pattern suggests a compartmentalized inflammatory response in which IL-6 is probably synthesized by activated resident cells and/or inflammatory cells that have migrated from the bloodstream to the peritoneal compartment (5b, 10, 15, 16). We recently reported a case of purulent pericarditis that presented a similar cytokine pattern (5b).

Several aspects of this case make it potentially noteworthy, in particular the fact that it illustrates the importance of considering peritonitis within the constellation of the clinical profiles of meningococcal disease. In addition, it demonstrates the potential of Neisseria meningitidis to penetrate a wide range of compartments other than the leptomeninges. To our knowledge, this is the first case of septic meningococcal peritonitis diagnosed by PCR analysis of peritoneal fluid. This is also the first case in which IL-6 activation has been identified in the peritoneal fluid of a patient with meningococcal infection. In conclusion, immunological and molecular techniques are powerful tools for the diagnostic evaluation and clarification of meningococcal septic phenomena, especially after the administration of antibiotics (5a, 5b). The use of these tools could provide new clues regarding the stormy interaction between the host and the catastrophic infection caused by Neisseria meningitidis.

 
We thank the Department of Pathology for helpful assistance.

All of the authors have read and approved the manuscript in its present form. In addition, the authors do not have a commercial or other association that might pose a conflict of interest.

 
* Corresponding author. Mailing address: Rua da Consolação, 2270 Ap 304, CEP 01302-001, São Paulo, SP, Brasil. Phone: 55 11 3066 7292. Fax: 55 11 30882267. E-mail: alexandre{at}emilioribas.sp.gov.br.

Published ahead of print on 20 September 2006.

Top Abstract References

 

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Clinical and Vaccine Immunology, November 2006, p. 1287-1290, Vol. 13, No. 11
1071-412X/06/$08.00+0     doi:10.1128/CVI.00269-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• de Souza, A. L., Seguro, A. C. (2008). Two centuries of meningococcal infection: from Vieusseux to the cellular and molecular basis of disease. J Med Microbiol 57: 1313-1321 [Abstract] [Full Text]  
• de Souza, A. L., Sztajnbok, J., Salgado, M. M., Romano, C. C., Alkmin, M. d. G. A., Duarte, A. J. S., Seguro, A. C. (2007). Severe Myalgia of the Lower Extremities as the First Clinical Feature of Meningococcal Purpura Fulminans. Am J Trop Med Hyg 77: 723-726 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by de Souza, A. L. Articles by Seguro, A. C. Search for Related Content PubMed PubMed Citation Articles by de Souza, A. L. Articles by Seguro, A. C.