SURGICAL INFECTIONS Volume 15, Number 3, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/sur.2012.211
Tumor Necrosis Factor-b Nco1 Polymorphism and Susceptibility to Sepsis following Major Elective Surgery Kavita Baghel,1 Rajeshwar Nath Srivastava,2 Abhijit Chandra,1 Saloni Raj,1 Sudhir K. Goel,4 Aditya Bhusan Pant,5 and Jyotsna Agrawal 3
Abstract
Background: Post-operative sepsis remains a substantial cause of morbidity and mortality. In injured patients, that a polymorphism of the gene for tumor necrosis factor-b (TNF-b) has been related to the development of sepsis. Genetic factors may also have a role in etio-pathogenesis of sepsis following surgery. We investigated the relationship of the polymorphism of the gene for TNF-b and the serum concentration of TNF-a to the development of sepsis after elective major surgery. Methods: The study population consisted of 211 patients undergoing major elective surgery. The NcoI polymorphism of TNF-b was studied in genomic DNA through the analysis of restriction fragments of Nco1digested DNA with the polymerase chain reaction (PCR). All patients were followed for 1 mo after surgery for any evidence of sepsis. Serum concentrations of TNF-a were measured pre- and post-operatively by enzyme linked immunosorbent assay (ELISA). Genotypes of TNF-b and the production of TNF-a were related to the occurrence of sepsis. Results: Post-operative sepsis developed in 21.8% (n = 46) of the patients. The overall mortality was 4.2% (n = 9). The overall allele frequency of the TNF-b genotype was 0.32 for TNFB1 and 0.68 for TNFB2. Within the TNF-b genotype, 11.84% (n = 25) of the patients were homozygous recessive for TNFB1, 41.23% (n = 87) were heterozygous, with TNFB1/TNFB2, and 46.91% (n = 99) were homozygous dominant for TNFB2. The incidence of post-operative sepsis was significantly (p = 0.01) higher in patients homozygous for the TNFB2 allele. When compared with patients carrying at least one TNFB1 allele (TNFB1 homozygous and heterozygous genotype), the TNFB2 homozygous genotype was associated with an odds ratio (OR) of 2.60 (p = 0.005; 95% CI 1.32–5.15) for the development of sepsis. As compared with that for the heterozygous genotype, the OR for the homozygous TNFB2 genotype was 3.00 (p = 0.003; 95% CI 1.39–6.44). In patients with post-operative sepsis, serum concentrations of TNF-a were significantly higher (p = 0.02) in TNFB2 homozygous individuals than in those of individuals of the other TNF-b genotypes. Conclusion: The development of sepsis was associated with a greater capacity to produce TNF-a after surgery. The Nco1 polymorphism of the TNF-b gene was associated with the development of post-operative sepsis with an increased serum concentration of TNF-a. In patients without post-operative sepsis, polymorphism of the TNF-b gene was not related to different levels of TNF-a production. This indicates an association between polymorphism of the TNF-b gene and post-operative sepsis, suggesting the TNFB2/B2 genotype as a high-risk factor for the development of sepsis after elective surgery.
Departments of 1Surgical Gastroenterology, 2Orthopaedic Surgery, and 3Microbiology, King George’s Medical University, Lucknow, India. 4 Department of Biochemistry, All India Institute of Medical Sciences, Bhopal, Madhya Pradhesh, India. 5 Invitro Toxicology Division, Indian Institute of Toxicology Research, Lucknow, India. King George’s Medical University, Lucknow, India. Presented at the Thirty-second Annual Meeting of the Surgical Infection Society, Dallas, Texas, April 18–21, 2012.
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patients who underwent major elective abdominal surgery during October 2010 to June 2012 in the surgical wing of the University Hospital (KGMU) were included in the study. Elective major surgery was defined as planned surgery requiring more than one hour of operating time and anesthesia or respiratory assistance. All patients enrolled in the study met the criteria of being 18–50 y of age and without preexisting infection, rheumatoid arthritis or a seronegative inflammatory arthropathy, malignant disease, or diabetes mellitus; not taking a steroid medication; and having no acquired or inherited immunodeficiencies. Informed consent was obtained for the withdrawal of blood samples for determining patients’ individual NcoI polymorphism for TNFb and serum cytokine analysis, and prospective collection of the patients’ data. All of the patients had surgery under conditions as similar as possible, as the first case of the day to undergo surgery in the operating room by the same operating team. The intra-operative blood loss and duration of surgery (incision to closure) for each patient were recorded. The patients; characteristics are shown in Table 1. All patients were given non-steroidal anti-inflammatory drugs for postoperative pain, and at the time of induction of anesthesia were given a single intravenous dose of a broad-spectrum antibiotic (a third-generation cephalosporin) for prophylaxis against surgical infection. All patients were followed for 1 mo after surgery for any evidence of sepsis. Sepsis was defined according to the criteria of Bone et al. [16] by a body temperature > 38.5C or < 35.6C, tachycardia > 90 beats/min, tachypnea > 20 breaths/min, leukocytosis > 12,000/ mcL, or leukopenia < 4,000/mcL.
he clinical presentations and outcomes of patients with surgical infection are highly diverse. In cases of similar infections, some patients recover uneventfully whereas others develop sepsis, septic shock, and multiple organ dysfunctions (MOD). The factors determining such variation are being studied. Sepsis describes a complex clinical syndrome resulting from a systemic inflammatory response to bacteria or bacterial products. It is the most common cause of death in adult intensive care units, and its diagnosis is being made with increasing frequency [1,2]. Although knowledge of the pathophysiologic events related to sepsis and MOD has grown enormously, and new approaches to its treatment are being developed, the clinical outcome in severe sepsis remains poor [3–5]. Cytokine networks are believed to be of major importance in the pathogenesis of infectious diseases, and tumor necrosis factor (TNF) in particular appears to play a crucial role in this [6,7]. The pro-inflammatory cytokine TNF-a is an essential component of the host immune response to infection [8], and is responsible for the release of other inflammatory mediators. Tumor necrosis factor-a also plays a major role in the clinical manifestations of septic shock [9], and its serum concentrations correlate inversely with survival in severe sepsis [10,11]. With regard to this, an inherited variability in cytokine production, including that of TNF-a, and a genetic predisposition to fatal infectious diseases have been suggested [12]. The variability in TNF-a synthesis was initially correlated with genotypes of class II human leukocyte antigens (HLA) [13,14]. However, the location of the gene that encodes TNF-a and TNF-b within the major histocompatibility complex (MHC) raised the possibility that genetic alterations in the loci for these cytokines may be involved directly in a high level of production of TNF-a. A restriction fragment length polymorphism (RFLP) present in the TNF-b gene is correlated with increased concentrations of TNF-a in plasma and a high mortality in patients with severe sepsis [15]. The mortality in severe sepsis was found to be statistically significantly greater in patients homozygous for the TNFB2 allele than in those heterozygous for the Nco1 poly-morphism. A statistically significant association was found for the genotype of biallelic Nco1 polymorphism of the TNF-b gene and the development of severe sepsis after major blunt trauma [15]. For this reason, it must also be considered possible that a genetically determined inflammatory response contributes to sepsis or other post-operative complications in elective surgeries. In view of this, it would be important to find a genetic marker that identifies surgical patients with an increased susceptibility to post-operative sepsis. Because of a strong association of TNF-b Nco1 polymorphism and the risk of severe sepsis after major trauma [15], we hypothesized that this polymorphism might have a role in the development of sepsis following major surgery. We therefore conducted a study to determine the relationship of the NcoI polymorphism of the gene for TNF-b and the occurrence of sepsis after major surgery.
Materials and Methods Patient characteristics and study protocol
Ethical approval for the study was obtained from the institutional ethics committee, King George’s Medical University (KGMU), Lucknow, India. Two hundred eleven
Genotype analysis of the Nco1 restriction fragment length polymorphism within the locus of the gene for tumor necrosis factor-b
The genotype of the TNF b gene according to Nco1 RFLP was determined through amplification with polymerase chain reaction (PCR) and enzymatic digestion of the products with Nco1 (Fermentas, ThermoFisher Scientific, Waltham, MA). For this, 3 mL of venous blood were drawn into ethylenediamine-tetra-acetic acid (EDTA)-containing Vacutainer tubes (Becton–Dickinson, Franklin Lakes, NJ), and each patient’s genomic DNA was extracted from EDTA-anti-coagulated whole blood with a commercially available deoxyribonucleic acid (DNA) isolation kit (QIAmp DNA Blood Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A 782-bp PCR product of the TNF-b gene was amplified through PCR withthe following pair of primers: forward: 5¢CCGTGCTTCGTGCTTTGGACTA-3¢ and reverse: 5¢AGAGGGGTGGATGCTTGGGTTC-3¢ [17]. The amplification reaction was done with 10–100 ng of genomic DNA, 0.2 micromol of primers, 200 micromol of deoxynucleotide triphosphates (dNTPs), 1.5 mmol of MgCl2, and 1 unit of Taq polymerase in a total volume of 20 mcL. The PCR conditions consisted of initial denaturation at 95C for 5 min followed by 35 cycles of denaturation at 94C for 1 min, annealing at 58C for 1 min, and extension at 72C for 1.5 minutes. This was followed by final extension at 72C for 10 min. A total of 10 mcL of the resulting PCR product was digested with NcoI for 10 min at 37C, and the resulting fragments were analyzed on 1% agarose gel (Fig. 1).
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Table 1. Characteristics of Patients with and without Post-Operative Sepsisa Patients with post-operative sepsis (n = 46) Age (y) Gender (female/male) Duration of surgery (h) Blood loss (mL) Hb level (g/dL) Pre-operative Post-operative WBC/mcL Pre-operative Post-operative TNF-a (pg/mL) Pre-operative Post-operative day 2 Post-operative day 4 Mortality
Patients without post-operative sepsis (n = 165)
36.24 – 11.94 26/20 2.67 – 0.58 125.21 – 78.87 11.11 – 10.83 –
35.90 – 11.05 102/63 2.16 – 1.65 42.56 – 55.92 11.27 – 11.17 –
1.48 1.68
p value* 0.86 0.51 0.04* < 0.001*
1.79 0.99
0.58 0.08
7,253.04 – 1,836.65 10,015.87 – 6,417.77
7,150.42 – 1,676.03 7,194.16 – 1,444.72
0.72 < 0.001*
52.57 – 40.16 98.59 – 59.37 180.24 – 91.28 8 (17.39)
51.87 – 32.80 55.47 – 22.37 56.35 – 20.45 1 (0.60)
0.90 < 0.001* < 0.001* < 0.001*
a Values are given as numbers (%) or mean – SD. Differences were tested with the w2 test and the Fisher exact test for frequencies and with the Student t-test for mean – SD. *A value of p < 0.05 was considered statistically significant. TNF-a = tumor necrosis factor-a.
Assay of tumor necrosis factor-a
Results
Serum was collected pre- and post-operatively from each of the patients in the study on post-operative days (POD) 2 and 4. Commercially available, highly sensitive enzymelinked immunosolvent assay (ELISA) kits for human TNF-a (Orgenium AviBion, Vantaa, Finland) were used to measure serum concentrations of TNF-a according to the manufacturer’s instructions.
Of the 211 patients enrolled in the study, 128 (60.66%) were female and 83 (39.33%) were male. The mean age of the patients was 35.97 – 11.22 y. Post-operative sepsis was diagnosed in 46 (21.80%) of the patients. The patients with and without post-operative sepsis were comparable in terms of parameters including age, gender, duration of surgery, blood loss, and mortality (Table 1). The duration of surgery (p = 0.04) and intraoperative blood loss (p < 0.001) were observed to be statistically significantly associated with the development of post-operative sepsis. Significantly increased white blood cell counts (WBC) (p < 0.001) and mortality (p < 0.001) were observed in patients with post-operative sepsis as compared with those without sepsis. The most common post-operative complication found in the study was surgical site infection (n = 24; 11.37%), followed by the other complications listed in Table 3. The site of each type of surgery included in the study and the relevant incidences of post-operative sepsis and mortality are shown in Table 4. Sepsis was diagnosed in three patients 2 d after surgery, in
Statistical analysis
Data were expressed as proportion or mean – standard deviation (SD), as appropriate. Testing for intergroup differences in proportions was done with the w2 test or Fisher exact test. Odds ratios (OR) for the estimation of relative risk were calculated with 95% confidence intervals (CI). For intergroup comparisons, unpaired or paired Student t-test and one-way analysis of variance (ANOVA) was used. A value of p < 0.05 was considered as statistically significant.
FIG. 1. Genotypic analysis of the tumor necrosis factor (TNF)-b gene. The polymerase chain reaction product of 782 bp was digested with the endonuclease Nco1. Lane 1: 100 bp ladder; lanes 2–4: heterozygous genotype (TNFB1/B2); lanes 5 and 7: TNFB2 homozygous genotype (TNFB2/B2); lane 6: TNFB1 homozygous genotype (TNFB1/B1).
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Table 2. Genotypic Distribution and Characteristics of Patientsa Genotype Age (y) Gender (female/male) Blood loss (mL) Length of surgery (h) Hb level (g/dL) Pre-operative Post-operative WBC/mcL Pre-operative Post-operative Mortality Sepsis
TNFB1/B1 (n = 25) 34.96 – 75.62 – 2.18 –
12.66 16/9 80.07 0.65
11.17 – 10.96 –
1.97 0.96
TNFB1/B2 (n = 87) 35.89 –
7,084 – 1,232.09 7,364 – 2,907.15 0 5 (20%)
48.13 – 2.15 –
10.96 53/34 62.80 0.61
11.33 – 11.11 –
1.63 1.04
6,942.87 – 1,692.31 7,793.50 – 3,024.86 2 ( 2.3%) 11 (12.64%)
TNFB2/B2 (n = 99) 36.30 –
p value
68.36 – 2.41 –
11.17 59/40 73.26 2.09
0.86 0.91 0.08 0.49
11.17 – 10.97 –
1.76 0.91
0.80 0.57
7,397.27 – 1,807.99 8,011.05 – 4,021.90 7 ( 7.07%) 30 (30.30%)
0.18 0.70 0.13 0.01*
a Values are given as frequency (%) or mean – SD. Differences were tested with the w2 test for frequencies and ANOVA for mean – SD. *A value of p < 0.05 was considered statistically significant. TNF = tumor necrosis factor.
five patients at 4 d after surgery, in two patients at 5 d after surgery, and in the remaining 36 patients at 8 d or more after surgery. The relevant microbiologic findings on blood culture for each case of post-operative sepsis are shown in Table 5. In all surgical site infections, the organism observed was Staphylococcus aureus. Allele frequency and genotype distribution
With regard to TNF-b genotype, 11.84% (n = 25) patients were homozygous recessive for TNFB1, 41.23% (n = 87) were heterozygous, with TNFB1/TNFB2, and 46.91% (n = 99) were homozygous dominant for TNFB2. The distribution of the TNF-b genotype subgroups among the patients was compared in terms of parameters including age, gender, duration of surgery, blood loss, and mortality (Table 2). Post-operative sepsis was observed in 5/25 (20%), 11/87 (12.64%), and 30/99 (30.30%) patients of the TNFB1 homozygous, TNFB1/B2 heterozygous, and TNFB2 homozygous genotypes, respectively. The overall allele frequency was 0.32 for TNFB1 and 0.68 for TNFB2. The allele frequencies of TNFB1 and TNFB2 were 0.22 and 0.78 in patients with post-operative sepsis and 0.35 and 0.65 in patients without post-operative sepsis.
Table 3. Post-Operative Complications in Study Patients Complication Infections Surgical site infection Multiple organ failure Anastomotic leak Pancreatic leak Intra-abdominal abscess Pneumonia Non-infections Cardio-respiratory arrest a
Values are given as number (%).
No. of patients
Mortality (no. of patients)
24 3 8 5 3 3
1 2 3 1 1 0
1
1
The genotype distribution in patients with an uncomplicated clinical course was statistically significantly (p < 0.01) different from that in patients with post-operative sepsis. The development of post-operative sepsis was significantly (p = 0.01) more frequent in patients homozygous for the TNFB2 allele. However, the likely source of significance is the TNFB2 homozygous versus the TNFB1/B2 heterozygous genotype, which was derived by logistic regression analysis (table not shown). When compared with patients carrying at least one TNFB1 allele (TNFB1 homozygous and heterozygous genotype), the TNFB2 homozygous genotype was associated with an OR of 2.60 (p = 0.005; 95% CI 1.32–5.15) for the development of sepsis. Compared with that for the heterozygous genotype, the OR for the homozygous TNFB2 genotype was 3.00 (p = 0.003; 95% CI 1.39–6.44). When the TNFB1 and TNFB2 homozygous genotypes were assessed, no significant (p = 0.35; 95% CI 0.18–1.86) association of either was found with the development of post-operative sepsis. Regarding overall mortality, 4.1% (9/211) patients expired during their post-operative hospital stay (eight of the nine patients had post-operative sepsis and one had no evidence of sepsis). In none of the nine patients who expired was a statistically significant difference (p = 0.13) observed among the TNF-b genotype subgroups. Similarly, in terms of the deaths of eight of the nine patients with postoperative sepsis, no statistically significant difference (p = 0.05; 95% CI
Table 4. Post-Operative Sepsis and Mortality in Relation to Site of Surgerya
Site of surgery
Post-operative sepsis (n = 46)
Esophageal (n = 14) 1 ( 7.14%) Gastrointestinal (n = 52) 8 (15.38%) Hepatobiliary (n = 101) 13 (12.87%) Pancreatic (n = 27) 13 (48.15%) Other (n = 17) 3 (17.64%)
Mortality following post-operative sepsis (n = 8) 1 3 2 1 1
(7.14%) (5.77%) (1.98%) (3.70%) (5.88%)
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Table 5. Blood Culture Profile of Patients with Post-Operative Sepsis (n = 46) Isolates
No. of patients (n = 46)
Sterile Enterobacter Micrococcus Acetinobacter Staphylococcus aureus Escherichia coli
26 3 5 3 4 5
0.02,1.27) was observed between the TNFB2 homozygous and TNFBI/B2 heterozygous subgroups. Serum concentrations of tumor necrosis factor-a
No significant difference (p = 0.90) in serum TNF-a concentration was observed in the pre-operative samples of patients who did and did not develop post-operative sepsis, whereas on both POD 2 and 4, serum concentrations of TNF-a were significantly higher (p < 0.001) in patients who developed sepsis following surgery (Table 1). Tumor necrosis factor-a production in relation to different TNF-b genotypes
To study whether TNF-a production was influenced by the TNF-b genotype, we grouped the patients with and without post-operative sepsis according to their TNF-b genotypes and compared the TNF-a production in the groups. A significant increase (p = 0.02) was observed in TNF-a production in the TNFB2 homozygous patients with sepsis as compared with that in patients in the other subgroups of TNF-b genotype, whereas no such increment was observed in patients without sepsis (Figs. 2 and 3). Discussion
Tumor necrosis factor-a is believed to be a pivotal proinflammatory mediator in the host defense against infection [18,19]. However, only a limited number of studies have been done of the relationship of cytokine production to genetic
susceptibility to sepsis [12,20]. Several polymorphic elements in the region of the TNF gene have been related to TNF production, but the precise location of the genetic elements controlling the TNF response remains unclear, and their expression has been shown to be regulated only in part by transcriptional events [21–23]. Two single nucleotide polymorphisms (SNP) in the human TNF-a/TNF-b gene that might influence TNF-a secretion have been described as being associated with clinically severe forms of infectious diseases [15,23–25]: the Nco1 polymorphism of the TNF-b gene and a G/A polymorphism at position - 308 in the promoter region of the TNF-a gene. Reports of an association between polymorphism of the TNF-b gene and increased susceptibility to severe sepsis [15,26,27] encouraged us to examine the distribution of the TNF-b genotype in patients undergoing elective abdominal operations and to determine their post-operative status in terms of the development of sepsis. In the view of the contradictory findings in previous studies [17,26,28], we studied the functionality of biallelic TNF-b Nco1 polymorphism in relation to susceptibility to post-operative sepsis. We found the allele frequencies and genotype distributions of TNF-b Nco1 polymorphisms to be similar to those previously described in a German population [26,28,29]. In our study, the TNFB2/B2 genotype was present in 46.91% of the population, in which the observed incidence of septic complications was 30%, and was associated with an OR of 2.60 for the development of post-operative sepsis as compared with the other genotypes of TNF-b. At the same time, the low prevalence (11.84%) of the TNFB1 homozygous genotype makes its estimation less accurate as a factor in reducing the risk of post-operative sepsis. Although the TNFB2/B2 homozygous genotype was commonly observed in the patients in our study who did not survive after surgery, the association of this genotype with mortality after surgery was not statistically significant (p = 0.05). Previous studies of the relationship to sepsis of the TNFB2/B2 genotype among the Nco1 polymorphisms of the TNF-b gene demonstrated a poor prognosis and an increased mortality for this genotype as compared with the other genotypes of TNF-b [15,26,28,30]. The possibility that variation in the immune response associated with different TNF-b genotypes may be
FIG. 2. Comparison in the pre- and post-operative concentrations of tumor necrosis factor (TNF)-a in patients with postoperative sepsis and in TNF-b genotype subgroups. Statistical analysis using one-way analysis of variance showed a significant difference (p = 0.02) in the serum concentration of TNF-a on post-operative day 4 in different TNF-b genotype subgroups, whereas no such significant difference was observed pre-operatively or on post-operative day 2 (p = 0.93 and p = 0.31, respectively).
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FIG. 3. Comparison in the pre- and post-operative concentrations of tumor necrosis factor (TNF)-a in patients without post-operative sepsis and in genotype subgroups. Statistical analysis using one-way analysis of variance showed an insignificant difference in the pre- and post-operative concentrations of TNF-a in the different genotype subgroups (preoperatively: p = 0.63; post-operative day 2: p = 0.34; post-operative day: p = 0.58. responsible for the development of sepsis cannot be ruled out. Given that a strict protocol for asepsis was used for all of the patients in our study for the entire duration of the study, we believe that the results of the study do not reflect a breach in anti-infective measures, but instead reflect genetic differences in the immune response to septic insult itself. Because most of the patients in our study who developed septic complications post-operatively were of the TNFB2/B2 genotype, we doubted that this genotype influenced the postoperative production of TNF-a by leukocytes in the blood. To assess this, we analyzed serum concentrations of TNF-a preand post-operatively in our patients who did and did not develop post-operative sepsis in relation to their TNF-b genotypes. In accord with the available literature on patients with severe sepsis [15,26], patients in our study who had a TNFB2/B2 homozygous genotype had a greater serum TNFa response post-operatively than that of the patients with other TNF-b gentoypes. Although an increased cytokine response may be related to surgical stress, Majetschak et al. [31] reported that the stress associated with surgery is transient and does not contribute significantly to an increased cytokine response for more than 24 h after surgery. In contrast to a previous report [32], we found that the degree of TNF-a production in whole blood in our patients who did not have complications was unrelated to the polymorphism of the TNF-b gene. Although the source of the measured serum concentration of TNF-a in patients with post-operative sepsis is unknown, the difference found in serum TNF-a concentrations in the TNF-b genotypic subgroups in our study may be associated with the distinct capacity of monocytes to produce TNF-a as an inflammatory cytokine that affects susceptibility to sepsis, and this genotype-related difference has been shown to be associated with the Nco1 polymorphism. The preoperative identification of patients at high-risk for developing post-operative sepsis may be useful in clinical practice for preventing post-operative sepsis. Our study suggests that the early identification of patients at high risk of developing severe post-operative sepsis can be achieved with the use of easily collectable markers. These markers represent an inexpensive way in which to identify the high-risk patients in whom the effects of early, aggressive, goaldirected antiseptic treatment can be evaluated in future studies.
It is well known that various factors related to the nature of an operation or to the patient will influence the occurrence of major post-operative complications. The same team operated the patients in the current study and as the first case of the day in the operating room. This aimed to maintain uniformity in the study. We found that the amount of intra-operative blood loss and duration of surgery statistically significantly influenced the development of postoperative sepsis (Table 1), a finding also reported by other investigators [32–35], but we found no association of this with TNF-b genotypes. Other patient-related factors, such as age and gender, had no influence on the development of post-operative sepsis. Although various studies indicate that pre-existing patient-related factors (e.g., advanced age, male gender) may influence the development of postoperative complications [33,34,36–40], the findings in this regard are inconsistent. Ansari et al. [41] reported that age influences the development of post-operative complications, whereas Tang et al. [33] could not identify age as a risk factor for the same type of surgery as in the study by Ansari et al. Some studies found male gender to be an independent risk factor for the development of post-operative complications such as sepsis [38,34], whereas others could not confirm these findings [39,40]. Vogel et al. [42] described the rates of sepsis for a variety of hospital-based surgical procedures. Gastrointestinal, cardiovascular, and thoracic procedures accounted for nearly 50% of cases, and the rates of post-operative sepsis were highest for esophageal, pancreatic, gastric, small bowel, hepatic, and biliary procedures. Hensler et al. [43] reported a greater frequency of post-operative sepsis after esophagectomy. In our study, pancreatic operations, among all major elective gastrointestinal procedures, accounted for most (30.43%) of the instances of post-operative sepsis. The major finding in the present study was that the biallelic Nco1 polymorphism of the TNF-b gene is associated significantly with the development of sepsis after major surgery. However, it cannot be ruled out that the Nco1 polymorphism is not itself related directly to susceptibility to sepsis, but rather serves as a marker linked to the major histocompatibility complex (MHC) because of its location in the class III region of the MHC [21,23].
TNF-b NCO1 POLYMORPHISM AND SEPSIS
A limitation of the present study is that the influence of TNF-b genotypes on the development of post-operative sepsis according to type of surgical procedure could not be established because of the small number of patients undergoing each type of surgery. Similarly, the small number of TNFB1 homozygous patients in the study makes the estimation of this genotype less accurate as a risk-reducing factor for sepsis, demanding a larger cohort for evidence of this, although the possibility of a high risk of the TNFB2 homozygous genotype for sepsis as compared with the risk of TNFB2 heterozygotes should not be dismissed. Our data provide evidence of a genetic susceptibility to sepsis in surgical patients. In our study, the development of sepsis was associated with a greater capacity to produce TNF-a after surgery. The Nco1 polymorphism of the TNF gene was associated with the development of post-operative sepsis through an increased serum concentration of TNF. Analysis of this polymorphism appears to be a useful means for identifying patients at high risk for sepsis after surgical trauma. Further studies of molecular pathways with larger cohorts are needed to investigate the mechanism of increased susceptibility to sepsis in relation to the TNF Nco1 polymorphism. Acknowledgments
We thank the Council of Science and Technology of the Government of Uttar Pradesh, Lucknow, India for financial support. Author Disclosure Statement
The authors declare no conflicts of interest in the work described in this paper. References
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8. Bazzoni F, Beutler B. The tumor necrosis factor ligand and receptor families. N Engl J Med 1996;334:1717–1725. 9. Wheeler AP, Bernard GR. Treating patients with severe sepsis. N Engl J Med 1999;340:207–214. 10. Debets JMH, Kampmeijer R, van der Linden MPMH, et al. Plasma tumor necrosis factor and mortality in critically ill septic patients. Crit Care Med 1989;17:489–494. 11. Waage A, Brandtzaeg P, Halstennsen A, et al. The complex pattern of cytokines in serum from patients with meningococcal septic shock. J Exp Med 1989;169:333–338. 12. Westendorp RGJ, Langermans JAM, Huizinga TWJ, et al. Genetic influence on cytokine production and fatal meningococcal disease. Lancet 1997;349:170–173. 13. Molvig J, Baek L, Christensen P, et al. Endotoxin stimulated human monocyte secretion of Interleukin1, tumor necrosis factor alpha and prostaglandin E2 shows stable interindividual differences. Scand J Immunol 1988;27: 705–716. 14. Jacob CO, Fronek Z, Lewis GD, et al. Heritable major histocompatibility complex class II-associated differences in production of tumor necrosis factor a: Relevance to genetic predisposition to systemic lupus erythematosus. Proc Natl Acad Sci USA 1990;87:1233–1237. 15. Stu¨ber F, Petersen M, Bokelmann F, Schade U. A genomic polymorphism within the tumor necrosis factor locus influences plasma tumor necrosis factor-a concentrations and outcome of patients with severe sepsis. Crit Care Med 1996;24:381–384. 16. Bone RC, Balk RA, Cerra FB, et al. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/ Society of Critical Care Medicine. Chest 1992;101:1644– 1655. 17. Schueller AC, Heep A, Kattner E, et al. Prevalence of two tumor necrosis factor gene polymorphisms in premature infants with early onset sepsis. Biol Neonate 2006;90: 229–232 18. Bone RC. The pathogenesis of sepsis. Ann Intern Med 1991;115:457–469. 19. Beutler B, Cerami A. Cachectin: More than a tumor necrosis factor. N Engl J Med 1987;316:379–385. 20. Sorensen TIA, Nielsen GG, Andersen PK, Teasdale TW. Genetic and environmental influences on premature death in adult adoptees. N Engl J Med 1988;318:727–732. 21. Pauli U. Control of tumor necrosis factor gene expression. Crit Rev Eukar Gen Expr 1994;4:323–344. 22. Trowsdale J. Genomic structure and function in the MHC. Trends Genet 1993;9:117–122. 23. McGuire W, Hill AVS, Allsopp CEM, et al. Variation in the TNF-a promoter region associated with susceptibility to cerebral malaria. Nature 1994;371:508–511. 24. Cabrera M, Shaw MA, Sharples C, et al. Polymorphism in tumor necrosis factor genes associated with mucocutaneous leishmaniasis. J Exp Med 1995;182:1259–1264. 25. Kroeger KM, Carville KS, Abraham LJ. The -308 tumor necrosis factor-alpha promoter polymorphism effects transcription. Mol Immunol 1997;34:391–399. 26. Majetschak M, Flohe S, Obertacke U, et al. Relation of a TNF gene polymorphism to severe sepsis in trauma patients. Ann Surg 1999:230:207–214. 27. Flach R, Majetschak M, Heukamp T, et al. Relation of ex vivo stimulated blood cytokine synthesis to post-traumatic sepsis. Cytokine 1999;11:173–178.
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28. Kahlke V, Schafmayer C, Schniewind B, et al. Are postoperative complications genetically determined by TNF-b NcoI gene polymorphism? Surgery 2004;135:365–373. 29. Riese J, Woerner K, Zimmermann P, et al. Association of a TNF-b gene polymorphism with complications after major abdominal operations. Shock 2003;19:1–4. 30. Waterer GW, Quasney MW, Cantor RM, et al. Septic shock and respiratory failure in community-acquired pneumonia have different TNF polymorphism associations. Am J Respir Crit Care Med 2001;163:1599–1604. 31. Majetschak M, Krehmeier U, Ostroverkh L, et al. Alterations in leukocyte function following surgical trauma: Differentiation of distinct reaction types and association with tumor necrosis factor gene polymorphisms. Clin Diagn Lab Immunol 2005;12:296–303. 32. Pociot F, Briant L, Jongeneel CV, et al. Association of tumor necrosis factor (TNF) and class II major histocompatibility complex alleles with the secretion of TNF-a and TNF-b by human mononuclear cells: A possible link to insulin-dependent diabetes mellitus. Eur J Immunol 1993; 23:224–231. 33. Tang R, Chen HH, Wang YL, et al. Risk factors for surgical site infection after elective resection of the colon and rectum: a single-center prospective study of 2,809 consecutive patients. Ann Surg 2001;234:181–189. 34. Offner PJ, Moore EE, Biffl WL. Male gender is a risk factor for major infections after surgery. Arch Surg 1999;134: 935–940. 35. Velasco E, Thuler LC, Martins CA, et al. Risk factors for infectious complications after abdominal surgery for malignant disease. Am J Infect Control 1996;24:1–6. 36. Sørensen LT, Jørgensen T, Kirkby LT, et al. Smoking and alcohol abuse are major risk factors for anastomotic leakage in colorectal surgery. Br J Surg 1999;86:927–931.
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37. Law WI, Chu KW, Ho JWC, Chan CW. Risk factors for anastomotic leakage after low anterior resection with total mesorectal excision. Am J Surg 2000;179:92–96. 38. Schroeder J, Kahlke V, Staubach KH, et al. Gender differences in human sepsis. Arch Surg 1998;133:1200– 1205. 39. Eachempati SR, Hydo L, Barie PS. Gender-based differences in outcome in patients with sepsis. Arch Surg 1999; 134:1342–1347. 40. Wichmann MW, Inthorn D, Andress HJ, et al. Incidence and mortality of severe sepsis in surgical intensive care patients: the influence of patient gender on disease process and outcome. Int Care Med 2000;26:167–172. 41. Ansari MZ, Collopy BT, Hart WG, et al. In-hospital mortality and associated complications after bowel surgery in Victorian public hospitals. Aust NZ J Surg 2000;70:6–10. 42. Vogel TR, Dombrovskiy VY, Carson JL, et al. Postoperative sepsis in the United States. Ann Surg 2010;252: 1065–1071. 43. Hensler T, Heidecke CD, Hecker H, et al. Increased susceptibility to postoperative sepsis in patients with impaired monocyte IL-12 production. J Immunol 1998;161:2655– 2659.
Address correspondence to: Dr. Rajeshwar Nath Srivastava Department of Orthopaedic Surgery King George’s Medical University Lucknow - 226003, U.P. India E-mail: [email protected]
Tumor Necrosis Factor-b Nco1 Polymorphism and Susceptibility to Sepsis following Major Elective Surgery Kavita Baghel,1 Rajeshwar Nath Srivastava,2 Abhijit Chandra,1 Saloni Raj,1 Sudhir K. Goel,4 Aditya Bhusan Pant,5 and Jyotsna Agrawal 3
Abstract
Background: Post-operative sepsis remains a substantial cause of morbidity and mortality. In injured patients, that a polymorphism of the gene for tumor necrosis factor-b (TNF-b) has been related to the development of sepsis. Genetic factors may also have a role in etio-pathogenesis of sepsis following surgery. We investigated the relationship of the polymorphism of the gene for TNF-b and the serum concentration of TNF-a to the development of sepsis after elective major surgery. Methods: The study population consisted of 211 patients undergoing major elective surgery. The NcoI polymorphism of TNF-b was studied in genomic DNA through the analysis of restriction fragments of Nco1digested DNA with the polymerase chain reaction (PCR). All patients were followed for 1 mo after surgery for any evidence of sepsis. Serum concentrations of TNF-a were measured pre- and post-operatively by enzyme linked immunosorbent assay (ELISA). Genotypes of TNF-b and the production of TNF-a were related to the occurrence of sepsis. Results: Post-operative sepsis developed in 21.8% (n = 46) of the patients. The overall mortality was 4.2% (n = 9). The overall allele frequency of the TNF-b genotype was 0.32 for TNFB1 and 0.68 for TNFB2. Within the TNF-b genotype, 11.84% (n = 25) of the patients were homozygous recessive for TNFB1, 41.23% (n = 87) were heterozygous, with TNFB1/TNFB2, and 46.91% (n = 99) were homozygous dominant for TNFB2. The incidence of post-operative sepsis was significantly (p = 0.01) higher in patients homozygous for the TNFB2 allele. When compared with patients carrying at least one TNFB1 allele (TNFB1 homozygous and heterozygous genotype), the TNFB2 homozygous genotype was associated with an odds ratio (OR) of 2.60 (p = 0.005; 95% CI 1.32–5.15) for the development of sepsis. As compared with that for the heterozygous genotype, the OR for the homozygous TNFB2 genotype was 3.00 (p = 0.003; 95% CI 1.39–6.44). In patients with post-operative sepsis, serum concentrations of TNF-a were significantly higher (p = 0.02) in TNFB2 homozygous individuals than in those of individuals of the other TNF-b genotypes. Conclusion: The development of sepsis was associated with a greater capacity to produce TNF-a after surgery. The Nco1 polymorphism of the TNF-b gene was associated with the development of post-operative sepsis with an increased serum concentration of TNF-a. In patients without post-operative sepsis, polymorphism of the TNF-b gene was not related to different levels of TNF-a production. This indicates an association between polymorphism of the TNF-b gene and post-operative sepsis, suggesting the TNFB2/B2 genotype as a high-risk factor for the development of sepsis after elective surgery.
Departments of 1Surgical Gastroenterology, 2Orthopaedic Surgery, and 3Microbiology, King George’s Medical University, Lucknow, India. 4 Department of Biochemistry, All India Institute of Medical Sciences, Bhopal, Madhya Pradhesh, India. 5 Invitro Toxicology Division, Indian Institute of Toxicology Research, Lucknow, India. King George’s Medical University, Lucknow, India. Presented at the Thirty-second Annual Meeting of the Surgical Infection Society, Dallas, Texas, April 18–21, 2012.
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patients who underwent major elective abdominal surgery during October 2010 to June 2012 in the surgical wing of the University Hospital (KGMU) were included in the study. Elective major surgery was defined as planned surgery requiring more than one hour of operating time and anesthesia or respiratory assistance. All patients enrolled in the study met the criteria of being 18–50 y of age and without preexisting infection, rheumatoid arthritis or a seronegative inflammatory arthropathy, malignant disease, or diabetes mellitus; not taking a steroid medication; and having no acquired or inherited immunodeficiencies. Informed consent was obtained for the withdrawal of blood samples for determining patients’ individual NcoI polymorphism for TNFb and serum cytokine analysis, and prospective collection of the patients’ data. All of the patients had surgery under conditions as similar as possible, as the first case of the day to undergo surgery in the operating room by the same operating team. The intra-operative blood loss and duration of surgery (incision to closure) for each patient were recorded. The patients; characteristics are shown in Table 1. All patients were given non-steroidal anti-inflammatory drugs for postoperative pain, and at the time of induction of anesthesia were given a single intravenous dose of a broad-spectrum antibiotic (a third-generation cephalosporin) for prophylaxis against surgical infection. All patients were followed for 1 mo after surgery for any evidence of sepsis. Sepsis was defined according to the criteria of Bone et al. [16] by a body temperature > 38.5C or < 35.6C, tachycardia > 90 beats/min, tachypnea > 20 breaths/min, leukocytosis > 12,000/ mcL, or leukopenia < 4,000/mcL.
he clinical presentations and outcomes of patients with surgical infection are highly diverse. In cases of similar infections, some patients recover uneventfully whereas others develop sepsis, septic shock, and multiple organ dysfunctions (MOD). The factors determining such variation are being studied. Sepsis describes a complex clinical syndrome resulting from a systemic inflammatory response to bacteria or bacterial products. It is the most common cause of death in adult intensive care units, and its diagnosis is being made with increasing frequency [1,2]. Although knowledge of the pathophysiologic events related to sepsis and MOD has grown enormously, and new approaches to its treatment are being developed, the clinical outcome in severe sepsis remains poor [3–5]. Cytokine networks are believed to be of major importance in the pathogenesis of infectious diseases, and tumor necrosis factor (TNF) in particular appears to play a crucial role in this [6,7]. The pro-inflammatory cytokine TNF-a is an essential component of the host immune response to infection [8], and is responsible for the release of other inflammatory mediators. Tumor necrosis factor-a also plays a major role in the clinical manifestations of septic shock [9], and its serum concentrations correlate inversely with survival in severe sepsis [10,11]. With regard to this, an inherited variability in cytokine production, including that of TNF-a, and a genetic predisposition to fatal infectious diseases have been suggested [12]. The variability in TNF-a synthesis was initially correlated with genotypes of class II human leukocyte antigens (HLA) [13,14]. However, the location of the gene that encodes TNF-a and TNF-b within the major histocompatibility complex (MHC) raised the possibility that genetic alterations in the loci for these cytokines may be involved directly in a high level of production of TNF-a. A restriction fragment length polymorphism (RFLP) present in the TNF-b gene is correlated with increased concentrations of TNF-a in plasma and a high mortality in patients with severe sepsis [15]. The mortality in severe sepsis was found to be statistically significantly greater in patients homozygous for the TNFB2 allele than in those heterozygous for the Nco1 poly-morphism. A statistically significant association was found for the genotype of biallelic Nco1 polymorphism of the TNF-b gene and the development of severe sepsis after major blunt trauma [15]. For this reason, it must also be considered possible that a genetically determined inflammatory response contributes to sepsis or other post-operative complications in elective surgeries. In view of this, it would be important to find a genetic marker that identifies surgical patients with an increased susceptibility to post-operative sepsis. Because of a strong association of TNF-b Nco1 polymorphism and the risk of severe sepsis after major trauma [15], we hypothesized that this polymorphism might have a role in the development of sepsis following major surgery. We therefore conducted a study to determine the relationship of the NcoI polymorphism of the gene for TNF-b and the occurrence of sepsis after major surgery.
Materials and Methods Patient characteristics and study protocol
Ethical approval for the study was obtained from the institutional ethics committee, King George’s Medical University (KGMU), Lucknow, India. Two hundred eleven
Genotype analysis of the Nco1 restriction fragment length polymorphism within the locus of the gene for tumor necrosis factor-b
The genotype of the TNF b gene according to Nco1 RFLP was determined through amplification with polymerase chain reaction (PCR) and enzymatic digestion of the products with Nco1 (Fermentas, ThermoFisher Scientific, Waltham, MA). For this, 3 mL of venous blood were drawn into ethylenediamine-tetra-acetic acid (EDTA)-containing Vacutainer tubes (Becton–Dickinson, Franklin Lakes, NJ), and each patient’s genomic DNA was extracted from EDTA-anti-coagulated whole blood with a commercially available deoxyribonucleic acid (DNA) isolation kit (QIAmp DNA Blood Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A 782-bp PCR product of the TNF-b gene was amplified through PCR withthe following pair of primers: forward: 5¢CCGTGCTTCGTGCTTTGGACTA-3¢ and reverse: 5¢AGAGGGGTGGATGCTTGGGTTC-3¢ [17]. The amplification reaction was done with 10–100 ng of genomic DNA, 0.2 micromol of primers, 200 micromol of deoxynucleotide triphosphates (dNTPs), 1.5 mmol of MgCl2, and 1 unit of Taq polymerase in a total volume of 20 mcL. The PCR conditions consisted of initial denaturation at 95C for 5 min followed by 35 cycles of denaturation at 94C for 1 min, annealing at 58C for 1 min, and extension at 72C for 1.5 minutes. This was followed by final extension at 72C for 10 min. A total of 10 mcL of the resulting PCR product was digested with NcoI for 10 min at 37C, and the resulting fragments were analyzed on 1% agarose gel (Fig. 1).
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Table 1. Characteristics of Patients with and without Post-Operative Sepsisa Patients with post-operative sepsis (n = 46) Age (y) Gender (female/male) Duration of surgery (h) Blood loss (mL) Hb level (g/dL) Pre-operative Post-operative WBC/mcL Pre-operative Post-operative TNF-a (pg/mL) Pre-operative Post-operative day 2 Post-operative day 4 Mortality
Patients without post-operative sepsis (n = 165)
36.24 – 11.94 26/20 2.67 – 0.58 125.21 – 78.87 11.11 – 10.83 –
35.90 – 11.05 102/63 2.16 – 1.65 42.56 – 55.92 11.27 – 11.17 –
1.48 1.68
p value* 0.86 0.51 0.04* < 0.001*
1.79 0.99
0.58 0.08
7,253.04 – 1,836.65 10,015.87 – 6,417.77
7,150.42 – 1,676.03 7,194.16 – 1,444.72
0.72 < 0.001*
52.57 – 40.16 98.59 – 59.37 180.24 – 91.28 8 (17.39)
51.87 – 32.80 55.47 – 22.37 56.35 – 20.45 1 (0.60)
0.90 < 0.001* < 0.001* < 0.001*
a Values are given as numbers (%) or mean – SD. Differences were tested with the w2 test and the Fisher exact test for frequencies and with the Student t-test for mean – SD. *A value of p < 0.05 was considered statistically significant. TNF-a = tumor necrosis factor-a.
Assay of tumor necrosis factor-a
Results
Serum was collected pre- and post-operatively from each of the patients in the study on post-operative days (POD) 2 and 4. Commercially available, highly sensitive enzymelinked immunosolvent assay (ELISA) kits for human TNF-a (Orgenium AviBion, Vantaa, Finland) were used to measure serum concentrations of TNF-a according to the manufacturer’s instructions.
Of the 211 patients enrolled in the study, 128 (60.66%) were female and 83 (39.33%) were male. The mean age of the patients was 35.97 – 11.22 y. Post-operative sepsis was diagnosed in 46 (21.80%) of the patients. The patients with and without post-operative sepsis were comparable in terms of parameters including age, gender, duration of surgery, blood loss, and mortality (Table 1). The duration of surgery (p = 0.04) and intraoperative blood loss (p < 0.001) were observed to be statistically significantly associated with the development of post-operative sepsis. Significantly increased white blood cell counts (WBC) (p < 0.001) and mortality (p < 0.001) were observed in patients with post-operative sepsis as compared with those without sepsis. The most common post-operative complication found in the study was surgical site infection (n = 24; 11.37%), followed by the other complications listed in Table 3. The site of each type of surgery included in the study and the relevant incidences of post-operative sepsis and mortality are shown in Table 4. Sepsis was diagnosed in three patients 2 d after surgery, in
Statistical analysis
Data were expressed as proportion or mean – standard deviation (SD), as appropriate. Testing for intergroup differences in proportions was done with the w2 test or Fisher exact test. Odds ratios (OR) for the estimation of relative risk were calculated with 95% confidence intervals (CI). For intergroup comparisons, unpaired or paired Student t-test and one-way analysis of variance (ANOVA) was used. A value of p < 0.05 was considered as statistically significant.
FIG. 1. Genotypic analysis of the tumor necrosis factor (TNF)-b gene. The polymerase chain reaction product of 782 bp was digested with the endonuclease Nco1. Lane 1: 100 bp ladder; lanes 2–4: heterozygous genotype (TNFB1/B2); lanes 5 and 7: TNFB2 homozygous genotype (TNFB2/B2); lane 6: TNFB1 homozygous genotype (TNFB1/B1).
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Table 2. Genotypic Distribution and Characteristics of Patientsa Genotype Age (y) Gender (female/male) Blood loss (mL) Length of surgery (h) Hb level (g/dL) Pre-operative Post-operative WBC/mcL Pre-operative Post-operative Mortality Sepsis
TNFB1/B1 (n = 25) 34.96 – 75.62 – 2.18 –
12.66 16/9 80.07 0.65
11.17 – 10.96 –
1.97 0.96
TNFB1/B2 (n = 87) 35.89 –
7,084 – 1,232.09 7,364 – 2,907.15 0 5 (20%)
48.13 – 2.15 –
10.96 53/34 62.80 0.61
11.33 – 11.11 –
1.63 1.04
6,942.87 – 1,692.31 7,793.50 – 3,024.86 2 ( 2.3%) 11 (12.64%)
TNFB2/B2 (n = 99) 36.30 –
p value
68.36 – 2.41 –
11.17 59/40 73.26 2.09
0.86 0.91 0.08 0.49
11.17 – 10.97 –
1.76 0.91
0.80 0.57
7,397.27 – 1,807.99 8,011.05 – 4,021.90 7 ( 7.07%) 30 (30.30%)
0.18 0.70 0.13 0.01*
a Values are given as frequency (%) or mean – SD. Differences were tested with the w2 test for frequencies and ANOVA for mean – SD. *A value of p < 0.05 was considered statistically significant. TNF = tumor necrosis factor.
five patients at 4 d after surgery, in two patients at 5 d after surgery, and in the remaining 36 patients at 8 d or more after surgery. The relevant microbiologic findings on blood culture for each case of post-operative sepsis are shown in Table 5. In all surgical site infections, the organism observed was Staphylococcus aureus. Allele frequency and genotype distribution
With regard to TNF-b genotype, 11.84% (n = 25) patients were homozygous recessive for TNFB1, 41.23% (n = 87) were heterozygous, with TNFB1/TNFB2, and 46.91% (n = 99) were homozygous dominant for TNFB2. The distribution of the TNF-b genotype subgroups among the patients was compared in terms of parameters including age, gender, duration of surgery, blood loss, and mortality (Table 2). Post-operative sepsis was observed in 5/25 (20%), 11/87 (12.64%), and 30/99 (30.30%) patients of the TNFB1 homozygous, TNFB1/B2 heterozygous, and TNFB2 homozygous genotypes, respectively. The overall allele frequency was 0.32 for TNFB1 and 0.68 for TNFB2. The allele frequencies of TNFB1 and TNFB2 were 0.22 and 0.78 in patients with post-operative sepsis and 0.35 and 0.65 in patients without post-operative sepsis.
Table 3. Post-Operative Complications in Study Patients Complication Infections Surgical site infection Multiple organ failure Anastomotic leak Pancreatic leak Intra-abdominal abscess Pneumonia Non-infections Cardio-respiratory arrest a
Values are given as number (%).
No. of patients
Mortality (no. of patients)
24 3 8 5 3 3
1 2 3 1 1 0
1
1
The genotype distribution in patients with an uncomplicated clinical course was statistically significantly (p < 0.01) different from that in patients with post-operative sepsis. The development of post-operative sepsis was significantly (p = 0.01) more frequent in patients homozygous for the TNFB2 allele. However, the likely source of significance is the TNFB2 homozygous versus the TNFB1/B2 heterozygous genotype, which was derived by logistic regression analysis (table not shown). When compared with patients carrying at least one TNFB1 allele (TNFB1 homozygous and heterozygous genotype), the TNFB2 homozygous genotype was associated with an OR of 2.60 (p = 0.005; 95% CI 1.32–5.15) for the development of sepsis. Compared with that for the heterozygous genotype, the OR for the homozygous TNFB2 genotype was 3.00 (p = 0.003; 95% CI 1.39–6.44). When the TNFB1 and TNFB2 homozygous genotypes were assessed, no significant (p = 0.35; 95% CI 0.18–1.86) association of either was found with the development of post-operative sepsis. Regarding overall mortality, 4.1% (9/211) patients expired during their post-operative hospital stay (eight of the nine patients had post-operative sepsis and one had no evidence of sepsis). In none of the nine patients who expired was a statistically significant difference (p = 0.13) observed among the TNF-b genotype subgroups. Similarly, in terms of the deaths of eight of the nine patients with postoperative sepsis, no statistically significant difference (p = 0.05; 95% CI
Table 4. Post-Operative Sepsis and Mortality in Relation to Site of Surgerya
Site of surgery
Post-operative sepsis (n = 46)
Esophageal (n = 14) 1 ( 7.14%) Gastrointestinal (n = 52) 8 (15.38%) Hepatobiliary (n = 101) 13 (12.87%) Pancreatic (n = 27) 13 (48.15%) Other (n = 17) 3 (17.64%)
Mortality following post-operative sepsis (n = 8) 1 3 2 1 1
(7.14%) (5.77%) (1.98%) (3.70%) (5.88%)
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Table 5. Blood Culture Profile of Patients with Post-Operative Sepsis (n = 46) Isolates
No. of patients (n = 46)
Sterile Enterobacter Micrococcus Acetinobacter Staphylococcus aureus Escherichia coli
26 3 5 3 4 5
0.02,1.27) was observed between the TNFB2 homozygous and TNFBI/B2 heterozygous subgroups. Serum concentrations of tumor necrosis factor-a
No significant difference (p = 0.90) in serum TNF-a concentration was observed in the pre-operative samples of patients who did and did not develop post-operative sepsis, whereas on both POD 2 and 4, serum concentrations of TNF-a were significantly higher (p < 0.001) in patients who developed sepsis following surgery (Table 1). Tumor necrosis factor-a production in relation to different TNF-b genotypes
To study whether TNF-a production was influenced by the TNF-b genotype, we grouped the patients with and without post-operative sepsis according to their TNF-b genotypes and compared the TNF-a production in the groups. A significant increase (p = 0.02) was observed in TNF-a production in the TNFB2 homozygous patients with sepsis as compared with that in patients in the other subgroups of TNF-b genotype, whereas no such increment was observed in patients without sepsis (Figs. 2 and 3). Discussion
Tumor necrosis factor-a is believed to be a pivotal proinflammatory mediator in the host defense against infection [18,19]. However, only a limited number of studies have been done of the relationship of cytokine production to genetic
susceptibility to sepsis [12,20]. Several polymorphic elements in the region of the TNF gene have been related to TNF production, but the precise location of the genetic elements controlling the TNF response remains unclear, and their expression has been shown to be regulated only in part by transcriptional events [21–23]. Two single nucleotide polymorphisms (SNP) in the human TNF-a/TNF-b gene that might influence TNF-a secretion have been described as being associated with clinically severe forms of infectious diseases [15,23–25]: the Nco1 polymorphism of the TNF-b gene and a G/A polymorphism at position - 308 in the promoter region of the TNF-a gene. Reports of an association between polymorphism of the TNF-b gene and increased susceptibility to severe sepsis [15,26,27] encouraged us to examine the distribution of the TNF-b genotype in patients undergoing elective abdominal operations and to determine their post-operative status in terms of the development of sepsis. In the view of the contradictory findings in previous studies [17,26,28], we studied the functionality of biallelic TNF-b Nco1 polymorphism in relation to susceptibility to post-operative sepsis. We found the allele frequencies and genotype distributions of TNF-b Nco1 polymorphisms to be similar to those previously described in a German population [26,28,29]. In our study, the TNFB2/B2 genotype was present in 46.91% of the population, in which the observed incidence of septic complications was 30%, and was associated with an OR of 2.60 for the development of post-operative sepsis as compared with the other genotypes of TNF-b. At the same time, the low prevalence (11.84%) of the TNFB1 homozygous genotype makes its estimation less accurate as a factor in reducing the risk of post-operative sepsis. Although the TNFB2/B2 homozygous genotype was commonly observed in the patients in our study who did not survive after surgery, the association of this genotype with mortality after surgery was not statistically significant (p = 0.05). Previous studies of the relationship to sepsis of the TNFB2/B2 genotype among the Nco1 polymorphisms of the TNF-b gene demonstrated a poor prognosis and an increased mortality for this genotype as compared with the other genotypes of TNF-b [15,26,28,30]. The possibility that variation in the immune response associated with different TNF-b genotypes may be
FIG. 2. Comparison in the pre- and post-operative concentrations of tumor necrosis factor (TNF)-a in patients with postoperative sepsis and in TNF-b genotype subgroups. Statistical analysis using one-way analysis of variance showed a significant difference (p = 0.02) in the serum concentration of TNF-a on post-operative day 4 in different TNF-b genotype subgroups, whereas no such significant difference was observed pre-operatively or on post-operative day 2 (p = 0.93 and p = 0.31, respectively).
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FIG. 3. Comparison in the pre- and post-operative concentrations of tumor necrosis factor (TNF)-a in patients without post-operative sepsis and in genotype subgroups. Statistical analysis using one-way analysis of variance showed an insignificant difference in the pre- and post-operative concentrations of TNF-a in the different genotype subgroups (preoperatively: p = 0.63; post-operative day 2: p = 0.34; post-operative day: p = 0.58. responsible for the development of sepsis cannot be ruled out. Given that a strict protocol for asepsis was used for all of the patients in our study for the entire duration of the study, we believe that the results of the study do not reflect a breach in anti-infective measures, but instead reflect genetic differences in the immune response to septic insult itself. Because most of the patients in our study who developed septic complications post-operatively were of the TNFB2/B2 genotype, we doubted that this genotype influenced the postoperative production of TNF-a by leukocytes in the blood. To assess this, we analyzed serum concentrations of TNF-a preand post-operatively in our patients who did and did not develop post-operative sepsis in relation to their TNF-b genotypes. In accord with the available literature on patients with severe sepsis [15,26], patients in our study who had a TNFB2/B2 homozygous genotype had a greater serum TNFa response post-operatively than that of the patients with other TNF-b gentoypes. Although an increased cytokine response may be related to surgical stress, Majetschak et al. [31] reported that the stress associated with surgery is transient and does not contribute significantly to an increased cytokine response for more than 24 h after surgery. In contrast to a previous report [32], we found that the degree of TNF-a production in whole blood in our patients who did not have complications was unrelated to the polymorphism of the TNF-b gene. Although the source of the measured serum concentration of TNF-a in patients with post-operative sepsis is unknown, the difference found in serum TNF-a concentrations in the TNF-b genotypic subgroups in our study may be associated with the distinct capacity of monocytes to produce TNF-a as an inflammatory cytokine that affects susceptibility to sepsis, and this genotype-related difference has been shown to be associated with the Nco1 polymorphism. The preoperative identification of patients at high-risk for developing post-operative sepsis may be useful in clinical practice for preventing post-operative sepsis. Our study suggests that the early identification of patients at high risk of developing severe post-operative sepsis can be achieved with the use of easily collectable markers. These markers represent an inexpensive way in which to identify the high-risk patients in whom the effects of early, aggressive, goaldirected antiseptic treatment can be evaluated in future studies.
It is well known that various factors related to the nature of an operation or to the patient will influence the occurrence of major post-operative complications. The same team operated the patients in the current study and as the first case of the day in the operating room. This aimed to maintain uniformity in the study. We found that the amount of intra-operative blood loss and duration of surgery statistically significantly influenced the development of postoperative sepsis (Table 1), a finding also reported by other investigators [32–35], but we found no association of this with TNF-b genotypes. Other patient-related factors, such as age and gender, had no influence on the development of post-operative sepsis. Although various studies indicate that pre-existing patient-related factors (e.g., advanced age, male gender) may influence the development of postoperative complications [33,34,36–40], the findings in this regard are inconsistent. Ansari et al. [41] reported that age influences the development of post-operative complications, whereas Tang et al. [33] could not identify age as a risk factor for the same type of surgery as in the study by Ansari et al. Some studies found male gender to be an independent risk factor for the development of post-operative complications such as sepsis [38,34], whereas others could not confirm these findings [39,40]. Vogel et al. [42] described the rates of sepsis for a variety of hospital-based surgical procedures. Gastrointestinal, cardiovascular, and thoracic procedures accounted for nearly 50% of cases, and the rates of post-operative sepsis were highest for esophageal, pancreatic, gastric, small bowel, hepatic, and biliary procedures. Hensler et al. [43] reported a greater frequency of post-operative sepsis after esophagectomy. In our study, pancreatic operations, among all major elective gastrointestinal procedures, accounted for most (30.43%) of the instances of post-operative sepsis. The major finding in the present study was that the biallelic Nco1 polymorphism of the TNF-b gene is associated significantly with the development of sepsis after major surgery. However, it cannot be ruled out that the Nco1 polymorphism is not itself related directly to susceptibility to sepsis, but rather serves as a marker linked to the major histocompatibility complex (MHC) because of its location in the class III region of the MHC [21,23].
TNF-b NCO1 POLYMORPHISM AND SEPSIS
A limitation of the present study is that the influence of TNF-b genotypes on the development of post-operative sepsis according to type of surgical procedure could not be established because of the small number of patients undergoing each type of surgery. Similarly, the small number of TNFB1 homozygous patients in the study makes the estimation of this genotype less accurate as a risk-reducing factor for sepsis, demanding a larger cohort for evidence of this, although the possibility of a high risk of the TNFB2 homozygous genotype for sepsis as compared with the risk of TNFB2 heterozygotes should not be dismissed. Our data provide evidence of a genetic susceptibility to sepsis in surgical patients. In our study, the development of sepsis was associated with a greater capacity to produce TNF-a after surgery. The Nco1 polymorphism of the TNF gene was associated with the development of post-operative sepsis through an increased serum concentration of TNF. Analysis of this polymorphism appears to be a useful means for identifying patients at high risk for sepsis after surgical trauma. Further studies of molecular pathways with larger cohorts are needed to investigate the mechanism of increased susceptibility to sepsis in relation to the TNF Nco1 polymorphism. Acknowledgments
We thank the Council of Science and Technology of the Government of Uttar Pradesh, Lucknow, India for financial support. Author Disclosure Statement
The authors declare no conflicts of interest in the work described in this paper. References
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Address correspondence to: Dr. Rajeshwar Nath Srivastava Department of Orthopaedic Surgery King George’s Medical University Lucknow - 226003, U.P. India E-mail: [email protected]
