Natural Inhibitor; Antithrombin and Protein S Levels among Neonates Suffering from Sepsis

Albara Ahmed¹, Babiker Mohammed ², Abdelhalim Nasr ³, Awadelkarim Abbas ^4, Mosab Nouraldein Mohammed Hamad^*

¹Assistant professor. Alfajr College for sciences and technology, Medical laboratory science program, department of hematology

² Professor. Karary University, faculty of medicine, department of pathology ⁽²⁾, Omdurman, Sudan

³ Associate professor. University of Bahri, faculty of medicine, department of pediatrics, Khartoum north, Sudan

⁴ Assistant professor. University of Khartoum, faculty of medical laboratory science, department of hematology.

*Corresponding Author: Mosab Nouraldein Mohammed Hamad, Head of Phylum of Medical Parasitology, Medical Laboratory Sciences Department, Faculty of Health Science, Elsheikh Abdallah Elbadri University, Berber, Sudan.

Received Date:  December 22, 2020

Publication Date:  January 01, 2021

Abstract
Neonatal sepsis is a fatal disease with significant neonatal morbidities and mortalities worldwide, despite preventive measures and efforts that target neonatal sepsis, morbidity and mortality statistics were not promising. Common clinical symptoms of sepsis make earlier diagnosis challenging.

Objectives: the study aimed to assess antithrombin (AT) and Protein S (PS) among Sudanese neonates with sepsis and compare them with healthy neonates to study the susceptible alteration in both natural inhibitors of hemostasis (AT and PS) considering different variables (gender, mode of delivery, gestational age, sepsis mode, outcome and causative bacterial agent).

Methods: a prospective case-control study achieved in the maternity hospital, Omdurman, Sudan in the period between Jun.2013 and Apr.2015 on a total of 100 samples divided into the case (neonates diagnosed with proven sepsis) and a control group of health neonates (50 for each) selected by non-probability sampling, Protein S was assessed by the clotting procedure using semi-automated coagulometer (Stago stat four), AT was assessed spectrophotometrically by the turbimetric method using semi-automated chemistry analyzer (Mindray BA-88A).

Results: the gender distribution was 23, 27 and 24, 26 males and females for case and control respectively, among case group; 17 neonates with early mode sepsis (0-7 days), and 33 with late onset (7-28 days). Considering the outcome in case group; 40 were recovered (80%) and 10 neonates dead (20%) of them; 4 (40%) with early onset sepsis, and 6 (60%) with late onset. Blood culture distributions were; Pseudomonas 23 (46%), Salmonella 9 (18), Klebsiella 7 (14%), Staph. epidermidis 3 (6%), Strep. fecailis 3 (6%), E. coli 2 (4%), Staph. aureus 2 (4%), and 1 Streptococci (Non group B) (2%).

AT was significantly decreased in the case compared to the control group (mean; 183.9 and 221.5 Mg/ml) P. value 0.003. PS was insignificantly decreased (33.4 and 34.7%) P. value 0.76.

Among the case group; None of the gender, mode of delivery, Mode of sepsis, etiologic agent and sepsis outcome showed significant correlation with AT or PS.

Conclusion: it has been concluded that antithrombin was significantly decreased in septic neonates than healthy ones (P. value 0.003). It can be used as a diagnostic marker to offer quick reliable useful test feedback for septic neonates.

Keywords:  Neonatal sepsis, natural inhibitor, Antithrombin, Protein S, Sudan.

Background: 

Neonatal Sepsis is a life-threatening response to infection which causes tissue damage, organ failure, and death in the first twenty-eight days of life. It remains one of the significant morbidity and mortality causes among developing countries (1) accounts for the third common cause of neonatal deaths of annual deaths of 225 000 (2). Worldwide highest rates of neonatal deaths in Sub-Saharan Africa (2, 3, 4, 5), although, it is declining of specific mortality rates annually (3). Neonatal sepsis deaths account 1.6 times deaths with malaria and four times HIV deaths (6). The pathophysiological mechanism of sepsis is not completely understood, but coagulation alterations are the general feature of the sepsis (7). Both inflammation and hemostasis are strongly linked pathophysiologically and significantly affect each other. Coagulation system activation caused by the inflammation that in turn also considerably stimulate an inflammatory response, strong tight links between two systems significantly participate in disease pathogenesis and/or progression (8). Activation of coagulation along with inhibition of fibrinolysis and increased possibility of organ dysfunction characterizes the sepsis and sepsis induced-DIC (9-11) a very serious condition results from coagulation activation followed by a continuation of coagulation which ends with consumption of coagulation elements later (consumption coagulopathy) (12, 13), the later alterations occur in the delayed stages of sepsis-induced DIC (12, 14-16). Antithrombin; a natural inhibitor plasma protein produce mainly in the liver, its main activity is to inactivate the thrombin which represents a sepsis important mediator (17, 18), AT also serves as a local anti-inflammatory role in sepsis (19) Protein C pathways (PC and PS) also serves several biologic functions like; anti-inflammatory function (20, 21). When activation of the hemostatic system changes from helpful to harmful represents an adequate moment for coagulation-targeted treatment or intervention where the immunological role of hemostasis is preserved and getting safe from the harmful effect of excessive activation of coagulation (22). Analyzing blood coagulation found to be of more clinical significance than other classical tests (23). Sepsis Diagnosis among critically ill septic patients is challenging, it can be complicated by inflammation (24).

The objectives of the study were to assess the natural inhibitor; antithrombin (AT) and protein S (PS) among Sudanese neonates with sepsis compared to healthy neonates, and to correlate gender, mode of delivery with that parameter among both Groups, and to correlate sepsis outcome, the onset of sepsis, and the etiologic agent of sepsis among case group.

Method:   

A prospective cross-sectional hospital-based study was conducted in the maternity hospital, Omdurman. Sudan between June.2013 to April 2015. on a total of 100 Sudanese neonates divided equally into; cases (neonates with proven sepsis by blood culture), and controls (healthy neonates). 

Blood culture for identification of microorganism was done, then positive culture included as a case sample. Venous neonatal blood collected and plasma prepared for AT and PS assessment. Blood culture, Gram stain, culture and sensitivity as well as biochemical tests for identification of microorganism was done for suspected neonates initially, then positive cultures were included as case samples. Platelet poor plasma collected was used for coagulation studies. 

AT Procedure

1/10 diluted plasma was prepared (90 microliters of 0.15 M Saline + 10 microliter of plasma), Sufficient time was allowed for frozen plasma to thaw before use, Antithrombin reagent (working reagent) was prepared by transfer reagent two content (glycine buffer) into reagent one (contains micro latex particles coated with rabbit anti-human AT antibodies), the materials were allowed to stand at room temperature for 15 minutes. Then 50 microliter of 1/10 diluted plasma was obtained, and 250 microliters of antithrombin reagent were added, mixed and incubated for exactly 20 minutes, the absorbance then measured at 590nm using semi-automated pre-calibrated chemistry analyzer (Mindray BA-88A), calibrator as a test control (Uricalibrator, Stago) used, as well as all test reagents used were supplied by Stago. France). The result was interpreted in Mg/ml.

PS Procedure

1/10 diluted plasma was prepared (180 microliters of Owren-Koller buffer + 20 microliter of plasma) and mixed. Sufficient time was allowed for frozen plasma to thaw before use. Lyophilized reagents were left for about 15 minutes at room temperature before use. Then 50 microliter of 1/10 diluted pre-warmed plasma was added, a piece of the metal ball (Stago) was obtained, and reagent was incubated to reach 37+/- 0.2 C, then 50 microliters of protein S deficient plasma, factor V, then 50 microliters of PS activator were added, incubated for exactly 120 seconds, then 0.02 M calcium chloride was added, and the clotting time was counted in seconds using semi-automated pre-calibrated coagulometer (Stago. France), calibrator as a test control (Uricalibrator, Stago) used, as well as all test reagents used were supplied by Stago. France). The result was interpreted in percentage.

Ethical clearance:

Ethical clearance was obtained from the research ethical committee of Omdurman maternity hospital. The principal investigator obtained an informed consent form from the neonates’ mothers who were included in the study before going on. 

Data analysis

Data was entered and tabulated in an EXCEL sheet, then to SPSS (a statistical program for social and sciences). Data were analyzed by use of SPSS (IBM SPSS Statistics 20).

Results and discussion:

A total of 100 venous neonatal blood samples (50 for both groups) were included. Gender distributions were; 26 females (52%), and 24 males (48%), 27 females (54%), and 23 (46%) for case and control group respectively. Table (1) 

Table (1): Distribution of study population according to their gender

Table (2): Distribution of study population according to their gestational age

Groups were categorized according to delivery mode; cesarean section and normal vaginal delivery neonates. 17 were delivered normally (34%), and 33 were delivered by cesarean sections 66%), 7 were delivered normally (14%), and 43 were delivered by cesarean sections (86%) for case and control. (Table 3)

Table (3): Distribution of study population regarding their mode of delivery

Mortality among sepsis onset distributed in case group into; early onset 4 (40%), late onset 6 (60%). The causative bacterial agents were distributed as; Pseudomonas 23 (46%), Salmonella 9 (18%), Klebsiella 7 (14%), 3 (6%) Staph.epidermidis 3 (6%) Strep. fecailis 2 (4%), E.coli 2 (4%), Staph.aureus 2 (4%), and 1 (2%) Streptococci (Non group B). Mortality caused by Pseudomonas 3 (30%), Salmonella 3 (30%), S.Epidermidis 2 (20%), Klebsiella 1 (10%), and E.Coli 1 (10%). Table (4).

Table (4): Frequency of bacterial isolates (n=50) considering its etiologic agent

AT mean showed a significant decrease in the case group compared to the control (183.9 and 221.5 Mg/ml) (P value 0.00). Table (5).

PS mean showed an insignificant decrease in the case compared to the control (33.4% and 34.7%) (P value 0.76). Table (5).

Table (5): Antithrombin and protein S mean and P. value among both groups

Mode of delivery didn’t show a significant decrease with AT or PS (AT mean; 176.8 and 189.1 Mg/ml and for normal vaginal delivery and caesarian section and PS; 32.5 and 34.1%). (P.value; 0.54, and 0.79). Among case sepsis, the outcome showed a significant decrease in PC between dead and recovered neonates (mean; 25.4 and 36.2% for dead and recovered). (P.value 0.04).

Fibrinogen, PS, or AT didn’t show a significant correlation with PC among the same group (mean; 517.3, 473.4 mg/dl in fibrinogen for dead and recovered septic neonates, 30.4 and 34.2% PS, 176.9 and 185.7 Mg/ml in AT). (P.value; 0.49, 0.61 and 0.72 for fibrinogen, PS and AT respectively).

Among case group, sepsis onset didn’t show significant decrease in fibrinogen, PC, PS or AT (mean; 491.2 and 477.5 mg/dl in fibrinogen for early and late onset sepsis respectively, 32.2 and 34.9% in PC, 28.7 and 35.9% PS, 173.7 and 189.2 Mg/ml in AT). (P value; 0.80, 0.56, 0.25 and 0.46).

The etiologic agents of sepsis in the case group didn’t show significant difference with fibrinogen, PC, PS or AT (mean was; 475.6 and 512.0 mg/dl in fibrinogen for Gram-negative and Gram-positive respectively, 35.5 and 27.4% in PC, 34.3 and 29.3% PS, 187.9 and 165.7 Mg/ml in AT). (P-value; 0.59, 0.16, 0.52 and 0.39).

Discussion:

Fibrinogen showed a significant increase in the case compared to the control (P.value 0.00), the result was in line with Krishna I et al (25), Charan P A et al (26), and Mondal et al (27) who concluded increased fibrinogen level in sepsis, fibrinogen is an acute-phase protein it increases in inflammatory conditions like sepsis.

AT showed a significant decrease in the case group compare to the control (P.value 0.00), this result was in line with Warkentin et al (28), Nishida et al (29), Elbeshlawy et al (30), and Hagag et al (31) who concluded reduced AT level in sepsis cases. Significant AT reduction in septic patients was suggested, it can be justified due to the consumption and impaired synthesis of AT in sepsis (28). AT can be useful as a biomarker in the diagnosis of neonates suffering from sepsis.

PC showed a significant decrease in dead septic neonates compared to the recovered septic (P.value 0.04), this result was matched with Griffin et al (32), M De La Torre-Prados et al (33), William L (34), Sundaram, et al (35) who correlated PC (low PC) with poor outcome and mortality in septic patients. Consumption of PC may be clarified the decreased PC level which accompanied with mortality, PC has an anti-inflammatory function, cytoprotective, antithrombotic (20, 21), anti-apoptotic function (21) among other functions i.e. regulating gene expression, these function gives PC to play a crucial role in sepsis progression. PC can be a useful marker in neonatal sepsis mortality.

The insignificant decrease in PC and PS among both groups (P.value; 0.41 and 0.76), the study decreased level of both parameter, the insignificance may due to the physiological decrease of healthy neonates (control) which confirmed in the study (normal PC in an adult population usually between 70% and 130% (33) (according to the used kit's manufacturer), whereas; most of the PC studies conducted on adult, relatively smaller sample size could also be contributed in PC insignificance, along with that a lot of studies reported PC and its cofactor PS reduced significantly in sepsis (28, 30, 34), and several studies confirmed it among septic neonates (30, 35) 

On my view, it is noted that PC correlation is a much stronger correlation with poor outcome and mortality more than the disease itself (sepsis), another meaning that I noted that several studies focused targeted PC in treatment and management of sepsis mortality than using PC as a marker in the diagnosis of sepsis, moreover; recombinant studies and approved as a novel therapy for sepsis (37), then later it withdrawn from the market (38) subsequently recombinant protein C introduced (39). Insufficient data reported to use of recombinant PC for the management of severe sepsis in neonates, and it’s reported that neonates shouldn’t be treated with recombinant PC (40). This discussion can reinforce the PC correlation with sepsis outcome (discussed above).

Conclusion: 

Fibrinogen showed a significant increase in septic neonates compared to healthy. AT showed a significant decrease in septic neonates compared to the healthy. PC showed a significant decrease in dead septic neonates compared to the recovered septic. Both fibrinogen and AT can be a useful biomarker in sepsis diagnosis, PC can be a useful biomarker in neonatal sepsis mortality. 

Conflict of interest:

Nothing to disclose.

References

1.Gabriel Kambale Bunduki, Yaw Adu-Sarkodie. “Clinical outcome and isolated pathogens among neonates with sepsis in Democratic Republic of the Congo: a cross-sectional study”. BMC research Notes. Volume 12: 303 (2019)

2. GBD 2016 Causes of Death Collaborators. “Global, regional, and national age-sex specific mortality for 264 causes of death, 1980-2016: a systematic analysis for the Global Burden of Disease Study 2016”. Lancet 2017; 390:1151-210.

3.Liu L, Oza, S, Hogan D et al. “Global, regional, and national causes of child mortality in 2000-13, with projections to inform post-2015 priorities: an updated systematic analysis”. Lancet 2015; 385: 430-40.

4.Asakura H, Takahashi H, Uchiyama T, Eguchi Y, Okamoto K, Kawasugi K, et al. DIC Subcommittee of the Japanese Society on Thrombosis and Hemostasis. “Thrombosis, hemostasis, proposal for new diagnostic criteria for DIC from the Japanese Society on Thrombosis and Hemostasis”. Thromb J 2016; 14:42.

5.UNICEF. “The state of the world's children 2009: maternal and newborn health”. New York: Unicef, 2008.

6.Seale AC, Blencowe H, Zaidi A, et al. “Neonatal severe bacterial infection impairment estimates in South Asia, sub-Saharan Africa, and Latin America for 2010”. Pediatr Res 2013; 74:73–85.

7.Mihajlovic D, Lendak D, Mitic G, Cebovic T, Draskovic B, Novakov A, Brkic S.  “Prognostic value of hemostasis-related parameters for prediction of organ dysfunction and mortality in sepsis”. Turk J Med Sci. 2015; 45(1):93-8.   

8. Sandra M. “Inflammation and hemostasis”. Biochemia Medica 2012;22 (1):49-62.

9.Okamoto K, Tamura T, Sawatsubashi Y. “Sepsis and disseminated intravascular coagulation”. J Intensive Care 2016; 4:23.

10. Iba T, Ito T, Maruyama I, Jilma B, Brenner T, Muller MC, et al. “Potential diagnostic markers for disseminated intravascular coagulation of sepsis”. Blood Rev 2016; 30:149 155.

11.Madoiwa S. “Recent advances in disseminated intravascular coagulation: endothelial cells and fibrinolysis in sepsis-induced DIC”. J Intensive Care 2015; 3:8.

12. Asakura H, Takahashi H, Uchiyama T, Eguchi Y, Okamoto K, Kawasugi K, et al. “DIC Subcommittee of the Japanese Society on Thrombosis and Hemostasis. Thrombosis, hemostasis, proposal for new diagnostic criteria for DIC from the Japanese Society on Thrombosis and Hemostasis”. Thromb J 2016; 14:42

13. Levi M, van der Poll T. “Disseminated intravascular coagulation: a review for the internist”. Intern Emerg Med 2013; 8:23–32.

14.Levi M. “Another step in improving the diagnosis of disseminated intravascular coagulation in sepsis”. Crit Care 2013; 17:448.

15. Wada H, Matsumoto T, Yamashita Y, Hatada T. “Disseminated intravascular coagulation: testing and diagnosis”. Clin Chim Acta 2014; 436:130–134.

16. Koami H, Sakamoto Y, Sakurai R, Ohta M, Imahase H, Yahata M, et al. “The thromboelastometric discrepancy between septic and trauma induced disseminated intravascular coagulation diagnosed by the scoring system from the Japanese Association for Acute Medicine”. Medicine (Baltimore) 2016; 95:e4514.

17. Levy, J.H.; Sniecinski, R.M.;Welsby, I.J.; Levi, M. “Antithrombin: Anti-inflammatory properties and clinical applications”. Thromb. Haemost. 2016, 115, 712–728.

18. Roemisch, J.; Gray, E.; Ho_mann, J.N.; Wiedermann, C.J. “Antithrombin: A new look at the actions of a serine protease inhibitor”. Blood Coagul. Fibrinolysis 2002, 13, 657–670.

19. Mehta, D.; Ravindran, K.; Kuebler, W.M. “Novel regulators of endothelial barrier function”. Am. J. Physiol. Lung Cell. Mol. Physiol. 2014, 307, L924–L935.

20. Griffin, J.H.; Zlokovic, B.V.;Mosnier, L.O. “Activated protein C: Biased for translation”. Blood 2015, 125, 2898–2907.

21. Choi, Q.; Hong, K.H.; Kim, J.E.; Kim, H.K. “Changes in plasma levels of natural anticoagulants in disseminated intravascular coagulation: High prognostic value of antithrombin and protein C in patients with underlying sepsis or severe infection”. Ann. Lab. Med. 2014, 34, 85–91.

22. Ecaterina Scarlatescu, Dana Tomescu, Sorin Stefan Arama. “Anticoagulant Therapy in Sepsis. The Importance of Timing”. J Crit Care Med (Targu Mures). 2017 Apr; 3(2): 63–69.

23. Jeff Simmons, Jean-Francois Pittet.  “The coagulopathy of acute sepsis”. Curr Opin Anaesthesiol. 2015 Apr; 28(2): 227–236.

24. Jean-Louis Vincent. “The Clinical Challenge of Sepsis Identification and Monitoring’. PLoS Med. 2016 May; 13(5): e1002022.

25. Krishna I, Chandran P, Jacob R. “Current and novel biochemical markers for detection and monitoring of sepsis”. Medlab. 2014;2.

26. Charan Pal A, Mukhopadhyay D, Kundu P. “Serum fibrinogen profile in neonatal septicemia. IOSR journal of dental and medical sciences”. May.- Jun. 2013,7(5): 1- 5.

27. Mondal SK, Roy Nag R, Bandyopadhyay R, Chakraborty D, SK Sinha. “Neonatal sepsis: Role of a battery of immunohematological tests in early diagnosis”. 2012; 2 (1) 43-47.

28. Warkentin, T.E. “Microvascular thrombosis and ischaemic limb losses in critically ill patients”. Hamostaseologie. 2019, 39, 6–19.

29. Nishida, O.; Ogura, H.; Egi, M.; Hayashi, Y.; Iba, T.; Imaizumi, H.; Inoue, S.; Kakihana, Y.; Kotani, J.; Kushimoto, S.; et al. “The Japanese Clinical Practice Guidelines for Management of Sepsis and Septic Shock”. 2016 (J-SSCG 2016). Acute Med. Surg. 2018, 5, 3–89.

30. El Beshlawy A, Alaraby I, Abou Hussein H, Abou-Elew HH, Mohamed Abdel Kader MS. “Study of protein C, protein S, and antithrombin III in newborns with sepsis”.  Pediatr Crit Care Med. 2010; 11(1):52-59.

31.Hagag AA, Elmahdy HS, Ezzat AA. “Prognostic value of plasma pro-adrenomedullin and antithrombin levels in neonatal sepsis”. Indian Pediatr. 2011 Jun; 48(6):471-3.

32. Griffin, J.H.; Zlokovic, B.V.; Mosnier, L.O. “Protein C anticoagulant and cytoprotective pathways”. Int. J.Hematol. 2012, 95, 333–345.

33. M De La Torre-Prados, A García-de la Torre, M Nieto-González, I Lucena-González,R Escobar-ConesaA García-Alcántara, A Enguix-Armada. “Activated protein C, severe sepsis and 28-day mortality”. Critical Care volume 16, Article number: P23 (2012).

34. William L Macias, David R Nelson. “Severe protein C deficiency predicts early death in severe sepsis”. June 2004. Critical Care Medicine 32(5 Suppl):S223-8

35. Sundaram Venkataseshan;Sourabh Dutta;Jasmina Ahluwalia;Anil Narang. “Low Plasma Protein C Values Predict Mortality in Low Birth Weight Neonates with Septicemia”. The Pediatric Infectious Disease Journal. 26(8):684-688, AUGUST 2007.

36. Guglielmone HA., Vides M.A.; “A novel functional assay of protein C in human plasma and it’s with amidolytic and anticoagulant assay”. Thromb. Haemostasis, 67, 46-49, 1992.

37. Bernard, G.R, Vincent, J.L, Laterre, P.F, LaRosa, S.P, Dhainaut, J.F, Lopez-Rodriguez, A, Steingrub, J.S, Garber, G.E, Helterbrand, J.D, Ely, E.W, et al. “Efficacy and safety of recombinant human activated protein C for severe sepsis”. N. Engl. J. Med. 2001, 344, 699–709.

38. Thachil, J.; Toh, C.H.; Levi, M.; Watson, H.G. “The withdrawal of Activated Protein C from the use in patients with severe sepsis and DIC” [Amendment to the BCSH guideline on disseminated intravascular coagulation]. Br. J. Haematol. 2012, 157, 493–494.

39. Kerschen, E.J, Fernandez, J.A, Cooley, B.C, Yang, X.V, Sood, R, Mosnier, L.O, Castellino, F.J, Mackman, N, Gri_n, J.H, Weiler, H. “Endotoxemia and sepsis mortality reduction by non-anticoagulantactivated protein”. C. J. Exp. Med. 2007, 204, 2439–2448.

40. Ranjit I Kylat,  Arne Ohlsson. “Recombinant human activated protein C for severe sepsis in neonates. Cochrane Systematic review- intervention”. 2012. doi.org/10.1002/14651858.CD005385.pub3.

Volume 2 Issue 1 January 2021

©All rights reserved by Mosab Nouraldein Mohammed Hamad