INTRODUCTION

Neonatal sepsis is still a major health problem worldwide, more common in developing countries, especially Indonesia. Neonatal sepsis causes high morbidity and death in infants due to difficulties in diagnosing and case managmint.¹ It cause 2.6 million deaths worldwide every year, 75% of which occur in the first week of life and almost all (99%) occur in middle to lower income countries.² In Indonesia, there is no specific data related to the incidence of neonatal sepsis, but Cipto Mangunkusumo Hospital in Jakarta reported that the incidence of neonatal sepsis is quite high, reaching 13.7% with a mortality rate of 14%.³ Non specific clinical signs and symptoms make problems in the diagnosis of neonatal sepsis. Distinctive symptoms that appear can not be distinguished whether caused by infectious or non-infectious causes, such as hypothermia, respiratory distress, poor feeding, lethargy and other clinical symptoms. Empirical antibiotics are given, if diagnosis is suspected based on clinical and laboratory findings. Delayed management of neonatal sepsis can lead to undesirable outcomes.⁴ Blood culture is  a gold standard, although it requires 48-72 hours to obtain the result.

In 1988, Rodwell developed a hematological scoring system to help clinicians utilize laboratory findings to establish the diagnosis of neonatal sepsis.⁵ This scoring system is based on the immunological processes that occur in neonates as a response to infection.^6–8 The scoring system composed of seven components, those are leucocyte count, immature polymorphonuclear (PMN) count, immature to mature neutrophil ratio (IM ratio), immature to total neutrophil ratio (IT ratio), total PMN count, platelet count and PMN degenerative changes. NLCR is a reliable marker of inflammation.⁹ The immune response to endotoxemia is characterized by an increase in neutrophils and a low number of lymphocytes circulating in the blood.¹⁰ NLCR was previously used as a predictor of the severity of bacteremia and community acquired pneumonia in children; but as a marker of early onset sepsis, the cut-off point of the NLCR is still controversial. The aim of this study is to compare the diagnostic value of RHSS and NLCR in diagnosing neonatal sepsis.

METHODS

Study Protocol

We conducted a cross-sectional, observational analytic study at Neonatology Division in Dr. Moewardi Hospital from October 2018 to October 2019. We used a consecutive sampling method. All neonates fulfilling the inclusion criteria were included in the sequential study according to the time of arrival of the patient until the research sample size was completed. All study subjects underwent history taking of neonatal sepsis risk factors, clinical examination and laboratory tests (hematological profile, IT ratio, peripheral blood smear and blood culture).

Inclusion criteria was all neonates born by spontaneous vaginal delivery or Cesarean section who were suspected for neonatal sepsis with an onset less than 72 hours after delivery and whom parents signed the informed consent for the study. Exclusion criteria was neonates with major congenital anomalies, required major or minor surgical procedures, gestational age less than 34 weeks, birth weight less than 1000 grams and/or born to immunodeficient mothers. Neonatal sepsis was suspected in presence of maternal and infant septic risk factors. Maternal risk factors were preterm delivery, chorioamnionitis, maternal fever (temperature >38.4°C), maternal urinary tract infection and premature rupture of membranes more than 18 hours. Infant risk factors were perinatal asphyxia and low birth weight.

All blood samples were taken by nurses on duty in the first 5-12 hours after neonates were born. Blood samples were put into the EDTA tubes and sent to the clinical pathology laboratory in Dr. Moewardi Hospital. All blood samples were analyzed using automatic hemoanalyses machine Siemens Advia 120 Hematology Analyzer. The examination of peripheral blood smear was done manually according to the standard procedure of clinical pathology laboratory in Dr. Moewardi Hospital.

Rodwell hematological scoring system and Neutrophil to Lymphocyte Count Ratio (NLCR) from all study subject were calculated from hematological profile and IT ratio. Assessment using RHSS described as  white blood cell count (≤ 5000 /μL or ≥ 25000/μL at birth or ≥ 30000/μL at 12-24 hour or ≥ 21000/μL day 2 onward =1), abnormal total polymorphonuclear cells (PMN)] count (1800-5400 =0, No mature PMN seen= 2, Increase/decrease =1), elevated immature to total PMN ratio (IT ratio > 0.12 =1), immature to mature neutrophil (IM) ratio ≥0.3 (=1), elevated immature PMN count(>600 =1), platelet count < 150,000/mm3(=1), and degenerative changes in PMNs (toxic granules or cytoplasmic vacuoles =1).^5,11 NLCR score was calculated as neutrophil (%) divided by lymphocyte (%).

Diagnostic values of Rodwell hematologic scoring system and NLCR for neonatal sepsis were taken by making a 2x2 table to obtain sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR) and negative likelihood ratio (NLR). NLCR was analyzed by receiver-operator characteristic (ROC) curve and measurement of the area under the curve (AUC) to determine the cut-off point. This study was approved by Ethical Committee of Dr. Moewardi Hospital.

Statistical Analysis

The data collected was analyzed and presented in the form of narratives, tables and graphs. The basic characteristics of the study subjects consisted of gender, maternal gestational age, birth weight, APGAR at the 5th minute, clinical manifestations of sepsis and risk factors for sepsis. Statistical data was presented as mean, median, standard deviation, minimum and maximum values and percentages. Data analysis was performed using SPSS 23 for Windows. The characteristic data obtained was analyzed by Chi-square test. The diagnostic value of each method was presented in 2x2 table form. Comparison of diagnostic values ​​was analyzed using the ROC (Receiving-Operating Characteristic) curve by calculating the area under the curve (AUC). The cut-off point value for NLCR was obtained from the ROC (Receiving-Operating Characteristic).

RESULT

Fifty neonates were included in this study. Of the 50 subjects, 23 (46%) subjects were proven to have neonatal sepsis, while 27 (54%) subjects had negative blood culture. Twenty one neonates (42%) were male and others were female. The mean gestational age was not different in the two groups (p = 0.968). There was no statistically significant difference in birth weight between the two groups (p = 0.688) as shown in Table 1.

Table1. Demographic characteristics of neonates

Notes : ^a Chi square test (nominal data),

^bMann whitney test (abnormal distribution numeric data),

^cindependent t test (normal distributin numeric data).

* significance if α =5 %

Table2. Laboratory characteristic

Notes : ^a Chi square test (nominal data),

^bMann whitney test (abnormal distribution numeric data),

^c independent t test (normal distributin numeric data).

* significance if α =5 %

Laboratory characteristics of patients with neonatal sepsis are presented in Table 2. Two groups of different tests were performed with chi square test for nominal data, independent T-test for numerical data with normal data distribution, and Mann Whitney test for numerical data with abnormal data distribution. It shows that group with positive blood cultures have leukocytes, polymorphonuclear values, IM ratios, IT ratios, immature polymorphonuclears, neutrophil counts and lymphocyte counts higher than group with negative blood cultures, with significant p-value (​​p <0.005). The mean platelet values ​​differed in the 2 groups, but the difference was not significant. The mean value of eosinophils and basophils in the positive culture group was lower than the negative culture group.

Rodwell scoring as a diagnostic test of neonatal sepsis in this study, with a cut-off point of 4 showing a sensitivity of 0.739; specificity 1.0; positive predictive value 1.0; and a negative estimated value of 0.818 with the area under the 0.937 curve (Figure 1). NLCR as a diagnostic test of neonatal sepsis in this study, with a cutoff of 2.495 showing a sensitivity of 0.652; specificity 0.704; positive predictive value of 0.652; and a negative value of 0.704 with an area under the 0.721 curve (Figure 2). The area value of AUC on the ROC NLCR chart in this study was 0.721. Compared to the NLCR, Rodwell`s hematological scoring has an AUC area on the ROC chart of 0.937 (Table 3).

DISCUSSION

Neonatal sepsis shows non specific symptoms in each neonate. The clinical signs often overlap with non-infectious causes, so clinician must exclude the possibility of sepsis.¹² Early diagnosis of neonatal sepsis is very important as the course of the disease is progressive, may be very rapid and even can lead to death.¹³ Although blood culture is still the gold standard to establish the diagnosis of sepsis, but there are many drawbacks, i.e. it takes 48-72 hours to obtain the results, antibiotics given before blood culture sampling may reduce the yield,¹⁴ results may also be affected by sampling technique. A repeat blood culture examination can be done to improve the test results, but the clinical condition of the neonate may not wait for the results of the next blood culture examination.  In view of the difficulty of establishing a diagnosis of neonatal sepsis, many studies have been done to look for various biomarkers that can help clinicians to make a diagnosis. 

In this study we evaluated the laboratory profile of neonates with suspicion of early onset sepsis. This study was conducted on 50 neonates with suspicion of early onset sepsis. Twenty three neonates proved to be sepsis by positive blood culture.

We evaluated the RHSS as the diagnostic tool of neonatal sepsis. In our study, we had a sensitivity of 73.9% and a specificity of 100% with the cut-off value of 4. This is in line with a previous study conducted by Pramana in Bali in 2016  and Ahlawat, et al. in India in 2017 .^6,7 However, it differs from research conducted by Mousa in Egypt in 2019 where Rodwell`s scoring was not superior, as compared to other hematological parameters such as RDW ( Red-cell Distribution Width ) and SNAP II ( Score for Neonatal Acute Physiology II ) with AUC of 0.56.¹⁵

Total leukocyte counts ​​and total neutrophil counts have long been used as markers of infection, as is the relationship between neutrophilia and pyogen bacterial infections.¹⁶ Inflammatory reactions caused by bacterial infections can increase the number of leukocytes and neutrophil counts. In response to the inflammatory process of endotoxins, there are some serial changes, such as an increase in T lymphocytes. The mechanism of underlying lymphopenia involves the release and distribution of lymphocytes in the lymphatic system and the acceleration of increased lymphocyte apoptosis. This mechanism explains the use of the lymphocyte neutrophil ratio as a marker of the sepsis.¹⁷

NLCR is a hematological parameter that is calculated from the ratio of neutrophils to lymphocytes in leukocyte differential counts. An increase in the value of NLCR can be caused by an increase in the number of neutrophils or a decrease in the number of lymphocytes, which increase is related to the incidence of inflammation. During the infection, the production of granulocytes by the bone marrow increases along with the production of other blood cells. Increased granulocyte production is shown by increasing the rate of proliferation of hematopetic cells and granulopoetic progenitors which is then followed by the process of differentiation.

In our study, NLCR as a diagnostic test of neonatal sepsis had an AUC of 0.721 at a cut-off of 2.495. NLCR showed sensitivity of 65.2% and specificity of 70.4%. In line with research conducted by Varal & Dogan in 2020 in Turkey, the NLCR can confirm neonatal sepsis with a sensitivity of 68% and a specificity of 82% and AUC of 0.723 at the 1.57 cut-off, while in a study of premature infants with slow onset sepsis, the NLCR gave an AUC of 0.78 at a cut-off point of 1.77.^18,19 In another study conducted by Ghrahani in 2019, the NLCR also had a strong correlation with CRP and  recommended  as an early marker of neonatal sepsis.²⁰

RHSS had a superior diagnostic value as compared to NLCR, with AUC 0.937 and 0.721, respectively in this study. This might have happened because there were more components in RHSS. One of Rodwell`s Scoring parameters assesses the immaturity of circulating neutrophils due to the systemic infection process in neonates, called I/T ratio, that describes ratio of immature to total neutrophil. It is in line with research by Saboohi et. al, where the I/T ratio ≥ 0.2 can be an early predictor of neonatal sepsis. The I/T ratio has a high specificity and NPV so that it can diagnose early onset neonatal sepsis.²⁰  While NLCR only depends on neutrophil and lymphocyte values ​​that may increase due to inflammatory processes, it not specific for infectious causes. In this study a significant difference was seen in the immaturity assessment component of the Rodwell scoring between the two groups (p <0.001).

Limitations of The Study

A limitation of our study was that many blood culture results were negative, inconsistent with clinical signs and symptoms in study subjects. More studies with large sample size should be conducted.

CONCLUSION

In summary, we conclude that Rodwell Hematological Scoring System was superior to NLCR in diagnosing early onset neonatal sepsis with 0.937 AUC, sensitivity 73.9%, specificity 100%, while NLCR has 0.721 AUC with cut-off point 2.495, sensitivity 65.2%, and specificity of 70.4%.

CONFLICT OF INTEREST

No specific grant from any funding agency in the public, commercial, or not-for-profit sectors was received by authors.

REFERENCES

1. Melvan JN, Bagby GJ, Weish DA, Nelson S, Zhang P. Neonatal sepsis and neutrophil insufficiencies. Int Rev Immunol. 2010;29(3):315–48.
2. Zea-Vera A, Ochoa TJ. Challenges in the diagnosis and management of neonatal sepsis. J Trop Pediatr. 2015;61(1):1–13.
3. Aminullah A. Sepsis pada bayi baru lahir. In: Kosim MS, Yunanto A, Dewi R, Sarosa GI, Usman A, editors. Neonatology Textbook. 1st ed. Jakarta: Badan Penerbit Ikatan Dokter Anak Indonesia; 2014. p. 170–87.
4. Shah BA, Padbury JF. Neonatal sepsis an old problem with new insights. Virulence. 2014;5(1):163–71.
5. Rodwell R, Leslie A, Tudehope D. Early diagnosis of neonatal sepsis using a haematological scoring system. J Pediatr. 1988;112(5):761–7.
6. Pramana KP, Kardana IM, Nilawati GAP. Diagnosis Accuracy of Hematological Scoring System in Early Identification of Neonatal Sepsis. Bali Med J [Internet]. 2016;5(3):139. Available from: http://balimedicaljournal.org/index.php/bmj/article/view/310
7. Ahlawat S, Chand N, Mehta S, Bhardwaj A., Yadav V, Jain R. Assesment of haematological scoring system in early diagnosis of neonatal sepsis in term and preterm neonates. ejpmr. 2017;4(3):387–90.
8. Medarametla V, Chakkirala N. Evaluation of Cord Blood - Haematological Scoring System as an Early Predictive Screening Method for the Detection of Early Onset Neonatal Sepsis. 2015;9(June 2014):2014–6.
9. Yuniaty T. Luaran perkembangan neonatus dengan sepsis neonatorum. In: Update on management of neonatal infection. 2017. p. 157–71.
10. Katleya F, Dadiyanto DW, Katleya F, Dadiyanto DW. Rasio Jumlah Neutrofil-Limfosit pada Awal Masuk Rawat sebagai Faktor Risiko Luaran Pneumonia Anak. 2015;17(1):47–51.
11. Makkar M, Pathak R, Garg S, Gupta C, Mahajan N. Performance evaluation of hematologic scoring system in early diagnosis of neonatal sepsis. J Clin Neonatol [Internet]. 2013;2(1):25. Available from: jcnonweb.com
12. Wynn JL. Defining neonatal sepsis. Curr Opin Pediatr. 2016;28(2):135–40.
13. Majumdar A, Bhattacharyya S, Biswas S, Jana A, Jana A. Hematologic scoring system (HSS): A guide to decide judicious use of antibiotics in neonatal septicemia in developing countries. J Appl Hematol [Internet]. 2013;4(3):110. Available from: http://www.jahjournal.org/text.asp?2013/4/3/110/123310
14. Delanghe JR, Speeckaert MM. Translational research and biomarkers in neonatal sepsis. Clin Chim Acta [Internet]. 2015;451(Pt A):46–64. Available from: http://dx.doi.org/10.1016/j.cca.2015.01.031
15. Mousa S, Moustafa A, Aly H. Prognostic value of red cell distribution width, platelet parameters, and the hematological scoring system in neonatal sepsis. Egypt J Haematol. 2019;44(3):183.
16. Gurol G, Ciftci IH, Terzi HA, Atasoy AR, Ozbek A, Koroglu M. Are There standardized cutoff values for neutrophil-lymphocyte ratios in bacteremia or sepsis? J Microbiol Biotechnol.