Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka s23

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA

Synopsis of Dissertation

STUDY OF HEMATOLOGICAL PARAMETERS IN THE EARLY DIAGNOSIS OF NEONATAL SEPSIS

Submitted by:

DR. SUSHMA H M, MBBS Post Graduate Student in Pathology

Department of Pathology Adichunchanagiri Institute of Medical Sciences, B.G. Nagara, Nagamangala Taluk, Mandya District -571448. PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. NAME OF THE CANDIDATE & DR. SUSHMA H M ADDRESS DEPARTMENT OF PATHOLOGY ADICHUNCHANAGIRI INSTITUTE OF MEDICAL SCIENCES B.G.NAGARA-571448 ADICHUNCHANAGIRI INSTITUTE OF 2. NAME OF THE INSTITUTION MEDICAL SCIENCES, B.G.NAGARA

3. COURSE OF STUDY & SUBJECT M.D. IN PATHOLOGY

4. DATE OF ADMISSION TO COURSE 13.07.2012

STUDY OF HEMATOLOGICAL 5. TITLE OF THE DISSERTATION PARAMETERS IN THE EARLY DIAGNOSIS OF NEONATAL SEPSIS

6. BRIEF RESUME OF THE INTENDED APPENDIX – I WORK 6.1 NEED FOR THE STUDY APPENDIX – IA 6.2 REVIEW OF LITERATURE APPENDIX – IB 6.3 OBJECTIVES OF THE STUDY APPENDIX – IC

7. MATERIALS AND METHODS APPENDIX – II

7.1 SOURCE OF DATA: APPENDIX – IIA

7.2 METHOD OF COLLECTION OF APPENDIX – IIB DATA: (INCLUDING SAMPLING PROCEDURE IF ANY)

7.3 DOES THE STUDY REQUIRE ANY INVESTIGATION OR YES INTERVENTIONS TO BE APPENDIX – IIC CONDUCTED ON PATIENTS OR OTHER ANIMALS, IF SO PLEASE DESCRIBE BRIEFLY.

7.4 HAS ETHICAL CLEARANCE BEEN YES OBTAINED FROM YOUR APPENDIX – IID INSTITUTION IN CASE OF 7.3. 8. LIST OF REFERENCES APPENDIX – III

2 9. SIGNATURE OF THE CANDIDATE

The study of hematological parameters in 10. REMARKS OF THE GUIDE neonatal sepsis helps in early diagnosis and in preventing morbidity and mortality. It also helps in guiding treatment. Hence the study is recommended.

11. 11.1 NAME AND DESIGNATION OF Dr. Y. H. KALEGOWDA. MBBS, DCP, MD GUIDE PROFESSOR AND H.O.D. DEPARTMENT OF PATHOLOGY ADICHUNCHANAGIRI INSTITUTE OF MEDICAL SCIENCES, B.G.NAGARA

11.2 SIGNATURE

11.3 CO- GUIDE (IF ANY) NO

11.4 SIGNATURE

11.5 HEAD OF THE DEPARTMENT Dr. Y. H. KALEGOWDA. MBBS, DCP, MD PROFESSOR AND H.O.D. DEPARTMENT OF PATHOLOGY ADICHUNCHANAGIRI INSTITUTE OF MEDICAL SCIENCES, B.G.NAGARA

11.6 SIGNATURE

12. 12.1 REMARKS OF THE CHAIRMAN The facilities required for the investigation will AND PRINCIPAL be made available by the college

Dr. M.G SHIVARAMU M.B.B.S., MD PRINCIPAL, AIMS, B.G. NAGARA.

12.2 SIGNATURE

3 APPENDIX-I

6. BRIEF RESUME OF THE INTENDED WORK:

APPENDIX-IA

6.1 NEED FOR THE STUDY

Neonatal sepsis is a clinical syndrome characterized by signs and symptoms of infection with or without accompanying bacteremia in the first month of life.

The incidence of neonatal sepsis according to the data from national neonatal perinatal database (NNPD, 2002-2003) is 30% per 1000 live births. Most common in preterm and low birth weight neonates because of low immunity to combat bacterial infections, term babies are not exclusion from the infection.

Early recognition and diagnosis of neonatal sepsis is a difficult task for the clinicians because such infants often present with variable and nonspecific clinical presentation of this condition, that leads to failure or delay in the treatment which causes significant morbidity and mortality.

Early diagnosis of neonatal sepsis is necessary to prevent the fatal outcome and for the guidance to prompt initiation of antibiotic therapy.

The “Gold standard” test for the diagnosis of neonatal infection is positive blood culture. Although a positive culture is obtained in 30-75% of the cases, it is time consuming procedure and requires 48-72 hours. Institution of antibiotics before culture often increases the problem of unnecessary exposure to antibiotics and bacterial resistance.

The current study is undertaken to study the hematological parameters in the early diagnosis of neonatal septicemia using Rodwell’s scoring system which includes simple laboratory tests such as total leucocyte count (TLC), absolute neutrophil count (ANC),

4 immature neutrophil count (I), immature to total neutrophil ratio (I: TNR), immature to mature neutrophil count ratio, platelet count, degenerative changes in neutrophils and C- reactive protein (CRP) .

APPENDIX –I B

6.2 REVIEW OF LITERATURE:

When pathogenic organisms gain entry into the blood stream, they may cause an overwhelming infection without much localization (septicemia), or may get predominantly localized to the lung (pneumonia), or the meninges (meningitis). Neonatal sepsis is one of most common condition which causes morbidity and mortality in the community. A panel of hematological tests helps in the diagnosis. Blood culture acts as a guide to treatment.

Hematological criteria to diagnose the case as septicemia include total leucocyte count < 5000/ cumm, absolute neutrophil count<1800/cu mm, immature neutrophil count to total neutrophil count more than 0.2 and C-reactive protein >1 mg/dl. Micro- ESR should be high (has moderate sensitivity).1

Parida et al in their study evaluated 20 neonates with proven septicemia and 12 neonates with suspected sepsis for assessing the role of TLC, percentage of total neutrophils and band cell count for early diagnosis of neonatal septicemia. They concluded that percentage of band cell count is highly sensitive and has high predictive value in the diagnosis of neonatal septicemia. Simplicity of the test makes its routine use feasible.2

Sharma A et al conducted a study to evaluate the role of sepsis screen in the diagnosis of neonatal septicemia. They evaluated the sensitivity and specificity of hematological parameters singly and in combination. 48% of septicemia could be evaluated by sepsis screen, only 20% cases were diagnosed by positive culture. This study showed that sepsis screen is helpful in avoiding overuse of antibiotics.3

5 Bandari V et al conducted prospective study at Yale- New Haven children hospital,for the duration of one year from May 2005 to July 2006, enrolling consecutive infants with suspected sepsis. All hematological parameters, neutrophil CD64 and neutrophil indices were correlated with the diagnosis of confirmed and suspected sepsis. They concluded that neutrophil CD64 was highly sensitive marker for neonatal sepsis. But the drawback in using this as routine use its availability and high cost.4

Kumar H and Narasimha A conducted a prospective study on 50 neonates with the aim of evaluating the significance of hematological scoring system of Rodwell’s in the diagnosis of neonatal sepsis. In that study they evaluated that an abnormal immature to total neutrophil ratio, followed by immature to mature neutrophil ratio were the most sensitive indicators in identifying sepsis. They put forth that hematological scoring system is a simple, quick, cost effective tool, which can be used as a screening test for early diagnosis of neonatal sepsis.5

Buch A C et al conducted a study to evaluate the role of hematological parameters as early indicators of neonatal sepsis. They used different hematological parameters such as total leucocyte count (TLC), absolute neutrophil count (ANC), immature to total neutrophil ratio (I:TNR), platelet count, C-Reactive protein and micro-erythrocyte sedimentation rate

(ESR) in different permutation and combination to diagnose and to evaluate neonatal sepsis.

These parameters were evaluated statistically based on standard reference values. They found that combination of 5 tests (I:TNR+ANC+CRP+ESR+Platelet count) when used in combination gives best results.6

In addition to the hematological parameters as per the Rodwell’s scoring system,

Santhosh et al studied the role of plasma fibrinogen level, IgM, micro- ESR, CRP as a promising early indicators of neonatal sepsis. The study included 62 patients with clinical suspicion of neonatal sepsis and 40 neonates without clinical suspicion of neonatal sepsis as

6 controls. They divided the cases into culture positive and culture negative. At the end of the study they concluded that m-ESR, immature to total neutrophil ratio (I/T), morphological changes in neutrophils and CRP were the most sensitive indicators of early neonatal sepsis.7

Setal BC, Viren V, Bimal BC conducted a prospective study to evaluate C-reactive protein as a screening tool for neonatal sepsis. Peripheral smears of 75 neonates were revised and CRP performed by semi quantitative latex agglutination method. Positive cultures and absolute neutrophils count were compared. The sensitivity and specificity of CRP was

92.30% and 85.71% respectively. CRP assay using semi quantitative latex agglutination method is valuable adjunct in screening for neonatal sepsis complementing clinical decision making.8

Sucilathangam et al conducted a prospective study at Tiruneveli medical college hospital, Tamilnadu for the duration of one month from July 2010 – Aug2010 to compare the diagnostic role of procalcitonin and CRP against hematological parameters and blood culture.

Study was done by collecting 50 blood samples from the neonates and data were expressed as mean ± SD and statistical significance was assessed by the Chi- square test. In that study they found that procalcitonin was superior not only in diagnosis but also in detecting severity of illness and in evaluation of response to antibiotic treatment.9

The peripheral leucocyte count is the most frequently used indicator of bacterial infection. In the absence of maternal hypertension, severe asphyxia, peri ventricular hemorrhage, maternal fever or hemolytic disease, absolute total neutropenia and elevated ratio of immature to total neutrophilic forms suggest bacterial infection. Repeating complete blood count within 24 hours of birth may increase the value of the test as a screening test

.The detection of IL-6 in serum, especially in conjunction with CRP or procalcitonin may be useful in the early diagnosis of sepsis.10

7 BhatR and Rao A conducted a retrospective study for the period of 4 years from Jan

2000 to Dec 2004. They considered only the neonates who were clinically suspected of having bacterial infection within 48 hours. They were subjected to various hematological tests and blood cultures. Those hematological parameters were assessed singly and in combination, in culture positive neonates. The statistical analysis was done. They found that sensitivities of traditional parameters and CRP was not satisfactory in the diagnosis of septicemia.11

Isoimmune neonatal neutropenia is the neutrophil equivalent of Rh hemolytic disease of the newborn. Clinical manifestation include fever soon after birth, pneumonia, skin infections, urinary infections, and septicemia. The most common pathogens are those of neonatal period and include Staphylococcus aureus and Group B Streptococcus. The average duration of neutropenia is 7 weeks, but a range of 2 to 17 weeks is reported.12

APPENDIX-IC

6.3 OBJECTIVES OF THE STUDY

1. To study the hematological parameters, peripheral smear and CRP in neonates

clinically suspicious of neonatal septicemia.

2. To compare all these parameters with the blood culture as the gold standard.

8 APPENDIX-II

7. MATERIALS AND METHODS:

APPENDIX-II A

7.1 SOURCE OF DATA

The present study will be conducted in department of Pathology, Adichunchanagiri

Institute of Medical sciences.

Study design- Cross sectional-descriptive study.

Sample size- This study will include all the blood samples of neonates which will be admitted to NICU, with clinically suspected septicemia, during December 2012 to May 2014

(i.e. for the duration of 18 months).

APPENDIX-II B

7.2 METHOD OF COLLECTION OF DATA

Blood samples of all neonates suspected of sepsis will be taken to study the hematological parameters. Clinical history and physical findings will be noted. Red blood cell count, hemoglobin, uncorrected WBC count, platelet count will be measured using automated analyser. Leishman stained peripheral smears will be examined for nucleated red blood cells, total white cell count, total neutrophil count, immature neutrophil count, immature to total neutrophil count ratio, immature to mature neutrophil count ratio, degenerative changes in neutrophils. Qualitative value of C-reactive protein (CRP) and blood culture reports will be noted from the Microbiology department, Adichunchanagiri Institute of Medical sciences.

The cases will be classified as per Rodwell’s hematological scoring system. The results will be analysed using percentage, proportion and Chi-square tests.

INCLUSION CRITERIA

9 1. All the neonates with features of sepsis.

2. All the neonates with history suggestive of sepsis.

EXCLUSION CRITERIA

1. Neonates with hemolytic jaundice.

2. Neonates with inborn errors of metabolism.

3. Neonates with suspicion of TORCH.

APPENDIX-II C

7.3 DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS

TO BE CONDUCTED ON PATIENTS OR OTHER ANIMALS; IF SO PLEASE

DESCRIBE BRIEFLY.

Blood samples which will be sent for the routine investigation with clinically suspected neonatal septicemia will be included in the study. Separate samples will not be drawn for the study purpose.

APPENDIX-II D

7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION

IN CASE OF 7.3?

PROFORMA APPLICATION FOR ETHICS COMMITTEE APPROVAL

10 SECTION A STUDY OF HEMATOLOGICAL a Title of the study PARAMETERS IN THE EARLY DIAGNOSIS OF NEONATAL SEPSIS

DR. SUSHMA H M Principle investigator DEPARTMENT OF PATHOLOGY b (Name and Designation) ADICHUNCHANAGIRI INSTITUTE OF MEDICAL SCIENCES B.G.NAGARA-571448 Co-investigator c NIL (Name and Designation) 1.DEPARTMENT OF PAEDIATRICS ADICHUNCHANAGIRI HOSPITAL AND RESEARCH CENTRE Name of the Collaborating B.G.NAGARA d Department/Institutions 2.DEPARTMENT OF MICROBIOLOGY ADICHUNCHANAGIRI HOSPITAL AND RESEARCH CENTRE B.G. NAGARA Whether permission has been obtained from e the heads of the collaborating departments YES & Institution Section – B APPENDIX II Summary of the Project Section – C APPENDIX IC Objectives of the study Section – D APPENDIX IIB Methodology Where the proposed study will be ADICHUNCHANAGIRI HOSPITAL AND A undertaken RESEARCH CENTRE, B.G. NAGARA B Duration of the Project 18 MONTHS C Nature of the subjects: Does the study involve adult patients? NO Does the study involve Children? YES (NEONATE) Does the study involve normal volunteers? NO Does the study involve Psychiatric patients? NO Does the study involve pregnant women? NO

D If the study involves health volunteers NA I. Will they be institute students? NO

11 II. Will they be institute employees? NO III. Will they be paid? NA IV. If they are to be paid, how much per NA session?

E Is the study a part of multi central trial? NO

F If yes, who is the coordinator? (Name and Designation) NA

Has the trial been approved by the ethics NA Committee of the other centers?

If the study involves the use of drugs please indicate whether.

I. The drug is marketed in India for the indication in which it will be used in the study. NA II. The drug is marketed in India but not for the indication in which it will be used in the study

III. The drug is only used for experimental use in humans.

IV. Clearance of the drugs controller of India has been obtained for:

 Use of the drug in healthy volunteers  Use of the drug in-patients for a new indication.  Phase one and two clinical trials  Experimental use in-patients and healthy volunteers.

G How do you propose to obtain the drug to NA

12 be used in the study? - Gift from a drug company - Hospital supplies - Patients will be asked to purchase - Other sources (Explain) H Funding (If any) for the project please state - None - Amount NONE - Source - To whom payable Does any agency have a vested interest in I NO the out come of the Project? Will data relating to subjects /controls be J YES stored in a computer? Will the data analysis be done by K - The researcher? YES - The funding agent NO L Will technical / nursing help be required from the staff of hospital. NO

If yes, will it interfere with their duties? NO

Will you recruit other staff for the duration of the study?

If Yes give details of NA I. Designation NA II. Qualification NA III. Number NA IV. Duration of Employment

13 M Will informed consent be taken? If yes NA Will it be written informed consent: NO Will it be oral consent? NO Will it be taken from the subject themselves? NO Will it be from the legal guardian? NO If no, give reason: NA

N Describe design, Methodology and techniques APPENDIX II

Ethical clearance has been accorded.

Chairman, P.G Training Cum-Research Institute, A.I.M.S., B.G.Nagara. Date :

PG Training-cum research committee

14 APPENDIX-III

LIST OF REFERENCES

1. Paul VK et al. Newborn infants. In: Ghai OP, Paul VK, Bhagya A. Ghai essential

pediatrics. 7th ed. 2009, New Delhi, CBS Publication, 2009; 96-159.

2. Parida SN, Verma IC, Singh M. Blood leucocyte changes for the early diagnosis of

neonatal sepsis. Indian J Pediatr 1982; 49: 653-57.

3. Sharma A, Kutty CVK, Sabharwal U, Rathee S, Mohan H. Evaluation of sepsis screen

for diagnosis of neonatal septicemia. Indian J Pediatr 1993; 60: 559-63.

4. Bhandari V, Wang C, Rinder C, Rinder H. Hematologic profile of sepsis in neonates:

Neutrophil CD64 as a diagnostic marker. Pediatrics 2008; 121: 129-34.

5. Narasimha A, Kumar HML. Significance of hematological scoring system (HSS) in

early diagnosis of neonatal sepsis. India J Hematol Blood Transfus 2011; 27: 14-17.

6. Buch AC, Srivastava V, Kumar H, Jadhav PS. Evaluation of hematological profile in

early diagnosis of clinically suspected cases of neonatal sepsis. Int J Basic App Sci

2011; 1:1-6.

7. Mondal SK, Nag DR, Bandyopadhyay R, Chakraborty D, Sinha SK. Neonatal sepsis:

Role of battery of immunohematological tests in early diagnosis. Int J App Basic Med

Res 2012; 2:43-47.

8. Setal BC, Viren V, Bimal CB. C-reactive protein (CRP) in early diagnosis of neonatal

septicemia. Nat J Med Res 2012; 2:276-78.

9. Sucilathangam G,Amuthavalli K, Velvizhi G, Ashihabegum MA, Jeyamurugan T,

Palaniappan N. Early diagnostic markers for neonatal sepsis: Comparing procalcitonin

(PCT) and C-reactive Protein (CRP). J Clin Diag Res 2012; 6: 627-31.

10.Schelonka RL, Freij BJ, Mccracken GH. Bacterial and fungal infections. In :

Macdonald MG, Mullett MD, Seshia MMK. Avery’s neonatology-Pathophysiology

15 and management of the newborn. 6th ed. Philadelphia, Lippincott Williams and

Wilkins, 2005; 1235-73.

11.Bhat R, Rao A. Sepsis screen in early neonatal sepsis. J Clin Diag Res 2010; 4: 3331-

12.Watts RG. Neutropenia. In: Greer JP, Foerster J, Rodgers GM, Paraskevas F, Gladder

B, Arber DA, et al. Wintrobe’s Clinical Heamatology.12th ed. Philadelphia, Lippincott

Williams and Wilkins, 2009; 1527-47.