Mr/Mrs ... are being asked consent for their child to be a participant in the research study titled

A DISSERTATION ON

ROLE OF UMBILICAL CORD BLOOD CULTURE IN PREDICTION OF EARLY ONSET SEPSIS AMONG NEWBORNS WITH HIGH RISK

FACTORS

Dissertation submitted to

THE TAMILNADU Dr .M .G .R .MEDICAL UNIVERSITY CHENNAI-600032

With partial fulfillment of the regulations for the award of the degree of

M.D.PEDIATRICS

COIMBATORE MEDICAL COLLEGE , COIMBATORE MAY 2020

UNIVERSITY REGISTER NUMBER : 201717304

CERTIFICATE

Certified that this dissertation entitled “ROLE OF UMBILICAL CORD BLOOD CULTURE IN PREDICTION OF EARLY ONSET SEPSIS AMONG NEWBORNS WITH HIGH RISK FACTORS” is a bonafide work done by Dr. S. VANI, M.D. Post graduate student of Pediatric Medicine, Coimbatore Medical College & Hospital, Coimbatore – 641 018 during the academic year 2017 – 2020.

Date: Guide,

Dr.V.BOOMA MD., Professor & Head

Department of Paediatrics Govt. Coimbatore Medical College & Hospital.

Date: Professor & Head Dr. V.BOOMA MD., Department of Paediatrics Govt. Coimbatore Medical College & Hospital.

Date: Dean

Dr. B.ASOKAN MS.,MCh Govt. Coimbatore Medical College & Hospital.

DECLARATION

I declare that this dissertation entitled “ ROLE OF UMBILICAL CORD BLOOD CULTURE IN PREDICTION OF EARLY ONSET SEPSIS AMONG NEWBORNS WITH HIGH RISK FACTORS ” has been conducted by me, Department of Pediatrics, Coimbatore Medical College and Hospital, under the guidance and supervision of my guide Prof. Dr.V. BOOMA, M.D .It is submitted in part of fulfillment of the award of the degree of M.D.Pediatrics for the MAY 2020 examination to be held under The TamilNadu Dr.M.G.R. Medical University, Chennai.

This has not been submitted previously by me for the award of any degree or diploma from any other university.

[Dr. S. VANI ]

ACKNOWLEDGEMENT

My sincere thanks to Prof.Dr. B.ASOKAN. M.S.Mch., DEAN, Coimbatore Medical College Hospital for allowing me to do this dissertation and to utilize the institutional facilities.

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to Prof.Dr. V.Booma MD, Professor of Pediatrics, Coimbatore Medical College and Hospital for permitting me to undertake this study and for her guidance, invaluable help, encouragement and support throughout the study.

I am extremely thankful to Prof. Dr.A.Lakshmanasamy MD.DCh, Prof. Dr.M.Geethanjali,MD.DCh, Prof. Dr.B.R.SASIKUMAR M.D.

DCh for their guidance, encouragement and support throughout the study.

I would like to thank our Registrar, Dr.P.THIYAGARAJAN.MD.DCh for his valuable guidance and support in doing this study.

I extend my sincere thanks to Assistant Professors Dr.N.Kumar MD, Dr.A.Uma Maheswari MD, Dr.S.Jayaprakash MD, Dr.P.Senthil kumar MD.DM, Dr.V.K.Sathyan .MD.DM, Dr.Suganya M.D, Dr. Menaka MD for their invaluable suggestion , help and support throughout the study.

I sincerely thank Mr.JOSHVA ALLEN SHEPHERD, for helping me in statistical analysis.

I sincerely thank all the parents of the children who participated in this study and rendered their extreme co operation.

I would also like to thank my beloved parents for their moral support throughout the study period without which this project would have been impossible.

CONTENTS

SL.NO TITLE PAGE NO:

1. INTRODUCTION 1

2. STUDY JUSTIFICATION 30

3. AIM & OBJECTIVE 31

4. METHODOLOGY 32

5. REVIEW OF LITERATURE 36

6. OBSERVATION & RESULTS 44

7. DISCUSSION 70

8. SUMMARY 71

9. CONCLUSION 72

10. BIBLIOGRAPHY 73

11. ABBREVIATIONS ANNEXURE

PROFORMA CONSENT FORM MASTER CHART

1

INTRODUCTION

Neonatal Septicemia is the leading cause of neonatal mortality and morbidity in India¹. It is estimated that 20% of all neonates develop sepsis and is responsible for 30-50% of total neonatal death in developing countries². Neonatal sepsis is a clinical syndrome of bacteremia characterized by systemic signs and symptoms of infection in the first month of life.

Neonatal sepsis can be classified into two sub types depending on the onset of symptoms related to sepsis. Early onset sepsis usually presents within the first 72 hours of life. Late onset sepsis usually presents after 72 hours of life. The mortality associated with early onset neonatal sepsis is higher than that of late onset sepsis³.

Accurate and timely diagnosis of neonatal sepsis still remains a major challenge to the pediatricians and neonatologists. Mortality due to neonatal sepsis is preventable andoutcome is better if diagnosed earlier.

Diagnosis of neonatal sepsis by blood culture collected from peripheral vein⁴ is the gold standard method. Umbilical cord (placental end) is a less commonly used site for collection of blood culture. Although the umbilical cord is infrequently used for blood collection, the procedure is painless and technically less challenging when compared to peripheral

2

veins. Moreover, it ensures adequate volume of blood for culture with less contamination⁵.

This study was carried out to evaluate role of umbilical cord blood culture in diagnosis of early-onset neonatal bacterial sepsis in neonates with risk factors.

Definition:

When pathogenic bacteria gain access into the blood stream of neonates, they may cause an overwhelming infection without much localization (septicemia) or may get predominantly localized to the lung (pneumonia) or the meninges (meningitis). Systemic bacterial infections are known by the generic term neonatal sepsis which incorporates septicemia, pneumonia and meningitis of the newborn¹³.

Based on the onset of symptoms and probable mode of transmission, neonatal sepsis may be of following categories¹⁴ :

a) Early onset sepsis.

b) Late onset sepsis.

3 Early onset sepsis (EOS) :

The signs and symptoms of sepsis present within 72 hours of life¹⁵ . EOS is often associated with organisms ascending from the maternal genital tract by vertical transmission or transplacental transmission resulting in chorio-amnionitis or fetal infection in- utero or by acquisition via passage through birth canal during labour. The potential risk factors for acquisition of EOS include :

 Prolonged rupture of membrane(PROM) >12 hours

 Prolonged labour >24 hours

 Febrile illness of mother prior to 2 weeks of delivery

 Low birth weight < 2.5 kg/ prematurity < 36 weeks of gestation

 Meconium stained liquor

 Foul smelling liquor

 Birth asphyxia

4 CHORIOAMNIONITIS;

-Intrauterine infection includes maternal fever with or without local or systemic signs of corioamnionitis

Uterine tenderness

Foul smelling discharge

Maternal leukocytosis(>15000 cells/mm3)

Maternal or fetaltachycardia

May be asymptomatic,diagnosed by pathologic examination of placenta or amniotic fluid analysis.Histologic findings of chorioamnionitis directly related to duration of membrane rupture

The usual isolates of EOS comprises of Streptococcus agalactiae, E. coli, Klebsiella and Listeria monocytogenes. S.aureus & CONS are less frequent causes.

Late onset sepsis (LOS):

The signs and symptoms of sepsis present later than 72 hours of life . The origin of infection is usually from the environment. Poor hand hygiene practices of health care personnel is most common source of Post-natal infection in hospital .

5

Coagulase negative staphylococci and other Staphylococci account for 30 to 60% of LOS owing to the use of intravascular devices especially in NICU setup. The other organisms are S.aureus, group B streptococci, Escherichia coli, Klebsiella, Enterobacter etc. The risk factors include prolonged hospitalisation, indiscriminate use of antibiotics, use of intravascular devices etc.

MODES OF TRANSMISSION AND PATHOGENESIS

Neonatal infections are unique for several reasons:

Infectious agents can be transmitted from the mother to the fetus or new born infants by diverse modes.

New born infants are less capable of responding to infection because of one or more immunological deficiencies.

Coexisting conditions often complicate the diagnosis and management of neonatal infections.

The clinical manifestations of newborn infections vary and include subclinical infection, mild to severe manifestation of focal or systemic infection and rarely congenital malformations resulting from infection in

6 1st trimester.

Maternal infection that is the source of transplacental fetal infection is often undiagnosed during pregnancy because the mother was either asymptomatic or had non specific symptoms at the time of acute infection.

Finally, with advance in neonatal intensive care, increasingly immature, very low birth weight newborns are surviving and remain in the hospital for a longer time, and environment that puts them at ongoing high risk for infection¹⁶.

PATHOGENESIS OF INTRAUTERINE INFECTION

Intrauterine infection is a result of clinical or subclinical maternal infection with a variety of agents (e.g. Cytomegalovirus, Treponema pallidum, Toxoplasma gondii, Rubella virus, Varicella virus, Parvo virus B19) and hematogenous transplacental transmission to the fetus.

Transplacental infection may occur anytime during gestation and signs and symptoms may be present at birth or be delayed for months or years.

The timing of infection during gestation affects the outcome. First trimester infection may alter embryogenesis with resulting congenital

7 Malformation (e.g. congenital rubella).

Third trimester infection often results in active infection at the time of delivery (toxoplasmosis, syphilis). Infections that occur late may lead to delay in clinical manifestation until birth (syphilis).

8 ETIOLOGY OF NEONATAL SEPSIS:

In general, the common bacterial causes of early onset sepsis (EOS) include :

Group B-streptococcus:

Most common cause of infection in neonate. Overall incidence is 0.72/1000 live births.Its a commensal of vagina and gastrointestinal tract.Serotypes based on capsular polysaccharide antigen Ia, Ib, II,I II, IV, V, VI, VII, VIII .types Ia, III, V are most common subtypes for early onset infection. Surface exposed protein antigen also reason for pathogenicity. Group B streptococci cross the intact fetal membrane. In 40% of newborn with GBS infection showed prolonged rupture of fetal membrane. Mortality and morbidity associated with GBS infection are high for preterm babies. Invasion of pulmonary endotheial cells by GBS cause release of eichosonoids, prostaglandin E2,prostacyclin.

CULTURE PROVEN GBS:

GBS isolated from blood or CSF

9

FACTORS THAT DECREASE THE YIELD OF CULTURE

1. Prior use of antibiotics 2. Inadequate blood volume 3. Low level bacteriemia

PROBABLE GBS INFECTION

Episode of clinical sepsis caused by GBS with clinical features but negative blood culture

RISK FACTORS

1. Preterm delivery <37 weeks 2. Prolonged rupture of membranes

3. Genital carriers of GBS infection during pregnancy

PRESENTATION OF GBS INFECTION

Signs occur within 12 hours of delivery

 Poor feeding

 Excessive sleepiness

 Tachypnoea

10

 Apnoea

 Cyanosis

 Respiratory failure

 Shock

INVESTIGATIONS

 Chest xray

 CRP

 Blood culture

Normal CRP levels does not exclude GBS infection Benzyl penicillin is the drug of choice

Recurrent GBS infection occurs in <1% of neonates.

DETECTION OF GBS

PCR is a diagnostic tool for detection of GBS.

PCR also used to detect bacterial proteins in swab and body fluids.

Prevention of Group B Streptococci infection:

11

 Antibiotic prophylaxis for mothers during labour

LISTERIA :

 Gram positive rod

 Intra and extra cellular organism

Fetal and neonatal infection caused by

 Transplacental infection

 Intrapartum infection-early onset

 Nosocomial infection –late onset

Transplacental infection :

Fetal infection occurs by hematogenous spread through the placenta,infection of the amniotic fluid.

Fetal death /miscarriage occurs during first and second trimester infection. Precipitate preterm labour if infections occur later .severe Pneumonia, Meningitis , Hepatosplenomegaly may be present

12 B) Escherichia coli

C) Streptococcus pneumoniae D) Viridans streptococci

E ) Enterococci

F) Haemophilus influenzae G) Neisseria meningitidis

H) Neisseria gonorrhoea

I) Listeria monocytogenes etc.,

Of these, Group B- streptococcus and Listeria monocytogenes are usually of maternal origin.

The common bacteria causing late onset sepsis include :

 Staphylococcus aureus

 Coagulase negative Staphylococci

 Enterococci

 Citrobacter

 Enterobacter

13

 Klebsiella pneumoniae

 Salmonella

NONBACTERIAL CAUSES OF SYSTEMIC NEONATAL INFECTIONS

 Viruses

 Adenovirus

 Cytomegalovirus

 Enteroviruses

 Herpes simplex virus

 HIV

 Parvovirus

 Rubella virus

 Varicella-zoster virus

 Fungi

 Candida species

 Protozoa

14

 Plasmodia

 Toxoplasma gondii

 Trypanosoma cruzi

CLINICAL MANIFESTATIONS

The earliest signs of sepsis are often subtle and nonspecific and need a high index of suspicion for early diagnosis. Babies with sepsis may present with one or more of the following symptoms¹⁷.

Non-specific features of sepsis

a) Hypothermia or fever

b) Lethargy, poor cry, refusal to suck

c) Poor perfusion, prolonged capillary refill time d) Hypoglycemia, hyperglycemia

e) Metabolic acidosis¹⁷.

Specific features related to various system

Gastrointestinal system

Feed intolerance, abdominal distention, vomiting, diarrhea,

15 hepatomegaly

Respiratory System

Apnea, dyspnea, tachypnea, retractions, flaring, grunting, cyanosis.

Renal System

Oliguria

Cardiovascular System

Pallor, mottling, cold, clammy skin, tachycardia, hypotension, bradycardia

Central nervous System

Irritability, lethargy, tremors, seizures, hyporeflexia, hypotonia, abnormal moro reflex, irregular respirations, full fontanel, high-pitched cry.

Hematological System

Jaundice, Splenomegaly, pallor, petechiae, purpura, bleeding¹⁸

DIAGNOSIS

The diagnosis of systemic bacterial infection must start with a careful evaluation of the newborn’s symptoms and signs, physical

16

examination, changes in vital signs and laboratory indicators and history including maternal history.

LABORATORY INVESTIGATION

SEPSIS SCREEN

All newborns suspected to have sepsis should have a septic screen to corroborate the diagnosis of sepsis. The various components of septic screen are haematological changes in the peripheral blood, estimation of acute phase reactants, micro ESR ,CBC,total count, IT ratio.

HAEMATOLOGICAL CHANGES

1. Total WBC count

-useful and rapid test, but non-specific.

Total count <5000

I/T Ratio normal 0.16 first 24 hours

0.14 -48 hours

0.13 -60 hours to 5 days 0.12 end of first month Band cell/total ratio>0.2or 0.3 – infection.

17 I:T ratio calculation =immature cells

Total(mature+immature)

The normal range of WBC counts in newborn infants is wide and this wide range should be taken into account in interpreting the values (Manroe et al). Thrombocytopenia, as well as elevated Prothrombin time, partial thromboplastin time and international normalized ratio (INR) and decrease fibrinogen will be found in severely ill infants¹⁹.

PLATELET COUNT:

Platelet count will fall below 1,00,000 in babies with bacterial infection

Common feature of NEC and infection is thrombocytopenia

Thrombocytopenia may be present in non infective conditions –HIE

Profound thrombocytopenia occurs in both congenital and acquired viral infection.

ACUTE PHASE REACTANTS

These proteins are produced by the liver under the influence of interleukin - 1 when inflammation from any cause is present. The most

18

important and widely used is C-reactive protein (CRP).

 C-reactive protein is a non-specific marker of inflammation or tissue necrosis It was the first protein to be discovered which behaves as an acute phase reactant. It was so named because of its ability to bind and precipitate the somatic C-polysaccharide of pneumococci.

Elevations in CRP are found in bacterial sepsis and meningitis. A single determination of CRP at birth lacks both sensitivity and specificity for infection, but serial CRP determination at birth and at 12 hours and beyond have been used to manage infants at risk for sepsis²⁰.

CRP levels fall quickly after efficient elimination of microbial stimulus, due to its short half life of 19 hours. Thus CRP may be used as a parameter to identify the time period when antibiotic therapy can safely be discontinued in case of suspected neonatal septicemia²¹. A good response to antibiotics is indicated by a rapid return to normal of CRP, whereas persistent elevations of serum CRP suggest that the treatment is inadequate or indicate some complication.

19 OTHER ACUTE PHASE REACTANTS

Fibronectin,prealbumin and transferrin are negative reactants.

Procalcitonin is produced in plasma of infected patients,

 physiologically elevated during 1^st three days of life

 normal value 0.5

 Indicator of late onset sepsis

 More reliable than C-Reactive protein.

BLOOD CULTURE:

To confirm sepsis blood sample should be sent for culture.Amount of blood needed for culture is lower than needed for adult .Neonates tend to have

1-log-higher concentration of bacteria in their bloodstream than adults.1 ml of blood needed for good yield.blood can be drawn from umbilical artery and umbilical vein.

20 CYTOKINES:

TNF AND IL-6

Elevation 0f TNF and IL-6 in plasma indicates sepsis,but also raised in non infective inflammation.

IL-6 better marker of EOS than sepsis

Another indicator of sepsis is IL -8 involved in neutrophil release and activation.

SERUM GRANULOCYTE COLONY STIMULATING FACTOR

 Endogenous and immunoreactive

 neonates able to produce it

 measurable in plasma

 inversely related to absolute neutrophil count

 plasma levels increased in infection and inflammation

 not specific and sensitive than CReactive protein.

21 MANNOSE BINDING LECTIN

 Protein of the innate immune system

 Binds with mannose units present on pathogens

o Activates complement after binding o Binding results in opsonisation

Low levelsof mannose binding lectin results in decreased ability to opsonise bacteria ,increased susceptibility to infection

Newborns with low levels of mannose binding lectin had increasd risk for culture positive sepsis.

IMMUNOLOGICAL TESTS:

ANTIGEN DETECTION TEST

Rapid screening for group B streptococci

Both maternal and neonatal colonisasation can be detected May provide reassurance of absence of GBS

22 ANTIBODY DETECTION TEST

GENETIC TECHNIQUES

MICRO-ESR

Inexpensive & easy screening test for neonatal sepsis.

Barrett (1980) described a micro-ESR method using 0.2 ml blood to fill a plastic disposable tube 230 mm long with a 1 mm internal bore.

Capillary blood values correlated well with venous blood micro-ESR and westegren-ESR value. It is simple, but not very reliable.Normal value increase with postnatal age.

A value >15mm in 1st hour is suggestive of infection.

Less sensitive.

SEPSIS SCREEN :

To rule out sepsis

Consists of :

 C-reactive protein

 Total leukocyte count

 Absolute neutrophil count

23

 Immature to total neutrophil count

 Micro erythrocyte sedimentation rate.

TREATMENT

Institution of prompt treatment is essential for ensuring optimum outcome of neonates with sepsis who often reach the healthcare facilities late and in a critical condition.

Components of treatment

a) Supportive care b) Antibiotics Supportive Care:

The purpose of supportive care is to normalize the temperature, stabilize the cardiopulmonary status, correct hypoglycemia and prevent bleeding tendency. This can be done as follows:

• Provide warmth, ensure consistently normal temperature

• Start intravenous line

• Infuse normal saline 10 ml per kg over 5 to 10 minutes, if perfusion is poor as evidenced by capillary refill time of more than 3

24

seconds. Repeat the same dose 1 to 2 times over the next 30 to 45 minutes, if perfusion continues to be poor.

• Infuse glucose (10 percent) 2 ml per kg stat.

• Inject vitamin K 1 mg intramuscularly.

• Start oxygen by hood or mask, if cyanosed or grunting.

• Provide gentle physical stimulation if apneic.

• Provide bag and mask ventilation with oxygen if breathing is inadequate.

• Avoid enteral feed if very sick, give maintenance fluids intravenously.

Antimicrobial Therapy

The choice of antibiotics depend on the prevailing flora responsible for sepsis and their antimicrobial susceptibility.

Early onset neonatal sepsis:

25

<48 hours to 1 week Benzylpenicillin with gentamycin Amoxicillin if listeria suspected Floxacillin if staphylococcus Aureus suspected.

>48 hours to 1 week

First line Floxacillin with gentamycin

Second line Vancomycin and gentamycin Third line Meropenam,ciprofloxacin Meningitis

First line Cefotaxime with amoxicillin or

Benzylpenicillin /gentamycin Second line Meropenam

Disadvantages of aminoglycosides:

 Ototoxicity and sensorineural hearing loss

26 Mechanism:

 Sustained high trough gentamycin concentration

 genetically determined ototoxicity-mitochondrial DNA mutation m.1555A-G.Profound and permanent hearing loss seen in carriers of this mutation,after receiving aminoglycosides even when drug levels are within therapeutic range.

STOPPING OF DRUGS:

 Should be stopped if cultures are negative.

 Culture that becomes positive after 48 hours is unlikely in an asymptomatic baby.

 By using BacT/Alert microbial detection system incubation period of 3 days enough to detect relevant infections.

MONITORING RESPONSE TO THERAPY:

 Baby remains sick or presense of thrombocytopenia ,abnormal labouratory parameters blood culture should be taken again.

 persistent positive blood culture

27 Resistant organism

Inadequate antibiotic regimen

Indwelling foreign body colonization Focal infection

Abscess formation

Osteomyelitis/endocarditic

FURTHER INVESTIGATIONS:

 CSF analysis

 Suprapubic urine aspirate culture

 skeletal radiographs

 Ultrasonogram to findout the focus

 echocardiogram

Duration of antibiotics:

Culture positive sepsis -14 days Culture positive /sepsis screen positive -7-10 days

Culture negative /sepsis screen negative 3-5 days

28

Pyogenic meningitis - 21 days

PREVENTION

 Good antenatal care

 Maternal infections diagnosed and treated early.

 Babies should be breastfed early.

 Infection control policies applied in the unit.

 Avoid unnecessary needle pricks,intravenous fluids.

 Hand washing before and after handling the baby.

Before delivery

 Diagnosis and treatment of infection in mother

 Strict asepsis in labor room

29 During delivery

 Clean hand

 Clean perineum

 Clean delivery surface

 Clean blade

 Clean cord care

30

JUSTIFICATION OF THE STUDY

Neonatal sepsis is a major cause of neonatal mortality. The clinical outcome mainly depends on the early diagnosis and peripheral venous blood culture.Despite recent advances in anti microbial therapy and the increasing sophistication of neonatal intensive care, the mortality from sepsis remains high. Peripheral venous blood culture is the most widely practiced diagnostic method. However, it is often difficult to obtain blood from preterm neonates.But if the umbilical cord blood is used to diagnose early onset sepsis, babies would not need to have blood drawn via peripheral vein and would experience less pain. Hence my study was carried out to evaluate the role of umbilical cord blood culture in neonates at high risk for early onset neonatal sepsis in comparison to peripheral venous blood culture.

31

AIM & OBJECTIVES

AIM :

To evaluate the role of umbilical cord blood culture in neonates at high risk of early onset sepsis

OBJECTIVES :

PRIMARY OUTCOME

-To stydy correlation of umbilical cord blood culture with peripheral venous blood culture.

SECONDARY OUTCOME

-To compare organisms grown in peripheral venous blood culture and umbilical cord blood culture

32

METHODOLOGY

STUDY DESIGN:

Prospective analytical study STUDY PLACE:

Neonatal intensive care unit, Coimbatore Medical College Hospital.

STUDY PERIOD:

Over a period of one year from March 2018 – February 2019

STUDY POPULATION:

Newborns delivered by labour natural/LSCS having two or more risk factors for sepsis were included in this study.

INCLUSION CRITERIA:

Newborns with two or more risk factors for sepsis

EXCLUSION CRITERIA:

 Newborns with congenital anomalies

 Outborn babies.

33

SAMPLE SIZE CALCULATION FORMULA

DESCRIPTION:

n = Required sample size

t = Confidence level of 95%(standard value of 1.96)

p = Expected Frequency of the Factor under study – 46%

m = Margin of error of 5%(standard value of 0.05)

n=382 contingency

34

The sample is further increased by 5% to account for contingencies such as non response or recording error

n+ 5% =382 + 5% = 400 samples

This study was approved by Ethical Committee. Informed consent in written format was obtained from parents. Neonates who were at risk of developing sepsis based on presence of two or more risk factors for early onset neonatal sepsis enlisted.

These risk factors were prematurity (<35 completed weeks), prolonged rupture of membrane (>18 hours), premature rupture of membrane, prolonged labour (> 24 hours), foul smelling liquor, maternal fever (>100.4 f), frequent vaginal examinations (>3) and birth asphyxia^10,11,12.

Umbilical cord blood, peripheral vein blood and sample for sepsis screen were collected within 24 hours of birth of neonates with presence of two or more risk factors for neonatal sepsis.

Method of blood collection

Umbilical cord blood collection

The umbilical cord was clamped on both placental and umbilical end and blood was drawn from placental end of umbilical vein by 22

35

gauge syringe, 2 ml blood was collected . The needle from syringe was replaced with a new sterile needle and the culture bottle top was wiped with alcohol. 1 mL of blood was injected in an aerobic blood culture bottle and sent to the laboratory.

Peripheral venous blood collection

After providing routine care, peripheral vein blood collection was done using sterile technique in a separate culture bottle and labeled. Both culture samples were immediately taken to microbiology laboratory.

The results were collected, tabulated and analysed.

36

REVIEW OF LITERATURE

1.Jothimeena et al .A prospective analytical method was conducted from May- October 2014 in south india at tertiary care hospital

Fourty neonates with two or more risk factors were included in the study & evaluated for sepsis by peripheralvenous blood culture ,umbilical cord blood culture,sepsis screen.Umbilical blood was collected immediately after delivery and sent for lab.After routine care peripheral venous blood was collected and sent for culture ,sepsis screen.All enrolled neonates were assessed for clinical sepsis.

Fourty neonates ,mean gestational age 36.6 weeks.cord blood CRP was negative in all neonatessepsis screen was positive in 11 newborns.Prolonged rupture of membrane ,prematurity ,frequent vaginal examination were the common risk factors both in sepsis screen positive and negative groups.

Among 40 neonates 3 babies had positive umbilical cord blood culture,one baby positive for peripheral venous blood culture,cord blood CRP was negative in all babies.sepsis screen positive in 11 babies.Most common risk factors were premature rupture of membrane, prematurity, frequent vaginal examination.

37

They have concluded that umbilical cord blood culture may be good alternative method for detection of early onset neonatal sepsis in neonates with risk factors.

2. Hansen et al conducted a prospective cohort study comparing cord blood and peripheral blood CBC,I:T ratio and culture

113 term babies were enrolled in this study.113 pairs of cord and venous blood for cbc,I:T ratio ,culrure sent

All cord and peripheral blood culture were negative,92% babies both cord and peripheral blood I:T ratio were <0.2.

WBC ,hematocrit,platelet counts were moderately correlated.

They have concluded that cord blood can be used alternatively for peripheral blood culture in routine sepsis screen.

3.Fos et al conducted a prospective analytical study in vega hospital from January 2006 to may 2007.

Out of 784 neonates 45 newborns had infection risk factors(

intrapartum fever, chorioamnionitis, neonatalfever in delivery room,abnormality in fetal heart rate,neonatal sepsis infant in previous delivery, premature rupture of membrane >18 hours)).Enrolled newborns were analysed on the basis of birth weight, mode of delivery ,maternal

38

age,gestational age,APGAR score,amniotic fluid nature,time of membrane rupture,maternal temperature,screening of GBS ,fetal temperature ,culture result,clinical evolution.

Out of 30 samples 13 were positive for cord blood culture.7 newborns positive for cord blood culture had characteristics of sepsis started on intravenous antibiotics.Three newborns blood culture were considered contamination .3 cases diagnosed as bacteriemia.cord blood culture had sensitivity of 100%,specificity of 74% positive predictive value of 54%

They have concluded that umbilical cord blood culture is a good way to increase the etiological diagnosis of sepsis

4. Aundhakar CD et al.⁶ cohort study was conducted in Krishna Institute of medical science, Neonatal Intensive Care Unit from December 2015 to May 2017. In this study,a cohort of 75 neonates with perinatal infection risk factorswas followed up prospectively. In delivery room,blood from umbilical cord and peripheral venous blood were collected and analysed.

Peripheral venous blood was also collected for culture. All enrolled neonates were followed till their hospital stay. Sepsis screen and UCBC were compared with PVBC to find out their sensitivity and specificity.

39

Among the 75 high risk neonates, sepsis screen was found positive in 24 (32%) while UCBC and PVBC were found positive in 13 (17.3%) and 4 (5.3%) respectively. Pseudomonas was the most commonly found organism in culture. Sepsis screen and UCBC had sensitivity of 100%

and 75% respectively while specificity was 71.83% and 85.92%

respectively when compared to PVBC. Hence they concluded that UCBC has good diagnostic validity for etiological diagnosis of bacterial sepsis in high risk neonates. Organisms grown in umbilical cord blood samples are comparable with venous blood culture samples. UCBC could be a painless, kinder, gentle approach instead of painful collection of blood by pricking the neonate.

5.Sonawane VB et al⁷. Conducted a hospital based study over three years from August 2005 to August 2008. Hundred and sixty neonates delivered in hospital, having risk factors for neonatal sepsis , along with those coming to hospital with signs and symptoms of sepsis upto 28 days of life ( study group ) along with normal newborns admitted to the postnatal ward without high risk factors ( control group ) were enrolled for this study. Comparative study on various diagnostic markers such as Blood culture, CBC, CRP, IT ratio and micro-ESR was carried out to know their sensitivity and specificity.

40

E coli was the most common organism responsible for sepsis. CRP was reported to be highly sensitive (84.21%) and CBC was highly specific (75%). They concluded that blood culture is the gold standard for the diagnosis of septicemia. CRP is the most sensitive while CBC is most specific marker in neonatal sepsis.

6.Mandot S et al⁸. done a Prospective analytical study in a Tertiary care hospital in North India. Study subjects were newborns delivered in labour room/ operation theatre over a period of 8 months from November 2015 - June 2016.

This study was conducted to evaluate the use of umbilical cord blood culture in the diagnosis of Early onset sepsis as compared to peripheral venous blood culture. 80 neonates with two or more risk factors for early onset neonatal sepsis were included in the study. Blood samples were collected from umbilical cord and peripheral vein for culture. Sepsis screen was done to corroborate the diagnosis of neonatal sepsis.

41

As a result, sepsis screen was positive in 23 babies. Among these, 4 had grown organism on umbilical cord blood culture only. While 2 babies had both positive umbilical cord blood culture and peripheral venous blood culture. Organisms grown on culture were E.coli, Pseudomonas, Klebsiella and Acinetobacter. Hence they concluded that umbilical cord blood culture is simple and convenient method for the diagnosis of Early onset neonatal sepsis compared to peripheral venous blood culture.

7. Kalathia, Mitul Babubhai et al⁹. Done a study in tertiary care teaching hospital during May-June 2012. A total of 45 newborns with presence of two or more risk factors of sepsis were included. This study was conducted to evaluate use of Umbilical cord blood culture (UCBC) in diagnosis of neonatal sepsis as compared to peripheral venous blood culture. Blood sample from placental end of umbilical cord was collected and cultured. Primary outcome was diagnosis of neonatal sepsis by use of umbilical cord blood sample as compared with venous blood sample.

Secondary outcome was to compare organisms identifi ed by UCBC and venous blood culture.

A total of 24.44% (11 out of 45) high-risk newborns had positive UCBC. A total of 17.8% (8 out of 45) newborns had positive blood

42

culture report. Organisms grown in UCBC were Pseudomonas (45%, 5 out of 11), Acinetobacter (27.27%, 3 out of 11), Escherichia coli (18.18%, 2 out of 11), and Klebsiella (9%, 1 out of 11). They concluded that UCBC is a good method for diagnosis of neonatal sepsis among high-risk newborns as compared to venous blood culture with a sensitivity of 80% and specifi city of 91.43%. Organisms grown are comparable to blood culture samples.

43

STATISTICAL ANALYSIS

Chi Square test was used as a test of signifi cance.

A p-value of less than 0.05 was considered statistically significant.

The validity of UCBC for diagnosis of early neonatal sepsis was measure by mean of sensitivity, specificity, positive predictive value and negative predictive value.

ROC curve was performed.

Pearson coefficient correlation was used to assess the relationship between the variables .

44

OBSERVATION AND RESULTS

DISTRIBUTION OF CASES –GESTATIONAL AGE

TABLE1

GESTATIONAL AGE DISTRIBUTION

GESTATIONAL AGE

No.of study

Subjects %

< 28 WKS 110 27.5%

28 – 34 140 35%

34 – 37 80 20%

>37 WKS 70 17.5%

TOTAL 400

Among 400 neonates 35%(140) were between 28-34 weeks 27.5%(110) were less than 28 weeks,18%(80)were between 34-37weeks, 17.5%(70)were > 37 weeks.

45 TABLE 2

MODE OF DELIVERY DISTRIBUTION

Mode FREQUENCY PERCENTAGE

NORMAL 130 32.5%

LSCS 270 67.5%

Out of 400 neonates 270(67.5%) were born by caesarian section

46 TABLE 3

BIRTH WEIGHT DISTRIBUTION

Birth Weight FREQUENCY PERCENTAGE

< 1 kg 99 24.75%

1.0 - 1.5 163 40.75%

1.5 - 2.5 90 22.5%

> 2.5 58 14.5%

Total

Among 400 neonates 163(40.7%) were between 1-1.5 kg, 99(24.75%) were <1kg, 90(22.5%) between 1.5 to 2.5kg, 58(14.5%) were >2.5kg

47 TABLE 4

MATURITY DISTRIBUTION

Out of 400 neonates, 242 babies(60.5%) were born as preterm and158(39.5%) were born as term

MATURITY FREQUENCY PERCENTAGE %

Term 158 39.5

pre Term 242 60.5

Total 400

48 TABLE 5

SEX DISTRIBUTION

SEX FREQUENCY PERCENTAGE

MALE 186 46.5

FEMALE 214 53.5

Out of 400 neonates,214(53.5%) were females and 186(46.5) were male babies

49 TABLE 6

PARITY DISTRIBUTION WITH STUDY SUBJECTS

OBSTETRICS SCORE No.of study Subjects %

PRIMI 226 57%

GRAVIDA 2 14 4%

GRAVIDA 3 141 35%

GRAVIDA 4 16 4%

GRAVIDA 5 3 1%

TOTAL 400

50 CHART 1

PARITY DISTRIBUTION WITH STUDY SUBJECTS

Among 400 newborns 56% were born to primi mother,35% gravid 3,4% gravid 4 & gravid 2,1% born to gravida 5.

51 TABLE 7

Prevalence of Risk factors

Risk factors Frequency %

PREMATURITY 251 63%

LBW 315 79%

PROLONGED LABOUR 154 39%

PROM 160 40%

>2 PV 109 27%

MATERNAL FEVER 5 1%

BIRTH ASPHYXIA 131 33%

FOUL SMELLING LIQUOR 1 0.3%

52 CHART – 2

PREVALENCE OF RISK FACTORS

This table shows the prevalence of risk factors with low birth weight being the most common risk factor followed by prematurity and preterm rupture of membranes. In most of the cases, more than 2 risk factors were

associated.

53

Out of 400 neonates, 30(7.5%) were positive for UCBC, 26(6.5%) were positive for PVBC

Result of Umblical cord Blood culture and Peripheral Venous Blood culture

Culture Result UCBC (%) PVBC (%)

Positive 30 7.5% 26 6.5%

Negative 370 92.5% 374 93.5%

Total 400 400

54 TABLE 9

DIAGNOSTIC EFFICIENCY OF SEPSIS SCREEN TO UCBC

UCBC

TOTAL

SEPSIS

SCREEN Positive (%) Negative (%)

Positive 23 19.8% 93 80% 116

Negative 7 2% 277 97.5% 284

Total 30 370

Out of 400 ,116 were positive for sepsis screen, Among 116 ,23 were positive for umbilical cord blood culture. Which is statistically significant.

Comparison of sepsis screen results with UCBC results found to have sepsis screen sensitivity of 76.6% and specificity 74.8%.

Positive predictive value 19.6%

Negative predictive value97.5%

55 CHART - 3

DIAGNOSTIC EFFICIENCY OF SEPSIS SCREEN TO UCBC

56 TABLE 10

DIAGNOSTIC EFFICIANCY OF SEPSIS SCREEN COMPARED TO PVBC

PVBC TOTAL

Sepsis screen Positive (%) Negative (%)

Positive 15 13s% 101 87% 116

Negative 11 4% 273 96% 284

Total 26 374

Out of 400, 116 were positive for sepsis screen , and among 116 , 15 were positive for blood culture, which is statistically insignificant. In comparison to PVBC, sensitivity of sepsis screen was found to be 57.6%

and specificity was 72%

Positive predictive value12%

Negative predictive value 96%

57 CHART - 4

DIAGNOSTIC EFFICIANCY OF SEPSIS SCREEN COMPARED TO PVBC

58 TABLE - 11

Among 400 neonates,192 were showed clinical features,among 192 ,15 babies were positive for peripheral venous blood culture,11 were negative for culture, which is statistically insignificant.

ASSOCIATION OF CF-PVBC

PVBC TOTAL

CF Positive (%) Negative (%)

Positive 15 8% 177 92% 192

Negative 11 5% 197 95% 208

Total 26 374

59 CHART - 5

ASSOCIATION OF CF-PVBC

60 TABLE 12

Among 400 neonates,192 (48%) were positive for sepsis screen,among 192 ,12.5% were positive for umbilical cord blood culture ,which is clinically significant.

ASSOCIATION OF CF -UCBC

UCBC TOTAL

CF Positive (%) Negative (%)

Positive 24 12.5% 168 93% 192

Negative 6 3% 202 97% 208

Total 30 370

61 CHART - 6

ASSOCIATION OF CF -UCBC

62 TABLE 12

Association of sepsis screen –clinical features

CF TOTAL

SEPSIS

SCREEN Positive (%) Negative (%)

Positive 86 74% 30 26% 116

Negative 77 27% 207 73% 284

Total 163 237

63 CHART - 7

Association of sepsis screen –clinical features

Among 400 neonates sepsis screen positive for 116,among 116 (74%) were showed clinical features ,which is clinically significant.

64 TABLE 14

UMBLICAL CORD BLOOD

CULTURE

PERIPHERAL VENOUS BLOOD CULTURE

POSITIVE NEGATIVE

FREQUENCY % FREQUENCY %

POSITIVE 22 84% 8 2%

NEGATIVE 4 15% 366 97.8%

TOTAL 26 374

IN Comparison to PVBC ,the sensitivity of UCBC was found to be 84%

and specificity was 97%

Positive predictive value 73%

Negative predictive value 98%

65 CHART - 8

Coefficient correlation r = +0.310,which is statistically significant

66 CHART - 9

The area under the curve 0.6, which is significant.

67

ORGANISM UCBC PVBC

FREQUENCY % FREQUENCY %

Group B

streptococci 1 .2% 1 .2%

Staphylococcus

aureus 6 1.5% 7 1.75%

Klebsiella 7 1.5% 8 2%

Acinetobacter 8 2% 7 1.75%

Pseudomonas 1 .2%

E.COLI 5 1.25% 2 .5%

CONS 2 .5% 1 .2%

68

Culture isolates in UCBC samples Group B Streptococci grown in 1sample both in UCBC and PVBC. Staphylococcus aureas was found in 6 UCBC sample and 7 in PVBC samples .Klebsiella was found in 7 samples in UCBC and found positive in 8 PVBC samples.

Acinetobacter grown in 8 UCBC samples and 7 in PVBC samples.

Pseudomonas found positive only in UCBC.E.coli was found in5 samples in UCBC and 2 samples in PVBC, 2 samples were positive for CONS in UCBC and 1 sample in PVBV.In UCBC 370 (92.5%) samples did not show any growth,in PVBC 374(93. ) samples did not show any growth 0rganisms grown in both umbilical cord blood culture and peripheral venous blood culture were comparable.

69

RISK FACTOR UCBC +(30) (Positive)

UCBC-(370) (Negative)

PREMATURITY 20(66.6%) 231(62.4%)

LBW 25(83.3%) 290(78.3%)

MATERNAL FEVER 1(3.3%) 4(1%)

PROM 16(53.3%) 144(38.9%)

PROLONGED LABOUR 18(60%) 136(36.7%)

BIRTH ASPHYXIA 13(43.3%) 118(31.8%)

FOULSMELLING LIQUOR 1(3.3%) 0

>2 PV EXAMINATION 18(60%) 91(24.5%)

This table showed that distribution of risk factors in umbilical cord blood culture positive newborns.Prematurity is more common among all risk factors.

70 DISCUSSION

The present prospective cohort study was conducted among 400 neonates having risk of developing early onset sepsis .

Among 400 neonates 116(29% ) were positive for sepsis screen ,sensitivity and specificity of sepsis screen was 76% and 75% respectively when compared to UCBC,57% &76% when compare to PVBC.

Among 400 neonates 30 (7.5%) found to have UCBC result positive while remaining did not show any growth and 26(6.5%) found to have PVBC positive while remaining did not show any growth

Culture isolates in UCBC samples Group B Streptococci grown in 1sample both in UCBC and PVBC. Staphylococcus aureas was found in 6 UCBC sample and 7 in PVBC samples .Klebsiella was found in 7 samples in UCBC and found positive in 8 PVBC samples. Acinetobacter grown in 8 UCBC samples and 7 in PVBC samples. Pseudomonas found positive only in UCBC.E.coli was found in5 samples in UCBC and 2 samples in PVBC, 2 samples were positive for CONS in UCBC and 1 sample in PVBV.In UCBC 370 (92.5%) samples did not show any growth,in PVBC 374(93. ) samples did not show any growth

71 SUMMARY

This study was done To evaluate the role of umbilical cord blood culture in neonates at high risk of early onset sepsis

And To stydy correlation of umbilical cord blood culture with peripheral venous blood culture,To compare organisms grown in peripheral venous blood culture and umbilical cord blood culture was done in the Neonatal intensive care unit, Coimbatore Medical College Among 400 neonates were included in the study of which 116 were positive for sepsis screen ,sensitivity and specificity of sepsis screen was 76% and 75% respectively when compared to UCBC,57%

&76% when compare to PVBC

Among 400 neonates 30 (7.5%) found to have UCBC result positive while remaining did not show any growth and 26(6.5%) found to have PVBC positive while remaining did not show any growth

72 CONCLUSION

Blood culture obtained from an umbilical cord has good diagnostic validity for etiological diagnosis of early onset sepsis in high risk neonates as compared with PVBC.

Organisms grown in umbilical cord blood samples are comparable with venous blood culture

73 BIBLIOGRAPHY

1. Gotoff SP. Neonatal Sepsis and meningi s: in: Nelson textbook of Paediatrics. (15th Edi on). Eds Behraman RE, Kleigman RM, Arbin AM. Philadelphia, WB saunders company 1996: 528537.

2. Agrawal R, Sarkar N, Deorary AK and Paul VK. Sepsis in newborn. Ind J Paediatr 2001;68:114347

3. Stoll BJ, Hansen NI, Sanchez PJ. Early onset neonatal sepsis: the burden of group B Streptococcal and E. coli disease continues.

Pediatr. 2011; 127:817-26

4. Chawla D, Agarwal R. Rational approach to diagnosis of neonatal sepsis. J Neonatol. 2006; 20:4-7

5. Hansen A, Forbes P, Buck R. Potential substitution of cord blood for infant blood in the neonatal sepsis evaluation. Neonatology.

2005; 88(1):12-8

6. Sonawane VB, Gaikwad SU, Kadam NN, Gavhane J. Comparative Study of Diagnostic Markers in Neonatal Sepsis. J Nepal Paediatr Soc 2014;34(2):111-114.

74

7. Mandot S, Gandhi JS. Umbilical cord blood culture versus peripheral venous blood culture in early onset neonatal sepsis. Int J Contemp Pediatr 2017;4:53-6.

8. Kalathia, Mitul Babubhai et al. “Study of Umbilical Cord Blood Culture in Diagnosis of Early-onset Sepsis Among Newborns with High-risk Factors.” Journal of clinical neonatology vol. 2,4 (2013):

169-72. doi:10.4103/2249-4847.123092

9. Liu L, Johnson HL, Cousens S. Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet. 2012;379:2151-61.

10. Committee on fetus and newborn. Available at http://pediatrics.aappublications.org/content/129/5/1 006.full.html.

Accessed on 10 May 2013

11. Chawla D, Agarwal R. Rational approach to diagnosis of neonatal sepsis. J Neonatol. 2006;20:47.

12. Ghai Essential Paediatrics – 6th Edition Pg. 161-163.

13. Infections of the Neonatal Infant’, Nelson Textbook of Pediatrcs.

(2011) 19th edition ;118, p 629-648.

75

14. B.P.Zakariya, Bhat V, Arun Babu T, Joseph NM. Neonatal sepsis in a Tertiary care hospital in South India: Bacteriological profile and antibiotic Sensitivity pettern. Indian J Pediatr.(2010).

15. Nelson Text book of Paediatrics – 17th Edition Pg 623 – 640

16. Rajiv Aggarwal, Nupur sarkar, Ashok K, deorari and Vinod K Paul Sepsis in the newborn. Indian Journal Pediatrics. Vol.68(2):

pages 1143 – 1147, 2001

17. Nelson Text book of Paediatrics – 17th Edition Pg 623 – 640

18. Manual of Neonatal Care – John P . Cloherty 5th Edition Pg. 288 – 293

19. William E. Benitz, Michael Y. Han, Ashima Madan and Pramela Ramachandra : Serial Serum C- reactive protein levels in the diagnosis of neonatal infection. Pediatrics Vol. 102 : 1998.

20. R.S. Jaswal, R.k. Kaushal, Asha Goel and Kushla Pathania : Role of C-Reactive Protein in deciding duration of antibiotic therapy in neonatal septicemia. Indian Pediatrics Vol.40: pages 880 – 883, 2003

PROFORMA MOTHER DETAILS

Name:

Age:

Obstetric score:

BABY DETAILS Mode of delivery:

Birth weight:

Gestational age:

Sex:

RISK FACTORS Prematurity/LBW:

Prolonged labour:

Prom:

Maternal fever:

Pervaginal examination:

foul smelling liquor:

Birth asphyxia:

POST NATAL

NICU: SEPSIS SCREENING CBC:

CRP:

ANC:

Blood culture:

Organism:

Cord Blood:

Organism:

CLINICAL FEATURES lethargy:

poor feeding:

tachycardia/bradycardia:

skin mottling:

prolonged crt:

abdominal distension:

CONSENT FORM

Mr/Mrs ... are being asked consent for their child to be a participant in the research study titled

“ROLE OF UMBILICAL CORD BLOOD CULTURE IN PREDICTION OF EARLY ONSET SEPSIS AMONG NEWBORNS WITH HIGH RISK FACTORS” conducted by Dr.Krishna Kumari, Post Graduate student in the department of Paediatrics, Coimbatore Medical College and Hospital. Your child satisfies the eligibility as per the inclusion criteria. You can ask any queries you may have before agreeing to participate.

PURPOSE OF RESEARCH:

To Evaluate Roll of Umbilical Cord blood Culture in Neonates with Risk factors in Neonatal intensive Care Unit, Coimbatore medical college and hospital.

PRIVACY AND CONFIDENTIALITY:

Privacy of individuals will be respected and any

information provided will be kept confidential.

AUTHORISATION TO PUBLISH RESULTS:

Results of the study may be published for scientific purposes and/or presented to scientific groups; however your child will not be identified.

STATEMENT OF CONSENT:

I Father/Mother/Guardian of... give consent to participate my child in this study. I have read the consent form/it has been read to me. The study has been fully explained to me and I understand that I am entitled to explanations regarding the study as and when necessary.

...

Signature /Left thumb impression of patient/legal guardian of

the child with date ...Signature

of witness with date

KEY TO MASTER CHART Maternal age

<19 1 19-24 2 24 -30 3

>30

Mode of delivery

Labour natural 1 LSCS 2

BIRTH WEIGHT

<1 1 1-1.5 2 1.5-2.5 3

>2.5 4 Sepsis screen

Positive 1 Negative 2

Cord blood culture

Positive 1 Negative 2

Peripheral venous blood culture Positive 1 Negative 2

Sl.No NAME MOTHERS AGE GRAVIDA AMENORRHEA PREMATURITY LBW PROLONGE

D LABOUR PROM >2 PV MATERNAL FEVER BIRTH ASPHYXIA FOUL SMELLING LIQUOR MODE B,WT TERM GA CF CRP BC UBC OUTCOME

1 B/O ANANDHI 2 1 3 1 1 2 2 1 2 2 2 2 3 2 2 1 1 2 2 1

2 B/O MOHANASUNDARI 2 3 3 1 1 1 2 2 2 2 2 2 3 2 1 2 2 2 2 1

3 B/O PAVITHRA 1 1 3 1 1 1 1 1 2 2 2 2 3 2 1 1 1 1 2 1

4 B/O UMAMAHESWARI 4 4 3 1 1 2 2 2 2 2 2 2 3 2 1 2 2 2 2 1

5 B/O SHAJITHA 2 1 3 1 1 2 2 2 2 2 2 2 3 2 1 1 1 2 2 1

6 B/O PRIYA 2 1 3 1 1 2 2 2 2 2 2 2 3 2 2 1 1 2 2 1

7 B/O NAGARANI 1 1 4 2 1 1 2 2 2 1 2 1 1 1 2 1 1 1 2 2

8 B/O USHARANI 4 4 2 1 1 2 1 2 2 2 2 2 1 2 1 2 2 2 2 1

9 B/O SAJIFUNISHA 2 3 3 1 1 1 2 2 2 2 2 2 1 2 1 2 1 2 2 2

10 B/O GIRIJA 3 1 2 1 1 2 1 2 2 2 2 2 2 2 2 2 1 2 2 1

11 B/O RAJESHWARI 3 4 4 2 1 1 2 2 2 2 2 2 1 1 1 2 2 2 2 1

12 B/O SATHYA 2 1 3 1 1 2 1 2 2 2 2 2 1 2 2 1 1 1 2 2

13 B/O MEENA 2 3 3 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 1

14 B/O NANDINI 3 1 4 2 1 2 1 2 2 1 2 2 2 1 2 2 1 2 2 1

15 B/O SANGEETHA 2 1 4 2 1 1 2 2 2 1 2 2 2 1 2 2 2 1 2 1

16 B/O RIFANA ABUDHAKIR 2 1 4 2 1 2 1 2 2 1 2 2 1 1 1 2 1 2 2 1

17 B/O JANANI 2 1 3 1 1 1 2 2 2 2 2 2 1 2 2 2 2 1 1 1

18 B/O NANDINI 2 3 4 2 2 2 1 2 2 1 2 1 2 1 1 2 1 2 2 2

19 B/O KEERTHANA 2 1 2 1 1 1 2 2 2 2 2 1 2 2 2 2 2 2 2 1

20 B/O ARUNADEVI 2 3 3 1 1 2 2 2 2 2 2 2 2 2 1 2 1 2 2 1