AETIOLOGY OF NEONATAL INFECTION IN SOUTH

ANISA ASIA (ANISA)

ANISA

STANDARD
OPERATING PROCEDURE

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CONTENTS
1.

2.

3.

4.

5.

Introduction and Overview

1.1

Background of the study

1.2

Project goal

1.3

Objectives of the study

1.4

Activities

1.5

Study overview

Pregnancy and Birth Surveillance

2.1

Objectives of field surveillance

2.2

Pregnancy surveillance activities

2.3

Antenatal care

2.4

Postnatal care

15

Sepsis screening

17

3.1

Objectives of sepsis screening

18

3.2

Post-natal visits for sepsis screening

19

3.3

Case Identification

20

3.4

Signs of very severe infection/disease

20

3.5

Clinical assessment of young infants

21

3.6

Case management

23

3.7

Operational Definitions

24

Specimen Collection and Transport

25

4.1

Objectives

26

4.2

Blood Collection by Venipuncture

26

4.3

CSF Collection by Lumbar Puncture

28

4.4

Nasopharyngeal-Throat Swab Pool Collection

30

4.5

Specimens Transport to site lab

31

4.6

Specimens transport to central lab

31

Microbiological Analyses of Specimens

32

5.1

Objectives

33

5.2

Specimen Processing

34

5.3

Identification of Bacteria

43

5.4

Pathogen detection by TLDA

53

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6.

Specimen Storage and Bio-banking

56

6.1

Objectives

57

6.2

Specimen banking

57

7.

Data Capture and Management

59

7.1

Objectives

60

7.2

Database development

60

7.3

Database management

62

8.

Quality Assurance and Quality Control

63

8.1

Objectives

64

8.2

Laboratory QA & QC

64

8.3

QA & QC for Data Management

68

8.4

Field QA &QC

69

8.5

Role of CHRF during the preparatory phase of the study

70

9.

Waste management

73

9.1

Objectives

74

9.2

Introduction

74

9.3

Categories of healthcare waste

74

10.

Site specific Roles and Responsibilities

77

10.1 Investigators

78

10.2 Field staffs

78

10.3 Laboratory staffs

80

10.4 Data Coordination Centre

82

Appendix I
Culture media preparation

83
83

Appendix II

91

Appendix III

92

Study management review

92

Review of subject records

92

Observation of key study procedures

92

Standardization

92

Site monitoring report and feedback

92

Appendix IV

93

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1. Introduction and Overview

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1.1 Background of the study

Neonatal mortality at present, accounts for about 40% of under-5 child deaths in three South Asian
countries- Bangladesh, India and Pakistan; which account for more than a third of all neonatal
deaths in the world. Infection is a leading cause of neonatal mortality. However, current knowledge
on the aetiology of neonatal infections in developing countries comes from a small number of
studies in which bacterial aetiology was confirmed by blood culture for only 5-10% of suspected
serious infections. Thus, aetiologies of about 90% of the suspected infection cases remain unknown.
Furthermore, most of these studies did not capture infants during the first 3 days of life. These were
conducted in hospital settings, and included hospital-acquired infections. Moreover, infants who
reach the hospital for care are self-selected and are not representative of the population and these
findings are unlikely to represent the distribution of pathogens that cause disease in a community
setting. Thus, although approximately half of the neonatal deaths occur at home and a significant
portion of these deaths are attributable to infections, there are almost no data on the aetiology of
community-acquired neonatal infections. This remains a major impediment for the design of
effective community-based intervention programs to prevent and manage newborn infections.
In recent years, different antibiotic regimens have been used to treat neonatal infections and
several other regimens are under evaluation. Most of these treatments, however, are presumptive
and the diagnosis is based on clinical algorithms, which cannot permit a reliable differentiation of
invasive bacterial infection from other causes of ill appearance in neonates, e.g. viral infection, birth
asphyxia, complications of pre-term birth, etc. Although the currently used treatment regimens are
effective in reducing neonatal mortality by 30 to 50%, they may not be optimal for treatment
against pathogens in the community and may encourage the emergence of multidrug resistance
among bacteria circulating in the community. Moreover, even a successful treatment regimen is the
second choice to primary prevention. To date, maternal tetanus vaccination and clean delivery are
among the few proven interventions.
Knowing the pathogens that cause newborn infections, their distributions, including rates and
trends of antibiotic resistance, and the relative contributions of vertical vs environmental
transmission, would help enormously in taking evidence-based policy decisions for prevention and
treatment of neonatal infections.

1.2 Project goal

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This project aims to determine the population-based incidence, aetiology and antibiotic resistance
profiles of community-acquired infections in neonates in three high mortality South Asian countries
- Bangladesh, India and Pakistan, using standard and new diagnostic tests. The project also aims to
identify the risk factors of infections in neonates and the most appropriate battery of clinical signs to
predict serious infections.

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1.3 Objectives of the study

1.

2.

3.
4.

To establish community-based surveillance in young infants (0-59 days) to identify cases with
suspected infections and collect specimens to determine the aetiology of community-acquired
newborn infections in high mortality developing countries of South Asia.
To use standard and new diagnostics to determine i) community level aetiology specific incidence
of bacterial and viral infections in neonates during 0-6 days and 7- 59 days of life, and ii) pattern
of antimicrobial resistance among the isolated bacterial pathogens
To identify risk factors for serious bacterial and viral infections.
To identify clinical predictors of serious bacterial infections to refine algorithms for determining
whether referral and/or antibiotic therapy is indicated

1.4 Activities
We have identified four sites (3 rural, 1 urban) in South Asia that represents high neonatal disease
burden in South Asia. They have a track record of successfully conducting surveillance for neonatal
infections, established laboratory capabilities to perform microbiological and molecular assays, are
positioned to influence local and regional policy makers and receive immediate past and present
investments from Bill and Melinda Gates Foundation (BMGF), Saving Newborn Lives, USAID and other
major donors in global neonatal health. Thus, this study builds on sites with concurrent funding and
established expertise in conducting surveillance for neonatal infections.

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The sites will enhance methods as needed to ensure active capture of all illness episodes among young
infants aged <60 days in the catchment area. Because days 0-2 of life are of particular importance,
each site will conduct formative research before initiating enrollment to understand community
concerns about having study personnel present at or immediately after the birth. This will also explore
possible ethical incentives that will encourage families to inform Community Health Workers (CHW),
TBAs and/or other study personnel of young infants that become ill. Formative research to understand
the pattern of illness recognition and care seeking has already been conducted in several sites as part
of earlier studies. Since illness recognition and care seeking are limited, we will institute active
antenatal and postnatal surveillance at all sites. The surveillance will be conducted for two years. This
duration is important to minimize the impact of inter-year variation or outbreak of any organism . Less
than 2 years of surveillance will have the risk of over-representing some aetiology, specifically viruses
such as Respiratory Syncytial Virus and Influenza viruses, or missing some important but uncommon
aetiologies (e.g., Group B Streptococci, Listeria spp.). CHWs will routinely conduct home-based
assessments of infants for signs of serious infection according to the WHO Young Infant Integrated
Management of Childhood Illness (IMCI) algorithm. Infants meeting these criteria upon reassessment
by a study physician will be enrolled as cases of suspected infection after informed consent are
obtained from the caregiver. Blood, nasopharyngeal, and throat specimens will be collected from
every case; among cases treated at hospitals, cerebrospinal fluid (CSF) will also be collected if feasible.
In addition to cases of suspected infection, study workers will also enroll a group of infants without
signs of disease, to measure the background rate of infections in the community.
We estimated that about 14,000-20,000 young infants will be enrolled at each site over the two years.
About 10% of the young infants will have signs of infection, and laboratory tests will be conducted in
about 70% of the young infants with suspected infections. It is anticipated that 5% and 30% of the
young infants will have tests that are positive for bacterial and viral infections, respectively. This
manual is the layout of the whole study and it will guide the study activities, the responsibilities of
each person, and the procedures to be followed at each step of the study.

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1.5 Study overview

1.5.1 Surveillance Methods:
1.5.1.1 Pregnancy and birth surveillance
ANISA will establish a community based surveillance to capture young infants with suspected
infection. The surveillance population will consist of all women of reproductive age (13-49) and all liveborn infants delivered in the surveillance catchment of each field site. Trained community health
workers (CHWs) will conduct home visits every 2 months to update the list of married women of
reproductive age. At every visit, the women under surveillance will be asked about the date of the
first day of the last menstrual period (LMP) and the date will be recorded. Pregnant women will be
identified based on the missed period. All pregnant women will be eligible for enrolment, and will be
consented for their participation and that of their newborn infant in the study. A detailed verbal
informed consent process will be undertaken to ensure that participating women understand the
nature of the home-based infant surveillance that will begin shortly after newborn delivery. All
pregnant women in the surveillance areas will be offered a standard package of antenatal care
(adapted for each site and meeting national standards). Data on potential risk factors for infections in
young infants will be collected at antenatal and postnatal visits. Data collected at the initial enrolment
visit will include mothers age, parity, gestational age at recruitment, literacy, occupation, husbands
age and occupation, and other information. All study sites will develop a process to ensure notification
of delivery as promptly as possible within the first 24 hours to the local CHW by a family member
and/or the NCP, TBA, or other skilled attendant assisting the delivery.

1.5.1.2 PNC visit and Sepsis surveillance

Surveillance for suspected sepsis among young infants will entail both active and passive components.
Active surveillance is based on frequent, scheduled CHW home visits to question caregivers and assess
young infants for clinical signs of sepsis. Passive surveillance captures cases that present to designated
health facilities due to parental care-seeking; caregivers will be encouraged to seek care at such
facilities if signs of illness arise between CHW visits. CHWs will aim to visit all newborns in the home
within 6 hours of birth and not later than 24 hours. If delivery occurs at a facility or outside the
surveillance area, babies will be eligible to be enrolled in the study if they return to their home within
seven days of birth. It is possible that some pregnant women will deliver in a location other than the
place in which the initial pregnancy enrolment visit occurred.. A field tracking system has been
developed at the study sites to identify new arrivals into the catchment area for potential enrolment.
For each newborn delivered, data on date and time of birth, sex, gestational age based on LMP, weight
of the infant, care given during and immediately after birth (bathing, massage, cord care,
breastfeeding practices) will be recorded.

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The general health status of the infant will be assessed by the CHW using the WHO Young Infant IMCI
guidelines. After first visit, the CHW will return on days 2, 6, 13, 20, 27, 34, 41, 48 and 59 after birth to
question the mother regarding any illness of the infant in the intervening period, and assess the status
of the infant using the WHO Young Infant IMCI guidelines. CHWs will refer infants meeting the IMCI
criteria for very severe disease to the nearest designated facility for care and further evaluation. If
caregivers accept referral, CHWs will arrange prompt transport to pre-designated hospitals/clinics that
are equipped to collect specimens and manage sick young infants. If caregivers refuse facility referral,
CHWs will revisit the family on the next day to convince them to comply with referral.

1.5.2 Specimen collection

Blood and respiratory specimens will be collected from the enrolled infants at the facility by trained
personnel. CSF specimens will be collected from hospitalized suspected sepsis cases by a trained study

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physician in the designated hospitals. All specimens will be promptly transferred to the laboratory. In
case of refusal to go to the facility, blood and respiratory specimens may be collected in the home, if
trained personnel can be deployed, consent is provided, treatment can be ensured and supplies are
available within the necessary timeframe.

1.5.2.1 Blood
Up to 3.0 ml of blood will be collected from each infant by a trained phlebotomist. The specimens will
be divided into 2 aliquots. One aliquot (0.5-1 ml) will be directly inoculated into a BACTEC paediatric
blood culture bottle. The signal positive culture bottles will be sub-cultured. The growth will be
identified, and tested for antibiotic susceptibility following Clinical Laboratory Science Institute (CLSI)
guidelines (2010) and preserved for future characterization for example of serotype, antibiotic
resistance mechanisms, or molecular finger printing. Second aliquot will be inoculated into a DNAse
0
and RNAse free tube containing anticoagulant and stored at 2-8 C. The specimen will be transported
to the field site laboratory within 3-4 hours of collection, where it will be further divided into 2 subaliquots if there is more than 2 mL of blood. One sub-aliquot will be used for detection of genetic
signatures of multiple pathogens. Molecular testing will be conducted at a central laboratory in each
country; laboratories will use a common automated extraction method and run PCR using customized
TLDA plates. Any remaining will be preserved for future collaborative work with BMGF. The other subaliquot will be stored in a biobank. The specimens will be stored at -70C as soon as possible after
collection to preserve fragile components (IL6, RNA etc) intact.

1.5.2.2 Respiratory Specimens

Remels dual swab (M6) with micro-tip and a traditional-tip flocked swab will be used to collect nasopharyngeal and throat specimens respectively. Both specimens will be pooled in the same transport
0
media, storedat 2-8 C, transported to the field site lab within 4-6 hours, aliquoted and preserved in
liquid nitrogen, until transfer to the central in-country laboratory for preservation at -70C. One of the
aliquots will be used for molecular detection of respiratory pathogens by a customized TLDA panel (fig:
4) and the other will be kept ina bio-repository.

1.5.2.3 Cerebrospinal Fluid (CSF)

Lumber puncture will be performed on hospitalized ANISA case-patients. Upto 2ml of CSF will be
collected for immediate plating on Chocolate, Blood and MacConkey agar media and analysis for cell
count and biochemical parameters. Left over specimens, if available, will be preserved at -70C and
tested for bacterial and viral aetiology using molecular techniques (Fig: 4).

1.5.3 Aetiology Detection Method

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The list of possible pathogens for infection in young infants is diverse and varies geographically.
Further, many of the pathogens are also commensals and colonizers. Therefore, it is difficult to make a
comprehensive list of pathogens for detection by targeted or pathogen-specific molecular assays.
ANISA investigators determined that the preferred molecular diagnostic approach is the TLDA
platform, using cards customized for the signatures of the organisms anticipated to cause neonatal
infections in the study setting.

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Pregnancy Surveillance

2 monthly
home visits
By CHW

Figure-1 Study Profile

PW Identified

PW Not Identified

Consent given

LMP-EDD

Enrolled

Demographic data

Basic antenatal intervention package

Visit-1
Collect
Within 24 hrs

Pregnancy Outcome

Birth

assessment

Postnatal follow-up visits

2
20
41

6
27
48

13
34
59

Continue Follow-up
visit

Refused
Revisit

Suspected
infection

Sepsis screening

Clinical algorithm

If denied

Accepted

If not
Routine care

Referred to facilities

Eligibility criteria
Assessed by doctor

Bio-banking
RS

Treatment
at Facility

Not eligible

PCR
2

Eligible

Consent taken
Send back to
home

Specimen
collected

3
1

Blood

6
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Bio-banking

CSF culture
Cytology

Blood Culture
PCR

CSF*

* Will be only
collected at
Hospital

Antigen test
PCR
Bio-banking
Back to CONTENTS

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2. Pregnancy and Birth Surveillance

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2.1 Objectives of field surveillance

1.

2.
3.
4.

To establish community-based surveillance in young infants to indentify cases of suspected

infection, and collect specimens from suspected infection cases to determine the aetiology of
community-acquired infections.
To collect demographic and behavioural information of the population to identify the risk
factors for neonatal infections.
To collect data on pregnancy, delivery complications, and signs of infections in young infants
for identifying clinical predictors of neonatal sepsis.
To promote antenatal /postnatal care for delivery of a healthy baby and healthy mother

Reproductive Married woman (13-49 yrs)

CHW

Registered

2-monthly
pregnancy
surveillance

LMP recorded

Pregnancy Identified

Pregnancy not identified

Consent taken
Enrolled

Refer
care

Data

If complication found
Counseling
Provide Iron & &
Folate

ANC visit-1

Pregnancy
Outcome

ANC visit-2

Birth
assessment
Still
Birth

Live
Birth

Sepsis surveillance

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Miscarriage

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2.2 Pregnancy surveillance activities

2.2.1 Two-monthly pregnancy surveillance
A CHW will visit all households within her assigned area
Objectives
every two months to identify pregnant women.We
To maintain an updated list of every
expect that the CHW will have 20 working days every
married women of reproductive age (15month for pregnancy surveillance activities. Each
49 years) (MWRA)
CHWs area will be divided into units to help her
complete one full round of surveillance in every two
To identify women in the study area in
months.
early pregnancy
The major responsibilities of CHWs during the 2monthly household visits include:
To enrol the pregnant women in the birth
1. Maintaining and updating a list of MWRA in the
surveillance
pre-printed MWRA Register.
To
collect
socio-economic
and
2. Asking every registered MWRA about the first day
demographic
information
of
the
of the last menstrual period and whether she is
household
and
of
the
enrolled
pregnant
currently pregnant, at the 2-monthly visits,
woman, and collect data on pregnancy
3. If the MWRA reports being pregnant, collecting
complications and care seeking behaviour
information on first day of Last menstrual period
(LMP), socio-economic and demographic variables,
and calculating the expected delivery date (EDD).
4. Recording the information in the MWRA Register and Pregnancy Enrolment Form (Form 02):
5. Sending the completed Form 02 to their supervisor.
6. Providing a 2-month supply of iron-folate supplements to the pregnant women
7. Encouraging the pregnant women to seek antenatal care from a designated provider
8. Scheduling the date of the first antenatal birth-preparedness home visit
9. Collecting data on pregnancy complication and history of care seeking behaviour.

2.2.2 Guidelines for pregnancy identification

CHW will ask, during every visit, about the first day of the last menstrual period of all women aged 1349 . A pregnancy will be suspected pregnant if she fulfils at least one of the following criteria:
1. A regularly menstruating woman aged 13-49 reporting missed menstrual date at the 2-monthly
home visit by the CHW;
2. A woman who discontinued using injectable contraceptive with the intention of becoming
pregnant is still amenorrhoeic, 6 months after discontinuation;
A woman with lactational amenorrhea, who suspects pregnancy and is still amenorrhoeic
An in-migrant woman with more than one month of amenorrhea.

2.3 Antenatal care

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2.3.1

Antenatal birth preparedness home visits

CHWs should deliver the first antenatal visit to the

The proposed antenatal visit schedules are:
enrolled pregnant woman preferably within 3-4
1st visit | 12 16 weeks of pregnancy
months of pregnancy. If a pregnancy is identified at
less than 3 months of gestation, the CHW will make
2nd visit | 28 30 weeks of pregnancy
the first antenatal visit on her next scheduled 2monthly visit to that household. If a pregnancy is identified in the second or third trimester (i.e., after
12 weeks), CHW will make the first antenatal visit at the same visit. If a pregnancy is identified after 24
weeks of pregnancy, the CHW will combine the two antenatal visits into one, and visit the pregnant

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woman within a week of identification; there will be no

Building rapport with the family
second antenatal visit. During these visits, CHW will advice
members
the family members on some essential skills and practices
When a CHW reaches the household of
for safe and clean delivery and newborn care, through
the pregnant women, she will exchange
demonstrations and interaction using printed materials
customary greetings with the family
and visual aids (i.e., birth preparedness card and doll). It is
members. She will introduce herself and
important that each CHW maintain a good communication
explain the purpose of her visit. She will
skill to establish a bond with the families and should be
identify the pregnant woman and ask
very careful so that the pregnant women do not feel shy
about her health, sit with the family
when talking with them. CHW should follow a sequence of
members and describe different care
listed activities so that the demonstration procedures
seeking behaviors.
remain uniform to all families. CHW will ask the mother
whether she has made any Ante-Natal Care (ANC) visits
and if yes, then how many. If no ANC visit has been made,
CHW will explain the importance and reasons of ANC with
the family.
1. ANC visits help to assess the condition of pregnant women
2. ANC visits increase the chances of a good outcome of pregnancy - a healthy baby and a healthy
mother
3. ANC visits help early identification of complication and its management, including appropriate
referral
ANC visits also educate the mothers and their families regarding proper homecare for the pregnant
women and the newborns.

2.3.2 Taking consent

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The first consent for this study will be taken when a woman is found pregnant during the 2-monthly
pregnancy surveillance visits to a household. This consent will be obtained at the first ante-natal home
visit by the CHW, whenever it takes place. In some cases, the pregnancy will not be identified before
the birth and thus the first consent will not be obtained. However, these babies will be included in the
study after taking consent at the first postnatal visit. It is important to include newborns at any age up
to day 59 days of life. Missing antenatal visits is not an exclusion criterion for this study. Furthermore,
a woman may have received antenatal visits in a different cluster. For example, a woman may register,
provide consent, and receive antenatal visit 1 and/or antenatal visit 2 in her home cluster, but gives
birth in another cluster or outside the catchment area before the CHW of that cluster knows about the
pregnancy. In such cases, where CHWs does not meet the woman until the First Post-natal care Visit
(PNC1) visit, they will obtain consent at the time of the first postnatal visit. The first consent will be
taken from a new pregnancy identified in a CHWs catchment area.
The second consent will be taken before specimen collection from ill babies or healthy babies selected
as controls. While taking consent, the CHW will provide a printed consent form (in the local language)
to the pregnant woman and request her to read. In the cases where the women cannot read, the
consent form will be read out loudly and clearly for her understanding about the study. The CHW will
ask if the woman has any queries. The CHW she will then ask the woman whether she agrees to be
enrolled in the study. The CHW will record the verbal consent of the woman in her register book, get
the signature or finger-print of the witness and countersign the consent form. Enrolment will be
voluntary. If a pregnant woman refuses to participate in the study, she will still be offered the routine
antenatal care package. If at a later stage (within 59 days of birth of the baby) the woman decides to
participate in the study, she may do so at any of the subsequent antenatal visits or after the birth of
the baby.

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Calculating the Expected Date of Delivery: CHW will ask the pregnant woman about the date of her
LMP and calculate the EDD using the calendar provided in Appendix II. CHW will use
Bangla/Hindi/Urdu-English conversion table where necessary. The expected delivery date will be
mentioned to the pregnant woman and her family members so that they can take preparations.

2.3.3 Provide Iron and folic acid to pregnant woman

The CHWs will provide required doses (a pack of
30 tablets for each month) of Iron and Folic acid
tablets and encourage pregnant women and
their family members so that the pregnant
women take the Iron and folic acid tablets once
daily during pregnancy till delivery. If the study
site does not provide any medicine, then the
CHW shall educate the mothers and family
members on how to get the medicines for free
or at low price.

Counselling
Iron and Folic acid tablets protect mothers
from anaemia.
The mother loses blood during delivery.
These iron and folic acid tablets
compensate for the loss of blood.
Supplements provide a protection against
blood loss and ensure a healthy mother and
a healthy baby.

2.3.4 Counsel on Tetanus Toxoid Vaccination

CHW will try to make sure that the pregnant woman has been properly vaccinated with Tetanus
Toxoid vaccine. She should help the pregnant mother understand that Tetanus is a dangerous disease,
and it can kill both the mother and the baby. CHW should also explain to the mother that taking
proper doses of Tetanus Toxoid vaccine according to national immunization schedule can protect the
mother and the baby from harmful effects of tetanus. Additionally, she will also describe the sources
from where pregnant women can get TT vaccine. CHW will follow up the TT vaccination in the
subsequent visits.

2.3.4.1 Recommended schedule of TT vaccination:

Immunization program
TT 1: at the age of 15 years
st
TT 2: after 1 month of 1 dose
nd
TT 3: after 6 months of 2 dose
rd
TT 4: after 12 months of 3 dose
th
TT 5: after 12 months of 4 dose

All women of reproductive age (13-49 years), regardless of

marital and/or pregnancy status, should have received the five
doses of TT vaccine according to immunization program.
Pregnant women who have not received TT vaccines earlier
should get two doses of TT vaccine at an interval of 4 weeks. The
CHW shall also promote TT vaccination at recommended
schedules:
If the mother comes later, then she could be given two
nd
doses but the 2 dose must be given one month before
Immunization schedule at pregnancy
st
th
1 dose: at 5 month of pregnancy
the EDD. If two injections have been given in the
nd
th
previous pregnancy, then only one is required. This can
2 dose: at 6 month of pregnancy
be given at any time during the pregnancy.

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2.3.5 Explain Proper Nutrition of Pregnant woman

CHW will explain to the pregnant woman and the family about the importance of taking extra food
during pregnancy.
CHW will do the following:
1. Explain that the mother needs a variety of foods available at home, including milk, fruits, green
leafy and other vegetables, meat, fish, eggs, etc.
2. Verify the family understands her messages about workload, rest and diet.
3. Give advice about the diet for a healthy mother and baby
4. Ask about the pregnant womans diet

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5.
6.

Verify the womans understanding of messages about diet

Suggest the foods and drinks that she should consume on a daily basis.

2.3.6 Counsel on proper rest and workload

CHW should discuss the importance of taking proper rest during pregnancy. She should enquire about
the present workload of the pregnant woman and guide her on what she needs to do and what she
should not do.
1. Ask about the pregnant womans activities and workload
2. Suggest adequate rest during and after pregnancy
3. Inform the family about the importance of rest during pregnancy and after child birth
4. Discuss with the family different ways to share or reduce her
Suggested food
workload
5. Encourage the pregnant woman to continue routine light Milk 2 cups (400 ml)
work during pregnancy. This includes cooking, washing, Pulses 2 cups
cleaning, etc.
Cereals 300 g
6. Emphasize on avoiding heavy work, eg. Washing clothes, Eggs 2
fetching water, moving heavy objects (>4 Kg), etc.
Meat / fish 2 servings
7. Encourage her to take rest for at least two hours during the Leafy vegetable 2 servings
day
Fruits 2 servings
8. Encourage family members to share the pregnant womans
work, especially the heavy work like fetching water, washing
clothes, etc.

2.3.7 Promoting recognition of danger signs during pregnancy

CHW will explain to the family members, especially the decision makers, about the danger signs during
pregnancy so that they can make a decision in case of emergency.

Explain the following danger signs for the mother and the newborn:
1. Severe headache, blurred vision, and/or swollen legs, hands, and face (during pregnancy, during
delivery, or during post natal period)
2. Convulsions (during pregnancy, during delivery, or during post natal period)
3. Vaginal bleeding during pregnancy and excessive bleeding during or after delivery
4. High Fever (>100.4F) and for three days or more. If there is concern about high temperature, the
family can call the CHW to have her measure the temperature
5. Reduced foetal movement

6. Severe lower abdominal pain

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If and when one or more of these danger signs are detected, advise the family members to seek
emergency medical treatment. Mention that if these danger signs are ignored, it can have severe
consequences, including the death of the mother or the child or both.CHW will ask the mother if she
has any of the danger signs. If any of the above danger signs are present, then she will refer the
woman to the hospital with referral slip.

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2.3.8 Birth and delivery preparedness

2.3.8.1 Counsel on safe and clean delivery place
CHW will counsel the pregnant woman and her family members to select a health facility for the safe
delivery and prepare necessary transport, blood donor and money. If the family is not willing to go to a
health facility and chooses home delivery, then suggest the family members to select a safe and clean
delivery place.
A delivery place should have the following criteria
1. The room should be clean with a flat surface
2. It should have adequate light and air
3. It should not be damp or moist (no need to put a layer of cow manure or anything like that)
4. If the floor is chosen for delivery, a mattress/blanket should be spread and covered with a clean
sheet
5. A Clean plastic sheet should be placed right under the perineum just before the delivery
6. Hands should be washed properly (see hand washing section) before spreading the sheet

2.3.8.2 Choosing a birth Attendant

In case of home delivery, CHW will encourage the family to select a skilled birth attendant well in
advance. If skilled birth attendant is not available, encourage the family to select a person with similar
experience.
2.3.8.2.1 Selecting a caregiver for the neonate right after birth
CHW will find out the roles of various family members during and after delivery (monitoring labour,
conducting the delivery, cutting the cord and delivering the placenta). She will also find out the person
who will be present at the time of birth and will be available to take care of the baby right after birth
regarding the following issues:
1. Drying and wrapping the baby
2. Helping the baby to breathe (if needed)
3. Ensuring the baby is breastfed after birth,
4. Clean cord cutting and healthy skin and cord care during delivery

2.3.8.3 Protecting the neonate from infection

CHW will emphasize on the importance of the Five Cleans that
Five cleans
will help to keep the mother and the baby free from infection.
Clean hands
CHW will help the mother and family members to understand
how they can maintain the five cleans.
Clean perineum
1. Discuss the equipment and supplies the family will need to
Clean delivery surface
take care of the mother and the baby during labor and
Clean blade and thread for
delivery.
cord cutting and clamping
2. Ask if the family has already bought/acquired a birth kit, or
Clean cloth for mother and
whether they have some other materials for the delivery
wrapping the baby
3. Explain the importance of having a clean birth kit
4. Advise the family to use a birth kit and clean dry clothes for the baby during and after the delivery

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13

2.3.8.4 Promoting clean birth/delivery kit

A birth kit should be prepared before the EDD. It should contain a clean and sterile blade to cut the
cord; three pieces (size should be sufficient to wrap up the baby) of clean and soft cloth for drying and
wrapping ,and the third to be used as a neck support if it is a non-breathing baby who might be
needing resuscitation.

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2.3.8.5 Promoting bathing care

CHW should ask the family members when they plan to give the baby its first bath, and who will bathe
the baby and how. She will explain the advantages of delaying (at least 72 hrs) the first bath and not
removing the white skin (vernix). Delaying the first bath keeps the vernix intact, helps maintain the
body temperature, and also prevents from getting infection.

2.3.8.6 Promoting cord care

CHW will explain to the senior family member (or the selected family member) about taking care while
cutting the cord. The cord should be kept dry and clean after cutting. Nothing should be applied to the
cord immediately after it is cut. Substances like mud, ashes, oil or cow dung may directly harm the
baby by increasing the chance of Infection. Umbilical cord should be cut very carefully maintaining
proper hygiene. The equipment usually used to cut the cord is a potential source of umbilical infection
if the cord is heavily soiled after cutting (with faeces or urine). The cord may be gently cleansed with
water. She should teach the senior member to abide by the following procedure:
1. Hands must be washed with soap before cutting the cord.
2. Before cutting the cord should be tied at three places, using a clean thread/ thread(s) from
Delivery Kit). The first thread should be tied about two finger widths from the abdominal wall. The
second should be one finger-width away from the first one, and the third should be tied four
finger widths away from the second.
3. Using a clean blade, the cord should be cut in the space between the last two threads, and about
one finger width away from the second one.
4. The cord stump should not be tightly bound and/or covered. The cord stump area should be left
open or covered loosely with a cloth. If a nappy or other cloth is used, it should be folded below
the cord stump.
Signs of cord infection include fever, redness of the skin around the umbilical stump, and oozing of pus
or fluid from the umbilical stump. These signs may be localized to the area around the stump or may
lead to serious illness and should be monitored. Care should be sought from health care providers in
such cases without delay

2.3.9 Second antenatal care visit

Focus should be on the roles of household members in newborn care during her second antenatal
visit. In counseling the household members, the CHW will regenerate rapport and ask about the
current roles of various female household members in safe and clean delivery, keeping the baby warm,
importance of cord care, breastfeeding, danger signs for both the mother and the neonate and bathing
the newborn. The CHW will Negotiate and participate in decision making on various issues of newborn
care, especially cleaning and birth asphyxia management. If needed, the CHW will make an extra effort
to counsel the male member/s of the household for emergency medical care.

2.3.10 Birth notification to the CHW

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14

The study sites will use their own mechanisms to notify the CHW of a birth.

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2.4 Postnatal care

Postnatal care visits will enable CHWs to
interact with female household members
and emphasize proper newborn care and
postpartum care for the mother. While
counseling the household members,
CHWs will build rapport and ask about the
current roles of various female household
members in feeding, changing clothes and
keeping the baby warm, cord care, and
bathing of the newborn baby. CHWs will
follow a similar technique of negotiation
and participation in decision-making on
various issues of newborn care as she
does in the Antenatal home visits. If
needed, she will make an extra effort to
counsel the male member(s) of the
household for emergency medical care.
CHW will counsel the mother and her
family members for postnatal visits to the
nearest health facility at least once in the
first week and two times within 6 weeks
of delivery. PNC visits will be aimed at
educating, preventing, and treating
postnatal illnesses of the mother and
baby.

Counseling during PNC Visit

1. Advice the mother to take Iron and Folic acid
tablets regularly for a month (one per day)
2. Advice the mother to take one vitamin A capsule
according to the study site schedule after the
delivery to make sure her breast milk has enough
Vitamin A for the baby.
3. Explain benefits of drinking boiled or purified
water
4. Recommend that the mother should eat an extra
handful of food at every meal, or eat an additional
meal everyday.
5. Advise the mother and the family members so the
mother includes additional food in her diet: i.e.
green leafy vegetables, vegetables with high
water content, fresh fruits, eggs, fish, meat, and
milk or milk products.
6. Advise the mother and the family members to use
iodized salt.
7. Ask the family members about who takes care of
household work during the postpartum period,
for example: cooking, washing, fetching water,
taking care of newborn and other children
8. Explain to the family the importance of rest for
the mother during the postpartum period.

Note: If the CHW is attending the delivery, then she will begin with the Newborn Assessment
guidelines (given separately in another document). In conducting the session, help the family
members practice some skills through counseling. A detailed description of messages and their
contents are provided in the following sections of this chapter for the counseling sessions during the
postnatal home visits.

2.4.1 Promoting Newborns care

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15

2.4.1.1 Teach how to keep the baby warm

Explain and educate the mother and her family members how to keep the baby warm. Baby care
should be done in a room which is warm and not damp. The baby should be in the same room and
sleep with the mother in the same bed. The baby should have warm clothes. The whole body should
be wrapped from head to toe, and an extra one or two layers of clothing and coverings should be
there for the baby - specifically if the baby is small (i.e., low birth weight). Advice the mother on the
importance of skin-to-skin contact, that it reduces the chances of hypothermia or simply protects from
getting cold. Educate the caregiver to hold the baby at all times and never to put it on the

ground.

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2.4.1.2 Bathing protocol

Ask the family member about the benefits of
delaying the first bath. Ask the family members to
take the options. If there is a national guideline
regarding this issue, then CHW will counsel the
pregnant woman and the family members
according to that. If any national guideline is not
available, then counsel them that option # 1 is the
best, option # 2 is better and option # 3 is good for
the health of the neonate.

Option # 1 Message: No bath within 72

hours of birth.
Option # 2 Message: Sponge cleaning as
delayed as possible.
Option # 3 Message: Bathing as delayed
as possible

2.4.1.3 Cleaning the newborn

Explain how to provide eye care. Advice the mother and her family members to keep the babys eyes
clean. Counsel the family members regarding bathing and the care of eye. Explain advantages of not
removing the white skin (vernix)

2.4.1.4 Care for skin, eye, and umbilical area

Counsel the bathing procedure and timing. Explain advantages of delaying the first bath and remind
the family and the mother about delaying the first bath. Also advice how to take care of the umbilical
area. Suggest alternatives to bath (wiping, etc.) Counsel the mother not to apply any Surma/Kajal,
lamp soot, etc. in the eyes, as these can irritate the eyes.

2.4.2 Promoting exclusive breastfeeding on demand

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16

Explain advantages of exclusive breastfeeding and problems with feeding other foods and drinks to
babies less than 6 months of age. Ask her to demonstrate breast feeding - check for position and
attachment, breast problem. Explain what Colostrums is and how it helps the baby in fighting diseases.
Ensure the understanding of the mother and other family members regarding the importance of
exclusive breastfeeding (only breast milk and nothing else - not even water). Mention that anything
else is bad for the health of the baby before 6 (six) months. Help the mother to solve any problems she
might have in breast feeding the baby (i.e. sore nipples, enlarged breasts, inverted nipples). (For
detailed instructions, follow the breastfeeding module.) Advice the mother to feed the baby at any
time of the day or night (whenever it cries).

Back to CONTENTS

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17

3. Sepsis screening

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3.1 Objectives of sepsis screening

1.

To follow-up the newborns up to 59 days on the scheduled dates to identify suspected serious
neonatal infections according to the inclusion and exclusion criteria
To refer the neonates with suspected infection to healthcare facilities for seeking care

2.

CHW

Newborn assessment
1st visit within 24 hrs
Data
Collected

Maternal
complications
Labour & delivery
details

Next Visit

New born assessment

Care seeking behaviour
Risk factor data

Data
Collected

48

6
13

Follow up
visit 1

20
nd

59

27

41

34
Healthy

Screening at every visit

If 2
referral
not accepted

Meet case definition

Referral not accepted
Next day visit

Referral to designated facility

Referral accepted

Reinforce for referral

Not eligible

Assessed by physician

Eligible

Consent taken

Specimen collected

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18

Accepted

Advice for hospitalization

Treatment at OPD

Not accepted

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3.2 Post-natal visits for sepsis screening

There will be twelve postnatal care visits in this study, and these will be scheduled for Day 0 (0 - 24
hours), Day 2, Day 6, Day 13, Day 20, Day 27, Day 34, Day 41, Day 48 and Day 59. Each of these visits
will commonly be denoted as PNC0, PNC2, PNC6, PNC13, PNC20, PNC27, PNC34, PNC41, PNC48 and
PNC59 respectively. PNC0 visit should be conducted as soon as possible (within 6 hours of birth is
preferred) after birth. However, it may be conducted within 24 hours of age. If the infant has already
reached 24 hours of age when the first PNC visit is made, PNC0 will be considered missed, and the
PNC2 will be conducted.
Schedule of Post-natal Care (PNC) visits
st
1
PNC0 on the 1 day (0 - 24 hours) of delivery
2
5
8

rd

PNC2 on 3 day of
delivery
st
PNC 20- on 21 day of
delivery
nd
PNC 41- on
42 of
delivery

3
6
9

th

PNC6 on 6 day of
delivery
th
PNC 27- on 28 day of
delivery
th
PNC 48- on 49 of
delivery

th

PNC 13- on 13 of delivery

PNC 34- on 35 day of

delivery
th
PNC 59- on 59 of delivery

10

th

3.2.1 Performing an examination of the baby

1.
2.
3.
4.
5.
6.
7.
8.

Seek permission to examine the baby.

Perform an examination of the newborn for any abnormalities according to the clinical algorithm.
Ensure the newborn is in the mothers arms while the examination continues.
Make an effort to do the job quickly so that there is no heat loss from the newborns body.
Identify the danger signs and seek care
Ask family about the danger signs they know (newborn and maternal postpartum signs)
Mention other danger signs they did not mention and explain what can happen if danger signs are
ignored, and why it is important to seek care
Verify their understanding of the danger signs

3.2.2 Postnatal danger signs of the mother

3.2.3 Danger signs of the newborn

1. Fever of 100.4F and for three days or more

2. Excessive bleeding (bleeding that increase rather than
decreases or large blood clots)
3. Foul smelling vaginal discharge
4. Fainting, fits, or convulsions
5. Severe pain in the abdomen or pain that keeps
increasing
6. Pain, swelling, and /or redness in the breast

1. Convulsions
2. Difficult breathing- Severe chest indrawing
3. Mother feels the baby is hot
4. Mother feels the baby is cold
5. Weak, abnormal or absent cry
6. Lethargic or less than normal movement
7. Feeding problem

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19

3.2.4 Caring for mother & newborns personal hygiene

Explain to the mother and the family members about the importance of regular bathing and
cleanliness of the baby and its caregiver (regular changing, washing and drying with home-made
sanitary napkins, washing hands with soap after using the toilet and before taking care of the baby
emphasize that all the members of the household who are involved in taking care of the newborn
should also wash their hands with soap. Explain that the baby should not be left in wet clothes for a

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long time. Explain that the cleanliness of the mother and others who hold the baby affects the health
and well being of the baby.

3.2.5 Immunization of the newborn

Inform the mother and her family members about scheduled visits to Immunization centers to get the
baby vaccinated on time. Explain the importance of vaccination for the survival of their baby. Also
inform them about the availability of Immunization services nearby.

3.2.5.1 Immunization schedule

Vaccination dates will be recorded in the immunization card and the child record. The mother will be
advised when to return for the next immunizations. CHW will counsel the pregnant woman about the
national immunization schedules. If there is no set immunization schedule, follow the following
schedule. CHW will counsel the pregnant woman about the national immunization schedule
Immediately after birth | BCG (if missed, then with DPT-1)
At 6 weeks

|DPT-1 (diphtheria, pertussis, and tetanus), HepatitisB -1, HiB and OPV-1

At 10 weeks

| DPT-2, Hepatitis- B -2, HiB and OPV-2

At 14 weeks

| DPT-3, hepatitis B -3, HiB and OPV-3

At 9 months

| Measles, OPV-4

3.3 Case Identification

Inclusion criteria
(Any one of the following seven)
1. history of or observed convulsion,
2. history of not feeding well (on the day of
evaluation)
confirmed
by
feeding
assessment,
3. fast breathing (respiratory rate 60 breaths
or more per minute on repeat count )
4. severe chest in-drawing,
o
o
5. axillary temperature >38.0 C (>100.4 F)
o
o
6. axillary temperature <35.5 C ( <95.9 F),
7. movement only when stimulated or no
movement at all

Exclusion criteria
1. Surgical conditions requiring urgent care would
be serious cleft palate (resulting in difficulty in
feeding),
bowel
obstruction
(Persistent
vomiting),
abdominal
wall
defects,
myelomeningocoele, burns, fractures, active
bleeding requiring transfusion e tc.
2. Hospitalization of 3 days for illness in the last
two weeks (14 days gap between last day of
hospitalization)
3. Previous enrolment in the study with <7 illness
free days between the two illness episodes.

3.4 Signs of very severe infection/disease

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20

1
2
3
4
5

Unconsciousness
Convulsions
Inability to feed
Apnoea
Inability to cry

6
7
8
9
10

Major bleeding
Surgical conditions
High fever
Cyanosis
Jaundice

11
12
13
14
15

Persistent vomiting
Prolonged capillary refill time
Bulged fontanelle
Major congenital malformations
Very low temperature

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3.5 Clinical assessment

ssessment of young infants
3.5.1 Recording axillary temperature
Recording the body temperature of a sick young infant is of
paramount importance to determine whether the baby has
fever or hypothermia. Temperature is recorded by a
thermometer. For this study, we will be using digital
thermometers. Perform the following steps to measure the
th
axillary temperature of a sick infant:
1.
2.
3.
4.
5.
6.
7.
8.
9.

Figure 3.1:
1: Digital thermometer
Carefully clean the probe with boiled water and the
body of the thermometer before use.
Put the batteries inside the device
If the thermometer does not turn on, then replace the batteries with a new set of batteries.
batterie
Turn ON the thermometer.
Place it gently in the childs armpit and hold firmly in place
Wait until the thermometer signals that the process is complete (usually it takes one minute).
Read the temperature on the display.
Turn OFF the thermometer. Immediately
tely record the readings in respective case report form.

Place the thermometer in axilla

Hold it firmly in place

Figure 3.2:
2: Measuring temperature with digital thermometer

Note: If the reading does not seem correct,

If the CHW finds that the thermometer is not working, she should inform the field supervisor. The field
supervisor will arrange a new thermometer

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21

3.5.2 Weighing the infant

Calculating body weight of a young
infant is crucial for determining low
birth weights as well as for calculating
calculat
the
appropriate
dosages
of
antibiotics. Procedure of weighing an
infant is as follows:
Taking weight of newborn baby needs
appropriate weighing scale e.g. handy

Figure 3.3:
3: Weighing scale

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digital scale. Before weighing the baby explain what you are going to do. Do not try measuring the
weight without the consent of the mother.
Place the weighing bag on the hook of
o the unit, before you turn the unit on.
Undress the baby before weighing. While undressing the baby take help of the mother. (Make sure
that everything the baby is wearing is removed, including the amulets)
To turn the scale on, press the ON/ TARE key once.
once. (If the machine is turned on without the bag, bags
weight will be added in newborns weight. If the mother is uncomfortable to undress her baby fully,
weigh the baby along with the quilt, keeping the quilt inside the bag before turning the machine on)
Once the scale is turned on, 0.00 reading will be displayed for a few seconds indicating that the scale
is self-adjusting
adjusting to zero. Do not place weight on the scale while it is displaying. Make sure that the
machine is at your eye level, when you take the reading. After a few seconds a 0 will be displayed.
The scale is now ready for use.
Now put the baby on the weighing bag and fix the bag on the hook of the scale to obtain a reading. Be
careful to keep the scale in a steady position throughout
th
the procedure.
dure. Do not attempt to hold the
bag while measuring instead,, fix the hook scale at a place in the house and keep it suspended.
Press the hold button to check the record displayed
Guiding notes on planning for Standardization
Standardiza
of scale would be helpful.

3.5.3 Assessment of sucking reflex

Figure 3.4: Sucking reflex examination

Sucking reflex, which triggers an infant to

forcibly suck on any object put in the mouth.
The sucking reflex is initiated when something
touches the roof of an infants mouth. Infants
have a strong sucking reflex which helps to
ensure they can latch onto a bottle or breast. On
digital exam* for one minute, poor suck is a
sucking effort that is perceived to be inadequate
for optimal breastfeeding. The test must be
done for one full minute, as it
i takes several
seconds for a healthy infant to initiate an active
act
suck. Unable to feed can be ascertained if
there is a complete suspension of sucking
motion and if the infant is consistently refusing
mothers breasts or other foods.

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22

3.5.4 Counting the respiratory rates using an ARI timer

For counting the respiratory rate
rat of an infant, follow these instructions:
1. Ensure the child is quiet, at rest, or sleeping.
2. Remove or lift the childs shirt so that the chest is easily observed.
3. Press the Start/Stop button at the middle of the timer device. A short beep will be heard. The
CHW will hear shorter pulses and another short beep at thirty seconds. CHW will hear two such
short beeps at 60 seconds and the timer will switch off.
4. Count the number of times the chest rises in 60 seconds.
5. Always count the rate in 60 seconds.
6. Do nott perform other examination maneuvers at the time that the respiratory rate is being
counted.
7. Record the number on respective form.
8. Repeat if the count is more than 60 breaths in one minute.

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3.5.5 Assessment of chest in-drawing

in
Severe chest in-drawing
drawing is the symptom of severe
pneumonia. Follow these steps to observe the chest inin
drawing:
1. Lift the babys clothing over the abdomen.
2. Look for chest in-drawing
drawing when the baby breathes IN.
3. Look at the lower chest wall (lower ribs).
The baby has chest in-drawing
drawing if the lower chest wall goes IN
when the baby breathes IN.

Figure 3.5:
5: Chest in-drawing
in

3.5.6 Assessment of level of consciousness

Lethargy or decreased level of consciousness in a young infant may indicate a serious health problem.
Less than normal movement is the condition of minimal spontaneous movement of the body.
However, for this study, lethargy can be reported if there is movement (limb movement or eye
opening) only on tactile stimulation, but the infant is otherwise not moving. An unconscious infant is a
young infant that cannot be wakened at all.

3.5.7 Assessment of Convulsions

During an episode of convulsion, the childs arms and legs stiffen because the muscles are contracting.
The child may loose consciousness or not be able to respond to spoken direction.
direction. There may also be
passing off urine, staring and turning the head. Ask the mother if the child has had convulsion during
this current illness. Use words that the mother can understand. For example: the mother may know
convulsion as Fits or Spasm.

3.6 Case management

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23

All cases with clinical signs of infection will be referred to the hospital. Hospitalized infants will be
treated using site specific standard treatment procedures. Where treatment procedure is not
followed, patient can be taken care of according
according to WHO guidelines (using One Intramascular Injection
of Gentamicin and Procaine-Penicillin
Penicillin injection per day). If the family refuses hospitalization, they will
be encouraged to come back for daily dosages of antibiotic injections for 7 days. All the
th cases will be
followed-up up to 59 days of birth. During this period, the neonates will be visited by the CHWs to
record the fate of the baby. If the baby develops illness matching the eligibility criteria, then the CHW
will refer the baby to the health facility again for care and specimen collection. The CHW will perform a
verbal autopsy if the baby dies during follow-up.
follow

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3.7 Operational Definitions

Fever: Axillary temperature 100.4F confirmed by a second reading.

Hypothermia: Axillary temperature 95.9F confirmed by a second reading.

Lethargy: Movement only with stimulation (limb movement or eye opening). Stimulation this
context is a gentle non-painful touching of the baby with enough pressure that would evoke
movement, sound-making, and eye-opening in a normal, healthy infant.

Unconsciousness: No response (limb movement or eye opening) even with a tactile

stimulation. An unconscious young infant cannot be wakened at all.

Severe chest in-drawing: Persistent, regular and easily perceived retraction of the lower
chest wall (lower ribs) when the baby breathes IN.

Apnoea: Cessation of respiration for more than 15 seconds accompanied by bradycardia

(heart rate less than 90 beats per minute), cyanosis, or pallor

Bradycardia: Heart rate less than 90 beats per minute

Difficulty in feeding: Unable to feed can be evaluated if there is a complete suspension of

sucking motion (on digital suck exam) and if the infant is consistently refusing to mothers
breasts or other foods.

Convulsion: History of one or more episodes of involuntary contractions of the voluntary

muscles, or directly observed seizures by a physician.

Inability to cry: Extremely feeble or absence of cry which is apparently a deviation from the
infants normal crying history.

Persistent vomiting: Vomiting following three attempts to feed the baby within hour.

Cyanosis: Bluish coloration of skin and mucous membrane, best noted at nail beds, lips or
conjunctivae.

Bulged fontanel: The fontanel is soft spot on the top of the young infants head where the
bones of head have not formed completely. Hold the young infant in an upright position. The
infant must not be crying. Then look and feel the fontanelle. If fontanelle is bulged rather
than flat, this may mean the young infant has meningitis.

Prolonged capillary refill time: If the required time is more than 2 seconds for the normal
skin color to reappear after a blanching pressure is applied on the back of the palm.

Jaundice: Yellowish coloration of sclera, mucosal membrane and skin of the palm or sole.

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24

Major congenital malformation: Congenital malformation that may require surgical

interventions or hospital referral (e.g. cleft lip & palate).

Back to CONTENTS

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25

4. Specimen Collection and Transport

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4.1 Objectives
1.
2.

To collect clinical specimens of appropriate quality and quantity for the study
To transport the specimens to the laboratories in required state.

4.2 Blood Collection by Venipuncture

4.2.1 Materials and instruments
Tourniquet or Velcro, Sterile Cotton balls,
balls Latex or Vinyl Gloves, 10% Povidone-Iodine,, Sterile 5 CC Syringe,
Biohazard bag, BACTEC PEDS PLUS/F blood culture bottle, Sterile with

acid-citrate-dextrose
dextrose tube, Adhesive

plaster (Band-Aid/ Elastoplast), 70% Isopropyl Alcohol and 2% Chlorhexidine Gluconate,

Gluconate Sterile No. 23 Butterfly
Needle, Waste disposal Box

4.2.2 Preparing the neonate for sampling

Chose a vein in the following order of preference:
1. Antecubital fossa
2. Dorsum of hands
3. Dorsum of feet
4. Greater saphenous vein at the ankle
5. Vein in centre of the volar aspect of the wrist
6. Scalp
7. Proximal greater saphenous vein
8. Neck.

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26

Vigorously wipe the skin of the chosen area with 70%

Alcohol, and swab with iodine tincture or povidonepovidone
iodine. Starting in the centre of the circle, apply 10%
povidone
ne iodine (betadine) in ever widening circles until
the entire circle is saturated with iodine. Chlorhexidine
Chlorhexid
swabs which are 2% chlorhexidine Gluconate and 70%
Isoporpyl Alcohol can be used in place of iodine. Rub the
selected area, and allow the area to dry for 60 seconds. If
the vein is palpated again, repeat the skin disinfection
procedure. When the skin iss completely dry, proceed
with the venipuncture procedure. The vein is palpated,
and to dilate the vein a tourniquet is wrapped around
the arm proximal to the intended site of puncture.
Immobilize the vein by applying traction on the skin
around the puncture site.

8
1
5
2
7

Figure 4.1:
.1: Preferred areas for venipuncture
in a neonates body (Numbers are described
in the text on the left side of the figure)

Figure 4.2:: Proper tourniquet application

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4.2.3 Procedure
1.
2.

3.

4.

5.

6.
7.
8.
9.
10.
11.
12.
13.
14.

Weigh the empty syringe with the attached

needle and record the weight in Lab CRF
Attach a sterile butterfly needle in place of
the normal needle at the tip of a sterile
syringe after the disinfectant has dried.
Insert the needle into the vein with the bevel
of the needle face up. Once the vein is
entered withdraw the blood by pulling back
the barrel of the syringe in a slow and steady
Figure 4.3: Venipuncture
manner.
Target to draw 3.0 ml blood. After obtaining
Blood
Blood in acid- In
BACTEC
blood, release the tourniquet and place a
volume
citrateblood Culture
sterile cotton ball over the puncture site
dextrose tube bottle
while holding the butterfly needle in place.
0.5 ml 1.0
Nil
Full volume
Withdraw the butterfly needle and have
ml
patient hold the cotton-ball firmly in place
>1.0 ml to 2
0.5 ml
The rest
until the wound has stopped bleeding.
ml
Put the Band-Aid on the puncture site.
>2 ml
1.0 ml
The rest
Remove the butterfly needle attach the
original normal needle to the syringe.
Weigh the syringe with needle and record the weight in Lab CRF..
Dispense appropriate volume of the blood drawn in blood culture bottle
according to the following table.
Weigh the syringe with blood left in the syringe and record the weight in
Lab CRF.
Dispense the rest of the blood in syringe in the acid-citrate-dextrose tube
tube.
Mix the contents of both the tube and the bottle by three gentle
inversions.
Dispose everything used for this procedure into appropriate waste disposal
boxes.
Figure 4.4: BACTEC
Stick the appropriately labeled sticker on all the blood collection tubes for
Peds Plus bottle
identification of the patient and the sample. Put a copy of the same label
on the CRF.

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27

Infection may be transmitted from patient to staff and from staff to patient during blood collection.
Viral agents are the greatest hazard and in some instances are potentially lethal. Of particular
importance are the viruses causing hepatitis and acquired immunodeficiency syndrome (AIDS). To
decrease the risk of transmission of these viral agents, follow the following recommendations:
1. Wear Latex or vinyl gloves that are impermeable to liquids.
2. Change gloves between patients.
3. Wash hands with soap and water immediately after removing gloves.

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4.2.4 Clinical Complications

1.
2.

3.

4.
5.

Use a new syringe and needle for each patient.

In the event of a needle--stick or other skin puncture
or wound, wash the wound liberally with soap and
water.
If the tip of the needle can not be felt to be in the
vein, remove the needle, and apply pressure to the
puncture site, and start the procedure again,
a
using
new equipment.
Do NOT recap the needles.
Do NOT change needles before injecting the blood
into vial(s).

Extravasations of blood and haematoma

may occur.

4.3 CSF Collection by Lumbar Puncture

4.3.1 Materials and instruments
1% Xylocaine, 22 Gauge Spinal Needle and Stylet,
Stylet 70% Isopropyl Alcohol and 2% Chlorhexidine
Gluconatee, Adhesive plaster (Band-Aid/
(Band
Elastoplasts), Central Hole Towel,
Towel Cotton Balls,
Disposal Box, Latex or Vinyl Gloves,
Gloves Mask, 10% Povidone-Iodine, Sterile Specimen
Spe
Tubes,
Waste disposal box

4.3.2 Preparing the neonate for sampling

An attendant restrains the baby in the lateral decubitus or sitting position. An incubated critically ill
baby should have lumbar puncture in the decubitus position. Sitting position may help
h
in cases where
fluid can not be obtained in the lateral position.

Figure 4.5A: Sitting position for lumbar puncture

Figure 4.5B: Lying position for lumbar puncture

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28

The airway patency should be maintained during the procedure and the neck should never be flexed. Only
the shoulders and legs should be held for the knee-chest
knee
position.

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4.3.3 Procedure
1.

2.

3.
4.
5.

Palpate the iliac crest with one hand and slide the fingers
of the other hand down the spine till it reaches the
Lumber 4 of the spine. Use the space between L4 and L5
as site for lumbar puncture.
Wear mask and gloves and wash hands thoroughly.
Clean the site with spirit and povidone-iodine,
povidone
and cover
the site with a central hole towel.
Inject about 0.2 ml of 1% Xylocaine into the skin of the
L4-L5
L5 area through the hole.
Insert the spinal needle in the midline aimed toward the
umbilicus.
Remove the stylet
et frequently to check CSF, as the pop
felt in older children and adults may be missing in
neonates.

Figure 4.6:: Lumbar puncture technique with needle and stylet

6.
7.
8.
9.

Collect 1 ml CSF in each of the two specimen tubes.

Reinsert the stylet and withdraw the needle. Apply a tincture benzoin seal over the site.
Seal the place with cotton ball and adhesive plaster.
Stick the appropriate label on the respective specimen tubes.

1.
2.
3.
4.

If the specimen is blood tinged, check the puncture site for the following situations:
If the bleeding clears, the tap was traumatic.
If blood does not clear but forms a clot, then a blood vessel is probably punctured.
If blood does not clear and do not form a clot, the baby probably had an intraventricular
intraventricular bleed.

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29

4.3.4 Clinical Complications

1. Maintain the Airway patency during the
procedure.
2. Always check for complication arising from the
procedure.
3. Do not ever flex the neck during the
procedure.
4. Do not hold the infant too tightly during the
procedure. The procedure may cause apnoea
and cardiac arythmia.

1. Hypoxia may occur due to stress caused by the

procedure and handling, especially in sick
babies.
2. If a plug of epithelial tissue is pushed into the
dura due to the use of a needle without a
stylet, intraspinal epidermoid tumour may
result from the procedure.
3. Bacteraemia may result if a blood vessel is
punctured.

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4.4 Nasopharyngeal-Throat
Nasopharyngeal
Swab Pool Collection
4.4.1

Materials and instruments

Remel M6 viral transport,

transport medium, Throat swab,
Nasopharyngeal flocked swab,
swab Latex or Vinyl Gloves,
Mask, Disposal Box, Gauge Pieces,
Pieces Waste disposal
box, Gown, Clipper / scissors,
scissors Tongue depressor

4.4.2 Preparing the neonate for sampling

An attendant restrains the baby in lying position so that
the baby can not move its hands and legs.

Figure 4.7:: Remel M6 viral transport

medium with throat swab and
nasopharyngeal flocked swab

4.4.3 Procedure
1.

6.
7.
8.
9.

Insert the flocked swab into one nostril straight back (not
upwards) and continue along the floor of the nasal passage
until reaching the nasopharynx (resistance will be met). The
distance from the nose to the ear gives an estimate of the
distance the swab should be inserted.
2. Rotate the swab gently for 5-10
10 seconds to loosen the
epithelial cells.
3. Remove swab and immediately inoculate viral transport
media by inserting the swab at least inch below the surface
of the media.
4. Clip the swab handle to fit thee transport medium tube.
Figure 4.8:: Collecting RS from
5. For collecting throat swab, hold the babys head firmly and
nasopharynx
illuminate the throat well, depress the tongue so that the
back of the throat can be seen.
Rub the swab up and down the back of the throat and against any white patches in the tonsillar
area. Avoid the tongue and the cheeks.
Clip swab shaft, leaving tip in the same tube, dipped at least inch below the surface of the
media.
Reattach the cap securely.
Stick the appropriate label to the tubes

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30

Avoid touching the tongue and the cheeks with swab or hand.Do not force the swab, if obstruction is
encountered before reaching the nasopharynx, remove the swab and try the other nostril.
nost The patient
may have a deviated septum.

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4.5 Specimens Transport to site lab

4.5.1 Materials & Instruments
Biohazard bag, Cool box, Alcohol thermometer, Ice packs

4.5.2 Procedure
1.
2.
3.
4.
5.
6.
7.
8.
9.

Put the Blood, CSF and NT swab specimen tubes inside separate biohazard bags.
Put an Alcohol Thermometers inside a Cool box.
Set the temperature of the box to 4-8C by varying the number of ice packs inside the box.
Put the biohazard bags with blood, CSF and NT swab specimen tubes inside the Cool box at 4-8C.
Keep the biohazard bag with inoculated BACTEC PEDS PLUS bottle at room temperature
Carry the Cool box and Inoculated BACTEC bottle to the site laboratory.
Enter the information about the samples in the sample reception ledger immediately after
reaching the site laboratory.
Put the BACTEC PEDS PLUS bottle inside the BACTEC 9050 following the instructions mentioned in
the Chapter 7 of this manual.
Put the biohazard bags with blood, CSF and NT swab specimen tubes inside a refrigerator
maintained at 4-8C.

4.6 Specimens transport to central lab

4.6.1 Materials & Instruments
Biohazard bag, Styrofoam box/ liquid nitrogen vapour chamber, Dry Ice pellet/ Liquid nitrogen

4.6.2 Procedure
1.
2.
3.
4.
5.

Bring out the biohazard bags with blood, CSF and NT swab specimen tubes from the refrigerator
and put inside a Styrofoam box filled with Dry Ice pellet.
Close the lid of the box tightly.
Carry the box to the central laboratory with caution avoiding any shaking and jerking.
Enter the sample information into the sample reception ledger immediately after reaching the
laboratory.
Put the specimen tubes in the designated shelves of a refrigerator at -80C.

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31

The Styrofoam box should be marked with the label "Infectious Substance Affecting Humans" at all
times.

Back to CONTENTS

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Page

32

5. Microbiological Analyses of Specimens

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5.1 Objectives
The purpose of microbiological analyses is to show the presence of organisms which may be causing
disease and to measure the sensitivity of the pathogens present to antimicrobial agents.
Precautions
1. Strictly follow aseptic techniques to avoid contamination. Wear gloves to protect yourself from
pathogens and to prevent contamination of blood from hands.
2. Take a handful 70% alcohol or isopropyl alcohol and rub properly. Wait till dry of the gloves
before start processing.
3. Use sterile consumables e.g. tips, Pasteur pipette.Heat-sterilize the wire loops in the flame of a
Bunsen burner before use. When cool, streak or spread the inoculum.

Field site in all countries

Site laboratory in Pakistan and India

Blood

Culture
Bottle

Tube

Aliquot

BACTEC

Site laboratory in Bangladesh

Isolat

Health
Facility/
Home

Positive

Field laboratory in Bangladesh

C/S
2b
a
2a
a

Serotyping

Preservation

Bio-banking
Nucleic acid
extraction

TLDA

Tube
Bio-banking
Specimen
Collection

Health
Facility

CSF

Aliquot
Cytology

C/S

Isolates
Culture negative CSF

Health
Facility/
Home

Serotyping

Preservation

Antigen
Nucleic acid
extraction

RS

VTM

Aliquot
Bio-banking

Page

33

Note: CSF=Cerebrospinal Fluid, RS= Respiratory specimen, VTM= Viral Transport Media, C/S= Culture & sensitivity test

TLDA

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5.2 Specimen Processing

5.2.1 Presumptive blood culture by BACTEC 9050
5.2.1.1 Principle
The BACTEC 9050 blood culture instrument is designed for
the rapid detection of microorganisms in clinical
specimens. The sample to be tested is entered into the
instrument for incubation and periodic reading. Each bottle
contains a sensor which responds
respo
to the concentration of
CO2 produced by the metabolism of microorganisms or the
consumption of oxygen needed for the growth of
microorganisms. The sensor is monitored by the
instrument every ten minutes for an increase in its
fluorescence, which is proportional
pr
to the increasing
amount of CO2 or the decreasing amount of O2 present in
the bottle. A positive reading indicates the presumptive
presence of viable microorganisms in the bottle.

Figure 5.1: BACTEC 9050 blood

culture machine and blood culture
bottle

Step-1

5.2.1.2 Entering data and loading instrument:

Take the new cultures to the instrument.
Switch ON the uninterrupted power supply.
Wait for the power to become stable.
Turn ON the switch located at the rear of the BACTEC 9050
machine.
Wait for the machine to start up and to load program.
Press the ROTOR HOME Soft Key in the front panel.

Rotor home

If an error E10 appears,, press the Soft Key below.

Press the ROTOR HOME Soft Key again to stop the rotor.
The rotor will stop and the program parameters will be
displayed.

Error 10

Open the door of the BACTEC 9050 machine.

Step-2

5.2.1.3 Entering new sample:

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34

1.

Press the BOTTLE ENTRY Soft Key. A RED light will glow inside
a railed cavity called the ALIGNMENT BLOCK located at the
front-bottom-right
right position indicating the Scanner is ready.

Bottle entry

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2.

3.

Take the inoculated bottle and place the bottle in the

ALIGNMENT BLOCK with the bar-coded
bar
label facing the
scanner. If necessary, rotate the bottle slightly so the scanner
can read the label. The system beeps once to indicate a good
scan. The bottle should be inserted into the WELL designated
by the machine. The designated WELL number will appear in
the top-middle
middle of the panel.
When inserting bottles into stations, carefully push the bottle
into the station assignments are calculated by the system

Alignment box

software to balance the rotor

roto
4. In order to maintain rotor balance, always enter bottles
into the instrument as described and place the bottles
where the system indicates
5.
6.
7.

8.

Press the green Soft Key just below the button OK

The machine will be ready again for another bottle intake.
Red light will glow in the scanner area.
Place & scan the bottle in the ALIGNMENT BLOCK,
designated WELL number will appear on the panel display.
Insert the bottle in the designated WELL.
Press against the shoulders to insure that the bottle is fully
seated in the station. Repeat the process until all the bottles
are inserted into the machine.

Bar-Code scanning

Exceptions

5.2.1.4 If the bottles have no Bar-Code:

Bar
Press the BOTTLE ENTRY Soft Key. A RED light will glow
inside the Scanner.
1. Press the No BAR-CODE Soft Key
No Bar code
2. This display appears. Press the UP and DOWN Soft
Keys to adjust the bottle type to PEDS PLUS/F.

Up and

Down

3. Press the OK Soft Key.

4. The display appears with the designated WELL for
inserting the bottle.
5. If you need to change the protocol, press the SETUP
Soft Key, edit as necessary by using the UP and DOWN
Accept
arrows. Press the ACCEPT Soft key.
Insert the bottle in the designated WELL.. Press against the shoulders to insure that the bottle is
fully seated in the station.

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35

Repeat the process until all the bottles are inserted into the machine.
Close the instrument Door immediately.
Press the BOTTLE ENTRY Soft Key. A RED light will glow inside the Scanner.

ANISA

1.

2.
3.

Once the bottles are placed in stations, do not twist or turn those. Bottles should not be
removed except in the following conditions:
Removal of positive
Removal of negative
Reassign if station becomes had
Identification of anonymous bottles
Immediately subculture any bottle reassigned to a new station.
Do NOT place bottles into the instrument without scanning the barcode. If bottles are placed
into the instrument without scanning, they will become ANONYMOUS BOTTLE.
An
anonymous bottle must be identified as soon as possible so that the instrument can display the
bottle's current status (i.e., Ongoing, Positive, etc.).

Step-3

5.2.1.5 Positive culture:

1.
2.

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36

An audible alarm sounds if a bottle is positive.

The NEW POSITIVE INDICATOR on the front-bottom-left
side of the instruments flashes RED.
3. The REMOVE POSITIVES soft key appears in the LCD display
when the instrument door is opened.
4. On the Main Status display, the station with the positive
bottle is displayed as a filled circle with a plus sign (+) in it
and the positive total in the Summary region reflects the
number of positives in the instrument.
5. To Remove the Positive Bottles:
6. Press the SILENCE ALARM key to acknowledge the alarm.
(Available only alarm volume is set to >0.)
7. Open the door of the instrument and press the REMOVE
POSITIVES soft key.
8. LCD display indicates the positive bottle by position and the
barcode number of the bottle.
9. Remove the bottle from the indicated station and scan the
barcode label on the bottle.
10. Repeat these steps until all of the positives are removed.
Three audible beeps indicate all positive bottles have been
removed.
11. Press the EXIT Soft Key.
12. Close the door of the instrument.

Figure 5.2: Difference in the

appearances of BACTEC Peds
PLUS/F blood culture broths
between
no
growth
and
pneumococcal growth

Exit SoftKey

Note: Perform a subculture and a Gram stain from each presumptive positive bottle. If the staining
reveals no Organism, treat the bottle as Smear Negative and return the bottle to the BACTEC 9050
instrument.

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Step-4

5.2.1.6 Processing a positive bottle

1.
2.
3.
4.
5.
6.
7.

Remove the bottle from the instrument and place in a biological safety cabinet.
Invert the bottle to mix the contents.
Observe Safety Precautions to vent each presumptive positive blood culture bottle. Use a venting
needle (BBL Venting Units catalog # 271056).
Remove an aliquot
liquot from the bottle for stain preparations (Gram and/or AFB).
Subculture bottles according to the Gram stain and/or AFB stain results.
Report preliminary results only after stain preparation.
Perform identification and susceptibility of organism(s) grown on solid media according to your
laboratory protocol.

Interpretation: Positive bottles will be determined by the BACTEC fluorescent series instrument and
identified as such (Bottles entered into the instrument will be automatically tested every ten minutes
for the duration of the testing protocol period). The sensor inside the bottle will not appear visibly
different in positive
itive and negative bottles however the BACTEC fluorescent series instrument can
determine a difference in fluorescence. At the end of the testing period, examine all the Negative
bottles for chocolatized blood, bulging septum, lysed and/or very darkened blood.
blood. If any of these are
visible, then subculture the content of the bottle/s and treat those as Presumptive Positive. For the
processing of blood culture positive cases please follow the instruction of Gram staining procedure
and if positive then blood culture
lture on solid media.

5.2.1.7 To Return "Smear Negative" Positive Bottles to the BACTEC 9050:

1.
2.

Open the door of the instrument and press the BOTTLE ENTRY soft key.
Scan the bottle barcode and place the bottle in the indicated station shown on the LCD display.
Thiss station may differ from the original station.

5.2.1.8 Negative Cultures:

1.
2.
3.

4.

5.

6.

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37

7.
8.

The system notifies the user of the presence of Negative presumptive bottles.
Ongoing negatives are displayed as a solid Black circle on the Main Status Display of the BACTEC
9050.
Out-of-protocol
protocol negatives in the BACTEC 9050 are
displayed as a solid circle with a minus sign (-) in the Main
Status Display. The summary region reflects the number of
Negative bottle sign
negatives in the instrument. Also, the REMOVE
NEGATIVES Soft Key appears when the door is opened.
Open the door to the instrument. Press the REMOVE
NEGATIVES soft key. The LCD display indicates the position
Remove negatives
and the barcode number of the negative bottles.
Remove the bottle from the station and scan the barcode
label. If the bottle was originally entered by selecting the
media type, the LCD displays the station location only.
Remove the bottle
tle from the specified station and press the
FORCE STATION AVAILABLE soft key.
Repeat the appropriate steps until all negatives are
removed.
Three audible beeps sound when the removal process is
completed. Press the EXIT soft key.

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5.2.2 Instrument Maintenance

The following checks should be performed daily:
1. Check the temperature readout on the LCD
display on the instrument.
2. Verify that the temperature of the instrument
and internal temperature bottle read 35C
35
1.5C.
3. If a printer is attached to the instrument, check
the paper supply.
4. Change or clean the air filters on both sides of
the instrument monthly.
5. Check the filters more frequently if the
laboratory's environment is dusty.
Figure 5.3:: BACTEC 9050 machine opening
Procedure: Remove the flip--off cap from BACTEC
PEDS PLUS/F bottle top and inspect the bottle for cracks, contamination, excessive cloudiness, and
bulging or indented stoppers. Before inoculating, swab the septum with 70% Isopropyl alcohol.
Aseptically inject 0.5 to 1.0 mL of specimen in a single bottle. Place the Inoculated aerobic bottles in
the BACTEC fluorescent series instrument as soon as possible for incubation and monitoring.

DO NOT refrigerate inoculated bottle. Keep in room temperature

tem
Troubleshooting: Refer to the BACTEC 9050 fluorescent series Users Manual for troubleshooting
If placement of an inoculated bottle into the instrument has been delayed, observe the bottle for
visible growth. If growth is apparent, Do NOT put the bottle into BACTEC fluorescent series instrument.
Subculture the content, Gram Stain and treat the bottle as a presumptively positive bottle.

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38

Figure 5.4: Control panel of BACTEC 9050

ANISA

5.2.3 Blood culture on Solid media

Signal given by the automated blood culture instrument or any turbidity or lysis of the erythrocytes
may be indicative of growth and subcultures should be made immediately. Simultaneously, gram
staining was done from the centrifuged broth to observe for any bacterial cells. Gram staining
procedure is described in section 7.2.2. If bacteria is found in gram staining that didnt grow on solid
culture media, preserve 1 ml centrifuged broth in liquid nitrogen vapour or dry ice (whichever
available) for the identification of bacteria.
Procedure:
Ensure all the plates are dry before use. Presence of
water inside the plate can cause contamination from
the peripheral part of the culture media.
Wear safety gloves. Rub hands with 70% alcohol. Label
each plate with ID and date.
Disinfect the top of the blood culture bottle rubber
stopper with alcohol and then flame the bottle on
burner.
Mix the content in the bottle by shaking and aspirate
0.5ml blood from the culture bottle by syringe
or aspirator.
Inoculate equal amount (approximately 150I) of
blood on each plate (choocolate, blood and
MacConkey agar plates).
Let them soak on the media for 15 minutes.
Flame the loop on burner and let it cool before
streaking.
Incubate the chocolate and blood agar plate up to 48
hours with 5% CO2, and the MacConkey agar plate
aerobically for 24 hours

1. Disinfection

2. Aspiration

3. Inoculation
Mc Conkey
Blood
Chocolate

4. Streaking

Page

39

Note:
When only one agar plate is used, it should be CAP, since CAP contains needed growth factors for
H.influenzae whereas BAP does not and MacConkey is selective for Gram negatives. Refer to section
7.6 for identifying the bacteria growing on the Blood Agar plate.
When bacterial growth has been confirmed by subculture of the blood culture bottle, there is no need
to continue incubation of the bottle. The bottle should be disposed of according to safety procedures.
If there is no growth on solid media, prepare another 1.0 ml aliquot of the presumptive positive
culture broth in a sterile plastic vial and refrigerate for transport to central lab.

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5.2.4 CSF analyses

5.2.4.1 CSF specimen processing:
CSF specimen is very difficult to collect. So, once collected, it should be handled properly. The volume
available of CSF for processing may vary on collection. So, it is important to plan priority based
synchronization of the experiments. The following table showed the minimum requirements of CSF
specimen for different diagnostic tests in the laboratory.
Table 5.1: Distribution of diagnostic tests of CSF specimen depending on the quantity of collection.
Collection of CSF Culture
Cytolog Biochemistry ICT
TLDA
Preserve
Collection of
(l)
y (l)
(l)
(l)
(l)
(l)
CSF
>0.8 ml
45
50
200
80
200
The rest
>0.8 ml
0.6-0.8ml
45
50
200
80
200
The rest
0.6-0.8ml
0.5ml or less
45
50
200
80
200
X
0.5ml or less
Culture is the gold standard tests which allows identification of the etiological a agent of particular
infection in the CSF and subsequently suggest the type of antibiotics for patient. Prioritizing the
culture, the subsequent tests will be cytology, biochemistry, LAT, ICT and PCR. When only one culture
plate is used, it should be CA, since it favors the growth of all pathogens including H. influenzae.

5.2.4.2 CSF cytology

Infections in the CSF can be predicted by
counting the leukocytes. Total and
differential cell count of CSF can be
performed by improved Neubouer Chamber.
The chamber consists of four large squares
at four corners and one at the centre.
Procedure:
1. Pour 50 l of CSF to Neubouer Counter
Chamber to observe the RBC and WBC.
2. Count the cells in the 5 large squares
and multiply by 2.
3. If there is RBC in the CSF, dilute the CSF
with 5% acetic acid (1:1) to lysis the
RBC. In addition to lysis of RBC, acetic
acid will also make the differentiation of
polymorphs and lymphocytes easier.

Figure 5.5: Blood cells counting by Neubouer Counter

Chamber

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40

Interpretation: The resultant number of count before adding acetic acid is the count of total cells per
3
cm . Multiply the number of cells with the dilution factor if the specimen is diluted. e.g. if a 1 in 4 CSF
dilution is used and 200 cells are counted in the 5 large squares the total number of cells will be 200 X
3
2 X 4=1600cm .

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5.2.5 CSF Bio-chemistry

5.2.5.1 Quantitative measurement of total protein:
Total protein in CSF can be measured using a colorimetric technique. Presence of bacterial metabolism
results in increased concentration of total protein in CSF. The following procedure should be followed
Procedure:
Tube
1
2
3
P-ID
1. Take 4 tubes and label them
ml of Trichloro-acetic 1.5
1.5
1.5
1.5
1, 2, 3 and patient ID.
acid 50 g/l
2. Pipette into each tubes as
ml of 0.44 g/l
0.5
described in the table.
ml of 0.88 g/l
0.5
3. Mix the contents of each
0.5
tube and leave for 5 ml of 1.10 g/l
ml
of
CSF
0.5
minutes.
4. Remix the solution in each tube and make an estimate of the approximate amount of protein in
the CSF by comparing the cloudiness in the Patients tube with that in each of the three standard
tubes.
5. Report the approximate protein concentration in grams per liter (g/l)
Result interpretation: The normal value of total protein in CSF is 0.15 to 0.40 g/l (15-40 mg%) and for
new born baby the value is 1.0 g/l (100 mg%). Only traces of globulin are found in the normal CSF
(Insufficient to give positive pandys test). An increase in total protein and a positive pandys test occurs
in all forms of meningitis, in amoebic and trypanosomiasis meningoencephalitis, cerebral malaria,
brain tumors, cerebral injury, spinal; cord compression, poliomyelitis, the Guelline Bare Syndrome
(often the only abnormality) and polyneuritis. When the total protein exceeds 2.0 g/l (200%) the
fibrinogen level is usually increased sufficiently to cause the CSF to clot. This may occur in severe
pyogenic meningitis and spinal block following haemorrhage.

5.2.5.2 Quantitative measurement of glucose in CSF:

Glucose present in the specimen (CSF) can be converted to gluconic acid with hydrogen peroxide
which can be detected by chromogenic acceptor (phenol 4-aminophenazone) in the presence of
peroxidase. The intensity of the color formed is proportional to the glucose concentration in the
sample.
Procedure:
1. Adjust the colorimeter in required wavelength and calibrate with blank (dist. water).
2. Pipette into a cuvette.
Assay conditions:
Tube

Blank
Standard Sample
Wavelength of the colorimeter: 490-550 nm.
R (mL)
1 mL
1 mL
1 mL
Cuvette: 1 cm, light path.
Standard (L)
--10 L
--Temperature: 15-25C
Sample (L)
----10 L

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41

3.
4.

Mix and incubate at 37C for 10 minutes or in room temperature for 30 minutes.
Read the absorbance (A) of the samples and standard, against the blank. The colour should be
stable for at least 30 minutes.

Calculation of concentration:
(Absorbance of sample) X Calibrator conc. = mg/dL glucose in the sample
(Absorbance of standard)
Conversion factor: mg/dL X 0.0555=mmol/L

ANISA

5.2.6 CSF culture

As there is no pre enrichment step before subculture
it must be processed immediately when it reaches to
the laboratory.
Procedure:
Dry agar plates properly before use. Water inside the
plate can be a potential source of contamination.
Wear gloves to protect yourself from pathogens and
to prevent contamination of blood from hands. Rub
hands with 70% alcohol. Label each plate with ID and
date.
Open the tube containing CSF and flame the top of
the tube on burner. Using sterile tips to aspirate the
required quantity of CSF from the bottom of the
tube. The pathogens, if there, may precipitate at the
bottom. Dispense 15 l each on chocolate, blood and
MacConkey agar plates.
CSF may contain antibiotic. Allow the CSF to be
soaked on the media for 5 minutes. Sterilize the loop
in the flame of the burner. Allow to cool before
streaking properly.
Incubate the chocolate agar and blood agar plate
anaerobically for upto 48 hours. Incubate the
MacConkey agar plate aerobically for 18 hours.

1. Disinfection

2. Aspiration

3. Inoculation
Mc Conkey
Chocolate

Blood

4. Streaking

5.2.7 Immuno-Chromatographic Test (ICT)

Principle: The Binax NOW Streptococcus pneumooniae test is an immunochromatographic membrane
assay used to detect pnumococcal soluble antigen in human CSF. Rabbit antiS. pneumoniae antibody,
the sample line, is absorbed on to nitro-cellulose membrane. Control antibody is absorbed onto the
same membrane as a second stripe. Both rabbit anti- S. pneumoniae and anti- species antibodies are
conjugated to visualize the particles that are dried onto an inert fibrous support. The resulting
conjugate pad and the striped membrane are combined to construct the test strip. That test strip and
a well to hold the swab specimen are mounted on opposite sides of a hinged, book shaped test device.

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42

Procedure:
1. Bring the sample to room temperature then swirl gently to mix.
2. Open pouch just prior use, remove test device and lay flat.
3. Insert the swab into the hole at the bottom of the device. Firmly push upwards so that the swab
tip is fully visible in the top hole. Do not remove swab.
4. Add 80l CSF on the swab. Hold reagent-A vial vertically to 1 inch above the device.
5. Slowly add 3 free falling drops to the bottom hole.
6. Immediately peel off adhesive liner from the right edge of the test device. Close and securely seal
the device.
7. Read the result in window 15 minutes after closing the device.
Result: Appearance of two bands, one band and no band indicate positive, negative and invalid result
respectively.

ANISA

5.3 Identification of Bacteria

5.3.1 Culture
Blood and CSF specimen are inoculated in Blood agar, MacConkey agar and Chocolate agar plate.
Before going to the primary identification of isolates, an algorithm for the pure culture needs to be
known.
1. If a pure culture has grown only on chocolate agar but not on other plates, it could be
2. Haemophilus influenzae.
3. If a pure culture has grown on both blood and chocolate agar plate, but not on MacConkey, it
could be N. meningitidis, Flavobacterium, Streptococcus or Staphylococcus.
4. If a pure culture has grown on all three plates, it could be Gram negative bacteria like Salmonella,
E. coli, Klebsiella, Serratia, Pseudomonas, and Acinetobacter, which can grow in the presence of
bile salt. (Appendix IV)

5.3.2 Gram staining

Principle
Bacterial cell wall contain peptidoglycan layer. Some group
contain thick layer of peptidoglycan where as others contain
thin. Depending on thickness of this layer, bacteria can be
divided by staining into two large groups, gram-positive and
gram-negative. Peptidoglycan layer can be stained with the
dye. The isolates were initially stained with a primary stain
(usually crystal violet) and fix it with a mordant (grams
iodine), then quick wash with alcohol dissolves the primary
stain and absorb the secondary stain (safranin) whose
peptidoglycan layer is thin. But other bacteria retain this stain
and cannot be stained further with secondary dye.

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43

Procedure:
1. Add two drops of normal saline onto the slide
2. Pick target colony with sterile loop and mix with
the normal saline on the slide. Let the
suspension dry.
3. Heat fixed the bacterial smear carefully. Do not
heat directly on the flame.
4. Add 3-4 drops of crystal violet dye onto the
smear of the slide. Wait 1 minute.
5. Wash off the basic dye with tap water.
6. Add 3-4 drops of iodine onto the smear. Wait
1min.
7. Wash off the mordant with tap water.
8. Add 3-4 drops of alcohol. Wait 10 sec.
9. Wash immediately. Do not wait more than 10
seconds; otherwise it will wash away all the dye.
10. Add safranin, keep standing for one minute.
11. Wash and observe the slide under microscope.

Figure 5.6: Contrast between Gram

negative and Gram positive
bacteria.

G +ve

G -ve
Fixation
3-4 drops

Crystal
1min

3-4 drops

Iodine treatment
1min

3-4 drops

Decolourization
10se

3-4 drops

Sefranin
10se

Figure 5.7: Gram staining procedure

ANISA

5.3.3 Gram positive bacteria

5.3.3.1 Identification of S. pneumoniae
S. pneumoniae appear as small, grayish mucoid colonies surrounded by a greenish zone of haemolysis
on Trypticase Soy blood agar. A 3X hand lens or a microscope (30X-50X) is a useful aid in differentiating
pneumococci from viridans streptococci, which also produce a greenish zone of haemolysis on TSA.
Young pneumococcal colonies appear raised, similar to viridans streptococci, but as the culture ages
24-48 h, the colony becomes flattened, and the central part of each colony may become depressed.
This does not occur with the Streptococci viridans. The degree of mucoidness of S. pneumoniae is
dependent upon the freshness of the medium and the incubation atmosphere. The fresher the
medium, the more mucoid the cultures appear. Some serotypes appear very mucoid. The dull gray flat
colony surrounded by a greenish zone of haemolysis is a pneumococcus and the raised gray-white
colony with no haemolytic action is a Staphylococcus. On chocolate agar that does not contain
inhibitors, S. pneumoniae appears as small grayish colonies surrounded by a greenish zone of
haemolysis. It is difficult to differentiate S. pneumoniae from S. viridans Group on chocolate agar.
5.3.3.1.1 Optochin sensitivity test
Presumptive identification of S. pneumoniae is made by determining the susceptibility of the strain to
optochin.
Procedure
An optochin disk with a diameter of 6 mm (containing 5 mg optochin) is required.
1. A suspected alpha-haemolytic colony is touched with a sterile bacteriologic loop and streaked onto
a tryptic soy agar plate containing 5% sheep blood.
2. After the loop is streaked across the agar, the optochin disk is placed aseptically on the end of the
streak where the wire loop was first placed.
3. Three to four colonies may be tested on the same plate.
4. The plates are incubated in a candle extinction jar at 35C for 18-24 h.
Interpretation: Alpha-haemolytic strains with a zone of inhibition of growth greater than 14 mm in
diameter are pneumococci. Alpha-haemolytic strains with no zones of inhibition are viridans
streptococci. Alpha-haemolytic strains with zones of inhibition ranging between 9 and 13 mm should
be tested for bile solubility.
5.3.3.1.2 Bile solubility test

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44

Principle: Bile solubility test is performed to differentiate S. pneumoniae from other alpha-haemolytic
streptococci which are not soluble in bile salts. S. pneumoniae is soluble in bile and bile salt; where as
other streptococci are not. If any dense suspension of S. pneumoniae is prepared in physiological
saline, it can be dissolved within 10-15 minutes after adding bile salt.
Procedure-1: 10% Sodium deoxycholate (bile salts) suspension is required to prepare the solution.
1. Take several loops of the strain from the blood agar plate and prepare a turbid suspension equal
to that of 1.0 McFarland density in 2.0 ml of sterile normal saline.
2. The suspension of cells should be equal to that of 1.0 McFarland density.
3. Divide the suspension in two equal amounts (1.0 ml in a separate tube).
4. Add 250 l of 2% deoxycholate (bile salts) in one tube and 250 l of normal saline to the other
tube.
5. The tubes are shaken gently and incubated at 35-37C for up to 15 minutes
Interpretation: A clearing of the tube or a loss in turbidity is a positive result.

ANISA

Procedure-2: 2% solution of sodium deoxycholate (pH 8.2). (ref-5)

(ref
1. Samples should be inoculated on to Mueller-Hinton blood agar
and incubated overnight at 37C
37 in an anaerobic environment
containing5%
5% added carbon dioxide.
2. A loop full of bile salt (2% sodium deoxycholate) should be
placed on the suspected pneumococcal colonies, and the plate
should be returned to the incubator. After 15 minutes, take out
the plate for reading.

Interpretation: The dissolution of treated colonies compared with

the adjacent ones could be clearly distinguished (Figure-24).If, after a
second period of 15 minutes' incubation,
incubatio the colony does not
dissolve, the test should be considered negative.

Before adding bile salt

After adding bile salt

Figure 5.8
8: Bile solubility test

Note: The most accurate and convenient identification of pneumococci based on the optochin and bile
solubility tests is the following: if the strain has a zone of inhibition to optochin of 14 mm or more it is a
pneumococcus. If the strain has a smaller but definite zone of inhibition of optochin and is bile soluble it
is also a pneumococcus. However, if the strain with a small zone of inhibition of optochin is not bile
soluble, itt is not a pneumococcus, and strains with no zones of inhibition of optochin are not
pneumococci.

5.3.3.2 Identification of Staphylococcus

Staphylococcus will be identified on the basis of its growth on blood and chocolate agar, no growth on
MacConKey agar, catalase
ase positive test and Gram positive cocci in cluster by Gram staining.
5.3.3.2.1 Catalase test
Principle
This test is used to differentiate catalase producing bacteria such as staphylococci from non-catalase
non
producing bacteria such as streptococci. Catalase acts as an enzyme in the breakdown of hydrogen
peroxide to oxygen and water. An organism is tested for catalase production by bringing it into contact
with hydrogen peroxide. Bubbles of oxygen are released if the organism is a catalase producer. The
culture should not be more than 24 hours old.
Procedure: 3% Hydrogen peroxide (H2O2) of 10 volume solution is required.
1. Pour two drops of the H2O2 solution onto a glass slide.
2.

3.

Using a sterile wooden stick (not a nichrome wire loop),

remove a colonies of test organism and immerse in the
H2O2 solution.
Care must be taken while testing an organism from blood
containing medium,because catalase is present in the red
blood cell. If any of the blood agar is removed with the
organism, a false positive reaction may occur.
occur

Figure 5.9:: Catalase test

Page

45

Interpretation: Immediate bubbling in the solution indicates that the organism is a catalase producer.

ANISA

5.3.3.2.2 Coagulase test by slide test

Principle
This test is used to identify S. aureus which produces the enzyme coagulase. Before performing it,
gram staining needs to be done to confirm the organism as a Gram positive coccus.
Coagulase causes plasma to clot by converting fibrinogen to fibrin. Two types of coagulase are
produced by most strains of S. aureus: free coagulase which is released and bound coagulase (or
clumping factor) which is bound to the cell wall. Free coagulase converts fibrinogen to fibrin by
activating a coagulase-reacting factor present in plasma. Free coagulase is detected by clotting in the
test tube. Bound coagulase converts fibrinogen directly to fibrin without requiring a coagulase-reacting
factor. It can be detected by the clumping of bacterial cells in the rapid slide test.
Procedure: Anticoagulated human plasma or rabbit plasma is
required to perform the test. The plasma should be allowed to warm
to room temperature before testing.
1. Place a drop of distilled water on each end of a slide.
2. Emulsify a colony of the test organism, grown on nutrient agar or
blood agar, in each of the drops to prepare a thick suspension.
3. Add a loop full of plasma to one of the suspensions, and mix
gently.
4. Observe carefully for clumping after 10 seconds.

Figure 5.10: Coagulase test

Interpretation: Presence of clumping into the plasma mixed suspension is the indication of coagulase
producing organism.

5.3.4 Gram negative bacteria

5.3.4.1 Identification of H. influenzae
H. influenzae is a fastidious organism requiring media containing haemin and nicotinamide adenine
dinucleotide (NAD, V-factor). Growth occurs on chocolate agar because NAD (V-factor) is released
during the heating process in the preparation of chocolate agar. Haemin is available from nonhaemolyzed as well as haemolyzed cells. H. influenzae is identified based on growth requirements for
X- and V-factors.
Procedure
1. Pick 6-8 colonies from fresh culture and prepare heavy suspension in Normal Saline (>1 McFarland
standards).
2. Ensure that no medium is transferred to the suspension; the smallest sample of agar will affect
the tests and may lead to misidentification of the bacteria.
3. Prepare a lawn over one-half of the plate using a sterile cotton swab stick on Mueller Hinton agar.
Place the paper disks containing both X and V factors on the inoculated plate after the inoculum
has dried.
Interpreation: If the suspected organism has grown only around the disk containing both X and V
factor, itis H. influenzae.

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46

Note: if separate X-and V-factor discs are applied, they should be placed approximately 2cm apart and
that H. influenzae will grow between the two discs.

ANISA

5.3.4.2 Identification of N. meningitidis,

meningitidis Flavobacterium
N. meningitidis and Flavobacterium grow on Blood Aagar and Chocolate Agar but not on MacConkey
Agar. Colony morphology- Pseudomonas and Acinetobacter are similar but can be differentiated by
oxidase test.
5.3.4.2.1 Oxidase test
Principle: Pseudomonadaceae,
Pseudomonadaceae Neisseria and Moraxellacan utilize oxygen for energy production using
oxidase enzyme. A disks/paper, impregnated with N,N,N,N-tetramethyl-p-phenylenediamine
phenylenediamine (TMPD),
act as a redox indicator in this test. If the organism is oxydase-producing,
oxydase producing, the phenylenediamine in the
reagent will be oxidized to a deep purple colour. Nesiserria does not grow on Mac conkey but
Pseudomonadaceae will grow. Typically Enterobacteriaceae are oxidase negative.
Procedures: Freshly prepared oxidase reagent (1% TMPD) is
required.
1. Place a piece of filter paper in a clean petri dish and add 2-3
2
drops of freshly prepared oxidase reagent.
2. Using a piece of stick, remove a colony of the test organism and
smear it onto the filter paper.
Interpretation: Development of blue-purple
blue
colour within 10
seconds will indicate a positive oxidase test.
Figure 5.11
1: Oxidase reaction

Instructions
Do not use the test organism grown on MacConkey and TCBS as acid produced from carbohydrate
fermentation inhibits oxidase enzyme activity. Subculture on nutrient agar can be used.
Do not use oxidized wire loop.
Do not use oxidized reagent that has turned to blue.

5.3.5

Gram negatives other than H. Influenzae and N. meningitidis

Triple Sugar Iron (TSI), Motility Indole Urea (MIU) and Simmons Citrate Medium is routinely used for
the presumptive identification of Gram negative enteric organisms.

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47

Procedure
1. Label a TSI, MIU and citrate media with appropriate ID.
2. Touch a part of the colony to be identified with a sterile needle.
3. Take TSI agar first and open the screw cap with ladies finger.
4. Stab the needle straight into the butt of TSI media carefully. During taking out the needle streak
on the slant.
5. Heat-sterilize
sterilize the needle and touch the same colony to inoculate the MIU tube. Uncap and stab
the needle straight into the tube. Hang the indole paper inside the MIU tube with cotton plug.
6. Heat -sterilize
sterilize the needle again and touch the same colony to inoculate the citrate tube. Inoculate
by streaking on the slant.
7. Incubate the tubes for 24-48
24 48 hours with loosen screw cap or vented with a needle filled with
wit
cotton, if the rubber tube is used.
Interpretation of results: Identification of the organism needs to be done by comparing the results of
the citrate, MIU and TSI with the features of the particular organism described in the table below.

ANISA

Take the Simmons citrate tube first. Observe the slant for any change in colour for growth. If the
colour turns blue it indicates the organism can utilize citrate as a sole source of carbon and look for the
organisms who gave the citrate positive test. If color was not changed, the organism could be
anything. As few Klebsiella, Enterobacter sp. may be citrate negative. Then observe the MIU for
motility, urease and indole production. If the test organism is motile it could not be Klebsiella. If urease
is produced by the test organism, then it might be Klebsiella. Observe TSI tube and note the change of
colour on butt and slant, production of gas and/or H2S. Gradually narrow your choice of suspected
bacteria by interpreting one after another tube.

5.3.6 Precautions
1.

2.
3.
4.

Do not use an inoculating loop to inoculate the media containing tube. While stabbing the butt,
mechanical splitting of the medium occurs, causing a false positive result for gas production.
(Macfadin 1985)
Inoculate from a single colony for all 3 tests.
Reading must be taken within 18-24 hours. Reactions start reversion after than due to continuous
growth of the organism.
Compare with non-inoculated tube as negative control before interpreting the results.

5.3.7 Serotyping of S. pneumoniae by Quellung reaction

Principle
S. pneumoniae has more than 90 different
serotypes. Quellung reaction is the conventional
method for pneumococcal serotyping. Specific
antibody bound to the pneumococcal capsular
polysaccharide causes a change in the refractive
index
(as
a
result
of
an
in
situ
immunoprecipitation) of the capsule so that it
appears "swollen" and more visible (see figure7.3). In addition, the bacteria agglutinate. The light
transmitted through the capsule appears brighter
than either the pneumococcal cell or the
background

Cross reactivity- In three antisera all crossreactions cannot be removed without

simultaneously absorbing too much of the
homologous antibody. So, the three antisera
listed are not group or type specific
Antisera
Cross-reactive type
Type 29

35B

Group 35

42,47F

Type 42

20,31,33A,35A,35B,35C

Precautions
Adjust antisera set at room temperature for 25-30 minutes before use.
Do not refrigerate the antisera if your work needs an interval.

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48

Procedure
Antiserum set for pneumococcal serotyping is required. The result is often more evident when
compared with a negative control. All the reactions except positive one can be considered as negative
control.

ANISA

1.

2.

Take 5-10 isolated healthy colonies of pneumococcal

cultured on Mueller-Hinton blood agar not more 18-24
Colony
hours old.
20/30 colonies
Make a suspension of pneumococcus in normal saline
Normal
(0.85% sodium chloride) equal to a 0.5 McFarland density
saline
standard. Optimum quelling reaction can be observed when
there are 100 to 150 cells in a microscopic field.
Dispense 2l of the cell suspension on the slide.
Suspension
Dispense equal amount of omniantisera and mix with
pippetting 5 times.
2
Immediately place a 22-mm
cover-slip on top of the mixture
OMNI SERA
(must not dry out).
AN
If positive, then go for
Examine the mixture under a
TI
next step
phase
contrast
microscope
(100X). The reaction is stable for
POOL SERA
Example:
A-I & P-T
half an hour.
AN If the sample
If the capsule becomes visible
TI
agglutinate
Check all the pool until
(the bacterium appears swollen)
positive result.
with pool A
the reaction is positive (see
figure-7.3-B).
Example:
TYPE SERA
If positive reaction observed,
AN Pool A contains 5 types
then repeat step 1-7 with the nine
(1, 2, 4, 5 &18). Positive
One pool sera may contain TI
traditional pools (A to I) until a
result with A pool will
different type. Check all the
positive reaction was observed.
Example:
Sometimes,
reaction
with
Type 18 has 4 factors.
FACTOR SERA
AN
omniantisera
gives
weak
Positive result with type 18
TI
reaction that might be wrongly
will be checked with all 4
Some types contain different
interpreted as negative. Please
factors. Check all the factors
factors as follows until
of the positive type until
note there is a very minimum
agglutination
agglutination.
chance of getting non-typable
invasive pneumococci. It is advised to test all pools before reporting the pneumococcus as nontypable.
.

........................ .....

.....

..................... ......... .......... .......... ..

......................... .......... ......... .......... ......

.................... .......... .......... ......... .......... .......... ...

.......................... .......... ...... ........ .......... ....

.......................... .......... .........

............................ ......... .......... ......... .....

3.
4.
5.

6.

7.

Page

49

8.

ANISA

Interpretation
The result is often more evident when compared with a negative control. Typing will then proceed by
testing the strain with antisera against those individual types or groups that were included in the serum pool
that gave a positive reaction (see the flow chart below). e.g.
.g. If the suspension give positive reaction in pool-C,
pool
test with 7, 20, 24, 31 and 40. Further if positive by serogroup 7, test with all factor sera specific for 7 (7b, 7c, 7e
& 7f).
QUELLUNG REACTION
A) Negative reaction

B) Positive
reaction.

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50

B
Figure 5.12: Quellung reaction

quelling

ANISA

5.3.8 Antibiotic Susceptibility of Isolated Organism

Sensitivity tests measures antimicrobial activity against bacteria under laboratory conditions (in vitro
activity).This test determines the susceptibility of the organisms against routinely administered specific
drugs/antibiotics. Doctors can select appropriate therapy based on the result of susceptibility test.

Antibiotic susceptibility tests can be performed by dilution method, disk diffusion and E-strip
diffusion. Mueller-Hinton agar is used for the test.

5.3.8.1 Preparation of 0.5 McFarland standard solution

A barium chloride 0.5 McFarland density standard solution is prepared by adding 0.5 ml of a 1.175%
(v/v) Barium Chloride dihydrate (BaCl2.2H20) solution to 99.5 ml of 1% sulfuric acid. The resulting
mixture is placed in a tube identical to that used for preparing the dilution for the antimicrobial
susceptibility tests. The same size tube (screw-capped) and volume of liquid must be used. It should be
stored in the dark, at room temperature when not in use. Prepare a fresh standard solution every 6
months. Mark the tube to indicate level of liquid, check before use to be sure that evaporation has not
occurred. If evaporation has occurred, prepare a fresh standard.

5.3.8.2 Disk Diffusion

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51

Principle: A filter paper disc impregnated with drug placed on culture agar media will diffuse the
drugs/antibiotics into the media. This diffusion will place the drug in the agar only around the disc. The
solubility of the drugs/antibiotics and its molecular size will determine the size of the area of
drugs/antibiotics infiltration around the disc. If an organism is placed on the agar it will not grow in the
area around the disc if it is susceptible to the drugs/antibiotics. This area of no growth around the disc
is known as a zone of inhibition.
Procedure
1. To prepare the bacterial suspension, touch a
single isolated colony from Gram negative
bacteria and Staphylococcus. Pick 1-2 and 3-5
well isolated colonies from H. influenzae and
3
Pneumococcus respectively.
1
2. Emulsify the bacteria in a normal saline to
achieve a turbidity equivalent to a 0.5
McFarland standard.
3. Use Mueller-Hinton agar alone, Mueller2
Hinton agar supplemented with 5% sheep
blood and Mueller-Hinton agar supplemented
with NAD (5mg/ml) plus 1% haemoglobin for
Disk
Gram negative bacteria, Pneumococcus and H.
influenzae respectively.
Susceptibility test
4
4. Dip a sterile, non-toxic swab into the inoculum
suspension having a turbidity equivalent to a Figure 5.13: Antibiotic Susceptibility test
0.5 McFarland standard. (1 in the figure)
5. Remove the excess fluid by rotating and pressing the swab firmly against the inside wall of the test
tube (2 in the figure).
6. Swab the entire agar surface. Repeat the swabbing procedure three times; rotating the plate
approximately 90 degrees each time to ensure an even distribution of inoculums (3 in the figure)
7. Allow excess moisture to be absorbed for about 10 minutes so that the surface is completely dry
before applying the discs containing specific amount of antibiotic.
8. Place maximum 6 discs containing specific antibiotics either by dispenser or by sterile forceps on

ANISA

9.

the media (4 in the figure)

Ensure the complete contact of disc with the agar surface. Agar plates can be incubated
0
immediately at 37 C. The incubation temperature and atmosphere selected for fastidious
bacteria, such as Haemophilus, and pneumococcus species is 37C for 18-24 hours at 5% C02 if
necessary for growth.

Interpretation: Zones of inhibition are read with calipers or metric rulers in millimeters. Either
measure the diameter between two edges of the growth or measure the radius from the centre of the
disc to one edge of the growth. Selecting the edges could vary from organism to organism. Record
results and compare with accompanying chart provided by CLSI manual.

5.3.8.3 E-strip

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52

Pick 3-5 well isolated colonies to be tested and emulsify in a suitable suspension medium to achieve a
turbidity equivalent to a 0.5 McFarland standard.
1. To prepare the bacterial suspension,
touch a single isolated colony from Gram
negative bacteria and Staphylococcus.
Pick 1-2 and 3-5 well isolated colonies
from H. influenzae and Pneumococcus
E-Strip
respectively.
2. Emulsify the bacteria in a normal saline
MIC of specific drug
to achieve a turbidity equivalent to a 0.5
Figure 5.14: Determination of MIC for a specific drug
McFarland standard.
3. Use Mueller-Hinton agar alone, MuellerHinton agar supplemented with 5% sheep blood and Mueller-Hinton agar supplemented with NAD
(5mg/ml) plus 1% haemoglobin for Gram negative bacteria, Pneumococcus and H. influenzae
respectively.
4. Dip a sterile, non-toxic swab into the inoculum suspension having a turbidity equivalent to a 0.5
McFarland standard.
5. Remove the excess fluid by rotating and pressing the swab firmly against the inside wall of the test
tube.
6. Swab the entire agar surface. Repeat the swabbing procedure three times; rotating the plate
approximately 90 degrees each time to ensure an even distribution of inoculum.
7. Allow excess moisture to be absorbed for about 10 minutes so that the surface is completely dry
before applying the discs containing specific amount of antibiotic.
8. Place an E-strip by sterile forceps on the media.
9. Ensure the complete contact of strip with the agar surface. Agar plates can be incubated
0
immediately at 37 C. The incubation temperature and atmosphere selected for fastidious
bacteria, such as Haemophilus, and pneumococcus is 37C for 18-24 hours at 5% C02 if necessary
for growth.

ANISA

5.4 Pathogen detection by TLDA

5.4.1 Nucleic Acid extraction from blood and respiratory specimen
Procedure:
1.

2.
3.
4.
5.
6.
7.

Resuspend the sample in 400 l of Resuspension Buffer R in a lysis reagent tube. For clinical
samples, use 400 l of NP swab if available and place it in a lysis reagent tube. If less volume of
clinical sample is used, bring up the volume to 400 l using the Resuspension Buffer R in a lysis
reagent tube.
Incubate at 65C for 10 min in a Thermomixer
KF strip setup:
followed by 95C for 10 min. Centrifuge briefly.
1. Add 400 l of Binding Buffer B6 to Tube A
After lysis, transfer the samples into the Tube A
2. Add 20 l of MAP Solution A to Tube A
of the King Fisher strip.
3. Add 800 l of Wash Buffer I to Tube B
Place the filled King Fisher tube strips into the
4. Add 800 l of Wash Buffer II to Tube C
King Fisher System.
5. Add 800 l of Wash Buffer II to Tube D
Place the King Fisher tips onto the magnetic
6. Add 120 l of Elution Buffer D to Tube E
track.
Run program: InviMAG_Virus_KFml.
Transfer the nucleic acids into a labeled tube.

5.4.2 Amplification and detection of pathogen

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53

1.
2.

Extract total nucleic acids (TNA)

Prepare the master mix (the example
below is for a full card). If the card is not
full, leave the unused wells empty and
calculate the appropriate amount of the
master mix. Always run a no template
control.
3. Combine 80 l of the master mix with 20
l of TNA and load on the TLDA card.
4. When the entire card is loaded,
centrifuge it TWICE in the Sorvall
centrifuge on preset #1.
5. Seal the card using the tool provided by
the Applied Biosystems.
6. Cut off the loading ports.
7. Use the Applied Biosystems 7900HT real
time instrument with the TLDA card
block.
8. Open SDS software.
9. Select New from the File menu.
10. Choose the following options: Assay
Standard Curve (AQ), Container 384
Wells Taqman Low Density Array, press
OK.
11. Wells A1 to B24 represent port one on
the card (the wells on the screen
represent the card laying down with its

Reagents
AgPath-ID 2x buffer
AgPath-ID enzyme
Water
Mastermix TOTAL
Extracted sample

TNA extraction
or

Per specimen
(1 sample) l
50
4
26
80
20l each

Per plate
(8.5 sample) l
425
34
221
360

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12.
13.
14.

15.
16.
17.
18.

ports to the right). To label the samples, highlight two rows at the time and name them in the box
on the right side of the screen.
Choose Add detector, select FAM MGB, click Copy to plate document, select VIC MGB,
click Copy to plate document, click Done.
Highlight the entire card by clicking on the box in the upper left corner of the card layout. Select
both detectors from the list on the right side.
Go to the Instrument tab, select Thermal cycler and set it to 45 repeats, 45C for 10 min, 94C
for 10 min, and then 45 cycles of 94C for 30 s and 60C for 1 min. Make sure the last two steps do
not have a bar in between them.
Go to the Real-time tab, Connect to the instrument, save the file, open/Close, load the card
with the barcode facing you, and Start run.
The run takes about 2.5 h.
When the run is completed, discard the TLDA card.
To analyze the data, click on the analysis setting (a button with a small green arrow and a blue
line on top), select restore factory settings from the bottom of the window, and select manual
Ct and manual threshold. Click OK.

5.4.3 Result Interpretation

A single TLDA card consists of 8 lanes, and there are 48 individual wells per lane. Pathogen
specific primers and probes are dried down in each well.

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54

Figure 5.15: Coding arrangement of primer and probe in TLDA card

Each lane consists of 21 sets of primers and probes for 21 target pathogens spotted in
duplicate wells, with 3 sets of control targets. Usually in a TLDA card the first lane is used for
a no template control. The rest of the lanes can be used for specimen testing. Therefore,
using a TLDA card, maximum of seven specimens can be tested at a time. A specimen is
considered positive if it has an S-shaped curve for any of the 21 sets of target pathogen
specific primers and probes. The test will be valid if any of the two genes GAPDH and RNP3
amplified and the negative controls are negative. If Ct values are positive for more than one
pathogen, the specimens are considered to have multiple infection/co-infection.

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General precaution
1. Clinical specimens are hazardous; always wear a suitable lab coat, disposable gloves, mask and
protective goggles.
2. It is necessary to check the necessary apparatus are available for use during the time of specimen
processing, i.e. some apparatus may be shared by a number of people. So, inform the other
persons about the time when this equipment will be occupied for your experiment. A tag must be
hanged over the equipments mentioning the time of use.
3. For some equipments, (water bath, centrifuge machine etc.) temperature may need to be set. For
these equipments, set the desired temperature and wait until the machine display the set
temperature.
4. Clean the working area with 70% alcohol and if possible allow UV radiation exposure for 15
minutes. Proper cleaning and UV exposure will ensure the quality of the extracted nucleic acid, i.e.
contamination by DNA/RNA will be minimum.
5. Take sufficient ice in a bucket if needed.
6. Take an empty specimens holder in ice bucket if required.
7. Take the specimens storage log book and pick the desired specimens from the freezer.
8. Put a mark on the log book mentioning the time, date and amount of specimens taken out.

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6. Specimen Storage and Bio-banking

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6.1 Objectives
1.
2.

To store collected clinical specimens for a considerably long period of time, in case further analysis
is needed for a particular specimen of interest.
To assure integrity and preservation of the specimens at the original concentration and form until
subsequent analyses are carried out.

Specimen aliquoting
1.
2.

Mix the plasma and the blood cells inside the acid-citrate-dextrose tube by gently inverting the
tube three times.
Using a Micropipette, transfer 0.5 ml of blood from the acid-citrate-dextrose tube into a sterile,
DNA/RNA and DNAse/RNAse free cryovials for molecular assays. Leave the rest of the blood in the
original tube.

6.2 Specimen banking

6.2.1 Site laboratory
Store the blood in acid-citrate-dextrose tube, respiratory specimen and CSF specimen inside a
refrigerator at 4-8C.

6.2.1.1 Blood
1.

2.
3.
4.
5.
6.

Label a cryovial using the pre-printed labels with identical number dedicated to the patient, which
is already assigned and applied on the specimen collection tubes and the accompanying manifest
identifying the patient.
Use a sterile dropper to draw all the blood out of the tube and transfer the blood to a sterile
DNA/RNA and DNAse/RNAse free cryovial.
Add roughly equal volume of 20 % DMSO solution into the same cryovial to adjust the final DMSO
concentration to 10%.
Tighten the cap of the cryovial as tight as possible to prevent leaking.
Mix the DMSO with whole blood by gently inverting the cryovial three times.
o
Keep the cryovial inside a refrigerator at 4-8 C for 30 minutes.

6.2.1.2 Respiratory specimen

1.
2.
3.
4.
5.

Mix the specimen inside the collection tube with viral transport medium by gently inverting the
tube three times.
Using a Micropipette, transfer 0.5 ml of the medium into the cryovial. Leave the rest of the RS in
the original tube.
Add 20 % DMSO solution into the cryovial to adjust the final DMSO concentration to 10%.
Tighten the cap of the cryovial as tight as possible to prevent leaking.
Mix the vial content by gently inverting cryovial three times. Keep the vial inside a refrigerator at
o
4-8 C for 30 minutes.

6.2.1.3 Storage of specimens

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57

Put all the cryovials and tubes containing the specimens inside vapour phase liquid nitrogen/ dry iIce
chamber and keep the vials there until transported to the central laboratory.

6.2.1.4 Transport to Central laboratory

Transport all the tubes and cryovials in liquid nitrogen vapour or in dry ice chamber.

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6.2.2 Central laboratory

Transfer all the cryovials (added with DMSO) to -80C freezer. If -80C freezer is not available or not
functional, store the cryovials in liquid nitrogen vapour. The specimens can be kept in LN2 vapour for
indefinite period of time without harming the specimen quality.

6.2.3 Thawing

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The final fates of the specimens are not yet clear. Therefore, prescribing a specific thawing procedure
in advance is not feasible. As a general rule, apply an appropriate thawing method which will assure
maximum preservation of the integrity of the specimens for their downstream use.

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7. Data Capture and Management

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7.1 Objectives
1
2
3
4
5

To capture and manage data efficiently and timely

To provide data in usable format as per requirement
To generate necessary reports
To ensure data security against loss, damage and undesired access to the data
To facilitate collation of data from all the sites

7.2 Database development

System Design, Development and software used
The database will be designed using Microsoft SQL Server 2005 and will use Visual Basic 6.0 for user
interface. It will be a Relational Database Management System (RDBMS) and all tables will be linked
with the unique keys (primary and secondary keys).
Figure 7.1.A : Management system of Data coordination centre
Data share

Data
5

One way
Data sharing
(Site Specific)

Accept
Request

System Design
&
Development

Request for
Data

Monitor for
any changes

Coordination
Centre

Request

Accept Data

Run Programs
Sites specific and
combined reports
for sharing

10

Data
synchronization

Share

Implementation
At all sites

Using Data

Provide reports for field verification

Sites

Run Programs
Generate
reports for data
consistency

Data
Transfer

Central
Database

Using Data

Maintain
Schedule
Backup

External
Drive

Hard

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Team
Member

CD/DVD

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Co-ordination
Centre
1

Verify the development on each step

Clarify with each

field sites during
development

Sites

System Design &

Development

Tools Used:
SQL Server 2005
Visual Basic 6.0
Crystal Report 8.0
Team Member:
Data Analyst
System Analyst
Sr. Programmer

Testing
Feedback
From
Sites

Update and
Finalize

System
Implementation

Packages:
Installation
Training
Guideline
User manual
Documentation

Figure 7.1.B: System Development on Data coordination centre

7.2.1 Client/Server (Technology supported)

There will be a central database server and the clients will access the central database from anywhere
within the network. It can be the same room, same building or different buildings. Client/user can
communicate with the database server over the dedicated wired connection (Local Area NetworkLAN), radio link or Wi-Fi. Any number of users can access the database simultaneously. .

7.2.2 Establishing the data entry system at the study sites

After developing common data capture software, the respective persons will make sites visits to
provide necessary training on data entry, backup and data security etc. Training on data transfer and
maintenance will also be provided.

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7.2.2.1 Data entry and validation

All possible ranges and consistency checks will be in-built with the data capture system. If the data
entry personnel provide any invalid or inconsistent value then the system will not accept that value
and give an alert message. An error correction procedure will initiate whenever the date error is
detected. Some of the errors can be solved immediately; others may need to initiate feedback
procedure (field verification). The correction will be made within a schedule time.

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7.3 Database management

7.3.1 Collating data and making the master data set
After completion of data entry of every month, all study sites will send the data files to the central
data centre (Coordination Centre). Then the team of data centre will organize the dataset (by sites and
pool the data files).

7.3.2 Policy for access to data, study sites Vs Coordination Centre

Coordination centre can use web based data sharing application (share folder etc) to exchange the
data with study sites. Sites will have full access to their own data and Data Coordination Centre will
have access to all.

7.3.3 Data security

The data capture system and the database will be password protected to ensure the safety of the
database. Each user will have a login ID and password to login to the system. Only the authorized
persons can access the database. According to specific responsibilities, different persons will have
different levels of permissions in accessing the database. To maintain the security of the database, the
access level of the Data Entry Operators will be different from the other users.

7.3.4 Database backup

A daily backup of the database will be maintained in a different media (removable hard disk, pen drive,
DVD, etc). Data will be backed up weekly in separate folders. The MS SQL Server database will also
allow an auto backup system as per defined schedule. A backup of the data will also be kept in a
separate place for the data safety.

7.3.5 Data audit system

We will provide a powerful data auditing system supported by the MS SQL server database. Database
will internally manage a table that will store each and every change made in data files. The database
will keep record of the audits and will store the old value, new value, table name, variable name,
date,time and user name of whoever makes the changes. There will be an option to view the audit
table from different angles like:

How many changes have been made to each table?

Which variable have changed the most?

What types of changes are made frequently? How frequently?

Which users have made most of the changes?

7.3.6 Reporting system

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We will use Crystal Report for the reporting purpose. According to the requirement, the system will
automatically generate reports for field monitoring, performance review and management etc.

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8. Quality Assurance and Quality Control

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8.1 Objectives
1.
2.
3.

To ensure quality in every step of the study

To ensure that the outcome of every procedure meets the desired standard
To ensure that the sub-standard outcomes of any activity do not compromise the quality of the
study

8.2 Laboratory QA & QC

8.2.1 QA & QC in Specimen Collection
8.2.1.1 QA & QC in Blood collection
1.
2.
3.
4.
5.
6.
7.

8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.

Be careful not to touch the rubber covers of the tubes and bottles to prevent contamination.
The phlebotomist should avoid touching the collection site with the gloved finger(s).
Swap the skin of collection site with disinfectant every time you palpate the vein.
Wipe the surfaces of the blood collection tubes, bottles, and the gloves with disinfectant prior to
collection of blood.
Ensure inoculated blood culture bottles are put to incubation within one hour of inoculation.
The acid-citrate-dextrose tubes with blood are to be refrigerated at 2-6C at all stages from
collection and transport to the central lab.
If the specimen is likely to be contaminated, report immediately to the Site Coordinator and put a
note in the Lab CRF. The site PI is most likely to discard the sample and order another sample
collection.
Check the expiry date on the side of the vial. Return any expired vials to the Lab Collection area.
These vials may be used for training purposes.
Before storage and incubation, check whether the bottle is appropriately labeled or not.
Report any contamination of the hands or body with blood; or any puncture, wound or cut to the
supervisor and seek treatment.
After collecting blood in acid-citrate-dextrose tube, mix the content thoroughly by three gentle
inversions.
Specimen containers should be individually and conspicuously labeled.
Thoroughly wipe any container with blood on the outside.
If the amount of blood drawn is insufficient, attempt another one venipuncture on the same day.
No other attempts can be made later.
Keep the inoculated BACTEC PEDS PLUS bottles between 20 to 25C inside a cool box until put to
incubation in BACTEC 9050.
Make certain the Labels go around the vials, but DO NOT OBSCURE THE BAR CODE and adjacent
sequence number on the original BACTEC PEDS PLUS vial or other specimen vials.
Blood must be inoculated into BACTEC PEDS PLUS blood culture bottle immediately to prevent
clotting inside the syringe.
Do NOT use alcohol with concentrations greater than 70%. Increased ethanol concentrations
result in decreased anti-bactericidal activity.

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8.2.1.2 QA & QC in CSF Collection

1.
2.
3.
4.
5.

CSF will be collected in the hospital level.

CSF will be collected by the clinician.
Collect the CSF in DNAse, RNAse free sterile tube.
Do not refrigerate the CSF specimen before doing culture.
Send the CSF specimen to the culture lab instantly.

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6.

Put the specimen tubes inside Cool Box immediately after sample collection. Keep the
temperature of Cool box between 2-6 Celsius.

8.2.1.3 QA & QC in NT Swab pool collection

1.
2.

3.
4.

Make sure the swabs remain moist from the specimen collection to reaching the lab for PCR. Dry
swabs are not suitable for PCR based diagnostics.
Put the Specimen tubes at 2-6 Celsius into Cool box immediately after sampling. Store the tubes
in refrigeration temperature, and send to the Central laboratory as soon as possible, within no
later than 5 days from the time of collection.
Thoroughly clean the scissors (for clipping swab shaft) with bleach prior to use to avoid potential
DNA contamination of a PCR specimen.
Clean the nostril with a swab if there is a lot of mucus.

8.2.2 QA & QC in Specimen Transport

1.
2.
3.

4.
5.

Avoid shaking of the specimens and Cool box at every stage of transport to reduce the chance of
lysis and sample loss.
Monitor the thermometer placed inside the Cool box at two hour intervals to check the
temperature stays between the specified ranges (2-6C).
Carry extra ice-packs in dry ice so that these ice-packs remain frozen. These ice packs can be used
to replace the older ice packs with specimen to ensure the specimens reach the lab at the desired
temperature.
Monitor the thermometer placed inside the cool box at two hour intervals to check the
temperature stays below within 2-6C.
Reject NT swab samples if the swabs are DRY when received at the central laboratory and advise
redraw of sample.

8.2.3 QA & QC Control in Specimen processing:

1.
2.
3.

The laboratory personnel who process specimen should receive training quarterly.
Blood culture broth spiked with known pathogen should be supplied for processing quarterly.
Separate laboratory person should process the same specimen once in a month.

8.2.4 QA & QC of Laboratory Equipments

1.
2.

3.
4.
5.

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6.

Temperature Chart for the refrigerator and freezer should be maintained. It should be recorded
twice a day, especially before leaving and after entering the laboratory.
The freezer inside the lab which is functioning need to defrost every quarterly. During defrosting,
a back-up of the low temperature needs to be ready. If there is no back-up freezer, arrange dry-ice
inside the thermo-coolant, and keep all the content in it.
Maintain temperature of the incubator and update it twice a day.
A log book should be maintained before and after the use of the equipments.
User manual should be read by the lab person and discussed among themselves.. The person who
will handle the equipment should understand every point in the manual.
Check the expiration dates of your reagents regularly, and write the opening date of the reagents
on the containers (bottles).Check the expiration dates of your reagents regularly, and write the
opening date of the reagents on the containers (bottles). Also put an initial of yourself while
opening.

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8.2.5 QC of the antibiotic disc and E-strip

Quality control tests for all the tests will be done using the ATCC stains of known MIC values. Each
batch of antibiotic disks and E-strips purchased, need to be checked for their efficacy after reaching
the lab and again quarterly until the expiry date is reached. This will ensure the quality of transport
and preservation of reagents.

8.2.6 & QC in blood culture by BACTEC

1. Do not use expired culture vials
2. Do not use culture vials that exhibit any cracks or defects; discard defective vial.
3.

4.

Extra vial sequence barcode labels are included in the instrument start-up kit. Use these labels to
replace damaged or unreadable labels on culture vials. The barcode labels contain sequence
numbers which uniquely identify each vial.
Quality Control Certificates are provided with each carton of media. Quality Control Certificates
list test organisms, including ATCC cultures specified in the CLSI Standard, Quality Control for
Commercially Prepared Microbiological Culture Media. The range of time-to-detection in hours
was 72 hours for each of the organisms listed on the Quality Control Certificate for this medium

8.2.7 QA & QC of the Culture media

8.2.7.1 pH and color
1.
2.

3.

Examine the pH of the media before autoclave.

Adjust pH if required. Examine the color of media
and take a decision for its accuracy and examine
any crystal formations or variations in color (for
agars).
The containers of media should be thoroughly
examined for cracks or defects, and all defective
units should be discarded.

Quadrant

8.2.7.2 Sterility testing

1.
2.
3.
4.

Perform sterility test on all media prepared in the

laboratory.
Incubate one plate from each batch, at 37C for 48 hours and
Incubate again on each successive 7 days until the supply in the refrigerator is depleted or the
expiry date is reached.
If expected results are not attained, the QA technologist must be informed.

8.2.7.3 Quality Control of solid media:

1.
2.
3.
4.

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5.

Use fresh ATCC culture (no later the 24 hours old culture) of S. aureus, E. coli, S.pneumoniae and
H. influezae.
Dry one media from each batch prepared.
Mark a quadrant on the backside of each plate.
Touch single colony from the above mentioned cultures and inoculate them in separate quadrant.
Incubate at 37C for 24 hours with 5% CO2.
Media will be tagged as QC pass, if the media yield the following growth.

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Table 11.1: Chart of the QC result

Media for QC
Batch no.
pH

S. aureus

E. coli

S.
pneumoniae

H.
influezae

Results

Blood agar

2010-###

7.2-7.6

Pass

Chocolate agar

2010-###

7.2-7.6

Pass

MacConkey agar

2010-###

7.2-7.6

Pass

Mueller-Hinton
blood agar
Mueller-Hinton
agar

2010-###

7.2-7.6

Pass

2010-###

7.2-7.6

Pass

8.2.8 QA & QC of the Biochemical media

Bio-chemical media used to identify the pathogens needs to prepare aseptically. Each batch of media
should go through a process of quality control.
Inoculate all three media with freshly prepared culture of Salmonella typhi, Proteus and Klebsiella.
Observe the reaction of each media and declare as Passed if they give the following reaction.
Strain

Simmons
Citrate

Motility Indole Urease (MIU)

Triple Sugar Iron (TSI)

Results

Motility

Indole

Urea

Slant

Butt

H2S

Gas

Klebsiella
oxytoca
Proteus
mirabilis
S. typhi

+89 -11

+78 -22

Pass

+57 -43

Pass

Pass

8.2.9 QC of Commercial blood culture broth

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Commercial blood culture broth from any reputed company has been prepared through an extensive
quality assurance and distributed after quality assurance. During transportation, the product quality
might be deteriorated under unexpected circumstances. After reaching to the laboratory each batch
needs to go through a quality control procedure. If the products pass the QC test, they will be used in
this study.
This quality control procedure should be done quantitatively to observe that:
A. All types of bacteria can grow in this media.
B. Even low magnitude of bacteria (10-100 cfu) can grow in this media.
The procedure is written below:
1. Take pure culture of ATCC strains of S. aureus, E. coli, H. influenzae & S. pneumoniae), and prepare
the suspension of 0.5 McFarland standard in normal saline.
-5
2. Dilute the suspension upto 10 fold.
-4
-5
3. Inoculate 100l of last two dilutions (10 & 10 ) of each organism in TSB broth (containing sheep
blood)
4. Spread 100l on solid media to get the count.
5. Incubate the liquid media on 37C and the solid media at 37C with 5% CO2 for 18hrs.
6. On day two, count the colonies on the solid plate, and subculture the incubated broth on qualified
solid media as described section 7.3.5.1.
7. On day three, correlate the colony counts with previous days count. Growth will obviously appear
on the plate from the broth if significant numbers of colonies grow on the plate from day1
suspension. Otherwise, the broth should be quarantined for its efficiency.

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8.2.10 QA & QC in Oxidase Test

1.
2.

Do not use oxidized reagent that has turned to blue.

Do not use oxidized wire loop.

8.2.11 QC of quantitative determination of Glucose in CSF

Controls are recommended to monitor the performance of assay procedures with control N and
control P.
If control values are found outside the defined range, check the instrument, reagents and calibration
for problems.
Each laboratory should establish its own Quality Control scheme and corrective actions if controls do
not meet the acceptable tolerances.
Perform inventory check of your stock (AT LEAST TWICE A YEAR).

8.2.12 QA & QC in Nucleic Acid analysis by TLDA

Although in TLDA card assay test quality control is in-built, the following parameters should be
carefully monitored to comply with the quality of the test.
The test will be valid if any of the two genes GAPDH and RNP3 are amplified and the threshold cycle
(CT) value of the target pathogen is less than 38 and the CT values of the reagent control and test
positive controls are within range. If CT values of more than two target pathogens are less the 38, then
the specimen has multiple infection/ co-infection.
1.

To check the quality of nucleic acid extraction, 3 universal human RNAs are tested. If the CT values
of these target sequences are within range then the quality of extraction will be considered
satisfactory. If the CT value of these sequences are above the range and other parameters of the
test are within limit, then the extraction system should be rechecked, including expiry date of the
extraction kits, equipment performance etc.

2.

For the validity of the test, CT value of reagent control should be above the cut off value (40
cycles), and CT value of the target control specimens should be within the specific range
mentioned by the provider. If this criterion is not met, the test will be not valid. In that case, check
expiry date of the positive controls and check expiry date of the reagent. If expiry date is okay
then repeat the test.

3.

If the CT values for a target pathogen differ more than 2 cycles in duplicate wells, specimen has
not mixed properly. In that case, mix the specimens with reagent by vortexing vigorously.

8.2.13 Laboratory Quality control

The Coordination Centre will ship 5% of all the positive specimens and 2% of all the negative
specimens to the CDC as part of the external quality control for laboratory examinations. CDC will
carry out the tests and compare the results with the site laboratory and central laboratory results. CDC
will share the results of the tests with the respective laboratories along with suggestions to improve
the quality of laboratory procedures.

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8.3 QA & QC for Data Management

To ensure the data quality, a portion of individual or household will be re-interviewed for all study
sites. The QA/QC data will be entered and will be compared with the original dataset. The results will
be shared with all the site investigators and the Coordination Centre. Coordination Centre will provide
the program and support for the QA/QC of data management. Subject enrolment and management
will be conducted by a number of trained staff who will be dedicated to the study to ensure
consistency of data collection. Study personnel from each site will be trained and supervised by the

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investigators to ensure quality data collection. Data centre will provide different types of reporting
option to monitor the field activities in each site which is integrated into the system.

8.3.1 Check for integrity of data sent by the sites

Data centre will provide different types of reporting option to monitor the field activities at each site
which is integrated into the system. The Coordination Centre data team will monitor whether the data
collection tools are functioning properly.

8.3.2 Provisions for protecting privacy and confidentiality

Questionnaires and data entry forms will be stored at each site in a secured place and only authorized
person can access the data forms. All enrolment study subject from each site will get a unique
identification number. Laboratory specimens will be labelled using a unique number that should link
with subject. The data entry personnel will enter data from the data collection forms into SQL
database using the identification number. All materials with enrolment subject identifiers will be
maintained in a secure locked local site under the supervision of the primary investigator at each site.
All study personnel will be trained in procedures for maintaining confidentiality. No personal
identifiers will be used in any reports or publications arising from this study.

8.3.3 Check for integrity of data sent by the sites

The Coordination Centre data team will monitor whether the data collection tools are functioning
properly. This supervision will include periodic review of data collection forms for completeness and
consistency, and periodic visits by supervisors to the field to observe interview techniques. The QC
data entry will be checked real time by the field coordinator. A data cleaning process will review the
overall quality of data, and share the quality control reports with the team, and take necessary steps
whenever required.

8.4 Field QA &QC

8.4.1 Monitoring
Monitoring is necessary to assure adequate protection of the rights of human subjects and the safety
of all subjects involved in clinical investigations and the quality and integrity of the resulting data.
CHRF will monitor the research studies on the basis of this document. There are some principles
recognized by the scientific community as desirable approaches to monitoring research involving
human subjects. These principles are not legal requirements but represent an acceptable standard of
practice.

8.4.1.1 A. Selection of a Monitor

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CHRF will be responsible, as part of its study management role, for monitoring of the research studies.
CHRF will assign a technical staff member with considerable experience in research and research
management and monitoring as the primary monitor for a site. The assigned CHRF staff member will
additionally take help from external temporary advisers with the required expertise from time to time
(collectively called the "Technical Advisory Group (TAG). The qualifications required from monitors will
possibly vary during the implementation of the study, given the changing needs of project.

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8.5 Role of CHRF during the preparatory phase of the study

8.5.1.1 Investigators' workshop
CHRF, through the assigned CHRF technical staff for each site, will be responsible for assuring that
investigators clearly understand and accept the obligations incurred in undertaking the research. CHRF
will hold investigators' workshops to finalize site-specific protocols, standard operating procedures,
consent forms and data collection instruments. This workshop will aim at ensuring standardization of
the preparation across sites. At the end of the workshop, the CHRF will ensure itself that the study
investigators at their sites:
1. Understand all the details of the research protocol.
2. Understand the requirements for an adequate and well-controlled study.
3. Understand and accept their obligations to obtain informed consent from the pregnant women
and parents/guardian of the enrolled neonates.

8.5.1.2 Training and standardization

The internal monitors will visit all the sites during the process of training and standardization. The
internal monitors will assure that the training and standardization is occurring in line with the plan
outlined in the Manual of Operation and Standard Operating Procedures. Internal monitors will also
organize a standardization session for key measurements and procedures during this visit.

8.5.1.3 Study initiation site visit

At the time just prior to participant recruitment, , the internal monitors will visit the sites to reassure
CHRF that the investigators:
1. Have access to an adequate number of suitable subjects to conduct the research.
2. Have adequate facilities for conducting the clinical research.
3. Have sufficient time from other obligations to carry out the responsibilities to which the
investigator is committed by signing agreements with CHRF
4. Have sufficient and adequate trained and standardized staff to help them conduct the research
activities
5. Have adequate supplies and equipment needed for conducting the study.

8.5.1.4 Monitoring periodic reports

It would include information about babies enrolled, exclusion criteria, eligibility, enrolment, progress
during follow-up 1-59 days (alive, dead, withdrawn from the study, lost to follow up, unknown status,
relapses and deaths). These reports will be sent to CHRF Study Coordinator, CHRF will provide
feedback within one week of receiving such a report. Furthermore, the sites will also provide
information about the progress of data entry (number of CRF sets completed, double data entered,
time lag between completion of data collection and entry, transmission of data to the Data
Coordination Centre etc.).

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8.5.1.5 Serious Adverse Events monitoring

Serious adverse events such as death during follow-up period of enrolled babies will be reported
within one week to the Study Coordinator, CHRF will then share it at periodic intervals with Technical
Advisory Group (TAG) and local Institutional Review Board (IRB) (as per local IRB requirement) and
CHRF Ethical Review Committee (ERC). Study Coordinator will provide feedback to sites within one
week of getting SAE reports.

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8.5.1.6 Data Coordination Centre's monthly reports

Double data entry will be done at country level, and routine range and consistency checks will be
applied at country level, with errors corrected before periodic and timely transmission of the data to
Data Coordination Centre (DCC) via a secure server. DCC will conduct further data quality control
checks, review and generate queries and give feedback to the sites/CHRF coordinator, get responses
and resolve conflicts to ensure that identical copies of the database are held within country and in
CHRF, Dhaka. DCC will provide progress reports to the Study Coordinator every month.

8.5.2 Site visit monitoring checklist

CHRF has also adopted a "Site Monitoring Checklist" for monitoring the research studies during site
visits (Appendix III). The aim of this checklist is to audit the quality of the study and to assure that each
person involved in the monitoring process carries out his or her duties in a comprehensive way during
a site visit.

8.5.2.1 Periodic site visits during study implementation

CHRF is responsible for assuring throughout the research that the investigators obligations are being
fulfilled, the facilities used in the investigation continue to be acceptable and that the study is being
implemented according to the approved protocol. The most effective way to achieve this assurance is
to maintain personal contact between TAG and the Project Investigator throughout the research
process. The Coordination Centre representatives will pay 3 visits per year to each of the study sites.
The monitoring team will do the following:

8.5.2.2 Study Management review

To assure that:
1. The facilities used by the investigator continue to be acceptable for purposes of the study.
2. The study protocol or investigational plan is being followed.
3. Changes to the protocol, if any, have been approved by the IRB/ERC and/or reported to the
sponsor and the IRB/ERC.
4. Accurate, complete, and current records are being maintained.
5. Accurate, complete, and timely reports are being made to CHRF, TAG and IRB/ERC.
6. The investigator is carrying out his/hers agreed-upon activities and has not delegated them to
other previously unspecified staff.

8.5.2.3 Review of Subject Records

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CHRF is responsible for assuring that the data are accurate and complete. The most effective way to
assure the accuracy of the data is to review individual subject records and other supporting documents
and compare those records with the reports prepared by the investigator for submission to CHRF, TAG
and/or IRB/ERC. Therefore, during the periodic visit, the monitor will compare a representative
number of subject records and other supporting documents with the investigators reports to
determine that:
1. The information recorded in the investigators report is complete, accurate, and legible.
2. There are no omissions or discrepancies in the reports of specific data elements such as serious
adverse events.
3. Missing visits or examinations are noted in the reports.
4. Subjects failing to complete the study and the reason for each failure are noted in the reports.
5. Informed consent has been documented.

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8.5.2.4 Observations of key procedures

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.

Observe/review screening, enrolment and randomization

Administering the consent form
Observe management of cases
Control selection
Consistency in data between various levels (CHW, Study Physician and Site Supervisor)
Study outcome assessment
Home visits to see some enrolled neonates and interview mothers/caregivers
Communication between families and study staff
Specimen collection
Laboratory procedures (blood culture, CSF analyses, TLDA)
Data management

8.5.2.5 Site monitoring reports and feedback to the Principal Investigators

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Coordination Centre representatives will prepare reports on the findings, conclusions, and actions
taken to correct deficiencies for each monitoring to an investigator. Such a report will document the
fulfillment of CHRF's obligations in monitoring the progress of the study. The report will include at
least the following elements such as:
1. The date of the visit.
2. The names of the Coordination Centre representatives who conducted the visit.
3. The names and addresses of the PIs visited.
4. A statement of the findings, conclusions and any actions taken to correct any deficiencies noted
during the visit.
5. Feedback provided to the PIs
6. Actions taken or to be taken, and/or actions recommended to secure compliance.
The follow-up on recommended actions in previous visits will be documented. Periodic reporting by
the sites on a pre-agreed format and agreed parameters will be an on-going process. If any unforeseen
problems are identified in between, an extra monitoring visit can be undertaken as and when needed.

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9. Waste management

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9.1 Objectives
The objectives of waste management are as follows:
1. To protect the patients from the risk of hazardous wastes.
2. To protect the healthcare providers from the risk of hazardous wastes.
3. To prevent dissemination of pathogenic microorganisms into the environment.
4. To reduce the volume of waste to be dumped.

9.2 Introduction
Waste management is the collection, transport, processing, recycling or disposal of waste
materials including solid, liquid, gaseous or radioactive substances. Waste management
practices differ for developed and developing nations, for urban and rural areas, and for
residential, industrial and clinical producers.
The management of healthcare wastes is discussed in this chapter with regards to
categorization, definition and management guidelines:

9.3 Categories of healthcare waste

9.3.1 Infectious Waste
Infectious waste is suspected to contain pathogens (bacteria, viruses, parasites, or fungi) in
sufficient concentration or quantity to cause disease in susceptible hosts. This category
includes:
1. Cultures and stocks of infectious agents from laboratory work;
2. Empty or full plastic tubes contaminated with pathological and clinical agents (gloves,
syringes, contaminated gauge, bandage, body parts, specimen tubes);
3. Waste from surgery and autopsies on patients with infectious diseases (e.g. absorbent
tissues, and materials or equipment that have been in contact with blood or other body
fluids);
4. Waste from infected patients like excreta, dressings from infected or surgical wounds,
clothes heavily soiled with human blood or other body fluids);
5. Any other instruments or materials that have been in contact with infected neonates;
6. All the culture media both solid and liquid, the biological samples like blood and serum,
suspension of cells and supernatant contain live cells.
Note: This category does not include Sharp objects namely, scalpels, scissors, ampoules,
needles.

9.3.1.1 Segregation

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Put in YELLOW BOX. Print a standard BIOHAZARD symbol on the box.

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9.3.1.2 Treatment
1. Collect the solid wastes in autoclavable bags labeled with bio-hazard warning symbol.
Close but do not seal the bag before autoclaving.
2. For live cultures and stocks, empty the vessels and pour the contents into autoclavable
metal container. Autoclave the containers.

9.3.1.3 Disposal
1. Drain off the liquid and semisolid waste through the existing drainage facility.
2. Send the autoclaved plastic containers for recycling.
3. Bury the sterilized solid wastes in soil.

9.3.2 Non infectious waste

This category includes the non-contaminated, recyclable and non-recyclable solid waste
normally deposited into office garbage receptacles like paper towels and tissues, carbon
paper, plastic wrappers and plastic ware etc.

9.3.2.1 Segregation
Put these wastes in BLACK BOX or BLACK labeled box.

9.3.2.2 Treatment
NO treatment is required for these wastes

9.3.2.3 Disposal
Discard these wastes into transparent plastic bag without any treatment with municipal
wastes.

9.3.3 Sharps
This category includes all the objects that have sharp edges irrespective of being infectious or
not. This category consists of the metal and glass wastes generated by activities in this study.
Metal: Hypodermic needles, scalpels and blades.
Glass: Small or large, intact or broken glassware, bottles and ampoules. Put in separate box.

9.3.3.1 Segregation
Empty all glass containers in puncture-resistant bottle containing disinfectant (bleach). Label
this container as SHARP DISCARD. Discard the glass and metal objects in a metal container
that contains disinfectant (bleach). Put standard BIOHAZARD symbols on the bottle and the
box.

9.3.3.2 Treatment
Autoclave all objects

9.3.3.3 Disposal

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Instruct service workers not to mix sharp waste with other solid wastes. Send the waste to
recycling plant.

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Do not use incinerator. It has given rise to serious environmental health concern all over the
world.
Do not recap any needle. Attempts may cause puncture in skin.

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10. Site specific Roles and Responsibilities

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10.1 Investigators
1.
2.
3.
4.
5.
6.
7.
8.
9.

To supervise the implementation of the study at the site

To perform the QA/QC activities to ensure the quality of field activities
To organize training for the field staff and study physicians
To maintain liaison with the government officials and the community leaders
To take care of the sub-studies taking place at the site
To update the progress to the Coordination Centre
To ensure the Coordination Centre recommendations are being implemented at the site
To cooperate the monitoring and evaluation activities regarding the study
To provide study reports on the dates scheduled by the Coordination Centre

10.2 Field staffs

10.2.1 Field Services Supervisor or equivalent
1.
2.
3.
4.
5.
6.
7.

To supervise CHWs activities

To ensure CHWs activities are according to CHWs work plan, and ensure household visits by
CHWs as per study protocol
To evaluate CHWs performance
To attend review meeting and assist CHWs in preparing report.
To ensure logistics for CHWs works
To identify weaknesses of CHWs and recommend for training, and also provide necessary field
based training to CHWs
To assist and work with other staff members as required and instructed by the designated
supervisor

10.2.2 Project Officer or equivalent

Under the supervision and guidance of the Field Research Officer, the Project Officer will be
responsible for overall field supervision and management, and implementation of the intervention
activities of the project at the field level
1.
2.
3.
4.
5.
6.
7.

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8.
9.

To be responsible for effective and successful implementation and management of the program
by CHWs at field level
To ensure strict adherence to project plan and the project protocol as approved by concerned
authority
To carry out continuous monitoring and supervisory field visits to support study activities being
implemented in the field
To prepare fortnightly/monthly reports, and interpret the MIS compilation results effectively to
monitor the success and failures in the field implementation of the study by CHWs
To have direct observation of quality, quantity and time management of FSS during field visit
To conduct assessment of the needs of the training, education and communication activities
related to the project.
To directly assist Field Research Officer and FRM (whenever required) in planning, implementation
and management of the field programs
To carry out Effective personnel management and supporting staff development activities for field
staff (CHW, FSS)
To maintain close liaison with government officials, local NGOs, other concerned organizations
and individuals to ensure smooth implementation of project activities in the field.

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10. To assist and work with other staff members as required and instructed by the designated
supervisor
11. To facilitate field visits of project officials and other visitors and explain study activities. To assist
and work with other staff members of the program as required and instructed by the Project
Coordinator and the Principal Investigator.

10.2.3 Senior Field Research Officer (SFRO) or equivalent

Under the direct supervision of the Field Research Manager, the Senior Field Research Officer (SFRO)
will be responsible for all kinds of field activities and will also assist in the coordination and
implementation of the field activities undertaken by the project at the field sites. S/He will conduct
frequent field visits and carry out the following:
1.
Assist in developing, planning, organizing and implementing research related activities at field
level.
2.
Assist in preparing questionnaires and other survey and/or MIS tools for field research activities
3.
Assist to organize staff training as well as to coordinate field activities with data management
section in Coordination Centre
4.
Assist project officers in supervision, ensuring quality of field data collection, field monitoring
and coordination of day-to-day activities at field sites
5.
Assist and work with other team members as required and instructed by the Project Coordinator
and the Principal Investigator.

10.2.4 Field Research Manager (FRM) or equivalent

Under the direct supervision of the Deputy Coordinator or the assigned delegated Supervisor, the Field
Research Manager (FRM) will be responsible for ensuring proper implementation, supervision and
monitoring of the field research activities undertaken by the project at the field sites. The duties
include:
1. To assist in developing, organizing and implementing research related activities for the study
2. To ensure implementation of the study including organization of field activities, training of field
staff.
3. To ensure quality assurance in data collection and timely collection of data
4. To coordinate, supervise, organize and monitor day-to-day field research activities and also to
coordinate all administrative and management support for study field sites.
5. To provide coaching and mentoring to the field staff and also provide adequate attention to
resolving problems related to field research.
6. To assist Project Coordinator and Deputy Coordinator in writing reports and preparation of project
documents, questionnaire and data collection instruments.

10.2.5 Study Physicians

1.

2.

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3.

4.
5.

To reassess the newborns referred to the health facility by the CHW for meeting the eligibility
criteria for enrolment in the study and to fill up the CRF. If a case does not meet the eligibility
criteria, then s/he will not enrol the newborn and will refer the baby to any healthcare facility or
will send the baby home following standard treatment. The physicians responsibility includes:
To enrol newborns into the study, according to the inclusion and exclusion criteria following
assessment of clinical symptoms.
To advise specimen collection for the identification of the causative agents of infection, and
provide medical treatment to the newborn. In some cases, the physicians have to oversee the
specimen collection, storage and transport activity.
To attend any training or workshop he is assigned to by the authority.
To fill up the respective CRFs.

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10.2.6 Community Health Worker or equivalent

Responsibilities of the Community Health worker will be:
1. To conduct pregnancy surveillance within the respective cluster to identify pregnant women
2. To work at the household and family level to conduct antenatal counseling
3. To promote essential newborn care during and/or immediately after birth
4. To team up with traditional/family birth attendants (BA) and Newborn Care Person (NCP)
identified by the family to promote a clean delivery and proper newborn care
5. To plan and make household visits during the first two months of life as per study protocol, and
refer sick newborns to a health facility.
6. To maintain records and documentation in the prescribed formats for each of the activities in a
clear, legible, consistent and valid manner.
7. To attend review meetings with all necessary preparations completed prior to the meetings.
To cooperate with the supervisors, while the latter is accompanying her to conduct on-site observation
of activities at the household level.

10.2.7 Phlebotomist
Responsibilities of the phlebotomist will be
1. To perform venipuncture for blood collection.
2. To collect nasopharyngeal swab and throat swab from the selected neonates.
3. To attend any training or workshop he is assigned to by the authority.
4. To discard the used materials in designated containers.

10.3 Laboratory staffs

10.3.1 Laboratory Manager or equivalent
Responsibilities of the Laboratory Manager will be:
1. To oversee the activities of site laboratory and central laboratory
2. To monitor all the laboratory activities
3. To report the progress and problems to the site coordinator
4. To perform compliance assessment with the Manual Of Operations
To place requisitions for the needs of the laboratory
5. To attend any training or workshop he is assigned to by the authority.

10.3.2 Laboratory Coordinators or equivalent

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Responsibilities of the Laboratory Coordinators will be:

1. To monitor the activities of the respective laboratory
2. To report the development to the laboratory manager
3. To play the role of Laboratory Manager in his/her absence
To send specimens and reports to the central laboratory and the coordination center
To update the inventory once in a month
4. To perform laboratory environment control activities
5. To attend any training or workshop he is assigned to by the authority.
6. To Manage and organize all documentations regarding the laboratory activities

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10.3.3 Microbiologist
Responsibilities of the Microbiologist will be:
1. To Process and store specimens for analysis
2. To carry out microbiological analyses for the identification of pathogens
3. To perform molecular biology assays (PCR, TLDA)
4. To fill up the respective laboratory CRFs
5. To prepare the weekly and monthly progress reports
6. To perform any diagnostic procedure assigned by the supervisor or the authority.
7. To attend any training or workshop he is assigned to by the authority.

10.3.4 Technologist
Responsibilities of the Technologist will be:
1.
To perform laboratory environment control activities
2.
To perform specimen storage activities
3.
To perform laboratory culture activities
4.
To perform any diagnostic procedure assigned to by the supervisor or the authority.
5.
To attend any training or workshop he is assigned to by the authority.
6.
To perform the blood culture by BACTEC 9050 blood culture system
7.
To perform CSF cytology and bio chemical tests
8.
To tabulate all the results of all the tests performed at the site laboratory
9.
To perform the Gram staining of the presumptive culture positive blood culture bottle
10. To perform the validation activities at scheduled dates
11. Manage and organize all documentation regarding the laboratory activities
12. To pack the cool box with ice packs, specimens and labels for transport to central lab

10.3.5 Laboratory assistants

Responsibilities of the Laboratory Assistants will be:
1. To assist the Physician, Phlebotomist and Technologist in every activity.
2. To prepare media, reagents, chemicals, necessary equipments and materials for any activity to be
performed by the Physician, Phlebotomist and Technologist. e.g., specimen collection, processing,
testing, culturing and transport.
3. To check the temperature of the refrigerators on scheduled dates.
4. To discard the used materials, cultures, tubes, and other pathological wastes in designated
containers.
5. To transport the prepared media and required reagents from central lab to site laboratory.

10.3.6 Carrier
Responsibilities of the Carrier will be:
1. To carry commodities or specimens from and to any place as ordered by the Physician,
Phlebotomist, Technologist and Laboratory Assistant.
2. To carry goods, reagents and other things between the site laboratory and the central laboratory.

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10.3.7 Cleaner
Responsibilities of the Cleaner will be:
1. To do the cleaning up activities routinely in the specimen collection facility and in the laboratory
as per the instruction of the supervisor.
2. To carry out the weekly disinfection procedures as per the instruction of the supervisor.
3. To treat and dispose the wastes as instructed in chapter 12 of this manual

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10.4 Data Coordination Centre

10.4.1 System Analyst:
Responsibilities of the System Analyst will be:
1. To manage the Data Management activities and ensure adequate data processing
2. System analysis, design and assist programmers to develop the customized computarized data
capture system, report etc.
3. To assist investigators to develop the data collection instruments
4. To coordinate data management staffs and trained essentials packages for data entry and data
analysis
5. To coordinate with the field staff/officers, and train field staff on appropriate data coding and
capture systems
6. To provide analytical support to the scientific staffs according to the requirement.
7. Visit different fields sites for installing the developed systems, and provide training for
appropriate data capture system
8. Ensure the security of database and questionnaires
9. Maintain and ensure the timely output

10.4.2 Senior programmer:

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Responsibilities of the Senior Programmer will be:

1. To design and develop the customized and computerized data capture systems
2. To prepare user manual and documentation for the data capture system, database and data files
3. To maintain communication with the study sites as per requirement
4. To prepare working files from the raw datasets according to the requirement of scientific staffs for
data analysis
5. To develop different types of report to monitor the field activities and progress
6. To incorporate automated data backup and auditing system
7. To ensure the safety and security of data at different levels.
8. To provide analytical support to the scientific staffs according to the requirement
9. To coordinate data management staffs and trained essentials packages for data entry and Also
provide them essential supports related to data entry

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Appendix I
Culture media preparation
Culture media for the isolation of blood and CSF specimen will be prepared at the central lab. Each
batch of media will go through a process of quality assurance (QA) during preparation and quality
control (QC) after then. Media will be distributed among the site labs only if the batch of prepared
media has passed quality control test. The procedure for QC test has described at section 7.5.

MacConkey Agar Plate (Mac)

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Principle
MacConkey agar is a selective media for most of the Gram-negative
bacteria except H. influenzae and N. meningitidis; it can also
differentiate the gram negative bacteria based on lactose
fermentation. MacConkey agar media contains bile salts that inhibit
most of the Gram-positive bacteria, except Enterococcus and some
species of Staphylococcus. It also contains Crystal violet dye which
inhibits certain Gram-positive bacteria and neutral red dye that
stains lactose fermenting microbes. By utilizing the lactose available
in the medium, Lactose producing bacteria (E. coli, Enterobacter and
Klebsiella) produce acid, which lowers the pH of the agar below 6.8
and results in the appearance of red/pink colonies. Non-Lactose
fermenting bacteria (Serratia and Citrobacter) use peptone, forms
ammonia, which raises the pH of the agar and leads to the
formation of white or colourless colonies.

MacConkey Agar plate

Preparation:
1. Suspend the dehydrated powder (Oxoid CM0115, Hampshire, England) with required amount of
distilled water according to manufacturers instruction.
2. In case of Oxoid product CM0115, suspend 51.5 gram of dehydrated media in 1 litre of distilled
water.
3. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
4. Measure the pH of the media. It should be within the range of 7.2-7.6 at room temperature.
Adjust the pH if required as described above.
5. Insert a dry, tight cotton plug on the top of the flask.
6. Autoclave the media at 121C at 15 PSI for 15 minutes.
7. Transfer the autoclaved media to a 50C water bath.
8. When the agar has cooled to 50C, dispense 20 ml onto a sterile petri dish inside the laminar flow
cabinet. Remove the bubbles from the plate if formed after dispensing.
9. Place the plate inside the leveled surface of the laminar flow and keep at room temperature to
solidify the media.
10. Label the plate with media name, date and batch number.
11. Send a randomly selected media for QA test. Store the rest at 2-8C in sealed plastic bags to
prevent the loss of moisture.

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Blood Agar (BA) Plate

Principle
Blood agar (BA) plate containing 5% sheep blood is a
differential growth medium that differentiates pathogenic
bacteria based on the interaction of sheep's blood,
bacterial hemolytic enzymes (appendix2) and morphology
of the colonies. The pH of media maintained at 7.3
stabilizes red blood corpuscles and favors the formation of
a clear haemolysis zone.
Blood Agar plate

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Procedure:
1. Suspend the dehydrated powder (Oxoid CM0055, Hampshire, England) with required amount of
distilled water according to manufacturers instruction. In case of Oxoid product CM0055, suspend
40g of dehydrated media in 1 litre of distilled water.
2. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
3. Measure the pH of the media. It should be within the range of 7.2-7.6 at room temperature.
Adjust the pH if required using 0.1 mol/l (N/10) sodium hydroxide when the medium is too acid,
and 0.1 mol/l (N/10) hydrochloric acid when too alkaline.
4. Insert a dry, tight cotton plug on the top of the flask.
5. Autoclave the media at 121C for 15 minutes.
6. Transfer the autoclaved media to a 50C water bath.
7. When the agar has cooled to 50C, aseptically add 5% sterile sheep blood and mix gently. During
mixing, avoid forming air bubbles.
8. Dispense 20 ml onto a sterile petri dish inside the laminar flow cabinet. Remove the bubbles from
the plate if formed after dispensing.
9. Place the plate inside the leveled surface of the laminar flow and keep at room temperature to
solidify the media.
10. Label the plate with media name, date and batch number.
11. Send a randomly selected media for QA test. Store the rest at 2-8C in sealed plastic bags to
prevent the loss of moisture.

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Chocolate agar (CA) plate

Principle
Chocolate agar (CA) plate is a boiled blood agar plate, in
which red blood cells have been lysed by heating very slowly
at 80C. These facilitate to extract two growth factor, NAD
and hematin inside from erythrocytes. Haemophilus
influenzae can grow on only chocolate agar plate due to
availability of these two growth factors in the media. Plate
does not chocolate; it is named chocolate agar only for its
color.

Chocolate Agar plate

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Procedure:
1. Suspend the dehydrated powder (Oxoid CM0055, Hampshire, England) with required amount of
distilled water according to manufacturers instruction. In case of Oxoid product CM0055, suspend
40g of dehydrated media in 1 liter of distilled water.
2. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
3. Measure the pH of the media. It should be within the range of 7.2-7.6 at room temperature.
Adjust the pH if required.
4. Insert a dry, tight cotton plug on the top of the flask.
5. Autoclave the media at 121C for 15 minutes.
6. Transfer the autoclaved media to a 50C water bath.
7. When the agar has cooled to 50C, aseptically add 5% sterile sheep blood and mix gently.
8. Heat the liquid media mixed blood at 80C for 15 minutes to lyse the blood. The red color of the
media will turn into chocolate.
9. Dispense 20 ml onto a sterile petri dish inside the laminar flow. Remove the bubbles from the
plate if formed after dispensing.
10. Place the plate inside the leveled surface of the laminar flow and keep at room temperature to
solidify the media.
11. Label the plate with media name, date and batch number.
12. Send a randomly selected media for QA test. Store the rest at 2-8C in sealed plastic bags to
prevent the loss of moisture.

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Mueller-Hinton Agar Plate (MA):

Principle
Mueller Hinton Media contains beef infusion and casamino
acids, as a source of energy and nutrients. It also contains
starch, acts as a colloid that provide protection against toxic
material in the medium. The levels of tetracycline and
sulfonamide inhibitors, thymidine, thymine, magnesium and
calcium ions are controlled so as not to interfere with
susceptibility testing (disk diffusion and E-strips) and to yield
good growth. Susceptibility testing is done using this media of
those organisms, which do not require blood for their growth.
E.g.- Klebsiella sp., Salmonella sp., E.coli, Acinetobacter,
Pseudomonas

Mueller-Hinton Agar plate

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Preparation:
1) Suspend the dehydrated powder (Oxoid CM0337, Hampshire, England) with required amount of
distilled water according to manufacturers instruction. In case of Oxoid product CM0337, suspend 38
g of dehydrated media in 1 liter of distilled water.
2) Mix thoroughly. Heat with frequent agitation, and boil for 1 minute to completely dissolve the
powder.
3) Measure the pH of the media. It should be within the range of 7.2-7.6 at room temperature. Adjust
the pH if required.
4) Insert a dry, tight cotton plug on the top of the flask.
5) Autoclave the media at 121C for 15 minutes.
6) Transfer the autoclaved media to a 50C water bath.
7) When the agar has cooled to 50C, dispense 20 ml onto a sterile petri dish. Remove the bubbles
from the plate if formed after dispensing.
8) Place the plate inside the leveled surface of the laminar flow and keep at room temperature to
solidify the media.
9) Label the plate with media name, date and batch number.
10) Send a randomly selected media for QA test. Store the rest at 2-8C in sealed plastic bags to
prevent the loss of moisture.

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Mueller-Hinton Blood Agar Plate (MB)

Principle
Mueller Hinton Media contains beef infusion and casamino acids, as a source of energy and nutrients.
It also contains starch, acts as a colloid that provide protection against toxic material in the medium.
The levels of tetracycline and sulfonamide inhibitors, thymidine, thymine, magnesium and calcium
ions are controlled so as not to interfere with susceptibility testing (disk diffusion and E-strips) and to
yield good growth. Susceptibility testing is done using this media of those organisms, which do not
require blood for their growth. E.g.- Streptococcus sp., Staphylococcus sp.,
Preparation:
1. Suspend the dehydrated powder (Oxoid CM0337, Hampshire, England) with required amount of
distilled water according to manufacturers instruction. In case of Oxoid product CM0337, suspend
38 g of dehydrated media in 1 litre of distilled water.
2. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
3. Measure the pH of the media. It should be within the range of 7.2-7.6 at room temperature.
Adjust the pH if required.
4. Insert a dry, tight cotton plug on the top of the flask.
5. Autoclave the media at 121C for 15 minutes.
6. Transfer the autoclaved media to a 50C water bath.
7. When the agar has cooled to 50C, aseptically add 5% sterile sheep blood and mix gently. During
mixing, avoid forming air bubbles.
8. Dispense 20 ml onto a sterile petri dish inside the laminar flow. Remove the bubbles from the
plate if formed after dispensing.
9. Place the plate inside the leveled surface of the laminar flow and keep at room temperature to
solidify the media.
10. Label the plate with media name, date and batch number.
11. Send a randomly selected media for QA test. Store the rest at 2-8C in sealed plastic bags to
prevent the loss of moisture.

10.4.3 Biochemical test media preparation

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Bio-chemical media is required for the primary identification of isolates. In our lab, we use 3 types of
biochemical media for most Gram negative clinical isolates. Further biochemical tests and serological
typing must be performed for definite identification and confirmation of organisms.
Each batch of prepared media will go through a process of quality assurance (QA) and quality control
(QC). Media will be distributed among the site labs only if the batch of prepared media has passed
quality control test. The procedure for QC test has described at Chapter 11.

ANISA

Kligler Iron Agar (KIA) /Triple Sugar Iron Agar (TSI)

Principle
KIA contain same composition of TSI are same except sucrose. Sucrose is
added to TSI to facilitate some sucrose-fermenting lactosenonfermenters such as Proteus and Citrobacterspp (ref-2, Macfadin
1985). Kligler Iron Agar, in addition to casein and meat peptones,
contains lactose and dextrose which enable the differentiation of species
of enteric bacilli due to color changes of the phenol red pH indicator in
response to the acid produced during the fermentation of these sugars.
The dextrose concentration is only 10% of the lactose concentration. The
combination of ferric ammonium citrate and sodium thiosulfate enables
the detection of hydrogen sulfide (H2S) production.

Kligler Iron Agar

Preparation
1. Suspend the dehydrated powder (Oxoid CM0277, Hampshire, England) of TSI with required
amount of distilled water according to manufacturers instruction. In case of Oxoid product
CM0277, suspend 13 gm of dehydrated media in 200 ml of distilled water.
2. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
3. Measure the pH of the media. It should be within the range of 6.8-7.2 at room temperature.
Adjust the pH if required.
4. Dispense 7 ml in each large size screw cap tubes (approx. 16160 mm) before doing autoclave.
5. Loosen the cap of tubes containing media and autoclave at 121C for 15 minutes.
6. Allow the medium to solidify in a sloped position to give a butt 25-30 mm deep and slope 20-25
mm long (the butt should be longer than the slope).
7. Label the plate with media name, date and batch number.
8. Send a randomly selected media for QC test. Store the rest at 2-8C in a dark place.

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88

Note Hydrogen sulfide production may be evident on KIA but negative on TSI agar in few cases. Studies
by Bulmash and Fulton (ref.-3, 1964) showed that the utilization of sucrose could suppress the
enzymatic mechanisms responsible for H2S production. Padron and Dockstader (ref.-4, 1972) found that
not all H2S -positive Salmonella are positive on TSI.

ANISA

MIU (Motility Indole Urease) Agar

Principle: MIU media contain urea and phenol red to detect urease enzyme produced
by organism. This media is a semi-solid media that confirm the motility of the organism
by changing the transparent appearance of the media into fuzzy.

MIU Agar

Preparation:
Suspend the dehydrated powder (HIMEDIA M1076, Mumbai, India) with required amount of
distilled water according to manufacturers instruction. In case of HIMEDIA product M1076,
suspend 3.6 gm of dehydrated media in 190 ml of distilled water.
2. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
3. Measure the pH of the media. It should be within the range of 6.6-7.0 at room temperature.
Adjust the pH if required.
4. Insert a dry, tight cotton plug on the top of the flask.
5. Autoclave the media at 121C for 15 minutes.
6. Transfer the autoclaved media to a 50C water bath.
7. When the agar has cooled to 50C, aseptically add 10 ml of sterile 40% urea solution in 190 ml
media and mix gently. During mixing, avoid forming air bubbles.
8. Dispense 5 ml in each tube (approx. 16150 mm).
9. Allow the media to solidify Place the tube in upright position and DO NOT MAKE ANY SLOPE IN
THIS MEDIA. Cover with cap or cotton at the top.
10. Label the plate with media name, date and batch number.
11. Send a randomly selected media for QC test. Store the rest at 2-8C.

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89

1.

ANISA

Simmons Citrate Agar (SCA)

Principle
Simmons citrate agar media allows the growth of the organism which can
exclusively depend on citrate as a sole source of energy. Bromophenol blue
change of color of the medium if citrate is utilized.

Simmons Citrate Agar

1.

2.
3.

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90

4.
5.
6.
7.
8.

Preparation: Suspend the dehydrated powder (Oxoid CM0155, Hampshire, England) with required
amount of distilled water according to manufacturers instruction. In case of Oxoid product
CM0155, suspend 4.6 gm of dehydrated media in 200 ml of distilled water.
Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
Measure the pH of the media. It should be within the range of 6.8-7.2 at room temperature.
Adjust the pH if required.
Dispense 7 ml in each large tube (approx. 16150 mm) before doing autoclave.
Loose the cap of the tube containing media and autoclave at 121C for 15 minutes.
Allow the medium to solidify in an oblique position to prepare only slant (no butt is required).
Label the plate with media name, date and batch number.
Send a randomly selected media for QC test. Store the rest at 2-8C.

Back to CONTENTS

ANISA

Appendix II
10.4.4 Table for EDD Calculation
Carefully figure out the LMP (First day of the last menstrual period) with the help of this table

LMP EDD LMP EDD LMP EDD LMP EDD LMP EDD LMP EDD LMP EDD LMP EDD LMP EDD LMP EDD LMP EDD LMP EDD

Page

91

Jan Oct Feb Nov Mar Dec Apr Jan May Feb Jun Mar Jul Apr Aug May Sep Jun Oct Jul Nov Aug Dec Sep

10

10

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11

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Jan Nov Feb Dec Mar Jan Apr Feb May Mar Jun Apr Jul May Aug Jun Sep Jul Oct Aug Nov Sep Dec Oct

** EDDs are indicated in BOLD letters

Back to CONTENTS

ANISA

Appendix III
Checklist for Site Monitoring Visit for the ANISA Study
(Adopted from WHO checklist for Neonatal Sepsis Study)
A pre- agreed agenda and timing of the visit with the investigators is necessary for the success of a
monitoring visit. Monitors will hold meetings with PIs and other study staff during the visits and
discuss aspects of the study progress. Monitors will do the following by review/observation/checking
Study management review
1. Availability of updated protocol with all the approved amendments, procedures
manual/standard operating procedures, consent forms and communications with the local
IRB and CHRF and their approvals
2. Storage space [locked cabinets and storage of case report forms (CRF)]
3. Progress of the study/enrolment and Fund utilization
4. Maintenance of screening and enrollment treatment follow-up registers / forms.
5. Transport system (for follow-up, supervision and specimen collection)
6. Local process/system of checking data quality
7. Process of feedback documentation and identification of gaps
8. CRF shipment to Data Coordination Centre and computerized data entry
9. Communication between various cadres of health workers/supervisors, with investigators and
with Data Coordination Centre and CHRF
10. Situation of supplies/reordering of supplies

Review of subject records

1. Check filled CRFs (randomly selected 10-20%) and Home visits to see some enrolled infants
and interview mothers/caretakers
2. Check all (or at least 50%) clinical assessment and serious adverse events forms CRFs

Observation of key study procedures

1. Screening, enrollment and randomization of study patients and administering the consent
form
2. Collection of used needles, syringes, medicine vials and bottles and proper storage/disposal
3. Maintenance of follow-up calendar/proper date of follow-up
4. Follow-up of neonates and completing follow up CRF's
5. Consistency in data between various levels (CHW, study physician, and study supervisor)
6. Communication between study staff and potential/enrolled patients' families
7. Data management (data entry progress, due, done, entered, double date entry, audit trail,
security, cleaning and data backup) and regular transmission of electronic data to the Data
Coordination Centre

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Standardization
1. Review presence of training materials and training/re-training records as per CHRF
expectations
2. Interviews with a sample of data collectors to confirm that processes being used are
according to the Manual of Operation.
3. Organize a standardization session for key measurements and procedures during this visit.

Site monitoring report and feedback

1. Feedback and actions for Principal Investigators
2. Recommendations and actions for CHRF
3. Report prepared and submitted
4. Date and signature

Back to CONTENTS

ANISA

Appendix IV
Table of colony characteristics of different bacteria
Organism
Media Plate
Color
S. pneumoniae
Blood Agar
Green

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93

H. influenzae

Size (mm)
1-2 mm

Shape
Round

Mucoid
No/Yes

Haemolysis
Alpha

Flat
convex
Low and
convex

Yes

No

Mucoid,
Gummy

No

Grayish

1-2 mm

N. meningitidis

Chocolate
Agar
Blood agar

Gray

1 mm

Flavobacterium
Streptococcus spp.

Blood agar

colourless

0.5-1.0mm

Staphylococcus

Blood Agar

Salmonella
E. coli
Klebsiella
Serratia

MacConkey
MacConkey
MacConkey
MacConkey

Pseudomonas
Acinetobacter

MacConkey
MacConkey

Yellow
to 1-2 mm
cream
or
occasionally
white
colourless
2-3 mm
pink
1-4 mm
pink
Red pigment
in
nutrient
agar at room
temperature
greenish
colourless

circular
circular

yes

haemolytic
May
be
Betahaemolytic

Yes

Table for Gram reaction of different bacteria under microscope

Organism
Gram reaction

Shape (form)

S. pneumoniae
H. influenzae
N. meningitidis
Flavobacterium
Streptococcus
Staphylococus
Listeria
Salmonella
E.coli
Klebsiella
Serratia
Pseudomonas
Acinetobacter

Cocci (Diplococci)
Cocco-bacilli or short rod
Cocci (Diplococci)
Rod
Cocci (Chain)
Cocci (Cluster)
Short rod or coccobacilli
Rod
Short Rod
Rod (Capsulated)
Rod
Rod
Coccobacilli or cocci

G+ve
G-ve
G-ve
G-ve
G+ve
G+ve
G+ve
G-ve
G-ve
G-ve
G-ve
G-ve
G-ve

ANISA

Table for Differentiation of certain Gram negative pathogens by biochemical tests

Indole
Urease Triple Sugar Iron medium
Organism
Simmons Motility
medium
Citrate
agar
Motility Indole Urea
Slant Butt H2S
Gas

Klebsiella pneumoniae

+79 -21

+75 -25

Klebsiella oxytoca

+89 -11

+78 -22

Enterobacter cloacae

+85 -15

Enterobacter aerogenes

+60 -40

Citrobacter freundii

+73 -27

+7 -93

+69 31

Salmonella spp.

+75 -25

Salomnella typhi

Salmonella paratyphi A

Edwardsiella tarda

Escherichia coli

+80 -20

+95 -5

+90 -10

Symbol analysis:
A = acid (yellow) reaction,
K = alkaline (red) reaction,
D = different biochemical types,
Numbers = positive, negative rate.
 = produced a little

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94

Note: Any organism that does not fit with the above mentioned characters should be sent to the
reference laboratory with specific notes.

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