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NeoOBS – Microbiology Manual, Version 1.0 15.08.2018 of 44

NeoOBS Microbiology Manual

Study Title NeoAMR Global Neonatal Sepsis Observational Study (NeoOBS): A

Prospective Cohort Study Of Sepsis In Hospitalised Neonates

Sponsor: GARDP / DNDi

Chief Investigator(s) Professor Mike Sharland and Prof Paul Heath (St George’s

University of London)

Lead Microbiologist(S) Professor Herman Goossens (University of Antwerp / COMBACTE

LAB-Net)

Study number NeoOBS 001


Contents

1. Abbreviations ............................................................................................................ 5

2. NeoOBS Study Organisation ....................................................................................... 6

2.1 Study Coordinators ........................................................................................................................6

2.2 Contact Information .......................................................................................................................6

3. Introduction to the study ........................................................................................... 8

4. Study design .............................................................................................................. 9

5. The Screening/Enrolment process for the Microbiology Cohort ................................. 10

How to identify/screen a potential baby for the NeoOBS Microbiology Cohort ...................... 10

Enrolement into the NeoOBS Microbiology Cohort .................................................................. 10

Communication of results to the clinical team ......................................................................... 11

6. Overview of the sample materials ............................................................................. 13

Description of Sample Materials ............................................................................................... 14

Labels ......................................................................................................................................... 15

Study ID ..................................................................................................................................... 15

7. Sample collection instructions – AS PER LOCAL ROUTINE LABORATORY PROCEDURES 17

Blood samples ........................................................................................................................... 17

Cerebrospinal fluid .................................................................................................................... 17

8. Sample processing procedures – AS PER LOCAL ROUTINE LABORATORY

PROCEDURES ................................................................................................................... 18

Blood samples ........................................................................................................................... 18

Cerebrospinal fluid .................................................................................................................... 19

9. Storage of isolates – ADDITIONAL STUDY PROCEDURE ............................................... 21

10. Shipment of isolates.................................................................................................. 23

11. Data Collection ......................................................................................................... 23

Screening log for Microbiology Cohort ..................................................................................... 23

Isolate Shipment log for all NeoOBS isolates ............................................................................ 24

NeoOBS Case Report Form 7.0 Microbiology Record and Microbiology Annex (Pathogen

specific) Forms .................................................................................................................................. 24

12. Quality assurance...................................................................................................... 25

Daily Temperature Log .............................................................................................................. 25

Reporting deviations ................................................................................................................. 25

External quality assurance panel ............................................................................................... 26


13. Appendices ............................................................................................................... 28

Figures Figure 1. Enrolment process for NeoOBS microbiology cohort. ........................................................... 12

Figure 2. Example of Isolate label with Patient's Study ID and blank space for additional microbiology

information. ........................................................................................................................................... 15

Figure 3. Example study ID numbers for NeoOBS clinical sepsis cohort and microbiology cohort. ..... 16

Appendices Appendix 1 Reordering form for microbanks ........................................................................................ 28

Appendix 2 Screening Log for Microbiology Cohort ............................................................................. 29

Appendix 3 Isolate Shipment Log .......................................................................................................... 30

Appendix 4 Daily Temperature Log ....................................................................................................... 31

Appendix 5 Microbiology deviation reporting form.............................................................................. 32

Appendix 6 Case Report Form 7: Microbiology Record ........................................................................ 34

Appendix 7. CRF 7.1 to 7.10 Microbiology Annex ................................................................................. 35


1. Abbreviations

AMR antimicrobial resistance

AST antimicrobial susceptibility testing

BSI bloodstream infection (bacteremia)

CLSI Clinical and Laboratory Standards Institute

COMBACTE Combatting Bacterial resistance in Europe

CR carbapenem-resistant

CRE carbapenem-resistant Enterobacteriaceae

CR-GNB carbapenem-resistant Gram-negative bacteria

CRO carbapenem-resistant organism

DNDi Drugs for Neglected Diseases initiative

eCRF electronic case report form

EMA European Medicines Agency

EQA external quality assurance

EUCAST European Committee on Antimicrobial Susceptibility Testing

FUP follow up

GARDP Global Antibiotic Research & Development Partnership

GCP Good Clinical Practice

GCLP Good Clinical Laboratory Practice

HIC high-income countries

ICH Intenational Council for Harmonization

ICU intensive care unit

IV intravenous

LMIC low- and middle income countries

MDR multidrug-resistant

MIC minimum inhibitory concentration

NICU neonatal intensive care unit

QC quality control

SGUL St. George's, University of London

SIV site initiation visit

UA University of Antwerp

WHO World Health Organization


2. NeoOBS Study Organisation

2.1 Study Coordinators

Sponsor Drugs for Neglected Diseases initiative (DNDi) /Global Antibiotic Research and Development Partnership (GARDP) Global Coordinating Investigator(s) Professor Mike Sharland and Professor Paul Heath Paediatric Infectious Disease Research Group Institute for Infection and Immunity St. George’s, University of London Clinical Laboratory Professor Herman Goossens and Dr. Tomislav Kostyanev Laboratory of Medical Microbiology Vaccine and Infectious Diseases Institute University of Antwerp, Belgium Statisticians Professor Sarah Walker and Dr. Wolfgang Stohr MRC Clinical Trials Unit at University College London Neonatal Sepsis Project Leader Sally Ellis GARDP Clinical Trial Manager Nathalie Khavessian GARDP Operations Manager Dr. Tatiana Munera Huertas St. George’s, University of London Data Manager Aislinn Cook St. George’s, University of London Clinical Research Associate Dr. Amy Riddell St. George’s, University of London Clinical Research Fellow Dr. Neal Russell St. George’s, University of London Contracts and Financial Manager Davide Bilardi PENTA Foundation Regulatory Officer Alessandra Nardone PENTA Foundation President PENTA Foundation Professor Carlo Giaquinto PENTA Foundation

2.2 Contact Information

Please address all queries to BOTH Dr. Tomislav Kostyanev ([email protected]) and

Dr. Amy Riddell ([email protected]).

mailto:[email protected]



Dr. Tomislav Kostyanev / Christine Lammens

COMBACTE LAB-Net

University of Antwerp, Laboratory of Medical Microbiology,

Universiteitsplein 1, S 620

2610 Wilrijk, BELGIUM

Email: [email protected]

Telephone: +32 3 265 24 62


Telephone: + 32 3 265 25 51

Dr. Amy Riddell

Clinical Research Associate

St. George’s University of London

Institute for Infection and Immunity

Paediatric Infectious Diseases Research Group

St. George’s University of London

Jenner Wing, Level 2.216F, Mail Point J2C,

London SW17 0RE


Telephone: +44 208 725 5382

Fax: +44 208 725 0716

Reordering supplies

Should you run out of microbanks supplied to you in the beginning of the study, you can reorder a refill

by completing the request form for microbanks (Appendix 1) and send it to the Central lab via email at

[email protected] or by fax.






3. Introduction to the study

Significant reductions in maternal and child deaths have been seen worldwide in the last 20 years, in

large part due to the activities resulting from the Millennium Development Goals. However, the

progress in reducing neonatal mortality has been relatively small. Amongst the 5.9 million deaths in

children under the age of 5 years globally in 2015, 45% were in the neonatal period. The main causes

of deaths in infants include congenital anomalies, preterm births, injuries and neonatal sepsis.

Multi-drug resistant Gram-negative bacteria are of particular concern due to the significant morbidity

and mortality associated with these infections, particularly in infants. The emergence of extended

spectrum beta-lactamase producing Enterobacteriaceae presents a further challenge in managing

neonatal sepsis and, although relatively uncommon, carbapenem-resistant Enterobacteriaceae (CRE)

are an emerging global public health threat.

The NeoAMR project will generate a robust evidence base for managing neonatal sepsis in settings

with high resistance rates to the WHO first-line empiric therapy of ampicillin and gentamicin and for

infections caused by MDR Gram-negative pathogens. A major objective of this observational study is

to collect high-quality observational data to inform study design and comparator selection for a clinical

trial(s) to assess the efficacy of novel antibiotic regimens in areas with high endemic rates of AMR.

There are 3 main justifications for the NeoAMR Observational (NeoOBS) study:

1. There is currently no validated universal definition for neonatal sepsis within the LMIC hospital

setting.

2. Etiological data from LMIC, particularly from urban and NICU based studies, are very limited. The

bacterial pathogens associated with neonatal sepsis and neonatal meningitis differ between HIC and

LMIC settings.

3. Finally, linked etiological and clinical data (such as risk factors and outcomes attributable to sepsis)

from LMIC are limited.

This study will gather observational data in hospitalized infants across each of the 5 WHO regions

(Africa, Americas, Europe, South East Asia and Western Pacific).


4. Study design

This study is a prospective, multinational, multicentre, observational cohort study of the inpatient

management of neonatal sepsis in approximately 15-20 partner institutions. This study is designed to

evaluate the use of healthcare resources, the current clinical practice, the outcomes and the risk

factors for poor outcomes of young infants with significant sepsis (including culture-negative, culture-

positive and culture-positive with AMR pathogens).

Hospitalised babies with significant sepsis will be treated with antibiotics as clinically indicated, at the

discretion of the clinical team, in line with local clinical practice or guidance. Babies will be followed up

until 28 days after enrolment and start of treatment with IV antibiotics for significant sepsis. This study

will also explore new approaches to community follow-up after discharge, in settings where follow-up

is anticipated to be a challenge. This study is expected to recruit over a period of approximately 12

months.

There are two ways a baby can be recruited into the study: through a clinical diagnosis of significant

sepsis (clinical sepsis cohort) or through a microbiology finding (microbiology cohort). For specific

details regarding the inclusion/exclusion criteria, screening, enrolment, and study processes, please

refer to the NeoOBS Protocol and the Manual of Procedures (MOP).

This manual will outline the processes performed by the site’s microbiology team. The processes

included:

The screening process for the Microbiology Cohort

Communicating microbiology results to the clinical study team

Processing samples as per local routine procedures

Labelling, storing, and shipment of isolates

Quality assurance and deviation reporting

This document should be used together with the current approved version of the protocol.


5. The Screening/Enrolment process for the Microbiology Cohort

The microbiology team will be responsible for screening and aiding study enrolment for the

NeoOBS Microbiology Cohort.

The good communication between the local clinical and microbiology teams is very critical for

the study and should be conducted on a daily basis.

How to identify/screen a potential baby for the NeoOBS Microbiology Cohort

Screening is conducted by the microbiology team at each site who will be reviewing the microbiology results of babies <60 days of age (postnatal age) for: o New episode of infection in which a Candida species is isolated from blood culture OR o New episode of infection in which a carbapenem-resistant organism (CRO) is isolated from blood culture OR o New episode of confirmed bacterial meningitis – defined as (A) or (B) below

(A) Isolation of a significant bacterial pathogen from cerebrospinal fluid (CSF)

(B) Isolation of a significant bacterial pathogen from blood cultures AND CSF white cells ≥20 cells/mm3 (for babies 0-28 days of age) or CSF white cells ≥ 10 cells/ mm3 (for babies 29-60 days of age)

Enrolement into the NeoOBS Microbiology Cohort

If the microbiology team identifies a baby meeting the microbiology criteria for this cohort, they will

contact the NeoOBS clinical study team who will confirm eligibility (outlined in section 3.2 in the

Manual of Procedures). If the baby is still alive and an inpatient at the time the clinical study team is

alerted to the baby’s microbiology result, the clinical study team will confirm that this is a new episode

of infection and obtain informed consent from the parent/guardian and then allocate the NEXT study

ID to this baby from the master list (found in the microbiology cohort enrolment log – Refer to section

5.3. in the Manual of Procedures). Allocation of study ID number is the formal time of enrolment.

Once the baby has been allocated a study ID number, the research team will need to contact the

microbiology team and provide this number so the microbiology team can complete the following:

The Microbiology Cohort Screening Log (Appendix 2)

The Isolate Shipment Log (Appendix 3)

The microbiology team will need to label and store the isolate(s) for that enrolled baby (See section

5.5 and 5.6 for labelling and section 8 storage details). Please refer to section 10 for instructions on

how to complete the logs.


Communication of results to the clinical team

The results from positive blood cultures and CSF samples are crucial for the enrolment of babies into

the microbiology cohort. These results need to be communicated timely to the clinical team. While the

clinical team is evaluating the inclusion and exclusion criteria and consenting the parents/guardians of

the baby, please keep the isolates from the blood and CSF samples refrigerated (as explained in

section 9). The clinical study team will allocate a study ID number to the enrolled baby and will

communicate it to the microbiology team. Then store the isolate in duplicate microbanks, as explained

in section 6 of this manual.

If a baby is not alive or has been discharged before screening or does NOT consent, the clinical study

team will need to contact the microbiology team to inform them that they can dispose of stored

isolate(s) for that baby. The reason for non-recruitment should be recorded in the Screening Log for

the Microbiology Cohort (See section 10). Figure 1 summaries the enrolment process for NeoOBS

microbiology cohort.


Figure 1. Enrolment process for NeoOBS microbiology cohort.


Overview of the sample materials

Initial laboratory supplies are provided to you by the Central Laboratory at the University of

Antwerp (UA). Initial laboratory supplies consist of External Quality Assurance (EQA) panel

strains and microbanks for storage of isolates collected within the study.

Sites should use their own sampling and laboratory materials in order to collect and process

samples according to their standard local procedures.

Sites will be provided with:

Central Laboratory supplies (provided by University of Antwerp):

o 20 EQA strains to assess the detection of Gram-negative pathogens (see Section 9 for

more details)

o 100 microbanks (Cryovials with beads)

o 2 empty box for storing isolate from the study

Pre-printed labels to label microbanks with isolates (provided by University of Antwerp)

Documents (provided by St George’s University of London):

o NeoOBS Study Protocol

o NeoOBS Microbiology Manual

o NeoOBS Screening log for the Microbiology Cohort (Appendix 2)

o NeoOBS Isolate Shipment log (Appendix 3)

o Temperature Log (Appendix 4)

o Microbiology Deviation Reporting Form (Appendix 5)

Please check the above list carefully against the supplies you have received. Please contact the Central

Laboratory at University of Antwerp and St George’s University of London should you have any

questions or concerns.


Description of Sample Materials

Storage conditions and expiration dates of supplies

External quality assurance panel strains should be stored at 2° - 8°C upon receipt until the local

laboratory starts processing them. The EQA panel strains should be processed within 4 weeks of

receipt. Please refer to Section 12.2 for further instructions.

Microbanks should be stored at 8-25°C until the expiry date shown on the outer label.

Reordering supplies

Should you run out of microbanks supplied to you in the beginning of the study, you can reorder a refill

by completing the request form for microbanks (Appendix 1) and send it to the Central Laboratory at

University of Antwerp via email at [email protected] or by fax.

Sample

Material

provided for

sample

collection

Handling at your

local lab Material for sample processing/storage

Blood samples/ Cerebro-

spinal fluid samples

Blood culture

bottles or other

sampling

material not

provided.

Please use your

own tubes/bottles.

Gram-stain Please use your local standard procedure

Culture

Identification

Susceptibility

testing

Isolates

preservation

Pathogen identification per local procedure

Pathogen susceptibility per local procedure

Microbanks (provided by UA)



Labels

Pre-printed labels will be provided to each site.

They should be used to label the provided microbanks in which pathogens from study isolates will

be stored.

Each microbank must have a pre-printed patient study ID number label and the following information

written onto the label:

Name of microorganism,

Morphology type (i.e. t1 for morphotype 1 or t2 for morphotype 2, in case more than one

microorganism of the same species are isolated from the sample),

Sample type (blood or CSF) and

Date of when samples was taken (use format dd/mmm/yy)

Study ID

Each baby should only have ONE study ID number. When labelling microbanks, make sure that the study

ID number on the label corresponds to the study ID number on the sample from which the pathogen was

isolated (or to the study ID number communicated to you by the clinical team).

The study IDs are formatted where the first two digits represent the hospital, the third digit represents the

cohort (1 for clinical sepsis cohort or 2 for microbiology cohort), the fourth, fifth and sixth digits are the

enrolment number (in ascending order from 001 to 250 for clinical sepsis cohort and from 001 to 150 for

microbiology cohort) and the last two digits are check letters (to form a back-up identifier when combined

with the enrolment number without having to use baby identifiers such as initials). Figure 3 shows an

example of a study ID number for each cohort.

Figure 2. Example of Isolate label with Patient's Study ID and blank space for additional microbiology information.

Neo OBS SG1001XA

Org. Name: …………………………………

Sample type: ………………………………

Collection date: ………………………….

Subject ID number

Write down the name of the organism,

morphotype (if more than one per organism),

sample type and sample colletion date

e.g.

K. pneumioniae t1

Blood

01/Oct/18


Figure 3. Example study ID numbers for NeoOBS clinical sepsis cohort and microbiology cohort.


6. Sample collection instructions – AS PER LOCAL ROUTINE

LABORATORY PROCEDURES

All clinical study samples (blood and cerebrospinal fluid) are to be obtained according to the routine local

procedures and will be considered as study samples.

All other samples (i.e. urine, skin, nasal swabs) are to be obtained according to the routine local procedures

and will not be considered as study samples.

Blood samples

a. Label blood culture bottles according to your standard laboratory procedures. If the baby’s study

ID number is known at the moment of collection, please add this number to the NeoOBS labels

provided.

b. Draw the baby’s blood as per routine protocol.

c. Transfer to the local laboratory according to the standard practice at your site after collection.

Cerebrospinal fluid

a. Label the CSF container according to your standard laboratory procedures. Ensure you are able to

identify the specimen and link it back to the baby’s study ID number and the specific study day. If

the baby’s study ID number is known at the moment of collection, please add this number to the

NeoOBS labels

b. Collect the CSF as per routine protocol.

c. Transfer to the local laboratory according to the standard practice at your site after collection.


7. Sample processing procedures – AS PER LOCAL ROUTINE

LABORATORY PROCEDURES

Upon receipt of samples in the lab, check if the patient’s study ID number is written on the labels.

Some blood and CSF samples from babies might have patient’s routine Hospital ID number only.

Blood and CSF samples from babies might not have a patient study ID number on them. Should

these samples yield pathogens such as CRO or Candida spp. or meningitis-causative agents (CSF),

please take into account that these babies might not be enrolled by the clinical team and could be

enrolled by the microbiology team. Therefore, the isolated pathogens need to be kept as explained

in Section 9.

Recommendations on how to process blood and cerebrospinal fluid samples are outlined below.

Blood samples

Incubation

Either an automated or a manual system can be used for blood culturing according to the SOPs of

the laboratory performing the testing.

If processing is delayed, do not refrigerate blood specimens. Keep bottles at room temperature.

Gram-stain and culture

Positive blood culture bottles should be Gram stained and sub-cultured to isolate and identify

suspected pathogens to the genus and species level.

Identification

Identification of pathogens isolated from blood cultures should be performed according to your

routine laboratory procedures.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing (AST) of pathogens from blood cultures should be performed

according to the routine laboratory procedures. The results are reported to the clinician via the

regular path of communication used at the local site. The Principal investigator or member of the

clinical study team will enter the required microbiology data (incl. AST profile) into the eCRF (see

section 8).

Store all pathogens from blood samples, as explained in Section 8.

Recommendations for processing blood specimens

If an automated system is not used, blood culture bottles should be examined daily for positive cultures

by the detection of hemolysis, turbidity, gas production, pellicle formation or clotting.


Primary agar plating media for blood specimens should include at least the following agar types (or similar)

to enable growth of a broad range of Gram-positive and Gram-negative pathogens, as well as yeasts:

Blood agar plate

Chocolate agar plate

MacConkey or Eosin Methylene Blue agar (EMB) plate

Sabouraud agar

Plates should be incubated for at least 72 hours at 35 to 37ºC under appropriate conditions (Blood

Agar, MacConkey or EMB in ambient atmosphere, Chocolate agar in 5% CO2).

Bacterial identification should be conducted from a pure culture. Note that infections can be polymicrobic,

and pure cultures need to be performed on all the pathogens that can be isolated from the specimens.

If carbapenem-resistant Gram-negative pathogens are susected, it is best to use isolation and

identification methods that will shorten the time to detection (i.e. rapid diagnostic tests, etc., if available

and as per your local routine procedures).

Cerebrospinal fluid

Gram-stain and culture

Use your routine laboratory procedures for Gram-staining and culturing.

Identification

Identification of pathogens isolated from CSF should be performed according to your routine

laboratory procedures.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing (AST) of pathogens from CSF should be performed according

to the routine laboratory procedures. The results are reported to the clinician via the regular path

of communication used at the local site. The Principal investigator or member of the clinical study

team will enter the required microbiology data (incl. AST profile) into the eCRF.

Store all pathogens from CSF samples, as explained in Section 8.

Recommendations for processing cerebrospinal fluid specimens

CSF samples should be cultured to at least the following agar types (or similar) to enable growth of a broad

range of Gram-positive and Gram-negative pathogens, as well as yeasts:

• Blood agar plate

• Chocolate agar plate

• MacConkey or Eosin Methylene Blue agar (EMB) plate

• Sabouraud agar

Broth media should include at least the following broth types:


Brain Heart Infusion (BHI), Trypticase Soy Broth (TSB) or other suitable media to support aerobic

growth under appropriate conditions.

Broth should be incubated for at least 72 to 96 hours at 35 to 37ºC. If aerobic bacteria are not present on

the primary culture plates, then streak BHI or other medium onto appropriate agar plates for isolation of

aerobic bacteria.

Bacterial identification should be conducted from a pure culture. Note that infections can be polymicrobic,

and pure cultures need to be performed on all the pathogens that can be isolated from the specimens.


8. Storage of isolates – ADDITIONAL STUDY PROCEDURE

For ALL pathogens from babies samples (age <60 days) – please keep pure culture of isolates

refrigerated for one week after processing in order to wait for clinician to alert you to study

enrolment, in case the babies have not been already enrolled in the study.

In case of different morphology types, store each of these.

Once the clinicians have consented patients – they will communicate to the microbiologist the Study

ID number that is linked with patient’s hospital number. Then store each isolate in two microbanks.

The Central Laboratory at University of Antwerp will organise the transport of strains after the export

licence is obtained locally (if applicable). First batch should be sent to UA six months after opening

the site.

All bacterial pathogens, as well as yeasts belonging to the Candida spp., from blood cultures and CSF

specimens from babies enrolled in the study should be stored. Each isolate should be stored in duplicate

microbanks and if possible, stored in a different freezer.

If blood cultures from babies, not enrolled yet in the study, yield one of the following pathogens:

carbapenem-resistant microorganism or Candida spp., this would make the babies potentially

eligible to be enrolled into the microbiology cohort.

If CSF samples from babies, not enrolled in the study, yield a meningitis-causative agent or there

is a positive blood culture and the CSF white cell count is elevated, this would make the babies

potentially eligible to be enrolled into the microbiology cohort.

In such cases, alert the clinical study team of eligibility of babies whose samples yielded the above-

mentioned pathogens. Keep the strain refrigerated until confirmation from the clinical team that the baby

is enrolled in the study. Once the baby is enrolled and a subject study ID number has been assigned, store

the pathogen(s) from the baby’s samples, as per the instructions described in this section of the

Microbiology Manual.

Refrigerated isolates can be kept for one week. If isolates have been kept refrigerated (i.e. until study ID

number has been assigned by the clinical team), please prepare a pure 20-24h culture before storing the

isolate in microbanks. Each isolate should be stored in duplicate microbanks, one to be sent to the Central

Laboratory and one to be stored at the local laboratory as a back-up.


Inoculation of microbanks

1. Grow bacteria under the recommended conditions for isolation.

2. Use two of the provided pre-printed labels to label two Microbanks for each isolated pathogen. Each

label will have a patient study ID number on it. On each of the two labels write down the species of the

isolate, morphotype (i.e. t1 or t2, if more than one type), the sample type (blood or CSF) and date of when

samples was taken on the label.

3. Open the screw cap under aseptic conditions.

4. Inoculate the microbank with colonies from a pure 20-24h culture using a sterile swab. Make the

suspension as dense as possible.

5. Close the vials tightly.

6. Vortex for 20 seconds (do not centrifuge).

7. Visually check to see that there is a dense inoculum of organisms in the microbank medium.

8. Open the vial again and remove excess fluid using a sterile Pasteur pipet, leaving the inoculated beads

as free of liquid as possible. Excess suspension left in the tube makes it more difficult to remove individual

beads when required.

9. Close the vial tight.

10. Store the vials, preferably in a -70◦C freezer. If a -70◦C freezer is not available, the vials may be stored

in a -20◦C freezer for a period of maximum 12 months.

The two prepared microbanks should be stored separately. Store one microbank in a box for the central

laboratory (one box can contain 81 microbanks). The second microbank can be stored in a separate box

with all the back-up microbanks. Both boxes are provided by the University of Antwerp. If possible store

the 2 boxes in separate freezers

As soon as study isolates have been stored, please ensure that the Isolate Shipment form has been

completed (Appendix 3).


9. Shipment of isolates

Pathogens should remain stored until further instruction from the Central Laboratory at University of

Antwerp and St George’s University of London. The shipment process will vary between sites e.g. some

sites will need an export licence and therefore sites will be given specific instructions of how and when to

ship isolates to each site. Not all sites will be shipping samples outside their country.

As part of the shipment process, please ensure that the Isolate Shipment Log (see section 10.2) (Appendix

3).

10. Data Collection

Screening log for Microbiology Cohort

There will be a Screening Log for Microbiology Cohort to be completed by the microbiology team and in

collaboration with the clinical study team. Please only complete this log for babies who meet the

microbiology cohort criteria (i.e. Candida BSI, CRO BSI, or Bacterial Meningitis) (Appendix 2).

Data that needs to be collected in this log includes:

Patient’s Hospital number

Internal Lab Number of sample (if applicable)

Study ID Number (if applicable) Sample Type (Blood or CSF)

Pathogen (Candida BSI, CRO BSI, or Bacterial Meningitis)

Date Sample Taken DD/MMM/YY

Date Of Positive Culture and Clinical Team Informed DD/MMM/YY

Consented (Yes or No) (if applicable)

Reason for not consenting (e.g. declined to take part in study, death, discharged, or transferred to

other hospital) Date of declined to take part in study, death, discharged, or transferred to other

hospital (DD/MMM/YY) (if applicable)

Sites will be expected to email a copy of this log to Dr. Amy Riddell ([email protected]) at the end of

each month. The first two columns must be removed (i.e. Patient’s Hospital number and Internal Lab

Number of sample (if applicable) before log being sent.



Isolate Shipment log for all NeoOBS isolates

There will be a NeoOBS Isolate Shipment Log to be completed by the microbiology team. ALL isolates from

ENROLLED babies from both cohorts (i.e. the NeoOBS Clinical Sepsis Cohort and NeoOBS Microbiology

Cohort) must be listed on this log (Appendix 3).

Data that needs to be collected in this log includes:

Patient’s Hospital number

Internal Lab Number of sample (not compulsory)

Position in storage box (eg. A1, A2)

Study ID Number

Sample Type (Blood or CSF)

Sample collection date DD/MMM/YY

Date of isolate freezing DD/MMM/YY

Pathogen Type

Morphological type (only required if more than one colony type is observed from the same

organism) (eg; t1, t2)

Storage temp

Date of shipment DD/MMM/YY Who and where was shipment sent to? (Institute, contact name,

address)

The log must be completed as isolates are labelled and stored. The Study ID Number must be written on

the isolate label.

Sites will be expected to email a copy of this log to Dr. Tomislav Kostyanev

([email protected]) and Dr. Amy Riddell ([email protected]) at the end of each

month. The first two columns must be removed (i.e. Patient’s Hospital number and Internal Lab Number

of sample (if applicable) before log being sent.

NeoOBS Case Report Form 7.0 Microbiology Record and Microbiology Annex (Pathogen specific)

Forms

The clinical research team will be recording data on microbiology findings for all enrolled babies. They will

complete Case Report Form 7.0 (Appendix 6) and the associated Microbiology Annex (Case Report Form

7.1-7.10) (Appendix 7) based on the routine final microbiology report. The microbiology team do not

complete the Case Report Form 7.0 Microbiology Record and Microbiology Annex (Pathogen specific)

forms but the microbiology team will need to ensure that microbiology results/reports of all babies

enrolled in both cohorts (Clinical Sepsis Cohort and Microbiology Cohort) are in the patient file/clinical

notes (paper or electronic) to help the clinical research team complete the forms in a timely manner.




11. Quality assurance

Daily Temperature Log

The microbiology team will be given a Daily Temperature Log (Appendix 4) which should recorded daily

for the following information:

Date (dd/mmm/yy)

Actual Temperature

Staff initials

Comments

If you have more than one freezer, please compete a Daily Temperature Log for each freezer and state the

unique freezer serial number on each log.

If you have your own log or a freezer monitoring system that collects all the information required, you do

not have to use the study specific log, you can continue using your log.

Reporting deviations

Microbiology deviations, for example, samples stored at the incorrect temperature, freezer temperature

deviations, lost samples, etc., should be documented using the Microbiology Deviations Reporting Form

(Appendix 5) and sent to BOTH Tomislav Kostyanev ([email protected]) at University

of Antwerp and Amy Riddell ([email protected]) at St George’s University London within 24 hours of

knowledge of event occurring.

All isolates must be stored at -80°C or -70°C. If the temperature increases to >-65°C then this is classed as

a temperature deviation.

If site cannot store in -80°C or -70°C freezer, then all samples must be kept at <-20°C at all times. If the

temperature increases to >-15°C then this is classed as a temperature deviation.

The Microbiology Deviations Reporting Form includes two sections, which must be completed will the

following information:

Section 1 - Details of deviation

Date & Time event occurred

Date & Time event reported

Site

Staff involved & reporting

Description of deviation

List all patient study IDs effected

Any documents attached to report? E.g. temp logs




Section 2 – Corrective action / prevention action

Action taken to correct deviation

Preventive action

Signature of member of core study team reporting the deviation

External quality assurance panel

COMBACTE LAB-Net will prepare and send to your laboratory a panel of EQA strains. EQA panels will be

sent to sites around the site initiation visit or later, depending on issuing of import license and custom

clearance.

The EQA panel aims to assess the readiness of each local laboratory to detect Gram-negative pathogens

and to process microbiological samples according to the local laboratory procedures.

Upon delivery, the strains should be processed as per the instruction in this section. Detailed instructions

will also be provided together with the shipped strains. Strains should be stored as per the instructions in

this section.

EQA strains

The panel includes 20 well-characterized Gram-negative strains which are numbered as “CR01” up to

“CR20”. These strains are transported to your laboratory refrigerated. Each of the 20 tubes, containing

EQA panel strains, is marked with a label with the inscription “NeoOBS CR strain”, your site number for

the study and the number of the panel strain (from CR01 to CR20). Store the vials at 2° - 8°C upon receipt

until your laboratory starts processing them.

For each of these strains, perform the following procedures:

Refreshing the strains:

• Subculture the EQA Panel strains on Mueller Hinton agar plate and incubate for 24h at 37°C

without CO2.

• Pick a well-isolated colony and make a new subculture on Mueller Hinton agar and incubate for

24h at 37°C without CO2. Subculture the strain also on a selective medium for detection of

carbapenem-resistant Gram-negatives (if used in your routine practice).

Identification and susceptibility testing:

• Identify all 20 strains according to your routine procedures.

• Determine their antimicrobial susceptibility to antibiotics according to your routine procedures.

• Perform any other routine diagnostic tests for detection of Gram-negatives.


Reporting results:

• You will receive access (username and password) to an online platform where the EQA results can

be entered. You will also receive detailed instructions how to enter the results in the online form.

• Perform all the necessary analysis and input the results within one month of receiving the strains.

Enter all the required results in the EQA online form.

After receiving the results, University of Antwerp will send feedback to all sites on their performance in

the EQA panel on carbapenem-resistant Gram-negatives.

Keep the transport tubes until further instructions. In case of discordant results with the strains, it might

be required that you retest some of the strains and/or send the transport tubes back to the University of

Antwerp.


12. Appendices

Appendix 1 Reordering form for microbanks

NeoOBS observational study

Reordering form for Microbanks

Date: No of pages faxed/sent:

Supplies Quantity

Microbanks ………… microbanks

Hospital Name: ……………………………………………..

Laboratory Name: ……………………………………………..

Delivery Address: …………………………………………….. …………………………………………….. …………………………………………….. Contact Name: ……………………………………………..

…………………………………………….. Tel: ……………………………………………..

Back-up contact: ……………………………………………..

E-mail address: ……………………………………………..

Tel of back-up person:……………………………………………..

Send this form to:

E-mail: [email protected]

Fax: +32 3 265 2663

University Antwerp, Medical Microbiology, Belgium



Appendix 2 Screening Log for Microbiology Cohort


Appendix 3 Isolate Shipment Log


Appendix 4 Daily Temperature Log


Appendix 5 Microbiology deviation reporting form


Appendix 6 Case Report Form 7: Microbiology Record


Appendix 7. CRF 7.1 to 7.10 Microbiology Annex