Management of samples in the laboratory to avoid infection

1. Ebola Laboratory Training

2. Introduction
1 Ebola or Marburg hemorrhagic fever outbreaks
constitute a major public health issue in Sub-
Saharan Africa
2 Between (1967 - 2011) in Sub-Saharan Africa
EVD/MVD caused 270(9%) among health-care
workers
3 Marburg and Ebola belong to the Filoviridae
family (filovirus)
4 Ebola virus is comprised of five distinct species:
• Zaïre
• Bundibugyo
• Côte d’Ivoire
• Sudan
• Reston
Overview of EVD/MVD Epidemiology and transmission
Ebola an enveloped virus with a lipid layer (fat-like
substance-water insoluble).
Envelope aids in attachment of the virus to the host cell

3. Ebola Virus Transmission

4. Transmission of EVD/MVD virus in human
Human to human transmission
1 EVD/MVD is started in human population when there is spill over occurs (animal – human
transmission) with case fatality of 25%–90%.
2 Person-to-person transmission occurs through direct contact with:
 Blood,
 Secretions
 Organs
 other body fluids of infected persons and materials
3 Burial ceremonies, health facilities and travel significantly contribute spread of EVD/MVD in the
human population
4 During EVD and MVD outbreaks, only strict compliance with biosafety guidelines:
 Appropriate laboratory practices
 Infection control precautions
 Barrier nursing procedures
 Use of personal protective equipment by health-care workers handling patients
 Disinfection of contaminated objects and areas
 Safe burials
Breaks chain of transmission and control and prevent of the epidemic

5. Case Definition:
Suspected case with positive
laboratory results for either
antigen of viral RNA detected by RT-
PCR or Viral antibodies against Ebola
On 20th September 2022, MoH
declared the index Ebola case in
Uganda (Mubende) by test
confirmation from UVRI (RT-PCR).
Ebola Confirmed Case definition

6. Syndromic Approach
Using WHO classification of acute syndromes,
this provides guidance on specimen type to
collected for;
a) Acute diarrheal syndrome
b) Acute haemorrhagic fever syndrome
c) Acute jaundice syndrome
d) Acute respiratory syndrome
e) Acute neurological syndrome
f) Acute dermatological syndrome
g) Acute ophthalmological syndrome
h) Acute systemic syndrome
• Ebola virus spreads by direct contact with
body fluids, excreta from an infected person.
• Early noticeable symptoms: Fever, Fatigue, Joint
and muscle pain, Headache, Sore throat, Cough.
• Disease progression symptoms: Vomiting,
Diarrhoea, Rash, Unexplained bruising, Internal and
external bleeding.

7. Biosafety Biosecurity
 The nature of work in facilities is potentially risky and
often exposes service providers to harm.
 Biorisk management involves principle techniques and
measures put in place for the safety of personnel,
community/environment and for the security of
valuable biological materials (VBM) and information
Laboratory biosafety:
 Practices and controls that reduce the risk of
unintentional exposure or release of biological materials.
Laboratory biosecurity:
 Practices and controls that reduce the risk of loss , theft,
misuse, diversion of, or intentional unauthorized release
of biological materials.
(ISO 35001: 2019 Clause 3.23, ISO 15190:2020 clause 4.3.1)

8.  Trained and deemed competent to implement bio risk management
measures
 Conduct thorough risk assessment exercise to understand the likely
hazards that they will face during the response activities and set
suitable mitigation measures to counter these risks.
 The choice of PPE to use and set up of the facility including
deployment of the teams should be based on a thorough risk
assessment.
 Little information known about the agent, the teams should apply
maximum containment principles and deploy highly competent
staff to handle any work in this area.
Biosafety considerations
BSBS Considerations
 Laboratory personnel safety practices and techniques must be
supplemented by recommended immunizations (EVD Zaire,
Hepatitis B virus, yellow fever), and appropriate facility design
and features, safety equipment, and management practices.
 Safety equipment should be deployed based a thorough risk
assessment process these include biological safety cabinets
(BSCs), centrifuges with caps, an enclosed container designed to
prevent aerosols from being released during centrifugation.
 Waste generated from all the procedures should be handled as per
the risk assessment

9.  Use barrier protection at all times.
 Use gloves for protection when working with or around
blood and body fluids.
 Change glove between patients.
 Use glasses, goggles, masks, shields, and waterproof
gowns/aprons to protect face from splashes.
 Wash hands if contaminated and after removing gloves.
 Use puncture-resistant sharps disposal containers (at
point of use).
 Do not recap, bend or break needles and handle all
sharps carefully.
 Use resuscitation equipment and devices for mouth-to-
mouth resuscitation.
 Minimize spills and spatters; use leak-proof containers;
appropriate biological safety cabinet.
 Decontaminate all surfaces and devices after use.
 Observe prudent laboratory practices.
 Use proper waste management/housekeeping.
 Promptly seek medical attention and counselling if
exposed to contaminated materials (see below).
 Only persons presenting with EVD cardinal signs
and symptoms is/are eligible for sample collection
& testing after DRRT / clinical verification.
 Laboratory staff must always don full PPE when
conducting EVD sample collection/ given test
 Routine procedures/tests will only be done after a
EVD negative confirmatory test.
 Laboratory assays/tests to be preformed at health
facility after EVD Negative laboratory
confirmation.
 Only suspects /patients in designated
isolation/holding units or ETU can have sample
collection or bedside tests done
Dos – Universal precautions Ebola Biosafety precuations
Biosafety precautions during routine laboratory services (Dos and Donts)

10.  A competent regional and national focal point person should be
identified to coordinate specimen repository
 Freezers used to store the specimens need to be maintained, kept
in secure rooms and connected to a reliable power supply
with a back-up generator available.
 Inventories must be made of all the specimens and these should
also be stored securely.
 Ensuring long-term specimen security and integrity,
consideration should be given to assuring laboratory managers’
control of laboratory security and biosecurity
 Personnel reliability should be regularly evaluated to assure their
continued suitability
 Training and competency in biosecurity should be conducted
for all the response team members
Biosecurity considerations
BSBS Considerations
 Access to specimen’s storage areas must be controlled and only
authorised person allowed
 Positive specimens from outbreaks be kept at the regional
repositories except for select agents of national importance
that will be kept at national specialized repositories.
 Biosecurity risk assessments should be carried out and
reviewed regularly including storage risks.
 During and after outbreaks, graded security should be set up for
laboratory and storage facilities even if the specimen are
shipped to alternative storage sit.
 Specimen storage facilities will require adequate funding to
ensure continued security and power supplies, and to prevent
degradation of either specimens or the surrounding security.

11. Organisation
Personnel
Equipment
Inventory Management
Documents and records
Occurrence Management
Information Management
Assessment
Process Improvement
QMS approach in context to EVD response
a). Pre-Examination:
• Alert verification made
• Suspect / patient identification
• Trained and competent staff
• Routinely serviced & certified equipment RT-PCR
• Availability of required supplies
• VHF case investigation form completely filled
• Biorisk assessment
• Test to be done
b). Examination:
• SOPs / protocol to be followed (read and understood by attestation)
• Internal Quality control (passed)
• Test performed by a trained and competent laboratory staff
• Laboratory test result verified against the controls
• Laboratory result signed by test performer
• Laboratory result verified by senior laboratory staff
c). Post-Examination:
• Results relay through MoH outbreak protocol to PHEOC
• Result dispatched to relevant authorities, respective DHO and requester
• Suspected case informed of laboratory findings
In accordance to ISO: 9001; 15189; 15190; 17025

12. Successful
laboratory
confirmation
of a disease
etiology and
it’s
determinants
depends on:
correct packaging
and rapid
transport to an
appropriate
laboratory
Advance
planning
The ability of the laboratory
to accurately perform the
diagnostic tests
Collection of appropriate
and adequate specimens
Proper biosafety and
decontamination procedures to
reduce the risk of further spread
of the disease.
Correct packaging and
rapid transport to an
appropriate laboratory
Confirmat
ion &
Prognosis
Role of Laboratory in outbreak management

13. Recommended PPE & IPC measures
The PPE level of protection should be appropriate for the pathogen
and the activity being performed. Below are minimum PPE
recommendations;
1. Blood-borne/Body Fluid Pathogens;
• Low Risk: gloves, gown, goggles;
• Medium Risk: gloves, gown, face shield;
• High Risk: double gloves, gown, full respiratory protection,
boots.
2. Airborne Pathogens;
• Low Risk: gloves, gown, goggles, partial respiratory
protection;
• Medium Risk: gloves, gown, goggles, full respiratory
protection;
• High Risk: double gloves, gown, full respiratory protection.
3. Vector Borne Pathogens;
• Use PPE appropriate for Blood-borne/Body Fluid
Pathogens;
• Insect repellant should also be used.

14. Donning PPE-Using coverall
1. Remove all personal items (jewelry, watches, cell phones, pens, etc.)
2. Put on the scrub suit and rubber boots* in the changing room.
3. Move to the clean area at the entrance of the isolation unit
4. Gather PPE beforehand. Select the right size coverall.
5. Putting on PPE under the guidance and supervision of a buddy
6. Perform hand hygiene.
7. Put on inner gloves (examination, nitrile).
8. Put on coverall.
9. Thumb (or middle finger) hole in the coverall sleeve or thumb loop.
10.Put on face mask.
11.Put on face protection (either face shield or goggles)
12.Put on head covering: Surgical bonnet or hood.
13.Put on disposable waterproof apron.
14.Put on outer gloves (examination, nitrile) over cuff.
15.Self-check in mirror.
16.Check buddy and write name/ occupation/ time of entry.

15. Preparation of chlorine concentrations-IPC
 Concentrations vary e.g. 3.5%, 3.65%, 3.7%, 5%, or
6%
 To make desired concentration, use this formula:
Available Concentration (%) - 1
Desired Concentration (%)
= parts of clean water to be added to one part of available
chlorine solution
e.g. if you want to mix 0.5% concentration and you have 6% Hypochlorite (Jik),
= (6% / 0.5%) - 1
= 12 - 1
= 11
 11 parts water + add 1 part chlorine
Application prepared chlorine concentrations:
a). 0.5% chlorine
• Disinfecting bodily fluids (vomit, diarrhea, urine)
• Disinfecting dead bodies
• Disinfecting toilets & bathrooms
• Disinfecting gloved hands
• Disinfecting floors
• Disinfecting beds & mattress covers
• Disinfecting footbaths
• Disinfecting patient chairs, wheel chairs
b). 0.05% chlorine:
• Disinfection of bare hands and skin
• Disinfection of medical equipment (e.g., thermometers)
• Disinfection of laundry
• Washing of plates and eating utensils

16. Laboratory Preparatory Phase
1. Define the cause and magnitude of the outbreak
2. Determine which sample types are required to confirm the cause of the outbreak
3. Decide who will be in the investigation team (RRT) for the laboratory
• Trained and competent staff - Blood, CSF, OP/NP swabs, animal or environmental samples
4. Gather the necessary equipment, sample containers, transport media, packaging and transport supplies
5. Contact the hub system or transportation network (Hub rider/vehicle or designated system)
6. Identify the Referral laboratories
• National laboratory, International laboratory, Animal or Environmental laboratory.
• Test to be performed
• Sample requirements-volume, transport temperature, labeling.
• Inform them of the sample types and numbers referred and expected delivery time
* Sample collection must be done by a trained personnel following an SOP in a designated isolation or Holding unit or ETU only.
NB: Take
samples before
the patient is
treated with
antibiotics

17. Ebola Documentation tools
Revised VHF Case Investigation Form (CIF) VHF Result Report Form

18. Other sample types: Skin or tissue biopsy, Arterial or cardiac blood draws, Rectal swabs
Additional tests: Hematological, Chemistry, Coagulation tests
* Sample collection must be done by a trained personnel following an SOP in a designated isolation or Holding unit or ETU only.
Ebola Sample collection
Sample Type Test Transport Media Recommended Temperature
Whole blood PCR N/A 2 to 8ºC
Plasma PCR N/A 2 to 8ºC
Serum Serology N/A 2 to 8ºC
Swab(OP/NP/Nasal) PCR UTM or VTM 2 to 8ºC
Swab Culture Amies or Cary Blair Media 2 to 8ºC
CSF Culture TI Media Ambient
Stool Culture Cary Blair Media 2 to 8ºC

19. Primary receptacle
• Watertight, leak-proof receptacle containing the sample.
• Package with enough absorbent material to absorb all fluid in
case of breakage or leakage and to stabilize samples during
transport.
Secondary packaging
• Durable, watertight, leak-proof packaging to enclose and protect
the primary receptacles.
• Several cushioned primary receptacles may be placed in one
secondary packaging.
Tertiary packaging
• Secondary packaging is placed in outer packaging with suitable
cushioning material.
• May contain ice or dry ice (if necessary) to maintain sample
temperature.
• Contains documentation (Lab request form / CIF).
• Place label UN3373 BIOLOGICAL SUBSTANCE CATEGORY B
Safe sample handling: Triple packaging

20. Triple packaging at Isolation unit or ETU

21. Inappropriate PPE for EVD sample collection
Wrong labelling prior to dispatch to testing laboratory
Appropriate PPE for EVD sample collection
Wrong practices Recommended practices
Shipment to testing lab

22. Ebola Testing Algorithm (Suspects)

23. Discharge Testing Criteria

24. • Rapid tests (diagnostic or screening)-Lateral flow immunoassays or molecular techniques.
• Depending on the country, the following may be diagnosed based on the result of an RDT; e.g.
• Malaria*
• Dengue
• Other diseases may use RDT initially but require laboratory confirmation by
 Culture,
 Serology or
 Molecular testing; e.g. Cholera, Ebola, CCHF
• Molecular Techniques (UVRI): RT-PCR (EVD*, Marburg, RVF, CCHF)
• Mobile Laboratories: (Bio-fire film array, Bio-Rad technologies)
• ETU Complementary laboratory testing (Clinical Labs) may include:
 Complete Blood Count – CBC
 ABO Blood and Rhesus grouping
 Coagulation tests – Prothrombin time, Activated partial thromboplastin time, D-dimer, and Fibrinogen
 Liver and Renal Functional test
Laboratory Diagnostics
Mobile Lab setup @ Mubende RRH

25. 1. Always remove PPE under the guidance and supervision of a trained observer
(colleague).
2. Enter decontamination area by walking through chlorine tray.
3. Perform hand hygiene on gloved hands (0.5% chlorine).
4. Remove apron taking care to avoid contaminating your hands by peeling it off.
5. Perform hand hygiene on gloved hands (0.5% chlorine)..
6. Remove hood or bonnet taking care to avoid contaminating your face.
7. Perform hand hygiene on gloved hands (0.5% chlorine)..
8. Remove coverall and outer pair of gloves:
• Tilt head back to reach zipper, unzip completely without touching any skin or scrubs, remove
coverall from top to bottom.
• After freeing shoulders, remove the outer gloves while pulling the arms out of the sleeves.
• With inner gloves roll the coverall, from the waist down and from the inside of the coverall,
down to the top of the boots.
• Use one boot to pull off coverall from other boot and vice versa, then step away from the
coverall and dispose of it safely.
9. Perform hand hygiene on gloved hands (0.5% chlorine)..
10. Remove the goggles or face shield from behind the head (keep eyes closed).
11. Perform hand hygiene on gloved hands (0.5% chlorine)..
12. Remove mask from behind the head (keep eyes closed).
13. Perform hand hygiene on gloved hands (0.5% chlorine).
14. Remove inner gloves with appropriate technique and dispose of safely.
15. Decontaminate boots appropriately and move to lower risk area one foot at a time.
16. Perform hand hygiene (0.05% chlorine).
Doffing PPE-Using coverall

26. Infectious waste can be disposed of in the following ways:
• Incineration
• Containment & burial
Infectious waste should be disinfected before disposal:
• Autoclave
• Chemical disinfection
*Biosafety and Biosecurity practices should always be adhered to at all times
Waste management

27. National Integrated Hub System

28. 1. Sending an alert to 6767:
a). Alert to 6767 (MoH PHEOC)
Type: Alert; Suspected disease; Age/Sex; Signs & Symptoms; Date of disease onset; Location of disease
origin (Village / Health facility, Sub county and District ); Senders Name; send to 6767
b). After verification of alert, sample collected and referred to Reference laboratory.
Type: Update; Sample from a suspected disease case; Age/Sex, Location has been collected and Transported
to Reference laboratory through Hub system; send to 6767
2. RDS (Ebola Results Management): Web based information systems
Must do:
 Always complete the filling of VHF CIF to enable quick upload of laboratory results into RDS
 Always check into the system for results as per TAT (within 24 hours)
 Only authorized personnel have access
Reporting

29. Result Management

30.  Information (Identification, Result etc.) concerning the patient or client is classified (confidential).
 Only approved or authorized systems are used for information sharing.
 Laboratory outcomes only dispensed by authorized personnel using authorized channels of reporting.
 Limited access to patient data through secure and approved MoH systems.
Ethical Considerations

31. Facility based
requests to DLFPs
Submissions
through DHO to
central stores (NMS)
Logistics Management and Reporting

32. Specimen Collection PPE
IPC:
 Disinfectant and soap for setting up hand-washing stations
 Supply of gloves
 Safety boxes for collecting and disposing of contaminated
supplies and equipment
 Waste bins and Biohazard bags (All Red)
Blood collection:
 Sterile needles, different sizes
 Sterile syringes
 Vacutainers
 Whole blood specimen container (purple top)
 Antiseptic – alcohol hub rub or 0.05% Jik (skin)
 Disinfectant – 0.5% Jik (Surfaces)
 Tourniquets
Specimen packaging
 A canister with a screw-on cap
 Cold chain box
 Ice packs
 Cotton wool for cushioning sample to avoid breakage
 Labels for addressing items to the lab
 Labels for marking "store in a refrigerator" on the outside of
the shipping box
 Case forms and line lists to act as specimen transmittal form
 Marking pen to mark tubes with patient’s Initials, ID number (if
assigned by the district), Ward, Date of sample collection, Test
 Single-use (disposable) impermeable coverall.
 Respiratory Protection (certified N95)
 Single-use (disposable) full face shield.
 Single-use (disposable) examination gloves with extended cuffs
 Gumboots
 Single-use (disposable) boot covers
 Single-use (disposable) apron
 Head cover
 2 pairs of goggles
 Hand sprayer
 Back sprayer
 Scotch of tapes
EVD tool kit

33. Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008, CDC
https://stacks.cdc.gov/view/cdc/11560
Guidelines for the collection of clinical specimens during field investigation of outbreaks,
WHO/CDS/CSR/EDC/2000.4
http://www.who.int/ihr/publications/WHO_CDS_CSR_EDC_2000_4/en/
CDC Infectious Diseases Laboratories Test Directory, https://www.cdc.gov/laboratory/specimen-
submission/list.html
Ebola out break, Laboratory Technical guidances, 2014
(http://www.who.int/csr/resources/publications/ebola/surveillance/en/index2.html)
Laboratory Biosafety and Biosecurity manual 2015
Walimu-WHO Rapid Response Team for Ebola training
http://who.int/csr/resources/publications/ebola/ppe-steps/en/
Reference

34. Acknowledgement