1. HOW TO INVESTIGATE EBOLA?
Dr. ROHIT JAIN
Department
of
Pathology & Transfusion Medicine

2. WHO Risk group classification For
Infectious microorganisms (2004)
Risk
Group
Individual risk Community risk
1 no, low no, low
2 moderate low
3 high low
4 high high

3. Ebola
• Risk group 4
•Lethal, pathogenic agent
•Readily transmittable
– direct, indirect
•Effective treatment and preventive
measures not usually available

4. DIAGNOSTIC CONSIDERATIONS

5. OTHER DISEASES SHOULD BE RULED
OUT
• Malaria
• Typhoid fever
• Shigellosis
• Cholera
• Leptospirosis
• Plague
• Rickettsiosis
• Relapsing fever
• Meningitis
• Hepatitis
• Other Viral hemorrgahic
fevers

6. CLINICAL CASE DEFINITION
SUSPECTED CASE
History of travel or close contact
with symptomatic person
travelling from EVD affected
areas in the past 21 days, with
high grade fever more that 101
degree F, along with the
following additional symptoms
• Headache
• Body ache
• Unexplained hemorrhage
• Abdominal pain
• Diarrhea
• Vomiting
CONFIRMED CASE
Laboratory confirmed diagnostic
evidence of Ebola virus infection
by any one of the following
• IgM capture ELISA
• Antigen capture ELISA
• RT PCR

7. Whom to test?
CDC RECOMMENDATIONS

8. 1. HIGH RISK EXPOSURE
Testing of all persons with onset of fever within 21 days
of having a high risk exposure which includes
• Percutaneous or mucous membrane exposure to body
fluids of a person with a suspected or confirmed case
of EVD
• Direct care or exposure to body fluids of a patient
with suspected or confirmed case of EVD without
appropriate PPE
• Laboratory processing of body fluids of suspected or
confirmed case of EVD without appropriate PPE or
Standard biosafety precautions
• Participation in funeral rites or other direct exposure to
human remains in the geographic area where the
outbreak is occurring without appropriate PPE

9. HIGH RISK EXPOSURE
For persons with HIGH – risk exposure but
without a fever
Testing is recommended only if
• There are other compatible clinical symptoms
present
• Laboratory findings are unknown or abnormal
 Platelet counts < 1.5 lakh / microlitre
 Elevated transaminases

10. 2. LOW RISK EXPOSURE
Testing persons with a low risk exposure
 Who develop fever with/without other clinical findings
 Have unknown or abnormal laboratory findings.
A low risk exposure includes:
• Providing patient care or casual contact without high-risk
exposure to EVD patients in health care facilities in outbreak
affected countries.
• Household members or other casual contact of an EVD patient
without high-risk exposures.
• Persons who had direct unprotected contact with bats or primates
from EVD affected countries.
Casual contact
1. Within 1 meter or room or care area of EVD patient for prolonged period
without wearing recommended PPE
2. Direct brief contact with an EVD patient without wearing recommended PPE

11. 3. FROM OUTBREAK AREA , NO DIRECT
EXPOSURE
Persons with no type of exposure and with no
other diagnosis but who have
• Fever with other signs or symptoms
• Unknown or abnormal laboratory findings
within 21 days of visiting EVD affected
countries

12. 4. ASYMPTOMATIC PERSONS
Asymptomatic persons with high- or low-risk
exposures
• should be monitored daily for fever and
symptoms for 21 days from the last known
exposure
• tested at the first indication of illness

13. SPECIMEN REQUEST FORM

16. When samples should be
collected for Ebola testing?

17. • Virus is detected in blood only after onset of
symptoms, most notably fever.
• Up to 3 days post onset of symptoms for the
virus to reach detectable levels.
• Specimen should be taken when a symptomatic
patient reports to a healthcare facility and is
suspected to have an EVD exposure.
* If the onset of the symptoms is <3 days, a subsequent sample is required to
completely rule out EVD.

18. SAMPLE TYPE
• Minimum 4 mL whole blood in EDTA
vial/Clot Vial/ Citrate vial in Plastic
collection tubes.
• BODY FLUIDS (saliva, urine, vomit,
stool, nasal secretions, GI secretions)
Antemortem
• Tissue Sample Liver,
spleen, bone marrow, kidney, lung
Postmortem and brain

19. HOW TO COLLECT SAMPLE
• Samples should be collected with all standard
biosafety precautions
• Should be accompanied with detailed history
as per the TRF.
• Before dispatching the sample disinfect the
outer surface of the vial using 1% sodium
hypochlorite or 5% lysol soultion.
• Bold labeling of SUSPECT EBOLA on all
vials

20. STORAGE OF SAMPLE
• Specimen should be stored at 4 degree
Centigrade or Frozen.

21. SAMPLE TRANSPORTATION

22. • Main goals
– protects the environment
– the carrier
– protects the sample
• arrival in good condition for analysis

23. Packaging of the Specimen
(Triple Packaging system)
• Primary receptacle (a
sealable bag) wrapped
with absorbent
material.
• Secondary receptacle
(watertight, Leak proof)
.
• Outer shipping
package.

24. If triple packaging not available
• Sample containing vials should be should be
kept in 1st plastic bag and either heat sealed
or tied with rubber bands so that the sample
if leaks does not come out of the bag.
• This plastic bag should be placed in a plastic
container and sealed with adhesive tape.
• This should be placed in 2nd plastic bag sealed
with rubber bands.
• 2nd plastic bag should be then placed in a
thermocol or vaccine container containing
ice.

25. • Sample should be transported within 24
hours
• If not ,
Serum should be stored at – 70 degree
centigrade.

26. DIAGNOSTIC MODALITIES

27. LABORATORY TESTS
• Antigen capture ELISA
• IgM capture ELISA
• Real time Reverse Transcription PCR
• Virus Isolation in Vero cells
Within a few days
after symptoms
Later in Disease • IgM and IgG capture ELISA
Course or after
recovery
• Immunohistochemistry Testing
• PCR
• Virus Isolation
Postmortem
Blood Specimens usually test positive on PCR one day before the symptoms appear.

28. IMAGING
• Introduction of an aerosolized filovirus into
the respiratory tract is unlikely to cause
discrete lesions that would produce focal
abnormalities on chest X-ray.
• Presence of pulmonary infiltrates might
suggest an alternative diagnosis.

29. OTHER INVESTIGATIONS FOR
MANAGEMENT OF CONFIRMED CASE
• LIVER FUNCTION TESTS
• RENAL FUNCTION TESTS
• ELECTROLYTES
• HEMATOCRIT
• REPEATED PLATELET COUNTS
• HEMOGLOBIN
• WBC

30. EMERGING TEST
MICROARRAY
• Cheaper
• Faster
• Accurate
• Awaiting FDA approval

31. BIOSAFETY LEVEL(BSL) CRITERIA
FOR
LABORATORIES
To protect:
• The patient
• Laboratory Workers
• The environment
• Level 1& 2
basic laboratories
• Level 3
containment laboratories
• Level 4
high containment
laboratories

32. BSL Laboratory type Laboratory
practices
Safety equipment
1 Basic teaching,
research
Good microbiological
techniques
None
Open bench work
2 Primary health
services; diagnostic
services, research
Good microbiological
techniques,
protective clothing,
biohazard sign
Open bench PLUS
biological safety cabinet for potential
aerosols
3 Special diagnostic
services, research
As BSL 2 PLUS
special clothing,
negative airflow.
Biological safety cabinet and/or other
primary devices for all activities.
Self closing double door controlled
access
4 Dangerous
pathogen units
As BSL 3 PLUS
airlock entry, shower
on exit, special waste
disposal
Class III biological safety cabinet,
positive pressure suits, double
ended autoclave (through the wall),
filtered air

33. BSL - 4 LABS
• Approximately 50 facilities in the entire world.
• 4 in India
• National Centre for Disease Control, Delhi
• National Institute of Virology ,Pune
• High Security Animal Disease Laboratory, Bhopal
• Centre for Cellular and Molecular Biology ,
Hyderabad

35. SUSPECTED EBOLA SAMPLES MAY BE
SENT IN INDIA TO
National Centre for Disease
Control
Directorate General of
Health Services
22, Sham Nath Marg
New Delhi-110 054
Phone: +91-11-23913148,
23946893
+91-11-23971
272/060/344/524/449/326
National Institute of Virology
20/ A,
Dr. Ambedkar Road.
Post Box No. 11,
Pune 411001
Tel.No. : 91-020-26127301 /
91-020-26006290