HOW TO INVESTIGATE EBOLA?

Dr. ROHIT JAIN

Department of

Pathology & Transfusion Medicine

Risk Group

Individual risk Community risk

1 no, low no, low

2 moderate low

3 high low

4 high high

WHO Risk group classification For Infectious microorganisms (2004)

Ebola• Risk group 4•Lethal, pathogenic agent

•Readily transmittable– direct, indirect

•Effective treatment and preventive measures not usually available

DIAGNOSTIC CONSIDERATIONS

OTHER DISEASES SHOULD BE RULED OUT

• Malaria• Typhoid fever• Shigellosis• Cholera• Leptospirosis• Plague

• Rickettsiosis• Relapsing fever• Meningitis• Hepatitis• Other Viral hemorrgahic

fevers

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

Whom to test?CDC RECOMMENDATIONS

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

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

2. LOW RISK EXPOSURETesting 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 PPE2. Direct brief contact with an EVD patient without wearing recommended PPE

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

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

SPECIMEN REQUEST FORM

When samples should be collected for Ebola testing?

• 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.

SAMPLE TYPE

Antemortem

• 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)

Postmortem

• Tissue Sample Liver, spleen, bone marrow, kidney, lung and brain

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

STORAGE OF SAMPLE

• Specimen should be stored at 4 degree Centigrade or Frozen.

SAMPLE TRANSPORTATION

• Main goals

– protects the environment

– the carrier

– protects the sample

• arrival in good condition for analysis

Packaging of the Specimen(Triple Packaging system)

• Primary receptacle (a sealable bag) wrapped with absorbent material.

• Secondary receptacle (watertight, Leak proof) .

• Outer shipping package.

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.

• Sample should be transported within 24 hours

• If not , Serum should be stored at – 70 degree

centigrade.

DIAGNOSTIC MODALITIES

LABORATORY TESTS

Within a few days after symptoms

• Antigen capture ELISA• IgM capture ELISA• Real time Reverse Transcription PCR• Virus Isolation in Vero cells

Later in Disease Course or after

recovery• IgM and IgG capture ELISA

Postmortem

• Immunohistochemistry Testing• PCR• Virus Isolation

Blood Specimens usually test positive on PCR one day before the symptoms appear.

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.

OTHER INVESTIGATIONS FOR MANAGEMENT OF CONFIRMED CASE

• LIVER FUNCTION TESTS• RENAL FUNCTION TESTS• ELECTROLYTES• HEMATOCRIT• REPEATED PLATELET COUNTS• HEMOGLOBIN• WBC

EMERGING TEST

MICROARRAY• Cheaper • Faster• Accurate• Awaiting FDA approval

BIOSAFETY LEVEL(BSL) CRITERIA FOR

LABORATORIES

To protect:

• The patient • Laboratory Workers• The environment

• Level 1& 2basic laboratories • Level 3

containment laboratories

• Level 4

high containment laboratories

BSL Laboratory type Laboratory practices

Safety equipment

1 Basic teaching, research

Good microbiological techniques

None

Open bench work

2 Primary healthservices; 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 PLUSspecial 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

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

SUSPECTED EBOLA SAMPLES MAY BE SENT IN INDIA TO

National Centre for Disease Control

Directorate General of Health Services22, Sham Nath MargNew 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