Diagnosing Ebola virus

The driving factor for an Ebola virus diagnosis is typically during an active outbreak when a confirmed epidemic is occurring. An isolated incidence which presents suspicious indications associated with Ebola virus, like haemorrhage accompanied by fever and other flu-like symptoms, anywhere in the world will also prompt case reporting and diagnosis. Risk factors will also be taken into consideration if an individual has recently been to or lives within an area prone to outbreaks, especially in West or Central Africa.

It is standard practice for several differential diagnostic procedures to be implemented when an unwell individual presents a set of symptoms similar to those experienced with an Ebola infection. Before a diagnosis of Ebola is made, it is likely that any collected samples may also be screened for other acute (sometimes fatal) illnesses like malaria, Marburg virus disease (previously known as Marburg haemorrhagic fever) and Lassa fever (also known as Lassa haemorrhagic fever).

Due to the devastating nature of the Ebola outbreaks experienced during the past four decades, especially those which resulted in a massive loss of life (between 2014 and 2016) any diagnosis of Ebola virus disease (EVD) made typically requires coordinated involvement between international authorities, including government health departments, the World Health Organisation (WHO) and the Centers for Disease Control and Prevention (CDC) in the USA. Any suspected case is taken very seriously and is handled according to strict outlined evaluation and management strategies.

How will suspected Ebola virus cases be handled during the diagnostic process?

A diagnosis of Ebola virus infection in individuals suspected to be infected with it is essential even though, to date, there are no formally approved therapies specifically for the treatment of the condition. The earlier a person is diagnosed; the sooner supportive care and appropriate treatment can be implemented so as to increase the odds of survival and reduce the risk of further transmission.

A diagnosis confirms what healthcare personnel are dealing with when providing care and enables the mobilisation of teams to ensure that strategies for larger population safety can be swiftly implemented.

1. Initial assessment steps for possible diagnosis

When a symptomatic individual is present at a consultation, the following general assessment steps will likely take place:

Medical review

A medical professional or physician will request some detail pertaining to the person’s overall health condition in order to determine all of the related information that will need to be taken into consideration before any diagnosis is made. This will include existing or recently experienced medical conditions, treatment procedures and medication / substance use. A vaccination history will also likely be discussed.

If the Ebola virus or another medical condition which results in a similar set of signs and symptoms is suspected, a medical professional may ask several questions relating to the person’s travel activity – this is to determine if a patient has recently visited a region or country (within the past 21 days) that either has a history of or a confirmed active Ebola outbreak. A medical doctor will also likely keep in mind any other potential conditions, especially infectious illnesses which may be experiencing high case numbers in such regions or countries.

A physician will likely ask questions relating to whether or not a patient has recently been exposed to an individual with a similar set of symptoms. If so, he or she will ask questions related to the nature of exposure to help identify any potential risk factors. This line of questioning may help to highlight any other people who may be at risk of infection (through possible exposure) and currently be classed as asymptomatic.

Physical examination

Medical professionals will look out for the tell-tale combination of signs and symptoms that are consistent with the Ebola virus (or possibly another condition). A physician will look out for the most common indications of illness and be particularly cautious if any signs or symptoms of haemorrhage are apparent at the time of examination. If an infectious condition like Ebola or another haemorrhagic virus is suspected, a physician will likely wear protective equipment (including a mask and gloves) as a precaution while examining the patient.

If Ebola is suspected during this process, a medical doctor will seek to determine if the patient’s condition is likely in the early or progressive stages of the disease. Symptomatic indications, like a low-grade fever, fatigue and other flu-like ailments, as well as gastrointestinal troubles may point to early disease development. A maculopapular rash is more typical around the 5th day following virus exposure and is often more clearly visible on fairer complexion individuals (such as Caucasian patients). Conjunctival injection (red eyes) may also indicate earlier stages of the disease.

Haemorrhage (bruising and bleeding), an expressionless facies (masked facial expressions), myocarditis (inflammation of the heart muscle) and pulmonary oedema (fluid on the lungs), as well as tachypnoea (abnormally fast breathing), hypotension (low blood pressure), anuria (failure of the kidneys to produce urine), organ dysfunction (multiple failures occur when a person becomes terminal) or meningoencephalitis (inflammation of the brain and surrounding tissues) will prompt immediate emergency care.

2. Differential diagnosis considerations

During medical evaluation, a medical doctor will take into careful consideration any other infectious or non-infectious conditions which may present similar symptoms. The process a doctor will follow largely depends on the individual’s risk factors (especially if a person resides in or has travelled to an outbreak prone region or country), as well as his / her predominant symptoms and vaccination history.

Other conditions that a doctor will consider (or keep in mind) for differential diagnosis will include:

• Influenza: Flu-like symptoms (including an acute onset of fever, headache, general malaise, fatigue and myalgia) are normally accompanied by respiratory ailments, like a sore throat, nasal congestion and / or discharge and a cough. Respiratory symptoms are not typically characteristic of the Ebola virus. During evaluation, a medical doctor may look for such symptoms in order to make a quick differentiation between infections. Should an influenza diagnosis be necessary, or a distinction need to be made, a doctor may make use of rapid results testing to detect viral antigens (e.g. influenza A or B viruses).
• Traveller’s diarrhoea: This condition typically develops during travel or up to 10 days following one’s return. General malaise, abdominal discomfort (cramping or pain) and a sudden onset of diarrhoea are typical. A person may also experience a low-grade fever, as well as nausea and vomiting. Differential diagnosis will involve determining whether diarrhoea is mostly confined to the gastrointestinal tract or if it is more aligned with that of a systemic illness and is just a part of a whole other set of characteristic symptoms. The latter may be considered as potentially a symptom of an Ebola infection, especially if the patient has been experiencing fever, fatigue or aching muscles (myalgia) for several days before seeking medical consultation.
• Malaria: A similar set of symptoms may indicate an infection with either the Ebola virus or malaria. Malaria can sometimes occur concurrently with EVD, especially if a person has recently travelled to Central or West African countries. Microscopic analysis of blood sample smears, as well as rapid antigen tests can help to differentiate between the two conditions and diagnose malaria.
• Lassa fever: Most common in West Africa, a person who has recently travelled to or resides in this part of the world will be considered a possible candidate for infection with this condition. Symptoms which are present are typically mild but can progress in severity. Testing will involve reverse-transcription polymerase chain reaction (RT-PCR), as well as blood serum sample analysis (serology) to determine a Lassa fever diagnosis.
• Marburg virus disease: This condition is more commonly seen in those who reside in or have travelled to countries in Central Africa. Symptoms are similar to that of Ebola. Reverse-transcription polymerase chain reaction (RT-PCR) testing will be done in order to confirm a diagnosis.
• Typhoid fever: A systemic (bacterial) condition, typhoid is most prevalent in areas which have poor access to sanitation. Fever and abdominal pain are characteristic symptoms of this condition, which is caused by Salmonella enterica serotype Typhi (previously known as S. typhi) infection. A blood culture is usually the best means of identifying the pathogen causing infection and is used to make a diagnosis of this condition.
• Meningococcal disease: This condition is caused by an infection with Neisseria meningitidis (a gram-negative bacterium) and can also present with symptoms such as fever and headache. Blood cultures in order to identify the bacterium and analysis of cerebrospinal fluid will be done in order to make a specific diagnosis.
• Measles (rubeola): The incubation periods of both conditions are fairly similar, as are early symptomatic indications of illness. In the case of measles, rhinitis (nasal stuffiness), conjunctivitis (an eye infection) and a cough typically develop, along with the characteristic (maculopapular) rash. Measles may be tested using a polymerase chain reaction (PCR) analysis and blood sample tests (to determine the presence of antibodies).

3. Determining an exposure risk category

Once initial assessments have been completed and other medical conditions ruled out, should an infection with the Ebola virus be strongly suspected, a medical professional will need to determine a patient’s exposure risk category (high, moderate, low or non-identifiable). This is necessary as it will guide the subsequent management and therapeutic actions of the attending medical professional/s. Determining the risk of exposure will apply to any individual who is symptomatic (showing symptoms) or asymptomatic (not showing symptoms).

The starting point is to assess any possible exposure that may have occurred within the previous 21 days, especially if symptoms are present. The level of exposure risk typically increases for healthcare workers with the number of probable or confirmed cases requiring care.

Ebola virus exposure risk categories
High	Moderate	Low (not zero)	Non-identifiable
Direct contact (touch) with the bodily fluids / the body of a symptomatic (or recently deceased) individual or contaminated objects and surfaces.	Direct contact with a symptomatic (a person showing symptoms) or deceased individual or bodily fluids while wearing appropriate personal protective equipment (PPE)	Travel to, visiting or residing in recognised outbreak regions within the past 21 days where no transmission cases have been recorded.	Direct or close contact with an asymptomatic individual who may have been exposed to another with confirmed Ebola virus disease.
Exposure to the bodily fluids of a symptomatic individual or the body of a recently deceased person who had an Ebola infection (without personal protective equipment / PPE).	Persons providing direct patient care within a treatment facility (a hospital, clinic or set up treatment care centre).	Brief direct contact (or casual contact – such as shaking hands) with an individual during the incubation period or whom is displaying very early signs of disease – without PPE.	Direct or close contact with an individual during the incubation stage (before Ebola symptoms develop).
People who reside in a household with a symptomatic individual – especially if the infected person has had direct contact with them, which is often involved in those providing care.	Close contact (within 1-meter to 3 feet) with a symptomatic individual for a prolonged period of time (without PPE) – including within households, treatment or healthcare facilities.	Briefly been close to or in the company (such as in the same room) of an individual who has fallen ill and is experiencing early indications of infection with the Ebola virus.	Visited a country more than 21 days prior to a confirmed outbreak (no known contact was made with a potentially infected individual).
Processing of bodily fluids (laboratory analysis) from a symptomatic individual without the use of PPE or within an environment that is not set up with standard biosafety precautionary measures.		Direct contact with a symptomatic individual, their bodily fluids or a deceased body while wearing PPE in a region or country that does not have an active, widespread outbreak (high record of transmission cases).	Visited or travelled to a country with confirmed EVD cases, but without widespread transmission (especially in urban areas) – no contact with individuals suspected as having the Ebola virus disease is made during the visiting period.
Healthcare workers providing direct care to symptomatic patients within a treatment facility (clinic or hospitals).		Close contact with an individual who is symptomatic while traveling on an aircraft, ship or other mode of transportation / or having been aboard the same mode of transportation (but not in direct / close contact with an already symptomatic individual).	Spent a prolonged period of time aboard a mode of transportation (e.g. a ship or aircraft) within an area or country with an active outbreak but having no direct contact with an infected individual.

 

Diagnostic considerations and Ebola infection control precautions based on exposure risk category
Symptomatic patients with identifiable risk (high, moderate or low)	Asymptomatic patients with identifiable risk (high, moderate or low)	Patients with no identifiable risk
Isolation / quarantine: A patient will be placed in an isolated, single room (if possible with a private bathroom, or alternatively that offers a covered bedside commode). If a suspected incidence is part of a possible Ebola outbreak, specific screening facilities will need to be set up in order to safely evaluate patients with as low a risk of further transmission exposure as possible.	Medical monitoring: Individuals who may have exposure risk but are not showing signs and symptoms of being infected with the Ebola virus at the time of evaluation are required to be monitored in the days and weeks that follow (i.e. at least 21 days from the date of potential exposure).	Medical monitoring: No monitoring or testing procedures will be required for the Ebola virus.
Notifying authorities: Local healthcare facilities (in which a patient may be being treated, as well as the staff working in these settings) and health departments will need to be promptly notified of a suspected or probable case of Ebola virus infection. International health organisations, such as WHO and CDC will also be required to be notified.	Onset of symptoms: Should infectious indications develop, such as fever or other flu-like symptoms, an affected individual must notify a medical professional immediately. Depending on the nature of the person’s condition and the level of exposure risk that is determined, a physician may request that the patient be admitted for direct observation or advise him / her on self-monitoring or care processes. Public health authorities will need to be notified if infection with the Ebola virus is suspected and diagnostic procedures followed.	Presence of symptoms: If a patient has any viral signs and symptoms which could be attributed to any other condition known to cause these (like influenza), appropriate evaluations will take place. A medical doctor will take into consideration the specific patient clinical indications of infection, as well as the suspected pathogens involved.
Standard contact and droplet precautions: In order to provide any care to a symptomatic individual, healthcare professionals will need to wear PPE – before and after an actual case is diagnosed as an Ebola virus infection. Infection control precautions will need to be strictly followed.	Movement considerations: Once a person is symptomatic, a medical doctor will consider transmission risk and likely request that the infected individual’s movement be restricted (including restricting travel) so as to avoid possibly exposing others to the infection. Total isolation or quarantine procedures may also be recommended during a period of testing or once an Ebola infection is confirmed.
Laboratory testing: Suspected cases of Ebola will need to be reported to local and international authorities. Samples will need to be collected and submitted for diagnostic testing – either to diagnose Ebola virus disease or rule out any other conditions which share similar symptoms.
Confirmed EVD: If test results confirm infection with the Ebola virus, a specialised Ebola treatment centre will need to be arranged for the patient to be transferred to in order to reduce the risk of exposure and transmission, as well as to provide sufficient medical care.

What tests are used in Ebola virus diagnosis?

1. Laboratory testing considerations – blood sample analysis

If infection with the Ebola virus is suspected, a medical professional may expect to find the following during laboratory testing of blood samples:

• Leukopenia (or Lymphocytopenia): This refers to a reduced number or leukocytes (white blood cells which form part of the immune system and help to fight infections) present in the body. This can occur during the early stages of infection, followed by a sharp increase in neutrophil cell (a type of white blood cell) counts after the initial few days of symptoms onset. Abnormalities in lymphocytes (another type of white blood cell) may also be present. Analysis of white blood cells may be done using blood smear samples.
• Thrombocytopenia: This refers to a low count of blood platelets (or thrombocytes). During acute infections (especially during early symptomatic stages), platelets can decrease considerably as a result of the virus. Platelet reduction may typically be lowest between 6 and 8 days of illness.
• Transaminase (enzymes) elevations: Hepatic or liver dysfunction can occur in Ebola virus infections. Signs of liver damage may be seen in elevated levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) – transaminase enzymes within cells, assessed via a blood sample analysis.
• Haematocrit abnormalities: The volume percentage of red blood cells within the blood may increase or decrease in individuals infected with the Ebola virus. Haematocrit blood tests can determine any volume abnormalities – either high or low results are indicative of illness. High volume percentages may indicate concerns such as dehydration, lung or heart dysfunction or infection which increases the production of red blood cells. Low volume percentages could indicate anaemia or deficiencies in vitamin and mineral supplies within the body.
• Coagulation (clotting) abnormalities: Such abnormalities may be most prominent in the progressive stages of Ebola virus infection and are indicative of haemorrhage which refers to the inability of blood to clot sufficiently and stop any bleeding that occurs within the body. Clotting factors which include proteins such as prothrombin (produced in the liver) assist with the clotting process. A lack of the protein can lead to coagulation disruptions. Testing will involve measurement tools such as prothrombin time (PT) and partial thromboplastin time (PTT) which assess the length of time it takes for blood to sufficiently clot.
• Renal abnormalities: Kidney tissue damage may occur as a result of abnormal quantities of protein in the urine (known as proteinuria). Elevations in blood urea nitrogen and creatinine are common during the early and progressive stages of Ebola virus disease. Excessive fluid loss as a result of vomiting and diarrhoea can also contribute to or worsen kidney dysfunction (caused by injury to the organ’s tissues).
• Electrolyte abnormalities: The severity of the gastrointestinal symptoms and problems experienced as a result of the Ebola virus tend to cause considerable abnormalities in the body’s electrolytes (nutrients in the body that play a role in a number of vital bodily functions from heart rate regulation to muscle contraction). This may result in abnormally low quantities of sodium in the blood (referred to as hyponatremia), as well as reduced levels of potassium (hypokalaemia), magnesium (hypomagnesemia) and calcium (hypocalcaemia). Abnormal elevations in potassium can also occur (hyperkalaemia). Cardiac arrhythmia (an irregular heartbeat) can occur if abnormalities are not sufficiently treated in order to restore balance in the body.

2. Diagnostic tests for Ebola Virus Disease (EVD)

Testing for Ebola virus infection is required in the diagnosis of the virus and involves coordinated efforts between the local health authorities where the incidence (or outbreak) is suspected to have occurred, as well as the CDC and WHO. The attending medical professional/s will also need to request the assistance of an infectious disease specialist as quickly as possible and implement isolation and barrier protection protocols.

Any testing that is required will be carefully handled by the medical personnel exposed to an individual suspected as having a condition that may be transmitted via human-to-human contact. Medical professionals will take precautions so as to avoid any direct contact with the bodily fluids of a patient, and thereby reduce the risk of transmission – even before a confirmed diagnosis is made.

All healthcare personnel and officials providing care to a patient suspected of being infected with Ebola will be required to wear personal protective equipment (PPE) or similar equipment that does not expose the skin. Such equipment can include things like surgical hoods (covering the head and neck), gloves (which must be disinfected after handling a patient or their bodily fluids), a facial shield and respirator (instead of just goggles and a mask). Equipment should never be adjusted while handling an infected patient.

Testing for Ebola is required for any patient who is assessed as symptomatic (showing symptoms) at any level of identifiable risk (high, moderate or low). Testing requirements involve:

• Tests which either diagnose Ebola or rule out the condition.
• Tests which diagnose or rule out an alternative infection or condition with a similar set of symptoms.
• Tests which diagnose or rule out a possible concurrent infection (such as malaria).
• Tests which are required to provide emergency care in order to save the life of a patient.

Testing is typically performed in maximum-containment laboratories (biosafety level 4) which can safely handle high-risk microbes (pathogens) associated with infectious diseases, like Ebola.

Laboratory personnel are required to wear positive-pressure protective suits equipped with umbilical-fed air supply.

Testing is not typically required…

• If a patient has been determined as having an identifiable exposure risk but does not show signs and symptoms of infectious disease. Instead monitoring of the patient will be prioritised – if symptoms develop, testing will then be required.
• If a patient has no identifiable exposure risk to the Ebola virus.

Tests which will be used for diagnostic purposes include:

• Reverse transcription polymerase chain reaction (RT-PCR): This test is used to detect and quantify RNA gene expression (or RNA sequence changes). The purpose is to identify and quantify the presence of the virus (viral antigens) in the body. The laboratory test is conducted from blood samples (or other bodily fluids, like saliva) and can be done within 3 days of symptom onset (this is when the virus is sufficiently detectable within the blood). Blood samples are the preferred fluid for testing as viral RNA levels can be better detected in these than in other bodily fluid samples. If testing is done in less than 3 days from symptom onset, RT-PCR may need to be repeated several times in order to check the consistency of the results. The results of these tests can be obtained within a 2 to 6-hour period. If a negative result is obtained at least 72 hours after the onset of symptoms, Ebola virus may not be diagnosed and another condition with similar symptoms may be then be considered and tested for in order to potentially make a diagnosis.
• Immunoassays (rapid chromatographic immunoassay / ReEBOV): This biochemical test is also able to identify and determine the presence or concentration of viral antigens in the blood or other bodily fluids. The test can determine viral antigens within as little as 15 minutes and is mostly used to support a RT-PCR positive test result. A ReEBOV is not typically used alone in order to make a confirmed Ebola diagnosis – this is because the test has not always been able to accurately detect viral antigens in the early stages of the disease.

Other tests which may be useful for viral detection include:

• Indirect fluorescent antibody test (IFAT): The test can be used to help identify viral antibodies but is not favoured as a diagnostic method as it has been associated with false-positive results in the past.
• Enzyme-linked immunosorbent assay (ELISA): This test may be useful for diagnosis. Immunoglobulin M (IgM)-capture ELISA is capable of detecting the IgM antibodies of Zaire ebolavirus strains when grown in Vero E6 cells (cells derived from the kidneys of an African green monkey which are often used in cell cultures). Immunoglobulin G (IgG)-capture ELISA can detect or identify IgC anti-Ebola antibodies when using non-ionic detergent-extracted viral antigens.
• Immunohistochemical tests: This testing procedure may be useful in determining or detecting the distribution of antigen tissues. In the case of the Ebola virus, skin tissue samples may be used for analysis once a patient has died. It can be used for diagnostic purposes (i.e. diagnosing an Ebola virus infection post-mortem).
• Electron microscopy: This process may be useful for the analysis of a tissue sample or specimen using a microscope. A beam of electrons magnifies a specimen, showing fine detail. As such the technique can help to identify filoviruses, like Ebola. This analysis technique alone is not typically used for diagnostic purposes, however.

Emergency use authorisation (EUA) of in vitro diagnostics ⁽⁷⁾

Emergency authorisation of in vitro testing (i.e. test in test tubes or cultures) used in the detection of the Ebola virus is granted by the FDA (Food and Drug Administration in the USA). Such emergency authorisations may be approved when medical personnel are presented with Ebola or similar illnesses which may be life-threatening when no other approved alternative testing means are available. In such cases unapproved medical products may be used for diagnostic purposes.

Means for diagnosis which may be used by specially trained individuals include:

• U.S. Department of Defence (DoD) EZ1 Real-time RT-PCR Assay: This test may be authorised in laboratories designated by the U.S. DoD.
• CDC Ebola Virus NP Real-time RT-PCR Assay: Emergency use may be authorised by qualified laboratories designated by the CDC.
• CDC Ebola Virus VP40 Real-time RT-PCR Assay: Emergency use may be authorised by qualified laboratories designated by the CDC.
• FilmArray Biothreat-E Test (BioFire Defence, LLC): This test may be used to identify Ebola virus in blood and urine samples and may be authorised by the U.S. (under federal regulatory standard) within moderate and high complexity laboratories.
• FilmArray NGDS BT-E Assay (BioFire Defence, LLC): This test may be authorised in laboratories designated by the U.S. DoD.
• RealStar® Ebolavirus RT-PCR Kit 1.0 (altona Diagnostics GmbH): This test may be authorised at moderate and high complexity laboratories within and outside of the U.S.
• LightMix® Ebola Zaire rRT-PCR Test (Roche Molecular Systems, Inc.): This test may be authorised at moderate and high complexity laboratories within the U.S. or at similarly run facilities outside of the USA.
• Xpert® Ebola Assay (Cepheid): This test may be authorised at moderate and high complexity laboratories within the U.S. or at similarly run facilities outside of the USA.
• Idylla™ Ebola Virus Triage Test (Biocartis NV): This test may be authorised at moderate and high complexity laboratories within the U.S. or at similarly run facilities outside of the USA.

Ebola and pregnancy

Precautions will be followed as with any other individual when it comes to pregnant women who are suspected of or confirmed as being infected with the Ebola virus. Pregnant women are not considered as being at a higher risk of infection than the general population. Over and above the standard precautions, medical professionals will, however, take into careful consideration additional risks to both a mother and her unborn baby – such as the loss of the baby or pregnancy-associated haemorrhage. Pregnant women may be more susceptible to severe illness and even death.

No female healthcare workers or personnel who are pregnant themselves are advised to handle any suspected or confirmed cases of the Ebola virus in pregnant women.

Any suspected cases will need to be thoroughly screened and diagnosed in the same way as any other non-pregnant individual. Additional monitoring and care will be provided for the safety of a pregnant woman and her unborn baby.

Reference:

^{7. U.S. Food and Drug Administration (FDA). Emergency Use Authorizations: https://www.fda.gov/medicaldevices/safety/emergencysituations/ucm161496.htm  [Accessed 23.05.2018]}