10. Ebola virus identification

One of the primary means of management of Ebola disease is to promptly diagnose the virus. Hence, early laboratory diagnosis for the confirmation of suspected individuals with haemorrhagic fever is paramount for the implementation of appropriate control measures. Reverse transcription-Polymerase chain reaction (RT-PCR) and viral isolation on Vero cells are definitive diagnostic methods for the detection of Ebola virus infections [48, 49]. Also, serological diagnosis based on enzyme-linked immunosorbent assays (ELISAs) to detect immunoglobulins such as IgG and IgM specific to Ebola virus antigens are effective diagnostic tools [50, 51]. Viral nucleic acid and antigen can be detected in blood as early as 3 days after symptoms starts to manifest and can be identified by the above diagnostic tools [52]. Prior to laboratory diagnosis, clinicians should examine patients for vital signs and symptoms such as high fever, severe headache, muscle pain, bleeding, bloody diarrhoea, blood in urine, vomiting, abdominal pain, diarrhoea, or unexplained haemorrhage, etc. in suspected individuals [53]. In addition to primary diagnosis of Ebola virus, secondary diagnosis, particularly in infected patients such as the presence of atypical lymphocytes, leucopenia (as low as 1000 cells/L), thrombocytopenia (50,000–100,000 cells/L), elevated aspartate aminotransferase and alanine aminotransferase level, prothrombin and partial thromboplastin time are necessary to manage related complications that may arise due to the infection [54]. The various diagnostic tests for Ebola are summarized in Table 2.

One of the major challenges in the diagnosis of Ebola virus is that the infection presents similar signs and symptoms to that of common diseases such as flu, malaria, yellow fever, typhoid, meningococcal meningitis and other bacterial and viral infections [24, 30]. As such, clinical diagnosis is usually not sufficient for routine screening especially during outbreaks where the infection rate is high. More so, definitive diagnosis by RT-PCR and viral culture are usually not


Table 2. Ebola diagnostic test.

feasible in Africa as most local health settings lack or do not have sufficient or adequate laboratory facilities for such molecular techniques. Another major challenge is the high cost for the molecular diagnostic tests (RT-PCR, ELISA). The cost per sample may cost between \$50 to \$100 which may not be affordable by majority of individuals in developing countries especially in endemic areas including West Africa, Sub-Saharan Africa and central East Africa. More so, though these molecular diagnostic tools are very reliable, analyses take about 2–6 h, which is too long for such acute infection. As such, there is considerable need for rapid diagnostic tests which can take just a few minutes for detection. Also, cell culture on vero E6 African monkey kidney cells which is a traditional gold standard test requires biosafety level 4 (BSL-4) containment, thus, restricts its use for routine diagnosis. More so, the test can last for up to 5 days from the moment of viral inoculation to microscopic visualization [55].

An immunochromatographic assay may be suitable for such effective and prompt diagnosis. In 1995, a colorimetric assay was developed by Dr. Sherif Zaki of the CDC for the identification of Ebola virus in skin biopsies preserved in formalin [56]. However in recent years, this diagnostic tool is not readily available.

#### 10.1. Rapid diagnostic test for Ebola virus disease

fulminant shock and eventually death which occurs between the 6 and 16 days of illness [46, 47]. However, a few patients may survive and recovery from the infection gradually

One of the primary means of management of Ebola disease is to promptly diagnose the virus. Hence, early laboratory diagnosis for the confirmation of suspected individuals with haemorrhagic fever is paramount for the implementation of appropriate control measures. Reverse transcription-Polymerase chain reaction (RT-PCR) and viral isolation on Vero cells are definitive diagnostic methods for the detection of Ebola virus infections [48, 49]. Also, serological diagnosis based on enzyme-linked immunosorbent assays (ELISAs) to detect immunoglobulins such as IgG and IgM specific to Ebola virus antigens are effective diagnostic tools [50, 51]. Viral nucleic acid and antigen can be detected in blood as early as 3 days after symptoms starts to manifest and can be identified by the above diagnostic tools [52]. Prior to laboratory diagnosis, clinicians should examine patients for vital signs and symptoms such as high fever, severe headache, muscle pain, bleeding, bloody diarrhoea, blood in urine, vomiting, abdominal pain, diarrhoea, or unexplained haemorrhage, etc. in suspected individuals [53]. In addition to primary diagnosis of Ebola virus, secondary diagnosis, particularly in infected patients such as the presence of atypical lymphocytes, leucopenia (as low as 1000 cells/L), thrombocytopenia (50,000–100,000 cells/L), elevated aspartate aminotransferase and alanine aminotransferase level, prothrombin and partial thromboplastin time are necessary to manage related complications that may arise due to the infection [54]. The various diagnostic tests for

One of the major challenges in the diagnosis of Ebola virus is that the infection presents similar signs and symptoms to that of common diseases such as flu, malaria, yellow fever, typhoid, meningococcal meningitis and other bacterial and viral infections [24, 30]. As such, clinical diagnosis is usually not sufficient for routine screening especially during outbreaks where the infection rate is high. More so, definitive diagnosis by RT-PCR and viral culture are usually not

Time line of infection Primary diagnostic tests Secondary diagnostic tests

Virus isolation on Vero cells

Immunohistochemistry testing

Virus isolation on Vero cells

Rapid diagnostic test IgM and IgG antibodies

IgM ELISA RT-PCR

RT-PCR

Immunohistochemistry testing Rapid diagnostic test Antigen-capture ELISA testing Atypical lymphocytes

Aspartate aminotransferase, alanine aminotransferase prothrombin and thromboplastin

Leucocytes Thrombocytes

presenting arthralgia and fatigue.

136 Current Topics in Tropical Emerging Diseases and Travel Medicine

10. Ebola virus identification

Ebola are summarized in Table 2.

Within a few days after symptoms

Later stage of disease course or

Table 2. Ebola diagnostic test.

Deceased patients Rapid diagnostic test

begin

after recovery

Following the 2014, outbreak in West Africa, several field trials on rapid diagnostic tests (RDTs) are ongoing and a few RDT kits have been approved by U.S. Food and drug Administration (FDA) and WHO on Emergency Use Authorization (EUA) status. These RDT kits are lateral flow immunoassays (LFIs) which basically detects viral protein antigens circulating in blood. Three of the recently approved RDTs include ReEBOV Antigen Rapid Test kit, OraQuick Ebola Rapid Antigen Test and SD Q Line Ebola Zaire Ag test [57].

The ReEBOV Antigen Rapid Test kit by Corgenix, Inc. was the first LFI for EVD to receive emergency use authorization (EUA) status from both FDA and WHO) [58, 59]. This chromatographic dipstick immunoassay kit is a RDT that detects the Ebola virus VP40 matrix protein of three species which includes EBOV, SUDV, and BDBV in whole blood, plasma or serum. Following a finger prick, a drop of blood is applied directly unto the nitrocellulose test strip. The nitrocellulose strip is then deepened into a tube containing reaction buffer which initiates the movement of the sample along the test strip by capillary action. The presence of VP40 in the sample leads to the formation of an immune complex between the VP40 matrix protein antigen and gold-labelled anti-antibodies against VP40 which is subsequently deposited along the strip boundary of anti-VP40 producing a pink-red line that is visible between 15 to 25 min after the analysis. Validation study for the performance of ReEBOV RDT conducted in Sierra Leone on venipuncture blood showed a 100% sensitivity and 92% specificity when compared with results obtained from RealStar Filovirus Screen RT-PCR kit by Altona Diagnostics [58].

The second RDT kit that has received approval from WHO and FDA on EUA status is the OraQuick Ebola Rapid Antigen Test manufactured by OraSure Technologies, Inc. [60, 61]. Just like the ReEBOV Antigen Rapid Test kit, this RDT kit detects VP40 matrix protein of EBOV, SUDV, and BDBV species with similar assay procedure. In addition to the use of whole blood, this kit also makes use of cadaveric oral fluid which is collected using an oral mucosa swab for the detection of Ebola virus. Similarly as ReEBOV RDT, the presence of Ebola virus antigens is visibly detected following immune complex between viral proteins and gold-labelled antibodies bound along the test line in less than 30 minutes. Validation of the test performance of the OraQuick RDT based on a retrospective study in Sierra Leone as reported by WHO showed the OraQuick RDT with a 84% sensitivity and 98% specificity compared to clinical real-time RT-PCR testing [60].

saturation and blood pressure, replenishment of nutrients, antivirals as well as antibiotics for concomitant infections [65]. Administration of sufficient fluids by oral or intravenous route serve to maintain circulatory stability and replenish electrolytes and fluids lost during the infection. A broad-spectrum of antibiotics are used to manage potential concomitant bacterial infections; antimalarials are used for the treatment of malaria while antiretrovirals are used to inhibit viral replication. Antipyretics and analgesics are frequently used for the control of fever or body temperature and pain respectively. Also, specific drugs could be administered for the

Ebola Virus Disease: Progress So Far in the Management of the Disease

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With no specific treatment against the disease, considerable efforts in research have been ongoing for the identification of possible drug candidates for therapeutic interventions. One of such clinical investigation was conducted in 1995 during the Ebola epidemic in Kikwit where blood of improving patients was transfused to eight Ebola patients as a means of passive immunization. Among the eight patients, seven of them successfully survived the infection while only one patient died [66]. However, subsequent in vitro assays showed antibodies not to have neutralizing action against Ebola virus. As such, clinical investigation based on passive immunization has not been conducted in subsequent outbreaks. Furthermore, in vitro assays showed monoclonal antibodies against the GP of Ebola virus to exhibit defensive and healing properties in mice but were unable to protect non- human primates [67, 68]. Also, immunoglobulins which were raised in goat and had undergone pre-clinical test on laboratory animals were administered to infected scientists with Ebola haemorrhagic fever during an outbreak showed some degree of protection against the disease. Thus, these immunoglobulins were suggested to be beneficial as an emergency cure for individuals inadvertently infected with Ebola virus [69]. More so, a series of nucleoside analogue inhibitors for carbocyclic 3 deazaadenosine and S-adenosylhomocysteine hydrolase were shown to avert

With such preliminary studies showing immunoglobulins to have protective effects against the infection, several researches have dueled on this aspect as well as other targets and several clinical trials have been ongoing to assess some potential drug candidates. The main classes of drugs which are being evaluated for potential therapeutic effect against Ebola virus infection include monoclonal antibodies such as ZMapp, nucleoside analogues, RNA inhibitor based (TKM-Ebola) agents, positively charged phosphorodiamidate morpholino oligomers as well as antisense-based (AVI-7537) drugs [71]. Among these drug candidates, ZMapp is one of the most promising therapeutic interventions against Ebola virus disease that affects viral replica-

ZMapp is an experimental drug by Mapp Biopharmaceutical, Inc., which comprises of a combination of 3 monoclonal humanized murine antibodies produced in mice infected with Ebola virus and subsequently generated in tobacco plants [71, 72]. In vivo pre-clinical animal study showed 43% of infected mice treated with Zmapp to survived infection [73]. Though pre-clinical studies had exhibited therapeutic effect of Zmapp against Ebola virus, the experimental drug came into the lamplight when two US citizens who were health workers in Liberia during the 2014 West-Africa outbreak became infected and were successfully treated with ZMapp in Atlanta USA. Following this success, the drug was then used as an experimental treatment in the 2014 West-Africa Ebola outbreak and several patients survived and recovered

death in infected mice by inhibiting Ebola virus replication [70].

control of organ failure.

tion inhibiting its expression.

SD Q Line Ebola Zaire Ag test by SD Biosensor, Inc. is the third and most recent RDT kit to be approved by WHO on EUA status [62]. Unlike ReEBOV and OraQuick RDTs which detects only VP40 in EBOV, SUDV, and BDBV species, SD Q Line Ebola Zaire Ag test is a chromatographic deep stick test that simultaneously detects GP, NP, and VP40 antigens of EBOV in whole blood, serum or plasma. In this test, the presence of the three antigens in the sample forms complex with their specific gold-labelled mouse monoclonal antibodies at three different test boundaries at which visible lines are seen. Thus, three drops of sample are added to a sample port on the assay device and visualized at 20–30 min. The presence of at least any of the three test lines is interpreted as positive result. A WHO validation study in Sierra Leone using a total of 446 specimens including 100 fresh venous whole blood and 346 frozen plasma showed SD Q Line Ebola Zaire Ag test with 84.9% sensitivity and 99.7% specificity when compared to the RealStar Filovirus Screen RT-PCR kit 1.0 as gold standard [62].

In a nutshell, these RDT kits are very effective in diagnosis Ebola virus and useful for field settings especially during outbreaks as results can be obtained within a very short time without the use of any electronic equipment and does not require refrigeration for storage. The approval of these RDTs is of major importance for public health management of the disease as prompt diagnosis especially in the field following Ebola outbreaks is key to effective treatment.

Recently, a new immunochromatographic strip and a smartphone reader based on Sudan virus (SUDV) glycoprotein monoplex which detects and semiquantifies Ebola-specific IgG antibodies in human survivors has been developed [63, 64]. When the point-of-care test was tested in freshly collected patient samples including 90 SUDV survivors and 31 non-infected controls in Uganda, it showed a sensitivity of 100% and a specificity of 98% compared to standard enzyme-linked immunosorbent assay (ELISA) of whole Ebola antigen [64]. More so, a multiplex test which simultaneous detects antibodies against three recombinant SUDV proteins has also been developed. A pilot study involving 15 survivors and 5 non-infected controls showed sensitivity and specificity of 100% compared to standard ELISA [64]. Also, another multiplex subtype assay for the identification of three Ebola species: BDBV, SUDV, and EBOV based on recombinant viral glycoproteins has been developed [64]. The advantage of this multiplex viral species test is that it could differentiate the host's immunity to specific viral species and also identify cross-reactive immunity in infected patients.
