Epidemiological association is the key to suspect ebola virus infection.
Endemic countries: Possible contact with bat/animal or visiting any sick patients or funeral.
Non-endemic countries: Travel history to country where active ebola virus transmission is going on.
Differential diagnosis: During the early phase of illness, the symptoms are similar to several other common tropical diseases like malaria, enteric fever, yellow fever, leptospirosis, etc. Therefore, even in the presence of epidemiological association, it is difficult to diagnose EVD solely on clinical basis particularly before the onset of hemorrhagic symptoms.
Lab Diagnosis
Samples
• Blood: Blood is the most important sample.
• Body fluids: Other body fluids, such as oral fluid, semen, urine can also be tested in absence of blood sample.
• Tissue: Tissue samples from various affected organs can be tested in absence of blood sample or for postmortem diagnosis.
All the clinical samples are tested after inactivation with either gamma irradiation or heat inactivation. Chemical treatment with guanidium isothiocyanate (GITC) is done before PCR testing to degrade the viral proteins.
Methods for EBOV Detection
Virus isolation: Isolation of virus by cell culture method is the conventional gold standard for confirmation of presence of virus.
African green monkey kidney cell line Vero E6 is commonly used. Identification of virus is done either by visualization of virus by electron microscopy or virus antigen detection by immunofluorescence method.
Disadvantages
• The process is time consuming and requires the facility of biosafety level 4.
• The test is less sensitive as compared to PCR.
• Not recommended as a diagnostic test in a clinical setting.
Advantages
• Helps in providing the virus isolates for sequencing.
• Helps in molecular epidemiological studies and virus research.
• Provides positive control for diagnostic tests.
• Used as a tool to evaluate new diagnostic tests.
Virus nucleic acid detection: Polymerase chain reaction (PCR) for the detection of viral nucleic acid has been developed and used by CDC since 1995 ebola virus outbreak in Kikwit.
Currently this is one of the standard and preferred methods for EBOV diagnosis because of its high sensitivity, specificity and ease of sample processing with chemical inactivation in biosafety level 2 facilities.
PCR test is done from blood and body fluids, such as oral fluid, semen, ocular fluid, breast milk, etc.
Both conventional RT-PCR and real-time PCR (for viral load quantitation) have been developed by CDC.
Various viral genes have been used as the target gene for detection. These are, L, GP, VP40 and NP genes. Target using L gene can detect all fioviruses, whereas other gene targets are species specific.
L and NP genes have been reported to detect the viral RNA in convalescent samples for several weeks after the cessation of symptoms.
RT-PCR may be negative during the first three days of illness due to low level of virus RNA in the blood. Therefore, a negative PCR result at this phase of illness should be repeated after a few days and does not rule out the infection.
Virus RNA can be detected 1–3 days before the antigen test becomes positive and persists 1–3 days after the disappearance of antigen.
Significance of viral load: Quantitative value of viral load determination can be done by real time PCR.
• Viral load determination in convalescent patient helps in taking decision in discharging the ebola positive patients.
• A CT value > 35 in convalescent patient is considered non-infectious.
• CT value < 40 is considered as positive.
• The exact clinical significance of low viral load in convalescent patients is not known as regarding their transmission potential.
• Viral load > 107 RNA copies/mL has been associated with high mortality.
Detection of antibody: This can be done by indirect fluorescence antibody test (IFAT) or ELISA. The later has almost replaced the IFAT which requires biosafety containment level 4.
IFAT: This test was in use since 1977. It detects the antibody by using the viral antigen prepared from the cell culture. The processing requires the biosafety level 4, poorly sensitive and specific. The test has largely been replaced by ELISA.
ELISA: Ebola virus specific IgM and IgG antibody can be detected by ELISA system. The high sensitivity, high specificity and high throughput applicability have made this test feasible for patient diagnosis. The test can be done in samples inactivated by gamma irradiation. Therefore, ELISA can be done in a biosafety level 2 facility.
Capture ELISA format is preferred for detection of IgM antibody. For detection of IgG, indirect ELISA has been developed by CDC.
In general, IgM antibody appears during 2–11 days after the onset of illness and persists beyond 30 days. IgG appears mostly during the second week of illness and persists for years.
Detection of ebola virus antigen: ELISA for detection of ebola virus antigen in the serum sample has been used by CDC. Antigen is detected by using pool of mouse monoclonal antibodies.
Antigen can be detected from day 1 of illness, and most of the symptomatic patients are positive by day 3 of illness. Positive antigen test indicates the acute illness.
The ELISA available for antigen detection has not been extensively used in clinical setting because of less availability and widely used RT-PCR.
Rapid immunochromatographic test: During the last ebola virus outbreak in 2014, a rapid immunochromatographic test has been developed for the detection of ebola virus antigen in field setting as point of care test.
• Test can be performed from whole blood (both from finger prick and venipuncture blood), plasma and serum samples.
• It detects the VP40 antigen of ebola virus with 91.8% sensitivity and 84.6% specificity as compared to PCR.
• Test result is given in 20–30 minutes.
• Positive rapid test gives a presumptive diagnosis.
• Three different rapid tests have been approved by WHO.
الاكثر قراءة في الفايروسات
اخر الاخبار
اخبار العتبة العباسية المقدسة
