Epstein barr virus diagnosis tests

They peak wk after symptom onset and then decline rapidly, although they may rarely persist for mo[ 35 , 48 ]. Despite these limitations, detecting heterophile antibodies can be helpful in the case of primary infection mainly because of the simplicity of the tests.

In routine clinical practice, these include agglutination tests using sheep, goat or horse red blood cells as antigens after absorption with guinea pig kidney extracts in order to remove natural antibodies against Forssman antigen the Paul Bunnel reaction , and newer tests based on latex particles with adherent specific bovine antigen for heterophile antibodies monospot assays.

Recently, multiplex flow immunoassays MFI have been proposed because they are more sensitive in the case of acute infections[ 49 ]. However, given the level of false negative results, negative findings need to be followed by a search for specific antibodies[ 2 , 48 ]. The type and preparation of the antigens used are probably responsible for the differences in the sensitivity and specificity of the various assays[ 42 , 53 , 54 ]. IFA has been used as the reference method, although its sensitivity is the same as or less than that of EIAs[ 43 ], automated versions of which allow a large number of samples to be tested and are commonly used in laboratories with a large routine workload.

In order to confirm the screening assays, various immunoblotting tests have been developed using viral lysates of EBV-transformed cells and recombinant antigens line blots [ 57 ].

It is considered that the latter is unaffected by antibodies against the cell material that can be found in patients with mononucleosis[ 23 , 58 ]. Kinetic studies have found that a strong IgG response to p72 is not observed until 20 d after disease onset. The IgG avidity test can assess the degree of IgG maturation. Avidity is low at the beginning of an acute infection, but increases when the immune response matures[ 59 - 61 ].

For example, the maturation kinetics of VCA IgG lasts several weeks and in some cases up to 3 mo after symptom onset[ 62 , 63 ]. Two aliquots of the same sample are tested in parallel for the presence of IgG antibodies: one is not treated, and the other is treated with substances that dissociate the antibodies from the antigens usually 8 M urea.

Since the dissociation depends on antibody avidity, the ratio between the treated and untreated sample defines the degree of avidity. A search for avidity can therefore be used to estimate the duration of a primary infection, and differentiate acute and past infection[ 23 , 57 , 63 - 65 ].

The published data depend on the examined antibody and therefore the type of antigen used for the test. Avidity varies depending on the kinetics of the various antibodies[ 63 , 66 ], e. Consequently, the manufacturer points out that a reduction in the intensity of the EA and IEA bands alone is not an index of recent infection[ 68 ].

The limitations of avidity testing are the individual maturation rates of antibodies and the fact that the tests cannot be used in newborns because of the presence of maternal antibodies. A number of different methods, techniques and protocols have been used to determine the presence of EBV DNA and measure viral load[ 69 - 72 ]. Dot blotting, Southern blotting, PCR and in situ hybridization have all been applied to various materials, but their differences in sensitivity and specificity have led to the results that need to be considered cautiously[ 28 ] as they vary from laboratory to laboratory[ 73 , 74 ].

More recent studies indicate that real-time PCR is particularly sensitive[ 28 ], and very useful for defining infection status, especially in immunocompromised patients[ 45 , 75 , 76 ] and those at risk of developing EBV-related disorders[ 45 ].

However, there is still no consensus concerning the best material to use, units of measurement, or the quantitative levels requiring intervention or predicting prognosis[ 16 , 74 , 77 - 79 ]. This means that particular care is necessary when comparing the data of different studies[ 73 ]: for example, the units of measurement include copies per milliliter, copies per microgram of DNA, copies per leukocytes, and copies per positive cell[ 77 ].

Furthermore, there is much debate concerning the material that should be used to search for EBV DNA, such as whole blood, peripheral blood mononuclear cells PBMCs , plasma or serum[ 13 , 80 ]. There is also the problem that incorrectly stored whole blood can cause EBV DNA to leave the intracellular compartment and give rise to false positive results in plasma or serum, and false negative results may be due to nucleases that are capable of partially degrading plasma EBV DNA[ 81 ].

In general, the best material used to search for EBV DNA depends on where it is, and varies during the course of the disease[ 13 ].

The virions produced during primary infection spread in peripheral blood[ 82 , 83 ], and it is also possible to determine the EBV-free or fragmented DNA coming from apoptotic cells[ 83 ], and the B cells transformed during the latent phase also pass into the bloodstream.

However, it must be kept in mind that there may be individual variations due to individual differences in kinetics, and viral load may increase after an initial decline, and in some cases, it may take as long as a year or more before it reaches stably low levels.

Finally, even when this level is reached, the blood of a healthy carrier contains copies of EBV DNA per million white blood cells, whereas EBV-DNA is almost always undetectable in plasma or serum[ 82 , 85 , 93 - 96 ].

A search for EBV DNA may be more sensitive than serology in the early stages of the disease[ 89 ], and some studies have found that it correlates better with clinical acute infection than the avidity of VCA IgG[ 86 ]. However, in immunocompetent patients with acute infection, it is not usually necessary to look for EBV DNA as serology is sufficient except in cases with negative or doubtful serological findings in which there is a strong clinical suspicion of infection[ 89 , 97 , 98 ].

A search for EBV DNA is particularly important in immunocompromised patients with an incomplete humoral response and patients who have received transfusions or immunoglobulins that confound serological test results[ 28 ]. It has been reported that immunocompromised patients have higher baseline viral levels than healthy carriers[ 99 , ], which decline after treatment.

In EBV-related cancers, episomal or naked EBV DNA from apoptotic tumor cells is found in serum and plasma[ 84 , ], which may also contain tumor cells with latent EBV infection[ 13 ] and virions from a small number of tumor cells undergoing lytic infection. These profiles cover the vast majority of situations found in routine laboratory practice.

However, the profile of a minority of cases may give rise to doubts or require confirmation. Nevertheless, it is recommended to assess the specificity of the result because it may be made aspecific by interfering rheumatoid factor and autoantibodies, or cross-reacting factors such as HCMV or parvovirus B19[ 67 ].

A search for these factors and immunoblotting for IgM may be as helpful as the determination of other parameters of acute infection such as heterophilic antibodies or HBV DNA[ 98 ].

In some cases, VCA IgM may not be produced or appear wk after VCA IgG, or present for a very short time or at low concentrations and it may not be detected by conventional tests[ 2 , 32 , , ]. Furthermore, even when they are produced, they may be lost over time particularly, but not exclusively, in immunocompromised patients[ 23 , , ].

However, this last option inevitably delays the diagnosis until the second sample is collected, and physicians tend to avoid it if the symptoms improve over time, especially in the case of children, they may find it traumatic, which means the second sampling usually involves only a small number of patients[ ]. As anti-p18 IgG can be considered equivalent in meaning to EBNA-1 IgG because they are both produced late during EBV infection , their presence indicates past infection, and their absence indicates an acute infection.

A VCA IgG avidity test can also be used to distinguish acute and past infection[ 64 , 67 ], which are respectively characterized by low and high levels of avidity; studies of patients with isolated VCA IgG have found low levels in about half of the cases, and high levels in the remaining half[ 75 , 86 ]. The detection of EBV DNA is particularly useful in the case of serologically indeterminate EBV infection[ 75 , 86 , 98 ] because its presence in serum or plasma indicates primary infection or reactivation[ 13 ].

A search for heterophile antibodies may be useful in patients with isolated VCA IgG if the result is positive, although there have been reports of their presence for mo[ 28 , 35 , 48 ]. However, nothing can be said in the absence of heterophile antibodies, especially in children aged less than 5 years, and other tests must be used.

Reactivation is still relatively rare and often short in immunocompetent subjects, and is generally considered of no clinical relevance[ , ]; however, it can cause serious complications in immunocompromised patients. Moreover, as false positive reactions may also result from the presence of autoantibodies or rheumatoid factor, it can be useful to look for these interfering factors. However, it needs to be pointed out that cold agglutinins, rheumatoid factor and autoantibodies can be found for a short period during the course of EBV mononucleosis because of the polyclonal activation of B-cells[ ], therefore, the presence of rheumatoid factor does not automatically mean a falsely positive VCA IgM result.

Finally, EBNA-1 IgG positivity in patients with primary infection may also result from aspecific reactivity, which can be detected by immunoblotting for IgG antibodies[ ]. Furthermore, the recently developed EIA for antibodies to EBNA-1 IgG based on recombinant or synthetic peptides may be more sensitive than its predecessors, and allow their identification early in the course of primary EBV infection[ ].

In addition to repeating the test after a reasonable period of time in order to detect any changes in the antibody profile, VCA IgG avidity has proved to be particularly useful because low levels of avidity have been found in the course of recent infection, and high levels in the course of past infection or reactivation[ 62 - 64 ].

In order to fully understand the IgG avidity test and evaluate the results in relation to antibody maturation time, it is important to know how long after the onset of symptoms the blood sample was taken. The use of molecular biology seems to be a rather delicate question.

Consequently, as EBV DNA is present in cases of reactivation or primary infection, it is unlikely that testing one sample will be able to distinguish the two situations[ 75 , 95 ]. Other antibodies have been found in cases of reactivation. These antibodies reach a peak level wk after primary infection and decline slowly, but may last indefinitely. High levels were also found in patients with immunodeficiencies, recurrent parotitis, multiple sclerosis or nasopharyngeal cancer, as well as in pregnant women and elderly subjects[ 26 , - ].

Consequently, a search for VCA IgA is considered useful only in the diagnosis and management of patients with nasopharyngeal carcinoma[ - ]. At clinical level, the problem is to discover whether the patient has experienced a past infection or is still susceptible.

As the doubt is to distinguish past infection and non-specificity, it is useless to search for heterophile antibodies or EBV DNA. One immunoblotting study found that such patients were not only all anti-p72 EBNA-1 positive, but also anti-p23 VCA positive, which was not detected by the screening tests using p18 antigen alone to detect VCA IgG[ ].

A number of tests have recently been developed that may help clarify doubtful results in the serological diagnosis of EBV infection, and it is now possible to reach a conclusion without having to wait for a second sample taken after a certain lapse of time. What tests should be used after screening depends on various factors in addition to their scientific and technical suitability; these include organizational and economic questions such as differences in costs and the reimbursements foreseen by the National Health Service or insurance companies , as well as the availability of space and adequately trained personnel.

Furthermore, the number of routine tests the number of samples with inconclusive results affects the decision to undertake further more or less expensive laboratory tests or to send the sample or the patient to a reference laboratory. Finally, the type of patient may also be decisive. In conclusion, considerable progress has been made in the serological diagnosis of EBV infection and, using appropriate algorithms and methodologies, it is possible to solve all of the problems that may arise during the course of routine laboratory practice.

Independentei , Sect. National Center for Biotechnology Information , U. Journal List World J Virol v. World J Virol. Published online Feb Massimo De Paschale and Pierangelo Clerici.

Author information Article notes Copyright and License information Disclaimer. All rights reserved. This article has been cited by other articles in PMC. Abstract Serological tests for antibodies specific for Epstein-Barr virus EBV antigens are frequently used to define infection status and for the differential diagnosis of other pathogens responsible for mononucleosis syndrome. Table 1 Types of latency in Epstein-Barr virus-associated diseases.

Open in a separate window. The virus probably survives on an object at least as long as the object remains moist. The first time you get infected with EBV primary EBV infection you can spread the virus for weeks and even before you have symptoms. Once the virus is in your body, it stays there in a latent inactive state. If the virus reactivates, you can potentially spread EBV to others no matter how much time has passed since the initial infection. Diagnosing EBV infection can be challenging because the symptoms are similar to other illnesses.

EBV infection can be confirmed with a blood test that detects antibodies. About nine out of ten of adults have antibodies that show that they have a current or past EBV infection. For more information, see Laboratory Testing. There is no vaccine to protect against EBV infection. You can help protect yourself by not kissing or sharing drinks, food, or personal items, like toothbrushes, with people who have EBV infection.

In the following, we give an overview of diagnostic methods to accurately determine EBV serostatus and viral load. We evaluate the advantages and disadvantages of each method and report on the diagnostic significance of each and how to resolve diagnostic problems in case of uncertainties.

For practical procedures, we refer to the detailed instruction manuals of the respective test kit manufacturers which have to be closely followed for reliable results. Laboratory Testing. Minus Related Pages. Healthcare providers can test for antibodies to the following EBV-associated antigens: This photomicrograph depicts leukemia cells that contain Epstein-Barr virus using an FA staining technique.

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