Serological diagnosis

S. Specter D. Jeffries
The introduction of rapid diagnostic techniques to detect viruses or viral antigens has vastly improved the isolation and/or identification of etiologic agents in viral diseases; however, there are still many instances for which serologic diagnosis is necessary. Agents that do not replicate well in cell culture or for which there are as yet no good reagents for direct detection of virus are routinely diagnosed using measurement of antibody. Routine testing for ARBOviruses, Epstein-Barr virus (EBV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human T lymphotropic viruses (HTLV) and in some circumstances cytomegalovirus (CMV), herpes simplex virus (HSV), varicella-zoster virus (VZV), measles and rubella is performed by serologic techniques. Serologic testing is performed both for screening of susceptible populations and for the diagnosis of acute or chronic diseases. The use of serologic testing in these circumstances is delineated in this chapter. (i.e. appearance of antibody) or an increase in titer as an indicator of recent infection; (3) a particular class of immunoglobulin to determine the nature of the infection; (4) responses to various antigens associated with the same virus to indicate disease progression; (5) a combination of antibody and antigen levels also to monitor disease state or progression, and (6)levels in different body fluids, such as serum versus CSF, to determine involvement of possible site of infection, such as CNS.

Qualitative assessment of immune status

The qualitative measurement of antibody presence or absence is a useful measure of immune status relative to a particular virus. This can confirm either exposure to a natural infection or successful vaccination of an individual. Immune status is extremely important in circumstances for which secondary exposure to a virus may have very different consequences than primary exposure. For a pregnant female, evidence of prior CMV infection may have devastating consequences for the fetus, whereas a reactivated latent infection is less likely to have pathologic consequences for the fetus. Similarly, screening tests for antibodies to rubella virus in pregnant women indicate that the mother will not likely get rubella if she is antibody positive and thus the fetus is also protected. In rare instances, reinfection with rubella has been recorded but this is an insignificant problem. Copyright 9 1996 Academic Press Ltd All rights of reproduction in any form reserved

Principles for serologic diagnosis and for screening

The detection of antibodies as a measure of infection by a particular virus may be used in one of several ways for diagnosis. One may measure the presence of antibody as: (1) an indication of immune status, following natural infection or vaccination; (2) seroconversion Virology Methods Manual ISBN 0-12-465330-8

Virology methods manual Screening tests are also important for a variety of other medical circumstances. Transplant donors and recipients must be screened for CMV immune status, and organs from CMV positive donors should not be used in CMV negative recipients, if this can be avoided, as there is a high likelihood of transmitting infection. Blood products are routinely screened for several viruses that are blood borne pathogens, including CMV, HBV, HCV, HIV and HTLV. This practice has severely reduced the incidence of disease caused by these viruses that is attributed to transfusion. It must be noted here that qualitative assessment of antibody is of limited value, merely indicating exposure to a particular virus. Although in many cases the presence of antibody can be equated with protective immunity, diagnosis of acute infection requires a quantitative analysis of antibody. to examine an acute serum for indicators of acute infection.

Immunoglobulin class assessment

IgM determination
The determination of immunoglobulin (Ig) M levels in a serum is often useful for rapid identification of acute, primary infection. Numerous methods have been devised to detect IgM and these are welt described (Hermann and Erdman 1992) (see chapter 7). More importantly the limitations of this approach must be noted. Considerable care must be taken in measuring IgM levels to a particular virus to be certain that the results are not confused by the presence of rheumatoid factor in the serum (Ziola et al 1979). A variety of strategies have been devised to avoid this problem and are also described by Herrmann and Erdman (1992) (see chapter 7). While IgM generally is diagnostic of primary infection this is not always so. With certain herpesviruses, particularly CMV and HSV, IgM levels may rise with reactivation of infection (Pass et al 1983; Lopez et al 1993). Thus, detection of IgM is not necessarily indicative of primary infection with these viruses and is only useful in this regard when IgM can be measured in the absence of significant amounts of IgG.

Quantitative analysis of antibody

The detection of antibodies in serum is not always diagnostic for an acute infection, because it is not possible to determine qualitatively whether these antibodies are due to a current, recent or past infection. Since several viruses can cause similar symptoms the presence of antibody in conjunction with clinical evidence of disease still is not sufficient for diagnosis. Thus, it is necessary to measure virus-specific antibody titers in acute and convalescent sera to determine the cause of an acute infection. Acute sera should be collected as soon as possible after onset of infection and convalescent serum should be collected 2-3 weeks later. A two tube (usually four-fold using two-fold dilutions) rise in titer is indicative of a significant change in antibody titer denoting a current infection. The disadvantage to this method is that it takes a considerable amount of time for antibodies to rise and hence for a meaningful result to be obtained; thus utility for diagnosing and, if necessary, treating an acute infection is severely hampered. An alternative to using the titer rise is 344

Immunoglobulin A
igA is important as an antibody in fluids associated with mucosal surfaces, since it is more stable than other Ig as secretory IgA in secretions such as saliva, nasal secretions, gastrointestinal secretions, etc. (Cremer 1985). Thus, measurement of IgA levels in such secretions may be an important and useful measurement of protective immunity against respiratory and enteric viral infections. It also is important in the assessment of generation of protective immunity on mucosal surfaces. By contrast C h a p t e r 17

Assessment of antibodies to different antigens

IgA is less important in systemic immunity since it is usually minimal compared to IgM and IgG responses, in fact, measurement of IgA in serum can often be obscured by high affinity IgG (Cremer 1985). Thus, IgA is not routinely measured in the diagnostic laboratory but is used only in special circumstances. One such circumstance is the measurement of IgA antibodies against the viral capsid antigen (IgA-VCA) of EBV. IgA is considered diagnostic for nasopharyngeal carcinoma (NPC) that is EBV induced (Lennette 1985). In such cases, IgA-VCA titers can be used prognostically in following the tumor burden due to NPC and to monitor the effectiveness of treatment. EBV, antibody responses are first detected against the VCA, followed by the early antigen (EA) then the Epstein-Barr nuclear antigen (EBNA) (Paar and Strauss 1992). Thus, high VCA and EA titers in the absence of EBNA would be indicative of a current acute infection, whereas a high EBNA titer along with high VCA but low EA would suggest a past EBV infection. When combined with comparing IgM-VCA (higher early in infection) to IgGVCA (higher later in infection) titers more specific information can be gained as to how early in infection the sample was collected. It should be noted that while these titers provide some very specific information about EBV infection, there is a very simple test for infectious mononucleosis. This test involves detection of the PauI-Bunneil heterophile antibody. This antibody is a common anti-red blood cell antibody that is induced in humans by EBV infection (Lennette 1985). When properly controlled, this test is a far less expensive way of diagnosing EBV induced infectious mononuclesosis than the specific anti-EBV antibody tests. Heterophile testing that gives a negative result may be indicative of CMV

Assessment of antibodies to different antigens

The onset of antibody production to various antigens of a particular virus can occur sequentially and quantitative testing for these different antibodies using one serum specimen can be diagnostic of the state of infection (Fig. 17.1). For infectious mononucleosis due to

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induced mononucleosis or may be a false negative. This can be confirmed by specific testing for antibodies to CMV or EBV. For the diagnosis of mumps infection the combination of antibodies to S and V antigens can be used in a similar manner to indicate current versus past infection with this virus (Kleiman 1985). Anti-S antibody rises first and persists for only 2-3 months, whereas anti-V rises later and persists for a significantly longer time period. tion is prognostic for disease progression. The presence of HBeAg indicates that a patient has infectious virus in their blood and is associated with a poor prognosis, while the presence of anti-HBe suggests that a patient is recovering. The conversion from HBsAg positive to antiHBs positive along with anti-HBe denotes complete recovery from HBV infection. Thus, by using these various markers one can diagnose HBV infection and can ascertain a prognosis for the disease.

Assessment of antibody and antigen combinations

Hepatitis B virus diagnosis is dependent upon the detection of a combination of viral antigens and anti-viral antibodies in serum (Fig. 17.2). Three antigens of HBV, the surface (s) antigen, core (c) antigen and e antigen are used for this testing as are the antibodies to these antigens (Escobar 1992). The presence of hepatitis B surface (HBsAg)is indicative of active infection and is used to screen for HBV. The detection of anti-HBs is indicative of recovery from HBV infection, while presence of HBc in the absence of either HBsAg or anti-HBs (the core 'window')indicates a late acute infection. The use of HBeAg and anti-HBe detecJ Symptoms ]

Detection of antibody in fluids other than serum

Cerebrospinal fluid
The measurement of antibodies in the CSF may be useful in the diagnosis of CNS disease. Antibodies may be present in high levels in the CSF as a result of CNS infection and an active immune response or because of leakage of serum into the CSF due to breakdown of the blood-brain barrier (BBB). The two situations can be distinguished by calculating CSF/serum ratios of albumin and IgG (Cremer 1985). The normal levels for albumin and IgG are 200-fold and 500-fold lower, respectively in

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D e t e c t i o n of a n t i b o d y in fluids o t h e r than s e r u m CSF. If there is antibody synthesis in the CSF, then only the IgG ratio changes but if there is BBB damage then both the IgG and albumin ratios between CSF and serum will change. The presence of increased levels of Ig in the CSF is indicative of local antibody synthesis and therefore local viral infection. For the most part this is an IgG response; however, in HSV encephalitis and chronic measles leading to subacute sclerosing panencephalitis IgM class antibody may also be detected (Doerr et al 1976). Additionally, one may compare ratios of antibody in serum versus CSF, such that low ratios, e.g. 8-32, are of diagnostic importance for CNS infection.

Saliva
The detection of antibodies in saliva is not a common laboratory practice. Nevertheless this source of antibody is being examined in circumstances where drawing blood for serum is impractical for reasons of both convenience and cost. There is currently a test kit that is commercially available for testing HIV antibodies in saliva and is based on a similar test that has been reported for testing anti simian immunodefiency virus antibodies in monkeys (Israel et a11993). While this is marketed in the United States for convenience use, the approach may be very useful in developing nations, where AIDS has become a severe problem and serum collection and testing is far too expensive for the large numbers of people that require testing. The reliability of such testing remains to be verified using large numbers of samples.

Serological d i a g n o s i s

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Confi rmatory testi ng

The need for confirmatory testing of serum antibodies is most notable for HIV. This is necessitated by the nature of the infection and the primary test used to detect antibody. Because there is a desire to eliminate false negative results, the HIV test is performed so that sensitivity is very high at the cost of decreased specificity. This results in a relatively high false positive rate, necessitating a confirmatory test. In the case of HIV, the primary test is most often performed using ELISA technology. Early tests had a false positive rate as high as 10% (Veronese et a11992), although later generation tests have reduced this significantly (<1% and for some tests below 0.1%). The most common confirmatory tests are performed using immunoblotting (Western blotting, Chapters 7 & 12) (Veronese et al 1992). The advantages of this latter test is that it is highly specific since a pattern of bands denoting HIV antigens is detected, indicating antibodies to several antigens. Alternative to the Western blot, confirmation may be performed using ELISA or immune fluorescence systems based on different principles than the initial testing. For example, viral lysates, recombinant antigens or peptide antigens can be used for confirmation of positivity. Other testing that requires confirmation generally results from the use of high sensitivity, lowered specificity testing. since virus is usually eliminated from the blood prior to the onset of overt disease.

Monitoring infection
Routine testing of serum for viral antigens is limited mainly to HBV and HIV infections. We have discussed above (page 346) the monitoring of HBV infection by antigen detection in concert with detection of the antibody to those antigens. The detection of the HBV antigens has been used more extensively than any other serum antigen detection. More recently, the detection of HIV p24 antigen has been used to monitor the progression of HIV from clinical latency through the development of overt AIDS (Fig. 17.3). While the detection of this antigen per se is not an indicator of the stage of development of HIV, when used in conjunction with other markers it can be used prognostically (Phillips et al 1991; Henrard et al 1992). Generally, in latent infection, there is little p24 present in blood. When virus replication increases and immune deficiency begins to appear p24 levels increase. This is usually associated with a decline in the number of CD4+ lymphocytes in circulation and these two indicators are prognostic for progressing disease. Additionally, the presence of p24 in a newborn from an HIV infected mother is diagnostic for HIV infection during pregnancy (Amirhessami-Aghili and Spector 1991). It must be noted that a significant proportion of HIV infected individuals may be p24 antigenemia negative during much of their infection, with negative results observed in 30% or more of HIV infected individuals in developed nations and higher percentages of negative samples seen from blood of African patients.

Antigen testing
The detection of viral antigen in serum may be used for blood-borne diseases in which there is a substantial period of infection of the blood, to monitor the presence of infection and the progress of that infection, and for the evaluation of drug therapy of such infections. Viruses for which this can be readily performed include HBV, HIV, and HTLV. Antigen testing of blood specimens is not particularly useful for viral infections in which there is a transient viremia, 348

C h a p t e r 17

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Evaluation of drug therapy

The detection of viral antigen to monitor drug therapy is again mainly limited to HBV, HCV and H IV. In the case of the hepatitis viruses the levels of HBsAg and HBeAg are measured following treatment of chronic HBV with interferon alpha (Davis and Hoofnagle 1986). These antigens are usually tested along with the antibodies to the same antigens, as well as liver enzyme levels and HBV DNA polymerase levels. For HCV testing, measurement of antibodies or, more sensitively, the presence of virus genome by PCR, is used to follow the efficacy of interferon therapy for chronic infection (Davis 1994). Therapy of HIV with azidothymidine (AZT) or other drugs is followed by the monitoring of p24 levels in conjunction with measuring the number of CD4+ lymphocytes in peripheral blood (Japour et al 1993). This method has been very useful for documenting efficacy of therapy. It must be noted that technology such as the development of quantitative PCR has diminished some testing for antigen since the former provides a more precise assessment of the amount of virus present (Persing et al 1993). This is especially true with techniques that can assess quantitatively the amounts of cell associated versus cell free virus in blood. Serological diagnosis

Use of commercial kits

The serologic assessment of antibodies and antigens using commercially prepared and marketed kits is commonplace. These kits are available for most of the common viruses and are highly reputable for the most part. They use ELISA, radioimmunoassay, immunofluorescence, immunobiotting, latex agglutination, hemagglutination and a variety of other methods for detection. The quality of such kits has improved over the past decade due to the availability of monoclonal antibodies, recombinant technologies and the automation of some testing procedures. The use of these kits in lieu of reagents developed in-house is encouraged because it adds to the standardization of testing and results in reduced time technologists are required to put in developing and testing reagents. With this in mind however, it is essential for the laboratory to have a thorough quality assurance/quality control program to insure that reagents are of the highest quality and are properly used. The list of available tests is extensive and constantly changing so it is of little value to list them here. Linscott's Directory provides information on available reagents and tests (Linscott 1992). 349

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Summary
The use of serology to detect viruses remains a vital adjunct to virus isolation and direct detection of virus for the diagnosis of infections. The wide variety of immunoassays is delineated in Chapter 7. This chapter has not dealt with the issue of which assay is best for each virus, since this is often a matter of pre-

ference and changes as new assays are introduced. The use of serology requires excellent quality assurance and control and when combined with other viral detection techniques provides the laboratory with the ability to assist the physician in the identification of etiologic agents and to monitor the progression of infection and the success of therapeutic intervention.

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References

References
Amirhessami-Aghili N, Spector SA (1991) J Virol 65: 2231-2236. Cremer NE (1985) In: Laboratory Diagnosis of Viral infections, Lennette EH (Ed.) Marcel Dekker, New York, pp. 73-85. Davis GL (1994) Am J Med 96: 41S-46S. Davis GL, Hoofnagle JH (1986) Hepatology 6: 10381041. Doerr HW, Gross G, Schmitz H (1976) Med Microbiol Immunol 162: 183-192. Escobar MR (1992) In: Clinical Virology Manual 2nd Ed. Specter S, Lancz G (Eds.) Elsevier Science, New York, pp. 397-424. Henrard DR, Mehaffey WF, Allain J-P (1992) AIDS Res Human Retroviruses 8: 47-52. Herrmann KL, Erdman DD (1992)In: Clinical Virology Manual 2nd Ed. Specter S, Lancz G (Eds.) Elsevier Science, New York, pp. 263-273. Israel ZR, Dean GA, Maul DH, O'Neil SP, Dreitz Mj, Mullins JI, Fultz PN, Hoover EA (1993) AIDS Res Human Retroviruses 9: 277-286. Japour AJ, Mayers DL, Johnson VA, Kuritzkes DR, Beckett LA, Arduino J-M, Lane J, Black RJ, Reichelderfer PS, D'Aquila RT, Crumpacker CS, The RV-43 Study Group and The AIDS Trials Clinical Group Virology Committee Resistance Working Group (1993) Antimicrob Agents Chemother 37: 1095-1101. Kleiman MB (1985) In: Laboratory Diagnosis of Viral Infections, Lennette EH (Ed.) Marcel Dekker, New York, pp. 369-384. Lennette ET (1985) In: Laboratory Diagnosis of Viral Infections, Lennette EH (Ed.) Marcel Dekker, New York, pp. 257-271. Lincott's Directory of Immunological and Biological Reagents, 7th Ed. 1992. Santa Rosa, CA p. 237. Lopez C, Arvin AM, Ashley R (1993)In: The Human Herpesvirus, Roizman B, Whitley Rj, Lopez C (Eds.) Raven Press, New York, pp. 397-425. Paar D, Straus SE (1992) In: Clinical Virology Manual 2nd Ed. Specter S, Lancz G (Eds.) Elsevier Science, New York, pp. 501-526. Pass RF, Griffiths PD, August AM (1983) J Infect Dis 147: 40-46. Persing DH, Smith TF, Tenover FC, White TJ (Eds.) (1993) Diagnostic Molecular Microbiology: Principles and Applications. ASM Press, Washington, DC, p. 641. Phillips AN, Lee CA, Elford J e t al (1991) AIDS 5: 1217-1222. Veronese EdiM, Lusso F, SchLipbach J, Gallo RC (1992) In: Clinical Virology Manual 2nd Ed. Specter S, Lancz G (Eds.) Elsevier Science, New York, pp. 585-625. Ziola B, Salmi A, Panelius M, Halonen P (1979) Clin Immunol immunopathol 13: 462-474.

Serological diagnosis

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