17 - serological diagnosis

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SerologicaldiagnosisS. SpecterD. JeffriesThe introduct ion of rapid diagnost ic tech-niques to detect viruses or viral antigens hasvastly improved the isolat ion and/or identi f ica-t ion of et iologic agents in viral diseases; how-ever, there are st i l l many instances for whichserologic diagnosis is necessary. Agents thatdo not repl icate well in cel l culture or for whichthere are as yet no good reagents for directdetect ion of vi rus are rout inely diagnosedusing measurement of ant ibody. Rout ine test-ing for ARBOviruses, Epstein-Barr virus (EBV),hepatitis B virus (HBV), hepatitis C virus (HCV),human immu node f iciency vi rus (HIV), human Tlym pho tropic viruses (HTLV) and in some cir-cumstances cytomegalovirus (CMV), herpessimplex virus (HSV), varicel la-zoster virus(VZV), measles and rubella is performed byserologic techniques.

Serologic test ing is per formed both forscreening of suscept ible populat ions and forthe diagnosis of acute or chronic diseases.The use of serologic test ing in these ci rcum-stances is del ineated in this chapter.

Principles for serologicdiagnosis and forscreeningThe detect ion of ant ibodies as a measure ofinfection by a part icular virus may be used inone of several ways for diagnosis. One maymeasure the presence of antibody as: (1) anindicat ion of immune status, fol lowing naturalinfection or vaccination; (2) seroconversionVirology Methods ManualISBN 0 -12-465330-8

(i.e. appearance of antibody) or an increase int i ter as an indicator of recent infection; (3) apart icular class of immunoglobul in to deter-mine the nature of the infection; (4) responsesto var ious ant igens associated wi th the samevirus to indicate disease progression; (5) acombinat ion of ant ibody and ant igen levelsalso to moni tor disease state or progression,and (6)levels in dif ferent body f luids, such asserum versus CSF, to determine involvementof possible site of infection, such as CNS.

Qualitative assessment ofimmune statusThe qual i tat ive measurement of ant ibody pre-sence or absence is a useful measure ofimmune status relat ive to a part icular virus.This can confirm either exposure to a naturalinfection or successful vaccination of an indi-vidual . Immune status is extremely importantin ci rcumstances for which secondary expo-sure to a virus may have very dif ferent conse-quences than pr imary exposure. For apregnant female, evidence of pr ior CMV infec-t ion may have devastat ing consequences forthe fetus, whereas a reactivated latent infec-t ion is less l ikely to have pathologic conse-quences for the fetus. Similar ly, screeningtests for antibodies to rubella virus in preg-nant women indicate that the mother wi l l notl ikely get rubella i f she is antibody posit ive andthus the fetus is also protected. In rareinstances, reinfection with rubella has beenrecorded but this is an insignif icant problem.

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V i r o l o g y m e t h o d s m a n u a lScreening tests are also important for a

variety of other medical circumstances. Trans-plant donors and recipients must be screenedfor CMV immune status, and organs f rom CMVposit ive donors should not be used in CMVnegative recipients, i f this can be avoided, asthere is a high l ikel ihood of transmitt ing infec-t ion. Blood products are routinely screened forseveral viruses that are blood borne patho-gens, including CMV, H BV , HC V, HIV andHTLV. This pra ctice has severely reduce d theincidence of disease caused by these virusesthat is attr ibuted to transfusion.

It must be noted here that quali tat iveassessment of antibody is of l imited value,merely indicating exposure to a part icularvirus. Although in many cases the presenceof ant ibody can be equated wi th protect iveimmunity, diagnosis of acute infectionrequires a quantitat ive analysis of antibody.

Quantitative analysis ofantibodyThe detection of antibodies in serum is notalways diagnostic for an acute infection,because it is not possible to determine quali-tat ively whether these antibodies are due to acurrent, recent or past infection. Since severalvi ruses can cause simi lar symptoms the pre-sence of antibody in conjunction with cl inicalevidence of disease st i l l is not suff icient fordiagnosis. Thus, i t is necessary to measurevirus-specif ic antibody t i ters in acute and con-valescent sera to determine the cause of anacute infection. Acute sera should be col lectedas soon as possible after onset of infectionand convalescent serum should be col lected2-3 weeks later. A two tube (usually four-foldusing two-fold di lut ions) r ise in t i ter is indica-t ive of a signif icant change in antibody t i terdenoting a current infection. The disadvan-tage to this method is that i t takes a consider-able amount of t ime for antibodies to r ise andhence for a meaningful result to be obtained;thus uti l i ty for diagnosing and, i f necessary,treating an acute infection is severely ham-pered. An alternative to using the titer rise is

to examine an acute serum for indicators ofacute infection.

Immunoglobulin classassessmentIgM determinationThe determinat ion of immuno globul in ( Ig) Mlevels in a serum is often useful for rapid iden-t i f icat ion of acute, pr imary infection. Numerousmethods have been devised to detect IgM andthese are welt described (Hermann andErdman 1992) (see chapter 7). More impor-tantly the l imitat ions of this approach mustbe noted. Considerable care must be taken inmeasuring IgM levels to a part icular virus to becertain that the results are not confused by thepresence of rheumatoid factor in the serum(Ziola et al 1979). A variety of strategies havebeen devised to avoid this problem and arealso described by Herrmann and Erdman(1992) (see chapter 7).

While IgM general ly is diagnostic of pr imaryinfection this is not always so. With certainherpesviruses, part icularly CM V and HSV, IgMlevels may r ise with reactivation of infection(Pass et al 1983; Lopez et al 1993). Thus,detection of IgM is not necessari ly indicativeof primary infection with these viruses and isonly useful in this regard when IgM can bemeasured in the absence of signif icantamounts of IgG.

Immunoglobulin AigA is important as an antibody in f luids asso-ciated with mucosal surfaces, since i t is morestable than other Ig as secretory IgA in secre-t ions such as sal iva, nasal secretions, gastro-intestinal secretions, etc. (Cremer 1985). Thus,measurement of IgA levels in such secretionsmay be an important and useful measurementof protective immunity against respiratory andenteric viral infections. It also is important inthe assessment of generat ion of protect iveimmunity on mucosal sur faces. By contrast

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Assessment of antibodies to different antigensIgA is less important in systemic immunitysince it is usually minimal compared to IgMand IgG responses, in fact, measurement ofIgA in serum can often be obscured by highaffinity IgG (Cremer 1985). Thus, IgA is notroutinely measured in the diagnostic labora-tory but is used only in special circumstances.One such circumstance is the measurementof IgA antibodies against the viral capsid anti-gen (IgA-VCA) of EBV . IgA is cons idered diag-nostic for nasopharyngeal carcinoma (NPC)that is EBV induc ed (Lennette 1 985 ). In suchcases, IgA-VCA titers can be used prognosti-cally in fol lowing the tumo r burden due to NPCand to monitor the effectiveness of treatment.

Assessment of antibodiesto different antigensThe onset of antibody production to variousantigens of a particular virus can occursequential ly and quantitative testing for thesedifferent antibodies using one serum specimencan be diagnostic of the state of infection (Fig.17.1). For infectious mononucleosis due to

EBV, antibody responses are first detectedagainst the VCA, fol lowed by the early antigen(EA) then the Epstein-Barr nuclear antigen(EBNA) (Paar and Strauss 1992 ). Thus, highVCA and EA titers in the absence of EBNAwould be indicative of a current acute infec-t ion, whereas a high EBNA titer along with highVCA but low EA would suggest a past EBVinfection. When combined with comparingIgM-VCA (higher early in infection) to IgG-VCA (higher later in infection) titers more spe-cif ic information can be gained as to how earlyin infection the sample was collected.

It should be noted that while these t i tersprovide some very specif ic information aboutEBV infection, there is a very simple test forinfectious mononucleosis. This test involvesdetection of the PauI-Bunneil heterophile anti-body. This antibody is a common anti-redblood cell antibody that is induced in humansby EBV infection (Lennette 1985). When prop-erly controlled, this test is a far less expensiveway of diagnosing EBV induced infectiousmononuclesosis than the specif ic anti-EBVantibody tests. Heterophile testing that givesa negative result may be indicative of CMV

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Virology methods manualinduced mon onuc leosis or may be a fa lsenegat ive. This can be conf i rmed by speci f ictest ing for ant ibodies to CMV or EBV.

For the d iagnosis of mum ps infect ion thecombinat ion of ant ibodies to S and V ant igenscan be used in a similar manner to indicatecurrent versus past in fect ion wi th th is v i rus(Kleiman 1985). Anti-S antibody r ises f irst andpersists for only 2-3 months, whereas ant i -Vrises later and persists for a signif icantly longert ime per iod.

t ion is prognost ic for d isease progression. Thepresence of HBeAg indicates that a pat ient hasinfect ious v i rus in their b lood and is associatedwith a poor prognosis, whi le the presence ofant i -HBe suggests that a pat ient is recover ing.The conversion f rom HBsAg posi t ive to ant i -HBs posi t ive a long wi th ant i -HBe denotescomplete recovery f rom HBV infect ion. Thus,by using these var ious markers one candiagnose HBV infect ion and can ascer ta in aprognosis for the d isease.

Assessment of antibodyand antigen combinations Detection of antibody influids other than serumHepat i t is B v i rus d iagnosis is dependent uponthe detect ion of a combinat ion of v i ra l ant igensand 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 thistest ing as are the ant ibodies to these ant igens(Escobar 1992) . The presence of hepat i t is Bsur face (HB sA g) i s indicat ive of act ive in fec-t ion and is used to screen for HBV. The detec-t ion of ant i -HBs is indicat ive of recovery f romHBV infect ion, whi le presence of HBc in theabsence of e i ther HBsAg or ant i -HBs ( thecore 'w i nd ow ' ) in d ic a te s a late acu te in fec-t ion. The use of HBeAg and ant i -HBe detec-

Cerebrospinal fluidThe measurement of ant ibodies in the CSFmay be usefu l in the d iagnosis of CNS dis-ease. Ant ibodies may be present in h ighlevels in the CSF as a result of CNS infectionand an act ive immune response or because ofleakage of serum into the CSF due to break-down of the b lood-bra in barr ier (BBB). The twosi tuat ions can be d ist inguished by calculat ingCSF/serum rat ios of a lbumin and IgG (Cremer1985). The normal levels for albumin and IgGare 200- fo ld and 500- fo ld lower, respect ive ly in

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F i g u r e 17 .2. Antigen and antibody responses in hepatitis B infection.346 Chapter 17


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D e t e c t i o n o f a n t i b o d y in f l u id s o t h e r t h a n s e r u mCSF. If there is antibody synthesis in the CSF,then only the IgG ratio changes but if there isBBB damage then both the IgG and albuminratios between CSF and serum wil l change.

The presence of increased levels of Ig in theCSF is indicative of local antibody synthesisand therefore local viral infection. For the mostpart this is an IgG response; however, in HSVencephalit is and chronic measles leading tosubacute sclerosing panencephalit is IgMclass antibody may also be detected (Doerret al 1976). Additionally, one may compareratios of antibo dy in serum versus CSF, suchthat low ratios, e.g. 8-32, are of diagnosticimportance for CNS infection.

SalivaThe detection of antibodies in saliva is not acommon laboratory practice. Nevertheless thissource of antibody is being examined in cir-cumstances where drawing blood for serum isimpractical for reasons of both convenienceand cost. There is currently a test kit that iscommercial ly available for testing HIV antibo-dies in saliva and is based on a similar test thathas been reported for testing anti simianimmunodefiency virus antibodies in monkeys(Israel et a11993). While this is marketed in theUnited States for convenience use, theapproach may be very useful in developingnations, where AIDS has become a severeproblem and serum collection and testing isfar too expensive for the large numbers ofpeople that require testing. The reliability ofsuch testing remains to be verified using largenumbers of samples.

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V i r o lo g y m e t h o d s m a n u a l

Confi rmatory testi ngThe need for confirmatory testing of serumantibodies is most notable for HIV. This isnecessitated by the nature of the infectionand the primary test used to detect antibody.Because there is a desire to eliminate falsenegative results, the HIV test is performed sothat sensit ivity is very high at the cost ofdecreased specificity. This results in a rela-tively high false positive rate, necessitating aconfirm atory test. In the case of HIV, the pri-mary test is most often performed using ELISAtechnology. Early tests had a false posit ive rateas high as 10% (Veronese et a11992), althoughlater generation tests have reduced this signifi-cantly (


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Figure 1 7.3 . Antig en and antibody responses during the cou rse of HIV infection.Evaluat ion of drug therapyThe detection of viral antigen to monitor drugtherapy is again m ainly l imited to H BV, HCVand H IV. In the case of the hepa titis virusesthe levels of HBsAg and HBeAg are measuredfollowing treatment of chronic HBV with inter-feron alpha (Davis and H oofnagle 1986). Theseantigens are usually tested along with the anti-bodies to the same antigens, as well as l iverenzyme levels and HBV DNA polymeraselevels. For HCV testing, measurement of anti-bodies or, more sensitively, the presence ofvirus genome by PCR, is used to fol low theeff icacy of interferon therapy for chronic infec-tion (Davis 1994).

Therapy of HIV with a zidothym idine (AZT) orother drugs is fol lowed by the monitoring ofp24 levels in conjunction with measuring thenumber of CD4+ lymphocytes in peripheralblood (Japour et al 1993). This method hasbeen very useful for documenting eff icacy oftherapy. It must be noted that technology suchas the development of quantitative PCR hasdiminished some testing for antigen since theformer provides a more precise assessment ofthe amount of virus present (Persing et al1993). This is especially true with techniquesthat can assess quantitatively the amounts ofcell associated versus cell free virus in blood.

Use of commercial kitsThe serologic assessment of antibodies andantigens using commercial ly prepared andmarketed kits is commonplace. These kitsare available for most of the common virusesand are highly reputable for the most part.They use ELISA, radioimmunoassay, immuno-fluorescence, immunobiott ing, latex agglutina-tion, hemagglutination and a variety of othermethods for detection. The quality of such kitshas improved over the past decade due to theavailabil i ty of monoclonal antibodies, recombi-nant technologies and the automation of sometesting procedures.

The use of these kits in lieu of reagentsdeveloped in-house is encouraged because itadds to the standardization of testing andresults in reduced time technologists arerequired to put in developing and testingreagents. With this in mind however, it isessential for the laboratory to have a thoroughquality assurance/quality control program toinsure that reagents are of the highest qualityand are properly used.

The list of available tests is extensive andconstantly changing so it is of little value to listthem here. Linscott 's Directory provides infor-mation on available reagents and tests (Lin-scott 1992).

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Virology methods manualSummaryThe use of serology to detect viruses remainsa vi tal adjunct to virus isolation and directdetection of virus for the diagnosis of infec-t ions. The wide var iety of immunoassays isdel ineated in Chapter 7. This chapter has notdealt with the issue of which assay is best foreach virus, since this is often a matter of pre-

ference and changes as new assays are intro-duced. The use of serology requires excel lentqual i ty assurance and contro l and when com-bined with other viral detection techniquesprovides the laboratory with the abi l i ty toassist the physician in the identi f ication ofet io logic agents and to moni tor the progres-sion of infect ion and the success of therapeu-tic intervention.

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References

ReferencesAmirhessam i-Aghili N, Spector SA (1991) J Virol 65:

2231-2236.Cremer NE (1985) In: Labo ratory Diagnosis of Viralinfections, Lennette EH (Ed.) Marcel Dekker, NewYork, pp. 73-85.Davis GL (1994) Am J Med 96: 41S -46S.Davis GL, Hoofnagle JH (1986) Hepato logy 6: 103 8-1041.Doerr HW, Gross G, Schmitz H (1976) Med MicrobiolImmunol 162: 183-192.Escobar MR (1992 ) In: Clinical V irology Manual 2ndEd. Spec ter S, La ncz G (Eds .) Elsevier Science,New York, pp. 3 97-424.Hen rard DR, Mehaffey WF, Allain J-P (1992 ) AIDSRes Human Retroviruses 8: 47-52.Herrmann KL, Erdman DD (1992)In: Clinical VirologyManual 2nd Ed. Specter S, Lancz G (Eds.) ElsevierScience, New York, pp. 263-273.Israel ZR, D ean GA, Maul DH, O'Neil SP, Dreitz M j,Mullins JI, Fultz PN, Hoover EA (1993) AIDS ResHuman Retroviruses 9: 277-286.Japour AJ, Mayers DL, Johnson VA, Kuritzkes DR,Becke tt LA, Arduino J-M, Lan e J, Black RJ,Reichelderfer PS, D'Aquila RT, Crumpacker CS,The R V- 43 Study Group and The AIDS TrialsClinical Group Virology Committee ResistanceWorking Group (1 99 3) Antimicrob AgentsChemother 37: 1095-1101.

Kleiman MB (1985) In: Laboratory Diagnosis of ViralInfections, Lennette EH (Ed.) Marcel D ekker, NewYork, pp. 369-384.Lennette ET (1985) In: Laboratory Diagnosis of ViralInfections, Lennette EH (Ed.) Marcel Dekker, NewYork, pp. 257-271.Lincott's Directory of Immunological and BiologicalReagents, 7th Ed. 199 2. Santa R osa, CA p. 237.Lopez C, Arvin AM, Ashley R (1993)In: The HumanHerpesvirus, Roizman B, Whitley Rj, Lopez C(Eds.) Raven Press, New York, pp. 397-42 5.Paar D, Straus SE (1992) In: Clinical V irology Manual2nd Ed. Specter S, Lancz G (Ed s.) ElsevierScience, New Yo rk, pp. 501-526 .

Pass RF, Griffiths PD, August AM (1983) J Infect Dis147: 40-46.Persing DH, Smith TF, Tenover FC, White TJ (Eds.)(1993) Diagnostic Molecular Microbiology: Prin-ciples and Applications. A SM Press, Washington,DC, p. 641.Phillips AN, Lee CA, Elford J e t al (1991) AIDS 5:1217-1222.Veronese EdiM, Lusso F, SchL ipbach J, Gallo RC(1992) In: Clinical Virology Manual 2nd Ed. Spec-ter S, Lancz G (Eds.) Elsevier Science, New York,pp. 585-625.Ziola B, Salmi A, Panelius M , Halonen P (1979) ClinImmunol immunopathol 13: 462-474.

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17 - serological diagnosis

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