med laboratory techniques in the investigation of...

Genitourin Med 1992;68:55-59

Laboratory techniques in the investigation oftoxoplasmosis

K F Barker, R E Holliman

IntroductionInfection by the protozoan Toxoplasma gondiiis one of the most common parasitic infectionsof warm blooded animals including man. Thedefinitive host is the cat in which the sexuallife cycle takes place. Human infection mayarise in utero or be acquired by ingestingtissue cysts in undercooked meat or by inges-tion of oocysts in soil or via contaminatedfoods. Seroconversion increases with age andvaries according to geographical area and eat-ing habit. Reported prevalence rates, as deter-mined from the presence of serum antibodies,are 20-40% in Great Britain, 50-60% in theUSA and 80-90% in France.`2Acute toxoplasma infection in the immuno-

competent patient is often asymptomatic butmay cause lymphadenopathy or a glandularfever-like illness. Congenital infection maycause foetal death or result in cerebral damageand retinochoroiditis in severely affected cases.Toxoplasmosis is now well recognised as animportant opportunistic infection of theimmunocompromised. Cancer patients,organ-transplant recipients and patients withthe acquired immune deficiency syndrome(AIDS) are all at risk of severe, sometimesfatal, toxoplasmosis. The infection in thesepatients has a predilection for the centralnervous system (CNS).3 Since the recognitionof AIDS, toxoplasmic encephalitis hasbecome one of the most common causes ofencephalitis in the USA.45 It is the mostcommon cause of intracerebral mass lesions inpatients with AIDS and is possibly the mostcommon opportunistic infection of the CNS.CNS toxoplasmosis is due principally to

reactivation of endogenous infection acquiredin the past and the risk of an AIDS patientwith positive toxoplasma serological testsdeveloping CNS infection has been estimatedat 30%.6 Published estimates of the incidenceof CNS toxoplasmosis in AIDS patients varyenormously and probably reflect prevalence ofparasite infection in different populations.Involvement of other organs is rare but pul-monary toxoplasmosis is said to occur in 1%of AIDS patients7 and a diffuse retino-choroiditis8 has been recorded.The early diagnosis of CNS toxoplasmosis

in AIDS patients requires a high index ofsuspicion. The patient may present with focalor generalised neurological abnormalities.Computed tomography (CT) is extremelyuseful for investigating suspected CNS toxo-plasmosis. Lesions, single or multiple, andisodense or hypodense are usually seen in the

cerebral hemispheres often with surroundingoedema and mass effect (fig). Contrast studiesreveal either ring or nodular enhancement inmost cases. Magnetic resonance imaging, ifavailable, may detect lesions not demonstratedby CT. Most often a diagnosis is made inAIDS patients on the basis of compatibleclinical, radiological and serological findingswhich is only confirmed after a clinical andradiological response to anti-parasite therapy.This article reviews the laboratory techniquesavailable for investigating toxoplasmosis withparticular reference to patients with AIDS.These techniques include serology, histology,culture and specific nucleic acid detection byDNA probe and the polymerase chain reac-tion (PCR). Clinical and radiological findingswill not be discussed further.

Serological techniquesDetection of toxoplasma specific antibody inserum is the investigation of choice in theimmunocompetent patient. Rising IgG titresand demonstrable IgM may be seen in cases ofacute toxoplasmosis but often the diagnosis isnot considered until late in the illness and it isthe persistence of specific IgG which confirmstoxoplasma exposure at some time. In themajority of cases toxoplasmosis in AIDS isassociated with a secondary reactivation of a

r.Bg lyp:cal uT appearance o0 lesions in CNStoxoplasmosis.

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Public HealthLaboratory Service,ToxoplasmaReference Laboratory,St George's Hospital,Blackshaw Road,London, SW17 OQT,UKK F BarkerR E HollimanAddress correspondence to:Dr K F BarkerAccepted for publication10 October 1991

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chronic, previously latent, infection. Primaryinfections with seroconversion, rising titresand specific IgM production are thought to berare, although in a recent study in this country10 of 20 documented cases of CNS toxoplas-mosis did have demonstrable IgM by one ormore assay.9 However, the absence of specificIgG antibody in the peripheral blood of AIDSpatients excludes the diagnosis of CNS toxo-plasmosis.6Antibody tests are of two major types

depending on the antigen source used. Assaysincorporating whole organisms include theSabin and Feldman dye test, direct agglutina-tion test and immunosorbent agglutinationassay. These predominantly detect antibodiesto membrane antigens and are most reactivein the early stages of infection. Cytoplasmicantigens are exposed by disrupting thetrophozoites and assays using this antigensource (complement fixation test, enzyme lin-ked immunosorbent assay, latex agglutinationand haemagglutination tests) are slower tobecome positive in the course of the infectionbut remain reactive for a greater length oftime. Antibody levels do not correlate withseverity of illness. Whilst many laboratoriesperform a latex agglutination test on serumsamples, confirmation of IgG and IgMpositivity and the investigation of theimmunocompromised and acute infectionduring pregnancy is best performed byreference centres. Assays commonly used bysuch laboratories will be discussed further.

The Sabin and Feldman dye test.'0This is the accepted reference assay for detec-ting toxoplasma specific antibody. The dyetest uses live, virulent trophozoites and istherefore not performed outside of referencelaboratories. Bound antibody fixes com-plement which then damages the parasite cellwall preventing the organism from retainingthe vital stain alkaline methylene blue. Titresare expressed in international units as com-pared with a reference serum. Unlike otherassays, the dye test detects both complementfixing IgG and IgM. Although not easy toperform the dye test is both highly sensitiveand specific. Patients become dye test positivewithin 1-2 weeks of exposure, reaching a peakby 8 weeks. Low titres commonly persist forlife.

Direct agglutination assayFirst described by Fulton and Turk," andinvolving the reaction of specific antibody withformalin fixed trophozoites to form a visibleagglutination pattern, this assay is still widelyused. Sensitivity and specificity have beenimproved by pre-treatment of trophozoiteswith trypsin to expose additional antigens andremoval of non-specific IgM respectively.Using the dye test findings as a reference onerecent study found the sensitivity andspecificity of the direct agglutination assay tobe 96% and 98% respectively.'2Having established the presence of toxo-

plasma specific antibody in the peripheralblood, thereby identifying those AIDS

patients at risk of reactivation, diagnosingthose with reactivated cerebral infection byserological means alone is difficult. It has beensuggested that calculating direct agglutinationto dye test titre ratios may be of value. In onestudy 12 of 16 patients with AIDS and CNStoxoplasmosis had ratios greater than 20,whilst in normal individuals with comparabledye test titres the ratio was less than 10.`3 Asubsequent report suggests a ratio > 5 isindicative of CNS toxoplasmosis althoughlower values do not rule out the diagnosis.'4The agglutination test may also be performedusing acetone rather than formalin fixed tro-phozoites. Titres in the two agglutinationreactions have been compared and when usedtogether had a sensitivity and specificity of70% and 93% respectively for the diagnosisof CNS toxoplasmosis in a group of 43biopsy-proven cases.`

Latex agglutination testsThe latex agglutination test is widely used inscreening for toxoplasmosis. Antigens derivedfrom disrupted trophozoites are fixed to a latexbead and when serum containing specificantibody is added visible agglutinationoccurs.'6 This test will not detect antibody forseveral weeks into the course of an infectionreflecting the predominantly cytoplasmicantigen source.

False positive reactions do occur with thelatex agglutination test when compared withdye test findings. In one analysis of 4450 serathere were 59 (1.3%) such discrepant latexagglutination test results.'7 These falsepositives were associated with an unspecifiedIgM antibody but not with CMV infection orhepatitis B virus "e" antigen as suggested byother workers.'8 19 False negative reactions areuncommon. One study found the sensitivityand specificity of the latex agglutination test tobe 99% and 81% respectively when using thedye test as a standard reference.'2

Whilst there is no evidence that false positiveand negative reactions are more common in theAIDS patient, misleading results have beenreported in heart transplant recipients.'8 It isadvisable to investigate the immunocom-promised by additional serological tests.

Enzyme linked immunosorbent assay (ELISA)ELISA technology is becoming increasinglypopular as a means ofmicrobiological diagnosisand a number of such assays are available on acommercial basis for detection of toxoplasmaspecific antibodies. The sensitivity of conven-tional ELISA methods can be improved by theuse of double sandwich assays (DS-ELISA).Such an assay is used for detecting toxoplasmaspecific IgM antibody.20 Class specific anti-human immunoglobulin is absorbed onto asolid phase to "capture" the IgM. Test serumis added and the IgM content is bound to thesolid phase. Antigen (disrupted trophozoite) isadded and the antigen bound to specific IgM isthen detected by an enzyme-conjugated specificantibody. False positive IgM reactionsassociated with rheumatoid factor, and false

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Laboratory techniques in the investigation of toxoplasmosis

negative results due to an excess of IgG in testsera are reduced in the DS-ELISA system.2`By measuring IgM levels in cases of acutetoxoplasmosis with known date of onset DS-ELISA reactivity has been shown, on average,to peak at 2 months and to persist for about 6months.20Toxoplasma specific IgG and IgA can also be

detected using these techniques. Specific IgAantibodies against P30, a major surface proteinof Toxoplasma gondii, may be useful markers inacute and congenital infections disappearingfrom the circulation earlier in the course ofinfection than anti-P30 IgM. The diagnosticvalue of specific IgA antibodies in AIDSpatients requires further evaluation but onlyone patient of a group of twenty individualswith biopsy-proven toxoplasmosis associatedwith AIDS had specific IgA demonstrable byELISA.2'

Immunosorbent agglutination assay (ISAGA)This assay detects human IgM antibodies totoxoplasma by an initial IgM capture using amonoclonal antibody to the CH2 domain of thehuman p-chain and subsequent agglutinationof whole toxoplasma trophozoites. Routinelyused in France for the diagnosis of acute andcongenital toxoplasmosis the ISAGA has nowbeen evaluated and introduced into certainlaboratories in this country. The ISAGA hasbeen found to be significantly more sensitivethan ELISA methods for detecting specificIgM, with comparable specificity.24 There is nodefinitive reference assay with which IgM testscan be compared.

Unlike ELISA reactivity, which rarelyremains positive more than 6 months afteracute exposure, the ISAGA will detect specificIgM for a longer period of time. In one studyreactivity persisted in 80% of patients for 12months after initial infection.25When investigating samples of serum from

AIDS patients with possible CNS toxoplas-mosis specific IgM should always be looked for.Primary infections are not usual in this group,but some cases of cerebral reactivation may beaccompanied by low levels of specific IgMantibodies. ISAGA being more sensitive thanDS-ELISA may be the assay of choice in thissituation.'

Antigen detection testsExperimental assays using ELISA and latexagglutination methodology to detect circulat-ing antigen in acute infections of animals andboth primary and reactivated infection ofhumans have been described.2627 However, theusual duration of parasitaemia following acuteexposure is brief, and the diagnostic value ofthese tests on serum and other samples remainsto be proven.

Western blottingThis technique involves transfer of proteinsonto a membrane, usually nitrocellulose. Byusing a class specific anti-human immuno-globulin and detection system it is possible toanalyse patterns of toxoplasma antigens onnitrocellulose strips recognised by antibodies

in human serum. Detailed investigations ofadult sera at various stages of infection havebeen performed and their antigenic profilescompared.2`" More work is needed to deter-mine whether this technique is of value in theinvestigation of congenital toxoplasmosis.'2 Tothe authors' knowledge little information hasbeen gained by comparing Western blots fromAIDS patients with biopsy-proven CNS toxo-plasmosis with asymptomatic dye test positiveAIDS patients as controls.

Interpretation of Western blots is com-plicated by naturally occurring antibodies innon-immune sera that are reactive with toxo-plasma antigens. The technique is not quan-titative and accurate measurement of antigenmolecular weights can be a problem.

Cerebrospinalfluid (CSF)The diagnostic value of CSF sampling inAIDS patients with possible CNS toxoplas-mosis is uncertain. Lumbar puncture is oftennot performed or contraindicated for clinicalreasons. When a sample is obtained analysisoften reveals a raised protein concentrationwith or without a lymphocytosis. Dye tests canbe performed on specimens of CSF and in onestudy 23 of 37 (62%) patients with AIDS andbiopsy-proven CNS toxoplasmosis had toxo-plasma specific IgG in the CSF compared tonone of 11 patients with AIDS alone." In the20 cases of toxoplasmosis associated withAIDS described by Holliman9 CSF samplingwas only performed on three patients. Twopatients had specific IgG in the CSF but theresult did not contribute to the final diagnosis.Clearly the absence of specific antibody in theCSF does not exclude cerebral infection.Chemical staining, culture and specific

nucleic acid detection can be performed onCSF samples and yield useful information.

HistologyHistological examination of enlarged lymphnodes from immunocompetent patients withacute toxoplasmosis can be useful. Normaltissue architecture is preserved, with follicularhyperplasia and collections of mononuclearcells at the node periphery as characteristicfindings.The use of invasive techniques such as brain

biopsy for diagnosing CNS toxoplasmosis inAIDS patients is controversial. The possibilityofCNS pathology of other infectious and non-infectious aetiology in dye test positive AIDScases cannot be ignored, particularly in certainpopulations of patients. However, the sen-sitivity and specificity of brain biopsy for diag-nosing CNS toxoplasmosis is variable. Areas ofwell demarcated necrosis with surroundinginflammatory infiltrate, vasculitis, oedema andmild astrocytosis have been reported.`4 Cysts,when present, are at the necrotic margins. Inone study trophozoites were not demonstrableby conventional haematoxylin and eosin stain-ing in more than 50% of 48 cases.'4 Immuno-histochemical studies improved the sensitivity.'4Pre-biopsy treatment will alter the histologicalappearance as organisation and chronic abscessformation take place. Open excisional biopsy,

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where possible, is preferable to a CT guidedneedle biopsy.34 Electron microscopy may alsobe used to demonstrate trophozoites in histo-logical specimens from AIDS patients.835

Diagnostic strategies involving brain biopsycan be considered "aggressive" or "con-servative". In the former routine biopsy isperformed in all AIDS patients with compati-ble clinical, serology and CT findings. Alter-natively, and more usually, an empirical trial ofanti-parasite therapy can be given (rather thanbrain biopsy) to these patients, and biopsyreserved for cases with negative serology,atypical CT findings, or when therapy fails.

Isolation of the parasiteToxoplasma gondii can be isolated from biopsyspecimens by intraperitoneal inoculation intoinfection-free mice. Definitive results may notbe available for 6-8 weeks. Inoculation of celllines (such as human embryonic lung) whichare then examined by immunofluorescence mayproduce more rapid results' but are less sen-sitive.37 Regulations in our laboratory do notpermit the injection of samples from AIDSpatients into animals, and using a cell culturetechnique on specimens from two of 20 AIDSpatients who underwent brain biopsy theparasite was isolated in neither case despitepositive histology and specific nucleic aciddetection.9 Cell culture isolation of toxoplasmafrom blood of a patient with AIDS has beenreported38 and in the rare cases of pulmonarytoxoplasmosis in AIDS inoculation of bron-choalveolar lavage into cell culture has beensuccessful.39The problem with these methods is that they

cannot differentiate active infection from thepresence of quiescent tissue cysts. The tech-niques are commonly used in the investigationof foetuses at risk of congenital infection butmay be underutilised, when compared withhistology, in patients with AIDS. Interpreta-tion of culture results in AIDS patients may bea problem when prophylactic regimes involv-ing antimicrobial agents with activity againstToxoplasma gondii have been used.

Molecular techniquesRecombinant DNA technology has led to greatdevelopments in microbiological diagnosis.`Specific nucleic acid detection in clinical sam-ples using DNA probes and a hybridisationreaction provides an altemative to culture andserological tests, and has been used successfullyfor several bacterial, viral and protozoan path-ogens. The smaller the quantity of specificDNA in a clinical specimen that can be detec-ted by a hybridisation reaction the greater thesensitivity of the system. When the number oforganisms in a specimen is small, sensitivitycan be inadequate. Target DNA amplificationby the polymerase chain reaction (PCR)dramatically improves the sensitivity of suchDNA assays. PCR involves repeated,automated cycles of denaturing double stran-ded to single stranded DNA, annealing specificoligonucleotide primers to regions either sideof the target sequence and extension of DNAsynthesis between the primers. This results in a

million fold amplification of a target DNAsequence within a few hours. The PCR productcan then be detected by agarose gel electro-phoresis, transfer of DNA onto membranes(Southern blotting) followed by hybridisationwith specific labelled DNA probes, or usingnew colorimetric methods more suitable formass screening. Signal amplification usingrecombinant RNA molecules that functionboth as specific probe and template for expon-ential amplification by the enzyme Qj3 replicasehas been described.42These molecular techniques have been

applied in the detection of Toxoplasma gondii.Savva4` has reported the construction of agenomic DNA library from T gondii but didnot reveal any repetitive sequences that wouldmake an ideal DNA probe. Using uncharacter-ised clonedDNA fragments and dot hybridisa-tion the minimum amount of Tgondii detectedby these assays corresponded to more than 104trophozoites which may be too insensitive forinfected tissue samples. Recently a PCR assayhas been developed that amplifies part of theP30 gene of T gondii DNA.' This techniquecan detect specific DNA in brains and bodyfluids from mice infected 72 hours previously.The minimum amount of DNA that can bedetected is 0.05 pg possibly corresponding to asingle organism." In certain clinical situationsthese assays may have a complementary role tothe more conventional diagnostic techniques.Foetal blood and products of conception maybe examined to assist in a diagnosis of congeni-tal infection.45 ThePCR can be applied to brainbiopsies from AIDS patients46 but unfortun-ately the demonstration of TgondiiDNA againdoes not help to distinguish active infectionfrom quiescent cysts. An understanding oftoxoplasma stage specific gene expression couldbe of value in developing techniques to distin-guish trophozoite from bradyzoite in the cystform.Although PCR has had an immediate and

dramatic impact on molecular diagnosis it isnot a technique free of problems which maylimit its use in the laboratory. The extra-ordinary sensitivity of the assay may bring intoquestion previous "gold standards" of diag-nosis, contamination and inhibitors can com-plicate matters, and PCR is not essentially aquantitative technique.

ConclusionsExisting laboratory methods for the diagnosisof toxoplasmosis utilise serology, histology,culture and molecular techniques. In theimmunosuppressed and the foetus, where anaccurate and rapid diagnosis is most critical, acombination of all these methods is often mostappropriate. To some extent this reflects thelimitations of the individual techniques.Demonstrating exposure to the organism atsome time does not pose a problem. However,establishing the exact timing of acute exposureand differentiating a reactivated from latentinfection is far more difficult. Addressing theseproblems is critical to the optimal managementof those most at risk from infection. At present

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a diagnosis in an AIDS patient must be made,without delay, on the basis of compatibleclinical, radiological and serological findings.This is confirmed by an early response toantitoxoplasma therapy, often within 48 hoursand rarely requiring more than 10 days ofspecific treatment.

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