Recent developments in the diagnosis of toxoplasmosis

Serodiagn. Immunother. Infect. Disease 1994; 6: 05-16

Guest Editorial

Recent developments in the diagnosis of toxoplasmosis

R E Holliman

PHLS Toxoplasma Reference Unit, St. George’s Hospital, Blackshaw Road, London SW17 OQT, UK

Summary

Conventional diagnostic methods are of limited value when estimating the duration of toxoplasma infection or investigating immunocompromised individuals. Avidity assays are helpful when investigating pregnant women in order to establish the relative risk of congenital infection. Congenital toxoplasmosis can be diagnosed by the presence of specific IgA in infant venous blood. Detection of the parasite using PCR is of clinical value for the investigation of immunocompromised patients including organ graft recipients, the foetus and HIV-infected persons provided the integrity of the sample is assured.

Key words: Toxoplasmosis, avidity, IgA, PCR

Introduction

Human infection with the protozoan parasite Toxoplasma gondii is a cause of significant morbidity and mortality. Infection of the immune competent is commonly asymptomatic or associated with a mild illness but toxoplasmosis in the immune suppressed, such as the foetus, AIDS patient and organ graft recipient, can be life threatening. Conventional diagnostic techniques rely on the cultivation of the parasite in animals and tissue culture or the measurement of specific antibody’. Despite the plethora of serological methods available, a number of diagnostic problems remain, notably the detection of reactivated infection in the immune suppressed, the identification of congenital infection in utero and during the neonatal period, and the precise estimation of the duration of infection of the pregnant woman. Secondary reactivation of previously chronic, quiescent toxoplasma infection has important clinical sequelae in the context of the HIV-infected person2 and ocular disease subsequent to congenital toxoplasmosis3. Following primary infection of a secondary host, including man, T. gondii undergoes asexual reproduction culminating in the

Received: 22 October 1993 Accepfed: 30 October 1993 Correspondence and reprint requests to: RE Holliman, PHLS Toxoplasma Reference Unit, St. George’s Hospital. Blackshaw Road, London SW17 OQT, UK 01994 Butterworth-Heinemann Ltd 0888-0786/94/OlOWS-12

formation of tissue cysts which persist throughout the life of the host. When the host defences are diminished due to HIV infection, malignancy or immune-suppress- ing drugs, breakdown of these tissue cysts release the active tachyzoite form resulting in clinical disease4. In congenital toxoplasmosis the parasite may infect the eye with or without initial retinal damage. Cyst formation in this immune-privileged site may be followed by episodes of reactivated infection and progressive ocular disease in later lifes. Although the patient with ocular toxoplasmosis or AIDS and cerebral toxoplasmosis can be shown to be exposed to the parasite by the presence of specific IgG, the acute episode of reactivation is often not associated with detectable IgM and the diagnosis must be based on clinical features, radiological findings and the response to specific therapy’.

The risk of congenital infection depends on the gestational age at the time of maternal parasitaemia. As the period of maternal parasitaemia following acute infection is usually limited to three weeks or less, infection of the woman prior to conception is rarely transmitted to the foetus although the precise incidence of this phenomenon is not established. As the gestational age advances the risk of acute maternal infection reaching the foetus increases, possibly due to the increasing size of the placenta and associated increased blood flow. Conversely the risk of the infected foetus suffering severe damage, apparent at birth, is greatest in early pregnancy. Consequently accurate estimation of the onset of maternal infection

6 Serodiagn. Immunother. Infect. Disease 1994; 6: No 1

allows the risk of congenital infection and severe neonatal disease to be calculatedh. Most cases of maternal toxoplasmosis remain asymptomatic so that the onset of infection is not clinically apparent. Acute infection can be diagnosed by serial IgG estimation and the detection of seroconversion as in the French programme of routine antenatal screening for toxoplasmosis. However, the harm-benefit ratio of this approach is not established in most countries so that routine antenatal screening cannot be justified’. Sporadic investigation of self-selected individuals, often first tested relatively late in pregnancy results in considerable diagnostic difficulties. The woman with detectable IgG has been exposed to the parasite but what is the duration of infection? The presence of specific IgM indicates infection of relatively recent onset but the persistence of IgM varies between individuals as does the sensitivity of different assays8. Even if the date of maternal infection can be established the crucial factor, the presence or absence of congenital infection, remains unresolved. Investigations in utero include ultrasound examination, which only detects the minority of babies with physical damagey and foetal blood sampling. Cordocentesis carries a risk of foetal mortality of 2% or more, depending on the experience of the opera- tor’“. Measurement of specific IgM in foetal blood has low sensitivity in the diagnosis of congenital infection” and absolute specificity cannot be ensured’?. Isolation of the parasite from the foetal blood cells remains the most sensitive measure of congenital infection but animal inoculation results may be delayed for up to six weeks”. Tissue culture provides a more rapid result but with reduced sensitivity’“. Similarly, detection of specific IgM and isolation of the parasite from blood during the neonatal period are relatively insensitive methods for the diagnosis of congenital infection and the status of the child is most often established following serial IgG estimation during the first year of life14. Compliance during this protracted period of investigation is poor15.

Novel diagnostic techniques have been developed in an attempt to overcome such difficulties and this review will consider three of these: IgG avidity, IgA measurement and the use of the polymerase chain reaction (PCR).

IgG Avidity

Established serological methods identify recent onset of infection by the demonstration of rising levels of IgG and the detection of specific IgM. An alternative approach is to study the interaction between antibody and antigen. When first exposed to an epitope, most antibody binds relatively weakly but the strength of this bonding increases as the immune response matures and the duration of infection increases. The strength of the interaction between an antibody and an epitope is known as ‘affinity’ and the combined strength of multiple antibody-epitope bonds consti-

tutes ‘avidity’. Low-avidity antibody predominates early in the course of an infection whereas high-avidity antibody is more common at a later date. The range of avidities within an antibody population does not follow a normal distribution; although both high- and low-avidity antibody are produced throughout the duration of an infection the proportion of low- to high-avidity antibody changes over a period of timerh. Measurement of IgG avidity has been applied to the diagnosis of viral infections including rubella’7Jg and hantavirus14. Preliminary studies suggest recent primary toxoplasma infection may be identified by assessment of IgG avidity’“-*?.

Two different methods have been described for the measurement of antibody avidity; the ‘bind and break’ assay and ‘prevention of bond formation’18. In the ‘bind and break’ method antigen-antibody bonds are allowed to form and then an attempt is made to disrupt these bonds using an elution agent”. The antigen is fixed to a solid phase and then a dilution of the patient’s serum is added. Specific antibody in the serum attaches to the immobilized antigen and forms hydrogen bonds. The antibody-antigen complex is then washed with a solution containing a hydrogen bond disrupting, elution agent. Depending on the avidity of the antibody-antigen interaction a proportion of the antibody is detached and the remaining bound antibody is measured using an identification system. Elution agents include urea’” and diethy- Iamine”. The optimum discrimination between low- and high-avidity antibody is determined by testing acute-phase and chronic-phase sera using a range of concentrations of the elution agent*O. Assays for the measurement of toxoplasma-specific IgG avidity have used 6M urea”.*’ but 8M urea has been incorporated into avidity tests for other pathogensi7Jy. Maturation of the avidity of rubella-specific IgM has been described” but only IgG avidity assays have been developed for the investigation of toxoplasmosis. Avidity measurement based on the indirect fluorescent antibody (IFA) test’” and enzyme-linked immunosorbent assay (ELISA)?” have been developed.

Calibration of avidity assays has been problematic due to the limited availability of reference sera. Ideally, serial samples demonstrating seroconversion should be used so that the precise duration of infection can be calculated. However such samples are rarely identified and few studies have incorporated a limited number of seroconversion series1”,Z3. Indirect parameters of acute and chronic infection have been used to establish assays of toxoplasma-specific IgG avidity. Acute infection has been associated with a fourfold rise of the specific IgG leve12”, the detection of specific IgM*2 or the presence of toxoplasma-associated lymphadenopathy for a specified duration”. Differences in the individual’s immune response to toxoplasma infection, persistence of IgM production, variation in the time between acquisition of infection and the onset and recognition of symptoms and the coexistence of asymptomatic toxoplasmosis with lymphadenopathy of a

Holliman: Recent developments in diagnosis of toxoplasmosis 7

separate aetioiogy may lead to inaccurate estimation of the duration of infection. Some of the apparently discordant avidity findings described may be explained by erroneous definition of the phase of infection.

Clinical upplication

Measurement of the avidity of toxoplasma-specific IgG has only been applied in one clinical situation, the determination of the duration of infection. It has been shown that patients with toxoplasma-associated lymphadenopathy symptomatic for less than three2’ or four months’” have specific IgG of low avidity, whereas those symptomatic for greater than six months have high-avidity antibody 21. Studies of serial samples after seroconversion have demonstrated maturation of the immune response to toxoplasma infection associated with increasing avidity of the IgG responsezfl.

Although absolute correlation between duration of infection and antibody avidity has been claimed by some groups?” others have demonstrated discordant findings. Two centres measured low-avidity antibody in sera taken from patients with symptomatic toxoplasmosis of greater than six months’ duratior+. Subsequent analysis of the data showed that semi- quantitative IgM assessment provided a more accurate estimation of the duration of infection than IgG avidityId. In certain cases dating the onset of toxoplasma infection may be complicated by the persistence of the IgM response. It has been suggested that IgG avidity assays may resolve this problem but extensive studies of IgG avidity in patients with.long- standing IgM production have not been performed. One study included two patients with toxoplasma- specific IgM detected by ELISA and lymphadenopathy present for over six months. Borderline avidity findings were found in each case*‘. It is not established if toxoplasma-specific IgG avidity increases in the expected pattern when IgM persists. The association of IgM production and the formation of high-avidity IgG to toxoplasma could be linked immune phenomena, in which case avidity assays would offer little advantage over conventional IgM tests.

It has been proposed that IgG avidity estimation could be used to identify pregnancies that are at risk of congenital toxoplasmosis’“.“‘. A prenatal screening programme to detect acute maternal toxoplasmosis in Finland included assessment of specific IgG avidity*“. A total of 25 women from over 16 000 tested were found to have low-avidity antibody. In particular, 11 of 13 women who were shown to have seroconverted during pregnancy were associated with low-avidity IgG. Of these 11 seroconverting with low-level avidity, 10 showed a maturation to high-level avidity within six months but the remaining woman still produced low- avidity IgG 11 months after seroconversion. The study concluded that measurement of IgG avidity should be performed after initial IgG and IgM testing of pregnant women had identified those with possible recent toxoplasma infection.

The value of antibody avidity measurement in the diagnosis of cerebral toxoplasmosis with AIDS or the distinction between passively-acquired and actively- produced specific IgG in the neonate with congenital infection has not been established.

Immunoglobulin-A is produced as part of the acute phase response to a pathogen and has recently been found to be useful in the diagnosis of infection*“. Human intestinal fluidZ6, breast milk” and serum?8 have been shown to contain specific IgA following toxoplasma infection. IgA production is thought to play a major role in the body’s defence against intestinal infectionZ9 and has been shown to agglutinate toxoplasma and to inhibit invasion of enterocytes by the parasite”‘. Concurrent exposure to cholera toxin amplifies the gut secretory IgA response to T. gondii so that appropriate adjuvants may be used to enhance immunity following ingestion of a toxoplasma vaccine”O. The toxoplasma antigens targeted by IgA have been analysed in animal models”‘J’ and human infectionJ*,33. Multiple antigens are recognized but the 30, 35, 40 and 50 kDa antigens predominate as antibody targets in human sera.

A number of assays have been described for the measurement of IgA including the immunofluorescent antibody test (IFA)2h, a-chain capture immunosorbent agglutination assay (IgA ISAGA)““-“” and Western blotting”3.37. In addition monoclona12x~3x and polyclonal~ 7h.3y a-chain capture ELISA have been developed. These assays have shown acceptable specificity”5 and reproducibility”h but the level of sensitivity varies with the methodology employed. The IgA ISAGA has superior sensitivity compared to a-chain capture ELISA and the enzyme-linked immunofiltration assay (ELIFA)74. Similar findings for IgM tests have been reported and it is proposed that the enhanced sensitivity of the ISAGA format reflects the greater proportion of membrane antigens compared to cytoplasmic antigen incorporated in the assays.

Clinical application

Clinical specimens tested for IgA include sera from pregnant womenJo and other adult9J7, cerebrospinal fluid (CSF)“JJX, foetal blood sample9 and neonatal sera36.40 . Clinical situations investigated by IgA measurement comprise the duration of toxoplasma infection”“, congenital toxoplasmosis”’ and cerebral reactivation associated with AIDS’“.

The kinetics of IgA production have been studied in animal models of toxoplasma infection. In rats. IgA levels were maximal 40-70 days after infection and could still be detected at day 300”. Similar kinetics were observed in mice where IgA was first detected in the serum on day 14 and in the intestinal secretions on day 21. Peak serum levels of IgA were recorded after 42 days and reactivity persisted beyond 120 days”.


Such detailed knowledge of the duration of infection is rare in human infection but one case of laboratory- acquired toxoplasmosis is of interest because the exact date of exposure was established”“. IgA was first detected after 28 days and maximal levels were recorded between 2842 days. The IgA response persisted beyond day 300. Comparative studies indicate IgA levels rise later than IgM” and that maximal levels of IgA are attained later than IgM?J. The persistence of detectable IgA is highly variable, reflecting the sensitivity of the assay employed, differences in the host immune response and possibly the virulence of the infecting strain of T. go&ii. Typically, IgA is detected in human serum for three to six months after infectionzR,3y but loss of IgA within one month and persistence beyond 18 months have been noted36. Details of studies of the kinetics of IgA production in human toxoplasma infection are presented in Table 1. Detection of specific IgA in the serum indicates toxoplasmosis of recent onset but the variation in the persistence of the IgA response and the diversity of IgA levels at a defined duration of infection prevent precise dating of the onset of infection.

A number of studies have investigated the diagnosis of congenital infection by measurement of toxoplasma-specific IgA.7”Jh.4” and all have shown that

detection of IgA is a more sensitive indicator of congenital toxoplasmosis than detection of IgM. In addition, the superior sensitivity of the ISAGA methodology compared to ELISA offers a diagnostic advantage in this context’h. Toxoplasma-specific IgA have been detected in the peripheral blood of two non-infected infants?“,Jx. In each case specific IgM was not present. These false positive reactions probably represent maternal contamination of the sample via a placental defect. As the IgA molecule is significantly smaller than pentameric IgM, maternal IgA may reach the neonatal circulation with greater frequency than IgM and an increased frequency of false positive IgA reaction may be recorded. Due to the reduced specificity of IgA estimation and the lack of absolute sensitivity, investigation of the neonate for suspected congenital infection should include measurement of IgA, IgM, IgG and parasite isolation or detection studies. Details of studies of IgA in the diagnosis of congenital toxoplasmosis are shown in Table 2. Preliminary investigations have shown that IgA can be detected in foetal blood from 26 weeks’ gestatiot+ but the relative sensitivity of IgA measurement compared to parasite isolation or IgM assay for the diagnosis of congenital toxoplasmosis is not established.

Table 1. Kinetics of IgA production in human toxoplasmosis

Reference Study population IgA assay Onset of IgA response

(weeks after exposure)

Persistence of 1gA

(weeks)

33

34

36

37

39

39

1 laboratory worker ELISA 4 >45

300 patients with acquired ISAGA >I2 toxoplasmosis

49 patients with toxoplasma- a-chain ELISA capture 2-4 <2 associated lymphadenopathy >70

18 pregnant lymphadenopathy women, P30 antigen immunoblot 120 and HIV-infected patients

12 pregnant women a-chain ELISA capture 15 231

10 toxoplasma lymphadenopathy a-chain ELISA capture 53 224 patients

Table 2. Detection of specific IgA and IgM in infants with congenital toxoplasmosis

Reference Cases of congenital W kd bM h@ toxoplasmosis assay detected assay detected

35

36

39

28

17

17

9

ISAGA

e-chain capture ELISA ISAGA

a-chain capture ELISA

40 46 a-chain capture ELISA (P30 antigen)

21 ISAGA 17

2 E-&&n capture 4

12 ISAGA 7

8 ISAGA 4

41 E;kc;n capture

(P30 antigen)

31


Primary toxoplasma infection in AIDS patients is associated with an IgA response similar to that seen in immune-competent persons”h. However, most cases of cerebral toxoplamosis with HIV infection result from secondary reactivation of the patient’s chronic, previously quiescent infection’. In these circumstances the value of IgA measurement is controversial. One group reported the detection of toxoplasma-specific antibody in cerebral toxoplasmosis with AIDS using immunoblot. Of 12 samples investigated, 10 had detectable IgM and 11 had detectable IgA, whereas only a single specimen was reactive using an IgM ELISA-?:. These findings were not confirmed in two studies where specific IgA and IgM could not be detected by ELISA3” or ISAGA”“. Further studies are required to resolve these discordant findings and the value of IgA estimation in HIV-infected patients remains uncertain.

Polymerase chain reaction (PCR)

The limitations of antigen detection and parasite isolation emphasise the need for a rapid, sensitive method for detecting the presence of T. gondii in clinical samples. Recombinant plasmids derived from a genomic DNA library for T. gun&i in pUC8 and radio- labelled with i?P using nick translation were used as DNA probes for the parasite“‘. The limit of sensitivity of this assay was ~10~ trophozoites, a level which was regarded as inadequate for clinical utility. A DNA probe based on a highly-repetitive sequence ABGTg4 was used to detect toxoplasma in the CSF of four AIDS patients with cerebral diseasej’. It was calculated that each CSF sample contained Z 6 X lo4 parasites mll’ and the heavy parasite load in this condition has been confirmed by the detection of T. gun&i using Wright’s staining of CSF and direct microscopy”‘. Raised intracranial pressure associated with toxoplasmosis in AIDS often precludes CSF sampling and other tissues may contain smaller numbers of the parasite. Consequently the PCR was utilized to amplify target sequences in the specimen under investigation in order to improve the sensitivity of DNA detectionti.15.

Advances in our understanding of the protein struc- ture and function of 7: gondii have been accompanied by the identification and sequencing of parts of the parasite genome’“. These sequences represent potential targets for PCR amplification. One study investigated 2X strains of toxoplasma (17 human and 11 animal isolates) and used PCR amplification of three genes (SAG, 850 and BS) followed by restriction fragment length polymorphism (RFLP) analysisj’. Ten strains virulent in mice (LD,,,, ~10) were found to have an identical idiotype. whereas the remaining non-virulent strains (LD,,,,, >I()‘) were polymorphic. The authors proposed that virulent strains of T. ~ondii are derived from a single clonal lineage which has remained genet- ically homogeneous despite wide geographical distribution. However-. the clinical relevance of a classification system based on virulence in an animal

model is questionable. Six of the eight toxoplasma strains isolated from patients with AIDS were classi- fied as non-virulent by animal studies even though cerebral toxoplasmosis is a life-threatening condition. Despite these limitations the work illustrates sufficient homology amongst toxoplasma isolates for consistent detection of the parasite using PCR to be practicable, as well as variation in certain regions of the genome which could be used to fingerprint strains in epidemi- ological studies. The earliest applications of PCR to the detection of toxoplasma used the Bl’” and P3(r”-5’5 genes as amplification targets. Subsequently a number of modified systems based on these”JJ and other nucleic acid sequences55.5s have been described. Details of methods for detecting toxoplasma using PCR are given in Table 3. Denaturation temperatures between 93” C and 95” C have been used. The lower denaturation temperature may be less harmful to the polymerase enzyme. The duration of the denaturation temperature required depends on the thermal proper- ties of the heating block and tubes. Annealing temperatures have varied between 45” c‘ and 64°C. The higher the annealing temperature the greater is the specificity of the reaction. Most assays have used the conventional extension temperature of 73” C‘.

The sensitivity of any PCR assay for toxoplasma detection depends on the efficiency of nucleic acid extraction, the nature of the target gene, the number of amplification cycles and the detection system employed. Several different techniques for the extraction of DNA from the host tissue have been utilized. Most groups have used conventional proteinase lysis and disruption of the tissue, solvent extraction and precipitation. This process is relatively complex and time consuming, requires the use of noxious chemicals and carries the risk of losing DNA at several points. Other groups have used less complex methods such as freezing/boiling”’ and proteinase K digestion alone’“. One study compared three methods of sample preparation: proteinase-K digestion, heating and guanidinium thiocyanate/silicai’. Heat treatment of clinical samples was found to be insufficient and was associated with false negative reactions after PCR. The guanidinium thiocyanate/silica method was shown to be inferior to proteinase treatment as the latter produced a more reproducible and sensitive assay when combined with PCR amplification. An animal model of toxoplasma infection showed that the sensitivity of parasite detection by PC’R was enhanced if clinical samples, particularly leucocyte preparations. were washed prior to amplification to reduce the haemaglobulin content5”. A different approach to avoiding inhibition of the Taq polymerase is to dilute the clinical sample serially. reducing non-specific enzyme inhibition but resulting in a decrease of target sequences per unit volume.

The sensitivity of a PCR assay based on a repetitive gene, such as BIJS or TCRIE” should be greater than that of an assay using a single copy gent target. such as P3(r”, assuming that the efficiency of amplification

Table

3.

M

etho

dolo

gy

for

the

dete

ctio

n of

tox

opla

sma

usin

g PC

R

Refe

renc

e DN

A Ta

rget

ex

tract

ion

gene

C

ycle

s De

natu

ratio

n An

neali

ng

Exte

nsio

n De

tect

ion

of

PCR

prod

uct

Sens

itivi

ty

(min

imum

nu

mbe

r pa

rasit

es

dete

cted

in

hu

man

DN

A)

93”

C;

1 m

in

55”

C;

1-2

min

72

” C

; 1.

5-3

min

Hy

brid

izatio

n IO

95

” C

; 1

min

60

” C

; 1

min

74

” C

; 3

min

G

el el

ectro

phor

esis

1 48

PP

CE

Bl

49,

50

PPCE

P3

0 55

30

x

2 (n

este

d pr

imer

s)

40

51

Prot

eina

se-K

He

atin

g 95

” C

; 15

m

in

Gua

nidi

nium

th

iocy

anat

ekilic

a PP

CE

Bl

Bl

Bl

Bl

94”

C;

1 m

in

55”

C;

2 m

in

72“

C;

3 m

in

72”

C;

3 m

in

Rea

ctiv

atio

n en

zym

e an

alys

is

and

shor

t bl

ot

IO

100

40

94”

C;

1 m

in

55”

C;

2 m

in

94”

C;

1 m

in

55”

C;

2 m

in

72”

C;

3 m

in

94”

C;

l-3

min

53

” C

; 2

min

72

” C

; 2.

5 m

in

40

30

x 2

(nes

ted

prim

ers)

30

30

30

35

Gel

elec

troph

ores

is IO

1-

16

52

53

54

55

56

57

58

PPCE

PP

CE

PPCE

N

ot

Stat

ed

Free

ze

and

boil

Prot

eina

se-K

Bl

P30

TGRI

, Ri

boso

mal

DN

A Ri

boso

mal

DN

A Ri

boso

mal

DN

A

94”

C;

1 m

in

55”

C;

1.5

min

72

” C

; 1

min

Hy

brid

izatio

n 1

95”

C;

1 m

in

45”

C;

2 m

in

72”

C;

3 m

in

Hybr

idiza

tion

100

94”

C;

1 m

in

55’C

; 2

min

72

” C

; 3

min

Hy

brid

izatio

n 1

95”

C;

10

set

64”

C;

30

set

70”

C;

1 m

in

Gel

elec

troph

ores

is 5

94”

C;

1 m

in

95”

C;

10

set

50”

C;

2 m

in

72”

C;

3 m

in

Gel

elec

troph

ores

is 1

64”

C;

30

set

70”

C;

1 m

in

Gel

elec

troph

ores

is 1

35

35

PPCE

=

Prot

einas

e di

gest

ion,

ph

enol-

chlor

ofor

m

extra

ction

, et

hano

l pr

ecipi

tatio

n.


and number of PCR cycles employed are similar. One study showed that 40 cycles of PCR using the Bl gene but not P30 was able to detect toxoplasma in ocular tissuehn. Nested PCR based on the Bl gene was found to be more sensitive than that based on P30 in the diagnosis of ovine infection”‘. Ribosomal DNA sequences can be used to enhance sensitivity as each toxoplasma trophozoite may contain 104 ribosomes, each a potential PCR target5h-58. Alternatively, increased amplification of target DNA may be achieved by increasing the number of PCR cycles incorporated in the assay, either using a single set of primers or by the use of two nested primer sets. Self priming and amplification may lead to false-positive reactions when a large number of PCR cycles is used with a single set of primers. Increasing the number of PCR cycles does not lead to a proportional increase in target amplification and further PCR cycles above an upper threshold for that assay produce no additional benefit. Up to 55 amplification cycles have been used in PCR for toxoplasmaJX. The sensitivity of PCR can be increased by subjecting the product of one round of amplification to a second round of PCR using a set of primers internal to the original pair. Nested PCR has been applied to the P3oj” and Bl gene5* to achieve the necessary level of sensitivity while limiting the number of PCR cycles in each round. The disadvantages of nested PCR are the extra manipulations, increased cost and additional risk of contamination. A one-tube nested PCR technique avoids some of these problems. Here, PCR is performed in two stages using two primer sets which anneal at differing temperatures. A longer, outer primer pair hybridizes at the higher temperature while the shorter. internal primer pair anneals at the lower temperature. By altering the temperature within the reaction mix, two rounds of PCR are performed without the need for manipulation between the first and second round. This technique has been applied to the diagnosis of tuberculosis6? and may be of value for the detection of toxoplasma.

Detection of the amplified PCR product is usually performed by agarose gel electrophoresis and visual- ization of the ethidium bromide stained band of the expected size under UV lightsX.h’. The specificity of the reaction can be confirmed by hybridization using a toxoplasma-specific DNA probe carrying a biotin or radiolabePK. PCR product detection by hybridization can also be used to enhance the sensitivity of the assayhJ. The sensitivity of PCR for toxoplasma detection has been reported for a number of different assays and brief details are presented in Table 3. Most groups have been able to detect a single parasite in a clean preparation but noted a reduced sensitivity when toxoplasma is suspended in human tissue such as white blood cells4x-J”. Incorporation of a multicopy gene target, nested PCR or detection of the amplified product by labelled probe hybridization in the assay technique has resulted in detection down to the level of a single parasite contained in a human tissue samples11~53.ix. Different strains of T. gondii are reliably

detected using amplification of the P30JyJJ and B148JI genes. The specificity of the various assays has been confirmed by the failure to amplify related organisms such as Hammondia, Cryptosporidium, Eimeria or Giardia using P30 gene primersJO or Sarcocystis, Neospora, Cryptococcus or Plasmodium using Bl gene primersJX. However, when applying Bl gene amplification to the investigation of clinical samples, it has been necessary to compare test findings to those obtained from negative controls as non-specific amplification may occur6-5. Contamination of the reaction mix leading to false positive results remains the greatest problem in any clinical test based on PCR. The risk of mislead- ing results is greatly reduced by the routine use of positive displacement pipettes, wearing of gloves and protective clothing, spatial separation of pre-amplification from post-amplification work and careful selection of negative and minimal content positive controls”“. Despite these precautions, random contamination can be problematich5 so that all positive reactions should be subject to repeat investigation for confirmation. False negative reactions may be associated with the presence of non-specific inhibitors of Taq polymerase in clinical tissues. This phenomenon can be detected by adding a restricted fragment of the PCR product, bound by the correct terminal sequences, to each sample. Unless the Taq polymerase is inhibited, this DNA fragment will be amplified during the PCR and acts as a positive control within each reaction mix.

Clinical application

PCR has been used to detect toxoplasma in the blood of animals acutely infected in the laboratory including mice54, rats’9 and rabbits”3 as well as brain tissue from chronically-infected mice49. Subsequently, this technique has been used to diagnose congenital toxoplasmosis in sheepM and horse@’ where examination of ocular tissue was found to be of particular value. The diagnosis of human toxoplasma infection has been performed in a range of clinical conditions by the examination of a variety of tissues (Table 4). The precision of diagnosis based on PCR has been compared to conventional methods including serol- ogy’j, histologyhX.74 and parasite isolation by animal inoculation and/or tissue culture@. Due to the non- uniform distribution of toxoplasma in tissue, discordant findings may reflect the presence or absence of the parasite in separate specimens examined by each method. In addition to sampling error and assay contamination. a positive PCR reaction associated with negative isolation studies may result from the presence of non-viable parasite fragments in clinical samples7”. False negative PCR findings may result from the small sample volume examined by this technique compared to conventional isolation studies(?.

Toxoplasma infection of the immune competent can be diagnosed by PCR amplification of parasite target sequences in lymph node biopsy tissue or peripheral blood samples hrl.hX. The sensitivity of this technique is

Table

4.

De

tect

ion

of

toxo

plas

ma

by

PCR

: cli

nica

l ap

plica

tion

Refe

renc

e L

ymph

aden

opat

h y

Preg

nant

wo

man

Ne

onat

e O

cula

r di

seas

e O

rgan

gr

aft

recip

ient

M

alig

nanc

y H

IV

infe

ctio

n

Hear

t Bo

ne

mar

row

68

Biop

sy

tissu

e 64

Ve

nous

blo

od

Veno

us

blood

Co

rd

blood

Ve

nous

blo

od

Biop

sy

tissu

e Am

niot

ic flu

id

Neon

atal

blo

od

Veno

us

blood

Br

ain

biop

sy

Foet

al

b1oo

.d

Plac

enta

55

Am

niot

ic flu

id

Bone

m

arro

w Fo

etal

blo

od

Veno

us

blood

56

, 65

Am

niot

ic flu

id

69

Foet

al

brai

n Br

ain

Brain

CS

F He

art

Skel

etal

m

uscle

70

Pl

acen

ta

Cord

blo

od

73

CSF

58

Aque

ous

hum

our

60

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lower than conventional histology due to the restricted numbers of toxoplasma in these tissues. In contrast, histological examination detects the associated immune reaction to the parasite in lymph node tissue, a more reliable indication of toxoplasma infection.

Toxoplasma can be detected in the peripheral blood of pregnant women by PCR although the value of this parameter for predicting transmission of infection to the foetus is not established. Contamination of pregnancy-associated samples, such as amniotic fluid, foetal blood, cord blood or placental tissue with maternal blood may result in a positive PCR reaction in the absence of congenital toxoplasmosi@“. At present the sensitivity of parasite gene detection exceeds the sensitivity of methods used to detect sample contamination such as the Kleihauer test. It may be necessary to use PCR for HLA typing and hybridization with allele- specific oligonucleotides to detect maternal contamination of foetal samples. Some studies have shown PCR investigation of amniotic fluid to be more sensitive than culture of amniotic fluid or foetal blood examination for the diagnosis of congenital toxoplas- mosis5h.hS, whereas other groups have found conventional methods more reliabless. Further studies are required to resolve this controversy. PCR investigation of the placenta and cord blood is of limited value due to potential contamination of these samples with maternal bloodM,70. In contrast detection of toxoplasma in neonatal CSF. blood or brain tissue by PCR is of clinical value in the diagnosis of congenital infec- tionhv.73. Negative PCR findings are useful in excluding congenital toxoplasmosis during the immediate postna- tal period”J-7cl.

PCR has been used to demonstrate the presence of toxoplasma in histological sections of infected ocular tissueM1. However. tissue samples are rarely made available during the investigation of suspected ocular toxoplasmosis. Examination of aqueous humour using PCR was found to be highly specific but of relatively low sensitivity due to the low volume samples obtainedsx. Calculation of the Witmer-Desmonts coefficient demonstrating intra-ocular formation of specific antibody remains the definitive investigation for ocular toxoplasmosis.

Toxoplasmosis is a life-threatening opportunistic infection in immunocompromised patients. At-risk groups include organ graft recipients, patients with malignancy and HIV-infected persons. In such cases the primary pathogenic mechanism is reactivation of previously quiescent cysts of T. go&i. Currently available PCR methods do not distinguish the presence of quiescent cysts from actively-dividing tachyzoites. Results of PCR assays must be interpreted with caution when investigating the immunocompromised patient. Quantitative PCR or amplification based on a stage-specific RNA target could be used to identify active toxoplasma infection, but neither technique is available in the clinical laboratory. Detection of toxoplasma using PCR was found to be more sensitive than histological examination of cardiac biopsy tissue’j.

Severe toxoplasma infection associated with heart, lung and liver transplantation is restricted to seroneg- ative recipients of seropositive donors and is prevented by the use of prophylactic pyrimethaminex’. Serious recipient infection is associated with an acute serological response including seroconversion, rising IgG levels and detection of specific lgMK2. Consequently, the role of PCR in solid organ transplantation is confined to the confirmation of the diagnosis of toxoplasma infection readily established by conventional methods. Conversely, toxoplasma infection of bone marrow transplant recipients is difficult to diagnose using conventional techniques. The infected recipient may fail to mount a detectable antibody response. Isolation of the parasite by tissue culture lacks sensitivity and animal inoculation does not produce results with sufficient rapidity to be of clinical valuex’. Toxoplasma has been detected in the bone marrow and venous blood of infected marrow graft recipients but not in uninfected controls”J~‘i.

Toxoplasma infection of patients with malignancy has been diagnosed by PCR examination of venous bloods? but the most frequent application of this technique has been in the investigation of HIV- infected persons. Initial reports demonstrated that PCR could be used to detect toxoplasma in brain biopsy samples taken from patients with cerebral disease7i,72. Subsequent investigations found that PCR amplification of target sequences in CSF was diagnostic in approximately half of patients with cerebral toxoplasmosis and consistently negative in AIDS patients with quiescent toxoplasma infection7h-7y. These findings are similar to the measurement of intrathecal specific antibody production where the proximity of toxoplasma lesions to the ventricular system is thought to determine the sensitivity of the assayXJ. In some cases toxoplasma can be visualized in the CSF by direct microscopyj3. The pathogenesis of cerebral toxoplasmosis with AIDS is not determined. The majority of cases are thought to result from reactivation of chronic, previously quiescent infection’. Reactivation may be limited to the cerebral tissues resulting in the formation of single or more often multiple lesions detected by computed tomography. Alternatively the breakdown of toxoplasma cysts in muscle and other tissue may lead to parasitaemia and secondary seeding of the brain, forming the characteristic lesions. Some support for the second theory is provided by the detection of parasites in the blood of AIDS patients with cerebral toxoplasmosis using PCR52.hJ.X’1 and by direct cultiva- tionx5. Pulmonary toxoplasmosis with AIDS has been diagnosed using detection of the parasite in bronchoalveolar lavage fluid with PCR5’,s’1.

Conclusions

Recently introduced assays may assist the diagnosis of toxoplasmosis in defined clinical situations. Measurement of IgG avidity is useful for determining the duration of toxoplasmosis and may help the

14 Serodiagn. Immunorher. Infect. Disease 1994; 6: No 1

accurate calculation of the risk of foetal infection. Specific IgA detection in the infant’s blood represents the most sensitive indicator of congenital infection available during early life. Detection of T. go&ii using PCR is the investigation of choice for the diagnosis of toxoplasmosis associated with bone marrow transplantation. PCR is of value for the examination of CSF, bronchoalveolar lavage fluid and venous blood taken from HIV-infected patients. Current molecular methods may not distinguish between active infection and the presence of quiescent cysts of the parasite. Contamination of placental, foetal blood and amniotic fluid samples with maternal blood may complicate the interpretation of PCR findings when investigating toxoplasmosis in pregnancy.

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Acknowledgement 22

I am grateful to Mrs D. Lyndon for the preparation of the typescript.

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The International Immunocompromised Host Society Announces the 8th International Symposium on Infections in the Immunocompromised Host 19-22 June 1994, Davos, Switzerland

The programme will include Plenary Sessions entitled Gene Therapy, HIV Infection in Children: a New and Future Problem, The Cellular Basis of Bacterial Invasion, Pattern Recognition Molecules in First Line Host Defense, Future Therapies for Tuberculosis, Growth Factors in the Management of the Compromised Host. Mini-Symposia will be held on: Fungal and Parasitic Infections; Cytokines/Anti-Cytokines; Prevention and Therapy in Cancer; Prevention and Therapy in Transplant Patients; AIDS; Disorders of Phagocytic Cells; Quantative and Qualitive; and Infections in the Critically Ill. For this first time, Round Table discussions will be held on BCG Vaccination to Prevent Tuberculosis, and White Blood Cell Transfusions in the Compromised Host.

Abstracts are solicited from the subject areas related to Mini-Symposia, Plenary Sessions, and Round Table Discussions. Deadline for submission is 15 February 1994. Each day there will be oral presentation of selected abstracts as well as poster sessions. For more information please contact the symposium secretariat:

P.O. Box 319 Comstock, MI 49041 USA

Tel: + + 1 (616) 329 5640 Fax: ++l (616) 327 7161

Documents

Recent developments in the diagnosis of toxoplasmosis