Experience of the P30 PCR in routine diagnosis

As in most parts of Scandinavia, the Gothenburg region is considered to be a low-endemic area with respect to T. gondii infections (Andersson et al. 1992; Bergström et al. 1998). The seroprevalence of IgG antibodies to T. gondii in blood donors in Göteborg is approximately 10% (unpublished observation). The PCR-based diagnosis of T. gondii infections in Göteborg was introduced in 1993 and 842 clinical specimens have been analysed to date (December 2001). The types of specimen received included EDTA blood for PBMC preparation (199 samples), serum (45 samples), vitreous body and fluid (40 samples), cerebrospinal fluid (356 samples), biopsies from various tissues (57 samples), placenta (81 samples), amniotic fluid (32 samples). Various other body fluids were examined such as urine, broncheoalveolar lavage, ascites and pericardial fluid (43 samples). The frequency of toxoplasma PCR positive samples was 1.2%, representing positive samples obtained from the 10 patients presented in Table 29.2. In contrast to PBMC or CSF samples no positive PCR signals were obtained from serum. The rapid laboratory confirmation of T. gondii infection was important. Besides identifying the etiological agent for a severe medical condition under investigation, it should be noted that in at least six of these cases, the early PCR diagnosis of T. gondii infection was of significance for the clinical management of the patient.

The specificity of the P30 PCR products was confirmed by hybridization of the positive samples obtained from each patient (10 cases). In addition, PCR amplicons from three cases (patient nos. 8, 9, 10; Table 29.2) were control sequenced, and the 375bp outer products of the P30 target gene were shown to vary in just one nucleotide, nt 293 (Accession no. S85174). At this position, the HIV positive case (patient no.10, from Lebanon) read GTT coding for valine, whilst the two congenital cases (patient nos. 8 and 9) sequenced as GCT for alanine. GenBank Blast analyses of the P30 sequences derived from the HIV positive case of cerebral toxoplasma indicated that it was similar to the RH strain at nt position 293. The two cases of congenital infection that resulted in neonatal death, gave P30 toxoplasma sequences identical to strains P and/or C described by Bulow and Boothroyd (1991). These findings emphasise the P30 target region as well conserved between the cases studied, despite

Table 29.2 PCR-positive cases with T. gondii infections diagnosed in Goteborg 1993-2001a

Patient

Gender, age

Serology Toxo Toxoplasma PCR-

Diagnosis

Underlying disease

No.

IgM/IgG

positive specimen

1

Neonate M, 9

Neg/Pos

PBMC

Congenital toxoplasmosis

days

2

F, 21 years

Pos/Pos

PBMC

Toxoplasmosis during

pregnancy, mother of pat. 1.

3

M, 14 years

Not available

Liver biopsy

Generalized toxoplasmosis

Liver transplanted

with hepatitis

4

M, 39 years

Neg/Pos

Vitreous body

Retinochoroiditis

5

F, 52 years

Neg/Neg

CSF

Cerebral toxoplasmosis

Liver transplanted

6

F, 57 years

Neg/Pos

Vitreous body

Retinochoroiditis

l

F, 66 years

Pos/Pos

PBMC

Generalized toxoplasmosis

Malignant

lymphoma

8

M, 3 dayst

Pos/Pos

PBMC

Congenital toxoplasmosis

9

M, 1 dayt

No data

PBMC, CSF

Congenital toxoplasmosis

10

M, 29 yearst

Neg/Pos

PBMC, CSF

Cerebral toxoplasmosis

Untreated HIV-1

infection

Note

a A total of 842 specimens were analysed.

a A total of 842 specimens were analysed.

their widely different geographical sources of infection. Therefore, the P30 primer based PCR provides a good detection system for diagnosis of toxoplasma infections, confirming previous observations. Access to rapid toxoplasma diagnosis is of special relevance for paediatricians engaged in the diagnosis of congenital infections. This is exemplified by a case of congenital toxoplasmosis where diagnosis by PCR amplification of T. gondii DNA from peripheral blood led to early treatment of the infant, and seemingly normal brain development on examination at age 2 years, despite presence of intra-cranial calcifications at birth (Bergström et al. 1998). The laboratory diagnosis of T. gondii infection was instigated by findings detected on careful ophthalmic investigation of the neonate. Rapid detection of T. gondii DNA in the baby's PBMC prompted immediate early treatment that was continued for 12 months. At 2 years of age, the boy showed normal development for his age, the opalescence of the vitreous body detected at birth had resolved bilaterally, and the chorioretinitic areas were demarcated. The child appeared to have visual impairment in the form of loss of visual fields in at least one eye. The mother, who experienced sub-clinical infection during pregnancy, was PCR-positive for toxoplasma DNA in a sample of peripheral blood taken nine days after delivery. This case of child and mother illustrates the usefulness of the PCR method for diagnosis once the clinical suspicion has been raised, indicating prompt treatment of the infection. Our PCR observations as well as results obtained in Stockholm (Fahnehjelm et al. 2000) using PKU cards for neonatal antibody screening, emphasize the importance of careful ophthalmic examination and rapid laboratory diagnosis of congenital toxoplasmosis of neonates, as treatment has been shown to reduce long-term sequelae.

In all, we detected three cases of congenital toxoplasmosis (1993-2001) by examining neonatal PBMC and/or CSF by the toxoplasma specific P30 PCR (Table 29.2). This should be viewed in the light of the Swedish study based on PKU sample testing (Evengard et al. 2001), giving a prevalence of 0.7 cases per 10000 births, and the 3.3 per 10000 (11 cases) found in Norway (Jenum et al. 1998). The two additional cases of congenital toxoplasmosis diagnosed by PCR in the Göteborg region exhibited the classical features of very severe congenital toxoplasma infection and resulted in neonatal death. Although the exposure of pregnant women to toxoplasma is rare in this area, the condition is regarded with informed concern among women attending the maternity clinics. These cases illustrate the significant contribution PCR can make in confirming the diagnosis of congenital toxoplasmosis, or past maternal infection. Speedy confirmation of the diagnosis is very important, not only in prompting rapid management of the baby, but also in providing reassurance for the women that subsequent pregnancies are unlikely to be affected (Chatterton 1992).

In our experience, serology is of limited value in the diagnosis of toxoplasma-induced retinochoroiditis. Levels of IgM antibodies to T. gondii are often too low to allow detection, and IgG antibodies also tend to be of low titre. In this field, PCR diagnosis based on samples of vitreous body/vitreous humour seems especially promising. Although the material presented is very small, the finding of two positive samples out of 40 examined (5%) indicates that vitreous body is a valuable test sample in cases of uveitis as has been observed previously (de Boer et al. 1996). These two positive findings were probably not due to the irrelevant detection of dormant parasites, since control material, vitreous body specimens taken on surgery in the absence of clinical suspicion of T. gondii infection (n=10), was found to be negative (unpublished observation).

The remaining four toxoplasma PCR positive cases in our study were found among immunosuppressed patients. It is interesting to note that following liver transplantation, one patient developed cerebral toxoplasmosis, probably after a primary infection as judged by the seronegative status at onset, and the other transplant case showed signs of toxoplasma-induced hepatitis. One elderly patient with acute myeloic leukaemia was a typical case of generalized toxoplasmosis where prolonged fever had resisted all therapeutic efforts until the PCR analysis of PBMC gave the correct diagnosis. This patient lived in very close contact with many stray cats. It should also be noted that only one case of AIDS-related toxoplasmosis was diagnosed, which may reflect the low prevalence, not only of T. gondii but also of HIV infection, in the Göteborg area. In this case, severe cerebral toxoplasmosis was detected in a newly arrived immigrant from the Lebanon. On admission to hospital, the young man was comatose, and despite rapid laboratory diagnosis of HIV positive antibodies, and strongly positive toxoplasma PCR signals in the CSF and PBMC, the patient succumbed to the infection.

These 10 cases describe our experience of the laboratory diagnosis of toxoplasma infection over 10 years. They typify the opportunistic character of T. gondii infection in humans, where a subtle shift in the balance between the immune competence of the host and the quiescent passenger state of the protozoa, can result in overwhelming infection. Accurate diagnosis requires alertness in the clinic and the laboratory.

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