¹Department of Haematology, Royal Free and University College Medical School, London, UK

²Department of Microbiology, Royal Free and University College Medical School, London, UK

³Department of Haematology, University School of Medicine, Ancona, Italy

Correspondence to: Dr C C Kibbler, Department of Microbiology, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK

Toxoplasma infection represents a rare but often fatal complication in bone marrow transplant (BMT) recipients. We report two cases of toxoplasmosis: one of successfully treated cerebral toxoplasmosis after peripheral blood stem cell transplantation, and a fatal case of pulmonary toxoplasmosis in a BMT recipient. We have systematically reviewed the 110 published cases of toxoplasmosis following BMT. We analyzed the pre-transplant and clinical features of BMT recipients developing toxoplasmosis, together with the diagnostic procedures used and treatment given. By univariate and multivariate statistical analysis we analyzed the risk factors for diagnosis (during life vs post-mortem) and Toxoplasma-related mortality. Ante-mortem diagnosis was made in 47% of cases. Site of infection (P = 0.02; odds ratio 10.8), presence of symptoms at onset (P = 0.01) and conditioning regimen (P = 0.04) were factors influencing whether the diagnosis was made before or after death. Overall mortality rate was 80% and that attributed to toxoplasmosis was 66%. Variables influencing outcome were: site of infection (P= 0.02; odds ratio 5.28), day of onset (P = 0.04) and conditioning regimen (P = 0.04). Underlying disease (P = 0.02; odds ratio 9.45), among patients diagnosed before death, was the most significant factor influencing outcome.

Bone Marrow Transplantation (2002) 29, 691-698. DOI: 10.1038/sj/bmt/1703425

toxoplasmosis; bone marrow transplantation; diagnosis; treatment and systematic review

Toxoplasma gondii is a ubiquitous obligate intracellular protozoan parasite causing clinical and latent infection in almost all known mammals. The incidence of toxoplasmosis shows marked geographical variation depending on local pet keeping habits, especially the presence of cats, and food contamination with cysts.¹

In immunocompetent individuals, T. gondii causes an asymptomatic infection or fever and lymphadenopathy (primary infection). In immunocompromised patients, it may cause life-threatening infection. Toxoplasmosis after bone marrow transplantation (BMT) is a rare but often fatal complication. In BMT recipients, it is usually the result of reactivation of latent infection rather than due to primary infection. A few cases have been reported in BMT recipients with negative Toxoplasma antibody titres, suggesting transmission of infection via marrow or blood products.²

The incidence of toxoplasmosis after BMT is not well established and ranges reported are from 0.3% in the USA³ to 5% in France.⁴ In 1997, Chandrasekar et al² in a review, reported a total of 55 cases of clinical toxoplasmosis following BMT. The most frequently involved organs were the central nervous system (CNS), lungs and heart. The infection was often disseminated, but isolated cerebral or pulmonary toxoplasmosis has been described.¹ ,² ,³ ,⁴ ,⁵ ,⁶ ,⁷ ,⁸ ,⁹ ,¹⁰ ,¹¹ ,¹² ,¹³ ,¹⁴ ,¹⁵ ,¹⁶ ,¹⁷ ,¹⁸ ,¹⁹ ,²⁰ ,²¹ ,²² ,²³ ,²⁴ ,²⁵ ,²⁶ ,²⁷ ,²⁸ ,²⁹ ,³⁰ ,³¹ ,³² ,³³ ,³⁴ ,³⁵ ,³⁶ ,³⁷ ,³⁸ ,³⁹ ,⁴⁰ ,⁴¹ ,⁴² ,⁴³ ,⁴⁴ ,⁴⁵ ,⁴⁶ ,⁴⁷ ,⁴⁸ ,⁴⁹ ,⁵⁰ ,⁵¹ ,⁵²

We report a case of cerebral toxoplasmosis successfully treated following diagnosis by PCR analysis of brain tissue and a case of fatal pulmonary toxoplasmosis diagnosed post-mortem. In addition, we have systematically reviewed the published cases of toxoplasmosis in BMT recipients to analyze the basic patient characteristics, the clinical features and management. By univariate and multivariate analysis, we studied the influence on mortality and diagnosis (post-mortem vs during life) of several risk factors.

Case report

Case 1

A 21-year-old Turkish man with a 10-year history of previously untreated myelodysplasia underwent an HLA matched sibling, non-T cell-depleted allogeneic peripheral blood stem cell transplant in March 2000. The conditioning regimen included total body irradiation, cyclophosphamide and fludarabine. Prophylaxis against graft-versus-host disease (GVHD) consisted of a short course of methotrexate and tacrolimus, instead of cyclosporin A, which had been discontinued following a severe reaction on day +2. The patient was seropositive for T. gondii and cytomegalovirus (CMV).

The post-transplant period was complicated (day +120) by grade III GVHD involving the skin, gut and liver, successfully treated with immunosuppressive therapy. On day +169 the patient presented with headache, nausea and vomiting and behavioural changes. He had no loss of memory, visual disturbance or fever and was fully orientated. Brain computed tomography (CT) scans showed numerous bilateral high-density lesions in the subcortical white matter. Multiple enhancing space occupying lesions involving the cerebellum, brainstem and cerebrum were seen on magnetic resonance imaging (MRI). A lumbar puncture yielded cerebrospinal fluid (CSF) with elevated protein concentration (1 g/l), leukocytosis (60 mononuclear cells/mm³) and reduced glucose level. CSF and blood cultured specifically for bacteria, fungi and mycobacteria were sterile. Viral tests on CSF were also negative. On day +171, stereotactic brain biopsy was performed but was not diagnostic. Based on radiological findings, CSF result, clinical presentation and positive pretransplant recipient T. gondii serostatus, pyrimethamine (75 mg once a day) and sulphadiazine (2 g three times a day) were added on day +172. Because of further neurological deterioration (impaired gait, left facial weakness, choreiform movements) and evidence of bilateral lentiform nuclear abnormalities on MRI, an open brain biopsy was performed on day +189. Macroscopically, caseous material was noted. Histological examination of the biopsy specimen revealed a perivascular lymphohistiocytic infiltrate with areas of necrosis. PCR amplification for T. gondii was performed on brain tissue. The Toxoplasma B1 gene sequence was amplified⁵³ and Southern blotting was used to detect PCR products. Using the positive control B1 gene PCR products as a probe, this gave a positive signal. By day +206, the patient had stabilized and a slow but steady improvement was observed thereafter. Therapy with pyrimethamine and sulphadiazine (P/S) was discontinued at day +223 because of pruritus and an erythematous macular rash and the patient was commenced on atovaquone, which was discontinued after 10 days following adverse reactions. He started treatment with 250 mg azithromycin every other day. At day +268, the patient had a further recurrence of his neurological symptoms. Head MRI scan showed multiple lesions, with an increase in size of previous lesions and cerebral oedema around some of these. He re-started (day +269) pyrimethamine and sulphadiazine. At last follow-up on day +379 post transplant his neurological symptoms had improved. A repeat MRI head scan showed a reduction in size of all lesions.

Case 2

A 30-year-old Greek man underwent an HLA matched unrelated, T cell-depleted allogeneic bone marrow transplant in December 1997. The patient had a 2-year history of drug-resistant acute myeloblastic leukemia. At the time of transplantation, his disease was not in remission, with 40% blasts in the marrow. Pretransplant recipient CMV serostatus was positive and T. gondii serostatus was not available. The conditioning regimen included total body irradiation, cyclophosphamide and Campath 1G i.v. On day -5, he had a reactivation of a previous presumed aspergillus sinusitis. He had high fever associated with pain in his left maxilla. A paranasal X-ray showed mucosal involvement of the sinuses. Antral washings cultured for bacteria and fungi were sterile. He was commenced on antifungal and antibacterial therapy and the fever and sinusitis improved. At day +5, he had fever again associated with severe mucositis. At day +8, a chest CT showed bilateral basal consolidation and MRSA was cultured from the sputum. Over the following days, he improved. At day +30 fever and sinus pain associated with shortness of breath and weakness developed. Three days later he developed sudden onset of tachypnoea, dyspnoea and severe hypoxaemia. In spite of antifungal, antibacterial, antiviral therapy and assisted ventilation his clinical condition deteriorated. He died on day +36 post transplant.

MRSA was isolated from bronchial washing (BAL) performed 2 days before death; no other bacterial or fungal agent was found. On cytological examination of the bronchial specimen, no malignant cells or viral cytopathic effect was seen. Post-mortem needle biopsy of the lungs revealed an interstitial lymphoid infiltrate with alveolar hyaline membrane formation indicating adult respiratory distress syndrome and macrophages containing multiple cysts of T. gondii, without evidence of other opportunistic infection. Autopsy was not performed.

Materials and methods

Literature search

We identified eligible studies by a Medline search from 1966 to December 2000. 'Toxoplasmosis' was combined with the following words as subject headings or text-words: 'BMT recipients', 'cerebral infections', 'diagnosis' and 'therapy'. This was complemented by a search of the references cited in the included studies or review articles in any language.

Statistical methods

All analyses were performed with the SPSS statistical package (SPSS, Chicago, IL, USA).

Descriptive statistics of the main patient characteristics were analyzed. We evaluated the influence on diagnosis (during life vs post-mortem) and mortality of these factors by univariate analysis. Univariate relationships between a diagnosis of or mortality from toxoplasmosis and the factors of interest were studied using ² statistics.⁵⁴ All significant variables were included in a multivariate analysis. For this analysis, we used a multiple logistic regression model with an enter approach.⁵⁵ Adjusted odds ratios were calculated and their statistical significance was evaluated according to the Wald ² test. A level of of 0.05 was used to denote statistical significance.

Results

By Medline search, a total of 45 case report papers and seven review articles were found. The majority of these were in the English language; one paper was in Spanish, one in Portuguese, one in German and one in French. All reported results came from the original papers. In one paper with 20 cases of cerebral toxoplasmosis,⁴⁷ we could not analyze mortality and risk factors because these data were not given in the paper. We contacted the author who was not able to provide them.

Including the two cases in this report, 110 cases of toxoplasmosis have been reported in bone marrow transplant recipients.^{1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52} Forty-three cases have been described in the USA (39%), two (2%) in Brazil, one (1%) in Australia and 64 (58%) in Europe (15 in France, 33 in Germany, five in Great Britain, three in The Netherlands, four in Spain and Portugal, two in Italy, one in Switzerland and one in Israel). The first case was described in the USA in 1981 by Emerson et al.⁸

Basic characteristics of reported cases

Patient characteristics are shown in Table 1. The infection occurred mainly in pre-transplant seropositive recipients and in only 12% of cases was it described in seronegative patients. Transmission of infection by marrow donor product was considered possible in two of these cases (positive donor serology) but in six cases donor serology was also negative. In two cases, donor serostatus was unknown.

Patients with a diagnosis of toxoplasmosis during life: In Table 1 are shown the features of patients with a diagnosis of toxoplasmosis during life. Their clinical characteristics were the same as the previously described cases. The only non-significant difference was age. The median age was 25 years (10-62).

Clinical features of toxoplasmosis

The clinical features of toxoplasmosis are summarized in Table 2. The CNS was the main site of infection, with 48% isolated cerebral cases and 76% brain involvement in patients with disseminated disease.

Diagnosis: Forty-two (47%) patients were diagnosed during life (Table 2). Table 3 summarizes the invasive procedures, radiological and laboratory findings that led to the diagnosis of toxoplasmosis. Some cases were diagnosed only on the basis of radiological findings (five patients with brain involvement) or response to pyrimethamine-sulphadiazine therapy (four patients). More recent cases were diagnosed by positive PCR results on tissues and/or biological fluids. In the two ocular cases, the diagnosis was made by fundoscopy. In four cases the parasite was identified in the blood by microscopy.

Statistical analysis: By univariate analysis, we studied the influence on diagnosis (during life vs post-mortem) of several factors (Table 4). Significant variables were site of infection (P = 0.02), presence of symptoms at the onset of toxoplasmosis (P = 0.01) and conditioning regimen (P = 0.04). More frequently, the diagnosis was made post- mortem in patients with disseminated toxoplasmosis (80% of cases) and in patients who had received TBI regimens (88%) vs no TBI (12%). All cases in which the diagnosis was made during life had symptoms at the onset. By multivariate analysis, the only significant variable was site of infection (P = 0.02). Logistic regression analysis showed that patients with disseminated toxoplasmosis had 10.8 times the odds of having the diagnosis made post-mortem compared with those with cerebral toxoplasmosis (95% CI 3.72 to 31.2).

Clinical presentation: Ninety-two percent of patients presented with one or more symptoms. Fever was the most common clinical feature, seen in 43% of all cases, 29% of cerebral and 48% of disseminated toxoplasmosis cases. The other main clinical symptoms were neurological (seizures 14%, headache 13%, lethargy 12%, confusion 13%, tremor 5%, hemiparesis 2%). These symptoms, more frequent in isolated cerebral toxoplasmosis, were also described in disseminated infection. In the same way, features of pulmonary infection (dyspnoea 12%, hypoxaemia 6%, tachypnoea 7% and cough 6%) were reported in some cases of disseminated infection. Seven patients presented with meningitis, one disseminated case with neck rigidity and positive CSF and six cases with brain involvement. In these patients with cerebral toxoplasmosis, two had neck rigidity and negative CSF and four had positive CSF without clinical symptoms of meningitis. Miscellaneous features included hemichorea (2 cases), aphasia (1 case), ataxia (1 case), muscle pain (1 case), behaviour change (1 case), difficulty in swallowing (1 case), tachycardia (1 case), arrhythmia (1 case), heart failure (2 cases), chest pain (1 case), lymphadenopathy (2 cases) and ARDS (1 case).

Treatment: Of the 110 cases reviewed, 88 had data on treatment (Table 2). Forty-one (47%) received a specific anti-toxoplasma therapy. Eighty percent of patients were treated with pyrimethamine and sulphadiazine (P/S) and in three of these patients, other antibiotics (clindamycin in 1 and spiramycin in 2 cases) were added. Twenty per cent of patients received other combinations of antibiotics: trimethoprim-sulphamethoxazole (TMP-SMZ) (2 cases), TMP-SMZ and clindamycin (1 case), sulphadiazine and TMP (1 case), or pyrimethamine and clindamycin (4 cases). In one treated patient, the specific type of antitoxoplasma antibiotic was unknown.

Outcome: Eighty-eight (80%) of 110 reported cases died a median of 87 days after BMT (range 12-730) (Table 2). Ninety of 110 cases had data allowing analysis of attributable mortality. The attributable mortality rate was 66% and the median day of death was 11.5 (range 1-460) post-toxoplasmosis diagnosis and 74 (12-730) post BMT.

Univariate analysis showed site of infection (P = 0.003), day of onset (P = 0.04) and conditioning regimen (TBI+CY vs TBI+CHT vs CHT) (P = 0.04) to be significant variables (Table 5). The outcome was best in cases of isolated cerebral involvement (60% of those who survived) and worst in disseminated infection (64% of fatal cases). Patients with the onset between 31 and 100 days after BMT had a poor prognosis (only 24% survived). They represented 74% of all fatal cases. Mortality and survival rates were similar in patients with onset of infection in the first 30 days (46% survived) and after 100 days (53% survived). Seventy-four percent of subjects who died received the conditioning regimen TBI+CY vs 4% (TBI+CHT) and 22% (CHT). On multivariate analysis (Table 5), the only significant variable was site of infection (P = 0.002). Logistic regression analysis showed that patients with disseminated toxoplasmosis had 5.28 times the odds of dying (of toxoplasmosis) compared with subjects with cerebral toxoplasmosis (95% CI 1.93 to 15.5).

Patients with the diagnosis made during life: We evaluated the clinical features in patients in whom the diagnosis was made ante-mortem (Table 2). All such cases were treated and the combination P/S had a better outcome (P = 0.05): 88% of these cases survived vs 12% receiving other treatments. The attributable mortality rate was 40% with a median day of death of 22.5 (2-460) days post-toxoplasmosis diagnosis and 92 (28-730) days post BMT. Univariate analysis showed underlying disease (P = 0.02) and day of onset (P = 0.04) to be significant variables. Outcome was better in the subjects with a late onset (>100 days) of whom 89% survived. Outcome was worst for cases with the onset between 31 and 100 days. They represented 80% of fatal cases. In the analysis of the role of underlying disease (CML vs acute leukaemia (AL) vs other), CML patients had a poor prognosis (69% died). In AL patients, only 18% died from toxoplasmosis. To further evaluate whether this resulted from other reasons, we analyzed the distribution of basic patient characteristics. We observed that 77% of CML patients were older than 30 years of age and 67% of AL patients were less than 30 (P = 0.02). In the multivariate model (Table 5), the only significant variable was underlying disease (P = 0.03). Patients with CML had 9.45 times the odds of dying compared with patients with AL (95% CI 1.20 to 74.1).

Cerebral and disseminated toxoplasmosis

After analyzing all the reported cases of toxoplasmosis in BMT recipients as a single group, we compared the clinical features of cerebral and disseminated toxoplasmosis. The median day of onset post BMT was 76 (8-689) days in cerebral toxoplasmosis and 55 (1-365) days in disseminated toxoplasmosis (P = 0.09).

Fifty-eight percent of cerebral cases survived. Fatal cases died a median 35 (5-460) days post diagnosis and 113 (48-730) days post BMT. In disseminated toxoplasmosis, 80% of cases died with a median day of death of 10 (1-235) days post diagnosis and 62.5 (12-290) days post BMT. There were statistically significant differences in mortality rates between cerebral and disseminated toxoplasmosis (P = 0.002 and P = 0.004 for attributable and crude mortality, respectively). To assess the reason for this difference, we compared basic patient characteristics in the two groups by ² test. There was no difference in gender, age, GVHD and immunosuppressive therapy, pre-BMT recipient or donor serostatus, type of transplant or conditioning regimen. The only statistically significant difference was underlying disease (P = 0.01): 41% of patients with brain involvement had CML, 33% had other diseases and 23% had AL. In disseminated infection, the most common underlying disease was AL.

Discussion

Toxoplasmosis is a rare opportunistic infection in BMT recipients. The incidence of this infection after BMT is not well established and it is probably underestimated in view of the difficulty in making a diagnosis. The incidence varies geographically with the frequency of seropositivity in the general population and ranges from 0.3% in the USA^3,2,15 to 5% in France,⁴ where the seropositivity of the population is 3-68%^56,57 and 50-80%,⁴⁸ respectively. In the UK, Pendry et al⁶ described the first cases in 1990 and reported an incidence of toxoplasmosis in BMT recipients of 1.25%. Including the two cases described here, five UK cases have been reported. In our centre, we have had only two confirmed cases of toxoplasmosis in 631 allogeneic transplants between January 1978 and December 2000 (an incidence of 0.32%).

Diagnosing toxoplasmosis in transplant recipients is often difficult because of the non-specific signs and symptoms of the infection^4,15,48 and because immunosuppressive therapy or irradiation impairs the recipient's immune response. When neurological symptoms, especially with fever, occur in BMT recipients with risk factors (positive pretransplant serology and GVHD and/or TBI in the conditioning regimen) cerebral toxoplasmosis should be suspected. In disseminated infection, the diagnosis is more difficult to make during life. In the logistic model, these patients had 10.8 times the risk of having the diagnosis made at autopsy. From our analysis, disseminated toxoplasmosis should be considered if pulmonary symptoms or signs and/or fever occur in recipients with risk factors, above all between 30 and 100 days post BMT. In 60% of reported cases, invasive procedures with a risk of serious complications in BMT recipients have been required to confirm the diagnosis. Sensitive and specific non-invasive diagnostic tests for detection of the parasite are required.

More recently, PCR amplification for T. gondii DNA in the blood, the tissues or the body fluids has been used. In HIV-positive patients with cerebral toxoplasmosis, PCR amplification in CSF or blood has a high specificity (100%) but a low sensitivity (30-50%).^59,60 In BMT patients, we do not have data concerning sensitivity or specificity, although Johnson et al⁵² reported one false-negative result. However, many authors consider that PCR testing contributes to a rapid diagnosis and potentially improves outcome, and also allows monitoring of efficacy of treatment.^1,2,5,21,31 The data, however, do not confirm an improvement in outcome in patients diagnosed by PCR. We analyzed the crude and attributable mortality and there was no significant correlation with diagnosis by PCR (P = 0.48). This may be explained by the small number of cases diagnosed by PCR because there is a suggestion of clinical benefit.

The prognosis of toxoplasmosis post BMT was poor, with 66% of patients dying from this infection. However, our data showed that, if treated, 60% of patients survived. This suggested that the high number of fatal cases with no ante-mortem diagnosis or specific antitoxoplasma treatment may have influenced the prognosis.

The most important factor influencing outcome was the site of infection, with the worst prognosis occurring in patients with disseminated infection. The reason for this would appear to be due to the difficulty in making an early diagnosis in disseminated cases who died without having specific therapy. In cerebral toxoplasmosis, 80% of cases had the diagnosis made during life and all of these were treated. In disseminated infection, 80% of cases were diagnosed post-mortem and none of these had received treatment. In fact, site of infection does not present a risk factor in patients with diagnosis during life. In this subgroup, we found that the only significant risk factor for mortality was CML as the underlying disease, which was associated with 9.45 times the risk of death compared with AL. The reason for this is not clear. It may be because these patients were older but also because CML is the most common indication for allogeneic BMT.⁵⁸

Pre-transplant Toxoplasma serology should be tested in both recipient and donor to identify individuals at risk of reactivation or infection. It would seem appropriate to give a course of treatment to recipients with evidence of recent infection (as evidenced by IgM antibodies) in donors or recipients. Prophylaxis should be considered for allogeneic BMT patients with raised IgG titres and other risk factors for reactivation (eg GVHD). It is unclear what constitutes optimal prophylaxis, although co-trimoxazole, given for Pneumocystis carinii prophylaxis may have some effect in this patient group. In 1994, Foot et al⁶¹ demonstrated that no proven cases of toxoplasmosis (or Pneumocystis carinii infection) occurred in 90 Toxoplasma seropositive recipients receiving weekly prophylaxis with pyrimethamine and sulfadoxine (Fansidar). This contrasts with a previous study,⁴ in which they documented four cases of toxoplasmosis in 54 seropositive recipients in the same institution. Unfortunately, this regimen is myelotoxic and there is an understandable reluctance to use these agents in the early post-BMT period. Indeed, this study documented marrow toxicity (requiring discontinuation) in 11.6% of cases.

In conclusion, toxoplasmosis is a relatively rare complication in BMT recipients but it contributes to post- transplant morbidity and mortality in highly endemic countries. Only with heightened awareness can we expect a reduction in incidence and an improvement in outcome. Diagnostic procedures and specific treatment must be considered in all allogeneic BMT recipients who develop prolonged fever of unknown origin or clinical features of infection of the respiratory tract or central nervous system.

Acknowledgements

The authors thank Dr Massimo Offidani (Department of Haematology, University School of Medicine, Ancona) for his assistance in reviewing the manuscript; Dr Richard Morris and Dr Alessandro Cozzi Lepri (Department of Primary Care and Population Services, Royal Free and University College Medical School, London) for their advice on statistical issues; Rita Drobner for review of the German literature and Clare Ling for her technical support (Department of Microbiology, Royal Free Campus and Royal Free Hospital, London). Dr Anna Mele is supported by a University Scholarship from the University School of Medicine, Ancona.

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Table 1 Demographic features of reported cases with toxoplasmosis

Table 2 Clinical features of toxoplasmosis

Table 3 Positive diagnostic tests in patients with diagnosis during life

Table 4 Factors found on univariate and multivariate analysis to influence the diagnosis (during life vs post-mortem)

Table 5 Factors found in univariate and multivariate analysis to influence outcome