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Diagnosis of Toxoplasmosis in Bone Marrow Transplant Recipients: Comparison of PCR-Based Results and Immunohistochemistry

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Diagnosis of Toxoplasmosis in Bone Marrow Transplant Recipients: Comparison of PCR-Based Results and Immunohistochemistry Bone Marrow Transplantation (2000) 25, 1257–1262 2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt Diagnosis of toxoplasmosis in bone marrow transplant recipients: comparison of PCR-based results and immunohistochemistry TK Held1, D Kru¨ger2, AR Switala3, J Beyer4, D Kingreen1, C Busemann1, K Janitschke2 and W Siegert1 1Klinik fu¨r Innere Medizin mit Schwerpunkt Ha¨matologie und Onkologie, Charite´/Campus Virchow-Klinikum, Humboldt-Universita¨t; 2Projekt Parasitologie/Mykologie, Robert Koch-Institut, Berlin; 3Institut fu¨r Pathologie, Charite´/Campus Virchow-Klinikum, Humboldt-Universita¨t, Berlin; and 4Abteilung fu¨rHa¨matologie, Philipps-Universita¨t, Marburg, Germany Summary: regions, depending on local pet habits, especially the presence of cats and food contamination with cysts.1 Toxoplasmosis in bone marrow transplant recipients is In contrast to patients with other forms of immuno- a rare but serious complication and if untreated, almost suppression (eg HIV infection), the incidence of tox- uniformly fatal. The diagnosis, however, remains diffi- oplasmosis in bone marrow transplant recipients is low, cult. We therefore compared serial determination of despite the profound and long lasting immunosuppression antibody titers specific for T. gondii before and after during and after bone marrow transplantation (BMT). Until transplantation, serial PCR for T. gondii DNA in serum, now, 55 cases of disseminated toxoplasmosis following PCR and nested PCR for T. gondii DNA in various BMT have been reported in the English language litera- tissues, conventional histology and immunohistochemis- ture.2 After BMT, the rate of toxoplasmosis diagnosed try for detection of parasites in three patients with auto- either intra vitam or at autopsy ranges from 0.3% in the psy-confirmed toxoplasmosis after bone marrow trans- United States3 to 5% in France.4 Non-invasive diagnosis of plantation. Immunohistochemistry demonstrated the toxoplasmosis after BMT is important since the course of presence of parasites in 13 out of 20 organs investigated untreated toxoplasmosis after BMT is rapid and almost uni- (65%), whereas PCR detected T. gondii-specific DNA in formly fatal.5 Serology after BMT, however, has not been 15 out of 20 organs (75%). Immunohistochemistry informative, since IgG titers do not rise, and may even revealed concordant results to PCR data in 60% of the decline, during infection6,7 and since an IgM response may specimens. With the use of a nested PCR protocol, eight be lacking.6 Alternatively, cysts and free tachyzoites could out of nine samples (89%) were positive for T. gondii- be visualized directly in bronchoalveolar lavage fluid specific DNA. The combination of both methods (BAL)8 or bone marrow aspirates,9 but this approach carries detected the presence of parasites in 90% of the speci- the risks associated with obtaining samples. Tissue culture mens. Serial PCR in serum did not yield positive results. methods have a long turnaround time, and animal inocu- Neither PCR nor immunohistochemistry was able to lation methods may substantially delay the diagnosis.6 detect parasites in all organs investigated, but both Polymerase chain reaction (PCR) has been evaluated as a methods together improved sensitivity to 90% and diagnostic tool suggesting that this method is more sensi- consequently, should be used jointly to maximize diag- tive in detecting T. gondii in infected body fluids than the nostic precision. Bone Marrow Transplantation (2000) 25, available microscopic methods.10 Here we report our inves- 1257–1262. tigations of three patients with disseminated toxoplasmosis Keywords: toxoplasmosis; bone marrow transplantation; after BMT in which we compared two sensitive methods PCR; immunohistochemistry; serology for diagnosing toxoplasmosis. Case reports Toxoplasma gondii is a protozoon affecting and persisting in almost all known mammals. In man, it is known to invade many cell types causing an asymptomatic infection Patient UPN 596 in the vast majority of immunocompetent individuals. A 20-year-old man with acute lymphocytic leukemia in Toxoplasmosis results from reactivation of latent infection second complete remission received a bone marrow trans- when the individual is immunocompromised. The incidence plant from his HLA-identical brother. Serological data for of toxoplasmosis and seropositivity varies considerably the recipient before BMT and donor for toxoplasmosis are between different countries and within distinct geographic given in Table 1. Preparative therapy included total body irradiation (3 ϫ 400 cGy), cyclophosphamide (100 mg/kg body weight) and etoposide (50 mg/kg body weight). After Correspondence: Prof W Siegert, Klinik fu¨r Innere Medizin mit Schwer- punkt Ha¨matologie und Onkologie, Charite´/Campus Virchow-Klinikum, BMT, antibiotic prophylaxis consisted of oral ciprofloxacin, Augustenburger Platz 1, 13353 Berlin, Germany amphotericin B, and acyclovir. Prophylaxis against graft- Received 10 December 1999; accepted 28 March 2000 versus-host disease (GVHD) was performed with cyclospo- Diagnosis of toxoplasmosis after bone marrow transplantation TK Held et al 1258 Table 1 Serology for T. gondii in bone marrow transplant recipients various organs including the lungs contained numerous T. before transplantation gondii pseudocysts. Patient Donor for Patient Patient UPN 596 Patient UPN 639a UPN 676a Patient UPN 676 UPN 56 A 37-year-old man with CML in second chronic phase IgG ϩϩϩϩreceived a bone marrow transplant from an unrelated donor IgM ϪϩϪϪwith one HLA-B antigen mismatch (recipient, HLA-B* IgA Ϫ ND ϩϪ35,Ϫ; donor, HLA-B*15,35). Serological data of the recipi- SFT 1:512 1:512 1:256 1:256 ent before BMT with regard to toxoplasmosis are given in Table 1. Conditioning regimen, post-transplant immuno- a Serology for bone marrow donors of patients UPN 639 and 676 was suppression and antimicrobial prophylaxis were identical to not known. SFT = Sabin–Feldman test; ND = not done. those given to patient UPN 639. Neutropenic fever occurred at day ϩ3 post-BMT. At day ϩ25, he developed grade III acute GVHD with massive watery diarrhea, skin rash, and an increase in serum bilirubin up to 6.0 mg/dl. Treatment rin (CsA) and a short course of methotrexate. The patient with methylprednisolone gradually improved the diarrhea, developed grade III acute GVHD at day ϩ16 after BMT and the rash as well as the bilirubinemia disappeared. and was treated with methylprednisolone. After initial res- After discharge from the hospital, the patient had a posi- olution of the symptoms, acute GVHD reappeared requiring tive PCR and APAAP reaction for CMV (day ϩ57). He increasing doses of corticosteroids and administration of developed fever and clinical and radiologic signs suggestive rabbit anti-thymocyte globulin (ATG). The symptoms of of interstitial pneumonia. Bronchoscopy done on day ϩ59 GVHD resolved again. At day ϩ85, the patient’s mental revealed inflamed bronchi. Bronchoalveolar lavage was status deteriorated, and CT and MRT scans of the brain negative for Pneumocystis carinii, fungi and acid-fast rods. revealed two focal lesions within the right frontal and the Gancyclovir treatment was instituted leading to a resolution temporal lobe, respectively. Toxoplasmosis of the brain of symptoms; PCR and APAAP reaction for CMV was suspected, and systemic therapy with pyrimethamin became negative. (50 mg/day) and clindamycin (4 ϫ 600 mg/day) was begun. At day ϩ76 after BMT, a cranial computed tomography In the cerebrospinal fluid (CSF), PCR for toxoplasma was was performed because of head-aches, nausea, and vomit- positive at day ϩ90. On the same day, positive PCR results ing, revealing four lesions which were consistent with for toxoplasma were obtained in the serum as well. Despite cerebral toxoplasmosis. Therapy was instituted with antiparasitic therapy, the patient expired on day ϩ92. pyrimethamin (75 mg daily), clindamycin (3 ϫ 600 mg), leukovorin, and dexamethasone, which led to a rapid disap- Patient UPN 639 pearance of the patient’s symptoms. Because of pancyto- penia, gancyclovir and pyrimethamin were discontinued at A 37-year-old man suffering from chronic myelogenous days ϩ85 and ϩ119 after BMT, respectively. Control CT leukemia (CML) was transplanted in second chronic phase scans of the brain at day ϩ90 and day ϩ127 did not show with bone marrow from an unrelated donor with one minor any significant changes in the lesions’ size and appearance. DRB1 mismatch. Serological data of the recipient before A stereotactic biopsy, performed at day ϩ104, revealed BMT with regard to toxoplasmosis are given in Table 1. necrotizing inflammatory changes but no microorganisms. Conditioning therapy included busulfan (10 mg/kg body PCR and immunohistochemistry for T. gondii done on the weight), cyclophosphamide (120 mg/kg body weight), and biopsy specimens were negative. PCR for T. gondii in the thiotepa (750 mg/m2) together with rabbit anti-thymocyte cerebrospinal fluid obtained at day ϩ80 and day ϩ111, globulin (ATG). Prophylaxis against GVHD consisted of however, was positive. CsA and a short course of methotrexate. Antimicrobial At day ϩ126 after BMT, the patient presented with signs prophylaxis was performed as described in patient UPN of extensive chronic GVHD. Skin changes resolved on 596. Engraftment was noted at day ϩ20 for neutrophils and prednisone treatment, however, liver function deteriorated. at day ϩ29 for platelets. At day ϩ19 post BMT, the patient At day ϩ161, he was readmitted with weakness, shortness
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