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

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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 showed symptoms of acute grade IV GVHD involving the of breath, loss of appetite, and edema of the feet. He skin and subsequently the gut and liver. Since treatment showed symptoms of liver failure and sepsis. Blood cul- with methylprednisolone at 3 mg/kg failed, high-dose tures were positive for group B streptococci. The patient methylprednisolone (3 ϫ 250 mg/day) and horse ATG were expired on day ϩ164 after BMT. administered leading to resolution of gut and skin symptoms. However, serum bilirubin remained elevated. At day Serology ϩ28, cytomegalovirus (CMV)-DNA was demonstrated in the peripheral blood by PCR, and antiviral therapy with Patient serum samples collected before BMT and at weekly gancyclovir was initiated. At day ϩ32, the patient intervals thereafter, stored frozen at Ϫ20°C, were available developed pneumonia and an acute respiratory distress syn- for retrospective analysis. Samples were analyzed by the drome which was thought to be related to reactivation of Sabin–Feldman test,11 and Toxoplasma-specific antibodies CMV infection. He died on day ϩ34 from progressive res- were detected using commercially available enzyme immu- piratory insufficiency and multi-organ failure. At autopsy, noassays (IMx-IgG, Abbott, Wiesbaden, Germany;

Bone Marrow Transplantation Diagnosis of toxoplasmosis after bone marrow transplantation TK Held et al 1259 VIDAS-IgM, BioMe´rieux, Marcy l’Etoile, France; Platelia- Oldendorf, Germany) and visualized by staining with ethid- IgA, Sanofi-Pasteur, Freiburg, Germany). ium bromide. To check for false positive reactions or con- taminations, two negative controls were included in each DNA extraction experiment using sterile water and EDTA blood from a healthy volunteer tested negative for T. gondii IgG and IgM For patients UPN 596 and 639, samples of formalin-fixed and were subjected to the DNA extraction procedure tissue (ie heart, brain, lung, intestine, spleen, liver and bone described above. No PCR product was obtained with the marrow) were removed from the fixative, stored at 4°C and negative controls in any experiment. allowed to dry for at least 1 week. Pieces of about 20– 40 mg of the dried tissues were then pulverized with a Histology and immunohistochemistry pestle and suspended in 400 ␮l TE9 lysis buffer12 sup- plemented with 1% SDS and 2 mg/ml proteinase K (Merck, At autopsy, several specimens were taken from brain, heart, Darmstadt, Germany) and were incubated at 55°C over- lungs, intestine, liver, spleen and bone marrow. Also, night. The amount of proteinase K was then increased to biopsies were taken from all macroscopically suspicious a final concentration of 6 mg/ml and the incubation was areas. Tissues were fixed in 5% formalin and embedded in continued for another 24 h. During this incubation pro- paraffin. Sections (4 ␮m) were cut and stained with cedure, the samples were vortexed extensively several hematoxylin/eosin, and in part also with the periodic acid- times. For patient UPN 676, unfixed native tissue was Schiff (PAS) reaction and trichrome light-green (Masson obtained at autopsy, cut into small pieces and lysed in TE9 stain). lysis buffer as described. Immunohistochemistry was performed using a standard Two hundred microliters of the lysate were submitted to alkaline phosphatase-anti alkaline phosphatase (APAAP) a DNA extraction procedure with spin columns (QIAamp method after pronase treatment. For detection of T. gondii, Blood Kit, Qiagen, Hilden, Germany) following the instruc- a polyclonal antibody directed against whole T. gondii para- tions of the manufacturer. Alternatively, a tissue DNA sites (Quartett, Berlin, Germany) was used. CMV-infected extraction kit (NucleoSpin CϩT, Macherey-Nagel, Du¨ren, cells were detected by use of a combination of a mono- Germany) was used for some samples. In order to exclude clonal mouse antibody against the CMV immediate–early possible false negative results, 200 ␮l aliquots of the lysates antigen (clone E13; Paesel ϩ Lorei, Hanau, Germany) and were spiked before DNA extraction with 25 or 50 toxo- a mouse anti-CMV antibody against the delayed early plasmic cells (BK strain) derived from the peritoneum of DNA-binding protein p52 (clone CCH2; Dako, Glostrup, infected NMRI mice. Infection and maintenance of these Denmark). Each antibody was diluted appropriately in Tris mice was done as described10 (obtained from the Zentral- buffer (Dako ChemMate Antibody Diluent; Dako). labor fu¨r das Bundesamt fu¨r gesundheitlichen Verbraucher- schutz und Veterina¨rmedizin, Berlin, Germany). For all three patients, serum and CSF samples were treated with Results the QIAamp Blood Kit. Serology Polymerase chain reaction Serial serology data for specific IgG showed a decline in PCR was performed in a total volume of 50 ␮l containing the amount of serum IgG against T. gondii down to the 1 U of DyNAzyme polymerase (Biometra, Go¨ttingen, cutoff level of the test, which was 3 IU/ml, in patients UPN Germany), 0.2 mm of each dNTP, 0.5 ␮m of each primer 596 and 676, respectively. Specific IgM was consistently 13 and 2.5 mm of MgCl2 in reaction buffer. Toxoplasma tar- below the limit of detection in every patient. In patient UPN get genes for PCR were the B1 gene14 and a 18S ribosomal 676, specific IgA became positive from day ϩ41 post DNA gene.15 The B1 gene sequence was amplified for 40 BMT. In all other patients, specific IgA was below the limit cycles using primers P7/P814 corresponding to positions of detection throughout the respective observation period. 864 to 883 and 694 to 714 of the gene, respectively, and Titers in the SFT were 1:256 in every patient before leading to a PCR product of 190 bp. Ribosomal DNA was BMT and declined to 1:64 in case 1 at day ϩ55 post BMT, amplified under the same conditions and with primers TG in case 2 at day 0 and in case 3 at day ϩ6 post BMT, IIb and TG III, published by Cazenave et al.16 The resulting respectively. product had a size of 88 bp. For nested PCR experiments with the B1 gene as a target, Polymerase chain reaction a first amplification was performed after initial denaturation for 5 min at 95°C for 35 cycles (95°C for 30 s, 55°C for The PCR results obtained with DNA extracted from the 10 s and 70°C for 100 s) with primers P3 and P7B histological material are summarized in Table 2. Of the 18 (positions 661 to 680 and 868 to 887), which yielded a organs investigated, 11 (61%) were positive with immuno- product of 227 bp as a target for inner primers P7/P8. After histochemistry, and nine (50%) yielded positive PCR the first 35 cycles, 1 ␮l of the sample was transferred into results with both target genes. Five organs (28%) gave posi- 49 ␮l of fresh master mix containing the inner primer pair tive PCR results with either one of the target genes used. and the procedure was repeated. In only two organs (11%), no parasites could be detected PCR products were detected after gel electrophoresis either by immunohistochemistry or by one of the PCR sys- (100 V, 70 min, 2% MetaPhor agarose, BIOzym, Hess, tems (Table 2). With specimens derived from lungs, intes-

Bone Marrow Transplantation Diagnosis of toxoplasmosis after bone marrow transplantation TK Held et al 1260 Table 2 PCR and immunohistochemistry for T. gondii in various there were also multiple small areas of necrosis in lungs, organs at autopsy liver, and spleen containing pseudocysts and isolated organisms of T. gondii. We also found single tachyzoits in the Patient Organ PCR results (target Immunohisto- bone marrow of patient UPN 596; patient UPN 676 showed UPN gene) chemistry isolated organisms within the liver. In patient UPN 639, pseudocysts of T. gondii were found within vital tissue of B1 rDNA Nested B1 the lungs, liver, and bone marrow; and isolated tachyzoits were detected in the intestinal mucosa. All three patients 596 Brain ϪϪ ϩ ϩ showed only a slight inflammatory reaction, if any, in the Liver ϩϪ ϩ ϩ affected regions. Spleen ϪϩND ϩ Lungsa ϩϩ ϩ ϩ Intestine ϩϩ ϩ Ϫ Comparison of immunohistochemistry with PCR results Heart ϩϩ ϩ ϩ Viewed separately, immunohistochemistry alone demon- 639 Brain ϪϪ ϩ ϩ Liver ϪϩND ϩ strated parasites in 13 out of 20 organs investigated (65%), Lungsa ϩϪ ϩ ϩ whereas PCR gave signals with either one or both systems Intestine ϩϩND ϩ in 15 out of 20 organs (75%). Immunohistochemistry Bone marrow ϩϪ ϩ ϩ revealed results concordant to PCR data in 60% of the specimens (12 organs, four of which were positive in both 676 Brain (left) ϩϩND ϩ Brain (right) ϪϩND ϩ PCR systems and in immunohistochemistry, six of which Liver ϪϪ Ϫ ϩ were positive in at least one PCR system and in immuno- Spleen ϩϩND Ϫ histochemistry, and two of which were found to be negative Lung (left)a ϩϩND Ϫ with all three methods) (Table 2). When PCR was positive Lung (right)a ϩϩND Ϫ Intestine ϪϪND Ϫ in both or either one of the systems, immunohistochemistry Heart ϪϪND Ϫ did not detect any parasites in five out of 20 organs investi- Bone marrow ϩϩND Ϫ gated (25%). In three specimens (15%), immunohistochemistry demonstrated the presence of parasites when PCR did aImmunohistochemistry was positive for CMV-antigen. not yield an amplification product in either system. When ϩ = positive result; Ϫ = negative result; ND = not done due to lack used together, however, PCR or immunohistochemistry of material. demonstrated the presence of parasites in 18 of 20 specimens (90%). The use of a nested PCR protocol for the B1 gene detected toxoplasmic DNA in eight of nine samples tine, and heart of patient UPN 596, positive and reproduc- (89%), and two of three samples, which were negative with ible PCR results could be obtained with primers for both conventional PCR but positive by immunohistochemistry, target genes. DNA from spleen and liver of patient UPN were positive by nested PCR (brains of patients 596 and 596 and from intestine, liver, and lungs of patient UPN 639 639). also showed positive signals after PCR of one target gene. However, these results were not reproduced with the respective second test system. Patient UPN 676 showed reproducible PCR results from brain, lungs, spleen and Discussion bone marrow. All samples gave positive signals when being spiked with 25 or 50 toxoplasmic cells before DNA Toxoplasmosis is a rare, but serious complication following extraction. BMT. It occurs as early as 30 days after BMT (our patient Retrospectively, some of the formalin-fixed samples UPN 639) as well as being a late complication up to 12 from UPN 596 and UPN 639 were tested with a nested months after BMT.9 The prevalence depends, among other PCR procedure for the B1 gene recently developed in our variables, on the frequency of autopsies performed and on laboratory. With this method, eight out of nine samples the diagnostic acumen of the physicians. For example, in were positive for Toxoplasma DNA which otherwise gave the largest series of patients reported, diagnosis was made contradictory results either by immunohistochemistry or intra vitam in only two out of 12 patients (17%).3 The between the two target genes by conventional PCR (Table second series, however, reported a diagnosis made during 2). Unfortunately, DNA from 11 samples was no longer life in five out of seven patients by demonstration of the available for testing with nested PCR. parasites in the blood, CSF and BAL in three out of seven patients and improvement after therapy with Conventional histology pyrimethamine/sulfadiazine in three out of seven patients as a diagnostic indicator.7 In our patients, diagnosis was Using hematoxylin/eosin as well as Masson staining and suggested by clinical signs and typical CT scans of the the PAS reaction, multiple large foci of cerebral necrosis brain in patients UPN 596 and 676, whereas in patient UPN were seen in patients UPN 596 and 676, respectively. 639 the diagnosis was made at autopsy. There was no dem- Within the necrotic areas as well as within vital tissue next onstration of parasites during the clinical course despite to the necroses, multiple pseudocysts of T. gondii and iso- various invasive diagnostic procedures in patient UPN 676. lated tachyzoits could be detected. In patient UPN 596, Definitive diagnosis was made in all patients at autopsy

Bone Marrow Transplantation Diagnosis of toxoplasmosis after bone marrow transplantation TK Held et al 1261 by demonstration of parasites and their DNA using both side. Our results indicate that PCR may have the potential immunohistochemistry and PCR. to be a definitive diagnostic procedure in combination with Invasive procedures in BMT recipients carry a high risk immunohistochemistry. Neither method, when used alone, for complications, and non-invasive methods for detection was able to detect parasites in all tissues investigated. When of T. gondii have long been sought. Although serology is used together, however, the presence of parasites was generally considered not to be helpful in BMT recipients, detected in 90% of the specimens. The use of a more sensi- one report discusses the value of declining IgG antibody tive nested PCR procedure may further increase the sensi- titers in a case of disseminated toxoplasmosis speculating tivity. New protocols are currently evaluated in our labora- that this might have been due to the short half life of circul- tory to improve the reproducibility of this method when ating specific antibodies during active infection and the not working with formalin-fixed samples.22 yet optimal production of these antibodies.17 Two of our patients who survived long enough after BMT displayed the same phenomenon of declining IgG antibody titers. This Acknowledgements observation, however, requires confirmation by large num- bers of patients before diagnostic clues can be obtained We gratefully acknowledge the technical assistance of Ms C Bug, from serology after BMT. E Kamal and G Stoffels. We also thank the nurses and physicians Other diagnostic methods rely on the demonstration of of our bone marrow transplant unit who excellently cared for the parasite in tissue specimens or BAL either by direct our patients. visualization of the parasites18 or by tissue culture methods using peripheral blood buffy coat cells6 and BAL,18 respectively. Disadvantages of these approaches are either low References parasite density resulting in decreased sensitivity when direct visualization is used or the delay of diagnosis with 1 Beaman MH, McCabe RE, Wong S-Y et al. Toxoplasma gon- tissue culture methods. Recently, PCR has been used to dii. In: Mandell GL, Bennett JE, Dolin R (eds). Principles and facilitate the detection of T. gondii.9 Although T. gondii has Practice of Infectious Diseases. Churchill Livingstone: New been detected by PCR in blood and buffy coat specimens,1 York, 1995, pp 2455–2475. 2 Chandrasekar PH, Momin F, the Bone Marrow Transplant in all three patients reported here, PCR in serum was con- Team. Disseminated toxoplasmosis in marrow recipients: a sistently negative with the exception of patient UPN 596, report of three cases and a review of the literature. Bone where it became positive 2 days prior to his death. It may Marrow Transplant 1997; 19: 685–689. be possible that T. gondii is separated from the serum, along 3 Slavin MA, Meyers JD, Remington JS et al. Toxoplasma gon- with the cellular blood components, by centrifugation. In a dii infection in marrow transplant recipients: a 20 year experi- prospective study, we now investigate whole EDTA blood ence. Bone Marrow Transplant 1994; 13: 549–557. for the presence of T. gondii DNA after BMT using the 4 Derouin F, Gluckman E, Beauvais B et al. Toxoplasma infec- nested PCR technique described above. Indeed, by using tion after human allogeneic bone marrow transplantation: whole blood rather than serum, we were able to detect para- clinical and serological study of 80 patients. Bone Marrow sitemia in a number of patients during their course after Transplant 1986; 1: 67–73. 5 Israelski DM, Remington JS. Toxoplasmosis in the non-AIDS BMT. At this stage, however, this approach is still investi- immunocompromised host. In: Remington JS, Schwartz M gational, and any conclusions about the routine use of PCR (eds). Current Clinical Topics in Infectious Diseases. of whole blood for monitoring patients at risk would be Blackwell Scientific: Boston, 1993, pp 322–356. premature. 6 Shepp DH, Hackman RC, Conley FK et al. Toxoplasma gondii Poor quality template DNA due to fixation of tissues with reactivation identified by detection of parasitemia in tissue cul- formalin19 may be a reason for discrepancies when compar- ture. Ann Intern Med 1985; 103: 218–221. ing negative PCR results with positive immunohistochem- 7 Derouin F, Devergie A, Auber P et al. Toxoplasmosis in bone ical findings. On the other hand, a positive DNA amplifi- marrow-transplant recipients: report of seven cases and cation which does not correlate with immunohistochemistry review. Clin Infect Dis 1992; 15: 267–270. (eg intestine samples of patient UPN 596 and lung and 8 Bottone EJ. Diagnosis of acute pulmonary toxoplasmosis by visualization of invasive and intracellular tachyzoites in spleen samples of patient UPN 676) may be due to the Giemsa-stained smears of bronchoalveolar lavage fluid. J Clin higher sensitivity of the PCR method. A false positive Microbiol 1991; 29: 2626–2627. result due to carryover contamination is unlikely because 9 Bretagne S, Costa JM, Kuentz M et al. Late toxoplasmosis precautions were strictly followed in our laboratory20 and evidenced by PCR in a marrow transplant recipient. Bone because a positive result with the negative controls was Marrow Transplant 1995; 15: 809–811. never observed. Contamination, however, may have 10 Roth A, Roth B, Ho¨ffken G et al. Application of the poly- occurred during autopsy, sample handling, or sample stor- merase chain reaction in the diagnosis of pulmonary tox- age in close contact with other tissues containing specific oplasmosis in immunocompromised patients. Eur J Clin DNA.21 Furthermore, it should be mentioned that in patient Microbiol Infect Dis 1992; 11: 1177–1181. UPN 596, a positive PCR result for Toxoplasma was 11 Bundesgesundheitsamt. Sabin-Feldman-Test (SFT) zum Nachweis von Antiko¨rpern gegen Toxoplasma gondii obtained in serum 2 days before death. Hence, contami- (Routinemethode). Bundesgesundheitsblatt 1989; 32: 553– nation of the intestine with toxoplasmic DNA via the blood- 555. stream can not be excluded in this patient. 12 Goelz SE, Hamilton SR, Vogelstein B. Purification of DNA This is, to our knowledge, the first report comparing two from formaldehyde fixed and paraffin embedded human tissue. methods of diagnosing toxoplasmosis after BMT side-by- Biochem Biophys Res Commun 1985; 130: 118–126.

Bone Marrow Transplantation Diagnosis of toxoplasmosis after bone marrow transplantation TK Held et al 1262 13 Kru¨ger D, Weise W, Bug C et al. Investigations on selected 18 Derouin F, Sarfati C, Beauvais B et al. Laboratory diagnosis red cell concentrates for Toxoplasma and Toxoplasma–DNA. of pulmonary toxoplasmosis in patients with acquired Infusionsther Transfusionsmed 1997; 24: 82–85. immunodeficiency syndrome. J Clin Microbiol 1989; 27: 14 Khalifa KES, Roth A, Roth B et al. Value of PCR for evaluat- 1661–1663. ing occurrence of parasitemia in immunocompromised 19 Crisan D, Mattson JC. Retrospective DNA analysis using patients with cerebral and extracerebral toxoplasmosis. J Clin fixed tissue specimens. DNA Cell Biol 1993; 12: 455–464. Microbiol 1994; 32: 2813–2819. 20 Kwok S, Higuchi R. Avoiding false positives with PCR. Nat- 15 Ellis JT, Luton K, Baverstock PR et al. Phylogenetic relation- ure 1989; 339: 237–238. ships between Toxoplasma and Sarcocystis deduced from a 21 Romero RL, Juston AC, Ballantyne J et al. The applicability comparison of 18S rDNA sequences. Parasitology 1995; 110: of formalin-fixed and formalin fixed paraffin embedded tissues 521–528. in forensic DNA analysis. J Forensic Sci 1997; 42: 708–714. 16 Cazenave J, Cheyrou A, Begueret J. Improvement of rDNA- 22 Shedlock AM, Haygood MG, Pietsch TW et al. Enhanced based PCR assay to detect Toxoplasma. Prenat Diag 1993; DNA extraction and PCR amplification of mitochondrial 13: 543. genes from formalin-fixed museum specimens. Bio Tech- 17 Heurkens AHM, Koelma IA, de Planque MM et al. Failure to niques 1997; 22: 394–400. diagnose fatal disseminated toxoplasmosis in a bone marrow transplant recipient: the possible significance of declining antibody titres. J Immunol 1989; 18: 283–288.

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