Real-Time Polymerase Chain Reaction for Early Diagnosis of Toxoplasmosis in Stem Cell Transplant Recipients: Ready for Prime Time?

Clinical Infectious Diseases, Jan 2005

P. H. Chandrasekar

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Real-Time Polymerase Chain Reaction for Early Diagnosis of Toxoplasmosis in Stem Cell Transplant Recipients: Ready for Prime Time?

P. H. Chandrasekar () 0 0 Division of Infectious Diseases, Wayne State University School of Medicine and Harper University Hospital , Detroit, Michigan - Toxoplasmosis is an uncommon but often fatal opportunistic infection that occurs after receipt of an allogeneic hematopoietic stem cell transplant (HSCT). A 20year study of 2000 HSCT recipients in North America found only 12 cases of toxoplasmosis, 10 of which were diagnosed at autopsy [1]. Thus far, there have been 150 reported cases of this protozoan infection in HSCT recipients worldwide [2 4]. The exact frequency distribution of toxoplasmosis in different countries is unknown; because the disease mostly occurs with reactivation of the latent parasite, the varying seroprevalence in different geographic regions appears to dictate disease frequency. The rate of toxoplasmosis reactivation in HSCT recipients is estimated to range from !0.5% in areas of low endemicity to 2%3% in areas with a higher seroprevalence, such as France [5]. The true incidence of toxoplasmosis in HSCT recipients is probably underestimated, if one considers the diagnostic difficulty. In contrast, Toxoplasma brain abscesses are recognized much more readily in patients with AIDS. In a French study of patients with AIDS, the incidence of toxoplasmosis was 24% for patients with CD4+ cell counts of !100 cells/mm3 [6]. Other situations in which there is a high risk of serious toxoplasmosis include vertical transmission from infected mother to fetus and solid organ transplantation. In contrast to diagnosis in patients with AIDS, the antemortem diagnosis of toxoplasmosis in HSCT recipients has been elusive and problematic. Most cases diagnosed at autopsy reveal widespread dissemination. The disease typically is seen within the first 6 months after transplantation. Clinical presentation is nonspecific; fever and altered mentation are the most common symptoms. Less common presentations are pneumonitis and myocarditis. Unfortunately, CT and/or MRI findings and results of CSF examinations are not diagnostic. Also, Toxoplasma gondii organisms cannot be directly observed in CSF or pleural fluid specimens. Although serological tests are useful in identifying persons at high risk of acquiring infection (e.g., IgG-seropositive HSCT recipients), they do not aid in the diagnosis of active infection. Histological examination of tissue specimens from the brain, lung, or heart has a high diagnostic yield; however, thrombocytopenia is a major limiting factor in obtaining tissue samples. In recent years, amplification of T. gondii nucleic acid by PCR has been reported as a promising tool for early diagnosis and perhaps may even influence outcome [7, 8]. PCR assays have principally targeted the B1 gene and, less commonly, the P30 (SAG1) gene or rDNA. With use of in-house PCR assays, congenital, ocular, cerebral, and disseminated toxoplasmosis have been diagnosed by detection of the organism in specimens of various body fluids and/or tissues, including amniotic fluid, aqueous humor, CSF, bronchoaveolar lavage fluid, and peripheral blood monocytes. Although the specificity is high (90% 100%), the sensitivity of the assay (33% 50%) in patients with AIDS and encephalitis has been lacking. Nested PCR assays with 2 sets of primers have improved sensitivity. In a survey of 52 European HSCT centers during the period of 19941998, 24 (46%) reported using a PCR technique for the diagnosis of toxoplasmosis [9]. Nevertheless, all PCR studies to date have used in-house methodologies; therefore, a lack of standardization has been a major concern. Furthermore, the problems of false-positive results associated with contamination and of false-negative results associated with polymerase inhibitors have persisted. A quantitative, real-time PCR technique based on LightCycler technology (Roche Molecular Biochemicals) has been developed for the rapid diagnosis of toxoplasmosis within 2 h after samples are obtained. In addition to its high sensitivity and specificity, the test procedure has minimized the possibility of false-positive and false-negative reactions [10]. With use of the real-time PCR technique, the investigators of the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation (EBMT-IDWP) prospectively evaluated 106 Toxoplasma-seropositive adult HSCT recipients during the first 6 months after transplantation. Results of their meticulous study appear in this issue of the journal [11]. The same group, incorporating the PCR application, had previously proposed definitions for Toxoplasma infection and for possible, probable, and definite disease, analogous to the definitions of cytomegalovirus (CMV) infection and disease that were developed to address a similar situation. In their present study [11], the group found Toxoplasma infection (defined as positive PCR results and fever without organ involvement) in 16 patients (16%), and they found probable disease in 6 of these 16 patients. Of these 6 patients, 2 had positive results of blood PCR coinciding with disease onset, whereas in the other 4 patients, positive results of blood PCR preceded clinical findings (5 days before onset of disease and 4 days before onset of infection). Notably, none of the 90 patients with negative PCR results developed disease. Statistical analysis revealed that cord blood transplantation was a risk factor; also, a trend towards infection/disease was seen in patients who were not receiving chemoprophylaxis. The study included high-risk HSCT recipientsnamely, 39% of patents had received transplants from matched, unrelated donors or mismatched donors; 41% had received a reduced-intensity conditioning regimen; 51% had received antithymocyte globulin or T celldepleted stem cells; and 53% had lymphocytopenia on day 30 after transplantation. In summary, the study showed that the real-time PCR was an effective tool for early detection of T. gondii reactivation before the development of toxoplasmosis in highrisk HSCT recipients. Data on quantitation of parasite load by PCR are preliminary and appear to be promising. The EBMT-IDWP deserves to be congratulated on the present study, which clearly has advanced the field of toxoplasmosis in HSCT recipients. Are we ready for routine use of real-time PCR for therapeutic intervention? Not quite. The study was not intended to address the value of preemptive therapy. Several questions are to be raised once real-time PCR commercial kits become available. The exact incidence of Toxoplasma disease developing in untreated HSCT recipients with positive PCR results is not known. A copy-number threshold indicating progression from infection to disease needs to be identified; the PCR kinetics remain to be delineated. The exact type of intervention for infectionthe type of drug and duration of therapyneeds to be defined. Similar to previous CMV studies that have been based on a preemptive PCR strategy, a large randomized study is needed to evaluate the efficacy of intervention. Also, the present study [11] included a substantial number of high-risk HSCT recipients; the incidence of infection and disease, as determined by PCR, in lower-risk HSCT recipients and in other at-risk populations may be different. Posttreatment monitoring and secondary prophylaxis strategies after preemptive therapy have to be explored. Encouragingly, a hypothetical cost analysis of using PCR for toxoplasmosis diagnosis in HSCT recipients has favored its use [7]. In the future, multiplex PCR may be routinely used to detect Toxoplasma species and other opportunists, such as CMV, human herpes virus type 6, and adenovirus, among others. A final comment on prophylaxis. In the population of patients with AIDS, chemoprophylaxis with trimethoprim-sulfamethoxazole (TMP-SMZ) is effective against toxoplasmosis, whereas in HSCT recipients, the drug is recommended, albeit with limited supportive data [12]. In the present study, 58 of 106 patients did not receive adequate prophylaxis with TMP-SMZ; of the 16 who had positive PCR results, 14 had not received TMPSMZ, and of the 6 who developed disease, none had received prophylaxis. TMPSMZ is an effective prophylactic agent against Pneumocystis species, and although breakthrough infections do occur, the drug does provide protection against Toxoplasma and Nocardia infection. All too often, therapy with this drug is discontinued without sound reasons in HSCT recipients. Because safer, more effective, and less expensive alternatives do not exist, the tendency to prematurely discontinue the use of TMP-SMZ needs to be resisted. Acknowledgment Potential conflicts of interest. P.H.C.: no conflicts. References

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P. H. Chandrasekar. Real-Time Polymerase Chain Reaction for Early Diagnosis of Toxoplasmosis in Stem Cell Transplant Recipients: Ready for Prime Time?, Clinical Infectious Diseases, 2005, 79-81, DOI: 10.1086/426449