Recipient of kidney from donor with asymptomatic infection by Paracoccidioides brasiliensis

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1 Medical Mycology February 2012, 50, Case Reports Recipient of kidney from donor with asymptomatic infection by Paracoccidioides brasiliensis MARJORIE V. BATISTA *, PAULA K. SATO, LIGIA C. PIERROTTI *, FLAVIO J. DE PAULA, GUSTAVO F. FERREIRA, DAISA S. RIBEIRO-DAVID #, WILLIAM C. NAHAS, MARIA I. S. DUARTE # & MARIA A. SHIKANAI-YASUDA * * Department of Infectious and Parasitic Diseases, Faculdade de Medicina, University of S ã o Paulo ( FMUSP ), Laboratory of Immunology ( LIM 48 ), FMUSP, Infections in Immunocompromised Host Group, Hospital das Cl í nicas da Faculdade de Medicina, University of S ã o Paulo ( HCFMUSP ), Renal Transplant Unit, Urological Division, HCFMUSP, and # Department of Pathology Diseases, FMUSP, S ã o Paulo, Brazil Introduction Paracoccidioidomycosis (PCM) is a chronic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. It is the most common systemic mycosis in South America, especially in the Southern and Southeastern regions of Brazil, with an estimated annual incidence of 1 3 cases per 100,000 inhabitants [1,2]. The main risk factors for infection are rural activities related to soil management. The infection occurs through inhalation of P. brasiliensis conidia that subsequently transform into yeast cells in host tissue. The mycosis can present in acute, subacute or chronic forms. In the latter, the lungs are the main organs affected, but the skin and mucous membranes may also be involved. Full resolution and return to health depends on an effective cellular immune response, generally associated with type 1 cytokine secretion pattern [1]. Received 9 January 2011 ; Received in final revised form 9 March 2011 ; Accepted 17 March Correspondence: Marjorie Vieira Batista, University of S ã o Paulo, Laboratory of Medical Investigation, Immunology (LIM48), Hospital das Cl í nicas, Faculdade de Medicina, S ã o Paulo, Brazil. marjorie. mi@gmail.com The increase in solid organ transplantations may soon create a rise in the occurrence of endemic fungal diseases, such as paracoccidioidomycosis, due to the lack of rigorous screening of donors from endemic areas. Here we present the first case of an immunocompetent and asymptomatic kidney donor who had Paracoccidioides brasiliensis infected-adrenal tissue but no glandular dysfunction. Keywords tropical disease, transplant infectious disease, endemic mycosis, Paracoccidioides brasiliensis, paracoccidioidomycosis Asymptomatic paracoccidiodomycosis has been documented through necropsy of individuals without signs or symptoms of paracoccidioidmycosis and by case reports of regressive pulmonary involvement without symptomatic disease [3]. Paracoccidioidomycosis is not a common event in the immunocompromised such as those with HIV infections or patients undergoing solid organ transplantation (SOT) [4 9]. In SOT recipients, PCM may occur as a primary infection in a susceptible recipient after exposure to fungal conidia or through donor-transmitted infection, as well as a reactivation of dormant PCM. There are four reports in the literature describing seven cases of PCM infection in SOT recipients which were diagnosed several years after transplantation (Table 1). The mean time from transplant to the diagnosis of PCM infection was 10 years (range, 5 14 years) after transplantation. Lungs, mucosa and skin were the organs most frequently involved and severe progression of disease occurred in some cases. The patients in two out of the seven reported cases died as a result of the reactivation of dormant infections. Here, we report a case of an immunocompetent male kidney donor with an asymptomatic P. brasiliensis - infection in the adrenal gland and the resulting follow-up of the recipient s status ISHAM DOI: /

2 188 Batista et al. Table 1 Reported cases with paracoccidioidomycosis after transplant. n Study, year Age (sex) Epid Country aonset (year) b Organ Immunos. regimens Diagnosis Radiologic finds (lungs) Treatment Outcome 1 Sugar et al, (M) Yes El Salvador 11 K Aza, Pred Lung bx/ BAL cul, Nod Ampho Alive CF ID 2 Reis et al, NA NA Brazil NA K NA NA NA NA Alive 3 NA NA Brazil NA K NA NA NA NA Alive 4 NA NA Brazil NA K NA NA NA NA Alive 5 NA NA Brazil NA K NA NA NA NA Dead 6 Shikanai-Yasuda 29 (F) Yes Brazil 5 K Aza, Pred Lung bx/ BAL cul, Cons AmB Dead et al Zavascki et al (M) Yes Brazil 14 K Aza, Cysp A, Pred CF CIE Skin bx, sputum cul. Cav AmB, Itr Alive NOTE. n, number of patients; a, Time of; b, Type of organ transplanted; Epid, Epidemiology; Immunos., Immunosuppressive; Aza, azathioprine; Cysp A, cyclosporine A; Pred, prednisone; Cul, culture; bx, biopsy; BAL, bronchoalveolar lavage; CF, Complement fixation reaction; CIE, counterimmunoelectrophoresis; Nod, Nodular; Cons, Consolidation; Cav, Cavity; AmB, amphotericin B (colloidal dispersion or lipid formulations); Itr, itraconazole; NA, not available. Case report Donor On 19 February 2008, a 53-year-old asymptomatic male bus driver, who was born and lived in Parana State in the southeast region of Brazil (an endemic area for paracoccidioidomycosis) was evaluated as a potential kidney transplant donor. The most pertinent aspect of his pre-transplant screening history was the fact that he had been involved in rural work in coffee and soybean fields for five years in the 1970s. There were no previous hospitalizations and his physical examination was normal. His preoperative abdominal CT revealed the presence of two nodules (2.0 cm and 1.5 cm) in the right adrenal gland but functional tests for adrenal sufficiency (cortisol and ACTH) showed no abnormalities. Serological tests for HCV, HIV, CMV, HTLV, syphilis, toxoplasmosis and Chagas disease were negative. Hepatitis B markers were indicative of controlled infection. On 1 October 2008, a right-side kidney and adrenal gland from the donor were removed for transplantation. At the time of its removal the gross surface of the adrenal gland was covered by fatty tissue, irregular, orange and with bleeding points. On sectioning, the cortex was normal in appearance except for the presence of whitish irregular, confluent nodules in the region of the spinal cord, the largest of which measured cm. Histopathology of the right adrenal gland revealed a chronic granulomatous process with giant cells and central caseous necrosis, with the presence of numerous fungal forms compatible with Paracoccidioides brasiliensis (Fig. 1.1 and 1.2). Recipient Also on 19 February 2008, a 54-year-old man born in Juquitiba-Sao Paulo, who had lived for five years in a rural area, but denied contact with crops and animals, was referred for kidney transplantation from a living donor. He had chronic renal failure caused by anti-inflammatory therapy for hyperuricemia. Since 2006 he had been on hemodialysis but did not have previous hospitalizations related to infectious diseases. Serology revealed prior contact with CMV. Prophylaxis with itraconazole 400 mg/day was prescribed for the recipient for 12 months after the transplant. On the day of transplantation, 1 October 2008, laboratory evaluation, chest roentgenogram and abdominal CT scan were performed and revealed no abnormalities. Immunological studies of the donor Immunohistochemistry of the adrenal gland from the donor revealed predominance of CD4 and CD8 T cells and expression of TGF- β, TNF-α, IL-10 and IL-4 cytokines in a large number of macrophages, suggesting a Th2 type response at the tissue level (Fig. 1.3, 1.4, 1.5, 1.6, 1.7, 1.8). In vivo and ex-vivo immunological studies of the donor and recipient While the donor did have a positive paracoccidioidin test, immunodiffusion (ID) and counter-immunoelectrophoresis (CIE) assays for P. brasiliensis were negative in both donor and recipient. We performed the more sensitive Western immunoblot and ELISA assays with P. brasiliensis antigens and found that the donor s sample was positive on both, but the recipient s results were all negative. Lymphoproliferation of mononuclear blood cells from the donor and recipient Peripheral blood mononuclear cells (PBMC) from the donor, recipient and one healthy non-sensitized volunteer

3 Kidney donor with infection by P. brasiliensis 189 Fig. 1 (1) Haematoxylin & eosin stain. Chronic granulomatous process with giant cells and central caseous necrosis; (2) Grocott stain. Presence of numerous fungal forms compatible with Paracoccidioides brasiliensis ; (3) predominance of CD4 cells expression in the inflammatory infiltrate; (4) CD8 cells expression in the inflammatory infiltrate; and (5) expression of TNF α ; (6) TGF β cytokine expression; (7) IL-10 cytokine expression. (8) IL4 cytokine expression at the tissular level. The predominance of TH2 type cytokines at the tissular level in the presence of a large number of macrophages, suggests a TH2 type response at this level.

4 190 Batista et al. with negative paracoccidioidin skin test and no P. brasiliensis antibodies, were separated with Ficoll-Hypaque by density gradient centrifugation. The 43 kda glycoprotein (gp43) from P. brasiliensis B-339 strain and a cell wall extract from 18 strain (PbAg) were purified as previously described [10]. Cells were grown in flat-bottom, 96-well microplates at 37 C and 5% CO 2 atmosphere, in the presence of phytohemaglutinin (PHA; 5 μ g/ml), gp43 (1 μg/ml), PbAg (75 μ g/ml) or medium for six days, pulsed with 0.5 μ Ci/well of methyl-[ 3 H] thymidine for 8 h and harvested. Cell-bound radioactivity was measured using a β -plate scintillation counter. Statistical significances were assessed by Kruskal-Wallis test by comparing the differences between the mean counts per minute (cpm) of the triplicates samples from stimulated and non-stimulated cultures from donor, recipient and healthy volunteer. Two months after the kidney transplant, the gp43 P. brasiliensis antigen induced significantly higher proliferation of PBMC from the donor as compared to recipient ( P 0.05). Twelve months after transplant, cellular proliferation of the recipient s PBMC showed normal response to PHA and depressed proliferation to gp43, while the donor s PBMC continued to proliferate in response to this antigen (Fig. 2). PbAg is a crude antigen preparation and no differences were observed as to the proliferation of PBMC derived from all subjects (data not shown). One year after transplantation the donor and the recipient remain asymptomatic with medical care. Discussion Adrenal involvement in PCM infection varies according to the disease severity. Autopsy studies described by Del Negro et al. [12] showed lesions in 50 90% of the patients and outpatient investigations described that about 30% of functional involvement compatible with Addison s disease. Isolated involvement of the adrenals is rare and can be interpreted, in most instances, as a metastatic dissemination from a primary asymptomatic lesion [11,12]. Such cases of adrenal involvement were also described by Del Negro et al. [12] in patients with impaired adrenal function. Histopathologic analysis of the adrenal glands affected by P. brasiliensis revealed the absence of giant cells and typical epithelioid reaction commonly seen in other organs and tissues. Furthermore, extensive necrosis is much more evident in adrenal glands than other organs of the same patient [12,13]. These descriptions were confirmed in our kidney donor, who showed extensive adrenal areas of necrosis associated with multinucleated giant cells, granuloma with epithelioid cells and numerous fungal forms including the presence of several buds. In concert with the pathological findings, immunohistochemical analysis of the donor adrenal gland revealed a Fig. 2 Proliferation of PBMC from donor, recipient and healthy volunteer two and twelve months after transplant. PBMC from donor, recipient and a healthy volunteer were stimulated with PHA and gp43 for 6 days or left untreated (Medium). Cell-bound radioactivity was measured and statistical significances were assessed by Kruskal-Wallis test by comparing the differences between the mean counts per minute (cpm) of the triplicates samples from stimulated and no stimulated cultures from donor, recipient and healthy volunteer. Results were expressed as median range, asterisks denote statistical significance compared to Medium and capped lines denote statistical significance between groups ( P 0.05). predominant tissue Th2 cytokine pattern, distinct from what we would expect for patients with the chronic form of PCM. Predominance of CD68 cells, TGF- β, IL-10 were observed in lymph nodes of patients with juvenile PCM rather than in the mucosa of those with the chronic form of the disease [14,15]. To the best of our knowledge no studies have previously reported cytokine analyses in the adrenal glands of PCM patients. The donor presented an asymptomatic form of PCM with calcified granulomas in the lung on CT scan, high levels of PBMC proliferation induced by gp43 and positive intradermal paracoccidioidin skin test. However, the immunohistochemical staining of the donor s adrenal tissue also indicated P. brasiliensis infection with the presence of multiple fungal particles and chronic granulomatous process with giant cells and central caseous necrosis. A high steroid concentration in adrenal gland would be one possible explanation for the presence of this local immunosuppression and absence of systemic disease. In our report, we emphasize that the donor, as an immunocompetent host, had previous host-parasite interaction leading to an asymptomatic infection, favoring the host. In effect, it appears that immunocompetent lymphocytes demonstrating high proliferation induced by P. brasiliensis antigen can protect infected individuals from clinical symptoms, indicating an effective Th1 immune response [16]. In contrast, similar responses are less frequent when an immunosuppressed recipient receives PCM-infected tissue. Immunosuppressed individuals are more susceptible to severe disease with lymphohematogenic dissemination of P. brasiliensis as described in HIV infected patients [9]. Our kidney transplant recipient s PBMC did not proliferate two months after transplantation when stimulated by PHA

5 Kidney donor with infection by P. brasiliensis 191 and gp43, likely due to high doses of immunosuppressive anti-graft rejection treatment. Additionally, this anergy in the recipient is common in active PCM, usually associated with reversible cellular antigen-specific anergy to stimulation with gp43 and PbAg [17]. However, other laboratory or imaging analyses of the recipient did not show any PCM involvement. Anergy of active PCM has been shown to be reversible after clinical cure, as seen in our recipient s lymphocyte proliferation to PHA [16,17]. Twelve months after transplant, the recipient s PBMC proliferation induced by PHA was reversed and hyporesponsiveness to gp43 remained. The absence of clinical and laboratory involvement attributable to PCM and the recovery of lymphoproliferation probably indicates that this kidney from a P. brasiliensis infected subject was unable to promote infection and dissemination. This interpretation is supported by the fact that the recipient has had a good clinical outcome without symptoms or signs of PCM. This evolution suggests that: (i) the graft did not have fungal particles, or (ii) even though fungal particles were present, they were unable to promote dissemination, and/or (iii) the immunosuppressive regimen required in this period to prevent rejection might have decreased the anti-gp43 response, and/or (iv) the prophylatic use of itraconazole by the recipient could enhance proliferation rather than prevent it. This is the first case report of a kidney donor with PCM infection, with isolated and asymptomatic involvement of the adrenal gland. Additionally, we demonstrated a Th2 cytokine profile in this tissue, consistent with the histopathologic findings described by Del Negro et al. [12], with this same pattern regardless of the clinical form of PCM. Asymptomatic infections in endemic areas for PCM lead to an additional concern with respect to the screening of donors and recipients of solid organ transplantation. The diagnostic tools should be based on knowledge of hostparasite interaction. Therefore, special attention with regard to a careful medical history should be performed, with a particular focus on previous infections and unusual exposures such as residence in endemic regions or travel to these areas, geographic diseases and work in agricultural occupations for living or deceased organ donors. A search for dormant infections should be included in the routine for screening of donors or recipients from endemic regions, e.g., monitoring for calcifications in sites such as adrenal glands, prostate, thyroid, hilar lymph nodes and spleen that could be indicative of past PCM or other granulomatous diseases [18 22]. Diagnostic tools such as imaging methods, skin tests and serology could be used routinely prior to transplantation in both the recipient and the living donor from endemic areas (Fig. 3). However, low levels of antibodies should be carefully interpreted since false-negative results can be observed in immunosuppressed patients. Finally, prevention of primary and/or reactivation infection in recipients of solid organ transplantation depends on the diagnosis of infection in the donor and careful followup of the recipient in the post-transplant period based on clinical suspicion and clinico-laboratory examination. Intradermal tests for diagnosis of primary infection, or presence of humoral antibodies indicative of active disease and laboratory and imaging (chest radiograph, CT scan) tests should be employed to monitor for primary or reactivated fungal infections as suggested in the algorithm shown in Fig. 3. In conclusion a high index of suspicion for possible graft infection should guide the approach to solid organ transplantation in areas of endemic infection. Acknowledgements We thank Richard B. Moss, MD, for helpful and critical manuscript review. Declaration of interest : The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References Fig. 3 (SOT). Screening of donors and recipients of solid organ transplantation 1 Shikanai-Yasuda MA, Telles Filho FQ, Mendes RP, Colombo AL, Moretti ML. Guidelines in paracoccidioidomycosis. Rev Soc Bras Med Trop 2006; 39 : Wanke B, Londero AT. Epidemiology and paracoccidioidomycosis infection. In: Franco M, Lacaz CS, Restrepo-Moreno A, Del Negro G (eds). Paracoccidioidomycosis. CRC Press, Boca Raton, p , Furtado T. Infection versus disease in South American blastomycosis. Int J Dermatol 1975; 14 :

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