Genetic diversity of Pneumocystis jirovecii from a cluster of cases of pneumonia in renal transplant patients: Cross-sectional study.

Pneumocystis jirovecii can cause severe potentially life-threatening pneumonia (PCP) in kidney transplant patients. Prophylaxis of patients against PCP in this setting is usually performed during 6 months after transplantation. The aim of this study is to describe the molecular epidemiology of a cluster of PCP in renal transplant recipients in Brazil. Renal transplant patients who developed PCP between May and December 2011 had their formalin-fixed paraffin-embedded (FFPE) lung biopsy samples analysed. Pneumocystis jirovecii 23S mitochondrial large subunit of ribosomal RNA (23S mtLSU-rRNA), 26S rRNA, and dihydropteroate synthase (DHPS) genes were amplified by polymerase chain reaction (PCR), sequenced, and analysed for genetic variation. During the study period, 17 patients developed PCP (only four infections were documented within the first year after transplantation) and six (35.3%) died. Thirty FFPE samples from 11 patients, including one external control HIV-infected patient, had fungal DNA successfully extracted for further amplification and sequencing for all three genes. A total of five genotypes were identified among the 10 infected patients. Of note, four patients were infected by more than one genotype and seven patients were infected by the same genotype. DNA extracted from FFPE samples can be used for genotyping; this approach allowed us to demonstrate that multiple P. jirovecii strains were responsible for this cluster, and one genotype was found infecting seven patients. The knowledge of the causative agents of PCP may help to develop new initiatives for control and prevention of PCP among patients undergoing renal transplant and improve routine PCP prophylaxis.

Acquired cystic kidney disease in allograft with long-standing poor function.

Acquired Cystic Kidney Disease (ACKD) is regarded as a common late condition of end stage renal damage and expresses its most important features when associated with long term hemodialysis. ACKD is also widely known as a premalignant lesion. Its occurrence in chronically rejected renal allografts is rare and its frequency and behavior in this setting are not well known. Herein we report a case of ACKD in a long standing nonfunctional allograft (215 months) which is not associated with malignancy and briefly review the related literature.

Acquired cystic kidney disease in allograft with long-standing poor function / Doença renal cística adquirida em um enxerto cronicamente disfuncional

Abstract Acquired Cystic Kidney Disease (ACKD) is regarded as a common late condition of end stage renal damage and expresses its most important features when associated with long term hemodialysis. ACKD is also widely known as a premalignant lesion. Its occurrence in chronically rejected renal allografts is rare and its frequency and behavior in this setting are not well known. Herein we report a case of ACKD in a long standing nonfunctional allograft (215 months) which is not associated with malignancy and briefly review the related literature.

Resumo A doença renal cística adquirida (ACKD) é considerada uma condição tardia relacionada à doença renal crônica terminal e manifesta-se de modo mais evidente no contexto de hemodiálise de longo prazo. ACKD é amplamente reconhecida como lesão pré-maligna. Sua ocorrência em enxertos renais cronicamente rejeitados é rara, de modo que a frequência e o comportamento da entidade nesse cenário não estão bem documentados. Relatamos a ocorrência de ACKD em um aloenxerto renal não funcionante sem malignidade após 215 meses de transplante e brevemente revisamos a literatura relacionada.

Absence of mycobacterial DNA in peripheral blood and artery specimens in patients with Takayasu arteritis.

The objective of this study was to demonstrate the presence of mycobacterial nucleic acid sequences in peripheral blood and arteries from patients with Takayasu arteritis (TA). Polymerase chain reaction was performed to detect mycobacterial DNA from three different nucleic acid sequences including the insertion sequence (IS) 6110, the 65-kDa heat shock protein gene (HSP65), and the 16S ribosomal RNA (rRNA) gene in peripheral blood from 32 TA patients and in arterial specimens from 10 TA patients. Twenty-eight HIV-negative patients with pulmonary tuberculosis prior to therapy were tested for IS6110 in peripheral blood as positive controls, and 24 blood donors were evaluated as healthy controls (HC). All TA patients were negative for the insertion sequence IS6110 and for HSP65 and 16S rRNA genes in blood samples and in arterial specimens. IS6110 sequence was found in peripheral blood from 22 (78.5 %) patients with pulmonary tuberculosis but not in HC. In conclusion, the strategy of mycobacterial-specific nucleic acid amplification in the peripheral blood and arterial specimens of TA patients was unable to lend support to the association between TA and tuberculosis long suggested in the literature.

Immunohistochemical assessment of cell populations in leprosy-spectrum lesions and reactional forms.

In situ immunophenotyping of leprosy lesions can improve our understanding of the biology of inflammatory cells during the immune response to Mycobacterium leprae antigens. In the present study, biopsies from 10 healthy controls and 70 leprosy patients were selected, 10 for each of the following conditions: clinical tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL), lepromatous (LL), reversal reaction (R1), and erythema nodosum leprosum (R2). Qualitative and quantitative immunohistochemical analyses were performed to detect CD3, CD4, CD8, FoxP3, CD20, CD138, CD1a, CD57, CD15, CD117, CD68, and CD163. In addition, histochemistry was employed to identify eosinophils. The amount of CD3+ and CD4+ T cells was higher in TT than in LL patients. CD8+ T cells were predominant in T lymphocyte infiltrations in the basal layer of the epidermis. The number of FoxP3+ cells was similar among different forms of the disease, but was higher in BL and LL than in R2 individuals. CD20+ lymphocytes were most abundant in TT samples, while CD138+ plasma cells displayed no detectable differences. Epithelioid macrophages from the center of TT and R1 granulomas exhibited the M1 phenotype (CD68+CD163-), whereas those in LL granulomas showed the M2 phenotype (CD68+CD163+). There was a gradual decrease in the amount of CD1a+ cells from the TT towards the LL form of the disease. A significant increase in the number of neutrophils was observed only in R2 samples. All the cells investigated, except eosinophils, participated in the immunopathogenesis of leprosy.

Immunohistochemical assessment of cell populations in leprosy-spectrum lesions and reactional forms

In situ immunophenotyping of leprosy lesions can improve our understanding of the biology of inflammatory cells during the immune response to Mycobacterium leprae antigens. In the present study, biopsies from 10 healthy controls and 70 leprosy patients were selected, 10 for each of the following conditions clinical tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL), lepromatous (LL), reversal reaction (R1), and erythema nodosum leprosum (R2). Qualitative and quantitative immunohistochemical analyses were performed to detect CD3, CD4, CD8, FoxP3, CD20, CD138, CD1a, CD57, CD15, CD117, CD68, and CD163. In addition, histochemistry was employed to identify eosinophils. The amount of CD3+ and CD4+ T cells was higher in TT than in LL patients. CD8+ T cells were predominant in T lymphocyte infiltrations in the basal layer of the epidermis. The number of FoxP3+ cells was similar among different forms of the disease, but was higher in BL and LL than in R2 individuals. CD20+ lymphocytes were most abundant in TT samples, while CD138+ plasma cells displayed no detectable differences. Epithelioid macrophages from the center of TT and R1 granulomas exhibited the M1 phenotype (CD68+CD163-), whereas those in LL granulomas showed the M2 phenotype (CD68+CD163+). There was a gradual decrease in the amount of CD1a+ cells from the TT towards the LL form of the disease. A significant increase in the number of neutrophils was observed only in R2 samples. All the cells investigated, except eosinophils, participated in the immunopathogenesis of leprosy.

Association of Exercise Training with Tobacco Smoking Prevents Fibrosis but has Adverse Impact on Myocardial Mechanics.

INTRODUCTION: There was no data for cardiac repercussion of exercise training associated with tobacco smoking. This issue is interesting because some smoking people can be enrolled in an exercise-training program. Thus, we evaluated swimming training effects on the function and structural myocardial in rats exposed to tobacco smoking. METHODS: Male Wistar rats were assigned to one of four groups: C, untrained rats without exposure to tobacco smoking; E, exercised rats without exposure to tobacco smoking; CS, untrained rats exposed to tobacco smoking; ECS, exercised rats exposed to tobacco smoking. Rats swam five times a week twice daily (60min per session) for 8 weeks. Before each bout exercise, rats breathed smoke from 20 cigarettes for 60min. Twenty-four hours after the last day of the protocol, papillary muscles were isolated for in vitro analysis of myocardial mechanics. The myocardial mass and nuclear cardiomyocyte volume were used as hypertrophy markers, and collagen content was determined by picrosirius red staining. RESULTS: There was a well-pronounced myocardial hypertrophic effect for two interventions. The exercise blunted myocardial collagen increases induced by tobacco smoking. However, exercise and tobacco-smoking association was deleterious to myocardial performance. Thereby, in vitro experiments with papillary muscles contracting in isometric showed impairment myocardial inotropism in exercised rats exposed to tobacco smoking. CONCLUSIONS: This work presents novel findings on the role of exercise training on cardiac remodeling induced by tobacco smoking. Although exercise has mitigated tissue fibrosis, their association with tobacco smoking exacerbated hypertrophy and in vitro myocardial dysfunction. IMPLICATIONS: This is first study to show that the association of an aerobic exercise training with tobacco smoking intensifies the phenotype of pathological cardiac hypertrophy. Therefore, the combination of interventions resulted in exacerbated myocardial hypertrophy and contractility dysfunction. These findings have significant clinical implication because some smoking people can be enrolled in an exercise-training program.

Subclinical Lesions and Donor-Specific Antibodies in Kidney Transplant Recipients Receiving Tacrolimus-Based Immunosuppressive Regimen Followed by Early Conversion to Sirolimus.

BACKGROUND: There is no evidence on the incidence of subclinical inflammation and scaring lesions in patients receiving tacrolimus (TAC) minimization and elimination immunosuppressive regimens. METHODS: This study analyzed preimplantation, 3 and 24 months protocol biopsies and anti-HLA donor-specific antibodies (DSA) in 140 low immunological risk kidney transplant recipients receiving reduced TAC exposure, prednisone, and mycophenolate, randomized at 3 months to be converted or not to sirolimus (SRL). RESULTS: Mean TAC concentrations were 6.0 ± 2.4 ng/mL and 5.8 ± 2.2 ng/mL at 3 and 24 months. The incidence of subclinical inflammation lesions at 3 months was 9.3%. The incidence of (interstitial fibrosis) IF/(tubular atrophy) TA at month 24 was 57.6%, higher in SRL compared to TAC group (68.8 vs 44.4%; P = 0.022). Patients converted to SRL showed higher incidence of acute rejection (7.3% vs 0%), proteinuria (59.6% vs 25%; P = 0.001), and DSA (17.8% vs 7.3%; P = 0.201), respectively. Biopsy-proven acute rejection (odds ratio [OR] 2.32, 95% confidence interval [95% CI], 0.979-5.518, P = 0.056), subclinical inflammation lesions at 3 months (OR, 11.75; 95% CI, 1.286-107.474; P = 0.029) and conversion to SRL (OR, 2.72; 95% CI, 1.155-6.383; P = 0.022) were associated with IF/TA at month 24. Black ethnicity (OR, 0.22; 95% CI, 0.058-0.873; P = 0.031), donor age (OR, 2.74; 95% CI, 1.329-5.649; P = 0.006), and conversion to SRL (OR, 2.34; 95% CI, 1.043-5.267; P = 0.039) were associated with inferior renal function at 24 months. CONCLUSIONS: In kidney transplant recipients receiving reduced TAC exposure, subclinical inflammation lesions at 3 months were associated with IF/TA at 24 months. Conversion from TAC to SRL was associated with inferior renal function, higher incidence of IF/TA, and trends to higher incidence of DSA at 24 months.

EXPERIMENTAL PARACOCCIDIOIDOMYCOSIS IN PREGNANT RATS.

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb), is the most prevalent systemic mycosis in Latin America. There are few reports in the literature about the disease damages during pregnancy and the consequences to the fetuses and breeding. This study evaluated the implications of PCM during pregnancy on offspring and mothers in Wistar rats. Groups of rats were submitted to systemic Pb infection, by intraperitoneal infusion, and mated 30 days after the infection date. Immediately after birth, rats and neonates were sacrificed to obtain organs for standard histological examination, morphometric analysis, fungi recovery by plating (CFU) and dosing of anti-Pb antibodies by ELISA. There were no stillbirths or miscarriages, however, the fetuses from infected pregnant rats had lower body and organ weight but the fertility rate was 100%. The largest number of CFU was recovered from the organ of pregnant rats, the pathological examination revealed more severe infection in the same group, further on the largest number of granulomas and fungal field. It can be concluded that the PCM was more severe in the group of pregnant rats, with implications to the weight of offspring.

Dose response effect of Paracoccidioides brasiliensis in an experimental model of arthritis.

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb) and corresponds to prevalent systemic mycosis in Latin America. The aim of the present work was to evaluate the dose response effect of the fungal yeast phase for the standardization of an experimental model of septic arthritis. The experiments were performed with groups of 14 rats that received doses of 103, 104 or 105 P. brasiliensis (Pb18) cells. The fungi were injected in 50 µL of phosphate-buffered saline (PBS) directly into the knee joints of the animals. The following parameters were analyzed in this work: the formation of swelling in knees infused with yeast cells and the radiological and anatomopathological alterations, besides antibody titer by ELISA. After 15 days of infection, signs of inflammation were evident. At 45 days, some features of damage and necrosis were observed in the articular cartilage. The systemic dissemination of the fungus was observed in 11% of the inoculated animals, and it was concluded that the experimental model is able to mimic articular PCM in humans and that the dose of 105 yeast cells can be used as standard in this model.

DOSE RESPONSE EFFECT OF Paracoccidioides brasiliensis IN AN EXPERIMENTAL MODEL OF ARTHRITIS / Efeito da dose resposta de Paracoccidioides brasiliensis no modelo experimental de artrite

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb) and corresponds to prevalent systemic mycosis in Latin America. The aim of the present work was to evaluate the dose response effect of the fungal yeast phase for the standardization of an experimental model of septic arthritis. The experiments were performed with groups of 14 rats that received doses of 103, 104 or 105 P. brasiliensis (Pb18) cells. The fungi were injected in 50 µL of phosphate-buffered saline (PBS) directly into the knee joints of the animals. The following parameters were analyzed in this work: the formation of swelling in knees infused with yeast cells and the radiological and anatomopathological alterations, besides antibody titer by ELISA. After 15 days of infection, signs of inflammation were evident. At 45 days, some features of damage and necrosis were observed in the articular cartilage. The systemic dissemination of the fungus was observed in 11% of the inoculated animals, and it was concluded that the experimental model is able to mimic articular PCM in humans and that the dose of 105 yeast cells can be used as standard in this model.

A paracoccidioidomicose (PCM) é causada pelo fungo dimórfico Paracoccidioides brasiliensis (Pb) e corresponde à micose sistêmica de maior prevalência na América Latina. O objetivo do presente trabalho foi avaliar a dose resposta de leveduras do fungo para padronização do modelo experimental de artrite séptica. Os experimentos foram realizados com grupos de 14 ratos que receberam doses de 103, 104 ou 105 células de P. brasiliensis (Pb18). Os fungos foram injetados em 50 µL de solução salina em tampão fosfatado (PBS) diretamente na articulação do joelho dos animais. Os seguintes parâmetros foram analisados neste trabalho: a formação de edema nos joelhos infundidos com as células das leveduras e alterações radiológicas, anatopalógicas além de titulação de anticorpos por Elisa. Após 15 dias de infecção, os sinais de inflamação foram evidentes. Aos 45 dias, algumas características de dano e necrose foram observadas na cartilagem articular. A disseminação sistêmica do fungo foi observada em 11% dos animais inoculados, concluiu-se que o modelo experimental é capaz de mimetizar a PCM articular em humanos e que a dose de 105 leveduras representa a dose padrão para o desenvolvimento do modelo.

Transplantation with kidneys retrieved from deceased donors with acute renal failure.

BACKGROUND: The discard rate of kidneys recovered from deceased donors with acute renal failure (ARF) is higher compared with those without ARF mainly due to the uncertainty regarding short-term and long-term outcomes. METHODS: We retrospectively analyzed 1-year patient, graft, and rejection-free survivals and renal function of transplantations performed with kidneys recovered from deceased donors with or without ARF, defined as serum creatinine level of more than 1.5 mg/dL. We performed multivariable analysis to evaluate whether ARF was an independent risk factor associated with inferior outcomes. RESULTS: Of a total of 1518 patients, 253 received kidneys from expanded-criteria donors (ECD; with ARF [n=116] and without ARF [n=137]) and 1265 from standard-criteria donors (SCD; with ARF [n=369] and without ARF [n=896]). The incidence of delayed graft function was higher in ECD (68.1% vs. 58.4%; P=0.072) and SCD (69.9% vs. 50.6%; P<0.001) recipients of kidneys with ARF compared with those without ARF, respectively. At 1 year, patient, graft, and rejection-free survivals were not statistically different in SCD or ECD recipients with or without ARF. Renal function at 1 year was similar in recipients of ECD (41.9±26.3 vs. 40.1±21.7 mL/min; P=0.565) or SCD (50.9±29.9 vs. 53.6±28.5 mL/min; P=0.131) kidneys with and without ARF, respectively. Compared with kidneys without ARF, receiving a kidney allograft with ARF was not associated with increased risk of death, graft lost, or inferior renal function 1 year after transplantation. CONCLUSION: In this cohort of patients, kidneys from deceased donors with ARF provided graft survival and renal function comparable with kidneys from donors without ARF 1 year after transplantation.

Basiliximab induction in patients receiving tacrolimus-based immunosuppressive regimens.

PURPOSE: The use of basiliximab induction increased significantly in recent years based on its superior efficacy and excellent safety profile demonstrated in studies with cyclosporine-based immunosuppression. However, its clinical utility in patients receiving tacrolimus-based immunosuppressive regimens is still uncertain. METHODS: We retrospectively reviewed data of 366 low immunological risk recipients of deceased donor kidney transplants. Of them, 134 received basiliximab and tacrolimus (TAC-IL2-RA), 100 received basiliximab and delayed tacrolimus(dTAC-IL2-RA), and 132 patients received tacrolimus without basiliximab(TAC-No). The endpoints were the incidence of acute rejection, graft function, and patient and graft survivals at 1 year. RESULTS: The incidence of acute rejection was higher in dTAC-IL2-RA compared to TAC-IL-2RA and TAC-No Groups (33 vs.14.9 vs. 14.3 %, p < 0.001). Inferior creatinine clearance was observed in dTAC-IL2-RA Group compared to TAC-IL2-RA and TAC-No Groups at months 1 (41.6 vs. 49.9 vs. 44.8 mL/min, p = 0.004), 3 (49.8 vs. 57.2 vs. 53.5 mL/min, p = 0.017), and 6 (53.1 vs. 61.8 vs. 57.0 mL/min, p = 0.001). Patients who received basiliximab (TAC-IL2-RA and dTAC-IL2-RA Groups) had lower incidence of posttransplant diabetes (24 vs.18 vs. 39.3 %, p = 0.009). Patient and graft survivals were similar among the groups. CONCLUSIONS: In low immunological risk kidney transplant recipients receiving tacrolimus, the use of basiliximab induction was not associated with lower rejection rates and did not allow delayed tacrolimus introduction.

Integrin-linked kinase (ILK) expression correlates with tumor severity in clear cell renal carcinoma.

Integrin-linked kinase (ILK) is an unique intracellular serine/threonine kinase and adapter protein. When dysregulated, it has been associated with increased cell proliferation, anchorage-independent cell growth, evasion of apoptosis, angiogenesis, invasion of surrounding tissues, downregulation of E-cadherin expression, nuclear translocation of ß-catenin and metastasis, all features of tumoral malignancy. The objective of the present work was to evaluate the expression of ILK in clear cell renal carcinomas (CCRC) as a possible prognostic indicator. ILK immunoexpression was evaluated in a tissue microarray (TMA) with 45 human CCRCs. In addition, the apoptotic and proliferative indices and the immuno-expression of ß-catenin and E-cadherin were also evaluated. E-cadherin expression was significantly decreased in tumors with positive ILK expression in relation to those with negative immunoexpression (p = 0.011). ILK immunostaining was significantly increased in high-grade in comparison to low-grade CCRCs (p = 0.0008). ILK expression was also associated with increased proliferative index (p = 0.020), tumor size >7.0 cm (p = 0.018) and with renal vein and capsule invasion (p = 0.003 and p = 0.00). Finally, tumors stage I and II (noninvasive) presented significantly reduced ILK immunoexpression when compared to stage III (locally invasive) (p = 0.0028). ILK immunoexpression in CCRC increases with loss of intercellular adhesion, nuclear grading, increased proliferative index and Robson stage. Altogether, our data suggest a possible role for ILK in the progression of CRCC.

[Posttransplant chronic anemia: parvovirus B19]. / Anemia crônica no pós-transplante renal: parvovirose B19.

Anemia is frequent in kidney transplant patients, and its prevalence varies according to posttransplant time and the adopted diagnostic criteria. Parvovirus B19 (PV B19) infection is an underdiagnosed cause of anemia in this particular population. To illustrate epidemiologic and clinical data regarding it, we present a case of PV B19 infection complicated by pure red cell aplasia (PRCA), pointing out the pitfalls we encountered in diagnosis and treatment. The use of viral DNA detection by polymerase chain reaction (PCR), and correct interpretation of morphological features of bone marrow histology are particularly important for the diagnosis of this condition in kidney transplant patients, who fail to develop a proper humoral response against PV B19, thus importantly decreasing the sensitivity of serological methods in this setting.

An experimental model for the study of collagen fibers in skeletal muscle.

PURPOSE: To examine histological and histomorphometric techniques for measuring collagen in skeletal muscle. METHODS: The following staining methods were used in the study: hematoxylin and eosin, Masson's trichrome, reticulin, and picrosirius red, and immunostaining for collagen types I, II, III, IV, and V. Histomorphometric measurements were performed using Corel PhotoPaint and UTHSCSA Image Tool 3.0 software. RESULTS: Both the Masson's trichrome and picrosirius red staining provided the best visualization for the measurement of collagen content. CONCLUSION: This methodology is important for the identification and quantification of the different types of collagen in muscles and can be used in the investigation of the qualitative and quantitative influence of collagen on physical activities, aging, and diseases.

An experimental model for the study of collagen fibers in skeletal muscle / Modelo experimental para o estudo de fibras de colágeno no músculo esquelético

PURPOSE: To examine histological and histomorphometric techniques for measuring collagen in skeletal muscle. METHODS: The following staining methods were used in the study: hematoxylin and eosin, Masson's trichrome, reticulin, and picrosirius red, and immunostaining for collagen types I, II, III, IV, and V. Histomorphometric measurements were performed using Corel PhotoPaint and UTHSCSA Image Tool 3.0 software. RESULTS: Both the Masson's trichrome and picrosirius red staining provided the best visualization for the measurement of collagen content. CONCLUSION: This methodology is important for the identification and quantification of the different types of collagen in muscles and can be used in the investigation of the qualitative and quantitative influence of collagen on physical activities, aging, and diseases.

OBJETIVO: Examinar os procedimentos empregados na quantificação do colágeno por métodos histológicos e histomorfométricos. MÉTODOS: Foram utilizadas colorações histológicas por HE, Tricrômico de Masson, Reticulina, Picrossírius e reação imuno-histoquímica para colágeno I, II, III, IV e V. A quantificação histomorfométrica foi realizada utilizando-se os programas Corel PhotoPaint e Image Tool versão 3.0. RESULTADOS: Os métodos histológicos de Masson e Picrossírius apresentaram uma maior facilidade na quantificação do colágeno. CONCLUSÃO: Este modelo é importante para que possa ser identificado e quantificado os diferentes tipos de colágenos nos músculos e relacionar com a atividade física, envelhecimento e doenças.

Anemia crônica no pós-transplante renal: parvovirose B19 / Posttransplant chronic anemia: parvovirus B19

A anemia é frequente em pacientes após o transplante renal (TxR) e sua prevalência varia conforme o tempo pós-transplante e os critérios diagnósticos empregados. A infecção pelo Parvovírus B19 (PV B19) é causa subdiagnosticada de anemia nesta população. Para ilustrar a epidemiologia e espectro clínico, apresentamos caso de PV B19 que evoluiu com aplasia pura de série vermelha (APSV), ressaltando as dificuldades do diagnóstico e tratamento. O emprego da detecção do DNA viral pela reação em cadeia da polimerase e do diagnóstico das alterações da morfologia da medula óssea são particularmente úteis para o diagnóstico no paciente transplantado imunossuprimido que falha na produção da resposta humoral contra o PV B19.

Anemia is frequent in kidney transplant patients, and its prevalence varies according to posttransplant time and the adopted diagnostic criteria. Parvovirus B19 (PV B19) infection is an underdiagnosed cause of anemia in this particular population. To illustrate epidemiologic and clinical data regarding it, we present a case of PV B19 infection complicated by pure red cell aplasia (PRCA), pointing out the pitfalls we encountered in diagnosis and treatment. The use of viral DNA detection by polymerase chain reaction (PCR), and correct interpretation of morphological features of bone marrow histology are particularly important for the diagnosis of this condition in kidney transplant patients, who fail to develop a proper humoral response against PV B19, thus importantly decreasing the sensitivity of serological methods in this setting.

DNA extraction and molecular analysis of non-tumoral liver, spleen, and brain from autopsy samples: the effect of formalin fixation and paraffin embedding.

The use of molecular biology in combination with morphological analysis is increasing because of the treatments by target therapies. However, to improve the methods for obtaining DNA for molecular analyses from formalin-fixed, paraffin-embedded (FFPE) tissue is a challenge. The aim of this study was to evaluate the DNA extracted from FFPE tissue blocks (non-tumoral liver, spleen, and brain), obtained from autopsy, 8-24 h post mortem, using three methods of DNA extraction. PCR of the ß-actin (136 pb) and human amelogenin (AMEL 212-218 bp/106-112 bp) genes, as well as short tandem repeat (STR) (100-400 bp fragments), reported in forensic scientific analysis, was performed to evaluate the effectiveness of the methods of DNA extraction. We used 28 archived (1 and 5 years) and 12 recent autopsy cases. The commercial kit showed reproducible and consistent results in the PCR amplification of the ß-actin and AMEL genes and in analysis by STR used in forensic analysis. This is the first report using non-tumoral samples from FFPE autopsy tissues, comparing the three most common methods of DNA extraction and using the STR previously described in forensics. Our study has clarified the challenges for pathologists in applying the molecular biology approach in combination with methods suited for morphology, which must be improved. The data provided here should be used in other molecular studies in FFPE samples.

Experimental model of arthritis induced by Paracoccidioides brasiliensis in rats.

Paracoccidioidomycosis (PCM), a disease caused by the fungus Paracoccidioides brasiliensis (Pb), is highly prevalent in Brazil, where it is the principal cause of death by systemic mycoses. The disease primarily affects men aged 30-50 year old and usually starts as a pulmonary focus and then may spread to other organs and systems, including the joints. The present study aimed to develop an experimental model of paracoccidioidomycotic arthritis. Two-month-old male Wistar rats (n = 48) were used, divided in 6 groups: test groups EG/15 and EG/45 (received one dose of 100 µl of saline containing 10(5) Pb viable yeasts in the knee); heat killed Pb-group HK/15 and HK/45 (received a suspension of 10(5) Pb nonviable yeasts in the knee) and control groups CG/15 and CG/45 (received only sterile saline in the knee). The rats were killed 15 and 45 days postinoculation. In contrast with the control rats, the histopathology of the joints of rats of the test groups (EG/15 and EG/45) revealed a picture of well-established PCM arthritis characterized by extensive sclerosing granulomatous inflammation with numerous multiple budding fungal cells. The X-ray examination revealed joint alterations in these groups. Only metabolic active fungi evoked inflammation. The experimental model was able to induce fungal arthritis in the knees of the rats infected with metabolic active P. brasiliensis. The disease tended to be regressive and restrained by the immune system. No evidence of fungal dissemination to the lungs was observed.