ABSTRACT Sporotrichosis is a neglected mycosis that affects human and animal hosts, including domestic cats. In Brazil, its most frequently diagnosed etiological agent is Sporothrix brasiliensis. Zoonotic transmission of S. brasiliensis occurs via direct contact between an infected cat and a susceptible human host. Notification of confirmed cases of feline sporotrichosis is not mandatory in Brazil. The metropolitan area of Goiania city can be considered a silent area for the occurrence of feline sporotrichosis. In this context, voluntary reporting of feline sporotrichosis cases is recommended for all healthcare professionals. This study aimed to report the first occurrence of S. brasiliensis in a cat from the metropolitan area of Goiania city. Cytopathology, mycology, thermal dimorphism and calmodulin gene amplification tests were performed. The mycological and molecular biological diagnoses corresponded to S. brasiliensis. The etiological agent of zoonotic sporotrichosis was detected in the metropolitan area of Goiania city, and therefore there is a risk of the emergence of new cases of cats infected with S. brasiliensis and the occurrence of zoonotic transmission of this fungus. hosts Brazil S host context professionals Cytopathology mycology performed fungus

ABSTRACT Candidemia is a significant cause of bloodstream infections (BSI) in nosocomial settings. The identification of species can potentially improve the quality of care and decrease human mortality. Quantitative PCR (qPCR) was evaluated for Candida albicans detection using culture suspensions containing C. albicans , spiked human blood, the cloned qPCR target fragment (ITS2 region) and the results of these assays were compared. The assays showed a good detection limit: C. albicans DNA extracted from yeast (sensitivity 0.2 CFU/µL), spiked human blood (sensitivity 10 CFU/mL), and cloned fragment of ITS2 region (sensitivity 20 target copies/μL). The efficiency of ITS2 fragment-qPCR ranged from 89.67 to 97.07, and the linearity (R2) of the standard curve ranged from 0.992 to 0.999. The results showed that this ITS2-qPCR has a great potential as a molecular prototype model for the development of a test to be applied in clinical practice, greatly reducing the time of candidemia diagnosis, which is extremely important in this clinical setting.

SUMMARY Severe anemia and cholestatic hepatitis are associated with bartonella infections. A putative vertical Bartonella henselae infection was defined on the basis of ultrastructural and molecular analyses in a three-year-old child with anemia, jaundice and hepatosplenomegaly since birth. Physicians should consider bartonellosis in patients with anemia and hepatitis of unknown origin.

Coccidioidomicose é uma doença emergente no Brasil; a manutenção adequada e autenticação de isolados de Coccidioides spp são essenciais para a pesquisa em diversidade genética de micro-organismos, bem como para a compreensão da doença em humanos. Dezessete isolados de Coccidioides preservados em óleo mineral desde 1975 na coleção de culturas do Instituto de Medicina Tropical de São Paulo (IMTSP) foram avaliados com relação à viabilidade, características morfológicas e genéticas, com o objetivo de autenticação das culturas fúngicas. Dos 17 isolados, apenas cinco foram viáveis após quase 30 anos mantidos em óleo mineral, apresentando características morfológicas e moleculares típicas do gênero, o sequenciamento utilizando os oligonucleotídeos Coi-F e Coi-R revelou identidade de 99% com isolados de Coccidioides. Estes cinco isolados foram preservados em nitrogênio líquido e água destilada esterilizada, e permaneceram viáveis após dois anos de armazenamento sob estas condições, mantendo as mesmas características.

Coccidioidomycosis is an emerging fungal disease in Brazil; adequate maintenance and authentication of Coccidioides isolates are essential for research into genetic diversity of the environmental organisms, as well as for understanding the human disease. Seventeen Coccidioides isolates maintained under mineral oil since 1975 in the Instituto de Medicina Tropical de São Paulo (IMTSP) culture collection, Brazil, were evaluated with respect to their viability, morphological characteristics and genetic features in order to authenticate these fungal cultures. Only five isolates were viable after almost 30 years, showing typical morphological characteristics, and sequencing analysis using Coi-F and Coi-R primers revealed 99% identity with Coccidioides genera. These five isolates were then preserved in liquid nitrogen and sterile water, and remained viable after two years of storage under these conditions, maintaining the same features.

Bactérias do gênero Bartonella constituem patógenos emergentes detectados em biópsias de linfonodos e secreções de gânglios provavelmente devido a maior concentração de bactérias. Vinte e três amostras de 18 pacientes com dados clínicos, laboratoriais e/ou epidemiológicos sugestivos de bartonelose foram submetidas a três amplificações duplas para a detecção de fragmento da proteína de choque térmico de 60-kDa (HSP), do espaçador interno 16S-23S rRNA (ITS) e da proteína de divisão celular (FtsZ) de Bartonella henselae, para melhorar a detecção em amostras clínicas. Na primeira amplificação, uma, quatro e cinco amostras, respectivamente, foram positivas pelo HSP (4,3%), FtsZ (17,4%) e pelo ITS (21,7%). Com a segunda amplificação foram identificadas seis amostras positivas pelo nested-HSP (26%), oito pelo nested-ITS (34,8%) e 18 pelo nested- FtsZ (78,2%), correspondentes a 10 amostras de sangue periférico, cinco biópsias de linfonodos, duas biópsias de pele e um aspirado de gânglio. A nested-FtsZ foi mais sensível que a nested-HSP e a nested-ITS (p < 0,0001), possibilitando a detecção de DNA de Bartonella henselae em 15 de 18 pacientes (83,3%). No presente estudo, três nested-PCR, consideradas específicas para a amplificação da Bartonella henselae, foram desenvolvidas, porém somente a nested-FtsZ não amplificou o DNA de Bartonella quintana. Concluímos que amplificações duplas aumentaram a detecção de DNA de B. henselae, e que a nested-FtsZ foi a mais sensível e a única específica para B. henselae em diferentes amostras biológicas. Como todas as amostras detectadas pelo HSP-nested e nested-ITS foram também pela nested-FtsZ, inferimos que, em nossa casuística, as infecções foram causadas por Bartonella henselae. A elevada positividade de amostras de sangue chamou a atenção para a utilização deste material biológico na investigação de bartoneloses, independentemente do estado imune dos pacientes. Este fato é importante no caso de pacientes criticamente enfermos e crianças pequenas para evitar procedimentos mais invasivos, como biópsias e punções de gânglios.

Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP), the internal transcribed spacer 16S-23S rRNA (ITS) and the cell division (FtsZ) of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%), FtsZ (17.4%) and ITS (21.7%), respectively. After the second round six positive samples were identified by nested-HSP (26%), eight by nested-ITS (34.8%) and 18 by nested-FtsZ (78.2%), corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001), enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%). In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. This fact is important in the case of critically ill patients and young children to avoid more invasive procedures such as lymph nodes biopsies and aspirates.

OBJECTIVES: Administering steroids before cardiopulmonary bypass in pediatric heart surgery modulates systemic inflammatory response syndrome and improves postoperative recovery. However, the use of steroids aggravates hyperglycemia, which is associated with a poor prognosis. Adult patients with systemic inflammatory response syndrome usually evolve with hyperglycemia and high insulin levels, whereas >90% of pediatric patients exhibit hyperglycemia and low insulin levels. This study aims to determine: A) the metabolic and inflammatory factors that are associated with hyperglycemia and low insulin levels in children who underwent cardiac surgery with cardiopulmonary bypass and who received a single high dose of methylprednisolone and B) the best predictors of insulin variation using a mathematical model. METHODS: This preliminary study recruited 20 children who underwent heart surgery with cardiopulmonary bypass and received methylprednisolone (30 mg/kg) immediately after anesthesia. Among the 20 patients initially recruited, one was excluded because of the absence of hyperglycemia and lower insulin levels after surgery. However, these abnormalities were confirmed in the remaining 19 children. The C-peptide, CRP, IL-6, and adrenomedullin levels were measured before surgery, immediately after cardiopulmonary bypass, and on the first, second, and third days after cardiac surgery. RESULTS: IL-6, CRP, and adrenomedullin increments were observed, whereas the C-peptide levels remained within reference intervals. CONCLUSION: The multiple regression model demonstrated that in addition to age and glycemia (two well-known factors that are directly involved in glucose metabolism), adrenomedullin and IL-6 levels were independent factors associated with lower insulin concentrations. These four parameters were responsible for 64.7% of the observed insulin variances. In addition, the fact that C-peptide levels did not fall together with insulin could have grounded the medical decision not to administer insulin to patients.

INTRODUCTION: Performance variation among PCR systems in detecting Toxoplasma gondii has been extensively reported and associated with target genes, primer composition, amplification parameters, treatment during pregnancy, host genetic susceptibility and genotypes of different parasites according to geographical characteristics. PATIENTS: A total of 467 amniotic fluid samples from T. gondii IgM- and IgG-positive Brazilian pregnant women being treated for 1 to 6 weeks at the time of amniocentesis (gestational ages of 14 to 25 weeks). METHODS: One nested-B1-PCR and three one-round amplification systems targeted to rDNA, AF146527 and the B1 gene were employed. RESULTS: Of the 467 samples, 189 (40.47%) were positive for one-round amplifications: 120 (63.49%) for the B1 gene, 24 (12.69%) for AF146527, 45 (23.80%) for both AF146527 and the B1 gene, and none for rDNA. Fifty previously negative one-round PCR samples were chosen by computer-assisted randomization analysis and re-tested (nested-B1-PCR), during which nine additional cases were detected (9/50 or 18%). DISCUSSION: The B1 gene PCR was far more sensitive than the AF146527 PCR, and the rDNA PCR was the least effective even though the rDNA had the most repetitive sequence. Considering that the four amplification systems were equally affected by treatment, that the amplification conditions were optimized for the target genes and that most of the primers have already been reported, it is plausible that the striking differences found among PCR performances could be associated with genetic diversity in patients and/or with different Toxoplasma gondii genotypes occurring in Brazil. CONCLUSION: The use of PCR for the diagnosis of fetal Toxoplasma infections in Brazil should be targeted to the B1 gene when only one gene can be amplified, preferably by nested amplification with primers B22/B23.

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, plays a significant role as a cofactor in the process of tumorigenesis, and has consistently been associated with a variety of malignancies especially in immunocompromised patients. Forty-four children and adolescents (21 liver transplant patients, 7 heart transplant, 5 AIDS, 3 autoimmune hepatitis, 2 nephritic syndromes, 2 medullar aplasia, 2 primary immunodeficiency disorder patients, 1 thrombocytopenic purpura and 1 systemic lupus erythematosus) presenting with chronic active EBV infection (VCA-IgM persistently positive; VCA-IgG > 20 AU/mL and positive IgG _ EBNA) had peripheral blood samples obtained during clinically characterized EBV reactivation episodes. DNA samples were amplified in order to detect and type EBV on the basis of the EBNA-2 sequence (EBNA2 protein is essential for EBV-driven immortalization of B lymphocytes). Although we have found a predominance of type 1 EBNA-2 virus (33/44; 75%), 10 patients (22.73%) carried type 2 EBNA-2, and one liver transplant patient (2.27%) a mixture of the two types, the higher proportion of type 2 EBV, as well as the finding of one patient bearing the two types is in agreement with other reports held on lymphoproliferative disorder (LPD) patients, which analyzed tumor biopsies. We conclude that EBNA-2 detection and typing can be performed in peripheral blood samples, and the high prevalence of type 2 in our casuistic indicates that this population is actually at risk of developing LPD, and should be monitored.

O estreptococo do grupo B (GBS) constitui a causa mais freqüente de sepse neonatal precoce. O teste de referência continua sendo o isolamento em cultura, apesar de apresentar problemas de sensibilidade. O objetivo do presente estudo foi validar uma técnica de dupla amplificação e determinar a possibilidade do uso de amostras residuais de urina colhidas por método não invasivo, não estéril, para a confirmação da sepse por GBS em recém-nascidos. As amostras foram amplificadas com primers do principal gene de superfície do GBS. A insuficiência de volume de material biológico para a realização de exames para suporte de vida, além de outros necessários à identificação do agente etiológico de infecções é muito freqüente em recém-nascidos. Mesmo assim, decidimos definir critérios bastante rigorosos para a inclusão de pacientes na casuística: os recém-nascidos deveriam apresentar sinais e sintomas compatíveis com infecção pelo GBS; deveriam ter tido ao menos uma amostra enviada para cultura, podendo ser sangue, urina ou líquor; disponibilidade de volumes residuais dessas amostras, ou de outras colhidas no dia da hospitalização, antes da introdução da antibioticoterapia, de forma a possibilitar a análise por PCR, e evolução favorável com a antibioticoterapia empírica. Em apenas um dos quatro recém-nascidos a infecção foi confirmada por cultura, enquanto nos outros três casos a infecção foi considerada presuntiva (pacientes preencheram os critérios de inclusão, mas o GBS não foi isolado). De um total de 12 amostras dos quatro pacientes (5 de sangue, 3 de líquor e 4 de urina), 8 foram testadas por cultura (2 foram positivas), 8 foram testadas por PCR (7 foram positivas), e apenas 4 pelos dois métodos simultaneamente (1 positiva por cultura e 3 por PCR). Concluímos que apesar do número restrito de pacientes e de amostras testadas, os resultados apresentados sugerem que a amplificação proposta poderia ser usada para o diagnóstico de sepse pelo GBS, uma vez que a amplificação foi possível nos três tipos de materiais biológicos testados (sangue, urina e líquor), e a PCR foi mais sensível que as culturas por ter conseguido confirmar a infecção na urina dos quatro pacientes, usando volumes residuais de amostras colhidas por método não invasivo, não estéril, o que é tecnicamente adequado uma vez que o GBS não faz parte da flora normal da urina, evitando procedimentos invasivos, tais como a punção supra-púbica da bexiga ou a cateterização transuretral. Ao mesmo tempo, o uso de urina em lugar de sangue ajuda a prevenir a espoliação sangüínea dos recém-nascidos.

Group B streptococcus (GBS) remains the most common cause of early-onset sepsis in newborns. Laboratory gold-standard, broth culture methods are highly specific, but lack sensitivity. The aim of this study was to validate a nested-PCR and to determine whether residue volumes of urine samples obtained by non invasive, non sterile methods could be used to confirm neonatal GBS sepsis. The nested-PCR was performed with primers of the major GBS surface antigen. Unavailability of biological samples to perform life supporting exams, as well as others to elucidate the etiology of infections is a frequent problem concerning newborn patients. Nevertheless, we decided to include cases according to strict criteria: newborns had to present with signs and symptoms compatible with GBS infection; at least one of the following biological samples had to be sent for culture: blood, urine, or cerebrospinal fluid; availability of residue volumes of the samples sent for cultures, or of others collected on the day of hospitalization, prior to antibiotic therapy prescription, to be analyzed by PCR; favorable outcome after GBS empiric treatment. In only one newborn GBS infection was confirmed by cultures, while infection was only presumptive in the other three patients (they fulfilled inclusion criteria but were GBS-culture negative). From a total of 12 biological samples (5 blood, 3 CSF and 4 urine specimen), eight were tested by culture methods (2/8 were positive), and 8 were tested by PCR (7/8 were positive), and only 4 samples were simultaneously tested by both methods (1 positive by culture and 3 by PCR). In conclusion, although based on a restricted number of neonates and samples, our results suggest that the proposed nested-PCR might be used to diagnose GBS sepsis as it has successfully amplified the three types of biological samples analyzed (blood, urine and cerebrospinal fluid), and was more sensitive than culture methods as PCR in urine confirmed diagnosis in all four patients. Moreover, PCR has enabled us to use residue volumes of urine samples collected by non invasive, non sterile methods, what is technically adequate as GBS is not part of the normal urine flora, thus avoiding invasive procedures such as suprapubic bladder punction or transurethral catheterization. At the same time, the use of urine instead of blood samples could help preventing newborns blood spoliation.

Trinta e quatro isolados de Candida foram analisados por amplificação aleatória de DNA polimórfico (primer OPG-10): 24 Candida albicans, 4 Candida tropicalis, 2 Candida parapsilosis, 2 Candida dubliniensis, 1 Candida glabrata e 1 Candida krusei. O coeficiente de correlação de Pearson-UPGMA calculou a distância genética entre os diferentes agrupamentos de Candida: amostras idênticas (100% de correlação), amostras muito relacionadas (90%), moderadamente relacionadas (80%), e não relacionadas (< 70%). Os resultados demonstram que a amplificação aleatória de DNA polimórfico proposta é capaz de classificar os isolados de forma coerente, ficando os de mesma espécie em um mesmo dendograma, com exceção dos dois isolados de Candida parapsilosis e o controle positivo (Holanda, 1973), provavelmente por serem atualmente classificadas em três espécies diferentes. Quanto ao único isolado de Candida tropicalis resistente ao fluconazol com genótipo diferente dos outros, os dados não são suficientes para afirmar que a única característica distinta fosse a sensibilidade ao fluconazol. Concluímos que a amplificação aleatória de DNA polimórfico proposta poderia ser usada para a confirmação das espécies de Candida identificadas nos testes microbiológicos.

Thirty-four Candida isolates were analyzed by random amplified polymorphic DNA using the primer OPG-10:24 Candida albicans; 4 Candida tropicalis; 2 Candida parapsilosis; 2 Candida dubliniensis; 1 Candida glabrata and 1 Candida krusei. The UPGMA-Pearson correlation coefficient was used to calculate the genetic distance between the different Candida groupings. Samples were classified as identical (correlation of 100%); highly related samples (90%); moderately related samples (80%) and unrelated samples (< 70%). The results showed that the RAPD proposed was capable of classifying the isolates coherently (such that same species were in the same dendrogram), except for two isolates of Candida parapsilosis and the positive control (Netherlands, 1973), probably because they are now recognized as three different species. Concerning the only fluconazole-resistant Candida tropicalis isolate with a genotype that was different to the others, the data were insufficient to affirm that the only difference was the sensitivity to fluconazole. We concluded that the Random Amplified Polymorphic DNA proposed might be used to confirm Candida species identified by microbiological methods.

Critical illness has a major impact on the nutritional status of both children and adults. A retrospective study was conducted to evaluate the incidence of hospital malnutrition at a pediatric tertiary intensive care unit (PICU). Serum concentrations of IL-6 in subgroups of well-nourished and malnourished patients were also evaluated in an attempt to identify those with a potential nutritional risk. METHODS: A total of 1077 patients were enrolled. Nutritional status was evaluated by Z-score (weight for age). We compared mortality, sepsis incidence, and length of hospital stay for nourished and malnourished patients. We had a subgroup of 15 patients with severe malnutrition (MN) and another with 14 well-nourished patients (WN). Cytokine IL-6 determinations were performed by enzyme-linked immunosorbent assay. RESULTS: 53% of patients were classified with moderate or severe malnutrition. Similar amounts of C- reactive protein (CRP) were observed in WN and MN patients. Both groups were able to increase IL-6 concentrations in response to inflammatory systemic response and the levels followed a similar evolution during the study. However, the mean values of serum IL-6 were significantly different between WN and MN patients across time, throughout the study (p = 0.043). DISCUSSION: a considerable proportion of malnourished patients need specialized nutritional therapy during an intensive care unit (ICU) stay. Malnutrition in children remains largely unrecognized by healthcare workers on admission. CONCLUSIONS: The incidence of malnutrition was very high. Malnourished patients maintain the capacity to release inflammatory markers such as CRP and IL-6, which can be considered favorable for combating infections On the other hand, this capacity might also have a significant impact on nutritional status during hospitalization.

Vinte e quatro amostras de sangue total e de soro foram colhidas durante seguimento por 36 meses de criança de oito anos de idade, imunodeprimida devido a transplante cardíaco. O paciente apresentou VCA-IgG e IgM positivos e EBNA-IgG negativo aos cinco anos de idade quando foi diagnosticada mononucleose infecciosa. Quatorze meses depois o VCA-IgG e o EBNA-IgG eram positivos e o VCA-IgM negativo. Este padrão sorológico persiste desde aquela época mesmo durante episódios sugestivos de reativação. As amostras de sangue total e de soro foram analisadas pela Reação em Cadeia da Polimerase (PCR) que amplificou fragmento oriundo da gp220 do EBV (detecção de 100 cópias virais). Todas as 24 amostras de sangue total e 12 amostras de soro foram positivas por PCR. Com o objetivo de verificar se a detecção de DNA do EBV em soro estaria associada à reativação da doença, os resultados de PCR foram analisados em relação à necessidade de hospitalização e uso de anti-viral. O teste de Kappa mostrou que existe concordância entre a presença de DNA do EBV em soro e a necessidade de hospitalização e tratamento com anti-virais (valor de 0,750; p < 0,001). Concluímos que a detecção de DNA do EBV em amostras de soro de pacientes imunosuprimidos poderia ser usada como marcador laboratorial de atividade da infecção quando técnicas quantitativas de amplificação não estiverem disponíveis.

Twenty-four whole blood and serum samples were drawn from an eight year-old heart transplant child during a 36 months follow-up. EBV serology was positive for VCA-IgM and IgG, and negative for EBNA-IgG at the age of five years old when the child presented with signs and symptoms suggestive of acute infectious mononucleosis. After 14 months, serological parameters were: positive VCA-IgG, EBNA-IgG and negative VCA-IgM. This serological pattern has been maintained since then even during episodes suggestive of EBV reactivation. PCR amplified a specific DNA fragment from the EBV gp220 (detection limit of 100 viral copies). All twenty-four whole blood samples yielded positive results by PCR, while 12 out of 24 serum samples were positive. We aimed at analyzing whether detection of EBV-DNA in serum samples by PCR was associated with overt disease as stated by the need of antiviral treatment and hospitalization. Statistical analysis showed agreement between the two parameters evidenced by the Kappa test (value 0.750; p < 0.001). We concluded that detection of EBV-DNA in serum samples of immunosuppressed patients might be used as a laboratory marker of active EBV disease when a Real-Time PCR or another quantitative method is not available.

OBJETIVO: Analisar a freqüência da mutação delta F508 (deltaF508) em 108 pacientes não aparentados, com fibrose cística e comparou os resultados com os dados de outros estudos brasileiros. A fibrose cística (CF) constitui a doença genética mais comum em populações caucasianas, sendo a deltaF508 a mais freqüente dentre as mutações relacionadas à doença. MÉTODO: A freqüência da deltaF508 foi analisada por meio da Reação em Cadeia da Polimerase (PCR) seguida de detecção em géis de poliacrilamida a 8%. RESULTADOS: Vinte e três dos 108 pacientes foram homozigotos para a mutação (21,3%), 50 foram heterozigotos (46,3%) e os 35 restantes não eram portadores da deltaF508 (32,4%). Em termos de alelos, foram observados 96 mutados (44,45%) e 120 do tipo selvagem (55,55%), ou seja, não portadores da mutação. CONCLUSÃO: A freqüência de 44,45% de alelos mutados encontrada no estudo é mais elevada que os 33,0% descritos em pesquisa realizada em São Paulo em 1993, e ligeiramente mais baixa que os 48,0% encontrados em relatos mais recentes. Além disso, nossos resultados corroboraram a idéia de que a freqüência da mutação deltaF508 é mais baixa no Brasil em comparação a países europeus e nos Estados Unidos da América (cerca de 70,0%), provavelmente devido a maior miscigenação racial. Estas observações terão que ser consideradas antes da implementação de testes genéticos de triagem no Brasil.

PURPOSE: To analyze the frequency of the delta F508 (deltaF508) deletion mutation in 108 unrelated cystic fibrosis patients and compare the results with the previously reported data for Brazilian patients. Cystic fibrosis is the leading cause of genetic disease in Caucasians, and the deltaF508 deletion is the most common mutation associated with the disease. METHOD: The frequency of the deltaF508 mutation was assessed by means of a polymerase chain reaction (PCR) followed by detection in 8% silver-stained polyacrylamide gels. RESULTS: Twenty-three of 108 patients (21.3%) were homozygous for the deltaF508 deletion, 50 were heterozygous (46.3%), and the remaining 35 (32.4%) were non-carriers. In terms of alleles, there were 96 mutated (96/216 or 44.45%) and 120 wild-type ones (120/216 or 55.5%). CONCLUSION: The 44.45% of affected alleles that were found is higher than the 33% first described in 1993, but slightly lower than the 48% recently reported. Moreover, our data corroborated the idea that the frequency of the deltaF508 mutation is lower in Brazil in comparison to that found in studies carried out in Europe and North American (circa 70.0%), probably due to increased racial miscegenation. These findings must be taken into account before any genetic screening of the population is proposed in Brazil.

O presente trabalho registra caso de histoplasmose em paciente de 5 anos, HIV negativo, natural e procedente da cidade de São Paulo, com lesões cutâneas não diagnosticadas clinicamente. Exame histopatológico negativo para infecção fúngica. Cultivos em duas ocasiões, positivos para Histoplasma capsulatum var. capsulatum (amostras 361 e 387). Sorologia negativa para anticorpos anti-Histoplasma capsulatum e Paracoccidioides brasiliensis pelas provas de Imunodifusão dupla e Contraimunoeletroforese. Ensaio imunoenzimático pesquisando o antígeno polissacarídico específico, negativo. A obtenção do exoantígeno de uma das amostras permitiu, através de provas de Imunodifusão, com soro de coelho anti-Histoplasma capsulatum var. capsulatum, detectar duas bandas de precipitação. Registre-se a negatividade da reação de Imunodifusão dupla do soro do paciente face ao exoantígeno produzido com a amostra dele isolada. Através da reação de Immunoblotting foi identificada uma fração com peso molecular de aproximadamente 94 kDa, além de outras. Os dois cultivos foram enviados ao Prof. Leo Kaufman, CDC, Atlanta, USA, o qual identificou as duas amostras como Histoplasma capsulatum var. capsulatum, através de provas de Imunodifusão dupla com os respectivos exoantígenos e sonda específica do fungo (DNA-probe). As colorações específicas para fungos, nos tecidos, foram negativas, incluindo prova de Imunoperoxidase para Histoplasma capsulatum var.capsulatum, a negatividade das provas sorológicas para histoplasmose clássica e o próprio aspecto clínico das lesões cutâneas eritêmato-violáceas infiltradas, mostram que o caso em apreço é realmente inusitado, correspondendo o cultivo a uma "variante aberrante" do Histoplasma capsulatum var. capsulatum. Face ao diagnóstico micológico de histoplasmose clássica, o paciente foi tratado com itraconazol 100 mg/dia, durante três meses, com regressão total das lesões.

A case of atypical disseminated cutaneous histoplasmosis in a five-year old, otherwise healthy child, native and resident in São Paulo metropolitan area is reported. Cutaneous lesions were clinically atypical. Histologic examination disclosed a granulomatous reaction but no fungal structures could be demonstrated by specific staining nor by immunohistochemical reaction. The fungus was isolated from biopsy material on two different occasions, confirming diagnosis of an unusual fungal infection. The fungus, originally thought to be a Sepedonium sp. due to the large sized, hyaline or brownish colored tuberculated macroconidia and to lack of dimorphism (yeast form at 37 °C) produce H and M antigens, visualized by the immunodiffusion with rabbit anti-Histoplasma capsulatum hyperimmune serum. Patient’s serum sample was non reactive with H. capsulatum antigen by immunodiffusion, counterimmunoelectrophoresis and complement fixation tests, and immunoenzymatic assay failed to detect the specific circulating antigen. This serum was tested negative by double immunodiffusion when antigen obtained from one of the isolated samples was used. Both cultures were sent to Dr. Leo Kaufman, Ph.D. (Mycoses Immunodiagnostic Laboratory, CDC-Atlanta/USA), who identified them as H. capsulatum by the exoantigen and gen-probe tests. Both clinic and mycologic characteristics of the present case were atypical, suggesting the fungus isolated is an “aberrant variant” of H. capsulatum var. capsulatum, as described by SUTTON et al. in 199719. Treatment with itraconazole 100 mg/day led to cure within 90 days