Oral colonization by yeasts in HIV-positive patients in Brazil

INTRODUCTION: In HIV-infected patients, colonization of the oral cavity by potential pathogenic yeast may lead to development of systemic fungemia. We evaluated the prevalence of yeast in the oral cavity of Brazilian HIV-positive patients and verified whether or not the species characterized were enzymatically active. Furthermore, the species identified were tested for their susceptibility to antifungal treatment. METHODS: Patient saliva and oropharyngeal candidiasis samples were collected from 60 seropositive HIV patients and identified by the API20C system. Enzymatic activity was evaluated by the production of proteinase and phospholipase. Susceptibility to antifungal treatments were determined using the broth microdilution method. RESULTS: the most commonly isolated species were C. albicans (51.56%) followed by non-albicans Candida species (43.73%), Trichosporon mucoides (3.12%) and Kodamaea ohmeri (1.56%). Oral colonization by association of different species was observed in 42% of the patients. Enzymatic activity was verified in most of species isolated, except for C. glabrata, C. lusitaniae and C. guilliermondii. Resistance to Fluconazole and Amphotericin B was observed in isolates of C. albicans, C. glabrata, C. parapsilosis, C. krusei, and K. ohmeri. CONCLUSION: HIV-positive patients are orally colonized by single or multiple species of yeast that are occasionally resistant to Fluconazole or Amphotericin B.

APPLICATION OF 6-NITROCOUMARIN AS A SUBSTRATE FOR THE FLUORESCENT DETECTION OF NITROREDUCTASE ACTIVITY IN Sporothrix schenckii

Introduction Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. Methods Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. Results All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. Conclusion The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.

AMINOGLYCOSIDE RESISTANCE GENES IN Pseudomonas aeruginosa ISOLATES FROM CUMANA, VENEZUELA

The enzymatic modification of aminoglycosides by aminoglycoside-acetyltransferases (AAC), aminoglycoside-adenyltransferases (AAD), and aminoglycoside-phosphotransferases (APH), is the most common resistance mechanism in P. aeruginosa and these enzymes can be coded on mobile genetic elements that contribute to their dispersion. One hundred and thirty seven P. aeruginosa isolates from the University Hospital, Cumana, Venezuela (HUAPA) were evaluated. Antimicrobial susceptibility was determined by the disk diffusion method and theaac, aadB and aph genes were detected by PCR. Most of the P. aeruginosa isolates (33/137) were identified from the Intensive Care Unit (ICU), mainly from discharges (96/137). The frequency of resistant P. aeruginosaisolates was found to be higher for the aminoglycosides tobramycin and amikacin (30.7 and 29.9%, respectively). Phenotype VI, resistant to these antibiotics, was the most frequent (14/49), followed by phenotype I, resistant to all the aminoglycosides tested (12/49). The aac(6´)-Ib,aphA1 and aadB genes were the most frequently detected, and the simultaneous presence of several resistance genes in the same isolate was demonstrated. Aminoglycoside resistance in isolates ofP. aeruginosa at the HUAPA is partly due to the presence of the aac(6´)-Ib, aphA1 andaadB genes, but the high rates of antimicrobial resistance suggest the existence of several mechanisms acting together. This is the first report of aminoglycoside resistance genes in Venezuela and one of the few in Latin America.

Isolation and functional characterization of mononuclear phagocytes from human lepromatous lesions

In the present study, inflammatory cells from human lepromatous lesions were isolated by enzymatic dissociation of tissue. They were maintained in culture up to five days and their morphologic, cythochemicaland functional characteristics were described.

Characterization of dengue virus serotype 1 in epidemics in Porto Velho, Rondônia, in 2001-2003

The first dengue fever epidemic in the State of Rondônia (western region of Brazil) was recorded in 1997, without laboratory confirmation. Following this, there was an epidemic in Manaus, in the neighboring State of Amazon, in 1998, in which DENV-1 and DENV-2 viruses were isolated from patients. In the present paper, the serotype characterization of the dengue virus isolated from patients with clinically suspected dengue in Porto Velho, Rondônia, between 2001 and 2003 is described. One hundred and fifty blood samples were collected between the first and fifth days of symptoms. Seventy samples of virus isolates were subjected to dengue identification by means of RT-PCR using universal primers for the NS1 gene of DENV, which amplifies a 419 bp fragment. The amplicons obtained were subjected to enzymatic digestion to characterize the viral serotypes. All the samples analyzed were DENV-1. A nucleotide sequence randomly selected from one amplicon, which was also DENV-1, presented 98% similarity to sequences from Southeast Asia that were obtained from GenBank.

Serum proteins and fractions, HDL-cholesterol and total IgG and IgE levels in cases of acute and chronic paracoccidioidomycosis

This study evaluated serum protein fractions, HDL-cholesterol, total immunoglobulin G and total immunoglobulin E levels in patients with acute and chronic paracoccidioidomycosis, by means of electrophoresis, enzymatic reaction and immunoenzymatic assay. The results demonstrated elevated levels of total immunoglobulin G, total immunoglobulin E, alpha-2 and gamma-globulins, which were more evident in acute than in chronic PCM, but no increase in HDL-cholesterol levels. There was a correlation between the levels of total immunoglobulin E and gamma-globulins and the alpha-2 and beta-globulin fractions in the acute form and between beta and gamma-globulins in both the acute and the chronic form. In conclusion, changes in total immunoglobulin G and immunoglobulin E levels and in the electrophoretic profile may be important markers for the prognosis and therapeutic follow-up of PCM cases, especially because protein electrophoresis is a simple laboratory test that can be applied when specific PCM serological tests are not available. In addition, levels of the gamma-globulin fraction greater than 2.0g/dl may suggest that the patient is developing a more severe form of PCM.

Candida dubliniensis does not show phospholipase activity: true or false?

INTRODUCTION: The phospholipase activity in Candida albicans and Candida dubliniensis isolated from oral candidiasis cases were studied. METHODS: The phospholipase activity was evaluated in egg yolk agar. RESULTS: All the C. albicans isolates (n = 48) showed phospholipase activity (mean Pz = 0.66). However, none of the C. dubliniensis isolates (n = 24) showed this activity. CONCLUSIONS: The authors discuss whether these findings are a true characteristic of C. dubliniensis or a consequence of the methodology employed, which includes the possibility that NaCl may have inhibited the enzymatic activity of C. dubliniensis.

Digoxin serum levels in patients with Chagas' cardiomyopathy and heart failure

INTRODUCTION: The purpose of this study was to determine digoxin serum concentrations in patients with Chagas' cardiomyopathy with chronic heart failure, because little is known concerning this laboratory test in patients with this condition. METHODS: This study focuses on 29 (29%) out of 101 patients with chronic heart failure secondary to Chagas' cardiomyopathy receiving digoxin therapy. Digoxin was measured by the immune-enzymatic method. RESULTS: New York Heart Association Functional Class III/IV was noted in 13 (45%) patients. The mean potassium serum level was 4.3± 0.5mEq/L, mean creatinine serum levels 1.4± 0.3dg/100ml, and left ventricular ejection fraction 34.7± 13.8%. The median digoxin serum level was 1.27 (0.55; 1.79)ng/ml. Sixteen (55%) patients had digoxin serum levels higher than 1.0ng/ml. Abnormal digoxin serum levels were verified in 13 (45%) patients. Digoxin serum levels correlated moderately with creatinine serum levels (r = 0.39; p< 0.03) and negatively with sodium serum levels (r= -0.38; p= 0.03). CONCLUSIONS: Digoxin serum concentration should be measured in patients with Chagas' cardiomyopathy with chronic heart failure receiving digoxin therapy due to the potential for digoxin toxicity.

Ação ovicida do extrato bruto enzimático do fungo Pochonia chlamydosporia sobre ovos de Ancylostoma sp

INTRODUÇÃO: Ancylostoma sp é um geo-helminto potencialmente zoonótico. MÉTODOS: O objetivo deste trabalho foi avaliar in vitro a ação do extrato bruto enzimático de Pochonia chlamydosporia (VC4) sobre ovos de Ancylostoma sp, em meio ágar-água 2% e em cultura de fezes. RESULTADOS: Observou-se um percentual de redução na eclosão dos ovos de Ancylostoma sp, de 76,8% na placas de Petri do grupo tratado em relação ao grupo controle. CONCLUSÕES: O extrato bruto enzimático de Pochonia chlamydosporia foi eficiente na redução da eclosão dos ovos de Ancylostoma sp, podendo ser utilizado como controlador biológico desse nematoide.

Restriction enzyme analysis of the human cytomegalovirus genome in specimens collected from immunodeficient patients in Belém, State of Pará, Brazil

INTRODUCTION: Human cytomegalovirus is an opportunistic betaherpesvirus that causes persistent and serious infections in immunodeficient patients. Recurrent infections occur due to the presence of the virus in a latent state in some cell types. It is possible to examine the virus using molecular methods to aid in the immunological diagnosis and to generate a molecular viral profile in immunodeficient patients. The objective of this study was to characterize cytomegalovirus genotypes and to generate the epidemiological and molecular viral profile in immunodeficient patients. METHODS: A total of 105 samples were collected from immunodeficient patients from the City of Belém, including newborns, hemodialysis patients, transplant recipients and HIV+ patients. An IgG and IgM antibody study was completed using ELISA, and enzymatic analysis by restriction fragment length polymorphism (RFLP) was performed to characterize viral genotypes. RESULTS: It was observed that 100% of the patients had IgG antibodies, 87% of which were IgG+/IgM-, consistent with a prior infection profile, 13% were IgG+/IgM+, suggestive of recent infection. The newborn group had the highest frequency (27%) of the IgG+/IgM+ profile. By RFLP analysis, only one genotype was observed, gB2, which corresponded to the standard AD169 strain. CONCLUSIONS: The presence of IgM antibodies in new borns indicates that HCMV continues to be an important cause of congenital infection. The low observed genotypic diversity could be attributed to the small sample size because newborns were excluded from the RFLP analysis. This study will be continued including samples from newborns to extend the knowledge of the general and molecular epidemiology of HCMV in immunodeficient patients.

Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide

INTRODUCTION: The capacity to overcome the oxidative stress imposed by phagocytes seems to be critical for Candida species to cause invasive candidiasis. METHODS: To better characterize the oxidative stress response (OSR) of 8 clinically relevant Candida sp., glutathione, a vital component of the intracellular redox balance, was measured using the 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB)-glutathione disulfide (GSSG) reductase reconversion method; the total antioxidant capacity (TAC) was measured using a modified method based on the decolorization of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid radical cation (ABTS*+). Both methods were used with cellular Candida sp. extracts treated or not with hydrogen peroxide (0.5 mM). RESULTS: Oxidative stress induced by hydrogen peroxide clearly reduced intracellular glutathione levels. This depletion was stronger in Candida albicans and the levels of glutathione in untreated cells were also higher in this species. The TAC demonstrated intra-specific variation. CONCLUSIONS: Glutathione levels did not correlate with the measured TAC values, despite this being the most important non-enzymatic intracellular antioxidant molecule. The results indicate that the isolated measurement of TAC does not give a clear picture of the ability of a given Candida sp. to respond to oxidative stress.

Fluconazole and amphotericin-B resistance are associated with increased catalase and superoxide dismutase activity in Candida albicans and Candida dubliniensis

Introduction Candida dubliniensis, a new species of Candida that has been recovered from several sites in healthy people, has been associated with recurrent episodes of oral candidiasis in AIDS and HIV-positive patients. This species is closely related to C. albicans. The enzymatic activity of C. dubliniensis in response to oxidative stress is of interest for the development of drugs to combat C. dubliniensis. Methods Fluconazole- and amphotericin B-resistant strains were generated as described by Fekete-Forgács et al. (2000). Superoxide dismutase (SOD) and catalase assays were performed as described by McCord and Fridovich (1969) and Aebi (1984), respectively. Results We demonstrated that superoxide dismutase (SOD) and catalase activities were significantly higher (p<0.05) in the fluconazole- and amphotericin B-resistant strains of C. dubliniensis and C. albicans than in the sensitive strains. The catalase and SOD activities were also significantly (p<0.01) higher in the sensitive and resistant C. albicans strains than in the respective C. dubliniensis strains. Conclusions These data suggest that C. albicans is better protected from oxidative stress than C. dubliniensis and that fluconazole, like amphotericin B, can induce oxidative stress in Candida; oxidative stress induces an adaptive response that results in a coordinated increase in catalase and SOD activities.

Relationship between plasma creatinine concentration and glomerular filtration in preterm newborn infants

Fluid management and dosage regimens of drugs in preterm infants should be based on the glomerular filtration rate. The current methods to determine glomerular flitration rate are invasive, time-consuming, and expensive. In contrast, creatinine clearance can be easy obtained and quickly determined. The purpose of this study was to compare plasma creatinine on the third and seventh day of life in preterm newborn infants, to evaluate the influence of maternal creatinine, and to demonstrate creatinine clearance can be used as a reliable indicator of glomerular filtration rate. We developed a prospective study (1994) including 40 preterm newborns (gestational age < 37 weeks), average = 34 weeks; birth weight (average) = 1840 g, in the first week of life. Inclusion criteria consisted of: absence of renal and urinary tract anomalies; O2 saturation 3 92%; adequate urine output (>1ml/kg/hr); normal blood pressure; absence of infections and no sympathomimetic amines in use. A blood sample was collected to determine plasma creatinine (enzymatic method) on the third and seventh day of life and creatinine clearance (CrCl) was obtained using the following equation: , k = 0.33 in preterm infant All plasma creatinine determinations showed normal values [third day: 0.78 mg/dl ± 0.24 (mean ± SD)and seventh day: 0.67 mg/dl ± 0.31 - (p>0.05)]. Also all creatinine clearance at third and seventh day of life were normal [third day: 19.5 ml/min ± 5.2 (mean ± SD) and seventh day: 23.8 ml/min ± 7.3 - (p>0,05)]. All preterm infants developed adequate renal function for their respective gestational age. In summary, our results indicate that, for clinical practice, the creatinine clearance, using newborn length, can be used to estimate glomerular filtration rate in preterm newborn infants.

Pheochromocytoma treated by laparoscopic surgery

OBJECTIVE: To evaluate the results of the laparoscopic technique in the treatment of adrenal pheochromocytoma. METHOD: Ten patients, 7 men and 3 women, between 10 and 67 years of age (mean 48) with pheochromocytoma underwent transperitoneal laparoscopic adrenalectomy and were evaluated retrospectively, based on clinical, laboratory, and pathological diagnosis. In all cases there was a solid unilateral adrenal tumor, 5 on the left side and 5 on the right side, whose greater diameter varied from 7 to 80 mm (mean 32). Nine of the 10 patients were chronically hypertensive or had already had hypertensive crises. One patient was normotensive, but presented metabolic alterations suggestive of adrenergic hyperfunction. RESULTS: No deaths occurred in this series. There were two (20%) conversions to open surgery, one due to venous bleeding and one due to the difficulty of dissection behind the vena cava in a patient presenting a partially retro-caval tumor. Surgical time in the 8 non-converted cases ranged from 70 to 215 minutes (mean 136). One patient (10%) received blood transfusion, and another (10%) presented two complications - acute renal failure and a subcutaneous infection. Both had been converted to open surgery. None of the non-converted cases was transfused or presented complications. Hospital discharge occurred between the 2nd and 11th post-operative day (mean 3). The pathological exam of the surgical specimens confirmed the diagnoses of pheochromocytoma in all 10 cases, one of them associated with an aldosterone-producing cortical tumor. CONCLUSIONS: Laparoscopic adrenalectomy for selected patients presenting pheochromocytoma is feasible and provides good results.

Laparoscopic versus open splenectomy in the management of hematologic diseases

Splenectomy is the best available treatment for severe forms of hereditary spherocytosis, idiopathic thrombocytopenic purpura, and other hematologic conditions when these prove refractory to conservative management. It has been employed for many decades with low mortality and favorable remission rates. The use of laparoscopic splenectomy in recent years has been rapidly and even enthusiastically adopted in this field. However, the exact role of laparoscopic versus open surgery for hematologic diseases is still debated. In this study of 58 adult patients, laparoscopic procedures were compared with conventional splenectomies for similar indications. METHODS: All patients were operated on within an 8-year period. Subjects underwent similar procedures under the supervision of the same surgical school and were compared regarding age, gender, body mass index, and diagnosis. Laparoscopically managed cases (Group I, n = 30) were prospectively followed according to a written protocol, whereas the same investigation was retrospectively done with regard to traditional laparotomy (Group II, n = 28). Methods included general and demographic findings, duration and technical steps of operation, blood loss, weight of spleen, need for conversion (in minimally invasive subjects), intraoperative and postoperative complications, time until realimentation, postoperative hospitalization, mortality, and late follow-up including recurrence rate. RESULTS: Idiopathic thrombocytopenic purpura was the surgical indication in over 50% of the patients in both groups, but familial spherocytosis, thalassemia, myelodysplasia, and lymphomas were also represented in this series. Laparoscopic procedures took more time to perform (P = 0.004), and postoperative hospitalization was 2 days shorter, but this difference was not statistically significant. Postoperative hematocrit and volume of blood transfusions was equivalent, although the laparoscopic cases had a somewhat lower preoperative hematocrit (NS) and displayed better recovery for this measurement (P = 0.03). More patients in Group I were able to accept oral food on the first day than subjects undergoing conventional operations (P < 0.05). Relatively few conversions were necessary during the minimally invasive surgeries (13.3%), and postoperative early and late complications as well as recurrences occurred in similar proportions. Also, the mean weight of the spleen was not statistically different between the groups, although there was a marked numerical tendency toward larger masses in conventional procedures. No spleen in Group I exceeded 2.0 kg, whereas in Group II values up to 4.0 kg occurred, and the mean weight was 50% higher in the latter group. CONCLUSIONS: 1) Minimally invasive splenectomy was essentially comparable to open surgery with regard to safety, efficacy, and late results; 2) Advantages concerning shorter postoperative hospitalization could not be shown, despite earlier food intake and a non-significant tendency toward earlier discharge; 3) This new modality should be considered an option in cases of hematologic conditions whenever the spleen is not hugely enlarged.