Role of iron in the nitric oxide-mediated fungicidal mechanism of IFN-gamma-activated murine macrophages against Paracoccidioides brasiliensis conidia

Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50U/mL of IFN-gamma or treated with 35 µM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS0(4). The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56%, respectively) in comparison with non-activated or untreated Mthetas (80%). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.

Cytokine and nitric oxide production by mouse macrophages infected with brazilian flaviviruses

The Flaviviridae family, Flavivirus genus includes viruses that are transmitted to vertebrates by infected mosquitoes or ticks. The genus Flavivirus includes a variety of viruses that cause diseases such as acute febrile illness, encephalitis, and hemorrhagic fever. Flaviviruses primarily infect blood monocytes and tissue macrophages, which have been shown to be permissive, supporting viral replication and serving as virus reservoirs. On the other hand, these cells may have an important antiviral activity related to modulation by cytokine production and by the capacity of these cells to synthesize reactive free radicals such as nitric oxide (NO) which can have a microbicidal effect. The present study was performed in order to determine the production of cytokines interleukin-1beta (IL-1β), tumor necrosis factor -alpha (TNF-α), transforming growth factor- beta (TGF-β) and interferon -alpha (IFN-α) and NO by macrophages infected with one of four Brazilian flaviviruses, Bussuquara virus (BUSV), Yellow Fever virus (YFV), Rocio virus (ROCV) and Encephalitis Saint Louis virus (SLEV), and to verify the possible antiviral effect of NO during macrophage infection with ROCV. Moreover, we asked if the different viruses were able to regulate bacterial lipopolysaccharide (LPS) induced cytokine production. Our results showed that YFV and SLEV reduced the production of IL-1β and TGF-β by LPS-stimulated macrophages, while ROCV only diminished LPS-stimulated TGF-β synthesis. On the other hand, BUSV more likely favored an enhancement of the LPS-induced production of IL-1β by macrophages. Additionally, while most of the viruses stimulated the production of IFN-α, none of them altered the production of TNF-α by murine macrophages. Interestingly, all viruses induced synthesis of NO that was not correlated with antiviral activity for ROCV.

SHIFT IN HUMAN ROTAVIRUS DISTRIBUTION IN BELO HORIZONTE, BRAZIL DETECTED BY RIBONUCLEIC ACID ELECTROPHORESIS

Rotavirus has been considered the main agent of infectious diarrhea especially among younger children. We addressed the prevalence of rotavirus-associated diarrhea and the diversity of circulating electropherotypes by immunochromatography and RNA electrophoresis. Stool samples were taken from 391 children (267 with diarrhea) from the lower socioeconomic stratum who sought treatment in the Hospital Infantil João Paulo II/Belo Horizonte, during 2005 and 2006. Rotavirus was detected in 79/20.2% of subjects, 64/24.0% with diarrhea and 15/12.1% with no diarrhea. The virus was strongly associated with diarrhea (p = 0.003). A total of 76/19.4% and 69/17.6% rotavirus-positive children were identified by immunochromatography and electrophoresis, respectively. Rotavirus-associated diarrhea was more frequently detected in dry months (p < 0.001) and almost exclusively in children aged up to three years. Long profile strains prevailed (54/78.3%) but a shift toward short electropherotype was identified. Despite the decrease seen in 2006, rotavirus infection is still very common in our area. Although viral RNA electrophoresis is useful as a typing method, it should not be used exclusively in the diagnosis of rotavirus infection. We confirmed a shift from long to short profile strains, as already described for other South American countries.

Comparação entre os métodos de Ziehl-Neelsen modificado e Acid-Fast-Trichrome para a pesquisa fecal de Cryptosporidium parvum e Isospora belli

Devido a crescente importância dos coccídios intestinais (Cryptosporidium, Isospora e Cyclospora) como parasitos oportunistas, é fundamental para os laboratórios diferenciar morfologicamente estes protozoários; a técnica de Ziehl-Neelsen modificada (ZNm) é amplamente utilizada para este fim; recentemente, foi proposto um novo procedimento, a coloração combinada do ácido tricrômico (Acid-Fast-Trichrome - AFT). O objetivo do presente estudo foi comparar os processos AFT e ZNm para a detecção destes coccídios em amostras fecais de pacientes portadores do vírus VIH. Foram selecionados dois grupos de indivíduos, para inclusão no estudo, segundo a presença (n=60) ou ausência de diarréia (n=60). As amostras de fezes foram coletadas em solução de formalina 10% e os esfregaços fecais preparados i) diretamente das fezes e ii) após concentração prévia a 500xg (10 minutos), foram submetidos aos diferentes processos de coloração. Considerando-se a positividade por técnica (AFT e ZNm), verificou-se a superioridade do procedimento de ZNm (n=19; 100% dos casos positivos) sobre o de AFT (n=8; 42,1%). Ambos possibilitaram a identificação dos 101 casos verdadeiramente negativos. Coccidiose intestinal foi mais frequente entre os pacientes que apresentaram diarréia (26,6%) em comparação à positividade observada entre os indíviduos assintomáticos (5%) sendo que C. cayetanensis não foi detectada em ambos os grupos. Foi de nosso interesse avaliar a aplicabilidade da técnica AFT para a coloração deste protozoário. Devido à sensibilidade e especificidade obtida neste estudo (100%), conclui-se que o método de ZNm continua sendo o mais indicado para o diagnóstico da criptosporidiose e isosporose, principalmente quando associado ao procedimento de centrífugo-concentração (500xg, 10 minutos). Embora a coloração AFT tenha baixo custo, faz-se necessário o seu aperfeiçoamento pois este procedimento permite o diagnóstico simultâneo dos coccídios intestinais (C. parvum, I. belli e C. cayetanensis) e dos microsporídios.

Nitric oxide levels are not changed in saliva of patients infected with hepatitis C virus

The aim of this investigation was to determine nitric oxide metabolite levels in saliva samples from hepatitis C virus-positive patients in an attempt to test the hypothesis if increased levels of nitric oxide metabolites correlates with the presence of HCV-RNA in saliva. Saliva of 39 HCV-positive patients and 13 HCV-negative patients, without clinical or laboratorial evidence of liver disease were tested for nitric oxide metabolites. HCV-RNA was detected in serum and saliva by a RT-PCR method and nitric oxide level was determined by evaluation of its stable degradation products, nitrate and nitrite. No differences were found between the concentration of nitrite in saliva from HCV patients and controls, in despite of the presence or not of HCV RNA in saliva. Patients with HCV and cirrhosis had higher concentrations of nitrite but not significantly different from the control group or the groups of anti-HCV patients without cirrhosis. Increased levels of nitrite were not detected in anti-HCV positive patients, an indirect indication that chronic sialoadenitis are infrequent in these patients or occurs with low intensity not sufficient to increase nitric oxide metabolite levels in saliva.

In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida

INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical isolates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3%) of isolates tested were phospholipase positive and 16 (44.4%) were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7%). Statistically significant differences were observed in relation to production of phospholipases among species (p<0,0001) and among the strains from different sites of origin (p=0.014). Regarding the production of acid protease, the isolates of C. parapsilosis tested presented a larger number of producers (69.2%). Among the species analyzed, the percentage of protease producing isolates did not differ statistically (χ2=1.9 p=0.5901 (χ2=1.9 p=0.5901). CONCLUSIONS: The majority of C. non-albicans and all C. albicans isolates were great producers of hydrolytic enzymes and, consequently, might be able to cause infection under favorable conditions.

A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru region, state of São Paulo, Brazil: case report

Amino acid insertions in the protease have rarely been described in HIVinfected patients. One of these insertions has recently been described in codon 35, although its impact on resistance remains unknown. This study presents a case of an HIV variant with an insertion in codon 35 of the protease, described for the first time in Bauru, State of Sao Paulo, Brazil, circulating in a 38-year-old caucasian male with asymptomatic HIV infection since 1997. The variant isolated showed a codon 35 insertion of two amino acids in the protease: a threonine and an aspartic acid, resulting in the amino acid sequence E35E_TD.

In vitro antifungal activity of fatty acid methyl esters of the seeds of Annona cornifolia A.St.-Hil. (Annonaceae) against pathogenic fungus Paracoccidioides brasiliensis

INTRODUCTION: Fatty acids are abundant in vegetable oils. They are known to have antibacterial and antifungal properties. METHODS: Antifungal susceptibility was evaluated by broth microdilution assay following CLSI (formerly the NCCLS) guidelines against 16 fungal strains of clinical interest. RESULTS: In this work, fatty acid methyl esters (FAME) was able to inhibit 12 clinical strains of the pathogenic fungus Paracoccidioides brasiliensis and were also active in the bioautographic assay against Cladosporium sphaerospermum. CONCLUSIONS: FAME was a more potent antifungal than trimethoprim-sulfamethoxazole against P. brasiliensis under the experimental conditions tested.

Evaluation of bacterial growth inhibition by mercaptopropionic acid in metallo-β-lactamase detection on multidrug-resistant Acinetobacter baumannii

INTRODUCTION: Metallo-β-lactamase (MBL) has been reported all over the world. METHODS: The inhibitory effect of mercaptopropionic acid (MPA) on bacterial growth was evaluated by comparison between disk diffusion and broth dilution methodology with determination of the minimum inhibitory concentration (MIC) for multidrug-resistant Acinetobacter baumanni strains. RESULTS: MPA significantly inhibited growth of the strains. CONCLUSIONS: The use of MPA can affect the results in phenotypic methods of MBL detection.

Larvicidal activity of crude extracts from Larrea cuneifolia (Zygophyllaceae) and of its metabolite nordihydroguaiaretic acid against the vector Culex quinquefasciatus (Diptera: Culicidae)

INTRODUCTION: The aim of the present study was to analyze the larvicidal activity of different crude extracts of Larrea cuneifolia and its most abundant lignan, nordihydroguaiaretic acid (NDGA), against Culex quinquefasciatus. METHODS: Chloroform, methanol, and aqueous extracts from L. cuneifolia and NDGA were tested against larvae of Cx. quinquefasciatus under laboratory conditions. RESULTS: The chloroform extract showed the highest larvicidal effect, with an estimated LC50 of 0.062 mg/ml. NDGA also demonstrated significant larvicidal activity with an estimated LC50 of 0.092 mg/ml. CONCLUSIONS: These results indicate that the chloroform extract of L. cuneifolia and NDGA are promising insecticides of botanical origin that could be useful for controlling Cx. quinquefasciatus.

Antioxidant factors, nitric oxide levels, and cellular damage in leprosy patients

Introduction The immune response caused by Mycobacterium leprae is a risk factor for the development of oxidative stress (OS) in leprosy patients. This study aimed to assess OS in leprosy patients before the use of a multidrug therapy. Methods We evaluated the nitric oxide (NO) concentration; antioxidant capacity; levels of malondialdehyde, methemoglobin and reduced glutathione; and the activity of catalase and superoxide dismutase (SOD) in leprosy patients. Results We observed lower SOD activity in these leprosy patients; however, the NO levels and antioxidant capacity were increased. Conclusions The infectious process in response to M. leprae could primarily be responsible for the OS observed in these patients.

Evaluation of three enzyme immunoassays and a nucleic acid amplification test for the diagnosis of Clostridium difficile-associated diarrhea at a university hospital in Brazil

Introduction Despite the known importance of Clostridium difficile as a nosocomial pathogen, few studies regarding Clostridium difficile infection (CDI) in Brazil have been conducted. To date, the diagnostic tests that are available on the Brazilian market for the diagnosis of CDI have not been evaluated. The aim of this study was to compare the performances of four commercial methods for the diagnosis of CDI in patients from a university hospital in Brazil. Methods Three enzyme immunoassays (EIAs) and one nucleic acid amplification test (NAAT) were evaluated against a cytotoxicity assay (CTA) and toxigenic culture (TC). Stool samples from 92 patients with suspected CDI were used in this study. Results Twenty-five (27.2%) of 92 samples were positive according to the CTA, and 23 (25%) were positive according to the TC. All EIAs and the NAAT test demonstrated sensitivities between 59 and 68% and specificities greater than 91%. Conclusions All four methods exhibited low sensitivities for the diagnosis of CDI, which could lead to a large number of false-negative results, an increased risk of cross-infection to other patients, and overtreatment with empirical antibiotics.

Inhaled nitric oxide in the management of persistent pulmonary hypertension of the newborn: a meta-analysis

OBJECTIVES: To evaluate the use of inhaled nitric oxide (NO) in the management of persistent pulmonary hypertension of the newborn. METHODS: Computerized bibliographic search on MEDLINE, CURRENT CONTENTS and LILACS covering the period from January 1990 to March 1998; review of references of all papers found on the subject. Only randomized clinical trials evaluating nitric oxide and conventional treatment were included. OUTCOMES STUDIED: death, requirement for extracorporeal membrane oxygenation (ECMO), systemic oxygenation, complications at the central nervous system and development of chronic pulmonary disease. The methodologic quality of the studies was evaluated by a quality score system, on a scale of 13 points. RESULTS: For infants without congenital diaphragmatic hernia, inhaled NO did not change mortality (typical odds ratio: 1.04; 95% CI: 0.6 to 1.8); the need for ECMO was reduced (relative risk: 0.73; 95% CI: 0.60 to 0.90), and the oxygenation was improved (PaO2 by a mean of 53.3 mm Hg; 95% CI: 44.8 to 61.4; oxygenation index by a mean of -12.2; 95% CI: -14.1 to -9.9). For infants with congenital diaphragmatic hernia, mortality, requirement for ECMO, and oxygenation were not changed. For all infants, central nervous system complications and incidence of chronic pulmonary disease did not change. CONCLUSIONS: Inhaled NO improves oxygenation and reduces requirement for ECMO only in newborns with persistent pulmonary hypertension who do not have diaphragmatic hernia. The risk of complications of the central nervous system and chronic pulmonary disease were not affected by inhaled NO.

Ursodeoxycholic acid does not interfere with in vivo Helicobacter pylori colonization

A low frequency of Helicobacter pylori in the gastric mucosa of patients with alkaline gastritis has been reported. At the same time, it can be noted that the growth of bacteria can be inhibited by bile acids. We studied 40 patients with chronic gastritis related to Helicobacter pylori in order to determine the effect of ursodeoxycholic acid on this infection. Diagnoses of the infection and the inflammatory process were obtained by histologic study of gastric biopsies collected during endoscopy. Two groups were studied: group I received ursodeoxycholic acid - 300 mg/day, and group II received the placebo, twice a day, both for 28 days. The colonization by Helicobacter pylori and the intensity of the mononuclear and polymorphonuclear inflammatory infiltrate were determined before (time 1) and after (time 2) treatment. Ursodeoxycholic acid had no effect on the Helicobacter pylori infection. A significant reduction in the intensity of the mononuclear inflammatory infiltrate of the gastric antrum mucosa was observed in patients from group I, when we compared not only times 1 and 2 but also groups I and II. However, this was not the case with the body mucosa. We concluded that ursodeoxycholic acid had no action on the colonization by Helicobacter pylori or on the polymorphonuclear inflammatory infiltrate, but it caused a significant reduction in the intensity of the mononuclear inflammatory infiltrate of the gastric antrum.