Influenza A(H1N1)pdm09 Resistance and Cross-Decreased Susceptibility to Oseltamivir and Zanamivir Antiviral Drugs

Neuraminidase inhibitors (NAIs) oseltamivir and zanamivir are currently the only effective antiviral drugs available worldwide for the management of influenza. The potential development of resistance is continually threatening their use, rationalizing and highlighting the need for a close and sustained evaluation of virus susceptibility. This study aimed to analyze and characterize the phenotypic and genotypic NAIs susceptibility profiles of A(H1N1)pdm09 viruses circulating in Portugal from 2009 to 2010/2011. A total of 144 cases of A(H1N1)pdm09 virus infection from community and hospitalized patients were studied, including three suspected cases of clinical resistance to oseltamivir. Oseltamivir resistance was confirmed for two of the suspected cases. Neuraminidase (NA) H275Y resistant marker was found in viruses from both cases but for one it was only present in 26.2% of virus population, raising questions about the minimal percentage of resistant virus that should be considered relevant. Cross-decreased susceptibility to oseltamivir and zanamivir (2-4 IC50 fold-change) was detected on viruses from two potentially linked community patients from 2009. Both viruses harbored the NA I223V mutation. NA Y155H mutation was found in 18 statistical non-outlier viruses from 2009, having no impact on virus susceptibility. The mutations at NA N369K and V241I may have contributed to the significantly higher baseline IC50 value obtained to oseltamivir for 2010/2011 viruses, compared to viruses from the pandemic period. These results may contribute to a better understanding of the relationship between phenotype and genotype, which is currently challenging, and to the global assessment of A(H1N1)pdm09 virus susceptibility profile and baseline level to NAIs.

HDL in HIV-1 infection: a quality perspective through paraoxonase-1 activities

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Nevirapine in an animal model of pre-diabetes: study of drug pharmacokinetic and its effects on fasting glycemia and insulin resistance

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Treatment with benznidazole in association with immunosuppressive drugs in mice chronically infected with Trypanosoma cruzi: investigation into the possible development of neoplasias

Benznidazole is recommended in Brazil for the treatment of Trypanosoma cruzi infection in acute and early chronic phases of Chagas' disease. Observations by others have indicated a higher incidence of neoplasias in immunosuppressed patients, presenting Chagas' disease reactivation, submitted to treatment with benznidazole. In the present study, we investigated whether there is a potentiation in the generation of lymphomas in chronically infected mice, treated with immunosuppressive drugs and benznidazole. For this, 142 Swiss mice chronically infected with the 21 SF strain of T. cruzi and 72 normal Swiss mice were used. Both infected and normal mice were divided into experimental groups and submitted to one of the following treatment regimens: benznidazole alone; immunosuppressive drugs (azathioprine, betamethasone and cyclosporin); a combination of immunosuppressive drugs and benznidazole; and untreated controls. In the infected group treated with benznidazole, one mouse developed a non-Hodgkin's lymphoma. This finding has been interpreted as a spontaneous tumor of mice. The study of the chronically infected mice treated with the combination of immunosuppressive drugs and benznidazole demonstrated an absence of lymphomas or other neoplasias. These findings support the indication of benznidazole, as the drug of choice, for immunosuppressed patients that develop a reactivation of Chagas' disease.

Longitudinal comparison between plasma and seminal HIV-1 viral loads during antiretroviral treatment

This study was designed to investigate the impact of anti-retroviral therapy on both plasma and seminal HIV-1 viral loads and the correlation between viral loads in these compartments after treatment. Viral load, CD4+ and CD8+ T-cell counts were evaluated in paired plasma and semen samples from 36 antiretroviral therapy-naïve patients at baseline and on days 45, 90, and 180 of treatment. Slopes for blood and seminal viral loads in all treated patients were similar (p = 0.21). Median HIV-1 RNA titers in plasma and semen at baseline were 4.95 log10 and 4.48 log10 copies/ml, respectively. After 180 days of therapy, the median viral load declined to 3.15 log10 copies/ml (plasma) and 3.2 log10 copies/ml (semen). At this timepoint 22 patients presented HIV-1 viral load below 400 copies/ml in either plasma or semen, but only 9 had viral loads below 400 copies/ml in both compartments.

The role of integrated home-based care in patient adherence to antiretroviral therapy

Non-adherence is one of the primary obstacles to successful antiretroviral therapy in HIV+ patients worldwide. In Brazil, the Domiciliary Therapeutic Assistance is a multidisciplinary and integrated home-based assistance program provided for HIV+ patients confined in their homes due to physical deficiency. This study investigated ADT's ability to monitor and promote appropriate adherence to ARV therapy. Fifty-six individuals were recruited from three study groups: Group 1 - patients currently in the ADT program, Group 2 - 21 patients previously treated by the ADT program, and Group 3 - 20 patients who have always been treated using conventional ambulatory care. Using multivariable self-reporting to evaluate adherence, patients in the ADT program had significantly better adherence than patients in ambulatory care (F = 6.66, p = 0.003). This effect was independent of demographic and socioeconomic characteristics as well as medical history. Patients in the ADT program also showed a trend towards greater therapeutic success than ambulatory patients. These results suggest the incorporation of characteristics of ADT in conventional ambulatory care as a strategy to increase adherence to ARV therapy.

Neurological disease in HIV-infected patients in the era of highly active antiretroviral treatment: a Brazilian experience

To study characteristics of neurological disorders in HIV/AIDS patients and their relationship to highly active antiretroviral treatment, a cross-sectional study was conducted in an infectious disease public hospital in Belo Horizonte, Brazil, between February 1999 and March 2000. Of the 417 patients enrolled, neurological disease was observed in 194 (46.5%) and a new AIDS-defining neurological event developed in 23.7% of individuals. Toxoplasmosis (42.3%), cryptococcosis meningitis (12.9%) and tuberculosis (10.8%) were the most common causes of neurological complications. The majority (79.3%) of patients were on highly active antiretroviral treatment and these individuals using HAART showed higher CD4 cell counts (p = 0.014) and presented stable neurological disease (p= 0.0001), although no difference was found with respect to the profile of neurological complications. The neurological diseases continue to be a frequent complication of HIV/AIDS and infections are still its main causes in Brazil, even in the highly active antiretroviral treatment era.

High frequency of resistance to the drugs isoniazid and rifampicin among tuberculosis cases in the city of Cabo de Santo Agostinho, an urban area in Northeastern Brazil

The objective of the present study was to investigate the frequency and risk factors for developing multidrug-resistant tuberculosis in Cabo de Santo Agostinho, PE. This was a prospective study conducted from 2000 to 2003, in which suspected cases were investigated using bacilloscopy and culturing. Out of 232 confirmed cases of tuberculosis, culturing and antibiotic susceptibility tests were performed on 174. Thirty-five of the 174 cultures showed resistance to all drugs. The frequencies of primary and acquired resistance to any drug were 14% and 50% respectively, while the frequencies of primary and acquired multidrug resistance were 8.3% and 40%. Previous tuberculosis treatment and abandonment of treatment were risk factors for drug resistance. The high levels of primary and acquired resistance to the combination of isoniazid and rifampicin contributed towards the difficulties in controlling tuberculosis transmission in the city.

Development of novel active pharmaceutical ionic liquids and salts based on antibiotics and anti-fungal drugs

Ionic Liquids (ILs) are class of compounds, which have become popular since the mid-1990s. Despite the fact that ILs are defined by one physical property (melting point), many of the potential applications are now related to their biological properties. The use of a drug as a liquid can avoid some problems related to polymorphism which can influence a drug´s solubility and thus its dosages. Also, the arrangement of the anion or cation with a specific drug might be relevant in order to: a) change the correspondent biopharmaceutical drug classification system; b) for the drug formulation process and c) the change the Active Pharmaceutical Ingredients’ (APIs). The main goal of this Thesis is the synthesis and study of physicochemical and biological properties of ILs as APIs from beta-lactam antibiotics (ampicillin, penicillin G and amoxicillin) and from the anti-fungal Amphotericin B. All the APIs used here were neutralized in a buffer appropriate hydroxide cations. The cation hydroxide was obtained on Amberlite resin (in the OH form) in order to exchange halides. The biological studies of these new compounds were made using techniques like the micro dilution and colorimetric methods. Overall a total of 19 new ILs were synthesised (6 ILs based on ampicillin, 4 ILs, based on amoxicillin, 6 ILs based on penicillin G and 4 ILs based on amphotericin B) and characterized by spectroscopic and analytical methods in order to confirm their structure and purity. The study of the biological properties of the synthesised ILs showed that some have antimicrobial activity against bacteria and yeast cells, even in resistant bacteria. Also this work allowed to show that ILs based on ampicillin could be used as anti-tumour agents. This proves that with a careful selection of the organic cation, it is possible to provoke important physico-chemical and biological alteration in the properties of ILs-APIs with great impact, having in mind their applications.

Nutritional assessment and lipid profile in HIV-infected children and adolescents treated with highly active antiretroviral therapy

INTRODUCTION: HIV-infected children and adolescents treated with highly active antiretroviral therapy (HAART) regimens that include a protease inhibitor (PI) can show significant improvements in clinical outcomes, nutritional status and quality of life. The study aimed to report nutritional and metabolic alterations for pediatric patients continuously exposed to HAART and for healthy controls for up to 1 year. METHODS: Clinical, anthropometric, lipid profile and food intake data were collected prospectively over approximately 12-months for each patient. RESULTS: Fifty-one individuals were studied, of these, 16 were healthy. After 12 months follow-up, HIV-positive individuals remained below the healthy control group parameters. No change was observed concerning food intake. Triglyceride serum levels were higher in patients using protease inhibitor at the onset of the study [PI groups: 114 (43 - 336), and 136 (63 - 271) versus control group: 54.5 (20 - 162); p = 0.003], but after twelve months follow-up, only the group using protease inhibitor for up to two months presented higher values [140 (73 - 273) versus 67.5 (33 - 117); p = 0.004]. HDL-cholesterol was lower in HIV-positive individuals [HIV-positive groups: 36 (27 - 58) and 36 (23 - 43); control 49.5 (34 - 69); p = 0.004]. CONCLUSIONS: HIV-infected children and adolescents treated with highly active antiretroviral therapy showed compromised nutritional parameters compared to a paired healthy control group. Individuals using protease inhibitor presented worse triglyceride serum levels compared to their healthy counterparts.

Lipodystrophy in HIV/AIDS patients with different levels of physical activity while on antiretroviral therapy

INTRODUCTION: Lipodystrophy is related to the use of highly active antiretroviral therapy (HAART) and can cause aesthetic stigma and increase the risk of developing cardiovascular diseases. Physical activity may be a valid alternative for the treatment and prevention of lipodystrophy. However, few studies address this issue. The objective of this study was to assess lipodystrophy related to highly active antiretroviral therapy in HIV/AIDS patients with different physical activity habits. METHODS: The sample was composed of 42 HIV/AIDS patients taking HAART medication who were visiting the Counseling and Testing Center (CTC) in Presidente Prudente. The level of physical activity was obtained using the International Physical Activity Questionnaire (IPAQ); lipodystrophy was diagnosed using a self-report questionnaire that was administered to the patient and then followed up by medical confirmation. The percentage of trunk fat was estimated by dual X-Ray absorptiometry (DEXA). Information about sex, age, length of HAART treatment, CD4+ T lymphocyte count (CD4) and viral load was also collected. RESULTS: A higher prevalence of lipodystrophy was observed in the sedentary group when compared to the physically active group, which indicates that physical activity may be a protective factor in relation to the occurrence of lipodystrophy. The group that had a higher CD4 had a higher proportion of lipodystrophy and a higher proportion of younger and physically active individuals. The patients with lipodystrophy had a higher percentage of trunk fat and were more sedentary than active individuals. CONCLUSIONS: A physically active lifestyle has a protective effect against the occurrence of lipodystrophy related to HAART.

Quality of essential drugs in tropical countries: evaluation of antimalarial drugs in the Brazilian Health System

INTRODUCTION: The emergence of drug resistance is one of the main problems concerning malaria treatment. The use of counterfeit and/or substandard antimalarial drugs can contribute to the development of parasite resistance. Thus, the aim of this study was to evaluate the quality of antimalarial drugs distributed in Brazil. METHODS: Samples containing chloroquine phosphate, mefloquine hydrochloride, primaquine phosphate, and quinine sulfate tablets were delivered to the Rio de Janeiro central storeroom (CENADI), state storerooms (SS), and Basic Health Units (BHUs) in the north region of Brazil - a total of 10 sample sets. After 5 months of storage, the samples were collected, and in vitro quality control analyses according to official and published methods were performed. RESULTS: Inadequate drug storage conditions were found in two SS and in all BHUs evaluated. There were no quality deviations found in the chloroquine samples. The quinine samples exhibited weight variation above the allowed limits. The primaquine samples were found to have packaging deficiency. The release of mefloquine in samples from some regions showed a statistically significant difference when compared with the CENADI samples. CONCLUSIONS: It is important to periodically evaluate the quality and storage conditions of essential drugs. The quality deviations found with the primaquine and quinine samples are not related to storage conditions and must be addressed urgently. The decreased mefloquine release from tablets is related to formulation problems or influenced by inadequate storage conditions, prompting further investigation. Even with the mentioned problems, the samples would probably not contribute to resistant parasite selection.

Enteric parasites in HIV-1/AIDS-infected patients from a Northwestern São Paulo reference unit in the highly active antiretroviral therapy era

INTRODUCTION: We describe the epidemiology of intestinal parasites in patients from an AIDS reference service in Northeastern São Paulo, Brazil. METHODS: Retrospective evaluation was done for all HIV-1/AIDS-positive patients whose Hospital de Base/São José do Rio Preto laboratorial analysis was positive for enteroparasites after diagnosis of HIV-1 infection, from January 1998 to December 2008. Statistical analysis was performed using the R statistical software version 2.4.1. The level of significance adopted was 5%. RESULTS: The most frequent protozoan was Isospora belli (4.2%), followed by Giardia lamblia (3.5%), Entamoeba coli (2.8%), and Cryptosporidium parvum (0.3%). Ancylostoma duodenale (1.4%) was the most frequently detected helminth, while Taenia saginata and Strongiloides stercoralis were found in 0.7% of the samples. The results showed that diarrhea was significantly associated with giardiasis and isosporiasis. However, no association was observed between CD4+ cell counts, viral load, and the characteristics of any particular parasite. CONCLUSIONS: Our data may be useful for further comparisons with other Brazilian regions and other developing countries. The data may also provide important clues toward improving the understanding, prevention, and control of enteric parasites around the world.

Liver fibrosis progression in HIV/hepatitis C virus coinfected patients with normal aminotransferases levels

INTRODUCTION: Approximately 30% of hepatitis C virus (HCV) monoinfected patients present persistently normal alanine aminotransferase (ALT) levels. Most of these patients have a slow progression of liver fibrosis. Studies have demonstrated the rate of liver fibrosis progression in hepatitis C virus-human immunodeficiency virus (HCV-HIV) coinfected patients is faster than in patients infected only by HCV. Few studies have evaluated the histological features of chronic hepatitis C in HIV-infected patients with normal ALT levels. METHODS: HCV-HIV coinfected patients (HCV-RNA and anti-HIV positive) with known time of HCV infection (intravenous drugs users) were selected. Patients with hepatitis B surface antigen (HBsAg) positive or hepatitis C treatment before liver biopsy were excluded. Patients were considered to have a normal ALT levels if they had at least 3 normal determinations in the previous 6 months prior to liver biopsy. All patients were submitted to liver biopsy and METAVIR scale was used. RESULTS: Of 50 studied patients 40 (80%) were males. All patients were treated with antiretroviral therapy. The ALT levels were normal in 13 (26%) patients. HCV-HIV co-infected patients with normal ALT levels had presented means of the liver fibrosis stages (0.77±0.44 versus 1.86±1.38; p<0.001) periportal inflammatory activity (0.62±0.77 versus 2.24±1.35; p<0.001) and liver fibrosis progression rate (0.058±0.043 fibrosis unit/year versus 0.118±0.102 fibrosis unit/year) significantly lower as compared to those with elevated ALT. CONCLUSIONS: HCV-HIV coinfected patients with persistently normal ALTs showed slower progression of liver fibrosis. In these patients the development of liver cirrhosis is improbable.