Gold nanoprobes for the detection of mutations associated with antibiotic resistance in Mycobacterium tuberculosis complex

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

Resistance to salts of lime and pozzolan mortars

International RILEM Workshop on Repairs Mortars for Historic Masonry, Technical University of Delft, 2009

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

Plasmodium vivax resistance to chloroquine (R2) and mefloquine (R3) in Brazilian Amazon region

We report for the first time a patient with malaria due to Plasmodium vivax who showed R2 resistance to chloroquine and R3 resistance to mefloquine in the Brazilian Amazon region based on WHO clinical criteria for diagnosis of malaria resistance. Failure was observed with unsupervised oral chloroquine, chloroquine under rigorous supervision and mefloquine in the same scheme. Finally, the patient was cured with oral artesunate.

Multiple lymphoid nodules in bone marrow biopsy in immunocompetent patient with cytomegalovirus infection: an immunohistochemical analysis

In Brazil, a high prevalence of cytomegalovirus (CMV) infection has been documented. In immunocompetent adults CMV infection is usually asymptomatic and therefore morphologic and immunophenotypic bone marrow changes have rarely been described. The authors report the case of a previously healthy patient who developed fever of undetermined origin. The diagnosis of acute CMV infection was based on serological testing. A computed tomographic scan showed mediastinal lymphadenopathy. A bone marrow biopsy revealed a hypercellular haematopoiesis with eosinophilia and large mixed T- and B-cell lymphoid aggregates. In spite of bcl-2 positivity, their reactive nature was demonstrated. Polymerase chain reaction (PCR) and immunohistochemistry were unable to detect CMV-DNA in paraffin-embedded bone marrow sections. Much like in other systemic disorders, the lymphoid nodules in this case seemed to be caused by immunological mechanisms, possibly due to cytokines released in response to the systemic infectious process.

The impact of high involvement human resources practices on resistance to change: The mediator effect of commitment to change and the influence of ethical leadership

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Mycetoma fungal infection: multiple organisms as colonizers or pathogens?

We describe a patient with mycetoma or Madura foot, in which histopathological stains of the bone and surface cultures suggested three different organisms including Nocardia species as the cause. Criteria for the diagnosis of the organisms, differentiation between colonizer and pathogen, and significance of mixed infections are discussed.

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.

Hepatitis B virus genotyping among chronic hepatitis B patients with resistance to treatment with lamivudine in the City of Ribeirão Preto, State of São Paulo

INTRODUCTION: Lamivudine is a nucleoside analogue that is used clinically for treating chronic hepatitis B infection. However, the main problem with prolonged use of lamivudine is the development of viral resistance to the treatment. Mutations in the YMDD motif of the hepatitis B virus DNA polymerase gene have been associated with resistance to drug therapy. So far, there have not been many studies in Brazil reporting on genotype-dependent development of resistance to lamivudine. Thus, the aim of the present study was to determine the possible correlation between a certain genotype and increased development of resistance to lamivudine among chronic hepatitis B patients. METHODS: HBV DNA in samples from 50 patients under lamivudine treatment was amplified by means of conventional PCR. Samples were collected at Hospital das Clínicas, FMRP-USP. The products were then sequenced and phylogenetic analysis was performed. RESULTS: Phylogenetic analysis revealed that 29 (58%) patients were infected with genotype D, 20 (40%) with genotype A and one (2%) with genotype F. Mutations in the YMDD motif occurred in 20% of the patients with genotype A and 27.6% of the patients with genotype D. CONCLUSIONS: Despite the small number of samples, our results indicated that mutations in the YMDD motif were 1.38 times more frequent in genotype D than in genotype A.

Antimicrobial resistance and investigation of the molecular epidemiology of Listeria monocytogenes in dairy products

INTRODUCTION: Listeria monocytogenes is a ubiquitous microorganism in nature and is responsible for listeriosis, an infectious disease caused by consumption of contaminated food. METHODS: Molecular characterization was performed on 19 strains of Listeria monocytogenes (serovars 1/2a, 1/2b, 4b and 4c), isolated from dairy products in Rio Grande do Sul, Brazil. The molecular techniques applied were random amplification of polymorphic DNA and restriction enzyme analysis. In addition to the molecular analysis, the antimicrobial resistance profile was determined. RESULTS: The strains studied showed a low degree of diversity. In relation to the antimicrobial resistance profile of those microorganisms from the samples analyzed, all of them were susceptible to the antimicrobials tested. CONCLUSIONS: The molecular techniques that were used presented good discriminatory power for the strains studied. Furthermore, all of the samples that were analyzed were susceptible to the antimicrobials tested.

PCR detection of multiple human herpesvirus DNA in saliva from HIV-infected individuals in Teresina, State of Piauí, Brazil

INTRODUCTION: Human herpesviruses are frequently associated with orofacial diseases in humans (HSV-1, EBV, CMV and HHV-8), some can also cause systemic disease (CMV and HHV-8). The transmission of these viruses occurs by contact with infected secretions, especially saliva. Human immunodeficiency virus infection is associated with an increased risk of HHVs and related diseases. METHODS: This work aimed to detect HSV-1, EBV, CMV and HHV-8 DNA in saliva of HIV-infected patients from Teresina, northeast Brazil, by PCR and compare these findings with age and sex matched HIV-seronegative individuals. RESULTS: No difference in prevalence was verified between HHV detection in the saliva of HIV-seropositive individuals and controls. The individual frequencies of these viruses in these two populations were different. HIV seropositivity correlated positively with the presence of CMV (OR: 18.2, p= 0.00032) and EBV (OR: 3.44, p= 0.0081). No association between CD4 counts and the prevalence of HHVs in the saliva was observed; however, a strong association was determined between seropositivity and the presence of multiple HHV DNAs in saliva (OR: 4.83, p = 0.0028). CONCLUSIONS: These findings suggest the asymptomatic salivary shedding of HHVs is a common event between HIV-seropositive and seronegative individuals from Teresina, Piauí, Brazil, and, especially for HIV-seropositive patients, saliva is a risk factor for the acquisition/transmission of multiple HHVs.

Trends in antimicrobial resistance among clinical isolates of enterococci in a Brazilian tertiary hospital: a 4-year study

INTRODUCTION: In the past two decades members of the genus Enterococcus have emerged as important nosocomial pathogens worldwide. This study prospectively analyzed the distribution of species and trends in antimicrobial resistance among clinical isolates of enterococci in a Brazilian tertiary hospital from 2006-2009. METHODS: Enterococcal species were identified by conventional biochemical tests. The antimicrobial susceptibility profile was performed by disk diffusion in accordance with the Clinical and Laboratory Standards Institute (CLSI). A screening test for vancomycin was also performed. Minimal inhibitory concentration (MIC) for vancomycin was determined using the broth dilution method. Molecular assays were used to confirm speciation and genotype of vancomycin-resistant enterococci (VRE). RESULTS: A total of 324 non-repetitive enterococcal isolates were recovered, of which 87% were E. faecalis and 10.8% E. faecium. The incidence of E. faecium per 1,000 admissions increased significantly (p < 0.001) from 0.3 in 2006 to 2.3 in 2009. The VRE rate also increased over time from 2.5% to 15.5% (p < 0.001). All VRE expressed high-level resistance to vancomycin (MIC >256µg/ mL) and harbored vanA genes. The majority (89.5%) of VRE belonged to E. faecium species, which were characteristically resistant to ampicillin and quinolones. Overall, ampicillin resistance rate increased significantly from 2.5% to 21.4% from 2006-2009. Resistance rates for gentamicin, chloramphenicol, tetracycline, and erythromycin significantly decreased over time, although they remained high. Quinolones resistance rates were high and did not change significantly over time. CONCLUSIONS: The data obtained show a significant increasing trend in the incidence of E. faecium resistant to ampicillin and vancomycin.

Antimicrobial Resistance of Shigella spp. isolated in the State of Pará, Brazil

INTRODUCTION: Shigella spp. are Gram-negative, nonsporulating, rod-shaped bacteria that belong to the family Enterobacteriaceae and are responsible for shigellosis or bacillary dysentery, an important cause of worldwide morbidity and mortality. METHODS: We studied the antibiotic resistance profiles of 122 Shigella spp. strains (81 S. flexneri, 41 S. sonnei, 1 S. boydii) isolated from patients (female and male from 0 to 80 years of age) presenting diarrhea in different districts of the State of Pará, in the North of Brazil. The antibiotic resistance of the strains, isolated from human fecal samples, was determined by the diffusion disk method and by using the VITEK-2 system. RESULTS: The highest resistance rate found was the resistance rate to tetracycline (93.8%), followed by the resistance rate to chloramphenicol (63.9%) and to trimethoprim/sulfamethoxazole (63.1%). Resistance to at least three drugs was more common among S. flexneri than S. sonnei (39.5% vs. 10%). Six (4.9%) strains were susceptible to all the antibiotics tested. All strains were susceptible to cefotaxime, ceftazidime, ciprofloxacin, nalidixic acid and nitrofurantoin. CONCLUSIONS: High rates of multidrug resistance in Shigella spp. are a serious public health concern in Brazil. It is extremely important to continuously monitor the antimicrobial resistances of Shigella spp. for effective therapy and control measures against shigellosis.

Drug resistance of Mycobacterium tuberculosis strains in southern Brazil

INTRODUCTION: The aim of this work was to evaluate the prevalence of Mycobacterium tuberculosis (MT) strains with mutations that could result in resistance to the main drugs used in treatment in a region with one of the highest numbers of tuberculosis (TB) cases in southern Brazil. METHODS: Deoxyribonucleic acid (DNA) from 120 sputum samples from different patients suspicious of pulmonary tuberculosis who attended the Municipal Public Laboratory for Mycobacterium sp. diagnosis was directly amplified and analyzed by PCR-SSCP. The DNA was amplified in known hotspot mutation regions of the genes rpoB, ahpC, embB, katG, inhA, and pncA. RESULTS: The percentage of samples positive by culture was 9.2% (11/120); 5% (6/120) were positive by bacilloscopy and MT-PCR, and DNA fragments of the aforementioned resistance genes could be amplified from seven (7) of the eleven (11) samples with positive results, either by culture or PCR/bacilloscopy. All presented a SSCP pattern similar to a native, nonresistant genotype, with the ATCC strain 25177 as control, except for one sample (0.01%), which presented a SSCP profile demonstrating mutation at the embB gene. CONCLUSIONS: These results are consistent with the empirical observations by physicians treating TB patients in our region of a low occurrence of cases that are refractory to conventional treatment schemes, in contrast to other parts of the country. Continued surveillance, especially molecular, is essential to detect and monitor the outbreak of MT-resistant strains.