Role of the gp85/Trans-Sialidases in Trypanosoma cruzi Tissue Tropism: Preferential Binding of a Conserved Peptide Motif to the Vasculature In Vivo

Background: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature. Methods: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied. Findings and Conclusions: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.

Interference with Hemozoin Formation Represents an Important Mechanism of Schistosomicidal Action of Antimalarial Quinoline Methanols

Background: The parasitic trematode Schistosoma mansoni is one of the major causative agents of human schistosomiasis, which afflicts 200 million people worldwide. Praziquantel remains the main drug used for schistosomiasis treatment, and reliance on the single therapy has been prompting the search for new therapeutic compounds against this disease. Our group has demonstrated that heme crystallization into hemozoin (Hz) within the S. mansoni gut is a major heme detoxification route with lipid droplets involved in this process and acting as a potential chemotherapeutical target. In the present work, we investigated the effects of three antimalarial compounds, quinine (QN), quinidine (QND) and quinacrine (QCR) in a murine schistosomiasis model by using a combination of biochemical, cell biology and molecular biology approaches. Methodology/Principal Findings: Treatment of S. mansoni-infected female Swiss mice with daily intraperitoneal injections of QN, and QND (75 mg/kg/day) from the 11(th) to 17(th) day after infection caused significant decreases in worm burden (39%-61%) and egg production (42%-98%). Hz formation was significantly inhibited (40%-65%) in female worms recovered from QN- and QND-treated mice and correlated with reduction in the female worm burden. We also observed that QN treatment promoted remarkable ultrastructural changes in male and female worms, particularly in the gut epithelium and reduced the granulomatous reaction to parasite eggs trapped in the liver. Microarray gene expression analysis indicated that QN treatment increased the expression of transcripts related to musculature, protein synthesis and repair mechanisms. Conclusions: The overall significant reduction in several disease burden parameters by the antimalarial quinoline methanols indicates that interference with Hz formation in S. mansoni represents an important mechanism of schistosomicidal action of these compounds and points out the heme crystallization process as a valid chemotherapeutic target to treat schistosomiasis.

Schistosoma mansoni Tegument Protein Sm29 Is Able to Induce a Th1-Type of Immune Response and Protection against Parasite Infection

Background: Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate. Methods and Findings: We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-gamma, TNF-alpha and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins. Conclusion: This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.

A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 angstrom X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7(XAC2622)) and its interaction with VirB9. NMR solution studies show that residues 27-41 of the disordered flexible N-terminal region of VirB7(XAC2622) interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7(XAC2622) has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7(XAC2622) oligomerizes through interactions involving conserved residues in the N0 domain and residues 42-49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB(7XAC2622) oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

Surveillance Programme for Healthcare Associated Infections in the State of Sao Paulo, Brazil. Implementation and the first three years` results

Governmental programmes should be developed to collect and analyse data on healthcare associated infections (HAIs). This study describes the healthcare setting and both the implementation and preliminary results of the Programme for Surveillance of Healthcare Associated Infections in the State of Sao Paulo (PSHAISP), Brazil, from 2004 to 2006. Characterisation of the healthcare settings was carried out using a national database. The PSHAISP was implemented using components for acute care hospitals (ACH) or long term care facilities (LTCF). The components for surveillance in ACHs were surgical unit, intensive care unit and high risk nursery. The infections included in the surveillance were surgical site infection in clean surgery, pneumonia, urinary tract infection and device-associated bloodstream infections. Regarding the LTCF component, pneumonia, scabies and gastroenteritis in all inpatients were reported. In the first year of the programme there were 457 participating healthcare settings, representing 51.1% of the hospitals registered in the national database. Data obtained in this study are the initial results and have already been used for education in both surveillance and the prevention of HAI. The results of the PSHAISP show that it is feasible to collect data from a large number of hospitals. This will assist the State of Sao Paulo in assessing the impact of interventions and in resource allocation. (C) 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Evaluation of biologic occupational risk control practices: Quality indicators development and validation

Background: There is growing demand for the adoption of qualification systems for health care practices. This study is aimed at describing the development and validation of indicators for evaluation of biologic occupational risk control programs. Methods: The study involved 3 stages: (1) setting up a research team, (2) development of indicators, and (3) validation of the indicators by a team of specialists recruited to validate each attribute of the developed indicators. The content validation method was used for the validation, and a psychometric scale was developed for the specialists` assessment. A consensus technique was used, and every attribute that obtained a Content Validity Index of at least 0.75 was approved. Results: Eight indicators were developed for the evaluation of the biologic occupational risk prevention program, with emphasis on accidents caused by sharp instruments and occupational tuberculosis prevention. The indicators included evaluation of the structure, process, and results at the prevention and biologic risk control levels. The majority of indicators achieved a favorable consensus regarding all validated attributes. Conclusion: The developed indicators were considered validated, and the method used for construction and validation proved to be effective.

Care during the continuation phase

As the patient`s treatment progresses, symptoms start to disappear and he or she becomes more familiar with the treatment. The standards in this section focus on the types of elements that need to be considered as the patient progresses from the intensive to the continuation phase of tuberculosis (TB) treatment, leading to less contact with the TB service and a resumption of `normal` activities. Social and psychological as well as physical factors need to be assessed to plan effective care and treatment for the continuation phase. Treatment for TB takes a minimum of 6 months, during which changes to the regimen and personal changes associated with making a recovery can create barriers to continuation of treatment. Lifestyle and other changes that may occur during 6 months of anybody`s life can complicate or be complicated by TB treatment. The patient may move to another location at any point during the course of treatment, in which case it may be necessary to transfer his or her care to another TB management unit. This process needs to be carefully managed to maintain contact with the patient and avoid any break in treatment; this is covered by the third standard in this chapter.

Care during the intensive phase: promotion of adherence

The standards in this chapter focus on maximising the patient`s ability to adhere to the treatment prescribed. Many people are extremely shocked when they are told they have TB, some refuse to accept it and others are relieved to find out what is wrong and that treatment is available. The reaction depends on many factors, including cultural beliefs and values, previous experience and knowledge of the disease. Even though TB is more common among vulnerable groups, it can affect anyone and it is important for patients to be able to discuss their concerns in relation to their own individual context. The cure for TB relies on the patient receiving a full, uninterrupted course of treatment, which can only be achieved if the patient and the health service work together. A system needs to be in place to trace patients who miss their appointments for treatment (late patients). The best success will be achieved through the use of flexible, innovative and individualised approaches. The treatment and care the patient has received will inevitably have an impact on his or her willingness to attend in the future. A well-defined system of late patient tracing is mandatory in all situations. However, when the rates are high (above 10%), any tracing system will be useless without also examining the service as a whole.

Starting treatment: caring for patients and their families

The standards presented in this section focus on providing physical, social and psychological care for the patient at the point he or she is diagnosed with tuberculosis (TB) and starts treatment. Detailed guidance is included with regard to organising directly observed treatment (DOT) safely and acceptably for both the patient and the management unit. The aim is to give the patient the best possible chance of successfully completing treatment according to a regimen recommended by the World Health Organization. If the health service where the patient is diagnosed cannot offer ongoing treatment and care due to a lack of facilities, overcrowding or inaccessibility, the patient needs to be referred to a designated TB management unit (BMU) elsewhere. The patient may also receive treatment from a facility outside a BMU. However care is organised, it is essential for all patients who are diagnosed with TB to be registered at an appropriate BMU so that their progress can be routinely monitored and programme performance can be assessed. To avoid the risk of losing contact with the patient at any stage of their care, good communication is essential between all parties involved, from the patient him/herself to the person supervising their DOT to the BMU.

City tuberculosis control coordinators` perspectives of patient adherence to DOT in Sao Paulo State, Brazil, 2005

SETTING: Thirty-six priority cities in Sao Paulo State, Brazil, with a high incidence of tuberculosis (TB) cases, deaths and treatment default. OBJECTIVE: To identify the perspectives of city TB control coordinators regarding the most important components of adherence strategies adopted by health care teams to ensure patient adherence in 36 priority cities in the State of Sao Paulo, Brazil. DESIGN: Qualitative research with semi-structured interviews conducted with the coordinators of the National TB Control Programme involved in the management of TB treatment services in the public sector. RESULTS: The main issues thought to influence adherence to directly observed treatment (DOT) by coordinators include incentives and benefits delivered to patients, patient-health care worker bonding and comprehensive care, the encouragement given by others to follow treatment (family, neighbours and health professionals), and help provided by health professionals for patients to recover their self-esteem. CONCLUSION: The main aspects mentioned by city TB control coordinators regarding patient adherence to treatment and to DOT in Sao Paulo are improvements in communications, relationships based on trust, a humane approach and including the patients in the decision-making process concerning their health.

Identifying an active case of tuberculosis

The best practice standards set out in chapter 2 of the Best Practice guide focus on the various aspects of identifying an active case of TB and aim to address some of the challenges associated with case detection. The importance of developing a good relationship with the patient from the start, when he or she is often most vulnerable, is emphasised. The first standard focuses on the assessment of someone who might have TB and the second gives detailed guidance about the collection of sputum for diagnosis. The standards are aimed at the health care worker, who assesses the patient when he or she presents at a health care facility and therefore needs to be familiar with the signs, symptoms and risk factors associated with TB. Having suspected TB, the health care worker then needs to ensure that the correct tests are ordered and procedures are followed so that the best quality samples possible are sent to the laboratory and all documentation is filled out clearly and correctly. The successful implementation of these standards can be measured by the accurate and prompt reporting of results, the registration of every case detected and the continued attendance of every patient who needs treatment.

Guidance for the implementation of best practice for the care of patients with tuberculosis

The first two chapters of Best practice for the care of patients with tuberculosis: a guide for low-income countries include an introduction and guidance regarding implementation of best practice. The background to how the guide was developed is significant, as it was developed in collaboration with nurses and other health workers working in the most challenging settings. It therefore provides realistic and practical guidance for best practice where patient loads are large and resources are stretched. Guidance regarding standard setting and clinical audit is an important part of enabling people to recognise the strengths that already exist in their practice and approach those areas that require change in a systematic and practical way. The guide itself consists of a series of standards covering different aspects of patient care, from the moment they seek health care with symptoms to their diagnosis to early stages of treatment, directly observed treatment, the continuation phase and transfer of treatment. There are also standards relating specifically to HIV testing and the care of patients co-infected with tuberculosis and HIV. The standards themselves will appear in full in the subsequent chapters of this series.

HIV testing and care of the patient co-infected with tuberculosis and HIV

Human immunodeficiency virus (HIV) infection poses one of the greatest challenges to tuberculosis (TB) control, with TB killing more people with HIV infection than any other condition. The standards in this chapter cover provider-initiated HIV counselling and testing and the care of HIV-infected patients with TB. All TB patients who have not previously been diagnosed with HIV infection should be encouraged to have an HIV test. Failing to do so is to deny people access to the care and treatment they might need, especially in the context of the wider availability of treatments that prevent infections associated with HIV A clearly defined plan of care for those found to be co-infected with TB and HIV should be in place., with procedures to ensure that the patient has access to this care before offering routine testing for HIV in persons with TB. It is acknowledged that people caring for TB patients should ensure that those who are HIV positive are transferred for the appropriate ongoing care once their TB treatment has been completed. In some cases, referral for specialised HIV-related treatment and care may be necessary during treatment for TB. The aim of these standards is to enable patients to remain as healthy as possible, whatever their HIV status.

Rapid detection of resistance to pyrazinamide in Mycobacterium tuberculosis using the resazurin microtitre assay

Objectives: The resazurin microtitre plate assay (REMA) was evaluated to determine the susceptibility of Mycobacterium tuberculosis to pyrazinamide, and was compared with the broth microdilution method (BMM), the absolute concentration method (ACM) and pyrazinamidase (PZase) determination. Methods: Thirty-four M. tuberculosis clinical isolates (26 susceptible and 8 resistant to pyrazinamide) and reference strains M. tuberculosis H37Rv ATCC 27294 and Mycobacterium bovis AN5 were tested. Results: REMA and BMM showed 100% specificity and sensitivity when compared with ACM; BMM, however, demanded more reading time. The PZase determination assay showed 87.50% and 100% sensitivity and specificity, respectively. Conclusions: All tested methods in this preliminary study showed excellent sensitivity and specificity for the determination of pyrazinamide susceptibility of M. tuberculosis, but REMA was faster, low-cost and easy to perform and interpret. Additional studies evaluating REMA for differentiating pyrazinamide-resistant and-susceptible M. tuberculosis should be conducted on an extended panel of clinical isolates.

Etiology of childhood diarrhea in the northeast of Brazil: significant emergent diarrheal pathogens

In a study conducted in Joao Pessoa, northeast of Brazil, 2344 Escherichia coli isolated from 290 infants with diarrhea and 290 healthy matched controls were analyzed for virulence traits. Enteroaggregative E. coli (EAEC) was the most prevalent pathogen associated to acute diarrhea. Based on the results of colony blot hybridization, serotyping, and HEp-2 cell adherence assays, strains were separated in categories as typical enteropathogenic E. coli (EPEC) (1.7%), atypical EPEC (a-EPEC) (9.3%), EAEC (25%), enterotoxigenic E. coli (10%), and enteroinvasive E. coli (EIEC) (1.4%). No enterohemorrhagic E. coli strains were isolated. Other enteropathogens were found, including Salmonella (7.9%), Shigella spp. (4.1%), thermophilic Campylobacter spp. (2.4%), Giardia lamblia (9.3%), and Entamoeba histolytica (5.8%). All enteropathogens were associated with diarrhea (P < 0.01). However, the association was lower for EPEC and EIEC (P < 0.03). Different pathogens associated with diarrhea may have been changing in Brazil where EAEC and a-EPEC seem to be the most prevalent pathogens among them. (C) 2010 Elsevier Inc. All rights reserved.